Endothelial Cell-Targeted Amatoxin Conjugates for Effective Therapy of Breast Cancer

摘要 拟议项目的总体目标是利用新发现的、活性的跨内皮细胞 转运途径，小窝泵送系统，为输送和毒性提供有效的解决方案 转移性乳腺癌治疗中的化疗问题。我们建议开发新的抗体- 利用我们新发现的内皮细胞(EC)小凹靶向系统的药物结合物(ADC) 目的提高现代化疗药物的给药效率和靶向性。我们已经确定欧共体 临床前模型中的小凹可以快速和特异地泵送Annexin A1(AnnA1)抗体并附着在 货物穿过血管内皮屏障直接进入实体肿瘤。基于这一发现，我们建议 设计新颖的EC小凹靶向ADC，可能会彻底改变转移性乳腺癌的治疗方法。我们 通过用鹅膏毒素武装hAnnA1创建了一个强大的递送平台，这是一种超强的毒素，可以 可以有效地杀死快速分裂和休眠的肿瘤细胞。其中包括α-Amanitin，一种源自 从“死亡帽”蘑菇和毛壳菌素中提取的一种抗增殖广谱毒素 丝状真菌Chaetomiun sp.我们的主要假设是小窝靶向抗体-药物结合物 (CTA)，将极大地增加超强毒素对肿瘤的特异性输送，从而需要 更低的剂量和显著提高癌症治疗的疗效。这一假设将由 具体目标如下：在目标1中，我们将设计并表征新型的hAnnA1-毒素CTAs。我们将优化 它们的物理化学性质、稳定性、在肿瘤间质中的释放动力学和结合亲和力 人类膜联蛋白A1蛋白。在目标2中，我们将评估治疗效果、药物输送和偏离目标 应用活体显微镜(IVM)观察候选CTA对人乳腺癌肿瘤模型的影响。在《目标3》中， 我们将在转移性肿瘤模型和患者来源的肿瘤模型中检查选择的CTA的治疗效果 异种移植(PDX)模型。我们还将为临床测试做好准备，成功的目标是 治疗公司将被选为未来第一阶段安全试验的候选者。肿瘤靶向、递送和 这些新疗法在啮齿类动物身上的累积将使用X射线/CT成像、IVM和组织学进行评估。 长期目标是将我们的关键基本发现转化为一个独特的创新交付平台，该平台可能 极大地改善了实体癌症的治疗。

SUMMARY The overall objective of the proposed project is to harness a newly discovered, active transendothelial transport pathway, the caveolae pumping system, to provide an effective solution to the delivery and toxicity problem of chemotherapeutics in metastatic breast cancer treatment. We propose to develop novel antibody- drug conjugates (ADCs) that exploit our newly discovered endothelial cell (EC) caveolae targeting system in order to improve the delivery and targeting of modern chemotherapeutics. We have established that EC caveolae in preclinical models can rapidly and specifically pump Annexin A1 (AnnA1) antibodies and attached cargo across the vascular endothelial barrier directly into solid tumors. Based on this discovery, we propose to design novel EC caveolae-targeted ADCs that may revolutionize treatments for metastatic breast cancer. We have created a robust delivery platform by arming hAnnA1 with amatoxins, a class of ultra-potent toxins that can effectively kill both rapidly dividing and dormant tumor cells. These include α-Amanitin, a toxin derived from the “death cap” mushroom and Chaetocin, an anti-proliferative, broad-spectrum toxin derived from the filamentous fungus Chaetomiun sp. Our main hypothesis is that caveolae-targeted antibody-drug conjugates (CTAs), will dramatically increase delivery of ultra-potent toxins specifically into tumors, thereby requiring much lower dosages and dramatically enhancing efficacy of cancer treatment. This hypothesis will be tested by the following specific aims: In Aim 1, we will design and characterize novel hAnnA1-toxin CTAs. We will optimize their physicochemical properties, stability, release kinetics in the tumor interstitium, and binding affinity towards the human Annexin A1 protein. In Aim 2, we will evaluate therapeutic efficacy, drug delivery and off-target effects of candidate CTAs in human tumor models of breast cancer using intravital microscopy (IVM). In Aim 3, we will examine the therapeutic efficacy of the select CTAs in metastatic tumor models and in patient-derived xenograft (PDX) models. We will also prepare a foundation for clinical testing, where successful targeted therapeutics will be selected as candidates for future Phase 1 safety trial. Tumor targeting, delivery and accumulation of these novel therapies will be assessed in rodents using x-ray/CT imaging, IVM, and histology. The long-term goal is to translate our key basic discoveries into a unique, innovative delivery platform that may vastly improve treatments for solid cancers.