Methods for treating diabetic kidney disease and glomerular disease

Abstract

Methods are provided for delaying onset or progression of kidney damage, or treating kidney disease, in a subject who has type 1 diabetes or type 2 diabetes or glomerular disease. The methods comprise administering to a subject with type 1 or type 2 diabetes or glomerular disease an effective amount of an agent capable of inhibiting NBL1 activity, and in particular, capable of inhibiting NBL1-mediated toxicity of human podocytes. In some embodiments, the agent is an antibody capable of binding to human NBL1. Monoclonal anti-NBL1 antibodies are also provided.

Description

METHODS FOR TREATING DIABETIC KIDNEY DISEASE AND GLOMERULAR DISEASE1. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 675,702, filed July 25, 2024, the disclosure of which is incorporated herein by reference in its entirety2. SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing and is hereby incorporated by reference in its entirety. Said XML copy, created on July 14, 2025, is named 63694WO_sequencelisting.xml, and is 601,375 bytes in size.3. BACKGROUND OF THE INVENTION

[0003] Diabetes mellitus type 1 (T1D) and type 2 (T2D) collectively affect hundreds of millions of people worldwide, and prevalence is increasing. Diabetes is characterized by hyperglycemia and complications that greatly impact patient quality and duration of life.

[0004] There is no current cure for T1D or T2D. Most therapies help patients manage the symptoms to a certain extent, but diabetics still face multiple long-term health complications. Among these complications is kidney damage, which can progress to end-stage renal disease (ESRD). Diabetes was the primary cause of kidney failure in 44% of all new cases in 2011. Given the prevalence and severity of complications associated with diabetes, in particular kidney disease and its progression, there is a need for therapeutic agents that delay onset and progression of kidney disease in diabetes.

[0005] Kidney damage also has other etiologies. There is a need for therapeutic agents that delay onset and progression of kidney disease caused by disorders other than diabetes. There is a particular need for therapeutic agents that treat glomerular disorders.4. SUMMARY OF THE INVENTION

[0006] As detailed in the experimental examples in this disclosure, we have discovered that the protein, neuroblastoma suppressor of tumorgenicity 1 (NBL1), is directly toxic to renal cells, including podocytes and tubular cells. The toxic effect is not mediated through inhibition of renal BMP proteins; we show that BMPs are not expressed in and are not secreted by kidney cells. Moreover, we have discovered that NBL1 is primarily secreted byintestinal cells. Neutralizing NBL1 with an antagonist, either soluble BMP2 or anti-NBLl monoclonal antibodies, prevents toxicity in vitro. Further, we have demonstrated that NBL1 is elevated in Type 1 Diabetes and Type 2 Diabetes, and that in streptozotocin (STZ)-induced diabetic mice as well as leptin receptor deficient mice (Db / Db), administration of anti-NBLl antibodies markedly reduce NBL1 -induced podocyte death. We have further demonstrated that anti-NBLl antibodies rescued the expression of podocyte markers in human kidney organoids.

[0007] In addition, , when administered in vivo, anti-NBLl antibodies prevented the increase of albumin and creatinine in urine commonly observed in diabetic mice. Moreover, NBL1 blockade reduced mesangial expansion, as observed in PAS-stained kidney sections. Finally, anti-NBLl ameliorated fibrosis and cell death by reducing expression of specific markers.

[0008] Inhibition of NBL1 is therefore a new therapeutic approach for preventing onset and progression of kidney damage caused by circulating NBL1, and in particular, in a patient with type 1 or type 2 diabetes. Inhibition of NBL1 will also be effective in treating non-diabetes glomerular diseases in which damage is mediated by NBL1.

[0009] Accordingly, in a first aspect, methods are provided for delaying onset or progression of kidney damage in a subject who has type 1 diabetes or type 2 diabetes or a glomerular disease. The method comprises administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity, in particular, capable of inhibiting NBL1 toxicity on human podocytes. In some embodiments, the method prevents onset of or slows decline in kidney function.

[0010] In a further aspect, methods are provided for slowing decline in kidney function in a subject who has type 1 or type 2 diabetes or a glomerular disease. The method comprises administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity, in particular, capable of inhibiting NBL1 toxicity on human podocytes.

[0011] In a further aspect, methods are provided for treating diabetic kidney disease (DKD) in a subject who has type 1 or type 2 diabetes or a glomerular disease. The method comprises administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity, in particular, capable of inhibiting NBL1 toxicity on human podocytes.

[0012] In some embodiments of these methods, the agent is capable of binding to NBL1. In some embodiments the agent is capable of binding to human NBL1.

[0013] In some embodiments the agent is an antibody, or an antigen binding fragment of an antibody, that is capable of binding to human NBL1.

[0014] In some embodiments the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs having sequences selected from: a) SEQ ID NOs: 3, 8, and 13 and SEQ ID NOs: 18, 23, and 28 (antibody E05a); b) SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53, and 58 (antibody H08); c) SED ID NOs: 63, 68, and 73 and SED ID NOs: 78, 83, and 88 (antibody F06); d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibodyA12); e) SED ID NOs: 123, 128, and 133 and SED ID NOs: 138, 143, and 148 (antibody G01); f) SED ID NOs: 153, 158, and 163 and SED ID NOs: 168, 173 and 178 (antibody El l); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); h) SED ID NOs: 213, 218, and 223 and SED ID NOs: 228, 233, and 238 (antibody DI 2); i) SED ID NOs: 243, 248, and 253 and SED ID NOs: 258, 263 and 268 (antibody H01); j) SED ID NOs: 273, 278, and 283 and SED ID NOs: 288, 293, and 298 (antibody Cl l); k) SED ID NOs: 303, 308, and 313 and SED ID NOs: 318, 323, and 328 (antibody E05b); l) SED ID NOs:333, 338, and 343 and SED ID NOs: 348, 353 and 358 (antibody F10); m) SED ID NOs: 363, 368 and 373 and SED ID NOs: 378, 383 and 388 (antibody G10); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04);o) SED ID NOs: 423, 428, and 433 and SED ID NOs: 438, 443, and 448 (antibody E07); p) SED ID NOs: 453, 458, and 463 and SED ID NOs: 468, 473, and 478 (antibody El 2); q) SED ID NOs: 483, 488, and 493 and SED ID NOs: 498, and 503, and 508 (antibody D08); r) SED ID NOs: 513, 518, and 523 and SED ID NOs: 528, 533, and 538 (antibody E10); s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SED ID NOs: 573, 578, and 583 and SED ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the said sets of CDR sequences (a) - (t) by at most two conservative amino acid changes in each CDR.

[0015] In some embodiments of the methods, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs with sequences identical to the said CDRs. In some embodiments the three heavy chain CDRs and three light chain CDRs have sequences selected from:SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53 and 58 (antibody H08); d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibody A12); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04); s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SEQ ID NOs: 573, 578, and 583 and SEQ ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the said sets of CDR sequences b), d), g), n) s), and t) by at most two conservative amino acid changes in each CDR.

[0016] In some embodiments the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs with sequences identical to the said CDRs.

[0017] In some embodiments the antibody framework regions are human antibody framework regions.

[0018] In some embodiments the antibody is a full length bivalent monospecific monoclonal antibody. In some embodiments the antibody comprises human IgGl, IgG2, or IgG4 heavy chain constant regions. In some embodiments the antibody comprises a human IgGl constant region. In some embodiments the antibody Fc region has engineered mutations that reduce antibody binding to at least one type of Fc receptor and / or reduce complement fixation. In some embodiments the antibody is a Fab, optionally wherein the Fab is PEGylated. In some embodiments the antibody or antigen binding fragment is further capable of binding to cynomolgus monkey NBL1. In some embodiments the antibody or antigen binding fragment is further capable of binding to mouse NBL1.

[0019] In some embodiments the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 100 nM. In some embodiments the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 10 nM. In some embodiments the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 5 nM. In some embodiments the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 1 nM.

[0020] In some embodiments, the agent comprises a bone morphogenetic protein (BMP) or soluble fragment thereof. In some embodiments the agent comprises a soluble fragment of human BMP-2. In some particular embodiments the agent further comprises a moiety that extends serum half-life. In some embodiments the half-life extension moiety is an antibody Fc domain. In some embodiments the half-life extension moiety is at least one covalently linked polyethylene glycol (PEG) moiety.

[0021] In some embodiments the agent is capable of inhibiting dimerization of NBL1.

[0022] In some embodiments the agent is capable of inhibiting NBL1 expression.

[0023] In some embodiments, the agent is administered parenterally. In some embodiments the agent is administered intravenously. In some embodiments the agent is administeredsubcutaneously. In some embodiments the agent is administered for at least 3 months. In some embodiments the agent is administered for at least 6 months. In some embodiments the agent is administered for at least 12 months.

[0024] In some embodiments the subject has elevated pre-treatment plasma levels of NBL1. In some embodiments the subject has type 1 diabetes. In some embodiments the subject has type 2 diabetes. In some embodiments the subject has a glomerular disease. In some embodiments the subject with glomerular disease does not have type 1 diabetes or type 2 diabetes. In some embodiments the glomerular disease is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), chronic glomerulopathies, hereditary nephritis, and minimal change disease.

[0025] In another aspect, antibodies or antigen binding fragments capable of binding to NBL1 and inhibiting NBL1 -induced toxicity of human podocytes are provided.

[0026] In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs having sequences selected from: a) SEQ ID NOs: 3, 8, and 13 and SEQ ID NOs: 18, 23, and 28 (antibody E05a); b) SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53, and 58 (antibody H08); c) SED ID NOs: 63, 68, and 73 and SED ID NOs: 78, 83, and 88 (antibody F06); d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibodyA12); e) SED ID NOs: 123, 128, and 133 and SED ID NOs: 138, 143, and 148 (antibody G01); f) SED ID NOs: 153, 158, and 163 and SED ID NOs: 168, 173 and 178 (antibody El l); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); h) SED ID NOs: 213, 218, and 223 and SED ID NOs: 228, 233, and 238 (antibody DI 2); i) SED ID NOs: 243, 248, and 253 and SED ID NOs: 258, 263 and 268 (antibody H01); j) SED ID NOs: 273, 278, and 283 and SED ID NOs: 288, 293, and 298 (antibody Cl 1);k) SED ID NOs: 303, 308, and 313 and SED ID NOs: 318, 323, and 328 (antibody E05b); l) SED ID NOs:333, 338, and 343 and SED ID NOs: 348, 353 and 358 (antibody F10); m) SED ID NOs: 363, 368 and 373 and SED ID NOs: 378, 383 and 388 (antibody GIO); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04); o) SED ID NOs: 423, 428, and 433 and SED ID NOs: 438, 443, and 448 (antibody E07); p) SED ID NOs: 453, 458, and 463 and SED ID NOs: 468, 473, and 478 (antibody El 2); q) SED ID NOs: 483, 488, and 493 and SED ID NOs: 498, and 503, and 508 (antibody D08); r) SED ID NOs: 513, 518, and 523 and SED ID NOs: 528, 533, and 538 (antibody E10); s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SED ID NOs: 573, 578, and 583 and SED ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the CDR sequences selected from (a) - (t) by at most two conservative amino acid substitutions in each CDR. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs with sequences identical to one of the said sets of CDRs (a)-(t).

[0027] In some embodiments, the three heavy chain CDRs and three light chain CDRs have sequences selected from:SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53 and 58 (antibody H08); d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibody A12); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04);s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SEQ ID NOs: 573, 578, and 583 and SEQ ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the said CDR sequences b), d), g), n) s), and t) by at most two conservative amino acid substitutions in each CDR. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs with sequences identical to the selected CDRs b), d), g), n), s), or t).

[0028] In some embodiments, the antibody or antigen-binding fragment framework regions are human antibody framework regions.

[0029] In some embodiments, the antibody or antigen binding fragment is a full length bivalent monospecific monoclonal antibody. In some embodiments, the antibody comprises human IgGl, IgG2, or IgG4 heavy chain constant regions. In some embodiments, the antibody comprises a human IgGl constant region. In some embodiments, the antibody Fc region has engineered mutations that reduce antibody binding to FcRy and / or reduce complement fixation.

[0030] In some embodiments, the antigen binding fragment is a Fab, optionally wherein the Fab is PEGylated.

[0031] In some embodiments, the antibody or antigen binding fragment is further capable of binding to cynomolgus monkey NBL1. In some embodiments, the antibody or antigen binding fragment is further capable of binding to mouse NBL1.

[0032] In some embodiments, the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 100 nM, 10 nM, 5 nM or 1 nM.

[0033] In another aspect, pharmaceutical compositions are provided. The compositions comprise the anti-NBLl antibody or antigen binding fragment, and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for parenteral administration. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for subcutaneous administration.5. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] These and other features, aspects, and advantages of the present invention will become better understood with regard to the following description, and accompanying drawings, where:

[0035] FIGs. 1A-1D are bar graphs summarizing cell death analysis in human kidney cells. FIG. 1 A shows data from human podocytes (HuPodo) cultured with escalating doses of recombinant human NBL1, from 0.2 pg / ml to 2 pg / ml. FIG. IB shows data from human mesangial cells (HuMRC) cultured in the presence of 2 pg / ml recombinant human NBL1. FIG. 1C shows data from human renal tubular cells (HuK2) cultured in the presence of 2 pg / ml recombinant human NBL1. FIG. ID shows data from control human umbilical vein endothelial cells (Huvec) cultured in the presence of 2 pg / ml recombinant human NBL1.

[0036] FIGs. 2A-2B depict representative images of confocal analysis conducted on human podocytes cultured with NBL1 at 2 pg / ml (FIG. 2A) or left untreated (FIG. 2B). The podocytes were stained with Apoptag, a marker for apoptosis, and Synaptopodin, a marker for podocytes. FIG. 2C is a bar graph quantifying the percentage of human podocytes that are double positive for Apoptag and Synaptopodin (Syn+Apo+). The experiment demonstrates NBLl-induced apoptosis of differentiated human podocytes (arrows in FIG. 2B, quantified in FIG. 2C).

[0037] FIG. 3 depicts changes in levels of apoptosis-related transcripts in human podocytes cultured with NBL1 at 2 pg / ml as compared to untreated cells.

[0038] FIG. 4 is a bar graph quantifying expression of NBL1 mRNA in human immune cells and cell lines. mRNA levels were normalized to beta-actin level.

[0039] FIGs. 5A-5B depict the high expression of NBL1 protein in T cells and various T cell subsets (CD3+, CD4+and CD8+T cells) and in myeloid cells (CD14+) using flow-cytometry analysis. FIG. 5 A depicts physical gating on monocytes (upper gate) and lymphocytes (lower gate). All analysis was conducted on peripheral blood mononuclear cells (PBMCs) isolated from blood samples of healthy volunteers. FIG. 5B is a bar graph showing percentage of NBL1 positive cells in cells having the indicated CD surface markers. FIGs. 5C-5D depict the results of an NBL1 Atlas study surveying NBL1 expression in various human tissues by immunohistochemistry. FIG. 5C shows images of various tissue samples illustrating thepresence of NBL1 through immunohistochemistry techniques. FIG. 5D is a bar graphing showing high expression of NBL1 protein in intestinal and muscular tissues.

[0040] FIG. 6 depicts the neutralizing effect of soluble BMP2 on NBL1 -mediated apoptosis. Human podocytes were cultured in the presence of NBL1 (2 pg / ml) and in the presence / absence of soluble BMP2 (1 pg / ml) for 48 hours.

[0041] FIGs. 7A-7B depict increased NBL1 serum levels measured by ELISA, with FIG. 7A comparing levels in patients with long-standing type 1 diabetes (T1D), type 2 diabetes (T2D), and non-diabetic subjects (CTRL), with FIG. 7B showing NBL1 serum levels in patients with diabetic kidney disease (DKD) stage 2-3 compared to those without DKD.

[0042] FIG. 8 is a bar graph displaying quantified cell death in human podocytes cultured in the presence of NBL1 (2 pg / ml) in the presence and absence of either anti-NBLl antibodies (20 pg / ml) or soluble BMP2 (1 pg / ml). The left-most bar shows data from cells incubated in medium alone, without NBL1, and without treatment with antibody or sBMP.

