Large Scale Synthesis of the Next Generation Synthetic Saponin Adjuvant TiterQuil

说明(申请人提供)：抗肿瘤和抗传染病疫苗需要佐剂才能获得最佳的免疫原性以及治疗和保护效果。QS-21是一种天然皂素组分，在诱导产生抗体和T细胞反应方面明显优于其他佐剂。它仍然是首选佐剂，并在许多癌症、传染病和退化性疾病疫苗试验中证明了其作为免疫佐剂的效力。阻碍QS-21进一步发展的问题包括不利的生物效应(限制了耐受剂量)、化学不稳定、Quillaja Saponaria树(QS-21是从Quillaja Saponaria树中提取的)的稀缺性以及这些树的收获产量的变异性。最近，我们对QS-21合成的半合成方法已经克服了所有这些缺陷。这种合成还使分子构建块易于用于合成修饰和截断的QS-21类似物。天然皂苷中不稳定的键被化学上强健的官能团取代，增强了分子的稳定性。结构变体结合了外围糖部分和酰链亚结构的系统简化，有助于确定安全和有效佐剂活性的最低结构要求，并将所需的合成步骤从全合成QS-21(SQS-21)减少到我们的先导合成类似物的50%以上。这些努力(现已完成)导致选择最佳的QS-21类似物TiterQuil-1-0-5-5进行更正式的临床前开发，因为其结构简化，稳定性提高，效力一致，毒性减弱。核心知识产权是我们的三萜皂素合成技术(TriSST)平台，涵盖了申请编号为12/420,803的专利申请“三萜皂苷、合成方法及其用途”下的物质的合成方法和新成分，以及我们受美国专利8,283,456保护的专有半合成技术。TriSST是由纪念斯隆-凯特琳癌症中心(MSKCC)的David Gin等人开发的，这项技术已获得Adjuvance Technologies，Inc.的独家许可。TriSST是一种高度收敛的合成方法，将QS-21中的四个结构域(支链三糖+三萜+直链四糖+脂肪酰链)分别合成并组装成目标分子。每个域都可以独立修改，然后组合在一起，以产生几乎无限数量的合理设计的QS-21类似物。在一期临床试验中，最初完全合成的QS-21(SQS-21)被证明是安全的和免疫活性的。然后，我们使用TriSST在一系列系统的序列研究中制备和测试了100多个新的类似物。我们评估QS-21及其类似物的模型包括针对一系列多肽和碳水化合物癌症抗原的KLH结合疫苗，针对这些抗原的抗体反应的测量，以及在某些情况下对MUC1和KLH的T细胞反应的测量。这几项检测的结果是平行跟踪的，没有一致的异常值。选定的类似物的效力已被证实对新的莱姆热SPC多肽和肺炎球菌Prevnar 13疫苗。在这里，我们建议通过在独立设施中扩大合成来进一步开发用于临床的TiterQuil-1-0-5-5。在这些研究的结论中，我们将证明TiterQuil-1-0-5-5合成可以扩大到接近商业所需的水平，并且与天然或合成的QS-21相比，它具有更高的纯度、稳定性、安全性、制造简易性和免疫效力(使用与传染病疫苗更相关的扩大的抗原小组)。这将为支持TiterQuil-1-0-5-5药物主文件(DMF)的第二阶段SBIR应用程序及其在针对各种公共卫生问题的疫苗中的更广泛使用提供基础。具体目标1：合成2克截短的QS-21类似物TiterQuil-1-0-5-5，足以满足目标2和3。具体目标2：确认这一放大批次的TiterQuil-1-0-5-5的纯度、稳定性和溶血能力，并根据DMF应用要求进行正式的药理学/毒理学研究。具体目标3：比较TiterQuil-1-0-5-5与天然和合成的QS-21(SQS-21)的免疫效力和安全性，该疫苗使用针对OspC多肽的疫苗以及商用的肺炎球菌结合物Prevnar-13。

DESCRIPTION (provided by applicant): Antitumor and anti-infectious disease vaccines require adjuvants in order to obtain optimal immunogenicity and therapeutic and protective efficacy. QS-21 is a natural saponin fraction that significantly out-performs other adjuvants in eliciting productive antibody and T-cell responses. It has remained the adjuvant of choice and has proven it's potency as immunological adjuvant in many cancer, infectious disease and degenerative disorder vaccine trials. Problems that have hampered the further development of QS-21 include adverse biological effects (that limit the tolerable dose), chemical instability, scarcity of the Quillaja saponaria tree (from which QS-21 is extracted) and variability of harvest yields from these trees. Recently, all of these liabilities have been overcome by our semisynthetic approach to QS-21 synthesis. This synthesis has also made molecular building blocks readily available for synthesis of modified and truncated QS-21 analogues. The replacement of unstable linkages within the native saponin with chemically robust functional groups has enhanced molecular stability. Structural variants incorporating systematic simplification of the peripheral sugar moieties and of the acyl chain substructure have aided in determining the minimal structural requirements for safety and potent adjuvant activity, and have decreased the required number of synthetic steps by over 50% from fully synthetic QS-21 (SQS-21) to our lead synthetic analogues. These efforts (now completed) have resulted