The data presented herein validate the TWEAK/Fn14 pathway as a target in oncology. We show that administration of TWEAK in vivo is efficacious in inhibiting tumor growth in a xenograft model. To therapeutically target the pathway, we developed a humanized agonistic anti-Fn14 antibody, BIIB036, which mimics many activities of TWEAK, including the ability to inhibit tumor growth. Importantly, BIIB036 is highly efficacious in inhibiting tumor growth in multiple xenograft models irrespective of its relative activity in vitro in the various cell lines. We found that the anti-tumor activity of BIIB036 can be enhanced by multimerization of the antibody, suggesting an effect of higher order cross-linking of Fn14 molecules on the cell surface. Indeed, we observed that maximal activity in vivo is achieved only with full effector function of the antibody, suggesting the role of Fc receptors in scaffolding the antibody or ADCC activity in vivo.
BIIB036 was developed as an anti-Fn14 agonistic antibody to mimic the activity of the natural ligand, TWEAK. Indeed, BIIB036 can induce chemokine release, activate NFκB pathway signaling, and, importantly, stimulate tumor cell death. However, BIIB036, the most potent agonist in our panel of anti-Fn14 antibodies, is significantly less potent than TWEAK. Notably, other anti-Fn14 antibodies that have been described also show reduced potency relative to TWEAK.16,32
The decreased potency might be a function of the dimeric interaction of an antibody with the Fn14 receptor in contrast to TWEAK which presumably engages in a trimer-trimer interaction with Fn14. It is also possible that a particular geometrical configuration of Fn14 engagement is unique to TWEAK. Nevertheless, as a therapeutic, BIIB036 is highly efficacious in multiple xenograft models. Moreover, as a monoclonal antibody, BIIB036 is a better drug candidate than soluble TWEAK. The adenoviral system, while delivering abundant quantities of soluble TWEAK, is not feasible for human use. Fc-TWEAK, consisting of a dimerizing Ig Fc domain and a trimerizing extra-cellular domain of TWEAK, can potentially fold into several oligomeric forms as observed by size exclusion chromatography (Hsu Y-M, unpublished observations). The minimal structure of Fc-TWEAK is expected to be a hexamer consisting of two TWEAK trimers and three dimeric Ig Fc domains. This complexity and the presence of heterogeneous populations of Fc-TWEAK, make it a poor drug candidate. Recombinant soluble TWEAK would also not be a desirable drug candidate due to an extremely short half-life in vivo (Burkly LC, data not shown). Finally, BIIB036 may be a safer alternative because TWEAK administration in vivo has been associated with significant weight loss in mice (reviewed in ref. 33
and Michaelson JS unpublished observations), whereas no weight loss was observed in response to BIIB036 treatment even with weekly administration at a high dose of 25.6 mg/kg.
Fn14 is emerging as a therapeutic target for the treatment of cancer, joining other TNF family member receptors, such as the CD40 and TRAIL receptors, that are currently under investigation. Similar to the robust anti-tumor activity we observed in xenograft models in response to TWEAK or BII036 treatment, efficacy was observed with recombinant soluble TRAIL and agonistic antibodies to the TRAIL receptors, (TRAIL-R1 (DR4) and TRAIL-R2 (DR5)) in numerous tumor xenograft models.2
Ongoing clinical trials with soluble TRAIL and anti-TRAIL receptor antibodies have thus far demonstrated acceptable safety and tolerability in humans,3
thereby providing precedence for safety with systemic administration of TNF family agonists in man. However, one notable distinction between Fn14 and TRAIL receptors is that Fn14 does not contain a death domain which is responsible for DR4- and DR5-mediated assembly of the death inducing signaling complex (DISC).1
The differential mechanism of inducing tumor cell death, along with the distinct pattern of receptor expression in human tumors, may allow for a differentiated opportunity for Fn14 agonists in the clinic.
In the absence of a death domain in the cytoplasmic region of Fn14, it is less clear how stimulation of the Fn14 pathway leads to death of tumor cells. We showed that, like TWEAK, BIIB036 is capable of inducing NFκB pathway activation. An early report suggested that TWEAK-induced killing is TNF-dependent in Kym-1 cells.23
However, Nakayama et al.22
reported that TWEAK-induced death of HT-29, HS-C or KATO-III cells does not exhibit TNF dependence, so it appears that not all Fn14-mediated cell death is TNF-dependent. Our analysis showed that tumor cell killing induced by Fn14 agonists is not TNF dependent in a majority of tumor cell lines evaluated. Specifically, we showed that only three (SKOV-3, MCF7 and Panc-1) of 12 cell lines were TNF dependent. Interestingly, Vince et al. showed that TWEAK killing is TNF dependent in the three cell lines (Kym-1, SKOV-3 and OVCAR) that they tested, and TNF was detectable in the supernatants and cell extracts of TWEAK-treated cells. However, in our evaluation of six cell lines, we could only detect TNF in supernatants or cell extracts from SKOV-3-treated cells, suggesting that the cell lines described in Vince et al. may not be representative. Studies in several other cell types have also documented that TWEAK signaling is independent of TNF, including pro-inflammatory responses in synoviocytes, dermal fibroblasts and keratinocytes.34,35
Taken together, the results suggest that TWEAK signaling in general, and TWEAK-induced tumor cell killing in particular, may be predominantly independent of TNF.
