For tumor specific co-stimulation via 4-1BB (CD137) a novel tumor-stroma targeted FAP-4-1BB ligand (FAP-4-1BBL) was designed composed of a trimeric split 4-1BBL, a tumor-stroma targeted 4B9 Fab moiety recognizing fibroblast activation protein (FAP) and a heterodimeric Fc-region devoid of FcgR binding but maintained FcRn binding. In presence of a TCR signal 1 either from a T cell receptor MHCI/peptide interaction or through TCR engagement by a T-cell bispecific antibody, FAP-4-1BBL provides co-stimulation to T cells strictly dependent on cross-linking by FAP-expressing fibroblasts. Similarly, FAP-4-1BBL can provide co-stimulation to NK cells with activated FcgRIIIa signaling. FAP-4-1BBL was tested as monotherapy and combined with CEA-TCB, an anti-CEA T-cell bispecific antibody, in the s.c. gastric MKN45 xenograft model co-grafted with NIH-3T3 fibroblasts in human stem cell engrafted (HSC) NSG mice in comparison to DP47-4-1BBL, an analogous untargeted 4-1BBL fusion protein. For use in syngeneic mouse models in immunocompetent mice muFAP-4-1BB, a murine surrogate made of a mu4-1BB agonistic surrogate antibody fused to the variable region of the FAP antibody 4B9, was used. muFAP-4-1BB was tested as monotherapy and combined with a murine specific CEA-TCB surrogate (muCEA-TCB) and the muPD-L1 specific surrogate antibody 6E11 in the s.c. colorectal MC38-CEA model in CEA transgenic (Tg) C57BL/6 mice. For combination with ADCC-mediating antibodies FAP-mu4-1BBL, a hybrid FAP-mu4-1BBL fusion protein, was generated. FAP-mu4-1BBL was tested as monotherapy and combined with the ADCC-mediating anti-HER2 antibody trastuzumab in the s.c. gastric N87 xenograft model in human CD16 Tg Scid mice. In the s.c. gastric MKN45 xenograft model co-grafted with NIH-3T3 fibroblasts in HSC-NSG mice FAP-4-1BBL resulted in combined anti-tumoral efficacy in combination with CEA-TCB, whereas the respective monotherapies as well as the combination with the untargeted DP47-4-1BBL did not improve anti-tumor efficacy. In the syngeneic s.c. MC38-CEA model in CEA Tg C56BL/6 mice, a model with natural FAP expression due to fibroblast infiltration, the muFAP-4-1BB surrogate antibody resulted in combined anti-tumor efficacy, both combined with muCEA-TCB and muPD-L1 antibodies including the induction of tumor remission. Finally, the hybrid FAP-mu4-1BBL surrogate resulted in improved anti-tumor efficacy combined with the ADCC-mediating antibody trastuzumab in the s.c. gastric N87 xenograft model in human CD16 Tg Scid mice. These data show that FAP-4-1BBL is a versatile combination partner for cancer immunotherapy mediating FAP-dependent co-stimulation to T and NK cells in combination with PD-L1 checkpoint inhibition, TCBs and ADCC-mediating antibodies. Clinical evaluation of this novel therapeutic approach is planned early 2018.

Citation Format: Johannes Sam, Christina Claus, Claudia Ferrara, Sabine Lang, Valeria Nicolini, Sara Colombetti, Volker Teichgräber, Stefan Evers, Marina Bacac, Pablo Umana, Christian Klein. FAP-4-1BBL: A novel versatile tumor-stroma targeted 4-1BB agonist for combination immunotherapy with checkpoint inhibitors, T-cell bispecific antibodies, and ADCC-mediating antibodies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 5621.