Oncolytic adenovirus and checkpoint inhibitor combination therapy

Abstract

To provide combination therapy for the treatment of cancer.SOLUTION: The present invention relates to combination therapy with oncolytic viruses, particularly oncolytic adenoviruses, and checkpoint inhibitors for the treatment of cancer, particularly to a combination comprising (a) an oncolytic adenoviral vector encoding tumor necrosis factor α (TNFα) and / or interleukin-2 (IL-2) as a transgene, and (b) one or more immune checkpoint inhibitors for use in the treatment of cancer.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates generally to virology, immunology and medicine. In particular, the present invention provides an oncolytic virus, particularly an oncolytic adenovirus, for the treatment of cancer. This study concerns the combination therapy of a virus with a checkpoint inhibitor. [Background technology]

[0002] Checkpoint inhibitors (CPIs) have revolutionized cancer treatment, providing an immunotherapy approach Unfortunately, reactions are seen only in a minority of patients. In non-melanoma solid tumors, some patients have benefited, albeit for a limited period of time. Most patients do not achieve a detectable response. Thus, CPI is certainly an approach. Although proven, only a small proportion of patients benefit, leaving an unmet clinical need still exists.

[0003] An example of such unmet need is the anti-PD-1 nivolumab, which is approved as a second-line treatment. In a randomized phase 3 trial, the confirmed response rate was , 21.5% in the CPI-treated group, compared with 21.5% in the everolimus (mammalian target of rapamycin) group. The median overall survival (OS) was 25.0 months in the BRCA1 / BRCA2 treatment group and 3.9% in the BRCA1 / BRCA2 treatment group. months and 19.1 months 1 In another clinical trial, the use of a CPI (atezolizumab, anti-PD-L1) When combined with an anti-VEGF (sunitinib) drug for this purpose, the overall response rate (ORR) was 32% (compared to anti-PD-L1 alone). The improvement was 25% with monotherapy and 29% with sunitinib monotherapy. 2 RCC has long been known as an "immunogen This tumor type is said to be "prone" to IL-2 therapy, but some patients respond to high doses of IL-2. 3 ,large Many do not respond to immunotherapy. ORR remains low in most tumor types (especially Melanoma 40% 4 , urothelial cancer 21.1% 5 , non-small cell lung cancer 19.4% 6 , hepatocellular carcinoma 14.3% 7 ), There is clearly room for improvement.

[0004] Patent document 1 describes an oncolytic agent for therapeutic use, either alone or in combination with a therapeutic composition. This is about denovirus vectors and cancer treatment. Oncolytic viruses are now beginning to be used as cancer treatments. Although there have been some discoveries regarding factors influencing the onset and efficacy of viral therapy, There is still a need to identify the pathways that determine the overall response. Antivirals have shown a favorable safety profile and promising efficacy. In patients with a large metastatic burden, there is still room for improvement in response. Further characterization of pathways involved in activation may improve the efficacy of viral therapy. This may reveal potential targets for furthering the [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2014 / 170389 Brochure [Non-patent literature]

[0006] [Non-Patent Document 1] Tumeh PC , Harview CL , Yearley JH , Shintaku IP , Taylor EJ , Robert L , Chmielowski B , Spasic M , Henry G , Ciobanu V , West AN , Carmona M , et al. ,”PD-1 blockade in responses by inhibiting adaptive immune resistance.” Nature ,2014,515,568-71.

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Summary of the Invention

Problems to be Solved by the Invention

[0007] The present invention provides an oncolytic adenosine viras encoding the cytokines TNFα and / or IL-2. and immune checkpoint inhibitors, anti-PD-L1 or anti-PD-1, in clinically relevant cancers. Co-administration in the model significantly altered immune activity against the treated cancer. The study found that both drugs offer a significant life-prolonging effect compared with administering either drug alone. Thus, in some embodiments, the present application provides a method for detecting a mammalian for use in the treatment and / or prevention of the establishment of cancer and / or metastasis in and / or for use in initiating, enhancing or prolonging an anti-tumor response in a mammal. The present invention provides a combination therapy for the treatment of a cancer, the combination therapy comprising (a) TNFα and / or (b) an oncolytic adenoviral vector encoding IL-2, and (c) an oncolytic adenoviral vector encoding IL-2, and (d) an oncolytic adenoviral vector encoding IL-2, and (e) an oncolytic adenoviral vector encoding IL-2, and (f) an oncolytic adenoviral vector encoding IL-2, and (g ... and one or more immune checkpoint inhibitors that selectively bind to PD-1. This includes simultaneous administration.

[0008] In certain embodiments, the oncolytic virus and immune checkpoint inhibitor are administered simultaneously to cancer patients. administration of α- and β-glucan-containing steroids, resulting in enhanced and even synergistic effects compared to either treatment alone. Provides anti-tumor immunity.

[0009] In other related aspects, the effects of checkpoint inhibitors are enhanced, potentiated, or prolonged. or reduce the toxicity, dose, or frequency of treatment of checkpoint inhibitors (a) encoding TNFα and / or IL-2 as transgenes, and (b) an oncolytic adenoviral vector that is preferably selective for PD-L1 or PD-1. and one or more immune checkpoint inhibitors that bind to the The method includes administering to a subject. [Brief explanation of the drawings]

[0010] [Figure 1] This figure shows that samples from patients with urological cancers respond to oncolytic virotherapy despite differences in T cell infiltration and suppression. Patient sample 1 is urothelial carcinoma, and patient samples 2 and 3 are clear cell renal cell carcinoma. (A) Hematoxylin and eosin staining performed on paraffin-embedded samples. (B) CD8 immunohistochemistry. (C) PD-L1 immunohistochemistry. (D) MTS viability assay after ex vivo processing of tissue cultures. Statistical significance is shown for day 7 results calculated by unpaired t-test with Welch's correction (**p<0.01; ***p<0.001). Means and standard errors of the mean (SEM) are shown. [Figure 2] Cytokine response to virotherapy and checkpoint inhibitor treatment over 7 days of treatment. Expression values ​​from tumor tissue cultures from three individual patients were pooled and plotted. (A) IFNg, (B) TNFα, (C) IL-2, (D) IFNb, (E) Granzyme B, (F) CXCL10, (G) IL-6, (H) TGFb, (I) Arginase. Statistical significance was determined by two-way ANOVA. (*p<0.05; **p<0.01). Means and standard errors of the mean (SEM) are shown. [Figure 3] Analysis of grouped cytokine responses to virotherapy and checkpoint inhibitor treatment on day 7. Expression values ​​from tumor tissue cultures from three different patients were plotted together. (A) Fold change of immunostimulatory cytokines. (B) Fold change of immunosuppressive effectors. (C) All suppressors (including IL-6, TGF-b, and arginase) vs. all activators (including IFNg, IFNb, granzyme B, and CXCL10). (D) Pearson's r correlation between expression of immunostimulatory cytokines and tumor tissue culture viability. [Figure 4] In vivo testing of virotherapy, enabling checkpoint inhibitor treatment. (A) Experimental design: Treatment began on day 0 in 66 animals bearing B16.OVA tumors. Thirty of these animals were sacrificed on day 7 of treatment (gray dashed line) to harvest tumors, while the remaining animals were kept alive for survival experiments and underwent multiple rounds of treatment (S: virus and anti-PD-L1 co-administration; PB: prime-boost) until complete tumor regression or death. (B) Overall survival (Kaplan-Meier, log-rank Mantel-Cox test). (CG) Individual tumor growth lines. (*p<0.05; **p<0.01, ***p<0.001). [Figure 5] Schematic diagram of adenoviral constructs expressing a single cytokine or two cytokines. The viral backbone is human adenovirus serotype 5, except for the fiber knob, which is derived from serotype 3. Both single and double transgenes (TNFα, IL-2, or TNFα and IL-2) are under the transcriptional control of the viral E3 promoter. In the case of double transgenes, an IRES sequence separates the two cytokines, resulting in independent synthesis of each cytokine. The transgenes are located in the gp19k and 6.7k deleted E3 region. The E1A protein contains a 24-amino acid deletion ("D24") in constant region 2, rendering viral E1A incapable of binding to Rb. E1A expression is under the control of the E2F promoter. E1B / 19k has a disabling deletion. [Figure 6] Development and validation of an in vivo model of resistance to anti-PD-1. (A) Experimental design: Seventeen mice were implanted with subcutaneous B16.OVA tumors. When tumors reached a maximum diameter of 4 mm, they were assigned to either the mock group (n=7) or the anti-PD-1 group (n=10). 0.1 mg of anti-PD-1 (or PBS) was administered every 3 days. Animals were sacrificed when tumors progressed to >10 mm. (B) Percentage of animals with tumors <10 mm after initiation of treatment. (C) Individual tumor growth curves for both groups. (Kaplan-Meier, log-rank Mantel-Cox test; ***p<0.001). [Figure 7]Figure 1 shows a comparison of gene expression levels in treatment-naïve, progressive tumors and tumors progressing after anti-PD-1 therapy. Animals treated as described in Figure 6 were sacrificed and tumors excised when tumors were deemed resistant to anti-PD-1. RNA was extracted and expression profiles of both groups were compared. (A) Heatmap and unsupervised clustering of samples. (B) Volcano plot comparing expression levels in treatment-naïve and anti-PD-1-treated tumors. (C) Significantly regulated genes related to immunity. (Differences in gene regulation were considered when the fold change was ≤-2 or ≥2 and the q-value was ≤0.001.) [Figure 8] This figure shows that the use of engineered adenoviruses can result in tumor growth control in anti-PD-1-resistant tumors. (A) Experimental design: 29 mice were implanted with subcutaneous B16.OVA tumors. When tumors reached a maximum diameter of 4 mm, 0.1 mg of anti-PD-1 was administered intraperitoneally every 3 days. When tumors progressed to >8 mm, animals were assigned to receive the same aPD-1 regimen (n=8), intratumoral treatment with 1 x 10 viral particles (vp) every 3 days (n=8), or both (n=8). Treatment continued until a complete response was observed or the criteria for sacrifice were met. (B) Cancer-specific survival. (C) Individual tumor growth curves for each group. (Kaplan-Meier, log-rank Mantel-Cox test; ***p<0.001). [Figure 9]Figure 1 shows the generation and analysis of tumor samples to study the mechanism of action of the treatment. (A) Experimental design: Twenty-seven mice bearing B16.OVA tumors were treated with aPD-1 until they developed resistance to the drug, as described above. Animals were then assigned to receive either the same aPD-1 regimen (n=9), intratumoral treatment with 1 x 10 viral particles (vp) every three days (n=9), or both (n=9). Animals received four rounds of treatment on days 0, 1, 3, and 6, when they were considered resistant, and were sacrificed for tumor harvest on day 7. (B) Mean tumor burden (and SEM) on days 0 (the day the tumors were considered resistant) and 7 (the day the tumors were removed). (C) Heatmaps after tumor analysis by CyTOF and subsequent data processing by FLOWSOM, yielding 64 distinct immune cell populations (CD45+). (Mann-Whitney test; ****p<0.0001). [Figure 10] Changes in major immune populations following virotherapy were assessed by mass cytometry and cluster analysis. Unbiased cell populations were generated from the CD45+ fraction, resulting in multiple clusters associated with cytotype or phenotype. The relative proportions of these clusters between experimental groups were compared using the Mann-Whitney test. Key markers for identifying cluster identity are shown. (A) Cluster 25. (B) Cluster 41. (C) Cluster 10. (D) Cluster 17. (E) Cluster 6. (F) Cluster 14. (G) Cluster 36. (H) Cluster 5. (I) Cluster 39. (J) Cluster 58. (K) Cluster 32. (L) Cluster 55. [Figure 11]Combining anti-PD-L1 with adenovirus carrying IL-2 and TNFα improves tumor growth control and survival in a poorly immunogenic (MOC2) mouse oral cancer model. (A) Normalized individual tumor volume at day 0; sample groups: PBS control (PBS), treatment with anti-PD-L1 antibody (aPD-L1), treatment with virus Ad5-CMV-IL2 + Ad5-CMV-TNFα (Virus), and combination treatment with anti-PD-L1 antibody and virus Ad5-CMV-IL2 + Ad5-CMV-TNFα (aPD-L1 + Virus). (B) Mean normalized tumor volume demonstrating improved tumor growth control by day 30. (C) Overall survival curves, including median survival times for each group. Tumor growth curve statistics were calculated using mixed-effects analysis with Tukey's post-hoc test (*p<0.05, ***p<0.001). Tumor volume data are presented as mean + SEM (standard error of the mean). [Figure 12] This figure shows that the combination of TILT-123 and anti-PD-L1 therapy rapidly killed tumor cells, regardless of the histology of patient-derived ovarian cancer samples. MTS viability assays were performed after ex vivo treatment of ovarian cancer tumor tissue cultures with 100 viral particles (vp) per cell of Ad5 / 3-E2F-D24-TNFα-IRES-IL2 (TILT-123), 20 μg / ml of anti-PD-L1 (aPD-L1), or both, or culture medium (vehicle). OVCA P1 is ovarian low-grade serous carcinoma (Stage IVB), OVCA P2 is ovarian high-grade serous carcinoma (Stage IIIC), and OVCA P3 is ovarian clear cell carcinoma (Stage IVB). Statistical significance was calculated by unpaired t-test with Welch's correction and is shown for day 1 (**p<0.01; ***p<0.001). All data are presented as mean + SEM (standard error of the mean). [Figure 13]Figure 1 shows that TILT-123 induces the killing of tumor cells from patients resistant to aPD-1 therapy. MTS viability assays were performed after ex vivo treatment of ovarian cancer tumor tissue cultures with 100 viral particles (vp) per cell of Ad5 / 3-E2F-D24-TNFα-IRES-IL2 (TILT-123), Ad5 / 3-E2F-D24, or culture medium (virus-free). SCCHN P1 is a brain metastasis from a patient with head and neck squamous cell carcinoma resistant to anti-PD-1 therapy. Statistical significance was calculated by unpaired t-test with Welch's correction and is shown for day 7 (*p<0.05; **p<0.01). All data are presented as mean + SEM (standard error of the mean). DETAILED DESCRIPTION OF THE INVENTION

