CN116710564A - Oncolytic viruses enhance T cell response to effective TIL therapy - Google Patents

Abstract

Methods of amplifying a tumor-infiltrating lymphocyte (TIL) population and methods of treating cancer using the amplified TIL population are disclosed.

Description

Oncolytic viruses enhance T cell response to effective TIL therapy

Technical Field

The present application claims the benefit of U.S. provisional application No. 63/106,215, the entire contents of which are incorporated herein by reference.

Background

Recent advances in cancer immunotherapy have drastically altered the treatment pattern. However, many cancer patients benefit little or no from available treatments, including Immune Checkpoint Inhibitors (ICI). Numerous clinical studies have shown that pre-existing tumor-reactive T cells are critical to achieving the benefit from checkpoint inhibitors. Thus, new methods to increase the number and function of tumor-reactive T cells are needed.

Disclosure of Invention

Methods and compositions related to expanding a tumor-infiltrating lymphocyte (TIL) population and using the TIL population to treat cancer are disclosed.

In one aspect, disclosed herein are methods of producing tumor-infiltrating lymphocytes comprising a) administering to a tumor cell an effective amount of an oncolytic virus that expresses one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF 6); and b) collecting tumor-infiltrating lymphocytes. In some aspects, the oncolytic virus may also 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). In some aspects, the TIL produced is obtained in a tumor microenvironment in which the oncolytic viral site is administered; however, TIL can also be obtained in a tumor microenvironment that is not infected with the oncolytic virus.

Also disclosed herein are methods of producing tumor-infiltrating lymphocytes of any of the preceding aspects, wherein the oncolytic virus is administered by intratumoral injection.

Also disclosed herein are methods of producing tumor-infiltrating lymphocytes of any of the preceding aspects, further comprising ex vivo expanding the harvested TILs.

In one aspect, disclosed herein is a method of expanding a tumor-infiltrating lymphocyte (TIL) population or a bone marrow-infiltrating lymphocyte (MILs) population, comprising: a) Collecting TIL or MILs from a subject having cancer; b) The harvested TIL or MIL is cultured in the presence of antigen presenting cells infected with an oncolytic virus that expresses one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF 6). In some aspects, the oncolytic virus may also 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).

Also disclosed herein are methods of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing cancer and/or metastasis in a subject comprising administering to the subject a therapeutically effective amount of the TIL or MILs of any of the preceding aspects. For example, disclosed herein are methods of treating, alleviating, inhibiting, reducing, ameliorating, and/or preventing cancer and/or metastasis in a subject comprising: a) Administering an effective amount of an oncolytic virus expressing one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF 6) into a tumor cell; b) Collecting tumor-infiltrating lymphocytes (TIL) and/or bone marrow-infiltrating lymphocytes (MILs); c) Amplifying the collected TIL and/or MIL ex vivo; and d) administering a therapeutically effective amount of the amplified TIL and/or MIL to the subject. In some aspects, the cancerous tumor or metastatic tumor treated, reduced, inhibited, reduced, ameliorated, and/or prevented is distant to a tumor that receives any oncolytic virus and/or TIL or MILs disclosed herein. In some aspects, the oncolytic virus may also 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).

Further, disclosed herein are methods of treating, alleviating, inhibiting, reducing, ameliorating, and/or preventing cancer and/or metastasis in a subject comprising a) harvesting tumor-infiltrating lymphocytes (TILs) and/or bone marrow-infiltrating lymphocytes (MILs) from a subject having cancer; culturing the harvested TIL or MILs in the presence of antigen presenting cells infected with an oncolytic virus expressing one or more exogenous immunostimulatory molecules; and administering a therapeutically effective amount of the amplified TIL and/or MILs to the subject. In some aspects, the oncolytic virus may also 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).

In one aspect, disclosed herein are methods of treating, alleviating, inhibiting, reducing, ameliorating, and/or preventing cancer and/or metastasis of any of the foregoing aspects, further comprising administering an anti-cancer agent to a subject.

Drawings

The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate various embodiments and, together with the description, explain the disclosed compositions and methods.

FIG. 1A shows B16-OVA cells were transduced with pLenti-Puro control lentiviruses or lentiviruses expressing mouse or human IFN beta, followed by puromycin selection. The same number of cells were plated, and after 2 days the supernatant was collected for ELISA to detect human and mouse IFNbeta. n=6, statistical analysis was performed using t-test of growth curve two-way ANOVA or OVA tetramer assessment. Statistical significance is expressed by p values as p <0.05, p <0.01, p <0.001.NS: is not significant.

FIG. 1B shows that wild-type or IFNAR1 KO (IFNAR) C57BL/6 mice received two rounds of B16-OVA-pLenti, B16-OVA-mouse IFNbeta or B16-OVA-human IFNP vaccine on day 0 and day 7 (100 Gy radiation was received on the cells prior to injection). Day 12, shown in MHCII in peripheral blood ^- And CD8 ⁺ OVA tetramers in cells ⁺ Is a percentage of (c). n=6, statistical analysis was performed using t-test of growth curve two-way ANOVA or OVA tetramer assessment. Statistical significance is represented by p-value as p<0.05，**p<0.01，***p<0.001.NS: is not significant.

FIG. 1C is the same as in (A) except that mice were challenged with 3e5 live B16-OVA cells on day 21. Tumor growth was determined as shown. n=6, statistical analysis was performed using t-test of growth curve two-way ANOVA or OVA tetramer assessment. Statistical significance is expressed by p values as p <0.05, p <0.01, p <0.001.NS: is not significant.

Fig. 2A is a schematic diagram of MEM 40.

FIG. 2B is a schematic diagram of MEM-288.

Figures 3A, 3B, 3C and 3D show that (3A) a549 cells or (3C) B16-F10 cells were infected with control GFP virus or oncolytic Ad-MEM-188 or 288, as shown at moi=250, for 2 days. Expression of GFP and MEM40 was determined by flow cytometry. After infection with the indicated OV in (3B) A549 cells or (3D) B16-F10 cells, IFN beta secretion was determined by ELISA.

FIGS. 4A, 4B and 4C show (4A) 344SQ (344), (4B) B16-F10 mouse cell line and (4C) A549 human cell line infected with designated OV AD-GFP (GFP), MEM-188 (188) or MEM-288 (288) at different MOI (1, 10, 100) for 2 days. Cell viability was determined by trypan blue staining assay 2 days after infection.

FIGS. 5A and 5B show the subcutaneous inoculation of 5e 5B 16-OVA cells from C57BL/6 mice. On day 12 and 16, these mice received two intratumoral injections of Ad-GFP, MEM-188 and MEM-288 (5A) 10e8 or (5B) 10e9 IU. The significance of the tumor size differences was shown compared to untreated control mice at the last time point.

FIG. 5C shows the percentage of OVA-specific MHCII-and CD8+ cells in peripheral blood at day 12 after intratumoral injection of l0e9 IU of MEM-188 and MEM-288 in B16-OVA. Statistical analysis was performed using t-test with two-way ANOVA or OVAT assessment of growth curves. Statistical significance is represented by p-value as p<0.05、**p<0.01、***p<0.001.NS: is not significant. FIG. 5D shows quantification of ELISPOT results for IFNγ from spleen CD 8T cells. C57BL/6WT, IFNAR1 KO and CD40 KO mice (3 per group) were inoculated subcutaneously with 5e 5B 16-OVA cells on the flank and injected with PBS (UT: untreated) or 10e9 IU MEM-288 on day 12 and day 16. The spleen of the mice was subjected to magnetic bead isolation of CD 8T cells according to the manufacturer's recommendations. Subsequently, 2×10 ⁵ CD8 cells and 1X 10 cells per well ⁵ The 50Gy irradiated B16-OVA cells per well were plated into triplicate wells and incubated in 96-well plates for 24 hours at 37 ℃. Stimulation of tumor cells with ifnγ increases MHC expression. T cells were also cultured alone or with concanavalin a (ConA) as negative and positive controls, respectively. The results are shown relative to ConA treatment for each group. Statistical analysis was performed using t-test. Statistical significance is represented by p-value as p<0.05、**p<0.01、***p<0.001.NS: is not significant.

Fig. 6A shows the treatment regimen for mice: c57BL/6 mice were inoculated subcutaneously with 5e 5B 16-F10 cells at the primary site and 2.5e 5B 16-F10 cells at the contralateral site. As shown, these mice were injected with 10e9 IU of MEM-288 to the primary tumor on day 12 and day 16, and anti-PD-1 and CTLA-4 antibodies to the primary tumor on day 16, day 19, day 23, and day 27.

FIGS. 6B and 6C show that tumor growth is determined at the primary site (6B) and the contralateral site (6C) as shown. Statistical analysis was performed using two-way ANOVA of the growth curve. Statistical significance is expressed by p values as p <0.05, p <0.01, p <0.001.NS: is not significant.

Fig. 6D shows Kaplan-Meier survival analysis showing overall survival of mice. Statistical significance is expressed by p values as p <0.05, p <0.01, p <0.001.NS: is not significant.

FIG. 7A shows that 129 mice were subcutaneously inoculated with 5e5 344 cells on the flank and 10e9 IU of Ad-GFP, MEM-188 or 288 was injected into the tumor on day 12 and day 16. Tumor growth was determined at the primary site as shown. Showing significance compared to the control UT group (PBS injection). Statistical significance is expressed by p values as p <0.05, p <0.01, p <0.001.NS: is not significant.

Figure 7B shows a typical H & E staining of lung tumors of tumor-bearing mice injected subcutaneously 344 in (7A) on day 38.

Figure 7C shows the quantification of tumors from individual mice from different groups, as shown. Statistical significance is expressed or represented by p values as p <0.05, < p <0.01, < p <0.001.NS: is not significant.

FIG. 7D shows a typical IHC staining of mouse lung CD 8T cells in (7A).

Figure 7E shows quantification of CD 8T cell density from individual mice from different groups, as shown. Statistical significance is expressed or represented by p values as p <0.05, < p <0.01, < p <0.001.NS: is not significant.

Figures 8A and 8B show that 129 mice injected subcutaneously 5e5 344 cells on the flank and 10e9 IU of MEM-288 on day 12 and day 16 and anti-PD-1 antibodies on day 16, day 19, day 23 and day 27 intraperitoneally. Fig. 8A shows ifnγ ELISPOT from spleen CD 8T cells of mice, and fig. 8B shows quantification of ELISPOT results from (a). Statistical analysis was performed using t-test. Statistical significance is expressed by p values as p <0.05, p <0.01, p <0.001.NS: is not significant.

FIG. 9A shows a flow cytometry analysis protocol of B16-OVA to detect the indicated macrophage, neutrophil, monocyte and DC populations.

FIG. 9B shows a flow cytometry analysis protocol of B16-OVA to detect populations of B cells, CD 4T cells, CD 8T cells, and OVA-specific CD 8T cells.

FIG. 10 shows that C57BL/6 mice were subcutaneously injected on the flank with 5e 5B 16-OVA or B16-OVA-ZsGreen cells. On day 14, tumor B16-OVA was analyzed by flow cytometry to detect ZsGreen positive populations of macrophages and DCs as identified in FIG. 9A. The percentage of positive cells is shown.

FIG. 11A shows that C57BL/6 mice were inoculated with 5e 5B 16-OVA or B16-OVA-ZsGreen cells subcutaneously on the flank. On day 14, flow cytometry analysis was performed to detect CD45 as indicated ^- ZsGreen in (A) ⁺ Tumor cells.

FIG. 11B shows that B16-OVA-ZsGreen tumors were injected into tumors on day 12 with 10e9 IU of PBS or MEM-288. ZsGreen from PBS and MEM-288 injected tumors ⁺ CD45 ^- Cells were analyzed by flow cytometry for CD 40L.

FIG. 12A shows MHC-II progression of the tumor of FIG. 11b ^{High height} Macrophages and DC (CD 11 b) ⁺ Flow cytometry analysis of CD80 and CD86 expression in DC 2). Injection of PBS and MEM-288 in tumors is shown.

Fig. 12B shows the Mean Fluorescence Intensity (MFI) of the result (a).

Fig. 13 shows DC populations in inguinal LNs. CD11c was detected ⁺ MHC-II ⁺ Two major populations of DCs. The MHC-II high population comprises migratory DC1 and migratory DC2. The MHC-II intermediate population comprises colonising DC1 and colonising DC2. As expected, mobility groupsThe body has a higher proportion of CD103 ⁺ DC1, while the colonising population has a higher proportion of CD8a ⁺ DC1。

FIG. 14 shows DC populations in LN of WT, CD40 KO and IFNAR1 KO mice. CD11c ⁺ MHC-II ⁺ Two major populations of DCs were detected in all genotype mice. The MHC-II high population comprises migratory DC1 and migratory DC2. The MHC-II intermediate population comprises colonising DC1 and colonising DC2.

