Methods for the treatment of lupus nephritis

Abstract

Disclosed are methods for non-invasively classifying an individual diagnosed with lupus nephritis (LN) as a responder, a partial responder or a non-responder to lupus nephritis induction therapy. The method generally comprises (a) comparing a first Renal Activity Index for Lupus (RAIL) score and a second RAIL score, based on the detection of neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molcculc-1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin and ceruloplasmin in a urine sample from the individual. Based on the RAIL score, the individual can be classified as a responder, a partial responder or a complete responder, and therapy can be appropriately adjusted.

Description

METHODS FOR THE TREATMENT OF LUPUS NEPHRITISCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application is a nonprovisional application which claims priority to and benefit of U.S. Serial No. 63 / 538,565, filed September 15, 2023, the contents of which are incorporated in their entirety for all purposes.STATEMENT REGARDING FEDERALLY-SPONSORED RESEARCH

[0002] This invention was made with government support under DK096418 awarded by the National Institutes of Health. The government has certain rights in the invention.BACKGROUND

[0003] Kidney involvement with systemic lupus erythematosus (SLE), or lupus nephritis (LN), continues to account for sizable morbidity, mortality, and kidney failure. An estimated 20% of SLE cases begin in childhood onset (cSLE), and LN occurs in 32-37% of those cases, more frequently than in adult onset SLE. Current clinical and laboratory measures fail to diagnose LN or capture LN activity reliably; thus, invasive kidney biopsies are required for diagnosis, treatment, and prognosis. A recent study indicates that approximately 30% of LN patients clinically classified as complete responders exhibit residual kidney inflammation upon repeat biopsy after completing induction therapy. This misclassification of non-responders as responders, especially during the induction phase, can lead to ineffective treatment and a missed opportunity for timely intervention. Consequently, misidentifying a patient as a complete responder during induction significantly reduces the chances of providing effective treatment and achieving a positive outcome. Therefore, there is a critical need to improve the identification of patients who, under current protocols, may be incorrectly categorized as complete responders but would benefit from a modification in their initial treatment regimen — particularly during the induction therapy phase. The present disclosure aims to address one or more of these unmet needs.BRIEF SUMMARY

[0004] Disclosed are methods for non-invasively classifying an individual diagnosed with lupus nephritis (LN) as a responder, a partial responder or a non-responder to lupus nephritisinduction therapy. The method generally comprises (a) comparing a first Renal Activity Index for Lupus (RAIL) score and a second RAIL score, based on the detection of neutrophil gelatinase- associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin and ceruloplasmin in at least two urine samples from the individual, obtained at two different time points. Based on a comparison of the two RAIL scores, the individual can be classified as a responder, a partial responder or a complete responder, and therapy can be appropriately maintained or adjusted.BRIEF DESCRIPTION OF THE DRAWINGS

[0005] 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.

[0006] FIG. 1 depicts UPCR in mg / mg (orange / open circles, r = 0.33) and pRAIL scores (blue / closed circles, r = 0.40) for all patients with active disease (NIH-AI) diagnosed by biopsy.

[0007] FIG. 2 depicts the change in absolute RAIL score by responder status. Complete responders had decrease in pRAIL scores, partial responders had no change, and non-responders had an increase in pRAIL scores

[0008] FIG. 3. Panel A depicts the NIH-AI score available during induction therapy by the pRAIL score. The blue / solid line shows pre-treatment values (r = 0.46) and the orange / dashed line shows post- treatment values (r = 0.37). Panel B depicts the available NIH-AI by UPCR in mg / mg during induction therapy. The blue / solid line shows pre-treatment values (r = 0) and the orange / dashed line shows posttreatment values (r = 0.1)

[0009] FIG. 4. ROC curve of creatinine-adjusted RAIL scores in both LN and active-SLE- groups. 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 4.56DETAILED DESCRIPTIONDEFINITIONS

[0010] 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. Exemplary embodiments including methods and / or 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 in the treatment of lupus nephritis, particularly during an induction phase of the treatment. In aspects, 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 in the diagnosis of an individual having lupus nephritis.

[0011] 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.

[0012] 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, e.g., 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, unlessotherwise stated the term “about” meaning within an acceptable error range for the particular value should be assumed.

[0013] 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.

[0014] The terms “individual,” “host,” “subject,” and “patient” are used interchangeably to refer to a mammal 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 aspects, the terms refer to humans. In further aspects, the terms may refer to children. In aspects, the individual is a child who is 18 years of age or younger.Induction Therapy and Identification of Responder Status

[0015] cSLE remains a diagnostic and therapeutic challenge, particularly lupus nephritis (LN). Current clinical and laboratory measure do not diagnose LN or capture LN activity reliably, thus requiring invasive kidney biopsies for diagnosis, treatment, and prognosis of LN. Disclosed are methods for the identification of responder status in an individual having LN. In aspects, the individual having LN has Class III. In aspects, the individual having LN has Class IV LN. In aspects, the individual having LN has Class V LN. In further aspects, the individual having LN has cSLE. As disclosed herein, responder status - which refers to the expected therapeutic trajectory of the individual to the administered therapeutic - may be determined in the individual during the induction phase of a therapy. Induction therapy (i.e., initial or induction phase treatment), typically involves an aggressive, short-term administration of a therapy with the aim of rapidly controlling or reducing the severity of a disease. In the context of LN, a severe manifestation of cSLE, induction therapy is generally involves an intensive treatment designed to control inflammation and mitigate kidney damage. Standard immunosuppressive therapies for active LN that may be administered during the induction stage include, for example, intravenouscyclophosphamide and oral mycophenolate mofetil, which are administered with the intent of achieving a complete therapeutic response. In general, the duration of induction therapy lasts about 3 months to 1 year, in which an individual is administered, for example, a glucocorticoid with either MMF or intravenous cyclophosphamide, such as administration of 0.5-1 mg / kg / d prednisolone (max. 80 mg / d) followed by a gradual tapering over a period of 3 to 6 months. Responses to these treatments, however, can vary significantly among patients, and an ineffective response to induction therapy can compromise patient care. That is, effective induction therapy ideally reduces kidney inflammation and risk of kidney damage, whereas administration of an ineffective treatment increases the likelihood of a negative outcome.

[0016] Prior to Applicant’s invention, there has been no reliable method for identifying patients who are responding to the induction therapy, but who have less than a full (“complete”) therapeutic response, nor has such a category of patients been previously recognized. Applicant has 1) identified this new subset of individuals, termed “partial responders,” who have less than a complete response to a therapy but which are not non-responders, and 2) have developed a protocol whereby these partial responders can be identified, based on detection and quantification of the RAIL biomarkers.

