WO1997035600A1 - Glycosylated amylins - Google Patents

Abstract

The invention discloses amylin and amylin-like peptides and the use of these peptides in assays for the diagnosis or evaluation of a subject. Also disclosed is the use of such assays to predict the onset of diabetes in patients who otherwise show normal glycemic control.

Description

GLYCOSYLATED AMYLINS

Field of the Invention

The present invention relates to the discovery of naturally-occurring,

glycosylated human amylin species and the use of assays for these glycoproteins for the diagnosis or evaluation of the state of insulin resistance in a subject, and for the use of

such assays to predict the onset of diabetes in patients who otherwise show normal

glycemic control.

Background ofthe Invention

Amylin

The structure and biology of amylin have previously been reviewed. See, for

example, Rink et al., Trends in Pharmaceutical Sciences, 14:113-118 (1993); Gaeta and Rink, Med. Chem. Res., 3:483-490 (1994); and, Pittner et al., J. Cell. Biochem., 55S:19-

28 (1994). Amylin is a 37 amino acid protein hormone. It was isolated, purified and chemically characterized as the major component of amyloid deposits in the islets of

pancreases of deceased human Type 2 diabetics (Cooper et aL, Proc. Natl. Acad. Sci.,

USA, 84:8628-8632 (1987)). The amylin molecule has two important post-translational

modifications: the C-terminus is amidated, and the cysteines in positions 2 and 7 are cross-linked to form an N-terminal loop. The sequence of the open reading frame of the

human amylin gene shows the presence of the Lys- Arg dibasic amino acid proteolytic cleavage signal, prior to the N-terminal codon for Lys, and the Gly prior to the Lys-Arg proteolytic signal at the CLAIMS-terminal position, a typical sequence for amidation by protein amidating enzyme, PAM (Cooper et aL, Biochem. Biophys. Acta, 1014:247-258 (1989)). Amylin is the subject of United States Patent No 5,367,052, issued on November 22, 1995.

In Type 1 diabetes, amylin has been shown to be deficient and combined replacement with insulin has been proposed as a preferred treatment over insulin alone in all forms of diabetes. The use of amylin and other amylin agonists for the treatment of

diabetes mellitus is the subject of United States Patent No. 5,175,145, issued on December 29, 1992. Pharmaceutical compositions containing amylin and amylin plus insulin are described in United States Patent No. 5,124,314, issued June 23, 1992.

Excess amylin action has been said to mimic key features of Type 2 diabetes and amylin blockade has been proposed as a novel therapeutic strategy. It has been disclosed in United States Patent No. 5,266,561, issued November 30, 1993, that amylin causes reduction in both basal and insulin-stimulated incoφoration of labeled glucose into glycogen in skeletal muscle. The latter effect was also disclosed to be shared by CGRP (see also Leighton, B. and Cooper, G.J.S., Nature, 335:632-635 (1988)). Amylin and CGRP were approximately equipotent, showing marked activity at 1 to 10 nM. Amylin is also reported to reduce insulin-stimulated uptake of glucose into skeletal muscle and reduce glycogen content (Young et al.. Amer. J. Physiol. 259:45746-1 (1990)). The treatment of Type 2 diabetes and insulin resistance with amylin antagonists is disclosed.

Both the chemical structure and the gene sequence of amylin have been said to

support the determination that it is a biologically active or "messenger" molecule. The chemical structure is nearly 50% identical to the calcitonin-gene-related peptides (CGRP), also 37 amino acid proteins which are widespread neurotransmitters with many potent-biological actions, including vasodilation. Amylin and CGRP share the ²Cys-⁷Cys disulphide bridge and the C-teπiiinal amide, both of which are essential for full biologic activity (Cooper et al. Proc. Natl. Acad. Sci., 857763-7766 (1988)).

Amylin reportedly may be one member of a family of related peptides which include CGRP, insulin, insulin-like growth factors, and the relaxins and which share common genetic heritage (Cooper, G.J.S.. et al.. Prog. Growth Factor Research 1:99-105 (1989)). The two peptides calcitonin and CGRP-1 share common parentage in the calcitonin gene where alternative processing of the primary mRNA transcript leads to the generation of the two distinct peptides, which share only limited sequence homology (about 30%) (Amara, S.G. et al.. Science, 229:1094-1097 (1985)). The amylin gene sequence is

typical for a secreted messenger protein, with the mRNA coding a prepropeptide with processing sites for production of the secreted protein within the Golgi or secretary

granules. Amylin is mainly co-localized with insulin in beta cell granules and may share the proteolytic processing enzymes that generate insulin from pro-insulin.

Amylin is primarily synthesized in pancreatic beta cells and is secreted in response to

nutrient stimuli such as glucose and arginine. Studies with cloned beta-cell tumor lines (Moore et aL, Biochem. Biophys. Res. Commun., 179(1) (1991)), isolated islets (Kanatsuka et aL, FEBS Lett., 259(1), 199-201 (1989)) and perfused rat pancreases (Ogawa et aL, J. Clin Invest., 85:973-976 (1990)) have shown that short pulses, 10 to 20

minutes, of nutrient secretagogues such as glucose and arginine, stimulate release of amylin as well as insulin. The molar amylin:insulin ratio of the secreted proteins varies between preparations from about 0.01 to 0.4, but appears not to vary much with acute stimuli in any one preparation. However, during prolonged stimulation by elevated glucose, the amylin:insul_n ratio can progressively increase (Gedulin et aL, Biochem. Biophys. Res. Commun., 180(l):782-789 (1991)). Thus, perhaps because gene expression

and rate of translation are independently controlled, amylin and insulin are not always

secreted in a constant ratio.

It has been discovered that certain actions of amylin are similar to known non- metabolic actions of CGRP and calcitonin; however, the metabolic actions of amylin discovered during investigations of this newly identified protein appear to reflect its primary biologic role. At least some of these metabolic actions are mimicked by CGRP, albeit at doses which are markedly vasodilatory (see, e.g.. Leighton et aL, Nature, 335:632-635 (1988)); Molina et aL, Diabetes, 39:260-265 (1990)).

The first discovered action of amylin was the reduction of insulin-stimulated incorporation of glucose into glycogen in rat skeletal muscle (Leighton et aL, Nature, 335:632-635 (1988)); the muscle was made "insulin-resistant". Subsequent work with rat

soleus muscle ex-vivo and in vitro has indicated that amylin reduces glycogen-synthase activity, promotes conversion of glycogen phosphorylase from the inactive b form to the active a form, promotes net loss of glycogen (in the presence or absence of insulin), increases glucose-6-phosphate levels, and can increase lactate output (see, e.g.. Deems et aL, Biochem. Biophys. Res. Commun., 181(1):116-120 (1991)); Young et aL, FEBS Letts, 281(1,2):149-151 (1991)). Amylin, like epinephrine, appears not to affect glucose transport per se ( e.g., Pitner et al.. FEBS Letts. 365(1):98-100 (1995)). Studies of amylin and insulin dose-response relations show that amylin acts as a noncompetitive or functional antagonist of insulin in skeletal muscle (Young et al.. Am. J. Physiol, 263(2).E274-E281 (1992)). Thus, at an effective concentration of amylin, no concentration of insulin can overcome amylin action. There is no evidence that amylin interferes with insulin binding to its receptors, or the subsequent activation of insulin

receptor tyrosine kinase (Follett et aL Clinical Research 39(1):39A (1991)); Koopmans et aL, Diabetologia, 34, 218-224 (1991)). The actions of amylin on skeletal muscle resemble those of adrenaline (epmephrine). However, while adrenaline's actions are believed to be mediated largely by cAMP, some workers have concluded that amylin's actions are not mediated by cAMP (see Deems et aL, Biochem. Biophys. Res. Commun., 181(1):116-120 (1991)). Others report that amylin does activate adenyl cyclase and increases cAMP in skeletal muscle (Pittner, et al., BBA 1267, 75-82 (1995); Moore and Rink, Diabetes 42:5,821 June (1993)), consistent with transduction of its effect on glycogen metabolism via cAMPdependent protein kinase phosphorylation of synthase and phosphorylase.

It is believed that amylin acts through receptors present in plasma membranes. It has

been reported that amylin works in skeletal muscle via a receptor-mediated mechanism that promotes glycogenolysis, by activating the rate-limiting enzyme for glycogen breakdown, phosphorylase a (Young, A. et __ FEBS Letts, 281:149-151 (1991)). Studies of amylin and CGRP, and the effect of selective antagonists, suggest that amylin acts via

its own receptor (Beaumont et al., Br. J. Pharmacol, 115(5), 713-715 (1995); Wang et aL, FEBS Letts., 219:195-198 (1991 b)), counter to the conclusion of other workers that amylin may act primarily at CGRP receptors (e.g.. Chantry et aL, Biochem. J., 277:139- 143 (1991)); Galeazza et aL, Peptides, 12:585-591 (1991)); Zhu et aL, Biochem. Biophys. Res. Commun., \11(2):11\116 (1991)). Recently, amylin receptors and their use in various methods for screening and assaying for amylin agonist and antagonist compounds were described in United States Patent No. 5, 264,372, issued November 23, 1993.

While amylin has marked effects on hepatic fuel metabolism in vivo, there is no general agreement as to what amylin actions are seen in isolated hepatocytes or perfused

liver. The available data do not support the idea that amylin promotes hepatic glycogenolysis, i.e., it does not act like glucagon (e.g.. Stephens, et al.. Diabetes, 40:395- 400 (1991)); Gomez-Foix et al.. Biochem J., 276:607-610 (1991)). It has been suggested that amylin may act on the liver to promote conversion of lactate to glycogen and to enhance the amount of glucose able to be liberated by glucagon (see Roden et al.. Diabetologia, 35:116-120 (1992)). Thus, amylin could act as an anabolic partner to insulin in liver, in contrast to its catabolic action in muscle.

The effect of amylin on regional hemodynamic actions, including renal blood flow, in conscious rats was recently reported (Gardiner et al.. Diabetes, 40:948-951 (1991)). The authors noted that infusion of rat amylin was associated with greater renal

vasodilation and less mesenteric vasoconstriction than is seen with infusion of human α-

CGRP. They concluded that, by promoting renal hyperemia to a greater extent than did

α-CGRP, rat amylin could cause less marked stimulation ofthe reninangiotensin system, and thus, less secondary angiotensin II-mediated vasoconstriction. It was also noted,

however, that during coninfusion of human α-⁸"³⁷ CGRP and rat amylin renal and mesenteric vasoconstrictions were unmasked, presumably due to unopposed vasoconstrictor effects of angiotensin II, and that this finding is similar to that seen during

coinfusion of human A-CGRP and human cc-^8'37 CGRP (id. at 951). In fat cells, contrary to its adrenalin-like action in muscle, amylin has no detectable actions on insulin-stimulated glucose uptake, incoφoration of glucose into triglyceride, C0₂ production (Cooper et aL, Proc. Natl Acad Sci., 85:7763-7766 (1988)) epinephrine-

stimulated lipolysis, or insulin-inhibition of lipolysis (Lupien J.R., and Young, A. A., "Diabetes Nutrition and metabolism - Clinical and Experimental", Vol. 6(1), pages 1318 (February 1993)). Amylin thus exerts tissue-specific effects, with direct action on

skeletal muscle, marked indirect (via supply of substrate) and perhaps direct effects on liver, while adipocytes appear "blind" to the presence or absence of amylin.

