Research Article Intravenous Injection of MVA Virus Targets CD8 þ Lymphocytes to Tumors to Control Tumor Growth upon Combinatorial Treatment with a TLR9 Agonist Laetitia Fend 1 , Tanja Gatard-Scheikl 1 , Jacqueline Kintz 1 , Murielle Gantzer 1 , Emmanuelle Schaedler 1 , Karola Rittner 1 , Sandrine Cochin 1 , Sylvie Fournel 2 , and Xavier Pr eville 1 Abstract Effector T-cell access to tumor tissue is a limiting step for clinical efficacy of antigen-specific T cell–based immunotherapies. Ectopic mouse tumor models, in which a subcutaneously (s.c.) implanted tumor is treated with s.c. or intramuscular therapeutic immunization, may not be optimal for targeting effector T cells to an organ-borne tumor. We used an orthotopic renal carcinoma model to evaluate the impact of injection routes on therapeutic efficacy of a Modified Vaccinia virus Ankara viral vector expressing the human mucin 1 tumor–associated xeno-antigen (MVA-MUC1). We show that intravenous (i.v.) administration of MVA-MUC1 displayed enhanced efficacy when compared with s.c. injection. Therapeutic efficacy of MVA-MUC1 was further enhanced by i.v. injection of a TLR9 agonist. In all cases, infiltration of tumor-bearing kidney by CD8 þ lymphocytes was associated with control of tumor growth. Biodistribution experiments indicate that, following i.v. injection, MVA-encoded antigens are quickly expressed in visceral organs and, in particular, in splenic antigen-presenting cells, compared with those following s.c. injection. This appears to result in a faster generation of MUC1-specific CD8 þ T cells. Lymphocytes infiltrating tumor-bearing kidneys are characterized by an effector memory phenotype and express PD-1 and Tim3 immune checkpoint molecules. Therapeutic efficacy was associated with a modification of the tumor microenvironment toward a Th1-type immune response and recruitment of activated lymphocytes. This study supports the clinical evaluation of MVA-based immunotherapies via the i.v. route. Cancer Immunol Res; 2(12); 1163–74. Ó2014 AACR. Introduction Modified Vaccinia virus Ankara (MVA) is a double-stranded DNA poxvirus derived from a Turkish smallpox vaccine strain through more than 570 passages in primary chicken embryo fibroblasts (1). Consequently, it has lost nearly 30 kb of genomic information and is unable to complete its replication cycle in most mammalian cells. MVA has several features that render it a good vector for targeted immunotherapy of cancer: (i) an excellent safety profile in humans (1); (ii) a large amount of foreign DNA (up to 20 kb) can be integrated into the MVA genome without loss of infectivity; (iii) viral DNA remains in the cytoplasm and, therefore, gene expression is cytoplasmic; and (iv) MVA has the ability to induce both humoral and cellular responses against the encoded foreign antigens (2, 3). On the basis of promising preclinical results, clinical trials for cancer immunotherapy have been and currently are being conducted using recombinant MVA that is injected subcutaneously (s.c.; ref. 1). Nevertheless, similar to most cancer immunotherapies, translation of preclinical efficacy to clinical benefit has remained below expectations (4). The generation of robust cellular immunity to tumor-associated antigens requires the induced tumor-specific T cells to traffic to and enter the tumors (5). T-cell infiltration is associated with patient survival in many cancers (6–8), and it is now accepted that one of the major challenges of immunotherapy is to target T cells to the tumors (9). In this context, most preclinical therapeutic cancer models, which are based on ectopic (s.c.) implantation of tumor cell lines from various tumor types, are poor represen- tative of the clinical situation. One of the differences is the tumor microenvironment at the anatomic site of injection resulting from the implanted tumor cells. For example, the implantation of colorectal adenocarcinoma cells under the skin would not recreate a tumor microenvironment reflecting that of spontaneous colon cancer. In addition, there is now evidence to suggest that the immunization route influences the subsequent migratory capacity of primed, antigen-specificT cells (10–12). Hence, s.c. or intramuscular (i.m.) immunizations of immunotherapeutic drugs may not be optimal for directing 1 Transgene S.A., Illkirch-Graffenstaden, France. 2 Laboratoire de Concep- tion et Application de Mol ecules Bioactives, Equipe de Biovectorologie, UMR 7199 CNRS-Universit e de Strasbourg, Facult e de Pharmacie, Illkirch- Graffenstaden, France. Note: Supplementary data for this article are available at Cancer Immu- nology Research Online (http://cancerimmunolres.aacrjournals.org/). Corresponding Author: Xavier Pr eville, Transgene S.A., 400 boulevard Gonthier d'Andernach, Parc d'innovation, CS80166, 67405 Illkirch-Graf- fenstaden, France. Phone: 33-388-279-242; Fax: 33-388-225-807; E-mail: preville@transgene.fr doi: 10.1158/2326-6066.CIR-14-0050 Ó2014 American Association for Cancer Research. Cancer Immunology Research www.aacrjournals.org 1163 on January 12, 2022. © 2014 American Association for Cancer Research. cancerimmunolres.aacrjournals.org Downloaded from Published OnlineFirst August 28, 2014; DOI: 10.1158/2326-6066.CIR-14-0050