A phase I clinical study of VB4-845: weekly intratumoral administration of an anti-EpCAM recombinant fusion protein in patients with squamous cell carcinoma of the head and neck.

VB4-845 is a scFv-Pseudomonas exotoxin A fusion construct that targets epithelial cell adhesion molecule (EpCAM). A phase I trial was conducted to determine the maximum tolerated dose (MTD) of VB4-845 when administered as weekly intratumoral (IT) injections to patients with squamous cell carcinoma of the head and neck (SCCHN). Secondary objectives included the evaluation of the safety, tolerability, pharmacokinetic profile, and immunogenicity, and a preliminary assessment of tumor response. Twenty patients with advanced, recurrent SCCHN were treated weekly for four weeks in ascending dose cohorts of 100, 200, 330, 500, 700, and 930 microg. The MTD was established as 930 microg with a dose limiting toxicity of elevated liver enzymes in two of five patients. VB4-845 therapy was well tolerated with common treatment-related adverse events of injection site reactions, fever, gastrointestinal disorders, and elevated liver enzyme levels. All patients developed antibodies to VB4-845 by the end of the study, but only seven patients had neutralizing antibodies. Preliminary efficacy data found 87.5% of EpCAM-positive patients had a positive response to VB4-845 therapy. Noninjected dermal metastases were also resolved in one patient. VB4-845 IT therapy is safe and feasible and warrants further clinical evaluation for the treatment of SCCHN.

Preclinical assessment of an anti-EpCAM immunotoxin: locoregional delivery provides a safer alternative to systemic administration.

VB4-845 is a recombinant immunotoxin that is comprised of a truncated form of Pseudomonas exotoxin A (ETA) genetically-linked to a humanized scFv fragment, (4D5MOCB), specific to epithelial cell adhesion molecule (EpCAM). EpCAM is overexpressed on a wide variety of human tumors and thus represents a suitable target antigen for immunotoxin therapy. Preclinical studies were used to evaluate the benefit of locoregional administration of an ETA-based immunotoxin versus systemic delivery. Repeated subcutaneous (s.c.) administration of VB4-845 (up to 77.8 microg/kg) in rats resulted in minimal adverse effects, except for injection-site reactions, while repeated systemic administration elicited symptoms consistent with vascular leak syndrome. S.c. weekly doses of the drug in cynomolgus monkeys resulted in minimal adverse effects limited to injection-site reactions and a transient elevation of liver enzymes in 1 animal. Toxicokinetics showed rapid clearance of the drug, with the development of an immune response by day 14 following repeated injections. These results argue that the local administration of VB4-845 has advantages with respect to safety over systemic administration and may be an effective alternative method for targeting those cancers that are amenable to local routes of administration.

Rcl is a novel ETV1/ER81 target gene upregulated in breast tumors.

ETV1 (ER81) is a transcription factor that can be activated by HER2/Neu, a proto-oncoprotein often overexpressed in metastatic breast tumors. Here, we demonstrate that ETV1 downregulation suppresses proliferation of HER2/Neu-positive MDA-MB-231 breast cancer cells in vitro and tumor formation in vivo, proving for the first time the existence of a critical role of ETV1 in breast cancer cell physiology. A screen for novel ETV1 target genes hinted at Rcl, an enzyme involved in nucleotide metabolism. To characterize the human Rcl gene, we cloned its promoter and found that ETV1 and HER2/Neu cooperated in activating the Rcl promoter, whereas a dominant-negative ETV1 molecule suppressed the Rcl promoter. Moreover, ETV1 and HER2/Neu synergized to upregulate the endogenous Rcl gene. ETV1 also bound to the Rcl promoter in vivo, indicating that Rcl is a bona fide target gene of ETV1. Hybridization of Rcl cDNA to a breast cancer array revealed that Rcl is overexpressed in approximately 40% of all breast tumors. Importantly, its expression significantly escalates with increasing tumor grade, strongly implicating that Rcl contributes to breast tumorigenesis. Since joint overexpression of Rcl with vascular endothelial growth factor, another target gene of ETV1, has been shown to induce tumor formation, Rcl may be one crucial effector of ETV1 and HER2/Neu in breast tumors. Furthermore, given its expression pattern and enzymatic function in nucleotide metabolism, Rcl presents itself as a novel target in breast cancer therapy via modulation of its activity by small molecule drugs.

Regulation of Her2/neu promoter activity by the ETS transcription factor, ER81.

Overexpression of the HER2/Neu receptor is correlated to a poor prognosis in tumor patients and leads to stimulation of mitogen-activated protein kinase (MAPK) signaling pathways, which in turn activate transcription factors, such as the ETS protein ER81. Here, we have analyzed whether, on the other hand, ER81 may regulate the Her2/neu gene. Indeed, ER81, together with its co-activators, p300 and CBP, activates the Her2/neu promoter, and this activation is enhanced upon stimulation of MAPK pathways as well as by oncogenic HER2/Neu protein. Furthermore, ER81 interacts with one ETS binding site in the Her2/neu promoter, whose mutation decreases ER81-mediated transcription. Activation of the Her2/neu promoter is also diminished upon mutation of MAPK-dependent phosphorylation sites in ER81 or upon deletion of ER81 transactivation domains. In addition, the ER81 DNA-binding domain on its own functions as a dominant-negative molecule, effectively repressing any stimulation of the Her2/neu promoter. Altogether, our results show that ER81 is a component of a positive regulatory feedback loop, in which the HER2/Neu protein activates ER81, as well as p300/CBP via MAPKs causing the upregulation of the Her2/neu gene.