[0017] Thus, the disclosed methods allow for the identification of a novel subset of patients, referred to as “partial responders,” who can be identified during the induction phase of a treatment for LN, in comparison to non-responders and complete responders. A “complete responder” is defined as an individual having a “complete response” to induction therapy. “Complete response to induction therapy,” or “complete kidney remission,” is defined as NIH-AI as < 4 on repeat kidney biopsy. In contrast, a “partial responder” is defined an individual having a “partial response” to induction therapy. “Partial response” to induction therapy, or “partial kidney remission,” is defined as UPCR improvement by > 50% plus at least stable eGFR compared to LN diagnosis.

[0018] Partial responders represent a previously unrecognized group who may benefit from an intcrv ention in the standard induction protocol due to an incomplete response to the therapy, particularly an induction phase of a therapy. These patients do not achieve a complete response to therapy but also cannot be classified as non-responders, in that the partial responders have someresponse to therapy as determined by kidney inflammation. Because partial responders mount a partial response, it is particularly challenging to identify these individuals using methods currently known in the ail. However, by identifying a partial responder via the disclosed methods, a change in therapy (such as an escalation of therapy including increased dosage or frequency of dosing, administration of an additional therapy, or administration of an alternative therapy) can be carried out during the induction phase of therapy to increase the odds of a successful outcome. By altering the therapy based on this newly identified status (i.e., based on identifying a patient as a partial responder) during the induction phase of a treatment, the individual can be more effectively treated via a different and / or additional and / or escalated regimen. Unlike prior methods, which failed to distinguish this group of patients, the disclosed methods may be used to recognize and address the needs of partial responders, ultimately leading to better health outcomes and a more refined approach to managing LN in the context of cSLE.RAIL Biomarkers

[0019] Applicant has previously described a composite score, termed the Renal Activity Index for Lupus (“RAIL”), based on the detection and measurement of the six following urine proteins: neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin and ceruloplasmin. Higher RAIL scores represent more active inflammation on biopsy, whereas lower RAIL scores represent less active inflammation on biopsy.

[0020] Use of the RAIL biomarkers and quantification thereof has been described, for example in Brunner, Hermine I et al. “Development of a Novel Renal Activity Index of Lupus Nephritis in Children and Young Adults.” Arthritis care & research vol. 68,7 (2016): 1003-11. doi:10.1002 / acr.22762. Each of the RAIL proteins may be detected and quantified based on methods known in the art, for example via an Enzyme-Linked Immunosorbent Assay (ELISA) assay, as described in the methods below. For example, NGAL may be quantified by ELISA (Human NGAL ELISA; Bioporto, Grusbakken, Denmark). MCP-1 may be quantified by ELISA (R&D Systems, Minneapolis, MN); Ceruloplasmin may be quantified by ELISA (Assaypro, St.Charles, MO); Adiponectin may be quantified using the Quantikine ELISA Human HMW Adiponectin / Acrp30 (R&D Systems, Minneapolis, MN); hemopexin may be quantified with theAssayMax Human Hemopexin ELISA Kit (Assaypro, St. Charles, MO). KIM-1 may be quantified using commercially available reagents (Duoset DY1750, R & D Systems, Minneapolis, MN) as described in Chaturvedi S, Fanner T, Kapke GF. Assay validation for KIM-1: human urinary renal dysfunction biomarker. Int J Biol Sci. 2009;5(2):128-134. doi:10.7150 / ijbs.5.128, also reproduced below. Urine creatinine may be quantified using a modified Jaffe reaction on a Dimension Xp and plus HM Clinical Analyzer (Siemens, Munich, Germany).

[0021] While use of the RAIL score is known, the use of RAIL scores to distinguish varying responses to induction therapy in patients with lupus nephritis (LN) has not been described. Herein, methods using a RAIL score to classify the individual into one of three categories: non-responder, complete responder, or partial responder, is disclosed, wherein the non-responder is effectively non-responsive to (not improving in response to) an induction therapy, the complete responder is effectively responsive to (maximally or close to maximally responding to) an induction therapy, and wherein the partial-responder is achieving a detectable level of response to the induction therapy, but whose response is minimally therapeutic or substantially sub-therapeutic such that a change in therapy is warranted. Thus, in aspects, the disclosed methods may be used to categorize individuals with cSLE and LN based on at least two RAIL scores determined from at least two urine samples in the individual, obtained at at least two time points, as described herein, for the purpose of personalizing treatment of the individual having LN.Detection of RAIL Biomarkers

[0022] Classification of the individual is determined, in part, based on the detection and quantification of the RAIL biomarkers at at least two time points, in at least two different samples obtained from the individual. For example, in aspects, the method comprises detecting the RAIL biomarkers in a first sample obtained at a first time point, and a second sample obtained at a second time point. The described methods may be carried out in conjunction with a typical induction therapy as would be understood by one of ordinary skill in the art.

[0023] In general, the RAIL biomarkers are detected in at least a first sample and a second sample, wherein RAIL scores are determined for each sample, and the RAIL scores are compared as described herein. However, it should be understood that the methods further contemplate detection of the RAIL biomarkers in a third sample, a fourth sample, a fifth sample, or more thanfive samples, for the purpose of determining additional RAIL scores which may be compared to a RAIL score of an earlier sample, such as the baseline sample, for the purpose of determining responder status.

[0024] In aspects, the first sample is the “baseline” sample, which is generally obtained at the outset (before, or at the beginning) of induction therapy. In general, the first sample (e.g., the “baseline” sample) is typically obtained at the outset of induction therapy, whereas later samples are obtained following initiation of the induction therapy at a point in time at which a response to the therapy is expected. For example, in aspects, the first sample may be obtained at a time point selected from at the time of renal biopsy, at initiation of induction therapy (e.g., simultaneously to beginning therapy or day of therapy initiation), prior to initiation of induction therapy, within a day of initiation of induction therapy, within two days of initiation of induction therapy, within three days of initiation of induction therapy, within four days of initiation of induction therapy, within five days of initiation of induction therapy, within six days of initiation of induction therapy, within seven days of initiation of induction therapy, within eight days of initiation of induction therapy, within nine days of initiation of induction therapy, within 10 days of initiation of induction therapy, within 11 days of initiation of induction therapy, within 12 days of initiation of induction therapy, within 13 days of initiation of induction therapy, or within 14 days of initiation of induction therapy, or within a month of initiation of induction therapy, or within two months of initiation of induction therapy, or within three months of initiation of induction therapy, or within four months of initiation of induction therapy, or within five months of initiation of induction therapy, or within six months of initiation of induction therapy, or within seven months of initiation of induction therapy, or within eight months of initiation of induction therapy, or within nine months of initiation of induction therapy.

