A Potent D-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo
A Potent D-Protein Antagonist of VEGF-A is Nonimmunogenic, Metabolically Stable, and Longer-Circulating in Vivo
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DOI：
10.1021/acschembio.5b01006
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发表时间：
2016-04-01
影响因子：
4
通讯作者：
Sidhu, Sachdev S.
中科院分区：
文献类型：
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作者：
Uppalapati, Maruti;Lee, Dong Jun;Sidhu, Sachdev S.
完全由D-氨基酸和非手性氨基酸甘氨酸（D-蛋白质）组成的多肽固有地具有被认为对于大分子治疗剂接近最佳的体内性质。具体而言，D-蛋白对蛋白酶降解具有抗性，并且预期是非免疫原性的。此外，D-蛋白是化学制造的，并且可以被工程化以具有其他期望的特性，例如改善的稳定性、亲和力和药代动力学。因此，设计良好的D蛋白治疗药物可能比L蛋白药物具有显着优势。为了开发D-蛋白治疗剂的目标，我们先前产生了RFX001.D，一种天然血管内皮生长因子A（VEGF-A）的D-蛋白拮抗剂，可抑制与其受体的结合。然而，RFX001.D在生理温度（T-m = 33 ℃）下不稳定。在这里，我们描述了RFX037.D，RFX001.D的变体，具有极高的热稳定性（T-m > 95摄氏度），对VEGF-A的高亲和力（Kd = 6 nM）和改善的受体阻断。RFX 037的两种对映体形式的比较显示，D-蛋白在小鼠、猴和人血浆中更稳定，并且在小鼠体内具有更长的半衰期。值得注意的是，RFX037.D在小鼠中是非免疫原性的，而L-对映体产生强烈的免疫应答。这些结果证实了合成D-蛋白作为治疗性抗体的替代品的潜在效用。
Polypeptides composed entirely of D-amino acids and the achiral amino acid glycine (D-proteins) inherently have in vivo properties that are proposed to be near-optimal for a large molecule therapeutic agent. Specifically, D-proteins are resistant to degradation by proteases and are anticipated to be non immunogenic. Furthermore, D-proteins are manufactured chemically and can be engineered to have other desirable properties, such as improved stability, affinity, and pharmacokinetics. Thus, a well-designed D-protein therapeutic would likely have significant advantages over L-protein drugs. Toward the goal of developing D-protein therapeutics, we previously generated RFX001.D, a D-protein antagonist of natural vascular endothelial growth factor A (VEGF-A) that inhibited binding to its receptor. However, RFX001.D is unstable at physiological temperatures (T-m = 33 degrees C). Here, we describe RFX037.D, a variant of RFX001.D with extreme thermal stability (T-m > 95 degrees C), high affinity for VEGF-A (K-d = 6 nM), and improved receptor blocking. Comparison of the two enantiomeric forms of RFX037 revealed that the D-protein is more stable in mouse, monkey, and human plasma and has a longer half-life in vivo in mice. Significantly, RFX037.D was nonimmunogenic in mice, whereas the L-enantiomer generated a strong immune response. These results confirm the potential utility of synthetic D-proteins as alternatives to therapeutic antibodies.