[0011] cytokine TNFα and / or Oncolytic adenoviral vectors encoding IL-2 and CPIs were prepared as described in the Examples section below. When used in combination as described above, immune activity (interferon gamma, i.e., IFNg, and A significant shift to CXCL10 (granzyme B) and an increase in T cell trafficking signals (CXCL10) were observed. The virus carries transgenes for TNFα and IL-2, which increases the levels of these cytokines. In vivo, our virus inhibited the anti-PD-L1 (CPI) was able to achieve a 100% complete response (hazard ratio vs. anti-PD-L1 alone 0.057 [0.007; 0.451], or a hazard ratio of 0.067 [0.011; 0.415] relative to virotherapy alone, which The group also showed a significant increase in activated CD8 T cells. Thus, tumor lytic tumors encoding TNFα and / or IL-2 as transgenes were Combination of a cytotoxic adenoviral vector with a checkpoint inhibitor that selectively binds to PD-L1 Therapeutic use of oncolytic viruses has been shown to provide unexpected improvements in the treatment of cancer. Simultaneous or sequential administration of oncolytic viruses and checkpoint inhibitors inhibits the Checkpoint inhibitors act cooperatively and even synergistically to prevent significant side effects or side effects. The survival rate was significantly improved compared to the administration of either component alone, without a decrease in virus titer. This unexpected effect allows the effective dose of each ingredient to be reduced, which may reduce side effects. This may lead to improved clinical efficacy of compounds and treatments.

[0012] In further experiments (Example 2 below), oncolytic adenoviruses encoding TNFα and IL-2 were used. The combination of virus and anti-PD-1 antibody was shown to be effective against CPI-resistant tumors.

[0013] In some embodiments, the method is used to treat cancer and / or establish metastases in a mammal. The combination therapy for the treatment of cancer comprises (i) an oncolytic adenosine monophosphate (TAP) encoding TNFα and / or IL-2; (ii) co-administering the virus vector in combination with an immune checkpoint inhibitor to a mammal. In a preferred embodiment, the checkpoint inhibitor is In a preferred embodiment, the oncolytic adenovirus selectively binds to PD-L1 or PD-1. The viral vector is administered simultaneously or sequentially with the immune checkpoint inhibitor.

[0014] Oncolytic viruses In a preferred embodiment, the oncolytic virus of the combination is an oncolytic adenovirus. It is.

[0015] As used herein, an "oncolytic adenoviral vector" refers to a vector that targets tumors versus normal tissue. By selectively replicating in cells, they can infect and kill cancer cells. It refers to an adenovirus vector. Patent Document 1 describes a transgene that can be used in the present invention. As a result, oncolytic adenoviral vectors encoding TNFα and / or IL-2 have been used. It has been disclosed.

[0016] Vectors can be prepared by any method known in the art, e.g., by incorporating any viral region. The vector can be modified by deletion, insertion, mutation, or alteration. They are made tumor-specific for replication. For example, adenoviral vectors are tumor-specific. Insertion of a specific promoter (e.g., to drive E1), a region (e.g., the region used in "D24"), E1, E2, E3 / gp19k, E3 / 6.7k) and transgene insertion. The vector may also contain modifications in E3 and / or E4. In one embodiment of the present invention, the adenoviral vector A contains an Ad5 nucleic acid backbone and an Ad3 fiber knob or an Ad5 / 3 chimeric fiber knob. It is d5 / 3.

[0017] As used herein, the term "adenovirus serotype 5 (Ad5) nucleic acid backbone" refers to The expression refers to the Ad5 genome.

[0018] "Ad5 / 3 vector" refers to a chimeric vector that contains parts of both the Ad5 vector and the Ad3 vector. In a specific embodiment of the present invention, the capsid modification of the vector is Ad5 / 3 chimera As used herein, an "Ad5 / 3 chimeric fiber knob" refers to a fiber knob. The knob portion is from Ad serotype 3, and the rest of the fiber is from Ad serotype 5. Specifically, in one embodiment, the construct is derived from Ad3. The remaining part of the genome is derived from Ad5 (SEQ ID NO:5). .

[0019] One method for generating tumor-specific oncolytic adenoviruses involves targeting E1 (SEQ ID N° 1) and The goal was to engineer a 24 base pair deletion (D24) in the constant region 2 (CR2) of the wild-type In adenoviruses, CR2 binds to the cellular Rb tumor suppressor / cell cycle regulator protein. Rb and E1 are involved in inducing the synthetic (S) phase, i.e., the DNA synthesis or replication phase. A interaction requires eight amino acids (121 to 127) in the conserved region of the E1A protein. However, in the present invention, these are deleted. The vector of the present invention is a vector according to the method described by Heise C. et al. (2000, Nature re Med 6, 1134-1139), the nucleotides corresponding to amino acids 122-129 of the vector were Viruses with D24 are impaired in their ability to overcome the G1-S checkpoint. , cells that do not require this interaction, e.g., most, if not all, human tumors. It is known to replicate efficiently only in tumor cells with defects in the Rb-p16 pathway, including tumors are.

[0020] Alternatively, the E1A endogenous viral promoter can be replaced with, for example, a tumor-specific promoter. In a specific embodiment of the invention, the E1A endogenous viral promoter Instead, the hTERT promoter is used.

[0021] In certain embodiments, a viral vector generally known to support the replication of adenoviral vectors is used. The E1B 19K gene (SEQ ID NO: 1) is replaced by the disabling deletion dE1B 19K in the vector of the present invention. (SEQ ID NO:2). Deletion of E1B 19K sensitizes cancer cells to TNFα. It is known that ATP promotes apoptosis (7).

[0022] The sequence of the wild-type E1B 19K gene is as follows (the region that can be deleted is underlined): (This is the case.) [ka] (SEQ ID NO: 1)

[0023] Therefore, in one embodiment, the sequence of dE1B 19K in the viral vector of the present invention is [ka] (SEQ ID NO: 2) is.

[0024] The E3 region is not essential for viral replication in vitro, but the E3 protein is involved in host immunity. It plays an important role in regulating responses, i.e., suppressing both innate and specific immune responses. The gp19k / 6.7K deletion in E3 is a deletion of 965 base pairs from the E3A region of adenovirus. The resulting adenoviral construct contains gp19k and 6.7K. Both genes are deleted (Kanelva A et al. 2005, Gene Therapy 12, 87-94). gp19 The k gene product binds to tumor histocompatibility complex I (MHC1, known in humans as HLA1) molecules. binds to and sequesters the virus in the endoplasmic reticulum, preventing cytotoxic T lymphocytes from recognizing infected cells. Many tumors lack HLA1 / MHC1, so deleting gp19k increases the virus The virus is eliminated from normal cells more quickly than the wild-type virus, resulting in increased tumor selectivity. The 6.7K protein is expressed on the cell surface and mediates TNF-related apoptosis. It is involved in the downregulation of TRAIL receptor 2 (TRAIL) receptor expression.

[0025] Both of these deletions have advantages: presentation of tumor epitopes, e.g., adoptively transferred To restore HLA / MHC expression for presentation to T cells, expression of gp19k protein is required. In fact, the loss of gp19k is necessary for increased HLA / MHC expression. In one embodiment of the present invention, the production of TNFα from a virus is one of its anti-tumor activities. Direct (to both transfected and non-transfected bystander cells) Since the effect of 6.7k is a direct antitumor proapoptotic effect, the presence of 6.7k is counterproductive.

[0026] In one embodiment of the present invention, the cytokine transgene or transgenes are driven by the E3 promoter. This allows expression of the transgene to be placed in the gp19k / 6.7k deleted E3 region below the viral The E3 promoter is restricted to tumor cells, allowing replication of the E3 promoter and subsequent activation of the E3 promoter. The promoter can be any exogenous promoter known in the art (e.g., CMV or E2F promoter, SE Q ID NO:3) or an endogenous promoter, in particular the endogenous E3 promoter. The E3 promoter is mainly activated by replication, but some expression is also activated by E1. The selectivity of D24 virus occurs after E1 expression (when E1 cannot bind to Rb). Therefore, these viruses express E1 in the normal cells into which they are introduced. To restrict promoter-driven transgene expression to tumor cells, E1 expression must also be regulated. It is very important to do so.

[0027] In another embodiment of the invention, E3 gp19k / 6.7k is carried in a vector, but is expressed in one or more Many other E3 regions have been deleted (e.g., E3 9-kDa, E3 10.2 kDa, E3 15.2 kDa, and and / or E3 15.3 kDa).

[0028] In a specific embodiment of the invention, the oncolytic adenoviral vector is a 5 / 3 chimeric phage. It is based on the adenovirus serotype 5 (Ad5) nucleic acid backbone, which contains the ribosome. The following: the E2F1 promoter for tumor-specific expression of E1A and the Rb binding of adenovirus E1 A 24-bp deletion (D24) in the fusion constant region 2 and the viral gp19k and 6.7k readings Deletion of the frame nucleic acid sequence and substitution of the deleted adenoviral gene gp19k / 6.7K in the E3 region and a nucleic acid sequence encoding at least one cytokine transgene, A transgene is inserted into the vector, resulting in expression of the transgene under the control of the viral E3 promoter. In one embodiment of the present invention, the adenoviral vector comprises: It is based on a human adenovirus.

[0029] The exact function of the adenovirus 3 early region (E3) protein is unknown. In general, in adenoviruses, these deletions do not impair replication, and It appears to affect the host's antiviral response to adenoviruses. The E3 of the virus genome is the highest among the six adenovirus species found in humans (AF). This genetic diversity is mainly due to the highly conserved E3-gp19K and Located between the open reading frames (ORFs) of E3-RIDα, this sequence is species-specific is encoded by the gene.

[0030] Cytotoxic T cell-mediated killing of virus-infected cells is regulated by E3-gp19K. This blocks the transport of MHC class I to the cell membrane and inhibits the formation of TAP-MHC class I complexes. This is achieved by harming

[0031] Thus, in one aspect of the present invention, the key molecule E3-gp19K is expressed in an adenoviral vector. - makes viral replication stealthier, leading to tumor regression and its beneficial effects In addition, by retaining E3-gp19K, anti-adenovirus cytotoxicity This suppresses the induction of T cells, resulting in the production of more anti-tumor T cells.

