Novel IGFR-like receptors and uses thereof

Abstract

Due to safety concerns and adverse effects of available diabetes treatments, and the lack of durable disease relief from any of the agents tested to date, new treatments are provided that can modulate the disease. The present invention provides antagonists or agonists of IGFR-like receptors, which are used in methods for the prophylactic and / or therapeutic treatment of diabetes.

Description

[Technical Field]

[0001] [background] Insulin / insulin-like growth factors (IGFs) constitute a network of ligands, cell surface receptors, and binding proteins that are involved in the regulation of multiple physiological and pathological processes. Insulin / IGFs play important developmental and metabolic roles at all stages of life. Insulin / IGF signaling also contributes to the regulation of lifespan, while dysregulation of signaling is implicated in tumors. Although the insulin receptor (InsR) and IGF-1 receptor (IGF1R) share most of their downstream cytoplasmic mediators, most experimental and clinical evidence is consistent with the notion that InsR activation (primarily by insulin) primarily leads to metabolic activity, whereas IGF1R activation (primarily by IGF-1 or IGF-2) leads to proliferation and differentiation events (Sarfstein R and Werner H Endocrinology. 2013 May;154(5):1672-9; Siddle KJ Mol Endocrinol. 2011 Jun 17;47(1):R1-10).

[0002] InsR and IGF1R belong to a family of transmembrane tyrosine kinase-containing receptors. In their mature forms, they exist as heterotetramers composed of two extracellular α-subunits and two transmembrane β-subunits with tyrosine kinase activity. IGF- and insulin receptors show a high degree of homology (84% in the tyrosine kinase domain, 45%-65% in the ligand-binding domain, and >50% in the overall amino acid sequence). In addition, these receptors display significant similarities in genomic organization (Sarfstein R and Werner H, loc. cit.; Arnalez F and Helman L. Hematol Oncol Clin North Am. 2012 Jun;26(3):527-42).

[0003] There is also a "hybrid" receptor (IRαβ bound to IGF1Rαβ) that consists of half an insulin receptor and half an IGF receptor. The hybrid binds IGF with an affinity similar to that of the IGFR, but binds insulin with a substantially lower affinity than the InsR. It is unclear whether the hybrid receptor has a distinct physiological role (Sarfstein R and Werner H, loc. cit.; Arnalez F and Helman L., loc. cit.).

[0004] The insulin receptor exists in two splice variant isoforms as a result of alternative splicing of the sequence encoded by exon 11. The "B" isoform recognizes only insulin, while the "A" isoform, which is the isoform most commonly expressed by tumors, recognizes both insulin and IGF1 and IGF2. Both isoforms are differentially expressed during development, with InsR-A being preferentially expressed in fetal tissues and InsR-B being preferentially expressed in adult tissues, particularly liver, muscle, and adipocytes. IGF1R exhibits the inverse pattern of expression, being absent from the liver, present at low levels in adipose tissue, and present at high levels in the brain. Additionally, consistent with its potent anti-apoptotic and pro-survival role, IGF1R is overexpressed in most tumors and malignant cells (Pollak M Nat Rev Cancer. 2012 Feb 16;12(3):159-69, Sarfstein R and Werner H, loc. cit.; Siddle K, loc. cit.).

[0005] IGF1 and IGF2 can be expressed in an endocrine, paracrine, or autocrine manner. The latter is common in transformed cells. The liver is their primary site of production. In contrast, insulin production is restricted to pancreatic β cells. Insulin and IGFs bind with high affinity to their specific receptors and, with the exception of IGF2, bind with low affinity to non-cognate receptors. IGF2 also binds with high affinity to InsR-A (Pollak M, loc cit.; Siddle K, loc. cit.).

[0006] Ligand binding induces conformational changes in the structure of InsR and IGF1R, activating their intrinsic tyrosine kinase activity. Although insulin and IGFs have distinct physiological roles, they utilize the same signaling pathways. Downstream signaling of InsR and IGF1R is mostly mediated by the MAPK / Ras-Raf-Erk pathway, the phosphatidylinositol-3-kinase / AKT / mTOR (PI3K / AKT) pathway, and the Janus kinase / signal transducer and activator of transcription (JAK / STAT) pathway. Ultimately, activation of IGF1R leads to increased cell proliferation and decreased apoptosis. Meanwhile, activation of InsR by insulin binding promotes the storage and synthesis of lipids, proteins, and carbohydrates and inhibits their breakdown and release into the circulation. The first step by which insulin increases energy storage or utilization is through the regulated transport of glucose into cells, mediated by the facilitative glucose transporter Glut4 (Chang et al. Mol Med. 2004 Jul-Dec;10(7-12):65-71.). Insulin expression is restricted to specialized pancreatic β-cells and, under normal circumstances, is tightly regulated by circulating glucose levels. Insulin-stimulated glucose uptake by classical insulin-sensitive organs (liver, muscle, and adipose tissue) reduces circulating glucose levels. Thus, β-cells are glucose "thermostats" that sense glucose and release insulin to maintain physiological glucose levels within a relatively narrow range. Disruption of the delicately balanced InsR signaling pathway leads to uncontrolled or impaired insulin secretion, dysregulated blood glucose levels, and ultimately destruction or loss of function of pancreatic beta cells, a condition commonly known as diabetes (Pollak M, loc. cit.; Siddle K, loc. cit.; Sarfstein R and Werner H, loc. cit.; Arnalez F and Helman L., loc. cit.).

[0007] Diabetes, affecting 8.3% of the adult population worldwide and increasing at a rapid rate, is one of the most common diseases of our time. The number of people with diabetes is projected to increase from 382 million in 2013 to 592 million by 2035, representing a net increase of 55%. The predominant form is type 2 diabetes (T2D), accounting for nearly 90% of all diabetes cases (Hameed et al. World J Diabetes. 2015 May 15; 6(4): 598-612).

[0008] Type 1 diabetes (T1D) is an autoimmune disorder affecting millions worldwide and results from organ-specific immune destruction of insulin-producing beta cells in the islets of Langerhans within the pancreas. Once these cells are destroyed, patients with type 1 diabetes lose control of their blood glucose, which can lead to acute symptoms (e.g., ketoacidosis and severe hypoglycemia) and secondary complications (including heart disease, blindness, and kidney failure). Type 1 diabetes is thought to develop as a result of a combination of genetic predisposition, largely unknown environmental factors, and stochastic events. However, the precise immunological, genetic, and physiological events that control the initiation and progression of the disease continue to be elucidated.

[0009] Early type 2 diabetes (T2D) results from insulin resistance in classical insulin target organs (i.e., reduced glucose uptake by normal insulin target cells, often induced by excessive caloric intake), leading to hyperinsulinemia. Initially, these increases in insulin levels are sufficient to overcome insulin resistance and avoid hyperglycemia. However, eventually, hyperglycemia occurs not only due to increased insulin resistance but also due to reduced insulin secretion by pancreatic β cells.

[0010] Controlling blood glucose levels is the primary goal of diabetes treatment. T1D is typically managed through insulin administration, dietary changes, and exercise. However, the lifelong requirement for insulin injections after nutritional intake can severely reduce patients' quality of life. Furthermore, the appropriate dose and timing of insulin injections are difficult to prove. Cure or prevention of T1D is severely hampered by the lack of biomarkers reliably linked to the pathogenic process, which results in a significant reduction in β-cell numbers at the time of diagnosis. Today, the goal of most clinical trials in type 1 diabetes is to improve the remaining functional β-cell mass by optimally inducing immune tolerance while preserving protective immune responses. By definition, this rarely cures the disease due to the significant β-cell destruction prior to treatment. Therefore, a reliable biomarker that is favorably expressed at the onset of disease would be highly desirable. Other approaches focus on transplantation of either the pancreas or pancreatic β-cells to reconstitute insulin-secreting function. However, this technique is hampered by a shortage of donor organs. Besides insulin, other non-insulin treatments for T2D are available, including synthetic hypoglycemic agents, which are often limited in terms of their practical effectiveness, ease of administration, and potential adverse reactions.

[0011] Safety concerns and adverse effects of available diabetes treatments, as well as the failure of any agent tested to date to provide durable relief of the disease, have led to growing interest in specific interventions that can modulate the disease.

[0012] SUMMARY OF THE INVENTION It is an object of the present invention to address the needs in the art. Summary of the Invention

[0013] The present inventors have observed that a protein encoded by the human KIAA1324 gene (also known as estrogen-inducible gene 121 protein (Q6UXG2)) colocalizes with IGF-1R, IGF-2R, and InsR, particularly in the pancreas. The estrogen-inducible gene 121 protein has been assigned functions of regulating autophagy and promoting cell survival under stress. A mutant protein encoded by estrogen-inducible gene 121 is also known in the art and designated MABA-1 (WO 2007 / 005987). MABA-1 differs from the protein encoded by estrogen-inducible gene 121 at one position. MABA-1 is thought to play a role in cancer, since blocking this protein results in the inhibition of cancer cell growth. However, nothing was known about the protein's role in metabolism, let alone speculation. This role was first attributed to the protein by the present inventors.

[0014] Because of its domain structure similar to that of IGFR, the inventors endowed this protein with receptor function, and therefore called it an IGFR-like receptor. The inventors also observed that the IGFR-like receptor was most highly expressed in the pituitary gland, hypothalamus, and pancreatic islet cells, which constitute the endocrine system that controls food intake and metabolism.

[0015] Furthermore, the present inventors have demonstrated that knockdown or knockout of IGFR-like receptors results in increased phosphorylation of InsR and IGFR1, as well as increased phosphorylation of AMPK, indicating that downstream signaling components become active when either IGFR or InsR, or both, transmit signals, such as insulin binding. Therefore, IGFR-like receptors are thought to negatively regulate InsR- and / or IGFR-mediated signaling. Furthermore, in light of the findings of the present invention, namely, that the protein encoded by the KIAA1324 gene actually acts as an IGFR-like receptor, the present inventors have conducted genome-wide association studies (GWAS) and found a strong association between SNPs in the KIAA1324 gene and type 2 diabetes, LDL cholesterol, and / or coronary artery disease.

[0016] In summary, the protein encoded by the human KIAA1324 gene, which the inventors have attributed the function of a modulator of IGFR2 and / or InsR, plays a role in metabolism, particularly in insulin signaling, and similarly in LDL cholesterol metabolism, as well as in diseases such as coronary heart disease. This finding is extremely important, as it was not previously thought that there might be additional players in insulin signaling apart from IGFR and InsR. Due to its putative negative regulatory function on IGFR and / or InsR, the IGFR-like receptor of the present invention is considered an attractive target for modulators, which could open new avenues for the development of pharmaceuticals for treating, for example, diabetes, LDL cholesterol-related disorders, and coronary artery disease, but particularly type II diabetes.

[0017] Antagonists of these receptors therefore open up new and urgently needed possibilities for reversing, for example, the insulin resistance commonly seen in the pathogenesis of type II diabetes. On the other hand, agonists can be used to block insulin signaling. Remarkably, the inventors have also been able to show that IGFR-like receptors are involved in the dedifferentiation of beta cells in the pancreas, an early event in the initiation of the disease that ultimately leads to the destruction or loss of function of beta cells. Therefore, IGFR-like receptors are also a facilitating diagnostic tool that allows for early diagnosis and treatment of diabetes before irreversible beta cell loss, and thus may pave the way for the treatment of type I diabetes.

[0018] Accordingly, the present invention provides an isolated DNA sequence encoding an IGF receptor (IGFR)-like receptor capable of reacting with an antibody raised against the IGFR-like receptor of SEQ ID NO:1, wherein said antibody is (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) Specifically binds to at least one epitope of

[0019] In particular, the isolated DNA sequence can encode an IGFR-like receptor comprising a sequence corresponding to SEQ ID NO:1.

[0020] The isolated DNA sequence may in particular comprise the sequence corresponding to SEQ ID NO: 7 (NM_0200775.4).

[0021] Further provided herein are vectors comprising the DNA sequences described herein. The vectors may further comprise genetic regulatory elements operably linked to the DNA sequence encoding the IGFR-like receptor. Host cells comprising the vectors are also contemplated.

[0022] Further provided herein is a binding agent capable of specifically binding to an IGFR-like receptor, wherein the IGFR-like receptor comprises a sequence corresponding to the sequence of SEQ ID NO: 1, for use as a diagnostic marker for diabetes or the risk of progression of diabetes.

[0023] Antagonists and agonists of the IGFR-like receptors of the present invention (e.g., the IGFR-like receptors include a sequence corresponding to SEQ ID NO: 1) are also provided herein. The antagonists and agonists are generally contemplated for use as pharmaceuticals. Specifically, the antagonists and agonists provided herein are intended for use in methods for the prophylactic and / or therapeutic treatment of diabetes, although antagonists are preferred.

[0024] The antagonists and agonists are expected to specifically bind to the IGFR-like receptor and can be selected from, inter alia, antibodies, siRNAs, nucleic acids, aptamers, peptides, proteins, or small organic compounds, etc. The antibodies can be monoclonal or polyclonal antibodies.

[0025] In particular, the antibody, preferably a monoclonal antibody, can be an antibody (e.g., a monoclonal antibody) that specifically binds to an epitope of the IGFR-like receptor, the epitope having the sequence: (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) It comprises or consists of:

[0026] For the diagnostic and / or therapeutic uses provided herein, diabetes is intended to include type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, insulin resistance, metabolic syndrome, and impaired glucose tolerance.

[0027] Treatment with antagonists or agonists (antagonists preferred) of IGFR-like receptors can prevent or reverse insulin resistance and / or reverse the dedifferentiation and / or loss of function of pancreatic β cells.

[0028] Further provided herein is a medicament or pharmaceutical composition comprising an antagonist or agonist (antagonists are preferred) of an IGFR-like receptor. In a preferred embodiment, the IGFR-like receptor comprises or consists of a sequence corresponding to SEQ ID NO: 1. In a preferred embodiment, the antagonist or agonist is a monoclonal antibody that specifically binds to the IGFR-like receptor.

[0029] It is envisaged that the preferred monoclonal antibodies will specifically bind to an epitope of the IGFR-like receptor, the epitope being of the sequence (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) or consisting of the same sequence.

[0030] Further, as used herein, the sequence (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) Monoclonal antibodies are provided that specifically bind to an epitope of an IGFR-like receptor.

[0031] Further provided herein is an in vitro screening assay for an antagonist or agonist of an IGFR-like receptor, comprising: (a) providing a stable cell line expressing said IGFR-like receptor; (b) contacting the cell line of (a) with a candidate antagonist or agonist; (c) measuring or detecting a downstream signaling event of an IGFR-like receptor, wherein an antagonist is identified by suppressing the downstream signaling event of the IGFR-like receptor and an agonist is identified by promoting the downstream signaling event of the IGFR-like receptor; Here, a method is provided wherein said IGFR-like receptor comprises, in a preferred embodiment, a sequence corresponding to the sequence of SEQ ID NO:1.

[0032] Also provided herein is an IGFR-like receptor antagonist or agonist that can be obtained by an in vitro screening assay, wherein the IGFR-like receptor antagonist or agonist is selected from an antibody, siRNA, nucleic acid, aptamer, peptide, protein, or small molecule organic compound.

[0033] The present invention further provides an in vitro diagnostic assay for detecting pancreatic islet cell degeneration in a subject, comprising: i) contacting a sample, e.g., a plasma sample, from the subject with a diagnostic binding agent, e.g., a monoclonal antibody, that specifically binds to an IGFR-like receptor; ii) detecting binding of the binding agent; An in vitro diagnostic assay is provided wherein detectable binding of the binding agent indicates dedifferentiation of pancreatic islet cells in a subject, wherein the IGFR-like receptor comprises a sequence corresponding to SEQ ID NO:1.

[0034] The diagnostic binding agent (which may in particular be an antibody of the invention, e.g., a monoclonal antibody) may, for example, be a binding agent of the sequence (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) It is assumed that the antibody specifically binds to an epitope of an IGFR-like receptor.

[0035] Dedifferentiation of pancreatic islet cells is thought to indicate diabetes or the risk of progression of diabetes.

[0036] The diagnostic binding agent may preferably be in the form of a composition, e.g., a diagnostic composition. Accordingly, the present invention also provides diagnostic compositions comprising a diagnostic binding agent as described herein, and optionally further comprising means for detecting binding of said diagnostic binding agent to its target, i.e., an IGFR-like receptor as described herein.

[0037] The present invention also relates to the use of the binding agents of the invention, and in particular the antibodies of the invention, for the in vitro detection of pancreatic islet cell degeneration. The use of these antibodies for the preparation of therapeutic or diagnostic compositions is also contemplated.