FIGS. 15A and 15B show that C57BL/6 mice were inoculated with 5e 5B 16-OVA cells subcutaneously on the flank. On days 12 and 16, tumors received injections of PBS (n=4), adenovirus-GFP (n=3) or MEM-288 (n=4) of 10e9 IU. On day 20, tumors were obtained for IHC to detect CD8 ⁺ The presence of Tumor Infiltrating Lymphocytes (TILs). CD 8T cell staining was scored on a scale of 0-3 by a pathologist blinded to each group. Fig. 15A shows representative tumor images of 3 treatment groups. Fig. 15B shows the scoring values of the 3 treatment groups.

Detailed Description

Before the present compounds, compositions, articles, devices, and/or methods are disclosed and described, it is to be understood that they are not limited to specific synthetic methods or specific recombinant biotechnology methods, or specific reagents, unless otherwise indicated, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting.

A. Definition of the definition

As used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a pharmaceutical carrier" includes mixtures of two or more such carriers, and the like.

Ranges may be expressed herein as from "about" one particular value, and/or to "about" another particular value. When such a range is expressed, another embodiment includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent "about," it will be understood that the particular value forms another embodiment. It will also be understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. It will also be understood that there are many values disclosed herein, and that each value is also disclosed herein as a particular value other than the "about" value itself. For example, if the disclosed value is "10," then "about 10" is also disclosed. It will also be understood that when a value is disclosed, "less than or equal to" the value, "greater than or equal to the value," and possible ranges between values are also disclosed, as would be well understood by one of ordinary skill in the art. For example, if the value "10" is disclosed, then "less than or equal to 10" and "greater than or equal to 10" are also disclosed. It should also be understood that throughout this application, data is provided in a variety of different formats, and that this data represents endpoints and starting points, and ranges for any combination of the data points. For example, if a particular data point "10" and a particular data point 15 are disclosed, it is understood that greater than, greater than or equal to, less than or equal to, and equal to 10 and 15, and 10-15 are considered disclosed. It should also be understood that each unit between two particular units is also disclosed. For example, if 10 and 15 are disclosed, 11, 12, 13 and 14 are also disclosed.

In this specification and in the claims that follow, reference will be made to a number of terms, which shall be defined to have the following meanings:

"optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where the event or circumstance occurs and instances where it does not.

As used herein, the term "antigen presenting cell" (APC) refers to a professional antigen presenting cell selected from the group consisting of dendritic cells, macrophages and B cells. In some embodiments, the APC is 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

An "increase" may refer to any change in the amount that results in a greater amount of symptoms, disease, composition, condition, or activity. The increase may be any individual, median or average increase in condition, symptom, activity, composition in a statistically significant amount. Thus, the increase may 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 increase is statistically significant.

"reduced" may refer to any change in the amount that results in less symptoms, disease, composition, condition, or activity. When the genetic output of a gene product having the substance is small relative to the output of a gene product without the substance, the substance is also understood to reduce the genetic output of the gene. Also, for example, the decrease may be a change in symptoms of the disorder, making the symptoms less than previously observed. The decrease may be any individual, median or average decrease in condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease may be a decrease of 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or 100% as long as the decrease is statistically significant.

"Inhibit", "Inhibit" and "inhibition" refer to a decrease in activity, response, condition, disease or other biological parameter. This may include, but is not limited to, complete elimination of activity, response, condition or disease. This may also include, for example, a 10% reduction in activity, response, condition or disease compared to a natural or control level. Thus, the decrease may be 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% or any decrease in between, as compared to a natural or control level.

"reduction" or other forms of the word, such as "reduce" or "reduction", refer to a decrease in an event or feature (e.g., tumor growth). It will be appreciated that this is typically associated with certain standards or expected values, in other words, it is relative, but it is not always necessary to refer to a standard or relative value. For example, "reducing tumor growth" refers to reducing the growth rate of a tumor relative to a standard or control.

"prevention" or other forms of words, such as "prevention" or "prevention," refer to the cessation of a particular event or feature to stabilize or delay the development or progression of the particular event or feature, or to minimize the chance of the particular event or feature occurring. Prevention does not need to be compared to control, as it is generally more absolute than, for example, reduction, etc. As used herein, something may be reduced but cannot be prevented, but reduction of something may also be prevented. Also, something can be prevented but cannot be reduced, but something that is prevented can also be reduced. It is to be understood that where the use is reduced or prevented, the use of other words is also expressly disclosed unless specifically stated otherwise.

The term "subject" refers to any individual to whom the target of administration or treatment is directed. The subject may be a vertebrate, for example a mammal. In one aspect, the subject may be a human, non-human primate, bovine, equine, porcine, canine, or feline. Thus, the subject may also be guinea pig, rat, hamster, rabbit, mouse or mole. Thus, the subject may be a human or veterinary patient. The term "patient" refers to a subject under treatment by a clinician (e.g., doctor).

The term "therapeutically effective amount" refers to an amount of the composition used sufficient to ameliorate one or more causes or symptoms of a disease or disorder. This improvement need only be reduced or altered and need not necessarily be eliminated.

The term "treatment" refers to the medical management of a patient with the aim of curing, ameliorating, stabilizing or preventing a disease, pathological condition or disorder. The term encompasses active treatments, i.e. treatments directed specifically to amelioration of a disease, pathological condition or disorder, as well as causal treatments, i.e. treatments directed specifically to elimination of the etiology of the associated disease, pathological condition or disorder. Furthermore, the term includes palliative treatment, i.e. treatment intended to alleviate symptoms rather than cure a disease, pathological condition or disorder; prophylactic treatment, i.e., treatment intended to minimize or partially or completely inhibit the development of a related disease, pathological condition, or disorder; and supportive treatment, i.e., treatment for supplementing another specific therapy aimed at ameliorating a related disease, pathological condition, or disorder.

"biocompatible" generally refers to materials and any metabolites or degradation products thereof that are generally non-toxic to the recipient and do not significantly adversely affect the subject.

"comprising" is intended to mean including the composition, method, etc. of the recited elements, but not excluding other elements. When used to define compositions and methods, "consisting essentially of … …" is meant to include the recited elements, but not other elements that are of significance to the combination. Thus, a composition consisting essentially of the elements defined herein does not exclude minor contaminants such as phosphate buffered saline, preservatives, and the like, both in isolation and purification methods, as well as in pharmaceutically acceptable carriers. "consisting of … …" means the substantial process steps excluding other ingredients beyond trace elements and for the administration of the compositions provided and/or claimed in the present disclosure. Embodiments are defined by each of these transitional terms within the scope of this disclosure.

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

An "effective amount" of an agent refers to an amount of the agent sufficient to provide the desired effect. The amount of an agent that is "effective" will vary from subject to subject, depending on a number of factors, such as the age and general condition of the subject, the particular agent or agents, and the like. Therefore, it is not always possible to specify an "effective amount" of an amount. However, a suitable "effective amount" in any subject case can be determined by one of ordinary skill in the art using routine experimentation. Furthermore, as used herein, unless specifically stated otherwise, an "effective amount" of an agent may also be meant to encompass both a therapeutically effective amount and a prophylactically effective amount. The "effective amount" of the agent necessary to achieve a therapeutic effect may vary depending on factors such as the age, sex, and weight of the subject. Dosage regimens may be adjusted to provide the optimum therapeutic response. For example, multiple divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the emergency state of the treatment situation.

A "pharmaceutically acceptable" component may refer to a component that is not biologically or otherwise undesirable, i.e., the component may be incorporated into a pharmaceutical formulation provided by the present disclosure and administered to a subject as described herein without causing a significant undesirable biological effect or interacting in a deleterious manner with any of the other components of the formulation in which it is contained. When used in reference to administration to humans, the term generally means that the ingredient has reached the required criteria for toxicology and manufacturing trials, or that it is contained in an inactive ingredient guide established by the U.S. food and drug administration (Food and Drug Administration).

"pharmaceutically acceptable carrier" (sometimes referred to as "carrier") refers to a carrier or excipient used in the preparation of generally safe and nontoxic pharmaceutical or therapeutic compositions, including veterinary and/or human pharmaceutical or therapeutic acceptable carriers. The term "carrier" or "pharmaceutically acceptable carrier" may include, but is not limited to, various types of phosphate buffered saline solutions, water, emulsions (e.g., oil/water or water/oil emulsions), and/or 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 material known in the art for use in pharmaceutical formulations and as further described herein.

"pharmacological activity" (or simply "activity"), as in a "pharmacologically active" derivative or analog, may refer to a derivative or analog (e.g., salt, ester, amide, conjugate, metabolite, isomer, fragment, etc.) that has the same type of pharmacological activity as the parent compound and is substantially equivalent in extent.

"therapeutic agent" refers to any composition having a beneficial biological effect. Beneficial biological effects include therapeutic effects (e.g., treating a disorder or other undesirable physiological condition) and prophylactic effects (e.g., preventing a disorder or other undesirable physiological condition (e.g., non-immunogenic cancer)). The term also includes pharmaceutically acceptable, pharmacologically active derivatives of the beneficial agents specifically mentioned herein, including but not limited to salts, esters, amides, precursors, active metabolites, isomers, fragments, analogs, and the like. When the term "therapeutic agent" is used, or when a particular agent is specifically identified, it is understood that the term includes the agent itself as well as pharmaceutically acceptable, pharmacologically active salts, esters, amides, precursors, conjugates, active metabolites, isomers, fragments, analogs, and the like.

A "therapeutically effective amount" or "therapeutically effective dose" of a composition (e.g., a composition comprising a pharmaceutical agent) refers to an amount effective to achieve a desired therapeutic result. In some embodiments, the desired therapeutic result is the management of type I diabetes. In some embodiments, the desired therapeutic result is the management of obesity. The therapeutically effective amount of a given therapeutic agent will generally vary with factors such as the type and severity of the disorder or disease being treated and the age, sex and weight of the subject. The term may also refer to an amount of a therapeutic agent or a rate of delivery of a therapeutic agent (e.g., an amount that varies over time) that is effective to promote a desired therapeutic effect, such as pain relief. The exact desired therapeutic effect will vary depending on the condition to be treated, the tolerance of the subject, the agent and/or agent formulation to be administered (e.g., the efficacy of the therapeutic agent, the concentration of the agent in the formulation, etc.), and various other factors understood by one of ordinary skill in the art. In some cases, the desired biological or medical response is achieved after administration of multiple doses of the composition to the subject over a period of days, weeks, or years.

Throughout this application, various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which they pertain. The disclosed references are also incorporated herein by reference, individually and specifically, where the materials contained therein are dependent upon being discussed in the sentences of the references.

B. Composition and method for producing the same

Disclosed are ingredients for preparing the disclosed compositions, as well as the compositions themselves for use in the methods disclosed herein. These and other materials are disclosed herein, and it is understood that when combinations, subsets, interactions, groups, etc. of these materials are disclosed that while specific reference of each various individual and collective combinations and permutation of these compounds may not be explicitly disclosed, each is specifically contemplated and described herein. For example, if a particular type 1 IFN and immunostimulant comprising an oncolytic virus are disclosed and discussed, and a number of modifications that can be made to a number of molecules comprising a type 1 IFN and an immunostimulant comprising an oncolytic virus are discussed, each and every combination and permutation of a type 1 IFN and an immunostimulant comprising an oncolytic virus, and potentially large modifications, are specifically contemplated unless specifically indicated to the contrary. Thus, if the classes of molecules A, B and C and the classes of molecules D, E and F and examples of combined molecules are disclosed, A-D are disclosed, then each individually and collectively are contemplated to mean a combination, even though each is not individually recited, and A-E, A-F, B-D, B-E, B-F, C-D, C-E and C-F are considered disclosed. Also, any subset or combination of these is disclosed. Thus, for example, the A-E, B-F and C-E subsets would be considered public. This concept applies to all aspects of the present application including, but not limited to, steps in methods of making and using the disclosed compositions. Thus, if there are a plurality of additional steps that can be performed, it should be understood that each of these additional steps can be performed with any particular embodiment or combination of embodiments of the disclosed methods.

