Oncolytic viruses enhance T cell responses for effective TIL therapy.

Abstract

Methods for expanding tumor infiltrating lymphocyte (TIL) populations and methods of using the expanded TIL populations to treat cancer are disclosed.

Description

[Technical Field]

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 106,215, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Recent advances in cancer immunotherapy have revolutionized the treatment paradigm. However, many cancer patients receive little to no benefit from available therapies, including immune checkpoint inhibitors (ICIs). Numerous clinical trials have shown that existing tumor-reactive T cells are critically important for benefiting from checkpoint inhibitors. Therefore, new methods are needed to enhance the number and function of tumor-reactive T cells. [Prior art documents] [Patent Documents]

[0003] [Patent Document 1] U.S. Patent No. 7,495,090 [Patent Document 2] U.S. No. 7,928,213 [Patent Document 3] U.S. Patent No. 8,138,310 [Non-patent literature]

[0004] [Non-Patent Document 1] Racher, AJ, Fooks, AR & Griffiths, JB Biotechnol Tech(1995) 9: 169 [Non-Patent Document 2] Senter et al., Bioconjugate Chem., 2:447-451 (1991) [Non-Patent Document 3] Bagshawe, KD, Br. J. Cancer, 60:275-281 (1989) [Non-Patent Document 4] Bagshawe et al., Br. J. Cancer, 58: 700 - 703 (1988) [Non - Patent Document 5] Senter et al., Bioconjugate Chem., 4: 3 - 9 (1993) [Non - Patent Document 6] Battelli et al., Cancer Immunol. Immunother., 35: 421 - 425 (1992) [Non - Patent Document 7] Pietersz and McKenzie, Immunolog. Reviews, 129: 57 - 80 (1992) [Non - Patent Document 8] Roffler et al., Biochem. Pharmacol, 42: 2062 - 2065 (1991) [Non - Patent Document 9] Hughes et al., Cancer Research, 49: 6214 - 6220 (1989) [Non - Patent Document 10] Litzinger and Huang, Biochimica et Biophysica Acta, 1104: 179 - 187 (1992) [Non - Patent Document 11] Brown and Greene, DNA and Cell Biology 10: 6, 399 - 409 (1991) [Non - Patent Document 12] Remington: The Science and Practice of Pharmacy (19th Edition) edited by A.R. Gennaro, Mack Publishing Company, Easton, PA 1995 [Non - Patent Document 13] Handbook of Monoclonal Antibodies, edited by Ferrone et al., Noges Publications, Park Ridge, NJ. (1985) ch. 22 and 303 - 357 [Non - Patent Document 14] Smith et al., Antibodies in Human Diagnosis and Therapy, edited by Haber et al., Raven Press, New York (1977), pp. 365 - 389

Summary of the Invention

Means for Solving the Problems

[0005] Disclosed are methods and compositions related to the proliferation of tumor infiltrating lymphocyte (TIL) populations and the use of such TIL populations for cancer treatment.

[0006] In one aspect, a method for generating tumor infiltrating lymphocytes is disclosed herein, comprising: a) administering to tumor cells an effective amount of a tumor-lytic virus that expresses one or more exogenous immune stimulatory molecules (e.g., CD40-L, MEM40, B7-1 (CD8*) / B7-2 (CD86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58 and / or SLAMF6, etc.); and b) recovering tumor infiltrating lymphocytes. In some aspects, the tumor-lytic virus may further express one or more type I interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω and / or IFN-ζ, etc.). TIL can be obtained even in the tumor microenvironment not infected with the tumor-lytic virus, but in some aspects, the generated TIL is obtained in the tumor microenvironment at the administration site of the tumor-lytic virus.

[0007] <> Also disclosed herein is a method for generating tumor infiltrating lymphocytes of any of the前述 aspects, wherein the tumor-lytic virus is administered via intratumoral injection.

[0008] Also disclosed herein is a method for generating tumor infiltrating lymphocytes of any of the前述 aspects, further comprising the step of ex vivo expanding the recovered TIL.

[0009] In one embodiment, a method for growing a population of tumor-infiltrating lymphocytes (TILs) or bone marrow-infiltrating lymphocytes (MILs) is disclosed herein, comprising the steps of: a) recovering TILs or MILs from a subject having cancer; and b) culturing the recovered TILs or MILs in the presence of antigen-presenting cells infected with an oncolytic virus expressing one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, B7-1(CD80) / B7-2(CD86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58 and / or SLAMF6, etc.). In some embodiments, the oncolytic virus may further express one or more type 1 interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and / or IFN-ζ, etc.).

[0010] Furthermore, this specification discloses a method for treating, reducing, inhibiting, decreasing, restoring, and / or preventing cancer and / or metastasis in a subject, comprising the step of administering a therapeutically effective amount of TIL or MIL in any of the embodiments described above. For example, this specification discloses a method for treating, reducing, inhibiting, decreasing, restoring, and / or preventing cancer and / or cancer metastasis in a subject, comprising the steps of: a) administering an effective amount of oncolytic virus expressing one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, B7-1(CD80) / B7-2(CD86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and / or SLAMF6, etc.) into tumor cells; b) recovering tumor-infiltrating lymphocytes (TILs) and / or bone marrow-infiltrating lymphocytes (MILs); c) growing the recovered TILs and / or MILs ex vivo; and d) administering a therapeutically effective amount of the grown TILs and / or MILs to the subject. In some embodiments, the cancerous or metastatic tumors to be treated, reduced, inhibited, decreased, restored, and / or prevented are abscopal to tumors receiving either TIL or MIL of the oncolytic viruses disclosed herein. In some embodiments, the oncolytic viruses may further express one or more type 1 interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and / or IFN-ζ, etc.).

[0011] Alternatively, the present invention discloses a method for treating, reducing, inhibiting, decreasing, restoring, and / or preventing cancer and / or metastasis in a subject, comprising the steps of: a) recovering tumor-infiltrating lymphocytes (TILs) and / or bone marrow-infiltrating lymphocytes (MILs) from a subject having cancer; culturing the recovered TILs or MILs in the presence of antigen-presenting cells infected with an oncolytic virus expressing one or more exogenous immunostimulatory molecules; and administering the proliferated TILs and / or MILs to the subject in a therapeutically effective amount. In some embodiments, the oncolytic virus may further express one or more type 1 interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and / or IFN-ζ, etc.).

[0012] In one embodiment, a method for treating, reducing, inhibiting, decreasing, restoring, and / or preventing cancer and / or metastasis of any of the above embodiments is disclosed herein, further comprising the step of administering an anticancer agent to a target.

[0013] The accompanying drawings incorporated herein and constituting part thereof illustrate several embodiments and illustrate compositions and methods disclosed together with this description. [Brief explanation of the drawing]

