NZ733478B2 - 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
NZ733478B2
NZ733478B2 NZ733478A NZ73347812A NZ733478B2 NZ 733478 B2 NZ733478 B2 NZ 733478B2 NZ 733478 A NZ733478 A NZ 733478A NZ 73347812 A NZ73347812 A NZ 73347812A NZ 733478 B2 NZ733478 B2 NZ 733478B2
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New Zealand
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seq
immunoglobulin
oxyntomodulin
region
derivative
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NZ733478A
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NZ733478A (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 NZ742400A priority Critical patent/NZ742400B2/en
Publication of NZ733478A publication Critical patent/NZ733478A/en
Publication of NZ733478B2 publication Critical patent/NZ733478B2/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

Discloses conjugates comprising oxyntomodulin variant peptides covalently linked to an immunoglobulin Fc region via a non-peptidyl polymer. Discloses a number of possible amino acid substitutions. Further discloses use of the conjugate for the treatment of obesity.

Description

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Many studies have been made to develop therapeutic agents for obesity which do not have the problems of the conventional anti—obesity drugs. Recently, glucagon derivatives have received much ion. Glucagon is produced by the pancreas when the level of e in the blood drops resulting from other medications or diseases, hormone or enzyme deficiencies. Glucagon stimulates glycogen breakdown in the liver, and facilitates glucose release to raise blood glucose levels to a normal range. In addition to the effect of increasing the blood e level, glucagon suppresses appetite and activates hormone—sensitive lipase(HSL) of adipocytes to facilitate sis, thereby showing anti-obesity effects. One of the glucagon tives, glucagon like peptide-l (GLP- l) 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 emptying, to increase glucose utilization, and to inhibit food intake. Exendin-4 is isolated from lizard venom that shares approximately 50% amino acid gy with GLP—1 and is also reported to activate the GLP-1 receptor, thereby ameliorating hyperglycemia in patients with diabetes. However, anti—obesity drugs ing GLP—l are reported to show side- effects such as vomiting and .
As an alternative to GLP-l, therefore, much attention has been focused on oxyn- tomodulin, a peptide derived from a glucagon sor, pre-glucagon that binds to the ors of two peptides, GLP-1 and on. Oxyntomodulin represents a potent anti—obesity therapy, because it inhibits food intake like GLP—l, promotes satiety, and has a lipolytic ty like glucagon.
Based on the dual function of the oxyntomodulin peptide, it has been actively studied as a drug for the treatment of obesity. For example, Korean Patent No. 925017 ses a pharmaceutical composition including oxyntomodulin as an active in- gredient for the treatment of overweight human, which is administered Via an oral, parenteral, mucosal, rectal, subcutaneous, or ermal 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 e 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 (DPP—IV) and by attaching a cholesterol moiety at the C- terminal to increase the blood half-life at the same time. ZP2929 (Zealand) is ed by tuting L—serine with D—serine at position 2 to enhance resistance to DPP—IV, substituting arginine with alanine at position 17 to enhance resistance to se, sub- stituting methionine with lysine at position 27 to e oxidative stability, and sub— stituting glutamine with aspartic acid and alanine at ons 20 and 24 and asparagine with serine at position 28 to enhance deamidation ity. However, even though the half—life of the dual agonist modulin (Merck) was ed to show half—life 8~l2 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. Unfor— tunately, oxyntomodulin or derivatives thereof have disadvantages of daily admin— istration of high dose due to the short half-life and low efficacy.
Disclosure of Invention Technical Problem Accordingly, the present inventors have made many efforts to develop a method for increasing the blood half-life of oxyntomodulin while maintaining its activity in vivo.
As a result, they found that a conjugate prepared by linking a carrier to oxyntomodulin using a non—peptidyl polymer show ed blood half-life while maintaining the activity in vivo so as to exhibit excellent anti-obesity effects, thereby completing the present ion.
Solution to Problem An object of the present invention is to provide a conjugate comprising oxyn- lin, an immunoglobulin Fc region, and non-peptidyl polymer wherein the conjugate being obtainable by covalently linking oxyntomodulin to immunoglobulin Fc region via non—peptidyl polymer.
Another object of the present invention is to provide a pharmaceutical composition for the prevention or treatment of obesity, comprising the conjugates.
Still another object of the present invention is to provide a method for preventing or treating y, comprising the step of administering the conjugate or the composition to a subject.
Still another object of the present invention is to provide use of the conjugate or the composition in the preparation of drugs for the prevention or treatment of obesity.
Advantageous Effects of Invention The conjugate comprising oxyntomodulin and the immunoglobulin Fc of the present invention reduces food intake, suppresses gastric emptying, and facilitates lipolysis without side—effects, unlike native oxyntomodulin, and also shows excellent receptor— activating s and long-term sustainability, ed to oxyntomodulin. Thus, it can be widely used in the ent of obesity with safety and cy. Unlike native oxyntomodulin, the novel peptide of the t invention reduces food intake, suppresses 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.
Brief Description of Drawings is a graph showing changes in food intake according to administration 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 ing oxyn- tomodulin and immunoglobulin Fc h a SOURCE lSQ purification column. is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 29) h a SOURCE S purification column. is a graph showing the result of purifying conjugates including oxyn— tomodulin derivative (SEQ ID NO. 29) 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. 30) through a SOURCE S cation . is a graph showing the result of peptide mapping of ed mono— PEGylated oxyntomodulin derivative (SEQ ID NO. 30). is a graph showing the result of purifying conjugates including oxyn— tomodulin derivative (SEQ ID NO. 30) and immunoglobulin Fc through a SOURCE lSQ purification column. 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 ing oxyn— tomodulin derivative (SEQ ID NO. 31) and immunoglobulin Fc through a SOURCE lSQ purification column. is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 2) through a SOURCE S cation column. is a graph showing the result of peptide mapping of purified mono- ted oxyntomodulin derivative (SEQ ID NO. 2). is a graph showing the result of purifying conjugates including oxyntomodulin tive (SEQ ID NO. 2) and immunoglobulin Fc through a SOURCE 15Q purification column. is a graph showing the result of purifying conjugates including oxyn- tomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc through a Source ISO purification column. is a graph g the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 3) through a SOURCE S purification . is a graph g 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 oxyn— tomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc through a Butyl FF pu- rification column. is a graph showing the result of purifying ates including oxyn— tomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc through a Source 15Q purification column. is a graph g the result of purifying a EGylated oxyntomodulin derivative (SEQ ID NO. 23) through a SOURCE S purification column; is a graph showing the result of purifying conjugates including oxyn— tomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc through a Source 15Q purification column; is a graph showing the result of purifying conjugates including oxyn- tomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc through a SOURCE ISO purification column; is a graph showing the result of purifying a EGylated oxyntomodulin derivative (SEQ ID NO. 24) through a SOURCE S purification column; is a graph showing the result of purifying conjugates including oxyn- tomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc through a Source 15Q purification column; [45 l is a graph showing the result of purifying conjugates including modulin 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 derivative (SEQ ID NO. 25) and immunoglobulin Fc through a Source 15Q purification column; c is a graph showing the result of purifying conjugates including oxyn- lin derivative (SEQ ID NO. 25) and immunoglobulin Fc through a SOURCE ISO purification column; a is a graph g the result of purifying a mono-PEGylated oxyn- tomodulin derivative (SEQ ID NO. 28) through a SOURCE S purification column; b is a graph showing the result of purifying conjugates including oxyn- tomodulin derivative (SEQ ID NO. 28) and globulin Fc through a Source 15Q purification column; FIG. llc is a graph showing the result of purifying conjugates ing oxyn- tomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc through a SOURCE ISO cation column; is a graph showing 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 one aspect to achieve the above s, the present invention provides a conjugate comprising oxyntomodulin, an immunoglobulin Fc region, and non-peptidyl polymer wherein the conjugate being obtainable by ntly g oxyntomodulin to im— munoglobulin 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 increased stability in blood, suspend emission through the kidney, or other useful effects. In the present invention, the factors can be immunoglobulin Fc region. Preferably, the conjugate 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 globulin Fc region via covalent bonds. Two terminal ends of non-peptidyl polymer can be linked to an amine group or thiol group of the im— munoglobulin Fc region and oxyntomodulin derivatives, respectively.
