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。如前所述，我们的TF颗粒现已在下拉实验中被证明与半乳糖凝集素-3和整合素复合物结合，与转移有关。它们还以某些特定的方式抑制胶原蛋白的入侵。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。初步数据显示，被激活的小鼠巨噬细胞中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。这是重新检查和改进，具体地说，tnf - α表达与非常特定的gycopeptide结构比其他更多。我们已经准备了三套不同大小的新颗粒，上面包裹着我们重要的抗原。这些在巨噬细胞系统中进行了检查，并显示细胞因子表达随着颗粒大小而急剧增加。我们是下一个。计划将此扩展到树突细胞，并探索将这些细胞再注射回动物体内的模型，以检查对肿瘤的反应，现在已经到位。此外，透射电镜实验正在确定小鼠巨噬细胞和树突状细胞对这些颗粒的摄取。我们开发的用于充分表征颗粒分子组成的测定方法运行良好。经过大量的改进，我们已经制定出了溶解少量纳米颗粒并定量回收其配体的程序，并将每个配体与可通过HPLC分析的荧光标记结合。我们也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，现在已经接近可以发表了。

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.