It has also been reported that amylin can have marked effects on secretion of insulin. In isolated islets (Ohsawa et al.. Biochem. Biophys. Res. Commun., 160(2):961-967

(1989)), in the perfused pancreas (Silvestre et al.. Reg. Pept., 31-23-31 (1991)), and in the intact rat (Young et al.. Mol. Cell Endocrinol^ 84:R1-R5 (1992)), some experiments indicate that amylin inhibits insulin secretion. Other workers, however, have been unable to detect effects of amylin on isolated β-cells, on isolated islets, or in the whole animal (see Broderick et al.. Biochem. Biophvs. Res. Comm.. Vol. 177:932-938 (1991) and references therein).

In preclinical experiments, a striking effect of amylin in vivo is stimulation of a rapid rise in plasma lactate, followed by a rise in plasma glucose (Young et aL, FEBS Letts,

281(1,2):149-151 (1991)). Evidence indicates that the increased lactate provides substrate for glucose production and that amylin actions can occur independent of changes in insulin or glucagon. In "glucose clamp" experiments, amylin infusions cause "insulin resistance", both by reducing peripheral glucose disposal, and by limiting insulin-mediated suppression of hepatic glucose output (e.g.. Frontoni et al.. Diabetes, 40:568-573 (1991)); Koopmans et al.. Diabetolosia. 34, 218-224 (1991)).

In lightly anesthetized rats which were fasted for 18 hours to deplete their stores of hepatic glycogen, amylin injections stimulated rises in plasma lactate from about 0.5 to 1.5 mM followed by a prolonged increase in plasma glucose levels from about 6 to 11 mM. These effects were observed for both intravenous and subcutaneous injections (Young et al.. FEBS Letts, 281(1,2):149-151 (1991)). The effects of amylin in fed animals differ quantitatively from its effects in fasted animals. In fed rats, with presumably normal liver glycogen stores, amylin causes a more pronounced and prolonged rise in plasma lactate; however, there is only a modest rise in plasma glucose. It has been suggested that amylin promotes the "return limb" of the Cori cycle, i.e.. muscle glycogen via breakdown to lactate provides substrate for hepatic gluconeogenesis

and glycogen production and probably triglyceride synthesis. Insulin drives the forward limb, __, uptake of glucose into muscle and production of muscle glycogen. Insulin and amylin can thus be seen as partners in regulating the "indirect" pathway of post-prandial hepatic glycogen repletion. "Insulin resistance" in muscle and liver may be under normal, physiologic regulation by amylin.

Amylin or amylin agonists potently inhibit gastric emptying in rats (Young et aL, Diabetologia 38 (6): 642-648 (1995)), dogs (Brown et al., Diabetes 43 (Suppl 1): 172A (1994))] and humans (Macdonald et aL, Diabetologia 38 (suppl 1): A32 (abstract 118)(1995)). The effect of amylin on gastric emptying appears to be physiological (operative at concentrations that normally circulate) since gastric emptying is accelerated in amylin-deficient type 1 diabetic BB rats (Young et aL, Diabetologia, supra: Nowak et al., JLab Clin Med 1994 Jan; 123 (1): 110-6 (1994)) and in rats treated with the selective amylin antagonist, ACI 87 (Gedulin et _ , Diabetologia 38 (suppl 1): A244 (1995)).

Control of nutrient release from the stomach is becoming recognized as an important component of overall fuel homeostasis. In human volunteers, over a range of carbohydrate concentrations in a liquid meal, energy release from the stomach was

remarkably constant at ~2 kcal/min (Brener et aL, Gastroenterology 85 (1): 76-82 (1983)), equivalent to ~500 mg glucose/min. This release rate is about the^" same as the rate of glucose disposal that insulin-sensitive individuals can attain at their peak plasma insulin concentrations (InM) (Young et aL, Am J Physiol 254 (2 Pt 1): E231-6 (1988)). Thus, the rate at which carbohydrate is released from the stomach and absorbed is normally matched to the rate at which it can be metabolized.

Several feedback loops may control nutrient efflux from the stomach. Peptides that are candidates as participants in such control loops should (1) change in response to meals, and (2) potently modulate gastric emptying. Peptides known to inhibit gastric

emptying are limited to amylin, cholecystokinin (CCK), glucagon-like peptide-I (GLP-1), secretin and gastrin releasing peptide/bombesin (GRP). GRP does not change with meals. Secretin is secreted in response to acid (but not nutrients) entering the duodenum. Ofthe peptides that are secreted in response to nutrients (amylin, CCK and GLP-1), only amylin and CCK are secreted in response to glucose ingestion. GLP-1 is secreted in response to fat ingestion. Ofthe hormones that might therefore mediate feedback control ofthe gastric release and subsequent absoφtion of glucose (amylin and CCK), amylin is the more potent (Young et al., Metabolism Clinical and Experimental 45 (1): 1-3 (1996)), and it appears that amylin may be a major regulator of carbohydrate absoφtion, at least in rodents (Young et al., Biochemical Society Transactions 23 (2): 325-331 (1995)).

Non-metabolic actions of amylin include vasodilator effects which may be mediated by interaction with CGRP vascular receptors. Reported in vivo tests suggest that amylin

is at least about 100 to 1000 times less potent than CGRP as a vasodilator (Brain et al.. Eur. J. Pharmacol, 183:2221 (1990); Wang et al.. FEBS Letts., 291:195-198 (1991)). Injected into the brain, or administered peripherally, amylin has been reported to suppress food intake (e.g.. Chance et al.. Brain Res., 539, 352-354 (1991)), an action shared with CGRP and calcitonin. The effective concentrations at the cells that mediate this action are not known. Amylin has also been reported to have effects both on isolated osteoclasts where it caused cell quiescence, and in vivo where it was reported to lower plasma calcium by up to 20% in rats, in rabbits, and in humans with Paget's disease (see, e.g.. Zaidi et aL. J. Bone Mineral Res., S293 (1990). From the available data, amylin seems to be 10 to 30 times less potent than human calcitonin for these actions. Interestingly, it was

reported that amylin appeared to increase osteoclast cAMP production but not to increase

cytosolic Ca²⁺, while calcitonin does both (Alam et al.. Biochem. Biophys. Res. Commun., 179(1): 134- 139 (1991)). It was suggested, though not established, that calcitonin may act via two receptor types and that amylin may interact with one of these.

Infusing amylin receptor antagonists may be used to alter glucoregulation. 8-37 CGRP is a demonstrated amylin blocker in vitro and in vivo (Wang et aL, Biochem.

Biophys. Res. Commun., 181(3):1288-1293 (1991)), and was found to alter glucoregulation following an arginine infusion in fed rats (Young et aL, Mo Cell. Endocrino., 84:R1-R5 (1992)). The initial increase in glucose concentration is attributed to arginine-stimulated glucagon secretion from islet alpha cells; the subsequent restoration of basal glucose is attributed to insulin action along with changes in other glucoregulatory hormones. When the action of amylin is blocked by preinfusion of *^{" 37}hCGRP, the initial glucose increase is not significantly different, but there is a

subsequent fall in glucose concentration to well below the basal level, which is restored only after some 80 minutes. Thus, glucoregulation following this challenge with an islet secretagogue was altered by infusion of an amylin receptor antagonist. Additionally, insulin concentrations were measured at half hour intervals and it was found that insulin concentration 30 minutes following the arginine infusion was almost twice as high in animals infused with an amylin receptor antagonist as in the normal controls. ^{8 37}CGRP is also an effective CGRP antagonist. However, very similar results were seen with another amylin antagonist, AC66, which is selective for amylin receptors compared with CGRP receptors (Young et __ Mol Cell Endocrino., 84:R1-R5 (1992)). These results are said to support the conclusion that blockade of amylin action can exert important therapeutic benefits in Type 2 diabetes.

It has also been discovered that, suφrisingly in view of its previously described renal vasodilator and other properties, amylin markedly increases plasma renin activity in intact rats when given subcutaneously in a manner that avoids any disturbance of blood pressure. This is important because lowered blood pressure is a strong stimulus to renin release. Amylin antagonists, such as amylin receptor antagonists, including those selective for amylin receptors compared to CGRP and/or calcitonin receptors, can be used to block the amylin-evoked rise of plasma renin activity. These unexpected findings support the determination that amylin antagonists will reduce plasma renin activity with consequent therapeutic benefit in hypertension and cardiac failure and other disorders associated with elevated, inappropriate or undesired renin activity. Moreover, the additional ability of amylin antagonists to favorably modulate insulin resistance and other common metabolic disorders frequently associated with hypertension and cardiac disease provides a particularly desirable therapeutic profile. The use of amylin antagonists to treat renin-related disorders is described in United States Patent No. 5,376,638, issued December 27, 1994.

Amylin Assays

Amylin-like immunoreactivity has been measured in circulating blood in rodents and humans by a variety of radioimmunoassays all of which use rabbit anti-amylin antiserum, and most of which use an extraction and concentration procedure to increase assay sensitivity. In normal humans, fasting amylin levels from 1 to lOpM and post-prandial or post-glucose levels of 5 to 20pM have been reported (e.g.. Hartter et aL, Diabetologia, 34:52-54 (1991)); Sanke et aL, Diabetologia, 34:129-132 (1991)); Koda et aL, The Lancet, 339:1179-1180 (1992)). In obese, insulin-resistant individuals, post-food amylin levels can go higher, reaching up to about 50pM. For comparison, the values for fasting and post-prandial insulin are 20 to 50pM, and 100 to 300 pM respectively in healthy

people, with perhaps 3-to 4-fold higher levels in insulin-resistant people.' In Type 1 diabetes, where beta-cells are destroyed, amylin levels are at or below the levels of detection and do not rise in response to glucose (Koda et __ The Lancet, 339, 1179-1180

(1992)). In normal mice and rats, basal amylin levels have been reported from 30 to lOOpM, while values up to 600pM have been measured in certain insulin-resistant, diabetic strains of rodents (__,, Huang _ aL, Hypertension, 19:1-101-1-109 (1991)); Gill et aL, Life Sciences, 48:703-710 (1991).

