NZ718999B2 - A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof - Google Patents

A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof Download PDF

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Publication number
NZ718999B2
NZ718999B2 NZ718999A NZ71899912A NZ718999B2 NZ 718999 B2 NZ718999 B2 NZ 718999B2 NZ 718999 A NZ718999 A NZ 718999A NZ 71899912 A NZ71899912 A NZ 71899912A NZ 718999 B2 NZ718999 B2 NZ 718999B2
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seq
immunoglobulin
oxyntomodulin
region
derivative
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NZ718999A
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NZ718999A (en
Inventor
In Young Choi
Sung Youb Jung
Dae Jin Kim
Se Chang Kwon
Sung Hee Park
Young Eun Woo
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Hanmi Science Co Ltd
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Priority to NZ733478A priority Critical patent/NZ733478B2/en
Publication of NZ718999A publication Critical patent/NZ718999A/en
Publication of NZ718999B2 publication Critical patent/NZ718999B2/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/22—Hormones
    • A61K38/26—Glucagons
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/56—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule
    • A61K47/59—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes
    • A61K47/60—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an organic macromolecular compound, e.g. an oligomeric, polymeric or dendrimeric molecule obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyureas or polyurethanes the organic macromolecular compound being a polyoxyalkylene oligomer, polymer or dendrimer, e.g. PEG, PPG, PEO or polyglycerol
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6801—Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
    • A61K47/6803—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates
    • A61K47/6811—Drugs conjugated to an antibody or immunoglobulin, e.g. cisplatin-antibody conjugates the drug being a protein or peptide, e.g. transferrin or bleomycin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/50—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates
    • A61K47/51—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent
    • A61K47/68—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient the non-active ingredient being chemically bound to the active ingredient, e.g. polymer-drug conjugates the non-active ingredient being a modifying agent the modifying agent being an antibody, an immunoglobulin or a fragment thereof, e.g. an Fc-fragment
    • A61K47/6889—Conjugates wherein the antibody being the modifying agent and wherein the linker, binder or spacer confers particular properties to the conjugates, e.g. peptidic enzyme-labile linkers or acid-labile linkers, providing for an acid-labile immuno conjugate wherein the drug may be released from its antibody conjugated part in an acidic, e.g. tumoural or environment
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A61P3/04—Anorexiants; Antiobesity agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/575—Hormones
    • C07K14/605—Glucagons
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Abstract

Disclosed is a conjugate comprising oxyntomodulin derivative, an immunoglobulin Fc region, and non-peptidyl polymer. The oxyntomodulin derivative is covalently linked to the immunoglobulin Fc region via a non-peptidyl polymer. The oxyntomodulin derivative comprises the amino acid sequence of Also disclosed is the use of the conjugate for prevention or treatment of obesity. isclosed is the use of the conjugate for prevention or treatment of obesity.

Description

Description Title of Invention: A CONJUGATE COMPRISING OXYNTOMODULIN AND AN IMMUNOGLOBULIN FRAGMENT, AND USE F Related applications This application is a onal ation ofNew Zealand Patent Application 618811 which claims ty to Korean Patent Application 10—2011-0058852, the entirety of which is hereby incorporated by reference.
Technical Field The present invention relates to a conjugate comprising oxyntomodulin and an immunoglobulin fragment, and the use thereof. More particularly, the present invention relates to a conjugate comprising oxyntomodulin, an immunoglobulin Fc region, and non-peptidyl r wherein the conjugate being obtainable by covalently linking oxyntomodulin to immunoglobulin Fc region via nonpeptidyl polymer, and a pharmaceutical ition for the prevention or treatment of y comprising the conjugate.
Background Art Recently, economic growth and changes in lifestyle are leading to changes in eating habits. The main causes of rising overweight and obesity rates in contemporary people are consumption of high-calorie foods such as fast foods and lack of exercise. World Health Organization (WHO) estimates that more than 1 billion people worldwide are overweight and at least 300 million of them are clinically obese. In particular, 250,000 people die each year in Europe and more than 2.5 million people worldwide die each year as a result of being overweight (World Health Organization, Global gy on Diet, Physical Activity and Health, 2004).
Being overweight and obese increases blood pressure and cholesterol levels to cause occurrence or exacerbation of various diseases such as cardiovascular disease, diabetes, and tis, and are also main causes of rising incidence rates of arteriosclerosis, hypertension, hyperlipidemia or cardiovascular disease in children or cents as well as in adults. y is a severe condition that causes various es worldwide. It is thought to be overcome by individual efforts, and it is also ed that obese patients lack self-control. However, it is difficult to treat obesity, because obesity is a complex disorder involving-appetite regulation and energy metabolism, For the ent of obesity, abnormal actions associated with appetite regulation and energy metabolism should be treated together with efforts of obese patients. Many attempts have been made to develop drugs capable of treating the abnormal actions. As the result of these efforts, drugs such as Rimonabant (Sanofi—Aventis), Sibutramin (Abbott), Contrave (Takeda) and at (Roche) have been developed, but they have the disadvantages of serious adverse s or very weak anti—obesity effects. For example, it was reported that Ri- monabant (Sanofi-Aventis) shows a side-effect of central nerve disorder, Sibutramine (Abbott) and Contrave (Takeda) show cardiovascular side-effects, and Orlistat (Roche) shows only 4 kg of weight loss when taken for 1 year. Unfortunately, there are no therapeutic agents for obesity which can be safely prescribed for obese patients. l7] Many studies have been made to develop therapeutic agents for obesity which do have the ms of the conventional anti-obesity drugs.
Recently, glucagon derivatives have received much ion. Glucagon is produced by the pancreas when the level of glucose in the blood drops resulting from other medications or diseases, e or enzyme deficiencies. on stimulates glycogen own in the liver, and facilitates glucose release to raise blood e levels to a normal range. In addition to the effect of increasing the blood glucose level, glucagon suppresses appetite and activates hormone~sensitive lipase(HSL) of adipocytes to facilitate sis, thereby showing anti—obesity s. One of the glucagon derivatives, glucagon like peptide-l (GLP—1) is under development as a therapeutic agent for hy- perglycemia in patients with diabetes, and it functions to stimulate insulin synthesis and secretion, to inhibit glucagon secretion, to slow gastric ng, to increase glucose utilization, and to inhibit food intake. Exendin-4 is isolated from lizard venom that shares approximately 50% amino acid homology with GLP~1 and is also reported to activate the GLP-l receptor, thereby ameliorating hyperglycemia in patients with diabetes. However, anti-obesity drugs including GLP-l are reported to show side— effects such as vomiting and nausea.
As an alternative to GLP—l , therefore, much ion has been focused on oxyn— tomodulin, a e derived from a glucagon precursor, pre-glucagon that binds to the ors of two peptides, GLP-1 and glucagon. Oxyntomodulin represents a potent anti—obesity y, because it inhibits food intake like GLP—l, promotes satiety, and has a lipolytic activity like glucagon.
Based on the dual function of the oxyntomodulin peptide, it has been actively studied as a drug for the ent of obesity. For example, Korean Patent No. 925017 discloses a pharmaceutical ition ing modulin as an active in- gredient for the treatment of overweight human, which is administered via an oral, parenteral, mucosal, rectal, subcutaneous, or transdermal route. However, it has been reported that this anti—obesity drug including oxyntomodulin has a short in vivo half- life and weak therapeutic efficacy, even though administered at a high dose three times a day. Thus, many efforts have been made to improve the in vivo half-life or therapeutic effect of oxyntomodulin on obesity by its modification.
For example, a dual agonist oxyntomodulin (Merck) is prepared by substituting L- serine with D-serine at position 2 of oxyntomodulin to increase a resistance to dipeptidyl peptidase-IV V) and by attaching a cholesterol moiety at the C- terminal to increase the blood half-life at the same time. ZP2929 (Zealand) is prepared by substituting L-serine with D-serine at on 2 to enhance resistance to DPP-IV, substituting arginine with alanine at position 17 to enhance resistance to protease, tuting methionine with lysine at position 27 to enhance oxidative stability, and tuting glutamine with aspartic acid and alanine at ons 20 and 24 and asparagine with serine at position 28 to enhance deamidation stability. However, even though the half-life of the dual agonist oxyntomodulin (Merck) was enhanced to show half-life 8-12 minutes longer than the native oxyntomodulin, it still has a very short in vivo half-life of 1.7 hr and its administration dose is also as high as several mg/kg.
Unfortunately, oxyntomodulin or derivatives thereof have disadvantages of daily administration of high dose due to the short half-life and low efficacy.
Disclosure of Invention Technical Problem Accordingly, the t inventors have made many efforts to develop a method for increasing the blood half-life of oxyntomodulin while maintaining its ty in vivo.
As a result, they found that a conjugate ed by linking a carrier to oxyntomodulin using a non-peptidyl polymer show improved blood ife while ining the activity in vivo so as to exhibit excellent anti-obesity effects, thereby completing the present ion.
Solution to Problem In an aspect a conjugate is provided comprising oxyntomodulin, an immunoglobulin Fc region, and non-peptidyl r wherein the conjugate being obtainable by covalently linking oxyntomodulin to immunoglobulin Fc region via non-peptidyl polymer. [14A] In an aspect, a conjugate comprising an oxyntomodulin derivative, an immunoglobulin Fc region, and a non-peptidyl polymer, wherein the oxyntomodulin derivative is covalently linked to the immunoglobulin Fc region via the non-peptidyl polymer, and wherein the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NOs: 34.
In another aspect a ceutical composition is provided for the prevention or treatment of obesity, comprising the conjugates, as described herein.
Still another aspect a method is provided for preventing or treating obesity, comprising the step of administering the conjugate or the composition to a subject.
Still another aspect a use of the conjugate or the composition is provided in the preparation of drugs for the prevention or treatment of obesity.
Advantageous Effects of Invention The conjugate comprising oxyntomodulin and the globulin PC of the t ion reduces food intake, suppresses gastric emptylng, and facilitates lipolysis without side-effects, unlike native oxyntomodulin, and also shows excellent receptor— ting effects and long~term nability, compared to oxyntomodulin. Thus, it can be widely used in the treatment of obesity with safety and efficacy. Unlike native oxyntomodulin, the novel peptide of the present invention reduces food intake, sses gastric emptying, and facilitates lipolysis t side-effects, and also shows excellent receptor-activating effects. Thus, it can be widely used in the treatment of obesity with safety and efficacy.