[0025] In aspects, the second (or subsequent, i.e., third, fourth, fifth, or more) urine sample may be obtained at a time point selected from a day after initiation of induction therapy, two days after initiation induction therapy, three days after initiation of induction therapy, four days after initiation of induction therapy, five days after initiation of induction therapy, six days after initiation of induction therapy, seven days after initiation of induction therapy, eight days after initiation of induction therapy, nine days after initiation of induction therapy, 10 days after initiation of induction therapy, 11 days after initiation of induction therapy, 12 days after initiationof induction therapy, 13 days after initiation of induction therapy, 14 days after initiation of induction therapy, a month after initiation of induction therapy, about two months after initiation of induction therapy, about three months after initiation of induction therapy, about four months after initiation of induction therapy, about five months after initiation of induction therapy, about six months after initiation of induction therapy, about seven months after initiation of induction therapy, about eight months after initiation of induction therapy, or about nine months or more than nine months after initiation of induction therapy. In aspects, the subsequent sample may be obtained following a given dose of the therapy, for example, after a first dose, after a second dose, after a third dose, after a fourth dose, after a fifth dose, after a sixth dose, after a seventh dose, after an eight those, after a ninth dose, or after the tenth dose.

[0026] In further aspects, the first and second (or subsequent) samples are obtained at a given interval. For example, the first and second (or subsequent) dose may be obtained at least two weeks apart, or at least three weeks apart, or at least four weeks apart, or at least five weeks apart, or at least six weeks apart, or at least seven weeks apart, or at least eight weeks apart, or at least nine weeks apart, or at least 10 weeks apart, or at least 11 weeks apart, or at least 12 weeks apart, or more than 3 months apart or more than 4 months apart or more than 5 months apart.RAIL Algorithm and RAIL Score

[0027] The method further comprises determination of a RAIL score for each sample obtained as described above, as calculated using a RAIL algorithm. The responder type of the individual may then be determined based on the RAIL score, as described herein.

[0028] In aspects, the pediatric RAIL score, or “pRAIL” score is employed, in which the pRAIL score of the first sample and second (or subsequent) sample is compared to classify the individual as a non-responder, a complete responders, or a partial responder based on that comparison. In general, the pRAIL score may be used for an individual of an age of birth to 18 years of age.

[0029] The pRAIL score is determined using the pRAIL algorithm, as provided below:Pediatric RAIL GpRAIL”) (with or without creatinine standardization) = -4.29 - 0.34 x -NGAL - 0.06 x ceruloplasmin + 0.89 x MCP-1 + 0.18 x adiponcctin - 0.65 x hemopexin + 0.62 x KIM-1

[0030] In aspects, the RAIL score is standardized. In other aspects, the RAIL score is nonstandardized. In aspects, the RAIL score may be standardized via normalization of the RAIL biomarkers to creatine in the urine sample obtained from the individual to account for variations in urine concentration and applied to the disclosed methods. In aspects, the RAIL score is determined and applied in the disclosed methods without being creatine normalized.Comparison of the RAIL Scores and Classification of Responder Status

[0031] In aspects, following the determination of a first (baseline) and second (subsequent) RAIL score, the scores are compared for classifying the individual for determining an appropriate treatment. In aspects, the method comprises, based on a comparison of the RAIL score of two urine samples obtained at two different time points, classifying an individual having active lupus nephritis as either a complete responder to therapy, a non-responder to therapy), or a partialresponder to therapy.

[0032] In certain aspects, an individual is classified as a complete responder when a second (or subsequent) RAIL score shows a statistically significant decrease compared to a prior (baseline) score. Specifically, an individual may be diagnosed as a complete responder when the RAIL score, based on the detection of biomarkers in a urine sample from the individual, demonstrates a decrease compared to the baseline / previous value. In some aspects, this decrease in the RAIL score is at least about 0.5, or greater than 0.5, compared to the baseline / previous value. Alternatively, in other aspects, the decrease is at least about 1, or greater than 1, compared to the baseline / previous value. Furthermore, in some aspects, the decrease in the RAIL score is at least 0.5, or at least 1, when compared to a value obtained during the induction phase, irrespective of the baseline value.

[0033] In certain aspects, an individual is classified as a non-responder when a second (or subsequent) RAIL score shows an increase compared to a prior RAIL score. Specifically, an individual may be diagnosed as a non-responder when the RAIL score, based on the detection ofbiomarkers in a urine sample from the individual, increases compared to the baseline / earlier value. In some aspects, the increase in the RAIL score is at least about 0.5, or greater than 0.5, compared to the baseline / earlier value. Alternatively, in other aspects, the increase in the RAIL score is at least about 1 , or greater than 1 , compared to the baseline / earlier value.

[0034] In aspects, an individual is classified as a partial responder when a second (or subsequent) RAIL score remains essentially unchanged compared to a prior score. Specifically, an individual may be classified as a partial responder when the change in a the RAIL score is less than 0.5 in either direction compared to the prior RAIL score, or less than 1 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than 0.1 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than 0.2 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than 0.3 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than 0.4 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than 0.5 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than 0.6 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than 0.7 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than 0.8 in either direction compared to the prior RAIL score. In further aspects, an individual may be classified as a partial responder when the change in the RAIL score is less than or 0.9 in either direction compared to the prior RAIL score. In aspects, the individual is classified as a partial responder where the individual is not classified as a responder or nonresponder in accordance with the disclosed methods.Administration of a Treatment Based on Responder Status

[0035] As noted above, Applicant has identified a previously unrecognized class of patients with LN who respond to induction therapy only in part, and are neither responders nor nonresponders. Thus, in aspects, the methods allow for a personalized treatment for this new subtype of patient. Based on determination of a RAIL score obtained at two different time points in therapy, an individual may be identified as a partial responder and may be administered an increased amount of an immunosuppressive therapy. For example, an individual may be identified as a partial responder and may be administered an increased amount of an initial induction therapy comprising administration of a glucocorticoid. Yet further, an individual may be identified as a partial responder and may be administered an increased amount of an initial induction therapy comprising administration of a glucocorticoid combined with cyclophosphamide and / or my cophenolate mofetil.