Binding of CD40 expressed on DCs by CD40L ligand results in the acquisition of a critical cross-priming function to activate CD 8T cells. Thus, the therapeutic use of CD40 agonists has the potential to trigger powerful anti-tumor T cell immunity. Recent clinical studies have shown that the combination of CD40 agonistic antibodies delivered systemically with ICI and chemotherapy has a good sensitization response, but this is also associated with significant toxicity. In addition to the role of type 1 IFNs in enhancing tumor immunogenicity by increasing expression of immune function genes in a variety of cell types, type 1 IFNs can act as direct activators of Dendritic Cell (DC) and T cell functions. This key immunostimulatory function of type 1 IFN has recently been exploited through the use of stimulators of type 1 IFN expression (e.g., STING and TLR9 agonists). There is a synergistic relationship between CD40 ligation and stimulators of type I IFN for robust CD 8T cell activation. However, the combined systemic administration of CD40 agonists and type 1 IFN activators is highly toxic.

In the intralesional immunotherapy approach, the tumor acts as a vaccine site, resulting in DC activation and subsequent T cell stimulation to generate systemic anti-tumor immunity capable of controlling the growth of distant untreated tumors. This approach aimed at avoiding systemic toxicity has recently been tested with STING and TLR9 agonists. It is shown herein that combined intralesional activation of CD40 and type 1 IFN signaling results in robust immune activation in the Tumor Microenvironment (TME), resulting in high levels of increase in systemic T cell responses.

Oncolytic Viruses (OVs) have been developed for their ability to replicate specifically in cancer cells. Recent studies have shown that stimulation of host anti-tumor immunity is critical to the mechanism of action of OV. OV also allows the ability to encode transgenes that can be used to express powerful immune response activators to further enhance anti-tumor immunity. It is shown herein that intravesically delivered OVs capable of activating type 1 IFN signaling in immunostimulatory molecules (e.g., such as CD 40) and/or TMEs can act as potent activators of systemic T cell responses. To test this, a conditionally replicating adenovirus type 5 that expresses chimeric CD40L ligand (MEM 40) and IFN beta was developed in concert with the company Memgen. We further demonstrated that MEM-288 induced high levels of CD40L and ifnβ expression and induced a robust systemic T cell response that was able to significantly reduce (curtaining) distant tumor growth in murine melanoma and lung tumor models.

In one aspect, disclosed herein are methods of producing tumor-infiltrating lymphocytes comprising a) administering an oncolytic virus expressing one or more type 1 Interferons (IFNs) (e.g., IIFN- α, IFN- β, IFN- κ, IFN- δ, IFN- ε, IFN- τ, IFN- ω, and/or IFN- ζ) and/or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF 6) into a tumor cell; b) Tumor infiltrating lymphocytes are collected. It is understood and contemplated herein that oncolytic viruses may be administered to a tumor by any means known in the art, including but not limited to intratumoral injection. While the above methods result in a significant increase in the number of TILs and MILs at the tumor site, it is also recognized that TILs and MILs can be produced and/or increased at tumor sites outside of the treated tumor microenvironment (i.e., distant effects).

Simply because the amount of TIL and MILs increases at the target site and/or distant tumor site of oncolytic virus administration does not mean that no further amplification occurs. In one aspect, it is understood and contemplated herein that the TIL in MILs may be further amplified if cultured ex vivo. Thus, also disclosed herein are methods of producing tumor-infiltrating lymphocytes, further comprising ex vivo expansion of the harvested TILs.

In one aspect, it is to be understood and contemplated herein that expansion of TIL and MILs is not necessarily limited to oncolytic viral methods in vivo, wherein tumors are infected with oncolytic viruses and harvested, but expansion of TIL can occur in vitro by harvesting TIL and MILs at a cancer site, and then culturing the harvested cells in the presence of oncolytic virus-infected antigen presenting cells. Thus, amplification of TIL and/or MILs occurs entirely in vitro. Accordingly, disclosed herein are methods of expanding a tumor-infiltrating lymphocyte (TIL) population or a bone marrow-infiltrating lymphocyte (MILs) population, comprising: a) Collecting TIL or MILs from a subject having cancer; b) Culturing the harvested TIL or MIL in the presence of antigen presenting cells infected with an oncolytic virus that expresses one or more type 1 Interferons (IFNs) (e.g., IIFN-alpha, IFN-beta, IFN-kappa, IFN-delta, IFN-epsilon, IFN-tau, IFN-omega, and/or IFN-zeta) and/or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF 6).

In certain embodiments, the invention provides a viral vector, such as a lentivirus (lentivirus), that expresses one or more type 1 Interferons (IFNs) (e.g., IIFN- α, IFN- β, IFN- κ, IFN- δ, IFN- ε, IFN- τ, IFN- ω, and/or IFN- ζ) and/or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, cluster of Differentiation (CD) 80, and/or CD86 (also referred to as B7-1 (CD 80) and B7-2 (CD 86)), OX40L, 4-1BB ligand (4-1 BBL), CD70, LIGHT, glucocorticoid-induced TNFR-related protein ligand (GITRL), LIGHT, T cell immunoglobulin, and mucin domain 4 (TIM-4), intracellular adhesion molecule-1 (ICAM-1), CD58, and/or Signaling Lymphocyte Activating Molecule (SLAM) family member 6 (SLAM 6)), or any combination thereof.

Other embodiments of the invention provide oncolytic viruses that express type I IFNs (e.g., IFN- α, IFN- β, IFN- ε, IFN- κ, and IFN- ω) as well as any combination of immunostimulatory molecules (e.g., CD40-L, MEM, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF 6), e.g., by one or more heterologous nucleic acid sequences encoding a combination of IFN- β and CD 40-L. Any oncolytic virus may be used. In some embodiments, the oncolytic virus may be an adenovirus, a respiratory enterovirus, a herpes virus, a picornavirus (including coxsackie virus, poliovirus, and saint card virus (Seneca Valley virus)), a paramyxovirus (including measles virus and Newcastle Disease Virus (NDV)), a parvovirus, a rhabdovirus (including Vesicular Stomatitis Virus (VSV)), or a vaccinia virus. Preferably, the oncolytic virus is replication competent.

Other embodiments of the invention provide methods of treating a malignancy in a subject by administering an oncolytic virus as disclosed herein to the subject in combination with administering a checkpoint inhibitor to the subject.

IFN- α can be represented by the sequence of SEQ ID NO:1, and the sequence of the polypeptide is expressed as human IFN-alpha. The IFN- α may be mammalian IFN- α, such as mouse, rat, rabbit, pig, feline, canine, or bovine IFN- α. Other embodiments of IFN- α from other mammals are known to the skilled artisan and such embodiments are within the scope of the invention. In certain embodiments, IFN- α has 80.00% to up to 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%) identity with human wild-type IFN- α (e.g., IFN- α of SEQ ID NO: 1).

IFN- β may be represented by the sequence of SEQ ID NO:2, and 2. The IFN- β may be a mammalian IFN- β, such as a mouse, rat, rabbit, pig, feline, canine, or bovine IFN- β. Other embodiments of IFN- β from other mammals are known to the skilled artisan and such embodiments are within the scope of the invention. In certain embodiments, IFN- β has 80.00% to up to 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%) sequence identity with a human wild-type IFN- β (e.g., IFN- β of SEQ ID NO: 2). In particular embodiments, IFN- β lacks the sequence of SEQ ID NO:2, and optionally further having 17 cysteine substitutions to obtain a 165 amino acid peptide having serine.

IFN-. Epsilon.may be represented by the sequence of SEQ ID NO:3, and 3. IFN- ε may be mammalian IFN- ε, e.g., mouse, rat, rabbit, pig, feline, canine, or bovine IFN- ε. Other embodiments of IFN- ε from other mammals are known to the skilled artisan and such embodiments are within the scope of the invention. In certain embodiments, IFN- ε has 80.00% to at most 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%) sequence identity with human wild-type IFN- ε (e.g., IFN- ε of SEQ ID NO: 3).

IFN-. Kappa.can be expressed by the sequence of SEQ ID NO:4, and the sequence of 4. IFN-kappa may be mammalian IFN-kappa, e.g., mouse, rat, rabbit, pig, feline, canine, or bovine IFN-kappa. Other embodiments of IFN- κ from other mammals are known to the skilled artisan and such embodiments are within the scope of the invention. In certain embodiments, IFN-kappa has 80.00% to up to 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%) sequence identity to human wild-type IFN-kappa (e.g., IFN-kappa of SEQ ID NO: 4).

IFN- ω can be represented by the sequence of SEQ ID NO:5, and the sequence of the sequence is expressed as human IFN-omega. IFN- ω can be mammalian IFN- ω, e.g., mouse, rat, rabbit, pig, feline, canine, or bovine IFN- ω. Other embodiments of IFN- ω from other mammals are known to the skilled artisan and such embodiments are within the scope of the invention. In certain embodiments, IFN- ω has 80.00% to up to 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%) sequence identity with human wild-type IFN- ω (e.g., IFN- ω of SEQ ID NO: 5).

CD40-L may be represented by SEQ ID NO:6, and the sequence of human CD40-L. The CD40-L may be mammalian CD40-L, such as mouse, rat, rabbit, pig, feline, canine, or bovine CD40-L. Other embodiments of CD40-L from other mammals are known to the skilled artisan and such embodiments are within the scope of the invention. In certain embodiments, CD40-L has 80.00% to up to 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%) sequence identity with human wild-type CD40-L (e.g., CD40-L of SEQ ID NO: 6).

CD40-L may be a chimeric CD40-L or a non-chimeric CD40-L polypeptide. In some embodiments, the CD40-L expressed in an oncolytic virus of the invention is chimeric CD40-L. Such chimeric CD40-L polypeptides comprise CD40-L domains or subdomains from at least two different species, such as human and mouse CD40-L. Chimeric CD40-L may provide a higher stimulation of immune responses than naturally occurring CD40-L. Examples of chimeric CD40-L suitable for use in the present invention are disclosed in U.S. Pat. Nos. 7,495,090, 7,928,213 and 8,138,310. Each of these patents is incorporated by reference herein in its entirety.

In certain embodiments of chimeric CD40-L, at least one domain or subdomain of CD40-L comprises a cleavage site for human CD40-L and is substituted with a corresponding domain or subdomain of non-human CD40-L, preferably murine CD40-L. In addition, chimeric CD40-L may comprise a domain or subdomain of human CD40-L that binds to the CD40-L receptor. Domains I to IV of human CD40-L (SEQ ID NO: 6) correspond to SEQ ID NO: amino acid portions 1-14, 14-45, 46-110 and 111-261 of 6. Based on sequence alignment of non-human CD40-L with human CD40-L, the skilled artisan can determine domains I through IV of non-human CD40-L. Certain domain positions for non-human CD40-L are provided in Table 1 of U.S. Pat. No. 7,495,090, which is incorporated herein by reference in its entirety.

In some embodiments, chimeric CD40-L comprises a first subdomain of non-human CD40-L, wherein the subdomain replaces the 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 comprise a subdomain of domain IV of non-human CD40-L. Furthermore, the first subdomain may also comprise domain III of non-human CD40-L, or subdomain or domain III. In certain embodiments, the first subdomain replaces a portion of the cleavage site of human CD40-L. In a further embodiment, the chimeric CD40-L comprises a domain II or a subdomain of domain II of a non-human CD40-L in addition to domain IV or a subdomain of domain IV, and optionally domain III or a subdomain of domain III. In addition, the first subdomain of chimeric CD40-L may comprise domain I or a subdomain of domain I of non-human CD40-L. Thus, in some chimeric CD40-L, the first subdomain comprises domains or subdomains of domains I, II, III, and IV of non-human CD40-L. In a preferred embodiment, the non-human CD40-L is murine CD40-L.

In a preferred embodiment, the chimeric human/mouse CD40 ligand has 92% amino acid sequence homology with human CD40L (SEQ ID NO: 12) (see, U.S. Pat. No. 7,495,090, incorporated herein by reference and referred to herein as "MEM 40"). "CD40 ligand" and "CD40-L" are used interchangeably herein and may also be referred to as "CD154". Specifically, domains I, II and III-regions comprising the intracellular, intramembrane and proximal extracellular domains, respectively, of this molecule-have been fully humanized. Of domain IV comprising the CD40 binding portion of the molecule, only those murine domains are retained that are necessary for optimal CD40 ligand expression in the cell. MEM40 is fully humanized at the 3' end of the molecule, wherein antibody binding neutralizes the activity of murine CD154 (CD 40 ligand) when administered to humans.

