Omics of Rituximab-resistance

Primary Membranous Nephropathy (MN) remains a leading cause of nephrotic syndrome (NS) in adults. MN is an autoimmune disorder where autoantibodies (abs) target antigens at the podocyte-glomerular basement membrane interface altering the renal filtration barrier, frequently leading to full-blown nephrotic syndrome (NS) and chronic kidney failure. NS, marked by massive proteinuria, hypoalbuminemia and generalized oedema, severely affects quality of life and can cause life-threatening complications. The discovery of pathogenic antibodies (Abs) against podocyte autoantigens - including the phospholipase A2 receptor (PLA2R) in 70% of primary MN, and more recently thrombospondin type-1 domain containing 7A (THSD7A)- has supported the use of targeted anti B-cell therapies. The anti-CD20 monoclonal Ab Rituximab (RTX) is increasingly used as first-line therapy for MN4, thanks to its better safety profile compared to glucocorticoids, alkylating agents or calcineurin inhibitors, which cause broad immunosuppression. RTX induces a profound circulating B-cell depletion, which persists for several months and is frequently followed by reduced Ab titre and NS remission. However, RTX is ineffective in up to 40% of cases, and about one third of initial responders relapse8, potentially leading to RTX-dependency and prolonged nephrotic range proteinuria with associated complications. Although RTX typically achieves complete peripheral blood B-cell depletion, the reasons for variable patient responses remain unclear. Proposed resistance mechanisms include anti-CD20 Ab-antigen complex internalization, anti-RTX Ab production, incomplete elimination of autoreactive B cell clone in lymphoid organs and failure to target CD20-negative plasma cells8. None of these mechanisms can be envisaged a priori to distinguish between RTX-sensitive and RTX-resistant patients. Therefore, almost half of MN patients are exposed to potential B cell depletion-related complications with negligible or no clinical benefit and high-risk of progression to end-stage kidney disease (ESKD). RTX-resistant patients might benefit from next-generation anti-CD20 monoclonal Abs such as obinutuzumab, which induces a longer and deeper B-cell depletion than RTX and depletes B cells in lymphoid tissues. In preliminary studies it was effective in several RTX-resistant MN patients9. The CONFUCIUS project aims to move beyond current trial-and-error strategies to a precision medicine framework, by integrating pharmacogenomics with other -omics markers that can predict the response to anti-CD20 treatments, ensuring that the right therapy is administered to the right patient at the right time. Consequently, RTX or obinutuzumab will be safely administered to patients exhibiting an adequate response profile, while avoiding unnecessary treatment in potentially anti-CD20 Ab-resistant patients. Subjects with this anti-CD20 antibody- resistant profile, may benefit from alternative strategies which could be identified through pharmacogenomic analyses. Potential strategies for anti-CD20-resistant patients may include targeting CD20-negative B cells, co-stimulatory pathways, or components of the complement system, offering new avenues for patients unresponsive to CD20+B cell-depleting agents. The favourable cost-effectiveness profile for these specific patient subgroups could offset the additional cost of these alternatives.

PERSONALIZED MEDICINE DIMENSION Due to significant variability in pathophysiology and clinical presentation, the treatment of MN remains challenging. MN patients usually receive optimal supportive care for 3-to-6 months, during which approximately one-third of them experience spontaneous remission. For those with persistent proteinuria and at risk of progressive kidney injury, immunosuppressive therapy, including RTX is recommended. However, up to 30-40% of patients are refractory to treatment, putting them at high risk of persistent NS and progression to end stage kidney disease (ESKD). For these patients, alternative therapeutic strategies are often introduced too late, after prolonged ineffective treatment and significant, treatment- and disease-related irreversible complications. By leveraging state-of-the-art methodologies on existing biobanked biological samples from well- characterized MN patients, this project aims to identify gene variants, proteomic and metabolomic biomarkers, and specific B or other immune cell signatures associated with RTX resistance. For patients resistant to both RTX and obinutuzumab, comprehensive re-analysis will be conducted to identify markers of anti-CD20 resistance and discover potential molecular targets to guide the development of alternative therapeutic strategies. This approach will facilitate the development of a standardized, robust, and specific algorithm for therapeutic decision-making. Results from this project will move beyond current trial-and-error strategies to a precision medicine framework, ensuring the right therapy is delivered to the right patient at the right time.

KNOWLEDGE GAP AND UNMET MEDICAL AND PATIENT NEED RTX is ineffective in a substantial subset of MN patients likely due to insufficient or transient depletion of anti-PLA2R abs. As RTX treatment fails in up to 40% of patients, the nephrologists face the dilemma of either exposing patients to the potential risks of B-cell depletion or providing conservative treatment with a high-risk of progression to ESKD. This issue is not trivial, because NS is associated with potentially life-threatening complications and substantially reduces the quality of life of the patient. The identification of markers predicting the response to RTX or to the novel anti-CD20 antibody obinutuzumab may guide choice of the best anti-CD20 approach, and will distinguish patients who are not likely to respond to CD20+B cell depletion. This will avoid unnecessary exposure to the risk of B cell-related immunosuppression and possibly offer anti- CD20 Ab- resistant patients alternative options with the potential of limiting disease progression.

The general aim of this project is to identify specific pharmacogenomics/multi-omics markers of response/resistance to RTX in MN by integrating pharmacogenomics with other -omics technologies, and to evaluate whether emerging anti-CD20 Abs represent a valuable option for patients with RTX resistance signature.

For patients resistant to obinutuzumab, the study will investigate alternative mechanisms of anti-CD20 Ab resistance and search potential therapeutic targets. The results will be instrumental in tailoring individualized anti-B cell or alternative therapeutic strategies, advancing personalized medicine and minimizing drug-related side effects, by preventing the use of ineffective treatments and optimizing healthcare resources.