Neoadjuvant Gene-Mediated Cytotoxic Immunotherapy for Non-Small-Cell Lung Cancer: Safety and Immunologic Activity

Jarrod D Predina, Andrew R Haas, Marina Martinez, Shaun O'Brien, Edmund K Moon, Patrick Woodruff, Jason Stadanlick, Christopher Corbett, Lydia Frenzel-Sulyok, Mitchell G Bryski, Evgeniy Eruslanov, Charuhas Deshpande, Corey Langer, Laura K Aguilar, Brian W Guzik, Andrea G Manzanera, Estuardo Aguilar-Cordova, Sunil Singhal, Steven M Albelda, Jarrod D Predina, Andrew R Haas, Marina Martinez, Shaun O'Brien, Edmund K Moon, Patrick Woodruff, Jason Stadanlick, Christopher Corbett, Lydia Frenzel-Sulyok, Mitchell G Bryski, Evgeniy Eruslanov, Charuhas Deshpande, Corey Langer, Laura K Aguilar, Brian W Guzik, Andrea G Manzanera, Estuardo Aguilar-Cordova, Sunil Singhal, Steven M Albelda

Abstract

Gene-mediated cytotoxic immunotherapy (GMCI) is an immuno-oncology approach involving local delivery of a replication-deficient adenovirus expressing herpes simplex thymidine kinase (AdV-tk) followed by anti-herpetic prodrug activation that promotes immunogenic tumor cell death, antigen-presenting cell activation, and T cell stimulation. This phase I dose-escalation pilot trial assessed bronchoscopic delivery of AdV-tk in patients with suspected lung cancer who were candidates for surgery. A single intra-tumoral AdV-tk injection in three dose cohorts (maximum 1012 viral particles) was performed during diagnostic staging, followed by a 14-day course of the prodrug valacyclovir, and subsequent surgery 1 week later. Twelve patients participated after appropriate informed consent. Vector-related adverse events were minimal. Immune biomarkers were evaluated in tumor and blood before and after GMCI. Significantly increased infiltration of CD8+ T cells was found in resected tumors. Expression of activation, inhibitory, and proliferation markers, such as human leukocyte antigen (HLA)-DR, CD38, Ki67, PD-1, CD39, and CTLA-4, were significantly increased in both the tumor and peripheral CD8+ T cells. Thus, intratumoral AdV-tk injection into non-small-cell lung cancer (NSCLC) proved safe and feasible, and it effectively induced CD8+ T cell activation. These data provide a foundation for additional clinical trials of GMCI for lung cancer patients with potential benefit if combined with other immune therapies.

Keywords: PD-1; PD-L1; adenovirus; checkpoint inhibitor; gene therapy; gene-mediated cytotoxic immunotherapy; immunotherapy; intratumoral immunotherapy; lung cancer; neoadjuvant clinical trial.

Conflict of interest statement

Declaration of Interests S.O., M.M., and S.M.A. were partially supported by a clinical trial agreement with Advantagene. L.K.A., B.W.G., A.G.M., and E.A.-C. are employees of Advantagene, who sponsored the trial. The remaining authors declare no competing interests.

Copyright © 2020 The Authors. Published by Elsevier Inc. All rights reserved.

