Anti-renalase antibodies for the treatment and prevention of diseases and disorders

Abstract

To provide compositions and methods for binding and inhibiting renalase.SOLUTION: In one embodiment, the renalase binding molecule inhibits renalase activity. Thus, in diseases and conditions where a reduction of renalase activity is beneficial, such inhibitory renalase binding molecules may act as therapeutics.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to anti-renalase antibodies for the treatment and prevention of diseases and disorders.

[0002] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 62 / 611,609, filed December 29, 2017, which is incorporated herein by reference in its entirety. [Background technology]

[0003] Background of the Invention Renalase (RNLS) is a protein produced primarily in the kidney, heart, skeletal muscle, and testis, and to a lesser extent in other tissues (Xu et al., 2005 J Clin Invest. 115 (5):1275-80, and Wang et al., 2008 Mol Biol Rep. 35(4):613-20). Two isoform variants of renalase, renalase-1 and renalase-2, have been described. These two forms of renalase differ due to differential splicing of the final exon. Renalase has been described as a novel flavin adenine dinucleotide-containing monoamine oxidase with selective deamination activity for the catecholamines epinephrine, norepinephrine, and dopamine. Deficiency of renalase in the plasma of patients with end-stage renal disease compared with healthy individuals has been described. Catecholamines play a major role in maintaining and regulating blood pressure, including pathological blood pressure, through their effects on cardiac output and vascular resistance. Infusion of recombinant renalase into rats causes a decrease in cardiac contractility, heart rate, and blood pressure. Patients with renal failure are characterized by elevated levels of circulating catecholamines, which correlate with hypertension and greater mortality from cardiovascular complications. Thus, the protein renalase may play a role in regulating and maintaining catecholamine-induced changes in blood pressure, and renalase deficiency observed in patients with renal disease may lead to adverse outcomes.

[0004] A deficiency of renalase in the plasma of patients with end-stage renal disease compared with healthy individuals has been described. Patients with renal failure are characterized by elevated levels of circulating catecholamines, which correlate with higher mortality from hypertension and cardiovascular complications. Thus, the protein renalase may play a role in regulating and maintaining catecholamine-induced changes in blood pressure, and renalase deficiency observed in patients with renal disease may lead to adverse outcomes. However, little is known about the role of renalase in cancer.

[0005] An essential feature of cancer is the dysregulation of cell senescence and death. Renalase (RNLS) is a secreted flavoprotein that protects against ischemic and toxic cell injury by signaling through the plasma membrane calcium ATPase PMCA4b to activate the PI3K / AKT and MAPK pathways.

[0006] Skin cancer is a common human malignancy, and its incidence is increasing in developing countries (Gray-Schopfer et al., 2007 Nature. 445:851-7; Lowe et al., 2014 Mayo Clinic Proceedings. 89:52-9; Lesinski et al., 2013 Future Oncology. 9:925-7). Melanoma is the most devastating form of skin cancer, and once unresectable, survival rates are low (Lowe et al., 2014 Mayo Clinic Proceedings. 89:52-9). It is a molecularly heterogeneous disease, and several key alterations in signaling pathways that contribute to disease development and progression have been identified. The Ras / Raf / MEK / ERK and PI3K / AKT signaling pathways play an important role in the pathogenesis of melanoma (Gray-Schopfer et al., 2007 Nature. 445:851-7; Lesinski et al., 2013 Future oncology. 9:925-7; Yajima et al., 2012 Dermatology research and practice. 2012:354191). Mutations in Ras, Raf, PI3K, or PTEN (a PI3K inhibitor) can lead to sustained activation of ERK and AKT, which in turn promotes cell survival and proliferation. Dankort et al. demonstrated this by suppressing BRaf expression in melanoma-free mice. V600E Conditional melanocyte-specific expression of β-glucanase (GlcNAc) has been well documented, but when combined with silencing of the Pten tumor suppressor gene, it revealed 100% penetrance of melanoma development (Dankort et al., 2009 Nature Genetics. 41:544-52). Elucidation of these pathogenic pathways has facilitated the development of specific inhibitors that target hyperactivated kinases. Although these agents have proven effective in treating select patients with metastatic melanoma, their beneficial effects are often short-lived, and therefore the identification of additional therapeutic targets is urgently needed.

[0007] RNLS expression is increased in melanoma tumors and specifically in CD163 + It is significantly increased in tumor-associated macrophages (TAMs). In a cohort of primary melanoma patients, disease-specific survival was inversely correlated with RNLS expression in the tumor mass, suggesting a role for RNLS in pathogenesis. Inhibition of RNLS signaling using siRNA, anti-RNLS antibodies, or RNLS-derived inhibitory peptides significantly reduces melanoma cell survival in vitro. Anti-RNLS therapy with monoclonal antibodies significantly inhibits melanoma tumor growth in xenograft mouse models. Treatment with m28-RNLS (previously also known as 1D-28-4) significantly reduces endogenous RNLS expression, as well as CD163 expression. + This resulted in a significant decrease in total and phosphorylated STAT3 in TAMs. Increased apoptosis in tumor cells was transiently associated with p38 MAPK-mediated activation of the B-cell lymphoma 2-associated protein Bax. Expression of the cell cycle inhibitor p21 was increased, and cell cycle arrest was reported. These results support the conclusion that CD163 + Increased RNLS production by TAMs promotes melanoma growth by activating STAT3, and inhibition of RNLS signaling points to potential therapeutic applications in the management of melanoma.

[0008] Improved methods for detecting renalase in body fluids and tissues could aid in the diagnosis and prognosis of renal disease, cardiovascular disease, and / or cancer. However, validation of renalase as a relevant biomarker requires highly selective reagents for its detection. Antibody-based techniques have been widely used for biomarker detection. To date, there are only a few reagent antibodies raised against renalase that are uncharacterized or minimally characterized.

[0009] Pancreatic cancer is one of the most lethal neoplasms, causing approximately 330,000 deaths worldwide and 40,000 deaths in the United States (World Cancer Report 2014. WHO Press; 2014). Pancreatic cancer is difficult to detect, and most cases are diagnosed at late stages (Nolen et al., 2014 PLoS ONE. 9(4):e94928). Although there have been some advances in the use of chemotherapy for this cancer, the disease remains highly resistant to all drug therapies (Hidalgo et al., 2010 New England Journal of Medicine. 362(17):1605-17). The overall 5-year survival rate for individuals with pancreatic cancer is <5% (Hidalgo et al., 2010 New England Journal of Medicine. 362(17):1605-17), indicating the need for additional therapeutic targets.

[0010] The development of pancreatic cancer relies on the gradual accumulation of genetic mutations (Jones et al., 2008 Science. 321(5897):1801-6), some of which cause aberrant MAPK, PI3K, and JAK-STAT signaling. The progression from minimally dysplastic epithelium to dysplasia to invasive carcinoma reflects the gradual accumulation of genetic mutations that either activate oncogenes (e.g., KRAS2) or inactivate tumor suppressor genes (e.g., CDKN2a / INK4a, TP53, and DPC4 / SMaD4) (Hidalgo et al., 2012 Annals of Oncology. 23(Suppl. 10):x135-x8). 95, 90, and 75% of pancreatic tumors harbor mutations in KRAS2, CDKN2a, and TP53, respectively. These mutations result in the sustained and unregulated proliferation that characterizes cancer development. The central signaling pathways underlying this mutation in pancreatic ductal adenocarcinoma (PDAC) have been defined by comprehensive genetic analysis of 24 advanced PDAC (Jones et al., 2008 Science. 321(5897):1801-6). These data indicate that most PDAC contain multiple genetic alterations, primarily point mutations, affecting approximately 12 cell signaling pathways.

[0011] The study also identified 541 genes that were overexpressed in PDAC by at least 10-fold in 90% of tumors, including a 2- to 4-fold increase in the recently characterized protein renalase (RNLS) in tumors or tumor-derived cell lines. RNLS, a novel secreted flavoprotein (Xu et al., 2005 J Clin Invest. 115(5):1275-80; Desir et al., 2012 J Am Heart Assoc. 1(e002634; Desir et al., 2012 J Am Soc Hypertens. 6(6):417-26; Li et al., 2008 Circulation. 117(10):1277-82), possesses NADH oxidase activity (Farzaneh-Far et al., 2010 PLoS One. 5(10):e13496; Beaupre et al., 2015 Biochemistry. 54(3):795-806), which explains its intrinsic enzymatic activity (Wang et al., 2014 Journal of the American Society of Nephrology. RNLS promotes cell and organ survival through a receptor-mediated process that is independent of RNLS-mediated signal transduction (DOI:10.1681 / asn.2013060665) (Lee et al., 2013 J Am Soc Nephrol. 24(3):445-55). RNLS rapidly activates protein kinase B (AKT), extracellular signal-regulated kinase (ERK), and mitogen-activated protein kinase (p38). Chemical inhibition of either ERK or AKT abolishes the protective effects of RNLS (Wang et al., 2014 Journal of the American Society of Nephrology. DOI:10.1681 / asn.2013060665).

[0012] Thus, there is a need for improved methods and compositions, such as antibodies, that bind to renalase for the detection, diagnosis, prevention, and treatment of diseases or disorders, including renal disease, cardiovascular disease, and cancer. The present invention addresses this need. Summary of the Invention

[0013] Summary of the Invention In one embodiment, the invention relates to a composition comprising an antibody or binding portion thereof that specifically binds to renalase.

[0014] In one embodiment, the antibody or binding portion thereof has at least 10 -6 It specifically binds to renalase with an affinity of M.

[0015] In one embodiment, the antibody or binding portion thereof specifically binds to a peptide comprising an amino acid sequence selected from SEQ ID NOs: 1-8.

[0016] In one embodiment, the renalase is human renalase.

[0017] In one embodiment, the antibody or binding portion thereof is a monoclonal antibody, a polyclonal antibody, a single chain antibody, an immunoconjugate, a defucosylated antibody, and a bispecific antibody.

[0018] In one embodiment, the immunoconjugate comprises a therapeutic agent or a detection moiety.

[0019] In one embodiment, the antibody or binding portion thereof is selected from a humanized antibody, a chimeric antibody, a fully human antibody, and an antibody mimetic.

[0020] In one embodiment, the antibody or binding portion thereof comprises at least one of: a) a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 155; b) a HC CDR2 comprising an amino acid sequence selected from SEQ ID NO: 156, SEQ ID NO: 162, and SEQ ID NO: 168; c) a HC CDR3 comprising an amino acid sequence selected from SEQ ID NO: 157 and SEQ ID NO: 163; d) a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 152; e) a LC CDR2 comprising the amino acid sequence of SEQ ID NO: 153; and f) a LC CDR3 comprising an amino acid sequence selected from SEQ ID NO: 154 and SEQ ID NO: 160.

[0021] In one embodiment, the antibody, or binding portion thereof, comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 155, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 156, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 157, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 152, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 153, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 154.

[0022] In one embodiment, the antibody or binding portion thereof comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 221. L Includes.

[0023] In one embodiment, the antibody or binding portion thereof comprises at least one of: a) an HC CDR1 comprising an amino acid sequence selected from SEQ ID NO: 197 and SEQ ID NO: 203; b) an HC CDR2 comprising an amino acid sequence selected from SEQ ID NO: 198 and SEQ ID NO: 204; c) an HC CDR3 comprising an amino acid sequence selected from SEQ ID NO: 199 and SEQ ID NO: 205; d) an LC CDR1 comprising an amino acid sequence selected from SEQ ID NO: 194 and SEQ ID NO: 200; e) an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 195; and f) an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 196.

[0024] In one embodiment, the antibody or binding portion thereof comprises an HC CDR1 comprising the amino acid sequence of SEQ ID NO: 197, an HC CDR2 comprising the amino acid sequence of SEQ ID NO: 198, an HC CDR3 comprising the amino acid sequence of SEQ ID NO: 199, an LC CDR1 comprising the amino acid sequence of SEQ ID NO: 194, an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 195, and an LC CDR3 comprising the amino acid sequence of SEQ ID NO: 196.

[0025] In one embodiment, the antibody or binding portion thereof comprises a V H and V comprising the amino acid sequence of SEQ ID NO: 225 L Includes.

[0026] In one embodiment, the invention relates to an isolated nucleic acid molecule comprising a sequence encoding at least one antibody or binding portion thereof that specifically binds to renalase.

[0027] In one embodiment, the molecule comprises at least one nucleic acid sequence that is at least 80% identical to at least one nucleic acid sequence selected from SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, and SEQ ID NO:224.

[0028] In one embodiment, the present invention relates to an expression vector comprising at least one nucleic acid sequence selected from SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, and SEQ ID NO:224.

[0029] In one embodiment, the present invention relates to a cell comprising at least one nucleic acid sequence selected from SEQ ID NO:218, SEQ ID NO:220, SEQ ID NO:222, and SEQ ID NO:224.

[0030] In one embodiment, the invention relates to a method of treating a disease or disorder in a subject in need thereof, comprising administering to the subject a composition containing an antibody or binding portion thereof that specifically binds to renalase.

[0031] In one embodiment, the method further comprises administering at least one additional agent to the subject.

[0032] In one embodiment, the disease or disorder is at least one selected from kidney disease, cardiovascular disease, pancreatitis, hepatitis, inflammatory disorders of the kidney, and cancer.

[0033] In one embodiment, the disease or disorder is cancer, and the cancer is pancreatic cancer or melanoma.

[0034] In one embodiment, the subject is a human. [Brief explanation of the drawings]

[0035] BRIEF DESCRIPTION OF THE DRAWINGS The following detailed description of the embodiments of the invention will be better understood when read in conjunction with the accompanying drawings, in which it is understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings.

[0036] [Figure 1] FIG. 1 shows the peptide antigens (SEQ ID NOs: 1-7) used to immunize rabbits.

[0037] [Figure 2] FIG. 2 shows the full-length renalase-1 protein sequence (SEQ ID NO: 8).

[0038] [Figure 3] Figure 3 shows the location of antigens within the renalase protein (SEQ ID NO: 8 and SEQ ID NO: 50). Antigens 1A / 1B, 1C, 1D, 1E, 1F, and 3A5 are shown.

[0039] [Figure 4-1] FIG. 4-1 shows the coding sequence (SEQ ID NO: 52) and amino acid sequence (SEQ ID NO: 9) of the anti-1D epitope monoclonal 1D-28-4 heavy chain. [Figure 4-2] FIG. 4-2 shows the coding sequence (SEQ ID NO: 52) and amino acid sequence (SEQ ID NO: 9) of the anti-1D epitope monoclonal 1D-28-4 heavy chain.

[0040] [Figure 5-1] FIG. 5-1 shows the coding sequence (SEQ ID NO: 53) and amino acid sequence (SEQ ID NO: 10) of the anti-1D epitope monoclonal 1D-28-4 light chain. [Figure 5-2] FIG. 5-2 shows the coding sequence (SEQ ID NO: 53) and amino acid sequence (SEQ ID NO: 10) of the anti-1D epitope monoclonal 1D-28-4 light chain.

[0041] [Figure 6-1]FIG. 6-1 shows the coding sequence (SEQ ID NO: 60) and amino acid sequence (SEQ ID NO: 17) of the anti-1D epitope monoclonal 1D-37-10 heavy chain. [Figure 6-2] FIG. 6-2 shows the coding sequence (SEQ ID NO: 60) and amino acid sequence (SEQ ID NO: 17) of the anti-1D epitope monoclonal 1D-37-10 heavy chain. [Figure 6-3] FIG. 6-3 shows the coding sequence (SEQ ID NO: 60) and amino acid sequence (SEQ ID NO: 17) of the anti-1D epitope monoclonal 1D-37-10 heavy chain.

[0042] [Figure 7] FIG. 7 shows the coding sequence (SEQ ID NO: 61) and amino acid sequence (SEQ ID NO: 18) of the anti-1D epitope monoclonal 1D-37-10 light chain.

[0043] [Figure 8-1] FIG. 8-1 shows the coding sequence (SEQ ID NO: 68) and amino acid sequence (SEQ ID NO: 25) of the anti-1F epitope monoclonal 1F-26-1 heavy chain. [Figure 8-2] FIG. 8-2 shows the coding sequence (SEQ ID NO: 68) and amino acid sequence (SEQ ID NO: 25) of the anti-1F epitope monoclonal 1F-26-1 heavy chain.

[0044] [Figure 9] FIG. 9 shows the coding sequence (SEQ ID NO: 69) and amino acid sequence (SEQ ID NO: 26) of the anti-1F epitope monoclonal 1F-26-1 light chain.

[0045] [Figure 10-1] FIG. 10-1 shows the coding sequence (SEQ ID NO: 76) and amino acid sequence (SEQ ID NO: 33) of the anti-1F epitope monoclonal 1F-42-7 heavy chain. [Figure 10-2] FIG. 10-2 shows the coding sequence (SEQ ID NO: 76) and amino acid sequence (SEQ ID NO: 33) of the anti-1F epitope monoclonal 1F-42-7 heavy chain.

[0046] [Figure 11] FIG. 11 shows the coding sequence (SEQ ID NO: 77) and amino acid sequence (SEQ ID NO: 34) of the anti-1F epitope monoclonal 1F-42-7 light chain.

[0047] [Figure 12-1] Figure 12-1 shows the coding sequence (SEQ ID NO: 84) and amino acid sequence (SEQ ID NO: 41) of the anti-renalase-2 epitope monoclonal 3A-5-2 heavy chain.

[0048] [Figure 12-2] Figure 12-2 shows the coding sequence (SEQ ID NO: 84) and amino acid sequence (SEQ ID NO: 41) of the anti-renalase-2 epitope monoclonal 3A-5-2 heavy chain.

[0049] [Figure 13] FIG. 13 shows the coding sequence (SEQ ID NO: 85) and amino acid sequence (SEQ ID NO: 42) of the anti-renalase-2 epitope monoclonal 3A-5-2 light chain.

[0050] [Figure 14] FIG. 14 shows that anti-renalase polyclonal antibodies raised against full-length renalase protein specifically bind to the bound renalase protein in an ELISA assay.

[0051] [Figure 15] Figure 15 shows that monoclonal antibodies 1D 28-4 and 1D 37-10, raised against the 1D peptide, bound to renalase-1 in a concentration-dependent manner. The 1D peptide is present in both renalase-1 and renalase-2. Monoclonal antibodies 1F 42-7 and 1F 26-1 were raised against the 1F peptide, which is present only in renalase-1. The 1F mAb bound to renalase-1 in a concentration-dependent manner.

[0052] [Figure 16]Figure 16 shows that to establish whether antibodies can be used to detect renalase by Western blotting, serial dilutions of recombinant renalase protein from bacterial or mammalian origin were run on SDS-PAGE, and Western blotting was performed using the Ren1D 28-4 antibody. Recombinant proteins from either origin were clearly identified by this method.

[0053] [Figure 17] Figure 17 shows that antibodies 1D-28-4, 1D-37-10, 1F-42-7, and 1F-26-1 bound to renalase-1 protein in a concentration-dependent manner by ELISA assay (Figure 15). However, when the same antibodies were used to detect recombinant renalase-2 in an ELISA plate assay, only 1D-28-4 and 1D-37-10 showed robust, concentration-dependent binding. Antibody 3A5-2 was observed to bind to renalase-2 isoforms in the ELISA assay. Thus, it can be seen that antibodies raised against peptides corresponding to one or other renalase isoforms exhibit specificity for the relevant full-length protein.

[0054] [Figure 18] FIG. 18 shows that monoclonal antibodies 1D 28-4 and 1D 37-10, raised against the 1D epitope, bound to the 1D-BSA protein in a concentration-dependent manner (filled circles: 1D 28-4, open circles: 1D 37-10).

[0055] [Figure 19] Figure 19 shows that biotinylated monoclonal antibodies 1D 28-4 (closed circles), 1D 37-10 (open circles), 1F 42-7 (closed triangles), and 1F 26-1 (open triangles) bound to renalase-1 in a concentration-dependent manner.

[0056] [Figure 20]Figure 20, including Figures 20A and 20B, shows that overlapping epitopes can be detected by competitive ELISA. (Figure 20A) Biotinylated 1D 37-10 was competed with unconjugated 1D 28-4 (filled circles); similarly, antibodies raised against 1F peptides were competed with each other; biotinylated 1F 26-1 was competed with unconjugated 1F 42-7 (filled triangles); the signal from the biotinylated monoclonal antibody was not reduced when incubated with unconjugated antibody from an unimmunized rabbit (open circles and open triangles). (Figure 20B) In a further example, polyclonal antibody E2930 raised against full-length renalase-1 showed competition with each of biotinylated monoclonal antibodies 1D 37-10 and 1F 26-1, as well as a mixture of these two biotinylated mAbs (filled circles, filled squares, and open diamonds); again, the mixture of the two biotinylated antibodies was not competed with by unconjugated antibody from an unimmunized rabbit (filled triangles); the competition observed with polyclonal antibody E2930 against the two biotinylated monoclonal antibodies suggests that this polyclonal antibody binds to multiple epitopes on the renalase polypeptide.

[0057] [Figure 21-1] Figure 21-1 shows that antibodies 1D-28-4, 1F-42-7, 1D-37-10, and 1F-26-1 all bound to renalase with high affinity—K values ​​ranging from 2.67 nM to 0.316 nM. A range of association and dissociation rates was observed among these antibodies, revealing the differential contributions of different antibody compositions. [Figure 21-2] Figure 21-2 shows that antibodies 1D-28-4, 1F-42-7, 1D-37-10, and 1F-26-1 all bound to renalase with high affinity—K values ​​ranging from 2.67 nM to 0.316 nM. A range of association and dissociation rates was observed among these antibodies, demonstrating the differential contributions of different antibody compositions.

[0058] [Figure 22] FIG. 22 shows a summary of anti-renalase antibody binding affinities.

[0059] [Figure 23] Figure 23 shows that two monoclonal antibodies raised against RNLS [clone # 28-4 (m28-RNLS) and 37-10 (m37-RNLS)] reduced the viability of all melanoma cell lines tested (a total of five), and representative examples are shown.

[0060] [Figure 24] FIG. 24 shows that m28-RNLS demonstrates increasing levels of cytotoxicity (p<0.05) that correlate with increasing therapeutic concentrations.

[0061] [Figure 25] Figure 25 shows tumor volume as a function of time between control (rabbit IgG) and anti-renalase (m28-RNLS) treatment groups. For in vivo studies, A375.S2 (human melanoma) cells were injected subcutaneously into athymic nude mice to generate tumors. Once tumors reached a volume of ∼50 mm3, animals were then treated with either control rabbit IgG or the RNLS-neutralizing monoclonal antibody, m28-RNLS. The antibody treatment did not appear to be toxic, as general animal health and activity were maintained throughout the study. Tumor size was measured every other day, and treatment with m28-RNLS reduced tumor volume at all time points tested (p<0.05).

[0062] [Figure 26] Figure 26 shows that IHC staining of sections from xenografted tumors with the cell proliferation marker Ki67 revealed a significant decrease in cell proliferation in tumors treated with anti-RNLS antibody versus those treated with rabbit IgG: 35.1±2.3 positive cells / high-power field in the control group versus 13.4±3.0 in the RNLS Ab-treated group, n=14, p=0.0004.

[0063] [Figure 27] FIG. 27 shows that m28-RNLS administration caused a significant reduction in tumor volume compared to rabbit IgG.

[0064] [Figure 28] FIG. 28 shows the inhibitory effects of m28-RNLS, m37-RNLS, and a commercially available polyclonal antibody (against a partial sequence of RP-220) on the growth of human pancreatic adenocarcinoma cells.

[0065] [Figure 29] FIG. 29 shows the inhibitory effects of m28-RNLS, m37-RNLS, and a commercially available polyclonal antibody (against a partial sequence of RP-220) on the growth of human pancreatic adenocarcinoma cells.

[0066] [Figure 30] FIG. 30 shows the inhibitory effects of m28-RNLS, m37-RNLS, and a commercially available polyclonal antibody (against a partial sequence of RP-220) on the growth of human pancreatic adenocarcinoma cells.

[0067] [Figure 31] FIG. 31 shows that m28-RNLS treatment caused a significant reduction in tumor volume compared to rabbit IgG.

[0068] [Figure 32] Figure 32 shows that five humanized m28 variants (m28-K2, m2-K5, m28-K13, m28-K14 and m28-K16, Table 2) showed increased binding to both human and mouse RNLS.

[0069] [Figure 33] Figure 33 shows that five humanized m28 variants (m28-K2, m2-K5, m28-K13, m28-K14 and m28-K16, Table 2) showed increased binding to both human and mouse RNLS.

[0070] [Figure 34]Figure 34 shows that five humanized m28 variants (m28-K2, m2-K5, m28-K13, m28-K14, and m28-K16, Table 2) also reduced the viability of human melanoma (SK-MEL-28) and pancreatic adenocarcinoma (BxPC3) cell lines (representative examples shown), indicating the potential therapeutic utility of m28 variants in human cancers.

[0071] [Figure 35] Figure 35 shows that five humanized m28 variants (m28-K2, m2-K5, m28-K13, m28-K14, and m28-K16, Table 2) also reduced the viability of human melanoma (SK-MEL-28) and pancreatic adenocarcinoma (BxPC3) cell lines (representative examples shown), indicating the potential therapeutic utility of m28 variants in human cancers.