The conjugate of the present invention means to have an improved in—vivo duration of efficacy, compared to native oxyntomodulin, and the long-acting ate may include oxyntomodulin prepared by cation, substitution, addition, or deletion of the amino acid sequences of the native modulin, 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 oxyn- tomodulin encapsulated in biodegradable nanoparticles, but the type of the long-acting conjugate is not d thereto.
As used herein, the term "oxyntomodulin" means a peptide derived from a glucagon precursor, pre—glucagon, and includes a native modulin, precursors, tives, fragments thereof, and variants thereof. Preferably, it can have the amino acid sequence of SEQ ID NO. 1 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).
The term, omodulin 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 on receptors, and it may be prepared by any one of substitution, addition, deletion, and modification or by a com- bination thereof in a part of the amino acid sequences of the native oxyntomodulin.
The term, "oxyntomodulin derivative" includes peptides, peptide derivatives or peptide cs 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 deleted at the N-terminus or the inus of the native oxyn— tomodulin, in which non-naturally occurring amino acids (for example, D—type amino acid) can be added, and has a on of activating both of the GLP-1 receptor and the on receptor.
Each of the preparation methods for the variants, derivatives, and fragments of oxyn- lin can be used dually or in combination. For example, the present invention includes a e that has one or more amino acids different 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 gine N Aspartic acid D Cysteine C Glutamic acid E Glutamine Q Glycine G Histidine 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 tive encompasses any peptide that is prepared by substitutions, additions, deletions or post translational modifications (e.g., ation, ion, ubiquitination, intramolecular covalent bonding) in the amino acid ce of oxyntomodulin FTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA, SEQ ID NO. 1) so as to activate the glucagon and GLP-1 receptors 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 turally occurring amino acids can be used. Com- mercially available sources of atypical amino acids include Sigma-Aldrich, ChemPep Inc., and Genzyme Pharmaceuticals. The peptides including these amino acids and atypical peptide 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.
Rl-X1—X2-GTFTSD—X3-X4—X5—X6-X7-X8—X9-X10-X1 X13-X14—X15-X16— X17—Xl8-X19—X20-X21-X22-X23-X24-R2 (Formula 1) wherein R1 is histidine, desamino—histidyl, dimethyl—histidyl (N—dimethyl—histidyl), beta-hydroxyirnidazopropionyl, 4-imidazoacetyl, beta-carboxy imidazopropionyl or tyrosine; X1 is Aib(aminosiobutyric acid), d—alanine, glycine, Sar(N-methylglycine), serine, or d-serine; X2 is glutamic acid or glutamine; X3 is leucine or tyrosine; X4 is serine or alanine; X5 is lysine or arginine; X6 is ine or tyrosine; X7 is leucine or nine; X8 is aspartic acid or glutamic acid; X9 is glutamic acid, serine, 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; X12 is alanine, arginine, serine, valine or is deleted; X13 is lysine, glutamine, arginine, alpha-methyl-glutamic acid or is deleted; X14 is aspartic acid, glutamic acid, leucine or is deleted; X15 is alanine or is deleted; X16 is isoleucine, valine or is d; X17 is alanine, cysteine, 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 deleted; X21 is asparagine or is deleted; X22 is alanine, glycine, threonine or is deleted; X23 is ne, lysine or is deleted; X24 is a e having 2 to 10 amino acids consisting of ations of alanine, glycine and serine, or is d; and R2 is KRNRNNIA (SEQ ID NO. 35), GPSSGAPPPS (SEQ ID NO. 36), GPSSGAPPPSK (SEQ ID NO. 37), HSQGTFTSDYSKYLD (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 1 is identical to that of SEQ ID NO. 1).
In order to enhance the activity of the wild—type oxyntomodulin for the glucagon receptor and the GLP-1 receptor, the peptide of the t invention may be sub- stituted with 4—imidazoacetyl where the alpha carbon of histidine at position 1 of amino acid sequence represented by SEQ ID NO. 1 is d, no-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 imida— zopropionyl 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 on, 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 modulin sequence may be substituted with a sequence stabilizing alpha helix. Preferably, amino acids at positions 10, l4, 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), Phe(4-Cl), CN), Phe(4—NOZ), Phe(4-NH2), Phg, Pal, Nal, Ala(2—thienyl) and Ala(benzothieny1) that are known to ize 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. Preferably, 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 ic acid or lysine, respectively so as to form rings, and there is no limitation on the number of rings to be inserted. Most preferably, the e may be a peptide having an amino acid sequence selected from the following Formulae 1 to 6.
In one specific embodiment, the oxyntomodulin derivative of the present 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- R1—A-R3 la 2) In another specific embodiment, the modulin derivative of the present invention is a novel peptide including the amino acid sequence of the ing Formula 3, which is prepared by linking a part of the amino acid sequence of oxyn- tomodulin and a part of the amino acid sequence of exendin or GLP—l Via a proper amino acid linker.
R1-B-C—R4 (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 ce of oxyntomodulin is substituted with an amino acid capable of enhancing the binding affinity to GLP-1 or, for example, Leu at position 26 which binds with GLP-1 receptor by hydrophobic in- teraction is tuted with the hydrophobic residue, Ile or Val.
