Metabolite Identification of Therapeutic Peptides and Proteins by Top-down Differential Mass Spectrometry and Metabolite Database Matching.

As metabolism impacts the efficacy and safety of therapeutic peptides and proteins (TPPs), understanding of the metabolic fate of TPPs is critical for their preclinical and clinical development. Despite the continued increase of new TPPs entering clinical trials, the metabolite identification (MetID) of these emerging modalities remains challenging. In the present study, we report an analytical workflow for MetID of TPPs. Using insulin detemir as an example, we demonstrated that top-down differential mass spectrometry (dMS) was able to distinguish and discover metabolites from complex biological matrices. For structural interpretation, we developed an algorithm to generate a complete and nonredundant theoretical metabolite database for a TPP of any topology (e.g., branched, multicyclic, etc.). Candidate structures of a metabolite were obtained by matching the monoisotopic mass of a dMS feature to the theoretical metabolite database. Finally, the MS/MS sequence tags enabled unambiguous characterization of metabolite structures when isobaric/isomeric candidates were present. This platform is widely applicable to TPPs with complex structures and will ultimately guide the structural optimization of TPPs in pharmaceutical development.

Development of Anti-CD74 Antibody-Drug Conjugates to Target Glucocorticoids to Immune Cells.

Glucocorticoids (GCs) are excellent anti-inflammatory drugs but are dose-limited by on-target toxicity. We sought to solve this problem by delivering GCs to immune cells with antibody-drug conjugates (ADCs) using antibodies containing site-specific incorporation of a non-natural amino acid, novel linker chemistry for in vitro and in vivo stability, and existing and novel glucocorticoid receptor (GR) agonists as payloads. We directed fluticasone propionate to human antigen-presenting immune cells to afford GR activation that was dependent on the targeted antigen. However, mechanism of action studies pointed to accumulation of free payload in the tissue culture supernatant as the dominant driver of activity and indeed administration of the ADC to human CD74 transgenic mice failed to activate GR target genes in splenic B cells. Suspecting dissipation of released payload, we designed an ADC bearing a novel GR agonist payload with reduced permeability which afforded cell-intrinsic activity in human B cells. Our work shows that antibody-targeting offers significant potential for rescuing existing and new dose-limited drugs outside the field of oncology.

Discovery of Pyrophosphate Diesters as Tunable, Soluble, and Bioorthogonal Linkers for Site-Specific Antibody-Drug Conjugates.

As part of an effort to examine the utility of antibody-drug conjugates (ADCs) beyond oncology indications, a novel pyrophosphate ester linker was discovered to enable the targeted delivery of glucocorticoids. As small molecules, these highly soluble phosphate ester drug linkers were found to have ideal orthogonal properties: robust plasma stability coupled with rapid release of payload in a lysosomal environment. Building upon these findings, site-specific ADCs were made between this drug linker combination and an antibody against human CD70, a receptor specifically expressed in immune cells but also found aberrantly expressed in multiple human carcinomas. Full characterization of these ADCs enabled procession to in vitro proof of concept, wherein ADCs 1-22 and 1-37 were demonstrated to afford potent, targeted delivery of glucocorticoids to a representative cell line, as measured by changes in glucocorticoid receptor-mediated gene mRNA levels. These activities were found to be antibody-, linker-, and payload-dependent. Preliminary mechanistic studies support the notion that lysosomal trafficking and enzymatic linker cleavage are required for activity and that the utility for the pyrophosphate linker may be general for internalizing ADCs as well as other targeted delivery platforms.