Chimeric and humanized versions of the anti-CD44 monoclonal antibody ARH460-16-2 have potent anti-tumor efficacy in established models of human breast and metastatic liver cancer

A74 CD44 is a widely distributed protein implicated in multiple normal physiological and pathological events including co-stimulation of T-lymphocytes, leukocyte re-circulation and tumor metastasis. CD44 interacts with hyaluronic acid and other extracellular matrix components and metalloproteases, and recently has been shown to be expressed in tumor cancer stem cells from several cancer types, including breast, colon, prostate, pancreas and AML. The association of CD44 with cancer makes it a very interesting candidate molecule for targeted therapy. The anti-CD44 monoclonal antibody ARH460-16-2, that was generated and identified with Arius’ FunctionFirst™ platform, has significant tumor growth inhibitory activity in in vivo animal models of breast, prostate and liver cancer. A chimeric version of the antibody (chARH460-16-2) demonstrated potent dose-dependent tumor growth inhibitory efficacy in an in vivo established subcutaneous model of breast cancer and in an established orthotopic model of metastatic liver cancer at all doses (20, 2 and 0.2mg/kg). In the MDA-MB-231 breast cancer model it was associated with tumor regression and with increased survival (time-to-endpoint) at all doses. IHC staining of colon tumor tissue sections revealed specific binding of ARH460-16-2 to tumor cells in 47% of patient samples (28/59), therefore extending the potential therapeutic benefit to human colon cancer. Previous work, carried out to elucidate mechanisms of action by which the ARH460-16-2 antibody mediates its tumor growth inhibitory activity, demonstrated its ability to induce tumor cell apoptosis in vitro and blocking of tumor cell binding to HA-coated surfaces. Early results from an in vitro kinase profiler screening study, carried out to address the direct impact of the antibody on intracellular signaling pathways, revealed suppression of phosphorylation of Tie-1 receptor tyrosine kinase upon stimulation of MDA-MB-231 cells with serum, after pre-incubation with chARH460-16-2. This result could lead to the identification of potential biomarkers for activity and efficacy of anti-CD44 targeted therapy to be used in clinical trials. Two humanized versions of the ARH460-16-2 antibody were generated and their affinity was comparable to those of the chimeric version and parent murine monoclonal antibody, as determined by Biacore (KD ranged from 3-6 nM). The tumor growth inhibitory potency of the two humanized variants of huARH460-16-2 is currently being assessed in established tumor models. Analysis of the binding of ARH460-16-2, by FACS and by IHC, to tissues from various species, including non-human primates, led to the selection of cynomolgus monkeys as a model for the in vivo toxicology studies with this antibody.