[0043] FIG. 9 is a bar graph summarizing caspase 3 and caspase 7 activation analysis in human podocytes cultured with recombinant human NBL1 at a concentration of 2 pg / ml in the presence and absence of either anti-NBLl antibodies (20 pg / ml) or soluble BMP2 (1 pg / ml). The left-most bar shows data from cells incubated in medium alone, without NBL1, and without treatment with antibody or sBMP.

[0044] FIGs. 10A-10B depict anti-NBLl monoclonal antibody in vitro test results for antibodies not previously presented in FIG 8. FIG. 10A is a bar graph summarizing caspase 3 and caspase 7 activation analysis in human podocytes cultured with recombinant human NBL1 at a concentration of 2 pg / ml in the presence and absence of either anti-NBLl antibodies (20 pg / ml) or soluble BMP2 (1 pg / ml). The left-most bar shows data from cells incubated in medium alone, without NBL1, and without treatment with antibody or sBMP. FIG 10B is a bar graph summarizing cell death analysis in human podocytes cultured with recombinant NBL1 at a concentration of 2 pg / ml in the presence and absence of either anti- NBLl antibodies (20 pg / ml) or soluble BMP2 (1 pg / ml). The left-most bar shows data from cells incubated in medium alone, without NBL1, and without treatment with antibody or sBMP.

[0045] FIGs. 11A-11B depict assays that assessed binding of various monoclonal anti-NBLl antibodies to native murine NBL1 found in murine plasma. FIG. 11 A illustrates the sandwich ELISA binding assay. FIG 1 IB is a bar graph quantifying the binding of various anti-NBLl antibodies to native murine plasma NBL1.

[0046] FIGs. 12A-12B depict assays that assessed binding of various monoclonal anti-NBLl antibodies to native murine NBL1 present in murine splenocytes. FIG. 12A illustrates the steps performed in the binding assay. FIG. 12B is a bar graph quantifying binding of various anti-NBLl antibodies to native murine NBL1 derived from murine splenocytes.

[0047] FIGs. 13A-13B depict assays that assessed binding of various monoclonal anti-NBLl antibodies to native human NBL1 found in human peripheral blood mononuclear cells (PBMCs). FIG. 13 A illustrates the steps performed in the binding assay. FIG 13B is a bar graph quantifying binding of various anti-NBLl antibodies bound to native human NBL1.

[0048] FIGs. 14A-14B summarize apoptotic assays performed using two isoforms of NBL1. FIG. 14A shows a full-length NBL1 protein (21KDa) and a c-terminus truncated NBL1 protein (19KDa). FIG. 14B are bar graphs quantifying cell death in human podocytes cultured in the presence of commercially available NBL1, full-length recombinant NBL1, and truncated NBL1 (2 pg / ml). The left-most bar shows data from cells incubated in medium alone, without NBL 1.

[0049] FIGs. 15A-15C are bar graphs monitoring the relative expression or detection of apoptotic transcripts in human podocytes cultured with NBL1 at 2 pg / ml, as compared to untreated control cells. FIG. 15A depicts the relative change in death-associated protein kinase (DAPK1). FIG. 15B depicts the relative change in FAS-associated death domain protein (FADD). FIG.15C depicts the relative change in TNF-receptor superfamily member 10A (TNFRSF10A).

[0050] FIGs. 16A-16C depict processes and expression of proapoptotic transcripts in kidney organoids. FIG. 16A illustrates the timeline of steps performed. FIG. 16B is a microscopic image of immature embryonic stem cells at day 0 and at day 17. FIG. 16C summarizes the quantification of relative mRNA expression of apoptotic markers in kidney organoids cultured in the presence or absence of NBL1.

[0051] FIGs. 17A-17F are bar graphs of the expression of proapoptotic and damage markers in kidney organoids cultured in the presence or absence of NBL1 for 48 hours, measured at day 14 (NBL1 added at day 12+48h). FIG. 17A is a bar graph of marker COL4A1 gene. FIG. 17B is a bar graph of marker GATM gene. FIG. 17C is a bar graph of marker N0X4 gene. FIG. 17D is a bar graph of marker TGFB1 gene. FIG. 17E is a bar graph of marker DAPK1 gene. FIG. 17F is a bar graph of marker FADD gene.

[0052] FIGs. 18A-18B depict assay methodology and expression of proapoptotic transcripts in kidney organoids. FIG. 18A illustrates the steps performed. FIG. 18B summarizes the quantification of change in expression of nephrotoxic markers in human podocytes cultured in NBL1 at a concentration of 2pg / ml, or NBL1 plus soluble BMP2 at Ipg / ml, versus control.

[0053] FIGs. 19A-19B illustrate two protocols for in vivo studies of acute nephrotoxicity of HuNBLl in C57BL6 mice. FIG. 19A illustrates acute protocol 1 where 500 pg of recombinant human NBL1 was injected into C57 / BL6 mice once daily for 3 days. FIG. 19B illustrates acute protocol 2 where 500 pg of recombinant human NBL1 was injected into C57 / BL6 mice once daily for 4 days.

[0054] FIGs. 20A-20C summarize urinary albumin and creatinine assessments from the two different protocols illustrated in FIG. 19, comparing effects of HuNBLl injected in vivo as compared to untreated C57BL6 mice. FIG. 20A summarizes the results from acute protocol 1. FIG. 20B summarizes the results from acute protocol 2 at T2. FIG. 20C summarizes results from acute protocol 2 at T3.

[0055] FIG. 21 depicts urinary volume over 12 hours measured in C57BL6 mouse following injection of HuNBLl at week 4 as compared to untreated animals.

[0056] FIGs. 22A-22B summarize analysis of glomerular pathology displayed following acute protocol 1. FIG. 22A is a microscopic image showing deposition of amorphous material within the glomeruli as compared to untreated animals. Magnification 20X. FIG. 22B is a bar graph quantifying abnormalities in matrix deposition.

[0057] FIG. 23 is a schematic of the protocol using an in vivo model of huNBLl -induced kidney damage and administration of anti-NBLl mAbs in C57 / BL6 mice.

[0058] FIGs. 24A-24B are line graphs illustrating the effect of anti-NBLl monoclonal antibodies (mAh) on creatinuria in huNBLl -induced kidney damage. FIG. 24 A graphs the effect of A12 anti-NBLl mAh on urinary creatinine. FIG. 24B graphs the effect of B06 anti- NBLl mAh on urinary creatinine.

[0059] FIG. 25 is a schematic illustration of the experimental protocol for testing treatment with anti-NBLl mAbs in streptozotocin (STZ)-induced kidney damage in vivo.

[0060] FIGs. 26A-26B are line graphs indicating the effect of anti-NBLl mAb A12 on renal function in STZ-induced diabetic kidney disease (DKD) model as evidenced by albuminuria (FIG. 26A) and creatinuria (FIG. 26B).

[0061] FIGs. 27A-27B are line graphs indicating the effect of anti-NBLl mAb B06 on renal function in STZ-induced diabetic kidney disease (DKD) model as evidenced by albuminuria (FIG. 27 A) and creatinuria (FIG. 27B).

[0062] FIG. 28A-28B are line graphs indicating the effect of anti-NBLl mAb E04 on renal function in STZ-induced diabetic kidney disease (DKD) model as evidenced by albuminuria (FIG. 28A) and creatinuria (FIG. 28B).

[0063] FIGs. 29A-29B are bar graphs demonstrating the effect of anti-NBLl -mAbs A12, B06, and E04 on renal function parameters in the STZ-induced DKD model. FIG. 29 A compares percentage reduction of urinary albumin. FIG. 29B compares percentage reduction of urinary creatinine.

[0064] FIGs. 30A-30E show the effect of select anti-NBLl mAbs on mesangial expansion in the STZ-induced DKD model. FIG. 30A is a histological representation mesangial cells of mice treated with STZ. FIG. 30B is a histological representation mesangial cells of mice treated with STZ and mAb A12. FIG. 30C is a histological representation mesangial cells of mice treated with STZ and mAb B06. FIG. 30D is a histological representation mesangial cells of mice treated with STZ and mAb E04. FIG. 30E is a bar graph indicating mesangial expansion in glomeruli in mice treated with STZ, and with and without mAbs A12, B06, and E04.

[0065] FIG. 31 is an illustration of the experimental protocol using the homozygous for diabetes mutations (Db / Db) mouse model to assess treatment with anti-NBLl mAbs.

[0066] FIGs. 32A-32B graph the effect of anti-NBLl mAh A12 on renal function in the Db / Db model. FIG. 32A shows the effect of anti-NBLl mAb A12 on urinary albumin. FIG. 32B shows the effect of anti-NBLl mAb A12 on urinary creatinine.

[0067] FIGs. 33A-33B graph the effect of anti-NBLl mAb E04 on renal function in the Db / Db model. FIG. 33 A show the effect of anti-NBLl mAb E04 on urinary albumin. FIG. 33B shows the effect of anti-NBLl mAb E04 on urinary creatinine.

[0068] FIGs. 34A-34D display the effect of anti-NBLl mAbs on mesangial expansion in the Db / Db mouse model. FIG. 34A is a histological section of glomeruli in the untreated Db / Db mouse at 25 weeks. FIG. 34B is a histological section of glomeruli in the Db / Db mouse model treated with mAb A12. FIG. 34C is a histological section of glomeruli in the Db / Db mouse model treated with mAb E04. FIG. 34D is a bar graph comparing mesangial expansion in Db / Db mouse model control versus mice subject to mAb A12 treatment.

[0069] FIGs. 35A-35D illustrate effects of anti-NBLl antibodies on podocytic markers. FIG. 35A illustrates the schedule of NBL1 administration and time points of analysis steps. FIG. 35B is a bar graph showing anti-NBLl monoclonal antibody (mAb) effect on podocytic marker podocalyxin (POD XL) in kidney organoids cultured in the presence and absence of NBL1. FIG. 35C is a bar graph showing anti-NBLl monoclonal antibody (mAb) effect on podocytic marker nephrin (NPHS1) in kidney organoids cultured in the presence and absence of NBL1. FIG. 35D is a bar graph representing anti-NBLl monoclonal antibody (mAb) effect on podocytic marker podocin (NPHS2) in kidney organoids cultured in the presence and absence of NBL1.

[0070] FIGs. 36A-36B are graphs displaying the effect of anti-NBLl mAbs A12 and B06 on fibrotic marker TGFP mRNA expression in human kidneys. FIG. 36A displays effect of anti- NBLl mAb A12 on fibrotic marker TGFP mRNA expression in human podocytes cultured with varying amounts of human NBL1. FIG. 36B displays effect of anti-NBLl mAbs A12 and B06 on fibrotic marker TGFP mRNA expression in human kidney organoids cultured with varying amounts of human NBL1.

[0071] FIGs. 37A-37C depict binding of anti-NBLl monoclonal antibodies (mAbs) to DAN family members. FIG. 37A-37B are bar graphs displaying quantified absorbance spectroscopy results of anti-NBLl monoclonal antibodies A12 (mAb 1) and E04 (mAb 3) binding to DAN (differential screening-selected gene aberrant in neuroblastoma) gene familymembers. FIG. 37 A shows binding A12 and E04 binding to GREM2 and NBL1. FIG. 37B shows binding A12 and E04 binding to SOST, CER1, GREM1, COCO and NBL1. FIG. 37C is a schematic of the assay approach.

[0072] FIG. 38 is a schematic illustration of a dose-response study for testing the effect of three ascending doses of once weekly anti-NBLl mAb A12 (low, mid and high), and placebo treatment in a homozygous diabetic mouse model (Db / Db). The low dose, also noted as dose 3, was 0.1 mg. The mid dose, also noted as dose 2, was 0.5 mg. The high dose, also noted as dose 3, was 11.25 mg / mouse). The study commenced when the mice were 11 weeks of age and terminated when the mice were 24 weeks of age. The study had a duration of 13 weeks.

[0073] FIGs. 39A-39B show the effect of A12 (low, mid, and high) and placebo treatment on urinary albumin in Db / Db mice. FIG. 39A is a line graph showing the level of urinary albumin produced along the course of the study in response to A12 (low, mid, and high) and placebo treatment. (All three A12 doses vs. placebo, p<0.0001). FIG. 39B shows the effect of A12 (low, mid, and high) and placebo treatment on urinary albumin production at 24 weeks of age, following the 13 week course of treatment.

[0074] FIGs. 40A-40B show the effect of A12 (low, mid, and high) and placebo treatment on urinary albumin / creatinine ratio (ACR) in Db / Db mice. FIG. 40A is a line graph showing the urinary ACR along the course of the study for A12 (low, mid, and high) and placebo treatment, (all A12 dosing vs placebo, p<0.0001). FIG. 40B shows the effect of A12 (low, mid, and high) and placebo treatment on urinary ACR at week 24, following the 13 week course of treatment.

[0075] FIGs. 41A-41B show the effect of A12 (low, mid, and high) and placebo treatment on the morphology of kidney cells in Db / Db mice at 24 weeks of age, following the 13-week course of treatment. FIG. 41A are images of cross-sections of kidney tissue of mice treated with A12 (low, mid, and high) and placebo. FIG. 41B is a bar graph showing the percentage of collagen positive area in response to A12 (low, mid, and high) and placebo treatment.

[0076] FIGs. 42A-42B are schematic illustrations of a protocol to study the effects of mAb treatment in Db / Db mice (FIG. 42A), versus the effects of GLP-1 agonist, liraglutide, (FIG 42B), in Db / Db mice.

[0077] FIGs. 43A-43B show the effect of A12, liraglutide, and placebo treatment on urinary albumin in Db / Db mice. FIG. 43A is a line graph showing the effect of A12 (high), liraglutide, and placebo along the course of the study. FIG. 43B shows the effect of A12 (high, mid, low), liraglutide, and placebo following the 13 -week course of treatment, at 24 weeks of age.

[0078] FIG. 44 is a bar graph showing the fold change in urinary albumin at 24 weeks of age versus baseline for A12 (low, mid, high), liraglutide, and placebo in Db / Db mice following the 13 -week course of treatment.

[0079] FIGs. 45A-45B show the effect of Al 2 administered at the high dose (high), liraglutide, and placebo treatment on urinary ACR in Db / Db mice. FIG. 45 A shows the effect of A12 (high), liraglutide, and placebo therapy on urinary ACR over the course of the study. FIG. 45B shows the effect of A12 (low, mid, high), liraglutide, and placebo at 24 weeks of age, following the 13 -week course of treatment.

[0080] FIG. 46 is a bar graph showing the effect of A12 (low, mid, high), and liraglutide, on fold reduction of urinary ACR as compared to placebo in Db / Db mice at 24 weeks of age following the course of treatment. (All A12 doses vs. placebo, p<0.0001).

[0081] FIG. 47A-47B show the effect of A12 (high), liraglutide, and placebo therapy on glomerular morphology in Db / Db mice at 24 weeks of age, following course of treatment. FIG. 47A are images of cross-sections of kidney tissue obtained from mice treated with A12 (high), liraglutide, and placebo therapy. FIG. 47B is a bar graph showing the percentage of collagen positive area in mice treated with A12 (high), liraglutide, and placebo therapy.

[0082] FIGs. 48A-48B are line graphs showing the effect of A12 (high, mid, low), liraglutide, and placebo treatment on glycemia (FIG. 48A), and weight loss (FIG. 48B) in Db / Db mice along the course of the study.

[0083] FIG. 49A is a schematic illustration of a study protocol to assess the effects of anti- NBL1 mAb administered at the middle dose (A12 mid) on Db / Db mice. FIG. 49B is a bar graph showing the fold change in circulating levels of NBL1 in heterozygyous Db / + mice (non-diabetic phenotype) versus homozygous Db / Db mice as measured following termination of the study. FIG. 49C is a bar graph showing the effect of A12 (mid) mAb therapy versus no treatment (control) on urine albumin levels in Db / Db mice at 13 weeks and 24 weeks of age.

[0084] FIGs. 50A-50B show the effect of anti-NBLl mAh (A12 (mid)) on collagen deposition in Db / Db mice. FIG. 50A shows images of cross-sections of kidney tissue obtained from mice treated with A12 (mid) (lower panel), versus untreated (top panel). FIG. 50B are bar graphs showing fibrosis quantification in mice treated with A12 (mid) versus no treatment. The upper panel shows fibrosis quantification as a measure of collagen positive area and the lower panel shows fibrosis quantification as a measure of collagen staining mean intensity.