[0072] [Figure 36] FIG. 36 shows an exemplary experiment evaluating the dose response and IC50 of humanized m28 variants. DETAILED DESCRIPTION OF THE INVENTION

[0073] Detailed Description The present invention relates to inhibiting at least one biological activity of renalase using an agent that binds to renalase. In various embodiments, the present invention is directed to compositions and methods for treating a renalase-associated pathology or a renalase-associated condition in an individual by administering an inhibitor of renalase to a subject in need thereof. In various embodiments, diseases and disorders that can be diagnosed, prevented, and treated using the compositions and methods of the present invention include acute renal failure (i.e., acute tubular necrosis, or ATN, an ischemic condition of the kidney), cardiovascular disease, pancreatitis, hepatitis, inflammatory disorders of the kidney, and cancer.

[0074] In one embodiment, the present invention broadly relates to the treatment, prevention, and diagnosis of renalase-associated conditions, such as cancer. In one embodiment, the present invention is directed to methods and compositions for the diagnosis, treatment, inhibition, prevention, or alleviation of cancer. In one embodiment, the present invention provides compositions and methods for modulating one or more of renalase level, production, and activity. In the context of cancer and related diseases and disorders, the present invention provides compositions and methods for reducing one or more of renalase level, production, and activity. Some aspects of the present invention provide methods and compositions for the treatment, prevention, diagnosis, or prognosis of cancer metastasis.

[0075] definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, preferred methods and materials are described.

[0076] Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and nucleic acid chemistry and hybridization are those well known and commonly employed in the art.

[0077] Standard techniques are used for nucleic acid and peptide synthesis. These techniques and procedures are generally carried out according to conventional methods in the art and various general references provided throughout this document (e.g., Sambrook and Russell, 2012, Molecular Cloning, A Laboratory Approach, Cold Spring Harbor Press, Cold Spring Harbor, NY, and Ausubel et al., 2012, Current Protocols in Molecular Biology, John Wiley & Sons, NY).

[0078] The nomenclature used herein and the laboratory procedures of analytical chemistry and organic synthesis described below are those well known and commonly employed in the art. Standard techniques, or modifications thereof, are used for chemical syntheses and chemical analyses.

[0079] The articles "a" and "an" are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, "an element" means one element or more than one element.

[0080] As used herein, "about" when referring to measurable values ​​such as amounts, durations, and the like, encompasses variations of ±20%, ±10%, ±5%, ±1%, or ±0.1% of the particular value, and as such variations are meant to be appropriate in practicing the disclosed methods.

[0081] The term "abnormal," when used in the context of an organism, tissue, cell, or component thereof, refers to an organism, tissue, cell, or component thereof that differs in at least one observable or detectable characteristic (e.g., age, treatment, time of day, etc.) from those organisms, tissues, cells, or components thereof that exhibit the "normal" (expected / normal) respective characteristic. A characteristic that is normal or expected for one cell, tissue type, or subject may be abnormal for a different cell or tissue type.

[0082] The term "analog" as used herein generally refers to a compound that is generally structurally similar to the compound of which it is an analog, or to the "parent" compound. Generally, an analog will retain certain characteristics of the parent compound, such as biological or pharmacological activity. Analogs may lack other less desirable characteristics, such as antigenicity, proteolytic instability, toxicity, etc. Analogs include compounds in which a particular biological activity of the parent is reduced, but one or more other biological activities of the parent are unaffected in the "analog." When applied to polypeptides, the term "analog" may have varying ranges of amino acid sequence identity to the parent compound, e.g., at least about 70%, more preferably at least about 80%-85%, or about 86%-89%, and even more preferably at least about 90%, about 92%, about 94%, about 96%, about 98%, or about 99% identity of the amino acids in a given amino acid sequence of the parent or a selected portion or domain of the parent. When applied to polypeptides, the term "analog" generally refers to a polypeptide comprised of a segment of at least about three amino acids that is substantially identical to at least a portion of a binding domain fusion protein. Analogs are typically at least 5 amino acids long, at least 20 amino acids or longer, at least 50 amino acids or longer, at least 100 amino acids or longer, at least 150 amino acids or longer, at least 200 amino acids or longer, and more typically at least 250 amino acids or longer. Some analogs lack substantial biological activity but may still be utilized in a variety of applications, such as as immunological reagents for raising antibodies against a predetermined epitope, detecting and / or purifying reactive antibodies by affinity chromatography, or as competitive or non-competitive agonists, antagonists, or partial agonists of binding domain fusion protein function.

[0083] The term "antibody" as used herein refers to an immunoglobulin molecule capable of specifically binding to a particular epitope of a binding partner molecule. Antibodies can be intact immunoglobulins from natural sources or from recombinant sources, and can be immunoreactive portions of intact immunoglobulins. Antibodies in the present invention may exist in a variety of forms, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab, Fab', F(ab)2 and F(ab')2, as well as single chain antibodies (scFv), heavy chain antibodies such as camelid antibodies, and humanized antibodies (Harlow et al., 1999, Using Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, NY; Harlow et al., 1989, Antibodies: A Laboratory Manual, Cold Spring Harbor, New York; Houston et al., 1988, Proc. Natl. Acad. Sci. USA 85:5879-5883; Bird et al., 1988, Science 242:423-426).

[0084] The term "antibody fragment" refers to at least a portion of an intact antibody and refers to the antigen-determining variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments, linear antibodies, sdAbs (V L or V H Camelidae V HHThe term "scFv" refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, as used herein, an scFv refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, wherein the light and heavy chain variable regions are contiguously linked via a short flexible polypeptide linker and can be expressed as a single polypeptide chain, and wherein the scFv retains the specificity of the intact antibody from which it is derived. Unless specified, an scFv as used herein refers to a fusion protein comprising at least one antibody fragment comprising a light chain variable region and at least one antibody fragment comprising a heavy chain variable region, in any order, e.g., relative to the N-terminus and C-terminus of the polypeptide, e.g., V L and V H The scFv may have a variable region, L -Linker-V H or V H -Linker-V L may include:

[0085] As used herein, "antibody heavy chain" refers to the larger of the two polypeptide chains present in antibody molecules in their naturally occurring conformations and typically determines the class to which the antibody belongs.

[0086] As used herein, "antibody light chain" refers to the smaller of the two polypeptide chains present in antibody molecules in their naturally occurring conformations. Kappa (κ) and lambda (λ) light chains refer to the two major antibody light chain isotypes.

[0087] By the term "synthetic antibody" as used herein is meant an antibody made using recombinant DNA techniques, such as, for example, an antibody expressed by a bacteriophage as described herein. The term should also be taken to mean an antibody made by synthesis of a DNA molecule encoding the antibody, as well as an antibody where the DNA molecule expresses an antibody protein, or an amino acid sequence specifying the antibody, where the DNA sequence or amino acid sequence is obtained using synthetic DNA or amino acid sequence techniques that are available and well known in the art.

[0088] "Chimeric antibody" refers to an engineered antibody type that contains naturally occurring variable regions (light and heavy chains) derived from a donor antibody combined with light and heavy chain constant regions derived from an acceptor antibody.

[0089] A "humanized antibody" refers to an engineered antibody type having its CDRs derived from a non-human donor immunoglobulin, with the remaining immunoglobulin-derived portion of the molecule derived from one or more human immunoglobulins. In addition, framework support residues may be altered to preserve binding affinity (see, e.g., 1989, Queen et al., Proc. Natl. Acad Sci USA, 86:10029-10032; 1991, Hodgson et al., Bio / Technology, 9:421). A suitable human acceptor antibody may be selected from conventional databases, such as the KABAT database, the Los Alamos database, and the Swiss Protein database, based on homology to the nucleotide and amino acid sequences of the donor antibody. A human antibody characterized by homology (on an amino acid basis) to the framework regions of the donor antibody is suitable for providing heavy chain constant and / or heavy chain variable framework regions for insertion of donor CDRs. A suitable acceptor antibody capable of donating light chain constant and variable framework regions may be selected in a similar manner. It should be noted that the acceptor antibody heavy and light chains do not have to originate from the same acceptor antibody. The prior art describes several ways of making such humanized antibodies (see, for example, EP-A-0239400 and EP-A-054951).

[0090] The term "donor antibody" refers to an antibody (monoclonal and / or recombinant) that contributes the amino acid sequence of its variable region, CDRs, or other functional fragment, or analogs thereof, to a first immunoglobulin partner to provide an altered immunoglobulin coding region and the resulting expressed altered antibody with the binding specificity and neutralizing activity characteristic of the donor antibody.

[0091] The term "acceptor antibody" refers to an antibody (monoclonal and / or recombinant) heterologous to the donor antibody that contributes all (or any portion, but in some embodiments all) of the amino acid sequences encoding its heavy and / or light chain framework regions and / or its heavy and / or light chain constant regions to the first immunoglobulin partner. In some embodiments, a human antibody is the acceptor antibody.

[0092] "CDR" is defined as the complementarity-determining region amino acid sequences of an antibody, which are the hypervariable regions of the immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain CDRs and three light chain CDRs (or CDR regions) in the variable portion of an immunoglobulin. Thus, as used herein, "CDR" refers to all three heavy chain CDRs or all three light chain CDRs (or, where appropriate, all heavy and all light chain CDRs). The structure and protein folding of an antibody may mean that other residues are considered part of the binding region, and will be so understood by those skilled in the art. See, e.g., Chothia et al. (1989) Conformations of immunoglobulin hypervariable regions; Nature 342, pp. 877-883.

[0093] The term "framework" or "framework sequence" refers to the remaining sequence of a variable region excluding the CDRs. Because the exact definition of a CDR sequence can be determined by different systems, the meaning of a framework sequence is interpreted differently accordingly. The six CDRs (CDRs L1, L2, and L3 in the light chain and CDRs H1, H2, and H3 in the heavy chain) also divide the framework regions on the light and heavy chains into four sub-regions (FR1, FR2, FR3, and FR4), where CDR1 is located between FR1 and FR2, CDR2 is located between FR2 and FR3, and CDR3 is located between FR3 and FR4. A specific sub-region is not identified as FR1, FR2, FR3, or FR4; the framework region, as otherwise referred to, represents the FR combination within the variable region of a single naturally occurring immunoglobulin chain. FR represents one of the four sub-regions, and FR represents two or more of the four sub-regions that make up the framework region.

[0094] As used herein, "immunoassay" refers to any binding assay that uses an antibody capable of specifically binding to a target molecule for the detection and quantitation of the target molecule.

[0095] The term "specific binding," as used herein with respect to an antibody, refers to an antibody that specifically recognizes a binding partner molecule but does not substantially recognize or bind to other molecules in a sample. For example, an antibody that specifically binds to a binding partner molecule from one species may also bind to that binding partner molecule from one or more species. However, such cross-species reactivity does not in itself change the classification of the identified antibody. In another example, an antibody that specifically binds to a binding partner molecule may also bind to different allelic forms of the binding partner molecule. However, such cross-reactivity does not in itself change the classification of the identified antibody.

[0096] In some cases, the terms "specific binding" or "specifically binds" can be used in reference to the interaction of an antibody, protein, or peptide with a second binding partner molecule, meaning that the interaction is dependent on the presence of a particular structure (e.g., an antigenic determinant or epitope) on the binding partner molecule; for example, an antibody recognizes and binds to a specific protein structure rather than proteins in general. If an antibody is specific for epitope "A," then the presence of a molecule containing epitope A (or free, unlabeled A) in a reaction containing labeled "A" and the antibody will reduce the amount of labeled A bound to the antibody. In some cases, the terms "specific binding" and "specifically binds" refer to selective binding, where the antibody recognizes a sequence or conformational epitope that is important for enhanced affinity binding to the binding partner molecule.

[0097] As used herein, the term "neutralizing" refers to neutralizing the biological activity of renalase when a binding protein specifically binds to renalase. Preferably, the neutralizing binding protein is a neutralizing antibody, and its binding to renalase results in inhibition of the biological activity of renalase. Preferably, the neutralizing binding protein binds to renalase and reduces the biological activity of renalase by at least about 20%, 40%, 60, 80%, 85% or more. In some embodiments, the renalase is human renalase.

[0098] The term "epitope" has its ordinary meaning of a site on a binding partner molecule recognized by an antibody or binding site thereof or other binding molecule, such as an scFv. Epitopes may be molecules or segments of amino acids, including segments representing small portions of an entire protein or polypeptide. Epitopes may be conformational (i.e., discontinuous); that is, they may be formed from amino acids encoded by non-contiguous portions of the primary sequence that are juxtaposed by protein folding.

[0099] As used herein, the phrase "biological sample" is intended to include any sample containing cells, tissues, or bodily fluids in which nucleic acid or polypeptide expression is detected. Examples of such biological samples include, but are not limited to, blood, lymph, bone marrow, biopsies, and smears. Samples that are liquid in nature are referred to herein as "bodily fluids." Biological samples can be obtained from a patient by a variety of techniques, including, for example, by scraping or swabbing an area or by using a needle to obtain a bodily fluid. Methods for collecting various biological samples are well known in the art.

[0100] The term "cancer," as used herein, is defined as a disease characterized by the abnormal growth of ectopic cells. Cancer cells can spread locally or to other parts of the body through the bloodstream and lymphatic system. Examples of various cancers include, but are not limited to, breast cancer, prostate cancer, ovarian cancer, cervical cancer, skin cancer (e.g., melanoma), pancreatic cancer, colorectal cancer, kidney cancer, liver cancer, brain cancer, lymphoma, leukemia, lung cancer, sarcoma, and the like.

[0101] As used herein, "conjugated" refers to the covalent attachment of one molecule to a second molecule.

[0102] The "coding region" of a gene consists of nucleotide residues on the coding strand of the gene and nucleotides on the non-coding strand of the gene that are homologous to or complementary to, respectively, the coding region of an mRNA molecule produced by transcription of the gene.

[0103] The "coding region" of an mRNA molecule also consists of nucleotide residues of the mRNA molecule that match the anticodon region of the transcribed RNA molecule or encode a stop codon during translation of the mRNA molecule. Thus, the coding region may include nucleotide residues, including codons for amino acid residues that are not present in the mature protein encoded by the mRNA molecule (e.g., amino acid residues in a protein export signal sequence).

[0104] "Complementary," as used herein with reference to nucleic acids, refers to the broad concept of sequence complementarity between regions of two nucleic acid strands or between two regions of the same nucleic acid strand. It is known that adenine residues in a first nucleic acid region can form specific hydrogen bonds ("base pairs") with residues in a second nucleic acid region antiparallel to the first region if the residue is thymine or uracil. Similarly, it is known that cytosine residues in a first nucleic acid region can form base pairs with residues in a second nucleic acid region antiparallel to the first region if the residue is guanine. A first region of nucleic acid is complementary to a second region of the same or a different nucleic acid if at least one nucleotide residue in the first region is capable of base pairing with a residue in the second region when the two regions are arranged in an antiparallel fashion. Preferably, the first region comprises a first portion and the second region comprises a second portion such that when the first and second portions are arranged in an antiparallel manner, at least about 50%, and preferably at least about 75%, at least about 90%, or at least about 95% of the nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion. More preferably, all nucleotide residues in the first portion are capable of base pairing with nucleotide residues in the second portion.

[0105] The term "derivative" as used herein includes chemical modifications of polypeptides, polynucleotides, or other molecules. In the context of the present invention, "derivative polypeptides," such as those modified by glycosylation, pegylation, or any similar process, retain binding activity. For example, the term "derivative" of a binding domain includes fusion proteins, variants, or fragments of a binding domain that have been chemically modified, such as by the addition of one or more polyethylene glycol molecules, sugars, phosphates, and / or other such molecules, where these one or more molecules are not naturally attached to the wild-type binding domain fusion protein. A "derivative" of a polypeptide also includes those polypeptides that are "derived" from a reference polypeptide by, for example, having amino acid substitutions, deletions, or insertions relative to the reference polypeptide. Thus, a polypeptide may be "derived" from a wild-type polypeptide or from any other polypeptide. As used herein, a compound, including a polypeptide, may also be "derived" from a particular source, such as a particular organism, tissue type, or from a particular polypeptide, nucleic acid, or other compound present in a particular organism or tissue type.

[0106] The term "DNA" as used herein is defined as deoxyribonucleic acid.

[0107] "Encoding" refers to the inherent property of a specific sequence of nucleotides in a polynucleotide, such as a gene, cDNA, or mRNA, to serve as a template for the synthesis of other polymers and macromolecules in biological processes, having either a defined sequence of nucleotides (i.e., rRNA, tRNA, and mRNA) or a defined amino acid sequence, and the biological properties resulting therefrom. Thus, a gene encodes a protein if transcription and translation of the mRNA corresponding to that gene produces the protein in a cell or other biological system. Both the coding strand, whose nucleotide sequence is identical to the mRNA sequence and is usually provided in a sequence listing, and the non-coding strand, which is used as a template for transcription of the gene or cDNA, can be referred to as encoding the protein or other product of the gene or cDNA.

[0108] Unless otherwise specified, a "nucleotide sequence encoding an amino acid sequence" includes all nucleotide sequences that are degenerate versions of each other and that encode the same amino acid sequence. The phrase nucleotide sequence encoding a protein or RNA also includes introns, to the extent that a nucleotide sequence encoding a protein may, in some forms, contain intron(s).

[0109] A "disease" is a state of health in an animal in which the animal is unable to maintain homeostasis and the animal's health continues to deteriorate if the disease is not ameliorated.

[0110] In contrast, an animal "disorder" is a health condition in which the animal is able to maintain homeostasis but in which the animal's health is less favorable than it would be without the disorder. If left untreated, the disorder does not necessarily cause further deterioration in the animal's health.

[0111] A disease or disorder is "alleviated" if the severity of a sign or symptom of the disease or disorder, the frequency with which the patient experiences such sign or symptom, or both, is reduced.

[0112] An "effective amount" or "therapeutically effective amount" of a compound is the amount of that compound sufficient to provide a beneficial effect to the subject to which it is administered.

[0113] The term "high affinity" with respect to the binding domain polypeptides described herein means an affinity of at least about 10 -6 M, preferably at least about 10 -7 M, more preferably at least about 10 -8 M or stronger, more preferably at least about 10 -9 M or stronger, more preferably at least about 10 -10 M or stronger, e.g., up to 10 -12 "High affinity" refers to a dissociation constant (Kd) of M or stronger, however, "high affinity" binding can vary with respect to other binding domain polypeptides.

[0114] As used herein, the term "inhibit" means to suppress or block an activity or function, for example, by about 10% relative to a control value. Preferably, activity is suppressed or blocked by 50% compared to a control value, more preferably by 75%, and even more preferably by 95%. As used herein, "inhibit" also means to measurably reduce or totally interfere with the level of expression, stability, function, or activity of a molecule, reaction, interaction, gene, mRNA, and / or protein. Inhibitors are compounds, such as antagonists, that, for example, bind to, partially or totally block activity, reduce, prevent, delay activation, inactivate, desensitize, or down-regulate the stability, expression, function, and activity of proteins, genes, and mRNAs.

[0115] As used herein, the terms "modulator" and "modulation" of a molecule of interest, in its various forms, are intended to encompass antagonism, agonism, partial antagonism, and / or partial agonism of the activity associated with the protease of interest. In various embodiments, a "modulator" may inhibit or stimulate the expression or activity of the protease. Such modulators include small molecule agonists and antagonists of protease molecules, antisense molecules, ribozymes, triplex molecules, and RNAi polynucleotides, among others.

[0116] As used herein, "instruction material" includes publications, records, diagrams, or any other medium of expression that can be used to communicate the usefulness of the compounds, compositions, vectors, or delivery systems of the invention in the kits to affect the alleviation of various diseases or disorders listed herein. Optionally, or alternatively, the instructions can describe one or more methods of alleviating a disease or disorder in a mammalian cell or tissue. The instructions for the kits of the invention can be, for example, affixed to a container containing the identified compound, composition, vector, or delivery system of the invention, or shipped together with a container containing the identified compound, composition, vector, or delivery system. Alternatively, the instructions can be shipped separately from the container, with the instructions and the compound intended to be used cooperatively by the recipient.

[0117] "Isolated" means altered or removed from the natural state. For example, a nucleic acid or peptide that is naturally present in its normal context in a living animal is not "isolated," but the same nucleic acid or peptide partially or completely separated from the coexisting materials of its natural context is "isolated." An isolated nucleic acid or protein can exist in substantially purified form, or can exist in a non-native environment, such as, for example, a host cell.

[0118] An "isolated nucleic acid" refers to a nucleic acid segment or fragment that has been separated from sequences that flank it in its naturally occurring state, i.e., a DNA fragment that has been removed from sequences that normally flank the fragment, i.e., sequences that flank the fragment in the naturally occurring genome. The term also applies to nucleic acids that have been substantially purified from other components that naturally accompany the nucleic acid, i.e., from the RNA or DNA or proteins that naturally accompany it in the cell. Thus, the term includes recombinant DNA that has been incorporated, for example, into a vector, an autonomously replicating plasmid or virus, or into the genomic DNA of a prokaryote or eukaryote, or that exists as a separate molecule free from other sequences (i.e., as a cDNA or genomic or cDNA fragment generated by PCR or restriction enzyme digestion). It also includes recombinant DNA that is part of a hybrid gene encoding additional polypeptide sequences.

[0119] In the context of the present invention, the following abbreviations are used for commonly occurring nucleobases: "A" refers to adenosine, "C" refers to cytosine, "G" refers to guanosine, "T" refers to thymidine, and "U" refers to uridine.

[0120] The term "polynucleotide" as used herein is defined as a chain of nucleotides. Furthermore, a nucleic acid is a polymer of nucleotides. Therefore, as used herein, nucleic acid and polynucleotide are interchangeable. Those skilled in the art have the general knowledge that nucleic acids and polynucleotides can be hydrolyzed into monomeric "nucleotides." Monomeric nucleotides can be hydrolyzed into nucleosides. As used herein, polynucleotide includes, but is not limited to, all nucleic acid sequences obtained by any means available in the art, including but not limited to recombinant means, i.e., cloning nucleic acid sequences from recombinant libraries or cell genomes using conventional cloning techniques and PCR, as well as by synthetic means.

[0121] As used herein, the terms "peptide," "polypeptide," and "protein" are used interchangeably and refer to compounds composed of amino acid residues covalently linked by peptide bonds. A protein or peptide must contain at least two amino acids and, without limitation, must be arranged with the maximum number of amino acids possible to form a protein or peptide sequence. A polypeptide includes any peptide or protein containing two or more amino acids linked to each other by peptide bonds. As used herein, the term refers to both short chains, commonly referred to in the art as peptides, oligopeptides, and oligomers, as well as long chains, of which there are many varieties, generally referred to in the art as proteins. "Polypeptide" includes, for example, biologically active fragments, particularly substantially homologous polypeptides, oligopeptides, homodimers, heterodimers, variants of polypeptides, modified polypeptides, derivatives, analogs, and fusion proteins. A polypeptide includes natural peptides, recombinant peptides, synthetic peptides, or combinations thereof.

[0122] The term "conservative substitution," when describing a polypeptide, refers to a change in the amino acid composition of a polypeptide that does not substantially alter the activity of the polypeptide, i.e., the substitution of an amino acid with another amino acid having similar properties. Conservative substitution tables providing functionally similar amino acids are well known in the art. The following six groups each contain amino acids that are generally understood to represent conservative substitutions for one another: (1) alanine (A), serine (S), threonine (T); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); and (6) phenylalanine (F), tyrosine (Y), tryptophan (W) (see also Creighton, 1984, Proteins, W.H. Freeman and Company). In addition to conservative substitutions as defined above, other modifications of amino acid residues can also result in "conservatively modified variants." For example, all charged amino acids, whether positive or negative, can be considered as substitutions for one another. Additionally, conservatively modified variants can also result from individual substitutions, deletions, or additions that alter, add, or delete a single amino acid or a small percentage, often less than 5%, of the amino acids in the encoded sequence. Furthermore, conservatively modified variants can be generated from recombinant polypeptides by substituting a codon for an amino acid utilized by the native or wild-type gene with a different codon for the same amino acid.

[0123] The term "RNA" as used herein is defined as ribonucleic acid.

[0124] The term "recombinant DNA" as used herein is defined as DNA that is made by joining pieces of DNA from different sources.

[0125] The term "recombinant polypeptide," as used herein, is defined as a polypeptide made by using recombinant DNA methods.

[0126] By "pharmaceutically acceptable" is meant a carrier, diluent, or excipient that is compatible with the other components of the formulation and generally safe for administration to the recipient thereof, e.g., a carrier, diluent, or excipient. As used herein, "carrier" includes any substance that, when combined with the conjugate, retains the activity of the conjugate and is non-reactive with the subject's immune system. Examples include, but are not limited to, standard pharmaceutical carriers such as phosphate-buffered saline, water, emulsions such as oil-in-water emulsions, and any of various types of wetting agents. Other carriers may also include sterile liquids, tablets, including coated tablets, and capsules. Typically, such carriers include excipients such as starch, lactose, certain types of clay, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavorings or coloring agents or other components. Compositions containing such carriers are formulated using well-known, conventional methods.

[0127] The terms "patient," "subject," "individual," and the like, are used interchangeably herein and refer to any animal, preferably a mammal, most preferably a human, having a complement system, including humans in need of treatment for a condition or its sequelae, or susceptible humans. Thus, individuals may include, for example, dogs, cats, pigs, cows, sheep, goats, horses, rats, monkeys, and mice, and humans.

