Novel treatment of diabetes and kidney disease by inhibition of D2D3, a proteolytic uPAR protein

Abstract

The present invention discloses a method for treating diseases characterized by elevated levels of urokinase plasminogen activator receptor (uPAR) protein D2D3, said diseases being one or more of chronic kidney disease, insulin-dependent diabetes mellitus or diabetic neuropathy.Furthermore, the present invention provides a method for restoring the number and function of pancreatic β cells in the pancreas of a subject diagnosed with insulin-dependent diabetes mellitus, said insulin-dependent diabetes mellitus being characterized by the presence of detectable levels of D2D3.Specifically, these methods include administration of a therapeutically effective amount of an agent that antagonizes D2D3 or removes D2D3 from the circulation of a subject, said agent comprising an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein.Alternatively, these methods include removing D2D3 protein from the circulation of a subject by extracorporeal treatment. TIFF2025510581000005.tif74128

Description

[Technical field]

[0001] Government Grant Reference This invention was made with Government support under several grants awarded by the National Institutes of Health, including 1RO1DK101350, 1RO1DK125858, 1RO1DK093773 and 1RO1DK087985. The Government has certain rights.

[0002] FIELD OF THEINVENTION The present invention relates to novel treatments for chronic kidney disease and diabetes. [Background technology]

[0003] 2. Background of the Invention Chronic kidney disease (CKD) affects hundreds of millions of people worldwide. The three common causes of CKD are diabetes mellitus, hypertension and glomerulonephritis. See Romagnani et al., “Chronic kidney disease,” (2017) Nat Rev Dis Primers 3: p. 17088. Diabetic nephropathy (DN) occurs in approximately 40% of diabetic patients and is the leading cause of end-stage renal disease (ESRD) worldwide. See Sulaiman, “Diabetic nephropathy: recent advances in pathophysiology and challenges in dietary management,” (2019) Diabetol Metab Syndr 11: p. 7. The pathogenesis of DN encompasses diverse molecular mechanisms including genetic, metabolic and hemodynamic factors such as glomerular hypertrophy and hypertension. Ibid. Histological and genetic data strongly suggest the involvement of podocyte dysfunction in the development of DN. See Dai et al., “Research Progress on Mechanism of Podocyte Depletion in Diabetic Nephropathy, (2017) J Diabetes Res 2615286.

[0004] Elevated levels of soluble urokinase-type plasminogen activator receptor (suPAR) have been associated with DN in patients with type 1 diabetes (T1D) and type 2 diabetes (T2D). Theilade et al., “Increased plasma concentrations of midregional proatrial natriuretic peptide is associated with risk of cardiorenal dysfunction in type 1 diabetes,” (2015) Am J Hypertens 28:pp.772-779;Guthoff et al., “Soluble urokinase receptor(suPAR) predicts microalbuminuria in patients at risk for type 2 diabetes. mellitus,”(2017)Sci Rep 7:p.40627;Haugaard et al.,“The immune marker soluble urokinase plasminogen activator receptor is associated with new-onset diabetes in non-smoking women and men,”(2012)Diabet Med 29:pp.479-487;Eugen-Olsen et al.,“Circulating soluble urokinase plasminogen activator receptor predicts cancer, cardiovascular disease, diabetes and mortality in the general population,"(2010)J Intern Med 268:pp.296-308(2010).

[0005] suPAR is generated by proteolytic shedding of membrane-bound uPAR from the surface of various cells of the innate immune system, such as macrophages, immature myeloid cells, and neutrophils. (Figure 12) See also Andolfo et al., “Metalloproteases cleave the urokinase type plasminogen activator receptor in the D1-D2 linker region and expose epitopes not present in intact soluble receptor,” (2002) Thromb Haemost 88:pp.298-306; van Veen et al., “Negative regulation of urokinase receptor activity by a GPI-specific phospholipase C in breast cancer cells,” (2017) Elife 6. The inventors have demonstrated that uPAR / suPAR regulates α-phospholipase C on glomerular podocytes. v β 3It has been shown to cause CKD by activating integrins, resulting in foot process (FP) effacement and proteinuria. Kugler et al., “Urokinase receptor and integrin interactions,” (2003) Curr Pharm Des 9:pp.1565-1574; Castillo et al., “Disruption of thyroid hormone activation in type 2 deiodinase knockout mice causes obesity with glucose intolerance and liver steatosis only at thermoneutrality,” (2011) Diabetes 60:pp.1082-1089;Dong et al.,“Multiple myeloma with a previous diagnosis of focal segmental glomerulosclerosis:A case report and review of the literature,”(2015)Oncol Lett 10:pp.2821-2827;Alfano et al.,“Full-length soluble urokinase plasminogen activator receptor down-modulates nephrin expression in podocytes,”Sci Rep 5:p.13647;Hahm See et al., “Bone marrow-derived immature myeloid cells are a main source of circulating suPAR contributing to proteinuric kidney disease,” (2017) Nat Med 23: pp. 100-106.

[0006] It is known that uPAR / suPAR consists of three homologous domains: D1, D2 and D3. uPAR proteolysis generates two additional circulating forms: the N-terminal D1 fragment and the C-terminal D2D3 protein, both of which are involved in cancer biology. (FIG. 12) See also Thuno et al., "suPAR: the molecular crystal ball," (2009) Dis Markers 27: pp. 157-172; Sidenius et al., "Shedding and cleavage of the urokinase receptor (uPAR): identification and characterization of uPAR fragments in vitro and in vivo," (2000) FEBS Lett 475: pp. 52-56. The D2D3 protein is in part an α v β 3 It is also known that activating integrins induces chemotaxis of cancer cells. v β 3 mediates chemotactic and haptotactic motility in human melanoma cells through different signaling pathways,” (1996) J Biol Chem 271: pp. 3247-3254. 3 Increased expression of integrins has been associated with diabetic nephropathy (DN). See Wilson et al., “The single-cell transcriptomic landscape of early human diabetic nephropathy,” (2019) Proc Natl Acad Sci USA 116: pp. 19619-19625.

[0007] Additionally, it has been disclosed that suPAR antagonists may be beneficial in treating acute kidney injury. See International Publication No. WO2020069498, the disclosure of which is incorporated by reference in its entirety.

[0008] However, little is known about the role of D2D3 in chronic kidney disease, advanced insulin-dependent diabetes mellitus, and its potential as a therapeutic target in these diseases. This application addresses these needs. Summary of the Invention

[0009] The present invention is based on the discovery of the presence of the C-terminal uPAR protein D2D3 in patients with diabetic nephropathy. D2D3-positive human serum inhibited glucose-stimulated insulin release in human pancreatic islets and was associated with patients requiring insulin therapy. D2D3 transgenic mice showed kidney disease characterized by proteinuria and foot process effacement. Furthermore, D2D3 transgenic mice developed diabetes characterized by reduced levels of insulin and C-peptide, impaired glucose-stimulated insulin secretion, reduced pancreatic beta cell mass and high fasting glucose. Recombinant D2D3 protein dysregulated glucose-induced cytoskeletal dynamics, impaired insulin granule maturation and trafficking, and inhibited beta cell bioenergetics in culture. Anti-uPAR antibodies restored beta cell function and number in D2D3 transgenic mice. The present invention identifies a causal role of D2D3 protein in the development of insulin-dependent diabetes mellitus through impaired insulin release and reduced beta cell number in the pancreas during the postnatal period. In addition, D2D3 protein also directly damages podocytes in renal glomeruli, causing kidney disease. Detecting D2D3 protein is of clinical value for risk stratification of kidney and diabetic patients. The present invention demonstrates that sequestering D2D3 protein in circulation improves insulin-dependent diabetes and kidney disease, thus establishing a unique dual therapeutic approach to kidney disease and insulin-dependent diabetes. Specifically, the present invention provides a method for treating D2D3-dependent diabetes mellitus and D2D3-dependent kidney disease based on novel molecular diagnosis and mechanism.

[0010] Thus, the present invention discloses a method for treating chronic kidney disease, comprising measuring or determining the presence of D2D3 protein in a biological sample from a subject, and administering a therapeutically effective amount of an agent that antagonizes D2D3 and / or removes D2D3 from the circulation of said subject if the presence of D2D3 protein is detected. In some embodiments, the agent can be an antibody or an antibody fragment. In several embodiments, the agent comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein. In some embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein is humanized. In other embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein is a monoclonal antibody. In other embodiments, it is contemplated that D2D3 can be removed from the circulation by extracorporeal processes such as plasmapheresis, dialysis, or immunoadsorption.

[0011] In any embodiment, the chronic kidney disease may be caused by diabetes mellitus, hypertension or glomerulonephritis, indicating the presence of D2D3.

[0012] The present invention also provides a method for treating any form of insulin-dependent diabetes or its consequences, such as diabetic neuropathy, comprising measuring or determining the presence of D2D3 protein in a biological sample from a subject, and administering a therapeutically effective amount of an agent that antagonizes D2D3 and / or removes D2D3 from the circulation of said subject if the presence of D2D3 protein is detected. In some embodiments, the agent may be an antibody or an antibody fragment. In several embodiments, the agent comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein. In some embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein is humanized. In other embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein is a monoclonal antibody. In other embodiments, it is contemplated that D2D3 may be removed from the circulation by a process such as plasmapheresis or immunoadsorption.

[0013] In yet another aspect, the present invention provides a method of restoring both the number of beta cells in the pancreas of an insulin-dependent diabetic patient in whom the presence of D2D3 has been detected. The method includes measuring or determining the presence of D2D3 protein in a biological sample from a subject, and, if the presence of D2D3 protein is detected, administering a therapeutically effective amount of an agent that antagonizes D2D3 and / or removes D2D3 from the circulation of the subject. In some embodiments, the agent can be an antibody or an antibody fragment. In several embodiments, the agent comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein. In some embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein is humanized. In other embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein is a monoclonal antibody. In other embodiments, it is contemplated that D2D3 can be removed from the circulation by extracorporeal processes such as plasmapheresis, dialysis, or immunoadsorption.

[0014] In any of the embodiments, the level of D2D3 is measured by any method known to those skilled in the art, such as mass spectrometry, immunoprecipitation combined with Western blot analysis or a D2D3-specific ELISA to specifically detect D2D3 in a biological sample.

[0015] In yet other embodiments, the method further comprises administration of an anti-soluble urokinase plasminogen activator receptor (suPAR) antibody or antibody fragment. [Brief description of the drawings]