[0085] FIGs. 51 A- 5 IB show the Wilms’ tumor gene 1 immunohistochemistry results for the effect of NBL1 blockade on podocytes in Db / Db mice at 25 weeks of age treated with anti- NBLl mAb A12 (mid) versus untreated mice. FIG. 51 A are images of cross-sections of kidney tissue obtained from mice treated with A12 (mid) (right panel) versus no treatment (left panel). FIG. 5 IB is a bar graph showing the effect of A12 (mid) on podocyte density as a measure of WT1 -positive area versus mice undergoing no treatment (control).

[0086] FIG. 52A shows the results of an NBL1 atlas study displaying NBL1 expression in various tissues (FIG. 52A). FIG. 52B is a bar graph showing observed levels of in vitro NBL1 secretion in intestinal cell lines in the presence / absence of high glucose levels.6. DETAILED DESCRIPTION OF THE INVENTION6.1. Definitions

[0087] Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this disclosure belongs.

[0088] The terms “individual” and “subject” are used interchangeably and refer to an animal to be treated, including but not limited to humans; non-human primates; rodents, including rats and mice; bovines; equines; ovines; felines; and canines.

[0089] The term “patient” refers to a human subject.

[0090] The terms “treating,” “treatment,” and grammatical variations thereof are used in the broadest sense understood in the clinical arts. Accordingly, the terms do not require cure or complete remission of disease and encompass obtaining any clinically desired pharmacologic and / or physiologic effect. As used herein, “treating diabetic kidney disease (DKD)” explicitly encompasses delaying onset of kidney damage, delaying progression ofkidney damage, and slowing decline in kidney function in patients with type 1 or type 2 diabetes or a glomerular disorder.

[0091] The phrase “therapeutically effective amount” refers to the amount of a compound that, when administered to a mammal or other subject for treating a disease, condition, or disorder, is sufficient to effect treatment of the disease, condition, or disorder, as treatment is defined herein. Determining the "therapeutically effective amount" is within the skill in the art.

[0092] Neuroblastoma suppressor of tumorigenicity 1 (NBL1) is a founding member of the DAN (differential screening-selected gene aberrant in neuroblastoma) gene family. Members of the DAN gene family are expressed during development and function as bone morphogenetic protein (BMP) antagonists; DAN proteins bind to BMPs and prevent them from interacting with BMP receptors. Neuroblastoma suppressor of tumorigenicity 1 (NBL1), also known as D1S1733E, DAN, DAND1, NB, and NO3, is identified by NCBI Gene ID: 4681. The protein sequence of NCBI Gene ID: 4681 is incorporated herein by reference.

[0093] Bone morphogenetic proteins (BMPs) are a group of growth factors originally identified by their ability to induce the formation of bone and cartilage, and now considered to constitute a group of pivotal morphogenetic signals, orchestrating tissue architecture throughout the body. Human BMP-2, also known as BDA2, BMP2A, SSFSC, and SSFSC1, is identified by NCBI Gene ID:650. The protein sequence of NCBI Gene ID:650 is incorporated herein by reference. Human BMP-4, also known as BMP2B, BMP2B1, MCOPS6, OFC11, and ZYME, is identified by NCBI Gene ID:652. The protein sequence of NCBI Gene ID:652 is incorporated herein by reference. Human BMP-7, also known as OP-1, is identified by NCBI Gene ID:655. The protein sequence of NCBI Gene ID:655 is incorporated herein by reference.

[0094] As used herein, the term “antibody” has its broadest art-recognized meaning, and therefore includes all known formats. The term specifically includes, without limitation, polyclonal antibodies, monoclonal antibodies, monospecific antibodies, multispecific antibodies (including bispecific antibodies), chimeric antibodies, humanized and fully human antibodies. Antibodies that include heavy chain constant region domains can be of any class, including IgG, IgE, IgM, IgD, and IgA and any subclass, including IgGl, IgG2, IgG3, IgG4,IgAl, and IgA2. “Antigen binding fragments” of antibodies are antibody fragments (and / or polypeptides that comprise antibody fragments) that retain the binding characteristics (e.g., specificity, monovalent affinity or bivalent avidity) of the antibody from which derived, and has its broadest art-recognized meaning. The term includes, without limitation, Fab, Fab’, F(ab’)2, Fv, scFv, (scFv)2, single domain antibody (including camelid VHH and shark VNAR formats), and multispecific antibodies formed from antibody fragments, including without limitation F(ab)2, and diabody.

[0095] As used herein, “chronic kidney disease” (CKD) has the meaning ascribed in the National Kidney Foundation KDOQI guidelines, and stages of CKD are defined as provided in the NKF KDOQI guidelines. “End stage kidney disease” (ESKD) and “end stage renal disease” (ESRD) are used interchangeably herein and have the meaning ascribed in the National Kidney foundation KDOQI guidelines.

[0096] In this disclosure, “comprises,” “comprising,” “containing,” “having,” “includes,” “including”, and linguistic variants thereof have the meaning ascribed to them in U.S. Patent law, permitting the presence of additional components beyond those explicitly recited.

[0097] Unless specifically stated or apparent from context, as used herein the term “or” is understood to be inclusive.

[0098] Unless specifically stated or apparent from context, as used herein, the terms “a”, “an”, and “the” are understood to be singular or plural.

[0099] As used herein, “conservative amino acid substitutions” are those substitutions in which the original and substituted amino acids have similar biochemical properties, or their biochemical effects are similarly maintained across substitutions, as set forth in Table A.6.2. Summary of experimental results

[0100] As detailed in the experimental examples in this disclosure, we have discovered that NBL1 is directly toxic to renal cells, including podocytes and tubular cells. The toxic effect is not mediated through inhibition of renal BMP proteins; we show that BMPs are not expressed in and are not secreted by kidney cells. Moreover, we have discovered that NBL1 is also not expressed in kidney cells, but is highly expressed in intestinal cells and circulating immune cells. Neutralizing NBL1 with an antagonist prevents toxicity. Importantly, we demonstrate that NBLl is elevated in Type 1 Diabetes and Type 2 Diabetes, and that anti-NBL1 monoclonal antibodies are capable of arresting NBL1 induced podocyte damage in diabetic mice.6.3. Methods of delaying onset or progression of kidney damage

[0101] Accordingly, in a first aspect, methods are presented for delaying onset or progression of kidney damage in a subject who has, or is at risk for, type 1 diabetes or type 2 diabetes or who has or is at risk for developing a glomerular disease. The method comprises administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity, and in particular, inhibiting NBL1 -mediated toxicity of human podocytes.

[0102] In various embodiments, the method delays onset or progression of damage to one or more of renal blood vessels, podocytes, renal tubular cells, or glomerular or tubular basement membrane. In some embodiments, the method delays onset or progression of thickening of glomerular and tubular basement membrane, increase in mesangial matrix, Kimmelstiel- Wilson nodules, microaneurysms, exudative or hyalinosis lesions, capsular drop or afferent and efferent arteriolar hyalinosis.

[0103] In some embodiments, the subject has type 1 diabetes. In some embodiments, the subject has type 2 diabetes. In some embodiments, the subject is pre-diabetic. In some embodiments, the subject is not prediabetic as measured by hemoglobin Ale levels or blood glucose levels but is at risk for type 1 diabetes or type 2 diabetes and has elevated NBL1 plasma levels.

[0104] In some embodiments, the subject has a glomerular disease. In certain embodiments, the subject with glomerular disease does not have type 1 diabetes or type 2 diabetes. In certain embodiments, the glomerular disease is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), chronic glomerulopathies, hereditary nephritis, and minimal change disease.

[0105] In some embodiments, the subject has pre-treatment plasma NBL1 levels at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% or 100% higher than normal subjects who do not have type 1 or type 2 diabetes or prediabetes. In some embodiments, the subjects to be treated have NBL1 plasma levels at least 2-fold, 3-fold, 4-fold, 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, or 10-fold higher than normal subjects who do not have type 1 or type 2 diabetes or prediabetes.

[0106] In some embodiments, the method effectively reduces the availability of circulating free NBL1 in subjects having type 1 or type 2 diabetes, or who are prediabetic, or who are at risk for type 1 diabetes or type 2 diabetes, and have elevated NBL1 plasma levels, as compared to normal subjects who do not have type 1 or type 2 diabetes or prediabetes. In some embodiments, the method effectively reduces the availability of circulating free NBL1 in subjects who have a glomerular disease. In certain embodiments, the method effectively reduces the availability of circulating free NBL1 in subjects who have a glomerular disease selected from the group consisting of focal segmental glomerulosclerosis (FSGS), chronic glomerulopathies, hereditary nephritis, and minimal change disease.

[0107] In some embodiments, the agent capable of inhibiting NBL1 activity is capable of binding to NBL1. In some embodiments, the binding occurs in the N-terminus of NBL1. In some embodiments, the binding occurs in the functional DAN domain of NBL1. In some embodiments, the binding occurs in the C-terminus of NBL1. In some embodiments, the binding is noncovalent. In some embodiments, the binding is covalent. In certain covalent embodiments, the inhibitor binds to NBL1 via disulfide bonds within the DAN domain.

[0108] In some embodiments, the agent capable of inhibiting NBL1 activity is capable of inhibiting dimerization of NBL1.

[0109] In some embodiments, the agent capable of inhibiting NBL1 activity is a dimerization inhibitor that prevents the formation of stabilizing hydrogen bonds between two NBL1 monomers. In some embodiments, the dimerization inhibitor disrupts at least one of the three disulfide bonds that form a ring-like structure known as cysteine-knot motif within each monomer, or the disulfide bond linking Fl to F2, where Fl and F2 are the first loop from the N terminus, finger 1, and the third loop from the N terminus, finger 2, respectively.6.3.1. Soluble BMP inhibitors

[0110] In some embodiments, the agent capable of inhibiting NBL1 activity comprises a bone morphogenetic protein (BMP) or soluble NBL1 -binding fragment thereof. In some embodiments, the BMP is BMP-2 or a soluble NBL1 -binding fragment thereof. In some embodiments, the BMP is BMP-4 or a soluble NBL1 -binding fragment thereof. In some embodiments, the BMP is BMP-7 or a soluble NBL1 -binding fragment thereof. In currently preferred embodiments, the BMP is a human BMP or soluble NBL1 -binding fragment thereof.[OHl] In some embodiments, the agent capable of inhibiting NBL1 further comprises a moiety that extends serum half-life.

[0112] In some embodiments, the moiety that extends serum half-life is provided through covalent chemical modification. In some embodiments, the moiety that extends serum halflife is at least one polyethylene glycol (PEG) moiety. In some embodiments, the PEG moiety is permanently attached to the agent. In some embodiments, the PEG moiety is a releasable carrier, provided through covalent chemical modification. In certain embodiments, the agent comprises a PEGylated BMP or soluble NBL1 -binding fragment thereof.

[0113] In some embodiments, the moiety that extends serum half-life is an antibody Fc domain. In some embodiments, the Fc domain is engineered to optimize the pH-dependent IgG Fc-FcRn interaction. In certain embodiments, the Fc domain is engineered to have the YTE triple mutation (M252Y / S254T / T256E). In certain embodiments, the Fc domain is engineered to have the M428L / N434S mutations. In certain embodiments, the Fc domain is fused in frame to a BMP or soluble NBL1 -binding fragment thereof.

[0114] In some embodiments, the moiety that extends serum half-life is a serum albumin molecule. In certain embodiments, the half-life extending moiety is a human serum albumin molecule fused in frame to a BMP or soluble NBL1 -binding fragment thereof.

[0115] In some embodiments, the moiety that extends serum half-life is an XTEN protein polymer covalently attached to the agent, as described in Podust et al., Protein Eng. Des. Sei. 26(11): 743-53 (2013), the disclosure of which is incorporated herein by reference in its entirety.6.3.2. Antibodies and antigen-binding fragments thereof

[0116] In some embodiments, the agent capable of inhibiting NBL1 activity is an antibody capable of binding NBL1, or an NBL1 -binding fragment thereof and inhibiting NBL1 activity, and in particular, inhibiting NBL1 -mediated toxicity of human podocytes. In preferred embodiments, the antibody or antigen-binding antibody fragment is capable of binding human NBL1.

[0117] In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within the N-terminal domain of NBL1. In some embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within the DAN domain of NBL1. Insome embodiments, the antibody or antigen-binding fragment thereof binds to an epitope within the C-terminal domain of NBL1.

[0118] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain and light chain CDRs selected from Tables 23 - 44 below, or the heavy chain and light chain CDRs selected from Tables 23-44 below with no more than 10 amino acid deletions, insertions, or conservative amino acid substitutions (as defined in Table A), as compared thereto.

[0119] In some embodiments, the amino acid deletions, insertions, or conservative substitutions are no more than 8. In some embodiments, the amino acid deletions, insertions, or conservative substitutions are no more than 6. In some embodiments, the amino acid deletions, insertions, or conservative substitutions are no more than 4.

[0120] In some embodiments, the antibody or antigen-binding fragment thereof comprises heavy chain V region and light chain V region selected from Tables 1 - 22 below.

[0121] In some embodiments, the antibody or antigen-binding fragment thereof comprises the heavy chain and light chain CDRs selected from Tables 23 - 44 and with no more than 10 amino acid deletions, insertions, or conservative amino acid substitutions (as defined in Table A), as compared thereto. In some embodiments, the amino acid deletions, insertions, or conservative substitutions are no more than 8. In some embodiments, the amino acid deletions, insertions, or conservative substitutions are no more than 6. In some embodiments, the amino acid deletions, insertions, or conservative substitutions are no more than 4.-25-38877 / 63694 / FW / 21628459.3-26-38877 / 63694 / FW / 21628459.3-27-38877 / 63694 / FW / 21628459.3-28-38877 / 63694 / FW / 21628459.3-29-38877 / 63694 / FW / 21628459.3-30-38877 / 63694 / FW / 21628459.3-31-38877 / 63694 / FW / 21628459.3-32-38877 / 63694 / FW / 21628459.3-33-38877 / 63694 / FW / 21628459.3-34-38877 / 63694 / FW / 21628459.3-35-38877 / 63694 / FW / 21628459.3-36-38877 / 63694 / FW / 21628459.3-37-38877 / 63694 / FW / 21628459.3-38-38877 / 63694 / FW / 21628459.3-39-38877 / 63694 / FW / 21628459.3-40-38877 / 63694 / FW / 21628459.3-41-38877 / 63694 / FW / 21628459.3-42-38877 / 63694 / FW / 21628459.3

[0123] In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs having sequences selected from: a) SEQ ID NOs: 3, 8, and 13 and SEQ ID NOs: 18, 23, and 28 (antibody E05a); b) SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53, and 58 (antibody H08); c) SED ID NOs: 63, 68, and 73 and SED ID NOs: 78, 83, and 88 (antibody F06); d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibody A12); e) SED ID NOs: 123, 128, and 133 and SED ID NOs: 138, 143, and 148 (antibody G01); f) SED ID NOs: 153, 158, and 163 and SED ID NOs: 168, 173 and 178 (antibody El l); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); h) SED ID NOs: 213, 218, and 223 and SED ID NOs: 228, 233, and 238 (antibody DI 2); i) SED ID NOs: 243, 248, and 253 and SED ID NOs: 258, 263 and 268 (antibody H01); j) SED ID NOs: 273, 278, and 283 and SED ID NOs: 288, 293, and 298 (antibody Cl l); k) SED ID NOs: 303, 308, and 313 and SED ID NOs: 318, 323, and 328 (antibody E05b); l) SED ID NOs:333, 338, and 343 and SED ID NOs: 348, 353 and 358 (antibody F10); m) SED ID NOs: 363, 368 and 373 and SED ID NOs: 378, 383 and 388 (antibody G10); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04);-43-38877 / 63694 / FW / 21628459.3o) SED ID NOs: 423, 428, and 433 and SED ID NOs: 438, 443, and 448 (antibody E07); p) SEQ ID NOs: 453, 458, and 463 and SED ID NOs: 468, 473, and 478 (antibody El 2); q) SED ID NOs: 483, 488, and 493 and SED ID NOs: 498, and 503, and 508 (antibody D08); r) SED ID NOs: 513, 518, and 523 and SED ID NOs: 528, 533, and 538 (antibody E10); s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SED ID NOs: 573, 578, and 583 and SED ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the selected CDR sequences (a) - (t) by at most two conservative amino acid substitutions in each CDR.