[0032] Cytokines act through various mechanisms, including recruitment of T cells to tumors, leading to immune responses. The nucleotide sequence encoding the cytokine transgene is expressed in human, monkey, and Any animal, such as rats, mice, hamsters, dogs, or cats, may be used. The nucleotide sequence encoding the transgene is typically human. They may be modified to enhance their effectiveness or may be unmodified, i.e., wild-type. That's fine.

[0033] Certain embodiments of the invention include a viral vector encoding at least one cytokine. In a specific embodiment of the invention, the cytokine is IL-2 or TNFα, Preferably, the viral vector encodes both cytokines. In embodiments, the viral vector contains IL-2 and / or TNFα, as well as additional sites. interferon α, interferon β, interferon γ, Complement C5a, CD40L, IL12, IL-23, IL15, IL17, CCL1, CCL11, CCL12, CCL13, CCL14-1, CCL 14-2, CCL14-3, CCL15-1, CCL15-2, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21 , CCL22, CCL23-1, CCL23-2, CCL24, CCL25-1, CCL25-2, CCL26, CCL27, CCL28, CCL3, C CL3L1, CCL4, CCL4L1, CCL5, CCL6, CCL7, CCL8, CCL9, CCR10, CCR2, CCR5, CCR6, CCR7 , CCR8, CCRL1, CCRL2, CX3CL1, CX3CR, CXCL1, CXCL10, CXCL11, CXCL12, CXCL13, CXCL 14, CXCL15, CXCL16, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7, CXCL8, CXCL9, CXCR 1, a cytokine selected from the group consisting of CXCR2, CXCR4, CXCR5, CXCR6, CXCR7, and XCL2 It codes in.

[0034] The cytokine TNFα (tumor necrosis factor α) attracts and activates T cells, resulting in tumor immunosuppression. On the other hand, IL-2 (interleukin 2) has the function of reducing the T cell graft. Therefore, IL-2 is not a side effect of T cell therapy, as is commonly done in T cell therapy. Produced locally in the tumor where it is needed, rather than injected systemically, which can cause side effects Therefore, a major problem with prior art therapies (i.e., systemic IL-2 toxicity) is overcome. This can be prevented by the embodiment.

[0035] Danger signals provided by oncolytic virus replication and viral DNA pathogenesis Activation of the associated molecular pattern recognition receptors, coupled with the action of the transgene, induces preconditioning. This reduces tumor immunosuppression to the point that tumor suppression therapy can be omitted. Preconditioning for chemotherapy and radiotherapy, which has been a major challenge in the prior art, This can avoid toxicity caused by

[0036] In one embodiment of the present invention, the viral vector contains an intracellular ribonucleotide sequence between the two transgenes. and optionally a ribosome entry site (IRES) or a ribosome shunt site 2A. ES or ribosomal shunt site 2A can be coupled to any cytokine, such as IL-2, preferably It may be present among other cytokines selected from the group of cytokines listed above. As used herein, "IRES" refers to an IRES that regulates the function of a messenger RNA sequence in protein synthesis. IRES refers to a nucleotide sequence that allows translation initiation during the transcription process. In one embodiment of the present invention, the IRES is derived from the encephalomyocarditis virus (EMCV). As used herein, "ribosomal shunt site 2A" refers to the site where the ribosome is 5' nontranslated. It means a translation initiation site that physically bypasses a part of the translation region to reach the start codon. Both ES and A2 viruses encode two transducing genes from a single promoter (E3 promoter). It allows genes to be produced.

[0037] Oncolytic adenoviral vectors encoding TNFα and / or IL-2 as transgenes An example of the detailed structure of the actuator is disclosed in Patent Document 1. See also FIG.

[0038] In summary, viral vectors containing at least one cytokine transgene are used. The main advantages of the present invention are: i) the cytokines and the virus itself stimulate T-cell ii) cytokines that induce a danger signal that recruits tumor cells and other immune cells to the tumor; iii) cytokines and viruses, which induce T cell proliferation in both tumors and local lymphoid organs itself induces T cells (both adoptive T cell grafts and natural innate antitumor T cells) to attack tumors. iv) cytokines and / or viruses can bind to antigens on cancer cells Induces increased expression of HLA-presenting molecules, sensitizing them to recognition and killing by T cells , v) Reduces immunosuppression and cellular allergies through cytokines and viral replication, and promotes tumor suppression. favorably alters the tumor microenvironment.

[0039] The viral vectors used in the present invention may contain modifications other than those described above. Any additional components or modifications may optionally be used, but are not essential to the invention. isn't it.

[0040] Insertion of exogenous factors can enhance the efficacy of vectors in target cells. The use of exogenous tissue- or tumor-specific promoters is a These are common and can be used in the present invention.

[0041] In summary, the present invention demonstrates that replication of oncolytic viruses recruits T cells and targets dangerous T cells in tumors. This study reveals that the compound can induce immune suppression and reduce cellular allergies by inducing immune signaling. These effects are due to the activation of pathogen-associated molecular pattern recognition receptors, i.e., receptors that mediate immune responses. The present invention is mediated by an evolutionarily conserved mechanism and does not induce resistance. The added benefit of an oncolytic platform that is replicable in normal cells but not in normal cells is Furthermore, the oncolytic effect itself is , which may enhance the overall antitumor effect in humans.

[0042] Checkpoint inhibitors Immune checkpoint proteins are proteins that send signals to T cells that inhibit their function. Cancer cells express checkpoint proteins at high levels on their surface. This can be harnessed by expressing it in the bell, thereby suppressing anti-cancer immune responses.

[0043] The checkpoint inhibitors (also referred to as CPIs) described herein are intended to inhibit immune checkpoint It is any compound that can inhibit the function of a target protein. This includes not only blocking the immune system but also reducing its function. These are human checkpoint proteins. Therefore, immune checkpoint inhibitors Preferably, it is a human immune checkpoint inhibitor.

[0044] Checkpoint proteins include, but are not limited to, CTLA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3, GAL9, LAG3, and VIST A, KIR, BTLA, TIGIT and / or IDO. LAG3, BTLA, B7-H3, B7-H4, TIM3 and KIR-mediated pathways are immune checkpoint pathways similar to CTLA-4 and PD-1-dependent pathways. It is recognized in the art that immune checkpoint inhibitors are involved in the CT pathway. LA-4, PD-1 (and its ligands PD-L1 and PD-L2), B7-H3, B7-H4, HVEM, TIM3 , GAL9, LAG3, VISTA, KIR, BTLA, TIGIT and / or IDO inhibitors. In some embodiments, the immune checkpoint inhibitor is an inhibitor of PD-L1 or PD-1. .

[0045] In some embodiments, the checkpoint inhibitor of the combination is an antibody. As used herein, the term "antibody" refers to naturally occurring antibodies and engineered antibodies, etc. and, for example, target immune checkpoints or epitopes (e.g., antigen-binding moieties). The present invention also encompasses a full-length antibody or a functional fragment or analog thereof capable of binding to a target antigen. Antibodies for use in the methods described herein include, but are not limited to, human, humanized, It may be from any source, including animal or chimeric, and is preferably of the IgG1 or IgG4 isotype. It may be of any isotype and may be glycosylated or non-glycosylated. The term antibody also includes any of the terms used herein. Antibodies that exhibit binding specificity also include bispecific or multispecific antibodies. Immune checkpoint inhibitors are monoclonal antibodies that selectively bind to PD-L1. more preferably BMS-936559, LY3300054, atezolizumab, durvalumab and Examples of monoclonal antibodies that bind to human PD-1 include: U.S. Patent Nos. 7,521,051, 8,008,449, and 8,354,509 Specific antibodies useful as PD-1 antagonists in this method of treatment are described herein. Human PD-1 mAbs include pembrolizumab (MK-3475), nivolumab (BMS-936558), etc. and humanized antibodies h409A11 and h409A16 described in International Publication No. 2008156712. and h409A17.

[0046] Humanized antibodies are non-human antibodies whose protein sequences have been altered to increase their similarity to human antibodies. A chimeric antibody refers to an antibody of one species (e.g., mouse, rat, etc.). or antibodies consisting of multiple components and one or more components of other species, e.g., human immunoglobulins. The term "human antibody" refers to a non-human antibody that comprises at least a portion of a globulin constant region (Fc).

[0047] Many forms of antibodies can be engineered for use in the combinations of the present invention, Representative examples include Fab fragments (monovalent fragments consisting of VL, VH, CL, and CHI domains), F (ab')2 fragment (linked by at least one disulfide bridge in the hinge region) a bivalent fragment containing two Fab fragments), an Fd fragment (consisting of a VH and a CHI domain), an Fv fragment ( a dAb fragment (a single variable domain fragment consisting of the VL and VH domains of a single arm of an antibody); (VH domain or VL domain), and the two VL and VH domains of the Fv fragment. Single-chain Fv (scFv) in which domains are fused together to form a single protein chain with a linker Examples include:

[0048] In some embodiments, the checkpoint inhibitor (also referred to as a CPI) of the combination therapy , an antibody or a compound that specifically binds to the immune checkpoint protein PD-L1 or PD-1, or a fragment thereof. In a particularly preferred embodiment, the immune checkpoint inhibitor is PD-L1 or a monoclonal antibody, fully human, capable of at least partially antagonizing PD-1. The antibody is a chimeric antibody, a humanized antibody or a fragment thereof.

[0049] cancer The recombinant vector of the present invention has replication ability in tumor cells. In this study, the vector was shown to be replication-competent in cells defective in the Rb pathway, specifically the Rb-p16 pathway. These defective cells include all tumor cells in animals and humans. As used herein, a "defect in the Rb pathway" refers to a defect in any gene or protein in the pathway. These defects can lead to the development of tumor cells. The cells overexpress E2F, resulting in the loss of Rb by E1A CR2, which is normally required for efficient replication. Further selectivity is mediated by the E2F promoter, which is Rb / p1 6 pathway is activated only in the presence of free E2F, as seen in defective cells. Without it, transcription of E1A does not occur and the virus does not replicate. , directly or indirectly, allowing expression of the transgene from the E3 promoter, thereby producing toxic This is important to prevent expression of E1A in normal tissues, which can cause cancer.

[0050] The present invention relates to an approach for treating cancer in a subject. In embodiments, the subject is a human or mammal, particularly a mammal or human patient, more particularly a mammal or human patient. Specifically, the subject is a human or mammal suffering from cancer.

[0051] This approach can be used to treat any cancer or tumor, including both malignant and benign tumors. This approach can be used to target both primary tumors and metastases. In one embodiment of the invention, the cancer is characterized by tumor-infiltrating lymphocytes. It is particularly attractive for the treatment of metastatic solid tumors characterized by infiltrating lymphocytes. In this form, T cell grafts are mediated by tumor- or tissue-specific T cell receptors of chimeric antigen receptors. It has been modified as follows.

[0052] As used herein, the term "treatment" or "cure" refers to not only complete cure but also for purposes including the prevention, amelioration, or alleviation of disorders or symptoms related to cancer or tumors, at least Both are oncolytic adenoviral vectors and checkpoints that selectively bind to PD-L1. Therapeutic effect means administering a therapeutically effective amount of a steroid inhibitor to a subject, preferably a mammalian or human subject. monitors the patient's symptoms, blood tumor markers, tumor size, or patient survival time. It can be evaluated by ringing.

