Developing an Anti-sialyl-Lewisa Diabody for ImmunoPET Imaging of Pancreas Cancer

描述（由申请人提供）：该研究项目的目标是开发和应用模块化策略来开发和应用正电子发射断层扫描（PET）显像剂，用于胰腺腺癌（PDAC）的诊断和分期，胰腺腺癌是全国第四大癌症死亡原因。目前的诊断方法缺乏特异性和敏感性来有效地识别PDAC病变，而缺乏适当的诊断工具导致PDAC的高死亡率，这代表了一个重要的未满足的医疗需求。在PET成像中使用抗体是实现高灵敏度和选择性的一种经过验证的方法，因此，开发一种PDAC诊断工具是一种合乎逻辑的选择，可以克服这些缺点。5B1是一种针对PDAC肿瘤细胞表面表达的一种抗原——唾液Lewisa （sLea）的全人抗体，在初步研究中显示出作为免疫pet显像剂的前景。然而，与全长抗体片段相比，抗体片段作为显像剂具有一定的优势。利用抗体工程技术，MabVax Therapeutics公司开发了由全人5B1抗体设计的糖尿病体（5B1Db），该抗体将使用模块化放射性标记策略开发为PET显像剂。为了确定最有希望用于最终临床转化的候选药物，将应用两种策略：直接方法和预靶向方法将在BxPC3胰腺癌细胞诱导的SCID小鼠中进行评估，以及在胰腺肿瘤中表达sLea的基因工程小鼠模型。在这两种方法中，将一系列放射性核素（18F和64Cu）偶联到5B1Db上，以找到放射性核素和标记策略的最佳组合，以补充5B1Db的药代动力学、药效学和生物分布特性。我们将通过PET成像比较5B1Db组合的肿瘤摄取和生物分布，以确定一种最佳候选药物，用于进一步的临床前开发。在第一种方法中，5B1Db结构体将与螯合剂结合，在PET成像中使用之前可以立即进行放射性标记。在第二种方法中，放射性标记将在体内实现，使用螯合物共轭的反式环烯（TCO）和四嗪（Tz）共轭的5B1Db之间的逆[4+2]环加成反应。我们的预靶向方法包括四个步骤：i)将5B1-Tz片段注射到血液中；Ii)抗体片段在肿瘤中积累，并随之从血液中清除；iii)将放射性标记的TCO偶联物注射到血液中；iv)放射性标记的TCO缀合物与5B1-Tz片段结合，随后快速清除未结合的放射性物质。这两种方法将提供确定进一步开发的构造的最佳机会。

DESCRIPTION (provided by applicant): The goal of the proposed research project is to develop and apply modular strategies to the development and application of positron emission tomography (PET) imaging agents for the diagnosis and staging of pancreatic adenocarcinoma (PDAC), which ranks as the fourth leading cause of cancer mortality nationally. Current diagnostic methodology lacks the specificity and sensitivity to efficiently identify PDAC lesions, and this lack of adequate diagnostic tools contributes to the high mortality of PDAC, representing a significant unmet medical need. The use of antibodies in PET imaging is a proven method for achieving high sensitivity and selectivity, and, thus, is a logical choice for th development of a diagnostic tool for PDAC that overcomes those shortfalls. 5B1, a fully-human antibody that targets an sialyl Lewisa (sLea), an antigen that is expressed on the surface of PDAC tumor cells, has shown promise in initial studies as an immunoPET imaging agent. However, antibody fragments can offer certain advantages as imaging agents, compared to their full-length counterparts. Using antibody engineering, diabodies (5B1Db) engineered from the fully-human 5B1 antibody have been developed by MabVax Therapeutics, which will be developed as a PET imaging agent using a modular radiolabeling strategy. Two strategies will be applied in order to identify the most promising candidate for eventual clinical translation: a direct approach and a pre-targeting approach will be assessed in SCID mice with tumors induced by BxPC3 pancreas cancer cells as well as a genetically engineered mouse model expressing sLea in pancreatic tumors. In both approaches, series of radionuclides (18F and 64Cu) will be conjugated to 5B1Db in order to find the best combination of radionuclide and labeling strategy that complement the pharmacokinetic, pharmacodynamics, and biodistribution properties of 5B1Db. We will compare tumor uptake and biodistribution of the combinations of 5B1Db via PET imaging in order to identify one optimal candidate(s) for further pre-clinical development. In the first approach, 5B1Db constructs will be conjugated to chelators that may be radiolabeled immediately prior to use in PET imaging. In the second approach, radiolabeling will be achieved in vivo, using the inverse [4+2] cycloaddition reaction between a chelator-conjugated trans-cylcooctene (TCO) and a tetrazine (Tz)-conjugated 5B1Db. Our pre-targeted method will involve four steps: i) the injection into the bloodstream of 5B1-Tz fragment; ii) accumulation of the antibody fragment in the tumor and concomitant clearance from the blood; iii) the injection into the bloodstream of the radiolabeled TCO conjugate; and iv) the binding of the radiolabeled TCO conjugate to the 5B1-Tz fragment followed by the rapid clearance of unbound radioactive material. These two approaches will provide the best chance of identifying a construct for further development.