Abstract 2829: A novel candidate for immunotherapy mediating the balance between the immune system and cancer
Authors: Pesce Elisa , Cordiglieri Chiara , Manara Cristina , Oliveto Stefania , Campagnoli Susanna , Donnarumma Tiziano , Martinelli Manuele , Crosti Mariacristina , De Camilli Elisa , Biffo Stefano , Abrignani Sergio , Bombaci Mauro , Grifantini Renata Maria , Gobbini Andrea 
Published: September 2020

Cytotoxic CD8+ T cells are considered as one of the main populations of effector immune cells in antitumor immunity. The absence of CD8+ T cells in the central tumor area has become a major obstacle for solid tumor immunotherapy. Thus, novel therapeutic strategies that could promote CD8+ T cells to accumulate in the central tumor area are urgently needed. To this aim, we want to propose a novel candidate INGM01 a highly glycosylated mucin-like protein localized on the cell surface and so far poorly characterized. High INGM01 expression is reported to be associated to a higher survival rate in some cancers (PreCOG database). We found that INGM01 is physiologically expressed on the surface of different cancer human cell lines and its over-expression by cDNA transfection significantly increased motility and migration phenotypes, such as improved scratch recovery in the wound healing assay, altered cytoskeleton organization with loss of actin branches, reduced E-cadherin expression and activated Fak pathway through phosphorylation of its Y925. We also found that INGM01 is specifically over-expressed in tumor-infiltrating CD8+ and CD4+ lymphocytes, as judged by IHC and IF analysis of cryopreserved tissues and by FACS analysis of T cells isolated from different cancers (e.g. colon, kidney), while it is marginally expressed in T-cells resident in adjacent normal tissues. Microscopy analysis showed that INGM01 localizes in anchoring sites of CD8+ T-cells attacking the cancer cells forming a cluster of lymphocytes on their surface. INGM01 expression in T lymphocyte is significantly induced by CD3/CD28 receptor activation and by the microenvironmental milieu conditioned by cancer cells, through the production of soluble factors. Indeed, INGM01 is localized in the uropod of both CD3/28 activated and ex vivo isolated tumor infiltrating CD4+ and CD8+ T cells confirming a role of INGM01 in motility of the T-cells. We found INGM01 co-expressed with other uropod-associated proteins, such as ICAM-I, LFA-1 and CXCR-3, involved in the acquisition of the polarity needed for T-cell chemotaxis and for migration. By Boyden chamber, with and without a HUVEC cell monolayer, we found that INGM01 positive CD8+ and Jurkat cells migrate towards wells containing conditioned medium of cancer cells and this process is significantly impaired by INGM01 silencing indicating its role in chemotaxis and trans-endothelial migration. Furthermore, INGM01 contributes to the cytotoxic function of CD8+ T-cells, since its silencing causes a reduction of expression of Th1 effector cytokines, such as IFN-γ, TNFα. Our hypothesis is that, after a deeper analysis of the interaction/s and ligand/s involving INGM01, it might be possible to generate affinity agents or bi-spefic antibodies able to enhance the intratumoral infiltration of cytotoxic CD8+ T-cells expressing INGM01 and their migration towards cancer cells, to promote their killing.

Citation Format: Elisa Pesce, Chiara Cordiglieri, Cristina Manara, Stefania Oliveto, Susanna Campagnoli, Tiziano Donnarumma, Manuele Martinelli, Mariacristina Crosti, Elisa De Camilli, Stefano Biffo, Sergio Abrignani, Mauro Bombaci, Renata Maria Grifantini, Andrea Gobbini. A novel candidate for immunotherapy mediating the balance between the immune system and cancer [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2829.