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导致异常聚糖链的生物合成。这些畸变随着肿瘤获得更具侵袭性的表型而变得更加显著。肿瘤细胞表面碳水化合物在许多不同癌细胞的运动和转移中起重要作用。此外，这些异常聚糖中的许多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TACA的相互作用还不清楚，因此迫切需要更好地表征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特征是多价性的概念：多价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。我们已经在非常特异的模板上制备了重要的T-F抗原（Gal（beta）1-3GalNAc（alpha）-O-Ser/Thr），以利用这种所谓的簇糖苷效应。如上一份报告所述，我们制备了含有糖衍生物的金自组装纳米球和量子点，并报告了其功能的初步细节。我们的金纳米球在小鼠体内的实验是相互矛盾的，所以我们退回到基础，进行了更严格的表征，并探索了一系列新的合成方法，可以生产更均匀的颗粒。我们从几种相关方法中系统地研究了最佳程序，这些方法提供了稳定性和均匀性方面最高质量的颗粒。我们仍在各种媒体中检查这些数据，以测试其稳定性。我们已经制备了不同背景下的TF抗原（连接到丝氨酸和苏氨酸），并将它们连接到颗粒上。如上所述，我们的TF颗粒现已在下拉实验中显示结合与转移相关的半乳糖凝集素-3和整联蛋白复合物。它们还以某些特定的方式通过胶原蛋白抑制侵袭。 我们非常重视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，并将其与各种浓度的接头和辅助性T细胞表位相结合，以构建可能作为新型免疫原的颗粒。我们制备了至少七种单独的颗粒，其具有二糖在肽上的各种位置，并且沿着有接头和C3 d的28个残基部分，C3 d是补体成分3的结构域和CD 21的配体，CD 21是一种B细胞表面蛋白，当接合时，降低B细胞活化的阈值。将这些颗粒注射到小鼠体内，并分析血清的免疫反应。在至少两个测试组中观察到统计学显著的免疫应答，并且我们用新鲜颗粒第二次加强动物。植入肿瘤并随访存活率。尽管一个特异性抗原组比其他组做得更好，但它们并不比只接受PBS的组做得更好。有几个参数可能导致低于预期的反应，我们现在正在调查这些参数。与霍华德杨合作开始的一项新研究使我们探索了细胞因子谱的调节，这些细胞因子谱是由具有不同抗原的颗粒以不同的化学伪装引起的。初始数据显示，来自活化的鼠巨噬细胞的几种细胞因子的水平用含有不同表面化学的颗粒增强或减弱。这是重新检查和完善，以表明具体地说，TNF-α表达打开非常具体的糖肽结构比其他更多。我们已经准备了三组不同大小的新颗粒，包被有我们的重要抗原。这些在巨噬细胞系统中进行了检查，并显示出细胞因子表达随颗粒大小的急剧增加。我们是下一个。现在，将其扩展到树突状细胞并探索将这些细胞重新注射回动物体内以检查对肿瘤反应的模型的计划正在进行中。此外，TEM实验正在定义这些颗粒在小鼠巨噬细胞和树突细胞中的摄取。 我们开发的用于充分表征颗粒分子组成的测定法工作良好。经过大量的改进，我们已经制定了溶解少量纳米颗粒并定量回收其配体并将其与可以通过HPLC分析的荧光标记结合的程序。我们也正在研究一种质谱方法，用于这些配体的半定量。这种方法已经改进了几次，现在即将出版。

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. As mentioned, our TF particles have now been shown in pull down experiments to bind to Galectin-3 and integrin complexes related to metastasis. They also inhibit invasion through collagen in some context-specific ways. 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 showed that levels of several cytokines from activated murine macrophages are either potentiated or attenuated with particles containing different surface chemistries. This was reexamined and refined to show that specifically, TNF-alpha expression was turned on much more with very specific gycopeptide constructs than others. We have prepared three new sets of particles of various sizes coated with our important antigens. These were examined in the macrophage system and showed a dramatic increase in cytokine expression with particle size. We are next. The plan to extend this to dendritic cells and explore models for reinjecting these cells back into animals to examine the response to tumor is now i place. In addition, TEM experiments are defining the uptake of these particles in mouse macrophages and dendritic cells. The assay we have developed to fully characterize the molecular composition of the particles is working well. After much refinement, we have worked out procedures for dissolving small amounts of the nanoparticles and recovering their ligands quantitatively and conjugating each to a fluorescent tag that can be analyzed by HPLC. We are also working on a mass spec method for semi quantitation of these ligands. This method has been refined several times and is now close to being ready for publication.