Therapeutic agent comprising recombinant oncolytic vaccinia virus and antibody-drug conjugate and use thereof

Abstract

A therapeutic agent comprising a recombinant oncolytic vaccinia virus and an antibody-drug conjugate, and use thereof in the preparation of a drug for treating tumors and / or cancers. The recombinant oncolytic vaccinia virus can be selectively replicated in tumor cells; an exogenous IL-21 gene is integrated into the genome of the recombinant oncolytic vaccinia virus, and the IL-21 gene can be expressed in the tumor cells. The recombinant oncolytic vaccinia virus can enhance the efficacy of the antibody-drug conjugate. The combined use of the recombinant oncolytic vaccinia virus and the antibody-drug conjugate can show an anti-tumor synergistic effect, and can reduce the toxic side effects of the recombinant oncolytic vaccinia virus and the antibody-drug conjugate, thereby improving safety.

Description

Therapeutic agents containing recombinant oncolytic poxviruses and antibody-drug conjugates and their uses Technical Field

[0001] This invention belongs to the field of biomedicine, and more specifically, relates to therapeutic agents comprising recombinant oncolytic poxvirus and antibody-drug conjugates and their uses. Background Technology

[0002] Cancer is a significant public health issue worldwide. With an aging population, unhealthy lifestyle habits, and various social and environmental factors, the number of cancer cases and deaths is projected to continue increasing over the next decade. Current treatments for malignant tumors primarily include surgery, radiotherapy, chemotherapy, biotherapy, and immunotherapy.

[0003] Oncolytic virus therapy falls under the category of biotherapy. As early as the late 19th century, it was discovered that various viruses could slow the progression of tumors, suggesting the potential of viruses in cancer treatment. With the development of gene technology, the genome structure of viruses can be altered to selectively replicate within tumor cells, enhancing their oncolytic targeting. In the past decade, researchers have used techniques such as gene recombination, gene transfer, and gene knockout to genetically modify viruses such as adenoviruses, herpesviruses, microRNA viruses, and poxviruses, developing a series of oncolytic virus products. To date, dozens of products have entered different stages of clinical trials. In 2005, the China Food and Drug Administration (CFDA) approved the genetically modified oncolytic adenovirus H101 from Shanghai Sanwei Biotechnology Co., Ltd. for the treatment of head and neck tumors, making it the world's first marketed oncolytic virus drug. Ten years later, in 2015, the second oncolytic virus drug, Amgen's genetically modified oncolytic herpes simplex virus T-Vec, was approved by the US FDA and the EU EMA for the treatment of advanced malignant melanoma. Furthermore,

[0004] In 2023, herpes simplex virus Delytact was approved in Japan for the treatment of advanced malignant gliosis. Currently, there are no genetically modified oncolytic poxviruses available as drugs on the market.

[0005] In addition to these, tumor immunotherapy is also a very important approach in the fight against cancer. It mainly includes antibody therapy, cell therapy, and tumor vaccines. Antibody drug conjugates (ADCs) are large-molecule targeted drugs that have shown excellent efficacy and potential in the field of cancer treatment, becoming a new hot topic in drug development. ADCs are typically composed of monoclonal antibodies conjugated to a payload (including small toxin molecules) via a linker, combining the high targeting power of monoclonal antibodies with the high activity of small-molecule drugs (e.g., small molecule drugs). Currently, 16 ADC drugs have been approved for marketing globally, and more than 140 ADC drug candidates are in clinical research stages.

[0006] Although ADCs have shown significant efficacy in treating hematologic malignancies, breast cancer, urothelial carcinoma, and gastric cancer, their efficacy in treating a wider range of indications remains unsatisfactory. For example, currently, there are over ten ADCs targeting pancreatic cancer in various stages of clinical trials. The clinical trial of trastuzumab DS-8201 in HER2-expressing advanced pancreatic cancer (NCT04482309) confirmed an objective response rate (ORR) of only 4%; trastuzumab emtansine T-DM1 underwent a phase II clinical trial (NCT02999672), but the trial was terminated early and no efficacy data for pancreatic cancer were published. Therefore, there is an urgent need to explore new treatment strategies to further improve the efficacy of ADCs in treating more indications.

[0007] Furthermore, due to the multiple combinations of antibody-linker-toxin molecules, ADC drugs have a higher incidence and severity of toxic side effects than general monoclonal antibodies and small molecules. Among the dozen or so ADC drugs approved by the FDA, only two did not have black box warnings, while the rest were warned due to side effects such as severe hepatotoxicity, ocular toxicity, severe skin reactions, neutropenia, progressive multifocal leukoencephalopathy, and interstitial lung disease (ILD). A meta-analysis covering 169 clinical trials and 22,492 patients (see “Zhu Y, et al. Treatment-related adverse events of antibody-drug conjugates in clinical trials: A systematic review and meta-analysis. Cancer. 2023 Jan 15; 129(2):283-295.”) showed that the overall incidence of adverse reactions to ADC treatment was 91.2%, of which the incidence of ≥ grade 3 adverse reactions was 46.1%. Currently, the dose-limiting toxicities of most ADCs are usually related to off-target toxicities, limiting the dose of ADCs to below the optimal anticancer dose level. Therefore, the challenge remains how to reduce the toxicity of ADC drugs while ensuring their efficacy, thereby improving their safety.

[0008] Currently, in the immunotherapy of tumors and / or cancers, the medical needs of patients for treatments with stronger efficacy and lower toxicity remain unmet, especially for patients with advanced tumors who are unresponsive to or resistant to existing therapies. Therefore, the development of innovative products and the exploration of more effective and safer new therapies are imperative. Summary of the Invention

[0009] To address the problems existing in the prior art, the present invention provides a therapeutic agent comprising recombinant oncolytic poxvirus and antibody-drug conjugate and its uses.

[0010] Specifically, the present invention provides:

[0011] (1) Use of a therapeutic agent comprising a recombinant oncolytic poxvirus and an antibody-drug conjugate in the preparation of a medicament for treating tumors and / or cancer; wherein the recombinant oncolytic poxvirus is capable of selectively replicating in tumor cells, and the genome of the recombinant oncolytic poxvirus is integrated with an exogenous IL-21 gene, and the IL-21 gene is capable of being expressed in the tumor cells.

[0012] (2) Use of recombinant oncolytic poxvirus in the preparation of a medicament for enhancing the sensitivity of an antibody-drug conjugate to tumors and / or cancer; wherein the recombinant oncolytic poxvirus and the antibody-drug conjugate are used in combination; and wherein the recombinant oncolytic poxvirus is capable of selectively replicating in tumor cells, and wherein the genome of the recombinant oncolytic poxvirus is integrated with an exogenous IL-21 gene, and the IL-21 gene is capable of being expressed in the tumor cells.

[0013] (3) According to the use described in (1) or (2), the recombinant oncolytic poxvirus is a TK gene and VGF gene defective type; the TK gene is defective by inserting a foreign nucleotide sequence, and / or the VGF gene is defective by gene knockout or insertion of a foreign nucleotide sequence; preferably, the foreign IL-21 gene is inserted into the TK gene, thereby causing the TK gene to be defective.

[0014] (4) As described in (1) or (2), the recombinant oncolytic poxvirus is a Wyeth strain or a WR strain.

[0015] (5) According to the use described in (1) or (2), the genome of the recombinant oncolytic poxvirus also integrates an exogenous selection gene, which includes the gpt gene but does not include the fluorescent protein gene.

[0016] (6) According to the use described in (1) or (2), the target of the antibody-drug conjugate is selected from: EGFR, HER2, HER3, TROP2, PSMA, TF, Nectin-4, FRα, CD19, CD20, CD22, CD33, CD79b, BCMA, CD30, F3, CEACAM5, FOLH1, AXL, IL2RA, GUCY2C, CLDN18, SLC39A6, ROR2, MET, ENPP3, CD70, GPR20, TPBG, SLC44A4, MUC1, CD276, CD38, CD37, CD22, CA9, NaPi2b, or combinations thereof.

[0017] (7) As described in (1) or (2), the antibody-drug conjugate is selected from: Gemtuzumab Ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox, Polotuzumab vedotin, Enfortumab Vedotin, Trastuzumab deruxtecan, Sacituzumab govitecan, Disitamab vedotin, Belantamab mafodotin, Loncastuximab tesirine, Tisotumab Vedotin), Mirvetuximab soravtansine, Cetuximab saratolacan sodium, Sacituzumab Tirumotecan, 9MW2821, A166, ABBV-011, ABBV-154, ABBV-319, ABBV-400, ABBV-637, ABL202, ADCT-602, ADCT-901, AGS62P1, ALT-P7, AMT-151, Anetumab ravtansine, AOC 1001, AOC 1020, AOC 1044, ARX305, ARX517, ARX788, ASN004, AURIXIM, AVID100, AZD8205, B003, BAT8006, BAT8008, BAT8009, BAT8010, BB-1701, BB-1705, BI-CON-02, BIO-106, BL-B01 D1, BL-M02D1, BL-M07D1, BYON3521, Cami (camidanlumab tesirine), CMG901, cofetuzumab pelidotin, CX-2029, datopotamabderuxtecan), DB-1303, DB-1305, DP303c, DS-6000a, DS-9606a, DX126-262, DXC-004, DXC-005, DXC-007, DYNE-101, DYNE-251, ESG-401, F0002-ADC, MORAb-202 (farletuzumab ecteribulin), FDA018, FDA022, FOR46, FS-1502, GB251, GQ1001, GQ1007, HDP-101, HS-20089, HS-20093, HS630, HTI-1066, IBI-343, I-DXd(ifinatamab deruxtecan), IKS03, IMGC936, IMGN151, indatuximab ravtansine, BMS-986352 (ispectamab debotansine), JBH492, JS107, JS108, JSKN-003, SGN-LIV1A (ladiratuzumab vedotin), L-DOS47, LM-102, W0101 (lonigutamab ugodotin), Luvelta (luveltamab tazevibulin), M1231, M9140, BA-3011 (mecbotamab vedotin), MGTA-117, ADCT-601 (mipasetamab uzoptirine), ABBV-155(mirzotamab clezutoclax), MRG001, MRG003, MRG004a, MT-8633, naratuximab emtansine, NBE-002, NBT508, NBT828, OBI-999, OBT076, OMTX705, ORM-5029, CAB-ROR2-ADC (ozuriftamab vedotin), HER3-DXd (patritumab deruxtecan), PF-06804103, IMGN632 (pivekimab sunirine), praluzatamab ravtansine, PRO1184, PSMA ADC, RC88, RC108, RC118, RC248, REGN5093-M114, RG7861, serclutamabtalirine), SGN-ALPV, SGN-B6A, SGN-B7H4V, SGN-CD228A, SGN-PDL1 V, SGN-STNV, SHR-A1201, SHR-A1904, SHR-A1912, SHR-A1921, SHR-A2009, SKB315, SOT102, STI-3258, STI-6129, STRO-001, SYD1875, SYS6002, SYSA1801, TAA013, TAC-001, TAK-164, TAK-500, telisotuzumab vedotin, TORL-1-23, TORL-2-307-ADC, TPX-4589, trastuzumab The following are listed: duocarmazine, BDC-1001 (trastuzumab imbotolimod), SHR-A1811 (trastuzumab rezetecan), trastuzumab vedotin, TRPH-222, TRS005, tusamitamab ravtansine, XMT-1536 (upifitamab rilsodotin), vobra duo (vobramitamab duocarmazine), XB002, XMT-1660, XMT-2056, YL201, ZW49 (zanidatamab zovodotin), MK-2140 (zilovertamab vedotin), and / or ZV0203.

