WO2022036453A1 - Treatment methods for systemic lupus erythematosus and lupus nephritis - Google Patents

Abstract

The present disclosure provides compositions and methods for treating kidney dysfunction associated with systemic lupus erythematosus (SLE) and other diseases and conditions.

Description

TREATMENT METHODS FOR SYSTEMIC LUPUS ERYTHEMATOSUS AND LUPUS NEPHRITIS

CROSS-REFERENCE TO RELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Application Number 63/068,956, filed August 21, 2020, the contents of which is incorporated by reference herein in its entirety.

SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ASCII format and is hereby incorporated by reference in its entirety. Said ASCII copy, created on August 10, 2021, is named 55409_704_601_SL.txt and is 14,313 bytes in size.

BACKGROUND

[0003] Between three hundred thousand and one million persons in the United States are diagnosed with or are suffering from systemic lupus erythematosus (SLE). SLE is a heterogenous autoimmune disorder that damages many parts of the body, including the joints, skin, heart, lungs, blood vessels, brain, and kidney. A subset of SLE patients manifest the clinical complication of lupus nephritis (LN); renal damage associated with LN is a major cause of mortality in these patients, with 10-20% of them developing end-stage renal disease (ESRD) within ten years of diagnosis. Unfortunately, there are relatively few treatment options for patients with LN. Accordingly, there remains an unmet need for methods and compositions that help treat LN in patients with SLE.

SUMMARY

[0004] The present invention addresses this need. Accordingly, the present disclosure provides methods and compositions that treat systemic lupus erythematosus (SLE), including lupus nephritis (LN) and/or associated with antigen presenting cell (APC) dysfunction.

[0005] An aspect of the present disclosure is a method of treating systemic lupus erythematosus (SLE). The method comprising a step of administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from SLE. In this aspect, the SLE is manifested in the subject as active lupus nephritis, and the HSPB5 protein is administered in an effective amount to treat, improve or ameliorate at least one symptom or indicator of reduced kidney function.

[0006] Another aspect of the present disclosure is a method of preserving or improving kidney function. The method comprising a step of administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from a decrease in normal kidney function or abnormal kidney function. In some examples, the subject is diagnosed or suffering from systemic lupus erythematosus (SLE) or lupus nephritis (LN); the subject exhibits tubular atrophy and the subject is diagnosed with or suffering from IgA nephropathies, focal segmental glomerulosclerosis, primary membranous nephropathy, or C3 glomerulopathy; or the subject exhibits glomerular depositions and the subject is diagnosed with or suffering from IgA nephropathies, focal segmental glomerulosclerosis, primary membranous nephropathy, or C3 glomerulopathy. In this aspect, the subject exhibits at least one symptom of compromised kidney function, such as increased protein in the urine (proteinuria) and/or a high blood urea nitrogen (BUN) and the subject’s kidney function is improved after administering HSPB5.

[0007] Yet another aspect of the present disclosure is a method of treating systemic lupus erythematosus (SLE). The method comprising a step of administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from SLE. In this aspect, the SLE is manifested in the subject as antigen presenting cell (APC) dysfunction, and wherein administering the HSPB5 protein results in one or more effects in the subject selected from the group consisting of an increase in the proportion of macrophages expressing PD-L1 (CD14+PD-L1+), an increase in IL- 10 secretion from splenic macrophages, a decrease in the number of splenocytes, an increase in dendritic cells, an induction of immunosuppressive macrophages, and any combination thereof, as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

[0008] In an aspect, the present disclosure provides a method of restoring antigen presenting cell (APC) function. The method comprising a step of administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from systemic lupus erythematosus (SLE). In this aspect, the subject exhibits an indication of APC dysfunction and administering the HSPB5 protein results in one or more effects in the subject selected from the group consisting of an increase in the proportion of macrophages expressing PD-L1, an increase in IL-10 secretion from splenic macrophages, a decrease in the number of splenocytes, an increase in dendritic cells, an induction of immunosuppressive macrophages, and any combination thereof, as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

[0009] In any of the herein-disclosed methods, a treatment with HSPB5 protein results in two or more features selected from the group consisting of a reduction in proteinuria, a reduction in BUN, a reduction in urine foaming, a reduction in glomerular deposits, a reduction in endocapillary proliferation, a reduction in glomerular lesions, a reduction in tubular atrophy, a reduction in splenocyte count, a reduction in average follicle diameter, a reduction in splenic T cells and an increase in number of splenic follicles. In embodiments, the treatment results in 3, 4, 5, or more than 5 of the features.

[0010] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The novel features of the invention are set forth with particularity in the appended claims. A better understanding of the features and advantages of the present invention will be obtained by reference to the following detailed description that sets forth illustrative embodiments, in which the principles of the invention are utilized, and the accompanying drawings of which:

[0012] Figure 1A shows dextran uptake of PBMCs pre-treated with 0 or 10 pg/ml HSPB5 overnight and stimulated with 1 pg/ml R848 for 24 hours.

[0013] Figure IB shows cytokine secretion of PBMCs pre-treated with 0 or 10 pg/ml HSPB5 overnight and stimulated with 1 pg/ml R848 for 24 hours.

[0014] Figure 2A show the study design for proteinuria measurements of MRL/lpr mice that were treated with HSPB5 (2.5, 10 or 20 pg) or PBS (vehicle).

[0015] Figure 2B shows the percent of mice with severe proteinuria (>3) for MRL/lpr mice that were treated with HSPB5 (2.5, 10 or 20 pg) or PBS (vehicle) over weeks 9-21 of the study.

[0016] Figure 2C shows terminal blood urea nitrogen (BUN) for MRL/lpr mice that were treated with HSPB5 (2.5, 10 or 20 pg) or PBS (vehicle).

[0017] Figure 2D shows serum anti-dsDNA antibody levels at 21 weeks of age for the MRL/lpr mice that were treated with HSPB5 (2.5, 10 or 20 pg) or PBS (vehicle).

[0018] Figure 3A to Figure 3F show HSPB5 treatment reduces the severity of symptoms of lupus nephritis in MRL/lpr mice. The figures show features of kidney structure of the MRL/lpr mice treated with HSPB5 (2.5, 10 or 20 pg) or PBS (vehicle): Fig. 3A total glomerular lesions, Fig. 3B glomerular deposits, Fig. 3C relative frequency of glomerular deposit scores, Fig. 3D endocapillary proliferation, Fig. 3E: tubular atrophy, Fig. 3F frequency distribution of tubular atrophy scores.

[0019] Figure 4A to Figure 4C shows reduced splenic pathology in spleens of HSPB5-treated MRL/lpr mice as assessed by: splenocyte count (Fig. 4A), splenic follicle diameter (Fig. 4B), number of follicles per area (Fig. 4C). [0020] Figure 4D-to Figure 4F show increased anti-inflammatory macrophages in spleens of HSPB5-treated MRL/lpr mice: Figure 4D shows the frequency of cell populations as analyzed by flow cytometric analysis of splenocytes, CD3 and B cells are shown as frequency of live cells. CD4, CD8 and DN cells are frequencies of CD3⁺ population. Regulatory T cells (Tregs,FoxP3+CD25+) are shown as % of CD3+CD4+ population; Figure 4E shows the frequency of splenic dendritic cells; Figure 4F shows the frequency of monocytes/macrophages (CD14+ cells); Figure 4G shows the percentage of monocytes/macrophages that are positive for PD-L1 ; Figure 4H shows IL- 10 expression by CD 14+ cells; Figure 41 shows frequency of regulatory B cells (Bregs, CD19+IL-10+).

[0021] Figure 5A to Figure 5C show MRL/lpr mice treated with HSPB5 and the levels of IL- 10, IL-6 and TNF as compared to vehicle treated mice. Fig. 5A. shows that the HSPB5 treated mice demonstrated significantly higher serum IL-10 and slightly lower TNFa cytokine levels in terminal (day 21) serum levels of cytokines; Fig. 5B shows relative mRNA expression for IL-10, IL-6 and TNF. Figure 5C shows a heat map of Pearson correlation coefficients for IL-6 expression with kidney pathology: BUN (p < 0.001), total glomerular lesions (p < 0.001), total kidney lesions (p< 0.001), tubular atrophy (p < 0.001).

[0022] Figure 6 shows qPCR results mapped against kidney histology and functional parameters, mouse per mouse.

