NZ751062B2 - Novel insulin analog and use thereof - Google Patents

Abstract

The present invention relates to an insulin analog conjugate, the insulin analogue having a reduced insulin titer and a reduced insulin receptor binding affinity compared to the native form for the purpose of increasing the blood half-life of insulin, the conjugate prepared by linking the insulin analog and a carrier, a long-acting formulation including the conjugate, and a method for preparing the conjugate. alog and a carrier, a long-acting formulation including the conjugate, and a method for preparing the conjugate.

Description

[DESCRIPTION] [Invention Title] Novel insulin analog and use thereof [Technical Field] The present invention s to an insulin analog that has a d insulin titer and a reduced insulin receptor binding ty compared to the native form for the purpose of increasing the blood half-life of insulin, a conjugate prepared by linking the insulin analog and a carrier, a longacting formulation including the ate, and a method for preparing the conjugate.

[Background Art] In vivo proteins are known to be eliminated via various routes, such as degradation by proteolytic s in blood, excretion through the kidney, or clearance by receptors. Thus, many efforts have been made to improve therapeutic efficacy by avoiding the protein clearance mechanisms and increasing halflife of physiologically active proteins.

On the other hand, insulin is a e secreted by the pancreas of the human body, which regulates blood glucose levels, and plays a role in maintaining normal blood glucose levels while carrying surplus glucose in the blood to cells to provide energy for cells. In diabetic patients, however, n does not function properly due to lack of insulin, resistance to insulin, and loss of beta-cell function, and thus glucose in the blood cannot be utilized as an energy source and the blood glucose level is ed, leading to hyperglycemia. Eventually, urinary excretion occurs, contributing to development of various complications.

Therefore, insulin y is essential for patients with abnormal insulin secretion (Type I) or insulin resistance (Type II), and blood glucose levels can be normally regulated by insulin administration. However, like other protein and peptide hormones, insulin has a very short in vivo ife, and thus has a disadvantage of repeated administration. Such frequent administration causes severe pain and discomfort for the patients. For this reason, in order to improve y of life by increasing in vivo half-life of the protein and ng the administration frequency, many studies on protein ation and chemical conjugation (fatty acid conjugate, polyethylene polymer conjugate) have been conducted.

Commercially available long-acting insulin es insulin glargine manufactured by Sanofi Aventis (lantus, lasting for about 20-22 hours), and insulin detemir (levemir, lasting for about 18-22 hours) and tresiba (degludec, lasting for about 40 hours) manufactured by Novo Nordisk. These long-acting insulin formulations produce no peak in the blood insulin concentration, and thus they are le as basal insulin.

However, because these formulations do not have sufficiently long ife, the antage of one or two injections per day still remains. Accordingly, there is a limitation in achieving the intended goal that administration frequency is remarkably reduced to improve convenience of diabetic patients in need of long-term administration.

The previous research ed a specific in vivo insulin clearance process; 50% or more of insulin is removed in the kidney and the rest is removed via a or mediated nce (RMC) process in target sites such as muscle, fat, liver, etc.

In this regard, many studies, including J Pharmacol Exp Ther (1998) 286: 959, Diabetes Care (1990) 13: 923, Diabetes (1990) 39: 1033, have reported that in vitro activity is reduced to avoid RMC of insulin, thereby increasing the blood level. However, these insulin s having reduced receptor binding affinity cannot avoid renal clearance which is a main clearance mechanism, although RMC is reduced. Accordingly, there has been a limit in remarkably increasing the blood half-life.

Under this background, the present inventors have made many s to se the blood half-life of insulin. As a result, they found that a novel insulin analog having no native insulin sequence but a non-native insulin sequence shows a reduced in-vitro titer and a d insulin receptor binding affinity, and therefore, its renal clearance can be reduced. They also found that the blood half-life of insulin can be r increased by linking the insulin analog to an immunoglobulin Fc nt as a representative carrier effective for half-life improvement, thereby completing the present invention.

[Disclosure] [Technical Problem] An object of the present invention is to provide an insulin analog that is prepared to have a reduced in-vitro titer for the purpose of prolonging in vivo half-life of insulin, and a conjugate prepared by linking a carrier thereto.

Specifically, one object of the present invention is to provide an insulin analog having a reduced insulin titer, compared to the native form.

Another object of the present invention is to provide an n analog conjugate that is prepared by linking the insulin analog to the carrier.

Still r object of the present invention is to provide a cting insulin formulation including the insulin analog conjugate.

Still another object of the present ion is to provide a method for preparing the insulin analog conjugate.

Still another object of the present invention is to provide a method for increasing in vivo half-life using the insulin analog or the insulin analog conjugate prepared by linking the insulin analog to the carrier.

Still another object of the present invention is to provide a method for treating insulin-related diseases, including the step of stering the insulin analog or the insulin analog conjugate to a subject in need f.

[Technical Solution] In one aspect to achieve the above objects, the present ion provides an insulin analog having a reduced insulin titer, compared to the native form, in which an amino acid of B chain or A chain is modified.

In one specific embodiment, the present invention provides an insulin analog having a d insulin receptor binding affinity.

In another specific embodiment, the present invention provides an insulin analog conjugate, in which an insulin analog having a reduced insulin titer compared to the native form, n an amino acid in B chain or A chain of insulin is modified by substituting one amino acid ed from the group consisting of 8th amino acid, 23rd amino acid, 24th amino acid, and 25th amino acid of B chain and 1st amino acid, 2nd amino acid, and 19th amino acid of A chain with alanine; is linked to FcRn-binding al.

In another specific embodiment, the present invention provides a non-native insulin analog, in which one amino acid selected from the group consisting of 8th amino acid, 23th amino acid, 24th amino acid, and 25th amino acid of B chain and 1th amino acid, 2th amino acid, and 19th amino acid of A chain is substituted with alanine, or in which 14th amino acid of A chain is tuted with glutamic acid or asparagine in the insulin analog according to the present invention.

In still another specific embodiment, the present invention provides an n analog, in which the insulin analog according to the t invention is selected from the group consisting of SEQ ID NOs. 20, 22, 24, 26, 28, 30, 32, 34 and 36.

In another aspect, the present invention provides an insulin analog conjugate that is prepared by linking the above described insulin analog to a carrier capable of prolonging half-life.

In one specific ment, the present invention provides an insulin analog conjugate, in which the n analog conjugate is prepared by linking (i) the above described insulin analog and (ii) an immunoglobulin Fc region via (iii) a peptide linker or a non-peptidyl linker selected from the group consisting of hylene glycol, polypropylene glycol, copolymers of ethylene -propylene glycol, polyoxyethylated polyols, polyvinyl alcohols, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid, and combination thereof.

In still another aspect, the present invention provides a long-acting insulin formulation including the above described insulin analog conjugate, in which in vivo duration and stability are increased.

In one specific embodiment, the present invention provides a cting formulation that is used for the treatment of diabetes.

In another embodiment, the present invention provides a method for ing the above described insulin analog conjugate.

In still another specific embodiment, the present invention provides a method for increasing in vivo ife using the insulin analog or the insulin analog conjugate that is prepared by linking the n analog and the carrier.

In still another specific embodiment, the present invention provides a method for ng insulin-related diseases, including the step of administering the insulin analog or the insulin analog conjugate to a subject in need thereof.

[Advantageous Effects] A non-native insulin analog of the present invention has a reduced insulin titer and a reduced insulin receptor binding affinity, compared to the native form, and thus avoids in vivo nce mechanisms. Therefore, the insulin analog has increased blood half-life in vivo, and an insulin analogimmunoglobulin Fc conjugate prepared by using the same shows remarkably increased blood half-life, thereby improving convenience of ts in need of insulin administration.

