Managing lupus nephritis treatment in adults by using a renal activity index for lupus (RAIL) score

Abstract

Disclosed are methods for managing a treatment in an adult individual having systemic lupus erythematosus (SLE) undergoing therapy for lupus nephritis. In aspects, the method comprises detecting a level of each of neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin, and ceruloplasmin in a urine sample obtained from the individual, the levels of which are used to determine a score at a first time point and a second time point. Whether the individual is responding to the therapy, in particular an induction phase of a therapy, is determined based on a comparison of the first and second score. The therapy may then be continued or adjusted based on that determination.

Description

MANAGING LUPUS NEPHRITIS TREATMENT IN ADULTS BY USING A RENAL ACTIVITY INDEX FOR LUPUS (RAIL) SCORECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. Provisional Patent Application 63 / 693,777, filed on September 12, 2024, the contents of which are incorporated in their entirety.STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under AR076316 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Lupus nephritis (LN) confers significant morbidity and mortality to patients with systemic lupus erythematosus (SLE) and confers a poor prognosis; in part because there is a lack of a non-invasive, sensitive and specific biomarkers to monitor renal activity in patients with LN. In patients with systemic lupus erythematosus (SLE), lupus nephritis (LN) is considered one of the most common, severe organ manifestations and a leading cause of morbidity and mortality. LN affects up to 40% of patients with SLE with a higher prevalence observed in women, individuals of lower socioeconomic status, and with African American, African Caribbean, Asian and Hispanic or Latino race / ethnicity.

[0004] Baseline renal biopsies are the gold standard for LN diagnosis and are used to guide initial treatment decisions, based on the International Society of Nephrology / Renal Pathology Society (ISN / RPS) classification and the extent of kidney damage. Repeat kidney biopsies are less established as they are invasive and costly, and hence not feasible in routine surveillance as part of clinical practice, or even in clinical trials. Further, although repeated kidney biopsies are needed to fully understand the nature and extent of treatment’s impact on the kidneys, there is no consensus on the timing for post-baseline biopsies. Thus, cunent methods are insufficient for evaluating a patient’s response to lupus nephritis (LN) therapy, in that repeated biopsies are invasive and therefore impractical. This limitation puts patients at a particular disadvantage, as assessment of LN activity is particularly advantageous during the induction phase of LN therapy, when higher doses or more frequent administration of therapy are used to achieve a rapid decreasein LN activity. Timely evaluation of the effectiveness of induction therapy efficacy is beneficial for enabling prompt adjustment of treatment if the initial therapy proves ineffective; a delay in tailoring the treatment increases the likelihood of a poor prognosis. Existing methods, which rely on kidney biopsy, cannot be performed often enough to inform timely therapy modifications.

[0005] The Renal Activity Index for Lupus (“RAIL”) is a composite score based on the detection and measurement of six urine proteins. The six urine proteins that are detected for determination of a RAIL score are: neutrophil gelatinase- associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin and ceruloplasmin, in which higher scores represent more active inflammation on biopsy. RAIL scores, with or without standardization of urine creatinine levels, may be calculated from the urine concentrations of urine proteins. While the RAIL score has been described for both pediatric (“p- RAIL”) and adult patients (“A-RAIL”), it is known that NGAL, KIM-1, and MCP-1 urine concentrations increase with age, and eGFR declines with age, such that it is not yet clear whether the A-RAIL score can successfully be used to determine the responsiveness of an adult individual to a therapy for lupus nephritis, particularly during the induction phase of treatment.

[0006] Yet further, it would be beneficial to distinguish between individuals likely to have a complete renal response versus those likely to have a partial renal response versus those likely to have no response, to a given therapy, particularly in adults, more particularly during the induction phase of a treatment, which may be used to determine whether the individual is responding to therapy.

[0007] Thus, there remains a need in the art for non-invasive methods for determination of LN status, particularly in response to a therapy, more particularly for treating adult LN patients. The instant disclosure seeks to address one or more of the aforementioned needs in the art.BRIEF SUMMARY

[0008] Disclosed are methods for managing a treatment in an adult individual having systemic lupus erythematosus (SLE) undergoing therapy for lupus nephritis. In aspects, the method comprises detecting a level of each of a biomarker set comprising, consisting of, or consisting essentially of the following biomarkers: neutrophil gelatinase associated lipocalin (NGAL), kidneyinjury molecule 1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin, and ceruloplasmin, in a urine sample obtained from the individual. The urine sample is obtained at a first time point and a subsequent time point, and a first RAIL score based on the first time point and a subsequent RAIL score based on the second time point are calculated. Whether the individual is a complete responder, partial responder, or non-responder to the therapy, in particular an induction therapy or induction phase of a therapy, is determined, based on a comparison of the first RAIL score and the subsequent RAIL score. The therapy is then continued or adjusted based on the comparison of the first RAIL score and the subsequent RAIL score. The method may be used to determine whether the adult individual will achieve no renal response, a complete renal response (CRR), or a partial renal response (PRR), based on the comparison of the first RAIL score and subsequent RAIL score, as measured during the induction phase. The first RAIL score may be determined based on a sample obtained prior to the initiation of induction therapy, at the same time as initiation of induction therapy, or immediately following (such as within one day, within 1-7 days or within 1-14 days of) initiation of induction therapy.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0010] FIG 1 depicts a ROC curve of creatinine-adjusted RAIL-scores in both active LN and active SLE-control groups without active LN. The statistically optimal threshold value to differentiate groups is represented by a diamond and indicates a sensitivity of 72% and a specificity of 78% based on a RAIL-score of 4.56. LN, lupus nephritis; RAIL, Renal Activity Index for Lupus; ROC, receiver operator characteristic.

[0011] FIG 2 illustrates differences in RAIL-score based on renal response. Cross-sectional comparison of the RAIL-score from baseline does not discriminate who will be a responder at week 12 but RAIL scores significantly differ at week 12. The same holds true for week 24 responses (a-c). A) Complete renal response. B) Partial renal response. C) UPCR decrease >50% from baseline. *Nominal P-values. CRR was defined as 24-hour UPCR ≤0.7 mg / mg, eGFR ≥60 mL / min / 1.73 m2 or no decrease ≥20%, no treatment discontinuation, and no restricted medicationuse. PRR was defined as improvement in 24-hour UPCR to <1.0 mg / mg (if baseline UPCR ≤3 mg / mg) or to ≤3.0 mg / mg with >50% improvement (if baseline UPCR >3 mg / mg) was required. CRR, complete renal response; eGFR, estimated glomerular filtration rate; PRR, partial renal response; RAIL, Renal Activity Index for Lupus; RAIL, longitudinal creatinine-standardized RAIL; UPCR, urine protein-creatinine ratio; UPCR50, UPCR decrease >50%. Cross-sectional comparison of the change in RAIL-score from baseline does not discriminate who will be a responder at baseline but RAIL scores significantly differ at to week 12. At week 12, RAIL scores are significantly lower in patients who are complete responders when compared to non-responders. The same holds true for week 24 responses. At week 24, RAIL scores are significantly lower in patients who are complete responders when compared to non-responders. At both week 12 and week 24, RAIL scores are also significantly lower in patients who are partial responders when compared to non-responders.

[0012] FIG. 3A-3G depicts baseline urine biomarkers levels in the active-LN-group (red) and the active-SLE-group.

[0013] FIG. 4 depicts ROC curves for comparison. Comparison of the performance of the RAIL score (corrected for urine creatinine) and the UPCR as well as eGFR to discriminate the active-SLE-group from the active-LN-group. The MODEL shows the performance of the RAIL for baseline differences between groups after adjustment for baseline differences in mycophenolate use, ethnicity, proteinuria and anti-dsDNA levels.

[0014] FIG. 5 depicts Box (SD) and whisker (95% CI) plots illustrating cross-sectional comparison of the change in RAIL-score from baseline to week 12, week 24, and week 52 for all the patients together (A), the placebo group alone (B), and the abatacept group alone (C). Absolute changes in RAIL- scores are depicted for patients considered at these time points to have one of the evaluated renal outcomes: CRR, complete renal response; PRR only, partial renal response but not CRR; NR, non-responder. Shown are RAIL- scores with renal response states during the study as described in Example 2. From left to right in each panel, NR (left), PRR only (middle), and CRR (right).

[0015] FIG. 6 depicts Ability of biomarkers and their combination to reflect current renal response State. Shown in FIG. 6: Receiver operating characteristic (ROC) curves of RAIL-scoreand other biomarkers alone or in combination to differentiate renal response status. Comparisons include CRR vs PRR-only +NR (A), CRR+PRR-only vs NR (B), and CRR vs NR (C). Horizontal dotted lines depict the sensitivity at 90%, 80% and 70%. eGFR has poor accuracy, irrespective of response status comparison. The RAIL score and the UPCR performed similarly well. After adjustment for baseline differences between groups in UPCR, eGFR, age, weight, sex and race, the accuracy of the RAIL- score was excellent in discriminating CRR from other renal response states. CRR= complete renal response, PRR=partial renal response, NR, non-responder; eGFR = estimated glomerular’ filtration rate (based on the CKD-EPI equations); UPCR = urine protein creatinine ratio; RAIL = renal activity index for lupus score.

[0016] FIG. 7 depicts Ability of biomarkers and their combination to reflect next renal response State. FIG. 7 shows Receiver operating characteristic (ROC) curves illustrating the sensitivity and specificity of each variable in predicting renal response for the next visit with the area under the curve (AUC) labeled for each. Comparisons include CRR vs PRR-only+NR (A), CRR+PRR-only vs NR (B), and CRR vs NR (C). Horizontal dotted lines depict the sensitivity at 90%, 80% and 70%. The eGFR has poor accuracy (AUC ≤ 0.51) , irrespective of response status comparison. The RAIL score unadjusted for baseline UPCR and eGFR had fair accuracy to anticipate the renal response status at the next visit, while the UPCR (blue) performed well. After adjustment for baseline differences between groups for UPCR, eGFR, age, weight, sex and race, the accuracy of the RAIL-score was excellent (AUC = ≥0.84) in discriminating CRR from other renal response states. CRR, complete renal response; PRR, partial renal response; NR, non- responder; eGFR = estimated glomerular filtration rate (based on the CKD-EPI equations); UPCR = urine protein creatinine ratio; RAIL = renal activity index for lupus score.

[0017] FIG. 8 depicts RAIL-score model comparisons to current renal function assessment scales at current visit. Shown in FIG. 8: Receiver operating characteristic (ROC) curves of RAIL- score and other biomarkers alone or in combination to differentiate renal response status Comparisons include CRR vs PRR-only +NR (A), CRR+PRR-only vs NR (B), and CRR vs NR (C). Horizontal dotted lines depict the specificity at 90%, 80% and 70%. The eGFR has poor accuracy, irrespective of response status comparison. The RAIL score and the UPCR performed similarly well. After adjustment for baseline differences between groups in UPCR, eGFR, age, weight, sex and race, the accuracy of the RAIL-score was excellent (AUC 0.83) in discriminatingCRR from other renal response states. CRR= complete renal response, PRR=partial renal response, NR, non-rcspondcr; cGFR = estimated glomerular filtration rate (based on the CKD-EPI equations); UPCR = urine protein creatinine ratio; RAIL = renal activity index for lupus scoreDETAILED DESCRIPTION

[0018] DEFINITIONS

[0019] Unless otherwise noted, terms are to be understood according to conventional usage by those of ordinary skill in the relevant art. In case of conflict, the present document, including definitions, will control. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein may be used in practice or testing of the present invention. All publications, patent applications, patents and other references mentioned herein are incorporated by reference in their entirety. The materials, methods, and examples disclosed herein are illustrative only and not intended to be limiting. The methods may comprise, consist of, or consist essentially of the elements of the compositions and / or methods as described herein, as well as any additional or optional element described herein or otherwise useful in methods of managing a treatment in an adult individual having system lupus erythematosus undergoing therapy for lupus nephritis (LN). In aspects, the methods may comprise, consist of, or consist essentially of the elements of or methods as described herein, as well as any additional or optional clement described herein or otherwise useful in methods of identifying a subset of patients having renal disease, such as system lupus erythematosus or lupus nephritis (LN), who are likely to be a non-responder, or a complete responder, or a partial responder.

[0020] As used herein and in the appended claims, the singular forms “a,” “and,” and “the” include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to “a method” includes a plurality of such methods and reference to “a dose” includes reference to one or more doses and equivalents thereof known to those skilled in the art, and so forth.

[0021] The term “about” or “approximately” means within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, for example, the limitations of the measurement system. For example, “about” may mean within 1 or more than 1 standard deviation, per the practice in the art.Alternatively, “about” may mean a range of up to 20%, or up to 10%, or up to 5%, or up to 1 % of a given value. Alternatively, particularly with respect to biological systems or processes, the term may mean within an order of magnitude, preferably within 5 -fold, and more preferably within 2- fold, of a value. Where particular values are described in the application and claims, unless otherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0022] As used herein, the term “effective amount” means the amount of one or more active components that is sufficient to show a desired effect. This includes both therapeutic and prophylactic effects. When applied to an individual active ingredient, administered alone, the term refers to that ingredient alone. When applied to a combination, the term refers to combined amounts of the active ingredients that result in the therapeutic effect, whether administered in combination, serially or simultaneously.

[0023] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to an animal that is the object of treatment, observation and / or experiment. Generally, the term refers to a human patient, but the methods and compositions may be equally applicable to non-human subjects such as other mammals. In some aspects, the terms refer to humans. In further embodiments, the terms may refer to children. In further aspects, the term refers to an adult. In aspects, the individual is 18 years of age or older.

[0024] Methods for Assessing Efficacy of Lupus Nephritis Induction Therapy in Adults

[0025] Measurement of biomarkers in the urine provides a non-invasive tool for estimating the degree of LN activity and the associated renal pathology in patients with LN, given that immune effectors mediating LN pathogenesis including autoantibodies, complement, inflammatory cytokines, immune-mediators and leukocytes, have been detected in urine samples from patients with LN. Currently, the presence and degree of proteinuria is among the principal parameters used to clinically diagnose LN, gauge the degree of LN activity (or inflammation), and to monitor treatment response. However, proteinuria can come from pre-existing renal disease, hypertension, or damage from LN. Further, some patients with marked kidney inflammation only have low levels of proteinuria. Likewise, other conventional SLE biomarkers, for example, complement proteins, and anti-double stranded (ds)DNA cannot differentiate active renal disease from damage.