[0023] Figure 7 illustrates a model of HSPB5-mediated therapeutic effects in SLE and LN; created with BioRender, com.

DETAILED DESCRIPTION OF THE INVENTION

[0024] The present disclosure is based, in part, on the discovery of methods and compositions that that are surprisingly effective in treating systemic lupus erythematosus (SLE), including symptoms associated with lupus nephritis (LN) and/or associated with antigen presenting cell (APC) dysfunction.

[0025] SLE is considered to be a prototypical autoimmune disease. Indeed, SLE is characterized by severe organ damage caused by anti-self-antibodies. In some cases, a triggering event causes antigen-presenting cells (APCs) to activate Thl7 and Thl cells that are reactive to self-antigens. Then, effector T cells activate B cells to produce anti-self-antibodies. These anti- self-antibodies form immune complexes that deposit in multiple organ systems, causing tissue damage through inflammation cascades. Some patient’s symptoms may be limited to skin rash and joint pain and require little medication. The other end of the spectrum includes patients who demonstrate severe and progressive kidney involvement (glomerulonephritis) that requires therapy with high doses of steroids and cytotoxic drugs, such as cyclophosphamide.

[0026] Lupus nephritis (LN) is inflammation of the kidney that is caused by SLE. Up to 60% of lupus patients will develop LN. When the kidneys are inflamed, they cannot function normally to filter toxins, byproducts, excess salts, excess fluid, and other impurities the blood. If not controlled, LN can lead to kidney failure. Even with treatment, loss of kidney function sometimes progresses. If both kidneys fail, people with LN may need dialysis. Ultimately, it may be necessary for the LN patient to receive a kidney transplant. In those cases, the patient will forever need additional immunosuppressive drugs to keep their immune system from rejecting the transplanted kidney. Symptoms of loss of or abnormal kidney function include increased amounts of protein in urine (proteinuria), foaming in a subject’s urine, and/or a higher level of blood urea nitrogen (BUN).

Heat shock proteins (HSPs)

[0027] HSPs are a diverse class of numerous proteins that are produced by cells in response to exposure to stressful conditions, e.g, heat, cold, infection, inflammation, UV, and toxins. HSPs are generally defined as molecular chaperones which bind misfolded or unfolded proteins and promote proper re-folding or degradation and/or sequestration of misfolded proteins. This class of proteins is diverse in terms of their size, subcellular localization, and functional mechanisms and members are found in all kingdoms of life. Generally, HSPs are grouped into categories based on their molecular weight. Major categories include the HSPlOOs, HSP90s (which includes gp96), HSP70s, HSP60s, HSP40s, HSP33s, and the small HSPs, such as HSP10, HSPBs (including HSP20, HSP27, HSPB6, HSPB4/alpha-A crystallin (CRYAA), and HSPB5/alpha-B crystallin (CRY AB).

[0028] The various HSPs differ in many aspects. Some HSPs, like the HSP70s and HSP90s, are ATP-dependent chaperones, whereas small HSPs, HSPB5 as an example, possess activity that is ATP -independent. Also, the small HSPs, including HSPB5, form high-molecular weight complexes (>400 kDa) whereas HSP70s and HSP90s do not form such complexes. HSP90, HSP84, HSP70, HSP27, HSP20, and HSPB5 have roles in the cardiovasculature; whereas HSPB5 and HSPB4 have roles in preserving the eye lens. Some HSPs are located in the endoplasmic reticulum (i.e., gp96) whereas other HSPs are found in the cytosol. Also, the HSPs have differing expression levels depending on the tissue; for example, HSP70s have different endogenous expression levels than endogenous HSPB5. Relatedly, higher levels of HSP90 expression have been measured in SLE patients in comparison to HSP70 or HSPB5 expression levels; indeed, HSPB5 is only detectable in about 8 to 15% of SLE patients. Finally, some exogenous HSPs have been shown to have therapeutic effects on a specific disorder whereas other exogenous HSPs lack any therapeutic effect on the specific disorder. As an example, exogenous HSP70 and gp96 (a HSP90 member) have therapeutic ability in reducing symptoms in a SLE model, whereas exogenous HSP60 has been shown to be ineffective in this model.

[0029] The differing aspects and activities of the various members of the HSPs made it surprising, as provided here in the present disclosure, that one small HSP family member HSPB5 is effective in treating SLE, including symptoms associated with lupus nephritis (LN) and/or associated with antigen presenting cell (APC) dysfunction. The present disclosure is based on the surprising discovery that one small HSP family member HSPB5 is effective in treating SLE, including symptoms associated with lupus nephritis (LN) and/or associated with antigen presenting cell (APC) dysfunction.

[0030] The human HSPB5 (also known as alphaB-crystallin, aB crystallin, and/or CRY AB) gene encodes a 175-amino acid protein with a molecular mass of about 20 kD. Methods of the present disclosure comprise administering a HSPB5 protein comprising an amino acid sequence of any one of SEQ ID NO: 1 to SEQ ID NO: 8, a variant thereof, or a functional fragment of a HSPB5 protein comprising a fragment of a sequence of any of SEQ ID NO: 1 to SEQ ID NO: 8. In embodiments, the HSPB5 protein is a variant that comprises a sequence which is at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% identical to one of SEQ ID NO: 1 to SEQ ID NO: 8. In embodiments, the functional fragment of HSPB5 is at least 9 amino acids in length, e.g., at least 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 25, 30, 40, 50, 60, 70, 80, 90, 100, 125, or 150, and any number amino acids therebetween. In some embodiments, a functional fragment of HSPB5 shares a functional or binding property with full-length HSPB5. In embodiments, the functional fragment of HSPB5 is one of SEQ ID NO: 9 to SEQ ID NO: 11.

TABLE: Illustrative HSPB5 Sequences

[0031] HSPB5 protein use has been investigated in animal models of chemically-induced neuroinflammation, autoimmune neuroinflammation, ischemic optic neuropathy, stroke, spinal cord injury, ischemia reperfusion, and chronic obstructive pulmonary disorders. However, HSPB5’s use in treating SLE, and especially LN, and its use in treating loss or decreased kidney function, has not previously been investigated. Treatment Methods

[0032] An aspect of the present disclosure is a method for treating systemic lupus erythematosus (SLE). The method comprising a step of administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from SLE. In this aspect, the SLE is manifested in the subject as active lupus nephritis (LN), and the HSPB5 protein is administered in an effective amount to treat, improve or ameliorate at least one symptom or indicator of reduced kidney function, such as proteinuria, a high blood urea nitrogen (BUN), a decrease in estimated glomerular fdtration rate (eGFR) and any combination thereof.

[0033] In embodiments for treating SLE manifested as LN, the level of proteinuria in the subject is maintained or reduced by the method as compared to the level prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care.

[0034] As used in the present disclosure, “historical control” means a measurement or assessment (such as a measurement or assessment of proteinuria, another marker or other symptom) in the same subject. A historical control may be a measurement or assessment taken prior to HSPB5 protein administration, prior to any treatment, or after SOC or other treatment but prior to HSPB5 protein administration. A “historical control” may also be a measurement or assessment (such as a measurement or assessment of proteinuria, other marker or other symptom) in a different subject and/or known (published) levels or amounts of a specific marker that is found in control subjects or in affected patients. In one example, a threshold level of a marker is known in which a subject having a level of the marker above the threshold is identified as having the relevant disorder.

[0035] In embodiments for treating SLE manifested as LN, the subject’s level of blood urea nitrogen (BUN) may be maintained or reduced by the method as compared to the level prior to administration of the HSPB5 protein or relative to a historical control. In various embodiments, the level of proteinuria, amount of urine foaming (an indicator of protein in urine), blood urea nitrogen, or a combination thereof is reduced over 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more than 7 weeks following administration of the HSPB5 protein or relative to the standard of care.

[0036] In embodiments, the structure of the kidney is preserved or improved by the method for treating SLE manifested as LN as compared to kidney structure prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care. The preservation or improvement of kidney structure may be assessed by a decrease in one or more of glomerular deposits, endocapillary proliferation, glomerular lesions, tubular atrophy or any combinations thereof as compared to kidney structure prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC). The preservation or improvement of kidney structure may be assessed by a decrease in blood urea nitrogen, a decrease in proteinuria, a reduction in foaming of urine (an indicator of protein in urine), an increase or maintenance (little to no further decrease) in estimated glomerular filtration rate (eGFR) or any combination thereof.

