Methods for treating lung cancer

Abstract

The present disclosure is directed to compositions and methods of treating a cancer (e.g., a lung cancer) in a subject using a virus, such as an adenovirus (e.g., aglatimagene besadenovec).

Description

METHODS FOR TREATING LUNG CANCERCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to and benefit of U.S. Provisional Application Number 63 / 651,153, filed May 23, 2024 and International (PCT) Patent Application No. PCT / US2024 / 048479, filed on September 25, 2024, the contents of each of which are incorporated by reference herein in their entirety.FIELD OF THE INVENTION

[0002] The present disclosure is directed to compositions and methods of lung cancer in a subject using a virus such as an adenovirus (e.g., aglatimagene besadenovec).BACKGROUND

[0003] Lung cancer is the most commonly diagnosed cancer in the U.S. with NSCLC representing over 80% of all lung cancer diagnoses (American Cancer Society Website, accessed Mar 2024). The majority of NSCLC patients without actionable mutations are treated with immune checkpoint inhibitors (ICI) as the first line of treatment. After one year of ICI treatment, 60% of anti-PDl treated patients will have progressive disease (Gandi et al. (2018) NEW ENGLAND J. MEDICINE 378:2078-92). In ICI inadequate responders treated with standard of care docetaxel, the median overall survival (mOS) is less than 12 months (Reckamp el al. (2022) J. Clin One. 40:2295-2306).

[0004] Therefore, there is a need for improved treatment methods for lung cancer, including methods that improve response to ICIs. There is also a need for determining the progress and efficacy of an ongoing lung cancer treatment to inform medical practitioners on whether to continue or modify a treatment regimen in a subject.SUMMARY

[0005] The disclosure relates, in part, to the discovery that delivery of a virus, such as aglatimagene besadenovec, to a disease positive lymph node, can be effective in extending survival of a subject. Administration to a disease-positive lymph node may be advantageous over administration to a lung tumor because lymph node administration can be done percutaneously which may be less invasive then intratumoral injection to a lung tumor.

[0006] The disclosure also relates, in part, to the discovery that treatment of subject with non-squamous lung cancer with a virus, such as aglatimagene besadenovec, results in improved survival of the subject as compared to a subject with non-squamous lung cancerreceiving standard of care chemotherapy. Subjects with advanced lung cancer can include subjects that have been diagnosed with one or more of stage IV lung cancer, lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1.

[0007] The disclosure also relates, in part, to the discovery that treatment of subject with advanced lung cancer with a virus, such as aglatimagene besadenovec, results in improved survival of the subject as compared to a subject receiving standard of care chemotherapy. Subjects with advanced lung cancer can include subjects that have been diagnosed with one or more of stage IV lung cancer, lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1.

[0008] The disclosure also relates, in part, to the discovery that treatment of a subject having a lung cancer with low or negative PD-L1 expression with a therapeutically effective amount (e.g., two doses) of a virus, such as aglatimagene besadenovec, can result in improved survival of the subject as compared to a subject receiving standard of care chemotherapy.

[0009] The disclosure also relates, in part, to the discovery of certain biomarkers associated with subjects experiencing an abscopal effect and / or extended survival. A subject can be tested for circulating expression of one or more of these biomarkers. If one or more of the biomarkers is present, a virus therapy, such as aglatimagene besadenovec, is likely to have an a positive effect on survival (e.g., extend survival). If none of the biomarkers is present, the virus therapy, such as aglatimagene besadenovec, can be administered again to induce an immune response that includes an increase in circulation of one or more of the biomarkers.

[0010] Accordingly, in one aspect, the disclosure provides a method of treating a subject with non-squamous non-small cell lung cancer in need thereof. The method can include administering to the subject a therapeutically effective amount of aglatimagene besadenovec, wherein administration of the aglatimagene besadenovec results in improved survival of the subject as compared to a subject receiving a standard of care treatment without the aglatimagene besadenovec (e.g., a chemotherapy such as docetaxel). In certain embodiments, the aglatimagene besadenovec is administered intratumorally to a primary tumor. In certain embodiments, the aglatimagene besadenovec is administered intratumorally to a secondaryintra-thoracic lesion in the lung tissue. In certain embodiments, the primary tumor is a lung tumor. Depending upon the circumstances, the subject may receive two administrations of the aglatimagene besadenovec.

[0011] In another aspect, the disclosure provides a method of modulating the immune system of a subject with non-resectable, non-small cell lung cancer in need thereof. The method can include administering to the subject a therapeutically effective amount of aglatimagene besadenovec, wherein administration of the aglatimagene besadenovec results in an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment. In certain embodiments, the aglatimagene besadenovec is administered intratumorally to a primary tumor. In certain embodiments, the aglatimagene besadenovec is administered intratumorally to a secondary intra-thoracic lesion in the lung tissue. In certain embodiments, the primary tumor is a lung tumor. Depending upon the circumstances, the subject may receive two administrations of the aglatimagene besadenovec.

[0012] In another aspect, the disclosure provides a method of treating a subject with lung cancer in need thereof. The method includes administering to a disease-positive lymph node of the subject a therapeutically effective amount of aglatimagene besadenovec, wherein administration of the aglatimagene besadenovec results in improved survival of the subject as compared to a subject receiving a standard of care treatment without the aglatimagene besadenovec (e.g., a chemotherapy such as docetaxel). In certain embodiments, the aglatimagene besadenovec is administered to the disease-positive lymph node percutaneously.

[0013] In another aspect, the disclosure provides a method of treating a subject with advanced lung cancer, e.g., where in the cancer is stage III or stage IV, in need thereof. The method can include administering a therapeutically effective amount of aglatimagene besadenovec to the subject, wherein administration of the aglatimagene besadenovec results in improved survival of the subject as compared to a subject receiving standard of care chemotherapy. In certain embodiments, the subject has been diagnosed with one or more of stage IV non-small cell lung cancer (e.g., squamous or non-squamous), lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1.

[0014] In certain embodiments, the aglatimagene besadenovec is administered to a lung tumor or site of resection in the lung.

[0015] Depending upon the circumstances, the subject has a survival of at least 10 months after initial treatment with the aglatimagene besadenovec, for example, a survival of at least one year, 18 months, or 24 months after initial treatment with the aglatimagene besadenovec.

[0016] In certain embodiments, the lung cancer selected for treatment has low or negative PD-L1 expression.

[0017] In certain embodiments, the subject’s cancer has progressed on immune checkpoint inhibitor therapy.

[0018] In another aspect, the disclosure provides a method of extending survival of a subject with non-resectable, non-small cell lung cancer in need thereof. The method can include administering to the subject a therapeutically effective amount of aglatimagene besadenovec, wherein administration of the aglatimagene besadenovec results in extended survival of the subject as compared to a subject receiving a standard of care treatment (e.g., docetaxel) without the aglatimagene besadenovec. The aglatimagene besadenovec can be administered intratumorally to a primary tumor, e.g., a lung tumor, or a site of tumor resection. The cancer can be stage III or stage IV non-small cell lung cancer. Alternatively or in addition, the cancer can be a squamous or non-squamous cancer.

[0019] In certain embodiments, the subject has been diagnosed with one or more of: stage IV non-small cell lung cancer, lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1.

[0020] In certain embodiments, the subject has a survival of at least 10 months after initial treatment with the aglatimagene besadenovec. In certain embodiments, the subject has a survival of at least one year, 18 months, or 24 months after initial treatment with the aglatimagene besadenovec.

[0021] In certain embodiments, the lung cancer selected for treatment has low or negative PD-L1 expression.

[0022] In another aspect, the disclosure provides a method of treating a subject with non- resectable, non-small cell lung cancer with low or negative PD-L1 expression. The methodcan inlcude administering to the subject a therapeutically effective amount of aglatimagene besadenovec, wherein administration of the aglatimagene besadenovec results in extended survival of the subject as compared to a subject with non-resectable, non-small cell lung cancer with low or negative PD-L1 expression receiving a standard of care treatment (e.g., docetaxel) without the aglatimagene besadenovec. The aglatimagene besadenovec can be administered intratumorally to a primary tumor. In certain embodiments, the aglatimagene besadenovec is administered intratumorally to a secondary intra-thoracic lesion in the lung tissue. The cancer can be stage III or stage IV non-small cell lung cancer. Alternatively or in addition, the cancer can be squamous or non-squamous cancer.

[0023] In certain embodiments, the subject has been diagnosed with one or more of: stage IV non-small cell lung cancer, lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1.

[0024] Depending upon the circumstances, the subject has a survival of at least 10 months after initial treatment with the aglatimagene besadenovec. In certain embodiments, the subject has a survival of at least one year, 18 months, or 24 months after initial treatment with the aglatimagene besadenovec.

[0025] In another aspect, the disclosure provides a method of treating a non-resectable, non-small cell lung cancer in a subject who has previously received an aglatimagene besadenovec treatment (e.g., one or two doses of aglatimagene besadenovec). The method can include administering to the subject one or more additional doses of the aglatimagene besadenovec when the subject has been determined not to exhibit an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment. The method can include not administering to the subject additional doses of the aglatimagene besadenovec when the subject exhibits an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment, because an increase in the frequency of circulating ICOS+ T helper and central memory cells may be indicative that the prior doses of aglatimagene besadenovec were sufficient.

