SCD2+ stromal cells for treating diabetic kidney disease

Abstract

Provided herein are compositions and methods for treating kidney disease, eg, diabetic kidney disease, using compositions comprising SDC2+ stromal stem cells.

Description

[Technical Field]

[0001] cross reference This application claims the benefit of U.S. Provisional Application No. 63 / 379,352, filed October 13, 2022, which is incorporated herein by reference in its entirety. [Background technology]

[0002] Diabetic kidney disease, or diabetic nephropathy, is currently defined as chronic kidney disease secondary to diabetes and is a complication of both type 1 and type 2 diabetes. Diabetic kidney disease affects the kidneys' ability to remove waste products and excess fluid from the body. In the United States, one in three people with diabetes has diabetic kidney disease. Over time, diabetic kidney disease can progress to kidney failure or end-stage renal disease, requiring dialysis and / or a kidney transplant. Summary of the Invention

[0003] Provided herein are methods for treating diabetic kidney disease in an individual. In some embodiments, the methods involve administering to the individual about 80×10 6 The present invention relates to a method for treating diabetic kidney disease, comprising administering a single dose of SDC2+ interstitial cells, thereby treating diabetic kidney disease. In some embodiments, the single dose is sufficient to improve renal function compared to an untreated individual. In some embodiments, the improvement in renal function is assessed by measuring glomerular filtration rate (mGFR) or estimating glomerular filtration rate (eGFR). In some embodiments, the measured glomerular filtration rate (mGFR) or estimated glomerular filtration rate (eGFR) is improved compared to a baseline measurement. In some embodiments, the measured glomerular filtration rate (mGFR) or estimated glomerular filtration rate (eGFR) does not decline compared to a baseline measurement. In some embodiments, the single dose treatment is effective for at least 18 months. In some embodiments, the single dose treatment prevents renal failure for at least 18 months. In some embodiments, the single dose treatment is effective ... + CD25 highFoxP3 + CD127 - ) is increased after a single dose of treatment in an individual compared to an untreated individual. In some embodiments, memory regulatory T cells (CD4 + CD25 high FoxP3 + CD127 - CD45RA - CD45RO + or CD4+Helios + CD95 + HLA-DR - In some embodiments, the frequency of natural killer T (NKT) cells (CD3+CD56+) is not increased compared to untreated individuals. In some embodiments, the frequency of intermediately activated monocytes (HLA-DR1) is increased after a single dose of treatment in an individual compared to untreated individuals. + CD33 + CD14 + CD16 + In some embodiments, the frequency of regulatory T cells, memory regulatory T cells, NKT cells, intermediately activated monocytes, and / or non-classical patrolling monocytes is suppressed or decreased after a single dose of treatment in the individual compared to an untreated individual. In some embodiments, the frequency of regulatory T cells, memory regulatory T cells, NKT cells, intermediately activated monocytes, and / or non-classical patrolling monocytes is measured after about 12 to about 18 months of treatment. In some embodiments, the individual has type 2 diabetes or is suffering from symptoms of type 2 diabetes. In some embodiments, the individual has a urinary albumin excretion (UAE) of at least 60 μg / min before treatment. In some embodiments, the individual has a urinary albumin / creatinine ratio (UACR) of at least 88 mg / g or 10 mg / mmol before treatment. In some embodiments, the individual receives at least 30-50 ml / min / 1.73 m prior to treatment. 2In some embodiments, the individual has an eGFR of at least 10 ml / min / 1.73 m for three years prior to treatment. 2 In some embodiments, the individual has experienced a decline in eGFR of at least 5 ml / min / 1.73 m2 in the 18 months prior to treatment.

[0004] Incorporation by Reference All publications, patents, and patent applications mentioned in this specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference. [Brief explanation of the drawings]

[0005] The features and advantages of the present invention will be understood 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:

