JP2008546373A - Human GLP-1 mimetibody, compositions, methods and uses - Google Patents

Abstract

  The present invention relates to an isolated nucleic acid encoding at least one GLP-1 mimetibody or specified portion or variant, GLP-1 mimetibody or specified portion or variant, vector, host cell, transgenic animal or plant At least one novel human GLP-1 mimetibody or designated portion or variant, as well as methods for making and using them, including therapeutic compositions, methods and devices.

Description

  The present invention relates to a mammalian GLP-1 mimetibody specific for biologically active proteins, fragments or ligands, designated portions and variants, nucleic acids encoding and complementary GLP-1 mimetibodies, hosts The invention relates to cells and methods of making and using them, including therapeutic formulations, administration and devices.

  Recombinant proteins are a class of new generations of therapeutic agents. Such recombinant therapeutics have led to advances in protein formulations and chemical modifications. Such modifications, such as by extending half-life (eg, by inhibiting their exposure to proteolytic enzymes), increasing biological activity, or reducing unwanted side effects, It can potentially enhance therapeutic utility. One such modification is the use of immunoglobulin fragments fused to a receptor protein, such as enteracept. Therapeutic proteins are also constructed using Fc domains to attempt to provide longer half-lives or to incorporate functions such as Fc receptor binding, protein A binding and complement fixation. Yes.

  Diabetes is an increasing epidemic that is estimated to affect over 300 million people by 2025 while waiting for effective medication. Type 2 diabetes accounts for 90-95% of all cases. Complications arising from sustained elevated plasma glucose levels include cardiovascular disease, nephropathy, neuropathy and retinopathy. In addition, during the late stages of type 2 diabetes, pancreatic β-cells die and thus stop secreting insulin. Current treatments for diabetes are associated with a variety of adverse side effects including hypoglycemia and weight gain. In addition, current treatment of type 2 diabetes does not cure the disease, but simply prolongs the time until the patient needs insulin therapy.

  Glucagon-like peptide-1 (GLP-1) is a 37 amino acid peptide secreted from intestinal L cells after oral glucose administration. Subsequent endogenous cleavage between the sixth and seventh positions yields a biologically active GLP-1 (7-37) peptide. The GLP-1 (7-37) peptide sequence can be divided into two structural domains. The amino terminal domain of the peptide is involved in signal transduction, while the rest of the peptide appears to bind to the extracellular loop of the helical conformation of the GLP-1 receptor. In response to glucose, active GLP-1 binds to the pancreatic GLP-1 receptor and causes an increase in insulin secretion (insulin secretion). In addition, GLP-1 has been shown to reduce gastric emptying, which can reduce the glucose bolus released into the circulation and reduce food intake. Both of these actions lower blood sugar levels. GLP-1 has also been shown to inhibit apoptosis and increase proliferation of pancreatic beta cells. Thus, GLP-1 is an attractive therapeutic for lowering blood glucose and preserving pancreatic β cells in diabetic patients. In addition, GLP-1 activity is controlled by blood glucose levels. If the blood glucose level falls to a certain threshold level, GLP-1 is not active. Thus, there is no risk of hypoglycemia associated with treatment with GLP-1.

The feasibility of GLP-1 therapy has been demonstrated in the office. Six weeks of GLP-1 infusion effectively reduced fasting and 8-hour mean blood glucose levels in patients with type 2 diabetes. GLP-1 therapy also resulted in improved beta cell function. Exenatide is a GLP-1 analog currently in clinical trials. Exenatide was first identified in saliva of the American lizard and is 53% identical to GLP-1. Exenatide can bind the GLP-1 receptor and initiate a signaling cascade responsible for many activities attributed to GLP-1 (7-37). To date, it has been shown to reduce HbA1c and serum fructosamine levels in patients with type 2 diabetes. In addition, it delayed gastric emptying and inhibited food intake in healthy volunteers.

  However, GLP-1 is rapidly inactivated by the protease, dipeptidyl peptidase IV (DPP-IV) in vivo. Thus, the usefulness of treatments involving GLP-1 peptides is limited by their rapid disappearance and short half-life. For example, GLP-1 (7-37) has a serum half-life of only 3 to 5 minutes. GLP-1 (7-36) amide has a duration of action of about 50 minutes when administered subcutaneously. Even analogs and derivatives that are resistant to cleavage of endogenous proteases do not have long enough half-lives to avoid repeated administration over 24 hours. For example, exenatide is resistant to DPP-IV, yet it still requires twice a day pre-meal administration due to its short half-life and great variability in pharmacokinetics in vivo. Another compound currently in clinical trial, NN2211, is a lipidated GLP-1 analog. It is expected to be administered once a day.

  The rapid disappearance of a therapeutic agent is inconvenient when it is desirable to maintain a high blood concentration of the agent over time. In that case, repeated administration will be necessary. In addition, long-acting compounds are particularly important for diabetic patients whose past treatment regimes required to take only oral medications. These patients often have a very difficult time to transition to a regimen that requires multiple injections of the drug. GLP-1 therapy with increased half-life appears to have significant advantages over other GLP-1 peptides and compounds under development.

  Accordingly, there is a need to provide improved and / or modified versions of GLP-1 therapeutic proteins that overcome these and other problems known in the art. Mimetibody technology provides a new delivery platform for peptide therapeutics. GLP-1 mimetibodies can provide a means to deliver GLP-1 peptides in a sustained manner and provide improvements over currently developed GLP-1 peptides. Moreover, based on its dimeric structure and its tissue distribution characteristics, GLP-1 mimetibody may have distinguishable characteristics with respect to insulin secretion, β-cell storage and food intake.

[Summary of Invention]
The present invention relates to modified immunoglobulins, cleavage products and other designated portions thereof as described and / or enabled herein in combination with those known in the art. Human GLP-1 mimetibodies, including variants, and compositions of GLP-1 mimetibodies, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and their Provide creation and usage.

  The invention also provides at least one isolated GLP-1 mimetibody or specified portion or variant as described herein and / or as known in the art. The GLP-1 mimetibody optionally has at least one partially variable region directly attached to any linker sequence (L) that is directly attached to at least one GLP-1 therapeutic peptide (P). Including at least one CH3 region directly coupled to at least one CH2 region directly coupled to at least a portion (H) of at least one hinge region or fragment thereof directly coupled to (V) obtain.

In a preferred embodiment, a pair of CH3-CH2-hinge-partial V region sequences-linker-therapeutic peptide sequences (the pair is optionally associated or not limited to at least one Cys-Cys). Linked by a covalent bond, which may include a disulfide bond or at least one CH4 or other immunoglobulin sequence). In one aspect, the GLP-1 mimetibodies are a variety of types that can include, but are not limited to, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, IgE, or any subclass thereof. Mimics the immunoglobulin molecule of formula (I):
a. (P (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
[Wherein P is at least one bioactive GLP-1 peptide, variant or derivative, and L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence that can be a polypeptide, V is at least a portion of the C-terminus of an immunoglobulin variable region, H is at least a portion of an immunoglobulin variable hinge region, and CH2 is an immunoglobulin CH2 constant region. At least a portion, CH3 is at least a portion of an immunoglobulin CH3 constant region, n is an integer from 1 to 10, and o, p, q, r, s and t are independently from 0 to 10 Can be an integer],
Or any combination thereof.

  The variable region of the antibody sequence is not limited, but includes PCT Publication No. WO 05/05604 (No. PCT) filed Jun. 24, 2004 and published Jan. 20, 2005 with the corresponding SEQ ID Nos. 1-9. US 04/19988) described in SEQ ID NOs: 47-55 or Table 1 further optionally comprising at least one substitution, insertion or deletion as further described in FIGS. However, at least a part of at least one of these fragments can be mentioned. The CH2, CH3 and hinge regions include, but are not limited to, PCT Publication No. WO 05/05604 (No. 05/05604, filed Jun. 24, 2004 and published Jan. 20, 2005 with corresponding SEQ ID NOs: 32-40. PCT US04 / 19988) as described in FIGS. 32-40 of the specification and further described in SEQ ID NOs: 56-64 or Table 1, optionally further comprising at least one substitution, insertion or deletion. And at least a portion of at least one of those fragments.

  Accordingly, the GLP-1 mimetibody of the present invention provides a GLP-1 therapeutic peptide and an antibody with its inherent properties and functions while providing its inherent or acquired in vitro, in vivo or in situ properties or activities Mimics at least part of the structure or function of an immunoglobulin. The various portions of the antibody and therapeutic peptide portions of the GLP-1 mimetibody of the invention can vary as described herein, along with those known in the art.

  The present invention also includes, but is not limited to, at least one biologically active GLP-1 peptide corresponding to the P moiety of formula (I) described herein or known in the art, or Also provided is at least one isolated GLP-1 mimetibody or specified portion or variant having at least one activity that can mention a known biological activity of the polypeptide.

In one aspect, the present invention relates to SEQ ID NO: 1, or at least one, optionally accompanied by one or more substitutions, deletions or insertions described herein or as known in the art. At least one isolated human GLP-1 mimetibody comprising a species polypeptide sequence is provided. In another aspect, the at least one GLP-1 mimetibody or designated portion or variant of the present invention comprises at least one epitope comprising at least 1 to 3, at least one ligand (eg, limited Mimics the binding of at least one GLP-1 peptide or a polypeptide corresponding to the P portion of the mimetibody in formula (I) to the entire amino acid sequence of (but not the GLP-1 receptor), or a fragment thereof The ligand binds to at least a portion of SEQ ID NO: 1, or optionally with one or more substitutions, deletions or insertions described herein or known in the art. . The at least one GLP-1 mimetibody optionally binds the GLP-1 receptor with an affinity of at least 10 ⁻⁹ M, at least 10 ⁻¹⁰ M, at least 10 ⁻¹¹ M, or at least 10 ⁻¹² M. obtain. GLP-1 mimetibodies can therefore be screened according to known methods for the corresponding activity which can include, but is not limited to, binding activity to the receptor or fragment thereof.

  The present invention further provides at least one anti-idiotypic antibody against at least one GLP-1 mimetibody of the present invention. The anti-idiotype antibody or fragment specifically binds at least one GLP-1 mimetibody of the present invention. Anti-idiotype antibodies include, but are not limited to, at least one complementarity determination of at least one heavy or light chain that competitively binds the GLP-1 ligand binding region of at least one GLP-1 mimetibody of the present invention. Including at least a portion of an immunoglobulin molecule that can include a region (CDR) or a ligand binding portion thereof, a heavy or light chain variable region, a heavy or light chain constant region, a framework region, or any portion thereof. Any protein or peptide containing molecule is included. Such idiotype antibodies of the invention can include or be derived from any mammal that can include, but is not limited to, humans, mice, rabbits, rats, rodents, primates, and the like. .

  In one aspect, the present invention provides at least one GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody comprising at least one specified sequence, domain, portion or variant thereof, or their Provided is an isolated nucleic acid molecule comprising, complementary to, having significant identity to, or hybridizing to a polynucleotide encoding a specified portion or variant. The present invention further comprises a recombinant vector comprising at least one nucleic acid molecule encoding said isolated GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody, such nucleic acid and / or recombinant vector As well as nucleic acids, vectors and / or methods of making and / or using such GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibodies.

  An isolated nucleic acid encoding at least one isolated mammalian GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody; an isolated nucleic acid vector comprising said isolated nucleic acid, Prokaryotic or eukaryotic host cells comprising the isolated nucleic acids and / or are also provided. The host cell may optionally be COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2 / 0, 293, HeLa, myeloma or lymphoma cell, or any of them Or at least one selected from an immortalized or transformed cell.

The invention also provides, under conditions where at least one GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody, or specified portion or variant is expressed in a detectable and / or recoverable amount. At least one GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody in a host cell comprising culturing a host cell as described in the literature and / or known in the art, or Also provided is at least one method of expression of a specified portion or variant. The GLP-1 mimetibody or GLP-1 mimetibody anti-antigen is expressed under in vitro, in vivo or in situ conditions such that the GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody is expressed in a detectable or recoverable amount. Also provided is a method of producing at least one GLP-1 mimetibody or GLP-1 mimetibody anti-idiotype antibody comprising translating a nucleic acid encoding an idiotype antibody.

  At least one of the present invention comprising providing a host cell or transgenic animal or transgenic plant capable of expressing GLP-1 mimetibody, or GLP-1 anti-idiotype antibody in a recoverable amount Also provided are methods of producing the isolated human GLP-1 mimetibody or GLP-1 anti-idiotype antibody.

  Further provided in the present invention is at least one GLP-1 mimetibody produced by the above method.

  The invention also includes (a) an isolated GLP-1 mimetibody or a nucleic acid and / or GLP-1 mimetibody encoding a specified portion or variant as described herein; and (b) a suitable carrier. Alternatively, at least one composition comprising a diluent is also provided. The carrier or diluent can in some cases be pharmaceutically acceptable by known methods. The composition may optionally further comprise at least one additional compound, protein or composition.

  Also provided is a composition comprising at least one isolated human GLP-1 mimetibody and at least one pharmaceutically acceptable carrier or diluent. The composition may optionally be a detectable label or reporter, an anti-infective agent, a drug associated with diabetes or insulin metabolism, a cardiovascular (CV) agent, a central nervous system (CNS) agent, an autonomic nervous system (ANS) ) Drugs, respiratory tract drugs, gastrointestinal (GI) tract drugs, hormonal drugs, fluid or electrolyte balance drugs, blood products, antitumor drugs, immunomodulators, eye, ear or nose drugs, topical drugs, nutritional drugs, TNF antagonist, anti-rheumatic drug, muscle relaxant, narcotic, non-steroidal anti-inflammatory drug (NTHE), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocker, antibacterial, anti-psoriatic, cortico Steroid, anabolic steroid, erythropoietin, immunization, immunoglobulin, immunosuppressant, growth hormone, hormone replacement agent, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma, Agonists, inhaled steroids, may include epinephrine or analog, further an effective amount of at least one compound or protein selected from the lowest one cytokine or cytokine antagonist.

  The invention also provides a therapeutically or prophylactically effective amount of at least one GLP-1 mimetibody or specified portion or variant of at least one composition, device and / or delivery method of the invention.

  The invention further includes at least one species in a cell, tissue, organ, animal or patient and / or before, after or during the associated condition as is known in the art and / or described herein. Provided is a method or composition of at least one GLP-1 mimetibody for administering a therapeutically effective amount to modulate or treat a condition associated with GLP-1.

The present invention further includes a number of cells, tissues, organs, animals or patients as known in the art and / or can include, but are not limited to, before, after, or between related diseases or treatment conditions. Therapeutically effective for modulation to treat or reduce symptoms of at least one metabolic, immune, cardiovascular, infectious, malignant and / or neurological disorder as required in a variety of conditions Provide at least one GLP-1 mimetibody, designated portion or variant in the method or composition when administered in an amount.

  The present invention further includes diabetes as required in many diverse conditions known in the art, including but not limited to, before, after, or between related diseases or treatment conditions. Or disorders related to insulin metabolism, bone and joint disorders, cardiovascular disorders, dental or oral disorders, dermatological disorders, ear, nose or throat disorders, endocrine or metabolic disorders, gastrointestinal disorders, gynecological disorders, liver or gallbladder Disorders, obstetric disorders, hematological disorders, immunological or allergic disorders, infectious diseases, musculoskeletal disorders, oncological disorders, neurological disorders, nutritional disorders, ophthalmic disorders, pediatric disorders, addiction disorders, mental disorders Disorders, kidney disorders, lung disorders, or any other known disorder (eg, The Merck Manual, 17th edition, Merck Research Labo for adjustment to treat or reduce at least one symptom of attories, Merck and Co., White House Station, NJ (1999), which is hereby incorporated by reference in its entirety. Provides at least one GLP-1 mimetibody, designated portion or variant in a method or composition when administered in a therapeutically effective amount.

  The invention also provides at least one composition, device and / or delivery method for diagnosing GPL-1 related conditions of at least one GLP-1 mimetibody of the invention.

  The invention further includes at least one species in cells, tissues, organs, animals or patients and / or before, during or between related conditions as known in the art and / or described herein. Provided is a method or composition of at least one GLP-1 mimetibody for diagnosing a condition associated with GLP-1.

(A) contacting or administering to a cell, tissue, organ or animal a composition comprising an effective amount of at least one isolated human GLP-1 mimetibody of the present invention, Also provided are methods of diagnosing or treating disease states in cells, tissues, organs or animals. The method optionally includes cells, tissues, per 0-24 hours, 1-7 days, 1-52 weeks, 1-24 months, 1-30 years, or any range or value therein. It may further comprise using an effective amount of 0.001-50 mg per kilogram of organ or animal. The method may optionally include parenteral, subcutaneous, intramuscular, intravenous, intraarterial, intrabronchial, intraabdominal, intracapsular, intrachondral, sinus, intraceliac, intracerebellar, Intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, Contact or administer by at least one mode selected from intraretinal, intrathecal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vagina, rectum, buccal, sublingual, intranasal or transdermal Can further be included. The methods include: a detectable label or reporter, an anti-infective drug, a drug associated with diabetes or insulin metabolism, a cardiovascular (CV) drug, a central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, an airway Drugs, gastrointestinal (GI) tract drugs, hormonal drugs, drugs for fluid or electrolyte balance, blood products, antitumor drugs, immunomodulators, eye, ear or nose drugs, topical drugs, nutritional drugs, TNF antagonists, anti Rheumatism, muscle relaxant, narcotic, non-steroidal anti-inflammatory drug (NSAID), analgesic, anesthetic, sedative, local anesthetic, neuromuscular blocking agent, antibacterial, anti-psoriatic, corticosteroid, anabolic Steroid, erythropoietin, immunization, immunoglobulin, immunosuppressant, growth hormone, hormone replacement, radiopharmaceutical, antidepressant, antipsychotic, stimulant, asthma, beta agonist, Contacting or administering (a) at least one composition comprising an effective amount of at least one compound or protein selected from at least one of steroids, epinephrine or analogs, cytokines or cytokine antagonists May optionally further comprise administering before, simultaneously or after.

  Also provided is a medical device comprising at least one isolated human GLP-1 mimetibody of the present invention, the device comprising parenteral, subcutaneous, intramuscular, intravenous, intraarterial, intrabronchial, intraperitoneal. (Intraabdominal), intracapsular, intrachondral, sinus, intraperitoneal, intracerebellum, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intrapericardial Intraperitoneal, intrapleural cavity, prostate, lung, rectal, intrarenal, intraretinal, intrathecal, bursa, intrathoracic, intrauterine, intravesical, bolus, vagina, rectum, buccal, tongue Suitable for contacting or administering at least one GLP-1 mimetibody by at least one mode selected from below, intranasally or transdermally.

  For human pharmaceutical or diagnostic use comprising a packaging material and a container comprising at least one isolated human GLP-1 mimetibody of the invention in solution or lyophilized form A product is also provided. The product may optionally be parenteral, subcutaneous, intramuscular, intravenous, intraarterial, intrabronchial, intraabdominal, intracapsular, intrachondral, sinus, intraceliac, intracerebellar, Intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, Intraretinal, intrathecal, bursa, intrathoracic, intrauterine, intravesical, bolus, vagina, rectum, buccal, sublingual, intranasal or transdermal delivery device or as a component of the system Can be included.

  The present invention further provides any invention described herein.

[Detailed Description of the Invention]
The invention comprises a composition comprising an isolated, recombinant and / or synthetic mimetibody or specified portion or variant, and at least one polynucleotide encoding at least one GLP-1 mimetibody. And encoding nucleic acid molecules. Such mimetibodies or designated portions or variants of the present invention include the nucleic acids and specific GLP-1 mimetibody sequences, domains, fragments and designated variants thereof, as well as therapeutic nucleic acids, methods and devices described above. Comprising how to create and use mimeti bodies or specified parts or variants.

  The invention also provides at least one isolated GLP-1 mimetibody or specified portion or variant as described herein and / or as known in the art. The GLP-1 mimetibody optionally has at least one partially variable region directly attached to any linker sequence (L) that is directly attached to at least one GLP-1 therapeutic peptide (P). It may comprise at least one CH3 region that is directly bound to at least one hinge region that is directly bound to (V) or at least one CH2 region that is directly bound to its fragment (H).

In a preferred embodiment, the GLP-1 mimetibody is a variety of types that can include, but are not limited to, IgG1, IgG2, IgG3, IgG4, IgA1, IgA2, IgM, IgD, IgE, or any subclass thereof. Mimics the immunoglobulin molecule of formula (I):
((P (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t), or any combination thereof, , P is at least one bioactive GLP-1 polypeptide, and L can be a polypeptide that provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence, V is at least part of the C-terminus of the immunoglobulin variable region, H is at least part of the immunoglobulin variable hinge region, and CH2 is immunoglobulin C
At least a portion of the H2 constant region, CH3 is at least a portion of an immunoglobulin CH3 constant region, and m, n, o, p, q, r, s, and t are independently between and including 0 and 10 Can be an integer.

  Accordingly, the GLP-1 mimetibodies of the present invention mimic antibody structures with their unique properties and functions while providing therapeutic peptides and their unique or acquired in vitro, in vivo or in situ properties or activities To do. In one preferred embodiment of t = 1, the monomeric CH3-CH2-hinge-partial J sequence-linker-therapeutic peptide can be associated or shared, including but not limited to a Cys-Cys disulfide bond. It can be linked to other monomers by a bond. The various portions of the antibody and the GLP-1 therapeutic peptide portion of at least one GLP-1 mimetibody of the present invention can vary as described herein along with those known in the art.

  The CH3-CH2-hinge portion can be extensively modified according to the present invention to form variants, however, binding to the rescue receptor is maintained. Such variants may remove one or more natural sites that provide structural features or functional activity not required by the fusion molecules of the invention. For example, these sites can be removed by substituting or deleting residues, inserting residues at sites, or cleaving the portion containing the site. The inserted or substituted residue can also be a peptidomimetic or an altered amino acid such as a D-amino acid. One variant of CH3-CH2-hinge is (1) disulfide bond formation, (2) incompatibility with selected host cells, (3) heterogeneity upon expression in selected host cells, (4 Affects or participates in :) glycosylation, (5) interaction with complement, (6) binding to non-rescue Fc receptors, or (7) antibody-dependent cellular cytotoxicity (ADCC) 1 It may lack one or more natural sites or residues. Exemplary CH3-CH2-hinge variants are: Sites involved in disulfide bond formation have been removed. Such removal may avoid reaction with other cysteine-containing proteins present in the host cell used to produce the molecules of the invention. For this purpose, cysteine residues can be removed or substituted with other amino acids (eg alanyl, seryl). Even when cysteine residues are removed, the single-stranded CH3-CH2-hinge domain may still form a dimeric CH3-CH2-hinge domain that is held together non-covalently; 2. The CH3-CH2-hinge region is modified to make it more compatible with the selected host cell. For example, if the molecule is expressed recombinantly in bacterial cells such as E. coli, the PA in the hinge that can be recognized by digestive enzymes in E. coli such as proline iminopeptidase. 2. can remove sequences; A portion of the hinge region is deleted or replaced with other amino acids to prevent heterogeneity when expressed in selected host cells; 4. Eliminates one or more glycosylation sites To do. Residues that are typically glycosylated (eg, asparagine) can confer a cytolytic response. 4. These residues can be deleted or replaced with residues that are not glycosylated (eg alanine); Sites involved in the interaction with complement such as the C1q binding site are removed. Complement recruitment may not be advantageous for the molecules of the invention and can therefore be avoided with such variants; Sites that affect binding to Fc receptors other than rescue receptors are removed. The CH3-CH2-hinge region may have sites for interaction with certain leukocytes that are not required for the fusion molecules of the invention and can therefore be removed; The ADCC site is removed. ADCC sites are known in the art; see, for example, Molec. Immunol. 29 (5): 633-9 (1992). These sites also include molecules and sequences that are not required for the fusion molecules of the invention and can therefore be removed.

A linker polypeptide provides structural flexibility by allowing the mimetibody to have alternative orientations and binding properties. When present, its chemical structure is not critical. The linker is preferably composed of amino acids linked together by peptide bonds. Thus, in a preferred embodiment, the linker is composed of 1 to 20 amino acids linked by peptide bonds, the amino acids being selected from the 20 naturally occurring amino acids. Several of these amino acids can be glycosylated as is well understood by those skilled in the art. In a more preferred embodiment, the 1-20 amino acids are selected from glycine, alanine, serine, proline, asparagine, glutamine and lysine. Even more preferably, the linker is composed of a majority of amino acids without steric hindrance such as glycine and alanine. Thus, preferred linkers are poly (Gly-Ser), polyglycine (especially (Gly) ₄ , (Gly) ₅ ), poly (Gly-Ala) and polyalanine. Other specific examples of linkers are: (Gly) ₃ Lys (Gly) ₄ (SEQ ID NO: 65), (Gly) ₃ AsnGlySer (Gly) ₂ (SEQ ID NO: 66), (Gly) ₃ Cys (Gly) ₄ (sequence) No. 67) and GlyProAsnGlyGly (SEQ ID NO: 68).

In order to describe the above nomenclature, for example, (Gly) ₃ Lys (Gly) ₄ means Gly-Gly-Gly-Lys-Gly-Gly-Gly-Gly. A combination of Gly and Ala is also preferred. The linkers shown here are exemplary; linkers within the scope of the invention may be much longer and may include other residues.

Non-peptide linkers are also possible. For example, an alkyl linker such as —NH— (CH 2) s —C (O) — (where s = 2 to 20) may be used. These alkyl linkers can be further substituted with any sterically unhindered group, such as lower alkyl (eg C ₁ -C ₆ ) lower acyl, halogen (eg Cl, Br), CN, NH 2, phenyl, and the like. An exemplary non-peptide linker is a PEG linker having a molecular weight of 100 to 5000 kD, preferably 100 to 500 kD. The peptide linker may be varied to form the derivative in the same manner as described above.

As used herein, “GLP-1 peptide” or “GLP-1 peptide, variant or derivative” refers to at least one GLP-1 peptide, GLP-1 fragment, GLP-1 homolog, GLP-1 It can be an analog or a GLP-1 derivative. GLP-1 peptides have about 25 homology to natural GLP-1 (7-37) such that they exhibit insulinotropic activity by binding to the GLP-1 receptor of pancreatic β cells. To about 45 naturally occurring or non-naturally occurring amino acids. GLP-1 (7-37) is SEQ ID NO: 15:
His-Ala-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp- Leu-Val-Lys-Gly-Arg-Gly
It has the amino acid sequence of

A GLP-1 fragment is a polypeptide obtained after cleavage of one or more amino acids from the N-terminus and / or C-terminus of GLP-1 (7-37) or an analog or derivative thereof. A GLP-1 homolog is a peptide in which one or more amino acids have been added to the N-terminus and / or C-terminus of GLP-1 (7-37) or a fragment or analog thereof. A GLP-1 analog is a peptide in which one or more amino acids of GLP-1 (7-37) have been modified and / or substituted. The GLP-1 analog has sufficient homology to GLP-1 (7-37) or a fragment of GLP-1 (7-37) such that the analog has insulinotropic activity. GLP-1 derivatives have the amino acid sequence of GLP-1 peptide, GLP-1 homolog or GLP-1 analog, but one of its amino acid side groups, α-carbon atoms, terminal amino groups or terminal carboxylic acid groups, It is defined as a molecule that additionally has further chemical modifications.

Numerous active GLP-1 fragments, analogs and derivatives are known in the art, and any of these analogs and derivatives may also be part of the GLP-1 mimetibody of the present invention. Several GLP-1 analogs and GLP-1 fragments known in the art are described in US Pat. Nos. 5,118,666, 5,977,071, and 5,545,618, As well as Adelhorst et al . Biol. Chem. 269: 6275 (1994). Examples include but are not limited to GLP-1 (7-34), GLP-1 (7-35), GLP-1 (7-36), Gln9-GLP-1 (7-37), D-Gln9 -GLP-1 (7-37), Thr16-Lys18-GLP-1 (7-37) and Lys18-GLP-1 (7-37) can be mentioned.

  “GLP-1 mimetibody”, “GLP-1 mimetibody part” or “GLP-1 mimetibody fragment” and / or “GLP-1 mimetibody variant” and the like include, but are not limited to, the lowest of SEQ ID NO: 1. At least one GLP-1 peptide, variant or derivative of which at least one can be mentioned, but at least one of which can mention ligand binding in vitro, in situ and / or preferably in vivo Has, mimics or stimulates biological activity. For example, a suitable GLP-1 mimetibody, specified portion or variant may also modulate, increase, modify, activate at least one GLP-1 receptor signaling or other measurable or detectable activity.

GLP-1 mimetibodies useful in the methods and compositions of the invention are characterized by having a suitable affinity for binding to a protein ligand, such as the GLP-1 receptor, and optionally and preferably low toxicity. In particular, the variable region, constant region (not including the CH1 portion) and part of the framework, or any part thereof (eg part of the J, D or V region of the variable heavy or light chain; at least one hinge region, constant GLP-1 mimetibodies in which individual components, such as at least a portion of the H chain or L chain, individually and / or collectively, optionally and preferably have low immunogenicity are useful in the present invention. The mimetibodies that can be used in the present invention are optionally characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and / or high affinity, as well as other undefined characteristics, can contribute to the therapeutic outcome achieved. “Low immunogenicity” is less than about 75% of patients being treated, or preferably about 50, 45, 40, 35, 30, 35, 20, 15, 10, 9, 8, 7, 6, 5, Produce a significant HAMA, HACA or HAHA response in less than 4, 3, 2 and / or 1%, and / or lower titers in the patient being treated (less than about 300 as measured in a dual antigen enzyme immunoassay, Preferably less than 100) as defined herein (see, eg, Elliott et al., Lancet 344: 1125-1127 (1994)).

