Patent Application
20110245174
The invention relates to a fusion polypeptide comprising a GLP peptide or functional variant thereof; dimers comprising said fusion polypeptide; and methods to treat diseases that would benefit from administration of said fusion polypeptide.
Glucagon-like peptide 1 [GLP-1] has various functions. For example GLP-1 stimulates the production of insulin by pancreatic β cells, enhances pancreatic β cell proliferation, inhibits pancreatic β cell apoptosis, lowers glucagon activity, slows gastric emptying and enhances insulin sensitivity. GLP-1 is derived from a larger polypeptide referred to as proglucagon which comprises glucagon [29 amino acids], GLP-1 [36 or 37 amino acid residues] and GLP-2 [34 amino acid residues],
There are a number of pathological conditions that result in hyperglycaemia; the most well known being diabetes mellitus. Diabetes mellitus can be of type I or type II. Type I diabetes is an autoimmune disease resulting in destruction of the pancreatic β cells which means the subject is unable to manufacture any insulin. Type II diabetes is a more complicated condition and can result from a number of associated ailments but commonly involves resistance to the metabolic actions of insulin. For example, type II diabetes is associated with age, obesity, a sedentary life style which results in insulin resistance. An associated condition is called Metabolic Syndrome which may predispose subjects to type II diabetes. The symptoms associated with this syndrome are high blood pressure, dyslipidemia, increased body fat deposition and cardiovascular disease. A further condition that results in insulin resistance is polycystic ovary syndrome which results in a failure to produce mature ova, androgen excess and hirsuitism. Hypoglycaemia [abnormally low levels of serum glucose] is also known and is typically the result of administration of an insulin overdose.
GLP-1 has been used as a therapeutic agent in the control of problem associated with native GLP-1 is that because of its small mass it is very rapidly cleared from the circulation having a pharmacokinetic half life of 2-5 mins. This means that to achieve a therapeutic effect a relatively large dose of GLP-1 has to be administered. This has lead to the development of long acting forms of GLP-1 and the use of DPP4 inhibitors. The former approach involves the production of fusion proteins comprising GLP-1; the latter utilizes DPP4 inhibitors which suffer from a lack of specificity due to the fact that the inhibitor will inactivate DPP4s that modify other peptide hormones leading to undesirable side effects. There is therefore a continued desire to address the problem of rapid digestion and/or renal clearance of GLP-1 and related molecules.
As mentioned above prior art approaches to reduce rapid GLP-1 clearance involves the creation of GLP-1 fusion proteins. For example, WO2007/016764 describes a fusion protein comprising GLP-1 and an autoimmune suppressor to decrease an autoimmmune reaction in type I diabetes. EP1 724 284 describes the fusion of GLP-1 to either the Fc portion of an immunoglobulin or to albumin. Similarly, WO2005/00892 describes fusion proteins comprising GLP-1 analogues and the Fc portion of IgG4 and their use in the treatment of diabetes, obesity and irritable bowel syndrome. In US2007/0111940 are disclosed conjugates comprising GLP-1 and a peptide carrier that includes modified amino acids that improve stability of GLP-1. In U.S. Pat. No. 7, 716, 278 the fusion of GLP-1 to transferrin is used to reduce renal clearance and treat diabetes and related conditions. In WO2008/061355 an alternative to fusing GLP-1 to a carrier protein/peptide is described which is an implantable hydrogel device which releases GLP-1 and analogues of GLP-1 in a sustained fashion over a defined period.
This disclosure relates to alternative fusion polypeptides comprising a GLP-1 peptide or functional analogue thereof. In one embodiment GLP-1 is linked to an extracellular domain of a GLP-1 receptor. Alternative embodiments include the fusion of GLP-1 to inactivated DDP4 and optionally inactive ADA.
According to an aspect of the invention there is provided a nucleic acid molecule comprising a nucleic acid sequence that encodes a polypeptide that has the activity of GLP-1 wherein said polypeptide comprises GLP-1, or a receptor binding part thereof, linked directly or indirectly to a polypeptide that naturally binds GLP-1.
According to an aspect of the invention there is provided a fusion the amino acid sequence of a GLP-1 peptide or functional analogue thereof, linked directly or indirectly to a polypeptide that naturally binds GLP-1.