[0006] [Figure 1] The difference in eGFR from baseline to 18 months after treatment is shown. [Figure 2] Difference in eGFR from 30 months to baseline and from baseline to 18 months post-treatment will be shown. [Figure 3A] The study flowchart of the NEPHSTROM cohort study is shown. [Figure 3B] The overall NEPHSTROM clinical treatment plan and follow-up are presented. [Figure 4A] Figure 4A shows the frequency of peripheral blood leukocytes during the study period. Numbers are shown as the percentage of CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Figure 4B shows CD4+ T cells. Values ​​are expressed as median (IQR). *P<0.05 (ANCOVA) between the ORBCEL-M group and the placebo group. §P<0.05 (Wilcoxon test) compared with pre-infusion. Lin-: CD3, CD14, CD16, CD19, CD20, and CD56. [Figure 4B]Figure 4B shows the frequency of peripheral blood leukocytes during the study period. Numbers are shown as the percentage of CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Figure 4B shows CD8+ T cells. Values ​​are expressed as median (IQR). *P<0.05 (ANCOVA) between the ORBCEL-M group and the placebo group. §P<0.05 (Wilcoxon test) compared with pre-infusion of the group. Lin-: CD3, CD14, CD16, CD19, CD20, and CD56. [Figure 4C] Figure 4C shows the frequency of peripheral blood leukocytes during the study period. Numbers are shown as the percentage of CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Figure 4C shows B cells. Values ​​are expressed as median (IQR). *P<0.05 (ANCOVA) between the ORBCEL-M group and the placebo group. §P<0.05 (Wilcoxon test) compared with pre-infusion of the group. Lin-: CD3, CD14, CD16, CD19, CD20, and CD56. [Figure 4D] Figure 4D shows the frequency of peripheral blood leukocytes during the study period. Numbers are shown as the percentage of CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Figure 4C shows Lin-HLADR+ dendritic cells. Values ​​are expressed as median (IQR). *P<0.05 (ANCOVA) between the ORBCEL-M group and the placebo group. §P<0.05 (Wilcoxon test) compared with pre-infusion. Lin-: CD3, CD14, CD16, CD19, CD20, and CD56. [Figure 4E] Figure 4D shows the frequency of peripheral blood leukocytes during the study period. Numbers are shown as the percentage of CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Figure 4E shows monocytes. Values ​​are expressed as median (IQR). *P<0.05 (ANCOVA) between ORBCEL-M and placebo groups. §P<0.05 (Wilcoxon test) compared with pre-infusion of the group. Lin-: CD3, CD14, CD16, CD19, CD20, and CD56. [Figure 4F]Figure 4F shows the frequency of peripheral blood leukocytes during the study period. Numbers are shown as the percentage of CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Figure 4F shows cytotoxic NK cells. Values ​​are expressed as median (IQR). *P<0.05 (ANCOVA) between the ORBCEL-M group and the placebo group. §P<0.05 (Wilcoxon test) compared with pre-infusion of the group. Lin-: CD3, CD14, CD16, CD19, CD20, and CD56. [Figure 4G] Figure 4G shows the frequency of peripheral blood leukocytes during the study period. Numbers are shown as the percentage of CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Figure 4G shows natural killer T cells. Values ​​are expressed as median (IQR). *P<0.05 (ANCOVA) between the ORBCEL-M group and the placebo group. §P<0.05 (Wilcoxon test) compared with pre-infusion. Lin-: CD3, CD14, CD16, CD19, CD20, and CD56. [Figure 5A] The frequencies of peripheral blood Tregs and Treg subpopulations during the study period are shown. Percentages of Tregs (Figure 5A) among peripheral blood CD3+CD4+ T cells in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). Tregs are regulatory T cells. *P<0.05 between the ORBCEL-M and placebo groups (ANCOVA). Tregs are regulatory T cells. [Figure 5B] The frequencies of peripheral blood Tregs and Treg subpopulations during the study period are shown. The percentage of CD45RA-RO+ memory Tregs (Figure 5B) among peripheral blood CD3+CD4+ T cells in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). Tregs are regulatory T cells. *P<0.05 between the ORBCEL-M and placebo groups (ANCOVA). Tregs are regulatory T cells. [Figure 5C]The frequencies of peripheral blood Tregs and Treg subpopulations during the study period are shown. Percentages of Helios+CD95+HLA-DR- memory Tregs (Figure 5C) among peripheral blood CD3+CD4+ T cells in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). Tregs are regulatory T cells. *P<0.05 between the ORBCEL-M and placebo groups (ANCOVA). Tregs are regulatory T cells. [Figure 5D] The frequencies of peripheral blood Tregs and Treg subpopulations during the study period are shown. The percentage of CD45RA+RO- naive Tregs (Figure 5D) among peripheral blood CD3+CD4+ T cells in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). Tregs are regulatory T cells. *P<0.05 between the ORBCEL-M group and the placebo group (ANCOVA). Tregs are regulatory T cells. [Figure 6A] The frequencies of peripheral blood monocyte subpopulations during the study period are shown. The percentage of HLADR+CD33+CD14+CD16- monocytes (Figure 6A) among CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). *P<0.05 between the ORBCEL-M and placebo groups (ANCOVA). [Figure 6B] The frequencies of peripheral blood monocyte subpopulations during the study period are shown. The percentage of HLADR+CD33+CD14-CD16+ monocytes (Figure 6B) among CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). *P<0.05 between the ORBCEL-M and placebo groups (ANCOVA). [Figure 6C] The frequencies of peripheral blood monocyte subpopulations during the study period are shown. The percentage of HLADR+CD33+CD14+CD16+ monocytes (Figure 6C) among CD45+ peripheral blood leukocytes in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). *P<0.05 between the ORBCEL-M and placebo groups (ANCOVA). [Figure 7A] Serum concentrations of the pro-inflammatory mediator TNFR1 (Figure 7A) were measured in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). P<0.05 (Wilcoxon test) compared with pre-infusion in the group. TNFR1 is soluble tumor necrosis factor type 1. [Figure 7B] Serum concentrations of the pro-inflammatory mediator NGAL (Figure 7B) were measured in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). † P<0.05 (Wilcoxon test) compared with pre-infusion in the group. NGAL is a neutrophil gelatinase-binding lipocalin. [Figure 7C] Serum concentrations of the pro-inflammatory mediator VCAM-1 (Figure 7C) were shown in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). § P<0.05 (Wilcoxon test) compared with pre-infusion in the group. VCAM-1 is vascular cell adhesion molecule 1. [Figure 7D] Serum concentrations of the pro-inflammatory mediator EGF (Figure 7D) were measured in participants randomized to ORBCEL-M or placebo during the follow-up period. Values ​​are expressed as median (IQR). P<0.05 (Wilcoxon test) compared with pre-infusion for each group. EGF, epidermal growth factor. [Figure 8] Progression of diabetic kidney disease in the two study arms. Risk of progression of diabetic kidney disease in participants randomized to ORBCEL-M or placebo is shown as change in 2-year risk of achieving end-stage renal disease from baseline to 18 months based on the validated Tangri 4-parameter kidney failure risk equation. Total participants were: n=12 in the ORBCEL-M group and n=4 in the placebo group at baseline; and n=10 in the ORBCEL-M group (2 died before the end of the study) and n=4 in the placebo group at 18 months. [Figure 9A]Correlation between the percentage of Tregs or CD45RA-RO+ memory Tregs and serum concentrations of inflammatory mediators in the entire study cohort. Correlation between the percentage of Tregs among peripheral blood CD3+CD4+ T cells in the entire study cohort (Figure 9A) and serum TNFR1 concentrations (r=-0.3690, P<0.001). TNFR1 is the tumor necrosis factor type 1 receptor. [Figure 9B] Correlation between the percentage of Tregs or CD45RA-RO+ memory Tregs and serum concentrations of inflammatory mediators in the entire study cohort is shown. Correlation between the percentage of CD45RA-RO+ memory Tregs among peripheral blood CD3+CD4+ T cells in the entire study cohort (Figure 9B) and serum TNFR1 concentrations (r=-0.4024, P<0.001). TNFR1 is the tumor necrosis factor type 1 receptor. [Figure 9C] Correlation between the percentage of Tregs or CD45RA-RO+ memory Tregs and serum concentrations of inflammatory mediators in the entire study cohort. Correlation between the percentage of Tregs among peripheral blood CD3+CD4+ T cells in the entire study cohort (Figure 9C) and serum NGAL concentrations (r=-0.3879, P<0.001). NGAL is a neutrophil gelatinase-binding lipocalin. [Figure 9D] Correlation between the percentage of Tregs or CD45RA-RO+ memory Tregs and serum concentrations of inflammatory mediators in the entire study cohort. Correlation between the percentage of CD45RA-RO+ Tregs among peripheral blood CD3+CD4+ T cells in the entire study cohort (Figure 9D) and serum NGAL concentrations (r=-0.3907, P<0.001). NGAL is a neutrophil gelatinase-binding lipocalin. [Figure 10A] Correlation between the percentage of Tregs or CD45RA-RO+ memory Tregs and estimated glomerular filtration rate in the entire study cohort. Correlation between the percentage of Tregs among peripheral blood CD3+CD4+ T cells in the entire study cohort and estimated glomerular filtration rate using the CKD-EPI (Figure 10A) equation (r=0.2235, P=0.035). CKD-EPI is the Chronic Kidney Disease Epidemiology Collaboration equation. [Figure 10B] Correlation between the percentage of Tregs or CD45RA-RO+ memory Tregs and estimated glomerular filtration rate in the entire study cohort. Correlation between the percentage of Tregs among peripheral blood CD3+CD4+ T cells in the entire study cohort and estimated glomerular filtration rate using the MDRD (Figure 10B) formula (r=0.2949, P<0.005). MDRD is the Modification of Diet in Renal Disease formula. [Figure 10C] Correlation between the percentage of Tregs or CD45RA-RO+ memory Tregs and estimated glomerular filtration rate in the entire study cohort. Correlation between the percentage of CD45RA-RO+ memory Tregs among peripheral blood CD3+CD4+ T cells in the entire study cohort and estimated glomerular filtration rate using the CKD-EPI (Figure 10C) equation (r=0.2578, P=0.015). CKD-EPI is the Chronic Kidney Disease Epidemiology Collaboration equation. [Figure 10D] Correlation between the percentage of Tregs or CD45RA-RO+ memory Tregs and estimated glomerular filtration rate in the entire study cohort. Correlation between the percentage of CD45RA-RO+ memory Tregs among peripheral blood CD3+CD4+ T cells in the entire study cohort and glomerular filtration rate estimated using the MDRD (Figure 10D) formula (r=0.3313, P<0.002). MDRD is the Modified Diet for Renal Disease formula. [Figure 11A] Correlation between serum concentrations of inflammatory mediators and measured or estimated glomerular filtration rate in the entire study cohort. Correlation between serum TNFR1 concentrations and glomerular filtration rate measured by iohexol plasma clearance (Figure 11A) in the entire study cohort (r=-0.3929, P=0.002). TNFR1 is the tumor necrosis factor type 1 receptor. [Figure 11B]Correlations between serum concentrations of inflammatory mediators and measured or estimated glomerular filtration rate in the entire study cohort are shown. Correlation between serum TNFR1 concentrations and glomerular filtration rate estimated by the CKD-EPI (Figure 11B) equation in the entire study cohort (r=-0.5493, P<0.001). CKD-EPI is the Chronic Kidney Disease Epidemiology Collaboration equation, and TNFR1 is tumor necrosis factor receptor type 1. [Figure 11C] Correlations between serum concentrations of inflammatory mediators and measured or estimated glomerular filtration rate in the entire study cohort are shown. Correlation between serum TNFR1 concentrations and glomerular filtration rate estimated by the MDRD (Figure 11C) equation in the entire study cohort (r=-0.5928, P<0.001). MDRD is the Modified Diet in Renal Disease equation; TNFR1 is tumor necrosis factor receptor type 1. [Figure 11D] Correlation between serum concentrations of inflammatory mediators and measured or estimated glomerular filtration rate in the entire study cohort is shown. Correlation between serum NGAL concentrations and glomerular filtration rate measured by iohexol plasma clearance (Figure 11D) in the entire study cohort (r=-0.5932, P<0.001). NGAL is a neutrophil gelatinase-binding lipocalin. [Figure 11E] Correlations between serum concentrations of inflammatory mediators and measured or estimated glomerular filtration rate (GFRT) in the entire study cohort are shown. Correlation between serum NGAL concentrations and glomerular filtration rate estimated by the CKD-EPI (Figure 11E) equation in the entire study cohort (r = -0.3486, P = 0.001). CKD-EPI is the Chronic Kidney Disease Epidemiology Collaboration equation, and NGAL is neutrophil gelatinase-binding lipocalin. [Figure 11F] Correlations between serum concentrations of inflammatory mediators and measured or estimated glomerular filtration rate (GFRT) in the entire study cohort are shown. Correlation between serum NGAL concentrations and GFRT estimated by the MDRD (Figure 11F) equation in the entire study cohort (r = -0.3626, P = 0.001). MDRD is the Modified Diet for Renal Disease equation; NGAL is neutrophil gelatinase-binding lipocalin. DETAILED DESCRIPTION OF THE INVENTION

[0007] Type 2 diabetes mellitus (DM) is a rapidly growing global healthcare challenge, estimated to affect 437 million people worldwide in 2019. Among its complications, diabetic kidney disease (DKD) affects 30%–40% of adults living with type 2 diabetes and accounts for approximately 40% of people with end-stage renal disease (ESKF) requiring renal replacement therapy in high-income countries. Clinically, DKD is typically characterized by the development of microalbuminuria, which can further progress to macroalbuminuria, followed by a decline in glomerular filtration rate (GFR), and ultimately uremia. A wide range of maladaptive processes, primarily driven by hyperglycemia, contribute to the pathobiology of DKD, including increased oxidative stress, chronic inflammation, accumulation of advanced glycation end products (ADGs), renal hypoxia, cellular apoptosis, and altered activation of the renin-angiotensin-aldosterone system.

[0008] Over the past few decades, medical advances have significantly improved the management of patients with DKD, thereby extending their survival. However, despite optimal treatment, including metabolic and blood pressure control, lipid management, and proteinuria, patients with DKD remain at high renal and cardiovascular risk. Recent clinical trials of sodium-glucose cotransporter 2 (SGLT2) inhibitors and other pharmacological agents have demonstrated the potential to slow the rate of renal function decline in type 2 diabetes-induced DKD. Halting DKD progression may require successfully targeting multiple injury pathways, including those mediating inflammation, oxidative stress, renal hypoxia, and fibrosis.

[0009] Among novel therapeutic strategies for DKD, cell therapy using MSCs has emerged as an option based on its potential to deliver or induce the production of a wide range of mediators that simultaneously target maladaptive processes contributing to kidney injury. Numerous studies in preclinical models of diabetes and diabetic nephropathy have demonstrated that MSCs exert beneficial renoprotective activity by locally and systemically modulating several key pathophysiological pathways that underpin DKD. However, clinical application of this cell therapy has been limited, despite promising results reported with intravenous infusion of allogeneic mesenchymal precursor cells (rexlemestrocel-L) in adults with type 2 diabetes and moderate to severe chronic kidney disease (CKD).

[0010] The stromal stem cells provided herein are isolated from a cell population based on the expression of the cell surface marker syndecan-2 (SDC2). Stromal stem cells are a population of immunomodulatory fibroblasts isolated from one or more of human bone marrow, adipose tissue, placenta, and umbilical cord tissue. In some cases, small amounts of stromal stem cells are isolated from these tissues and cultured in vitro or ex vivo, where they proliferate as plastic-adherent cells and form fibroblast colonies (CFU-F). Stromal stem cells act as immune system regulators.