[0053] In another embodiment of the present invention, the cancer or tumor is nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, Kidney cancer, connective tissue cancer, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain Cancer, pharyngeal cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, brown Chromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lynn Dow disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, urinary ductal carcinoma, brain tumor, oligodendroglioma, neuroblastoma, meningioma, spinal tumor, bone cancer, chondroma, chondrosarcoma , Ewing's sarcoma, cancer of unknown primary site, carcinoid, gastrointestinal carcinoid, fibrosarcoma, breast Cancer of the cervix, Paget's disease, cervical cancer, colon cancer, rectal cancer, esophageal cancer, gallbladder cancer, head and neck cancer , eye cancer, kidney cancer, Wilms' tumor, liver cancer, Kaposi's sarcoma, prostate cancer, lung cancer, testicular cancer Cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, Ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penile cancer, ptosis Body cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymic cancer, thyroid cancer, cystic leukemia Mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulin tumour, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, pulp cancer Membrane cancer, oral cancer, nerve cancer, palate cancer, parotid cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, Preferably, the cancer to be treated is selected from the group consisting of salivary gland cancer, tongue cancer, and tonsil cancer. Or the tumor is kidney cancer, ovarian cancer, bladder cancer, prostate cancer, breast cancer, colon cancer, lung cancer ( small cell lung cancer, non-small cell lung cancer, squamous cell non-small cell lung cancer, etc.), gastric cancer, classic Hojiki In a more preferred embodiment, the cancer is selected from the group consisting of lymphoma, mesothelioma, and liver cancer. Preferably, the cancer or tumor type is head and neck cancer, most preferably human head and neck cancer.

[0054] Before classifying a human or animal patient as suitable for the therapy of the present invention, the clinician must screen the patient. Results that deviate from normal and that reveal tumors or cancer can be examined. Based on this, the clinician can recommend the treatment of the present invention to the patient.

[0055] In an embodiment of the invention, the subject or patient has had prior chemotherapy or immunotherapy, such as CPI treatment. The patient has already failed at least one prior therapy, meaning the patient's cancer is at a checkpoint In a preferred embodiment, the present invention relates to a CPI-resistant tumor. Without wishing to be bound by theory, the results of this study suggest that CD8+ T cells These genes mediate the activity of T cells, resulting in the expression of genes related to immune activity, such as T cell precursor GZMG and and GMZF genes, and genes for T cell proteins involved in interactions with other cell types, such as KLRC2. genes for immune components such as complement and CD46, and T cell activity, particularly TNFSF18 / GITRL and EAR2 The expression of regulatory factor genes was significantly increased in CPI-resistant cancer cells compared to cells that had not undergone CPI therapy. This gene expression change is therefore a sign of resistance. Therefore, in one embodiment, the present invention is directed to the function of CD8+ T cells. Intended for the treatment of CPI-resistant cancers that may mediate or cause incompetence and / or inactivity do.

[0056] Pharmaceutical Composition The pharmaceutical composition of the present invention comprises at least one viral vector of the present invention. The present invention relates to a method for treating cancer by combining (a) an oncolytic virus with (b) a checkpoint inhibitor. The present invention also provides a pharmaceutical composition comprising the pharmaceutical composition for use in the treatment of cancer. Further, the composition may comprise at least two, three, or four different vectors. In addition to the vector and the checkpoint inhibitor, the pharmaceutical composition may also include , other therapeutically active agents, pharmaceutically acceptable carriers, buffers, excipients, adjuvants, additives Any other agents such as additives, preservatives, antiseptics, fillers, stabilizers and / or thickeners and / or any ingredients normally found in the corresponding product. The selection of suitable ingredients and suitable manufacturing methods for formulation is within the general knowledge of a person skilled in the art. do.

[0057] The pharmaceutical composition may be in any form suitable for administration, such as solid, semi-solid, or liquid. may be selected from the group consisting of a solution, an emulsion, a suspension, a tablet, a pellet, and a capsule. The compositions of the present invention are not limited to specific formulations. Instead, the composition can be in any known pharmaceutically acceptable formulation. The pharmaceutical composition can be prepared by any conventional process known in the art. It can be made.

[0058] The pharmaceutical kit of the present invention comprises a tumor necrosis factor (TNFα)-encoding gene and / or a tumor necrosis factor (IL-2)-encoding gene. A lytic adenoviral vector and one or more molecules that selectively bind to PD-L1 or PD-1. and several immune checkpoint inhibitors. Transgenes include TNFα and / or IL-2. An oncolytic adenoviral vector encoding the one or more immune checkpoint inhibitors selectively bind to PD-1 are In another embodiment of the invention, the first formulation and the second formulation are formulated into a formulation. In another embodiment, the compounds are for administration to a subject simultaneously or sequentially in any order. The kit is for use in treating cancer or tumors.

[0059] Administration The vectors or pharmaceutical compositions of the present invention can be administered to any mammalian subject. In certain embodiments of the invention, the subject is a human. Mammals include pets, livestock and production animals. It may be selected from the group consisting of:

[0060] Any conventional method can be used to administer the vector or composition to a subject. The route of administration depends on the formulation or form of the composition, the disease, the location of the tumor, the patient, coexisting conditions, and other factors. Therefore, the dosage and frequency of administration of each therapeutic agent in the combination will depend on the following factors: The type of treatment will depend in part on the particular therapeutic agent, the severity of the cancer being treated, and patient characteristics. Or, the dosing regimen should be tailored to each individual drug delivered to the patient while maintaining an acceptable level of side effects. In a preferred embodiment, the amount of the checkpoint inhibitor is about 2 mg / kg It is administered in an amount of about 2 mg / kg to 25 mg / kg, more preferably about 50 mg / kg to 50 mg / kg.

[0061] In one embodiment of the present invention, (a) a transgene encoding TNFα and / or IL-2 is used. and (b) an oncolytic adenoviral vector that preferably selectively binds to PD-L1 or PD-1. One or more immune checkpoint inhibitors, each of which is combined with other immune checkpoint inhibitors, may be administered simultaneously or sequentially in any order. and administered separately to a subject. This means that (a) and (b) are administered separately as a single or may be provided in unit doses of 100 mg / kg or for administration at the same time or at staggered intervals. This means that the ingredients may be provided as separate entities (e.g., in separate containers). The difference may be between 1 hour and 1 week, preferably between 12 hours and 3 days, more preferably at most It may be 24 hours or 48 hours. In a preferred embodiment, the adenoviral vector The first dose of the checkpoint inhibitor is administered before the first dose of the checkpoint inhibitor. It is also possible to administer the virus using a method other than that of the blockade inhibitor. In this study, either the virus or the checkpoint inhibitor was administered intratumorally, and the other was administered systemically. In a particularly preferred embodiment, the drug is administered orally. The virus is administered intratumorally and the checkpoint inhibitor is administered intravenously. The virus and checkpoint inhibitor are administered as separate compounds. Combination treatments are also possible.

[0062] As used herein, "separate administration" or "separate" refers to (a) a transgene and (b) an oncolytic adenoviral vector encoding TNFα and / or IL-2; and Preferably, one or more immune checkpoint inhibitors that selectively bind to PD-L1 or PD-1. refers to a situation where the harmful agent is two different products or compositions that are distinct from one another. do.

[0063] (a) Oncolytic adenovirus encoding TNFα and / or IL-2 as transgenes (b) one or more immunoglobulins that selectively bind to PD-L1 or PD-1, Achieving therapeutic effects by administering a checkpoint inhibitor in combination with a single dose During administration, the following may be done depending on the patient and the type, severity, or location of the cancer: In one embodiment of the present invention, (a) TNFα and / or IL-2 (b) an oncolytic adenoviral vector encoding as a transgene, preferably P one or more immune checkpoint inhibitors that selectively bind to D-L1 or PD-1; Between successive doses, the period is from 1 minute to 4 weeks, specifically from 1 to 10 days, more specifically 1 to 5 days, most specifically for a maximum period of 24 or 48 hours; and and / or (a) an oncolytic enzyme encoding TNFα and / or IL-2 as a transgene. a denoviral vector and (b) one or more antibodies that selectively bind to PD-L1 or PD-1, preferably or multiple doses of multiple immune checkpoint inhibitors. an oncolytic adenoviral vector encoding TNFα and / or IL-2 as vectors; (b) one or more immune checkpoints that preferably selectively bind to PD-L1 or PD-1 The frequency of administration of the oncolytic adenovirus inhibitor may vary during the course of treatment. Vectors or checkpoint inhibitors may be administered for the first 2 weeks, 4 weeks, monthly, or during the treatment period. In one embodiment of the invention, the vector or Administration of any of the compositions is 3 to 7 times over the first 2 weeks, then every 4 weeks, and then monthly. In certain embodiments of the invention, administration is four times over the first two weeks, then four weeks, then monthly. In another specific embodiment, the adenoviral vector is administered three times over the first four weeks. (In one embodiment, the first administration is intravenous, and the second and third administrations are intratumoral), Checkpoint inhibitors are administered once or twice over the first four weeks, and then the virus Both the vector and checkpoint inhibitor are administered once a month. The length of treatment varies. The duration may vary, for example, from 2 months to 12 months or more.

[0064] In a particular embodiment of the invention, (a) a transgene encoding TNFα and / or IL-2 is used. and (b) an oncolytic adenoviral vector that is preferably selective for PD-L1 or PD-1. One or more immune checkpoint inhibitors that bind to the Lytic adenoviral vectors can be used for, for example, one to six months, or for longer than 12 months. The treatment is administered weekly, every two weeks, every three weeks, or every month.

[0065] In one embodiment of the present invention, the administration of the oncolytic virus is by intratumoral, intra-arterial, intravenous, intrathoracic, or intravenous route. It is administered by intrathecal, intravesical, intravesical, or intraperitoneal injection, or by oral administration. Any combination of administration is also possible. In this way, despite the local injection, Checkpoint inhibitors can be administered intravenously or intratumorally. In one embodiment, administration of the checkpoint inhibitor can be intratumoral, intra-arterial, or intra-arterial. It is administered by intravenous, intrapleural, intravesical, intravesical, or peritoneal injection, or by oral administration. It can be done.

[0066] The effective amount of the vector will depend at least on the subject needing treatment, the type of tumor, the location of the tumor, and The dose varies depending on the type and stage of the tumor. 8 virus particles (VP) ~Approx. 1×10 14 VPs, specifically, approximately 5 x 10 9VP ~ approx. 1 x 10 13 Individual VPs, more specifically , about 3×10 9 VP ~ approx. 2 x 10 12 In one embodiment, at least one VP may be changed. Oncolytic adenoviral vectors encoding cytokines were administered in 1 × 10 10 -1×10 14 pieces In another embodiment of the invention, the dose is about 5 x 10 10 -5×10 1 1 Within the range of VP.

[0067] In addition to the therapy of the present invention, any other treatment or combination of treatments may be used. In certain embodiments, the method or use of the present invention is in combination with concurrent or sequential radiation therapy, chemotherapy, angiogenesis inhibitors, targeted therapies, e.g., alkylating agents, nucleoside analogues, cytotoxic agents, Other anti-cancer drugs, such as cytoskeleton modifiers, cytostatics, monoclonal antibodies, and kinase inhibitors The method further includes administering to the subject an agent or intervention (including surgery).

[0068] The terms "treat" or "increase" as used herein and their derivatives The term "effective" does not necessarily imply 100% or complete cure or enhancement. However, the degree to which those skilled in the art recognize potential benefits or therapeutic effects varies.

[0069] Other embodiments The present invention also provides (a) a method for the treatment of cancer or tumors, comprising administering to a subject a therapeutic agent selected from the group consisting of TNFα and TNF-α-as transgenes; and (b) an oncolytic adenoviral vector encoding one or more of: Multiple immune checkpoint inhibitors are also contemplated, preferably in human cancers or tumors. be.

[0070] Reference throughout this specification to an embodiment or an embodiment is a reference to that embodiment. The particular features, structures, or characteristics described in the series are at least one embodiment of the present invention. Thus, in various places throughout this specification, the terms "in one embodiment" and "in one embodiment" are used. Appearances of the phrase "in one embodiment" do not necessarily all refer to the same embodiment. Furthermore, when referring to numerical values ​​using terms such as about or substantially, The exact figures are also disclosed.

[0071] In this book, the verbs "comprise" and "include" are used as follows: as an open limitation that neither excludes nor requires the presence of uncited features Features recited in dependent claims may not be used interchangeably unless expressly stated otherwise. Furthermore, in this book, "a" or "an" i.e., a single It will be understood that the use of numeral forms does not exclude a plurality.