[0026] While biomarkers have some utility, given the diversity of histological findings on kidney biopsy, it is considered unlikely that a single biomarkcr measured in the urine suffices to estimate LN activity. The Renal Activity Index for Lupus (RAIL) was developed whose scores are informed by the urine concentrations of six urine biomarkers. The RAIL biomarkers are neutrophil gelatinase associated lipocalin (NGAL), monocyte chemotactic protein 1 (MCP-1), ceruloplasmin, adiponectin, hemopexin and kidney injury molecule 1 (KIM-1). Higher RAIL-scores reflect higher active inflammation in LN and changes in pediatric patients RAIL score reproduce the course of LN, i.e. response to treatment and LN flare. Two different RAIL algorithms have been developed, one for adults and one for pediatric LN patients. When used in pediatric patients, the pediatric RAIL-scores were also able to predict National Institutes of Health Activity Index (NIH-AI) scores with >92% accuracy and Tubulointerstitial Activity Index scores with >80% accuracy. When prospectively validated in an adult LN cohort, the RAIL has been found to accurately discriminate the level of LN activity as assessed by NIH-AI scores. Furthermore, improvement of RAIL scores in pediatric LN patients captures and even precedes clinical response of LN by at least 3 months, with over 90% accuracy. However, the ability of the RAIL biomarkers to monitor therapeutic response over time has not yet been established in adults.

[0027] Applicant has found that the six urinary RAIL biomarkers (NGAL, KIM-1, MCP-1, adiponectin, hemopexin, and ceruloplasmin) can be used to determine a RAIL score which can be assessed during an induction phase of treatment to accurately detect active LN in adult patients with SLE. Applicant has further found that the RAIL score may be used to differentiate treatment response, agnostic of treatment type, over time, in adult patients with LN. Thus, the present disclosure provides methods for managing treatment in an adult individual having systemic lupus erythematosus (SLE) undergoing treatment for lupus nephritis (LN), via determination of a RAIL score during an induction therapy treatment, which may be used to determine whether the individual is responding to therapy. Thus, the RAIL score may be used for non-invasive assessment of LN activity to optimize management of patients with LN.

[0028] In aspects, the method may be used to manage or assess a treatment in an adult individual having systemic lupus erythematosus (SLE) undergoing therapy for lupus nephritis. In aspects, the therapy is the induction phase of a therapy.

[0029] In aspects, the individual for whom a treatment is being managed or assessed is an individual having, or suspected of having, lupus nephritis. In aspects, the individual has active lupus nephritis. In aspects, the individual is diagnosed with LN secondary to SLE (Systemic Lupus Erythematosus). In aspects, the individual is an adult individual. In aspects, the individual is an adult individual diagnosed with SLE, and who presents with active LN. . In aspects, the individual is an adult having active Class V LN. In aspects, the individual is an adult individual having active class IV LN. In aspects, the individual is an adult individual having class III LN. In aspects, the individual is an adult individual having an overlap of class III / V. In aspects, the individual is an adult individual having an overlap of class IV / V LN. In aspects, the individual is an adult individual having biopsy-proven active Class III and IV LN. In aspects, the individual is an adult individual having biopsy-proven active Class III and IV LN with Class V overlap. In aspects, the individual is an adult individual having biopsy-proven active Class III and IV LN without Class V overlap. In aspects, the individual has not yet received treatment for LN. In aspects, the individual is receiving an induction therapy for LN. In aspects, the individual is receiving an experimental treatment for LN. In aspects, the individual is receiving an induction therapy, in addition to standard of care treatment such as MMF and corticosteroids. In aspects, the individual is receiving an experimental induction therapy, in addition to standard of care treatment such as MMF and corticosteroids.

[0030] In aspects, the individual for whom the treatment is being monitored may have LN with a National Institute of Health-Index Score (NIH-AI) score of less than 2. In further aspects, the individual for whom the treatment is being monitored may have LN with an NIH-AI score of 2- 10. In aspects, the individual for whom the treatment is being monitored may have LN with an NIH-AI score of greater than 10.

[0031] In aspects, the disclosed methods improve management and / or assessment of an induction treatment for LN in an individual in need thereof. For example, in aspects, the method allows for determining whether an individual is a non-responder, a partial responder, or a complete responder to an administered induction therapy.

[0032] In one aspect, the method comprises:detecting a level of each of a set of biomarkers, the set of biomarkers comprising, consisting of, or consisting essentially of neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin, and ceruloplasmin, in a urine sample obtained from the individual at a first time point and a subsequent time point; calculating a first RAIL score based on levels of each biomarker detected at the first time point; calculating a second RAIL score based on levels of each biomarker detected at the subsequent time point; determining whether the individual is responding to the therapy based on a comparison of the first RAIL score and the subsequent RAIL score; and continuing, ceasing, or adjusting the therapy based on the comparison of the first RAIL score and subsequent RAIL score.

[0033] Determination of the RAIL score is based on the detection of RAIL biomarkers in a sample obtained from the individual at predetermined time points. The RAIL biomarkers may be detected using methods known in the art and as described in the literature. In aspects, the detecting may be carried out in a single sample via a multiplex assay, wherein each biomarker is detected in a single urine sample. In aspects, the detecting may be carried out in a single sample via a single plex assay, via ELISA. In aspects, the detecting may be carried out in a single sample via a Luminex technique. In aspects, the detecting may be carried out in a single sample via a MSD technique. In further aspects, the detecting may be using a multiplex assay such as the Milliplex assay. In further aspects, the detecting may be using a multiplex assay such as the mesoscale discovery (MSD) technology. Milliplex and MSD technologies are described in, for example, Cody EM, Sproles A, Rose J, et al. Development and Validation of Multiplex Assays for Lupus Nephritis Activity Biomarkers. Kidney Int Rep. 2025;10(7):2255-2264. Published 2025 Apr 21. doi:10.1016 / j.ekir.2025.04.013, incorporated herein by reference.

[0034] The RAIL score (or, “A-RAIL” score as described below) can be achieved via application of one or more equations as set forth herein. In aspects, the RAIL (or A-RAIL) scoreis creatine adjusted. In aspects, the RAIL (or A-RAIL) score is albumin adjusted. In aspects, the RAIL (or A-RAIL) score is based on absolute amounts of the RAIL biomarkcr and is not adjusted. Exemplary equations useful for carrying out the disclosed methods are provided below.

[0035] Table 1. Determination of RAIL Score: A-RAIL Equations

[0036] In aspects, the RAIL score is determined using methods known in the art according to the following equation: RAIL = -0.21 +0.67*NGAL +0.28*MCP-1 -0.12*Ceruloplasmin +0.88*Adiponectin -0.25* Hemopexin -0.05*KIM-l. In aspects, the RAIL score is creatine adjusted, i.e., normalized to a measured creatine value.

[0037] In aspects, the RAIL score is determined using methods known in the art according to the following equation: -5.05 +0.56*NGAL +0.12*MCP1 -0.29*Ceruloplasmin+0.88*Adiponectin +0.01*Hemopexin +0.02*KIMl, and is not normalized to creatine or albumin.

[0038] In aspects, the RAIL score is determined using methods known in the art according to the following equation: 3.47 +0.43*NGAL +0.16*MCP1 -0.23*Ceruloplasmin+0.81*Adiponectin -0.05*Hemopexin -0.62*KIM1. In aspects, the RAIL score is albumin adjusted, i.e., normalized to a measured albumin value.

[0039] Determination of a Renal Response

[0040] The disclosed methods may be used to determine whether an individual is likely to be a complete responder, a partial responder, or a non-responder to a therapy, particularly during administration of an induction therapy for treatment of LN. For example, in aspects, the method may further comprise, based on determination of a first and / or a second (or subsequent) RAIL score, determining whether the individual will achieve no renal response to the administeredtherapy, a complete renal response (CRR) to the administered therapy, or a partial renal response (PRR) to the administered therapy, based on the comparison of the first RAIL score and a subsequent RAIL score. The determination of the status of the individual may be used to determine a course of action for the individual, for example continuation of treatment in a responder or partial responder, or a change of treatment in a non-responder or partial responder. As used herein, a change of treatment can include an increase in the amount of a given therapy, an administration of a further treatment in addition to the initial treatment, or cessation of the first treatment in favor of administration of a different treatment.

[0041] Detection of the RAIL biomarkers may be carried out at multiple time points for determining a change in RAIL score indicating the response to treatment in the individual. For example, in aspects, the first time point at which a sample is taken is at day 0 (one day prior to administration of the induction therapy) or at day 1 (the first day of the administration of the induction therapy), and the subsequent time point may one or more days following the initiation of the induction therapy. In further aspects, the first time point may be at the initiation of an induction therapy (e.g., day 0, day 1, day 2, or within one week of the initiation of therapy), and the subsequent time point may be later in the induction therapy, for example at least one week, or two weeks, or three weeks, or four weeks, or five weeks, or six weeks, or seven weeks, or eight weeks, or nine weeks, or 10 weeks, or 11 weeks, or 12 weeks, or 16 weeks, or 20 weeks, or 24 weeks after initiation (e.g., day 1 ) of an induction therapy. In these aspects, a first RAIL score is determined at the onset of therapy, and a subsequent RAIL score is determined after the onset of therapy. The resulting RAIL scores arc then compared for determination of the responder status of the individual in response to the induction therapy.

[0042] In aspects, a RAIL score is determined at two time points, or more than two time points, for example at three time points, or four time points, or five time points, or more than five time points. In this aspect, the RAIL score of any of the time points may be compared to a RAIL score of an earlier time point to provide a comparison that is used to determine the response status of the individual. Based on the response status, the determination may be made to adjust, continue, or cease the induction therapy based on that status.

[0043] In further aspect, the first and subsequent time point may be at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months, or at least seven months, at least eight months, at least nine months, at least ten months, at least 11 months, or at least 12 months apart.

[0044] In general, the RAIL score of the first time point and that of a subsequent time point is calculated using the same equation.

[0045] In aspects, based on the comparison of at least two RAIL scores as described above, the individual is identified as having, or likely to have, a complete renal remission (CRR) in response to a therapy. In aspects, complete renal remission (CRR) in response to a therapy is defined as 24-hour UPCR ≤0.7 mg / mg, eGFR ≥60 mL / min / 1.73m2or no decrease ≥20%, no treatment discontinuation, and no restricted medication use, including a mandatory oral glucocorticoid taper to a dosage of ≤15 mg / day by Week 12 or ≤15 mg / day by Week 24.

[0046] In aspects, based on the comparison of at least two RAIL scores, the individual is identified as having, or likely to have, a partial renal response (PRR) in response to a therapy. In aspects, partial renal response (PRR) is defined as improvement in 24-hour UPCR to <1.0 mg / mg (if baseline UPCR ≤3 mg / mg) or to ≤3.0 mg / mg with >50% improvement (if baseline UPCR >3 mg / mg).

[0047] In aspects, based on the comparison of at least two RAIL scores, the individual is identified as having no renal response (e.g., as a “non-responder”) in response to a therapy.

[0048] Following calculation of the RAIL score in a second, or subsequent RAIL score, the response to therapy can be determined. For example, a decrease of about 0.6 in the RAIL score indicates that the individual is a partial responder to the induction therapy, whereas a decrease of 1.2 in the RAIL score indicates that the individual is a complete responder to the induction therapy. In further aspects, a decrease of about 0.4 to about 0.8, or about 0.5 to about 0.7, or about 0.55 to about 0.65 in the RAIL score indicates that the individual is a partial responder to the induction therapy, whereas a decrease of 1 to about 1.4, or about 0.5 to about 2, or about 0.7 to about 1.8 in the RAIL score indicates that the individual is a complete responder to the induction therapy.

[0049] In aspects, the change in RAIL score from baseline to a second or subsequent RAIL score is decreased by at least 1, or at least 1.1, or at least 1.2, or at least 1.3, at week 12 following initiation of a therapy, and the individual is identified as being a complete responder likely to have complete renal remission (CRR).

[0050] In aspects, the change in RAIL score from baseline to a second or subsequent RAIL score is decreased by at least 2, or at least 2.1, or at least 2.2, or at least 2.3, at week 24 following initiation of a therapy, and the individual is identified as being a complete responder likely to have complete renal remission (CRR).

[0051] In aspects, the change in RAIL score from baseline to a second or subsequent RAIL score is decreased by no more than 0.4, or no more than 0.39, at week 12 following initiation of a therapy, and the individual is identified as being a non-responder.

[0052] In aspects, the change in RAIL score from baseline to a second or subsequent RAIL score is decreased by no more than 0.9, or no more than 0.8, at week 24 following initiation of a therapy, and the individual is identified as being a non-responder.

[0053] In aspects, the biomarkers (NGAL, KIM-1, MCP-1, adiponectin, hemopexin, ceruloplasmin) are detected in a biological sample obtained from the individual undergoing an induction therapy for LN, wherein the biological sample is a urine sample obtained from the individual. In aspects, the urine sample is a fresh (i.e. not previously frozen) urine sample. In aspects, the urine sample is a previously frozen urine sample. In aspects, the urine sample is a previously frozen urine sample, wherein the sample was frozen within 24 hours from collection. In aspects, the RAIL biomarkers are detected using a single plex assay. In aspects, the RAIL biomarkers are detected using a multiplex assay such as described in Cody EM, Sproles A, Rose J, et al. Development and Validation of Multiplex Assays for Lupus Nephritis Activity Biomarkers. Kidney Int Rep. 2025;10(7):2255-2264. Published 2025 Apr 21. doi: 10.1016 / j.ekir.2025.04.013.

[0054] The methods disclosed herein can be used with any type of therapy being administered. For example, the disclosed methods may be used to assess the efficacy of the induction therapy, wherein the induction therapy is mycophenolate mofetil (MMF). In further aspects, the disclosedmethods may be used to assess the efficacy of the induction therapy, wherein the induction therapy is a RAAS blocker. In further aspects, the disclosed methods may be used to assess the efficacy of the induction therapy, wherein the induction therapy is cyclophosphamide. In further aspects, the disclosed methods may be used to assess the efficacy of the induction therapy, wherein the induction therapy is anifrolumab. In further aspects, the disclosed methods may be used to assess the efficacy of the induction therapy, wherein the induction therapy is one or more of corticosteroid, antimalarial, azathioprine, mizoribine, mycophenolate mofetil (MMF), mycophenolic acid, methotrexate, and combinations thereof. In further aspects, the disclosed methods may be used to assess the efficacy of the induction therapy, wherein the induction therapy comprises an induction dose of anifrolumab of 300 mg, or 900 mg for the first three doses and then 300 mg thereafter, every four weeks. In further aspects, the disclosed methods may be used to assess the efficacy of the induction therapy, wherein the induction therapy is any of the aforementioned therapies, further comprising MMF and corticosteroid.

[0055] In further aspects, the disclosed methods may be used to assess the efficacy of the induction therapy, wherein the induction therapy is a therapy of interest which has not yet been approved for use with LN. In this aspect, the method may be used with an experimental therapy. In further aspects, the method may be used in the context of a clinical trial.