[0036] In a further aspect, an individual may be identified as a partial responder and the individual may be administer an entirely different therapy as compared to the initial therapy administered during the induction phase. For example, the initial therapy may be discontinued and the individual may be administered belimumab. In a further example, the initial therapy may be discontinued and the individual may be administered voclosporin. In a further example, the initial therapy may be discontinued and the individual may be administered obinutuzumab. In a further example, the initial therapy may be discontinued and the individual may be administered rituximab. Rituximab, a monoclonal antibody targeting the CD20 antigen, and responsible for B cells depletion, is typically not used as initial induction therapy. In a further example, the initial therapy may be discontinued and the individual may be administered a calcineurin inhibitors (CNI) such as one or both of tacrolimus and voclosporin. In a further example, the initial therapy may be discontinued and the individual may be administered belimumab. Belimumab is a IgGl -lambda monoclonal antibody which blocks the connection of soluble human B lymphocyte stimulator protein with receptors on B lymphocyte, resulting the apoptosis of B lymphocytes, which finally leads to the decrease of the autoimmune response. In a further example, the initial therapy may be discontinued and the individual may be administered obinutuzumab, a new generation anti-CD20 monoclonal antibody. In a further example, the initial therapy may be discontinued and the individual may be administered anifrolumab, a human monoclonal antibody against the type Iinterferon receptor. Exemplary alternative therapies that may be administered after identifying an individual as a partial responder arc described in, for example, Xagas, Efstathios ct al. “Evidence based treatment for lupus nephritis: present perspectives and challenges.” Frontiers in nephrology vol. 4 1417026. 6 Aug. 2024, doi:10.3389 / fneph.2024.1417026.

[0037] In a further example, the initial therapy may be continued, and the individual identified as a partial-responder may be administered, in addition to the initial therapy, an additional active as listed above.

[0038] 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.Example 1

[0039] Patients aged 23 and younger diagnosed with LN were recruited (IRB #2008-0635). Diagnosis was made according to ACR criteria for cSLE, with renal biopsy confirmation of LN. Urine was collected at diagnosis and end of induction. Complete responders were defined by urine protein to creatinine ratio <0.5 mg / mg, absence of hematuria, and normal glomerular filtration rate. Partial responders had decrease in proteinuria by 50% and GFR stable to improved from diagnosis. Response was also defined as improved activity index on follow up biopsy. Descriptive analyses, ANOVA, paired and student t-tests were performed for analyses.

[0040] This study shows the pRAIL score (both absolute and standardized) distinguishes complete responders versus partial responders during induction therapy. Complete responders pRAIL score decrease by mean of 1 point, whereas partial responders had no change. Notably, standardized pRAIL scores yielded more pronounced differences during induction therapy among both partial and complete responders.

[0041] The Renal Activity Index for Lupus (RAIL) is a biomarker panel comprising at least one, or at least two, or at least three, or at least four, at least five, or in some instances, six biomarkers, the biomarkers being selected from neutrophil gelatinase-associated lipocalin (NG AL), kidney injury molecule- 1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin and ceruloplasmin. The RAIL biomarkers can be detected in the urine of an individual having Lupus Nephritis (LN) to determine a treatment for an individual.

[0042] A total of 25 patients aged 23 or younger were recruited at the stall of induction therapy. 15 of the patients were complete responders, 7 were partial responders. Table 1 shows the characteristics and pRAIL scores of the complete and partial responders pre and post therapy. Among the three non-responders, standardized and non- standardized pRAIL scores numerically increased during induction therapy. The mean+SD of pRAIL scores significantly decreased in complete responders by 1.07 ± 1.7 (p=0.03) and were unchanged in partial responders (0.07 ± 1.56, p= 0.91), as shown in FIG. 1.Table 1: Characteristics and Biomarkers of Complete, Partial and Non-Responders in Pediatric Patients during Induction Therapyf Values are mean+ SI): f standardization by urine creatinine # paired t- test; complete responder: UPCR < 0.5 AND GFR >90 ml / min / 1.73m2plus no hematuria; partial responder UPCR < 1 OR at least stable GFR but < 90 ml / min / 1.73m2. GFR provided by cystatin C or calculated by modified Schwartz equation; {Visit 1 : diagnosis of LN at the time of kidney biopsy ; Visit 2: at end of induction therapy around month 6-9Example 2

[0043] The Renal Activity Index for Lupus (RAIL) consists of urine protein assessment of neutrophil gelatinase-associated lipocalin, kidney injury molecule- 1, monocyte chemotactic protein 1, adiponectin, hemopexin, and ceruloplasmin, which non-invasively identifies lupus nephritis (LN). RAIL scores with inactive versus active LN and changes over time with response to LN induction therapy were delineated. 128 pediatric patients with cSLE and age-matched healthy controls were recruited in a prospective case control study, with kidney biopsy confirmation of LN. Laboratory and clinical information was recorded and urine collected at diagnosis and end of induction and during maintenance therapy. Response to therapy was assessed by repeat kidney biopsy or laboratory parameters. Urine was assayed for RAIL biomarkers and the RAIL score calculated. Pediatric RAIL (pRAIL) scores from 128 children and young adults with cSLE (with / without LN: 70 / 38) including 25 during LN induction therapy, differentiated clinically active LN from inactive LN or without LN, and controls (all p < 0.0017). pRAIL scores significantly decreased with complete LN remission by 1.07 ± 1.7 (p = 0.03). It was concluded that the RAIL biomarkers differentiate LN patients based on activity of kidney disease, with decreases of > 1 in pRAIL scores indicating complete response to induction therapy. Significantly lower RAIL scores in healthy controls and in SLE patients without known LN raise the possibility of subclinical kidney disease.

[0044] Patients. Clinical data and urine samples of patients with cSLE were included in this study. Study participants were pediatric patients diagnosed with SLE by the 1997 or the 2019American College of Rheumatology (ACR) classification criteria, irrespective of degree of SLE activity, prcscncc / abscncc of LN. Those with a diagnosis of other kidney diseases, or other autoimmune or inflammatory conditions besides SLE were excluded. Age-matched healthy controls were recruited and studied at CCHMC (IRB 2017-0585). SLE patients were classified as having LN based on the result of a kidney biopsy. Patients with LN contributed two or more visits within a 1-year period, while cross-sectional assessment occurred for all patients. Sub-group analysis included LN patients undergoing induction therapy, who had to be enrolled at time of LN diagnosis and followed for 6-9 months, with urine collected at the time of biopsy and at 6-month intervals until the end of induction therapy. REDCap (Research Electronic Data Capture, Vanderbilt University, Nashville TN USA), a secure, web-based application designed to support data capture.