  Usability. Isolated nucleic acids of the present invention include, but are not limited to, diabetes related disorders, insulin metabolism related disorders, immune disorders or diseases, cardiovascular disorders or diseases, infectious, malignant and / or neurological disorders or diseases, As well as modulating, treating, alleviating, preventing the occurrence of, or preventing the condition associated with at least one protein selected from at least one of the conditions associated with other known or specified proteins, or Used to produce at least one GLP-1 mimetibody, fragment thereof or specified variant that can be used to achieve a reduction in symptoms in cells, tissues, organs or animals, including mammals and humans Can do.

  Such methods include at least one GLP-1 mimetibody or designated portion or variant for cells, tissues, organs, animals or patients in need of such modulation, treatment, alleviation, prevention or reduction of symptoms, effects or mechanisms Administering an effective amount of a composition comprising or a pharmaceutical composition. An effective amount is about 0.0001 to 500 mg / kg per single or multiple doses, as performed and determined using known methods described herein or known in the relevant art. Or an amount to achieve a serum concentration of 0.01-5000 μg / ml serum concentration or any effective range or value therein per single or multiple doses.

Quote. All publications or patents cited in this specification are to be cited to illustrate the prior art at the time of the present invention and / or to provide a description and applicability of the present invention. Is incorporated herein in its entirety. A publication refers to any scholarly or patent publication, or any other information available in any media format, including all recordings, video recordings, electronic or print formats. The following references are incorporated herein by reference in their entirety: edited by Ausubel et al., Current.
Protocols in Molecular Biology, John Wiley & Sons, Inc. New York, NY (1987-2003); Sambrook et al., Molecular Cloning: A Laboratory.
Manual, 2nd edition, Cold Spring Harbor, NY (1989); Harlow and Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); edited by Colligan et al., Current Protocols in Immunology. New York (1994-2003); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, New York, NY (1997-2003).

  The mimeti body of the present invention. The GLP-1 mimetibody optionally has at least one partially variable region directly attached to any linker sequence (L) that is directly attached to at least one GLP-1 therapeutic peptide (P). Directly bound to at least one CH2 region directly bound to at least a portion (H) of at least one hinge region fragment such that it comprises at least one core hinge region directly bound to (V). May contain at least one CH3 region. In a preferred embodiment, a pair of CH3-CH2-HVLP can be linked or linked by a covalent bond that can include, but is not limited to, a Cys-Cys disulfide bond. Accordingly, the GLP-1 mimetibodies of the present invention mimic antibody structures with their unique properties and functions while providing therapeutic peptides and their unique or acquired in vitro, in vivo or in situ properties or activities To do. The various portions of the antibody and the therapeutic peptide portion of at least one GLP-1 mimetibody of the present invention can vary as described herein along with those known in the art.

  The mimetibodies of the present invention thus include, but are not limited to, extended half-life, increased activity, more specific activity, increased affinity, increased or decreased off rate, Known proteins that can include at least one of a selected or more suitable subset of activity, less immunogenicity, at least one desired therapeutic effect increased quality or duration, fewer side effects, etc. Provides at least one suitable property compared to

Fragments of mimetibodies of formula (I) may be produced by enzymatic cleavage, synthesis or recombinant techniques as known in the art and / or described herein. Mimetibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. The various parts of the mimetibody can be joined together chemically by conventional techniques, or can be produced as a continuous protein using genetic engineering techniques. For example, a nucleic acid encoding at least one of the constant regions of a human antibody chain can be expressed to produce a continuous protein for use in the mimetibody of the present invention. See, for example, Ladner et al., US Pat. No. 4,946,778 and Bird, R., et al. E. Science , 242: 423-426 (1988).

As used herein, the term “human mimetibody” refers to substantially any portion of the protein (eg, GLP-1 peptide, _CH domain (eg, C _H 2, C _H 3), hinge, V) refers to an antibody that is expected to be substantially non-immunogenic in humans with only minor sequence changes or variations. Such changes or variations optionally and preferably retain or reduce human immunogenicity with respect to unmodified human antibodies or mimetibodies of the invention. Accordingly, the human antibodies and corresponding GLP-1 mimetibodies of the present invention are different from chimeric or humanized antibodies. It is pointed out that GLP-1 mimetibodies can be produced by non-human animals or cells capable of expressing human immunoglobulin (eg heavy and / or light chain) genes.

  Human mimetibodies that are specific for at least one of these protein ligands are bound to a suitable ligand such as an isolated GLP-1 receptor or a portion thereof (including synthetic molecules such as synthetic peptides). Can be designed. The production of such mimetibodies is carried out using known techniques for identifying and characterizing the ligand binding region or sequence of at least one protein or portion thereof.

  In a preferred embodiment, the at least one GLP-1 mimetibody or designated portion or variant of the present invention is a clone of at least one cell line, mixed cell line, immortalized cell, or immortalized and / or cultured cell. Produced by a population. Immortalized protein-producing cells can be produced using suitable methods. Preferably, at least one GLP-1 mimetibody or specified portion or variant is functionally rearranged or can undergo functional rearrangement, and is not limited in the art. In this specification, mention may be made of the known formula (I), wherein the C-terminal variable region part may be used for V, the hinge region for H, CH2 for CH2 and CH3 for CH3. Produced by providing a nucleic acid or vector comprising DNA derived from or having a sequence substantially similar to at least one human immunoglobulin locus further comprising a mimetibody structure as described.

  As used herein, the term “functionally rearranged” refers to one immunoglobulin chain (eg, heavy chain) or any portion thereof that has undergone V (D) J recombination. Refers to a segment of nucleic acid from an immunoglobulin locus that gives rise to an immunoglobulin gene encoding. Functionally rearranged immunoglobulin genes can be encoded by, for example, nucleotide sequencing, hybridization using probes that can anneal to the coding junctions between gene segments (eg, Southern blotting, Northern blotting), or coding between gene segments. Identification can be made directly or indirectly using suitable methods such as enzymatic amplification of immunoglobulin genes (eg, polymerase chain reaction) using primers that can anneal to the junction. Whether a cell produces a GLP-1 mimetibody or portion or variant comprising a particular variable region or a variable region comprising a particular sequence (eg, at least one P sequence) also uses suitable methods Can be determined.

  The mimetibodies, designated portions and variants of the present invention provide transgenic animals or mammals such as goats, cows, horses, sheep, etc. that produce such mimetibodies or designated portions or variants in their milk As such, nucleic acids encoding at least one GLP-1 mimetibody or specified portion or variant may also be used. Such animals can be provided using known methods as applied to antibody-encoding sequences. For example, but not limited to, U.S. Pat. Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; No. 5,616, No. 5,565,362; No. 5,304,489, each of which is incorporated herein by reference in its entirety.

  The mimetibodies, designated portions and variants of the present invention include, but are not limited to, transgenic plants and cultured plant cells that produce such mimetibodies, designated portions or variants in plant parts or cells cultured therefrom. Can be produced using nucleic acids encoding at least one GLP-1 mimetibody or specified portion or variant. As a non-limiting example, transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large quantities of recombinant proteins, for example using inducible promoters. See, for example, Cramer et al., Curr. Top. Microbol. Immunol. 240: 95-118 (1999) and references cited therein. In addition, transgenic maize, or corn, with biological activity equivalent to that produced by other recombinant systems or purified from natural sources, can be used to produce mammalian proteins at commercial production levels. Used to express. See, for example, Hood et al., Adv. Exp. Med. Biol. 464: 127-147 (1999) and references cited therein. Antibodies including antibody fragments such as single chain mimetibody (scFv) are also produced in large quantities from transgenic plant seeds including tobacco seeds and potato tubers. For example, Conrad et al., Plant Mol. Biol. 38: 101-109 (1998) and references cited therein. Thus, mimetibodies, designated portions and variants of the present invention can also be produced using transgenic plants according to known methods. See, for example, Fischer et al., Biotechnol. Appl. Biochem. 30: 99-108 (Oct, 1999), Ma et al., Trends Biotechnol. 13: 522-7 (1995); Ma et al., Plant Physiol. 109: 341-6 (1995); Whiteram et al., Biochem. Soc. Trans. 22: 940-944 (1994); and references cited therein. The above references are incorporated herein by reference in their entirety.

The mimetibodies of the present invention can bind human protein ligands with a wide range of affinities (K _D ). In a preferred embodiment, the at least one human GLP-1 mimetibody of the present invention can optionally bind at least one protein ligand with high affinity. For example, least one GLP-1 mimetibody of the present invention is about ^{10 -7} M to equal to or less a _{K D} or more preferably about, 0.1 to 9.9 (or any of them Range or value) × 10 ⁻⁷ , 10 ⁻⁸ , 10 ⁻⁹ , 10 ⁻¹⁰ , 10 ⁻¹¹ , 10 ⁻¹² or 10 ⁻¹³ M equal to or less than K _D , or any of them A range or value can bind at least one protein ligand.

The affinity, or affinity, of a GLP-1 mimetibody for at least one protein ligand is, for example, the affinity of antibody-antigen binding (af
It can be measured experimentally using any suitable method that is used to measure fidelity, ie, affinity. (For example, Fundamental
Immunology , Paul, W.M. E. Ed., Raven Press: New York, NY (1984), Berzofsky et al., “Antibody-Antigen Interactions,”; Kuby, Janis Immunology , W. H. Freeman and Company: New York, NY (1992); and methods described therein). The measured affinity of a particular GLP-1 mimetibody-ligand interaction may vary when measured under different conditions (eg, salt concentration, pH). Therefore, measurement of affinity and other ligand-binding parameters (e.g. _{K D, K a, K d} ) are preferably, or either standardized solutions of GLP-1 mimetibody and ligand, and is described herein This is done using standardized buffers such as those known in the art.

  Nucleic acid molecule. A nucleotide sequence encoding at least one of SEQ ID NOs: 1 and 6, and at least 90-100% of contiguous amino acids of at least a portion of an antibody (SEQ ID NO: 1), further comprising the designated fragment, variant or consensus sequence Inserted as a P sequence of formula (I) to provide a GLP-1 mimetibody of the invention, or a deposit comprising at least one of these sequences The nucleic acid molecules of the invention that encode at least one GLP-1 mimetibody or specified portion or variant using the generated vectors are described herein or as known in the art. Can be obtained using the method.

  The nucleic acid molecules of the present invention may comprise RNA, such as mRNA, hnRNA, tRNA or any other form, or, but not limited to, cDNA and genomic DNA obtained by cloning or synthetically produced. It can be the form of DNA that can be mentioned, or any combination thereof. The DNA can be triple stranded, double stranded or single stranded, or any combination thereof. Any portion of at least one strand of DNA or RNA can be the coding strand, also known as the sense strand, or it can be the non-coding strand, also referred to as the antisense strand.

  An isolated nucleic acid molecule of the invention optionally comprises a nucleic acid molecule comprising an open reading frame (ORF) comprising one or more introns, a GLP-1 mimetibody or a coding sequence of a specified portion or variant A nucleic acid molecule comprising: and a nucleotide sequence substantially different from that described above, but described herein by virtue of the degeneracy of the genetic code and / or as known in the art Nucleic acid molecules that still encode one GLP-1 mimetibody may be included. Of course, the genetic code is known in the art. Accordingly, it will be routine to those skilled in the art to generate such degenerate nucleic acid variants that encode a particular GLP-1 mimetibody or specified portion or variant of the present invention. See, for example, Ausubel et al., Supra, and such nucleic acid variants are encompassed by the present invention.

As shown herein, a nucleic acid molecule of the invention comprising a nucleic acid encoding a GLP-1 mimetibody or specified portion or variant includes, but is not limited to, the amino acids of a GLP-1 mimetibody fragment A coding sequence for the GLP-1 mimetibody in whole or in part thereof; a coding sequence for the GLP-1 mimetibody, fragment or part, and at least one signal leader or, but not limited to, transcription Non-coding 5 'and 3' sequences such as transcribed and untranslated sequences that play a role in mRNA processing, including splicing and polyadenylation signals (eg-mRNA ribosome binding and stability). Minimum 1 together with additional non-coding sequences that
Additional sequences such as coding sequences for fusion peptides with or without the aforementioned additional coding sequences such as species introns; additions encoding additional amino acids such as those that provide additional functionality Specific coding sequences. Thus, a sequence encoding a GLP-1 mimetibody or specified portion or variant may purify a specified portion or variant comprising a fused GLP-1 mimetibody, or GLP-1 mimetibody fragment or portion. It can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates.

  A polynucleotide that selectively hybridizes to a polynucleotide as described herein. The present invention relates to an isolated polynucleotide that hybridizes under selective hybridization conditions to others disclosed herein, including a polynucleotide disclosed herein, or a designated variant or portion thereof. Nucleic acids are provided. Thus, the polynucleotides of this embodiment can be used to isolate, detect and / or quantify nucleic acids comprising such polynucleotides.

  Low or moderate stringency hybridization conditions are typically but not exclusively used with sequences having reduced sequence identity with respect to the complementary sequence. Moderate and high stringency conditions may be used in some cases for sequences of greater identity. Low stringency conditions allow selective hybridization of sequences having about 40-99% sequence identity and can be used to identify orthologous or paralogous sequences.

  In some cases, a polynucleotide of the invention can encode at least a portion of a GLP-1 mimetibody or specified portion or variant encoded by a polynucleotide described herein. The polynucleotides of the invention include nucleic acid sequences that can be used for selective hybridization to a polynucleotide encoding a GLP-1 mimetibody or specified portion or variant of the invention. See, eg, Ausubel, supra; Colligan, supra (each incorporated by reference in its entirety).

  Nucleic acid construction. Isolated nucleic acids of the invention can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof as are known in the art.

  The nucleic acid may conveniently comprise a sequence in addition to the polynucleotide of the present invention. For example, a multiple cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in polynucleotide isolation. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the invention. For example, the hexahistidine marker sequence provides a convenient means for purifying the proteins of the present invention. The nucleic acid of the present invention (excluding the coding sequence) is optionally a vector, adapter or linker for cloning and / or expression of the polynucleotide of the present invention.

  Additional sequences may be used in order to optimize their function in cloning and / or expression, to assist in the isolation of the polynucleotide, or to improve the introduction of the polynucleotide into the cell. Alternatively, it can be added to the expression sequence. The use of cloning vectors, expression vectors, adapters and linkers is known in the art. See, eg, Ausubel, supra; or Sambrook, supra.

A method for recombinant construction of nucleic acids. An isolated nucleic acid composition of the present invention, such as RNA, cDNA, genomic DNA, or any combination thereof, is a biological source using any number of cloning methodologies known to those of skill in the art. Can be obtained from In some embodiments, oligonucleotide probes that selectively hybridize to the polynucleotides of the invention under suitable stringency conditions are used to identify the desired sequence in a cDNA or genomic DNA library. RNA isolation and construction of cDNA and genomic libraries are known to those skilled in the art. (See, eg, Ausubel, supra; or Sambrook, supra).

  A method for the synthesis and construction of nucleic acids. Isolated nucleic acids of the invention can also be produced by direct chemical synthesis by known methods (see, eg, Ausubel et al., Supra). Chemical synthesis generally produces a single stranded oligonucleotide which can be converted to double stranded DNA by hybridization with a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template. One skilled in the art will recognize that chemical synthesis of DNA can be limited to sequences of about 100 or more bases, while longer sequences can be obtained by linking shorter sequences.

  Recombinant expression cassette. The invention further provides a recombinant expression cassette comprising the nucleic acid of the invention. A recombinant expression cassette that can be introduced into at least one desired host cell using a nucleic acid sequence of the invention, eg, a cDNA or genomic sequence encoding a GLP-1 mimetibody of the invention or a designated portion or variant. Can be built. A recombinant expression cassette typically includes a polynucleotide of the invention operably linked to a transcription initiation control sequence capable of directing transcription of the polynucleotide in the intended host cell. it can. Both heterologous and non-heterologous (ie endogenous) promoters can be used to direct the expression of the nucleic acids of the invention.

  In some embodiments, the isolated nucleic acid serving as a promoter, enhancer or other element can be adapted to a non-heterologous form of a polynucleotide of the invention so as to up- or down-regulate expression of the polynucleotide of the invention. Can be introduced at any location (upstream, downstream or in an intron). For example, an endogenous promoter can be altered in vivo or in vitro by mutations, deletions and / or substitutions as are known in the art. The polynucleotides of the invention can be expressed in either sense or antisense orientation as desired. It will be appreciated that control of gene expression in either sense or antisense orientation can have a direct impact on observable characteristics. Another suppression method is sense suppression. Introduction of nucleic acids arranged in sense orientation has been shown to be an effective means for inhibiting transcription of target genes.

  Vectors and host cells. The present invention also includes a vector comprising an isolated nucleic acid molecule of the present invention, a host cell genetically engineered with the recombinant vector, and a recombinant technology known in the art. It also relates to the production of one GLP-1 mimetibody or specified portion or variant. See, for example, Sambrook et al., Supra; Ausubel et al., Supra (incorporated herein by reference in their entirety).

  The polynucleotide may optionally be linked to a vector containing a selectable marker for growth in the host. In general, plasmid vectors are introduced into cells using suitable known methods such as electroporation, and other known methods include precipitation as calcium phosphate precipitation or complex with charged lipids. Includes the use of vectors in. If the vector is a virus, it can be packaged in vitro using an appropriate packaging cell line and then transduced into host cells.

  The DNA insert should be effectively linked to a suitable promoter. The expression construct may further optionally contain a site for transcription initiation, at least one of termination, and a ribosome binding site for translation in the transcribed region. The coding portion of the mature transcript expressed by the construct preferably includes a stop codon (eg UAA, UGA or UAG) appropriately positioned at the beginning and end of translation of the mRNA to be translated. UAA and UAG are preferred for mammalian or eukaryotic cell expression.

The expression vector is preferably but can optionally include at least one selectable marker. Such markers include, but are not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, US Pat. No. 4,399,216; 4,634,665 for eukaryotic cell cultures. No. 4,656,134; No. 4,956,288; No. 5,149,636; No. 5,179,017, ampicillin, neomycin (G418), mycophenolic acid or Glutamine synthase (GS, US Pat. Nos. 5,122,464; 5,770,359; 5,827,739) resistance, and E. coli and other bacteria or Tetracycline or ampicillin resistance genes for culturing in prokaryotes (the above patent is incorporated by reference in its entirety) Suitable media and culture conditions for the host cells described above are known in the art, suitable vectors will be readily apparent to those skilled in the art, and the vector constructs into the host cells. Can be accomplished by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid mediated transfection, electroporation, transduction, infection or other known methods such as Sambrook. , Supra, chapters 1-4 and 16-18; Ausubel, supra, chapters 1, 9, 13, 15, 16 described in the art.

  At least one GLP-1 mimetibody or specified portion or variant of the invention can be expressed in a modified form, such as a fusion protein, and not only a secretion signal, but also an additional heterologous functional region Can also be included. For example, GLP-1 mimetibody or a region of additional amino acids, especially a charged amino acid, to improve stability and refractory properties in host cells, during purification, or during subsequent handling and storage. It can be added to the N-terminus of a specified portion or variant. In addition, peptide moieties may be added to the GLP-1 mimetibodies or specified moieties or variants of the present invention to facilitate purification. Such regions can be removed prior to final preparation of the GLP-1 mimetibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals such as Sambrook, supra, 17.29-17.42 and 18.1-18.74; Ausubel, supra, chapters 16, 17 and 18. It is described in.

  Those skilled in the art are aware of numerous expression systems that can be used to express nucleic acids encoding the proteins of the invention.

It is mammalian cells that specifically describe cell cultures useful for the production of mimetibodies, specified portions or variants thereof. Mammalian cell lines can often be in the form of a monolayer of cells, although mammalian cell suspensions or bioreactors can also be used. A number of suitable host cell lines capable of expressing intact glycosylated proteins have been developed in the art and include COS-1 (eg, ATCC CRL 1650), COS-7 (eg, ATCC CRL- 1651), HEK293, BHK21 (eg ATCC CRL-10), CHO (eg ATCC CRL 1610, DG-44) and BSC-1 (eg ATCC CRL-26) cell lines, hepG2 cells, P3X63Ag8.653, SP2 / 0- Ag14, 293 cells, HeLa
These include cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, VA. Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC accession number CRL-1580) and SP2 / 0-Ag14 cells (ATCC accession number CRL-1851).

  Expression vectors for these cells include, but are not limited to, origins of replication; promoters (eg late or early SV40 promoter, CMV promoter (eg US Pat. No. 5,168,062; US Pat. No. 5,385,839) ), HSV tk promoter, pgk (phosphoglycerate kinase) promoter, EF-1α promoter (eg, US Pat. No. 5,266,491), at least one human immunoglobulin promoter; enhancer, and / or Includes one or more expression control sequences that can include processing information sites such as ribosome binding sites, RNA splice sites, polyadenylation sites (eg, SV40 large T Ag poly A addition site), and transcription termination sequences. For example, Au See, for example, Ubel et al., supra; Sambrook et al., supra. (Www.atcc.org) or other known or commercial sources.

  When using eukaryotic host cells, a polyadenylation or transcription termination sequence is typically incorporated into the vector. An example of a termination sequence is a polyadenylation sequence from the bovine growth hormone gene. Sequences for precise splicing of transcripts can also be included. An example of a splicing sequence is the VP1 intron from SV40 (Sprague et al., J. Virol. 45: 773-781 (1983)). In addition, gene sequences that control replication in host cells can be incorporated into vectors as is known in the art.

Purification of GLP-1 mimetibody or specified portions or variants thereof. GLP-1 mimetibodies or designated portions or variants include, but are not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction It can be recovered and purified from recombinant cell cultures by known methods, including chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification. For example, Colligan, Current
Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, New York, NY (1997-2003), for example, Chapters 1, 4, 6, 8, 9, 10 (hereby incorporated by reference in their entirety) See).

The mimetibody or specified portion or variant of the present invention is a eukaryotic host that includes naturally purified products, products of chemical synthesis procedures, and recombinant techniques such as yeast, higher plant, insect and mammalian cells. Including products made from Depending on the host used in the recombinant production procedure, the GLP-1 mimetibody or specified portion or variant of the invention can be glycosylated or unglycosylated, with glycosylation being preferred. Such methods are described in Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, 10, 12, 13, 16, 18, and 20, Colligan, Protein Science, supra, Chapters 12-14 (
All are described in many standard laboratory manuals, such as those incorporated herein by reference in their entirety.

  Mimetibody, specified fragment and / or variant. An isolated mimetibody of the invention is a GLP-1 mimetibody or specified portion or variant encoded by any one of the polynucleotides of the invention, as fully discussed herein, or any Isolated or produced GLP-1 mimetibody or a designated portion or variant thereof.

  Preferably, the GLP-1 mimetibody or ligand binding moiety or variant binds at least one GLP-1 protein ligand, and thereby the biological of at least one GLP-1 of the corresponding protein or fragment thereof. Provides activity. A variety of therapeutically or diagnostically significant proteins are known in the art, and suitable assays or biological activities of such proteins are also known in the art.

Non-limiting examples of GLP-1 peptides, variants and derivatives suitable for the present invention are: Xaa15-Xaa16-Xaa17-Xaa18-Xaa19-Xaa20-Xaa21-Phe-Xaa23-Xaa24-Xaa25-Xaa26-Xaa27-Xaa28-Xaa29-Xaa30-Xaa31 (wherein Xaa2 is Ala, T , Val, Glu, Asp or Lys; Xaa3 is Glu, Asp or Lys; Xaa5 is Thr, Ala, Gly, Ser, Leu, Ile, Val, Arg, His Xaa6 is Phe, His, Trp, or Tyr; Xaa7 is Thr or Asn; Xaa8 is Ser, Ala, Gly, Thr, Leu, Ile, Val, Glu, Asp, or Lys. Xaa9 is Asp or Glu; Xaa10 is Val, Ala, Gly, Ser, Thr, Leu, Ile, Met, Tyr, Trp, His, Phe, Glu, Asp or Lys; Xaa11 is Ser, Val, Ala, Gly, Thr, Leu, Ile, Glu, Asp or Lys; Xaa12 is Ser, Val, Ala, Gly, Thr, Leu, Ile, Glu, Asp or Lys; Xaa13 is Tyr, Phe, Trp, Glu, Xaa14 is Leu, Ala, Met, Gly, Ser, Thr, Leu, Ile, Val, Glu, Asp or Lys; Xaa15 is Glu, Ala, Thr, Ser, Gly, Gln, Asp or Xaa16 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Gln, Asn, Arg, Cys, Glu, Asp or Lys; Xaa17 is Gln, Asn, Arg, His, Glu, Asp or Xaa18 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Arg, Glu, Asp or Lys; Xaa19 is Ala, Gly, Ser, Thr, Leu, Ile, Val, Met, Glu, Asp or Lys Xaa20 is Lys, Arg, His, Gln, Trp, Tyr, Phe, Glu or Asp; Xaa21 is Glu, Leu, Ala, His, Phe, Tyr, Trp, Arg, Gln, Thr, Ser, Gly Xaa23 is Ile, Ala, Val, Leu or Glu; Xaa24 is Ala, Gly, Ser, Thr, Leu, Ile, Val, His, Glu, Asp or Lys; Xaa25 is Trp , Phe, Tyr, Glu, Asp or Lys; Xaa26 is Leu, Gly, Ala, Ser, Thr, Ile, Val, Glu, Asp or Lys; Xaa27 is Val, Leu, Gly, Ala, Ser, Thr , Ile, Arg Glu, be Asp or Lys; Xaa28 is L
ys, Asn, Arg, His, Glu or Asp; Xaa29 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Arg, Trp, Tyr, Phe, Pro, His, Glu, Asp or Lys; Xaa30 is Arg, His, Thr, Ser, Trp, Tyr, Phe, Glu, Asp or Lys; and Xaa31 is Gly, Ala, Ser, Thr, Leu, Ile, Val, Arg, Trp, Tyr, Phe, His , Glu, Asp, and Lys).

  Another preferred group of GLP-1 peptides, variants or derivatives is SEQ ID NO: 6: His-Xaa2-Xaa3-Gly-Thr-Xaa6-Xaa7-Xaa8-Xaa9-Xaa10-Ser-Xaa12-Tyr-Xaa14-Glu-Xaa16 -Xaa17-Xaa18-Xaa19-Lys-Xaa21-Phe-Xaa23-Ala-Trp-Leu-Xaa27-Xaa28-Gly-Xaa30 (where Xaa2 is Ala, Gly or Ser; Xaa3 is Glu or Asp; Xaa6 is Phe or Tyr; Xaa7 is Thr or Asn; Xaa8 is Ser, Thr or Ala; Xaa9 is Asp or Glu; Xaa10 is Val, Leu, Met young Xaa12 is Ser or Lys; Xaa14 is Leu, Ala, or Met; Xaa16 is Gly, Ala, Glu, or Asp; Xaa17 is Gln or Glu; Xaa18 is Ala or Lys; Xaa19 is Ala, Val, Ile, Leu or Met; Xaa21 is Glu or Leu; Xaa23 is Ile, Ala, Val, Leu or Glu; Xaa27 is Val or Lys; Xaa28 is Lys or Asn Yes; and Xaa30 is Arg or Glu).

  These peptides can be produced by methods disclosed and / or known in the art. In the sequence (and throughout this specification unless otherwise specified) Xaa includes the designated amino acid residues, their derivatives or modified amino acids. GLP protected from DPP-IV activity in the context of mimetibodies because the enzyme dipeptidyl peptidase IV (DPP-IV) can be the incumbent on observed rapid in vivo inactivation of administered GLP-1 -1 peptides, homologues, analogs and derivatives are preferred.

  A GLP-1 mimetibody or a designated portion or variant thereof that partially or preferably substantially provides at least one biological activity of GLP-1 is capable of binding a GLP-1 ligand and thereby May provide GLP-1 binding to at least one ligand, such as the GLP-1 receptor, or at least one activity mediated otherwise by other protein-dependent or mediated mechanisms. . The term “GLP-1 mimetibody activity” as used herein refers to at least one GLP-1-dependent activity of about 20-10,000%, preferably at least about 60, depending on the assay. 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000% or more GLP-1 that can be adjusted or caused Refers to mimeti body.

The ability of a GLP-1 mimetibody or specified portion or variant to provide at least one protein dependent activity is preferably at least one as described herein and / or as known in the art. A suitable protein biological assay. The human GLP-1 mimetibody or designated portion or variant of the invention may be similar to any class (IgG, IgA, IgM, etc.) or isotype and may include at least a portion of a kappa or lambda light chain. In one embodiment, the human GLP-1 mimetibody or specified portion or variant comprises at least one IgG heavy chain variable fragment of an isotype, eg, IgG1, IgG2, IgG3 or IgG4, hinge region, CH2 and CH3.

  At least one GLP-1 mimetibody or specified portion or variant of the invention binds at least one ligand, subunit, fragment, portion or any combination thereof. At least one GLP-1 mimetibody of the invention, at least one GLP-1 peptide, variant or derivative of a specified portion or variant may optionally bind at least one specified epitope of a ligand. . The binding epitope may comprise any combination of at least 1 to 3 amino acids of contiguous amino acids of the protein ligand sequence, such as the GLP-1 receptor or portions thereof, or at least one amino acid sequence of the entire specified portion. .

  Such mimetibodies encode GLP-1 mimetibodies using known techniques of recombinant DNA technology by joining together the various portions of formula (I) of GLP-1 mimetibody using known techniques. It can be produced by producing and expressing at least one nucleic acid molecule or by using any other suitable method such as chemical synthesis.

Mimetibodies that bind to human GLP-1 ligands such as receptors and comprise defined heavy or light chain variable regions or portions thereof are phage display (Katsusube, Y. et al., Int J Mol . Med , 1 (5): 863-868 (1998)) or can be produced using methods using transgenic animals as known in the art. The GLP-1 mimetibody, specified portion or variant may be expressed using the encoding nucleic acid or portion thereof in a suitable host cell.