[0026] In another aspect, the disclosure provides a method of treating non-resectable, non-small cell lung cancer in a subject who previously received aglatimagene besadenovectreatment. The method can include administering to the subject one or more additional doses of the aglatimagene besadenovec when the subject has been determined not to exhibit one or more of features (a) - (e):(a) an increase in circulating activated cytotoxic T cells relative to the frequency of circulating activated cytotoxic T cells prior to the treatment;(b) an increase in circulating T helper cells relative to the frequency of circulating T helper cells prior to the treatment;(c) an increase in circulating central memory T cells relative to the frequency of circulating central memory T cells prior to the treatment;(d) an increase in circulating effector memory T cells relative to the frequency of circulating effector memory T cells prior to the treatment; and(e) an increase in circulating B cells relative to the frequency of circulating B cells prior to the treatment; and, optionally, not administering to the subject the aglatimagene besadenovec when the subject exhibits at least one of features (a) - (e).

[0027] In another aspect, the disclosure provides a method of treating non-resectable, non-small cell lung cancer in a subject who has previously received an aglatimagene besadenovec treatment. The method can include administering to the subject an effective amount of the aglatimagene besadenovec when the subject has been determined to exhibit one or more of:(a) a low frequency of OX40+CD8+T cells;(b) a low frequency of CD56+gd+T cells;(c) high numbers of Tim3+ central memory T cells; and(d) high numbers of CD11C+CD8+ naive and memory T cells; and, optionally, not administering to the subject the aglatimagene besadenovec when the subject does not exhibit at least one of features (a) - (d).

[0028] In another aspect, the disclosure provides a method of treating non-resectable, non-small cell lung cancer in a subject who has previously received an aglatimagene besadenovec treatment. The method can include administering to the subject an effectiveamount of the aglatimagene besadenovec when the subject has been determined to exhibit one or more of:(a) a low frequency of CD8+ and CD4+ T cells;(b) a low frequency of effector T helper cells; and(c) a high level of a protein associated with tumor growth (e.g., ANGPT2, HGF, or CD70); and, optionally, not administering to the subject the aglatimagene besadenovec when the subject does not exhibit at least one of features (a) - (c).

[0029] Depending upon the circumstances, aglatimagene besadenovec can be administered to a disease-positive lymph node of the subject or a lung tumor or site of resection in the lung. Furthermore, the aglatimagene besadenovec can be administered percutaneously or via bronchoscopy. In certain embodiments, the cancer is stage III or stage IV non-small cell lung cancer. Alternatively or in addition, the non-small cell lung cancer is squamous or non-squamous.

[0030] In certain embodiments, the subject has been diagnosed with one or more of stage IV non-small cell lung cancer e.g., squamous or non-squamous), lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1.

[0031] Depending upon the circumstances, the subject has a survival of at least 10 months after initial treatment with the aglatimagene besadenovec, for example, a survival of at least one year, 18 months, or 24 months after initial treatment with the aglatimagene besadenovec.

[0032] In certain embodiments, the lung cancer selected for treatment has low or negative PD-L1 expression.

[0033] In certain embodiments of any of the above aspects, the method further comprises administering to the subject a substrate of Herpes Simplex Virus thymidine kinase (HSV- TK), such as valacyclovir, ganciclovir, acyclovir, penciclovir, valganciclovir, and famciclovir. In certain embodiments, the substrate of HSV-TK is valacyclovir.

[0034] In another aspect, the disclosure provides a method of monitoring treatment efficacy in a subject who has or previously had a lung cancer wherein the subject has received at least two doses of an aglatimagene besadenovec therapy. The method includes determining whether the subject exhibits an increase in the frequency of circulating ICOS+ Thelper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment. When the subject does not exhibit an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment, the subject can be administered additional doses of the aglatimagene besadenovec therapy to induce an immune response (e.g., an increase the frequency of circulating ICOS+ T helper and central memory cells). When the subject exhibits an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment, the subject may not receive further treatment with the aglatimagene besadenovec, because the increase in circulating ICOS+ T helper and central memory cells suggests that the treatment has been effective.

[0035] In another aspect, the disclosure provides a method of monitoring treatment efficacy in a subject who has or previously had a non-resectable, non-small cell lung cancer wherein the subject has received at least two doses of an aglatimagene besadenovec therapy. The method includes determining whether the subject exhibits one or more of features (a) -(e):(a) an increase in circulating activated cytotoxic T cells relative to the frequency of circulating activated cytotoxic T cells prior to the treatment;(b) an increase in circulating T helper cells relative to the frequency of circulating T helper cells prior to the treatment;(c) an increase in circulating central memory T cells relative to the frequency of circulating central memory T cells prior to the treatment;(d) an increase in circulating effector memory T cells relative to the frequency of circulating effector memory T cells prior to the treatment; and(e) an increase in circulating B cells relative to the frequency of circulating B cells prior to the treatment.When the subject does not exhibit one or more of (a) through (e), the subject can be administered additional doses of the aglatimagene besadenovec therapy to induce an immune response (e.g., an increase in one or more of (a) through (e)). When the subject exhibits one or more of (a) through (e), the subject the subject may not need further treatment with the aglatimagene besadenovec, because the increase in one or more of features (a) through (e) suggests that the treatment has been effective.

[0036] In another aspect, the disclosure provides a method of monitoring treatment efficacy in a subject who has or previously had a lung cancer wherein the subject has received at least two doses of an aglatimagene besadenovec therapy. The method includes determining whether the subject exhibits one or more of features (a) - (d):(a) a low frequency of OX40+CD8+T cells;(b) a low frequency of CD56+gd+T cells;(c) high numbers of Tim3+ central memory T cells; and(d) high numbers of CD11C+CD8+ naive and memory T cells.When the subject exhibits one or more of (a) through (d), the subject the subject is administered additional doses of the aglatimagene besadenovec therapy to induce at least one of features (a) - (d), and when the subject does not exhibit one or more of (a) through (d), the subject does not receive further treatment with the aglatimagene besadenovec.

[0037] In another aspect, the disclosure provides a method of monitoring treatment efficacy in a subject who has or previously had a lung cancer wherein the subject has received at least two doses of an aglatimagene besadenovec therapy. The method includes determining whether the subject exhibits one or more of features (a) - (c):(a) a low frequency of CD8+ and CD4+ T cells;(b) a low frequency of effector T helper cells; and(c) a high level of a protein associated with tumor growth (e.g., ANGPT2, HGF, or CD70).When the subject exhibits one or more of (a) through (c), the subject the subject is administered additional doses of the aglatimagene besadenovec therapy to induce at least one of features (a) - (c), and when the subject does not exhibit one or more of (a) through (c), the subject does not receive further treatment with the aglatimagene besadenovec.

[0038] In certain embodiments of any of the methods disclosed herein, the subject has been treated previously with an immune checkpoint inhibitor (ICI), and in certain embodiments, the subject is ICI refractory. In certain embodiments of any of the methods disclosed herein, the subject has stable disease or progressing disease.

[0039] These and other aspects and features of the invention are described in the following detailed description and claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0040] The invention can be more completely understood with reference to the following drawings.

[0041] Figure (FIG.) 1A is a schematic representation of the potential biological mechanism of action of CAN-2409 (also referred to as aglatimagene besadenovec).

[0042] FIG. IB is a schematic representation of NCT04495153, an open-label, phase 2 clinical trial of CAN-2409 + valacyclovir in combination with continued immune checkpoint inhibitor (ICI) in patients with non-resectable, stage III / IV non-small cell lung cancer (NSCLC), refractory or resistant to anti-PD-(L)l. N refers to sample size.

[0043] FIG. 2 is a schematic representation of patient cohorts included in the clinical trial(NCT04495153). 'Safety population: Received at least 1 injection of CAN-2409.2Per protocol evaluable patients are those who received 2 courses of CAN-2409 + prodrug (valacyclovir) and completed the 12- week treatment period.

[0044] FIG. 3 is a swimmer plot of evaluable patients in Cohorts 1 and 2 (n=46).

[0045] FIG. 4 is a graph showing the best overall percentage change in tumor size for target lesions from baseline. PD refers to progressive disease.

[0046] FIG. 5A (top) is a Kaplan Meier curve for patients in cohorts 1 and 2. FIG. 5A (bottom) is a chart of the number of patients at risk at each timepoint.

[0047] FIG. 5B (top) is a Kaplan Meier curve for patients in cohort 2. FIG. 5B (bottom) is a chart of the number of patients at risk at each timepoint.

[0048] FIG. 5C (top) is a Kaplan Meier curve for patients based on PD-L1 status. FIG. 5C (bottom) is a chart of the number of patients at risk at each timepoint, grouped by PD-L1 status.

[0049] FIG. 6A is a schematic representation of a case study (PA-003 : partial response extended in lung mass with durable after treatment tumor regression after CAN-2409 treatment (> 30 mOS, ongoing)).

[0050] FIG. 6B is a schematic representation of a case study (MU-002: patient with partial response and evidence of abscopal effect and OS 30.5 months (ongoing)).

[0051] FIGs. 7 A and 7B are graphs showing the number of patients with a detectable abscopal response (any decrease and >5% response, respectively).

[0052] FIGs. 8A and 8B are graphs showing changes in circulating T cells after CAN- 2409 treatment.