[0045] Demographic, clinical, and laboratory data of patients. Besides demographics, information needed to score the SLE Disease Activity Index (SLED Al) and the renal domain score of the SLEDAI (renal SLEDAI) was extracted. This included results of urinalysis and measured urine protein to creatinine ratio (UPCR, in mg / mg) in spot urine at each visit. The estimated glomerular filtration rates (eGFR) were calculated by modified Schwartz equation in the younger subjects and by CKD-EPI for patients above 18. For patients with LN, the results of kidney biopsies were recorded. Kidney histology was interpreted using the International Society of Nephrology / Renal Pathology Society (ISN / RPS) classification system, which included histologic activity and chronicity as measured by NIH-AI and NIH-CI.

[0046] Biomarker measurements and RAIL scores. Urine collected was spun at 2200 xg for 10 min and stored at -80 °C for no longer than 5 years. Human NGAL and human urine creatinine were measured on a Roche Cobas c 311 clinical chemistry analyzer using a commercially available assay (BioPorto, Denmark, Catalog KIT ST001RA for NGAL; Roche Diagnostics, Indianapolis, IN, Reference 03,263,991 190 for Creatinine). NGAL had a lower limit of detection of 9.8 ng / mL while creatinine had a lower limit of detection of 1.1 mg / dL. The remaining biomarkers were measured via ELISA, all done in duplicate. Human urinary KIM-1 (R&D Systems, Minneapolis, MN, DKM100) had a mean minimal detectable dose of 0.009 ng / mL. Human MCP-1 (R&D Systems, Minneapolis, MN, DCP00), diluted 1:1, had a mean minimal detectable dose of 1.7 pg / mL. Human adiponectin (R&D Systems, Minneapolis, MN,DRP300), diluted 1 :5, had a mean minimal detectable dose of 0.246 ng / mL. Human ceruloplasmin (Assaypro LLC, St. Charles, MO, EC4201-1), diluted 1:50, had a mean minimal detectable dose of 0.085 ng / mL. Human hemopexin (Assaypro LLC, St. Charles, MO, EH2001-1), diluted 1:20, had a mean minimal detectable dose of 4.2 ng / mL. KIM-1 and MCP-1 used a four-parameter logistic curve to fit the standard curve. Adiponectin, ceruloplasmin, and hemopexin used a log / log curve to fit the standard curve. Analyte concentrations that were above or below the limits of detection were imputed by 50% of the level of lower limit of detection and 50% over the upper limit of detection, respectively.

[0047] Definitions of active LN and course of LN. Currently, there is no consensus as to how to define active LN based on the NIH-AI scores; scores from 2 to 6 have been proposed previously. For this analysis, Applicant defined active LN as an NIH-AI score of > 4 within 3 months of a study visit. Patients who had an NIH-AI score of < 4 were classified as inactive nephritis, regardless of ISN / RPS class. All LN patients required a kidney biopsy with urine collection at disease onset to be included in the study induction arm. In the setting of unavailable kidney biopsy within 3 months of a study visit, active LN was defined as a UPCR of > 0.5 mg / mg and / or hematuria > 5 RBC / HPF and / or eGFR < 90 mL / min / 1.73 m2. Accordingly, inactive LN was defined as NIH-AI score < 4 and UPCR < 0.5 plus no hematuria (0-4 RBC / HPF) when NIH-AI scores were or were not available within 3 months of a study visit. Complete response to induction therapy, or complete kidney remission, was defined as NIH-AI as<4 on repeat kidney biopsy. If repeat biopsy was not available, then complete response was defined as UPCR <0.5 plus GFR >90 mL / min / 1.73 m2plus inactive urine sediment (no hematuria). Partial response to induction therapy, or partial kidney remission, was defined as UPCR improvement by > 50% plus at least stable eGFR compared to LN diagnosis. Those patients with LN who failed to achieve a partial or complete response to induction therapy were considered non-responders.

[0048] Statistical analysis. In patient- level variables, descriptive analyses are reported and compared by Student’s t-test (mean ± SD) or ANOVA test (mean ± SD). The Wilcoxon test (median, IQR) or Kruskal-Wallis test (median, IQR) was performed to compare group differences for continuous variables for patients with LN (active or inactive LN), SLE patients without known LN (no-LN), and age-matched healthy controls. Matching controls were recruited to address potential sources of bias. Study size was determined by prior studies using biomarkers. Fisher’sexact or chi-square tests, as appropriate, were performed for testing of group differences of categorical variables. Correlation was established via Pearson’s correlation coefficient, with r of 0.1-0.39 representing weak correlation, 0.4-0.69 representing moderate correlation, 0.7-0.89 representing strong correlation, and greater than 0.9 representing very strong correlation. Baseline (at the time of enrollment) demographics and SLE characteristics were compared for each clinical group (LN, no-LN, and control). Visit-level variables were compared by patient’s age and urinary biomarkers by clinical groups by general estimating equation (GEE) to account for repeated measures within patients. Paired t-test was performed in evaluating patient changes of pRAIL scores from the start to the end of induction therapy for LN patients classified as LN responders (complete and partial) during induction therapy, but not for non-responders. Two-sided p-values < 0.05 were considered statistically significant. Prior to use in the RAIL algorithm, biomarker concentrations were natural-log transformed. The pRAIL algorithm was used only for pediatric and young adult cSLE patients up to age 23 years at enrollment, as previously published. The pRAIL produced standardized and non-standardized scores. The standardized RAIL score refers to the score that utilized creatinine standardized urinary biomarkers, while the nonstandardized score used absolute values of the urinary biomarkers. All statistics were performed using SAS (version 9.4, Cary, NC, USA).

