Deprecated: The each() function is deprecated. This message will be suppressed on further calls in /home/zhenxiangba/zhenxiangba.com/public_html/phproxy-improved-master/index.php on line 456
NZ618811B2 - A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof - Google Patents
[go: Go Back, main page]

NZ618811B2 - 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

Info

Publication number
NZ618811B2
NZ618811B2 NZ618811A NZ61881112A NZ618811B2 NZ 618811 B2 NZ618811 B2 NZ 618811B2 NZ 618811 A NZ618811 A NZ 618811A NZ 61881112 A NZ61881112 A NZ 61881112A NZ 618811 B2 NZ618811 B2 NZ 618811B2
Authority
NZ
New Zealand
Prior art keywords
seq
immunoglobulin
oxyntomodulin
region
group
Prior art date
Application number
NZ618811A
Other versions
NZ618811A (en
Inventor
In Young Choi
Sung Youb Jung
Dae Jin Kim
Se Chang Kwon
Sung Hee Park
Young Eun Woo
Original Assignee
Hanmi Science Co Ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hanmi Science Co Ltd filed Critical Hanmi Science Co Ltd
Priority to NZ718999A priority Critical patent/NZ718999B2/en
Priority claimed from PCT/KR2012/004722 external-priority patent/WO2012173422A1/en
Publication of NZ618811A publication Critical patent/NZ618811A/en
Publication of NZ618811B2 publication Critical patent/NZ618811B2/en

Links

Classifications

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

Abstract

Disclosed is a conjugate comprising oxyntomodulin, 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.