Non-limiting examples of chimeric CD40-L useful in the present invention include the following sequences:

SEQ ID NO：7

MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：8

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：9

MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：10

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSVHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：11

MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：12

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：13

MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIVGLWLKPSSGSERILLKAANTHSSAKPCGQQSIHLGGVFELQPGASCFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：14

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIVGLWLKPSSGSERILLKAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：15

MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：16

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSSQLCEQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：17

MIETYSQPSPRSVATGLPASMKIFMYLLTVFLITQMIGSVLFAVYLHRRLDKVEEEVNLHEDFVFIKKLKRCNKGEGSLSLLNCEEMRRQFEDLVKDITLNKEEKKENSFEMQRGDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

SEQ ID NO：18

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKDEDPQIAAHVVSEANSNAASVLQWAKKGYYTMKSNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREPSSQRPFIVGLWLKPSSGSERILLKAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

encoding SEQ ID NO: certain nucleotide sequences of chimeric CD40-L of 7 to 18 are disclosed in U.S. Pat. Nos. 7,495,090, 7,928,213 and 8,138,310. These nucleotide sequences are incorporated herein by reference, and the use of such nucleotide sequences is contemplated herein.

Oncolytic viruses of the invention may comprise a nucleic acid encoding an IFN- α, wherein the IFN- α comprises human wild-type IFN- α (SEQ ID NO: 1) or has 80.00% to at most 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%) sequence identity to human wild-type IFN- α (e.g., IFN- α of SEQ ID NO: 1).

Oncolytic viruses of the invention may comprise a nucleic acid encoding an IFN- β, wherein the IFN- β comprises a human wild-type IFN- β (SEQ ID NO: 2) or has 80.00% to at most 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%) sequence identity to a human wild-type IFN- β (e.g., IFN- β of SEQ ID NO: 2). In particular embodiments, the nucleic acid sequence encodes an IFN- β lacking the sequence of SEQ ID NO:2, and optionally further having 17 cysteine substitutions to obtain a 165 amino acid peptide having serine.

Oncolytic viruses of the invention may comprise a nucleic acid encoding IFN- ε, wherein IFN- ε comprises human wild-type IFN- ε (SEQ ID NO: 3) or has 80.00% to 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%) sequence identity to human wild-type IFN- ε (e.g., IFN- β of SEQ ID NO: 3).

Oncolytic viruses of the invention may comprise a nucleic acid encoding an IFN-kappa, wherein the IFN-kappa comprises human wild-type IFN-kappa (SEQ ID NO: 4) or has 80.00% to at most 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%) sequence identity to human wild-type IFN-kappa (e.g., IFN-kappa of SEQ ID NO: 4).

Oncolytic viruses of the invention may comprise a nucleic acid encoding an IFN- ω, wherein the IFN- ω comprises a human wild-type IFN- ω (SEQ ID NO: 5) or has 80.00% to at most 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%) sequence identity to a human wild-type IFN- ω (e.g., IFN- ω of SEQ ID NO: 5).

Oncolytic viruses of the invention may comprise a nucleic acid encoding CD40-L, wherein the CD40-L comprises human wild-type CD40-L (SEQ ID NO: 6) or CD40-L having 80.00% to at most 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%) sequence identity to human wild-type CD40-L (e.g., CD40-L of SEQ ID NO: 6). The oncolytic viruses of the present invention may comprise a nucleic acid encoding a chimeric CD40-L, wherein the chimeric CD40-L has a sequence selected from the group consisting of SEQ ID NOs: 7 to 18 or with a sequence having a sequence selected from SEQ ID NOs: the chimeric CD40-L of the sequences of 7 to 18 has a CD40-L that comprises 80.00% to at most 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%) sequence identity.

In a preferred embodiment, the oncolytic virus of the present invention may comprise a nucleic acid encoding MEM 40. For example, in a preferred embodiment, the oncolytic virus of the invention comprises an IFN beta having at least 80% sequence identity to a human IFN beta (SEQ ID NO: 2) and has the sequence of SEQ ID NO:12, the chimeric CD40-L has at least 80% sequence identity to CD40-L.

One or more heterologous nucleic acid sequences encoding a combination of type I IFN and CD40-L may be present in one or more viral constructs. Non-limiting examples of viral constructs include adenovirus constructs, adeno-associated virus constructs (AAV), poxvirus constructs, lentivirus constructs, alpha virus constructs (alphaviral construct), herpes virus constructs, retrovirus constructs, vaccinia virus constructs, vesicular stomatitis virus constructs, or herpes simplex virus constructs.

In addition to nucleic acids encoding type I interferon (e.g., IFNP) and CD40-L (chimeric human/mouse CD 40L), oncolytic viruses according to the invention may also comprise other modifications in their genome. For example, it may contain additional DNA inserted into a gene that has been inactivated or substituted for the deleted gene. Oncolytic viruses may also incorporate one or more promoters therein that confer an enhanced level of tumor cell specificity to the virus. In this way, oncolytic viruses can be targeted to specific cancer types using cancer cell-specific promoters. The term "tumor cell-specific promoter" or "tumor cell-specific transcriptional regulatory sequence" or "tumor-specific promoter" or "tumor-specific transcriptional regulatory sequence" means transcriptional regulatory sequences, promoters and/or enhancers that are present in a target cancer cell at a higher level than in a normal cell. For example, oncolytic viruses may be used in the present invention under the control of exogenously added modulators.

In a preferred embodiment, the oncolytic virus is an adenovirus (Ad). Ad is a large (about 36 kb) DNA virus that infects humans, but also shows a broad host range. Physically, adenoviruses are icosahedral viruses that comprise a double-stranded linear DNA genome. Human adenoviruses are of about 50 serotypes, and are divided into six families according to molecular, immunological and functional criteria. By adulthood, nearly everyone is infected with adenovirus serotypes with the more common primary effect being cold-like symptoms.

Adenovirus infection of the host cell results in the maintenance of adenovirus DNA in free form, which reduces the potential genotoxicity associated with integrating vectors. In addition, adenoviruses are structurally stable and no genomic rearrangement is detected after extensive amplification. Adenoviruses can infect most epithelial cells, regardless of the stage of their cell cycle. Up to now, adenovirus infection appears to be associated with only mild diseases (e.g. acute respiratory diseases in humans).

The adenovirus infection cycle occurs in two steps: early stages before the start of adenovirus genome replication allow the production of regulatory proteins and proteins involved in viral DNA replication and transcription, as well as late stages leading to the synthesis of structural proteins. Early genes are distributed in 4 regions scattered in the adenovirus genome, designated as E1 to E4 ("E" means "early"). The early region contains at least six transcription units, each with its own promoter. The expression of early genes is itself regulated, with some genes being expressed before others. The three regions E1, E2 and E4 are critical for viral replication. Thus, if one of these functions of the adenovirus is defective, the protein must be provided in trans, otherwise the virus will not replicate.

The E1 early region is located 5' to the adenovirus genome and comprises 2 viral transcription units E1A and E1B. The protein encoded by this region is involved in the very early stages of the viral cycle and is critical for the expression of almost all other genes of adenovirus. In particular, the E1A transcription unit encodes a protein that transactivates transcription of other viral genes, inducing transcription from the promoters of the E1B, E2A, E2B, E3 and E4 regions and late genes.

Adenovirus enters the permissive host cell through a cell surface receptor and is then internalized. Viral DNA associated with certain viral proteins required in the first step of the replication cycle enters the nucleus of the infected cell where transcription begins. Replication of adenovirus DNA occurs in the nucleus of the infected cell and cell replication is not required. After the new viral particles or virions are assembled, they are released from the infected cells and may infect other allowed cells.

Adenoviruses are an attractive delivery system. Embodiments of the present disclosure may utilize manufacturing processes that may be free or substantially free of proteins, serum, and animal derived components, making them suitable for use in a wide range of prophylactic and therapeutic vaccine products.

If the adenovirus has been mutated to replicate conditionally (replication-competent under certain conditions), helper cells may be required for viral replication. When desired, the helper cell line may be derived from a human cell, such as a human embryonic kidney cell, muscle cell, hematopoietic cell, or other human embryonic mesenchymal or epithelial cell. Alternatively, the helper cells may be derived from cells of other mammalian species that are permissive for human adenovirus. Such cells include, for example, vero cells or other monkey embryo mesenchymal cells or epithelial cells. In certain aspects, the helper cell line is 293. Various methods of culturing host cells and helper cells can be found in the art, for example (Racher, a.j., fooks, a.r., and Griffiths, j.b. biotechnol Tech (1995) 9:169).

1. Delivery of drug carriers/drug products

As noted above, the compositions may also be administered in vivo in a pharmaceutically acceptable carrier. By "pharmaceutically acceptable" is meant a material that is biologically or otherwise undesirable, i.e., the material may be administered to a subject with a nucleic acid or vector without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. The carrier will naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as is well known to those skilled in the art.

The compositions may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically, etc., including topical intranasal administration or inhalation administration. As used herein, "topical intranasal administration" refers to delivery of a composition into the nose and nasal passages through one or both nostrils, and may include administration by a spray mechanism or a droplet mechanism, or by nebulization of a nucleic acid or carrier. Administration of the composition by inhalation may be by nasal or oral delivery by a spray or droplet mechanism. It may also be delivered directly to any area of the respiratory system (e.g., the lungs) through a cannula. The exact amount of the composition required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity of the allergic disease being treated, the particular nucleic acid or vector used, its mode of administration, and the like. Thus, it is not possible to specify an exact amount for each composition. However, the appropriate amount may be determined by one of ordinary skill in the art using only routine experimentation given the teachings herein.

Parenteral administration of the composition, if used, is typically characterized by injection. Injections may be prepared in conventional forms, such as liquid solutions or suspensions, solid forms of solutions suitable for suspension in a liquid prior to injection, or emulsions. A recently modified method for parenteral administration involves the use of slow or sustained release systems to maintain a constant dose. See, for example, U.S. Pat. No. 3,610,795, incorporated herein by reference.

The material may be a solution, suspension (e.g., incorporated into microparticles, liposomes, or cells). These may be targeted to a particular cell type by antibodies, receptors or receptor ligands. The following references are examples of using this technique to target specific proteins 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); pieteersz and McKenzie, immunolog.Reviews,129:57-80, (1992); roffer, et al, biochem. Pharmacol,42:2062-2065, (1991)). Vectors such as "stealth" (stealth) and other liposome-coupled antibodies (including lipid-mediated colon cancer targeting drugs), receptor-mediated DNA targeting by cell-specific ligands, lymphocyte-directed tumor targeting, and highly specific therapeutic retroviral targeting of mouse glioma cells in vivo. The following references are examples of targeting specific proteins to tumor tissue using this technique (Hughes et al, cancer Research,49:6214-6220, (1989), and Litzinger and Huang, biochimica et Biophysica Acta,1104:179-187, (1992)). In general, receptors are involved in the endocytic pathway, either constitutive or ligand-induced. These receptors accumulate in clathrin-coated cells, enter the cells through clathrin-coated vesicles, pass through acidified endosomes in which the receptors are classified, and then are recycled to the cell surface for storage in the cell or degradation in lysosomes. The internalization pathway has a variety of functions, such as nutrient uptake, removal of activated proteins, clearance of macromolecules, opportunistic entry of viruses and toxins, dissociation and degradation of ligands, and receptor level modulation. Many receptors follow more than one intracellular pathway, depending on the cell type, receptor concentration, ligand type, ligand valency and ligand concentration. The molecular and cellular mechanisms of receptor-mediated endocytosis have been reviewed (Brown and Greene, DNA and Cell Biology 10:6, 399-409 (1991)).

a) Pharmaceutically acceptable carrier

The compositions, including antibodies, may be used in combination with a pharmaceutically acceptable carrier.

Suitable carriers and formulations thereof are described in "leimington: pharmaceutical science and practice (Remington: the Science and Practice of Pharmacy) (19 th edition) is described in Easton, pa., 1995, by the company Mack publishing, A.R. Gennaro. Typically, an appropriate amount of a pharmaceutically acceptable salt is used in the formulation to render the formulation 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 from about 5 to about 8, more preferably from about 7 to about 7.5. Other carriers include sustained release formulations such as semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, liposomes, or microparticles. It will be apparent to those skilled in the art that certain carriers may be more preferred depending, for example, on the route of administration and the concentration of the composition to be administered.

Drug carriers are known to those skilled in the art. These are most typically standard carriers for administration to humans, including solutions such as sterile water, saline, and buffered solutions at physiological pH. The composition may be administered intramuscularly or subcutaneously. Other compounds will be administered according to standard procedures used by those skilled in the art.

In addition to the selected molecules, the pharmaceutical compositions may contain carriers, thickeners, diluents, buffers, preservatives, surfactants and the like. The pharmaceutical composition may also contain one or more active ingredients, such as antimicrobial agents, anti-inflammatory agents, anesthetics, and the like.