[0014] [Figure 1A] This figure shows the results of transduction of B16-OVA cells with pLenti-Puro control lentivirus or lentivirus expressing mouse or human IFNβ, followed by puromycin selection. The same number of cells were seeded, and the supernatant was collected two days later for ELISA to detect human and mouse IFNβ. Statistical analysis was performed on n=6 using two-way ANOVA for growth curves or t-tests for OVA tetramer evaluation. Statistical significance is indicated by p-values ​​as *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 1B]This figure shows the results of subjecting wild-type or IFNAR1 KO (IFNAR) C57BL / 6 mice to two rounds of B16-OVA-pLenti, B16-OVA mouse IFNβ, or B16-OVA human IFNβ vaccine on D0 and D7 (cells were irradiated with 100 Gy before injection). The percentage of OVA tetramers in MHCII- and CD8+ cells in peripheral blood on D12 is shown. Statistical analysis of n=6 was performed using two-way ANOVA for growth curves or t-tests for OVA tetramer evaluation. Statistical significance is indicated by p-values ​​as *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 1C] Figure 1C is identical to (A), except that mice were exposed to 3e5 live B16-OVA cells on D21. Tumor growth was determined as instructed. Statistical analysis was performed on n=6 using two-way ANOVA for growth curves or t-tests for OVA tetramer evaluation. Statistical significance is indicated by p-values ​​as *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 2A] This is a schematic diagram of MEM40. [Figure 2B] This is a schematic diagram of MEM-288. [Figure 3A] This figure shows the results of infecting (3A)A549 or (3C)B16-F10 cells with control GFP virus or oncolytic Ad-MEM-188 or 288 at MOI=250 for 2 days as instructed. GFP and MEM40 expression were determined by flow cytometry. [Figure 3B] This figure shows (3A) A549 or (3C) B16-F10 cells infected with control GFP virus or oncolytic Ad-MEM-188 or 288 at MOI=250 for 2 days as instructed. IFNβ secretion was determined by ELISA after infection with the instructed OV in (3B) A549 cells or (3D) B16-F10 cells. [Figure 3C]This figure shows the results of infecting (3A)A549 or (3C)B16-F10 cells with control GFP virus or oncolytic Ad-MEM-188 or 288 at MOI=250 for 2 days as instructed. GFP and MEM40 expression were determined by flow cytometry. [Figure 3D] This figure shows (3A) A549 or (3C) B16-F10 cells infected with control GFP virus or oncolytic Ad-MEM-188 or 288 at MOI=250 for 2 days as instructed. IFNβ secretion was determined by ELISA after infection with the instructed OV in (3B) A549 cells or (3D) B16-F10 cells. [Figure 4A] This figure shows the results of infecting (4A)344SQ(344), (4B)B16-F10 mouse cell lines and (4C)A549 human cell line with the indicated OV, AD-GFP(GFP), MEM-188(188), or MEM-288(288) at various MOIs (1, 10, 100) for 2 days. Cell viability was determined by trypan blue staining assay 2 days after infection. [Figure 4B] This figure shows the results of infecting (4A)344SQ(344), (4B)B16-F10 mouse cell lines and (4C)A549 human cell line with the indicated OV, AD-GFP(GFP), MEM-188(188), or MEM-288(288) at various MOIs (1, 10, 100) for 2 days. Cell viability was determined by trypan blue staining assay 2 days after infection. [Figure 4C] This figure shows the results of infecting (4A)344SQ(344), (4B)B16-F10 mouse cell lines and (4C)A549 human cell line with the indicated OV, AD-GFP(GFP), MEM-188(188), or MEM-288(288) at various MOIs (1, 10, 100) for 2 days. Cell viability was determined by trypan blue staining assay 2 days after infection. [Figure 5A]This figure shows C57BL / 6 mice inoculated with 5e5 B16-OVA cells in sc. On days D12 and D16, these mice were subjected to two intratumoral injections of Ad-GFP, MEM-188, and MEM-288 at (5A)10e8 or (5B)10e9 IU. The significance of the difference in tumor size is shown compared to untreated control mice at the final time. [Figure 5B] This figure shows C57BL / 6 mice inoculated with 5e5 B16-OVA cells in sc. On days D12 and D16, these mice were subjected to two intratumoral injections of Ad-GFP, MEM-188, and MEM-288 at (5A)10e8 or (5B)10e9 IU. The significance of the difference in tumor size is shown compared to untreated control mice at the final time. [Figure 5C] This figure shows the percentage of OVA-specific MHCII and CD8+ cells in peripheral blood on D12, as shown after intratumoral injection of 10e9 IU of MEM-188 and MEM-288 in B16-OVA. Statistical analysis was performed using two-way ANOVA for growth curves or t-tests for OVAT evaluation. Statistical significance is indicated by p-values ​​as *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 5D]This figure shows the quantification of IFNγ ELISPOT derived from spleen CD8 T cells. 5e5 B16-OVA cells were inoculated sc onto the flanks of C57BL / 6 WT, IFNARl KO, and CD40 KO mice (3 mice per group), and 109 IU of PBS (UT: untreated) or MEM-288 was administered intratumorally on D12 and D16. Mouse spleens were subjected to magnetic bead isolation of CD8 T cells according to the manufacturer's recommendations. Subsequently, 2 × 10⁵ / well of CD8 cells and 1 × 10⁵ / well of 50 Gy-irradiated B16-OVA cells were seeded into triple wells and incubated in a 96-well plate at 37°C for 24 hours. Tumor cells were stimulated with IFNγ to increase MHC expression. T cells were also cultured alone or with concavalin A (ConA) as negative and positive controls, respectively. Results for each group treated with ConA are shown. Statistical analysis was performed using the t-test. Statistical significance is indicated by the p-value, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 6A] This figure shows the treatment plan in mice. C57BL / 6 mice were inoculated with 5e5 B16-F10 cells in the primary site and 2.5e5 B16-F10 cells in the contralateral site using sc. As shown, these mice were injected with MEM-288 at 10e9 IU into the primary tumor on D12 and D16, and with anti-PD-1 and CTLA-4 antibodies via ip on D16, D19, D23, and D27. [Figure 6B] This figure shows the determination of tumor growth in the primary site (6B) and the contralateral site (6C), as indicated. Statistical analysis was performed using two-way ANOVA for the growth curves. Statistical significance is indicated by the p-value, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 6C] This figure shows the determination of tumor growth in the primary site (6B) and the contralateral site (6C), as indicated. Statistical analysis was performed using two-way ANOVA for the growth curves. Statistical significance is indicated by the p-value, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 6D] This figure shows the Kaplan-Meier survival curves for overall mouse survival. Statistical significance is indicated by the p-value, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not statistically significant. [Figure 7A] This figure shows the results of inoculating 344 cells of 5e5 in the flanks of 129 mice with sc, followed by intratumoral injection of 10e9 IU of Ad-GFP, MEM-188, or MEM-288 on D12 and D16. Tumor growth at the primary site was determined as shown. Significance is shown compared to the control UT group (PBS injection). Statistical significance is indicated by p-values, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 7B] This figure (7A) shows typical H&E staining of tumors in the lungs of 344 tumor-bearing mice using sc at D38. [Figure 7C] This figure shows the quantitative determination of tumor metastasis in individual mice from various groups as instructed. Statistical significance is indicated by the p-value, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 7D] (7A) This figure shows typical IHC staining of CD8 T cells in the lungs of a mouse. [Figure 7E] This figure shows the quantification of CD8 T cell density in individual mice from various groups as instructed. Statistical significance is indicated by the p-value, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 8A] This figure shows the results of inoculating 344 cells of 5e5 in the flanks of 129 mice with sc, administering 10e9 IU of MEM-288 intratumorally on D12 and D16, and treating with anti-PD-1 antibody via ip on D16, D19, D23, and D27. Figure 8A shows IFNγ ELISPOT by mouse spleen CD8 T cells, and Figure 8B shows the quantification of ELISPOT resulting from (A). Statistical analysis was performed using the t-test. Statistical significance is indicated by the p-value, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 8B] This figure shows the results of inoculating 344 cells of 5e5 in the flanks of 129 mice with sc, administering 10e9 IU of MEM-288 intratumorally on D12 and D16, and treating with anti-PD-1 antibody via ip on D16, D19, D23, and D27. Figure 8A shows IFNγ ELISPOT by mouse spleen CD8 T cells, and Figure 8B shows the quantification of ELISPOT resulting from (A). Statistical analysis was performed using the t-test. Statistical significance is indicated by the p-value, where *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. [Figure 9A] This figure shows the flow cytometry analysis scheme of B16-OVA for detecting populations of macrophages, neutrophils, monocytes, and DCs. [Figure 9B] This figure shows the flow cytometry analysis scheme for B16-OVA to detect populations of B cells, CD4 T cells, CD8 T cells, and OVA-specific CD8 T cells. [Figure 10] This figure shows the flanks of C57BL / 6 mice inoculated with 5e5 B16-OVA or B16-OVA-ZsGreen cells via sc. At D14, flow cytometry analysis of tumor B16-OVA cells was performed to detect the ZsGreen-positive population of macrophages and DCs, as shown in Figure 9A. The percentage of positive cells is shown. [Figure 11A] This figure shows the flanks of C57BL / 6 mice inoculated with 5e5 B16-OVA or B16-OVA-ZsGreen cells via sc. Flow cytometry analysis was performed on D14, and ZsGreen+ tumor cells were detected in CD45- as shown. [Figure 11B] This figure shows B16-OVA-ZsGreen tumors subjected to intratumoral injection of PBS or 10e9 IU of MEM-288 on D12. Flow cytometry analysis of CD40L against ZsGreen+CD45- cells was performed on the PBS and MEM-288 injected tumors. [Figure 12A]This figure shows flow cytometry analysis of CD80 and CD86 expression in MHC-II hypermacrophages and DCs (CD11b+DC2) performed on the tumor shown in Figure 1B. It also shows injections of PBS and MEM-288 into the tumor. [Figure 12B] This figure shows the average fluorescence intensity (MFI) of result (A). [Figure 13] This figure shows the DC population in the inguinal neuropathy (LN). Two main populations of CD11c+MHC-II+DC were detected. The high MHC-II population consisted of migratory DC1 and DC2. The moderate MHC-II population consisted of resident DC1 and DC2. As expected, the migratory population had a higher proportion of CD103+DC1, while the resident population had a higher proportion of CD8a+DC1. [Figure 14] This figure shows the DC population in the lnx of WT, CD40 KO, and IFNAR1 KO mice. Two main populations, CD11c+MHC-II+DC, were detected in mice of all genotypes. The high MHC-II population consists of migratory DC1 and DC2. The moderate MHC-II population consists of commensal DC1 and DC2. [Figure 15A] This figure shows the flank ventral region of C57BL / 6 mice inoculated with 5e5 B16-OVA cells in sc. Tumors were subjected to 109 IU injections of PBS (n=4), adenovirus GFP (n=3), or MEM-288 (n=4) on D12 and D16. On day 20, tumors were obtained and IHC was performed to detect the presence of CD8+ tumor-infiltrating lymphocytes (TILs). CD8 T-cell staining was scored on a scale of 0-3 by pathologists who were not informed of the group. Figure 15A shows typical images of tumors for the three treatment groups. [Figure 15B]This figure shows the flank ventral region of C57BL / 6 mice inoculated with 5e5 B16-OVA cells in sc. Tumors were subjected to 109 IU injections of PBS (n=4), adenovirus GFP (n=3), or MEM-288 (n=4) on D12 and D16. On day 20, tumors were obtained and IHC was performed to detect the presence of CD8+ tumor-infiltrating lymphocytes (TILs). CD8 T-cell staining was scored on a scale of 0 to 3 by pathologists who were not informed of the group. Figure 15B shows the scored values ​​for the three treatment groups. [Modes for carrying out the invention]

[0015] Before disclosing and describing the compounds, compositions, articles, apparatus, and / or methods of the present invention, it should be understood that, unless otherwise specified, they are not limited to specific synthesis methods or specific recombinant biotechnological methods, or, unless otherwise specified, specific reagents, and these may, of course, vary. Furthermore, it should be understood that the terminology used herein is for the purpose of describing only specific embodiments and is not intended to be limiting.

[0016] A.Definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include multiple references unless the context clearly indicates otherwise. Thus, for example, a reference to "pharmaceutical carrier" includes two or more mixtures of such carriers.

[0017] A range may be expressed herein as from one specific "approximate" value to and / or another specific "approximate" value. When such a range is expressed, in another embodiment, it includes from one specific value to and / or the other specific value. Similarly, when a value is expressed as an approximation, it is understood that by using the antecedent "approximately," a specific value forms another embodiment. Furthermore, it is understood that both endpoints of each range are important with respect to and independently of the other endpoint. Also, when a number of values ​​are disclosed herein, it is understood that each value is disclosed herein not only as the value itself but also as this specific "approximate" value. For example, when the value "10" is disclosed, "approximately 10" is also disclosed. Also, when a value "less than or equal to" that value is disclosed, it is understood that "greater than or equal to that value" and possible ranges between values ​​are also disclosed, as will be appropriately understood by those skilled in the art. For example, when the value "10" is disclosed, "less than or equal to 10" and "greater than or equal to 10" are also disclosed. Furthermore, it is understood that through this application, data is provided in numerous different formats, and moreover, this data represents endpoints and starting points, as well as ranges of any combination of data points. For example, if a specific data point "10" and a specific data point "15" are disclosed, it is understood that not only are the ranges greater than and greater than 10 and 15, less than and less than 10 and 15, and equivalents thereof also disclosed, but the range from 10 to 15 is also disclosed. It is also understood that each unit between two specific units is disclosed. For example, if 10 and 15 are disclosed, then 11, 12, 13 and 14 are also disclosed.

[0018] In this specification and the subsequent claims, numerous terms are used that should be defined as having the following meanings:

[0019] "Optional" or "optional" means that the following phenomenon or situation may or may not occur, and the description includes both cases in which the phenomenon or situation occurs and cases in which it does not occur.

[0020] As used herein, the term “antigen-presenting cell” (APC) refers to a professional antigen-presenting cell, which is selected from dendritic cells, macrophages, and B cells. In some embodiments, the APC is a DC. In some embodiments, the APC is a mammalian cell. In some embodiments, the APC, e.g., DC, macrophage, or B cell, is a human cell.

[0021] "Increase" can refer to any change resulting in a large amount of symptoms, disease, composition, symptoms, or activity. An increase can be any individual, median, or average increase in a statistically significant amount of symptoms, symptoms, activity, or composition. Thus, an increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase, as long as the increase is statistically significant.

[0022] "Reduction" can refer to any change resulting in a small amount of symptom, disease, composition, condition, or activity. It is also understood that a substance reduces the genetic output of a gene if the genetic output of the gene product by the substance is less than the genetic output of the gene product without the substance. For example, a reduction could be a change in the symptoms of a disorder, such that the symptoms are less severe than previously observed. A reduction can be any individual, median, or average reduction in a statistically significant amount of symptom, condition, activity, or composition. Therefore, a reduction can be a reduction of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100%, as long as the reduction is statistically significant.

[0023] "Inhibition" means reducing activity, response, symptom, disease, or other biological parameters. This may include, but is not limited to, the complete disappearance of activity, response, symptom, or disease. It may also include, for example, a 10% reduction in activity, response, symptom, or disease compared to natural or control levels. Thus, the reduction may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or any amount between these, compared to natural or control levels.

[0024] "Reduce," or other forms of the word, such as "reducing" or "reduction," means to decrease a phenomenon or characteristic (e.g., tumor growth). This is typically relative to some standard or expected value, in other words, it is understood that the reference standard or relative value is not always necessary. For example, "to reduce tumor growth" means that the rate of tumor growth decreases compared to a standard or control.

[0025] "Prevention," or other forms of the word, such as "preventing" or "prevention," means to halt a particular phenomenon or characteristic, stabilize or delay the onset or exacerbation of that phenomenon or characteristic, or minimize the opportunity for that phenomenon or characteristic to occur. Prevention is typically absolute, for example, more so than reduction, and therefore does not require comparison with a control. As used herein, some things can be reduced but not prevented, while others can be reduced and preventable. Similarly, some things can be prevented but not reduced, while others can be prevented and reduced. When using reduction or prevention, it is understood that the use of other words is also explicitly disclosed unless otherwise indicated in detail.

[0026] The term "subject" refers to any individual that is the target of administration or treatment. The subject may be a vertebrate, such as a mammal. In one embodiment, the subject may be a human, a non-human primate, a cattle, a horse, a pig, a dog, or a cat. Alternatively, the subject may be a guinea pig, a rat, a hamster, a rabbit, a mouse, or a mole. Therefore, the subject may be a human or a diseased animal. The term "patient" refers to a subject under the treatment of a clinician, such as an internist.

[0027] The term "therapeutably effective" means that the amount of the composition used is sufficient to restore one or more causes or symptoms of a disease or disorder. Such restoration does not necessarily require elimination, but only reduction or alteration.

[0028] The term “treatment” refers to the medical management of a patient with the intention of curing, restoring, stabilizing, or preventing a disease, condition, or disorder. This term includes active treatment, i.e., treatment directed more specifically to improve the disease, symptoms, or disorder, and causal treatment, i.e., treatment directed to eliminate the cause of the associated disease, condition, or disorder. In addition, this term includes palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder; preventive treatment, i.e., treatment directed to minimize or partially or completely inhibit the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement other specific therapies directed to improve the associated disease, condition, or disorder.

[0029] "Biocompatibility" generally refers to substances that are generally non-toxic to the recipient and do not cause significant adverse effects on the target, as well as any metabolites or degradation products thereof.