Description

Description Title of Invention: A CONJUGATE COMPRISING OXYN- TOMODULIN AND AN IMNIUNOGLOBULIN FRAGMENT, AND USE THEREOF Technical Field The present invention relates to a conjugate comprising oxyntomodulin and an im- munoglobulin fragment, and the use f. More particularly, the present invention relates to a conjugate comprising oxyntomodulin, an immunoglobulin Fc region, and non-peptidyl polymer wherein the conjugate being obtainable by covalently linking oxyntomodulin to immunoglobulin Fc region via ptidyl polymer, and a pharma- ceutical composition for the prevention or treatment of obesity comprising the conjugate.
Background Art l3] Recently, economic growth and changes in lifestyle are g to changes in eating habits. The main causes of rising overweight and obesity rates in contemporary people are ption of high-calorie foods such as fast foods and lack of exercise. World Health Organization (WHO) estimates that more than 1 billion people ide are overweight and at least 300 million of them are clinically obese. In particular, 250,000 people die each year in Europe and more than 2.5 million people worldwide die each year as a result of being overweight (World Health Organization, Global gy on Diet, Physical Activity and Health, 2004).
Being overweight and obese ses blood pressure and cholesterol levels to cause occurrence or exacerbation of various diseases such as cardiovascular disease, diabetes, and tis, and are also main causes of rising incidence rates of arte- riosclerosis, hypertension, hyperlipidemia or cardiovascular disease in children or ado— lescents as well as in adults.
Obesity is a severe condition that causes various diseases worldwide. It is thought to be overcome by individual efforts, and it is also believed that obese patients lack self control. However, it is difficult to treat obesity, because obesity is a complex disorder involving appetite regulation and energy metabolism. For the treatment of obesity, abnormal actions associated with appetite tion and energy metabolism should be d er with efforts of obese patients. Many attempts have been made to develop drugs capable of treating the abnormal actions. As the result of these effons, drugs such as Rimonabant (Sanofi—Aventis), Sibutramin (Abbott), Contrave (Takeda), and Orlistat (Roche) have been developed, but they have the antages of serious adverse effects or very weak besity effects. For example, it was reported that Ri— monabant (Sanofi—Aventis) shows a side-effect of central nerve disorder, Sibutramine (Abbott) and Contrave (Takeda) show cardiovascular side-effects, and Orlistat ) shows only 4 kg of weight loss when taken for 1 year. unately, there are no therapeutic agents for obesity which can be safely prescribed for obese patients.
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 tives have received much attention. Glucagon is produced by the pancreas when the level of glucose in the blood drops resulting from other medications or diseases, hormone or enzyme deficiencies. Glucagon ates 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 glucose level, glucagon suppresses te and activates hormone—sensitive lipase(HSL) of adipocytes to facilitate lipolysis, thereby showing anti—obesity effects. One of the glucagon derivatives, glucagon like peptide—l (GLP— 1) is under development as a therapeutic agent for hy— perglycemia in patients with es, and it ons 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 homology with GLP-1 and is also ed to activate the GLP-1 receptor, thereby ameliorating lycemia in patients with diabetes. However, anti—obesity drugs including GLP—l are reported to show side— effects such as vomiting and nausea.
As an alternative to GLP—l, therefore, much attention has been focused on oxyn- tomodulin, a peptide derived from a glucagon sor, pre—glucagon that binds to the receptors of two peptides, GLP—1 and glucagon. modulin ents a potent anti-obesity therapy, because it inhibits food intake like GLP—l, promotes satiety, and has a lipolytic activity like on.
Based on the dual function of the oxyntomodulin peptide, it has been actively d as a drug for the treatment of obesity. For example, Korean Patent N0. 925017 discloses a pharmaceutical composition including oxyntomodulin as an active in— gredient for the treatment of overweight human, which is administered via an oral, eral, mucosal, rectal, subcutaneous, or transdermal route. However, it has been reported that this anti—obesity drug including oxyntomodulin has a short in vivo half- life and weak therapeutic efficacy, even though administered at a high dose three times a day. Thus, many efforts have been made to improve the in vivo half-life or eutic effect of oxyntomodulin on obesity by its modification.
For example, a dual agonist oxyntomodulin (Merck) is prepared by substituting L— serine with ne at position 2 of oxyntomodulin to increase a resistance to dipeptidyl peptidase-IV (DPP-IV) and by ing a cholesterol moiety at the C- terminal to increase the blood half—life at the same time. ZP2929 (Zealand) is prepared by substituting L-serine with D-serine at on 2 to enhance resistance to DPP-IV, substituting arginine with alanine at on 17 to enhance resistance to protease, substituting methionine with lysine at position 27 to enhance oxidative stability, and substituting glutamine with aspartic acid and alanine at positions 20 and 24 and asparagine with serine at position 28 to enhance deamidation stability. However, even though the half-life of the dual t oxyntomodulin (Merck) was enhanced to show half—life 8-12 minutes longer than the native modulin, it still has a very short in vivo half—life of l .7 hr and its administration dose is also as high as several mg/kg.
Unfortunately, oxyntomodulin or derivatives thereof have disadvantages of daily administration of high dose due to the short ife and low efficacy.
Disclosure of Invention Technical Problem Accordingly, the present inventors have made many s to develop a method for sing 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 improved blood half-life while ining the activity in vivo so as to t excellent anti-obesity effects, thereby completing the present invention. on to Problem In an aspect, a conjugate is ed comprising modulin, 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. [14A] In an aspect, a conjugate is provided a conjugate comprising an oxyntomodulin derivative, an immunoglobulin Fc region, and non—peptidyl polymer wherein the oxyntomodulin derivative is covalently linked to immunoglobulin Fc region via non- peptidyl polymer, and wherein the oxyntomodulin derivative comprising the amino acid sequence selected from SEQ ID NOS: 24-26 and 28.
In another aspect a pharmaceutical composition is provided for the prevention or treatment of obesity, comprising the conjugates, as described herein.
Still another aspect a method is provided for preventing or ng obesity, comprising the step of administering the conjugate or the composition to a subject.
Still r aspect a use of the ate is provided 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 PC of the present ion reduces food intake, suppresses gastric emptylng, and facilitates lipolysis without side-effects, unlike native oxyntomodulin, and also shows excellent or- activating effects and long—term sustainability, compared to oxyntomodulin. Thus, it can be widely used in the treatment of obesity with safety and efficacy. Unlike native oxyntomodulin, the novel peptide of the present invention reduces food intake, suppresses gastric emptylng, and facilitates lipolysis without side-effects, and also shows ent receptor-activating effects. Thus, it can be widely used in the treatment of obesity with safety and efficacy.
Any discussion of documents, acts, materials, s, articles or the like which has been included in the t specification is not to be taken as an ion that any or all of these matters form part of the prior art base or were common general knowledge in the field relevant to the present disclosure as it d before the ty date of each claim of this application.
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 h a SOURCE S purification column. is a graph showing the result of e mapping of purified mono— PEGylated oxyntomodulin. is a graph showing the result of purifying conjugates including oxyntomodulin 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. 29) through a SOURCE S purification column. is a graph showing the result of purifying conjugates including oxyntomodulin tive (SEQ ID NO. 29) and immunoglobulin Fc h a SOURCE 15Q purification . is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 30) through a SOURCE S purification column. is a graph showing the result of peptide mapping of purified mono— PEGylated oxyntomodulin tive (SEQ ID NO. 30). is a graph showing the result of purifying conjugates including modulin derivative (SEQ ID NO. 30) 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. 31) through a SOURCE S purification column. is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 31) and immunoglobulin Fc through a SOURCE 15Q purification . is a graph showing the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 2) through a SOURCE S purification column. is a graph showing the result of peptide mapping of purified mono- PEGylated oxyntomodulin derivative (SEQ ID NO. 2). is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin F0 through a SOURCE 15Q purification column. is a graph showing the result fying conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc through a Source ISO purification column. is a graph showing the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 3) h a SOURCE S purification column. is a graph showing the result of peptide mapping of purified mono— PEGylated oxyntomodulin derivative (SEQ ID NO. 3). is a graph showing the result of ing conjugates including oxyntomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc through a Butyl FF purification column. is a graph showing the result of purifying conjugates including oxyntomodulin tive (SEQ ID NO. 3) and immuneglobulin Fc through a Source 15Q purification column. is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 23) through a SOURCE S purification ; is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fe h a Source lSQ purification column; is a graph g the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 23) and globulin Fc through a SOURCE ISO ation column; is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 24) through a SOURCE S purification column; is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 24) and globulin Fc through a Source 15Q ation column; is a graph g the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc through a SOURCE ISO purification column; a is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 25) through a SOURCE S purification column; b is a graph showing the result of ing conjugates including oxyn- tomodulin derivative (SEQ ID NO. 25) and immunoglobulin Fc through a Source lSQ ation column; 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 column; a is a graph showing the result of purifying a mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 28) through a SOURCE S purification column; lb is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 28) and globulin Fc through a Source 15Q purification column; 0 is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc through a SOURCE ISO purification column; 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 s in body weight of mice according to the type and administration dose of oxyntomodulin derivative—immunoglobulin Fc conjugates.