[0016] [Figure 1]Figure 1A-H shows that D2D3 protein discriminates between DN patients on insulin therapy and those not on insulin therapy. (Figure 1A) Schematic of human sample analysis. (Figure 1B and Figure 1C) Detection of suPAR and D2D3 protein by IP-WB in serum (Figure 1B) or urine (Figure 1C) of DN patients. Controls: recombinant human suPAR or chymotrypsin digested suPAR corresponding to D2D3 protein (Figure 1D). Characteristics of the test patients. (Figure 1E) Comparison between patients who were on insulin therapy or who were not on insulin therapy (area under the curve, (AUC) values). (Figure 1F) Pie chart showing the distribution (%) of D2D3 positive and D2D3 negative sera in DN patients who were on insulin therapy or who were not on insulin therapy. (Figure 1G) Scatter dot plot representing the levels of GSIS from human islets in the presence of human serum (n=3 experiments). Healthy donor islets were incubated in 10% healthy serum (HS), D2D3 positive serum (D2D3 PS, pooled from 8 patients), D2D3 negative serum (D2D3 NS, pooled from 6 patients) or D2D3 PS (D2D3 DS) immunodepleted using anti-uPAR antibody (R4). Where indicated, 10 ng / ml of recombinant hD2D3 or suPAR was added. (Figure 1H) Scatter dot plot showing activation of b3 integrin on human podocytes (cells >35) treated with serum as described in (G) except for a hD2D3 concentration of 2.5 ng / ml. Immunofluorescence analysis was performed using anti-paxillin antibody (focal adhesion) and AP5 antibody (activated β3 integrin). Figure 1G and Figure 1H, error bars, mean ± SEM (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, unpaired t-test). ns, not significant. [Diagram 2]Figure 2A-J shows that D2D3-Tg mice exhibit glomerular damage and insulin deficiency. All animals were fed a high-fat diet (HFD) and measurements were performed on 6-month-old animals unless otherwise stated. (Figure 2A) Schematic of the D2D3-Tg construct. (Figure 2B) qPCR analysis of D2D3 and D1 in adipose tissue normalized to Gapdh (n=3 biological replicates). (Figure 2C) D2D3 detection in adipose tissue of D2D3-Tg mice using anti-Myc antibody (D2D3 is Myc-tagged). Control: tissue stained with primary or secondary antibodies only. (Figure 2D) Mouse serum suPAR and D2D3 protein levels were determined using uPAR-specific ELISA (n=8). (Figure 2E) Detection of Myc-tagged D2D3 in mouse serum. Serum was immunoprecipitated with anti-uPAR antibody and detected by WB using anti-Myc antibody. Control: recombinant Myc-tagged mD2D3 (lanes 3, 4, 7, 8; + / - N-glycanase treated). (Figure 2F) Serum IL-6 (WT, n=11; D2D3-Tg, n=7) and CPR (n=7, each group) levels. (Figure 2G) Fasting blood glucose (n=9, each group), C-peptide (n=6 for WT; n=10 for D2D3-Tg) and insulin (n=7 for WT; n=8 for D2D3-Tg) levels. (Figure 2H) In vivo GSIS. Overnight fasted animals were intraperitoneally injected with glucose (2 g / kg body weight) and their blood insulin levels were measured by ELISA (n=6-8 for each group). (Figure 2I) Scatter dot plots representing albumin creatinine ratio (ACR) or serum creatinine (n=7-10 for each group) levels. (Fig. 2J) TEM of PAS-stained kidney (left) and foot process (right). (Fig. 2C, Fig. 2J). For all results, error bars, mean ± SEM, (*P<0.05; **P<0.01; ***P<0.001. Unpaired t-test). ns, not statistically significant. [Diagram 3]Figure 3A-N shows that D2D3-Tg mice exhibit insulin-dependent diabetes due to impaired pancreatic function and β-cell mass. All animals were fed a normal diet. (Figure 3A) Scatter dot plots representing fasting C-peptide and blood insulin levels (n=5-10 mice in each group) (Figure 3B). In vivo GSIS. Overnight fasted animals were intraperitoneally injected with glucose (2 g / kg body weight), and their blood insulin levels were measured by ELISA (WT, n=5; D2D3-Tg, n=8). (Figure 3C) In vitro GSIS. Pancreatic islets from 2-month-old animals (n=3) were subjected to in vitro GSIS. (Figure 3D) Glucose tolerance test (GTT). Adult (5-6 months) and aged (12 months) mice were fasted overnight and then intraperitoneally injected with glucose (2 g / kg body weight). Blood glucose was measured at the indicated time points (n = 5-6 animals per condition). (Figure 3E) Scatter dot plots representing fasting blood glucose levels (n = 8-10 mice per condition). (Figure 3F) Representative immunohistochemistry of pancreatic islets from 2-month-old mice stained with anti-CD4, anti-CD8 and anti-B220 antibodies. Spleens were used as positive controls for T-cell and B-cell staining (n = 4 mice per condition). (Figure 3G) Representative immunohistochemistry of pancreases from 2-month-old mice stained with anti-insulin antibodies (n = 5 mice per condition). (Figure 3H) Representative immunohistochemistry of pancreatic islets using anti-glucagon (α-cells) and anti-insulin (β-cells) antibodies (n = 5 mice per condition). (Figure 3I) Scatter dot plots representing β-cell mass and β-cell area / pancreas area ratio. Data were generated using the images shown in (Figure 3H). Where indicated, D2D3-Tg mice were treated with anti-uPAR-Ab or IgG isotype control (IgG) for 4 weeks starting at 2 months of age. These data were collected on 3-month-old animals (n=5 for WT and D2D3-Tg; n=6 for D2D3-Tg treated with either IgG or uPAR-Ab). Six to eight sections from each tissue were analyzed. (Figure 3J) Scatter dot plots representing the composition of islets in animals treated as described in (Figure 3I).Islet composition was determined by counting the total number of β-cells (green) and α-cells (red) and expressing them as a percentage of the total cells counted within a single islet. (Figure 3K) Scatter dot plots representing the level of apoptosis determined by TUNEL staining or cell proliferation determined by Ki67 positive staining of mouse islets (n=5 mice per condition). Each dot represents an average of 1000-1350 insulin positive cells counted per animal. (Figure 3L) In vivo GSIS using 2-month-old D2D3-Tg treated twice within 1 week with either IgG or anti-uPAR-Ab. All animals were male (n=7-10 animals per condition). (Figure 3M and Figure 3N) D2D3-Tg mice were treated with either anti-uPAR-Ab or IgG for 4 weeks starting at 2 months of age. Experiments were performed at 3 months of age. All animals were male. Scatter dot plots representing blood insulin levels are shown in (Figure 3M) (n=8 mice per condition). Glucose tolerance tests are shown in (Figure 3N) (n=6 mice per condition). For all results, error bars, mean ± SEM, (*P<0.05; **P<0.01; ***P<0.001. Unpaired t-test). ns, not statistically significant. Additionally, P values ​​are shown in red or blue, where appropriate. [Figure 4]Figures 4A-L show that D2D3 protein impairs multiple aspects of beta cell physiology. (Figure 4A) Graph showing GSIS by MIN-6 cells incubated with or without BSA, mD2D3 (100 ng / ml) and anti-uPAR Ab (n=3). (Figure 4B) Graph showing GSIS by mouse islets incubated with or without BSA, mD2D3 (100 ng / ml) and anti-uPAR Ab (n=3). (Figure 4C) Graph showing GSIS by human islets incubated with or without BSA, hD2D3 (100 ng / ml) (n=3). (Figure 4D) PR-EM micrograph showing the cytoskeleton of MIN6 cells. MIN6 cells were grown as described in (Figure 4A), except that glucose levels were increased to 20 nM. Where indicated, mD2D3 protein (100 ng / ml) was added for 24 h. Actin bundles (pink), hyperbundles (purple) and microtubules (MT, green). (Figure 4E) TEM micrograph of MIN-6 cells treated as described in (D). Dense-core vesicles (LDCVs) localized close (yellow) to the cell membrane (pink) and more than 500 nm away (green) from the cell membrane. (Figure 4F and Figure 4G) Graphs represent the average LDCV diameter (Figure 4F), the number of LDCVs per cell and the distribution based on location (Figure 4G) in the cells shown in (Figure 4E). In (F), more than 75 vesicles were counted in 10 or more cells per treatment. Of note, the total number of LDCVs per cell was similar, ranging from 17 to 57, regardless of the treatment. Error bars, mean ± SD. (Figure 4H) Schematic of the experimental setup for glycolysis and oxygen consumption rate (OCR) measurements in MIN-6 cells. (Figure 4I-J) Extracellular acidification rate (ECAR) was measured in non-treated (CTL) and D2D3-treated cells using a Seahorse XFe24 analyzer. Where indicated, cells were treated with hD2D3. (Figure 4K-L) OCR curves and measurement of bioenergetic parameters for mitochondrial respiration of treated cells as described in (H). Each OCR value was normalized to cell number and expressed as pmol / min / 100,000 cells (three experiments).For Figures 4J and 4L, error bars mean ± SEM (*P<0.05, **P<0.01, ***P<0.001, one-way ANOVA). ns, not statistically significant. [Diagram 5] Figure 5A,B shows uPAR-specific peptides in human samples. (Figure 5A) Amino acid sequence of human uPAR protein isoform 1 (SEQ ID NO:1). The different domains, domain 1 (D1), domain 2 (D2) and domain 3 (D3), are labeled. The linker and GPI anchor between D1 and D2 are underlined and labeled. The bold letters demarcate the boundaries of suPAR-specific peptides detected by mass spectrometry, the details of which are given herein. Specifically, fragment-1 to fragment-5 are shown. *An additional fragment was generated within fragment-4. Fragment-3 spans the D2 and D3 domains and is therefore marked as 3 / 3. (Figure 5B) Coomassie gel showing proteins immunoprecipitated from serum using anti-uPAR antibody (R4). Serum samples (10 ml) from 14 DN patients who also had D2D3-like fragments in their serum were used for the experiment. The protein bands shown in the figure were excised from the gel and analyzed using mass spectrometry. [Figure 6]Figure 6A-D show that recombinant hD2D3 protein activates β3 integrin on human podocytes. (Figure 6A) Representative images of human podocytes stained with phalloidin (F-actin) and AP5 (an antibody that recognizes the activated form of β3 integrin). It can be seen that serum-free medium (SFM) and healthy serum (HS) do not activate β3 integrin, but the addition of hD2D3 (2.5 ng / ml) to HS resulted in an AP5 signal. Scale bar, 5 μm. (Figure 6B) Graph showing the concentration dependence of the ability of hD2D3 to activate β3 integrin on human podocytes grown in the presence of healthy human serum. 20-101 cells were analyzed per treatment. Error bars = mean ± SEM. It can be seen that at lower protein concentrations (1-approximately 3 ng / ml), the activation appears cooperative. (Figure 6C) Scatter dot plot representing the ratio between activated β3 integrin (AP5) and total focal adhesions determined by paxillin staining. It can be seen that the addition of hD2D3 to SFM activated β3 integrin on human podocytes. This demonstrates that hD2D3 activates β3 integrin on human podocytes even in the absence of any other serum proteins. For experiments with SFM, 62–79 cells were analyzed. For experiments with HS, 43–46 cells were analyzed. Data are shown as mean ± SEM. Unpaired two-tailed t-tests were performed to determine statistical significance (****P<0.0001). (Figure 6D) Scatter dot plot showing the levels of activated β3 integrin on human podocytes (38–53 cells) treated with 10% healthy serum (HS), D2D3 positive serum (D2D3 PS), D2D3 negative serum (D2D3 NS) or D2D3 PS (D2D3 DS) immunodepleted using anti-uPAR antibody (R4). Where indicated, 2.5 ng / ml hD2D3 was added. Data are presented relative to control (HS or SFM). All results are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey's multiple comparisons. [Figure 7]Figures 7A-C show that the ELISA detects mouse suPAR and mD2D3 proteins. (Figure 7A) Coomassie gel showing recombinant mouse suPAR and mD2D3 proteins expressed and purified from HEK-293 cells. It can be seen that the protein purification resulted in highly pure proteins. Both proteins are glycosylated and therefore migrate as broad bands. (Figure 7B) Table summarizing the MW of mouse proteins. The MW of mD2D3 detected in the plasma of D2D3-Tg mice was similar to that of the purified recombinant protein. (Figure 7C) Mouse-specific ELISA (R&D) detected full-length suPAR and mD2D3 equally well, but with lower sensitivity. BCA was used to determine the protein concentrations used in the ELISA. [Figure 8]Figures 8A-I show that D2D3-Tg mice fed a normal diet do not develop glomerular injury. (Figure 8A) Scatter dot plot showing fasting blood insulin levels in WT and mice expressing mouse suPAR isoform 1 (suPAR-Tg) (n=15 for each group). (Figure 8B) Scatter dot plot comparing body weight of animals fed a normal diet and a high fat diet (HFD). It can be seen that expression of mD2D3 did not affect the body weight of animals regardless of diet. (Figure 8C) qPCR analysis of D2D3 and D1 in adipose tissue. D2D3 and D1 mRNA levels were normalized to Gapdh mRNA levels (n=3 biological replicates). The data show that mRNA for D2D3 was detected in adipose tissue even when animals were fed a normal diet. (Figure 8D) mD2D3 protein is expressed in adipose tissue of animals fed a normal diet. As D2D3-Tg carries a c-Myc tag, immunohistochemistry was performed using rabbit anti-Myc antibody. D2D3 was observed in adipocytes of D2D3-Tg mice, but not in control mice. Tissues stained with primary or secondary antibodies alone were used as negative controls. Scale bar, 50 μm. (Figure 8E) The levels of suPAR and suPAR / D2D3 increased as animals aged. Scatter dot plots representing protein levels determined using mouse-specific ELISA (n = 5–11 for each group). (Figure 8F) Body weights of WT and D2D3-Tg mice (n = 5–8 for each group). (Figure 8G) Renal function was not affected in D2D3-Tg mice when fed a normal diet. Renal function was assessed by measuring ACR, serum creatinine and serum BUN levels (n = 5–12 for each group). (FIG. 8H) Immunohistochemistry of pancreatic islets using anti-CD4 and anti-CD8 antibodies in 12-month-old animals. (FIG. 8I) Scatter dot plots comparing pancreatic weights between WT (n=5) and D2D3-Tg (n=5) and D2D3-Tg animals treated with IgG (n=6) or anti-uPAR-Ab (n=6). All results were expressed as mean ± SEM. Statistical analysis was determined using unpaired two-tailed Student's t-test.*P<0.05; **P<0.01; ***P<0.001. ns, not significant. [Figure 9] Figure 9A-D show that neonatal D2D3-Tg mice have normal β-cell mass. (Figure 9A) Immunohistochemistry of pancreas isolated from neonatal mice (P0) stained with anti-insulin and anti-glucagon antibodies. (Figure 9B) The stains indicated in A were used to determine β-cell area / pancreas area ratio (n=5 for each group) and β-cell mass (n=5 for each group). (Figure 9C) Scatter dot plot showing islet composition (n=5 for each group) and pancreas weight (n=5 for each group) in neonatal mice. (Figure 9D) GSIS of MIN6 cells cultured in the presence or absence of BSA (100 ng / ml), IgG (100 ng / ml), mD2D3 protein or mouse suPAR (n=3). All results are expressed as mean ± SEM. Statistical analysis was determined using unpaired two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001. ns, not significant. [Figure 10] Figure 10A,B shows that mD2D3 fragments impair glucose-stimulated reorganization of the actin cytoskeleton in MIN6 cells. (Figure 10A) MIN6 cells were grown in low glucose (5 mM) and then stimulated by addition of high glucose (20 mM) in the presence or absence of mD2D3 protein (100 ng / ml). The status of F-actin was examined by phalloidin staining. (Figure 10B) Representative PR-EM micrographs of MIN6 cells, focusing on the organization and status of the actin cytoskeleton. Cells were grown as described in (Figure 10A). It can be seen that mD2D3 inhibited high glucose-induced disassembly of actin filaments. [Figure 11]Figure 11A-C shows that mD2D3 impairs insulin granule maturation and trafficking. Representative TEM micrographs of MIN6 cells. Where indicated, cells were grown in low glucose (5 mM) and then stimulated by addition of high glucose (5 mM) in the presence or absence of mD2D3 protein (100 ng / ml) for 24 h. Images shown were taken at increasing magnification. The cell membrane is colored pink and the insulin-transporting dense-core vesicles (LDCVs) are colored yellow. In MIN6 cells grown in the presence of mD2D3 protein, LDCVs are smaller in size and less associated with the cell membrane. [Figure 12] FIG. 12 is a schematic depicting the proteolytic shedding of membrane-bound uPAR into suPAR, D1 and D2D3 proteins. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0017] Detailed Description of the Invention The present invention discloses a method for treating chronic kidney disease, comprising measuring or determining the presence of D2D3 protein in a biological sample from a subject, and administering a therapeutically effective amount of an agent that antagonizes D2D3 and / or removes D2D3 from the circulation of said subject if the presence of D2D3 protein is detected. In some embodiments, the agent can be an antibody or an antibody fragment. In several embodiments, the agent comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein. In some embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein is humanized. In other embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3 protein is a monoclonal antibody. In other embodiments, it is contemplated that D2D3 can be removed from the circulation by a process such as plasmapheresis or immunoadsorption.

[0018] In any embodiment, the chronic kidney disease may be caused by diabetes mellitus, hypertension or glomerulonephritis, indicating the presence of D2D3.

[0019] The present invention also provides a method for treating any form of insulin-dependent diabetes or its consequences, such as diabetic neuropathy, comprising measuring or having measured the presence of D2D3 protein in a biological sample from a subject, and administering a therapeutically effective amount of an agent that antagonizes D2D3 and / or removes D2D3 from the circulation of the subject if the presence of D2D3 protein is detected. In some embodiments, the agent can be an antibody or an antibody fragment. In several embodiments, the agent comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein. In some embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein is humanized. In other embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein is a monoclonal antibody. In other embodiments, it is contemplated that D2D3 can be removed from the circulation by an extracorporeal process, such as plasmapheresis, dialysis, or immunoadsorption.

[0020] In yet another aspect, the present invention provides a method of restoring both the number of beta cells in the pancreas of an insulin-dependent diabetic patient in whom the presence of D2D3 has been detected. The method includes measuring or determining the presence of D2D3 protein in a biological sample from a subject, and, if the presence of D2D3 protein is detected, administering a therapeutically effective amount of an agent that antagonizes D2D3 and / or removes D2D3 from the circulation of the subject. In some embodiments, the agent can be an antibody or an antibody fragment. In several embodiments, the agent comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein. In some embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein is humanized. In other embodiments, the anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein is a monoclonal antibody. In other embodiments, it is contemplated that D2D3 can be removed from the circulation by extracorporeal processes such as plasmapheresis, dialysis, or immunoadsorption.

[0021] In any of the embodiments, the level of D2D3 is measured by any method known to those skilled in the art, such as mass spectrometry, immunoprecipitation combined with Western blot analysis or a D2D3-specific ELISA to specifically detect D2D3 in a biological sample.

[0022] In yet other embodiments, the method further comprises administration of an anti-soluble urokinase plasminogen activator receptor (suPAR) antibody or antibody fragment.

[0023] Definitions used throughout this disclosure The term "antibody" as used herein refers to a whole antibody that interacts with a D2D3 epitope (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) and inhibits signal transduction. A naturally occurring "antibody" is a glycoprotein that includes at least two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Each heavy chain is composed of a heavy chain variable region (abbreviated herein as VH) and a heavy chain constant region. The heavy chain constant region is composed of three domains CHI, CH2 and CH3. Each light chain is composed of a light chain variable region (abbreviated herein as VL) and a light chain constant region. The light chain constant region is composed of one domain CL. The VH and VL regions can be further subdivided into hypervariable regions called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain binding domains that interact with antigens. The constant regions of the antibody may mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. The term "antibody" includes, for example, monoclonal antibodies, human antibodies, humanized antibodies, camelized antibodies, chimeric antibodies, single-chain Fvs (scFvs), disulfide-linked Fvs (sdFvs), Fab fragments, F(ab') fragments, and anti-idiotypic (anti-Id) antibodies (including, for example, anti-Id antibodies to the antibodies of the present invention), as well as epitope-binding fragments of any of the above. Antibodies can be of any isotype (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGI, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass. Both the light and heavy chains are divided into regions of structural and functional homology. The terms "constant" and "variable" are used functionally. In this regard, it will be understood that the variable domains of both the light chain portion (VL) and the heavy chain portion (VH) determine antigen recognition and specificity.Conversely, the constant regions of the light (CL) and heavy chains (CHI, CH2 or CH3) confer important biological properties such as secretion, transplacental transfer, Fc receptor binding, complement fixation, etc. By convention, the numbering of constant region domains increases as they become more distal from the antigen binding site or amino-terminus of the antibody. At the N-terminus are the variable regions and at the C-terminus are the constant regions; the CH3 and CL domains actually comprise the carboxy-terminus of the heavy and light chains, respectively.

[0024] The term "antibody fragment" as used herein refers to one or more parts of an antibody that specifically interacts with D2D3 epitopes (e.g., by binding, steric hindrance, stabilization / destabilization, spatial distribution) and retains the ability to inhibit signal transduction.Examples of binding fragments include, but are not limited to, Fab fragment, a monovalent fragment consisting of VL, VH, CL and CHI domains; F(ab)2 fragment, a bivalent fragment that comprises two Fab fragments linked by disulfide bridges at the hinge region; Fd fragment consisting of VH and CHI domains; Fv fragment consisting of the VL and VH domains of a single arm of an antibody; dAb fragment consisting of VH domain (Ward et al, (1989) Nature 341: pp. 544-546); and isolated complementarity determining region (CDR).

[0025] Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be linked by a synthetic linker that allows them to be produced using recombinant methods as a single protein chain in which the VL and VH regions pair to form a monovalent molecule (known as single-chain Fv (scFv)). See, for example, Bird et al, (1988) Science 242:pp. 423-426; and Huston et al, (1988) Proc. Natl. Acad, Sci, 85: pp. 5879-5883. Such single chain antibodies are also intended to be encompassed by the term "antibody fragment". These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as intact antibodies. Antibody fragments can also be incorporated into single domain antibodies, maxibodies, minibodies, intrabodies, diabodies, triabodies, tetrabodies, v-NARs, and bis-scFvs. See, for example, Hollinger and Hudson, (2005) Nature Biotechnology 23: pp. 1126-1136. Antibody fragments can be grafted onto polypeptide-based scaffolds such as fibronectin type III (Fn3). See U.S. Patent No. 6,703,199, which describes fibronectin polypeptide monobodies.

[0026] The antibody fragment can be assembled into a single chain molecule that comprises a pair of tandem Fv segments (VH-CHI-VH-CH1) that form a pair of antigen-binding regions together with complementary light chain polypeptides. See, for example, Zapata et al., (1995) Protein Eng. 8:1057-1062, and U.S. Patent No. 5,641,870.

