Receptor tyrosine kinase EphA5 is a functional molecular target in human lung cancer.

Lung cancer is often refractory to radiotherapy, but molecular mechanisms of tumor resistance remain poorly defined. Here we show that the receptor tyrosine kinase EphA5 is specifically overexpressed in lung cancer and is involved in regulating cellular responses to genotoxic insult. In the absence of EphA5, lung cancer cells displayed a defective G1/S cell cycle checkpoint, were unable to resolve DNA damage, and became radiosensitive. Upon irradiation, EphA5 was transported into the nucleus where it interacted with activated ATM (ataxia-telangiectasia mutated) at sites of DNA repair. Finally, we demonstrate that a new monoclonal antibody against human EphA5 sensitized lung cancer cells and human lung cancer xenografts to radiotherapy and significantly prolonged survival, thus suggesting the likelihood of translational applications.

In vitro potency, pharmacokinetic profiles, and pharmacological activity of optimized anti-IL-21R antibodies in a mouse model of lupus.

Using phage display, we generated a panel of optimized neutralizing antibodies against the human and mouse receptors for interleukin 21 (IL-21), a cytokine that is implicated in the pathogenesis of many types of autoimmune disease. Two antibodies, Ab-01 and Ab-02, which differed by only four amino acids in V(L) CDR3, showed potent inhibition of human and mouse IL-21R in cell-based assays and were evaluated for their pharmacological and pharmacodynamic properties. Ab-01, but not Ab-02, significantly reduced a biomarker of disease (anti-dsDNA antibodies) and IgG deposits in the kidney in the MRL-Fas(lpr) mouse model of lupus, suggesting that anti-IL-21R antibodies may prove useful in the treatment of lupus. Ab-01 also had a consistently higher exposure (AUC(0-infinity)) than Ab-02 following a single dose in rodents or cynomolgus monkeys (2-3-fold or 4-7-fold, respectively). Our data demonstrate that small differences in CDR3 sequences of optimized antibodies can lead to profound differences in in vitro and in vivo properties, including differences in pharmacological activity and pharmacokinetic profiles. The lack of persistent activity of Ab-02 in the MRL-Fas(lpr) mouse lupus model may have been a consequence of faster elimination, reduced potency in blocking the effects of mouse IL-21R, and more potent/earlier onset of the anti-product response relative to Ab-01.

Novel agonist monoclonal antibodies activate TrkB receptors and demonstrate potent neurotrophic activities.

Tyrosine kinase receptor B (TrkB) mediates neurotrophic effects of brain-derived neurotrophic factor (BDNF) to increase neuronal survival, differentiation, synaptic plasticity, and neurogenesis. The therapeutic potential of TrkB activation using BDNF has been demonstrated well in several preclinical models of CNS diseases, validating TrkB as a promising drug target. Therefore, we aimed to develop TrkB-specific receptor agonists by using a monoclonal antibody approach. After generation of hybridoma clones and assessment of their binding and functional activity, we identified five mouse monoclonal antibodies that show highly selective binding to TrkB and that induce robust activation of TrkB signaling. Epitope mapping studies using competition analysis showed that each of the monoclonal antibodies recognizes a unique binding site on TrkB, some of which are distinct from BDNF docking sites. These antibodies behave as true agonists based on their ability to both activate proximal and secondary signaling molecules downstream of TrkB receptors and promote neuronal survival and neurite outgrowth. The binding affinities and the functional efficacy of these antibodies are comparable to those of BDNF, whereas they do not bind to the p75 low-affinity neurotrophin receptor at all. Therefore, they could represent novel reagents to explore the pathophysiological roles of TrkB and its potential therapeutic utility in treating CNS disorders.

Anti GPVI human antibodies neutralizing collagen-induced platelet aggregation isolated from a combinatorial phage display library.

Glycoprotein VI is a type I membrane protein identified as a key platelet receptor for collagen. In vitro binding of the GPVI receptor with collagen leads to activation and ultimately to aggregation of platelets. In vivo, GPVI-collagen interactions could cause formation of occlusive thrombi within vessels with damaged endothelial barriers. GPVI antagonists are therefore important therapeutics in patients suffering from collagen-mediated ischemic disorders such as myocardial infarction or stroke. Polyclonal antibodies to GPVI prepared from one patient serum have previously been described. However, only their monovalent Fab fragments, incapable of receptor crosslinking, were found to protect platelets from collagen-mediated aggregation. Here we describe GPVI-neutralizing human antibodies derived from a combinatorial phage display library of single-chain antibodies. By selecting phage on GPVI-expressing U937 cells, we isolated five specific antibodies - A4, A9, A10, C3 and C9. Of the set A10 and C3 specifically blocked GPVI binding to collagen-rich adventitial layers in aorta sections. The higher affinity antibody A10 inhibited binding of snake-venom convulxin to GPVI. It also specifically protected human platelets from collagen-induced aggregation in vitro. A10-bound platelets could still be activated by ADP or thrombin suggesting that this human scFv may represent an original anti-platelet agent for the treatment of collagen-mediated thrombotic diseases.