The pharmaceutical compositions may be administered in a variety of ways depending on whether local or systemic treatment and the area to be treated is desired. Administration may be topical (including ocular, vaginal, rectal, intranasal), oral, by inhalation, or parenteral, for example by intravenous drip injection, subcutaneous injection, intraperitoneal injection, or intramuscular injection. The disclosed antibodies can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.

Formulations for parenteral administration include sterile aqueous or nonaqueous solutions, suspensions and emulsions. Examples of nonaqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil and injectable organic esters such as ethyl oleate. The aqueous carrier comprises water, an alcohol/water solution, an emulsion or a suspension, including saline and a buffer medium. Parenteral carriers include sodium chloride solution, ringer's dextrose, dextrose and sodium chloride, ringer's lactate solution, or fixed oils. Intravenous infusion includes liquid and nutritional supplements, electrolyte supplements (such as those based on ringer's dextrose), and the like. Preservatives and other additives may also be present such as, for example, antimicrobials, antioxidants, chelating agents, and inert gases and the like.

Formulations for topical administration may include ointments, lotions, creams, gels, drops, suppositories, sprays, liquids and powders. Conventional pharmaceutical carrier matrices, aqueous matrices, powder matrices or oily matrices, thickeners, and the like may be necessary or desirable.

Compositions for oral administration comprise powders or granules, suspensions or solutions in water or non-aqueous media, capsules, sachets or tablets. Thickeners, flavoring agents, diluents, emulsifiers, dispersing aids or binders may be required.

Certain compositions may potentially be administered as pharmaceutically acceptable acid or base addition salts formed by reaction with inorganic acids such as hydrochloric, hydrobromic, perchloric, nitric, thiocyanate, sulfuric and phosphoric acids, and organic acids such as formic, acetic, propionic, glycolic, lactic, pyruvic, oxalic, malonic, succinic, maleic and fumaric acids, or by reaction with inorganic bases such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, tri-and aryl amines and substituted ethanolamines.

b) Therapeutic use

The effective dosage and schedule of administration of the composition can be determined empirically and making such decisions is within the purview of those skilled in the art. The compositions are administered in a dosage range large enough to produce the desired effect affecting the symptoms of the disorder. The dosage should not be so large as to cause deleterious side effects such as undesired cross-reactions, allergic reactions, and the like. Generally, the dosage will vary with the age, condition, sex and extent of disease of the patient, the route of administration or whether other drugs are included in the regimen, and can be determined by one skilled in the art. In the case of any contraindications, the individual physician can adjust the dosage. The dosage may vary and may be administered in one or more doses per day for one or more days. Guidance regarding the proper dosage of a given class of drugs can be found in the literature. Guidance for selection of appropriate doses of antibodies can be found, for example, in the literature for antibody therapeutic use, e.g., in monoclonal antibody handbook (Handbook of Monoclonal Antibodies), ferrone et al eds, noges Publications, park Ridge, n.j. (1985) ch.22 and pp.303-357; smith et al, antibodies in human diagnosis and therapy (Antibodies in Human Diagnosis and Therapy), haber et al, eds., raven Press, new York (1977) pp.365-389. Typical daily doses of antibody used alone may range from about 1 μg/kg to up to above 100mg/kg body weight per day, depending on the factors described above.

C. Methods of treating cancer

The disclosed compositions may be used to treat any disease in which uncontrolled cell proliferation occurs, such as cancer. A representative but non-limiting list of cancers that the disclosed compositions can be used to treat is as follows: lymphomas; b cell lymphoma; t cell lymphomas; mycosis fungoides; hodgkin's disease; myeloid leukemia; bladder cancer; brain cancer; cancers of the nervous system; cancer of head and neck; squamous cell carcinoma of head and neck; lung cancer, such as small cell lung cancer and non-small cell lung cancer; neuroblastoma/glioblastoma; ovarian cancer; skin cancer; liver cancer; melanoma; squamous cell carcinoma of the mouth, throat, and lungs; cervical cancer; cancer of the cervix; breast cancer; epithelial cancer; renal cancer; genitourinary system cancer; lung cancer; esophageal cancer; cancer of the head and neck; colorectal cancer; hematopoietic cancers; testicular cancer; colon cancer; rectal cancer; prostate cancer or pancreatic cancer.

In one aspect, disclosed herein is a method of treating, reducing, inhibiting, reducing, ameliorating, and/or preventing cancer and/or cancer metastasis (including distant tumors) in a subject comprising administering to the subject any of the amplified TILs or MILs disclosed herein. For example, disclosed herein are methods of treating, alleviating, inhibiting, reducing, ameliorating, and/or preventing cancer and/or cancer metastasis in a subject comprising: 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) and/or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF 6) into a tumor cell; b) Collecting tumor-infiltrating lymphocytes (TIL) and/or bone marrow-infiltrating lymphocytes (MILs); c) Amplifying the collected TIL and/or MIL ex vivo; and d) administering the amplified TIL and/or MIL to the subject. In some aspects, the cancerous or metastatic tumor treated, reduced, inhibited, reduced, ameliorated, and/or prevented is distant from a tumor that receives any oncolytic virus and/or TIL or MILs disclosed herein.

Alternatively, TIL or MIL may be first harvested from a tumor and then expanded 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 administering the amplified TIL and/or MILs to the subject. The amplified TIL or MILs may then be sequentially administered (adoptive transfer) to a subject with cancer. Thus, in one aspect, disclosed herein are methods of treating, alleviating, inhibiting, reducing, ameliorating, and/or preventing cancer and/or cancer metastasis in a subject comprising a) harvesting tumor-infiltrating lymphocytes (TILs) and/or bone marrow-infiltrating lymphocytes (MILs) from a subject having cancer; culturing the harvested TIL or MILs in the presence of antigen presenting cells infected with an oncolytic virus that expresses 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) and/or one or more exogenous immunostimulatory molecules (e.g., CD40-L, MEM40, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF 6); and administering the amplified TIL and/or MILs to the subject.