[0027] The phrases "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to polypeptides, including antibodies, antibody fragments, bispecific antibodies, and the like, having substantially identical amino acid sequences or derived from the same genetic source. The term also includes preparations of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0028] The phrase "human antibody" or "humanized antibody" as used herein includes antibodies having variable regions in which both framework and CDR regions are derived from sequences of human origin. Furthermore, if the antibody contains a constant region, the constant region is also derived from such a human sequence, for example, an antibody containing a human germline sequence or a mutated version of a human germline sequence or a consensus framework sequence derived from human framework sequence analysis, for example, as described in Knappik et al., (2000) J Mol Biol 296: pp. 57-86. The structure and position of immunoglobulin variable domains, for example, CDRs, can be defined using well-known numbering schemes, for example, the Kabat numbering scheme, the Chothia numbering scheme, or a combination of Kabat and Chothia. For example, Kabat et al., “Sequences of Proteins of Immunological Interest,” 5th edit., NIH Publication no. 91-3242, USDepartment of Health and Human Services (1991; Lazikani et al., (1997) J.Mol.Bio.273:pp.927-948); Chothia et al. al., (1987) J.Mol.Biol.196:pp.901-917; Chothia et al.. (1989) Nature 342:pp.877-883; Al-Lazikani et al., (1997) J.Mol.Biol.273:pp.927-948. The human antibodies of the invention may include amino acid residues not encoded by human sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo, or conservative substitutions to facilitate stability or manufacturing).

[0029] The phrase "human monoclonal antibody" as used herein refers to an antibody exhibiting a single binding specificity having variable regions in which both the framework and CDR regions are derived from human sequences. In one embodiment, a human monoclonal antibody is produced by a hybridoma comprising a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, whose genome comprises human heavy and light chain transgenes, fused to an immortalized cell.

[0030] The phrase "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant means, such as antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, antibodies isolated from host cells, e.g., transfectomas, transformed to express human antibodies, antibodies isolated from recombinant combinatorial human antibody libraries, and antibodies prepared, expressed, produced or isolated by any other means including splicing of all or a portion of a human immunoglobulin gene, sequence to other DNA sequences. Such recombinant human antibodies have variable regions in which the framework and CDR regions are derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies may be subjected to in vitro mutagenesis (or, when using animals transgenic for human Ig sequences, in vivo somatic mutagenesis) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies are derived from and related to human germline VH and VL sequences, but are sequences that may not naturally occur within the human antibody germline repertoire in vivo.

[0031] The specific binding between the two entities is at least 10 2 M -1 , at least 5 × 10 2 M -1 , at least 10 3 M -1 , at least 5 × 10 3 M-1 , at least 10 4 M -1 , at least 5 × 10 4 M -1 , at least 10 5 M -1 , at least 5 × 10 5 M -1 , at least 10 6 M -1 , at least 5 × 10 6 M -1 , at least 10 7 M -1 , at least 5 × 10 7 M -1 , at least 10 8 M -1 , at least 5 × 10 8 M -1 , at least 10 9 M -1 , at least 5 × 10 9 M -1 , at least 10 10 M -1 , at least 5 × 10 10 M -1 , at least 10 11 M -1 , at least 5 × 10 11 M -1 , at least 10 12 M -1 , at least 5 × 10 12 M -1 , at least 10 13 M -1 , at least 5 × 10 13 M -1 , at least 10 14 M -1 , at least 5 × 10 14 M -1 , at least 10 15 M -1 , or at least 5 × 10 15 M -1 Equilibrium constant (KA) (k on / k off ) means bonding at

[0032] The phrase "specifically (or selectively) binds" to an antibody (e.g., a D2D3-binding antibody) refers to a binding reaction that determines the presence of the cognate antigen (e.g., human D2D3 protein) in a heterogeneous population of proteins and other biologics. In addition to the equilibrium constant (KA) described above, the D2D3-binding antibodies of the present invention typically have an equilibrium constant of 5×10 -2 Less than M, 10 -2 Less than M, 5×10 -3 Less than M, 10 -3 Less than M, 5×10 -4 Less than M, 10 -4 Less than M, 5×10 -5 Less than M, 10 -5 Less than M, 5×10 -6 Less than M, 10 -6 Less than M, 5×10 -7 Less than M, 10 -7 Less than M, 5×10 -8 Less than M, 10 -8 Less than M, 5×10 -9 Less than M, 10 -9 Less than M, 5×10 -10 Less than M, 10 -10 Less than M, 5×10 -11 Less than M, 10 -11 Less than M, 5×10 -12 Less than M, 10 -12 Less than M, 5×10 -13 Less than M, 10 -13 Less than M, 5×10 -14 Less than M, 10 -14 Less than M, 5×10 -15 Less than M or 10 -15 Dissociation rate constant (KD) less than or equal to M (k off / k on ) which binds to D2D3 with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (eg, HSA).

[0033] In one embodiment, the antibody or fragment thereof has a dissociation constant (Ka) of less than 3000 pM, less than 2500 pM, less than 2000 pM, less than 1500 pM, less than 1000 pM, less than 750 pM, less than 500 pM, less than 250 pM, less than 200 pM, less than 150 pM, less than 100 pM, less than 75 pM, less than 10 pM, less than 1 pM, as assessed using methods described herein or known to one of skill in the art (e.g., BIACORE assay, ELISA, FACS, SET) (Biacore International AB, Uppsala, Sweden). As used herein, "K assoc " or "K a The term "K" refers to the association rate of a particular antibody-antigen interaction and is used herein. j " or "K d The term "KD" refers to the dissociation rate of a particular antibody-antigen interaction. a K for j (i.e., K j / K a ) and expressed as a molar concentration (M). The KD value of an antibody can be determined using methods well established in the art. Methods for determining the KD of an antibody are by using surface plasmon resonance or by using a biosensor system such as the BIACORE system.

[0034] The term "affinity" as used herein refers to the strength of interaction between an antibody and an antigen at a single antigenic site. Within each antigenic site, the variable regions of the antibody "arms" interact with the antigen at multiple sites through weak non-covalent forces; the greater the interaction, the stronger the affinity.

[0035] The term "binding activity" as used herein refers to the informative measure of the overall stability or strength of antibody-antigen complex.Binding activity is controlled by three main factors: antibody epitope affinity; both antigen and antibody valency; and the structural arrangement of interacting moieties.Finally, these factors define antibody specificity, i.e., the likelihood that a particular antibody binds to a precise antigen epitope.

[0036] The term "valency" as used herein refers to the number of potential target binding sites in a polypeptide. Each target binding site specifically binds one target molecule or specific site (i.e., epitope) on a target molecule. When a polypeptide contains more than one target binding site, each target binding site can specifically bind the same or different molecules (e.g., can bind different molecules, such as different antigens, or different epitopes on the same molecule).

[0037] As used herein, the phrase "antagonist antibody" refers to an antibody that binds to D2D3 protein and neutralizes the biological activity of D2D3 signaling, e.g., by removing circulating D2D3 levels in the blood, thereby decreasing, reducing and / or inhibiting signaling activity induced by D2D3.

[0038] The phrase "isolated antibody" refers to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds D2D3 or D2D3 protein is substantially free of antibodies that specifically bind antigens other than D2D3). However, an isolated antibody that specifically binds D2D3 or D2D3 protein may have cross-reactivity to other antigens. Additionally, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0039] The phrase "conservatively modified variants" applies to both amino acid and nucleic acid sequences. With respect to a particular nucleic acid sequence, "conservatively modified variants" refers to nucleic acids that code for the same or essentially identical amino acid sequences, or, if the nucleic acid does not code for an amino acid sequence, to essentially identical sequences. Due to the degeneracy of the genetic code, a large number of functionally identical nucleic acids code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations," and silent variations are a type of conservatively modified variation. All nucleic acid sequences herein that code for a polypeptide also describe all possible silent variations of the nucleic acid. Those skilled in the art will recognize that each codon in a nucleic acid (except AUG, which is usually the only codon for methionine, and TGG, which is usually the only codon for tryptophan) can be modified to obtain a functionally identical molecule. Accordingly, each silent variation of a nucleic acid that encodes a polypeptide is implicit in each described sequence.

[0040] In the case of polypeptide sequences, "conservatively modified variants" include individual substitutions, deletions, or additions to a polypeptide sequence that result in the replacement of an amino acid with a chemically similar amino acid. Conservative substitution tables providing functionally similar amino acids are well known in the art. Such conservatively modified variants are in addition to, and do not exclude, the polymorphic variants, interspecies homologs, and alleles of the present invention. The following eight groups contain amino acids that are conservative substitutions for one another: 1) alanine (A), glycine (G); 2) aspartic acid (D), glutamic acid (E); 3) asparagine (N), glutamine (0); 4) arginine (R), lysine (K); 5) isoleucine (I), leucine (L), methionine (M), valine (V); 6) phenylalanine (F), tyrosine (Y), tryptophan (W); 7) serine (S), threonine (T); and 8) cysteine ​​(C), methionine (M). See, e.g., Creighton, Proteins (1984). In some embodiments, the term "conservative sequence modifications" is used to refer to amino acid modifications that do not significantly affect or significantly alter the binding characteristics of the antibody containing the amino acid sequence.

[0041] The terms "cross-compete" and "cross-competing" are used interchangeably herein to refer to the ability of an antibody or other binding agent to interfere with the binding of other antibodies or binding agents to D2D3 in a standard competitive binding assay.

[0042] The ability or extent to which an antibody or other binding agent can interfere with the binding of another antibody or binding molecule to D2D3, and therefore can be said to cross-compete according to the present invention, can be determined using standard competitive binding assays.One suitable assay includes the use of BIACORE technology (for example, by using BIACORE 3000 instrument (Biacore, Uppsala, Sweden)), which can measure the degree of interaction using surface plasmon resonance technology.Another assay for measuring cross-competition uses ELISA-based approach.

[0043] The term "optimized" as used herein refers to a nucleotide sequence that has been modified to encode an amino acid sequence using codons that are preferred in a production cell or organism, typically a eukaryotic cell, such as a Pichia cell, a Trichoderma cell, a Chinese Hamster Ovary cell (CHO) or a human cell. An optimized nucleotide sequence is engineered to completely or as far as possible retain the amino acid sequence originally encoded by the starting nucleotide sequence, also known as the "parent" sequence.

[0044] Standard assays for evaluating the binding ability of antibody to various species of D2D3 are known in the art, including, for example, ELISA, Western blot and RIA.The binding kinetics (e.g., binding affinity) of antibody can also be evaluated by standard assays known in the art, such as by BIACORE analysis or FACS relative affinity (Scatchard).The assay for evaluating the effect of antibody on the functional properties of D2D3 known in the art can be used.

[0045] The terms "polypeptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply not only to naturally occurring and non-naturally occurring amino acid polymers, but also to amino acid polymers in which one or more amino acid residues are artificial chemical mimics of corresponding naturally occurring amino acids. Unless otherwise indicated, a particular polypeptide sequence also implicitly encompasses its conservatively modified variants.

[0046] "Measuring" or "measurement" means assessing the presence, absence, quantity or amount (which may be an effective amount) of a given substance in a sample, including deriving a qualitative or quantitative concentration level of the given substance in the sample, or otherwise assessing the value or classification of a clinical parameter of interest. Alternatively, the term "detecting" or "detection" may be used and is understood to encompass all measuring or measurements described herein.

[0047] The term "sample" or "biological sample" as used herein refers to a biological fluid, tissue or cell sample in a healthy and / or pathological state obtained from a subject. Such samples include, but are not limited to, blood, bronchial washings, sputum, saliva, urine, amniotic fluid, lymphatic fluid, tissue or fine needle biopsy samples, peritoneal fluid, cerebrospinal fluid, nipple aspirates, including cell lysates, lysed cells, cell extracts and supernatants from nuclear extracts. In some embodiments, the whole blood sample is further processed into a serum or plasma sample. Preferably, the biological sample is selected from serum, plasma, saliva and urine. See also the Examples herein and Fernandez-Botran et al., "The levels of soluble urokinase plasminogen activator receptor (suPAR) in saliva are influenced by acute stress," (2021) Biological Psychology 165: p. 108147.

[0048] "Treating", "treat" or "treatment" within the context of this invention means alleviating the symptoms associated with a disorder or disease, or halting the further progression or worsening of those symptoms, or preventing or prophylaxis of a disease or disorder.

[0049] The term chronic kidney disease (CKD) is a chronic and progressive condition that occurs when one or both of the following conditions are present: i) there is evidence of renal damage lasting for at least 3 months, defined as structural or functional abnormalities of the kidney, with or without a reduced glomerular filtration rate (GFR), demonstrated by pathological abnormalities or by markers of renal damage, including urinary or blood abnormalities or imaging abnormalities, and / or ii) a GFR less than 60 ml / min / 1.73 m for at least 3 months, with or without renal damage. 2 Currently, in the United States, nearly 22% of the adult population has CKD, making it a very common disease process. CKD is classified by the level of GFR and the presence or absence of proteinuria. Stage 1 includes patients with no reduction in GFR but with renal abnormalities. Stage 2 includes patients with a GFR of 60 ml / min / 1.73 m 2 ~89ml / min / 1.73m 2 Stage 3 includes patients with mild CKD and renal abnormalities with an estimated GFR (eGFR) of 30 ml / min / 1.73 m 2 ~59ml / min / 1.73m 2 Patients with an eGFR of 1.0 ml / min / 1.73 m were included, and stage 4 patients were those with an eGFR of 1.0 ml / min / 1.73 m 2 ~29ml / min / 1.73m 2 Stage 5 is renal failure; this includes an eGFR of less than 15 ml / min / 1.73 m 2 This includes patients with an eGFR of 100-150%. After patients start dialysis, there is about a 20% 1-year mortality rate, and nearly a 75% mortality rate at 5 years. CKD can result from a myriad of conditions, including diabetes (type 1, type 2), hypertension, and glomerulonephritis. In either case, the presence of D2D3 is indicative of CKD.

[0050] As used herein, "type 1 diabetes" or "insulin-dependent diabetes" refers to a chronic illness characterized by the body's inability to produce insulin due to autoimmune destruction of beta cells in the pancreas. Although onset frequently occurs in childhood, the disease can also develop in adults. Insulin-dependent diabetes is characterized by hyperglycemia and is distinct from "type 2 diabetes," which refers to a set of dysfunctions resulting from a combination of resistance to insulin action, insufficient insulin secretion, and excessive or inappropriate glucagon secretion. The presence of D2D3 is indicative of insulin-dependent diabetes.

[0051] D2D3 Detection In some embodiments, the presence of D2D3 in a biological sample is determined. D2D3 determination can be performed at any time, for example, before or after a medical procedure.

[0052] In some embodiments, the presence of D2D3 can be detected from biological samples of subjects.The detection of the presence of D2D3 in biological samples can be carried out by any method known to those skilled in the art.The method for detecting the presence of D2D3 includes but is not limited to enzyme-linked immunosorbent assay (ELISA), Western blot, immunoprecipitation, immunohistochemistry, radioimmunoassay (RIA), radioreceptor assay, proteomics method, mass spectrometry-based detection (SRM or MRM) or quantitative immunostaining method.

[0053] In any embodiment, the presence of D2D3 determines whether or not an agent that antagonizes D2D3 is administered to the subject. In some embodiments, the agent is an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3. In some embodiments, the anti-D2D3 antibody is a commercially available anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3, or an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to D2D3.

[0054] D2D3 Removal The present invention also contemplates that, in various embodiments, D2D3 may be removed from the circulation of a patient in which D2D3 is detected by any extracorporeal means known to those of skill in the art, including, but not limited to, plasmapheresis, i.e. therapeutic plasma exchange, dialysis, immunoadsorption, or any combination of treatments capable of removing D2D3 protein from the circulation.

[0055] Pharmaceutical Compositions To prepare a pharmaceutical or sterile composition comprising a D2D3 binding antibody (intact or binding fragment), the D2D3 binding antibody (intact or binding fragment) is mixed with a pharma- ceutically acceptable carrier or excipient. The composition may further contain one or more other therapeutic agents suitable for treating or preventing diabetes.

[0056] Formulations of therapeutic and diagnostic agents can be prepared, for example, by mixing with a physiologically acceptable carrier, excipient, or stabilizer in the form of a lyophilized powder, a slurry, an aqueous solution, a lotion, or a suspension (see, e.g., Hardman et al., (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical See Dosage Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY).

[0057] Selecting the dosage regimen for a therapeutic agent depends on several factors, including the serum or tissue turnover rate of the entity, the level of symptoms, the immunogenicity of the entity, and the accessibility of target cells in the biological matrix.In some embodiments, the dosage regimen maximizes the amount of therapeutic agent delivered to the patient, consistent with an acceptable level of side effects.Therefore, the amount of biologic delivered depends, in part, on the individual entity and the severity of the condition being treated. Guidance on selecting appropriate doses of antibodies, cytokines and small molecules is available (e.g., Wawrzynczak (1996) Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK; Kresina (ed.) (1991) Monoclonal Antibodies, Cytokines and Arthritis, Marcel Dekker, New York, NY; Bach (ed.) (1993) Monoclonal Antibodies and Peptide Therapy in Autoimmune Diseases, Marcel Dekker, New York, NY; Baert et al, (2003) New Engl. J. Med. 348:601-608; Milgrom et al, (1999) New Engl. J. Med. 341:1966-1973; Slamon et al, (2001) New (see Engl. J. Med. 344:783-792; Beniaminovitz et al, (2000) New Engl. J. Med. 342:613-619; Ghosh et al, (2003) New Engl. J. Med. 348:24-32; Lipsky et al, (2000) New Engl. J. Med. 343:1594-1602).