Best Mode for Carrying out the Invention In an aspect, the present invention provides a conjugate comprising oxyntomodulin, an immunoglobulin Fc region, and non—peptidyl polymer wherein the conjugate being obtainable by covalently linking oxyntomodulin to globulin Fc region via non-peptidyl polymer.
As used herein, the term "conjugate" means a conjugate sing oxyntomodulin and other factors. Other factors can be any nce which can induce increased stability in blood, suspend emission through the kidney, or other useful effects. In the present invention, the factors can be globulin Fc region. In an embodiment, the conjugate can be comprised of an oxyntomodulin, and an immunoglobulin Fc region, which are linked by a ptidyl polymer. The non-peptidyl polymer can link an oxyntomodulin and an immunoglobulin Fc region via covalent bonds. Two terminal ends of non—peptidyl polymer can be linked to an amine group or thiol group of the immunoglobulin Fc region and oxyntomodulin derivatives, respectively.
The ate of the present ion means to have an ed o duration of efficacy, compared to native oxyntomodulin, and the long—acting conjugate may include oxyntomodulin prepared by modification, substitution, addition, or deletion of the amino acid sequences of the native oxyntomodulin, oxyntomodulin conjugated to a biodegradable polymer such as polyethylene glycol (PEG), oxyntomodulin conjugated to a long-acting n such as albumin or immunoglobulin, oxyntomodulin conjugated to fatty acid having the ability of binding to albumin in the body, or oxyntomodulin encapsulated in biodegradable nanoparticles, but the type of the long- acting conjugate is not limited thereto.
As used herein, the term ”oxyntomodulin" means a peptide derived from a on precursor, pre-glucagon, and includes a native oxyntomodulin, precursors, derivatives, fragments thereof, and variants thereof. In an embodiment, it can have the amino acid sequence of SEQ ID NO. 1 (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA).
The term, "oxyntomodulin variant" is a peptide having one or more amino acid sequences different from those of native oxyntomodulin, and means a peptide that retains the function of activating the GLP—l and glucagon receptors, and it may be prepared by any one of substitution, addition, deletion, and ation or by a combination thereof in a part of the amino acid sequences of the native oxyntomodulin.
The term, "oxyntomodulin derivative" includes peptides, peptide derivatives or peptide mimetics that are ed by addition, on or substitution of amino acids of oxyntomodulin so as to activate both of the GLP—l receptor and the glucagon receptor at a high level, compared to the native modulin.
The term, "oxyntomodulin fragment means a fragment having one or more amino acids added or deleted at the N—terminus or the C—terminus of the native oxyntomodulin, in which non-naturally occurring amino acids (for example, D—type amino acid) can be added, and has a function of activating both of the GLP- 1 receptor and the glucagon receptor.
Each of the preparation methods for the variants, derivatives, and fragments of oxyntomodulin can be used individually 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 or and the glucagon receptor.
Amino acids mentioned herein are iated according to the nomenclature rule of lUPAC-IUB as follows: Alanine A Arginine R Asparagine N Aspartic acid D Cysteine C ic acid B Glutamine Q Glycine G Histidine H lsoleucine 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 modulin derivative encompasses any peptide that is prepared by substitutions, additions, deletions or post translational modifications (e.g., ation, acylation, ubiquitination, intramolecular covalent bonding) in the amino acid ce of oxyntomodulin (HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA, 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 ns, as well as atypical or non-naturally ng amino acids can be used.
Commercially available sources of atypical amino acids include Sigma-Aldrich, ChemPep Inc., and Genzyme Pharmaceuticals. The peptides ing 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 ).
In one specific embodiment, the oxyntomodulin derivative of the present invention is a novel peptide including the amino acids of the following Formula 1.
R1-X1-X2-GTFTSD-X3-X4-X5-X6-X7-X8—X9-XlO-Xl 1-X12-X13-X14-X15-X16- X17-Xl8-X19-X20-X21—X22—X23-X24-R2 (Formula 1) wherein R1 is histidine, desamino~histidyl, dimethyl-histidyl (N—dimethyl-histidyl), beta—hydroxyimidazopropionyl, 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; X5 is lysine or arginine; X6 is glutamine or tyrosine; X7 is leucine or methionine; X8 is aspartic acid or glutamic acid; X9 is glutamic acid, serine, alpha-methyl-glutamic acid or is d; X10 is glutamine, glutamic acid, lysine, arginine, serine or is deleted; X11 is alanine, arginine, valine or is deleted; X12 is e, arginine, serine, valine or is deleted; X13 is lysine, glutamine, arginine, methyl—glutamic acid or is deleted; X14 is aspartic acid, glutamic acid, leucine or is deleted; X15 is phenylalanine or is d; X16 is isoleucine, valine or is deleted; X17 is alanine, ne, glutamic acid, lysine, glutamine, alpha-methyl-glutamic acid or is deleted; X18 is tryptophan or is deleted; X19 is alanine, isoleucine, leucine, serine, valine or is deleted; X20 is e, lysine, methionine, ine, arginine or is deleted; X21 is asparagine or is deleted; X22 is alanine, glycine, threonine or is d; X23 is cysteine, lysine or is deleted; X24 is a peptide having 2 to 10 amino acids consisting of ations of alanine, glycine and serine, or is deleted; and R2 is IA (SEQ ID NO. 35), GPSSGAPPPS (SEQ ID NO. 36), GPSSGAPPPSK (SEQ ID NO. 37), HSQGTFTSDYSKYLD (SEQ ID NO. 38), 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 present invention may be substituted with 4—imidazoacetyl where the alpha carbon of histidine at position 1 of amino acid sequence represented by SEQ ID NO. 1 is deleted, desamino—histidyl where the N-terminal amino group is deleted, yl—histidyl (N—dimethyl-histidyl) where the inal amino group is modified with two methyl groups, beta—hydroxy imidazopropionyl where the N—terminal amino group is substituted with a hydroxyl group, or beta-carboxy imidazopropionyl where the N—terminal amino group is substituted with a carboxyl group. In addition, the GLP—l receptor-binding region may be substituted with amino acids that enhance hydrophobic and ionic bonds or combinations 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. r, a part of the oxyntomodulin ce may be substituted with a sequence stabilizing alpha helix. In an embodiment, amino acids at positions 10, 14, 16, 20, 24 and 28 of the amino acid sequence of Formula 1 may be substituted with amino acids or amino acid tives ting of Tyr(4-Me), Phe, Phe(4~Me), Phe(4—ll), Phe(4- CN), Phe(4—N02), Phe(4-NH2), Phg, Pal, Nal, Ala(2—thienyl) and Ala(benzothienyl) that are known to stabilize alpha helix, and there are no limitations on the type and number of alpha helix—stabilizing amino acid or amino acid derivatives to be inserted.
In an embodiment, amino acids at positions 10 and 14, 12 and 16, 16 and 20, 20 and 24, and 24 and 28 may be also substituted with ic acid or lysine, respectively so as to form rings, and there is no limitation on the number of rings to be inserted. The peptide may be a peptide having an amino acid sequence selected from the following Formulae 1 to 6.
In one specific embodiment, the oxyntomodulin tive of the t invention is a novel peptide including the amino acid sequence of the following Formula 2 Where the amino acid sequence of oxyntomodulin is substituted with that of exendin or GLP- 1.
Rl—A-R3 (Formula 2) In another specific embodiment, the oxyntomodulin derivative of the t invention is a novel peptide including the amino acid sequence of the following Formula 3, which is prepared by linking a part of the amino acid sequence of oxyntomodulin and a part of the amino acid sequence of exendin or GLP-l Via a proper amino acid linker.
Rl-B—C-R4 (Formula 3) In still another specific embodiment, the oxyntomodulin tive of the present invention is a novel e including the amino acid sequence of the following Formula 4, wherein a part of the amino acid sequence of oxyntomodulin is substituted with an amino acid capable of enhancing the binding affinity to GLP-1 receptor, for example, Leu at on 26 which binds with GLP—1 or by hydrophobic interaction is substituted with the hydrophobic e, lle or Val.
R1—SQGTFTSDYSKYLD-D l-D2-D3-D4-D5-LFVQW-D6-D7-N—D8-R3 (Formula 4) In still another specific embodiment, the oxyntomodulin derivative of the present invention is a novel peptide including the following Formula 5, wherein a part of the amino acid sequence is deleted, added, or substituted with other amino acid in order to enhance the ties of native oxyntomodulin on GLP-1 receptor and glucagon receptor.
R1-E1-QGTFTSDYSKYLD-E2-E3-RA-E4-E5—FV-E6—WLMNT-E7-R5 (Formula 5) In Formulae 2 to 5, R1 is the same as in the ption of Formula 1; A is selected from the group consisting of SQGTFTSDYSKYLDSRRAQD— FVQWLMNT (SEQ ID NO. 41), SDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO. 42), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO. 43), GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO. 44), GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO. 45), GEGTFTSDL— SRQMEEEAVRLFIEWAA (SEQ ID NO. 46), and SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO. 47); B is selected from the group consisting of SQGTFTSDYSKYLDSRRAQD—iFVQWLMNT (SEQ ID NO. 41), SDYSKYLDEEAVRLFIEWLMNT (SEQ ID NO. 42), SQGTFTSDYSKYLDERRAQDFVAWLKNT (SEQ ID NO. 43), GQGTFTSDYSRYLEEEAVRLFIEWLKNG (SEQ ID NO. 44), GQGTFTSDYSRQMEEEAVRLFIEWLKNG (SEQ ID NO. 45), GEGTFTSDLSRQMEEEAVRLFIEWAA (SEQ ID NO. 46), SQGTFTSDYSRQMEEEAVRLFIEWLMNG (SEQ ID NO. 47), GEGTFTSDLSRQMEEEAVRLFIEW (SEQ ID NO. 48), and SQGTFTSDYSRYLD (SEQ ID NO. 49); C is a peptide having 2 to 10 amino acids consisting of combinations of alanine, glycine and serine; D1 is serine, glutamic acid or arginine; D2 is 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 methionine, arginine or glutamine; D8 is ine, 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 ic acid or glutamic acid; E6 is glutamine, cysteine or lysine; E7 is cysteine, lysine or is d; R3 is IA (SEQ ID NO. 35), GPSSGAPPPS (SEQ ID NO. 36) or GPSSGAPPPSK (SEQ ID NO. 37); R4 is HSQGTFTSDYSKYLD (SEQ ID NO. 38), HSQGTFTSDYSRYLDK (SEQ IDNO. 39) or HGEGTFTSDLSKQMEEEAVK (SEQ ID NO. 40); and, R5 is KRNRNNLA (SEQ ID NO. 35), GPSSGAPPPS (SEQ ID NO. 36), GPSSGAPPPSK (SEQ ID NO. 37) or is deleted (excluded if the amino acid sequences of Fonnulae 2 to 5 are identical to that of SEQ ID NO. 1).