[0128] As used herein, "identical" or "identity" in the context of two or more nucleic acid or polypeptide sequences means that the sequences have a specified percentage of residues that are the same over a specified region. This percentage can be calculated by optimally aligning the two sequences, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matched positions, dividing the number of matched positions by the total number of positions in the specified region, and multiplying this result by 100 to obtain the percentage of sequence identity. In cases where the two sequences are of different lengths, or where alignment results in one or more cohesive ends, and the specified region being compared contains only a single sequence, the residues of the single sequence are included in the denominator but not the numerator of this calculation. When comparing DNA and RNA, thymine (T) and uracil (U) can be considered equivalent. Identity can be performed manually or by using a computer sequence algorithm, such as BLAST or BLAST 2.0.

[0129] The phrases "percent (%) identity" or "percent identical" refer to the percentage of sequence similarity found in a comparison of two or more amino acid sequences. Percent identity can be determined electronically using any suitable software. Similarly, "similarity" between two polypeptides (or one or more portions of either or both) is determined by comparing the amino acid sequence of one polypeptide with the amino acid sequence of a second polypeptide. Any suitable algorithm suitable for such comparisons can be adapted for application in the context of the present invention.

[0130] A "therapeutic" treatment is a treatment administered to a subject who exhibits signs of pathology with the intent of reducing or eliminating those signs.

[0131] A "therapeutically effective amount" is an amount of a compound of the invention that, when administered to a patient, ameliorates the symptoms of the disease. The amount of a compound of the invention that constitutes a "therapeutically effective amount" will vary depending on the compound, the condition and its severity, the age of the patient being treated, and the like. A therapeutically effective amount can be determined routinely by one of ordinary skill in the art having regard to their own knowledge and this disclosure.

[0132] The terms "treat," "treating," and "treatment" refer to therapeutic or prophylactic measures described herein. Methods of "treatment" utilize the administration of a composition of the invention to a subject in need of such treatment, e.g., a subject afflicted with or who will ultimately acquire a disease or disorder, to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of a disorder or the recurrence of a disorder, or to prolong the subject's survival beyond that expected in the absence of such treatment.

[0133] As used herein, "variant" refers to a nucleic acid sequence or peptide sequence that differs in sequence from a reference nucleic acid sequence or peptide sequence, respectively, but retains essential biological properties of the reference molecule. Sequence changes in nucleic acid variants may not alter the amino acid sequence of the peptide encoded by the reference nucleic acid, or may result in amino acid substitutions, additions, deletions, fusions, and truncations. Sequence changes in peptide variants are limited or conservative, and typically the sequences of the reference peptide and variant are closely similar overall and, in many regions, identical. The variant and reference peptide can differ in amino acid sequence by one or more substitutions, additions, or deletions, in any combination. Nucleic acid or peptide variants can be naturally occurring, such as allelic variants, or variants not known to occur naturally. Non-naturally occurring variants of nucleic acids and peptides may be generated by mutagenesis techniques or direct synthesis.

[0134] Ranges: Throughout this disclosure, various aspects of the invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as inflexibly limiting the scope of the invention. Accordingly, the description of a range should be considered to specifically set forth all the possible subranges as well as individual numerical values ​​within that range. For example, the description of a range such as 1 to 6 should be considered to specifically set forth subranges such as 1 to 3, 1 to 4, 1 to 5, 2 to 4, 2 to 6, 3 to 6, etc., as well as individual numerical values ​​within that range, e.g., 1, 2, 2.7, 3, 4, 5, 5.3, and 6. This applies regardless of the breadth of the range.

[0135] explanation The present invention relates to the binding and inhibition of renalase using substances that specifically bind to renalase. In various embodiments, the present invention is directed to compositions and methods for treating a renalase-associated disease or disorder in an individual by administering an inhibitor of renalase to a subject in need thereof. In some embodiments, the renalase inhibitor is a renalase-binding molecule. In some embodiments, the renalase-binding molecule is an antibody. In various embodiments, diseases and disorders that can be diagnosed, prevented, and treated using the compositions and methods of the present invention include acute renal failure (i.e., acute tubular necrosis, or ATN, an ischemic condition of the kidney), cardiovascular disease, pancreatitis, hepatitis, inflammatory disorders of the kidney, and cancer.

[0136] In one embodiment, the present invention broadly relates to the treatment, prevention, and diagnosis of cancer. In one embodiment, the present invention is directed to methods and compositions for diagnosing, staging, treating, inhibiting, preventing, or alleviating cancer. In one embodiment, the present invention provides compositions and methods for modulating one or more of renalase levels, production, and activity. In the context of cancer and related diseases and disorders, the present invention provides compositions and methods for reducing one or more of renalase levels, production, and activity. Some aspects of the present invention provide methods and compositions for the treatment, prevention, diagnosis, or prognosis of cancer metastasis.

[0137] Renalase inhibitor compositions and methods of use - Patents.com In various embodiments, the present invention includes renalase inhibitor compositions and methods for treating or preventing diseases or disorders in which a decrease in renalase levels or activity is desirable. One non-limiting example of a disease or disorder in which a decrease in renalase levels or activity is desirable that can be treated or prevented by the compositions and methods of the present invention includes cancer. In various embodiments, the renalase inhibitor compositions and treatment or prevention methods of the present invention reduce the amount of active renalase polypeptide, the amount of active renalase peptide fragment, the amount of renalase enzymatic activity, the amount of renalase substrate binding activity, the amount of renalase receptor binding activity, or a combination thereof.

[0138] Those skilled in the art will understand, based on the disclosure provided herein, that a decrease in the level of active renalase encompasses a decrease in renalase expression, including transcription, translation, or both, and also encompasses promoting the degradation of renalase, including at the RNA level (e.g., RNAi, shRNA, etc.) and protein level (e.g., ubiquitination, etc.). Once the subject matter of the present invention is operational, those skilled in the art will also understand that a decrease in the level of active renalase includes a decrease in renalase activity (e.g., enzymatic activity, substrate-binding activity, receptor-binding activity, etc.). Thus, a decrease in the level or activity of renalase includes, but is not limited to, a decrease in the transcription, translation, or both of a nucleic acid encoding renalase; it also includes a decrease in any activity of a renalase polypeptide or a peptide fragment thereof. The renalase inhibitor compositions and methods of the present invention can selectively inhibit renalase, or can inhibit both renalase and another molecule.

[0139] Inhibition of renalase can be assessed using a variety of methods, including those disclosed herein as well as methods known in the art or hereafter developed. That is, a routineer will understand, based on the disclosure provided herein, that a decrease in renalase level or activity can be readily assessed by using a method that assesses the level of nucleic acid (e.g., mRNA) encoding renalase, the level of a renalase polypeptide or a peptide fragment thereof, the level of renalase activity (e.g., enzymatic activity, substrate-binding activity, receptor-binding activity, etc.), or a combination thereof, present in a biological sample.

[0140] Based on the disclosure provided herein, one of skill in the art will understand that the present invention is useful for treating or preventing in a subject in need thereof, regardless of whether the subject is being treated with other medications or therapies. Furthermore, based on the content provided herein, one of skill in the art will further understand that the disease or disorder treatable by the compositions and methods described herein includes any disease or disorder in which renalase plays a role and in which reduced renalase levels or activity promotes a positive therapeutic outcome. In various embodiments, diseases or disorders treatable or preventable using the compounds and methods of the invention include acute renal failure (i.e., acute tubular necrosis, or ATN, an ischemic condition of the kidney), cardiovascular disease or disorder (e.g., hypertension, pulmonary hypertension, systolic hypertension, diabetic hypertension, asymptomatic left ventricular dysfunction, chronic congestive heart failure, myocardial infarction, cardiac rhythm disorders, atherosclerosis, etc.), cancer, cardiac disease or disorder, renal disease or disorder, gastrointestinal disease or disorder, liver disease or disorder, pulmonary disease or disorder, pancreatic disease or disorder (e.g., pancreatitis), psychiatric disease or disorder (e.g., depression, anxiety, etc.), or neurological disease or disorder.

[0141] In another embodiment, the renalase inhibitors of the present invention can be administered to patients being treated with exogenous renalase, recombinant renalase, renalase fragments, and / or renalase activators to control, titrate, reduce, or stabilize the level or activity of endogenous and / or exogenous renalase in the patient.

[0142] The renalase inhibitor compositions and methods of the present invention that reduce the level or activity (e.g., enzymatic activity, substrate-binding activity, receptor-binding activity, etc.) of renalase or a renalase fragment include, but should not be construed as being limited to, chemical compounds, proteins, peptides, peptidomimetics, antibodies, antibody fragments, antibody mimetics, ribozymes, small molecule chemical compounds, short hairpin RNAs, RNAi, antisense nucleic acid molecules (e.g., siRNA, miRNA, etc.), nucleic acids encoding antisense nucleic acid molecules, nucleic acid sequences encoding proteins, renalase receptors, renalase receptor fragments, or combinations thereof. In some embodiments, the inhibitor is an allosteric inhibitor. Based on the disclosure provided herein, one of ordinary skill in the art will readily understand that the renalase inhibitor composition encompasses any chemical compound that reduces the level or activity of renalase or a fragment thereof. In addition, the renalase inhibitor composition encompasses chemically modified compounds and derivatives, as is well known to those skilled in the chemical arts.

[0143] The renalase inhibitor compositions and methods of the present invention that reduce the level or activity (e.g., enzymatic activity, substrate-binding activity, receptor-binding activity, etc.) of renalase or renalase fragments include antibodies and fragments thereof. Antibodies of the present invention include various forms of antibodies, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)2, single-chain antibodies (scFv), heavy-chain antibodies (e.g., camelid antibodies), synthetic antibodies, chimeric antibodies, and humanized antibodies. In one embodiment, an antibody of the present invention is an antibody that specifically binds to renalase. In some embodiments, an antibody of the present invention is a bispecific antibody, in which a first specificity is for renalase and a second specificity is for a target molecule on a cell or tissue to guide the bispecific antibody to the anatomical location where the target molecule is present and renalase binding is desired. In some embodiments, the antibodies of the present invention are bispecific antibodies, in which the first specificity is for renalase and the second specificity is for a second binding partner molecule (i.e., payload) that is carried by the secondary specificity of the antibody and deployed to an anatomical location where renalase binding is desired.

[0144] In some embodiments, administration of a renalase inhibitor (e.g., a renalase-binding molecule) of the present invention to a subject for the treatment of cancer serves to initiate and / or supplement an immune response by the subject's immune system against the cancer. The subject's immune response against the cancer can be any host defense or response, including an innate immune response, a humoral immune response, a cellular immune response, or a combination thereof.

[0145] Furthermore, those skilled in the art will understand that, armed with the present disclosure and methods exemplified herein, renalase inhibitor compositions include such inhibitors yet to be discovered as may be identified by criteria well known in the art of pharmacology, such as the physiological consequences of renalase inhibition detailed herein and / or known in the art. Thus, the present invention is not limited in any way to the specific renalase inhibitor compositions exemplified or disclosed herein; rather, the present invention encompasses those inhibitor compositions known in the art and yet to be discovered that will be appreciated by the ordinary worker as useful.

[0146] Additional methods for identifying and producing renalase inhibitor compositions are well known to those of skill in the art and include, but are not limited to, obtaining inhibitors from naturally occurring sources (e.g., Streptomyces species, Pseudomonas species, Stylotella aurantium, etc.). Alternatively, renalase inhibitors can be chemically synthesized. Furthermore, the practitioner will understand, based on the teachings provided herein, that renalase inhibitor compositions can be obtained from recombinant organisms. Compositions and methods for chemically synthesizing renalase inhibitors, as well as for obtaining them from natural sources, are well known and described in the art.

[0147] Those skilled in the art will understand that inhibitors can be administered as chemical compounds, proteins, peptides, peptidomimetics, antibodies, antibody fragments, antibody mimetics, ribozymes, small molecule chemical compounds, short hairpin RNAs, RNAi, antisense nucleic acid molecules (e.g., siRNA, miRNA, etc.), nucleic acids encoding antisense nucleic acid molecules, nucleic acid sequences encoding proteins, renalase receptors, renalase receptor fragments, or combinations thereof. Many vectors and other compositions and methods are known for administering proteins or protein-encoding nucleic acid constructs to cells or tissues. Thus, the present invention includes methods for administering proteins or protein-encoding nucleic acids that are inhibitors of renalase (Sambrook et al., 2012, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York; Ausubel et al., 1997, Current Protocols in Molecular Biology, John Wiley & Sons, New York).

[0148] Those skilled in the art will recognize that reducing the amount or activity of molecules that themselves increase renalase levels or activity can be useful in the compositions and methods of the present invention for reducing renalase levels or activity.

[0149] Antisense oligonucleotides are DNA or RNA molecules that are complementary to a portion of an RNA molecule. When present in a cell, antisense oligonucleotides hybridize to the existing RNA molecule and inhibit translation into a gene product. Inhibition of gene expression using antisense oligonucleotides is well known in the art (Marcus-Sekura, 1988, Anal. Biochem. 172:289), as is the method of expressing antisense oligonucleotides in cells (Inoue, U.S. Pat. No. 5,190,931). The method of the present invention includes the use of antisense oligonucleotides to reduce the amount of renalase or to reduce the amount of molecules that cause an increase in the amount or activity of renalase, thereby reducing the amount or activity of renalase.

[0150] The present invention contemplates antisense oligonucleotides that are synthesized and delivered to cells by methods well known to those skilled in the art. For example, antisense oligonucleotides can be synthesized to be about 10 to about 100 nucleotides in length, more preferably about 15 to about 50 nucleotides in length. The synthesis of nucleic acid molecules is well known in the art, as is the synthesis of antisense oligonucleotides modified to improve biological activity compared to unmodified antisense oligonucleotides (Tullis, 1991; U.S. Pat. No. 5,023,243).

[0151] Similarly, gene expression may be inhibited by hybridization of an antisense molecule to the promoter or other regulatory elements of the gene, thereby affecting transcription of the gene. Methods for identifying promoters or other regulatory elements that interact with a gene of interest are well known in the art and include methods such as the yeast two-hybrid system (Bartel and Fields, eds., The Yeast Two Hybrid System, Oxford University Press, Cary, NC).

[0152] Alternatively, inhibition of genes expressing renalase or genes expressing proteins that increase the level or activity of renalase can be achieved by the use of ribozymes. The use of ribozymes to inhibit gene expression is well known to those skilled in the art (see, e.g., Cech et al., 1992, J. Biol. Chem. 267:17479; Hampel et al., 1989, Biochemistry 28:4929; Altman et al., U.S. Pat. No. 5,168,053). Ribozymes are catalytic RNA molecules capable of cleaving other single-stranded RNA molecules. Ribozymes are known to be sequence-specific and can therefore be modified to recognize specific nucleotide sequences (Cech, 1988, J. Amer. Med. Assn. 260:3030), allowing for the selective cleavage of specific mRNA molecules. Given the nucleotide sequence of this molecule, one of ordinary skill in the art can synthesize the antisense oligonucleotide or ribozyme without undue experimentation, as provided in this disclosure and the references incorporated herein.

[0153] Alternatively, inhibition of genes expressing renalase or genes expressing proteins that increase renalase levels or activity can be achieved through the use of short hairpin RNAs or antisense RNAs, including siRNAs, miRNAs, and RNAis. Given the nucleotide sequence of the molecule, one of skill in the art can synthesize short hairpin RNAs or antisense RNAs without undue experimentation, as provided in this disclosure and the references incorporated herein.

[0154] Those skilled in the art will understand that inhibitors of renalase or renalase fragments can be administered acutely (e.g., over a short period of time, such as a day, a week, or a month) or chronically (e.g., over an extended period of time, such as several months or a year or more). Those skilled in the art will understand that inhibitors of renalase can be administered alone or in combination with other agents. Furthermore, renalase inhibitors can be administered alone or in any combination in a temporal sense, in that they are administered simultaneously with, before, and / or after each other. Those skilled in the art will understand, based on the disclosure provided herein, that renalase inhibitor compositions can be used to treat or prevent diseases or disorders in subjects in need thereof, and that the inhibitor compositions can be used alone or in any combination with another inhibitor to affect a therapeutic outcome.

[0155] In various embodiments, any inhibitor of renalase or renalase fragments herein described herein can be administered alone or in combination with other inhibitors of other molecules associated with cancer.

[0156] Those skilled in the art, when enabled by the present disclosure, including the methods described herein, will understand that the present invention is not limited to the treatment of established diseases or disorders, such as cancer. In particular, the disease or disorder need not be symptomatic to the point of being harmful to the subject; in fact, the disease or disorder need not be detected in the subject before treatment is administered. That is, significant disease or disorder must not have occurred before the present invention provides benefit. Thus, the present invention includes methods of preventing disease or disorder in a subject, in that, as discussed above elsewhere herein, a renalase inhibitor composition can be administered to a subject prior to the onset of the disease or disorder, thereby preventing the disease or disorder from developing. The prophylactic methods described herein also include the treatment of a subject in remission with respect to preventing the recurrence of the disease or disorder.

[0157] Those skilled in the art will understand that, when enabled by the disclosure herein, prevention of a disease or disorder encompasses administering a renalase inhibitor composition to a subject as a preventative measure against a disease or disorder, including cancer. As discussed more fully elsewhere herein, methods of reducing renalase levels or activity encompass a wide variety of techniques that not only reduce renalase activity, but also reduce expression of nucleic acids encoding renalase, including reducing transcription, reducing translation, or both.

[0158] Additionally, as disclosed elsewhere herein, those skilled in the art will understand that, once enabled by the teachings provided herein, the present invention encompasses methods for preventing a wide variety of diseases, disorders, and pathologies in which a decrease in renalase expression and / or activity mediates, treats, or prevents the disease, disorder, or pathology. Methods for assessing whether a disease is associated with renalase levels or activity are known in the art. Furthermore, the present invention encompasses the treatment or prevention of such diseases yet to be discovered.

[0159] The present invention encompasses the administration of inhibitors of renalase to practice the methods of the present invention; one of skill in the art will know, based on the disclosure provided herein, how to formulate and administer a suitable renalase inhibitor to a subject. However, the present invention is not limited to any particular method of administration or treatment regimen.

[0160] The present invention provides compositions that bind to renalase. In one embodiment, the renalase binding agent inhibits the level or activity of renalase. Thus, in diseases and conditions where reduced renalase activity is beneficial, such inhibitory renalase binding agents can potentially act as therapeutic agents.

[0161] In addition to its potential therapeutic role, in some cases, renalase can be used as a diagnostic marker for diseases or disorders, including, but not limited to, acute renal failure (i.e., acute tubular necrosis, or ATN, an ischemic condition of the kidney), cardiovascular disease, pancreatitis, hepatitis, inflammatory disorders of the kidney, and cancer. Patients who do not have properly functioning kidneys have lower levels of renalase. Thus, the present invention also includes methods for diagnosing susceptibility to conditions, disorders, and diseases related to the cardiovascular system, heart, kidney, gastrointestinal, liver, lung, pancreas, and psychiatric and neurological systems, including cancer, based on the detection and / or quantification of renalase using the renalase binding agents of the present invention. For example, cardiovascular conditions, disorders, and diseases such as hypertension, asymptomatic left ventricular dysfunction, chronic congestive heart failure, myocardial infarction, cardiac rhythm disorders, and atherosclerosis; psychiatric conditions, disorders, and diseases such as depression and anxiety; and cardiac conditions, disorders, and diseases such as pulmonary hypertension can all be diagnosed, evaluated, and monitored by determining renalase levels, such as renalase protein levels. For example, decreased levels of renalase protein may be a diagnostic marker for disorders associated with increased symptomatic outcomes. The compositions and methods of the present invention can be used to treat, prevent, reduce, or ameliorate hypertension, including systolic hypertension, isolated systolic hypertension, and diabetic hypertension. Furthermore, the same benefit is expected for rarer hypertensive disorders, such as pulmonary hypertension and pancreatitis. Pulmonary hypertension is a rare vascular disorder of the lungs in which pressure in the pulmonary arteries (blood vessels connecting the heart to the lungs) rises above normal levels and can become life-threatening. The similar development of elevated blood pressure in the pulmonary bed that accompanies the rise in systemic blood pressure in diabetic hypertension and in isolated systolic hypertension suggests that similar mechanisms are involved.

[0162] The renalase inhibitor compositions of the present invention that reduce the level or activity (e.g., enzymatic activity, substrate-binding activity, receptor-binding activity, etc.) of renalase include, but should not be construed as being limited to, chemical compounds, proteins, peptides, peptidomimetics, antibodies, antibody fragments, antibody mimetics, ribozymes, small molecule chemical compounds, short hairpin RNAs, RNAi, antisense nucleic acid molecules (e.g., siRNA, miRNA, etc.), nucleic acids encoding antisense nucleic acid molecules, nucleic acid sequences encoding proteins, renalase receptors, renalase receptor fragments, or combinations thereof. In some embodiments, the inhibitor is an allosteric inhibitor. Based on the disclosure provided herein, one skilled in the art will readily understand that renalase inhibitor compositions encompass chemical compounds that reduce the level or activity of renalase. In addition, renalase inhibitor compositions encompass chemically modified compounds and derivatives, as is well known to those skilled in the chemical arts.

[0163] Renalase inhibitor compositions of the present invention that reduce renalase levels or activity (e.g., enzymatic activity, substrate-binding activity, receptor-binding activity, etc.) include antibodies and fragments thereof. Antibodies of the present invention include various forms of antibodies, including, for example, polyclonal antibodies, monoclonal antibodies, intracellular antibodies ("intrabodies"), Fv, Fab and F(ab)2, single-chain antibodies (scFv), heavy-chain antibodies (e.g., camelid antibodies), synthetic antibodies, chimeric antibodies, and humanized antibodies. In one embodiment, an antibody of the present invention is an antibody that specifically binds to renalase. In some embodiments, an antibody of the present invention is a bispecific antibody, in which a first specificity is for renalase and a second specificity is for a target molecule that directs the bispecific antibody to an anatomical location where renalase binding is desired. In some embodiments, an antibody of the present invention is a bispecific antibody, in which a first specificity is for renalase and a second specificity is for a second binding partner molecule that is delivered and deployed to an anatomical location where renalase binding is desired.

[0164] Antibodies of the present invention, including renalase-binding fragments thereof, in some embodiments include the antibody amino acid sequences disclosed herein encoded by any suitable polynucleotide, or any isolated or formulated antibody. Furthermore, antibodies of the present disclosure include antibodies having structural and / or functional characteristics of the anti-renalase antibodies described herein. In one embodiment, the anti-renalase antibody binds to renalase and thereby partially or substantially alters at least one biological activity of renalase (e.g., enzymatic activity, substrate-binding activity, receptor-binding activity, etc.). In some embodiments, the renalase is human renalase.

[0165] In one embodiment, the anti-renalase antibody of the present invention immunospecifically binds to at least one specified epitope specific to a renalase protein, peptide, subunit, fragment, portion, or any combination thereof, and does not specifically bind to other polypeptides, other than renalase, from other species. The at least one epitope can comprise at least one antibody binding region comprising at least a portion of the renalase protein. As used herein, the term "epitope" refers to a protein determinant capable of binding to an antibody. Epitopes usually consist of chemically active surface groupings of molecules such as amino acids or sugar side chains, and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics. Conformational and non-conformational epitopes are distinguished in that the binding to the former is lost in the presence of denaturing solvents, while the latter is not.

[0166] In some embodiments, the present invention includes compositions containing antibodies (e.g., binding portions of antibodies) that specifically bind to renalase. In one embodiment, the anti-renalase antibody is a polyclonal antibody. In another embodiment, the anti-renalase antibody is a monoclonal antibody. In some embodiments, the anti-renalase antibody is a chimeric antibody. In further embodiments, the anti-renalase antibody is a humanized antibody. In some embodiments, the renalase is human renalase. In some embodiments, the antibodies of the present invention specifically bind to at least one of SEQ ID NOs: 1-7, 8, 50, 92, 94, and fragments thereof.

[0167] A binding portion of an antibody includes one or more fragments of an antibody that retain the ability to specifically bind to a binding partner molecule (e.g., renalase). It has been shown that the binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "binding portion" of an antibody include (i) V L , V H , C L and C H (ii) a Fab fragment, which is a monovalent fragment consisting of one domain; (iii) a F(ab')2 fragment, which is a bivalent fragment containing two Fab fragments linked by a disulfide bridge at the hinge region; H and C H (iv) a V fragment of one arm of an antibody; L and V H (v) an Fv fragment consisting of a V domain; H (vi) isolated complementarity-determining regions (CDRs); and (vi) isolated complementarity-determining regions (CDRs). In addition, two domains of the Fv fragment, V, and VD, are included. L and V H are encoded by individual genes, among which V L and V HThese regions can be joined using recombinant methods by synthetic linkers that allow them to be produced as single-chain proteins, in which case they pair to form monovalent molecules (known as single-chain Fvs (scFvs); see, e.g., Bird et al. (1988) Science 242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single-chain antibodies are also intended to be encompassed by the term "binding portion" of an antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are secreted for use in the same manner as intact antibodies. Binding portions can be produced by recombinant DNA techniques or by enzymatic or chemical cleavage of intact immunoglobulins.

