Use of Bacteriophages to Prevent, Diagnose, and Treat Diseases

本项目旨在通过T4噬菌体内外蛋白展示结合，利用哺乳动物内吞途径，创造一种新的给药方法。噬菌体T4被广泛用于蛋白质展示。两种辅助衣壳蛋白，Hoc和Soc，它们对衣壳的形成不是必需的。人们可以将其他非噬菌体蛋白与Hoc或Soc或两者融合，以便在噬菌体头表面显示融合蛋白。这是通过在细菌宿主中培养T4阴性的Hoc或Soc突变体来实现的，这些突变体从质粒中表达Hoc和Soc融合到单链抗体基因中，这些基因已被证明与特定癌细胞结合以展示，在噬菌体成熟过程中导致融合抗体附着在头部颗粒上16。Hoc可以在其N或C端显示，而SOC可以同时在其N和C端显示，从而允许大量显示蛋白质，或者显示蛋白质的变化。T4成熟的头部也包含，除其他外，内部蛋白质称为IPI， IPII和IPIII。后三种蛋白可以通过连接子序列融合以包含外源蛋白，该连接子序列被蛋白gp21通过称为CTS的切割序列切割，使两个被切割的产物游离。这一发现使得12-160kD的多种蛋白质可以融合到CTS中。这个过程发生在噬菌体头体内。在注射DNA的过程中，内部蛋白质全部释放到噬菌体宿主中。外源蛋白注入细菌后具有明显的生物活性。内吞作用。迄今为止，哺乳动物细胞内吞作用的许多途径已被阐明。它们一般分为网格蛋白或小泡依赖途径、巨噬细胞作用、ICAM-1（细胞间粘附分子-1）途径等。其中，ICAM-1途径涉及内吞作用，形成相对较大的囊泡（可达500nm），移动到晚期核内体和回收室，以及溶酶体运输。途径的多样性允许内化货物的降解或内化受体和信号蛋白的再循环回到细胞膜。癌细胞在膜相关蛋白的过度表达方面与正常细胞不同，包括ICAM-1受体（作为转移活性的指标）。这些分子的过度表达使得针对表面蛋白和其他基于亲和力的蛋白的抗体能够特异性地靶向癌症，消灭恶性细胞，同时保持健康细胞相对不受影响。

The purpose of this project is to create a new method of drug delivery to cancer cells through a combination of internal and external protein display on bacteriophage T4 and utilizing mammalian endocytosis pathways. Phage T4 has been used extensively for protein display. The two accessory capsid proteins, Hoc and Soc that are non-essential for capsid formation. One can fuse other non-phage proteins to either Hoc or Soc or both in order to display the fusion protein on the phage head surface. This is achieved by growing mutants of T4 negative for Hoc or Soc in bacterial host that expresses from plasmid(s) HOC and SOC fused to single chain antibody genes that have been shown to bind to specific cancer cells for display, causing attachment of fused antibody to head particle during phage maturation16. Hoc can display at either its N or C terminus, while SOC can display at its N and C termini concurrently, allowing for large numbers of displayed proteins, or variations in the proteins being displayed. The mature head of T4 also contains, among others, internal proteins called IPI, IPII, and IPIII. The latter three proteins can be fused to contain foreign proteins through linker sequence, which is cleaved by protein gp21through the cleavage sequence, termed CTS making the two cleaved products free. This discovery has allowed a variety of proteins from 12-160kD to be fused to the CTS. The process takes place inside the phage head article. The internal proteins are all released into the phage host during DNA injection. The foreign proteins have demonstrable biological activity after injection into the bacteria. Endocytosis. A number of endocytosis pathways in mammalian cells have been elucidated to date. They are generally categorized as clathirin or caveolae dependent pathways, macropinocytosis, ICAM-1 (intercellular adhesion molecule -1) pathway, etc. Of these, the ICAM-1 pathway involves endocytosis with formation of relatively large vesicles (up to 500nm), movement to late endosomes and recycling compartments, as well as lysosomal trafficking. The variety in the pathway allows for degradation of the internalized cargo or recycling of internalized receptors and signaling proteins back to the cell membrane. Cancer cells look different from normal cells in overexpression of membrane associated proteins, including ICAM-1 receptor (as an indicator of metastatic activity). The overexpression of these molecules allows antibodies against the surface proteins and other affinity-based proteins to target cancer specifically, wiping out malignant cells while leaving healthy cells relatively untouched.