[0011] When stromal stem cells are exposed to an inflammatory and apoptotic environment containing CD95 / Fas Ligands and Granzyme B / Perforins, they can activate flippases such as TMEM30a / CDC50a and caspases, which display phosphatidylserine (PS) on the stromal stem cell surface and cleave pannexin 1 channels. Cleavage of pannexin 1 by caspases triggers the release of specific immunometabolites, including but not limited to the polyamine spermidine, UDP, ATP, and lactate, which act as a "find me" signal to attract circulating phagocytes via ATP-dependent P2Y receptors. The released ATP can be processed by approaching phagocytes expressing CD39 / CD73, which can then locally produce adenosine, which suppresses inflammation via adenosine receptors. This also leads to the expression of anti-inflammatory and antipyretic molecules such as Nr4a1, Nr4a2, arginase, and thrombospondin. Activation of TMEM30a / CDC50a results in the presentation of PS on the surface of stromal stem cells as an "eat me" signal to attracted phagocytes. Efferocytosis of PS+ stromal stem cells leads inflammatory phagocytes to accumulate ingested metabolites, such as lactate, polyamines / spermidine, and ATP. The accumulation of polyamines / spermidine, lactate, and ATP in efferocytic phagocytes can induce hypusination of eukaryotic translation factor 5A (eIF5A). The natural amino acid hypusine (Nε-4-amino-2-hydroxybutyl (lysine)) is derived from the polyamine spermidine and is present only in a single family of cellular proteins, the eIF5A isoform. Hypusine is formed by the attachment of the aminobutyl moiety of spermidine to a specific lysine residue in this protein. Post-translational synthesis of hypusine involves two enzymatic steps catalyzed by deoxyhypusine synthase (DHPS) and deoxyhypusine hydroxylase (DOHH). Hypusine is essential for the efficient activity of eIF5A. Therefore, spermidine is required to hypusinate the translation factor eIF5A. Hypusinated eIF5A (eIF5A H) promotes efficient expression of a subset of mitochondrial proteins involved in the TCA cycle and oxidative phosphorylation (OXPHOS). Some of these enzymes have a mitochondrial targeting sequence (MTS), such as succinate coenzyme A ligase (SUCLG1), methylmalonyl-CoA mutase (MCM), and succinate dehydrogenase (SDHA), and eIF5A. H This, in part, increases their dependency on polyamines. In monocytes / macrophages / phagocytes, a metabolic switch between OXPHOS and glycolysis underpins divergent functional fates stimulated by activation signals. In these phagocytes, hypusination of eIF5A appears to be dynamically regulated after activation, promoting the expression of degradative proteins such as Nr4a1, Nr4a2, arginase, IDO1, and amphiregulin. Inhibiting this hypusination blunts OXPHOS-dependent phagocyte polarization / alternative activation. Without being limited to any one theory, stromal stem cells undergo Fas / GrB-mediated apoptosis and release spermidine / ATP via caspase-activated pannexin 1, allowing inflammatory phagocytes to uptake spermidine / ATP, potentially therapeutically controlling phagocyte activation by targeting the polyamine-eIF5A-hypusin axis.

[0012] Stromal stem cells also secrete proteins and extracellular vesicles (exosomes) containing important immunosuppressive factors, such as transforming growth factor β1 (TGFβ1), indoleamine 2,3-dioxygenase 1 (IDO1), TNF-stimulated gene 6 (TSG6), and the purinergic enzymes CD39 and CD73. Without being limited to any one theory, this collection of factors is believed to induce large numbers of regulatory T cells, suppress the proliferation of both T helper T cells and cytotoxic T cells, reduce the production of the proinflammatory cytokine interferon gamma (IFN-γ), reduce the production of tumor necrosis factor alpha (TNF-α) and IL-2, inhibit the activation of natural killer cells, arrest B cell maturation, and inhibit dendritic cell maturation, resulting in reduced expression of antigens and costimulatory molecules required to activate T cells. Accordingly, methods for treating kidney diseases, such as diabetic kidney disease or diabetic nephropathy, are disclosed herein.

[0013] Treatment for diabetic kidney disease Provided herein are methods for treating diabetic kidney disease in an individual. In some embodiments, the methods comprise administering to an individual a dose of about 80×10 6The method includes administering a single dose of syndecan-2 positive (SDC2+) stromal cells to an individual, thereby treating diabetic kidney disease. In some embodiments, the SDC2+ stromal cells are a population of SDC2+ stromal cells in which at least 10% of the cells are SDC2+. In some embodiments, at least 15% of the cells are SDC2+. In some embodiments, at least 20% of the cells are SDC2+. In some embodiments, at least 25% of the cells are SDC2+. In some embodiments, at least 30% of the cells are SDC2+. In some embodiments, at least 35% of the cells are SDC2+. In some embodiments, at least 40% of the cells are SDC2+. In some embodiments, at least 50% of the cells are SDC2+. In some embodiments, at least 60% of the cells are SDC2+. In some embodiments, at least 70% of the cells are SDC2+. In some embodiments, at least 80% of the cells are SDC2+. In some embodiments, at least 90% of the cells are SDC2+. In some embodiments, at least 95% of the cells are SDC2+. In some embodiments, at least 99% of the cells are SDC2+. In some embodiments, 100% of the cells are SDC2+. In some embodiments, a single administration is sufficient to improve renal function compared to an untreated individual. In some embodiments, the improvement in renal function is assessed by measuring glomerular filtration rate (mGFR). Alternatively, or in combination, the improvement in renal function is assessed by estimating glomerular filtration rate (eGFR). In some embodiments, the mGFR is improved compared to a baseline measurement. Alternatively, or in combination, the eGFR is improved compared to a baseline measurement.

[0014] In another aspect, provided herein is a method for treating diabetic kidney disease in an individual, comprising administering to said individual a dose of about 80×10 6A composition comprising SDC2+ interstitial cells. In some embodiments, a single administration is sufficient to improve renal function compared to an untreated individual. In some embodiments, the improvement in renal function is assessed by measuring glomerular filtration rate (mGFR). Alternatively, or in combination, the improvement in renal function is assessed by estimating glomerular filtration rate (eGFR). In some embodiments, the mGFR is improved compared to a baseline measurement. Alternatively, or in combination, the eGFR is improved compared to a baseline measurement.

[0015] In some embodiments of the methods of treating diabetic nephropathy provided herein, renal function is improved by about 5%, about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, about 100%, about 120%, about 150%, about 200%, or more compared to untreated or placebo-treated individuals.In some embodiments, renal function is about 5% to about 10%, about 5% to about 20%, about 5% to about 30%, about 5% to about 40%, about 5% to about 50%, about 5% to about 60%, about 5% to about 70%, about 5% to about 80%, about 5% to about 90%, about 5% to about 100%, about 5% to about 120%, about 5% to about 150%, about 5% to about 200%, about 10% to about 20%, about 10% to about 30%, about 10% to about 40%, about 10% to about 50%, about 10% to about 60%, about 10% to about 70%, about 10% to about 80%, about 10% to about 90%, about 10% to about 100%, about 10% to about 120%, about 10% to about 150%, about 10 ... % to about 150%, about 10% to about 200%, about 20% to about 30%, about 20% to about 40%, about 20% to about 50%, about 20% to about 60%, about 20% to about 70%, about 20% to about 80%, about 20% to about 90%, about 20% to about 100%, about 20% to about 120%, about 20% to about 150%, about 20% to about 200%, about 30% to about 40%, about 30% to about 50%, about 30% to about 60%, about 30% to about 70%, about 30% to about 80%, about 30% to about 90%, about 30% to about 100%, about 30% to about 120%, about 30% to about 150%, about 30% to about 200%, about 40% to about 5 0%, approximately 40% to approximately 60%, approximately 40% to approximately 70%, approximately 40% to approximately 80%, approximately 40% to approximately 90%, approximately 40% to approximately 100%, approximately 40% to approximately 120%, approximately 40% to approximately 150%, approximately 40% to approximately 200%, approximately 50% to approximately 60%, approximately 50% to approximately 70%, approximately 50% to approximately 80%, approximately 50% to approximately 90%, approximately 50% to approximately 100%, approximately 50% to approximately 120%, approximately 50% to approximately 150%, approximately 50% to approximately 200%, approximately 60% to approximately 70%, approximately 60% to approximately 80%, approximately 60% to approximately 90%, approximately 60% to approximately 100%, approximately 60% to approximately 120%, approximately 60% to approximately 150%, approximately 60% to approximately 200 %, about 70% to about 80%, about 70% to about 90%, about 70% to about 100%, about 70% to about 120%, about 70% to about 150%, about 70% to about 200%, about 80% to about 90%, about 80% to about 100%, about 80% to about 120%, about 80% to about 150%, about 80% to about 200%, about 90% to about 100%, about 90% to about 120%, about 90% to about 150%, about 90% to about 200%, about 100% to about 120%, about 100% to about 150%, about 100% to about 200%, about 120% to about 150%, about 120% to about 200%, or about 150% to about 200% improvement.

[0016] In another aspect of the methods of treatment herein, renal function does not decline compared to baseline measurements. In some embodiments, mGFR does not decline compared to baseline measurements. In some embodiments, eGFR does not decline compared to baseline measurements. In some embodiments, renal function does not decline by more than about 1%, about 2%, about 5%, about 10%, or about 20% compared to baseline measurements. In some embodiments, renal function is maintained at about 90%, about 95%, about 99%, or about 100% compared to baseline measurements.

[0017] In another aspect of the methods of treatment herein, a single dose of treatment is effective for at least about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, or more, hi some embodiments, a single dose of treatment prevents renal failure for at least about 6 months, about 12 months, about 18 months, about 24 months, about 36 months, or more.

[0018] In another aspect of the treatment methods provided herein, the frequency of one or more immune cells is altered in treated individuals compared to untreated individuals or placebo-treated individuals. In some embodiments, the frequency of regulatory T cells (CD4+CD25highFoxP3+CD127-) is increased after a single dose of treatment in individuals compared to untreated individuals. In some embodiments, memory regulatory T cells (CD4+CD25highFoxP3+CD127-CD45RA-CD45RO+ or CD4+Helios + CD95 + HLA-DR - In some embodiments, the frequency of natural killer T (NKT) cells (CD3+CD56+) is not increased compared to untreated individuals. In some embodiments, the frequency of intermediate activated monocytes (HLA-DR1) is increased after a single dose of treatment in individuals compared to untreated individuals. + CD33 + CD14 + CD16 +The frequency of immune cells is suppressed in an individual after a single dose of treatment, compared to an untreated individual. In some embodiments, the change in the frequency of one or more immune cells is observed for about 12 to about 18 months after treatment.

[0019] In some embodiments, serum cytokines NGAL and sTNFR1 are reduced after a single dose of treatment in an individual compared to an untreated individual.

[0020] In another aspect of the methods of treatment provided herein, the individual has type 2 diabetes. In some embodiments, the individual is suffering from symptoms of type 2 diabetes. In some embodiments, the individual has a urinary albumin excretion (UAE) of at least 60 μg / min before treatment. In some embodiments, the individual has a urinary albumin / creatinine ratio (UACR) of at least 88 mg / g or 10 mg / mmol before treatment. In some embodiments, the individual has a urinary albumin / creatinine ratio (UACR) of at least 30-50 ml / min / 1.73 ml before treatment. 2 In some embodiments, the individual has an eGFR of at least 10 ml / min / 1.73 m over the three years prior to treatment. 2 In some embodiments, the individual has experienced a decline in eGFR of at least 5 ml / min / 1.73 m over the 18 months prior to treatment. 2 have experienced a decline in eGFR.