The invention also relates to mimetibodies, ligand binding fragments and immunoglobulin chains comprising amino acids in a sequence that is substantially identical to the amino acid sequences described herein. Preferably, such mimetibodies or ligand binding fragments thereof can bind human GLP-1 ligands such as receptors with high affinity (eg, a K _{D of} less than or equal to about 10 ⁻⁷ M). Amino acid sequences that are substantially identical to the sequences described herein include sequences comprising conservative amino acid substitutions and amino acid deletions and / or insertions. A conservative amino acid substitution involves the replacement of a first amino acid with a second amino acid having chemical and / or physical properties (eg, charge, structure, polarity, hydrophobicity / hydrophilicity) that are similar to those of the first amino acid. Point to. Conservative substitutions are the following groups: lysine (K), arginine (R) and histidine (H); aspartic acid (D) and glutamic acid (E); asparagine (N), glutamine (Q), serine (S) , Threonine (T), tyrosine (Y), K, R, H, D and E; alanine (A), valine (V), leucine (L), isoleucine (I), proline (P), phenylalanine (F) , Tryptophan (W), methionine (M), cysteine (C) and glycine (G); F, W and Y; substitution of one amino acid by another within C, S and T.

  Amino acid symbol. The amino acids that make up the mimetibodies or specified portions or variants of the invention are often abbreviated. Amino acid designations may be indicated by naming the amino acid by its one letter symbol, its three letter symbol, name, or three nucleotide codon (s) as well understood in the art (Alberts, B , Et al., Molecular Biology of The Cell, 3rd edition, Garland Publishing, Inc., New York, 1994).

One-letter symbol Three-letter symbol Name Three nucleotide codons
(One or more)
A Ala Alanine GCA, GCC, GCG, GCU
C Cys Cysteine UGC, UGU
D Asp Aspartic acid GAC, GAU
E Glu Glutamic acid GAA, GAG
F Phe Phenylalanine UUC, UUU
G Gly Glycine GGA, GGC, GGG, GGU
H His histidine CAC, CAU
I Ile Isoleucine AUA, AUC, AUU
K Lys Lysine AAA, AAG
L Leu leucine UUA, UUG, CUA, CUC,
CUG, CUU
M Met Methionine AUG
N Asn Asparagine AAC, AAU
P Pro Proline CCA, CCC, CCG, CCU
Q Gln Glutamine CAA, CAG
R Arg Arginine AGA, AGG, CGA, CGC,
CGG, CGU
S Ser serine AGC, AGU, UCA, UCC,
UCG, UCU
T Thr Threonine ACA, ACC, ACG, ACU
V Val Valin GUA, GUC, GUG, GUU
W Trp Tryptophan UGG
Y Tyr tyrosine UAC, UAU

  The GLP-1 mimetibody or specified portion or variant of the invention has one or more amino acid substitutions, deletions or deletions from any of the natural mutations or human manipulations specified herein. Additions can be included. These or other sequences that may be used in the present invention are not limited, but the partial variable regions of the antibody sequence include, but are not limited to, the corresponding SEQ ID NOs: 1-9 June 24, 2004. At least one substitution, insertion or deletion as further described in FIGS. 1-9 of the application and published PCT Publication No. WO 05/05604 (PCT US04 / 1998), published January 20, 2005. In a designated portion of SEQ ID NOs: 47-64, which may further include at least a portion of at least one of SEQ ID NOs: 47-55 or fragments thereof as described in Table 1 Mention may be made of the sequences presented in Table 1 as shown correspondingly. The CH2, CH3 and hinge regions include, but are not limited to, PCT Publication No. WO 05/05604 (No. 05/05604, filed Jun. 24, 2004 and published Jan. 20, 2005 with corresponding SEQ ID NOs: 32-40. PCT US04 / 19988) at least one of SEQ ID NOs: 56-64, further comprising at least one substitution, insertion or deletion as further described in FIGS. 32-40 of the specification. Mention may be made of some or fragments thereof as described in Table 1. Of course, the number of amino acid substitutions that one of ordinary skill in the art would make depends on a number of factors, including those described above. Generally speaking, the number of at least one amino acid substitution, insertion or deletion of a GLP-1 mimetibody is as indicated by 1-30 or any range or value therein as specified herein. No more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 amino acids Can do.

  In the formula I ((P (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t) of the present invention, V, H, CH2 of formula I , The CH3 moiety can be any suitable human or human compatible sequence, eg, as presented in Table 1, where the partial variable regions of the antibody sequence are not limited, but correspond Further described in FIGS. 1-9 of the specification of PCT Publication No. WO 05/05604 (No. PCT US04 / 1998), filed June 24, 2004 and published January 20, 2005 with SEQ ID NOS: 1-9. To list at least a portion of at least one of SEQ ID NOs: 47-55, or a fragment thereof as described in Table 1, further comprising at least one substitution, insertion or deletion, as the case may be. Yes; Here, the CH2, CH3 and hinge regions are not limited, but PCT Publication No. WO 05/05604 filed June 24, 2004 and published January 20, 2005 with the corresponding SEQ ID NOs 32-40. No. (PCT US04 / 1998), further described in FIGS. 32-40, or as known in the art, optionally further comprising at least one substitution, insertion or deletion. At least a portion of at least one of SEQ ID NOs: 56-64 or a fragment thereof as set forth in Table 1, or any combination or consensus sequence thereof, or preferably administered to or of human origin Cite any of those fusion proteins engineered to minimize the immunogenicity of the case Can do.

  The P moiety is known in the art, which can include, but is not limited to, that presented in SEQ ID NO: 1, or any combination or consensus sequence thereof, or any fusion protein thereof, or It may comprise at least one GLP-1 therapeutic peptide as described herein. In a preferred embodiment, the P moiety comprises at least one GLP-1 peptide having at least one sequence of SEQ ID NO: 6, or any combination or consensus sequence thereof, or any fusion protein thereof. obtain.

  The optional linker sequence can be any suitable peptide linker known in the art. Preferred sequences include any combination of G and S, such as X1-X2-X3-X4 -...- Xn, where X can be G or S and n is 5-30 obtain. Non-limiting examples include GS, GGS, GGGS (SEQ ID NO: 16), GSGGGS (SEQ ID NO: 17), GGSGGGS (SEQ ID NO: 18), GGSGGGSGGG (SEQ ID NO: 19) and GGGSGGGSGG (SEQ ID NO: 20);

Amino acids in the GLP-1 mimetibody of the invention or specified portion or variant that are essential for function are site-directed mutagenesis or alanine scanning mutagenesis (eg, Ausubel, supra, chapters 8, 15; Cunningham and And can be identified by methods known in the art such as Wells, Science 244: 1081-1085 (1989)). The latter treatment introduces single alanine mutations at every residue in the molecule. The resulting mutant molecule is then tested for biological activity that may include, but is not limited to, at least one protein-related activity as specified herein or known in the art. To do. Sites critical to GLP-1 mimetibody or specified portion or variant binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al., J Mol.Biol.224: 899-904 (1992) and de Vos et al., Science.
255: 306-312 (1992)).

The mimetibody or specified portion or variant of the invention may comprise at least a portion of at least one of SEQ ID NOs: 1 and 6, for example, but not limited to, the P portion of formula (I). The GLP-1 mimetibody or specified portion or variant may further optionally include at least one functional portion of at least one polypeptide, at least one of SEQ ID NOs: 1 and 6, as the P portion of formula (I). It may contain a minimum of 90-100%. Non-limiting variants that can enhance or maintain at least one of the activities listed above, but are not limited, do not significantly affect the appropriate biological activity or function of the GLP-1 mimetibody Any of the above polypeptides further comprising at least one mutation corresponding to at least one substitution, insertion or deletion may be mentioned.

  In one embodiment, the amino acid sequence of P, or a portion thereof, has about 90-100% identity (ie 90, 91, 92, 93, 94) to the corresponding amino acid sequence of the corresponding portion of at least one of SEQ ID NOs: 1 and 6. 95, 96, 97, 98, 99, 100 or any range or value therein. Preferably, 90-100% amino acid identity (ie 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein) is known in the art. Determined using a suitable computer algorithm known in the art.

  A mimetibody or specified portion or variant of the present invention may comprise any number of consecutive amino acid residues from the GLP-1 mimetibody or specified portion or variant of the present invention, the number of which is the GLP-1 It is selected from the group of integers consisting of 10-100% of the number of consecutive residues in the mimetibody. In some cases, this subsequence of contiguous amino acids is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids, or any range or value therein. Furthermore, the number of such subsequences must be at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 or more It can be any integer selected from the group consisting of 1 to 20, such as

  As those skilled in the art will appreciate, the present invention encompasses at least one biologically active GLP-1 mimetibody or specified portion or variant of the present invention. A biologically active mimetibody or specified portion or variant is a specific activity of a natural (non-synthetic), endogenous or related and known inserted or fused protein or specified portion or variant. It has a specific activity of at least 20%, 30% or 40%, and preferably at least 50%, 60% or 70%, and most preferably at least 80%, 90% or 95% to 1000%. Methods for assaying and quantifying enzyme activity and substrate specificity are known to those skilled in the art.

  In another aspect, the invention relates to human mimetibodies and ligand-binding fragments as described herein that are modified by covalent attachment of an organic moiety. Such modifications can result in GLP-1 mimetibodies or ligand-binding fragments with improved pharmacokinetic properties (eg, increased in vivo serum half-life). The organic moiety can be a linear or branched hydrophilic polymer group, a fatty acid group or a fatty acid ester group. In certain embodiments, the hydrophilic polymer group can have a molecular weight of about 800 to about 120,000 daltons and can be a polyalkane glycol (eg, polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl. It may be pyrrolidone and the fatty acid or fatty acid ester group may contain from about 8 to about 40 carbon atoms.

The modified mimetibodies and ligand-binding fragments of the invention can comprise one or more organic moieties that are covalently linked directly or indirectly to the GLP-1 mimetibody or specified portion or variant. Each organic moiety attached to the GLP-1 mimetibody or ligand binding fragment of the present invention can independently be a hydrophilic polymer group, a fatty acid group or a fatty acid ester group. As used herein, the term “fatty acid” includes monocarboxylic and dicarboxylic acids. A “hydrophilic polymer group” as the term is used herein refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, GLP-1 mimetibodies modified by covalent attachment of polylysine are encompassed by the present invention. Hydrophilic polymers suitable for modifying mimetibodies of the present invention can be linear or branched and include, for example, polyalkane glycols (eg, PEG, monomethoxypolyethylene glycol (mPEG), PPG, etc.), carbohydrates (eg, dextran). , Cellulose, oligosaccharides, polysaccharides, etc.), polymers of hydrophilic amino acids (eg polylysine, polyarginine, polyaspartic acid etc.), polyalkane oxides (eg polyethylene oxide, polypropylene oxide etc.) and polyvinylpyrrolidone. Preferably, the hydrophilic polymer that modifies the GLP-1 mimetibody of the present invention has a molecular weight of about 800 to about 150,000 daltons as a separate molecular entity. For example, PEG ₂₅₀₀ , PEG ₅₀₀₀ , PEG ₇₅₀₀ , PEG ₉₀₀₀ , PEG ₁₀₀₀₀ , PEG ₁₂₅₀₀ , PEG ₁₅₀₀₀ and PEG _20,000 (where the subscript number is the average molecular weight (Dalton) of the polymer) can be used.

  The hydrophilic polymer group can be substituted with 1 to about 6 alkyl, fatty acid or fatty acid ester groups. Hydrophilic polymers that are substituted with a fatty acid or fatty acid ester group can be prepared by using suitable methods. For example, a polymer comprising an amine group can be conjugated to a fatty acid or a carboxylate of a fatty acid ester and activated with a carboxylate on the fatty acid or fatty acid ester (eg, activated with N, N-carbonyldiimidazole) ) May be bonded to hydroxyl groups on the polymer.

Fatty acids and fatty acid esters suitable for modifying mimetibodies of the invention can be saturated or can contain one or more unsaturated units. Fatty acids suitable for modifying mimetibodies of the present invention include, for example, n-dodecanoate (C ₁₂ , laurate), n-tetradecanoate (C ₁₄ , myristate), n-octadecanoate (C ₁₈ , steer). rate), n-Eikosanoeto _{(C 20,} arachidates), n-Dokosanoeto _{(C 22,} behenate), n-triacontanyl hexanoate _(C 30), n- tetraconta hexanoate _(C 40), _cis-Δ9- octadecanoate _{(C 18,} oleate), all cis-Δ5,8,11,14- eicosatetraenoic enoate _{(C 20,} arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid Etc. Suitable fatty acid esters include monoesters of dicarboxylic acids comprising linear or branched lower alkyl groups. The lower alkyl group may contain 1 to about 12, preferably 1 to about 6 carbon atoms.

Modified human mimetibodies and ligand-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. A “modifier” as the term is used herein refers to a suitable organic group comprising an activating group (eg, hydrophilic polymer, fatty acid, fatty acid ester). An “activating group” is a chemical moiety or functional group that can react with a second chemical group under appropriate conditions, thereby forming a covalent bond between the modifier and the second chemical group. For example, amine reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl ester (NHS), and the like. Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfide, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like. Aldehyde functional groups can be attached to molecules containing amines or hydrazides, and azide groups can be reacted with trivalent phosphorus groups to form phosphoramidate or phosphorimide linkages. Suitable methods for introducing activating groups into the molecule are known in the art (see, eg, Hermanson, GT, Bioconjugate Technologies, Academic Press: San Diego, Calif. (1996)). Activating groups can be substituted directly on organic groups (eg hydrophilic polymers, fatty acids, fatty acid esters) or linker moieties, eg one or more carbon atoms can be replaced by heteroatoms such as oxygen, nitrogen or sulfur. It can be bound by a divalent C ₁ -C ₁₂ group. Suitable linker moieties are, for example, tetraethylene glycol, — (CH ₂ ) ₃ —, —NH— (CH ₂ ) ₆ —NH—, — (CH ₂ ) ₂ —NH— and —CH ₂ —O—CH ₂ —. CH _{2 -O-CH 2} encompasses -CH 2 -O-CH-NH-. Modifiers comprising a linker moiety include, for example, mono-Boc-alkyldiamines (eg mono-Boc-ethylenediamine, mono-Boc-diaminohexane) and 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide ( Can be prepared by reacting with a fatty acid in the presence of EDC) to form an amide bond between the free amine and the carboxylate of the fatty acid. The Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose the primary amine, which can be coupled to another carboxylate as described, or It can be reacted with maleic anhydride and the resulting product can be cyclized to give activated maleimide derivatives of fatty acids (eg, Thompson et al., WO 92/16221, the entire teachings of which are incorporated by reference). See incorporated herein)).

The modified mimetibody of the present invention can be produced by reacting human GLP-1 mimetibody or a ligand-binding fragment with a modifying agent. For example, by using an amine-reactive modifier, such as an NHS ester of PEG, the organic moiety can be attached to the GLP-1 mimetibody in a site-nonspecific manner. Modified human mimetibodies or ligand-binding fragments can also be produced by reducing disulfide bonds (eg, intrachain disulfide bonds) of GLP-1 mimetibody or ligand-binding fragments. The reduced GLP-1 mimetibody or ligand-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified GLP-1 mimetibody of the present invention. Modified human mimetibodies and ligand-binding fragments comprising an organic moiety attached to a specific site of a GLP-1 mimetibody or specified portion or variant of the present invention can be obtained by reverse proteolysis (Fisch et al., Bioconjugate Chem. 3: 147-153 (1992); Werlen et al., Bioconjugate Chem. , 5: 411-417 (1994); Kumaran et al., Protein Sci. 6 (10): 2233-3241 (1997); Itoh et al . , Bioorg. , 24 (1): 59-68 (1996); Capellas et al . , Biotechnol.Bioeng. , 56 (4): 456-463 (1997)), and Hermanson, G. et al. T.A. Bioconjugate Technologies , Academic Press: San Diego, Calif. (1996).

  GLP-1 mimetibody composition. The present invention is also provided in a non-naturally occurring composition, mixture or form, as described herein and / or as known in the art, at least 1, at least 2, at least 3, at least 4, at least 5. Also provided is a composition of at least one GLP-1 mimetibody or specified portion or variant comprising at least 6 or more mimetibodies or specified portions or variants thereof. The percentage of such compositions is the weight, volume, concentration, molarity as a liquid or dry solution, mixture, suspension, emulsion or colloid as known in the art or as described herein. Or it is a molar concentration.

Such compositions are liquids known in the art or as described herein,
Can include 0.00001 to 99.9999 wt, volume, concentration, molarity or percent molarity as a gas or dry solution, mixture, suspension, emulsion or colloid, any range or value therein For example, but not limited to 0.00001, 0.00003, 0.00005, 0.00009, 0.0001, 0.0003, 0.0005, 0.0009, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5,. 6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45 , 50, 55, 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90 91, 92, 93, 94, 95, 96, 97, 98, 99, 99.1, 99.2, 99.3, 99.4, 99.5, 99.6, 99.7, 99.8 99.9%. Such compositions of the invention thus include, but are not limited to, 0.00001-100 mg / ml and / or 0.00001-100 mg / g.

  The composition may optionally comprise a drug associated with diabetes or insulin metabolism, an anti-infective drug, a cardiovascular (CV) drug, a central nervous system (CNS) drug, an autonomic nervous system (ANS) drug, a respiratory tract drug, Select from at least one of gastrointestinal (GI) tract drugs, hormone drugs, fluid or electrolyte balance drugs, blood products, antitumor drugs, immunomodulators, eye, ear or nose drugs, topical drugs, nutritional drugs, etc. It may further comprise an effective amount of at least one compound or protein that is made. Such drugs, including the respective formulations, indications, dosing and administration presented herein are known in the art (see, eg, Nursing 2001 Handbook of Drugs, 21st Edition, Springhouse Corp., Spring, PA). House, 2001; Health Professional's Drug Guide 2001, Shannon, Wilson, Stan ed., Pentice-Hall, Inc., Upper Saddle River, New Jersey; Pharmcotherapy Handbook, p. See incorporated herein by reference) .

  Diabetes-related drugs include glitazone, insulin and derivatives, sulfonylurea, meglitinide, biguanide, α-glucosidase inhibitor, protein tyrosine phosphatase-1B, glycogen synthase kinase 3, gluconeogenesis inhibitor, pyruvate dehydrogenase kinase (PDH) Inhibitors, lipolysis inhibitors, fatty acidization inhibitors, carnitine palmitoyltransferase I and / or II inhibitors, β-3 adrenergic receptor agonists, sodium and glucose cotransporter (SGLT) inhibitors, or: autoimmune suppression, Immunoregulation, T cell activation, proliferation, migration and / or suppressor cell function, inhibition of T cell receptor / peptide / MHC-II interaction, induction of T cell anergy, elimination of autoreactive T cells, blood brain Low transport across the barrier Lower, one or at least one of pre-inflammatory (Th1) and immunoregulatory (Th2) cytokine balance changes, inhibition of matrix metalloprotease inhibitors, neuroprotection, reduction of gliosis, promotion of remyelination It may be at least one of the compounds acting above.

Anti-infective drugs include amoeba drugs or antiprotozoal drugs, anthelmintic drugs, antifungal drugs, antimalarial drugs, antituberculosis drugs or anti-leak drugs, aminoglycosides, penicillins, cephalosporins, tetracyclines, sulfonamides, fluoroquinolones, antivirals It may be at least one selected from drugs, macrolide antiinfectives and miscellaneous antiinfectives. The CV drug may be at least one selected from inotropic drugs, antiarrhythmic drugs, antianginal drugs, antihypertensive drugs, antihyperlipidemic drugs and miscellaneous cardiovascular drugs. CNS drugs are at least one selected from non-narcotic analgesics, or antipyretic drugs, non-steroidal anti-inflammatory drugs, narcotic or opioid analgesics, analgesic sleep drugs, anticonvulsants, antidepressants, anxiolytics, It may be at least one selected from antipsychotics, central nervous system stimulants, antiparkinsonian drugs and miscellaneous central nervous system drugs. ANS drugs are selected from cholinergic drugs (parasympathomimetic drugs), anticholinergics, adrenergic drugs (sympathomimetic drugs), adrenergic blockers (sympathetic blockers), skeletal muscle relaxants and neuromuscular blockers Can be at least one. The respiratory tract drug may be at least one selected from antihistamines, bronchodilators, expectorants or antitussives, and miscellaneous respiratory drugs. The GI tract drug may be at least one selected from antacids, adsorbents, antiflatulents, digestive enzymes, gallstone solubilizers, antidiarrheals, laxatives, antiemetics and antiulcer drugs. Hormonal drugs are at least selected from corticosteroids, androgens, anabolic steroids, estrogens, progestins, gonadotropins, antidiabetics, at least one glucagon, thyroid hormone, thyroid hormone antagonist, pituitary hormone and parathyroid-like drug There can be one. The drug for liquid and electrolyte balance may be at least one selected from diuretics, electrolytes, replacement solutions, acidifying agents and alkalizing agents. The blood product can be at least one selected from hematopoietics, anticoagulants, blood derivatives and thrombolytic enzymes. The antitumor drug may be at least one selected from alkylating agents, antimetabolites, antibiotic antitumor drugs, antitumor drugs that change hormone balance, and miscellaneous antitumor drugs. The immunomodulator can be at least one selected from immunosuppressants, vaccines, toxins, antitoxins, anti-snake venoms, immune sera and biological response modifiers. Eye, ear and nose drugs are selected from ocular antiinfectives, ocular anti-inflammatory drugs, miotics, mydriatics, ocular vasoconstrictors and miscellaneous ophthalmics, otics, nasal drugs Can be at least one. The topical drug may be at least one selected from topical anti-infectives, anti-scabies, lice killing agents and topical corticosteroids. The nutrient can be at least one selected from vitamins, minerals and pyrogens. See, for example, Nursing 2001 Drug Handbook, supra.

The at least one amoebicidal or antiprotozoal agent can be at least one selected from atovaquone, chloroquine hydrochloride, chloroquine phosphate, metronidazole, metronidazole hydrochloride, and pentamidine isethionate. The at least one anthelmintic can be at least one selected from mebendazole, pyrantel pamoate and thiabendazole. At least one antifungal agent is amphotericin B, amphotericin B cholesteryl sulfate complex, amphotericin B lipid complex, amphotericin B liposome preparation, fluconazole, flucytosine, griseofulvin microparticle (microsize), griseofulvin ultrafine particle (ultramicrosizole), itraconazole , At least one selected from nystatin and terbinafine hydrochloride. The at least one antimalarial drug may be at least one selected from chloroquine hydrochloride, chloroquine phosphate, doxycycline, hydroxychloroquine sulfate, mefloquine hydrochloride, primaquine phosphate, pyrimethamine, and pyrimethamine with sulfadoxine. The at least one anti-tuberculosis or anti-leukemic agent can be at least one selected from clofazimine, cycloserine, dapsone, ethambutol hydrochloride, isoniazid, pyrazinamide, rifabutin, rifampin, rifapentine and streptomycin sulfate. The at least one aminoglycoside may be at least one selected from amikacin sulfate, gentamicin sulfate, neomycin sulfate, streptomycin sulfate and tobramycin sulfate. At least one penicillin is amoxicillin / potassium clavulanate, amoxicillin trihydrate, ampicillin, ampicillin sodium, ampicillin trihydrate, ampicillin sodium / sulbactam sodium, cloxacillin sodium, dicloxacillin sodium, mezlocillin Sodium, nafcillin sodium, oxacillin sodium, penicillin G benzathine, penicillin G potassium, penicillin G procaine, penicillin G sodium, penicillin V potassium, piperacillin sodium, piperacillin sodium / tazobactam sodium, ticarcillin disodium and ticarcillin disodium / potassium clavulanate It may be at least one selected from At least one cephalosporin is cefaclor, cefadroxyl, cefazolin sodium, cefdinir, cefepime hydrochloride, cefixime, cefmetazole sodium, cefoniside sodium, cefoperazone sodium, cefotaxime sodium, cefotetan disodium, cefoxitin sodium, cef Select from at least one of podoxime proxetyl, cefprozil, ceftazidime, ceftibutene, ceftizoxime sodium, ceftriaxone sodium, cefuroxime axetil, cefuroxime sodium, cephalexin hydrochloride, cephalexin monohydrate, cefradine, and loracarbef Can be at least one selected. The at least one tetracycline may be at least one selected from demeclocycline hydrochloride, doxycycline calcium, doxycycline hydrate, doxycycline hydrochloride, doxycycline monohydrate, minocycline hydrochloride, and tetracycline hydrochloride. The at least one sulfonamide may be at least one selected from co-trimoxazole, sulfadiazine, sulfamethoxazole, sulfisoxazole, sulfisoxazole acetyl. At least one fluoroquinolone is selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin mesylate There may be one. At least one fluoroquinolone is selected from alatrofloxacin mesylate, ciprofloxacin, enoxacin, levofloxacin, lomefloxacin hydrochloride, nalidixic acid, norfloxacin, ofloxacin, sparfloxacin, trovafloxacin mesylate There may be one. At least one antiviral drug is abacavir sulfate, acyclovir sodium, amantadine hydrochloride, amprenavir, cidofovir, delavirdine mesylate, didanosine, efavirenz, famciclovir, fomivirsen sodium, foscarnet sodium, ganciclovir, sulfate At least one selected from indinavir, lamivudine, lamivudine / zidovudine, nelfinavir mesylate, nevirapine, oseltamivir phosphate, ribavirin, rimantadine hydrochloride, ritonavir, saquinavir, saquinavir mesylate, stavudine, valaciclovir hydrochloride, zalcitabine, zanamivir, zidovudine obtain. At least one macroline anti-infective is at least one selected from azithromycin, clarithromycin, dirithromycin, erythromycin base, erythromycin estrate, erythromycin ethyl succinate, erythromycin lactobionate, erythromycin stearate obtain. At least one miscellaneous anti-infective agent is aztreonam, bacitracin, chloramphenicol sodium succinate, clindamycin hydrochloride, clindamycin palmitate, clindamycin phosphate, imipenem and cilastatin sodium, meropenem, nitropenem It may be at least one selected from large crystals of furantoin, nitrofurantoin microcrystals, quinupristin / dalfopristin, spectinomycin hydrochloride, trimethoprim, vancomycin hydrochloride (see, for example, pp. 24-214 of Nursing 2001 Drug Handbook). .)

The at least one inotropic agent may be at least one selected from amrinone lactate, digoxin, and milrinone lactate. At least one antiarrhythmic drug is adenosine, amiodarone hydrochloride, atropine sulfate, bretylium tosylate, diltiazem hydrochloride, disopyramide, disopyramide phosphate, esmolol hydrochloride, flecainide acetate, ibutilide fumarate, lidocaine hydrochloride, mexiletine hydrochloride, moricidin hydrochloride, phenytoin , Phenytoin sodium, procainamide hydrochloride, propaphenone hydrochloride, propranolol hydrochloride, quinidine hydrogen sulfate, quinidine gluconate, quinidine polygalacturonic acid, quinidine sulfate, sotalol, tocainide hydrochloride, verapamil hydrochloride. At least one anti-anginal drug is amlodipine besylate, amyl nitrite, bepridil hydrochloride, diltiazem hydrochloride, isosorbide nitrate, isosorbide mononitrate, nadolol, nicardipine hydrochloride, nifedipine, nitroglycerin, propranolol hydrochloride, verapamil, verapamil hydrochloride There may be at least one selected. At least one antihypertensive drug is acebutol hydrochloride, amlodipine besylate, atenolol, benazepril hydrochloride, betaxolol hydrochloride, bisoprolol fumarate, candesartan cilexetil, captopril, carteolol hydrochloride, carvedilol, clonidine, clonidine hydrochloride, diazoxide hydrochloride, diltiazem hydrochloride, Doxazosin mesylate, enalaprilate, enalapril maleate, eprosartan mesylate, felodipine, fenoldopam mesylate, fosinopril sodium, guanadazole acetate, guanadrel sulfate, guanfacine hydrochloride, hydralazine hydrochloride, irbesartan, isradipine, labetalol hydrochloride, ricinopril methyl sulpridol hydrochloride , Methyldopate hydrochloride, metoprolol succinate Metoprolol tartrate, minoxidil, moexipril hydrochloride, nadolol, nicardipine hydrochloride, nifedipine, nisoldipine, sodium nitroprusside, penbutolol sulfate, perindopril erbumine, phentolamine mesylate, pindolol, prazosin hydrochloride, propranolol hydrochloride, quinapril hydrochloride, ramipril hydrochloride, telmisartan hydrochloride, telmisartan hydrochloride It may be at least one selected from timolol acid, trandolapril, valsartan, verapamil hydrochloride. At least one antihyperlipidemic drug is selected from atorvastatin calcium, cerivastatin sodium, cholestyramine, colestipol hydrochloride, fenofibrate (fine powder), fluvastatin sodium, gemfibrozil, lovastatin, niacin, pravastatin sodium, simvastatin There can be at least one. At least one miscellaneous CV drug is at least one selected from abciximab, alprostadil, albutamine hydrochloride, cilostazol, clopidogrel bisulfate, dipyridamole, eptifibatide, midodrine hydrochloride, pentoxifylline, ticlopidine hydrochloride, tirofiban hydrochloride Get (eg, Nursing 2001 Drug)
Handbook pp. 215-336. ).

