Targeting Pancreatic Cancer with Aptamers to the CCK-B Receptor

描述（由申请人提供）：癌症治疗的进展都来自于对特定癌症的生物学和/或遗传组成的理解。当这些因素被确定后，治疗就可以通过靶向癌症相关受体来实现肿瘤特异性的“个性化”，并减少来自非部位损伤的毒性。与其他实体瘤不同，G蛋白偶联受体（GPCR）已被证明在介导胃肠道肿瘤生长中发挥关键作用。我们已经确定了胰腺癌的受体，CCK-B受体，与正常组织相比，它在癌症中过度表达，并负责生长刺激。我们的研究小组已经表明，胃泌素通过这种GPCR以自分泌方式介导其作用，从而刺激胰腺癌的生长。我们已经证明，如果胃泌素在受体上的相互作用可以被阻断，胰腺癌就被抑制。一个问题是开发一种选择性阻断这种受体的药物，这种药物可能用于治疗。我们实验室和其他实验室曾试图提出针对CCK-B受体的胃泌素结合位点的单克隆抗体，但由于人和小鼠序列之间的相似同源性而未成功。因此，我们采用了一种不同的策略，使用DNA适体靶向胰腺癌中的CCK- B受体。DNA适体-高度结构化的寡核苷酸，其以与抗体相当或更好的亲和力选择性地结合受体靶标，在外周血中比单克隆抗体更稳定，并且它们没有免疫原性。我们合成了两个肽对应的胃泌素结合位点的细胞外，N-末端的CCK-B受体。使用一种称为指数富集配体系统进化（SELEX）的技术，我们鉴定了与CCK-B受体的外部胃泌素结合位点结合的特异性DNA适体。我们假设，选择性的CCK-B受体的DNA适体可以特异性地靶向胰腺癌，并通过阻断胃泌素的作用来抑制生长。为了检验这一假设，我们提出了以下目标：1）表征我们在体外使用胰腺癌细胞鉴定的一组适体的结合亲和力（Kd）和IC 50，以选择具有最大亲和力的适体用于生长研究。2)研究适体抑制培养物和携带原位胰腺癌的小鼠中胰腺癌生长的功效。还将研究CCK-B靶向适体对细胞凋亡的作用。我们研究的长期目标是改善胰腺癌患者的治疗和生存。

DESCRIPTION (provided by applicant): Advances in cancer therapy have all come from understanding the biology and or genetic make-up of a particular cancer. When these factors are identified, treatments can then be 'personalized' with tumor specificity by targeting of cancer-related receptors, and less toxicity from off-site injury. Unlike other solid tumors, G-protein coupled receptors (GPCRs) have been shown to play a critical role in mediation of growth of gastrointestinal cancers. We have identified a receptor on pancreatic cancer, the CCK-B receptor, which is over-expressed in cancer compared to normal tissues and responsible for growth stimulation. Our research team has shown that gastrin stimulates growth of pancreatic cancer by mediating its effects in an autocrine fashion through this GPCR. We have shown that if gastrin's interaction at the receptor can be blocked, pancreatic cancer is inhibited. One problem has been in developing an agent to selectively block this receptor that can potentially be used therapeutically. Our lab and others have attempted to raise a monoclonal antibody to the gastrin binding site of the CCK-B receptor but have been unsuccessful due to the similar homology between human and mouse sequences. Therefore, we have undertaken a different strategy using DNA aptamers to target the CCK- B receptor in pancreatic cancer. DNA aptamers - highly structured oligonucleotides that bind selectively to receptor targets with affinities comparable to or better than antibodies, are more stable in the peripheral blood than monoclonal antibodies, and they are not immunogenic. We synthesized two peptides corresponding to the gastrin binding site on the extracellular, N-terminus of the CCK-B receptor. Using a technique called Systematic Evolution of Ligands by Exponential Enrichment (SELEX) we identified specific DNA aptamers that bind to the external gastrin binding site of the CCK-B receptor. We hypothesize that selective DNA aptamers to the CCK-B receptor can specifically target pancreatic cancer and inhibit growth by blocking the actions of gastrin. In order to test this hypothesis we propose the following aims: 1) Characterize the binding affinities (Kd) and IC50 of a panel of aptamers we have identified using pancreatic cancer cells in vitro to select aptamers with the greatest affinities for growth studies. 2) Study the efficacy of aptamers to inhibit growth of pancreatic cancer in culture and in mice bearing an orthotopic pancreatic cancer. The role of CCK-B targeted aptamers on apoptosis will also be investigated. The long term goal of our research is to improve treatment and survival of patients with pancreatic cancer.