[0021] Preparation of therapeutic stromal stem cells Therapeutic stromal stem cells are isolated from human umbilical cord tissue (UCT) or bone marrow by selecting cells expressing CD362 (SDC2).

[0022] The method for preparing interstitial stem cells for the treatment of diabetic kidney disease herein optionally includes obtaining an umbilical cord tissue sample. Often, the method includes sterilizing the umbilical cord sample. Optionally, the method includes dividing the umbilical cord sample into portions of known weight. Often, the method includes further cutting each portion of the umbilical cord into smaller pieces. Optionally, the method includes cutting each portion of the umbilical cord into 0.1 mm pieces. 2 , 0.2mm 2 , 0.3mm 2 , 0.4mm 2 , 0.5mm 2 , 0.6mm 2 , 0.7mm 2 , 0.8mm 2 , 0.9mm 2 , or 1.0 mm 2 Optionally, the method involves cutting each section of the umbilical cord into pieces of about 0.5 to about 1 mm. 2 The method includes cutting the umbilical cord into small pieces. Optionally, the method includes removing all blood from the umbilical cord sample. Optionally, the method includes mixing the umbilical cord pieces with a protease, such as collagenase, trypsin, proteinase K, or other proteases. Often, the umbilical cord sample is incubated with the protease for at least 30, 35, 40, 45, 50, 55, or 60 minutes. Optionally, the method includes quenching the protease reaction with cell culture medium containing serum. Often, the method includes filtering the protease-treated umbilical cord sample through a cell strainer, such as a 100 μm cell strainer, to obtain a solution containing umbilical cord-derived cells.

[0023] As described herein, a method for preparing interstitial stem cells for the treatment of diabetic kidney disease includes labeling dissociated umbilical cord cells with an agent that binds to SDC2, such as an anti-SDC2 antibody. Optionally, the cells labeled with the anti-SDC2 antibody are separated from unlabeled umbilical cord cells. Optionally, the labeled cells are separated from unlabeled cells using fluorescence-activated cell sorting. Optionally, the labeled cells are separated from unlabeled cells using a magnetic cell sorter. Optionally, the labeled cells are separated from unlabeled cells using a MACSQuant Tyto instrument. Often, dead cells are removed from the cells during the cell separation process. Optionally, the cell separation process includes enrichment sorting. Optionally, the cell separation process includes enrichment sorting and purity sorting. Optionally, the sorted cells are counted. Often, the method includes culturing the sorted cells to expand the cell number.

[0024] Interstitial stem cells for the treatment of diabetic kidney disease are optionally mixed with an excipient and stored prior to use. In some cases, the excipient includes a cryoprotectant or cryopreservation agent. Cryoprotectants include, but are not limited to, DMSO, glycerol, polyethylene glycol, propylene glycol, glycerin, polyvinylpyrrolidone, sorbitol, dextran, trehalose, and commercially available preparations such as BioLife Solutions' CryoStor. The stromal stem cell compositions herein retain their potency even when frozen using specialized freezing protocols. These protocols include rapid freezing, programmable rate freezers, and freezing in insulated containers. The stromal stem cell compositions are optionally frozen in a buffer or culture medium supplemented with a cryoprotectant. Buffers include physiologically acceptable buffers such as phosphate buffer, histidine buffer, citrate buffer, acetate buffer, BioLife Solutions' Hypothermasol, and other suitable buffers.

[0025] definition "Stromal stem cells," "mesenchymal stem cells," "SDC2+ stromal stem cells," "ORBCEL-M™," or "ORBCEL-C™," used interchangeably, are SDC2+ cells isolated from umbilical cord tissue or bone marrow that have therapeutic properties, such as for the treatment of inflammatory diseases, such as inflammatory liver disease, and wounds, such as non-healing wounds.

[0026] "SDC2," also known as syndecan-2, CD362, S2, or fibroglycan, generally refers herein to the SDC2 polypeptide encoded by the SDC2 gene locus. Syndecan-2, or "SDC2 protein," or simply SDC2, is a transmembrane type I heparin sulfate proteoglycan. Synonyms for "SDC2 protein" or syndecan-2 other than SDC2 include HSPG, CD362, HSPG1, and SYND2. Generally, as used herein, SDC2 refers to the protein or a recognizable fragment thereof, unless otherwise specified, e.g., "SDC2 gene," "SDC2 transcript," or "SDC2 antibody." An SDC2 fragment refers to any set of consecutive residues of SDC2 that uniquely or recognizably maps to the SDC2 polypeptide sequence. In some cases, an SDC2 fragment retains some or all of the activity of the SDC2 protein or acts as an inhibitor of full-length or native SDC2. SDC2 may also be used herein informally to refer to the locus or gene encoding the SDC2 protein. If one skilled in the art cannot distinguish the relatedness of SDC2, it is assumed that the term is used herein in reference to the protein or polypeptide, rather than the gene, transcript, or antibody produced against or bound to SDC2. In mammals, there is a family of syndecan proteins. SDC2 is used interchangeably, particularly to refer to mammalian syndecan-2 or human SDC2. If one skilled in the art cannot distinguish the relatedness of SDC2, it is assumed that the term is used herein in reference to the human protein or polypeptide.

[0027] The terms "recipient," "individual," "subject," "host," and "patient" are used interchangeably herein and refer to any mammalian subject, particularly humans, for whom diagnosis, treatment, or therapy is desired, as the case may be. A "mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domestic and farm animals, and laboratory, zoo, sport, or pet animals such as dogs, horses, cats, cows, sheep, goats, pigs, mice, rats, rabbits, guinea pigs, monkeys, and the like. Sometimes the mammal is a human.

[0028] As used herein, the terms "treatment," "treating," "ameliorating symptoms," and the like refer to administering a drug or performing a procedure, as the case may be, for the purpose of achieving an effect. The effect may be preventative, in that it completely or partially prevents a disease or its symptoms, and / or therapeutic, in that it results in a partial or complete cure of the disease and / or its symptoms. "Treatment," as used herein, may include the treatment of tumors in mammals, particularly humans, and includes (a) preventing a disease or disease symptoms from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed with it (including, for example, diseases that may be related to or caused by a primary disease), (b) inhibiting the disease, i.e., preventing its development, and (c) palliating the disease, i.e., causing regression of the disease. Treatment may refer to any indication of success in treating or ameliorating or preventing cancer, including any objective or subjective parameter, such as relief, remission, reduction of symptoms, or making the condition more tolerable to the patient, slowing the rate of degeneration or debilitation, or making the end point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters, including the results of a physician's examination. Thus, the term "treating" includes administering a compound or agent of the present invention to prevent or delay, alleviate, or prevent or inhibit the onset of symptoms or conditions associated with cancer or other diseases. The term "therapeutic effect" refers to the reduction, elimination, or prevention of a disease, disease symptoms, or disease side effects in a subject. An "untreated individual" refers to an individual who has kidney disease (e.g., diabetic kidney disease) and / or suffers from symptoms of kidney disease (e.g., diabetic kidney disease) and is receiving placebo treatment, or an individual who has kidney disease (e.g., diabetic kidney disease) and / or suffers from symptoms of kidney disease (e.g., diabetic kidney disease) and is not receiving any treatment.

[0029] The terms "pharmaceutically acceptable," "physiologically acceptable," and grammatical variations thereof, referring to compositions, carriers, diluents, and reagents, are used interchangeably and indicate that the material can be administered to a human without producing undesirable physiological effects, in some cases to an extent that would prohibit administration of the composition.

[0030] In some cases, a "therapeutically effective amount" means the amount that, when administered to a subject for treating a disease, is sufficient to treat the disease or ameliorate the symptoms.

[0031] In some cases, a "baseline measurement" refers to a quantitative or qualitative measurement taken before treatment is provided or initiated.

[0032] As used herein, the term "about" a numerical value refers to a range from 10% less than that numerical value to 10% more than that numerical value, including values ​​within that range, such as the numerical value itself.

[0033] As used herein, the term "comprising" claim element(s) refers to those elements but does not exclude the inclusion of additional element(s).

[0034] "Diabetic kidney disease" or "diabetic nephropathy," used interchangeably herein, refers to a complication of type 1 and type 2 diabetes caused by damage to the blood vessels of the kidneys that filter waste products from the blood. This results in persistent albuminuria, a progressive decline in glomerular filtration rate, and elevated arterial blood pressure.

[0035] As used herein, "about" a numerical value refers to a range that includes the numerical value and extends from 10% below to 10% above the numerical value. A range of "about" refers to a range that spans 10% below the lower limit of the range and 10% above the upper limit of the range.

[0036] Example The following examples are given for the purpose of illustrating various embodiments of the present invention and are not intended to limit the invention in any way. The examples, together with the methods described herein, are representative of presently preferred embodiments and are exemplary and are not intended to limit the scope of the invention. Those skilled in the art will envision modifications therein and other uses which are encompassed within the spirit of the invention as defined by the claims.

[0037] Example 1: Treatment of diabetic nephropathy Patients with type 2 diabetes and advanced diabetic kidney disease received 80 × 10 6 Patients received a single infusion of SDC2+ interstitial stem cells (ORBCEL-M) or placebo and were subsequently monitored for 18 months. Measured glomerular filtration rate (mGFR) and estimated glomerular filtration rate (eGFR) were assessed at baseline, 6 months, 12 months, and 18 months. Table 1 shows mGFR data at baseline, 6 months, 12 months, and 18 months. Table 2 shows eGFR data at baseline, 6 months, 12 months, and 18 months. Figure 1 shows the difference in eGFR from baseline to 18 months after treatment. Figure 2 shows the difference in eGFR from -30 months to baseline and from baseline to 18 months. Table 3 shows the annual rate of decline in renal function from baseline to 18 months of follow-up. Select metabolic parameters (Table 4), arterial blood pressure and heart rate (Table 5), and urinary albumin-to-creatinine ratio (UACR) were also investigated during the study period. Individuals treated with ORBCEL-M experienced a significantly smaller decline in eGFR from baseline to 18 months post-treatment (Figure 1, Figure 2, Table 2 and Table 3).

