JP7842751B2 - Oncolytic viruses enhance T cell responses for effective TIL therapy. - Google Patents

Description

This application claims the interests of U.S. Provisional Patent Application No. 63/106,215, which is incorporated herein by reference in its entirety.

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-responsive T cells are critically important for benefiting from checkpoint inhibitors. Therefore, new methods are needed to enhance the number and function of tumor-responsive T cells.

U.S. Patent No. 7,495,090 U.S. No. 7,928,213 U.S. Patent No. 8,138,310

Racher, A.J., Fooks, A.R. & Griffiths, J.B. Biotechnol Tech(1995) 9: 169 Senter et al., Bioconjugate Chem., 2:447–451 (1991) Bagshawe, K.D., 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: pp. 57–80 (1992) Roffler et al., Biochem. Pharmacol, 42:2062-2065 (1991) Hughes et al., Cancer Research, 49: pp. 6214–6220 (1989) Litzinger and Huang, Biochimica et Biophysica Acta, 1104:179-187 (1992) Brown and Greene, DNA and Cell Biology 10:6, pp. 399–409 (1991). Remington: The Science and Practice of Pharmacy (19th edition), edited by A.R. Gennaro, Mack Publishing Company, Easton, PA 1995. Handbook of Monoclonal Antibodies, edited by Ferrone et al., Noges Publications, Park Ridge, NJ. (1985), pp. 22 and 303–357. Smith et al., Antibodies in Human Diagnosis and Therapy, in Haber et al. (eds.), Raven Press, New York (1977), pp. 365-389.

This document discloses methods and compositions relating to the proliferation of tumor-infiltrating lymphocyte (TIL) populations and the use of this TIL population for cancer treatment.

In one embodiment, a method for generating tumor-infiltrating lymphocytes is disclosed herein, 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; and b) recovering tumor-infiltrating lymphocytes. 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.). TILs can be obtained even in the tumor microenvironment not infected with oncolytic viruses, but in some embodiments, the generated TILs are obtained in the tumor microenvironment at the site of oncolytic virus administration.

Furthermore, this specification discloses a method for generating tumor-infiltrating lymphocytes in any of the aforementioned embodiments, by administering an oncolytic virus via intratumor injection.

Furthermore, this specification discloses a method for generating tumor-infiltrating lymphocytes in any of the aforementioned embodiments, further comprising the step of growing the recovered TILs ex vivo.

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). 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.).

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, and/or 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.).

Alternatively, 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: 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.).

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

The accompanying drawings incorporated herein and constituting part thereof illustrate several embodiments and illustrate the compositions and methods disclosed together with this description.

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. 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 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. This is a schematic diagram of MEM40. This is a schematic diagram of MEM-288. 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. 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. 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. 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. 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. 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. 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. This figure shows C57BL/6 mice inoculated with 5e5 B16-OVA cells via s.c. At 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 point. This figure shows C57BL/6 mice inoculated with 5e5 B16-OVA cells via s.c. At 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 point. 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. 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 was administered as intratumoral injection of PBS (UT: untreated) or MEM-288 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. 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 s.c. As shown, these mice were injected with MEM-288 at 10e9 IU in the primary tumor on D12 and D16, and with anti-PD-1 and CTLA-4 antibodies i.p. on D16, D19, D23, and D27. 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. 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. 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. This figure shows the results of inoculating 344 cells of 5e5 in the flanks of 129 mice with s.c., 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 as *p<0.05, **p<0.01, and ***p<0.001. NS: Not significant. This figure (7A) shows typical H&E staining of tumors in the lungs of 344 tumor-bearing mice by s.c. in D38. 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. (7A) This figure shows typical IHC staining of CD8 T cells in the lungs of a mouse. 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. This figure shows the results of inoculating 344 cells of 5e5 in the flanks of 129 mice with s.c., administering 10e9 IU of MEM-288 intratumorally on days D12 and D16, and treating with anti-PD-1 antibody using i.p. on days 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. This figure shows the results of inoculating 344 cells of 5e5 in the flanks of 129 mice with s.c., administering 10e9 IU of MEM-288 intratumorally on days D12 and D16, and treating with anti-PD-1 antibody using i.p. on days 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. This figure shows the flow cytometry analysis scheme of B16-OVA for detecting populations of macrophages, neutrophils, monocytes, and DCs. 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. This figure shows the flanks of C57BL/6 mice inoculated with 5e5 B16-OVA or B16-OVA-ZsGreen cells via s.c. Flow cytometry analysis of tumor B16-OVA cells to detect the ZsGreen-positive population of macrophages and DCs was performed on D14, as shown in Figure 9A. The percentage of positive cells is shown. 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. 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. 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. This figure shows the average fluorescence intensity (MFI) of result (A). 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. 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. This figure shows the flanks of C57BL/6 mice inoculated with 5e5 B16-OVA cells in sc. Tumors were treated with PBS (n=4), adenovirus GFP (n=3), or MEM-288 (n=4) injections at ^10⁹ IU 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. 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 15B shows the scored values for the three treatment groups.