[0038] Further provided herein is a method of treating diabetes, comprising administering to a subject an antagonist or agonist of an IGFR-like receptor, wherein the IGFR-like receptor comprises a sequence corresponding to SEQ ID NO:1. [Brief explanation of the drawings]

[0039] The invention will be better understood by reference to the detailed description when considered in conjunction with the non-limiting examples and the accompanying drawings, which illustrate embodiments of the method of the invention. [Figure 1]

[0033] A diagram of sequences listed throughout the specification. SEQ ID NO: 1 represents the long transcript of an IGFR-like receptor. The predicted growth factor domain is underlined in red. The mannose-6-phosphate receptor binding domain is underlined in green. The transmembrane region is underlined in black. The cytoplasmic region is underlined in blue. The five epitopes corresponding to SEQ ID NOs: 2-6 against which antibodies were generated are highlighted in bold underlined text. [Figure 2] Pancreatic expression of IGFR-like receptors adjacent to IGF-1, IGF-2, and insulin ligands. Genepaint.org in situ hybridization of E14.5 mouse embryos shows specific expression of IGFR-like receptor mRNA in the pancreas (B). IGF1 and IGF2 mRNA are expressed in tissue adjacent to the pancreatic epithelium (A, C). Insulin mRNA, on the other hand, is present in the endocrine compartment of the pancreas (D). [Figure 3]IGFR-like receptor knockout mice die early after birth at normal weight. Schematic diagram of the EUCOMM allele (A). The EUCOMM allele is a conditionally potent knockout first reporter-tagged insert (promoter-driven cassette) (Skarnes WC et al. 2011; Nature). The L1L2_Bact_P cassette was inserted into chromosome 3 at position 108489484 (upstream of a critical exon) (Build GRCm38). This cassette consists of an FRT site followed by a lacZ sequence and a loxP site. This first loxP site is followed by neomycin, SV40 polyA, a second FRT site, and a second loxP site under the control of the human beta-actin promoter. The third loxP site is inserted downstream of the target exon at position 108488602. The critical exon is flanked by loxP sites. A "conditionally ready" (loxP-introduced) allele can be generated in mice carrying this allele by expressing flp recombinase. Subsequent cre expression results in knockout mice. If cre expression occurs without flp expression, reporter knockout mice will result (http: / / www.informatics.jax.org / allele / MGI:4436415). IGFR-like receptor knockout mice appear normal at birth but do not lactate, as evidenced by the absence of milk in their stomachs (B, star). They are sluggish and exhibit respiratory distress, a sign of metabolic dysfunction. Mendelian ratios at various stages show a normal distribution of the three genotypes until birth, but the probability of surviving knockout mice decreases significantly from birth to weaning age. Negligible survival is observed at weaning age, likely due to compensation mechanisms and / or splicing near the inserted cassette resulting in an incomplete knockout (C). Histograms show normal body weights of knockout mice compared to wild-type and heterozygous littermate controls at P0 (D). [Figure 4]Growth, proliferation, and endocrine differentiation in the pancreas of IGFR-like receptor KO mice. Confocal images of pancreatic sections show normal endocrine differentiation at E16.5, as indicated by immunofluorescently labeled β-cells using an insulin antibody (green). (Similar results were obtained at E14.5 and E18.5; not shown.) Normal proliferation of the pancreatic epithelium is indicated by incorporation of EdU (red). Quantification of EdU-positive cells relative to total cell number (stained with DAPI) at E14.5, E16.5, and E18.5 showed no significant difference in proliferation between knockout and wild-type pancreases (at least three sections were counted from different regions of one pancreas / stage; error bars represent standard error of the mean (SEM)). [Figure 5] Pancreatic gene expression in prenatal IGFR-like receptor KO mice. Real-time qPCR shows modest changes in relative mRNA expression of select genes important for endocrine β-cell differentiation, maturation, proliferation, and function in the knockout pancreas compared to wild-type controls at E18.5. At this stage, embryos are still dependent on maternal nutrition (N=4, error bars represent SEM). Actin was used as a housekeeping gene for normalization. [Figure 6] Changes in pancreatic gene expression in IGFR-like receptor knockout mice after birth, when placental nutrient supply is interrupted and InsR-A is switched to InsR-B. The relative mRNA expression of select genes important for endocrine β-cell differentiation (Foxa2, Pdx1, Pax6, Neurod1, Nkx2-2, Nkx6-1), maturation (MafA, MafB, Ucn3), proliferation (Ccnd1, Ccnd2, Cdk4, Cdkn1a, Cdkn1b), and function (Slc2a2, Slc30a8, Gjd3, Kcnj11, Smarca1, Abcc8) is significantly altered in the knockout pancreas compared with wild-type controls immediately after birth, as demonstrated by real-time qPCR (N = 2, error bars represent SEM). [Figure 7]IGFR-like receptors mediated Akt and AMPK signaling in the pancreas and Min6 mouse insulinoma cells. Western blots of whole pancreatic tissue lysates showed phosphorylation of Akt and AMPK using phospho-specific antibodies (A). A significant number of mutant embryos showed increased Akt (mt2, 3, 5, 6) and AMPK phosphorylation (mt1, 2, 3) (A). Similarly, in (B), knockdown of IGFR-like receptors in Min6 cells resulted in increased Akt phosphorylation, as shown by Western blot. Min6 cells were transfected with siRNA targeting IGFR-like receptor mRNA or scrambled siRNA as a control. [Figure 8] Effect of IGFR-like receptor knockdown on insulin / IGF signaling in Min6 cells. Knockdown of IGFR-like receptors in Min6 mouse insulinoma cells resulted in increased phosphorylation of IR / IGF1R under normal growth conditions, as shown by Western blot analysis using a phospho-specific antibody against IR / IGF1R, and appeared to be independent of starvation conditions. The total levels of IR, IGF1R, and IRS-2 (a downstream adaptor molecule for IR signaling) remained unchanged. Meanwhile, IGFR-like receptors were strongly reduced in the knockdown samples, confirming the efficacy of siRNA knockdown (A). Western blot analysis of downstream signaling molecules, such as AKT and mTOR, which are key molecules commonly phosphorylated as a result of IR activation. Knockdown of IGFR-like receptors in Min6 cells resulted in increased phosphorylation of AKT and mTOR, but not ERK or S6RP. Starvation conditions appear to have no effect on Akt, mTOR, and ERK phosphorylation in IGFR-like receptor knockdown samples compared to controls at the same time points, as indicated by a time-dependent decrease in phospho-S6RP (B). [Figure 9]Localization of IGFR-like receptors in endocrine, ductal, and exocrine cells of the pancreas. Confocal images of E18.5 pancreatic sections stained with antibodies against IGFR-like receptors (green), insulin (red), and E-cadherin (cyan) show immunolocalization of IGFR-like receptors in both the exocrine and endocrine compartments, where they partially colocalize with insulin. Nuclei were stained with DAPI. [Figure 10] Subcellular localization of IGFR-like receptors in Min6 cells. Immunofluorescence images of Min6 cells stained with antibodies against IGFR-like receptors and the Golgi marker GM130 show partial localization of IGFR-like receptors in the Golgi complex, similar to IGF2R. [Figure 11] Localization of IGFR-like receptors upon starvation. Confocal images of Min6 cells immunofluorescently labeled with IGFR-like receptors (red) and GM130 (green) show a redistribution of IGFR-like receptors concentrated in the cis-Golgi region after 2 hours of starvation compared to normal growth conditions. [Figure 12] Localization of AP-2 binding and fractionation signals of IGFR-like receptors. The figure shows the amino acid sequences of SEQ ID NO:11, SEQ ID NO:12, and part of SEQ ID NO:1. [Figure 13] Interaction of IGFR-like receptors with AP2 under starvation conditions. Western blot analysis demonstrates coimmunoprecipitation of the adaptin b subunit (part of the AP2 complex) with IGFR-like receptors in Min6 cells upon starvation, as demonstrated by immunoprecipitation using antibodies against IGFR-like receptors and adaptin b. Virtually no adaptin b was co-precipitated under normal conditions. [Figure 14] IGFR-like receptors, but not InsR, are transported into cells upon nutrient deprivation. Surface biotinylation followed by neutravidin pulldown and Western blot demonstrated a time-dependent decrease in surface expression of IGFR-like receptors, but not InsR, in Min6 cells under starvation conditions. [Figure 15]IGFR-like receptor knockdown likely does not alter the protein expression of autophagy-related proteins under basal conditions. Western blot analysis of autophagy-related molecules shows no apparent changes in expression upon IGFR-like receptor knockdown in Min6 cells under normal growth conditions. [Figure 16] Expression of IGFR-like receptors in mouse diabetic pancreatic islets and exocrine tissues. LSM images of wild-type (<120 mg / dL glucose) and diabetic (>500 mg / dL glucose) adult pancreases stained with antibodies against IGFR-like receptors (red), insulin (cyan), and E-cadherin (green) (A), and IGFR-like receptors (red), glucagon (cyan), and urocortin 3 (green) (B) show increased immunoreactivity of IGFR-like receptor antibodies in the diabetic pancreas in both the endocrine and exocrine compartments. The diabetic state was evidenced by decreased insulin and urocortin 3 staining in the diabetic pancreas. The images on the right show the areas marked with white squares at higher magnification. [Figure 17] Detection of IGFR-like receptors in pancreatic islet cells using antibodies against the intracellular and extracellular domains of IGFR-like receptors. Western blot experiments show specific recognition of a protein band of approximately 130 kDa in islet cell lysates. This recognition is absent in HEK cell lysates by antibodies against the extracellular domain of SEQ ID NO: 4 and 6. By comparison, the same band is specifically recognized by antibodies against the intracellular domain of SEQ ID NO: 2 in wild-type and heterozygous total pancreatic lysates, but not in mutant embryos and islets, and not in HEK cells. [Figure 18] IGF3R expression in human islets of Langerhans. Confocal images of islets from a human donor immunofluorescently labeled with antibodies against IGFR-like receptors (red), IGF2R (M6PR) (green), and insulin (cyan) show typical IGFR-like receptor distribution in the Golgi compartment and partial colocalization with IGF2R and insulin. [Figure 19] KIAA1324 / IGFR-like encoding SNPs in the extracellular domain, CRD and CTD. [Figure 20] Association of KIAA1324 with SNPs with insulin sensitivity (A), adipokines and inflammation (B), and proinsulin conversion (C). [Figure 21] Hypothetical model of IGFR-like receptor function in the insulin pathway. Schematic of a proposed mechanism by which IGFR-like receptors may perform their function. In one hypothesis, IGFR-like receptors may act as modulators of IR signaling and recycling. Upon insulin binding to IR, the assembly of the complex triggers the recruitment of IGFR-like receptors, which then target the complex to early endosomal / lysosomal compartments in a time- and dose-dependent manner. The ligand would be degraded, and IR, along with the IGFR-like receptor, would be shunted to the plasma membrane. Another hypothesis is that binding of insulin ligands to IGFR-like receptors triggers the internalization of the complex and the degradation of insulin in lysosomes, thereby allowing IGFR-like receptors to act as scavenger molecules to remove excess pericellular amounts of insulin released under certain circumstances. Finally, IGFR-like receptors may be involved in the biogenesis and maturation of insulin granules by the correct guidance and trafficking of secretory vesicles. [Figure 22] IGFR-like receptors indicate pancreatic beta cell dysfunction in humans. LSM image of a human pancreas section stained with antibodies against IGFR-like receptors (red) and insulin (green). Nuclear DAPI staining (blue) shows decreased insulin and increased IGFR-like receptor expression in diabetic patients (6.9% HbA1c) compared to non-diabetic patients (4.6% HbA1c). (HbA1c, used as an indicator of diabetic status, is defined as the percentage of glycated hemoglobin (HbA1c) in plasma. Normal range: <5.9%) [Figure 23]IGFR-like receptor knockout and knock-in strategies for studying receptor function in cell culture and mice. (1) Schematic of the CRISPR / Cas9-mediated knockout strategy targeting the core promoter and transcription start site. (2) Schematic of the CRISPR / Cas9-mediated knock-in fusion of the Venus fluorescent reporter by removing the translation stop sequence and fusing Venus in-frame to the IGFR-I open reading frame. The IGFR-I gene is located at 108455694-108536536 on mouse chromosome 3. [Figure 24] Subcellular localization of IGFR-like receptors in wild-type and knockout Min6 cells. Immunofluorescence images of Min6 cells stained with antibodies against IGFR-like receptors (red) and insulin (green). Nuclei were stained with DAPI (blue). [Figure 25] Confirmation of IGFR-like receptor knockout in Min6 cells. (A) Genomic PCR around the transcription start site of IGFR-I shows a homozygous internal deletion of a large fragment. (B) Western blot confirms the IGFR-I null mutation and shows that IGFR-I protein is completely absent in the knockout Min6 clone, confirming the immunocytochemistry results. [Figure 26] IGFR-like receptor knockout in Min6 cells. Western blot analysis of phosphorylated insulin receptor (IR) and downstream signaling molecules, such as AMPK, AKT, and mTOR, which are key molecules commonly phosphorylated as a result of IR activation. IGFR-like receptor knockout in Min6 cells resulted in increased phosphorylation of IR and consequently, increased AMPK phosphorylation, but not Akt or mTor phosphorylation. Min6 control and knockout clones were cultured under growth conditions (10% FCS and high glucose). [Figure 27]IGFR-like Venus fusion knock-in Min6 cell line. IGFR-I was N-terminally fused in frame to the Venus fluorescent reporter. Colocalization of endogenous IGFR-I (anti-IGFR-I, red) and IGFR-I-Venus (Venus detected by anti-GFP, green) is shown in the Golgi and trans-Golgi regions. Small molecule compounds and biologics that inhibit homodimerization and heterodimerization via the transmembrane domain (TM) or cysteine-rich domain (CRD) of growth factor receptors (IPR009030) or that alter subcellular localization in the trans-Golgi, Golgi, lysosomal, and plasma membrane compartments can be identified using a high-content screening approach with this knock-in Min6 cell line. [Figure 28] IGFR-I is strongly upregulated in PDX1+ / NKX6.1+ endocrine precursor cells differentiated from human induced pluripotent stem cells (iPSCs). [Figure 29] Detection of IGFR-like receptors in mouse Min6 insulinoma cells and human MCF7 breast cancer cells using antibodies against the extracellular domain of IGFR-like receptors. Western blot experiments show specific recognition of a protein band of approximately 130 kDa in Min6 (weak) and MCF7 (strong) cell lysates by antibodies against the extracellular domain of SEQ ID NO: 4 (EIG-B 18B2 and 10D9) and SEQ ID NO: 6 (EIG-D 26D7). The EIG-B clone is a monoclonal antibody raised in rats against the peptide of SEQ ID NO: 4. Meanwhile, the EIG-D clone is a monoclonal antibody raised in mice against SEQ ID NO: 6. [Figure 30] IGFR-like receptors were associated with type 2 diabetes in a genome-wide association meta-study. The human KIAA1324 gene, which encodes an IGFR-like receptor and is located on chromosome 1, is associated with type 2 diabetes. Figure shown from reference www.type2diabetesgenetics.com. [Figure 31]SNPs in IGFR-like receptors are highly associated with coronary artery disease, LDL cholesterol, and type 2 diabetes in genome-wide association meta-studies. Zoomed in from 109.55 to 109.85 Mb within human chromosome 1. Blue bars marked by dots indicate a positive association with the disease. Alternatively, red bars without dots indicate a negative association. Bar sizes (none, small, medium, and large) indicate significant differences. Figure taken from reference www.type2diabetesgenetics.com.

[0040] [Detailed explanation] The present inventors surprisingly discovered a novel IGFR-like receptor (IGFR) expressed adjacent to IGF-1, IGF-2, and insulin ligands in the pancreas and demonstrated that this IGFR-like receptor negatively regulates InsR- and / or IGF1R-mediated signaling. Therefore, antagonists and agonists of this receptor open up new and urgently needed possibilities for reversing insulin resistance, a common pathogenesis of diabetes. Meanwhile, agonists could be used to block insulin signaling. Remarkably, the present inventors also demonstrated that IGFR-like receptors are involved in the dedifferentiation of beta cells in the pancreas, an early event in the initiation of disease. This dedifferentiation ultimately leads to the destruction or loss of function of beta cells. Therefore, IGFR-like receptors are also promising diagnostic tools that enable early diagnosis and treatment of diabetes, before irreversible beta cell loss occurs.

[0041] The present inventors have pioneered the elucidation of the function of the human KIAA1324 gene, which encodes the IGFR-like receptor described herein. The protein product of KIAA1324, previously referred to as the UPF0577 protein KIAA1324 or estrogen-inducible gene 121 (EIG121) protein, was generally known as a cancer marker. However, the present inventors are the first to elucidate a central metabolic role for this protein. The terms "IGFR-like receptor," "IGF-like receptor," "IGF3 receptor," and "IGF3R" are used interchangeably herein.

[0042] Thus, in a first aspect, the present invention provides an isolated DNA sequence encoding an IGF receptor (IGFR)-like receptor capable of reacting with an antibody raised against the IGFR-like receptor of SEQ ID NO: 1, wherein said antibody is (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) and specifically binds to at least one epitope comprising:

[0043] In particular, the isolated DNA sequence is contemplated to encode the UPF0577 protein KIAA1324 (SEQ ID NO: 1) of Uniprot Acc. No. Q6UXG2, entry version 95 (July 22, 2015), or a functional variant thereof, which protein is also referred to herein as an "IGFR-like receptor."

