WO2009009782A2 - Protéines il-17 du macaque cynomolgus, anticorps, compositions, procédés et utilisations - Google Patents

Protéines il-17 du macaque cynomolgus, anticorps, compositions, procédés et utilisations Download PDF

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Publication number: WO2009009782A2
Authority: WO; WIPO (PCT)
Prior art keywords: antibody; drug; protein; nucleic acid; antibodies
Prior art date: 2007-07-12

Application number

PCT/US2008/069915

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English (en)

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WO2009009782A9 (fr

Inventor

Leslee Conrad

Michael Naso

Original Assignee

Centocor, Inc.

Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)

2007-07-12

Filing date

2008-07-14

Publication date

2009-01-15

2008-07-14 Application filed by Centocor, Inc. filed Critical Centocor, Inc.

2009-01-15 Publication of WO2009009782A2 publication Critical patent/WO2009009782A2/fr

2009-07-23 Publication of WO2009009782A9 publication Critical patent/WO2009009782A9/fr

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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/52—Cytokines; Lymphokines; Interferons
- C07K14/54—Interleukins [IL]
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides

Definitions

the present invention relates to at least one cynomolgus derived interleukin-17 (IL- 17) protein or fragment thereof, and antibodies, including specified portions or variants, specific therefore, as well as nucleic acids encoding such cynomolgous IL-17 proteins, fragments, antibodies, complementary nucleic acids, vectors, host cells, and methods of making and using thereof, including therapeutic formulations, administration and devices.
IL- 17 cynomolgus derived interleukin-17
IL-17 is a 155 amino acid secreted glycoprotein, that exists as a 35 kDa homodimer (Mosely, TA, 2003). IL-17 is mainly produced by activated memory CD4+ T cells and potentially is also secreted by CD8 T cell, eosinophils and neutrophils.
the cytokine can be found in synovial fluid of RA patients, can be produced by cartilage tissue of OA patients and can be found in bronchial alveolar lavage fluid and sputum of patients with airway diseases..
IL-17 induces secretion of proinflammatory cytokine IL-6 and IL-8 and is also involved in nitric oxide (NO) production pathways.
NO nitric oxide
the receptor for IL 17 is expressed in the bone marrow, spleen, lung, thymus, muscle, heart, kidney, lung, and liver. However, it is expressed at a lower level in many other tissues.
the receptor is a single pass type I transmembrane glycoprotein of approximately 130 kDa, and signals through NF -kB and JAK/STAT1. Inhibition of ILl 7 bioactivity has been shown to decrease collagen break-down in RA synovium, and IL 17 has been shown to play a major role in airway diseases.
Animal IL-17 proteins can be used to generate surrogate antibodies that can be used in animal studies for various uses in discovery and development of antibodies specific to human IL-17, or to find antibodies that bind both human and animal IL-17..
the present invention provides isolated cynomolgus IL-17 proteins, antibodies, immunoglobulins, cleavage products and other specified portions and variants thereof, as well as cynomologus IL-17 protein or antibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and
cynomologus monkeys Pre-clinical studies are often performed by testing potential human therapeutics in cynomologus monkeys. Obtaining cynomologus IL- 17 DNA sequence data will aid in the engineering, expression and purification of recombinant protein. Subsequently, one will be able to test cross-reactivity of recombinant cynomologus IL-17 to any anti-IL-17 therapeutics. This will enable researchers to account for potential differences in therapeutic activity which may be related to a difference between human and cynomologus genes.
the present invention also provides at least one isolated IL- 17 antibody as described herein.
An antibody according to the present invention can include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) (also termed the hypervariable region or HV) of a heavy or light chain variable region, or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, wherein the antibody can be incorporated into an antibody of the present invention.
CDR complementarity determining region
An antibody of the invention can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, or any combination thereof, and the like.
the present invention provides, in one aspect, isolated nucleic acid molecules comprising, complementary, or hybridizing to, a polynucleotide encoding specific IL- 17 proteins or antibodies, comprising at least one specified sequence, domain, portion or variant thereof.
the present invention further provides recombinant vectors comprising at least one of said IL- 17 protein or antibody encoding or complementary nucleic acid molecules, host cells containing such nucleic acids and/or recombinant vectors, as well as methods of making and/or using such antibody nucleic acids, vectors and/or host cells.
At least one antibody of the invention binds at least one specified epitope specific to at least one IL- 17 protein, subunit, fragment, portion or any combination thereof.
the at least one epitope can comprise at least one antibody binding region that comprises at least one portion of said protein, which epitope is preferably comprised of at least 1-5 amino acids of at least one portion thereof, such as but not limited to, at least one functional, extracellular, soluble, hydrophilic, external or cytoplasmic domain of said protein, or any portion thereof.
the at least one antibody can optionally comprise at least one specified portion of at least one complementarity determining region (CDR) (e.g., CDRl, CDR2 or CDR3 of the heavy or light chain variable region) and optionally at least one constant or variable framework region or any portion thereof.
CDR complementarity determining region
the at least one antibody amino acid sequence can further optionally comprise at least one specified substitution, insertion or deletion as described herein or as known in the art.