[0037] In embodiments, treatment with HSPB5 provides improved kidney function in conditions and diseases that manifest in a loss or reduction of kidney function such as IgA nephropathies, Focal segmental glomerulosclerosis, primary membranous nephropathy, or C3 glomerulopathy. In embodiments, the administration of HSPB5, preserves or improves the structure of the kidney as compared to kidney structure prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care. The preservation or improvement of kidney structure may be assessed by a decrease in one or more of glomerular deposits, endocapillary proliferation, glomerular lesions, tubular atrophy or any combinations thereof as compared to kidney structure prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC). The preservation or improvement of kidney structure may be assessed by a decrease in blood urea nitrogen, a decrease in proteinuria, a reduction in foaming of urine (an indicator of protein in urine) or any combination thereof.

[0038] In embodiments, administering HSPB5 for treating SLE manifested as LN extends the life of a patient’s kidney relative to the standard of care, such as extending the duration the kidney can function before a transplant may be available or needed. In embodiments, administering HSPB5 along with the standard of care treatment extends the life of a patient’s kidney with LN relative to a therapy with the standard of care alone.

[0039] In embodiments, administering HSPB5 for treating SLE manifested as LN reduces the need for a kidney transplant relative to the standard of care. In embodiments, administering HSPB5 along with the standard of care treatment reduces the need for a kidney transplant relative to a therapy with the standard of care alone.

[0040] In embodiments, a subject administered HSPB5 requires fewer dialysis treatments relative to the standard of care for treating SLE manifested as LN. In embodiments, a subject administered HSPB5 along with the standard of care requires fewer dialysis treatments relative to a therapy with the standard of care. [0041] In the methods for treating SLE manifested as LN, the structure of a subject’s kidney may be assayed via anon-invasive imaging method (e.g., X-ray, MRI, CT Scan, ultrasound, a urine sample, and the like) or by an invasive method, e.g, a tissue biopsy, optionally, coupled with a morphological and/or immunological analysis. Such methods may be used to assess the initial state of the subject’s kidney and the response of the kidney to treatment with HSPB5.

[0042] In embodiments, treatment with HSPB5 provides an improved spleen pathology of the subject as compared to spleen pathology prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC). In some cases, an improved spleen pathology comprises one or more of a reduction in splenocyte count, a reduction in average follicle diameter, an increase in the number of splenic follicles per area, or a combination thereof.

[0043] In embodiments, treatment with HSPB5 provides an increase in dendritic cells, an induction of immunosuppressive macrophages, a decrease in T cells in spleen or any combination thereof as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

[0044] In the methods for treating SLE manifested as LN, the state of a subject’s spleen and immune cells present in the spleen may be assayed via a non-invasive imaging method (e.g., X- ray, MRI, CT Scan, ultrasound, a urine sample, and the like) or by an invasive method, e.g., a tissue biopsy, optionally, coupled with a morphological and/or immunological analysis. In embodiments, morphological and/or immunological analysis is, splenic follicle diameter, splenic follicle density, spleen derived total splenocyte count, percentage of specific cell types such as dendritic cells, T cells, B cells, macrophages and cell types by cell surface markers such as CD4+ T cells, CD8+ T cells, CD3+ T cells, Treg cells (CD4+FoxP3+CD25+), aberrant DN T cells (CD3+CD19-CD4-CD8-), and any combination thereof.

[0045] Another aspect of the present disclosure is a method for preserving or improving kidney function. The method comprising a step of administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from a decrease in normal kidney function or from abnormal kidney function. In some embodiments, the subject is diagnosed or suffering from systemic lupus erythematosus (SLE) or lupus nephritis (LN). In some embodiments, the subject is diagnosed or suffering from a condition or disease that manifests as a decrease in normal kidney function or from abnormal kidney function such as IgA nephropathies, Focal segmental glomerulosclerosis, primary membranous nephropathy, or C3 glomerulopathy. In this aspect, the subject exhibits at least one symptom of compromised kidney function, such as increased protein in the urine (proteinuria), or a high blood urea nitrogen (BUN), and the subject’s kidney function is improved after administering HSPB5. In aspects, the subject also exhibits at least one symptom of compromised kidney function, wherein the symptom presents as manifestations of kidney structure such as glomerular deposits, endocapillary proliferation, glomerular lesions, tubular atrophy, and the subject’s kidney function is improved after administering HSPB5.

[0046] In various embodiments of the method for preserving or improving kidney function, the level of proteinuria in the subject is reduced by the method as compared to the level prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care. The subject’s level of blood urea nitrogen (BUN) may be reduced by the method as compared to the level prior to administration of the HSPB5 protein or relative to a historical control. In various embodiments, the level of proteinuria, urine foaming, blood urea nitrogen, or a combination thereof is reduced over 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more than 7 weeks following administration of the HSPB5 protein.

[0047] In some embodiments, the structure of the kidney is preserved or improved by the method for preserving or improving kidney function as compared to kidney structure prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care. The preservation or improvement of kidney structure may be assessed by a decrease in one or more of glomerular deposits, endocapillary proliferation, glomerular lesions, tubular atrophy, a kidney biopsy, or any combinations thereof as compared to kidney structure prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC). The preservation or improvement of kidney structure may be assessed by a decrease in blood urea nitrogen, a decrease in proteinuria, a reduction in foaming of urine (an indicator of protein in urine), an increase or maintenance (little to no further decrease) of estimated glomerular filtration rate (eGFR) or any combination thereof.

[0048] In some embodiments, the subject is diagnosed or suffering from a condition or disease that manifests as tubular atrophy of the kidney, such as IgA nephropathies, Focal segmental glomerulosclerosis, primary membranous nephropathy, or C3 glomerulopathy and the tubular atrophy is prevented, ameliorated or reduced after administering HSPB5. In some embodiments, the subject is diagnosed or suffering from a condition or disease that manifests as glomerular deposits in the kidney, such as IgA nephropathies, Focal segmental glomerulosclerosis, primary membranous nephropathy, or C3 glomerulopathy and the glomerular deposits are prevented, ameliorated or reduced after administering HSPB5. [0049] In embodiments, administering HSPB5 for preserving or improving kidney function extends the life of a patient’s kidney relative to the standard of care, such as extending the duration the kidney can function before a transplant may be available or needed. In embodiments, administering HSPB5 along with the standard of care treatment extends the life of a patient’s kidney relative to a therapy with the standard of care alone.

[0050] In embodiments, administering HSPB5 for preserving or improving kidney function reduces the need for a kidney transplant relative to the standard of care. In embodiments, administering HSPB5 along with the standard of care treatment reduces the need for a kidney transplant relative to a therapy with the standard of care alone.

[0051] In embodiments, a subject administered HSPB5 requires fewer dialysis treatments relative to the standard of care for preserving or improving kidney function. In embodiments, a subject administered HSPB5 along with the standard of care requires fewer dialysis treatments relative to a therapy with the standard of care.

[0052] In the methods for preserving or improving kidney function, the structure of a subject’s kidney may be assayed via anon-invasive imaging method (e.g., X-ray, MRI, CT Scan, ultrasound, urine analysis and the like) or by an invasive method, e.g, a tissue biopsy, optionally, coupled with a morphological and/or immunological analysis.

[0053] In the methods for treating and improving kidney structure and function, the state of a subject’s spleen and immune cells present in the spleen may be monitored to assess changes in the subject’s health. Such changes may be assayed via a non-invasive imaging method (e.g., X- ray, MRI, CT Scan, ultrasound, a urine analysis and the like) or by an invasive method, e.g, a tissue biopsy, optionally, coupled with a morphological and/or immunological analysis. In embodiments, morphological and/or immunological analysis is spleen derived splenocyte count, splenic follicle diameter, splenic follicle density, spleen derived total splenocyte count, percentage of specific cell types such as dendritic cells, T cells, B cells, macrophages and cell types by cell surface markers such as CD4+ T cells, CD8+ T cells, CD3+ T cells, Treg cells (CD4+FoxP3+CD25+), Bregs (CD19+IL-10+), aberrant DN T cells (CD3+CD19-CD4-CD8-), and any combination thereof.