In a preferred embodiment of the invention the polypeptide that naturally binds GLP-1 is the GLP-1 binding domain of the GLP-1 receptor.
In an alternative preferred embodiment of the invention the polypeptide that naturally binds GLP-1 is an enzymatically inactive GLP-1 dipeptidyl peptidase.
In a preferred embodiment of the invention said inactive GLP-1 dipeptidyl peptidase is modified by addition, deletion or substitution of at least one amino acid residue wherein said modification is to the active site of GLP-1 dipeptidyl peptidase.
Preferably said modification is to amino acid residue 630 of the amino acid sequence represented in
In a preferred embodiment of the invention said fusion polypeptide comprises or consists of the amino acid sequence represented in
In a preferred embodiment of the invention said fusion polypeptide further comprises a polypeptide that naturally binds said GLP-1 dipeptidyl peptidase wherein said polypeptide is an enzymatically inactive adenosine deaminase.
In a preferred embodiment of the invention said inactive adenosine deaminase is modified by addition, deletion or substitution of at least one amino acid residue wherein said modification is to the active site of said inactive adenosine deaminase.
Preferably, said modification is to amino acid residues 295 and/or 296 of the amino acid sequence represented in
In a preferred embodiment of the invention said fusion polypeptide comprises or consists of the amino acid sequence represented in
In a preferred embodiment of the invention said fusion polypeptide comprises a GLP-1 peptide comprising or consisting of the amino acid sequence: HAEGTFTSDVSSYLEGQAAKEFIAWLVKGR, or a modified GLP-1 peptide wherein said modified peptide varies from said amino acid sequence by addition, deletion or substitution of at least one amino acid residue wherein said modified GLP-1 peptide retains or has enhanced GLP-1 activity when compared to an unmodified GLP-1 peptide.
In a preferred embodiment of the invention said GLP-1 peptide comprises the amino acid sequence:
In a preferred embodiment of the invention said fusion polypeptide comprises an amino acid sequence:
In a preferred embodiment of the invention GLP-1 is linked to a polypeptide that naturally binds GLP-1 by a peptide linker.
In a preferred embodiment of the invention GLP-1 is linked to an inactive GLP-1 dipeptidyl peptidase GLP-1 by a peptide linker.
In a preferred embodiment of the invention GLP-1 is linked to an inactive adenosine deaminase by a peptide linker.
In a further preferred embodiment of the invention fusion inactive GLP-1 dipeptidyl peptidase is linked to an inactive adenosine deaminase by a peptide linker.
Preferably said peptide linker is a flexible peptide linker.
In a preferred embodiment of the invention said peptide linking molecule comprises at least one copy of the peptide Gly Gly Gly Gly Ser.
In a preferred embodiment of the invention said peptide linking molecule comprises 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 or 12 copies of the peptide Gly Gly Gly Gly Ser.
The polypeptide domains of the fusion polypeptide according to the invention typically are linked by peptide linkers as herein described, for example Gly4Ser linkers. The number of copies of Gly4Ser can vary. For example fusion of GLP-1 to DPP4 can vary between 0 and 10 copies, preferably 5-7 copies. The fusion of DDP4 to ADA domains can also vary from between 0 and 12 copies, preferably 7 or 8 copies. The fusion of GLP-1 to the ectodomain can vary between 0-8 copies, preferably 2-5 copies.
In an alternative preferred embodiment of the invention GLP-1 is linked to a polypeptide that naturally binds GLP-1 by a single peptidic bond.
In a preferred embodiment of the invention GLP-1 is linked to an inactive GLP-1 dipeptidyl peptidase GLP-1 by a single peptidic bond.
In a preferred embodiment of the invention GLP-1 is linked to an inactive adenosine deaminase by a single peptidic bond.
In a preferred embodiment of the invention inactive GLP-1 dipeptidyl peptidase is linked to an inactive adenosine deaminase by a single peptidic bond.
In an alternative preferred embodiment of the invention said peptide linker molecule comprises or consists of one copy of the glycosylation motif Asn-Xaa-Ser or Asn-Xaa-Thr where X is any amino acid except proline.
In a preferred embodiment of the invention said peptide linker molecule comprises at least 5 amino acid residues.
In a preferred embodiment of the invention said peptide linker comprises 5-50 amino acid residues.