Rl—SQGTFTSDYSKYLD—D1—D2-D3—D4—D5-LFVQW—D6—D7-N—D8-R3 (Formula In still another ic 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—E2—E3-RA-E4—E5—FV—E6—WLMNT-E7—R5 (Formula 5) In ae 2 to 5, R1 is the same as in the description of Formula 1; A is ed from the group consisting of SQGTFTSDYSKYLDSRRAQD— FVQWLMNT (SEQ ID NO. 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO. 42), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO. 43), GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO. 44), GQGTFTSDYS- RQMEEEAVRLFIEWLKNG (SEQ ID NO. 45), GEGTFTSDL- SRQMEEEAVRLFIEWAA (SEQ ID NO. 46), and SDYSRQMEEEAVRL- FIEWLMNG (SEQ ID NO. 47); B is selected from the group consisting of SDYSKYLDSRRAQD— NT (SEQ ID NO. 41), SQGTFTSDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO. 42), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO. 43), GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO. 44), GQGTFTSDYS- RQMEEEAVRLFIEWLKNG (SEQ ID NO. 45), GEGTFTSDL- SRQMEEEAVRLFIEWAA (SEQ ID NO. 46), SQGTFTSDYSRQMEEEAVRL— FIEWLMNG (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 ations of alanine, glycine and ; D1 is serine, glutamic acid or arginine; D2 is arginine, 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 nine, arginine or glutamine; D8 is threonine, glycine or alanine; E1 is serine, Aib, Sar, d-alanine or d-serine; E2 is serine or glutamic acid; E3 is arginine or lysine; E4 is glutamine or lysine; E5 is aspartic 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 ID NO. 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).
Preferably, the oxyntomodulin derivative of the present ion may be a noverl peptide of the ing Formula 6.
Rl-X1—X2-GTFTSD—X3-X4—X5—X6-X7-X8—X9-X10-X1 1—X12—X13-X14—X15-X16— X17-X18-X19—X20-X21-X22-X23—X24-R2 (Formula 6) wherein R1 is histidine, desamino-histidyl, 4-imidazoacetyl or tyrosine; X1 is Aib(aminosiobutyric acid), glycine or serine; X2 is ic acid or glutamine; X3 is leucine or tyrosine; X4 is serine or alanine; 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, alpha—methyl-glutamic acid or is d; X10 is glutamine, glutamic acid, , arginine or is deleted; X11 is alanine, arginine or is deleted; X12 is alanine, valine or is deleted; X13 is lysine, glutamine, arginine, alpha-methyl-glutamic acid or is deleted; X14 is ic acid, glutamic acid, leucine or is deleted; X15 is phenylalanine or is deleted; X16 is isoleucine, valine or is deleted; X17 is alanine, cysteine, glutamic acid, glutamine, alpha-methyl-glutamic acid or is deleted; X18 is tryptophan or is deleted; X19 is e, isoleucine, leucine, valine or is deleted; X20 is alanine, , methionine, arginine or is deleted; X21 is gine or is deleted; X22 is ine 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), HSQGTFTSDYSKYLD (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) More preferably, the oxyntomodulin derivative of the present invention may be ed from the group consisting of the es of SEQ ID NOs. 2 to 34. Much more ably, the oxyntomodulin derivative of the present invention may be an oxyn— tomodulin derivative described in Table 1 of Example 2-1.
Oxyntomodulin has the activities of two peptides, GLP-1 and glucagon. GLP—l decreases blood glucose, reduces food intake, and suppresses gastric ng, and glucagon increases blood glucose, facilitate lipolysis and decreases body—weight by in— ng energy metabolisms. The different ical effects of the two peptides can cause undesired effects like increasing blood glucose if glucagon shows a more nt 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 on receptor in Example 12 inspite of its low efficacy in relation to the GLP-1 receptor. Therefore, the oxyntomodulin derivatives 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 modified at positions 1 and 11 of modulin, which are known to suppress the activity of glucagon, to control the activity ratio between glucagon and GLP-1.
The conjugates of the present invention can induce increased stability in blood, suspend emission through the kidney, and change affinity to ors by linking a carrier to oxyntomodulin Via a covalent bond or forming phere. The carrier that can form a conjugate ning oxyntomodulin can be ed from the group consisting of albumin, transferrin, antibodies, dy frangments, elastin, heparin, polysaccharide such as chitin, fibronectin and most favorably immunoglobulin Fc region all of which can increase the blood half-life of the conjugates when bound to oxyntomodulin.
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 globulin, 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 t invention may contain a part or all of the Fc region including the heavy-chain constant region 1 (CH1) and/or the light—chain constant 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 deletion 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 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 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 ), 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 ion 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 ce due to a deletion, an insertion, a nservative or conservative substitution or combinations thereof of one or more amino acid residues. For example, in an IgG Fc, amino acid es 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 g 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. r, to remove effector ons, a deletion may occur in a complement-binding site, such as a Clq-binding site and an ADCC (antibody dependent cell mediated cyto— toxicity) site. Techniques of ing such sequence derivatives of the im— munoglobulin Fc region are disclosed in WO 97/34631 and WO 96/32478.
Amino acid exchanges in proteins and es, which do not generally alter the activity of the proteins or peptides, are known in the art (H. h, R. L. Hill, The ns, 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/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/lle, Leu/Val, Ala/Glu and Asp/Gly, in both directions. In on, the Fc region, if desired, may be modified by phosphorylation, sulfation, acrylation, ylation, methylation, farnesylation, acetylation, amidation, and the like.
The aforementioned Fc derivatives are derivatives that have a ical activity identical to the Fc region of the present ion or improved structural stability, for example, against heat, pH, or the like.
In addition, these PC regions may be obtained from native forms ed from humans and other animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or derivatives thereof, obtained from transformed animal cells or microorganisms. Herein, they may be obtained from a native im- munoglobulin by isolating whole immunoglobulins from human or animal organisms and treating them with a proteolytic enzyme. Papain digests the native immunoglobulin 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 chromatography to isolate Fc or pF'c. Preferably, a human-derived Fc region is a re- ant 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 ed 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 genetic engineering method using a microorganism. The removal of sugar chains from an Fc region results in a sharp decrease in binding affinity to the Clq part of the first complement component Cl and a decrease or loss in dy— dependent ediated cytotoxicity or complement-dependent cytotoxicity, y not inducing unnecessary immune responses in—vivo. In this regard, an im— munoglobulin Fc region in a deglycosylated or aglycosylated form may be more suitable to the object of the present invention as a drug carrier.
As used , 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, ably E. coli.