[0058] The determination of the appropriate dose is made by the clinician, for example, using parameters or factors known or suspected in the art to affect treatment, or predicted to affect treatment. Generally, the dose is started at an amount somewhat less than the optimal dose, and then increased by small increments until the desired or optimal effect is achieved relative to any negative side effects.

[0059] The actual dosage level of the active ingredient in the pharmaceutical composition of the present invention may be varied to obtain an amount of the active ingredient effective to achieve the desired therapeutic response for a particular patient, composition and mode of administration without being toxic to the patient.The dosage level selected depends on various pharmacokinetic factors, including the activity of the particular composition of the present invention used, or its ester, salt or amide, the route of administration, the time of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors known in the medical field.

[0060] The composition comprising the antibody or fragment thereof of the present invention may be provided by continuous infusion or by doses at intervals of, for example, one day, one week or 1-7 times per week. The doses may be provided intravenously, subcutaneously, topically, orally, nasally, rectally, intramuscularly, intracerebrally, or by inhalation. A specific dose protocol is one that includes the maximum dose or dose frequency that avoids significant undesirable side effects. The total weekly dose can be at least 0.05 pg / kg body weight, at least 0.2 pg / kg, at least 0.5 μg / kg, at least 1 pg / kg, at least 10 pg / kg, at least 100 pg / kg, at least 0.2 mg / kg, at least 1.0 mg / kg, at least 2.0 mg / kg, at least 10 mg / kg, at least 25 mg / kg, at least 30 mg / kg, at least 40 mg / kg, or at least 50 mg / kg (see, e.g., Yang et al, (2003) New Engl. J. Med. 349:427-434; Herold et al, (2002) New Engl. J. Med. 346:1692-1698; Liu et al, (1999) J. Neurol. Neurosurg. Psych. 67:451-456; Portielji et al, (2003) Cancer (see Immunol, Immunother. 52:133-144).

[0061] The desired dose of multiple antibodies or fragments thereof is roughly the same as that of a single antibody or polypeptide, on a moles / kg body weight basis. The desired plasma concentration of the antibody or fragment thereof, on a moles / kg body weight basis, is about. The dose can be at least 15 pg, at least 20 pg, at least 25 pg, at least 30 pg, at least 35 pg, at least 40 pg, at least 45 pg, at least 50 pg, at least 55 pg, at least 60 μg, at least 65 pg, at least 70 pg, at least 75 pg, at least 80 pg, at least 85 pg, at least 90 pg, at least 95 pg, or at least 100 pg. The dose administered to a subject can be at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12, or at least more than that. In the case of an antibody or fragment thereof of the present invention, the dosage administered to a patient may be 0.0001 mg / kg to 100 mg / kg of patient's body weight. Dosages may be 0.0001 mg / kg to 20 mg / kg of patient's body weight, 0.0001 mg / kg to 10 mg / kg of patient's body weight, 0.0001 mg / kg to 5 mg / kg of patient's body weight, 0.0001 to 2 mg / kg of patient's body weight, 0.0001 to 1 mg / kg of patient's body weight, 0.0001 mg / kg to 0.75 mg / kg of patient's body weight, 0.00 The dose may be 0.01 mg / kg to 0.5 mg / kg of patient's body weight, 0.0001 mg / kg to 0.25 mg / kg of patient's body weight, 0.0001 to 0.15 mg / kg of patient's body weight, 0.0001 to 0.10 mg / kg of patient's body weight, 0.001 to 0.5 mg / kg of patient's body weight, 0.01 to 0.25 mg / kg of patient's body weight or 0.01 to 0.10 mg / kg of patient's body weight.

[0062] Dosages of antibodies or fragments thereof of the invention can be calculated using the patient's weight in kilograms (kg) multiplied by the dose to be administered in mg / kg. Dosages of antibodies or fragments thereof of the invention can be up to 150 μg / kg of patient's body weight, up to 125 μg / kg of patient's body weight, up to 100 μg / kg of patient's body weight, up to 95 μg / kg of patient's body weight, up to 90 μg / kg of patient's body weight, up to 85 μg / kg of patient's body weight, up to 80 μg / kg of patient's body weight, up to 75 μg / kg of patient's body weight, up to 70 μg / kg of patient's body weight, up to 65 μg / kg of patient's body weight, up to 60 μg / kg of patient's body weight, up to 55 μg / kg of patient's body weight, up to 50 μg / kg of patient's body weight, up to 45 μg / kg of patient's body weight. The dose may be 1 μg / kg or less, 40 μg / kg or less of patient's body weight, 35 μg / kg or less of patient's body weight, 30 μg / kg or less of patient's body weight, 25 μg / kg or less of patient's body weight, 20 μg / kg or less of patient's body weight, 15 μg / kg or less of patient's body weight, 10 μg / kg or less of patient's body weight, 5 μg / kg or less of patient's body weight, 2.5 μg / kg or less of patient's body weight, 2 μg / kg or less of patient's body weight, 1.5 μg / kg or less of patient's body weight, 1 μg / kg or less of patient's body weight, 0.5 μg / kg or less of patient's body weight, or 0.5 μg / kg or less of patient's body weight.

[0063] A unit dose of the antibody or fragment thereof of the present invention may be 0.1 mg to 20 mg, 0.1 mg to 15 mg, 0.1 mg to 12 mg, 0.1 mg to 10 mg, 0.1 mg to 8 mg, 0.1 mg to 7 mg, 0.1 mg to 5 mg, 0.1 to 2.5 mg, 0.25 mg to 60 mg, 0.25 mg to 40 mg, 0.25 mg to 20 mg, 0.25 mg to 15 mg, 0.25 mg to 12 mg, 0.25 mg to 10 mg, 0.25 mg to 8 mg, 0.25 mg to 7 mg, 0.25 mg to 5 mg, 0.5 mg to 2.5 mg, 1 mg to 20 mg, 1 mg to 15 mg, 1 mg to 12 mg, 1 mg to 10 mg, 1 mg to 8 mg, 1 mg to 7 mg, 1 mg to 5 mg, or 1 mg to 2.5 mg.

[0064] Administration of an antibody or fragment thereof of the invention may achieve a serum titer in a subject of at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 μg / ml, at least 225 μg / ml, at least 250 μg / ml, at least 275 μg / ml, at least 300 μg / ml, at least 325 μg / ml, at least 350 μg / ml, at least 375 μg / ml or at least 400 μg / ml. Alternatively, the administered amount of the antibody or fragment thereof of the invention may achieve a serum titer in the subject of at least 0.1 μg / ml, at least 0.5 μg / ml, at least 1 μg / ml, at least 2 μg / ml, at least 5 μg / ml, at least 6 μg / ml, at least 10 μg / ml, at least 15 μg / ml, at least 20 μg / ml, at least 25 μg / ml, at least 50 μg / ml, at least 100 μg / ml, at least 125 μg / ml, at least 150 μg / ml, at least 175 μg / ml, at least 200 pg / ml, at least 225 pg / ml, at least 250 pg / ml, at least 275 pg / ml, at least 300 pg / ml, at least 325 pg / ml, at least 350 pg / ml, at least 375 pg / ml or at least 400 pg / ml.

[0065] Doses of the antibody or fragment thereof of the invention may be repeated and administrations may be separated by at least 1 day, at least 2 days, at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 15 days, at least 30 days, at least 45 days, at least 2 months, at least 75 days, at least 3 months or at least 6 months.

[0066] The effective amount for a particular patient may vary depending on factors such as the condition being treated, the patient's overall health, the route and dose of administration, and the severity of side effects (see, e.g., Maynard et al., (1996) A Handbook of SOPs for Good Clinical Practice, Interpharm Press, Boca Raton, Fla.; Dent (2001) Good Laboratory and Good Clinical Practice, Urch PubL, London, UK).

[0067] The route of administration may be, for example, by topical or dermal application, intravenous, intraperitoneal, intracerebral, intramuscular, intraocular, intraarterial, intracerebrospinal, intralesional injection or infusion, or by sustained release systems or implants. See, for example, Sidman et al., (1983) Biopolymers 22:547-556; Langer et al., (1981) J. Biomed. Mater. Res. 15:167-277; Langer (1982) Chem. Tech. 12:98-105; Epstein et al., (1985) Proc. Natl. Acad. Sci, USA 82:3688-3692; Hwang et al., (1980) Proc. Natl. Acad. Sci, USA 77:4030-4034; U.S. Patent Nos. 6,350,466 and 6,316,024.) If necessary, the composition may also include a solubilizing agent and a local anesthetic, such as lidocaine, to ease pain at the site of the injection. In addition, pulmonary administration can also be used, for example by using inhaler or nebulizer, and by formulating with aerosolizing agent.See, for example, U.S. Patent No. 6,019,968, U.S. Patent No. 5,985,320, U.S. Patent No. 5,985,309, U.S. Patent No. 5,934,272, U.S. Patent No. 5,874,064, U.S. Patent No. 5,855,913, U.S. Patent No. 5,290,540 and U.S. Patent No. 4,880.078; and International Publication No. WO 92 / 19244, International Publication No. WO 97 / 32572, International Publication No. WO 97 / 44013, International Publication No. WO 98 / 31346 and International Publication No. WO 99 / 66903, or any similar reference known to those skilled in the art.

[0068] The compositions of the present invention may also be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by those skilled in the art, the route and / or mode of administration will vary depending on the desired outcome. The selected administration route for the antibody or fragment thereof of the present invention includes intravenous, intramuscular, intradermal, intraperitoneal, subcutaneous, spinal or other parenteral administration routes, for example, by injection or infusion. Parenteral administration may refer to a mode of administration other than enteral and topical administration, usually by injection, and includes, but is not limited to, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion. Alternatively, the compositions of the present invention may be administered via a parenteral route, such as a topical, epidermal or mucosal administration route, for example, intranasally, orally, vaginally, rectally, sublingually or topically. In one embodiment, the antibody or fragment thereof of the present invention is administered by infusion.

[0069] In another embodiment, the multispecific epitope binding proteins of the invention are administered subcutaneously. If the antibodies or fragments thereof of the invention are administered in a controlled or sustained release system, a pump can be used to achieve controlled or sustained release. See, e.g., Langer et al., supra; Sefton, (1987) CRC Crit. Ref Biomed. Eng. 14:20; Buchwald et al., (1980), Surgery 88:507; Saudek et al, (1989) N. Engl. J. Med. 321:574).

[0070] Polymeric materials may be used to achieve controlled or sustained release of the therapeutics of the invention. See, e.g., Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Fla. (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, (1983) J. Macromol. Sci. Rev. Macromol. Chem. 23:61; Levy et al., (1985) Science 228:190; During et al, (1989) Ann. Neurol. 25:351; Howard et al., (1989) Ann. Neurol. 25:351; al, (1989) J. Neurosurg. 71:105; U.S. Patent No. 5,679,377; U.S. Patent No. 5,916,597; U.S. Patent No. 5,912,015; U.S. Patent No. 5,989,463; U.S. Patent No. 5,128,326; WO 99 / 15154; and WO 99 / 20253. Examples of polymers used in sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. In one aspect, the polymers used in the sustained release formulations are inert, free of leachable impurities, stable on storage, sterile, and biodegradable. Controlled or sustained release systems can be placed near the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose.See, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984).

[0071] Controlled release systems are discussed in the review by Langer, (1990), Science 249:1527-1533. Any technique known to those of ordinary skill in the art can be used to produce sustained release formulations containing one or more antibodies or fragments thereof of the invention. See, e.g., U.S. Pat. No. 4,526,938, WO 91 / 05548, WO 96 / 20698, Ning et al, (1996), Radiotherapy & Oncology 39:179-189, Song et al, (1995) PDA Journal of Pharmaceutical Science & Technology 50:372-397, Cleek et al, (1997) Pro. Intl Symp. Control. Rel. Bioact. Mater. 24:853-854, and Lam et al, (1997) Proc. Intl Symp. Control Rel. Bioact. Mater. 24:759-760.

[0072] When the antibody or fragment thereof of the present invention is administered topically, the antibody or fragment thereof of the present invention can be formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form known to those skilled in the art. For example, see Remington's Pharmaceutical Sciences and Introduction to Pharmaceutical Dosage Forms, 19th ed., Mack Pub. Co., Easton, Pa. (1995). For topical dosage forms that are not sprayable, a viscous to semi-solid or solid form that is compatible with topical application and optionally contains a carrier or one or more excipients with a dynamic viscosity greater than water is typically used. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which are sterilized, if desired, or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for affecting various properties, such as osmotic pressure. Other suitable topical dosage forms include the sprayable aerosol preparations, in which the active ingredient, optionally combined with solid or liquid inert carrier, is placed in a mixture with pressurized volatile material (e.g., gaseous propellant such as Freon) or in a squeeze bottle.If desired, moisturizer or humectant can also be added to pharmaceutical compositions and dosage forms.Examples of such additional components are well known in the art.

[0073] When the composition containing antibody or its fragment is administered intranasally, it can be formulated in aerosol form, spray, mist, or in the form of droplets.In particular, the prophylactic or therapeutic agent for use according to the present invention can be conveniently delivered in the form of aerosol spray presentation from pressurized pack or nebulizer using suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas).In the case of pressurized aerosol, dosage unit can be determined by providing a valve to deliver a metered amount.Capsules and cartridges (e.g., made of gelatin) for use in inhaler or insufflator can be formulated, containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0074] Methods for co-administration or treatment with a second therapeutic agent are known in the art. An effective amount of a therapeutic agent may reduce symptoms by at least 10%; at least 20%; at least about 30%>; at least 40%>, or at least 50%. Additional therapies (e.g., prophylactic or therapeutic agents) that can be administered in combination with the antibody or fragment thereof of the present invention may be administered less than 5 minutes apart, less than 30 minutes apart, 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours apart, or about 9 hours apart. The treatments may be administered at intervals of 10 to about 9 hours, at intervals of 9 to about 10 hours, at intervals of 10 to about 11 hours, at intervals of 11 to about 12 hours, at intervals of 12 to 18 hours, at intervals of 18 to 24 hours, at intervals of 24 to 36 hours, at intervals of 36 to 48 hours, at intervals of 48 to 52 hours, at intervals of 52 to 60 hours, at intervals of 60 to 72 hours, at intervals of 72 to 84 hours, at intervals of 84 to 96 hours, or at intervals of 96 to 120 hours. Two or more treatments may be administered within one and the same patient visit.

[0075] The antibody or fragment thereof of the present invention and other therapies can be administered cyclically. Cyclic therapy includes administering a first therapy (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administration of a second therapy (e.g., a second prophylactic or therapeutic agent) for a period of time, optionally followed by administration of a third therapy (e.g., a prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration, i.e., cycle, in order to reduce the occurrence of resistance to one of the therapies, to avoid or reduce the side effects of one of the therapies, and / or to improve the efficacy of the therapies.

[0076] In some embodiments, the antibody or fragment of the present invention can be formulated to ensure proper distribution in vivo. For example, the blood-brain barrier (BBB) ​​excludes many highly hydrophilic compounds. To ensure that the therapeutic compound of the present invention crosses the BBB (if desired), it can be formulated, for example, in liposomes. For methods of producing liposomes, see, for example, U.S. Patent Nos. 4,522,811; 5,374,548; and 5,399.331. Liposomes can contain one or more moieties that are selectively transported into specific cells or organs, thus enhancing targeted drug delivery (see, for example, Ranade, (1989) J.Olin.Pharmacol.29:685).

[0077] The present invention provides a protocol for administering a pharmaceutical composition comprising the antibody or fragment of the present invention alone or in combination with other treatments to a subject in need of administration of the pharmaceutical composition comprising the antibody or fragment of the present invention alone or in combination with other treatments. The treatments (e.g., prophylactic or therapeutic agents) of the combination therapy of the present invention can be administered simultaneously or sequentially to a subject. The treatments (e.g., prophylactic or therapeutic agents) of the combination therapy of the present invention can also be administered cyclically. Cyclic therapy involves administering a first treatment (e.g., a first prophylactic or therapeutic agent) for a period of time, followed by administration of a second treatment (e.g., a second prophylactic or therapeutic agent) for a period of time, and repeating this sequential administration, i.e., cycle, to reduce the development of resistance to one of the treatments (e.g., agents), to avoid or reduce the side effects of one of the treatments (e.g., agents), and / or to improve the effectiveness of the treatment.

[0078] The treatments (e.g., prophylactic or therapeutic agents) of the combination therapy of the present invention may be administered to a subject simultaneously. The term "simultaneously" is not limited to administering the treatments (e.g., prophylactic or therapeutic agents) at exactly the same time, but rather means that the pharmaceutical compositions containing the antibodies of the present invention or fragments thereof are administered to a subject in sequence and within a time interval such that the antibodies of the present invention can act together with the other treatments to provide increased benefit than if the antibodies of the present invention were otherwise administered. For example, each treatment may be administered to a subject at the same time or at different points in time, sequentially in any order, but if not administered simultaneously, they should be administered sufficiently close in time to provide the desired therapeutic or prophylactic effect. Each treatment may be administered separately to a subject in any suitable form and by any suitable route.