[0018] (8) According to the use described in (1) or (2), wherein the therapeutic agent comprises 1 × 10 2 -5×10 9 The recombinant oncolytic poxvirus was administered at a PFU / day dose.

[0019] (9) According to the use described in (1) or (2), wherein the recombinant oncolytic poxvirus is formulated for administration by intratumoral injection, intraperitoneal administration, intrapleural administration or intravenous administration; and the antibody-drug conjugate is formulated for administration by intravenous administration.

[0020] (10) According to the use described in (1) or (2), the tumor and / or cancer includes: lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphoma, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, and testicular cancer.

[0021] (11) A therapeutic agent comprising a recombinant oncolytic poxvirus and an antibody-drug conjugate; wherein the recombinant oncolytic poxvirus is capable of selectively replicating in tumor cells, and wherein the genome of the recombinant oncolytic poxvirus is integrated with an exogenous IL-21 gene, and the IL-21 gene is capable of being expressed in the tumor cells.

[0022] (12) The therapeutic agent according to (11), wherein the recombinant oncolytic poxvirus is a TK gene and VGF gene defective type; the TK gene is defective by inserting a foreign nucleotide sequence, and / or the VGF gene is defective by gene knockout or insertion of a foreign nucleotide sequence; preferably, the foreign IL-21 gene is inserted into the TK gene, thereby causing the TK gene to be defective.

[0023] (13) The therapeutic agent according to (11), wherein the recombinant oncolytic poxvirus is Wyeth strain or WR strain.

[0024] (14) According to the therapeutic agent described in (11), the genome of the recombinant oncolytic poxvirus also integrates an exogenous selection gene, which includes the gpt gene but does not include the fluorescent protein gene.

[0025] (15) According to the therapeutic agent described in (11), the target of the antibody-drug conjugate is selected from: EGFR, HER2, HER3, TROP2, PSMA, TF, Nectin-4, FRα, CD19, CD20, CD22, CD33, CD79b, BCMA, CD30, F3, CEACAM5, FOLH1, AXL, IL2RA, GUCY2C, CLDN18, SLC39A6, ROR2, MET, ENPP3, CD70, GPR20, TPBG, SLC44A4, MUC1, CD276, CD38, CD37, CD22, CA9, NaPi2b, or combinations thereof.

[0026] (16) The therapeutic agent according to (11), wherein the antibody-drug conjugate is selected from: Gemtuzumab Ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox, Polotuzumab vedotin, Enfortumab Vedotin, Trastuzumab deruxtecan, Sacituzumab govitecan, Disitamab vedotin, Belantamab mafodotin, Loncastuximab tesirine, Tisotumab Vedotin), Mirvetuximab soravtansine, Cetuximab saratolacan sodium, Sacituzumab Tirumotecan, 9MW2821, A166, ABBV-011, ABBV-154, ABBV-319, ABBV-400, ABBV-637, ABL202, ADCT-602, ADCT-901, AGS62P1, ALT-P7, AMT-151, Anetumab ravtansine, AOC 1001, AOC 1020, AOC 1044, ARX305, ARX517, ARX788, ASN004, AURIXIM, AVID100, AZD8205, B003, BAT8006, BAT8008, BAT8009, BAT8010, BB-1701, BB-1705, BI-CON-02, BIO-106, BL-B01 D1, BL-M02D1, BL-M07D1, BYON3521, Cami (camidanlumab tesirine), CMG901, cofetuzumab pelidotin, CX-2029, datopotamabderuxtecan), DB-1303, DB-1305, DP303c, DS-6000a, DS-9606a, DX126-262, DXC-004, DXC-005, DXC-007, DYNE-101, DYNE-251, ESG-401, F0002-ADC, MORAb-202 (farletuzumab ecteribulin), FDA018, FDA022, FOR46, FS-1502, GB251, GQ1001, GQ1007, HDP-101, HS-20089, HS-20093, HS630, HTI-1066, IBI-343, I-DXd(ifinatamab deruxtecan), IKS03, IMGC936, IMGN151, indatuximab ravtansine, BMS-986352 (ispectamab debotansine), JBH492, JS107, JS108, JSKN-003, SGN-LIV1A (ladiratuzumab vedotin), L-DOS47, LM-102, W0101 (lonigutamab ugodotin), Luvelta (luveltamab tazevibulin), M1231, M9140, BA-3011 (mecbotamab vedotin), MGTA-117, ADCT-601 (mipasetamab uzoptirine), ABBV-155(mirzotamab clezutoclax), MRG001, MRG003, MRG004a, MT-8633, naratuximab emtansine, NBE-002, NBT508, NBT828, OBI-999, OBT076, OMTX705, ORM-5029, CAB-ROR2-ADC (ozuriftamab vedotin), HER3-DXd (patritumab deruxtecan), PF-06804103, IMGN632 (pivekimab sunirine), praluzatamab ravtansine, PRO1184, PSMA ADC, RC88, RC108, RC118, RC248, REGN5093-M114, RG7861, serclutamabtalirine), SGN-ALPV, SGN-B6A, SGN-B7H4V, SGN-CD228A, SGN-PDL1 V, SGN-STNV, SHR-A1201, SHR-A1904, SHR-A1912, SHR-A1921, SHR-A2009, SKB315, SOT102, STI-3258, STI-6129, STRO-001, SYD1875, SYS6002, SYSA1801, TAA013, TAC-001, TAK-164, TAK-500, telisotuzumab vedotin, TORL-1-23, TORL-2-307-ADC, TPX-4589, trastuzumab The following are listed: duocarmazine, BDC-1001 (trastuzumab imbotolimod), SHR-A1811 (trastuzumab rezetecan), trastuzumab vedotin, TRPH-222, TRS005, tusamitamab ravtansine, XMT-1536 (upifitamab rilsodotin), vobra duo (vobramitamab duocarmazine), XB002, XMT-1660, XMT-2056, YL201, ZW49 (zanidatamab zovodotin), MK-2140 (zilovertamab vedotin), and / or ZV0203.

[0027] (17) The therapeutic agent according to (11), wherein the therapeutic agent comprises 1×10 2 -5×10 9 The recombinant oncolytic poxvirus was administered at a PFU / day dose.

[0028] (18) The therapeutic agent according to (11), wherein the recombinant oncolytic poxvirus is formulated for administration by intratumoral injection, intraperitoneal administration, intrapleural administration or intravenous administration; and the antibody-drug conjugate is formulated for administration by intravenous administration.

[0029] (19) A method for treating tumors and / or cancer, comprising:

[0030] 1) Administration of recombinant oncolytic poxvirus according to any one of (1)-(10) to patients with tumors and / or cancer; and

[0031] 2) Administer the antibody-drug conjugate according to any one of (1)-(10) to the patient with the tumor and / or cancer.

[0032] (20) The method according to (19) includes the following steps performed in sequence:

[0033] 1) Administer the recombinant oncolytic poxvirus to the patient with the tumor and / or cancer;

[0034] 2) After administration of the recombinant oncolytic poxvirus, the antibody-drug conjugate is administered to the tumor and / or cancer patient.

[0035] (21) According to the method of (19), the tumor and / or cancer includes: lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphoma, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, and testicular cancer.

[0036] Compared with the prior art, the present invention has the following advantages and positive effects:

[0037] This invention combines recombinant oncolytic poxvirus with antibody-drug conjugates (ADCs) to treat tumors and / or cancers. This combined strategy integrates the direct tumor lysis and immune activation functions of the recombinant oncolytic poxvirus described in this invention with the targeted killing properties of the antibody-drug conjugate. Furthermore, the recombinant oncolytic poxvirus enhances the sensitivity of the antibody-drug conjugate to tumor cells, thereby enhancing therapeutic efficacy and improving safety. The main mechanisms of action and effects of this invention include: 1) For tumors with high expression of the antibody-drug conjugate target, the recombinant oncolytic poxvirus, after infecting and lysing tumor cells, alters the tumor microenvironment, increasing the killing effect of the antibody-drug conjugate on tumors; 2) For tumors with low expression of the antibody-drug conjugate target, the recombinant oncolytic poxvirus, after infecting and lysing tumor cells, increases the target exposure of the antibody-drug conjugate, thereby enhancing the sensitivity of the antibody-drug conjugate to tumor cells that are insensitive due to low target expression; 3) The recombinant oncolytic poxvirus improves the tumor microenvironment by activating the immune system, thus facilitating the ADCC (antibody-dependent cell-mediated antibody-drug conjugate). The recombinant oncolytic poxvirus provides support for antibody-dependent cell-mediated cytotoxicity (ADCC) effects; 4) after infecting tumor cells, the recombinant oncolytic poxvirus shows the ability to activate NK cells and increase NK cell granzyme secretion, thereby enhancing NK cell-mediated ADCC; 5) after altering the tumor microenvironment structure, the recombinant oncolytic poxvirus enhances the bystander effect of toxins released by ADCs; 6) the recombinant oncolytic poxvirus significantly increases the diffusion of co-administered antibody-drug conjugates in tumor tissues, while allowing it to exert its original direct lysis and killing effect on tumor cells, thereby further improving the antitumor effect.

[0038] Therefore, this invention discovers that: 1) When recombinant oncolytic poxvirus is used in combination with antibody-drug conjugates (ADCs), the recombinant oncolytic poxvirus can directly induce tumor lysis and has immune-activating function, thereby increasing the sensitivity of the ADC to tumor cells, enhancing its efficacy, and expanding its indications. Therefore, the recombinant oncolytic poxvirus described in this invention can assist the efficacy of ADCs, and the combination of the two can demonstrate a synergistic anti-tumor effect. 2) Even when recombinant oncolytic poxvirus and ADCs are used at relatively low doses, significant synergistic effects can be observed. Therefore, this invention can reduce the toxic side effects of recombinant oncolytic poxvirus and ADCs respectively, improving their safety.

[0039] In particular, the combination strategy of this invention has shown significant synergistic inhibitory effects in the treatment of pancreatic cancer and gastric cancer, respectively. The present invention combines recombinant oncolytic poxvirus with antibody-drug conjugates, resulting in significantly better anti-tumor growth effects in a human pancreatic cancer model than either recombinant oncolytic poxvirus alone or antibody-drug conjugate alone; and the combination of recombinant oncolytic poxvirus with antibody-drug conjugates also shows significantly better anti-tumor effects in a human gastric cancer model than either recombinant oncolytic poxvirus alone or antibody-drug conjugate alone. Furthermore, this combination therapy has a good safety profile. Attached Figure Description

[0040] Figure 1 shows the weight changes of mice in each group in Example 1 of this application. The X-axis represents the time after drug administration, and the Y-axis represents the mouse weight.

[0041] Figure 2 shows the average tumor volume growth trend of mice in each group in Example 1 of this application. The X-axis represents the time after drug administration, and the Y-axis represents the tumor volume.

[0042] Figure 3 shows the relative tumor volume growth trend of each group of mice in Example 1 of this application. The X-axis represents the time after drug administration, and the Y-axis represents the relative tumor volume (RTV).

[0043] Figure 4 shows the trend of relative tumor proliferation rate (T / C%) in each group of mice in Example 1 of this application. The X-axis represents the time after drug administration, and the Y-axis represents the T / C (%).

[0044] Figure 5 shows the average tumor weight of mice in each group on day 24 in Example 1 of this application. The X-axis represents different groups, and the Y-axis represents tumor weight.