The oxyntomodulin derivative of the present invention may be a noverl peptide of the following Formula 6.
R1-Xl-X2-GTFTSD-X3-X4-X5-X6—X7—X8—X9-XlO-Xl l-Xl2-X13-Xl4-X15-Xl6- X17-Xl8-Xl9-X20-X21-X22-X23-X24—R2 (Formula 6) wherein R1 is histidine, no-histidyl, 4-imidazoacetyl or tyrosine; X1 is inosiobutyric acid), glycine or serine; X2 is glutamic 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 deleted; X10 is glutamine, glutamic acid, lysine, arginine or is d; X11 is alanine, ne or is deleted; X12 is alanine, valine or is deleted; X13 is , glutamine, arginine, alpha-methyl—glutamic acid or is deleted; X14 is aspartic acid, glutamic acid, leucine or is deleted; X15 is phenylalanine or is deleted; X16 is isoleucine, valine or is deleted; X17 is alanine, cysteine, glutamic acid, glutamine, alpha—methyl—glutamic acid or is deleted; X18 is tryptophan or is deleted; X19 is alanine, isoleucine, leucine, valine or is deleted; X20 is alanine, lysine, methionine, arginine or is d; X21 is asparagine or is deleted; X22 is threonine or is deleted; X23 is cysteine, lysine or is deleted; X24 is a peptide having 2 to 10 amino acids ting 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) The oxyntomodulin tive of the present invention may be selected from the group consisting of the peptides of SEQ ID NOs. 2 to 34. In an embodiment, the oxyntomodulin derivative of the present invention may be an modulin derivative described in Table l of Example 2-]. modulin has the activities of two peptides, GLP-1 and glucagon. GLP-l decreases blood glucose, reduces food intake, and suppresses gastric emptying, and glucagon increases blood glucose, facilitate lipolysis and decreases body—weight by increasing energy lisms. The different ical effects of the two peptides can cause undesired effects like sing 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 e 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 ates in on to the glucagon 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 d at positions 1 and ll of oxyntomodulin, 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 receptors by linking a carrier to modulin Via a covalent bond or forming microsphere. The carrier that can form a conjugate containing modulin can be selected from the group consisting of albumin, transferrin, dies, antibody frangments, elastin, heparin, polysaccharide such as chitin, fibronectin and most favorably immunoglobulin Fc region all of which can se 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 chain constant region 3 (CH3) of an immunoglobulin, excluding the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CH1) and the light-chain constant region 1 (CLl) of the immunoglobulin. It may further include a hinge region at the heavy-chain constant region. Also, the immunoglobulin Fc region of the present invention may contain a part or all of the Fc region including the heavy-chain constant region 1 (CH1) and/or the light—chain constant region 1 (CLl), except for the variable regions ofthe heavy and light , as long as it has a logical 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 immunoglobulin Fc region of the present invention may comprise l) a CHl domain, a CH2 , 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 region), and 6) a dimer of each domain of the heavy— chain constant regions and the light—chain constant region.
The immunoglobulin Fc region of the present invention es a native amino acid sequence, and a sequence derivative (mutant) thereof. An amino acid sequence derivative is a sequence that is different from the native amino acid sequence due to a on, an insertion, a non—conservative or conservative substitution or combinations thereof of one or more amino acid es. For example, in an IgG Fc, amino acid residues known to be important in binding, at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, may be used as a suitable target for ation.
Also, other various derivatives are possible, including one in which a region capable of forming a disulfide bond is deleted, or certain amino acid residues are eliminated at the N-terminal end of a native Fc form or a methionine residue is added thereto. Further, to remove effector functions, a deletion may occur in a complement—binding site, such as a Clq-binding site and an ADCC (antibody dependent cell mediated cytotoxicity) site. Techniques of ing such sequence derivatives of the immunoglobulin Fc region are disclosed in WO 97/34631 and WO 96/32478.
Amino acid exchanges in proteins and peptides, which do not generally alter the activity of the proteins or peptides, are known in the art (H. Neurath, R. L. Hill, The Proteins, Academic Press, New York, 1979). The most commonly occurring exchanges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, n, l, y, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, Ala/Glu and Asp/Gly, in both directions. In addition, the Fc region, if d, may be modified by phosphorylation, ion, acrylation, glycosylation, ation, famesylation, acetylation, amidation, and the like.
The aforementioned Fc derivatives are derivatives that have a biological activity identical to the Fc region of the present invention or improved structural stability, for example, against heat, pH, or the like.
In addition, these PC regions may be obtained from native forms isolated from humans and other animals including cows, goats, pigs, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or derivatives thereof, obtained from transformed animal cells or microorganisms. Herein, they may be obtained from a native immunoglobulin by isolating whole globulins from human or animal organisms and treating them with a proteolytic enzyme. Papain digests the native immunoglobulin into Fab and Fe regions, and pepsin treatment results in the production of pF'c and F(ab)2 nts. These fragments may be subjected, for example, to size exclusion chromatography to isolate PC or pF'c. In an embodiment, a human-derived Fc region is a inant 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 , sed 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 F0 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 C1 and a decrease or loss in antibody- ent cell-mediated cytotoxicity or complement-dependent xicity, thereby not ng unnecessary immune responses in-Vivo. In this regard, an immunoglobulin Fc region in a deglycosylated or aglycosylated form may a suitable drug carrier.
As used herein, the term "deglycosylation" refers to enzymatically removing sugar moieties from an Fc region, and the term "aglycosylation" means that an Fc region is produced in an unglycosylated form by a prokaryote, for example but not limited E. coli.
Meanwhile, the immunoglobulin Fc region may be derived from humans or other animals including cows, goats, pigs, mice, rabbits, rs, rats and guinea pigs.
In addition, the immuneglobulin 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 s thereof. In an embodiment, it is derived from IgG or IgM, which are among the most abundant proteins in human blood, which is known to enhance the half-lives of ligand-binding proteins.
On the other hand, the term "combination", 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 multimer may be formed from two or more fragments selected from the group consisting of IgG Fc, IgA Fc, IgM Fc, IgD Fe, and IgE 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 ptidyl polymer may be interchangeably used with the non-peptidyl linker.
The non—peptidyl polymer useful in the t ion may be selected from the group consisting of a biodegradable polymer, a lipid polymer, chitin, onic acid, and a combination f. In an embodiment, the biodegradable polymer may be polyethylene glycol, polypropylene glycol, ethylene glycol—propylene glycol mer, polyoxyethylated polyol, polyvinyl alcohol, polysaccharide, n, polyvinyl ethyl ether, polylactic acid (PLA) or polylactic—gly colic acid (PLGA) or polyethylene glycol (PEG). In addition, derivatives thereofknown in the art and tives 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 proteolytic enzyme, and thus a sufficient effect of increasing the serum half-life of the active drug by a carrier cannot be obtained as expected. r, in the t invention, the polymer having resistance to the proteolytic enzyme can be used to maintain the serum half—life of the peptide being similar to that of the carrier.
Therefore, any non-peptidyl polymer can be used without 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, or 1 to 20 kDa. The non-peptidyl polymer of the t invention, linked to the immunoglobulin Fc , may be one polymer or a ation of different types of polymers.
The non-peptidyl polymer used in the present invention has a reactive group capable ofbinding to the immunoglobulin Fc region and protein drug. The non-peptidyl polymer has a reactive group at both ends, which may be selected from the group consisting of a reactive aldehyde, a propionaldehyde, a butyraldehyde, a maleimide and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxy succinimidyl, succinimidyl ymethyl, or succinimidyl carbonate. In particular, when the non-peptidyl polymer has a reactive aldehyde group at both ends thereof, it is ive in linking at both ends with a physiologically active polypeptide and an immunoglobulin with minimal non-specific reactions. A final product generated by reductive alkylation by an aldehyde bond is much more stable than that linked by an amide bond. The aldehyde reactive group selectively binds to an N—terminus at a low pH, and binds to a lysine residue to form a covalent bond at a high pH, such as pH 9.0. The ve groups at both ends of the non—peptidyl polymer may be the same or different. For e, the non—peptidyl polymer may possess a maleimide group at one end, and an aldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. When a polyethylene glycol having a reactive 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 cially available d 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 globulin Fc region and oxyntomodulin tives, respectively.
The non-peptidyl polymer has a reactive group at both ends, which may be selected from the group ting of a reactive aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl, or imidyl ate.