[0168] Antibodies that bind to renalase of the present invention are antibodies that inhibit, block, or interfere with at least one renalase activity (e.g., enzymatic activity, substrate binding activity, receptor binding activity, etc.) in vitro, in situ, and / or in vivo. Suitable anti-renalase antibodies, specified portions, or variants can also optionally affect at least one renalase activity or function, including, but not limited to, RNA, DNA, or protein synthesis, protein release, renalase signaling, renalase cleavage, renalase activity, renalase receptor binding, renalase production and / or synthesis.

[0169] In one embodiment, the antibody of the present invention binds to renalase. In one embodiment, the antibody specifically binds to renalase-1. In another embodiment, the antibody specifically binds to renalase-2. In yet another embodiment, the antibody specifically binds to both renalase-1 and renalase-2. In addition, epitope-specific antibodies have been generated. Preferred antibodies of the present invention include monoclonal antibodies 1C-22-1, 1D-28-4, 1D-37-10, 1F-26-1, 1F-42-7, and 3A-5-2. Examples of bispecific antibodies, e.g., antibodies that recognize both renalase-1 and renalase-2, include antibodies 1C-22-1, 1D-28-4, 1D-37-10, and the polyclonal antibodies described herein. Examples of renalase-type-specific antibodies include 1F-26-1 and 1F-42-7, which are specific for renalase-1. 3A-5-2 is specific for renalase-2. Antibody fragments of the invention comprise humanized heavy and light chain Fv regions, such as those set forth in SEQ ID NOs: 219, 221, 223, and 225. In various embodiments of the invention, anti-renalase antibodies or antibody fragments comprise humanized heavy and light chain Fv regions, or fragments or portions thereof, such as those set forth in SEQ ID NOs: 219, 221, 223, and 225. Sequences encoding anti-renalase monoclonal antibodies are shown in Figures 4 through 13 and in Example 2.

[0170] The nucleic acid (SEQ ID NO:52) and amino acid (SEQ ID NO:9) sequences of the heavy chain coding sequence of monoclonal antibody 1D-28-4 are found in Figure 4. The nucleic acid (SEQ ID NO:53) and amino acid (SEQ ID NO:10) sequences of the light chain coding sequence of monoclonal antibody 1D-28-4 are found in Figure 5.

[0171] The nucleic acid (SEQ ID NO:60) and amino acid (SEQ ID NO:17) sequences of the heavy chain coding sequence of monoclonal antibody 1D-37-10 are found in Figure 6. The nucleic acid (SEQ ID NO:61) and amino acid (SEQ ID NO:18) sequences of the light chain coding sequence of monoclonal antibody 1D-37-10 are found in Figure 7.

[0172] The nucleic acid (SEQ ID NO:68) and amino acid (SEQ ID NO:25) sequences of the heavy chain coding sequence of monoclonal antibody 1F-26-1 are found in Figure 8. The nucleic acid (SEQ ID NO:69) and amino acid (SEQ ID NO:26) sequences of the light chain coding sequence of monoclonal antibody 1F-26-1 are found in Figure 9.

[0173] The nucleic acid (SEQ ID NO:76) and amino acid (SEQ ID NO:33) sequences of the heavy chain coding sequence of monoclonal antibody 1F-42-7 are found in Figure 10. The nucleic acid (SEQ ID NO:77) and amino acid (SEQ ID NO:34) sequences of the light chain coding sequence of monoclonal antibody 1F-42-7 are found in Figure 11.

[0174] The nucleic acid (SEQ ID NO:84) and amino acid (SEQ ID NO:41) sequences of the heavy chain coding sequence of monoclonal antibody 3A-5-2 are found in Figure 12. The nucleic acid (SEQ ID NO:85) and amino acid (SEQ ID NO:42) sequences of the light chain coding sequence of monoclonal antibody 3A-5-2 are found in Figure 13.

[0175] If some monoclonal antibodies can bind to the renalase protein, their V H and V L Sequences can be "mixed and matched" to create other anti-renalase binding molecules of the present disclosure. Renalase binding of such "mixed and matched" antibodies can be tested using the binding assays (e.g., immunoblotting, Bia-Core, etc.) described above and in the Examples. Preferably, V H and V L When chains are mixed and matched, a particular V H / V L V derived from pairing H The sequence is structurally similar to V H Also preferably, a particular V H / V L V derived from pairing L The sequence is structurally similar to V L It is replaced by an array.

[0176] Thus, in some embodiments, the invention is an antibody or antibody fragment, wherein the antibody or antibody fragment comprises at least one polypeptide sequence encoding a novel heavy chain variable sequence, hi other embodiments, the invention is an antibody or antibody fragment, wherein the antibody or antibody fragment comprises at least one polypeptide sequence encoding a novel light chain variable sequence.

[0177] In one embodiment, the first V H The complementarity determining region (HC CDR1) comprises the amino acid sequence set forth in SEQ ID NO: 155; the second V H The complementarity determining region (HC CDR2) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 156, SEQ ID NO: 162, and SEQ ID NO: 168; H The complementarity determining region (HC CDR3) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 157 and SEQ ID NO: 163; L The complementarity determining region (LC CDR1) comprises the amino acid sequence set forth in SEQ ID NO: 152; the second V L The complementarity determining region (LC CDR2) comprises the amino acid sequence set forth in SEQ ID NO: 153; L The complementarity determining region (LC CDR3) comprises an amino acid sequence selected from the group of SEQ ID NO: 154 and SEQ ID NO: 160. In one embodiment, HC CDR1 comprises SEQ ID NO: 155; HC CDR2 comprises SEQ ID NO: 156; HC CDR3 comprises SEQ ID NO: 157; LC CDR1 comprises SEQ ID NO: 152; LC CDR2 comprises SEQ ID NO: 153; and LC CDR3 comprises SEQ ID NO: 154. In one embodiment, V H comprises SEQ ID NO: 219; L comprises SEQ ID NO: 221. In one embodiment, these two polypeptides are linked by a linker to form a single chain variable region (scFv), wherein the orientation of the polypeptides relative to the linker is V H -Linker-V L , and V L -Linker-V H is selected from the group:

[0178] In one embodiment, the first V H The complementarity determining region (HC CDR1) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 197 and SEQ ID NO: 203; H The complementarity determining region (HC CDR2) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 198 and SEQ ID NO: 204; H The complementarity determining region (HC CDR3) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 199 and SEQ ID NO: 205; L The complementarity determining region (LC CDR1) comprises an amino acid sequence selected from the group consisting of SEQ ID NO: 194 and SEQ ID NO: 200; L The complementarity determining region (LC CDR2) comprises the amino acid sequence set forth in SEQ ID NO: 195; L The complementarity determining region (LC CDR3) comprises the amino acid sequence set forth in SEQ ID NO: 196. In one embodiment, HC CDR1 comprises SEQ ID NO: 197; wherein HC CDR2 comprises SEQ ID NO: 198; wherein HC CDR3 comprises SEQ ID NO: 199; wherein LC CDR1 comprises SEQ ID NO: 194; wherein LC CDR2 comprises SEQ ID NO: 195; and wherein LC CDR3 comprises SEQ ID NO: 196. In one embodiment, V H comprises SEQ ID NO: 223; L comprises SEQ ID NO: 225. In one embodiment, these two polypeptides are linked by a linker to form a single chain variable region (scFv), wherein the orientation of the polypeptides relative to the linker is V H -Linker-V L , and V L -Linker-V H is selected from the group:

[0179] In various embodiments, the novel heavy chain variable sequences further comprise at least one additional polypeptide sequence. In various embodiments, the novel light chain variable sequences further comprise at least one additional polypeptide sequence. In one embodiment, V H The polypeptide further comprises a heavy chain C H Contains 1 domain; V L The polypeptide further comprises a light chain CL In one embodiment, the V H The polypeptide further comprises a heavy chain C H 1 domain, heavy chain C H 2 domain, and heavy chain C H Contains three domains that form antibody heavy chains; V L The polypeptide further comprises a light chain C L The antibody heavy chain comprises a domain, forming an antibody light chain. In one embodiment, an antibody heavy chain and an antibody light chain are linked to form a half antibody. In one embodiment, this half antibody is linked to another half antibody to form an antibody. In various embodiments, the linkage of the heavy chain to the light chain to form the half antibody, and the linkage of these two half antibodies is achieved using a disulfide bond. In other embodiments, the linkage of the heavy chain to the light chain to form the half antibody, and the linkage of the two half antibodies, is achieved using a bond or linkage that is not a disulfide bond.

[0180] In another aspect, the present disclosure provides an isolated monoclonal antibody or binding portion thereof, comprising: (a) a heavy chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 9, 17, 25, 33, and 41; and (b) a light chain variable region comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 18, 26, 34, and 42, wherein the antibody specifically binds to a renalase protein.

[0181] Preferred heavy and light chain combinations include: (a) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 9 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 10; or (b) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 17 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; or (c) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 25 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 26; or (d) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 33 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 34; or (e) a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 41 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 42.

[0182] In another aspect, the disclosure provides an antibody comprising the heavy and light chain CDR1, CDR2 and CDR3 of 1D-28-4, 1D-37-10, 1F-26-1, 1F-42-7 or 3A-5-2 or a combination thereof. H The amino acid sequences of CDR1 are incorporated into the sequences shown in SEQ ID NOs: 11, 19, 27, 35, and 43, respectively. H The amino acid sequences of CDR2 are incorporated into the sequences shown in SEQ ID NOs: 12, 20, 28, 36, and 44, respectively. H The amino acid sequences of CDR3 are incorporated into the sequences shown in SEQ ID NOs: 13, 21, 29, 37, and 45, respectively. The amino acid sequences of VK CDR1 of 1D-28-4, 1D-37-10, 1F-26-1, 1F-42-7, and 3A-5-2 are incorporated into the sequences shown in SEQ ID NOs: 14, 22, 30, 38, and 46, respectively. The amino acid sequences of VK CDR2 of 1D-28-4, 1D-37-10, 1F-26-1, 1F-42-7, and 3A-5-2 are incorporated into the sequences shown in SEQ ID NOs: 15, 23, 31, 39, and 47. The amino acid sequences of the VK CDR3s of 1D-28-4, 1D-37-10, 1F-26-1, 1F-42-7, and 3A-5-2 are incorporated into the sequences shown in SEQ ID NOs: 16, 24, 32, 40, and 48, respectively. The CDR regions are indicated using the Kabat system (Kabat, EA, (1991) Sequences of Proteins of Immunological Interest, 5th ed., US Department of Health and Human Services, NIH Publication No. 91-3242).

[0183] Each of these antibodies can bind to a renalase family member, and given that the binding specificity is provided primarily by the CDR1, CDR2, and CDR3 regions, VH CDR1, CDR2, and CDR3 sequences and V L CDR1, CDR2, and CDR3 sequences may be "mixed and matched" (i.e., CDRs from different antibodies may be mixed and matched, but each antibody may have a V H CDR1, CDR2, and CDR3 and V L Other anti-renalase binding molecules of the disclosure are generated based on the V and VL sequences (which must contain CDR1, CDR2, and CDR3). Renalase binding of such "mixed and matched" antibodies can be tested using the binding assays described above and in the Examples (e.g., immunoblotting, Biacore® analysis, etc.). Preferably, V H When CDR sequences are mixed and matched, a particular V H The CDR1, CDR2 and / or CDR3 sequences from the sequence are replaced with structurally similar CDR sequence(s). L When CDR sequences are mixed and matched, a particular V L The CDR1, CDR2 and / or CDR3 sequences derived from the sequence are preferably replaced with structurally similar CDR sequence(s). H and V L The sequence may contain one or more V H and / or V L It will be readily apparent that CDR region sequences can be generated by substituting structurally similar sequences from the CDR sequences disclosed herein for monoclonal antibodies 1D-28-4, 1D-37-10, 1F-26-1, 1F42-7, or 3A-5-2.

[0184] Thus, in another aspect, the present invention provides an isolated monoclonal antibody or binding portion thereof comprising at least one selected from: (a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 19, 27, 35, and 43; (b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 20, 28, 36, and 44; (c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 21, 29, 37, and 45; (d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 22, 30, 38, and 46; (e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 23, 31, 39, and 47; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 24, 32, 40, and 48, wherein the antibody specifically binds to renalase.

[0185] In another embodiment, the antibody comprises at least one CDR selected from: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 11; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 12; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 13; (d) a light chain variable region CDR1 comprising SEQ ID NO: 14; (e) a light chain variable region CDR2 comprising SEQ ID NO: 15; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 16.

[0186] In another embodiment, the antibody comprises at least one CDR selected from: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 19; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 20; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 21; (d) a light chain variable region CDR1 comprising SEQ ID NO: 22; (e) a light chain variable region CDR2 comprising SEQ ID NO: 23; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 24.

[0187] In another embodiment, the antibody comprises at least one CDR selected from: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 27; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 28; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 29; (d) a light chain variable region CDR1 comprising SEQ ID NO: 30; (e) a light chain variable region CDR2 comprising SEQ ID NO: 31; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 32.

[0188] In another embodiment, the antibody comprises at least one CDR selected from: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 35; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 36; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 37; (d) a light chain variable region CDR1 comprising SEQ ID NO: 38; (e) a light chain variable region CDR2 comprising SEQ ID NO: 39; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 40.

[0189] In another embodiment, the antibody comprises at least one CDR selected from: (a) a heavy chain variable region CDR1 comprising SEQ ID NO: 43; (b) a heavy chain variable region CDR2 comprising SEQ ID NO: 44; (c) a heavy chain variable region CDR3 comprising SEQ ID NO: 45; (d) a light chain variable region CDR1 comprising SEQ ID NO: 46; (e) a light chain variable region CDR2 comprising SEQ ID NO: 47; and (f) a light chain variable region CDR3 comprising SEQ ID NO: 48.

[0190] The foregoing isolated anti-renalase antibody CDR sequences establish a novel family of renalase binding proteins, comprising polypeptides isolated in accordance with the present invention and comprising the listed CDR sequences. To generate and select CDRs of the present invention having renalase-binding and / or renalase-detection and / or renalase-neutralizing activity, standard methods known in the art for generating binding proteins of the present invention and assessing the renalase and / or renalase-binding and / or renalase-detection and / or neutralization characteristics of those binding proteins can be used, including, but not limited to, the methods specifically described herein.

[0191] Preferably, the renalase-binding molecules (e.g., antibodies) of the present invention exhibit a high ability to detect and bind renalase in complex mixtures of salts, compounds, and other polypeptides, as assessed, for example, by any one of several in vitro and in vivo assays known in the art. Those skilled in the art will understand that the renalase-binding molecules (e.g., antibodies, etc.) described herein as useful in methods of diagnosing, treating, and preventing disease will also be useful in the techniques and methods of the present invention, including, but not limited to, immunochromatographic assays, immunodot assays, Luminex assays, ELISA assays, ELISPOT assays, protein microarray assays, Western blot assays, mass spectrometry assays, radioimmunoassays (RIA), radial immunodiffusion assays, liquid chromatography-tandem mass spectrometry assays, Ouchterlony immunodiffusion assays, reverse-phase protein microarrays, rocket immunoelectrophoresis assays, immunohistochemistry assays, immunoprecipitation assays, complement fixation assays, FACS, protein chip assays, separation and purification processes, and affinity chromatography (see also, 2007, Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay Handbook, Gulf Professional Publishing; 1996, Diamandis and Christopoulos, Immunoassay, Academic Press). Press; 2005, Joos, Microarrays in Clinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, Proteomics Today, John Wiley and Sons; 2007).

[0192] More preferably, the renalase-binding molecules (e.g., antibodies, etc.) of the present invention exhibit a high ability to neutralize renalase activity (e.g., enzymatic activity, substrate-binding activity, receptor-binding activity, etc.) as assessed by any one of several in vitro and in vivo assays known in the art. For example, these renalase-binding molecules (e.g., antibodies, etc.) neutralize renalase-associated or renalase-mediated diseases or disorders. Preferably, the renalase-binding molecules (e.g., antibodies, etc.) of the present invention also exhibit a high ability to reduce or neutralize renalase activity. In some embodiments, the renalase is human renalase.

[0193] As used herein, a renalase-binding molecule (e.g., an antibody, etc.) that "specifically binds to a renalase protein" is intended to refer to a renalase-binding molecule (e.g., an antibody, etc.) that binds to the renalase protein of any animal. In some embodiments, the antibody binds to human renalase. Preferably, the renalase-binding molecule (e.g., an antibody, etc.) binds to a renalase protein of 1×10 -6 M or less, preferably 1×10 -7 M or less, preferably 1×10 -8 M or less, preferably 5×10 -9 M or less, preferably 1×10 -9 M or less, or even more preferably 3×10 -10 The term "does not substantially bind" to a protein or cell as used herein means that the antibody does not bind to the protein or cell or does not bind with high affinity, i.e., binds to the renalase protein with a KD of 1 x 10 or less. 6 M or more, preferably 1×10 5 M or more, preferably 1×10 4 M or more, preferably 1×10 3 M or more, and even more preferably 1×10 2It means binding with a KD greater than M or more. As used herein, the term "KD" is intended to refer to the dissociation constant, which is obtained by the ratio of Kd to Ka (i.e., Kd / Ka) and is expressed as a molar concentration (M). KD values ​​for renalase-binding molecules (e.g., antibodies, etc.) can be determined using methods well established in the art. A preferred method for determining the KD of a binding molecule (e.g., antibody, etc.) is by using surface plasmon resonance, preferably by using a biosensor system such as a Biacore® system.

[0194] As used herein, the term "high affinity" with respect to an IgG antibody means an antibody with a binding affinity of 1×10 with respect to the target binding partner molecule. -7 M or less, preferably 5×10 -8 M or less, and even more preferably 1×10 -8 M or less, and even more preferably 5×10 -9 M or less, and even more preferably 1×10 -9 "High affinity" refers to an antibody with a KD of 10 M or less. However, "high affinity" binding can vary for other antibody isotypes. For example, "high affinity" binding for an IgM isotype is defined as binding of 10 M or less. -6 M or less, preferably 10 -7 M or less, and even more preferably 10 -8 This refers to an antibody with a KD of M or less.

[0195] In some embodiments, the antibody comprises a heavy chain constant region, such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM, or IgD constant region. Preferably, the heavy chain constant region is an IgG1 heavy chain constant region or an IgG4 heavy chain constant region. Additionally, the antibody can comprise a light chain constant region, either a kappa light chain constant region or a lambda light chain constant region. Preferably, the antibody comprises a kappa light chain constant region. Alternatively, the antibody portion can be, for example, a Fab fragment or a single-chain Fv fragment.

[0196] Preparation of anti-renalase antibodies The present invention provides compositions that bind to renalase. The renalase molecules disclosed herein are a class of molecules that include those with high and / or significant sequence identity to other polypeptides disclosed herein. More specifically, a putative renalase will share at least about 40% sequence identity with a nucleic acid having the sequence of SEQ ID NO: 49 or 51. More preferably, the nucleic acid encoding renalase has at least about 45% identity, or at least about 50% identity, or at least about 55% identity, or at least about 60% identity, or at least about 65% identity, or at least about 70% identity, or at least about 75% identity, or at least about 80% identity, or at least about 85% identity, or at least about 90% identity, or at least about 95% identity, or at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 49 or 51 disclosed herein. Even more preferably, the nucleic acid is SEQ ID NO: 49 or 51 or 93 or 95. The term "renalase" also includes renalase isoforms. The renalase gene contains nine exons spanning 310,188 bp on chromosome 10 of the human genome. The renalase clone disclosed herein (SEQ ID NO: 49, GenBank Accession No. BC005364) is a gene containing exons 1, 2, 3, 4, 5, 6, and 8. There are at least two additional alternatively spliced ​​forms of the renalase protein represented in the human genome database. One alternatively spliced ​​form contains exons 1, 2, 3, 4, 5, 6, and 9, identified by clones in the human genome database as GenBank Accession Nos. AK002080 and NMJ18363, the sequences of which are expressly incorporated herein by reference. The other alternatively spliced ​​form contains exons 5, 6, 7, and 8, identified by a clone in the human genome database under GenBank accession number BX648154, the sequence of which is expressly incorporated herein by reference.Unless otherwise specified, "renalase" includes all known renalases (e.g., rat renalase and human renalase), as well as renalases to be discovered, including, but not limited to, human renalase and chimpanzee renalase, having the characteristics and / or physical properties of the renalases disclosed herein.

[0197] Additionally, the putative renalase shares at least about 60% sequence identity with a polypeptide having the sequence of SEQ ID NO: 8 or 50. More preferably, the renalase has at least about 45% identity, or at least about 50% identity, or at least about 55% identity, or at least about 60% identity, or at least about 65% identity, or at least about 70% identity, or at least about 75% identity, or at least about 80% identity, or at least about 85% identity, or at least about 90% identity, or at least about 95% identity, or at least about 98%, or at least about 99% sequence identity to SEQ ID NO: 8 or 50 disclosed herein. Even more preferably, the renalase polypeptide has the amino acid sequence of SEQ ID NO: 8 or 50 or 92 or 94.

[0198] In one embodiment, antibodies of the present invention are made by using peptides derived from the renalase sequence to immunize an animal, causing the animal to produce antibodies against the immunogen. Exemplary immunogens include peptides derived from renalase. That is, peptides having fragments of the renalase sequence can be used in the present invention. Peptides can be made in a variety of ways, including expression as recombinant peptides, expression as larger polypeptides, and enzymatic or chemical cleavage. Alternatively, they may be made synthetically, as known in the art. Preferred peptides used to make affinity reagents of the present invention are shown in Figure 1 (SEQ ID NOs: 1-7).

[0199] Anti-renalase antibodies of the invention can optionally be produced by a variety of techniques, including the standard somatic cell hybridization technique (hybridoma method) of Kohler and Milstein (1975) Nature 256:495. In the hybridoma method, a mouse or other suitable host animal, such as a hamster or macaque, is immunized as described herein to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0200] Methods for producing and screening specific antibodies using hybridoma technology are conventional and well known in the art. In one embodiment, the present invention provides a method for producing monoclonal antibodies, as well as antibodies produced by a method comprising culturing hybridoma cells secreting the antibodies of the present invention, wherein the hybridomas are preferably produced by fusing splenocytes isolated from mice, rabbits, or other species immunized with a polypeptide or peptide of the present invention with myeloma cells, and then screening for hybridomas resulting from the fusion of hybridoma clones that secrete antibodies capable of binding to the polypeptide of the present invention. Briefly, mice can be immunized with a renalase polypeptide or a peptide thereof. In a preferred embodiment, the renalase polypeptide or a peptide thereof is administered with an adjuvant to stimulate the immune response. Such adjuvants include complete or incomplete Freund's adjuvant, RIBI (muramyl dipeptide), or ISCOM (immunostimulating complex). Such adjuvants may protect the polypeptide from rapid dispersal by trapping it in a local deposit, or they may contain substances that stimulate the host to secrete factors that are chemotactic for macrophages and other components of the immune system. Preferably, if a polypeptide is administered, the immunization schedule will involve two or more administrations of the polypeptide, spread out over several weeks.

[0201] Alternatively, rabbits can be immunized with a renalase polypeptide or a peptide thereof. In this embodiment, either the full-length renalase protein or a peptide derived from renalase can be used as the immunogen.

[0202] The renalase used in the present invention can take various forms. For example, these can include purified renalase protein or fragments thereof, or recombinantly produced renalase or fragments thereof. In some embodiments, the renalase is human renalase. When recombinant renalase is used, it can be produced in eukaryotic or prokaryotic cells, as known in the art. Booster immunogens include renalase-derived peptides. That is, peptides having fragments of the renalase sequence can be used in the present invention. Peptides can be produced in various ways, including expression as a recombinant peptide or as a larger polypeptide, and can be enzymatically or chemically cleaved. Alternatively, they can be produced synthetically, as known in the art. Preferred peptides used to make affinity reagents of the present invention are shown in Figure 1 (SEQ ID NOS: 1-7). The full-length amino acid sequence of human renalase is shown in SEQ ID NOS: 8, and known polymorphisms are possible, as noted herein (compare SEQ ID NOS: 92). The amino acid sequence of renalase-2 is found in SEQ ID NO:50, and again, as shown here, known polymorphisms are possible (compare SEQ ID NO:94). It is understood that other polymorphisms exist, and these are also included in the definition of renalase. In some embodiments, the renalase binding molecules of the present invention specifically bind to at least one of SEQ ID NOs:1-7, 8, 50, 92, 94, and fragments thereof.

[0203] The anti-renalase antibodies can also optionally be produced by immunization of transgenic animals (e.g., mice, rats, hamsters, non-human primates, etc.) capable of producing a repertoire of human antibodies as described herein and / or known in the art. Cells producing human anti-renalase antibodies can be isolated from such animals and immortalized using suitable methods, such as those described herein. Alternatively, the antibody coding sequence can be cloned, by methods described herein and known in the art, into a suitable vector and used to transfect host cells for antibody expression and isolation.

