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.

肿瘤发生的既定标志是由于肿瘤组织中糖化酶表达的变化，异常聚糖链的生物合成。随着肿瘤获得更具侵略性的表型，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性和转移中起着重要作用。此外，这些异常的聚糖中的许多是与肿瘤相关的碳水化合物抗原（TACA），并且已用于肿瘤疫苗的发育。由于大多数与塔卡斯的细胞相互作用尚不清楚，因此迫切需要更好地表征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，同时聚集单体会提高这种亲和力多达一百万倍。我们已经在非常具体的模板上准备了重要的Thomsen-Friedenreich（TF）抗原（GAL（beta）1-3GALNAC（Alpha）-o-Ser/Thr），以利用这种所谓的糖苷糖苷效应。正如上一报告所述，我们已经准备了金纳米球和量子点，这些纳米球和量子点含有糖衍生物，并报告了有关其功能的初步细节。在小鼠中使用金纳米球进行的体内实验是冲突的，因此我们撤退到基础知识并进行了更严格的特征，并探索了许多新合成，这些合成允许产生更均匀的颗粒。我们从几种相关方法中系统地研究了最佳过程，这些方法就稳定性和均匀性提供了最高质量的颗粒。我们仍在各种媒体中检查这些数据以测试稳定性。我们已经在不同的情况下（连接到丝氨酸和苏氨酸）准备了TF抗原，并将其与颗粒联系起来。现在，我们的TF颗粒已在下拉实验中显示，以与转移相关的Galectin-3和整联蛋白复合物结合。这项工作仍在我们的合作者Vladislav Glinskii，他有兴趣定义TF-Glycome，即确定具有O-Linked TF TF抗原的前列腺癌细胞中的所有蛋白质。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佳抗原的颗粒，来自肿瘤相关的细胞表面粘蛋白的糖肽，并将其与各种浓度的接头和T螺旋体表位相结合，以构建可能充当新型免疫原子的颗粒。我们制备了至少七个单独的颗粒，这些颗粒具有各种二糖在肽上的位置，以及连接器和28个残基的部分C3D，C3D（补体组件3的一个结构域）和CD21的配体B-cell表面蛋白，B细胞表面蛋白，当接合时，这些粒子被降低了这些粒子的阈值。在至少两个测试组中观察到具有统计学意义的免疫反应，我们第二次用新鲜颗粒增强了动物。植入肿瘤并遵循存活。尽管一个特定的抗原组比其他抗原组更好，但它们的表现并不比仅接受PBS的组更好。有几个参数可能导致比所需的响应低，我们现在正在研究这些响应。一项与霍华德·杨（Howard Young）合作的新研究使我们探索了细胞因子谱的调节，这是由不同化学套管中具有不同抗原变化的颗粒引起的。初始数据非常令人鼓舞，因为活化鼠巨噬细胞的几种细胞因子的水平被含有不同表面化学的颗粒增强或减弱。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入动物以检查对肿瘤的反应的模型。此外，TEM实验定义了这些颗粒在小鼠巨噬细胞和树突状细胞中的摄取，我们还探索了其他方法，用于合成TF和其他具有优化的接头，以连接到纳米平台上。开发了新型的接头化学，并将基于lipoic酸的末端二硫化物用作与黄金和其他表面的共轭。准备了各种构造，以比较颗粒的均匀性与表面化学的变化。 最后，我们开发了一种测定法，以充分表征我们制备的颗粒的分子组成。在文献中如何实现这一目标的详细信息很少且不令人满意，我们决定开发自己的方法。我们已经制定了少量的纳米颗粒，将它们完全剥离的配体，并将每个纳米颗粒剥离为荧光标签，并通过HPLC和荧光分光光度法进行定量。该方法已被完善，并准备了详细介绍这些实验的手稿。