Sculpting a Uniquely Reactive Cysteine Residue for Site-Specific Antibody Conjugation.
Sculpting a Uniquely Reactive Cysteine Residue for Site-Specific Antibody Conjugation.
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DOI：
10.1021/acs.bioconjchem.2c00146
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发表时间：
2022-06-15
影响因子：
4.7
通讯作者：
Rader, Christoph
中科院分区：
文献类型：
--
作者：
Hwang, Dobeen;Nilchan, Napon;Park, HaJeung;Roy, Raktim N.;Roush, William R.;Rader, Christoph
催化抗体38C2及其人源化版本h38C2在11-Å深囊底部有一个独特的活性赖氨酸，允许位点特异性偶联β-二酮-，β-内酰胺-和杂芳基甲基磺酰基功能化的小分子和大分子。多种双变量结构域（DVD）格式将肿瘤靶向抗体与h38C2配对，以实现抗体-药物偶联物（adc）的精确、快速和稳定组装。在这里，我们通过将h38C2的活性赖氨酸突变为半胱氨酸来扩展这种ADC组装策略的范围。该点突变体h38C2_K99C的x射线晶体学证实了深埋的未配对半胱氨酸。用马来酰亚胺、单溴代亚胺和二溴代亚胺衍生物探测h38C2_K99C显示出高度不同的共轭效率和稳定性。二溴代亚胺作为一种合适的亲电试剂，可以用h38C2_K99C模块精确、快速、高效、稳定地组装adc。质谱分析表明存在硫代单溴代亚胺连接，这进一步支持了硅对接研究。利用高效微管蛋白聚合抑制剂MMAF （monomethyl auristatin F）的二溴代亚胺衍生物，发现h38c2_k99c基adc与h38c2基adc具有同样的强效，为单载荷和双载荷adc的组装提供了新的途径。
Catalytic antibody 38C2 and its humanized version h38C2 harbor a uniquely reactive lysine at the bottom of a 11-Å deep pocket that permits site-specific conjugation of β-diketone-, β-lactam-, and heteroaryl methylsulfonyl-functionalized small and large molecules. Various dual variable domain (DVD) formats pair a tumor-targeting antibody with h38C2 to enable precise, fast, and stable assembly of antibody-drug conjugates (ADCs). Here we expand the scope of this ADC assembly strategy by mutating h38C2’s reactive lysine to a cysteine. X-ray crystallography of this point mutant, h38C2_K99C, confirmed a deeply buried unpaired cysteine. Probing h38C2_K99C with maleimide, monobromomaleimide, and dibromomaleimide derivatives of a fluorophore revealed highly disparate conjugation efficiencies and stabilities. Dibromomaleimide emerged as a suitable electrophile for precise, fast, efficient, and stable assembly of ADCs with the h38C2_K99C module. Mass spectrometry indicated the presence of a thio-monobromomaleimide linkage which was further supported by in silico docking studies. Using a dibromomaleimide derivative of the highly potent tubulin polymerization inhibitor monomethyl auristatin F (MMAF), h38C2_K99C-based ADCs were found to be as potent as h38C2-based ADCs and afford a new assembly route for ADCs with single and dual payloads.
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DOI：
10.3390/molecules26195847
发表时间：
2021-09-27
期刊：
Molecules (Basel, Switzerland)
影响因子：
--
作者：
Tong JTW;Harris PWR;Brimble MA;Kavianinia I
通讯作者：
Kavianinia I
影响因子：
5.5
作者：
Hwang, Dobeen;Rader, Christoph
通讯作者：
Rader, Christoph
影响因子：
15
作者：
Zhu, Xueyong;Tanaka, Fujie;Wilson, Ian A.
通讯作者：
Wilson, Ian A.
影响因子：
46.9
作者：
Lyon, Robert P.;Setter, Jocelyn R.;Senter, Peter D.
通讯作者：
Senter, Peter D.
影响因子：
4.7
作者：
Hwang D;Tsuji K;Park H;Burke TR Jr;Rader C
通讯作者：
Rader C