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。如前所述，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 B 细胞表位相结合，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响应低于预期，我们现在正在研究这些参数。最近在《生物共轭化学》上发表了一篇关于这项工作的完整论文，其中一些构建体的抗肿瘤活性已通过多种方法进行了评估，本文由 Kate Rittenhouse-Olson 撰写，即将提交。本研究周期该领域的进一步工作是采用上述最佳构建体（称为 MUC4-5TF）并制备多克隆抗体血清。我们制备的这种构建体与 KLH 缀合，并由 Precision Antibodies Inc. 给小鼠接种疫苗。针对我们的特定糖肽抗原的滴度在数万范围内，并将血清提供给北卡罗来纳大学的 Pinku Mukherjee 教授在夏洛特。该血清没有对任何可用的组织进行染色，但我们正在寻找更合适的具有异常 MUC4 明显表达的肿瘤样本。这项新研究是与上一份年度报告中提到的霍华德·杨(Howard Young)合作开始的，继续探索由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。初步数据显示，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。经过重新检查和完善，结果表明，与其他糖肽构建体相比，非常特定的糖肽构建体更能激活 TNF-α 表达。我们准备了三组不同大小的新颗粒，这些颗粒涂有我们的重要抗原。在巨噬细胞系统中对它们进行检查，结果显示细胞因子表达随颗粒尺寸的增加而显着增加。因此，尺寸为 3、16、25 和 40 nm 的颗粒对细胞因子基因表达具有显着不同的影响，这也取决于配体的活性。此外，我们还在突变型 MyD88 敲除巨噬细胞系中测试了所有构建体。 MyD88 是一种接头蛋白，参与 Toll 样受体蛋白表达的信号转导级联。在敲除细胞系中，纳米颗粒诱导的细胞因子表达大大减少，表明Toll样受体参与了纳米颗粒表达细胞因子基因的过程。 完成这项研究后，我们将重点放在 MUC4-5TF 构建体（见上文）上，以磨练引发炎症细胞因子基因表达的实际化学要求。上一份报告中提到的关于评估最佳前体的手稿制备具有多种配体的金纳米颗粒的研究成果发表在今年的Journal of Colloid and Interface Science杂志上。我们与英国利物浦大学生物化学家/细胞生物学家Lugang YU发起了合作，研究抑制通过涂有非常特异性的 TF 抗原呈递的金颗粒进行转移。 Yu 博士发表了关于肿瘤细胞上 MUC1 粘蛋白上的 galectin-3 和 TF 抗原相互作用的文章，我们的颗粒是有效抑制这种相互作用并从而抑制转移的理想系统。距离我们向于教授发送三个构建体并测试它们作为 Galectin-3-TF 相互作用的抑制剂还有几天的时间