[Description of Drawings] shows the result of analyzing purity of an insulin analog by protein electrophoresis, which is the result of the representative insulin analog, Analog No. 7 (Lane 1: size marker, Lane 2: native insulin, Lane 3: insulin analog (No. 7); shows the result of analyzing purity of an insulin analog by high pressure chromatography, which is the result of the representative insulin analog, Analog No. 7 ((A) RP-HPLC, (B) SE-HPLC); shows the result of peptide g of an insulin analog, which is the result of the representative insulin analog, Analog No. 7 ((A) native insulin, (B) insulin analog (No. 7)); shows the result of analyzing purity of an n analog-immunoglobulin Fc ate by protein electrophoresis, which is the result of the representative insulin analog, Analog No. 7 (Lane 1: size marker, Lane 2: insulin analog (No. 7)-immunoglobulin Fc conjugate); shows the result of analyzing purity of an insulin analog-immunoglobulin Fc conjugate by high pressure tography, which is the result of the representative insulin analog, Analog No. 7 ((A) RP-HPLC, (B) SE-HPLC, (C) IE-HPLC); and shows the result of analyzing pharmacokinetics of native insulin-immunoglobulin Fc conjugate and insulin immunoglobulin Fc ate in normal rats, which is the result of the entative insulin analog, Analog No. 7 (○: native insulin-immunoglobulin Fc conjugate (21.7 nmol/kg), ●: native insulin-immunoglobulin Fc conjugate (65.1 nmol/kg), □: n analog-immunoglobulin Fc conjugate (21.7 nmol/kg), ■: insulin analog-immunoglobulin Fc conjugate (65.1 nmol/kg). (A) native insulin-immunoglobulin Fc conjugate and insulin analog (No. 7)-immunoglobulin Fc conjugate, (B) native insulinimmunoglobulin Fc conjugate and n analog (No. 8)- immunoglobulin Fc conjugate, (C) native insulin-immunoglobulin Fc conjugate and insulin analog (No. 9)-immunoglobulin Fc conjugate).

[Best Mode] The present invention relates to an insulin analog having a reduced in-vitro titer. This insulin analog is characterized in that it has the non-native insulin sequence and therefore, has a reduced insulin or binding affinity, compared to the native insulin, and consequently, or-mediated clearance is remarkably reduced by increased dissociation constant, resulting in an increase in blood half-life.

As used herein, the term "insulin analog" includes various analogs having reduced insulin titer, compared to the native form.

The insulin analog may be an insulin analog having reduced insulin titer, compared to the native form, in which an amino acid of B chain or A chain of insulin is modified.

The amino acid sequences of the native insulin are as follows.

-A chain: Gly-Ile-Val-Glu-Gln-Cys-Cys-Thr-Ser-Ile-Cys-Ser-Leu-Tyr- u-Glu-Asn-Tyr-Cys-Asn (SEQ ID NO. 37) -B chain: Phe-Val-Asn-Gln-His-Leu-Cys-Gly-Ser-His-Leu-Val-Glu-Ala- r-Leu-Val-Cys-Gly-Glu-Arg-Gly-Phe-Phe-Tyr-Thr-Pro-Lys- Thr (SEQ ID NO. 38) The insulin analog used in Examples of the present invention is an insulin analog prepared by a genetic recombination technique. However, the present invention is not limited thereto, but includes all insulins having d invitro titer. Preferably, the insulin analog may include inverted insulins, insulin ts, insulin fragments or the like, and the preparation method may include a solid phase method as well as a genetic recombination que, but is not limited thereto.

The insulin analog is a peptide retaining a function of controlling blood glucose in the body, which is identical to that of insulin, and this peptide includes insulin agonists, derivatives, fragments, variants thereof or the like.

The insulin agonist of the present ion refers to a substance which is bound to the in vivo receptor of n to exhibit the same biological activities as insulin, regardless of the structure of insulin.

The insulin analog of the t invention denotes a peptide which shows a sequence gy of at least 80% in an amino acid sequence as compared to A chain or B chain of the native insulin, has some groups of amino acid residues altered in the form of chemical substitution (e.g., alpha-methylation, alpha-hydroxylation), removal (e.g., ation) or modification (e.g., ylation), and has a function of controlling blood glucose in the body. With respect to the objects of the present invention, the insulin analog is an insulin analog having a reduced insulin receptor binding affinity, compared to the native form, and insulin analogs having a d insulin titer compared to the native form are included without limitation.

As long as the insulin analog is able to exhibit low receptor-mediated internalization or receptor-mediated clearance, its type and size are not particularly limited. An insulin analog, of which major in vivo nce mechanism is the receptor-mediated internalization or receptor-mediated clearance, is suitable for the objects of the present invention.

The insulin fragment of the t invention denotes the type of insulin in which one or more amino acids are added or deleted, and the added amino acids may be non-native amino acids (e.g., D-type amino acid). Such insulin fragments retain the function of lling blood e in the body.

The insulin variant of the present invention denotes a peptide which differs from insulin in one or more amino acid sequences, and retains the function of controlling blood glucose in the body.

The respective methods for preparation of insulin agonists, derivatives, fragments and variants of the present invention can be used independently or in combination. For example, peptides of which one or more amino acid sequences differ from those of insulin and which have deamination at the amino-terminal amino acid residue and also have the function of lling blood e in the body are included in the present invention.

Specifically, the insulin analog may be an insulin analog in which one or more amino acids ed from the group consisting of 8th amino acid, 23th amino acid, 24th amino acid, and 25th amino acid of B chain and 1th amino acid, 2th amino acid, 14th amino acid and 19th amino acid of A chain are substituted with other amino acid, and preferably, in which one or more amino acids selected from the group consisting of 8th amino acid, 23th amino acid, 24th amino acid, and 25th amino acid of B chain and 1th amino acid, 2th amino acid, and 19th amino acid of A chain are tuted with alanine or in which 14 th amino acid of A chain is substituted with glutamic acid or asparagine. In addition, the insulin analog may be selected from the group ting of SEQ ID NOs. 20, 22, 24, 26, 28, , 32, 34 and 36, but may e any insulin analog having a reduced insulin receptor binding affinity without limitation.

According to one embodiment of the present invention, the insulin analogs of SEQ ID NOs. 20, 22, 24, 26, 28, 30, 32, 34 and 36, in particular, the representative insulin analogs, 7, 8, and 9 (SEQ ID NOs. 32, 34, and 36) were found to have reduced insulin receptor binding affinity in vitro, compared to the native form (Table 4).

In another aspect, the present invention provides an insulin analog conjugate that is prepared by g the insulin analog and a carrier.

As used herein, the term "carrier" denotes a substance capable of increasing in vivo ife of the linked insulin analog. The n analog according to the present invention is characterized in that it has a remarkably reduced insulin receptor binding ty, compared to the native form, and avoids receptor-mediated clearance or renal clearance.

Therefore, if a carrier known to increase in vivo half-life when linked to the known various physiologically active polypeptides is linked with the insulin analog, it is apparent that in vivo half-life can be improved and the resulting conjugate can be used as a cting formulation.