[0054] Yet another aspect of the present disclosure is a method for treating systemic lupus erythematosus (SLE). The method comprising a step of administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from SLE. In this aspect, the SLE is manifested in the subject as antigen presenting cell (APC) dysfunction, and wherein administering the HSPB5 protein results in one or more effects in the subject selected from the group consisting of an increase in the proportion of macrophages expressing PD-L1 (CD14+PD-L1+), an increase in IL- 10 by splenic macrophages, a decrease in the number of splenocytes, an increase in dendritic cells (CDllc+CD14-), an increase in Tregs (CD4+FoxP3+CD25+), an increase in Bregs (CD19+IL-10+), a reduction in spleen derived macrophages (CD14+), a decrease in spleen derived total T cell count, or a decrease in one or more of CD4+ T cells, CD8+ T cells, CD3+ T cells, aberrant DN T cells (CD3+CD19-CD4-CD8-), B cells, a decrease in the serum level of TNFa, an increase in the serum level of IL-10, a decrease in IL-6 expression level in kidney, and any combination thereof, as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

[0055] In various embodiments, administration of HSPB5 protein does not result in significant change in the serum levels of auto-anti-dsDNA antibodies as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

[0056] In some embodiments, administering the HSPB5 protein for treating SLE manifested as APC dysfunction results in at least two of the effects, e.g., three or more of the effects, in a treated subject.

[0057] In various embodiments, the method for treating SLE manifested as APC dysfunction comprises administering the HSPB5 protein one or more times to a subject. In various embodiments, the HSPB5 protein is administered at least two times to the subject. The HSPB5 protein may be administered two times, three times, four times, five times or more than five times to the subject. The HSPB5 protein may be administered to the subject daily, every other day, once per week, twice per week, once per month or twice per month. The HSPB5 protein may be administered to the subject more than once a day.

[0058] The method for treating SLE manifested as APC dysfunction may further comprise administering to the subject an additional therapy. The additional therapy may be a steroid, an anti-inflammatory, an immunosuppressant, a chemotherapeutic agent, or a combination thereof. The additional therapy may be a standard of care (SOC) for a subject with the relevant disease/disorder.

[0059] In an aspect, the present disclosure provides a method for restoring antigen presenting cell (APC) function. The method comprising a step of administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from systemic lupus erythematosus (SLE). In this aspect, the subject exhibits an indication of APC dysfunction and administering the HSPB5 protein results in one or more effects in the subject selected from the group consisting of an increase in the proportion of macrophages expressing PD-L1 (CD14+PD-L1+), an increase in IL- 10 secretion from splenic macrophages, a decrease in the number of splenocytes, an increase in dendritic cells (CDllc+CD14-), an increase in Tregs (CD4+FoxP3+CD25+), an increase in Bregs (CD19+IL-10+), a reduction in spleen derived macrophages (CD14+), a decrease in spleen derived total T cell count, or a decrease in one or more of CD4+ T cells, CD8+ T cells, CD3+ T cells, aberrant DN T cells (CD3+CD19-CD4-CD8-), B cells and any combination thereof.

[0060] In some embodiments, administering the HSPB5 protein for restoring APC function comprises results in at least two of the effects, e.g., three or more of the effects.

[0061] In various embodiments, the method for restoring APC function comprises administering HSPB5 protein is at least once to the subject. In various embodiments, the HSPB5 protein is administered at least two times to the subject. The HSPB5 protein may be administered two times, three times, four times, five times or more than five times to the subject. In embodiments, the HSPB5 protein is administered to the subject daily, every other day, once per week, twice per week, once per month or twice per month. The HSPB5 protein may be administered to the subject more than once a day.

[0062] The method for restoring APC function may further comprises administering to the subject an additional therapy. The additional therapy may be a steroid, an anti-inflammatory, an immunosuppressant, a chemotherapeutic agent, or a combination thereof. The additional therapy may be a standard of care (SOC) for a subject with the relevant disease/disorder.

[0063] In any of the herein-disclosed methods, a treatment with HSPB5 protein results in two or more features selected from the group consisting of a reduction in proteinuria, a reduction in BUN, a reduction in urine foaming, a reduction in glomerular deposits, a reduction in endocapillary proliferation, a reduction in glomerular lesions, a reduction in tubular atrophy, a reduction in splenocyte count, a reduction in average follicle diameter, a reduction in splenic T cells and an increase in number of splenic follicles. In embodiments, the treatment results in 3, 4, 5, or more than 5 of the features.

Compositions

[0064] The HSPB5 used in methods of the present disclosure may be formulated into a composition (e.g, a pharmaceutical composition) for in vivo administration to a subject. Such compositions can optionally comprise a suitable amount of a pharmaceutically acceptable excipient to provide the form for proper administration. In embodiments, acceptable excipients in the pharmaceutical compositions are preferably nontoxic to subjects at the dosages and concentrations employed. Pharmaceutical excipients can be liquids, such as water or saline. Acceptable excipients may include buffers such as phosphate, citrate, Ringer’s, TBS, PBS, HEPES, HBSS, and TAE, antioxidants such as ascorbic acid and methionine, preservatives such as hexamethonium chloride, octadecyldimethylbenzyl ammonium chloride, resorcinol, and benzalkonium chloride, proteins such as human serum albumin, gelatin, and immunoglobulins, hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, histidine, and lysine, dried skim milk, glycerol, and carbohydrates such as starch, glucose, dextran, lactose, mannose, sucrose, sorbitol, glycerol, glycerol monostearate, and/or glycol. In embodiments, A pharmaceutical excipient may comprise sodium chloride, propylene, ethanol and the like. Pharmaceutical compositions of the disclosure may be administered locally or systemically using an injectable formulation. Pharmaceutical compositions for injection can be formulated using a sterile solution or any pharmaceutically acceptable liquid as a vehicle. Pharmaceutically acceptable vehicles comprise, but are not limited to, sterile water and physiological saline. In a preferred embodiment, a pharmaceutical composition provided herein is administered by injection or infusion.

[0065] A pharmaceutical composition can be in an acceptable diluent, or can comprise a slow release matrix in which the HSPB5 undergoes delayed release.

[0066] Dosage forms suitable for parenteral administration (e.g., intravenous injection or infusion, intraarterial injection or infusion, intra-lymphatic injection or infusion, intramuscular injection, intraperitoneal injection, subcutaneous injection, intra-dermal injection, epi-cutaneous injection, intra-peritoneal, and intra-arterial injection or infusion) or enteral administration (e.g, rectal) include, for example, solutions, suspensions, dispersions, emulsions, and the like.

[0067] Furthermore, pharmaceutical compositions disclosed herein may be formulated according to different methods of delivery. For examples, the pharmaceutical compositions can be formulated for inhalation administration, intratracheal administration, parenteral administration, subcutaneous administration, epi-cutaneous administration, intra-dermal administration, intravenous administration, intra-lymphatic administration, intramuscular administration, intra-arterial administration, intrathecal administration, intra-peritoneal administration, or intraperitoneal administration. The pharmaceutical composition may also be formulated for, or administered via, nasal, spray, oral, aerosol, rectal, or vaginal administration. Exemplary tissue targets may include kidney, liver, skeletal muscle, lung, vascular endothelium, epithelial, and/or hematopoietic cells.

[0068] In embodiments, a route of administering the HSPB5 protein is selected from the group consisting of mucosal, intra-nasal, oral, intra-vaginal, pulmonary, transdermal, intra-venous, sublingual, intra-dermal, epi-cutaneous, intra-lymphatic, intra-peritoneal, rectal, and intramuscular.

[0069] The dosage of a HSPB5 protein can depend on several factors including the characteristics of the subject to be administered. Examples of characteristics include species, strain, sex, age, weight, health, and/or disease status. In any case, the dosage of HSPB5 is understood to be an effective amount. The term “effective amount” or a “therapeutically effective amount” refers to that amount of HSPB5 described herein that is sufficient to affect the intended application, including but not limited to treating SLE or a symptom thereof, as defined herein. The therapeutically effective amount may vary depending upon the intended treatment application, or the subject and disease condition being treated, e.g., the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinary skill in the art. The specific dose will vary depending on the particular composition chosen, the dosing regimen to be followed, whether it is administered in combination with other compounds (e.g, a steroid, an anti-inflammatory, or a combination thereof), timing of administration, the tissue to which it is administered, and the physical delivery system in which it is carried. Dosage may be adjusted to provide the optimum therapeutic response. A dosage may be an amount that effectively treats a disease without inducing significant toxicity and/or inducing an undesirable immune response. Dosages and timings of administrations can be determined on a case-by-case basis.