[0030] "Contains" is intended to mean that a composition, method, etc., includes the elements listed, but does not exclude others. "Essentially consists of," when used to define a composition and method, means that it includes the elements listed, but does not exclude other elements that have any essential significance to this combination. Accordingly, compositions essentially consisting of the elements defined herein do not exclude trace amounts of impurities from isolation and purification methods, as well as pharmaceutically acceptable carriers, such as phosphate-buffered saline, preservatives, etc. "Consists of" means that elements in amounts exceeding trace amounts of other components, as well as substantial method steps for administering the compositions provided and / or claimed in this disclosure. Embodiments defined by each of these conversion terms are within the scope of this disclosure.

[0031] A "control" is an alternative subject or sample used in an experiment for comparative purposes. A control may be "positive" or "negative."

[0032] The “effective dose” of a drug refers to the amount of drug sufficient to produce the desired effect. The “effective” amount of drug varies from subject to subject depending on many factors, such as the subject’s age and general condition, and one or more specific drugs. Therefore, it is not always possible to identify a quantifiable “effective dose.” However, the appropriate “effective dose” for any subject can be determined by those skilled in the art using routine experimental methods. Also, as used herein, and unless otherwise specified in detail, the “effective dose” of a drug may also refer to an amount that includes both a therapeutically effective amount and a prophylactically effective amount. The “effective dose” of a drug required to achieve a therapeutic effect may vary depending on factors such as the subject’s age, sex, and weight. The administration plan can be adjusted to produce the optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the urgency of the treatment situation.

[0033] A “pharmaceutically acceptable” ingredient may mean an ingredient that is not biologically or otherwise unsuitable, i.e., an ingredient that can be incorporated into the pharmaceutical formulations provided herein and administered to subjects as described herein without causing significantly unsuitable biological effects or adverse interactions with any other ingredients in the formulation containing it. When used in reference to administration to humans, this term generally means that the ingredient meets the necessary standards for toxicological and manufacturing testing or is included in the inactive ingredient guideline created by the U.S. Food and Drug Administration.

[0034] A “pharmaceutically acceptable carrier” (sometimes referred to as “carrier”) generally means a carrier or excipient that is safe and non-toxic and useful in the preparation of pharmaceutical or therapeutic compositions, and includes carriers that are acceptable for pharmaceutically or therapeutic use in animals and / or humans. The terms “carrier” or “pharmaceutically acceptable carrier” may include, but are not limited to, phosphate-buffered salt solutions, water, emulsions (e.g., oil / water or water / oil emulsions) and / or various wetting agents. As used herein, the term “carrier” includes, but is not limited to, any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other substances well known in the art for use in pharmaceutical formulations and further described herein.

[0035] "Pharmacologically active" (or simply "active") in the context of "pharmacologically active" derivatives or analogues may refer to derivatives or analogues (e.g., salts, esters, amides, conjugates, metabolites, isomers, fragments, etc.) that possess the same type of pharmacological activity as the parent compound and are of approximately the same degree.

[0036] "Therapeutic agent" refers to any composition having beneficial biological effects. Beneficial biological effects include both therapeutic effects, e.g., treatment of disorders or other inappropriate physiological conditions, and prophylactic effects, e.g., prevention of disorders or other inappropriate physiological conditions (e.g., non-immunogenic cancer). The term also encompasses, but is not limited to, pharmaceutically acceptable and pharmacologically active derivatives of beneficial agents as described in detail herein, including salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, etc. When using the term "therapeutic agent," or when specifically identifying a particular agent, it should be understood that the term includes not only the agent itself but also pharmaceutically acceptable and pharmacologically active salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc.

[0037] The “therapeutic effective amount” or “therapeutic effective dose” of a composition (i.e., a composition containing a drug) refers to the amount effective in achieving a desired therapeutic outcome. In some embodiments, the desired therapeutic outcome is the regulation of type 1 diabetes. In some embodiments, the desired therapeutic outcome is the regulation of obesity. A given therapeutic effective amount of a therapeutic agent typically varies depending on the type and severity of the disorder or disease being treated, as well as factors such as the patient's age, sex, and weight. The term also refers to the desired therapeutic effect, for example, the amount of the therapeutic agent or the rate of delivery of the therapeutic agent (e.g., amount over time) that is effective in promoting analgesia. The exact desired therapeutic effect varies depending on the symptom being treated, the patient's tolerance, the drug and / or drug formulation being administered (e.g., the potency of the therapeutic agent, the concentration of the drug in the formulation, etc.), and a variety of other factors understood by those skilled in the art. In some cases, the desired biological or medical response is achieved after administration of multiple doses of the composition to the subject over several days, weeks, or years.

[0038] This application references various papers. The entirety of the disclosures in such papers is incorporated herein by reference to further adequately describe the prior art in the field to which they pertain. Furthermore, the materials contained in the disclosed references are incorporated herein by reference individually and in detail, and this is discussed in the texts that rely on the references.

[0039] B. Composition Disclosed are compositions, as well as components used in the preparation of the compositions themselves used in the methods disclosed herein. When these and other substances are disclosed herein, and when combinations, subsets, interactions, groups, etc., of such substances are disclosed, it is understood that specific references to various individual and collective combinations and permutations of such compounds may not be explicitly disclosed, and each will be considered in detail and described herein. For example, when disclosing and considering a specific type 1 IFN and immunostimulant-containing oncolytic virus, and considering numerous modifications that can be made to numerous molecules including type 1 IFN and immunostimulant-containing oncolytic virus, all possible combinations and permutations of type 1 IFN and immunostimulant-containing oncolytic virus, as well as the opposite possible modifications unless specifically indicated, will be considered in detail. Therefore, when disclosing examples of certain molecules A, B, and C, and certain molecules D, E, and F, and combinations of molecules, we consider disclosing A-D, and then disclosing each of the combinations A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F, which are considered individually and collectively, without having to enumerate each one individually. Similarly, we also disclose any subsets or combinations of these. Therefore, we consider disclosing, for example, subgroups of A-E, B-F, and C-E. This concept applies to all aspects of this application and includes, but is not limited to, steps in methods for producing and using the disclosed compositions. Therefore, where there are various possible further steps, it is understood that each of these further steps can be implemented by any particular embodiment or combination of embodiments of the methods of this disclosure.

[0040] Engagement of CD40 expressed on dendritic cells (DCs) by CD40L ligands leads to the acquisition of a crucial cross-presentation function that activates CD8 T cells. Consequently, therapeutic use of CD40 agonists has the potential to induce potent antitumor T cell immunity. Recent clinical trials have shown promising response rates to systemic delivery of a combination of CD40 agonist antibodies and chemotherapy via ICI, although this was associated with significant toxicity. In addition to these roles in enhancing tumor immunogenicity by increasing the expression of immune function genes in multiple cell types, type 1 IFNs can function as direct activators of both dendritic cell (DC) and T cell function. This key immunostimulatory function of type 1 IFNs has recently been developed with the use of stimulants of type 1 IFN expression, such as STING and TLR9 agonists. A synergistic relationship arises between CD40 ligation and type 1 IFN stimulants for robust CD8 T cell activation. However, systemic administration of a combination of CD40 agonists and type 1 IFN activators carries high toxicity.

[0041] In intrafocal immunotherapy, the tumor acts as a vaccine site, leading to systemic antitumor immunity capable of regulating the growth of distal, untreated tumors through dendritic cell (DC) activation and subsequent T cell stimulation. Such approaches, aimed at avoiding systemic toxicity, have recently been tested with STING and TLR9 agonists. Here, we demonstrate that combined intrafocal activation of CD40 and type 1 IFN signaling results in robust immune activation within the tumor microenvironment (TME), leading to a high level of elevation in the systemic T cell response.

[0042] Oncolytic viruses (OVs) have been developed for their ability to specifically replicate in cancer cells. Recent studies have shown that stimulation of host anti-tumor immunity is a key mechanism of OV action. Furthermore, OVs can be made capable of encoding transgenes that can be used to further enhance anti-tumor immunity by expressing potent activators of the immune response. This specification shows that intralesional-delivered OVs capable of activating immunostimulatory molecules (e.g., CD40) and / or type 1 IFN signaling in TMEs can function as potent activators of the systemic T cell response. To investigate this, we collaborated with Memgen, Inc. to develop a conditionally replicable adenovirus type 5 expressing a chimeric CD40L ligand (MEM40) and IFNβ. We further demonstrated that MEM-288 induces high levels of CD40L and IFNβ expression, and a robust systemic T cell response that can significantly reduce distal tumor growth in mouse melanoma and lung tumor models.

[0043] In one embodiment, a method for generating tumor-infiltrating lymphocytes is disclosed herein, comprising the steps of: a) administering an oncolytic virus expressing one or more type 1 interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω, and / or IFN-ζ, etc.) and / or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, B7-1(CD80) / B7-2(CD86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and / or SLAMF6, etc.) into tumor cells; and b) recovering tumor-infiltrating lymphocytes. It is understood and discussed herein that oncolytic viruses can be administered to tumors by any means known in the art, including but not limited to intratumoral injection. While the above method results in a dramatic increase in the number of TILs and MILs at the tumor site, it is also recognized that TILs and MILs can be generated and / or increased at the tumor site outside the treated tumor microenvironment (i.e., abscopal effect).

[0044] The mere fact that the number of TILs and MILs increases at the target site and / or abscopal tumor site after oncolytic virus administration does not mean that further proliferation cannot occur. In one embodiment, it is understood that these TILs and MILs can be further proliferated when cultured ex vivo, and this is discussed herein. Accordingly, a method for generating tumor-infiltrating lymphocytes, further comprising the step of proliferating recovered TILs ex vivo, is also disclosed herein.

[0045] In one embodiment, the proliferation of TILs and MILs is not limited to in vivo oncolytic virus methods, which involve injecting tumors with oncolytic viruses and then recovering them. Rather, it is understood and discussed herein that TIL proliferation can occur ex vivo by recovering TILs and MILs at the cancer site and then culturing the recovered cells in the presence of antigen-presenting cells infected with oncolytic viruses. Therefore, the proliferation of TILs and / or MILs occurs entirely ex vivo. Therefore, a method for growing a population of tumor-infiltrating lymphocytes (TILs) or bone marrow-infiltrating lymphocytes (MILs) is disclosed herein, comprising the steps of: a) recovering TILs or MILs from a subject having cancer; and b) culturing the recovered TILs or MILs in the presence of antigen-presenting cells infected with an oncolytic virus expressing one or more type 1 interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω and / or IFN-ζ, etc.) and / or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, B7-I (CD80) / B7-2 (CD86), OX40L, 4-I BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58 and / or SLAMF6, etc.).

[0046] In certain embodiments, one or more type 1 interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω and / or IFN-ζ, etc.) and / or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, surface antigen classification (CD)80 and / or CD86 (also known as B7-1 (CD80) and B7-2 (CD86))), OX40L, 4-IBB ligand (4 The present invention provides a viral vector expressing (1BBL), CD70, LIGHT, glucocorticoid-induced TNFR-related protein ligand (GITRL), LIGHT, T-cell immunoglobulin and mucin domain 4 (TIM-4), intracellular adhesion molecule 1 (ICAM-1), CD58, and / or signal transduction lymphocyte activating molecule (SLAM) family member 6 (SLAMF6), etc., or any combination thereof, such as a lentivirus.

[0047] Further embodiments of the present invention provide oncolytic viruses that express any combination of type 1 IFNs, such as IFN-α, IFN-β, IFN-ε, IFN-κ, and IFN-ω, and immunostimulatory molecules (e.g., CD40-L, MEM40, B7-1(CD80) / B7-2(CD86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and / or SLAMF6, etc.) via one or more heterogeneous nucleic acid sequences encoding, for example, a combination of IFN-β and CD40-L. Any oncolytic virus can be utilized. In some embodiments, the oncolytic virus may be an adenovirus, reovirus, herpesvirus, picornavirus (including coxsackievirus, poliovirus, and Seneca Valley virus), paramyxovirus (including measles virus and Newcastle disease virus (NDV)), parvovirus, rhabdovirus (including vesicular stomatitis virus (VSV)), or vacciniavirus. Preferably, the oncolytic virus is replicable.

[0048] Further embodiments of the present invention provide a method for treating malignant lesions in a subject by administering an oncolytic virus disclosed herein in combination with a checkpoint inhibitor administered to the subject.