Meanwhile, the immunoglobulin Fc region may be derived from humans or other animals ing cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, and preferably from humans.
In addition, 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 thereof. Preferably, it is derived from IgG or IgM, which are among the most abundant proteins in human blood, and most preferably from IgG, which is known to enhance the half—lives of ligand-binding ns.
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 different origin to form a dimer or multimer. That is, a dimer or er may be formed from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fc, and lgE Fc fragments.
The term “non—peptidyl polymer”, refers to a biocompatible polymer 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 ptidyl linker.
The non-peptidyl polymer useful in the present ion may be selected from the group consisting of a radable polymer, a lipid polymer, chitin, hyaluronic acid, and a combination thereof, and preferably, the biodegradable polymer may be polyethylene glycol, polypropylene glycol, ethylene -propylene glycol mer, polyoxyethylated polyol, polyvinyl l, polysaccharide, dextran, polyvinyl ethyl ether, polylactic acid (PLA) or polylactic-glycolic acid (PLGA), and more preferably, is polyethylene glycol (PEG). In on, derivatives thereof known in the art and derivatives easily prepared 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 obtained by a conventional inframe fusion method has drawbacks in that it is easily in-vivo cleaved by a pro— teolytic enzyme, and thus a ient effect of increasing the serum half-life of the active drug by a r 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. ore, any non-peptidyl polymer can be used without limitation, as long as it is a polymer having the aforementioned function, that is, a polymer having resistance to the in-vivo proteolytic enzyme. The non-peptidyl polymer has a molecular weight in the range of l to 100 kDa, and preferably of l to 20 kDa. The non-peptidyl polymer of the present invention, linked to the immunoglobulin Fc , may be one polymer or a combination of different types of polymers.
The non—peptidyl polymer used in the present invention has a reactive group capable of binding to the immunoglobulin Fc region and protein drug. The non—peptidyl polymer has a ve group at both ends, which is preferably 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, imidyl carboxymethyl, or succinimidyl carbonate. In particular, when the non—peptidyl polymer has a reactive aldehyde group at both ends thereof, it is effective in linking at both ends with a physiologically active polypeptide and an immunoglobulin with l non-specific reactions. A final product generated by reductive tion 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 e 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 bu— tyraldehyde group at the other end. When a polyethylene glycol having a ve hydroxy group at both ends thereof is used as the non-peptidyl r, 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 is preferably selected from the group consisting of a reactive aldehyde group, a propionaldehyde group, a ldehyde group, a maleimide group and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, y imidyl, suc- cinimidyl carboxymethyl, or succinimidyl carbonate.
The two reactive terminal groups of the non-peptidyl 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 aldehyde group, a propionaldehyde group or a bu- tyraldehyde group at the other end. For example, when the non-peptidyl polymer has a reactive aldehyde group at a terminal group, and a maleimide group at the other al group, it is effective in linking at both ends with a physiologically active polypeptide and an immunoglobulin with minimal non—specific reactions. According to Examples of the present invention, conjugates were ed by linking the oxyn- tomodulin or tive thereof and the immunoglobulin Fc region Via a covalent bond using PEG that is a non-peptidyl polymer including the propionaldehyde group alone or both the ides group and the aldehyde group.
The conjugates of the present invention show excellent activity on GLP-1 receptor and glucagon or, compared to native oxyntomodulin, and the blood half-life is sed by linking with the Fc region so as to in in vivo activity for a long period of time.
In still another aspect, the present invention provides a pharmaceutical composition for the tion or treatment of obesity comprising the e.
As used herein, the term "prevention" means all of the actions by which the ence of the disease is restrained or retarded. In the present invention, "prevention" means that the occurrence of obesity from such factors as an increase in body weight or body fat is restrained or retarded by stration of the conjugates of the t invention.
As used herein, the term "treatment" means all of the actions by which the symptoms of the disease have been alleviated, improved or ameliorated. In the present invention, "treatment" means that the symptoms of obesity are ated, improved or ame- liorated by administration of the conjugates of the present invention, resulting in a reduction in body weight or body fat.
As used herein, the term "obesity" implies accumulation 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 metabolic disease that affects the whole body, and increases the risk for diabetes, hyperlipidemia, sexual dys- function, arthritis, and cardiovascular diseases, and in some cases, is associated with incidence of cancer.
The conjugates of the t invention, which are prepared by g oxyn- tomodulin or a tive thereof with the globulin Fc region, show excellent binding affinity to glucagon and GLP-1 receptors (Table 3) and excellent resistance to in-vivo proteolytic enzymes so as to exhibit the in vivo activity for a long period of time, thereby showing excellent anti-obesity effects such as reductions in body weight ().
The pharmaceutical composition of the present invention may further include a phar— maceutically 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 y 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 ncy, duration of treatment, drugs used in combination or coincident With the composition of this invention, and other factors known in medicine.
The pharmaceutical composition including the derivative of the present invention may further include a pharmaceutically acceptable carrier. For oral administration, the carrier may include, but is not limited to, a , a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a colorant, and a nt. For injectable preparations, the carrier may include a buffering agent, a preserving agent, an sic, 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 stration, the pharmaceutical composition may be formulated into tablets, troches, capsules, elixirs, suspensions, syrups or . For in- jectable preparations, the ceutical composition may be formulated into an ampule as a single dosage form or a multidose container. The pharmaceutical com- position may also be formulated into solutions, suspensions, tablets, pills, capsules and long-acting preparations.
On the other hand, examples of the carrier, the excipient, and the diluent suitable for the pharmaceutical formulations include lactose, se, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, te, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, nylpyrrolidone, water, methylhydroxybenzoate, hydroxybenzoate, talc, magnesium stearate and mineral oils. In addition, the pharmaceutical formulations may further include s, oagulating agents, lubricants, humectants, flavorants, and antiseptics.
Further, the pharmaceutical composition of the t invention may have any for- mulation selected from the group consisting of tablets, pills, powders, granules, capsules, suspensions, liquids for al use, emulsions, syrups, sterile aqueous solutions, non—aqueous solvents, lyophilized ations and suppositories.
Further, the composition may be formulated into a single dosage form le for the patient's body, and preferably is formulated into a preparation useful for peptide 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, intra- arterial, intramedullary, intramedullary, intraventricular, ary, transdermal, sub- cutaneous, intrapen'toneal, intranasal, intracolonic, topical, sublingual, vaginal, or rectal administration, but is not limited thereto.
The composition may be used by blending with a variety of pharmaceutically ac- ceptable carriers such as physiological saline or organic solvents. In order to increase the stability or tivity, carbohydrates such as glucose, sucrose or dextrans, an- tioxidants such as ascorbic acid or hione, chelating agents, low molecular weight proteins or other stabilizers may be used.
