The identification of factors associated with the response to anti-TNF therapy is a major objective for treatment personalization in RA. Diagnostic autoantibodies, like rheumatoid factor and anti-CCP, are appealing for this task since they are already integrated into the standard clinical routine. However, conflicting results have been reported and their association to treatment response to anti-TNF is yet not clear. We hypothesized that this inconsistency could be due to the presence of interaction effects between the autoantibodies. In the present study we have tested this hypothesis for the first time. Using a prospective cohort of RA patients starting anti-TNF therapy, we have found that the interaction between anti-CCP and RF and the interaction between anti-CarP and anti-PAD4 antibodies are both strongly associated with the clinical response at week 12. The present results suggest that interactions between antibodies are important in the response to anti-TNF therapy, and provide an explanation for the previous conflicting evidence.
Our study shows that the presence of both anti-CCP and RF antibodies is needed for a favorable response to anti-TNF therapy. Several previous studies have analyzed the association of either antibody in relation with the response to anti-TNF drugs. The results, however, have been largely inconsistent or inconclusive . In those few studies where both antibodies were determined, the presence of interactions was not evaluated. Here we show that, when the interaction is considered, a strong and positive association between these two classic antibodies and the clinical response emerges. From a statistical perspective, when interaction effects are present and are strong, not taking them into account in the association model can lead to inconsistent findings . Failing to take this into account could therefore explain the lack of reproducibility of previous studies with anti-CCP and RF and treatment response.
The interaction association identified between anti-CCP and RF with the response to anti-TNF therapy is in accordance with recent findings at the functional level. In a recent study, macrophages -the main producers of TNF in the RA joint- have been shown to secrete much higher TNF cytokine levels when stimulated with both anti-CCP and RF antibodies than when stimulated with anti-CCP alone . According to these results, disease activity in RA patients that express both anti-CCP and RF might be partially due to the overexpression of TNF by macrophages reacting strongly to the combination of the two antibodies. Instead, in patients expressing only one of the antibodies or in seronegative patients, this synergic production of TNF by the synovial macrophage will not occur, and other inflammatory pathways will therefore have a more predominant role in disease activity. Our results show that, although still effective in some patients, anti-TNF therapy has a much less pronounced therapeutic effect in patients with only one antibody compared to patients positive for both anti-CCP and RF.
In our study we also found that the interaction between anti-CarP and anti-PAD4 is associated to anti-TNF response. In this case, we found that the higher the expression of both antibodies, the worse the patients responded to TNF blocking. Compared to anti-CCP and RF, these two antibodies do not physically interact at the molecular level. However, the two antibodies share a strong association to neutrophil activity. PAD4 is responsible for most of the citrullinated epitopes in RA  and is specifically expressed in neutrophils. Protein carbamylation is caused by an increase in tissue cyanate due to the activity of neutrophil myeloperoxidase during inflammation in RA. A higher abundance of both antibodies therefore suggests a more predominant role of neutrophils in RA pathology. According to our results, patients with a strong neutrophil-mediated inflammation are less sensitive to therapeutic TNF blocking. This is in line with recent experimental evidence showing that neutrophil activation and TNF have independent effects in RA pathology . New therapies that directly affect neutrophil activation like, granulocyte-macrophage colony-stimulating factor blocking, are currently under way in RA . Our results suggest that the simultaneous analysis of anti-PAD4 and anti-CarP antibodies could be a useful biomarker of response in this new therapeutic approach.
The present study has limitations. Despite most previous studies analyzing the association of antibodies to anti-TNF response have used similar or smaller sample sizes, the number of patients used in our prospective study is relatively modest. Having a larger patient cohort would have enabled a more precise estimation of the interaction effects, with narrower confidence intervals. To this regard, while the quantitative nature of the ΔDAS28 can help increase the power to identify biomarkers of drug efficacy, small improvements in DAS28 might not have a translation into clinically meaningful responses. More patient data on the four antibodies will help to better define this boundary. Finally, the comparison of the interaction association between different types of anti-TNF drugs could not be explored. There is evidence that TNF blocking agents work through biological mechanisms that are not entirely overlapping [21, 22]. An individual analysis of each drug type might reveal stronger interactions and better biomarker utility. For this aspect to be adequately tested, larger drug-specific prospective patient cohorts will need to be analyzed.