For example, because half-life improvement is the first priority, the carrier to be linked with the novel insulin having a reduced titer is not limited to the immunoglobulin Fc . The carrier includes a biocompatible material that is able to g in vivo half-life by linking it with any one biocompatible material, capable of ng renal clearance, selected from the group consisting of various polymers (e.g., polyethylene glycol and fatty acid, n and fragments thereof, particular amino acid ce, etc.), albumin and fragments thereof, albumin-binding materials, and polymers of repeating units of particular amino acid sequence, antibody, antibody nts, FcRn-binding materials, in vivo connective tissue or derivatives thereof, nucleotide, fibronectin, transferrin, saccharide, and polymers, but is not limited thereto. In addition, the method for linking the biocompatible material capable of prolonging in vivo half-life to the insulin analog having a reduced titer es genetic recombination, in vitro conjugation or the like. Examples of the biocompatible material may include an FcRn-binding material, fatty acid, polyethylene glycol, an amino acid fragment, or albumin. The FcRn-binding material may be an globulin Fc .

The insulin analog and the biocompatible material as the carrier may be linked to each other via a peptide or a nonpeptidyl polymer as a linker.

The n conjugate may be an insulin analog conjugate that is prepared by linking (i) the insulin analog and (ii) the immunoglobulin Fc region via (iii) a peptide linker or a non-peptidyl linker selected from the group consisting of hylene glycol, polypropylene glycol, copolymers of ethylene glycol-propylene glycol, polyoxyethylated polyol, polyvinyl l, polysaccharides, n, polyvinyl ethyl ether, biodegradable polymer, lipid polymers, chitins, hyaluronic acid and combination thereof.

In one specific embodiment of the insulin analog conjugate of the present invention, a non-peptidyl polymer as a linker is linked to the amino terminus of B chain of the n analog. In another specific embodiment of the conjugate of the present invention, a non-peptidyl polymer as a linker is linked to the residue of B chain of the insulin analog. The modification in A chain of insulin leads to a reduction in the activity and . In these embodiments, therefore, the non-peptidyl polymer as a linker is linked to B chain of insulin, thereby maintaining insulin activity and improving safety.

As used herein, the term "activity" means the ability of insulin to bind to the insulin receptor, and means that insulin binds to its or to exhibit its action. Such binding of the non-peptidyl polymer to the amino terminus of B chain of insulin of the present invention can be achieved by pH control, and the preferred pH range is 4.5 to 7.5.

As used herein, the term "N-terminus" can be used interchangeably with "N-terminal region".

In one specific e, the present inventors prepared an insulin analog-PEG-immunoglobulin Fc conjugate by linking PEG to the inus of an globulin Fc region, and ively coupling the N-terminus of B chain of insulin thereto. The serum half-life of this insulin analog-PEG- immunoglobulin Fc conjugate was increased, compared to nonconjugate , and it showed a hypoglycemic effect in disease animal models. Therefore, it is nt that a new longacting insulin ation maintaining in vivo activity can be prepared.

The immunoglobulin Fc region is safe for use as a drug carrier because it is a biodegradable polypeptide that is in vivo metabolized. Also, the immunoglobulin Fc region has a relatively low molecular weight, as compared to the whole immunoglobulin molecules, and thus, it is advantageous in terms of preparation, purification and yield of the conjugate.

The immunoglobulin Fc region does not contain a Fab fragment, which is highly non-homogenous due to different amino acid sequences ing to the antibody sses, and thus it can be expected that the immunoglobulin Fc region may greatly increase the homogeneity of substances and be less antigenic in blood.

As used herein, the term "immunoglobulin Fc region" refers to a protein that ns the heavy-chain constant region 2 (CH2) and the heavy-chain constant region 3 (CH3) of an immunoglobulin, excluding the variable regions of the heavy and light chains, the heavy-chain constant region 1 (CH1) and the light-chain constant region 1 (CL1) of the immunoglobulin.

It may further include a hinge region at the heavy-chain constant region. Also, the immunoglobulin Fc region of the t invention may contain a part or all of the Fc region including the chain nt region 1 (CH1) and/or the light-chain constant region 1 (CL1), except for the variable regions of the heavy and light chains of the immunoglobulin, as long as it has an effect ntially similar to or better than that of the native form. Also, it 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 include 1) a CH1 domain, a CH2 domain, a CH3 domain and a CH4 domain, 2) a CH1 domain and a CH2 domain, 3) a CH1 domain and a CH3 domain, 4) a CH2 domain and a CH3 domain, 5) a combination of one or more domains and an immunoglobulin hinge region (or a portion of the hinge ), and 6) a dimer of each domain of the heavy-chain constant regions and the light-chain constant . r, the immunoglobulin Fc region of the present invention includes a sequence derivative (mutant) thereof as well as a native amino acid sequence. An amino acid sequence derivative has a sequence that is different from the native amino acid ce due to a deletion, an insertion, a nonconservative or conservative substitution or combinations thereof of one or more amino acid residues. For example, in an IgG Fc, amino acid residues known to be important in binding, at positions 214 to 238, 297 to 299, 318 to 322, or 327 to 331, may be used as a suitable target for modification.

In addition, other various derivatives are possible, including derivatives having a deletion of a region capable of g a disulfide bond, a deletion of several amino acid residues at the N-terminus of a native Fc form, or an on of methionine residue to the N-terminus of a native Fc form.

Furthermore, to remove or functions, a deletion may occur in a complement-binding site, such as a nding site and an ADCC (antibody dependent cell mediated cytotoxicity) site. Techniques of preparing such sequence derivatives of the immunoglobulin Fc region are disclosed in WO 31 and WO 96/32478.

Amino acid exchanges in proteins and peptides, which do not generally alter the activity of molecules, are known in the art (H.Neurath, R.L.Hill, The Proteins, ic Press, New York, 1979). The most commonly occurring ges are Ala/Ser, Val/Ile, Asp/Glu, Thr/Ser, Ala/Gly, Ala/Thr, Ser/Asn, Ala/Val, Ser/Gly, Thy/Phe, Ala/Pro, Lys/Arg, Asp/Asn, Leu/Ile, Leu/Val, u, Asp/Gly, in both directions.

The Fc region, if d, may be modified by phosphorylation, sulfation, tion, glycosylation, methylation, farnesylation, acetylation, amidation or the like.

The aforementioned Fc derivatives are derivatives that have a biological activity identical to that of the Fc region of the present invention or improved structural stability against heat, pH, or the like.

In addition, these Fc regions may be obtained from native forms isolated from humans and other animals including cows, goats, swine, mice, rabbits, hamsters, rats and guinea pigs, or may be recombinants or derivatives thereof, obtained from transformed animal cells or microorganisms. Here, they may be obtained from a native immunoglobulin by isolating whole immunoglobulins from human or animal organisms and treating them with a proteolytic enzyme. Papain digests the native immunoglobulin into Fab and Fc regions, and pepsin treatment results in the production of pF’c and F(ab)2. These nts may be subjected to size-exclusion tography to isolate Fc or pF’c.

Preferably, a human-derived Fc region is a recombinant immunoglobulin Fc region that is obtained from a microorganism.

In on, the immunoglobulin Fc region may be in the form of having native sugar chains, increased sugar chains ed to a native form or decreased sugar chains compared to the native form, or maybe in a deglycosylated form. The increase, decrease or removal of the globulin Fc sugar chains may be achieved by methods common in the art, such as a chemical method, an enzymatic method and a genetic engineering method using a microorganism. Here, the removal of sugar chains from an Fc region results in a sharp decrease in binding affinity to the complement (c1q) and a decrease or loss in antibody-dependent cell-mediated cytotoxicity or complement-dependent xicity, thereby not inducing ssary immune responses in-vivo. In this regard, an immunoglobulin Fc region in a deglycosylated or aglycosylated form may be more suitable to the object of the present invention as a drug carrier.

The term "deglycosylation", as used herein, means to enzymatically remove sugar moieties from an Fc region, and the term "aglycosylation" means that an Fc region is produced in an unglycosylated form by a yote, preferably, E. coli.

