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

肿瘤发生的一个既定标志是由于肿瘤组织中糖加工酶表达的变化而引起的异常聚糖链的生物合成。当肿瘤获得更具侵袭性的表型时，这些畸变变得更加明显。肿瘤细胞表面碳水化合物在多种肿瘤细胞的运动和转移中起着重要作用。此外，许多这些异常聚糖是肿瘤相关碳水化合物抗原（TACA），并已用于肿瘤疫苗的开发。由于大多数细胞与TACAs的相互作用尚不清楚，因此迫切需要更好地表征在这些事件中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很好理解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，而单体的聚类将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(β)1-3GalNAc(α)-O-Ser/Thr)，以利用这种所谓的簇糖苷效应。如上一篇报道所述，我们制备了含糖衍生物的金自组装纳米球和量子点，并报道了其功能的初步细节。我们的金纳米球在小鼠体内的实验结果相互矛盾，因此我们回归基础，进行了更严格的表征，并探索了一系列新的合成方法，以生产更均匀的颗粒。我们开始系统地研究最佳程序，从几个相关的方法，提供最高质量的颗粒在稳定性和均匀性。我们仍在各种媒介中检查这些数据，以测试其稳定性。我们制备了不同情况下的TF抗原（分别附着在丝氨酸和苏氨酸上），并将它们连接到颗粒上。我们的TF颗粒现在已经在下拉实验中被证明可以结合半乳糖凝集素-3和与转移相关的整合素复合物。我们的合作者Vladislav Glinskii正在继续这项工作，他对定义TF-糖苷感兴趣，即识别前列腺癌细胞中具有o -连接TF抗原的所有蛋白质。我们正在研究一种蛋白质组学方法，以帮助他在明年确定这些分子。我们将重点放在制备包含我们认为最好的抗原（来自肿瘤相关细胞表面粘蛋白的糖肽）的颗粒上，并将其与不同浓度的连接体和t辅助表位结合，以构建可能作为新型免疫原的颗粒。我们制备了至少7个独立的颗粒，在肽上有不同的双糖位置，以及连接器和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，可以降低b细胞的激活阈值。将这些颗粒注射到小鼠体内，分析其血清的免疫反应。至少在两个实验组中观察到统计上显著的免疫反应，我们用新鲜颗粒第二次刺激动物。植入肿瘤并随访存活情况。虽然一个特定抗原组比其他组表现更好，但他们并不比只接受PBS的组表现更好。有几个参数可能导致低于预期的响应，我们现在正在研究这些参数。我们与霍华德·杨（Howard Young）合作开展了一项新研究，探索细胞因子谱的调节，这种调节是由具有不同化学伪装的不同抗原的颗粒引起的。初始数据非常令人鼓舞，因为来自活化小鼠巨噬细胞的几种细胞因子的水平被含有不同表面化学物质的颗粒增强或减弱。他们的计划是复制这些数据，将其扩展到树突细胞，并探索将这些细胞重新注射回动物体内的模型，以检查对肿瘤的反应。此外，透射电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，并优化了连接物以附着在纳米平台上。利用硫辛酸基末端二硫化物作为金和其他表面的共轭基团，开发了新的连接化学。制备了各种结构体，以比较颗粒的均匀性与表面化学的变化。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。关于如何在文献中实现这一点的细节很少，也不令人满意，我们决定开发我们自己的方法。我们已经制定出了少量纳米颗粒的程序，将它们完全剥离配体，并将每个纳米颗粒结合到可以通过HPLC和荧光分光光度法定量的荧光标签上。这种方法已得到改进，目前正在准备详细说明这些实验的手稿。

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.