PTD-Mediated Protein or Drug Delivery for Cancer Therapy

描述（由申请人提供）：到目前为止，药物治疗在治疗癌症方面取得的成功很少，主要是由于缺乏临床疗效和药物诱导毒性的存在。在之前的项目中，我们制定了克服这些障碍的原则，以实现最大疗效和最小药物诱导毒性的理想药物治疗。该药物传递系统（DDS）由两部分组成：[1]是靶向部分，由肝素与单抗连接组成；[2]是药物部分，由药物与细胞穿透肽（CPP）偶联组成。这两种成分可以通过肝素和CPP之间的电荷相互作用自组装成紧密的复合物。CPP与肝素结合会抑制其在细胞表面的吸附，从而掩盖其穿透细胞的功能。药物复合物提供药物前行为，阻止正常组织对cpp介导的摄取，减轻药物引起的副作用。在通过mAb靶向肿瘤后，将给予鱼精蛋白以解除肝素抑制并恢复释放的pcp - drug的活性，仅诱导肿瘤细胞凋亡。在过去的资助期内取得了显著进展，发表了36篇论文，有12名博士/博士后学员毕业。所有目标均已实现，并证实了该DDS的实用性/适用性。尽管前景看好，但两个关键因素-抗体靶向的有效性和药物前保护的程度-被发现需要改进，以实现临床启用的药物治疗。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，被证明在结肠癌中过度表达，但在正常组织中表达有限。其次，我们发现注射的T84.66（一种针对人CEA的小鼠单抗）通过抗原介导的消除途径在结直肠肿瘤靶点积累了b> 85%。第三，最近有报道称，蛋白酶豆科蛋白在结直肠肿瘤表面深度表达，而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来跨越上述障碍。简而言之，一种有效的毒素（gelonin）或siRNA药物将通过S-S键与LMWP（一种已证实无毒的CPP）共价结合。该LMWP-Drug将通过含有豆类可降解特异性AANL序列的肽连接物进一步与T84.66连接。由于LMWP嵌入在抗体和药物分子之间，因此药物复合物在肿瘤靶向过程中表现出前药行为，因此无法穿过肿瘤细胞发挥细胞毒性作用。通过T84.66靶向积累肿瘤后，表面表达的豆科蛋白通过切割AANL连接体在肿瘤表面释放lmwp -药物。然后，低分子蛋白药物通过低分子蛋白介导的细胞进入作用内化到肿瘤细胞中，启动肿瘤凋亡。具体目的是通过使用成熟的LS174T异种移植小鼠模型进行广泛的体外和体内研究，验证该系统在治疗特异性结直肠癌方面的临床前能力；从而为今后的临床翻译铺平道路。

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.