the present invention also provides at least one isolated IL- 17 protein or antibody as described herein, wherein the antibody has at least one activity, such as, but not limited to know IL-17 activities.
A(n) IL- 17 protein antibody can thus be screened for a corresponding activity according to known methods, such as but not limited to, at least one biological activity towards an IL-17 protein or protein related function.
the present invention further provides at least one IL- 17 anti-idiotype antibody to at least one IL- 17 antibody of the present invention.
the anti-idiotype antibody includes any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion thereof, that can be incorporated into an antibody of the present invention.
CDR complementarity determining region
An antibody of the invention can include or be derived from any mammal, such as but not limited to a human, a mouse, a rabbit, a rat, a rodent, a primate, and the like.
the present invention provides, in one aspect, isolated nucleic acid molecules comprising, complementary, or hybridizing to, a polynucleotide encoding at least one IL- 17 anti-idiotype antibody, comprising at least one specified sequence, domain, portion or variant thereof.
the present invention further provides recombinant vectors comprising said IL- 17 anti-idiotype antibody encoding nucleic acid molecules, host cells containing such nucleic acids and/or recombinant vectors, as well as methods of making and/or using such anti-idiotype antibody nucleic acids, vectors and/or host cells.
the present invention also provides at least one method for expressing at least one IL- 17 protein or antibody, or IL- 17 anti-idiotype antibody, in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one IL- 17 antibody is expressed in detectable and/or recoverable amounts.
the present invention also provides at least one composition
a composition comprising (a) an isolated IL- 17 protein or antibody encoding nucleic acid and/or protein or antibody as described herein; and (b) a suitable carrier or diluent.
the carrier or diluent can optionally be pharmaceutically acceptable, such as but not limited to known carriers or diluents.
the composition can optionally further comprises at least one further compound, protein or composition.
the present invention further provides at least one IL- 17 protein or antibody method or composition, for administering a therapeutically effective amount to modulate or treat at least one IL-
the present invention also provides at least one composition, device and/or method of delivery of a therapeutically or prophylactically effective amount of at least one IL- 17 protein or antibody, according to the present invention.
the present invention further provides at least one IL- 17 protein or antibody method or composition, for diagnosing at least one IL- 17 related condition in a cell, tissue, organ, animal or patient and/or, prior to, subsequent to, or during a related condition, as known in the art and/or as described herein.
the present invention also provides at least one composition, device and/or method of delivery for diagnosing of at least one IL-17 protein or antibody, according to the present invention.
the present invention provides at least one isolated mammalian IL- 17 protein, comprising the amino acid sequences as part of at least one of SEQ ID NO:1.
an isolated nucleic acid encoding at least one isolated mammalian IL- 17 protein; an isolated nucleic acid vector comprising the isolated nucleic acid, and/or a prokaryotic or eukaryotic host cell comprising the isolated nucleic acid.
the host cell can optionally be at least one selected from prokaryotic or eukaryotic cells, or fusion cells thereof, e.g., but not limited to, mammalian, plant or insect, such as but not limited to, CHO, myeloma, or lymphoma cells, bacterial cells, yeast cells, silk worm cells, or any derivative, immortalized or transformed cell thereof.
a method for producing at least one IL- 17 protein comprising translating the protein encoding nucleic acid under conditions in vitro, in vivo or in situ, such that the IL- 17 protein is expressed in detectable or recoverable amounts.
compositions comprising at least one isolated mammalian IL- 17 protein and at least one pharmaceutically acceptable carrier or diluent.
the composition can optionally further comprise an effective amount of at least one compound or protein selected from at least one of a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a nonsteroid inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epine
Also provided is a method for diagnosing or treating a IL- 17 related condition in a cell, tissue, organ or animal comprising (a) contacting or administering a composition comprising an effective amount of at least one isolated mammalian IL- 17 protein of the invention with, or to, the cell, tissue, organ or animal.
the method can optionally further comprise using an effective amount of 0.0000001-500 mg/kilogram of the cells, tissue, organ or animal.
the method can optionally further comprise using the contacting or the administrating by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous,
the method can optionally further comprise administering, prior, concurrently or after the (a) contacting or administering, at least one composition comprising an effective amount of at least one compound or protein selected from at least one of a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, an anti-inflammatory, a non-steroid inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog
the device is suitable to contacting or administering the at least one IL- 17 protein by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
parenteral subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intrac
an article of manufacture for human pharmaceutical or diagnostic use comprising packaging material and a container comprising a solution or a lyophilized form of at least one isolated mammalian IL- 17 protein of the present invention.
the article of manufacture can optionally comprise having the container as a component of a parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal
CEN05189 PCT 5 cell capable of expressing in recoverable amounts the protein. Further provided in the present invention is at least one IL-17 protein produced by the above method.