[0204] The use of transgenic mice carrying human immunoglobulin (Ig) loci in their germline configuration provides for the isolation of high-affinity, fully human monoclonal antibodies directed against a variety of targets, including human self-antigens, to which the normal human immune system is tolerant (Lonberg, N. et al., U.S. Pat. Nos. 5,569,825, 6,300,129, and 1994, Nature 368:856-9; Green, L. et al., 1994, Nature Genet. 7:13-21; Green, L. and Jakobovits, 1998, Exp. Med. 188:483-95; Lonberg, N. and Huszar, D., 1995, Int. Rev. Immunol. 13:65-93; Kucherlapati et al., U.S. Pat. No. 6,713,610; Bruggemann, M. et al., 1991, Eur. J. Immunol. 13:65-93). Immunol. 21:1323-1326; Fishwild, D. et al., 1996, Nat. Biotechnol. 14:845-851; Mendez, M. et al., 1997, Nat. Genet. 15:146-156; Green, L., 1999, J. Immunol. Methods 231:11-23; Yang, X. et al., 1999, Cancer Res. 59:1236-1243; Bruggemann, M. and Taussig, M J., Curr. Opin. Biotechnol. 8:455-458, 1997; Tomizuka et al., WO02043478). The endogenous immunoglobulin loci in such mice can be disrupted or deleted, resulting in the loss of the animal's ability to produce antibodies encoded by endogenous genes. Additionally, companies such as Abgenix (Fremont, CA) and Medarex (San Jose, CA) can be engaged to provide human antibodies directed against partner molecules (e.g., antigens) that bind to selected targets using technology described elsewhere herein.

[0205] In another embodiment, human antibodies are selected from phage libraries, where the phage comprise human immunoglobulin genes and the libraries express human antibody binding domains, e.g., as single-chain antibodies (scFv), Fab, or some other construct displaying paired or unpaired antibody variable regions (Vaughan et al., Nature Biotechnology 14:309-314 (1996); Sheets et al., PITAS (USA) 95:6157-6162 (1998)); Hoogenboom and Winter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol., 222:581 (1991)). Human monoclonal antibodies of the invention can also be prepared using phage display methods for screening human immunoglobulin gene libraries. Such phage display methods for isolating human antibodies are established in the art. See, for example, U.S. Patent Nos. 5,223,409; 5,403,484; and 5,571,698 to Ladner et al.; U.S. Patent Nos. 5,427,908 and 5,580,717 to Dower et al.; U.S. Patent Nos. 5,969,108 and 6,172,197 to McCafferty et al.; and U.S. Patent Nos. 5,885,793; 6,521,404; 6,544,731; 6,555,313; 6,582,915; and 6,593,081 to Griffiths et al.

[0206] Preparation of immunogenic antigens and monoclonal antibody production can be carried out using any suitable technique, including recombinant protein production. The immunogenic antigen can be administered to the animal in the form of purified protein or protein mixtures containing whole cells or cell or tissue extracts, or the antigen can be formed de novo in the animal's body from nucleic acid encoding the antigen or a portion thereof.

[0207] The isolated nucleic acids of the present invention can be produced using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or a combination thereof, as is well known in the art. DNA encoding monoclonal antibodies is readily isolated and sequenced using methods known in the art (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of murine antibodies). When hybridomas are generated, such cells serve as a source of such DNA. Alternatively, display technologies, such as phage or ribosome display libraries, in which the coding sequence and translation product are linked are used, simplifying the selection of binders and nucleic acids. After phage selection, the antibody coding regions from the phage are isolated and used to generate whole antibodies, including human antibodies, or any other desired binding fragments, which are expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria.

[0208] humanized antibodies The present invention further provides humanized immunoglobulins (or antibodies) that bind to human renalase. Humanized forms of immunoglobulins have variable framework region(s) substantially derived from a human immunoglobulin (referred to as the acceptor immunoglobulin) and CDRs substantially derived from a non-human mAb that specifically binds to renalase. The constant region(s), if present, are also substantially derived from a human immunoglobulin. The humanized antibodies have a K for renalase. D , at least about 10 -6 M (1 μM), approximately 10 -7 The binding affinity of humanized antibodies may be greater or less than that of the murine antibody from which they are derived. Substitutions in either the CDR residues or human residues can be made to affect and improve the affinity of the humanized antibody for renalase.

[0209] The source for the generation of humanized antibodies that bind to renalase is preferably the 1D-28-4, 1D-37-10, 1F-26-1, 1F-42-7, or 3A-5-2 rabbit monoclonal antibodies, the production, isolation, and characterization of which are described in the Examples provided herein, although other antibodies that compete with the 1D-28-4, 1D-37-10, 1F-26-1, 1F42-7, or 3A-5-2 antibodies for binding to renalase can also be used. The identified CDRs set forth in the Sequence Listing can be the starting point for the humanization process. For example, any one or more of the following amino acid sequences (and their corresponding nucleic acid sequences) can be the starting point for the humanization process: (a) a heavy chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 11, 19, 27, 35, and 43; (b) a heavy chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 12, 20, 28, 36, and 44; (c) a heavy chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 13, 21, 29, 37, and 45; (d) a light chain variable region CDR1 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 14, 22, 30, 38, and 46; (e) a light chain variable region CDR2 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 15, 23, 31, 39, and 47; and (f) a light chain variable region CDR3 comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 24, 32, 40, and 48.

[0210] Substitution of rabbit or mouse CDRs into a human variable domain framework is likely to result in retention of their correct spatial orientation, provided that the human variable domain framework adopts the same or similar conformation as the parent variable framework from which the CDRs originated. This is achieved by obtaining human variable domains from human antibodies whose framework sequences exhibit a high degree of sequence identity with the parent variable framework domains from which the CDRs are derived. The heavy and light chain variable framework regions can be derived from the same or different human antibody sequences. The human antibody sequences can be sequences of naturally occurring human antibodies, sequences derived from human germline immunoglobulin sequences, or consensus sequences of several human antibody and / or germline sequences.

[0211] Suitable human antibody sequences are identified by computer comparison of the amino acid sequences of rabbit or mouse variable regions with the sequences of known human antibodies. This comparison is performed separately for the heavy and light chains, although the principles are similar.

[0212] In one example, the amino acid sequence of an anti-renalase mAb is used to query a human antibody database compiled from public antibody sequence databases. The heavy chain variable region can be used to find the human variable region with the highest sequence identity. The light chain variable region can similarly be used to find the human variable region with the highest sequence identity. A DNA construct is prepared for each parent variable region in which the region encoding one CDR of the heavy chain variable region from the parent mAb donor is transferred to the selected human heavy chain variable sequence in place of the CDR of the human variable region.

[0213] The non-native proximity of the parent CDR regions with the human variable framework regions creates non-native conformational constraints that, if not corrected by substitution of certain amino acid residues, will lead to loss of binding affinity. As noted above, the humanized antibodies of the present invention comprise variable framework region(s) substantially derived from a human immunoglobulin and CDRs substantially derived from a parent (e.g., rabbit or mouse) immunoglobulin. Having identified the CDRs of the parent antibody and a suitable human acceptor immunoglobulin sequence, the next step is to determine which, if any, residues from these components should be substituted to optimize the properties of the resulting humanized antibody. Generally, substitution of human amino acid residues with the parent should be minimized, since the introduction of parent residues increases the risk of the antibody eliciting an immune response in humans. Amino acids are selected for substitution based on their potential effect on CDR conformation and / or binding to the target bound to the partner molecule. Study of such potential effects can be performed by modeling, examining the characteristics of the amino acid at specific positions, or empirical observation of the effect of specific amino acid substitutions or mutagenesis. With respect to empirical methods, it has been found particularly advantageous to generate libraries of variant sequences that can be screened for desired activity, binding affinity, or specificity. One format for generating such libraries of variants is a phage display vector. Alternatively, variants can be generated using other methods for variation of nucleic acid sequences encoding targeted residues within the variable domains.

[0214] Another method for determining whether further substitutions are necessary and the selection of amino acid residues for substitution can be achieved using computer modeling. Computer hardware and software for generating three-dimensional images of immunoglobulin molecules are widely available. Generally, molecular models are created starting from a resolved structure of an immunoglobulin chain or its domain. The chains to be modeled are compared with the chains or domains of the resolved three-dimensional structure for amino acid sequence similarity, and those chains or domains showing the greatest sequence similarity are selected as the starting point for building the molecular model. The resolved starting structure is modified to allow for differences between the actual amino acids in the immunoglobulin chain or domain to be modeled and those in the starting structure. The modified structure is then assembled into a composite immunoglobulin. Finally, the model is refined by energy minimization and by verifying that all atoms are within appropriate distances from each other and that bond lengths and angles are within chemically acceptable limits.

[0215] Generally, the CDR regions in a humanized antibody are substantially identical, and more usually identical, to the corresponding CDR regions in the parent antibody from which they are derived. Although not usually desirable, it is sometimes possible to make one or more conservative amino acid substitutions of CDR residues without appreciably affecting the binding affinity of the resulting humanized immunoglobulin. In some cases, substitutions in the CDR regions can enhance binding affinity.

[0216] Other than the specific amino acid substitutions discussed above, the framework regions of humanized immunoglobulins are usually substantially identical, more usually identical, to the framework regions of the human antibodies from which they are derived. Of course, many of the amino acids in framework regions make little or no direct contribution to the specificity or affinity of an antibody. Thus, many individual conservative substitutions of framework residues can be tolerated without appreciable change in the specificity or affinity of the resulting humanized immunoglobulin.

[0217] Due to code degeneracy, a variety of nucleic acid sequences will encode each immunoglobulin amino acid sequence. The desired nucleic acid sequence can be generated by solid-phase DNA synthesis or by PCR mutagenesis of an earlier prepared variant of the desired polynucleotide. All nucleic acids encoding the antibodies described in this application are expressly included in the present invention.

[0218] The variable segments of humanized antibodies produced as described above are typically linked to at least a portion of a human immunoglobulin constant region. The antibody will contain both light and heavy chain constant regions. The heavy chain constant region usually contains CH1, hinge, CH2, and CH3 domains, and sometimes a CH4 domain.

[0219] The humanized antibody may comprise any type of constant domain from any antibody class, including IgM, IgG, IgD, IgA, and IgE, and any subclass (isotype), including IgG1, IgG2, IgG3, and IgG4. If it is desired that the humanized antibody exhibit cytotoxicity, the constant domain will usually be a complement-fixing constant domain, typically of class IgG1. If such cytotoxicity is not desired, the constant domain may be of the IgG2 class. The humanized antibody may comprise sequences from more than one class or isotype.

[0220] Nucleic acids encoding the humanized light and heavy chain variable regions, optionally linked to constant regions, are inserted into an expression vector. The light and heavy chains can be cloned in the same or different expression vectors. The DNA segments encoding immunoglobulin chains are operably linked to control sequences in the expression vector(s) that ensure expression of immunoglobulin polypeptides. Such control sequences include signal sequences, promoters, enhancers, and transcription termination sequences (see Queen et al., Proc. Natl. Acad. Sci. USA 86, 10029 (1989); WO 90 / 07861; Co et al., J. Immunol. 148, 1149 (1992), which are incorporated by reference in their entirety for all purposes).

[0221] Methods of Using Renalase-Binding Molecules Given the properties of the renalase-binding molecules (e.g., antibodies) of the present invention, the renalase-binding molecules are suitable as diagnostic, therapeutic and prophylactic agents for diagnosing, treating or preventing renalase-associated conditions in humans and animals.

[0222] Generally, uses involve administering a therapeutically or prophylactically effective amount of one or more monoclonal antibodies or binding fragments of the invention to a susceptible subject or a subject exhibiting a condition in which renalase activity is known to have pathological sequelae, such as tumor growth and metastasis. Active forms of renalase binding molecules, including antibody Fab and F(ab')2 fragments, can be administered.

[0223] Preferably, the renalase-binding molecule used is compatible with the recipient species so that an immune response to the renalase-binding molecule does not result in an unacceptably short circulating half-life or induces an immune response to the renalase-binding molecule in the subject. Preferably, the administered renalase-binding molecule performs several secondary functions, such as binding to the subject's Fc receptor and activating the ADCC mechanism.

[0224] Treatment of an individual may involve administering a therapeutically effective amount of a renalase-binding molecule of the present invention. The renalase-binding molecule may be provided in a kit as described below. The renalase-binding molecules may be used or administered, for example, as a mixture in equal amounts, or may be provided individually in sequence or all at once. When providing a renalase-binding molecule to a patient, the dose of the drug administered will vary depending on factors such as the patient's age, weight, height, sex, general medical condition, and medical history.

[0225] Generally, when administering a systemic dose of a renalase-binding molecule, it is desirable to provide the recipient with a dose of the renalase-binding molecule in the range of about 1 ng / kg to 100 ng / kg, 100 ng / kg to 500 ng / kg, 500 ng / kg to 1 μg / kg, 1 μg / kg to 100 μg / kg, 100 μg / kg to 500 μg / kg, 500 μg / kg to 1 mg / kg, 1 mg / kg to 50 mg / kg, 50 mg / kg to 100 mg / kg, or 100 mg / kg to 500 mg / kg of recipient body weight, although lower or higher doses may be administered. Doses as low as about 1.0 mg / kg are expected to show some efficacy. Preferably, about 5 mg / kg is an acceptable dose, although dosage levels up to about 50 mg / kg are also preferred, particularly for therapeutic use. Alternatively, a specific amount of renalase-binding molecule administered may be given without being based on patient weight, such as an amount ranging from 1 μg to 100 μg, 1 mg to 100 mg, or 1 g to 100 g. For example, site-specific administration can be to a body compartment or sinus, such as intra-articular, intrabronchial, intra-abdominal, intracapsular, intrachondral, intra-sinus, intracavity, intracerebral, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, transvaginal, transrectal, buccal, sublingual, intranasal, intraocular, or transdermal means.

[0226] The renalase-binding molecule compositions are intended for parenteral (subcutaneous, intramuscular or intravenous) or any other administration, particularly in the form of a liquid solution or suspension; for vaginal or rectal administration, particularly in semi-solid form, such as but not limited to creams and suppositories; for oral or sublingual administration, such as but not limited to in the form of tablets or capsules; or for intranasal administration, such as but not limited to powders, nasal drops or sprays or certain medications; or for the treatment of dental diseases, such as but not limited to eye drops; or for the treatment of dental diseases; or with chemical enhancers, such as dimethyl sulfoxide, which either modify the skin structure or increase the drug concentration in transdermal patches, or with oxidizing agents (WO) that allow the application of protein and peptide-containing formulations to the skin. Transdermal delivery systems, including but not limited to gels, ointments, lotions, suspensions, or patch delivery systems, can be formulated in conjunction with other methods, such as 98 / 53847, or by application of an electric field to create a transient transport pathway, such as electroporation, or by application of ultrasound to enhance the movement of charged drugs through the skin, such as iontophoresis, or sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402).

[0227] In a similar approach, another therapeutic use of the renalase-binding molecules of the present invention is the active immunization of patients with anti-idiotypic antibodies raised against one of the present monoclonal antibodies. Immunization with anti-idiotypes that mimic the structure of the epitope can induce an active anti-renalase response (Linthicum, D. S. and Farid, N. R., Anti-idiotypes, Receptors, and Molecular Mimicry (1988), pp. 1-5 and 285-300).

[0228] The renalase-binding molecules of the present invention can be formulated according to known methods to prepare pharmaceutically useful compositions, whereby these materials, or their functional derivatives, are mixed and combined with a pharmaceutically acceptable carrier vehicle. Suitable vehicles and their formulations, including other human proteins such as human serum albumin, are described, for example, in "Remington's Pharmaceutical Sciences" (16th ed., Osol, A., ed., Mack, Easton Pa. (1980)). To form a pharmaceutically acceptable composition suitable for effective administration, such compositions contain an effective amount of the compound described above together with an appropriate amount of carrier vehicle. Additional formulation methods may be utilized to control the duration of action. Controlled-release preparations may be achieved through the use of polymers to complex or absorb the compound. Another possible method for controlling the duration of action of a controlled-release preparation is by incorporating the compound of the present invention into particles of polymeric materials, such as polyesters, polyamino acids, hydrogels, poly(lactic acid), or ethylene vinyl acetate copolymers. Alternatively, instead of incorporating these substances into polymeric particles, it is possible to entrap these substances in microcapsules prepared, for example, by interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively, or in colloidal drug delivery systems, such as liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules or macroemulsions.

[0229] This treatment may be given as a single dose schedule, or preferably as a repeated dose schedule, in which an initial course of treatment is given in 1 to 10 individual doses, followed by another dose at subsequent time intervals as needed to maintain or strengthen the response, e.g., a second dose given at 1 to 4 months, and if necessary, subsequent dose(s) at several months thereafter. Examples of suitable treatment schedules include: (i) 0, 1 month, and 6 months, (ii) 0, 7 days, and 1 month, (iii) 0 and 1 month, (iv) 0 and 6 months, or any other schedule sufficient to elicit the desired response expected to alleviate disease symptoms or reduce disease severity.

[0230] The present invention also provides kits useful for practicing the present invention. The kit includes a first container containing or packaged in association with the antibody described above. The kit may also include separate containers containing or packaging associated solutions necessary for or facilitating practicing the present invention. These containers may be made of glass, plastic, or foil, and may be vials, bottles, pouches, tubes, bags, or the like. The kit may also include written information, such as procedures for practicing the present invention, or analytical information, such as the amounts of reagents contained in the first container means. The container, along with the written information, may be within another container device, such as a box or bag.

[0231] Yet another embodiment of the present invention is a kit for detecting renalase in a biological sample. The kit includes a container holding one or more renalase-binding molecules that bind to an epitope of renalase, and instructions for using the renalase-binding molecules to bind to renalase to form a complex, and detecting the formation of the complex, the presence or absence of which correlates with the presence or absence of renalase in the sample. An example of a container includes a multi-well plate, which allows for simultaneous detection of renalase in multiple samples.

[0232] Combination Therapy The renalase binding molecule compositions of the present invention can be used in combination with other therapeutic treatments or agents to treat diseases or disorders. For example, the renalase binding molecules of the present invention can be administered alone or in combination with one or more therapeutically effective agents or treatments. The other therapeutically effective agents can be conjugated to the renalase binding molecules of the present invention, incorporated into the same composition as the renalase binding molecules of the present invention, or administered as separate compositions. The other therapeutic agents or treatments can be administered before, during, and / or after administration of the antibodies or related compounds of the present invention.

[0233] In some embodiments, the renalase binding molecule of the present invention is co-administered with one or more other therapeutic agents or treatments. In other embodiments, the renalase binding molecule of the present invention is administered independently of the administration of one or more other therapeutic agents or treatments. For example, the renalase binding molecule of the present invention is administered first, followed by the administration of one or more other therapeutic agents or treatments. Alternatively, one or more other therapeutic agents are administered first, followed by the administration of the renalase binding molecule of the present invention. As another example, a treatment (e.g., surgery, radiation, etc.) is performed first, followed by the administration of the renalase binding molecule of the present invention.

[0234] Other therapeutically effective agents / treatments include surgery, anti-neoplastic agents (including chemotherapeutic agents and radiation), anti-angiogenic agents, antibodies against other targets, small molecules, photodynamic therapy, immunotherapy, immunoenhancing therapy, cytotoxic agents, cytokines, chemokines, growth inhibitory agents, antihormones, kinase inhibitors, cardioprotective agents, immunostimulatory agents, immunosuppressants, and agents that promote hematopoietic cell proliferation.

[0235] In one embodiment, an "another therapeutic agent" as used herein is a second separate therapeutic agent or anti-cancer agent, i.e., a therapeutic agent or anti-cancer agent "other than" the renalase-binding molecule of the present invention. Any secondary therapeutic agent can be used in the combination therapy of the present invention. Similarly, the secondary therapeutic agent or "second anti-cancer agent" may be selected with a view to achieving additive, greater additive, and potentially synergistic effects according to the following guidelines.

[0236] To practice combined antitumor therapy, the renalase-binding molecule of the present invention will be administered to an animal or patient in combination with another, i.e., second, individual anticancer agent in a manner effective to produce their combined antitumor effect in the animal or patient. Thus, these agents will be provided in amounts and for periods effective to produce their combined or simultaneous presence within the tumor or tumor vasculature and their combined effect within the tumor environment. To achieve this goal, the renalase-binding molecule of the present invention and the second, individual anticancer agent may be administered to an animal substantially simultaneously, either in a single composition or as two separate compositions using different routes of administration.

[0237] Alternatively, the renalase-binding molecule of the present invention can precede or follow the second, separate anti-cancer agent by an interval ranging, for example, from minutes to weeks. In some embodiments in which the renalase-binding molecule of the present invention and the second, separate anti-cancer agent are administered to an animal separately, it is ensured that no significant period of time will expire between the time of each delivery, so that each agent can still exert its beneficial combined effect on the tumor. In such cases, contact of both agents with the tumor within about 5 minutes to about 1 week of each other, more preferably within about 12 to 72 hours of each other, is contemplated, with a delay of no more than about 12 to 48 hours being most preferred.

[0238] The second therapeutic agent for the individualized time-directed combination therapy may be selected based on several criteria, including those discussed elsewhere herein. However, the preference for selecting one or more second individual anti-cancer agents prior to or consecutive with administration does not preclude their use in substantially simultaneous administration, if desired. The second individual anti-cancer agent is selected to be administered "before" the primary therapeutic agent of the present invention and is designed to achieve enhanced and potentially synergistic effects.

[0239] Secondary individual anti-cancer agents selected for administration "sequentially" to the primary therapeutic agent of the present invention and designed to achieve enhanced and potentially synergistic effects include agents that benefit from the action of the primary therapeutic agent. Thus, effective secondary individual anti-cancer agents for sequential administration include angiogenesis inhibitors that inhibit metastasis; agents that target necrotic tumor cells, such as antibodies specific for intracellular binding partner molecules that become accessible to malignant cells in vivo (U.S. Patent Nos. 5,019,368, 4,861,581, and 5,882,626, each specifically incorporated herein by reference); chemotherapeutic agents; and anti-tumor cell immunoconjugates that attack any tumor cells.

[0240] The renalase-binding molecules of the present invention can also be administered in combination with cancer immunotherapies. Cancer immunotherapies can be designed to elicit a humoral immune response against the subject's cancer cells, or a cell-mediated immune response against the subject's cancer cells, or a combination of a humoral response and a cell-mediated response against the subject's cancer cells. Non-limiting examples of cancer immunotherapies useful in combination with the renalase-binding molecules of the present invention include cancer vaccines, DNA cancer vaccines, adoptive cell therapy, adoptive immunotherapy, CAR T cell therapy, antibodies, immune-enhancing compounds, cytokines, interleukins (e.g., IL-2, etc.), interferons (e.g., IFN-α, etc.), and checkpoint inhibitors (e.g., PD-1 inhibitors, CTLA-4 inhibitors, etc.).

[0241] In some situations, it may be desirable to extend the treatment period significantly, where several days (2, 3, 4, 5, 6, or 7), weeks (1, 2, 3, 4, 5, 6, 7, or 8), or even months (1, 2, 3, 4, 5, 6, 7, or 8) pass between administrations. This may be advantageous in situations where one treatment, such as the primary therapeutic agent of the present invention, is intended to substantially destroy the tumor, and another treatment, such as administration of an angiogenesis inhibitor, is intended to prevent micrometastases or tumor regrowth. However, the antiangiogenic agent should be administered carefully after surgery to allow wound healing to take effect. The angiogenesis inhibitor may then be administered for the life of the patient.

[0242] It is also envisioned that administration of two or more of either the renalase-binding molecule of the present invention or a second individual anti-cancer agent may be utilized. The renalase-binding molecule of the present invention and the second individual anti-cancer agent may be administered interchangeably every other day or every other week; or a course of one agent may be given followed by a course of treatment with the other. In any event, all that is required to achieve tumor regression using combination therapy is that both agents be delivered in a combined amount effective to exert an anti-tumor effect, regardless of the time of administration.

[0243] Chemotherapeutic agents can be used in combination with the renalase inhibitors of the present invention to kill proliferating tumor cells and enhance the necrotic areas produced by the overall treatment.