[0124] In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs with sequences identical to the selected CDRs (a)-(t). In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs having sequences that differ from the selected CDR sequences (a) - (t) by at most two conservative amino acid substitutions in each CDR, or by at most 1 conservative amino acid substitution in each CDR. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs having sequences that differ from the selected CDR sequences (a) - (t) by 6 conservative amino acid changes in total across all 6 CDRs, 5 conservative amino acid substitutions in total across all 6 CDRs, 4 conservative amino acid substitutions in total across all 6 CDRs, 3 conservative amino acid substitutions in total across all 6 CDRs, 2 conservative amino acid substitutions in total across all 6 CDRs, or 1 conservative amino acid substitution total across all 6 CDRs.

[0125] In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs having sequences selected from: b) SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53 and 58 (antibody H08);d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibody A12); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04); s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SEQ ID NOs: 573, 578, and 583 and SEQ ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the selected CDR sequences b), d), g), n) s), and t) by at most two conservative amino acid substitutions in each CDR.

[0126] In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs with sequences identical to the selected CDRs (b), (d), (g), (n), (s) or (t). In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs having sequences that differ from the selected CDR sequences (b), (d), (g), (n), (s) or (t) by at most two conservative amino acid substitutions in each CDR, or by at most 1 conservative amino acid substitution in each CDR. In some embodiments, the antibody or antigen-binding fragment comprises three heavy chain CDRs and three light chain CDRs having sequences that differ from the selected CDR sequences (b), (d), (g), (n), (s) or (t) by 6 conservative amino acid changes in total across all 6 CDRs, 5 conservative amino acid substitutions in total across all 6 CDRs, 4 conservative amino acid substitutions in total across all 6 CDRs, 3 conservative amino acid substitutions in total across all 6 CDRs, 2 conservative amino acid substitutions in total across all 6 CDRs, or 1 conservative amino acid substitution total across all 6 CDRs.

[0127] In some embodiments, the antibody framework regions are human antibody framework regions. In some embodiments, the antibody is a full length bivalent monospecific monoclonal antibody. In some embodiments, the antibody comprises human IgGl, IgG2, or IgG4 heavy chain constant regions. In some embodiments, the antibody comprises a human IgGl constant region. In some embodiments, the antibody Fc region has at least one engineered mutation that reduces antibody binding to at least an Fc y receptor. In some embodiments, the mutation is N297A. In some embodiments, the antibody Fc regionhas at least one engineered mutation that reduces complement fixation. In some embodiments, the mutation is K322A.

[0128] In some embodiments, the antibody is a Fab, optionally wherein the Fab is PEGylated.

[0129] In some embodiments, the antibody or antigen binding fragment is further capable of binding to cynomolgus monkey NBL1. In some embodiments, the antibody or antigen binding fragment is further capable of binding to mouse NBL1.

[0130] In some embodiments, the antibody is an IgG monoclonal antibody. In particular embodiments, the antibody is an IgGl or IgG4 monoclonal antibody.

[0131] In some embodiments, the antibody is a human monoclonal antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a chimeric mouse-human antibody. In some embodiments, the agent comprises an NBL1- binding antigen binding fragment selected from a Fab, Fab’, F(ab’)2, Fv, scFv, Fd, or diabody.

[0132] In some embodiments the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 100 nM. In some embodiments the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 10 nM. In some embodiments the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 5 nM. In some embodiments the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 1 nM.

[0133] In certain embodiments, the NBL1 dimerization inhibitor is an antibody. In some of these embodiments, the antibody is a monoclonal antibody. In some of these embodiments, the antibody is polyclonal. In some embodiments the dimerization inhibitor binds to at least one of the synonymous P-strands from each NBL1 monomer.6.3.3. NBL1 Expression inhibitors

[0134] In some embodiments, the agent is capable of inhibiting NBL1 expression.

[0135] In certain embodiments, the agent inhibits transcription of the NBL1 gene. In certain embodiments, the agent causes degradation of the NBL1 mRNA. In certain embodiments,the agent inhibits translation of the NBL1 mRNA. In certain embodiments, the agent targets NBL1 protein for degradation.

[0136] In specific embodiments, the agent is an antisense oligonucleotide. In specific embodiments, the agent mediates RNA interference. In particular RNA interference embodiments, the agent is a short hairpin RNA (shRNA) or short interfering RNA (siRNA). In specific embodiments, the agent is a microRNA (miRNA). In specific embodiments, the agent is a sequence-specific mRNA interferase.6.4. Methods of slowing decline in kidney function

[0137] In a further aspect, methods are provided for slowing decline in kidney function in a subject who has, or is at risk for, type 1 or type 2 diabetes, or who has or is at risk for developing a glomerular disease. The method comprises administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity. In various embodiments, the agent capable of inhibiting NBL1 activity is an inhibitor as described in Section 6.3.1, 6.3.2, or 6.3.3 above.

[0138] In some embodiments, the method slows progression of microalbuminuria, slows progression of macroalbuminuria, slows progression of proteinuria, or slows reduction of glomerular filtration rate (GFR).

[0139] In some embodiments, the method prevents onset of or slows progressive kidney function decline (PKFD). In some embodiments, the agent inhibits the progression of at least one or more symptoms associated with PKFD. In some embodiments, the rate of decline in kidney function of the subject in relation to an untreated control group is reduced by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90%.

[0140] In some embodiments, the method prevents onset of or slows progression to end-stage kidney disease (ESKD). In some embodiments, the method inhibits the progression of at least one or more symptoms associated with ESKD. In some embodiments, the method inhibits progression to required dialysis.

[0141] In some embodiments, the method slows progression of chronic kidney disease (CKD) stage 1 to stage 2, stage 2 to stage 3, stage 3 A to stage 3B, stage 3B to stage 4, stage 4 to stage 5, or progression from stage 5 without dialysis to stage 5 with dialysis.

[0142] In some embodiments, the subject has type 1 diabetes. In some embodiments, the subject has type 2 diabetes. In some embodiments, the subject is pre-diabetic. In some embodiments, the subject is not prediabetic as measured by hemoglobin Ale levels or blood glucose levels but is at risk for type 1 diabetes or type 2 diabetes and has elevated NBL1 plasma levels. In some embodiments, the subject has a glomerular disorder. In particular embodiments, the subject has a glomerular disorder and does not have type 1 or type 2 diabetes. In particular embodiments, the glomerular disorder is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), chronic glomerulopathies, hereditary nephritis, and minimal change disease.

[0143] In some embodiments, the method effectively reduces the availability of circulating free NBL1 in subjects having type 1 or type 2 diabetes, or who are prediabetic, or who are at risk for type 1 diabetes or type 2 diabetes and have elevated NBL1 plasma levels, as compared to normal subjects who do not have type 1 or type 2 diabetes or prediabetes. In some embodiments, the method effectively reduces the availability of circulating free NBL1 in subjects who have a glomerular disease. In certain embodiments, the method effectively reduces the availability of circulating free NBL1 in subjects who have a glomerular disease selected from the group consisting of focal segmental glomerulosclerosis (FSGS), chronic glomerulopathies, hereditary nephritis, and minimal change disease.

[0144] In some embodiments, the method effectively lowers or reduces urinary albumin / creatinine ratio (ACR) in a subject who has type 1 or type 2 diabetes or a glomerular disease.

[0145] In some embodiments, the method is effective to lower or reduce ACR in the subject compared too pretreatment ACR levels by about 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%,67%, 66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%,51%, 50%, 49%, 48%, 47%, 46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%,35%, 34%, 33%, 32%, 31%, 30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%,19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, or 10%.

[0146] In some embodiments, the method reduces the percentage collagen positive area in the kidney of the subject compared to the pre-treatment percentage collagen positive area in the kidney of the subject.

[0147] In some embodiments, the method is effective to collagen positive area compared to the pre-treatment collagen positive area in the kidney of the subject by about 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%, 72%, 71%, 70%, 69%, 68%, 67%, 66%, 65%, 64%, 63%,62%, 61%, 60%, 59%, 58%, 57%, 56%, 55%, 54%, 53%, 52%, 51%, 50%, 49%, 48%, 47%,46%, 45%, 44%, 43%, 42%, 41%, 40%, 39%, 38%, 37%, 36%, 35%, 34%, 33%, 32%, 31%,30%, 29%, 28%, 27%, 26%, 25%, 24%, 23%, 22%, 21%, 20%, 19%, 18%, 17%, 16%, 15%,14%, 13%, 12%, 11%, or 10%.

[0148] In various embodiments, the method does not induce body weight loss in subjects.

[0149] In some embodiments, the subject does not experience weight loss from their pretreatment body weight while adhering to a standard diet regimen over the course of treatment.

[0150] In some embodiments, the subject experiences a reduction in body weight compared to pretreatment of about 1%, 2%, 3%, 4%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%, while adhering to a standard diet regimen over the course of treatment.

[0151] In various embodiments, the agent is an agent described in Section 6.3.1, 6.3.2, or 6.3.3 above, incorporated herein by reference.6.5. Methods of treating diabetic kidney disease or a glomerular disease

[0152] In a further aspect, methods are presented for treating diabetic kidney disease (DKD) in a subject who has type 1 or type 2 diabetes or who has a glomerular disease. The method comprises administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity.

[0153] In some embodiments, the subject has type 1 or type 2 diabetes and one or more of glomerular hypertrophy, glomerulosclerosis, tubulointerstitial inflammation, fibrosis, glomerular hyperfiltration, progressive albuminuria, declining GFR, and ESKD.

[0154] In some embodiments, the subject has a glomerular disease. In certain embodiments, the subject has a glomerular disease and does not have type 1 or type 2 diabetes. In certain embodiments, the glomerular disorder is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), chronic glomerulopathies, hereditary nephritis, and minimal change disease.

[0155] In various embodiments, the agent is an agent described in Section 6.3.1, 6.3.2, or 6.3.3 above, incorporated here by reference.6.6. Dose regimen

[0156] In some embodiments of the methods described herein, the agent is administered parenterally. In particular embodiments, the agent is administered intravenously. In specific embodiments, the subject is on dialysis and the agent is administered intravenously. In particular embodiments, the agent is administered subcutaneously.

[0157] In some embodiments, the agent is administered once. In some embodiments, the agent is administered more than one. In particular embodiments, the agent is administered for at least 3 months. In particular embodiments, the agent is administered for at least 6 months. In particular embodiments, the agent is administered for at least 12 months.6.7. Antibodies and pharmaceutical formulations thereof

[0158] In another aspect, antibodies or antigen binding fragments are provided that are capable of binding to NBL1 and inhibiting NBL1 -induced toxicity of human podocytes. Embodiments include all antibodies and antigen-binding fragment embodiments described in Section 6.3.2 above, which is incorporated herein by reference.

[0159] In a further aspect, pharmaceutical compositions are provided. The pharmaceutical compositions comprise the antibody or antigen binding fragment described herein and a pharmaceutically acceptable carrier. In some embodiments, the composition is formulated for parenteral administration. In some embodiments, the composition is formulated for intravenous administration. In some embodiments, the composition is formulated for subcutaneous administration.6.8. Additional Embodiments

[0160] Additional embodiments are set out in the following numbered clauses.

[0161] In one aspect, the techniques described herein relate to a method for delaying onset or progression of kidney damage in a subject who has type 1 diabetes or type 2 diabetes or a glomerular disease, the method including: administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity.

[0162] In some embodiments, the techniques described herein relate to a method, wherein the agent is capable of binding to NBL1.

[0163] In some embodiments, the techniques described herein relate to a method, wherein the agent is capable of binding to human NBL1.

[0164] In some embodiments, the techniques described herein relate to a method, wherein the agent is an antibody, or an antigen binding fragment of an antibody, that is capable of binding to human NBL1.

[0165] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment includes three heavy chain CDRs and three light chain CDRs having sequences selected from: a) SEQ ID NOs: 3, 8, and 13 and SEQ ID NOs: 18, 23, and 28 (antibody E05a); b) SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53, and 58 (antibody H08); c) SED ID NOs: 63, 68, and 73 and SED ID NOs: 78, 83, and 88 (antibody F06); d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibody A12); e) SED ID NOs: 123, 128, and 133 and SED ID NOs: 138, 143, and 148 (antibody G01); f) SED ID NOs: 153, 158, and 163 and SED ID NOs: 168, 173 and 178 (antibody El l); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); h) SED ID NOs: 213, 218, and 223 and SED ID NOs: 228, 233, and 238 (antibody D12); i) SED ID NOs: 243, 248, and 253 and SED ID NOs: 258, 263 and 268 (antibody H01); j) SED ID NOs: 273, 278, and 283 and SED ID NOs: 288, 293, and 298 (antibody Cl 1); k) SED ID NOs: 303, 308, and 313 and SED ID NOs: 318, 323, and 328 (antibody E05b); 1) SED ID NOs:333, 338, and 343 and SED ID NOs: 348, 353 and 358 (antibody F10); m) SED ID NOs: 363, 368 and 373 and SED ID NOs: 378, 383 and 388 (antibody G10); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04); o) SED ID NOs: 423, 428, and 433 and SED ID NOs: 438, 443, and 448 (antibody E07); p) SEQ ID NOs: 453, 458, and 463 and SED ID NOs: 468, 473, and 478 (antibody E12); q) SED ID NOs: 483, 488, and 493 and SED ID NOs: 498, and 503, and 508 (antibody D08); r) SED ID NOs: 513, 518, and 523 and SED ID NOs: 528, 533, and 538 (antibody E10); s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SED ID NOs: 573, 578, and 583 and SED ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the selected CDR sequences (a) - (t) by at most two conservative amino acid substitutions in each CDR.

[0166] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment includes three heavy chain CDRs and three light chain CDRs with sequences identical to the selected CDRs.

[0167] In some embodiments, the techniques described herein relate to a method, wherein the three heavy chain CDRs and three light chain CDRs have sequences selected from: b) SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53 and 58 (antibody H08); d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibody A12); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04); s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SEQ ID NOs: 573, 578, and 583 and SEQ ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the selected CDR sequences b), d), g), n) s), and t) by at most two conservative amino acid substitutions in each CDR.

[0168] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment includes three heavy chain CDRs and three light chain CDRs with sequences identical to the selected CDRs.

[0169] In some embodiments, the techniques described herein relate to a method, wherein the antibody framework regions are human antibody framework regions.

[0170] In some embodiments, the techniques described herein relate to a method, wherein the antibody is a full length bivalent monospecific monoclonal antibody.

[0171] In some embodiments, the techniques described herein relate to a method, wherein the antibody includes human IgGl, IgG2, or IgG4 heavy chain constant regions.

[0172] In some embodiments, the techniques described herein relate to a method, wherein the antibody includes a human IgGl constant region.

[0173] In some embodiments, the techniques described herein relate to a method, wherein the antibody Fc region has engineered mutations that reduce antibody binding to at least one type of Fc receptor and / or reduce complement fixation.

[0174] In some embodiments, the techniques described herein relate to a method, wherein the antibody is a Fab, optionally wherein the Fab is PEGylated.

[0175] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment is further capable of binding to cynomolgus monkey NBL1.

[0176] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment is further capable of binding to mouse NBL1.

[0177] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 100 nM.

[0178] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 10 nM.

[0179] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 5 nM.

[0180] In some embodiments, the techniques described herein relate to a method, wherein the agent includes a bone morphogenetic protein (BMP) or soluble fragment thereof.

[0181] In some embodiments, the techniques described herein relate to a method, wherein the agent includes a soluble fragment of human BMP-2.

[0182] In some embodiments, the techniques described herein relate to a method, wherein the agent further includes a moiety that extends serum half-life.

[0183] In some embodiments, the techniques described herein relate to a method, wherein the half-life extension moiety is an antibody Fc domain.

[0184] In some embodiments, the techniques described herein relate to a method, wherein the half-life extension moiety is at least one covalently linked polyethylene glycol (PEG) moiety.

[0185] In some embodiments, the techniques described herein relate to a method, wherein the agent is capable of inhibiting dimerization of NBL1.

[0186] In some embodiments, the techniques described herein relate to a method, wherein the agent is capable of inhibiting NBL1 expression.

[0187] In some embodiments, the techniques described herein relate to a method, wherein the agent is administered parenterally.

[0188] In some embodiments, the techniques described herein relate to a method, wherein the agent is administered intravenously.

[0189] In some embodiments, the techniques described herein relate to a method, wherein the agent is administered subcutaneously.

