ValCitGlyPro-dexamethasone antibody conjugates selectively suppress the activation of human monocytes.

Glucocorticoids (GCs) are effective in treating autoimmune and inflammatory disorders but come with significant side effects, many of which are mediated by non-immunological cells. Therefore, there is rapidly growing interest in using antibody drug conjugate (ADC) technology to deliver GCs specifically to immune cells, thereby minimizing off-target side effects. Herein, we report the study of anti-CD11a, anti-CD38, and anti-TNFα ADCs to deliver dexamethasone to monocytes. We found that anti-CD11a and anti-CD38 were rapidly internalized by monocytes, while uptake of anti-TNFα depended on pre-activation with LPS. Using these antibodies were attached to a novel linker system, ValCitGlyPro-Dex (VCGP-Dex), that efficiently released dexamethasone upon lysosomal catabolism. This linker relies on lysosomal cathepsins to cleave after the ValCit sequence, thereby releasing a GlyPro-Dex species that undergoes rapid self-immolation to form dexamethasone. The resulting monocyte-targeting ADCs bearing this linker payload effectively suppressed LPS-induced NFκB activation and cytokine release in both a monocytic cell line (THP1) and in human PBMCs. Anti-TNFα_VCGP-Dex and anti-CD38_VCGP-Dex were particularly effective, suppressing ∼60-80% of LPS-induced IL-6 release from PBMCs at 3-10 µg mL-1 concentrations. In contrast, the corresponding isotype control ADC (anti-RSV) and the corresponding naked antibodies (anti-CD38 and anti-TNFα) resulted in only modest suppression (0-30%) of LPS-induced IL-6. Taken together, these results provide further evidence of the ability of glucocorticoid-ADCs to selectively suppress immune responses, and highlight the potential of two targets (CD38 and TNFα) for the development of novel immune-suppressing ADCs.

Synthesis and Optimization of 1-Substituted Imidazo[4,5-c]quinoline TLR7 Agonists.

TLR7 agonists have significant therapeutic potential in a variety of oncology and autoimmune applications. We recently reported a potent TLR7 selective agonist 1 that could be delivered by antibody-drug conjugate (ADC) technology to elicit potent anticancer activity. Herein we report synthetic chemistry and structure-activity relationship studies to develop TLR7 agonists with improved potency for next-generation ADC efforts. We found that the addition of hydrophobic acyl tails to parent compound 1 generally resulted in retained or improved TLR7 agonist activity without sacrificing the permeability or the selectivity over TLR8. In contrast, the addition of a simple alkyl tail at the same position resulted in a dramatic loss in potency. Molecular modeling was performed to provide a rationale for this dramatic loss in potency. We ultimately identified compounds 17b, 16b, and 16d as highly potent TLR7 agonists that potently induced the activation of mouse macrophages and hPBMCs at low-nanomolar concentrations.

Evaluation of an ester-linked immunosuppressive payload: A case study in understanding the stability and cleavability of ester-containing ADC linkers.

In spite of their value in prodrug applications, the use of esters in antibody-drug-conjugate (ADC) payloads and linkers has generally been avoided due to the ubiquitous and promiscuous nature of human esterases. ADCs generally have a long circulating half life (3-7 days) that makes them susceptible to esterase-mediated metabolism. Moreover, it is largely unclear whether lysosomal and cytosolic esterases cleave ester-containing linkers upon ADC internalization. Due to our interest in the targeted delivery of immune-modulators, our team has recently prepared a series of ester-linked dexamethasone ADCs. Herein, we report our studies of the functional activity of these ADCs, with a particular focus on their catabolism in various biological milieu. We found that esters are selectively but inefficiently cleaved upon cellular uptake, likely by cytosolic esterases. Lysosomal catabolism studies indicate that, in spite of the strong proteolytic activity, very little cleavage of ester-containing linkers occurs in the lysosome. However, ADCs bearing the ester-linked payloads are active in various immune-suppressive assays, suggesting that cytosolic cleavage is taking place. This was confirmed through LCMS quantitation of the payload following cell lysis. Finally, the stability of the ester linkage was evaluated in mouse and human plasma. We found, similar to other reports, there is a significant site-dependence on the cleavage. Esters attached at highly exposed sites, such as 443C, were rapidly cleaved in plasma while esters at more hindered sites, such at 334C, were not. Together, these results help to unravel the complexities of ester-incorporation into ADC linkers and pave a path forward for their utility in ADC applications.