[0244] One aspect of the present invention provides methods for treating or preventing cancer using the renalase inhibitors of the present invention. Those skilled in the art will understand that treating or preventing cancer in a patient includes, by way of non-limiting example, killing or destroying cancer cells, as well as slowing the rate of cancer cell growth or cell division. Those skilled in the art will also understand that cancer cells can be, by way of non-limiting example, primary cancer cells, cancer stem cells, or metastatic cancer cells. The following are non-limiting examples of cancers that may be treated by the disclosed methods and compositions: acute lymphoblastic; acute myeloid leukemia; adrenocortical carcinoma; adrenocortical carcinoma, pediatric; appendix cancer; basal cell carcinoma; extrahepatic bile duct cancer; bladder cancer; bone cancer; osteosarcoma and malignant fibrous histiocytoma; brain stem glioma, pediatric; brain tumor, adult; brain tumor, brain stem glioma, pediatric; brain tumor, central nervous system atypical teratoid / rhabdoid tumor, pediatric; central nervous system embryonal tumor; cerebellar astrocytoma; cerebral astrocytoma / malignant glioma; craniopharynx tumors; ependymoblastoma; ependymoma; medulloblastoma; medulloepithelioma; pineal parenchymal tumor of intermediate differentiation; supratentorial primitive neuroectodermal tumor and pineoblastoma; gliomas of the visual pathway and hypothalamus; brain and spinal cord tumors; breast cancer; bronchial tumors; Burkitt's lymphoma; carcinoid tumor; carcinoid tumor, gastrointestinal tract; central nervous system atypical teratoid / rhabdoid tumor; central nervous system embryonal tumors; central nervous system lymphoma; cerebellar astrocytoma, cerebral astrocytoma / malignant glioma, children; cervical cancer; chordoma, Pediatric; chronic lymphocytic leukemia; chronic myeloid leukemia; chronic myeloproliferative syndrome; colon cancer; colorectal cancer; craniopharyngioma; cutaneous T-cell lymphoma; esophageal cancer; Ewing's sarcoma family tumors; extragonadal germ cell tumors; extrahepatic bile duct cancer; eye cancer, intraocular melanoma; eye cancer, retinoblastoma; gallbladder cancer; gastric (gastrointestinal) cancer; gastrointestinal carcinoid tumors; gastrointestinal stromal tumors (GIST); extracranial germ cell tumors; extragonadal germ cell tumors; ovarian germ cell tumors; gestational trophoblastic tumors; gliomas; pediatric brainstem gliomas; gliomas Glioma, pediatric cerebral astrocytoma; Glioma, pediatric visual conduction and hypothalamic; Hairy cell leukemia; Head and neck cancer; Hepatocellular (liver) carcinoma; Langerhans cell histiocytosis; Hodgkin's lymphoma; Hypopharyngeal carcinoma; Hypothalamic and visual conduction glioma; Intraocular melanoma; Islet cell tumor; Kidney (renal cell) carcinoma; Langerhans cell histiocytosis; Laryngeal carcinoma; Leukemia, acute lymphoblastic; Leukemia, acute myeloid; Leukemia, chronic lymphocytic; Leukemia, chronic myeloid; Leukemia, hairy cell; Lip and oral cavity cancer;Liver cancer; Lung cancer, non-small cell; Lung cancer, small cell; Lymphoma, AIDS-related; Lymphoma, Burkitt; Lymphoma, cutaneous T-cell; Lymphoma, Hodgkin; Lymphoma, non-Hodgkin; Lymphoma, primary central nervous system; Macroglobulinemia, Waldensteroma; Malignant fibrous histiocytoma and osteosarcoma of bone; Medulloblastoma; Melanoma; Melanoma, intraocular (eye); Merkel cell carcinoma; Mesothelioma; Metastatic squamous neck cancer of unknown primary; Oral cancer; Multiple endocrine neoplasia syndrome (pediatric); Multiple myeloma / phenotype Mycosis fungoides; Myelodysplastic syndromes; Myelodysplastic / myeloproliferative disorders; Myeloid leukemia, chronic; Myeloid leukemia, adult acute; Myeloid leukemia, childhood acute; Myeloma, multiple; Myeloproliferative disorders, chronic; Nasal cavity and paranasal sinus cancer; Nasopharyngeal carcinoma; Neuroblastoma; Non-small cell lung cancer; Oral cancer; Oral cavity cancer; Oropharyngeal cancer; Osteosarcoma and malignant fibrous histiocytoma of bone; Ovarian cancer; Ovarian epithelial cancer; Ovarian germ cell tumor; Ovarian low malignant potential tumor; Pancreatic cancer; Pancreatic cancer, islet cell tumor; Papillomatosis; Parathyroid carcinoma; Penile cancer; Pharyngeal cancer; Chromosome Aphelocytoma; Paraganglioma; Pineal parenchymal tumor of intermediate differentiation; Pineoblastoma and supratentorial primitive neuroectodermal tumor; Pituitary tumor; Plasmacytoma / multiple myeloma; Pleuropulmonary blastoma; Primary central nervous system lymphoma; Prostate cancer; Rectal cancer; Renal cell (kidney) carcinoma; Renal pelvis and ureter, transitional cell carcinoma; Airway carcinoma involving the NUT gene on chromosome 15; Retinoblastoma; Rhabdomyosarcoma; Salivary gland carcinoma; Sarcoma, Ewing family tumor; Sarcoma, Kaposi's; Sarcoma, soft tissue; Sarcoma, uterine; Sézary syndrome; Skin cancer (non- Melanoma); Skin cancer (melanoma); Skin carcinoma, Merkel cell; Small cell lung cancer; Small intestine cancer; Soft tissue sarcoma; Squamous cell carcinoma, squamous neck carcinoma of unknown primary, metastatic; Gastrointestinal (gastric) cancer; Supratentorial primitive neuroectodermal tumor; T-cell lymphoma, skin; Testicular cancer; Thymoma and thymic carcinoma; Thyroid cancer; Transitional cell carcinoma of the renal pelvis and ureter; Trophoblastic tumor, gestational; Urethral cancer; Uterine cancer, endometrium; Uterine sarcoma; Vaginal cancer; Vulvar cancer; Waldenstrom's macroglobulinemia; and Wilms' tumor.

[0245] In one embodiment, the present invention provides a method of treating cancer comprising treating a subject with a complementary cancer therapy, such as surgery, chemotherapy, chemotherapeutic agents, radiation therapy, or hormone therapy, or a combination thereof, prior to, simultaneously with, or sequentially with, administration of a renalase-binding molecule of the present invention.

[0246] Chemotherapeutic agents include cytotoxic agents (e.g., 5-fluorouracil, cisplatin, carboplatin, methotrexate, daunorubicin, doxorubicin, vincristine, vinblastine, oxorubicin, carmustine (BCNU), lomustine (CCNU), cytarabine USP, cyclophosphamide, estramutine sodium phosphate, altretamine, hydroxyurea, ifosfamide, procarbazine, mitomycin, busulfan, cyclophosphamide, mitoxantrone, carboplatin, cisplatin, interferon α- 2a recombinant, paclitaxel, teniposide, and streptozocin), cytotoxic alkylating agents (e.g., busulfan, chlorambucil, cyclophosphamide, melphalan, or ethyl sulfonate), alkylating agents (e.g., asaley, AZQ, BCNU, busulfan, bisulfan, carboxyphthalatoplatinum, CBDCA, CCNU, CHIP, chlorambucil, chlorozotocin, cisplatin, clomesone, cyanomorpholinodoxorubicin, cyclodisone, cyclophosphamide, dianhydraglactitol, fluorouracil, dopant, hepsulfam, hycanthone, ifosfamide, melphalan, methyl CCNU, mitomycin C, mitozolamide, nitrogen mustard, PCNU, piperazine, piperazinedione, pipobroman, porfiromycin, spirohydantoin mustard, streptozotocin, teroxylon, tetraplatin, thiotepa, triethylenemelamine, uracil nitrogen mustard, and Yoshi-864), antimitotic drugs (e.g., allocolchicine, halichondrin M, colchicine, colchicine derivatives, dolastatin 10, mycobacterial danshen, rhizoxin, paclitaxel derivatives, paclitaxel, thiocolchicine, trityl cysteine, vinblastine sulfate, and vincristine sulfate), plant alkaloids (e.g., actinomycin D, bleomycin, L-asparaginase, idarubicin, vinblastine sulfate, vincristine sulfate, mithramycin, mitomycin, daunorubicin, VP-16-213, VM-26, navelbine, and taxotere), biologics (e.g., alpha interferon, BCG, G-CSF, GM-CSF, and interleukin-2),These include topoisomerase I inhibitors (e.g., camptothecin, camptothecin derivatives, and morpholinodoxorubicin), topoisomerase II inhibitors (e.g., mitoxantrone, amonafide, m-AMSA, anthrapyrazole derivatives, pyrazoloacridine, bisantrene HCl, daunorubicin, deoxydoxorubicin, menogaril, N,N-dibenzyldaunomycin, oxanthrazole, rubidazone, VM-26, and VP-16), and synthetic products (e.g., hydroxyurea, procarbazine, o,p'-DDD, dacarbazine, CCNU, BCNU, cis-diamminedichloroplatinum, mitoxantrone, CBDCA, levamisole, hexamethylmelamine, all-trans retinoic acid, gliadel, and porfimer sodium).

[0247] Antiproliferative agents are compounds that reduce cell proliferation. Antiproliferative agents include alkylating agents, antimetabolites, enzymes, biological response modifiers, miscellaneous agents, hormones and antagonists, androgen inhibitors (e.g., flutamide and leuprolide acetate), antiestrogens (e.g., tamoxifen citrate and its analogs, toremifene, droloxifene, and roloxifene). Additional examples of specific antiproliferative agents include, but are not limited to, levamisole, gallium nitrate, granisetron, sargramostim strontium-89 chloride, filgrastim, pilocarpine, dexrazoxane, and ondansetron.

[0248] The renalase-binding molecules of the present invention can be administered alone or in combination with other anti-tumor agents, including cytotoxic / antineoplastic agents and angiogenesis inhibitors. Cytotoxic / antineoplastic agents are defined as agents that attack and kill cancer cells. Some cytotoxic / antineoplastic agents are alkylating agents that alkylate genetic material in tumor cells, such as cisplatin, cyclophosphamide, nitrogen mustard, trimethylenethiophosphoramide, carmustine, busulfan, chlorambucil, verstine, uracil mustard, chromafazine, and dacabazine. Other cytotoxic / antineoplastic agents are tumor cell antimetabolites, such as cytosine arabinoside, fluorouracil, methotrexate, mercaptopurine, azathioprim, and procarbazine. Other cytotoxic / antineoplastic agents include antibiotics such as doxorubicin, bleomycin, dactinomycin, daunorubicin, mithramycin, mitomycin, mitomycin C, and daunomycin. Many liposomal formulations of these compounds are commercially available. Still other cytotoxic / antineoplastic agents include mitotic inhibitors (vinca alkaloids). These include vincristine, vinblastine, and etoposide. Mixed cytotoxic / antineoplastic agents include taxol and its derivatives, L-asparaginase, antitumor antibodies, dacarbazine, azacitidine, amsacrine, melphalan, VM-26, ifosfamide, mitoxantrone, and vindesine.

[0249] Angiogenesis inhibitors are well known to those skilled in the art. Suitable angiogenesis inhibitors for use in the methods and compositions of the present disclosure include anti-VEGF antibodies, including humanized and chimeric antibodies, anti-VEGF aptamers, and antisense oligonucleotides. Other known angiogenesis inhibitors include angiostatin, endostatin, interferon, interleukin 1 (including α and β), interleukin 12, retinoic acid, and tissue inhibitors of metalloproteinases-1 and -2 (TIMP-1 and -2). Small molecules with anti-angiogenic activity, including topoisomerase II inhibitors, such as razoxane, can also be used.

[0250] Other anti-cancer agents that can be used in combination with the disclosed compounds include, but are not limited to, acivicin; aclarubicin; acodazole hydrochloride; acronine; adozelesin; aldesleukin; altretamine; ambomycin; amethanthrone acetate; aminoglutethimide; amsacrine; anastrozole; anthramycin; asparaginase; asperlin; azacitidine; azetepa; azotomycin; batimastat; benzodepa; bicalutamide; bisantrene hydrochloride; bisnafide dimesylate; bizelesin; b Rheomycin sulfate;Brequinar sodium;Bropirimine;Busulfan;Cactinomycin;Calsterone;Caracemide;Carbetimer;Carboplatin;Carmustine;Carubicin hydrochloride;Carzelesin;Cedefingol;Chlorambucil;Ciloremycin;Cisplatin;Cladribine;Crisnatol mesylate;Cyclophosphamide;Cytarabine;Dacarbazine;Dactinomycin;Daunorubicin hydrochloride;Decitabine;Dexormaplatin;Dezaguanine;Dezaguanine mesylate;Diaziquone;Docetaxel;Doxorubicin;Doxorubicin Rubicin hydrochloride;Droloxifene;Droloxifene citrate;Dromostanolone propionate;Duazomycin;Edatrexate;Eflornithine hydrochloride;Elsamiturcin;Enloplatin;Empromate;Epipropizine;Epirubicin hydrochloride;Elburozole;Esorubicin hydrochloride;Estramustine;Estramustine sodium phosphate;Etanidazole;Etoposide;Etoposide phosphate;Etoprine;Fadrozole hydrochloride;Fazarabine;Fenretinide;Floxuridine;Fludarabine phosphate;Fluorouracil; Fluorocitabine; foskidone; fostriecin sodium; gemcitabine; gemcitabine hydrochloride; hydroxyurea; idarubicin hydrochloride; ifosfamide; ilmofosine; interleukin II (including recombinant interleukin II, or rIL2), interferon alpha-2a; interferon alpha-2b; interferon alpha-n1; interferon alpha-n3; interferon beta-Ia; interferon gamma-Ib; iproplatin; irinotecan hydrochloride; lanreotide acetate; letrozole; leuprolide acetate; liarozole hydrochloride;Lometrexol sodium; Lomustine; Losoxantrone hydrochloride; Masoprocol; Maytansine; Mechlorethamine hydrochloride; Megestrol acetate; Melengesterol acetate; Melphalan; Menogaril; Mercaptopurine; Methotrexate; Methotrexate sodium; Metoprine; Meturedepa; Mitindomide; Mitocalcin; Mitochromine; Mitogillin; Mitomarcin; Mitomycin; Mitospel; Mitotane; Mitoxantrone hydrochloride; Mycophenolic acid; Nocodazole; Noga Ramicycin; Ormaplatin; Oxisuran; Paclitaxel; Albumin-bound paclitaxel; Pegaspargase; Periomycin; Pentamustine; Peplomycin sulfate; Perfosfamide; Pipobroman; Piposulfan; Piroxantrone hydrochloride; Plicamycin; Promestane; Porfimer sodium; Porfiromycin; Prednimustine; Procarbazine hydrochloride; Puromycin; Puromycin hydrochloride; Pirazofurin; Ribopurin; Rogletimide; Safingol ;Safingol hydrochloride;Semustine;Simtrazene;Sparfosate sodium;Sparsomycin;Spirogermanium hydrochloride;Spiromustine;Spiroplatin;Streptonigrin;Streptozocin;Sulofenur;Tallysomycin;Tecogalan sodium;Tegafur;Teroxantrone hydrochloride;Temoporfin;Teniposide;Teroxylon;Testolactone;Thiamiprine;Thioguanine;Thiotepa;Tiazofurin;Tirapazamine;Toremifene citrate;Trestron acetate ;Triciribine phosphate;Trimetrexate;Trimetrexate glucuronate;Triptorelin;Tubrozole hydrochloride;Uracil mustard;Uredepam;Vapreotide;Verteporfin;Vinblastine sulfate;Vincristine sulfate;Vindesine;Vindesine sulfate;Binepidine sulfate;Vingrisinate sulfate;Vinleurosine sulfate;Vinorelbine;Vinorelbine tartrate;Vinrocidine sulfate;Vinzolidine sulfate;Vorozole;Zeniplatin;Zinostatin;Zorubicin hydrochloride.Other anticancer drugs include, but are not limited to:20-epi-1,25-dihydroxyvitamin D3;5-ethynyluracil;Abiraterone;Aclarubicin;Acylfulvene;Adecipenol;Adozelesin;Aldesleukin; ALL-TK antagonist; altretamine; ambamustine; amidox; amifostine; aminolevulinic acid; amrubicin; amsacrine; anagrelide; anastrozole; andrographolide; angiogenesis inhibitors; antagonist D; antagonist G; antarelix; anti-dorsal morphogenetic protein-1; antiandrogenic substances, prostate cancer; antiestrogens; antineoplastons; antisense oligonucleotides; aphidicolin glycinate; apoptosis gene modulators; apoptosis regulators; apurinic acid; ara-CDP-DL-PTBA; arginine deaminase; asulaculin; atamestane; atlimustine; axinastatin 1; a Xinastatin 2; Axinastatin 3; Azasetron; Azatoxin; Azatyrosine; Baccatin III derivatives; Balanol; Batimastat; BCR / ABL antagonists; Benzochlorins; Benzoylstaurosporines; β-lactam derivatives; β-arretin; Betaclamycin B; Betulinic acid; bFGF inhibitors; Bicalutamide; Bisantrene; Bisaziridinylspermine; Bisnafide; Bistraten A; Bizelesin; Breflate; Bropirimine; Budotitane; Buthionine sulfoximine; Calcipotriol; Calphostin C; Camptothecin derivatives; Canarypox IL-2; Capecitabine; Carboxamido-amino-triazoles; Carboxamidotriazoles; CaRest M3; CARN 700; cartilage-derived inhibitor; carzelesin; casein kinase inhibitor (ICOS); castanospermine; cecropin B; cetrorelix; chlorine; chloroquinoxaline sulfonamide; cicaprost; cis-porphyrin; cladribine; clomiphene analog; clotrimazole; colismycin A; colismycin B; combretastatin A4; combretastatin analog; conagenin; clambecidin 816; crisnatol; cryptophycin 8; cryptophycin A derivative; curacin A; cyclopentaneseraquinone; cycloplatam; sipemycin; cytarabine ocfosfate; cytolytic factor; cytostatin; dacliximab; decitabine; dehydrodydemnin B; deslorelin; dexamethasone; dexphosphamide; dexrazoxane; dexverapamil;Diaziquone; Didemnin B; Didox; Diethylnorspermine; Dihydro-5-azacytidine; 9-dihydrotaxol; Dioxamycin; Diphenylspiromustine; Docetaxel; Docosanol; Dolasetron; Doxifluridine; Droloxifene; Dronabinol; Duocarmycin SA; Ebselen; Ecomustine; Edelfosine; Edrecolomab; Eflornithine; Elemene; Emiteflu; Epirubicin; Epristeride; Estramustine analogs; Estrogen agonists; Estrogen antagonists; Etanidazole; E Toposide phosphate; Exemestane; Fadrozole; Fazarabine; Fenretinide; Filgrastim; Finasteride; Flavopiridol; Flezelastine; Fluasterone; Fludarabine; Fluorodaunornithine hydrochloride; Forfenimex; Formestane; Fostriecin; Fotemustine; Gadolinium texaphyrin; Gallium nitrate; Galocitabine; Ganirelix; Gelatinase inhibitors; Gemcitabine; Glutathione inhibitors; Hepsulfame; Heregulin; Hexamethylenebisacetamide; Hypericin; Ibandronate; Ida Rubicin; Idoxifene; Idramanton; Ilmofosine; Ilomastat; Imidazoacridone; Imiquimod; Immunostimulatory peptides; Insulin-like growth factor 1 receptor inhibitors; Interferon agonists; Interferons; Interleukins; Iobenguane; Iododoxorubicin; 4-Ipomeanol; Iloprakt; Irsogladine; Isobengazole; Isohomohalichondrin B; Itasetron; Jasplakinolide; Kahalalide F; Lamellarin-N triacetate; Lanreotide; Leinamycin; Lenograstim; Lentinan sulfate Salt; leptolstatin; letrozole; leukemia inhibitory factor; leukocyte alpha interferon; leuprolide + estrogen + progesterone; leuprorelin; levamisole; liarozole; linear polyamine analogs; lipophilic disaccharide peptides; lipophilic platinum compounds; lisoclinamide 7; lobaplatin; lombricin; lometrexol; lonidamine; losoxantrone; lovastatin; loxoribine; lurtotecan; lutetium texaphyrin; lisofylline; cytolytic peptides; maytansine; mannostatin A; marimastat; massoprocol; maspin;Matrilysin inhibitors; matrix metalloproteinase inhibitors; menogaril; mervalone; meterelin; methioninase; metoclopramide; MIF inhibitors; mifepristone; miltefosine; millimostim; mismatched double-stranded RNA; mitoguazone; mitolactol; mitomycin analogs; mitonafide; mitotoxin fibroblast growth factor-saporin; mitoxantrone; mofalotene; molgramostim; monoclonal antibodies, human chorionic gonadotropin; monophosphoryl lipid A + Myobacterium cell wall sk; mopi Damor; multidrug resistance gene inhibitors; multiple tumor suppressor 1-based therapy; mustard anticancer drugs; mycaperoxide B; mycobacterial cell wall extract; myriaporone; N-acetyldinaline; N-substituted benzamides; nafarelin; nagressip; naloxone + pentazocine; napavine; naphterpine; nartograstim; nedaplatin; nemorubicin; neridronic acid; neutral endopeptidase; nilutamide; nisamycin; nitric oxide modulators; nitric oxide antioxidants; nitrulline; O6-benzylguanine; octreotide; oxalate Senon; Oligonucleotides; Onapristone; Ondansetron; Ondansetron; Oracin; Oral cytokine inducers; Ormaplatin; Osateron; Oxaliplatin; Oxaunomycin; Paclitaxel; Paclitaxel analogs; Paclitaxel derivatives; Palauamine; Palmitoylrhizoxin; Pamidronic acid; Panaxytriol; Panomyphen; Parabactin; Pazeliptin; Pegaspargase; Perdecin; Pentosan polysulfate sodium; Pentostatin; Pentrozole; Perflubron; Perfosfamide; Perillylal Cole; Phenazinomycin; Phenylacetate; Phosphatase inhibitors; Picibanil; Pilocarpine hydrochloride; Pirarubicin; Piritrexim; Prasetin A; Prasetin B; Plasminogen activator inhibitors; Platinum complexes; Platinum compounds; Platinum-triamine complexes; Porfimer sodium; Porfiromycin; Prednisone; Propylbisacridone; Prostaglandin J2; Proteasome inhibitors; Protein A-based immunomodulators; Protein kinase C inhibitors; Protein kinase C inhibitors, microalgae;Protein tyrosine phosphatase inhibitors; purine nucleoside phosphorylase inhibitors; purpurins; pyrazoloacridines; pyridoxylated hemoglobin polyoxyethylene conjugates; raf antagonists; raltitrexed; ramosetron; ras farnesyl protein transferase inhibitors; ras inhibitors; ;ras-GAP inhibitor;demethylated leteriptin;rhenium Re186 etidronate;rhizoxin;ribozyme;RII retinamide;logletimide;rohitukin;romurtide;roquinimex;rubiginone B1;ruboxil;safingol;saintopine;SarCNU;sarcophytol A;sargramostim;Sdi1 mimetic;semustine;senescence-derived inhibitor 1;sense oligonucleotide;signal transduction inhibitor;signal transduction modulator;single-chain antigen-binding protein;sizofiran;sobzoxane;bozoxane Locaptate sodium; Sodium phenylacetate; Sorbetol; Somatomedin-binding proteins; Sonermin; Sparfosic acid; Spicamycin D; Spiromustine; Splenopentin; Spongestatin 1; Squalamine; Stem cell inhibitors; Stem cell division inhibitors; Stipiamide; Stromelysin inhibitors; Sulfinosine; Superactive vasoactive intestinal peptide antagonists; Sladista; Suramin; Swainsonine; Synthetic glycosaminoglycans; Talimustine; Tamoxifen methiodide; Tauromustine; Tazarote Tecogalan sodium; Tegafur; Terlapyrylium; Telomerase inhibitors; Temoporfin; Temozolomide; Teniposide; Tetrachlorodecaoxide; Tetrazomine; Taliblastine; Chicoraline; Thrombopoietin; Thrombopoietin mimetics; Thymalfasin; Thymopoietin receptor agonists; Thymotrin; Thyroid-stimulating hormone; Tin ethyl etiopurpurin; Tirapazamine; Titanocene dichloride; Topsentin; Toremifene; Totipotent stem cell factor; Translation inhibitors; Tretinoin; Triacetyluridine; Trisilicate Vin; trimetrexate; triptorelin; tropisetron; turosteride; tyrosine kinase inhibitors; tyrphostins; UBC inhibitors; ubenimex; urogenital sinus-derived growth inhibitory factor; urokinase receptor antagonists; vapreotide; variolin B; vector systems, erythrocyte gene therapy; veraresol; veramine; verudin; verteporfin; vinorelbine; vinxartin; vitaxin; vorozole; zanoteron; zeniplatin; zilascorub; imilimumab; mirtazapine; BrUOG 278; BrUOG 292; RAD0001; CT-011; Forfirinox;Tipifarnib; R115777; LDE225; calcitriol; AZD6244; AMG655; AMG479; BKM120; mFOLFOX6; NC-6004; cetuximab; IM-C225; LGX818; MEK162; BBI608; MEDI4736; vemurafenib; ipilimumab; ivolumab; nivolumab; panobinostat; leflunomide; CEP-32496; alemtuzumab; bevacizumab; ofatumumab; panitumumab; pembrolizumab; rituximab; trastuzumab; STAT3 inhibitors (e.g., STA-21, LLL-3, LLL) 12, XZH-5, S31-201, SF-1066, SF-1087, STX-0119, cryptotanshinone, curcumin, diferuloylmethane, FLLL11, FLLL12, FLLL32, FLLL62, C3, C30, C188, C188-9, LY5, OPB-31121, pyrimethamine, OPB-51602, AZD9150, etc.; hypoxia-inducible factor 1 (HIF-1) inhibitors (e.g., LW6, digoxin, laurenditerpenol, PX-478, RX-0047, vitexin, KC7F2, YC-1, etc.) and zinostatin stimalamer. In one embodiment, the anticancer agent is 5-fluorouracil, taxol, or leucovorin.

[0251] Diagnostic methods In some embodiments, a change (e.g., an increase) in the level of renalase or a renalase fragment in a subject's cells, tissues, or body fluids compared to a comparator is used in the methods of the present invention as a marker for diagnosing a disease or disorder, assessing the severity of a disease or disorder, and monitoring the effect or effectiveness of a treatment for a disease or disorder. In various embodiments, the disease or disorder is acute renal failure (i.e., acute tubular necrosis, or ATN, a renal ischemic condition), cardiovascular disease or disorder (e.g., hypertension, pulmonary hypertension, systolic hypertension, diabetic hypertension, asymptomatic left ventricular dysfunction, chronic congestive heart failure, myocardial infarction, cardiac rhythm abnormalities, atherosclerosis, etc.), cancer, cardiac disease or disorder, kidney disease or disorder, gastrointestinal disease or disorder, liver disease or disorder, pulmonary disease or disorder, pancreatic disease or disorder (e.g., pancreatitis), psychiatric disease or disorder (e.g., depression, anxiety, etc.), or nervous system disease or disorder.

