Carbohydrate Antigen-bearing Nanoparticles for Anti-adhesives and Tumor Vaccines

肿瘤发生的一个公认的标志是由于肿瘤组织中糖处理酶表达的变化而导致异常糖链的生物合成。随着肿瘤获得更具侵袭性的表型，这些像差变得更加明显。肿瘤细胞表面的碳水化合物在许多不同癌细胞的运动和转移中起着重要作用。此外，这些异常多糖中的许多是肿瘤相关碳水化合物抗原(TACA)，并已被用于肿瘤疫苗的开发。由于大多数细胞与TACA的相互作用还不是很清楚，因此迫切需要更好地描述在这些事件期间发生的特定分子相互作用。碳水化合物与大分子结合的一个众所周知的特征是多价性的概念：单体碳水化合物与蛋白质的结合非常弱，而单体的聚集使这种亲和力提高了一百万倍。我们已经在非常特定的模板上制备了重要的Thomsen-Friedenreich(TF)抗原(Gal(Beta)1-3GalNAc(α)-O-Ser/Thr)，以利用这种所谓的簇苷效应。正如上一份报告中提到的，我们已经制备了含有糖衍生物的金自组装纳米球和量子点，并报告了它们的功能的初步细节。我们的金纳米球在小鼠身上的体内实验相互矛盾，所以我们退回到基础上，进行了更严格的表征，并探索了一系列新的合成方法，允许生产更均匀的颗粒。我们从几种相关的方法出发，系统地研究了提供最高质量颗粒的稳定性和均匀性的最佳工艺。我们仍在各种媒体上检查这些数据，以测试其稳定性。我们在不同的环境中制备了转铁蛋白抗原(既附着于丝氨酸又附着于苏氨酸)，并将它们连接到颗粒上。我们的转铁蛋白颗粒现在已经在下拉实验中被证明与Galectin-3和与转移相关的整合素复合体结合。我们的合作者Vladislav Glinskii正在继续这项工作，他对定义转铁蛋白糖蛋白感兴趣，即识别前列腺癌细胞中具有O-连接转铁蛋白抗原的所有蛋白质。我们正在研究一种蛋白质组学方法，以帮助他在明年定义这些分子。我们着重于制备包含我们认为最好的抗原的颗粒，这是一种来自肿瘤相关细胞表面粘蛋白的糖肽，并将其与不同浓度的连接物和T辅助表位相结合，构建可能作为新型免疫原的颗粒。我们制备了至少七个独立的颗粒，它们在多肽上有不同的位置，以及连接子和C3d的28个残基部分，补体成分3的结构域和CD21的配体，CD21是一种B细胞表面蛋白，当参与时，降低B细胞激活的阈值。这些颗粒被注射到小鼠体内，并分析了血清的免疫反应。在至少两个试验组中观察到了统计上显著的免疫反应，我们用新鲜颗粒第二次增强了动物。移植肿瘤，并随访生存情况。尽管一个特定抗原组比其他组表现更好，但他们并没有比只接受PBS的组表现更好。有几个参数可能会导致反应低于预期，我们现在正在调查这些参数。与Howard Young合作开始的一项新研究，让我们探索了不同化学伪装下具有不同抗原的颗粒对细胞因子谱的调制。最初的数据非常令人鼓舞，因为含有不同表面化学成分的颗粒可以增强或减弱激活的小鼠巨噬细胞中几种细胞因子的水平。该计划是复制这些数据，将其扩展到树突状细胞，并探索将这些细胞重新注入动物体内的模型，以检查对肿瘤的反应。此外，透射电子显微镜实验正在确定这些颗粒在小鼠巨噬细胞和树突状细胞中的摄取情况。我们还探索了其他方法来合成TF和其他糖肽抗原，并将优化的连接物连接到纳米平台上。发展了新的连接物化学，并使用基于硫辛酸的末端二硫化物作为金和其他表面的偶联部分。制备了不同的结构，以比较颗粒的均匀性和表面化学的变化。最后，我们开发了一种分析方法来充分表征我们制备的颗粒的分子组成。文献中关于如何做到这一点的细节很少，也不令人满意，我们决定开发我们自己的方法。我们已经制定了获取少量纳米颗粒的程序，完全剥离它们的配体，并将每个纳米颗粒连接到一个荧光标记上，该标记可以通过高效液相色谱和荧光分光光度进行定量。这种方法已经得到了改进，正在准备一份详细描述这些实验的手稿。

An established hallmark of tumorigenesis is the biosynthesis of aberrant glycan chains due to changes in the expression of glycoprocessing enzymes in tumor tissue. These aberrations become more marked as the tumor acquires a more aggressive phenotype. Tumor cell-surface carbohydrates play important roles in the motility and metastasis of many different cancer cells. In addition, many of these aberrant glycans are tumor-associated carbohydrate antigens (TACA) and have been used in the development of tumor vaccines. Since most of the cellular interactions with TACAs are not well understood, there is an urgent need to better characterize the specific molecular interactions that occur during these events. One feature of carbohydrate binding to macromolecules that is well understood is the concept of multivalency: Monomer carbohydrates bind to proteins very weakly while clustering of a monomer raises this affinity as much as a million-fold. We have prepared the important Thomsen-Friedenreich (Tf) antigen (Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr) on very specific templates to take advantage of this so-called cluster glycoside effect. As mentioned in the last report, we have prepared gold self-assembled nanospheres and quantum dots containing sugar derivative and reported preliminary details on their function. The in vivo experiments with our gold nanospheres in mice were conflicting, so we retreated to basics and performed more rigorous characterization and explored a host of new syntheses that allowed for production of more uniform particles. We proceeded to systematically study the optimum procedure, from several related methods, that offered the highest quality particles with regards to stability