Any discussion of documents, acts, materials, devices, articles or the like which has been included in the t specification is not to be taken as an admission that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present sure as it existed before the priority date of each claim of this application.
Brief Description of Drawings is a graph showing changes in food intake according to stration dose of oxyntomodulin or oxyntomodulin derivative. is a graph showing the result of purifying mono—PEGylated oxyntomodulin through a SOURCE S purification column. is a graph showing the result of peptide mapping of purified mono- PEGylated oxyntomodulin. is a graph showing the result of purifying conjugates including modulin and immunoglobulin Fc through a SOURCE lSQ purification column. is a graph showing the result of purifying a mono—PEGylated oxyntomodulin tive (SEQ ID NO. 29) through a SOURCE S purification column. is a graph g the result of purifying ates including oxyntomodulin derivative (SEQ ID NO. 29) and immunoglobulin Fc through a SOURCE 15Q purification column. is a graph showing the result of purifying a EGylated oxyntomodulin derivative (SEQ ID NO. 30) through a SOURCE S purification column. is a graph showing the result of peptide mapping of purified mono— PEGylated modulin derivative (SEQ ID NO. 30). is a graph showing the result of purifying ates including oxyntomodulin derivative (SEQ ID NO. 30) and immunoglobulin Fc through a SOURCE 15Q purification . is a graph showing the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 31) through a SOURCE S purification column. is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 31) and globulin Fc through a SOURCE 15Q purification column. is a graph showing the result of ing a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 2) through a SOURCE S purification column. is a graph showing the result of peptide mapping of purified mono- PEGylated oxyntomodulin derivative (SEQ ID NO. 2). is a graph g the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc through a SOURCE 15Q purification column. is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc through a Source ISO purification column. is a graph showing the result of purifying a monoePEGylated oxyntomodulin tive (SEQ ID NO. 3) through a SOURCE S purification column. is a graph showing the result of peptide mapping of purified mono- PEGylated oxyntomodulin derivative (SEQ ID NO. 3). is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc through a Butyl FF purification column.
FIG. ”id is a graph showing the result of ing conjugates including modulin derivative (SEQ ID NO. 3) and immunoglobulin Fc through a Source 15Q purification column. is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 23) through 21 SOURCE S cation column; is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc through a Source 15Q purification column; is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc through a SOURCE ISO purification column; is a graph showing the result of ing a mono—PEGylated modulin derivative (SEQ ID NO. 24) through a SOURCE S purification column; is a graph showing the result of ing conjugates including oxyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc through a Source lSQ purification column; is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc through a SOURCE ISO purification column; a is a graph showing the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 25) through a SOURCE S purification column; b is a graph g the result of purifying conjugates including oxyn— tomodulin tive (SEQ ID NO. 25) and immunoglobulin Fc through a Source 15Q purification column; c is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 25) and immunoglobulin Fc through a SOURCE ISO purification column; a is a graph showing the result of purifying a mono-PEGyiated oxyntomodulin derivative (SEQ ID NO. 28) through 21 SOURCE S purification column; FIG. llb is a graph showing the result of ing ates including oxyntomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc through a Source 15Q purification column; FIG. llc is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc through a SOURCE ISO purification ; is a graph g changes in body weight of mice according to the type and administration dose of oxyntomodulin derivative—immunoglobulin Fc conjugates. is a graph showing changes in body weight of mice according to the type and administration dose of oxyntomodulin derivative—immunoglobulin Fc conjugates.
Best Mode for Carrying out the Invention In an aspect, the present invention provides a conjugate comprising oxyntomodulin, an immunoglobulin Fc region, and non—peptidyl polymer n the conjugate being obtainable by covalently linking oxyntomodulin to immunoglobulin Fc region via non—peptidyl polymer.
As used herein, the term "conjugate" means a conjugate comprising oxyntomodulin and other factors. Other factors can be any substance which can induce sed stability in blood, suspend emission through the kidney, or other useful s. In the present invention, the factors can be globulin Fc . In an ment, the ate can be comprised of an oxyntomodulin, and an immunoglobulin Fc region, which are linked by a non-peptidyl polymer. The non-peptidyl polymer can link an oxyntomodulin and an immunoglobulin Fc region Via covalent bonds. Two terminal ends of non-peptidyl polymer can be linked to an amine group or thiol group of the immunoglobulin Fc region and oxyntomodulin derivatives, respectively.
The conjugate of the present invention means to have an improved in—vivo duration of cy, compared to native oxyntomodulin, and the long—acting conjugate may include oxyntomodulin prepared by modification, substitution, addition, or deletion of the amino acid sequences of the native oxyntomodulin, oxyntomodulin conjugated to a biodegradable polymer such as polyethylene glycol (PEG), oxyntomodulin conjugated to a long—acting protein such as albumin or immunoglobulin, oxyntomodulin conjugated to fatty acid having the ability of binding to albumin in the body, or oxyntomodulin ulated in biodegradable nanoparticles, but the type of the long— acting conjugate is not limited thereto.
As used herein, the term "oxyntomodulin" means a peptide derived from a glucagon precursor, ucagon, and includes a native oxyntomodulin, precursors, derivatives, fragments thereof, and variants thereof. In an embodiment, it can have the amino acid sequence of SEQ ID NO. 1 (HSQGTPTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).
The term, ”oxyntomodulin variant" is a peptide having one or more amino acid sequences different from those of native oxyntomodulin, and means a peptide that retains the function of activating the GLP—1 and glucagon receptors, and it may be prepared by any one of substitution, addition, deletion, and modification or by a combination thereof in a part of the amino acid sequences of the native oxyntomodulin.
The term, "oxyntomodulin derivative" includes peptides, e derivatives or peptide mimetics that are prepared by addition, deletion or substitution of amino acids of oxyntomodulin so as to activate both of the GLP~1 receptor and the glucagon receptor at a high level, compared to the native oxyntomodulin.
The term, "oxyntomodulin fragment means a fragment having one or more amino acids added or d at the N—terminus or the inus of the native oxyntomodulin, in which turally occurring amino acids (for example, D-type amino acid) can be added, and has a function of activating both of the GLP- 1 or and the glucagon receptor.
Each of the preparation methods for the variants, derivatives, and fragments of modulin can be used individually or in combination. For example, the present invention includes a peptide that has one or more amino acids ent from those of native peptide and deamination of the N-terminal amino acid residue, and has a function of activating both of the GLP- 1 receptor and the glucagon receptor.
Amino acids mentioned herein are abbreviated according to the nomenclature rule of IUPAC-IUB as follows: Alanine A Arginine R Asparagine N Aspartic acid D Cysteine C Glutamic acid E Glutamine Q e G ine H Isoleucine I Leucine L Lysine K Methionine M Phenylalanine F Proline P Serine S Threonine T Tryptophan W Tyrosine Y Valine V In the present invention, the oxyntomodulin derivative encompasses any peptide that is prepared by substitutions, additions, deletions or post ational modifications (e.g., methylation, acylation, ubiquitination, intramolecular covalent g) in the amino acid sequence of oxyntomodulin FTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA, SEQ ID NO. 1) so as to activate the glucagon and GLP—1 ors at the same time. Upon substitution or addition of amino acids, any of the 20 amino acids commonly found in human proteins, as well as atypical or non-naturally occurring amino acids can be used.
Commercially available sources of atypical amino acids include Sigma—Aldrich, p Inc., and Genzyme Pharmaceuticals. The es including these amino acids and atypical e sequences may be synthesized and purchased from commercial suppliers, for example, American Peptide Company or Bachem (USA) or Anygen (Korea).
In one specific embodiment, the oxyntomodulin derivative of the present invention is a novel peptide including the amino acids of the following Formula 1.
R1—X1-X2—GTFTSD-X3—X4—X5-X6-X7—X8—X9-XlO-Xl 1—X12—X13—X14-X15-X16— S-Xl9—X20-X21-X22—X23-X24—R2 (SEQ ID N0254) (Formula 1) wherein R1 is ine, desamino~histidyl, dimethyl—histidyl (N~dimethy1—histidyl), beta—hydroxyimidazopropionyl, 4~imidazoacetyl, beta~carboxy irnidazopropionyl or tyrosine; X1 is Aib(aminosiobutyric acid), d- alanine, glycine, methylglycine), serine, or d—serine; X2 is glutamic acid or glutamine; X3 is leucine or tyrosine; X5 is lysine or arginine; X6 is glutamine or tyrosine; X7 is leucine or methionine; X8 is aspartic acid or glutamic acid; X9 is glutamic acid, serine, alpha-methyl-glutamic acid or is deleted; X10 is glutamine, glutamic acid, lysine, arginine, serine or is deleted; X11 is alanine, arginine, valine or is deleted; r—ir—ir—r—Ir—I \O 00 bl100]96] X12 is alanine, arginine, serine, valine or is deleted; X13 is lysine, ine, arginine, methyl-glutamic acid or is deleted; X14 is aspartic acid, glutamic acid, leucine or is deleted; X15 is phenylalanine or is deleted; X16 is isoleucine, valine or is deleted; X17 is alanine, ne, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid or is deleted; X18 is tryptophan or is deleted; X19 is alanine, isoleucine, leucine, serine, valine or is deleted; X20 is alanine, lysine, methionine, glutamine, arginine or is d; X21 is asparagine or is deleted; X22 is alanine, glycine, threonine or is deleted; X23 is cysteine, lysine or is deleted; X24 is a peptide having 2 to 10 amino acids consisting of combinations of e, glycine and serine, or is deleted; and R2 is IA (SEQ ID NO. 35), GPSSGAPPPS (SEQ ID NO. 36), GPSSGAPPPSK (SEQ ID NO. 37), HSQGTFTSDYSKYLD (SEQ ID NO. 38), HSQGTPTSDYSRYLDK (SEQ ID NO. 39), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO. 40) or is d (excluded if the amino acid sequence of a 1 is identical to that of SEQ ID NO. 1).
