Multivalent Nanophage Engineered as Dual Receptor Cancer Theranostic Agents.

描述(申请人提供)：几乎所有癌症在最初的形式都是可以治疗的，但一旦发生转移，往往是致命的。癌症诊断和治疗的改进将来自一种更直接的方法，在这种方法中，针对肿瘤的特异性或过度表达的抗原是有针对性的。尽管针对单一抗原的治疗已被证明是适度成功的，但较差的特异性和亲和力，加上耐药性，限制了它们的使用。目标是 这项研究的目的是开发一种基于噬菌体的平台，用于靶向多种癌症抗原，而不考虑空间或生物相关性。双重靶向的优点包括通过降低同时解离率来增加亲和力，以及通过多抗原结合来增加选择性。许多受体已被证明在肿瘤发生中发挥关键作用，但被证明很难靶向。例如，ErbB2和ErbB3受体在早期和耐药癌中都存在，但在正常组织中不存在或低水平存在。尽管受体可以异二聚化，但大多数受体不能也不足以被当前的异价平台靶向，例如双特异性抗体构建。噬菌体提供了一种跨越数百纳米的支架，并展示了多个靶向多肽副本，这些多肽可用于结合抗原，而不考虑相互作用或分离。假设在病毒粒子的相对末端展示目标多肽的噬菌体可以同时结合多个肿瘤抗原(如ERB2和Erb3)，而不考虑它们的接近或相互作用状态。这种异多价(HMV)噬菌体被设计成合适的长度，将被用于一种新的、放大的三步预靶向方法。截短的、生物素化的HMV噬菌体将被注射用于体内肿瘤靶向。噬菌体清除后，荧光链霉亲和素包裹的二氧化硅纳米颗粒将被用于靶向肿瘤结合噬菌体上的多个生物素。这种纳米粒子表现出快速的血液清除，并将允许多模式成像和第二水平的信号放大。1，4，7，10-四氮杂环十二烷-1，4，7，10-四氮杂十二烷-1，4，7，10-四乙酸(DOTA)螯合放射性核素(177Lu)的生物素除了提供高剂量的肿瘤局部β辐射外，还将提供高水平的治疗试剂，可同时用于单光子发射计算机断层扫描(SPECT)成像。其具体目的包括：1)利用基因工程技术获得显示ErbB2和ErbB3靶向多肽的离散长度(50-750 nm)的杂多价(HMV)纳米噬菌体，2)鉴定HMV纳米噬菌体对ErbB2/ErbB3的亲和力、特异性和亲和力，以及3)在ErbB2+/ErbB3+BT-474人乳腺癌移植瘤小鼠中用扩增的三步预靶向方法表征纳米噬菌体的肿瘤靶向性和药代动力学。这些噬菌体将产生一个新的成像和治疗平台，具有高亲和力、选择性和最佳药代动力学。此外，该噬菌体可以被设计成展示针对任何所选抗原(S)的靶向肽或抗体，作为一种新型的治疗药物提供了极大的通用性。

DESCRIPTION (provided by applicant): Nearly all cancers are treatable in their earliest forms, but often fatal once metastasis has occurred. Improved diagnosis and therapy of cancer will result from a more directed approach in which antigens specific to or over expressed on tumors are targeted. Although therapies targeted to a single antigen have proven moderately successful, poor specificity and affinity, coupled with resistance have limited their use. The goal of this research is to develop a bacteriophage (phage)-based platform for targeting multiple cancer antigens regardless of spatial or biological relatedness. The advantage to dual targeting includes both increased avidity through decreased simultaneous dissociation rates, and increased selectivity through multiple antigen binding. Numerous receptors have been shown to play key roles in tumorigenesis but have proven difficult to target. For example, the ErbB2 and ErbB3 receptors are both present in early stage and resistant carcinomas, but absent or present at low levels in normal tissues. Although the receptors can heterodimerize, the majority do not and are not close enough for targeting by current heterovalent platforms, such as bispecific antibody constructs. Phage provide a scaffold that spans hundreds of nanometers and display multiple copies of targeting peptides, which can be utilized to bind antigens regardless of interaction or separation. It is hypothesized that phage displaying targeted peptides at opposite tips of the virion can simultaneously bind multiple cancer antigens (such as Erb2 and Erb3) regardless of their proximity or interaction status. Such heteromultivalent (HMV) phage, engineered to appropriate lengths, will be employed in a novel, amplified 3-step pretargeting approach. Truncated, biotinylated HMV phage will be injected for in vivo tumor targeting. Following phage clearance, fluorescent streptavidin-coated silica nanoparticles will be used to target the multiple biotins on tumor-bound phage. The nanoparticles have demonstrated rapid blood clearance and will allow for multimodal imaging and a second level of signal amplification. Biotin conjugated to the 1,4,7,10- tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA)-chelated radionuclide [177Lu] lutetium will deliver high levels of a theranostic agent that can be simultaneously used for single photon emission computed tomography (SPECT) imaging in addition to providing a high dose of tumor-localized beta radiation. The specific aims include: 1) genetically engineer heteromultivalent (HMV) nanophage of discrete lengths (50-750 nm) displaying ErbB2 and ErbB3 targeting peptides, 2) characterize HMV nanophage affinity, specificity, and avidity to ErbB2/ErbB3, and 3) characterize the tumor targeting and pharmacokinetics of the nanophage using the amplified three-step pretargeting approach in ErbB2+/ErbB3+ BT-474 human breast cancer xenografted mice. These phage will yield a new imaging and therapeutic platform with high affinity, selectivity and optimum pharmacokinetics. Furthermore, the phage can be engineered to display targeting peptides or antibodies for any antigen(s) of choice, providing great versatility as a novel theranostic agent.