[0045] Figure 6 shows the weight changes of mice in each group in Example 2 of this application. The X-axis represents the time after drug administration, and the Y-axis represents the mouse weight.

[0046] Figure 7 shows the average tumor volume growth trend of mice in each group in Example 2 of this application. The X-axis represents the time after drug administration, and the Y-axis represents the tumor volume.

[0047] Figure 8 shows the relative tumor volume growth trend of each group of mice in Example 2 of this application. The X-axis represents the time after drug administration, and the Y-axis represents the relative tumor volume (RTV).

[0048] Figure 9 shows the trend of relative tumor proliferation rate (T / C%) in each group of mice in Example 2 of this application. The X-axis represents time after drug administration, and the Y-axis represents the relative tumor proliferation rate (%).

[0049] Figure 10 shows the average tumor weight of mice in each group on day 56 in Example 2 of this application. The X-axis represents different groups, and the Y-axis represents tumor weight.

[0050] Figure 11 shows the expression levels of granzyme B protein in each group in Example 3 of this application. The X-axis represents different groups, and the Y-axis represents the expression level of granzyme B protein.

[0051] Figure 12 shows the expression levels of granzyme B protein in each group in Example 4 of this application. The X-axis represents different groups, and the Y-axis represents the expression level of granzyme B protein.

[0052] In the figure, * indicates p < 0.05, ** indicates p < 0.01, and *** indicates p < 0.001. Detailed Implementation

[0053] The present invention will be further described below with reference to the accompanying drawings through specific embodiments. However, this is not a limitation of the present invention. Those skilled in the art can make various modifications or improvements based on the basic idea of ​​the present invention, but as long as they do not depart from the basic idea of ​​the present invention, they are all within the scope of the present invention.

[0054] In this invention, the terms “tumor,” “cancer,” “tumor cell,” and “cancer cell” encompass the meanings commonly understood in the art.

[0055] The terms "oncolytic virus" or "oncolytic poxvirus" used in this article refer to viruses or poxviruses that can selectively replicate in and lyse tumor cells.

[0056] As used herein, the term "therapeuticly effective amount" refers to the amount of a functional pharmaceutical agent or composition that enables it to exhibit a detectable therapeutic or inhibitory effect, or to achieve an antitumor effect. This effect can be detected by any known testing method in the art.

[0057] As used in this article, the terms “drug administration” or “application” refer to the provision of a compound, complex, or composition (including viral and antibody-drug conjugates) to a subject.

[0058] As used herein, the term "patient" refers to a human or non-human organism. Therefore, the methods and compositions described herein are applicable to both human and animal diseases. In some embodiments, the patient suffers from a tumor and / or cancer. In some examples, the patient simultaneously suffers from one or more types of tumors and / or cancers.

[0059] The term "synergistic effect" as used in this article refers to the combined effect of two or more drugs, which is greater than the sum of the individual effects of each drug.

[0060] The term “pfu” or “plague forming unit” as used in this article refers to the amount of virus that produces one plaque, which is called a plaque forming unit (pfu).

[0061] The term "MOI" or "Multiplicity of Infection" used in this article refers to the ratio of virus to cells, which is the number of virus particles used to initiate viral infection in each cell. MOI = PFU / cell, that is, number of cells × MOI = total PFU.

[0062] The term "antibody drug conjugate" (ADC) used in this article refers to a combination of monoclonal antibody targeted therapy and traditional chemotherapy, possessing both high selectivity for targeting and the potent killing power of chemotherapy. These drugs are designed to provide precise, targeted anti-cancer therapy, specifically attacking and destroying cancer cells. An ADC consists of three parts: an antibody, a payload (e.g., cytotoxin), and a linker connecting the two. The antibody is selective for tissue-specific antigens that are poorly expressed or absent on normal cells. The cytotoxin has high broad-spectrum cytotoxicity; after being phagocytosed by target cells, it is released to kill the target cells. The linker stabilizes the ADC drug in the bloodstream; upon entering the target cells, it is cleaved in lysosomes, rapidly releasing the toxin to kill the target cells. The mechanism of action of ADCs generally consists of three steps: First, the antibody portion of the ADC binds to the antigen on the target cell; second, the target cell engulfs the ADC, releasing the toxin; finally, the toxin damages DNA or inhibits cell division, ultimately killing the target cell.

[0063] This invention designs a combination immunotherapy for treating tumors and / or cancers (e.g., solid tumors, and even advanced solid tumors) using recombinant oncolytic poxviruses (e.g., recombinant human IL-21 oncolytic poxvirus injection (hV01)) in combination with antibody-drug conjugates (e.g., trastuzumab and vedicetuzumab). This combination strategy combines the direct tumor lysis and immune activation functions of the recombinant oncolytic poxviruses described in this invention with the targeted killing properties of antibody-drug conjugates. Furthermore, the recombinant oncolytic poxviruses described in this invention can enhance the sensitivity of antibody-drug conjugates to tumor cells, thereby enhancing the therapeutic effect, improving safety, and demonstrating a synergistic anti-tumor effect.

[0064] Therefore, one aspect of the present invention provides the use of a therapeutic agent comprising a recombinant oncolytic poxvirus and an antibody-drug conjugate in the preparation of a medicament for treating tumors and / or cancer; wherein the recombinant oncolytic poxvirus is capable of selectively replicating in tumor cells, and wherein the genome of the recombinant oncolytic poxvirus is integrated with an exogenous IL-21 gene, and the IL-21 gene is capable of being expressed in the tumor cells.

[0065] Another aspect of the present invention provides the use of a recombinant oncolytic poxvirus in the preparation of a medicament for enhancing the sensitivity of an antibody-drug conjugate to tumors and / or cancers; wherein the recombinant oncolytic poxvirus and the antibody-drug conjugate are used in combination; and wherein the recombinant oncolytic poxvirus is selectively capable of replicating in tumor cells, and wherein the genome of the recombinant oncolytic poxvirus is integrated with an exogenous IL-21 gene, and the IL-21 gene is capable of being expressed in the tumor cells.

[0066] The drugs used to enhance the sensitivity of antibody-drug conjugates to tumors and / or cancers include, for example, antibody-drug conjugate adjuvants, antibody-drug conjugate enhancers, antibody-drug conjugate combination agents, etc., which are intended to enable the recombinant oncolytic poxvirus of the present invention to be used in combination with the antibody-drug conjugates to achieve a synergistic effect between the two.

[0067] Another aspect of the present invention provides a therapeutic agent comprising a recombinant oncolytic poxvirus and an antibody-drug conjugate; wherein the recombinant oncolytic poxvirus is capable of selectively replicating in tumor cells, and the genome of the recombinant oncolytic poxvirus integrates an exogenous IL-21 gene, which is capable of being expressed in tumor cells.

[0068] Another aspect of the present invention provides a method for treating tumors and / or cancer, comprising:

[0069] 1) Administration of the recombinant oncolytic poxvirus to patients with tumors and / or cancer; and

[0070] 2) Administer the antibody-drug conjugate to the tumor and / or cancer patient.

[0071] In various aspects of the invention, preferably, the recombinant oncolytic poxvirus is a TK gene and VGF gene defective type.

[0072] The term "functional defect" used in this invention when referring to the gene of an oncolytic virus means that the oncolytic virus is unable to perform the function that the gene should have, i.e., loss of function, which can be achieved, for example, by inserting a foreign fragment into the gene or knocking out the gene.

[0073] Therefore, a foreign nucleotide sequence can be inserted into the TK gene, thereby causing it to be functionally defective. Similarly, a foreign nucleotide sequence can be inserted into the VGF gene and / or the gene can be knocked out, thereby causing it to be functionally defective.

[0074] Preferably, the exogenous IL-21 gene is inserted into the TK gene, which causes the TK gene to be functionally defective and to express the IL-21 gene after infecting tumor cells. The specific preparation method is as described in Chinese Patent Publication CN109554353A, the entire text of which is incorporated herein by reference.

[0075] In various aspects of the present invention, the poxviruses that can be used include Wyeth strains or WR strains, an example of which is VSC20.

[0076] In a preferred embodiment, the recombinant oncolytic vaccinia virus is obtained by genetically modifying the VSC20 vaccinia virus. The VSC20 vaccinia virus is a vaccinia virus with the VGF gene deleted, wherein the LacZ gene is inserted at the C11R site. The preparation method can be found in the scientific literature: "McCart, JA, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res (2001) 61: 8751–8757." The genetic modification involves inserting an exogenous IL-21 gene into the TK gene of the VSC20 vaccinia virus, thereby rendering the TK gene functionally defective. Plasmids for inserting the IL-21 gene into the TK gene of the VSC20 vaccinia virus are known, and the insertion is achieved through a recombination mechanism.

[0077] In various aspects of the present invention, the genome of the recombinant oncolytic poxvirus may also integrate exogenous selection genes, including the gpt (guanine phosphoribosyltransferase) gene and / or the LacZ gene, but excluding fluorescent protein genes, in order to avoid safety risks caused by the expression of fluorescent proteins in patients.

[0078] The genome of the recombinant oncolytic poxvirus may also not contain integrated exogenous selection genes.

[0079] In some embodiments, the present invention constructs an oncolytic virus by using the vaccinia virus early / late promoter p7.5 to control the gpt gene and the artificially synthesized vaccinia virus early promoter pSEL to control the exogenous IL-21 gene, and by using in vitro intracellular recombination technology to insert the gpt and IL-21 genes into the TK gene region of the vaccinia virus VSC20 strain. The two promoters initiate the expression of their respective regulated genes in a back-to-back manner.

[0080] In various aspects of the present invention, the exogenous IL-21 gene can be inserted into the TK gene to cause it to be functionally defective, and the VGF gene of the oncolytic poxvirus can also be functionally defective, thereby enabling the oncolytic virus to selectively replicate in tumor cells, kill tumor cells, and induce a subsequent immune response. Furthermore, the exogenous IL-21 gene carried by the oncolytic poxvirus can directly induce anti-tumor immune effects.

[0081] Preferably, the exogenous IL-21 gene is derived from mice or humans.

[0082] The recombinant oncolytic poxvirus described in this invention can be obtained using known methods in the field of bioengineering.

[0083] In various aspects of this invention, the targets of the antibody-drug conjugates described herein include (but are not limited to): EGFR, HER2, HER3, TROP2, PSMA, TF (tissue factor), Nectin-4, FRα (folate receptor α), CD19, CD20, CD22, CD33, CD79b, BCMA, CD30, F3, CEACAM5, FOLH1, AXL, IL2RA, GUCY2C, CLDN18, SLC39A6, ROR2, MET, ENPP3, CD70, GPR20, TPBG(5T4), SLC44A4, MUC1, CD276, CD38, CD37, CD22, CA9, NaPi2b, etc., or combinations thereof.

[0084] In various aspects of the present invention, the linkers of the antibody-drug conjugates of the present invention include (but are not limited to): cleavable or non-cleavable.

[0085] In various aspects of this invention, the toxic molecules of the antibody-drug conjugates described herein include (but are not limited to): microtubule inhibitors (including orlistatins and maytansine alkaloids (DMs)); DNA synthesis inhibitors (including calicheamicin, duocarmycin, and pyrrolobenzodiazepines). Inhibitors include: pyrrolobenzodiazepine, etc.; topoisomerase I inhibitors and RNA polymerase II inhibitors (including camptothecin derivatives and α-amaminine, etc.); targeted small molecules (e.g., Bcl-xL inhibitors); and immunomodulators (e.g., TLR and STING agonists).

