Biology subjects

Barfield, R. M.

Publications and source records attributed to Barfield, R. M..

3 recordsLinked to original sources

bioRxiv2021

Tandem-Cleavage Linkers Improve the In Vivo Stability and Tolerability of Antibody-Drug Conjugates

Although peptide motifs represent the majority of cleavable linkers used in clinical-stage antibody-drug conjugates (ADCs), the sequences are often sensitive to cleavage by extracellular enzymes, such as elastase, leading to systemic release of the cytotoxic payload. This action reduces the therapeutic index by causing off-target toxicities that can be dose-limiting. For example, a common side-effect of ADCs made using peptide-cleavable linkers is myelosuppression, including neutropenia. Only a few reports describe methods for optimizing peptide linkers to maintain efficient and potent tumor payload delivery while enhancing circulating stability. Herein, we address these critical limitations through the development of a tandem-cleavage linker strategy, where two sequential enzymatic cleavage events mediate payload release. We prepared dipeptides that are protected from degradation in the circulation by a sterically-encumbering glucuronide moiety. Upon ADC internalization and lysosomal degradation, the monosaccharide is removed and the exposed dipeptide is degraded, liberating the attached payload inside the target cell. We used CD79b-targeted monomethyl auristatin E (MMAE) conjugates as our model system, and compared the stability, efficacy, and tolerability of ADCs made with tandem-cleavage linkers to ADCs made using standard technology with the vedotin linker. The results--where rat studies showed dramatically improved tolerability in the hematopoietic compartment--highlight the role that linker stability plays in efficacy and tolerability, and offer a means of improving an ADCs therapeutic index for improved patient outcomes.

pharmacology and toxicology↗

bioRxiv2020

A novel HER2-targeted antibody-drug conjugate offers the possibility of clinical dosing at trastuzumab-equivalent exposure levels

Trastuzumab and the related antibody-drug conjugate (ADC), ado-trastuzumab emtansine (T-DM1), both target HER2-overexpressing cells. Together, these drugs have treatment indications in both early-stage and metastatic settings for HER2+ breast cancer. T-DM1 retains the antibody functionalities of trastuzumab and adds the potency of a cytotoxic maytansine payload. Interestingly, in the clinic, T-DM1 cannot always replace the use of trastuzumab plus chemotherapy administered together as single agents. We hypothesize that this failure may be due in part to the limited systemic exposure achieved by T-DM1 relative to trastuzumab because of toxicity-related dosing constraints on the ADC. We have developed a trastuzumab-based ADC site-specifically conjugated to maytansine through a noncleavable linker. This construct, termed CAT-01-106, has a drug-to-antibody ratio (DAR) of 1.8, approximately half the average DAR of T-DM1, which comprises a mixture of antibodies variously conjugated with DARs ranging from 0-8. The high DAR species present in T-DM1 contribute to its toxicity and limit its clinical dose. CAT-01-106 showed superior in vivo efficacy compared to T-DM1 at equal payload dosing and was equally or better tolerated compared to T-DM1 at equal payload dosing up to 120 mg/kg in Sprague-Dawley rats and 60 mg/kg in cynomolgus monkeys. CAT-01-106 also showed improved pharmacokinetics in rats relative to T-DM1, with 40% higher ADC exposure levels. Together, the data suggest that CAT-01-106 may be sufficiently tolerable to enable clinical dosing at trastuzumab-equivalent exposure levels, combining the functions of both the antibody and the payload in one drug and potentially improving patient outcomes.

pharmacology and toxicology↗

bioRxiv2019

Systematic identification of regulators of antibody-drug conjugate toxicity using CRISPR-Cas9 screens

Antibody-drug conjugates (ADCs) selectively deliver highly toxic chemotherapeutic agents to target antigen-expressing cells and have become an important cancer treatment in recent years. However, the molecular mechanisms by which ADCs are internalized and activated within cells remain unclear. Here we use CRISPR-Cas9 screens to identify genes that control the toxicity of ADCs. Our results demonstrate critical roles for a range of known and novel endolysosomal trafficking regulators in ADC toxicity. We identify and characterize C18orf8/RMC1 as a regulator of ADC toxicity through its role in endosomal maturation. Through comparative analysis of CRISPR screens with ADCs bearing a noncleavable linker versus a cleavable valine-citrulline (VC) linker, we show that a subset of late endosomal and lysosomal regulators are selectively essential for toxicity of noncleavable linker ADCs. We further show that cleavable VC linkers are rapidly processed upon internalization and therefore surprisingly appear to bypass the requirement of lysosomal delivery. Lastly, we show that inhibition of sialic acid biosynthesis sensitizes cells to ADC treatment by increasing the rate of ADC internalization. This sensitization was observed using several ADCs targeting different antigens in diverse cancer cell types, including the FDA-approved ADC trastuzumab emtansine (T-DM1) in Her2-positive breast cancer cells. Together, these results reveal novel regulators of endolysosomal trafficking, provide important insights to guide future ADC design, and identify candidate combination therapy targets as well as potential mechanisms of ADC resistance.

cancer biology↗