Carbohydrate Antigen-bearing Nanoparticles for Antitumor Therapy

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导致异常聚糖链的生物合成。这些畸变随着肿瘤获得更具侵袭性的表型而变得更加显著。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起重要作用。此外，这些异常聚糖中的许多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TACA的相互作用还不清楚，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特征是多价性的概念：多价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。我们已经在非常特异的模板上制备了重要的T-F抗原（Gal（beta）1-3GalNAc（alpha）-O-Ser/Thr），以利用这种所谓的簇糖苷效应。多年来，我们已经制备了许多优化的金纳米颗粒，其具有附着于苏氨酸和丝氨酸氨基酸残基的TF抗原，在N-末端胺基上具有封端乙酸酯。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半乳糖凝集素-3（TF抗原的体内受体）表达阳性或阴性。对Gal-3呈阴性的细胞不对我们的颗粒产生应答，这表明细胞毒性确实通过Gal-3介导的途径。我们最新的迭代利用了一种新型的更稳定的连接体，它赋予了人类血清中颗粒令人难以置信的稳定性，并在本周期中发表。我们的初始疫苗构建体先前已描述，并且这种3组分疫苗对携带TF抗原的糖肽产生良好的免疫应答，所述TF抗原源自粘蛋白-4（MUC 4）的串联重复序列，粘蛋白-4是一种在胰腺导管腺癌（PDAC）细胞上过表达的大蛋白，并且是基于PDAC的肿瘤的生物标志物。本研究鉴定了称为MUC 4 - 5 TF的最佳构建体，并且该特异性抗原与KLH缀合，并且多克隆血清显示出对用于免疫的糖肽的高度特异性。我们随后与罗克兰免疫化学公司合作，制备了一种单克隆抗体（mAb），发现该抗体作为PDACS的诊断剂和可能的治疗剂非常有用。我们已经证明该试剂1）对组织阵列中的肿瘤具有特异性，2）仅与我们接种的序列结合，3）不与针对非糖基化序列产生的商业抗体交叉反应，并且完全不与我们的接头技术反应。该试剂现在是罗克兰的商业产品。此外，我们重新设计了疫苗结构，并制备了具有β-1，3葡聚糖涂层的金纳米颗粒。这些是葡萄糖多糖，与抗原呈递细胞（APC）上的特定C型凝集素结合，如巨噬细胞和称为Dectin-1的树突状细胞。这有助于进入APC并将附着的抗原呈递给T细胞。我们表明，小鼠接种疫苗后，抗原的抗体滴度非常高，细胞因子谱与抗肿瘤反应相关，并产生与抗原反应的CD 4 + T细胞。我们正在优化这种结构用于治疗。此外，我们用罗克兰生产的mAb已经显示出在体内选择性地分布到MUC 4阳性肿瘤。还制备了抗体药物缀合物（ADC），并且其在体外对MUC 4阳性肿瘤显示出高毒性。我们已经制备了一个单链片段（ScFv），现在正在开发一种新的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.