[0056] EXAMPLES

[0057] The following non-limiting examples are provided to further illustrate embodiments of the invention disclosed herein. It should be appreciated by those of skill in the art that the techniques disclosed in the examples that follow represent approaches that have been found to function well in the practice of the invention, and thus may be considered to constitute examples of modes for its practice. However, those of skill in the art should, in light of the present disclosure, appreciate that many changes may be made in the specific embodiments that are disclosed and still obtain a like or similar result without departing from the spirit and scope of the invention.

[0058] EXAMPLE 1. The Renal Activity Index for Lupus Identifies Active Renal Disease and Treatment Response in Adult Patients with Systemic Lupus Erythematosus and Lupus Nephritis

[0059] Lupus nephritis (LN) confers significant morbidity and mortality to patients with systemic lupus erythematosus (SLE); in part because there is a lack of a non-invasivc, sensitive, and specific biomarkers to monitor renal activity in patients with LN. The Renal Activity Index for Lupus (RAIL) was developed as a non-invasive measure of LN activity based on the concentration of 6 urinary biomarkers (NGAL, KIM-1, MCP-1, adiponectin, hemopexin, ceruloplasmin) and was shown to accurately detect histologically active pediatric and adult patients with SLE and LN. Applicant confirmed the high accuracy of the RAIL score to detect active LN in urine samples from adult patients with SLE. Further, Applicant demonstrate for the first time the ability of RAIL score to differentiate treatment response (agnostic of treatment type) over time in adult patients with LN. The RAIL allows for the non-invasive assessment of LN activity that could be readily utilized in the real-world clinical setting to optimize management of patients with LN. These findings support the clinical utility of the RAIL biomarkers and the RAIL score to non-invasively assess kidney inflammation and response to therapy in adult LN, and thus allowing a personalized monitoring approach.Methods: Urine samples were collected from adults with biopsy-proven active Class III and IV LN from TUL1P-LN (Treatment of Uncontrolled Lupus via the Interferon Pathway-Lupus Nephritis, active-LN-group; NCT02547922) and adults with active, non-renal SLE from TULIP- 1 (Treatment of Uncontrolled Lupus via the Interferon Pathway-1, active-SLE-group; NCT02446912). RAIL-scores were calculated from the creatinine-adjusted RAIL biomarkers (NGAL, KIM-1, MCP-1, adiponectin, hemopexin, ceruloplasmin) measured in the urine at baseline (both studies); and at Week 12 and Week 24 for TULIP-LN only. The LN and active-SLE groups were compared cross-sectionally, and changes in RAIL-scores from baseline in the active- LN-group were compared between non-responders and responders over time, i.e. those with complete renal response likely to have complete renal remission (CRR), partial renal response (PRR) and urine protein-creatine ratio decrease ≥50% (UPCR50).

[0060] Results: At baseline, median (interquartile range (IQR)) concentrations of individual RAIL biomarkers were significantly higher (P<0.01) in the active-LN-group (n=128) versus the SLE-control group (n=59), as were RAIL-scores 5.6 (4.3-6.5) versus 3.6 (2.8— 4.5) (P<0.001) with an odds ratio of 2.0 (95% confidence interval: 1.6-2.6). At Week 12 / Week 24 there were 25 / 31 patients achieving CRR, 39 / 54 with PRR and 41 / 63 with UPCR50, respectively. RAIL-scoresdecreased over time, irrespective of LN response. However, changes of R AIL-scores from baseline to Week 12 / Wcck 24 significantly differed between non-rcspondcrs and responders (PRR, CRR, UPCR50: all P<0.0006) with lower scores in responders. For CRR versus non-response, median (IQR) RAIL-scores decreased by -1.3 (-3.64 / -0.21) versus -0.39 at Week 12 and -2.30 (-3.63 / - 1.03) versus -0.88 (-2.20 / 0.33) at Week 24, respectively.

[0061] In patients with systemic lupus erythematosus (SLE), lupus nephritis (LN) is considered one of the most common, severe organ manifestations and a leading cause of morbidity and mortality. [1,2] LN affects up to 40% of patients with SLE with a higher prevalence observed in women, individuals of lower socioeconomic status, and with African American, African Caribbean, Asian and Hispanic or Latino race / ethnicity.[l, 3-6]

[0062] There is a lack of non-invasive, accurate biomarkers to assess disease activity and evaluate response to treatment in patients with LN. [7, 8] Baseline renal biopsies are the gold standard for LN diagnosis and are used to guide initial treatment decisions, based on the International Society of Nephrology / Renal Pathology Society (ISN / RPS) classification and the extent of kidney damage.[l, 9, 10] Repeat kidney biopsies are less established as they are invasive and costly, hence not feasible in routine surveillance as part of clinical practice, or even in clinical trials. [1, 11, 12] Further, although repeated kidney biopsies are needed to fully understand the nature and extent of treatment impact on the kidneys, there is no consensus on the timing for post- baseline biopsies. [1, 13-15]

[0063] Measurement of biomarkers in the urine may provide a non-invasive tool for estimating the degree of LN activity and the associated renal pathology in patients with LN, given that immune effectors mediating LN pathogenesis including autoantibodies, complement, inflammatory cytokines, immune-mediators and leukocytes, have been detected in urine samples from patients with LN.

[0012] Currently, the presence and degree of proteinuria is among the principal parameters used to clinically diagnose LN, gauge the degree of LN activity (or inflammation), and monitor treatment response. [1, 16] However, proteinuria can come from pre- existing renal disease, hypertension, or renal damage; and some patients with marked kidney inflammation only have low levels of proteinuria.[l, 16] Likewise, other conventional SLEbiomarkers, for example, complement proteins, and anti-double stranded [ds]DNA cannot differentiate active renal disease from damage. [11, 17]

[0064] Given the diversity of histological findings on kidney biopsy, it is unlikely that a single biomarker measured in the urine suffices to estimate LN activity. Thus, the Renal Activity Index for Lupus (RAIL) was developed whose scores are informed by the urine concentrations of six urine biomarkers. [18, 19] RAIL biomarkers are neutrophil gelatinase associated lipocalin [NGAL] , monocyte chemotactic protein 1 [MCP-1], ceruloplasmin, adiponectin, hemopexin, and kidney injury molecule 1 [KIM-1].

[0018] Higher RAIL scores reflect higher active inflammation in LN and changes in pediatric patients’ RAIL score reproduce the course of LN, that is, response to treatment and LN flare. [18, 19] Two different RAIL algorithms have been developed, one for adults and one for pediatric patients with LN.

[0020] When used in pediatric patients, the pediatric RAIL scores were also able to predict National Institutes of Health Activity Index (NIH-AI) scores with >92% accuracy and Tubulointerstitial Activity Index scores with >80% accuracy. [18, 20] When prospectively validated in an adult LN cohort, the RAIL has been found to accurately discriminate the level of LN activity as assessed by NIH-AI scores.

[0020] Furthermore, improvement of RAIL scores in pediatric patients with LN captures and even precedes clinical response of LN by at least 3 months, with over 90% accuracy.

[0021] However, the ability of the RAIL biomarkers to monitor therapeutic response over time has not yet been investigated in adults.

[0065] In the phase 3 Treatment of Uncontrolled Lupus via the Interferon Pathway (TULIP- 1) trial (ClinicalTrials.gov: NCT02446912) anifrolumab, a type I interferon receptor targeting therapy, was well tolerated and provided therapeutic benefit in patients with moderate-to- severe SLE, excluding patients with severe, active LN.

[0022] This has led to anifrolumab subsequently receiving regulatory approval by the US Food and Drug Administration, European Medicines Evaluation Agency, as well as others regions for moderate-to-severe SLE treatment. [23-27] In the phase 2, TULIP-Lupus Nephritis (TULIP-LN) study (ClinicalTrials.gov: NCT02547922), anifrolumab was shown to have a consistent safety profile, and the intensified dosing regimen led to clinically meaningful improvements in LN endpoints in patients with SLE and active LN.

[0028]

[0066] In this study, Applicant utilized data and samples from TULIP- 1 and TULIP-LN to investigate the ability of the RAIL to discriminate patients requiring therapy for active LN fromother patients with SLE and to differentiate LN treatment responders from non-responders in adult patients, irrespective of LN treatment rendered.

[0067] MATERIALS AND METHODS

[0068] Patients

[0069] Full study design details including patient inclusion and exclusion criteria for TULIP- 1 and TULIP-LN have been previously published. [22, 28] In this analysis, the active-LN-group comprised 128 adult patients (18-70 years of age) from TULIP-LN with biopsy-proven (biopsy within 3 months) active Class III and IV LN, with or without coexistent Class V LN according to the World Health Organization or ISN / RPS 2003 criteria. [10, 28] Eligible patients had 24-hour urine-protein creatinine ratios (UPCR) >1 mg / mg (113.17 mg / mmol), estimated glomerular filtration rate (eGFR) ≥35 mL / min / 1.73m2, and fulfilled ≥4 of the 11 American College of Rheumatology (ACR) SLE 1997 classification criteria.[28, 29]

[0070] The comparator for this analysis (active-SLE-group) comprised 59 adult patients (18- 70 years of age) from TULIP- 1 with a diagnosis of moderate-to-severe active SLE despite standard therapy but without active renal disease that required additional treatment, that is, renal British Isles Lupus Assessment Group (BILAG) 2004 domain scores of C, D or E.[22, 29], Applicant included these comparator group to study the ability of the RAIL to discriminate patients with active SLE from those with active LN requiring intensive therapy for their active kidney disease.

[0071] As this was a secondary analysis of anonymized data, no ethics committee or institutional review board approvals were required. All such approvals were obtained in the original trials which were conducted in accordance with the Declaration of Helsinki and the International Conference on Hamionisation Good Clinical Practice Guidelines [22, 28]; independent ethics committee or independent institutional review board approvals were obtained, and all patients provided written informed consent. [22, 28]

[0072] Study design

[0073] TULIP- 1 and TULIP-LN were both randomized, placebo-controlled, double-blind clinical trials of anifrolumab.[22, 28] At baseline, in TULIP- 1, patients received standard of caretreatment, including corticosteroids, antimalarials, azathioprine, mizoribine, mycophenolate mofctil [MMF] or mycophenolic acid, or methotrexate for their active SLE. During TULIP-LN, patients received either placebo or anifrolumab every 4 weeks through Week 48, in addition to standard of care treatment, that is, MMF and corticosteroids. [22, 28] Patients in TULIP-LN were required to discontinue treatment if they experienced predefined worsening of LN defined as an LN-related, confirmed eGFR decrease >30% from baseline to <60 mL / min / 1.73m2at any time, eGFR decrease <75% from baseline to <60 mL / min / 1.73m2at Week 12 or Week 24, or nephrotic range UPCR at Week 12 or Week 24 (>3.5 mg / mg or <60% improvement in patients >3 mg / mg at baseline).

[0028]

[0074] For the study, a subset of patients enrolled in TULIP- 1 with available urine samples were included and all patients participating in the TULIP-LN for at least 24 weeks, irrespective of treatment allocation. For all patients, Applicant utilized urine samples obtained at baseline, and for the TULIP-LN participants also urine samples collected at Weeks 12 and Week 24. [22, 28]

[0075] Course of renal disease during the study period

[0076] In the TULIP-LN study, complete renal response (CRR) was defined as 24-hour UPCR ≤0.7 mg / mg, eGFR ≥60 mL / min / 1.73m2or no decrease ≥20%, no treatment discontinuation, and no restricted medication use, including a mandatory oral glucocorticoid taper to a dosage of ≤15 mg / day by Week 12 or <15 mg / day by Week 24. Accordingly, for partial renal response (PRR), improvement in 24-hour UPCR to <1.0 mg / mg (if baseline UPCR ≤3 mg / mg) or to ≤3.0 mg / mg with >50% improvement (if baseline UPCR >3 mg / mg) was required.

[0077] Biomarker measurement

[0078] Spun urine samples were frozen within 24 hours from collection prior to testing in individual RAIL protein assays. RAIL urine biomarkers were quantified using single-plex assays per the manufacturers’ instructions.

[0079] A Roche Cobas c 311 clinical chemistry analyser, using a commercially available assay (BioPorto, Denmark, Catalog KIT ST001RA for NGAL; Roche Diagnostics, Indianapolis, IN, Reference 03263991 190 for Creatinine), was used to measure both human urine NGAL and urinecreatinine. NGAL had a lower limit of detection (LLOD) of 9.8 ng / mL and creatinine had a LLOD of 1.1 mg / dL.

[0080] Enzyme-linked immunosorbent assays were used to measure the remaining biomarkers; all sample testing was done in singlicate. Human urinary KIM-1 (R&D Systems, Minneapolis, MN, DKM100) had a LLOD of 0.009 ng / mL. Human MCP-1 (R&D Systems, Minneapolis, MN, DCP00) had a LLOD of 1.7 pg / mL. Human adiponectin (R&D Systems, Minneapolis, MN, DRP300) had a LLOD of 0.246 ng / mL. Human ceruloplasmin (Assaypro LLC, St. Charles, MO, EC4201-1) had a LLOD of 0.085 ng / mL. Human hemopexin (Assaypro LLC, St. Charles, MO, EH2001-1) had a LLOD of 4.2 ng / mL.

[0081] KIM-1 and MCP-1 used a four-parameter logistic curve to fit the standard curve, and adiponectin, ceruloplasmin, and hemopexin instead used a log / log curve. Analyte concentrations that were above or below the limits of detection were imputed by 50% of the level of LLOD and 50% over the upper limit of detection, respectively. Natural log-transformed quantities of NGAL, adiponectin, hemopexin and ceruloplasmin are reported in ng / mL, while those of KIM- 1 and MCP- 1 are reported in pg / mL.

[0082] RAIL score derivation for use in adult populations

[0083] Applicant has shown in the past that the RAIL biomarkers arc produced locally in the kidney with active LN

[0030] and that standardization of biomarker quantities by urine creatinine but not unspecific proteinuria results in the best reflection of histologic activity of LN as per renal biopsies (NIH-AI score), in both children and adults with LN. [18, 20, 30]

[0084] RAIL scores were developed in pediatric patients with SLE and then validated in adult patients previously. [18, 20] NGAL, KIM-1, and MCP-1 urine concentrations have been shown to increase with age, whereas adiponectin concentrations decrease with age.[l l, 31] Significantly higher concentrations of NGAL, hemopexin, and ceruloplasmin have been found in urine samples from females compared with males.[l l, 31] Further, eGFR declines with age. Given these differences, Applicant has developed two RAIL algorithms for use in pediatric and adult populations, respectively.

[0032]

[0085] As previously published, the RAIL score for adults was calculated from the urine quantities of the RAIL biomarkers, after natural logarithmic (In) transformation of values after standardization by urine creatinine concentrations as follows: RAIL = -0.21 +0.67*NGAL +0.28*MCP1 -0.12*Ceruloplasmin +0.88*Adiponectin -0.25*Hemopexin -0.05*KIM-l.