The administration dose and frequency of the pharmaceutical ition of the present 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, , and body weight, and disease severity.
The total effective dose of the composition of the present invention may be ad- ered to a patient in a single dose, or may be administered for a long period of time in multiple doses according to a fractionated treatment protocol. In the - ceutical composition of the present invention, the content of active ingredient may vary depending on the disease severity. ably, the total daily dose of the peptide of the present invention may be approximately 0.0001 Mg to 500 mg per 1 kg of body weight of a patient. However, the effective dose of the peptide is determined ering s factors including patient's age, body , health conditions, gender, disease severity, diet, and ion rate, in addition to administration route and treatment frequency of the pharmaceutical composition. In View of this, those d in the art may easily determine an effective dose suitable for the particular use of the pharma- ceutical composition of the present ion. The pharmaceutical composition according to the present invention is not particularly limited to the formulation, and ad— ministration route and mode, as long as it shows the effects of the present ion.
The pharmaceutical composition of the present invention shows excellent in-vivo duration of efficacy and titer, thereby remarkably reducing the number and frequency of administration thereof. er, the pharmaceutical ition may be administered alone or in com- bination or coincident with other pharmaceutical formulations showing prophylactic or therapeutic effects on obesity. The pharmaceutical formulations showing prophylactic or therapeutic effects on obesity are not particularly limited, and may include a GLP—1 receptor agonist, a leptin receptor agonist, a DPP—IV inhibitor, a Y5 receptor an— tagonist, a Melanin-concentrating hormone (MCH) receptor antagonist, a Y2/3 receptor agonist, a MC3/4 receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2c t, a [53A receptor agonist, an Amylin receptor agonist, a Ghrelin an- tagonist, and/or a Ghrelin receptor antagonist.
In still another aspect, the present invention es a method for preventing or treating 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 pre— determined substance into a t by a certain suitable method. The composition of the t 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, in- travenous, uscular, subcutaneous, intradermal, oral, topical, intranasal, intra- pulmonary, or intrarectal administration. However, since peptides are digested upon oral administration, active ingredients of a composition for oral administration should be coated or formulath for protection t degradation in the h.
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 treated with the peptide or the pharmaceutical composition of the present invention is included without limitation.
The pharmaceutical composition including the peptide of the present invention is ad— ministered to a subject suspected of having y, thereby treating the subject ef- fectively. The obesity is as described above.
The therapeutic method of the present invention may include the step of admin— istering the composition ing the peptide at a pharmaceutically effective amount.
The total daily dose should be determined through appropriate l judgment by a physician, and administered once or several times. With respect to the objects of the present invention, 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 r other agents are used ith or not, the patient’s age, body weight, health ion, gender, and diet, the time and route of administration, the secretion rate of the composition, the time period of therapy, other drugs used in combination or co— nt with the composition of this invention, and like factors well known in the medical arts.
In still another aspect, the present invention provides a use of the conjugate or the pharmaceutical ition including the same in the preparation of drugs for the prevention or treatment of obesity.
Mode for the Invention Hereinafter, the present invention 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 activated 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 Reading Frame) in cDNA (OriGene Technologies, Inc. USA) of human GLP-1 receptor gene as a template, and the following forward and reverse s including each of the HindIII and EcoRI ction sites so as to obtain a PCR product.
Forward primer: GGCCCCCGCGGCCGCTATTCGAAATAC—3'(SEQ ID NO. 47) Reverse primer: 5‘-GAACGGTCCGGAGGACGTCGACTCTTAAGATAG-3'(SEQ ID NO. 48) The PCR product was cloned into the known animal cell expression vector hfr to prepare a recombinant vector XOGC/GLPlR.
CHO DG44 cell line cultured in DMEM/F12 (10% FBS) medium was transfected with the inant vector x0GC/GLP1R using Lipofectamine (Invitrogen, USA), and cultured in a selection 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 therefrom.
Example 1-2: Production of cell line showing cAMP response to glucagon PCR was performed using a region ponding to ORF in cDNA (OriGene Tech- nologies, Inc. USA) of human glucagon receptor gene as a template, and the following forward and reverse primers ing each of the EcoRI and XhoI restriction sites so as to obtain a PCR product. [25 8] Forward primer: 5'-CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC—3'(SEQ ID NO. 49) Reverse : 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/Fl2 (10% FBS) medium was transfected with the recombinant vector XOGC/GCGR using Lipofectamine, and cultured in a selection 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 glucagon in a concentration-dependent manner was finally selected therefrom.
Example 2. Test on in vitro activity of oxyntomodulin derivatives e 2-1: Synthesis of oxyntomodulin tives In order to measure in vitro activities of oxyntomodulin tives, oxyntomodulin derivatives having the following amino acid sequences were synthesized (Table 1).