The two reactive terminal groups of the non—peptidyl polymer may be the same as or different from each other. For example, the ptide polymer may possess a maleimide group at one end and an aldehyde group, a propionaldehyde group or a butyraldehyde group at the other end. For example, when the non-peptidyl polymer has a reactive aldehyde group at a al group, and a maleimide group at the other terminal group, it is effective in linking at both ends with a physiologically active polypeptide and an immunoglobulin with minimal non-specific reactions. According to Examples of the present invention, conjugates were prepared by linking the oxyntomodulin 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 maleimides group and the de group.
The conjugates of the present invention show excellent ty on GLP-1 receptor and glucagon receptor, compared to native oxyntomodulin, and the blood half-life is increased by linking with the Fc region so as to in in vivo activity for a long period of time.
In still another aspect, the present invention provides a pharmaceutical composition for the tion ortreatment of obesity comprising the peptide.
As used herein, the term "prevention" means all of the actions by which the occurrence 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 administration of the conjugates of the present invention.
As used herein, the term "treatment" means all of the actions by which the symptoms of the disease have been alleviated, ed or ameliorated. In the present invention, "treatment" means that the symptoms of y are ated, improved or rated 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 e 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 es, hyperlipidemia, sexual dysfunction, arthritis, and cardiovascular diseases, and in some cases, is associated with incidence of .
The conjugates of the present invention, which are prepared by linking oxyntomodulin or a derivative thereof with the immunoglobulin Fc region, show excellent binding affinity to glucagon and GLP-1 receptors (Table 3) and 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 t invention may further include a pharrnaceutically acceptable carrier, excipient, or diluent. As used herein, the term "pharmaceutically acceptable" means that the composition is sufficient to achieve the therapeutic effects without deleterious side effects, and may be readily determined depending on the type of the diseases, the patient's age, body , health conditions, , and drug sensitivity, administration route, administration mode, administration frequency, duration of treatment, drugs used in combination or coincident with the composition of this ion, and other factors known in medicine.
The pharmaceutical composition including the derivative of the present invention may further include a ceutically acceptable r. For oral administration, the carrier may e, but is not limited to, a binder, a lubricant, a disintegrant, an excipient, a lizer, a dispersing agent, a stabilizer, a suspending agent, a colorant, and a flavorant. For in] ectable preparations, the carrier may include a buffering agent, a preserving agent, an analgesic, a solubilizer, an isotonic agent, and a stabilizer. For preparations for l 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 ceutically acceptable carriers.
For example, for oral administration, the pharmaceutical composition may be formulated into tablets, troches, es, s, suspensions, syrups or wafers. For injectable preparations, the pharmaceutical composition may be formulated into an ampule as a single dosage form or a multidose container. The pharmaceutical composition may also be formulated into solutions, sions, 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, dextrose, sucrose, sorbitol, mannitol, l, erythritol, maltitol, starch, acacia rubber, alginate, n, calcium phosphate, calcium silicate, cellulose, cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils. In addition, the pharmaceutical ations may further include fillers, anti—coagulating agents, lubricants, humectants, flavorants, and antiseptics.
Further, the pharmaceutical composition of the present invention may have any formulation selected from the group consisting of tablets, pills, powders, granules, es, suspensions, liquids for internal use, emulsions, syrups, sterile aqueous ons, non-aqueous solvents, lyophilized formulations and itories.
Further, the composition may be formulated into a single dosage form suitable for the patient's body, and maybe 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, intraarterial, intramedullary, intramedullary, entricular, pulmonary, transderrnal, subcutaneous, eritoneal, intranasal, olonic, topical, sublingual, vaginal, or rectal administration, but is not limited thereto.
The composition may be used by blending with a variety of pharmaceutically acceptable carriers such as physiological saline or organic solvents. In order to increase the stability or tivity, carbohydrates such as glucose, sucrose 0r dextrans, antioxidants such as ascorbic acid or glutathione, chelating agents, low molecular weight proteins or other stabilizers may be used.
The administration dose and frequency of the pharmaceutical ition of the present ion are determined by the type of active ingredient, together with various s such as the disease to be treated, administration route, patient's age, gender, and body weight, and disease severity.
The total effective dose of the composition of the present invention may be administered to a t in a single dose, or may be stered for along period of time in multiple doses according to a fractionated treatment protocol. In the pharmaceutical composition of the present invention, the t of active ingredient may vary depending on the disease severity. In an ment, the total daily dose of the peptide of the present invention may be approximately 0.0001 jig to 500 mg per 1 kg ofbody weight of a patient. However, the effective dose of the peptide is determined considering various factors including patient‘s age, body weight, health conditions, gender, disease severity, diet, and secretion rate, in addition to administration route and treatment frequency of the pharmaceutical ition. In view of this, those skilled in the art may easily determine an effective dose suitable for the particular use of the pharmaceutical composition of the present invention. The pharmaceutical ition according to the t invention is not particularly limited to the formulation, and administration route and mode, as long as it shows the effects of the present invention.
The ceutical 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 composition may be administered alone or in combination or coincident with other pharmaceutical formulations showing prophylactic or therapeutic effects on obesity. The pharmaceutical formulations showing prophylactic or eutic s 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 antagonist, a Melanin—concentrating hormone (MCH) receptor antagonist, a Y2/3 receptor agonist, a MC3/4 receptor agonist, a gastric/pancreatic lipase inhibitor, a 5HT2C agonist, a 03A receptor t, an Amylin receptor agonist, a Ghrelin antagonist, and/or a Ghrelin receptor antagonist.
In still another aspect, the present invention provides a method for 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 predetermined nce into a patient by a certain suitable method. The composition of the present invention may be administered via any of the common routes, as long as it is able to reach a d tissue, for example, but is not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, l, intranasal, intrapulmonary, or intrarectal stration. However, since peptides are ed upon oral administration, active ients of a composition for oral administration should be coated or formulated for protection against degradation in the h.
In the present invention, the term ”subj ect" is those suspected of having obesity, which means mammals including human, mouse, and livestock having obesity or having the ility 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 stered to a subject suspected of having obesity, thereby treating the subject effectively. The obesity is as described above.
The therapeutic method of the present invention may include the step of administering the composition including the peptide at a pharmaceutically effective amount. The total daily dose should be ined through appropriate medical judgment by a physician, and administered once or l times. The specific therapeutically effective dose level for any particular patient may vary depending on various factors well known in the medical art, including the kind and degree of the response to be achieved, concrete compositions according to whether other agents are used therewith or not, the patient's age, body weight, health condition, , 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 coincident 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 composition ing 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. r, these Examples are for illustrative purposes only, 2012/004722 and the invention is not intended to be d 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 ponding 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 restriction sites so as to obtain a PCR product.
Forward : 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 XOGC/dhfr to prepare a recombinant vector XOGC/GLPlR.
CHO DG44 cell line cultured in DMEM/Fl2 (10% FBS) medium was transfected with the recombinant vector XOGC/GLPlR 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 que, 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 on PCR was performed using a region con‘esponding to ORF in CDNA (OriGene Tech- nologies, Inc. USA) of human glucagon or gene as a template, and the following forward and reverse primers including each of the EcoRI and XhoI restriction sites so as to obtain a PCR product.
Forward primer: 5'—CAGCGACACCGACCGTCCCCCCGTACTTAAGGCC—3'(SEQ ID NO. 49) Reverse primer: 5'-CTAACCGACTCTCGGGGAAGACTGAGCTCGCC—3'(SEQ ID NO. 50) The PCR product was cloned into the known animal cell expression vector XOGC/dhfr to prepare a recombinant vector xOGC/GCGR.
CHO DG44 cell line cultured in DMEM/Fl2 (10% FBS) medium was transfected with the recombinant vector XOGC/GCGR using Lipofectarnine, and cultured in a selection medium containing 1 mg/mL G418 and 10 11M methotraxate. Single clone cell lines were selected therefrom by a limit dilution que, and a cell line showing excellent CAMP se to glucagon in a concentration-dependent manner was finally selected therefrom.
Example 2. Test on in vitro activity of oxyntomodulin derivatives Example 2-1: Synthesis of modulin derivatives In order to measure in vitro activities of oxyntomodulin derivatives, oxyntomodulin derivatives having the following amino acid sequences were synthesized (Table 1).