[0020] Herein, higher scores reflect higher LN activity and a score of ≤-0.97 has been shown to reflect histologic activity as measured by an NIH-AI score of >10[20, 32] with a sensitivity of 80% and a specificity of 76.3%.

[0020]

[0086] Statistical analyses and predictive model

[0087] Data were pooled agnostic of treatment received in TULIP- LN or TULIP- 1, given that treatment arm assignment did not influence RAIL score change. Using the RAIL algorithm for adults, RAIL scores presented are calculated from the urinary biomarker concentrations, standardized by urine creatinine, as previously published.

[0020] Clinical characteristic and urinary biomarkers comparisons at baseline between the active-LN-group and the active-SLE-group were performed using Wilcoxon rank-sum test. To assess the accuracy of the RAIL score, receiver operator characteristics (ROC) curve analyses were done to assess the ability of RAIL scores to distinguish active-LN-group from the active-SLE-group and calculated the area under the ROC curve (AUC; range 0 - 1). In general, values of the AUC are considered as outstanding, excellent, good, fair, or poor if for AUC of 0.90-1.00, .81-0.90, 0.71-0.80, 0.61-0.70 or 0.51-0.60, respectively. Longitudinal analysis only included patients from the TULIP-LN study. Herein, Applicant compared RAIL scores between responders and non-responders (a) at baseline; and the again RAIL-score at Weeks 12 and 24, respectively. LN responses considered were CRR, PRR, and UPCR decrease of >50% (UPCR50). Groups were compared using logistic regression models, adjusted for sex, race, baseline RAIL score, and SLE duration.

[0088] RESULTS

[0089] Patients

[0090] This analysis included 128 patients with SLE with active LN (active-LN-group) and 59 patients with SLE without clinically meaningful renal activity, that is, this SLE group consisted of 59 patients with a BILAG renal domain score of C / D / E |C=mild, stable disease, n=11; D=previousinvolvement with no current activity, n=25; or E=no previous involvement, n=23]. [22, 29], No information about the Class of LN or time since kidney biopsy was available for the patients with BILAG renal domain score of C or D. Demographics and baseline disease characteristics of the active-LN-group and the active-SLE-group are presented in Table 2. Despite no more than mild, stable kidney disease (BILAG renal Domain C or lower), there was proteinuria with a mean+SD UPCR of 1.9±2.2.

[0091] At baseline, out of those with available LN class information, almost two-thirds of the patients in the active-LN-group had Class IV (76 / 118 = 64.41%), approximately one-quarter had Class III (26 / 118 = 22.03%) and the remainder of the active-LN-group were an overlap of either Class III / V or Class IV / V (16 / 118 = 13.56%). Further, In the active-LN-group, the median age and the proportion of females were lower while the proportion of Hispanic or Latino (46.56% vs 15.25%) was greater compared with the active-SLE-group. As expected from the study eligibility criteria, median renal SLE Disease Activity Index-2000 (SLEDAI-2K) score and median spot UPCR were both higher and median eGFR lower in the active-LN-group compared with the active- SLE-group. Lower complement C3 and higher anti-dsDNA were observed in the active-LN-group compared with the active-SLE-group (P<0.01 and P< 0.001, respectively), but there were no differences in C4 levels between groups. Also expected was the more frequent use of MMF in the active-LN-group compared with active-SLE-group as all patients in TULIP-LN were required to take MMF as background standard of care from randomization onwards.Table 2. Patient demographics and disease characteristics. C, complement; dsDNA, double- stranded DNA; eGFR, estimated glomerular filtration rate; IQR, interquartile range; LN, lupus nephritis; MMF, mycophenolate mofetil; SD, standard deviation; SLE, systemic lupus erythematosus; SLEDAI-2K, SLE Disease Activity Index-2000; UPCR, urine protein-creatinine ratio. *Data presented as n (% of N), unless otherwise specified,+All patients started MMF upon randomization as pail of the study protocol.Table 3. Baseline urine biomarkers. Data presented as median (IQR). *Nominal P-values, Wilcoxon rank-sum test,+Scores are based on the algorithm for adults and standardized for urine creatinine. IQR, interquartile range; KIM, kidney injury molecule; LN, lupus nephritis; MCP,monocyte chemotactic protein; NGAL, neutrophil gelatinase-associated lipocalin; RAIL, Renal Activity Index for Lupus.

[0092] ROC analyses of RAIL performance to identify active LN requiring treatment

[0093] ROC analyses were conducted to evaluate the ability of the RAIL to identify active LN requiring treatment. When comparing the RAIL scores of the active-LN-group to those of the active-SLE-group in ROC analysis, the area under the ROC curve (AUC) was 0.8004 [95% CI: 0.740 - 0.867], with an odds ratio (OR) of 2.0 [95% confidence interval (CI): 1.6-2.6] (FIG. 1). A RAIL score of 4.56 (statistically optimal threshold value) had a sensitivity of 72% and a specificity of 78% to discriminate between the active-LN-group and the active-SLE-group. After adjusting for baseline differences in mycophenolate use, UPCR, anti-ds-DNA antibodies and ethnicity between groups, the RAIL was outstanding in differentiating between the active-LN-group and the active-SLE-group [AUC= 0.91, 95% CI: 0.868 - 0.952], Applicant also explored the ability of the UPCR and anti-ds-DNA antibodies to differentiate between groups and determined that the AUCwas 0.687 [95% CI: 0.600-0.773] and 0.689 [95% CI: 0.610 - 0.784], respectively, suggesting a fair ability to discriminate groups (sec FIG. 4).

[0094] Association of RAIL scores with baseline activity and longitudinal renal response

[0095] Renal response was studied using urine samples collected at baseline, Week 12 and Week 24 of the treatment period. There were 25, 39, and 41 patients who achieved CRR, PRR, and UPCR50 at Week 12; and 31, 54, and 63 patients with CRR, PRR, and UPCR50 at Week 24, respectively. At baseline, there were no significant differences in RAIL scores between subsequent responders (CRR, PRR, UPCR50) and non-responders at Weeks 12 and 24. FIG. 2, Panel A compares side-by-side baseline RAIL scores and week 12 (or 24) for patients achieving CRR at week 12 (or 24). Using a Wilcoxon rank-sum test, RAIL scores were similar between groups at baseline, but there were significant differences in the RAIL scores between patients achieving CRR and non-responders at Weeks 12 (P < 0.0003), and the same held true for week 24 (P<0.0001). Indeed, compared with baseline, patients with CRR had a median [IQR] decrease in RAIL score -1.3 [-3.64 / -0.21] at Week 12; and by -2.30 [-3.63 / -1.03] at Week 24. FIG. 2, Panel B show significant differences in the RAIL score between those achieving PRR compared to non- responders at week 12 ( P<0.0001 ) and, respectively, week 24 (P<0.0001). Again, there were no differences in RAIL scores at baseline in patients achieving PRR versus those categorized as non- responders at week 12 or week 24. As shown in FIG. 2, Panel C, when considering the response measure UPCR50, the RAIL-score of those achieving UPCR50 versus non-responders significantly differed at week 12 (P<0.0006), and also at week 24(P<0.0001), while RAIL scores were similar at baseline. Table 4 summarizes the absolute change in UPCR, eGFR, and SLEDAL 2K over time in the active-LN-group for those (not) achieving CCR at Week 12 and 24, respectively. Notably, while non-responders (those not achieving CRR) and those achieving CRR at week 24 had similar changes in UPCR and eGFR (both P < 0.20), changes in RAIL scores were significantly larger in those achieving CRR compared to non-responders (P = 0.004). Similar observations were made for CRR at week 12. Table 5 provides the respective information for PRR, again suggesting a significant decrease of the RAIL score at week 12 and 24 in those with PRR compared to non-responders. For the UPCR50 outcome similar results were observed (data not shown). Decrease in RAIL score was less substantial for those with PRR or UPCR50 comparedto CRR at week 24 but differences in RAIL scores between those reaching PRR as compared to CRR did not reach statistical significance.

[0096] In sensitivity analyses, Applicant removed the restricted medication usage criterion when determining course of LN over time. As summarized in Table 5, no important differences in RAIL scores between responders and non-responders compared with the primary analysis were observed.

[0097] Table 4. Change from baseline of proteinuria, eGFR, disease activity and RAIL- scores in the active-LN-group during the study when achieving CRR or non-responders. *Medians and interquartile ranges are shown and patients with PRR were excluded from the comparison.+Scores are based on the algorithm for adults and standardized for urine creatinine. *Nominal P-values, Wilcoxon rank-sum test. CRR, complete renal remission; eGFR, estimated glomerular filtration rate; LN, lupus nephritis; RAIL, Renal Activity Index for Lupus; SLEDAI-2K, SLE Disease Activity Index- 2000; UPCR, urine protein-creatinine ratio.

[0098] Table 5. Change from baseline of proteinuria, eGFR, disease activity and RAIL scores in the LN group during the study when achieving CRR or not (non-responders) after omission of prednisone tapering requirements in the TULIP-LN study. Medians and interquartile ranges are shown. P= 0.01 CRR, complete renal remission; eGFR, estimated glomerular filtration rate; IQR, interquartile range; LN, lupus nephritis; RAIL, Renal Activity Index for Lupus; SLEDAI-2K, SLE Disease Activity Index-2000; UPCR, urine protein-creatinine ratio.

[0099] Table 6. Change from baseline of proteinuria, eGFR, disease activity and RAIL scores in the LN group during the study when achieving CRR or not (non-responders) after omission of prednisone tapering requirements in the TULIP-LN study. Medians and interquartile ranges are shown. CRR, complete renal remission; eGFR, estimated glomerular filtration rate; IQR, interquartile range; LN, lupus nephritis; RAIL, Renal Activity Index for Lupus; SLEDAI-2K, SLE Disease Activity Index-2000; UPCR, urine protein-creatinine ratio.

[0100] Tables 7a-7c. GEE model of responder status considering baseline patient characteristics and RAIL-scores7a. CRR Model7b. PRR Model7c. UPCR50 model

[0101] DISCUSSION

[0102] Despite therapeutic advances in SLE management, LN remains a common severe disease manifestation, conferring significant morbidity and mortality.[l, 3-5] The RAIL score based on a set of urine biomarkers has been developed as a non-invasive tool to help diagnose LN and monitor the course of pediatric LN. [18, 20] Here, Applicant newly demonstrate the ability of individual and composite RAIL biomarkers to differentiate adult patients requiring treatment for active LN from other patients with SLE. Further, Applicant newly show that absolute change in RAIL scores differentiated LN responders from non-responders in the TULIP-LN study, irrespective of the clinical definition of LN response considered (CRR, PRR, UPCR50). Collectively, these results suggest the RAIL is an effective, non-invasive tool for assessing kidney activity and treatment response over time in adult patients with LN.

[0103] In this analysis, a RAIL-score of 4.56 or higher differentiated patients with active LN from those with SLE with at most, mild renal involvement with 80% accuracy, consistent with Applicant’s earlier findings. [18, 20] This observation could be exploited in clinical care for the surveillance of LN, especially in patients with known proteinuria from kidney damage. This is because Applicant has shown in the past that the RAIL biomarkers do not reflect kidney damage. [19, 20]

[0104] Change in RAIL scores over time, rather than RAIL score at baseline, differentiated LN responders from non-responders at Week 12 and 24. Similarly, in pediatric patients with LN, a significant decrease of pediatric RAIL scores also standardized by urine creatinine by at least 1.1 was associated with a complete response to LN induction therapy, irrespective of the type of immunosuppressive therapy rendered.

[0033]

[0105] RAIL scores decreased from baseline during the LN study even in treatment non- rcspondcrs. This might be expected as disease activity decreased in the patient population during TULIP-LN, as did the UPCR

[0028] ; despite this, improvement in RAIL scores were smaller than in responders.

[0106] Proteinuria and eGFR are included in current LN compositve response measures, including those of TULIP-LN (CRR,PRR, UPCR50). UPCR levels only significantly differed with or CRR at week 12 but not week 24, and for PRR at both time points.

[0107] The active- SLE-group had proteinuria when entering TULIP- 1 at baseline despite the fact that there was at most mild, stable kidney inflammation (BILAG C / D / E). The shortcoming of proteinuria to gauge LN activity has long been recognized

[0034] . For examples proteinuria can be a reflection of kidney damage or even comorbid disease, such as hypertension and diabetes mellitus [35,36]. Indeed, in this study, the change UPCR in the active-LN-group at week 12 was similar in those considered having PRR or even CRR, further supporting the need for a more robust biomarker of LN activity besides proteinuria.

[0108] RAIL biomarkers levels and RAIL score at baseline

[0109] At baseline, individual RAIL biomarkers were all significantly higher (P<0.007) in the urine samples from the activc-LN-group compared with the activc-SLE-group (Table 2). Urine creatinine concentration did not significantly differ between groups (P=0.059). Median [IQR] RAIL scores were significantly higher in the active-LN-group compared with the active-SLE- group (5.59 [4.31-6.47] vs 3.57 [2.78-4.47]; P<0.001). FIG. 3 (A-G) depict the distribution of RAIL-scores and RAIL biomarkers for the active LN-group and active-SLE-group, respectively.

[0110] Example 1 References

[0111] 1. Anders HJ, Saxena R, Zhao MH, Parodis I, Salmon JE, Mohan C. Lupus nephritis.Nat Rev Dis Primers. 2020;6(l):7. Epub 2020 / 01 / 25. doi: 10.1038 / s41572-019-0141-9. PubMed PMID: 31974366.

[0112] 2. Yurko vich M, Vostretsova K, Chen W, Avina-Zubieta JA. Overall and cause- specific mortality in patients with systemic lupus erythematosus: a meta-analysis of observationalstudies. Arthritis Care Res (Hoboken). 2014;66(4):608-16. Epub 2013 / 10 / 10. doi: 10.1002 / acr.22173. PubMcd PMID: 24106157.

[0113] 3. Hanly JG, O'Keeffe AG, Su L, Urowitz MB, Romero-Diaz J, Gordon C, Bae SC,Bernatsky S, Clarke AE, Wallace DJ, Merrill JT, Isenberg DA, Rahman A, Ginzler EM, Fortin P, Gladman DD, Sanchez-Guerrero J, Petri M, Bruce IN, Dooley MA, Ramsey-Goldman R, Aranow C, Alarcon GS, Fessler BJ, Steinsson K, Nived O, Sturfelt GK, Manzi S, Khamashta MA, van Vollenhoven RF, Zoma AA, Ramos-Casals M, Ruiz-Irastorza G, Lim SS, Stoll T, Inane M, Kalunian KC, Kamen DL, Maddison P, Peschken CA, Jacobsen S, Askanase A, Theriault C, Thompson K, Farewell V. The frequency and outcome of lupus nephritis: results from an international inception cohort study. Rheumatology (Oxford). 2016;55(2):252-62. Epub 2015 / 09 / 06. doi: 10.1093 / rheumatology / kev311. PubMed PMID: 26342222; PMCID: PMC4939728.