[0072] As technology advances, it will be apparent to those skilled in the art that the concept of the present invention can be implemented in a variety of ways. It will be apparent that the present invention and its embodiments are not limited to the examples described below. However, it may vary within the scope of the claims.

[0073] [Table 1] [Example]

[0074] Example 1 Materials and Methods Human tumor tissue cultures from urinary tumor samples Urinary tract samples were collected from surgically removed kidneys and single-cell cultures were prepared according to previously described methods (9). Single-cell cultures were incubated with 100 viral particles (vp) of Ad5 / 3-E2F-d24-h suspension per cell. TNFα-IRES-hIL2, 20 μg / mL anti-human PD-L1 (atezolizumab, Roche), or both Cytokine production and cell viability were assessed after 1, 3, and 7 days. Ta.

[0075] Pathological tissue analysis Hematoxylin and eosin (H&E) and CD8 (clone 4B11, CD8-4B11-L-CE- H, Novocastra) staining was performed on patient samples and analyzed by trained pathologists. PD-L1 expression was assessed by a trained pathologist. Histopathological analysis from mouse samples was performed by a veterinary pathologist. The experiment was carried out as previously described (10).

[0076] Cell viability assay Human tumor tissue cultures were treated for up to 7 days (as described above). Cell viability was assessed by Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega, G3582) was used. The viability of mock-treated cells was determined as 100%.

[0077] In vivo experiments To examine changes in tumors due to treatment, 2.5 × 10 5 B16.OVA melanoma cells were cultured in 4-6 week old mice. The mice were subcutaneously implanted into female C57BL / 6JOlaHsd mice (Envigo Labs). 11 days after implantation, the animals were They were randomly assigned to each group (n = 12-14 / group). Then, they received 0.1 mg of anti-PD-L1 (clone 10F.9G2, BE010 1, BioXCell) was administered systemically at 1 × 10 on days 0, 1, 3, and 6. 8 vp (the same amount of Ad5-C Intratumoral injection of the MV-mIL2 virus and Ad5-CMV-mTNFa virus (which do not replicate in mice) into the tumor The group that did not receive the virus was injected with PBS into the tumor. On the 7th day, 6 mice in each group were Animals were sacrificed and tumors were collected to examine immune cell phenotypes and cytokine signatures. The remaining animals (n = 6-8 / group) continued on the OS study for 90 days, and the maximum tumor size ( Treatment continued until the tumor reached a size of 18 mm or completely regressed.

[0078] Cell lines and viruses The mouse melanoma cell line B16.OVA was cultured under recommended conditions (8). For the construction and production of the mouse adenoviruses (Ad5-CMV-mIL2 and Ad5-CMV-mTNFa), , as previously described (12), and was used for in vivo experiments. For tissue culture experiments, oncolytic Ad5 / 3-E2F-d24-hTNFα-IRES-hIL2 (known as TILT-123) was used. (11) was used.

[0079] Cytokine analysis Cell culture supernatants from tumor tissue cultures were collected after 1, 3, and 7 days. The sample size for cytokine analysis was limited by the availability of tumor samples ( ck; n=4, aPD-L1; n=6, virus; n=3, virus + aPD-L1(S); n=2, virus + aPD-L 1 (PB); n = 0). Cytokine levels in the samples (IFNg, TNFa, IL-2, IFNb, granulocyte colony-stimulating factor (GFR)-1 (IFN-g), erythrocyte sedimentation factor (TNF-α), erythrocyte sedimentation factor (IL-2 ... Immunoglobulin B, CXCL10, IL-6, arginase, and TGF-b1 were measured using a custom Legendplex panel (Bioleg End) and Free Active / Total TGF-b1 Detection Kit (Products 740488, 740486 and 740487, Biolegen) The evaluation was performed using Cytometric Bead Array Mouse Th1 / Th2 / Th17 Cytokine Kit ( Mouse tumor samples were examined using the 5'-amino acid sequence (560485, BD) as previously described (8). The bead arrays were analyzed using Accuri® (BD). The cytokine values ​​obtained were The concentrations were normalized to the total protein concentration of the sample.

[0080] PD-L1 / 2 expression assay To investigate the dynamics of PD-L1 expression, B16.OVA cells were used as a known inducer of PD-L1 expression. The cells were treated with 1000 U / mL of mouse IFNg (315-05, Peprotech). Mock control cells were left untreated. After 24 hours of culture, PD-L1 expression was confirmed in some cells, and PD-L1 expression in the remaining cells was confirmed. The cells were washed twice with BS and transferred to duplicate 12-well plates. Treatment was discontinued in some of the mice (the "discontinuation" group), while treatment continued in others (the "IFNg maintenance" group). Plates were analyzed 24 and 72 hours after plating.

[0081] Similarly, IFNg (1000 U / mL), TNFa (1000 U / mL and 10,000 U / mL), IL-2 (1000 U / mL and and 10000U / mL), Ad5-luc (1 and 100vp / cell), Ad5-CMV-mIL2 (1 and 100vp / cell), Ad5-CMV-mTNFα (1 and 100 vp / cell), or different combinations thereof, were administered to B16.OVA cells. The effect of PD-L1 and PD-L2 expression on tumor cells was investigated. The subjects were examined at 0 and 72 hours.

[0082] Flow cytometry Cell cultures and tumor samples were processed and labeled as described elsewhere ( 8 ). Anti-CD4-FITC (clone GK1.5, 100406, Biolegend), anti-CD3e-PE (clone 145-2C11, 1 2-0031-82, eBioscience), anti-CD69-PE-Dazzle (clone H1.2F3, 104536, Biolegend Anti-CD8-PE-Cy5 (clone 53.6-7, 100710, Biolegend), anti-PD-1-PE-Cy-7 (clone Anti-CD45-FITC (clone 30-F11, 103107, Biolegend) End), anti-PD-L2-PE (clone MIH5, 558091, BD), anti-Gr-1-PE-Dazzle (RB6-8C5, 1084 52, Biolegend), anti-CD11b-PE-Cy5 (M1 / 70, 101210, Biolegend), anti-PD-L1-PE-Cy7 (TY-25, 107214, Biolegend) for flow cytometry analysis according to the manufacturer's instructions. The analysis was performed using an SH800Z cytometer (SONY).

[0083] statistical analysis Tumor growth progression was measured using SPSS Statistics 25 (IBM) as a function of logarithmic transformation of tumor volume. The data were analyzed using mixed model analysis. GraphPad was used to analyze the hazard ratios (HRs) and 95% confidence intervals (CIs). In addition, GraphPad was used to analyze cytometry or cytochemistry. Evaluation of differences between groups in the analysis of the data (unpaired t-test with Welch's correction), correlation analysis between variables ( Pearson's r), time course of variables (two-way ANOVA), and linear regression were performed. The effect was calculated using the FTV (Fraction Tumor Volume) method, and a P value of <0.05 was considered statistically significant.

[0084] result Oncolytic adenovirus-mediated tumorigenesis was observed in patient-derived urinary tumor tissue cultures. Lysis of tumor cells How oncolytic virus therapy responds to checkpoint inhibitors in solid tumors Surgically removed tissues were analyzed pathologically to understand the mechanisms underlying the response. These tumors were pathologically evaluated by hematoxylin and eosin staining. The clinical grades were 3 (patient sample 2) or 4 (patient samples 1 and 3) (Figure 1A). One of the main effects of the cargo carried by the virus is to affect CD8+ T cells, and thus its existence The presence of HIV was also assessed (Fig. 1B), revealing two samples with an immune-exclusion ("cold") phenotype ( Patient samples 1 and 2) and one sample with an immune-inflammatory ("hot") phenotype (patient sample 3) Furthermore, in relation to anti-PD-L1 targeting, an assay for determining PD-L1 positivity was also shown. As a result, PD-L1 expression on immune cells was less than 5% in all samples. , which was shown to be negative according to the test guidelines (Figure 1C).

[0085] After processing the samples, measure how the treatment affects the viability of the tumor tissue cultures To confirm this, a viability assay was performed (Figure 1D). By day 7, all three samples had Statistically significant tumor growth was observed with virotherapy compared with mock or anti-PD-L1 monotherapy At that time, survival in the viral treatment group was significantly lower than that in the mock group (p<0.01). 62% (95% CI = [54.19; 69.89]) compared with the anti-PD-L1 treated sample and 56% (95% CI = [38 When the virus and anti-PD-L1 were administered together, the two samples showed a significant reduction in the PD-L1 response rate. A significant decrease in cell viability was observed already from day 1.

[0086] Oncolytic virus therapy induces broad immunostimulatory responses in human urinary tumor tissue cultures cause We also examined tissue cultures for the effect of treatment on cytokine levels (Figure 2). Because tumors in real patients are generally heterogeneous, cytokine levels vary between samples. However, in the group receiving virotherapy, the immunostimulatory cytokines A clear trend towards increased expression of (IFNg, TNFα, IL-2, granzyme B, and CXCL10) For IFNg, Ad5 / 3-E2F-d24-hTNFα-IRES-hIL2 was used in combination with anti-PD-L1 When administered with anti-PD-L1 alone, the Mock group (p=0.0182) and the Anti-PD-L1 alone group (p=0.0181) The production amount increased compared to the "virus" group. A similar trend was observed in the "virus" alone group (p=0.06 8) TNFα and IL-2 production also increased in the virus group, and the difference was not significant when the samples were analyzed individually. On the other hand, IFNb production peaked on the third day of dual therapy, but The effects were not as clear as those of other immunostimulatory cytokines.

[0087] Granzyme B and CXCL10 were highly expressed in the virus-treated group. The expression of these cytokines was correlated with the expression of IFNg (IFNg / Granzyme B: r = 0.629 [0. 376; 0.792], p<0.001. IFNg / CXCL10: r=0.494 [0.198; 0.708], p= 0.002). Expression was significantly higher in the “U” group compared to the “Mock” ​​group (p= 0.003) and the “Anti-PD-L1” group (p= 0.002). Treatment with aPD-L1 alone significantly increased the immune response compared to the mock group. There was no effect on the expression of active cytokines.

[0088] For immunosuppressive mediators (IL-6, TGF-b, and arginase), stimulatory The therapeutic effect was not as clear as that of mediators (Figure 2A-F). The “1” group and “virus + anti-PD-L1” group were significantly different from the “mock” and “virus + anti-PD-L1” groups in two of the three samples. A statistically significant decrease in IL-6 was observed compared to the "virus" group. In the “anti-PD-L1 + anti-PD-L1” group, a significant decrease was observed in all samples, but in the “anti-PD-L1” group, a significant decrease was observed in only one sample. Arginase is an enzyme that affects immune activity, not cytokines. This enzyme was included in the panel despite the fact that it is an anti-PD-L In the "1" group and the "virus + anti-PD-L1" group, 1 Expression was decreased in the samples.

[0089] Treatment-induced production of immunostimulatory cytokines is associated with decreased viability in solid tumor specimens. Related Compare differences in cytokine and arginase expression in tumor tissue cultures at day 7 Therefore, the average fold change compared to “mock” was plotted side by side (Fig. 3A and B). Of the six stimulatory cytokines analyzed, increased expression of TNFα and IL-2 was at least partially responsible for the This is thought to be primarily related to viral expression of the transgene and cannot be distinguished from endogenous production. It is noteworthy that the expression of TNFα increased by more than 1000 times and that of IL-2 increased by about 100 times. For IFNg, the virus-treated group showed a more than 100-fold increase compared to the mock condition. Interestingly, adding anti-PD-L1 to viral therapy significantly improved survival compared to viral therapy alone. In all cases, 10-fold increased expression was observed (1000-fold compared to the "mock" case). Treatment with CXCL10 increased CXCL10 levels by an average of 25-fold.