[0086] In various aspects of this invention, the antibody-drug conjugates described herein include commercially available or candidate antibody-drug conjugates.

[0087] More specifically, the antibody-drug conjugates described in this invention include (but are not limited to): Gemtuzumab Ozogamicin (available from Pfizer / Wyeth), Brentuximab vedotin (available from Seattle Genetics / Takeda), Trastuzumab emtansine (available from Genentech / Roche), Inotuzumab ozogamicin (available from Pfizer / Wyeth), Moxetumomab pasudotox (available from AstraZeneca), Polotuzumab vedotin (available from Genentech / Roche), Enfortumab Vedotin (available from Astellas / Seagen), and Trastuzumab... deruxtecan (available from AstraZeneca / Daiichi Sankyo), Sacituzumab govitecan (available from Gilead Sciences), Disitamab vedotin (available from Rongchang Biotechnology), Belantamab mafodotin (available from GlaxoSmithKline), Loncastuximab tesirine (available from ADC Therapeutics), Tisotumab vedotin (available from Seagen / Genmab), Mirvetuximab soravtansine (available from ImmunoGen), Cetuximab saratolacan sodium (available from Rakuten Medical), and Sacituzumab... Tirumotecan (available from Kelun Biotech), etc.

[0088] The antibody-drug conjugates described in this invention also include (but are not limited to): 9MW2821, A166, ABBV-011, ABBV-154, ABBV-319, ABBV-400, ABBV-637, ABL202, ADCT-602, ADCT-901, AGS62P1, ALT-P7, AMT-151, Rastar-anetumab ravtansine, AOC 1001, AOC 1020, AOC1044, ARX305, ARX517, ARX788, ASN004, AURIXIM, AVID100, AZD8205, B003, BAT8006, BAT8008, BAT8009, BAT8010, BB-1701, BB-1705, BI-CON-02, BIO-106, BL-B01 D1, BL-M02D1, BL-M07D1, BYON3521, Cami (camidanlumab tesirine), CMG901, cofetuzumab pelidotin, CX-2029, datopotamab deruxtecan), DB-1303, DB-1305, DP303c, DS-6000a, DS-9606a, DX126-262, DXC-004, DXC-005, DXC-007, DYNE-101, DYNE-251, ESG-401, F0002-ADC, MORAb-202 (farletuzumab ecteribulin), FDA018, FDA022, FOR46, FS-1502, GB251, GQ1001, GQ1007, HDP-101, HS-20089, HS-20093, HS630, HTI-1066, IBI-343, I-DXd(ifinatamab deruxtecan), IKS03, IMGC936, IMGN151, indatuximab ravtansine, BMS-986352 (ispectamab debotansine), JBH492, JS107, JS108, JSKN-003, SGN-LIV1A (ladiratuzumab vedotin), L-DOS47, LM-102, W0101 (lonigutamab ugodotin), Luxleta (luveltamab tazevibulin), M1231, M9140, BA-3011 (mecbotamabvedotin), MGTA-117, ADCT-601 (mipasetamab uzoptirine), ABBV-155 (mirzotamab clezutoclax), MRG001, MRG003, MRG004a, MT-8633, Naratuximab (naratuximab emtansine), NBE-002, NBT508, NBT828, OBI-999, OBT076, OMTX705, ORM-5029, CAB-ROR2-ADC (ozuriftamab vedotin), HER3-DXd (patritumab deruxtecan), PF-06804103, IMGN632 (pivekimab) sunirine), praluzatamab ravtansine, PRO1184, PSMA ADC, RC88, RC108, RC118, RC248, REGN5093-M114, RG7861, Serclutamab talirine, SGN-ALPV, SGN-B6A, SGN-B7H4V, SGN-CD228A, SGN-PDL1 V, SGN-STNV, SHR-A1201, SHR-A1904, SHR-A1912, SHR-A1921, SHR-A2009, SKB315, SOT102, STI-3258, STI-6129, STRO-001, SYD1875, SYS6002, SYSA1801, TAA013, TAC-001, TAK-164, TAK-500, Terisotuzumab vedotin), TORL-1-23, TORL-2-307-ADC, TPX-4589, trastuzumab duocarmazine, BDC-1001 (trastuzumab imbotolimod), SHR-A1811 (trastuzumab rezetecan), trastuzumab vedotin, TRPH-222, TRS005, tusamitamab ravtansine, XMT-1536 (upifitamab rilsodotin), vobra duo (vobramitamab duocarmazine), XB002, XMT-1660, XMT-2056, YL201, ZW49 (zanidatamab)zovodotin), MK-2140 (zilovertamab vedotin), and / or ZV0203, etc.

[0089] In some embodiments of the present invention, the antibody-drug conjugate includes trastuzumab. Trastuzumab (also known as: Dexitrastuzumab, English name: trastuzumab deruxtecan, trade name: Other names: DS-8201 (T-DXd) is an antibody-drug conjugate targeting HER2, jointly developed and commercialized by AstraZeneca and Daiichi Sankyo. It consists of three parts: the antibody portion is a humanized anti-HER2 IgG1 monoclonal antibody with an amino acid sequence identical to trastuzumab, enabling it to specifically bind to the surface of HER2-positive cancer cells; the linker is a cleavable tetrapeptide linker based on maleimide-glycine-phenylalanine-glycine (GGFG); and the payload is a topoisomerase I inhibitor DXd (MAAA-1181a), a camptothecin derivative. Approximately eight DXd molecules are linked to each antibody molecule, resulting in a drug-antibody ratio (DAR) of approximately 8.

[0090] In some embodiments of the present invention, the antibody-drug conjugate includes vedotin. Vidicetumab (RC48, Disitamab Vedotin) is an anti-HER2 ADC drug developed by Rongchang Biotechnology Co., Ltd., composed of a humanized monoclonal antibody targeting HER2, a cleavable polypeptide Mc-VC-PAB linker, and the cytotoxic MMAE (Monomethyl auristatin E), with random cysteine ​​conjugation and an average DAR of 4.

[0091] In a preferred embodiment, the therapeutic agent comprises a therapeutically effective amount of the recombinant oncolytic poxvirus. For example, the therapeutic agent comprises 1 × 10⁻⁶ 2 -5×10 9 The recombinant oncolytic poxvirus, administered at a dose of pfu / day, can be given once daily for 1-6 consecutive times, or once every 2-7 days for 2-6 times.

[0092] In a preferred embodiment, the therapeutic agent comprises a therapeutically effective amount of the antibody-drug conjugate. For example, the dosage of the antibody-drug conjugate varies depending on the specific antibody-drug conjugate and can be determined based on the actual situation. In some cases, the antibody-drug conjugate is administered in the range of 0.1-10 mg / kg / dose, for example, at doses of 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, ..., 1.5, ..., 2.0, ..., 2.5, ..., 3.0, ..., 3.5, ..., 4.0, ..., 4.5, ..., 5.0, ..., 5.5, ..., 6.0, ..., 6.5, ..., 7.0, ..., 7.5, ..., 8.0, ..., 8.5, ..., 9.0, ..., 9.5, ..., 10.0 mg / kg / dose; it can be administered once every 7-28 days, for example, once every 1, 2, 3, or 4 weeks.

[0093] In some embodiments of the invention, the therapeutic agent comprises trastuzumab, the dose of which may be in the range of 1.0-6.4 mg / kg / dose, for example, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, ..., 3.0, ..., 3.5, ..., 4.0, ..., 4.5, ..., 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4 mg / kg / dose; and may be administered once every 7-28 days, for example, once every 1, 2, 3, or 4 weeks.

[0094] In some embodiments of the present invention, the therapeutic agent comprises vedicetumab, the dose of which may be in the range of 0.5-3 mg / kg / dose, for example, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 mg / kg / dose; and may be administered once every 7-28 days, for example, once every 1, 2, 3, or 4 weeks.

[0095] Preferably, the recombinant oncolytic poxvirus and the antibody-drug conjugate exist independently in the therapeutic agent and are not mixed with each other.

[0096] In various aspects of the present invention, the oncolytic virus may be administered using methods commonly employed in the art, such as intratumoral injection, intraperitoneal administration, intrapleural administration, or intravenous administration.

[0097] In various aspects of the invention, the antibody-drug conjugate may also be administered using methods of administration commonly employed in the art, such as intravenous administration.

[0098] In various aspects of the present invention, the therapeutic agents of the present invention may also contain other active ingredients known in the art, such as interleukin-2 (IL-2), IL-15, IL-18, granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α), etc., and the dosage and administration method may be carried out in accordance with their respective conventional methods.

[0099] Those skilled in the art will understand that the therapeutic agents of the present invention may also contain suitable pharmaceutically acceptable excipients.

[0100] In some embodiments, the therapeutic agents of the present invention comprise one or more pharmaceutically acceptable carriers. Pharmaceutical formulations can be prepared using methods known in the art. For example, the active ingredient, such as a compound, can be formulated with common excipients, diluents (e.g., phosphate-buffered saline or physiological saline), tissue culture media, and carriers (e.g., autologous plasma or human serum albumin) and administered as a suspension. Other carriers may include liposomes, micelles, nanocapsules, polymeric nanoparticles, and solid lipid particles (see, for example, E. Koren and V. Torchilin, Life, 63:586-595, 2011). Specific formulation methods of the therapeutic agents of the present invention can be found in descriptions in scientific and patent literature, such as the latest edition of Remington's Pharmaceutical Sciences, Maack Publishing, Easton PA ("Remington's").

[0101] In various aspects of this invention, the tumors and / or cancers include, but are not limited to: lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphoma, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, and testicular cancer.

[0102] In some embodiments of the present invention, the method of the present invention includes the following steps performed in sequence:

[0103] 1) Administer the recombinant oncolytic poxvirus to the patient with the tumor and / or cancer;

[0104] 2) After administration of the recombinant oncolytic poxvirus, the antibody-drug conjugate is administered to the tumor and / or cancer patient.

[0105] Preferably, the recombinant oncolytic poxvirus is administered first, followed by the antibody-drug conjugate at intervals of 1-10 days, for example, at intervals of 1, 2, 3, 4, 5, 6, 7, 8, 9, and 10 days. For example, the recombinant oncolytic poxvirus is first administered on day 0, and then the antibody-drug conjugate is first administered again on day 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 after administration. Alternatively, the time interval between the first administration of the antibody-drug conjugate and the first administration of the recombinant oncolytic poxvirus may be 1-10 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days), or the time interval between the first administration of the antibody-drug conjugate and the most recent prior administration of the recombinant oncolytic poxvirus may be 1-10 days (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 days).

[0106] In a further invention, the therapeutic agent described in this invention has shown significant synergistic inhibitory effects in the treatment of pancreatic cancer and gastric cancer, respectively.

[0107] Pancreatic cancer is a highly aggressive cancer with a rising incidence and a 5-year relative survival rate of only about 12%. Pancreatic ductal adenocarcinoma (PDA) is the most common and aggressive type, accounting for approximately 90% of pancreatic cancers. Chemotherapy remains the best treatment for metastatic, unresectable PDA, with a median survival of only 10-12 months for patients. Given the limited efficacy of current combination chemotherapy, research focus in pancreatic cancer treatment has shifted to targeted therapy and antibody-based therapies. PDA is one of the most immunologically resistant tumor types. Its unique genome is formed by oncogenic drivers that promote immunosuppression from early stages of tumor development, disrupting adaptive T-cell immunity. To date, single-agent immunomodulatory agents have proven clinically ineffective, and alternative modalities targeting immunotherapy resistance mechanisms remain necessary.

