Fcμ-Receptor (FcμR; TOSO/FAIM3) Mediated Internalization of Antibody-Drug Conjugates: A Novel Approach to Selectively Target Chronic Lymphocytic Leukemia Cells

Abstract 734

The therapeutic principle of antibody-drug conjugates (ADCs) in cancer relies on an antibody to deliver a cytotoxic agent into malignant cells that express a tumor-associated antigen. Recent progress in the field has shown that the use of such conjugates can increase drug activity and reduce cytotoxicity through selective targeting. Recently, TOSO/FAIM3 was identified as the long sought FcR for IgM (FcμR). FcμR is a transmembrane protein expressed on CD19⁺ B cells, on some CD4⁺/CD8⁺ T cells, and weakly on CD56⁺/CD3⁻ NK cells. We and others have shown that FcμR is consistently overexpressed on chronic lymphocytic leukemia (CLL) cells compared to normal B-cells. In addition, we detected FcμR expression in the mantle cell lymphoma cell line Mino. Using immunofluorescence staining, we found that FcμR can rapidly internalize IgM and transport it through the endocytic pathway to the lysosome. Interestingly, aggregation of FcμR with IgM lead to rapid internalization (>80% internalized within 5 minutes, n=5) whereas mAb bound FcμR was not internalized (n=3). Because of its restricted expression on CLL cells, its cell surface localization and its rapid internalization, FcμR represents an excellent target to deliver a cytotoxic drug into CLL cells using an ADC. To make use of the rapid internalization of FcμR when bound by the Fc-portion of IgM, we engineered a protein scaffold derived from the CH3-CH4 IgM constant region, and inserted a C-terminal selenocysteine, that allows site specific and covalent conjugation of drugs to the protein scaffold. Using gel filtration, we found that purified CH3-CH4 exists mostly as a multimer including pentameric and hexameric forms, similar to native IgM. First, we verified that the CH3-CH4 protein scaffold also binds FcμR, is internalized and addressed to the lysosomes. Next, we conjugated the CH3-CH4 protein scaffold to cemadotin, a synthetic anti-mitotic agent that inhibits tubulin polymerization. This CH3-CH4-cemadotin conjugate was designed to specifically release the drug inside the cell by inserting a peptide linker that can by cleaved by cathepsin B in the lysosomal compartment. In vitro cell killing showed that this CH3-CH4-cemadotin conjugate potently and selectively killed FcμR expressing cells (IC(50) <0.16 μM) while it was about 200-fold less efficient for cells with undetectable FcμR (IC(50) >31 μM). Control experiments showed equal, i.e. non-selective cytotoxicity of free cemadotin against FcμR expressing and non-expressing cells and absence of cell killing with the unconjugated CH3-CH4 protein scaffold. Taken together, these data identify FcμR as a promising therapeutic target in CLL and possibly select other malignanices. IgM-derived scaffolds constitute ideal carriers for drugs or toxins as they are rapidly internalized. In addition, trafficking of IgM scaffolds to the lysosome provides an additional layer of selectivity as a precursor inactive drug can be activated through lysosomal peptidases. Ongoing efforts aim to test modifications of the scaffold and evaluate additional cytotoxic drugs. Furthermore, we are scaling up production of the lead scaffold drug conjugate in order to test its efficacy in xenograft mouse models.

This work was supported by the Intramural Research Program of the National, Heart, Lung and Blood Institute.