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Single Domain Antibodies as Carriers for Intracellular Drug Delivery: A Proof of Principle Study.

ABSTRACT: Antibody-drug conjugates (ADCs) are currently used for the targeted delivery of drugs to diseased cells, but intracellular drug delivery and therefore efficacy may be suboptimal because of the large size, slow internalization and ineffective intracellular trafficking of the antibody. Using a phage display method selecting internalizing phages only, we developed internalizing single domain antibodies (sdAbs) with high binding affinity to rat PDGFRβ, a receptor involved in different types of diseases. We demonstrate that these constructs have different characteristics with respect to internalization rates but all traffic to lysosomes. To compare their efficacy in targeted drug delivery, we conjugated the sdAbs to a cytotoxic drug. The conjugates showed improved cytotoxicity correlating to their internalization speed. The efficacy of the conjugates was inhibited in the presence of vacuolin-1, an inhibitor of lysosomal maturation, suggesting lysosomal trafficking is needed for efficient drug release. In conclusion, sdAb constructs with different internalization rates can be designed against the same target, and sdAbs with a high internalization rate induce more cell killing than sdAbs with a lower internalization rate in vitro. Even though the overall efficacy should also be tested in vivo, sdAbs are particularly interesting formats to be explored to obtain different internalization rates.

SUBMITTER: Pronk SD

PROVIDER: S-EPMC8301836 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Assessment of microcirculatory functional capacity is considered to be of prime importance for therapy guidance and outcome prediction in critically ill intensive care patients. Here, we show determination of skin microcirculatory oxygen delivery and consumption rates to be a feasible approach at the patient's bedside. Real time laser-doppler flowmetry (LDF) and white light spectrophotometry (WLS) were used for assessment of thenar skin microperfusion, regional Hb and postcapillary venous oxygen saturation before and after forearm ischemia. Adapted Fick's principle equations allowed for calculation of microcirculatory oxygen delivery and uptake. Patient groups with expected different microcirculatory status were compared [control (n = 20), sepsis-1/2 definition criteria identified SIRS (n = 10) and septic shock patients (n = 20), and the latter group further classified according to sepsis-3 definition criteria in sepsis (n = 10) and septic shock (n = 10)], respectively. In otherwise healthy controls, microcirculatory oxygen delivery and uptake approximately doubled after ischemia with maximum values (mDO2max and mVO2max) significantly lower in SIRS or septic patient groups, respectively. Scatter plots of mVO2max and mDO2max values defined a region of unphysiological low values not observed in control but in critically ill patients with the percentage of dots within this region being highest in septic shock patients. LDF and WLS combined with vasoocclusive testing reveals significant differences in microcirculatory oxygen delivery and uptake capacity between control and critically ill patients. As a clinically feasible technique for bedside determination of microcirculatory oxygen delivery and uptake, LDF and WLS combined with vasoocclusive testing holds promise for monitoring of disease progression and/or guidance of therapy at the microcirculatory level to be tested in further clinical trials.ClinicalTrials.gov: NCT01530932.

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Single Domain Antibodies as Carriers for Intracellular Drug Delivery: A Proof of Principle Study.

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