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.

A. Definitions 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.

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, the use of the antecedent "approximately" is understood to mean that 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. Furthermore, when a value "less than or equal to" that value is disclosed, it is understood, as will be appropriately understood by those skilled in the art, that "greater than or equal to" and the possible range between values are also disclosed. 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 that this data represents endpoints and starting points, as well as ranges of any combination of data points. For example, when disclosing a specific data point "10" and a specific data point "15," 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 their equivalents 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, when disclosing 10 and 15, 11, 12, 13, and 14 are also disclosed.

This specification and subsequent claims refer to numerous terms that should be defined as having the following meanings:

"Optional" or "optional" means that the phenomenon or situation described thereafter may or may not occur, and the description includes both cases where this phenomenon or situation occurs and cases where it does not.

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.

"Increase" can refer to any change resulting in a large amount of symptoms, disease, composition, symptoms, or activity. An increase may be any individual, median, or average increase in a statistically significant amount of symptoms, symptoms, activity, or composition. Therefore, an increase may 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.

"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 due to the substance is less than the genetic output of the gene product without the substance. For example, a reduction may also be a change in the symptoms of a disorder, such as the symptoms being 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 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.

"Inhibition" means reducing activity, response, symptoms, disease, or other biological parameters. This may include, but is not limited to, the complete disappearance of activity, response, symptoms, or disease. It may also include, for example, a 10% reduction in activity, response, symptoms, or disease compared to natural or control levels. Therefore, 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.

"Reduce," or other forms of the word, such as "reducing" or "reduction," means to decrease a phenomenon or characteristic (e.g., tumor growth). It 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.

"Prevention," or other forms of the word, such as "preventing" or "prevention," means to halt a particular phenomenon or characteristic, stabilize or delay its onset or exacerbation, or minimize the opportunity for a particular phenomenon or characteristic to occur. Prevention is typically absolute, more so than reduction, and therefore does not require comparison with a control. In its use 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.

The term "subject" refers to any individual that is the target of administration or treatment. The subject may be a vertebrate, e.g., 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, e.g., an internist.

The term "therapeutably effective" refers to a composition used in an amount sufficient to restore one or more causes or symptoms of a disease or disorder. Such restoration does not necessarily require elimination, but rather reduction or alteration.

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 specifically towards improvement of the disease, symptoms, or disorder; causal treatment, i.e., treatment directed towards the elimination of the cause of the associated disease, condition, or disorder; and additionally, palliative treatment, i.e., treatment designed to alleviate symptoms rather than cure the disease, condition, or disorder; preventive treatment, i.e., treatment directed towards minimizing or partially or completely inhibiting the onset of the associated disease, condition, or disorder; and supportive treatment, i.e., treatment used to complement other specific therapies directed towards improvement of the associated disease, condition, or disorder.

"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.

