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

用于抗粘连剂和肿瘤疫苗的携带碳水化合物抗原的纳米颗粒

• 批准号: 7965330
• 负责人: Joseph John Barchi
• 金额: $ 36.39万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7965330
• 关键词: Adhesives; Affinity; Anabolism; Animals; Antigens; Attenuated; B-Cell Activation; B-Lymphocytes; Back; Binding; Biological Assay; Cancer Vaccines; Carbohydrates; Cell Surface Proteins; Cell surface; Cells; Chemicals; Chemistry; Collaborations; Complement 3; Complement 3d; Complement 3d Receptors; Complex; Conflict (Psychology); Data; Dendritic Cells; Development; Diagnostic Imaging; Disaccharides; Disulfides; Drug Delivery Systems; Enzymes; Epitopes; Event; Extramural Activities; Fluorescence Spectrophotometry; Galectin 3; Glycopeptides; Glycosides; Gold; High Pressure Liquid Chromatography; Hybrids; Immune response; Implant; Integrins; Ligands; Link; Literature; Magnetism; Malignant Neoplasms; Malignant neoplasm of prostate; Manuscripts; Medical Research; Metals; Methods; Modeling; Molecular; Mucins; Mus; Nanosphere; Neoplasm Metastasis; Organic Synthesis; Peptides; Pharmaceutical Preparations; Phenotype; Play; Polysaccharides; Procedures; Production; Proteins; Proteomics; Quantum Dots; Reporting; Research Personnel; Role; Serine; Serum; Surface; Testing; Therapeutic Agents; Thioctic Acid; Thompson-Friedenreich Antigen; Threonine; Time; Toxic effect; Tumor Tissue; Tumor-Associated Carbohydrate Antigens; Work; analog; base; cancer cell; cell motility; cytokine; design; gag Gene Products; in vivo; interest; macromolecule; macrophage; mimetics; monomer; nano; nanoparticle; neoplastic cell; novel; novel vaccines; particle; physical property; research study; response; stability testing; sugar; tumor; tumorigenesis; uptake

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.

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生物合成异常聚糖链。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许多不同癌细胞的运动和转移中发挥重要作用。此外，许多异常聚糖是肿瘤相关糖抗原（TACA），已用于肿瘤疫苗的开发。由于大多数细胞与 TACA 的相互作用尚不清楚，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众所周知的特征是多价概念：单体碳水化合物与蛋白质的结合非常弱，而单体的聚集将这种亲和力提高了数百万倍。我们在非常特定的模板上制备了重要的 Thomsen-Friedenreich (Tf) 抗原（Gal(beta)1-3GalNAc(alpha)-O-Ser/Thr），以利用这种所谓的簇糖苷效应。正如上一篇报告中提到的，我们制备了金自组装纳米球和含有糖衍生物的量子点，并报告了其功能的初步细节。我们的金纳米球在小鼠身上进行的体内实验是相互矛盾的，因此我们退回到基础知识并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。我们继续从几种相关方法中系统地研究最佳程序，以提供在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稳定性。我们在不同的环境下制备了 TF 抗原（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 颗粒现已在 Pull down 实验中证明可以与与转移相关的 Galectin-3 和整合素复合物结合。我们的合作者 Vladislav Glinskii 正在继续这项工作，他对定义 TF-糖组感兴趣，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，以帮助他在明年定义这些分子。我们非常重视制备包含我们认为最好的抗原（一种来自肿瘤相关细胞表面粘蛋白的糖肽）的颗粒，并将其与不同浓度的接头和 T 辅助表位相结合，以构建可以充当新型免疫原的颗粒。我们制备了至少七个单独的颗粒，其中二糖在肽上的不同位置，以及接头和 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，会降低 B 细胞激活的阈值。这些颗粒被注射到小鼠体内，并分析血清的免疫反应。在至少两个测试组中观察到统计学上显着的免疫反应，并且我们用新鲜颗粒对动物进行了第二次加强。植入肿瘤并追踪存活情况。尽管一个特定抗原组的表现优于其他组，但它们并不比仅接受 PBS 的组表现更好。有几个参数可能会导致低于预期的响应，我们现在正在研究这些参数。与 Howard Young 合作开展的一项新研究让我们探索了由具有不同化学形式的不同抗原的颗粒引起的细胞因子谱的调节。最初的数据非常令人鼓舞，因为含有不同表面化学物质的颗粒可以增强或减弱来自活化的鼠巨噬细胞的几种细胞因子的水平。该计划是重现这些数据，将其扩展到树突状细胞，并探索将这些细胞重新注射回动物体内以检查对肿瘤的反应的模型。此外，TEM 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。我们还探索了合成 TF 和其他糖肽抗原的其他方法，并具有优化的连接器以连接到纳米平台。开发了新型连接化学，并使用基于硫辛酸的末端二硫化物作为金和其他表面的缀合部分。制备了各种构造来比较颗粒的均匀性与表面化学的变化。 最后，我们开发了一种测定方法来充分表征我们制备的颗粒的分子组成。文献中关于如何实现这一点的细节很少且不能令人满意，因此我们决定开发自己的方法。我们已经制定了提取少量纳米颗粒的程序，将它们完全剥离配体，并将每个纳米颗粒与荧光标签结合，可以通过 HPLC 和荧光分光光度法进行定量。该方法已经得到完善，并且正在准备详细介绍这些实验的手稿。