Figures

Graphical abstract
Graphical abstract
Figure 1
Figure 1
CT Scans of Tumor before and after AdV-tk Administration Thoracic CT scans from PT 3LU02P show a reduction in size of the pleural-based tumor (white arrows) when the scan from pre-AdV-tk administration (A) is compared to the scan immediately prior to surgery 3 weeks later (B).
Figure 2
Figure 2
Effect of Neoadjuvant GMCI on Types of TILs Left panels: samples from baseline tumor needle biopsies taken at the time of vector injection (called the “pre-Rx” sample) are compared to the surgical specimens (called the “post-Rx” sample). The percentage of specific types of T cells of live cells in tumor biopsy samples are plotted. The following markers are plotted: CD3+ cells, CD8+ cells, CD4+ cells, and CD4+ regulatory T cells (CD4+/FOXP3+ cells). Paired t tests were applied to define the statistical significance (p value) of the changes. Right panels: the percentages of specific types of T cells of live cells in the tumor biopsy sample from the current study (Ad.TK) were compared with values from a recent independent study where TIL phenotype and function were analyzed in 44 early-stage lung cancers using the same flow cytometry panels and protocols (called “control” samples). Student’s t tests were applied to define the statistical significance (p value) of the differences.
Figure 3
Figure 3
Effect of Neoadjuvant GMCI on Activation Markers and Cytokine Production in CD8+ TILs Using the same samples as described in Figure 2, the expression of three proliferation/activation markers (Ki67, CD38, and HLA-DR) on the CD8+ TILs in the pre-Rx samples were compared to post-Rx tumor samples. Paired t tests were applied to define the statistical significance (p value) of the changes. The upper right panel shows the comparison between the Ki67 expression in this trial compared to our lung cancer control samples. A Student’s t test was applied to define the statistical significance (p value) of the differences.
Figure 4
Figure 4
Effect of Neoadjuvant GMCI on Inhibitory/Activation Markers in CD8+ TILs Left panels: using the same samples as described in Figure 2, the expression of five inhibitory/activation markers (PD-1, CD39, CTLA-4, TIM3, and TIGIT) on CD8+ T cells in the pre-Rx samples were compared to post-Rx tumor samples. Paired t tests were applied to define the statistical significance (p value) of the changes. Right panels: the percentages of specific markers on CD8+ T cells from the current study (Ad.TK) were compared with values from a recent independent study, where TIL phenotype and function were analyzed in 44 early-stage lung cancers using the same flow cytometry panels and protocols (control samples). Student’s t tests were applied to define the statistical significance (p value) of the differences.
Figure 5
Figure 5
Effects of Neoadjuvant GMCI on T Cells in the Blood Expression of T cell markers was assessed on baseline peripheral blood mononuclear cells (PBMCs) obtained from each patient prior to vector injection and compared to PBMCs obtained at the time of surgery (19–22 days after vector delivery). Paired t tests were applied to define the statistical significance (p value) of the change. (A) The percentages of specific types of T cells within the live PBMC population are plotted: upper left, CD3+ T cells; upper right, CD8+ T cells; lower left, CD4+ T cells; lower right, CD4+ regulatory T cells (CD4+/FOXP3+ cells). (B) The percentage of CD8+ T cells expressing specific activation/proliferation markers are plotted: upper left, CD38; upper right, HLA-DR; lower left, Ki67 (proliferation marker); lower right, 41BB (CD137). (C) The percentage of CD8+ T cells expressing specific inhibitory/activation markers are plotted: upper left, PD-1; upper right, CD39; lower left, CTLA-4; lower right, TIGIT.
Figure 6
Figure 6
Flow Cytometry Tracings Showing Activation Markers on CD8+ T Cells in PBMCs from Patient 3LU13P Examples of flow tracings from the CD8+ T cells of pre-treatment (day 0, upper tracings) and post-treatment (day 21, lower tracings) in the PBMCs from one patient (3LU13P) are shown. Expression of HLA-DR, Ki67, and CD38 are shown as marked.