"Contains" is intended to mean that a composition, method, etc., includes the elements listed, but does not exclude others. "Essentially consisting of" (as 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. Therefore, 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" (as defined herein) means excluding elements in amounts exceeding trace amounts of other components, as well as substantial method steps for administering the compositions provided and/or claimed herein. Embodiments defined by each of these conversion terms are within the scope of this disclosure.

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

The “effective dose” of a drug refers to the amount of drug sufficient to produce the desired effect. The “effective” dose varies from subject to subject depending on many factors, such as the subject’s age and general condition, and the specific drug being administered (one or more drugs). Therefore, it is not always possible to determine 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. Furthermore, as used herein, and unless otherwise specified, the “effective dose” of a drug may also refer to an amount encompassing both therapeutically effective and prophylactically effective doses. 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. Dosage plans can be adjusted to produce an 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.

A “pharmaceutically acceptable” ingredient may mean an ingredient that is not biologically or otherwise unsuitable, i.e., an ingredient that does not cause significantly unsuitable biological effects or adverse interactions with any other ingredients in the formulation containing it, and that can be incorporated into the pharmaceutical formulations provided herein and administered to subjects as described herein. 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 guidelines created by the U.S. Food and Drug Administration.

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.

"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 approximately equivalent in degree.

"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). Furthermore, this term 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 this 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.

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 the 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. This term also refers to the desired therapeutic effect, e.g., the amount of the therapeutic agent effective in promoting analgesia, or the rate of delivery of the therapeutic agent (e.g., amount over time). The exact desired therapeutic effect varies depending on the symptoms 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 various 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 target over several days, weeks, or years.

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 within the context of the references.

B. Compositions The compositions disclosed herein, as well as the components used in the preparation of the compositions themselves used in the methods disclosed herein, are 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 herein, will be considered in detail. Therefore, when disclosing certain molecules A, B, and C, and certain molecules D, E, and F, and examples of combinations of molecules, we consider disclosing A through D, and then disclosing each of the combinations A through E, A through F, B through D, B through E, B through F, C through D, C through E, and C through 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 through E, B through F, and C through 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.

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 anti-tumor 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 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 explored through 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.

In intrafocal immunotherapy, the tumor acts as a vaccine site, leading to dendritic cell (DC) activation and subsequent T cell stimulation, resulting in systemic antitumor immunity capable of regulating the growth of distal, untreated tumors. 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 leads to robust immune activation in the tumor microenvironment (TME), resulting in a high level of elevation in the systemic T cell response.

Oncolytic viruses (OVs) have been developed for their ability to specifically replicate in cancer cells. Recent studies indicate that stimulation of host anti-tumor immunity is a key mechanism of OV action. Furthermore, OVs can encode transgenes that can be used to further enhance anti-tumor immunity by expressing potent activators of the immune response. This specification demonstrates that intralesional-delivered OVs capable of activating immunostimulatory molecules (e.g., CD40) and/or type 1 IFN signaling in the tumor melanoma (TME) 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, resulting in a robust systemic T cell response capable of significantly reducing distal tumor growth in mouse melanoma and lung tumor models.

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 dramatically increases the number of TILs and MILs at the tumor site, it is also recognized that TILs and MILs can be generated and/or increased outside the treated tumor microenvironment at the tumor site (i.e., abscopal effect).

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. Therefore, a method for generating tumor-infiltrating lymphocytes, further comprising the step of proliferating recovered TILs ex vivo, is also disclosed herein.

In one embodiment, the proliferation of TILs and MILs is not limited to in vivo oncolytic virus methods, which involve injecting oncolytic viruses into tumors 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, this specification discloses a method for growing a population of tumor-infiltrating lymphocytes (TILs) or bone marrow-infiltrating lymphocytes (MILs), comprising the steps of: a) recovering TILs or MILs from a subject with 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.).

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 (e.g., lentivirus) or any combination thereof, such as (-1BBL), CD70, LIGHT, glucocorticoid-inducing 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.

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 varicella stomatitis virus (VSV)), or vaccinia virus. Preferably, the oncolytic virus is replicable.