The at least one non-narcotic analgesic or antipyretic may be at least one selected from acetaminophen, aspirin, choline magnesium trisalicylate, diflunisal, magnesium salicylate. At least one non-steroidal anti-inflammatory drug is celecoxib, diclofenac potassium, diclofenac sodium, etodolac, fenoprofen calcium, flurbiprofen, ibuprofen, indomethacin, indomethacin sodium trihydrate, ketoprofen, ketorolac tromethamine, It may be at least one selected from nabumetone, naproxen, naproxen sodium, oxaprozin, piroxicam, rofecoxib, sulindac. At least one narcotic or opioid analgesic is alfentanil hydrochloride, buprenorphine hydrochloride, butorphanol tartrate, codeine phosphate, codeine sulfate, fentanyl citrate, fentanyl transdermal system, fentanyl transmucosal, hydromorphone hydrochloride, meperidine hydrochloride, methadone hydrochloride , Morphine hydrochloride, morphine sulfate, morphine tartrate, nalbuphine hydrochloride, oxycodone hydrochloride, oxycodone pectate, oxymorphone hydrochloride, pentazocine hydrochloride, pentazocine hydrochloride, naloxone hydrochloride, pentazocine lactate, propoxyphene hydrochloride, propoxyphene napsylate, remifentanil hydrochloride, citric acid It may be at least one selected from sufentanil and tramadol hydrochloride. The at least one sedative hypnotic may be at least one selected from chloral hydrate, estazolam, flurazepam hydrochloride, pentobarbital, pentobarbital sodium, phenobarbital sodium, secobarbital sodium, temazepam, triazolam, zalepron, zolpidem tartrate . At least one anticonvulsant is acetazolamide sodium, carbamazepine, clonazepam, chlorazepate dipotassium, diazepam, divalproex sodium, ethosuccid, phosphenytoin sodium, gabapentin, lamotrigine, magnesium sulfate, phenobarbital sodium, phenytoin, It may be at least one selected from phenytoin sodium, phenytoin sodium (sustained release), primidone, tyagabine hydrochloride, topiramate, sodium valproate, and valproic acid. At least one antidepressant is amitriptyline hydrochloride, amitriptyline pamoate, amoxapine, bupropion hydrochloride, citalopram hydrobromide, clomipramine hydrochloride, desipramine hydrochloride, doxepin hydrochloride, fluoxetine hydrochloride, imipramine hydrochloride, imipramine pamoate, mirtazapine hydrochloride, nefazodone hydrochloride And at least one selected from nortriptyline hydrochloride, paroxetine hydrochloride, phenelzine sulfate, sertraline hydrochloride, tranylcypromine sulfate, trimipramine maleate, and venlafaxine hydrochloride. At least one anti-anxiety drug includes alprazolam, buspirone hydrochloride, chlordiaz GLP-1 xide, chlordiaz GLP-1 xide, dipotassium chlorazepate, diazepam, doxepin hydrochloride, hydroxyzeneene It may be at least one selected from bonate, hydroxyzine hydrochloride, hydroxyzine pamoate, lorazepam, meflovamate, midazolam hydrochloride, and oxazepam. At least one antipsychotic is chlorpromazine hydrochloride, clozapine, fluphenazine decanoate, flufenazine enanthate, fluphenazine hydrochloride, haloperidol, haloperidol decanoate, haloperidol lactate, loxapine hydrochloride, loxapine succinate, mesoridazine besylate, morindon hydrochloride Olanzapine, perphenazine, pimozide, prochlorperazine, quetiapine fumarate, risperidone, thioridazine hydrochloride, thiothixene, thiothixene hydrochloride, and trifloperazine hydrochloride. The at least one central nervous system stimulant may be at least one selected from amphetamine sulfate, caffeine, dextroamphetamine sulfate, doxapram hydrochloride, methamphetamine hydrochloride, methylphenidate hydrochloride, modafinil, pemoline, phentermine hydrochloride. . At least one antiparkinsonian drug is amantadine hydrochloride, benztropine mesylate, biperidene hydrochloride, biperidene lactate, bromocriptine mesylate, carbidopa-levodopa, entacapone, levodopa, pergolide mesylate, pramipexole dihydridol broth hydrochloride And at least one selected from selegiline hydrochloride, tolcapone, and trihexyphenidyl hydrochloride. At least one miscellaneous central nervous system drug is bupropion hydrochloride, donepezil hydrochloride, droperidol, fluvoxamine maleate, lithium carbonate, lithium citrate, naratriptan hydrochloride, nicotine polacrilex, nicotine transdermal system, propofol, lisa benzoate It may be at least one selected from triptan, sibutramine hydrochloride monohydrate, sumatriptan succinate, tacrine hydrochloride, zolmitriptan (see, for example, pp. 337-530 of Nursing 2001 Drug Handbook).

  The at least one cholinergic agonist (eg, parasympathomimetic) may be at least one selected from betanecol chloride, edrophonium chloride, neostigmine bromide, neostigmine methylsulfate, physostigmine salicylate, pyridostigmine bromide. At least one anticholinergic agent is at least one selected from atropine sulfate, dicyclomine hydrochloride, glycopyrrolic acid, hyoscyamine, hyoscyamine sulfate, propanthel bromide, scopolamine, butyl scopolamine, scopolamine hydrobromide. It can be a seed. The at least one adrenergic agonist (sympathomimetic) may be at least one selected from dobutamine hydrochloride, dopamine hydrochloride, metallaminol hydrogen tartrate, norepinephrine hydrogen tartrate, phenylephrine hydrochloride, pseudoephedrine hydrochloride, and pseudoephedrine sulfate. The at least one adrenergic blocker (sympathetic blocker) can be at least one selected from dihydroergotamine mesylate, ergotamine tartrate, methysergide maleate, propranolol hydrochloride. The at least one skeletal muscle relaxant may be at least one selected from baclofen, carisoprodol, chlorzoxazone, cyclobenzaprine hydrochloride, dantrolene sodium, metcarbamol, tizanidine hydrochloride. At least one neuromuscular blocking agent is atraclium besylate, cisatracurium besylate, doxacurium chloride, mivacurium chloride, pancuronium bromide, pipecuronium bromide, rapacuronium bromide, rocuronium bromide, succinylcholine chloride, tubocurarine chloride, bromide It can be at least one selected from vecuronium (see, eg, Nursing 2001 Drug Handbook, pp. 531-84).

At least one antihistamine is brompheniramine maleate, cetirizine hydrochloride, chlorpheniramine maleate, clemastine fumarate, cyproheptadine hydrochloride, diphenhydramine hydrochloride, fexofenadine hydrochloride, loratadine, promethazine hydrochloride, promethazine hydrochloride and triproate hydrochloride It may be at least one selected from lysine. At least one bronchodilator is albuterol, albuterol sulfate, aminophylline, atropine sulfate, ephedrine sulfate, epinephrine, epinephrine hydrogen tartrate, epinephrine hydrochloride, ipratropium bromide, isoproterenol, isoproterenol hydrochloride, isoproterenol sulfate, hydrochloric acid It may be at least one selected from levalbuterol, metaproterenol sulfate, oxytriphilin, pyrbuterol acetate, salmeterol xinafoate, terbutaline sulfate, and theophylline. The at least one expectorant or antitussive may be at least one selected from benzonate, codeine phosphate, codeine sulfate, dextramethorphan hydrobromide, diphenhydramine hydrochloride, guaifenesin, hydromorphone hydrochloride. At least one miscellaneous respiratory drug is acetylcysteine, beclomethasone dipropionate, veracant, budesonide, calfactorant, cromolyn sodium, dornase alpha, GLP-1 prostenol sodium, flunisolide, It may be at least one selected from fluticasone propionate, montelukast sodium, nedocromil sodium, palivizumab, triamcinolone acetonide, zafirlukast, zileuton (see, for example, Nursing 2001 Drug Handbook, pp. 585-642). .

At least one antacid, adsorbent or antiflatulent is at least one selected from aluminum carbonate, aluminum hydroxide, calcium carbonate, magaldrate, magnesium hydroxide, magnesium oxide, dimethicone and sodium bicarbonate obtain. The at least one digestive enzyme or gallstone solubilizer may be at least one selected from pancreatin, pancrelipase and ursodiol. At least one antidiarrheal agent is at least one selected from attapulgite, bismuth subsalicylate, calcium polycarbophil, diphenoxylate hydrochloride or atropine sulfate, loperamide, octreotide acetate, opium tincture, and opium tincture (camphorated) possible. At least one laxative is bisacodyl, calcium polycarbophil, Cascara Sagrada, Cascara Sagrada fragrance extract, Cascara Sagrada extract, castor oil, docusate calcium, docusate sodium, glycerin, lactulose, magnesium citrate, It may be at least one selected from magnesium hydroxide, magnesium sulfate, methylcellulose, mineral oil, polyethylene glycol or electrolyte solution, psyllium, senna, sodium phosphate. At least one antiemetic is chlorpromazine hydrochloride, dimenhydrinate, dolasetron mesylate, dronabinol, granisetron hydrochloride, meclizine hydrochloride, metocloproamide hydrochloride, ondansetron hydrochloride, perphenazine, prochlorperazine, prochloridicyl chloride It may be at least one selected from perazine, prochlorperazine maleate, promethazine hydrochloride, scopolamine, thiethylperazine maleate, and trimethobenzamide hydrochloride. The at least one anti-ulcer drug may be at least one selected from cimetidine, cimetidine hydrochloride, famotidine, lansoprazole, misoprostol, nizatidine, omeprazole, rabeprazole sodium, lantidine bismuth citrate, ranitidine hydrochloride, sucralfate. (See, for example, Nursing 2001 Drug Handbook, pp. 643-95.) At least one corticosteroid is betamethasone, betamethasone acetate or betamethasone sodium phosphate, betamethasone sodium phosphate, cortisone acetate, dexamethasone acetate, dexamethasone acetate, Dexamethasone sodium phosphate, fludrocortisone acetate, hydrocortisone, hydrocortisone acetate, hydrocortisone cypionate, hydrocortisone sodium phosphate, hydrocortisone sodium succinate, methylprednisolone, methylprednisolone acetate, methylprednisolone sodium succinate, prednisolone acetate, prednisolone acetate Prednisolone sodium, prednisolone tebutate, prednisone, Riamushinoron, triamcinolone acetonide, can be at least one selected from triamcinolone acetate. At least one androgen or anabolic steroid is from danazol, fluoxymesterone, methyl testosterone, nandrolone decanoate, nandrolone fenpropionate, testosterone, testosterone cypionate, testosterone enanthate, testosterone propionate, testosterone transdermal system There may be at least one selected. At least one estrogen or progestin consists of esterified estrogens, estradiol, estradiol cypionate, estradiol / norethindrone acetate transdermal system, estradiol valerate, estrogen (conjugation), estropipete, ethinyl estradiol, ethinyl estradiol and desogestrel, ethinyl estradiol Ethinodiol acetate, ethinylestradiol and desogestrel, ethinylestradiol and etinodiol acetate, ethinylestradiol and levonorgestrel, ethinylestradiol and norethindrone, ethinylestradiol and norethindrone acetate, ethinylestradiol and norgestimate, ethinylestradiol and norgestrel, Rajioru and norethindrone and acetate and ferrous fumarate, levonorgestrel, medroxyprogesterone acetate, mestranol and norethindrone, norethindrone, norethindrone acetate, norgestrel, can be at least one selected from progesterone. The at least one gonadotrophin may be at least one selected from ganirelix acetate, gonadorelin acetate, histrelin acetate, and menotropin. At least one antidiabetic drug or glucaone is at least one selected from acarbose, chlorpropamide, glimepiride, glipizide, glucagon, glyburide, insulin, metformin hydrochloride, miglitol, pioglitazone hydrochloride, repaglinide, rosiglitazone maleate, troglitazone It can be. The at least one thyroid hormone may be at least one selected from levothyroxine sodium, liothyronine sodium, liotrix, thyroid preparation. The at least one thyroid hormone antagonist is selected from methimazole, potassium iodide, potassium iodide (saturated solution), propylthiouracil, radioactive iodine (sodium iodide ¹³¹ I), concentrated iodine solution. There can be at least one. The at least one pituitary hormone can be at least one selected from corticotropin, cosyntropin, desmofresin acetate, leuprolide acetate, rGLP-1 site, corticotropin, somatrem, somatropin, vasopressin. The at least one parathyroid-like drug may be at least one selected from calcifediol, calcitonin (human), calcitonin (salmon), calcitriol, dihydrotaxosterol, etidronate disodium (eg, Nursing 2001). (See Drug Handbook, pp. 696-796).

At least one diuretic is selected from acetazolamide, acetazolamide sodium, amiloride hydrochloride, bumetanide, chlorthiaridone, sodium ethacrynate, ethacrynic acid, furosemide, hydrochlorothiazide, indapamide, mannitol, metrazone, spironolactone, torsemide, triamterene, urea There can be at least one. At least one electrolyte or replenisher solution is calcium acetate, calcium carbonate, calcium chloride, calcium citrate, gluconate calcium, glucoceptor calcium, calcium gluconate, calcium lactate, calcium phosphate (second), calcium phosphate (third ), Dextran (high molecular weight), dextran (low molecular weight), hetastarch, magnesium chloride, magnesium sulfate, potassium acetate, potassium bicarbonate, potassium chloride, potassium gluconate, Ringer's solution, Ringer's solution (lactate added), and sodium chloride There can be at least one. The at least one acidifying or alkalizing agent may be at least one selected from sodium bicarbonate, sodium lactate, tromethamine (eg, Nursing 2001).
Drug Handbook pp. See 797-833. ).

At least one hematopoietic agent is at least one selected from iron fumarate, iron gluconate, iron sulfate, iron sulfate (dried), iron dextran, iron sorbitol, polysaccharide-iron complex, and iron gluconate sodium complex. possible. The at least one anticoagulant may be at least one selected from ardeparin sodium, dalteparin sodium, danaparoid sodium, enoxaparin sodium, heparin calcium, heparin sodium, warfarin sodium. At least one blood product consists of albumin 5%, albumin 25%, antihemophilic factor, anti-inhibitor coagulant complex, antithrombin III (human), factor IX (human) , A factor IX complex, or a plasma protein fraction. The at least one thrombolytic enzyme may be at least one selected from alteplase, anistreplase, reteplase (recombinant), streptokinase, urokinase (see, eg, Nursing 2001 Drug Handbook, pp. 834-66). I want to be.)

  The at least one alkylating agent is at least one selected from busulfan, carboplatin, carmustine, chlorambucil, cisplatin, cyclophosphamide, ifosfamide, lomustine, mechlorethamine hydrochloride, melphalan, melphalan hydrochloride, streptozocin, temozolomide, thiotepa It can be a seed. The at least one antimetabolite can be at least one selected from capecitabine, cladribine, cytarabine, floxuridine, fludarabine phosphate, fluorouracil, hydroxyurea, mercaptopurine, methotrexate, methotrexate sodium, thioguanine. At least one antibiotic antitumor drug is bleomycin sulfate, dactinomycin, daunorubicin citrate liposome preparation, daunorubicin hydrochloride, doxorubicin hydrochloride, doxorubicin hydrochloride liposome preparation, epirubicin hydrochloride, idarubicin hydrochloride, mitomycin, pentostatin, pricamycin, It may be at least one selected from valrubicin. At least one antitumor agent that alters hormone balance is anastrozole, bicalutamide, estramustine phosphate sodium, exemestane, flutamide, goserelin acetate, letrozole, leuprolide acetate, megestrol acetate, nilutamide, tamoxifen citrate, It may be at least one selected from a test lactone and toremifene citrate. At least one miscellaneous antitumor agent is asparaginase, bacilli Calmette-Guerin (BCG) (live bacteria in the bladder), dacarbazine, docetaxel, etoposide, etoposide phosphate, gemcitabine hydrochloride, irinotecan hydrochloride, mitotane, mitoxantrone hydrochloride, It may be at least one selected from paclitaxel, pegaspargase, porfimer sodium, procarbazine hydrochloride, rituximab, teniposide, topotecan hydrochloride, trastuzumab, tretinoin, vinblastine sulfate, vincristine sulfate, vinorelbine tartrate (eg, Nursing 2001 Drug Handbo .867-963).

The at least one immunosuppressive drug may be at least one selected from azathioprine, basiliximab, cyclosporine, daclizumab, lymphocyte immunoglobulin, muromomab-CD3, mycophenolate mofetil, mycophenolate mofetil hydrochloride, sirolimus, tacrolimus . At least one vaccine or toxin such as BCG vaccine, cholera vaccine, diphtheria and tetanus toxin (adsorption), diphtheria and tetanus toxin and acellular pertussis vaccine adsorption, diphtheria and tetanus toxin and whole cell pertussis vaccine, hemophilia Disease b conjugate vaccine, hepatitis A vaccine (inactivated), hepatitis B vaccine (recombinant), influenza virus vaccine 1999-2000 trivalent A and B (purified surface antigen), influenza virus vaccine 1999-2000 three Titers A and B (subvirion or purified subvirion), influenza virus vaccine 1999-2000 trivalent A and B (all virions), Japanese encephalitis virus vaccine (inactivated), Lyme disease vaccine (recombinant OspA), measles and Fashion Papillitis and rubella virus vaccine (live), measles and epidemic parotitis and rubella virus vaccine (live, attenuated), measles virus vaccine (live, attenuated), meningococcal polysaccharide vaccine, epidemic ear Lower adenitis virus vaccine (live), plague vaccine, pneumococcal vaccine (multivalent), poliovirus vaccine (inactivated), poliovirus vaccine (live, oral, trivalent), rabies vaccine (adsorption), rabies vaccine (human) Diploid cells), rubella and mumps virus vaccine (live), rubella virus vaccine (live, attenuated), tetanus toxin (adsorption), tetanus toxin (liquid), typhoid vaccine (oral), It may be at least one selected from typhoid vaccine (parenteral), typhoid Vi polysaccharide vaccine, varicella virus vaccine, yellow fever vaccine. The at least one antitoxin or anti-snake venom may be at least one selected from the black spider anti-snake venom, the Crotalidae anti-snake venom (multivalent), the diphtheria anti-toxin (horse), and the American coral snake ( Micurus fulvius ) anti-snake venom ). At least one immune serum is cytomegalovirus immunoglobulin (intravenous), hepatitis B immunoglobulin (human), immunoglobulin muscle, immunoglobulin intravenous, rabies immunoglobulin (human), RS virus immunoglobulin intravenous (Human), Rh ₀ (D) immunoglobulin (human), Rh ₀ (D) immunoglobulin intravenous (human), tetanus immunoglobulin (human), varicella / zoster herpes immunoglobulin obtain. At least one biological response modifier is aldesleukin, GLP-1 etin alpha, filgrastim, glatiramer acetate for injection, interferon alphacon-1, interferon alpha-2a (recombinant) , Interferon α-2b (recombinant), interferon β-1a, interferon β-1b (recombinant), interferon γ-1b, levamisole hydrochloride, oprelbequin, sargramostim (eg, Nursing 2001) (See Drug Handbook, pp. 964-1040).

  At least one ocular anti-infective agent is bacitracin, chloramphenicol, ciprofloxacin hydrochloride, erythromycin, gentamicin sulfate, ofloxacin 0.3%, polymyxin B sulfate, 10% sodium sulfacetamide, sulfaceta It can be selected from mid sodium 15%, sulfacetamide sodium 30%, tobramycin, vidarabine. At least one ophthalmic anti-inflammatory drug is dexamethasone, dexamethasone sodium phosphate, diclofenac sodium 0.1%, fluorometholone, flurbiprofen sodium, ketorolac tromethamine, prednisolone acetate prednisolone phosphate sodium It may be at least one selected from (solution). The at least one miotic drug may be at least one selected from acetylcholine chloride, carbachol (intraocular), carbachol (topical), iodothiothiophate, pilocarpine, pilocarpine hydrochloride, pilocarpine nitrate. The at least one mydriatic agent may be at least one selected from atropine sulfate, cyclopentrate hydrochloride, epinephrine hydrochloride, epinephryl borate, homatropine hydrobromide, phenylephrine hydrochloride, scopolamine hydrobromide, tropicamide. . The at least one ocular vasoconstrictor may be at least one selected from naphazoline hydrochloride, oxymetazoline hydrochloride, and tetrahydrozoline hydrochloride. At least one miscellaneous ophthalmic drug is apraclonidine hydrochloride, betaxolol hydrochloride, brimonidine tartrate, carteolol hydrochloride, dipivefrin hydrochloride, dorzolamide hydrochloride, emedastine fumarate, sodium fluorescein, ketotifen fumarate, latanoprost, levobunolol hydrochloride, metipranolol hydrochloride It may be at least one selected from roll, sodium chloride (hypertonic), timolol maleate. The at least one otic agent may be at least one selected from boric acid, carbamide peroxide, chloramphenicol, triethanolamine polypeptide amine-condensate. At least one nasal drug is selected from beclomethasone propionate, budesonide, ephedrine sulfate, epinephrine hydrochloride, flunisolide, fluticasone propionate, naphazoline hydrochloride, oxymetazoline hydrochloride, phenylephrine hydrochloride, tetrahydrozoline hydrochloride, triamcinolone acetonide, xylometazoline hydrochloride (See, for example, Nursing 2001 Drug Handbook, pp. 1041-97).

At least one topical anti-infective drug is acyclovir, amphotericin B, azelaic acid cream, bacitracin, butconazole nitrate, clindamycin phosphate, clotrimazole, econazole nitrate, erythromycin, gentamicin sulfate, ketoconazole, mafenide acetate, metronidazole It may be at least one selected from (local), miconazole nitrate, mupirocin, naphthyfin hydrochloride, neomycin sulfate, nitrofurazone, nystatin, silver sulfadiazine, terbinafine hydrochloride, terconazole, tetracycline hydrochloride, thioconazole, and tolnaftate. The at least one anti-scabicide or lice killing agent can be at least one selected from crotamiton, lindane, permethrin and pyrethrin. At least one topical corticosteroid is: betamethasone dipropionate, betamethasone valerate, clobetasol propionate, desonide, desoxymethasone, dexamethasone, dexamethasone sodium phosphate, diflorazone acetate, fluocinolone acetonide, fluocinonide, fluland It may be at least one selected from lenolide, fluticasone propionate, harsionide, hydrocortisone, hydrocortisone acetate, hydrocortisone butyrate, hydrocortisone valerate, mometasone furoate, triamcinolone acetonide (see, for example, Nursing 2001 Drug Handbook pp. 1098- 1136).

  At least one vitamin or mineral is vitamin A, vitamin B group, cyanocobalamin, folic acid, hydroxocobalamin, leucovorin calcium, niacin, niacinamide, pyridoxine hydrochloride, riboflavin, thiamine hydrochloride, vitamin C, vitamin D, cholecalciferol, ergo Calciferol, vitamin D analog, doxel calciferol, paricalcitol, vitamin E, vitamin K analog, phytonadione, sodium fluoride, sodium fluoride (topical), trace elements, chromium, copper, iodine, manganese, selenium, zinc It may be at least one selected from At least one kind of pyrogen is amino acid infusion (crystal), D-glucose amino acid infusion, amino acid infusion containing electrolyte, amino acid infusion containing D-glucose electrolyte, amino acid infusion for liver failure, highly metabolically invasive An amino acid infusion for amino acids, amino acid infusion for renal failure, D-glucose, fat emulsion, medium chain triglycerides (see, eg, Nursing 2001 Drug Handbook, pp. 1137-63). .)

The present invention also provides a suitable and / or effective amount of at least one of any composition or pharmaceutical composition comprising at least one GLP-1 mimetibody or specified portion or variant. Optionally, at least one TNF antagonist (including but not limited to a TNF chemical or protein antagonist, a TNF monoclonal or polyclonal antibody or fragment, a soluble TNF receptor (eg, p55, p70 or p85) or fragment, Their fusion polypeptides, or small molecule TNF antagonists such as TNF binding protein I or II (TBP-1 or TBP-II), nerellimonmab, infliximab, enteraccept, CDP-571, CDP-87 Anti-rheumatic drugs (eg methotrexate, auranofin, gold thioglucose, azathioprine, etanercept, sodium gold thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasaldin), muscle relaxant, narcotic Non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, local anesthetics, neuromuscular blocking agents, antibacterial drugs (eg aminoglycosides, antifungals, antiparasitic drugs, antiviral drugs, carbapenem, cephalo Sporin, flurolquinolone, macrolide, penicillin, sulfonamide, tetracycline, another antibacterial agent), anti-psoriatic agent, corticosteroid, anabolic steroid, diabetes related drug, mineral, nutritional drug, thyroid drug, vitamin, calcium Related hor , Antipruritics, antitussives, antiemetics, anti-ulcers, laxatives, anticoagulants, erythropoietin (eg, epoetin alfa), filgrastim (eg, G-CSF, Neupogen), sargramostim (GM-CSF, Leukine), immunity , Immunoglobulin, immunosuppressant (eg basiliximab, cyclosporine, daclizumab), growth hormone, hormone replacement agent, estrogen receptor modulator, mydriatic, ciliary muscle modulator, alkylating agent, antimetabolite, thread Mitotic inhibitor, radiopharmaceutical, antidepressant, antidepressant, antipsychotic, anxiolytic, sleeping pill, sympathomimetic, stimulant, donepezil, tacrine, asthma drug, beta agonist, inhaled steroid, leukotriene inhibitor, Methylxanthine, cromolyn, epinephrine or analog, Dornase α (Pulm ozyme), an effective amount of at least one additional compound, protein or composition selected from cytokines or cytokine antagonists. Non-limiting examples of such cytokines include, but are not limited to, any of IL-1 to IL-23. Suitable dosages are known in the art. For example, edited by Wells et al., Pharmacotherapy Handbook, 2nd edition, Appleton and Lange, Stanford, Connecticut (2000); PDR
See Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Luxury Edition, Tarascon Publishing, Lomarinda, Calif. (2000), each of which is incorporated herein by reference in its entirety.

Such compositions can also include toxin molecules that are associated, bound, co-formulated or co-administered with at least one antibody or polypeptide of the invention. Toxins can in some cases act to selectively kill diseased cells or tissues. The pathological cell can be a cancer or other cell. Such toxins include, but are not limited to, purified or recombinant toxins selected from, for example, at least one of ricin, diphtheria toxin, snake venom, or bacterial toxin, or at least one functional cytotoxicity of the toxin Mention may be made of toxin fragments comprising a sex domain. The term toxin is also used by any naturally occurring mutant or recombinant bacterium or virus that can cause any pathological condition in humans and other mammals, including toxic shock that can result in death. Also included are both endotoxins and exotoxins that are produced. Such toxins include, but are not limited to, heat-labile enterotoxin of enterotoxigenic E. (E.coli) (LT), heat-stable enterotoxin (ST), Shigella (Shigella) cytotoxin, Aeromonas (Aeromonas ) Enterotoxins, toxic shock syndrome toxin-1 (TSST-1), staphylococcal enterotoxin A (SEA), B (SEB) or C (SEC), streptococcal enterotoxins, and the like. Such bacteria include, but are not limited to, Enterotoxigenic Escherichia coli ( E. coli ) (ETEC), Enterohemorrhagic Escherichia coli ( E. coli ) (eg, serotype 0157: strain of H7) species, Staphylococcus (Staphylococcus) species (e.g., Staphylococcus aureus (Staphylococcus aureus), Staphylococcus Pojensu (Staphylococcus pyogenes)), Shigella (Shigella) species (e.g., Shigella dysenteriae (Shigella dysenteriae), Shigella flexneri (Shigella flexneri), voids dysentery bacteria (Shigella boydii) and sonnei Shigella (Shigella sonnei)), Salmonella (Sal onella) species (for example Salmonella typhi (Salmonella typhi), pig cholera bacteria (Salmonella cholera-suis), Salmonella enteritidis (Salmonella enteritidis)), Clostridium (Clostridium) species (for example Clostridium perfringens (Clostridium perfringens), Clostridium difficile (Clostridium dificile ), botulinum (Clostridium botulinum)), Kang furo genus (Camphlobacter) species (e.g., cans furo Citrobacter jejuni (Camphlobacter jejuni), Kang furo Acetobacter Fetsusu (Camphlobacter f tus)), Helicobacter (Heliobacter) species (e.g., Helicobacter pylori (Heliobacter pylori)), Aeromonas (Aeromonas) species (e.g., Aeromonas Soburia (Aeromonas sobria), Aeromonas hydrophila (Aeromonas hydrophila), Aeromonas Kabie (Aeromonas caviae)), Pureisomonasu Shigeroidesu (Pleisomonas shigelloides), Yersinia Enterobacter coli Tikka (Yersina enterocolitica), Vibrio (Vibrios) species (e.g., Vibrio cholerae (Vibrios cholerae), Vibrio Parahemoritiku (Vibrios parahemolyticus)), Klebsiella (Klebsiella) species, may be mentioned strains of Pseudomonas aeruginosa (Pseudomonas aeruginosa) and Streptococcus (Streptococci). For example, Stein, INTERNAL MEDICINE, 3rd edition, pp 1-13, Little, Brown and Co. Boston, (1990); edited by Evans et al., Bacterial Infections of Humans: Epidemiology and Control, 2nd edition, pp 239-254, Plenum.
Medical Book Co. , New York (1991); Mandell et al., Pr inciples and Practice of Infectious Diseases , 3rd Edition, Churchill Livingstone, New York (1990); Berkow et al., Eds., The Merck Manual, 16th edition, Merck and Co. Lowe, New Jersey, 1992; Wood et al., FEMS Microbiology Immunology, 76: 121-134 (1991); Marrack et al., Science, 248: 705-711 (1990) (the contents of those references are directly incorporated by reference). (Incorporated herein).

The GLP-1 mimetibody or designated portion or variant compositions of the present invention include, but are not limited to, diluents, binders, stabilizers, buffers, salts, lipophilic solvents, preservatives, adjuvants, and the like. It may further comprise at least one of any suitable auxiliary substances that can. Pharmaceutically acceptable auxiliary substances are preferred. Non-limiting examples of such sterilized solutions and methods of manufacture are not limited, but are described by Gennaro, Remington's Pharmaceutical Sciences , 18th Edition, Mack Publishing Co. (Easton, Pa.) 1990 can be mentioned in the art. Pharmaceutically acceptable carriers suitable for the mode of administration, solubility and / or stability of GLP-1 mimetibody compositions as known in the art or as described herein may be routinely selected.