[0053] FIGs. 9A and 9B are graphs showing changes in circulating proinflammatory mediators and cytotoxic enzymes.

[0054] FIG. 10A is a graph showing a significant increase in ICOS+ T helper and central memory T helper cells in long survivors (overall survival (OS)>24 months). Increase was measured between week 8 (after 2nd injection) and baseline.

[0055] FIG. 10B is a graph showing a significant increase in activated cytotoxic T cells, T helper cells, central and effector memory T cells in long survivors (OS >24 months). Increase was measured between week 8 (after 2nd injection) and week 6 (pre 2nd injection).

[0056] FIGs. 10C and 10D are graphs showing an increase in circulating levels of granzyme H (p<0.03) (FIG. 10C) and B (n.s.) (FIG. 10D) in long survivors. Increase was measured between week 8 (after 2nd injection) and week 6 (pre 2nd injection).

[0057] FIG. 10E is a graph showing significant upregulation of activated cytotoxic T cells, T helper cells, central and effector memory T cells in patients with abscopal response as compared to patients without abscopal response.

[0058] FIG. 11A is a heatmap illustrating the contribution of different data modalities to each Multi-Omics Factor Analysis (MOFA) identified factor.

[0059] FIG. 11B is a graph showing principal component analysis (PCA) interrogating the ability of factor 2 and 21 to separate patients according to survival (each dot represents a patient).

[0060] FIG. 11C is a heatmap representing r (Pearson Correlation Coefficient) for each factor and specific clinical feature or measure of outcome.

[0061] FIG. 12A is a survival curve for patients with squamous cell carcinoma differentiated based on site of injection. “Lung_lung” indicates patients injected twice in the lung (at the same or different sites). “Lymph lymph” indicates patients injected twice in the lymph node (in the same or different lymph nodes). “Peri_Peri” indicates patients injected twice in peripheral metastases (in the same or different peripheral metastases). “Different”indicates patients injected once at one site and a second time a different site (e.g., once at a lung site and once at a lymph node site).

[0062] FIG. 12B is a survival curve for patients with non-squamous cell carcinoma differentiated based on site of injection. “Lung_lung” indicates patients injected twice in the lung. “Lymph_lymph” indicates patients injected twice in the lymph node. “Peri_Peri” indicates patients injected twice in peripheral metastases. “Different” indicates patients injected once at one site and a second time a different site.

[0063] FIG. 13 is a survival curve for patients with stage IV non-squamous cell carcinoma differentiated based on site of injection. “Lung lesion” indicates patients injected twice in the lung (at the same or different sites). “Lymph node” indicates patients injected twice in the lymph node (in the same or different lymph nodes). “Peripheral metastasis” indicates patients injected twice in peripheral metastases (in the same or different peripheral metastases).DETAILED DESCRIPTIONI. Definitions

[0064] Unless defined otherwise, technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art to which the claimed subject matter belongs. For example, nomenclatures utilized in connection with, and techniques of, e.g., molecular and immunology, etc. described herein are those well-known and commonly used in the art.

[0065] As used herein, the singular forms “a,” “an,” and “the” include plural referents unless context clearly dictates otherwise.

[0066] The use of any and all examples, or exemplary language herein, for example, “such as” or “including,” is intended merely to illustrate better the present disclosure and does not pose a limitation on the scope of any invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of any invention disclosed herein.

[0067] It should be understood that the expression “at least one of’ includes individually each of the recited objects after the expression and the various combinations of two or more of the recited objects unless otherwise understood from the context and use.

[0068] The use of the term “include,” “includes,” “including,” “have,” “has,” “having,” “contain,” “contains,” or “containing,” including grammatical equivalents thereof, should be understood generally as open-ended and non-limiting, for example, not excluding additional unrecited elements or steps, unless otherwise specifically stated or understood from the context.

[0069] As used herein, the expression “and / or” in connection with two or more recited objects includes individually each of the recited objects and the various combinations of two or more of the recited objects, unless otherwise understood from the context and use.

[0070] Where the use of the term “about” is before a quantitative value, the present invention also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term “about” refers to a ±10% variation from the nominal value unless otherwise indicated or inferred.

[0071] The term “determine,” “determined,” or “determining” includes any means of detecting, including direct and indirect detection. Depending upon the circumstances, the foregoing terms also include receiving results from a test or assessment.

[0072] The term “gene” encompasses both the regions coding a gene product as well as regulatory regions for that gene, such as a promoter or enhancer, unless otherwise indicated.

[0073] The term “virus,” as used herein, can refer to a virus that can be used therapeutically, such an adenovirus (e.g., an Ad5 adenovirus). Any instance of the term “virus” herein can include aglatimagene besadenovec.

[0074] The terms “patient” and “subject” are used interchangeably herein and may include any animal, including mammals, preferably mice, rats, other rodents, rabbits, dogs, cats, swine, cattle, sheep, horses, non-human primates, and humans. The compositions of the invention can be administered to a mammal, such as a human, but can also be administered to other mammals such as an animal in need of veterinary treatment, e.g., domestic animals (e.g., dogs, cats, and the like), farm animals (e.g., cows, sheep, pigs, horses, and the like) and laboratory animals (e.g., rats, mice, guinea pigs, non-human primates, and the like).

[0075] As used herein, the terms “administering” and “administration” refer to any method of providing an agent to the subject. Such methods are known to those skilled in the art, and include, but are not limited to, intratumoral administration, oral administration, transdermal administration, administration by inhalation, nasal administration, topical administration,intravaginal administration, ophthalmic administration, intra-aural administration, intracerebral administration, administration to spinal cord, administration to intracerebral fluid, rectal administration, parenteral administration, intravenous administration, intraarterial administration, intramuscular administration, intrathecal administration, systemic administration, and subcutaneous administration. Administration can be continuous or intermittent. In some instances a virus described herein can be administered therapeutically or prophylactically, such as administered for treatment of a disease or condition (e.g., a lung cancer) in a subject, or for improvement of one or more functions in a subject.

[0076] Throughout the description, where compositions are described as having, including, or comprising specific components, or where processes and methods are described as having, including, or comprising specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist essentially of, or consist of, the recited components, and that there are processes and methods according to the present disclosure that consist essentially of, or consist of, the recited processing steps. Similarly, throughout the description, where compositions are described as consisting essentially of specific components, or where processes and methods are described as consisting essentially of specific steps, it is contemplated that, additionally, there are compositions of the present disclosure that consist of the recited components, and that there are processes and methods according to the present disclosure that consist of the recited processing steps.

[0077] Throughout the text, where an element or component is said to be included in and / or selected from a list of recited elements or components, it should be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0078] Further, it should be understood that elements and / or features of a composition or a method described herein can be combined in a variety of ways without departing from the spirit and scope of the present disclosure, whether explicit or implicit herein. For example, where reference is made to a particular compound, that compound can be used in various embodiments of compositions of the present disclosure and / or in methods of the present disclosure, unless otherwise understood from the context. In other words, within this application, embodiments have been described and depicted in a way that enables a clear and concise application to be written and drawn, but it is intended and will be appreciated thatembodiments may be variously combined or separated without parting from the present teachings and any invention provided herein. For example, it will be appreciated that all features described and depicted herein can be applicable to all aspects of any invention described.

[0079] Tt should be understood that the order of steps or order for performing certain actions is immaterial so long as disclosed invention(s) remain operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0080] As used herein, all numerical values or numerical ranges include whole integers within or encompassing such ranges and fractions of the values or the integers within or encompassing ranges unless the context clearly indicates otherwise. Thus, for example, reference to a range of 90%-100%, includes 90%, 91%, 92%, 93%, 94%, 95%, 95%, 97%, 98%, 99% or 100%.IL Aglatimagene Besadenovec

[0081] Aglatimagene besadenovec (also referred to as CAN-2409) is an adenoviral replication-defective engineered gene construct encoding the thymidine kinase gene derived from herpes simplex virus (HSV). (See, e.g., Chen et al. (1994) PROC. NATL. ACAD. Set.USA 91:3054-2057; Trask et al. (2000) MOLECULAR THERAPY 1(2): 195-203.) Aglatimagene besadenovec can be injected directly into tumor or target tissue. It is contemplated that localized injection can minimize the chance of development of anti-drug antibodies and systemic toxicides associated with systemic administration. Further, as disclosed herein, delivery of a virus, such as aglatimagene besadenovec, to a disease positive lymph node, is as effective as intertumoral administration in extending survival of a subject. Administration to a disease-positive lymph node may be advantageous over administration to a lung tumor because lymph node administration can be done percutaneously which may be less invasive then intratumoral injection to a lung tumor.

[0082] As shown in FIG. 1A, the adenoviral construct serves as a vector to transport the HSV-thymidine kinase gene into tumor cells at the site of injection. These tumor cells can then express HSV-thymidine kinase, which converts orally administered anti-herpes prodrugs, such as valacyclovir, ganciclovir, acyclovir, penciclovir, valganciclovir, and famciclovir, into a toxic nucleotide analogue, which blocks DNA synthesis in dividing cells. Cells exposed to the toxic nucleotide analogue in the tumor microenvironment have been observed to undergo immunogenic cell death. At the same time, the adenoviral serotype 5capsid protein elicits a strong pro-inflammatory signal in the tumor microenvironment. This induces a CD8+ T cell mediated response against the injected tumor and uninjected distant metastases for broad and systemic anti-tumor activity.III. Pharmaceutical Compositions, Medicaments, and Routes of Administration

[0083] For therapeutic use, a virus described herein is preferably combined with a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers include buffers, carriers, and excipients suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as a phosphate buffered saline solution, water, emulsions (e.g., such as an oil / water or water / oil emulsions), and various types of wetting agents.