Table 1 [Table 1] Oxyntomodulin and oxyntomodulin delivatives SEQ ID NO. 1 HSQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO. 2 CA—SQGTFTSDYSKYLDEEAVRLFIEWLMNTKRNRNNIA SEQ ID NO. 3 CA-SQGTFTSDYSKYLDERRAQDFVAWLKNTGPSSGAPPP SEQ ID NO. 4 CA-GQGTFTSDYSRYLEEEAVRLFIEWLKNGGPSSGAPPPS SEQ ID NO. 5 CA—GQGTFTSDYSRQMEEEAVRLFIEWLKNGGPSSGAPPP SEQ ID NO. 6 TFTSDLSRQMEEEAVRLFIEWAAHSQGTFTSDYS KYLD SEQ ID NO. 7 CA—SQGTFTSDYSRYLDEEAVRLFIEWLMNTK SEQ ID NO. 8 TFTSDLSRQLEEEAVRLFIEWLMNK SEQ ID NO. 9 CA~GQGTFTSDYSRYLDEEAVXLFIEWLMNTKRNRNNIA SEQ ID NO. 10 CA-SQGTFTSDYSRQMEEEAVRLFIEWLMNGGPSSGAPPP SEQ ID NO. 11 CA—GEGTFTSDLSRQMEEEAVRLFIEWAAHSQGTFTSDYS RYLDK SEQ ID NO. 12 CA—SQGTFTSDYSRYLDGGGHGEGTFTSDLSKQMEEEAV SEQ ID NO. 13 CA—SQGTFTSDYSRYLDXEAVXLFIEWLMNTK SEQ ID NO. 14 CA—GQGTFTSDYSRYLDEEAVXLFIXWLMNTKRNRNNIA SEQ ID NO. 15 CA—GQGTFTSDYSRYLDEEAVRLFIXWLMNTKRNRNNIA SEQ ID NO. 16 CA—SQGTFTSDLSRQLEGGGHSQGTFTSDLSRQLEK SEQ ID NO. 17 CA—SQGTFTSDYSRYLDEEAVRLFIEWIRNTKRNRNNIA SEQ ID NO. 18 CA—SQGTFTSDYSRYLDEEAVRLFIEWIRNGGPSSGAPPPS SEQ ID NO. 19 CA-SQGTFTSDYSRYLD _E_ EAV _E_ LFIEWIRN— TKRNRNNIA SEQ ID NO. 20 CA—SQGTFTSDYSRYLD _E EAV E LFIEWIRNGG— PSSGAPPPSK W0 2012/173422 SEQ ID NO. 25 HAibQGTFTSDYSKYLD E KRA K EFVQWLMNTC SEQ ID NO. 26 HAibQGTFTSDYS K YLD E KRAKEFVQWLMNTC SEQ ID NO. 27 HAibQGTFTSDYSKYLD E QAA E EFICWLMNT SEQ ID NO. 28 HAibQGTFTSDYSKYLDEKRAKEFVQWLMNT SEQ ID NO. 29 H(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO. 30 CA—SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNNIA SEQ ID NO. 31 CA-(d)SQGTFTSDYSKYLDSRRAQDFVQWLMNTKRNRNN SEQ ID NO. 32 QGTFTSDYSKYLDEKRAKEFVQWLMNTC SEQ ID NO. 33 HAibQGTFTSDYAKYLDEKRAKEFVQWLMNTC SEQ ID NO. 34 YAibQGTFTSDYSKYLDEKRAKEFVQWLMNTC In Table 1, amino acids in bold and underlined represent ring formation, and amino acids represented by X mean a non-native amino acid, alpha—methyl—glutamic acid. In addition, CA represents 4—imidazoacetyl, and DA represents desamino-histidyl.
Example 2-2: Test on in vitro activity of oxyntomodulin derivatives In order to measure besity efficacies of the oxyntomodulin derivatives syn ed 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 s human GLP—1 receptor gene and glucagon receptor gene, respectively. Thus, they are suitable to measure GLP-1 and glucagon activities. Therefore, the activity of each oxyntomodulin derivative was measured using each transformed cell line.
Specifically, each cell line was sub—cultured twice or three time a week, and aliquoted in each well of a 96—well plate at a density of 1 X 105, ed by cul- on for 24 hours.
The cultured cells were washed with KRB buffer and suspended in 40 ml of KRB WO 73422 buffer ning 1 mM IBMX, and left at room temperature for 5 minutes. Oxyn— tomodulin (SEQ ID NO. 1) and oxyntomodulin derivatives (represented 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°C for 1 hour in a CO; 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. ECSO values were calculated therefrom, and compared to each other. ECSO values are shown in the following Table Table 2 W0 2012/173422 [Table 2] Comparison of in vitro activities for GLP—1 receptor and glucagon receptor between modulin and oxyntomodulin derivatives SEQ ID No. 2 51.8 SEQ ID No. 3 >1,000 SEQ ID 13 SEQ ID NO. 24 1.43 6.95 SEQ ID NO. 25 As shown in Table 2, there were oxyntomodulin derivatives showing 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 oxyntomodulin activates both the GLP—1 or and glucagon receptor to suppress appetite, facilitate sis, and e satiety, thereby showing anti—obesity effects. The oxyntomodulin derivatives according to the present 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 oxyntomodulin.
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 ic ob/ob mice, commonly used to test the efficacies of therapeutic agents for y and diabetes, were fasted for 16 hours, and administered with l or 10 mg/kg of oxyntomodulin, or 0.02, 0.1, 1 or 10 mg/kg of the oxyn- tomodulin derivative of SEQ ID NO. 2. Then, food intake was examined for 2 hours (. is a graph showing changes in food intake according to administration dose of oxyntomodulin or oxyntomodulin derivative. As shown in admin— istration of 1 mg/kg of oxyntomodulin derivative showed more ent inhibitory effects on food intake than administration of 10 mg/kg of oxyntomodulin.
Taken together, the modulin derivatives of the t invention have much higher anti—obesity s 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 sequence of oxyntomodulin (SEQ ID NO. 1) with 3.4 K PropionALD(2) PEG (PEG with two propylaldehyde groups, NOF, Japan), the oxyntomodulin and 3.4 K PropionALD(2) PEG were d 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 cyanoborohydride (cyanoborohydride (SCB, NaCNBH3), NaCNBH3) was added thereto as a reducing agent. After tion of the reaction, the reaction mixture was d to a SOURCE S (XK16, am Biosciences) to purify modulin having mono-pegylated lysine (column: SOURCE S (XK16, Amersham Biosciences), flow rate: 2.0 nil/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 h a SOURCE S pu- rification 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.
Next, the purified EGylated oxyntomodulin and globulin Fc were reacted at a molar ratio of 1 : 10 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was d to a SOURCE 15Q pu— ion column to purify conjugates including modulin and immunoglobulin Fc (column: SOURCE 15Q (XK16, Amersham Biosciences), flow rate: 2.0 , gradient: A 0 —> 20% 100 min B (A: 20mM Tris-HCl, pH 7.5, B: A + 1M NaCl)) (). is a graph g the result of purifying conjugates including oxyn- tomodulin and immunoglobulin Fc.
Example 5: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 29) and immunoglobulin Fc Firstly, for PEGylation of lysine residue at on 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 29) with 3.4 K PropionALD(2) PEG, the oxyntomodulin derivative (SEQ ID NO. 29) and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of l : 12 with the protein concentration of 5 mg/ml at 4°C for 4.5 hours. At this time, the reaction was conducted in a solvent mixture of 100 mM Na—Borate buffer (pH 9.0) and 45% isopropanol, and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE Sto purify the oxyntomodulin derivative having mono—pegylated lysine (Column: SOURCE S, flow rate: 2.0 ml/rnin, gradient: A 0 —)3% l min B —> 40% 222 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KC1)) (). is a graph showing the result of purifying a EGylated oxyntomodulin derivative (SEQ ID NO. 29) through a SOURCE S purification column.
Next, the d EGylated oxyntomodulin derivative (SEQ ID NO. 29) 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 mixture was applied to a SOURCE lSQ purification column to purify conjugates including oxyntomodulin tive (SEQ ID NO. 29) and immunoglobulin FC (column: SOURCE lSQ, flow rate: 2.0 ml/min, gradient: A 0 —9 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 modulin derivative (SEQ ID NO. 29) and immunoglobulin Fc.
Example 6: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 30) and immunoglobulin 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 tive (SEQ ID NO. 30) and 3.4 K PropionALD(2) PEG were d 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 on, 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. 30) through a SOURCE S purification column. Mono-PEGylation of the eluted peaks was ed by SDS-PAGE, and lysine ivity was ed 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 d 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 ate 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 derivative (SEQ ID NO. 30) and immunoglobulin Fc (column: SOURCE lSQ, 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. 30) and immunoglobulin Fc. e 7: Preparation of conjugates including oxyntomodulin 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. 3]) with 3.4 K PropionALD(2) PEG, the oxyntomodulin derivative (SEQ ID NO. 31) and 3.4 K PropionALD(2) PEG were reacted at a molar ratio of l : 15 with the protein concentration of 3 mg/ml at 4°C for 4.5 hours. At this time, the reaction was ted in a solvent mixture of 100 mM HEPES buffer (pH 7.5) and 45% isopropanol, and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin tive having mono— pegylated lysine (Column: SOURCE S, flow rate: 2.0 ml/min, gradient: A 0 —>3% 1 min B —> 40% 222 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KCD) (). is a graph showing the result of purifying a mono—PEGylated oxyntomodulin tive (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 d 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. 31) and immunoglobulin Fc (column: SOURCE lSQ, flow rate: 2.0 nil/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 oxyntomodulin derivative (SEQ ID NO. 31) and globulin Fc.
Example 8: Preparation of conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc y, 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 1 : 10 with the protein concentration of 3 mg/ml at 4°C for 4 hours. At this time, the reaction was conducted in a solvent 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 ication 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% l, B: A + 1M KC1)) (). is a graph showing the result of purifying a EGylated oxyntomodulin derivative (SEQ ID NO. 2) through a SOURCE S purification column.
EGylation of the eluted peaks was examined by SDS-PAGE, and lysine se— lectivity was examined by e mapping using Asp—N protease (). is a graph showing the result of peptide g of purified mono—PEGylated oxyn— tomodulin derivative (SEQ ID NO. 2).
Next, the purified mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc were reacted at a molar ratio of 1 : 8 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was conducted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After tion of the reaction, the reaction e was applied to a SOURCE 15Q purification column (Column : SOURCE 15Q, flow rate : 2.0 mllmin, gradient : A 0 —> 4% 1 min B —~> 20% 80 min B (A: 20mM Tiis-HCl, pH 7.5, B: A + 1M NaCl)) () and a Source ISO purification column (Column: SOURCE ISO (XK16, Amersham Biosciences), flow rate : 2.0 ml/min, gradient: A 0 —> 100% 100 min B, (A: 20mM Tris—HCl, pH 7.5, B: A + 1.3M AS))() to purify conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc. is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 2) and immunoglobulin Fc through a Source ISO purification column, and is a graph showing the result of ing conjugates including modulin derivative (SEQ ID NO. 2) and immunoglobulin Fc h a Source ISO ation 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 tration 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 o as a ng 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 KC1)) (). is a graph showing the result of purifying a mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 3) h a SOURCE S purification column.
Mono—PEGyIation 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. 3).