[0044] Importantly, while expression of the "EIG121" gene was known in the prior art primarily for its role in tumor growth associated with estrogen excess, it was not, or could not be, predicted that the protein product of that gene, i.e., the IGFR-like receptor described herein, would regulate IGFR / IR signaling and be specifically recognized by an antibody that binds to an epitope comprising a sequence selected from SEQ ID NOs: 1, 2, 3, 4, 5, or 6, or could be targeted by an antagonist or agonist for the treatment of diabetes.

[0045] Also encompassed within the term "IGFR-like receptor" are functional variants of the IGFR-like receptors disclosed herein that have a threshold sequence identity or sequence homology to the IGFR-like receptors disclosed herein. It is contemplated that the functional variants have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the UPF0577 protein KIAA1324 (SEQ ID NO: 1) of Uniprot Acc. No. Q6UXG2, entry version 95 (July 22, 2015), and are capable of reacting with antibodies raised against the IGFR-like receptor of SEQ ID NO: 1, wherein the antibodies have a sequence (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) and specifically binds to at least one epitope comprising:

[0046] It is further assumed that the functional variant preferably exhibits the same properties as the UPF0577 protein KIAA1324, i.e., inhibits or reduces InsR- and / or IGF1R-mediated signal transduction, in particular (i) Akt phosphorylation and / or (ii) AMPK phosphorylation and / or (iii) mTOR phosphorylation, which can be accessed by routine methods shown in the accompanying examples. As used herein, the term "% identity" or "% sequence identity" refers to the percentage of pairwise identical residues in the longer of the two sequences, determined by (homologous) alignment of the sequence of the polypeptide of the present invention with the sequence of interest. The % identity is determined by dividing the number of identical residues by the total number of residues and multiplying by 100. The term "homology" is used herein in its ordinary sense and includes identical amino acids at equivalent positions in the linear amino acid sequences of the two proteins, as well as amino acids that are considered to be conservative substitutions (e.g., replacement of a glutamic acid residue with an aspartic acid residue). Preferably, the amino acid sequence shown in SEQ ID NO: 1 is preferred as a "reference sequence." SEQ ID NO: 1 represents the human UPF0577 protein KIAA1324, also referred to herein as an IGFR-like receptor. The terms "reference sequence" and "wild-type sequence" (of an IGFR-like receptor) are used interchangeably herein. Alternatively, the amino acid sequence of SWISS-PROT / UniProt databank accession number Q6UXG2 (entry version 95 of July 22, 2015) can be used as a reference sequence.

[0047] Percent sequence homology or sequence identity can be determined, for example, herein using BLASTP, version blastp2.2.5 (November 16, 2002, see Altschul, SF et al. (1997) Nucl. Acids Res. 25, 3389-3402). In this embodiment, percent homology is preferably based on an alignment of the entire polypeptide sequence, including the propeptide sequence, in a pairwise comparison using the wild-type protein scaffold (matrix: BLOSUM62; gap cost: 11.1). It is calculated as the percentage of "positive" (homologous amino acids) results in the BLASTP program output divided by the total number of amino acids selected by the program for the alignment.

[0048] In the context of the present invention, the expression "a position corresponding to another position" (e.g., a region, fragment, nucleotide, or amino acid position, etc.) is based on numbering conversion based on nucleotide or amino acid position number and then aligning the sequences in a manner that maximizes percent sequence identity. Not all positions within a given "corresponding region" need be identical, as unmatched positions within a corresponding region can be considered "corresponding positions." Thus, as used herein, reference to an "amino acid position corresponding to amino acid position [X]" of a particular protein sequence represents a reference to the amino acid position of the particular protein sequence as well as to the collection of equivalent positions in other recognized proteins, structural homologs, and families. The same applies mutatis mutandis to the expression "a sequence corresponding to a sequence." That is, reference to a sequence "corresponding to" a particular protein sequence [X] represents a reference to the sequence of the particular protein sequence as well as to the collection of equivalent positions in other recognized proteins, structural homologs, and families.

[0049] The terms "InsR" or "IR" refer to insulin receptor and generally include both the IR-A (also known as the "short isoform") and IR-B (also known as the "long isoform") isoforms. InsR occurs as a tetramer consisting of two α chains containing insulin-binding regions and two β chains containing kinase domains linked by disulfide bonds. InsR is a receptor tyrosine kinase that is activated by the binding of insulin, IGF-1, and IGF-2, ultimately resulting in signal transduction via the MAPK / Ras-Raf-Erk pathway, the phosphatidylinositol-3-kinase / AKT / mTOR (PI3K / AKT) pathway, and / or the Janus kinase / signal transducer and activator of transcription (JAK / STAT) pathway. More precisely, ligand binding to the InsR ectodomain α chain induces a conformational change within the receptor, leading to autophosphorylation of various tyrosine residues within the intracellular tyrosine kinase domain of the β chain, resulting in the recruitment and phosphorylation of multiple intracellular substrates, including insulin receptor substrates (IRS1, 2, 3, and 4), SHC, GAB1, CBL, and other signaling intermediates. Each of these phosphorylated proteins functions as a docking protein for other signaling proteins containing Src-homology-2 domains (SH2 domains), including the p85 regulatory subunits of PI3K and SHP2. Phosphorylation of IRS proteins leads to the activation of two major signaling pathways: the PI3K-AKT / PKB pathway (which is responsible for most of the metabolic effects of insulin) and the Ras-MAPK pathway (which regulates the expression of certain genes and, in cooperation with the PI3K pathway, controls cell proliferation and differentiation). Binding of PI3K to phosphotyrosines in IRS1 and subsequent activation of PI3K leads to the phosphorylation and activation of AKT, AMPK, and mTOR, signaling pathways that regulate metabolism and coordinate insulin signaling. Activation of InsR by ligand binding also triggers the Ras / RAF / MAP2K / MAPK pathway via phosphorylation of IRS1 and recruitment of GRB2 / SOS.This pathway is primarily involved in insulin mediating cell proliferation, survival, and cell differentiation.

[0050] Examples of InsR are human InsR and variants thereof of Uniprot Acc. No. P06213 (entry version 216 of July 22, 2015). The insulin receptor in the context of the present invention is preferably capable of inducing (i) Akt phosphorylation and / or (ii) AMPK phosphorylation and / or (iii) mTOR phosphorylation upon binding of its ligand, in particular insulin.

[0051] The terms "IGF-receptor 1" or "IGF1R" or "IGFRI" are used herein to refer to insulin-like growth factor 1 receptor tyrosine kinase. IGF1R binds IGF1 with high affinity and IGF2 and insulin (INS) with lower affinity. Ligand binding activates the receptor kinase, leading to receptor autophosphorylation and phosphorylation of multiple substrates, including insulin receptor substrates (IRS1 / 2), Shc, and 14-3-3 proteins, ultimately resulting in the activation of three major signaling pathways: the PI3K-AKT / PKB pathway, the Ras-MAPK pathway, and the JAK / STAT pathway. Activated IGF1R is involved in regulating cell proliferation and survival. Thus, InsR and IGF1R are recruited into similar signaling pathways, but InsR-mediated signaling preferentially regulates metabolism, while IGF1R signaling is involved in cell proliferation and survival.

[0052] Examples of IGF1R are human IGF1R and variants thereof of Uniprot Acc. No. P08069 (entry version 185 of July 22, 2015). IGF1R in the context of the present invention is preferably capable of inducing (i) Akt phosphorylation and / or (ii) AMPK phosphorylation and / or (iii) mTOR phosphorylation upon binding of its ligand, in particular IGF1.

[0053] The term "IGF1" or "IGF1" refers to insulin-like growth factor I, a protein structurally and functionally related to insulin, but with greater growth-promoting activity. An example is human IGF1, Uniprot Acc. No. P05019 (entry version 186 of July 22, 2015). "IGF1" in the context of the present invention is preferably capable of binding to the IGF1 receptor and triggering IGF1R signaling as described elsewhere herein.

[0054] The terms "IGF2R" or "IGF2R" or "IGFRII" are used herein to refer to the insulin-like growth factor 2 / mannose-6-phosphate (IGF-2 / M6P) receptor. IGF2R is a single transmembrane protein composed of a large extracytoplasmic (i.e., extracellular) domain, a single transmembrane region, and a short cytoplasmic tail that lacks intrinsic catalytic activity. The receptor binds IGF-2 with higher affinity than IGF-1, but not insulin. IGF2R has been reported to interact with lysosomal enzymes and various other M6P-containing ligands through distinct sites, modulating signaling through the growth-stimulatory IGF-1 receptor pathway by regulating extracellular IGF-2 concentrations.

[0055] An example of an IGF2R is the human IGF2R of Uniprot Acc. No. P11717 (entry version 174 of July 22, 2015) and variants thereof.

[0056] The term "IGF2" or "IGF II" refers to insulin-like growth factor II. An example is human IGF2 and its variants of Uniprot Acc. No. P01344 (entry version 199 of July 22, 2015). "IGF2" in the context of the present invention is preferably capable of binding to the IGF2 receptor.

[0057] The isolated DNA sequence provided herein may include a sequence corresponding to the sequence of the human KIAA1324 gene set forth in SEQ ID NO:7 (NCBI Gene ID 57535, updated July 15, 2015) or a variant thereof. A variant of the human KIAA1324 gene may include an ortholog. An ortholog or orthologous gene is a gene that has a portion of its sequence similar to a portion of a known gene, but has a sequence found in a different species than the known gene. Orthologs and known genes are derived from a common ancestral gene by vertical inheritance.

[0058] As used herein, a variant or ortholog of the human KIAA1324 gene is intended to encode an IGFR-like receptor or a functional variant thereof, i.e., preferably capable of inhibiting or reducing InsR- and / or IGF1R-mediated signaling, in particular (i) Akt phosphorylation and / or (ii) AMPK phosphorylation and / or (iii) mTOR phosphorylation, and to have at least about 60%, 65%, 70%, 75%, 80%, 90%, 95%, 97%, 98%, 99%, 99.5%, or 100% sequence identity to the human KIAA1324 gene (NCBI Gene ID 57535, updated July 15, 2015), i.e., sequence identity to the coding sequence of the human KIAA1324 gene set forth in SEQ ID NO: 7. The coding sequence of the human KIAA1324 gene (NG_032763.1) is identified as follows: nucleotides 222..374, 47932..48052, 50537..50729, 57904..58051, 58526..58606, 59512..59617, 59726..59875, 71083..71171, 74215..74392, 75104..75232, 75304..75306, 75308..75308, 75309..75309 ... This is obtained by combining 5630..75720, 77404..77509, 77764..77901, 78649..78912, 80459..80632, 83512..83692, 84017..84113, 84611..84712, 85892..86018, 86097..86275, 86773..86938, 88982..89050.

[0059] As used herein, the term "isolated DNA sequence" means a DNA molecule that has been purified or substantially purified from endogenous materials, including other nucleic acid sequences, proteins, peptides, lipids, etc., that are naturally present in the cell and / or organism from which the DNA sequence is obtained, and includes DNA purified by standard purification techniques as well as DNA prepared by recombinant techniques and chemically synthesized DNA.

[0060] vector The nucleic acids of the invention can be in the form of, present in and / or part of a vector.

[0061] The term "vector" refers to a nucleic acid molecule used as a vehicle for transporting (exogenous) genetic material into a host cell, and includes, but is not limited to, plasmids, viruses, cosmids, and artificial chromosomes, such as bacterial artificial chromosomes (BACs) and yeast artificial chromosomes (YACs). Engineered vectors typically contain an origin of replication, a multiple cloning site, and a selectable marker. The vector itself is typically a nucleotide sequence, typically a DNA sequence, that contains an insert (transgene) and a larger sequence that functions as the "backbone" of the vector. In addition to the transgene insert and backbone, a vector can contain additional elements, including gene regulatory elements, genetic markers, antibiotic resistance, reporter genes, targeting sequences, or protein purification tags. Expression vectors (expression constructs) for expressing a transgene in a host cell are particularly contemplated within the context of the present invention. These expression vectors typically contain gene regulatory sequences in addition to the transgene.

[0062] An expression vector is generally a vector that is capable of providing expression of an IGFR-like receptor in vitro and / or in vivo (i.e., in an appropriate host cell, host organism, and / or expression system). One of skill in the art will readily appreciate that the selection of a particular vector will depend, for example, on the host cell, the intended copy number of the vector, whether transient or stable expression of the IGFR-like receptor is envisioned, etc.

[0063] "Transient expression" occurs by the introduction of a nucleic acid (e.g., a linear or non-linear DNA or RNA molecule) or vector that is not capable of autonomous replication in a recipient host cell. Expression of the transgene occurs by the transient expression of the introduced sequence.

[0064] However, "stable expression" of the nucleic acid sequences described herein will often be preferred and can be achieved either by stably integrating the nucleic acid sequence into the genome of the host cell or by introducing a vector comprising a nucleic acid sequence of the invention and capable of autonomous replication within the host cell.

[0065] It is specifically contemplated that the vectors provided herein contain genetic regulatory elements operably linked to the DNA sequence encoding the IGFR-like receptor.

[0066] The term "genetic regulatory element" refers to a DNA sequence essential for the expression of an operably linked coding sequence in a particular host organism. The term "genetic regulatory element" includes control transcriptional promoters, operators, enhancers, silencers, transcription terminators, 5' and 3' untranslated regions that interact with host cell proteins to effect transcription and translation, and other elements (including initiation and termination codons) that can control gene expression. The exact nature of the regulatory regions required for gene expression can vary between organisms. Genetic regulatory elements in prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site (RBS). In contrast, genetic regulatory elements for eukaryotic cells include a promoter, a polyadenylation (polyA) signal, and an enhancer.

[0067] A genetic regulatory element is considered to be "operably linked" to a gene to be expressed, i.e., in a functional relationship with the gene. For example, a promoter or enhancer is "operably linked" to a coding nucleic acid sequence if it affects the transcription of the sequence. "Operably linked" DNA sequences can be contiguous or non-contiguous. Linking is typically accomplished by ligation at convenient restriction sites or by synthetic oligonucleotide adapters or linkers.

[0068] host cells Further provided herein is a host cell comprising the vectors described herein.

[0069] A variety of host cells can be utilized to express the nucleic acid sequences encoding the IGFR-like receptors described herein. Host cells can be prepared using genetic engineering methods known in the art. The method of introducing a vector into a recipient host cell is hereinafter also referred to as "transformation" or "transfection." These terms are used interchangeably herein.

[0070] Transformation of host cells typically involves the creation of transient pores or "holes" in the cell wall and / or membrane to allow for the uptake of materials. Illustrative transformation protocols include the use of calcium phosphate, electroporation, cell squeezing, dendrimers, liposomes, cationic polymers such as DEAE-dextran or polyethyleneimine, sonoporation, optical transfection, impalefection, nanoparticles (gene guns), magnetofection, particle bombardment, alkali cations (cesium, lithium), enzymatic digestion, shaking with glass beads, viral vectors, etc. The choice of method generally depends on the type of cell to be transformed, the vector to be introduced into the cell, and the conditions under which the transformation will be carried out.

[0071] As used herein, the term "host cell" refers to any cell or cell culture that can serve as a recipient for a vector or isolated nucleic acid sequence encoding an IGFR-like receptor described herein. Suitable host cells include prokaryotic or eukaryotic cells, including, but not limited to, bacteria, yeast cells, fungal cells, plant cells, and animal cells, e.g., insect cells and mammalian cells, e.g., mouse, rat, macaque, or human cells.

[0072] For example, IGFR-like receptors can be produced in bacteria. In addition to prokaryotes, eukaryotic microbes such as filamentous fungi or yeast are suitable cloning or expression hosts for the IGFR-like receptors of the present invention. Examples include Saccharomyces cerevisiae, Schizosaccharomyces pombe, Kluyveromyces hosts such as K. lactis, K. fragilis (ATCC 12424), K. bulgaricus (ATCC 16045), K. wickeramii (ATCC 24178), K. waltii (ATCC 56500), K. drosophilarum (ATCC 36906), K. thermotolerans, and K. marxianus; yarrowia (EP 402226); Pichia pastoris (EP 183070); Candida; Trichoderma reesia (EP 244234); Neurospora crassa; Schwanniomyces, e.g., Schwanniomyces occidentalis; and filamentous fungi such as Neurospora, Penicillium, Tolypocladium, and Aspergillus hosts such as A. nidulans and A. niger.

[0073] Suitable host cells for expressing the glycosylated antibody constructs of the present invention can also be obtained from multicellular organisms. Examples of invertebrate cells include plant and insect cells. Numerous baculovirus strains and variants have been identified, along with corresponding optional insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Drosophila melanogaster (fruit fly), and Bombyx mori. Various virus strains for transfection, such as the L-1 variant of Autographa californica NPV and the Bm-5 strain of Bombyx mori NPV, are publicly available.

[0074] Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, Arabidopsis, and tobacco can also be used as hosts. Cloning and expression vectors useful for protein production in plant cell culture are known to those of skill in the art.

