PTD-Mediated Protein or Drug Delivery for Cancer Therapy

描述（由申请人提供）：迄今为止，药物治疗在控制癌症方面几乎没有取得成功，主要是由于缺乏临床疗效和存在药物诱导的毒性。在上一个项目中，我们制定了克服这些障碍的原则，以实现最大治疗效果和最小药物诱导毒性的理想药物治疗。该药物递送系统（DDS）由两个组件组成：[1]由连接至mAb的肝素组成的靶向部分，以及[2]由与细胞穿透肽（CPP）偶联的药物组成的药物部分。这两种组分可以通过肝素和CPP之间的电荷相互作用自组装成紧密的复合物。将CPP与肝素结合将由于抑制其吸附到细胞表面而掩盖其细胞穿透功能。药物复合物提供前药行为并防止CPP介导的正常组织摄取，减轻药物诱导的副作用。在通过mAb靶向肿瘤后，将施用鱼精蛋白以揭开肝素抑制并恢复释放的CPP药物的活性，仅诱导肿瘤细胞凋亡。在过去的赠款期间取得了非凡的进展，发表了36篇论文，12名博士/博士后学员毕业。所有目标均已实现，并确认了该DDS的实用性/适用性。尽管前景看好，但发现两个关键因素-抗体靶向的功效和前药保护的程度-需要改进以实现临床上可行的药物治疗。结直肠癌研究的最新进展似乎为这两个问题提供了解决方案。首先，癌胚抗原（CEA），一种已知与肿瘤生长/转移相关的表面抗原，在结肠癌中过度表达，但在正常组织中表达有限。其次，我们发现>85%的注射的T84.66（一种针对人CEA的小鼠mAb）经由抗原介导的消除途径在结肠直肠肿瘤靶标处积累。第三，最近报道蛋白酶legumain在结直肠肿瘤表面有很深的表达，但在正常组织中几乎检测不到。利用这些信息，我们进一步设计了一个高度改进的DDS，以跨越上述障碍。简而言之，有效的毒素（白树毒素）或siRNA药物将通过S-S键与LMWP（一种经证实但无毒的CPP）共价连接。该LMWP-药物将通过含有豆类蛋白可降解的特异性AANL序列的肽接头进一步连接至T84.66。然后，药物复合物将在肿瘤靶向期间表现出前药行为，因为LMWP嵌入在抗体和药物分子之间，因此不能使药物穿过肿瘤细胞以发挥细胞毒性作用。在通过T84.66靶向的肿瘤积累后，LMWP-药物从T84.66的释放将通过表面表达的豆荚蛋白切割AANL接头在肿瘤表面上发生。LMWP-药物然后将通过LMWP介导的细胞进入作用内化到肿瘤细胞中，引发肿瘤细胞凋亡。具体目的是通过使用成熟的LS 174 T异种移植小鼠模型进行广泛的体外和体内研究，验证该系统在专门管理结直肠癌方面的临床前能力;从而为未来的临床转化铺平道路。

DESCRIPTION (provided by applicant): Drug therapy has thus far attained little success in managing cancers, primarily due to a lack clinical efficacy and presence of drug-induced toxicity. In the previous project, we instituted principles to overcome such obstacles in achieving an ideal drug therapy of maximum therapeutic efficacy with minimal drug-induced toxicity. This drug delivery system (DDS) is comprised of two components: [1] a targeting part composed of heparin linked to a mAb, and [2] a drug part, comprised of a drug coupled with a cell penetrating peptide (CPP). These two components can self-assemble into a tight complex via charge interaction between heparin and CPP. Binding CPP with heparin would mask its cell-penetrating function due to inhibition of its adsorption to the cell surfaces. The drug complex provides a prodrug behavior and prevents CPP-mediated uptake by normal tissues, alleviating drug-induced side effects. Following tumor targeting via the mAb, protamine will be administered to unmask heparin inhibition and restore the activity of the released CPP-Drug, inducing apoptosis to only tumor cells. Exceptional progress was made during the past grant period, with publication of 36 papers and graduation of 12 PhD/Postdoctoral trainees. All aims were accomplished, and utility/applicability of this DDS was confirmed. Despite promise, two crucial factors - efficacy of antibody targeting and extent of prodrug protection - were found in need of improvement in order to realize a clinically enabled drug therapy. Recent advancement in colorectal cancer research seems to offer a solution to these two issues. First, carcinoembryonic antigen (CEA), a surface antigen known to associate with tumor growth/metastasis, was shown to be overly expressed in colonic cancers but limited in normal tissues. Secondly, we discovered that >85% of the injected T84.66, a mouse mAb against human CEA, accumulated at the colorectal tumor target via an antigen-mediated elimination pathway. Thirdly, the protease legumain was recently reported to express profoundly on the surface of colorectal tumors but is almost undetectable in normal tissues. Using this information, we further engineered a highly improved DDS to cross the afore-cited barriers. Briefly, a potent toxin (gelonin) or siRNA drug will be covalently linked with LMWP, a proven but non-toxic CPP, via S-S bond. This LMWP-Drug will be further linked to T84.66 through a peptide linker containing the legumain-degradable specific AANL sequence. The drug complex would then exhibit a prodrug behavior during tumor targeting, since LMWP is embedded between the antibody and drug molecule, thus unable to transverse the drug through tumor cells in exerting cytotoxic effects. Following tumor accumulation via T84.66 targeting, release of LMWP-Drug from T84.66 will take place on tumor surface via cleavage of the AANL linker by surface-expressed legumain. LMWP-Drug will then internalize into tumor cells via LMWP-mediated cell entry action, initiating tumor apoptosis. The specific aims are to validate the pre-clinical capability of this system in managing specifically colorectal cancers, through extensive in vitro and in vivo studies using a well-established LS174T xenograft mouse model; thus paving the road for future clinical translation.