Patients with Type 1 diabetes, in addition to a lack of insulin, are reported to have marked amylin deficiency. As noted above, data show that amylin expression and

secretion by pancreatic beta-cells is absent or well below normal in Type 1 diabetes. In several animal models of Type 1 diabetes, amylin secretion and gene expression are depressed (Cooper et aL, Diabetes.497-500 (1991); Ogawa et aL, J. Clin. Invest., 85:973- 976 (1990)). Measurements of plasma amylin in Type I diabetic patients show that amylin is deficient in these patients after an overnight fast, and that a glucose load does not elicit any increase in amylin levels (Koda et al.. The Lancet, 339:1179-1180 (1992)).

Two monoclonal antibody-based sandwich assays have been developed for

measurement of amylin immunoreactive material. Fineman, et al., Diabetologia 1994; 37 (Suppl 1): A52 (abstract #197); Percy et aL, Diabetologia 1994, 37 (Suppl 1): A117 (abstract #455). These assays use the same detection antibody (F025), which binds to the C-terminal region of amylin, but use different capture antibodies with distinct eptiope specificity. See "Antibody Assay for Amylin," Application No. PCT/US93/04651, published November 25, 1993. Capture antibody F024 binds to rat and human amylin and requires an intact disulfide bond between residues 2 and 7 for binding. Capture antibody F002 binds only to human amylin, which differs significantly from the rat sequence between residues 23 to 29. Characterization of immunoreactive material in human plasma measured by each assay revealed that the F024 assay measures only human amylin. The F002 assay, however, measured human amylin and at least two

additional species of higher molecular weight "amylin-like" peptides (i.e., peptides that are immunoreactive with F002 anti-amylin antibodies). These additional peptides were reportedly 5000-8000 daltons in molecular weight as assessed by SDS-PAGE and Western blot, and were postulated to be incompletely processed pro-amylin molecules. Fineman et al.. supra.

Using these two assays as well as assays measuring proinsulin and 32,33 split proinsulin, hormone levels were measured in 20 normal subjects and 13 newly diagnosed patients^" with Type II diabetes mellitus, before and after an oral glucose load. Id. Analysis of fasting plasma samples indicated that, in newly diagnosed patients with Type II diabetes compared with normal controls, high molecular weight "amylin-like" peptides were elevated along with insulin, proinsulin and 32,33 split proinsulin. It was concluded that increased levels of incompletely processed insulin are accompanied by increased levels of high molecular weight "amylin-like" peptides in newly diagnosed Type II

diabetes.

The heterogeneity of plasma amylin immunoreactive material was confirmed by reversed phase HPLC of extracted human plasma and pancreas. S_ee Pittner et al., J. Cell.

Biochem. 55S:19-28 (1994). From both sources a peak corresponding to amylin was preceded by two peaks of material that were bound by antibodies to epitopes of the mid (F002) and C-terminal (F025) region of the amylin molecule, but not the NH₂-teπninal (F024) region. The early-eluting molecules were found to constitute about 60% of circulating amylin immunoreactivity in normal fasting and glucose-dosed individuals. Summary ofthe Invention

The assay using antibodies F024 and F025 is a measure of amylin in plasma, while the assay using antibodies F002 and F025 assay measures total plasma amylin immunoreactivity, that is, amylin plus other, higher molecular weight immunoreactive

peptides. We have discovered that these early eluting amylin immunoreactive materials are not incompletely processed proamylin, but comprise three predominant glycosylated species of human amylin having O-linked carbohydrate groups in the NH₂-terminal region. The carbohydrate group of one glycosylated amylin species was discovered to be a monosialated pentasaccharide linked at the threonine-9 amino acid and having the following core disaccharide structure: Gal(βl-3)-GalNac(α-Thr-9). The second glycosylated amylin has a similar oligosaccharide structure linked at the threonine-6 amino acid, while the third has similar oligosaccharide structures linked at both the

threonine-6 and threonine-9 amino acid residues. Suφrisingly these glycosylated amylins were found to be inactive at concentrations up to 37 nM in amylin receptor binding and other bioassays for human amylin.

In one aspect, the invention is directed to glycosylated amylins, including but not limited to human glycosylated amylins, such as threonine-6, threonine-9, and threonine- 6/threonine-9 glycosylated amylins.

In another aspect, the invention is directed to amylins, including but not limited to human amylins, with threonine-linked monosialated pentasaccharides having the following structure: GlcNAc(βl-3) NeuAc(α2-6)

\ \

Gal(β 1 -3)-GalNAc(α 1 -Threonine)

/

GlcNAc(βl-ό)

Alternative carbohydrate structures are possible, but are believed to be unlikely. A possible alternative structure for the threonine-linked monosialated pentasaccharides is:

NeuAc(α2-6)

\

GalN Ac(α 1 -3)-GalN Ac(β 1 -3)-Gal(β 1 -3)- GalNAc(α 1 -Threonine)

In another aspect, the invention is directed to the preparation of antibodies and immunoassays specific to these glycosylated amylins, particularly the threonine-6, threonine-9, and threorι e-6/threonine-9 glycosylated amylins. The present invention provides novel antibodies, preferably monoclonal antibodies, and antibody fragments which can be produced in mice or by recombinant cell lines or by hybrid cell lines, the antibodies being characterized in that they have certain predetermined specificity to the herein described glycosylated amylins, including the threonine-6, threonine-9, and tru"eonine-6/threonine-9 glycosylated amylins. By virtue of their specificity, such

antibodies and antibody fragments are useful in methods for the purification of glycosylated amylins, including the threonine-6, threonine-9, and threonine-6/threonine-9 glycosylated amylins, and in the immunoassay of these target antigens to determine the presence or amount of glycosylated amylins in a test sample. In another aspect, antibodies which are not specific to glycosylated amylins are used for detection by immunoassay. Thus, antibodies which are neither specific to non- glycosylated amylin nor to the glycosylated amylins, such as the threonine-6, threonine-9, and threonine-6/threonine-9 glycosylated amylins, are useful in the immunoassay of total

amylin immunoreactivity. Such antibodies include those described herein for the F002 assay, which bind to the C-terminus of amylin and to the middle portion of the amylin molecule. Using the results from such a total amylin immunoreativity assay, and the results from an amylin-specific assay, such as the F024 assay described herein, it is possible to determine the presence or amount of the glycosylated amylins in a test

sample.

In still another aspect, the invention is directed to the use of an immunoassay or immunoassays to determine the presence or amount of these glycosylated amylins, including the threonine-6, threonine-9, and threoιιine-6/threonine-9 glycosylated amylins

to diagnose or evaluate the state of insulin resistance in a subject, for example, in the diagnosis or evaluation of gestational diabetes. Additionally, and importantly, the invention provides for the use of such assays to predict the onset of diabetes in patients who otherwise show normal glycemic control.

Brief Description ofthe Drawings

FIGURE 1 shows the heterogeneity of amylin immunoreactive material in extracts of human pancreas (Fig. IA) and human plasma (Fig. IB) detected by HPLC fractionation. The arrows denote the retention time of synthetic human amylin. The peaks of amylin immunoreactive material are designated peaks 1, 2 and 3. FIGURE 2 shows a the final microbore HPLC purification step. for the pancreatic high molecular weight amylin-like peptides. Traces 1 and 2 show peaks 1 A/B and peak 2, respectively. The small peak labeled 2m, also was immunoreactive.

FIGURE 3 is a Western Blot of purified pancreatic amylin immunoreactive materials from peaks 1, 2 and 3. The two outer lanes ("AMLN") are synthetic human

amylin.

FIGURE 4 shows the results of a reversed phase HPLC of fragments 1-11 and 12-37

from tryptic digests of peak IB amylin immunoreactive material and synthetic amylin.

FIGURE 5 shows the results from the electrospray mass spectrometry of tryptic 12- 37 fragments from amylin peak IB (Fig. 5 A) and synthetic amylin (Fig. 5B).

FIGURE 6 shows the results of an HPLC fractionation of peak IB amylin immunoreactive material following treatment with a chemical deglycosylation agent. Shown are HPLC profiles of: A, untreated peak IB; B, TFMSA-digested peak IB; C, TFMS A-digested synthetic human amylin.

FIGURE 7 is a laser desoφtion mass spectrum (MALTI-ITMS) of material from

peak IB.

FIGURE 8 is a measure of amylin immunoreactivity in response to a 75 gram oral glucose load in 112 pregnant women. The solid lines are data from the specific amylin

assay (F024) and the dashed lines are from the HMW-ALPS assay (F002). Diamond symbols represent subjects that were diagnosed with gestational diabetes (n=56) by WHO criteria. The square symbols represent data from age and BMI matched controls (n=56). Results are graphed as means +/- SEM. FIGURE 9 is a measure of amylin immunoreactivity in response to a 75 gram oral glucose load at the baseline visit The solid lines are data from the specific amylin assay

(F024) and the dashed lines are from the HMW-ALPS assay (F002). Square symbols represent subjects that remained normoglycemic (n=219) at the 4 year follow up visit. Diamonds represent data from subjects that became impaired glucose tolerant or Type II diabetic at follow up (n=l 9). Results are graphed as means +/- SEM.

Detailed Description of the Invention

The present invention contemplates the isolation and purification of high molecular weight amylin-like peptides, the use of these peptides, and the use of assays for their detection and/or measurement in the diagnosis or evaluation of insulin resistance and prediabetes. As used herein, the terms "high molecular weight amylin-like peptides" and "HMW-ALPS" refer to the amylin immunoreactive materials which have been discovered to exist in the human pancreas and human plasma, and found to be glycosylated amylins, predominantly the threonine-6, threonine-9, and threonine- 6/threonine-9 glycosylated amylins described herein.

Glycosylation has been found in a number of larger peptide and protein hormones, i.e., those having a molecular weight of about 18,000Kd or greater. These include: leuteinizing hormone, thyrotropin, activin, human chorionic gonadotropin, hepatocyte growth factor, human granulocyte colony stimulating factor, and human tumor necrosis factor beta. In the latter four there is at least one O-linked oligosaccharide. In the case of human granulocyte colony stimulating factor, hormonal potency and stability are reduced upon removal of the O-glycan chain (Nissen, Eur. J. Cancer, 30A Suppl. 3:S12-4 (1994)). In fact it has been suggested that the activities of different cytokines are regulated by glycosylation, through changes in diffusability, tissue distribution, pharmacokinetics, and by targeting different populations of responsive cells (Opdenakker, et al., "Cells regulate the activities of cytokines by glycosylation," FASEB Journal 9:453-457 (1995)). This is, however, believed to be the first report of glycosylation of a small peptide hormone, amylin having a molecular weight of about 3900 Kd.

Plasma amylin from large numbers of normal, glucose intolerant, and diabetic subjects was measured using two types of assays (the F002 and the F024 assays) in parallel, in a number of experimental protocols. The term "F024 assay" refers to an immunoassay that measures only human amylin. The term "F002 assay" refers to an immunoassay that can measure both amylin and HMW-ALPS. Methods for the

preparation and use of amylin-specific and amylin total reactivity immunoassays, including assays such as the F024 and F002 immunoassays, are described in "Antibody Assay for Amylin," Application No. PCT/US93/04651, published November 25, 1993. Results from several such studies are described in Examples Twelve and Thirteen. We discovered that the elevations of plasma concentrations of high molecular weight amylin- like peptides are more closely associated with diabetic and prediabetic states. That is, that values from total amylin assays that can measure both amylin and HMW-ALPS (e.g., the F002 assay) correlate better with disease than values from an amylin-specific assay that does not measure high molecular weight amylin-like peptides (e.g., the F024 assay).

