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: 8552700
• 负责人: Joseph John Barchi
• 金额: $ 43.57万
• 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/8552700
• 关键词: Adhesives; Affinity; Anabolism; Animals; Annual Reports; Antibodies; Antigens; Area; Attenuated; B-Cell Activation; B-Lymphocyte Epitopes; B-Lymphocytes; Binding; Biological Assay; Biomedical Engineering; Cancer Vaccines; Carbohydrates; Cell Line; Cell Surface Proteins; Cell surface; Cells; Chemicals; Chemistry; Collaborations; Colloids; Complement 3; Complement 3d; Complement 3d Receptors; Complex; Conflict (Psychology); Cytokine Gene; Data; Development; Diagnostic Imaging; Disaccharides; Drug Delivery Systems; Enzymes; Epitopes; Evaluation; Event; Extramural Activities; Galectin 3; Gene Expression; Glycopeptides; Glycosides; Gold; Great Britain; Hybrids; Immune response; Implant; Inflammatory; Integrins; Journals; Keyhole Limpet Hemocyanin; Knock-out; Ligands; Link; Magnetism; Malignant Neoplasms; Malignant neoplasm of pancreas; Manuscripts; Medical Research; Metals; Methods; Mucin-1 Staining Method; Mucins; Mus; Nanosphere; Nanotechnology; Neoplasm Metastasis; North Carolina; Organic Synthesis; Paper; Particle Size; Peptides; Pharmaceutical Preparations; Phenotype; Play; Polysaccharides; Preparation; Procedures; Process; Production; Proteins; Publishing; Quantum Dots; Reporting; Research; Research Personnel; Role; Sampling; Science; Serine; Serum; Signal Transduction; Staining method; Stains; Surface; System; Testing; Therapeutic Agents; Thompson-Friedenreich Antigen; Threonine; Time; Tissues; Toll-like receptors; Toxic effect; Tumor Necrosis Factor-alpha; Tumor Tissue; Tumor-Associated Carbohydrate Antigens; Universities; Vaccinated; Work; Writing; adapter protein; analog; cancer cell; cancer site; cell motility; cytokine; design; gag Gene Products; human TNF protein; in vivo; inhibitor/antagonist; interest; macromolecule; macrophage; malignant breast neoplasm; mimetics; monomer; mutant; nanoparticle; neoplastic cell; novel; novel vaccines; particle; physical property; polyclonal antibody; professor; receptor; research study; response; stability testing; sugar; tumor; tumorigenesis; vaccine development

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. 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 B-cell epitopes 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 response, and we are looking into these now. A full paper in Bioconjugate Chemistry was recently published on this work, and the antitumor activity of some of these constructs has been evaluated by several methods and this paper is written with Kate Rittenhouse-Olson and close to submission. Further work in this area for this research cycle has been to take the aforementioned best construct, called MUC4-5TF, and prepare polyclonal antibody sera. This construct as prepared by us was conjugated to KLH and mice were vaccinated by Precision Antibodies Inc. Titers against our specific glycopeptides antigen are in the tens-of-thousand range and the sera were given to Professor Pinku Mukherjee, at the University of North Carolina at Charlotte. This sera did not stain any of the tissue that was available, but we are looking at more appropriate tumor samples with distinct expression of aberrant MUC4. The new study started in collaboration with Howard Young mentioned in the last annual report, continues to explore 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 glycopeptide 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. Thus particles from sizes of 3, 16, 25 and 40 nm have dramatically different effects on cytokine gene expression, which is also dependent on the ligand for activity. In addition, we tested al the constructs in a mutant MyD88 knockout macrophage cell line. MyD88 is an adapter protein involved in the signal transduction cascade for the expression of toll-like receptor proteins. The cytokine expression induced by the nanoparticles was greatly reduced in the knockout cell line, suggesting that toll-like receptors are involved in the process of cytokine gene expression by the nanoparticles. Yo complete this study, we are focusing on the MUC4-5TF construct (vide supra) to hone in on the actual chemical requirements for eliciting gene expression of inflammatory cytokines.The manuscript mentioned in the last report on the evaluation of the optimum precursors for the preparation gold nanoparticles with a variety of ligands was published in the Journal of Colloid and Interface Science this year.We have initiated a collaboration with Lugang YU, a biochemist/cell biologist at the University of Liverpool in Great Britain on the inhibition of metastasis by gold particles coated with very specific presentations of TF antigen. Dr Yu has published on the interaction of galectin-3 and TF antigen on MUC1 mucin on tumor cells, and our particles are ideal systems to potently inhibit this interaction and hence metastasis. We are days away from sending three constructs to Professor Yu and testing them as inhibitors of Galectin-3-TF interactions

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。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b细胞表位结合，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。最近在《生物偶联化学》上发表了一篇关于这项工作的论文全文，其中一些结构体的抗肿瘤活性已经通过几种方法进行了评估，这篇论文是与Kate Rittenhouse-Olson共同撰写的，即将提交。本研究周期在该领域的进一步工作是采用上述最佳构建物MUC4-5TF制备多克隆抗体血清。我们制备的该构建体与KLH结合，由Precision antibody Inc.接种小鼠。针对我们的特定糖肽抗原的滴度在数万的范围内，血清被提供给北卡罗来纳大学夏洛特分校的Pinku Mukherjee教授。这种血清没有染色任何可用的组织，但我们正在寻找具有明显异常MUC4表达的更合适的肿瘤样本。这项新研究是与Howard Young合作开始的，在上一份年度报告中提到，该研究继续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表达与非常特定的糖肽结构相比，更多地开启。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。因此，3、16、25和40 nm大小的颗粒对细胞因子基因表达的影响显著不同，而细胞因子基因表达也依赖于配体的活性。此外，我们在突变型MyD88敲除巨噬细胞系中测试了所有构建物。MyD88是参与toll样受体蛋白表达信号转导级联的一种适配蛋白。在敲除细胞系中，纳米颗粒诱导的细胞因子表达显著降低，提示toll样受体参与了纳米颗粒介导细胞因子基因表达的过程。为了完成这项研究，我们将重点关注MUC4-5TF构建体（见上），以了解引发炎症细胞因子基因表达的实际化学需求。上一篇关于用各种配体制备金纳米颗粒的最佳前体的评价报告中提到的手稿发表在今年的《胶体与界面科学杂志》上。我们已经开始与英国利物浦大学的生物化学家/细胞生物学家Lugang YU合作，研究涂有TF抗原特异性表达的金颗粒对转移的抑制作用。Yu博士发表了关于半乳糖凝集素-3和TF抗原在肿瘤细胞MUC1粘蛋白上的相互作用的文章，我们的颗粒是有效抑制这种相互作用并因此转移的理想系统。再过几天，我们就会把三个结构体交给余教授，测试它们是否能抑制半乳糖凝集素-3- tf相互作用