In order to enhance the activity of the wild— type oxyntomodulin for the glucagon or and the GLP—1 receptor, the e of the present invention may be substituted with 4—imidazoacetyl where the alpha carbon of histidine at position 1 of amino acid sequence represented by SEQ ID NO. 1 is deleted, desamino—histidyl where the N—terminal amino group is deleted, dimethyl-histidyl (N—dimethyl-histidyl) where the N-terminal amino group is modified with two methyl groups, beta-hydroxy imidazopropionyl where the N~terminal amino group is substituted with a hydroxyl group, or beta—carboxy imidazopropionyl where the N—terminal amino group is substituted with a carboxyl group. In addition, the GLP—l receptor-binding region may be substituted with amino acids that enhance hydrophobic and ionic bonds or ations thereof. A part of the oxyntomodulin sequence may be substituted with the amino acid sequence of GLP-1 or Exendin~4 to enhance the activity on GLP- 1 receptor.
Further, a part of the oxyntomodulin sequence may be substituted with a sequence stabilizing alpha helix. In an embodiment, amino acids at positions 10, 14, 16, 20, 24 and 28 of the amino acid sequence of Formula 1 may be substituted with amino acids or amino acid derivatives consisting of Tyr(4-Me), Phe, Phe(4-Me), ll), Phe(4- CN), Phe(4-N02), NH2), Phg, Pal, Nal, Ala(2—thienyl) and Ala(benzothienyl) that are known to stabilize alpha helix, and there are no limitations on the type and number of alpha helix-stabilizing amino acid or amino acid derivatives to be inserted.
In an embodiment, amino acids at positions 10 and 14, 12 and 16, 16 and 20, 20 and 24, and 24 and 28 may be also substituted with glutamic acid or lysine, respectively so as to form rings, and there is no limitation on the number of rings to be inserted. The peptide may be a peptide having an amino acid sequence selected from the following Formulae l to 6.
In one specific embodiment, the oxyntomodulin derivative of the t invention is a novel peptide including the amino acid sequence of the following Formula 2 where the amino acid sequence of oxyntomodulin is substituted with that of exendin or GLP- 1.
Rl-A-R3 (SEQ ID NO: 55) (Formula 2) In r specific ment, the modulin derivative of the present invention is a novel e including the amino acid sequence of the following Formula 3, which is prepared by linking a part of the amino acid sequence of oxyntomodulin and a part of the amino acid sequence of exendin or GLP—l via a proper amino acid linker.
Rl-B—C-R4 (SEQ ID NO: 56) (Formula 3) In still another specific embodiment, the oxyntomodulin derivative of the present invention is a novel peptide including the amino acid sequence of the following Formula 4, wherein a part of the amino acid sequence of oxyntomodulin is tuted with an amino acid capable of enhancing the binding affinity to GLP—1 receptor, for example, Leu at position 26 which binds with GLP—1 or by hydrophobic interaction is substituted with the hydrophobic residue, lie or Val.
TFTSDYSKYLD-D1-D2-D3-D4-D5-LFVQW-D6-D7-N—D8-R3 (SEQ ID NO: 57) (Formula 4) In still another specific embodiment, the oxyntomodulin derivative of the present invention is a novel peptide including the following Formula 5, wherein a part of the amino acid sequence is deleted, added, or substituted with other amino acid in order to enhance the activities of native oxyntomodulin on GLP-1 receptor and glucagon receptor.
R1-El—QGTFTSDYSKYLD—EZ-E3—RA-E4—E5—FV-E6—WLMNT—E7—R5 (SEQ ID NO: 58) (Formula 5) In ae 2 to 5, R1 is the same as in the description of a 1; A is selected from the group ting of SQGTFTSDYSKYLDSRRAQD- NT (SEQ ID NO. 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO. 42), SQGTFI‘SDYSKYLDERRAQDFVAWLKNT (SEQ ID NO. 43), GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO. 44), GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO. 45), GEGTFI‘SDLSRQMEEEAVRLFIEWAA (SEQ ID NO. 46), and SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO. 47); B is selected from the group consisting of SQGTFTSDYSKYLDSRRAQD-wFVQWLMNT (SEQ ID NO. 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO. 42), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO. 43), GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO. 44), GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO. 45), GEGTFTSDLSRQMEEEAVRLFIEWAA (SEQ ID NO. 46), SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO. 47), GEGTFTSDLSRQMEEEAVRLFIEW (SEQ ID NO. 48), and SQGTFTSDYSRYLD (SEQ ID NO. 49); C is a peptide having 2 to 10 amino acids consisting of combinations of alanine, glycine and serine; D1 is serine, glutamic acid or arginine, D2 is ne, glutamic acid or serine; D3 is arginine, alanine or valine; D4 is arginine, valine or serine; D5 is glutamine, arginine or lysine; D6 is isoleucine, valine or serine; D7 is methionine, arginine or glutamine; D8 is threonine, glycine or alanine; E1 is serine, Aib, Sar, d—alanine or d—serine; E2 is serine or glutarnic acid; E3 is arginine or lysine; [I45] E4 is ine or lysine; E5 is ic acid or glutamic acid; E6 is glutamine, cysteine or lysine; E7 is cysteine, lysine or is deleted; R3 is KRNRNNIA (SEQ ID NO. 35), GPSSGAPPPS (SEQ ID NO. 36) or GPSSGAPPPSK (SEQ ID NO. 37); R4 is HSQGTFTSDYSKYLD (SEQ ID NO. 38), HSQGTFTSDYSRYLDK (SEQ IDNO. 39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO. 40); and, R5 is KRNRNNIA (SEQ ID NO. 35), GPSSGAPPPS (SEQ ID NO. 36), GPSSGAPPPSK (SEQ ID NO. 37) or is deleted (excluded if the amino acid sequences of Formulae 2 to 5 are identical to that of SEQ ID NO. 1); The oxyntomodulin derivative of the present invention may be a noverl peptide of the following Formula 6.
[155] Rl-XI—X2—GTFTSD—X3—X4-X5-X6—X7~X8-X9-XIO-Xl l-X12-X13—X14— 6- X17—Xl8—X19—X20~X21-X22-X23-X24—R2 (SEQ ID NO: 59) (Formula 6) wherein R1 is histidine, desamino—histidyl, 4—imidazoacetyl or tyrosine; X1 is Aib(aminosiobutyric acid), glycine or serine; X2 is glutamic acid or glutamine; X3 is leucine or ne; X4 is serine or alanine; X5 is lysine or arginine; X6 is ine or tyrosine; X7 is leucine or methionine; X8 is aspartic acid or glutamic acid; X9 is glutamic acid, alpha-methyl—glutamic acid or is deleted; X10 is glutamine, glutamic acid, lysine, arginine or is deleted; X11 is alanine, ne or is deleted; X12 is alanine, valine or is deleted; X13 is lysine, glutamine, arginine, alpha—methyl—glutamic acid or is d; X14 is aspartic acid, glutamic acid, leucine or is deleted; X15 is phenylalanine or is deleted; X16 is isoleucine, valine or is d; X17 is alanine, cysteine, glutamic acid, ine, alpha~methyl~glutamic acid or is deleted; X18 is tryptophan or is deleted; X19 is alanine, isoleucine, leucine, valine or is deleted; X20 is alanine, lysine, methionine, arginine or is deleted; X21 is gine or is deleted; X22 is threonine or is deleted; X23 is cysteine, lysine or is deleted; X24 is a peptide having 2 to 10 amino acids consisting of glycine or is deleted; and R2 is KRNRNNIA (SEQ ID NO. 35), GPSSGAPPPS (SEQ ID NO. 36), GPSSGAPPPSK (SEQ ID NO. 37), TSDYSKYLD (SEQ ID NO. 38), HSQGTFTSDYSRYLDK (SEQ ID NO. 39), HGEGTFTSDLSKQMEEEAVK (SEQ ID NO. 40) or is deleted (excluded if the amino acid sequence of Formula 6 is identical to that of SEQ ID NO. 1) The oxyntomodulin derivative of the present invention may be selected from the group consisting of the peptides of SEQ ID NOS. 2 to 34. In the embodiment, the oxyntomodulin tive of the present invention may be an oxyntomodulin derivative described in Table 1 of Example 2—1.
Oxyntomodulin has the activities of two peptides, GLP-1 and glucagon. GLP—l decreases blood e, reduces food , and suppresses c emptying, and glucagon increases blood glucose, facilitate lipolysis and decreases body—weight by increasing energy metabolisms. The different biological effects of the two peptides can cause undesired effects like increasing blood glucose if glucagon shows a more dominant effect than GLP-l, or causing nausea and vomiting if GLP-l shows more dominant effect than glucagon. For example, the conjugate that was produced in Example 10 below showed greater affinity to GLP-1 receptor than the one produced in Example 12, but the efficacy of the former was lower than the latter as shown in the in vivo experiment in Example 18. This might be due to the increased efficacy of the conjugates in relation to the glucagon receptor in Example 12 inspite of its low efficacy in on to the GLP— 1 receptor. Therefore, the oxyntomodulin tives and their conjugates of the present invention are not limited to those derivatives which Show for unconditional increase of activities. For example, the amino acids can be ed at positions 1 and 11 of oxyntomodulin, which are known to suppress the activity of glucagon, to control the ty ratio between glucagon and GLP-1.
The conjugates of the present ion can induce increased stability in blood, suspend emission through the kidney, and change ty to receptors by linking a carrier to oxyntomodulin Via a covalent bond or forming microsphere. The carrier that can form a conjugate containing oxyntomodulin can be selected from the group consisting of albumin, transferrin, antibodies, antibody ents, n, heparin, polysaccharide such as chitin, fibronectin and most favorably immunoglobulin Fc region, all of which can increase the blood half—life of the ates when bound to modulin.
The term "immunoglobulin FC region" as used herein, refers to a protein that contains the heavy—chain constant region 2 (CH2) and the heavy—chain constant region 3 (CH3) of an immunoglobulin, excluding the variable regions of the heavy and light chains, the heavy—chain constant region 1 (CH1) and the light—chain constant region 1 (CLl) of the immunoglobulin. It may further include a hinge region at the heavy-chain constant region. Also, the immunoglobulin Fc region of the present invention may contain a part or all of the Fc region including the heavy~chain constant region 1 (CH1) and/or the light—chain nt region 1 (CLl), except for the variable regions of the heavy and light chains, as long as it has a physiological function substantially similar to or better than the native protein. Also, the immunoglobulin Fc region may be a fragment having a on in a relatively long portion of the amino acid sequence of CH2 and/or CH3. That is, the globulin Fc region of the present invention may comprise l) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 , 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains and an immunoglobulin hinge region (or a portion of the hinge region), and 6) a dimer of each domain of the heavy— chain constant regions and the light-chain constant region.