Table l [Table 1] Oxyntomodulin and oxyntomodulin derivatives SEQ ID NO. Amino acid sequence Note SEQ ID NO. 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRN RNNIA SEQ ID NO. 2 FTSDYSKYLDEEAVRLFIEWLMNTKRNR NNIA SEQ ID NO. 3 CASQGTFTSDYSKYLDERRAQDFVAWLKNTGPSS GAPPPS SEQ ID NO. 4 CAGQGTFTSDYSRYLEEEAVRLFIEWLKNGGPSSG APPPS SEQ ID NO. 5 CAGQGTFTSDYSRQMEEEAVRLFIEWLKNGGPSS GAPPPS SEQ ID NO. 6 CAGEGTFTSDLSRQMEEEAVRLFIEWAAHSQGTFT SDYSKYLD SEQ ID NO. 7 CA-SQGTFTSDYSRYLDEEAVRLFIEWLMNTK SEQ ID NO. 8 CA-SQGTFTSDLSRQLEEEAVRLFIEWLMNK SEQ ID NO. 9 CAGQGTFTSDYSRYLDEEAVXLFIEWLMNTKRNR NNIA SEQ ID NO. 10 CASQGTFTSDYSRQMEEEAVRLFIEWLMNGGPSS GAPPPSK SEQ ID NO. 11 CAGEGTFTSDLSRQMEEEAVRLFIEWAAHSQGTFT SDYSRYLDK SEQ ID NO. 12 CA- SQGTFTSDYSRYLDGGGHGEGTFTSDLSKQME EEAVK SEQ ID NO. 13 CA-SQGTFTSDYSRYLDXEAVXLFIEWLMNTK SEQ ID NO. 14 CAGQGTFTSDYSRYLDEEAVXLFIXWLMNTKRNR NNIA SEQ ID NO. 15 CAGQGTFTSDYSRYLDEEAVRLFIXWLMNTKRNR NNIA SEQ ID NO. 16 FTSDLSRQLEGGGHSQGTFTSDLSRQLEK SEQ ID NO. 17 CASQGTFTSDYSRYLDEEAVRLFIEWIRNTKRNRN SEQ ID NO. 18 CASQGTFTSDYSRYLDEEAVRLFIEWIRNGGPSSG APPPSK SEQ ID NO. 19 CA- Ring Formation SQGTFTSDYSRYLDEEAVKLFIEWIRNTKRNRN SEQ ID NO. 20 CA- Ring Formation SQGTFTSDYSRYLDEEAVKLFIEWIRNGGPSSG APPPSK SEQ ID NO. 21 CASQGTFTSDYSRQLEEEAVRLFIEWVRNTKRNRN SEQ ID NO. 22 DA-SQGTFTSDYSKYLDEKRAKEFVQWLMNTK Ring Formation SEQ ID NO. 23 HAibQGTFTSDYSKYLDEKRAKEFVCWLMNT SEQ ID NO. 24 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO. 25 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC Ring Formation SEQ ID NO. 26 TFTSDYSKYLDEKRAKEFVQWLMNTC Ring Formation SEQ ID NO. 27 HAibQGTFTSDYSKYLDEQAAKEFICWLMNT Ring Formation SEQ ID NO. 28 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNT SEQ ID NO. 29 H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTK RNRNNIA SEQ ID NO. 30 CASQGTFTSDYSKYLDSRRAQDFVQWLMNTKRN RNNIA SEQ ID NO. 31 CA- (d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKR NRNNIA SEQ ID NO. 32 CA- Ring Formation AibQGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO. 33 HAibQGTFTSDYAKYLDEKRAKEFVQWLMNTC Ring Formation SEQ ID NO. 34 YAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC Ring Formation In Table 1, the amino acids in bold and underlined in each of SEQ ID NOS: 19, 20, 22, 25, 26, 27, 32, 33, and 34, taken er, form a ring, and the amino acids represented by X mean a non-native amino acid, 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 derivatives synthesized in Example 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 Ovary) to express human GLP-1 receptor gene and glucagon or gene, respectively. Thus, they are suitable to measure GLP-1 and glucagon activities. ore, the activity of each oxyntomodulin tive 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 1 X 105, followed by ation for 24 hours.
The cultured cells were washed with KRB buffer and suspended in 40 ml of KRB buffer ning 1 mM IBMX, and left at room temperature for 5 minutes. Oxyn- tomodulin (SEQ ID NO. 1) and oxyntomodulin derivatives sented by SEQ ID NOs. 2-6, 8, 10-13, 17, 18, 23-25, 27, 28 and 32-34) were diluted from 1000 nM to 0.02 nM by 5-fold serial dilution, and each 40 mL thereof was added to the cells, and cultured at 37 0C for 1 hour in a C02 incubator. Then, 20 mL of cell lysis buffer was added for cell lysis, and the cell lysates were applied to a CAMP assay kit (Molecular Device, USA) to measure cAMP concentrations. EC50 values were calculated therefrom, and compared to each other. EC50 values are shown in the following Table Table 2 [Table 2] Comparison of in vitro activities for GLP-1 receptor and glucagon or between oxyntomodulin and oxyntomodulin derivatives As shown in Table 2, there were oxyntomodulin derivatives g excellent 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 oxyntornodulin activates both the GLP-1 receptor and glucagon receptor to suppress appetite, tate lipolysis, and promote satiety, thereby g anti-obesity effects. The modulin derivatives according to the t invention show higher in vitro activities on both the GLP-1 receptor 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 modulin.
Example 3. Test on in vivo activity of oxyntomodulin derivatives In order to measure in vivo therapeutic activity of oxyntomodulin derivatives, 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 l or 10 mg/kg of oxyntomodulin, or 0.02, 0.1, l or 10 mg/kg of the oxyn- tomodulin tive 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 tive showed more excellent inhibitory 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 modulin (SEQ ID NO. 1) with 3.4 K PropionALD(2) PEG (PEG with two propylaldehyde groups, NOF, Japan), the modulin and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of 1 : 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 sodium orohydride (cyanoborohydride (SCB, NaCNBH3), NaCNBHg) was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE S (XK16, Amersham Biosciences) to purify oxyntomodulin having mono-pegylated lysine (column: SOURCE S (XK16, am 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 + lM KCl)) (). is a graph showing the result of purifying mono-PEGylated oxyntomodulin through a SOURCE S pu- rification . EGylation of the eluted peaks was examined by SDS- PAGE, and lysine selectivity was examined by peptide mapping using Asp—N protease (). is a graph showing the result of peptide mapping of purified mono- PEGylated oxyntomodulin.
Next, the purified mono-PEGylated oxyntomodulin and globulin Fc were reacted at a molar ratio of l : 10 with the n 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 pu— tion column to purify conjugates including oxyntomodulin and immunoglobulin Fc (column: SOURCE 15Q (XK16, Amersham Biosciences), flow rate: 2.0 ml/min, gradient: A O —> 20% 100 min B (A: 20mM Tris—HCl, pH 7.5, B: A + 1M NaCl)) (). is a graph showing the result of purifying ates including oxyn- tomodulin and immunoglobulin Fc.
Example 5: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 29) and immunoglobulin Fc Firstly, for PEGylation of lysine residue at position 30 of the amino acid sequence of modulin derivative (SEQ ID NO. 29) with 3.4 K nALD(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 n 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 reaction mixture was applied to a SOURCE Sto purify the oxyntomodulin derivative having mono-pegylated lysine n: 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)) (). is a graph g the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 29) through a SOURCE S purification column.
Next, the purified EGylated oxyntomodulin derivative (SEQ ID NO. 29) and globulin 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 (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 conjugates including oxyntomodulin derivative (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 including oxyntomodulin derivative (SEQ ID NO. 29) and immunoglobulin Fc.
Example 6: ation of conjugates ing modulin derivative (SEQ ID NO. 30) and globulin Fc Firstly, for PEGylation of lysine residue at position 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 30) with 3.4 K PropionALD(2) PEG, the oxyntomodulin 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 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 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)) (). is a graph showing the result of purifying a mono-PEGylated oxyntomodulin tive (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 peptide mapping using Asp-N protease (). is a graph showing the result of peptide 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 reacted at a molar ratio of l : 10 with the protein con- tion 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 tive (SEQ ID NO. 30) 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 ing conjugates including oxyntomodulin tive (SEQ ID NO. 30) and immunoglobulin Fc.
