Main objective of the SPOT Action was the development of active tumor targeting platforms based on nanoparticles, for bioimaging as well as for drug delivering applications. Despite the excessive research performed in the field of nanomedicine, the application of nanoparticles in clinics falls still well short of their expectations with only a very small number of FDA approved approaches. An active targeting (receptor-mediated targeting) of tumors using NP-based formulations is currently not approved by the FDA, neither for bioimaging nor drug-delivering purposes. Since antibodies (Ab) are currently the most potent compounds for active targeting, a possibility to overcome this current lack of clinical translation in nanomedicine could be the combination of cancer-specific Ab with NP based imaging or drug-delivering vectors. Direct conjugation of the Abs to the NPs showed to be not an optimal option, resulting in many cases in a similar or in an even lower tumor accumulation compared to a passive accumulation of the non-functionalized NPs in comparison to their Ab functionalized counterparts. A possible explanation is the drastic increase in the hydrodynamic diameter of the NP size once they are functionalized with Abs which then affects their distribution or EPR based tumor uptake. The second explanation is the enhanced recognition of Ab-functionalized NPs by the reticuloendothelial system (RES). In fact, some reports suggest that properly shielded NPs can feature longer circulation times compared to Ab functionalized NPs and result in a higher tumor accumulation compared to those functionalized with targeting Abs.
A possible solution that is showing promising results in primary in vitro as well as in vivo tests is the so-called pretargeted imaging or pretargeted drug delivery which has enormous potential in clinical applications and was thus thoroughly studied during the SPOT Action. In pretargeted approaches, an active targeting molecule, such as an Ab or a peptide, is previously injected into an organism resulting in an abundant accumulation at the targeted site. After its accumulation, an imaging probe or a drug-delivering system, which is known to be well shielded from the RES and thus has a long circulation time, is subsequently injected. Both parts, the targeting molecule as well as the subsequently injected nanoparticle, expose reactive functional groups which can undergo a covalent conjugation within the living organism. The applied reactions must be highly bioorthogonal as well as fast proceeding and high yielding, even at low concentrations and in presence of complex biological media. In this vein, different types of click reactions have been developed which are suitable for in vitro and even in vivo applications. Most prominent click reaction for in vivo applications is the reaction between trans-cyclooctene (TCO) with tetrazines, reaching more suitable second order reaction rate constants. Despite the broad application of TCO and tetrazines, a major disadvantage is the instability of TCO which can undergo isomerization to the more stable but almost unreactive cis-cyclooctene (CCO) isomer. Most suitable alternative, reaching reaction rates in a similar order but providing better stability, is the reaction between chlorosydnone (Cl-Syd) derivatives and DBCO or 3,3,6,6-tetramethylthiacycloheptyne (TMTH). Aiming to establish a more stable alternative to the commonly used click pair of tetrazine and TCO in pretargeted approaches, a thorough investigation of the Cl-Syd and DBCO pair was done during the SPOT Action.
As targeting moieties, Abs were aimed to be used since they are the state-of-the-art for tissue recognition. However, not only on the side of the nanoparticles but also for the Ab targeting molecules, tumor penetration was aimed to be enhanced. To overcome size-related drawbacks of intact Abs for active tumor targeting, small but multivalent Ab-fragments, which fall in the “ideal tumor targeting zone” were proposed in the SPOT Action. F(ab)2 fragments are significantly smaller compared to their intact counterparts but still maintain avidity.
