ABSTRACT

CXCR1 and CXCR2 signaling play a critical role in neutrophil migration, angiogenesis, and tumorigenesis and are therefore an attractive signaling axis to target in a variety of indications. In human, a total of seven chemokines signal through these receptors and comprise the ELR⁺CXC chemokine family, so named because of the conserved ELRCXC N-terminal motif. To fully antagonize CXCR1 and CXCR2 signaling, an effective therapeutic should block either both receptors or all seven ligands, yet neither approach has been fully realized clinically. In this work, we describe the generation and characterization of LY3041658, a humanized monoclonal antibody that binds and neutralizes all seven human and cynomolgus monkey ELR⁺CXC chemokines and three of five mouse and rat ELR⁺CXC chemokines with high affinity. LY3041658 is able to block ELR⁺CXC chemokine-induced Ca²⁺ mobilization, CXCR2 internalization, and chemotaxis in vitro as well as neutrophil mobilization in vivo without affecting other neutrophil functions. In addition to the in vitro and in vivo activity, we characterized the epitope and structural basis for binding in detail through alanine scanning, crystallography, and mutagenesis. Together, these data provide a robust preclinical characterization of LY3041658 for which the efficacy and safety is being evaluated in human clinical trials for neutrophilic skin diseases.

摘要

CXCR1 和 CXCR2 信号在中性粒细胞迁移、血管生成和肿瘤发生中起关键作用，因此是一个有吸引力的信号轴，可用于多种适应症。在人类中，共有七种趋化因子通过这些受体发出信号，包括 ELR ⁺ CXC 趋化因子家族，之所以如此命名，是因为保守的 ELRCXC N 端基序。为了完全拮抗 CXCR1 和 CXCR2 信号传导，有效的治疗方法应该阻断这两种受体或所有七种配体，但临床上尚未完全实现这两种方法。在这项工作中，我们描述了 LY3041658 的生成和表征，这是一种人源化单克隆抗体，可结合并中和所有七种人和食蟹猴 ELR ⁺ CXC 趋化因子以及五种小鼠和大鼠 ELR ^{+ 中的}三种CXC 趋化因子具有高亲和力。LY3041658 能够在体外阻断 ELR ⁺ CXC 趋化因子诱导的 Ca ²⁺动员、CXCR2 内化和趋化性以及体内中性粒细胞动员，而不影响其他中性粒细胞功能。除了体外和体内活性外，我们还通过丙氨酸扫描、晶体学和诱变详细描述了结合的表位和结构基础。总之，这些数据为 LY3041658 提供了强有力的临床前表征，正在对中性粒细胞皮肤病的人体临床试验评估其有效性和安全性。