[0049] IFN-α may be human IFN-α represented by the sequence of SEQ ID NO: 1. IFN-α may be mammalian IFN-α, e.g., mouse, rat, rabbit, pig, cat, dog, or bovine IFN-α. Further embodiments of IFN-α derived from other mammals are known to those skilled in the art, and such embodiments are within the scope of the present invention. In certain embodiments, IFN-α has sequence identity with respect to human wild-type IFN-α, e.g., IFN-a of SEQ ID NO: 1, ranging from 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0050] IFN-β may be human IFN-β represented by the sequence of SEQ ID NO: 2. IFN-β may be mammalian IFN-β, e.g., mouse, rat, rabbit, pig, cat, dog, or bovine IFN-β. Further embodiments of IFN-β from other mammals are known to those skilled in the art, and such embodiments are within the scope of the present invention. In certain embodiments, IFN-β has sequence identity with human wild-type IFN-β, e.g., IFN-β of SEQ ID NO: 2, containing 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%). In certain embodiments, IFN-β lacks the first 22 amino acids of SEQ ID NO: 2 and optionally further has the substitution of cysteine ​​17 with serine in the resulting 165-amino acid peptide.

[0051] IFN-ε may be human IFN-ε represented by the sequence of SEQ ID NO: 3. IFN-ε may be mammalian IFN-ε, e.g., mouse, rat, rabbit, pig, cat, dog, or bovine IFN-ε. Further embodiments of IFN-ε derived from other mammals are known to those skilled in the art, and such embodiments are within the scope of the present invention. In certain embodiments, IFN-ε has sequence identity with respect to human wild-type IFN-ε, e.g., IFN-ε of SEQ ID NO: 3, ranging from 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0052] IFN-κ may be human IFN-κ represented by the sequence of SEQ ID NO: 4. IFN-κ may be mammalian IFN-κ, e.g., mouse, rat, rabbit, pig, cat, dog, or bovine IFN-κ. Further embodiments of IFN-κ derived from other mammals are known to those skilled in the art, and such embodiments are within the scope of the present invention. In certain embodiments, IFN-κ has sequence identity with respect to human wild-type IFN-κ, e.g., IFN-κ of SEQ ID NO: 4, ranging from 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0053] IFN-ω may be human IFN-ω represented by the sequence of SEQ ID NO: 5. IFN-ω may be mammalian IFN-ω, e.g., mouse, rat, rabbit, pig, cat, dog, or bovine IFN-ω. Further embodiments of IFN-ω derived from other mammals are known to those skilled in the art, and such embodiments are within the scope of the present invention. In certain embodiments, IFN-ω has sequence identity with respect to human wild-type IFN-ω, e.g., IFN-ω of SEQ ID NO: 5, ranging from 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0054] CD40-L may be human CD40-L represented by the sequence of Sequence ID No. 6. CD40-L may be mammalian CD40-L, such as mouse, rat, rabbit, pig, cat, dog, or bovine CD40-L. Further embodiments of CD40-L from other mammals are known to those skilled in the art, and such embodiments are within the scope of the present invention. In certain embodiments, CD40-L has sequence identity with respect to human wild-type CD40-L, such as CD40-L of Sequence ID No. 6, ranging from 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0055] CD40-L may be a chimeric CD40-L or a non-chimeric CD40-L polypeptide. In some embodiments, the CD40-L expressed in the oncolytic virus of the present invention is a chimeric CD40-L. Such a chimeric CD40-L polypeptide contains domains or subdomains of CD40-L from at least two different species, e.g., human and mouse CD40-L. Chimeric CD40-L results in a higher immune response compared to naturally occurring CD40-L. Examples of chimeric CD40-L suitable for use in the present invention are disclosed in U.S. Patents 7,495,090, 7,928,213, and 8,138,310. Each of these patents is incorporated herein by reference in its entirety.

[0056] In certain embodiments of chimeric CD40-L, at least one domain or subdomain of CD40-L, including a cleavage site of human CD40-L, is replaced with a corresponding domain or subdomain of non-human CD40-L, preferably mouse CD40-L. In addition, chimeric CD40-L may include a domain or subdomain of human CD40-L that binds to the CD40-L receptor. Domains I-IV of human CD40-L (SEQ ID NO: 6) correspond to amino acid portions 1-14, 14-45, 46-110, and 111-261 of SEQ ID NO: 6. Those skilled in the art can determine domains I-IV of non-human CD40-L based on the sequence alignment of non-human CD40-L with human CD40-L. Specific domain positions of non-human CD40-L are presented in Table 1 of U.S. Patent No. 7,495,090, which is incorporated herein by reference in its entirety.

[0057] In some embodiments, the chimeric CD40-L includes a first subdomain of non-human CD40-L that replaces a subdomain with a cleavage site of human CD40-L, and a second subdomain of human CD40-L that binds to the CD40-L receptor.

[0058] The first subdomain may include a subdomain of domain IV of non-human CD40-L. In addition, the first subdomain may further include domain III or a subdomain of domain III of non-human CD40-L. In certain embodiments, the first subdomain replaces a portion of the cleavage site of human CD40-L. In further embodiments, in addition to domain IV or a subdomain of domain IV of non-human CD40-L, and optionally domain III or a subdomain of domain III, the chimeric CD40-L further includes domain II or a subdomain of domain II. Furthermore, the first subdomain of the chimeric CD40-L may include domain I or a subdomain of domain I of non-human CD40-L. Thus, in certain chimeric CD40-L, the first subdomain includes domains or subdomains of domains I, II, III, and IV of non-human CD40-L. In preferred embodiments, non-human CD40-L is mouse CD40-L.

[0059] In a preferred embodiment, the chimeric human / mouse CD40 ligand has 92% amino acid sequence homology to human CD40L (SEQ ID NO: 12) (see U.S. Patent No. 7,495,090, which is incorporated herein by reference and referred to herein as "MEM40"). "CD40 ligand" and "CD40-L" may be used interchangeably herein and may also be referred to as "CD154". In detail, domains I, II, and III, which contain the intracellular, intramembrane, and proximal extracellular domains of the molecule, respectively, are fully humanized. Domain IV, which contains the CD40 binding portion of the molecule, retains only the mouse domain necessary for optimal CD40 ligand expression in cells. MEM40 is fully humanized at the 3' end of the molecule, where, upon administration to humans, antibody binding neutralizes the activity of mouse CD154 (CD40 ligand).

[0060] Non-limiting examples of chimeric CD40-L useful in the present invention include the following sequences. Sequence ID 7

[0061] [ka]

[0062] Sequence ID 8

[0063] [ka]

[0064] Sequence ID 9

[0065] [ka]

[0066] Sequence ID 10

[0067] [ka]

[0068] Sequence ID 11

[0069] [ka]

[0070] Sequence ID 12

[0071] [ka]

[0072] Sequence ID 13

[0073] [ka]

[0074] Sequence ID 14

[0075] [ka]

[0076] Sequence ID 15

[0077] [ka]

[0078] Sequence ID 16

[0079] [ka]

[0080] Sequence ID 17

[0081] [ka]

[0082] Sequence ID 18

[0083] [ka]

[0084] The specific nucleotide sequences encoding the chimeric CD40-L of Sequence IDs 7-18 are disclosed in U.S. Patents 7,495,090, 7,928,213, and 8,138,310. Such nucleotide sequences are incorporated herein by reference, and the use of such nucleotide sequences is assumed herein.

[0085] The oncolytic virus of the present invention may include nucleic acids encoding IFN-α, wherein IFN-α includes human wild-type IFN-α (SEQ ID NO: 1), or IFN-α having sequence identity with human wild-type IFN-α, for example, IFN-α of SEQ ID NO: 1, comprising 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0086] The oncolytic virus of the present invention may include a nucleic acid encoding IFN-β, wherein IFN-β includes human wild-type IFN-β (SEQ ID NO: 2), or IFN-β having sequence identity with human wild-type IFN-β, for example, IFNP of SEQ ID NO: 2, comprising 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%). In certain embodiments, the nucleotide sequence encodes IFN-β lacking the first 22 amino acids of SEQ ID NO: 2, and optionally further comprises the substitution of cysteine ​​17 with serine in the resulting 165-amino acid peptide.

[0087] The oncolytic virus of the present invention may include nucleic acids encoding IFN-ε, wherein IFN-ε includes human wild-type IFN-ε (SEQ ID NO: 3), or IFN-ε having sequence identity with human wild-type IFN-ε, for example, IFN-ε of SEQ ID NO: 3, comprising 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0088] The oncolytic virus of the present invention may include a nucleic acid encoding IFN-κ, wherein IFN-κ includes human wild-type IFN-κ (SEQ ID NO: 4), or IFN-κ having sequence identity with respect to human wild-type IFN-κ, for example, IFN-κ of SEQ ID NO: 4, ranging from 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0089] The oncolytic virus of the present invention may include a nucleic acid encoding IFN-ω, wherein IFN-ω includes human wild-type IFN-ω (SEQ ID NO: 5), or IFN-ω having sequence identity with respect to human wild-type IFN-ω, for example, IFN-ω of SEQ ID NO: 5, ranging from 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%).

[0090] The oncolytic virus of the present invention may include a nucleic acid encoding CD40-L, wherein CD40-L includes human wild-type CD40-L (SEQ ID NO: 6), or CD40-L having sequence identity ranging from 80.00% to a maximum of 99.99% (e.g., 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) relative to human wild-type CD40-L, for example, CD40-L of SEQ ID NO: 6. Furthermore, the oncolytic virus of the present invention may also contain nucleic acids encoding chimeric CD40-L, where the chimeric CD40-L is a sequence selected from SEQ ID NOs. 7 to 18, or a CD40-L having sequence identity ranging from 80.00% to a maximum of 99.99% (for example, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) to a chimeric CD40-L having a sequence selected from SEQ ID NOs.

[0091] In preferred embodiments, the oncolytic virus of the present invention may include nucleic acid encoding MEM40. For example, in preferred embodiments, the oncolytic virus of the present invention includes IFNβ having at least 80% sequence identity to human IFNβ (SEQ ID NO: 2), and CD40-L having at least 80% sequence identity to chimeric CD40-L having the sequence of SEQ ID NO: 12.

[0092] One or more heterogeneous nucleic acid sequences encoding a combination of type 1 IFN and CD40-L may be present in one or more viral constructs. Non-exclusive examples of viral constructs include adenovirus constructs, adeno-associated virus constructs (AAVs), poxvirus constructs, lentivirus constructs, alphavirus constructs, herpesvirus constructs, retrovirus constructs, vacciniavirus constructs, vesicular stomatitis virus constructs, or herpes simplex virus constructs.

[0093] In addition to nucleic acids encoding type 1 interferon (e.g., IFNP) and CD40-L (chimeric human / mouse CD40-L), the oncolytic viruses according to the present invention may further include other modifications in their genome. For example, this may include additional DNA inserted into already inactivated genes or substituted for deleted genes. The oncolytic viruses may also incorporate one or more promoters that confer enhanced levels of tumor cell specificity to the virus. In this way, the oncolytic viruses may target specific cancer types using cancer cell-specific promoters. The terms “tumor cell-specific promoter,” “tumor cell-specific transcriptional regulatory sequence,” “tumor-specific promoter,” or “tumor-specific transcriptional regulatory sequence” refer to transcriptional regulatory sequences, promoters, and / or enhancers that are present in higher levels in target cancer cells than in normal cells. For example, the oncolytic viruses for use in the present invention may exist under the regulation of exogenously added regulatory factors.

[0094] In a preferred embodiment, the oncolytic virus is an adenovirus (Ad). Ad is a large (approximately 36 kb) DNA virus that infects humans, but also exhibits a broad host range. Physically, adenoviruses are icosahedral viruses containing a double-stranded linear DNA genome. There are approximately 50 serotypes of human adenoviruses, which are divided into six families based on molecular, immunological, and functional criteria. By adulthood, virtually all humans have been infected with one of the more common adenovirus serotypes, the primary effect being a flu-like symptom.

[0095] Adenovirus infection of host cells leads to the maintenance of adenovirus DNA in the episome, which reduces the potential genotoxicity associated with vector integration. In addition, adenoviruses are structurally stable, and no genomic rearrangements have been detected after large-scale amplification. Adenoviruses can infect most epithelial cells regardless of the cell cycle stage. To date, adenovirus infection appears to be associated only with mild illnesses in humans, such as acute respiratory illnesses.