[0079] In various embodiments, the therapies (e.g., prophylactic or therapeutic agents) are administered to a subject less than 15 minutes apart, less than 30 minutes apart, less than 1 hour apart, about 1 hour apart, about 1 hour to about 2 hours apart, about 2 hours to about 3 hours apart, about 3 hours to about 4 hours apart, about 4 hours to about 5 hours apart, about 5 hours to about 6 hours apart, about 6 hours to about 7 hours apart, about 7 hours to about 8 hours apart, about 8 hours to about 9 hours apart, about 9 hours to about 10 hours apart, about 10 hours to about 11 hours apart, about 11 hours to about 12 hours apart, 24 hours apart, 48 hours apart, 72 hours apart, or one week apart. In other embodiments, two or more therapies (e.g., prophylactic or therapeutic agents) are administered within the same patient visit.

[0080] The prophylactic or therapeutic agents of the combination therapy may be administered to a subject in the same pharmaceutical composition. Alternatively, the prophylactic or therapeutic agents of the combination therapy may be administered to a subject simultaneously in separate pharmaceutical compositions. The prophylactic or therapeutic agents may be administered to a subject by the same or different routes of administration. Having fully described the invention, the invention is further illustrated by the following examples and claims, which are illustrative and not intended to be further limiting.

[0081] Throughout this disclosure, various quantities, such as amounts, sizes, dimensions, ratios, etc., are presented in a range format. It should be understood that the description of quantities in a range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of any embodiment. Thus, the description of a range should be considered to specifically disclose all possible subranges and all individual numerical values ​​within that range, unless the context clearly dictates otherwise. For example, the description of a range such as 1-6 should be considered to specifically disclose subranges such as 1-3, 1-4, 1-5, 2-4, 2-6, 3-6, etc., as well as individual values ​​within that range, such as 1.1, 2, 2.3, 4.62, 5, and 5.9. This applies regardless of the breadth of the range. The upper and lower limits of these intervening ranges may be independently included in the smaller ranges and are also encompassed within the disclosure, subject to any specifically excluded limits in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure, unless the context clearly dictates otherwise.

[0082] The terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting of any embodiment. As used herein, the singular forms "a", "an" and "the" are intended to include the plural unless the context clearly dictates otherwise. It will be further understood that the terms "includes", "comprises", "including" and / or "comprising" as used herein specify the presence of the stated features, integers, steps, operations, elements and / or components, but do not exclude the presence or addition of one or more other features, integers, steps, operations, elements, components and / or groups thereof. As used herein, the term "and / or" includes any and all combinations of one or more of the associated listed items. It will be further understood that an item in a list of the form "at least one of A, B and C" can mean (A); (B); (C); (A and B); (B and C); (A and C); or (A, B and C). Similarly, an item listed in the form "at least one of A, B, or C" can mean (A)(B);(C);(A and B);(B and C);(A and C); or (A, B and C);.

[0083] As used herein, unless specifically stated otherwise or clear from the context, the term "about" in reference to a number or range of numbers is understood to mean the recited number and number + / - 10% thereof, or for values ​​recited for a range, 10% below the recited lower limit and 10% above the recited upper limit.

[0084] General Materials and Methods Human samples : A total of 25 human serum and corresponding urine samples were obtained from diabetic nephropathy (DN) patients seen at Renal Associates, MGH by Dr. Kristin M. Corapi under MGH IRB protocol #2014P001943 during the period 2014-2018. Additionally, during the period 2018-2020, we obtained 57 sera from Mass General Brigham Biobank. 40 patients were diagnosed with DN and were not receiving insulin therapy when serum samples were collected. 42 patients were diagnosed with DN and were receiving insulin therapy when serum samples were collected. Statistical analysis was performed using SPSS 27 (IBM, Armony NY). We used chi-square tests to compare the prevalence of hD2D3 (reported as %) between DN patients on or off insulin. To assess whether hD2D3 was independently associated with insulin use, we used a logistic regression model with insulin therapy as the dependent categorical variable, and the presence of hD2D3, hemoglobin Alc% and suPAR (log-transformed base 2) as independent variables.Finally, we calculated the area under the curve (AUC) for hD2D3 and suPAR in separate models and combined to evaluate their ability to distinguish between those on or off insulin therapy.AUC was compared using the Delong test.A two-sided P value of <0.05 was adopted to indicate statistical significance.

[0085] reagent : RPMI 1640 medium (11875-093), CMRL medium (21540-026); FBS (10082-147), Penicillin-Streptomycin (15140-122), Antibiotic / Antimycotic (15240-096) (Penicillin, Streptomycin and Amphtericin B) were obtained from Gibco. EZ-link Micro Sulfo-NHS-Biotinylation Kit (21925), Zeba™ Spin Desalting Column and Pierce™ IP RIPA Buffer (89901) were obtained from Thermo Fisher Scientific. β-Mercaptoethanol (M6250) and ITS (Insulin-Transferrin-Sodium Selenite) Medium Supplement (I3146) were obtained from Sigma-Aldrich. Reagents used for in vitro GSIS (MIN6 cells, mouse islets and human islets) were obtained from Sigma-Aldrich: sodium pyruvate (Corning, 10-013-CV); protease inhibitor cocktail tablets (Roche, 11836170001); chymotrypsin (Roche, 11 418 467 001); uPAR (R&D systems, 807-UK / CF); streptavidin Mag Sepharose beads (GE Healthcare, 28-9857-99); N-glycanase (PROzyme, GKE-5006A).

[0086] cell culture : Mouse MIN6 cells (kindly provided by Dr. Decheng Ren, University of Chicago) were grown as previously described. See Ren et al., “Role of BH3-only molecules Bim and Puma in beta-cell death in Pdx1 deficiency,” (2014) Diabetes 63: pp. 2744-2750. Immortalized human podocytes were cultured according to published protocols (Saleem et al., 2002). Mouse islets were cultured in RPMI 1640 medium containing 10% FBS, 1% penicillin / streptomycin and 50 μM β-mercaptoethanol. Human islets were cultured in CMRL medium containing 10% FBS and 1% penicillin / streptomycin.

[0087] antibody : Rabbit anti-c-Myc antibody (Sigma-Aldrich, PLA0001), guinea pig anti-insulin antibody (Abcam, ab7842), mouse anti-glucagon antibody (Sigma-Aldrich, G2654). The secondary antibody for insulin labeling was Alexa Fluor 488-conjugated goat anti-guinea pig IgG (Invitrogen), and the secondary antibody for glucagon labeling was Alexa Fluor 594-conjugated chicken anti-rabbit IgG (Invitrogen). uPAR(R4)-BSA Free (Novusbio, NBP2-41379); rabbit anti-uPAR (Bethyl, A304-462A); mouse uPAR polyclonal antibody (R&D systems, AF534); anti-goat IgG HRP antibody (Thermo Fisher, HAF109); anti-rabbit IgG HRP antibody (Thermo Fisher, G-21234); AP5 antibody (Blood Center of Wisconsin); paxillin antibody Y-113 (Abcam, ab32081).

[0088] Standard Procedure : Mouse islet isolation was performed as described. See Zhu et al., “Kindlin-2 modulates MafA and beta-catenin expression to regulate beta-cell function and mass in mice,” (2020) Nat Commun 11: p. 484. Islets were manually harvested under an operating microscope (Nikon Instruments Inc, Melville, NY, USA). Pancreatic beta cell area and beta cell mass were calculated as described above. AP5 assay using human podocytes was performed as described. See Hayek et al., “A tripartite complex of suPAR, APOL1 risk variants and alphavbeta3 integrin on podocytes mediates chronic kidney disease,” (2017) Nat Med 23: pp. 945-953.

[0089] Immunoprecipitation coupled with Western blot analysis (IP-WB) : Human serum and urine samples: Human serum or urine samples were diluted (1:1) in RIPA buffer containing a protease inhibitor cocktail tablet (RIPA-PI) and pre-cleared using Streptavidin Mag Sepharose beads. Human uPAR (R4) antibody (Novusbio, NBP2-41379) was biotinylated using the EZ-link Micro Sulfo-NHS-Biotinylation Kit. Biotinylated uPAR (R4) antibody was added to the pre-cleared samples. Streptavidin Mag Sepharose beads were subsequently added to the spinning samples. Total immunoprecipitation (IP) time to generate samples for mass spectrometry was 24 h, and time for IP-WP was 3–4 h. Magnetic beads were washed with RIPA buffer and bound fractions were deglycosylated using N-glycanase. Proteins were analyzed using SDS-PAGE. Western blot analysis was performed using polyclonal rabbit anti-uPAR (Bethyl, A304-462A). Recombinant human uPAR (R&D systems, 807-UK / CF), chymotrypsin-digested uPAR or recombinant hD2D3 were used as positive controls. For mass spectrometry analysis, proteins immunoprecipitated from patient serum or urine were deglycosylated and subjected to SDS PAGE, and protein bands were excised from the gels and submitted to the Taplin Biological Mass Spectrometry Facility at Harvard Medical School.

[0090] Mouse serum Mouse sera were diluted, pre-cleared, and immunoprecipitated as described above for human sera. The antibody used to immunoprecipitate mouse proteins was a polyclonal mouse uPAR antibody (R&D systems, AF534). Recombinant mouse suPAR and recombinant mouse D2D3 were used as positive controls. Western blots were performed with rabbit anti-c-myc antibody (Sigma-Aldrich, PLA0001) and rabbit IgG-HRP antibody.

[0091] β3 integrin activation assay using human podocytes : Experiments were performed as described elsewhere (see Hayek et al., 2017). Where indicated, cells were treated with serum-free medium ± hD2D3 for one set and with HS ± hD2D3 for another set. Images were acquired using a Zeiss microscope. AP5 or paxillin intensity was quantified using Fiji, ImageJ (NIH). Non-specific nuclear staining was ignored while performing quantitative analysis. Data are expressed as the ratio of AP5 / paxillin intensity relative to control. Unpaired two-tailed t-tests were performed using Prism (GraphPad) to compare these treatments.

[0092] Cloning, expression and purification of recombinant proteins : The gene encoding mouse uPAR isoform 1 (GenBank NM_011113.4) was amplified using total RNA isolated from cultured mouse podocytes using known forward and reverse primers. The PCR product was digested with restriction enzymes HindIII and EcoRI and subcloned into pSecTag2A vector (Thermo Fisher, V90020) containing a C-terminal Myc / His tag. FreeStyle™ 293-F cells (Thermo Fisher, 12347-019) were transiently transfected with pSecTag2A-suPAR / D2D3 plasmid. Recombinant protein was purified from culture medium using Pierce anti-c-Myc agarose (Thermo Fisher, 20168) according to the manufacturer's protocol.

[0093] mouse : Mice expressing full-length suPAR (suPAR-Tg) were previously described; see Hahm et al., 2017. Mice expressing D2D3 protein (D2D3-Tg) were generated at the Transgene Facility at the University of Miami. To achieve adipocyte-specific expression of the fragment, DNA encoding the C-terminal D2D3 protein of mouse suPAR isoform 1 (uniprot.org / uniprot / P35456), corresponding to NM_011113.4 in GenBank and encompassing amino acids 117-298 of the mature form, was placed under the control of an aP2 promoter cassette (Wang et al., 2010). In addition, to ensure that the fragment is secreted into circulation, DNA encoding a secretory signal peptide (Igk) was placed at the N-terminal alanine 117 of the D2D3 protein. The signal peptide is post-translationally cleaved between a glycine (G) and an aspartic acid (D, underlined). Furthermore, the DNA encoding the GPI-anchor was replaced with DNA encoding a Myc-tag (see, below, FIG. 5A and FIG. 7A to FIG. 7C).

[0094] To establish the colony, genotyped positive founder mice (D2D3-Tg) were backcrossed to C57BL / 6 mice for at least five generations. D2D3-Tg mice were viable and fertile. Mice were maintained on either a normal diet or a high-fat diet (HFD). All animal experiments were performed in accordance with the NIH Guide for Care and Use of Experimental Animals and approved by the Rush University Institutional Animal Care and Use Committee (IACUC) protocol #19-014.

[0095] Real-time RT-PCR (qPCR) : RNA was isolated with TRIzol (Thermo Fisher, 15596-026) and cDNA was generated using the High Capacity cDNA Reverse Transcription Kit (Thermo Fisher, 4368814). qPCR was performed using SsoAdvanced Universal SYBR Green Supermix (BioRad, 172-5271). All PCR data were normalized by Gapdh gene expression levels. qPCR primer sequences for identifying D2D3 (including part of the Myc tag) were used.

[0096] Immunohistochemical staining : Paraffin-embedded adipose or pancreatic sections (5 μm thick) were deparaffinized in xylene and rehydrated using a graded ethanol series (100%, 95%, 70%) followed by rinsing in distilled water. Antigen retrieval was performed by boiling the slides in a sodium citrate solution (pH 6.0) in a microwave. After incubation with blocking solution (2% BSA, 0.3% Triton X-100 in PBS) for 1 h at room temperature, sections were stained with anti-c-Myc antibody (1:2000), anti-insulin antibody (1:500) or anti-glucagon antibody (1:300) for D2D3 protein detection, followed by secondary antibodies. Images were acquired using an LSM 700 confocal microscope (Carl Zeiss).

[0097] Cell proliferation and apoptosis : Pancreatic sections from each group of mice were stained with antibodies against insulin or Ki67 as previously described (see Zhu et al., 2020). For quantitative analysis, the number of DAPI-positive cells in the insulin-stained area was counted using ImageJ (NIH). At least 1000 insulin-positive cells were counted per mouse to determine Ki67-positive cells. Ki67-positive cells were normalized to total insulin-positive cells in the same area. Cell survival was assessed using the In Situ Cell Death Detection Kit (Roche Applied Sciences, 11684795910) according to the manufacturer's instructions. Briefly, after removal of wax, antigen retrieval was performed using proteinase K (10 μg / ml in 10 mM Tris / HCl, pH 7.4) for 15 min at room temperature. Slides were washed twice with 1× PBS and incubated in TUNEL reaction mixture (50 μl enzyme solution + 450 μl Label Solution) for 60 min at 37 °C. Pancreatic sections were further stained with antibodies insulin and DAPI. Sections were examined using an LSM 700 laser scanning fluorescence confocal microscope running ZEN software (Zeiss). TUNEL positive cells were normalized to total insulin positive cells in the same area as we did for Ki67 quantification.

[0098] Assessment of renal function and insulin levels : Urinary albumin was measured using a mouse albumin ELISA (Bethyl Labs, E99-134) and creatinine was measured using an enzymatic assay kit (Cayman Chemical, 500701). Albumin to creatinine (ACR) ratios were calculated. Renal function was determined by measuring blood urea nitrogen levels (BUN) using BioAssay Systems (DIUR-500) and serum creatinine levels were determined using a Crystal Chem assay (80350). Pancreatic function was determined by measuring levels of C-peptide using a mouse C-peptide ELISA kit (90050) and insulin levels using an ultrasensitive mouse insulin ELISA kit (90080) from Crystal Chem. Levels of IL-6 were measured by R&D Systems (M6000B) and CRP was measured using a Crystal Chem assay (80350).

[0099] In vivo glucose tolerance test (GTT) and glucose stimulated insulin secretion GSIS assay : For both assays, animals were fasted overnight. Glucose was administered by intraperitoneal injection at a concentration of 2 g / kg body weight. Blood samples were collected at the indicated times by tail nicking as indicated time points. Blood glucose and serum insulin levels were measured using a glucose meter (Bayer HealthCare) and ELISA assay (Crystal Chem, 90080), respectively.

[0100] In vitro GSIS assay : GSIS was performed on MIN6 cells and isolated mouse or human islets. MIN6 cells were maintained in culture medium containing 1 g / L D-glucose. Recombinant mD2D3 or mouse suPAR at the indicated concentrations were added to the cells for 24 h. Before the initiation of GSIS, cells were washed twice with KRBH (137 mM NaCl, 4.7 mM KCl, 1.2 mM KH2PO4, 1.2 mM MgSO4, 2.5 mM CaCl2-2H2O, 25 mM NaHCO3, 10 mM HEPES) supplemented with 0.2% BSA and 1 g / L (5.5 mM) D-glucose. Cells were placed in low glucose KRBH medium (5.5 mM glucose, 0.2% BSA) for 30 min at 37°C, followed by KRBH (0.2% BSA) medium containing either 5.5 mM or 20 mM glucose for 1 h at 37°C. For mouse islets, after overnight culture in 5.5 mM RPMI, size-matched islets were treated with BSA, IgG, mD2D3 or suPAR protein as indicated in the figure legend. Islets were then placed in KRBH buffer containing 2.8 mM glucose for 1 hour and subjected to 2.8 mM and 16.7 mM glucose stimulation. For human islets obtained from the UVA Islet Microfluidic Laboratory at the University of Virginia or the ADI IsletCore Alberta Diabetes Institute at the University of Alberta (Edmonton, Canada), after overnight culture in CMRL medium, islets were incubated in 10% human serum for 4 days.

