Fibroblast-like synoviocyte targeting antibodies are associated with failure to reach early and sustained remission or low disease activity after first-line therapy in rheumatoid arthritis

Abstract

Objective: To discover antibody biomarkers that can predict a lack of response to first-line therapy in rheumatoid arthritis (RA) patients.

Methods: Two RA cDNA phage display libraries were screened for novel antibodies in baseline RA sera from the Care in early RA (CareRA) trial, differentiating between patients who did or did not reach remission after first-line therapy (n=20 each). Antibody reactivity to identified University Hasselt (UH)-RA antigens was validated in baseline samples from 136 additional CareRA participants. The novel antibodies' potential to predict failure to reach remission or low disease activity (LDA), according to the Disease Activity Score 28-joint C-reactive protein/erythrocyte sedimentation rate (DAS28CRP/ESR) and Clinical/Simplified Disease Activity Index (CDAI/SDAI), was studied by multivariate analyses. The presence of the antibody targets in RA synovial tissue and the fibroblast-like synoviocyte (FLS) cell line SW982 was determined by immunofluorescence.

Results: We identified antibodies to 41 novel antigens. Antibodies against any of three antigens, UH-RA.305/318/329, discriminated between RA patients not reaching week (w)8 DAS28CRP remission and those that did (36% vs 13%,p=0.0031). In all patients, anti-UH-RA.305/318/329 antibody reactivity was associated with failure to reach week 8 DAS28CRP and DAS28ESR remission (OR 3.63,p=0.0031; OR 2.92,p=0.016; respectively), SDAI/CDAI sustained remission (OR 5.59,p=0.039 for both) and DAS28CRP and DAS28ESR sustained LDA (OR 3.7,p=0.009; OR 2.76,p=0.042; respectively). In rheumatoid factor/anti-citrullinated protein antibody (RF/ACPA) seronegative patients, these antibodies were strongly associated with failure to achieve week 8 DAS28CRP remission (OR 17.3,p=0.0029). Anti-UH-RA.305/329 antibodies were shown to target FLS in RA synovial tissue and SW982 cells.

Conclusion: We identified three antibody biomarkers that are associated with failure to achieve remission/LDA after first-line RA therapy.

Keywords: Antibodies; Arthritis, Rheumatoid; Autoimmune Diseases; Therapeutics.

Figure 1. Flow chart of the antigens, RA baseline serum samples and disease activity measures used during SAS and subsequent determination of antibody reactivity in pooled and individual samples. Two human RA synovial cDNA phage display libraries containing 1.2×10⁷ recombinant clones were used to screen for novel antibodies in pooled baseline serum from week 16 DAS28CRP non-rem patients (n=20). During SAS, baseline samples from 20 additional week 16 DAS28CRP rem patients were used for counterselection. Next, antibody reactivity against 41 UH-RA antigens identified during SAS was determined in four week 16 DAS28CRP non-rem serum pools (each consisting of 10 baseline samples, with in total 20 samples from non-rem SAS pool, and 20 additional non-rem samples), and 5 week 16 DAS28CRP rem serum pools (each consisting of 10 baseline samples, with in total 20 samples from rem SAS pool, and 30 additional rem samples). Finally, antibody reactivity against 6 selected UH-RA antigens was determined in individual baseline serum samples from 136 RA patients (new samples; not included in the SAS pool or the plasma pool). Dotted arrow indicates patients included in the SAS pool. Regular arrows indicate new patients who are not included elsewhere. Ab, antibody; BL, baseline; CDAI, Clinical Disease Activity Index; DAS28CRP, Disease Activity Score in 28 joints with C-reactive protein; DAS28ESR, DAS28 with erythrocyte sedimentation rate; LDA, patient reaching low disease activity; non-rem, patient not reaching disease remission; rem, patient reaching disease remission; RA, rheumatoid arthritis; SAS, serological antigen selection; SDAI, Simplified Disease Activity Index; sust, sustained; UH, University Hasselt; w, week.

Figure 2. Receiver operating characteristic (ROC) curve for the relationship between failure to achieve remission according to week 8 DAS28CRP in all RA patients and anti-UH-RA.305/318/329 antibody reactivity with age, sex, RF/ACPA seronegativity, treatment type and disease duration as covariates in the multivariate logistic regression. The yellow line in the plot is a straight line at a 45° angle tangent to the ROC curve, and the contact point with the ROC curve shows the optimal cut-off value. A p<0.05 was considered to be statistically significant. ACPA, anti-citrullinated protein antibody; AUC, area under the curve; DAS28CRP, Disease Activity Score 28-joint C-reactive protein; RF, rheumatoid factor.

Figure 3. Representative staining of anti-UH-RA.305 and anti-UH-RA.329 antibodies in synovial RA tissue. IF showing colocalization of vimentin (green, FLS marker) and UH-RA.305 (A) or UH-RA.329 (B) (red, using human purified Ab). Magnification ×20, scale bar represents 5 µm. FLS, fibroblast-like synoviocyte; IF, immunofluorescence; RA, rheumatoid arthritis; UH, University Hasselt.

Figure 4. Representative staining of anti-UH-RA.305 and anti-UH-RA.329 antibodies in SW982 cells. IF showing costaining of (A) UH-RA.305 (red, using human purified Ab) and CD90 (green, FLS marker), (B) UH-RA.305 (red, using human purified Ab) and vimentin (green), (C) UH-RA.329 (red, using human purified Ab) and CD90 (green) and (D) UH-RA.329 (red, using rabbit polyclonal Ab) and vimentin (green) in SW982 cells. Magnification ×40, scale bar represents 50 µm. Ab, antibody; FLS, fibroblast-like synoviocyte; IF, immunofluorescence; RA, rheumatoid arthritis; UH, University Hasselt.