Sussman D, Westendorf L, Meyer D W, Leiske C I, Anderson M, Okeley N M, Alley S C, Lyon R, Sanderson R J, Carter P J, Benjamin D R
Seattle Genetics, Inc. 21823 30th Drive SE, Bothell, WA 98021, USA.
Department of Antibody Engineering, Genentech, Inc., 1 DNA Way, South San Francisco, CA 94080, USA.
Protein Eng Des Sel. 2018 Feb 1;31(2):47-54. doi: 10.1093/protein/gzx067.
Antibody-drug conjugates (ADCs) are fulfilling the promise of targeted therapy with meaningful clinical success. An intense research effort is directed towards improving pharmacokinetic profiles, toxicity and chemical stability of ADCs. The majority of ADCs use amide and thioether chemistry to link potent cytotoxic agents to antibodies via endogenous lysine and cysteine residues. While maleimide-cysteine conjugation is used for many clinical stage ADC programs, maleimides have been shown to exhibit some degree of post-conjugation instability. Previous research with site-directed mutagenic incorporation of cysteine residues for conjugation revealed that the stability of the drug-antibody linkage depends on the site of conjugation. Here we report on a collection of engineered cysteine antibodies (S239C, E269C, K326C and A327C) that can be site-specifically conjugated to potent cytotoxic agents to produce homogenous 2-loaded ADCs. These ADCs confirm that site of conjugation impacts maleimide stability and present a novel mechanism of thioether stabilization, effectively unlinking stability from either local chemical environment or calculated solvent accessibility and expanding the current paradigm for ADC drug-linker stability. These ADCs show potent in vitro and in vivo activity while delivering half of the molar equivalent dose of drug per antibody when compared to an average 4-loaded ADC. In addition, our lead engineered site shields highly hydrophobic drugs, enabling conjugation, formulation and clinical use of otherwise intractable chemotypes.
抗体药物偶联物（ADCs）正凭借显著的临床成功实现靶向治疗的前景。大量研究工作致力于改善ADC的药代动力学特征、毒性和化学稳定性。大多数ADC利用酰胺和硫醚化学方法，通过内源性赖氨酸和半胱氨酸残基将强效细胞毒性药物与抗体连接。虽然马来酰亚胺-半胱氨酸偶联用于许多临床阶段的ADC项目，但已表明马来酰亚胺在偶联后会表现出一定程度的不稳定性。先前关于通过定点诱变引入半胱氨酸残基进行偶联的研究表明，药物-抗体连接的稳定性取决于偶联位点。在此，我们报告了一系列工程化半胱氨酸抗体（S239C、E269C、K326C和A327C），它们可以位点特异性地与强效细胞毒性药物偶联，以产生均一的双负载ADC。这些ADC证实偶联位点会影响马来酰亚胺稳定性，并呈现出硫醚稳定的新机制，可以有效地将稳定性与局部化学环境或计算出的溶剂可及性脱钩，扩展了当前关于ADC药物连接子稳定性的范式。与平均四负载的ADC相比，这些ADC在体外和体内均表现出强效活性，同时每个抗体输送的药物摩尔当量剂量减半。此外，我们的主要工程位点可屏蔽高度疏水的药物，从而实现原本难以处理的化学类型的偶联、制剂和临床应用。