On the other hand, the immunoglobulin Fc region may be derived from humans or other animals including cows, goats, swine, mice, rabbits, hamsters, rats and guinea pigs, and preferably humans. In addition, the immunoglobulin Fc region may be an Fc region that is d from IgG, IgA, IgD, IgE and IgM, or that is made by combinations thereof or hybrids f. ably, it is derived from IgG or IgM, which is among the most abundant proteins in human blood, and most preferably, from IgG which is known to e 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 ting of IgG Fc, IgA Fc, IgM Fc, IgD Fc and IgE Fc fragments.

The term "hybrid", as used herein, means that sequences encoding two or more immunoglobulin Fc regions of different origin are present in a single-chain immunoglobulin Fc region.

In the present invention, various types of hybrids are possible. That is, domain hybrids may be composed of one to four domains selected from the group consisting of CH1, CH2, CH3 and CH4 of IgG Fc, IgM Fc, IgA Fc, IgE Fc and IgD Fc, and may include the hinge .

On the other hand, IgG is divided into IgG1, IgG2, IgG3 and IgG4 subclasses, and the present invention includes combinations and hybrids f. Preferred are IgG2 and IgG4 subclasses, and most preferred is the Fc region of IgG4 rarely having effector functions such as CDC (complement dependent cytotoxicity). That is, as the drug carrier of the present invention, the most preferable globulin Fc region is a human IgG4-derived non-glycosylated Fc . The humanderived Fc region is more preferable than a non-human derived Fc region which may act as an antigen in the human body and cause undesirable immune responses such as the production of a new antibody against the n.

In the specific embodiment of the insulin analog conjugate, both ends of the non-peptidyl polymer may be linked to the N-terminus of the immunoglobulin Fc region and the amine group of the N-terminus of B chain of the insulin analog or the ε-amino group of the internal lysine residue or the thiol group of B chain, respectively.

The Fc region-linker-insulin analog of the present ion is made at various molar ratios. That is, the number of the Fc fragment and/or linker linked to a single insulin analog is not limited.

In addition, the linkage of the Fc region, a certain linker, and the insulin analog of the present invention may include all types of covalent bonds and all types of noncovalent bonds such as hydrogen bonds, ionic interactions, van der Waals forces and hydrophobic interactions when the Fc region and the insulin analog are sed as a fusion protein by genetic recombination. However, with respect to the physiological activity of the insulin analog, the e is preferably made by covalent bonds, but is not limited thereto.

On the other hand, the Fc region of the t invention, a n linker and the insulin analog may be linked to each other at an N-terminus or C-terminus, and preferably at a free group, and ally, a nt bond may be formed at an amino terminal end, an amino acid residue of lysine, an amino acid residue of histidine, or a free cysteine residue.

In addition, the linkage of the Fc region of the present invention, a certain linker, and the insulin analog may be made in a certain direction. That is, the linker may be linked to the N-terminus, the C-terminus or a free group of the immunoglobulin Fc region, and may also be linked to the N- terminus, the C-terminus or a free group of the insulin analog.

The non-peptidyl linker may be linked to the inal amine group of the immunoglobulin fragment, and is not limited to any of the lysine residue or cysteine residue of the immunoglobulin fragment sequence.

Further, in the specific embodiment of the insulin analog conjugate, the end of the non-peptidyl polymer may be linked to the internal amino acid residue or free ve group capable of binding to the reactive group at the end of the non-peptidyl polymer, in on to the N-terminus of the immunoglobulin Fc region, but is not limited thereto.

In the present invention, the non-peptidyl polymer means a patible polymer including two or more repeating units linked to each other, in which the repeating units are linked by any covalent bond excluding the peptide bond. Such nonpeptidyl polymer may have two ends or three ends.

The non-peptidyl polymer which can be used in the present invention may be selected from the group consisting of polyethylene , polypropylene glycol, copolymers of ethylene glycol and propylene , polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, nyl ethyl ether, biodegradable polymers such as PLA (poly(lactic acid)) and PLGA (polylactic-glycolic acid), lipid polymers, chitins, hyaluronic acid, and combinations thereof, and preferably, polyethylene glycol. The derivatives thereof well known in the art and being easily prepared within the skill of the art are also ed 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 blood half-life of the active drug by a carrier cannot be obtained as expected.

In the present invention, however, the conjugate can be ed using the non-peptidyl linker as well as the peptide linker. In the non-peptidyl linker, the polymer having resistance to the proteolytic enzyme can be used to maintain the blood 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 r having the aforementioned function, that is, a polymer having resistance to the in-vivo proteolytic enzyme. The non-peptidyl polymer has a molecular weight g from 1 to 100 kDa, and preferably, g from 1 to 20 kDa.

The non-peptidyl polymer of the present invention, linked to the immunoglobulin Fc region, may be one r or a ation of different types of polymers.

The non-peptidyl polymer used in the present invention has a ve group capable of binding to the immunoglobulin Fc region and the protein drug.

The ptidyl polymer has a reactive group at both ends, which is preferably selected from the group consisting of a reactive aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative. The succinimide derivative may be succinimidyl propionate, hydroxy succinimidyl, succinimidyl carboxymethyl, or succinimidyl carbonate. In particular, when the nonpeptidyl polymer has a reactive aldehyde group at both ends thereof, it is ive in g 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 de reactive group selectively binds to an N-terminus at a low pH, and binds to a lysine residue to form a covalent bond at a high pH, such as pH 9.0.

The reactive groups at both ends of the non-peptidyl polymer may be the same as or different from each other. For example, the non-peptide polymer may possess a maleimide group at one end, and an aldehyde group, a propionaldehyde group or a 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 polymer, the hydroxy group may be activated to various reactive groups by known al reactions, or a polyethylene glycol having a cially available modified reactive group may be used so as to e the single chain insulin analog conjugate of the present ion.

The insulin analog conjugate of the present invention maintains in vivo activities of the tional insulin such as energy metabolism and sugar metabolism, and also increases blood half-life of the insulin analog and markedly increases duration of in-vivo efficacy of the peptide, and therefore, the conjugate is useful in the treatment of es.

In one Example of the present invention, it was confirmed that the insulin analog having a reduced insulin or binding affinity exhibits much higher in vivo half-life than the native insulin conjugate, when linked to the carrier e of prolonging in vivo half-life (.

In another aspect, the present invention provides a longacting insulin formulation including the insulin analog conjugate. The long-acting insulin formulation may be a longacting insulin formulation having increased in vivo duration and stability. The long-acting formulation may be a pharmaceutical composition for the treatment of diabetes.

The pharmaceutical composition including the conjugate of the present invention may include ceutically acceptable carriers. For oral administration, the pharmaceutically able carrier may include a binder, a lubricant, a disintegrator, an excipient, a solubilizer, a sing agent, a stabilizer, a suspending agent, a coloring agent, a perfume or the like. For able ations, the pharmaceutically acceptable carrier may include a buffering agent, a preserving agent, an analgesic, a solubilizer, an isotonic agent, and a stabilizer. For preparations for topical administration, the pharmaceutically acceptable carrier may include a base, an excipient, a lubricant, a preserving agent or the like. The pharmaceutical composition of the present invention may be formulated into a variety of dosage forms in combination with the entioned pharmaceutically acceptable carriers. For example, for oral administration, the pharmaceutical composition may be formulated into tablets, troches, capsules, elixirs, suspensions, syrups or wafers. For able preparations, the pharmaceutical composition may be formulated into -dose ampule or multidose container. The pharmaceutical composition may be also formulated into solutions, suspensions, tablets, pills, capsules and sustained release preparations.

On the other hand, examples of carriers, excipients and ts suitable for formulation e lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia, te, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, hydroxybenzoate, talc, magnesium stearate, mineral oils or the like.