Example 7: Preparation of conjugates including modulin derivative (SEQ ID NO. 31) and immunoglobulin Fc 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 modulin derivative (SEQ ID NO. 31) and 3.4 K nALD(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 on mixture was applied to a SOURCE Spurification 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 rate, pH 3.0 + 45% l, B: A + 1M KCl)) (). is a graph showing the result of ing 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 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 on, the reaction mixture was applied to a SOURCE 15Q purification column to purify conjugates including oxyntomodulin tive (SEQ ID NO. 31) 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 ing conjugates including modulin derivative (SEQ ID NO. 31) and immunoglobulin Fc.
Example 8: Preparation 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 derivative (SEQ ID NO. 2) with 3.4 K PropionALD(2) PEG, the oxyn- tomodulin derivative (SEQ ID NO. 2) and 3.4 K PropionALD(2) 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 t 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 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)) (). 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 examined 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 l : 8 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 completion of the reaction, the reaction mixture was applied to a SOURCE 15Q cation 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 (XK16, Amersham Biosciences), flow rate : 2.0 , gradient: A 0 —> 100% 100 min B, (A: 20mM Tris-HCl, pH 7.5, B: A + 1.3M AS))() to purify ates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc. is a graph g the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc through a Source ISO cation column, and 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.
Example 9: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc Firstly, for PEGylation of lysine residue at position 27 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 3) with 3.4 K PropionALD(2) PEG, the oxyn- tomodulin derivative (SEQ ID NO. 3) and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of 1 : 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 on mixture was applied to a SOURCE Spurification 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 + lM KC1)) (). is a graph showing the result of purifying a mono—PEGylated modulin 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 mapping using Asp-N protease (). is a graph showing the result of peptide mapping of purified EGylated oxyn— tomodulin tive (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 1 : 8 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 Butyl FF purification column (Column: Butyl FF(XK16, Amersham Biosciences), flow rate : 2.0 , gradient : B 0 —> 100% 5 min A(A: 20mM Tris-HCl, pH 7.5, B: A + 1.5M NaCl)) () and a SOURCE 15Q purification column n : 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)) () to purify conjugates including oxyn— tomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc. is a graph showing the result of purifying conjugates including modulin derivative (SEQ ID NO. 3) and immunoglobulin Fc through a Butyl FF purification , and is a graph showing the result of purifying conjugates ing oxyntomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc through a SOURCE 15Q pu- rification column.
Example 10: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at position 24 of the amino acid sequence of oxyntomodulin tive (SEQ ID NO. 23) with MAL-10K-ALD PEG (NOF., Japan), the modulin derivative (SEQ ID NO. 23) and MAL-10K-ALD PEG were reacted 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 thereto. After completion of the reaction, the reaction mixture was d to a SOURCE Spurification column to purify the oxyntomodulin derivative having egylated 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)) (). is a graph showing the result of purifying a mono—PEGylated oxyntomodulin tive (SEQ ID NO. 23) through a SOURCE S purification column.
Next, the purified mono-PEGylated oxyntomodulin tive (SEQ ID NO. 23) 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 conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added o 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 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 nil/min, nt: B 0 —> 100% 100 min A, (A: 20mM Tris-HCl, pH 7.5, B: A + 1.1M AS)) (FIG. SC) to purify conjugates including oxyntomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc. is a graph showing the result of purifying conjugates including 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 immunoglobulin Fc through a Source ISO purification column. e 11: Preparation of ates including oxyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at on 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 24) with MAL-lOK—ALD PEG, the oxyn- tomodulin derivative (SEQ ID NO. 24) and MAL—10K-ALD PEG were reacted 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% panol, 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)) (). is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 24) through a SOURCE S cation column.
Next, the purified mono-PEGylated modulin 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 on 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 , nt: 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, nt: 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. 24) and immunoglobulin Fc. is a graph showing the result of purifying conjugates including modulin 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 modulin 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 position 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 25 ) with MAL-lOK-ALD PEG, the oxyn- tomodulin derivative (SEQ ID NO. 25) and MAL-IOK-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 1M guanidine was added thereto. After completion of the reaction, the reaction mixture was applied to a SOURCE ication 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 rate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (a). a is a graph g 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 ID NO. 25) 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 reaction was ted 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 15Q purification column n: 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 + 1M NaCl)) (b) 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-HCl, pH 7.5, B: A + 1.1M AS)) (c) to purify conjugates including modulin derivative (SEQ ID NO. 25 ) and immunoglobulin Fc. b is a graph g the result of purifying ates including oxyntomodulin derivative (SEQ ID NO. 25 ) and im— munoglobulin Fc through a SOURCE 15Q purification , and 0 is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 25 ) and immunoglobulin Fc through a Source ISO purification .
Example 13: Preparation 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 ID NO. 28) and MAL-lOK-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 (pH 9.0) and 2M guanidine was added thereto. 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 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 purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 28) through a SOURCE S purification column.
Next, the purified mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 28) 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 thereto as a reducing agent. After completion of the reaction, the reaction e was applied to a SOURCE 15Q cation 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)) (b) and a Source ISO purification column (column: SOURCE ISO, flow rate: 2.0 ml/min, nt: B 0 —> 100% 100 min A, (A: 20mM Tris—HCl, pH 7.5, B: A + 1.1M AS)) (c) to purify conjugates including oxyntomodulin tive (SEQ ID NO. 28) and immunoglobulin Fc. b is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 28) 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. 28) and immunoglobulin Fc through a Source ISO purification .
Example 14: Preparation of conjugates including modulin 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-lOK-ALD PEG, the oxyn- lin derivative (SEQ ID NO. 32) and MAL—10K—ALD PEG were reacted at a molar ratio of 1 : 3 with the protein 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 KCl)).
Next, the ed mono-PEGylated modulin derivative (SEQ ID NO. 32) and globulin 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 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 e 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-HCl, pH 7.5, B: A + lM NaCl)) and a Source ISO cation column (column: 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. 32) and immunoglobulin Fc.
Example 15: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 33) and immunoglobulin Fc y, for PEGylation of ne residue at position 30 of the amino acid ce of oxyntomodulin derivative (SEQ ID NO. 33) with MAL-lOK—ALD PEG, the oxyn- tomodulin derivative (SEQ ID NO. 33) and MAL—lOK-ALD PEG were reacted at a molar ratio of l : l with the protein 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 thereto. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having egylated 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. 33) 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 (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 + lM 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—HCl, pH 7.5, B: A + 1.1M AS)) to purify conjugates including oxyntomodulin tive (SEQ ID NO. 33) and immunoglobulin Fc.
Example 16: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 34) and immunoglobulin Fc Firstly, for tion 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—lOK-ALD PEG were reacted at a molar ratio of l : l with the n tration 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 tion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the modulin derivative having mono-pegylated ne (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 immunoglobulin Fc were d 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 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% l min B —> 20% 80 min B(A: 20mM Tris-HCl, pH 7.5, B: A + 1M NaCl)) and a Source ISO cation column (column: 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. 34) and immunoglobulin Fc.
