Monoclonal Antibodies Targeting LecLex-Related Glycans with Potent Antitumor Activity

Abstract

Purpose: To produce antitumor monoclonal antibodies (mAbs) targeting glycans as they are aberrantly expressed in tumors and are coaccessory molecules for key survival pathways.Experimental Design: Two mAbs (FG88.2 and FG88.7) recognizing novel tumor-associated Lewis (Le) glycans were produced by immunizations with plasma membrane lipid extracts of the COLO205 cell line.Results: Glycan array analysis showed that both mAbs bound Le The mAbs demonstrated excellent antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC), in addition to direct tumor cell killing via a caspaseindependent mechanism. Scanning electron microscopy revealed antibody-induced pore formation. In addition, the mAbs internalized, colocalized with lysosomes, and delivered saporin that killed cells with subnanomolar potency. In vivo, the mAbs demonstrated potent antitumor efficacy in a metastatic colorectal tumor model, leading to significant long-term survival.Conclusions: The mAbs direct and immune-assisted tumor cell killing, pan-tumor reactivity, and potent in vivo antitumor efficacy indicate their potential as therapeutic agents for the treatment of multiple solid tumors. In addition, internalization of saporin conjugates and associated tumor cell killing suggests their potential as antibody drug carriers.

Monoclonal Antibody Targeting Sialyl-di-Lewisa–Containing Internalizing and Noninternalizing Glycoproteins with Cancer Immunotherapy Development Potential

“…Both 129 mAbs (FG129 (mIgG1) as well as CH129 (chimeric hIgG1) displayed high functional sialyl Lewis a binding affinity: K d $0.2 nmol/L and 20-50 nmol/L on high-and low-density surfaces (SPR), respectively, likely reflecting the target density-dependent differential binding modes (bivalent versus monovalent). ELISA experiments yielded similar EC 50 values compared with SPR, whereas cell surface binding functional affinity was slightly reduced, albeit still in the nanomolar range, presumably due to the more fluid nature of the cell membrane and the occurrence of other biological processes such as internalization (27). The findings may also reflect the different target antigen utilized (sialyl-Lewis a -APD-HSA in ELISA and SPR vs. complex glycoproteins on the cell surface).…”

“…Cancer cells (1 Â 10 5 ) or whole blood (50 mL/well) was incubated with primary mAbs (at 33.3 nmol/L) for 1 hour at 4 C, as described previously (27); followed by 1-hour incubation at 4 C with anti-mouse FITC-labeled secondary antibody, lysis of red blood cells (for whole blood binding analysis; Cal-Lyse, Invitrogen), and fixing in 0.4% formaldehyde (Sigma). Cells were run on Beckman Coulter FC-500 and analyzed using WinMDI 2.9.…”

“…Tumor and normal tissue binding was assessed on tissue microarrays (TMA) as described previously (27). In brief, after antigen retrieval and blocking of endogenous peroxidase activity and nonspecific binding sites, the slides were incubated with FG129 (7.4 nmol/L) at room temperature for 1 hour, followed by detection with a biotinylated secondary mAb (Vector Laboratories).…”

Glycan Microarrays as Chemical Tools for Identifying Glycan Recognition by Immune Proteins

“…As noted above, anti-glycan antibodies can be characterized on the array platforms, and many have been studied over the years on the CFG glycan array (Agrawal-Gamse et al, 2011;Zipser et al, 2012;Noble et al, 2013;Falkowska et al, 2014;Chua et al, 2015;Mickum et al, 2016;Tati et al, 2017;Nkurunungi et al, 2019), such as characterization of a new Lewis x antibody (Mandalasi et al, 2013) and multiple iterations of an anti-Tn antigen antibody (Chaturvedi et al, 2008;Tati et al, 2017). While antibody specificities are not the main focus of this review, it is clear from past work that the glycan microarrays have been and will continue to be very useful tools for their characterization, especially as the glycans and the types of linkers diversify.…”

Characterization of H type 1 and type 1 N-acetyllactosamine glycan epitopes on ovarian cancer specifically recognized by the anti-glycan monoclonal antibody mAb-A4

“…Also, anti-HESCA-2 stains primarily a single 250-kDa band on Western blotting of hESC lysate (27), whereas mAb-A4 stains multiple bands on hESC, indicating that these mAbs target different epitopes (data not shown). FG-88 also did not react with OVCAR3 (22), whereas mAb-A4 did (Table 1). Anti-SSEA-5, reported as binding H type 1 trisaccharide (40), also failed to recognize SKOV3 and HEYA8 via FACS, whereas mAb-A4 bound strongly to them (supplemental Fig.…”