In a further preferred embodiment of the invention said peptide linker consists of 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 amino acid residues.
In a preferred embodiment of the invention said peptide linker molecule comprises at least one copy of the motif (Xaa1 Xaa2 Xaa3 Xaa4 Xaa5) wherein said motif comprises the glycosylation motif Asn-Xaa-Ser or Asn-Xaa-Thr.
In a preferred embodiment of the invention said peptide linker comprises at least one copy of an amino acid motif selected from the group consisting of:
Asn1-Xaa2-Ser3 Xaa4 Xaa5 wherein Xaa2 is any amino acid except proline;
Xaa1 Asn2-Xaa3-Ser4 Xaa5 wherein Xaa3 is any amino acid except proline;
Xaa1 Xaa2 Asn3-Xaa4-Ser5 wherein Xaa4 is any amino acid except proline;
Asn1-Xaa2-Thr3 Xaa4 Xaa5wherein Xaa2 is any amino acid except proline;
Xaa1 Asn2-Xaa3-Thr4 Xaa5 wherein Xaa3 is any amino acid except proline; and
Xaa1 Xaa2 Asn3-Xaa4-Thr5wherein Xaa4 is any amino acid except proline.
Preferably said peptide linker comprises at least one copy of a motif selected from the group consisting of:
Asn1-Xaa2-Ser3 Gly4 Ser5 wherein Xaa2 is any amino acid except proline;
Gly1 Asn2-Xaa3-Ser4 Ser5wherein Xaa3 is any amino acid except proline;
Gly1 Gly2 Asn3-Xaa4-Ser5 wherein Xaa4 is any amino acid except proline;
Asn1-Xaa2-Thr3 Gly4 Ser5 wherein Xaa2 is any amino acid except proline;
Gly1 Asn2-Xaa3-Thr4 Ser5 wherein Xaa3 is any amino acid except proline; and
Gly1 Gly2 Asn3-Xaa4-Thr5wherein Xaa4 is any amino acid except proline.
In an alternative preferred embodiment of the invention said peptide linker comprises at least one copy of a motif selected from the group consisting of:
Asn1-Xaa2-Ser3 Ser4 Gly5 wherein Xaa2 is any amino acid except proline;
Ser1 Asn2-Xaa3-Ser4 Gly5wherein Xaa3 is any amino acid except proline;
Ser1 Ser2 Asn3-Xaa4-Ser5 wherein Xaa4 is any amino acid except proline;
Asn1-Xaa2-Thr3 Ser4 Gly5wherein Xaa2 is any amino acid except proline;
Ser1 Asn2-Xaa3-Thr4 Gly5wherein Xaa3 is any amino acid except proline; and
Ser1 Ser2 Asn3-Xaa4-Thr5wherein Xaa4 is any amino acid except proline.
In a preferred embodiment of the invention said peptide linker molecule comprises at least one copy of the motif (Xaa1, Xaa2 Xaa3 Xaa4 Xaa5) wherein said motif comprises the glycosylation motif Asn-Xaa-Ser or Asn-Xaa-Thr and at least one copy of the motif (Gly Gly Gly Gly Ser) wherein said peptide linker is 5-50 amino acids.
In a preferred embodiment of the invention said peptide linker comprises at least one copy of the motif (Xaa1 Xaa2 Xaa3 Xaa4 Xaa5) wherein said motif comprises the glycosylation motif Asn-Xaa-Ser or Asn-Xaa-Thr and a copy of the motif (Ser Ser Ser Ser Gly) wherein said peptide linker is 5-50 amino acids.
In a preferred embodiment of the invention said fusion polypeptide linker is modified by the addition of at least one sugar selected from the group consisting of: mannose, galactose, N-acetyl glucosamine, N-acetyl neuraminic, acid N-glycolyl neuraminic acid, N-acetyl galactosamine, fucose, glucose, rhamnose, xylose, or a combinations of sugars, for example in an oligosacharide or scaffolded system.