Example 17: In Vitro activity of oxyntomodulin derivative-immunoglobulin Fc conjugates In order to measure anti-obesity efficacies of the conjugates including the oxyn- tomodulin or oxyntomodulin derivative and the immunoglobulin PC that were prepared in the above es, experiments were performed in the same manner as in Example 2-2.
Specifically, each of the ormants prepared in es 1-1 and 1-2 was sub— cultured two or three times a week, and aliquoted in each well of a 96—well plate at a density of 1 X 105, ed by cultivation for 24 hours. Each of the cultured trans— formants was washed with KRB buffer and suspended 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 nM 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 lysates were applied to a cAMP assay kit (Molecular Device, USA) to measure cAMP concentrations using a Victor (Perkin Elmer, USA). EC50 values were calculated therefrom, and compared to each other (Table 3).
Table 3 [Table 3] In Vitro activity of oxyntomodulin derivative-immunoglobulin Fc conjugates SEQ ID NO.
CHO/GLP- 1R CHO/GCGR GLP-l Glucagon SEQ ID NO. 23 - Fc conjugates SEQ ID NO. 24 - Fc conjugates SEQ ID NO. 25 — Fc conjugates SEQ ID NO. 32 — Fc conjugates SEQ ID NO. 33 — Fc conjugates SEQ ID NO. 34 — Fc ates As shown in Table 3, the oxyntomodulin derivative—immunoglobulin Fc conjugates were found to show the in Vitro ty to GLP—1 and glucagon ors.
Example 18: In Vivo activity of oxyntomodulin derivative-immun0globulin conjugates It was examined whether the oxyntomodulin derivative-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 administered with oxyntomodulin derivative (SEQ ID NO. 23, 24 or )-immunoglobulin Fc conjugates at a dose of 0.03 or 0.06 week for 3 weeks.
Thereafter, changes in the body weight of the mice were measured ( and ). and are graphs showing s 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 derivative—immunoglobulin Fc conjugates was sed, the body weight was reduced in direct proportion, even though there were differences between the types of the oxyntomodulin derivative—immunoglobulin Fc conjugates, suggesting that the oxyntomodulin derivative-immunoglobulin Fc conjugates reduce the body weight in a dose-dependent manner.

Claims (25)

Claims 1.
1. A conjugate comprising: an oxyntomodulin derivative comprising the amino acid sequence of SEQ ID NO: 24, 25, or 26, an immunoglobulin Fc region, and a ptidyl polymer wherein the non-peptidyl polymer covalently links the oxyntomodulin derivative to the immunoglobulin Fc region.
2. The conjugate according to claim 1, wherein the oxyntomodulin derivative comprises the amino acid sequence of SEQ ID NO: 24.
3. The conjugate according to claim 1, wherein the oxyntomodulin derivative ses the amino acid sequence of SEQ ID NO: 25.
4. The conjugate according to claim 3, wherein the amino acid at positions 16 and 20 form a ring.
5. The conjugate according to claim 1, wherein the oxyntomodulin derivative comprising the amino acid sequence of SEQ ID NO: 26
6. The conjugate ing to claim 5, wherein the amino acid at positions 12 and 16 form a ring.
7. The ate according to any one of the previous claims 1 to 6, wherein the oxyntomodulin derivative is e of activating GLP-1 receptor and on
8. The conjugate according to any one of the previous claims 1 to 7, wherein the conjugate has besity effects.
9. The conjugate according to any one of the previous claims 1 to 8, wherein the nonpeptidyl polymer is polyethylene glycol, polypropylene glycol, an ethylene glycol/ propylene glycol copolymer, a polyoxyethylated polyol, polyvinyl alcohol, a polysaccharide, polyvinyl ethyl ether, polylactic acid (PLA), polylactic-glycolic acid (PLGA), a lipid polymer, hyaluronic acid or combinations thereof.
10. The conjugate according to claim 9, wherein the non-peptidyl polymer is polyethylene glycol.
11. The conjugate according to claim 9, wherein the polysaccharide is n, a chitin, or a combination thereof.
12. The ate according to any one of the previous claims 1 to 11, wherein one end of the non-peptidyl polymer is linked to an amine group or a thiol group of the immunoglobulin Fc region and the other end of the non-peptidyl polymer is linked to an amine group or a thiol group of the oxyntomodulin derivative.
13. The conjugate according to any one of the previous claims 1 to 12, wherein the nonpeptidyl polymer has reactive groups capable of g with the immunoglobulin Fc region and the oxyntomodulin derivative at both ends.
14. The conjugate according to claim 13, wherein the reactive group is an aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group or a succinimide derivative.
15. The conjugate according to claim 13, wherein the ve groups at both ends are the same as or different from each other.
16. The conjugate according to any one of the us claims 1 to 15, wherein the immunoglobulin Fc region is a non-glycosylated Fc region.
17. The conjugate according to any one of the previous claims 1 to 16, wherein the immunoglobulin Fc region is a CH1 , a CH2 domain, a CH3 domain and a CH4 domain; a CH1 domain and a CH2 domain; a CH1 domain and a CH3 ; a CH2 domain and a CH3 domain; a ation of one or more domains and an immunoglobulin hinge region or a portion thereof ; or a dimer of ea ch domain of the heavy-chain constant s and the light-chain constant region.
18. The conjugate according to any one of the previous claims 1 to 17, wherein the immunoglobulin Fc region is a derivative in which a region capable of forming a ide 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 d, or an antibody dependent cell mediated cytotoxicity (ADCC) site is deleted.
19. The conjugate according to any one of the previous claims 1 to 18, wherein the immunoglobulin Fc region is an Fc region derived from an IgG, IgA, IgD, IgE, or IgM.
20. The conjugate according to claim 19, wherein the immunoglobulin Fc region is an IgG4 Fc region.
21. The conjugate according to any one of the previous claims 1 to 20, wherein the immunoglobulin Fc region is a human IgG4-derived non-glycosylated Fc region.
22. A pharmaceutical composition for the prevention or treatment of obesity, comprising the conjugate of any one of claims 1 to 21.
23. The pharmaceutical composition according to claim 22, further comprising a ceutically acceptable carrier.
24. The pharmaceutical composition according to claim 22 or 23, wherein the composition is to be administered alone or is to be stered in combination with other pharmaceutical formulations g lactic or therapeutic effects on obesity.
25. The pharmaceutical composition according to claim 24, wherein the other pharmaceutical formulation is a GLP-1 receptor agonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a n-concentrating hormone (MCH) or antagonist, a Y
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