The immunoglobulin Fc region of the present invention includes a native amino acid sequence, and a sequence derivative (mutant) thereof. An amino acid sequence derivative is a sequence that is different from the native amino acid sequence due to a deletion, an insertion, a non—conservative or conservative substitution or combinations thereof of one or more amino acid residues. For example, in an IgG Fc, amino acid residues known to be important in binding, at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, may be used as a suitable target for modification.
Also, other various derivatives are possible, including one in which a region capable of forming a disulfide bond is deleted, or certain amino acid residues are eliminated at the N—terminal end of a native Fc form or a methionine residue is added thereto. Further, to remove effector functions, a deletion may occur in a ment~binding site, such as a Clq—binding site and an ADCC (antibody dependent cell mediated cytotoxicity) site. Techniques of ing such sequence derivatives of the globulin Fc region are disclosed in WO 97/34631 and WO 96/32478.
Amino acid exchanges in proteins and peptides, which do not generally alter the activity of the ns or peptides, are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Va1, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, n, Leu/Ile, 1, AlalGlu and Asp/Gly, in both directions. In addition, the Fc region, if desired, may be modified by , phosphorylation, sulfation, acrylation, glycosylation, methylation, famesylation, acetylation, amidation, and the like.
The aforementioned Fc derivatives are tives that have a ical activity identical to the Fc region of the t invention or improved structural stability, for example, against heat, pH, or the like.
In addition, these Fc regions may be ed from native forms isolated from humans and other animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or derivatives thereof, ed from transformed animal cells or microorganisms. , they may be ed from a native immunoglobulin by isolating whole immunoglobulins from human or animal organisms and treating them with a proteolytic enzyme. Papain digests the native globulin into Fab and Fc regions, and pepsin treatment results in the production of pF'c and F(ab)2 fragments. These fragments may be subjected, for example, to size exclusion tography to isolate PC or pF'c. In an embodiment, a human—derived Fc region is a recombinant immunoglobulin Fc region that is obtained from a microorganism.
In addition, the immunoglobulin Fc region of the present invention may be in the form of having native sugar chains, increased sugar chains compared to a native form or decreased sugar chains compared to the native form, or may be in a deglycosylated form. The increase, decrease or removal of the immunoglobulin Fc sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method and a c engineering method using a microorganism. The removal of sugar chains from an Fc region results in a sharp decrease in g affinity to the Clq part of the first complement component C l and a decrease or loss in antibody— dependent ediated cytotoxicity or complement—dependent cytotoxicity, thereby not inducing unnecessary immune responses in—vivo. In this regard, an immunoglobulin Fc region in a deglycosylated or sylated form may be a suitable drug carrier.
As used herein, the term ”deglycosylation" refers to enzymatically removing sugar moieties from an Fc region, and the term "aglycosylation" means that an Fc region is produced in an unglycosylated form by a prokaryote, for example but not limited to E. coli.
Meanwhile, the immunoglobulin Fc region may be derived from humans or other animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs.
In on, the immunoglobulin Fc region may be an Fc region that is derived from IgG, IgA, IgD, IgE and IgM, or that is made by combinations thereof or hybrids f. In an embodiment, it is derived from IgG or IgM, which are among the most abundant proteins in human blood, which is known to enhance the half-lives of ligand— binding proteins.
On the other hand, the term nation", as used herein, means that polypeptides encoding single-chain immunoglobulin Fc regions of the same origin are linked to a single-chain polypeptide of a ent origin to form a dimer or multimer. That is, a dimer or multimer may be formed from two or more fragments selected from the group ting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and IgE Fc fragments.
The term "non—peptidyl r", refers to a biocompatible r including two or more repeating units linked to each other by any covalent bond excluding a peptide bond. In the present invention, the non-peptidyl polymer may be interchangeably used with the non-peptidyl linker.
The non—peptidyl polymer useful in the present invention may be selected from the group consisting of a biodegradable polymer, a lipid polymer, chitin, hyaluronic acid, and a combination thereof. In an ment, the radable polymer may be polyethylene glycol, polypropylene glycol, ne glycol-propylene glycol copolymer, yethylated polyol, polyvinyl alcohol, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA) or polylactic-gly colic acid (PLGA) or polyethylene glycol (PEG). In addition, derivatives thereof known in the art and derivatives easily ed by a method known in the art may be included in the scope of the present invention.
The peptide linker which is used in the fusion protein ed by a conventional inframe fusion method has drawbacks in that it is easily in- vivo d by a proteolytic enzyme, and thus a ient effect of increasing the serum half-life of the active drug by a carrier cannot be obtained as expected. However, in the present invention, the polymer having resistance to the proteolytic enzyme can be used to maintain the serum half—life of the peptide being similar to that of the carrier.
Therefore, any non-peptidyl polymer can be used without tion, as long as it is a polymer having the aforementioned function, that is, a polymer having ance to the in- vivo proteolytic enzyme. The non-peptidyl polymer has a molecular weight in the range of l to 100 kDa, or 1 to 20 kDa. The non—peptidyl polymer of the present invention, linked to the immunoglobulin Fc region, may be one polymer or a combination of different types of polymers.
The non—peptidyl polymer used in the present ion has a reactive group capable of binding to the immunoglobulin Fc region and protein drug. The non—peptidyl polymer has a reactive group at both ends, which may be selected from the group consisting of a reactive aldehyde, a propionaldehyde, a butyraldehyde, a maleimide and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate. In particular, when the non—peptidyl polymer has a reactive aldehyde group at both ends f, it is ive in linking at both ends with a physiologically active polypeptide and an immunoglobulin with minimal non—specific reactions. A final product ted by reductive alkylation by an aldehyde bond is much more stable than that linked by an amide bond. The aldehyde reactive group selectively binds to an N~terminus at a low pH, and binds to a lysine residue to form a covalent bond at a high pH, such as pH 9.0. The reactive groups at both ends of the non-peptidyl polymer may be the same or different. For example, the non-peptidyl polymer may possess a maleimide group at one end, and an aldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. When a hylene glycol having a reactive hydroxy group at both ends f is used as the non-peptidyl polymer, the hydroxy group may be activated to various reactive groups by known chemical reactions, or a polyethylene glycol having a commercially available modified reactive group may be used so as to prepare the long acting conjugate of the present invention.
The conjugate of the present invention, can be which both ends of the non—peptidyl polymer having two reactive terminal groups are linked to an amine group or thiol group of the immunoglobulin Fc region and oxyntomodulin derivatives, respectively.
The non-peptidyl polymer has a reactive group at both ends, which may be selected from the group consisting of a reactive aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative. The succinimide derivative may be imidyl propionate, hydroxy imidyl, succinimidyl carboxymethyl, or succinimidyl carbonate.
The two ve terminal groups of the ptidyl polymer may be the same as or different from each other. For example, the non-peptide polymer may possess a maleimide group at one end and an de group, a propionaldehyde group or a butyraldehyde group at the other end. For example, when the non—peptidyl polymer has a reactive de group at a terminal group, and a maleimide group at the other terminal group, it is effective in linking at both ends with a physiologically active polypeptide and an immunoglobulin with l non—specific reactions. According to Examples of the present invention, conjugates were ed by linking the oxyntomodulin or derivative thereof and the immunoglobulin Fc region via a covalent bond using PEG that is a ptidyl polymer including the propionaldehyde group alone or both the maleimides group and the aldehyde group.
The ates of the present invention show excellent activity on GLP—1 receptor and glucagon receptor, compared to native oxyntomodulin, and the blood half—life is increased by linking with the Fc region so as to maintain in vivo activity for a long period of time.
In still another aspect, the present invention provides a pharmaceutical ition for the prevention or treatment of obesity comprising the peptide.
As used herein, the term "prevention" means all of the actions by which the occurrence of the disease is ined or retarded. In the present invention, "prevention" means that the occurrence of obesity from such s as an increase in body weight or body fat is restrained or retarded by administration of the ates of the present invention.
As used herein, the term "treatment" means all of the s by which the symptoms of the disease have been alleviated, improved or ameliorated. In the present invention, ment" means that the symptoms of obesity are alleviated, improved or ameliorated by administration of the conjugates of the present invention, resulting in a ion in body weight or body fat.
As used herein, the term "obesity" implies lation of an excess amount of adipose tissue in the body, and a body mass index (body weight (kg) divided by the square of the height (m)) above 25 is to be regarded as obesity. Obesity is usually caused by an energy imbalance, when the amount of dietary intake exceeds the amount of energy expended for a long period of time. Obesity is a lic disease that affects the whole body, and increases the risk for diabetes, hyperlipidemia, sexual dysfunction, arthritis, and cardiovascular diseases, and in some cases, is associated with incidence of cancer.
The ates of the present invention, which are prepared by linking oxyntomodulin or a derivative thereof with the immunoglobulin Fc region, show excellent binding affinity to glucagon and GLP-1 receptors (Table 3) and ent resistance to in- vivo proteolytic enzymes so as to t the in vivo activity for a long period of time, thereby g excellent anti-obesity effects such as reductions in body weight ().
The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier, excipient, or diluent. As used herein, the term "pharmaceutically acceptable" means that the composition is sufficient to achieve the therapeutic effects without deleterious side effects, and may be readily determined depending on the type of the diseases, the patient's age, body weight, health conditions, gender, and drug sensitivity, administration route, administration mode, administration frequency, duration of treatment, drugs used in combination or coincident with the composition of this invention, and other factors known in medicine.
The pharmaceutical ition including the derivative of the present invention may further include a pharmaceutically acceptable carrier. For oral stration, the carrier may include, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a ding agent, a colorant, and a flavorant. For injectable preparations, the carrier may include a buffering agent, a preserving agent, an analgesic, a solubilizer, an isotonic agent, and a stabilizer. For preparations for topical administration, the carrier may include a base, an excipient, a lubricant, and a preserving agent.
The composition of the present invention may be formulated into a variety of dosage forms in combination with the aforementioned pharmaceutically acceptable carriers.
For example, for oral administration, the ceutical composition may be formulated into tablets, troches, capsules, s, suspensions, syrups or wafers. For injectable preparations, the pharmaceutical composition may be formulated into an ampule as a single dosage form or a multidose container. The pharmaceutical composition may also be formulated into solutions, suspensions, s, pills, capsules and long-acting preparations.