Suitable carbohydrate moieties include monosaccharides, oligosaccharides and polysaccharides, and include any carbohydrate moiety that is present in naturally occurring glycoproteins or in biological systems. For example, optionally protected glycosyl or glycoside derivatives, for example optionally-protected glucosyl, glucoside, galactosyl or galactoside derivatives. Glycosyl and glycoside groups include both α and β groups. Suitable carbohydrate moieties include glucose, galactose, fucose, GlcNAc, GalNAc, sialic acid, and mannose, and oligosaccharides or polysaccharides comprising at least one glucose, galactose, fucose, GlcNAc, GalNAc, sialic acid, and/or mannose residue.
Any functional groups in the carbohydrate moiety may optionally be protected using protecting groups known in the art (see for example Greene et al, “Protecting groups in organic synthesis”, 2nd Edition, Wiley, New York, 1991, the disclosure of which is hereby incorporated by reference). Suitable protecting groups for any —OH groups in the carbohydrate moiety include acetate (Ac), benzyl (Bn), silyl (for example tert-butyl dimethylsilyl (TBDMSi) and tert-butyldiphenylsilyl (TMDPSi)), acetals, ketals, and methoxymethyl (MOM). Any protecting groups may be removed before or after attachment of the carbohydrate moiety to the peptide linker.
In a preferred embodiment of the invention said sugars are unprotected.
Particularly preferred carbohydrate moieties include Glc(Ac)4β-, Glc(Bn)4β-, Gal(Ac)4β-, Gal(Bn)4β-, Glc(Ac)4α(1,4)Glc(Ac)3α(1,4)Glc(Ac)4β-, β-Glc, β-Gal, -Et-β-Gal,-Et-β-Glc, Et-α-Glc, -Et-α-Man, -Et-Lac, -β-Glc(Ac)2, -β-Glc(Ac)3, -Et-α-Glc(Ac)2, -Et-α-Glc(Ac)3, -Et-α-Glc(Ac)4, -Et-β-Glc(Ac)2, -Et-β-Glc(Ac)3, -Et-β-Glc(Ac)4, -Et-α-Man(Ac)3, -Et-α-Man(Ac)4, -Et-β-Gal(Ac)3, -Et-β-Gal(Ac)4, -Et-Lac(Ac)5, -Et-La and their deprotected equivalents.
Preferably, any saccharide units making up the carbohydrate moiety which are derived from naturally occurring sugars will each be in the, naturally occurring enantiomeric form, which may be either the D-form (e.g. D-glucose or D-galactose), or the L-form (e.g. L-rhamnose or L-fucose). Any anomeric linkages may be α- or β- linkages.
According to a further aspect of the invention said fusion polypeptide is encoded by a nucleic acid molecule selected from the group consisting of:
i) a nucleic acid sequence as represented in
ii) a nucleic acid sequence as represented in
iii) a nucleic acid sequence as represented in
iv) a nucleic acid sequence as represented in
v) a nucleic acid sequence as represented in
vi) a nucleic acid sequence as represented in
vii) a nucleic acid sequence as represented in
viii) a nucleic acid sequence as represented in
ix) a nucleic acid sequence as represented in
x) a nucleic acid sequence as represented in
xi) a nucleic acid sequence as represented in
xii) a nucleic acid sequence as represented in
xiii) a nucleic acid sequence as represented in
xiv) a nucleic acid sequence as represented in
xv) a nucleic acid sequence as represented in
xvi) a nucleic acid sequence as represented in
xvii) a nucleic acid sequence as represented in
xviii) a nucleic acid sequence as represented in
xix) a nucleic acid sequence as represented in
xx) a nucleic acid sequence as represented in
xxi) a nucleic acid sequence as represented in
xxii) a nucleic acid sequence as represented in
xxiii) a nucleic acid sequence as represented in
xxiv) a nucleic acid sequence as represented in
In a preferred embodiment of the invention said nucleic acid molecule encodes a polypeptide that has agonist activity.
There are a number of pathological conditions result in hyperglycaemia and would benefit from a GLP-1 agonist the most well known being diabetes mellitus. Diabetes mellitus can be of type 1 or type 2. Type 1 diabetes is an autoimmune disease resulting in destruction of the pancreatic β cells which means the subject is unable to manufacture any insulin. Type 2 diabetes is a more complicated condition and can result from a number of associated ailments but commonly involves resistance to the metabolic actions of insulin. For example, type 2 diabetes is associated with age, obesity, a sedentary life style which results in insulin resistance. An associated condition is called Metabolic Syndrome which may predispose subjects to type 2 diabetes. The symptoms associated with this syndrome are high blood pressure, dyslipidemia, increased body fat deposition and cardiovascular disease.
