Carbohydrate Antigen-bearing Nanoparticles for Antitumor Therapy

肿瘤发生的一个公认的标志是由于肿瘤组织中糖处理酶表达的变化而导致异常糖链的生物合成。随着肿瘤获得更具侵袭性的表型，这些像差变得更加明显。肿瘤细胞表面的碳水化合物在许多不同癌细胞的运动和转移中起着重要作用。此外，这些异常多糖中的许多是肿瘤相关碳水化合物抗原(TACA)，并已被用于肿瘤疫苗的开发。由于大多数细胞与TACA的相互作用还不是很清楚，因此迫切需要更好地描述在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，而单体的聚集使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(Beta)1-3GalNAc(α)-O-Ser/Thr)，以利用这种所谓的簇苷效应。多年来，我们已经制备了许多优化的金纳米颗粒，其转铁蛋白抗原同时附着在苏氨酸和丝氨酸氨基酸残基上，在N-端胺基团上封端有醋酸酯。对不同肿瘤细胞系中这些颗粒的细胞毒性进行了检测，并与每种构建物进行了比较。这些细胞被选为抗凋亡蛋白Galectin-3的阳性或阴性表达，Galectin-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是通过Gal-3介导的途径进行的。我们最新的迭代使用了一种新的、更稳定的连接物，它为人血清中的颗粒提供了令人难以置信的稳定性，并在本周期中发表。我们最初的疫苗构建如前所述，这种三组分疫苗对含有TF抗原的糖肽有良好的免疫应答，该糖肽来自粘蛋白-4(MUC4)的串联重复序列，粘蛋白-4是一种在胰腺导管腺癌(PDAC)细胞上过度表达的大蛋白，是基于PDAC的肿瘤的生物标志物。本研究确定了一种最佳的构建物，命名为MUC4-5tf，并将其与KLH偶联，多克隆血清对用于免疫的糖肽显示出高度的特异性。随后，我们与Rockland免疫化学公司合作，制备了一种单抗，该抗体被发现作为PDACS的诊断和可能的治疗剂非常有用。我们已经证明，这种试剂1)对组织阵列中的肿瘤具有特异性，2)只与我们接种的序列结合，3)与商业抗体不发生交叉反应，被提高到未糖基化的序列，并且完全不与我们的接头技术发生反应。该试剂现在是Rockland的商业产品。此外，我们对疫苗结构进行了重新改造，并制备了包裹有β-1，3葡聚糖的金纳米颗粒。这些是葡萄糖多糖，可以与抗原提呈细胞(APC)上的特定C型凝集素结合，如巨噬细胞和树突状细胞，称为Dectin-1。这有助于进入APC并将附着的抗原呈递给T细胞。我们发现，在小鼠接种疫苗后，对该抗原的抗体效价非常高，细胞因子谱与抗肿瘤反应有关，并产生了与该抗原反应的CD4+T细胞。我们正在优化这种结构以用于治疗。此外，我们与Rockland共同生产的mAb已被证明在体内选择性地分布于MUC4阳性肿瘤。还制备了一种抗体药物结合物(ADC)，它在体外对MUC4阳性肿瘤显示出较高的毒性。我们已经制备了单链抗体(ScFv)，目前正在开发一种由该抗体产生的新型CAR-T细胞。

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. Over the years, we have prepared many optimized gold nanoparticles bearing the TF antigen attached to both 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. Our latest iteration utilized a novel and more stable linker that imparts incredible stability to the particles in human serum and was published in this cycle. Our initial vaccine construct was previously described, and this 3-component vaccine gave a good immune response to glycopeptides bearing the TF antigen that were derived from a tandem repeat sequence from Mucin-4 (MUC4), a large protein which is overexpressed on Pancreatic Ductal Adenocarcinoma (PDAC) Cells and is a biomarker for PDAC-based tumors. This study identified a best construct, called MUC4-5TF, and this specific antigen was conjugated to KLH and polyclonal sera showed high specificity for the glycopeptide used for immunization. We subsequently collaborated with Rockland Immunochemicals to prepare a monoclonal antibody (mAb) which was found to be highly useful as a diagnostic and possibly a therapeutic agent against PDACS. We have shown this reagent to be 1) specific for tumor in tissue arrays, 2) Bind only to the sequence we vaccinated with, 3) Not cross react with a commercial antibody raised to an unglycosylated sequence and to not react at all with our linker technology. The reagent is now a commercial product with Rockland. In addition, we have re-tooled our vaccine construct and prepared gold nanoparticles with a coating of beta-1,3 glucans. These are glucose polysaccharides that bind to a specific C-type lectin on antigen presenting cells (APCs) such as macrophages and dendritic cells called Dectin-1. This facilitates entry into the APC and presentation of the attached antigen to T-cells. We showed after vaccination of mice that antibody titers to the antigen were very high, cytokine profiles were relevant to an antitumor response and CD4+ T-cells that react with the antigen are generated. We are in the process of optimizing this construct for therapeutic use. In addition, the mAb we produced with Rockland has been shown to distribute selectively to MUC4 positive tumors in vivo. An antibody drug conjugate *ADC) was also prepared and it shows high toxicity to MUC4 positive tumors in vitro. We have prepared a single chain fragment (ScFv) and are now developing a novel CAR-T cell is from this antibody.