Preparation of site-specific antibody-drug conjugates by proximity-based sortase ligation

人们对使用抗体药物偶联物 (ADC) 治疗癌症越来越感兴趣，因为 越来越多的数据表明，与 未标记抗体与化疗联合给药。1-4 目前正在开发的两种 ADC 经 FDA 批准，brentuximab vedotin (Adcetris) 和 ado-trastuzumab emtansine (T-DM1; Kadcyla) 是 就药物的位置和每个 IgG 的药物数量而言，这两种混合物都是异质混合物。最近 有证据表明，差异标记的抗体，即在不同位置和用不同的标记进行标记 数字，可以具有独特的治疗和药代动力学特性5，并且一些亚群可以显示 大部分毒性的治疗活性（如果有的话）也很少。6-8 因此，已经出现了一项运动 致力于开发位点特异性 ADC，在预先定义的位置精确标记药物。 目前，有四种通用方法用于生成位点特定的 ADC。这些包括 将半胱氨酸标签引入 IgG 的遗传密码中，6, 9 使用非天然氨基酸，10 添加 被酶识别和修饰的肽标签（例如甲酰甘氨酸生成酶或 转谷氨酰胺酶），8, 11 和聚糖修饰（例如通过糖转移酶）。12 虽然每种方法都有其自己的 独特的优点，缺点可能包括产物产量低、药物缀合效率低、不稳定和/或 疏水性抗体-药物接头化学，或与糖基化抗体不相容。我们建议 进一步开发一种新的位点特异性生物缀合方法，基于邻近的分选酶连接（PBSL）， 可以高产率生产 ADC，允许将不同的药物特定位点添加到重链和/或轻链中 链，与糖基化 IgG 兼容，并为抗体-药物接头化学提供无限的灵活性。 因此，我们相信这项技术将为现场制作提供一种新的、有利的途径—— 制药行业感兴趣的特定 ADC。该提案的具体目标是： 目标 1：使用 PBSL 生产位点特异性抗前列腺特异性膜抗原 (PSMA)-MMAE ADC 描述它们的特性；目标 2：评估位点特异性抗 PSMA-MMAE ADC 的结合和功效 体外

There has been growing interest in the use of antibody drug conjugates (ADCs) for the treatment of cancer as mounting data suggests an increase in anti-tumor effectiveness and reduced toxicity, compared with the administration of unlabeled antibodies in combination with chemotherapy.1-4 The two ADCs that are currently approved by the FDA, brentuximab vedotin (Adcetris) and ado-trastuzumab emtansine (T-DM1; Kadcyla) are both heterogeneous mixtures in terms of both the location of the drug and the number of drugs per IgG. Recent evidence has shown that differentially labeled antibodies, i.e. labeled at different locations and with different numbers, can have distinct therapeutic and pharmacokinetic properties5 and some subpopulations can show little, if any, therapeutic activity yet account for most of the toxicity.6-8 Therefore, there has been a movement towards the development of site-specific ADCs, which are precisely labeled with drugs at pre-defined locations. Currently, there are four general approaches for the generation of site-specific ADCs. These include the introduction of cysteine-tags into the genetic code of IgG,6, 9 the use of unnatural amino acids,10 the addition of peptide tags that are recognized and modified with enzymes (e.g. formylglycine generating enzyme or transglutaminase),8, 11 and glycan modifications (e.g. via glycotransferase).12 While each approach has its own unique advantages, shortcomings can include low product yields, inefficient drug conjugation, unstable and/or hydrophobic antibody-drug linker chemistry, or incompatibility with glycosylated antibodies. We propose to further develop a new site-specific bioconjugation approach, Proximity-Based Sortase Ligation (PBSL), that can produce ADCs in high yields, allows different drugs to be site-specifically added to the heavy and/or light chains, is compatible with glycosylated IgG, and offers unlimited flexibility in antibody-drug linker chemistry. Therefore, we believe that this technology will provide a new, favorable approach for the production of site- specific ADCs that will be of interest to the pharmaceutical industry. The specific aims for this proposal are: Aim 1: Use PBSL to produce site-specific anti-prostate specific membrane antigen (PSMA)-MMAE ADCs and characterize their properties; Aim 2: Evaluate the binding and efficacy of site-specific anti-PSMA-MMAE ADCs in vitro