[0108] Gastric cancer is a global health problem and a leading cause of cancer-related deaths. Recent data shows that China has approximately 479,000 new cases of gastric cancer and 374,000 deaths annually, both ranking third among malignant tumors in China. Although statistics show a declining trend in age-standardized incidence and mortality rates of gastric cancer in China, the overall burden of gastric cancer remains heavy, accounting for about half of the cases in East Asian countries, and the prognosis is poor, with a 5-year overall survival rate of less than 40%. This is closely related to the high proportion of advanced gastric cancer patients in China and the strong heterogeneity in tumor biology and clinical presentation. In recent years, gastric cancer immunotherapy has injected new vitality into personalized precision treatment, becoming a significant breakthrough in the field of gastric cancer treatment. Tumor cells achieve immune evasion by activating immune checkpoints. Therefore, a series of immune checkpoint inhibitors targeting PD-L1 / PD-1 and CTLA-4 have been widely used in clinical trials. Currently, more effective treatment options and drugs developed from them are still needed to further improve patient survival rates and quality of life.

[0109] The combined administration strategy of this invention has demonstrated significant synergistic inhibitory effects in the treatment of pancreatic and gastric cancers. In this invention, the combined administration of recombinant oncolytic poxvirus and antibody-drug conjugates (ADCs) significantly enhances the anti-tumor growth effect in human pancreatic cancer models compared to either recombinant oncolytic poxvirus alone or ADC alone; and in human gastric cancer models, the combined administration of recombinant oncolytic poxvirus and ADCs significantly enhances the anti-tumor effect compared to either recombinant oncolytic poxvirus alone or ADC alone. Furthermore, this combination therapy exhibits good safety.

[0110] The following examples further explain or illustrate the content of the present invention, but these examples should not be construed as limiting the scope of protection of the present invention.

[0111] example

[0112] Unless otherwise specified, the experimental methods used in the following examples all follow standard experimental procedures, operations, materials, and conditions in the field of bioengineering.

[0113] Unless otherwise specified, the percentage concentration (%) of each reagent refers to the volume percentage concentration (% (v / v)) of that reagent.

[0114] The biological materials used in the following examples are as follows:

[0115] 1. hV01: This recombinant oncolytic vaccinia virus was obtained by genetically modifying the backbone vector VSC20 vaccinia virus. VSC20 is a vaccinia virus with the VGF gene deleted. The preparation method can be found in the scientific literature: "McCart, JA, et al. Systemic cancer therapy with a tumor-selective vaccinia virus mutant lacking thymidine kinase and vaccinia growth factor genes. Cancer Res (2001) 61: 8751–8757." The genetic modification of VSC20 included using the artificially synthesized early / late vaccinia virus promoter pSEL to regulate the exogenous human IL21 gene, and using intracellular recombination technology to insert the human IL21 gene into the TK gene region of the vaccinia virus VSC20 strain, thereby constructing the oncolytic vaccinia virus DDvv-hIL21 (the construction method can be found in Chinese Patent Application Publication CN109554353A, the entire contents of which are incorporated herein by reference), i.e., hV01.

[0116] 2. Oncolytic poxvirus ddvv-RFP is known to belong to the WR strain of oncolytic poxvirus (see, for example, the scientific literature: "X Song, et al. T-cell Engager-armed Oncolytic Vaccinia Virus Significantly Enhances Antitumor Therapy Molecular Therapy. (2014); 22 1, 102-111"). This virus has defective TK and VGF genes and carries an exogenous red fluorescent protein (RFP) gene. Since the RFP gene only serves a screening / reporter function, the antitumor function of oncolytic poxvirus ddvv-RFP is essentially equivalent to that of oncolytic poxviruses with defective TK and VGF genes. Oncolytic poxvirus ddvv-RFP can also be obtained by genetically modifying VSC20 poxvirus using conventional techniques in this field. The genetic modification involves using the synthetic early / late promoter pSEL of vaccinia virus to regulate the exogenous DsRed gene (i.e., the RFP gene), and using in vitro intracellular recombination technology to insert the DsRed gene into the TK gene region of vaccinia virus strain VSC20, thereby constructing the oncolytic vaccinia virus ddvv-RFP.

[0117] 3. Tumor cells: The sources of tumor cells are shown in Table A below.

[0118] Table A

[0119] 4. Mice can be purchased from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.

[0120] 5. Trastuzumab for injection (T-Dxd, DS-8201), available from Daiichi-Sankyo / AstraZeneca, National Drug Approval Number SJ20230005.

[0121] 6. Vidicetuximab for injection (Aidixi, RC48), available from Rongchang Biopharmaceutical (Yantai) Co., Ltd., National Drug Approval Number S20330017.

[0122] 7. Virus preservation solution: The formula is 10% glycerol and 20mM Tris-base.

[0123] 8. Sodium chloride injection (0.9%), available from Sichuan Kelun Pharmaceutical Co., Ltd., product number: National Drug Approval Number H51021158.

[0124] 9. Glucose injection (5%): available from Zhejiang Guojing Pharmaceutical Co., Ltd., National Drug Approval Number H33022607.

[0125] 9. 1×PBS: Available from Cellmax, catalog number CBS101.05.

[0126] 10. Recombinant human IL-21 protein (rhIL-21), available from R&D Company, catalog number: 8879-IL.

[0127] 11. NK cells were cultured and prepared by Hangzhou Kangwanda Pharmaceutical Technology Co., Ltd. The preparation method of these human NK cells is as follows: Using conventional methods in the art, peripheral venous blood was collected from healthy individuals by puncturing the elbow vein with a lancet, and whole immune cells (PBMCs) were extracted. Irradiated K562 feeder cells (purchased from Hangzhou Dingyun Biotechnology Co., Ltd.) were used to expand NK cells using autologous plasma culture. The final NK cell purity reached 90%, NK cell viability reached 90%, and the in vitro NK cell killing rate reached 85%.

[0128] 12. FVS780: Available from eBioscience, part number 65-0867-14.

[0129] 13. DMEM culture medium: available from Gibco, catalog number: C11875500.

[0130] 14. X-VIVO15 medium: available from Lonza, catalog number 04-418Q.

[0131] 15. L-15 medium: available from Gibco, catalog number 11415-064.

[0132] Experiment 1: Detection of HER2 expression on the surface of different human tumor cell lines using flow cytometry

[0133] Trypsin digested NCI-N87 (human gastric cancer cell line), C-33A (human cervical cancer cell line), HeLa (human cervical cancer cell line), PANC-1 (human pancreatic cancer cell line), BxPC-3 (human pancreatic cancer cell line), and CFPAC-1 (human pancreatic cancer cell line) in logarithmic growth phase, with approximately 2 × 10⁻⁶ cells per cell line. 6 Cells were washed once with 1×PBS, centrifuged at 250×g for 3 min, and the supernatant was discarded. The cell pellet was resuspended in 200 μL of PBS and divided into 4 aliquots of 50 μL each: blank control, FVS780 staining group, mouse IgG1 + FVS780 staining group, and PE anti-human CD340 (erbB2 / HER-2) antibody (Biolegend, catalog number 324406) + FVS780 staining group. Each staining group was stained with FVS780 staining solution and corresponding antibody. The blank control was stained with 50 μL PBS and incubated at room temperature in the dark for 20 min. Each tube was washed once with 1 mL of PBS buffer containing 2% FBS (fetal bovine serum), centrifuged at 250×g for 3 min, and the supernatant was discarded. The cell pellet was resuspended in 100 μL of PBS buffer containing 2% FBS. The cell suspension was transferred to flow cytometry tubes and analyzed using a flow cytometer (Agilent, NovoCyte). The expression level of HER2 was analyzed using the NovoExpress flow cytometry software.

[0134] The mean fluorescence intensity (MFI) of HER2 expression on the surface of each cell is shown in Table 1.

[0135] Table 1. Average fluorescence intensity of HER2 expression in each tumor cell line.

[0136] The results showed that the gastric cancer cell line NCI-N87 had the highest HER2 expression, with its mean fluorescence intensity (MFI) being more than 30 times higher than that of the other cell lines. The next highest expression was found in cervical cancer C-33A, HeLa, and human pancreatic cancer CFPAC-1, all of which showed similar HER2 expression. Human pancreatic cancer BxPC-3 showed slightly lower expression, while PANC-1 cells showed the lowest expression.

[0137] Example 1: Antitumor effect of combined administration of recombinant oncolytic poxvirus and antibody-drug conjugate on mouse subcutaneous transplantation of human pancreatic cancer PDX model

[0138] The mice used in this embodiment were BALB / c nude mice (6-8 weeks old, female, obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.). BALB / c nude mice are thymus-deficient mice with T lymphocyte dysfunction, partial loss of B cell function, and normal NK cell function. Human tumors transplanted from these mice showed good growth, thus the inhibitory effect of the test substance on human tumor growth was investigated.

[0139] The recombinant oncolytic poxvirus used in this embodiment is hV01, and the antibody-drug conjugate is trastuzumab for injection (T-DXd, DS-8201).

[0140] First, a subcutaneous tumor-bearing model of human pancreatic cancer PDX BALB / c nude mice was established, including: 1) Sample processing, in which freshly removed tumors were washed with 10 ml of 1×PBS (pre-cooled) to remove blood and other impurities, and transferred to a culture dish containing 1×PBS (pre-cooled). The washing was repeated twice. During this process, blood vessels and fibers were removed, and the specimens were cut to a size of 3 mm × 3 mm × 3 mm with scissors; 2) Tumor tissue was implanted into mice using a sterile trocar; 3) Mice with good growth status, no tumor ulceration, and a tumor volume of 300-1000 mm² were selected. 3 Tumor-bearing mice were used as donor animals. The mice were euthanized, and tumor tissue was collected and cut into 3mm×3mm×3mm pieces. 4) Steps 2)-3) were repeated in mice to reach the P3 generation of tumor-bearing cells. Tumor growth was observed daily after inoculation. When the tumor volume reached 150mm... 3 Screening will be conducted around 20 days later.

[0141] The screening process yielded subcutaneous tumors with the required volume (tumor volume 150-200 mm). 3 Twelve tumor-bearing mice were used in the experiment. They were randomly divided into four groups of three mice each. The drug regimen for each group was as follows:

[0142] The first group was the "negative control" group (virus preservation solution + 5% glucose injection, also known as the "vehicle control" group). Each mouse in the group was injected intratumorally with virus preservation solution (50 μL / mouse) on day 0 of administration (the day the animals were grouped), and was injected intravenously with 5% glucose injection (10 μL / g) on ​​days 7, 14 and 21 after administration.

[0143] The second group was the "hV01 monotherapy" group (1×10). 2 PFU / mouse), each mouse in the group was injected intratumorally with hV01 on day 0 of drug administration (dose volume 50 μL / mouse, dose 1×10). 2PFU was administered intravenously on days 7, 14, and 21 after administration of 5% glucose solution (administration volume of 10 μL / g).

[0144] The third group was the "T-DXd" single drug group (2mg / kg). Each mouse in the group was injected intratumorally with virus preservation solution (50μL / mouse) on day 0 of administration, and T-DXd was injected intravenously on days 7, 14 and 21 after administration (10μL / g, dose 2mg / kg).

