Directed Evolution of Therapeutic Antibodies Targeting Glycosylation in Cancer

Abstract

Glycosylation patterns commonly change in cancer, resulting in expression of tumor-associated carbohydrate antigens (TACA). While promising, currently available anti-glycan antibodies are not useful for clinical cancer therapy. Here, we show that potent anti-glycan antibodies can be engineered to acquire cancer therapeutic efficacy. We designed yeast surface display to generate and select for therapeutic antibodies against the TACA SLea (CA19−9) in colon and pancreatic cancers. Elite clones showed increased affinity, better specificity, improved binding of human pancreatic and colon cancer cell lines, and increased complement-dependent therapeutic efficacy. Molecular modeling explained the structural basis for improved antibody functionality at the molecular level. These new tools of directed molecular evolution and selection for effective anti-glycan antibodies, provide insights into the mechanisms of cancer therapy targeting glycosylation, and provide major methodological advances that are likely to open up innovative avenues of research in the field of cancer theranostics.

Biomolecular recognition of the glycan neoantigen CA19-9 by distinct antibodies

“…Being relatively small and hydrophilic makes CA19-9 a challenging target for molecular binding and for conventional optimization strategies that target the complementarity-determining region for mutation. We previously used library screening to identify variants of 1116NS19.9 that can bind to CA19-9 more tightly [18]. In this current study, we extended our efforts and used structural data together with innovative computational design to produce AbLIFT-15.…”

“…The flanking regions contained 5′-NdeI and 3′-BamHI restriction enzyme cloning site inframe with the scFv. Then, EBY100 yeast cells were transformed with each synthesized scFv and NdeI BamHI digested plasmid for in vivo ligation, as described [18]. The resulting scFv contained N’ HA and C’ c-Myc tags (encoded in the plasmid) that allowed to monitor surface expression.…”

“…Induced scFv-YSD cells were functionally analyzed for antigen binding by FACS, as described [18], with some modifications. Briefly, we used target antigens in a nanoparticle expression mode with multivalent expression on ~30 kDa polyacrylamide polymers carrying biotin tags (PAA-Bio; biotin ~every 5th amide group with 7–9 glycans per particle).…”

“…A potential obstacle for using anti-carbohydrate antibodies for theranostics is their low affinity and low specificity compared to antibodies targeting proteins [15,16]. This limitation prompted development of tools to better define such antibody-antigen interactions [17] and enhance their affinity [18]. Thus, detailed structural information for the CA19-9 and its recognition by mAbs is a step towards the design of more efficient reagents in the fight against some of the most devastating cancer types.…”

Targeting the “Sweet Side” of Tumor with Glycan-Binding Molecules Conjugated-Nanoparticles: Implications in Cancer Therapy and Diagnosis

“…Indeed, despite the high spectrum of antiglycan mAbs—refers to the Database for Anti-Glycan Reagents (DAGR) containing more than 1000 unique anti-glycan monoclonal antibody entries ( are quite a few antiglycan antibodies which are commercially available [ 53 ]. The development of highly selective and specific monoclonal antibodies to glycans/glycoproteins, then, remains a challenge and represents an obstacle for the progress of the glycoscience field [ 62 ]. The major hurdles are related to both the intrinsically poor glycan immunogenicity and similarities in carbohydrate sequences.…”