CEA-4-1BBL: Design and Optimization of a Novel CEACAM5-Targeted 4-1BB Ligand Antibody Fusion Protein to Provide Costimulation in Cis in Combination with T Cell Bispecific Antibodies

Background/Objectives: T cell bispecific antibodies (TCBs) activate T cells for the killing of tumor cells upon binding to tumor antigens resulting in the activation of T cell receptor signaling providing the so-called signal 1 to T cells. In order to enhance and sustain the activity of T cells a co-stimulatory signal 2 is required. Here we describe CEACAM5-targeted 4-1BBL antibody fusion protein for combination with the CEA-TCB cibisatamab investigating the relationship of CEACAM5 epitope and activity. Methods: CEACAM5-targeted bispecific 4-1BBL antibody fusion proteins (CEA-4-1BBL) were generated using 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 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 xenograft models T84.66-LCHA-4-1BBL showed overall the best anti-tumor efficacy. Concluions: In vitro assays and in vivo studies lead to different conclusions showing that in order to select the best candidate screening with different cell assays and in vivo efficacy studies together with the potential combination partner are essential. Based on the totality of data the T84.66-LCHA-4-1BBL antibody fusion protein based on the CEACAM5 antibody T84.66-LCHA was selected as optimal costimulatory antibody fusion protein for the combination with the CEA-TCB cibisatamab.