We have also discovered that the best disease correlation is found with plasma levels of high molecular weight amylin-like peptides calculated as the difference between the

values ofthe two assays.

An assay that measures high molecular weight amylin-like peptides concentrations directly would give the same value obtained by the latter subtraction method, but would have greater accuracy and precision and be easier to perform. The utility of measuring high molecular weight amylin-like peptides and the development and use of an improved direct assay for high molecular weight amylin-like peptides is described herein. Also described are the purification and characterization of high molecular weight amylin-like peptides from natural sources, and their use in production of glycosylated amylin-specific

monoclonal antibodies and development of a specific immunoassay for high molecular weight amylin-like peptides. Such glycosylated amylin-specific monoclonal antibodies may also be used to detect glycosylated amylins in tissue by immunochemical staining.

Examples One through Three describes a set of experimentals wherein three high

molecular weight amylin-like peptides, as well as unglycosylated amylin, were purified to homogeneity from extracts of human pancreas by a series of steps, including combined immunoaffinity chromatography and reversed phase HPLC. The unglycosylated species was found to be identical to synthetic human amylin as assessed by HPLC retention time, NH₂-terminal amino acid sequencing, Western Blotting, electrospray mass spectrometry, and tryptic peptide mapping. The three high molecular weight amylin-like peptides were discovered to be apparently normally processed amylin, but with O-linked mucin-type carbohydrate groups attached to one or both, respectively, of two closely-spaced threonines (amino acid residues 6 and 9) of the NH₂-terminal region of amylin. This identification was based on mass spectrometry, amino terminal amino acid sequencing, chemical deglycosylation, Western Blotting, and glycosidase digestion studies, all as described in Examples Five through Nine.

A detailed study of one high molecular weight amylin-like peptide (corresponding to

the material in peak IB of the HPLC trace attached as Figure 2, the most abundant glycosylated amylin species, showed that the carbohydrate was a monosialated pentasaccharide linked to the amylin peptide at the ninth amino acid (threonine-9) having the core disaccharide structure, Gal(βl-3)-GalNac(α-threonine-9), and the following

formula:

GlcN Ac(β 1 -3)[GlcNAc(β 1 -6)]Gal(β 1 -3) | euAc(α2-6)]GalNAc(α--threorιine-9)

The two other purified glycosylated amylins have similar or identical carbohydrate structures, as previously described, but are linked to the sixth amino acid of amylin (threonine-6), corresponding to the material in peak 2A of the HPLC trace attached as Figure 2, or at both threonine-6 and at the threonine-9, corresponding to the material in peak 1 A of the HPLC trace attached as Figure 2.

High molecular weight amylin-like peptides species were purified to homogeneity from human pancreas and may be prepared as set forth herein. The course of the

purification, described in Examples One through Three, was monitored by immunoassay. Purified unglycosylated amylin (peak 3) had the same retention time and co- chromatographed with synthetic human amylin by reversed phase chromatography. Additionally both had the same mass as determined by electrospray mass spectrometry. Three ofthe more abundant amylin immunoreactive species (material from peaks 1 A, IB, and 2) were isolated. Several other less abundant immunoreactive species also were observed on many of the HPLC traces, indicating microheterogeneity. Western Blotting

of the purified amylin species after separation by gel electrophoresis in the presence of

SDS is shown in Figure 3. As noted, the peak 3 amylin immunoreactive material co- migrated with synthetic amylin at an approximate molecular weight 4000, while the isolated material from peaks IB and 2A both appeared larger by about 1000 Kd, and the

peak IA material larger by about 2000 Kd. The sites of modification ofthe predominant endogenous amylin immunoreactive materials are depicted in Table 1 below.

TABLE 1. Sites of modification of endogenous human amylins. The amino terminal region of amylin is shown with vertical bars indicating those threonine residues where modifications are located.

PEAK ~MW 1 10

3 4000 K-C- N- T- A- T- C- A- T- Q-R- L-

IA 6000 K-C-N-T-A--C-C-A-Ψ-Q-R-L-

IB 5000 K-C-N-T- A-T- C-A--0-Q-R-L-

2A 5000 K-C- N- T- A- -fr- C- A- T- O-R- L-

In further characterization, amylin immunoreactive material from peak IB and synthetic amylin were both digested with trypsin, and the products of any cleavage, which would be expected to occur at Arg-11 of amylin, were isolated by reversed phase HPLC. A large fragment (12-37) from both peptides was identical by retention time (as shown in Figure 4), by mass as measured by electrospray mass spectrometry (as shown in Figure 5), and by amino terminal amino acid sequencing. However the (1-11) fragment from peak 1 B (confirmed by amino acid sequence) eluted in an earlier, broader peak than the corresponding fragment from synthetic amylin (Figure 4).

The high molecular weight amylin-like peptides were then subjected to chemical

deglycosylation/exoglycosidase digestion, as described in Examples Eight and Nine. Material from peak IB was digested with trifluoromethanesulfonic acid (TFMSA), a reagent that efficiently removes all sugars except the peptide-linked sugar from O-linked glycopeptides (Edge, et al., Anal. Biochem. 118:131-137 (1981)). This was followed by

digestion with α-N-acetylgalactosaminidase, the intent being to remove any remaining sugar residues. As shown in Figure 6, TFMSA-treated material from peak IB eluted later by reversed phase HPLC. But it also eluted earlier than synthetic amylin, and did not result in recovery of the F024 epitope. Western Blotting of the isolated HPLC peak of TFMSA-digested material from peak IB was carried out as described in Example 5, and confirmed that the molecular weight was lower, but still above that of synthetic amylin. Attempted removal of any residual sugar from the TFMSA-digested amylin

immunoreactive material from peak IB with α-N-acetylgalactosaminidase resulted in unmasking of the F024 epitope as evidenced by development of positivity in the F024/F025 immunoassay (see Table 3 in Example Nine), and α-N-a«tylgalactosamine

was identified as the peptide-linkage sugar. However, untreated material from peak IB was resistant to this exoglycosidase digestion, apparently due to the presence of substituent sugars. The F024 epitope was also restored in material from peak IA after it was digested with TFMSA and the treated with α-N-acetylgalactosaminidase. On the assumption that the peak 1 A material has carbohydrate groups attached at both threonines 6 and 9, it was determined that α-N-acetylgalactosamine (α-GalNAc) is the peptide- iinkage sugar at both positions. Using these methods, it was established that the materials of peaks IB and IA were glycosylated, and that the peptide linkage sugars were O-linked α-N-acetyl-D-galactosamine.

The high molecular weight amylin-like peptide were also studied by mass ^"spectrometry. Material from peaks IA and IB were analysed by matrix-assisted laser desoφtion/ionization quadrupole ion trap mass spectrometry. The technique produces preferential fragmentation at glycosidic bonds, removing variable numbers of sugar

molecules from the parent glycopeptide. The resulting MALDI/ITMS spectrum showed several peaks with masses differing by the discrete masses ofthe removed sugar residues, the lowest mass being that of the unsubstituted peptide. The spectrum from the peak IB material analysis is shown in Figure 7. The masses of five species are identified that of

unsubstituted human amylin at 3903.73, those of amylin plus a Hex-HexNAc core disaccharide with and without one and two additional HexNAcs, and an additional N- acetyl neuraminic acid (sialic acid) to give the starting glycopeptide mass of 4966.64. The profile indicates that the oligosaccharide ofthe peak IB material is a pentasaccharide containing a hexose-HexNAc core disaccharide (consistent with the above assignment of α-GalNAc as the peptide linkage sugar), a terminally located N-acetylneuraminic acid (sialic acid), and two N-acety exosamines (HexNAcs) that could occupy either terminal or substituted positions. An essentially identical spectrum was obtained with peak 2 material (not shown), and it was determined that the material from peaks 2 and IB may have the same oligosaccharide, but attach to different residues of the peptide (threonine-6 vs. threonine-9).

Further characterization of the carbohydrate structure of the peak IB material was obtained by digestions with mixtures of specific glycosidases, the primary endpoint being

recovery ofthe FO24 epitope dependent upon complete removal of all sugar residues. As shown in Table 4 of Example Nine, incubation of peak IB material with a mixture of two glycosidases, sialidase and O-glycosidase (endo-α-N-acetylgalactosaminidase), was sufficient to effect complete deglycosylation of the peak IB material, confirπύng the presence ofthe sialic acid detected by mass spectrometry. The strong cleavage observed with NANase III or A. ureafaciens sialidase combined with trace or no cleavage with C.

perfringens and Newcastle Disease virus sialidases helped in the identification of an α2-6 linkage, probably to the α-galNAc core sugar, although an α2-3 or 2-4 linkage remains a possibility. Digestion by O-glycosidase supported the conclusion that the hex-hexNAc core unit detected by mass spectrometry is gal(βl-3)galNAc(α-), for O-glycosidase

specifically removes this disaccharide unit. This is consistent with the determination in the TFMSA/ α-N-acetylgalactosaminidase digestion study reported above that the peptide linkage sugar is α-galNAc. It was suφrising that complete cleavage proceeded in the absence of added N-acety exosaminidase, since O-glycosidase would not be expected to cleave a core disaccharide having two hexNAc substituents as identified by mass spectrometry. A possible explanation for this finding is that O-glycosidase can, in fact, cleave certain substituted core disaccharides. Another explanation is that an N- acetylhexosaminidase is present as a contaminant in the sialidase or glycosidase preparations. However, different sources and combinations of sialidase and O- glycosidase (both natural and recombinant) were equally effective in carbohydrate

removal (Table 4). Since the maximum unmasking of the F024 epitope obtained was 38%, another possibility is that the peak IB material is heterogeneous, containing a proportion of structures that lack the peripheral N-acetylhexosamines. However, the expected molecular weight heterogeneity was not revealed by Western Blot, and the addition of various types of N-acety exosaminidases to the glycosidase digestion mixtures failed to produce greater unmasking of the F024 epitope. The following proposed partial structure for peak IB is consistent with the glycosidase digestion and mass spectrometry data:

[NeuAc, HexNAc₂]Gal(βl-3) GalNAc(αl-)Thr-9

The glycosylated amylins were tested in various receptor binding and bioassays as

described in Example Eleven and found to be inactive, at least at physiologically relevant concentrations. Isolated high molecular weight amylin-like peptides and unglycosylated amylin were tested for binding to three receptors of the calcitonin family in purified cell membranes. As shown in Table 5 in Example Eleven, neither the peak 1 nor the peak 2 material at 37 nM showed significant binding to any receptor, while the IC₅₀ for the peak 3 material and synthetic amylin ranged from 200 to 400 pM in the amylin receptor assay

(rat nucleus accumbens), indicating that glycosylated amylins are neither agonists nor antagonists at physiological concentrations mediated through the tested receptors (amylin, calcitonin, or calcitonin gene related peptide). Glycosylated amylins were additionally tested for their ability to inhibit glycogen synthesis in insulin-stimulated rat soleus muscle (Young, et al., Am. J. Physiol. 263, E274-E281 (1992)). No inhibition of glycogen synthesis by a sample containing 20 nM

of mixed amylin immunoreactive material from peaks 1 and 2 was observed, while the same concentration of synthetic amylin and purified peak 3 amylin showed strong

inhibition. Also 80 nM glycosylated amylins exhibited no antagonism against 5 nM amylin in the assay.