[0190] In some embodiments, the techniques described herein relate to a method, wherein the agent is administered for at least 3 months.

[0191] In some embodiments, the techniques described herein relate to a method, wherein the agent is administered for at least 6 months.

[0192] In some embodiments, the techniques described herein relate to a method, wherein the agent is administered for at least 12 months.

[0193] In some embodiments, the techniques described herein relate to a method, wherein the subject has elevated pre-treatment plasma levels of NBL1.

[0194] In some embodiments, the techniques described herein relate to a method, wherein the subject has type 1 diabetes.

[0195] In some embodiments, the techniques described herein relate to a method, wherein the subject has type 2 diabetes.

[0196] In some embodiments, the techniques described herein relate to a method, wherein the subject has a glomerular disease.

[0197] In some embodiments, the techniques described herein relate to a method, wherein the subject with glomerular disease does not have type 1 diabetes or type 2 diabetes.

[0198] In some embodiments, the techniques described herein relate to a method, wherein the glomerular disease is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), chronic glomerulopathies, hereditary nephritis, and minimal change disease.

[0199] In some embodiments, the techniques described herein relate to a method, wherein the method prevents onset of or slows decline in kidney function.

[0200] In another aspect, the techniques described herein relate to a method of treating diabetic kidney disease (DKD) or a glomerular disease in a subject who has type 1 or type 2 diabetes or a glomerular disease, the method including: administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity.

[0201] In some embodiments, the techniques described herein relate to a method, wherein the agent is an antibody, or antigen binding fragment of an antibody, that is capable of binding to human NBL1.

[0202] In another aspect, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody is capable of binding to NBL1 and inhibiting NBL1- induced toxicity of human podocytes.

[0203] In some embodiments, the techniques described herein relate to an antibody, wherein the antibody or antigen-binding fragment includes three heavy chain CDRs and three light chain CDRs having sequences selected from: (a) SEQ ID NOs: 3, 8, and 13 and SEQ ID NOs: 18, 23, and 28 (antibody E05a); (b) SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53, and 58 (antibody H08); (c) SED ID NOs: 63, 68, and 73 and SED ID NOs: 78, 83, and 88 (antibody F06); (d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibody A12); (e) SED ID NOs: 123, 128, and 133 and SED ID NOs: 138, 143, and 148 (antibody G01); (f) SED ID NOs: 153, 158, and 163 and SED ID NOs: 168, 173 and 178 (antibody El l); (g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); (h) SED ID NOs: 213, 218, and 223 and SED ID NOs: 228, 233, and 238 (antibody D12); (i) SED ID NOs: 243, 248, and 253 and SED ID NOs: 258, 263 and 268 (antibody H01); (j) SED ID NOs: 273, 278, and 283 and SED ID NOs: 288, 293, and 298 (antibody Cl 1); (k) SED ID NOs: 303, 308, and 313 and SED ID NOs: 318, 323, and 328 (antibody E05b); (1) SED ID NOs:333, 338, and 343 and SED ID NOs: 348, 353 and 358(antibody F10); (m) SED ID NOs: 363, 368 and 373 and SED ID NOs: 378, 383 and 388 (antibody GIO); (n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04); (o) SED ID NOs: 423, 428, and 433 and SED ID NOs: 438, 443, and 448 (antibody E07); (p) SED ID NOs: 453, 458, and 463 and SED ID NOs: 468, 473, and 478 (antibody E12); (q) SED ID NOs: 483, 488, and 493 and SED ID NOs: 498, and 503, and 508 (antibody D08); (r) SED ID NOs: 513, 518, and 523 and SED ID NOs: 528, 533, and 538 (antibody E10); (s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and (t) SED ID NOs: 573, 578, and 583 and SED ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the selected CDR sequences (a) - (t) by at most two conservative amino acid substitutions in each CDR.

[0204] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody or antigen-binding fragment includes three heavy chain CDRs and three light chain CDRs with sequences identical to the selected CDRs.

[0205] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment or claim 44, wherein the three heavy chain CDRs and three light chain CDRs have sequences selected from: b) SEQ ID NOs: 33, 38, and 43 and SEQ ID NOs: 48, 53 and 58 (antibody H08); d) SEQ ID NOs: 93, 98, and 103 and SEQ ID NOs: 108, 113, and 118 (antibody A12); g) SEQ ID NOs: 183, 188, and 193 and SEQ ID NOs: 198, 203, and 208 (antibody B06); n) SEQ ID NOs: 393, 398, and 403 and SEQ ID NOs: 408, 413, and 418 (antibody E04); s) SEQ ID NOs: 543, 548, and 553 and SEQ ID NOs: 558, 563, and 568 (antibody D06); and t) SEQ ID NOs: 573, 578, and 583 and SEQ ID NOs: 588, 593, and 598 (antibody E01), or having sequences that differ from the selected CDR sequences b), d), g), n) s), and t) by at most two conservative amino acid substitutions in each CDR.

[0206] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody or antigen-binding fragment includes three heavy chain CDRs and three light chain CDRs with sequences identical to the selected CDRs.

[0207] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody framework regions are human antibody framework regions.

[0208] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody is a full length bivalent monospecific monoclonal antibody.

[0209] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody includes human IgGl, IgG2, or IgG4 heavy chain constant regions.

[0210] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody includes a human IgGl constant region.

[0211] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody Fc region has engineered mutations that reduce antibody binding to FcRy and / or reduce complement fixation.

[0212] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody is a Fab, optionally wherein the Fab is PEGylated.

[0213] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody or antigen binding fragment is further capable of binding to cynomolgus monkey NBL1.

[0214] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody or antigen binding fragment is further capable of binding to mouse NBL1.

[0215] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 100 nM.

[0216] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 10 nM.

[0217] In some embodiments, the techniques described herein relate to an antibody or antigen binding fragment, wherein the antibody or antigen binding fragment has a binding affinity (KD) for human NBL1 of less than 5 nM.

[0218] In another aspect, the techniques described herein relate to a pharmaceutical composition, including: the antibody or antigen binding fragment, and a pharmaceutically acceptable carrier.

[0219] In some embodiments, the techniques described herein relate to a pharmaceutical composition, wherein the composition is formulated for parenteral administration.

[0220] In some embodiments, the techniques described herein relate to a pharmaceutical composition, wherein the composition is formulated for intravenous administration.

[0221] In some embodiments, the techniques described herein relate to a pharmaceutical composition, wherein the composition is formulated for subcutaneous administration.

[0222] In another aspect, the techniques described herein relate to a method for lowering or reducing urinary albumin / creatinine ratio in a subject who has type 1 or type 2 or a glomerular disease, the method including: administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity.

[0223] In some embodiments, the techniques described herein relate to a method, wherein the agent is an antibody, or an antigen binding fragment of an antibody, that is capable of binding to human NBL1.

[0224] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment includes the following three heavy chain CDRs: SEQ ID NOs: 93, 98, and 103, three light chain CDRs SEQ ID NOs: 108, 113, and 118 (antibody A 12), or having sequences that differ from the selected CDR sequences by at most two conservative amino acid substitutions in each CDR.

[0225] In some embodiments, the techniques described herein relate to a method, wherein the antibody or antigen-binding fragment includes three heavy chain CDRs and three light chain CDRs with sequences identical to the selected CDRs.

[0226] In some embodiments, the techniques described herein relate to a method, wherein the collagen positive area in the kidney of the subject is reduced from pre-treatment anti-NBLl monoclonal antibody A12 levels.

[0227] In some embodiments, the techniques described herein relate to a method, wherein the subject suffers no weight loss from pre-treatment measured weight while adhering to a standard diet regimen.

[0228] In some embodiments, the techniques described herein relate to a method, wherein the subject suffers about a 1% weight loss from pre-treatment measured weight while adhering to a standard diet regimen.

[0229] In some embodiments, the techniques described herein relate to a method, wherein the subject suffers less than a 5% weight loss from pre-treatment measured weight while adhering to a standard diet regimen.

[0230] In some embodiments, the techniques described herein relate to a method, wherein the subject suffers less than a 10% weight loss from pre-treatment measured weight while adhering to a standard diet regimen.6.9. Examples

[0231] Below are examples of specific embodiments for carrying out the present disclosure. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present disclosure in any way. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0232] The practice of the present disclosure will employ, unless otherwise indicated, conventional methods of protein chemistry, biochemistry, recombinant DNA techniques and pharmacology. Such techniques are explained fully in the literature.6.9.1. Example 1. NBL1 is directly toxic to podocytes and renal tubular cells

[0233] Increased DAN protein (Greml, Grem2, Grem3, Cerberus, NBL1, SOST, and USG1) levels have been associated with severe disease-states in adult kidneys. Wen et cd.. Biochimie 160: 113-121 (2019).

[0234] To assess whether the increased presence of a DAN protein has a causal effect on kidney damage, we cultured human podocytes (HuPodo), human mesangial cells (HuHMRC), and human renal tubular cells (HuK2) in vitro for 48 hours in the presence or absence of human NBL1. Human umbilical vein endothelial cells (Huvec) were used as a control. Podocytes were cultured in increasing concentrations of NBL1 : 0.2 pg / ml, 1.0 pg / ml, and 2.0 pg / ml. Mesangial cells and tubular cells were cultured in 2 pg / ml NBL1. Human umbilical vein endothelial cells were cultured in increasing concentrations of 0.2 pg / ml, 2 pg / ml, and 10 pg / ml NBL1.

[0235] FIGs. 1 A-1D are bar graphs summarizing cell death analysis data, quantified using arbitrary units (AU) for convenience. The results show increased apoptosis / death of human podocytes, and to a lesser degree mesangial and tubular cells, directly correlated to the added presence of NBL1 to culture media. Cell death was undetectable in NBL1 -cultured human umbilical vein endothelial cells (Huvec), even at higher concentrations.

[0236] FIGs. 2A-2B show representative images of confocal analysis conducted on human podocytes left untreated (FIG. 2 A) or cultured with NBL1 at 2 pg / ml (FIG. 2B), stained with Synaptopodin and Apoptag. Merged pictures are presented. These micrographs show evidence of NBL1 -induced apoptosis of human podocytes. The arrows highlight the colocalization of Apoptag and Synaptopodin, which are markers for apoptosis and podocytes respectively. The image emphasizes that the majority of podocytes are undergoing apoptosis. FIG. 2C is a bar graph quantifying the percentage of Apoptag+Synaptopodyn+double positive human podocytes in the presence / absence of NBL1 (n=3). The results indicate increased level of apoptotic differentiated human podocytes in the presence of NBL1.

[0237] FIG. 3 shows transcriptome analysis of apoptosis-related genes of human podocytes cultured with NBL1 at 2 pg / ml or left untreated. The data show significant increase in expression of apoptosis-related genes upon exposure to NBL-1 in vitro.

[0238] Collectively, these experiments show that NBLl is directly toxic to podocytes and renal tubular cells, with exposure to NBL1 causing apoptosis of human podocytes and renal tubular cells.6.9.2. Example 2. BMP proteins are not expressed in and are not secreted by kidney cells in vitro

[0239] Like other DAN proteins, NBL1 is known to interact with bone morphogenetic proteins. Hung etal., Biol, of Reprod. (2012) 86(5): 158, 1-9.

[0240] To determine whether NBL1 exerts its toxicity through inhibition of BMPs within the kidney, we analyzed whether BMP2, BMP4 and BMP7 were expressed as mRNA in human podocytes. We did not detect any mRNA expression of any of the BMPs. Next, we assessed whether BMP2, BMP4 or BMP7 are secreted from podocytes into the supernatant, thus allowing an autocrine / paracrine pro-survival effect. We did not detect any of the BMPs in the supernatants of human podocytes, thus confirming that podocytes are not able to synthesize and secrete BMPs in vitro (data not shown).

[0241] The toxic effect of NBL1 on podocytes is thus independent of the presence of BMPs; NBLl’s pro-apoptotic effect is not mediated via inhibition of BMPs.6.9.3. Example 3. NBL1 is not expressed in kidney cells, but is expressed in circulating immune cells, intestinal and muscle tissue.

[0242] Having determined that NBL1 is directly toxic to podocytes and renal tubular cells, we sought its physiological origin by measuring mRNA expression of NBL1 in a number of human cell lines, including kidney cell lines, using RT-PCR with expression normalized to beta actin expression. FIG. 4 shows that NBL1 was undetectable in kidney-derived cells. NBL1 was also undetectable in various other cell lines. However, it was highly expressed in immune cells, such as CD14+monocytes and CD4+and CD8+T cells (see FIG. 4).

[0243] Additional analysis using flow-cytometry of peripheral blood mononuclear cells (PBMCs) isolated from blood samples of healthy volunteers confirmed high expression of NBL1 protein in myeloid (CD14+) cells and various T cell subsets (CD3+, CD4+and CD8+T cells) (see FIGs. 5 A-5B). Human PBMCs were purified from 8 ml blood samples collected from non-diabetic subjects (n=5) at the ASST Sacco-FBF in Milan (Italy) by using Lymphoprep (07801, Stem Cell Technologies, Cambridge, MA) and cells were then stained for flow-cytometry analysis with anti-human CD3 (300330), anti-CD45 (560178), anti-CD4 (561030), anti-CD8 (560774), anti-CD14 (561707) from Biolegend (San Diego, CA) and BD Biosciences (San Jose, CA) to quantify surface expression. Rabbit polyclonal anti-NBLl (Sigma, HPA007394) followed by donkey anti-rabbit AlexaFluor488 antibody(ThermoFisher Scientific) was used to stain for NBL1. Cells were analyzed using a BD FACS Celesta (BD Biosciences).

[0244] To further explore the expression of NBL1, a comprehensive immunostaining study of NBL1 human tissue expression was performed. In this study, NBL1 expression was detected by immunohistochemistry and flow cytometry on human tissue specimens. NBL1 immunoreactivity was semi-quantitatively scored based on the percentage of NBL1 positive cells in the total cells per region of interest. The scoring was as follows: < 10%, 1+; 10-30%, 2+; 30-50%, 3+; 50-80%, 4+, and > 80%, 5+ Archival formalin-fixed, paraffin-embedded tissue samples of non-diabetic subjects from Pathology Unit, University of Parma were used forNBLl immunostaining (anti-NBLl primary antibody #HPA007394, Merck). A further magnification of each panel is shown in the insert on the top right black square, in which black arrows highlight the positive staining. Original magnification 20X, scale bar 100 um (See FIG. 5C).

[0245] A semi-quantitative score was also applied to NBL1 expression detected in T cells (CD3+NBL1+), B cells (CD19+NBL1+) and monocytes (CD14+NBL1+) (See FIG. 5A) based on the quantification of percentage of double positive cells as follows: < 15%, 1+; 15-45%, 2+; 45-75%, 3+; 75-90%, 4+, and > 90%, 5+

[0246] The semi-quantitative scores illustrating NBL1 protein expression in human tissues are illustrated in the bar graph presented in FIG. 5D. For histocytochemistry, the mean value of the score calculated in n=3 separate slides are presented. For flow cytometry, scores representing T cells, B cells and monocytes, mean values obtained in n=5 samples analyzed are presented. (See FIG. 5D).

[0247] The results of the study demonstrate that NBL1, which is not expressed in kidney, is expressed in circulatory immune cells, notably T cells and monocytes, in various intestinal tissues, and muscle, with lesser expression in reproductive tissues, bone and bone marrow..6.9.4. Example 4. Neutralizing NBL1 with an antagonist prevents toxicity

[0248] Having shown that NBL1 is toxic to podocytes and renal tubular cells, that this toxicity is directly mediated, and that NBL1 is not produced locally in renal cells but is instead expressed in circulating immune cells and other non-renal tissues, we tested whetherNBL1 could be an appropriate target for direct therapeutic intervention to protect kidney cells from damage.