We observed robust anti-tumor efficacy with BIIB036 in multiple xenograft models regardless of relative activity in vitro. Notably, in the NCI-N87 and MDA-MB-231 cell lines, BIIB036 exhibited negligible anti-tumor activity in MTT assays, yet in vivo a 75% reduction in tumor growth was observed. This lack of correlation between in vitro and in vivo sensitivity highlights the limitations of making predictions based on cell-based assays alone. Stromal cells, tumor architecture and host-tumor interactions likely contribute to the anti-tumor response in vivo. Indeed, one explanation for the enhanced activity that we observed in vivo with BIIB036 is the contribution of Fc effector function. Fc receptor expressing immune effector cells can bind the Fc portion of BIIB036 and may effectively induce ADCC, as we showed in the in vitro reconstituted ADCC assay. Moreover, binding of BIIB036 by Fc receptor expressing cells may result in effective multimerization of BIIB036 leading to higher order cross-linking of Fn14 molecules and thereby an enhanced anti-tumor effect. Indeed, we show that multimerization through protein A or secondary antibody cross-linking of BIIB036 resulted in significantly enhanced activity in MTT assays and signaling studies (Amatucci A and Michaelson JS, unpublished observations). This is consistent with a recent report36
showing that Fcγ receptors are capable of driving antibody-dependent signaling in target cells in the context of agonistic DR5 and CD40 antibodies, indicating that Fcγ receptor-dependent mechanisms contribute to the activity of antibodies directed against other TNF family member receptors as well. Our in vivo studies, demonstrating that effectorless versions of BIIB036 exhibited reduced activity in xenograft models, confirmed the contribution of Fc effector function to BIIB036 activity. Residual tumor inhibition was nevertheless observed with the fully effectorless versions of BIIB036 in xenograft models, suggesting that not all of the activity is Fc-mediated. Our data suggest that this is due to signaling through Fn14 on tumor cells (Michaelson JS, unpublished observations), although, given species cross-reactivity of BIIB036 to mouse Fn14, a direct effect of BIIB036 binding to non-tumor cells cannot be ruled out.
It is important to note that a monoclonal antibody targeting Fn14, PDL192 (Facet Biotech/Abbott), is currently undergoing evaluation in a Phase 1 clinical study. One difference between PDL192 and BIIB036 is that the binding affinity, as determined by Biacore, is five times greater for BIIB036 compared to that of PDL192.16
While it is possible that there may not be a direct correlation between binding affinity and activity, nevertheless there could be implications in terms of relative competition for Fn14 binding to TWEAK. Like BIIB036, PDL192 exhibited anti-tumor activity in multiple xenograft models.16
BIIB036 was administered at significantly lower doses and on a less frequent dosing schedule compared with PDL192, suggesting that BIIB036 may be a more potent anti-tumor agent. It is also likely that BIIB036 and PDL192 have different binding epitopes on Fn14. Indeed, a notable feature of BIIB036 is an ability to bind Fn14 across species, with equivalent affinity to rodent, monkey and human Fn14. Perhaps this is due to the fact that BIIB036 was selected from a panel of anti-Fn14 antibodies that were generated in Fn14-deficient mice, which would favor cross-reactivity to rodent Fn14. The species cross-reactivity is critical for drug development because it provides a path forward for informative toxicology studies in rodents and non-human primates.
In considering BIIB036 as a therapeutic for the treatment of cancer, it is important to note that high expression of Fn14 has been reported in multiple tumor types, including breast, pancreatic, glioma and non-small cell lung.7,11–16
Likewise, our xenograft studies, in which we observed robust efficacy, also spanned a range of tumor types, including colon, breast and gastric. Moreover, upregulated Fn14 expression was specifically observed in liver and bone metastases,16
and indeed BIIB036 demonstrated impressive anti-metastatic activity in the MDA-MB-231 model. In several tumor types, including glioma, breast and esophageal, a significant correlation between Fn14 expression and tumor grade and/or prognosis has been noted.13–15,17,37
The upregulated expression of Fn14 in tumors may be due to some of the other features of the TWEAK/Fn14 pathway, such as the ability to promote migration, invasion and survival.38
Interestingly, although these tumor promoting features, as well as a capacity to induce angiogenesis,9,10,39–42
have been attributed to TWEAK, it is important to emphasize that no enhancement in tumor growth was observed following BIIB036 treatment in any xenograft models tested to date. Thus, the fact that Fn14 is expressed in a wide variety of cancer types, and particularly in more aggressive tumors, together with our observation of in vivo anti-tumor efficacy in multiple tumor types, suggest that BIIB036 could be efficacious in the treatment of a broad range of tumors, and specifically in patients with advanced disease who are most in need of effective therapy.