In addition, the pharmaceutical formulations may further include fillers, anti-coagulating agents, lubricants, ants, perfumes, antiseptics or the like.

In still another aspect, the present ion provides a method for ng insulin-related diseases, including administering the insulin analog or the insulin analog conjugate to a subject in need f.

The conjugate according to the present invention is useful in the treatment of diabetes, and therefore, this disease can be treated by administering the pharmaceutical composition including the same.

The term istration", as used herein, means introduction of a predetermined substance into a patient by a certain le method. The conjugate of the present invention may be administered via any of the common routes, as long as it is able to reach a desired tissue. Intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrapulmonary and intrarectal administration can be performed, but the present invention is not limited thereto. However, since peptides are digested upon oral administration, active ingredients of a composition for oral stration should be coated or formulated for protection against degradation in the stomach. Preferably, the present composition may be administered in an injectable form.

In addition, the pharmaceutical ition may be administered using a certain apparatus capable of transporting the active ingredients into a target cell.

Further, the pharmaceutical composition of the present invention may be determined by several related factors including the types of diseases to be d, administration routes, the patient’s age, gender, weight and severity of the illness, as well as by the types of the drug as an active ent. Since the pharmaceutical composition of the present invention has excellent in vivo duration and titer, it has an advantage of greatly reducing administration frequency of the pharmaceutical formulation of the present ion.

In still another aspect, the present invention provides a method for preparing the n analog ate, including preparing the insulin analog; preparing the carrier; and linking the insulin analog and the carrier.

In still another aspect, the present invention provides a method for increasing in vivo half-life using the insulin analog or the insulin analog conjugate which is prepared by linking the insulin analog and the carrier.

[Mode for Invention] Hereinafter, the present invention will be described in more detail with reference to Examples. However, these Examples are for rative purposes only, and the invention is not intended to be limited by these Examples.

Example 1: Preparation of single chain insulin analogexpressing vector In order to e insulin analogs, each of them having a modified amino acid in A chain or B chain, using the native insulin-expressing vector as a template, d and reverse oligonucleotides were synthesized (Table 2), and then PCR was carried out to amplify each analog gene.

In the following Table 1, amino acid sequences modified in A chain or B chain and analog names are given. That is, Analog 1 represents that 1st glycine of A chain is tuted with alanine, and Analog 4 represents that 8th glycine of B chain is substituted with alanine.

[Table 1] Primers for insulin analog ication are given in the following Table 2.

[Table 2] PCR for insulin analog amplification was carried out under conditions of 95°C for 30 seconds, 55°C for 30 seconds, 68°C for 6 minutes for 18 cycles. The insulin analog fragments obtained under the conditions were inserted into pET22b vector to be expressed as intracellular ion bodies, and the resulting expression vectors were designated as -insulin analogs 1 to 9. The expression vectors contained nucleic acids encoding amino acid ces of insulin analogs 1 to 9 under the control of T7 promoter, and insulin analog proteins were expressed as inclusion bodies in host cells.

DNA sequences and protein sequences of insulin analogs 1 to 9 are given in the following Table 3.

[Table 3] l Ammog Sequence SEQIDNO. l Analog 1 DNA TlC (BTT AAC CAA CAC TUB TGT (SGC TCA CAC C76 (3T6 (3AA GCT 19 l CTC TAC CTA GT6 TGC (EGG GAA CGA (JGC TTC TTC TAC ACA CCC l AAG ACC C(SC CGG GAG («A GAS (SAC CTG CAO (ETC: (366 {AG i (3T6 (3A6 (:16 GGC GGG (SGC CC"? (SGT GCA GGC AGE CTG CA6 CCC WC) GO: (TO GAG (366 TU: CTG CAG AAG (GT GUS AW GTG l l3AA CAA TGC lG'l' ACC AGE AK TL’aC TCC CTC TAC CA6 CTG GAG i LAAC TAC TGC AAC l Pwe Val Asn Gln His Leu CyS (filly Set His Leu Val Glu Ala Leu Tyl Lem l Pnflem Val Cy; (Sly GIL: Alt) (Sly le Plus Tyr ’l'hr Pm Lye Tln' Arr.) Avg (Elm Ala l (Blu Asp Leu Calm Val Gly Gin Val Glu Leu (Sly Gly Gly Pro (Ely Ala Gly i Ser Loeu (Eln Pro Leu Ala Leu Cwlu Gly ‘39! ten Gln Lys Arg Ala lle Val g E(31m Gin Cys Cys Tlxr Se: lle Lys Ber LE‘U Tyr Gln Lem Calm Asn Tyr Cys l ASH lAnalqvg 2 'DNA TTC C C A}; (A? TTG €in (SEE—C TC A CA—(f (17:61:31 (3ij GET 21 l CTC TAC {TA (3T6 W3C C166 GAA CCSA 136C TFC TTC TAC ACA CCC l :AAG ACC CBC 036 GAG GCA GAG GAC (TC: (LAG GT6 EGG CAG i (3T6 (3A6 CTG GGC (366 C161: (ZCT (BGT ("CA (36C AGC (T6 ("AG E CCC WC: (301‘. C TO GAG C1663 TICC C713 CA6 AAC: CGT (36C (ECG GTG 6AA {AA TGC TGT ACE AGC ATC TGC TCC Cle TAC CAG CTG l GAG AAC TAC TQC AN] l ‘ l I Wotan Pm; Val Asn (nln H15 Lea! Ly$ Lxly Ber HlS Len Val blu Ala Leu Tyr Leu_ , _ _ . , . l rVal Cys (Sly GILL Arg (Sly Phe Phe Ty: th Pro. Lys Tl'n Arg Avg GIL] Ala '6li Asp leu Gln Val iSly Gln Val Glu Let: (Sly Gly (Sly Pro (Sly Ala: Gly l Sar Leu Culn Pro Leu Ala Leu Glu (Sly Ser Leu Calm Lys Arg Gly Ala Val l Glu Gln Cys Cys Thr 39: lle Cys Ser' Leu Tyr Gln Leu‘ (33m ASH Tyr Cys l l Analog 3 .DNA THC GTT AAC (AA CAC TTL’B NET 66:: TCA CAC CTG GT6 (3AA GCT IV 9: l CTC TAC CTA (ETC: TEE: (EGG 6AA CGA GGC FTC TTC TAL’ ACA CCC AAG ACC CGC COG GAG GCA GAG GAC CTG (AG (3R3 C160 (AG l (ETC: GAG CTG (36C (366 (36C CCT GGT (ECA Get AGC (TIC: CA6 (ICC TTG GUI CTG GAG (366 TU: (.‘TG CAG AAG CTL’ST GGC ATT GTG l (3AA (AA TGC TGT ACC AGC ATC TGC TCC (TC TAC CA6 CTG GAG l AAC (ECG TGC AAC g Pnflem Pm; Val Asn Gln His Leu Cys Gly Ser His Leu Val Glu Ala Leu Ty! Leu l Val Cys Gly (Slu Arg Gly Phe Pile Tyl Tlu Pm Lys th A113 Arg (3le Ala Glu Asp Lou Gin Val Ely Gin Val GIL: Leu (Sly (Sly (Sly Pro Gly Al.) Gly l 391' Leu (Sln Pm Lem Ala lgeu Glu Gly Sel Len Gin Lys Arg (Sly Ile Val l ,Glu Gln Cys Cys Tlir' $9! Ile Eys SBI Lem Ty; Gln Lem lilu Asn Ala ifys Analog 4 DNA TTC GTT AAC {AA CAC TTG TGT GUS TCA CAC (TC: (,3th 6AA GCT ZS CTC TAC CTA GTO TESC 6(3er GAA CGA (SGC T—TC TTC TAC ACA CCC AAG ACC CCIC CGG GAG GCA GAG GAC CTG CAL-1 GTG 666 (AG GT6 (3A6 CTG GGC (366 GGC CCT GGT (ECA GGC AGC. CTG; (:AG CCC TTG GCC CTG (SAG 6.th TCL’ CH} CAG AAG CGT GGC ATT GTG (3AA CAA TGC TGT ACC AGC ATC TGC ICC (TC TA(‘ (AG L'ITG GAG AAC TAC TGC AAC Protein Pile Val Am (31m His Leu Cys Ala Ser His Leu Val Glu Ala Lem Tyr Leu [V o.