[0038] [Table 1]

[0039] [Table 2]

[0040] [Table 3]

[0041] [Table 4-1]

[0042] [Table 4-2]

[0043] [Table 4-3]

[0044] [Table 5-1]

[0045] [Table 5-2]

[0046] [Table 6]

[0047] Example 2: Safety and Efficacy of ORBCEL-M Therapy in Diabetic Nephropathy The primary objective was to investigate the safety and feasibility of cell therapy using a next-generation human bone marrow-derived, antibody-purified (CD3621) MSC population (ORBCEL-M) in individuals with type 2 diabetes and advanced DKD, with a secondary objective of preliminary evaluation of efficacy.

[0048] A Phase 1b / 2a multicenter, randomized, placebo-controlled clinical trial (NEPHSTROM trial) was conducted. This study reports the results of the first ORBCEL-M / placebo cohort recruited into NEPHSTROM.

[0049] method

[0050] Study design and participants

[0051] The NEPHSTROM (Novel Stromal Cell Therapy for Diabetic Kidney Disease) trial is a pilot, exploratory, investigator-initiated, European, multicenter, randomized, double-blind, placebo-controlled clinical trial conducted at three academic clinical centers in Ireland (University of Galway), Italy (Azienda Socio-Sanitaria Territoriale Papa Giovanni XXIII, Bergamo, ASST-PG23), and the UK (University Hospital Birmingham NHS Foundation Trust, Birmingham, UHBFT). The trial was coordinated by the Istituto di Ricerche Farmacologiche Mario Negri IRCCS (IRFMN), Bergamo, Italy. A second UK clinical center (Belfast Health and Social Care Trust, Belfast, BHSCT), which initially acted as a clinical trial registry, declined but remained in the NEPHSTROM trial as a central laboratory for screening and monitoring anti-HLA antibodies in study participants. The local ethics committee and competent authorities approved the study protocol and investigational medicinal product (IMP). For these regulatory approvals, the NEPHSTROM consortium followed the Voluntary Harmonization Procedure ([VHP1038][VHP2017011]). The study was registered with the European Union Clinical Trials Register (EUDRACT N° 2016-000661-23) and ClinicalTrials.gov (NCT02585622). Written informed consent was obtained from all participants.

[0052] Participants were aged 40–85 years, had type 2 diabetes for 3 years or more, and were managed by a clinician who was required to follow national guidelines. Other inclusion criteria were: (1) a urinary albumin-to-creatinine ratio (UACR) of ≥ 88 mg / g (≥ 10 mg / mmol) in a morning spot urine specimen, and (2) an eGFR of 1.73 m. 2 (3) eGFR of 1.73 m or less in the past 3 years; 2 A documented decline of 10 ml / min or more per year in eGFR was recorded, or the rate of decline in eGFR was 1.73 m / year on three or more consecutive measurements at least 90 days apart during the past 18 months prior to the consent date. 2 Patients were required to have a documented blood pressure of ≥5 ml / min per 1000 mg / day or a moderate or high 5-year risk of progression to ESKF (dialysis or transplantation) based on the validated Tangri 4-variable (age, sex, eGFR, urinary albumin / creatinine ratio) kidney failure risk equation for patients with CKD stages 3–5. Key exclusion criteria were: (1) resting systolic blood pressure ≥150 mmHg or diastolic blood pressure ≥90 mmHg in a clinical setting despite treatment with three antihypertensive drugs from different classes; (2) hemoglobin A1c (HbA1c) ≥75 mmol / mol (≥9%); (3) fasting total cholesterol ≥7 mmol / L; (4) fasting total triglycerides ≥3.5 mmol / L; and (5) a positive anti-HLA antibody screening test (mean fluorescence intensity >1500). Patients with chronic pulmonary or hepatic disease, a cardiovascular event within 6 months before enrollment, currently enrolled with or with a history of New York Heart Association (NYHA) class III or IV heart failure within 6 months before enrollment, patients with active malignancy, or women of childbearing potential, pregnant, or breastfeeding who were not using an acceptable method of contraception were also excluded.

[0053] Given the vast majority of interventional treatment trials for DKD, none of the participants enrolled in the NEPHSTROM trial underwent a kidney biopsy at study entry to confirm the presence of pathological changes consistent with diabetic nephropathy and to rule out non-DKD. Of the 16 enrolled participants, one had a documented prior kidney biopsy (which identified pathological abnormalities consistent with diabetic nephropathy), and one had a previous attempted biopsy that failed to yield diagnostic tissue.

[0054] Procedures and Evaluation

[0055] The NEPHSTROM trial followed a phase 1b / 2a dose-escalation design aimed to recruit 48 participants with type 2 diabetes and DKD who provided written informed consent. An equal number of participants were planned to be enrolled from each center; however, if enrollment was difficult at one or more centers, additional recruitment could be conducted at other participating centers. Study participants received three doses of ORBCEL-M (80 × 10 6 cells, 160 x 10 6 cells, 240 x 10 6 Participants were randomly assigned 3:1 to receive a single double-blind intravenous infusion of one of the 80 × 10 cells or a placebo. Each of the three cohorts consisted of 16 participants (12 receiving ORBCEL-M and 4 receiving placebo [Cryostor CS10]). Because the NEPHSTROM trial is a preliminary safety study, the first cohort of participants received the lowest dose (80 × 10 cells). 6 Patients were given either 100 mg of 1000 cells or a placebo.

[0056] If the Data Monitoring and Safety Committee indicates that the study can proceed beyond this dose, the next 16 participants will be enrolled in the next dose of ORBCEL-M, i.e., 160 × 10, in the absence of dose-limiting toxicity events. 6 Finally, after allocation to Cohort 2 was completed, the subsequent 16 participants were randomized to the final 240 × 10 cell dose or placebo. 6Patients would have been assigned to either the cell dose or placebo (Cohort 3). As detailed herein, enrollment in Cohort 2 was terminated early (13 of 16 patients) following discussion between the investigators and the sponsor because the coronavirus disease 2019 (COVID-19) pandemic prevented further NEPHSTROM study activities. Additionally, Cohort 3 was not conducted due to study delays caused by the COVID-19 pandemic and the material inability to further extend validation of the cell / placebo bags used for intravenous infusion.

[0057] Active and placebo treatments were administered in the context of ongoing independent standard-of-care medical management of blood glucose, blood pressure, lipid levels, and other clinical problems by specialists blinded to treatment randomization.

[0058] At each clinical center, patients with type 2 diabetes were prescreened for potential eligibility by trained study personnel based on ongoing outpatient evaluations and medical record review. After obtaining informed consent from participants who met the inclusion criteria and agreed to participate in the study, screening tests were conducted to confirm eligibility for randomization. Prerandomization tests consisted of basic blood parameters, UACR, serum anti-HLA antibody screening (Luminex bead assay), and pregnancy testing for women of childbearing potential. Participants confirmed to meet the eligibility criteria were randomized to ORBCEL-M or placebo infusion according to a computer-generated randomization procedure through the IRFMN's Clinical Trial Coordination Center. The randomization list was created by an independent statistician (Giovanni Antonio Giuliano) at the Biostatistics Laboratory of the IRFMN's Clinical Research Center for Rare Diseases Aldo e Cele Dacco (Lanica, Bergamo, Italy).

[0059] Each randomized participant was admitted to the Clinical Research Facility (CRF) for a baseline evaluation, including systolic and diastolic blood pressure, electrocardiogram, fasting glucose, HbA1c, lipid profile, and a hematology and biochemistry panel with GFR estimation by the CKD-EPI and Modification of Diet in Renal Disease (MDRD) formulas. UACR was measured using a morning spot urine specimen, and GFR was measured using plasma iohexol clearance. Blood and urine specimens were also processed and stored for profiling of immune cell subsets and inflammatory biomarkers in the blood. The trial infusion (ORBCEL-M or placebo) was administered within 48 hours (Figure 3B). Participants were closely monitored at the CRF during the infusion and for 8 hours thereafter.

[0060] At 1 day, 7 days, and 1 month after infusion, participants returned to the CRF, where they were interviewed regarding symptoms and adverse events (AEs) (early post-IMP / placebo monitoring). At these time points, participants underwent measurements of blood pressure, serum creatinine, fasting glucose, hematology and biochemistry panels, and UACR. Blood and urine samples were collected at 7 days and 1 month and processed for immune monitoring and measurement of inflammation-related soluble mediators.

[0061] CRF follow-up visits at 3, 6, 12, and 18 months after ORBCEL-M / placebo infusion included a physical examination, a medical history regarding intercurrent AEs, and routine laboratory tests (later post-dose monitoring). At 6, 12, and 18 months, renal function (GFR by plasma iohexol clearance, eGFR by CKD-EPI and MDRD equations) was assessed, UACR was measured in a morning spot urine specimen, and blood was drawn for immune / inflammatory mediator monitoring. Study follow-up ended 18 months after infusion.

[0062] There were no restrictions on concomitant treatment in this study and it was left to the discretion of the participants' physicians and specialists. Concomitant treatments were reviewed and recorded at randomization and at each subsequent follow-up visit.

[0063] Preparation, administration, and post-infusion monitoring of ORBCEL-M

[0064] ORBCEL-M was manufactured under license (Orbsen Therapeutics Ltd, Galway, Ireland) from bone marrow aspirates of healthy donors at three Good Manufacturing Practice (GMP) facilities (Centro di Terapia Cellulare Gilberto Lanzani, ASST-PG23, Bergamo, Italy; Center for Cellular Manufacturing CCMI, Galway, Ireland; and NHS Blood and Transplant NHSBT, Liverpool, UK). A fourth GMP facility, Academish Ziekenhuis Leiden-Leids Universitair Medisch Centrum LUMC, Leiden, the Netherlands, served as the lead isolation and preparation facility responsible for the manufacturing protocol. Bone marrow aspirates were collected from screened healthy adult volunteers by consultant hematologists at the Irish HPRA-accredited Galway Blood and Tissue Establishment and transported to Leiden University Medical Center. Here, ORBCEL-M cells were antibody-enriched using a CliniMACS isolation system with GMP-grade anti-CD362 antibody and primarily expanded in tissue culture flasks until passage 1 (P1). P1 cells were harvested using recombinant enzymes and reseeded into cryoprecipitate-coated hollow-fiber bioreactors (Quantum Cell Expansion System [Terumo BCT Europe NV, Belgium]), where they were further expanded for up to 7 days and harvested by enzyme release. Aliquots of cells were cryopreserved as an intermediate ORBCEL-M product and expanded a second time in the Quantum Cell Expansion System bioreactor before being shipped to three other GMP teams for harvesting, formulation into individual cryobags, and final release of the GMP product. Each of the GMP facilities in Leiden, Bergamo, Galway, and Liverpool holds a national license for the manufacture of advanced therapeutic medicines, including MSC products.On the day of infusion, ORBCEL-M or placebo was frozen in 40 ml sterile bags and shipped frozen to CRFs in Bergamo, Galway, and Birmingham. GMP facilities meet the following criteria: (1) positivity for cell surface markers CD73, CD90, and CD105 by FACS analysis is ≥95% and positivity for CD45 and CD34 is ≤1%; (2) mycoplasma, gram-positive bacteria, gram-negative bacteria, and fungi are negative; (3) endotoxin is <10 EU / ml by chromogenic assay; (4) viability is ≥70% by trypan blue and manual counting; and (5) viable cell count is ≥350 × 10 cells. 6 ORBCEL-M was released according to specific criteria, including (6) karyotype G-banding and karyotype Q-banding (no clonal abnormalities and no more than three individual abnormalities), which conforms to the International Society for Cell Therapy's definition of multipotent MSCs.