In one aspect, it is understood and contemplated herein that successful treatment of cancer in a subject is important and that doing so may include administration of additional treatments. Accordingly, it is contemplated that the methods of treating, inhibiting, alleviating and/or preventing cancer disclosed herein may be enhanced by any cancer treatment, including, but not limited to, surgical, radiation and/or pharmaceutical treatment of cancer. Thus, the treatment of the present disclosure may include and/or further include any anti-cancer therapy known in the art, including but not limited to Abemacili (Abemacilib), abiraterone acetate, methotrexate (methotrexate), albumin paclitaxel (paclitaxel albumin stabilized nanoparticle formulation), ABVD, ABVE, ABVE-PC, AC-T, bentuximab (vitamin B), ADE, trastuzumab-maytansine conjugate, doxorubicin (doxorubicin hydrochloride), alfutilin dimaleate, feitinib (everolimus), ozagrypitant (netupitant and palonosetron hydrochloride), imiquimod (Imiqumod), aldesinterleukin, ai Leti-ni (Alatinib), aletinib, alemtuzumab, praecorata (disodium), coupananic (gemcitabine hydrochloride (354235), injection use of MALALABRANL (melphalan hydrochloride), MALALALALALALAL (melem), palonosetron hydrochloride), panoramide (palonosetron hydrochloride), 84 (buntinib), fluzamide (benzogline), prandine (Alexazobactam, asprine (Alexamide), aspride (Alexazobactam), asprine (Alexamide), alexazopran (Alexazobactam, alexade (Alexazopran), alexade (Alexazopraline), alexazopran (Alexaratan), alexaratan (Alexazone, alexapraline (Alexa) and Alexapraline (Alexa), altilizumab, avastin (bevacizumab), avistuzumab, axitinib, azacytidine, avistuzumab (bavendio/Avelumab), BEACOPP, carmustine (Becenum/Carmustine), beliostat (beloodaq/Belinostat), beliostat (Belinostat), bendamustine hydrochloride, BEP, oxbeuzumab (Besponsa/Inotuzumab Ozogamicin), bevacizumab, bexarotene, bucking (tositumomab and iod [131I ] tositumomab), bicalutamide, biCNU (Carmustine), bleomycin, bolimab, belitude (bortezomib), bortezomib (Bosutinib/Bosutinib), bosutinib, beniximab, buzanib, busulfan white sulfebuxine, cabazitaxel, cabazithromycin (cabazithromycin malate), cabazithromycin, CAF, canpase (alemtuzumab), irinotecan (irinotecan hydrochloride), capecitabine, CAPOX, fluorouracil (fluorouracil-topical), carboplatin-paclitaxel, carfilzomib, carmustine (carmubis/Carmustine), carmustine implant, constantan (bicalutamide), CEM, ceritinib, secatinib hydrochloride (daunorubicin hydrochloride), donepezil (recombinant HPV bivalent vaccine), cetuximab, CEV, chlorambucil-prednisone, CHOP, cisplatin, cladribine, cyclophosphamide (Clafen/cyclophospamide), clofarabine, clofarex/Clofarabine, CMF, cobratinib, cabazithromycin (cabazithromycin malate), copperac acid, COPDAC, COPP, COPP-ABV, actinomycin (Cosmegen/Dactinomycin), cobratinib (cobmetinib), crizotinib, CVP, cyclophosphamide, cyfos (ifosfamide), and Ramopirumab (Cyramza/Ramucicrumab), cytarabine liposome, sidamasca-U (cytarabine), cyclophosphamide (Cytoxan/Cyclophosphamide), dabrafenib, dacarbazine, decitabine (Dacog/Decistabine), actinomycin, darimumab (Darzalex/Daratumumab), dasatinib, daunorubicin hydrochloride, daunorubicin daunorubicin hydrochloride and cytarabine liposomes, decitabine, sodium defibrinate (Defitelio/Defibrotide Sodium), degarelix, dimesleukin, denolizumab, liposomal cytarabine (DepoCyt/Cytarabine Liposome), dexamethasone, dexrazoxane hydrochloride, denotuximab, docetaxel, doxycycline (doxorubicin hydrochloride liposomes), doxorubicin hydrochloride liposomes, dox-SL (doxorubicin hydrochloride liposomes), dacarbazine injection (dacarbazine), devaluzumab, fluoxetine (fluorodioxetine-topical), irinotecan (labyrin), epirubicin hydrochloride, erltuzumab, lenxadine (oxaliplatin), oxepin, etrazopraline, izomet, embodiment (aprepitant), embotuzumab (Emplititi/Elotuzumab), ensibirin mesylate, enzalutamine, epirubicin hydrochloride, EPOCH, erbitux (cetuximab), eribulin mesylate, vermod gedy/Vismodytab, erlotinib hydrochloride, yiwann (EmbH asparaginase), amiforin (amifostine), etoposide (etoposide phosphate), etoposide, doxorubicin (doxorubicin hydrochloride) liposomes, everolimus, raloxifene (raloxifene hydrochloride), uvulvonine (melphalan hydrochloride), exemestane, 5-FU (fluorouracil injection) 5-FU (fluorouracil-topical), fabryostat (toremifene), prabinostat (Farydak/Panobinostat), fulvestrant (Falodex/Fulvestrant), FEC, letrozole (Femara/Letrozole), fegrid, fludarabine (fludarabine phosphate), fludarabine phosphate, fluorouracil (fluorouracil-topical), fluorouracil injection, fluorouracil-topical, fluotamide, fluoxetine (methotrexate), folex PFS (methotrexate), irinotecan-bevacizumab, irinotecan-cetuximab, oxaliplatin (LFIRINOX), oxaliplatin (FOLFOX), prasufloxacin (Foloton/Pralarexa), FU-LV, fluoretinide, additional Wei Miao (recombinant HPV tetravalent vaccine), jiujiujiadaprocess (recombinant HPV nine vaccine), ozyme You Tuozhu mab (Gazyva/Obenutuzumab), gefitinib, gemcitabine hydrochloride, gemcitabine cisplatin, gemcitabine-cisplatin, gemtuzumab, gemcitabine hydrochloride, ji Tairui (bimetanide), glibenc (imatinib mesylate), gliclazide (carmustine implant), carmustine implant (carmustine implant), carboxypeptidase, goserelin acetate, eskibulin mesylate (Halaven/Eribulin Mesylate), propranolol hydrochloride (hemageol/Propranolol Hydrochloride), herceptin (trastuzumab), HPV bivalent vaccine, recombinant HPV nine vaccine recombinant HPV tetravalent vaccine, recombinant, cancer-condine (topotecan hydrochloride), hydroxyurea (hydroea/Hydroxyurea), hydroxyurea, high dose-CVAD, ebosin (Pabosutinib), temozolomide, ibrutinib, ICE, ponatinib (Prazotinib hydrochloride), idarubicin (idarubicin hydrochloride), idarubicin hydrochloride, ideranine, encidipine (Encidipine mesylate), ifex (Ifosfamide), ifosfamide (Ifosfamide/Ifosfamide), IL-2 (aldinterleukin), imatinib mesylate, ibutenib, devaluzumab (Imfinzi/Durvalumab), imiquimod, encidipine (Encidipine mesylate), ifex (Ifosfamide), ifosfamide, and other pharmaceutical compositions, imlygic (La-Tarillim (Talimogene Laherparepvec)), axitinib (Inlyta (Axitinib), orituximab, recombinant interferon alpha-2 b, recombinant interferon-2 (Aldiliginet), gan Le-energy A (recombinant interferon alpha-2 b), iodine [131I ] toxilimumab and toxilimumab, ipilimumab, iridamide (gefitinib), irinotecan hydrochloride liposome, romidepsin (Istodax/Romidepsin), ixabepilone, igafungin citrate, ixempron (Ixempra/Ixabepilone), ponitinib (ruxotinib phosphate), JEB, cabazitaxel (Jevtan/Cabazitaxel), trastuzumab (o-Adtrastuzumab-mevalnovel conjugate), and Raloxifene (raloxifene hydrochloride), paliff (Kepimobendan/Palifemin)), coryda (pembrolizumab), rebamiphene (Kisqali/Ribociclib), s Li Fuming (Kymriah/Tisamplelec Leucel), carfilzomib (Kyprolis/Carfilzomib), lanreotide acetate, lapatinib tosylate, olanizumab (Lartruvo/Olamaumab), lenalidomide, lenvatinib mesylate, lenvatinib (Levalatinib mesylate), letrozole, calcium folinate, octonine (Leukeran/Choramb), leuprorelin acetate, cladribine (Leustaine/Cladribine), levan (aminolevulinic acid), linezolid (Onconine), lipodox (doxorubicin hydrochloride), lenaline (Leustoride), the pharmaceutical compositions comprise roflumidine, troluridine (troluridine and tepirimidine hydrochloride), leuprolide acetate (Lupron/Leuprolide Acetate), leuprolide acetate microspheres (Lupron Depot) (leuprolide acetate), leuprolide acetate microspheres for injection (Lupron Depot-peg) (leuprolide acetate), olaparib (lynparaza/Olaparib), vincristine sulfate liposome injection (vincristine sulfate liposome), methylbenzyl hydrazine (methylbenzyl hydrazine hydrochloride), nitrogen mustard, megestrol acetate, trimetinib (Mekinist/trametinb), melphalan hydrochloride, mercaptopurine, mesna, mesnex/Mesna), temozolomide (Methalfastone/Temozolomide), methotrexate methotrexate LPF (Methotrexate), methylnaltrexone bromide, methotrexate (Mexate/methotrexa), methotrexate-AQ (Methotrexate), midostaurin, mitomycin C, mitoxantrone hydrochloride, mitomycin (Mitozytrex) (mitomycin C), MOPP, plexafu (Mozobil/plaixfor), nitrogen mustard (nitrogen mustard hydrochloride), mitomycin (mitomycin C), busulfan (mylaran/Busulfan), azacytidine (azacytidine), gemtuzumab (Mylotarg/Gemtuzumab Ozogamicin), paclitaxel nanoparticles (paclitaxel albumin stabilized nanoparticle formulation), noveltamitraz (vinorelbine tartrate), rituximab, nelarabine, cyclophosphamide (Neosar), nevirapine, lenatinib maleate, lenatinib (lenatinib maleate), netupitant and palonosetron hydrochloride, pefepristine (neuassta/Pegfilgrastim), fepristine (neuogen/Filgirstim), sorafenib tosylate (Nexavar/Sorafenib Tosylate), nilamide (niladron/Nilutamide), nilotinib, nilamide, i Sha Zuo m (Sha Zuo m), nilapamide monohydrate, niwuzumab, tamoxifen (tamoxifen citrate), romisettin (nplatte/romiplos), oxybutynin You Tuozhu monoclonal antibody, sonidegin (Odomzo/sonideginb), OEPA, ofloxamu monoclonal antibody, OFF, olaparib, homoharringtonine, asparaginase (Oncapar/pespargase), hydrochloride irinotecan hydrochloride liposomes (Onivyde/Irinotecan Hydrochloride Liposome), dimesleukin (Ontak/Denileukin Diftitox), nivolumab (Opdivo/Nivolumab), OPPA, ornitanib, oxaliplatin, paclitaxel albumin stabilized nanoparticulate formulations, PAD, palbociclib, palivomim, palonosetron hydrochloride and netupitant, pamidronate disodium, panitumumab, panitustat, pazopanib (Carboplatin), carboplatin (Paraplatin/Carboplatin), pazopanib hydrochloride, PCV, PEB, peginase, pefegliptin, pegylated alpha-2 b interferon, petaloenergy (pegylated alpha-2 b interferon), pembrolizumab, pemetrexed disodium, pertuzumab (Pertuta/jezumab), pertuzumab, cisplatin/cispratin, cisplatin-AQ (Cisplatin), praisafu, pomalidomide (Pomalidomide/Pomalidomide), ponatinib monohydrochloride, cetuximab (portraza/necitramulab), pralatrexed, prednisone, procarbazine hydrochloride, aldinterleukin (promeukin/Aldesleukin), pra Luo Li (dieselimab), eltrapopap (Ai Qubo pamine), propranolol hydrochloride, plaguezide (profenoki-T), mercaptopurine (pureethhol/mercaptepurine), mercaptopurine (puroxan/mercaptoprene), 223 dichloro, raloxifene hydrochloride, lei Molu monoclonal antibody, labyrinase, R-CHOP, R-CVP, recombinant human papilloma virus (bivalent HPV) vaccine recombinant Human Papillomavirus (HPV) nine-valent vaccine, recombinant Human Papillomavirus (HPV) tetravalent vaccine, recombinant interferon alpha-2 b, rigefenamide, bromomethylnaltrexone (methylnaltrexone bromide), R-EPOCH, lenalidomide (Revlimid/Lenalidomide), methotrexate (Rheumatrex/methotrex), rabocillin, R-ICE, rituximab (Rituximab/Rituximab), rituximab and hyaluronidase compositions (Rituximab and human hyaluronidase), rituximab and human hyaluronidase, roller-triptan hydrochloride, romide, romidepsin, erythromycins (daunorubicin hydrochloride), capenib (Rikappapyrisylate), the pharmaceutical composition comprises, in combination, ruxofenadine, ruxotinib phosphate, midostaurin (rydactyline/Midosatin), sterile talc aerosol (talc), rituximab, praline, lanreotide acetate (somatine Depot/Lanreotide Acetate), sonian gebub, sorafenib, dasatinib (Sprycel/Dasatinib), doxorubin (STANFORD V), sterile talc (talc), sterile talc particles (talc), regoramide (Stivarga/Regorafeib), sunitinib malate, sunitinib (sunitinib), polyethylene glycol interferon (polyethylene glycol alpha-2 b interferon), stetuximab (Sylvatin/Siltuximab), tricuspenside alkali (Synbot/Omacetaxine Mepesuccinate), thioguanine (Tableid/Thioguraine), talartinib (Talartinib/Dataenib), olanil), oxhide (Tafinamib), oxhide (Taxazin), tafmatin (Tafmatin/Tafmatin), tavanil (Tavanb), tavanil (Tavanneamide (Tavanb), talbox (Talbzocine/F), talbzocine (Talbox), talbox (Talbox), talbzocine (Talbox), talbox (Talbzob), talbzocine (Talbox) and other drugs (Talbuted/P-2 b), talbutinib (Talbuted), talbutinac (Talbuted) and (Talbuted) may be used in combination (Talbutinb) and may be used as drugs Topotecan hydrochloride, toremifene, temsirolimus (Torisel/Temsirimus), tositumomab and iodine [131I ] tositumomab, dexrazoxane (dexrazoxane hydrochloride), TPF, trabectedin, trametinib, trastuzumab, bendamustine (bendamustine hydrochloride), trofloxuridine and tepirimidine hydrochloride, arsenic Trioxide (Trisenox/Arsenic Trioxide), lapatinib (Lapatinib xylene sulfonate), denotuximab (Unituxin/Dinutuximab), uridine triacetate, VAC, vandetanib, VAMP, tarpitan (Lapidem hydrochloride), panitumumab (Vectibix/Panitumab), velp, vinblastine sulfate (Velban/Vinblastine Sulfate), velcade (bortezomib) vinblastine sulfate (Velsar/Vinblastine Sulfate), vemurafenib tablet, vinpockel (Venclexta/Venetoclax), venegram, glass fiber cilin (Verzenio/Abemaciclib), leuprorelin acetate implant (Leuprolide acetate), vedazab (azacytidine), vinblastine sulfate, vincristine PFS (vincristine sulfate), vincristine sulfate liposome, vinorelbine tartrate, VIP, vemoji, uridine triacetate (Vistogard/Uridine Triacetate), carboxypeptidase (Voraxaze/Glucoarpidase), vorinostat, pazopanib hydrochloride), daunorubicin+cytarabine composition (Vyxeos) (daunorubicin hydrochloride and cytarabine liposome), leucocyte (Wellcovarin/Leucovorin Calcium), folic acid, sicorydaline, XELIRI, XELOX, dirnosema (Xgeva/Denosumab), radium 223Dichloride (Xofigo/Radium 223 Dichloride), enzalutamide (Xtandi/Enzalutamide), yiprinima (Yervoiy/Ipain), trobin/trabectin, abelmosidene (Zaltrap/Ziv-Aflibeccept), femagistin (Zarcio/Filgastrim), then le (Nilapatinib monohydrate), zobevacine (vitamin Mo Feini), zevalin (Tivalin) (temozolomab), dex (dextrorotatum), abelmoschus, pivoxine (danserin hydrochloride), noridem (acetic acid), zoledronic acid (Zondatine/Vortide), or Albizizane acetate (Zidedate/Zibdate), and Albizizane. Chemotherapeutic agents (e.g., PD-1 inhibitors (Lambrolizumab), nivolumab, pembrolizumab, pilizumab, BMS-936559, atilizumab, dewaruzumab, or avermectin) are also contemplated herein as PDl/pdl blocking inhibitors.

D. Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and evaluate the compounds, compositions, articles, devices, and/or methods claimed herein, and are intended to be purely exemplary and are not intended to limit the disclosure. Efforts have been made to ensure accuracy with respect to numbers (e.g., amounts, temperature, etc.), but some errors and deviations should be accounted for. Unless otherwise indicated, parts are parts by weight, temperature is at or below ambient temperature, and pressure is at or near atmospheric pressure.

Example 1: immunostimulatory activity of human IFNbeta in a mouse model of melanoma

Animal studies using the same reagents as human trials are critical. However, this represents a biological problem, for example, the cross-reactivity between different human and mouse cytokines is very different for the use of cytokines. As noted above, it would be desirable to develop an OV expressing CD40L and human IFN beta as an anti-cancer therapeutic. IFNbeta in mice trigger signal transduction, although less than mouse IFN beta proficiency. Prior to OV development, it was first determined whether human IFN beta could trigger an anti-tumor T cell response in mice, as this is the primary focus of the present study. For this, we utilized a recently developed assay, where we found that irradiated B16-F10 or B16-OVA melanoma expressing mouse ifnβ could act as a potent prophylactic vaccine, with protection depending on CD8 and CD 4T cells (unpublished results). This approach is functionally similar to GM-CSF which expresses tumor cells (GVAX). B16-OVA cells were transduced with pLenti-Puro control lentiviruses or lentiviruses expressing mouse or human IFNbeta, followed by puromycin selection. The same number of cells were plated, after 2 days, the supernatant was collected for ELISA to detect human IFN beta and mouse IFN beta. Both human and mouse ifnβ were expressed at high levels, but higher levels of mouse ifnβ were detected (fig. 1A). Notably, both mouse and human ifnβ were found to be very effective in inducing T cell responses against tumor-encoded model antigens, although the mouse ifnβ induced a higher response (fig. 1B). The higher response to mouse ifnβ was likely due in part to higher expression (fig. 1A). Furthermore, this effect of human IFNbeta is strictly dependent on the presence of type 1 IFN receptor alpha 1 (IFNAR 1) in the host (FIG. 1B; similar results were obtained with independent experiments with mouse IFNbeta). Functionally, mouse and human IFNbeta are very effective in preventing tumor growth after challenge with live B16-OVA (FIG. 1C). These results indicate that human IFNbeta is also able to induce an anti-tumor T cell response in mice using OV.