[0084] The compositions also can include stabilizers and preservatives. For examples of carriers, stabilizers and adjuvants, see, e.g., Adejare (2020) REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY. 23rd ed. Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is known in the art.

[0085] In some embodiments, a pharmaceutical composition may contain formulation materials for modifying, maintaining or preserving, for example, the pH, osmolarity, viscosity, clarity, color, isotonicity, odor, sterility, stability, rate of dissolution or release, adsorption or penetration of the composition. In such embodiments, suitable formulation materials include, but are not limited to, amino acids (such as glycine, glutamine, asparagine, arginine or lysine); antimicrobials; antioxidants (such as ascorbic acid, sodium sulfite or sodium hydrogen-sulfite); buffers (such as borate, bicarbonate, Tris-HCl, citrates, phosphates or other organic acids); bulking agents (such as mannitol or glycine); chelating agents (such as ethylenediamine tetraacetic acid (EDTA)); complexing agents (such as caffeine, polyvinylpyrrolidone, beta-cyclodextrin or hydroxypropyl-beta-cyclodextrin); fillers; monosaccharides; disaccharides; and other carbohydrates (such as glucose, mannose or dextrins); proteins (such as serum albumin, gelatin or immunoglobulins); coloring, flavoring and diluting agents; emulsifying agents; hydrophilic polymers (such as polyvinylpyrrolidone); low molecular weight polypeptides; salt- forming counterions (such assodium); preservatives (such as benzalkonium chloride, benzoic acid, salicylic acid, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid or hydrogen peroxide); solvents (such as glycerin, propylene glycol or polyethylene glycol); sugar alcohols (such as mannitol or sorbitol); suspending agents; surfactants or wetting agents (such as pluronics, PEG, sorbitan esters, polysorbates such as polysorbate 20, polysorbate, triton, tromethamine, lecithin, cholesterol, tyloxapal); stability enhancing agents (such as sucrose or sorbitol); tonicity enhancing agents (such as alkali metal halides, preferably sodium or potassium chloride, mannitol sorbitol); delivery vehicles; diluents; excipients; and / or pharmaceutical adjuvants (see, Gennaro (1990) REMINGTON’S PHARMACEUTICAL SCIENCES, 18thed. (Mack Publishing Company).

[0086] In some embodiments, a pharmaceutical composition may contain nanoparticles, e.g., polymeric nanoparticles, liposomes, or micelles (see, Anselmo et al. (2016) BIOENG. TRANSL. MED. 1: 10-29).

[0087] In some embodiments, a pharmaceutical composition may contain a sustained- or controlled-delivery formulation. Techniques for formulating sustained- or controlled- delivery means, such as liposome carriers, bio-erodible microparticles or porous beads and depot injections, are also known to those skilled in the art. Sustained-release preparations may include, e.g., porous polymeric microparticles or semipermeable polymer matrices in the form of shaped articles, e.g., films, or microcapsules. Sustained release matrices may include polyesters, hydrogels, polylactides, copolymers of L-glutamic acid and gamma ethyl-L- glutamate, poly (2-hydroxyethyl-inethacrylate), ethylene vinyl acetate, or poly-D(-)-3- hydroxybutyric acid. Sustained release compositions may also include liposomes that can be prepared by any of several methods known in the art.

[0088] Pharmaceutical compositions containing a virus disclosed herein can be presented in a dosage unit form and can be prepared by any suitable method. A pharmaceutical composition should be formulated to be compatible with its intended route of administration. Examples of routes of administration are intratumoral, intravenous (IV), intradermal, inhalation, transdermal, topical, transmucosal, intrathecal, rectal administration, oral, buccal, intranasal, intradermal, intrathecal, intracistemal, and intralesional. One of skill in the art would readily appreciate that the various routes of administration described herein would allow for the viruses or compositions to be delivered on, in, or near the tumor or targeted cancer cells. One of skill in the art would also readily appreciate that various routes ofadministration described herein will allow for the vectors and compositions described herein to be delivered to a region in the vicinity of the tumor or individual cells to be treated. “In the vicinity” can include any tissue or bodily fluid in the subject that is in sufficiently close proximity to the tumor or individual cancer cells such that at least a portion of the vectors or compositions administered to the subject reach their intended targets and exert their therapeutic effects. A virus can be administered to a site of surgical resection in a subject by, for example, injection directly into the surgical bed after resection of a primary tumor, either before or after closing of the surgical site. Alternatively, as is discussed above, the viruses can be injected directly into tumors or lymph nodes.

[0089] Useful formulations can be prepared by methods known in the pharmaceutical art. For example, see Adejare (2020) REMINGTON: THE SCIENCE AND PRACTICE OF PHARMACY. 23rd ed. Formulation components suitable for parenteral administration include a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol, or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as EDTA; buffers such as acetates, citrates, or phosphates; and agents for the adjustment of tonicity such as sodium chloride or dextrose.

[0090] Pharmaceutical formulations preferably are sterile. Sterilization can be accomplished by any suitable method, e.g., filtration through sterile filtration membranes. Where the composition is lyophilized, filter sterilization can be conducted prior to or following lyophilization and reconstitution.

[0091] It is to be understood that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.IV. Methods of Treating a Tumor with a Virus Such as Aglatimagene Besadenovec

[0092] The disclosure also features methods of treating lung cancer, e.g., a non- resectable, non-small cell lung cancer, in a subject in need thereof, the method comprising: administering a therapeutically effective amount of a virus, such as aglatimagene besadenovec. The virus can be administered to a lung tumor, e.g., intratumorally, or a disease-positive lymph node, e.g., by percutaneous injection. The method can result in improved survival of the subject as compared to a subject receiving standard of care that does not include the aglatimagene besadenovec (e.g., a chemotherapy such as docetaxel). The method can further include administering a substrate of HSV-TK such as valacyclovir,ganciclovir, acyclovir, penciclovir, valganciclovir, and famciclovir. In certain embodiments, the substrate of HSV-TK is valacyclovir. The subject can be a subject who has been treated with an immune checkpoint inhibitor (ICI). The subject can be a subject who is ICI refractory. The subject can be a subject who has stable disease or progressing disease. In certain embodiments, the cancer is stage III or stage IV non-small cell lung cancer (e.g., squamous or non-squamous). In certain embodiments, the subject has been diagnosed with one or more of stage IV non-small cell lung cancer e.g., squamous or non-squamous), lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1. In certain embodiments, the subject has a survival of at least 10 months after initial treatment with the aglatimagene besadenovec, for example, a survival of at least one year, at least 13 months, at least 14 months, at least 15 months, at least 16 months, at least 17 months, at least 18 months, at least 19 months, at least 20 months, at least 21 months, at least 22 months, at least 23 months, at least 24 months, or more after initial treatment with the aglatimagene besadenovec e.g., one or two doses). In certain embodiments, the lung cancer selected for treatment has low or negative PD-L1 expression.

[0093] The disclosure also relates to methods of treating a subject with advanced lung cancer with a virus, such as aglatimagene besadenovec, wherein the treatment results in improved survival of the subject as compared to a subject receiving standard of care that does not include the aglatimagene besadenovec (e.g., a chemotherapy such as docetaxel). Subjects with advanced lung cancer can include subjects that have been diagnosed with one or more of stage IV lung cancer, lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1. The ECOG (Eastern Cooperative Oncology Group) Performance Status Scale describes a subject’s level of functioning, for example, their ability to care for themselves, daily activity, and physical activity. (See, Azam et al. (2019) CASE REP ONCOL. 12(3):728-736.) ECOG performance status 0 describes a subject who is “fully active, able to carry on all pre-disease performance without restriction.” ECOG performance status 1 describes a subject who is “restricted in physically strenuous activity but ambulatory and able to carry out work of a light or sedentary nature, e.g., light house work, office work.” (Id.) The subject can be a subject who has been treated with an immune checkpoint inhibitor (ICI). The subject can be a subject who is ICI refractory. The subject can be a subject who has stable disease or progressing disease. In certain embodiments, the subject has a survivalof at least 10 months after initial treatment with the aglatimagene besadenovec, for example, a survival of at least one year, 18 months, or 24 months after initial treatment with the aglatimagene besadenovec (e.g., one or two doses). In certain embodiments, the lung cancer selected for treatment has low or negative PD-L1 expression.

[0094] The method can further include administering a substrate of HSV-TK such as valacyclovir, ganciclovir, acyclovir, penciclovir, valganciclovir, and famciclovir. In certain embodiments, the substrate of HSV-TK is valacyclovir.

