Gética 2020

[ F I T C á n c e r - 6 ] 55 COMUNICACIONES PÓSTERES and FACS. The anti-PD-L1 BiTE induced expression of CD69 activation marker in T cells only when in- cubated with PD-L1 expressing cells, and cytotoxic responses against luciferase expressing cell lines with both high and low levels of PD-L1, being es- pecially efficient with the first. A genetic construction coding the anti-EGFR LiTE and the anti-PD-L1 BiTE joined by a viral 2A self-cleaving peptide (F2A) was cloned in a lentiviral vector. Liposome-transfected HEK293 coexpressed both anti-EGFR and anti-PD-L1 BiTEs, detected by WB with a migration pattern according to the MW calculated from their amino acid sequence (MW of the anti-EGFR LiTE was slightly higher than that of the original one due of a terminal segment re- siduary from F2A sequence after cleavage). No high MW bands correspondent to unprocessed protein were observed, demonstrating a high cleavage efficiency. FACS analysis demonstrated that both bsAbs maintained its ability to specifical- ly detect antigens in a cellular context. Conclusions: Our anti-PD-L1 x anti-CD3 BiTE is efficiently secreted as a soluble functional protein and promotes killing of PD-L1+ cells being more ef- fective than conventional TAA-specific BiTEs at in- ducing cytotoxicity of tumor cells expressing high levels of PD-L1. Simultaneous in situ production of this anti-PD-L1 and anti-TAA bsAbs is possible and could improve the efficacy of T cell-redirection strategies in solid tumors. References: 1. Mølgaard K, Harwood SL, Compte M, et al. Bispecific light T-cell engagers for gene-based immunothera- py of epidermal growth factor receptor (EGFR)-pos- itive malignancies. Cancer Immunol Immunother 2018;67:1251-60. CP13. Small tumor-targeted 4-1BB agonists for cancer immunotherapy Hangiu, Oana 1,2 ; Compte Grau, Marta 3 ; Blanco Durango, Belén 1,2 ; Domínguez Alonso, Carmen 1,2 ; Erce-Llamazares, Ainhoa 1,2 ; Álvarez-Vallina, Luis 1,2,4 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 Department of Antibody Engineering. Leadartis SL. Madrid, Spain. 4 Immunotherapy and Cell Engineering Laboratory. Aarhus University. Aarhus, Denmark Introduction: In order to generate a tumour-specific responses, cancer immunotherapy includes different approaches such as vaccines, therapy based on onco- lytic virus, adoptive transfer of ex vivo activated T and natural killer cells, and administration of antibodies or recombinant proteins that either costimulate cells or block the immune checkpoint pathways. The costimulatory receptors that belongs to the su- perfamily of tumour necrosis factor receptors (TNFR) such as 4-1BB (CD137), are interesting targets for can- cer immunotherapy, as these receptors are not consti- tutively expressed on resting T lymphocytes, but they are expressed when the cells are activated. The binding of 4-1BB through its ligand or an ago- nist antibodies promotes the proliferation, produc- tion of cytokines and cytotoxicity in T lymphocytes. Immunotherapy based on costimulatory agonistic antibodies has demonstrated antitumor response in clinical trials. The main drawback of using anti- body-based therapies is the toxicity associated with Fc g receptor (Fc?R)-mediated interactions. Objective: The aim of this study was the gener- ation and characterization of smaller Fc-free tu- mor-targeted 4-1BB agonists called light T cell-co- stimulatory (LiTCO) antibodies, consisting of an anti-4-1BB single-chain fragment variable (scFv) fused in tandem to an EGFR-specific single-domain V HH antibody. Methods: Transfection of human HEK293T cells with the EGFR-4-1BB LiTCO encoding expression vector resulted in the secretion of functional an- tibody that was able to recognize the cognate antigens as demonstrated by ELISA, against plas- tic immobilized mouse-4-1BB or human-EGFR, and by FACS on cells that express EGFR or 4-1BB on the cellular surface. We proceeded to generate a modified prototype bearing a N-terminal FLAG and StrepII tag (FS) (EGFR-4-1BB FS LiTCO) and we puri- fied it using the Strep-tactin chromatography. Structural oligomeric characterization was deter- mined by SECMALS, and the costimulatory activity in vitro was assayed by human IFN g ELISA in acti-