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。现在，我们有了一种新的、优化的、可重复的金纳米颗粒合成方法，该金纳米颗粒带有附着在苏氨酸和丝氨酸氨基酸残基上的 TF 抗原，并在 N 端胺基上有一个乙酸封端。已经对这些颗粒在各种肿瘤细胞系中的细胞毒性进行了检查，并与每种构建体进行了比较。这些细胞被选择为抗凋亡蛋白 Galectin-3（TF 抗原的体内受体）表达呈阳性或阴性。 Gal-3 呈阴性的细胞不会对我们的颗粒产生反应，这表明细胞毒性确实通过 Gal-3 介导的途径进行。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，当接合时，会降低 B 细胞激活的阈值。将这些颗粒注射到小鼠体内，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，小鼠由 Precision Antibodies Inc 进行疫苗接种。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夏洛特分校的 Pinku Mukherjee 教授。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。然而，在这个周期中，我们测试了多克隆血清与我们的糖肽构建体的结合，我们将其置于由我们的合作者 Jeff Gildersleeve 开发的糖肽阵列上。该血清仅对我们打印的糖肽进行染色，而不对任何其他环境中的 TF 抗原进行染色（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单克隆抗体，我们认为这将是检测肿瘤细胞上异常 MUC4 的极其有用的试剂。这项新研究是与上一份年度报告中提到的 Howard Young 合作开始的，继续探索由不同化学形式的不同抗原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。经过重新检查和改进，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。为了完成这项研究，我们将重点放在 MUC4-5TF 构建体（见上文）上，以研究引发炎症细胞因子基因表达的实际化学需求。上一篇报告中提到的关于评估用多种配体制备金纳米粒子的最佳前驱体的手稿今年发表在《胶体与界面科学杂志》上。我们已经证明，一些颗粒通过细胞凋亡机制具有细胞毒性，现在我们想要定义为什么多价平台在细胞中起作用，但在简单蛋白质上不起作用。这可能是由于 gal-3 在细胞中和在简单缓冲溶液中的聚集状态非常不同，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。我们发现，TF抗原展示在苏氨酸残基上的纳米构建体比其附着在丝氨酸残基上的纳米构建体更有活性。这是一个令人着迷的结果，我们现在正尝试用各种不同的纳米粒子在小鼠中重现这一结果。一项使用统计上显着数量的动物的新小鼠研究即将开始，该研究使用具有各种连接策略的新型颗粒。我们正在探索将糖与颗粒偶联的不同方法是否会影响糖的生物活性。