In an alternative preferred embodiment of the invention said nucleic acid molecule encodes a polypeptide that has antagonist activity.
Hypoglycaemia [abnormally low levels of serum glucose] is also known and is typically the result of administration of an insulin overdose. This would benefit from the administration of a GLP-1 antagonist. There are also diseases that result in excess insulin secretion resulting in a hypoglycaemic state. For example, insulinoma is a cancer of the pancreatic β cells resulting in over production of insulin. Other examples that may benefit from GLP-1 antagonism include hyperinsulinism, anorexia and controlling glucagon secretion in type 1 diabetes.
Hybridization of a nucleic acid molecule occurs when two complementary nucleic acid molecules undergo an amount of hydrogen bonding to each other. The stringency of hybridization can vary according to the environmental conditions surrounding the nucleic acids, the nature of the hybridization method, and the composition and length of the nucleic acid molecules used. Calculations regarding hybridization conditions required for attaining particular degrees of stringency are discussed in Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 2001); and Tijssen, Laboratory Techniques in Biochemistry and Molecular Biology-Hybridization with Nucleic Acid Probes Part I, Chapter 2 (Elsevier, New York, 1993). The Tm is the temperature at which 50% of a given strand of a nucleic acid molecule is hybridized to its complementary strand. The following is an exemplary set of hybridization conditions and is not limiting:
Very High Stringency (Allows Sequences that Share at least 90% Identity to Hybridize)
Hybridization: 5× SSC at 65° C. for 16 hours
Wash twice: 2× SSC at room temperature (RT) for 15 minutes each
Wash twice: 0.5× SSC at 65° C. for 20 minutes each
High Stringency (Allows Sequences that Share at least 80% Identity to Hybridize)
Hybridization: 5×-6× SSC at 65° C.-70° C. for 16-20 hours
Wash twice: 2× SSC at RT for 5-20 minutes each
Wash twice: 1× SSC at 55° C.-70° C. for 30 minutes each
Low Stringency (Allows Sequences that Share at least 50% Identity to Hybridize)
Hybridization: 6× SSC at RT to 55° C. for 16-20 hours
Wash at least twice: 2×-3× SSC at RT to 55° C. for 20-30 minutes each.
According to a further aspect of the invention there is provided a polypeptide encoded by a nucleic acid molecule according to the invention.
According to an aspect of the invention there is provided a polypeptide comprising an amino acid sequence selected from the group consisting of:
According to an aspect of the invention there is provided a homodimer consisting of two polypeptides according to the invention.
According to a further aspect of the invention there is provided a nucleic acid molecule that encodes a polypeptide according to the invention.
According to a further aspect of the invention there is provided a vector comprising a nucleic acid molecule according to the invention.
In a preferred embodiment of the invention said vector is an expression vector adapted to express the nucleic acid molecule according to the invention.
A vector including nucleic acid (s) according to the invention need not include a promoter or other regulatory sequence, particularly if the vector is to be used to introduce the nucleic acid into cells for recombination into the genome for stable transfection. Preferably the nucleic acid in the vector is operably linked to an appropriate promoter or other regulatory elements for transcription in a host cell. The vector may be a bi-functional expression vector which functions in multiple hosts. By “promoter” is meant a nucleotide sequence upstream from the transcriptional initiation site and which contains all the regulatory regions required for transcription. Suitable promoters include constitutive, tissue-specific, inducible, developmental or other promoters for expression in eukaryotic or prokaryotic cells. “Operably linked” means joined as part of the same nucleic acid molecule, suitably positioned and oriented for transcription to be initiated from the promoter. DNA operably linked to a promoter is “under transcriptional initiation regulation” of the promoter.
In a preferred embodiment the promoter is a constitutive, an inducible or regulatable promoter.
According to a further aspect of the invention there is provided a cell transfected or transformed with a nucleic acid molecule or vector according to the invention.
Preferably said cell is a eukaryotic cell. Alternatively said cell is a prokaryotic cell.