Additional therapies

[0070] In any of the herein-disclosed methods, e.g, for treating SLE manifested as LN, preserving or improving kidney function, for treating SLE manifested as APC dysfunction, and/or for restoring APC function, a subject may further be administered an additional therapy. The additional therapy may be a standard of care (SOC) for a subject with the relevant disease/disorder.

[0071] In some embodiments, the additional therapy is a steroid, a chemotherapeutic agent, an immunosuppressive, an anti-inflammatory, or a combination thereof.

[0072] The steroid may be a corticosteroid. The steroid may be bethamethasone, (Celestone), cortisone, dexamethasone (Dexamethasone), hydrocortisone (Cortef), methylprednisolone (Medrol, Depo-Medrol, Solu-Medrol), prednisolone (Orapred, Prelone), prednisone (Prednisone Intensol), or triamcinolone (Aristospan Intra- Articular, Aristospan Intralesional, Kenalog).

[0073] The anti-inflammatory may be a nonsteroidal anti-inflammatory drug (NSAID). Examples of NSAIDs are aspirin, celecoxib (Celebrex), diclofenac (Cambia, Cataflam, Voltaren- XR, Zipsor, Zorvolex), diflunisal, etodolac, ibuprofen (Motrin, Advil), indomethacin (Indocin), ketoprofen, ketorolac, nabumetone, naproxen (Aleve, Anaprox, Naprelan, Naprosyn), oxaprozin (Daypro), piroxicam (Feldene), salsalate (Disalsate), sulindac, and tolmetin.

[0074] The chemotherapeutic agent may be cyclophosphamide.

[0075] The immunosuppressive may be azathioprine, my cophenolate mofetil (MMF; CellCept), cyclosporin, or cyclosporin analogs (voclosporin).

[0076] The additional therapy may comprise hydroxychloroquine.

[0077] The additional therapy may be administered before the HSPB5 protein, the additional therapy may be administered after the HSPB5 protein, or the additional therapy may be administered contemporaneous with the HSPB5 protein.

[0078] The additional therapy may be administered via the same route as the HSPB5 protein or may be administered via a different route from the HSPB5 protein. Any route described herein may be used to administer the additional therapy.

[0079] Any formulation (e.g., pharmaceutical composition) described herein may be used to administer the additional therapy.

Subjects

[0080] The term “subject” as used herein refers to a vertebrate, preferably a mammal, more preferably a human. The methods described herein can be useful in human therapeutics, veterinary applications, and/or preclinical studies in animal models of a disease or condition.

[0081] In embodiments, the subject is a mammal, e.g, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, rabbit, sheep, or non-human primate, such as a monkey, chimpanzee, or baboon.

[0082] In embodiments, the subject is a non-human animal, and therefore the invention pertains to veterinary use. In a specific embodiment, the non-human animal is a companion animal, including a household pet, such as a cat or dog. In another specific embodiment, the non-human animal is a livestock animal.

[0083] In embodiments, the mammal is a human.

[0084] In embodiments, the human is an adult human. In embodiments, the adult human is between about 15 and 44 years old, e.g, about 15 to about 20 years old, from about 20 to about 25 years old, from about 25 to about 30 years old, from about 30 to about 35 years old, from about 35 to about 40 years old, or from about 40 to about 45 years old. The adult human may be between about 20 and about 40 years old. In some embodiments, the adult human has a “late onset” disease, which occurs after the age 50; in these embodiments, the adult human is from about 50 to about 55 years old, from about 55 to about 60 years old, from about 60 to about 65 years old, from about 65 to about 70 years old, from about 70 to about 75 years old, from about 75 to about 80 years old, from about 80 to about 85 years old, from about 85 to about 90 years old, from about 90 to about 95 years old or from about 95 to about 100 years old, or older. In embodiments, the human has an age in a range of from about 10 to about 15 years old or from about 45 to about 50 years old. In embodiments, the human is a juvenile. In embodiments, the human has an age in a range of from less than a year to about 10 years old.

[0085] Several embodiments are described herein with reference to example applications for illustration. It should be understood that numerous specific details, relationships, and methods are set forth to provide a full understanding of the features described herein. One having ordinary skill in the relevant art, however, will readily recognize that the features described herein can be practiced without one or more of the specific details or with other methods. The features described herein are not limited by the illustrated ordering of acts or events, as some acts can occur in different orders and/or concurrently with other acts or events. Furthermore, not all illustrated acts or events are required to implement a methodology in accordance with the features described herein.

DEFINITIONS

[0086] The terminology used herein is for the purpose of describing particular cases only and is not intended to be limiting.

[0087] As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0088] The terms “comprise”, “comprising”, “contain,” “containing,” “including”, “includes”, “having”, “has”, “with”, or variants thereof as used in either the present disclosure and/or in the claims, are intended to be inclusive in a manner similar to the term “comprising.”

[0089] Ranges can be expressed herein as from “about” or “approximately” one particular value, and/or to “about” or “approximately” another particular value. When such a range is expressed, another case includes from the one particular value and/or to the other particular value. Similarly, when values are expressed as approximations, by use of the antecedent “about” or “approximately”, it will be understood that the particular value forms another case. It will be further understood that the endpoints of each of the ranges are significant both in relation to the other endpoint, and independently of the other endpoint. The term “about” or “approximately” as used herein refers to a range that is 15% plus or minus from a stated numerical value within the context of the particular usage. For example, about 10 would include a range from 8.5 to 11.5. In another example, “about” can mean within 1 or more than 1 standard deviation, per the practice in the given value. The term “about” or “approximately” also accounts for typical error or imprecision in measurement of values.

[0090] The terms “administer,” “administering”, “administration,” and the like, as used herein, can refer to the methods that are used to enable delivery of a therapeutic (e.g., a HSPB5 protein and/or an additional therapy) or a pharmaceutical composition comprising the therapeutic to a subject, and includes delivery systemically and locally to the desired site of biological action.

[0091] As used herein, a “historical control” is where previous-obtained data is used to compare with new data. Information, e.g, the standard, median, normal, or pre-treatment condition, is essentially borrowed from historical data. The historical data is usually from a subject with the same characteristics, e.g, species, sex, age, weight, size, health, and/or disease status. The historical data may be from a subject who was administered the standard of care (SOC) treatment for his/her specific disease/disorder. The historical data may be from the subject but before being administered a treatment according to the present disclosure.

[0092] Any aspect or embodiment described herein can be combined with any other aspect or embodiment as disclosed herein.

EXAMPLES

[0093] The following examples are given for the purpose of illustrating various embodiments of the invention and are not meant to limit the present invention in any fashion. The present examples, along with the methods described herein are presently representative of preferred embodiments, are exemplary, and are not intended as limitations on the scope of the invention. Changes therein and other uses which are encompassed within the spirit of the invention as defined by the scope of the claims will occur to those skilled in the art.

[0094] Statistical tests for Examples herein: Data are presented as means ± standard error of the mean (SEM). All data were assessed for normality by Shapiro-Wilk test. Data that did not show normal distribution (e.g, proteinuria scores, glomerular deposits, endocapillary proliferation, skin lesions, etc.) were analyzed by the Mann- Whitney nonparametric test. Data with normal distribution (e.g, spleen cell numbers, number and diameter of follicles, splenocyte cell profiles, etc.) were assessed by Student’s t-test. All p-values are included in the figures to appreciate trends of statistical significance (p < 0.05).