[0101] Where indicated, 10 ng / ml of human recombinant suPAR or hD2D3 protein was added to human serum. GSIS was initiated by the end of day 4 as described above for mouse islets. Released insulin was determined in the supernatant using a mouse insulin ELISA kit (Crystal Chem, 90080) or a human insulin ELISA kit (Crystal Chem, 90095). Total protein was extracted by sonication for 2 min in 500 μl of acid / ethanol (0.18 M HCl in 95% ethanol) solution as previously described. See Zhu et al., 2020.

[0102] The study protocol for working with human pancreatic islets was approved by the Institutional Review Board at Rush University Medical Center (IRB protocol #14051401-IRB01-AM07).

[0103] Antibody treatment : Two-month-old D2D3-Tg mice were treated with mouse uPAR antibody (R&D Systems, AF534) at 0.5 mg kg-1 body weight injected intraperitoneally twice a week for 4 weeks.

[0104] Extracellular flux analysis : Experiments were performed as described in (Altintas et al., 2021). See Altintas et al., “Metabolic Changes in Peripheral Blood Mononuclear Cells Isolated From Patients With End Stage Renal Disease,” (2021) Front Endocrinol (Lausanne) 12:629-239. Cellular oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) were detected by a Seahorse XFe24 Analyzer (Agilent Technologies, Santa Clara, CA) using the Cell Mito Stress Test and Glycolysis Stress Test kits (both from Agilent), respectively. Mouse insulinoma 6 (MIN6) cells (6 × 104 cells) were seeded onto each well of a 24-well assay plate (Agilent), allowed to attach overnight, and grown in regular culture medium for 24 hours. The medium was then replaced with low glucose (2.8 mM) culture medium and cells were treated with 10 and 100 ng / ml of mouse D2D3 protein for 24 h. On the day of the assay, the medium was replaced with bicarbonate-free XF DMEM assay medium, pH 7.4 (Agilent), supplemented with 1 mM sodium pyruvate, 2 mM glutamine and 2.8 mM glucose, to optimize respiratory conditions. The plates were incubated in a non-CO2 incubator for 30-60 min and then transferred to the analyzer. For mitochondrial respiration analysis, the first three measurements of OCR were recorded under basal conditions. To stimulate cellular oxygen consumption, a 175 mM glucose solution was injected (final concentration in each well: 20 mM) and the OCR was measured for another 60 min. This was followed by sequential injections of 10x concentrations of oligomycin (complex V inhibitor; final concentration in each well: 1.0 μM), FCCP (uncoupler; 1.25 μM), rotenone and antimycin A (inhibitors of complex I and complex III, respectively; each, 0.5 μM). Three readings of the OCR were recorded after each injection, with all recordings separated by 8 min.This protocol allowed the estimation of basal respiration, proton leak, ATP production, maximal respiration, spare respiratory capacity and non-mitochondrial respiration rates as previously described. They were calculated as described ibid. For glycolysis assays, XF DMEM assay medium was supplemented with only 2 mM glutamine, but glucose (10 mM), oligomycin (1 μM) and 2-deoxy-glucose (2DG; 50 mM) were injected sequentially after the basal ECAR reading. Three readings of ECAR were recorded after each injection, all recordings were 8 min apart. Basal acidification (ECAR before glucose injection), glycolysis (difference between ECAR before the addition of glucose and ECAR after the addition of glucose) and non-glycolytic acidification (residual ECAR after the addition of oligomycin and 2DG) rates were calculated to determine the nature of glycolysis. Extracellular flux readings were normalized to the protein content in each well. OCR and ECAR measurements are therefore expressed as pmol / min / μg protein and mpH / min / mg protein, respectively.

[0105] Transmission electron microscopy (TEM) of kidney samples : Mouse kidney tissue was cut into 2 × 2 mm pieces. Tissue was fixed in Trump's fixative (EMS, 11750), dehydrated in graded ethanol, dried using an 850 Critical Point Dryer (EMS), gold-coated with a Cressington 108 Auto Sputter Coater (Ted Pella), and post-fixed in 1% OsO4 for 1 h on ice. Tissue was then washed in 0.1 M cacodylate buffer, dehydrated, and embedded in Epon812 (EMS). Ultrathin (70 nm) sections were collected on Formvar-coated Ni slot grids (EMS) and stained in 5% uranyl acetate and 0.1% lead citrate for 15 min. Electron micrographs were taken with a Sigma HDVP Electron Microscope (Zeiss).

[0106] Platinum replica electron microscopy (PR-EM) and ultrathin section electron microscopy (UTS-EM) on MIN6 cells : MIN6 cells were grown on glass coverslips coated with poly-L-lysine (Sigma, P4707) for PR-EM or on ACLAR plastic sheets for UTS-EM. GSIS was performed as described above. For PR-EM, cells were extracted with detergent and processed as described. See Svitkina “Imaging Cytoskeleton Components by Electron Microscopy,” (2016) Methods Mol Biol 1365: pp. 99-118. Samples were dehydrated in a critical point drying apparatus Autosamdri-815 (Tousimis), coated with a 2 nm layer of platinum, and stabilized with 5 nm of carbon using an EM ACE600 sputter coater (Leica). Platinum-carbon replicas were peeled off from glass coverslips on 10% hydrofluoric acid and picked up onto EM grids. For UTS-EM, cells were extracted with detergent and fixed with 2.5% glutaraldehyde, 1.25% paraformaldehyde and 0.03% picric acid in 0.1 M sodium cacodylate buffer, pH 7.4, for 1 h at room temperature. Cells were then rinsed three times in 0.1 M sodium cacodylate buffer, followed by post-fixation with 1% osmium tetroxide, OsO4 and 1.5% potassium ferrocyanide, K3(Fe(CN)6) at room temperature for 1 h. Samples were dehydrated as for PR-EM, and subsequently embedded in TAAB Epon (Marivac Canada Inc) and polymerized at 60 °C for 48 h. After polymerization, ACLAR was peeled off and ultrathin sections (~50–70 nm) were cut on a Reichert ultracut S microtome (Leica), picked onto EM grids and stained with lead citrate. Samples for PR-EM and UTS-EM were examined using a JEM 1011 model TEM (JEOL) at an accelerating voltage of 80 kV. Images were captured by a CCD camera (Gatan) and presented with inverted contrast. Vesicles and membranes were colored using the brush tool at 50% opacity in Photoshop. Vesicle sizes were measured using the ruler tool in ImageJ software (NIH).

[0107] See further the experimental examples below. EXAMPLES

[0108] The following examples are provided for the purpose of illustrating various aspects of the present invention and are not intended to limit the disclosure in any way. The examples, together with the methods described herein, are representative of currently preferred embodiments and are provided by way of example only and are not intended as limitations on the scope of the present invention. Those skilled in the art will envision modifications therein and other uses encompassed within the spirit of the disclosure as defined by the scope of the claims.

[0109] Example 1 D2D3 in patients with diabetic neuropathy (DN) Increased expression of β3 integrin has been associated with DN. See Wilson et al., “The single-cell transcriptomic landscape of early human diabetic nephropathy,” Proc Natl Acad Sci USA 116: pp. 19619-19625 (2019). Therefore, we first sought to determine whether patients with DN have circulating D2D3 protein (Figure 1A).

[0110] Figure 1A-1H show that D2D3 protein discriminates between DN patients on insulin therapy and those not on insulin therapy. (Figure 1A) Schematic of human sample analysis. (Figure 1B and Figure 1C) Detection of suPAR and D2D3 protein by IP-WB in serum (Figure 1B) or urine (Figure 1C) of DN patients. Controls: recombinant human suPAR or chymotrypsin-digested suPAR corresponding to D2D3 protein. (Figure 1D) Characteristics of the study patients. (Figure 1E) Comparison between patients who were on insulin therapy or who were not on insulin therapy (area under the curve, (AUC) values). (Figure 1F) Pie chart showing the distribution (%) of D2D3-positive and D2D3-negative sera in DN patients who were on insulin therapy or who were not on insulin therapy. (Figure 1G) Scatter dot plot representing the levels of GSIS from human islets in the presence of human serum (n=3 experiments). Healthy donor islets were incubated in 10% healthy serum (HS), D2D3 positive serum (D2D3 PS, pooled from 8 patients), D2D3 negative serum (D2D3 NS, pooled from 6 patients) or D2D3 PS (D2D3 DS) immunodepleted using anti-uPAR antibody (R4). Where indicated, 10 ng / ml of recombinant hD2D3 or suPAR was added. (Figure 1H) Scatter dot plot showing activation of b3 integrin on human podocytes (cells >35) treated with serum as described in (G) except for a hD2D3 concentration of 2.5 ng / ml. Immunofluorescence analysis was performed using anti-paxillin antibody (focal adhesions) and AP5 antibody (activated b3 integrin). Figure 1G and Figure 1H, error bars, mean ± SEM (*P<0.05, **P<0.01, ***P<0.001, ****P<0.0001, unpaired t-test). ns, not significant.

[0111] Figure 5A, Figure 5B show suPAR-specific peptides in human samples. (Figure 5A) Amino acid sequence of human uPAR protein isoform 1 (SEQ ID NO:1). The different domains, domain 1 (D1), domain 2 (D2) and domain 3 (D3), are labeled. The linker and GPI anchor between D1 and D2 are underlined and labeled. The bold letters demarcate the boundaries of the suPAR-specific peptides detected by mass spectrometry, the details of which are given herein. Specifically, fragment-1 to fragment-5 are shown. *An additional fragment was generated within fragment-4. Fragment-3 spans the D2 and D3 domains and is therefore marked as 3 / 3. (Figure 5B) Coomassie gel showing proteins immunoprecipitated from serum using anti-uPAR antibody (R4). Serum samples (10 ml) from 14 DN patients who also had D2D3-like fragments in their serum were used for the experiment. The protein bands shown in the figure were excised from the gel and analyzed using mass spectrometry.

[0112] Human uPAR protein and target fragments : TIFF2025510581000002.tif71151TIFF2025510581000003.tif175152

[0113] D2D3 protein with linker :D2D3 protein lacks the D1 domain and the GPI anchor. See Barinka et al., "Structural Basis of Interaction between Urokinase-Type Plasminogen Activator and Its Receptor," (2006) Mol Biol. 363(2): pp. 482-495. TIFF2025510581000004.tif24151

[0114] The presence of D2D3 was examined by modifying immunoprecipitation (IP) coupled to Western blot (WB) analysis originally developed for cancer patients. See Sidenius et al. 2000; Mustjoki et al., “Enhanced release of soluble urokinase receptor by endothelial cells in contact with peripheral blood cells,” (2000) FEBS Lett 486: pp. 237-242. As expected, high levels of suPAR were detected in DN patient serum. Importantly, a protein similar in molecular weight to a proteolytically generated human D2D3 (hD2D3) fragment in the serum of some DN patients was identified (Figure 1B). We also detected hD2D3-like proteins in the urine of DN patients, as previously found in the urine of cancer patients (Figure 1C). See Mustjoki et al., "Soluble urokinase receptor levels correlate with number of circulating tumor cells in acute myeloid leukemia and decrease rapidly during chemotherapy," (2000) Cancer Res 60:7126-7132. However, hD2D3 was not detected in serum or urine from healthy individuals. See Figure 5A; Sidenius et al. 2000; Mustjoki et al. 2000.

[0115] To verify that our IP-WB assay was indeed detecting hD2D3 protein, we performed mass spectrometry (MS) analysis on proteins immunoprecipitated from pooled serum samples from 14 different DN patients (Figure 5B). MS identified a suPAR domain 2-specific peptide in a protein band corresponding to the molecular weight of D2D3 protein (Figure 5B).

[0116] Example 2 hD2D3 in patients with diabetic neuropathy with and without insulin therapy Diabetic neuropathy (DN) is a consequence of type 1 and type 2 diabetes. Moreover, an increasing number of adults are diagnosed with late-onset insulin-dependent diabetes, often called latent autoimmune diabetes in adults (LADA). See Pozzilli et al., “Latent Autoimmune Diabetes in Adults: Current Status and New Horizons,” (2018) Endocrinol Metab (Seoul) 33: pp. 147-159. Since type 1 diabetes and LADA in adults are highly heterogeneous and often misdiagnosed as type 2 diabetes, we focused on DN patients with or without insulin therapy and investigated whether hD2D3 is preferentially present in one of the two patient populations.

[0117] Two patient groups matched for age, sex, glycated hemoglobin A1c (HgbA1c) levels, estimated glomerular filtration rate (eGFR) and urinary albumin to creatinine ratio (MALB / Cre) were analyzed (Figure 1D). Circulating hD2D3 was present in 55% of DN patients requiring insulin therapy, in contrast to only 20% of DN patients not requiring insulin therapy (Figure 1F). In a logistic regression model adjusted for HgbA1c and suPAR, the presence of hD2D3 was associated with a 4.28-fold (95% CI [1.54-11.88]) increased odds of patients using insulin. The presence of hD2D3 protein in serum was an excellent discriminator between insulin-using and insulin-nonusing DN patients (area under the curve (AUC) = 0.674) (Figure 1E).

[0118] We then decided to measure suPAR levels in the same patient population to see if combining the levels of D2D3 and full-length suPAR could better distinguish insulin-dependent from non-dependent DN patients. We first expressed and purified recombinant hD2D3 (Figure 6A, Figure 6B) and used it as a control to determine whether currently available suPAR ELISAs could detect hD2D3 protein.

[0119] Figure 6A-6D show that recombinant hD2D3 protein binds β- 3 (Figure 6A) Phalloidin (F-actin) and AP5 (β 3 Representative images of human podocytes stained with hD2D3 (an antibody that recognizes the active form of integrin). It can be seen that serum-free medium (SFM) and healthy serum (HS) do not activate β3 integrin, but the addition of hD2D3 (2.5 ng / ml) to HS resulted in an AP5 signal. Scale bar, 5 μm. (Figure 6B) β on human podocytes grown in the presence of healthy human serum. 3 Graph showing concentration dependence of hD2D3's ability to activate integrins. 20-101 cells were analyzed per treatment. Error bars = mean ± SEM. It can be seen that at lower protein concentrations (1-~3 ng / ml), activation appears to be cooperative. (Figure 6C) Activated β 3 Scatter dot plots representing the ratio between integrin (AP5) and total focal adhesions determined by paxillin staining. Addition of hD2D3 to SFM increases the expression of β-adhesins on human podocytes. 3 It was found that hD2D3 activated β integrins on human podocytes even in the absence of any other serum proteins. 3We demonstrate that β-activating integrins are involved in the regulation of endothelial function. For experiments with SFM, 62–79 cells were analyzed. For experiments with HS, 43–46 cells were analyzed. Data are shown as mean ± SEM. Unpaired two-tailed t-tests were performed to determine statistical significance (****P<0.0001). (Figure 6D) Activated β-activating integrins on human podocytes (38–53 cells) treated with 10% healthy serum (HS), D2D3 positive serum (D2D3 PS), D2D3 negative serum (D2D3 NS) or D2D3 PS (D2D3 DS) immunodepleted using anti-uPAR antibody (R4). 3 Scatter dot plots showing integrin levels. Where indicated, 2.5 ng / ml hD2D3 was added. Data are presented relative to control (HS or SFM). All results are presented as mean ± SEM. Statistical significance was assessed using one-way ANOVA with Tukey's multiple comparisons.

[0120] Both commercial ELISAs detected recombinant hD2D3 protein in addition to full-length suPAR, suggesting that these assays report the combined levels of fragments and full-length proteins in serum when present. Using the same logistic regression model, we found that suPAR levels measured by ELISA together with D2D3 protein identified using IP-WB had better discrimination power than measuring either one of them individually (Figure 1E). Taken together, these data suggest that circulating hD2D3 may be a previously unrecognized molecular link between kidney disease and insulin-dependent diabetes.

[0121] Example 3 Role of hD2D3 in glucose-stimulated insulin secretion by β-cells It is well established that loss of β-cell function and reduced glucose-stimulated insulin secretion (GSIS) underlie the pathogenesis of insulin-dependent diabetes. Therefore, we next investigated whether the presence of hD2D3 in serum inhibits GSIS in human islets. To allow performing multiple assays using an identical set of sera, we pooled sera from eight DN patients on insulin therapy (D2D3-positive sera) and six DN patients who did not require insulin therapy (D2D3-negative sera), which contained hD2D3 protein. Compared to sera from healthy donors (HS), D2D3-positive sera impaired GSIS in human islets (Figure 1G). This phenotype was reverted after hD2D3 and suPAR were immunodepleted using anti-uPAR antibodies (D2D3 DS in the figure). Re-addition of recombinant hD2D3, but not suPAR, resulted in impaired GSIS, convincingly demonstrating a direct role for hD2D3 in blocking insulin release by β-cells.