Example 2: MEM-288 production and in vitro testing

We generated chimeric CD40L expression driven by the CMV promoter (fig. 2A) and added an expression cassette to drive human IFN beta expression (MEM-288) (fig. 2B) type 5 oncolytic adenovirus. As a control, we used adenoviruses expressing only GFP (Ad-GFP) and chimeric CD40L (MEM-188). All 3 viruses used contained a deletion of E1A D to allow specific replication and lysis of cancer cells (see figure 4 below). Infection of human A549 lung cancer strains with MEM-188 or MEM-288 induced high CD40L expression, as determined by flow cytometry, whereas infection with Ad-GFP resulted in high GFP expression (FIG. 3A). High secretion of IFNbeta into the culture supernatant was observed only after MEM-288 infection (FIG. 3B). Similar results were obtained in a number of other human cell lines. Studies on the B16-F10 melanoma cell line also demonstrated high transgene expression after MEM-288 infection (FIG. 3C) and secretion of IFN beta into culture supernatants (FIG. 3D). Cell death by OV is due to the replication of the virus in the infected cancer cells resulting in cell lysis. Type 1 IFN does not impair adenovirus replication. Notably, the mouse cells did not support adenovirus replication, consistent with our findings that Δ24-deleted viruses did not induce lysis of the mouse B16-F10 and 344SQ cell lines (fig. 4), whereas human a549 cells were readily lysed (fig. 4; MEM-288 is shown). These results indicate that in vivo mouse studies, the anti-tumor response induced by OV injection is more likely to be due to immunostimulation rather than oncolysis. Despite the lack of oncolytic effects in mouse cells, we refer to these viruses as OV to demonstrate their use in humans.

Example 3: study of murine melanoma model

One of the main preconditions for the use of intralesional methods is the possibility of generating a systemic anti-tumor immune response that can target non-injected lesions, i.e. trigger a distant effect. Thus, the main objective of these studies was to determine whether combined activation of CD40 and type 1 IFN signaling could generate a strong anti-tumor T cell response, thereby enabling inhibition of growth of injected and non-injected tumors. Studies of adenovirus with no replication capacity of non-oncolytic cells expressing MEM40/CD40L showed activity in the B16-F10 melanoma model. However, 4 injections of this virus were required to observe activity as a single agent or in combination with ICI.

We first studied in B16-OVA melanoma to allow tracking of OVA-reactive CD8T cells. Typical doses of adenovirus used in mice to determine the effect of anti-tumor therapy are 10e9-10 Infectious Units (IU). Lower dose 1×l0 ⁸ Ad-GFP, MEM-188 and MEM-288 of (A) were used for intratumoral injection in the first experiment (FIG. 5A). Interestingly, MEM-288 showed a more significant reduction in tumor growth compared to Ad-GFP and MEM-188 (FIG. 5A). In the next experiment we used a 10e9 IU dose that was 10 times the previous dose of all 3 viruses, with MEM-288 injection resulting in the most significant reduction in tumor growth (fig. 5B). The above studies show that after 4 injections with adenovirus expressing MEM40/CD40L, significant activity was demonstrated. However, we did not see similar activity after two injections of MEM-188, suggesting that more durable CD40L expression may be required by multiple viral injections. MEM-288 also resulted in an increase in circulating OVA-specific CD8T cells, whereas MEM-188 did not (fig. 5C).

a) Effects of MEM-288 on systemic T cell responses: CD40 and IFNAR1 role.

For these studies we compared with PBS control using MEM-288 alone. The administration of MEM-288 into B16-OVA tumors resulted in a significant increase in the number of tumor-reactive CD 8T cells in the spleen of mice, as determined by ifnγ secretion (ELISPOT assay) (fig. 5D). To evaluate the role of CD40 and type 1 IFN signaling therein, we used CD40 and IFNAR1 KO mice carrying B16-OVA tumors. Importantly, the CD 8T cell response was significantly reduced in both IFNAR1 and CD40 KO mice (fig. 5D), indicating that both pathways are independently required for MEM-288 activity. The studies presented in the present application accurately determine how these 2 pathways play a role in activating T cell responses by studying APC and T cell regulation.

b) ICI-treated MEM-288 activity.

We wanted to determine the effect of MEM-288 intratumoral injection on injected tumor and on the growth of non-injected contralateral tumors (fig. 6A). Importantly, MEM-288 inhibited growth of both the injected tumor and the contralateral tumor (fig. 6B-6C). In contrast, ICI anti-CTLA 4 and anti-PD-1 therapies had minimal impact on tumor growth as known from this tumor model (fig. 6B-6C). These results indicate that MEM-288 has better efficacy in this tumor model compared to anti-ctla4+pd-1 treatment. Importantly, the combination of anti-ctla4+pd-1 and MEM-288 significantly reduced tumor growth compared to MEM-288 alone, indicating the benefits of ICI are more pronounced when combined with MEM-288 (fig. 6B-6C). Finally, MEM-288 alone increased the survival of mice, which effect was further enhanced when combined with ICI (fig. 6D). Thus, these results demonstrate that (a) topical MEM-288 administration induces strong distal activity as a single agent, and (b) ICI treatment benefits are manifested when used in combination with MEM-288 in ICI resistant tumor models.

Example 4: pulmonary metastasis model study

Subcutaneous injections of KRAS and TP53 mutant murine 344SQ (344) lung tumor cells were injected at the primary site to form tumors, followed by metastatic spread to the lung and other sites. We found that this model was resistant to PD-1 checkpoint blockade. The key issue we want to solve is the effectiveness of MEM-288 as a single agent in PD-1 refractory tumors. We show here whether MEM-288 induced T cell activation and expansion prevents metastatic spread and/or reduces primary tumor growth. Two doses of 10e9 IU of Ad-GFP, MEM-188 and MEM-288 were administered intratumorally 12 days after inoculation. Only MEM-288 administration resulted in a significant reduction in tumor growth by subcutaneous injection (fig. 7A). Tumor lesions in the lung were determined 38 days after tumor inoculation. As expected, mice injected subcutaneously with PBS into tumors had massive lung metastases (lungs mets), as was the case with mice injected with Ad-GFP and MEM-188 (FIGS. 7B-7C). In sharp contrast, MEM-288 injected mice had significantly reduced lung tumor lesions (fig. 7B-7C). Furthermore, tumor lesion size also appeared to be significantly smaller than in MEM-288 and MEM-188 injected mice. CD8 IHC further revealed a significantly higher TIL density in MEM-288 lung tumors and a lesser degree of MEM-188 treated mice (FIGS. 7D-7E). These results indicate that the addition of IFNbeta to CD40L may enhance the immunostimulatory function in this model. We next determined whether this is true and additionally studied the efficacy of MEM-288 with anti-PD-1. Importantly, we found that robust increase of tumor cell-reactive CD8T cells in tumor cells was determined by ifnγ ELISPOT in the spleen of MEM-288 injected mice (fig. 8A-8B). On the other hand, anti-PD-1 did not significantly increase tumor-reactive T cells. We conclude that local MEM-288 injection has the potential to trigger a strong systemic T cell response, probably as a single agent in PD-1 resistant tumors is able to control tumor growth. In other ongoing studies, we determined the synergistic effect of anti-tumor activity when MEM-288 and ICI (anti-PD-1 +/-anti-CTLA 4) were combined in this model.

Example 5: research of immune stimulation mechanism of CD40L and IFN beta in tumor microenvironment

The results indicate that the CD40L and IFNb expressing virus (MEM-288) is capable of inducing an effective systemic anti-tumor T cell response, thereby controlling the growth of distant tumor lesions. In addition to the above viruses, we can also use human IFNb alone to conduct the studies presented herein. We can determine the effect of CD40L and IFNb expression in TME on various immune cell types, especially DCs. Macrophages represent another major target of CD40L activity, which can lead to their enhanced antitumor activity. These studies included an in-depth assessment of the effect of OV on DCs, macrophages and T cells, the key goal of which was to determine the unique and synergistic effects of CD40L and IFNb. In addition to the IFNAR1 and CD40KO mice mentioned above, we can also use the bat f3 KO mice lacking DC1 (see below). We show above that human IFNb triggers T cell activation in mice in an IFNAR 1-dependent manner (fig. 1). However, human IFNb can elicit a more substantial response in the setting of the human IFNAR1/2 receptor in mouse cells. Importantly, harari et al generated a transgenic mouse strain designated HyBNAR (hybrid IFNAR) with a human IFNAR1/2 extracellular ligand binding domain and a mouse IFNAR1/2 receptor signaling domain. In this study, it was shown that although human IFNb induced signaling in wild-type mice, consistent with the results, this response was enhanced in HyBNAR mice.

The primary objective here was to show the effect of intralesional OV administration on TME in B16 melanoma and 344 lung tumor models. We define here the unique and complementary effects of CD40E and IFNb receptor binding on immune cells in TME and can also study their underlying mechanisms by using mice lacking CD40 or IFNAR 1. We believe that DC represents the primary target of MEM-288 activity, for which we can conduct the most intensive study to investigate the effect of OV on the function of DC1 and DC2 subsets.

a) Effects of OV on TME: description of CD40L and IFN beta specific role

The main objective was to determine the mechanism of action of CD40L and IFNbeta in TME, for which we can use Ad-GFP, ad-IFNbeta, MEM-188 and MEM-288OV. These exploratory studies can help to assess the broad effects of the above OVs on the main populations of bone marrow cells, DCs, T cells and B cells. As shown in FIG. 9A, by flow cytometry analysis of B16-OVA tumors, we easily detected macrophages (F4/80 ⁺ Ly6C ⁺ CD11b ⁺ MHC-II ⁺ ) Monocytes and neutrophils (F4/80) ^- CD11b ⁺ Ly6C ⁺ Or Ly6G ⁺ ) Including DC1 (CD 11 c) ⁺ CD11b ^- MHC-II ⁺ CD103 ⁺ ) And DC2 (CD 11 c) ⁺ CD11b ⁺ MHC-II ⁺ CD103 ^- ) Is a DC of (c). We also detected B cells, CD4 ⁺ T cells, CD8 ⁺ T cells and OVA-specific CD 8T cells (fig. 9B). The main emphasis can be on The effect of OV on DC activation in tumors was determined. Tumor antigens are readily taken up by resident macrophages and DCs, which can be detected by using tumors expressing GFP or ZsGreen. Recent studies have also demonstrated that antigen uptake functionally reprograms DCs. We generated B16-OVA and B16-F10 expressing ZsGreen to track antigen uptake by tumor resident APCs (FIG. 10). As shown in FIG. 10, zsGreen ⁺ Populations of macrophages and DCs were readily detected in ZsGreen-expressing tumors compared to parental non-expressing tumors. ZsGreen expression for identification of tumor cells (ZsGreen ⁺ CD45 ^- ) Among them, we could also detect CD40L expression 2 days after a single MEM-288 injection (FIGS. 11A-11B). Importantly, we also found that both DC and MHC-II were found in the same tumor after MEM-288 injection ^{High height} CD80/CD86 increase in macrophages (FIGS. 12A-12B). While these results indicate that MEM-288 induces activation of macrophages and DCs, we show that they demonstrate the feasibility of detecting CD40L expression and activation markers in APC, rather than as a practical finding. We are currently further determining the kinetics of CD40L transgene expression by testing tumors in the B16 and 344 models on days 2, 4 and 6 after a single injection. We can select the time point with the highest expression level for the following study.

b) Effects of OV on TME.