Notwithstanding their suφrising lack of bioactivity in these assays, the high molecular weight amylin-like peptides may serve an important but as yet unproved puφose m vivo. For example, glycosylation may aid in the transport of amylin across the blood-brain barrier. In any event, the high molecular weight amylin-like peptides are useful for the preparation of antibodies which bind to them, and these antibodies are in turn useful in immunoassays for their detection.

Thus, the present invention also contemplates antibodies and immunoassays useful

for detecting the presence or amount of the high molecular weight amylin-like peptides,

for example, the threonine-6, threonine-9, and threon e-6/threonine-9 glycosylated amylins, described herein. The general methodology and steps of antibody assays are described by David and Greene, U.S. Patent 4,376,110, entitled "Immunometric Assays Using Monoclonal Antibodies; Antibodies. A Laboratory Manual. Cold Spring Harbor Laboratory, Chapter 14 (1988); Radioimmunoassay and related methods", A. E. Bolton and W.M. Hunter, Chapter 26 of Handbook of Experimental Immunology. Volume I, Immunochemistry, edited by D.M. Weir, Blackwell Scientific Publications, 1986; "Enzyme immunoassays: heterogeneous and homogeneous systems", Nakamura, et al., Chapter 27 of Handbook of Experimental Immunology. Volume 1, Immunochemistry, edited by D.M. Weir, Blackwell Scientific Publications, 1986; and, Current Protocols in

Immunology. Chapter 2, Section I, Edited by John E. Coligan, et aL, (1991). These publications, and all other publications referenced herein, are hereby incoφorated in their entirety by reference. In all such assays controls may be included which are designed to

give positive and/or negative results. For example, and depending on the nature of the immunoassy, the test may include a known amount of one or more ofthe high molecular weight amylin-like peptides as a positive control, or unglycosylated amylin as a negative control, or both controls.

Preferably, the immunoassay is a sandwich immunoassay, and comprises the steps of

(1) reacting an immobilized high molecular weight amylin-like peptides antibody, preferably a monoclonal antibody, and a labeled high molecular weight amylin-like peptides antibody, preferably a monoclonal antibody, which recognizes a different site

from that recognized by the immobilized antibody, with a sample containing or suspected of containing high molecular weight amylin-like peptides so as to form a complex of immobilized antibody high molecular weight amylin-like peptides/labeled antibody, and

(2) detecting the presence or amount of high molecular weight amylin-like peptides by determining the presence or amount of label in the complex. In this process the reaction of the immobilized antibody and labeled antibody with the sample may be carried out either simultaneously or separately.

Suitable anti-high molecular weight amylin-like peptides antibodies, preferably monoclonal antibodies, in accordance with the present invention are specific to high molecular weight amylin-like peptides over unglycosylated amylin. Such antibodies can be prepared from hybridomas by the following method. High molecular weight amylin- like peptides or fragments thereof, including those fragments described in Example 10, in an amount sufficient to promote formation of antibodies, are emulsified in an adjuvant such as Freund's complete adjuvant. The immunogen may be either crude or partially purified, and is administered to a mammal, such as mice or rats, by intravenous, subcutaneous, intradermic, intramuscular, or intraperitoneal injection. After completion of an appropriate immunization protocol, Le., one that is sufficient to promote an immune

response to the immunogen in the animal, animal spleens are harvested. Myeloma cells having a suitable marker such as 8-azaguanine resistance are used as parent cells, which are then fused with the antibody-producing spleen cells to prepare hybridomas. Suitable media for the preparation of hybridomas according to the present invention include media such as Eagle's MEM, Dulbecco's modified medium, and RPMI- 1640. Myeloma parent cells and spleen cells can be suitably fused at a ratio of approximately 1 :4. Polyethylene glycol (PEG) can be used as a suitable fusing agent, typically at a concentration of about 35% for efficient fusion. Resulting cells may be selected by the HAT method described in Littlefield, J. W., Science 145:709 (1964). Screening of obtained hybridomas can be performed by conventional methods including an immunoassay using culture supernatant from the hybridomas to identify a hybridoma clone producing the objective immunoglobulin. The obtained antibody-producing hybridoma is then cloned using known methods such as the limiting dilution method. In order to produce, for example, high molecular weight amylin-like peptides monoclonal antibodies of the present invention, the hybridoma obtained above may be cultured either in vitro or in vivo. If the hybridoma is cultured in vitro, the hybridoma may be cultured in the above-mentioned media supplemented with fetal calf serum (FCS) for 3-5 days and monoclonal antibodies recovered from the culture supernatant. If the hybridoma is cultured in vivo, the hybridoma may be implanted in the abdominal cavity of a mammal, and after 1-3 weeks

the ascitic fluid collected to recover monoclonal antibodies therefrom. Much larger quantities ofthe monoclonal antibodies can efficiently be obtained using in vivo cultures than in vitro cultures and, thus, in vivo cultures are preferred. The monoclonal antibody obtained from the supernatant or ascitic fluids can be purified by conventional methods

such as ammonium sulfate-fractionation, Protein G-Sepharose column chromatography, or their combinations.

In addition to the above methods for generating monoclonal antibodies specific for the high molecular weight amylin-like peptides ofthe invention, one or more monoclonal

antibodies that are specific for the oligosaccharide side chains of the high molecular weight amylin-like peptides may be prepared and used in a monoclonal antibody-based sandwich immunoassay or other type of immunoassay or for chromatographic puφoses. Monoclonal antibodies specific to the predominant oligosaccharide of glycosylated amylins are generated using the basic procedures described above for immunization of mice, serum testing, fusions with myeloma cells, clonal expansion, and screening for specific, high affinity antibody-producing cells, but using the oligosaccharide side chain coupled to an appropriate carrier as immunogen. One form of immunogen, for example, is a synthetic amylin oligosaccharide coupled to thyroglobulin. Another useful immunogen is a mixture of glycosylated amylins partially purified from extracts of human pancreas using methods described herein or, for example, immunoaffinity chromatography. Screening for antibodies to glycosylated amylin carbohydrate in serum and hybridoma cell supernatants may be carried out by immobilization of the test antibodies on goat anti-mouse IgG-coated microtiter wells. After incubation with purified or partially purified ^,2ϊI-labeled glycosylated amylin, detection of bound

glycopeptide may be effected by ^12JI detection. An alternative screening method comprises capture of antibodies by immobilized glycosylated amylins followed by detection using an anti-mouse IgG reagent. Alternatively, existing monoclonal antibodies known to bind to specific sugars may be evaluated for binding to the high molecular weight amylin-like peptides ofthe invention for use in immunoassays or chromatographic procedures, for example. A specific monoclonal sandwich immunoassay may comprise one amylin oligosaccharide-specific monoclonal antibody paired with F025-27 or another human amylin-specific monoclonal antibody.

Antibodies, or the desired binding portions thereof including F(ab) and Fv fragments, along with antibody-based constructs such as single chain Fv's can also be generated using processes which involve cloning an immunoglobulin gene library in vivo. See, e.g., Huse et al.. Generation of a Large Combinatorial Library of the Immunoglobulin Repertoire in Phage Lambda. (1989) Science 246:1275-1281. Using these methods, a vector system is constructed following a PCR amplification of messenger RNA isolated from spleen cells with oligonucleotides that incoφorate restriction sites into the ends of the amplified product Separate heavy chain and light chain libraries are constructed and may be randomly combined to coexpress these molecules together and screened for antigen binding. Single chain antibodies and fragments may also be prepared by this

method. As noted above, a sandwich immunoassay for high molecular weight amylin-like peptides can suitably be prepared using an immobilized anti-high molecular weight amylin-like peptides monoclonal antibody and a labeled anti-high molecular weight

amylin-like peptides monoclonal antibody. Additionally, however., anti-amylin antibodies which recognize other than the N-terminal region in the area of amino acids 6 to 9, for example, such as those that recognize the middle portion of the C-terminal portion of amylin and can therefore bind to the high molecular weight amylin-like peptides of the invention, can also be used as the immobilized or labeled antibody in conjunction with an anti-high molecular weight amylin-like peptides monoclonal antibody to form the antibody/antigen/antibody sandwich. If an anti-amylin antibody is used in the sandwich immunoassay to form a part ofthe antibody pair, it is preferably the labeled antibody.

Antibodies according to the present invention are suitably immobilized on commercially available carriers for the antigen-antibody reaction including beads, balls,

tubes, and plates made of glass or synthetic resin. Suitable synthetic resins include

polystyrene and polyvinyl chloride. Anti-high molecular weight amylin-like peptides antibodies, and anti-amylin monoclonal antibodies, as the case may be, are suitably absorbed onto the carrier by allowing them to stand at 2-8°C overnight in 0.05M carbonate buffer, pH 9-10, preferably about pH 9.5. The immobilized anti-high molecular weight amylin-like peptides and/or anti-amylin monoclonal antibody can be stored cold in the presence of preservatives such as sodium azide. Both monoclonal and polyclonal antibodies can be immobilized onto carriers using this method. Labeled anti-high molecular weight amylin-like peptides and anti-amylin antibodies in accordance with the present invention can suitably be prepared by labeling anti-high molecular weight amylin-like peptides and anti-amylin antibodies with any substance

commonly used for an immunoassay including radioisotopes, enzymes, and fluorescent substrates. Radioisotopes and enzymes are preferably used. When radioisotopes are used

as labels, the antibody is preferably labeled with ^,25I using conventional methods such as the CMoramine T method. Hunter et al.. Nature 194:495 (1962). When enzymes are used as labels, the antibody is labeled with an enzyme such as horseradish peroxidase, β- D-galactosidase, or alkaline phosphatase by conventional methods including the

maleimide method and the Hingi method. Ishikawa et al, J. Immunoassay 4:1(1983).

The activity of the label can be detected by conventional methods. If radioisotopes are used as labels, the activity ofthe label can be detected using an appropriate instrument such as a scintillation counter. If enzymes are used as labels, the activity ofthe label can be detected by measuring absorbance, fluorescence intensity, or luminescence intensity after reacting the enzyme with an appropriate substrate.

