University of Oulu

Clubb JHA, Kudling TV, Heiniö C, Basnet S, Pakola S, Cervera Carrascón V, Santos JM, Quixabeira DCA, Havunen R, Sorsa S, Zheng V, Salo T, Bäck L, Aro K, Tulokas S, Loimu V and Hemminki A (2022) Adenovirus Encoding Tumor Necrosis Factor Alpha and Interleukin 2 Induces a Tertiary Lymphoid Structure Signature in Immune Checkpoint Inhibitor Refractory Head and Neck Cancer. Front. Immunol. 13:794251. doi: 10.3389/fimmu.2022.794251

Adenovirus encoding tumor necrosis factor alpha and interleukin 2 induces a tertiary lymphoid structure signature in immune checkpoint inhibitor refractory head and neck cancer

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Author: Clubb, James H. A.1,2,3; Kudling, Tatiana V.1,3; Heiniö, Camilla1,3;
Organizations: 1Cancer Gene Therapy Group, Faculty of Medicine, University of Helsinki, Helsinki, Finland
2TILT Biotherapeutics Ltd, Helsinki, Finland
3Translational Immunology Research Program (TRIMM), Research Program Unit (RPU), University of Helsinki, Helsinki, Finland
4Department of Oral and Maxillofacial Diseases, Clinicum, University of Helsinki, Helsinki, Finland
5Cancer and Translational Medicine Research Unit, University of Oulu, Oulu, Finland
6Oulu University Central Hospital, Oulu, Finland
7Department of Oncology, Comprehensive Cancer Centre, Helsinki University Hospital and University of Helsinki, Helsinki, Finland
8Department of Otorhinolaryngology – Head and Neck Surgery, Helsinki Head and Neck Center, Helsinki University Hospital and University of Helsinki, Helsinki, Finland
Format: article
Version: published version
Access: open
Online Access: PDF Full Text (PDF, 11.7 MB)
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Language: English
Published: Frontiers Media, 2022
Publish Date: 2022-09-26


Immune checkpoint inhibitors (ICI) have provided significant improvement in clinical outcomes for some patients with solid tumors. However, for patients with head and neck cancer, the response rate to ICI monotherapy remains low, leading to the exploration of combinatorial treatment strategies. In this preclinical study, we use an oncolytic adenovirus (Ad5/3) encoding hTNFα and hIL-2 and non-replicate adenoviruses (Ad5) encoding mTNFα and mIL-2 with ICI to achieve superior tumor growth control and improved survival outcomes. The in vitro effect of Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 was characterized through analyses of virus replication, transgene expression and lytic activity using head and neck cancer patient derived cell lines. Mouse models of ICI naïve and refractory oral cavity squamous cell carcinoma were established to evaluate the local and systemic anti-tumor immune response upon ICI treatment with or without the non-replicative adenovirus encoding mTNFα and mIL-2. We delineated the mechanism of action by measuring the metabolic activity and effector function of CD3⁺ tumor infiltrating lymphocytes (TIL) and transcriptomic profile of the CD45⁺ tumor immune compartment. Ad5/3-E2F-D24-hTNFa-IRES-hIL-2 demonstrated robust replicative capability in vitro across all head and neck cell lines screened through potent lytic activity, E1a and transgene expression. In vivo, in both ICI naïve and refractory models, we observed improvement to tumor growth control and long-term survival when combining anti-PD-1 or anti-PD-L1 with the non-replicative adenovirus encoding mTNFα and mIL-2 compared to monotherapies. This observation was verified by striking CD3⁺ TIL derived mGranzyme b and interferon gamma production complemented by increased T cell bioenergetics. Notably, interrogation of the tumor immune transcriptome revealed the upregulation of a gene signature distinctive of tertiary lymphoid structure formation upon treatment of murine anti-PD-L1 refractory tumors with non-replicative adenovirus encoding mTNFα and mIL-2. In addition, we detected an increase in anti-tumor antibody production and expansion of the memory T cell compartment in the secondary lymphoid organs. In summary, a non-replicative adenovirus encoding mTNFα and mIL-2 potentiates ICI therapy, demonstrated by improved tumor growth control and survival in head and neck tumor-bearing mice. Moreover, the data reveals a potential approach for inducing tertiary lymphoid structure formation. Altogether our results support the clinical potential of combining this adenovirotherapy with anti-PD-1 or anti-PD-L1.

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Series: Frontiers in immunology
ISSN: 1664-3224
ISSN-E: 1664-3224
ISSN-L: 1664-3224
Volume: 13
Article number: 794251
DOI: 10.3389/fimmu.2022.794251
Type of Publication: A1 Journal article – refereed
Field of Science: 3122 Cancers
1182 Biochemistry, cell and molecular biology
Funding: This study received funding from the European Union’s Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 813453. This study was supported by Jane and Aatos Erkko Foundation, HUCH Research Funds (VTR), Finnish Cancer Organizations, University of Helsinki, Novo Nordisk Foundation, Päivikki and Sakari Sohlberg Foundation, TILT Biotherapeutics Ltd. We thank Albert Ehrnrooth and Karl Fazer for research support. The funders were not involved in the study design, collection, analysis, interpretation of data, the writing of this article or the decision to submit it for publication.
Copyright information: © 2022 Clubb, Kudling, Heiniö, Basnet, Pakola, Cervera Carrascón, Santos, Quixabeira, Havunen, Sorsa, Zheng, Salo, Bäck, Aro, Tulokas, Loimu and Hemminki. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.