Example 1: HSPB5 treats and reduces symptoms of SLE and LN in PBMC in vitro model

[0095] The anti-inflammatory effects of HSPB5 in the context of TLR7/8 or TLR9 activation of Peripheral blood mononuclear cells (PBMCs) were assessed. Peripheral blood mononuclear cells (PBMCs) were isolated from healthy donor blood by density centrifugation over Lymphoprep (Stemcell). Briefly, whole blood was diluted 1:1 in PBS + 2% FBS + ImM EDTA, and gently layered over Lymphoprep in centrifuge tubes. The layered blood was centrifuged for 30 minutes at room temperature with brakes off on a Sorvall ST 16R. The PBMC layer was carefully removed with a serological pipette, and washed with additional PBS + 2% FBS + ImM EDTA. Isolated PBMCs were pelleted by centrifuging at 300 x g for 5 minutes. PBMCs were cultured at the density of 1 x 10⁶ cells/ml in complete RPMI medium. In the R848 experiments, PBMC were pre-treated with HSPB5 (0 or 10 pg/ml), then activated with 1 pg/ml R848 (Resiquimod, a synthetic agonist of TLR7/TLR8, Sigma- Aldrich) or media for 24h. Cells were collected for direct staining for flow cytometry using the following antibodies: CD14-FITC (clone M5E2; BD Pharmingen), CDl lc-PerCP-Cy5.5 (clone B-ly6; BD Pharmingen), MHC class II (HLA-DR) (clone G46-6; BD Pharmingen). For dextran uptake, cells were incubated in PBS + 2% fetal bovine serum (Stemcell Technologies) with 100 pg/ml Dextran-FITC (Sigma) and CDllc-PerCP-Cy5.5 (clone B-ly6; BD Pharmingen) for 30 minutes at 37°C, then washed and fixed with eBioscience™ Intracellular Fixation & Permeabilization Buffer Set (Thermo Fisher). Events were acquired by Atune NxT flow cytometer (ThermoFisher) and data were analyzed using FlowJo vlO software (BD). Results are representative of three similar experiments. Secreted cytokines in R848-treated PBMC culture media were assessed by human cytokine bead array kit according to the manufacturer’s instructions (BD). Data show means ± SEM from three independent experiments.

[0096] Figure 1A shows R848-treated PBMCs incubated with 100 pg/ml dextran and stained with CD11c prior to fixation and assessment by flow cytometry. The less mature status of the cells was further evident by an increased phagocytosis of dextran with HSPB5 treatment.

[0097] Figure IB shows secreted cytokines in R848-treated PBMC culture media assessed by human cytokine bead array kit. The levels of secreted pro-inflammatory cytokines in the culture media supernatants were measured. In R848-stimulated cultures, there was a slight decrease in IFNy and IL-6, but no change in TNF. [0098] These data demonstrate HSPB5 treatment attenuates pro-inflammatory responses in healthy human PBMCs.

Example 2: HSPB5 treats and reduces symptoms of SLE and LN in PBMC in vivo model

[0099] MRL/lpr mice were randomized into study groups at 11 weeks of age (average proteinuria score was 0.8). MRL/lpr mice are homozygous for the lymphoproliferation spontaneous mutation (Fas^lpr), and show systemic autoimmunity, massive lymphadenopathy associated with proliferation of aberrant T cells, arthritis, and immune complex glomerulonephrosis. Mice are useful as a model to determine the etiology of systemic lupus erythematosus (SLE) and Sjorgren (Sicca) syndrome and to evaluate therapies. Each experimental group had 15 mice, which were assigned to groups in a balanced manner to achieve similar average body weights and proteinuria scores. Recombinant HSPB5 (obtained from Delta Crystalion) was formulated in sterile PBS and kept at -80°C until use. At 11 weeks of age and confirmed average proteinuria score of > 0.8, mice received intravenous (i.v.) injections of 200 pL HSPB5 formulations (2.5, 10 or 20 pg) or PBS (vehicle control) twice per week for 10 weeks (study ended at 21 weeks of age). Figure 2A illustrates the overall steps of the study.

[0100] Proteinuria was scored semi-quantitatively as follows: 0 (no protein), 1 (traces of protein equivalent to < 30 mg/dL), 2 (30-100 mg/dL), 3 (100-500 mg/dL), and 4 (> 500 mg/dL). Severe proteinuria was defined as scores >3. Disease progression was monitored by body weight measurements, proteinuria scores, and observations of animal appearance and behavior. Data shown are means ± SEM; 13-15 mice per group. The concentrations of anti-dsDNA antibodies in mouse sera were determined using ELISA. Briefly, the plates were pre-coated with poly-L-lysine and coated with calf thymus DNA (DI 501, Sigma) prior to testing sera from mice.

[0101] In these mice, proteinuria was assessed once weekly by measuring the concentration of protein in urine using Roche Diagnostics Chemstrip 2GP (cat. no. 11895397160). Approximately 10 pl of urine was manually expressed from the bladder of each mouse and applied directly to the reagent strip. After ten seconds, the color of the test reagent strip was compared to the standard color chart to determine the proteinuria score.

[0102] Figure 2B shows weekly proteinuria scores from urine collected over the course of the experiment (weeks 9 to 21). The plot shows percent of mice with severe proteinuria (>3) for each experimental group. The frequency of mice with severe proteinuria (scores 3 and 4) was 67% in the vehicle group and mice treated with 20 pg HSPB5. On the contrary, 50% of mice had severe proteinuria when treated with 2.5 pg HSPB5 and only 31% of mice had severe proteinuria when treated with 10 pg HSPB5. The divergence of severe proteinuria scores was evident as early as 13 weeks, which is 2 weeks after study start, in 2.5 and 10 pg HSPB5 treatment groups and maintained until the study end. Terminal mean proteinuria score of all mice treated with 10 pg HSPB5 was 1.77 compared to a mean score of 2.70 in the vehicle group. Longitudinal proteinuria scores in the 10 pg HSPB5 treatment group were significantly different from the control group.

[0103] At 21 weeks of age, mice were sacrificed; tissues and blood were collected. Terminal blood was processed to serum to assess the concentration of blood urea nitrogen (BUN). To measure BUN, terminal whole blood was collected from anesthetized mice by cardiac puncture and serum was isolated via centrifugation at 1500xg for ten minutes at 4°C. The concentration of BUN in serum was measured by IDEXX Laboratories (test code #6207).

[0104] Figure 2C shows blood urea nitrogen (BUN) concentration in serum from mice at the end of the experiment (week 21). Terminal BUN levels in serum were significantly lower in mice treated with 2.5 pg HSPB5 at 29.64 mg/dL and 10 pg HSPB5 at 30.85 mg/dL versus 59.07 mg/dL in the vehicle group. Terminal BUN levels in serum were lower in mice treated with 20 pg HSPB5 versus the vehicle group.

[0105] The concentrations of anti-dsDNA antibodies in mouse sera were determined using ELISA. Briefly, the plates were pre-coated with poly-L-lysine and coated with calf thymus DNA (DI 501, Sigma) prior to testing sera from mice. Figure 2D shows changes in the serum levels of anti-dsDNA antibodies at 21 weeks of age were not detected, and the improvement in kidney function was not dependent on these antibodies.

[0106] These data demonstrate treatment with HSPB5 attenuates symptoms of lupus nephritis.

Example 3: Treatment with HSPB5 improves kidney pathology in MRL/lpr mice

[0107] Renal pathology was assessed using the same sample set of mice described in Example 2, with kidneys from the treated or vehicle mice characterized.

[0108] At 21 weeks, after euthanasia, kidneys were collected, weighed, and stained for histological scoring of the severity of glomerulonephritis. The collected kidneys were dissected, fixed, and embedded in paraffin. Two kidney cross-sections per mouse were mounted on a slide and were stained with periodic acid-Schiff (PAS). For this, slides were deparaffinized, placed in 0.5% periodic acid solution for five minutes, rinsed, and then placed in Schiff reagent for fifteen minutes. Slides were then rinsed and placed in Mayer's hematoxylin solution for one minute to counterstain. Briefly, glomerular changes (mesangial expansion, endocapillary and extracapillary proliferation, glomerular deposits) and tubular/interstitial changes (tubular atrophy, interstitial infiltration, interstitial fibrosis) were analyzed. The severity of lesions was graded semi- quantitatively as follows: 0 (no change), 1 (mild), 2 (moderate), and 3 (severe). Total histological scores represent the sum of the individual scores. The diameter of 10 randomly selected follicles and the number of follicles per 10⁸ pixel area were determined for each mouse. All histological analyses were performed by a pathologist blinded to the experimental groups and clinical data.

[0109] Figure 3A shows total glomerular lesions score. Upon HSPB5 treatment, total glomerular lesions were reduced in all HSPB5 treatment groups with the 2.5 pg HSPB5 group reaching statistical significance.

[0110] Figure 3B shows glomerular deposits score. There was a significant reduction in glomerular deposits in the 2.5 and 20 pg HSPB5 treatment groups. There was a reduction in glomerular deposits in the 10 pg HSPB5 treatment group.