The present invention also provides a kit for assaying the amount of high molecular weight amylin-like peptides present in a sample, including for example both biological samples and samples of high molecular weight amylin-like peptides proteins. The kit of the present invention comprises an immobilized anti-high molecular weight amylin-like peptides monoclonal antibody and a labeled anti-high molecular weight amylin-like peptides monoclonal antibody. When high molecular weight amylin-like peptides are assayed using this kit, high molecular weight amylin-like peptides become sandwiched between the immobilized monoclonal antibody and the labeled monoclonal antibody. As noted above, anti-amylin antibodies can also be used as the immobilized or labeled antibody to form one half of the antibody pair in conjunction with an anti-high molecular

weight amylin-like peptides monoclonal antibody.

To assist in understanding the present invention, the following Examples are provided which describe the results of a series of experiments. The experiments relating to this invention should not, of course, be construed as specifically limiting the invention

and such variations of the invention, now known or later developed, which would be within the purview of one skilled in the art are considered to fall within the scope of the invention as described herein and hereinafter claimed.

The isolation of amylin immunoreactive material from human pancreases was carried

out as described in the following Examples One through Four. The purification of the high molecular weight amylin-like peptides was monitored at each step using double monoclonal sandwich immunoassays using enzymatic detection (IEMAs) as described elsewhere (Fineman, et al., Diabetologia 1994; 37 (Suppl 1): A52 (abstract #197); Percy

et al., Diabetalogja 1994, 37 (Suppl 1): A117 (abstract #455). Antibody pairs used for assay included an assay F002/F025 that measures total immunoreactive amylin, and a

second assay F024 F025 that detects only full-length unhigh molecular weight amylin- like peptides and not glycosylated amylins. Assays were carried out using synthetic COOH-terminally amidated amylin (1-37) standards diluted in buffer (PBS pH 7.4/0.1% Triton X- 100/0.1% fish gelatin (Sigma)) The F002 antibody binds to the middle region of the human amylin molecule, and is specific to human amylin over other amylin species, aside from cat amylin. The F024 antibody binds to the N-terminal region of most amylins, and requires the presence of an intact disulfide bond. The F025 antibody

binds at the C-terminus of most amylins, but requires the presence of an amidated C- terminaus and does not bind to amylin acid, amylin-gly, or to proamylin

EXAMPLE ONE

Neutralized crude extract of human pancreas were prepared as follows. Human pancreas from normal adult subjects was obtained from the National Disease Research Interchange, Philadelphia, PA, prepared as for organ transplant, then frozen within 6

hours post-mortem and shipped on dry-ice to our laboratory. Extracts of human pancreas were prepared within 72 hours by placing the frozen tissue into a blender cup containing 5 to 10 wt/volume of precooled 0.2 N HCl prepared in 50% acetonitrile/50% H₂O (V:V). The tissue was broken up in the blender using stop/start pulses, then blended at high speed until for a total of 4 min. in 1 minute intervals with 2 minutes ice cooling between. Further operations were carried out at 4°. The extract was incubated for 3 hr, then centrifuged at 3,500 g for 40 min. The supernatant was filtered through a 127 mm glass fiber filter (Gelman #66084) under vacuum, and the volume reduced two-fold by vacuum

centrifugation. The volume was adjusted with water to give final 0.15 N HCl, and Tween-20 was then added to final 0.1% V/V. The pH was adjusted while stirring to about 6.5 by dropwise addition of liquid N-ethyl moφholine (Pierce Chemical Co.). At this stage the material could be stored at -70° for several months. In addition to the above source, Analytical Biological Services, Inc. (Wilmington, DE) provided extracts of human pancreas, which were taken through the centrifugation step then stored and shipped within 5 days on dry-ice for further processing in our laboratory. Its procedure differed from that described above in that tissue, a short time after death, was placed into

and temporarily stored in liquid nitrogen.

EXAMPLE TWO

Immunoafrinity chromatography of the neutralized extract from human pancreas

prepared in accordance with Example One was carried out as follows. The neutralized extract was frozen and thawed three times, then clarified by centrifugation. The supernate was passed at room temperature at 5 to 7 ml/min successively through a 2 ml bed column of F024-Emphaze beads, followed by a 0.5 ml bed column of F002-Emphaze

beads. Using an FPLC System (Pharmacia) both columns were separately washed, and bound amylins were eluted as follows: 75 ml PBS-T, 30 ml each PBS-T containing 0.5 M NaCl, PBS-T, PBS-T containing 0.5 M NaCl, PBS-T, 45 ml 50 mM sodium acetate, pH 4.0, containing 0.1% Tween-20. A final wash of 5 ml of 10 mM acetic acid without Tween was followed by elution at 0.2 ml/min with 20% acetonitrile/80% water (V:V) containing 10 mM acetic acid. The eluate from the F024 beads contained amylin (peak 3), while that from F002 beads was highly enriched in high molecular weight amylin-like peptides.

EXAMPLE THREE

Further purification of the material isolated as described in Examples One and Two was carried out as follows. The high molecular weight amylin-like peptides fractions eluted from the F002-immunoaffinity column were pooled and dried by vacuum centrifugation. These were taken up in 0.4 ml of 37.5% acetonitrile/67.5% water containing 0.068% TFA. After centrifugation the supernatant was passed through a 100

mg SepPak C18 cartridge (Waters) followed by the same solvent to remove hydrophobic contaminants. After discarding the dead volume (100 ml) the next 600 μl, containing the majority of glycosylated amylins, was collected.

After excess acetonitrile was removed from the SepPak pass material by vacuum

centrifugation, it was injected onto a Vydak 2.1 X 150 mm C4, 300A column (#214TP5215) using an Applied Biosystems Model MOB microbore HPLC system with absorbence monitoring at 210 nm. Elution was carried out at 200μl/min in 0.1% TFA/5% hexafluoroisopropanol using an ascending methanol gradient. Fractions were collected at

1 min intervals..

Individual immunoreactive fractions from the first HPLC procedure were evaporated as above and applied to a Synchrom 1.0 X 50 mm C8, 1000A [MB1C8R110-5] and eluted at 50 μl/min in 0.09% TFA with an acetonitrile gradient. Peaks containing high molecular weight amylin-like peptides were collected and identified by immunoassay,

characteristic retention times, and molecular weight estimates by Western Blotting using the F025 monoclonal antibody for detection. UV absorbence profiles from the material isolated by the final microbore HPLC step are shown in Figure 3.

The purification steps and results of Examples One, Two, and Three are summarized

below: Immunoreactive amylin

[pmol] Glycosylated

Volume A B amylin percent [ml] F002/F025 F024/F025 [A-B] yield

Extract, crude filtered 1330 34961.7 27379.4 7582.3 100.0

Extract, neutralized 1330 33827.2 22720.4 11106.8 146.5

F024-bead pass-through 1330 6784.7 204.7 6580.0 86.8

F002-immunoaffinity eluate 4 6435.3 233.9 6201.4 81.8

SepPak C18 pass-through 0.6 3430.3 119.7 3310.6 43.7

HPLC #1 (Narrowbore C4)

Peak l 1996.9 0.0 1996.9 26.3 Peak 2 1017.0 28.7 988.3 13.0

HPLC #2 (Microbore Cδ)

Peak 1A 155.6 0.0 155.6 2.0 Peak 1B 593.9 0.0 593.9 7.8

Peak 1A+1B (incompletely resolved) 47.0 0.0 47.0 0.6

Peak 2 290.8 0.0 290.8 3.8

EXAMPLE FOUR

Extraction of amylin immunoreactive materials from human plasma was

accomplished as follows. Blood from a normal fasting subject was collected in the

presence of protease inhibitors (final concentrations: antipain diHCl 2 μg/ml, leupeptin

0.5 mg/ml, and elastatinal 25 μg/m) and EDTA 0.5 mg/ml and chilled immediately.

Plasma (10 ml) was extracted using a 1 g SepPak C18 cartridge (Waters/Millipore) pre-

wetted by 2-propanol and washed with 0.1% TFA. An equal volume of 1% TFA was

mixed with the plasma, and it was passed through the cartridge and washed with 10 ml

0.1% TFA, followed by 10 ml 25% acetonitrile/0.5% TFA. Amylins were eluted from

the cartridge with 5 ml 50% acetonitrile 0.5% TFA. For HPLC fractionation the eluate

was reduced in volume by vacuum centrifugation and injected directly onto a reversed phase HPLC column (Vydak C4, 4.6 x 250 mm, 214TP54). Elution was carried out on a Waters 510 HPLC system at 0.6 ml/min with an acetonitrile gradient in 0.1% TFA. One

minute fractions were collected and dried by vacuum centrifugation, taken up in 0.5 ml immunoassay buffer, and assayed for amylin as described above. The results are shown

in Figure 1.

EXAMPLE FIVE

The high molecular weight amylin-like peptides were subjected to gel electrophoresis and Western blotting. SDS-gel electrophoresis of amylin immunoreactive materials was carried out using Novex Tris-Tricine 16% or 10- 20% polyacrylamide gels, sample and electrode buffers, following the manufacturer's basic instructions. Samples were prepared either by dilution into sample buffer or by drying and resuspension in the

sample buffer. Unless specified otherwise, no reducing agent was used. Volumes of

samples applied per lane ranged from 5 to 15 μl. The amylin bands were electophoretically transferred to PVDF membranes (Millipore) for lhr at 50 volts in

transfer buffer (Novex). The membranes were then incubated in a series of solutions with gentle mixing at room temperature. They were washed 3 times with TBS/T between each

solution. The solutions were 5% non-fat dry milk (Carnation) suspended in TBS T for lhr at RT or overnight at 4°, primary antibody FO25 at 5 (ug/ml) in TBS T for 1 hr, alkaline phosphatase-conjugated goat anti-mouse immunoglobulin [F(ab')₂]. The blots were developed with alkaline phosphatase-substrate solution (BioRad kit #170-6432), to produce dark blue staining of immunoreactive amylin. From these results, it was determined that the high molecular weight amylin-like peptides had molecular weights greater than that of amylin and were not fragments of amylin.

EXAMPLE SIX

The peptide samples from Example Three were sequenced as follows. Peptides purified in accordance with Examples One through Three were in 0.1% TFA containing varied acetonitrile concentrations. Each was evaporated to 10-20 μl by vacuum

centrifugation. Amino terminal amino acid sequence was obtained by automated Edman degradation using an Applied Biosystems (ABI) Model 470A gas phase sequencer. All reagents and sample supports were supplied by ABI. The instrument was equipped with a microcartridge and utilized reaction and conversion cycles optimized to accommodate

this modification. Samples were loaded onto Biobrene treated TFA/filters in 14μl portions, each aliquot in turn being dried under nitrogen. Residues were identified on a 120 A PTH-amino acid analyzer with the aid of a 610A data system. Separation of PTH- amino acids was achieved with an optimized gradient installed with the instrument. The

results are shown in Table 2 below

TABLE 2. Amino-Terminal Amino Acid Sequence Analyses of Purified Pancreatic Amylins. Recoveries (in picomoles) are given in parentheses. Analyses were quantitated for the number of cycles shown.