[0090] This treatment did not have a dramatic effect on the expression levels of IL-6, TGF-b, or arginase. Anti-PD-L1 therapy appeared to reduce the levels of IL-6 and TGF-β, but not the levels of α-PD-L1. The expression of arginase was significantly increased when both virus and checkpoint inhibitors were administered together. To compare the therapeutic effects on endogenously produced immunosuppressants and immunostimulators, To do this, we compared the mean values ​​of IL-6, TGF-b, and arginase with those of IFNg, IFNb, granzyme B, and The combined treatments were plotted against the mean values ​​of CXCL10 and CXCL10 (Figure 3C). The reduction of immunosuppression brought about by checkpoint inhibitors and the immunostimulation brought about by viral therapy There was a relationship between the production of immunostimulatory cytokines and the viability of tumor tissue culture cells. An inverse correlation was observed (r = -0.716 [-0.914; -0.241], p = 0.009). No correlation was found between the levels of erythrocyte sedimentation and immunosuppressive cytokines.

[0091] Adenoviruses expressing TNFα and IL-2 enable in vivo anti-PD-L1 therapy , with a complete response rate of 100% Second, the immune stimulation caused by the virus and the suppression achieved by anti-PD-L1. We aimed to assess whether the reduction in tumor growth factor receptor β-glucosidase activity could be reproduced in vivo. From this perspective, we wanted to examine the impact of immune remodeling on tumors (Figure 4A). To understand the effects, two groups were treated with the virus and a checkpoint inhibitor, but the results were different. Different dosing regimens were used: one group received both treatments simultaneously (S), and the other group received both treatments simultaneously (S). , "prime boost" (PB) method, check only after two rounds of viral treatment. A cross-point inhibitor was given.

[0092] The two groups that received the virus and anti-PD-L1 together had better survival outcomes. (Figure 4B). In particular, when the virus (intratumoral) was administered simultaneously with anti-PD-L1 (intraperitoneal), 100 The complete response rate of 100% was achieved in the "virus + aPD-L1" group. The survival time was significantly longer in the "virus + aPD-L1" group than in any other group. The time to response was longer (p<0.001 vs. "Mock", p=0.007 vs. "aPD-L1", p=0.007 vs. "Virus"). " and "virus + aPD-L1(PB)" (p=0.005 and p=0.025, respectively). The hazard ratio for the "L1" strategy was superior to any other group examined (HR for "Mock" = 0. 0.033 [0.006; 0.181], HR for "aPD-L1" = 0.057 [0.007; 0.451], and HR for "virus" = 0.026 [0.006; 0.181]. HR for "virus + aPD-L1(PB)" = 0.067 [0.011; 0.415], and HR for "virus + aPD-L1(PB)" = 0.104 [0.014; 0.752]).

[0093] The "prime-boost" approach resulted in significantly longer overall survival (OS) (p<0.001). The hazard ratio (HR) was also lower than mock (0.059 [0.012; 0.290]). Or checkpoint inhibitor monotherapy resulted in a complete response rate of approximately 33%. The OS with chemotherapy alone was statistically improved compared to mock (p=0.016), with an HR of 0.180 [0.044; 0.7 27]) was also reduced. In addition, the individual tumor volumes were plotted (Figure 4C). Synergistic effects were observed as early as day 5 after the initiation of chemotherapy. Complete response was observed by day 90 after the initiation of therapy. Some of the affected animals had scar tissue in the area around the tumor. The scar was collected and analyzed by a pathologist, who found melanophages, plasma cells, and lymphocytes. However, no malignant cells were reported.

[0094] discussion In this study, we demonstrate how a viral platform encoding TNFα and IL-2 mediates immune responses. It significantly impacts the tumor microenvironment, resulting in higher resistance in the setting of anti-PD-L1 checkpoint blockade. Similar results were obtained in tissue cultures of human urinary clinical samples (renal cells) This has been observed both in cancer and urothelial carcinomas and in vivo, resulting in tumor growth control and survival. It is noteworthy that each monotherapy had a positive effect. When used in combination, the two drugs demonstrated a synergistic effect in terms of tumor control.

[0095] Complete responses are extremely rare in patients, with partial responses being more common, but Even in most solid tumor types, ORRs with CPIs ranged from 10-40% after monotherapy. However, the presence of TILs (1, 2) and increased expression of proinflammatory cytokine signatures (3-5) are key factors. is also known to be a strong predictor (6). The researchers found that the tumors were effectively transformed into "hot" tumors to improve response rates and survival. The potential importance of this approach is that it has enabled more efficient CPI therapy.

[0096] Example 2 material and method In vivo experiments In vivo experiments were performed using C57BL / 6OlaHsd female mice (E. coli, Huntingdon, UK) aged 4-6 weeks at the start of the experiment. (purchased from Nvigo Labs) 2.5 x 10 5 B16.OVA melanoma cell line in the left lower flank After subcutaneous implantation, the presence of a palpable tumor measuring at least 4 mm in diameter was confirmed. Once tumor size criteria were met, animals were randomly assigned to different treatment groups. The volume of the blood was measured daily and general health was assessed. Animals with ulcers at the site of the tumor were immediately euthanized. The maximum tumor volume allowed was 18 mm; Animals without observable tumors were considered to be free of tumor recurrence. To confirm, patients were allowed to survive for at least 90 days after receiving their first treatment.

[0097] Antibodies and viruses The treatment method is shown for each specific experiment. Anti-PD-1 antibody (aPD-1) treatment was performed using 0.1 ml of PD-1 diluted in PBS. g and delivered systemically (intraperitoneally) Lebanon, New Hampshire, USA). Virotherapy treatment consists of x10 8 virus particles (containing equal amounts of Ad5-CMV-mIL2 virus and Ad5-CMV-mTNFa virus, (not replicated in mice).

[0098] Transcriptome analysis Tumors collected in the in vivo experiments were treated with RNAlater (R0901, Sigma-Aldrich, USA). The DNA was stabilized in RNeasy (74104, Qiagen, St. Louis, MO) and stored at -20°C. RNA was purified from these tumor samples according to the kit manufacturer's instructions (Bio-Rad, Inc., Hilden, Germany). A spectrophotometer (Biophotometer, Eppendorf, Weston, NY, USA) was used. The concentrations were adjusted after measurements were performed at Tver. Sequencing of the RNA samples was performed at BGI Tech. The data was then cleaned and quantitatively analyzed in a single-blind manner by Solutions (Tai Po, Hong Kong). It was done by law.

[0099] CyTOF Tumors harvested from in vivo experiments were processed into single cell suspensions and analyzed by mass cytometry. The tissue was then stored in freezing medium (containing 10% dimethyl sulfoxide) until staining.

[0100] statistical analysis GraphPad Prism 8 (GraphPad Software, Sandy, CA, USA) Using the EGO analysis tool, the log-rank Mantel-Kopf analysis of Kaplan-Meier survival curves was performed. Performed the Kuss test and Mann-Whitney test and calculated the mean for graphical display of the data. As mentioned above 8 As such, it is used to analyze the progression of tumor growth based on daily tumor diameter measurements. The software used was SPSS Statistics 25 (IBM Corporation, Arlington, NY, USA). Monk). Statistical significance was claimed when p-value was less than 0.05.

[0101] result The B16.OVA tumor model responded to anti-PD-1 but did not achieve a long-term response To investigate the mechanism of resistance to aPD-1, we used an inhibitory antibody 8 Limited response to We selected B16.OVA, a model that demonstrates the inherent causes of drug resistance. or to understand whether tumors are adapting after being subjected to survival pressures from drugs. In this context, a tumor growth criterion was adopted (Figure 6). In this sense, the tumor must first grow to at least Animals that reached 4 mm were randomly assigned to either the "mock" or "aPD-1" group. Animals in the "aPD-1" group then received systemic PD-1 blocking antibody treatment once every three days (total (at least 5 rounds). Tumors in all animals were immediately cultured until they reached a maximum diameter of at least 10 mm. Animals whose tumors exceeded that threshold were euthanized and excluded for further analysis. Tumors were harvested.

[0102] The tumor size at the start of the treatment period and at the time of euthanasia was the same until the second threshold was reached. The time to tumor growth was significantly different (p = 0.0008) (Fig. 6B). There was a significant (p=0.0002) benefit, with 1 in 10 animals treated with anti-PD-1 remaining alive by day 30. Even though the treatment slowed the progression of the tumor, 90% The tumor eventually recurred, reaching a second threshold (Fig. 6C).

[0103] These results demonstrate the presence of antitumor efficacy after PD-1 blockade, but do not predict long-term adverse effects. It also demonstrates a lack of response.

[0104] Tumors that no longer respond to anti-PD-1 have a different gene expression profile from untreated tumors. Show file Tumors were harvested and then processed for RNA extraction as described in Figure 6. Total RNA sequencing Sampling was performed to quantify the gene expression levels in each sample. Four samples belonging to the "" group and six samples from the "aPD-1" group were randomly selected. After screening, the samples were arranged in a heatmap (Figure 7A) and clustered based on the similarity between the samples. By this method, the samples from both groups were separated with a reasonable degree of accuracy. By comparing the files, the expression of 357 genes was specifically increased or decreased. Among these genes, 19 had significant immune properties (Fig. 7B). (Figure 7C).

[0105] Of the immune-related genes with downregulated expression and established functions, 75% are T cell-related. These genes include T cell precursors (GZMG and GMZF), T cell activation regulators (TN FSF18 [also known as GITRL] and EAR2), other T cell proteins involved in interactions with other cell types In addition to T cells, NK cells also contain immune components such as complement (CD46). Other lymphocyte populations, such as leukocytes or B cells, are unlikely to be affected by this downregulation. .

[0106] For genes with increased expression, the B cell compartment is a population with a high number of genes. (CD19, CD20, CR2, MMP8, and LY6D), followed by neutrophils (NGP, MMP8, and CXCL3). Among the genes whose expression levels were elevated, complement-related genes (C1S2, CR2) were prominent. In contrast to the down-regulated expression of many T cell-related genes in invasive tumors, this cell population The only gene that was clearly associated with increased expression was FOXP3.

[0107] The full list of genes differentially expressed in aPD-1-resistant tumors includes genes encoding T cell suppressors. There was an observable trend towards inhibition. Other cell populations were also affected, but the results were different. It's not as clear cut as that.

[0108] Anti-PD-1 resistant tumors respond to anti-PD-1 using T cell-activating viral therapy It becomes like this. Addressing the downregulation of T cells present in aPD-1-resistant tumors may improve response to therapy To see if a response could be achieved, two cytokines that target antitumor T cell activity were administered. Adenoviruses encoding TNFα and IL-2 were used. Similarly, animals bearing subcutaneous tumors were treated with aPD-1 until they were deemed resistant to the drug. After developing a resistant state, the animals were divided into two groups: the "aPD-1" group, in which they continued to receive aPD-1; A "viral" group received virotherapy alone, or aPD-1 plus additional virotherapy ("less"). Patients were randomly assigned to either the "aPD-1 + virus" group or the "cue treatment" group. until the maximum tumor volume allowed (18 mm) or as described in Figure 8A. Treatment was continued until an apparent complete response (no visible lesions in the area) was achieved.

[0109] We examined tumor-specific survival rates after reaching the resistance threshold (Fig. 8B). Cell-activating viral therapy significantly (p=0.0009) increased survival and further increased survival by 50%. It was shown that viral therapy alone could induce complete responses with aPD-1 monotherapy. Although no significant improvement in survival time was observed when compared with the above-mentioned virus-based methods, A similar conclusion was reached from the analysis of individual tumor growth curves. can also be derived (Fig. 8C).

[0110] Furthermore, in this experiment, animals in the "aPD-1" group continued to show antibody resistance even after reaching the threshold of resistance. Both survival and tumor growth data were previously unreported, suggesting that continued treatment with EGFR conferred no additional benefit. This demonstrates the tumor resistance to aPD-1 that had been hypothesized by previous studies.

[0111] We will examine aPD-1-resistant tumor samples treated with viral therapy to understand the effects of therapy on immune cells. I understood To generate samples for tumor immunophenotyping studies, follow the same experimental design as in Figure 8A. However, this time, the "rescue treatment" given after a resistant state was achieved was continued for seven days. (Figure 9A). Tumors were then collected and processed for analysis. Even though the tumors were similar, by day 7, the aPD-1 group and the viral therapy group showed When comparing animals in the treated groups, there was a statistically significant (p<0.001) reduction in tumor volume ( Figure 9B).