Here we determined that different OV pairs tumor infiltration CD45 in the B16-OVA, B16-F10 and 344 models ⁺ The number and phenotypic effects of immune cells. By determining CD45 per gram of tumor ⁺ Percentage of cells and number of cells to count each cell type. The main population is shown in figure 9, but NK cell assays with nk1.1ab were added, as NK cells are known targets for CD40L and type 1 IFN, while tregs (with intracellular FoxP3 expression) also play a role in regulating T cell responses after OV injection. Groups of mice bearing ZsGreen expressing tumors (see also statistical programs) can be treated with PBS or 4 OVs above. We can use 5 mice per treatment in 2 independent experiments. 2 days after treatment (or at various time points based on the highest CD40L expression in the study described above), tumors can be digested for flow cytometry,to determine the differences in immune cells following injection of different OVs. Particular attention may be paid to the tumor antigen-specific CD 8T cells in B16-OVA tumors. In mice implanted with B16-OVA treated with MEM-288, we easily detected an increase in anti-tumor T cells in blood (fig. 5C) and spleen (fig. 5D). We can determine here if MEM-288 treatment resulted in the highest level of sum in tumor and OVA-specific CD 8T cells (as detected in fig. 9B). We next determined how different OVs affected uptake of ZsGreen by macrophages and DCs. In a key study, the expression of MHC-II and activation markers CD80, CD86 and CD40 in DC1 and DC2 could be determined. We can also determine ZsGreen ⁺ And ZsGreen ^- Activation state of the cells. Furthermore, we can determine the effect of treatment on CCR7 expression in DCs, as it plays a critical role in DEN transport of DCs. IL-12p70 is a key Th 1-response promoting cytokine secreted by DCs and is also a known target for CD 40L. In addition, tumor DC expression of IL-12p70 is critical for T cell anti-tumor immune responses. As described, we can determine the expression of the IL-12p40 subunit by intracellular flow in DC after administration of brefeldin A in mice. In summary, these studies can help determine how different transgenes, i.e., CD40L, IFN β and cd40l+ ifnβ, affect the total number and activation status of DC1 and DC 2. For example, these studies could help determine if the increased CD80/CD86 expression we observed after MEM-288 injection (FIG. 12) was due to CD40L, IFNP or both. And (3) statistical inspection: for comparison of the two groups, student's t-test can be used to determine mean differences. The mean differences (e.g., specific cell types or activation marker expression) for multiple treatment groups can be determined by the Tukey method.

c) The specific effects of CD40L and IFNbeta are defined using the receptor KO mice.

OVs expressing different transgenes have different effects on broader TME and T cell/DC activation. Furthermore, MEM-288 has the most robust stimulatory effect due to the combined expression of CD40L and IFN beta. Using the complementary approach, we can use the lack of CD40 and IFNAR1 mice in MEM-288 injection after observed CD40L and IFN beta on key phenotype alone effect. Although the above studies indicate that CD40 and IFNAR1 are important for T cell responses (fig. 5D), we herein determine the role of DCs in this response. In the context of MEM-288 therapy, this approach may help provide additional insight into the unique and overlapping functions of the two approaches. These studies can be performed in the B16-F10 and B16-OVA models to match the C57BL/6 genetic background of CD40 and IFNAR1 KO.

d) Determination of the Effect of OV on DC transport to DLN and T cell stimulatory Capacity

As shown in fig. 10, both macrophages and DCs readily absorbed ZsGreen. However, DCs have the unique ability to transport from tumors to DLNs where they present tumor antigens to the naive T cells to trigger their activation. Shown herein is how different OVs affect DC migration to DLN and their ability to initiate T cells. For these studies, we can track migration of DC1 and DC2 to DLN by ZsGreen, as shown in a recent study, and evaluate their ability to activate T cells. These studies help to determine how different transgenes affect DC migration and T cell sensitization.

As shown in FIG. 13, MHC-II ⁺ CD11c ⁺ 2 major populations of cells were detected in inguinal DLN. MHC (major histocompatibility complex) ^{High height} Populations are considered to contain migratory DC1 and DC2, as expected, with higher CD103 ⁺ DC and CD11b ⁺ DC percentage. Has moderate level of MHC-II (MHC) ^int ) CD11c of the population ⁺ The population contains colonising DC1 and DC2 and has lower CD103 ⁺ DC percentage. Most of ZsGreen ⁺ DC colonise MHC ^{High height} In the population. We can determine here the effect of different OVs on the distribution and activation phenotype of these different populations. MEM-288 administration may result in the highest DLN transport and these DCs have the strongest activation phenotype. We can also determine the ability of MEM-288 to induce DC migration in CD40 and IFNAR1 KO mice. As shown in fig. 14, the LN population of DCs appeared to be unaffected by the deletion of CD40 and IFNAR 1. Nevertheless, it is not clear whether the absence of CD40 and IFNAR1 would affect migration of DC from tumor to DLN.

We can next determine the ability of LN DCs to activate T cells. While studies have shown that MEM-288 greatly enhances T cell sensitization, these studies can specifically determine if this is due at least in part to DC activation. For this, we can use mice bearing B16-OVA tumors for injection of all 4 OVs. Next, we can use DC1 and DC2, selected from DLN cells, for co-culture with CFSE-labeled naive cells OT-1cd 8T cells to perform the CFSE dilution assay described herein. These studies can determine whether MEM-288 induced DC activation resulted in the highest level of T cell proliferation. Furthermore, we can conduct the ELISPOT assay to determine ifnγ secretion by culturing DCs with OT-1cd 8T cells. Using MEM-288, we could next use CD40 and IFNAR1 KO mice to determine the specific role that these 2 transgenes play in the ability of DC to activate T cells. Finally, using BATF3 KO mice, we can determine the effect of DC1 on MEM-288-induced T cell proliferation and increased IFNγ secretion.

Example 6: oncolytic viral treatment of tumors

To demonstrate the effect of oncolytic viruses on tumors in vivo, C57BL/6 mice were inoculated subcutaneously with 5e 5B 16-OVA cells on the flank. Tumors were PBS, 10 on day 12 and day 16 ⁹ IU adenovirus-GFP or MEM-288 injection. On day 20, tumors were obtained for IHC to detect CD8 ⁺ The presence of Tumor Infiltrating Lymphocytes (TILs). Mice treated with PBS or GFP-expressing oncolytic viruses showed a similar amount of TIL. In contrast, the TIL of tumors vaccinated with MEM-288 had a nearly 3-fold increase (FIG. 15).

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Sequence(s)

SEQ ID NO:1 is the amino acid sequence of human wild-type interferon-alpha (UniProtKB reference number P05014):

MALSFSLLMAVLVLSYKSICSLGCDLPQTHSLGNRRALILLAQMGRISHFSCLKDRHDFGFPEEEFDGHQFQKAQAISVLHEMIQQTFNLFSTEDSSAAWEQSLLEKFSTELYQQLNDLEACVlQEVGVEETPLMNEDSILAVRKYFQRITLYLTEKKYSPCAWEVVRAE1Lv1RSLSFST NLQKRLRRKD

SEQ ID NO:2 is the amino acid sequence of human wild-type interferon- β (UniProtKB reference number P01574):

MTNKCLLQIALLLCFSTTALSMSYNLLGFLQRSSNFQCQKLLWQLNGRLEYCLKDRMNFDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHLKTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFINRLTGYLRN

SEQ ID NO:3 is the amino acid sequence of human wild-type interferon-epsilon (UniProtKB reference number Q9P0W 0):

MSTKPDMIQKCLWLEILMGIFIAGTLSLDCNLLNVHLRRVTWQNLRHLSSMSNSFPVECLRENIAFELPQEFLQYTQPMKRDIKKAFYEMSLQAFNIFSQHTFKYWKERHLKQIQIGLDQQAEYLNQCLEEDKNENEDMKEMKENEMKPSEARVPQLSSLELRRYFHRIDNFLKEKKYSDCAWEIVRVEIRRCLYYFYKFTALFRRK

SEQ ID NO:5 is the amino acid sequence of human wild-type interferon- ω (UniProtKB reference number P05000):

MALLFPLLAALVMTSYSPVGSLGCDLPQNHGLLSRNTLVLLHQMRRISPFLCLKDRRDFRFPQEMVKGSQLQKAHVMSVLHEMLQQIFSLFHTERSSAAWNMTLLDQLHTGLHQQLQHLETCLLQVVGEGESAGAISSPALTLRRYFQGIRVYLKEKKYSDCAWEVVRMEIMKSLFLSTNMQ ERLRSKDRDLGSS

SEQ ID NO:6 is the amino acid sequence of human CD40-L (ETniprotKB reference number P29965):

MIETYNQTSPRSAATGLPISMKIFMYLLTVFLITQMIGSALFAVYLHRRLDKIEDERNLHEDFVFMKTIQRCNTGERSLSLLNCEEIKSQFEGFVKDIMLNKEETKKENSFEMQKGDQNPQIAAHVISEASSKTTSVLQWAEKGYYTMSNNLVTLENGKQLTVKRQGLYYIYAQVTFCSNREASSQAPFIASLCLKSPGRFERILLRAANTHSSAKPCGQQSIHLGGVFELQPGASVFVNVTDPSQVSHGTGFTSFGLLKL

Claims (20)

1. a method of producing tumor-infiltrating lymphocytes, comprising:

a. administering an oncolytic virus expressing one or more exogenous immunostimulatory molecules into a tumor cell; and

b. tumor infiltrating lymphocytes are collected.

2. The method of producing tumor-infiltrating lymphocytes according to claim 1, wherein the oncolytic virus further expresses one or more type 1 interferons.

3. The method of producing tumor-infiltrating lymphocytes according to claim 2, wherein the one or more type 1 interferons comprise IFN- α, IFN- β, IFN- κ, IFN- δ, IFN- ε, IFN- τ, IFN- ω, and/or IFN- ζ.

4. The method of producing tumor-infiltrating lymphocytes according to any one of claims 1-3, wherein the one or more immunostimulatory molecules comprise CD40-L, MEM, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF6.

5. The method of producing tumor-infiltrating lymphocytes according to any one of claims 1-4, wherein the oncolytic virus is MEM288.

6. The method of producing tumor-infiltrating lymphocytes according to any one of claims 1-5, wherein the oncolytic virus is administered by intratumoral injection.

7. The method of producing tumor-infiltrating lymphocytes according to any one of claims 1-6, further comprising ex vivo expansion of the harvested TILs.

8. A method of expanding a tumor-infiltrating lymphocyte (TIL) population or a bone marrow-infiltrating lymphocyte (MIL) population, comprising:

a. collecting TIL or MILs from a subject having cancer;

b. the harvested TIL or MILs are cultured in the presence of antigen presenting cells infected with an oncolytic virus expressing one or more exogenous immunostimulatory molecules.

9. The method of expanding a population of tumor-infiltrating lymphocytes (TIL) or a population of bone marrow-infiltrating lymphocytes (MILs) according to claim 8, wherein the oncolytic virus further expresses one or more type 1 interferons.

10. The method of expanding a population of tumor-infiltrating lymphocytes (TIL) or bone marrow-infiltrating lymphocytes (MIL) according to claim 9, wherein the one or more type 1 interferons comprise IFN- α, IFN- β, IFN- κ, IFN- δ, IFN- ε, IFN- τ, IFN- ω, and/or IFN- ζ.

11. The method of expanding a population of tumor-infiltrating lymphocytes (TIL) or bone marrow-infiltrating lymphocytes (MILs) according to any one of claims 8-10, wherein the one or more immunostimulatory molecules comprise CD40-L, MEM, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58, and/or SLAMF6.

12. The method of expanding a tumor-infiltrating lymphocyte (TIL) population or a bone marrow-infiltrating lymphocyte (MIL) population according to any one of claims 8-11, wherein the oncolytic virus is MEM-288.

13. A method of treating cancer in a subject comprising administering the TIL or MIL of any of claims 1-12 to the subject.

14. A method of treating cancer in a subject, comprising:

a. administering an oncolytic virus expressing one or more immunostimulatory molecules into a tumor cell;

b. collecting tumor-infiltrating lymphocytes (TIL) and/or bone marrow-infiltrating lymphocytes (MILs);

c. amplifying the collected TIL and/or MIL ex vivo; and

d. the amplified TIL and/or MILs are administered to a subject.

15. A method of treating cancer in a subject, comprising:

a. collecting tumor-infiltrating lymphocytes (TILs) and/or bone marrow-infiltrating lymphocytes (MILs) from a subject having cancer;

b. culturing the harvested TIL or MILs in the presence of antigen presenting cells infected with an oncolytic virus expressing one or more exogenous immunostimulatory molecules; and

c. the amplified TIL and/or MI are administered to subject L.

16. The method of treating cancer in a subject according to claim 14 or 15, wherein the oncolytic virus further expresses one or more type 1 interferons.

17. The method of treating cancer according to claim 16, wherein the one or more type 1 interferons comprise IFN- α, IFN- β, IFN- κ, IFN- δ, IFN- ε, IFN- τ, IFN- ω, and/or IFN- ζ.

18. The method of treating cancer according to any one of claims 14-17, wherein the one or more immunostimulatory molecules comprise CD40-L, MEM40, B7-1 (CD 80)/B7-2 (CD 86), OX40L, 4-1BBL, CD70, GITRL, LIGHT, TIM-4, ICAM-1, CD58 and/or SLAMF6.

19. The method of treating cancer according to any one of claims 14-18, wherein the oncolytic virus is MEM-288.

20. The method of treating cancer according to any one of claims 13-19, further comprising administering an anti-cancer agent to the subject.