[0096] The adenovirus infection cycle consists of two phases: an early phase that precedes the initiation of adenovirus genome replication and enables the production of regulatory proteins and proteins involved in viral DNA replication and transcription; and a late phase that triggers the synthesis of structural proteins. Early genes are distributed in four regions scattered throughout the adenovirus genome, named E1-E4 ("E" stands for "early"). Each early region contains at least six transcription units, each with its own promoter. The expression of early genes is self-regulated, with some genes being expressed before others. Three regions, E1, E2, and E4, are essential for viral replication. Therefore, if an adenovirus has a defect in one of these functions, this protein must be supplied to the trans, or the virus will be unable to replicate.

[0097] The E1 early region is located at the 5' end of the adenovirus genome and contains two viral transcription units, E1A and E1B. This region is involved in the viral cycle very early and encodes proteins essential for the expression of almost all other adenovirus genes. In particular, the E1A transcription unit encodes proteins that transactivate the transcription of other viral genes, including those regulated by promoters in the E1B, E2A, E2B, E3, and E4 regions, as well as late-stage genes.

[0098] Adenoviruses enter permissible host cells via cell surface receptors and then translocate internally. The viral DNA, along with specific viral proteins required for the first step of the replication cycle, enters the nucleus of the infected cell, where transcription begins. Adenovirus DNA replication occurs in the nucleus of the infected cell and does not require cell replication. New viral particles or virions, after association, are released from the infected cell and can infect other permissible cells.

[0099] Adenoviruses are an attractive delivery system. Embodiments of this disclosure may utilize manufacturing processes that are free from or essentially free from proteins, serums, and animal-derived components, making them suitable for both broad prophylactic and therapeutic vaccine formulations.

[0100] If an adenovirus has mutated to be conditionally replicable (replicable under specific conditions), it may require helper cells for viral replication. If so, the helper cell line may be derived from human cells, such as human embryonic kidney cells, muscle cells, hematopoietic cells, or other human embryonic mesenchymal or epithelial cells. Alternatively, the helper cells may be derived from cells of other mammalian species tolerant of human adenovirus. Examples of such cells include Vero cells or other monkey embryonic mesenchymal or epithelial cells. In certain embodiments, the helper cell line is 293. Various methods for culturing host and helper cells can be found in the art (e.g., Racher, AJ, Fooks, AR & Griffiths, JB Biotechnol Tech (1995) 9: 169).

[0101] 1. Delivery of pharmaceutical carriers / pharmaceutical preparations As described above, the composition can also be administered in vivo by a pharmaceutically acceptable carrier. "Pharmaceutically acceptable" means a substance that is not biologically or otherwise unsuitable. That is, the substance can be administered to a subject together with a nucleic acid or vector without causing any unsuitable biological effects or harmful interactions with any of the other components of the pharmaceutical composition it contains. The carrier is, as is well known to those skilled in the art, naturally selected to minimize any degradation of the active ingredient and any harmful side effects in the subject.

[0102] The composition may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, intraperitoneal injection, percutaneously, extracorporeally, topically, etc., including topical intranasal administration or administration by inhalation. As used herein, "topical intranasal administration" means delivery of the composition to the nose and nasal cavity through one or both nostrils, and may include delivery by a spray or droplet mechanism of nucleic acid or vector, or by aerosolization. Administration of the composition by inhalation may be by administration through the nose or mouth via a spray or droplet mechanism. Delivery may also be to any area of ​​the respiratory system (e.g., lungs) via intubation. The exact amount of composition required will vary from subject to subject, depending on the species, age, weight and general condition, the severity of the allergic disorder being treated, the specific nucleic acid or vector used, and the method of administration. Therefore, it is impossible to specify the exact amount of all compositions. However, an appropriate amount can be determined by those skilled in the art, taking into account the teachings herein and using only routine experimental methods.

[0103] Parenteral administration of compositions, when used, is generally characterized by injection. Injectable preparations can be prepared in conventional forms, either as a solution or suspension, in a solid form suitable for a suspension in liquid before injection, or as an emulsion. More recent revisions to parenteral administration techniques include the use of sustained-release or continuous-release systems to maintain a constant dose. See, for example, U.S. Patent No. 3,610,795, which is incorporated herein by reference.

[0104] The substances may exist in solutions or suspensions (e.g., incorporated into microparticles, liposomes, or cells). They may target specific cell types via antibodies, receptors, or receptor ligands. The following references provide examples of the use of this technique, which targets proteins specific to tumor tissue (Senter et al., Bioconjugate Chem., 2:447-451 (1991); Bagshawe, KD, Br. J. Cancer, 60:275-281 (1989); Bagshawe et al., Br. J. Cancer, 58:700-703 (1988); Senter et al., Bioconjugate Chem., 4:3-9 (1993); Battelli et al., Cancer Immunol. Immunother., 35:421-425 (1992); Pietersz and McKenzie, Immunolog. Reviews, 129:57-80 (1992); and Roffler et al., Biochem. Pharmacol, 42:2062-2065 (1991)). The media used include, for example, “Stealth” and other antibody-conjugated liposomes (including liquid-mediated drug targeting of colon cancer), receptor-mediated targeting of DNA by cell-specific ligands, lymphocyte-specific tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references provide examples of the use of this technique to target proteins specific to tumor tissue (Hughes et al., Cancer Research, 49:6214-6220 (1989); and Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187 (1992)). Generally, receptors are involved in either constitutive or ligand-induced endocytosis pathways. Such receptor clusters within clathrin-coated pits enter cells via clathrin-coated vesicles, pass through acidified endosomes where receptors are sorted, and then recirculate to the cell surface, are stored intracellularly, or are degraded by liposomes.Internal translocation pathways perform diverse functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, ligand dissociation and degradation, and receptor-level regulation. Many receptors follow one or more intracellular pathways depending on cell type, receptor concentration, ligand type, ligand binding titer, and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, pp. 399-409 (1991)).

[0105] a) A pharmaceutically acceptable carrier Compositions containing antibodies can be used therapeutically in combination with pharmaceutically acceptable carriers.

[0106] Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th edition), edited by AR Gennaro, Mack Publishing Company, Easton, PA 1995. Typically, a suitable amount of pharmaceutically acceptable salt is used in the formulation to make it isotonic. Examples of pharmaceutically acceptable carriers include, but are not limited to, saline solution, Ringer's solution, and dextrose solution. The pH of the solution is preferably about 5 to about 8, more preferably about 7 to about 7.5. Furthermore, the carrier comprises a sustained-release formulation, for example, a semipermeable matrix of a solid hydrophobic polymer containing an antibody, the matrix in the form of a molded product, such as a film, liposome, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferable, for example, depending on the route of administration and concentration of the composition to be administered.

[0107] Pharmaceutical carriers are known to those skilled in the art. The most typical of these are standard carriers for the administration of drugs to humans, and include solutions such as sterile water, saline, and buffer solutions at physiological pH. Compositions can be administered intramuscularly or subcutaneously. Other compounds are administered according to standard procedures used by those skilled in the art.

[0108] The pharmaceutical composition may include, in addition to the selected molecule, a carrier, a thickener, a diluent, a buffer, a preservative, a surfactant, etc. Furthermore, the pharmaceutical composition may include one or more active ingredients, such as an antibacterial agent, an anti-inflammatory agent, an anesthetic, etc.

[0109] The pharmaceutical composition may be administered by local or systemic treatment, and by a number of methods depending on the area to be treated. Administration may be local (including intraocular, intravaginal, intrarectal, and intranasal), oral by inhalation, or parenteral by, for example, intravenous infusion, subcutaneous, intraperitoneal, or intramuscular injection. The disclosed antibodies may be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavitarially, or percutaneously.

[0110] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solutions include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and organic esters for injection such as ethyl oleate. Aqueous carriers include water, alcohol / aqueous solutions, emulsions, or suspensions, and also include saline and buffered media. Parenteral media include sodium chloride solutions, ringer's dextrose, dextrose and sodium chloride, Ringer's lactate, or fixative oils. Intravenous media include liquids and nutritional supplements, electrolyte supplements (e.g., those based on ringer's dextrose), etc. Preservatives and other additives, such as antimicrobial agents, antioxidants, chelating agents, and inert gases, may also be present.

[0111] Formulations for topical administration may include ointments, lotions, creams, gels, droplets, suppositories, sprays, liquids, and powders. Conventional drug carriers, aqueous, powder, or oily bases, thickeners, etc., may be necessary or desirable.

[0112] Compositions for oral administration include powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets, or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersion aids, or binders may be desirable.

[0113] Parts of the composition may be administered as pharmaceutically acceptable acids or base addition salts formed by reactions with inorganic acids, such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids, such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reactions with inorganic bases, such as sodium hydroxide, ammonium hydroxide, and potassium hydroxide, and organic bases, such as mono, di, trialkyl, and arylamines and substituted ethanolamines.

[0114] b) Therapeutic use The effective dosage and schedule for administering a composition can be determined empirically, and such determinations are within the scope of the art. The dosage range for administering a composition is broad enough to produce the desired effect on which the symptoms of the disorder are affected. The dosage should not be so broad as to cause adverse side effects, such as unnecessary cross-reactions or anaphylactic reactions. Generally, the dosage can be determined by those skilled in the art, depending on age, symptoms, sex, the severity of the disease in the patient, the route of administration, or whether other drugs are included in the plan. The dosage can be adjusted by the physician in cases of contraindications. The dosage may vary and may be administered once or multiple times daily for one or several days. Guidance on appropriate dosages for a given type of pharmaceutical preparation can be found in the literature. For example, guidance in selecting an appropriate antibody dose can be found in the literature on the therapeutic use of antibodies, e.g., Handbook of Monoclonal Antibodies, edited by Ferrone et al., Noges Publications, Park Ridge, NJ (1985), pp. 22 and 303-357; and Smith et al., Antibodies in Human Diagnosis and Therapy, edited by Haber et al., Raven Press, New York (1977), pp. 365-389. A typical daily dose of an antibody used alone may range from approximately 1 μg / kg to a maximum of 100 mg / kg or more per day of body weight, depending on the factors mentioned above.

[0115] C. Methods of treating cancer The compositions of this disclosure can be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. A representative but non-limiting list of cancers in which the compositions of this disclosure can be used for treatment is as follows: lymphoma, B-cell lymphoma, T-cell lymphoma, mycosis fungoides, Hodgkin's disease, myeloid leukemia, bladder cancer, brain cancer, nervous system cancer, head and neck cancer, squamous cell carcinoma of the head and neck, lung cancer, such as small cell lung cancer and non-small cell lung cancer, neuroblastoma / glioblastoma, ovarian cancer, skin cancer, liver cancer, melanoma, squamous cell carcinoma of the mouth, throat, pharynx, and lung, cervical cancer, uterine cancer, breast cancer, and epithelial cancer, kidney cancer, genitourinary cancer, lung cancer, esophageal cancer, head and neck cancer, colorectal cancer, hematopoietic cancer; testicular cancer; colon cancer, rectal cancer, prostate cancer, or pancreatic cancer.

[0116] In one embodiment, a method for treating, reducing, inhibiting, decreasing, restoring, and / or preventing cancer and / or metastasis (including abscopal tumors) in a subject is disclosed herein, comprising the step of administering either a proliferated TIL or MIL disclosed herein to the subject. For example, a) administering an oncolytic virus expressing one or more type 1 interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω and / or IFN-ζ, etc.) and / or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, B7-I (CD80) / B7-2 (CD86), OX40L, 4-I BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58 and / or SLAMF6, etc.) into tumor cells; b) recovering tumor-infiltrating lymphocytes (TILs) and / or bone marrow-infiltrating lymphocytes (MILs); and c) ex... Disclosed herein are methods for treating, reducing, inhibiting, decreasing, restoring, and / or preventing cancer and / or cancer metastasis in a subject, comprising the steps of: d) growing the virus in vivo and d) administering the grown TIL and / or MIL to the subject. In some embodiments, the cancerous or metastatic tumor to be treated, reduced, inhibited, decreased, restoring, and / or prevented is an abscopal to a tumor receiving either the oncolytic virus and / or TIL or MIL disclosed herein.