In a preferred embodiment of the invention said cell is selected from the group consisting of; a fungal cell (e.g. Pichia spp, Saccharomyces spp, Neurospora spp); insect cell (e.g. Spodoptera spp); a mammalian cell (e.g. COS cell, CHO cell); a plant cell.
According to a further aspect of the invention there is provided a pharmaceutical composition comprising a polypeptide according to the invention including an excipient or carrier.
In a preferred embodiment of the invention said pharmaceutical composition is combined with a further therapeutic agent.
When administered the pharmaceutical composition of the present invention is administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salt, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents.
The pharmaceutical compositions of the invention can be administered by any conventional route, including injection. The administration and application may, for example, be oral, intravenous, intraperitoneal, intramuscular, intracavity, intra-articular, subcutaneous, topical, dermal (e.g a cream lipid soluble insert into skin or mucus membrane), transdermal, or intranasal.
Pharmaceutical compositions of the invention are administered in effective amounts. An “effective amount” is that amount of pharmaceuticals/compositions that alone, or together with further doses or synergistic drugs, produces the desired response. This may involve only slowing the progression of the disease temporarily, although more preferably, it involves halting the progression of the disease permanently. This can be monitored by routine methods or can be monitored according to diagnostic methods.
The doses of the pharmaceutical compositions administered to a subject can be chosen in accordance with different parameters, in particular in accordance with the mode of administration used and the state of the subject (i.e. age, sex). When administered, the pharmaceutical compositions of the invention are applied in pharmaceutically-acceptable amounts and in pharmaceutically-acceptable compositions. When used in medicine salts should be pharmaceutically acceptable, but non-pharmaceutically acceptable salts may conveniently be used to prepare pharmaceutically-acceptable salts thereof and are not excluded from the scope of the invention. Such pharmacologically and pharmaceutically-acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric, hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic, citric, formic, malonic, succinic, and the like. Also, pharmaceutically-acceptable salts can be prepared as alkaline metal or alkaline earth salts potassium or calcium salts.
The pharmaceutical compositions may be combined, if desired, with a pharmaceutically-acceptable carrier. The term “pharmaceutically-acceptable carrier” as used herein means one or more compatible solid or liquid fillers, diluents or encapsulating substances that are suitable for administration into a human. The term “carrier” denotes an organic or inorganic ingredient, natural or synthetic, with which the active ingredient is combined to facilitate the application. The components of the pharmaceutical compositions also are capable of being co-mingled with the molecules of the present invention, and with each other, in a manner such that there is no interaction that would substantially impair the desired pharmaceutical efficacy.
The pharmaceutical compositions may contain suitable buffering agents, including: acetic acid in a salt; citric acid in a salt; boric acid in a salt; and phosphoric acid in a salt.
The pharmaceutical compositions also may contain, optionally, suitable preservatives, such as: benzalkonium chloride; chlorobutanol; parabens and thimerosal.
The pharmaceutical compositions may conveniently be presented in unit dosage form and may be prepared by any of the methods well-known in the art of pharmacy. All methods include the step of bringing the active agent into association with a carrier that constitutes one or more accessory ingredients. In general, the compositions are prepared by uniformly and intimately bringing the active compound into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product.
Compositions suitable for parenteral administration conveniently comprise a sterile aqueous or non-aqueous preparation that is preferably isotonic with the blood of the recipient. This preparation may be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation also may be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example, as a solution in 1, 3-butane diol. Among the acceptable solvents that may be employed are water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono-or di-glycerides. In addition, fatty acids such as oleic acid may be used in the preparation of injectables. Carrier formulation suitable for oral, subcutaneous, intravenous, intramuscular, etc. administrations can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.
According to a further aspect of the invention there is provided a method to treat a human subject suffering from hyperglycaemia comprising administering an effective amount of at least one polypeptide according to the invention.
In a preferred method of the invention said polypeptide is administered intravenously.
In an alternative preferred method of the invention said polypeptide is administered subcutaneously.
In a further preferred method of the invention said polypeptide is administered at two day intervals; preferably said polypeptide is administered at weekly, 2 weekly or monthly intervals.
In a preferred method of the invention said hyperglycaemic condition is diabetes mellitus.
In a preferred method of the invention diabetes mellitus is type I.
In a preferred method of the invention diabetes mellitus is type II.
