CEA-4-1BBL: CEACAM5-Targeted 4-1BB Ligand Fusion Proteins for Cis Co-Stimulation with CEA-TCB

Background/Objectives: T cell bispecific antibodies (TCBs) result in the activation of T cell receptor signaling upon binding to tumor antigens providing signal 1 to T cells. To enhance and sustain their activity a co-stimulatory signal 2 is required. Here CEACAM5-targeted 4-1BBL antibody fusion proteins for combination with CEA-TCB (cibisatamab, RG7802) are described to investigate the relationship of CEACAM5 epitope and activity. Methods: CEACAM5-targeted bispecific 4-1BBL antibody fusion proteins (CEA-4-1BBL) were generated based on different CEACAM5 antibodies and characterized in vitro in Jurkat-4-1BB reporter and PBMC cell assays. The impact of shed CEA on in vitro activity and cynomolgus cross-reactivity was studied. In vivo efficacy was assessed in human stem cell humanized NSG mice xenograft models bearing MKN-45 and HPAFII tumors. Results: MFE23-4-1BBL and Sm9b-4-1BBL showed superior functional activity in Jurkat-4-1BB reporter and in primary T cell assays when combined with the CD3 antibody V9, whereas T84.66-LCHA-4-1BBL and A5B7-4-1BBL performed better when combined with CEA-TCB. In humanized NSG mice MKN-45 and HPAFII xenograft models T84.66-LCHA-4-1BBL mediated the best anti-tumor efficacy. Conclusions: For the assessment of the combination of CEA-TCB with CEA-4-1BBL co-stimulatory antibody fusion proteins in vitro assays are not sufficient to fully capture the complex relationships affecting efficacy. Thus, screening with different cell assays and in vivo efficacy studies in combination with CEA-TCB is essential to select the best candidate. Based on the totality of data the T84.66-LCHA-4-1BBL antibody fusion protein comprising the CEACAM5 antibody T84.66-LCHA was selected as optimal combination partner for CEA-TCB.