[0122] Example 4 Role of hD2D3 in podocyte injury following insulin secretion stimulated by DN glucose by .BETA.-cells The present inventors demonstrated that suPAR mediates the expression of α v β 3 We previously showed that activating integrins induces glomerular disease. Hayek et al., “A tripartite complex of suPAR, APOL1 risk variants and α v β 3 integrin on podocytes mediates chronic kidney disease,” (2017) Nat Med 23: pp. 945-953. Using immunofluorescence (IF) microscopy, the presence of hD2D3 was detected by β 3 We tested whether hD2D3 mediates the ability of serum to activate integrins. Perhaps due to higher levels of suPAR, DN serum, compared to healthy serum (HS), showed significantly lower levels of ββ1 activation, regardless of the presence of hD2D3.3 HS activated integrins (Figure 1H). Immunodepletion of suPAR and hD2D3 abolished the activated phenotype, which was re-established upon addition of hD2D3 protein (Figure 1H). Similarly, addition of hD2D3 converted non-activated HS to activated serum in a concentration-dependent manner, resulting in a β-activated HS in serum-free medium (SFM). 3 was sufficient to induce integrin activation (Figure 6C,D). These observations implicate D2D3 in the podocyte injury that underlies DN and suggest that D2D3 can directly damage two organ-specific cells, glomerular podocytes and pancreatic β-cells.

[0123] Example 5 Role of D2D3 impairment in D2D3 transgenic mice To test the hypothesis that D2D3 can damage both glomeruli and pancreas, we generated D2D3 transgenic mice (D2D3-Tg) in which several studies were performed to verify the data. In the transgenic mouse model, the expression of Myc-tagged mouse D2D3 (mD2D3, FIG. 6B) was driven by the AP2 promoter (FIG. 2A).

[0124] Figures 2A-J show that D2D3-Tg mice exhibit glomerular damage and insulin deficiency. All animals were fed a high-fat diet (HFD) and measurements were performed on 6-month-old animals unless otherwise stated. (Figure 2A) Schematic of the D2D3-Tg construct. (Figure 2B) qPCR analysis of D2D3 and D1 in adipose tissue normalized to Gapdh (n=3 biological replicates). (Figure 2C) D2D3 detection in adipose tissue of D2D3-Tg mice using anti-Myc antibody (D2D3 is Myc-tagged). Control: tissue stained with primary or secondary antibodies only. (Figure 2D) Mouse serum suPAR and D2D3 protein levels were determined using uPAR-specific ELISA (n=8). (Figure 2E) Detection of Myc-tagged D2D3 in mouse serum. Serum was immunoprecipitated with anti-uPAR antibody and detected by WB using anti-Myc antibody. Control: recombinant Myc-tagged mD2D3 (lanes 3, 4, 7, 8; + / - N-glycanase treated). (Figure 2F) Serum IL-6 (WT, n=11; D2D3-Tg, n=7) and CPR (n=7, each group) levels. (Figure 2G) Fasting blood glucose (n=9, each group), C-peptide (n=6 for WT; n=10 for D2D3-Tg) and insulin (n=7 for WT; n=8 for D2D3-Tg) levels. (Figure 2H) In vivo GSIS. Overnight fasted animals were intraperitoneally injected with glucose (2 g / kg body weight) and their blood insulin levels were measured by ELISA (n=6-8 for each group). (Figure 2I) Scatter dot plots representing albumin creatinine ratio (ACR) or serum creatinine (n=7-10 for each group) levels. (Fig. 2J) TEM of PAS-stained kidney (left) and foot process (right). (Fig. 2C, Fig. 2J). For all results, error bars, mean ± SEM, (*P<0.05; **P<0.01; ***P<0.001. Unpaired t-test). ns, not statistically significant.

[0125] Figures 7A-C show that the ELISA detects mouse suPAR and mD2D3 proteins. (Figure 7A) Coomassie gel showing recombinant mouse suPAR and mD2D3 proteins expressed and purified from HEK-293 cells. It can be seen that the protein purification resulted in highly pure proteins. Both proteins are glycosylated and therefore migrate as broad bands. (Figure 7B) Table summarizing the MW of mouse proteins. The MW of mD2D3 detected in the plasma of D2D3-Tg mice was similar to that of the purified recombinant protein. (Figure 7C) Mouse-specific ELISA (R&D) detected full-length suPAR and mD2D3 equally well, but with lower sensitivity. BCA was used to determine the protein concentrations used in the ELISA.

[0126] D2D3-Tg animals were viable, fertile, and born in normal Mendelian ratios. RT-qPCR detected elevated mD2D3-specific mRNA levels in adipose tissue, while D1-specific mRNA levels were unchanged (Figure 2B). Immunohistochemistry using anti-Myc antibodies detected the presence of Myc-tagged mD2D3 in adipose tissue (Figure 2C). Mouse suPAR-specific ELISA detected an approximately two-fold increase in suPAR levels in the serum of transgenic animals (Figure 2D). To confirm whether mouse suPAR-specific ELISA detected both mD2D3 and suPAR in serum, we expressed and purified recombinant mouse Myc-tagged suPAR and mD2D3 as controls (Figures 7A-C). Indeed, both suPAR and mD2D3 were detected by ELISA, but with lower sensitivity (Figure 7D). To identify and quantitate the levels of mD2D3 in the circulation, IP-WB was performed to detect mD2D3 using an anti-Myc antibody. mD2D3 was detected at a concentration of 12 ± 8 ng / ml only in the serum of D2D3-Tg but not in control animals (Figure 2E).

[0127] Consistent with the presence of hD2D3 protein in serum from DN patients, D2D3-Tg animals showed higher levels of IL-6, a pro-inflammatory cytokine that is often increased in diabetic patients (Figure 2F). See Wegner et al., “Association between IL-6 concentration and diabetes-related variables in DM1 patients with and without microvascular complications,” (2013) Inflammation 36:pp.723-728. Although serum CRP or blood glucose levels were not significantly increased (Figure 2F), D2D3-Tg animals showed lower levels of insulin and C-peptide, suggesting impaired pancreatic b-cell function (Figure 2G). As observed using D2D3-positive human serum and human islets, D2D3-Tg mice showed impaired GSIS (Figure 2H). This phenotype was unique to mD2D3, as transgenic mice overexpressing full-length mouse suPAR (suPAR-Tg) (see Wei et al., “uPAR isoform 2 forms a dimer and induces severe kidney disease in mice,” (2019) J Clin Invest 129:1946-1959) did not show reduced levels of insulin (Figure 8A). These data further confirm the role of D2D3 protein in inhibiting b-cell function.

[0128] Figure 8A-I show that D2D3-Tg mice fed a normal diet do not develop glomerular damage. (Figure 8A) Scatter dot plot showing fasting blood insulin levels in WT and mice expressing mouse suPAR isoform 1 (suPAR-Tg) (n=15 for each group). (Figure 8B) Scatter dot plot comparing body weight of animals fed a normal diet and a high fat diet (HFD). It can be seen that expression of mD2D3 did not affect the body weight of animals regardless of diet. (Figure 8C) qPCR analysis of D2D3 and D1 in adipose tissue. D2D3 and D1 mRNA levels were normalized to Gapdh mRNA levels (n=3 biological replicates). The data show that mRNA for D2D3 was detected in adipose tissue even when animals were fed a normal diet. (Figure 8D) mD2D3 protein is expressed in adipose tissue of animals fed a normal diet. As D2D3-Tg carries a c-Myc tag, immunohistochemistry was performed using rabbit anti-Myc antibody. D2D3 was observed in adipocytes of D2D3-Tg mice, but not in control mice. Tissues stained with primary or secondary antibodies alone were used as negative controls. Scale bar, 50 μm. (Figure 8E) The levels of suPAR and suPAR / D2D3 increased as animals aged. Scatter dot plots representing protein levels determined using mouse-specific ELISA (n = 5–11 for each group). (Figure 8F) Body weights of WT and D2D3-Tg mice (n = 5–8 for each group). (Figure 8G) Renal function was not affected in D2D3-Tg mice when fed a normal diet. Renal function was assessed by measuring ACR, serum creatinine and serum BUN levels (n = 5–12 for each group). (FIG. 8H) Immunohistochemistry of pancreatic islets using anti-CD4 and anti-CD8 antibodies in 12-month-old animals. (FIG. 8I) Scatter dot plots comparing pancreatic weights between WT (n=5) and D2D3-Tg (n=5) and D2D3-Tg animals treated with IgG (n=6) or anti-uPAR-Ab (n=6). All results were expressed as mean ± SEM. Statistical analysis was determined using unpaired two-tailed Student's t-test.*P<0.05; **P<0.01; ***P<0.001. ns, not significant.

[0129] Alpha on human podocytes v β 3 Since we observed hD2D3-dependent activation of integrins, we investigated the kidney health of D2D3-Tg mice. Indeed, D2D3-Tg animals developed glomerular damage similar to the pattern seen in DN: foot process (FP) effacement, elevated serum creatinine, progressive microalbuminuria with glomerular hypertrophy and mesangial expansion (Figure 2I and Figure 2J). Combined with the data using human serum, these studies provide compelling evidence for a direct role of proteolytic D2D3 protein in inducing glomerular damage by activating avb3 on podocytes and insulin deficiency by impairing β-cell function.

[0130] Example 6 Role of D2D3 physiology in D2D3 transgenic mice fed a normal diet The phenotypic analysis in Figure 2 was performed on 6-month-old mice fed a high-fat diet (HFD), which allows for higher levels of mD2D3 expression. Since HFD can induce chronic low-grade systemic inflammation (see Laurentius et al., “High-fat diet-induced obesity causes an inflammatory microenvironment in the kidneys of aging Long-Evans rats,” (2019) J Inflamm (Lond) 16: p. 14), we next examined the effect of mD2D3 on animal physiology when the animals were fed a normal diet. As expected, there was a significant difference in the body weight of the animals on different diets (Figure 8B). Even on a normal diet, D2D3-Tg mice expressed mD2D3 protein (Figure 8C, Figure 8D), and suPAR / D2D3 levels increased as the animals aged and gained weight (Figure 8E, Figure 8F). Interestingly, in contrast to the DN type of glomerular injury on a HFD, D2D3-Tg mice on a normal diet did not develop a significant renal injury phenotype (Figure 8G), suggesting that renal injury requires the presence of higher levels of mD2D3 protein in the circulation or that mD2D3 protein needs to cooperate with a HFD to induce renal injury.

[0131] Example 7 The role of diet on the D2D3 pancreatic phenotype Diet affected the D2D3-mediated renal phenotype but not the mD2D3-induced pancreatic phenotype. D2D3-Tg mice fed a normal diet showed reduced levels of C-peptide and blood insulin as well as impaired in vivo GSIS even as early as 2 months of age (Fig. 3, A and B).

[0132] Figure 3A-3N show that D2D3-Tg mice exhibit insulin-dependent diabetes due to impaired pancreatic function and β-cell mass. All animals were fed a normal diet. (Figure 3A) Scatter dot plots representing fasting C-peptide and blood insulin levels (n=5-10 mice in each group) (Figure 3B). In vivo GSIS. Overnight fasted animals were intraperitoneally injected with glucose (2 g / kg body weight), and their blood insulin levels were measured by ELISA (WT, n=5; D2D3-Tg, n=8). (Figure 3C) In vitro GSIS. Pancreatic islets from 2-month-old animals (n=3) were subjected to in vitro GSIS. (Figure 3D) Glucose tolerance test (GTT). Adult (5-6 months) and aged (12 months) mice were fasted overnight and then intraperitoneally injected with glucose (2 g / kg body weight). Blood glucose was measured at the indicated time points (n = 5-6 animals per condition). (Figure 3E) Scatter dot plots representing fasting blood glucose levels (n = 8-10 mice per condition). (Figure 3F) Representative immunohistochemistry of pancreatic islets from 2-month-old mice stained with anti-CD4, anti-CD8 and anti-B220 antibodies. Spleens were used as positive controls for T-cell and B-cell staining (n = 4 mice per condition). (Figure 3G) Representative immunohistochemistry of pancreases from 2-month-old mice stained with anti-insulin antibodies (n = 5 mice per condition). (Figure 3H) Representative immunohistochemistry of pancreatic islets using anti-glucagon (α-cells) and anti-insulin (β-cells) antibodies (n = 5 mice per condition). (Figure 3I) Scatter dot plots representing β-cell mass and β-cell area / pancreas area ratio. Data were generated using the images shown in (Figure 3H). Where indicated, D2D3-Tg mice were treated with anti-uPAR-Ab or IgG isotype control (IgG) for 4 weeks starting at 2 months of age. These data were collected on 3-month-old animals (n=5 for WT and D2D3-Tg; n=6 for D2D3-Tg treated with either IgG or uPAR-Ab). Six to eight sections from each tissue were analyzed. (Figure 3J) Scatter dot plots representing the composition of islets in animals treated as described in (Figure 3I).Islet composition was determined by counting the total number of β-cells (green) and α-cells (red) and expressing them as a percentage of the total cells counted within a single islet. (Fig. 3K) Scatter dot plots representing the level of apoptosis determined by TUNEL staining or cell proliferation determined by Ki67 positive staining of mouse islets (n=5 mice per condition). Each dot represents an average of 1000-1350 insulin positive cells counted per animal. (Fig. 3L) In vivo GSIS using 2-month-old D2D3-Tg treated twice within 1 week with either IgG or anti-uPAR-Ab. All animals were male (n=7-10 animals per condition). (Fig. 3M, Fig. 3N) D2D3-Tg mice were treated with either anti-uPAR-Ab or IgG for 4 weeks starting at 2 months of age. Experiments were performed at 3 months of age. All animals were male. Scatter dot plots representing blood insulin levels are shown in (Figure 3M) (n=8 mice per condition). Glucose tolerance tests are shown in (Figure 3N) (n=6 mice per condition). For all results, error bars, mean ± SEM, (*P<0.05; **P<0.01; ***P<0.001. Unpaired t-test). ns, not statistically significant. Additionally, P values ​​are shown in red or blue, where appropriate.

[0133] Pancreatic islets isolated from D2D3-Tg mice exhibited impaired ex vivo GSIS (Figure 3C), demonstrating that the observed phenotype was intrinsic to pancreatic function. Concomitant with reduced insulin secretion, D2D3-Tg mice exhibited impaired glucose disposal capacity, as evidenced by glucose tolerance test (GTT) (Figure 3D). Finally, fasting blood glucose levels increased by 12 months of age (Figure 3E). Taken together, these data indicate that D2D3-Tg mice develop insulin-dependent diabetes.

[0134] One of the hallmarks of insulin-dependent diabetes is the decline of pancreatic β-cells, due in part to the infiltration of autoreactive CD4(+) and CD8(+) T cells. See Kelly et al., “Molecular aspects of type 1 diabetes,” (2003) Mol Pathol 56:pp.1-10. Interestingly, D2D3-Tg animals did not show a massive infiltration of CD4(+), CD8(+) T cells or B220(+) B cells at either 2 months of age (Fig. 3F) or 12 months of age (Fig. 8H), indicating the involvement of different mechanisms governing the D2D3-mediated injury. Therefore, we further investigated the islet structure and cellular distribution by staining the pancreas for insulin (a β-cell marker) and glucagon (a α-cell marker) (Fig. 3G and Fig. 3H). Although D2D3-Tg animals did not exhibit reduced total pancreatic weight (Figure 8I), surprisingly, we observed a smaller β-cell area and reduced β-cell mass (Figure 3I). Furthermore, a higher percentage of α-cell population was observed, which altered the overall composition of the islets (Figure 3J). Of note, these phenotypes were only observed in postnatal animals, as neonatal wild-type and D2D3-Tg mice showed similar distribution of pancreatic β- and α-cells (Figure 9A-C).

[0135] Figures 9A-D show that neonatal D2D3-Tg mice have normal β-cell mass. (Figure 9A) Immunohistochemistry of pancreas isolated from neonatal mice (P0) stained with anti-insulin and anti-glucagon antibodies. (Figure 9B) The stains indicated in A were used to determine the β-cell area / pancreas area ratio (n=5 for each group) and β-cell mass (n=5 for each group). (Figure 9C) Scatter dot plot showing islet composition (n=5 for each group) and pancreas weight (n=5 for each group) in neonatal mice. (Figure 9D) GSIS of MIN6 cells cultured in the presence or absence of BSA (100 ng / ml), IgG (100 ng / ml), mD2D3 protein or mouse suPAR (n=3). All results are expressed as mean ± SEM. Statistical analysis was determined using unpaired two-tailed Student's t-test. *P<0.05; **P<0.01; ***P<0.001. ns, not significant.

[0136] Given that reduced β-cell mass was only observed in post-neonatal D2D3-Tg animals, we next examined the proliferation potential of β-cells using the Ki67 marker, as well as the level of apoptosis using TUNEL staining of mouse islets. See Zhu et al., “Kindlin-2 modulates MafA and β-catenin expression to regulate β-cell function and mass in mice,” (2020) Nat Commun 11, 484. No difference was observed in the levels of apoptotic cells in the islets of wild-type and D2D3-Tg mice, but β-cell proliferation was impaired in D2D3-Tg mice (Figure 3K). Taken together, these data demonstrate that the presence of D2D3 in the circulation impairs β-cell proliferation, which results in reduced β-cell mass in postnatal D2D3-Tg mice compared to their wild-type counterparts.