In a preferred method of the invention said hyperglycaemia is the result of insulin resistance.
In a preferred method of the invention said hyperglycaemia is the result of Metabolic Syndrome.
According to an aspect of the invention there is provided the use of a polypeptide according to the invention for the manufacture of a medicament for the treatment of diabetes mellitus.
In a preferred embodiment of the invention diabetes mellitus is type I.
In a preferred embodiment of the invention diabetes mellitus is type I
According to a further aspect of the invention there is provided a monoclonal antibody that binds the polypeptide or dimer according to the invention.
Preferably said monoclonal antibody is an antibody that binds the polypeptide or dimer but does not specifically bind GLP-1 or GLP-1 receptor individually.
The monoclonal antibody binds a conformational antigen presented either by the polypeptide of the invention or a dimer comprising the polypeptide of the invention.
In a further aspect of the invention there is provided a method for preparing a hybridoma cell-line producing monoclonal antibodies according to the invention comprising the steps of:
Preferably, the said immunocompetent mammal is a mouse. Alternatively, said immunocompetent mammal is a rat.
The production of monoclonal antibodies using hybridoma cells is well-known in the art. The methods used to produce monoclonal antibodies are disclosed by Kohler and
Milstein in Nature 256, 495-497 (1975) and also by Donillard and Hoffman, “Basic Facts about Hybridomas” in Compendium of Immunology V.II ed. by Schwartz, 1981, which are incorporated by reference.
According to a further aspect of the invention there is provided a hybridoma cell-line obtained or obtainable by the method according to the invention.
According to a further aspect of the invention there is provided a diagnostic test to detect a polypeptide according to the invention in a biological sample comprising:
In a preferred embodiment of the invention said ligand is an antibody; preferably a monoclonal antibody.
Throughout the description and claims of this specification, the words “comprise” and “contain” and variations of the words, for example “comprising” and “comprises”, means “including but not limited to”, and is not intended to (and does not) exclude other moieties, additives, components, integers or steps.
Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.
Features, integers, characteristics, compounds, chemical moieties or groups described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein unless incompatible therewith.
An embodiment of the invention will now be described by example only and with reference to the following figures:
a is the nucleic acid sequence and amino acid sequence of human GLP-1 and human GLP-1 precursor;
a is the full length amino acid sequence of human GLP-1 receptor;
a is the amino acid sequence of human DPP4;
a is the amino acid sequence of human ADA; Figure b is the amino acid sequence of inactive ADA;
a is the full length amino acid sequence of IL4ss-GLP1-(G4S)4-GLP1R(24-145) fusion polypeptide and
a is the full length amino acid sequence of IL4ss-GLP1-(G4S)5-DPP4(39-766; S630A) fusion polypeptide and
a is the full length amino acid sequence of IL4ss-GLP1-(G4S)5-DPP4(39-766; S630A)-(G4S)8-ADA D295E, D296A) fusion polypeptide and
a is the full length amino acid sequence of IL4ss-GLP1-(G4S)7-ADA D295E, D296A)-(G4S)8-DPP4(39-766; S630A) fusion polypeptide and amino acid sequence of IL4ss-Ex4(9-39)-(G4S)7-ADA D295E, D296A)-(G4S) 8-DPP4(39-766; S630A) antagonist fusion polypeptide and
a is the full length amino acid sequence of GLP1Rss-GLP1R(24-145) -(G4S)2 -LVPR- GLP1 fusion polypeptide and
a is the full length amino acid sequence of HGHss-DPP4(39-766; S630A) -(G4S)5-LVPR-GLP1 fusion polypeptide and
a is the full length amino acid sequence of HGHss-DPP4 (39-766; S630A) -(G4S)8-ADA D295E, D296A)-(G4S)7-GLP1 fusion polypeptide and
a is the full length amino acid sequence of HGHss-ADA D295E, D296A) -(G4S)8-DPP4(39-766; S630A)-(G4S)5-LVPR-GLP1 fusion polypeptide and fusion polypeptide and
a is the full length amino acid sequence of IL4ss-GLP1-(G4S)4-GLP1R (24-145) fusion polypeptide;
a is the full length amino acid sequence of GLP1Rss-GLP1R(24-145) -(G4S)2-LVPR-GLP1;
a is the full length amino acid sequence of IL4ss-GLP1-(G4S)4-GLP1R (24-145) fusion polypeptide;
a is the full length amino acid sequence of GLP1Rss-GLP1R(24-145) -(G4S)2-LVPR-GLP1;
a is the full length amino acid sequence of IL4ss-GLP1-(G4S)4- GLP1R (24-145) fusion polypeptide;
a is the full length amino acid sequence of GLP1Rss-GLP1R(24-145) -(G4S)2 -LVPR-GLP1; -antagonist fusion polypeptide;
a is the full length amino acid sequence of IL4ss-GLP1-(G4S)4-GLP1R (24-145) fusion polypeptide;
a is the full length amino acid sequence of GLP1Rss-GLP1R(24-145) -(G4S)2 -LVPR-GLP1;
a is the nucleic acid sequence of the IL4 signal sequence;
Immunoassays that measure the binding of insulin to polyclonal and monoclonal antibodies are known in the art. Commercially available insulin antibodies are available to detect insulin in samples and also for use in competitive inhibition studies. For example monoclonal antibodies can be purchased at http://www.ab-direct.com/index AbD Serotec.
