Phage Display for Improved Peptide-Based Tumor Targeting and Imaging Agents

 描述（由申请人提供）： 癌症是美国退伍军人的主要健康问题。虽然大多数癌症在早期是可以治疗的，但与性别有关的癌症，包括卵巢癌、乳腺癌和乳腺癌， 当在晚期和/或转移性阶段诊断时，前列腺通常是致命的。据估计，到2015年，这些癌症将导致普通人群每年超过80，000人死亡，退伍军人的发病率明显更高。许多患者最初对治疗有反应，但经常复发。复发可能是由于肿瘤的异质性、各种信号传导途径的失调、异常的凝集素-碳水化合物或侵袭性和耐药性肿瘤的发展。最好的方法是通过实施更个性化的诊断和/或治疗方法来识别和治疗这些癌症，其中采用靶向癌细胞上多种生物标志物的药物。然而，令人惊讶的是，很少有肿瘤相关抗原或生物标志物显示与癌症类型或阶段相关。我们的实验室以前利用噬菌体展示技术来鉴定生物标志物，并分离识别卵巢癌、乳腺癌和前列腺癌的基于肽的靶向分子。已经获得了靶向ErbB-2和ErbB-3受体的肽、碳水化合物（Erbsen-Friedenreich抗原，TF）、凝集素（半乳糖凝集素-3，gal-3）和CD 44剪接变体CD 44 v6。这些生物标志物参与细胞粘附、血管生成和信号传导。TF在超过90%的所有腺癌中表达，gal-3是侵袭性和转移潜力的标志物，ErbB-2是侵袭性转移的标志物，ErbB-3通常与不良预后和治疗抗性相关，并且CD 44 v6与侵袭性癌症起始（干）细胞（CIC）相关。在体内检测这种生物标志物的能力 将有助于辨别肿瘤进展的各个阶段，并可能有助于监测对治疗的反应。 癌症生物标志物的靶向和成像通常依赖于放射性标记抗体（Ab）的使用。不幸的是，Ab非常大，并且经常引起免疫原性相关的毒性和耐药性。小肽在人类中耐受性良好，但具有低肿瘤摄取、不稳定性和高肾滞留的缺点。在小支架或单链抗体片段（scFv）的背景下展示的肽由于其高亲和力、稳定性和有利的体内药代动力学而可以为开发有效的肿瘤靶向剂提供理想的平台。假设噬菌体展示选择的gal-3/TF、ErbB- 2/ErbB-3和CD 44 v6肽可以被工程化为高亲和力、稳定的靶向支架，以用于卵巢癌、乳腺癌和前列腺癌的成像和对治疗的响应。 这些研究的目标是将ErbB-2/ErbB-3和gal-3/TF靶向肽以及新鉴定的CD 44 v6靶向肽开发成个性化的癌症检测或治疗剂。这项研究的目的是：1）功能性表征肽的中断， 体外和人乳腺癌、卵巢癌或前列腺癌内的信号通路和增殖; 2）通过以下方式开发适合于放射成像/治疗应用的新一代肽：i）合成反转和多抗原肽构建体，ii）工程化肽 3）使用荧光成像同时靶向多种生物标志物，以简化癌症检测探针的开发，和4）用64 Cu（或68 Ga）标记的支架进行体内生物分布和正电子发射断层扫描（PET）成像，以评估体内放射诊断和/或对治疗剂的反应。 我们的工作将对医疗保健产生积极影响，因为我们正在开发用于检测和治疗癌症的靶向药物。肽类药物在体内耐受性良好，有助于改善退伍军人的生活。这项研究与BLR&D妇女健康和基因组医学的特殊利益相吻合。

 DESCRIPTION (provided by applicant): Cancer is a leading health problem for United States Veterans. While most cancers are treatable in their earliest forms, gender-related cancers including those of the ovary, breast, and prostate are often fatal when diagnosed at advanced and/or metastatic stages. It is estimated that in 2015, these cancers will account for over 80,000 annual deaths in the general population, with morbidity rates significantly higher in the Veteran population. Many patients initially respon to treatment, but often relapse. Relapse may be due to the heterogeneous nature of the tumor, dysregulation of various signaling pathways, aberrant lectin- carbohydrates, or development of aggressive and drug resistant tumors. The best was to identify and treat these cancers may be through implementation of a more personally tailored diagnostic and/or therapeutic approach in which agents that target multiple biomarkers on cancer cells are employed. However, surprisingly few tumor-associated antigens or biomarkers have been shown to associate with cancer type or stage. Our laboratory has previously utilized bacteriophage (phage) display technology to identify biomarkers and isolate peptide-based targeting molecules that recognize ovarian, breast, and prostate carcinomas. Peptides that target the ErbB-2 and ErbB-3 receptors, carbohydrates (Thomsen-Friedenreich antigen, TF), lectins (galectin- 3, gal-3), and the CD44 splice variant CD44v6, have been obtained. These biomarkers are involved in cell adhesion, angiogenesis, and signaling. TF is expressed in over 90% of all adenocarcinomas, gal-3 is a marker of invasiveness and metastatic potential, ErbB-2 is a marker of aggressive metastasis, ErbB-3 often associates with poor prognosis and treatment resistance, and CD44v6 associates with aggressive cancer initiating (stem) cells (CIC). The ability to detect such biomarkers in vivo will help discern various stages of tumor progression and may help monitor response to therapy. The targeting and imaging of cancer biomarkers has generally relied on the use of radiolabeled antibodies (Abs). Unfortunately, Abs are very large and often cause immunogenic related toxicity and drug resistance. Small peptides are well tolerated in humans but suffer from low tumor uptake, instability, and high kidney retention. Peptides displayed in the context of small scaffolds or single chain Ab fragments (scFv) may provide ideal platforms for development of efficacious tumor targeting agents due to their high affinity, stability, and favorable in vivo pharmacokinetics. It is hypothesized that the phage display selected gal-3/TF, ErbB- 2/ErbB-3, and CD44v6 peptides can be engineered into high affinity, stable, targeting scaffolds to be used in the imaging and response to therapy of ovary, breast, and prostate cancer. The goal of these studies is to develop ErbB-2/ErbB-3 and gal-3/TF-targeting peptides and newly identified CD44v6-targeting peptides into personalized cancer detection or therapy agents. The objectives of this proposed research are to: 1) functionally characterize the peptides for the interruption of signaling pathways and proliferation in vitro and within human breast, ovary, or prostate carcinomas; 2) develop new generation of peptides tailored to radioimaging/therapy applications by: i) synthesis of retro-inverso and multiple antigen peptide constructs, ii) engineering peptides into novel scFv scaffolds, and iii) generating phage display scFv libraries for affinity maturation 3) simultaneous target multiple biomarkers using fluorescence imaging for streamlining the development of cancer detection probes, and 4) perform in vivo biodistribution and positron emission tomography (PET) imaging with 64Cu (or 68Ga)-labeled scaffolds to evaluate as in vivo radiodiagnostic and/or response to therapy agents. Our work will positively impact healthcare in that we are developing targeted agents for the detection and treatment of cancer. The peptide-based agents will be well tolerated in vivo and help improve the lives of our Veterans. This research coincides with BLR&D special interests of Women's Health and Genomic Medicine.