and uniformity. We are still examining these data in various media to test for stability. We have prepared the TF antigen in different contexts (attached to both serine and threonine) and linked them to particles. Our TF particles have now been shown in pull down experiments to bind to Galectin-3 and integrin complexes related to metastasis. This work is continuing with our collaborator Vladislav Glinskii, who is interested in defining the TF-glycome, i.e. identify all the proteins in prostate cancer cells that have O-linked TF antigen. We are working on a proteomic method to help him define these molecules in the next year. We put a heavy emphasis on preparing particles that encompassed what we consider the best antigen, a glycopeptide from tumor associated cell-surface mucins, and combined that with various concentrations of linker and T-helper epitope to construct particles that may act as novel immunogens. We prepared at least seven separate particles with various placements of the disaccharide on the peptide, and along with linker and a 28-residue portion of C3d, a domain of complement component 3 and a ligand of CD21, a B-cell surface protein that, when engaged, lowers the threshold of B-cell activation.. These particles were injected into mice and the sera were analyzed for immune responses. A statistically significant immune response was observed in at least two test groups, and animals we boosted a second time with fresh particles. Tumors were implanted and survival was followed. Although one specific antigen group did better than the others, they did not do better than the group that received only PBS. There are several parameters that could have led to a lower than desired reponse, and we are looking into these now. A new study started in collaboration with Howard Young, has us exploring the modulation in cytokine profiles that is elicited by particles with varying antigens in different chemical guises. Initial data is very encouraging as the levels of several cytokines from activated murine macrophages are either potentiated or attenuated with particles containing different surface chemistries. The plan is to reproduce these data, extend this to dendritic cells and explore models for reinjecting these cells back into animals to examine the response to tumor. In addition, TEM experiments are defining the uptake of these particles in mouse macrophages and dendritic cells We also have explored other methods for synthesizing TF and other glycopeptide antigens with optimized linkers to attach to nano-platforms. Novel linker chemistry was developed and the use of lipoic acid-based terminal disulfides as conjugating moieties to gold and other surfaces. Various constructs were prepared to compare the uniformity of the particles with changes in surface chemistry. Finally, we have developed an assay to fully characterize the molecular composition of the particles we prepared. Details of how this can be accomplished in the literature were scant and unsatisfying and we decided to develop our own method. We have worked out procedures for taking small amounts of the nanoparticles, stripping them completely of their ligands and conjugating each to a fluorescent tag that can quantitated by HPLC and fluorescence spectrophotometry. This method has been refined and a manuscript detailing these experiments is being prepared.