Monoclonal ACPA-IgG Feature Extensive Fab Glycosylation

Katy A. Lloyd¹, Johanna Steen¹, Philip J. Titcombe^2,3, Khaled Amara⁴, Diana Zhou¹, Lena Israelsson⁵, Susanna L. Lundström⁶, Daniel L. Mueller³, Lars Klareskog¹, Vivianne Malmström¹ and Caroline Grönwall⁷, ¹Dept. of Medicine, Rheumatology Unit, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden, ²Dept. of Medicine, Rheumatology Unit, Karolinska Institutet, Karolinska University Hospital, Sweden, Sweden, ³Dept. of Medicine, University of Minnesota Medical School, Minneapolis, MN, ⁴Department of Medicine, Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden, ⁵Dept. of Medicine, Rheumatology Unit, Karolinska Institutet, Stockholm, Sweden, ⁶Dept. of Medical Biochemistry and Biophysics, Division of Physiological Chemistry, Karolinska Institutet, Stockholm, Sweden, ⁷Dep. of Medicine, Rheumatology Unit, Karolinska Institutet, Karolinska University Hospital, Stockholm, Sweden

Meeting: 2017 ACR/ARHP Annual Meeting

Date of first publication: September 18, 2017

Keywords: anti-CCP antibodies, anti-citrullinated protein/peptide antibodies (ACPA), glycoproteins, immunoglobulin (IG) and rheumatoid arthritis (RA)

Session Information

Date: Sunday, November 5, 2017

Title: Rheumatoid Arthritis – Human Etiology and Pathogenesis Poster I

Session Type: ACR Poster Session A

Session Time: 9:00AM-11:00AM

Background/Purpose:

Fab-glycosylation is found in ~15-25% serum IgG and while its exact consequence remains unknown, it may alter IgG functionality. Recent data revealed elevated Fab-glycosylation in polyclonal anti-citrullinated protein autoantibodies (ACPA) from rheumatoid arthritis (RA) patients. Herein, we characterize the Fab-glycan profile of monoclonal ACPA.

Methods:

A total of 14 recombinant human ACPA, derived from RA synovial plasma cells or circulating memory, were evaluated for predicted N-linked glycosylation sites using the NetNGlyc server. Fab-glycosylation was verified with enzymatic digestion, Western blot, lectin-ELISA, and mass spectrometry. Antigen binding was investigated by CCP3 ELISA. VH-VL structure models were generated using the PIGS tool and the GlyProt server. The frequency of predicted VH-VL sites was compared to single-cell paired heavy and light chains from extensively mutated mAbs (>15 mutation in VH or VL): 51 expressed non-ACPA synovial B cells from seropos. RA, and 27 from seroneg. RA, and 198 bone marrow (BM) plasma cells, and 27 clones from healthy control circulating memory. These were compared to 19 highly-mutated broadly-neutralizing (bn) HIV mAbs and 103 plasmodium faciparium (PF) specific mAbs from the literature. Fisher’s exact test or Kruskal-Wallis test was used in statistical analysis.

Results:

The majority of ACPA exhibited variable region N-linked motifs (85.7%), compared to 18.5% in control (p<0.0001), 21.2% in RA BM plasma cells (p<0.0001), 31.4% non-ACPA synovial RA mAbs (p=0.0005), 7.4% of clones from seroneg. RA (p<0.0001), 25.2% in PF mAbs (p<0.0001), and 63.2% of HIV bnAbs (p=0.24), featured in both framework and CDRs generated by somatic hypermutation (SHM). Indeed, ACPA displayed high level of SHM (average 30 VL and 52 VH), yet when adjusted for SHM, N-linked motifs were significantly elevated in ACPA compared to all groups including bnAbs. IgG mAb characterization revealed that N-linked motifs were indeed glycosylated, although preliminary data suggested that glycans had no striking effect on antigen-binding. Homology-based structures predicted glycans to be primarily positioned outside of the potential antigen-binding site. Lectin analysis and mass spectrometry suggested that ACPA mAb Fab-glycan composition was distinctly different from Fc-glycans, and could have high sialic acid content.

Conclusion:

The results support that Fab glycosylation is a key feature of ACPA. Significant increases in N-linked motifs in ACPA compared to other highly-mutated antibodies signifies that this is not solely associated to mutation frequency. Future studies are merited to further investigate the selection mechanisms and functional role of Fab-glycosylated autoantibodies

Disclosure: K. A. Lloyd, None; J. Steen, None; P. J. Titcombe, None; K. Amara, None; D. Zhou, None; L. Israelsson, None; S. L. Lundström, None; D. L. Mueller, None; L. Klareskog, None; V. Malmström, None; C. Grönwall, None.

To cite this abstract in AMA style:

Lloyd KA, Steen J, Titcombe PJ, Amara K, Zhou D, Israelsson L, Lundström SL, Mueller DL, Klareskog L, Malmström V, Grönwall C. Monoclonal ACPA-IgG Feature Extensive Fab Glycosylation [abstract]. Arthritis Rheumatol. 2017; 69 (suppl 10). https://acrabstracts.org/abstract/monoclonal-acpa-igg-feature-extensive-fab-glycosylation/. Accessed .

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