[0249] Human podocytes were cultured for 48 hours in the presence of NBL1 (2 pg / ml) in the presence or absence of soluble BMP2 in a 1 : 1 ratio (NBL1 :sBMP2 = 1 : 1). Cell death was detected by ELISA. Incubation with soluble BMP2 significantly reduced the cell death effects experienced by human podocytes cultured in the presence of NBL1, indicating that soluble BMP2 is capable of partially neutralizing NBLl’s pro-apoptotic effect on human podocytes in vitro. Results for the above experiments are shown in FIG. 6. Three independent experiments were run in duplicate. Data are presented as mean ±SEM. A commercial anti-NBLl tool antibody (Sigma) showed a smaller effect (data not shown).6.9.5. Example 5. Human anti-NBLl antibody discovery campaign

[0250] Naive human phage display libraries were panned to discover Fabs capable of binding human NBL1 (UniProt ID P41271), and further screened for potential inter species cross reactivity to mouse NBL1 (UniProt ID Q61477) and / or cynomolgus (“cyno”) NBL1 (UniProt ID A0A2K5WIY3). The top 25 Fabs were cloned into expression vectors and expressed as full length human IgGl antibodies. Antigen binding of the IgGl formatted antibodies was tested by ELISA and EC50 values were calculated. Twenty (20) IgGl antibodies were shown to bind to human, mouse, and cyno NBL1 with EC50 in the ELISA of between 10 ng / mL and 100 ng / mL (~0 to 600 pM).

[0251] Tables presented in Section 6.3.2 above provide the VH and VL sequences, and separately, the CDR sequences, of the 20 IgGl antibodies.6.9.6. Example 6. Monoclonal anti-NBLl antibodies rescue NBL1- mediated apoptosis of human podocytes in vitro.

[0252] Having demonstrated that NBLl is directly toxic to podocytes and renal tubular cells, and that the toxicity can be averted using sBMP2, which is capable of specific binding to NBL1, we decided to assess the effects of monoclonal anti-NBLl antibodies on the cell death effects experienced by human podocytes cultured in the presence of NBL1. Of the monoclonal antibodies generated in Example 5, we tested 16 antibodies and assessed their ability to prevent cell death and apoptosis in human podocytes cultured in vitro.

[0253] Human podocytes were cultured in RPMI supplemented with 10% FBS and with ITS (IX). Human NBL1 recombinant protein was obtained from Genscript (Piscataway, NJ). Human monoclonal NBL1 antibodies obtained by cloning NBL1 -binding phage-displayed Fabs into a human IgGl format (Example 5 above) were tested at a concentration of 20 pg / ml.

[0254] The human podocytes were cultured for 48 hours in the presence / absence of human NBL1 (2 pg / ml) and in the presence / absence of our generated anti-NBLl mAbs (20 pg / ml) at a ratio of 1 : 1 (mAbs:NBLl). Cell lysates were collected at 48 hours of culture, and cell death / apoptosis was assessed by using ELISA (Roche Diagnostics GmbH, 11544675001, Mannheim, Germany). Quantification of cell death was normalized to untreated cells.

[0255] We observed that following incubation in the presence of these newly generated mAbs, a number of the mAbs were able to prevent / reduce NBL1 -induced cell death (p<0.0001) to varying degrees. Most importantly, 6 out of the 16 mAbs (YU1018-H08, YU1019-B06, YU1018-E01, YU1018-E04, YU1019-A12, YU1018-D06) were able to reduce the cell death effects experienced by human podocytes cultured in the presence of NBL1 to levels at or below the negative control (left-most bar, “Medium”), demonstrating that these mAbs are capable of completely neutralizing NBLl’s pro-apoptotic effect on human podocytes in vitro, which further indicates that among the tested antibodies, these antibodies will most potently be able to reduce progression of glomerular / renal disease in subjects with elevated levels of circulating NBL1, including human subjects with T1D and T2D. Results are shown in FIG. 8.

[0256] In the same experiment, we tested the ability of soluble BMP2 (1 pg / ml, at a 1 : 1 BMP2:NBL1 ratio), to neutralize the effects of NBL1. As shown in FIG. 8, mAbs H08, B06, and E01 were significantly more robust in the reduction of NBL1 -mediated apoptotic effects on human podocytes than sBMP2. This highlights the beneficial effectiveness of anti-NBLl antibodies in protecting podocytes from NBL1 -mediated toxicity and damage.6.9.7. Example 7. NBL1 is elevated in Type 1 Diabetes and Type 2 Diabetes

[0257] In order to determine whether NBL1 contributes to kidney damage in diabetes, NBL1 serum levels were assessed using an immunotargeted assay. NBL1 serum levels were measured in patients with long-standing type 1 diabetes (T1D, n=150) and compared withNBL1 serum levels observed in sera of non-diabetic subjects (n=l 5). NBL1 serum levels of patients with long-standing type 1 diabetes displayed a significant increase in NBL1. A parallel comparison of NBL1 serum levels in patients with type 2 diabetes (T2D, n=70) versus levels in non-diabetic subjects also displayed a significant increase in NBL1. Results are shown in FIG. 7A.

[0258] Subsequently, patients with T1D or T2D, and who had also received a diagnosis of diabetic kidney disease (DKD), were selected and we analyzed their NBL1 serum levels. The analysis demonstrated that patients with chronic kidney disease (CKD) at stage 2-3 (eGFR<60 ml / min / m2, n=60) had a 3-fold increase in NBL1 serum levels as compared to non-diabetic subjects and a 2-fold increase in NBL1 serum levels as compared to diabetic patients without CKD (see FIG. 7B).

[0259] Inhibition of NBL1 is therefore a new therapeutic approach for preventing onset and progression of kidney damage in a patient with type 1 or type 2 diabetes. Inhibition of NBL1 will also be effective in treating non-diabetes glomerular diseases in which damage is mediated by NBL1.6.9.8. Example 8. Identification of monoclonal anti-NBLl antibodies that are effective in reducing and / or neutralizing NBL1 activation of pro-apoptotic caspase-3 and caspase 7, and NBL1 induced cell death, in human podocytes in vitro.

[0260] Caspase-3 and caspase-7 perform effector functions in the execution phase of apoptosis. Having demonstrated that our monoclonal anti-NBL-1 antibodies are capable of rescuing NBLl-mediated apoptosis of human podocytes in-vitro, we decided to assess their effect on the activation of caspase 3 and caspase 7 in human podocytes cultured in the presence of NBL1.

[0261] Human podocytes were cultured in RPMI supplemented with 10% FBS and ITS (IX). Human NBL1 recombinant protein was obtained from Genscript (Piscataway, NJ). Human monoclonal NBL1 antibodies obtained by cloning NBLl-binding phage-displayed Fabs into a human IgGl format (Example 5 above) were tested at a concentration of 20 pg / ml.

[0262] The human podocytes were cultured for 48 hours in the presence / absence of human NBL1 (2 pg / ml) and in the presence / absence of our generated anti-NBLl mAbs (20 pg / ml) at a molar ratio of 1 : 1 (mAbs:NBLl). The human podocytes were also cultured for 48 hours in the presence of soluble BMP2 (1 pg / ml, at a 1 :1 BMP2:NBL1 ratio) and NBL1 (2 pg / ml) in the absence of our generated anti-NBLl mAbs. Similarly to Example 6, we also tested the ability of soluble BMP2 (1 pg / ml, at a 1 : 1 BMP2:NBL1 molar ratio) to neutralize effects of NBL1, this time with respect to NBL1 mediated caspase activation.

[0263] Cell lysates were collected at 48 hours of culture, and cell death / apoptosis was assessed by using ELISA (Roche Diagnostics GmbH, 11544675001, Mannheim, Germany). Quantification of caspase 3 and caspase 7 activation was normalized to untreated cells.

[0264] Results are shown in FIG. 9 and 10A. We observed that following incubation in the presence of our generated mAbs, a number of the mAbs were able to reduce NBLl-induced activation of caspase 3 and caspase 7 to varying degrees.

[0265] We also assayed mAbs El l, F06, E07, E05b, and the non-binding control mAb G09, for their ability to counter NBLl-induced cell death using the assay described in Example 6. Results are shown in FIG. 10B.

[0266] mAbs YU1018-E04 (E04), YU1019-A12 (A12), and YU1019-B06 (B06) were able to reduce the cell death effects experienced by human podocytes cultured in the presence of NBL1 to levels at or below the negative control (FIG. 8), and also modestly reduce the activation of pro-apoptotic effectors caspase 3 and caspase 7 (FIG. 9).

[0267] These antibodies were therefore selected for further testing.6.9.9. Example 9. Generated mAbs recognize native NBL1

[0268] Having demonstrated that recombinant NBL1 is directly toxic to podocytes and renal tubular cells, that the toxicity can be averted using our newly generated mAbs, and that the mAbs also reduce and / or neutralize the activation of pro-apoptotic effectors caspase 3 and caspase7, we assessed the binding capability of our monoclonal anti-NBLl antibodies on native NBL1.

[0269] Binding of the mAbs to native murine plasma NBL1 was performed using a sandwich ELISA, as illustrated in FIG. 11 A. Binding of the mAbs to murine splenocyte NBL1 was performed using an immunotarget assay as illustrated in FIG. 12 A, with NBL1 released from permeabilized murine splenocytes, and binding assessed by FACS.

[0270] The results demonstrate that mAbs A12 and H08 showed the greatest ability to bind murine plasma NBL1 (FIG. 11B) (p<0.01 vs negative control, unrelated protein), and that mAbs E04, A12 and B06 displayed the greatest binding signal to murine splenocyte NBL1 (FIG. 12B).

[0271] Second, having displayed our mAbs are effective in binding to native murine NBL1, we assessed whether the same was true for native human NBL1. PBMCs were isolated from blood samples of healthy volunteers. As illustrated in FIG. 13 A, the cells were permeabilized and mAb binding to native human NBLlwas assessed using an immunotargeted assay and the results quantified using FACS as illustrated in FIG. 13A. The resulting data indicated that mAbs E04, A12 and B06 provided the greatest binding signal. See FIG 13B.6.9.10. Example 10. Selection of monoclonal anti-NBLl antibodies

[0272] We narrowed down our selection of antibodies to E04, A12 and B06 based on semi- quantitative scoring using statistical analysis. The quantitative scores are arbitrary and used to indicate the differences among mAbs. See the table below.6.9.11. Example 11. NBL1 increases expression of apoptotic markers in kidney cells.

[0273] We proceeded to further confirm the toxic effect of NBL1 in human podocytes. We analyzed the expression of proapoptotic markers DAPK1, FADD and TNFRSF10A by mRNA quantification in human podocytes (HuPodo) treated with and without 2 pg / ml recombinant human NBL1 (HuNBLl).

[0274] We observed a 3-fold increase in mRNA expression of the three markers upon treatment with HuNBLl when compared to untreated cells. See FIG. 15A-C. This provided confirmation that NBL1 induces apoptosis / cell death in kidney cells.6.9.12. Example 12. NBL1 induces expression of proapoptotic and nephrotoxic markers in kidney organoids and affects kidney organoid development.

[0275] Using newly generated kidney organoids, three in vitro protocol treatments were established to address whether addition of NBL1 may affect the expression of lineage markers (podocytic, tubular and endothelial) at an earlier timepoint (8 days) or later on in organoid development (12 and 16 days).

[0276] Kidney organoids were differentiated from human embryonic stem cells (ESC) CHB8. ESCs were thawed in mTeSRTM medium (Stem Cell Technologies #85851 #85852), supplemented with ROCK inhibitor Y-27632 (Millipore #688001) and maintained in culture as colonies on matrigel-coated plates. Kidney organoids were generated using the STEMdiff™ Kidney organoid kit (Stem Cell Technologies #05160) according to manufacturer’s instructions. ESCs were collected and plated onto Matrigel-coated 96-well plates as single cell suspensions. Twenty -four hours later, cell monolayer was covered by a second layer of matrigel. After a full medium change, organoid differentiation began following the addition of Stage 1 medium, prepared diluting STEMdiff™ Kidney Basal Medium and STEMdiff™ Kidney supplement SG. After 36 hours, Stage 1 was substituted by Stage 2 medium, prepared diluting STEMdiff™ Kidney Basal Medium and STEMdiff™ Kidney supplement DM and changed every 2-3 days. After 8, 12 and 16 days from the beginning of differentiation respectively, kidney organoids were treated with human NBL1 (2 pg / ml). Finally, cells were collected for further use. Human podocytes and kidney organoids were treated with human NBL1 at 2 pg / ml, by addition to the medium (for 48 hours at 37°C, 5% CO2).

[0277] Human podocytes were established as conditionally immortalized cell line 28-29, grown, and maintained in RPMI medium (Euroclone #ECB9006LX10) supplemented with insulin-transferrin-selenium (ITS, Sigma Aldrich #13146), 100 units / ml of penicillin and 100 pg / ml of streptomycin (pen / strep, Thermo Fisher Scientific #15140122), 2 mM L-alanyl-L- glutamine dipeptide (glutamax, Thermo Fisher Scientific #35050087) and 10% fetal bovine serum (FBS, Cliniscience). Cells were cultured at 33°C / 5%CO2 until they reached 50-60% of confluence and then transferred to 37°C, 5%CO2 to trigger differentiation.

[0278] Human NBL1 was purchased from Aviva System Biology (OPCD00888) or custom produced by Genscript.

[0279] The choice of time points is reflective of the development timeline of kidney organoids: day 8, the organoid commences the development of nephron structures; day 12, the organoid expresses the podocytic, tubular and endothelial markers; day 16, the organoid displays differentiation and maturation. mRNA expression of podocytic markers POD XL and SYN, Tubular marker AQP1, and endothelial marker PEC AM were assessed at each timepoint. See FIG. 16A-B.

[0280] On day 12, mRNA expression of podocytic markers PODX and SYN demonstrated a reduction in expression, while tubular marker AQP1 was upregulated in the presence of HuNBLl (FIG. 16C). Interestingly, day 12 was also the time point mRNA expression of proapoptotic genes DAPK1 and FADD, and damaging nephrotoxic genes TGFB1 (transforming growth factor beta 1), GATM, N0X4 and COL41 was upregulated the most, thereby indicating this as key timepoint in which damage on podocytic structure is more evident. See FIG 17.

[0281] Also, note that as summarized in FIG. 18, soluble BMP2 cultured in the presence of NBL1 was able to arrest the increased expression of nephrotoxic markers COL4A1, GATM, and TNFRSF10A and to levels below normalization showing application for use as a therapeutic tool against NBL1 -mediated nephrotoxicity.

[0282] In summary, our data shows that NBL1 is toxic to human podocytes in vitro and that this is associated with the upregulation of mRNA expression of proapoptotic markers DAPK1 FADD and TNFRSF10A. The data also show that podocytic marker POD XL is downregulated after addition of NBL1 when organoids are treated at day 12, which is anearly timepoint of organoid development in which podocytic and tubular structure start to be formed, providing confirmation that in the kidney organoids model, the development of podocytes and related structure is targeted by NBL1 early on and the effect is more evident as compared to that observed on endothelial and tubular markers. Finally, we have shown that NBL1 promotes apoptosis in kidney organoids in vitro and that at day 12 of development, podocytes differentiate and addition of NBL1 has an effect on podocyte markers.6.9.13. Example 13. NBL1 induces nephrotoxicity in vivo.

[0283] Next, to characterize NBLl-induced nephrotoxicity in vivo, we administered human recombinant NBL1 protein to wild-type C57BL6 / J mice (Fig. 19). Eight- to ten-week-old male wild type C57BL / 6J were purchased from Charles River and housed in a pathogen-free, temperature-controlled environment with 12-h day and night cycles. They received water and food ad libitum.

[0284] An acute kidney injury model was established by injecting 500 pg of human recombinant NBL1, diluted in PBS, intraperitoneally (z. / z), once daily, for 3 days (Acute protocol 1) or 4 days (Acute protocol 2). FIG. 19. Before and after treatment, mice were housed in metabolic cages for maximum 12 hours and urine was collected for further analysis. At sacrifice, kidneys were collected for histopathological examination. Two groups were tested for each protocol. Schedule of treatment, animal numerosity and time points for analysis are indicated in FIG.19. Human NBL1 was custom produced by Genscript.

[0285] In a first set of experiments, we injected 500 pg of recombinant protein thrice (on day 1, day 2 and day 3, total amount injected 1.5 mg, Acute protocol 1). Alternatively, we treated mice with 500 pg of HuNBLl four times, once daily (on days 1, 2, 3 and 4, total amount injected 2 mg, Acute protocol 2). We quantified urine albumin and creatinine as surrogate markers of renal functions. Of note, two weeks after treatment (T2) with 1.5 mg (protocol 1) or 2 mg of HuNBLl (protocol 2), mice exhibited a significant increase of urine albumin and creatinine concentrations when compared to vehicle-treated mice. Moreover, proteinuria persisted after a month (T3) from injection of HuNBLl (2 mg, protocol 2, FIG. 20). Concomitantly, 30 days after administration, HuNBLl administration significantly reduced urine volume (510 ± 83.81 ml vs. 1044 ± 127.8, p<0.05, FIG. 21).