[0111] Figure 3C shows relative frequency percent of glomerular deposits. HSPB5-treated groups had substantially higher percentage of mice with no detectable glomerular deposits: 50% with 2.5 pg HSPB5, 46% with 10 pg HSPB5 and 62% with 20 pg HSPB5, compared to only 7% of mice in the vehicle group.

[0112] Figure 3D shows endocapillary proliferation score. Endocapillary proliferation was significantly reduced upon treatment with 10 pg HSPB5. Endocapillary proliferation was reduced upon treatment with 2.5 pg HSPB5 and 20 pg HSPB5.

[0113] Figure 3E shows tubular atrophy score. Tubular atrophy, which is a manifestation of chronic renal injury, was reduced with all doses of HSPB5 and nearly diminished in mice treated with 20 pg HSPB5.

[0114] Figure 3F shows tubular atrophy frequency distribution. 12 out of 13 mice (92%) had no signs of tubular atrophy when treated with either 10 or 20 pg HSPB5; only 1 mouse (8%) had a score of 2 in the 10 pg HSPB5 group and 1 mouse had a score of 1 when treated in the 20 pg HSPB5 group. In contrast, 40% of mice in the vehicle group had developed tubular atrophy (7% score 3, 7% score 2, 26% score 1).

[0115] These data demonstrate treatment with HSPB5 improves kidney pathology including improvements in glomerular lesions, glomerular depositions, endocapillary proliferation and tubular atrophy. Example 4: Reduced spleen pathology and increased anti-inflammatory macrophages in spleens of HSPB5-treated MRL/lpr mice.

[0116] Using the same sample set of mice described in Examples 2 and 3, spleens from the treated or vehicle mice were characterized. Approximately 100 mg of spleen was processed to cell suspensions and treated with ammonium-chloride-tris (ACT) lysis buffer to remove red blood cells. T and B lymphocytes as well as co-stimulatory/maturation markers on DCs and macrophages were profded to gain an insight about the immune cell populations. For lymphocyte assessment, cells were stained with the following fluorochrome-conjugated antibodies: CD3-PE-Cy5 (2C11, Biolegend), CD4-AlexaFluor 700 (RM4-5, Biolegend), CD8- PE-Dazzle 594 (5.3-6.7, Biolegend), CD25-FITC (PC61, Biolegend), FoxP3-APC (FJK-16s, Invitrogen), CD19-PerCP-Cy5.5 (1D3, BD), CD27-PE-Cy7 (LG.3A10, Biolegend), IgD-PE (11- 26c.2a, Biolegend). For assessment of antigen-presenting cells and intracellular IL-10 expression, splenocytes were stimulated for four hours with phorbol 12-myristate 13-acetate (PMA) and ionomycin. Cytokine secretion was inhibited with brefeldin A and monensin. After stimulation, splenocytes were stained with the following antibodies: CD14-FITC (Sal 4-2, Biolegend), IL-10-PE (JESS-16E3, Biolegend), CDllc-PerCP-Cy5.5 (HLC, Biolegend), CD86- A1 exaFluor 700 (GL-1, Biolegend), B220-PE-Dazzle 594 (R43-6B2, Biolegend), PD-Ll-PE-Cy7 (10 F.9G2, Biolegend), CD19-PE-Cy5 (6D5, Biolegend). Live/dead cell discrimination was determined using Fixable Viability Dye eFluor 780 (eBioscience). Data was acquired using an LSR II flow cytometer (BD) and analysed with FlowJo (vlO) software (BD).

[0117] Spleens were weighed and a portion of the tissue was formalin-fixed, paraffin embedded (FFPE). One cross section per spleen was stained with hematoxylin and eosin (H&E) for histological analysis. The diameter of 10 randomly selected follicles and the number of follicles per 10⁸ pixel area were determined for each mouse. Spleens sections (approximately 100 mg) were processed to splenocytes and the numbers of cells were recorded as the number of splenocytes/lOOmg of spleen. All histological analyses were performed by a pathologist blinded to the experimental groups and clinical data.

[0118] Figure 4A shows the number of splenocytes/lOOmg spleen section from mice administered vehicle or HSPB5. Histopathological analysis revealed a significant decrease in the splenocyte counts in all treatment groups.

[0119] Figure 4B shows follicle diameter from mice administered vehicle or HSPB5 and Figure 4C shows the number of follicles per 10⁸-pixel area from these mice. Histopathological analysis revealed reduction in the average follicle diameter (Figure 4B) and a significant increase in the number of splenic follicles per area (Figure 4C) in mice treated with 10 pg HSPB5. However no statistically significant reduction was observed in the other treatment groups compared to control.

[0120] Figure 4D shows flow cytometric analysis of splenocytes from mice administered vehicle or HSPB5. T cells (CD3+) and B cells are shown as frequency of live cells. CD4, CD8, and DN cells are shown as percentage of the CD3+ population. Tregs (FoxP3+CD25+) are shown as a percentage of CD3+CD4+ population. Treatment with 10 pg HSPB5 significantly decreased the frequency of total T cells in spleens, but it did not change the percentages of T cell populations, CD4+, CD8+ or aberrant DN T cells (CD3+CD19-CD4-CD8-). A slight (2%) increase in Tregs (CD4+FoxP3+CD25+) was observed in the same treatment group, but no substantial changes in the frequency of total B cells. Treatment with 20 pg HSPB5 gave similar effects in most cell types observed, although an increase in Tregs was not observed at this dose.

[0121] Figure 4E shows the percentage of splenic dendritic cells (CD1 lc+CD14-) in a splenocyte sample. Dendritic cells (CDl lc+) and monocytes/macrophages (CD14+), the cells of the innate immune system that interface with the T and B lymphocytes and modulate immune responses, were examined. There was a significant increase in the frequency of dendritic cells in mice treated with 10 pg HSPB5, but no change with respect to their activation status. There was an increase in the frequency of dendritic cells in mice treated with 20 pg HSPB5 and no substantial change in the frequency of dendritic cells in mice treated with 2.5 pg HSPB5.

[0122] Figure 4F shows the percentage of monocytes/macrophages (CD14+) in the splenocyte sample. There was a 25% reduction in the frequency of macrophages in mice treated with 10 pg HSPB5 and in mice treated with 20 pg HSPB5. There was no substantial change in the frequency of monocytes/macrophages in mice treated with 2.5 pg HSPB5.

[0123] Figure 4G shows the percentage of monocytes/macrophages that are positive for PD-L1 in the splenocyte sample. There was an increase in the subpopulation expressing PD-L1 upon treatment with HSPB5.

[0124] Figure 4H shows the mean fluorescent intensity (MFI) of IL-10 expression in monocytes/macrophages in the splenocyte sample. The expression of intracellular IL-10, a classic marker of regulatory antigen-presenting cells, was significantly increased in macrophages of mice treated with 10 pg HSPB5, and also slightly increased in the 2.5 pg HSPB5 and 20 pg HSPB5 treated mice. [0125] Figure 41 shows the percentage of Bregs (CD19+IL-10+) in the splenocyte sample. There was an increase in the frequency of Bregs in mice treated with 2.5 pg and 10 pg HSPB5. There was no substantial change in the frequency of Bregs in mice treated with 20 pg HSPB5.

[0126] These results demonstrate an immunomodulatory role for HSPB5 that manifests in induction of immunosuppressive macrophages and reduced spleen pathology in spleens of SLE model, MRL/lpr mice.

Example 5: MRL/lpr mice treated with HSPB5 demonstrate higher serum IL- 10 and lower IL-6 cytokine levels.

[0127] Using the same sample set of mice described in Examples 2 to 4, cytokines in mouse sera were measured by Cytometric Bead Array Flex sets (BD) with reactivity to mouse IL- 1 , IL-2, IL-4, IL-6, IL- 10, IL- 17 A, and TNF and according to the manufacturer’s instructions. The beads were acquired using Attune NxT cytometer and software (Thermo Fisher) and analyzed by FlowJo vlO (BD).

[0128] Figure 5A shows terminal (day 21) serum levels of cytokines assessed by mouse cytokine flex set bead array and profded by flow cytometry. Serum level of IL-6 was not reduced, TNF a was decreased, and IL-10 was significantly increased upon HSPB5 treatment.

