The size, well-defined structure, and relatively high folding energies of most proteins allow them to recognize disease-relevant receptors that present a challenge to small molecule reagents. While multiple challenges must be overcome in order to fully exploit the use of protein reagents in basic research and medicine, perhaps the greatest challenge is their intracellular delivery to a particular diseased cell. Here, we describe the genetic and enzymatic manipulation of prostate cancer cell-penetrating M13 bacteriophage to generate nanocarriers for the intracellular delivery of functional exogenous proteins to a human prostate cancer cell line.
We report a rationally designed nanobody activation immunotherapeutic that selectively redirects anti-dinitrophenyl (anti-DNP) antibodies to the surface of HER2-positive breast cancer cells, resulting in their targeted destruction by antibody-dependent cellular cytotoxicity. Since nanobodies are relatively easy to express, stable, can be humanized, and can be evolved to potently and selectively bind virtually any disease-relevant cell surface receptor, we anticipate broad utility of this therapeutic strategy.
Protein transduction domains (PTDs) are reagents that facilitate the delivery of diverse cargo to the interior of mammalian cells. We identified a PTD called "Ypep" (N-YTFGLKTSFNVQ-C), with cell penetration selectivity and potency profiles that are tightly controlled by multivalency effects. Pentavalent display of Ypep on M13 bacteriophage enables selective uptake of this phage in PC-3 human prostate cancer cells at low picomolar concentration and in the presence of human blood. All Ypep-dependent delivery is nontoxic and proceeds through energy-dependent endocytosis. Collectively, our results establish Ypep-displaying phage as a cell-penetrating platform with selectivity and potency profiles that compare to, or exceed, antibodies and their fragments. Our findings may have broader implications on the design of PTD technologies generated from phage display, as well as the use of Ypep-displaying phage as a prostate cancer cell-selective delivery platform.
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