[0286] Next, we evaluated HuNBLl -induced structural damage to glomeruli by performing a histopathological analysis of kidney tissues after periodic acid-Schiff staining (PAS). Notably, HuNBLl administration altered glomeruli morphology and caused their apparent shrinkage. In addition, HuNBLl induced the deposition of amorphous material surrounding glomeruli, particularly in the acute protocol 1 (FIGs. 22A-B).

[0287] In conclusion, the morphological and functional data provide that elevated levels of HuNBLl injected in mice reduced renal functions, as evidenced by progression to proteinuria, and caused morphological changes of glomeruli.6.9.14. Example 14: Anti-NBLl mAbs reduce podocytopathy and the progression of kidney injury.

[0288] Eight- to ten-week-old male wild type C57BL / 6J were purchased from Charles River and housed in a pathogen-free, temperature-controlled environment with 12-h day and night cycles. They received water and food ad libitum.

[0289] Acute kidney injury was established by injecting 500 pg of human recombinant NBL1, diluted in PBS, intraperitoneally (i.p. once daily, for 4 days. Anti-NBLl mAbs A12 and B06 were administered the same day of the first injection at a dose of 0.5 mg / mouse and at day 4, and then once a week 0.25 mg / mouse until day 28 (FIG. 23). Before and after treatment, mice were housed in metabolic cages for maximum 12 hours and urine was collected for further analysis. At sacrifice, kidneys were collected for histopathological examination. Human NBL1 was custom produced by Genscript.

[0290] Urinary albumin was measured by using a mouse albumin ELISA kit from Novus Biologies (NBP2-60484), Biotechne, Italy as per manufacturer’s instructions. Urinary creatinine was measured by using a Mouse Creatinine Assay Kit (#80350, Crystalchem, The Netherlands). Urine samples were collected overnight from mice being maintained in single metabolic cages.

[0291] Morphological analyses were conducted on renal samples obtained when mice were euthanized. The various samples were fixed in a 10% buffered formalin solution and routinely embedded in paraffin tissue blocks from which 3-5-pm-thick histological sections were obtained from each case. One slide was stained with hematoxylin and eosin (H&E) for general morphological evaluation, while additional unstained slides were used forimmunohistochemical analysis. In short, sections were dewaxed in xylene, rehydrated through graded alcohols, and after inhibition of the endogenous peroxidase with a 3% H2O2 water solution, the specimens were incubated with a protein block (Ready to Use Dako Biotin Blocking System, Carpenteria, CA).

[0292] Note, the methodology for urinary albumin and creatinine study provided above was followed in other related urinary studies that occur in later Examples 15, 16, and 18.

[0293] Results indicated a reduction in creatinuria evident in A12-treated mice and in B06-treated mice (FIG. 24A-24B). A smaller reduction was observed in the latter. No effect was observed in albuminuria and at the histopathological analysis (not shown).

[0294] In summary, our results indicate that administration of anti-NBLl mAbs counteracted the detrimental effect of HuNBLl on renal function.6.9.15. Example 15: Targeting NBL1 pathway improves renal morphology and function in STZ-induced kidney damage in murine model of diabetic kidney disease in vivo.Materials and Methods'.

[0295] Eight- to ten-week-old male wild type C57BL / 6J were purchased from Charles River and housed in a pathogen-free, temperature-controlled, environment with 12-h day and night cycles. They received water and food ad libitum.

[0296] Streptozotocin (STZ) was injected into naive mice to induce diabetes and diabetic kidney disease. Streptozotocin was injected (150 mg) once intraperitoneally (i.p.) at day 0 and animals were treated with anti-NBLl mAbs A12 or B06 or E04 the following day at a dose of 0.5 mg / mouse and then every week with a dose of 0.25 mg / mouse until day 24. A group of mice was treated with Streptozotocin alone. Glycemia was monitored 2 days / week. Animals were housed in metabolic cages for urine collection at 1 week and at the end of the study, when animals were euthanized, and kidney were collected for pathology (FIG. 25).

[0297] NBL1 protein peripheral levels were tested in the STZ-treated mice. NBL1 protein levels were increased in mice developing DKD as compared to those who did not. Anti- NBLl mAbs were then administered at a dose of 0.5 and 0.25 mg on a weekly basis. The mice demonstrated an improvement of renal function parameters albuminuria and creatinuriaat the end of the study. A morphological analysis also proved an improvement in mesangial expansion when anti-NBLl mAbs were administered. FIG. 26A-26B.

[0298] A reduction in albuminuria and creatinuria was evident in animals treated with each of the 3 anti-NBLl mAbs at the latest timepoint (FIG. 26A-26B, FIG. 27A-27B, and FIG. 28A-28B), with A12 and E04 being those with the most powerful effect as compared to B06, particularly in the albuminuria reduction (FIG. 29A-29B). The pathology analysis also revealed a reduced mesangial expansion in animals treated with anti-NBLl mAbs, particularly in the groups receiving A12 or E04, while B06 results showed less effect (FIG. 30A-30D). A quantification based on a semiquantitative score further proved that kidneys of mice treated with A12 had areas with no mesangial expansion and comparable to nondiabetic kidney (FIG. 30E). These data emphasize that anti-NBLl mAbs improves diabetic kidney damage both morphologically and functionally.

[0299] In summary, this study demonstrates that the anti-NBLl mAbs have a positive effect in preventing DKD onset in the STZ-induced diabetes model.6.9.16. Example 16: Targeting NBL1 pathway improves renal morphology and function in Db / Db in vivo model of murine diabetic kidney disease

[0300] Mice homozygous for the diabetes (Leprdb) mutations were used as a study model. Db / Db mice exhibit obesity, hyperglycemia, hyperinsulinemia, and pancreatic beta cell atrophy and progress to a final stage of DKD. Db / Db mice were purchased from Charles River through The Jackson Laboratories.

[0301] We injected A12 and E04 in Db / Db mice starting at 9 weeks of age at 0.5 mg / mouse and on a weekly basis at a dose of 0.25 mg / mouse until 24 weeks of age. A group of untreated controls was also included. Glycemia was monitored 2 days / week. Animals were housed in metabolic cages for urine collection at 9 weeks of age (baseline), at 13 weeks of age and at 24 weeks of age, when animals were euthanized and kidneys were collected for pathology (FIG. 31). The Db / Db-mice NBL1 protein peripheral levels were tested. NBL1 protein levels were increased in mice developing DKD as compared to those who did not. A group of Db / Db mice received anti-NBLl mAb A12 and a group received anti-NBLl mAb E04 at the following doses: 0.5 mg at day 0, then 0.25 mg once a week until week 24.

[0302] Results'. A reduction in albuminuria and creatinuria was evident in animals treated with A12 anti-NBLl mAbs at the early timepoint (week 13), when the hyperglycemia- mediated kidney damage starts to develop, and at the end of the study (week 24) when the damage is more evident (FIG. 32A-32B). Conversely, no effect was observed in animals treated with E04 mAb on either albuminuria or creatinuria at 24 weeks (FIG. 33A-33B), therefore suggesting that A12 showed a more powerful effect in this specific model of DKD.6.9.17. Example 17. Anti-NBLl antibodies reduced mesangial expansion in NBL1 treated mice kidneys in vivo

[0303] Pathology analysis of the Db / Db animals in Example 16 revealed reduced mesangial expansion in animals treated with A12 anti-NBLl mAb (FIG. 34B). A lesser reduction was observed in those that received the E04 anti-NBLlmAb (FIG. 34C). Remarkably, quantification based on a semi quantitative score further proved that kidneys of mice treated with A12 anti-NBLl mAb had areas with no mesangial expansion and were comparable to non-diabetic kidney (FIG. 34D). This analysis was not available for E04 due to the lack of glomeruli available for the evaluation and the tissue damage in the samples collected.

[0304] These data emphasize that anti-NBLl mAbs improves diabetic kidney damage both morphologically and functionally in the gold-standard model of in vivo studies in DKD.6.9.18. Example 18: Anti-NBLl mAbs reduce expression of podocytic and fibrotic markers in the presence of NBL1

[0305] To confirm the positive effect of anti-NBLl mAbs, we analyzed the expression of the podocytic markers PODXL, NPHS1, NPHS2 in kidney organoids by mRNA quantification at day 12 cultured for 48 hours with / without recombinant HuNBLl (2 pg / ml).

[0306] Kidney organoids were differentiated as earlier described in Example 12. After 12 days from the beginning of differentiation, kidney organoids were treated with human NBL1 (2 pg / ml) and with anti-NBLl human mAbs A12, B06, E04 (1 : 1 ratio with NBL1, 20 ug / ml). Finally, cells were collected for qRT-PCR analysis.

[0307] qRT-PCR: RNA from kidney organoids was extracted using Trizol Reagent (Thermo Fisher Scientific #15596026) and RNA retrotranscribed using the High-Capacity cDNA Reverse Transcription Kit (Thermo Fisher Scientific # 4368813). Thereafter, gene specific amplification was performed using TaqMan gene-specific assays (Thermo Fisher Scientific #4331182) according to the manufacturer’s instructions on a QuantStudio S6 Real Time System. Finally, threshold cycle (Ct) values of genes of interest were subtracted to Ct values of the housekeeping gene ACTB generating ACts; fold change of expression were calculated.

[0308] Kidney organoids were cultured with / without NBL1 in vitro and with / without antibodies A12, E04, or B06. The results indicated that the expression of podocytic markers PODXL, NPHS1 and NPHS2, were downregulated in presence of NBL1, but were restored when anti-NBLl mAbs are added to cultured organoids. These results confirm that NBL1 blockade has a positive effect in promoting the development of the podocytic component of kidney organoids. FIG. 35B-35D.

[0309] Kidney organoids and human podocytes were cultured with human NBL1 with and without anti-NBLl mAbs A12 or B06. The procedure was in accordance with similar related methodology already provided above. Fibrotic marker TGFp mRNA expression was monitored. Our results indicate that anti-NBLl mAbs are effective in reducing TGFP mRNA expression even in the presence of hNBLl in human podocytes and kidney organoids. FIG. 36.6.9.19. Summary of experimental results

[0310] Our results have demonstrated: (i) that anti-NBLl monoclonal antibodies markedly reduced NBL1 -induced podocyte death in vitro and rescued the expression of podocyte markers in human kidney organoids, (ii) that when administered in vivo, anti-NBLl mAbs prevented the increase of albumin and creatinine in urine commonly observed in diabetic mice; (iii) that NBLl blockade reduced mesangial expansion as observed in PAS-stained kidney sections; and (iv) finally, that anti-NBLl ameliorated fibrosis and cell death by reducing expression of specific markers.6.9.20. Example 19: Anti-NBLl mAb All and E04 preferentially bindNBL1 amongst other DAN proteins

[0311] To determine specificity for NBLl, we performed absorbance spectroscopy of anti- NBLl antibodies against NBL1 and other DAN protein family members. See FIG 37C. The antibodies were tested against GREM2 and NBL1 and then against a broader set of DAN protein family members of SOST, CER1, GREM1 and COCO. We observed that anti-NBLlantibody A 12, and E04 to a lesser extent, preferentially bind NBL1 among DAN family proteins: GERM2, SOST, CER1, GRM1 and COCO. See FIGs. 37A-37B.6.9.21. Example 20: Additional Materials and Methods a. Ex vivo analysis

[0312] Urinary albumin was measured by using a mouse albumin ELISA kit from Abeam (#ab 108792) Italy as per manufacturer’s instructions. Urinary creatinine was measured by using a Mouse Creatinine Assay Kit (#80350, Crystalchem, The Netherlands). Urine samples were collected overnight from mice being maintained in single metabolic cages. Morphological analyses were conducted on renal samples obtained when mice were euthanized. The various samples were fixed in a 10% buffered formalin solution and routinely embedded in paraffin tissue blocks from which 3-5-pm-thick histological sections were obtained from each case. One slide was stained with Masson Trichrome staining for general morphological evaluation and collagen positivity / deposition. In short, sections were dewaxed in xylene, rehydrated through graded alcohols, and after inhibition of the endogenous peroxidase with a 3% H2O2 water solution, the specimens were incubated with a protein block (Ready to Use Dako Biotin Blocking System, Carpenteria, CA). b. NBL1 atlas immunofluorescence study

[0313] Anti-human NBL1 (Atlas antibodies #HPA007394) and anti-human TDG (Abeam# ab 154192) specific antibody were coupled, in house, with APC and PE fl orescent molecules, respectively, by using the conjugation kit - lightning link and following manufacturer’s instructions (Abeam #ab201807 and #abl02918). HuPodo were plated onto glass coverslips and fixed using 4% formaldehyde diluted in PBS for 30 minutes at room temperature. After permeabilization with PBS / 0.25% triton, aspecific binding sites were saturated by incubation with PBS / 5% BSA for 30 minutes at room temperature. Cells were incubated with the anti-NBLl and anti-TDG antibodies diluted in PBS / 5% BSA overnight at 4°C. After washing, nuclei were stained with DAPI and images were acquired at the confocal microscope Leica TCS SP8. c. Wilms’ tumor immunohistochemistry experiment-n-

[0314] Harvested mouse kidney tissue were washed with PBS and antigen retrival was performed by boiling slices in sodium citrate (pH 6) buffer. Immunohistochemical staining was performed by using the UltraTek HRP anti-polyvalent (DAB) kit (Histo-line laboratories #AMF080) according to manufacturer’s instructions. Briefly, aspecific binding sites were blocked with peroxy block and super block reagents. Then, slices were stained with anti-WTl antibody (Abeam, #ab89901) diluted in PBS / BSA 1% for 20 hours at 4°C. After washing with PBS, slices were incubated for 10 minutes with ultratek polyvalent biotinylated antibody. Finally, the addition of streptavidin-HRP (horse radish peroxidase) and DAB (3,3'- diaminobenzidinesubstrate) allowed detection of Wilms’ tumor 1 gene (WT-1) positive regions.

[0315] Analysis was carried out by using ImageJ software. Regions WT-1 positive (or number of podocytes) were counted after color deconvolution and thresholding and number of podocytes was normalized to glomerular areas.6.9.22. Example 21: 13-week in-vivo response study for effects of anti- NBL1 A12 antibody in diabetic mice.

[0316] A 13-week in-vivo response study was performed to assess the effects of 3 separate ascending doses of A12 anti-NBLl mAb on the Db / Db mouse model. Mice homozygous for the diabetes (Leprdb) mutations, Db / Db, exhibit obesity, hyperglycemia, hyperinsulinemia, and pancreatic beta cell atrophy. Untreated, these Db / Db mice progress to a final stage of diabetic kidney disease (DKD).

[0317] In this study, the mice were separated into 4 treatment groups: A12 high dose, A12 mid dose, A12 low dose, and placebo. There were 10 mice in each treatment group. The study commenced when the mice were 11 weeks old and was ran to time 24 weeks of age for each mouse. The mice were dosed once a week by injection for 13 weeks.

[0318] The anti-NBLl mAb A12 doses were as follows:Dose 1, high dose: each mouse received an initial one-time weekly dose of 1.5 mg at 11 weeks of age. Thereafter the mice received a weekly dose of 0.75 mg up until 24 weeks of age.Dose 2, mid dose: each received an initial one-time weekly dose of 0.5 mg at 11 weeks of age. Thereafter the mice received a weekly dose of 0.25 mg up until 24 weeks of age.Dose 3, the low dose: each received an initial one-time weekly dose of 0.1 mg at 11 weeks of age. Thereafter the mice received a weekly dose of 0.1 mg up until 24 weeks of age.The placebo dose was phosphate buffered saline (PBS) solution.

