Pan Lucy Yan, Salas-Solano Oscar, Valliere-Douglass John F
Department of Analytical Sciences, Seattle Genetics, 21823 30th Drive SE, Bothell, Washington 98021, United States.
Anal Chem. 2015 Jun 2;87(11):5669-76. doi: 10.1021/acs.analchem.5b00764. Epub 2015 May 15.
We present the results of a hydrogen/deuterium exchange mass spectrometric (HDX-MS) investigation of an antibody-drug conjugate (ADC) comprised of drug-linkers conjugated to cysteine residues that have been engineered into heavy chain (HC) fragment crystallizable (Fc) domain at position 239. A side-by-side comparison of the HC Ser239 wild type (wt) monoclonal antibody (mAb) and the engineered Cys239 mAb indicates that site directed mutagenesis of Ser239 to cysteine has no impact on the HDX kinetics of the mAb. According to the crystal structure of a homologous immunoglobulin G1 (IgG1) antibody (PDB: 1HZH ), the backbone amide of Ser239 is hydrogen-bonded to Val264 backbone amide in the wt-mAb studied here. Replacing Ser239 with a Cys residue does not alter the exchange kinetics of the backbone amide of Val264 suggesting that either Ser or Cys at position 239 has similar amide-hydrogen bonding with Val264. However, a small segment in CH2 domain of the ADC ((264)VDVS) was found to have a slightly increased HDX rate compared to the wt- and C239-mAb constructs. The slightly increased HDX rate of the segment (264)VDVS in ADCs indicates that the further modification of Cys239 with drug-linkers only attenuates the local backbone amide hydrogen-bonding network between Cys239 and Val264. All other regions which are proximal to the site of drug conjugation are unaffected. The results demonstrate that the site-specific drug conjugation at the engineered Cys residue at the position 239 of HC does not impact the structural integrity of antibodies. The results also highlight the utility of applying HDX-MS to ADCs to gain a molecular level insight into the impact of site-specific conjugation technologies on the higher-order structure (HOS) of mAbs. The methodology can be applied generally to site-specific ADC modalities to understand the individual contributions of site-mutagenesis and drug-linker conjugation on the HOS of therapeutic candidate ADCs.
我们展示了一项氢/氘交换质谱（HDX-MS）研究的结果，该研究针对一种抗体药物偶联物（ADC），其由与半胱氨酸残基缀合的药物连接体组成，这些半胱氨酸残基已被工程改造到重链（HC）的可结晶片段（Fc）结构域的第239位。对HC Ser239野生型（wt）单克隆抗体（mAb）和工程化的Cys239 mAb进行的并列比较表明，将Ser239定点突变为半胱氨酸对mAb的HDX动力学没有影响。根据同源免疫球蛋白G1（IgG1）抗体的晶体结构（PDB：1HZH），在此研究的wt-mAb中，Ser239的主链酰胺与Val264的主链酰胺形成氢键。用半胱氨酸残基取代Ser239不会改变Val264主链酰胺的交换动力学，这表明第239位的Ser或Cys与Val264具有相似的酰胺-氢键。然而，发现ADC的CH2结构域中的一个小片段（(264)VDVS）与wt-和C239-mAb构建体相比，HDX速率略有增加。ADC中(264)VDVS片段HDX速率的略有增加表明，用药物连接体对Cys239进行进一步修饰只会减弱Cys239和Val264之间的局部主链酰胺氢键网络。所有其他靠近药物偶联位点的区域均未受影响。结果表明，在HC的第239位工程化半胱氨酸残基处进行位点特异性药物偶联不会影响抗体的结构完整性。结果还突出了将HDX-MS应用于ADC以在分子水平上深入了解位点特异性偶联技术对mAb高阶结构（HOS）影响的实用性。该方法通常可应用于位点特异性ADC模式，以了解位点诱变和药物连接体偶联对候选治疗性ADC的HOS的各自贡献。