[0145] The fourth group is the "hV01 combined T-DXd" group (1×10 2 PFU + 2 mg / kg, abbreviated as "hV01+T-DXd"), each mouse in the group was injected intratumorally with hV01 on day 0 of administration (administration volume 50 μL / mouse, dose 1×10). 2 T-DXd (administered via intravenous injection at a volume of 10 μL / g and a dose of 2 mg / kg) was administered on days 7, 14, and 21 after drug administration. Subcutaneous tumor volume and body weight were measured in each mouse starting from the day of grouping, and the measurements were recorded twice a week.

[0146] The relative tumor volume (RTV) is calculated using the following formula: RTV = Vt / V0, where Vt is the tumor volume measured in each instance, and V0 is the initial tumor volume (before drug administration). The relative tumor proliferation rate (T / C%) is calculated using the following formula: T / C% = (average RTV of the treatment group / average RTV of the control group) × 100%. If T / C% ≤ 40%, and the tumor volume in the treatment group is statistically significantly lower than that in the control group (p < 0.05), then there is an effective inhibition of tumor growth. Conversely, if T / C% > 40%, then there is no inhibition of tumor growth.

[0147] Tumor growth data were analyzed using the Bonferroni post-hoc test in two-way ANOVA; tumor weight data were analyzed using the Newman-Keuls multiple comparison test in one-way ANOVA. Statistical validity was defined as: *p<0.05, **p<0.01, ***p<0.001.

[0148] result

[0149] No mice died during the experiment. No obvious abnormalities were observed in the appearance, behavior, food intake, or water consumption of any of the mice. The average weight of the mice in each group increased, and the experiment ended on day 24 (D24). Post-mortem examination of the euthanized animals revealed no obvious abnormalities in the mice's organs. The weight changes of the mice in each group are shown in Figure 1.

[0150] In the solvent control group, the tumor volume of mice remained stable, and the average tumor volume (TV) of this group on day 24 was 3396.85 ± 354.09 mm. 3 The relative tumor volume was 18.79 ± 0.65 mm. The test products hV01, T-DXd, and the combination of hV01 and T-DXd all showed varying degrees of inhibitory effects on the tumor (see Figures 2-4). The tumor volumes of the animals in each group on D24 were 1090.91 ± 150.10 mm. 3 (hV01 group), 2959.84±132.64mm 3 (T-DXd group) and 442.44±92.18mm 3 (hV01+T-DXd group) (see Figure 2); the relative tumor volume (RTV) was 6.00±1.17 (hV01 group), 16.40±0.35 (T-DXd group), and 2.46±0.30 (hV01+T-DXd group) (see Figure 3). The relative tumor proliferation rate (T / C%) of each group was 31.93% (hV01 group), 87.3% (T-DXd group), and 13.07% (hV01+T-DXd group) (see Figure 4), among which the T / C% of hV01 group and hV01+T-DXd group was <40%, achieving effective tumor inhibition.

[0151] Statistical analysis showed that, compared with the solvent control group, T-DXd monotherapy did not show effective tumor inhibition. The tumor volume and relative tumor volume of the hV01 and hV01+T-DXd combination groups were significantly reduced (p<0.001, p<0.001). However, compared with the hV01 group, the tumor volume and relative tumor volume of the hV01+T-DXd combination group were significantly further reduced (p<0.001 and p<0.001).

[0152] The growth trend of average tumor volume in each group is shown in Figure 2, the change in relative tumor volume (RTV) is shown in Figure 3, the change in relative tumor proliferation rate (T / C%) is shown in Figure 4, and the statistical data of average tumor volume are shown in Table 2.

[0153] Table 2. Efficacy evaluation of the BALB / c nude mouse model of human pancreatic cancer using PDX tumor model (D24) a: Mean ± standard error; b: Comparison with solvent control group; c: Comparison with hV01 group; d: Comparison with T-DXd group; ns: No significant difference

[0154] At the end of the experiment, mice in each group were euthanized on day 24, and tumors were removed and weighed. The average tumor weight of mice in the solvent control group was 1.98±0.22g, while the average tumor weights of the other groups were 0.59±0.15g (hV01 group), 1.80±0.18g (T-DXd group), and 0.22±0.06g (hV01+T-DXd group), respectively. Statistical analysis showed that compared with the solvent control group, the tumor weight of the hV01 group and the hV01+T-DXd group was significantly reduced (p<0.001); compared with the hV01 group and the T-DXd group, the tumor weight of the hV01+T-DXd group was significantly reduced (p<0.05, p<0.001). The average tumor weight of each group is shown in Figure 5, and the statistical data of the average tumor weight of each group are shown in Table 3.

[0155] Table 3. Mean tumor weight for each group (D24) a: Mean ± standard error; b: Comparison with solvent control group; c: Comparison with T-DXd group; ns: No significant difference

[0156] Throughout the entire administration period, tumor-bearing mice tolerated hV01 oncolytic poxvirus, T-DXd, and hV01 combined with T-DXd well. A single intratumoral injection of hV01 combined with multiple intravenous injections of T-DXd significantly inhibited the growth of subcutaneous human pancreatic cancer PDX model in BALB / c nude mice, and was superior to the single-drug group.

[0157] Example 2: Antitumor effect of combined administration of recombinant oncolytic poxvirus and antibody-drug conjugate on mouse subcutaneous human gastric cancer NCI-N87 tumor model.

[0158] The mice used in this example were BALB / c nude mice (6-8 weeks old, female, obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.). Each mouse was subcutaneously inoculated with 1×10 7 Eleven days after engraftment of NCI-N87 cells, 30 tumor cells with tumor sizes ranging from 201 to 297 mm were selected. 3 Mice with good modeling quality were randomly divided into 6 groups, with 5 mice in each group.

[0159] The recombinant oncolytic poxvirus used in this embodiment is hV01, and the antibody-drug conjugate is vidicetuzumab for injection (Aidixi, RC48). The dosing regimen for each group is as follows:

[0160] The first group was the "negative control" group (virus preservation solution + 0.9% physiological saline, also known as the "vehicle control" group). Each mouse in the group was injected intratumorally with virus preservation solution (dose volume of 50 μL / mouse) on day 0 of drug administration (the day the animals were grouped). On days 4, 11, 18, 25, 32, 39, 46, and 53 after drug administration, 0.9% physiological saline was injected intravenously (dose volume of 10 μL / g).

[0161] The second group was the "hV01" single-drug group (1×10). 2 PFU / mouse), each mouse in the group was injected intratumorally with hV01 on day 0 of drug administration (dose volume 50 μL / mouse, dose 1×10). 2 PFU was administered via intravenous injection of 0.9% saline (dose volume 10 μL / g) on ​​days 4, 11, 18, 25, 32, 39, 46, and 53 after administration.

[0162] The third group was the "RC48" low-dose single-drug group (0.5 mg / kg) (referred to as the "RC48(L)" group). Each mouse in the group was injected intratumorally with virus preservation solution on day 0 of drug administration (drug volume of 50 μL / mouse). RC48 was then injected intravenously on days 4, 11, 18, 25, 32, 39, 46, and 53 after drug administration (drug volume of 10 μL / g, dose of 0.5 mg / kg).

[0163] The fourth group was the "RC48" high-dose single-drug group (1 mg / kg) (abbreviated as "RC48(H)" group). Each mouse in the group was injected intratumorally with virus preservation solution on day 0 of drug administration (drug volume of 50 μL / mouse), and RC48 was injected intravenously on days 4, 11, 18, 25, 32, 39, 46 and 53 after drug administration (drug volume of 10 μL / g, dose of 1 mg / kg).

[0164] The fifth group was the "hV01 combined with low-dose RC48" group (1×10 2 PFU + 0.5 mg / kg (abbreviated as "hV01 + RC48(L)") was administered to each mouse in the group via intratumoral injection of hV01 on day 0 of drug administration (dose volume: 50 μL / mouse, dose: 1 × 10⁻⁶). 2 RC48 was administered intravenously on days 4, 11, 18, 25, 32, 39, 46, and 53 after administration (dose volume 10 μL / g, dose 0.5 mg / kg).

[0165] The sixth group was the "hV01 combined with high-dose RC48" group (1×10 2PFU + 1 mg / kg (abbreviated as "hV01 + RC48(H)") was administered to each mouse in the group via intratumoral injection of hV01 on day 0 of drug administration (dose volume: 50 μL / mouse, dose: 1 × 10⁻⁶). 2 RC48 was administered intravenously on days 4, 11, 18, 25, 32, 39, 46, and 53 after administration (dose volume 10 μL / g, dose 1 mg / kg).

[0166] Starting from the day of grouping, the subcutaneous tumor volume of each mouse was measured and the mouse weight was weighed, and the measurements were recorded twice a week.

[0167] The methods for calculating relative tumor volume (RTV) and relative tumor proliferation rate (T / C%), as well as the statistical analysis of the data, are the same as in Example 1.

[0168] result

[0169] No mice died during the experiment. No obvious abnormalities were observed in the appearance, behavior, food intake, or water consumption of any of the mice. The average weight of the mice in each group increased. The experiment ended on day 56 (D56). Autopsy of the euthanized animals revealed no obvious abnormalities in the mice's organs. The weight changes of the mice in each group are shown in Figure 6.

[0170] The tumor volume in the solvent control mice remained stable, with the average tumor volume (TV) of the D56 solvent control mice being 3103.55 ± 384.73 mm. 3 The relative tumor volume was 13.13 ± 1.48 mm. The test products hV01, RC48, and the combination of hV01 and RC48 all showed varying degrees of inhibitory effects on the tumor (Figures 7-9). The tumor volumes in each group of D56 were 2544.24 ± 498.44 mm. 3 (hV01 group), 2340.32±295.87mm 3 (RC48_L group), 1954.69±358.91mm 3 (RC48_H group), 1682.52±270.58mm 3 (hV01+RC48_L group) and 620.94±204.18mm 3(hV01+RC48_H group). The D56 relative tumor volume (RTV) was 10.51±1.76 (hV01 group), 9.94±1.64 (RC48_L group), 8.16±1.27 (RC48_H group), 7.11±1.11 (hV01+RC48_L group) and 2.74±0.91 (hV01+RC48_H group). Statistical analysis showed that, compared with the solvent control group, neither hV01 nor RC48 monotherapy showed effective tumor inhibition. The tumor volume and relative tumor volume of the hV01+RC48_L and hV01+RC48_H combination therapy groups were significantly reduced (P<0.001, P<0.001). Compared with the hV01 group, the tumor volume of the hV01+RC48_L and hV01+RC48_H groups was significantly reduced (P<0.05, P<0.001). Compared with the RC48_L group, there was no significant difference in tumor volume in the hV01+RC48_L group. Compared with the RC48_H group, the tumor volume of the hV01+RC48_H group was significantly reduced (P<0.001). The relative tumor proliferation rates (T / C%) of the D56 groups were 80.01% (hV01 group), 75.68% (RC48_L group), 62.16% (RC48_H group), 54.15% (hV01+RC48_L group), and 20.84% ​​(hV01+RC48_H group), respectively. Among them, the T / C% of the hV01+RC48_H group was <40%, which achieved an effective tumor-suppressing effect.

[0171] The growth trend of average tumor volume in each group is shown in Figure 7, the change in relative tumor volume is shown in Figure 8, the change in relative tumor proliferation rate (T / C%) is shown in Figure 9, and the statistical data of average tumor volume is shown in Table 4.