[0114] 4. Hoover PJ, Costenbader KH. Insights into the epidemiology and management of lupus nephritis from the US rheumatologist's perspective. Kidney Int. 2016;90(3):487-92. Epub 2016 / 06 / 28. doi: 10.1016 / j.kint.2016.03.042. PubMed PMID: 27344205; PMCID: PMC5679458.

[0115] 5. Pryor KP, Barbhaiya M, Costenbader KH, Feldman CH. Disparities in Lupus andLupus Nephritis Care and Outcomes Among US Medicaid Beneficiaries. Rheum Dis Clin North Am. 2021 ;47(1):41-53. Epub 2021 / 05 / 28. doi: 10.1016 / j.rdc.2020.09.004. PubMed PMID: 34042053; PMCID: PMC8171807.

[0116] 6. Suzon B, Louis-Sidney F, Aglae C, Henry K, Bagoee C, Wolff S, Moinet F, Emal- glae V, Polomat K, DeBandt M, Deligny C, Couturier A. Good long-term prognosis of lupus nephritis in the high-income Afro-Caribbean population of Martinique with free access to healthcare. J Clin Med. 2022;l l(16):4860. doi: 10.3390 / jcml 1164860. PubMed PMID: 36013099; PMCID: PMC9410092.

[0117] 7. Bertsias GK, Tektonidou M, Amoura Z, Aringer M, Bajema I, Berden JH, Boletis J,Cervera R, Domer T, Doria A, Ferrario F, Floege J, Houssiau FA, loannidis JP, Isenberg DA, Kallenberg CG, Lightstone L, Marks SD, Martini A, Moroni G, Neumann I, Praga M, Schneider M, Starra A, Tesar V, Vasconcelos C, van Vollenhoven RF, Zakharova H, Haubitz M, Gordon C,Jayne D, Boumpas DT, European League Against R, European Renal Association-European D, Transplant A. Joint European League Against Rheumatism and European Renal Association- European Dialysis and Transplant Association (EULAR / ERA-EDTA) recommendations for the management of adult and paediatric lupus nephritis. Ann Rheum Dis. 2012;71(11): 1771-82. Epub 2012 / 08 / 02. doi: 10.1136 / annrheumdis-2012-201940. PubMed PMID: 22851469; PMCID: PMC3465859.

[0118] 8. Davidson A, Aranow C, Mackay M. Lupus nephritis: challenges and progress. CurrOpin Rheumatol. 2019;31(6):682-8. Epub 2019 / 08 / 08. doi: 10.1097 / BOR.0000000000000642. PubMed PMID: 31389814; PMCID: PMC6812494.

[0119] 9. Bajema IM, Wilhelmus S, Alpers CE, Bruijn JA, Colvin RB, Cook HT, D'Agati VD,Ferrario F, Haas M, Jennette JC, Joh K, Nast CC, Noel LH, Rijnink EC, Roberts ISD, Seshan SV, Sethi S, Fogo AB. Revision of the International Society of Nephrology / Renal Pathology Society classification for lupus nephritis: clarification of definitions, and modified National Institutes of Health activity and chronicity indices. Kidney Int. 2018;93(4):789-96. Epub 2018 / 02 / 21. doi: 10.1016 / j.kint.2017.11.023. PubMed PMID: 29459092.

[0120] 10. Weening JJ, D'Agati VD, Schwartz MM, Seshan SV, Alpers CE, Appel GB, BalowJE, Bruijn JA, Cook T, Ferrario F, Fogo AB, Ginzler EM, Hebert L, Hill G, Hill P, Jennette JC, Kong NC, Lcsavrc P, Lockshin M, Looi LM, Makino H, Moura LA, Nagata M, International Society of Nephrology Working Group on the Classification of Lupus N, Renal Pathology Society Working Group on the Classification of Lupus N. The classification of glomerulonephritis in systemic lupus erythematosus revisited. Kidney Int. 2004;65(2):521-30. Epub 2004 / 01 / 14. doi: 10.1111 / j.1523-1755.2004.00443.x. PubMed PMID: 14717922.

[0121] 11. Bennett MR, Ma Q, Ying J, Devarajan P, Brunner H. Effects of age and gender on reference levels of biomarkers comprising the pediatric Renal Activity Index for Lupus Nephritis (p-RAIL). Pediatr Rheumatol Online J. 2017;15(l):74. Epub 2017 / 10 / 17. doi: 10.1186 / s 12969- 017-0202-0. PubMed PMID: 29029629; PMCID: PMC5640910.

[0122] 12. Morell M, Perez-Cozar F, Maranon C. Immune- related urine biomarkers for the diagnosis of lupus nephritis. Int J Mol Sci. 2021 ;22(13):7143. Epub 20210701. doi: 10.3390 / ijms22137143. PubMed PMID: 34281193; PMCID: PMC8267641.

[0123] 13. De Rosa M, Azzato F, Toblli JE, De Rosa G, Fuentes F, Nagaraja HN, Nash R,Rovin BH. A prospective observational cohort study highlights kidney biopsy findings of lupus nephritis patients in remission who flare following withdrawal of maintenance therapy. Kidney Int. 2018;94(4):788-94. Epub 2018 / 07 / 27. doi: 10.1016 / j.kint.2018.05.021. PubMed PMID: 30045812.

[0124] 14. Malvar A, Pirruccio P, Alberton V, Lococo B, Recalde C, Fazini B, Nagaraja H,Indrakanti D, Rovin BH. Histologic versus clinical remission in proliferative lupus nephritis. Nephrol Dial Transplant. 2017;32(8): 1338-44. Epub 2015 / 08 / 08. doi: 10.1093 / ndt / gfv296. PubMed PMID: 26250434; PMCID: PMC5837387.

[0125] 15. Zickert A, Sundelin B, Svenungsson E, Gunnarsson I. Role of early repeated renal biopsies in lupus nephritis. Lupus Sci Med. 2014;l(l):e000018. Epub 2014 / 11 / 08. doi: 10.1136 / lupus-2014-000018. PubMed PMID: 25379188; PMCID: PMC4213828.

[0126] 16. Chedid A, Rossi GM, Peyronel F, Menez S, Atta MG, Bagnasco SM, Arend LJ,Rosenberg AZ, Fine DM. Low-Level Proteinuria in Systemic Lupus Erythematosus. Kidney Int Rep. 2020;5(12):2333-40. Epub 2020 / 12 / 12. doi: 10.1016 / j.ekir.2020.09.007. PubMed PMID: 33305127; PMCID: PMC7710831.

[0127] 17. Soliman S, Mohan C. Lupus nephritis biomarkers. Clin Immunol. 2017;185:10-20.Epub 2016 / 08 / 09. doi: 10.1016 / j.clim.2016.08.001. PubMed PMID: 27498110.

[0128] 18. Brunner HI, Bennett MR, Abulaban K, Klein-Gitelman MS, O'Neil KM, TuckerL, Ardoin SP, Rouster-Stevens KA, Onel KB, Singer NG, Anne Eberhard B, Jung LK, Imundo L, Wright TB, Witte D, Rovin BH, Ying J, Devarajan P. Development of a Novel Renal Activity Index of Lupus Nephritis in Children and Young Adults. Arthritis Care Res (Hoboken). 2016;68(7): 1003-11. Epub 2015 / 10 / 17. doi: 10.1002 / acr .22762. PubMed PMID: 26473509; PMCID: PMC4834060.

[0129] 19. Brunner HI, Bennett MR, Mina R, Suzuki M, Petri M, Kiani AN, Pendl J, WitteD, Ying J, Rovin BH, Dcvarajan P. Association of noninvasivcly measured renal protein biomarkers with histologic features of lupus nephritis. Arthritis Rheum. 2012;64(8):2687-97. Epub 2012 / 02 / 14. doi: 10.1002 / art.34426. PubMed PMID: 22328173; PMCID: PMC3381849.

[0130] 20. Gulati G, Bennett MR, Abulaban K, Song H, Zhang X, Ma Q, Brodsky SV,Nadasdy T, Haffner C, Wiley K, Ardoin SP, Devarajan P, Ying J, Rovin BH, Brunner HI. Prospective validation of a novel renal activity index of lupus nephritis. Lupus. 2017;26(9):927- 36. Epub 2016 / 01 / 01. doi: 10.1177 / 0961203316684212. PubMed PMID: 28361601; PMCID: PMC5380595.

[0131] 21. Brunner HI, Bennett MR, Gulati G, Abulaban K, Klein-Gitelman MS, Ardoin SP,Tucker LB, Rouster-Stevens KA, Witte D, Ying J, Devarajan P. Urine Biomarkers to Predict Response to Lupus Nephritis Therapy in Children and Young Adults. J Rheumatol. 2017;44(8): 1239-48. Epub 2017 / 06 / 18. doi: 10.3899 / jrheum.l61128. PubMed PMID: 28620062; PMCID: PMC6719540.

[0132] 22. Furie RA, Morand EF, Bruce IN, Manzi S, Kalunian KC, Vital EM, LawrenceFord T, Gupta R, Hiepe F, Santiago M, Brohawn PZ, Berglind A, Tummala R. Type I interferon inhibitor anifrolumab in active systemic lupus erythematosus (TULIP- 1): a randomised, controlled, phase 3 trial. Lancet Rheumatol. 2019;l(4):c208-cl9. doi: 10.1016 / S2665- 9913(19)30076-1.

[0133] 23. Burki TK. FDA approval for anifrolumab in patients with lupus. LancetRheumatol. 2021;3:e689.

[0134] 24. Rogers H, Adeniyi O, Ramamoorthy A, Bailey S, Pacanowski M. Clinical pharmacology studies supporting oligonucleotide therapy development: An assessment of therapies approved and in development between 2012 and 2018. Clin Transl Sci. 2021;14(2):468- 75. Epub 20210127. doi: 10.1111 / cts.12945. PubMed PMID: 33278337; PMCID: PMC7993268.

[0135] 25. Shadid M, Badawi M, Abulrob A. Antisense oligonucleotides: absorption, distribution, metabolism, and excretion. Expert Opin Drug Metab Toxicol. 2021 ; 17(11 ): 1281- 92. Epub 20211022. doi: 10.1080 / 17425255.2021.1992382. PubMed PMID: 34643122.

[0136] 26. European Medicines Agency (EMA). Guideline on the investigation of drug interactions. CPMP / EWP / 560 / 95 / Rcv. 1 Corr. 2. 2012 [cited 2024 March 14]. Available from:

[0137] 27. Food and Drug Administration (FDA). In vitro drug interaction studies - cytochrome P450 enzyme- and transporter-mediated drug interactions, guidance for industry. 2020 [cited 2024 March 14]. Available from: hUps: / / www.fda.gov / media / 134582 / download.

[0138] 28. Jayne D, Rovin B, Mysler EF, Furie RA, Houssiau FA, Trasieva T, KnagenhjelmJ, Schwetje E, Chia YL, Tummala R, Lindholm C. Phase II randomised trial of type I interferon inhibitor anifrolumab in patients with active lupus nephritis. Ann Rheum Dis. 2022;81(4):496- 506. Epub 2022 / 02 / 12. doi: 10.1136 / annrheumdis-2021-221478. PubMed PMID: 35144924; PMCID: PMC8921596.

[0139] 29. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum. 1997;40(9): 1725. PubMed PMID: 9324032.

[0140] 30. Sammaritano LR, Askanase A, Bermas BL, Dall'Era M, Duarte-Garcia A, HirakiLT, Rovin BH, Son MBF, Alvarado A, Aranow C, Bamado A, Broder A, Brunner HI, Chowdhary V, Contreras G, Felix C, Ferucci ED, Gibson KL, Hersh AO, Izmirly PM, Kalunian K, Kamen D, Rollins B, Smith BJ, Thomas A, Timlin H, Wallace DJ, Ward M, Azzam M, Bartels CM, Cunha JS, DeQuattro K, Fava A, Figueroa-Parra G, Garg S, Greco J, Cuellar-Gutierrez MC, Iyer P, Johannemann AS, Jorge A, Kasturi S, Kawtharany H, Khawandi J, Kirou KA, Legge A, Liang KV, Lockwood MM, Sanchez-Rodriguez A, Turgunbaev M, Williams JN, Turner AS, Mustafa RA. 2024 American College of Rheumatology (ACR) Guideline for the Screening, Treatment, and Management of Lupus Nephritis. Arthritis Rheumatol. 2025. Epub 20250507. doi: 10.1002 / art.43212. PubMed PMID: 40331662.

[0141] 31. Suzuki M, Wiers K, Brooks EB, Greis KD, Haines K, Klein-Gitelman MS, OlsonJ, Onel K, O'Neil KM, Silverman ED, Tucker L, Ying J, Devarajan P, Brunner HI. Initial validation of a novel protein biomarker panel for active pediatric lupus nephritis. Pediatr Res.2009;65(5):530-6. Epub 2009 / 02 / 17. doi: 10.1203 / PDR.0b013e31819e4305. PubMed PMTD: 19218887; PMCID: PMC2737257.

[0142] 32. Aljaberi N, Bennett M, Brunner HI, Devarajan P. Proteomic profiling of urine: implications for lupus nephritis. Expert Rev Proteomics. 2019;16(4):303-13. Epub 2019 / 03 / 12. doi: 10.1080 / 14789450.2019.1592681. PubMed PMID: 30855196; PMCID: PMC6693508.

[0143] 33. Austin HA, 3rd, Muenz LR, Joyce KM, Antonovych TT, Balow JE. Diffuse proliferative lupus nephritis: identification of specific pathologic features affecting renal outcome. Kidney Int. 1984;25(4):689-95. Epub 1984 / 04 / 01. doi: 10.1038 / ki.1984.75. PubMed PMID: 6482173.

[0144] 34. Cody EM, Wenderfer SE, Sullivan KE, Kim AHJ, Figg W, Ghumman H, Qiu T,Huang B, Devarajan P, Brunner HI. Urine biomarker score captures response to induction therapy with lupus nephritis. Pediatr Nephrol. 2023;38(8):2679-88. Epub 20230130. doi: 10.1007 / s00467- 023-05888-z. PubMed PMID: 36715772; PMCID: PMC10393841.

[0145] 35. Weeding E, Fava A, Magder L, Goldman D, Petri M. One-third of patients with lupus nephritis classified as complete responders continue to accrue progressive renal damage despite resolution of proteinuria. Lupus Sci Med. 2022;9(l):e000684. doi: 10.1 136 / lupus- 2022- 000684. PubMed PMID: 35512816; PMCID: PMC9047706.