On the other hand, examples of the r, the excipient, and the diluent suitable for the pharmaceutical formulations include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium ate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, ium stearate and mineral oils, In addition, the pharmaceutical ations may r include fillers, anti—coagulating agents, lubricants, humectants, flavorants, and antiseptics.
Further, the ceutical composition of the present invention may have any formulation selected from the group ting of tablets, pills, powders, granules, capsules, suspensions, liquids for internal use, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, lyophilized formulations and suppositories.
Further, the composition may be formulated into a single dosage form suitable for the patient‘s body, and may be formulated into a preparation useful for e drugs according to the typical method in the pharmaceutical field so as to be administered by an oral or parenteral route such as through skin, intravenous, intramuscular, intraarterial, intramedullary, intramedullary, intraventricular, pulmonary, ermal,subcutaneous, intraperitoneal, intranasal, intracolonic, topical, sublingual, l, or rectal administration, but is not limited thereto.
The composition may be used by blending with a variety of pharmaceutically able carriers such as physiological saline or organic solvents. In order to increase the ity or tivity, carbohydrates such as glucose, sucrose or dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other izers may be used.
The administration dose and frequency of the pharmaceutical composition of the t invention are determined by the type of active ingredient, together with various factors such as the disease to be treated, administration route, patient's age, gender, and body weight, and disease severity.
. The total effective dose of the composition of the present ion may be administered to a patient in a single dose, or may be administered for a long period of time in le doses according to a fractionated treatment protocol. In the pharmaceutical composition of the present ion, the content of active ingredient may vary depending on the disease severity. In an embodiment, the total daily dose of the e of the present invention may be approximately 0.0001 jig to 500 mg per 1 kg of body weight of a patient. However, the effective dose of the peptide is ined considering various factors including patient's age, body weight, health conditions, gender, disease ty, diet, and secretion rate, in addition to administration route and treatment frequency of the pharmaceutical composition. In view of this, those skilled in the art may easily determine an effective dose suitable for the particular use of the pharmaceutical composition of the present invention. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, and administration route and mode, as long as it shows the effects of the present invention.
The pharmaceutical ition of the present invention shows excellent in- vivo duration of efficacy and titer, thereby remarkably reducing the number and ncy of administration thereof.
Moreover, the pharmaceutical composition may be administered alone or in combination or coincident with other pharmaceutical formulations showing prophylactic or therapeutic effects on y. The pharmaceutical formulations g prophylactic or therapeutic s on obesity are not ularly limited, and may include a GLP—1 or agonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a Melanin—concentrating hormone (MCH) or antagonist, a Y2/3 receptor agonist, a MC3/4 receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2c agonist, a [33A receptor t, an Amylin receptor agonist, a Ghrelin antagonist, and/or a Ghrelin receptor antagonist.
In still another aspect, the present invention provides a method for ting or ng obesity, comprising the step of administering to a subject the conjugate or the pharmaceutical composition including the same.
As used herein, the term "administration" means introduction of an amount of a predetermined substance into a patient by a certain suitable method. The composition of the present invention may be administered via any of the common routes, as long as it is able to reach a desired tissue, for example, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary, or intrarectal administration. However, since peptides are ed upon oral administration, active ingredients of a composition for oral administration should be coated or formulated for protection against degradation in the stomach.
In the present invention, the term "subject" is those suspected of having obesity, which means mammals including human, mouse, and livestock having obesity or having the possibility of obesity. However, any subject to be d with the peptide or the pharmaceutical composition of the present invention is ed t limitation.
The pharmaceutical composition including the peptide of the present invention is administered to a subject suspected of having obesity, thereby treating the subject effectively. The obesity is as described above.
The therapeutic method of the present invention may include the step of stering the composition including the peptide at a pharmaceutically effective amount. The total daily dose should be determined through appropriate medical judgment by a physician, and administered once or several times.
The specific therapeutically effective dose level for any particular patient may vary depending on various factors well known in the medical art, including the kind and degree of the response to be achieved, concrete compositions according to whether other agents are used therewith or not, the patient's age, body weight, health condition, gender, and diet, the time and route of stration, the secretion rate of the ition, the time period of therapy, other drugs used in combination or coincident with the composition of this invention, and like factors well known in the medical arts.
In still another aspect, the t invention es a use of the conjugate or the pharmaceutical composition including the same in the preparation of drugs for the prevention or treatment of obesity.
Mode for the Invention Hereinafter, the present ion will be described in more detail with reference to the following Examples. However, these Examples are for illustrative purposes only, and the invention is not intended to be limited by these Examples.
Example 1. Production of in vitro ted cell line Example 1-1: Production of cell line showing CAMP response to GLP-l PCR was performed using a region corresponding to ORF (Open g Frame) in cDNA (OriGene Technologies, Inc. USA) of human GLP—1 receptor gene as a template, and the following forward and e primers including each of the HindIII and EcoRI restriction sites so as to obtain a PCR product.
Forward primer: 5’—CCCGGCCCCCGCGGCCGCTATTCGAAATAC—3’(SEQ ID NO. 47) Reverse primer: 5‘—GAACGGTCCGGAGGACGTCGACTCTTAAGATAG—3’(SEQ ID NO. 48) The PCR product was cloned into the known animal cell sion vector hfr to prepare a recombinant vector XOGC/GLPIR.
CHO DG44 cell line cultured in DMEM/F12 (10% FBS) medium was transfected with the recombinant vector XOGC/GLPlR using ctamine (Invitrogen, USA), and cultured in a ion medium containing 1 mg/mL G418 and 10 nM methotraxate. Single clone cell lines were selected therefrom by a limit dilution technique, and a cell line showing excellent CAMP response to GLP-l in a con- centration—dependent manner was finally selected rom.
Example 1-2: tion of cell line showing CAMP response to glucagon PCR was performed using a region corresponding to ORF in cDNA (OriGene Tech— nologies, Inc. USA) of human glucagon receptor gene as a template, and the following forward and reverse primers including each of the EcoRI and XhoI restriction sites so as to obtain a PCR product.
Forward primer: 5'—CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC—3'(SEQ ID NO. 49) Reverse primer: 5'—CTAACCGACTCTCGGGGAAGACTGAGCTCGCC~3'(SEQ ID NO. 50) The PCR product was cloned into the known animal cell expression vector XOGC/dhfr to prepare a recombinant vector XOGC/GCGR.
CHO DG44 cell line cultured in DMEM/F12 (10% FBS) medium was transfected with the recombinant vector XOGC/GCGR using Lipofectamine, and ed in a selection medium containing 1 mg/mL G418 and 10 nM methotraxate. Single clone cell lines were ed therefrom by a limit dilution technique, and a cell line g excellent CAMP response to glucagon in a concentration-dependent manner was finally selected therefrom.
Example 2. Test on in vitro activity of oxyntomodulin derivatives Example 2-1: Synthesis of oxyntomodulin derivatives In order to measure in vitro activities of oxyntomodulin derivatives, oxyntomodulin derivatives having the following amino acid sequences were synthesized (Table 1).
Table 1 [Table 1] modulin and oxyntomodulin derivatives SEQ ID NO. 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO. 2 CA—SQGTFTSDYSKYLDEEAVRLFIEWLMNTKRNRNNIA KYLD SEQ ID NO. 7 CA—SQGTFTSDYSRYLDEEAVRLFIEW’LMNTK SEQ ID NO. 8 CA-SQGTFTSDLSRQLEEEAVRLFIEWLMNK SEQ ID NO. 9 CA—GQGTFTSDYSRYLDEEAVXLFIEWLMNTKRNRNNIA RYLDK SEQ ID NO. 18 CA-SQGTFTSDYSRYLDEEAVRLFIEWIRNGGPSSGAPPPS SEQ ID NO. 19 CA-SQGTFTSDYSRYLD E EAV K LFIEWIRN- TKRNRNNIA SEQ ID NO. 20 CA-SQGTFTSDYSRYLD E EAV E LFIEWIRNGG- PSSGAPPPSK SEQ ID NO. 21 CA-SQGTFTSDYSRQLEEEAVRLFIEWVRNTKRNRNNIA SEQ ID NO. 22 DA-SQGTFTSDYSKYLD E KRA K EFVQWLMNTK SEQ ID NO. 27 HAibQGTFTSDYSKYLD E QAA K EFICWLMNT SEQ ID NO. 28 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNT SEQ ID NO. 29 H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO. 30 TFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO. 31 CA—(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNN SEQ ID NO. 32 CA-AibQGTFISDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO. 33 HAibQGTFTSDYAKYLDEKRAKEFVQWLMNTC SEQ ID NO. 34 TFTSDYSKYLDEKRAKEFVQWLMNTC In Table 1, amino acids in bold and ined represent ling formation, and amino acids represented by X mean a non-native amino acid, alpha-methyl—glutamic acid. In addition, CA represents 4-imidazoacetyl, and DA represents desamino—histidyl.
Example 2-2: Test on in vitro activity of oxyntomodulin derivatives In order to measure anti-obesity efficacies of the oxyntomodulin tives syn- thesized in e 2-1, cell activity was measured in Vitro using the cell lines prepared in Examples 1—1 and 1—2.
The cell lines were those prepared by transfecting CHO (Chinese Hamster Ovaiy) to express human GLP-1 receptor gene and glucagon or gene, respectively. Thus, they are suitable to measure GLP—1 and glucagon activities. Therefore, the activity of each oxyntomodulin derivative was measured using each transformed cell line.
Specifically, each cell line was sub—cultured twice or three time a week, and aliquoted in each well of a 96—well plate at a density of l X 105, followed by cul- tivation for 24 hours.