[0075] Examples of useful mammalian cell lines include SV40-transformed monkey kidney CV1 line (COS-7, ATCC CRL1651); human embryonic kidney line (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL10); Chinese hamster ovary cells / -DHFR (CHO), mouse Sertoli cells (TM4); monkey kidney cells (CVI ATCC CCL70); African green monkey kidney cells (VERO-76, ATCC CRL1587); human cervical carcinoma cells (HELA, ATCC CCL2); canine kidney cells (MDCK, ATCC CCL34); Buffalo rat liver cells (BRL 3A, ATCC CRL1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, 1413 8065); mouse mammary carcinoma (MMT 060562, ATCC CCL5 1); TRI cells; MRC5 cells; FS4 cells; and human hepatoma cells (Hep G2).

[0076] Ligand The inventors have identified several domains in the IGFR-like receptors described herein. The IGFR-like receptors may have binding sites for potential ligands. These binding sites can serve as templates for providing, inter alia, the diagnostic binding agents and antagonists or agonists of the present invention. The domains include two insulin-like growth factor-binding domains (Domain 1: amino acids 273-413 of SEQ ID NO:1; Domain 2: amino acids 579-659 of SEQ ID NO:1), a mannose-6-phosphate receptor-binding domain (M6P domain, amino acids 655-857 of SEQ ID NO:1), a transmembrane domain (amino acids 908-930 of SEQ ID NO:1), and a cytoplasmic domain (amino acids 932-1013 of SEQ ID NO:1). The domains can also be derived from Figure 1. Thus, the present invention also relates to binding agents, such as antibodies (preferably monoclonal antibodies), that specifically bind to at least one of the above domains.

[0077] Proteins containing insulin-like growth factor binding domains (InterPro Acc. No. IPR009030) are also known to bind to insulin-like growth factor binding proteins (IGFBP1-6), type 1 insulin-like growth factor receptor (IGF-1R), receptor protein tyrosine kinases Erbb-2, Erbb-3, and Erbb-4 (ErbB2, -3, -4), ephrin type A / B receptors, epidermal growth factor receptor (EGFR), EGFR-like binding proteins, CYR61, matrilin-2, -3, and -4, delta-like protein 1, cubilin, slit homolog 1 and 3 proteins, and multiple epidermal growth factor-like domains. Main protein 6, low-density lipoprotein receptor-associated protein 4, WNT1-inducible signaling pathway protein 2, WNT1-inducible signaling pathway protein 1, WNT1-inducible signaling pathway protein 3, EGF-containing fibulin-like extracellular matrix protein 2, low-density lipoprotein receptor, epidermal growth-promoting factor, complement component C9, thrombomodulin, vitamin K-dependent protein S, complement component C8 alpha chain, uromodulin, furin, bone morphogenetic protein 1, nidogen-1, insulin receptor-associated protein Protein, fibulin-1 and -2, proprotein convertase subtilisin / kexin type 6, connective tissue growth factor, fibrillin-1, -2, and -3, neurogenic locus notch homolog protein 1, protein NOV homolog, CD97 antigen, cartilage oligomeric matrix protein, keratin, type II keratinocyte Hb3, protein Jagged-1, protein Crumb homolog 1, serine protease HTRA4, serine protease HTRA3, low-density lipoprotein receptor-related protein 2, neurogenic locus notch homolog protein Protein 2, tumor necrosis factor receptor superfamily member 9, pro-low density lipoprotein receptor-related protein 1, EGF-containing fibulin-like extracellular matrix protein 1, nidogen-2, scavenger receptor class F member 1, adhesion G protein-coupled receptor E1, growth arrest-specific protein 6, keratin, type I keratinocyte Ha2, latent transforming growth factor beta-binding protein 1, latent transforming growth factor beta-binding protein 2, R-spondin-1, -2, and -4, Sushi, von Willebrand factor type A,EGF and pentraxin domain-containing protein 1, uromodulin-like 1, mechelin, protein ishat homolog, proprotein convertase subtilisin / kexin type 4, Sushi domain-containing protein 1, cysteine-rich containing EGF-like domain protein 2, nephronectin, protocadherin Fat4, BMP / retinoic acid-inducible neuron-specific protein 3, extracellular matrix protein FRAS1, epidermal growth factor-like protein 6, signal peptide CUB and EGF-like domain-containing protein 1, signal peptide CUB and EGF-like domain-containing protein 3, latent transforming growth factor beta-binding protein 4, hemicentin-2, delta and notch-like epithelial growth factor Growth factor-related receptor, insulin-like growth factor binding protein-like 1, serine protease HTRA1, proprotein convertase subtilisin / kexin type 5, protein kinase C-binding protein NELL1, von Willebrand factor C, and EGF domain-containing protein, scavenger receptor class F member 2, cysteine-rich-containing EGF-like domain protein 1, Kazal-type serine protease inhibitor domain-containing protein 1, hemicentin-1, protein kinase C-binding protein NELL2, neurogenic locus notch homolog protein 4, R-spondin-3, adhesion G protein-coupled receptor E3, mucin-13, endosialin, cadherin EGF These include, but are not limited to, LAG 7-transmembrane G-type receptor 2, complement component C1q receptor, endothelial cell-specific molecule 1, signal peptide CUB and EGF-like domain-containing protein 2, delta-like protein 4, latent transforming growth factor beta-binding protein 3, delta-like protein 3, cadherin EGF LAG 7-transmembrane G-type receptor 1, low-density lipoprotein receptor-related protein 1B, cysteine-rich motor neuron 1 protein, fibulin-5, epidermal growth factor-like protein 7, adhesion G protein-coupled receptor E2, neurogenic locus Notch homolog protein 3, protein Jagged-2, and the like.

[0078] Thus, potential ligands of the IGFR-like receptors described herein include ligands of the aforementioned proteins, such as IR, IGF1R, insulin, IGF1, IGF2, ephrin-B1, ephrin-B2, EGF, EGFR, TGFA / TGF-alpha, amphiregulin, epigen / EPGN, BTC / betacellulin, epiregulin / EREG, HBEGF / heparin-binding EGF, GP30, ALB, MB, kappa and lambda light chains, TF, hemoglobin, These include GC, SCGB1A1, APOA1, high density lipoprotein, GIF-cobalamin complex, LRP2, LGALS3, AGRIN, IL-1, IL-2, TNF, collagen I and IV, perlecan, laminin, heparin, integrins, fibronectin, protein C, EFNA5, EFNB1, EFNB2, EFNB3, Jagged 1, Jagged 2, and Delta 1, neuregulin, NTAK, CSPG5, TNFSF9 / 4-1BBL, Ac-LDL, AXL, TYRO3 MER, NRG1, NRG2, NRG3, NRG4, BTC, EREG, HBEGF, GR4, LGR5, LGR6, C1q, mannose-binding lectin (MBL2), pulmonary surfactant protein A (SPA), TGFBR2, and fragments and variants thereof.

[0079] Proteins containing a mannose-6-phosphate receptor binding domain (InterPro Acc. No. IPR009011) include, but are not limited to, cation-independent mannose-6-phosphate receptor, glucosidase 2 subunit beta, cation-dependent mannose-6-phosphate receptor, protein OS-9, endoplasmic reticulum lectin 1, and N-acetylglucosamine-1-phosphotransferase subunit gamma.

[0080] Thus, potential ligands for IGFR-like receptors provided herein include ligands of the aforementioned proteins, such as IGF2, DPP4, phosphomannosyl, TRPV4, IGF2, lysosomal enzymes, TGFβ, leukemia inhibitory factor (LIF), proliferin, thyroglobulin, prorenin, granzyme B, and retonic acid.

[0081] The present inventors found that knockdown of IGFR-like receptors resulted in increased phosphorylation of IGF1R and IR, as well as the downstream signaling proteins Akt, mTOR, and AMPK. Therefore, the present inventors propose that IGFR-like receptors can inhibit or attenuate the activation and / or downstream signaling of IGF1R and / or IR. Without being bound by any particular theory, it is believed that IGFR-like receptors may function as "insulin scavenger" receptors, for example, depleting insulin from the blood and transporting it to endosomal and lysosomal compartments, where it may be degraded. It is also speculated that IGFR-like receptors may associate with the insulin receptor (InsR) and remove it from the cell surface. In any event, both scenarios can explain the reduction of InsR activation and InsR- and / or IGF1R-mediated signaling (including phosphorylation of InsR, Akt, mTOR, and / or AMPK) in the presence of a functional IGFR-like receptor. Thus, insulin, InsR, and / or the insulin-IR complex are specifically contemplated as ligands for the IGFR-like receptors provided herein.

[0082] Antagonists and Agonists Additionally, agonists and antagonists of the IGFR-like receptors described herein are provided. As described herein and illustrated in the accompanying Examples, IGFR-like receptors have been found to negatively regulate, i.e., inhibit or decrease, InsR- and / or IGF1R-mediated signaling, and in particular, (i) Akt phosphorylation, (ii) AMPK phosphorylation, and (iii) mTOR phosphorylation.

[0083] The term "antagonist" refers to a receptor ligand that inhibits or diminishes an agonist-mediated biological response rather than eliciting a biological response itself upon binding to the receptor. Antagonists have affinity for the receptor but essentially no efficacy. This term also includes antagonists that bind to the receptor's active (orthosteric) or allosteric sites and / or other binding sites not normally involved in receptor function. The term "antagonist" generally encompasses full and partial antagonists, reversible and irreversible antagonists. According to the present invention, antagonists preferably specifically bind to IGFR-like receptors. Herein, a novel function of the UPF0577 protein KIAA1324 has been elucidated. Those skilled in the art will readily be able to identify antagonists of IGFR-like receptors, for example, by applying the screening assays described herein. As discussed above, IGFR-like receptor antagonists are contemplated to abolish receptor function and interfere with, i.e., inhibit or reduce, InsR and / or IGFR signaling, and can be particularly useful, for example, in reversing insulin resistance. Specifically, IGFR-like receptor antagonists are contemplated to increase InsR- and / or IGF1R-mediated signaling, and in particular, phosphorylation of IGF1R, InsR, Akt, mTOR, and / or AMPK, which can be determined using routine methods known in the art and described in the accompanying Examples.

[0084] As used herein, the term "agonist" generally refers to a receptor ligand that activates a receptor upon binding, resulting in a biological response. In contrast to an antagonist, an agonist has both affinity and efficacy for its receptor. The term "agonist" generally includes full and partial agonists, reversible and irreversible agonists. According to the present invention, an agonist preferably specifically binds to an IGFR-like receptor. As mentioned above, an agonist of an IGFR-like receptor is expected to cause or enhance a biological response of the IGFR-like receptor, i.e., to reduce and / or inhibit InsR activation and downstream signaling. It is therefore contemplated that IGFR-like agonists may reduce InsR- and / or IGF1R-mediated signaling, and in particular, phosphorylation of InsR, IGF1R, Akt, mTOR, and / or AMPK, which may be determined using routine methods known in the art and described in the accompanying examples.

[0085] An "antagonist" or "agonist" of the present invention can generally be any molecule, e.g., an antibody, siRNA, nucleic acid, aptamer, peptide, protein, or small organic compound, that binds to or specifically binds to an IGFR-like receptor identified herein or a variant or fragment thereof and either blocks or reduces a biological response mediated by the IGFR-like receptor (i.e., acts as an antagonist) or elicits or enhances a biological response mediated by the IGFR-like receptor (i.e., acts as an agonist).

[0086] Agonists and antagonists of IGFR-like receptors can be readily discovered, for example, using the screening assays provided herein. Those skilled in the art will readily recognize that, for example, protein ligands containing an insulin-like growth factor binding domain and / or a mannose-6-phosphate receptor binding domain (exemplary proteins and ligands are described in the "Ligands" section above) can be used as templates for preparing agents capable of binding to IGFR-like receptors and exhibiting agonistic or antagonistic activity. For example, in the case of protein or peptide ligands, variants and fragments thereof can be readily prepared using routine methods of genetic engineering. It is envisioned that the agonists and antagonists of the present invention specifically bind to the IGFR-like receptors described herein (i.e., preferably do not exhibit cross-reactivity with targets other than IGFR-like receptors), as can be readily tested, for example, by assessing antibody binding in IGFR-like receptor knockdown host cells (see the accompanying Examples).

[0087] antibody The antagonists or agonists provided herein can be antibodies. The antibodies provided herein preferably exhibit the desired biological activity, i.e., specifically bind to an IGFR-like receptor described herein. Specifically, it is contemplated that the antagonistic antibodies of the present invention can increase InsR- and / or IGF1R-mediated signaling, and in particular, phosphorylation of IGF1R, InsR, Akt, mTOR, and / or AMPK, which can be confirmed using routine methods known in the art and described in the accompanying Examples. It is also contemplated that the antagonistic antibodies of the present invention bind to the epitopes (SEQ ID NOS: 2-6). Thus, the present invention also relates to antibodies that (a) specifically bind to at least one epitope set forth in SEQ ID NOS: 2-6 and (b) increase phosphorylation of IGF1R, InsR, Akt, mTOR, and / or AMPK. Thus, "increases" indicates that the respective signal in the presence of the antibody is increased compared to the absence of the antibody in each detection method used to detect and / or quantify said increase. Furthermore, the present invention relates to antibodies that (a) bind to one of the aforementioned growth factor binding domains and / or mannose-6-phosphate receptor binding domains (shown in FIG. 1 ) and (b) increase phosphorylation of IGF1R, InsR, Akt, mTOR, and / or AMPK. Thus, "increases" indicates that the respective signal in the presence of the antibody is increased compared to the absence of the antibody in each detection method used to detect and / or quantify said increase. Notably, IGFR-I contains an extracellular domain that shows high similarity to the HER2 dimerization region, which is targeted by the antibody trastuzumab (Herceptin®). HER2 is constitutively active, acts in a ligand-independent manner, and can dimerize with other HER family members, targeting HER2 by trastuzumab, blocking its activity and inhibiting cancer growth. Furthermore, IGFR-I contains a GxxxG dimerization motif in its transmembrane domain, which is found in EGFR family members.This suggests that IGFR-I homo- and heterodimerizes with EGFR and IR family members to regulate metabolic versus mitogenic cell signaling outcomes. Therefore, it is anticipated that antibodies of the present invention targeting IGFR-like receptors may also interfere with receptor homo- and heterodimerization and / or ligand binding.

[0088] Methods for producing such antibodies are well known to those skilled in the art (e.g., WO 2014 / 124020) and are exemplified below. The IGFR-I ectodomain and full-length receptor can be highly purified using mammalian expression systems to produce receptors that are fully N-glycosylated and correctly folded in their native conformation. Receptor reconstitution in synthetic membranes not only allows receptor dimerization within a lipid bilayer, but also provides the receptor as an antigen in a membrane environment, making it highly suitable for producing antibodies of the present invention, and in particular therapeutic antibodies of the present invention. Because IGFR-I proteoliposomes are synthetic mimics of cell membranes, they are ideal for producing, for example, monoclonal antibodies. These antibodies are directed only against the extracellular domain of the receptor and can interfere with receptor homo- and heterodimerization and / or ligand binding. It is therefore also envisaged that the antibodies of the present invention are capable of binding to glycosylated IGFR-like receptors, preferably to their glycosylated ectodomains.

[0089] As is well known in the art, an antibody is an immunoglobulin molecule capable of specifically binding to a target (epitope) via at least one epitope recognition site located in the variable region of the immunoglobulin molecule. As used herein, the term "antibody" includes monoclonal and polyclonal antibodies, as well as fragments or variants thereof (natural or synthetic). These fragments and variants include fusion proteins comprising an antibody portion containing an antigen-binding fragment of the required specificity and any other modified antibody configuration containing an antigen-binding site or fragment (epitope recognition site) of the required specificity. Examples include dAbs, nanobodies, affibodies, Fab, Fab', F(ab'), Fv, single-chain Fv (scFv), diabodies, and minibodies comprising an scFv linked to a CH3 domain. It will be understood that other antibody frameworks or scaffolds containing an "antigen-binding site" can be utilized in accordance with the present invention. Therefore, the term "antibody" also encompasses these scaffolds. The scaffolds mentioned include, for example, non-immunoglobulin antibodies and scaffolds onto which the CDRs of an antibody can be grafted. Such scaffolds include, for example, anticalins, avimers, affilins, etc.

[0090] The antibody can be a chimeric antibody (or antigen-binding variant or fragment thereof). The term "chimeric antibody" refers to antibodies in which a portion of the heavy and / or light chain is identical to or homologous to corresponding sequences in antibodies from a particular species or belongs to a particular antibody class or subclass, while the remainder of the chain is identical to or homologous to corresponding sequences in antibodies from another species or belongs to another antibody class or subclass, and to fragments of such antibodies.

[0091] The antibody can be a humanized antibody (or an antigen-binding variant or fragment thereof). The term "humanized antibody" refers to an antibody that contains minimal sequence derived from a non-human antibody. Generally, humanized antibodies are human immunoglobulins comprising residues from a hypervariable region of an immunoglobulin obtained from a non-human species, such as mouse, rat, rabbit, or non-human primate (donor antibody), grafted into a human immunoglobulin (the "recipient antibody"). In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in either the recipient antibody or the donor antibody. These modifications can further refine antibody performance. Generally, a humanized antibody will comprise substantially at least one, and typically all, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.