Synthetic

Cycle Amylin Peak 3 Peak lA Peak IB Peak 2

1 Lys Lys (8.28) Lys (6.04) Lys (6.48)) Lys (5.97)

2 Cys (...) (...) (-) (...)

3 Asn Asn (8.72) Asn (1.92) Asn (5.38)) Asn (3.40)

4 Thr Thr (4.62) Thr (0.95) Thr (3.47) Thr (3.31)

5 Ala Ala (7.16) Ala (2.28) Ala (4.79) Ala (4.70)

6 Thr Thr (3.55) Thr (0.19) Thr (3.18) (0.00)

7 Cys (-) (-) <-) (_.)

8 Ala Ala (4.88) Ala (1.40) Ala (4.08) Ala (3.34)

9 Thr Thr (2.86) (0.00) (0.00) (1.99)

10 Gin Gin (3.72) Gin (1.07 Gin (3.15) Gin (2.55)

11 Arg Arg (2.07) Arg (0.49) Arg (1.87) Arg (1.00)

12 Leu Leu (4.31) Leu (1.42) Leu (3.42) Leu (2.72)

13 Ala Ala (3.46) Ala (1.01) Ala(2.54) Ala (2.18)

14 Asn Asn (2.34) Asn (0.57) Asn (2.22) Asn (1.60)

15 Phe Phe (2.88) Phe (0.77) Phe (2.60) Phe (1.79)

16 Leu Leu (3.44) Leu (1.00) Leu (2.70) Leu (2.28)

17 Val Val (2.01) Val (0.59) Val(1.56) Val (1.17)

18 His His (0.38) His (0.41) His (0.66) His (0.50)

19 Ser Ser (0.47) Ser (0.53) Ser (0.29)

20 Ser Ser (0.37) Ser (0.70) Ser (0.93

21 Asn Asn (0.54) Asn (0.76)

22 Asn Asn (1.23) Asn (1.24)

23 Phe Phe (0.32) Phe (0.66)

24 Gly Gly (0.52) Gly (0.46)

25 Ala Ala (0.72) Ala (0.44)

26 He He (0.73)

27 ^• Leu Leu (0.44)

EXAMPLE SEVEN

Electrospray mass spectrometry was performed on a VG Trio 2000 single quadruple mass spectrometer (Fisons-VG Biotech, Altrincham, UK) using electrospray ionization at atmospheric pressure. Instrument calibration was accomplished by comparing multiply- charged ion peaks generated by a 10 ml (25 pmol) injection of horse heart myoglobin (Sigma Chemical Co., St. Louis, MO) against the theoretical m/z ratios while scanning

over the m/z range of 500-1500 amu. Samples contained 20-50 pmol of peptide in 2-5μl 50/50 acetonitrile/water with 0.05% acetic acid/approximately 0.4% TFA. They were

introduced into the electrospray source at a flow rate of 6.67μl min of 50/50 acetonitrile/water with 1% acetic acid. Data were collected over the range of 500-1500 amu. Instrument control, data acquisition and analysis were accomplished using Mass

Lynx software (VG Biotech, Altrincham, UK). Matrix assisted laser

desoφtion/ionization quadrupole ion trap mass spectrometry (MALDI-ITMS) was carried out as described by Cox, K.A., Willams, J.D., Cooks, R.G., Kaiser, R.E., Jr. (1992) Biol. Mass. Spectrom. 21:226-241. The results of the electrospray mass spectrometry procedure are shown in Figure 5, and the results of the MALDI-ITMS are shown in Figure 7.

EXAMPLE EIGHT

Chemical deglycosylation of the peptide samples obtained in Example Three was

accomplished as follows. Carbohydrates were chemically removed using a commercial deglycosylation kit (Glycofree, Oxford Glycosy stems) following the manufacturer's instructions, except that volumes of all reagents were reduced by a factor of five to be

commensurate with the lower, picomole amounts of glycopeptide being digested. Purified high molecular weight amylin-like peptides were lyophilized in glass autosampler vials and then digested for 2 or 4 hr (as indicated) at -20° with 20 μl trifluoromethane sulfonic acid (TFMSA) reagent. The acid was neutralized with 25 μl pyridine reagent plus μl H₂O, and submitted to microbore reversed phase chromatography

as described in Example Three above. In cases where samples were to be digested with

glycosidase, the TFMSA digestion was carried out in 10 μl reagent for 2 hr at -20°, followed by neutralization with 30 μl pyridine reagent. They were next extracted with 5 mg of bulk SepPak C18 matrix as follows: Ten μl of 0.1% TFA were added to the 40 μl of digested/neutralized samples to give final 50 μl. A slurry containing 5 mg of 2- propanol-wetted SepPak C18 matrix was placed in a 1.2 ml capacity polypropylene microdilution tube (USA Scientific Plastics, Ocala, FL). The matrix was settled by brief centrifugation and washed twice with 200 μl of 0.1% TFA. The neutralized TFMSA digestion product was added to the packed matrix, followed by 20 μl 0.1% TFA that had been used to rinse the reaction vial. After incubation 10 minutes at room temperature

with occasional gentle mixing, it was centrifuged, and the super discarded. The matrix was rinsed 3 times with 200 μl 0.1% TFA. The peptide was eluted from the packed

matrix by three extractions with 20 μl each of 0.4% TFA in 60% ACN/40% H₂O (V:V). The extracts were pooled in siliconized polypropylene microtubes (Fisher), dried by

vacuum centrifugation, and stored at -70° pending glycosidase digestions.

EXAMPLE NINE Glycosidase digestions ofthe peptide samples obtained in Example Eight were done according to the following methods. The samples (1-6 pmol) were dried by vacuum centrifugation in siliconized 0.5 ml polypropylene tubes (Fisher). For digestion with N-

acetylgalactosaminidase-α, they were taken up in 5 μl l 00 mM sodium citrate-phosphate

buffer pH 4.0 containing 0.1% Tween-20, 1 mM Pefabloc, 2 μg/ml antipain DiHCl, 0.5 mg/ml EDTA, 0.5 μg/ml leupeptin, and 25 μg/ml elastatmal. Digestion was initiated by addition of 5 μl containing 5 mU of N-acetylgalacosaminidase-α (chicken liver, Oxford Glycosytems) freshly dissolved in digestion buffer, with incubation at room temperature. At indicated times aliquots were diluted and assayed for amylin immunoreactivity. Digestions with all other glycosidases were carried out essentially the same as above, but in 100 mM sodium citrate-phosphate pH 5.0 containing 0.1% fish gelatin (Sigma) plus EDTA and all protease inhibitors as described in the above buffer, but not containing Tween-20. Reaction volumes were 18 μl. Concentrations of individual enzymes were: neuraminidase (NANase III, Glyko) 0.28 mU/μl, O-glycosidase (Glyko) 0.17 mU/μl, and N-acetylglucosaminidase (HEXase I, Glyko) 7.0 mU/μl. As shown in Tables 3 and 4,

results of the TFMSA and glycosidase procedures described in Examples Eight and Nine showed that treatment ofthe high molecular weight amylin-like peptides with appropriate sugar-removing chemicals/enzymes led to the ability of the F024 antibody to bind to these peptides.

TABLE 3. Restoration of FO24 binding after digestion of TFMSA-treated IB with

N-acetylglucosaminidase-α. Values given are amylin concentrations (pM) measured in the F002/25-27 IEMAs following 800-fold dilution into immunoassay buffer.

Pea k 1B Peak 1A Synthetic Amylin

Digestion TFMSA-Treated Untreated TFMSA-Treated Untreated Untreated

Time (hr) F002 F024 F002 F024 F002 F024 F002 F024 F002 F024

0 74.8 0.0 51.9 0.7 77.0 0.3 66.7 0.0 31.8 28.5

4 54.9 7.9 46.3 0.6 64.5 3.2 68.2 0.1 29.9 26.4

7 - - 47.3 0.5 - - - - -

8 52.3 13.2 - - 59.0 7.9 63.6 0.0 34.5 34.0

11 53.2 14.0 - - 58.7 12.4 - - 35.2 29.8

25 45.7 16.6 - - 51.1 13.8 67.8 0.2 33.7 29.8

TABLE 4 Restoration of the F024 epitope after complete oligosaccharide removal from

peak IB by a glycosidase mixture.

Sialidase O-Glycosidase F024 F002

NANase ffl Glyko 038

NANase UI S. pneumoniae 0.25

A. ureafaciens S. pneumoniae 0.24

C. perfringens Glyko 0.01

C. perfringens S. pneumoniae 0.02

NDV* Glyko 0.00

NANase UI - 0.00

- S. pneumoniae 0.00

- - 0.00

*Newcastle Disease Virus EXAMPLE TEN

Tryptic digestions of the high molecular weight amylin-like peptides isolated pursuant to Examples One through Three, followed by HPLC separation of digestion products, were accomplished as follows. Samples were dried in siliconized

microcentrifuge tubes by vacuum centrifugation. Each was solubilized in 1 μl 0.025%

trifluoroacetic acid (TFA) in 40% acetonitrile/60% H₂O. 3 μl of just-mixed 1 volume of 0.2 mg ml trypsin stock in 0.01% TFA and 2 volumes of 35 mM NaHCO₃ were then added. After incubation at room temperature for 15-20 min, the digestion was stopped by

addition of 18 μl of 0.2% TFA and the products separated by direct injection onto a microbore reversed phase HPLC column (Vydak C4, 5 μm, 300A, 0.8 x 50 mm). Elution was effected at 30 μl/min in 0.1% TFA in a 1%/min acetonitrile gradient starting from 0% at 8 minutes. The results are shown in Figure 5.

EXAMPLE ELEVEN

The high molecular weight amylin-like peptides isolated by the procedures described in Examples One through Three were tested for amylin and calcitonin receptor binding by the following methods. Binding of purified pancreatic amylins to isolated membranes of cells expressing calcitonin family receptors was estimated through displacement of radioiodinated peptide tracers essentially as described previously: amylin receptors in rat nucleus accumbens membranes (Beaumont et al.,. "High affinity amylin binding sites in rat brain," Mol Pharmacol. 44:493-497 (1993)), human calcitonin receptors in cultured MCF-7-7 cells, and calcitonin gene related peptide (CGRP) in SKNMC cell (Beaumont et

al., Can. J. Physiol Pharmacol. 73:1025-1029 (1995))). Purified amylins were prepared for assay by taking to dryness by vacuum centrifugation, followed by solubilization in 20

μl of dimethyl sulfoxide, and final dilution into 1.0 ml of assay buffer. The data are

shown in Table 5 below, and are expressed either as the concentration showing 50%

tracer displacement (IC_J0)or, in the case of weak or no displacement, as percent displacement at the- highest concentration tested.