Val Cys (Sly Glu Arg (sly Phe Phe Tyr Tlu' Pro Lys Thr Arg Arg Glu Ala Glu Asp Leu Gln Val (Sly (Zulu Val Glu Leu Gly Gly (Sly Pro (Sly Ala (Bly Ser Lem Gln Pro Leu Ala Leu Glu (Ely Ser Leu (Elm Lys Arg (31y lle Val GIL} (Sln Cys Cys Thr Ser lle Cyg Ser' Lem Tyr Caln Leu GIL: ASH Tyr Cys Analog 5 DNA 4'TTC GTT AAL’ CAA {AC TTG TGT GGC TCA CAL" CTG GT6 GAA GCT 27 (TC TAC CTA OTC: TGC C166 (3AA (:GA L303 TTC TTC TA( ACA CCC AAQ ACC CGC (K36 GATT: GCA GAG CIAC {Tl} CA6 (ST6 (366 CAG GT6 GAG CTG (36C (36C: GGC (CT GGT GCA GGC AGC CTG CAT} CCC TTG (SEC CTG GAG £166 TCC CTG CAL} AAG (GT GGC ATT (ETC: (3AA CAA TGC TGT ACC AGE ATC TCaC TCC ETC TAC (IAG CTG GAG AAC TAC TGC AAC n Phe Val Asn Gin His Len Cys Gly Ser Hi5 Leu Val Glu Ala Leu Tyr Leu Val Cys Gly (3k: Arg Ala Phe Phe Ty: Thr Pro. Lys Tlrr Ar'g Arg Glu Ala (Zulu Asp Leu Gin Val (Ely C1311 Val Calu Leu Gly Gly Gly Pro My Ala (Sly Sex Lem Calm Pro Leu Ala Leu Glu (Sly Ser Len Gln Lys Arg Gly Ile Val Glu Gln Cys Cys Thr $er lle Cys Ber Leu Tyr Calm Leu Glu Asn Tyr Cys Analog l3 DNA T'T'L' GTT AAC {AA (AC TTG TGT (SGT: TCA CAC CTG (3H3 6AA GET CTC TA': CTA (3T6 TGC (366 (3AA 013A GCaC GCC: TTC TAT: ACA CCC AAG ACT: (6C CGG GAG GCA GAG GAC (TC) CA6 GTG (366 CAG (3T6 (3A6 CTG GGC GGG (36C CCT (SGT GCA GCuC AGC CTG CA6 (CC TTG (fiCC CTG (SAC: GGG TCC CTG CA6 AAG CGT (36C ATT GT6 (3AA CAA TGC TGT ACT; AGC ATC TGC TCC (TTC TAC CAG LC TG GAG AAC TAC TGC AAC Protein Phe Val Asn Gln His Leu Cys Gly 891 His Leu Val Glu Ala Leu Tyr Leu Val Cys [Sly lilu Arg (Sly Ala Phi? Tyr Thr Pro Lys Thr Arq Arg Glu Ala Glu Aap Leu Gln Val Gly Gln Val Glu Len (Sly (31y (fily Pro Gly Ala Gly SEW Leu Gln Pro Leu Ala Leu Glu 6!)! Ser Leu Gln Lys Arg (Ely lle Val (STU Gln Cys Cys Thr Se: lle Cys SEW Len Tyr kiln Leu Glu Asn Tyr Cys Example 2: sion of recombinant insulin analog fusion peptide Expressions of recombinant insulin s were carried out under the control of T7 promoter. E.coli BL21-DE3 (E. coli B F-dcm ompT hsdS(rB-mB-) gal λDE3); Novagen) was transformed with each of the recombinant n analog-expressing vectors.

Transformation was performed in accordance with the recommended protocol (Novagen). Single colonies transformed with each inant expression vector were collected and ated in 2X Luria Broth (LB) containing ampicillin (50 µg/ml) and cultured at 37°C for 15 hours. The recombinant strain culture broth and 2X LB medium containing 30% glycerol were mixed at a ratio of 1:1 (v/v). Each 1 ml was dispensed to a cryotube and stored at -140°C, which was used as a cell stock for production of the recombinant fusion protein.

To express the recombinant insulin analogs, 1 vial of each cell stock was thawed and inoculated in 500 ml of 2X Luria broth, and cultured with shaking at 37°C for 14~16 hours.

The cultivation was terminated, when OD600 reached 5.0 or . The e broth was used as a seed culture broth.

This seed culture broth was inoculated to a 50 L fermentor (MSJ-U2, B.E.MARUBISHI, Japan) ning 17 L of fermentation medium, and initial bath fermentation was started. The culture conditions were maintained at a temperature of 37°C, an air flow rate of 20 L/min (1 vvm), an agitation speed of 500 rpm, and at pH 6.70 by using a 30% ammonia solution. Fermentation was carried out in fed-batch mode by adding a g solution, when nutrients were depleted in the culture broth. Growth of the strain was monitored by OD value. IPTG was introduced in a final concentration of 500 µM, when OD value was above 100.

After uction, the cultivation was r carried out for about 23~25 hours. After terminating the cultivation, the recombinant strains were harvested by centrifugation and stored at -80°C until use.

Example 3: Recovery and Refolding of recombinant insulin analog In order to change the recombinant insulin analogs expressed in Example 2 into soluble forms, cells were disrupted, followed by ing. 100 g (wet weight) of the cell pellet was re-suspended in 1 L lysis buffer (50 mM Tris- HCl (pH 9.0), 1 mM EDTA (pH 8.0), 0.2 M NaCl and 0.5% Triton X-100). The cells were disrupted using a microfluidizer processor M-110EH (AC Technology Corp. Model M1475C) at an operating pressure of 15,000 psi. The cell lysate thus disrupted was centrifuged at 7,000 rpm and 4°C for 20 minutes.

The supernatant was discarded and the pellet was re-suspended in 3 L washing buffer (0.5% Triton X-100 and 50 mM Tris-HCl (pH 8.0), 0.2 M NaCl, 1 mM EDTA). After centrifugation at 7,000 rpm and 4°C for 20 minutes, the cell pellet was ended in distilled water, followed by centrifugation in the same manner. The pellet thus obtained was re-suspended in 400 ml of buffer (1 M e, 3.78 g Cysteine-HCl, pH 10.6) and d at room temperature for 1 hour. To recover the inant insulin analog thus re-suspended, 400 mL of 8M urea was added and stirred at 40°C for 1 hour. For refolding of the solubilized recombinant insulin analogs, centrifugation was carried out at 7,000 rpm and 4°C for 30 minutes, and the supernatant was obtained. 2 L of distilled water was added thereto using a altic pump at a flow rate of 1000 ml/hr while stirring at 4°C for 16 hours.

Example 4: Cation binding chromatography purification The sample refolded was loaded onto a Source S (GE healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer ning 45% ethanol, and then the insulin analog proteins were eluted in 10 column s with a linear gradient from 0% to 100% 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol.

