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

Abstract

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.

Targeted Desialylation Overcomes Glyco-Immune Checkpoints and Potentiates the Anticancer Immune Response in Vivo

“…Siglec receptors are expressed across a broad range of innate and adaptive immune cells and Siglec-9 + CD8 + TILs co-express immune checkpoint inhibitors 17 , indicating that T-Sia 2 may synergize with immune checkpoint agents. Recent evidence also suggests that removal of surface sialic acid increases the internalization rate of ADCs, indicating that targeted desialylation could enhance their cytotoxic effects 61 . We anticipate that glycan-editing antibody therapies will prove a potent tool amongst the wider arsenal of new anticancer therapies moving towards clinical translation.…”

Targeted Desialylation Overcomes Glyco-Immune Checkpoints and Potentiates the Anticancer Immune Response in Vivo

“…Siglec receptors are expressed across a broad range of innate and adaptive immune cells and Siglec-9 + CD8 + TILs co-express immune checkpoint inhibitors 17 , indicating that T-Sia 2 may synergize with immune checkpoint agents. Recent evidence also suggests that removal of surface sialic acid increases the internalization rate of ADCs, indicating that targeted desialylation could enhance their cytotoxic effects 60 . We anticipate that glycan-editing antibody therapies will prove a potent tool amongst the wider arsenal of new anticancer therapies moving towards clinical translation.…”

Performance of large scale pooled CRISPR screens is dependent on Cas9 expression levels

“…The simplicity, speed and low-cost of CRISPR technology has led to its widespread application in biomedical research in recent years. In particular, large scale pooled CRISPR screening technologies have yielded many new discoveries in a variety of research fields (1)(2)(3)(4). Since its establishment, the technology has undergone many improvements, and the CRISPR toolbox has been significantly expanded (5,6).…”