TABLE 5. Testing of glycosylated amylins for binding to receptors of the calcitonin

family. The data are expressed either as the concentration showing 50% tracer

displacement (IC50) or, in the case of weak or no displacement, as percent displacement at the highest conentration tested.

Rat Nucleus MCF-7-7 cell SKNMC cell Accumbens membranes (hu (CGRP (amylin receptor) calcitonin receptor) receptor) highest cone % dis¬ % dis¬ % dis¬

SAMPLE tested (nM) placement IC50 (pM) placement IC50 (pM) placement

Amylin peak 1 (A+B mixed) 36.5 10 >36000 25 >36000 10

Amylin peak 2 36.9 39 >10000 11 >37000 10

Amylin peak 3 20.4 387 1625 19 synthetic amylin (HPLC effluent) 17.4 262 1622 13 synthetic amylin (diluted stock) 31.1 197 1687 24 mobile phase (HPLC effluent) 0 19 10 10 mobile phase (new) 0 10 14 10 EXAMPLE TWELVE

Plasma amylin immunoreactivity was measured during a third trimester Oral Glucose Tolerance Test in 56 women with gestational diabetes (GDM) and 56 age and body mass index (BMI) matched pregnant controls. Plasma samples were drawn fasting as well as at 30, 90 and 120 minutes post glucose challenge, and both the total amylin immunoreactivity assay (F002) and the amylin specific assay (F024) were used for the measurements of amylin immunoreactivity. Using the F024 assay, there was no significant difference in plasma concentration between subject groups (diabetic vs. controls) at any timepoint or in the total area under the curve (AUC). With the F002 assay, however, there were extremely significant elevations of amylin immunnoreactivity in the diabetic group over the control group at every timepoint as well as with the AUC (pθ.001). These results demonstrate the ability of the HMW-ALPS assay for the measurement or evaluation of insulin resistance in patients.

EXAMPLE THIRTEEN

Plasma amylin immunoreactivity was measured during a standard 75 gram 2 hour Oral Glucose Tolerance Test in 238 non diabetic subjects. Plasma samples were drawn fasting as well as at 30 and 120 minutes post glucose challenge, and both the total amylin immunoreactivity assay (F002) and the amylin specific assay (F024) were used for the measurements of amylin immunoreactivity. Four years after their original visits, the

subjects under went a second oral glucose tolerance test to access their current glucose control status by the WHO criteria. 219 subjects had retained their normal glycemic status, while 16 subjects converted to a condition known as Impaired Glucose Tolerance (IGT), a known prediabetic state, and 2 converted to diabetic status. Both assays showed

a significant increase in the median area under the curve (AUC) for the converted group

compared to the subjects that remained normoglycemic as can be seen on the table below.

While both assays met the criteria for statistical differences, the F002 assay results were superior.

ASSAY Converted Normal P value

Mean AUC pM*min Mean AUC pM*min Mann- Whitney Test

F002 2412 1783 0.01

F024 899 632 0.05

Various modifications of the invention in addition to those shown and described

herein will become apparent to those skilled in the art from the foregoing description and fall within the scope ofthe following claims.

Claims

WE CLAIM:

1. A composition comprising an isolated glycosylated amylin peptide.

2. The composition of claim 1 wherein said isolated glycosylated amylin peptide is a human glycosylated amylin peptide.

3. The composition of claim 2 wherein said isolated glycosylated amylin peptide is an amylin having an oligosaccahride chain attached to the position 6 threonine residue.

4. The composition of claim 2 wherein said isolated glycosylated amylin peptide is an amylin having an oligosaccharide chain attached to the position 9 threonine residue.

5. The composition of claim 2 wherein said isolated glycosylated amylin peptide is an amylin having an oligosaccharide chain attached to the position 6 threonine residue and to the position 9 threonine residue.

6. The compositions of any of claims 3-5 wherein said oligosaccharide is a monosialated pentasaccharide.

7. The composition of claim 6 wherein said monosialated pentasaccharide has the

formula: GlcNAc(β 1 -3)[GIcNAc(β 1 -6)]Gal(β 1 -3)[NeuAc(α2-6)]GalNAc(α-threonine)

8. A composition for use as an immunogen for the generation of an immune response in an experimental animal, which comprises a immnogenically effective amount ofthe composition of any of claims 1-4 or 5 in association with a carrier.

9. A monoclonal antibody which binds to a high molecular weight amylin-like peptide, and is specific for said high molecular weight amylin-like peptide over unglycosylated amylin.

10. The monoclonal antibody of claim 9 wherein said high molecular . weight amylin-like peptide is a human high molecular weight amylin-like peptide.

11. The monoclonal antibody of claim 10 wherein said human high molecular weight amylin-like peptide is selected from the group consisting of amylin having an oligosaccharide chain attached to the position 6 threonine residue, an amylin having an oligosaccharide chain attached to the position 9 threonine residue, and an amylin having

an oligosaccharide chain attached to the position 6 threonine residue and to the position 9 threonine residue.

12. An assay using a monoclonal antibody for detecting the presence or amount of high molecular weight amylin-like peptide, comprising the steps of:

(a) contacting said high molecular weight amylin-like peptide with said monoclonal antibody, wherein said monoclonal antibody binds to said high molecular weight amylin-like peptide and is specific for said high molecular weight amylin-like peptide over unglycosylated amylin, and

(b) determining the presence or amount of said high molecular weight amylin-

like peptide.

13. The assay of claim 12 wherein a second monoclonal antibody to said high

molecular weight amylin-like peptide or a polyclonal antibody to said high molecular weight amylin-like peptide is used in said assay.

14. The assay of claim 13 wherein said second monoclonal antibody or polyclonal

antibody is detectably labeled.

15. The assay of claim 12 wherein said assay is a competitive assay.

16. The assay of claim 12 wherein said assay is.a sandwich assay.

17. An assay for determining the presence or amount of a high molecular weight amylin-like peptide in a sample suspected of containing a high molecular weight amylin- like peptide, comprising the steps of

(a) contacting said sample suspected of containing a high molecular weight amylin-like peptide with a monoclonal antibody according to any of claims 9, 10 or 11;

(b) contacting positive and/or negative control samples with a monoclonal

antibody according to any of claims 9, 10 or 11 ; and

(c) determining the presence or amount of said high molecular weight amylin- like peptide.

18. The assay according to claim 17 wherein said assay is a competitive assay.

19. The assay according to claim 17 wherein said assay is a sandwich assay.

20. The assay according to claim 17 wherein said monoclonal antibody is detectably labeled and wherein said labeled monoclonal antibody binds to said high molecular weight amylin-like peptide and is used to determine the presence or amount of said high molecular weight amylin-like peptide.

21. An assay for determining the presence or amount of a high molecular weight amylin-like peptide in fluid sample suspected of containing a high molecular weight amylin-like peptide, comprising the steps of:

(a) contacting said sample with a measured amount of a first antibody directed to said high molecular weight amylin-like peptide to form a soluble complex of said first antibody and said high molecular weight amylin-like peptide present in said sample, said first antibody being labeled; (b) contacting said soluble complex with a second antibody directed to said high molecular weight amylin-like peptide, said second antibody being bound to a solid carrier, to form a ternary complex of said first antibody, said high molecular weight amylin-like peptide and said second antibody bound to said solid carrier;

(c) separating said solid carrier from said sample and unreacted labeled first antibody;

(d) measuring either the amount of labeled first antibody associated with said solid carrier or the amount of unreacted labeled first antibody; and

(e) relating the amount of labeled antibody with the amount of labeled antibody measured for a control sample prepared in accordance with steps (a)-(d), said control sample being known to be free of said high molecular weight amylin-like peptide, to determine the presence of high molecular weight amylin-like peptide in said fluid sample,

or relating the amount of labeled antibody measured with the amount of labeled antibody measured for samples containing known amounts of high molecular weight amylin-like peptide prepared in accordance with steps (a)-(d) to determine the amount of high molecular weight amylin-like peptide in said fluid sample, either the first antibody or the second antibody or both being an antibody according

to claim 9.

22. An assay for determining the presence or amount of high molecular weight amylin-like peptide in a fluid sample suspected of containing said high molecular weight

amylin-like peptide, comprising the steps of:

(a) contacting said sample with a first antibody directed to said high molecular weight amylin-like peptide, wherein said first antibody is bound to a solid carrier, to form a complex of said high molecular weight amylin-like peptide present in said sample and said first antibody;

(b) separating unreacted sample from said complex;

(c) contacting said complex with a measured amount of a second antibody

directed to said high molecular weight amylin-like peptide, wherein said second antibody is labeled;

(d) measuring either the amount of labeled second antibody associated with said complex or the amount of unreacted labeled second antibody; and

(e) relating the amount of labeled antibody with the amount of labeled antibody measured for a control sample prepared in accordance with steps (a)-(d), said control sample being known to be free of high molecular weight amylin-like peptide, to determine the presence of high molecular weight amylin-like peptide in said fluid sample, or relating the amount of labeled antibody measured with the amount of labeled antibody

measured for samples containing known amounts of high molecular weight amylin-like peptide prepared in accordance with steps (a)-(d) to determine the amount of high molecular weight amylin-like peptide in said fluid sample, either the first antibody or the second antibody or both being an antibody according to claim 9.

23. In an immunometric assay to determine the presence or amount of high

molecular weight amylin-like peptide in a sample suspected of containing high molecular weight amylin-like peptide, the improvement comprising employing a monoclonal antibody according to any one of claims 9, 10 or 11.

24. The assay of any of claims 12-22 or 23 wherein said high molecular weight amylin-like peptide is a human high molecular weight amylin-like peptide.

25. A kit comprising a monoclonal antibody which binds to a high molecular weight amylin-like peptide, wherein said monoclonal antibody is specific for said high molecular weight amylin-like peptide over unglycosylated amylin.

26. A kit according to claim 25 which further comprises suitable control samples.

27. A substantially pure high molecular weight amylin-like peptide.

28. A pure high molecular weight amylin-like peptide.

29. The high molecular weight amylin-like peptide of claim 28 which is a human

high molecular weight amylin-like peptide.

30. A method of evaluating insulin resistance in asubject, which comprises determining in said patient the plasma level of high molecular weight amylin-like

peptides.

31. The method of claim 30 wherein said subject shows normal glycemic control.

32. The method of claim 30 wherein said subject shows abnormal glycemic control.

33. A method of determining whether a subject who shows normal glycemic control as determined, for example, by oral glucose tolerance test, is at risk for becoming diabetic, which comprises determining in said subject the plasma level of high molecular

weight amylin-like peptides.

34. A method of determining whether a subject who shows normal glycemic control as determined, for example, by oral glucose tolerance test, is at risk for becoming diabetic, which comprises determining in said subject the plasma level of unglycosylated

amylin.