in selection of the optimal QS-21 analogue TiterQuil-1-0-5-5 for more formal preclinical development because of its simplified structure, improved stability, consistent potency and attenuated toxicity. The core intellectual property is our Triterpene Saponin Synthesis Technology (TriSST) platform covering both the method of synthesis as well as novel compositions of matter under the patent application "TRITERPENE SAPONINS, METHODS OF SYNTHESIS, AND USES THEREOF" application number 12/420,803 as well as our proprietary semisynthetic technology which is protected by US patent U.S. Patent No. 8,283,456. TriSST was developed by Dr. David Gin et al at Memorial Sloan-Kettering Cancer Center (MSKCC) and this technology has been exclusively licensed by Adjuvance Technologies, Inc. TriSST is a highly convergent synthetic approach in which the four domains in QS-21 (branched trisaccharide + triterpene + linear tetrasaccharide + fatty acyl chain) are synthesized separately and then assembled to produce the target molecule. Each of the domains can be modified independently and then combined to produce a virtually infinite number of rationally designed QS-21 analogs. Initially fully synthetic QS-21(SQS-21) was shown to be safe and immunologically active in a Phase 1 clinical trial. We then used TriSST to prepare and test over 100 novel analogues in a systematic sequential series of studies. Our model for evaluating QS-21 and analogues has involved KLH-conjugate vaccines against a series of peptide and carbohydrate cancer antigens, measurement of antibody responses against these antigens, and in some cases measurement of T-cell responses against MUC1 and KLH. Results in these several assays have tracked in parallel with no consistent outliers. The potency of selected analogues has been confirmed against novel Lyme fever OspC peptides and pneumococcal Prevnar 13 vaccines. Here we propose to further develop TiterQuil-1-0-5-5 for clinical use by scaling up synthesis in an independent facility. At the conclusion of these studies we will have demonstrated that TiterQuil-1-0-5-5 synthesis can be scaled up to near commercially required levels and that it has improved purity, stability, safety, ease of manufacture, and immunologic potency (using an expanded panel of antigens more relevant for infectious disease vaccines) when compared to natural or synthetic QS-21. This will provide the basis for a Phase 2 SBIR application supporting a TiterQuil-1-0-5-5 drug master file (DMF) and its wider use in vaccines that target diverse public health concerns. Specific Aim 1: Synthesize 2 grams of truncated QS-21 analogue TiterQuil-1-0-5-5, a sufficient quantity for Aims 2 and 3. Specific Aim 2: Confirm purity, stability and erythrolytic capacity of this scaled up batch of TiterQuil-1- 0-5-5, and perfrm a formal pharmacology/toxicology study as required for a DMF application. Specific Aim 3: Compare the immunologic potency and safety of TiterQuil-1-0-5-5 with natural and synthetic QS-21(SQS-21) using vaccines targeting OspC peptide as well as the commercially available pneumococcal conjugate Prevnar-13.