PDF(Pubmed)
Abstract：
Monoclonal antibodies (mAbs) as therapeutics necessitate favorable pharmacokinetic properties, including extended serum half-life, achieved through pH-dependent binding to the neonatal Fc receptor (FcRn). While prior research has mainly investigated IgG-FcRn binding kinetics with a focus on single affinity values, it has been shown that each IgG molecule can engage two FcRn molecules throughout an endosomal pH gradient. As such, we present here a more comprehensive analysis of these interactions with an emphasis on both affinity and avidity by taking advantage of switchSENSE technology, a surface-based biosensor where recombinant FcRn was immobilized via short DNA nanolevers, mimicking the membranous orientation of the receptor. The results revealed insight into the avidity-to-affinity relationship, where assessing binding through a pH gradient ranging from pH 5.8 to 7.4 showed that the half-life extended IgG1-YTE has an affinity inflection point at pH 7.2, reflecting its engineering for improved FcRn binding compared with the wild-type counterpart. Furthermore, IgG1-YTE displayed a pH switch for the avidity enhancement factor at pH 6.2, reflecting strong receptor binding to both sides of the YTE-containing Fc, while avidity was abolished at pH 7.4. When compared with classical surface plasmon resonance (SPR) technology and complementary methods, the use of switchSENSE demonstrated superior capabilities in differentiating affinity from avidity within a single measurement. Thus, the methodology provides reliable kinetic rate parameters for both binding modes and their direct relationship as a function of pH. Also, it deciphers the potential effect of the variable Fab arms on FcRn binding, in which SPR has limitations. Our study offers guidance for how FcRn binding properties can be studied for IgG engineering strategies.
摘要：
单克隆抗体（mAb）作为治疗剂需要良好的药代动力学特性，包括延长血清半衰期,通过与新生儿Fc受体（FcRn）的pH依赖性结合来实现。虽然先前的研究主要研究了IgG-FcRn结合动力学，重点是单一亲和力值，已经显示,每个IgG分子可以在整个内体pH梯度中接合两个FcRn分子。因此，我们在这里通过利用switchSENSE技术对这些相互作用进行了更全面的分析，重点是亲和力和亲和力。一种基于表面的生物传感器，其中重组FcRn通过短DNA纳米杠杆固定，模仿受体的膜取向。结果显示了对亲合力与亲和力关系的洞察力，其中通过pH范围为5.8至7.4的pH梯度评估结合表明，半衰期延长的IgG1-YTE在pH7.2具有亲和力拐点，反映了其与野生型对应物相比改善的FcRn结合的工程。此外，IgG1-YTE在pH6.2时显示出亲和力增强因子的pH转换，反映了受体与含YTE的Fc的两侧的强结合，而在pH7.4时亲和力被取消。当与经典的表面等离子体共振（SPR）技术和互补方法相比，switchSENSE的使用证明了在单个测量中区分亲和力和亲和力的优异能力。因此，该方法为两种结合模式及其作为pH函数的直接关系提供了可靠的动力学速率参数。此外，它破译可变Fab臂对FcRn结合的潜在影响，其中SPR有局限性。我们的研究为如何研究FcRn结合特性用于IgG工程策略提供了指导。