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

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 human wild-type IFN-α, e.g., IFN-a of SEQ ID NO: 1, 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%).

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 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-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.

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 human wild-type IFN-ε, e.g., IFN-ε of SEQ ID NO: 3, 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%).

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 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 human wild-type IFN-κ, e.g., IFN-κ of SEQ ID NO: 4, 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%).

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 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 human wild-type IFN-ω, e.g., IFN-ω of SEQ ID NO: 5, 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%).

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 human wild-type CD40-L, such as CD40-L of Sequence ID No. 6, 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%).

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.

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.

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.

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. Therefore, 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.

In preferred embodiments, 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, 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". Specifically, 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 its 3' end, where, upon administration to humans, antibody binding neutralizes the activity of mouse CD154 (CD40 ligand).

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

Sequence ID 8

Sequence ID 9

Sequence ID 10

Sequence ID 11

Sequence ID 12

Sequence ID 13

Sequence ID 14

Sequence ID 15

Sequence ID 16

Sequence ID 17

Sequence ID 18

The specific nucleotide sequences encoding the chimeric CD40-L in SEQ ID NOs. 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.

The oncolytic virus of the present invention may contain 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%).

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, 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, the nucleotide sequence encodes IFN-β lacking the first 22 amino acids of SEQ ID NO: 2, and optionally further having a substitution of serine at cysteine 17 in the resulting 165-amino acid peptide.

The oncolytic virus of the present invention may contain 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%).

The oncolytic virus of the present invention may contain nucleic acids encoding IFN-κ, where IFN-κ includes human wild-type IFN-κ (SEQ ID NO: 4), or IFN-κ 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 IFN-κ, for example, SEQ ID NO: 4.

The oncolytic virus of the present invention may contain nucleic acids encoding IFN-ω, wherein IFN-ω includes human wild-type IFN-ω (SEQ ID NO: 5), or IFN-ω 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 IFN-ω, for example, SEQ ID NO: 5.

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 comprises 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% (e.g., 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.

In preferred embodiments, the oncolytic virus of the present invention may contain nucleic acid encoding MEM40. For example, in preferred embodiments, the oncolytic virus of the present invention contains 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.

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.

In addition to nucleic acids encoding type 1 interferon (e.g., IFNP) and CD40-L (chimeric human/mouse CD40-L), the oncolytic virus according to the present invention may further include other modifications in its genome. For example, this may include additional DNA inserted into already inactivated genes or substituted for deleted genes. Furthermore, the oncolytic virus may incorporate one or more promoters that confer enhanced levels of tumor cell specificity to the virus. Thus, the oncolytic virus 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 at higher levels in target cancer cells than in normal cells. For example, the oncolytic virus for use in the present invention may exist under the regulation of exogenously added regulatory factors.

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. Approximately 50 serotypes of human adenoviruses exist, 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, with the primary effect being a flu-like symptom.

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.

The adenovirus infection cycle consists of two phases: an early phase preceding the initiation of adenovirus genome replication, which 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 across four regions of the adenovirus genome, designated E1–E4 ("E" signifying "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.

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.

Adenoviruses enter 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 host cells.

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 a wide range of prophylactic and therapeutic vaccine formulations.

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, A.J., Fooks, A.R. & Griffiths, J.B. Biotechnol Tech (1995) 9: 169).

1. Delivery of Pharmaceutical Carriers/Pharmaceutical Preparations As described above, the composition may 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 may 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 in which it is contained. The carrier is, of course, selected to minimize any degradation of the active ingredient and to minimize any harmful side effects in the subject, as is well known to those skilled in the art.

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 via 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 depending on the species, age, weight and general condition of the subject, 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, the appropriate amount can be determined by those skilled in the art, considering the teachings herein and using only routine experimental methods.

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 pre-injection suspension, 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.