The components of the fusion proteins were generated by PCR using primers designed to anneal to the ligand or receptor and to introduce suitable restriction sites for cloning into the target vector (
Alternatively, the linker with flanking sequence, designed to anneal to the ligand or receptor domains of choice, was initially synthesised by creating an oligonucleotide duplex and this processed to generate double-stranded DNA (
Expression was carried out in a suitable system (e.g. mammalian CHO cells, E. coil) and this was dependant on the vector into which the insulin-fusion gene was generated. Expression was then analysed using a variety of methods which could include one or more of SDS-PAGE, Native PAGE, western blotting, ELISA well known in the art. Once a suitable level of expression was achieved the insulin fusions were expressed at a larger scale to produce enough protein for purification and subsequent analysis. Purification was carried out using a suitable combination of one or more chromatographic procedures such as ion exchange chromatography, hydrophobic interaction chromatography, ammonium sulphate precipitation, gel filtration, size exclusion and/or affinity chromatography (using nickel/cobalt-resin, antibody-immobilised resin and/or ligand/receptor-immobilised resin)._Purified protein was analysed using a variety of methods which could include one or more of Bradford's assay, SDS-PAGE, Native PAGE, western blotting, ELISA.
The fusion polypeptides include signal sequences that are processed during manufacture of the polypeptide. It will be apparent to one skilled in the art that signal sequences can be selected from a variety of sources appropriate for the particular expression system used [e.g. bacterial, mammalian]. In the not limiting examples disclosed we use the signal sequence of IL4 and growth hormone for expression in mammalian cells. For bacterial expression appropriate periplasmic signal sequences are selected.
Denaturing PAGE, native PAGE gels and western blotting were used to analyse the fusion polypeptides and western blotting performed with antibodies non-conformationally sensitive to the insulin fusion. Native solution state molecular weight information can be obtained from techniques such as size exclusion chromatography using a Superose G200 analytical column and analytical ultracentrifugation.
Two groups were compared with a Student's test if their variance was normally distributed or by a Student-Satterthwaite's test if not normally distributed. Distribution was tested with an F test. One-way ANOVA was used to compare the means of 3 or more groups and if the level of significance was p<0.05 individual comparisons were performed with Dunnett's tests. All statistical tests were two-sided at the 5% level of significance and no imputation was made for missing values.
GLP-1 LR-Fusion Expression: Western Blot of 10A1 and 10G1 from Transient Expressions in CHO Flpin Cells.
50 μl of sample concentrated and then run on and SDS-PAGE gel;
50 μl of sample concentrated and then run on and SDS-PAGE gel,
| Number | Date | Country | Kind |
|---|---|---|---|
| 0815248.0 | Aug 2008 | GB | national |
| 0907794.2 | May 2009 | GB | national |
| 0913901.5 | Aug 2009 | GB | national |
| Filing Document | Filing Date | Country | Kind | 371c Date |
|---|---|---|---|---|
| PCT/GB2009/002006 | 8/18/2009 | WO | 00 | 3/16/2011 |
| Number | Date | Country | |
|---|---|---|---|
| 61090813 | Aug 2008 | US |