Example 5: Trypsin and Carboxypeptidase B treatment Salts were removed from the eluted samples using a desalting column, and the buffer was exchanged with a buffer (10 mM Tris-HCl, pH 8.0). With respect to the obtained sample protein, trypsin corresponding to 1000 molar ratio and carboxypeptidase B corresponding to 2000 molar ratio were added, and then stirred at 16°C for 16 hours. To terminate the reaction, 1 M sodium citrate (pH 2.0) was used to reduce pH to 3.5.

Example 6: Cation binding chromatography purification The sample thus reacted was loaded onto a Source S (GE healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% l, and then the insulin analog proteins were eluted in 10 column volumes with a linear gradient from 0% to 100% 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol. e 7: Anion binding chromatography purification Salts were removed from the eluted sample using a ing column, and the buffer was exchanged with a buffer (10 mM Tris-HCl, pH 7.5). In order to isolate a pure insulin analog from the sample ed in e 6, the sample was loaded onto an anion exchange column (Source Q: GE healthcare) equilibrated with 10 mM Tris (pH 7.5) buffer, and the insulin analog protein was eluted in 10 column volumes with a linear gradient from 0% to 100% 10 mM Tris (pH 7.5) buffer containing 0.5 M sodium chloride.

Purity of the insulin analog thus purified was analyzed by protein electrophoresis (SDS-PAGE, and high pressure chromatography (HPLC) (, and modifications of amino acids were identified by peptide mapping ( and molecular weight analysis of each peak.

As a result, each insulin analog was found to have the d modification in its amino acid sequence.

Example 8: Preparation of insulin analog (No. 7)- immunoglobulin Fc ate To te the N-terminus of the beta chain of the insulin analog using 3.4K ALD2 PEG (NOF, Japan), the insulin analog and PEG were reacted at a molar ratio of 1:4 with an insulin analog concentration of 5 mg/ml at 4°C for about 2 hours. At this time, the reaction was performed in 50 mM sodium citrate at pH 6.0 and 45% isopropanol. 3.0 mM sodium cyanoborohydride was added as a reducing agent and was allowed to react. The reaction solution was purified with SP-HP (GE Healthcare, USA) column using a buffer containing sodium citrate (pH 3.0) and 45% ethanol, and KCl concentration gradient.

To prepare an insulin analog-immunoglobulin Fc fragment conjugate, the ed mono-PEGylated insulin analog and the immunoglobulin Fc fragment were d at a molar ratio of 1:1 to 1:2 and at 25°C for 13 hrs, with a total protein concentration of about 20 mg/ml. At this time, the reaction buffer conditions were 100 mM HEPES at pH 8.2, and 20 mM sodium cyanoborohydride as a reducing agent was added thereto.

Therefore, PEG was bound to the inus of the Fc fragment.

After the reaction was terminated, the reaction solution was loaded onto the Q HP (GE Healthcare, USA) column with Tris-HCl (pH 7.5) buffer and NaCl concentration gradient to separate and purify unreacted immunoglobulin Fc fragment and mono-PEGylated insulin analog.

Thereafter, Source 15ISO (GE care, USA) was used as a secondary column to remove the remaining globulin Fc fragment and the conjugate, in which two or more insulin analogs were linked to the immunoglobulin Fc fragment, thereby obtaining the insulin analog-immunoglobulin Fc fragment conjugate. At this time, elution was carried out using a concentration gradient of ammonium sulfate containing Tris-HCl (pH 7.5), and the insulin analog-immunoglobulin Fc conjugate thus eluted was analyzed by protein electrophoresis (SDS-PAGE, and high pressure chromatography （HPLC) (. As a result, the conjugate was found to have almost 99% purity.

Example 9: Comparison of insulin receptor binding affinity n native insulin, n analog, native insulin-immunoglobulin Fc ate, and insulin analogimmunoglobulin Fc conjugate In order to measure the insulin or binding affinity of the insulin analog-immunoglobulin Fc ate, Surface plasmon resonance (SPR, BIACORE 3000, GE healthcare) was used for analysis. Insulin receptors were immobilized on a CM5 chip by amine coupling, and 5 dilutions or more of native insulin, insulin analog, native n-immunoglobulin Fc conjugate, and insulin analog-immunoglobulin Fc conjugate were applied thereto, independently. Then, the insulin receptor binding ty of each substance was examined. The binding ty of each substance was calculated using BIAevaluation software.

At this time, the model used was 1:1 Langmuir binding with baseline drift.

As a , compared to human insulin, insulin analog (No. 6) showed receptor binding affinity of 14.8%, insulin analog (No. 7) showed receptor binding affinity of 9.9%, insulin analog (No. 8) showed receptor binding affinity of 57.1%, n analog (No. 9) showed receptor binding affinity of 78.8%, native insulin-immunoglobulin Fc conjugate showed receptor binding affinity of 3.7-5.9% ing on experimental runs, insulin analog (No. 6)-immunoglobulin Fc conjugate showed receptor binding affinity of 0.9% or less, insulin analog (No. 7)-immunoglobulin Fc ate showed or binding affinity of 1.9%, insulin analog (No. 8)- immunoglobulin Fc conjugate showed receptor binding affinity of 1.8%, and insulin analog (No. 9)-immunoglobulin Fc conjugate showed receptor binding ty of 3.3% (Table 4).

As such, it was observed that the insulin analogs of the present invention had reduced insulin receptor binding affinity, compared to the native insulin, and the insulin analog-immunoglobulin Fc conjugates also had remarkably d insulin receptor binding affinity.

[Table 4] Example 10: ison of in-vitro efficacy between native insulin-immunoglobulin Fc conjugate and n analogimmunoglobulin Fc conjugate In order to te in vitro efficacy of the insulin analog-immunoglobulin Fc conjugate, mouse-derived differentiated 3T3-L1 ytes were used to test glucose uptake or lipid synthesis. 3T3-L1 cells were sub-cultured in % NBCS (newborn calf -containing DMEM (Dulbeco’s Modified Eagle’s Medium, Gibco, Cat.No, 12430) twice or three times a week, and maintained. 3T3-L1 cells were suspended in a differentiation medium (10% FBS-containing DMEM), and then ated at a density of 5 x 104 per well in a 48-well dish, and cultured for 48 hours. For adipocyte differentiation, 1 µg/mL human insulin (Sigma, Cat. No. I9278), 0.5 mM IBMX (3- isobutylmethylxanthine, Sigma, Cat. No.I5879), and 1 µM Dexamethasone (Sigma, Cat. No. D4902) were mixed with the differentiation medium, and 250 µl of the mixture was added to each well, after the previous medium was removed. After 48 hours, the medium was exchanged with the differentiation medium supplemented with only 1 µg/mL of human insulin.

Thereafter, while the medium was exchanged with the differentiation medium supplemented with 1 µg/mL of human insulin every 48 hours, induction of adipocyte differentiation was ed for 7-9 days. To test glucose uptake, the differentiated cells were washed with serum-free DMEM medium once, and then 250 µl was added to induce serum depletion for 4 hours. Serum-free DMEM medium was used to carry out 10-fold serial dilutions for Human insulin from 2 µM to 0.01 µM, and for native insulin-immunoglobulin Fc conjugate and n analog-immunoglobulin Fc conjugates from 20 µM to 0.02 µM.