[0092] The antibody can be a human antibody. A "human antibody" is one having an amino acid sequence corresponding to that of an antibody produced by a human and / or prepared using any technique for preparing human antibodies. This definition of a human antibody specifically excludes humanized antibodies containing non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, including phage display libraries (Hoogenboom and Winter, J. Mol Biol, 227:381 (1991); Marks et al., J. Mol Biol, 222:581 (1991)). For the preparation of human monoclonal antibodies, the methods described in Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol, 147(1):86-95 (1991) can also be used. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol, 5: 368-374 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse (see, e.g., U.S. Pat. Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology), which have been engineered to produce such antibodies in response to antigen challenge but whose endogenous gene loci have been disabled. See also, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006), for human antibodies produced by human B cell hybridoma technology.

[0093] Chemical modification Antibodies or antigen-binding variants or fragments thereof used in accordance with the present invention can be modified. Exemplary modifications contemplated in the context of the present invention include, for example, chemical modifications described below.

[0094] Generally, all types of modifications are contemplated so long as they do not destroy the ability of the antibody or antigen-binding variant or fragment thereof to specifically bind to an IGFR-like receptor and act as an antagonist or agonist of the IGFR-like receptor as described elsewhere herein.

[0095] Possible chemical modifications of antibodies or antigen-binding variants or fragments thereof include acylation or acetylation of the amino terminus, or amidation or esterification of the carboxy terminus, or both. Modifications can also be made to the amino group in the side chain of lysine or the hydroxyl group of threonine. Other suitable modifications include, for example, extension of amino groups by polypeptide chains of variable length (e.g., XTEN technology or PASylation®), N-glycosylation, O-glycosylation, and chemical attachment of carbohydrates, such as hydroxyethyl starch (e.g., HESylation®) or polysialic acid (e.g., PolyXen® technology). Chemical modifications, such as alkylation (e.g., methylation, propylation, butylation), arylation, and etherification, may be possible and are also contemplated.

[0096] As described elsewhere herein, multiple domains have been identified in the IGFR-like receptors of the present invention. Antibodies that act as antagonists or agonists of IGFR-like receptors are, in principle, contemplated to bind, preferably, to any location in or between the extracellular domains of IGFR-like receptors (e.g., the insulin-like growth factor binding domain, the mannose-6-phosphate receptor binding domain). Also, known antibodies that bind to proteins containing one or more of these domains (exemplary proteins are listed in the "Ligands" section above), and specifically to epitopes in the insulin-like growth factor binding domain or the mannose-6-phosphate receptor binding domain of such proteins, are generally contemplated as antagonists or agonists, and their antagonistic or agonistic behavior can be readily monitored in the screening assays provided herein.

[0097] Additionally, antibodies to IGFR-like receptors can be prepared as described in the accompanying Examples and tested for their agonistic or antagonistic activity in the screening assays provided herein.

[0098] The antibodies provided as antagonists or agonists according to the invention may in particular be those having the sequence (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) It is assumed that the antibody can bind to an epitope of an IGFR-like receptor, including

[0099] The antibodies preferably bind specifically to the IGFR-like receptors described herein, i.e., do not exhibit cross-reactivity with non-target molecules, such as InsR, IGF1R, and IGF2R. Specific binding of antibodies recognizing epitopes containing SEQ ID NOs: 2, 3, 4, 5, and 6 is demonstrated in the accompanying examples. For example, it was demonstrated that antibody binding was abolished by knockdown of the IGFR-like receptor, indicating that non-specific binding did not occur.

[0100] The term "epitope" generally refers to a site on an antigen to which a binding domain, such as an antibody or immunoglobulin, or a derivative or fragment of an antibody or immunoglobulin, specifically binds. An "epitope" is antigenic. Therefore, the term epitope is sometimes referred to herein as an "antigen structure" or "antigenic determinant." Thus, a binding domain is an "antigen interaction site." The binding / interaction is also understood to define "specific recognition." The term "epitope" encompasses linear epitopes and conformational epitopes. A linear epitope is a contiguous epitope contained in a primary sequence of amino acids, typically comprising at least three or at least four, and more usually at least five, at least six, or at least seven, e.g., about 8 to about 10, amino acids in a unique sequence. A conformational epitope is formed by non-contiguous amino acids juxtaposed by protein folding. Methods of determining epitope conformation include, but are not limited to, x-ray crystallography, two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy, and site-directed spin labeling and electron paramagnetic resonance (EPR) spectroscopy.

[0101] Other molecules are also contemplated herein as antagonists or agonists of IGFR-like receptors.

[0102] siRNAs and nucleic acids are particularly useful as IGFR-like receptor antagonists that reduce or inhibit the expression of the human KIAA1324 gene (or its mutants or orthologs). The term "siRNA" is used interchangeably with "small interfering RNA" or "silencing RNA." siRNAs are typically double-stranded "antisense" RNA molecules that contain a sequence of at least 20 consecutive nucleotides that has at least 95% sequence identity to the complementary strand of the sequence of a target nucleic acid, e.g., the coding sequence of the human KIAA1324 gene, but may also be directed against the regulatory sequences of the gene (including the promoter sequence and transcription termination and polyadenylation signals).

[0103] Other nucleic acids capable of reducing and / or inhibiting the expression of IGFR-like receptors include aptamers, Spiegelmers®, nc-RNA (antisense RNA, L-RNA Spiegelmer), silencer RNA, microRNA (miRNA), short hairpin RNA (shRNA), small interfering RNA (siRNA), repeat-associated small interfering RNA (rasiRNA), and molecules or RNAs that interact with Piwi proteins (piRNA). Such non-coding nucleic acid molecules can be used, for example, to target IGFR-like receptor mRNA for degradation or to disrupt IGFR-like receptor mRNA translation. The individual reactants, in particular siRNA molecules, can in principle be synthesized directly in the host cell or can be introduced into the host cell.

[0104] Peptides and proteins can generally be used as antagonists or agonists of IGFR-like receptors, depending on whether they suppress (antagonists) or induce (agonists) biological responses mediated by IGFR-like receptor signaling. The terms "polypeptide" and "protein" are used interchangeably herein. Proteins and peptides are expected to specifically bind to IGFR-like receptors. As previously indicated herein, those skilled in the art can easily find peptide and protein antagonists or agonists capable of specifically binding to IGFR-like receptors, for example, using known ligands of proteins containing insulin-like growth factor binding domains and / or mannose-6-phosphate receptor binding domains (exemplary proteins are listed in the "Ligands" section above). The ligands can be used as templates for preparing mutants or fragments thereof using known methods of genetic engineering. The proteins or peptides can then be tested for their agonistic or antagonistic activity, for example, using the screening assays provided herein. Small organic molecules can also act as either antagonists or agonists of IGFR-like receptors. It is assumed that small organic molecules specifically bind to IGFR-like receptors. High-throughput screening assays for small organic molecules are readily available in the art and can be used to find ligands of the IGFR-like receptors provided herein that may exhibit agonistic or antagonistic activity.

[0105] specific binding As provided herein, specific binding of binding agents to IGFR-like receptors is preferred, particularly the antagonists and agonists provided herein, e.g., antibodies. The terms "bind to" and "recognize," in all grammatical forms, are used interchangeably herein.

[0106] The term "specifically binds" generally indicates that a binding agent, particularly an antagonist or agonist, e.g., an antibody, binds with higher affinity to its intended target (i.e., an IGFR-like receptor described herein) than to its non-target molecule. Non-target molecules include IGF receptors, particularly human IGF1R of Uniprot Acc. No. P08069 (entry version 185 of July 22, 2015), human IGF2R of Uniprot Acc. No. P11717 (entry version 174 of July 22, 2015), and human InsR of Uniprot Acc. No. P06213 (entry version 216 of July 22, 2015), and functional variants thereof. Preferably, the affinity of an agonist or antagonist will be at least about 5-fold, preferably 10-fold, more preferably 25-fold, even more preferably 50-fold, and most preferably 100-fold or more, greater for the target molecule than its affinity for a non-target molecule. Preferred antibodies have an affinity of at least about 10 7 M -1 , and preferably about 10 8 M -1 ~about 10 9 M -1 , about 10 9 M -1 ~about 10 10 M -1 , or about 10 10 M -1 ~about 10 12 M -1 It binds with an affinity of

[0107] Thus, preferably, the term "specifically binds" indicates that antagonists and agonists, eg, antibodies, bind exclusively to their intended target (ie, an IGFR-like receptor).

[0108] treatment It is anticipated that antagonists and agonists of IGFR-like receptors will be particularly useful in the treatment and diagnosis of diabetes. As used herein, "diabetes" refers to a broad category of disorders characterized by impaired insulin production and glucose tolerance, and generally includes type 1 and type 2 diabetes (also called juvenile and adult-onset, respectively), gestational diabetes, prediabetes, insulin resistance, metabolic syndrome, and impaired glucose tolerance. Diabetes results from the dysfunction or impairment of insulin-producing beta cells, alone or in combination with insulin resistance.

[0109] The term "metabolic syndrome" includes abdominal (central) obesity, elevated blood pressure, elevated fasting blood glucose, high serum triglycerides, low high-density lipoprotein (HDL), and / or high low-density lipoprotein (LDL) levels. Furthermore, metabolic syndrome is associated with a risk of progressing to type 2 diabetes and / or cardiovascular disease, including coronary heart disease.

[0110] The terms "β cell," "beta cell," and "pancreatic islet cell" are used interchangeably herein to refer to pancreatic β cells located in the islets of Langerhans, whose primary function is to store and release insulin.

[0111] Defective insulin secretion underlies all forms of diabetes. While β-cell destruction is the cause of type 1 diabetes (T1D), both a reduction in β-cell mass and loss of secretory function contribute to type 2 diabetes (T2D). Emerging results suggest that functional defects due to dedifferentiation of mature β-cells to a more precursor-like state may be an important driver of secretory impairment in T2D.

[0112] It is contemplated that the antagonists and agonists of the present invention can be advantageously used to prevent β-cell dedifferentiation and / or reverse loss of β-cell function. Accordingly, the antagonists and agonists described herein are contemplated for use as pharmaceuticals. In particular, the antagonists and agonists are intended for use in methods for the prophylactic and / or therapeutic treatment of diabetes.

[0113] Type 1 diabetes is also known as insulin-dependent diabetes mellitus (IDDM) and juvenile-onset diabetes mellitus. These terms are used interchangeably herein. This form accounts for 5-10% of diabetes cases and is thought to be caused by cell-mediated autoimmune destruction of pancreatic β cells, resulting in little or no insulin secretion. The IGFR-like receptor antagonists and agonists provided herein can prevent or even reverse β-cell dedifferentiation and / or loss of function. Therefore, it is anticipated that the present invention will open up new possibilities for preventive or regenerative treatment of T1D.

[0114] Type 2 diabetes, also known as adult-onset diabetes, accounts for approximately 90-95% of all diabetes cases. Insulin resistance in target tissues and a relative lack of insulin secretion from pancreatic β cells are key features of type 2 diabetes (TD2). The term "insulin resistance" is used herein to refer to a condition characterized by the inability of target cells to respond to insulin, resulting in hyperglycemia. Pancreatic β cells in the pancreas subsequently increase their production of insulin, resulting in hyperinsulinemia. Without being bound by any particular theory, it is contemplated that the IGFR-like receptors described herein act as scavengers for either insulin or the insulin receptor. For example, IGFR-like receptors may bind to insulin receptors and cause their internalization (insulin receptor scavengers). IGFR-like receptors may also bind to insulin, causing its internalization and possible lysosomal degradation (insulin scavengers). Inhibiting these IGFR-like receptor functions with the aid of the antagonists described herein increases InsR and / or IGF1R-mediated signaling, thereby restoring insulin sensitivity.

[0115] As further shown in the accompanying Examples, the inventors have demonstrated that the IGFR-like receptors described herein negatively regulate downstream insulin signaling. Therefore, it is expected that IGFR-like receptor antagonists will promote or restore downstream InsR signaling, while IGFR-like receptor agonists will inhibit or attenuate InsR signaling.

[0116] Thus, antagonists of IGFR-like receptors can advantageously improve insulin sensitivity in insulin-resistant cells, preferably by increasing (i) InsR- and / or IGF1R-mediated Akt phosphorylation, (ii) InsR- and / or IGF1R-mediated AMPK phosphorylation, and / or (iii) mTOR phosphorylation.

[0117] diagnosis The present inventors have surprisingly discovered that expression of the IGFR-like receptors identified herein is associated with dedifferentiation of pancreatic β cells. β cell dedifferentiation is a term used to describe a change in β cell phenotype back to the multipotent progenitor cells from which β cells are derived or another reversible dedifferentiated state. Pancreatic β cell dedifferentiation is thought to result in the loss of critical functions, including insulin secretion. As a result of the loss of critical β cell functions, dysregulated insulin secretion is seen as a fundamental part of the pathogenesis of diabetes (including T1D and T2D). Hyperglycemia, a process also known as glucose toxicity, is thought to be a major cause of pancreatic β cell dedifferentiation, which leads to impaired insulin secretion.

[0118] Until the filing of the present application, reliable biomarkers for the onset and progression of the disease have not been available. The inventors have discovered that the IGFR-like receptors described herein are not only potentially useful and potent targets for the treatment of diabetes, but are also involved in the pathogenesis at a very early stage. Thus, monitoring the expression of the IGFR-like receptors provided herein may enable early diagnosis of diabetes and may also enable (preventive) treatment before the function of β-cells is lost or severely impaired.

[0119] Thus, the present invention provides a binding molecule capable of specifically binding to an IGFR-like receptor described herein for use as a diagnostic marker for diabetes or the risk of developing diabetes. Preferably, said IGFR-like receptor comprises a sequence corresponding to the sequence of SEQ ID NO: 1, although variants of said IGFR-like receptor are also envisaged.

[0120] In view of the foregoing, there is also provided herein an in vitro diagnostic assay (i.e., a diagnostic method) for detecting pancreatic islet cell dedifferentiation in a patient, the assay comprising: i) contacting a sample obtained from the subject with a diagnostic binding agent that specifically binds to a soluble IGFR-like receptor; ii) detecting binding of the diagnostic binding agent; wherein detectable binding of said diagnostic binding agent is indicative of dedifferentiation of pancreatic islet cells in the subject, and wherein the IGFR-like receptor comprises a sequence corresponding to SEQ ID NO:1.

[0121] As indicated elsewhere herein, dedifferentiation of pancreatic beta cells is believed to be indicative of diabetes or the risk of developing diabetes. The sample can generally be any sample, in particular a plasma sample.

[0122] Generally, to provide a diagnostic binding agent, one skilled in the art can follow the same principles as those illustrated in the context of providing antagonists and agonists of IGFR-like receptors. Similarly, it is envisioned that the diagnostic binding agents provided herein specifically bind to the IGFR-like receptors provided herein. Diagnostic binding agents can exhibit antagonistic or agonistic activity (which allows, for example, stimulatory diagnosis and (prophylactic) treatment), or may have no effect on IGFR-like receptor signaling at all. In preparing a diagnostic binding agent, one skilled in the art can use, for example, ligands and proteins comprising an insulin-like growth factor binding domain and / or a mannose-6-phosphate receptor binding domain, as described elsewhere herein, and use the ligand as a "template" to generate a diagnostic binding agent capable of specifically binding to an IGFR-like receptor. Exemplary proteins and ligands are described in the "Ligands" section herein. This approach is described in the context of preparing antagonists and agonists of IGFR-like receptors and is entirely applicable to diagnostic binding agents, except that such binding agents do not necessarily have to exhibit agonistic and / or antagonistic properties.

[0123] Particularly useful binding agents for use in in vitro diagnostic binding assays are monoclonal antibodies, e.g., antibodies of the sequence (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) The present invention also includes antibodies that specifically recognize epitopes of IGFR-like receptors, including:

[0124] The diagnostic binding agents utilized in the methods of the present invention further comprise a detectable label attached to the diagnostic binding agent. The detectable label can be attached (preferably covalently) to the diagnostic binding agent either directly or via a spacer of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used to practice the present invention.

[0125] The term "label" or "label group" refers to any detectable label. Labels suitable for use with diagnostic binding agents include (i) isotopic labels (which can be radioisotopes or heavy isotopes), such as radioisotopes or radionuclides (e.g., 3 H, 14 C. 15 N, 35 S, 89 Zr, 90 Y, 99 Tc, 111 In, 125 I, 131 (i) (ii) magnetic labels (e.g., magnetic particles), (iii) redox-active moieties, (iv) optical dyes (including, but not limited to, chromophores, fluorophores, and fluorescent fluorophores), such as fluorescent labels (e.g., FITC, rhodamine, lanthanide fluorophores), chemiluminescent labels, and fluorescent fluorophores (which can be either "small molecule" fluorophores or proteinaceous fluorophores), (v) enzymatic groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase), (vi) biotinylation groups, and (vii) predetermined polypeptide epitopes recognized by secondary reporters (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.).

