Discovery and validation of novel biomarkers for diagnosis and outcome of autoimmune diseases: anti-LIN28A IgG and IgA antibodies are highly specific for Systemic Lupus Erythematosus and are associated with histological activity and treatment response in Lupus Nephritis.

Introduction: Currently known autoantibodies in Systemic Lupus Erythematosus (SLE) either lack specificity (e.g. ANA) or show high specificity but low sensitivity (e.g. anti dsDNA and anti Sm autoantibodies). At the same time, several autoantibodies are present in SLE patients whose specificity have yet to be determined. In a preliminary study involving patients with different autoimmune diseases and healthy controls, anti-LIN28A IgG and IgA antibodies have been identified as antibodies with a high specificity for SLE. Objective: We aim at studying the presence and prevalence of anti LIN28A IgG and IgA in a cohort of active lupus nephritis (LN) patients. Methods: Adult patients, fulfilling the revised 1997 ACR classification criteria and with a biopsy-proven LN classified according to the ISN/RPS 2003 criteria, were enrolled between 2017 and 2023 from two Lupus referral centers. Clinical, histological and laboratory data were collected at baseline. Patients were followed up clinically and laboratoristically at 3, 6 and 12 months. Sera from patients were collected at baseline. A subgroup of patients underwent a second per-protocol kidney biopsy at 12 months together with a second blood withdrawal. Patients’ sera were analyzed via KREX technology-based microarrays (i-Ome Discovery; Sengenics) and screened for circulating IgG and IgA autoantibodies against 1609 proteins. Log2-transformed mean net signal intensities of anti-LIN28A IgG and IgA were selected, median-normalized and batch-corrected together with 39 healthy controls’ (HCs) sera which were already available and analyzed. A positivity cut-off for anti-LIN28A IgG and IgA was defined as median plus two interquartile ranges (IQRs) of the distribution levels of the antibodies in HCs. Data were analyzed and compared using one-way ANOVA and t-test for continuous, normally-distributed, variables or X^2 test and Mann-Whitney U test for non-parametric variables. Spearman’s correlation (r) was used to assess correlation between non-normally distributed variables. Results: Thirty-six patients with active, biopsy-proven, LN were enrolled. At baseline, levels of anti-LIN28A IgG and IgA were significantly elevated in LN patients relative to HC (p < 0.0005). Among LN patients, 87% and 77.8% were positive for anti-LIN28A IgG and IgA respectively. IgG negative patients had significantly lower Activity Index (AI) levels at the baseline kidney biopsy (p<0.005). There was a strong correlation between total AI and anti-LIN28A IgG levels (r: 0.47, p:0.011). IgG levels strongly correlated with the severity of endocapillary proliferation and tubulointerstitial mononuclear cell infiltration (r 0.46 and 0.45, respectively. p<0.05). Moreover, patients with the presence of cellular crescents at baseline kidney biopsy showed significantly higher titers of anti LIN28A IgG and IgA (p: 0.0145 and p:0.04 respectively). At twelve months, autoantibody levels of patients with repeated sera withdrawal and per protocol kidney-biopsy (18 patients), were significantly reduced for both antibody isotypes (p<0.001). Among patients who underwent a per protocol-biopsy, 2 patients showed persistence of inflammation at the histological level (AI> 0) and displayed a significantly milder decrease in Anti-LIN28A IgG levels from LN onset (p<0.05). Conclusions: Anti-LIN28A antibodies are markedly elevated in our cohort of active LN patients. Anti-LIN28A IgG showed a significant association with inflammation at the histological level, especially with the degree of endocapillary proliferation and tubulointerstitial inflammatory infiltrate. What is more, failure to negativize the AI at the histological level was mirrored by a lower reduction in anti LIN28A IgG titers. Our preliminary data point to a potential role for Anti LIN28A IgG antibodies as diagnostic and prognostic markers of LN.