Background/Purpose: RFs are polyclonal autoantibodies which bind the Fc domain of IgGs. Patients with RA and high RF levels experience reduced serum drug concentrations and disease control when treated with Fc-containing biological DMARDs (bDMARD). Fc-free certolizumab pegol (CZP) exhibits stable serum drug concentrations and efficacy independent of patient RF levels1-4. Previously, we have shown that monoclonal recombinant RF IgMs bind to Fc-containing bDMARDs in vitro and form protein complexes that are cleared by human macrophages. Fc-free CZP is not bound and avoids clearance5-6. In this study, serum samples from RA patients were classified according to RF level, and the ability of human sera RFs to bind to and clear Fc-containing anti-TNF-α bDMARDs was investigated.

Methods: RF levels in human patient sera were quantified by RF latex agglutination and the sera was classified as “high RF” (>188 IU/ml), “low RF” (14-50 IU/ml) or “RF negative” ( < 14 IU/ml). bDMARDs [adalimumab (ADA), infliximab (IFX), golimumab (GLM), etanercept (ETN), CZP] were expressed and purified as previously described5-6. The CZP variable region sequence was incorporated into a full length human IgG1 to produce Fc-containing certolizumab hIgG1 (CZ hIgG1). ADA was digested with FabRICATOR (Genovis) to produce Fc-free ADA di-Fab (ADA F(ab’)2). RF binding to bDMARDs was assessed by ELISA. Live cell imaging of primary human macrophages was performed with an Incucyte microscope (Sartorius).

Results: In low and high RF patient sera, RFs bound to Fc-containing ADA by ELISA but not to Fc-free CZP. The magnitude of RF binding to ADA was greater in high RF sera than in the low RF sera. RF negative patient sera had no detectable binding to either bDMARD (Figure 1). Human sera RFs formed protein complexes with Fc-containing ADA, IFX, and GLM in the presence of TNF-α, but not with ETN or Fc-free CZP. No complexes formed in RF negative sera. The binding of human sera RFs to bDMARDs was Fc-dependent. Protein complexes formed in high RF sera with Fc-containing ADA and CZ hIgG1, but not with Fc-free CZP or ADA F(ab’)2. RF-bDMARD-TNF-α protein complexes were rapidly cleared by primary human macrophages (Figure 2).

Conclusion: In RA patient sera, RFs bound to Fc-containing bDMARDs, but not to Fc-free CZP. Serum RF levels corresponded with the magnitude of binding observed. RF binding to Fc-containing bDMARDs drove protein complex formation in the presence of TNF-α, which were cleared by macrophages in vitro. These findings provide mechanistic insights into why high RF levels negatively impact Fc-containing bDMARD efficacy, while CZP efficacy is independent of patient RF levels.References: 1. Martínez-Feito A. Clin Exp Rheumatol. 2024;42(5):999-1005; 2. Smolen J S. Rheumatol. 2024;63(11):3015-24 3. Smolen J S. Arthritis Rheumatol. 2024;76(suppl 9) 4. Miyazaki Y. Mod Rheumatol. 2025;35:S56 5. Bidgood S. Ann Rheum Dis. 2024; 83(1):727 6. Bidgood S. Arthritis Rheumatol. 2024;76(suppl 9)

Figure 1. In RA patient sera, RFs bound to Fc-containing ADA but not Fc-free CZP. ELISA assays showed (A) no detectable binding of RF IgM in RF negative ( < 14 IU/mL) patient sera to ADA (blue) or CZP (orange), (B & C) binding of RF IgM in low RF (14-50 IU/mL) and high RF (>188 IU/mL) patient sera to ADA (blue) but not to CZP (orange). (n=10-14/patient group; n=2 independent experiments; mean±SD)

Protein complexes formed between patient sera RFs, Fc-containing bDMARDs and TNF-α were cleared by human macrophages in vitro. (A) High RF patient sera, bDMARDs and TNF-α were mixed and incubated with primary human macrophages at 4°C. Cells were washed and remaining protein complexes detected with Fab anti-human IgM Fc5μ FITC. Cells were moved to 37°C and imaged for 160 minutes (scale bar=100 μm). Quantification of the protein complex area in the images presented as area of protein complex per μm2 at (B) timepoint 0 minutes (random sampling; n=8/bDMARD; mean±SD), and (C-D) over time (non-random sampling; n=4/bDMARD; mean±SD), * P < 0.05, ** P < 0.01, *** P < 0.001 by (B) paired t-test and (C-D) two-way ANOVA with Dunnett’s multiple comparison test (compared to CZP group) on log-transformed data.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/rheumatoid-factors-rfs-in-ra-patient-sera-do-not-bind-to-fc-free-certolizumab-pegol-but-do-bind-to-fc-containing-anti-tnf-%ce%b1-biological-dmards-driving-immune-complex-formation-and-cellular-cle/