RG7386, a Novel Tetravalent FAP-DR5 Antibody, Effectively Triggers FAP-Dependent, Avidity-Driven DR5 Hyperclustering and Tumor Cell Apoptosis

Abstract

Dysregulated cellular apoptosis and resistance to cell death are hallmarks of neoplastic initiation and disease progression. Therefore, the development of agents that overcome apoptosis dysregulation in tumor cells is an attractive therapeutic approach. Activation of the extrinsic apoptotic pathway is strongly dependent on death receptor (DR) hyperclustering on the cell surface. However, strategies to activate DR5 or DR4 through agonistic antibodies have had only limited clinical success. To pursue an alternative approach for tumor-targeted induction of apoptosis, we engineered a bispecific antibody (BsAb), which simultaneously targets fibroblast-activation protein (FAP) on cancerassociated fibroblasts in tumor stroma and DR5 on tumor cells. We hypothesized that bivalent binding to both FAP and DR5 leads to avidity-driven hyperclustering of DR5 and subsequently strong induction of apoptosis in tumor cells but not in normal cells.Here, we show that RG7386, an optimized FAP-DR5 BsAb, triggers potent tumor cell apoptosis in vitro and in vivo in preclinical tumor models with FAP-positive stroma. RG7386 antitumor efficacy was strictly FAP dependent, was independent of FcR crosslinking, and was superior to conventional DR5 antibodies. In combination with irinotecan or doxorubicin, FAP-DR5 treatment resulted in substantial tumor regression in patient-derived xenograft models. FAP-DR5 also demonstrated single-agent activity against FAP-expressing malignant cells, due to cross-binding of FAP and DR5 across tumor cells. Taken together, these data demonstrate that RG7386, a novel and potent antitumor agent in both mono-and combination therapies, overcomes limitations of previous DR5 antibodies and represents a promising approach to conquer tumor-associated resistance to apoptosis.

A Single-Agent Dual-Specificity Targeting of FOLR1 and DR5 as an Effective Strategy for Ovarian Cancer

“…Next we co-cultured GFP + Colo-205 cells with murine MC38 cells and treated with 50 nM LK26-AMG-655 bispecific antibody. The loss of GFP in Figure 4I (most right lane) confirms bispecific antibody functioning to engage muFOLR1 to “trans” activate huDR5, similar to described for RG7386 (Brunker et al, 2016). Next, we compared the activity of trans-engaging DR5 bispecific antibody against the BaCa strategy using serial dilutions.…”

“…Therefore, to achieve a higher enzymatic activity (Apoptosis) for a low affinity substrate, a higher enzyme (BaCa) antibody concentration is essential to increase the rate of reaction. Since stromal cell engaging antibodies such as RG7386 primarily works in “trans”, our results with LK26-AMG-655 bispecific antibody rationally indicate the higher therapeutic dose requirement for trans-engaging antibodies to achieve effective cytotoxic response as compared to BaCa antibody (Brunker et al, 2016). If a higher therapeutic dose will have a higher probability of toxicity and acquired resistance compared to a lower effective dose, need to be seen in clinical trials (Day and Read, 2016; Zuch de Zafra et al, 2016).…”

“…A dual specificity antibody capable of engaging DR5 on tumor cells and fibroblast activating protein (FAP) receptor on stromal cells has been shown to improve DR5 activity (Brunker et al, 2016). Since FAP is also over-expressed in the disease-associated stroma of wound healing tissues and multipotent bone marrow stem cells, FAP targeting does not give specificity to the tumor.…”

“…The clinical data at biochemical levels have accounted for insufficient tumor specific cell death signaling due to sub-optimal clustering of DR5 receptor (Merchant et al, 2012; Niyazi et al, 2009). As one alternative, trans-engaging (stromal cell and tumor cell) antibodies have been described to enhance DR5 clustering (Brunker et al, 2016). However, in addition to fundamental dependency on another cell type, the described fibroblast activation protein (FAP) engaging antibodies represent critical safety concerns such as severe cachexia and bone toxicity due to non-specific targeting (Tran et al, 2013).…”

Selective Homogeneous Assay for Circulating Endopeptidase Fibroblast Activation Protein (FAP)

“…Clinical development of an enzyme inhibitor as a therapeutic agent can be significantly facilitated by the ability to determine the activity of the enzyme in biological samples in a homogenous assay format. First, identification of patients with high levels of active enzyme may support the discovery of a patient sub-population who might benefit most from the use of an inhibitor or an enzyme-targeted anti-tumor agent, such as the recently described anti-DR5/FAP bispecific antibody 50 . This aspect of drug development has become ever more important in the era of precision medicine.…”