[0095] The disclosure also relates to methods of treating a subject having a lung cancer with low or negative PD-L1 expression with a therapeutically effective amount of a virus, such as aglatimagene besadenovec. The method can result in improved survival of the subject as compared to a subject receiving standard of care that does not include the aglatimagene besadenovec (e.g., docetaxel). The method can further include administering a substrate of HSV-TK such as valacyclovir, ganciclovir, acyclovir, penciclovir, valganciclovir, and famciclovir. In certain embodiments, the substrate of HSV-TK is valacyclovir. The subject can be a subject who has been treated with an immune checkpoint inhibitor (ICI). The subject can be a subject who is ICI refractory. The subject can be a subject who has stable disease or progressing disease. In certain embodiments, the subject has a survival of at least 10 months after initial treatment with the aglatimagene besadenovec, for example, a survival of at least one year, 18 months, or 24 months after initial treatment with the aglatimagene besadenovec (e.g., one or two doses).

[0096] The disclosure also provides methods of treating a subject with non-squamous non-small cell lung cancer with a therapeutically effective amount of aglatimagene besadenovec. Administration of the aglatimagene besadenovec can, in some embodiments, result in improved survival of the subject as compared to a subject receiving a standard of care treatment without the aglatimagene besadenovec (e.g., a chemotherapy such as docetaxel). Depending upon the circumstance, the aglatimagene besadenovec can be administered intratumorally to a primary tumor (e.g., a lung tumor) or to a secondary intra- thoracic lesion in the lung tissue. In certain embodiments, the aglatimagene besadenovec is administered at least twice to a lung tumor. In certain embodiments, the aglatimagene besadenovec is administered at least twice to the same lung tumor. In certain embodiments, the aglatimagene besadenovec is administered at least twice, at least once to two different lung tumors. Depending upon the circumstances, the aglatimagene besadenovec isadministered to a lymph node. In certain embodiments, the aglatimagene besadenovec is administered at least twice to a lymph node. In certain embodiments, the aglatimagene besadenovec is administered at least twice to the same lymph node. In certain embodiments, the aglatimagene besadenovec is administered at least twice, at least once to two different lymph nodes.

[0097] The disclosure also provides a method of modulating the immune system of a subject with non-resectable, non-small cell lung cancer in need thereof. The method can include administering to the subject a therapeutically effective amount of aglatimagene besadenovec, wherein administration of the aglatimagene besadenovec results in an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment. Administration of the aglatimagene besadenovec can also result in (a) an increase in circulating activated cytotoxic T cells relative to the frequency of circulating activated cytotoxic T cells prior to the treatment, (b) an increase in circulating T helper cells relative to the frequency of circulating T helper cells prior to the treatment, (c) an increase in circulating central memory T cells relative to the frequency of circulating central memory T cells prior to the treatment, (d) an increase in circulating effector memory T cells relative to the frequency of circulating effector memory T cells prior to the treatment, and / or (e) an increase in circulating B cells relative to the frequency of circulating B cells prior to the treatment.

[0098] Modulation of the immune system reflecting an increase in immune activity can be indicative that the treatment is effective, and a subject may continue with the treatment. Alternatively, an increase in immune activity can be indicative that a subject has received sufficient therapy, and the treatment can be discontinued. A lack of modulation of the immune system can be indicative that the treatment is not effective, and the subject may require additional doses of aglatimagene besadenovec for the treatment to be effective. Accordingly, in certain embodiments, a subject may be determined to exhibit no modulation of the immune system, and the subject may be administered additional doses of the aglatimagene besadenovec. Alternatively, in certain embodiments, a lack of modulation of the immune system can be indicative that the treatment is not effective, and an alternative treatment is administered to the subject.

[0099] The disclosure also relates to a method of treating non-resectable, non-small cell lung cancer in a subject who has previously received an aglatimagene besadenovec treatment.The method can include administering to the subject an effective amount of the aglatimagene besadenovec when the subject has been determined to exhibit one or more of:(a) a low frequency of OX40+CD8+T cells;(b) a low frequency of CD56+gd+T cells;(c) high numbers of Tim3+ central memory T cells; and(d) high numbers of CD11C+CD8+ naive and memory T cells; and, optionally, not administering to the subject the aglatimagene besadenovec when the subject does not exhibit at least one of features (a) - (d). Exhibition of one or more of (a) - (d) may be indicative that the subject requires additional doses of aglatimagene besadenovec for the treatment to be effective.

[0100] The disclosure also relates to a method of treating non-resectable, non-small cell lung cancer in a subject who has previously received an aglatimagene besadenovec treatment. The method can include administering to the subject an effective amount of the aglatimagene besadenovec when the subject has been determined to exhibit one or more of:(a) a low frequency of CD8+ and CD4+ T cells;(b) a low frequency of effector T helper cells; and(c) a high level of a protein associated with tumor growth (e.g., ANGPT2, HGF, or CD70); and, optionally, not administering to the subject the aglatimagene besadenovec when the subject exhibits at least one of features (a) - (c).

[0101] The term “therapeutically effective amount” as used herein refers to the amount of an active agent (e.g., a virus according to the present disclosure) sufficient to effect beneficial or desired results in a subject. A therapeutically effective amount can be an amount of an active agent to treat a tumor e.g., a lung tumor) in a subject in need thereof. A therapeutically effective amount can be an amount of an active agent sufficient to effect treatment of a tumor, e.g., amounts that are effective to reduce a targeted tumor size or load, or otherwise hinder the growth rate of tumor cells. More particularly, such terms refer to amounts of the virus that is effective, at the necessary dosages and periods of treatment, to achieve a desired result. For example, in the context of treating a cancer, an effective amount of the compositions described herein is an amount that induces remission, reduces tumor burden, and / or prevents tumor spread or growth of the cancer. Effective amounts may varyaccording to factors such as the subject’s disease state, age, gender, and weight, as well as the pharmaceutical formulation, the route of administration, and the like, but can nevertheless be routinely determined by one skilled in the art. A therapeutically effective amount is not intended to be limited to a particular formulation or administration route.

[0102] As used herein, “treat,” “treating,” and “treatment” refer to the treatment of a disease, disorder, or symptom or manifestation of such (e.g., a lung cancer) in a subject, e.g.. in a human. This includes: (a) preventing a disease or disorder, (b) inhibiting the disease, disorder, etc., i.e., slowing or arresting its progress or development; and (b) relieving the disease, disorder, etc., i.e., causing regression of the disease state.

[0103] The viruses described herein can be administered intratumorally, intravenously, intradermally, by inhalation, transdermally, topically, transmucosal, intrathecally, rectally, orally, buccally, intranasally, intradermally, intrathecally, intracisternally, or intralesionally. One of skill in the art would readily appreciate that the various routes of administration described herein would allow for the virus to be delivered on, in, or near the tumor or targeted cancer cells. One of skill in the art would also readily appreciate that various routes of administration described herein will allow for the virus described herein to be delivered to a region in the vicinity of the tumor or individual cells to be treated. “In the vicinity” can include any tissue or bodily fluid in the subject that is in sufficiently close proximity to the tumor or individual cancer cells such that at least a portion of the virus administered to the subject reach their intended targets and exert their therapeutic effects. A virus can be administered to a site of surgical resection in a subject by, for example, injection directly into the surgical bed after resection of a primary tumor, either before or after closing of the surgical site. Alternatively, as discussed in the Examples herein, the viruses can be injected directly into tumors.

[0104] The amount of virus administered will depend on variables such as the type and extent of disease or indication to be treated, the overall health of the subject, the in vivo potency of the active component, the pharmaceutical formulation, and the route of administration. The initial dosage can be increased beyond the upper level in order to rapidly achieve the desired blood-level or tissue-level. Alternatively, the initial dosage can be smaller than the optimum, and the daily dosage may be progressively increased during the course of treatment. Human dosage can be optimized, e.g., in a conventional Phase I dose escalation study. Dosing frequency can vary, depending on factors such as route ofadministration, dosage amount, and the disease being treated. In certain embodiments, the virus is administered in an amount between about 106PFU and about IO10PFU per subject. In certain embodiments, the virus is administered in an amount between about 5xl09and about 5xl013viral particles (vp) per subject. In certain embodiments, the virus is administered in an amount between about 5xlO10and about 5xl012viral particles (vp) per subject. In certain embodiments, the virus is administered in an amount of about 5x10*1viral particles (vp) per subject. In certain embodiments, one, two, three, four, five, six, seven, eight, nine, ten, or more doses of virus are administered. In certain embodiments, the method includes administering at least two doses of a virus (e.g., aglatimagene besadenovec). In certain embodiments, the method includes administering at least two doses of a virus (e.g., aglatimagene besadenovec) wherein administration of each dose is separated by at least four weeks. In certain embodiments, administration of a virus (e.g., aglatimagene besadenovec) is followed by administration of an effective amount of a substrate for HSV-TK (e.g., valacyclovir). In certain embodiments, the substrate for HSV-TK is administered at a dose of about two grams, three times a day for 15 days after each administration of a virus (e.g., aglatimagene besadenovec).V. Methods of Monitoring a Subject Receiving a Viral Therapy

[0105] The disclosure further relates, in part, to the discovery of biomarkers useful for monitoring, e.g., monitoring the effectiveness or efficacy of an ongoing virus therapy in a subject. The presence of specific markers of immune and / or inflammatory response disclosed herein are indicative of efficacy (e.g., increased survival) in a patient receiving a virus treatment. Accordingly, such biomarkers can be monitored, for example, by measuring the biomarker before and after treatment with the virus. In certain circumstances, the presence of the one or more biomarkers after treatment with the virus is indicative of the effectiveness of the treatment, whereas the absence of at least one of the biomarkers is indicative that the treatment is not effective. This information can be used by a medical practitioner treating the subject on whether to continue, modify, or stop a particular treatment regimen.