References

    1. Siegel R.L., Miller K.D., Jemal A. Cancer statistics, 2017. CA Cancer J. Clin. 2017;67:7–30.
    1. Aliperti L.A., Predina J.D., Vachani A., Singhal S. Local and systemic recurrence is the Achilles heel of cancer surgery. Ann. Surg. Oncol. 2011;18:603–607.
    1. Pignon J.P., Tribodet H., Scagliotti G.V., Douillard J.-Y., Shepherd F.A., Stephens R.J., Dunant A., Torri V., Rosell R., Seymour L., LACE Collaborative Group Lung adjuvant cisplatin evaluation: a pooled analysis by the LACE Collaborative Group. J. Clin. Oncol. 2008;26:3552–3559.
    1. Wakelee H.A., Dahlberg S.E., Keller S.M., Tester W.J., Gandara D.R., Graziano S.L., Adjei A.A., Leighl N.B., Aisner S.C., Rothman J.M., ECOG-ACRIN Adjuvant chemotherapy with or without bevacizumab in patients with resected non-small-cell lung cancer (E1505): an open-label, multicentre, randomised, phase 3 trial. Lancet Oncol. 2017;18:1610–1623.
    1. Reck M., Rodríguez-Abreu D., Robinson A.G., Hui R., Csőszi T., Fülöp A., Gottfried M., Peled N., Tafreshi A., Cuffe S. Updated analysis of KEYNOTE-024: pembrolizumab versus platinum-based chemotherapy for advanced non-small-cell lung cancer with PD-L1 tumor proportion score of 50% or greater. J. Clin. Oncol. 2019;37:537–546.
    1. Gandhi L., Rodríguez-Abreu D., Gadgeel S., Esteban E., Felip E., De Angelis F., Domine M., Clingan P., Hochmair M.J., Powell S.F., KEYNOTE-189 Investigators Pembrolizumab plus chemotherapy in metastatic non-small-cell lung cancer. N. Engl. J. Med. 2018;378:2078–2092.
    1. van der Burg S.H., Arens R., Ossendorp F., van Hall T., Melief C.J.M. Vaccines for established cancer: overcoming the challenges posed by immune evasion. Nat. Rev. Cancer. 2016;16:219–233.
    1. Hammerich L., Bhardwaj N., Kohrt H.E., Brody J.D. In situ vaccination for the treatment of cancer. Immunotherapy. 2016;8:315–330.
    1. Sheen M.R., Fiering S. In situ vaccination: harvesting low hanging fruit on the cancer immunotherapy tree. Wiley Interdiscip. Rev. Nanomed. Nanobiotechnol. 2019;11:e1524.
    1. Aguilar L.K., Guzik B.W., Aguilar-Cordova E. Cytotoxic immunotherapy strategies for cancer: mechanisms and clinical development. J. Cell. Biochem. 2011;112:1969–1977.
    1. Shaw A.R., Suzuki M. Immunology of adenoviral vectors in cancer therapy. Mol. Ther. Methods Clin. Dev. 2019;15:418–429.
    1. Eastham J.A., Chen S.H., Sehgal I., Yang G., Timme T.L., Hall S.J., Woo S.L., Thompson T.C. Prostate cancer gene therapy: herpes simplex virus thymidine kinase gene transduction followed by ganciclovir in mouse and human prostate cancer models. Hum. Gene Ther. 1996;7:515–523.
    1. Predina J.D., Judy B., Aliperti L.A., Fridlender Z.G., Blouin A., Kapoor V., Laguna B., Nakagawa H., Rustgi A.K., Aguilar L. Neoadjuvant in situ gene-mediated cytotoxic immunotherapy improves postoperative outcomes in novel syngeneic esophageal carcinoma models. Cancer Gene Ther. 2011;18:871–883.
    1. Predina J.D., Kapoor V., Judy B.F., Cheng G., Fridlender Z.G., Albelda S.M., Singhal S. Cytoreduction surgery reduces systemic myeloid suppressor cell populations and restores intratumoral immunotherapy effectiveness. J. Hematol. Oncol. 2012;5:34–45.
    1. Speranza M.C., Passaro C., Ricklefs F., Kasai K., Klein S.R., Nakashima H., Kaufmann J.K., Ahmed A.K., Nowicki M.O., Obi P. Preclinical investigation of combined gene-mediated cytotoxic immunotherapy and immune checkpoint blockade in glioblastoma. Neuro-oncol. 2018;20:225–235.
    1. Predina J.D., Judy B., Fridlender Z.G., Aliperti L.A., Madajewski B., Kapoor V., Cheng G., Quatromoni J., Okusanya O., Singhal S. A positive-margin resection model recreates the postsurgical tumor microenvironment and is a reliable model for adjuvant therapy evaluation. Cancer Biol. Ther. 2012;13:745–755.
