Thiolation of Q295: Site-Specific Conjugation of Hydrophobic Payloads without the Need for Genetic Engineering
Thiolation of Q295: Site-Specific Conjugation of Hydrophobic Payloads without the Need for Genetic Engineering
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DOI：
10.1021/acs.molpharmaceut.9b00323
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发表时间：
2019-06-01
影响因子：
4.9
通讯作者：
Tumey, L. Nathan
中科院分区：
文献类型：
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作者：
Benjamin, Samantha R.;Jackson, Courtney P.;Tumey, L. Nathan
位点特异性偶联技术通常依靠抗体工程将稀有或非天然氨基酸结合到蛋白质的一级序列中。然而，当初级序列未知或抗体工程不可行时，位点特异性蛋白质修饰的选择非常有限。我们开发了一种转谷氨酰胺酶介导的偶联物，在去糖基化抗体的“特权”位置结合硫醇（Q295）。也许令人惊讶的是，这种结合使用了一种报道的转谷氨酰胺酶抑制剂，胱胺，作为关键的酶底物。在Q295位点的硫醇化学结合允许通过马来酰亚胺化学对大量常用和商业上可用的有效载荷进行位点特异性附着。在这里，我们通过比较这些位点特异性抗体-药物偶联物（adc）与类似的内源性半胱氨酸偶联物的偶联性、血浆稳定性和体外效力来证明这种方法的实用性。使用这种方法制备的细胞毒性adc显示出与随机半胱氨酸偶联物相当的体外功效，同时显示出显着改善的血浆稳定性和偶联性。特别是，我们注意到，这种技术似乎是有用的高度疏水连接剂有效载荷的合并，而不添加PEG修饰剂。我们通过使用两个已知对局部疏水环境敏感的荧光探针探测Q295位点的局部环境，假设了这一特征的可能机制。总之，我们描述了一种高度实用的基因非工程抗体位点特异性偶联的方法，这种方法可以产生具有低内在疏水性的血浆稳定adc。我们相信这项技术将在ADC社区中得到广泛的应用。
Site-specific conjugation technology frequently relies on antibody engineering to incorporate rare or non natural amino acids into the primary sequence of the protein. However, when the primary sequence is unknown or when antibody engineering is not feasible, there are very limited options for site-specific protein modification. We have developed a transglutaminase-mediated conjugation that incorporates a thiol at a "privileged" location on deglycosylated antibodies (Q295). Perhaps surprisingly, this conjugation employs a reported transglutaminase inhibitor, cystamine, as the key enzyme substrate. The chemical incorporation of a thiol at the Q295 site allows for the site-specific attachment of a plethora of commonly used and commercially available payloads via maleimide chemistry. Herein, we demonstrate the utility of this method by comparing the conjugatability, plasma stability, and in vitro potency of these site-specific antibody-drug conjugates (ADCs) with analogous endogenous cysteine conjugates. Cytotoxic ADCs prepared using this methodology are shown to exhibit comparable in vitro efficacy to stochastic cysteine conjugates while displaying dramatically improved plasma stability and conjugatability. In particular, we note that this technique appears to be useful for the incorporation of highly hydrophobic linker payloads without the addition of PEG modifiers. We postulate a possible mechanism for this feature by probing the local environment of the Q295 site with two fluorescent probes that are known to be sensitive to the local hydrophobic environment. In summary, we describe a highly practical method for the site-specific conjugation of genetically nonengineered antibodies, which results in plasma-stable ADCs with low intrinsic hydrophobicity. We believe that this technology will find broad utility in the ADC community.