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, K.D., 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 tumor tissue-specific proteins (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. A review article has been published on the molecular and cellular mechanisms of receptor-mediated endocytosis (Brown and Greene, *DNA and Cell Biology* 10:6, pp. 399–409 (1991)).

a) A composition containing a pharmaceutically acceptable carrier antibody may be used therapeutically in combination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy (19th edition), edited by A.R. 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, 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.

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.

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

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

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 (e.g., olive oil), and organic esters for injection (e.g., ethyl oleate). Aqueous carriers include water, alcohol/aqueous solutions, emulsions, or suspensions, and also include saline and buffer 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.

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

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.

A portion of the composition may be potentially administered as a pharmaceutically acceptable acid or base addition salt 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.

b) Therapeutic Use The effective dose and schedule for administering the composition can be determined empirically, and such determination is within the scope of the art. The dosage range for administering the 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, and 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 Antibodies in Human Diagnosis and Therapy, edited by Smith 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.

C. Methods for treating cancer The compositions of this disclosure can be used to treat any disease in which uncontrolled cell proliferation occurs, for example, 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, e.g., 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.

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... 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: 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.

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 TILs or MILs in the presence of antigen-presenting cells infected with oncolytic viruses expressing (e.g., OX40L, 4-IBBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF6), and administering the proliferated TILs and/or MILs to the subject.

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-apple) (Salt), Copanlisib hydrochloride, COPDAC, COPP, COPP-ABV, Cosmegen (dactinomycin), Cotellic (cobimetinib), crizotinib, CVP, cyclophosphamide, Cyfos (ifosfamide), Cyramza (ramucirumab), cytarabine, cytarabine liposome, Cytosar-U (cytarabine), cytoxane (cyclophosphamide) Famide), 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, depot Site (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, Eloxatin (oxaliplatin), eltrombopagolamine, Emend (aprepitant), Empliciti (elotuzumab), enacidenib mesylate, enzalutamide, epirubicin hydrochloride, EPOCH, Erbitux (cetuximab), eribulin mesylate, Elivege (bismo Degib, Erlotinib hydrochloride, Erwinaze (Asparaginase from Black Leg Disease), Ethyol (Amiphostin), Etopophos (Etoposide phosphate), Etoposide, Etoposide phosphate, Evacet (Doxorubicin hydrochloride liposome), Everolimus, Evista (Raloxifene hydrochloride), Evomela (Melfa Fluorouracin hydrochloride), exemestane, 5-FU (fluorouracil injection), 5-FU (fluorouracil - topical), Fareston (toremifene), Faridac (panobinostat), Faslodex (fulvestrant), FEC, Femara (letrozole), filgrastim, Fludara (fludarabine phosphate), fludarabine phosphate, Fluoroplex (fluorouracil - topical), fluorouracil injection, fluorouracil - topical, flutamide, Forex (methotrexate), Forex PPS (methotrexate), Forfiri, Forfiri-bevacizumab, Forfiri-cetuximab, Forfirinox, Forfox, 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 2-valent vaccine, recombinant HPV 9-valent 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), ixabepyrone, ixazomib citrate, Ixempra (ixabepyrone), Jakafi (ruxolitinibrine), JEB, Jevtana (cabazitaxel), Kadcyla (Ado-trastuzumab emtansine), Keoxifene (raloxifene hydrochloride), Kepivans (palifermin), Keytruda (pembrolizumab), Kisqali (ribociclib), Kymriah (tisagenlecleucel), Kyprolis (carfilzo Mib), lanreotide acetate, lapatinib nitrate, Lartruvo (olaratumab), lenalidomide, lenvatinib mesylate, Lenvima (lenvatinib mesylate), letrozole, leucovorin calcium, Leukeran (chlorambucil), leuprolide acetate, leustatin (cladribine), Levulan (aminolevulinate), linfollizin (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, mes Mesnex (Mesna), Methazolastone (Temozolomide), Methotrexate, Methotrexate LPF (Methotrexate), Methylnaltrexone bromide, Mexate (Methotrexate), Mexate-AQ (Methotrexate), Midostaurin, Mitomycin C, Mitoxantrone hydrochloride, Mitoditrex (Mitozyt rex) (Mitomycin C), MOPP, Mozovir (Prelixafor), Mustargen (Mechloretamine hydrochloride), Mutamycin (Mitomycin C), Myrelan (Busulfan), Mylosar (Azacitidine), Mylotarg (Gemtuzumab Ozogamicin), Nanoparticle Paclitaxel (Paclitaxel albumin-stabilized nanoparticles) Drugs, 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, Odomzo (Sonidegib), OEPA, Ofatumumab, OFF, Olaparib, Oraratumumab, O Macetaxin mepesuccinate, Oncaspar (peguaspar gauze), ondansetron hydrochloride, Onivyde (irinotecan hydrochloride liposome), Ontak (denileukin difutitox), Opdivo (nivolumab), OPPA, osimertinib, oxaliplatin, paclitaxel, paclitaxel albumin-stabilized nanoparticle formulation, PAD, palbociclib, parifermin, palonosetron hydrochloride, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panobinostat, Paraplat (carboplatin), Paraplatin (carboplatin), pazopanib hydrochloride, PCV, PEB, peguaspar gauze, pegfilgrastim, pegylated interferon alpha-2b, PEG-intron (pegylated interferon Alpha-2b), pembrolizumab, pemetrexed disodium, Perjeta (pertuzumab), pertuzumab, Platinol (cisplatin), Platinol-AQ (cisplatin), prelixafor, pomalidomide, Pomalist (pomalidomide), ponatinib hydrochloride, Portraza (nesitumumab), 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 alpha-2b), Sylvant (siltuximab), Synribo (omacetaxin mepesuccinate), Tabloid (thioguanine), TAC, Tafinlar (dabrafenib), Tagrisso (osimertinib), talc, Tarimozine Laharpa Lepbec, tamoxifen citrate, tarabine (Ta rabine)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 nitrate), Unituxin (dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, Barbit (lorapitant hydrochloride), Vectibix (panitumumab), VeIP, Velban (vinblastine sulfate), Velcade (bortezomi). Velar (vinblastine sulfate), vemurafenib, venetoclax (venetoclax), venetoclax, abemaciclib (avemaciclib), Viadur (leuprolide acetate), azacitidine (azacitidine), vinblastine sulfate, Vincasar PFS (vincristine sulfate), vincristine sulfate, vincristine sulfate liposome, vinorelbine tartrate, VIP, bismodegib, Vistagoard (uridine triacetate), voraxaze (glucarpidase), vo Rinostat, Botrient (pazopanib hydrochloride), 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), Zartrap (Ziv-aflibercept), Zarxio (filgrastim), Zejura ( This may include any anticancer therapies known in the art, including 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.

