Gética 2020

[ F I T C á n c e r - 6 ] 39 Introduction and objective: Redirection of T cell activity using bispecific antibodies (bsAb) by cross-linking of tumour cells and T cells, inde- pendently of TCR (T-cell receptor), is one of the most promising immunotherapy protocols against cancer. Recently, cancer immunotherapies that block the PD-1/PD-L1 pathway have demonstrated unprecedented efficacy. By adapting the tandem trimer body approach, we have generated a tu- mour-targeted immune checkpoint inhibitor and T cell-engaging antibody to simultaneous blocking the PD-1/PD-L1 pathway, a tumour-associated an- tigen, and the CD3? chain of the TCR/CD3 complex. The molecule will trigger T cell activation and pro- liferation, and enhance the polyclonal recruitment of tumour-infiltrating T cells. Methods: The tetravalent and trispecific antibody is generated by fusing an anti-PD-L1 single-chain fragment variable (scFv), two anti-EGFR single-do- main V HH and one anti-CD3 scFv on a single-chain trimer body format. The antibody was purified from conditioned medium from transfected HEK-293 cells by chromatography which yielded proteins > 95% pure, with an estimated molecular weight of 120 kDa, as determined by SDS-PAGE. Results: The binding kinetics were assessed by Bi- olayer interferometry (BLI) and revealed that the an- tibody was capable of binding concurrently to EGFR and PD-L1 with high affinity and low dissociation rates. Gene-modified luciferase expressing HeLa and MDA-MB-231 cells were seeded in triplicate in 96well microtiter plate, and co-cultured with human CD3 + T cells at a 5:1 E:T ratio, in the presence of purified an- tibodies, alone or in combination with an anti-PD-L1 blocking antibody. After 48 hours, viability was mea- sured adding D-luciferin. Percent tumour cell viability was calculated, and cytotoxicity effect was then plot- ted against the effector molecule concentration. Conclusions: Altogether, these data suggest that antibody forms intramolecular homotrimers, is eas- ily produced and purified from mammalian cell sys- tems and is multispecific as demonstrated from the specific target recognition analysis. Furthermore, in the context of PD-L1 expressing tumour cells the antibody is more effective than the combination of EGFR-specific T cell-engagers and an anti-PD-L1 blocking antibodies. Sources of founding: Instituto de Salud Carlos III, Mi- nisterio de Ciencia, Innovación y Universidades. COMUNICACIONES ORALES CO01. Next-generation therapeutic antibody for simultaneous and tumor-specific immune checkpoint blockade and T cell-redirection Erce-Llamazares, Ainhoa 1 ; Domínguez-Alonso, Carmen 1 ; Lykkemark, Simon 2 ; Harwood, Seandean L. 2 ; Álvarez-Vallina, Luis 3 ; Compte Grau, Marta 4 1 Inmuno-Oncología e Inmunoterapia. Instituto de Investigación 12 de Octubre (I+12). Madrid, Spain. 2 Unidad de Inmunoterapia del Cáncer (UNICA). Servicio de Inmunología. Hospital Universitario 12 de Octubre. Madrid, Spain. 3 Immunotherapy and Cell Engineering Laboratory. Aarhus University. Aarhus, Denmark. 4 Department of Antibody Engineering. Leadartis SL. Madrid, Spain