[0049] Construction of KIM-1 sandwich ELISA (R&D Cat# DY1750, Minneapolis, MN): Wells of Nunc-Maxisorp EIA plates are coated by diluting the capture antibody (72 pg / mL) to a working concentration of 0.4 pg / mL in PBS with 100 pL in each well. The plate is sealed and incubated overnight at room temperature. Each well is aspirated and washed using an automated microplate washer (Bio-Tek) with 400 pL of Wash Buffer (0.05% Tween-20 in PBS), repeating the process two times for a total of three washes. The plates are blocked by adding 300 pL of reagent diluent (1% BSA in PBS, 0.2 pm filtered) to each well and incubated at room temperature for 2 hours. After washing as in the previous step, 100 pL of standard recombinant human KIM-1 (0-2000 pg / mL), control, and urine sample is pipetted to the designated well, covered with an adhesive strip, and placed on the orbital shaker at 400 rpm at room temperature for 2 hours. The plate is washed using the same wash protocol as before, and 100 pL of the biotinylated goat antihuman KIM-1 detection antibody diluted in reagent diluent to a working concentration of 400 ng / mL was added to each well. The plate is covered with a new adhesive strip, incubated at room temperature for 2 hours with continued shaking at 400 rpm. Washing step was repeated and 100pL of streptavidin-HRP diluted to a working dilution is added to each well. The plate was protected from light, covered with a new adhesive strip, shaken at 400 rpm, and incubated at room temperature for 20 minutes. After washing, 100 pL substrate solution is added to all wells, protected from light, covered with an adhesive strip, shaken at 400 rpm, and incubated at room temperature for 7 minutes. The reaction is stopped by adding 50 pL of stop solution to all wells. The absorbance is measured using a plate reader (BioTek Elx800) at 450 nm with an absorbance correction at 540 nm. The urinary KIM-1 concentration is calculated based on the standard curve and expressed in absolute terms (pg / mL).

[0050] Results

[0051] A total of 128 pediatric or young adult patients (< 24 years of age; LN: 70; no-LN: 38, controls: 20) were included for the analyses of changes of pRAIL scores. The study duration was from June 2019 to December 2021. Among 70 lupus nephritis patients, 32 patients were undergoing induction therapy for LN, of which 25 met our criteria for active LN, while the remaining 7 did not due to NIH-AI scores < 4.

[0052] Patient disposition and baseline characteristics of the pediatric and young adult patients

[0053] Table 1 shows the baseline characteristics of the pediatric and young adult patients included in pRAIL analyses. The median age at diagnosis of cSLE was 14 years, and most were female. Disease activity was significantly higher in the pediatric LN group as compared to the pediatric no-LN group (median total SLEDAI score; 10 vs 2, p < 0.0001). Most pediatric and young adult LN patients had proliferative LN with a median NIH-AI score of 7 (IQR: 2, 12) and NIH-CI score of 1 (IQR: 0, 3) on kidney biopsy, respectively.

[0054] Table 1. Baseline demographics and lupus characteristics of pediatric population n (% of N) (A) (B) (C) p- value"OR median (IQR) LN (N=70) no-LN (7V= 38) Controls (Age at visit (years) 16.0 (13.0, 19.0) 16.9 (15.6, 19.11 ) 10 (7.5, 13.5) 0.30 <.0001Sex -Female 59 (85.5%) 32 (88.9%) 8 (34%) 0.63 <.0001Ethnicity — % Hispanic 19 (27.1%) 1 (2.6%) 1 (5%) 0.0004 <.0001Race — % White 18 (25.7%) 19 (50%) 10 (50%) 0.01 0.0007SLEDAI total score 10 (2, 22) 2 (0, 6) - <.0001 -Renal SLEDAI 4 (0, 12) 0 (0, 0)Prednisone (mg / day) 26 / 15.5 (10, 40) 16 / 20 (10, 25.5) - 0.97Disease duration 0.1 (0, 3.12) 1.68 (0.5, 3.13) - < 0.05ISN / RPS class, n (% of 70)Class I / II 9 (12.9%)Class III 18 (25.7%)Class IV 24 (34.3%)Pure class V 5 (7.1%)Class III / IV+V 8 (11.4%)Unknown 6 (8.6%)NIH-AI score (n = 41) $ 7 (2, 12)NIH-CI score (ra = 41) $ 1 (0, 3)By Wilcoxon rank-sum test or Kruskal-Wallis tests for continuous variables, and by chi-square test used for categorical variables. *NIH-AI and NIH-CI scores were available for only 41 of the 70 patients with LN

[0055] Urine biomarker concentrations and RAIL score by LN status

[0056] The pRAIL scores significantly differed by LN status (active LN, inactive LN, no-LN) and by healthy controls. Table 2 summarizes individual urine biomarker concentrations contributing to the pRAIL score, with statistically significant differences with active versus inactive LN, active LN versus no-LN, and SLE (active LN, inactive LN, no-LN) versus controls. Only urine levels of NGAL, adiponectin, and KIM-1 were statistically significantly higher in the no-LN group compared to healthy controls (all p < 0.03). The pRAIL scores differed between the active LN group and other groups but not between the inactive LN group versus the non-LN group,irrespective of whether pR AIL scores were standardized by urine creatinine or not. There was only a weak correlation between pRAIL scores and UPCR, with an r value of 0.14. Considering the subset of active LN patients with available NIH-AI scores, pRAIL scores were moderately correlated (r = 0.4; p = 0.0054) and UPCR weakly correlated (r = 0.33; p = 0.02) with NIH-AI scores as shown in FIG. 1.

[0057] Table 2. Urine biomarker and pRAIL scores by LN status and in healthy controls

[0058] Table 3. Characteristics and biomarkers of complete and partial responders in pediatric patients during induction therapy.

[0059] Changes in RAIL scores with induction therapy

[0060] There were 32 analyzable pediatric or young adult LN patients who enrolled at the time of a kidney biopsy, among them 7 patients whose LN was classified as “inactive” (NIHAI score < 4) and therefore excluded, and 25 patients who had active LN at the start of induction therapy.Immunosuppressive therapy for active LN included intravenous cyclophosphamide (n = 14) and oral mycophcnolatc mofctil (n = 11). There were 22 responders to induction therapy, of whom 15 patients met criteria for complete response, 7 patients were partial responders, and 3 were nonresponders to induction therapy. Distribution of age at baseline, gender, race, ethnicity, and ISN / RPS classes were all similar across groups. Proliferative nephritis was evenly distributed across response status. Of the 17 patients with class III or IV LN, 2 patients (66.67%) were nonresponders, 5 patients (71%) were partial responders, and 10 patients (66.67%) were complete responders. Finally, baseline pRAIL scores were similar’ between partial and complete responders (Table 3). Among the three non-responders, standardized and nonstandardized pRAIL scores numerically increased during induction therapy (see FIG. 2). Conversely, mean ± SD of pRAIL scores showed a statistically significant decrease in complete responders by 1.07 ± 1.7 (p = 0.03). For partial responders, there was no change of the pRAIL score during induction therapy (0.07 ± 1.56, p = 0.91).