[0129] Total kidney mRNA was assessed for the transcriptional changes in IL-10, IL-6 and TNF by qPCR. Flash-frozen kidneys from MRL/lpr mice were homogenized using a CryoGrinder (OPS Diagnostics) and RNA was extracted using a ReliaPrep RNA Tissue Miniprep (Promega). RNA was reverse transcribed to cDNA using a GoScript Reverse Transcription System kit (Promega). The resulting cDNA was used to set-up qPCR reactions (50ng RNA-equivalents per reaction) using a GoTaq Probe mastermix (Promega) and TaqMan probe/primer sets (ThermoFisher). Each reaction was run in triplicate, with probes and primers against TATA-Binding Protein (TBP) included in each reaction as an internal control. The primer and probe sets used were targeted against IL-6 (assay ID: Mm00446190_ml), IL-10 (assay ID: Mm01288386_ml), TNF (assay ID: Mm00443258_ml) and TBP (assay ID: Mm00446973_ml). Probes against genes of interest were labeled with FAM, while the probe targeting TBP was labeled with VIC.

[0130] Figure 5B shows total kidney mRNA assessed for the transcriptional changes in IL-10, IL-6 and TNF by qPCR. The bar graph shows relative expression levels after normalization to the internal control (TBP). Substantial changes in IL- 10 and TNF were not detected, whereas IL- 6 expression levels were reduced in kidneys upon treatment with HSPB5 treatment. [0131] Figure 5C shows IL-6 expression strongly correlated with kidney pathology (r > 0.7): BUN (r = 0.82, p = 8.20e-8), tubular atrophy (r = 0.89, p = 1.77e-10) and total kidney lesions (r = 0.79, p = 6.93e-007). None of the other cytokines assessed were found to have strong correlations with any of the renal pathology parameters.

[0132] Figure 6 shows qPCR results mapped against kidney histology and functional parameters, mouse per mouse. There was increased frequency of mice expressing relatively moderate levels of IL-6 (Cq < 30) in the vehicle group compared to the HSPB5-treated group (27% vs 8%).

[0133] Figure 7 illustrates a model of HSPB5-mediated therapeutic effects in SLE and LN; created with BioRender, com

[0134] While embodiments of the present invention have been shown and described herein, it will be obvious to those skilled in the art that such embodiments are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments of the invention described herein may be employed in practicing the invention. It is intended that the following claims define the scope of the invention and that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

CLAIMS What is claimed is:

1. A method of treating systemic lupus erythematosus (SLE) comprising: administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from SLE, wherein the SLE is manifested in the subject as active lupus nephritis, and wherein the HSPB5 protein is administered in an effective amount to treat, improve or ameliorate at least one symptom or indicator of reduced kidney function.

2. A method of preserving or improving kidney function comprising: administering an aB-crystallin (HSPB5) protein to a subject diagnosed with or suffering from a decrease in normal kidney function or from abnormal kidney function, wherein the subject exhibits at least one symptom of compromised kidney function, and wherein the subject’s kidney function is improved after administering HSPB5.

3. The method of claim 2, wherein, the subject is diagnosed or suffering from systemic lupus erythematosus (SLE) or lupus nephritis (LN).

4. The method of claim 2, wherein, the subject exhibits tubular atrophy and the subject is diagnosed with or suffering from IgA nephropathies, focal segmental glomerulosclerosis, primary membranous nephropathy, or C3 glomerulopathy.

5. The method of claim 2, wherein, the subject exhibits glomerular depositions and the subject is diagnosed with or suffering from IgA nephropathies, focal segmental glomerulosclerosis, primary membranous nephropathy, or C3 glomerulopathy.

6. The method of any one of claims 1 to 5, wherein the at least one symptom or indicator is increased protein in the urine (proteinuria).

7. The method of claim 6, wherein the level of proteinuria in the subject is reduced as compared to the level prior to administration of the HSPB5 protein or relative to a historical control.

8. The method according to any one of claims 1-7, wherein the subject’s level of blood urea nitrogen (BUN) is reduced as compared to the level prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

- 28 -

9. The method according to any one of claims 1- 8, wherein amount of foaming in a subject’s urine is reduced as compared to the amount of foaming prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

10. The method of any one of claims 6-9, wherein the level of proteinuria, level of blood urea nitrogen, or amount of urine foaming, or a combination thereof is reduced over 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks or more than 7 weeks following administration of the HSPB5 protein.

11. The method according to any one of claims 1-10, wherein the structure of the kidney is preserved or improved as compared to kidney structure prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

12. The method of claim 11, wherein preservation or improvement of kidney structure is assessed by a decrease in one or more of glomerular deposits, endocapillary proliferation, glomerular lesions, tubular atrophy and any combinations thereof as compared to kidney structure prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

13. The method of any one of claims 1-12, wherein the treatment results in an improved spleen pathology of the subject as compared to spleen pathology prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

14. The method of claim 13, wherein improved spleen pathology comprises one or more of a reduction in splenocyte count, a reduction in average follicle diameter, an increase in the number of splenic follicles per area, or a combination thereof.

15. The method of any one of claims 1-14, wherein the treatment results in an increase in dendritic cells, an induction of immunosuppressive macrophages, a decrease in T cells in spleen or any combination thereof as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

16. A method of treating systemic lupus erythematosus (SLE) comprising: administering an HSPB5 protein to a subject diagnosed with or suffering from SLE, wherein the SLE is manifested in the subject as antigen presenting cell (APC) dysfunction, and wherein administering the HSPB5 protein results in one or more effects in the subject selected from the group consisting an increase in the proportion of macrophages expressing PD-L1 (CD14+PD- L1+), an increase in IL-10 secretion from splenic macrophages, a decrease in the number of splenocytes, an increase in dendritic cells, an induction of immunosuppressive macrophages, and any combination thereof as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

17. A method of restoring antigen presenting cell (APC) function, comprising: administering an HSPB5 protein to a subject diagnosed with or suffering from systemic lupus erythematosus (SLE), wherein the subject exhibits an indication of APC dysfunction, and wherein administering the HSPB5 protein results in one or more effects in the subject selected from the group consisting of an increase in the proportion of macrophages expressing PD-L1, an increase in IL- 10 secretion from splenic macrophages, a decrease in the number of splenocytes, an increase in dendritic cells, an induction of immunosuppressive macrophages, and any combination thereof as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

18. The method of any one of claims 1-17, wherein the treatment results in a decrease in the serum level of TNFa, an increase in the serum level of IL-10 or a combination thereof as compared to the level prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

19. The method of any one of claims 1-18, wherein the treatment results in a decrease in IL-6 expression level in kidney as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

20. The method of any one of claims 1-19, wherein the administration of HSPB5 protein does not result in significant change in the serum levels of auto-anti-dsDNA antibodies as compared to prior to administration of the HSPB5 protein or relative to a historical control, including a subject treated with the standard of care (SOC).

21. The method according to any one of claims 1-20, wherein the HSPB5 protein is administered at least two times to the subject.

22. The method according to any one of claims 1-20, wherein the HSPB5 protein is administered two times, three times, four times, five times or more than five times to the subject.

23. The method according to any one of claims 1- 22, wherein the HSPB5 protein is administered to the subject daily, every other day, once per week, twice per week, once per month or twice per month.

24. The method according to any one of claims 1-23, wherein the subject is further administered an additional therapy.

25. The method of claim 24, wherein the additional therapy comprises a standard of care (SOC) treatment.

26. The method of claim 24 or claim 25, wherein the additional therapy is a steroid, an antiinflammatory, a chemotherapy, an immunosuppressive, or a combination thereof.

27. The method of any one of claims 24-26, wherein the additional therapy is one or more of cyclophosphamide, a cyclosporin, and hydroxychloroquine.

28. The method of any one of claims 1-27, wherein the treatment results in two or more features selected from the group consisting of a reduction in proteinuria, a reduction in BUN, a reduction in urine foaming, a reduction in glomerular deposits, a reduction in endocapillary proliferation, a reduction in glomerular lesions, a reduction in tubular atrophy, a reduction in splenocyte count, a reduction in average follicle diameter, a reduction in splenic T cells and an increase in number of splenic follicles.

29. The method of claim 28, wherein the treatment results in three of the features.

30. The method of claim 28, wherein the treatment results in 3, 4, 5, or more than 5 of the features.