Various TNF and TNF receptor (TNFR) superfamily members have been identified as being essential elements in tumor growth and development (25
). In addition, they have also been implicated in the response and resistance of tumor cells to therapeutic agents (28
). For many of these reasons, TNF family member ligands and their cognate receptors seem to be attractive targets for intervention and for the development of targeted therapeutics for the treatment of cancer and immunologic diseases (28
). One particular TNF:TNFR pair, TWEAK and Fn14, has been implicated in several important processes associated with tumor growth and metastasis (5
), and a recent report has shown that administration of an anti-Fn14 mAb can inhibit tumor growth in xenograft assays (8
The use of antibodies to interfere with growth factors and their receptors present on tumors has gained wide acceptance as a therapeutic strategy (34
). Targeting the HER1, HER2, and VEGF pathways with antibodies is now considered an essential component in the clinical therapeutic management of a variety of tumor types (37
). There are also numerous groups developing antibody conjugates for the directed delivery of highly cytotoxic payloads such as small molecules—auristatin, caliecheamycin, or maytansinoids (38
), protein toxins, that is, RTA, PE, and gelonin (22
), or other cytotoxic human enzymes such as serine proteases (44
). The current study shows a unique Fn14 receptor–targeted antibody–toxin conjugate designated ITEM4-rGel that is shown to be capable of specifically delivering rGel to Fn14-expressing tumor cells in vitro
and in vivo.
As described previously, activation of TWEAK signaling by either ligands or antibodies has the potential to elicit progrowth effects in addition to defined cytotoxic properties. The use of cytotoxic conjugates targeting Fn14 has the potential to augment the cytotoxic effects of pathway activation while eliminating the growth stimulatory properties.
The antibody ITEM-4 and its corresponding immunoconjugate ITEM4-rGel was shown to selectively bind the Fn14 receptor on tumor cells. The cytotoxicity of ITEM4-rGel on Fn14-expressing tumor cells was investigated extensively in vitro.
ITEM4-rGel was found to be selectively toxic to a wide range of human tumor cell lines expressing the Fn14 receptor (). Cells overexpressing Fn14 were highly sensitive to the ITEM4-rGel, whereas cells expressing no Fn14 were no more sensitive to the conjugate than they were to free rGel. Surprisingly, some cell lines expressing high (Calu-3) or intermediate (ME-180 and HT-29) levels of Fn14 showed relatively low levels of sensitivity to the construct. Alternatively, Eb1 breast tumor cells expressing low levels of Fn14 showed extreme sensitivity to the construct. The observed differences in cellular response to the conjugate may be the result of differences in cellular protein synthesis rates, receptor recycling, immunotoxin internalization efficiency (45
), or intracellular trafficking and release from endosomal compartments (47
). All of these factors may play a role in determining the ultimate response of cells to the ITEM4-rGel construct.
Our previous studies with rGel-based constructs showed that some constructs induced apoptosis (48
), but some did not, and this seemed to be highly dependent on the cell type under investigation (49
). We found that the ITEM4-rGel induced cell death with clear mitochondrial dependent apoptosis. We also showed that when T-24 cells were treated with either ITEM-4 alone or ITEM4-rGel, the protein HMGB1 was released into the media. The effect of unconjugated ITEM-4 on HMGB1 release was unexpected, as ITEM-4 showed no effect on the growth of these cells in culture. The effect of ITEM-4 on HMGB1 release by tumor cells has not been reported previously, but a study by Kalinina and colleagues (50
) reported that treatment with the Fn14 ligand TWEAK caused an upregulation of HMGB1 synthesis and secretion by normal cells as part of the inflammatory process. It is possible that ITEM-4 agonistic effect on the Fn14 receptor could cause release of HMGB1 in certain cell types.
Fn14 is gathering much attention at this time because of its strong overexpression in many of the most prevalent and deadly solid tumor types. We used the T-24 bladder model because the in vitro response to the cytotoxic effects of the conjugate and the relative expression levels of the Fn14 receptor (via Western blot) seemed to be intermediate in the range of cell lines tested. The results of our studies using the T-24 xenograft model show that an immunoconjugate targeting Fn14 can suppress tumor growth. The maximum tolerated dose (MTD) for ITEM4-rGel with this schedule was determined to be 65 mg/kg (data not shown) and we selected 20 and 30 mg/kg total dosages (30% and 50% of the MTD, respectively) for these initial studies. The in vivo data show that effective tumor cell killing (by induction of apoptosis as assayed by TUNEL assay) can be achieved with ITEM4-rGel at these doses without any observable side effects in mice suggest that targeting of the conjugate to normal mouse tissues is minimal. The immunohistochemical studies showed that ITEM4-rGel uniformly distributed in tumor xenografts after intravenous administration, although additional studies are ongoing to examine the pharmacokinetics and the relative uptake kinetics of ITEM4-rGel into tumor compared with normal organs. The results from these studies may define a new schedule of administration to optimize tumor uptake of the immunoconjugate.
In summary, this study shows a proof of concept that Fn14 seems to have an excellent potential for targeted therapy approaches and ITEM-4–based therapeutic agents seem to warrant further development. Although therapeutic studies with ITEM4-rGel show in vivo efficacy, generating a human or humanized version of ITEM-4 is essential for long-term clinical administration. The immunogenicity of the rGel component of the conjugate is of potential concern, although clinical studies of an immunoconjugate with the anti-CD33 antibody HuM195 have shown limited antigenicity of the rGel component even with repeated administration (Cortes and colleagues, unpublished data). Studies of anti-Fn14 constructs containing second-generation payloads such as deimmunized rGel toxin (designated drGel) or fully human granzyme B as a replacement for full-length rGel are in progress.