[0065] Immediately after thawing, a dose of ORBCEL-M or placebo (40 ml volume) was administered intravenously over 10–20 minutes into a peripheral arm vein of each randomized participant using a 200 mm transfusion filter, according to randomization. The premedication regimen consisted of oral acetaminophen (1 g, 1 hour before infusion) and intravenous chlorpheniramine and hydrocortisone (10 mg and 100 mg, respectively, immediately before infusion). Baseline temperature, pulse, blood pressure, respiratory rate, oxygen saturation, and chest auscultation were recorded and then monitored continuously during the infusion, every 15 minutes for the first hour, and hourly for 7 hours after infusion. Study participants were also closely monitored for signs of other adverse reactions, such as rash, hives, or wheezing. All events were recorded during the 8-hour observation period, after which participants were allowed to withdraw from the CRF if no adverse events occurred. Although ORBCEL-M and placebo doses were indistinguishable on the label and instructions for use, to avoid identification of active IMP due to turbidity in the cell suspension, participants as well as their physicians and research nurses remained blinded and shielded from view of the infusion bag and tubing throughout the study, whereas infusion verification, thawing, and setup were performed by a separate (unblinded) team of technicians, pharmacists, and research nurses.

[0066] Test results

[0067] The primary study outcomes were the number and severity of prespecified cell infusion-related events, as well as the overall number and frequency of adverse events and unexpected serious adverse events, during early (up to 1 month) and late (from 2 to 18 months) follow-up periods in ORBCEL-M recipients compared with placebo recipients. Secondary outcomes included the efficacy of ORBCEL-M compared with placebo in slowing DKD progression, assessed using the following variables: (1) change in glomerular filtration rate (GFR) (ΔGFR and slope of GFR decline, assessed by serial measurements of mGFR via plasma iohexol clearance), (2) serum creatinine-based eGFR (ΔeGFR and slope of eGFR decline) according to the CKD-EPI and MDRD equations, and (3) absolute and percent changes in UACR in spot urine samples from baseline to 18 months post-infusion. Other secondary outcomes were the effect of ORBCEL-M compared with placebo on other relevant clinical parameters, including glycemic control (fasting blood glucose and HbA1c), lipid control (total cholesterol, LDL cholesterol, and triglycerides), and the proportion of study participants within target ranges for blood pressure control. Finally, additional secondary outcomes included the effect of ORBCEL-M compared with placebo on immune and inflammatory profiles, assessed by the following variables: (1) expression of anti-HLA antibodies, (2) percentages / total numbers of circulating lymphocyte (T cells, B cells, and NK cells) and myeloid cell (monocyte and dendritic cell) subsets, and (3) plasma / serum immunoassay concentrations of inflammatory biomarkers.

[0068] Clinical laboratory measurements

[0069] Clinical chemistry was performed according to the study monitoring protocol. Blood and urine specimens were analyzed in local clinical laboratories at the three participating centers. Measured glomerular filtration rate (mGFR) was determined by an established protocol for quantifying plasma clearance of unlabeled iohexol. 34 For this purpose, serial plasma samples were collected and initially stored at -80°C in each CRF. They were then transported to the laboratory of the Clinical Trials Coordinating Center, IRFMN, in Lanica, Italy, for centralized measurements. Iohexol plasma concentrations were measured by high-performance liquid chromatography. Serum anti-HLA antibody measurements were also performed centrally at the Belfast HSC Trust Histocompatibility and Immunogenetics Laboratory in Belfast, UK. This procedure consisted of two Luminex assays: an initial antibody screening with Luminex multiantigen beads to detect class I and class II MHC antibodies, and, if necessary, a Luminex single-antigen bead assay to determine the specificity of any detected antibodies. Longitudinal profiling of peripheral blood lymphocyte and myeloid subsets was performed centrally at the Laboratory of Immunology and Organ Transplantation (IRFMN) at the Italian Clinical Trial Coordinating Center. Using three antibody panels, the phenotypes of (1) CD4+ and CD8+ T cell subsets, B cell subsets, NK cells, and monocytes, (2) regulatory CD4+ T cells (Tregs), and (3) Lin-HLADR+ dendritic cells were analyzed using a FACS LSR Fortessa X-20 (Becton Dickinson) with FlowJo software. For evaluation of inflammatory biomarkers, longitudinal samples collected at the three participating clinical centers were centrally monitored at the University of Galway, Ireland. Soluble tumor necrosis factor receptor 1 (sTNFR1), neutrophil gelatinase-binding lipocalin (NGAL), vascular cell adhesion molecule-1 (VCAM-1), and epidermal growth factor (EGF) (severity of DKD)

[0070] and biomarkers with well-documented prognostic relevance) were quantified using DuoSet ELISA kits (R&D Systems, Minneapolis) according to the manufacturer's instructions.

[0071] Sample size estimation

[0072] Although this is a phase 1 trial primarily aimed at determining feasibility and safety, a sample size calculation was performed according to Cocks and Torgerson to assess the initial efficacy of ORBCEL-M in slowing the rate of GFR decline. It was determined that at least 36 participants needed to be included to detect a change from a 5.1 (SD 4.3) ml / min annual GFR decline in the placebo group to a 3.4 ml / min annual GFR decline in the active treatment group (power = 80%, α = 0.05, two-sided). This calculation was based on previously reported interim data from participants with diabetes enrolled in the Prevention of ESRD in Overt Nephropathy in Type 2 Diabetes (VALID) trial, taking into account the 3:1 randomization. Therefore, we planned to recruit 48 consenting participants with type 2 diabetes and advanced DKD.

[0073] statistical analysis

[0074] It was pre-determined that safety and efficacy analyses would primarily be performed on an "all-treater" basis, i.e., including all participants randomized to the study who received an ORBCEL-M or placebo infusion, regardless of whether treatment was initiated. Additionally, secondary safety analyses would also be performed on a "safety set" basis, i.e., including only participants randomized to the study who received some or all of the ORBCEL-M or placebo infusion. All statistical analyses were performed using SAS version 9.4 (SAS Institute Inc., Cary, NC) and STATA version 15 (StataCorp., College Station, TX). Mixed-effects models with random intercepts were used to determine the slopes of mGFR and eGFR, estimated by restricted maximum likelihood. Results were expressed as mean ± SD or median (interquartile range) or number (%), as appropriate. All P values ​​were two-sided, with significance set at P < 0.05.

[0075] result

[0076] Participant enrollment and baseline characteristics

[0077] The study protocol consisted of three cohorts: Cohort 1, 80 × 10 6 cells or placebo, Cohort 2, 160 x 10 6 cells or placebo, Cohort 3, 240 × 10 6 The cohorts were planned to include either 100 cells or placebo. The results presented here pertain to the enrollment, treatment, and completed follow-up of Cohort 1. The detailed rationale for unblinding and data analysis of this cohort is described elsewhere herein.

[0078] Between March 2018 and January 2020, 23 consented patients were screened for final eligibility, of which 16 were randomized to study cohort 1. Of the seven patients who failed screening, three were due to not meeting the criteria for eGFR decline and two were due to a screening eGFR of 1.73 m2 One patient had a screening eGFR of 1.73m 2 One patient had a blood pressure of less than 25 ml / min per day, and one had a positive anti-HLA screening assay (see study flowchart, Figure 3A). The number of participants randomized and treated at each center was n = 8 in Bergamo, Italy; n = 4 in Galway, Ireland; and n = 4 in Birmingham, UK.

[0079] Table 7 summarizes the key baseline demographic, clinical, and laboratory characteristics of the 16 participants randomized to treatment with ORBCEL-M or placebo. The median age was 69 years (interquartile range 66–73 years) in the cell therapy group and 59 years (interquartile range 54–66 years) in the placebo group. All participants were male. Systolic and diastolic blood pressures were well controlled with antihypertensive treatment in both groups. Consistent with the inclusion criteria, participants in both groups had similar moderate to severe CKD based on both mGFR and eGFR. Glycemic control, measured by HbA1c, was similar between the two groups, with no significant differences in median fasting plasma glucose levels, lipid parameters, or UACR. Only one of the 16 participants was prescribed an SGLT2 inhibitor at the time of randomization.

[0080] [Table 7-1]

[0081] [Table 7-2]

[0082] primary result

[0083] Of the 16 randomized participants, 14 completed the 18-month follow-up according to protocol, but follow-up for two (both in the ORBCEL-M treatment group) was terminated early due to an SAE resulting in death (detailed below).

[0084] Early Safety Monitoring

[0085] Per protocol, all randomized participants were in stable health at the time of study treatment. Fifteen of the 16 participants experienced no adverse reactions to cell or placebo infusion. Accordingly, body temperature, pulse rate, respiratory rate, blood pressure, and oxygen saturation remained stable during the infusion and the subsequent 8-hour observation period. One placebo-treated participant experienced moderate bronchospasm immediately after the completion of the infusion. In this case, the participant fully recovered approximately 50 minutes later with appropriate medication. No other adverse reactions occurred between the time of infusion and the 1-month follow-up (Table 8).

[0086] [Table 8]

[0087] Late-stage safety monitoring

[0088] As summarized in Table 8, between months 2 and 18 of follow-up, a total of 11 additional SAEs occurred in four participants (all cell infusion recipients): seven in one, two in another, and one each in the remaining two. None of the late SAEs were determined to be related to the study drug, ORBCEL-M. Two of these participants died from non-treatment-related SAEs: one due to congestive heart failure after 15 months of study follow-up, and the second due to multiple acute complications of newly diagnosed multiple myeloma after 15 months of follow-up.