  Pharmaceutical excipients and additives useful in the present compositions include, but are not limited to, proteins, peptides, amino acids, lipids and carbohydrates (eg, monosaccharides, 2, 3, 4 and oligosaccharides) Sugars; derivatized sugars such as alditols, aldonic acids, esterified sugars; and polysaccharides or sugar polymers), which can be present alone or in combination, alone or in combination from 1 to 99 .99% by weight or volume%. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein and the like. Representative amino acids / GLP-1 mimetibody or designated portion or variant components that can also function in buffering capacity include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, Includes valine, methionine, phenylalanine, aspartame and the like. One preferred amino acid is glycine.

  Suitable carbohydrate excipients for use in the present invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose; disaccharides such as lactose, sucrose, trehalose, cellobiose; Polysaccharides such as raffinose, melezitose, maltodextrin, dextran, starch and the like; and alditols such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glutitol), myo-inositol and the like. Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose and raffinose.

The GLP-1 mimetibody composition may also include a buffer or pH modifier; typically, the buffer is a salt prepared from an organic acid or base. Exemplary buffering agents include salts of organic acids such as citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid or phthalic acid salts; Tris, tromethamine hydrochloride or phosphate buffer. Preferred buffering agents for use in the present composition are organic acid salts such as citrate.

  In addition, the GLP-1 mimetibody or specified portion or variant composition of the present invention comprises polyvinylpyrrolidone, ficoll (polymer sugar), a dextrate (such as 2-hydroxypropyl-β-cyclodextrin). Cyclodextrins), polyethylene glycol, flavoring agents, antibacterial agents, sweeteners, antioxidants, antistatic agents, surfactants (eg polysorbates such as “TWEEN 20” and “TWEEN 80”), lipids (eg phospholipids, Fatty acid), steroids (eg cholesterol) and polymeric excipients / additives such as chelating agents (eg EDTA).

  These and additional known pharmaceutical excipients and / or additives suitable for use in the GLP-1 mimetibody compositions of the invention are described, for example, in “Remington: The Science & Practice of Pharmacy”, 19th edition, Williams & Williams, (1995) and “Physician's Desk Reference”, 52nd edition, Medical Economics, Montvale, NJ (1998), the disclosures of which are hereby incorporated by reference in their entirety. As known in the art. Preferred carrier or excipient materials are carbohydrates (eg monosaccharides and alditols) and buffering agents (eg citrate) or polymeric agents.

Formulation. As indicated above, the present invention provides a physiology suitable for pharmaceutical or veterinary use comprising at least one GLP-1 mimetibody or specified portion or variant in a pharmaceutically acceptable formulation. A stable formulation that can preferably include a suitable buffer containing saline or a selected salt, as well as any stored solutions and formulations containing preservatives, and multi-use stored formulations To do. The formulation to be stored is at least one known or at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrate, phenoxyethanol, in an aqueous diluent. Arbitrarily selected from the group consisting of formaldehyde, chlorobutanol, magnesium chloride (eg hexahydrate), alkylparabens (methyl, ethyl, propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal Contains preservatives, or mixtures thereof. Although not limited, 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03, 0.05, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1. 5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4. 0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9 or any range or value therein may be mentioned 0.001-5% or Any suitable concentration or mixture known in the art, such as any range or value It may be used. Non-limiting examples include no preservative, 0.1-2% m-cresol (eg, 0.2, 0.3.0.4, 0.5, 0.9, 1.0%), 0.1 3% benzyl alcohol (eg 0.5, 0.9, 1.1, 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (eg 0.005 0.01), 0.001 to 2.0% phenol (for example, 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005 to 1.0 % Alkylparaben (s) (e.g., 0.00075, 0.0009, 0.001, 0.002, 0.005, 0.0075, 0.009, 0.01, 0.02, 0.05) 0.075, 0.09, 0.1, 0.2, 0.3, 0.5, 0,.
75, 0.9, 1.0%) and the like.

  As indicated above, the present invention provides for at least one GLP-1 mimetibody or specified portion or variant of the packaging material, and optionally a formulated buffer and / or preservative in an aqueous diluent. Providing a product comprising at least one vial comprising a solution, wherein the packaging material comprises 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30 36, 40, 48, 54, 60, 66, 72, and a label indicating that it can be held for 72 hours or more. The invention further includes a packaging material, a first vial comprising at least one lyophilized GLP-1 mimetibody or a designated portion or variant, and an aqueous diluent of a formulated buffer or preservative. Wherein the packaging material comprises 24 hours of reconstitution of the at least one GLP-1 mimetibody or specified portion or variant in an aqueous diluent or It comprises a label that instructs the patient to form a solution that can be held over.

  At least one GLP-1 mimetibody or designated portion or variant used in the present invention is a mammalian cell or transgenic preparation as described herein or known in the art. Or can be purified from other biological sources.

  The range of the amount of at least one GLP-1 mimetibody or specified portion or variant in the product of the present invention is from the amount produced upon reconstitution, ie about 1.0 μg / ml in a wet / dry system. Although encompassing concentrations up to about 1000 mg / ml, lower and higher concentrations are operable and depend on the intended delivery vehicle, for example, solution formulations are transdermal patches, lungs, transmucosal Or different from osmotic or micropump methods.

  Preferably, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Preferred preservatives are phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkyl parabens (such as methyl, ethyl, propyl, butyl), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and Includes those selected from the group consisting of thimerosal, or mixtures thereof. The concentration of preservative used in the formulation is sufficient to produce an antimicrobial effect. Such concentrations depend on the preservative selected and are readily determined by those skilled in the art.

  Other excipients, such as isotonic agents, buffers, antioxidants, storage enhancers, may optionally and preferably be added to the diluent. Isotonic agents such as glycerin are generally used at known concentrations. A physiologically tolerable buffer is preferably added to provide improved pH control. The formulation can encompass a wide range of pH, such as from about pH 4 to about pH 10, and a preferred range from about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0. Preferably, the formulations of the present invention have a pH between about 6.8 and about 7.8. Preferred buffering agents include phosphate buffer, most preferably sodium phosphate, especially phosphate buffered saline (PBS).

Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxy) Pharmaceutically acceptable solubilizers such as ethylene polyoxypropylene block copolymers) and PEG (polyethylene glycol), or non-polysorbates 20 or 80 or poloxamers 184 or 188, PluronicO polyhydric alcohols, other block copolymers Ionic surfactants and other additives such as chelating agents such as EDTA and EGTA are optionally added to the formulation or composition to reduce aggregation. It can be. These additives are particularly useful when the formulation is administered using a pump or plastic container. The presence of a pharmaceutically acceptable surfactant alleviates the tendency of proteins to aggregate.

  The formulations of the present invention comprise at least one GLP-1 mimetibody or specified moiety or variant, as well as phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, Propyl, butyl, etc.), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or a mixture thereof in an aqueous diluent. obtain. Mixing at least one GLP-1 mimetibody or specified portion or variant and preservative in an aqueous diluent is performed using conventional dissolution and mixing procedures. In order to produce a suitable formulation, for example, a measured amount of at least one GLP-1 mimetibody or specified portion or variant in a buffer solution is sufficient to provide the desired concentration of protein and preservative. Combine with the desired preservative in buffer solution in an amount. Variations on this method will be recognized by those skilled in the art. For example, the order in which ingredients are added, whether additional additives are used, the temperature and pH at which the formulation is manufactured, are all factors that can be optimized for the concentration and means of administration used.

  The claimed formulation contains water, preservatives and / or excipients, preferably phosphate buffer and / or saline and selected salts as clear solutions or in aqueous diluents Can be provided to the patient as two vials comprising a vial of at least one GLP-1 mimetibody or specified portion or variant that is reconstituted in a second vial. Either a single solution vial or two vials that require reconstitution can be reused multiple times and may be sufficient for single or multiple cycles of patient treatment and are therefore more convenient than currently available Treatment regimens may be provided.

  The claimed product is useful for administration immediately or over 24 hours or more. Thus, the presently claimed product offers significant benefits to the patient. The formulations of the present invention can optionally be safely stored at temperatures from about 2 to about 40 ° C. and retain the biological activity of the protein for extended periods of time, so that the packaging labels are 6, 12, 18, 24 , 36, 48, 72 or 96 hours or more can be shown to show that the solution can be retained and / or used. When using stored diluents, such labels may include use of at least one of 1-12 months, half a year, one and a half years and / or two years.

  The solution of at least one GLP-1 mimetibody or specified portion or variant of the invention comprises mixing at least one GLP-1 mimetibody or specified portion or variant in an aqueous diluent. It can be produced by a method. Mixing is performed using conventional dissolution and mixing procedures. In order to produce a suitable diluent, for example, a measured amount of at least one GLP-1 mimetibody or specified portion or variant in water or buffer is added to the desired concentration of protein and optionally a preservative or Combine in an amount sufficient to provide diluent. Variations on this method will be recognized by those skilled in the art. For example, the order in which ingredients are added, whether additional additives are used, the temperature and pH at which the formulation is manufactured, are all factors that can be optimized for the concentration and means of administration used.

The claimed product may contain at least one lyophilized GLP-1 mimetibody or specified portion or variant vial reconstituted as a clear solution or in a second vial containing an aqueous diluent. It can be provided to the patient as two vials comprising. Either a single solution vial or two vials that require reconstitution can be reused multiple times and may be sufficient for single or multiple cycles of patient treatment and are therefore more convenient than currently available An effective treatment regimen.

  The claimed product comprises at least one lyophilized GLP-1 mimetibody or specified portion or variant vial reconstituted with a clear solution or a second vial containing an aqueous diluent Can be provided to the patient indirectly by providing two vials of The clear solution in this case can be up to 1 liter in size or even larger, with at least one GLP-1 mimetibody or a specified part or a smaller part of the variant solution more A large reservoir is provided that can be removed one or more times for transfer to a small vial and provided to their customers and / or patients.

These comprise a single vial systems recognized ^{device, Humaject (R), NovoPen (} R), B-D (R) Pen, delivery of a solution such as AutoPen ^(R), and OptiPen ^(R) Including a pen injector device. Comprising two vials system recognized device includes a pen injector device for reconstructing the drug was lyophilized in a cartridge for delivery of the reconstituted solution such as HumatroPen ^(R) .

  Currently claimed products include packaging materials. The packaging material provides the conditions under which the product can be used in addition to the information required by the regulatory authorities. The packaging material of the present invention reconstitutes at least one GLP-1 mimetibody or specified portion or variant in an aqueous diluent to form a solution for two vials or wet / dry products and the solution Provides instructions to the patient for use for 2 to 24 hours or longer. For single vial solution products, the label indicates that such a solution can be used for 2-24 hours or longer. The presently claimed product is useful for use in human pharmaceutical products.

  The formulation of the present invention is a mixture of phosphate buffer containing at least one GLP-1 mimetibody or specified portion or variant and a selected buffer, preferably physiological saline or a selected salt. Can be produced by a method comprising Mixing at least one GLP-1 mimetibody or specified portion or variant and buffer in an aqueous diluent is performed using conventional dissolution and mixing procedures. To produce a suitable formulation, for example, to provide a measured amount of at least one GLP-1 mimetibody or specified portion or variant in water or buffer to the desired concentration of protein and buffer. Combine with the desired buffer in water in sufficient quantity. Variations on this method will be recognized by those skilled in the art. For example, the order in which ingredients are added, whether additional additives are used, the temperature and pH at which the formulation is manufactured, are all factors that can be optimized for the concentration and means of administration used.

  The claimed stable or stored formulation is a lyophilized minimum reconstituted as a clear solution or in a second vial containing a preservative or buffer and excipients in an aqueous diluent. The patient may be provided as two vials comprising one GLP-1 mimetibody or a vial of a specified portion or variant. Either a single solution vial or two vials that require reconstitution can be reused multiple times and may be sufficient for single or multiple cycles of patient treatment and are therefore more convenient than currently available An effective treatment regimen.

  At least one GLP-1 mimetibody or specified portion or variant in any of the stable or preserved formulations or solutions described herein is an SC or IM as known in the art. Infusion; administered to a patient according to the present invention via a variety of delivery methods including transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micropump or other means recognized by those skilled in the art. obtain.

  Therapeutic application. The present invention for mimetibodies also includes, but is not limited to, insulin resistance, hyperglycemia, hypoglycemia, pancreatitis, sushing syndrome, melanosis, lipoatrophy in cells, tissues, organs, animals or patients Related signs and symptoms that can include diabetes, retinopathy, nephropathy, polyneuropathy, mononeuropathy, autonomic neuropathy, ulcers, foot ulcers, joint problems, infections (eg fungi or bacteria), etc. Also provided are methods for the regulation or treatment of diabetes, i.e. type I or type II diabetes, including adult-onset or juvenile-onset, insulin-dependent, insulin-independent.

  The invention also includes, but is not limited to, insulin resistance, hyperglycemia, hypoglycemia, pancreatitis, soothing syndrome, melanosis, lipotrophic diabetes, retinopathy, nephropathy, polyneuropathy Adult-onset or juvenile-onset, insulin dependent, including related signs and symptoms, which may include mononeuropathy, autonomic neuropathy, ulcers, foot ulcers, joint problems, infections (eg fungi or bacteria), etc. Modulation or treatment of at least one diabetes-related immune-related disease in a cell, tissue, organ, animal or patient, including at least one type I or type II diabetes, including type, non-insulin dependent, etc. A method is also provided. For example, Merck Manual, 12th to 17th edition, Merck & Company, Lowway, New Jersey (1972, 1977, 1982, 1987, 1992, 1999), Pharmacotherapy Handbook, Wells et al., 2nd edition, Applet and Lange (1998, 2001), each incorporated by reference in its entirety).

  Such methods optionally involve effective amounts of at least one GLP-1 mimetibody or at least one composition or pharmaceutical composition comprising a specified portion or variant in such modulation, treatment or therapy. Administration to a cell, tissue, organ, animal or patient in need thereof.

The present invention also includes, but is not limited to, stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, bleeding, arteriosclerosis, atherosclerosis, diabetic arteriosclerotic disease, hypertension , Arterial hypertension, renovascular hypertension, syncope, shock, cardiovascular syphilis, heart failure, pulmonary heart, primary pulmonary hypertension, cardiac arrhythmia, ectopic contraction of arteries, atrial flutter , Atrial fibrillation (persistent or paroxysmal), disordered or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrhythmia, ventricular fibrillation, His bundle arrhythmia, atrioventricular block, leg block , Myocardial ischemic disorder, coronary artery disease, angina pectoris, myocardial infarction, cardiomyopathy, dilated congestive cardiomyopathy, restrictive cardiomyopathy, valvular heart disease, endocarditis, pericardial disease, heart tumor, aorta and peripheral Aneurysm, aortic dissection, aorta Inflammation, obstruction of the abdominal aorta and its side branches, peripheral vascular disorders, obstructive arterial disorders, peripheral atherosclerotic disease, occlusive thrombotic vasculitis, functional peripheral arterial disorders, Raynaud's phenomenon and Raynaud's disease, extremity cyanosis Erythema pain, venous disease, venous thrombosis, varicose veins, arteriovenous fistula, lymphedema, fatty edema, unstable angina, reperfusion injury, post-pump syndrome, ischemia-reperfusion injury, etc. Also provided is a method of modulating or treating at least one cardiovascular disease in a cell, tissue, organ, animal or patient, which can include at least one. Such a method involves the use of an effective amount of a composition or pharmaceutical composition comprising at least one GLP-1 mimetibody or a specified portion or variant, such as cells, tissues, organs in need of such modulation, treatment or therapy. , May optionally include administering to an animal or patient.

  Any of the methods of the present invention provides an effective amount of a composition or pharmaceutical composition comprising at least one GLP-1 mimetibody or a specified portion or variant, in such cells in need of such modulation, treatment or therapy. Administration to a tissue, organ, animal or patient. Such methods may optionally involve administration of said at least one GLP-1 mimetibody, specified portion or variant thereof, with at least one TNF antagonist (including but not limited to TNF antibody or fragment, soluble TNF receptor). Body or fragments, fusion proteins thereof, or small molecule TNF antagonists), anti-rheumatic drugs, muscle relaxants, narcotics, non-steroidal anti-inflammatory drugs (NSAIDs), analgesics, anesthetics, sedatives, Local anesthetics, neuromuscular blockers, antibacterials (eg aminoglycosides, antifungals, antiparasitics, antivirals, carbapenem, cephalosporins, fluorquinolones, macrolides, penicillins, sulfonamides, tetracyclines, other antibacterials Drugs), anti-psoriatic drugs, corticosteroids, protein Anabolic steroids, diabetes-related drugs, minerals, nutritional drugs, thyroid drugs, vitamins, calcium-related hormones, antidiarrheals, antitussives, antiemetics, anti-ulcer drugs, laxatives, anticoagulants, erythropoietin (eg GLP-1 ethin (GLP- 1 etin) α), filgrastim (eg G-CSF, Neupogen), salgramostim (GM-CSF, Leukine), immunization, immunoglobulin, immunosuppressant (eg basiliximab, cyclosporine, daclizumab), growth hormone, hormone replacement , Estrogen receptor modulators, mydriatics, ciliary muscle modulators, alkylating agents, antimetabolites, mitotic inhibitors, radiopharmaceuticals, antidepressants, antidepressants, antipsychotics, anxiolytics, Hypnotic, sympathomimetic, stimulant, donepezil, tacrine, asthma medicine, beta agonist, inhalation Such immunity further comprising administering at the same time and / or after at least one selected from steroids, leukotriene inhibitors, methylxanthine, cromolyn, epinephrine or analogs, Dornase alpha (Pulmozyme), cytokines or cytokine antagonists It may further include co-administration or combination therapy to treat the disease. Suitable dosages are known in the art. For example, edited by Wells et al., Pharmacotherapy Handbook, 2nd edition, Appleton and Lange, Stamford, Connecticut (2000); PDR Pharmacopoeia, Tarascon Pocket Copodia 2000, Reference Palomar, Paramar, P Each of which is incorporated herein by reference in its entirety.

The mimetibody can also be used ex vivo, such as in autologous bone marrow cultures. Briefly, bone marrow is removed from the patient prior to chemotherapy and treated with TPO and / or GLP-1 optionally with mimetibody and optionally with one or more additional cytokines. The treated bone marrow is then returned to the patient after chemotherapy to accelerate bone marrow recovery. In addition, TPO alone and in combination with GLP-1 mimetibody and / or GLP-1 can also be used for ex vivo growth of bone marrow or peripheral blood precursor (PBPC) cells. Prior to chemotherapy treatment, the bone marrow can be stimulated with stem cell factor (SCF) or G-CSF to release early precursor cells into the peripheral circulation. These progenitor cells are optionally collected and concentrated from peripheral blood and then optionally, but not limited to, SCF, G-CSF, IL-3, GM-CSF, IL-6 or IL- 11 can be treated in culture with TPO and mimetibody in combination with one or more other cytokines, which can be differentiated and expanded into high density megakaryocyte cultures, which are optionally Return to patient after dose chemotherapy. The dose of TPO for ex vivo treatment of bone marrow can range from 100 pg / ml to 10 ng / ml, preferably from 500 pg / ml to 3 ng / ml. The dose of mimetibody can be comparable in activity against GLP-1, from 0.1 units / ml to 20 units / ml, preferably from 0.5 units / ml to 2 units / ml, or any of them Can be used within these ranges or values.

  TNF antagonists suitable for the compositions, combination therapies, co-administrations, devices and / or methods of the invention (which further comprise at least one antibody of the invention, specified portions and variants thereof) are limited Although not, anti-TNF antibodies, their ligand-binding fragments, and receptor molecules that specifically bind to TNF; thalidomide, tenidap, phosphodiesterase inhibitors (eg, pentoxyphyllin and rolipram), A2b adenosine receptor agonists and A2b adenosine receptors Compounds that prevent and / or inhibit TNF synthesis, TNF release or its effect on target cells, such as body potentiators; prevent and / or inhibit TNF receptor signaling such as mitogen-activated protein (MAP) kinase inhibitors / Or inhibit Compounds; compounds that block and / or inhibit membrane TNF cleavage such as metalloproteinase inhibitors; compounds that block and / or inhibit TNF activity such as angiotensin converting enzyme (ACE) inhibitors (eg captopril); and MAP kinases Mention may be made of compounds that block and / or inhibit the production and / or synthesis of TNF, such as inhibitors.

  As used herein, a “tumor necrosis factor antibody”, “TNF antibody”, “TNFα antibody” or fragment or the like reduces, blocks, blocks TNFα activity in vitro, in situ and / or preferably in vivo. Obstruct, abolish or obstruct. For example, suitable TNF human antibodies of the invention can bind TNFα and include anti-TNF antibodies, antigen binding fragments thereof, and designated variants or domains thereof that specifically bind to TNFα. Suitable TNF antibodies or fragments are also TNF RNA, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and / or synthesis also block, block, abolish, prevent And / or may inhibit.

  The chimeric antibody cA2 consists of a high-affinity neutralizing mouse anti-human TNFα IgG1 antibody antigen-binding variable region called A2, and a human IgG1 constant region κ immunoglobulin. The human IgG1 Fc region improves the effector function of alloantibodies, prolongs circulating serum half-life, and reduces antibody immunogenicity. The affinity and epitope specificity of the chimeric antibody cA2 is derived from the variable region of the mouse antibody A2. In one particular embodiment, one preferred source of nucleic acid encoding the variable region of mouse antibody A2 is the A2 hybridoma cell line.

Chimeric A2 (cA2) neutralizes the cytotoxic effects of both natural and recombinant human TNFα in a dose-dependent manner. From the binding assay of chimeric antibody cA2 and recombinant human TNFα, the affinity constant of chimeric antibody cA2 was calculated to be 1.04 × 10 ¹⁰ M ⁻¹ . A preferred method for measuring the specificity and affinity of monoclonal antibodies by competitive inhibition is described by Harlow et al., Antibodies: A Laboratory Manual, Cold.
Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; edited by Colligan et al., Current Protocols in Immunology, Green Publishing Assoc. and Wiley Interscience, New York, (1992-2003); Kozbor et al . , Immunol. Today , 4: 72-79 (1983); edited by Ausubel et al., Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-2003); and Muller, Meth. Enzymol. 92: 589-601 (1983), the references of which are hereby incorporated by reference in their entirety.

Additional examples of monoclonal anti-TNF antibodies that can be used in the present invention have been described in the art (eg, US Pat. No. 5,231,024; Moeller, A. et al., Cytokine 2 (3). : 162-169 (1990); U.S. Patent Application No. 07 / 943,852 (filed September 11, 1992); Rathjen et al., International Patent Publication No. WO 91/02078 (February 21, 1991). Rubin et al., GLP-1 Patent Publication No. 0 218 868 (published on April 22, 1987); Yone et al., GLP-1 Patent Publication No. 0 288 088 (1988 October) month 26 days); Liang et al., Biochem.Biophys.Res.Comm 137:. 847-854 (1986 ); Meager et al. Hybridoma 6: 305-311 (1987); Fendly et al., Hybridoma 6: 359-369 (1987) ; Bringman , et al., Hybridoma 6:. 489-507 (1987 ); and Hirai et al, J.Immunol.Meth 96: 57- 62 (1987), which references are incorporated herein by reference in their entirety).

TNF receptor molecule. Preferred TNF receptor molecules useful in the present invention bind TNFα with high affinity (see, eg, Feldmann et al., International Patent Publication No. WO 92/07076 (published 30 April 1992); Schall et al., See Cell 61: 361-370 (1990); and Loetscher et al., Cell 61: 351-359 (1990), which references are hereby incorporated by reference in their entirety. Have low immunogenicity. In particular, 55 kDa (p55 TNF-R) and 75 kDa (p75 TNF-R) TNF cell surface receptors are useful in the present invention. See truncated forms of these receptors comprising the extracellular domain (ECD) of the receptor or a functional part thereof (eg Corcoran et al . , Eur. J. Biochem. 223: 831-840 (1994)) . Is also useful in the present invention. A truncated TNF receptor comprising ECD has been detected as a 30 kDa and 40 kDa TNFα inhibitory binding protein in urine and serum (Engelmann, H. et al., J. Biol. Chem. 265: 1531-1536 . (1990)). Multimeric molecules of TNF receptors and TNF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof, are additional examples of TNF receptor molecules that are useful in the methods and compositions of the invention. TNF receptor molecules that can be used in the present invention are characterized by their ability to treat patients for extended periods with good to excellent relief of symptoms and low toxicity. Low immunogenicity and / or high affinity, as well as other undefined properties, can contribute to the therapeutic outcome achieved.

  Multimeric molecules of TNF receptors useful in the present invention include two or more TNF receptors linked via one or more polypeptide linkers or other non-peptide linkers such as polyethylene glycol (PEG). It comprises all or part of the body's ECD. The multimeric molecule may further comprise a signal peptide of a secreted protein that directs expression of the multimeric molecule. These multimeric molecules and methods for their production are described in US patent application Ser. No. 08 / 437,533 (filed May 9, 1995), the contents of which are hereby incorporated by reference in their entirety. It is described in.

TNF immunoreceptor fusion molecules useful in the methods and compositions of the invention comprise at least a portion of one or more immunoglobulin molecules and all or a functional portion of one or more TNF receptors. . These immunoreceptor fusion molecules can be assembled as monomers, or hetero- or homomultimers. Immunoreceptor fusion molecules can also be monovalent or multivalent. An example of such a TNF immunoreceptor fusion molecule is the TNF receptor / Ig
G fusion protein. TNF immunoreceptor fusion molecules and methods for their production are described in the art (Lesslauer et al . , Eur. J. Immunol. 21: 2883-2886 (1991); Ashkenazi et al., Proc. Natl. Acad. Sci. USA 88: 10535) . Peppel et al . , J. Exp. Med. 174: 1483-1489 (1991); Kolls et al., Proc. Natl. Acad. Sci. USA 91: 215-219 (1994); Butler et al., Cytokine 6 (6): 616-623 (1994) ; Baker et al., Eur.J.Immunol 24:. 2040-2048 (1994 ); Beutler et al., U.S. Pat. No. 5,447,851; and U.S. Patent application No. 08 442,133 Pat (May 16, 1995 filed) is described in (each of those references entirely incorporated herein by reference)). Methods for producing immunoreceptor fusion molecules are described in Capon et al., US Pat. No. 5,116,964; Capon et al., US Pat. No. 5,225,538; and Capon et al., Nature 337: 525-531. (1989) (these references are incorporated herein by reference in their entirety).

A functional equivalent, derivative, fragment or region of a TNF receptor molecule is functionally similar to a TNF receptor molecule that can be used in the present invention (eg, binds TNFα with high affinity and exhibits low immunogenicity). Refers to the portion of the TNF receptor molecule or the portion of the TNF receptor molecule sequence that encodes the TNF receptor molecule that is of sufficient size and sequence. A functional equivalent of a TNF receptor molecule is also modified to be functionally similar to a TNF receptor molecule that can be used in the present invention (eg, binds TNFα with high affinity and has low immunogenicity). Also included are TNF receptor molecules. For example, a functional equivalent of a TNF receptor molecule can be a “silent” codon, or one or more amino acid substitutions, deletions or additions (eg, the use of another amino acid in place of one acidic amino acid; or Use of another codon encoding the same or different hydrophobic amino acid instead of one codon encoding one hydrophobic amino acid. Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Assoc. and
See Wiley-Interscience, New York (1987-2003).

  Cytokines include, but are not limited to, all known cytokines. For example, Copywith Cytokines. com. Cytokine antagonists can include, but are not limited to, any antibody, fragment or mimetic, any soluble receptor, fragment or mimetic, any small molecule antagonist, or any combination thereof.

  Any of the methods of the present invention provides an effective amount of a composition or pharmaceutical composition comprising at least one GLP-1 mimetibody or a specified portion or variant, in such cells in need of such modulation, treatment or therapy. A method of treating a protein-mediated disorder comprising administering to a tissue, organ, animal or patient. Such a method is such that administration of said at least one GLP-1 mimetibody, specified portion or variant thereof is at least 1 such as IL-3, -6 and -11; stem cell factor; G-CSF and GM-CSF. It may optionally further comprise a co-administration or combination therapy to treat such immune diseases, further comprising administering at least one selected from other cytokines of the species prior to and / or after.

Typically, treatment of a pathological condition is a minimum of about 0.0001 to 500 milligrams minimum per kilogram of patient per dose on average, depending on the specific activity contained in the composition. One GLP-1 mimetibody or specified portion or variant,
And preferably at least about 0.001 to 100 milligrams of GLP-1 mimetibody per kilogram of patient per single or multiple doses or at least one GLP-1 mimetibody composition that falls within the specified portion or variant range This is accomplished by administering an effective amount or dosage of. Alternatively, the effective serum concentration may comprise a serum concentration of 0.001-5000 μg / ml per single or multiple doses. Suitable dosages are known to medical practitioners and can of course depend on the particular disease state, the specific activity of the composition being administered, and the particular patient being treated. In some instances, to achieve the desired therapeutic amount, repeated administration, i.e. repeated individual administration of a specific monitor or measured dose, which repeats individual administration until the desired daily dose or effect is achieved. It may be necessary to provide

  Preferred doses are optionally 0.0001, 0.0002, 0.0003, 0.0004, 0.0005.0.0006, 0.0007, 0.0008, 0.0009, 0.001, 0.001 per administration. 002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 0.09, 0.1, 0.2, 0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0. 9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 and / or 30 mg / kg, or any range, value or fraction thereof, Or 0.0001, 0.0002, 0.0003, 0.0004, 0.0005.0.0006, 0.0007, 0.0008, 00009, 0.001, 0.001 per single or multiple doses. 002, 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 0.09, 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2. 0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5, 15. 9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20. 9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400 and / or young May be included to achieve a serum concentration of 500 μg / ml, or any range, value or fraction thereof.

