Targeting of phage particles towards endothelial cells by antibodies selected through a multi-parameter selection strategy

Abstract

One of the hallmarks of cancer is sustained angiogenesis. Here, normal endothelial cells are activated, and their formation of new blood vessels leads to continued tumour growth. An improved patient condition is often observed when angiogenesis is prevented or normalized through targeting of these genomically stable endothelial cells. However, intracellular targets constitute a challenge in therapy, as the agents modulating these targets have to be delivered and internalized specifically to the endothelial cells. Selection of antibodies binding specifically to certain cell types is well established. It is nonetheless a challenge to ensure that the binding of antibodies to the target cell will mediate internalization. Previously selection of such antibodies has been performed targeting cancer cell lines; most often using either monovalent display or polyvalent display. In this article, we describe selections that isolate internalizing antibodies by sequential combining monovalent and polyvalent display using two types of helper phages, one which increases display valence and one which reduces background. One of the selected antibodies was found to mediate internalization into human endothelial cells, although our results confirms that the single stranded nature of the DNA packaged into phage particles may limit applications aimed at targeting nucleic acids in mammalian cells.

Lytic bacteriophages interact with respiratory epithelial cells and induce the secretion of antiviral and proinflammatory cytokines

“…Phage identity was confirmed by PCR using phage-specific primers (Table S1). Phage particle concentration was obtained by measuring absorbance at 269 and 320 nm and calculated using the (A 269 -A 320 ) x 6 x 10 16 / number of bases per virion, as described (71). Preparations were tested for endotoxins using the Limulus amoebocyte lysate test (ThermoFisher Scientific) and bacterial DNA by amplification with 16S rRNA gene primers (72).…”

Lytic bacteriophages induce the secretion of antiviral and proinflammatory cytokines from human respiratory epithelial cells

“…Phage identity was confirmed by PCR using phage-specific primers ( S1 Table ). Phage particle concentration was obtained by measuring absorbance at 269 and 320 nm and calculated using the (A269—A 320 ) × 6 × 10 16 /number of bases per virion, as described [ 76 ]. Preparations were tested for endotoxins using the Limulus amoebocyte lysate test (Thermo Fisher Scientific) and bacterial DNA by amplification with 16S rRNA gene primers [ 77 ].…”

Development of a new affinity maturation protocol for the construction of an internalizing anti-nucleolin antibody library

“…Nucleolin’s full length tertiary structure is yet to be fully characterized 39 , which hinders the use of the isolated protein for panning. In this regard, whole cell-based phage display has the advantage of presenting proteins in their native conformation and level of expression at the cell surface 40 , takes into account interaction with neighboring proteins, while adding the possibility of selecting both internalizing 41 – 44 or non-internalizing agents 45 – 48 . Screening by phage internalization is an attractive strategy to select antibodies against intracellular targets, or antibodies that act as intracellular carriers, such as antibody–drug conjugates.…”