[0117] Alternatively, TILs or MILs may be first recovered from a tumor, then grown ex vivo in the presence of antigen-presenting cells infected with an oncolytic virus expressing one or more type 1 interferons (IFNs) and / or one or more exogenous immunostimulatory molecules, and the grown TILs and / or MILs may be administered to a target. The grown TILs or MILs may then be administered (adoptive introduction) to a target with cancer. Thus, in one embodiment, a) a step of recovering tumor-infiltrating lymphocytes (TILs) and / or bone marrow-infiltrating lymphocytes (MILs) from a target with cancer, and one or more type 1 interferons (IFNs) (e.g., IFN-α, IFN-β, IFN-κ, IFN-δ, IFN-ε, IFN-τ, IFN-ω and / or IFN-ζ, etc.) and / or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, B7-I (CD80) / B7-2 (CD This specification discloses a method for treating, reducing, inhibiting, decreasing, restoring, and / or preventing cancer and / or metastasis in a subject, comprising the steps of culturing recovered TIL or MIL in the presence of antigen-presenting cells infected with an oncolytic virus expressing (86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58 and / or SLAMF6, etc., and administering the grown TIL and / or MIL to the subject.

[0118] In one embodiment, success in cancer treatment in the subject is important, and such success may include further treatment procedures, as is understood and discussed herein. Accordingly, it is intended herein that any cancer treatment procedure, including, but not limited to, surgery, radiation, and / or pharmacotherapy, can be enhanced by the disclosed methods for treating, inhibiting, reducing, and / or preventing cancer. Therefore, the disclosed treatments are not limited to abemaciclib, abiraterone acetate, abitrexate (methotrexate), Abraxane (paclitaxel albumin-stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC, AC-T, Adcetris (brentuximab vedotin), ADE, Ado-trastuzumab emtansine, Adriamycin (doxorubicin hydrochloride), afatinib maleate, Afinitor (everolimus), Akynzeo (netupitant and palonosetron hydrochloride), Aldara (imiquimod), aldesleukin, Alecensa (alecinib), alectinib, Alemtuzumab, Alimta (pemetrexed disodium), Aliqopa (copanlisib hydrochloride), Alkeran for injection (melphalan hydrochloride), Alkeran tablets (melphalan), Aloxi (palonosetron hydrochloride), Alumbrig (brigutinib), Ambochlorin (chlorambucil), Ambochlorin chlorambucil, Amifostine, aminolevulinate, Anastrozole, Aprepitant, Aredia (pamidronate disodium), Arimidex (anastrozole), Aromasin (exemestane), Arranon (nelarabine), Arsenic trioxide, Arzera (ofatumumab), Black Leg Disease (Erwinia) Chrysanthemi) Asparaginase, Atezolizumab, Avastin (Bevacizumab), Avelumab, Axitinib, Azacitidine, Bavencio (Avelumab), BEACOPP, Becenum (Carmustine), Beleodaq (Belinostat), Belinostat, Bendamustine hydrochloride, BEP, Besponsa (Inotuzumab ozogamicin), Bevacizumab, Bexarotene,Bexal (tositumomab and iodine I 131 tositumomab), bicalutamide, BiCNU (carmustine), bleomycin, blinatumomab, Blincyto (blinatumomab), bortezomib, Bosulif (bosutinib), bosutinib, brentuximab vedotin, brigutinib, BuMel, busulfan, busulfex (busulfan), cabazitaxel, cabometyx (cabozantinib-S-malate), cabozantinib-S-malate, CAF Campus (alemtuzumab), Campto Camptosar (irinotecan hydrochloride), capecitabine, CAPOX, Carac (fluorouracil - topical), carboplatin, carboplatin-taxol, carfilzomib, Carmubris (carmustine), carmustine, carmustine implant, Casodex (bicalutamide), CEM, ceritinib, Cerubidine (daunorubicin hydrochloride), Cervarix (recombinant HPV (Bivalent vaccine), cetuximab, CEV, chlorambucil, chlorambucil-prednisone, CHOP, cisplatin, cladribine, Clafen (cyclophosphamide), clofarabine, Clofarex (clofarabine), Clolar (clofarabine), CMF, cobimetinib, Cometriq (cabozantinib-S-malate), copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosph Famide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposome, Cytosar-U (cytarabine), cytoxane (cyclophosphamide), dabrafenib, dacarbazine, Dacogen (decitabine), dactinomycin, daratumumab, darazalex (daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin hydrochloride and cytarabine liposome, decitabine, defibrotide sodium, Defitelio (defibrotide sodium), degarelix, denileukin difutitox, denosumab,Depotite (cytarabine liposome), dexamethasone, dexrazoxane hydrochloride, dinutuximab, docetaxel, Doxil (doxorubicin hydrochloride liposome), doxorubicin hydrochloride, doxorubicin hydrochloride liposome, Dox-SL (doxorubicin hydrochloride liposome), DTIC-Dome (dacarbazine), durvalumab, Efudex (fluorouracil - topical), Elitek (rasburicase), Ellence (epirubicin hydrochloride), elotuzumab, elotuzumab Xatin (Eloxatin) (Oxaliplatin), Eltrombopagolamine, Emend (Aprepitant), Emliciti (Elotuzumab), Enasidenib mesylate, Enzalutamide, Epirubicin hydrochloride, EPOCH, Erbitux (Cetuximab), Eribulin mesylate, Elivege (Bismodegib), Erlotinib hydrochloride, Erwinaze (Asparaginase for Black Leg Disease), Ethyol (Amiphostin), Etopophos (Etoposidori) Fluorouracil (5-FU), etoposide, etoposide phosphate, Evacet (doxorubicin hydrochloride liposome), everolimus, Evista (raloxifene hydrochloride), Evomela (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Fareston (toremifene), Faridac (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, fludara (flu Darabine phosphate, fludarabine phosphate, Fluoroplex (fluorouracil - topical), fluorouracil injection, fluorouracil - topical, flutamide, Folex (methotrexate), Folex PPS (methotrexate), Folfiri, Folfiri-bevacizumab, Folfiri-cetuximab, Folfirinox, Folfox, Forotin (pralatrexate), FU-LV, fulvestrant, Gardasil (recombinant HPV 4-valent vaccine), Gardasil 9 (recombinant HPV 9-valent vaccine), Gazyva (obinutuzumab), gefitinib, gemcitabine hydrochloride,Gemcitabine-cisplatin, gemcitabine-oxaliplatin, gemtuzumab ozogamicin, gemzar (gemcitabine hydrochloride), Gilotrif (afatinib dimaleate), Gleevec (imatinib mesylate), Gliadel (carmustine implant), Gliadel wafer (carmustine implant), glucarpidase, goserelin acetate, Halaven (eribulin mesylate), Hemangeol (propranolol hydrochloride), Herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV 9valent vaccine, recombinant HPV Quadrivalent vaccine, recombinant Hycamtin (topotecan hydrochloride), Hydrea (hydroxyurea), hydroxyurea, Hyper-CVAD, Ibrance (palbociclib), ibritumomab tiuxetan, ibrutinib, ICE, Iclusig (ponatinib hydrochloride), Idamycin (idarubicin hydrochloride), idarubicin hydrochloride, idelalisib, Idhifa (enasidenib mesylate), Ifex (ifosfamide), ifosfamide, ifosfamide Ifosfamidum (Ifosfamid), IL-2 (Aldesleukin), Imatinib mesylate, Ibruvica (Ibrutinib), Imfinzi (Durvalumab), Imiquimod, Imlygic (Tarimogene Laharpa Repbec), Inlyta (Axitinib), Inotuzumab Ozogamicin, Interferon Alpha-2b, Recombinant Interleukin-2 (Aldesleukin), Intron A (Recombinant Interferon Alpha-2b), Iodine I 131 Tositumomab and tositumomab, ipilimumab, Iressa (gefitinib), irinotecan hydrochloride, irinotecan hydrochloride liposome, Istodax (romidepsin), ixabepirone, ixazomib citrate, Ixempra (ixabepirone), Jakafi (ruxolitinibrine), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivans (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib),Kymriah (tisagenlecleucel), Kyprolis (carfilzomib), lanreotide acetate, lapatinib nitrate, Lartruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, leustatin (cladribine), Levulan (aminolevulinate), Linfolizin (chlorambucil), LipoDox (doxorubicin hydrochloride liposome), lomustine, Lonsurf (trifluridine and tipiracil hydrochloride), Lupron (leuprolide acetate), Lupron Depot Leupron Depot (leuprolide acetate), Leupron Depot-Ped (leuprolide acetate), Lynparza (olaparib), Marqibo (vincristine sulfate liposome), Matulane (procarbazine hydrochloride), mechloretamine hydrochloride, megestrol acetate, Mekinist (trametinib), melphalan, melphalan hydrochloride, mercaptopurine, Mesna, Mesnex (mesna), Methazolastone (temozolomide), methotrexate, methotrexate LPF (methotrexate), methylnaltrexone bromide, Mexate (methotrexate), Mexate-AQ (methotrexate), midostaurine, Maitomy Syn C, Mitoxantrone hydrochloride, Mitozytrex (Mitomycin C), MOPP, Mozovir (Prelixafor), Mustargen (Mechloretamine hydrochloride), Mutamycin (Mitomycin C), Myrelan (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel Albumin Stabilized Nanoparticle Formulation), Navelbine (Vinorelbine Tartrate), Necitumumab, Nelarabine, Neosar (Cyclophosphamide), Neratinib Maleate, Nerlynx (Neratinib Maleate), Netupitant and Palonosetron Hydrochloride,Neulasta (pegfilgrastim), Newpogen (filgrastim), Nexavar (sorafenib tosylate), Nilandron (niltamide), nilotinib, niltamide, Ninlaro (ixazomib citrate), niraparib tosylate monohydrate, nivolumab, Nolvadex (tamoxifen citrate), Nplate (romiplostim), obinutuzumab, Od Odomzo (sonidegib), OEPA, ofatumumab, OFF, olaparib, olalatumab, omacetaxine mepecocilate, Oncaspar (peguaspargase), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontak (deniroikin difutitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclita Xelalbumin-stabilized nanoparticle formulations, PAD, palbociclib, parifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, pegaspargaze, pegfilgrastim, peginterferon alpha-2b, PEG-1 Ntron (pegylated interferon alpha-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), prelixafor, pomalidomide, Pomalist (pomalidomide), ponatinib hydrochloride, Portraza (necitumumab), pralatrexate, prednisone, procarbazine hydrochloride, proleukin (, Proleukin (Aldesleukin), Prolia (Denosumab), Promacta (Eltrombopagolamine), Propranolol hydrochloride, Provenge (Ciproisel T), Prinethol (Mercaptopurine), Prixan (Mercaptopurine), Radium-223 chloride, Raloxifene hydrochloride, Ramucirumab, Rasburicase, R-CHOP, R-CVP, Recombinant human papillomavirus (HPV) bivalent vaccine, recombinant human papillomavirus (HPV) 9-valent vaccine, recombinant human papillomavirus (HPV) 4-valent vaccine, recombinant interferon alpha-2b, regorafenib, Relistol (methylnaltrexone bromide), R-EPOCH, Revlimid (lenalidomide), Rheumatrex (methotrexate), ribociclib, R-ICE, Rituxan (rituximab), Rituxan Hysela (Rituxan Hysela) Hycela (rituximab and human hyaluronidase), rituximab, rituximab and human hyaluronidase, lorapitant hydrochloride, romidepsin, romiplostim, rubidomycin (daunorubicin hydrochloride), Rubraca (rucaparibucansylate), rucaparibucansylate, ruxolitinibulinate, Rydapt (midostaurine), Sclerosol intrapleural aerosol (talc), siltuximab, ciproisel T, somatuline depot (lanreotide acetate), sonidegib, sorafenib tosylate, Sprycel (dasatinib), STANFORD V, Sterile talc powder (talc), Steritalc (talc), Stivarga (regorafenib), sunitinib malate, Sutent (sunitinib malate), Sylatron (pegylated interferon alfa-2b), Sylvant (siltuximab), Synribo (omacetaxin mepesuccinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, Tarimozine Laharpa Repbec, tamoxifen citrate, Tarabine PFS (cytarabine), Tarceva (erlotinib hydrochloride),Targretin (bexarotene), Tasigna (nilotinib), Taxol (paclitaxel), Taxotere (docetaxel), Tecentriq (atezolizumab), Temodar (temozolomide), temozolomide, temsirolimus, thalidomide, thalomid (thalidomide), thioguanine, thiotepa, tisagenlecleucel, Tolak (fluorouracil - topical), topotecan hydrochloride, toremifene, Torisel (temsirolimus), tositumomab and iodine I 131 Tositumomab, Totect (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, Treanda (bendamustine hydrochloride), trifluridine and tipiracil hydrochloride, Trisenox (arsenic trioxide), Tykerb (lapatinib nitosylate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Val Varubi (lorapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomib), Velsar (vinblastine sulfate), vemurafenib, Veneclexta (venetoclax), venetoclax, Verzenio (abemaciclib), Viadur (leuprolide acetate), Vidar Za (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, bismodegib, Vistagard (uridine triacetate), Voraxaze (glucarpidase), vorinostat, Botrient (pazopanib hydrochloride), Bixeo Vyxeos (daunorubicin hydrochloride and cytarabine liposome), Wellcovorin (leucovorin calcium), Xalkori (crizotinib), Xeloda (capecitabine), Xeliri, Xelox, Xgeva (denosumab), Xofigo (radium-223 chloride), Xtandi (enzalutamide), Yervoy (ipilimumab), Yondelis (trabectedin),This may include any anticancer therapies known in the art, including Zartrap (Ziv-aflibercept), Zarxio (filgrastim), Zejura (niraparib tosylate monohydrate), Zelboraf (vemurafenib), Zevalin (ibritumomab tiuxetan), Zinecard (dexrazoxane hydrochloride), Ziv-aflibercept, Zofran (ondansetron hydrochloride), Zoladex (goserelin acetate), zoledronic acid, Zolinza (vorinostat), Zometa (zoledronic acid), Zydelig (idelalisib), Zykadia (ceritinib), and / or Zytiga (abiraterone acetate), and / or further include these. Furthermore, chemotherapeutic agents that are PDl / PDL blockade inhibitors (e.g., lambrolizumab, nivolumab, pembrolizumab, pidilizumab, BMS-936559, atezolizumab, durvalumab, or avelumab, etc.) will be examined in this specification. [Examples]