The cultured cells were washed with KRB buffer and suspended in 40 ml of KRB buffer containing 1 mM IBMX, and left at room temperature for 5 minutes. Oxyn— tomodulin (SEQ [D NO. 1) and oxyntomodulin derivatives (represented by SEQ 1]) NOs. 2—6, 8, 10-13, 17, 18, 23-25, 27, 28 and 32-34) were diluted from 1000 11M to 0.02 nM by 5-fold serial dilution, and each 40 mL f was added to the cells, and cultured at 37°C for 1 hour in a C02 incubator. Then, 20 mL of cell lysis buffer was added for cell lysis, and the cell lysates were d to a CAMP assay kit (Molecular Device, USA) to measure CAMP concentrations. ECSO values were calculated therefrom, and compared to each other. ECSO values are shown in the following Table Table 2 [Table 2] Comparison of in vitro ties for GLP—1 receptor and glucagon receptor between oxyntomodulin and oxyntomodulin derivatives m['1']1C) 2.0 m5? ’5‘E CHO/GLP—lR CHO/GCGR SEQ ID NO. 1 (J!O ’ N HC 10 - 43 SEQ ID NO. 2 L]! P—‘ 00 12.8 SEQ ID NO. 3 V 3—-OOC 637.7 SEQ ID No. 4 LII IL}! >1 ,OOO SEQ ID No. 5 3"\o >1 ,000 SEQ ID NO. 6 500.1 >1,000 SEQ 11) NO. 8 419.6 >1 ,000 SEQ ID NO. 10 >1,000 >1 ,000 SEQ ID NO. 11 >1,000 >1,000 SEQ ID NO. 12 > 1 ,000 >1 ,000 SEQ ID NO. 13 >1,000 >1,000 SEQ ID NO. 17 97.9 >1,000 SEQ lD NO. 18 96.3 >1,000 SEQ ID NO. 23 2.46 Li] 00 SEQ ID NO. 24 1.43 6.95 SEQ ID NO. 25 f"\O H DJ SEQ ID NO. 27 2.8—5.5 3.1-5.6 SEQ ID NO. 28 5” H .0 o.) SEQ ID NO. 32 14.25 17.3 SEQ 1]) No. 33 2.20 oo .0 to SEQ ID NO. 34 12.5 1.0 As shown in Table 2, there were oxyntomodulin den'vatives showing ent in vitro activities and different ratios of activities on GLP-1 receptor and glucagon receptor, compared to native oxyntomodulin of SEQ ID NO. 1.
It is known that modulin activates both the GLP-1 receptor and glucagon receptor to suppress appetite, facilitate lipolysis, and promote satiety, thereby showing anti-obesity effects. The oxyntomodulin derivatives ing to the present invention show higher in vitro activities on both the GLP-1 or and glucagon receptor than the wild-type oxyntomodulin, and therefore can be used as a therapeutic agent for obesity with higher efficacies than the known oxyntomodulin.
Example 3. Test on in vivo activity of modulin derivatives In order to measure in vivo therapeutic activity of oxyntomodulin tives, changes in food intake by administration of oxyntomodulin derivatives were examined in ob/ob mouse using native oxyntomodulin as a control.
Specifically, obese diabetic ob/ob mice, commonly used to test the efficacies of therapeutic agents for obesity and diabetes, were fasted for 16 hours, and administered with 1 or 10 mg/kg of oxyntomodulin, or 0.02, 0.1, l or 10 mg/kg of the oxyn— tomodulin derivative of SEQ ID NO. 2. Then, food intake was examined for 2 hours (. is a graph showing changes in food intake according to administration dose of oxyntomodulin or oxyntomodulin derivative. As shown in admin- istration of 1 mg/kg of oxyntomodulin derivative showed more excellent tory effects on food intake than administration of 10 mg/kg of oxyntomodulin.
Taken together, the oxyntomodulin derivatives of the present invention have much higher anti-obesity effects than the wild-type oxyntomodulin, even though ad- ministered at a lower dose, indicating improvement in the problems of the wild—type oxyntomodulin that shows lower anti—obesity effects and should be administered at a high dose three times a day.
Example 4: Preparation of conjugates including oxyntomodulin and im- munoglobulin Fc Firstly, for PEGylation of lysine residue at position 30 of the amino acid ce of oxyntomodulin (SEQ ID NO. 1) with 3.4 K PropionALD(2) PEG (PEG with two propylaldehyde groups, NOF, Japan), the oxyntomodulin and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of l : 12 with the protein concentration of 5 mg/ml at 4°C for 4.5 hours. At this time, the on was conducted in a t mixture of 100 mM Na—Borate buffer (pH 9.0) and 45% panol, and 20 mM sodium orohydiide borohydiide (SCB, NaCNBH3), NaCNBl-l3) was added thereto as a reducing agent, After tion of the reaction, the reaction mixture was applied to a SOURCE S (XKl 6, Amersham Biosciences) to purify modulin having mono-pegylated lysine (column: SOURCE S (XK16, Amersham Biosciences), flow rate: 2.0 ml/min, gradient: A 0 —>3% 1 min B —> 40% 222 min B (A: 20 mM Na— citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (). is a graph showing the result of purifying mono-PEGylated oxyntomodulin through a SOURCE S pu- rification column. Mono-PEGylation of the eluted peaks was ed by SDS- PAGE, and lysine selectivity was examined by peptide g using Asp-N protease ()). is a graph showing the result of e mapping of purified mono- PEGylated oxyntomodulin.
Next, the purified mono—PEGylated oxyntomodulin and immunoglobulin Fc were reacted at a molar ratio of 1 : 10 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE lSQ pu— ion column to purify ates including oxyntomodulin and immunoglobulin Fc (column: SOURCE 15Q (XK16, Amersham Biosciences), flow rate: 2.0 ml/min, nt: A 0 ~—> 20% 100 min B (A: 20mM Tris-HCl, pH 7.5, B: A + 1M NaCl)) (). is a graph showing the result of purifying conjugates including oxyn- tomodulin and immunoglobulin Fc.
Example 5: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 29) and immunoglobulin Fc Firstly, for tion of lysine residue at position 30 of the amino acid sequence of oxyntomodulin tive (SEQ ID NO. 29) with 3.4 K PropionALD(2) PEG, the oxyntomodulin derivative (SEQ ID NO. 29) and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of l : 12 with the protein concentration of 5 mg/ml at 4°C for 4.5 hours. At this time, the reaction was conducted in a solvent mixture of 100 mM Na—Borate buffer (pH 9.0) and 45% isopropanol, and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the on mixture was applied to a SOURCE Sto purify the oxyntomodulin derivative having egylated lysine (Column: SOURCE S, flow rate: 2.0 ml/min, gradient: A 0 ~93% l min B —> 40% 222 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (). is a graph showing the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 29) through a SOURCE S purification column.
Next, the purified mono-PEGylated modulin derivative (SEQ ID NO. 29) and immunoglobulin Fc were reacted at a molar ratio of l : 10 with the n con— centration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE 15Q purification column to purify ates including oxyntomodulin den’vative (SEQ ID NO. 29) and immunoglobulin Fc (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A 0 —+ 20% 100 min B (A: 20mM Tris—HCl, pH 7.5, B: A + 1M NaCl)) (). is a graph showing the result of purifying conjugates ing oxyntomodulin derivative (SEQ ID NO. 29) and immunoglobulin Fc.
Example 6: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 30) and immunoglobulin Fc y, for PEGylation of lysine e at position 30 of the amino acid sequence of oxyntomodulin tive (SEQ ID NO. 30) with 3.4 K PropionALD(2) PEG, the oxyntornodulin derivative (SEQ ID NO. 30) and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of l : 15 with the protein concentration of 3 mg/ml at 4°C for 4.5 hours. At this time, the reaction was conducted in a solvent mixture of 100 mM HEPES buffer (pH 7.5) and 45% isopropanol, and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the modulin derivative having mono- pegylated lysine (Column: SOURCE S, flow rate: 2.0 ml/min, gradient: A 0 —->3% 1 min B —a 40% 222 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (). is a graph showing the result of ing a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 30) through a SOURCE S purification column. Mono-PEGylation of the eluted peaks was examined by SDS-PAGE, and lysine selectivity was examined by e mapping using Asp—N protease (). is a graph showing the result of e mapping of purified mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 30).
Next, the purified mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 30) and immunoglobulin Fc were d at a molar ratio of 1 : 10 with the protein con— centration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was ted in 100 mM potassium phosphate buffer (pl-I 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE 15Q purification column to purify conjugates including oxyntomodulin tive (SEQ ID NO. 30) and immunoglobulin Fe (column: SOURCE 15Q, flow rate: 2.0 nil/min, gradient: A 0 -9 20% 100 min B (A: 20mM Tris—HCl, pH 7.5, B: A + lM NaCl)) (). is a graph showing the result of pun'fying conjugates including oxyntomodulin tive (SEQ ID NO. 30) and immunoglobulin Fc.
Example 7: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 31) and immunoglobulin Fe Firstly, for PEGylation of lysine residue at position 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 31) with 3.4 K PropionALD(2) PEG, the oxyntomodulin derivative (SEQ ID NO. 31) and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of 1 : 15 with the protein concentration of 3 mg/ml at 4°C for 4.5 hours. At this time, the on was conducted in a solvent mixture of 100 mM HEPES buffer (pH 7.5) and 45% panol, and 20 mM SCB was added thereto as a reducing agent. After tion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono- pegylated lysine (Column: SOURCE 8, flow rate: 2.0 ml/min, gradient: A 0 —>3% 1 min B —) 40% 222 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (). is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 31) through a SOURCE S purification column.
Next, the purified mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 31) and immunoglobulin Fc were reacted at a molar ratio of 1 : 10 with the protein con» centration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added o as a reducing agent. After completion of the on, the reaction mixture was applied to 3.
SOURCE 15Q purification column to purify conjugates including modulin derivative (SEQ ID NO. 31) and immunoglobuiin Fc (column: SOURCE 15Q, flow rate: 2.0 ml/min, nt: A O -> 20% 100 min B (A: 20mM Tris-HCI, pH 7.5. B: A + 1M NaCl)) (). is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 31) and immunoglobulin Fc.
Example 8: ation of conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc Firstly, for PEGylation of lysine residue at position 30 of the amino acid sequence of oxyntomodulin tive (SEQ ID NO. 2) with 3.4 K PropionALD(2) PEG, the oxyn- tornodulin derivative (SEQ ID NO. 2) and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of l : 10 with the n concentration of 3 mg/ml at 4°C for 4 hours. At this time, the reaction was conducted in a solvent mixture of 100 mM HEPES buffer (pH 7.5) and 45% isopropanol, and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin tive having mono- pegylated lysine (Column: SOURCE S, flow rate: 2.0 ml/min, gradient: A 0 —>3% 1 min B —> 40% 222 min B (A: 20mM Na-citrate, pH 3.0 + 45% ethanol, B: A + 1M KCD) (). is a graph showing the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 2) through a SOURCE S purification column.