D. Examples The following examples are provided to the art for the full disclosure and description of the compounds, compositions, articles, apparatus, and/or methods claimed herein and for the evaluation of such compounds, compositions, articles, apparatus, and/or methods, and are intended to be illustrative only and not to limit the disclosure. Although 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.

(Example 1)
Immunostimulatory Activity of Human IFNβ in Mouse Melanoma Models It is important to conduct animal studies using the same drugs that are tested in humans. However, this presents problems for biological agents, for example, in 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 with less capacity 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 function as a potent prophylactic vaccine, and this protective effect depends 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.

(Example 2)
Generating MEM-288 and In vitro Testing We generated oncolytic adenovirus type 5 (Figure 2A) expressing chimeric CD40L driven by the CMV promoter by adding an expression cassette (MEM-288) (Figure 2B) that drives human IFNβ expression. As controls, we used adenoviruses expressing GFP (Ad-GFP) and only chimeric CD40L (MEM-188). All three viruses used contained the E1A D24 deletion, which enables 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 studies 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. OV-induced cell death is due to viral replication in infected cancer cells, which causes cell lysis. Type 1 IFN did not impair adenovirus replication. Notably, in mouse cells, adenovirus replication was not supported, and consistent with this, 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 than to oncolytic effects. Despite lacking oncolytic activity in mouse cells, we refer to such viruses as OVs and discuss their potential applications in humans.