[0252] In one embodiment, the present invention is a method for diagnosing a disease or disorder in a subject by assessing the level of renalase or a renalase fragment in a biological sample from the subject. In one embodiment, the biological sample from the subject is a cell, tissue, or bodily fluid. Non-limiting examples of bodily fluids in which the level of renalase or a renalase fragment can be assessed include, but are not limited to, blood, serum, plasma, and urine. In various embodiments, the level of renalase or a renalase fragment in the subject's biological sample is compared to the level of renalase or a renalase fragment in a comparator. Non-limiting examples of comparators include, but are not limited to, a negative control, a positive control, the subject's expected normal background value, the subject's historical normal background value, the subject's expected normal background value for a population of which the subject is a member, or the subject's historical normal background value for a population of which the subject is a member. In various embodiments, the disease or disorder is acute renal failure (i.e., acute tubular necrosis, or ATN, a renal ischemic condition), a cardiovascular disease or disorder (e.g., hypertension, pulmonary hypertension, systolic hypertension, diabetic hypertension, asymptomatic left ventricular dysfunction, chronic congestive heart failure, myocardial infarction, cardiac rhythm abnormalities, atherosclerosis, etc.), cancer, a cardiac disease or disorder, a renal disease or disorder, a gastrointestinal disease or disorder, a liver disease or disorder, a pulmonary disease or disorder, a pancreatic disease or disorder (e.g., pancreatitis), a psychiatric disease or disorder (e.g., depression, anxiety, etc.), or a nervous system disease or disorder. In some embodiments, the diagnostic method includes the further step of treating the patient for the diagnosed disease or disorder.

[0253] In another embodiment, the present invention is a method for assessing the severity of a disease or disorder in a subject by assessing the level of renalase or a renalase fragment in a biological sample from the subject. In one embodiment, the subject's biological sample is a cell, tissue, or bodily fluid. Non-limiting examples of bodily fluids in which the level of renalase or a renalase fragment can be assessed include, but are not limited to, blood, serum, plasma, and urine. In various embodiments, the level of renalase or a renalase fragment in the subject's biological sample is compared to the level of renalase or a renalase fragment in a comparator. Non-limiting examples of comparators include, but are not limited to, a negative control, a positive control, the subject's expected normal background value, the subject's historical normal background value, the subject's expected normal background value for a population of which the subject is a member, or the subject's historical normal background value for a population of which the subject is a member. In various embodiments, the disease or disorder is acute renal failure (i.e., acute tubular necrosis, or ATN, a renal ischemic condition), a cardiovascular disease or disorder (e.g., hypertension, pulmonary hypertension, systolic hypertension, diabetic hypertension, asymptomatic left ventricular dysfunction, chronic congestive heart failure, myocardial infarction, cardiac rhythm abnormalities, atherosclerosis, etc.), cancer, a cardiac disease or disorder, a renal disease or disorder, a gastrointestinal disease or disorder, a liver disease or disorder, a pulmonary disease or disorder, a pancreatic disease or disorder (e.g., pancreatitis), a psychiatric disease or disorder (e.g., depression, anxiety, etc.), or a nervous system disease or disorder. In some embodiments, the method of assessing severity includes the further step of treating the patient for the disease or disorder.

[0254] In another embodiment, the present invention is a method for monitoring the effect of a treatment for a disease or disorder in a subject by assessing the level of renalase or a renalase fragment in the subject's biological sample. In one embodiment, the subject's biological sample is a cell, tissue, or bodily fluid. Non-limiting examples of bodily fluids in which the level of renalase or a renalase fragment can be assessed include, but are not limited to, blood, serum, plasma, and urine. In various embodiments, the level of renalase or a renalase fragment in the subject's biological sample is compared to the level of renalase or a renalase fragment in a comparator. Non-limiting examples of comparators include, but are not limited to, a negative control, a positive control, the subject's expected normal background value, the subject's historical normal background value, the subject's expected normal background value for a population of which the subject is a member, or the subject's historical normal background value for a population of which the subject is a member. In various embodiments, the disease or disorder is acute renal failure (i.e., acute tubular necrosis, or ATN, a renal ischemic condition), a cardiovascular disease or disorder (e.g., hypertension, pulmonary hypertension, systolic hypertension, diabetic hypertension, asymptomatic left ventricular dysfunction, chronic congestive heart failure, myocardial infarction, cardiac rhythm abnormalities, atherosclerosis, etc.), cancer, a cardiac disease or disorder, a renal disease or disorder, a gastrointestinal disease or disorder, a liver disease or disorder, a pulmonary disease or disorder, a pancreatic disease or disorder (e.g., pancreatitis), a psychiatric disease or disorder (e.g., depression, anxiety, etc.), or a nervous system disease or disorder. In some embodiments, the method of monitoring the effect of a treatment includes the further step of treating the patient for the disease or disorder.

[0255] In various embodiments, the subject is a human subject and can be of any race, sex, and age. Exemplary subjects include those suspected of experiencing a disease or disorder, those diagnosed as experiencing a disease or disorder, those diagnosed as having a disease or disorder, and those at risk of developing a disease or disorder.

[0256] The information obtained from the inventive methods disclosed herein can be used alone or in combination with other information from the subject or from a biological sample obtained from the subject (e.g., disease state, medical history, vital signs, blood chemistry, etc.).

[0257] In the diagnostic methods of the present invention, a biological sample obtained from a subject is evaluated for the level of renalase or a renalase fragment contained therein. In one embodiment, the biological sample is a sample containing at least a fragment of a renalase polypeptide useful in the methods described herein.

[0258] In various other embodiments of the methods of the present invention, the level of renalase or a renalase fragment is determined to be increased if the level is increased by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 100%, at least 200%, at least 300%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, at least 900%, or at least 1000% compared to a comparator control. In various embodiments, an increased level of renalase or a renalase fragment is indicative of a disease or disorder. In various embodiments, the disease or disorder is acute renal failure (i.e., acute tubular necrosis, or ATN, a renal ischemic condition), cardiovascular disease, pancreatitis, hepatitis, inflammatory disorders of the kidney, and cancer.

[0259] In the methods of the present invention, a biological sample from a subject is assessed for the level of renalase or a renalase fragment in the biological sample obtained from the patient. The level of renalase or a renalase fragment in the biological sample can be determined by assessing the amount of renalase polypeptide or fragment in the biological sample, the amount of renalase mRNA or fragment in the biological sample, the amount of renalase activity (e.g., enzymatic activity, substrate binding activity, receptor binding activity, etc.) in the biological sample, or a combination thereof. In some embodiments, the level of renalase in the biological sample is determined in an assay using at least one of the renalase-binding molecules of the present invention described elsewhere herein.

[0260] In various embodiments of the methods of the present invention, methods for measuring renalase levels in a biological sample obtained from a patient include, but are not limited to, immunochromatographic assays, immunoblot assays, Luminex assays, ELISA assays, ELISPOT assays, protein microarray assays, Western blot assays, mass spectrometry assays, radioimmunoassays (RIA), radial immunodiffusion assays, liquid chromatography-tandem mass spectrometry assays, Ouchterlony immunodiffusion assays, reversed-phase protein microarrays, rocket immunoelectrophoresis assays, immunohistochemistry assays, immunoprecipitation assays, complement fixation assays, FACS, enzyme-substrate binding assays, enzyme assays, enzyme assays utilizing detectable molecules such as chromophores, fluorophores, or radioactive substrates, substrate binding assays utilizing such substrates, substrate displacement assays utilizing such substrates, and protein chip assays (also see, 2007, Van Emon, Immunoassay and Other Bioanalytical Techniques, CRC Press; 2005, Wild, Immunoassay Handbook, Gulf Professional (See also, Diamandis and Christopoulos, Immunoassay, Academic Press; 2005, Joos, Microarrays in Clinical Diagnosis, Humana Press; 2005, Hamdan and Righetti, Proteomics Today, John Wiley and Sons; 2007.) In some embodiments, the level of renalase in a biological sample is measured by an assay using at least one of the renalase-binding molecules of the invention described elsewhere herein.

[0261] kit The present invention also includes kits comprising a renalase-binding molecule (e.g., an antibody) of the present invention, or a combination thereof, and instructions for administering the renalase-binding molecule or combination thereof to an individual, for example, as a therapeutic treatment or for non-therapeutic uses as described elsewhere herein. In embodiments, the kit further comprises a (preferably sterile) pharmaceutically acceptable carrier suitable for dissolving or suspending a therapeutic composition containing the renalase-binding molecule or combination thereof of the present invention, e.g., prior to administration of the renalase-binding molecule of the present invention to an individual. Optionally, the kit comprises an applicator for administering the renalase-binding molecule. [Example]

[0262] Experimental Examples The invention will now be described with reference to the following examples, which are provided for illustrative purposes only, and the invention should in no way be construed as being limited to these examples, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0263] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the compounds of the present invention and practice the claimed methods. The following working examples therefore, specifically point out preferred embodiments of the present invention, and are not to be construed as limiting in any way the remainder of the disclosure.

[0264] Example 1: Novel Compositions and Methods for Cancer Treatment The results described herein provide data supporting the utility of novel compositions and methods for treating cancer.

[0265] The materials and methods utilized in these experiments are described herein.

[0266] Synthesis of anti-renalase monoclonal antibodies in rabbits. Protein antigen preparation: Renalase-1 cDNA was subcloned into the pET27 expression vector (Novagen) and then used to transform the bacterial strain BL21 DE3 (Novagen). Renalase protein was isolated from bacterial inclusion bodies by standard procedures. Briefly, bacterial inclusion bodies were isolated and solubilized in a combination of chaotropic salt and reducing agent. The protein was refolded by dilution with the solubilization buffer. After final dialysis of the solubilized and refolded protein into phosphate-buffered saline (PBS), the protein was used as an antigen to immunize rabbits.

[0267] Peptide antigen preparation: Peptides for immunization were designed to cover several regions of the renalase-1 or 2 polypeptide and contain N- or C-terminal cysteine ​​residues for use in subsequent conjugation. The peptides were generated using standard peptide synthesis procedures and conjugated via the cysteine ​​residue to either bovine serum albumin (BSA) for screening purposes or keyhole limpet hemocyanin (KLH), an adjuvant for animal immunization.

[0268] Immunization and Antibody Selection: Using standard procedures, six rabbits were immunized using a multi-dose injection protocol with KLH-conjugated peptide antigen and whole protein antigen, respectively. Pre- and post-immunization test bleeds from each rabbit were tested for anti-renalase titers using a standard ELISA protocol (see protocol below) with either BSA-peptide conjugate or refolded whole protein as the coated antigen. Rabbits with the highest anti-renalase titers and antisera with the desired detection characteristics for endogenous renalase in Western blot analysis were selected for further analysis. Selected animals were either used for terminal bleeding for the production of anti-renalase polyclonal antibodies, or the spleens of selected rabbits were used for lymphocyte collection. After standard cell fusion, hybridoma pools from each animal were screened using the same peptide and whole protein ELISA format described above, using conditioned cell culture medium. Hybridomas with the highest anti-renalase titers and favorable endogenous renalase binding characteristics were selected and subcloned. Cloned hybridomas were subsequently expanded and monoclonal antibodies were purified from conditioned cell supernatants.

[0269] Hybridoma culture and expansion: Hybridoma cells were cultured in Hybridoma-SFM serum-free medium (Gibco / Invitrogen) supplemented with 55 μM 2-mercaptoethanol. For antibody purification, 10 hybridoma cells suspended in 15 mL of culture medium were applied to the cell compartment of a CELLine 1000 bioreactor flask (Wilson Wolf), and the upper compartment was filled with 1 L of culture medium. After 7 days of continuous culture, the contents of the cell compartment (including cells and secreted antibody) were removed, and the cells were pelleted by centrifugation at 1000 g for 5 minutes. The supernatant was collected, centrifuged at 10,000 g for 15 minutes, and filtered (0.2 μM) to remove cell debris prior to antibody purification.

[0270] Antibody purification: For anti-renalase monoclonal antibodies, pre-cleared and conditioned hybridoma supernatant was pumped at constant pressure onto a pre-equilibrated Protein A affinity column (GE Life Sciences). The column was washed with 20 column volumes of PBS (pH 7.4). The antibody was eluted from the column in 0.1 M glycine (pH 2.5) and immediately neutralized with Tris buffer. The pure antibody was dialyzed against an excess of PBS (pH 7.4), filter-sterilized, and stored in aliquots at either -80°C or 4°C. For anti-renalase polyclonal antibodies raised against either the peptide-conjugate or whole renalase protein immunogen, terminal rabbit bleed serum was first passed over a Protein A affinity column. The entire purified IgG pool from this Protein A column was dialyzed against PBS (pH 7.4). Anti-renalase antibodies were further purified by passing the pool through an Actigel ALD column (Sterogene) conjugated with either the relevant peptide antigen or the whole renalase protein (Actigel-antigen conjugation protocol was completed according to the manufacturer's instructions). The column was washed with PBS, and bound IgG was eluted with 0.1 M glycine (pH 2.5). Purified IgG was neutralized with Tris buffer and dialyzed against PBS.

[0271] General ELISA screening assay: 100 ng / well of recombinant renalase protein or renalase peptide-BSA conjugate was bound to a 96-well microtiter plate by overnight incubation at 4°C. The plate was blocked with a 5% milk solution in phosphate-buffered saline (PBST) containing 0.05% Tween-20 at 33°C for 1 hour. Renalase antibody bleed was diluted in PBS, and 50 μL was added to each well. After incubation at 33°C for 1 hour, the plate was rinsed three times with PBST. 50 μL of anti-rabbit-horseradish peroxidase (HRP)-conjugated antibody (Dako, diluted to 0.25 μg / mL in PBST) was added to each well, and the plate was incubated at 33°C for 45 minutes. The plate was washed three times with PBST and once with PBS. 100 μL of 3,3',5,5'-tetramethylbenzidine (TMB) substrate was added to each well, and the plate was incubated at room temperature for approximately 5 minutes. To stop the reaction, 100 μL of 2N H2SO4 was added to each well. The plate was measured in a spectrophotometer at 450 nm.

[0272] General sandwich ELISA assay: Typically, 400 ng / well of capture antibody in phosphate-buffered saline (PBS) was bound to a 96-well microtiter plate by overnight incubation at 4°C. The plate was blocked with a solution of 8% nonfat dry milk (NFDM) in PBS for 1 hour at 33°C. A sample containing renalase was diluted in PBS containing 0.05% Tween-20 (PBST), and 50 μL was added to each well. After incubation at 33°C for 1 hour, the plate was rinsed three times with PBST. A biotinylated antibody probe was diluted to 4 μg / mL in PBST, and 50 μL was added to each well. After incubation at 33°C for 1 hour, the plate was washed three times with PBST. For detection, 50 μL of horseradish peroxidase-conjugated (HRP) neutravidin (NA-HRP) diluted to 0.4 μg / mL was added to each well, and the plate was incubated at room temperature for 45 minutes. After incubation, the plate was washed three times with PBST and once with PBS. 100 μL / well of HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added, and the plate was incubated at room temperature for approximately 5 minutes. To stop the reaction, 100 μL of 2N H2SO4 was added to each well. The plate was measured at 450 nm in a spectrophotometer.

[0273] Western blotting procedure: Protein samples were resolved on a 4-20% Tris-glycine gradient gel (Invitrogen). Proteins were transferred from the gel to a polyvinyl difluoride (PVDF) membrane using an XCell II blot module (Invitrogen). The PVDF membrane was blocked with a solution of 5% nonfat dry milk in phosphate-buffered saline (PBST) containing 0.1% Tween-20 for 1 hour at room temperature. The blocking buffer was then removed, and the membrane was incubated with the detection antibody, diluted in 20 mL of 5% milk / PBST. After 1 hour of incubation at room temperature, the membrane was washed three times with PBST for 10 minutes each. 20 mL of anti-rabbit IgG horseradish peroxidase (HRP) conjugate (Dako, diluted to 0.25 μg / mL in 5% milk / PBST) was applied to the membrane, followed by an additional 1 hour of incubation at room temperature. The membrane was washed three times with PBST for 10 minutes each. Excess PBST was aspirated from the membrane, and enough enhanced chemiluminescence (ECL) plus reagent (GE Lifesciences) to cover the membrane's surface was applied and incubated for 1 minute. Excess ECL plus reagent was then aspirated, and the membrane was wrapped in plastic film. Protein bands were visualized by exposing the membrane to Hyperfilm-ECL (GE Lifesciences), which was processed using an automatic film developer (Konica).

[0274] Peptide-antigen specificity ELISA: Bovine serum albumin (BSA)-conjugated renalase peptide antigen, diluted to 100 ng / well in PBS, was bound to a 96-well microtiter plate by overnight incubation at 4°C. The plate was blocked with a solution of 8% NFDM in PBS for 1 hour at 33°C. The peptide antigen was probed with antibodies raised against it or serial dilutions of antibodies raised against different antigens. The starting antibody concentration was typically 5 μg / mL, prepared in two-fold dilution steps in PBS containing 0.05% Tween-20 (PBST). After 1 hour of incubation at 33°C, the plate was washed three times with PBST. Antibody binding was assayed by adding 50 μL / well of 50 ng / mL HRP-conjugated anti-rabbit IgG. The plate was incubated at room temperature for 40 minutes. After incubation, the plate was washed three times with PBST and once with PBS. 100 μL / well of HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added, and the plate was incubated at room temperature for approximately 5 minutes. 100 μL of 2N H2SO4 was added to each well to stop the reaction. The plate was measured at 450 nm in a spectrophotometer.

[0275] Epitope binding specificity: Bacterially expressed renalase isoforms were diluted to 100 ng / well in PBS and bound to 96-well microtiter plates by overnight incubation at 4°C. The plates were blocked with a solution of 8% NFDM in PBS for 1 hour at 33°C. The renalase protein was probed with serial dilutions of unconjugated antibodies, typically starting at 1 μg / mL, mixed with different biotinylated antibodies. The concentration of biotinylated antibodies was kept constant at 125 ng / well. Both antibodies were mixed together in PBST and applied as a single addition of 50 μL / well. After 1 hour of incubation at 33°C, the plates were rinsed three times with PBST. Binding of biotinylated antibodies to renalase was assayed by adding 0.4 ng / mL HRP-conjugated neutravidin (HRP-NA) at 50 μL / well. The plate was incubated at room temperature for 40 minutes. After incubation, the plate was washed three times with PBST and once with PBS. 100 μL / well of HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added, and the plate was incubated at room temperature for approximately 5 minutes. To stop the reaction, 100 μL of 2N H2SO4 was added to each well. The plate was measured at 450 nm in a spectrophotometer.

[0276] Biacore measurements of antibody affinity: Binding studies were performed using a Biacore T100 essentially as described (Guo X et al., 2016, Scientific Reports, 6:22996). Assays were completed at 25°C using 25 mM Tris (pH 8), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.005% Tween-20, and 0.1 mg / mL BSA as the running buffer. Biotinylated antibodies were captured and purified on individual streptavidin sensor chip flow cells, and recombinant renalase-1 was injected over the surface of the chip.

[0277] Synthesis and characterization of humanized anti-renalase monoclonal antibodies Humanization strategy and protocol: The complementarity-determining regions (CDRs) from M28 and M42 were grafted onto the 4D5 (Herceptin) framework in a Fab antibody fragment format in phagemids using standard methods (Nelson B, 2012, Methods in molecular biology, 899:27-41). The oligonucleotides used for this CDR grafting, including the resulting amino acid composition, are listed in Table 1. In addition, HC-A71 (numbered according to Chothia (Chothia C et al., 1987, Journal of molecular biology, 196(4):901-17)) had K71 or R71 changed to M28 and M42, respectively, to accommodate the parent M28 and M42 CDR-H2. Oligonucleotide-directed mutagenesis was used to construct affinity maturation libraries of M28 and M42 variants. Individual M28 CDR-L3, -H1, -H2, and -H3 and M42 CDR-L1, -H1, -H2, and -H3 were targeted for diversification as separate libraries. Applicable CDRs were first replaced with STOP codons using the oligonucleotides listed in Table 1 to generate STOP templates for affinity maturation libraries. These STOP templates were then used to generate affinity maturation libraries (4 for M28 and 4 for M42) using the oligonucleotides listed in Table 1, which were diversified using a "soft randomization" strategy in which the nucleotide ratios at degenerate positions were adjusted to 70% of the parental nucleotide and 10% of each other, using standard protocols (Nelson B, 2012, Methods in molecular biology, 899:27-41).

[0278] Selection of higher affinity variants cross-reactive with human and mouse renalase and directed by phage ELISA was performed as described (Nelson B, 2012, Methods in molecular biology, 899:27-41; Reshetnyak AV et al., 2013, Proc of Nat Acad Sci of the USA, 110(44):17832-7). Briefly, library phage pools were cycled through 3-5 rounds of binding selection using alternating rounds of human renalase (hRNLS) or mouse renalase (mRNLS) coated onto 96-well Maxisorp plates (Nunc). After 3-5 rounds of selection, specific binding of individual clones was assessed by phage ELISA as described (Reshetnyak AV et al., 2013, Proc of Nat Acad Sci of the USA, 110(44):17832-7). Clones that showed at least 10-fold greater signals for hRNLS and mRNLS binding compared to streptavidin (New England Biolabs) were subjected to DNA sequencing to decode the sequences of the phage-displayed Fabs.

[0279] Three cross-reactive M28 variants were isolated, two with changes to multiple CDRs, indicating recombination during library construction or antibody selection. Variant CDRs were systematically combined using standard methods to derive 23 unique variants with changes ranging from one to four CDRs. All 23 variants were rank-ordered using a solution-based competitive ELISA (Reshetnyak AV et al., 2013, Proc of Nat Acad Sci of the USA, 110(44):17832-7). Six variants, M28-K2, M28-K5, M28-K9, M28-13, M28-14, and M28-19, were selected for rearrangement against mouse IgG1 (chimera with human Fv) for multipoint ELISA, cell-based testing, surface plasmon resonance, and in vivo testing.

[0280] Eight cross-reactive M42 variants were isolated, each with changes to one CDR. Multipoint ELISA was used to rank-order two CDR-H2 variants and two CDR-H3 variants. One variant for each CDR-H2 and CDR-H3 was selected, and the CDRs were combined for a total of 11 variants. These 11 variants were subjected to in-solution competitive ELISA and ranked. Three variants, M42-K31, M42-K34, and M42-35, were selected for rearrangement against mouse IgG1 (chimera with human Fv).

[0281] IgG1 Conversion, Expression, and Purification: Light chain Fv fragments derived from the variants were PCR-amplified to include a 5' EcoRI site and a 3' BstAPI site and used for cloning into the pFUSE2ss-CLIg-mk vector (InvivoGen) for conversion to mouse IgGκ light chain expression (human-mouse chimera). Light chain Fv fragments derived from the variants were PCR-amplified to include a 5' EcoRI site and a 3' BsiWI site and used for cloning into the pFUSE2ss-CLIg-hk vector (InvivoGen) for conversion to human IgGκ light chain expression. Heavy chain Fv fragments were PCR-amplified to include a 5' EcoRI site and a 3' NheI site and used for cloning into pFUSEss-CHIg-mG1 (InvivoGen) for conversion to mouse IgG1 heavy chain expression (human-mouse chimera) or into pFUSEss-CHIg-hG4 (InvivoGen) for conversion to human IgG4 heavy chain expression. Heavy and light chain vectors were co-transfected into Expi293F cells (ThermoFisher) according to the manufacturer's instructions. Cell cultures were incubated for 5 days and then purified from the supernatant using Protein A Sepharose beads (GE Healthcare).

[0282] The results of these experiments are now described.

[0283] Renalase antigen selection, immunization and antibody production Renalase is an FAD-containing protein implicated in the regulation and maintenance of blood pressure (Xu, J. et al., 2005, J Clin Invest, 115(5):1275-80). The lack of high-affinity, high-specificity anti-renalase antibodies has hindered research and development of both renalase biology and potential renalase-related therapies. To date, several polyclonal research-use antibodies have been developed, but these have low affinity and have failed to robustly detect endogenous renalase in either tissue or body fluid samples. Therefore, the development of a set of renalase antibodies with specificity for various isoforms of renalase, as well as for different peptide sequences within the intact protein, would enable the unambiguous detection and characterization of renalase gene products. Furthermore, the selection of only high-affinity antibodies would enable the detection of potentially low levels of renalase, potentially leading to the development of protein-based diagnostics for renalase levels.

[0284] Two methods were used to generate various antibodies with high affinity and specificity for renalase. In the first method, polyclonal antibodies were generated using the full-length renalase-1 protein as an immunogen. The renalase-1 cDNA was subcloned into the pET27 expression vector (Novagen) and then used to transform the bacterial strain BL21 DE3 (Novagen). The renalase protein was isolated from bacterial inclusion bodies using standard procedures. Briefly, bacterial inclusion bodies were isolated and solubilized in a combination of chaotropic salts and reducing agents. The protein was refolded by dilution with the solubilization buffer, followed by final dialysis against phosphate-buffered saline (PBS).