[0146] 36. Kochi M, Kohagura K, Oshiro N, Zamami R, Nagahama K, Nakamura K, OhyaY. Association of blood pressure and hyperuricemia with proteinuria and reduced renal function in the general population. Hypertens Res. 2023;46(7): 1662-72. Epub 20230329. doi: 10.1038 / s41440-023-01250-w. PubMed PMID: 36991065.

[0147] 37. Khoury CC, Chen S, Ziyadeh FN. Chapter 19 - Pathophysiology of diabetic nephropathy. In: Kimmel PL, Rosenberg ME, editors. Chronic Renal Disease (Second Edition): 11 Academic Press; 2020. p. 279-96.

[0148] EXAMPLE 2. Renal Activity Index for Lupus: Validation for Prediction of Kidney Inflammation in Adult Patients with Lupus Nephritis

[0149] The following example was carried out to evaluate the ability of the Renal Activity Index for Lupus (RAIL) score to capture and predict the course of active LN in adult patients.

[0150] Methods: Available serial urine samples collected up to week 52 from a subset of adults with active biopsy-proven proliferative LN participating in the double-blind randomized ALLURE trial of abatacept (NCT01714817) were used to calculate RAIL-scores from the creatinine adjusted urine biomarkers (NGAL, KIM-1, MCP-1, adiponectin, hemopexin, ceruloplasmin). Using statistics for diagnostic tests and mixed models, RAIL-scores over time were compared between those of urine protein-to-creatinine- ration (UPCR) and kidney function (eGFR) by renal response states, i.e. complete renal response (CRR), partial renal response but not CRR (PRR-only), and non-response (NR).

[0151] The results of the study are as follows. The analysis included 240 patients who contributed 599 samples. At weeks 12 / 24 / 52 there were 44 / 22 / 15 patients with PRR-only, 27 / 33 / 18 with CRR, and 127 / 61 / 15 with NR. RAIL-scores, eGFR and UPCR all improved over time, irrespective of abatacept use but were significantly lower with CRR compared to NR. The eGFR alone had only poor accuracy [area under the receiver operating characteristic curve ( AUC) <0.51] to discriminate renal response Only after correction of baseline UPCR and eGFR, the RAIL score had excellent accuracy to reflect CRR from other renal response states at the current (AUC=0.83-0.84) and next visit (AUC=0.84-0.85) and performed numerically better than the UPCR.

[0152] Net, Applicant found that RAIL-scores identify active LN and longitudinally and predicts the course of LN in adults with LN.

[0153] Abatacept, a fusion protein of the Fc region of IgG 1 and the extracellular domain of CTLA-4, prevents T cell activation. This medication was approved for several rheumatic diseases, including rheumatoid arthritis and juvenile idiopathic arthritis. The potential benefits of abatacept for the treatment of LN were studied in two Phase 2 trials (NCT00430677 and NCT00774852). Findings of these trials prompted the Phase 3 ALLURE trial (NCT01714817) to further evaluate the benefits of abatacept when used for the treatment of LN.

[0154] In this exploratory biomarker study, data and available samples collected during the ALLURE trial were analyzed to investigate the ability of RAIL to differentiate LN treatment responders from nonresponders in adult patients, as well as to predict future kidney disease flare, under consideration of abatacept exposure.

[0155] METHODS

[0156] Study design and patients

[0157] The ALLURE trial (NCT01714817) was a 104-week, Phase 3 randomized, double- blind, placebo controlled Study to Evaluate the Efficacy and Safety of abatacept in patients with active Class III or IV LN, with or without Class V. 28-30 Upon enrollment, eligible patients were randomized 1 : 1 to receive monthly infusions of either placebo or abatacept together with stable background treatment that included MMF and corticosteroids which could be tapered during the study. The abatacept group received 30 mg / kg (based on body weight at baseline of intravenous abatacept Days 1, 15, 29, and 57. This was followed, starting at week 12, by a weight-tiered monthly dose of abatacept at 500 mg, 750 mg or 1000 mg for patients weighing < 60 kg, 60 kg to 100 kg and > 100 kg, respectively throughout the remainder of the study. The primary endpoint was complete renal response (CRR) measured week 52, and secondary endpoints included the achievement of partial renal response (PRR) at week 52.

[0158] Eligible patients were > 18 years of age, had a kidney biopsy within 12 months of screening that indicated the presence of active proliferative LN as per the 2003 International Society of Nephrology / Renal Pathology Society (ISN / RPS) Classification, 31 and had evidence of active LN within 3 months of screening, a baseline urine-protein creatinine ratios (UPCR) >1 mg / mg (113.17 mg / mmol) and serum creatinine of < 3mg / dL.

[0159] Definition of renal outcomes

[0160] Complete Renal Response (CRR) was defined as meeting ALL (a - c) of the following: (a) estimated GFR (eGFR based on the CKD-EPI equations) normal (>= 60 mL / min / 1.73m2) OR at least 85% of the eGFR at baseline; (b) urine protein / creatinine ratio (UPCR) < 0.5; (c) inactive urinary sediment defined as a urine red blood cell (RBC) casts present < 5 and urine RBC < 10. Accordingly, Partial Renal Response (PRR) was defined by meeting ALL (A-C) three criteria: (A)eGFR > 85% of baseline eGFR; (B) UPCR < 0.5, OR UPCR decreased as > 50% to value of < 1 , for baseline UPCR < 3, OR, > 50% reduced from baseline to UPCR < 3 if baseline UPCR > 3; and (C) urine RBC <10 and urine RBC casts <5. Patients not achieving at least PRR were considered non-responders (NR).

[0161] Urine biomarker measurement

[0162] Urine samples were frozen within 24 hours from collection prior to testing in individual RAIL protein assays. RAIL urine biomarkers were quantified using single-plex assays per the manufacturers’ instructions. Enzyme-linked immunosorbent assays were used to measure the remaining biomarkers; all sample testing was done in singlicate. Human urinary KIM-1 (R&D Systems, Minneapolis, MN, DKM100), had a lower limit of quantitation (LLOQ) of 0.009 ng / ml. Human MCP- 1 (R&D Systems, Minneapolis, MN, DCP00), had a lower limit of detection (LLOD) of 1.7 pg / mL. Human adiponectin (R&D Systems, Minneapolis, MN, DRP300), had a LLOD of 0.246 ng / ml. Human ceruloplasmin (Assaypro LLC, St. Charles, MO, EC 4201-1), had a LLOD of 0.085 ng / mL. Human hemopexin (Assaypro LLC, St. Charles, MO, EH2001-1), had a LLOD of 4.2 ng / ml. KIM-1 and MCP-1 used a four-parameter logistic curve to fit the standard curve, and adiponectin, ceruloplasmin, and hemopexin instead used a log / log curve. Analyte concentrations that were above or below the limits of detection were imputed by 50% of the level of LLOD and 50% over the upper limit of detection, respectively. Natural log transformed quantities of NGAL, adiponectin, hemopexin and ceruloplasmin are reported in ng / mL, while those of KIMI and MCP- 1 are reported in pg / mL. A Roche Cobas c 311 clinical chemistry analyser, using a commercially available assay (BioPorto, Denmark, Catalog KIT ST001RA for NGAL; Roche Diagnostics, Indianapolis, IN, Reference 03263991 190 for Creatinine), was used to measure both human NGAL and urine creatinine. NGAL had a LLOQ of 9.8 ng / ml while creatinine had a LLOQ of 1.1 mg / dL.

[0163] RAIL- score calculation

[0164] As previously published, the RAIL- score for adults was calculated from the urine quantities of the RAIL biomarkers, after natural logarithmic (In) transformation of values after normalization by urine creatinine concentrations as follows:

[0165] RAIL = -0.21 +0.67*NGAL +0.28*MCPl -0.12*Ceruloplasmin +0.88*Adiponectin-0.25*Hcmopcxin -0.05*KIMl.

[0166] Statistical analysis

[0167] Summary statistics of baseline sample characteristics were reported for treatment arms by frequency and corresponding percentage for categorical variables; median, and interquartile ranges (IQR) were reported for numerical variables. LN responses were classified into CRR, PRR only (excluding CRR responders) and NR, for all patients at each study visit (12, 24 and 52 weeks). The RAIL-scores were compared between groups of responders (PRR only or CRR) and NR at weeks 12, 24 and 52. To understand the minimum clinically important differences, relative (or percentage) changes in RAIL-scores were calculated between two adjacent visits (0 to 12, 12 to 24, and 24 to 52 weeks) expressed as the percentage of the previous values of RAIL-score. The mean difference and its corresponding standard deviation (SD) between responders (CRR, PRR only) and NR were reported for all and by treatment arm at each follow-up visit. Standardized mean difference (SMD) was calculated to express the group mean differences relative to the standard deviation of each group. Student T-tests were used for group comparisons.

[0168] To evaluate the ability of the RAIL-score to discriminate between the responder and NR groups, logistic regressions were used to model RAIL-score, compared against the performance of traditional kidney function markers cGFR (estimated glomerular filtration rate based on the CKD-EPI equations), or UPCR (urine protein creatinine ratio) at baseline with and without adjustment for baseline UPCR and eGFR, as well as patient age, weight, sex and race. The area under the receiver operating characteristics (ROC) curves (AUC), odds ratio (OR) and their corresponding 95% CI were calculated for each marker (RAIL-score, eGFR, UPCR). Comparisons considered were CRR plus PRR-only vs. NR, CRR vs. PRR only plus NR, and finally CRR vs. NR in all and by treatment arm. In general, values of AUC are considered as outstanding, excellent, good, fair, or poor accuracy if for AUC of 0.90-1.00, 0.81-0.90, 0.71-0.80, 0.61-0.70 or 0.51— 0.60, respectively. Two sets of analyses were performed, one modelled concurrent RAIL-score, and the other modelled the RAIL-score from the previous visit.

[0169] Results

[0170] Patients and Samples

[0171] This analysis included 240 patients enrolled in the ALLURE trial for which urine samples are available for future research at baseline. Urine samples at week 12, 24, and 52, were available for 198, 116, and 48 patients (96, 57, and 18 remaining in the abatacept group; and 102, 59, and 30 in the placebo group), respectively. Demographics and baseline disease characteristics are presented in Table 8, with 121 patients randomized to IV abatacept and 119 to placebo, respectively. Kidney biopsy results (Class only) were available for a total of 197 patients. At baseline, there were no statistically significant differences (all p-values > 0.52) in kidney parameters (UPCR, eGFR, urine RBCs) between treatment groups. However, the proportion of white patients and patients with Class IV (or IV / V) LN was higher in the abatacept group than the placebo group, while RAIL-scores were comparable between groups (p = 0.75).

[0172] Table 8. Patient characteristics by treatment arm at baseline (week 0). values are medians and interquartile ranges unless stated otherwise SD = standard deviation; UPCR = urine protein / creatinine ratio; eGFR = estimated glomerular filtration rate; RAIL-score = Renal Activity Index for Lupus score

[0173] Kidney parameters at weeks 12, 24, and 52 by treatment group

[0174] Traditional kidney measures showed trends of improvement from baseline by week 12 and continued to further improve at weeks 24 and 52, respectively. Only at week 12 but not at weeks 24 or 52 was the UPCR significantly lower in the abatacept group than the placebo group [median (interquartile range); abatacept vs. placebo: 1.13 (0.55, 2.29) vs. 1.55 (0.72, 3.22); p= 0.04], while other conventional kidney measures (eGFR, UPCR) were similar throughout the study (Table 9).

[0175] Table 9. Kidney responses by study visits and treatment arm. Wilcoxon two- sample test used for continuous variables, and Chi-square test used for categorical variables, f Values are medians and interquartile ranges unless stated otherwise; BL baseline; J RBCs and RBC casts; NR = non-responder; PRR only = partial response but not CRR; CRR = complete response; nl = normal

[0176] Prednisone use was similar in the abatacept group and the placebo group.

[0177] Renal response status and RAIL-scores and their changes over time by renal response status There were 13.6% and 22.2% of patients with CRR and PRR-only at week 12; then 28.5% CRR and 19% PRR only at week 24. At week 52, only 28 patients remained in the study, of which 37.5% and 31.3% met CRR and PRR-only responses. RAIL-scores decreased over time, generally without important differences between treatment groups. Compared to NR, mean (SD) RAIL- scores were lower in patients achieving CRR or PRR-only (Table 10); these differences only reached statistical significance for patients who achieved CRR at weeks 12, 24, and 52, and for those who achieved PRR-only at week 52. These absolute RAIL-scores are shown graphically in FIG. 5. Differences in RAIL-score based per treatment group show similar trends (Table I L).

[0178] Table 10. Absolute RAIL scores by renal response status at each follow-up timepoint. T test was used to compare the means of CRR or PRR vs. NR. Standardized meandifference = [mean(CRR or PRR-only) - mean(NR)] I SD NR = non-responder; PRR only = partial response but not CRR; CRR = complete response; ref = reference value; rcf= reference value.

[0179] Table 11. Absolute RAIL scores by renal response status and treatment group. T test was used to compare the means of two groups. Standardized mean difference = [mean(CRR or PRR only)- mean(NR)] I SD NR = non-responder; PRR only = partial response but not CRR; CRR = complete response; ref = reference value

[0180] Relative changes in RAIL-scores since baseline and the preceding visit were also assessed. Changes were and generally lacked statistically significance, irrespective of whether changes from baseline or since the prior visit were considered (Tables 12 and 13).

[0181] Table 12. Percentage change in RAIL scores from baseline by renal response state.% change = [RAIL(t) - RAIL(t-l)] / RAIL(t). # T test was used to compare the means of two groups. Standardized mean difference = [mean(CRR or PRR only)- mean(NR)] I SD; NR = non- responder; PRR only = partial response but not CRR; CRR = complete response; ref = reference value

[0182] Table 13. Percentage change in RAIL scores between time points by renal response state. Since the prior visit at time (t-1) % change = [RAIL(t) - RAIL(t-l)] / RAIL(t) with t=baseline, week 12, 24 and 52. # T test was used to compare the mean % change of RAIL scorefor the CRR or PRR vs. NR. Standardized mean difference = [mean(% change CRR or PRR only)- mcan(NR)] / SD; NR = non-responder; PRR only = partial response but not CRR; CRR = complete response; ref= reference value

[0183] Accuracy of the RAIL-score, UPCR and eGFR to discriminate LN response status

[0184] As summarized in Table 14, the eGFR was a poor predictor of renal response (AUC all <0.51, p>=0.71 ), irrespective of the comparison (CRR vs PRR-only plus NR; CRR plus PRR-only vs. NR; CRR vs NR) made. Without correction of baseline differences of renal response groups, the RAIL-score had fair accuracy in differentiating response groups, irrespective of the comparison made. However, after adjusting for baseline UPCR, eGFR, age, weight, sex and race, the RAIL- score outperformed the UPCR and had excellent accuracy for differentiating CRR from other renal response states (AUC> 0.83) and had still good accuracy (AUC=0.77) to discriminate patients who achieved at least PRR from NR (FIG. 6 (A+C)). Additional details about RAIL-score values for various levels of sensitivity and specificity are shown in FIG. 8.