[0137] Example 8 The role of circulating D2D3 on pancreatic β cells To further confirm that the observed pancreatic injury phenotype was a direct effect of D2D3, we tested whether neutralizing circulating D2D3 by anti-uPAR antibody (uPAR-Ab) could ameliorate the injury phenotype. We first treated 2-month-old condition-matched male D2D3-Tg mice twice within 1 week with either mouse anti-uPAR or IgG (control) antibody. Treatment with uPAR-Ab significantly restored islet function in D2D3-Tg mice, as demonstrated by improved in vivo GSIS (Figure 3L). We continued to treat the mice twice a week for the following 4 weeks and observed that animals treated with uPAR-Ab showed increased fasting blood insulin levels and improved glycemic excursion during glucose tolerance test in D2D3-Tg mice treated with uPAR-Ab (Figure 3M and Figure 3N). The observed positive effect of uPAR-Ab on pancreatic function was due to improved β-cell area and mass and total islet composition (Figure 3I and Figure 3J). Taken together, these data identify a novel mechanism underlying insulin-dependent diabetes mellitus in which circulating D2D3 protein directly damages pancreatic β-cells and suggest that this pathogenic mechanism may be ameliorated by blocking D2D3 protein using a specific anti-uPAR-Ab.

[0138] Example 9 Use of a mouse insulinoma cell line with pancreatic beta cell characteristics to examine the effects of D2D3 Next, we used MIN6 cells, a mouse insulinoma-derived cell line with pancreatic β-cell characteristics, to further elucidate the molecular mechanism by which D2D3 affects β-cell physiology. See Ishihara et al., “Pancreatic β-cell line MIN6 exhibits characteristics of glucose metabolism and glucose-stimulated insulin secretion similar to those of normal islets,” (1993) Diabetologia 36:1139-1145. Addition of recombinant mD2D3 impaired GSIS in MIN6 cells in a dose- and time-dependent manner (Fig. 9D), and this effect was inhibited by addition of anti-uPAR-Ab (Fig. 4A). This effect was D2D3-specific, since addition of full-length suPAR did not inhibit GSIS (Fig. 9D). The same trend was observed in isolated mouse as well as human islets (Fig. 4B, 4C). Taken together, these data demonstrate that D2D3 directly impairs the ability of β-cells to release insulin upon glucose stimulation and establish MIN6 cells as a suitable cell line to study the effects of D2D3 on insulin secretion.

[0139] Example 10 Determining which step in insulin release is inhibited by mD2D3 To clarify which step in insulin release was inhibited by mD2D3, we next examined the effect of D2D3 on the cytoskeleton using platinum replica electron microscopy (PR-EM) (Figures 10A and 10B).

[0140] Figure 10A,B shows that mD2D3 fragments impair glucose-stimulated reorganization of actin cytoskeleton in MIN6 cells. (Figure 10A) MIN6 cells were grown in low glucose (5 mM) and then stimulated by addition of high glucose (20 mM) in the presence or absence of mD2D3 protein (100 ng / ml). F-actin status was examined by phalloidin staining. (Figure 10B) Representative PR-EM micrographs of MIN6 cells, focusing on the organization and status of actin cytoskeleton. Cells were grown as described in (Figure 10A). It can be seen that mD2D3 inhibited high glucose-induced disassembly of actin filaments.

[0141] It is well documented that high glucose leads to microtubule (MT) polymerization, which aids in the transport of secretory vesicles from the cell periphery to the plasma membrane, and concomitant depolymerization of the actin network. See Wang et al., “Mechanisms of biphasic insulin-granule exocytosis-roles of the cytoskeleton, small GTPases and SNARE proteins,” (2009) J Cell Sci 122: pp. 893-903. Limited analysis suggests that mD2D3 inhibited both processes: impaired MT polymerization and inhibited actin network depolymerization (Figure 4D).

[0142] Because dysregulated cytoskeletal dynamics are expected to have profound effects on the trafficking and maturation of insulin-transporting dense-core vesicles (LDCVs) in β-cells, we next visualized LDCVs using transmission electron microscopy (TEM) (Figures 11A-C).

[0143] 11A-11C show that mD2D3 impairs insulin granule maturation and trafficking. Representative TEM micrographs of MIN6 cells. Where indicated, cells were grown in low glucose (5 mM) and then stimulated by addition of high glucose (5 mM) in the presence or absence of mD2D3 protein (100 ng / ml) for 24 h. Images shown were taken at increasing magnification. The cell membrane is colored pink and the insulin-transporting dense-core vesicles (LDCVs) are colored yellow. In MIN6 cells grown in the presence of mD2D3 protein, LDCVs are smaller in size and less associated with the cell membrane.

[0144] As seen in β-cells, glucose stimulation increased the diameter of LDCVs (vesicle maturation) and the number of membrane-bound LDCVs (vesicle trafficking) (Figure 4E-G). See Ferri et al., “Insulin secretory granules labeled with phogrin-fluorescent proteins show alterations in size, mobility and responsiveness to glucose stimulation in living β-cells,” (2019) Sci Rep 9: p. 2890; Zhang et al., “Visualizing insulin vesicle neighborhoods in β-cells by cryo-electron tomography,” (2020) Sci Adv 6. Addition of mD2D3 impaired both of these processes, demonstrating that mD2D3 protein impairs multiple steps of insulin secretion by dysregulating glucose-induced cytoskeletal dynamics.

[0145] Example 11 Effects of mD2D3 on glycolysis and mitochondrial respiration in MIN6 cells upon glucose stimulation Since insulin secretion is also directly linked to glycolysis and oxidative metabolism (32, 33), we investigated whether mD2D3 affects glycolysis and mitochondrial respiration in MIN6 cells upon glucose stimulation. See Rutter et al., “Pancreatic β-cell identity, glucose sensing and the control of insulin secretion,” (2015) Biochem J 466: pp. 203-218; Nicholls, “The Pancreatic β-Cell: A Bioenergetic Perspective,” (2016) Physiol Rev 96: pp. 1385-1447. To this end, we used a Seahorse XFe24 extracellular flux analyzer to measure extracellular acidification (ECAR) and oxygen consumption rate (OCR) in real time in MIN6 cells under basal conditions and in response to sequential injections of glycolysis or mitochondrial inhibitors. To mimic glucose stimulation, cells were grown in low glucose (2.8 mM) for 24 h in the presence of increasing concentrations of D2D3, then switched to high glucose (20 mM) and the experiment was initiated (Figure 4H). The presence of mD2D3 reduced glycolysis, basal and non-glycolytic acidification (Figure 4I and Figure 4J). Furthermore, mD2D3 reduced several bioenergetic parameters, including basal and maximal respiration, spare respiratory capacity and ATP production (Figure 4K and Figure 4L). The inability to increase OCR in response to glucose would be expected to impair insulin granule maturation and trafficking, as well as the proliferative potential of β-cells. Thus, these data identify a novel molecular mechanism by which D2D3, a circulating pathogenic factor, alters glucose-dependent β-cell physiology, including glycolysis, OCR, insulin granule maturation and trafficking, cytoskeleton dynamics, and insulin secretion, ultimately impairing β-cell proliferation in the postnatal period.

[0146] summary Of the US population, 34.2 million people of all ages have diabetes. Although type 2 is the most common form of diabetes, an increasing number of adults are being diagnosed with either type 1 diabetes or latent autoimmune diabetes of adults (LADA). Initially, it was suggested that the main difference between type 1 diabetes and LADA is the age at which the disease manifests, with type 1 diabetes typically occurring in childhood, whereas LADA is diagnosed in adulthood. Recent data suggest that the main difference is not age, but the rate at which these diseases progress, thus implying different molecular mechanisms for each disease. See Laugesen et al., “Danish Diabetes Academy, S.Workshop,Latent autoimmune diabetes of the adult: current knowledge and uncertainty,” (2015) Diabet Med 32: pp. 843-852; Jorns et al., “Pancreas Pathology of Latent Autoimmune Diabetes in Adults (LADA) in Patients and in a Rat Model Compared With Type 1 Diabetes,” (2020) Diabetes 69: pp. 624-633. Despite these differences, loss of pancreatic β cells is a central feature of diabetes, regardless of its etiology. Recently, signaling by the cell death receptor TMEM219 expressed on β cells through its circulating ligand insulin-like growth factor binding protein 3 (IGFBP3) has been implicated in the loss of β cells through the induction of apoptosis. See D'Addio et al., “The IGFBP3 / TMEM219 pathway regulates β-cell homeostasis,” (2022) Nat Commun 13:p.684. It has been suggested that blocking the IGFBP3 / TMEM219 signaling pathway may present a novel therapeutic option for both type 1 and type 2 diabetes.This study identifies a novel molecular mechanism of diabetes whereby a distinct circulating protein, the D2D3 protein of suPAR, causes damage to β-cells by impairing insulin secretion and β-cell proliferation in the postnatal period through inhibition of glucose-dependent cellular metabolism.

[0147] Moreover, previous studies have pointed to a β-cell-specific mechanism of injury. Id.; see Levitsky et al., “Role of growth factors in control of pancreatic β-cell mass: focus on betatrophin,” (2014) Curr Opin Pediatr 26: pp. 475-479. In contrast, the present study shows that D2D3 protein can simultaneously damage two organs. Treatment with uPAR-Ab attenuated D2D3-driven pancreatic injury in in vivo and ex vivo mouse models, and immunodepletion of hD2D3 from DN patient serum mitigated the pathogenic phenotype in human podocytes and pancreatic islets. Thus, D2D3-specific antibodies offer a unique dual therapeutic option for chronic kidney disease and insulin-dependent diabetes.

[0148] D2D3 protein is generated by proteolysis of uPAR, an effector of the innate immune system since it is expressed by macrophages, neutrophils and immature myeloid cells (see Figure 12). See Hahm et al., 2017. Thus, this study identifies a novel mechanism by which chronic inflammation leads to autoimmune diabetes mellitus. In light of the central role of suPAR as a major risk factor in COVID-19-associated kidney injury and adverse outcomes in diabetes mellitus patients, this study warrants attention to the increased emergence of innate autoimmune diabetes in the post-COVID era worldwide. Rovina et al., “Soluble urokinase plasminogen activator receptor(suPAR)as an early predictor of severe respiratory failure in patients with COVID-19 pneumonia,”(2020)Crit Care 24:p.187;Azam et al.,“Soluble Urokinase Receptor(SuPAR)in COVID-19-Related AKI,”J Am Soc Nephrol 31:pp.2725-2735; Vasbinder et al., “Inflammation, Hyperglycemia, and Adverse Outcomes in Individuals with Diabetes Mellitus Hospitalized for COVID-19,” (2022) Diabetes Care 45:pp.692-700.

[0149] As will be understood from the description herein, a wide variety of aspects and embodiments are contemplated by the present disclosure, examples of which include, but are not limited to, the aspects and embodiments disclosed herein.

[0150] Although embodiments of the present disclosure have been described herein, those skilled in the art should understand 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 present invention. It should be understood that various alternatives to the embodiments of the present invention described herein can be used in implementing the present invention. It is intended that the following claims define the scope of the present invention, and that methods and structures within the scope of these claims and their equivalents are covered thereby.

Claims

1. A pharmaceutical product for use in a method of treating chronic kidney disease in a subject, The aforementioned disease is characterized by the presence of detectable levels of urokinase plasminogen activator receptor (uPAR) protein D2D3, The pharmaceutical product comprises a therapeutically effective amount of an active substance that antagonizes and / or removes the D2D3 protein from the circulation of the target, The aforementioned method, (i) A step of detecting the presence of D2D3 protein in a biological sample derived from the subject, (ii) The step of administering a therapeutically effective amount of the active substance to the subject. Pharmaceuticals, including

2. The pharmaceutical product according to claim 1, wherein the biological sample is selected from serum, plasma, saliva, and urine.

3. The pharmaceutical product according to claim 1, wherein the active substance comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein.

4. The pharmaceutical product according to claim 3, wherein the anti-D2D3 antibody or its antigen-binding fragment is humanized.

5. The pharmaceutical product according to claim 3, wherein the anti-D2D3 antibody or its antigen-binding fragment is a monoclonal antibody.

6. The pharmaceutical product according to any one of claims 1 to 5, wherein the method further comprises administering an antisoluble urokinase plasminogen activator receptor (suPAR) antibody or an antigen-binding fragment thereof.

7. A pharmaceutical product for use in a method of treating insulin-dependent diabetes in a subject, The aforementioned insulin-dependent diabetes is characterized by the presence of detectable levels of urokinase plasminogen activator receptor (uPAR) protein D2D3, The pharmaceutical product comprises a therapeutically effective amount of an active substance that antagonizes and / or removes the D2D3 protein from the circulation of the target, The aforementioned method, (i) A step of detecting the presence of D2D3 protein in a biological sample derived from the subject, (ii) The step of administering a therapeutically effective amount of the active substance to the subject. Pharmaceuticals, including

8. The pharmaceutical product according to claim 7, wherein the biological sample is selected from serum, plasma, saliva, and urine.

9. The pharmaceutical product according to claim 7, wherein the active substance comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein.

10. The pharmaceutical product according to claim 9, wherein the anti-D2D3 antibody or its antigen-binding fragment is humanized.

11. The pharmaceutical product according to claim 9, wherein the anti-D2D3 antibody or its antigen-binding fragment is a monoclonal antibody.

12. The pharmaceutical product according to any one of claims 7 to 11, wherein the method further comprises administering an antisoluble urokinase plasminogen activator receptor (suPAR) antibody or an antigen-binding fragment thereof.

13. A pharmaceutical product for use in a method of treating diabetic neuropathy in a subject, The aforementioned diabetic neuropathy is characterized by the presence of detectable levels of urokinase plasminogen activator receptor (uPAR) protein D2D3, The pharmaceutical product comprises a therapeutically effective amount of an active substance that antagonizes and / or removes the D2D3 protein from the circulation of the target, The aforementioned method, (i) A step of detecting the presence of D2D3 protein in a biological sample derived from the subject, (ii) The step of administering a therapeutically effective amount of the active substance to the subject. Pharmaceuticals, including

14. The pharmaceutical product according to claim 13, wherein the biological sample is selected from serum, plasma, saliva, and urine.

15. The pharmaceutical product according to claim 13, wherein the active substance comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein.

16. The pharmaceutical product according to claim 15, wherein the anti-D2D3 antibody or its antigen-binding fragment is humanized.

17. The pharmaceutical product according to claim 15, wherein the anti-D2D3 antibody or its antigen-binding fragment is a monoclonal antibody.

18. The pharmaceutical product according to any one of claims 13 to 17, wherein the method further comprises administering an antisoluble urokinase plasminogen activator receptor (suPAR) antibody or an antigen-binding fragment thereof.

19. A pharmaceutical product for use in a method for restoring the number and function of pancreatic β-cells in the pancreas of a person diagnosed with insulin-dependent diabetes, The aforementioned insulin-dependent diabetes is characterized by the presence of detectable levels of urokinase plasminogen activator receptor (uPAR) protein D2D3, The pharmaceutical product comprises a therapeutically effective amount of an active substance that antagonizes and / or removes the D2D3 protein from the circulation of the target, The aforementioned method, (i) A step of detecting the presence of D2D3 protein in a biological sample derived from the subject, (ii) The step of administering a therapeutically effective amount of the active substance to the subject. Pharmaceuticals, including

20. The pharmaceutical product according to claim 19, wherein the biological sample is selected from serum, plasma, saliva, and urine.

21. The pharmaceutical product according to claim 19, wherein the active substance comprises an anti-D2D3 antibody or an antigen-binding fragment thereof that specifically binds to the D2D3 protein.

22. The pharmaceutical product according to claim 21, wherein the anti-D2D3 antibody or its antigen-binding fragment is humanized.

23. The pharmaceutical product according to claim 21, wherein the anti-D2D3 antibody or its antigen-binding fragment is a monoclonal antibody.

24. The pharmaceutical product according to any one of claims 19 to 23, wherein the method further comprises administering an antisoluble urokinase plasminogen activator receptor (suPAR) antibody or an antigen-binding fragment thereof.

25. The pharmaceutical product according to any one of claims 19 to 23, wherein the function of pancreatic β-cells is determined by glucose-stimulated insulin release.

26. A composition comprising a substance that antagonizes the urokinase plasminogen activator receptor (uPAR) protein D2D3 in a therapeutically effective amount for treating diseases characterized by the presence of D2D3.

27. The pharmaceutical product according to any one of claims 3 to 5, 9 to 11, 15 to 17, and 21 to 23, wherein the anti-D2D3 antibody or its antigen-binding fragment binds to a certain position in any one of SEQ ID NO:1, SEQ ID NO:3, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:10, SEQ ID NO:11, and SEQ ID NO:12.

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