[0089] Overall, 56 AEs were recorded in the cell therapy group, 11 of which were judged to be serious (Table 9). In the placebo group, 13 AEs occurred, one of which was judged to be serious and treatment-related (see above). The 57 AEs not classified as serious are summarized in Table 10.

[0090] [Table 9]

[0091] [Table 10-1]

[0092] [Table 10-2]

[0093] Predefined secondary comparisons

[0094] Effects on renal function

[0095] As summarized in Table 11, baseline mGFR (measured by plasma iohexol clearance) was similar between the ORBCEL-M and placebo groups. In both groups, mGFR declined during the 18-month follow-up period. The mean changes in mGFR compared with baseline at 6, 12, and 18 months were numerically smaller in the ORBCEL-M-treated group than in the placebo-treated group, but the differences were not statistically significant (P = 0.709, P = 0.443, and P = 0.236, respectively). In contrast, the mean changes in eGFR, calculated by either the CKD-EPI or MDRD equations, were significantly smaller in the ORBCEL-M group than in the placebo group (at 12 months, P = 0.015 and P = 0.018 for the CKD-EPI and MDRD equations, respectively; at 18 months, P = 0.012 and P = 0.014, respectively). When changes in renal function during study follow-up were calculated as a rate of decline per year, very similar results were observed (Table 12). For mGFR, the annual rate of decline was numerically lower in the cell-treated group compared with the placebo-treated group, but not significantly lower (P=0.467), and for eGFR, the annual rate of decline was significantly lower in the ORBCEL-M-treated group compared with the placebo-treated group (P=0.034 for eGFR in both CKD-EPI and MDRD).

[0096] [Table 11-1]

[0097] [Table 11-2]

[0098] [Table 12]

[0099] The trends in UACR during the study follow-up period are summarized in Table 13. As shown in the table, UACR values ​​fluctuated significantly in both groups at baseline and at 6, 12, and 18 months of follow-up, but there were no significant differences between the groups at any time point. Of note, despite a lower rate of decline in eGFR in the cell-treated group, there was no evidence of a decline in UACR in participants receiving ORBCEL-M.

[0100] [Table 13]

[0101] Effects on metabolic parameters and blood pressure

[0102] The values ​​of blood glucose, HbA1c, serum total cholesterol, serum triglycerides, and serum C-reactive protein at baseline and after 6, 12, and 18 months of follow-up are summarized for both groups in Table 14, along with the mean changes from baseline at the three follow-up time points. As shown in the table, these parameters remained generally stable in both groups throughout the study period, were within clinically acceptable ranges, and no differences were observed. Similar observations were made for blood pressure parameters and heart rate (Table 15).

[0103] [Table 14-1]

[0104] [Table 14-2]

[0105] [Table 15]

[0106] Effects on immunological and inflammatory parameters

[0107] Screening for anti-HLA class I and anti-HLA class II antibodies was negative at all scheduled visits for 15 of the 16 participants. One ORBCEL-M-treated participant tested positive for low-level anti-HLA class I antibodies starting 12 months after infusion, but this anti-HLA class I antibody persisted to the end of study follow-up without clinically relevant consequences.

[0108] Longitudinal PBMC profiling by flow cytometry revealed no significant changes or differences between groups in the percentages of CD4+ and CD8+ T cells (Figure 4A-B). There were small but significant differences in B cell frequencies between ORBCEL-M and placebo recipients at 1 and 12 months, but no clear trends were observed over time (Figure 4C). The percentages of dendritic cells, total monocytes, and cytotoxic NK cells were similar between both groups throughout the follow-up period (Figure 4D-F). In contrast, natural killer T cell frequencies were similar in both groups at baseline but significantly lower in ORBCEL-M-treated participants throughout the post-infusion follow-up period (Figure 4G).

[0109] Analysis of the proportion of regulatory T cells (Tregs) among total CD3+CD4+ T cells showed a significantly higher proportion in ORBCEL-M-treated participants at 6 months (Figure 5A). Furthermore, subanalysis showed that the proportion of memory Tregs (defined as CD45RA-RO+) decreased over time in placebo-treated participants but remained stable in ORBCEL-M-treated participants, with the proportions significantly different between 6 and 18 months (Figure 5B). This was particularly evident in the memory Treg subpopulation defined as Helio s+CD95+HLA-DR- (Figure 5C). The proportion of naive Tregs (defined as CD45RA+RO- Tregs) remained relatively low throughout the study in both groups (Figure 5D).

[0110] Analysis of the monocyte repertoire was performed using the well-recognized three-subset classification of classical monocytes, non-classical monocytes, and intermediate monocytes, and expressed as a percentage of total CD45+ cells. As shown in Figure 6A, the proportion of the most abundant classical monocyte subset remained stable and very similar between the two groups throughout the study follow-up period. In contrast, the proportions of non-classical and intermediate subsets tended to increase between 6 and 18 months in the placebo-treated group but remained stable throughout the study period in the ORBCEL-M-treated group, becoming significantly lower for non-classical monocytes at 18 months and for intermediate monocytes at 12 and 18 months (Figure 6B, Figure 6C).

[0111] Longitudinal analysis of serum levels of the inflammatory biomarkers sTNFR1, NGAL, and VCAM-1 showed no significant differences between groups over the 18-month follow-up period, with a trend toward increasing levels (Figures 7A-C). Serum levels of EGF remained stable in both groups (Figure 7D).

[0112] Other exploratory comparisons

[0113] In a post-hoc analysis, CKD progression relative to the transition from baseline to 18 months in the 2-year risk category for reaching ESKD was compared between the two groups using the kidney failure risk equation. As shown in Figure 8, the baseline 2-year risk category was intermediate for 11 of 12 participants in the ORBCEL-M group and 4 of 4 participants in the placebo group. One participant receiving ORBCEL-M was classified as high-risk at baseline. Of the 10 participants who received cell therapy and completed 18 months of follow-up, 8 remained in the moderate-risk category, 1 transitioned from moderate to high risk, and 1 remained in the high-risk category. In contrast, all four participants receiving placebo transitioned from the moderate-risk category to the high-risk category.

[0114] In correlation analyses of the combined immune / inflammatory profiling dataset, we observed that the peripheral blood proportions of total Tregs and CD45RA-RO+ memory Tregs across the entire cohort were significantly correlated with concurrent serum concentrations of sTNFR1 (Figures 9A and 9B). In subgroup analyses, these correlations remained statistically significant in ORBCEL-M-treated participants (P < 0.002 for both cell subsets) but not in placebo-treated participants (P = 0.364 and P = 0.063 for Tregs and CD45RA-RO+ memory Tregs, respectively). A negative correlation was also observed between the proportion of total Tregs or CD45RA-RO+ memory Tregs and concurrent serum NGAL concentrations (Figures 9C and 9D). In the entire cohort, a significant positive correlation was observed between the percentage of total or CD45RA-RO+ memory Tregs and concurrent GFR estimated by both the CKD-EPI and MDRD equations (Figures 10A-10D). Finally, serum sTNFR1 and NGAL concentrations were negatively correlated with both mGFR and eGFR (CKD-EPI and MDRD) in the entire cohort (Figures 11A-11F) and in the ORBCELM-treated group (sTNFR1 vs. CKD-EPI and MDRD, P < 0.001, vs. mGFR, P = 0.003; NGAL vs. CKD-EPI, MDRD, and mGFR, P < 0.001).

[0115] Consideration

[0116] In the initial cohort of this Phase 1b / 2a multicenter, randomized, double-blind, placebo-controlled clinical trial, 80 × 10 6A single intravenous infusion of next-generation bone marrow-derived, anti-CD362-selected allogeneic MSCs (ORBCEL-M) was observed to be well tolerated in participants with type 2 diabetes and advanced DKD. This acceptable safety profile persisted over the subsequent weeks to months until the end of the 18-month follow-up period. Importantly, two patients died due to serious adverse events (SAEs) that occurred at longer time intervals after cell administration. However, these events were not considered related to the study drug, given the short in vivo persistence of intravenously administered MSCs and the well-recognized possibility of additional causative factors related to medical comorbidities associated with type 2 diabetes and DKD. In fact, the participant who died from an acute cardiovascular event had received the cell infusion more than 14 months prior. Rarely associated acute thromboembolic events have only been reported at the end of or several days after intravenous administration of MSC-derived products, so this late time point would rule out a role for coagulation activation. In this regard, a recent meta-analysis of the reported results of 55 randomized clinical trials enrolling over 2,600 participants with a variety of serious diseases (e.g., cardiovascular, neurological, renal, and hepatic diseases) showed that MSC administration was associated with an increased risk of fever compared with a control group that did not receive cell therapy, but not with an increased risk of non-febrile acute infusion toxicity, thrombotic / embolic events, malignancies, or death. None of the included clinical trials was terminated prematurely due to safety concerns.

[0117] One theoretical concern regarding culture-expanded progenitor cell therapies, such as MSC therapy, is the possibility that such cells may transform during culture and acquire the potential to generate tumors in recipients after infusion. Importantly, while malignant transformation has been demonstrated in mouse MSCs, no such events have been reported with human MSC-based therapies. Furthermore, an autopsy study of 18 patients who received allogeneic MSCs for hematological malignancies or solid tumors and died 3–408 days after the last MSC infusion revealed no evidence of MSC-derived ectopic tissue formation or malignancies by macroscopic or histological examination. Additionally, despite its immunomodulatory properties, MSC therapy has not been associated with an increased risk of malignant tumor development in solid organ transplant recipients receiving long-term immunosuppressive medications. Regarding the trial participant who died of myeloma, the lack of prior reports of new or progressive cancers among thousands of recipients of MSC therapy for diverse clinical indications (including allogeneic hematopoietic stem cell transplants for myeloid malignancies, organ transplants, and many immunosuppressed patients with autoimmune diseases), the short duration of the culture expansion protocol for ORBCEL-M manufacturing, and the strict standards for GMP release of cell products led us to conclude that this condition was highly unlikely to have been caused or exacerbated by the trial intervention. Nevertheless, it is possible, albeit unlikely, that this participant had a preclinical plasma cell disorder associated with a circulating monoclonal protein prior to enrollment. This highlights the possibility of non-diabetes-related renal pathology in patients enrolled in clinical trials for DKD (which typically rely on clinical rather than biopsy-based diagnosis of diabetic nephropathy) and indicates the need to consider screening for monoclonal gammopathy, especially in trials of cell therapy and other immunomodulatory therapies for kidney disease.