[0112] Using tumors harvested on day 7, the re-emergence of responses to aPD-1 was confirmed by examining the tumor microenvironment. We investigated whether this is related to the reorganization of the immune compartment that leads to the development of 28 The samples were then subjected to mass cytometry analysis using the following cell markers: For the CD45+ fraction, 64 cell populations were identified using the FlowSOM algorithm. These populations were then depicted in a heatmap (Figure 9C) to further reveal the cell types and phenotypes they represent. I confirmed with.

[0113] By combining viral therapy with anti-PD-1, the immune microenvironment of anti-PD-1 resistant tumors becomes anti-tumor. Restructured to favor response Cell populations obtained by mass cytometry were individually examined and identified as the most likely cell type. The clusters that were most clearly associated with specific cell populations are shown in Figure 10.

[0114] Of the 64 cell populations, 29 were T cell populations based on co-expression of CD45, CD3e, and TCRb. Of these T cell populations, 22 were CD8+ (CD4-) and 4 were C Furthermore, one population (number 4) was CD3e+ TCRb+ CD8+ CD4+, The other two (populations 42 and 43) were CD3e+ TCRb+ CD8- CD4-. The significant change was CD8 It was observed only in the T cell population, but not in the CD4, double positive, or double negative T cell populations. Overall, 14 distinct CD8 T cell subsets expressed aPD-1 and virus-specific phenotypes (Fig. 10A–H). The phenotype associated with migration to inflammation sites (CC) was significantly increased in tumors receiving both chemotherapeutic agents. To observe how T cells with R2 markers (based on R2 markers) appear more abundantly after dual therapy Not only did T cell trafficking increase, but effector / memory CD8 T cells also proliferate. cells (based on CD44 and Ki-67 markers), activated and proliferating cells (based on Ki-67 and TIM-3 markers) A wide range of T cells, including not only naive T cells (based on the CD44 marker) but also naive T cells (based on the CD44 marker), The presence of CD8 T cells in tumors is increasing. While virotherapy alone does not provide the same level of efficacy, it consistently improves the distribution of HIV-1.

[0115] Regarding the relevant myeloid populations, M2 macrophages and MDSCs are involved in the treatment of viral infections. The addition of the above combinations reduces the expression of M1 macrophages or dendritic cells in tumors. Although no significant changes were observed in the dendritic cells, the dendritic cells showed a significant change when comparing viral therapy with aPD-1 monotherapy. It decreases in comparison.

[0116] discussion aPD-1-resistant tumors did not respond to aPD-1 monotherapy, but did respond to T By incorporating viral therapy using viruses encoding NFα and IL-2, These complete responses were associated with tumor growth control and even complete responses. In addition to being resistant, tumors initially treated with checkpoint inhibitors showed This is seen as a challenge to reject tumors that have expanded approximately eight-fold. Interestingly, survival results and tumor samples The biological analysis of these compounds suggests that they may have alternative mechanisms of action to circumvent aPD-1 resistance and promote antitumor responses. Rather than exploiting the tumor's suppressive state, viral therapy may mitigate the effects of PD-1 blockade. This shows that it is easy to accept.

[0117] Example 3 In vivo experiments Antitumor efficacy of viral therapy and checkpoint inhibitors in a murine head and neck cancer model To check, 1×10 5 MOC2 mouse oral cancer cells were cultured in 4-6 week old female C57BL / 6JOlaHsd mice. Twenty days after implantation, the animals were randomly assigned to groups (n = 1). 7-8 / group). Then, every 3 days (starting on day 0), 0.1 mg of anti-PD-L1 (clone 10F.9G2 , BE0101, BioXCell) on days 0, 1, 3, 6, 9 1 x 10 on days 1, 12, 15, and 18 8 Virus particles (vp) (equivalent to Ad5-CMV- mIL2 virus and Ad5-CMV-mTNFα virus (which do not replicate in mice) are injected into the tumor. The group that did not receive the virus was injected with PBS into the tumor. Tumor growth was monitored on day 30. Animal survival was followed up to day 90, and anti-PD-L1 treatment was administered once every 3 days until maximum tumor size was reached. Treatment was continued until the tumor size reached 18 mm or complete tumor regression.

[0118] Cell lines and viruses MOC2, a mouse oral squamous cell carcinoma cell line, was cultured under the recommended conditions. Construction and production of mouse adenoviruses (Ad5-CMV-mIL2 and Ad5-CMV-mTNFa) The markers were used for in vivo experiments as previously described (12).

[0119] statistical analysis GraphPad Prism 8 (GraphPad Software, Sandy, CA, USA) Log-rank Mantel analysis of Kaplan-Meier survival curves using the EGO analysis tool Cox and Mann-Whitney tests were performed and mean values ​​were used for graphing data. As previously described (8), the progression of tumor growth was estimated based on daily measurements of tumor diameter. The software used for the analysis was SPSS Statistics 25 (IBM Corporation, New York, USA). (Armonk, NY). Statistical significance was claimed when p-value was less than 0.05.

[0120] result Viral therapy and anti-PD-L1 therapy were the most effective antitumor agents in a mouse head and neck cancer model Demonstrate efficacy and survival Therapeutic synergy of adenovirus encoding TNFα and IL-2 with anti-PD-L1 in melanoma In fact, in a mouse oral cancer model, this combination The antitumor effect of the combined treatment was tracked. As expected, the growth curves of individual tumors showed that the tumors in the PBS group were significantly higher than those in the control group. Intravenous injection of anti-PD-L1 (aPD-L1) had little effect on tumor volume (Figure 11A). Administration of Ad5-CMV-mIL2+Ad5-CMV-mTNFa (virus) resulted in tumor-bearing mice. Interestingly, aPD-L1 provided some additional therapeutic benefit over time. The combination of the virus and aPD-L1 resulted in long-term tumor volume control (Figure 11A). demonstrated longer tumor growth control with fewer recurrences than any other therapy tested. This clearly shows that the combination therapy significantly improved the efficacy of the steroids compared with the control or monotherapy (Figure 11A). This also allows for significantly better antitumor efficacy (Figure 11B). Mice given virus and anti-PD-L1 survived the longest, outperforming mice treated with PBS or single agents. The survival rate was higher than that of the control group (Fig. 11C).

[0121] discussion Overall, these results suggest that the combination of adenovirus encoding TNFα and IL-2 with anti-PD-L1 The synergistic effect of the inhibitor combination has been demonstrated in other in vivo tumor models besides melanoma. , showing that surprisingly powerful therapeutic effects can be obtained.

[0122] Example 4 material and method Human tumor tissue culture of ovarian cancer samples Ovarian cancer samples collected from surgically treated patients were purified by single-cell suspension according to a previously described method (9). The cells were thawed and frozen at -140°C in a freezing medium containing 10% DMSO. 3.5 x 10 5 100 viral particles (vp) of Ad5 / 3-E2F-d2 per cell were seeded. 4-hTNFa-IRES-hIL2, 20 μg / mL anti-human PD-L1 (Avelumab, Evidentic), or both So, I treated it in four consecutive sessions.

[0123] Measurement of cell viability Human tumor tissue cultures were treated (as described above) for up to 7 days. Day 1, Day 5, Day 7 Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega, G3582 ) was used to assess cell viability according to the manufacturer's instructions. Viability of mock-treated cells The rate was set to 100%.

[0124] virus For human tumor tissue culture experiments, oncolytic Ad5 / 3-E2F-d24-hTNFα-IRES-hIL2 (TILT-123 (11) was used.

[0125] histopathology After resection, ovarian cancer tissues from the patients were processed for histopathological analysis. was confirmed by a gynecologic pathologist.

[0126] statistical analysis GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) Using the analysis tool of the GO, an unpaired t-test with Welch's correction was performed and the data was plotted in a graph. Mean values ​​were calculated for presentation. Statistical significance was claimed when p-values ​​were less than 0.05.

[0127] result Combining TILT-123 with anti-PD-L1 therapy resulted in increased expression of PD-L1 in patient-derived ovarian cancer tumor tissue cultures. This allows rapid and potent killing of tumor cells.

[0128] In addition, a combination of Ad5 / 3-E2F-D24-TNFα-IRES-IL-2 (TILT-123) and anti-PD-L1 (aPD-L1) was used. The treatment was tested in tumor tissue cultures established from patient-derived ovarian cancer samples. This allows us to develop clinically relevant tumor models while taking into account the histological heterogeneity of human cancers. We were able to validate our combinatorial strategy in tissue culture of established ovarian cancer tumors. Treatment with the nutrient (including tumor cells and immune cells) resulted in a combination of Therapies can kill tumor cells faster than vehicle and / or single-agent treatments. In this experiment, OVCA P1 ovarian low-grade serous carcinoma (stage DiIVB), OVCA P2 ovarian high-grade serous carcinoma (stage IIIC), and OVCA P3 ovarian clear cell There were three different types of histology of cancer (stage IVB).

[0129] discussion Overall, these results suggest that TILT-123 may be effective in combination with another anti-PD-L1 inhibitor (avelumab). The surprising results showed that it promoted potent antitumor efficacy in other indications, such as ovarian cancer. Importantly, the effects of this therapy are related to the tissue of origin. It is independent of the weave structure.

[0130] Example 5 material and method Human tumor tissue cultures obtained from brain metastases of patients with head and neck squamous cell carcinoma resistant to anti-PD-1 therapy thing Brain metastasis samples were collected from patients with head and neck squamous cell carcinoma resistant to anti-PD-1 therapy who underwent surgery. Single-cell suspension was prepared according to the method described previously (9). 5 100 cells were newly plated onto a 96-well plate. The cells were seeded with 100 viral particles (vp) of Ad5 / 3-E2F-d24-hTNFα-IRES-hTNFα cells. Treatments were performed in triplicate with IL2 or Ad5 / 3-E2F-d24, or with medium (no virus).

[0131] Cell viability assay Human tumor tissue cultures were treated (as described above) for up to 7 days. Days 3, 5, and 7 Cell Titer 96 AQueous One Solution Cell Proliferation Assay (Promega, G3582 Cell viability was assessed using a ELISA kit according to the manufacturer's instructions. The rate was set to 100%.

[0132] virus For human tumor tissue culture experiments, oncolytic Ad5 / 3-E2F-d24-hTNFα-IRES-hIL2 (TILT-123 ) or oncolytic Ad5 / 3-E2F-D24 ( 11 ).

[0133] histopathology Prior to undergoing resection, the patient had a confirmed grade 3 primary tumor at the base of the tongue. The tissue structure was confirmed by a pathologist.

[0134] statistical analysis GraphPad Prism 8 (GraphPad Software, San Diego, CA, USA) Using the analysis tool of the GO, an unpaired t-test with Welch's correction was performed and the data was plotted in a graph. Mean values ​​were calculated for presentation. Statistical significance was claimed when p-value was less than 0.05.

[0135] result Single-agent TILT-123 kills brain metastases in patients resistant to anti-PD-1 therapy In the setting of head and neck squamous cell carcinoma resistant to anti-PD-1 therapy, TILT-123 exhibits moderate antitumor activity. (Figure 13). Of note, TILT-123 showed a 7% increase in IL-123 compared to the virus-free control. By day 1, the tumor cell content in the tissue culture was significantly reduced (Figure 1). 13).

[0136] discussion Overall, these data suggest that tumor cells from patients resistant to anti-PD-1 therapy Although the antitumor activity of single-agent TILT-123 is high, its activity is limited. The latter is expected to increase the efficacy of therapy in these types of tumors by combining TILT-123 and Checkpoint inhibitors. This emphasizes the need for concomitant use of an entero-inhibitor.

Claims

1. an effective amount of (a) an oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene; (b) one or more immune checkpoint inhibitors; 20. A method of treating cancer in a subject in need thereof, comprising administering

2. The one or more immune checkpoint inhibitors selectively bind to PD-L1 or PD-1. The method of claim 1 , wherein

3. The oncolytic adenoviral vector is administered intratumorally, intravenously, intraarterially, or intraperitoneally. The method of claim 1 , wherein the

4. The method of claim 3, wherein the oncolytic adenoviral vector is administered intratumorally. 。

5. The immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-L1. and preferably BMS-936559, LY3300054, atezolizumab, durvalumab, and abet 10. The method of claim 1, wherein the compound is selected from the group consisting of lumab.