[0106] The disclosure also relates to methods of administering a virus to a subject who has been determined to exhibit one or more biomarkers following treatment with a virus (e.g., following an initial treatment with the virus), wherein the one or more biomarkers are indicative that the virus therapy is effective. In certain embodiments, the presence of one ormore biomarkers is indicative that the therapy has resulted in or is resulting in extended survival as compared to a therapy that does not include the virus. In certain embodiments, the presence of one or more biomarkers is indicative that the therapy has resulted in or is resulting in an abscopal effect. Alternatively, for a subject who has been determined not to exhibit one or more such biomarkers following treatment (e.g., following an initial treatment with the virus), a decision can be made around whether the subject should receive another round of virus therapy to induce an increase in the biomarkers associated with extended survival and / or abscopal effect.

[0107] Biomarkers useful in the methods discussed herein include:• an increase in the frequency of circulating ICOS-, CM-, and / or TIGIT-positive T helper and central memory cells relative to the frequency of circulating ICOS-, CM-, and / or TIGIT-positive T helper and central memory cells prior to the treatment;• an increase in circulating activated cytotoxic T cells relative to the frequency of circulating activated cytotoxic T cells prior to the treatment;• an increase in circulating T helper cells relative to the frequency of circulating T helper cells prior to the treatment;• an increase in circulating central memory T cells relative to the frequency of circulating central memory T cells prior to the treatment;• an increase in circulating effector memory T cells relative to the frequency of circulating effector memory T cells prior to the treatment;• an increase in circulating B cells relative to the frequency of circulating B cells prior to the treatment;• an increase in the level of TNFRSF4 and / or TNF-a relative to the relative to the level of TNFRSF4 and / or TNF-a prior to the treatment; and• an increase in CD83 and MCP3 levels in peripheral blood relative to the level of CD83 and MCP3 prior to the treatment.

[0108] As used herein, “frequency” of a given cell type (e.g., a T cell) refers to a measure of the number of cells (e.g., T cells) of that type present in a sample as compared to the total number of cells in the sample. The number of cells in the sample can be determined by anymeans known in the art, for example, using flow cytometry or mass cytometry (e.g., mass cytometry by time-of-flight, “CyTOF”).

[0109] It has now been discovered that an increase in the frequency of circulating ICOS+ T helper and central memory cells, in a subject who previously received a first dose and a second “booster dose” of aglatimagene hesadenovec, relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment is indicative that the treatment will result in extended survival as compared to a subject receiving a standard of care treatment that does not include aglatimagene besadenovec (e.g., a chemotherapy such as docetaxel). Accordingly, the disclosure provides a method of treating lung cancer in a subject who previously received aglatimagene besadenovec treatment (e.g., one or two doses of aglatimagene besadenovec), including administering to the subject an effective amount of the aglatimagene besadenovec when the subject has been determined not to exhibit an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment; and not administering to the subject the aglatimagene besadenovec when the subject exhibits an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment.

[0110] It has also been discovered that an increase in the level of CD83 and MCP3 in the peripheral blood in a subject who previously received a first dose and a second “booster dose” of aglatimagene besadenovec, relative to the level of CD83 and MCP3 in the peripheral blood prior to the treatment is indicative that the treatment will result in extended survival as compared to a subject receiving a standard of care treatment that does not include aglatimagene besadenovec (e.g., a chemotherapy such as docetaxel). Accordingly, the disclosure provides a method of treating lung cancer in a subject who previously received aglatimagene besadenovec treatment (e.g., one or two doses of aglatimagene besadenovec), including administering to the subject an effective amount of the aglatimagene besadenovec when the subject has been determined not to exhibit an increase level of CD83 and MCP3 in the peripheral blood relative to the level of CD83 and MCP3 in the peripheral blood prior to the treatment; and not administering to the subject the aglatimagene besadenovec when the subject exhibits an increase in the level of CD83 and MCP3 in the peripheral blood relative to the level of CD 83 and MCP3 in the peripheral blood prior to the treatment.

[0111] It has also been discovered that an increase in one or more of features (a) - (e) below in a subject who previously received a first dose and a second “booster dose” of aglatimagene besadenovec is indicative that the treatment has resulted in or is resulting in an abscopal effect (i.e., regression (reduction in size) of an untreated, distant tumor).Accordingly, in another aspect, the disclosure provides a method of treating lung cancer in a subject who previously received aglatimagene besadenovec treatment (e.g., one or two doses of aglatimagene besadenovec), including administering to the subject an effective amount of the aglatimagene besadenovec when the subject has been determined not to exhibit one or more of features (a) - (e):(a) an increase in circulating activated cytotoxic T cells relative to the frequency of circulating activated cytotoxic T cells prior to the treatment;(b) an increase in circulating T helper cells relative to the frequency of circulating T helper cells prior to the treatment;(c) an increase in circulating central memory T cells relative to the frequency of circulating central memory T cells prior to the treatment;(d) an increase in circulating effector memory T cells relative to the frequency of circulating effector memory T cells prior to the treatment; and(e) an increase in circulating B cells relative to the frequency of circulating B cells prior to the treatment; and not administering to the subject the aglatimagene besadenovec when the subject has been determined to exhibit at least one of features (a) - (e).

[0112] In another aspect, the disclosure relates to a method of monitoring treatment efficacy in a subject who has or previously had a lung cancer wherein the subject has received at least two doses of an aglatimagene besadenovec therapy. The method includes determining whether the subject exhibits an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment. When the subject does not exhibit an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment, the subject may not have responded to the administered aglatimagene besadenovec, and may benefit from one or more doses of aglatimagene besadenovec to induce immune activation.When the subject does exhibit an increase in the frequency of circulating ICOS+ T helper and central memory cells relative to the frequency of circulating ICOS+ T helper and central memory cells prior to the treatment, the subject may have responded sufficiently and not need further treatment with the aglatimagene besadenovec.

[0113] In another aspect, the disclosure relates to a method of monitoring treatment efficacy in a subject who has or previously had a lung cancer wherein the subject has received at least one dose (e.g., at least two doses) of an aglatimagene besadenovec therapy. The method includes determining whether the subject exhibits one or more of features (a) -(e):(a) an increase in circulating activated cytotoxic T cells relative to the frequency of circulating activated cytotoxic T cells prior to the treatment;(b) an increase in circulating T helper cells relative to the frequency of circulating T helper cells prior to the treatment;(c) an increase in circulating central memory T cells relative to the frequency of circulating central memory T cells prior to the treatment;(d) an increase in circulating effector memory T cells relative to the frequency of circulating effector memory T cells prior to the treatment; and(e) an increase in circulating B cells relative to the frequency of circulating B cells prior to the treatment.When the subject does not exhibits one or more of (a) through (e), the subject may not have responded to the administered aglatimagene besadenovec, and may benefit from one or more doses of aglatimagene besadenovec to induce immune activation. When the subject does exhibit one or more of (a) through (e), the subject may have responded sufficiently and not need further treatment with the aglatimagene besadenovec at that time.

[0114] It has also been discovered that an increase in one or more of features (a) - (d) below in a subject who previously received a first dose and a second “booster dose” of aglatimagene besadenovec inversely correlates with clinical effects, such as overall survival and abscopal effect. Accordingly, the disclosure further relates to a method of monitoring treatment efficacy in a subject who has or previously had a non-resectable, non-small cell lung cancer wherein the subject has received at least two doses of an aglatimagene besadenovec therapy, the method comprising: determining whether the subject exhibits one or more of features (a) - (d):(a) a low frequency of OX40+CD8+T cells;(b) a low frequency of CD56+gd+T cells;(c) high numbers of Tim3+ central memory T cells; and(d) high numbers of CD11C+CD8+ naive and memory T cells,

[0115] wherein, when the subject exhibits one or more of (a) through (d), the subject the subject is administered additional doses of the aglatimagene besadenovec therapy to induce at least one of features (a) - (d), and when the subject does not exhibit one or more of (a) through (d), the subject does not receive further treatment with the aglatimagene besadenovec. As discussed in Example 2 herein, features (a) - (d) are inversely correlated with overall survival and abscopal response. Accordingly, if a subject is determined to exhibit one or more of features (a) - (d), it may be indicative that the treatment is not effective, and the subject may require additional doses of aglatimagene besadenovec for the treatment to be effective. Accordingly, in certain embodiments, a subject is determined to exhibit one or more of (a) - (d), and the subject is administered additional doses of the aglatimagene besadenovec. In certain embodiments, a subject is determined to exhibit one or more of (a) - (d), which can be indicative that the treatment is not effective, and an alternative treatment is administered to the subject.

[0116] If one or more of (a) - (d) is not exhibited by a subject, it may be indicative that the treatment is effective, and the subject may continue with the treatment. Alternatively, a subject not exhibiting one or more of (a) - (d) can indicate that a subject has received sufficient therapy, and the treatment can be discontinued.