[0319] At selected timepoints - 11 weeks of age, 13 weeks and 24 weeks of age - mice were housed in metabolic cages for 12 hours and urine was collected for functional analysis. At sacrifice (post-24 weeks of age), kidneys were collected for histopathological examination. Plasma samples were also collected and analyzed. FIG. 38.6.9.23. Example 22: Positive effect of anti-NBLl A12 antibody on urinary albumin and mesangial expansion in diabetic mice is dose-responsive

[0320] The effects of anti-NBLl A12 antibody treatment on levels of urinary albumin were determined in the Db / Db mice. The results indicated that all three A12 doses resulted in a significant reduction of urinary albumin versus placebo. We note that the comparative differences versus placebo significantly increased along the length of the study. FIG. 39A. Additionally, at 24 weeks of age, the A12 high dose showed improved reduction of urinary albumin compared to A12 low and A12 mid doses. FIG. 39B. This result demonstrated a dose response effect of anti-NBLl mAb A12 on urinary albumin.

[0321] Next, we sought to confirm the protective effects of NBL1 blockade in the reduction of mesangial expansion and improvement on glomerular damage. A Masson staining analysis was performed on kidney cells obtained from the mice at 24 weeks of age. The results showed that collagen deposition, as measured by percentage collagen positive area, is particularly reduced with the A12 high and A12 mid doses compared to placebo. In comparison to placebo, the A12 low dose showed less reduction in collagen positive area compared to A12 high and A12 mid doses. FIG. 41B. The A12 high dose significantly improved the glomerular morphology and reduced the mesangial expansion as compared to the A12 low and A12 mid doses. FIGs. 41 A-41B. The quantitative data showed a dose response effect of mAb A12 for mesangial expansion.6.9.24. Example 23: Anti-NBLl A12 antibody is effective to reduce urinary ACR levels compared to placebo in diabetic mice.

[0322] We assessed the protective effects of A12 low, A12 mid, and A12 high doses versus placebo on renal function via their effect on urinary albumin / creatinine ratio (ACR). UrinaryACR represents the main parameter measured in patients with diabetic kidney disease (DKD) to evaluate their stage of disease.

[0323] Results showed that the A12 low, A12 mid, and A12 high doses were all able to reduce the urinary ACR along the course of study (FIG. 40 A), particularly at 24 weeks of age (13 weeks of dosing) as compared to placebo (FIG. 40B). A clear dose response was not observed. However, the data shows a trend for greater urinary ACR reduction at A12 high compared to A12 low. FIG. 40B.6.9.25. Example 24: Anti-NBLl mAb A12 high dose outperforms gold standard for DKD treatment, liraglutide, in reducing urinary albumin in diabetic mice.

[0324] We compared anti-NBLl mAb A12 (mid dose) versus GLP-1 receptor agonist, liraglutide, in Db / Db diabetic mice. Liraglutide is the current gold standard for treatment of diabetic kidney disease in humans and select animals.

[0325] The mice receiving the anti-NBLl mAb A12 were dosed according to the mid-dose schedule described in section 6.9.22, also illustrated in FIG. 42A.

[0326] Liraglutide (Saxenda®) was administered at 20 ug / mouse injected intraperitoneally (i.p.) daily. In a 10 week study, liraglutide treatment began at 11 weeks of age and terminated at 20 weeks of age. FIG. 42B. Notably, the dose of 20 ug in a mouse weighing 40-50 g is comparable to the maximum daily dose administered in human patients with diabetes (1.8 mg, up to 2.4 mg, subcutaneously for a diabetic individual of 70 Kg weight and low need for weight loss). Regarding the choice of route of administration, we had generated data (not shown) that i.p. administration of liraglutide (20 ug / mouse daily for 4 weeks) in C57BL6 / J mice in which diabetes was induced through single injection of Streptozotocin (150 mg / Kg) was effective in decreasing blood glucose levels (mean: 347 vs. 186 mg / dl).

[0327] The protocols used for these experiments have been described in Fiorina et al., J Am Soc Nephrol, 2014. This reference is incorporated herein by reference in its entirety.

[0328] Urine was collected for functional analysis at the following selected timepoints: 10, 13, 17 and 24 weeks of age. Mice were sacrificed following the 24thweek of age. Kidney samples were collected for histopathological examination. Plasma samples were also collected and analyzed.

[0329] Having shown that anti-NBLl mAh is effective to reduce urinary albumin, urinary ACR, and mesangial expansion in Db / Db mice, we compared how well mAb A12 affected DKD parameters in comparison to the current gold standard therapy, liraglutide.

[0330] Notably, the comparative results showed that A12 dose (high) was more effective than liraglutide in reducing urinary albumin along the course of treatment. FIG. 43 A. A comparison of urinary albumin reduction at 24 weeks of age demonstrated that A12 (mid) and A12 (low) doses lowered urinary albumin similarly to liraglutide. It is noted that the A12 (high) dose had a more powerful effect than liraglutide. FIG. 43B. These surprising results were further confirmed by comparison of fold change in urinary albumin versus baseline data. FIG. 44.

[0331] We also measured the effects of the treatments on urinary ACR. The results showed an improvement in ACR for liraglutide and for all three doses of anti-NBLl mAb A12 compared to placebo over the course of study. FIG. 45B. At 24 weeks of age, liraglutide and all anti-NBLl A12 mAb doses significantly reduced ACR in comparison to placebo. FIG. 45A-45B. Note that at 24 weeks of age, no statistically significant difference was observed among the different doses for A12 compared to liraglutide. FIG. 45B.

[0332] To confirm the results, we plotted fold change difference in ACR at 24 weeks of age in each treatment group and confirmed that all A12 doses, especially the high dose, provides a comparative ACR reduction benefit versus liraglutide treatment. FIG. 46.6.9.26. Example 25: Anti-NBLl mAb A12 doses achieve comparable results to liraglutide, the gold standard for reducing mesangial expansion in diabetic mice.

[0333] Next, we then analyzed the effects of the A12 high dose, liraglutide, and placebo therapy on mesangial expansion by using Masson Trichrome staining to evaluate collagen positivity within the glomeruli in samples taken at 24 weeks of age. Not surprising, the results showed that mesangial expansion is reduced following liraglutide therapy as compared to placebo. However, the results also showed that the A12 high dose was more powerful and statistically significantly different compared to liraglutide. FIG. 47A-7B.

[0334] Finally, to prove the relevance of our models, we demonstrated that while liraglutide was able to reduce blood glucose levels along the course of the study as compared to placebo-treated animals, the anti-NBLl A12 antibody demonstrated a specific effect on nephrotoxicity independent of glucose levels modulation. FIG. 48A. Applicants also demonstrated that mice on liraglutide therapy demonstrated significant weight loss over time as compared to placebo-treated mice. However, mice on anti-NBLl A12 doses did not show reduction in weight. FIGs. 48A-48B. Note that measurement of murine NBL1 (mNBLl) levels in serum confirmed elevated levels in untreated and placebo-treated animals as compared to non-diabetic Db / Db animals (data not shown).6.9.27. Example 26: Anti-NBLl A12 antibody prevents albuminuria inDb / Db mice

[0335] The homozygous Db / Db mouse model was used to study the effect of anti-NBLl mAbs on albuminuria in diabetic conditions that model human Type 2 diabetes. FIG. 49A. Mice were administered A12 mid antibody dose once a week. Mice in the control group received no treatment. Treatment began in the mice at 11 weeks of age and terminated at the end of their 24thweek of age, after which they were sacrificed and kidney samples collected and analyzed.

[0336] The results showed that circulating NBL1 levels were greatly increased in homozygous Db / Db mice versus heterozygous Db / + mice. FIG. 49B. The results also showed that anti-NBLl antibody A12 mid dose was effective to lower urine albumin levels in Db / Db mice versus control at 13 weeks of age and 24 weeks of age in the study.6.9.28. Example 27: Anti-NBLl A12 antibody prevents collagen deposition in Db / Db mice

[0337] Masson staining analysis performed on cross-section of glomeruli histology slides obtained from mice following termination of the study at week 25 showed that mice treated with anti-NBLl antibody A12 mid dose displayed improved glomerular morphology compared to control. In detail, the improved glomerular morphology showed reduced mesangial expansion and collagen deposition stems, resulting in improved glomerular structure. FIG. 50A. Furthermore, treated mice showed reduced collagen positive area and collagen staining mean intensity when compared to control, FIG. 50B upper and lower panel respectively.6.9.29. Example 28: NBL1 blockade by anti-NBLl antibody A12 antibody preserves podocytes in Db / Db mice

[0338] The Wilms tumor 1 gene (WT1) plays a crucial role in kidney development and function and maintenance of podocytes. We decided to examine the impact of anti-NBLl A12 (mid) on WT1 expression in Db / Db mice versus untreated mice.

[0339] Mice were dosed with A12 for 24 weeks. Mice received an initial one-time weekly dose of 0.5 mg at 11 weeks of age. Thereafter they received a weekly dose of 0.25 mg up until 24 weeks of age. The untreated mice represented the control group. At 25 weeks of age, the mice were sacrificed and organs harvested.

[0340] We observed that in kidneys of untreated mice, WT-1 positive cells were very few in the glomeruli. However, in the A12 treated mice, the number of WT-1+ cells per glomerulus / per area were significantly recovered at week 25. FIG. 51A-51B. (p<0.0001). These results suggest that anti-NBLl antibodies provide protective effects directly on podocytes in anti-NBLl treated animals.6.9.30. Example 29: NBL1 is primarily released by cells of the small intestine

[0341] Finally, we performed an additional in vitro Atlas study to map NBL1 secretion in human tissues. We had previously demonstrated that NBL1 is highly expressed in circulatory immune cells, notably T cells and monocytes, in various intestinal, muscle, and reproductive tissues, as well as bone and bone marrow. WO 2023 / 006850A1. This reference is herein incorporated by reference in its entirety. We now performed an in-depth study to better understand NBL1 secretion. Major primary cells and / or cell lines were screened for NBL1 secretion in vitro. The cells screened included blood and intestinal epithelial cells, cardiomyocytes and myocytes and renal cells by ELISA. The results demonstrated that, NBL1 is primarily released by cells of the small intestine, and to a lesser extent by mesangial cells. FIG. 52A. We then further tested whether inflammatory cytokines and / or high glucose levels, known features of DKD, may increase NBL1 secretion in intestinal cells. Human intestinal epithelial cell-6 (HIEC) and human colon epithelial cells (Caco-2) were exposed to high concentrations of glucose (35mM) and inflammatory cytokines (interleukin- lb (IL- 1 (3) and interferon-gamma (IFN-y), at 2 ng / ml and 1,000 U / ml respectively) for 48 hours. The results showed that significant secretion of NBL1 under high levels of glucose and inflammatory cytokines - supporting that intestinal epithelial cells are the primary source of NBL1. FIG. 52B. This observation suggests that NBL1 is mainly released by the smallintestine and that the diabetogenic environment increases NBL1 release into circulation and may explain elevated levels found in patients with DKD.7. EQUIVALENTS AND INCORPORATION BY REFERENCE

[0342] While the invention has been particularly shown and described with reference to a preferred embodiment and various alternate embodiments, it will be understood by persons skilled in the relevant art that various changes in form and details can be made therein without departing from the spirit and scope of the invention.US provisional application no. 63 / 675,702, filed July 25, 2024, is incorporated herein by reference in its entirety for all purposes. Furthermore, all references, issued patents and patent applications cited within the body of the instant specification are hereby incorporated by reference in their entirety, for all purposes.

Claims

WHAT IS CLAIMED IS:

1. A method for lowering or reducing urinary albumin / creatinine ratio in a subject who has type 1 or type 2 diabetes or a glomerular disease, the method comprising: administering to the subject an effective amount of an agent capable of inhibiting NBL1 activity.

2. The method of claim 1, wherein the agent is an antibody, or an antigen binding fragment of an antibody, that is capable of binding to human NBL1.

3. The method of claim 2, wherein the antibody or antigen-binding fragment comprises three heavy chain variable region (VH) CDRs and three light chain variable region (VL), wherein(a) the three VH CDRs respectively have the sequences of SEQ ID NO: 93, SEQ ID NO: 98, and SEQ ID NO: 103, and(b) the three VL CDRs respectively have the sequences of SEQ ID NOs: 108, 113, and 118; or having sequences that differ from said CDR sequences by at most two conservative amino acid substitutions in each CDR,4. The method of claim 3, wherein the antibody or antigen-binding fragment comprises three VH CDRs and three VL CDRs with sequences identical to the selected CDRs.

5. The antibody or antigen binding fragment thereof of claim 4, comprising a VH having the sequence of SEQ ID NO: 668.

6. The antibody or antigen binding fragment thereof of claim 4, comprising a VL having the sequence of SEQ ID NO:669.

7. The antibody or antigen binding fragment thereof of claim 4, comprising a VH having the sequence of SEQ ID NO:668 and a VL having the sequence of SEQ ID NO:669.

8. The method of any one of claims 2-7, wherein the antibody framework regions are human antibody framework regions.

9. The method of any one of claims 2-8, wherein the antibody is a full length bivalent monospecific monoclonal antibody.

10. The method of claim 9, wherein the antibody comprises human IgGl, IgG2, or IgG4 heavy chain constant regions.

11. The method of claim 10, wherein the antibody comprises a human IgGl constant region.

12. The method of any one of claims 2-11, wherein the antibody comprises a moiety that extends serum half-life.

13. The method of claim 12, wherein the half-life extension moiety is at least one covalently linked polyethylene glycol (PEG) moiety to the Fc region of the antibody.

14. The method of any one of claims 2-13, wherein the antibody Fc region has at least one engineered mutation that reduces antibody binding to at least one type of Fc receptor.

15. The method of claim 14, wherein the engineered mutation is N297A.

16. The method of any one of claims 2-15, wherein the antibody Fc region has at least one engineered mutation that reduces complement fixation.

17. The method of claim 16, wherein the engineered mutation is K322A18. The method of any one of claims 2-8, wherein the antibody is a Fab, optionally wherein the Fab is PEGylated.

19. The method of any one of claims 2-18, wherein the antibody or antigen binding fragment thereof has a binding affinity (KD) for human NBL1 of less than 100 nM.

20. The method of claim 19, wherein the antibody or antigen binding fragment thereof has a binding affinity (KD) for human NBL1 of less than 10 nM.

21. The method of claim 19, wherein the antibody or antigen binding fragment thereof has a binding affinity (KD) for human NBL1 of less than 5 nM.

22. The method of claim 1, wherein the agent comprises a bone morphogenetic protein (BMP) or soluble fragment thereof.

23. The method of claim 22, wherein the agent comprises a soluble fragment of human BMP-2.

24. The method of claim 1, wherein the agent is capable of inhibiting dimerization of NBL1.

25. The method of any one of claims 1-24, wherein the agent is administered parenterally.

26. The method of claim 25, wherein the agent is administered intravenously.

27. The method of claim 25, wherein the agent is administered subcutaneously.

28. The method of any one of claims 1-27, wherein the agent is administered for at least 3 months.

29. The method of claim 28 wherein the agent is administered for at least 6 months.

30. The method of claim 29, wherein the agent is administered for at least 12 months.

31. The method of any one of claims 1-30, wherein the subject has clinically determined elevated pre-treatment plasma levels of NBL1.

32. The method of claim 31, wherein the subject has clinically determined elevated pretreatment levels of NBL1 in kidney tissue.

33. The method of any one of claims 1-32, wherein the subject has type 1 diabetes.

34. The method of any one of claims 1-28, wherein the subject has type 2 diabetes.

35. The method of any one of claims 1-34, wherein the subject has a glomerular disease.

36. The method of claim 35, wherein the subject with glomerular disease does not have type 1 diabetes or type 2 diabetes.

37. The method of claim 35 or claim 36, wherein the glomerular disease is selected from the group consisting of focal segmental glomerulosclerosis (FSGS), chronic glomerulopathies, hereditary nephritis, and minimal change disease.

38. The method of any one of claims 1-37, wherein the method prevents onset of or slows decline in kidney function.

39. The method of any one of claims 1-38, wherein the method reduces the percentage collagen positive area in the kidney of the subject compared to the pre-treatment percentage collagen positive area in the kidney of the subject.

40. The method of any one of claims 1-39, wherein the subject suffers no weight loss from their pre-treatment measured weight while adhering to a standard diet regimen over the course of treatment.

41. The method of claim 40, wherein the subject suffers about a 1% weight loss from their pre-treatment measured weight while adhering to a standard diet regimen over the course of treatment.

42. The method of claim 41, wherein the subject suffers less than a 5% weight loss from their pre-treatment measured weight while adhering to a standard diet regimen over the course of treatment.

43. The method of claim 42, wherein the subject suffers less than a 10% weight loss from pre-treatment measured weight while adhering to a standard diet regimen over the course of treatment.

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