ABSTRACT

The long circulating half-life and inherently bivalent architecture of IgGs provide an ideal vehicle for presenting otherwise short-lived G-protein-coupled receptor agonists in a format that enables avidity-driven enhancement of potency. Here, we describe the site-specific conjugation of a dual agonist peptide (an oxyntomodulin variant engineered for potency and in vivo stability) to the complementarity-determining regions (CDRs) of an immunologically silent IgG4. A cysteine-containing heavy chain CDR3 variant was identified that provided clean conjugation to a bromoacetylated peptide without interference from any of the endogenous mAb cysteine residues. The resulting mAb-peptide homodimer has high potency at both target receptors (glucagon receptor, GCGR, and glucagon-like peptide 1 receptor, GLP-1R) driven by an increase in receptor avidity provided by the spatially defined presentation of the peptides. Interestingly, the avidity effects are different at the two target receptors. A single dose of the long-acting peptide conjugate robustly inhibited food intake and decreased body weight in insulin resistant diet-induced obese mice, in addition to ameliorating glucose intolerance. Inhibition of food intake and decrease in body weight was also seen in overweight cynomolgus monkeys. The weight loss resulting from dosing with the bivalently conjugated dual agonist was significantly greater than for the monomeric analog, clearly demonstrating translation of the measured in vitro avidity to in vivo pharmacology.

摘要

IgG的长循环半衰期和固有的二价结构提供了一种理想的载体，可以以亲和力驱动的效价增强形式呈递其他形式的短寿命G蛋白偶联受体激动剂。在这里，我们描述了双重激动剂肽（为增强效力和体内稳定性而设计的催产调节蛋白变体）与免疫沉默IgG4的互补决定区（CDR）的位点特异性结合。鉴定出含有半胱氨酸的重链CDR3变体，其提供与溴代乙酰化肽的干净结合，而不受任何内源性mAb半胱氨酸残基的干扰。所得的mAb肽同型二聚体对两种靶受体（胰高血糖素受体，GCGR和胰高血糖素样肽1受体，GLP-1R）由肽的空间定义呈递所提供的受体亲和力增加驱动。有趣的是，在两个靶受体上的亲和力作用是不同的。单一剂量的长效肽偶联物除了改善葡萄糖耐量外，还可以有效抑制胰岛素抵抗饮食诱导的肥胖小鼠的食物摄入并降低体重。在超重食蟹猴中也发现抑制食物摄入和减轻体重。用二价缀合的双重激动剂给药引起的重量损失明显大于单体类似物的重量损失，清楚地证明了所测得的体外亲和力向体内药理学的转化。两个靶受体的亲和力作用不同。单一剂量的长效肽偶联物除了改善葡萄糖耐量外，还可以有效抑制胰岛素抵抗饮食诱导的肥胖小鼠的食物摄入并降低体重。在超重食蟹猴中也发现抑制食物摄入和减轻体重。用二价缀合的双重激动剂给药引起的重量损失明显大于单体类似物的重量损失，清楚地证明了所测得的体外亲和力向体内药理学的转化。两个靶受体的亲和力作用不同。单一剂量的长效肽结合物除了可改善葡萄糖耐量外，还可以有效抑制胰岛素抵抗饮食诱导的肥胖小鼠的食物摄入并降低体重。在超重食蟹猴中也发现抑制食物摄入和减轻体重。用二价缀合的双重激动剂给药引起的重量损失明显大于单体类似物的重量损失，清楚地证明了所测得的体外亲和力向体内药理学的转化。在超重食蟹猴中也发现抑制食物摄入和减轻体重。用二价缀合的双重激动剂给药引起的重量损失明显大于单体类似物的重量损失，清楚地证明了所测得的体外亲和力向体内药理学的转化。在超重食蟹猴中也发现抑制食物摄入和减轻体重。用二价缀合的双重激动剂给药引起的重量损失明显大于单体类似物的重量损失，清楚地证明了所测得的体外亲和力向体内药理学的转化。