Development and preclinical characterization of a humanized antibody targeting CXCL12.

PURPOSE: Our goal was to develop a potent humanized antibody against mouse/human CXCL12. This report summarized its in vitro and in vivo activities. EXPERIMENTAL DESIGN: Cell surface binding and cell migration assays were used to select neutralizing hamster antibodies, followed by testing in several animal models. Monoclonal antibody (mAb) 30D8 was selected for humanization based on its in vitro and in vivo activities. RESULTS: 30D8, a hamster antibody against mouse and human CXCL12α, CXCL12ß, and CXCL12γ, was shown to dose-dependently block CXCL12α binding to CXCR4 and CXCR7, and CXCL12α-induced Jurkat cell migration in vitro. Inhibition of primary tumor growth and/or metastasis was observed in several models. 30D8 alone significantly ameliorated arthritis in a mouse collagen-induced arthritis model (CIA). Combination with a TNF-α antagonist was additive. In addition, 30D8 inhibited 50% of laser-induced choroidal neovascularization (CNV) in mice. Humanized 30D8 (hu30D8) showed similar in vitro and in vivo activities as the parental hamster antibody. A crystal structure of the hu30D8 Fab/CXCL12α complex in combination with mutational analysis revealed a "hot spot" around residues Asn(44)/Asn(45) of CXCL12α and part of the RFFESH region required for CXCL12α binding to CXCR4 and CXCR7. Finally, hu30D8 exhibited fast clearance in cynomolgus monkeys but not in rats. CONCLUSION: CXCL12 is an attractive target for treatment of cancer and inflammation-related diseases; hu30D8 is suitable for testing this hypothesis in humans.

Identification and analysis of in vivo VEGF downstream markers link VEGF pathway activity with efficacy of anti-VEGF therapies.

PURPOSE: The aim of this study was to identify conserved pharmacodynamic and potential predictive biomarkers of response to anti-VEGF therapy using gene expression profiling in preclinical tumor models and in patients. EXPERIMENTAL DESIGN: Surrogate markers of VEGF inhibition [VEGF-dependent genes or VEGF-dependent vasculature (VDV)] were identified by profiling gene expression changes induced in response to VEGF blockade in preclinical tumor models and in human biopsies from patients treated with anti-VEGF monoclonal antibodies. The potential value of VDV genes as candidate predictive biomarkers was tested by correlating high or low VDV gene expression levels in pretreatment clinical samples with the subsequent clinical efficacy of bevacizumab (anti-VEGF)-containing therapy. RESULTS: We show that VDV genes, including direct and more distal VEGF downstream endothelial targets, enable detection of VEGF signaling inhibition in mouse tumor models and human tumor biopsies. Retrospective analyses of clinical trial data indicate that patients with higher VDV expression in pretreatment tumor samples exhibited improved clinical outcome when treated with bevacizumab-containing therapies. CONCLUSIONS: In this work, we identified surrogate markers (VDV genes) for in vivo VEGF signaling in tumors and showed clinical data supporting a correlation between pretreatment VEGF bioactivity and the subsequent efficacy of anti-VEGF therapy. We propose that VDV genes are candidate biomarkers with the potential to aid the selection of novel indications as well as patients likely to respond to anti-VEGF therapy. The data presented here define a diagnostic biomarker hypothesis based on translational research that warrants further evaluation in additional retrospective and prospective trials.

Modeling and predicting clinical efficacy for drugs targeting the tumor milieu.

Disappointing results from most late-stage clinical trials of cancer therapeutics indicate a need for improved and more-predictive animal tumor models. This insufficiency of models, combined with the advent of a class of drugs that target the tumor microenvironment rather than the tumor cell, presents new challenges for designing and interpreting preclinical efficacy studies. A comparison of the clinical efficacy of anti-angiogenic drugs with their corresponding preclinical studies over the past two decades offers many lessons that can inform and improve the design of experiments in existing mouse models. In addition, technological and logistical advances in mouse models of human cancer over the past five years have the potential to increase the clinical translatability of animal studies.

Differential drug class-specific metastatic effects following treatment with a panel of angiogenesis inhibitors.

Inhibiting angiogenesis has become an important therapeutic strategy for cancer treatment but, like other current targeted therapies, benefits experienced for late-stage cancers can be curtailed by inherent refractoriness or by acquired drug resistance, requiring a need for better mechanistic understanding of such effects. Numerous preclinical studies have demonstrated that VEGF pathway inhibitors suppress primary tumour growth and metastasis. However, it has been recently reported that short-term VEGF and VEGFR inhibition can paradoxically accelerate tumour invasiveness and metastasis in certain models. Here we comprehensively compare the effects of both antibody and small molecule receptor tyrosine kinase (RTK) inhibitors targeting the VEGF-VEGFR pathway, using short-term therapy in various mouse models of metastasis. Our findings demonstrate that antibody inhibition of VEGF pathway molecules does not promote metastasis, in contrast to selected small molecule RTK inhibitors at elevated-therapeutic drug dosages. In particular, a multi-targeted RTK inhibitor, sunitinib, which most profoundly potentiated metastasis, also increased lung vascular permeability and promoted tumour cell extravasation. Mechanistically, sunitinib, but not anti-VEGF treatment, attenuated endothelial barrier function in culture and caused a global inhibition of protein tyrosine phosphorylation, including molecules important for maintaining endothelial cell-cell junctions. Together these findings indicate that, rather than a specific consequence of inhibiting the VEGF signalling pathway, pharmacological inhibitors of the VEGF pathway can have dose- and drug class-dependent side-effects on the host vasculature. These findings also advocate for the continued identification of mechanisms of resistance to anti-angiogenics and for therapy development to overcome it.