The study of autoantibodies to DNA has shown that antigen binding can be mediated primarily by the IgH regardless of the IgL

The study of autoantibodies to DNA has shown that antigen binding can be mediated primarily by the IgH regardless of the IgL.21 To better understand the independent roles of the mutated heavy and light chains in antigen binding, monoclonals were generated that combined mutated IgH or IgL with either their corresponding germline light and heavy chains or irrelevant chains from a monoclonal with no reactivity to PADs. Similar to the germline forms of antibodies 10, 17 and 104, reversion to germline of either IgL or IgH retained antigen binding (Fig. are not polyreactive, and arise from PAD4-reactive precursors. Somatic mutations increase the agonistic activity of these antibodies at low calcium concentrations by facilitating their interaction with structural epitopes that modulate calcium-binding site 5 in PAD4. Conclusions PAD4-activating antibodies directly amplify a key process in disease pathogenesis, making them unique among other autoantibodies in RA. Understanding the molecular basis for their functionality may inform the design of future MPC-3100 PAD4 inhibitors. Keywords: Rheumatoid Arthritis, peptidylarginine deiminase 4, autoantibody, monoclonal antibody, citrullination Introduction The study of monoclonal antibodies derived from patients with autoimmune diseases has provided important insights to understanding the origin of the humoral response against self-antigens1C5 and mechanisms of autoantibody pathogenicity.6C8 While autoantibodies in rheumatic diseases were first described in rheumatoid arthritis (RA),9 the molecular characterization of RA-specific antibodies lagged significantly behind other diseases. MPC-3100 The discovery that citrullinated proteins are major targets of the humoral response in RA renewed interest on further characterizing the autoantibody repertoire in this disease.3,10C14 Citrullinated proteins are generated post-translationally by the peptidylarginine deiminases (PADs), a family of five calcium-dependent enzymes (termed PAD1C4 and 6) that convert arginine residues to citrulline residues.15 At least Rabbit Polyclonal to p53 two major groups of autoantibodies have been linked to the pathogenic role of citrullination in RA: 1) anti-citrullinated protein antibodies (ACPAs), which likely drive effector functions by forming immune complexes16 and 2) anti-PAD4 antibodies that assist the production of citrullinated antigens by enhancing the activity of PAD4.17 Antibody-mediated PAD4 activation is driven by a subgroup of anti-PAD4 antibodies initially recognized MPC-3100 by their cross-reactivity with PAD3.17 These PAD3/PAD4 cross-reactive antibodies (anti-PAD3/4XR) enhance PAD4 activity by increasing the sensitivity of the enzyme to calcium. Together, ACPAs and antibodies to PAD4 may create a feed-forward loop that promotes effector functions via immune complex formation and autoantigen production. Initial studies on disease-specific monoclonal antibodies from patients with RA have been centered on ACPAs.3,11C14,18 A major conclusion from these studies is that ACPAs may originate from polyreactive precursors with their specificities generated as result of antigen-driven affinity maturation.3,11,18 To gain further mechanistic insights into the development and function of the anti-PAD4 autoantibody repertoire, we determined the molecular features and antigen binding properties of monoclonal antibodies to PAD4 cloned from memory B cells of patients with RA. Materials and Methods Patients All samples MPC-3100 were collected after obtaining signed informed consent in accordance with the Johns Hopkins Institutional Review Board. Patients with RA were recruited from the Johns Hopkins Arthritis Center and screened for anti-PAD4 and anti-PAD3/4XR antibodies in serum by immunoprecipitation (IP) as previously described. 17 Peripheral blood anti-PAD4 single memory B cells were purified from three patients who had with high titer anti-PAD4 and anti-PAD3/4XR antibodies, and used to generate monoclonal antibodies. Detailed descriptions are available in the online supplementary methods. Results Sequence analysis of anti-PAD4 antibodies cloned from single memory B cells We isolated single anti-PAD4 memory B cells by flow cytometry using recombinant PAD4 tagged with the monomeric fluorescent protein mWasabi (Fig. S1). In total, 44 single memory B cells with high binding to mWasabi-PAD4 were collected from three patients with RA. Heavy chain (IgH) and corresponding light chain (IgL) variable gene sequences were amplified from 32 cells and used to generate IgG1 monoclonal antibodies. The antibodies were screened by immunoprecipitation (IP) (Fig. 1A), identifying five monoclonal antibodies with specificity to PAD4. Clones 10, 16 and 17 derived from patient #1 were cross-reactive with PAD3, while clones 102 and 104 from patient #3 only recognized PAD4 (anti-PAD4-only antibodies). No antibodies to PAD4 were isolated from patient #2. None of the anti-PAD4 antibodies recognized PAD2 or the PAD from (PPAD) (Fig. 1B). Open in a separate window Fig. 1 Characterization of anti-PAD4 monoclonal antibodies derived from RA memory B cells. (A) Monoclonal antibodies with reactivity to PAD3/4 or PAD4 were screened by IP using [35S]methionine-labeled PAD3 and PAD4 as antigens..