[0119] D. Examples The following examples are provided to those skilled in the art to provide a complete disclosure and description of the compounds, compositions, articles, apparatus, and / or methods claimed herein, and are intended to be illustrative only and not to limit the disclosure. While attempts have been made to ensure accuracy with respect to numbers (e.g., quantities, temperatures, etc.), some errors and deviations should be explained. Unless otherwise indicated, fractions are fractions by weight, temperatures are in °C or ambient temperature, and pressures are atmospheric pressure or near atmospheric pressure.

[0120] (Example 1) Immunostimulatory activity of human IFNβ in a mouse melanoma model It is important to conduct animal studies using the same drugs that are tested in humans. However, this presents a problem for biological agents; for example, with the use of cytokines, cross-reactivity varies considerably between various human and mouse cytokines. As discussed above, we hope to develop an OV expressing CD40L and human IFNβ as an anticancer drug. IFNβ induces signaling in mice, albeit to a lesser degree than mouse IFNβ. Before OV development, as this is the primary focus of this study, we first determined whether human IFNβ can induce an antitumor T cell response in mice. To achieve this objective, we utilized a recently developed assay, in which we found that irradiated B16-FlO or B16-OVA melanoma expressing mouse IFNβ functioned as a potent prophylactic vaccine, and this protective effect depended on both CD8 and CD4 T cells (results not published). This method is functionally similar to GM-CSF expressing tumor cells (GVAX). B16-OVA cells were transduced with either a pLenti-Puro control lentivirus or a lentivirus expressing mouse or human IFNβ, followed by puromycin selection. Equal numbers of cells were seeded, and the supernatant was collected two days later for ELISA to detect human and mouse IFNβ. Both human and mouse IFNβ were expressed at high levels, but higher levels of mouse IFNβ were detected (Figure 1A). Notably, we found that both mouse and human IFNβ were highly effective in inducing T cell responses to tumor-coding model antigens, although a higher response was induced by mouse IFNβ (Figure 1B). The high response to mouse IFNβ may be partly due to high expression (Figure 1A). Furthermore, this effect of human IFNβ was strictly dependent on the presence of type 1 IFN receptor α1 (IFNAR1) in the host (Figure 1B; similar results obtained were from separate experiments with mouse IFNβ). Functionally, both mouse and human IFNβ were highly effective in preventing tumor growth after exposure with live B16-OVA (Figure 1C). These results suggest that, by using OV, it is also possible to induce an antitumor T cell response in mice with human IFNβ.

[0121] (Example 2) Generation of MEM-288 and in vitro testing We generated oncolytic adenovirus type 5 expressing chimeric CD40L driven by the CMV promoter (Figure 2A) by adding an expression cassette (MEM-288) that drives human IFNβ expression (Figure 2B). As controls, we used adenoviruses expressing GFP (Ad-GFP) and only chimeric CD40L (MEM-188). All three viruses used contained an E1A D24 deletion that enabled specific replication and lysis of cancer cells (see Figure 4 below). Infection of human A549 lung cancer strains containing either MEM-188 or MEM-288 induced high CD40L expression as determined by flow cytometry, while infection with Ad-GFP induced high GFP expression (Figure 3A). High levels of IFNβ secretion into the culture supernatant were observed only after MEM-288 infection (Figure 3B). Similar results were obtained in several other human cell lines. Furthermore, in tests using the B16-F10 melanoma cell line, high transgene expression (Figure 3C) and secretion of IFNβ into the culture supernatant after MEM-288 infection (Figure 3D) were demonstrated. Cell death induced by OV is due to viral replication in infected cancer cells, which causes cell lysis. Adenovirus replication was not impaired by type 1 IFN. Notably, in mouse cells, adenovirus replication was not supported, and consistent with this, we found that Δ24 deletion virus did not induce lysis in mouse B16-F10 and 344SQ cell lines (Figure 4), while human A549 cells were readily lysed (Figure 4; indicating MEM-288). These results suggest that, in in-vivo mouse studies, the antitumor response induced by OV injection is more likely due to immunostimulatory effects rather than oncolytic effects. Despite the lack of oncolytic activity in mouse cells, we refer to such viruses as OV and discuss their applications in humans.

[0122] (Example 3) Tests in a mouse melanoma model The primary premise for using the intralesional approach is the potential to generate a systemic antitumor immune response capable of targeting non-injectable lesions, i.e., inducing abscopal action. Therefore, the main objective of the study was to determine whether combined activation of CD40 and type 1 IFN signaling could evoke a potent antitumor T-cell response capable of reducing tumor growth in both injectable and non-injectable tumors. In a study using a non-oncolytic, non-replicating adenovirus expressing MEM40 / CD40L, activity was demonstrated in a B16-F10 melanoma model. However, four injections of this virus were required to observe its activity as a monotherapy or in combination with an ICI.

[0123] We were the first to conduct trials in B16-OVA melanoma, enabling the tracking of OVA-responsive CD8 T cells. The typical dose of adenovirus used in mice to determine antitumor therapeutic activity was 10 e9 to 10 infection units (IU). 8 Low doses of Ad-GFP, MEM-188, and MEM-288 were used for intratumoral injection in the first experiment (Figure 5A). Interestingly, MEM-288 showed a significant reduction in tumor growth compared to Ad-GFP and MEM-188 (Figure 5A). In the next experiment, we used all three viruses at a 10-fold higher dose of 10e9 IU, where MEM-288 injection induced the most significant reduction in tumor growth (Figure 5B). The above tests showed notable activity after four injections with MEM40 / CD40L-expressing adenovirus. However, we did not observe similar activity after two injections with MEM-188, suggesting that sustained CD40L expression may be required with multiple viral injections. Furthermore, MEM-288 induced an increase in circulating OVA-specific CD8 T cells, which was not induced by MEM-188 (Figure 5C).

[0124] a) MEM-288 influences systemic T cell response: the roles of CD40 and IFNAR1 In this type of test, we used MEM-288 alone compared to a PBS control. MEM-288 administration into B16-OVA tumors induced a dramatic increase in tumor-responsive CD8 T cell counts in mouse spleens, as determined by IFNγ secretion (ELISPOT assay) (Figure 5D). To evaluate the role of CD40 and type 1 IFN signaling in this, we used CD40 and IFNAR1 knockout mice with B16-OVA tumors. Importantly, the CD8 T cell response was significantly reduced in both IFNAR1 and CD40 knockout mice (Figure 5D), demonstrating that both pathways are independently required for MEM-288 activity. The test proposed in this application will investigate the regulation of both APCs and T cells to precisely determine how these two pathways function in activating the T cell response.

[0125] b) MEM-288 activation by ICI treatment We wanted to determine the effect of intratumoral injection of MEM-288 on the growth of contralateral tumors, both injectable and non-injectable (Figure 6A). Importantly, MEM-288 suppressed the growth of both the injected and contralateral tumors (Figures 6B-6C). In contrast, as is known for this tumor model, ICI anti-CTLA4 and anti-PD-1 therapy had minimal effect on tumor growth (Figures 6B-6C). These results indicate that MEM-288 has superior efficacy in this tumor model compared to anti-CTLA4 + PD-1 therapy. Importantly, the combination of anti-CTLA4 + PD-1 and MEM-288 significantly reduced tumor growth compared to MEM-288 alone, and the benefits of ICI were even more evident when combined with MEM-288 (Figures 6B-6C). Ultimately, MEM-288 alone improved mouse survival, which was further enhanced when combined with ICI (Figure 6D). Therefore, these results indicate that (a) localized administration of MEM-288 induces potent abscopal activity as a single agent, and (b) when combined with MEM-288, the benefits of ICI therapy are evident in other ICI-resistant tumor models.

[0126] (Example 4) Tests in lung metastasis models Subcutaneous injection of KRAS and TP53 mutant mouse 344SQ(344) lung tumor cells resulted in tumor formation at the primary injection site, followed by metastatic dissemination to the lungs and other sites. We found this model to be resistant to PD-1 checkpoint blockade. A key question we wish to address is the efficacy of MEM-288 as a monotherapy in PD-1 resistant tumors. We hereby demonstrate whether MEM-288-induced T cell activation and expression prevent metastatic dissemination and / or reduce primary tumor growth. Two doses of 10e9 IU each of Ad-GFP, MEM-188, and MEM-288 were administered intratumorally 12 days post-inoculation. MEM-288 administration alone induced a significant reduction in sc tumor growth (Figure 7A). Tumor lesions in the lungs were determined 38 days post-inoculation. As expected, mice injected with PBS into the tumor via sc had a higher number of lung metastases, and the same was true for mice injected with Ad-GFP and MEM-188 (Figures 7B-7C). In contrast, lung tumor lesions were significantly reduced in mice injected with MEM-288 (Figures 7B-7C). Furthermore, the size of the tumor lesions appeared to be substantially smaller in mice injected with MEM-288 and MEM-188. CD8 IHC further revealed significantly higher TIL density in lung tumors of mice treated with MEM-288 and slightly more with MEM-188 (Figures 7D-7E). These results suggest that the addition of IFNβ to CD40L may enhance the immunostimulatory function in this model. Next, we determined whether this was indeed the case and, in addition, investigated the efficacy of MEM-288 in parallel with anti-PD-1. Importantly, we found a robust elevation of tumor-reactive CD8 T cells in the spleen of MEM-288-injected mice, as determined by IFNγELISPOT (Figures 8A-8B). In contrast, anti-PD-1 did not substantially increase tumor-reactive T cells. We conclude that localized MEM-288 injection has the potential to induce a potent systemic T cell response, suggesting it may be able to modulate tumor growth as a single agent in PD-1-resistant tumors.In ongoing further trials, we will determine the synergistic effect on antitumor activity when MEM-288 and ICI (anti-PD-1+ / -anti-CTLA4) are combined in this model.

