Development of caveolae-targeted antibody-drug conjugates

项目2总结 项目2的总体目标是利用新发现的活跃的跨内皮细胞转运途径， 小窝抽水系统，以提供一个有效的解决输送和毒性问题 化疗药物在转移性乳腺癌治疗中的应用为了解决这些问题并显著 改善治疗效果我们建议开发新型抗体-药物结合物(ADC)，以利用我们的 新发现的内皮细胞(EC)小窝靶向系统，以规避被动递送问题 现代化疗药物的最新进展。我们已经证实，临床前模型中的EC小凹可以迅速和 特异性地将抗Annexin A1抗体和附着的货物泵过血管内皮细胞屏障 直接转化为实体瘤。基于这一发现，我们建议设计新的EC小窝靶向ADC 极大地增强了对乳腺癌肿瘤的破坏效果。我们的主要假设是 充分利用EC小窝泵送系统优势的免疫结合物将显著 增加化疗药物对肿瘤的输送，从而需要更低的剂量和 极大地提高了治疗效果。这一假设将通过以下具体目标进行检验： 目的1.设计合成铂(II)-羧甲基右旋糖苷(CMD)ADC，并对其进行优化 CMD的衍生化，共轭化学和负载铂(II)，以最大限度地结合亲和力 人Annexin A1蛋白。我们将测试和优化物理化学性质，储存稳定性，在 人血浆和肿瘤间质中铂(II)的释放动力学。在目标2中，我们将评估治疗效果 在转移性癌症模型中的ADC。我们的靶向递送系统的效果将在 Mda-231-LM2-4和13762乳腺癌细胞肺转移大鼠肿瘤模型的建立 用X光/CT进行动物成像。在目标3中，我们将检查ADC对肿瘤的治疗效果 使用活体显微镜(IVM)观察人体血管。我们将监测肿瘤大小对治疗的反应 使用BT-474肿瘤球体的人对人IVM模型，以及在患者来源的异种移植(PDX)中的IVM 模特。在这个目标中，我们还将把我们精选的临床前候选项目转化为临床测试，如果成功的话 靶向治疗将被选为cGMP生产的临床候选药物(核心B) 在项目3的第一阶段安全试验中进行测试。用于结合形成ADC的抗体将由Core提供 B.核心B还将提供ADC的质量控制分析和抗体结合亲和力的确认 在共轭之后。这些新疗法的肿瘤靶向、传递和积累将在#年进行评估。 使用Core C提供的多种成像服务的啮齿动物，包括X射线/CT、SPECT-CT、IVM、 和组织学。核心E将对项目过程中产生的数据进行生物统计分析。 我们的长期目标是将我们的关键基本发现转化为独特的创新交付平台，以便 提高人类癌症治疗的疗效。

PROJECT 2 SUMMARY The overall objective of the Project 2 is to utilize a newly discovered, active transendothelial transport pathway, the caveolae pumping system, in order to provide an effective solution to the delivery and toxicity problem of chemotherapeutics in metastatic breast cancer treatment. In order to address these problems and significantly improve therapeutic outcome we propose to develop novel antibody-drug conjugates (ADCs) that exploit our newly discovered endothelial cell (EC) caveolae targeting system in order to sidestep passive delivery problem of modern chemotherapeutics. We have established that EC caveolae in preclinical models can rapidly and specifically pump anti-Annexin A1 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 for greatly enhanced efficacy in tumor destruction in breast cancer. Our main hypothesis is that immunoconjugates that fully utilize the advantages of the EC caveolae-pumping system will dramatically increase delivery of chemotherapeutic drugs into tumors, thereby requiring much lower dosages and dramatically enhancing efficacy of treatment. This hypothesis will be tested by the following specific aims: In Aim 1, we will design and synthesize Pt(II)-carboxymethyl dextran (CMD) ADCs and optimize carboxylate derivatization of CMD, conjugation chemistry, and Pt(II) loading in order to maximize binding affinity toward human Annexin A1 protein. We will test and optimize physicochemical properties, stability on storage, in human plasma, and Pt(II) release kinetics in the tumor interstitium. In Aim 2 we will assess therapeutic efficacy of the ADCs in metastatic cancer models. The efficacy of our targeted delivery system will be examined in MDA-231-LM2-4, and rat tumor models with 13762 breast cancer cells metastasized to lung using whole-body animal imaging with X-ray/CT. In Aim 3 we will examine the therapeutic efficacy of the ADCs in tumors with human blood vessels using intravital microscopy (IVM). We will monitor tumor size in response to therapy in human-on-human IVM model using BT-474 tumor spheroids, and in patient-derived xenografts (PDX) IVM model. In this Aim we will also translate our select preclinical candidate for clinical testing, where successful targeted therapeutic will be selected as clinical candidates for cGMP production (Core B) for subsequent testing in a Phase 1 safety trial in Project 3. Antibodies for conjugation to form ADCs will be provided by Core B. Core B will also provide quality control analysis of ADCs and confirmation of antibody binding affinity following conjugation. Tumor targeting, delivery and accumulation of these novel therapies will be assessed in rodents using multi-modality imaging services provided by Core C, which includes X-ray/CT, SPECT-CT, IVM, and histology. Core E will handle the biostatistical analysis of data generated during the course of the project. The long-term goal is to translate our key basic discoveries into unique, innovative delivery platform in order to improve therapeutic efficacy of human cancer therapy.