[0126] "Fluorescent label" means any molecule that can be detected by virtue of its inherent fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malachite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes, Eugene, OR), FITC, rhodamine, and Texas Red (Pierce, Rockford, Suitable optical dyes, including fluorophores, include, but are not limited to, Cy5, Cy5.5, and Cy7 (Amersham Life Science, Pittsburgh, PA). Suitable optical dyes, including fluorophores, are described in Molecular Probes Handbook by Richard P. Haugland. Exemplary proteinaceous fluorophore labels also include, but are not limited to, green fluorescent protein (including GFP, EGFP from Renilla, Ptilosarcus, or Aequorea species), blue fluorescent protein (BFP), enhanced yellow fluorescent protein (EYFP), luciferase, and β-galactosidase.

[0127] It is within the knowledge of one of ordinary skill in the art to select an appropriate label depending on the type of diagnostic binding agent, its chemical properties, and the intended detection method. Diagnostic binding agents particularly suitable for use in the methods of the present invention are preferably monoclonal antibodies that specifically recognize the IGFR-like receptors described herein.

[0128] Advantageously, pancreatic β-cell dedifferentiation can be visualized by utilizing diagnostic binding agents capable of specifically recognizing IGFR-like receptors as described herein. Dedifferentiation of said β-cells is thought to indicate diabetes or the risk of developing diabetes and to precede β-cell dysfunction. Thus, the present invention provides, for the first time, a diagnostic assay that allows for the diagnosis of diabetes (including type 1, type 2, gestational diabetes, metabolic syndrome, and prediabetes) at a very early stage of disease onset, thereby opening new possibilities for early intervention of destructive mechanisms that lead to β-cell dysfunction and loss or defective insulin production.

[0129] By finding that the gene product of the human KIAA1324 gene plays a role in diabetes, the present invention makes it possible to assign any of the known SNPs in the KIAA1324 gene as to whether one or more SNPs may actually be associated with a propensity to develop diabetes, particularly type II diabetes, or may be at risk for developing the same.

[0130] The present invention also enables the use of known SNPs in the human KIAA1324 gene (including exons, introns, 5'- and 3'-untranslated regions) to determine whether a subject may be prone to or at risk of developing diabetes, particularly type II diabetes. Preferred SNPs for such determination are listed in the table below.

[0131] [Table 1]

[0132] If a subject has a different nucleotide at a position listed in the table compared to the wild-type human KIAA1324 gene, the subject may be prone to or at risk of developing diabetes, particularly type II diabetes. That is, the SNPs at the positions listed in the table may indicate a tendency to or at risk of developing diabetes, particularly type II diabetes.

[0133] The wild-type human KIAA1324 gene is that of NCBI GeneID 57535 (updated July 15, 2015), shown as the nucleotide sequence of GenBank accession number NG_032763.1.

[0134] The locations mentioned in the above table are taken from GeneID 57535 for KIAA1324 (updated 27 Aug 2016), annotation release 108, assembly GRCh37.p13 (GCF_000001405.25), chromosome 1, location NC_000001.10 (109656585..109749403).

[0135] patient As used herein, the term "patient" or "subject" refers to a human or non-human animal, generally a mammal. Mammals such as rabbits, mice, rats, guinea pigs, hamsters, dogs, cats, pigs, cows, goats, sheep, horses, monkeys, apes, or preferably humans are particularly contemplated. Thus, the methods, uses, and compounds described herein are generally applicable to diseases of both humans and vertebrates.

[0136] treatment The term "treatment" in all its grammatical forms includes therapeutic or prophylactic treatment. "Therapeutic or prophylactic treatment" includes prophylactic treatment aimed at preventing clinical and / or pathological symptoms altogether, or therapeutic treatment aimed at alleviating or reducing the clinical and / or pathological symptoms of the disease. Thus, the term "treatment" also includes the alleviation or prevention of diabetes.

[0137] In the context of the present invention, the term "therapeutic effect" generally refers to the desired or beneficial effects of treatment, such as the alleviation or reduction of disease symptoms. The term "sign" of a disease is used herein to describe its perceptible manifestations, and includes both clinical signs (defined below as signs (i.e., symptoms) of a disease that can be detected during a medical examination and / or are perceptible by the patient) and pathological signs (meaning manifestations of a disease at the cellular and molecular level). The therapeutic effect of treatment with IGFR-like antagonists and agonists can be evaluated using methods routine in the art, for example, by measuring insulin and / or glucose levels in a patient's blood sample. Additionally or alternatively, the general appearance (e.g., activity, health) of each patient can also be evaluated, which will help the expert assess whether the therapeutic effect is occurring. Those skilled in the art will be aware of numerous other methods suitable for monitoring the therapeutic effect of the compounds of the present invention.

[0138] dose Preferably, a therapeutically effective amount of a compound described herein is administered. "Therapeutically effective amount" refers to the amount of a compound described herein that elicits a therapeutic effect. The exact dose of an IGFR-like receptor antagonist or agonist will depend on the purpose of treatment (e.g., maintaining disease remission vs. treating acute symptoms) and can be ascertained by one of ordinary skill in the art using known techniques. Adjustments may be necessary for route of administration, age, weight, general health, sex, diet, timing of administration, drug interactions, and severity of symptoms, and can be ascertained by one of ordinary skill in the art using routine trial and error.

[0139] Administration Various routes are applicable to administering the compounds of the present invention, including but not limited to oral, topical, transdermal, subcutaneous, intravenous, intraperitoneal, intramuscular, or intraocular, although any other route can be easily selected by one skilled in the art if desired.

[0140] composition It is contemplated that the IGFR-like antagonist or agonist will be administered in the form of a pharmaceutical composition. Preferably, the antagonist or agonist is present in the pharmaceutical composition in a therapeutically effective amount. Particularly preferred antagonists or agonists for use in the pharmaceutical compositions provided herein are monoclonal antibodies that specifically bind to the IGFR-like receptors described herein. The antibodies have the sequence (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) It is envisioned that the antibody specifically binds to an epitope of an IGFR-like receptor, including

[0141] The term "pharmaceutical composition" particularly refers to a composition suitable for administration to humans, i.e., a composition that is preferably sterile and / or contains pharmaceutically acceptable ingredients. However, compositions suitable for administration to non-human animals are also contemplated herein. Preferably, a pharmaceutical composition comprises an IGFR-like receptor antagonist or agonist together with one or more pharmaceutical excipients. The term "excipient" includes fillers, binders, disintegrants, coatings, adsorbents, anti-adherents, lubricants, preservatives, antioxidants, flavors, colorants, sweeteners, solvents, cosolvents, buffers, chelating agents, viscosity modifiers, surfactants, diluents, wetting agents, carriers, diluents, preservatives, emulsifiers, stabilizers, or osmotic tonicity modifiers. The pharmaceutical compositions of the present invention can be formulated in various forms, for example, solid, liquid, gaseous, or lyophilized forms, and in particular in the form of ointments, creams, transdermal patches, gels, powders, tablets, liquids, aerosols, granules, pills, suspensions, emulsions, capsules, syrups, solutions, elixirs, extracts, tinctures, or fluid extracts, or in any other form particularly suited for administration in the desired manner.

[0142] The pharmaceutical compositions of the present invention may further comprise one or more additional agents. Preferably, the agents are therapeutically effective in treating the diseases described herein and are present in the composition in a therapeutically effective amount. Examples include, but are not limited to, metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin and insulin derivatives (insulin glulisine, insulin lispro, insulin aspart, insulin glargine, insulin detemir, insulin isophane), and combinations thereof.

[0143] Therefore, in view of the above, the present invention also provides pharmaceutical compositions comprising an IGFR-like antagonist or agonist, said pharmaceutical compositions being particularly intended for use in methods for the therapeutic and / or prophylactic treatment of diabetes.

[0144] kit Also provided herein are kits. The kits can be kits of two or more parts and include an IGFR-like receptor antagonist or agonist, preferably in a therapeutically effective amount and in a pharmaceutically acceptable form. The components of the kit can be contained in a container or vial. It is envisioned that the kits will include an additional agent useful for treating diabetes, as described elsewhere herein. Exemplary additional agents include, but are not limited to, metformin, sulfonylureas, meglitinides, thiazolidinediones, DPP-4 inhibitors, GLP-1 receptor agonists, SGLT2 inhibitors, insulin and insulin derivatives (insulin glulisine, insulin lispro, insulin aspart, insulin glargine, insulin detemir, insulin isophane), and combinations thereof.

[0145] The IGFR-like antagonist or agonist and the additional agent can be administered to the patient simultaneously or sequentially.

[0146] Screening Assay Method As used herein, the term "screening assay" is used equivalently to the term "screening method." Such assays or methods are preferably performed "in vitro," although they can also be performed "in vivo."

[0147] Antagonists and agonists of IGFR-like receptors can be identified using the in vitro screening assay provided herein, which comprises the steps of: (a) providing a stable cell line expressing the IGFR-like receptor, (b) contacting the cell line of (a) with a candidate antagonist or agonist, and (c) measuring downstream signaling events of the IGFR-like receptor, wherein antagonists are identified by increasing downstream signaling events of the IGFR-like receptor and agonists are identified by decreasing downstream signaling events of the IGFR-like receptor, wherein the IGFR-like receptor comprises a sequence corresponding to the sequence of SEQ ID NO:1.

[0148] Preferred downstream signaling events are InsR phosphorylation, AMPK phosphorylation, mTOR phosphorylation, AKT phosphorylation, ERK phosphorylation, and / or S6K phosphorylation.

[0149] Optionally, in the screening method described herein, a cell line expressing the IGFR-like receptor, preferably the same cell line as that used in the screening method of the present invention, is not contacted with a candidate antagonist or agonist, respectively. Such a cell line serves as a control. In such a control cell line, downstream signaling events of the IGFR-like receptor are measured and compared with those measured in the cell line that is (or was) contacted with the candidate antagonist or agonist.

[0150] A preferred example of the cell line expressing the IGFR-like receptor is Min6 or PDDX1+ / NKX6.1+ iPSC.

[0151] As described herein, downstream signaling events of IGFR-like receptors are believed to result in negative regulation of InsR- and / or IGF1R-mediated signaling, and in particular, inhibition or reduction of (i) Akt phosphorylation, (ii) AMPK phosphorylation, and / or (iii) mTOR phosphorylation. Furthermore, in the screening assays provided herein, binding agents that promote or increase (i) Akt phosphorylation, (ii) AMPK phosphorylation, and / or (iii) mTOR phosphorylation are likely to be antagonists of IGFR-like receptors. In the screening assays provided herein, binding agents that have no effect or that further decrease (i) Akt phosphorylation, (ii) AMPK phosphorylation, and / or (iii) mTOR phosphorylation are likely to be agonists of IGFR-like receptors.

[0152] The stable cell line can be any cell line suitable for expressing a functional IGFR-like receptor, where a "functional IGFR-like receptor" is particularly envisioned to negatively regulate InsR- and / or IGF1R-mediated signaling, as can be determined by reducing or inhibiting (i) Akt phosphorylation, (ii) AMPK phosphorylation, and / or (iii) mTOR phosphorylation.

[0153] The present invention also relates to antagonists or agonists that can be obtained by the screening method.

[0154] The present invention also contemplates a method for detecting whether an IGFR-like receptor homodimerizes or heterodimerizes with IGFR-1, IGFR-2, and / or InsR, comprising detecting whether a tagged IGFR-like receptor homodimerizes or heterodimerizes. The heterodimer may comprise an IGFR-like receptor and InsR, IGFR-1, and / or IGF-R2.

[0155] A preferred tag is a fluorescent protein, such as GFP or Venus, a genetic variant of green fluorescent protein (GFP) that has an emission peak at 527 nm.

[0156] Tagging of the IGFR-like receptor is preferably achieved by fusing the tag in-frame to said IGFR-like receptor, which can be achieved by techniques known in the art, for example by knocking in a nucleotide sequence encoding the tag into a nucleotide sequence encoding said IGFR-like receptor, for example by genome editing, for example using the CRISPR / Cas system.

[0157] The present invention is also characterized by the following features. 1. An isolated DNA sequence encoding an IGF receptor (IGFR)-like receptor capable of reacting with an antibody raised against the IGFR-like receptor of SEQ ID NO:1, wherein said antibody is (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) specifically binds to at least one epitope comprising Isolated DNA sequence.

[0158] 2. Encoding an IGFR-like receptor comprising a sequence corresponding to SEQ ID NO: 1; Isolated DNA sequence.

[0159] 3. A DNA sequence according to item 1 or 2, vector.

[0160] 4. The vector according to item 3, further comprising a gene regulatory element located upstream of the DNA sequence encoding the IGFR-like receptor.

[0161] 5. A vector comprising the vector according to item 3 or 4. Host cells.

[0162] 6. A diagnostic binding agent capable of specifically binding to an IGFR-like receptor for use in a method for diagnosing diabetes or the risk of progression of diabetes, wherein the IGFR-like receptor comprises a sequence corresponding to the sequence of SEQ ID NO: 1. Diagnostic binders.

[0163] 7. An antagonist or agonist of an IGFR-like receptor for use as a medicament, wherein the IGFR-like receptor comprises a sequence corresponding to SEQ ID NO: 1. Antagonists or agonists.

[0164] 8. An antagonist or agonist of an IGFR-like receptor, wherein the IGFR-like receptor comprises a sequence corresponding to SEQ ID NO: 1, for use in a method for the preventive and / or therapeutic treatment of diabetes. Antagonists or agonists.

[0165] 9. The antagonist or agonist according to item 7 or 8, wherein the antagonist specifically binds to the IGFR-like receptor.

[0166] 10. The antagonist or agonist according to any one of items 7 to 9, wherein the antagonist or agonist is selected from an antibody, siRNA, nucleic acid, aptamer, peptide, protein, or small molecule organic compound.

[0167] 11. The antagonist or agonist of paragraph 10, wherein the antibody includes antibodies, antibody variants, and antibody fragments.

[0168] 12. The antagonist or agonist according to item 10 or 11, wherein the antibody is a monoclonal or polyclonal antibody.

[0169] 13. The antibody is a monoclonal antibody that specifically binds to an epitope of the IGFR-like receptor, the epitope having the sequence: (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) Item 13. The antagonist or agonist according to Item 12, comprising:

[0170] 14. The antagonist or agonist according to any one of items 7 to 13, wherein the diabetes includes type 1 diabetes, type 2 diabetes, gestational diabetes, prediabetes, insulin resistance, metabolic syndrome, and impaired glucose tolerance.

[0171] 15. The antagonist or agonist of any one of paragraphs 8 to 15, wherein the treatment prevents or reverses insulin resistance.

[0172] 16. The antagonist or agonist of any one of paragraphs 8 to 16, wherein treatment prevents or reverses dedifferentiation of pancreatic islet cells.

[0173] 17. An antagonist or agonist of an IGFR-like receptor comprising a sequence corresponding to SEQ ID NO: 1, wherein the antagonist is a monoclonal antibody that specifically binds to the IGFR-like receptor. Pharmaceutical compositions.

[0174] 18. The antibody specifically binds to an epitope of the IGFR-like receptor, the epitope having the sequence: (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) Item 18. The pharmaceutical composition according to Item 17, comprising:

[0175] 19. Array (i) TSKRTPDGFDSVPLKT (SEQ ID NO: 2) and / or (ii) CHQCDPDKYSE (SEQ ID NO: 3) and / or (iii) MYKWAKPKICSEDLEG (SEQ ID NO: 4) and / or (iv) FQRTTFHEASRKYTN (SEQ ID NO: 5) and / or (v) CTFSRNTPTRTFNY (SEQ ID NO: 6) specifically binds to an epitope of an IGFR-like receptor, Monoclonal antibodies.

[0176] 20. An in vitro screening assay for antagonists or agonists of IGFR-like receptors, comprising: (a) providing a stable cell line expressing said IGFR-like receptor; (b) contacting the cell line of (a) with a candidate antagonist or agonist; (c) measuring downstream signaling events of an IGFR-like receptor, wherein an antagonist is identified by suppressing the downstream signaling events of the IGFR-like receptor and an agonist is identified by promoting the downstream signaling events of the IGFR-like receptor; wherein the IGFR-like receptor comprises a sequence corresponding to the sequence of SEQ ID NO: 1; method.

[0177] 21. An antagonist or agonist of an IGFR-like receptor obtainable by the method according to paragraph 20, wherein the IGFR antagonist is selected from an antibody, an siRNA, a nucleic acid, an aptamer, a peptide, a protein, or a small organic compound.

[0178] 22. An in vitro diagnostic assay for detecting pancreatic islet cell degeneration in a subject, comprising: i) contacting a sample obtained from said subject with a diagnostic binding agent that specifically binds to an IGFR-like receptor; ii) detecting binding of the binding agent; wherein detectable binding of said diagnostic binding agent is indicative of dedifferentiation of pancreatic islet cells in the subject; wherein the IGFR-like receptor comprises a sequence corresponding to SEQ ID NO: 1; In vitro diagnostic assays.

[0179] 23. The in vitro diagnostic assay according to paragraph 22, wherein dedifferentiation of pancreatic islet cells indicates diabetes or the risk of developing diabetes.