[0127] (Example 5) Investigation of the immune stimulation mechanisms of CD40L and IFNβ in the tumor microenvironment These results demonstrate that a virus expressing CD40L and IFNβ (MEM-288) can induce a potent systemic antitumor T cell response capable of modulating the proliferation of abscopal tumor lesions. In addition to the above virus, we can also utilize a virus expressing human IFNb alone for the tests proposed herein. We can determine the effects of CD40L and IFNb expression on multiple immune cell types, particularly on TMEs on DCs. Macrophages represent another major target of CD40L activity and can trigger enhancements of these antitumor activities. This test includes a thorough evaluation of the effects of OV on DCs, macrophages, and T cells by key targets defining the unique and synergistic contributions of CD40L and IFNb. In addition to the IFNAR1 and CD40 KO mice described above, we can also use BATF3 KO mice lacking DC1 (see below). We have shown above that human IFNb induces T cell activation in mice in an IFNAR1-dependent manner (Figure 1). However, human IFNb may potentially evoke a more substantial response in the establishment of the human IFNAR1 / 2 receptor in mouse cells. Importantly, Harari et al. created a genetically modified mouse line called HyBNAR (Hybrid IFNAR) that possesses both a human IFNAR1 / 2 extracellular ligand-binding domain and a mouse IFNAR1 / 2 receptor signaling domain. In this study, it was shown that human IFNb induces signaling in wild-type mice, and consistent with these results, this response is enhanced in HyBNAR mice.

[0128] The main goal here is to show the impact of intratumoral OV administration on the TME in the B16 melanoma and 344 lung tumor models. We here define the unique and complementary actions of CD40L and IFNβ receptor engagement on immune cells in the TME and can investigate the underlying mechanisms by using mice lacking CD40 or IFNAR1. We consider DCs to represent the main targets of MEM-288 activity and, for this purpose, we can conduct the most thorough tests to investigate the impact of OV on the functionality of the DC1 and DC2 subsets.

[0129] a) Impact of OV on the TME: Explanation of the specific roles of CD40L and IFNβ The main goal is to determine the mechanisms of action of CD40L and IFNβ in the available TME using Ad-GFP, Ad-IFNβ, MEM-188, and MEM-288 OVs. Such exploratory tests can serve to evaluate the broad impact of the above OVs on the major populations of myeloid cells, DCs, T, and B cells. As shown in Figure 9A, we have macrophages (F4 / 80 + Ly6C + CD11b + MHC-II + ), monocytes, and neutrophils (F4 / 80 - CD11b + Ly6C + or Ly6G + ) and DCs including DC1 (CD11c + CD11b - MHC-II + CD103 + ) and DC2 (CD11c + CD11b + MHC-II + CD103 - ) readily detected by flow cytometry analysis of B16-OVA tumors. We also have B cells, CD4 + T cells, CD8 +T cells and OVA-specific CD8 T cells were detected (Figure 9B). A primary focus may be to determine the effect of OV on DC activation in tumors. Tumor antigens are readily taken up by resident macrophages and DCs, and these can be detected by using tumors expressing GFP or ZsGreen. Recent studies have also demonstrated that antigen uptake functionally reprograms DCs. We generated B16-OVA and B16-F10 expressing ZsGreen to track antigen uptake by tumor-resident APCs (Figure 10). As shown in Figure 10, ZsGreen in macrophages and DCs. + The population was easily detected in ZsGreen-expressing tumors compared to non-expressing tumors in parents. Using ZsGreen expression, tumor cells (ZsGreen + CD45 - We identified ) and were also able to detect CD40L expression 2 days after a single MEM-288 injection (Figures 11A-11B). Importantly, we were able to detect DC and MHC-II expression in the same tumor after MEM-288 injection. 高 We also observed an increase in CD80 / CD86 in macrophages (Figures 12A-12B). While these results indicate that MEM-288 induces activation in both macrophages and DCs, we present them not as practical findings, but to demonstrate the feasibility of detecting CD40L expression and activation markers in APCs. We are currently further determining the dynamics of CD40L transgene expression by examining tumors at 2, 4, and 6 days after a single injection in both B16 and 344 models. We can select the time point with the highest expression for the following tests.

[0130] b) Effects of OV on TME Here, we discuss tumor-infiltrating CD45 in B16-OVA, B16-FlO, and 344 models. + Determine the effects of various OVs on the number and phenotype of immune cells. Enumerate each cell type using CD45. +This can be done by determining the cell proportion and the number of cells per gram of tumor. The main population may be as shown in Figure 9, but we add NK cell detection by NK1.1Ab, as NK cells are known targets of both CD40L and type 1 IFN, and also by Treg (with intracellular FoxP3 expression) as they also play a role in regulating the T cell response after OV injection. A group of mice with ZsGreen-expressing tumors (see also statistical design) can be treated with PBS or the four OVs above. We may use 5 mice per treatment in two independent experiments. Tumors can be degraded 2 days after treatment (or at different time points based on the highest CD40L expression in the above tests), and flow cytometry can be performed to determine the difference in immune cells after injection of various OVs. A specific focus may be on tumor antigen-specific CD8 T cells in B16-OVA tumors. In B16-OVA transplanted mice treated with MEM-288, we readily detected an increase in anti-tumor T cells in the blood (Figure 5C) and spleen (Figure 5D). We can determine here whether MEM-288 treatment generates the highest levels of total and OVA-specific CD8 T cells in the tumor (detected as shown in Figure 9B). Next, we will determine how various OVs affect ZsGreen uptake by macrophages and DCs. In key tests, we can determine the expression of MHC-II and the activation markers CD80, CD86, and CD40 in DC1 and DC2. We will also determine ZsGreen + and ZsGreen -The activation state in both cells can be determined. In addition, we can determine the therapeutic effect of CCR7 expression in DCs, as CCR7 expression plays a crucial role in DC-DLN translocation. IL-12 p70 is a definitive Th1 response-promoting cytokine secreted by DCs and is a known target of CD40L. Furthermore, IL-12 p70 expression by tumor DCs is important for the T-cell anti-tumor immune response. We can determine the expression of the IL-12p40 subunit by intracellular flow in DCs after administration of brefeldin A to mice, as described. In summary, such tests can help determine how various transgenes, namely CD40L, IFNβ, and CD40L+IFNβ, affect the total number and activation state of DC1 and DC2. For example, such tests can help determine whether the increase in CD80 / CD86 expression we observed after MEM-288 injection (Figure 12) is due to CD40L, IFNβ, or both. Statistical testing: In two-group comparisons, Student's t-test can be used to determine differences in treatment methods. Differences in treatment methods (e.g., specific cell type or activation marker expression) in multiple treatment groups can be determined using Tukey's method.

[0131] c) Definition of the specific roles of CD40L and IFNβ using receptor knockout mice OVs expressing various transgenes exhibit different effects depending on the broad range of TME and T cell / DC activation. Furthermore, MEM-288 has the most robust stimulating effect due to the combined expression of CD40L and IFNβ. Using complementary methods, we can determine the individual roles of CD40L and IFNβ in key phenotypes observed after MEM-288 injection in mice lacking CD40 and IFNAR1. While the above studies have shown that CD40 and IFNAR1 are important in the T cell response (Figure 5D), we here determine the role of DCs in this response. This method may help provide further insight into the unique and overlapping functions of these two pathways in the context of MEM-288 therapy. Such studies can be performed in B16-F10 and B16-OVA models to match the C57BL / 6 genetic background of CD40 and IFNAR1 KOs.

[0132] d) Definition of the effect of OV on DC migration to DLN and T cell stimulating ability As shown in Figure 10, both macrophages and DCs readily take up ZsGreen. However, DCs have a unique ability to migrate from tumors to the DLN, where they can present tumor antigens to naive T cells and trigger their activation. This specification describes how various OVs affect DC migration to the DLN and their ability to stimulate T cells. In such studies, we can track the migration of DC1 and DC2 to the DLN by ZsGreen, as shown in recent studies, and also evaluate their ability to activate T cells. Such studies help determine how various transgenes affect DC migration and T cell stimulation.

[0133] As shown in Figure 13, MHC-II + CD11c + Two main cell populations are detected in the inguinal DLN. MHC 高 The group is thought to consist of migratory DC1 and DC2, which, as expected, have a high proportion of CD103. + DC and CD11b +It has DC. Intermediate level MHC-II (MHC 中 ) CD11c with a population + The population consists of resident DC1 and DC2 cells, with a low proportion of CD103 cells. + It has DC. The majority of ZsGreen+DCs are MHC 高 They exist in the population. Here we can determine the effects of various OVs on the distribution and activated phenotype of such distinct populations. MEM-288 administration may result in the highest DLN migration, and these DCs may have the most potent activated phenotype. We can also determine the ability of MEM-288 to induce DC migration in CD40 and IFNAR1 KO mice. As shown in Figure 14, the LN population of DCs does not appear to be affected by the absence of CD40 and IFNAR1. Nevertheless, it is unknown whether the absence of CD40 and IFNAR1 affects DC migration from tumor to DLN.

[0134] Next, we can determine the ability of LN DCs to activate T cells. While tests have already shown that T cell stimulation is greatly enhanced by MEM-288, such tests can specifically determine whether this is at least partially due to DC activation. For this purpose, we can use mice with B16-OVA tumors and inject them with all four OV cells. Next, we can perform the CFSE dilution assay described herein by co-culturing CFSE-labeled naive OT-1 CD8 T cells using DC1 and DC2 selected from DLN cells. Such tests can determine whether the highest level of T cell population is produced by MEM-288-induced DC activation. In addition, we can determine IFNγ secretion by performing the ELISPOT assay by culturing DCs with OT-1 CD8 T cells. Next, using MEM-288, we can determine the specific roles that the two transgenes play in the ability of DCs to activate T cells using CD40 and IFNAR1 KO mice. Ultimately, using BATF3 KO mice, we can determine the role of DC1 in the elevation of T cell population and IFNγ secretion induced by MEM-288.

[0135] (Example 6) Oncolytic virus treatment for tumors To demonstrate the in vivo effects of oncolytic viruses on tumors, 5e5 B16-OVA cells were inoculated into the flanks of C57BL / 6 mice. The tumors were treated with PBS, adenovirus GFP, or MEM-288 injections on D12 and D16. 9 It was administered via IU. On the 20th day, a tumor was found, and IHC was performed, resulting in a CD8 + The presence of tumor-infiltrating lymphocytes (TILs) was detected. Mice treated with PBS or oncolytic viruses expressing GFP showed a similar number of TILs. In contrast, TIL levels were nearly three times higher in tumors vaccinated with MEM-288 (Figure 15).

[0136] E. References [References] TIFF0007842751000013.tif190160TIFF0007842751000014.tif224160TIFF0007842751000015.tif223160TIFF00078427510 00016.tif223160TIFF0007842751000017.tif231160TIFF0007842751000018.tif228160TIFF0007842751000019.tif158160

[0137] array Sequence ID 1 is the amino acid sequence of human wild-type interferon-alpha (UniProtKB reference number P05014).

[0138] [ka]

[0139] Sequence ID 2 is the amino acid sequence of human wild-type interferon-beta (UniProtKB reference number P01574).

[0140] [ka]

[0141] Sequence ID 3 is the amino acid sequence of human wild-type interferon ε (UniProtKB reference number Q9P0W0).

[0142] [ka]

[0143] Sequence ID 5 is the amino acid sequence of human wild-type interferon ω (UniProtKB reference number P05000).

[0144] [ka]

[0145] Sequence ID 6 is the amino acid sequence of human CD40-L (EtniProtKB reference number P29965).

[0146] [ka]

Claims

1. A composition for use in the treatment of cancer, comprising tumor-infiltrating lymphocytes (TILs), wherein the TILs are a. A step of administering the oncolytic virus adenovirus type 5 into tumor cells in the target, b. Process for recovering TIL Obtained by a method including, Oncolytic adenovirus type 5 expresses chimeric CD40L of sequence number 12 under the control of the CMV promoter and human IFN-β under the control of the SV40 promoter. composition.

2. A composition comprising an oncolytic virus for use in the treatment of cancer in a subject, wherein the treatment is a. A step of administering the composition containing the oncolytic virus adenovirus type 5 into tumor cells in a subject, b. A process for collecting tumor-infiltrating lymphocytes (TILs), c. A process for growing the recovered TILs ex vivo, d. The process of administering the proliferated TILs Includes, Oncolytic adenovirus type 5 expresses chimeric CD40L of sequence number 12 under the control of the CMV promoter and human IFN-β under the control of the SV40 promoter. composition.

3. The composition according to claim 2, which is administered in combination with an anticancer agent.

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