Carbohydrate Antigen-bearing Nanoparticles for Antitumor Therapy

用于抗肿瘤治疗的碳水化合物抗原纳米颗粒

• 批准号: 10262091
• 负责人: Joseph John Barchi
• 金额: $ 68.44万
• 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/10262091
• 关键词: Acetates; Adaptor Signaling Protein; Adhesives; Affect; Affinity; Amines; Amino Acids; Anabolism; Animals; Annual Reports; Antibodies; Antigens; Apoptotic; Area; Attenuated; B-Cell Activation; B-Lymphocyte Epitopes; B-Lymphocytes; Binding; Biological; Biological Assay; Buffers; CCR; Cancer Vaccines; Carbohydrates; Cell Line; Cell Surface Proteins; Cell surface; Cells; Chemicals; Chemistry; Collaborations; Colloids; Complement 3; Complement 3d; Complement 3d Receptors; Conflict (Psychology); Coupling; Cytokine Gene; Data; Detection; Development; Diagnostic Imaging; Disaccharides; Drug Delivery Systems; Enzymes; Epitopes; Evaluation; Event; Extramural Activities; Galectin 3; Gene Expression; Glycopeptides; Glycosides; Gold; Hybrids; Immune response; Implant; Inflammatory; Journals; Keyhole Limpet Hemocyanin; Knock-out; Ligands; Magnetism; Malignant Neoplasms; Manuscripts; Mediating; Medical Research; Metals; Methods; Monoclonal Antibodies; Mucins; Mus; N-terminal; Nanosphere; Neoplasm Metastasis; North Carolina; Organic Synthesis; Paper; Particle Size; Pathway interactions; Peptides; Pharmaceutical Preparations; Phenotype; Play; Polysaccharides; Preparation; Procedures; Process; Production; Proteins; Publishing; Quantum Dots; Reagent; Reporting; Reproducibility; Research; Research Personnel; Role; Sampling; Science; Serine; Signal Transduction; Stains; Surface; System; TNF gene; Testing; Therapeutic Agents; Thompson-Friedenreich Antigen; Threonine; Time; Tissues; Toll-like receptors; Toxic effect; Tumor Cell Line; Tumor Tissue; Tumor-Associated Carbohydrate Antigens; Universities; Vaccinated; Work; analog; animal imaging; biophysical techniques; cancer cell; cell motility; cell type; cytokine; cytotoxic; cytotoxicity; design; experimental study; fascinate; gag Gene Products; imaging program; in vivo; interest; macromolecule; macrophage; mimetics; monomer; mutant; nano; nanoGold; nanoparticle; neoplastic cell; novel; novel therapeutics; novel vaccines; particle; physical property; polyclonal antibody; professor; receptor; response; sugar; tumor; tumorigenesis

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 have now a new, optimized and reproducible synthesis for gold nanoparticles bearing the TF antigen attached to both eth threonine and serine amino acid residues with a capping acetate at the N-terminal amine group. Examination of these particles in various tumor cell lines for cytotoxicity has been performed and compared against each construct. These cells were chose to be either positive or negative for expression of the anti-apoptotic protein Galectin-3, an in vivo receptor for the TF antigen. Cells that are negative for Gal-3 do not respond to our particles, which suggest that cytotoxicity does go through a Gal-3 mediated pathway. 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. However, in this cycle, we tested the polyclonal sera for binding to our glycopeptide constructs that we put on a glycopeptide array developed by our collaborator Jeff Gildersleeve. This sera stained ONLY the glycopeptides we printed, and not to the TF antigen in ANY OTHER CONTEXT (there are about 30 on Jeff's array). Thus we have a company preparing a monoclonal antibody for us that we feel will be an extremely useful reagent for detection of aberrant MUC4 on tumor cells. 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. To 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 shown that some of the particles are cytotoxic through an apoptotic mechanism, and we now want to define why the multivalent platforms work in cells but not on simple proteins. This is probably due to the aggregation state of gal-3 being very different in cells and in while in a simple buffer solution, and we are designing experiments to help unravel this mystery. We are looking at various cell types that either express or do not express Gal-3 with Chand Khann of the CCR as well as performing antimetastaic studies in vivo in collaboration with the Small Animal Imaging Program of Leidos Biomedical. We have found that nano-constructs where the TF antigen is displayed on a threonine residue are are active than those where it is attached to a serine residue. This was a fascinating result and we are now trying to reproduce this in mice with a variety of different nanoparticles. A new mouse study using a statistically significant number of animals is just about to begin with novel particles with various linker strategies. We are exploring whether or not different methods for coupling the sugars to the particles will affect the biological activity of the saccahrides.

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。我们现在有了一个新的，优化的和可重复的合成金纳米颗粒，其TF抗原附着在eth苏氨酸和丝氨酸氨基酸残基上，在n端胺基上有一个醋酸盖。对这些颗粒在各种肿瘤细胞系中的细胞毒性进行了检查，并与每种结构进行了比较。这些细胞被选择为抗凋亡蛋白半乳糖凝集素-3的阳性或阴性表达，半乳糖凝集素-3是TF抗原的体内受体。Gal-3阴性的细胞对我们的颗粒没有反应，这表明细胞毒性确实通过Gal-3介导的途径进行。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。然而，在这个循环中，我们测试了多克隆血清与我们的糖肽构建物的结合，我们把糖肽构建物放在我们的合作者Jeff Gildersleeve开发的糖肽阵列上。这种血清只染色我们打印的糖肽，而不染色任何其他情况下的TF抗原（Jeff的阵列上大约有30个）。因此，我们有一家公司为我们准备了一种单克隆抗体，我们认为这将是一种非常有用的检测肿瘤细胞上异常MUC4的试剂。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，我们将重点放在MUC4-5TF构建体（见上）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。我们已经证明了一些颗粒通过凋亡机制具有细胞毒性，我们现在想要定义为什么多价平台在细胞中起作用，而不是在简单的蛋白质上起作用。这可能是由于gal-3的聚集状态在细胞和在一个简单的缓冲溶液中是非常不同的，我们正在设计实验来帮助解开这个谜团。我们正在与CCR的Chand Khann一起研究表达或不表达Gal-3的各种细胞类型，并与Leidos Biomedical的小动物成像项目合作进行体内抗转移研究。我们已经发现，TF抗原在苏氨酸残基上显示的纳米结构比那些附着在丝氨酸残基上的纳米结构更有活性。这是一个令人着迷的结果，我们现在正试图用各种不同的纳米颗粒在老鼠身上重现这一结果。一项新的小鼠研究使用了统计上显著数量的动物，即将开始使用具有各种连接策略的新颗粒。我们正在探索将糖与颗粒偶联的不同方法是否会影响糖苷的生物活性。