Next, the purified mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 3) and 2012/004722 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 ml/min, gradient : B 0 -—> 100% 5 min A(A: 20mM Tris—HCl, pH 7.5, B: A + 1.5M NaCl)) () and a SOURCE 15Q cation 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)) () to purify conjugates including oxyn- tomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc. is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 3) and immunoglobulin Fc through a Butyl FF purification column, and is a graph showing the result of ing conjugates including oxyntomodulin tive (SEQ ID NO. 3) and immunoglobulin Fc through a SOURCE 15Q pu— rification column.
Example 10: Preparation of conjugates including modulin derivative (SEQ ID NO. 23) and globulin Fc Firstly, for PEGylation of cysteine residue at position 24 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 23) with MAL-10K-ALD PEG (NOF., Japan), the oxyntomodulin derivative (SEQ ID NO. 23) 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 45% panol, and 1M guanidine was added thereto. After tion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin tive having mono-pegylated cysteine (column: SOURCE S, flow rate: 2.0 m1/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. 23) through a SOURCE S pu1ification column.
Next, the purified mono-PEGylated oxyntomodulin derivative (SEQ ID NO. 23) and immunoglobulin Fc were reacted at a molar ratio of l : 5 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the 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 mixture was applied to 3.
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—HCI, 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)) (FIG. SC) to purify conjugates ing modulin tive (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 .
Example 11: Preparation of ates including oxyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at position 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 24) with MAL~10K~ALD PEG, the oxyn— tomodulin derivative (SEQ ID NO. 24) and MAL~lOK-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 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 modulin derivative (SEQ ID NO. 24) through a SOURCE S purification .
Next, the purified mono~PEGylated oxyntomodulin tive (SEQ ID NO. 24) and immunoglobulin Fc were reacted at a molar ratio of 1 : 5 with the protein concentration of 20 mg/ml at 4°C for 16 hours. At this time, the reaction was ted in 100 mM potassium phosphate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE 15Q purification column (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A O —> 4% l min B —> 20% 80 min B(A: 20mM Tris-HCl, pH 7.5, B: A + 1M NaCl)) () and a Source ISO purification column (column: SOURCE ISO, flow rate: 2.0 ml/min, gradient: B 0 ——> 100% 100 min A, (A: 20mM Tris—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 ing oxyntomodulin derivative (SEQ ID NO. 24) and im- munoglobulin Fc through a SOURCE 15Q purification column, and is a graph showing the result of purifying ates including oxyntomodulin derivative (SEQ ID NO. 24) and immunoglobulin Fc through a Source ISO purification column.
Example 12: Preparation of conjugates including oxyntomodulin tive (SEQ ID NO. 25) and immunoglobulin Fc y, for PEGylation of cysteine e at position 30 of the amino acid sequence of modulin derivative (SEQ ID NO. 25) with MAL- lOK-ALD PEG, the oxyn— tomodulin derivative (SEQ ID NO. 25) and MAL—lOK—ALD PEG were reacted at a molar ratio of l : 3 with the protein concentration of 3 mg/ml at room temperature for 3 hours. At this time, the reaction was conducted in 50 mM Tris buffer (pH 8.0) and 1M guanidine was added thereto. After completion of the reaction, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono—pegylated cysteine (column: SOURCE S, flow rate: 2.0 ml/ min, gradient: A 0 ——>100% 50 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KCl)) (a). a is a graph showing the result of purifying a mono— PEGylated oxyntomodulin derivative (SEQ ID NO. 25) through a SOURCE S pu— rification .
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 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 e was applied to a SOURCE 15Q ation column n: 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)) (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 ates including oxyntomodulin derivative (SEQ ID NO. 25) and immunoglobulin Fc. b is a graph showing the result of purifying conjugates including oxyntomodulin derivative (SEQ ID NO. 25) and im— munoglobulin Fc through a SOURCE 15Q purification column, and c is a graph showing the result of purifying conjugates including modulin derivative (SEQ ID NO. 25) and immunoglobulin Fc h a Source ISO purification column. e 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-10K-ALD PEG were reacted at a molar ratio of l : 5 with the protein concentration of 3 mg/rnl 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 completion of the reaction, the reaction mixture was d 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 Net-citrate, pH 3.0 + 45% ethanol, B: A + 1M KC1)) (a). a is a graph showing the result of purifying a mono-PEGylated oxyntornodulin 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 111M SCB was added thereto as a reducing agent. After completion of the on, the reaction mixture was applied to 21 SOURCE 15Q purification column (column: SOURCE 15Q, flow rate: 2.0 nil/min, gradient: A 0 —> 4% 1 min B —> 20% 80 min B(A: 20mM Tris-HCI, 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-HCI, pH 7.5, B: A + 1.1M AS)) (c) to purify conjugates including oxyntomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc. b is a graph showing the result of purifying ates including oxyntomodulin derivative (SEQ ID NO. 28) and im— munoglobulin Fc through a SOURCE 15Q purification column, and c is a graph g the result of ing conjugates including oxyntomodulin derivative (SEQ ID NO. 28) and immunoglobulin Fc through a Source ISO purification .
Example 14: Preparation of conjugates ing oxyntomodulin derivative (SEQ ID NO. 32) and immunoglobulin Fc Firstly, for PEGylation of cysteine residue at position 30 of the amino acid sequence of oxyntomodulin tive (SEQ ID NO. 32) with K—ALD PEG, the oxyn— tomodulin 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 thereto. After completion of the reaction, the on mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono-pegylated ne (column: SOURCE S, flow rate: 2.0 ml/ min, nt: 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. 32) 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 SOURCE 15Q purification column (column: SOURCE 15Q, flow rate: 2.0 , gradient: A 0 —> 4% l min B —> 20% 80 min B(A: 20mM Tris-HCI, pH 7.5, B: A + 1M NaCl)) and a Source ISO purification column n: SOURCE ISO, flow rate: 2.0 , gradient: B 0 —> 100% 100 min A, (A: 20mM Tris—HCl, pH 7.5, B: A + 1.1M AS)) to purify ates including oxyntomodulin derivative (SEQ ID NO. 32) and immunoglobulin Fc.
Example 15: Preparation of conjugates including modulin derivative (SEQ ID NO. 33) and globulin Fe Firstly, for PEGylation of cysteine residue at position 30 of the amino acid sequence of oxyntomodulin derivative (SEQ ID NO. 33) with MAL—10K—ALD PEG, the oxyn— tomodulin derivative (SEQ ID NO. 33) and MAL-IOK—ALD PEG were reacted at a molar ratio of 1 : 1 with the 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 mono-pegylated cysteine (column: SOURCE S, flow rate: 2.0 ml/ min, gradient: A 0 —>100% 50 min B (A: 20mM Na—citrate, pH 3.0 + 45% ethanol, B: A + 1M KC1)).
Next, the purified mono—PEGylated oxyntomodulin derivative (SEQ ID NO. 33) and globulin 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 conducted in 100 mM potassium ate buffer (pH 6.0) and 20 mM SCB was added thereto as a reducing agent. After completion of the reaction, the reaction mixture was applied to a SOURCE 15Q purification column (column: SOURCE 15Q, flow rate: 2.0 ml/min, gradient: A 0 —> 4% 1 min B —> 20% 80 min B(A: 20mM Tn’s-HCl, pH 7.5, B: A + 1M NaCl)) and a Source ISO purification column (column: SOURCE ISO, flow rate: 2.0 ml/min, gradient: B 0 —} 100% 100 min A, (A: 20mM Tris~HCl, pH 7.5, B: A + 1.1M AS)) to purify conjugates ing oxyntomodulin derivative (SEQ ID NO. 33) and immunoglobulin Fc.
Example 16: Preparation of conjugates including oxyntomodulin tive (SEQ ID NO. 34) and immunoglobulin Fc Firstly, for PEGylation of cysteine e 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—IOK—ALD PEG were reacted at a molar ratio of l : l with the n 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 ine was added thereto. After completion of the on, the reaction mixture was applied to a SOURCE Spurification column to purify the oxyntomodulin derivative having mono-pegylated cysteine (column: SOURCE S, flow rate: 2.0 ml/ min, gradient: A 0 ——>100% 50 min B (A: 20le 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 reacted at a molar ratio of 1 : 5 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 on, 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 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 derivative (SEQ ID NO. 34) and immunoglobulin Fc.
Example 17: In vitro activity of oxyntomodulin derivative-immunoglobulin Fc ates 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 Examples, experiments were performed in the same manner as in Example 2—2.
Specifically, each of the transformants prepared in Examples 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 l X 10‘, followed by cultivation for 24 hours. Each of the cultured trans— ts was washed with KRB buffer and suspended in 40 ml of KRB buffer containing 1 mM IBMX, and left at room ature for 5 s. 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 ed 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 n Elmer, USA). EC50 values were calculated therefrom, and compared to each other (Table 3).
Table 3 [Table 3] In vitro activity of oxyntomodulin delivative—immunoglobulin Fc conjugates As shown in Table 3, the oxyntomodulin derivative—immunoglobulin Fc conjugates were found to show the in Vitro activity to GLP-l and glucagon receptors.
Example 18: In vivo activity of oxyntomodulin derivative-immunoglobulin conjugates It was examined r the oxyntomodulin derivative—immunoglobulin Fc conjugates show excellent body weight—reducing s 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 den'vative (SEQ ID NO. 23, 24 or )-immunoglobulin Fc ates at a dose of 0.03 or 0.06 mg/kg/week for 3 weeks. fter, changes in the body weight of the mice were measured ( and ). and are graphs showing changes in body weight of mice ing to the type and administration dose of oxyntomodulin derivative-im- munoglobulin Fc conjugates. As shown in and , as the administration dose of the oxyntomoduiin derivative-immunoglobulin Fc conjugates was increased, 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—imrnunoglobulin Fc ates reduce the body weight in a dose—dependent manner.
WO 73422