[0061] Ten patients underwent a repeat kidney biopsy upon completion of induction therapy, including nine with complete renal response and 1 non-responder. The mean ± SD of NIHAI scores decreased from 9.89 ± 3.92 to 1.1 ± 1.45 among complete responders during induction therapy (p = 0.0007). Overall, mean ± SD absolute pRAIL scores of complete responders verified by repeat kidney biopsy decreased by 1.08 ± 1.13, and standardized pRAIL scores by 0.43 ± 0.31, respectively. The average UPCR pre-therapy was 4.56 ± 4.74 and the average UPCR post-biopsy was 1.29 ± 2.04. As shown in FIG. 3, the pRAIL score demonstrates better correlation with NIHAI both pre- and post-treatment (r = 0.46 and 0.37, respectively), while UPCR had no correlation with NIH-AI at either time point, with r values of 0 and 0.1, respectively.

[0062] The pRAIL score, both absolute and standardized, can distinguish active, inactive, and absent lupus nephritis in the pediatric population. It is now shown that the change in pRAIL score can also be used to detect improvement in disease activity during induction therapy. The pRAIL difference score corresponds with the change in the NIH-AI on biopsy that may not be detected by current biochemical measures, including UPCR measurements. Applicant has discovered that this score can be utilized during induction therapy to guide treatment decisions, enabling clinicians to 1) modify the therapy — by either increasing the dose of the current treatment or switching to an alternative therapy — in cases where the patient is a non-responder or partial-responder; introducesupplemental therapy for patients who are not fully responding to the initial treatment; or 3) continue the current therapy in patients who arc showing a positive response.

[0063] Discussion

[0064] In this study, the ability of the pRAIL score to detect response to induction therapy was assessed. Applicant found that both absolute and standardized pRAIL scores differentiate patients with active LN from those with inactive LN, or SLE patients without kidney involvement. Healthy controls have significantly lower RAIL scores than SLE patients, even those without known kidney disease. Complete response of active LN to induction therapy is associated with a mean decrease of the standardized pRAIL score of at least 0.5, irrespective of the degree of the baseline histological activity. Finally, the pRAIL score is superior to proteinuria in correlating with activity as found on biopsy.

[0065] Both absolute and standardized pRAIL scores were similarly well suited to discriminate moderate to highly active LN (NIH-AI > 10) from LN with lesser degrees of inflammation as seen on kidney biopsy. Using current standard clinical measures of LN, pRAIL scores clearly differentiate active from inactive LN. The change in pRAIL scores are reflective of partial and complete response to induction therapy. Given that decreases in standardized rather than absolute pRAIL scores were consistent, irrespective of baseline kidney inflammation (NIHAI score), standardized pRAIL scores may be better suited to assess response to LN therapy.

[0066] Currently, there are no generally accepted, standard definitions for complete or partial response to induction therapy in either pediatric or adult LN. Choices of the response definitions influence the frequency of LN response in adult LN patients as used in clinical care and research, and the same holds true for pediatric LN. In this context, RAIL scores may be used to provide additional support for the biologic relevance of the observed response of LN to a given treatment intervention. Importantly, corrected for treatment response, changes in RAIL scores are not influenced by the type of concurrent therapies, including the use of proteinuria sparing agents.

[0067] The degree of histological activity on kidney biopsy at the time of LN diagnosis or on repeat biopsy is an established prognostic factor for the longer-term outcomes of LN. Given that RAIL scores reflect the level of inflammation, measuring RAIL biomarkers may provide a non-invasive measure to intervene and provide additional therapies, with the goal to improve kidney outcomes in LN. Proteinuria, particularly following treatment of active inflammation, may reflect chronic damage to the kidney as a result of their presentation that may respond to nonimmunosuppressant therapy. Combining the RAIL score with urine protein measurement may provide insight into the health of the kidney following induction therapy. Conversely, there is a subset of patients who may have resolution of proteinuria but have active disease on repeat biopsy and the addition of the RAIL score into practice may assist with decisions to continue immunosuppressive therapies. In this study, levels of urine biomarkers and RAIL scores were lower in age-matched healthy controls compared to SLE patients, irrespective of kidney involvement. Lower levels of kidney biomarkers and RAIL scores could be considered a reflection of common, subclinical kidney inflammation in SLE patients, even those without abnormal urinalysis. In conclusion, a composite urinary biomarker score and RAIL may be used to support the response of LN to induction therapy.

[0068] Exemplary Combinations

[0069] All percentages and ratios are calculated by weight unless otherwise indicated.

[0070] All percentages and ratios are calculated based on the total composition unless otherwise indicated.

[0071] 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.

[0072] 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 such dimension is intended to mean both the recited value and a functionally equivalent rangesurrounding that value. For example, a dimension disclosed as “20 mm” is intended to mean “about20 mm.”

[0073] 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 (e.g., 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 ail 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.

[0074] 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.

[0075] Exemplary Combinations

[0076] 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 filingsrelated 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.

[0077] Example 1

[0078] A method for non-invasively classifying an individual diagnosed with lupus nephritis (LN) as a partial responder or non-responder to LN induction therapy, comprising:(a) comparing a first Renal Activity Index for Lupus (RAIL) score to a second RAIL score; and(b) classifying the individual as a partial responder or non-responder based on the comparison in step (a); the first RAIL score being derived from the measured concentrations of a plurality of biomarkers in a first urine sample, the first urine sample being collected from the individual before or at the start of induction therapy; the second RAIL score being derived from the measured concentrations of the same plurality of biomarkers in a second urine sample collected from the individual after the initiation of induction therapy; and the plurality of biomarkers comprising neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule- 1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin, and ceruloplasmin.

[0079] Example 2

[0080] The method of Example 1 , wherein the RAIL score is creatine normalized.

[0081] Example 3

[0082] The method of any preceding Example, wherein the RAIL score is calculated from the equation: - 4.29 - 0.34* - NGAL - 0.06*ceruloplasmin + 0.89*MCP-1 + 0.18*adiponectin - 0.65*hemopexin + 0.62*KIM-l (“pRAIL” algorithm).

[0083] Example 4

[0084] The method of Example 3, wherein the biomarker concentration is natural-log transformed prior to calculation of the RAIL score.

[0085] Example 5

[0086] The method of Example 4, wherein the biomarker concentration is natural-log transformed prior to calculation of the RAIL score.

