A comparison of anti-HER2 IgA and IgG1 in vivo efficacy is facilitated by high N-glycan sialylation of the IgA

Abstract

Monomeric IgA has been proposed as an alternative antibody format for cancer therapy. Here, we present our studies on the production, purification and functional evaluation of anti-HER2 IgA antibodies as anti-cancer agents in comparison to the anti-HER2 IgG1 trastuzumab. MALDI-TOF MS analysis showed profound differences in glycosylation traits across the IgA isotypes and cell lines used for production, including sialylation and linkage thereof, fucosylation (both core and antennary) and the abundance of high-mannose type species. Increases in sialylation proved to positively correlate with in vivo plasma half-lives. The polymerization propensity of anti-HER2 IgA2m2 could be suppressed by an 18-aa deletion of the heavy chain tailpiece -coinciding with the loss of high-mannose type N-glycan species -as well as by 2 cysteine to serine mutations at positions 320 and 480. The HER2 F(ab')2-mediated antiproliferative effect of the IgA2m1 and IgA2m2 subtypes was similar to IgG1, whereas the IgA1 isotype displayed considerably lower potency and efficacy. The Fc-mediated induction of antibody-dependent cell-mediated cytotoxicity (ADCC) using human whole blood ADCC assays did not demonstrate such clear differences between the IgA isotypes. However, the potency of the anti-HER2 IgA antibodies in these ADCC assays was found to be significantly lower than that of trastuzumab. In vivo anti-tumor activity of the anti-HER2 IgA antibodies was compared to that of trastuzumab in a BT-474 breast cancer xenograft model. Multiple dosing and sialylation of the IgA antibodies compensated for the short in vivo half-life of native IgA antibodies in mice compared to a single dose of IgG1. In the case of the IgA2m2 antibody, the resulting high plasma exposure levels were sufficient to cause clear tumor stasis comparable to that observed for trastuzumab at much lower plasma exposure levels.

IgA: Structure, Function, and Developability

“…As mentioned previously, the half-life of IgA can also be extended by decreasing clearance by ASGPR in the liver, which can be achieved by sialylation of the IgA glycans [138]. A higher sialylation of the N-glycans in the IgA anti-HER2 did not interfere in the anti-tumour response and lead to the decrease in tumour growth in FcαRI transgenic mice, while increasing the antibody half-life [140]. In another study, the removal of two glycosylation sites and two free cysteines, together with a stabilised HC and LC linkage, created a new IgA2 anti-EGFR mAb with a longer half-life than the wild-type antibody, and higher efficacy due to Fab-mediated effects and interaction with myeloid cells expressing FcαRI [139].…”

“…This shorter half-life is in part due to clearance mediated by the ASGPR, which recognises terminal galactose residues on the glycans of IgA. Efforts have been made to extend half-life by removing N-linked glycosylation sites [139], generating IgA with higher terminal sialylation of N-glycans [140], by attaching an albumin-binding domain to either the LC or HC in order to facilitate binding to the neonatal Fc receptor FcRn [141], or by engineering in FcRn binding by generating an IgG-IgA Fc fusion [133].…”

“…It has long been recognised that IgA production suffers from low expression levels and heterogeneous glycosylation. Systems enabling increased expression of IgA have been developed [140,142,143], and advances in general expression systems for other Igs are likely also to bring benefits [144,145]. There is interest in using plant-based systems to express IgA [146][147][148], but the implications for glycosylation must be borne in mind, especially since it is known that IgA glycosylation is impacted by expression system [149,150].…”

High-throughput Serum N-Glycomics: Method Comparison and Application to Study Rheumatoid Arthritis and Pregnancy-associated Changes

“…Glycosylation is a critical and ubiquitous co-and posttranslational protein modification which affects a wide variety of biological functions (1,2). Manipulation of protein N-glycosylation has shown to be effective for influencing protein half-life and receptor interaction in molecules as diverse as gammaimmunoglobulins (IgG) and alpha-immunoglobulins (IgA), as well as erythropoietin (3)(4)(5). Furthermore, glycans of pathogens and cancer cells, as well as their receptors, are promising targets for both small molecule drugs and biopharmaceuticals (6 -8).…”

Antibody Isotypes for Tumor Immunotherapy

“…To overcome this limitation, an Fc-engineered IgA2 molecule was developed, which demonstrated improved stability and a longer serum half-life translating into higher in vivo efficacy of the engineered compared to the parental IgA molecule [43]. Also IgA antibodies against human epidermal growth factor receptor 2/neu (HER2/neu) or CD20 demonstrated in vitro and in vivo efficacy [40,44]. Despite these promising preclinical activities, IgA antibodies have currently not been introduced into clinical studies.…”