Microenvironment and Immunology T Cells Redirected to a Minor Histocompatibility Antigen Instruct Intratumoral TNFa Expression and Empower Adoptive Cell Therapy for Solid Tumors Teresa Manzo 1,2 , Tabea Sturmheit 1,2 , Veronica Basso 1 , Elisabetta Petrozziello 1,2 , Rodrigo Hess Michelini 1 , Michela Riba 3 , Massimo Freschi 4 , Angela R. Elia 1 , Matteo Grioni 1 , Flavio Curnis 5 , Maria Pia Protti 1 , Ton N. Schumacher 6 , Reno Debets 7 , Melody A. Swartz 8,9 , Angelo Corti 2,5 , Matteo Bellone 1 , and Anna Mondino 1 Abstract Donor-derived allogeneic T cells evoke potent graft versus tumor (GVT) effects likely due to the simultaneous recognition of tumor-specific and host-restricted minor histocompatibility (H) antigens. Here we investigated whether such effects could be reproduced in autologous settings by TCR gene–engineered lym- phocytes. We report that T cells redirected either to a broadly expressed Y-encoded minor H antigen or to a tumor-associated antigen, although poorly effective if individually transferred, when simultaneously administered enabled acute autochthonous tumor debulking and resulted in durable clinical remission. Y- redirected T cells proved hyporesponsive in peripheral lymphoid organs, whereas they retained effector function at the tumor site, where in synergy with tumor-redirected lymphocytes, they instructed TNFa expression, endothelial cell activation, and intra- tumoral T-cell infiltration. While neutralizing TNFa hindered GVT effects by the combined T-cell infusion, a single injection of picogram amounts of NGR-TNF, a tumor vessel–targeted TNFa derivative currently in phase III clinical trials, substituted for Y-redirected cells and enabled tumor debulking by tumor-redir- ected lymphocytes. Together, our results provide new mechanistic insights into allogeneic GVT, validate the importance of targeting the tumor and its associated stroma, and prove the potency of a novel combined approach suitable for immediate clinical imple- mentation. Cancer Res; 77(3); 658–71. Ó2016 AACR. Introduction Allogeneic hematopoietic stem cell transplantation (Allo- HSCT) is the most widely used form of adoptive immunotherapy in treating cancer. To date, it is the only curative treatment for several high-risk hematologic malignancies, such as chronic and acute myeloid leukemia (1, 2), and can exert therapeutic effects in a proportion of patients with solid tumors, such as renal, breast, colorectal, ovarian, and pancreatic cancer (3). Graft versus leuke- mia (GVL) and graft versus tumor (GVT) effects are evoked by mature T cells, which could be present in the donor graft, or administered after transplantation as donor lymphocyte infu- sions (DLI; refs. 1, 2). Donor-derived T cells are polyclonal and can recognize tumor-specific antigens, as well as host-restricted self-antigens, known as minor histocompatibility (H) antigens, which could be expressed only by cells of the hematopoietic lineage (e.g., progenitors, T and B lymphocytes, monocytes), or also by epithelial (keratinocytes, fibroblast, gut, liver) and endo- thelial cells. T lymphocytes specific for host minor H antigens contribute to therapeutic GVL/GVT and have been used to treat relapsing leukemia in clinical settings (4), and solid and hema- tologic malignancies in mouse models (5, 6). In some instances, reactivity towards broadly expressed minor H antigens can cause a reaction to healthy tissues and organs known as "graft versus host disease" (GVHD; ref. 1). Much effort has been directed to reduce transplant-related toxicity and identify alternative and safer strategies. Among these, adoptive T-cell therapy (ACT) with tumor-reactive autologous T cells has reported promising clinical results. Tumor-reactive cells can be isolated and expanded from tumor lesions (tumor-infil- trating lymphocytes, TIL), or produced by genetic engineering of peripheral blood T cells with T-cell receptors (TCR) or chimeric 1 Division of Immunology, Transplantation and Infectious Disease, San Raffaele Scientific Institute, Milan, Italy. 2 Universit a Vita-Salute San Raffaele, Milan, Italy. 3 Center for Translational Genomics and Bioinformatics, San Raffaele Scientific Institute, Milan, Italy. 4 Department of Pathology, San Raffaele Scientific Institute, Milan, Italy. 5 Division of Experimental Oncology, San Raffaele Scientific Institute, Milan, Italy. 6 Division of Immunology, The Netherlands Cancer Institute, Amster- dam, the Netherlands. 7 Laboratory of Tumor Immunology, Department of Medical Oncology, Erasmus MC Cancer Institute, Rotterdam, the Netherlands. 8 Institute of Bioengineering,  Ecole Polytechnique F ed erale de Lausanne, Lau- sanne, Switzerland. 9 Institute for Molecular Engineering, University of Chicago, Chicago, Illinois. Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/). T. Manzo and T. Sturmheit contributed equally to this article. Current address for T. Manzo: Department of Genomic Medicine, MD Anderson Cancer Center, Houston, Texas; current address for T. Sturmheit, Fraunhofer EMB, L€ ubeck 23562, Germany; and current address for R. Hess Michelini, Genomics Institute of the Novartis Research Foundation, San Diego, California. Corresponding Author: Anna Mondino, Division of Immunology, Transplanta- tion and Infectious Diseases, San Raffaele Scientific Institute, Via Olgettina 58, Milan 20132, Italy. Phone: 3902-2643-4801; Fax: 3902-2643-4844; E-mail: anna.mondino@hsr.it doi: 10.1158/0008-5472.CAN-16-0725 Ó2016 American Association for Cancer Research. Cancer Research Cancer Res; 77(3) February 1, 2017 658 Downloaded from http://aacrjournals.org/cancerres/article-pdf/77/3/658/2760975/658.pdf by guest on 20 June 2022