6. The immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-1. , preferably the group consisting of pembrolizumab (MK-3475) and nivolumab (BMS-936558) The method of claim 1 , wherein the compound is selected from the group consisting of:

7. The virus is about 10 6 -10 14 VP, 10 6 -10 12 VP, 10 8 -10 14 VP, 10 8 -10 12 pieces VP or 10 10 -10 12 The method of claim 1, wherein the VP is administered in an amount of 100 mg / kg.

8. The method of claim 1, wherein the checkpoint inhibitor is administered in an amount of about 2 mg / kg to 25 mg / kg. How to post.

9. The subjects are those with hepatocellular carcinoma, colon cancer, renal cell carcinoma, bladder cancer, lung cancer (non-small cell lung cancer), (including cerebrospinal fluid), stomach cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, and ovarian cancer 10. The method of claim 1, wherein the patient has a cancer selected from breast cancer, cervical cancer, and liver cancer. 。

10. 10. The method of claim 9, wherein the subject has renal cell carcinoma or head and neck cancer.

11. The subject has received prior chemotherapy or immunotherapy, such as treatment with a checkpoint inhibitor.

10. The method of claim 1, wherein the patient has failed at least one prior treatment.

12. 12. The subject has a cancer capable of mediating CD8+ T cell dysfunction. The method described below.

13. The subject has a gene associated with immune activity selected from the group consisting of GZMG, GMZF, KLRC2, and CD46. and a T cell activity regulator selected from the group consisting of TNFSF18 / GITRL and EAR2.

12. The method of claim 11, wherein the patient has a cancer in which expression of at least one gene associated with the disease is decreased. The method described.

14. 12. The subject has an anti-PD-1 resistant cancer, the cancer preferably being melanoma. The method described below.

15. Radiation therapy, chemotherapy, antiangiogenic agents, or targeted therapy, e.g., alkylating agents, nucleoside analogs, leucoside analogs, cytoskeleton regulators, cytostatic agents, monoclonal antibodies, kinase inhibitors 10. The method of claim 1, further comprising administering to the subject an additional therapy selected from the group consisting of a steroid drug and a steroid inhibitor.

16. The method of claim 1 , wherein the subject is a human.

17. a first dose of the oncolytic adenoviral vector and a second dose of the immune checkpoint inhibitor.

10. The method of claim 1, wherein a first dose of the inhibitor is administered to the subject simultaneously.

18. a first dose of the oncolytic adenoviral vector and a second dose of the immune checkpoint inhibitor.

10. The method of claim 1, wherein the first dose of the inhibitor is administered sequentially within a 24-hour period.

19. The oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene. The vector is -5 / 3 Chimera Fiber Knob and - the E2F1 promoter for tumor-specific expression of E1A, - a 24 bp deletion (D24) in the Rb-binding constant region 2 of adenovirus E1, - deletion of nucleic acid sequences in the reading frames of viral gp19k and 6.7k, - Instead of the E3-deleted gp19k / 6.7K, at least TNFα and and / or a nucleic acid sequence encoding IL-2, The method of claim 1, wherein expression of the offspring is controlled in a replication-related manner.

20. The oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene.

20. The method of claim 19, wherein the vector further comprises a deletion of E1B19k.

21. (a) Oncolytic adenoviruses encoding TNFα and / or IL-2 as transgenes A pharmaceutical composition comprising (a) a vector and (b) one or more immune checkpoint inhibitors.

22. The one or more immune checkpoint inhibitors selectively bind to PD-L1 or PD-1.

22. The composition of claim 21 .

23. The oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene. The vector is -5 / 3 Chimera Fiber Knob and - the E2F1 promoter for tumor-specific expression of E1A, - a 24 bp deletion (D24) in the Rb-binding constant region 2 of adenovirus E1, - deletion of nucleic acid sequences in the reading frames of viral gp19k and 6.7k, - Instead of the E3-deleted gp19k / 6.7K, at least TNFα and and / or a nucleic acid sequence encoding IL-2, The pharmaceutical composition of claim 21 , wherein expression of the gene is regulated in a replication-related manner.

24. A kit comprising a first container, a second container, and a package insert, One container contains an oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene. the second container contains at least one dose of a pharmaceutical composition comprising a viral vector; at least one dose of a pharmaceutical composition containing a checkpoint inhibitor, The insert contains instructions for treating an individual with cancer using the pharmaceutical composition. tt.

25. The oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene. The vector is -5 / 3 Chimera Fiber Knob and - the E2F1 promoter for tumor-specific expression of E1A, - a 24 bp deletion (D24) in the Rb-binding constant region 2 of adenovirus E1, - deletion of nucleic acid sequences in the reading frames of viral gp19k and 6.7k, - Instead of the E3-deleted gp19k / 6.7K, at least TNFα and and / or a nucleic acid sequence encoding IL-2, 25. The kit of claim 24, wherein expression of the offspring is controlled in a replication-related manner.

26. The oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene. The composition of claim 21 or claim 24, wherein the vector further comprises a deletion of E1B19k. The kit according to claim 1.

27. for use in the treatment of cancer or tumors, in combination with immune checkpoint inhibitors; Oncolytic adenoviral vectors encoding TNFα and / or IL-2 as transgenes Tar.

28. 27. The immune checkpoint inhibitor selectively binds to PD-L1 or PD-1. TN as a transgene, in combination with an immune checkpoint inhibitor, for the use described in Oncolytic adenoviral vectors encoding Fα and / or IL-2.

29. The oncolytic adenoviral vector is administered intratumorally, intravenously, intraarterially, or intraperitoneally.

29. The method of claim 27 or 28, wherein the immune checkpoint inhibitor is administered to a subject. The combined use of oncolytic adenoviruses encoding TNFα and / or IL-2 as transgenes Viral vector.

30. Any of claims 27 to 29, wherein the oncolytic adenoviral vector is administered intratumorally. a transgene for use according to any one of claims 1 to 4, in combination with an immune checkpoint inhibitor; Oncolytic adenoviral vectors encoding TNFα and / or IL-2 as the agonist.

31. The immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-L1. and preferably BMS-936559, LY3300054, atezolizumab, durvalumab, and avelumab. For use according to any one of claims 27 to 30, selected from the group consisting of velumab of TNFα and / or I as transgenes in combination with immune checkpoint inhibitors. Oncolytic adenoviral vector encoding L-2.

32. The immune checkpoint inhibitor is a monoclonal antibody that selectively binds to PD-1. and preferably consisting of pembrolizumab (MK-3475) and nivolumab (BMS-936558). The immune checkpoint inhibitor for use according to any one of claims 27 to 30, selected from the group consisting of: transgenes encoding TNFα and / or IL-2 in combination with cross-linking inhibitors. Oncolytic adenoviral vector.

33. The virus is about 10 6 -10 14 VP, 10 6 -10 12 VP, 10 8 -10 14 VP, 10 8 -10 12 pieces VP or 10 10 -10 12 The use according to any one of claims 27 to 32, wherein the amount of VP is administered. TNFα and TNF-α transgenes in combination with immune checkpoint inhibitors for and / or an oncolytic adenoviral vector encoding IL-2.

34. The checkpoint inhibitor is administered in an amount of about 2 mg / kg to 25 mg / kg. Introduction of a method for the treatment of a patient with a rheumatoid arthritis, for use according to any one of claims 3 to 5, in combination with an immune checkpoint inhibitor. Oncolytic adenoviral vectors encoding TNFα and / or IL-2 as genes 。

35. The subjects are those with hepatocellular carcinoma, colon cancer, renal cell carcinoma, bladder cancer, and lung cancer (including non-small cell lung cancer). (including), stomach cancer, esophageal cancer, sarcoma, mesothelioma, melanoma, pancreatic cancer, head and neck cancer, ovarian cancer, 35. The patient according to claim 27, having a cancer selected from breast cancer, cervical cancer, and liver cancer. A transgene for use in combination with an immune checkpoint inhibitor according to any one of claims 1 to 4. Oncolytic adenoviral vectors encoding TNFα and / or IL-2 as vectors.

36. 36. The method of claim 35, wherein the subject has renal cell carcinoma or head and neck cancer. TNFα and / or IL-2 as transgenes in combination with immune checkpoint inhibitors An oncolytic adenoviral vector encoding

37. The subject has received prior chemotherapy or immunotherapy, such as treatment with a checkpoint inhibitor. The use according to any one of claims 27 to 36, wherein the patient has failed at least one treatment for TNFα and / or TNFα transgenes in combination with immune checkpoint inhibitors for is an oncolytic adenoviral vector encoding IL-2.

38. 38. The subject has a cancer capable of mediating CD8+ T cell dysfunction. TN as a transgene, in combination with an immune checkpoint inhibitor, for the use described in Oncolytic adenoviral vectors encoding Fα and / or IL-2.

39. The subject has a gene associated with immune activity selected from the group consisting of GZMG, GMZF, KLRC2, and CD46. and a T cell activity regulator selected from the group consisting of TNFSF18 / GITRL and EAR2. 37 or 38, wherein the patient has a cancer in which expression of at least one gene associated with the disease is decreased.

38. A transgene for use in combination with an immune checkpoint inhibitor according to claim 38. Oncolytic adenoviral vectors encoding TNFα and / or IL-2 as the agonist.

40. 3. The subject has an anti-PD-1 resistant cancer, preferably melanoma. 7 to 39 for use in combination with an immune checkpoint inhibitor. , an oncolytic adenoviral vector encoding TNFα and / or IL-2 as a transgene; Kutar.

41. Radiation therapy, chemotherapy, angiogenesis inhibitors, or targeted therapy, e.g., alkylating agents, nucleoside analogs, Nucleoside analogues, cytoskeleton regulators, cytostatic agents, monoclonal antibodies, kinases and administering to the subject an additional therapy selected from the group consisting of a steroid inhibitor, a steroid drug ...

1. A method for the treatment of a rheumatoid arthritis, comprising administering to a patient a therapeutically effective amount of a rheumatoid arthritis vaccine ... Oncolytic adenoviral vectors encoding TNFα and / or IL-2 as genes.

42. The immunotherapy for use according to any one of claims 27 to 41, wherein the subject is a human. Transgene encoding TNFα and / or IL-2 in combination with checkpoint inhibitors Oncolytic adenoviral vectors that deliver

43. a first dose of said oncolytic adenoviral vector and said immune checkpoint 43. The method of any one of claims 27 to 42, wherein a first dose of the inhibitor is administered to the subject simultaneously. TNF as a transgene in combination with an immune checkpoint inhibitor for the described use. Oncolytic adenoviral vectors encoding α and / or IL-2.

44. a first dose of said oncolytic adenoviral vector and said immune checkpoint 44. Any one of claims 27 to 43, wherein the first dose of the inhibitor is administered sequentially within 24 hours. TN as a transgene, in combination with an immune checkpoint inhibitor, for the use described in Oncolytic adenoviral vectors encoding Fα and / or IL-2.

45. The oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene. The vector is -5 / 3 Chimera Fiber Knob and - the E2F1 promoter for tumor-specific expression of E1A, - a 24 bp deletion (D24) in the Rb-binding constant region 2 of adenovirus E1, - deletion of nucleic acid sequences in the reading frames of viral gp19k and 6.7k, - Instead of the E3-deleted gp19k / 6.7K, at least TNFα and and / or a nucleic acid sequence encoding IL-2, The use according to any one of claims 27 to 44, wherein expression of the gene is controlled in a replication-related manner. TNFα and / or TNFα transgenes in combination with immune checkpoint inhibitors for is an oncolytic adenoviral vector encoding IL-2.

46. The oncolytic adenovirus encoding TNFα and / or IL-2 as a transgene.

46. ​​The immune control vector for use according to claim 45, wherein the vector further comprises a deletion of E1B19k. transgene encoding TNFα and / or IL-2 in combination with a checkpoint inhibitor Oncolytic adenoviral vector.

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