  Alternatively, the dosage administered will depend on the pharmacokinetic characteristics of the particular agent, and its mode of administration and route; recipient age, health and weight; nature and extent of symptoms, type of treatment, It can vary depending on the frequency, as well as known factors such as the desired effect. Usually, the dosage of active ingredient can be about 0.0001 to 100 milligrams per kilogram of body weight. Usually 0.001 to 10 and preferably 0.001 to 1 milligram per kilogram per dose or in sustained release form is effective to achieve the desired result.

By way of non-limiting example, human or animal treatment can be performed using single, infusion, or repeated doses, using 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28
, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39 or 40 days or at least 1, 2, 3, 4, 5, 6, 7, 8 , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 weeks, or any combination thereof, at least one GLP-1 of the present invention 0.0002, 0.0003, 0.0004, 0.0005.0.0006, 0.0007, 0.0008, 00009, 0.001, 0.002, mimetibody or specified part or variant per day 0.003, 0.004, 0.005, 0.006, 0.007, 0.008, 0.009, 0.01, 0.02, 0.03, 0.04, 0.05.0. 06, 0.07, 0.08, 0.0 0.1, 0.2, 0.3, 0.4, 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8 , 9 or 10 mg / kg, or 0.0001 to 100 mg / kg as temporary or regular dosages.

  Dosage forms (composition) suitable for internal administration generally contain from about 0.0001 milligram to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions, the active ingredient can usually be present in an amount of about 0.5 to 99.999% by weight, based on the total weight of the composition.

  For parenteral administration, the GLP-1 mimetibody or specified portion or variant may be formulated as a solution, suspension, emulsion or lyophilized powder with a pharmaceutically acceptable parenteral vehicle, or Can be provided separately. Examples of such vehicles are water, saline, Ringer's solution, D-glucose solution and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils can also be used. The vehicle or lyophilized powder may contain additives that maintain isotonicity (eg, sodium chloride, mannitol) and chemical stability (eg, buffers and preservatives). The formulation is sterilized by known or suitable techniques.

  Suitable pharmaceutical carriers are standard reference textbooks in the field, Remington's Pharmaceutical Sciences, A.M. It is described in the latest version of Osol.

  Therapeutic administration. Many known and developed modalities can be used to administer a pharmaceutically effective amount of at least one GLP-1 mimetibody or specified portion or variant of the present invention. The GLP-1 mimetibody of the present invention can be solution in a carrier using any of a variety of devices and methods suitable for inhalation or administration by other modes described herein or known in the art. Can be delivered as an emulsion, colloid or suspension, or as a powder.

Parenteral formulation and administration. Formulations for parenteral administration may contain sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalene, and the like as common excipients. Aqueous or oily suspensions for injection can be prepared according to known methods by using suitable emulsifying or wetting agents and suspending agents. Agents for injection can be non-toxic, non-orally administrable diluents such as aqueous solutions or sterile injectable solutions or suspensions in solvents. Usable vehicles or solvents include water, Ringer's solution, isotonic saline, and the like; sterile, fixed oils can be used as conventional solvents or suspending solvents. For these purposes, any type of non-volatile oil and fatty acid may be used, including natural or synthetic or semi-synthetic fatty oils or fatty acids; natural or synthetic or semi-synthetic mono or di or triglycerides. Parenteral administration is known in the art and includes, but is not limited to, conventional injection means, gas pressurized needleless injection as described in US Pat. No. 5,851,198. Mention may be made of the apparatus and the laser perforator apparatus as described in US Pat. No. 5,839,446, which is hereby incorporated by reference in its entirety.

  Alternative delivery. The invention further provides parenteral, subcutaneous, intramuscular, intravenous, bolus, vaginal, rectal, buccal, sublingual, intranasal or transdermal means of at least one GLP-1 mimetibody or specified portion or variant. Related to administration. Protein or GLP-1 mimetibody or specified part or variant composition is in particular in the form of a liquid solution or suspension for parenteral (subcutaneous, intramuscular or intravenous); vaginal or rectal administration Especially in the form of semi-solids such as creams and suppositories; especially in the form of tablets or capsules for buccal or sublingual administration; or in particular powders, nasal drops or aerosols or certain types Including chemical enhancers such as dimethyl sulfoxide for either intranasally in the form of an agent; or to modify skin structure or increase drug concentration in transdermal patches ("Drug Permeation"). Enhancement "; Hsieh, DS edited by Junginger et al., Pp. 59- 90 (Marcel Dekker, Inc. New York 1994 (incorporated herein by reference in its entirety))) or application of a formulation containing protein and peptide onto the skin (WO 98/53847) ) Or the application of an electric field to create a transitory transport path such as electroporation or to increase the mobility of charged drugs through the skin such as iontophoresis, or ultrasound such as sonophoresis (US Patents 4,309,989 and 4,767,402) (the above publications and patents are incorporated herein by reference in their entirety) In particular in the form of gel, ointment, lotion, suspension or patch delivery system It can be prepared for use in transdermal.

Pulmonary / nasal administration. For pulmonary administration, preferably at least one GLP-1 mimetibody or specified portion or variant composition is delivered in a particle size effective to reach the lower respiratory tract or sinus of the lung. According to the present invention, at least one GLP-1 mimetibody or specified portion or variant may be delivered by any of a variety of inhalation or nasal devices known in the art for administration of therapeutic agents by inhalation. These devices capable of dGLP-1 sitting of aerosolized formulations in a patient's nasal sinus or alveoli include metered dose inhalers, nebulizers, dry powder generators, nebulizers, and the like. Other devices suitable for directing pulmonary or nasal administration of a GLP-1 mimetibody or specified portion or variant are also known in the art. All such devices may use a formulation suitable for administration for dispensing a GLP-1 mimetibody or specified portion or variant in an aerosol. Such aerosols can consist of either solutions (both aqueous and non-aqueous) or solid particles. Ventolin ^(R) metered dose inhaler, such as a metered dose inhaler, typically use a propellant gas and require activation during inspiration (eg No. WO 94/16970, the first WO No. 98/35888). Devices marketed by ^{Inhale Therapeutics, Turbuhaler TM (Astra)} , Rotahaler (R) (Glaxo), Diskus (R) (Glaxo), dry powder inhalers, such as Spiros ^TM inhaler (Dura), and Spinhaler ^{(R )} Powder inhalers (Fisons) use breath activation of mixed powders (US Pat. No. 4,668,218 Astra, European Patent EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, US Patent No. US 5458135 Inhale, WO 94/06498 Fisons (incorporated herein by reference in its entirety). AERx ^TM Aradigm, Ultravent ^(R) nebulizer (Mallinckrodt) and Acorn II ^(R) nebulizer (Marquest Medical Products) by references (U.S. Patent No. US 5,404,871 No. Aradigm, No. WO 97/22376) (above cited Nebulizers, such as those incorporated herein in their entirety, produce aerosols from solution, while metered dose inhalers, dry powder inhalers, etc. produce small particle aerosols. These specific examples of commercially available inhalation devices are intended to be representative of specific devices suitable for the practice of the present invention, and are not intended to limit the scope of the invention . Preferably, the composition comprising at least one GLP-1 mimetibody or specified portion or variant is delivered by a dry powder inhaler or nebulizer. There are several desirable features of an inhalation device for administering at least one GLP-1 mimetibody or specified portion or variant of the present invention. For example, delivery by an inhalation device is advantageously reliable, reproducible and accurate. The inhalation device can in some cases deliver small dry particles, eg less than about 10 μm, preferably about 1-5 μm, for good respirability.

  Administration of a GLP-1 mimetibody or specified portion or variant composition as a spray. A spray comprising a GLP-1 mimetibody or a designated portion or variant composition protein allows a nozzle to pass under pressure through a suspension or solution of at least one GLP-1 mimetibody or a designated portion or variant It can be caused by. Nozzle size and configuration, applied pressure, and liquid feed rate can be selected to achieve the desired output and particle size. For example, the electric spray can be generated by an electric field with a capillary or nozzle feed. Advantageously, the particles of at least one GLP-1 mimetibody or designated portion or variant composition protein delivered by a nebulizer are less than about 10 μm, preferably about 1 μm to 5 μm, and most preferably about 2 μm to about It has a particle size in the range of 3 μm.

  A formulation of at least one GLP-1 mimetibody or designated portion or variant composition protein suitable for use in a nebulizer is typically from about 1 mg to about 20 mg of at least one GLP-1 mimetibody or 1 ml of solution or Include a GLP-1 mimetibody or a specified part or variant composition protein in aqueous solution at a concentration of the specified part or variant composition protein. The formulation may include excipients, buffers, isotonic agents, preservatives, surfactants and preferably agents such as zinc. The formulation may also include excipients or agents for the stabilization of GLP-1 mimetibody or specified portion or variant composition proteins, such as buffers, reducing agents, bulk proteins or carbohydrates. Bulk proteins useful in formulating GLP-1 mimetibodies or designated portion or variant composition proteins include albumin, protamine, and the like. Exemplary carbohydrates useful in formulating GLP-1 mimetibodies or designated portion or variant composition proteins include sucrose, mannitol, lactose, trehalose, glucose and the like. The GLP-1 mimetibody or specified part or variant composition protein formulation also exhibits surface-induced aggregation of the GLP-1 mimetibody or specified part or variant composition protein caused by atomization of the solution in the formation of an aerosol. Surfactants that can be reduced or prevented can also be included. A variety of conventional surfactants can be used, such as polyoxyethylene fatty acid esters and alcohols and polyoxyethylene sorbitol fatty acid esters. The amount can generally range between 0.001 and 14% by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Additional agents known in the art for formulation of proteins such as mimetibodies or designated portions or variants may also be included in the formulation.

Administration of GLP-1 mimetibody or specified portion or variant composition by nebulizer. The GLP-1 mimetibody or specified portion or variant composition protein may be administered by a nebulizer such as a jet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air supply is used to create a high velocity air jet through the orifice. As the gas expands beyond the nozzle, a low pressure region is created that draws a solution of the GLP-1 mimetibody or specified portion or variant composition protein through a capillary tube connected to a liquid reservoir. The liquid stream from the capillary is sheared into unstable filaments and droplets as it exits the tube, creating an aerosol. A range of configurations, flow rates and baffle types can be used to achieve the desired performance characteristics from a given jet nebulizer. In ultrasonic nebulizers, high frequency electrical energy is used, typically a piezoelectric transformer, to create vibratory mechanical energy. This energy is transmitted to the GLP-1 mimetibody or specified portion or variant composition protein formulation, either directly or by a coupling fluid, so that the GLP-1 mimetibody or specified portion or variant composition protein Create an aerosol containing Advantageously, the particles of GLP-1 mimetibody or specified portion or variant composition protein delivered by the nebulizer are less than about 10 μm, preferably from about 1 μm to less than 5 μm, and most preferably from about 2 μm to about 3 μm. Having a particle size of

  A formulation of at least one GLP-1 mimetibody or specified portion or variant suitable for use in either a jet or ultrasonic nebulizer typically has a minimum of about 1 mg to about 20 mg of 1 mg / ml solution. Include the GLP-1 mimetibody or specified portion or variant composition protein in aqueous solution at a concentration of the GLP-1 mimetibody or specified portion or variant protein. The formulation may include excipients, buffers, isotonic agents, preservatives, surfactants, and preferably agents such as zinc. The formulation also includes an excipient or agent for stabilization of at least one GLP-1 mimetibody or specified portion or variant composition protein, such as a buffer, reducing agent, bulk protein or carbohydrate. Can do. Bulk proteins useful in formulating at least one GLP-1 mimetibody or designated partial or variant composition protein include albumin, protamine, and the like. Exemplary carbohydrates useful in formulating at least one GLP-1 mimetibody or specified portion or variant include sucrose, mannitol, lactose, trehalose, glucose and the like. At least one GLP-1 mimetibody or specified portion or variant formulation exhibits surface-induced aggregation of at least one GLP-1 mimetibody or specified portion or variant caused by atomization of the solution in the formation of an aerosol. Surfactants that can be reduced or prevented can also be included. A variety of conventional surfactants such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters may be used. The amount can generally range between 0.001 and 4% by weight of the formulation. Particularly preferred surfactants for the purposes of the present invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, and the like. Additional agents known in the art for formulation of a protein, such as at least one GLP-1 mimetibody or specified portion or variant protein, may also be included in the formulation.

Administration of GLP-1 mimetibody or specified part or variant composition by metered dose inhaler. In a metered dose inhaler (MDI), a propellant, at least one GLP-1 mimetibody or specified portion or variant, and any excipients or other additives, including liquefied compressed gas As contained in the canister. Actuation of the metering valve releases the mixture as an aerosol containing particles preferably in a size range of less than about 10 μm, preferably from about 1 μm to about 5 μm, and most preferably from about 2 μm to about 3 μm. The desired aerosol particle size can be obtained by formulating GLP-1 mimetibody or a specified partial or variant composition protein produced by a variety of methods known to those skilled in the art including jet milling, spray drying, critical point condensation, etc. It can be obtained by use. Preferred metered dose inhalers include those manufactured by 3M or Glaxo and using hydrofluorocarbon propellants.

  Formulations of at least one GLP-1 mimetibody or specified part or variant for use in a metered dose inhaler device will generally include at least one GLP-1 mimetibody or specified part or variant, eg, an interface Fine powders can be included containing as a suspension in a non-aqueous medium suspended in a propellant with the aid of an active agent. Propellants are trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluoroalkane-134a), HFA-227 (hydrofluoroalkane-227) Any conventional material used for this purpose such as chlorofluorocarbons, hydrochlorofluorocarbons, hydrofluorocarbons or hydrocarbons may be included. Preferably the propellant is a hydrofluorocarbon. Surfactants are selected to stabilize at least one GLP-1 mimetibody or specified portion or variant as a suspension in propellant, protect the active ingredient against chemical degradation, etc. be able to. Suitable surfactants include sorbitan trioleate, soy lecithin, oleic acid and the like. In some cases, a solution aerosol using a solvent such as ethanol is preferred. Additional agents known in the art for the formulation of proteins such as proteins can also be included in the formulation.

  One skilled in the art will recognize that the methods of the invention can be achieved by pulmonary administration of at least one GLP-1 mimetibody or a specified portion or variant composition via a device not described herein. Let's go.

  Mucosal formulation and administration. Compositions and methods of administering at least one GLP-1 mimetibody or specified portion or variant for absorption through the mucosal surface promote absorption through the mucosal surface by achieving mucoadhesion of the emulsion particles , Emulsions comprising a plurality of submicron particles, mucoadhesive macromolecules, bioactive peptides and an aqueous continuous layer (US Pat. No. 5,514,670). Mucosal surfaces suitable for application of the emulsions of the present invention may include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomatic, intestinal and rectal routes of administration. Formulations for vaginal or rectal administration such as suppositories may contain, for example, polyalkylene glycol, petrolatum, cocoa butter and the like as excipients. Formulations for intranasal administration can be solid and can contain, for example, lactose as an excipient, or can be an aqueous or oily solution of nasal drops. For buccal administration, excipients include sugar, calcium stearate, magnesium stearate, laked starch, and the like (US Pat. No. 5,849,695).

  Oral formulation and administration. Formulations for oral use are of auxiliary substances (such as resorcinol and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecyl polyethylene ether) to artificially increase the permeability of the intestinal wall. Rely on co-administration and co-administration of enzyme inhibitors (eg pancreatic trypsin inhibitor, diisopropylfluorophosphate (DFF) and trasilol) to inhibit enzymatic degradation. The active ingredients of solid dosage forms for oral administration are sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starch, agar, alginate, chitin, chitosan, pectin, tragacanth gum, gum arabic , Gelatin, collagen, casein, albumin, synthetic or semi-synthetic polymers, and at least one additive including glycerides. These dosage forms include other types of additive (s) such as inert diluents, lubricants such as magnesium stearate, parabens, sorbic acid, ascorbic acid, α-tocopherol and the like. Preservatives, antioxidants such as cysteine, disintegrants, binders, thickeners, buffers, sweeteners, flavorings, fragrances and the like may also be included.

  Tablets and pills can be further processed into enteric-coated formulations. Liquid formulations for oral administration include emulsions, syrups, elixirs, suspensions and solution formulations acceptable for medical use. These formulations may contain an inert diluent commonly used in the art, such as water. Liposomes have also been described as drug delivery systems for insulin and heparin (US Pat. No. 4,239,754). More recently, mixed amino acid artificial polymers (proteinoids) microspheres have been used to deliver pharmaceuticals (US Pat. No. 4,925,673). In addition, the carrier compounds described in US Pat. Nos. 5,879,681 and 5,5,871,753 are used to deliver biologically active ingredients orally. Known in the field.

  Transdermal formulation and administration. For transdermal administration, at least one GLP-1 mimetibody or specified portion or variant is assembled into liposomes or polymer nanoparticles, microparticles, microcapsules or microspheres (unless otherwise stated). Encapsulated in a delivery device (sometimes referred to as microparticles). Synthetic polymers such as polyhydroxy acids, polyorthoesters, polyanhydrides and polyphosphazenes such as polylactic acid, polyglycolic acid and copolymers thereof, and collagen, polyamino acids, albumin and other proteins, alginates and other A number of suitable devices are known (US Pat. No. 5,814,599), including natural polymers such as polysaccharides, as well as microparticles made from combinations thereof.

  Long-term administration and formulation. It may sometimes be desirable to deliver a compound of the invention to a subject over a long period of time from a single administration, for example from 1 week to 1 year. A variety of sustained release dGLP-1t or implant dosage forms may be utilized. For example, the dosage form may be a pharmaceutically acceptable non-toxic salt of a compound having a low degree of solubility in body fluids, such as (a) phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, Acid addition salts with polybasic acids such as alginic acid, polyglutamic acid, naphthalene mono- or disulfonic acid, polygalacturonic acid, etc .; (b) zinc, calcium, bismuth, barium, magnesium, aluminum, copper, cobalt, nickel, cadmium A salt with a polyvalent metal cation such as, for example, N, N′-dibenzyl-ethylenediamine or an organic cation formed from ethylenediamine; or a combination of (a) and (b) such as zinc tannate May be contained. In addition, compounds of the invention, or preferably relatively insoluble salts such as those just described, can be formulated in gels, such as aluminum monostearate gels, with sesame oils suitable for injection. Particularly preferred salts are zinc salts, zinc tannates, pamoates and the like. Another type of sustained release dGLP-1t formulation for injection is a slowly degrading non-toxic, such as polylactic acid / polyglycolic acid polymer as described in US Pat. No. 3,773,919. The compound or salt encapsulated or dispersed in the non-antigenic polymer. The compounds, or preferably relatively insoluble salts such as those described above, may also be formulated in cholesterol matrix silastic pellets, especially for use in animals. Additional sustained release dGLP-1t or implant formulations such as gaseous or liquid liposomes are known in the literature (US Pat. No. 5,770,222 and “Sustained and Controlled Release Drug Delivery Systems”, J R. Robinson, Marcel Dekker, Inc., New York, 1978).

  Having generally described the invention, it will be more readily understood with reference to the following examples, which are provided as specific illustrations and are not intended to be limiting.

Cloning and Expression of GLP-1 Mimetibody in Mammalian Cells A typical mammalian expression vector comprises at least one promoter element, GLP-1 mimetibody or specified portion or variant, that mediates the initiation of transcription of mRNA. Contains the coding sequence and signals required for transcription termination and transcript polyadenylation. Additional elements include enhancers, Kozak sequences, and intervening sequences flanked by donor and acceptor sites for RNA splicing. Highly efficient transcription can be achieved with the early and late promoters from SV40, the terminal repeats from retroviruses such as RSV, HTLVI, HIVI (LTRS), and the cytomegalovirus (CMV) early promoter. However, cellular elements can also be used (eg the human actin promoter). Expression vectors suitable for use in the practice of the present invention include, for example, pIRES1neo, pRetro-Off, pRetro-On, PLXSN or pLNCX (Clonetech Labs, Palo Alto, Calif.), PcDNA3.1 (+/−), pcDNA / Zeo (+ /-) Or pcDNA3.1 / Hygro (+/-) (Invitrogen), vectors such as PSVL and PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109) Is included. Mammalian host cells that can be used are human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells. Is included.

  Alternatively, the gene can be expressed in stable cell lines containing the gene integrated into the chromosome. Co-transfection with a selectable marker such as dhfr, gpt, neomycin or hygromycin allows the identification and isolation of the transfected cells.

  The transfected gene can also be amplified to express large amounts of the encoded GLP-1 mimetibody or specified portion or variant. DHFR (dihydrofolate reductase) markers are useful for developing cell lines carrying hundreds or even thousands of copies of the gene of interest. Another useful selectable marker is the enzyme glutamine synthetase (GS) (Murphy et al., Biochem. J. 227: 277-279 (1991); Bebbington et al., Bio / Technology 10: 169-175 (1992)). Using these markers, mammalian cells are grown in selective media and the cells with the highest resistance are selected. These cell lines contain the amplified gene (s) integrated into the chromosome. Chinese hamster ovary (CHO) and NSO cells are often used for the production of GLP-1 mimetibodies or specified portions or variants.

  The expression vectors pC1 and pC4 are the Rous sarcoma virus strong promoter (LTR) (Cullen et al., Molec. Cell. Biol. 5: 438-447 (1985)) and one fragment of the CMV-enhancer (Boshart et al., Cell 41: 521-530 (1985)). For example, a multiple cloning site containing restriction enzyme cleavage sites BamHI, XbaI and Asp718 facilitates cloning of the gene of interest. In addition to the 3 'intron, the vector contains polyadenylation and termination signals for the rat preproinsulin gene.

Cloning and expression in CHO cells. The vector pC4 is used for expression of the GLP-1 mimetibody or specified portion or variant. Plasmid pC4 is a derivative of plasmid pSV2-dhfr (ATCC accession number 37146). The plasmid contains the mouse DHFR gene under the control of the SV40 early promoter. Chinese hamster ovary or other cells lacking dihydrofolate activity, transfected with these plasmids, are selected in selective media supplemented with the chemotherapeutic agent methotrexate (eg, α-MEM, Life Technologies, Gatorsburg, Md.). Selection can be made by growing the cells. Amplification of the DHFR gene in cells resistant to methotrexate (MTX) has been well documented (eg, FW Alt et al., J. Biol. Chem. 253: 1357-1370 (1978); J.L. See Hamlin and C. Ma, Biochem. Et Biophys. Acta 1097: 107-143 (1990); and MJ Page and MA Sydenham, Biotechnology 9: 64-68 (1991)). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme DHFR as a result of amplification of the DHFR gene. When the second gene is linked to the DHFR gene, it is usually co-amplified and overexpressed. It is known in the art that this approach can be used to develop cell lines that carry over 1,000 copies of amplified gene (s). Subsequently, when the methotrexate is removed, a cell line is obtained containing the amplified gene integrated into one or more chromosomes of the host cell.

Plasmid pC4 is a strong promoter for Rous sarcoma virus long terminal repeat (LTR) (Cullen et al., Molec. Cell. Biol. 5: 438-447 (1985)) and human cytomegalovirus for expressing the gene of interest. A fragment isolated from the enhancer of the immediate early gene of (CMV) (Boshart et al., Cell
41: 521-530 (1985)). Downstream of the promoter are BamHI, XbaI, and Asp718 restriction enzyme cleavage sites that allow gene integration. Behind these cloning sites, the plasmid contains a 3 ′ intron and a polyadenylation site for the rat preproinsulin gene. Other high efficiency promoters such as the human b-actin promoter, SV40 early or late promoter, or terminal repeats from other retroviruses such as HIV and HTLVI may also be used for expression. Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express GLP-1 in a regulated manner in mammalian cells (M. Gossen and H. Bujard, Proc. Natl.Acad.Sci.USA 89: 5547-5551 (1992)). For polyadenylation of mRNA, other signals from, for example, human growth hormone or globin genes can be used as well. Stable cell lines carrying the gene of interest integrated into the chromosome can also be selected upon cotransfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to initially use one or more selectable markers such as G418 and methotrexate.

  Plasmid pC4 is digested with restriction enzymes and then dephosphorylated using calf intestinal phosphatase by procedures known in the art. The vector is then isolated from a 1% agarose gel.

  The DNA sequence encoding the complete GLP-1 mimetibody or specified portion or variant, corresponding to the HC and LC variable regions of the GLP-1 mimetibody of the present invention, is used according to known method steps. Isolated nucleic acids encoding suitable human constant regions (ie, HC and LC regions) are also used in the constructs.

  The isolated DNA encoding the variable and constant regions and the dephosphorylated vector are then ligated using T4 DNA ligase. E. coli HB101 or XL-1 Blue cells are then transformed, and bacteria containing the fragment inserted into plasmid pC4 are identified, for example using restriction enzyme analysis.

  Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection. 5 μg of expression plasmid pC4 is co-transfected with 0.5 μg of plasmid pSV2-neo using lipofection. Plasmid pSV2neo contains the neo gene from Tn5 which encodes an enzyme that confers resistance to a dominant selectable marker, ie, a group of antibiotics including G418. Cells are seeded in α-MEM supplemented with 1 μg / ml G418. Two days later, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in α-MEM supplemented with 10, 25 or 50 ng / ml methotrexate and 1 μg / ml G418. After about 10-14 days, single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using various concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones that grow at the highest concentration of methotrexate are then transferred to a new 6-well plate containing still higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is repeated until clones are obtained that grow at a concentration of 100-200 mM. Expression of the desired gene product is analyzed, for example, by SDS-PAGE and Western blot or reverse phase HPLC analysis.

Non-limiting example of GLP-1 mimetibody of the invention GLP-1 is a 37 amino acid peptide secreted from intestinal L cells after oral glucose administration. A mimetibody construct incorporating a biologically active GLP-1 (7-37) peptide, variant or derivative provides a new therapy for extending the in vivo life of the peptide and lowering blood glucose in patients with type 2 diabetes That is expected. A native GLP-1 (7-37) peptide or a peptide encoding a DPP-IV resistant analog can be incorporated into the mimetibody scaffold structure. Some of these molecules were made and the resulting mimetibodies showed activity in functional in vitro cell-based assays. It should be noted that a variety of in vitro assays and in vivo models may be used in these studies, and potency may not be comparable to each other or to the results presented herein.

  To generate a variant of GLP-1 mimetibody, GLP-1 peptide, linker, hinge or CH2 and CH3 sequences in the mimetibody are deleted, added, substituted to improve expression, potency, stability or effector function, Can be mutated or modified.

  Wild-type GLP-1 sequences as well as DDP-IV resistant GLP-1 variants such as GLP-1 (A2S) or GLP-1 (A2G) can be incorporated into mimetibody scaffold structures. Mutations of the peptide can be made to improve the properties of GLP-1 mimetibody. For example, mutations in the amino terminal residue can improve signal transduction, while mutations in the helical domain can stabilize the helix and thereby improve mimetibody binding to the receptor and / or stability. .

The length and composition of the linker can be mutated to vary the flexibility or stability of the bond between the GLP-1 peptide and the Fc region. A variety of isotypes can be incorporated into the hinge region of the molecule. In addition, mutations can be made in the hinge region of the mimetibody to stabilize the molecule. For example, a human IgG4 hinge can be mutated to create a Ser ^228- > Pro variant to stabilize interchain disulfide bonds in mimetibodies. Variations within the Fc portion of the mimetibody can be made to improve the stability of the molecule and alter effector functions such as FcR binding. For example, human or mouse isotypes (or variants of these molecules) such as IgG4 with Ala / Ala mutations can be used.

GLP-1 mimetibody of the present invention. A specific non-limiting example of the present invention is the formula (I):
((P (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t), where P is one copy of biologically active GLP-1 Peptide (7-36), L is the tandem repeat of either Gly-Ser or Gly-Gly-Gly-Ser flexible linker, and V is the C-terminus of the V _H sequence, ie the J region of naturally occurring IgG H is the complete IgG1 hinge region and CH2 and CH3 are of the IgG1 isotype subclass]
The GLP-1 mimetibody construct (SEQ ID NO: 2). It is expected that the half-life of this construct will be many times that of the GLP-1 peptide alone or variants or derivatives thereof and will be similar to that of IgG.

  In addition to the basic structure described above, variants with potentially favorable biological characteristics are described. These include constructs that may have a reduced tendency to self-associate, reduced immune effector function or reduced immunogenicity. Other modifications that confer desired characteristics, such as improved conformation of biologically active peptides and movement across the blood brain barrier, are envisioned. The proposed variants and modifications can be combined in any manner to produce a construct with the desired activity.

  Using recombinant DNA methods, the GLP-1 peptide was inserted into the intermediate vector between the immunoglobulin signal peptide and the human J sequence. This was done using complementary synthetic oligonucleotides with ends compatible with the restriction sites present in the vector. These oligonucleotides contained a flexible linker composed of the coding sequence of the GLP-1 peptide and two GGGS repeats. The restriction fragment containing the aforementioned functional elements was then transferred into an expression vector. This vector comprises anti-CD4 immunoglobulin promoters and enhancers, as well as human IgG1 hinge sequences, HC constant region 2 (CH2) and constant region 3 (CH3) coding sequences, and replication and selection of plasmids in bacteria and mammals. It contained the elements necessary for selection of stable expressers in cells.

  This plasmid was introduced into HEK293E cells and expression of wt GLP-1 MMB was achieved in transiently transfected cells. Purification of GLP-1 MMB was achieved by standard protein A and Superose 12 affinity chromatography, yielding approximately 1.5 mg per liter of transfected cells. This protein was the starting material for the experiments described below.