[0185] Table 14. Accuracy of the RAIL-score, UPCR and eGFR to discriminate LN response status concurrently or at the next visit. NR = non-responder; PRR only = partial response but not CRR; CRR = complete response; ref = reference value; f adjusted for baseline differences in UPCR, eGFR, age, weight, sex and race with renal response status

[0186] Using RAIL-scores to anticipate future renal response based on the current RAIL-score

[0187] Without adjustment for baseline differences of patients with different response states (CRR, PRR only, NR), the RAIL-score had good accuracy (AUC= 0.70 - 0.71) to discriminate CRR from other response states (Table 14). As shown in FIG. 8, after adjusting for baseline eGFR, UPCR, age, weight, sex and race, the accuracy of the RAIL-score to anticipate CRR at the next visit was excellent (AUC=0.84-0.85, FIG 7 (A+ C)) and had very good ability to differentiate NR from other response states (AUC=0.77; FIG. 8, B). Herein, the eGFR again was not useful to predict response (AUC<0.51) inferior to the UPCR (Table 14).

[0188] DISCUSSION

[0189] Despite therapeutic advances in SLE management, LN remains a common severe disease manifestation, conferring significant morbidity and mortality.1,3-5The RAIL-score, based on a set of urine biomarkers, was developed as a non-invasive tool to help diagnose LN and monitor the course of pediatric LN.19,21Here, Applicant newly demonstrate the ability of the RAIL biomarkers from adult patients with LN to predict future renal response state. Further, it was confirmed that absolute change in RAIL-scores since baseline differentiates renal response states (PRR, CRR, NR). Collectively, these results suggest that the RAIL-score is an effective, non- invasive tool for assessing kidney activity and treatment response over time in adult LN patients.

[0190] Active LN can be discriminated from inactive or absent LN for scores that are 4.25 or higher with 80% accuracy. Patients with CRR could be discriminated from non-rcspondcrs for scores of 4.18 or lower. Different from prior studies in adults with LN, Applicant newly shows that high RAIL scores at a given visit are predictors of future non-response of adults with LN. This finding is consistent with prior observations in pediatric patients with LN that changes in RAIL- score anticipated the clinically observed course at least 3 months earlier. Together, the findings might be exploited in clinical care for the surveillance of LN, especially in patients with known proteinuria from kidney damage in whom treatment resistance to the current treatment regimen is suspected. Indeed, RAIL-scores that remain 6.5 or higher may indicate the need to adjust immunosuppressive therapy or explore non-adherence.

[0191] The shortcoming of proteinuria to gauge LN activity has long been recognized34. For example, proteinuria can reflect kidney damage or even comorbid disease, such as hypertension and diabetes mellitus.35,36Current definition of renal response used in the clinical trial are reliant on changes in proteinuria, eGFR and abnormal urine sediment.27The latter two components were either very rarely abnormal, i.e. urine sediment, or did not contribute to gauging renal response as was the case for the eGFR. In the ALLURE trial, no participant that reached the 1-year endpoint had active sediment. In this study the UPCR performed similarly well as the RAIL-score to predict or identify patients who experienced PRR or CRR as opposed to NR. The observed result might be expected as the definition of CRR and PRR was based on a composite measure that included the UPCR, eGFR, and RBC or RBC casts, with the latter occurring only rarely in the study population. Thus, the RAIL-score provides additional value to the assessment of LN, as the RAIL biomarkers do not reflect kidney damage, different from the UPCR.

[0192] The ALLURE trial in LN was stopped after the assessment of the primary endpoint. Herein Applicant provides supportive evidence for this decision, given that the RAIL-scores in the two treatment arms (abatacept, placebo) remained almost identical over time and at baseline. Notably sustained CRR rates were initially higher with abatacept.

[0193] REFERENCES

[0194] 1. Anders HJ, Saxena R, Zhao MH, et al. Lupus nephritis. Nat Rev Dis Primers2020;6:7. doi: 10.1038 / s41572-019-0141-9

[0195] 2. Yurkovich M, Vostretsova K, Chen W, et al. Overall and cause-specific mortality in patients with systemic lupus erythematosus: a mcta-analysis of observational studies. Arthritis Care Res (Hoboken) 2014;66:608-16. doi: 10.1002 / acr.22173

[0196] 3. Hanly JG, O'Keeffe AG, Su L, et al. The frequency and outcome of lupus nephritis: results from an international inception cohort study. Rheumatology (Oxford) 2016;55:252-62. doi: 10.1093 / rheumatology / kev311

[0197] 4. Hoover PJ, Costenbader KH. Insights into the epidemiology and management of lupus nephritis from the US rheumatologist's perspective. Kidney Int 2016;90:487-92. doi: 10.1016 / j.kint.2016.03.042

[0198] 5. Pryor KP, Barbhaiya M, Costenbader KH, et al. Disparities in Lupus and LupusNephritis Care and Outcomes Among US Medicaid Beneficiaries . Rheum Dis Clin North Am 2021;47:41-53. doi: 10.1016 / j.rdc.2020.09.004

[0199] 6. Suzon B, Louis-Sidney F, Aglae C, et al. Good Long-Term Prognosis of LupusNephritis in the High-Income Afro-Caribbean Population of Martinique with Free Access to Healthcare. J Clin Med 2022;l l. doi: 10.3390 / jcml 1164860

[0200] 7. Bertsias GK, Tektonidou M, Amoura Z, et al. Joint European League AgainstRheumatism and European Renal Association-European Dialysis and Transplant Association (EULAR / ERA-EDTA) recommendations for the management of adult and paediatric lupus nephritis. Ann Rheum Dis 2012;71:1771-82. doi: 10.1136 / annrheumdis-2012-201940

[0201] 8. Davidson A, Aranow C, Mackay M. Lupus nephritis: challenges and progress. CurrOpin Rheumatol 2019;31:682-88. doi: 10.1097 / BOR.0000000000000642

[0202] 9. Bajema IM, Wilhelmus S, Alpers CE, et al. Revision of the International Society of Nephrology / Renal Pathology Society classification for lupus nephritis: clarification of definitions, and modified National Institutes of Health activity and chronicity indices. Kidney Int 2018;93:789-96. doi: 10.1016 / j.kint.2017.11.023

[0203] 10. Weening JJ, D'Agati VD, Schwartz MM, et al. The classification of glomerulonephritis in systemic lupus crythematosus revisited. Kidney Int 2004;65:521-30. doi: 10.1111 / j.1523-1755.2004.00443.x

[0204] 11. Bennett MR, Ma Q, Ying J, et al. Effects of age and gender on reference levels of biomarkers comprising the pediatric Renal Activity Index for Lupus Nephritis (p-RAIL). Pediatr Rheumatol Online J 2017; 15:74. doi: 10.1186 / sl2969-017-0202-0

[0205] 12. Morell M, Perez-Cozar F, Maranon C. Immune-Related Urine Biomarkers for theDiagnosis of Lupus Nephritis. Int J Mol Sci 2021;22. doi: 10.3390 / ijms22137143

[0206] 13. De Rosa M, Azzato F, Toblli IE, et al. A prospective observational cohort study highlights kidney biopsy findings of lupus nephritis patients in remission who flare following withdrawal of maintenance therapy. Kidney Int 2018;94:788-94. doi: 10.1016 / j.kint.2018.05.021

[0207] 14. Malvar A, Pirruccio P, Alberton V, et al. Histologic versus clinical remission in proliferative lupus nephritis. Nephrol Dial Transplant 2017;32:1338-44. doi: 10.1093 / ndt / gfv296

[0208] 15. Zickert A, Sundelin B, Svenungsson E, et al. Role of early repeated renal biopsies in lupus nephritis. Lupus Sci Med 2014; l :e000018. doi: 10.1136 / lupus-2014-000018

[0209] 16. Chedid A, Rossi GM, Peyronel F, et al. Low-Level Proteinuria in Systemic LupusErythematosus. Kidney Int Rep 2020;5:2333-40. doi: 10.1016 / j.ekir.2020.09.007

[0210] 17. Soliman S, Mohan C. Lupus nephritis biomarkers. Clin Immunol 2017;185:10-20. doi: 10.1016 / j.clim.2016.08.001

[0211] 18. Brunner HI, Bennett MR, Abulaban K, et al. Development of a Novel RenalActivity Index of Lupus Nephritis in Children and Young Adults. Arthritis Care Res (Hoboken) 2016;68:1003-11. doi: 10.1002 / acr.22762

[0212] 19. Brunner HI, Bennett MR, Mina R, et al. Association of noninvasively measured renal protein biomarkers with histologic features of lupus nephritis. Arthritis Rheum 2012;64:2687-97. doi: 10.1002 / art.34426

[0213] 20. Gulati G, Bennett MR, Abulaban K, et al. Prospective validation of a novel renal activity index of lupus nephritis. Lupus 2017;26:927-36. doi: 10.1177 / 0961203316684212

[0214] 21. Brunner HI, Bennett MR, Gulati G, et al. Urine Biomarkers to Predict Response toLupus Nephritis Therapy in Children and Young Adults. J Rheumatol 2017;44:1239-48. doi: 10.3899 / jrheum.161128

[0215] 22. Furie RA, Morand EF, Bruce IN, et al. Type I interferon inhibitor anifrolumab in active systemic lupus erythematosus (TULIP-1): a randomised, controlled, phase 3 trial. Lancet Rheumatol 2019;l:e208-el9. doi: 10.1016 / S2665-9913(19)30076-l

[0216] 23. Burki TK. FDA approval for anifrolumab in patients with lupus. The LancetRheumatology 2021;3:e689. doi: 10.1016 / S2665-9913(21)00291-5

[0217] 24. Rogers H, Adeniyi O, Ramamoorthy A, et al. Clinical Pharmacology StudiesSupporting Oligonucleotide Therapy Development: An Assessment of Therapies Approved and in Development Between 2012 and 2018. Clin Transl Sci 2021;14:468-75. doi: 10.1111 / cts.12945

[0218] 25. Shadid M, Badawi M, Abulrob A. Antisense oligonucleotides: absorption, distribution, metabolism, and excretion. Expert Opin Drug Metab Toxicol 2021 ;17: 1281-92. doi: 10.1080 / 17425255.2021.1992382

[0219] 26. European Medicines Agency (EMA). Guideline on the Investigation of DrugInteractions. CPMP / EWP / 560 / 95 / Rev. 1 Corr. 2. London, 2012. https: / / www.ema.europa.eu / en / documents / scientific-guideline / guideline-investigation-drug- interactions-revision-l_en.pdf. Accessed March 14, 2024.

[0220] 27. Food and Drug Administration (FDA). In Vitro Drug Interaction Studies -Cytochrome P450 Enzyme- and Transporter-Mediated Drug Interactions. Guidance for Industry. January 2020. https: / / www.fda.gov / media / 134582 / download. Accessed March 2024.

[0221] 28. Jayne D, Rovin B, Mysler EF, et al. Phase II randomised trial of type I interferon inhibitor anifrolumab in patients with active lupus nephritis. Ann Rheum Dis 2022;81:496-506. doi: 10.1136 / annrheumdis-2021-221478

[0222] 29. Hochberg MC. Updating the American College of Rheumatology revised criteria for the classification of systemic lupus erythematosus. Arthritis Rheum 1997;40:1725. doi: 10.1002 / art.1780400928

[0223] 30. Suzuki M, Wiers K, Brooks EB, et al. Initial validation of a novel protein biomarker panel for active pediatric lupus nephritis. Pediatr Res 2009;65:530-6. doi: 10.1203 / PDR.0b013e31819e4305

[0224] 31. Aljaberi N, Bennett M, Brunner HI, et al. Proteomic profiling of urine: implications for lupus nephritis. Expert Rev Proteomics 2019;16:303-13. doi: 10.1080 / 14789450.2019.1592681

[0225] 32. Austin HA, 3rd, Muenz LR, Joyce KM, et al. Diffuse proliferative lupus nephritis: identification of specific pathologic features affecting renal outcome. Kidney Int 1984;25:689-95. doi: 10.1038 / ki.1984.75

[0226] 33. Cody EM, Wenderfer SE, Sullivan KE, et al. Urine biomarker score captures response to induction therapy with lupus nephritis. Pediatr Nephrol 2023;38:2679-88. doi: 10.1007 / s00467-023-05888-z

[0227] 34. Weeding E, Fava A, Magder L, et al. One-third of patients with lupus nephritis classified as complete responders continue to accrue progressive renal damage despite resolution of proteinuria. Lupus Science & Medicine 2022;9(l):e000684.

[0228] 35. Kochi M, Kohagura K, Oshiro N, et al. Association of blood pressure and hyperuricemia with proteinuria and reduced renal function in the general population. Hypertension Research 2023;46(7): 1662-72.

[0229] 36. Khoury CC, Chen S, Ziyadeh FN. Pathophysiology of diabetic nephropathy.Chronic Renal Disease: Elsevier 2020:279-96.

[0230] 37. Vodehnal S, Mohan C. Urinary biomarkers for active Lupus Nephritis that have survived independent validation across cohorts. Kidney International 2024;106:1135-45.

[0231] All percentages and ratios are calculated by weight unless otherwise indicated.

[0232] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0233] Exemplary Combinations

[0234] The following examples relate to various non-exhaustive ways in which the teachings herein may be combined or applied. It should be understood that the following examples are not intended to restrict the coverage of any claims that may be presented at any time in this application or in subsequent filings of this application. No disclaimer is intended. The following examples are being provided for nothing more than merely illustrative purposes. It is contemplated that the various teachings herein may be arranged and applied in numerous other ways. It is also contemplated that some variations may omit certain features referred to in the below examples. Therefore, none of the aspects or features referred to below should be deemed critical unless otherwise explicitly indicated as such at a later date by the inventors or by a successor in interest to the inventors. If any claims are presented in this application or in subsequent filings related to this application that include additional features beyond those referred to below, those additional features shall not be presumed to have been added for any reason relating to patentability.