    1. Predina J., Eruslanov E., Judy B., Kapoor V., Cheng G., Wang L.C., Sun J., Moon E.K., Fridlender Z.G., Albelda S., Singhal S. Changes in the local tumor microenvironment in recurrent cancers may explain the failure of vaccines after surgery. Proc. Natl. Acad. Sci. USA. 2013;110:E415–E424.
    1. Wheeler L.A., Manzanera A.G., Bell S.D., Cavaliere R., McGregor J.M., Grecula J.C., Newton H.B., Lo S.S., Badie B., Portnow J. Phase II multicenter study of gene-mediated cytotoxic immunotherapy as adjuvant to surgical resection for newly diagnosed malignant glioma. Neuro-oncol. 2016;18:1137–1145.
    1. Chiocca E.A., Aguilar L.K., Bell S.D., Kaur B., Hardcastle J., Cavaliere R., McGregor J., Lo S., Ray-Chaudhuri A., Chakravarti A. Phase IB study of gene-mediated cytotoxic immunotherapy adjuvant to up-front surgery and intensive timing radiation for malignant glioma. J. Clin. Oncol. 2011;29:3611–3619.
    1. Aguilar L.K., Shirley L.A., Chung V.M., Marsh C.L., Walker J., Coyle W., Marx H., Bekaii-Saab T., Lesinski G.B., Swanson B. Gene-mediated cytotoxic immunotherapy as adjuvant to surgery or chemoradiation for pancreatic adenocarcinoma. Cancer Immunol. Immunother. 2015;64:727–736.
    1. Ayala G., Satoh T., Li R., Shalev M., Gdor Y., Aguilar-Cordova E., Frolov A., Wheeler T.M., Miles B.J., Rauen K. Biological response determinants in HSV-tk + ganciclovir gene therapy for prostate cancer. Mol. Ther. 2006;13:716–728.
    1. Aggarwal C., Haas A.R., Metzger S., Aguilar L.K., Aguilar-Cordova E., Manzanera A.G., Gómez-Hernández G., Katz S.I., Alley E.W., Evans T.L. Phase I study of intrapleural gene-mediated cytotoxic immunotherapy in patients with malignant pleural effusion. Mol. Ther. 2018;26:1198–1205.
    1. Kruklitis R.J., Singhal S., Delong P., Kapoor V., Sterman D.H., Kaiser L.R., Albelda S.M. Immuno-gene therapy with interferon-β before surgical debulking delays recurrence and improves survival in a murine model of malignant mesothelioma. J. Thorac. Cardiovasc. Surg. 2004;127:123–130.
    1. O’Brien S.M., Klampatsa A., Thompson J.C., Martinez M.C., Hwang W.T., Rao A.S., Standalick J.E., Kim S., Cantu E., Litzky L.A. Function of human tumor-infiltrating lymphocytes in early stage non-small cell lung cancer. Cancer Immunol. Res. 2019;7:896–909.
    1. Miller J.D., van der Most R.G., Akondy R.S., Glidewell J.T., Albott S., Masopust D., Murali-Krishna K., Mahar P.L., Edupuganti S., Lalor S. Human effector and memory CD8+ T cell responses to smallpox and yellow fever vaccines. Immunity. 2008;28:710–722.
    1. Griscelli F., Opolon P., Saulnier P., Mami-Chouaib F., Gautier E., Echchakir H., Angevin E., Le Chevalier T., Bataille V., Squiban P. Recombinant adenovirus shedding after intratumoral gene transfer in lung cancer patients. Gene Ther. 2003;10:386–395.
    1. Nemunaitis J., Swisher S.G., Timmons T., Connors D., Mack M., Doerksen L., Weill D., Wait J., Lawrence D.D., Kemp B.L. Adenovirus-mediated p53 gene transfer in sequence with cisplatin to tumors of patients with non-small-cell lung cancer. J. Clin. Oncol. 2000;18:609–622.
    1. Swisher S.G., Roth J.A., Komaki R., Gu J., Lee J.J., Hicks M., Ro J.Y., Hong W.K., Merritt J.A., Ahrar K. Induction of p53-regulated genes and tumor regression in lung cancer patients after intratumoral delivery of adenoviral p53 (INGN 201) and radiation therapy. Clin. Cancer Res. 2003;9:93–101.
    1. Nemunaitis J., Meyers T., Senzer N., Cunningham C., West H., Vallieres E., Anthony S., Vukelja S., Berman B., Tully H. Phase I trial of sequential administration of recombinant DNA and adenovirus expressing L523S protein in early stage non-small-cell lung cancer. Mol. Ther. 2006;13:1185–1191.
    1. Duhen T., Duhen R., Montler R., Moses J., Moudgil T., de Miranda N.F., Goodall C.P., Blair T.C., Fox B.A., McDermott J.E. Co-expression of CD39 and CD103 identifies tumor-reactive CD8 T cells in human solid tumors. Nat. Commun. 2018;9:2724.
