Arthritis & Rheumatism, Volume 62,
November 2010 Abstract Supplement

Abstracts of the American College of
Rheumatology/Association of Rheumatology Health Professionals
Annual Scientific Meeting
Atlanta, Georgia November 6-11, 2010.


HD6: A Novel Antibody That Recognises Cell Surface HLA-B27 Homodimers.

McHugh3,  Kirsty, Payeli1,  Sravan K., Kollnberger2,  Simon, Thiel1,  Markus, Shaw2,  Jacqueline, Kleber1,  Sascha, Wadle1,  Andreas

Department of Oncology, University of Zurich
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford
MRC Human Immunology Unit, Weatherall Institute of Molecular Medicine, Oxford, Oxfordshire, United Kingdom

Background:

Possession of HLA-B27 is strongly associated with development of the spondyloarthropathies, including Ankylosing Spondylitis (AS). The mechanism by which HLA-B27 confers this susceptibility is unclear. HLA-B27 forms both heterotrimers (B27) associated with peptide and beta-2-microglobulin, and also heavy chain homodimers (B272). A pathogenic role for these homodimers has been proposed. However, determination of the extent, distribution and triggers of B272 expression have been hampered by the lack of a specific detection reagent. In order to investigate the role of homodimers in AS, we generated an antibody to B272 using phage display technology.

Methods:

Phage display technology was used to generate monoclonal antibodies specific for B272. Biotinylated recombinant B272 complexes were used for positive selection and heterotrimeric B27 for negative selection of a phage Fab library. One clone selected for further characterisation, HD6, was then sub-cloned to generate a chimeric antibody comprising human Fab2 and murine IgG1 Fc. ELISA was used to confirm its specificity for B272 complexes. For recognition of cell-expressed B272, human B cell lines transfected with HLA-B27 or control HLAs, and AS patient and control peripheral blood mononuclear cells were used and results analysed by FACS. Inhibition of the interaction of B272 with the immunoreceptors KIR3DL1, KIR3DL2 and LILRB2 was determined using transfected and FACS-sorted cell lines.

Results:

1) In ELISA, HD6 specifically recognised recombinant B272 but not HLA-A2, B7 or B27 heterotrimers. 2) HD6 bound in FACS to LBL721.220 cells transfected with HLA-B27, which express B272 cell surface homodimers, but not to LBL721.220 B7 or to B27 with Cys 67 mutated to serine (which do not express B272). HD6 also bound the C1R human B cell line transfected with HLA-B27 but not with HLA-A2. 3) Cell surface HD6-reactive HLA-B27 molecules were sensitive to treatment with papain (in 3 independent experiments). 4) HD6 bound in FACS to peripheral blood monocytes from AS patients but not controls. B272 expression on monocytes from AS patients (n=7) was significantly higher when compared to monocytes from B27+ (p=0.01, n=4) or B27- (p=0.06, n=7) healthy individuals, respectively. Low level binding to B27+ B but not T lymphocytes was also observed. 5) HD6 inhibited the binding of the immunoreceptors KIR3DL1, KIR3DL2 and LILRB2 to B272.

Conclusions:

A novel phage display-derived monoclonal antibody recognises both recombinant and cell-expressed B272. HD6 stains monocytes from AS patients, implicating these cells in AS pathogenesis. HD6 will be a powerful tool to address the potential pathogenic role of B272 in SpA and may additionally have therapeutic potential.

To cite this abstract, please use the following information:
McHugh, Kirsty, Payeli, Sravan K., Kollnberger, Simon, Thiel, Markus, Shaw, Jacqueline, Kleber, Sascha, et al; HD6: A Novel Antibody That Recognises Cell Surface HLA-B27 Homodimers. [abstract]. Arthritis Rheum 2010;62 Suppl 10 :1973
DOI: 10.1002/art.29738

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