(Example 3)
The primary premise of using the intralesional approach in the mouse melanoma model 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 the growth of both injectable and non-injectable tumors. In the study using a non-oncolytic, non-replicating adenovirus expressing MEM40/CD40L, activity was demonstrated in the 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.

We first conducted trials in B16-OVA melanoma to enable tracking of OVA-responsive CD8 T cells. The typical dose of adenovirus used in mice to determine antitumor therapeutic activity was 10 e9–10 infection units (IU). Low doses of 1 × ^10⁸ 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 10 e9 IU, where MEM-288 injection induced the most significant reduction in tumor growth (Figure 5B). In the above trials, noteworthy activity was shown after four injections with MEM40/CD40L-expressing adenovirus. However, we did not observe similar activity after two injections with MEM-188, and further sustained CD40L expression may be required after multiple viral injections. In addition, while MEM-188 did not induce this, MEM-288 caused an increase in circulating OVA-specific CD8 T cells (Figure 5C).

a) MEM-288 influences systemic T cell response: Roles of CD40 and IFNAR1. In this type of study, we used MEM-288 alone compared to a PBS control. Administration of MEM-288 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 KO mice with B16-OVA tumors. Importantly, the CD8 T cell response was significantly reduced in both IFNAR1 and CD40 KO mice (Figure 5D), demonstrating that both pathways are independently required for MEM-288 activity. By examining the regulation of both APCs and T cells through the study proposed in this application, we can precisely determine how these two pathways function in activating the T cell response.

b) MEM-288 activity by ICI therapy 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 tumor and the contralateral tumor (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, demonstrating that the benefits of ICI are 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 an 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 become apparent in other ICI-resistant tumor models.

(Example 4)
Tests in a Lung Metastasis Model: 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 studies, we will determine the synergistic effect of combining MEM-288 with an ICI (anti-PD-1+/-anti-CTLA4) in antitumor activity in this model.

(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 regulating 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 in 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 previously demonstrated 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 setting of human IFNAR1/2 receptors in mouse cells. Importantly, Harari et al. created a genetically modified mouse line called HyBNAR (Hybrid IFNAR) possessing both a human IFNAR1/2 extracellular ligand-binding domain and a mouse IFNAR1/2 receptor signaling domain. In this study, we showed that human IFNb induces signaling in wild-type mice, and, consistent with our results, this response is enhanced in HyBNAR mice.

The primary objective here is to demonstrate the effects of intralesional ovulation (OV) administration on TME in B16 melanoma and 344 lung tumor models. We define the unique and complementary engagement of CD40L and IFNb receptors with immune cells in TME, and the underlying mechanisms can be investigated using mice lacking CD40 or IFNAR1. We consider DCs to represent the primary target of MEM-288 activity, and for this purpose, we can conduct the most thorough studies to investigate the effects of OV on the functionality of DC1 and DC2 subsets.

a) Effects of OV on TME: Explanation of the specific roles of CD40L and IFNβ The main objective is to determine the mechanisms of action of CD40L and IFNβ in TMEs where Ad-GFP, Ad-IFNβ, MEM-188, and MEM-288 OV are available. Such exploratory studies can be used to evaluate the broad effects of the above OV on major populations of myeloid cells, DCs, T cells, and B cells. As shown in Figure 9A, we readily detected macrophages (F4/80 ⁺ Ly6C ⁺ CD11b ⁺ MHC-II ⁺ ), monocytes and neutrophils (F4/80 ^- CD11b ⁺ Ly6C ⁺ or Ly6G ⁺ ), as well as DCs including DC1 (CD11c ⁺ CD11b ^- MHC-II ⁺ CD103 ⁺ ) and DC2 (CD11c ⁺ CD11b ⁺ MHC-II ⁺ CD103 ^- ) by flow cytometry analysis of B16-OVA tumors. Furthermore, we detected B cells, CD4 ⁺ T cells, CD8 ⁺ T cells, and OVA-specific CD8 T cells (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 ZsGreen-expressing B16-OVA and B16-F10 to track antigen uptake by tumor-resident APCs (Figure 10). As shown in Figure 10, the ZsGreen ⁺ population of macrophages and DCs was readily detected in ZsGreen-expressing tumors compared to parental non-expressing tumors. Using ZsGreen expression, we identified tumor cells (ZsGreen ^{+ CD45-} ), where we were also able to detect CD40L expression 2 days after a single MEM-288 injection (Figures 11A–11B). Importantly, we also found elevated CD80/CD86 levels in DCs and MHC-II ^{hypermacrophages} in the same tumor after MEM-288 injection (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.

