In vitro affinity optimization of an anti-BDNF monoclonal antibody translates to improved potency in targeting chronic pain states in vivo.

The role of brain-derived neurotrophic factor (BDNF) signaling in chronic pain has been well documented. Given the important central role of BDNF in long term plasticity and memory, we sought to engineer a high affinity, peripherally-restricted monoclonal antibody against BDNF to modulate pain. BDNF shares 100% sequence homology across human and rodents; thus, we selected chickens as an alternative immune host for initial antibody generation. Here, we describe the affinity optimization of complementarity-determining region-grafted, chicken-derived R3bH01, an anti-BDNF antibody specifically blocking the TrkB receptor interaction. Antibody optimization led to the identification of B30, which has a > 300-fold improvement in affinity based on BIAcore, an 800-fold improvement in potency in a cell-based pERK assay and demonstrates exquisite selectivity over related neurotrophins. Affinity improvements measured in vitro translated to in vivo pharmacological activity, with B30 demonstrating a 30-fold improvement in potency over parental R3bH01 in a peripheral nerve injury model. We further demonstrate that peripheral BDNF plays a role in maintaining the plasticity of sensory neurons following nerve damage, with B30 reversing neuron hyperexcitability associated with heat and mechanical stimuli in a dose-dependent fashion. In summary, our data demonstrate that effective sequestration of BDNF via a high affinity neutralizing antibody has potential utility in modulating the pathophysiological mechanisms that drive chronic pain states.

Program death-1 engagement upon TCR activation has distinct effects on costimulation and cytokine-driven proliferation: attenuation of ICOS, IL-4, and IL-21, but not CD28, IL-7, and IL-15 responses.

The program death 1 (PD-1) receptor and its ligands, PD-1 ligand (PD-L)1 and PD-L2, define a novel regulatory pathway with potential inhibitory effects on T, B, and monocyte responses. In the present study, we show that human CD4(+) T cells express PD-1, PD-L1, and PD-L2 upon activation, and Abs to the receptor can be agonists or antagonists of the pathway. Under optimal conditions of stimulation, ICOS but not CD28 costimulation can be prevented by PD-1 engagement. IL-2 levels induced by costimulation are critical in determining the outcome of the PD-1 engagement. Thus, low to marginal IL-2 levels produced upon ICOS costimulation account for the greater sensitivity of this pathway to PD-1-mediated inhibition. Interestingly, exogenous IL-2, IL-7, and IL-15 but not IL-4 and IL-21 can rescue PD-1 inhibition, suggesting that among these cytokines only those that activate STAT5 can rescue PD-1 inhibition. As STAT5 has been implicated in the maintenance of IL-2Ralpha expression, these results suggest that IL-7 and IL-15 restore proliferation under conditions of PD-1 engagement by enhancing high-affinity IL-2R expression and hence, IL-2 responsiveness.