Generation of antibody-drug conjugates by proximity-based sortase-mediated 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 在化疗的同时给予未标记抗体。最近的证据表明， 不同标记的抗体，即标记在不同位置和具有不同数量的抗体，可以具有不同的 治疗和药代动力学特性和某些亚群可以显示很少的治疗活性(如果有的话) 然而，大部分的毒性都是由它造成的。因此，出现了一场向遗址发展的运动- 特定的ADC，在预定义的位置精确地贴上药物标签。 我们最近开发了两种新的方法来制备高度均匀的ADC，一种是特定于部位的 生物偶联方法，基于邻近的索尔特酶介导的蛋白质连接(PBS-PL)，其中索尔特酶是 用于将药物连接到已被引入抗体骨架的多肽标签，并且一个区域- 特定的生物偶联方法，基于邻位的山梨酸酶异肽连接(PBS-IL)，它允许 与目前的赖氨酸/半胱氨酸残基标记相比，变异性降低的天然抗体的标记 接近了。这两种方法都高产率地产生ADC，与糖基化的免疫球蛋白兼容，并提供 抗体-药物连接物化学的无限灵活性。因此，我们相信这些技术将提供 制药业感兴趣的生产ADC的新的、有利的方法。 在这项提案中，我们与积分分子和宾夕法尼亚大学合作开发了 抗Claudin-18的ADC(CLDN 18.2)。CLD18.2在几种癌症中异位表达，包括 胰腺癌，这是这项提案的重点。胰腺癌患者的预后很差， 由于缺乏有效的治疗方式，5年存活率为8%。因此，发展新的 治疗是临床上的必需品。CLDN18.2在肿瘤中的高选择性表达，未检测到 在任何可被抗体访问的健康成人组织上表达，使其成为具有吸引力的选择 靶向治疗。我们将使用PBS-PL和PBS-IL制备各种ADC，并将鉴定其偶联 基于血清的有望最有利于临床翻译的方法和ADC配方 稳定性、药代动力学和疗效。将对一例原位胰腺肿瘤进行临床前测试 同基因小鼠模型。该提案的具体目标是：目标1：生产和表征反 使用PBS-PL和PBS-IL的CLDN18.2-vcMMAE ADC；目的2：评价抗-IL的结合和疗效 目的3.测定抗CLDN18.2抗体的药代动力学和药效 ADC在小鼠肿瘤模型中的作用

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. Recent evidence has shown that differentially labeled antibodies, i.e. labeled at different locations and with different numbers, can have distinct therapeutic and pharmacokinetic properties and some subpopulations can show little, if any, therapeutic activity yet account for most of the toxicity. Therefore, there has been a movement towards the development of site- specific ADCs, which are precisely labeled with drugs at pre-defined locations. We have recently developed two new approaches for the preparation of highly uniform ADCs, one site-specific bioconjugation approach, Proximity-Based Sortase-mediated protein Ligation (PBS-PL), whereby sortase is used to ligate drugs to a peptide tag that has been introduced into the antibody backbone, and one region- specific bioconjugation approach, Proximity-based Sortase Isopeptide Ligation (PBS-IL), which allows for the labeling of native antibodies with reduced variability compared with current lysine/cysteine residue labeling approaches. Both methods produce ADCs in high yields, are compatible with glycosylated IgG, and offer unlimited flexibility in antibody-drug linker chemistry. Therefore, we believe that these technologies will provide new, favorable approaches for the production of ADCs that will be of interest to the pharmaceutical industry. In this proposal, we have partnered with Integral Molecular and the University of Pennsylvania to develop ADCs against claudin-18 (CLDN 18.2). CLD18.2 is ectopically expressed in several cancers including pancreatic cancer, which is the focus of this proposal. Outcomes for pancreatic cancer patients are poor with a 5-year survival of <8% due to a lack of effective treatment modalities. Therefore, the development of new therapies are a clinical necessity. The highly selective expression of CLDN18.2 in cancer, with no detectable expression on any healthy adult tissues that are accessible to antibodies, make it an attractive option for targeted therapy. We will prepare various ADCs using PBS-PL and PBS-IL and will identify the conjugation approach and ADC formulation that is expected to be most favorable for clinical translation, based on serum stability, pharmacokinetics, and efficacy. Pre-clinical testing will be performed in an orthotopic pancreatic tumor model in syngeneic mice. The specific aims for the proposal are: Aim 1: Produce and characterize anti- CLDN18.2-vcMMAE ADCs using PBS-PL and PBS-IL; Aim 2: Evaluate the binding and efficacy of anti- CLDN18.2-vcMMAE ADCs in vitro; Aim 3. Determine the pharmacokinetics and efficacy of anti-CLDN18.2 ADCs in a murine tumor model