A human monoclonal antibody targeting the stem cell factor receptor (c-Kit) blocks tumor cell signaling and inhibits tumor growth.

Stem cell factor receptor (c-Kit) exerts multiple biological effects on target cells upon binding its ligand stem cell factor (SCF). Aberrant activation of c-Kit results in dysregulated signaling and is implicated in the pathogenesis of numerous cancers. The development of more specific and effective c-Kit therapies is warranted given its essential role in tumorigenesis. In this study, we describe the biological properties of CK6, a fully human IgG1 monoclonal antibody against the extracellular region of human c-Kit. CK6 specifically binds c-Kit receptor with high affinity (EC 50 = 0.06 nM) and strongly blocks its interaction with SCF (IC 50 = 0.41 nM) in solid phase assays. Flow cytometry shows CK6 binding to c-Kit on the cell surface of human small cell lung carcinoma (SCLC), melanoma, and leukemia tumor cell lines. Furthermore, exposure to CK6 inhibits SCF stimulation of c-Kit tyrosine kinase activity and downstream signaling pathways such as mitogen-activated protein kinase (MAPK) and protein kinase B (AKT), in addition to reducing tumor cell line growth in vitro. CK6 treatment significantly decreases human xenograft tumor growth in NCI-H526 SCLC (T/C% = 57) and Malme-3M melanoma (T/C% = 58) models in vivo. The combination of CK6 with standard of care chemotherapy agents, cisplatin and etoposide for SCLC or dacarbazine for melanoma, more potently reduces tumor growth (SCLC T/C% = 24, melanoma T/C% = 38) compared with CK6 or chemotherapy alone. In summary, our results demonstrate that CK6 is a c-Kit antagonist antibody with tumor growth neutralizing properties and are highly suggestive of potential therapeutic application in treating human malignancies harboring c-Kit receptor.

Pleiotropic stromal effects of vascular endothelial growth factor receptor 2 antibody therapy in renal cell carcinoma models.

The benefits of inhibiting vascular endothelial growth factor (VEGF) signaling in cancer patients are predominantly attributed to effects on tumor endothelial cells. Targeting non-endothelial stromal cells to further impact tumor cell growth and survival is being pursued through the inhibition of additional growth factor pathways important for the survival and/or proliferation of these cells. However, recent data suggest that VEGF receptor (VEGFR)-specific inhibitors may target lymphatic vessels and pericytes in addition to blood vessels. Here, in fact, we demonstrate that DC101 (40 mg/kg, thrice a week), an antibody specific to murine VEGFR2, significantly reduces all three of these stromal components in subcutaneous (SKRC-29) and orthotopic (786-O-LP) models of renal cell carcinoma (RCC) established in nu/nu athymic mice. Sunitinib (40 mg/kg, once daily), a receptor tyrosine kinase inhibitor of VEGFR2 and other growth factor receptors, also caused significant loss of tumor blood vessels in RCC models but had weaker effects than DC101 on pericytes and lymphatic vessels. In combination, sunitinib did not significantly add to the effects of DC101 on tumor blood vessels, lymphatic vessels, or pericytes. Nevertheless, sunitinib increased the effect of DC101 on tumor burden in the SKRC-29 model, perhaps related to its broader specificity. Our data have important implications for combination therapy design, supporting the conclusion that targeting VEGFR2 alone in RCC has the potential to have pleiotropic effects on tumor stroma.

BDNF-mediated enhancement of inflammation and injury in the aging heart.