Mono-PEGylation of the eluted peaks was ed by SDS-PAGE, and lysine se- lectivity was examined by peptide mapping using Asp-N protease (). is a graph showing the result of peptide mapping of purified mono-PEGylated oxyn— tomodulin derivative (SEQ ID NO. 2).
Next, the purified mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc Were reacted at a molar ratio of 1 : 8 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was ted in 100 mM potassium phosphate buffer (pl-I 6.0) and 20 mM SCB was added o as a reducing agent. After completion of the reaction, the reaction e was applied to a SOURCE 15Q purification column (Column : SOURCE 15Q, flow rate : 2.0 ml/rnin, gradient : A 0 —> 4% l min B —-> 20% 80 min B (A: 20mM Tris—HCl, pH 7.5, B: A + 1M NaCl)) () and a Source ISO purification column (Column: SOURCE ISO (XK16, Amersham Biosciences), flow rate : 2.0 ml/min, gradient: A 0 —> 100% 100 min B, (A: 20mM Tris—HCI, pH 7.5, B: A + 1.3M AS))() to purify conjugates including modulin derivative (SEQ ID NO. 2) and immunoglobulin Fc. is a graph showing the result of purifying conjugates including modulin derivative (SEQ ID NO. 2) and immunoglobulin Fc through a Source ISO purification column, and is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc through a Source ISO purification column. e 9: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc y, for PEGylation of lysine residue at position 27 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 3) with 3.4 K nALD(2) PEG, the oxyn— tomodulin derivative (SEQ ID NO. 3) and 3.4 K nALDQ) PEG were reacted at a molar ratio of l : 10 with the protein concentration of 3 mg/ml at 4°C for 4 hours. At this time, the reaction was conducted in a solvent e of 100 mM HEPES buffer (pH 7.5) and 45% isopropanol, and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin tive having mono- pegylated lysine (Column: SOURCE 8, flow rate: 2.0 ml/min, gradient: A 0 —>3% 1 min B -—> 40% 222 min B (A: 20mM Na-citrate, pH 3.0 + 45% ethanol, B: A + 1M KC1)) (). is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 3) through a SOURCE S purification column.
Mono-PEGylation of the eluted peaks was examined by SDS-PAGE, and lysine se— lectivity was examined by peptide g using Asp-N protease (). is a graph showing the result of peptide g of purified mono-PEGyIated oxyn- tomodulin derivative (SEQ ID NO. 3).
Next, the purified mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc were reacted at a molar ratio of l : 8 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the on was ted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a Butyl FF purification column (Column: Butyl FF(XK16, Amersham Biosciences), flow rate : 2.0 ml/min, gradient : B 0 ——> 100% 5 min A(A: 20mM Tris-HCl, pH 7.5, B: A + 1.5M NaCl)) () and a SOURCE lSQ purification column (Column : SOURCE lSQ, flow rate : 2.0 ml/min, gradient : A 0 —> 4% l min B -—«> 20% 80 min B(A: 20mM Tris— HCl, pH 7.5, B: A + lM NaCl)) () to purify conjugates including oxyn- tomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc. is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 3) and globulin Fc through a Butyl FF purification column, and is a graph showing the result of purifying ates including oxyntomodulin derivative (SEQ ID NO. 3) and globulin Fc through a SOURCE 15Q pu— rification column.
Example 10: Preparation of ates including oxyntomodulin tive (SEQ ID NO. 23) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at on 24 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 23) with MAL-lOK-ALD PEG (NOF., Japan), the oxyntomodulin derivative (SEQ ID NO. 23) and MAL-10K~ALD PEG were reacted at a molar ratio of l : 3 with the protein concentration of 3 mg/ml at room temperature for 3 hours. At this time, the reaction was conducted in 50 mM Tris buffer (pH 8.0) and 45% isopropanol, and 1M guanidine was added thereto. After completion of the on, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono-pegylated cysteine (column: SOURCE S, flow rate: 2.0 nil/min, gradient: A 0 —>100% 50 min B (A: 20mM Na- e, pH 3.0 + 45% ethanol, B: A + lM KCl)) (). is a graph showing the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 23) through a SOURCE S purification column.
Next, the purified mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc were reacted at a molar ratio of l : 5 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the on was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added o as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE lSQ purification column (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A 0 —> 4% l min B —> 20% 80 min B(A: 20mM Tris—HCl, pH 7.5, B: A + 1M NaCl)) () and a Source ISO purification column (column: SOURCE ISO, flow rate: 2.0 ml/min, gradient: B 0 -—> 100% 100 min A, (A: 20mM Tris—HCI, pH 7.5, B: A + 1.1M AS)) () to purify conjugates including oxyntomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc. is a graph showing the result of purifying ates ing oxyntomodulin derivative (SEQ ID NO. 23) and im- munoglobulin Fc through a SOURCE 15Q purification column, and is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 23) and giobulin Fc through a Source ISO purification column. e 11: Preparation of ates ing oxyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fe Firstly, for PEGylation of cysteine residue at position 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 24) with MAL-10K~ALD PEG, the oxyn— tomoduiin derivative (SEQ ID NO. 24) and MAL—IOK-ALD PEG were d at a molar ratio of 1 : 3 with the protein concentration of 3 mg/ml at room temperature for 3 hours. At this time, the reaction was conducted in 50 mM Tris buffer (pH 8.0) and 45% isopropanol, and 1M guanidine was added o. After completion of the reaction, the reaction mixture was d to a SOURCE Spurification column to purify the oxyntomodulin tive having mono~pegy1ated cysteine (column: SOURCE S, flow rate: 2.0 , gradient: A 0 —>100% 50 min B (A: 20mM Na- citrate, pH 3.0 + 45% ethanol, B: A + 1M KC1)) (). is a graph showing the result of purifying a mono—PEGyIated oxyntomodulin derivative (SEQ ID NO. 24) through a SOURCE S purification column.
Next, the purified mono—PEGylated ortyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc were reacted at a molar ratio of 1 : 5 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was ted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE ISQ purification column (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A 0 -—~> 4% 1 min B ~—> 20% 80 min B(A: 20mM Tris-HCl, pH 7.5, B: A + 1M NaCl)) () and a Source ISO purification column (column: SOURCE ISO, flow rate: 2.0 ml/min, gradient: B O ——> 100% 100 min A, (A: 20mM Tiis-HCI, pH 7.5, B: A + 1.1M AS)) () to purify conjugates including oxyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc. is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 24) and im- munoglobulin Fc through a SOURCE 15Q purification column, and is a graph showing the result of purifying conjugates including oxyntomoduiin derivative (SEQ ID NO. 24) and immunoglobulin Fc through a Source ISO purification column.
Example 12: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 25) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at on 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 25) with MAL-IOK-ALD PEG, the oxyn- tomodulin derivative (SEQ ID NO. 25) and MAL—lOK-ALD PEG were reacted at a molar ratio of l : 3 with the protein concentration of 3 mg/ml at room ature for 3 hours. At this time, the reaction was conducted in 50 mM Tris buffer (pH 8.0) and 1M guanidine was added thereto. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono—pegylated cysteine (column: SOURCE S, flow rate: 2.0 ml/ min, gradient: A 0 ——>100% 50 min B (A: 20mM Na~citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (a). a is a graph showing the result of purifying a mono— PEGylated modulin derivative (SEQ ID NO. 25) through a SOURCE S pu— rification column.
Next, the purified mono—PEGylated oxyntomodulin derivative (SEQ 1]) NO. 25) and globulin Fc were reacted at a molar ratio of 1 : 5 with the protein tration of 20 mg/ml at 4°C for 16 hours. At this time, the on was conducted in 100 mM ium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was d to a SOURCE 15Q purification column (column: SOURCE 15Q, flow rate: 2.0 , gradient: A 0 ——> 4% l min B —> 20% 80 min B(A: 20mM Tris—HCl, pH 7.5, B: A + 1M NaCl)) (b) and a Source ISO purification column (column: SOURCE ISO, flow rate: 2.0 , nt: B 0 ———> 100% 100 min A, (A: 20lel Tris—HCl, pH 7.5, B: A + 1.1M AS)) (c) to purify conjugates including oxyntomodulin den‘vative (SEQ ID NO. 25) and immunoglobulin Fc. b is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 25) and im— munoglobulin Fc through a SOURCE 15Q purification column, and c is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 25) and immunoglobulin Fc through a Source ISO purification column.
Example 13: ation of conjugates including oxyntomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc Firstly, for PEGylation of lysine residue at position 20 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 28) with 3.4 K PropionALD(2) PEG, the oxyntomodulin derivative (SEQ 1D NO. 28) and MAL—IOK—ALD PEG were reacted at a molar ratio of 1 : 5 with the protein concentration of 3 mg/ml at 4°C for 3 hours. At this time, the reaction was conducted in 50 mM Na-Borate buffer (pl-I 9.0) and 2M guanidine was added thereto. After completion of the reaction, the reaction mixture was applied to a SOURCE cation column to purify the oxyntomodulin derivative having mono-pegylated lysine (column: SOURCE S, flow rate: 2.0 ml/min, gradient: A 0 —>3% 1 min B —> 40% 222 min B (A: 20mM Na-citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (a). a is a graph showing the result of ing a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 28) through a SOURCE S purification column.
Next, the purified mono—PEGylated modulin derivative (SEQ ID NO. 28) and immunoglobulin Fc were reacted at a molar ratio of l : 10 with the protein con- centration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was conducted in 100 mM potassium phosphate buffer (pl-I 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE 15Q ation column (column: SOURCE lSQ, flow rate: 2.0 ml/min, gradient: A 0 —> 4% l min B -—> 20% 80 min B(A: 20mM Tris-HCI, pH 7.5, B: A + 1M NaCl)) (PIG. 11b) and a Source ISO purification column (column: SOURCE ISO, flow rate: 2.0 ml/min, gradient: B 0 -—-> 100% 100 min A, (A: 20mM Tris-HCI, pH 7.5, B: A + 1.1M AS)) (c) to purify conjugates ing oxyntomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc. b is a graph g the result of purifying ates ing oxyntomodulin derivative (SEQ ID NO. 28) and im- obulin Fc through a SOURCE lSQ purification column, and 0 is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc through a Source ISO purification column. [3321 Example 14: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 32) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at position 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 32) with MAL—10K~ALD PEG, the oxyn- tomodulin derivative (SEQ ID NO. 32) and MAL—lOK—ALD PEG were reacted at a molar ratio of l : 3 with the protein concentration of 1 mg/ml at room temperature for 3 hours. At this time, the reaction was ted in 50 mM Tris buffer (pH 8.0) and 2M guanidine was added thereto. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono-pegylated cysteine (column: SOURCE S, flow rate: 2.0 ml/ min, nt: A 0 —>]00% 50 min B (A: 20mM Na-citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)).