[0172] Table 4. Efficacy evaluation of the drug in the BALB / c nude mouse model of human gastric cancer NCI-N87 (D56) a: Mean ± standard error; b: Comparison with solvent control group; c: Comparison with hV01 group; d: Comparison with RC48_H group; ns: No significant difference

[0173] At the end of the experiment, mice in all groups were euthanized on day 56. Tumors were dissected and weighed. The average tumor weight in the solvent control group was 2.12 ± 0.24 g. The average tumor weights in the other groups were 1.77 ± 0.25 g (hV01 group), 1.46 ± 0.30 g (RC48_L group), 1.38 ± 0.21 g (RC48_H group), 1.20 ± 0.23 g (hV01+RC48_L group), and 0.42 ± 0.14 g (hV01+RC48_H group), respectively. Statistical analysis showed that compared with the solvent control group, the tumor weight in the hV01+RC48_H group was significantly reduced (P < 0.001). Compared with the hV01 group and the RC48_H monotherapy group, the tumor weight in the hV01+RC48_H group was also significantly reduced (P < 0.01, P < 0.05). The average tumor weight of each group is shown in Figure 10, and the statistical data of the average tumor weight of each group is shown in Table 5.

[0174] Table 5. Mean tumor weight for each group (D56) a: Mean ± standard error; b: Comparison with solvent control group; c: Comparison with hV01 group; d: Comparison with RC48_H group; ns: No significant difference

[0175] Throughout the entire administration period, tumor-bearing mice tolerated hV01 oncolytic poxvirus, RC48, and hV01 combined with RC48 well. A single intratumoral injection of hV01 combined with multiple intravenous injections of RC48 significantly inhibited the growth of NCI-N87 human gastric cancer in BALB / c nude mice, and was superior to the single-drug group.

[0176] Example 3: Effects of recombinant oncolytic poxvirus on human pancreatic cancer cells and NK cells

[0177] Logarithmic growth phase human pancreatic cancer cells PANC-1 were subjected to 3×10 4 Cells were seeded in 24-well plates and cultured overnight (500 μL of DMEM containing 10% FBS (PAN) + 1% P / S per well). The culture medium in each 250 μL well was discarded, and then 250 μL of 3.6 × 10⁶ cells / well was added to each well. 4 PFU / mL of the test virus hV01 or the control virus DDvv-RFP (Mock) was used to achieve an infection concentration of 0.3 MOI and incubated for 4 hours, or 250 μL of DMEM serum-free medium was added and cultured for 4 hours. The medium was then discarded and replaced with 500 μL of immune cell medium (X-VIVO15 containing 5% Gibco FBS) and cultured overnight. The next day, the number of cells in each well was counted using a cell counter. Based on the following grouping design, 500 μL of NK cells with an effector-target ratio (E / T, NK cells to tumor cells) of 0.2:1 or rhIL-21-pretreated NK cells were added respectively. rhIL-21The cells were cultured in 450 μL of rhIL-21 (0.5 μg / mL) plus 450 μL of immunocellular culture medium, or 500 μL of immunocellular culture medium. After culturing for 48 h, the cell culture medium was collected, and the granzyme B content was detected by ELISA. This experiment included 9 groups: NC group (negative control group), rhIL-21 group, NK group, NK... rhIL-21 Group, Mock group, Mock virus + NK group, Mock virus + NK rhIL-21 The study included three groups: the hV01 virus group, the hV01 virus + NK cell group, and the hV01 virus + NK cell group, with three replicates for each group. The NC (negative control group) consisted of PANC-1 cells supplemented with 500 μL of immune cell culture medium (containing no virus and no NK cells); the NK cell group... rhIL-21 The preparation method is as follows: 1.8 mL of cells with a concentration of 1.11 × 10⁻⁶ 6 NK cells per mL were placed in a 6 cm culture dish, and 200 μL of 0.5 μg / mL rhIL-21 was added to achieve a final rhIL-21 concentration of 50 ng / mL. The cells were cultured overnight to obtain NK cells. rhIL-21 .

[0178] Figure 11 shows the results of ELISA detection of granzyme B protein expression, which indicates that NK rhIL-21 The granzyme B protein expression level in the hV01 combined with NK group (10.16±0.75 pg / mL) was significantly higher than that in the NK-only group (1.14±0.66 pg / mL) (n=3, p<0.01). The granzyme B protein expression level in the hV01 combined with NK group (hV01+NK: 68.41±0.79 pg / mL) was significantly higher than that in the control virus DDvv-RFP combined with NK group (Mock+NK: 17.32±1.19 pg / mL) (n=3, p<0.001); it was also significantly higher than that in the DDvv-RFP combined with NK group. rhIL-21 Granulase B protein expression level in group (Mock+NK) rhIL-21 :24.67±0.84pg / mL) (n=3, p<0.001).

[0179] Therefore, the replication process of hV01 oncolytic poxvirus in human pancreatic cancer cells can effectively activate NK cells to release granzyme B, which may enhance the NK cell-mediated ADCC effect when hV01 is used in combination with antibody-drug conjugates.

[0180] Example 4: Effects of recombinant oncolytic poxvirus on human breast cancer cells and NK cells

[0181] Logarithmically growing human breast cancer cells MDA-MB-231 were used at 3×10 4Cells were seeded into 24-well plates and cultured overnight (500 μL of medium per well, L-15 medium containing 10% FBS + 1% P / S). The medium in each well was discarded (250 μL of medium was then added to each well). 3 PFU / mL of the test virus hV01 or the control virus DDvv-RFP (Mock) was added to achieve an infection concentration of 0.02 MOI and incubated for 4 hours, or 250 μL of L-15 serum-free medium was added and cultured for 4 hours. The medium was then discarded and replaced with 500 μL of immune cell medium (X-VIVO15 containing 5% Gibco FBS) and cultured overnight. The next day, the number of cells in each well was counted using a cell counter. Based on the following grouping design, 500 μL of NK cells with an effector-target ratio (E / T, NK cells to tumor cells) of 0.1:1 or rhIL-21-pretreated NK cells were added. rhIL-21 The cells were cultured in 450 μL of rhIL-21 (0.5 μg / mL) plus 450 μL of immunocellular culture medium, or 500 μL of immunocellular culture medium. After culturing for 48 h, the cell culture medium was collected, and the granzyme B content was detected by ELISA. This experiment included 9 groups: NC group (negative control group), rhIL-21 group, NK group, NK... rhIL-21 Group, Mock group, Mock virus + NK group, Mock virus + NK rhIL-21 The study included three groups: the NC group, the hV01 virus group, and the hV01 virus + NK group, with three replicates for each group. The NC (negative control group) consisted of MDA-MB-231 cells supplemented with 500 μL of immune cell culture medium (containing no virus and no NK cells); the NK... rhIL-21 The preparation method is as follows: 1.8 mL of cells with a concentration of 1.11 × 10⁻⁶ 6 NK cells per mL were placed in a 6 cm culture dish, and 200 μL of rhIL-21 working solution (0.5 μg / mL) was added to achieve a final rhIL-21 concentration of 50 ng / mL. The cells were cultured overnight to obtain NK cells. rhIL-21 .

[0182] Figure 12 shows the results of ELISA detection of granzyme B protein expression, which indicates that NK rhIL-21The expression level of granzyme B protein in the hV01 combined with NK group (4.07±0.43 pg / mL) was higher than that in the NK alone group (2.60±0.77 pg / mL); the expression level of granzyme B protein in the hV01 combined with NK group (hV01+NK: 71.98±2.07 pg / mL) was significantly higher than that in the control virus DDvv-RFP combined with NK group (Mock+NK: 9.25±0.93 pg / mL) (n=3, p<0.001); it was also significantly higher than that in the DDvv-RFP combined with NK group. rhIL-21 Granulase B protein expression level in group (Mock+NK) rhIL-21 :16.10±0.78pg / mL) (n=3, p<0.001).

[0183] Therefore, the replication process of hV01 oncolytic poxvirus in human breast cancer cells can effectively activate NK cells to release granzyme B, which may enhance the NK cell-mediated ADCC effect when hV01 is used in combination with antibody-drug conjugates.

[0184] Example 5: Effects of recombinant oncolytic poxvirus on tumor cells with low expression of antibody-drug conjugate targets

[0185] The mice used in this embodiment were BALB / c nude mice (6-8 weeks old, female, obtained from Zhejiang Vital River Laboratory Animal Technology Co., Ltd.). BALB / c nude mice are thymus-deficient mice, characterized by T lymphocyte dysfunction, partial loss of B cell function, and intact NK cell function. Human tumors transplanted from these mice showed good growth.

[0186] The recombinant oncolytic poxvirus used in this embodiment is hV01.

[0187] First, a subcutaneous tumor-bearing model of human pancreatic cancer PDX BALB / c nude mice was established, including: 1) Sample processing, in which freshly removed tumors were washed with 10 ml of 1×PBS (pre-cooled) to remove blood and other impurities, and transferred to a culture dish containing 1×PBS (pre-cooled). The washing was repeated twice. During this process, blood vessels and fibers were removed, and the specimens were cut to a size of 3 mm × 3 mm × 3 mm with scissors; 2) Tumor tissue was implanted into mice using a sterile trocar; 3) Mice with good growth status, no tumor ulceration, and a tumor volume of 300-1000 mm² were selected. 3 Tumor-bearing mice were used as donor animals. The mice were euthanized, and tumor tissue was collected and cut into 3mm×3mm×3mm pieces. 4) Steps 2)-3) were repeated in mice to reach the P3 generation of tumor-bearing cells. Tumor growth was observed daily after inoculation. When the tumor volume reached 150mm... 3Around day 20, 33 tumor-bearing mice were selected for drug administration. Among them, 3 tumor-bearing mice served as the baseline control group, and the remaining 30 tumor-bearing mice received intratumoral injection of hV01 (1×10⁻⁶). 2 PFU / mouse); 5) On days 1, 2, 3, 4, 5, 6, 7, 8, 9 and 10 after administration of hV01, 3 mice were sacrificed each day, and tumor samples were collected to detect changes in Her-2 expression.

Claims

1. Use of a therapeutic agent comprising a recombinant oncolytic poxvirus and an antibody-drug conjugate in the preparation of a medicament for treating tumors and / or cancer; wherein the recombinant oncolytic poxvirus is capable of selectively replicating in tumor cells, and wherein the genome of the recombinant oncolytic poxvirus is integrated with an exogenous IL-21 gene, and the IL-21 gene is capable of being expressed in the tumor cells.

2. Use of recombinant oncolytic poxvirus in the preparation of a medicament for enhancing the sensitivity of an antibody-drug conjugate to tumors and / or cancer; wherein the recombinant oncolytic poxvirus and the antibody-drug conjugate are used in combination; and wherein the recombinant oncolytic poxvirus is selectively capable of replicating in tumor cells, and wherein the genome of the recombinant oncolytic poxvirus is integrated with an exogenous IL-21 gene, and the IL-21 gene is capable of being expressed in the tumor cells.

3. The use according to claim 1 or 2, wherein the recombinant oncolytic poxvirus is a TK gene and VGF gene defective type; the TK gene is defective by inserting a foreign nucleotide sequence, and / or the VGF gene is defective by gene knockout or insertion of a foreign nucleotide sequence; preferably, the foreign IL-21 gene is inserted into the TK gene, thereby causing the TK gene to be defective.

4. The use according to claim 1 or 2, wherein the recombinant oncolytic poxvirus is the Wyeth strain or the WR strain.

5. In accordance with the use described in claim 1 or 2, the genome of the recombinant oncolytic poxvirus further integrates an exogenous selection gene, the exogenous selection gene including the gpt gene but excluding the fluorescent protein gene.

