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

人们对使用抗体药物结合物(Adc)治疗癌症的兴趣日益增长。 越来越多的数据表明，与对照组相比，抗肿瘤效果有所提高，毒性有所降低 联合化疗给药未标记抗体1-4目前的两个ADC 经FDA批准，Brentuximab vedotin(Adcetris)和ado-trastuzumab emtansine(T-DM1；Kadcyla)是 这两种混合物在药物的位置和每个免疫球蛋白的药物数量方面都是不同的。近期 有证据表明，不同标记的抗体，即在不同的位置标记和不同的 数字，可具有不同的治疗和药代动力学特性5，一些亚群可以显示 很少的治疗活动，如果有的话，仍然解释了大部分的毒性。6-8因此，有一种运动 用于开发特定部位的ADC，这些ADC在预定义的位置精确地标记药物。 目前，有四种生成特定于站点的ADC的一般方法。其中包括 将半胱氨酸标签引入到免疫球蛋白的遗传密码中，6，9使用非天然氨基酸，10添加 用酶识别和修饰的多肽标签(例如甲甘氨酸生成酶或 转谷氨酰胺酶)、8、11和葡聚糖修饰(例如通过糖转移酶)12，而每种方法都有自己的方法 独特的优点和缺点包括产品产量低、药物偶联效率低、不稳定和/或 疏水抗体-药物连接物化学，或与糖基化抗体不相容。我们建议 进一步发展了一种新的位点特异性生物连接方法，即基于邻近的索尔糖酶连接(PBSL)，该方法 可以高产率地生产ADC，允许将不同的药物特定地添加到重质和/或轻质 链，与糖基化的免疫球蛋白兼容，并在抗体-药物连接物化学方面提供了无限的灵活性。 因此，我们相信这项技术将为现场生产提供一种新的、有利的途径-- 制药行业将感兴趣的特定ADC。这项建议的具体目标是： 目的1：用PBSL制备位点特异性抗前列腺特异性膜抗原(PSMA)-MMAE-ADC和 鉴定它们的特性；目标2：评价位点特异性的抗PSMA-MMAE ADC的结合和有效性 体外培养

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