Nanobodies have recently been introduced to the field of photodynamic therapy (PDT) as a very promising strategy to target photosensitizers selectively to cancer cells. Nanobodies are known for their characteristic small size (15 kDa), high specificity, and high binding affinities. These features allow rapid accumulation of nanobody-photosensitizer conjugates at the tumor site and rapid clearance of unbound fractions, and thus illumination for activation is possible 1 or 2 h postinjection. Preclinical studies have shown extensive tumor damage after nanobody-targeted PDT . This chapter addresses the first steps toward preparing nanobody-photosensitizer conjugates, which are the nanobody production and purification. The protocol for nanobody production addresses either medium- or large-scale bacterial expression, while the nanobody purification is described for two main strategies: affinity chromatography and ion-exchange chromatography. For the first strategy, protocols are described for different affinity tags and purification from either medium-scale or large-scale productions. For the second strategy, the protocol given is for purification from a large-scale production.
Keywords: Affinity chromatography; Ion-exchange chromatography; Nanobody production; Nanobody purification.
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