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.

Development of fine-tuned top-down mass spectrometry strategies in the chromatographic time scale (LC-TD-MS) for the complete characterization of an anti EGFR single domain antibody-drug conjugate (sdADC)

“…For all these reasons, sdAbs have found their way into different applications such as infectious diseases, 18 cancer therapy, 19 and central nervous system (CNS) disorders. 20 Akin to mAbs, nanobodies can be subjected to covalent modifications allowing incorporation of cargo molecules with different properties allowing the use of conjugated nanobodies as drug carriers 21 (single domain antibody-drug conjugate, sdADC) or imaging probes. 22 Similar to other biotherapeutic proteins, the primary structure of sdAbs, and sdADCs has to be thoroughly assessed prior to their commercialization including sequence assessment, identification of post-translational modifications, and the precise localization of the position of the cargo molecules (for conjugated formats) through the development of tailored analytical methods.…”

In Vitro and In Silico Evaluation of Antiproliferative Activity of New Isoxazolidine Derivatives Targeting EGFR: Design, Synthesis, Cell Cycle Analysis, and Apoptotic Inducers

“…Current ineffective treatments against cancer including surgery, immunosuppression chemotherapy, and radiotherapy have many adverse effects and high drug resistance accompanied with high rates of incidence, mortality, and morbidity [ 8 , 9 ]. In contrast, chimeric antigen receptor T (CAR-T) cells and antibody–drug conjugates (ADCs) currently remain the most popular immunotherapies strategies in oncology for anticancer drug development [ 10 , 11 ]. Therefore, there is a need to discover and develop a new and effective anticancer agent with selectivity to kill this anarchic development of cells or inhibit their proliferation with fewer side effects.…”