Antibodies to TWEAK receptor inhibit human tumor growth through dual mechanisms.

PURPOSE: Targeted therapeutics have significantly changed the outcome for patients diagnosed with cancer. Still, effective therapeutic intervention does not exist for many cancers and much remains to be done. The objective of this study was to identify novel genes that potentially regulate tumor growth, to target these gene products with monoclonal antibodies, and to examine the therapeutic potential of these antibodies. EXPERIMENTAL DESIGN: Using cDNA microarray analysis, we identified genes overexpressed in several solid malignancies. We generated a mouse monoclonal antibody, 19.2.1, and its humanized counterpart, PDL192, to one such target, TweakR (TWEAK receptor, Fn14, TNFRSF12A, CD266), and characterized the antitumor activities in vitro and in mouse xenograft models. RESULTS: Both 19.2.1 (mouse IgG2a) and PDL192 (human IgG1), like TWEAK, the natural ligand of TweakR, inhibited the growth of several TweakR-expressing cancer cell lines in anchorage-dependent and anchorage-independent assays in vitro. Both antibodies showed significant antitumor activity in multiple mouse xenograft models. PDL192 and 19.2.1 also induced antibody-dependent cellular cytotoxicity (ADCC) of cancer cell lines in vitro. A chimeric version of 19.2.1 containing the mouse IgG1 Fc region (19.2.1 x G1) exhibited significantly less ADCC than 19.2.1. However, 19.2. 1x G1 showed differential activity in vivo, with activity equivalent to 19.2.1 in one model, but significantly less efficacy than 19.2.1 in a second model. These results indicate that PDL192 and 19.2.1 mediate their antitumor effects by signaling through TweakR, resulting in reduced tumor cell proliferation, and by ADCC.

Volociximab, a chimeric integrin alpha5beta1 antibody, inhibits the growth of VX2 tumors in rabbits.

Angiogenesis, the process by which new blood vessels form from existing vasculature, is critical for tumor growth and invasion. Growth factors, such as VEGF, initiate signaling cascades resulting in the proliferation of resting endothelial cells. Blockade of growth factor pathways has proven effective in inhibiting angiogenesis and tumor growth in vivo. Integrins, including the integrin alpha5beta1, are also important mediators of angiogenesis and these adhesion molecules also regulate cancer cell growth and migration in vitro. Volociximab is a high affinity, function-blocking antibody against integrin alpha5beta1 that is currently in multiple Phase II oncology clinical trials. Volociximab displays potent anti-angiogenic activity in a monkey model of choroidal neovascularization. In this study, we explored the consequences of integrin alpha5beta1 blockade on tumorigenesis. Because volociximab does not cross-react with rodent alpha5beta1, the syngeneic rabbit VX2 carcinoma model was utilized as an alternative to standard mouse xenograft models for the assessment of anti-tumor activity of volociximab. Volociximab administered intravenously to rabbits bearing VX2 tumors is detectable on tumor cells and vasculature 45 min post-administration. Volociximab was found to significantly inhibit the growth of tumors growing subcutaneously or intramuscularly, despite a 20-fold lower affinity for rabbit integrin, relative to human. This effect was found to correlate with decreased blood vessel density within these tumors. These results support the use of volociximab in the intervention of malignant disease.

A function blocking anti-mouse integrin alpha5beta1 antibody inhibits angiogenesis and impedes tumor growth in vivo.

BACKGROUND: Integrins are important adhesion molecules that regulate tumor and endothelial cell survival, proliferation and migration. The integrin alpha5beta1 has been shown to play a critical role during angiogenesis. An inhibitor of this integrin, volociximab (M200), inhibits endothelial cell growth and movement in vitro, independent of the growth factor milieu, and inhibits tumor growth in vivo in the rabbit VX2 carcinoma model. Although volociximab has already been tested in open label, pilot phase II clinical trials in melanoma, pancreatic and renal cell cancer, evaluation of the mechanism of action of volociximab has been limited because this antibody does not cross-react with murine alpha5beta1, precluding its use in standard mouse xenograft models. METHODS: We generated a panel of rat-anti-mouse alpha5beta1 antibodies, with the intent of identifying an antibody that recapitulated the properties of volociximab. Hybridoma clones were screened for analogous function to volociximab, including specificity for alpha5beta1 heterodimer and blocking of integrin binding to fibronectin. A subset of antibodies that met these criteria were further characterized for their capacities to bind to mouse endothelial cells, inhibit cell migration and block angiogenesis in vitro. One antibody that encompassed all of these attributes, 339.1, was selected from this panel and tested in xenograft models. RESULTS: A panel of antibodies was characterized for specificity and potency. The affinity of antibody 339.1 for mouse integrin alpha5beta1 was determined to be 0.59 nM, as measured by BIAcore. This antibody does not significantly cross-react with human integrin, however 339.1 inhibits murine endothelial cell migration and tube formation and elicits cell death in these cells (EC50 = 5.3 nM). In multiple xenograft models, 339.1 inhibited the growth of established tumors by 40-60% (p < 0.05) and this inhibition correlates with a concomitant decrease in vessel density. CONCLUSION: The results herein demonstrate that 339.1, like volociximab, exhibits potent anti-alpha5beta1 activity and confirms that inhibition of integrin alpha5beta1 impedes angiogenesis and slows tumor growth in vivo.

Preclinical evaluation of an anti-alpha5beta1 integrin antibody as a novel anti-angiogenic agent.

Integrin alpha5beta1, the principal fibronectin receptor, is an important survival factor, playing a key role in angiogenesis. Angiogenesis is critical for tumor growth, and anti-angiogenic therapies have met clinical success. To validate the therapeutic potential of an anti-alpha5beta1 strategy, we generated volociximab (M200) a chimeric human IgG4 version of the alpha5beta1 function-blocking murine antibody IIA1; and F200, the Fab derivative. Volociximab, F200 and IIA1 showed similar activity by ELISA (EC50= 0.2nM), Biacore (Kd= 0.1-0.4nM) and inhibition of fibronectin binding (IC50= 2-3nM). The inhibitory potential of alpha5beta1 antibodies was compared to HuMV833, an anti-VEGF antibody. Both volociximab and HuMV833 inhibited HUVEC proliferation (IC50 of volociximab = 0.2-0.5nM; IC50 of HuMV833 = 45nM). However, IIA1, volociximab and F200 were also potent inhibitors of an in vitro model of angiogenesis (HUVEC tube formation assay), unlike HuMV833. Additionally, volociximab inhibited in vitro tube formation induced by VEGF and/or bFGF, suggesting a mechanism of action independent of growth factor stimulus. In fact, inhibition of alpha5beta1 function by volociximab induced apoptosis of actively proliferating, but not resting, endothelial cells. Volociximab does not cross-react with rodent alpha5beta1, therefore in vivo validation of an anti-alpha5beta1 approach was conducted in a cynomolgus model of choroidal revascularization. Volociximab and F200 were potent inhibitors of neovessel formation in this model. These data demonstrate that volociximab has therapeutic potential in diseases in which new vessel formation is a component of the pathology.