Claims (20)

    Claims
  1. A conjugate comprising an oxyntomodulin derivative, an immunoglobulin Fc region, and non-peptidyl polymer wherein the modulin derivative is covalently linked to immunoglobulin Fc region via non—peptidyl polymer, and wherein the oxyntomodulin derivative comprising the amino acid sequence selected from SEQ ID NOs: 24-26 and 28.
  2. The conjugate ing to claim 1, n the oxyntomodulin derivative is capable of activating GLP-1 receptor and glucagon receptor.
  3. The conjugate according to claim 1 or claim 2, wherein the conjugate has anti-obesity effects.
  4. The conjugate according to any one of claims 1 to 3, n the oxyntomodulin derivative comprises SEQ ID NO: 25 or 26,
  5. The conjugate according to any one of claims 1 to 4, wherein the non-peptidyl polymer is selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol and propylene glycol, polyoxyethylated s, polyvinyl alcohol, polysaccharides, dextran, nyl ethyl ether, polylactic acid (PLA), ctic-glycolic acid (PLGA), lipid rs, chitins, onic acid, polysaccharide and combinations thereof.
  6. The conjugate according to any one of claims 1 to 5, wherein one functional group selected form the group consisting of an amine group and a thiol group of the immunoglobulin Fc region is attached to one end of the non-peptidyl polymer, and one functional group selected from the group consisting of an amine group and a thiol group of the oxyntomodulin derivative is ed to the other end of the non—peptidyl polymer.
  7. The conjugate according to any one of claims 1 to 6, wherein the conjugate is prepared by covalently linking an oxyntomodulin derivative to an immunoglobulin Fc region via a non—peptidyl polymer, the non—peptidyl polymer having, prior to forming the conjugate, reactive functional groups at both ends.
  8. The conjugate according to claim 7, wherein the reactive group is selected from the group consisting of an aldehyde group, a naldehyde group, a ldehyde group, a maleimide group and a succinimide derivative.
  9. The conjugate according to claim 7, wherein the reactive groups at both ends are the same as or different from each other.
  10. 10. The conjugate according to any one of claims 1 to 9, wherein the immunoglobulin Fc region is a non-glycosylated Fc region.
  11. ll. The conjugate according to any one of claims 1 to 10, wherein the immunoglobulin Fc region is selected from the group consisting of a CH1 domain, a CH2 domain, a CH3 domain and a CH4 ; a CH1 domain and a CH2 domain; a CH1 domain and a CH3 domain; a CH2 domain and a CH3 domain; a combination of one or more domains and an immunoglobulin hinge region (or a portion of the hinge region); and a dimer of each domain of the heavy-chain nt s and the light—chain constant region.
  12. 12. The conjugate according to any one of claims 1 to 11, wherein the immunoglobulin Fc region is a derivative in which a region capable of forming a disulfide bond is deleted, certain amino acid residues are eliminated at the N-terminal end of a native Fc form, a methionine residue is added at the N—terminal end of a native Fc form, a complement-binding site is d, or an antibody dependent cell mediated cytotoxicity (ADCC) site is deleted.
  13. 13. The conjugate ing to any one of claims 1 to 12, wherein the immunoglobulin Fc region is an Fc region d from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, lgE, and IgM.
  14. 14. The conjugate according to claim 13, wherein the immunoglobulin Fc region is an IgG4 Fc region.
  15. 15. The conjugate according to claim 13, n the immunoglobulin Fc region is a human IgG4—derived non-glycosylated Fc region.
  16. 16. The conjugate ing to any one of claims 1 to 15, wherein the non—peptidyl polymer is linked via a covalent bond to the oxyntomodulin derivative at the cystein residue of the amino acid sequence of SEQ ID NO:24.
  17. 17. A pharmaceutical composition for the prevention or treatment of obesity, comprising the conjugate of any one of claims 1 to 16.
  18. 18. The pharmaceutical composition according to claim 17, further comprising a pharmaceutically acceptable carrier.
  19. 19. The ceutical composition according to claim 17, wherein the composition is administered alone or in combination with other pharmaceutical ations showing prophylactic or therapeutic effects on obesity.
  20. 20. The pharmaceutical composition according to claim 19, wherein the pharmaceutical formulation is selected from the group consisting of a GLP-1 receptor agonist, a leptin receptor agonist, a DPP-IV inhibitor, a Y5 receptor antagonist, a Melaninconcentrating hormone (MCH) receptor antagonist, a Y
NZ618811A 2011-06-17 2012-06-15 A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof NZ618811B2 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
NZ718999A NZ718999B2 (en) 2011-06-17 2012-06-15 A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
KR20110058852 2011-06-17
KR10-2011-0058852 2011-06-17
PCT/KR2012/004722 WO2012173422A1 (en) 2011-06-17 2012-06-15 A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof

Publications (2)

Publication Number Publication Date
NZ618811A NZ618811A (en) 2016-05-27
NZ618811B2 true NZ618811B2 (en) 2016-08-30

Family

ID=

Similar Documents

Publication Publication Date Title
US11872283B2 (en) Conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
KR102395856B1 (en) glucagon derivative, a conjugate thereof, and a composition comprising the same, and a therapeutic use thereof
EP3322437B1 (en) Glucagon derivative and a composition comprising a long acting conjugate of the same
KR20140058104A (en) Composition for treating diabetes or obesity diabetes comprising oxytomodulin derivative
NZ618811B2 (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
HK40009561B (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
HK40009561A (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
NZ718999B2 (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
NZ742400B2 (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
NZ733478B2 (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
HK40003883B (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
HK40003883A (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof
HK1248129B (en) Glucagon derivative and a composition comprising a long acting conjugate of the same
HK1193828A (en) A conjugate comprising oxyntomodulin and an immunoglobulin fragment, and use thereof