b) Effects of OV on TME Here we determine the effects of various OVs on the number and phenotype of tumor-infiltrating CD45 ⁺ immune cells in B16-OVA, B16-FlO, and 344 models. Enumeration of each cell type can be done by determining the percentage of CD45 ⁺ cells 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, as well as 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 differences 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 now determine whether MEM-288 treatment results in 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. Key tests will allow us to determine the expression of MHC-II and the activation markers CD80, CD86, and CD40 in DC1 and DC2. We can also determine the activation state in both ZsGreen ⁺ and ^ZsGreen- cells. In addition, we can determine the therapeutic effect of CCR7 expression in DCs, as CCR7 expression plays a crucial role in DC DLN migration. IL-12 p70 is a key 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-12 p40 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 means. Differences in means (e.g., specific cell type or activation marker expression) between multiple treatment groups can be determined by Tukey's method.

c) Defining the specific roles of CD40L and IFNβ using receptor KO mice. OV mice 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.

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 DLNs, where they can present tumor antigens to naive T cells and trigger their activation. This specification describes how various OVs affect DC migration to DLNs and their ability to stimulate T cells. In such studies, we can track DC1 and DC2 migration to DLNs with 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.

As shown in Figure 13, two main populations of MHC-II ⁺ CD11c ⁺ cells are detected in the inguinal DLN. The ^high- MHC population is thought to consist of migratory DC1 and DC2 cells, which, as expected, have a high proportion of CD103 ⁺ DCs and CD11b ⁺ DCs. The CD11c ⁺ population with a moderate level of MHC-II ( ^medium- MHC) consists of resident DC1 and DC2 cells and has a low proportion of CD103 ⁺ DCs. The majority of ZsGreen+ DCs are located in the ^high -MHC population. Here, we can determine the distribution of these distinct populations and the effects of various OVs on their activation phenotypes. MEM-288 administration may result in the highest DLN migration, and these DCs may have the most potent activation 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 is not thought 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 tumors to DLNs.

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. Then, 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. Then, 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.

(Example 6)
Oncolytic virus therapy for tumors
To demonstrate the in vivo effects of oncolytic viruses on tumors, 5e5 B16-OVA cells were inoculated sc onto the flanks of C57BL/6 mice. Tumors were treated with PBS, adenovirus GFP, or MEM-288 injections at ^10⁹ IU on days 12 and 16. On day 20, tumors were obtained and IHC was performed to detect the presence of CD8 ⁺ tumor-infiltrating lymphocytes (TILs). Mice treated with PBS or GFP-expressing oncolytic viruses showed comparable numbers of TILs. In contrast, TIL levels in MEM-288-vaccinated tumors were nearly threefold higher (Figure 15).

E. References [References]

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

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

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

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

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

Claims (3)

A composition for use in the treatment of cancer, comprising tumor-infiltrating lymphocytes (TILs), wherein TILs are
a . A step of administering the oncolytic virus adenovirus type 5 into tumor cells in the target ,
b. Obtained by a method including a step of recovering TIL ,
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 . A composition for use in the treatment of cancer in a subject, comprising an oncolytic virus, the treatment being:
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 of growing the recovered TI L ex vivo,
d. The step of administering the proliferated TI L to the target ,
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 . The composition according to claim 2 , which is administered in combination with an anticancer agent .