Abstract B012: A novel in vitro approach elucidates a new mode of cytotoxic action of bispecific antibodies on hematologic malignancies
Authors: Primo Daniel , Hernandez Pilar , Gorrochategui Julian , Vicente Maria L. , Martinez-Cuadron David , Montesinos Pau , Mártinez-López Joaquin , Ballesteros Joan 
Published: August 2018

Abstract
Objectives: Bispecific antibodies (BsAbs) act through the formation of an immunologic synapse between T-cells (CD3) and a tumor-associated surface antigen (TAA) leading to T-cell activation and serial lysis of tumor cells. The aim of the present study is to explore the mechanism of action (MOA) and the in vitro effect of BsAbs on hematologic samples with the PharmaFlow platform. Methods: Fresh whole bone marrow (BM) and peripheral blood (PB) from 41 samples from 3 different hematologic diseases (31 AML, 3 ALL, and 7 CLL) and two AML cell lines were tested with CD3-CD123 (for AML patients and cell lines) or CD3-CD19 (for ALL and CLL patients) BsAbs in the PharmaFlow platform, an innovative proprietary method that uses flow cytometry (FCM) to efficiently count the number of tumor cells killed by activated T-cells. We analyzed the populations of leukemic cells, activated T-cells, and residual normal cells. Additional key parameters were also used to explore the MOA after BsAb exposure at different time incubations (24h-144h), such as the effective E:T ratio (the number of T cells that kill a number of leukemic cells), real basal E:T ratio, tumor antigen expression, T-cell expansion, and expression of immune checkpoint proteins on target and effector cells before and after cell culture. For some experiments, fluorescence-activated cell sorting (FACS) was performed to evaluate T-cell cytotoxicity after BsAb exposure. Results: Most of the samples demonstrated T-cell activation and effective lysis of tumor cells after BsAb exposure independent of TAA expression and in a dose-response manner. Once sorted, these T cells could kill tumor cells in the absence of BsAb, as well as tumor cells that did not express the TAA target. Interestingly, these activated T cells selectively killed tumor cells with low cytotoxicity in residual normal cells from the same patients. Moreover, differential T-cell cytotoxicity was observed between samples. We observed samples with leukemic resistance or no T-cell activity (especially in CLL with CD3-CD19), as well as others with higher T-cell cytotoxicity and minimal number of activated T cells (especially in AML with CD3-CD123). The integration of all the predictive parameters (E:T ratios, Tumor-Specific Antigen (TSA) expression, etc.) allowed us to generate an in vitro response model and select samples with higher T-cell cytotoxicity after the BsAb exposure. Conclusion: Our findings are consistent with a model where, in addition to the standard MOA inducing tumor cells lysis by proximity, BsAbs can highly enrich cytotoxic clonal T-cell subsets with TSA and induce strong activation and proliferation of T cells capable of killing tumor cells in an effective and selective manner. This differential in vitro T-cell cytotoxicity effect between patients could allow us to select better candidates for adoptive antitumor immunotherapy with BsAbs.
Citation Format: Daniel Primo, Pilar Hernandez, Julian Gorrochategui, Maria L. Vicente, David Martinez-Cuadron, Pau Montesinos, Joaquin Mártinez-López, Joan Ballesteros. A novel in vitro approach elucidates a new mode of cytotoxic action of bispecific antibodies on hematologic malignancies [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2017 Oct 26-30; Philadelphia, PA. Philadelphia (PA): AACR; Mol Cancer Ther 2018;17(1 Suppl):Abstract nr B012.