[0087] Example 6

[0088] The method of Example 4, wherein the biomarker concentration is creatinine-adjusted.

[0089] Example 7

[0090] The method of Example 4, wherein the biomarker concentration is albumin-adjusted.

[0091] Example 8

[0092] The method of Example 1, wherein when the first and second RAIL score arc substantially unchanged, the individual is classified as a non-responder to induction therapy.

[0093] Example 9

[0094] The method of Example 1 , wherein the second RAIL score decreases as compared to the first RAIL score, the individual is classified as a complete responder to induction therapy.

[0095] Example 10

[0096] The method of any preceding claim, wherein when the second RAIL score, as compared to the first RAIL score, decreases by at least 1, the individual is classified as a complete responder to induction therapy.

[0097] Example 11

[0098] The method of Example 10, wherein the RAIL score is determined based on a nonadjusted (absolute) biomarkcr concentration.

[0099] Example 12

[0100] The method of Example 10, wherein the RAIL score is determined based on a biomarker concentration that is creatinine-adjusted.

[0101] Example 13

[0102] The method of Example 10, wherein the RAIL score is determined based on a biomarker concentration that is albumin-adjusted.

[0103] Example 14

[0104] The method of Example 1, wherein the individual is classified as having LN based on a kidney biopsy.

[0105] Example 15

[0106] The method of Example 1 , wherein the individual has an NIH- Al score of greater than or equal to 4 prior or at the time of initiation of induction therapy.

[0107] Example 16

[0108] The method of Example 1, wherein the induction therapy is an immunosuppressive therapy.

[0109] Example 17

[0110] The method of Example 16, wherein the immunosuppressive therapy is one or both of cyclophosphamide and my cophenolate mofetil.

[0111] Example 18

[0112] The method of Example 1 , wherein the individual is classified as a partial responder, further comprising administering one or both of an increased dose of induction therapy, or an increased frequency of induction therapy.

[0113] Example 19

[0114] The method of Example 1, wherein the individual is classified as a non-responder and administered a treatment different from the induction therapy.

[0115] Example 20

[0116] The method of Example 1, wherein the individual is classified as having active LN at the start of induction therapy.

[0117] Example 21

[0118] The method of Example 1, wherein the individual is diagnosed with systemic lupus erythematosus (SLE).

[0119] Example 22

[0120] The method of Example 1, wherein the individual is classified as having inactive LN at start of induction therapy.

[0121] Example 23

[0122] The method of Example 1, wherein the individual is at least 18 years of age.

[0123] Example 24

[0124] The method of Example 1, wherein the individual is less than 18 years of age.

[0125] Example 25

[0126] The method of Example 1, wherein the individual is less than or equal to 23 years of age.

[0127] Example 26

[0128] The method of Example 1, wherein the individual is male.

[0129] Example 27

[0130] The method of Example 1, wherein the individual is female.

Claims

CLAIMSWhat is claimed is:

1. A method for non-invasively classifying an individual diagnosed with lupus nephritis (LN) as a partial responder or non-responder to LN induction therapy, comprising:(a) comparing a first Renal Activity Index for Lupus (RAIL) score to a second RAIL score; and(b) classifying the individual as a partial responder or non-responder based on the comparison in step (a); the first RAIL score being derived from the measured concentrations of a plurality of biomarkers in a first urine sample, the first urine sample being collected from the individual before or at the start of induction therapy; the second RAIL score being derived from the measured concentrations of the same plurality of biomarkers in a second urine sample collected from the individual after the initiation of induction therapy; and the plurality of biomarkers comprising neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1), monocyte chemotactic protein 1 (MCP-1), adiponectin, hemopexin, and ceruloplasmin.

2. The method of claim 1, wherein the RAIL score is creatine normalized.

3. The method of any preceding claim, wherein the RAIL score is calculated from the equation: - 4.29 - 0.34* - NGAL - 0.06*ceruloplasmin + 0.89*MCP-1 + 0.18*adiponectin - 0.65*hemopexin + 0.62*KIM-l (“pRAIL” algorithm).

4. The method of claim 3, wherein the biomarker concentration is natural-log transformed prior to calculation of the RAIL score.

5. The method of claim 4, wherein the biomarkcr concentration is natural-log transformed prior to calculation of the RAIL score.

6. The method of claim 4, wherein the biomarker concentration is creatinine-adjusted.

7. The method of claim 4, wherein the biomarker concentration is albumin-adjusted.

8. The method of claim 1, wherein when the first and second RAIL score are substantially unchanged, the individual is classified as a non-responder to induction therapy.

9. The method of claim 1, wherein the second RAIL score decreases as compared to the first RAIL score, the individual is classified as a complete responder to induction therapy.

10. The method of any preceding claim, wherein when the second RAIL score, as compared to the first RAIL score, decreases by at least 1, the individual is classified as a complete responder to induction therapy.

11. The method of claim 10, wherein the RAIL score is determined based on a non-adjusted (absolute) biomarker concentration.

12. The method of claim 10, wherein the RAIL score is determined based on a biomarker concentration that is creatinine-adjusted.

13. The method of claim 10, wherein the RAIL score is determined based on a biomarker concentration that is albumin-adjusted.

14. The method of claim 1, wherein the individual is classified as having LN based on a kidney biopsy.

15. The method of claim 1 , wherein the individual has an NTH- AT score of greater than or equal to 4 prior or at the time of initiation of induction therapy.

16. The method of claim 1, wherein the induction therapy is an immunosuppressive therapy.

17. The method of claim 16, wherein the immunosuppressive therapy is one or both of cyclophosphamide and my cophenolate mofetil.

18. The method of claim 1, wherein the individual is classified as a partial responder, further comprising administering one or both of an increased dose of induction therapy, or an increased frequency of induction therapy.

19. The method of claim 1, wherein the individual is classified as a non-responder and administered a treatment different from the induction therapy.

20. The method of claim 1, wherein the individual is classified as having active LN at the start of induction therapy.

21. The method of claim 1, wherein the individual is diagnosed with systemic lupus erythematosus (SLE).

22. The method of claim 1, wherein the individual is classified as having inactive LN at start of induction therapy.

23. The method of claim 1, wherein the individual is at least 18 years of age.

24. The method of claim 1, wherein the individual is less than 18 years of age.

25. The method of claim 1, wherein the individual is less than or equal to 23 years of age.

26. The method of claim 1, wherein the individual is male.

27. The method of claim 1, wherein the individual is female.

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