Aging is associated with shifts in autocrine and paracrine pathways in the cardiac vasculature that may contribute to the risk of cardiovascular disease in older persons. To elucidate the molecular basis of these changes in vivo, phage-display biopanning of 3- and 18-mo-old mouse hearts was performed that identified peptide epitopes with homology to brain-derived neurotrophic factor (BDNF) in old but not young phage pools. Quantification of cardiac phage binding by titration and immunostaining after injection with BDNF-like phage identified a twofold increased density of the BDNF receptor, truncated Trk B, in the aging hearts. Studies focused on the receptor ligand using a rat model of transient myocardial ischemia revealed increases in cardiac BDNF associated with local mononuclear infiltrates in 24- but not 4-mo-old rats. To investigate these changes, both 4- and 24-mo-old rat hearts were treated with intramyocardial injections of BDNF (or PBS control), demonstrating significant inflammatory increases with activated macrophage (ED1+) in BDNF-treated aging hearts compared with aging controls and similarly treated young hearts. Additional studies with permanent coronary occlusion following intramyocardial growth factor pretreatment revealed that BDNF significantly increased the extent of myocardial injury in older rat hearts (BDNF 35 +/- 10% vs. PBS 16.2 +/- 7.9% left ventricular injury; P < 0.05) without affecting younger hearts (BDNF 15 +/- 5.1% vs. PBS 14.5 +/- 6.0% left ventricular injury). Overall, these studies suggest that age-associated changes in BDNF-Trk B pathways may predispose the aging heart to increased injury after acute myocardial infarction and potentially contribute to the enhanced severity of cardiovascular disease in older individuals.

PDGF-AB-based functional cardioprotection of the aging rat heart.

Recent studies have demonstrated that targeting of an age-associated impairment in platelet-derived growth factor (PDGF-AB) pathways could reduce histological measures of myocardial infarction in aging rat hearts. To facilitate preclinical developments of this approach, non-invasive measures of cardiac function were investigated in a 24-month-old rat myocardial infarction model employing intramyocardial PDGF-AB (100 ng) or vehicle control pretreatment. Electrocardiographic recordings post-coronary occlusion revealed ST segment elevation-myocardial injury patterns in both groups, which was confirmed histologically 2 weeks later by Masson's trichrome stains (PDGF-AB, 14.6+/-2.8% of left ventricular area (LVA) vs. control, 27.9+/-9.2%; P<0.05). Echocardiographic fractional shortening (FS) measurements revealed greater preservation of cardiac function in PDGF-AB-treated hearts compared with controls (PDGF-AB FS: 27.3+/-3.7% vs. control--16.7+/-4.1% (ANOVA P=0.005) vs. sham operation--34.5+/-6.7%), with a significant inverse relationship between FS and extent of myocardial injury (m=-0.68; r=-0.84). Notably, exercise testing did not correlate with myocardial injury. These findings provide an important functional foundation in preclinical translations of PDGF-AB-based cardioprotective treatment strategies. Moreover, demonstration of respective roles of electrocardiography and echocardiography in the confirmation and correlation of myocardial injury in the aging rat heart may serve to facilitate both PDGF-AB-based and other age-targeted approaches in large animal models of aging and cardiovascular disease.

Synergistic targeting with bone marrow-derived cells and PDGF improves diabetic vascular function.

Diabetes mellitus is associated with an increased risk of vascular disease, with significant alterations in systemic endothelial progenitor cells (EPCs) and peripheral vascular function. To identify the contribution of the different vascular compartments in the diabetic impairment of vascularization, we employed streptozotocin- and control-treated 3-mo-old C57Bl/6 mice in an isogeneic pinnal cardiac allograft model, revealing a significant delay in vascularization of wild-type cardiac tissue transplanted into diabetic mice. To investigate the basis of this impairment, the function of diabetic bone marrow cells was tested by transplantation of bone marrow cells isolated from diabetic and control mice into intact, unirradiated 18-mo-old C57Bl/6 mice, which have impaired function of both EPCs and peripheral endothelial cells. Importantly, cells derived from control, but not diabetic, bone marrow integrated into transplanted cardiac allografts. To assess the contribution of diabetic changes in the local vasculature, diabetic mice were treated with pinnal injections of platelet-derived growth factor (PDGF)-AB, which promotes cardiac angiogenesis in wild-type mice. However, whereas PDGF-AB enhanced allograft function in control mice, the activity of the cardiac transplants in the PDGF-AB-treated diabetic mice was significantly decreased. To decipher the potential interactions between systemic bone marrow-derived cells and local vascular pathways, diabetic mice were transplanted with wild-type bone marrow cells with or without PDGF-AB pinnal pretreatment, resulting in improved allograft function and donor cell recruitment only in the combination treatment arm. Overall, these studies show that the diabetic impairment in cardiac angiogenesis can be reversed by targeting the synergism between local trophic pathways and systemic cell function.