Harnessing the Fcμ Receptor for Potent and Selective Cytotoxic Therapy of Chronic Lymphocytic Leukemia

Abstract

Chronic lymphocytic leukemia (CLL) is a B-cell malignancy in need of new, effective, and safe therapies. The recently identified IgM receptor FcμR is overexpressed on malignant B cells in CLL and mediates the rapid internalization and lysosomal shuttling of IgM via its Fc fragment (Fcμ). To exploit this internalization and trafficking pathway for targeted drug delivery, we engineered an IgM derived protein scaffold (Fcμ) and linked it with the cytotoxic agent monomethylauristatin F. This Fcμ-drug conjugate was selectively toxic for FcμR expressing cell lines in vitro and for CLL cells but not autologous normal T cells ex vivo. Notably, the cytotoxic activity of the Fcμ-drug conjugate was maintained in CLL cells carrying a 17p deletion, which predicts resistance to standard chemotherapy. Next, we tested the possible therapeutic application of the Fcμ-drug conjugate in immunodeficient NSG mice engrafted with peripheral blood cells from leukemia patients. Three intravenous injections of the Fcμ-drug conjugate over a 10 day period were well tolerated and selectively killed the human CLL cells but not the co-engrafted autologous human T cells. In summary, we developed a novel strategy for targeted cytotoxic therapy of CLL based on the unique properties of FcμR. FcμR-targeted drug delivery showed potent and specific therapeutic activity in CLL, thus providing proof-of-concept for FcμR as a valuable therapeutic target in CLL and for IgM-based antibody drug conjugates as a new targeting platform.

Stable and Potent Selenomab-Drug Conjugates

“…Our previous studies predominantly used maleimide derivatives for Michael addition with Sec under conditions that prevented concurrent conjugation to Cys (Cui et al, 2012; Hofer et al, 2009; Hofer et al, 2008; Thomas et al, 2012; Thomas et al, 2008; Vire et al, 2014). However, similar to maleimide-thiol adducts (succinimide thioethers) at solvent accessible Cys residues (such as Ser396Cys) in the antibody component (Alley et al, 2008; Shen et al, 2012), maleimide-selenol adducts (succinimide selenoethers) rapidly decay in human plasma by undergoing a retro-Michael/Michael reaction sequence that transfers the payload from the antibody to HSA, presumably to its free Cys residue at position 34 (Figure 1) (Patterson et al, 2014).…”

“…Our previous studies exclusively used first-generation selenomabs with one (Cui et al, 2012; Hofer et al, 2009; Hofer et al, 2008; Li et al, 2015; Patterson et al, 2014; Thomas et al, 2012; Thomas et al, 2008; Vire et al, 2014) or two C -terminal Sec residues (Li et al, 2014). This position mirrors the C -terminal location of the Sec residue in the natural selenoprotein thioredoxin reductase 1 (TXNRD1).…”

“…Subsequent studies revealed that the stability of thiomab-drug conjugates was inversely related to the solvent accessibility of the engineered Cys residues (Shen et al, 2012). Similar in concept to thiomabs, we have developed a site-specific conjugation technology based on antibodies with engineered selenocysteine (Sec) residues (Cui et al, 2012; Hofer et al, 2009; Hofer et al, 2008; Vire et al, 2014), which we named selenomabs.…”

Assessment of reagents for selenocysteine conjugation and the stability of selenocysteine adducts

“…25 Based on this property, we have introduced a unique class of selenocysteine (Sec)-modified antibodies (SELENOMABS) that allow site-specific antibody modification leading to homogeneous ADCs. 26, 27 Furthermore, we demonstrated that SELENOMAB-oxadiazole conjugates had enhanced stability relative to maleimide counterparts. 26 We subsequently incorporated two selenocysteine residues into one antibody 28 for increased drug loading and also successfully labeled antibodies with two different reporter groups via engineered Cys and Sec residues.…”

Chimeric antigen receptor T cells targeting Fc μ receptor selectively eliminate CLL cells while sparing healthy B cells

“…Instead of the anti-FcmR scFv for binding, the IgM Cm2-Cm3-Cm4 constant domain, which is the natural ligand for FcmR, may be used as described for an anti-FcmR immune toxin, which showed preferential elimination of CLL cells. 32 For the treatment of CLL, however, we think that targeting by an antibody is more selective because the IgM constant domain also binds to the polymeric immunoglobulin receptor and the FCAMR (Fca/m receptor), 21 which would diminish the selectivity in CAR targeting. Alternatively, ROR-1 was also proposed for CAR T-cell targeting of CLL cells.…”