  The amino acid sequence of GLP-1 mimetibody is shown in FIG. The functional domain is noted above the peptide coding sequence. The J sequence causes the GLP-1 dimer to conform properly and also protrudes from the globular structure of the immunoglobulin and penetrates the crevice between the two GLP-1 receptors. It is believed that it would provide even greater flexibility for. There are 3 cysteines in the IgG1 hinge region. The first is usually paired with an immunoglobulin light chain (LC), and the other two appear to participate in the interchain bonds between the two HCs. CH2 and CH3 regions constitute the bulk of the protein. One of the reasons that immunoglobulins are thought to have a long serum half-life is their ability to bind FcRn, extending serum half-life by returning phagocytosed immunoglobulins to the extracellular space. The binding site of FcRn overlaps the junction of the CH2 and CH3 regions (Shields et al., 2001, J. Biol. Chem., Vol. 276 (9), 6591-6604).

The two IgG heavy chains are known to assemble during cell processing through disulfide bonds between cysteines located in the hinge region to form homodimers. It is expected that this will also occur between the modified peptides to form an assembled GLP-1 mimetibody construct. In addition, it is expected that an intrachain disulfide bond between the two cysteines in the GLP-1 peptide will also occur. The expected structure of a GLP-1 mimetibody contains two GLP-1 peptides. The spatial arrangement of the N-terminal peptide along with the flexibility of the binding sequence should allow the peptide to form a bioactive dimer.

FACS binding assay The activity of GLP-1 mimetibody was tested in an in vitro FACS binding assay. To determine whether GLP-1 MMB binds GLP-1R, HEK293 cells overexpressing GLP-1R (1 × 10 ⁶ cells) were incubated with GLP-1 MMB (20 nM) at 4 ° C. for 2 hours. . Cells were washed and fluorescently labeled secondary detection antibody (1 μg / mL goat anti-human IgG, Fcγ specific) was added at 4 ° C. for 30 minutes. Cell fluorescence intensity was monitored via flow cytometry. FIG. 2A shows that GLP-1 MMB binds to HEK293 cells overexpressing GLP-1R (grey, GLP-1 MMB but no secondary; black, secondary only; red, negative control MMB and secondary Blue, GLP-1 MMB and secondary). FIG. 2B shows that GLP-1 MMB does not bind to control HEK293 cells (grey, GLP-1 MMB but no secondary; black, secondary only; blue, GLP-1 MMB and secondary). FIG. 2C shows that the GLP-1 peptide analog (A2S) can compete with GLP-1 MMB for binding to HEK293 cells overexpressing GLP-1R (grey, GLP-1 MMB but two None; black, GLP-1 MMB and secondary; orange, GLP-1 MMB, 0.2 nM competitor, secondary; blue, GLP-1 MMB, 20 nM competitor, secondary; red, GLP-1 MMB, 100 nM competitor, secondary).

cAMP Assay GLP-1 binds to its receptor, a G protein-coupled receptor, resulting in a dose-dependent increase in the signaling molecule 3 ′, 5′-cyclic AMP (cAMP). cAMP can be measured in an in vitro assay (Applied Biosystems) in cells expressing GLP-1R. Briefly, Rinm cells (100,000 cells) were incubated with increasing concentrations of GLP-1 peptide (0-30 nM) or GLP-1 MMB (0-100 nM). Cells were lysed and, using a competitive assay using alkaline phosphatase labeled cAMP conjugate and a chemiluminescent substrate ^{(Tropix (R) CDPD (R} )), to determine the amount of cAMP. The concentration-dependent cAMP activity of wt GLP-1 MMB (FIG. 3A) is comparable to the GLP-1 peptide (FIG. 3B) (EC ₅₀ = 11 nM vs. 0.4 nM, respectively). In a similar experiment, GLP-1 (A2G) MMB in the IgG4 scaffold structure (FIG. 3C) and GLP-1 (A2S) MMB in the IgG4 scaffold structure (FIG. 3D) are both cAMP in the Rinm cells. Level in wt in IgG4 scaffold structure
Increased to a significantly higher level than GLP-1 MMB.

DPP-IV Cleavage Assay Since GLP-1 is rapidly inactivated by DPP-IV, an in vitro assay was established to quantify intact (ie uncleaved) GLP-1 MMB. Briefly, GLP-1 MMB or peptide (1.2 nM) was incubated with DPP-IV (1 μg / mL, R & D Systems) at room temperature. After various times (0, 5, 10, 15, 20, 30, 40 minutes), DPP-IV inhibitor (100 μM, Linco)
Was added to quench the reaction. The amount of intact GLP-1 MMB or peptide was measured using GLP-1 Active ELISA (Linco) and GLP-1 MMB or peptide for each standard curve. FIG. 4 shows that GLP-1 MMB was significantly more resistant to cleavage by DPP-IV for the GLP-1 peptide.

Human serum stability assay To confirm that no other serum protease was able to cleave and inactivate GLP-1 MMB, the stability of GLP-1 MMB in serum was also measured. Briefly, GLP1 peptide or GLP1 MMB (30 nM) was incubated at 37 ° C. in human serum. After various times, the reaction was quenched with a DPP-IV inhibitor (100 μM, Linco) and samples were analyzed using a GLP-1 Active ELISA from Linco. FIG. 5 shows that GLP-1 MMB is stable in human serum for 24 hours while the peptide is rapidly disintegrated.

GLP-1 MMB causes insulin secretion in RINm cells To test the effect of GLP-1 MMB on insulin secretion, RINm cells were tested at increasing concentrations of GLP-1 (7-36) peptide (0-5 nM), Treatment with exendin-4 peptide (0-5 nM), or various GLP-1 mimetibodies (5 or 50 nM), and the amount of insulin secreted was measured via ELISA. All GLP-1 MMBs tested were active in stimulating insulin secretion in RINm cells (FIG. 6). At 50 nM, MMB had activity comparable to that of wild type GLP-1 (7-36) peptide.

GLP-1 MMB reduces glucose levels in db / db mice 6 week old db / db mice are fasted for 2 hours and then vehicle, GLP-1 peptide or GLP-1 (A2S) MMB is administered intravenously did. Blood glucose was monitored at 0.5, 1, 2, 3 and 4 hours after administration. GLP-1 peptide decreased blood glucose at 30 minutes, but by 60 minutes, blood glucose began to increase again, likely due to the short half-life of GLP-1 peptide. In comparison, a dose of GLP-1 (A2S) MMB that was 100 times lower than the dose of GLP-1 peptide induced a decrease in blood glucose throughout the 4 hours (FIG. 7A). In addition, the decrease in blood glucose was dose dependent (FIG. 7B).

Pharmacokinetics of GLP-1 MMB in mice and cynomolgus monkeys To measure the pharmacokinetics of four GLP-1 mimetibodies (A2G, A2S, exedin-cap and wt), 1 mg / kg MMB was used in C57 / B16 mice. Was administered intravenously. Plasma was obtained via cardiac puncture after mice were killed at various time points. A variety of ELISAs were used to measure Fc, total MMB, active MMB, and active peptides as they were metabolized in animals. Active MMB reflects the intact N-terminus of the peptide that is still bound to the Fc region of mimetibody. Replacement of the second amino acid (alanine) in the peptide with either serine or glycine extended the lifetime of active MMB in the circulation.

Cynomolgus monkeys were intravenously infused with 4 different GLP-1 MMB constructs at 1.0 mg / kg and serum samples were collected at various time points from 10 minutes to 5 days after dosing. Serum samples were evaluated by ELISA to quantify intact MMB. As specifically illustrated in FIG. 8, all four MMBs represent a rapid distribution period followed by a slower disappearance period. Pharmacokinetic constants were calculated for each of the constructs and showed a T1 _{/ 2 of} approximately 3 days with similar exposure determined by AUC from T = 0 to T = 120 hours.

Effect of GLP-1 MMB during oral glucose tolerance test in diabetic mice Eight week old diabetic db / db mice were fasted for 6 hours prior to subcutaneous injection of GLP-1 MMB (0.02 to 2 mg / kg). . Mice were fasted for an additional 6 hours after dosing and basal fasting blood glucose was measured. At t = 0, mice were given 1.0 mg / g glucose by oral gavage and blood glucose was measured at various time points. The results shown in FIG. 9 indicate that GLP-1 MMB was effective in reducing glucose excursion during the oral glucose tolerance test at all doses tested.

Effect of GLP-1 MMB on fasting blood glucose during chronic administration to diabetic mice. 10 weeks old diabetic db / db mice were subcutaneously administered with vehicle or GLP-1 MMB (1 mg / kg) daily for 6 weeks. Fasting blood glucose was measured twice a week during the course of the study. Fasting blood glucose is reduced with respect to controls in treated animals throughout the study (FIG. 10) and by 6 weeks the difference is greater than 200 mg / dL (466 vs. 221 mg / dL, control and treated animals, respectively). It was.

Effect of GLP-1 MMB on oral glucose tolerance test after chronic administration to diabetic mice As in Example 11, 10 weeks old diabetic db / db mice were treated with vehicle or GLP-1 MMB (1 mg / kg) every day. Weekly administration. 40 days after administration, mice were subjected to an oral glucose tolerance test. Briefly, at t = 0, mice received oral gavage of 1.0 mg / g glucose and blood glucose was measured at various time points. The results shown in FIG. 11 show that GLP-1 MMB was effective in reducing glucose excursions during the oral glucose tolerance test, and mice treated chronically with GLP-1 MMB were more efficient with respect to control animals This suggests that it is possible to eliminate the glucose load.

Effect of GLP-1 MMB on reduction of HbA1c after chronic administration to diabetic mice As in Examples 11 and 12, diabetic db / db mice at 10 weeks of age were given daily vehicle or GLP-1 MMB (1 mg / kg). Administration was for 6 weeks. Whole blood samples were taken before and after 6 weeks of administration and analyzed for percent HbA1c. As shown in FIG. 12, HbA1C of GLP-1 treated animals increased 109 percent at 6 weeks, while control treated animals increased 142 percent. This data suggests that treated animals are better able to control their blood glucose over the chronic phase with respect to controls.

Effect of GLP-1 MMB on oral glucose tolerance test in normal cynomolgus monkeys An oral glucose tolerance test (OGTT) was performed on normal cynomolgus monkeys before and 6 days after a single dose of GLP-1 MMB (1 mg / kg). Briefly, at t = 0, monkeys were given an oral gavage of 2.0 mg / g glucose and blood glucose was measured at various time points. Blood glucose levels were significantly reduced with OGTT performed 6 days after administration (FIG. 13A), and insulin levels were significantly increased (FIG. 13B). This suggests that GLP-1 MMB is causing insulin secretion from the pancreas at elevated glucose concentrations.

GLP-1 for insulin staining in islets of diabetic mice (db / db) after a single dose
Effect of MMB Twelve week old diabetic mice (db / db) were treated with a single subcutaneous dose of GLP-1 MMB (1.5 mg / kg) and pancreas was collected 4 weeks later. The pancreas was cut and stained for the presence of insulin. As shown in FIG. 14, there was significantly more insulin staining in the treated animals relative to the control animals.

GLP-1 MMB delays gastric emptying in normal dogs Gastric cannula was surgically implanted into female beagle dogs (10-15 kg) under general anesthesia and allowed to recover for a minimum of 2 weeks. The dog was fasted for 24 hours, after which water was freely available. The gastric cannula was opened and the gastric juice and the rest of the food were removed with 40-50 ml of boiling water. A group of 6 dogs received the α2 agonist, lidamidine (0.63 mg / kg) subcutaneously 60 minutes before the diet (positive control for delayed gastric emptying). Dogs receiving vehicle control or GLP-1 MMB (0.1 mg / kg) were administered intravenously into the cephalic vein 15 minutes prior to diet. The gastric cannula was opened 5 minutes before the diet to measure the amount of fluid present in the basal stomach and fluid was reintroduced rapidly. A test meal consisting of 250 ml glucose solution (5 g / l) was then administered via cannula and allowed to remain in the stomach for 30 minutes. After 30 minutes, the stomach contents were drained from the stomach and the total volume was measured. 1 ml of gastric contents was retained for analysis and the remaining volume was reintroduced into the stomach via cannula. Gastric content volume assessment and sample collection were repeated at 60, 90 and 120 minutes. The collected samples were evaluated for glucose concentration and used to determine the absolute amount of glucose remaining in the stomach at each time point. The percentage of glucose retained in the stomach was determined from the starting value and the concentration of glucose at each time point and plotted as a function of time. The time that 50% of the stomach contents were retained was determined by fitting the curve to a monoexponential function. As shown in FIG. 15, 50% of gastric contents were excreted after 12.35 ± 3.69 min in dogs receiving vehicle, while dogs receiving lidamidine positive control and GLP-1 MMB were gastric emptying. A significant delay (30.60 ± 6.47 and 59.23 ± 14.46, respectively).

GLP-1 MMB lowers blood glucose after oral glucose tolerance test (OGTT) in diet-induced obese mice To develop a mouse model of diet-induced obesity, mice were maintained on a high fat diet for a minimum of 27 weeks. Mice were obese and determined to be diabetic when fasting blood glucose levels exceeded 120 mg / dl. To evaluate the effect of GLP-1 MMB therapy on postprandial blood glucose levels, diet-induced obese mice were fasted overnight and administered 0.02, 0.2, or 2 mg / kg GLP-1 MMB or vehicle control subcutaneously. . Six hours after administration, the mice were given gavage of 1.5 mg / g glucose. Blood glucose levels were measured using tail vein blood at t = 0, 15, 30, 60, 90, 120, 150 and 180 minutes prior to MMB administration. As shown in FIG. 16, a GLP-1 MMB dose-dependent decrease in fasting blood glucose levels was observed at t = 0 and all subsequent time points. The area under the curve was calculated between t = 0 and t = 180 and showed a significant decrease in glucose disposition at all doses.

GLP-1 MMB reduces blood glucose in db / db mice in the intraperitoneal glucose tolerance test (IPGTT) Approximately 13-15 weeks old male db / db mice were treated with 6 mice based on fasting blood glucose levels Randomized. Mice were given 0.02 mg / kg or 0.1 mg / kg G
Either LP-1 MMB or 0.1 mg / kg negative control MMB was administered 6 hours prior to the glucose tolerance test. Five minutes before the glucose tolerance test, glucose measurements were taken from tail vein blood with a portable blood glucose meter. Mice were then administered intraperitoneally with 1 mg / g D-glucose and blood glucose levels were monitored at 10, 20, 30, 60, 90, 120, 150 and 180 minutes. As illustrated in FIG. 17A, blood glucose levels were significantly lower throughout the time course in both groups treated with GLP-1 MMB. Additional groups of animals treated in the same manner were killed at t = 10 minutes for measurement of insulin levels. There is a dose-dependent increase in the amount of insulin released 10 minutes after GLP-1 MMB administration.

GLP-1 MMB inhibits cytokine-induced apoptosis in a dose-dependent manner RIN-m cells were seeded at 50,000 cells / well in 96-well plates and incubated overnight at 37 ° C. The next day, cells were treated with a dose range of GLP-1 MMB (serially diluted from 100 nM) for 30 minutes before addition of cytokines TNFα (10 ng / mL) or IL-1β (4 ng / mL). Plates were incubated for 16 hours at 37 ° C. To assay for apoptosis, the Cell Death ELISA-Plus assay kit (Roche Applied Science, catalog number 119202085001) was used. The medium was aspirated from the wells, 200 μL of lysis buffer was added to each well, and the plate was incubated for 30 minutes at room temperature. After incubation, 20 μL of lysate was transferred to a streptavidin-coated 96 well microtiter plate. 80 μL of immunoreagent (20-fold dilution of anti-histone-biotin and anti-DNA-peroxidase in the provided incubation buffer) was also added to each well. 100 μL of the suspension was then incubated for 2 hours at room temperature with gentle shaking. After 2 hours the plate was washed 3 times with incubation buffer and the color development solution was added to the plate. Absorbance was read at 405 nm after approximately 5 minutes. The data show that GLP-1 MMB protects RIN-m cells from apoptosis induced by TNF-α or IL-1β (FIG. 18).

  In type 1 diabetes (T1D), there is a specific destruction of pancreatic β cells that secrete insulin. Although the exact molecular mechanism underlying β-cell destruction is not known, sera from T1D patients have been shown to promote apoptosis, suggesting that apoptosis plays a role in the development of diabetes Suggest. Treatment of patients with recent onset, those in remission, and patients still prone to diabetes with GLP-1 MMB can inhibit apoptosis and preserve beta cell mass.

  In T1D, β-cell apoptosis appears to play a role in the development of diabetes at two distinct phases of the disease. First, the wave of occurrence of islet apoptosis can affect the biology of T cells that ultimately affect the development of autoimmune diabetes. Second, islet apoptosis is also a mode of cell death that ultimately leads to large-scale destruction of beta cells. The use of GLP-1 MMB to inhibit β-cell apoptosis may therefore be useful in preventing the development of autoimmunity of diabetes or progression to overt diabetes by preserving β-cell mass.

T1D is the result from an irreversible loss of beta cells that secrete insulin. However, insulin secretion is detectable in some people with years of T1D, indicating that either a small population of living β cells or continuous regeneration of β cells dominates ongoing autoimmune destruction . T1D has been shown to have beta cells that undergo continuous apoptosis after replication. Beta cell apoptosis is about twice as frequent in T1D as in control subjects. Most people with years of T1D have beta cells that continue to be destroyed. The mechanism underlying increased β-cell death appears to involve both ongoing autoimmunity and glucose toxicity. The presence of β-cells despite ongoing apoptosis means that by definition, concomitant new β-cell formation should be occurring even after many years of T1D. We conclude that T1D can be prevented or abolished by targeted GLP-1 MMB inhibition targeted to beta cell destruction.

  In multi-organ donors, pancreatic islets are exposed to apoptosis and necrosis during events leading to brain death of post-mortem organ donors that continue during their clinical course prior to organ donation. In addition, islets are exposed to apoptosis during the islet isolation process in preparation for transplantation of insulin producing cells. Furthermore, when islets are transplanted into the liver of diabetic recipients, there is a cascade of cytokine activation (especially TNFα, IL1B and γINF). The use of GLP-1 MMB during the isolation process and in donor islet transplantation during and after transplantation reduces apoptosis and improves islet function. It also reduces the number of islets required for clinical benefit and provides an opportunity for the use of surviving donors in islet transplantation.

GLP-1 MMB increases glucose-dependent insulin secretion in INS-1E cells INS-1E cells in RPMI 1640 + 10% FBS + 1% L-glutamine + 1% sodium pyruvate + 1% non-essential amino acid + 50 μM β-mercaptoethanol medium Plated in 24-well plates at a cell density of 200,000 cells / well. Cells were grown for 7 days at 37 ° C. and 5% CO ₂ and fed on days 3 or 4. Cells were washed twice with 0.4 ml KRBH buffer / 3 mM glucose and allowed to incubate in this buffer for 30-60 minutes. The medium was removed from the cells and 0.4 ml of test article in KEBH containing the appropriate amount of glucose was added per well. Twenty microliters of supernatant was removed from the wells for insulin measurement after T = 0 and after 60 minutes incubation at 37 ° C., 5% CO ₂ . Insulin concentration was measured using a rat standard curve (12.8-0.1 ng / ml) using an Ultra Sensitive Rat Insulin ELISA kit (Crystal Chem). Five microliters of a 50-fold dilution of the time point supernatant was used in the assay to obtain the value that was on the standard curve. The ELISA plate was read at OD ₄₅₀ , subtracting OD ₆₃₀ with a Molecular Devices SpectraMax 340 PC plate reader. As shown in FIG. 19, GLP-1 MMB increased insulin secretion in a dose-dependent manner, but only in the presence of elevated glucose.

GLP-1 MMB increases glucose-dependent insulin secretion in rat and human islets Rat islets were obtained by collagenase digestion followed by Ficoll gradient purification. Human islets were isolated using enzyme digestion and density gradient purification detailed in Ricordi et al., Diabetes (1998) 37; 4, 413-420. Islets were grown 20 × 100 mm in medium with CMRL-1066 (Gibco), penicillin / streptomycin (5,000 units / 5,000 μg), 10% FBS, 1% L-glutamine 25 mM HEPES, pH 7.2-7.4. Plated overnight in petri dishes and incubated for 18-24 hours at 37 ° C. in 5% CO ₂ . The next day, islets were agitated in the center of the dish and collected in a 50 mL conical tube. The islets were allowed to settle to the bottom of the tube by gravity for approximately 10-15 minutes. The supernatant was removed, functional / viability medium (MediaTech, catalog number 99-786-CV) was added, and the islets were allowed to settle to the bottom of the tube by gravity. The islets were washed a total of 3 times. The supernatant is removed and the islets are placed in a functional / viability medium containing 1% BSA, 1% L-glutamine, 1% penicillin / streptomycin and 0.5 mM glucose solution at a density of 20 islets per mL. Resuspended. 1 mL of cell suspension (approximately 20 islets) was added to each well of a 24-well plate and incubated for 2 hours at 37 ° C. in 5% CO ₂ . After 2 hours, 1% BSA, 1% L-glutamine, 1% penicillin / streptomycin, GLP-1 MMB (50 nM final concentration; 100 nM initial concentration), and 3.5 mM (n = 12) or 15 mM (n = 12) 1 mL of functional / viability medium containing a final concentration of glucose was added. 25 μL of supernatant was collected as time 0 (insulin basis) and 4 hours after the addition of treatment. The supernatant was stored at −20 ° C. until an insulin ELISA was performed. Rat insulin was quantified using an ultrasensitive ELISA assay kit (Catalog Number 90060) from Crystal Chem (Downers Grove, Ill.). Human insulin was quantified using a human insulin ELISA kit from Linco Research (St. Charles, Michigan, catalog number EZHI-14K). FIG. 20 shows that GLP-1 MMB significantly increases insulin secretion from rats (FIG. 20A) and human islets (FIG. 20B).

  Both type 1 and type 2 diabetes are ultimately due to an insufficient amount of insulin secreted by the individual's beta cells. Their ability to increase insulin secretion by treating individuals with diabetes with GLP-1 MMB appears to ameliorate or eliminate their diabetic condition. Importantly, the increase in insulin secretion is glucose dependent and increases only in the presence of elevated glucose levels. Thus, the risk of patients experiencing dangerous hypoglycemic episodes is minimized with the use of GLP-1 MMB.

  Similarly, increased insulin secretion may also benefit islet transplantation procedures. By increasing the ability of transplanted islets to secrete insulin, the number of islets required to provide clinical benefit can be effectively reduced. Together with the limited amount of post-mortem pancreas available to support islet transplantation, the use of GLP-1 MMB is expected to significantly increase the number of patients that can benefit from islet transplantation procedures. Furthermore, the ability to increase insulin secretion can lead to the development of diabetes in donors due to insufficient insulin secretion capacity without intervention with GLP-1 MMB, without the risk of depleting their insulin secretion capacity from them. The donor population can be expanded to live donors.

Effect of GLP-1 MMB on blood glucose in normal mice Normal C57BLK / 6 mice were randomized into 2 groups, each group having 13 animals. Group 1 received daily intraperitoneal (IP) administration of PBS vehicle or GLP-1 MMB (0.5 mg / kg). Both groups were treated for 10 days. Mice were monitored daily for body weight and blood glucose was monitored for 32 days (the study was terminated at this point) (LifeScan, CA).

  Mice receiving GLP-1 MMB treatment showed lower blood glucose during the course of 10 days treatment compared to PBS treated mice (FIG. 21). Blood glucose returned to the level of the PBS group once treatment was terminated. Both groups showed the same weight gain throughout the course of the study (data not shown).

  Hyperglycemia is present both with type 1 and type 2 diabetes and is responsible for the progression of destructive secondary complications of the disease. The ability of GLP-1 MMB to lower blood glucose without adversely affecting the recipient's overall health (specifically indicated by the same progressive weight gain in the untreated control above) is type 1 and It appears to be beneficial for both type 2 patients.

GLP-1 MMB protects the reduction of β cells in diabetic mice Mice (12 weeks old db / db) were fasted overnight. On the first day of the study, mice (4 groups; n = 5 / group) were given s. c. Administered. On the fourth day of the test, fasting blood glucose was measured and OGTT (oral glucose tolerance test) was performed. Blood glucose levels were measured at 15, 30, 60, 90, 120, 150 and 180 minutes using tail vein blood. Mice were euthanized and the pancreas was removed and fixed by immersion in 10% neutral buffered formalin. The pancreas from each mouse was embedded in paraffin and sliced for staining with hematoxylin and eosin and immunohistochemistry. Morphometric analysis of insulin staining showed significantly higher intensity of insulin staining in GLP-1 MMB treated mice versus controls. The experiment was repeated with mice killed 14 days after dosing to assess the lifespan of a single dose of GLP-1 MMB. Again, morphometric analysis of insulin staining showed higher intensity of insulin staining in GLP-1 MMB treated mice 2 weeks after a single dose. The intensity of insulin staining is directly related to the amount of stored insulin in the islets, suggesting that GLP-1 MMB treatment increases insulin secretion and storage in the islets of this diabetic animal model.

  In type 2 diabetes, insufficient insulin synthesis and storage leads to hyperglycemia that is responsible for the development and progression of secondary complications of the disease. The ability of GLP-1 MMB to increase insulin synthesis and storage appears to lower hyperglycemia in type 2 patients, thereby limiting the onset and progression of secondary complications.

Effect of GLP-1 MMB on the effect of transplantation of a minimal amount of human islets into diabetic immunocompromised mice Athymic nu / nu (nude) mice were purchased from Harlan Laboratoris. Animals were housed in microisolation cages with free access to sterile food and water in a virus-free antibody chamber. All experiments are guided by the guidelines for the management and use of laboratory animals.
the Care and Use of Laboratory Animals).

  Diabetes was induced by intravenous administration of streptozotocin (200 mg / kg) freshly dissolved in citrate buffer. Diabetes induction was confirmed by monitoring non-fasting blood glucose levels after challenge, and only animals with values greater than 350 mg / dL were used as recipients. After transplantation of a minimum islet volume (approximately 50 IEQ / g body weight), meaning an amount that is not sufficient to immediately bring the recipient to normoglycemia, non-fasting blood glucose levels for the first week after transplantation, and every day thereafter, and up to three times thereafter The animals were monitored for graft function by measuring. Interventions that improve islet engraftment, survival and function can reduce the time to achieve normoglycemia in animals transplanted with minimal islet volume. Two groups of minimal human islet recipients were included; one group received daily IP infusions of GLP-1 MMB (0.5 mg / kg) and the control group received vehicle IP infusions.

  Animals were considered normoglycemic if the non-fasting blood glucose level was> 200 mg / dL for a minimum of 2 consecutive measurements. In addition, to study glucose loss after glucose administration, graft function was assessed by a glucose tolerance test (1.5 g / kg) performed on treated and control animals after overnight fasting over 30 days after transplantation. Animals that achieved normoglycemia underwent graft removal to eliminate the remaining function of natural islets.

As shown in FIG. 22, GLP-1 MMB treated mice achieved normoglycemia more rapidly than control mice after transplantation of a minimal amount of islets. In addition, GLP-1 MMB treated mice showed a more efficient loss of glucose administration. These data are GLP-1
MMB treatment improves the engraftment, viability and function of transplanted human islets, and the use of GLP-1 MMB in both donors (including survival and brain death) and recipients undergoing islet transplantation treatment I strongly suggest that it be supported. These results show the beneficial effects that GLP-1 MMB has on human islets transplanted into diabetic recipients. Furthermore, the data suggests that treatment with GLP-1 MMB would improve islet engraftment, survival and function and would be a valuable adjunct therapy in the context of islet transplantation.

GLP-1 for insulin secretion in response to glucose in non-human primate islets
Effect of MMB Non-human primate islets were isolated using enzymatic digestion and density gradient purification and divided into two groups and in a 175 cm ² flask (Corning, Mass.) That had not been treated with tissue culture. Incubated at 37 ° C. in a humidified mixed 95% air / 5% CO ₂ at a density of about 20,000 IE. One group of islets is cultured alone and the other group is 50 nM GLP-1.
Incubated in MM1 supplemented with MMB.

Islets were collected after 2 days of culture, and islet samples (2 from the control group and 1 from the GLP-1 MMB group) were subjected to a dynamic stimulation assay to assess the effect of glucose concentration on islet insulin secretion I went to. The islets were washed with a chromatography column (Bio-gel) with a buffer containing 125 mM NaCl, 5.9 mM KCl, 1.28 mM CaCl ₂ , 1.2 mM MgCl ₂ , 25 mM HEPES, 0.1% bovine serum albumin and 3 mM glucose. (Fine 45-90 nm; Bio-Rad) at 37 ° C. for 20 minutes. The islets were refluxed for 10 minutes in the same buffer and then sequentially exposed to 11 mM and 3 mM glucose. Perfusate fractions were collected with 3 mM glucose every 2 minutes during perfusion and every minute during stimulation. The concentration of insulin in each fraction was assayed by ELISA.

  Islets incubated in the presence of GLP-1 MMB showed a significant increase in glucose-stimulated insulin secretion, in contrast to untreated controls (FIG. 23). This data strongly supports the positive effect that GLP-1 MMB has in vitro on insulin synthesis and secretion and further supports its use prior to islet transplantation. Increasing the potency of islets with GLP-1 MMB prior to transplantation reduces the number of reduced islets required to achieve the same therapeutic effect in individuals with type 1 diabetes undergoing islet transplantation treatment Should bring. Furthermore, it can be predicted from these results that treatment of individuals with GLP-1 MMB can increase islet potency in vivo. This further supports the treatment of both donor and recipients of islet transplantation to increase the function of the donor (including survival and postmortem) from a minimal amount of islet.