Each 250 µl of the s thus prepared were added to cells, and cultured in a 5% CO2 incubator at 37°C for 24 hours. In order to measure the residual amount of glucose in the medium after incubation, 200 µl of the medium was taken and diluted -fold with D-PBS, followed by GOPOD (GOPOD Assay Kit, Megazyme, Cat. No. K-GLUC) assay. Based on the absorbance of glucose standard solution, the concentration of glucose remaining in the medium was ted, and EC50 values for glucose uptake of native insulin-immunoglobulin Fc conjugate and insulin analog-immunoglobulin Fc conjugates were calculated, respectively.

As a result, compared to human insulin, native insulinimmunoglobulin Fc conjugate showed glucose uptake of 11.6%, insulin analog (No. 6)-immunoglobulin Fc conjugate showed e uptake of 0.43%, insulin analog (No. 7)-immunoglobulin Fc conjugate showed e uptake of 1.84%, insulin analog (No. 8)-immunoglobulin Fc conjugate showed glucose uptake of 16.0%, insulin analog (No. 9)-immunoglobulin Fc conjugate showed glucose uptake of 15.1% (Table 5). As such, it was observed that the insulin analog (No. 6)-immunoglobulin Fc ate and insulin analog (No. 7)-immunoglobulin Fc conjugate of the present invention had remarkably reduced in vitro titer, ed to native insulin-immunoglobulin Fc conjugate, and insulin analog (No. 8)-immunoglobulin Fc conjugate and insulin analog (No. 9)-immunoglobulin Fc conjugate had in vitro titer similar to that of the native insulin-immunoglobulin Fc conjugate.

[Table 5] Example 11: Pharmacokinetics of insulin analogimmunoglobulin Fc conjugate In order to examine pharmacokinetics of the insulin analog-immunoglobulin Fc ates, their blood concentration over time was compared in normal rats (SD rat, male, 6-week old) adapted for 5 days to the laboratory. 21.7 nmol/kg of native insulin-immunoglobulin Fc conjugate and 65.1 g of insulin analog-immunoglobulin Fc conjugate were subcutaneously injected, respectively. The blood was ted at 0, 1, 4, 8, 24, 48, 72, 96, 120, 144, 168, 192, and 216 hours. At each time point, blood concentrations of native insulinimmunoglobulin Fc conjugate and insulin analog-immunoglobulin Fc conjugate were measured by enzyme linked immunosorbent assay (ELISA), and Insulin ELISA (ALPCO, USA) was used as a kit. However, as a detection antibody, mouse anti-human IgG4 HRP conjugate (Alpha Diagnostic Intl, Inc, USA) was used.

The results of examining pharmacokinetics of the native insulin-immunoglobulin Fc conjugate and the n immunoglobulin Fc conjugate showed that their blood concentrations increased in proportion to their administration concentrations, and the insulin analog-immunoglobulin Fc conjugates having low insulin receptor binding affinity showed highly sed half-life, compared to the native insulin-Fc conjugate (.

These results suggest that when the insulin analogs of the present invention modified to have reduced insulin receptor binding affinity are linked to immunoglobulin Fc region to prepare conjugates, the conjugates can be provided as stable insulin formulations due to remarkably sed in vivo blood half-life, and thus effectively used as therapeutic agents for diabetes. Furthermore, since the insulin analogs ing to the present invention themselves also have reduced insulin receptor binding affinity and reduced titer, the insulin analogs also exhibit the same effect gh they are linked to other various carriers.

Based on the above description, it will be apparent to those skilled in the art that various modifications and changes may be made without departing from the scope and spirit of the invention. ore, it should be understood that the above ment is not limitative, but illustrative in all aspects. The scope of the invention is defined by the appended claims rather than by the description preceding them, and therefore all changes and modifications that fall within metes and bounds of the , or equivalents of such metes and bounds are therefore intended to be embraced by the claims.

Claims (20)

Claims

1. An insulin analog conjugate, in which (i) an insulin analog having a reduced n titer compared to the native form, wherein an amino acid in B chain of insulin is modified by substituting one amino acid selected from the group consisting of 24th amino acid and 25th amino acid of B chain with alanine; is linked to (ii) a patible material, wherein the biocompatible material is an immunoglobulin Fc region.

2. The insulin analog according to claim 1, wherein the insulin analog is selected from the group consisting of SEQ ID NOs. 30, and 32.

3. The insulin analog conjugate according to claim 1, wherein the insulin analog and the FcRn-binding material are linked to each other via a e or a nonpeptidyl polymer as a linker.

4. The insulin analog conjugate according to claim 1, wherein (i) the insulin analog is linked to (ii) the immunoglobulin Fc region via (iii) a peptide linker or a nonpeptidyl linker selected from the group consisting of polyethylene glycol, polypropylene glycol, copolymers of ethylene glycol-propylene glycol, polyoxyethylated polyols, polyvinyl alcohol, polysaccharides, dextran, polyvinyl ethyl ether, biodegradable polymers, lipid polymers, chitins, hyaluronic acid and combination thereof.

5. The insulin analog conjugate according to claim 4, n the non-peptidyl linker is linked to the N-terminus of B chain of the insulin analog.

6. The insulin analog conjugate according to claim 4, wherein one end of the non-peptidyl polymer is linked to the N-terminus of the immunoglobulin Fc region and the other end of the non-peptidyl polymer is linked to the N-terminal amine group of the insulin analog or the ε-amino group of the al lysine residue or the thiol group of B chain, respectively.

7. The insulin analog conjugate according to claim 4, wherein the immunoglobulin Fc region is aglycosylated.

8. The insulin analog ate ing to claim 4, wherein the immunoglobulin Fc region is composed of 1 domain to 4 domains selected from the group ting of CH1, CH2, CH3 and CH4 domains.

9. The insulin analog conjugate according to claim 4, wherein the immunoglobulin Fc region is an Fc region derived from IgG, IgA, IgD, IgE or IgM.

10. The insulin analog ate according to claim 9, wherein each domain of the immunoglobulin Fc region is a hybrid of domains having different s and being derived from an immunoglobulin selected from the group consisting of IgG, IgA, IgD, IgE and IgM.

11. The n analog conjugate according to claim 4, wherein the immunoglobulin Fc region further includes a hinge region.

12. The insulin analog conjugate according to claim 9, wherein the immunoglobulin Fc region is a dimer or a multimer consisting of single-chain immunoglobulins composed of s of the same origin.

13. The insulin analog conjugate according to claim 9, n the immunoglobulin Fc region is an IgG4 Fc region.

14. The insulin analog conjugate according to claim 13, wherein the globulin Fc region is a human IgG4-derived aglycosylated Fc region.

15. The insulin analog conjugate according to claim 4, wherein a reactive group of the non-peptidyl linker is selected from the group consisting of an aldehyde group, a propionaldehyde group, a butyraldehyde group, a maleimide group and a succinimide derivative.

16. The insulin analog conjugate according to claim 15, n the succinimide tive is succinimidyl propionate, succinimidyl carboxymethyl, hydroxy succinimidyl, or succinimidyl carbonate.

17. The insulin analog conjugate according to claim 4, wherein the non-peptidyl linker has reactive aldehyde groups at both ends thereof.

18. A long-acting n formulation having improved in vivo duration and stability, comprising the insulin analog conjugate of claim 1.

19. The long-acting insulin formulation according to claim 18, n the formulation is a therapeutic agent for es.

20. A method for preparing the n analog conjugate of claim 1, comprising: linking an insulin analog having a reduced insulin titer compared to the native form, wherein an amino acid in B chain of insulin is modified by substituting one amino acid selected from the group consisting of 24th amino acid and 25th amino acid of B chain with alanine; to a biocompatible material, wherein the biocompatible material is an immunoglobulin Fc region. [ [ [ (A) Glu-C cleavage fragment of native insulin sequence (B) Glu-C cleavage fragment of n analog (No. 7) sequence [ [ [