[0117] The disclosure further relates to a method of monitoring treatment efficacy in a subject who has or previously had a non-resectable, non-small cell lung cancer wherein the subject has received at least two doses of an aglatimagene besadenovec therapy, the method comprising determining whether the subject exhibits one or more of features (a) - (c):(a) a low frequency of CD8+ and CD4+ T cells;(b) a low frequency of effector T helper cells; and(c) a high level of a protein associated with tumor growth (e.g., ANGPT2, HGF, or CD70),wherein, when the subject exhibits one or more of (a) through (c), the subject the subject is administered additional doses of the aglatimagene besadenovec therapy to induce at least one of features (a) - (c), and when the subject does not exhibit one or more of (a) through (c), the subject does not receive further treatment with the aglatimagene besadenovec.

[0118] As discussed in Example 2 herein, features (a) - (c) are inversely correlated with overall survival and abscopal response. Accordingly, if a subject is determined to exhibit one or more of features (a) - (c), it may be indicative that the treatment is not effective, and the subject may require additional doses of aglatimagene besadenovec for the treatment to be effective. Accordingly, in certain embodiments, a subject is determined to exhibit one or more of (a) - (c), and the subject is administered additional doses of the aglatimagene besadenovec. In certain embodiments, a subject is determined to exhibit one or more of (a) - (c), which can be indicative that the treatment is not effective, and an alternative treatment is administered to the subject.

[0119] As used herein, the term “low frequency” refers to a frequency of cells in a sample from a subject that is less than the median frequency of cells calculated from samples from a group of at least 3 patients (e.g., 42 patients) receiving an equivalent aglatimagene besadenovec treatment. As used herein, “high numbers of’ a given cell type refers to a number of cells in a sample from a subject that is greater than the median number of cells calculated from samples from a group of at least 3 patients (e.g., 42 patients) receiving an equivalent aglatimagene besadenovec treatment. As used herein, “high level of a protein” refers to an amount of protein in a sample from a subject that is greater than the median protein level calculated from samples from a group of at least 3 patients (e.g., 42 patients) receiving an aglatimagene besadenovec treatment.

[0120] Methods for detecting circulating cell (e.g., T cell) frequency and / or numbers of circulating cells are known in the art, and include, for example, flow cytometry and CyTOF (see, for example, EXAMPLE 8 hereinbelow).EXAMPLES

[0121] The disclosure is further illustrated by the following examples. The examples are provided for illustrative purposes only, and are not to be construed as limiting the scope or content of the disclosure in any way.Example 1: Treatment of Non-resectable, Stage III / IV Non-small Cell Lung Cancer (NSCLC) with Aglatimagene Besadenovec (CAN-2409) Plus Valacyclovir and Continued Immune Checkpoint Inhibitor (ICI) in ICI Refractory Patients Resulted in Extended Overall Survival (OS)

[0122] Patients with non-resectable, stage III / IV non-small cell lung cancer (NSCLC) with an inadequate response to immune checkpoint inhibitors (ICI) have very limited therapeutic options. In patients with progressive disease despite ICI treatment, median overall survival (mOS) is less than 12 months with standard of care docetaxel chemotherapy. The present example describes methods of treating non-resectable, stage III / IV NSCLC which improve median overall survival (mOS) in patients treated with two injections. Immunological changes after the second administration (“the booster”) are predictive of subsequent prolonged survival.

[0123] The results show mOS of 20.6 months achieved in ICI refractory patients with progressive disease after two administrations of aglatimagene besadenovec (CAN-2409) plus prodrug (in comparison to a mOS of 11.6 months in a similar patient population receiving a standard of care docetaxel-based chemotherapy (Reckamp (2022), supra)). Improved survival was observed across both PD-L1 positive and PD-L1 negative tumors. A beneficial effect on both injected and uninjected tumors in more than 70% of the patients with metastatic disease and at least one uninjected tumor was also observed. In addition, a significant increase in circulating CD8+ cytotoxic and CD4+ effector and central memory T cells and increased soluble granzyme B levels in peripheral blood after the second (“booster”) injection of CAN-2409, associated with subsequent prolonged survival. In addition, a significant increase in CD83 and MCP3 levels in peripheral blood after the second (“booster”) injection of CAN-2409 were associated with subsequent prolonged survival.

[0124] FIG. IB shows the protocol for an open-label, phase 2 clinical trial of CAN-2409 + valacyclovir in combination with continued ICI in patients with non-resectable, stage III / IV NSCLC, refractory or resistant to anti-PD-(L)l. CAN-2409 is a replication-defective adenovirus encoding the Herpes Simplex Virus thymidine kinase (HSV-TK) gene which converts a substrate, valacyclovir, into a toxic nucleotide analogue, which blocks DNA synthesis in dividing cells. Patients were enrolled in 2 cohorts (C) depending on disease status at enrollment: Cl, stable disease or C2, progressive disease. Two doses of CAN-2409 (5xl0! 1vp) were given 5-7 weeks apart via bronchoscopic or percutaneous injection into lungtumor, disease-positive lymph node, or peripheral metastasis, each followed by oral prodrug. Patients were assessed for safety, immunologic biomarkers, and OS.

[0125] FIG. 2 is a schematic representation of patient enrollment. The safety population received at least 1 injection of CAN-2409. Evaluable patients were those who received 2 courses of CAN-2409 + valacyclovir and completed the 12- week treatment period. In addition to patients evaluable for efficacy, the safety population included two cohort 1 patients who did not complete treatment due to adverse events (one pneumonitis possibly study drug related, and one pulmonary embolism unrelated to study drug). In cohort 2, four patients did not complete treatment due to AE (one possibly related empyema, one case of pneumonia, pre-syncope, and bullous dermatitis unrelated to study drug), 15 due to disease progression before they completed the treatment course, two who had incomplete treatment with valacyclovir, and four who withdrew for personal reasons. TABLE 1 summarizes the patient demographics of the study population.TABLE 1: Patient demographics

[0126] TABLE 2 summarizes the safety and tolerability profile of CAN-2409.TABLE 2: Most common TRAEs in >5% of patients

[0127] FIG. 3 is a swimmer plot of long-term survival in CAN-2409 treated patients, across cohorts 1 and 2. Similarly, in FIG. 4 and TABLE 3 show evidence that CAN-2409 can control disease. Data shown based on blinded independent central review (BICR) of radiographic data per RECIST 1 .1 in evaluable patients. Median (range) for DoR and SD duration. “+” indicates response was ongoing at date of last follow up. PR: partial response, SD: stable disease, PD: progressive disease, ORR: objective response rate, DCR: disease control rate, DoR: duration of response.TABLE 3: Evidence that CAN-2409 can control disease

[0128] FIGs. 5A-C show CAN-2409 improves overall survival in patients presenting with poor response to ICI. Kaplan Meier curves here show survival in evaluable patients (2 injections and complete 12-week treatment window). Cohorts 1 and 2 safety population mOS is 14.3 mos (mFU 19.8 mos); cohort 2 safety mOS is 13.7 mos (mFU19.2 mos). FIGs.6A-6B show case studies, PA-003 and MU-002. PA-003 exhibited a partial response extended in lung mass with durable after treatment tumor regression after CAN-2409 treatment (> 30 mOS, ongoing). MU-002 showed a partial response and evidence of abscopal effect and OS 30.5 months (ongoing).

[0129] FIGs. 7A-7B demonstrate local injection of CAN-2409 induces systemic antitumor activity. Systemic response or abscopal response (decrease of non-injected lesions) was measured on all evaluable patients with at least 1 non-injected lesion (n=35). Abscopal response (any decrease in non-injected lesion) was observed in two-thirds of patients; when using a threshold of >10% decrease still more than 50% of patients showed abscopal response. Abscopal response was associated with improved survival.

[0130] FIGs. 8A-8B demonstrate CAN-2409 induces significant increases in circulating T helper and cytotoxic T cells. CyTOF and flow cytometry analysis (FIGs. 8A-8B; 29 evaluable patients) unveiled significant changes after first and second CAN-2409 injections in both T helper and cytotoxic T cells population. FIGs. 9A-9B demonstrates CAN-2409 induces a significant increase in circulating inflammatory mediators including granzyme A, B and H. As such, OLINK proteomic analysis revealed significant increases in proinflammatory mediators and cytotoxic enzymes (granzyme A, B and H).

[0131] FIGs. 10A-10D are charts of immune activation after second CAN-2409 injection (‘booster’) and show an association with prolonged survival. CyTOF analysis (FIG. 10A) demonstrated significant increase in ICOS+ T helper and central memory T helper cells inlong survivors (OS >24 months). Increase was measured between week 8 (after 2nd injection) and baseline. Flow cytometry analysis (FIG. 10B) revealed a significant increase in activated cytotoxic T cells, T helper cells, central and effector memory T cells in long survivors (OS >24 months). Increase was measured between week 8 (after 2nd injection) and week 6 (pre 2nd injection). FIGs. IOC and 10D show OLINK analysis which revealed an increase in circulating levels of granzyme H (p<0.03) (FIG. IOC) and granzyme B (n.s.) (FIG. 10D) in long survivors. Increase was measured between week 8 (after 2nd injection) and week 6 (pre 2nd injection). Volcano plots represent t-tests performed for all markers. Above the red line are represented statistically significant results (uncorrected for multiple testing). Meanwhile, CAN-2409 injections into lung lesions (LL) and disease-positive lymph nodes (LN) were associated with more CM- and TIGIT-positive central memory T cells, as well as more co-stimulatory and co-inflammatory molecules including TNFRSF4 and TNF-a, as compared to peripheral metastasis injections (data not shown).

