Abstract 5570: Identification of novel mechanisms of resistance against the redirection of T-cell effector function for cancer therapy
Authors: Lahuerta Enrique Javier Arenas , Martínez-Sabadell Alex , Ruiz Irene Rius , Morancho Beatriz , Román Macarena , Bernadó Cristina , Arribas Joaquín 
Published: September 2020

Abstract
In the recent times the way to tackle cancer has been broadly expanded to new opportunities, being the immunotherapy the spearhead of this revolution. Two of the different novel strategies to boost the immune response against tumor cells are the T Cell Bispecific antibodies (TCBs) and the Chimeric Antigen Receptors (CARs), both able to redirect the T cells of the host against the tumor cells. While the TCBs consist of an engineered antibody that binds both to a tumor associated antigen and a T cell receptor, activating it; the CARs consist of a modified T cell that exhibit the binding site to the antigen fused to the intracellular signaling motifs that activate the T cell. Clinical evidence shows that either TCBs or CARs are effective to treat cancer. These can be directed to a vast variety of tumor antigens. Some examples are the HER2-TCB, directed against the HER2 receptor tyrosine kinase overexpressed in 20% of the breast cancers. However, the therapeutic effectivity in tumors is lower than expected and the mechanisms of resistance to these promising immunotherapies have not been explored yet. Therefore, there is a need to anticipate them. In this project, we have developed a immunoresistant model against HER2-TCB, by using the HER2+ breast cancer cell line BT474 as a model. To generate immunoresistant cells, cells were exposed with the TCBs and peripheral blood mononuclear cells (PBMCs) for several months in order to obtain resistance. Indeed, these resistant cells were resistant also in a 3D organoid model and in an in vivo humanized PBMC model. Furthermore, these resistant cells were also resistant to a CAR expressing HER2. Interestingly, a cytokine array that assess more than 80 cytokines did not show a dramatic change in the secretome of resistant cells, in basal conditions and in presence of PBMCs and HER2-TCB, suggesting that the mechanism of resistant is intrinsic on the resistant cells. In addition, these resistant cells did not lose the expression of the antigen (HER2). Therefore, we aimed to identify intrinsic mechanisms of resistance by means of RNA-seq. Although we did not identify the cause of the resistance yet, transcriptomic analysis showed a dramatic change between sensitive and resistant cells, with more than 50 genes acutely up-or downregulated in resistant cells (≥4-fold; p &lt; 10-5). Gene set enrichment analysis identified numerous differences in biological processes between both populations. In parallel, we have also generated the very same model in vivo, with serial passagues in humanized PBMC mice treated with HER2-TCB. The next step will be to identify the mechanism of resistance by gain and loss of function approaches and to validate it in a cohort of human samples and in our collection of Patient Derived Xenografts. In conclusion, we have generated an interesting resistant model to immunotherapies that can be used as a tool to identify unknown mechanisms of resistance and with potential clinical applications that will reinforce the use of immunotherapy in the clinics.
Citation Format: Enrique Javier Arenas Lahuerta, Alex Martínez-Sabadell, Irene Rius Ruiz, Beatriz Morancho, Macarena Román, Cristina Bernadó, Joaquín Arribas. Identification of novel mechanisms of resistance against the redirection of T-cell effector function for cancer therapy [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 5570.