    1. Simoni Y., Becht E., Fehlings M., Loh C.Y., Koo S.-L., Teng K.W.W., Yeong J.P.S., Nahar R., Zhang T., Kared H. Bystander CD8+ T cells are abundant and phenotypically distinct in human tumour infiltrates. Nature. 2018;557:575–579.
    1. Sterman D.H., Recio A., Vachani A., Sun J., Cheung L., DeLong P., Amin K.M., Litzky L.A., Wilson J.M., Kaiser L.R., Albelda S.M. Long-term follow-up of patients with malignant pleural mesothelioma receiving high-dose adenovirus herpes simplex thymidine kinase/ganciclovir suicide gene therapy. Clin. Cancer Res. 2005;11:7444–7453.
    1. Rojas-Martínez A., Manzanera A.G., Sukin S.W., Esteban-María J., González-Guerrero J.F., Gomez-Guerra L., Garza-Guajardo R., Flores-Gutiérrez J.P., Elizondo Riojas G., Delgado-Enciso I. Intraprostatic distribution and long-term follow-up after AdV-tk immunotherapy as neoadjuvant to surgery in patients with prostate cancer. Cancer Gene Ther. 2013;20:642–649.
    1. Hall S.J., Mutchnik S.E., Chen S.H., Woo S.L., Thompson T.C. Adenovirus-mediated herpes simplex virus thymidine kinase gene and ganciclovir therapy leads to systemic activity against spontaneous and induced metastasis in an orthotopic mouse model of prostate cancer. Int. J. Cancer. 1997;70:183–187.
    1. Sterman D.H., Alley E., Stevenson J.P., Friedberg J., Metzger S., Recio A., Moon E.K., Haas A.R., Vachani A., Katz S.I. Pilot and feasibility trial evaluating immuno-gene therapy of malignant mesothelioma using intrapleural delivery of adenovirus-IFNα combined with chemotherapy. Clin. Cancer Res. 2016;22:3791–3800.
    1. Chen C.-Y., Hutzen B., Wedekind M.F., Cripe T.P. Oncolytic virus and PD-1/PD-L1 blockade combination therapy. Oncolytic Virother. 2018;7:65–77.
    1. Woller N., Gürlevik E., Fleischmann-Mundt B., Schumacher A., Knocke S., Kloos A.M., Saborowski M., Geffers R., Manns M.P., Wirth T.C. Viral infection of tumors overcomes resistance to PD-1-immunotherapy by broadening neoantigenome-directed t-cell responses. Mol. Ther. 2015;23:1630–1640.
    1. Kuryk L., Møller A.S.W., Jaderberg M. Combination of immunogenic oncolytic adenovirus ONCOS-102 with anti-PD-1 pembrolizumab exhibits synergistic antitumor effect in humanized A2058 melanoma huNOG mouse model. Oncoimmunology. 2019;8:e1532763.
    1. Shin S.-P., Seo H.-H., Shin J.-H., Park H.-B., Lim D.-P., Eom H.S., Bae Y.S., Kim I.H., Choi K., Lee S.J. Adenovirus expressing both thymidine kinase and soluble PD1 enhances antitumor immunity by strengthening CD8 T-cell response. Mol. Ther. 2013;21:688–695.
    1. Fransen M.F., Schoonderwoerd M., Knopf P., Camps G.M., Hawinkels L., Keneilling M., van Hall T., Ossendorp F. Tumor draining lymph nodes are pivotal in PD-1/PD-L1 checkpoint therapy. JCI Insight. 2018;3:e124507.
    1. Chévez-Barrios P., Chintagumpala M., Mieler W., Paysse E., Boniuk M., Kozinetz C., Hurwitz M.Y., Hurwitz R.L. Response of retinoblastoma with vitreous tumor seeding to adenovirus-mediated delivery of thymidine kinase followed by ganciclovir. J. Clin. Oncol. 2005;23:7927–7935.
    1. National Institutes of Health . 2010. Common terminology criteria for adverse events (CTCAE) v4.0.
    1. Quatromoni J.G., Singhal S., Bhojnagarwala P., Hancock W.W., Albelda S.M., Eruslanov E. An optimized disaggregation method for human lung tumors that preserves the phenotype and function of the immune cells. J. Leukoc. Biol. 2015;97:201–209.
    1. Klampatsa A., O’Brien S.M., Thompson J.C., Rao A.S., Stadanlick J.E., Martinez M.C., Liousia M., Cantu E., Cengel K., Moon E.K. Phenotypic and functional analysis of malignant mesothelioma tumor-infiltrating lymphocytes. Oncoimmunology. 2019;8:e1638211.

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