6. In the use according to claim 1 or 2, the target of the antibody-drug conjugate is selected from: EGFR, HER2, HER3, TROP2, PSMA, TF, Nectin-4, FRα, CD19, CD20, CD22, CD33, CD79b, BCMA, CD30, F3, CEACAM5, FOLH1, AXL, IL2RA, GUCY2C, CLDN18, SLC39A6, ROR2, MET, ENPP3, CD70, GPR20, TPBG, SLC44A4, MUC1, CD276, CD38, CD37, CD22, CA9, NaPi2b, or combinations thereof.

7. The use according to claim 1 or 2, wherein the antibody-drug conjugate is selected from: Gemtuzumab Ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox, Polotuzumab vedotin, Enfortumab Vedotin, Trastuzumab deruxtecan, Sacituzumab govitecan, Disitamab vedotin, Belantamab mafodotin, Loncastuximab tesirine, and Tisotumab. Vedotin), Mirvetuximab soravtansine, Cetuximab saratolacan sodium, Sacituzumab Tirumotecan, 9MW2821, A166, ABBV-011, ABBV-154, ABBV-319, ABBV-400, ABBV-637, ABL202, ADCT-602, ADCT-901, AGS62P1, ALT-P7, AMT-151, Anetumab ravtansine, AOC 1001, AOC 1020, AOC 1044, ARX305, ARX517, ARX788, ASN004, AURIXIM, AVID100, AZD8205, B003, BAT8006, BAT8008, BAT8009, BAT8010, BB-1701, BB-1705, BI-CON-02, BIO-106, BL-B01 D1, BL-M02D1, BL-M07D1, BYON3521, Cami (camidanlumab tesirine), CMG901, cofetuzumab pelidotin, CX-2029, datopotamabderuxtecan), DB-1303, DB-1305, DP303c, DS-6000a, DS-9606a, DX126-262, DXC-004, DXC-005, DXC-007, DYNE-101, DYNE-251, ESG-401, F0002-ADC, MORAb-202 (farletuzumab ecteribulin), FDA018, FDA022, FOR46, FS-1502, GB251, GQ1001, GQ1007, HDP-101, HS-20089, HS-20093, HS630, HTI-1066, IBI-343, I-DXd(ifinatamab deruxtecan), IKS03, IMGC936, IMGN151, indatuximab ravtansine, BMS-986352 (ispectamab debotansine), JBH492, JS107, JS108, JSKN-003, SGN-LIV1A (ladiratuzumab vedotin), L-DOS47, LM-102, W0101 (lonigutamab ugodotin), Luvelta (luveltamab tazevibulin), M1231, M9140, BA-3011 (mecbotamab vedotin), MGTA-117, ADCT-601 (mipasetamab uzoptirine), ABBV-155(mirzotamab clezutoclax), MRG001, MRG003, MRG004a, MT-8633, naratuximab emtansine, NBE-002, NBT508, NBT828, OBI-999, OBT076, OMTX705, ORM-5029, CAB-ROR2-ADC (ozuriftamab vedotin), HER3-DXd (patritumab deruxtecan), PF-06804103, IMGN632 (pivekimab sunirine), praluzatamab ravtansine, PRO1184, PSMA ADC, RC88, RC108, RC118, RC248, REGN5093-M114, RG7861, serclutamabtalirine), SGN-ALPV, SGN-B6A, SGN-B7H4V, SGN-CD228A, SGN-PDL1 V, SGN-STNV, SHR-A1201, SHR-A1904, SHR-A1912, SHR-A1921, SHR-A2009, SKB315, SOT102, STI-3258, STI-6129, STRO-001, SYD1875, SYS6002, SYSA1801, TAA013, TAC-001, TAK-164, TAK-500, telisotuzumab vedotin, TORL-1-23, TORL-2-307-ADC, TPX-4589, trastuzumab The following are listed: duocarmazine, BDC-1001 (trastuzumab imbotolimod), SHR-A1811 (trastuzumab rezetecan), trastuzumab vedotin, TRPH-222, TRS005, tusamitamab ravtansine, XMT-1536 (upifitamab rilsodotin), vobra duo (vobramitamab duocarmazine), XB002, XMT-1660, XMT-2056, YL201, ZW49 (zanidatamab zovodotin), MK-2140 (zilovertamab vedotin), and / or ZV0203.

8. The use according to claim 1 or 2, wherein the therapeutic agent comprises 1 × 10 2 -5×10 9 The recombinant oncolytic poxvirus was administered at a PFU / day dose.

9. The use according to claim 1 or 2, wherein the recombinant oncolytic poxvirus is formulated for administration by intratumoral injection, intraperitoneal administration, intrapleural administration or intravenous administration; and the antibody-drug conjugate is formulated for administration by intravenous administration.

10. The use according to claim 1 or 2, wherein the tumor and / or cancer comprises: Lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphoma, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer.

11. A therapeutic agent comprising a recombinant oncolytic poxvirus and an antibody-drug conjugate; wherein the recombinant oncolytic poxvirus is capable of selectively replicating in tumor cells, and wherein an exogenous IL-21 gene is integrated into the genome of the recombinant oncolytic poxvirus, and the IL-21 gene is capable of being expressed in the tumor cells.

12. The therapeutic agent of claim 11, wherein the recombinant oncolytic poxvirus is a TK gene and VGF gene defective type; the TK gene is defective by inserting a foreign nucleotide sequence, and / or the VGF gene is defective by gene knockout or insertion of a foreign nucleotide sequence; preferably, the foreign IL-21 gene is inserted into the TK gene, thereby causing the TK gene to be defective.

13. The therapeutic agent according to claim 11, wherein the recombinant oncolytic poxvirus is the Wyeth strain or the WR strain.

14. The therapeutic agent according to claim 11, wherein the genome of the recombinant oncolytic poxvirus further integrates an exogenous selection gene, the exogenous selection gene including the gpt gene but excluding the fluorescent protein gene.

15. The therapeutic agent according to claim 11, wherein the target of the antibody-drug conjugate is selected from: EGFR, HER2, HER3, TROP2, PSMA, TF, Nectin-4, FRα, CD19, CD20, CD22, CD33, CD79b, BCMA, CD30, F3, CEACAM5, FOLH1, AXL, IL2RA, GUCY2C, CLDN18, SLC39A6, ROR2, MET, ENPP3, CD70, GPR20, TPBG, SLC44A4, MUC1, CD276, CD38, CD37, CD22, CA9, NaPi2b, or combinations thereof.

16. The therapeutic agent of claim 11, wherein the antibody-drug conjugate is selected from: Gemtuzumab Ozogamicin, Brentuximab vedotin, Trastuzumab emtansine, Inotuzumab ozogamicin, Moxetumomab pasudotox, Polotuzumab vedotin, Enfortumab Vedotin, Trastuzumab deruxtecan, Sacituzumab govitecan, Disitamab vedotin, Belantamab mafodotin, Loncastuximab tesirine, and Tisotumab. Vedotin), Mirvetuximab soravtansine, Cetuximab saratolacan sodium, Sacituzumab Tirumotecan, 9MW2821, A166, ABBV-011, ABBV-154, ABBV-319, ABBV-400, ABBV-637, ABL202, ADCT-602, ADCT-901, AGS62P1, ALT-P7, AMT-151, Anetumab ravtansine, AOC 1001, AOC 1020, AOC 1044, ARX305, ARX517, ARX788, ASN004, AURIXIM, AVID100, AZD8205, B003, BAT8006, BAT8008, BAT8009, BAT8010, BB-1701, BB-1705, BI-CON-02, BIO-106, BL-B01 D1, BL-M02D1, BL-M07D1, BYON3521, Cami (camidanlumab tesirine), CMG901, cofetuzumab pelidotin, CX-2029, datopotamabderuxtecan), DB-1303, DB-1305, DP303c, DS-6000a, DS-9606a, DX126-262, DXC-004, DXC-005, DXC-007, DYNE-101, DYNE-251, ESG-401, F0002-ADC, MORAb-202 (farletuzumab ecteribulin), FDA018, FDA022, FOR46, FS-1502, GB251, GQ1001, GQ1007, HDP-101, HS-20089, HS-20093, HS630, HTI-1066, IBI-343, I-DXd(ifinatamab deruxtecan), IKS03, IMGC936, IMGN151, indatuximab ravtansine, BMS-986352 (ispectamab debotansine), JBH492, JS107, JS108, JSKN-003, SGN-LIV1A (ladiratuzumab vedotin), L-DOS47, LM-102, W0101 (lonigutamab ugodotin), Luvelta (luveltamab tazevibulin), M1231, M9140, BA-3011 (mecbotamab vedotin), MGTA-117, ADCT-601 (mipasetamab uzoptirine), ABBV-155(mirzotamab clezutoclax), MRG001, MRG003, MRG004a, MT-8633, naratuximab emtansine, NBE-002, NBT508, NBT828, OBI-999, OBT076, OMTX705, ORM-5029, CAB-ROR2-ADC (ozuriftamab vedotin), HER3-DXd (patritumab deruxtecan), PF-06804103, IMGN632 (pivekimab sunirine), praluzatamab ravtansine, PRO1184, PSMA ADC, RC88, RC108, RC118, RC248, REGN5093-M114, RG7861, serclutamabtalirine), SGN-ALPV, SGN-B6A, SGN-B7H4V, SGN-CD228A, SGN-PDL1 V, SGN-STNV, SHR-A1201, SHR-A1904, SHR-A1912, SHR-A1921, SHR-A2009, SKB315, SOT102, STI-3258, STI-6129, STRO-001, SYD1875, SYS6002, SYSA1801, TAA013, TAC-001, TAK-164, TAK-500, telisotuzumab vedotin, TORL-1-23, TORL-2-307-ADC, TPX-4589, trastuzumab The following are listed: duocarmazine, BDC-1001 (trastuzumab imbotolimod), SHR-A1811 (trastuzumab rezetecan), trastuzumab vedotin, TRPH-222, TRS005, tusamitamab ravtansine, XMT-1536 (upifitamab rilsodotin), vobra duo (vobramitamab duocarmazine), XB002, XMT-1660, XMT-2056, YL201, ZW49 (zanidatamab zovodotin), MK-2140 (zilovertamab vedotin), and / or ZV0203.

17. The therapeutic agent of claim 11, wherein the therapeutic agent comprises 1 × 10 2 -5×10 9 The recombinant oncolytic poxvirus was administered at a PFU / day dose.

18. The therapeutic agent of claim 11, wherein the recombinant oncolytic poxvirus is formulated for administration by intratumoral injection, intraperitoneal administration, intrapleural administration or intravenous administration; and the antibody-drug conjugate is formulated for intravenous administration.

19. A method for treating tumors and / or cancer, comprising: 1) Administration of recombinant oncolytic poxvirus according to any one of claims 1-10 to patients with tumors and / or cancer; as well as 2) Administer the antibody-drug conjugate according to any one of claims 1-10 to the tumor and / or cancer patient.

20. The method of claim 19, comprising the following steps performed sequentially: 1) Administer the recombinant oncolytic poxvirus to the patient with the tumor and / or cancer; 2) After administration of the recombinant oncolytic poxvirus, the antibody-drug conjugate is administered to the tumor and / or cancer patient.

21. The method of claim 19, wherein the tumor and / or cancer comprises: Lung cancer, melanoma, head and neck cancer, liver cancer, brain cancer, colorectal cancer, bladder cancer, breast cancer, ovarian cancer, uterine cancer, cervical cancer, lymphoma, stomach cancer, esophageal cancer, kidney cancer, prostate cancer, pancreatic cancer, leukemia, bone cancer, testicular cancer.

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