Next, the purified mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 32) and immunoglobulin Fc were reacted at a molar ratio of 1 : 8 with the protein concentration of 20 mg/m] at 4°C for 16 hours. At this time, the reaction was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE 15Q purification column (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A 0 —> 4% 1 min B ——> 20% 80 min B(A: 20mM Tris-HCI, pH 7.5, B: A + 1M NaCl)) and a Source ISO purification column (colurrm: SOURCE ISO, flow rate: 2.0 ml/min, nt: B 0 -a 100% 100 min A, (A: 20mM Tris—HG], pH 7.5, B: A + 1.1M AS)) to purify ates including oxyntomodulin derivative (SEQ ID NO. 32) and immunoglobulin Fc.
Example 15: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 33) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at position 30 of the amino acid sequence of modulin derivative (SEQ ID NO. 33) with MAL-lOK—ALD PEG, the oxyn— tomodulin derivative (SEQ ID NO. 33) and MAL-IOK-ALD PEG were reacted at a molar ratio of 1 : 1 with the n concentration of 1 mg/ml at room temperature for 3 hours. At this time, the reaction was conducted in 50 mM Tris buffer (pH 8.0) and 2M guanidine was added o. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono-pegylated cysteine (column: SOURCE S, flow rate: 2.0 ml/ min, gradient: A 0 —->100% 50 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KC1)).
Next, the ed mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 33) and immunoglobulin Fc were reacted at a molar ratio of l : 5 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the on was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After tion of the reaction, the reaction mixture was applied to a SOURCE 15Q purification column (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A O —-> 4% 1 min B —> 20% 80 min B(A: 20mM Tris-HCl, pH 7.5, B: A + 1M NaCl)) and a Source ISO purification column n: SOURCE ISO, flow rate: 2.0 ml/min, gradient: B 0 —> 100% 100 min A, (A: 20mM Tris—HCl, pH 7.5, B: A + 1.1M AS)) to purify conjugates including oxyntomodulin derivative (SEQ ID NO. 33) and immunoglobulin Fc.
Example 16: Preparation of conjugates ing modulin derivative (SEQ ID NO. 34) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at position 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 34) with MAL-lOK-ALD PEG, the oxyn- tomodulin derivative (SEQ ID NO. 34) and MAL-10K-ALD PEG were reacted at a molar ratio of l : 1 with the protein concentration of 3 mg/ml at room temperature for 3 hours. At this time, the reaction was conducted in 50 mM Tris buffer (pH 8.0) and 1M guanidine was added thereto. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono-pegylated cysteine (column: SOURCE S, flow rate: 2.0 ml/ min, gradient: A 0 —>100% 50 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)).
Next, the purified mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 34) and globulin Fc were reacted at a molar ratio of 1 : 5 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE 15Q purification column n: SOURCE lSQ, flow rate: 2.0 , gradient: A 0 —~> 4% 1 min B —> 20% 80 min B(A: 20mM Tris—HCI, pH 7.5, B: A + 1M NaCl)) and a Source ISO purification column (column: SOURCE ISO, flow rate: 2.0 nil/min, gradient: B 0 —-> 100% 100 min A, (A: 20mM Tris-HCl, pH 7.5, B: A + 1.1M AS)) to purify conjugates including oxyntomodulin derivative (SEQ ID NO. 34) and immunoglobulin Fc.
Example 17: In vitro activity of oxyntomodulin tive-immunoglobulin Fc conjugates In order to measure anti—obesity efticacies of the conjugates including the oxyn- tomodulin or oxyntomodulin derivative and the immunoglobulin Fc that were prepared in the above Examples, experiments were performed in the same manner as in Example 2—2.
Specifically, each of the transformants prepared in Examples l~1 and 1—2 was sub- ed two or three times a week, and ted in each well of a 96—well plate at a density of 1 X 105, followed by cultivation for 24 hours. Each of the cultured trans- formants was washed with KRB buffer and ded in 40 ml of KRB buffer containing 1 mM IBMX, and left at room temperature for 5 minutes. GLP—l, glucagon, and oxyntomodulin derivative (SEQ ID NO. 23, 24, 25, 32, 33 or 34)—immunoglobulin Fc conjugates were diluted from 1000 11M to 0.02 nM by 5—fold serial dilution, and each 40 ml thereof was added to each transformant, and cultured at 37°C for 1 hour in a C02 incubator. Then, 20 ml of cell lysis buffer was added for cell lysis, and the cell s were applied to a CAMP assay kit (Molecular Device, USA) to measure CAMP concentrations using a Victor (Perkin Elmer, USA). ECso values were calculated therefrom, and compared to each other (Table 3).
Table 3 [Table 3] In vitro activity of oxyntomodulin derivative-immunoglobulin Fc ates SEQ ID NO. ECso (nM) SEQ ID NO. 24 — Fc conjugates 8 4 - SEQ ID No. 25 - Fc conjugates 5.5 9 SEQ ID NO. 32 - Fc conjugates 68.7 SEQ ID NO. 33 — Fc conjugates 11.7 SEQ ID No. 34 — Fc conjugates 168.0 _ As shown in Table 3, the oxyntomodulin derivative-immunoglobulin Fc conjugates were found to show the in vitro activity to GLP-1 and glucagon receptors.
Example 18: In vivo activity of oxyntomodulin derivative-immunoglobulin ates It was examined whether the oxyntomodulin detivative—immunoglobulin Fc conjugates show excellent body weight—reducing effects in vivo.
Specifically, 6-week—old normal C57BL/6 mice Were fed a high fat diet of 60 kcal for 24 weeks to increase their body weight by approximately 50 g on average, and subcu— taneously stered with oxyntomodulin derivative (SEQ ID NO. 23, 24 or )~immunoglobulin Fc conjugates at a dose of 0.03 or 0.06 mg/kg/week for 3 weeks.
Thereafter, changes in the body weight of the mice were measured ( and ). and are graphs g changes in body weight of mice according to the type and administration dose of oxyntomodulin derivative-im— munoglobulin Fc conjugates. As shown in and , as the administration dose of the oxyntomodulin detivative—immunoglobulin Fc conjugates was increased, the body weight was reduced in direct proportion, even though there were ences between the types of the modulin delivative—immunoglobulin Fc conjugates, suggesting that the oxyntomodulin tive—immunoglobulin Fc conjugates reduce the body weight in a dose—dependent manner.

Claims (20)

Claims 1.
1. A conjugate sing an oxyntomodulin derivative, an immunoglobulin Fc region, and a non-peptidyl polymer, n the modulin derivative is covalently linked to the immunoglobulin Fc region via the non-peptidyl polymer, and wherein the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID 34.
2. The conjugate according to claim 1, wherein the oxyntomodulin derivative is capable of activating GLP-1 receptor and glucagon receptor.
3. The conjugate according to claim 1 or claim 2, wherein the conjugate has antiobesity effects.
4. The conjugate ing to any one of claims 1 to 3, wherein the amino acid pairs at positions 16 and 20 of the oxyntomodulin derivative form a ring.
5. The conjugate ing to any one of claims 1 to 4, wherein the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, polylactic acid (PLA), polylacticglycolic acid (PLGA), lipid polymers, chitins, hyaluronic acid, polysaccharide and ations f.
6. The conjugate according to claim 5, wherein the non-peptidyl polymer comprises polyethylene glycol.
7. The conjugate according to any one of claims 1 to 6, wherein one functional group selected from the group consisting of an amine group and a thiol group of the immunoglobulin Fc region is ed to one end of the non-peptidyl polymer, and one functional group selected from the group consisting of an amine group and a thiol group of the modulin derivative is attached to the other end of the non-peptidyl polymer.
8. The conjugate according to any one of claims 1 to 7, wherein the conjugate is prepared by covalently linking an modulin derivative to an immunoglobulin Fc region via a ptidyl polymer, the non-peptidyl polymer having, prior to forming the conjugate, reactive functional groups at both ends.
9. The ate according to claim 8, wherein the ve group is selected from the group consisting of an aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative.
10. The conjugate according to claim 9, n the reactive groups at both ends are the same as or different from each other.
11. The conjugate according to any one of claims 1 to 10, wherein the immunoglobulin Fc region is a non-glycosylated Fc region.
12. The conjugate according to any one of claims 1 to 11, wherein the immunoglobulin Fc region is selected from the group consisting of a CH1 domain, a CH2 , a CH3 domain and a CH4 domain; a CH1 domain and a CH2 domain; a CH1 domain and a CH3 domain; a CH2 domain and a CH3 domain; a ation of one or more domains and an immunoglobulin hinge region (or a portion of the hinge region); and a dimer of each domain of the heavy-chain constant regions and the light-chain constant region.
13. The conjugate according to any one of claims 1 to 12, wherein the immunoglobulin Fc region is a derivative in which a region capable of forming a disulfide bond is deleted, certain amino acid residues are eliminated at the N-terminal end of a native Fc form, a methionine residue is added at the N-terminal end of a native Fc form, a complement-binding site is deleted, or an antibody dependent cell mediated cytotoxicity (ADCC) site is deleted.
14. The conjugate according to any one of claims 1 to 13, wherein the globulin Fc region is an Fc region derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.
15. The conjugate according to claim 14, wherein the immunoglobulin Fc region is an IgG4 Fc region.
16. The conjugate according to claim 15, n the immunoglobulin Fc region is a human IgG4-derived non-glycosylated Fc region.
17. A pharmaceutical composition for the prevention or treatment of obesity, comprising the conjugate of any one of claims 1 to 16.
18. The pharmaceutical composition according to claim 17, r comprising a ceutically acceptable carrier.
19. The pharmaceutical composition ing to claim 17 or 18, wherein the composition is administered alone or in combination with other pharmaceutical formulations showing prophylactic or therapeutic effects on obesity.
20. The pharmaceutical composition according to claim 19, wherein the pharmaceutical ation is selected from the group consisting of a GLP-1 receptor agonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a melanin-concentrating hormone (MCH) receptor antagonist, a Y
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