[0118] Limited clinical trials have examined the safety, tolerability, and potential benefits of MSC-based therapies in patients with type 2 diabetes. These trials used autologous or allogeneic MSCs harvested from different tissue sources and infused intravenously or via the pancreatic artery. Most often, these trials involved cells isolated and prepared by plastic adhesion, resulting in heterogeneous / unselected stromal cell products. Extensively characterized MSC products prepared from more selective primary tissue precursors may offer better and more consistent therapeutic effects and may be better suited to meeting future regulatory standards for advanced cell products. Only one early-phase clinical trial, reported by Packham et al., tested the efficacy of antibody-purified allogeneic stromal cell products, specifically mesenchymal progenitor cells (MPCs, rexlemestrocel-L, Mesoblast) selected to express the surface marker Stro3, in patients with established DKD. The ORBCEL-M product investigated in the NEPHSTROM trial was also produced from primary bone marrow stromal cells, but was selected to express a different surface marker (CD362, also known as syndecan-2) and cultured in hollow fiber bioreactors to ensure cells were at an early passage number at the time of administration. The ORBCEL-M product has a distinct therapeutic effect compared to other stromal cell therapies tested in patients with type 2 diabetes. The results reported herein demonstrate that ORBCEL-M and rexlemestrocel-L have comparable safety and tolerability profiles, with preliminary evidence of clinically relevant efficacy. Whether ORBCEL-M may offer advantages such as superior efficacy, higher effectiveness, or lower cost over other MSC-based cell therapies investigated in diabetes and DKD cannot be determined until larger or comparative trials are conducted. Regarding the metabolic effects of ORBCEL-M, no significant changes in glycemic parameters (fasting blood glucose, HbA1c) were observed 18 months after cell infusion. Preclinical and some clinical trials suggest that MSC therapy may improve glycemic control, and these results are consistent with those of other early-phase clinical trials in similar patients with type 2 diabetes.This may in part reflect the fact that study participants had very good glycemic control at enrollment. Of note, administration of the allogeneic cell product, ORBCEL-M, proved to have minimal immunological sensitization potential, as evidenced by the absence of detectable anti-HLA antibodies during 18 months of follow-up in all participants except one, who developed low levels of anti-HLA class I antibodies beginning 12 months after infusion. These observations are consistent with findings from other clinical trials reporting that allogeneic bone marrow-derived MSC products can be safely administered to humans without eliciting clinically relevant immunological responses. Directly relevant to the results reported herein, two reported results from clinical trials of the allogeneic mesenchymal progenitor cell product, rexlemstrocel-L, in patients with type 2 diabetes or diabetic nephropathy also demonstrate the absence of persistent de novo donor-specific antibody development. The lack of high frequency or high levels of sensitization to allogeneic HLA is particularly important in light of the future role of ORBCEL-M, or other allogeneic MSC-derived products, in the setting of kidney disease, particularly in light of the potential need for subsequent kidney transplantation. The completed 18-month follow-up of NEPHSTROM Cohort 1 showed that the rate of decline in mGFR was numerically lower, but not significantly lower, in patients receiving ORBCEL-M than in those receiving placebo, while a similar trend in eGFR reached statistically significant levels. This occurred in the context of comparable relevant baseline characteristics and similarly acceptable metabolic and blood pressure control between the two treatment groups. The additional observation that the 2-year risk category for achieving ESKF based on KRFE worsened in all placebo-treated patients but remained stable in most evaluable ORBCEL-M recipients also supports the cautious conclusion that these two groups of patients with a history of rapidly progressive DKD had different renal function trajectories after treatment. Regarding their mechanism of action, MSCs are currently thought to exert their therapeutic effects primarily through the inducible secretion of paracrine mediators and the reprogramming of myeloid and lymphoid immune cells.For example, multiple lines of evidence suggest that MSCs inhibit the differentiation of Th1 and Th17 cells, thereby promoting IL-10 production by T cells and thereby inducing the generation of Tregs. Furthermore, MSCs may act indirectly to promote the induction and proliferation of Tregs and other anti-inflammatory mediators by modulating the activity of monocytes, macrophages, and dendritic cells. Therefore, it is interesting to note that differences in the trends of circulating memory Tregs were observed between the two study groups, with placebo recipients (who experienced a greater decline in eGFR) experiencing a decrease in their proportion over time, whereas ORBCEL-M recipients maintained a more stable proportion. Although causality cannot be concluded from these findings, the observed positive correlation between Treg / memory Tregs and eGFR, as well as the observed inverse correlation between concurrent serum concentrations of sTNFR1 and NGAL, established DKD-associated inflammatory biomarkers, and eGFR in the entire cohort (and in the ORBCEL-M-treated group), tend to support the hypothesis that infusion of allogeneic MSC products is associated with sustained immunomodulatory / anti-inflammatory effects that may modulate pathophysiological aspects of advanced DKD. Also consistent with this hypothesis is the discrepancy in the percentage of circulating intermediate monocytes between cell therapy recipients and placebo recipients at later time points in the study. Circulating intermediate monocytes are proinflammatory and associated with the rate of eGFR decline in cardiovascular disease and CKD. Clinically applicable testing of serum, plasma, and urinary biomarkers related to systemic and intrarenal inflammation and fibrosis may provide value as indicators of response to MSC therapy. However, the interindividual variability observed for selected serum biomarkers in this cohort suggests that larger participant numbers are needed to meaningfully investigate this question.Finally, it should be acknowledged that the possible anti-inflammatory / immunomodulatory effects observed in patients receiving ORBCEL-M and documented in many other preclinical and clinical studies pose at least a theoretical risk of promoting or exacerbating infections and cancer, and this possibility must continue to be investigated in a careful and unbiased manner in trials such as this one.

[0119] In conclusion, the results reported here for the completed cohort of the multicenter, randomized, double-blind, placebo-controlled NEPHSTROM trial demonstrate that 80 × 10 6 These results demonstrate the safety and tolerability of a single infusion of ORBCEL-M. Furthermore, these findings confirm that this MSC-based cell therapy product is unlikely to sensitize recipients to allogeneic HLA and reveal preliminary evidence of potential renal protective and immunomodulatory effects over an 18-month observation period following infusion. These results, along with the ongoing need for novel disease-modifying therapies to preserve renal function in patients with advanced DKD, support further investigation of ORBCEL-M in an appropriately sized and powered Phase 2b trial.

[0120] While preferred 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 occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the embodiments described herein may be employed. The following claims define the scope of the invention, and it is intended that methods and structures within the scope of these claims and their equivalents be covered thereby.

Claims

1. 1. A method of treating diabetic kidney disease in an individual, said method comprising administering to said individual about 80×10 ６ a single administration of SDC2+ interstitial cells, thereby treating said diabetic kidney disease, wherein said single administration is sufficient to improve renal function compared to an untreated individual.

2. 10. The method of claim 1, wherein the improvement in renal function is assessed by measuring glomerular filtration rate (mGFR) or estimating glomerular filtration rate (eGFR).

3. 3. The method of claim 2, wherein the measured glomerular filtration rate (mGFR) or estimated glomerular filtration rate (eGFR) is improved compared to the baseline measurement.

4. 3. The method of claim 2, wherein the measured glomerular filtration rate (mGFR) or estimated glomerular filtration rate (eGFR) does not decrease compared to baseline measurements.

5. 5. The method of any one of claims 1 to 4, wherein the single dose treatment is effective in improving the renal function for at least 18 months.

6. 6. The method of any one of claims 1 to 5, wherein the single dose treatment prevents renal failure for at least 18 months.

7. Regulatory T cells (CD4 ＋ CD25 ｈｉｇｈ FoxP3 ＋ CD127 － 7. The method of any one of claims 1-6, wherein the frequency of (i) is increased after said single dose of treatment in said individual compared to an untreated individual.

8. Frequency of memory regulatory T cells (CD4 ＋ CD25 ｈｉｇｈ FoxP3 ＋ CD127 － CD45RA － CD45RO ＋ or CD4+ Helios ＋ CD95 ＋ HLA-DR － 8. The method of any one of claims 1-7, wherein the level of IL-16 (as measured by IL-16) is increased after said single dose of treatment in said individual compared to an untreated individual.

9. The method according to any one of claims 1 to 8, wherein the frequency of natural killer T (NKT) cells (CD3+CD56+) is not increased compared to untreated individuals.

10. Intermediately activated monocytes (HLADR ＋ CD33 ＋ CD14 ＋ CD16 ＋ 10. The method of any one of claims 1-9, wherein the frequency of HLADR+CD33+CD14-CD16+ and / or non-classical patrolling monocytes is suppressed or reduced after said single dose of treatment in said individual compared to an untreated individual.

11. 11. The method of any one of claims 1 to 10, wherein serum cytokines NGAL and sTNFR1 are reduced after said single dose treatment in said individual compared to an untreated individual.

12. 12. The method of any one of claims 7 to 11, wherein the frequencies of regulatory T cells, memory regulatory T cells, NKT cells, intermediately activated monocytes, and / or non-classical patrolling monocytes are monitored for about 12 months to about 18 months after treatment.

13. The method of any one of claims 1 to 12, wherein the individual has type 2 diabetes or is suffering from type 2 diabetes symptoms.

14. 14. The method of any one of claims 1 to 13, wherein the individual has a urinary albumin excretion (UAE) of at least 60 μg / min prior to the treatment.

15. 15. The method of any one of claims 1 to 14, wherein the individual has a urinary albumin / creatinine ratio (UACR) of at least 88 mg / g or 10 mg / mmol prior to the treatment.

16. The individual has an average blood flow of at least 30-50 ml / min / 1.73 m before the treatment. ２ The method of any one of claims 1 to 15, wherein the eGFR is

17. The individual has a blood glucose level of at least 10 ml / min / 1.73 m for three years prior to the treatment ２ The method of any one of claims 1 to 16, wherein the patient is experiencing a decrease in eGFR of 1 or more.

18. The individual has a blood glucose level of at least 5 ml / min / 1.73 m for 18 months prior to the treatment ２ The method of any one of claims 1 to 17, wherein the patient is experiencing a decrease in eGFR of 1 or more.

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