[0180] 24. The in vitro diagnostic assay of paragraph 22 or 23, wherein the sample is a plasma sample.

[0181] 25. The in vitro diagnostic assay of any one of paragraphs 22 to 24, wherein the diagnostic binding agent is a monoclonal antibody.

[0182] 26. A method of treating diabetes, comprising administering to a subject an IGFR antagonist, wherein the IGFR-like receptor comprises a sequence corresponding to the sequence of SEQ ID NO: 1. method.

[0183] 27. Use of an antagonist or agonist of an IGFR-like receptor in a method for treating diabetes, wherein the IGFR-like receptor comprises a sequence corresponding to the sequence of SEQ ID NO: 1. use.

[0184] It is noted that, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a reagent" includes one or more of such different reagents. Reference to "the method" includes reference to equivalent steps and methods known to those skilled in the art that may modify or substitute for the methods described herein.

[0185] Unless otherwise specified, the term "at least" preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain without undue experimentation, many equivalents to the specific embodiments of the invention described herein. Such equivalents are intended to be encompassed by this invention.

[0186] The term "and / or" as used anywhere in this specification includes the meaning of "and", "or" and "all or any other combination of the elements connected by said term".

[0187] As used herein, the term "about" or "approximately" means within 20%, preferably within 10%, and more preferably within 5% of a given value or range. However, the term is inclusive. For example, "about 20" includes 20.

[0188] The terms "less than" or "greater than" are inclusive. For example, less than 20 means less than or equal to. Similarly, more than or greater than means more than or equal to or greater than or equal to, respectively.

[0189] Throughout this specification and the claims that follow, unless the context requires otherwise, the word "comprise" and transition words such as "comprises" and "comprising" will be understood to include the inclusion of a stated integer or step or group of integers or steps and not to exclude any other integer or step or group of integers or steps. As used herein, the term "comprising" can be replaced by the word "containig" or "including," or, as sometimes used herein, by the word "having."

[0190] As used herein, "consisting of" excludes any element, step, or ingredient not specified in the claim element. As used herein, "consisting essentially of" does not exclude materials or steps that do not materially affect the basic and novel characteristics of the claim.

[0191] It is to be understood that this invention is not limited to the particular methodology, protocols, materials, reagents, and substances, etc., described herein, as these may vary. The terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention, which is defined only by the claims.

[0192] All publications and patents (including all patents, patent applications, scientific publications, manufacturing specifications, instructions, etc.) cited throughout the text of this specification, whether supra or infra, are incorporated by reference in their entirety. Nothing herein should be construed as an admission that the present invention is not entitled to antedate such disclosure by virtue of prior invention. To the extent that information incorporated by reference contradicts or is inconsistent with the present specification, the present specification will take precedence over any such information.

[0193] A better understanding of the present invention and its advantages will be gained from the following examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. [Example]

[0194] Example 1: Materials and Methods Generation of IGFR-like receptor knockout mice All mice were housed in a central facility at the HMGU in accordance with the German Animal Welfare Act and the recognized guidelines of the Society of Laboratory Animals (GV-SOLAS) and the Federation of Laboratory Animal Science Associations (FELASA). The sacrifice of mice at the embryonic stage did not require regulatory approval.

[0195] Targeted ES cell clones derived from the C57BL / 6N parental cell line JM8.N4 were obtained from the EUCOMM Cell Depository (EuMMCR) (Helmholtz Zentrum München GmbH, Alleles produced for the EUCOMM and EUCOMMTools projects by the Helmholtz Zentrum München GmbH (Hmgu). MGI Direct Data Submission. 2010-2015) and injected into CD1 blastocysts for chimera generation. The resulting chimeras were mated with CD1 or C57BL / 6J mice. Offspring were screened by PCR to confirm germline transmission. Heterozygous mice carrying the targeted mutation were intercrossed to generate homozygous mutant mice in either the CD1 or C57BL / 6J background.

[0196] Genotypes were confirmed by PCR using genomic DNA extracted from the tails of control and knockout mice. The primers used for PCR were as follows: forward primer: 5'TGGGTAGCCTTTCTGTATGG-3' (SEQ ID NO: 8) and reverse primer: 5'GACATAGGGCAGATTTGTGG-3' (SEQ ID NO: 9).

[0197] Proliferation assay To evaluate cell proliferation in mutant embryos, pregnant females were injected subcutaneously with 0.1 mg EdU / gram body weight. Two hours later, the animals were sacrificed, and pancreata from E14.5, E16.5, and E18.5 embryos were excised and fixed overnight at 4°C in 4% PFA in PBS pH 7.4. The following day, organs were subjected to a 7.5%, 15%, and 30% sucrose gradient, embedded in Tissue Freezing Medium (Leica, Germany), and stored at -80°C. Twenty-micrometer cryosections were stained using the Click-iT® EdU Imaging Kit (Life Technology) according to the manufacturer's instructions. The kit includes an antibody against insulin (1:250 guinea pig, Thermo Scientific; PA1-26938). Images were acquired using a Leica SP5 confocal microscope equipped with a 20x objective. Data analysis was performed using IMARIS software (Bitplane, Switzerland).

[0198] RT-qPCR For gene expression analysis, mRNA was isolated from the pancreases of knockout and control embryos using the miRNeasy mini kit (Qiagen, Germany) by real-time quantitative PCR. cDNA was synthesized using the GoScript™ Reverse Transcription System (Promega) and then used for qPCR using TaqMan® Low Density Arrays and run on a ViiA 7 system (Applied Biosystems). The probes used are listed in Table 1.

[0199] [Table 2]

[0200] Generation of monoclonal antibodies against estrogen-inducible gene 121 protein (EIG)

[0201] A peptide from the human EIG protein was synthesized and conjugated to OVA (Peps4LS, Heidelberg, Germany). Lou / c rats or C57BL / 6 mice were immunized subcutaneously and intraperitoneally with a mixture of 50 μg peptide-OVA, 5 nmol CPG oligonucleotide (Tib Molbiol, Berlin), 500 μl PBS (100 μl per mouse), and 500 μl incomplete Freund's adjuvant (100 μl per mouse). An adjuvant-free booster was given 6 weeks after the initial injection. Fusion was performed using standard techniques. Supernatants were tested in differential ELISA with biotinylated EIG peptide and an unrelated biotinylated peptide on avidin-coated ELISA plates. MAbs that specifically reacted with the EIG peptide were further analyzed by Western blot.

[0202] Rabbit polyclonal antibodies were purchased from Pineda Antikorper Service, Berlin, Germany.

[0203] cell culture Min6 is a pancreatic β-cell line that has been well characterized for its ability to secrete insulin upon glucose stimulation. This cell line was from Susumu Seino's Lab.

[0204] Min6 cells were maintained in Gibco's high glucose Dulbecco's modified Eagle's medium (41966-052) supplemented with 10% fetal bovine serum (FBS), 1% penicillin / streptomycin, and 2-mercaptoethanol.

[0205] HEK293 cells were purchased from the global biosource center ATCC.

[0206] IGFR-like receptor knockdown Min6 cells were cultured at a cell density of 5 × 10 4 pieces / cm 2 Cells were seeded at 100°C and cultured in high-glucose DMEM medium. After a single transfection of IGFR-like receptor siRNA on days 2 and 3, cells were lysed on ice in RIPA buffer at a cell density of 50-60% on day 4. Lipofectamine (Lipofectamine 2000, Life Technologies, #11668019)-based transfection was performed according to the Life Technologies protocol. 200 pmol of the following siRNA (Dharmacon, GE Healthcare) was used: On-Target plus mouse 5330417C22Rik (229722) siRNA-SMARTpool L-048745-01-0020 and control siRNA On-target plus non-targeting siRNA #1 D-001810-01-20.

[0207] IGFR-like receptor knockout CRISPR / Cas9-mediated IGFR-I knockout strategy in Min6 cells targeting the core promoter and transcription start site (Figure 23).

[0208] IGFR-like receptor knock-in Venus fusion CRISPR / Cas9-mediated knock-in fusion of the Venus fluorescent reporter by removing the translation stop sequence and fusing Venus in-frame to the IGFR-I open reading frame (Figure 23). Cas9 fused in-frame to the Venus fluorescent reporter was co-transfected into Min6 cells with guidance RNA (gRNA) flanking the transcription start site of IGFR-I. After 2 days, fluorescent cells were flow-sorted. Limiting dilutions were plated onto 10 cm dishes. After 10-14 days, single clones were picked into 96-well plates and analyzed for IGFR-I expression (staining and WB) and internal deletions by genomic PCR.

[0209] Min6 starvation assay Min6 cells were cultured at a cell density of 5 × 10 4 pieces / cm 2 Min6 cells were seeded at 100°C and cultured in high-glucose DMEM medium. After IGFR-like receptor knockdown, Min6 cells were washed six times with 1x PBS. Cells were starved with HBSS buffer containing 0.2% BSA for 15, 30, 60, and 120 minutes. Cells were then lysed in RIPA buffer on ice.

[0210] Western Blot (Semi-Dry Immunoblot) Cells and tissues were lysed in RIPA buffer (50 mM Tris pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% IGEPAL, 0.1% SDS, 0.1% Na-deoxycholate) containing a cocktail of protease inhibitors (1:100, Sigma, P8340) and phosphatase inhibitors (1:100, Sigma Aldrich, #2 P0044 and #3 P5726). Cell or tissue lysates were washed by centrifugation at 13,000 rpm for 10–30 min. Proteins (10–20 μg) were separated on 6.5–15% SDS-PAGE based on the size of the detected proteins and transferred to PVDF membranes. Primary antibodies in 5% milk were added to the membranes and incubated overnight at 4°C. Protein bands were visualized on Hyperfilms (GE healthcare, 28906837) or Blaufilme CEA-RP (Ernst Christiansen GmbH, EC84A) by chemiluminescence detection (ECL, Millipore, WBKLS0500).

[0211] The following antibodies were used at the corresponding dilutions:

[0212] IGFR-like receptor SEQ ID NO:2 (1:5000, rabbit, Pineda, Berlin, Germany), IGFR-like receptor SEQ ID NO:2 (1:100, rat and mouse), IGFR-like receptor SEQ ID NO:4 and 6 (1:10, rat and mouse), Akt (1:5000, Cell Signaling, 4691), P-Akt (1:5000, Cell Signaling, 4060), Erk (1:5000, Cell Signaling, 4695), P-Erk (1:5000, Cell Signaling, 4370), m-Tor (1:1000, Cell Signaling, 2972), P-mTor (1:1000, Cell Signaling, 5536), Ampk (1:1000, Cell Signaling, Cell Signaling, 2532), P-Ampk (1:2500, Cell Signaling, 2535), IRS-2 (1:1000, Cell Signaling, 4502), IRβ (1:1000, Cell Signaling, 3025), S6rp (1:5000, Cell Signaling, 2217S), P-S6RP (1:5000, Cell Signaling, 2211S), P-IR / IGFR (1:1000, Millipore, 07-841), α-tubulin (1:5000, Sigma, T7451), and mouse adaptin beta clone 74 (1:5000, BD Bioscience, 610382). Autophagy-related proteins were detected using the Cell Signaling Autophagy Antibody Sampler Kit (#4445, 1:1000).

[0213] Immunofluorescence and imaging For immunocytochemistry, Min6 cells were plated in Ibidi (Munich, Germany) 8-well chamber dishes (treated) at a cell density of 5 × 10 4Cells were seeded at 1000 cells / well. Three days after seeding, cells were directly fixed with 4% PFA for 10 minutes, followed by incubation in permeabilization solution (0.1 M glycine, 0.2% Triton-X100 in 1x PBS) for 10 minutes. After 1 hour of blocking (10% donkey serum, 1% BSA, 5% FCS, 0.5% Tween-20 in 1x PBS), cells were incubated overnight at 4°C in blocking solution containing primary antibodies: IGFR-like receptor SEQ ID NO:2 (rat and mouse 1:10), GM130 (1:300, BD, 610822), and IGF2R (1:100, ThermoScientific, PA3-850). The following secondary antibodies were added in blocking solution for 2 hours at room temperature: Donkey anti-rabbit IgG-555 (1:800, Invitrogen, A31572), donkey anti-mouse IgG-488 (1:800, Invitrogen, A21202), and donkey anti-rat IgG647 (1:400, Dianova, 712-605-150). After DAPI staining (50 ng / ml), samples were mounted with elvanol. Images were acquired on a Leica laser-scanning SP5 confocal microscope equipped with a 63x objective.

[0214] For immunocytochemistry in mouse tissues, pancreases were excised, fixed in 4% PFA in PBS for 2 hours at 4°C, and cryoprotected in a gradient series of sucrose solutions (7.5, 15, and 30% sucrose in PBS) for at least 2 hours in each solution. Finally, organs were embedded in Tissue Freezing Medium (Leica 14020108926) and stored at -80°C. 20 μm sections were used for immunostaining. Briefly, the sections were washed in PBS, permeabilized in 0.1% Triton X-100, 0.1 M glycine in PBS for 15 minutes, and then blocked in blocking solution (10% FCS, 3% donkey serum, 0.1% BSA, and 0.1% Tween-20 in PBS) for 1 hour at room temperature. Primary antibodies were diluted in blocking solution and incubated overnight at 4°C. The next day, sections were washed three times for 5 min each in PBST (0.1% Tween-20 in PBS), incubated with secondary antibodies diluted in blocking solution at room temperature for 3–5 h, stained with DAPI (4′,6-diamidine-2-phenylindole), and mounted in ProLong Gold antifade embedding medium (Life Technologies).

[0215] For whole-mount staining of human islets, islets were fixed in 4% PFA in PBS for 15 min at RT and directly incubated overnight at 4°C with primary antibodies diluted in blocking solution, followed by three washes in PBST and then incubation with secondary antibodies for 3–5 h at RT as described above.

[0216] For staining of human tissue sections, snap-frozen pancreatic sections from human donors were embedded in Tissue Freezing medium (Leica 14020108926) and stored at -80°C. 10 μm-thick sections were fixed in 4% PFA in PBS for 20 min at RT, washed twice with PBS, and permeabilized on ice for 5 min. Antibody staining procedures were performed as described above.

[0217] The primary antibodies used were anti-IGFR-like receptor rabbit, SEQ ID NO:2, 1:100; anti-IGFR-like receptor, SEQ ID NO:2 and SEQ ID NO:4 rat, 1:10; IGFR-like receptor, SEQ ID NO:2 and SEQ ID NO:6 mouse, 1:10; anti-insulin (rabbit, 1:250, Cell Signaling; 3014); anti-insulin (guinea pig, 1:250; Thermo Scientific; PA1-26938); anti-E-cadherin (rat, 1:500; DECMA Kremmer); anti-mannose 6P receptor (rabbit, 1:200; Thermo Scientific; PA3-850); and anti-urocortin 3 (rabbit, 1:300; Phoenix Pharmaceuticals; H-019-29). The secondary antibodies used were anti-rabbit Alexa-555 (1:800; Invitrogen; A31572), anti-rabbit Alexa-488 (1:800; Invitrogen; A21206), anti-rat Alexa-488 (1:800; Life Technologies, A-21208), anti-rat Alexa-647 (1:800; Dianova, 712-605-150), and anti-guinea pig Alexa-647 (1:800; Dianova; 706-495-148), and anti-mouse Alexa-555 (1:800; Invitrogen; A31570). Images were acquired on a Leica DMI 6000 microscope.

[0218] Immunoprecipitation assay For immunoprecipitation assays, Min6 cells were starved for 15, 30, 60, and 120 min in fatty acid-free HBSS containing 0.2% BSA and lysed for 20 min on ice in immunoprecipitation buffer (2% CHAPS, 50 mM HEPES pH 7.5, 200 mM NaCl, 2 mM EDTA) containing a cocktail of protease inhibitors (1:100, Sigma, P8340). The lysate was washed by centrifugation at 14,000 rpm for 30 min at 4°C. Approximately 500 μg of whole cell lysate was incubated with a 1:10 diluted IGFR-like receptor SEQ ID NO:2 antibody produced in rats for 1 h at 4°C, followed by incubation with protein G Sepharose 4 Fast Flow (GE Healthcare) for 16 h at 4°C. The precipitates were washed five times with immunoprecipitation buffer and denatured at 95°C for 5 minutes in 2x SDS sample buffer (100 mM Tris-HCl, 4% SDS, 20% glycerol, 0.2% bromophenol blue) containing 100 mM DTT and 5% 2-mercaptoethanol, and then subjected to Western blot analysis.

[0219] Surface biotinylation assay Min6 cells were incubated with 2 mM EZ-Link® Sulfo-NHS-LC-Biotin (Thermo Scientific) in PBS pH 8.0 at room temperature for 10 minutes. The biotinylation reaction was then stopped with 100 mM glycine in PBS. The cells were then washed with ice-cold PBS and lysed in RIPA buffer containing a cocktail of protease inhibitors. After centrifugation at 14,000 rpm for 30 minutes at 4°C, the supernatant was incubated with NeutrAvidin beads (Thermo Scientific / Pierce) overnight at 4°C. The next day, after five washes in RIPA buffer, biotin-labeled proteins were eluted from the beads by boiling in 2x SDS sample buffer containing 100 mM DTT and 5% 2-mercaptoethanol and subjected to Western blot analysis.

