Use of Bacteriophages to Prevent, Diagnose, and Treat Diseases

该项目的目的是通过在噬菌体T4上结合内外蛋白展示和利用哺乳动物的内吞作用途径来创造一种新的给药方法。噬菌体T4已被广泛用于蛋白质展示。衣壳蛋白两种辅助衣壳蛋白，对衣壳的形成不是必需的。为了在噬菌体头部表面展示融合蛋白，人们可以将其他非噬菌体蛋白融合到60C或SOC上，或者两者都融合。这是通过在细菌宿主中培养HOC或SOC的T4阴性突变体来实现的，该突变体从(S)HOC和SOC融合到单链抗体基因中表达，这些单链抗体基因已被证明与特定的癌细胞结合用于展示，导致融合抗体在噬菌体成熟期间附着在头部颗粒上。HOC可以在其N端或C端显示，而SOC可以同时在其N端和C端显示，从而允许大量显示的蛋白质或所显示的蛋白质的变化。除其他外，T4成熟头部还含有称为IPI、IPII和IPIII的内部蛋白质。后三种蛋白可以通过连接序列融合成含有外源蛋白的外源蛋白，该连接序列被蛋白gp21通过裂解序列切割，称为CTS，使两种裂解产物游离。这一发现使12-160kD的各种蛋白质能够与CTS融合。这一过程发生在噬菌体头部物品内部。在DNA注射过程中，体内的蛋白质全部释放到噬菌体宿主体内。外源蛋白注入细菌后具有明显的生物活性。内吞作用。到目前为止，哺乳动物细胞中的许多内吞途径已经被阐明。它们通常被归类为依赖于小泡或小泡的途径、巨噬细胞吞噬作用、ICAM-1(细胞间黏附分子-1)途径等。其中，ICAM-1途径涉及内吞作用，形成相对较大的小泡(长达500 nm)，运动到较晚的内小体和回收隔室，以及溶酶体运输。该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看起来与正常细胞不同，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针对癌症，在清除恶性细胞的同时相对保持健康细胞不受影响。

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.