[0285] The sequence of the full-length renalase protein used as the immunogen can be seen in Figure 2. This protein was used as an antigen to immunize six rabbits. Antisera from these animals were screened for renalase-binding specificity by ELISA assay, in which the antigen was the same refolded renalase protein. The antisera were also selected based on their ability to detect endogenous renalase in human tissue lysates by Western blot. The terminal bleed antiserum with the highest anti-renalase titer and greatest specificity for endogenous renalase was selected for antibody purification. Total IgG was purified from the antisera using protein G affinity chromatography, and specific anti-renalase antibodies were further purified on a column conjugated with recombinant renalase protein. As can be seen in the latter example, antibodies generated in this manner were shown to bind to renalase and to multiple epitopes on renalase.

[0286] In a second approach to raising anti-renalase antibodies, peptides were used as immunogens. These peptides ranged from 9 to 21 amino acids and corresponded to regions of the renalase-1 and renalase-2 proteins. All of these peptides had an N- or C-terminal cysteine ​​residue. The sequences of these peptides can be seen in Figure 1, where those peptides corresponding to the renalase-1 or renalase-2 sequences are revealed in the sequence alignment in Figure 3. As can be seen, the renalase-1-specific peptides are designated 1A-F, and the renalase-2-specific peptide is designated 3A5. Each peptide was conjugated via cysteine ​​to the adjuvant KLH and used to immunize six rabbits. The antisera from each animal were screened for anti-renalase antibody titers by ELISA assay using either the relevant peptide (BSA-conjugated) or full-length renalase-1 or -2. The antisera were also tested for their ability to detect endogenous renalase in tissue lysates by Western blot. Using these screening criteria, animals producing antibodies with desirable characteristics were selected. In some cases and for some peptides, several animals produced antibodies with the required specificity. In these cases, one animal had a terminal antiserum bleed for polyclonal antibody production, and one or, in some cases, two additional animals were used for splenic lymphocyte collection. In other cases, one animal had a terminal bleed and splenectomy. Polyclonal antibodies produced against all of these peptides were generated by purification of total IgG from the terminal bleed by protein G chromatography, followed by further purification on peptide affinity chromatography. Furthermore, using standard procedures, lymphocytes from the spleens of selected animals were fused to myeloma cells to generate hybridomas. The hybridoma supernatants were screened for binding to both peptides raised against them and, additionally, against the whole renalase protein. Selected hybridomas were subcloned and expanded for antibody purification.These monoclonal antibodies were purified from conditional hybridoma culture supernatants by protein A affinity chromatography. The peptides for which the monoclonal antibodies were raised can be seen in Figure 1.

[0287] Nucleotide and amino acid sequences of anti-renalase antibodies Monoclonal antibodies 1D-28-4, 1D-37-10, 1F-26-1, 1F-42-7, and 3A-5-2 were selected for their renalase binding specificity and high affinity (see below). Using standard polymerase chain reaction techniques and degenerate primer sets, cDNAs for the antibody heavy and light chain variable regions of these antibodies were amplified from subcloned hybridomas. The nucleotide and amino acid sequences of the variable regions are shown in Figures 4 through 13. In this manner, the composition of antibodies with favorable characteristics is demonstrated.

[0288] The antibody shows specificity for renalase in both ELISA and Western blot. As can be seen in Figure 14, anti-renalase polyclonal antibodies raised against the full-length renalase protein specifically bind to the bound renalase protein in an ELISA assay. Bacterially expressed renalase-1 diluted in PBS at 100 ng / well was bound to a 96-well microtiter plate by overnight incubation at 4°C. After blocking with NFDM, the renalase protein was probed with serial dilutions of polyclonal antibody E2930, starting at a concentration of 1 μg / mL. Antibody binding was assayed by the addition of HRP-conjugated anti-rabbit IgG. The plate was measured at 450 nm in a spectrophotometer after reaction with the HRP substrate TMB.

[0289] In a second example, all polyclonal antibodies and selected monoclonal antibody clones were bound to the peptide antigens against which they were raised. For example, as can be seen in Figure 15, monoclonal antibodies 1D 28-4 and 1D 37-10, which were raised against the 1D peptide, bound to renalase-1 in a concentration-dependent manner. The 1D peptide is present in both renalase-1 and renalase-2. Monoclonal antibodies 1F 42-7 and 1F 26-1 were raised against the 1F peptide, which is present only in renalase-1. The 1F mAb bound to renalase-1 in a concentration-dependent manner.

[0290] In a further example, to establish whether renalase can be detected by Western blotting using antibodies, serial dilutions of recombinant renalase protein of bacterial or mammalian origin were run on SDS-PAGE, and Western blotting was performed using the Ren1D 28-4 antibody. Recombinant proteins of either origin were clearly identified in this manner, as can be seen in Figure 16. In this manner, anti-renalase antibodies specific for the full-length renalase protein (SEQ ID NO: 8, 50, 92, or 94) or the peptide antigens set forth in SEQ ID NOs: 1-7 were generated.

[0291] The antibodies are specific for either renalase-1 or renalase-2 In this example, monoclonal antibodies raised against specific peptides of either renalase-1 or renalase-2 demonstrated absolute specificity for the relevant full-length renalase isoform. Antibodies 1D-28-4 and 1D-37-10 were raised against the 1D peptide and should therefore bind to both renalase-1 and renalase-2. Antibodies 1F-42-7 and 1F-26-1 were raised against the 1F peptide (see Figure 3 for alignment) and should therefore be specific only for renalase-1. Antibody 3A5-2 was raised against peptide 3A and should therefore be specific for renalase-2.

[0292] Bacterially expressed renalase-1 and -2 isoforms were diluted to 100 ng / well in PBS and bound to a 96-well microtiter plate. The plate was blocked with a solution of 8% NFDM in PBS at 33°C for 1 hour. Renalase protein was probed with serial dilutions of antibodies 1D-28-4, 1D-37-10, 1F-42-7, 1F-26-1, and 3A5-2. The starting antibody concentration was typically 1 μg / mL, prepared in 2-fold dilutions in PBS containing 0.05% Tween-20 (PBST). After incubation at 33°C for 1 hour, the plate was rinsed three times with PBST. Antibody binding was assayed by adding 50 μL per well of 50 ng / mL HRP-conjugated anti-rabbit IgG. The plate was incubated at room temperature for 40 minutes. After incubation, the plate was washed three times with PBST and once with PBS. 100 μL of HRP substrate 3,3',5,5'-tetramethylbenzidine (TMB) was added per well, and the plate was incubated at room temperature for approximately 5 minutes. To stop the reaction, 100 μL of 2N H2SO4 was added to each well. The plate was measured in a spectrophotometer at 450 nm.

[0293] Antibodies 1D-28-4, 1D-37-10, 1F-42-7, and 1F-26-1 bound to renalase-1 protein in a concentration-dependent manner by ELISA assay (Figure 15). However, when the same antibodies were used to detect recombinant renalase-2 in an ELISA plate assay, only 1D-28-4 and 1D-37-10 showed robust, concentration-dependent binding (Figure 17). Antibody 3A5-2 was observed to bind to renalase-2 isoforms in the ELISA assay (Figure 17). Therefore, it can be seen that antibodies raised against peptides corresponding to one or other renalase isoforms exhibit specificity for the relevant full-length protein.

[0294] Epitope-specific anti-renalase antibodies The renalase peptide used for immunization was synthesized and then conjugated to bovine serum albumin (BSA). The conjugated peptide was diluted in PBS at 100 ng / well and bound to a 96-well microtiter plate by overnight incubation at 4°C. After blocking with NFDM, the BSA-conjugated peptide was probed with serial dilutions of antibody. Antibody binding was assayed by adding HRP-conjugated anti-rabbit IgG. After reaction of the HRP with the substrate TMB, the plate was measured spectrophotometrically at 450 nm. Monoclonal antibodies 1D 28-4 and 1D 37-10, raised against the 1D epitope, bound to the 1D-BSA protein in a concentration-dependent manner (Figure 18, filled circles for 1D 28-4, open circles for 1D 37-10). However, these monoclonals demonstrated very weak binding to the 1F antigen (semi-black diamonds for 1D 28-4 and 1D 37-10, inset). Monoclonal antibodies 1F 42-7 and 1F 26-1, raised against the 1F epitope, bound to the 1F-BSA protein in a concentration-dependent manner (black squares for 1F 26-1, open squares for 1F 42-7 binding). However, these 1F mAbs demonstrated very weak binding to the 1D peptide (semi-black circles for 1F 26-1 and 1F 42-7, inset). In further examples, the same epitope specificity was observed for antibody 3A5-2 on peptide 3A and for antibody 1C-22-1 on peptide 1C. In yet a further example, purified polyclonal antibodies raised against peptides 1A, 1B, 1C, 1D, 1E, 1F and 3A all specifically bound to the relevant BSA-peptide fusions.

[0295] Thus, multiple examples of antibodies raised against peptide epitopes within the renalase protein have demonstrated absolute epitope specificity. The amino acid compositions of both these epitopes and the monoclonal antibodies that bind to them are recognized and described herein.

[0296] Anti-renalase antibody conjugation Renalase-specific antibodies can be conjugated to several entities that aid in the use of antibodies as renalase detection reagents or renalase-directed therapeutics. These conjugations can be accomplished without interfering with the relevant epitope specificity or affinity of the antibodies. Examples of conjugates include, but are not limited to, radioactive ions, colored, metallic, or fluorescent labels, tags, or toxins. In this example, anti-renalase antibodies were conjugated to the vitamin biotin. Biotin is a small, naturally occurring vitamin that binds with extremely high affinity to the proteins avidin, streptavidin, and their variants. Conjugation is accomplished via an N-hydroxysuccinimide (NHS) ester bond of biotin to primary amino groups on the antibody. Excess unconjugated biotin can be removed by dialysis.

[0297] In a further example, anti-renalase antibodies conjugated in this manner were found to maintain their ability to bind to renalase protein in an ELISA assay. Bacterially expressed renalase-1 was bound to a 96-well microtiter plate by diluting it at 100 ng / well in PBS and incubating overnight at 4°C. After blocking with NFDM, the renalase protein was probed with serial dilutions of biotinylated antibody starting at a concentration of 1 μg / mL. The presence of bound biotinylated antibody was assayed by the addition of HRP-conjugated neutravidin. The plate was read at 450 nm in a spectrophotometer after reaction of the HRP with the substrate TMB. Biotinylated monoclonal antibodies 1D 28-4 (FIG. 19, closed circles), 1D 37-10 (open circles), 1F 42-7 (closed triangles), and 1F 26-1 (open triangles) bound to renalase-1 in a concentration-dependent manner.

[0298] Multiple antibodies can compete for binding to the same epitope on renalase In this example, various combinations of both monoclonal and polyclonal anti-renalase antibodies were shown to compete with each other for binding to renalase, revealing overlapping epitopes of multiple antibodies. In a further example, a polyclonal antibody was observed to compete with two different monoclonal antibodies, revealing the multiple epitope specificity of this polyclonal. Bacterially expressed renalase-1 was bound to 96-well microtiter plates by diluting in PBS at 100 ng / well and incubating overnight at 4°C. After blocking, the protein was probed with serial dilutions of unconjugated antibody, typically starting at a concentration of 1 μg / mL, and mixed with different biotinylated antibodies. The concentration of the biotinylated antibody was kept constant at 125 ng / well. Both antibodies were mixed together in PBST and applied to the pre-blocked renalase plate in a single addition of 50 μL / well. After 1 hour of incubation at 33°C, binding of biotinylated antibodies to renalase was assayed by adding 50 µL / well of 0.4 ng / mL HRP-conjugated neutravidin (HRP-NA). HRP substrate TMB was added to visualize bound biotinylated antibodies. Plates were then read at 450 nm in a spectrophotometer.

[0299] The specificity of the anti-renalase antibodies was demonstrated by the reduction in signal when the biotinylated antibodies were incubated with unconjugated antibodies sharing the same epitope. An example is shown in Figure 20A, using monoclonal antibodies raised against the 1D peptide. Biotinylated 1D 37-10 was competed with unconjugated 1D 28-4 (filled circles, Figure 20A). Similarly, antibodies raised against the 1F peptide also competed with each other: biotinylated 1F 26-1 was competed with unconjugated 1F 42-7 (filled triangles, Figure 20A). The signal from the biotinylated monoclonal antibodies was not reduced when incubated with unconjugated antibodies from unimmunized rabbits (open circles and triangles, Figure 20A).

[0300] In further examples, and as can be seen in Figure 20B, polyclonal antibody E2930 raised against full-length renalase-1 was shown to compete with each of biotinylated monoclonal antibodies 1D 37-10 and 1F 26-1, as well as a mixture of these two biotinylated mAbs (filled circles, filled squares, and open diamonds, Figure 20B). Again, the mixture of the two biotinylated antibodies was not competed with by unconjugated antibody from an unimmunized rabbit (filled triangles, Figure 20B). The competition observed by polyclonal antibody E2930 against the two biotinylated monoclonal antibodies suggests that this polyclonal antibody binds to multiple epitopes on the renalase polypeptide.

[0301] Antibodies bind to renalase with high affinity and different binding kinetics When an antibody is used to detect an endogenous protein, it must exhibit high affinity for its target. To date, the binding kinetics for anti-renalase antibodies have not been described. In this example, several anti-renalase antibodies were found to have low or subnanomolar affinity for full-length renalase in solution binding assays. Purified anti-renalase antibodies were biotinylated and bound to a streptavidin CM5 sensor chip for a Biacore T100 instrument. Pure recombinant renalase-1 was injected over the surface of the chip. Binding studies were performed at 25°C using 25 mM Tris (pH 8), 150 mM NaCl, 1 mM EDTA, 10% glycerol, 0.005% Tween-20, and 0.1 mg / mL BSA as the running buffer. The binding kinetics of renalase to the immobilized antibody can be measured in this manner. As can be seen in Figure 21 and summarized in Figure 22, antibodies 1D-28-4, 1F-42-7, 1D-37-10 and 1F-26-1 all bind to renalase with high affinity -K D The values ​​ranged from 2.67 nM to 0.316 nM. A wide range of association and dissociation was observed between these antibodies, revealing different contributions of different antibody compositions.

[0302] In a xenograft mouse model, anti-RNLS therapy with the monoclonal antibody m28-RNLS significantly inhibited melanoma tumor growth (Hollander L et al., 2016, Cancer Research, 76(13):3884-94) Two monoclonal antibodies raised against RNLS [clone #28-4 (m28-RNLS), 37-10 (m37-RNLS)] reduced the viability of all melanoma cell lines tested (a total of five), and representative examples are shown in Figure 23. m28-RNLS demonstrated increasing levels of cytotoxicity that correlated with increasing treatment concentrations (p<0.05, Figure 24).

[0303] For in vivo studies, A375.S2 (human melanoma) cells were injected subcutaneously into athymic nude mice to form tumors. Once tumors reached a volume of ∼50 mm 3Once the tumor size reached 100 μg / mL, the animals were then treated with either control rabbit IgG or the RNLS-neutralizing monoclonal antibody m28-RNLS. This antibody treatment did not appear to be toxic, as overall animal health and activity were maintained throughout the study. Tumor size was measured every other day, and treatment with m28-RNLS reduced tumor volume at all time points tested (p<0.05, Figure 25). The animals were sacrificed on day 11 due to overall tumor size and ulceration in some animals. IHC staining of sections from xenografted tumors with the cell proliferation marker Ki67 revealed a significant decrease in cell proliferation in tumors treated with anti-RNLS antibody compared with those treated with rabbit IgG: 35.1 ± 2.3 positive cells / high-power field in the control group vs. 13.4 ± 3.0 in the RNLS Ab-treated group, n=14, p=0.0004 (Figure 26). To test the efficacy of anti-RNLS therapy in immunocompetent mice, B16f10 cells (a murine melanoma line) were injected subcutaneously into C57BL / 6 mice. Once tumors reached a volume of ∼500 mm, 3 Once tumor volume reached 0.05, animals were treated with either control rabbit IgG or the RNLS-neutralizing monoclonal antibody m28-RNLS and sacrificed on day 7 due to the very large tumor burden in the control group. As shown in Figure 27, m28-RNLS administration caused a significant reduction in tumor volume compared to rabbit IgG.

[0304] Anti-RNLS therapy with monoclonal antibody m28-RNLS significantly inhibits tumor growth in pancreatic adenocarcinoma in xenograft mouse models (Guo X et al., 2016, Scientific Reports, 6:22996) From a panel of rabbit monoclonal antibodies against RP-220, two clones, m28-RNLS and m37-RNLS, were selected based on their high binding affinity (K D The inhibitory effects of m28-RNLS, m37-RNLS, and a commercially available polyclonal antibody (against a partial sequence of RP-220) on the growth of human pancreatic adenocarcinoma cells are shown in representative examples in Figures 28, 29, and 30.

[0305] To evaluate the therapeutic potential of the inhibitory antibodies, BxPC3 cells were injected subcutaneously into athymic nude mice, which were treated with either control rabbit IgG or m28-RNLS, and tumor volume was measured for up to 3 weeks. As shown in Figure 31, m28-RNLS treatment caused a significant reduction in tumor volume compared with rabbit IgG.

[0306] Anti-RNLS therapy with the m28-RNLS humanized variant inhibits tumor growth Melanoma cells (human: SK-MEL-28, mouse: YUMM1.7) and pancreatic adenocarcinoma cells (human: BxPC3) were cultured in a 6-well culture plate at a density of 5 × 10 4 Cells were seeded at 1000 cells / well. The percentage of cell proliferation and viability was determined using a trypan blue assay. The cells were washed twice with PBS, trypsinized, and stained with 0.5% trypan blue dye. A Bio-Rad TC10 automated cell counter (Hercules, CA, USA) was used to assess the total number of cells, the total number of viable cells, and the percentage of viable cells in each sample. The recorded numbers were used to determine the percentage of proliferation and viability.

[0307] Five humanized m28 variants (m28-K2, m2-K5, m28-K13, m28-K14, and m28-K16, Table 2) showed increased binding to both human and mouse RNLS (Figures 32 and 33). These m28 variants also reduced the viability of human melanoma (SK-MEL-28) and pancreatic adenocarcinoma (BxPC3) cell lines (representative examples are shown in Figures 34 and 35), indicating the potential therapeutic utility of m28 variants in human cancers.

[0308] Example 2: Sequences [Table 1-1] [Table 1-2] [Table 1-3] [Table 2]

[0309] SEQ ID NO:218 M28-Humanized Fv Sequence Heavy Chain Nucleotide GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAATCTGAGCAGCTTCGCCGTTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCAATCATCAGCAGCGTTGGCATCACCCGCTATGCC GATAGCGTCAAGGGCCGTTTCACTATAAGCAAAGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCTATGGCTATAGCGGCGACGTGAACCGCCTGGACCTGTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO:219 M28-Humanized Fv Sequence Heavy Chain Amino Acids (M28 CDRs and HC71 grafts are underlined) EVQLVESGGGLVQPGGSLRLSCAASGFN LSSFAV HWVRQAPGKGLEWVA IISSVGITR YADSVKGRFTIS K DTSKNTAYLQMNSLRAEDTAVYYCAR YGYSGDVNRLDL WGQGTLVTVSS SEQ ID NO:220 M28-humanized Fv sequence light chain nucleotide GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGAGCGTGTATGACAACAACAACGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACGGCGCCAGCACCCTCTAC TCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCTGGGCGAATTCAGCTGCAGCAGCGCTGACTGCTTCGCCTTCGGACAGGGTACCAAGGTGGAGATCAAACGA SEQ ID NO:221 M28-humanized Fv sequence light chain amino acids (M28 CDRs and HC71 grafts are underlined) DIQMTQSPSSLSASVGDRVTITCRASQ SVYDNNN VAWYQQKPGKAPKLLIY GAST LYSGVPSRFSGSRSGTDFLTISSLQPEDFATYYC LGEFSCSSADCFA FGQGTKVEIKR SEQ ID NO:222 M42 humanized Fv sequence heavy chain nucleotide GAGGTTCAGCTGGTGGAGTCTGGCGGTGGCCTGGTGCAGCCAGGGGGCTCACTCCGTTTGTCCTGTGCAGCTTCTGGCTTCAACCTGACCACCTACGGCGTTCACTGGGTGCGTCAGGCCCCGGGTAAGGGCCTGGAATGGGTTGCACTGATCGGCGATCGCGGCACCACCTAT TATGCCGATAGCGTCAAGGGCCGTTTCACTATAAGCCGCGACACATCCAAAAACACAGCCTACCTACAAATGAACAGCTTAAGAGCTGAGGACACTGCCGTCTATTATTGTGCTCGCGGCAGCGGCTATGGCGCTCGCATCTGGGGTCAAGGAACCCTGGTCACCGTCTCCTCG SEQ ID NO:223 M42 humanized Fv sequence heavy chain amino acids (M42 CDRs and HC71 grafts are underlined) EVQLVESGGGLVQPGGSLRLSCAASGFN LTTYGV HWVRQAPGKGLEWVA LIGDRGTTY YADSVKGRFTISRDTSKNTAYLQMNSLRAEDTAVYYCAR GSGYGARI WGQGTLVTVSS SEQ ID NO:224 M42 humanized Fv sequence light chain nucleotide GATATCCAGATGACCCAGTCCCCGAGCTCCCTGTCCGCCTCTGTGGGCGATAGGGTCACCATCACCTGCCGTGCCAGTCAGACCGTGTATAACAACAACTACGTAGCCTGGTATCAACAGAAACCAGGAAAAGCTCCGAAGCTTCTGATTTACGAACCAGCAAACTC TACTCTGGAGTCCCTTCTCGCTTCTCTGGTAGCCGTTCCGGGACGGATTTCACTCTGACCATCAGCAGTCTGCAGCCGGAAGACTTCGCAACTTATTACTGTCAGGGCGGCTACAGCGGCGTGGACTTCATGGCTTTCGGACAGGGTACCAAGGTGGAGATCAAACGA SEQ ID NO:225 M42 humanized Fv sequence light chain amino acids (M42 CDRs and HC71 grafts are underlined) DIQMTQSPSSLSASVGDRVTITCRASQ TVYNNNY VAWYQQKPGKAPKLLIY ETSKL YSGVPSRFSGSRSGTDFLTISSLQPEDFATYYC QGGYSGVDFM AFGQGTKVEIKR

[0310] The disclosures of each and every patent, patent application, and publication cited herein are hereby incorporated by reference in their entirety. While the present invention has been described with reference to specific embodiments, it will be apparent that other embodiments and variations of the present invention may be devised by those skilled in the art without departing from the true spirit and scope of the present invention. It is intended that the appended claims be construed to include all such embodiments and equivalent variations.

Claims

1. a) a heavy chain (HC) CDR1 comprising the amino acid sequence of SEQ ID NO: 191; b) a HC CDR2 comprising the amino acid sequence of SEQ ID NO: 192; c) a HC CDR3 comprising the amino acid sequence of SEQ ID NO: 193; d) a light chain (LC) CDR1 comprising the amino acid sequence of SEQ ID NO: 188; e) an LC CDR2 comprising the amino acid sequence of SEQ ID NO: 189; and f) a LC CDR3 comprising the amino acid sequence of SEQ ID NO: 190; An antibody or a binding site thereof which specifically binds to renalase.

2. At least 10 -6 2. The antibody or binding portion thereof of claim 1, which specifically binds to renalase with an affinity of M.

3. The antibody or binding portion thereof according to claim 1, which specifically binds to a peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 and 8.

4. The antibody or binding portion thereof according to claim 1, wherein the renalase is human renalase.

5. The antibody or binding portion thereof of claim 1 , wherein the antibody or binding portion thereof is selected from the group consisting of a monoclonal antibody, a single-chain antibody, a defucosylated antibody, and a bispecific antibody.

6. An immune complex comprising the antibody or binding site thereof according to claim 1, The immunoconjugates described above, which comprise a therapeutic agent or a detection moiety.

7. The antibody or binding portion thereof of claim 1 , wherein the antibody or binding portion thereof is selected from the group consisting of a humanized antibody and a chimeric antibody.

8. An isolated nucleic acid molecule comprising a sequence encoding the antibody or binding portion thereof according to any one of claims 1 to 7.

9. An expression vector comprising the nucleic acid molecule of claim 8.

10. A cell comprising the nucleic acid molecule of claim 8.

11. A pharmaceutical composition comprising an antibody or binding portion thereof according to any one of claims 1 to 7 for treating a disease or disorder in a subject.

12. 12. The pharmaceutical composition of claim 11, further comprising at least one additional pharmaceutical agent.

13. 12. The pharmaceutical composition according to claim 11, wherein the disease or disorder is at least one selected from the group consisting of renal disease, cardiovascular disease, pancreatitis, hepatitis, inflammatory disorders of the kidney, and cancer.

14. The pharmaceutical composition according to claim 11, wherein the disease or disorder is cancer, and the cancer is pancreatic cancer or melanoma.

15. The pharmaceutical composition of claim 11 , wherein the subject is a human.

16. 10. Use of an antibody or binding portion thereof according to any one of claims 1 to 7 in the manufacture of a medicament for treating a disease or disorder.

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