[0220] Example 2: Pancreatic expression of IGFR-like receptors adjacent to IGF-1, IGF-2, and insulin ligands Briefly, E14.5 mouse embryos were obtained and subjected to in situ hybridization (ISH) using GenePaint™ (Tecan).

[0221] Gene paint in situ hybridization of E14.5 mouse embryos demonstrates the specific expression of IGFR-like receptor mRNA in the pancreas (Fig. 2B). IGF1 and IGF2 mRNA are expressed in tissues adjacent to the pancreatic epithelium (Fig. 2A, C). Insulin mRNA, on the other hand, is present in the endocrine compartment of the pancreas (Fig. 2D).

[0222] Example 3: IGFR-like receptor knockout mice IGFR-like receptor knockout mice were generated as described in Example 1.

[0223] IGFR-like receptor KO mice appear normal at birth but do not lactate, as evidenced by the absence of milk in their stomachs (Figure 3B, star). They are sluggish and exhibit respiratory distress. Mendelian ratios at various stages show a normal distribution of the three genotypes until birth, but the probability of surviving knockout mice decreases significantly from birth to weaning age. Negligible survival is observed at weaning age, likely due to compensatory mechanisms and / or splicing near the inserted cassette resulting in an incomplete knockout (Figure 3C). Histograms show normal body weights of knockout mice compared with wild-type and heterozygous littermate controls at P0 (Figure 3D).

[0224] Example 4: Growth, proliferation, and endocrine differentiation in the pancreas of IGFR-like receptor KO mice Briefly, IGFR-like receptor knockout mice were generated, and the pancreas was removed and subjected to immunohistochemistry using an antibody against insulin. Cell proliferation was measured using EdU. A detailed description of the materials and methods used is provided in Example 1.

[0225] Confocal images of pancreatic sections show normal endocrine differentiation at E16.5, as indicated by immunofluorescently labeled β-cells using an insulin antibody (green). (Similar results were obtained at E14.5 and E18.5, not shown.) Normal proliferation of the pancreatic epithelium is indicated by the incorporation of EdU (red). Quantification of EdU-positive cells relative to the total cell number (stained with DAPI) at E14.5, E16.5, and E18.5 showed no significant difference in proliferation between knockout and wild-type pancreases (at least three sections were counted from different regions of one pancreas / stage; error bars represent the standard error of the mean) (Figure 4).

[0226] Example 5: Pancreatic gene expression in prenatal IGFR-like receptor KO mice Briefly, IGFR-like receptor knockout mice were generated as previously described. The pancreases of KO mice and wild-type controls were removed before and after birth, mRNA was isolated, and real-time quantitative PCR was performed. A detailed description of the materials and methods used is provided in Example 1.

[0227] Real-time qPCR showed modest changes in the relative mRNA expression of select genes important for endocrine β-cell differentiation, maturation, proliferation, and function in the knockout pancreas compared to wild-type controls at E18.5, a stage when the embryos are still dependent on maternal nutrition (Figure 5). Actin was used as a housekeeping gene for normalization. The relative mRNA expression of select genes important for endocrine β-cell differentiation (Foxa2, Pdx1, Pax6, Neurod1, Nkx2-2, Nkx6-1), maturation (MafA, MafB, Ucn3), proliferation (Ccnd1, Ccnd2, Cdk4, Cdkn1a, Cdkn1b), and function (Slc2a2, Slc30a8, Gjd3, Kcnj11, Smarca1, Abcc8) was significantly altered in the knockout pancreas compared to wild-type controls immediately after birth, as shown by real-time qPCR (Figure 6).

[0228] Example 7: IGFR-like receptors mediated Akt and AMPK signaling in the pancreas and Min6 Briefly, IGFR-like receptor knockout mice were generated and the pancreas was removed. Min6 cells were cultured and harvested as previously described. IGFR-like receptor knockdown in Min6 cells was achieved by transfection with siRNA targeting IGFR-like receptor mRNA or scrambled siRNA as a control. Cells and tissues were subjected to Western blotting using phospho-specific antibodies against Akt and AMPK. A detailed description of the materials and methods used is provided in Example 1.

[0229] Western blots of whole pancreatic tissue lysates using phospho-specific antibodies show phosphorylation of Akt and AMPK (Figure 7A). A significant number of mutant embryos show an increase in Akt (mt2, 3, 5, 6) and AMPK phosphorylation (mt1, 2, 3) (Figure 7A). Similarly, (Figure 7B) knockdown of IGFR-like receptors in Min6 cells results in increased Akt phosphorylation.

[0230] Example 8: Effect of IGFR-like receptor knockdown on insulin / IGF signaling in Min6 Briefly, Min6 cells were cultured, transfected with siRNA targeting IGFR-like receptor mRNA, and studied by Western blot using phospho-specific antibodies against IR / IGF1R and the downstream signaling molecules AKT, mTOR, ERK, and S6RP. A detailed description of the materials and methods used is provided in Example 1.

[0231] Knockdown of IGFR-like receptors in Min6 cells resulted in increased phosphorylation of IR / IGF1R under normal growth conditions, which appeared to be independent of starvation. Total levels of IR and IGF1R, as well as IRS-2 (a downstream adaptor molecule for IR signaling), remained unchanged. Meanwhile, IGFR-like receptors were strongly reduced in the knockdown samples, confirming the efficacy of siRNA knockdown (Figure 8A). Western blot analysis of downstream signaling molecules, such as AKT and mTOR, which are key molecules commonly phosphorylated as a result of IR activation, demonstrated that knockdown of IGFR-like receptors in Min6 cells resulted in increased phosphorylation of Akt and mTOR, but not ERK or S6RP. Starvation conditions appeared to have no effect on Akt, mTOR, and ERK phosphorylation in IGFR-like receptor knockdown samples compared to controls at the same time points, as indicated by a time-dependent decrease in phospho-S6RP (Figure 8B).

[0232] Example 9: Localization of IGFR-like receptors in endocrine, ductal, and exocrine cells of the pancreas Briefly, E18.5 pancreata were excised and subjected to immunohistochemistry using antibodies against IGFR-like receptors, insulin, and E-cadherin. A detailed description of the materials and methods used is provided in Example 1.

[0233] Confocal images of E18.5 pancreatic sections stained with IGFR-like receptors (green), insulin antibodies (red), and antibodies against E-cadherin (cyan) show immunolocalization of IGFR-like receptors in both the exocrine and endocrine compartments, where they partially colocalize with insulin (Figure 9). Nuclei were stained with DAPI.

[0234] Example 10: Subcellular localization of IGFR-like receptors Briefly, Min6 cells were cultured as previously described and subjected to immunocytochemistry using antibodies against IGFR-like receptors and GM130. A detailed description of the materials and methods used is provided in Example 1.

[0235] Example 11: Subcellular localization of IGFR-like receptors Briefly, immunofluorescence staining and imaging were performed as described in Example 1.

[0236] Immunofluorescence images of Min6 cells stained with immunofluorescently labeled antibodies against IGFR-like receptors and the Golgi marker GM130 show partial localization of IGFR-like receptors in the Golgi complex, similar to IGF2R (Figures 10 and 11).

[0237] Compared to normal growth conditions, after 2 hours of starvation a redistribution of IGFR-like receptors concentrated in the cis-Golgi region can be observed (FIG. 11).

[0238] Example 11: Interaction of IGFR-like receptors with AP-2 under starvation conditions Briefly, Min6 cells were cultured as previously described and subjected to immunoprecipitation and Western blotting using antibodies against IGFR-like receptors and adaptin β (a subunit of the AP2 complex). A detailed description of the materials and methods used is provided in Example 1.

[0239] Western blot analysis demonstrated co-immunoprecipitation of the adaptin β subunit (part of the AP2 complex) with IGFR-like receptors in starvation-induced Min6 cells, as evidenced by immunoprecipitation using antibodies against the IGFR-like receptors and adaptin β. Under normal starvation conditions, adaptin β was not substantially co-precipitated (Figure 13).

[0240] Example 12: IGF3R, but not InsR, is transported into cells upon nutrient deprivation Briefly, Min6 cells were cultured as previously described and subjected to surface biotinylation and Western blotting using antibodies against IGFR-like receptors and adaptin β (a subunit of the AP2 complex). A detailed description of the materials and methods used is provided in Example 1.

[0241] Surface biotinylation followed by neutravidin pulldown and Western blot demonstrated a time-dependent decrease in surface expression of IGFR-like receptors, but not IR, in Min6 cells under starvation conditions.

[0242] Example 14: Effect of IGFR-like receptor knockdown on the expression of autophagy-related proteins Briefly, Min6 cells were cultured and transfected as previously described and subjected to Western blot analysis using antibodies against ATG16L1, ATG7, ATG3, Beclin-1, LC3A / B, and ATG5 (i.e., autophagy-related proteins). A detailed description of the materials and methods used is provided in Example 1.

[0243] IGFR-like receptor knockdown likely does not alter the protein expression of autophagy-related proteins under basal conditions. Western blot analysis of autophagy-related molecules showed no apparent changes in expression upon IGFR-like receptor knockdown in Min6 cells under normal growth conditions (Figure 15).

[0244] Example 15: Expression of IGFR-like receptors in diabetic mouse pancreatic islets and exocrine tissues Briefly, pancreata from wt and diabetic mice were excised and subjected to immunocytochemistry using antibodies against IGFR-like receptors, insulin, and E-cadherin. A detailed description of the materials and methods used is provided in Example 1.

[0245] LSM images of wild-type (<120 mg / dL glucose) and diabetic (>500 mg / dL glucose) adult pancreases stained with antibodies against IGFR-like receptors (red), insulin (cyan), and E-cadherin (green) (A), and IGFR-like receptors (red), glucagon (cyan), and urocortin 3 (green) (B), show increased immunoreactivity of IGFR-like receptor antibodies in the diabetic pancreas, both in the endocrine and exocrine compartments. The diabetic state was evidenced by decreased insulin and urocortin 3 staining in the diabetic pancreas (Figure 16).

[0246] Example 16: Detection of IGFR-like receptors in pancreatic islet cells using antibodies against the extracellular domain of IGFR-like receptors Briefly, HEK293 cells were cultured and human pancreatic islets were obtained as previously described. Pancreata from wild-type, heterozygous, and mutant (IGFR-like receptor knockout) mice were excised. Cells and tissues were subjected to Western blotting using anti-IGFR-like receptor antibodies that recognize SEQ ID NOs: 2, 4, and 6.

[0247] Western blot experiments show specific recognition of a protein band of approximately 130 kDa in islet cell lysates, which is absent in HEK293 cell lysates, by antibodies against SEQ ID NOs: 4 and 6. By comparison, the same band is specifically recognized by antibodies against SEQ ID NO: 2 in wild-type and heterozygous total pancreatic lysates, but not in mutant embryonic and islet cells, and not in HEK cells (Figure 17).

[0248] Example 17: Distribution of IGFR-like receptors in human pancreatic islets Briefly, human pancreatic islets were obtained and subjected to immunocytochemistry using antibodies against IGFR-like receptors, IGF2R, and insulin. A detailed description of the materials and methods used is provided in Example 1.

[0249] Expression of IGFR-like receptors in human islets of Langerhans. Confocal images of islets from a human donor immunofluorescently labeled with antibodies against IGFR-like receptors (red), IGF2R (M6PR) (green), and insulin (cyan) show typical IGFR-like receptor distribution in the Golgi compartment and partial colocalization with IGF2R and insulin (Figure 18).

[0250] Example 18: IGFR-like receptors indicate pancreatic beta cell dysfunction in humans Briefly, human pancreatic islets were obtained from healthy and diabetic donors and subjected to immunocytochemistry using antibodies against IGFR-like receptors and insulin. A detailed description of the materials and methods used is provided in Example 1.

[0251] LSM images of human pancreatic sections stained with antibodies against IGFR-like receptors (red) and insulin (green), with nuclei stained with DAPI (blue), show decreased insulin and increased IGFR-like receptor expression in diabetic (6.9% HbA1c) patients compared to non-diabetic (4.6% HbA1c) patients (HbA1c was used as an indicator of diabetic status, defined as the percentage of glycated hemoglobin in plasma; normal range: <5.9%) (FIG. 22).

[0252] Example 19: Subcellular localization of IGFR-like receptors in wild-type and knockout Min6 cells. Immunofluorescence images of Min6 cells stained with antibodies against IGFR-like receptors and insulin. Briefly, wild-type and knockout Min6 clones were obtained and examined by genomic PCR and Western blot. It is shown that IGFR-I protein is completely absent in the knockout Min6 clone (Figures 25A and B). WT and KO were then subjected to immunocytochemistry using antibodies against IGFR-like receptors (red) and insulin (green). Nuclei were stained with DAPI (blue) (Figure 24). Immunocytochemistry confirms the results of genomic PCR and Western blot. A detailed description of the materials and methods used is provided in Example 1.

[0253] Example 20: Effects of IGFR-like receptor knockout on insulin / IGF signaling in Min6(C22RIK) Briefly, Min6 knockout cells were generated by the CRISPR / Cas9-mediated knockout strategy described in Example 1 and studied by Western blot analysis using phospho-specific antibodies against IR / IGF1R and the downstream signaling molecules Ampk, AKT, and mTOR.

[0254] IGFR-like receptor knockout in Min6 cells leads to increased phosphorylation of IR and consequently AMPK, but not Akt or mTor (Figure 26). Min6 control and knockout clones were cultured under growth conditions (10% FCS and high glucose).

[0255] Example 21: IGFR-like Venus fusion knock-in Min6 cell line Briefly, IGFR-I Venus fusion knock-in Min6 cell lines were generated as described in Example 1 and subjected to immunohistochemistry using antibodies against IGFR-like receptors and the Venus fluorescent reporter.

[0256] IGFR-I Venus fusions (Venus detected by anti-GFP, green) colocalize with endogenous IGFR-I (anti-IGFR-I, red) in the Golgi and trans-Golgi regions. Small molecule compounds and biologics that inhibit homodimerization and homo- and heterodimerization via the transmembrane domain (TM) or cysteine-rich domain (CRD) of growth factor receptors (IPR009030), or that alter subcellular localization in the trans-Golgi, Golgi, lysosomal, and plasma membrane compartments, can be identified using a high-content screening approach with this knock-in Min6 cell line (Figure 27).

[0257] Example 22: Localization of IGFR-like receptors in endocrine precursor cells differentiated from induced pluripotent stem cells (iPSCs). Briefly, PDX1 + / NKX6.1 + Endocrine precursor cells were obtained and subjected to immunohistochemistry using antibodies against PDX1, NKX6.1, and IGFR-like receptors.

[0258] Confocal images of endocrine precursor cells stained with antibodies against PDX1 (light blue), NKX6.1 (red), and IGFR-like receptors (green) show immunolocalization of IGFR-like receptors with PDX1 and NKX6.1 (Figure 28). Nuclei were stained with DAPI (blue).

[0259] Example 23: Antibodies can be generated against peptides B and D of the IGFR-I ectodomain in murine Min6 insulinoma cells and the MCF7 human breast cancer cell line Rat monoclonal antibodies EIG-B 18B2 and EIG-B 10D9 were raised and used in Western blots against peptide B of the IGFR-I ectodomain, representing the epitope of SEQ ID NO: 4. Mouse monoclonal antibody EIG-D 26D7 was raised against peptide D of the IGFR-I ectodomain, representing the epitope of SEQ ID NO: 6.

[0260] These antibodies (EIG-B and EIG-D) show specific recognition of a protein band of approximately 130 kDa in Min6 (weak) and MCF7 (strong) cell lysates.

[0261] Example 24: SNPs in IGFR-like receptors are highly associated with coronary artery disease, LDL cholesterol, and type 2 diabetes in genome-wide association meta-studies Association of the human KIAA1324 gene encoding IGFR-I on chromosome 1 and IGFR-I SNPs with certain diseases, such as coronary artery disease, LDL cholesterol, and type 2 diabetes (Figure 31). These studies represent genome-wide association meta-studies of 500,000 subjects.

Claims

[Claim 1] 1. An in vitro method for screening for an antagonist of an IGFR-like receptor, comprising: (a) providing a stable cell line expressing said IGFR-like receptor; (b) contacting the cell line of (a) with a candidate antagonist; (c) measuring downstream signaling events of an IGFR-like receptor, wherein an antagonist is identified by an increase in said downstream signaling events of the IGFR-like receptor; A method comprising: the IGFR-like receptor comprises a sequence corresponding to SEQ ID NO: 1; the downstream signaling event is InsR phosphorylation, AMPK phosphorylation, mTOR phosphorylation, and / or AKT phosphorylation; the antagonist specifically binds to the IGFR-like receptor; said measuring of said downstream signaling events of said IGFR-like receptor in step (c) is compared to downstream signaling events of said IGFR-like receptor measured in a control cell line that has not been contacted with said candidate antagonist; The antagonist is an antibody or an siRNA. method.

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