[0132] FIG. 10E shows the abscopal response is associated with increase in circulating effector T cells. Flow cytometry demonstrated significant upregulation in activated cytotoxic T cells, T helper cells, central and effector memory T cells in patients with abscopal response as compared to patients without abscopal response. Identified markers largely overlap with markers associated with long term survival. Volcano plots represent t-tests performed for all markers. Above the red line are represented statistically significant results (uncorrected for multiple testing).Example 2: Advanced analytics identify distinct immunological profiles associated with histologic subset and site of injection for CAN-2409 treatment in patients with progressive NSCLC despite ICI treatment

[0133] As described in Example 1, two injections of CAN-2409 + prodrug may prolong overall survival (OS) in patients with non-resectable, stage 111 / IV non-small cell lung cancer (NSCLC) progressing after immune checkpoint inhibitor (ICI) treatment, nearly doubling median OS compared to historical second line chemotherapy. The data from Example 1 showed that CAN-2409 injections into lung lesions (LL) or disease positive-lymph nodes (LN) are associated with improved OS. Non-squamous (NSq) and squamous cell (Sq) carcinomas represent distinct subtypes of NSCLC with different responses to ICI treatment. Here, advanced analytics were used to profile baseline characteristics and immunologicalchanges associated with specific CAN-2409 sites of injection and histologic subtypes, and their relationship to OS.

[0134] A machine learning algorithm, Multi-Omics Factor Analysis (MOFA) (Argelaguet et al. (2020) “M0FA+: a statistical framework for comprehensive integration of multi-modal single-cell data,” GENOME BIOL. 21 : 111), was applied to biomarker data including flow cytometry, CyTOF, and Olink proteomics based on analysis of biosamples from 41 patients who received two doses of CAN-2409 into LL (n=15), LN (n=13), peripheral metastasis (PM) (n=9), or different sites (DS) (n=4). 3,097 features derived from available data were integrated into 22 low-dimensional mathematical representations of multi-omic biological program “factors” across different modalities associated with clinical features and responses, including OS and abscopal response.

[0135] FIG. 11A shows a heatmap illustrating contribution of different data modalities to each MOFA identified factor. Principal component analysis was used to interrogate the ability of factor 2 and 21 to separate patient according to survival, and the results are shown in FIG. 11B.

[0136] MOFA identified certain factors differently associated with either histologic subtype, site of injection, or overall survival (FIG. 11C; heatmap representing Pearson r). Among these, factor 2 and 21 positively correlated with Sq histology and inversely correlated with NSq, LL injection and clinical outcomes (OS and abscopal response). Factor 2 captured integrated features including low frequency of OX40+CD8+Tcells and CD56+gd+T cells (CyTOF), high numbers of Tim3+ central memory T cells and CDl lc+CD8+ naive and memory T cells. Factor 21 captured, in post treatment samples, low frequency of CD8+ and CD4+T cells and effector T helper cells (CyTOF and flow) and high levels of proteins associated with tumor growth, including ANGPT2, HGF and CD70 (Olink). There was a trend towards improved mOS in NSq vs. Sq (mOS 24.5 months vs. mOS 14.3 months, respectively; p=0.135) (FIGs. 12A-B). Lung lesion delivery further improved mOS compared to other injection sites in NSq (24-month mOS for NSq= 76.9% in LL, 29.6% in LN, 27.8% in PM and 0% in DS), but not in Sq (FIGs. 12A-B). Lymph node delivery also improved OS relative to peripheral metastasis injection.

[0137] FIG. 13 and TABLE 4 show a survival curve and present survival data, respectively, in the Cohort 2 patients with non-resectable, stage IV non-squamous NSCLCprogressing after immune checkpoint inhibitor (ICI) treatment, which demonstrated that lung and lymph node delivery improved OS relative to peripheral metastasis injection.TABLE 4: Cohort 2 evaluable stage IV non-squamous by injection site

[0138] To determine whether a patient exhibited low frequencies or high numbers of a particular cell type, the median of flow frequencies and CyTOF frequencies for specific cell types was calculated and exemplary data for Factor 2 is shown in TABLE 5 and TABLE 6. Patients exhibiting low frequency of a given cell type exhibited a frequency below the median. Patients exhibiting a high number of a given cell type exhibited more than the median number of cells.

[0139] TABLE 5 provides median of flow cytometry frequencies used in the MOFA analysis, obtained from PBMC samples from 42 patients with Stage III and IV NSCLC, nonresponding to PD-1, treated with 2 injections of CAN-2409 and valacyclovir. Samples were obtained at different time points. TO is a baseline sample prior to CAN-2409 trial, T1 is a sample obtained after the 1st injection, T3 is a sample obtained after the 2nd injection, and T4 and T5 are follow up samples.TABLE 5: Median of flow cytometry frequencies

[0140] TABLE 6 provides the median of CyTOF frequencies obtained from PBMC samples from 42 patients with Stage III and IV NSCLC, non-responding to PD-1, treated with 2 injections of CAN-2409 and valacyclovir. Samples were obtained at different time points. TO is a baseline sample prior to CAN-2409 trial, T1 is a sample obtained after the 1st injection, T3 is a sample obtained after the 2nd injection, and T4 and T5 are follow up samples.TABLE 6: Median of CyTQF frequencies obtained from PBMC samples

[0141] CAN-2409+prodrug is associated with prolonged survival in NSCLC patients with progressive disease despite ICI treatment. Multimodal analysis revealed that patients with non-squamous histology who received two injections into lung lesions had a distinct immunologic response and further improved survival compared to patients with squamous histology.INCORPORATION BY REFERENCE

[0142] The entire disclosure of each of the patent documents and scientific articles cited herein is incorporated by reference for all purposes.EQUIVALENTS

[0143] The disclosure can be embodied in other specific forms without departing from the essential characteristics thereof. The foregoing embodiments therefore are to be considered illustrative rather than limiting on the disclosure described herein. The scope of the disclosure is indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

CLAIMS1. A method of treating a subject with non-resectable, non-small cell lung cancer in need thereof, the method comprising: administering to a disease-positive lymph node of the subject a therapeutically effective amount of aglatimagene besadenovec, wherein administration of the aglatimagene besadenovec results in improved survival of the subject as compared to a subject receiving a standard of care treatment (e.g., docetaxel) without the aglatimagene besadenovec.

2. The method of claim 1 , wherein the aglatimagene besadenovec is administered to the disease-positive lymph node percutaneously.

3. The method of claim 1 or 2, wherein the cancer is stage III or stage IV non-small cell lung cancer.

4. The method of claim 3, wherein the histology of the cancer is squamous or non-squamous.

5. The method of claim 4, wherein the histology of the cancer is non-squamous.

6. The method of any one of claims 3-5, wherein the subject has been diagnosed with one or more of: stage IV non-small cell lung cancer, lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1.

7. The method of any one of claims 1-6, wherein the subject has a survival of at least 10 months after initial treatment with the aglatimagene besadenovec.

8. The method of claim 7, wherein the subject has a survival of at least one year, 18 months, or 24 months after initial treatment with the aglatimagene besadenovec.

9. The method of any one of claims 1-8, wherein lung cancer selected for treatment has low or negative PD-L1 expression.

10. The method of any one of claims 1-9, wherein the subject’s cancer has progressed on immune checkpoint inhibitor therapy.

11. The method of any one of claims 1-10, wherein the method further comprises administering to the subject a substrate of Herpes Simplex Virus thymidine kinase (HSV- TK).

12. The method of claim 11, wherein the substrate is valacyclovir.

13. Aglatimagene besadenovec for use in treating a subject with non-resectable, non-small cell lung cancer, the use comprising: administering to a disease-positive lymph node of the subject a therapeutically effective amount of aglatimagene besadenovec, wherein administration of the aglatimagene besadenovec results in improved survival of the subject as compared to a subject receiving a standard of care treatment (e.g., docetaxel) without the aglatimagene besadenovec.

14. The use of claim 13, wherein the aglatimagene besadenovec is administered to the disease-positive lymph node percutaneously.

15. The use of claim 13 or 14, wherein the cancer is stage III or stage IV non-small cell lung cancer.

16. The use of claim 15, wherein the histology of the cancer is squamous or non-squamous.

17. The use of claim 16, wherein the histology of the cancer is non-squamous.

18. The use of any one of claims 15-17, wherein the subject has been diagnosed with one or more of: stage IV non-small cell lung cancer, lymph node involvement, pleural effusion, bone metastasis, adrenal metastases, brain metastases, liver metastases, involvement of three or more organs, and ECOG performance status of 0 or 1.

19. The use of any one of claims 13-18, wherein the subject has a survival of at least 10 months after initial treatment with the aglatimagene besadenovec.

20. The use of claim 19, wherein the subject has a survival of at least one year, 18 months, or 24 months after initial treatment with the aglatimagene besadenovec.

21. The use of any one of claims 13-20, wherein lung cancer selected for treatment has low or negative PD-L1 expression.

22. The use of any one of claims 13-21, wherein the subject’s cancer has progressed on immune checkpoint inhibitor therapy.

23. The use of any one of claims 13-22, wherein the use further comprises administering to the subject a substrate of Herpes Simplex Virus thymidine kinase (HSV-TK).

24. The use of claim 23, wherein the substrate is valacyclovir.

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