Development of caveolae-targeted antibody-drug conjugates

项目2概要 项目2的总体目标是利用新发现的主动跨内皮转运途径， 为了提供一种有效的解决输送和毒性问题的方法， 转移性乳腺癌治疗中的化学治疗剂。为了解决这些问题， 改进治疗结果我们提出开发新的抗体-药物缀合物（ADC），其利用我们的 一种新发现的内皮细胞（EC）小窝靶向系统，以避免被动递送问题 现代化学疗法。我们已经确定，在临床前模型中，EC小窝可以快速， 特异性泵送抗膜联蛋白A1抗体和附着的货物穿过血管内皮屏障 直接植入实体瘤基于这一发现，我们提出设计新型EC小窝靶向ADC， 大大增强了乳腺癌中肿瘤破坏的功效。我们的主要假设是 充分利用EC小窝泵送系统的优点的免疫缀合物将显著地 增加化疗药物向肿瘤中的递送，从而需要低得多的剂量， 大大提高了治疗效果。这一假设将通过以下具体目标进行检验： 目的1、设计合成Pt（II）-羧甲基葡聚糖（CMD）ADC，并优化其羧酸酯结构 CMD的衍生化、缀合化学和Pt（II）负载，以最大化对 人膜联蛋白A1蛋白。我们将测试和优化理化性质，储存稳定性， 人血浆和肿瘤组织中Pt（II）的释放动力学。在目标2中，我们将评估治疗效果 ADC在转移性癌症模型中的作用。我们的靶向给药系统的疗效将在 MDA-231-LM 2 -4和使用全身给药的具有13762个乳腺癌细胞转移到肺的大鼠肿瘤模型 X线/CT动物成像。在目标3中，我们将检查ADC在具有以下特征的肿瘤中的治疗功效： 使用活体显微镜（IVM）的人血管。我们将监测肿瘤的大小，以应对治疗， 使用BT-474肿瘤球状体的人对人IVM模型，以及在患者来源的异种移植物（PDX）IVM中 模型在这个目标中，我们还将翻译我们选择的临床前候选药物进行临床试验，如果成功的话， 将选择靶向治疗剂作为cGMP生产的临床候选物（核心B），用于随后的 在项目3的1期安全性试验中进行测试。用于缀合以形成ADC的抗体将由核心抗体库提供。 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、递送和累积将在 啮齿动物使用Core C提供的多模态成像服务，包括X射线/CT、SPECT-CT、IVM， 和组织学。核心E将处理项目过程中生成的数据的生物统计分析。 长期目标是将我们的关键基础发现转化为独特的创新交付平台， 提高人类癌症治疗疗效。

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.