[0235] Example 1

[0236] A method treating an adult individual for active lupus nephritis (LN) comprising detecting a biomarker level for each of a biomarker set comprising, consisting of, or consisting essentially of neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule 1 (KIM- 1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin, and ceruloplasmin in a urine sample obtained from the individual at a first time point and a subsequent time point; calculating a first RAIL score based on the biomarker level detected at the first time point; calculating a subsequent RAIL score based on the biomarker level detected at the subsequent time point; determining whether the individual is responding to the therapy based on a comparison of the first RAIL score and the subsequent RAIL score; and continuing, ceasing, or adjusting the therapy based on the comparison of the first RAIL score and subsequent RAIL score.

[0237] Example 2

[0238] The method of example 1 , further comprising determining whether the individual will achieve no renal response, a complete renal response (CRR), or a partial renal response (PRR), based on the comparison of the first RAIL score and subsequent RAIL score.

[0239] Example 3

[0240] The method of example 1 or 2, wherein one or both of the first and subsequent time points are during administration of an induction therapy to the individual.

[0241] Example 4

[0242] The method of any preceding example, where the first time point is immediately prior to, or at the initiation of an induction therapy, and the subsequent time point is at least or about 12 weeks after initiation of an induction therapy.

[0243] Example 5

[0244] The method of any preceding example, where the first time point is immediately prior to, or at the initiation of an induction therapy, and the subsequent time point is at least or about 24 weeks after initiation of an induction therapy.

[0245] Example 6

[0246] The method of any preceding example, the first and subsequent time point being at least one week apart, or at least two weeks apart, or at least three weeks apart, or at least four weeks apart, or more than four weeks apart.

[0247] Example 7

[0248] The method of any preceding example, the first and subsequent time point being at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months, or at least seven months, or at least eight months apart, or at least nine months apart, or at least ten months apart, or more than ten months apart.

[0249] Example 8

[0250] The method of any preceding example, the individual having LN with a National Institute of Health-Index Score (NIH-AI) score of less than 2.

[0251] Example 9

[0252] The method of any preceding example, the individual having LN with an NIH-AI score of 2-10.

[0253] Example 10

[0254] The method of any preceding example, the individual having LN with an NIH-AI score of greater than 10.

[0255] Example 11

[0256] The method of any preceding example, wherein the RAIL score is calculated according to the following equation RAIL = -0.21 +0.67*NGAL +0.28*MCP-l -0.12*Ceruloplasmin +0.88*Adiponectin -0.25*Hemopexin -0.05*KIM-l.

[0257] Example 12

[0258] The method of any preceding example, wherein a decrease of from about 0.2 to about 1.0 or about 0.3 to about 0.9, or about 0.4 to about 0.8, or about 0.5 to about 0.7 about 0.6 in the RAIL scores indicates that the individual is a partial responder to the induction therapy.

[0259] Example 13

[0260] The method of any preceding example, wherein a decrease of about 1.2 in the RAIL scores indicates that the individual is a complete responder (likely to have complete renal response) to the induction therapy.

[0261] Example 14

[0262] The method of example 13, the induction therapy being my cophenolate mofetil (MMF).

[0263] Example 15

[0264] The method of example 13, the induction therapy being RAAS blocker.

[0265] Example 16

[0266] The method of example 13, the induction therapy being cyclophosphamide.

[0267] Example 17

[0268] The method of example 13, the induction therapy being anifrolumab.

[0269] Example 18

[0270] The method of any preceding example, the induction therapy comprising one or more of anifrolumab, abatacept, corticosteroid, antimalarial, azathioprine, mizoribine, mycophenolate mofetil (MMF), mycophenolic acid, methotrexate, rituximab, and combinations thereof.

[0271] Example 19

[0272] The method of example 13, the induction therapy comprising an induction dose of anifrolumab of 300 mg, or 900 mg for the first three doses and then 300 mg thereafter, every four weeks.

[0273] Example 20

[0274] The method of example 19, further comprising MMF and corticosteroid.

[0275] Example 21

[0276] The method of any preceding example, the individual having active Class III LN.

[0277] Example 22

[0278] The method of any preceding example, the individual having active Class IV LN.

[0279] Example 23

[0280] The method of any preceding example, the individual having active Class V LN.

[0281] Example 24

[0282] The method of any preceding example, wherein the detecting is carried out in a single sample via a multiplex assay, wherein each biomarker is detected in a single urine sample.

[0283] Example 25

[0284] The method of any preceding example, wherein the detecting is canned out in a single sample via a single plex assay, via ELISA.

[0285] Example 26

[0286] The method of any preceding example, wherein the detecting is carried out in a single sample via a Luminex technique.

[0287] Example 27

[0288] The method of any preceding example, wherein the detecting is carried out in a single sample via a MSD technique.

[0289] Example 28

[0290] A method for discriminating active LN from inactive or absent LN in an individual diagnosed with LN, comprising determining a RAIL score in the individual, and determining that the individual has active LN when the individual has a RAIL score of 4.25 or higher.

[0291] Example 29

[0292] The method of example 28, further comprising administering a treatment for LN to the individual having active LN.

[0293] Example 30

[0294] A method for determining that an individual having LN is likely to have a complete renal response (CRR) to a therapy, comprising determining a RAIL score in the individual, and determining that the individual is likely to have a CRR when the individual has a RAIL score of 4.18 or lower.

[0295] Example 31

[0296] The method of example 30 wherein the individual is an adult.

[0297] Example 32

[0298] The method of example 30 or 31 , wherein the RAIL score is determined based on a urine sample obtained from the individual.

[0299] Example 33

[0300] The method of any one of examples 30-32, wherein the RAIL score is determined prior to initiation of a therapy for LN and / or during administration of a therapy for LN.

[0301] Example 34

[0302] A method for determining that an individual is likely to be a non-responder to a therapy, comprising determining a RAIL score in the individual, and determining that the individual is likely to be a non-responder when the RAIL score is 6.5 or higher.

[0303] Example 35

[0304] The method of example 34 wherein the individual is an adult.

[0305] Example 36

[0306] The method of example 34 or 35, wherein the RAIL score is determined based on a urine sample obtained from the individual.

[0307] Example 37

[0308] The method of any one of examples 34-36, wherein the RAIL score is determined prior to initiation of a therapy for LN and / or during administration of a therapy for LN.

[0309] Example 38

[0310] A method of treating an individual for LN, comprising determining a RAIL score in the individual, and, where the RAIL score is 6.5 or higher, adjust a therapy being administered to the individual or explore non-adherence to therapy in the individual.

[0311] Example 39

[0312] The method of example 38 wherein the individual is an adult.

[0313] Example 40

[0314] The method of example 38 or 39, wherein the RAIL score is determined based on a urine sample obtained from the individual.

[0315] Example 41

[0316] The method of any one of examples 38-40, wherein the RAIL score is determined prior to initiation of a therapy for LN and / or during administration of a therapy for LN.

[0317] Example 42

[0318] The method of any one of examples 30-41, the therapy being selected from one or more of anifrolumab, abatacept, mycophenolate mofetil (MMF), a RAAS blocker, cyclophosphamide, corticosteroid, antimalarial, azathioprine, mizoribine, mycophenolate mofetil (MMF), mycophenolic acid, methotrexate, rituximab, and combinations thereof.

[0319] It should be understood that every maximum numerical limitation given throughout this specification includes every lower numerical limitation, as if such lower numerical limitations were expressly written herein. Every minimum numerical limitation given throughout this specification will include every higher numerical limitation, as if such higher numerical limitations were expressly written herein. Every numerical range given throughout this specification will include every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein.

[0320] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each suchdimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about 20 mm.”

[0321] Every document cited herein, including any cross referenced or related patent or application, is hereby incorporated herein by reference in its entirety unless expressly excluded or otherwise limited. All accessioned information (for example, as identified by PUBMED, PUBCHEM, NCBI, UNIPROT, or EBI accession numbers) and publications in their entireties are incorporated into this disclosure by reference in order to more fully describe the state of the art as known to those skilled therein as of the date of this disclosure. The citation of any document is not an admission that it is prior art with respect to any invention disclosed or claimed herein or that it alone, or in any combination with any other reference or references, teaches, suggests or discloses any such invention. Further, to the extent that any meaning or definition of a term in this document conflicts with any meaning or definition of the same term in a document incorporated by reference, the meaning or definition assigned to that term in this document shall govern.

[0322] While particular embodiments of the present invention have been illustrated and described, it would be obvious to those skilled in the art that various other changes and modifications may be made without departing from the spirit and scope of the invention. It is therefore intended to cover in the appended claims all such changes and modifications that are within the scope of this invention.

Claims

CLAIMSWhat is claimed is:

1. A method of treating an adult individual for active lupus nephritis (LN) comprising(a) detecting a biomarker level for each of a biomarker set comprising, consisting of, or consisting essentially of neutrophil gelatinase associated lipocalin (NGAL), kidney injury molecule 1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin, and ceruloplasmin in a urine sample obtained from the individual at a first time point and a subsequent time point;(b) calculating a first RAIL score based on the biomarker level detected at the first time point;(c) calculating a subsequent RAIL score based on the biomarker level detected at the subsequent time point;(d) determining whether the individual is responding to a therapy based on a comparison of the first RAIL score and the subsequent RAIL score; and(e) continuing, ceasing, or adjusting the therapy based on the comparison of the first RAIL score and subsequent RAIL score.

2. The method of claim 1 , further comprising determining whether the individual will achieve no renal response, a complete renal response (CRR), or a partial renal response (PRR), based on the comparison of the first RAIL score and subsequent RAIL score.

3. The method of claim 1 or 2, wherein the therapy is an induction therapy and wherein one or both of the first and subsequent time points are during administration of the induction therapy to the individual.

4. The method of any preceding claim, wherein the therapy is an induction therapy and wherein the first time point is immediately prior to, or at an initiation of an inductiontherapy, and the subsequent time point is at least or about 12 weeks after initiation of the induction therapy.

5. The method of any preceding claim, wherein the therapy is an induction therapy and wherein the first time point is immediately prior to, or at the initiation of the induction therapy, and the subsequent time point is at least or about 24 weeks after initiation of the induction therapy.

6. The method of any preceding claim, the first and subsequent time point being at least one week apart, or at least two weeks apart, or at least three weeks apart, or at least four weeks apart, or more than four weeks apart.

7. The method of any preceding claim, the first and subsequent time point being at least one month, or at least two months, or at least three months, or at least four months, or at least five months, or at least six months, or at least seven months, or at least eight months apart, or at least nine months apart, or at least ten months apart, or more than ten months apart.

8. The method of any preceding claim, the individual having LN with a National Institute of Health-Index Score (NIH-AI) score of less than 2.

9. The method of any preceding claim, the individual having LN with an NIH-AI score of 2-10.

10. The method of any preceding claim, the individual having LN with an NIH-AI score of greater than 10.

11. The method of any preceding claim, wherein the RAIL score is calculated according to the following equation RAIL = -0.21 +0.67*NGAL +0.28*MCP-l -0.12*Ceruloplasmin +0.88*Adiponectin -0.25*Hemopexin -0.05*KIM-l.

12. The method of any preceding claim, wherein a decrease of from about 0.2 to about 1.0 or about 0.3 to about 0.9, or about 0.4 to about 0.8, or about 0.5 to about 0.7 about 0.6 in the RAIL scores indicates that the individual is a partial responder to the induction therapy.

13. The method of any preceding claim, wherein a decrease of about 1.2 in the RAIL scores indicates that the individual is a complete responder (likely to have complete renal response) to the induction therapy.

14. The method of claim 13, the induction therapy being mycophenolate mofetil (MMF).

15. The method of claim 13, the induction therapy being RAAS blocker.

16. The method of claim 13, the induction therapy being cyclophosphamide.

17. The method of claim 13, the induction therapy being anifrolumab.

18. The method of any preceding claim, the induction therapy comprising one or more of anifrolumab, abatacept, corticosteroid, antimalarial, azathioprine, mizoribine, mycophenolate mofetil (MMF), mycophenolic acid, methotrexate, rituximab, and combinations thereof.

19. The method of claim 13, the induction therapy comprising an induction dose of anifrolumab of 300 mg, or 900 mg for a first three doses and then 300 mg thereafter, every four weeks.

20. The method of claim 19, further comprising MMF and corticosteroid.

21. The method of any preceding claim, the individual having active Class III LN.

22. The method of any preceding claim, the individual having active Class IV LN.

23. The method of any preceding claim, the individual having active Class V LN.

24. The method of any preceding claim, wherein the detecting is carried out in a single sample via a multiplex assay, wherein each biomarker is detected in a single urine sample.

25. The method of any preceding claim, wherein the detecting is carried out in a single sample via a single plex assay, via ELISA.

26. The method of any preceding claim, wherein the detecting is carried out in a single sample via a Luminex technique.

27. The method of any preceding claim, wherein the detecting is carried out in a single sample via a MSD technique.

28. A method for discriminating active LN from inactive or absent LN in an individual diagnosed with LN, comprising determining a RAIL score in the individual, and determining that the individual has active LN when the individual has a RAIL score of 4.25 or higher.

29. The method of claim 28, further comprising administering a therapy for LN to the individual having active LN.

30. A method for determining that an individual having LN is likely to have a complete renal response (CRR) to a therapy, comprising determining a RAIL score in the individual, and determining that the individual is likely to have a CRR when the individual has a RAIL score of 4.18 or lower.

31. The method of claim 30 wherein the individual is an adult.

32. The method of claim 30 or 31, wherein the RAIL score is determined based on a urine sample obtained from the individual.

33. The method of any one of claims 30-32, wherein the RAIL score is determined prior to initiation of a therapy for LN and / or during administration of a therapy for LN.

34. A method for determining that an individual is likely to be a non-responder to a therapy, comprising determining a RAIL score in the individual, and determining that the individual is likely to be a non-responder when the RAIL score is 6.5 or higher.

35. The method of claim 34 wherein the individual is an adult.

36. The method of claim 34 or 35, wherein the RAIL score is determined based on a urine sample obtained from the individual.

37. The method of any one of claims 34-36, wherein the RAIL score is determined prior to initiation of a therapy for LN and / or during administration of a therapy for LN.

38. A method of treating an individual for LN, comprising determining a RAIL score in the individual, and, where the RAIL score is 6.5 or higher, adjust a therapy being administered to the individual or explore non-adherence to therapy in the individual.

39. The method of claim 38 wherein the individual is an adult.

40. The method of claim 38 or 39, wherein the RAIL score is determined based on a urine sample obtained from the individual.

41. The method of any one of claims 38 through 40, wherein the RAIL score is determined prior to initiation of a therapy for LN and / or during administration of a therapy for LN.

42. The method of any one of claims 30 through 41, the therapy being selected from one or more of anifrolumab, abatacept, mycophenolate mofetil (MMF), a RAAS blocker, cyclophosphamide, corticosteroid, antimalarial, azathioprine, mizoribine, mycophenolate mofetil (MMF), mycophenolic acid, methotrexate, rituximab, and combinations thereof.

Images