Caroline Grönwall1, Anatoly Dubnovitsky1, Philip Titcombe2, Daniel Mueller2 and vivianne malmström1, 1Karolinska Institutet, Stockholm, Sweden, 2University of Minnesota, Minneapolis, MN
Background/Purpose: Anti-citrullinated protein antibodies (ACPA) are a hallmark of rheumatoid arthritis (RA) and the clonality and antigen targets of ACPA positive B cells can be studied on monoclonal level following single B cell isolation, immunoglobulin (Ig) sequencing and recombinant protein expression. Antigen-specific B-cells were isolated from PBMCs of an RA patient using the RA candidate antigen CEP1 peptide as a tetramer, whereby many recombinant human ACPA monoclonal antibodies could be generated (Titcombe et al 2018). Two of these monoclonal ACPA, C22 and C40, from two clonally related B cells, had Ig/BCR sequences differing by only five amino acid residues.
Methods: ELISA and a peptide array was used to compare the antigen-reactivities of the two ACPAs, including the CCP2 and CCP3 assays. Fab fragments of the two ACPAs were purified for crystal structure determination of the antigen-binding capacity to the CEP-1 peptide.
Results: In spite of only 5 amino acid differences between the two ACPAs, the two clones demonstrate strikingly different reactivity.
The C40 clone displayed broad multi-reactivity to a variety of citrullinated peptides while C22 had a restricted binding pattern, including different levels of CCP2 positivity. Next, the C40 mAb was expressed as variants where the amino acid substitutions from C22 was introduced one by one. These variants displayed lower citrulline reactivity. Last, to dissect in detail how the ACPA recognise citrulline, protein structure determination of C40 and C22 fab-fragments were performed, demonstrating that the citrulline had the same position in the variable region, but the rest of the peptide was distinctly different in its position.
Conclusion: Our data show that the characteristic citrulline multireactivity and recognition of distinct small linear citrulline motifs by different ACPA+ B cells can be modulated by minor structural changes and amino acid substitution in the evolution of B cell clonal clades. It further suggests that many of the somatic hypermutations that ACPA clones have been shown to accumulate do not contribute to their citrulline reactivity.
Disclosures: C. Grönwall, None; A. Dubnovitsky, None; P. Titcombe, None; D. Mueller, None; v. malmström, Eli Lilly, Pfizer, Janssen.