Impact of GLP-1 MMB on allogeneic islet engraftment and long-term survival in a cynomolgus monkey minimal dose model The use of a nonhuman primate minimal dose model may allow for reversal of diabetes in a reduced number of islets Makes it possible to study transplantation approaches with In the minimal dose model, approximately 5,000 IEQ / kg, ie half the number required to achieve normoglycemia (usually 10,000 IEQ / kg) is implanted. By utilizing islets obtained from the same allogeneic donor, donor and isolation variables are eliminated. Increased islet engraftment in monkeys treated with the agent compared to monkeys that do not receive agents designed to enhance islet function by transplanting pairs of monkeys with similar pre-transplant insulin requirements Can be observed and early islet loss can be limited.

Recipients were 2-3 year old cynomolgus monkeys of Mauritius origin; donors of the same strain were over 4 years old. Diabetes was induced with 1250 mg / m ² of streptozotocin. Diabetic animals were treated with NPH insulin before breakfast and NPH / Lantus before lunch in an attempt to maintain blood glucose levels between 150-300 mg / dl. Four weeks after induction of diabetes, monkeys received glucagon to confirm that no stimulated c-peptide was produced.

  Donor organs were collected and islets were isolated via the semi-automated method cited in Example A and Example B and then cultured overnight for two phases (initially at 37 ° C. and then at 22 ° C.). Control monkey islets were cultured in standard MM1 medium and experimental monkey islets were cultured in MM1 supplemented with 50 nM GLP-1 MMB. Monkeys underwent mini-laparotomy and performed islet transplantation into the liver via the portal vein. A pair of monkeys received approximately 5,000 IEQ / kg (cultured as described above) from the same donor; one monkey received GLP-1 MMB (0.2 mg / kg) subcutaneously twice per week; And no control monkeys. Both control and GLP-1 MMB-treated mice prevent rejection with clinically used steroid-free immunosuppression (abbreviated as SFIS, consisting of low-dose FK506, high-dose rapamycin, anti-IL2R induction therapy) did. Animals are monitored twice daily to measure fasting and postprandial blood glucose levels (heel stick, blood glucose meter) and to maintain blood glucose in the 100-200 mg / dl range after transplantation. Treated with insulin as required. Fasting c-peptide was measured every other week, and intravenous glucose tolerance test (IVGTT) was performed every 8 weeks to assess graft function.

  GLP-1 MMB treated animals had excellent glucose regulation with a significant reduction in exogenous insulin demand (FIG. 24A). Improved glucose regulation was more specifically explained by the rapid decrease in HgbA1c in GLP-1 MMB treated animals compared to untreated controls (FIG. 24B). This study further supports the use of GLP-1 MMB in type 1 diabetic patients undergoing islet transplantation procedures.

  Advantages: The use of the novel molecules as therapeutic agents to treat type 2 diabetes offers several advantages over other GLP-1 analogs. For example, it is likely to extend the half-life of the GLP-1 peptide. Also, the wild type GLP-1 peptide in the mimetibody scaffold structure is resistant to protease degradation, particularly DPP-IV. This can be expected to be treated with the wild type GLP-1 peptide rather than the mutant peptide. Because GLP-1 is a natural peptide, there may be less immune response in patients treated with GLP-1 mimetibody than in patients treated with mutant GLP-1 analogs. In addition, the large size of GLP-1 MMB can be excluded from crossing the blood brain barrier. This appears to provide an advantage because nausea and anxiety are associated with GLP-1 binding GLP-1R in the brain. In addition, the mimetibody platform results in the expression of two peptides on each mimetibody molecule. This appears to allow GLP-1 peptides to interact with each other, forming a dimeric ligand that can increase the affinity for cell surface GLP-1 receptors.

  It will be apparent that the invention may be practiced otherwise than as particularly described in the foregoing description and examples.

  Many modifications and variations of the present invention are possible in light of the above teachings and, therefore, are within the scope of the invention.

The nucleotide and peptide sequences of GLP-1 MMB in the IgG1 scaffold structure showing important functional domains are specifically described. The FACS binding assay of GLP-1 MMB is specifically described. FIG. 2A shows that GLP-1 MMB binds to HEK293 cells that overexpress GLP-1R. Gray area: GLP-1 MMB but no secondary; Gray line: secondary only; dotted line, negative control MMB and secondary; Black line: GLP-1 MMB and secondary. FIG. 2B shows that GLP-1 MMB does not bind to control HEK293E cells. Gray area: GLP-1 MMB but no secondary; black line: secondary only; gray line: GLP-1 MMB and secondary. FIG. 2C shows that GLP-1 peptide analog (A2S) can compete with GLP-1 MMB for binding to HEK293E cells overexpressing GLP-1R. Gray area: GLP-1 MMB but no secondary; black line: GLP-1 MMB and secondary; dashed line: GLP-1 MMB, 0.2 nM competitor, secondary; dotted line: GLP-1 MMB, 20 nM competition Body, secondary; gray line: GLP-1 MMB, 100 nM competitor, secondary). The cAMP assay of GLP-1 MMB is specifically described. 3A: wt GLP-1 MMB in IgG1 scaffold structure; FIG. 3B: GLP-1 peptide; FIG. 3C: GLP-1 (A2G) MMB in IgG4 (Ala / Ala, Ser-> Pro) scaffold structure; 3D: GLP-1 (A2S) MMB in IgG4 (Ala / Ala, Ser-> Pro) scaffold structure; FIG. 3E: wt GLP-1 MMB in IgG4 (Ala / Ala, Ser-> Pro) scaffold structure. The resistance of GLP-1 MMB to DPP-IV cleavage is specifically described. 2 shows the stability of GLP-1 MMB in serum. FIG. 5 shows that GLP-1 MMB causes insulin secretion in RINm cells. FIG. 6A shows that GLP-1 (7-36) peptide and exendin-4 peptide stimulate insulin release in RINm cells. FIG. 6B shows GLP-1 (A2S) MMB in either IgG1 or IgG4 (Ala / Ala, Ser-> Pro) scaffold structure or GLP-1 in IgG4 (Ala / Ala, Ser-> Pro) scaffold structure. (A2G) MMB is active in stimulating insulin secretion in RINm cells. FIG. 5 shows that GLP-1 MMB reduces glucose (FIG. 7A) in a dose-dependent manner (FIG. 7B). Figure 2 shows the pharmacokinetic profiles of four GLP-1 MMBs (A2G, A2S, Ex-cap and wild type) in cynomolgus monkeys. Figure 3 shows the effect of GLP-1 MMB during oral glucose tolerance test in diabetic mice. Figure 3 shows the effect of GLP-1 MMB on fasting blood glucose during chronic administration to diabetic mice. The effect of GLP-1 MMB on the oral glucose tolerance test after chronic administration to diabetic mice is shown. FIG. 6 shows the effect of GLP-1 MMB on the reduction of HbAlc after chronic administration to diabetic mice. FIG. 9 shows the effect of GLP-1 MMB on blood glucose (FIG. 13A) and insulin (FIG. 13B) levels in an oral glucose tolerance test in normal cynomolgus monkeys. FIG. 6 shows the effect of GLP-1 MMB on insulin staining of islets of diabetic mice after a single dose. FIG. 5 shows that GLP-1 MMB delays gastric emptying in normal dogs. FIG. 5 shows that GLP-1 MMB reduces blood glucose after oral glucose tolerance test in diet-induced obese mice. FIG. 5 shows that GLP-1 MMB reduces blood glucose (FIG. 17A) and insulin levels (FIG. 17B) in an intraperitoneal glucose tolerance test in diabetic mice. 4 shows that GLP-1 mimetibody inhibits cytokine-induced apoptosis in a dose-dependent manner. The percent apoptosis relative to the untreated control is plotted against the concentration of GLP-1 MMB. FIG. 5 shows that GLP-1 mimetibody increases glucose-dependent insulin secretion in INS-1E cells. A. The bar graph shows the amount of insulin secreted at each concentration of GLP-1 MMB in the presence of 5.5 mM glucose. In addition, the insulin secreted by only 3 and 7.5 mM glucose is plotted. B. The amount of insulin secreted is plotted against the concentration of GLP-1 MMB. The data was fitted to the Hill equation and provided an EC ₅₀ of 0.07 nM. FIG. 4 shows that GLP-1 mimetibody increases glucose-dependent insulin secretion in rats (FIG. 20A) and human islets (FIG. 20B). The effect of GLP-1 MMB treatment on the blood glucose level of normal C57 / BLK6 mice is shown. FIG. 10 shows the effect of GLP-1 MMB treatment on blood glucose levels (FIG. 22A) and glucose tolerance (FIG. 22B) in STZ-treated diabetic nude mice transplanted with a minimal amount of human islets. In FIG. 22A, blood glucose was plotted against time (days) for control mice (×) and GLP-1 MMB treated mice (■) treated with PBS receiving daily IP infusions. A minimal amount of human islets (50 IEQ / g) was transplanted on day 0 and the animals' blood glucose was subsequently monitored. The arrow indicates when the transplanted islet was removed that resulted in a rapid return to the diabetic state. In FIG. 22B, blood glucose was plotted against time (minutes) for control mice treated with PBS (x) and GLP-1 MMB (0.5 mg / kg) (■). IVGTT was performed after mice received daily IP infusions for more than 30 days. The animals were fasted overnight and then IV infused with glucose (1.5 g / kg). Mice were monitored for blood glucose and plotted against time after injection. The inset represents the area under the curve of control and GLP-1 MMB treated animals. Figure 2 shows the effect of GLP-1 MMB on glucose-induced insulin secretion in non-human primate (NHP) islets. FIG. 25 shows the effect of GLP-1 MMB on exogenous insulin requirements (FIG. 24A) and hemoglobin A1c (HgbA1c) (FIG. 24B) of STZ diabetic NHP implanted with a minimal amount.

Claims (49)

  At least one GLP-1 CH1-deleted mimetibody nucleic acid comprising at least one polynucleotide encoding the amino acid sequence of SEQ ID NO: 2 or 4, or a polynucleotide complementary thereto.   At least one GLP-1 CH1 deficiency comprising at least one polynucleotide encoding an amino acid sequence comprising at least one selected from SEQ ID NOs: 7 to 14, or a polynucleotide complementary thereto Lost mimetibody nucleic acid. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Where P is at least one bioactive GLP-1 peptide, variant or derivative, and L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence that can be a polypeptide, V is at least a portion of the C-terminus of an immunoglobulin variable region, H is at least a portion of an immunoglobulin variable hinge region, and CH2 is an immunoglobulin CH2 constant region. And CH3 is at least part of an immunoglobulin CH3 constant region, n is an integer from 1 to 10, and o, p, q, r, s and t are independently from 0 to 10 Can be an integer of
At least one GLP-1 CH1-deleted mimetibody nucleic acid comprising at least one polynucleotide encoding a polypeptide of At least one GLP-1 comprising all of the contiguous amino acids of SEQ ID NO: 2 or 4
CH1 deletion mimetibody polypeptide.   At least one GLP-1 CH1 deletion mimetibody polypeptide comprising all of at least one contiguous amino acid of SEQ ID NOs: 7-14. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Wherein P is at least one bioactive GLP-1 peptide selected from SEQ ID NOs: 1 and 6, and L is GS, GGS, GGGS (SEQ ID NO: 16), GSGGGS (SEQ ID NO: 17), GGSGGGS ( SEQ ID NO: 18), GGSGGGSGG (SEQ ID NO: 19) and GGGSGGGSGG (SEQ ID NO: 20); V is GTLVTVSS (SEQ ID NO: 21), GTLVAVSS (SEQ ID NO: 22), GTAVTVSS (SEQ ID NO: 23), TVSS (SEQ ID NO: 23) 24) and AVSS (SEQ ID NO: 25); H is EPKSCDKTHTCPPCPAPELLGGP (SEQ ID NO: 26), CH2 is SVFLFPPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTTKPREEQYNSTY A BuibuiesubuierutibuierueichikyudidaburyueruenuGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 43), CH3 is JikyuPiaruiPikyubuiwaitieruPiPiesuarudiierutikeienukyubuiesuerutishierubuikeijiefuwaiPiesudiaieibuiidaburyuiesuenujikyuPiienuenuwaikeititiPiPibuierudiesudijiesuefuefueruwaiesukeierutibuidikeiesuarudaburyukyukyujienubuiFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44), n is an integer from 1 to 10, as well as, o, p, q, r, s and t is from 0 independently 10 Can be an integer up to,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Wherein P is at least one biologically active GLP-1 peptide of SEQ ID NO: 6, L is GS, GGS, GGGS (SEQ ID NO: 16), GSGGGS (SEQ ID NO: 17), GGSGGGS (SEQ ID NO: 18), Selected from GGSGGGSGG (SEQ ID NO: 19) and GGGSGGGGSGG (SEQ ID NO: 20); V is GTLVTVSS (SEQ ID NO: 21), GTLVAVSS (SEQ ID NO: 22), GTAVTVSS (SEQ ID NO: 23), TVSS (SEQ ID NO: 24) and AVSS ( H is ESKYGPPCPSCPAPEFLGGGP (SEQ ID NO: 27) and CH2 is SVFLFPPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTTYRVVSVLTVLDH A EnujiKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 45), CH3 is JikyuPiaruiPikyubuiwaitieruPiPiesukyuiiemutikeienukyubuiesuerutishierubuikeijiefuwaiPiesudiaieibuiidaburyuiesuenujikyuPiienuenuwaikeititiPiPibuierudiesudijiesuefuefueruwaiesuaruerutibuidikeiesuarudaburyukyuijienubuiFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 46), n is an integer from 1 to 10, as well as, o, p, q, r, s and t is from 0 independently 10 Can be an integer up to,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Wherein P is at least one biologically active GLP-1 peptide of SEQ ID NO: 6, L is GS, GGS, GGGS (SEQ ID NO: 16), GSGGGS (SEQ ID NO: 17), GGSGGGS (SEQ ID NO: 18), Selected from GGSGGGSGG (SEQ ID NO: 19) and GGGSGGGGSGG (SEQ ID NO: 20); V is GTLVTVSS (SEQ ID NO: 21), GTLVAVSS (SEQ ID NO: 22), GTAVTVSS (SEQ ID NO: 23), TVSS (SEQ ID NO: 24) and AVSS ( H is ESKYGPPCPPCPAPEAAGGP (SEQ ID NO: 28) and CH2 is SVFLFPPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLDH A EnujiKEYKCKVSNKGLPSSIEKTISKAK (SEQ ID NO: 45), CH3 is JikyuPiaruiPikyubuiwaitieruPiPiesukyuiiemutikeienukyubuiesuerutishierubuikeijiefuwaiPiesudiaieibuiidaburyuiesuenujikyuPiienuenuwaikeititiPiPibuierudiesudijiesuefuefueruwaiesuaruerutibuidikeiesuarudaburyukyuijienubuiFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 46), n is an integer from 1 to 10, as well as, o, p, q, r, s and t is from 0 independently 10 Can be an integer up to,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Where P is at least one bioactive GLP-1 peptide, variant or derivative, and L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence that can be a polypeptide, V is at least a portion of the C-terminus of an immunoglobulin variable region, H is at least a portion of an immunoglobulin variable hinge region, and CH2 is SVFLFPPPKKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVGDGVHVHNAKTTKPREQYN
A EsutiwaiarubuibuiesubuierutibuierueichikyudidaburyueruenuGKEYKCKVSNKALPAPIEKTISKAK (SEQ ID NO: 43), CH3 is JikyuPiaruiPikyubuiwaitieruPiPiesuarudiierutikeienukyubuiesuerutishierubuikeijiefuwaiPiesudiaieibuiidaburyuiesuenujikyuPiienuenuwaikeititiPiPibuierudiesudijiesuefuefueruwaiesukeierutibuidikeiesuarudaburyukyukyujienubuiFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 44), n is an integer from 1 to 10, as well as, o, p, q, r, s and t is from 0 independently 10 Can be an integer up to,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Where P is at least one bioactive GLP-1 peptide, variant or derivative, and L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence that may be a polypeptide, V is at least a portion of the C-terminus of an immunoglobulin variable region, H is at least a portion of an immunoglobulin variable hinge region, and CH2 is SVFLFPPPKPDKTLKSLKYK45 And CH3 is GQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYP A DiaieibuiidaburyuiesuenujikyuPiienuenuwaikeititiPiPibuierudiesudijiesuefuefueruwaiesuaruerutibuidikeiesuarudaburyukyuijienubuiFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 46), n is an integer from 1 to 10, as well as, o, p, q, r, s and t can be an integer from 0 to independently to 10,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Where P is at least one bioactive GLP-1 peptide of SEQ ID NO: 6 and L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence that can be a polypeptide, V is at least a portion of the C-terminus of an immunoglobulin variable region, H is at least a portion of an immunoglobulin variable hinge region, and CH2 is an immunoglobulin CH2 constant region. At least a portion, CH3 is at least a portion of an immunoglobulin CH3 constant region, n is an integer from 1 to 10, and o, p, q, r, s and t are independently from 0 to 10 Can be an integer,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Wherein P is at least one biologically active GLP-1 peptide, variant or derivative, and L is GS, GGS, GGGS (SEQ ID NO: 16), GSGGGS (SEQ ID NO: 17), GGSGGS (SEQ ID NO: 18), Selected from GGSGGGSGG (SEQ ID NO: 19) and GGGSGGGSGGG (SEQ ID NO: 20); V is at least part of the C-terminus of the immunoglobulin variable region, H is at least part of the immunoglobulin variable hinge region, and CH2 is the immunoglobulin CH2 At least a portion of the constant region, CH3 is at least a portion of an immunoglobulin CH3 constant region, n is an integer from 1 to 10, and o, p, q, r, s and t are independently from 0 Can be an integer up to 10,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Where P is at least one bioactive GLP-1 peptide, variant or derivative; L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties Is at least one linker sequence that can be a polypeptide; V is GTLVTVSS (SEQ ID NO: 21), GTLVAVSS (SEQ ID NO: 22), GTAVTVSS (SEQ ID NO: 23), TVSS (SEQ ID NO: 24) and AVSS (SEQ ID NO: 25). H is at least part of an immunoglobulin variable hinge region; CH2 is at least part of an immunoglobulin CH2 constant region; CH3 is at least part of an immunoglobulin CH3 constant region; n is from 1 to 10 And o, p, q, r, s and t are German Can be an integer from 0 to 10,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Where P is at least one bioactive GLP-1 peptide, variant or derivative, and L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence that can be a polypeptide, V is at least a portion of the C-terminus of an immunoglobulin variable region, and H is EPKSCDKTHTCPPCPAPELLGPGP (SEQ ID NO: 26), ESKYGPPPCPSCPAPEFLGGGP (SEQ ID NO: 27) and ESKYGPPPCPPCPAPEAAGGP (SEQ ID NO: 27) 28), CH2 is at least part of an immunoglobulin CH2 constant region, CH3 is at least part of an immunoglobulin CH3 constant region, n is an integer from 1 to 10, and o p, q, r, s and t can be an integer from 0 to independently to 10,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Where P is at least one bioactive GLP-1 peptide, variant or derivative, and L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence that can be a polypeptide, V is at least a portion of the C-terminus of the immunoglobulin variable region, and H is EPKSADKTHTCPPCPAPEAGAGGP (SEQ ID NO: 29), EPKSADKTHTCPPCPAPELAGGP (SEQ ID NO: 30), EPKSADKTHTCPPCPAPEALGGGP (SEQ ID NO: 31), EPKSADKTHTCPPCPAPELEGGP (SEQ ID NO: 32), EPKSSDKTHTCPPCPAPEFLGGGP (SEQ ID NO: 33), EPKSADKTHACPPCPAPELLGG (SEQ ID NO: 34), EPKSADKAHTCPPCPAPELLGGP (SEQ ID NO: 35) and EPKSADKTHTCPPCPAPELLGGP (SEQ ID NO: 36), ADKTHTCPPCPAPELLGGP (SEQ ID NO: 37), THT
Selected from CPPCPAPELLGGP (SEQ ID NO: 38), ESKYGPPCPSCPAPEAAGGP (SEQ ID NO: 39), ESKYGPPCPPCPAPELLGP (SEQ ID NO: 40), CPPCPAPELLGGP (SEQ ID NO: 41) and CPPCPAPEAAGGP (SEQ ID NO: 42), where CH2 is at least part of an immunoglobulin CH2 constant region , CH3 is at least a portion of an immunoglobulin CH3 constant region, n is an integer from 1 to 10, and o, p, q, r, s and t can independently be integers from 0 to 10 ,
At least one GLP-1 CH1-deficient mimetibody polypeptide comprising the polypeptide. Formula (I):
(Pep (n) -L (o) -V (p) -H (q) -CH2 (r) -CH3 (s)) (t),
Where P is at least one bioactive GLP-1 peptide, variant or derivative, and L provides structural flexibility by allowing the mimetibody to have alternative orientation and binding properties. At least one linker sequence that can be a polypeptide, V is at least a portion of the C-terminus of an immunoglobulin variable region, H is at least a portion of an immunoglobulin variable hinge region, and CH2 is an immunoglobulin CH2 constant region. At least a portion, CH3 is at least a portion of an immunoglobulin CH3 constant region, n is an integer from 1 to 10, and o, p, q, r, s and t are independently from 0 to 10 Can be an integer,
At least one GLP-1 CH1-deficient mimetibody polypeptide.   The CLP-1 CH1-deleted mimetibody nucleic acid according to any one of claims 1 to 16, or the GLP-1 CH1-deleted mimetibody polypeptide (the polypeptide is at least one P polypeptide). Active).   An anti-idiotype monoclonal or polyclonal antibody, a fusion protein or a fragment thereof that specifically binds at least one GLP-1 CH1-deleted mimetibody polypeptide according to any of claims 4-16.   A coding for at least one GLP-1 CH1-deleted mimetibody polypeptide or CLP-1 CH1-deleted mimetibody antibody according to any of claims 1-3 or any of claims 4-18. A GLP-1 CH1-deleted mimetibody nucleic acid, or a polynucleotide complementary thereto.   20. A GLP-1 CH1-deleted mimetibody vector comprising at least one isolated nucleic acid according to claim 19.   20. A GLP-1 CH1-deficient mimetibody host cell comprising the isolated nucleic acid of claim 19.   The host cell is COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2 / 0, 293, NSO, DG44 CHO, CHO K1, HeLa, myeloma or lymphoma cell, The GLP-1 CH1-deficient mimetibody host cell according to claim 21, which is at least one selected from a derivative thereof, an immortalized or transformed cell thereof. 20. Translating the nucleic acid of claim 19 under in vitro, in vivo or in situ conditions such that the GLP-1 CH1-deleted mimetibody or antibody is expressed in detectable or recoverable amount A method for producing at least one GLP-1 CH1-deleted mimetibody polypeptide or GLP-1 CH1-deleted mimetibody antibody.   20. A composition comprising at least one GLP-1 CH1-deleted mimetibody nucleic acid, GLP-1 CH1-deleted mimetibody polypeptide, or GLP-1 CH1-deleted mimetibody antibody according to any of claims 1-19. object.   25. The composition of claim 24, wherein the composition further comprises at least one pharmaceutically acceptable carrier or diluent.   Drugs related to diabetes or insulin metabolism, detectable labels or reporters, TNF antagonists, anti-infective drugs, cardiovascular (CV) drugs, central nervous system (CNS) drugs, autonomic nervous system (ANS) drugs, respiratory tract drugs Gastrointestinal (GI) tube drugs, hormone drugs, fluid or electrolyte balance drugs, blood products, antitumor drugs, immunomodulators, eye, ear or nose drugs, topical drugs, nutritional drugs, cytokines or cytokine antagonists 25. The composition of claim 24, further comprising at least one composition comprising a therapeutically effective amount of at least one compound, composition or polypeptide selected from at least one.   25. The composition of claim 24 in at least one form selected from a liquid, gas or dry matter, solution, mixture, suspension, emulsion or colloid, lyophilized preparation or powder. (A) a composition comprising an effective amount of at least one GLP-1 CH1-deficient mimetibody nucleic acid, polypeptide or antibody according to any one of claims 1 to 19 as a cell, tissue, organ or animal; A method of diagnosing or treating a GLP-1 related condition in said cells, tissues, organs or animals comprising contacting or administering to said cells.   30. The method of claim 28, wherein the GLP-1-related condition is diabetes or congestive heart failure.   The effective amount is 0.0001-50 mg of GLP-1 CH1-deficient mimetibody antibody per kilogram of the cells, tissues, organs or animals; 0.1-500 mg of the GLP-1 CH1-deficient mimetibody; 29. The method of claim 28, wherein said method is 0001-100 [mu] g of said GLP-1 CH1-deleted mimetibody nucleic acid.   The contacting or administering can be parenteral, subcutaneous, intramuscular, intravenous, intraarterial, intrabronchial, intraabdominal, intracapsular, intrachondral, sinus, intracelial, Intracerebellum, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, At least one mode selected from intrarenal, intraretinal, intrathecal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vagina, rectum, buccal, sublingual, intranasal or percutaneous 30. The method according to claim 28. Before, simultaneously with or after the contact or administration of (a) above, a drug related to diabetes or insulin metabolism, a detectable label or reporter, a TNF antagonist, an anti-infective drug, a cardiovascular (CV) drug, a central drug Nervous system (CNS) drugs, autonomic nervous system (ANS) drugs, respiratory tract drugs, gastrointestinal (GI) tract drugs, hormonal drugs, drugs for fluid or electrolyte balance, blood products, antitumor drugs, immunomodulators, eyes, Administering at least one composition comprising an effective amount of at least one compound or polypeptide selected from at least one of ear or nose drugs, topical drugs, nutritional drugs, cytokines or cytokine antagonists. 30. The method of claim 28, further comprising.   30. The method of claim 28, wherein the effective amount reduces blood glucose levels in an animal in need thereof.   30. The method of claim 28, wherein the effective amount increases insulin secretion from insulin producing cells.   30. The method of claim 28, wherein the effective amount prevents apoptosis of insulin producing cells.   30. The method of claim 28, wherein the effective amount increases the proliferation of insulin producing cells.   20. A composition comprising an effective amount of at least one GLP-1 CH1-deficient mimetibody nucleic acid, polypeptide or antibody according to any one of claims 1 to 19 in contact with a cell, tissue, organ or animal. Or a method for the treatment of said cells, tissues, organs or animals comprising administering to them.   The effective amount is 0.0001-50 mg of GLP-1 CH1-deficient mimetibody antibody per kilogram of the cells, tissues, organs or animals; 0.1-500 mg of the GLP-1 CH1-deficient mimetibody; 38. The method of claim 37, wherein said method is 0001-100 [mu] g of said GLP-1 CH1-deleted mimetibody nucleic acid.   The contacting or administering can be parenteral, subcutaneous, intramuscular, intravenous, intraarterial, intrabronchial, intraabdominal, intracapsular, intrachondral, sinus, intracelial, Intracerebellum, intraventricular, intracolonic, intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, intrapelvic, intraperitoneal, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, At least one mode selected from intrarenal, intraretinal, intrathecal, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vagina, rectum, buccal, sublingual, intranasal or percutaneous 38. The method of claim 37.   Before, simultaneously with or after the contact or administration of (a) above, a drug related to diabetes or insulin metabolism, a detectable label or reporter, a TNF antagonist, an anti-infective drug, a cardiovascular (CV) drug, a central drug Nervous system (CNS) drugs, autonomic nervous system (ANS) drugs, respiratory tract drugs, gastrointestinal (GI) tract drugs, hormonal drugs, drugs for fluid or electrolyte balance, blood products, antitumor drugs, immunomodulators, eyes, Administering at least one composition comprising an effective amount of at least one compound or polypeptide selected from at least one of ear or nose drugs, topical drugs, nutritional drugs, cytokines or cytokine antagonists. 38. The method of claim 37, further comprising.   38. The method of claim 37, wherein the effective amount reduces blood glucose levels in an animal in need thereof.   38. The method of claim 37, wherein the effective amount increases insulin secretion from insulin producing cells.   38. The method of claim 37, wherein the effective amount prevents apoptosis of insulin producing cells.   38. The method of claim 37, wherein the effective amount increases the proliferation of insulin producing cells.   20. A device comprising at least one isolated GLP-1 CH1-deleted mimetibody polypeptide, antibody or nucleic acid according to any of claims 1 to 19, wherein the device is parenteral, Subcutaneous, intramuscular, intravenous, intraarterial, intrabronchial, intraabdominal, intracapsular, intrachondral, sinus, intraceliac, intracerebellum, intraventricular, intracolonic, intracervical, intragastric Intrahepatic, intramyocardial, intraosseous, pelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intrathecal, intrasynovial, GLP-1 CH1-deficient mimetibody polypeptide, antibody by at least one mode selected from intrathoracic, intrauterine, intravesical, intralesional, bolus, vagina, rectum, buccal, sublingual, intranasal or transdermal Or nuclear A device as described above, suitable for contacting or administering said at least one acid. Packaging material and at least one isolated GLP-1 according to any of claims 1-19
A product for human pharmaceutical or diagnostic use comprising a container comprising a CH1-deleted mimetibody polypeptide, antibody or nucleic acid.   The container is parenteral, subcutaneous, intramuscular, intravenous, intraarterial, intrabronchial, intraabdominal, intracapsular, intrachondral, sinus, intraceliac, intracerebellum, intraventricular, colon Intracervical, intragastric, intrahepatic, intramyocardial, intraosseous, pelvic, intrapericardial, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, marrow 48. Intracavity, intrasynovial, intrathoracic, intrauterine, intravesical, intralesional, bolus, vagina, rectal, buccal, sublingual, intranasal or transdermal delivery device or system component Product listed.   Providing at least one host cell, transgenic animal, transgenic plant, plant cell capable of expressing the polypeptide, antibody or nucleic acid in detectable or recoverable amounts. Item 20. A method for producing a polypeptide, antibody or nucleic acid of at least one isolated GLP-1 CH1-deleted mimetibody according to any one of Items 1 to 19.   49. A polypeptide, antibody or nucleic acid of at least one GLP-1 CH1-deleted mimetibody produced by the method of claim 48.

Images