The display of an anti-CS1 nanobody by small extracellular vesicles does not improve disease targeting in multiple myeloma

Introduction: Multiple myeloma (MM) is an incurable plasma cell cancer predominantly residing in the bone marrow (BM). Due to inevitable refractory disease, novel therapeutic options are needed. Small extracellular vesicles (sEVs) are promising therapeutic cargo delivery vehicles due to their high biocompatibility and ability to traverse biological barriers. This study aims to engineer HEK293-derived sEVs to display a nanobody (Nb) targeting CS1, a well-established surface marker of MM cells, to increase sEV specificity to MM-associated organs and MM cells.
Methods: HEK293 cells were stably transfected with Nb-SDC1CTF fusion proteins, linked with the juxtamembranal domain of CD4 to prevent membrane cleavage. Western blot and confocal microscopy confirmed sEVs sorting and correct cell membrane topology. Control sEVs without Nb and with an irrelevant Nb were included. Binding of anti-CS1 Nb-displaying sEVs to CS1 was evaluated by incubating sEVs with soluble CS1 and analyzing size-exclusion chromatography (SEC) fractions for co-elution of CS1 with sEVs by western blot. To evaluate the effect of the anti-CS1 Nb on sEV biodistribution, DiR-labeled sEVs were intravenously injected in 5T33MM mice. After 24h, organs were imaged with a Fluobeam800 camera. MM cell-specificity was determined by measuring DiR fluorescence by flow cytometry.
Results: Cleavage-resistant constructs were expressed with the correct topology and were highly enriched in sEVs. Both human and murine anti-CS1 Nb-displaying sEVs showed binding to soluble forms of CS1. Mononuclear cells isolated from spleen, spine and legs showed no enhanced sEV specificity and/or selectivity towards MM cells. Interestingly, in myeloma-bearing mice, anti-CS1 Nb display increased sEV accumulation in liver and lungs.
Conclusion: Anti-CS1 Nbs were successfully displayed on the sEV surface. While these EVs efficiently bind soluble forms of CS1, they do not improve MM targeting in vivo. Further work will explore suborgan distribution of sEVs in the lungs and expand the flow cytometric panel to include immune cell populations.