the present invention provides at least one isolated mammalian IL- 17 antibody, comprising at least one human CDR, wherein the antibody specifically binds at least one epitope comprising at least 1-3, to the entire amino acid sequence of SEQ ID NOS: 1.
the at least one antibody can optionally further at least one of: bind IL- 17 with an affinity of at least one selected from at least 10 '9 M, at least 10 '10 M, at least 10 ' ⁇ M, or at least 10 '12 M; substantially neutralizes at least one activity of at least one IL- 17 protein.
an isolated nucleic acid encoding at least one isolated mammalian IL- 17 antibody; an isolated nucleic acid vector comprising the isolated nucleic acid, and/or a prokaryotic or eukaryotic host cell comprising the isolated nucleic acid.
the host cell can optionally be at least one selected from prokaryotic or eukaryotic cells, or fusion cells thereof, e.g., but not limited to, mammalian, plant or insect, such as but not limited to, CHO, myeloma, or lymphoma cells, bacterial cells, yeast cells, silk worm cells, or any derivative, immortalized or transformed cell thereof.
a method for producing at least one IL- 17 antibody comprising translating the antibody encoding nucleic acid under conditions in vitro, in vivo or in situ, such that the IL- 17 antibody is expressed in detectable or recoverable amounts.
compositions comprising at least one isolated mammalian IL- 17 antibody and at least one pharmaceutically acceptable carrier or diluent.
the composition can optionally further comprise an effective amount of at least one compound or protein selected from at least one of a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a nonsteroid inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a growth hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epine
the present invention further provides an anti-idiotype antibody or fragment that specifically binds at least one isolated mammalian IL- 17 antibody of the present invention. Also provided is a method for diagnosing or treating a IL-17 related condition in a cell, tissue, organ or animal, comprising
composition comprising an effective amount of at least one isolated mammalian IL- 17 antibody of the invention with, or to, the cell, tissue, organ or animal.
the method can optionally further comprise using an effective amount of 0.0001-500 mg/kilogram of the cells, tissue, organ or animal.
the method can optionally further comprise using the contacting or the
CEN05189 PCT 6 administrating by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
parenteral subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, in
the method can optionally further comprise administering, prior, concurrently or after the (a) contacting or administering, at least one composition comprising an effective amount of at least one compound or protein selected from at least one of a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, an anti-inflammatory, a non-steroid inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local anesthetic, a neuromuscular blocker, an antimicrobial, an antipsoriatic, a corticosteriod, an anabolic steroid, an erythropoietin, an immunization, an immunoglobulin, an immunosuppressive, a hormone, a hormone replacement drug, a radiopharmaceutical, an antidepressant, an antipsychotic, a stimulant, an asthma medication, a beta agonist, an inhaled steroid, an epinephrine or analog
At least one medical device comprising at least one isolated mammalian IL- 17 antibody of the invention, wherein the device is suitable to contacting or administering the at least one IL- 17 antibody by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal.
parenteral subcutaneous, intramuscular, intravenous, intraarticul
an article of manufacture for human pharmaceutical or diagnostic use comprising packaging material and a container comprising a solution or a lyophilized form of at least one isolated mammalian IL- 17 antibody of the present invention.
the article of manufacture can optionally comprise having the container as a component of a parenteral, subcutaneous, intramuscular, intravenous, intraarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal
CEN05189 PCT 7 Also provided is a method for producing at least one isolated mammalian IL- 17 antibody of the present invention, comprising providing a host cell or transgenic animal or transgenic plant or plant cell capable of expressing in recoverable amounts the antibody. Further provided in the present invention is at least one IL- 17 antibody produced by the above method. Lhe present invention further provides any invention described herein.
Lhe present invention provides isolated, recombinant and/or synthetic cynomolgus protein muteins as IL- 17 protein and IL- 17 antibodies, including human, primate, rodent, mammalian, chimeric, humanized or CDR-grafted, anti-IL-17 antibodies and IL- 17 anti-idiotype antibodies thereto, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least one IL-17 antibody or anti-idiotype antibody.
Lhe present invention further includes, but is not limited to, methods of making and using such nucleic acids and antibodies and anti-idiotype antibodies, including diagnostic and therapeutic compositions, methods and devices.
an "Interleukin-13 muteins antibody,” “IL-17 antibody,” and the like include any protein or peptide containing molecule that comprises at least a portion of an immunoglobulin molecule, such as but not limited to at least one complementarity determining region (CDR) of a heavy or light chain or a ligand binding portion thereof, a heavy chain or light chain variable region, a heavy chain or light chain constant region, a framework region, or any portion , fragment or variant thereof, or at least one portion of an IL- 17 receptor or binding protein, which can be incorporated into a IL-17 antibody of the present invention.
Antibodies can include one or more of at least one
CDR CDR
variable region at least one constant region
heavy chain e.g., ⁇ i, ⁇ 2 , 7 3 , ⁇ 4 , ⁇ , oci, Oc 2 , ⁇ , ⁇
light chain e.g., K and ⁇
Light chains typically have a molecular weight of about 25Kd and heavy chains typically range from 50K-77Kd.
Light chains can exist in two distinct forms or isotypes, kappa (K) and lambda ( ⁇ ), which can combine with any of the heavy chain types. All light chains have at least one variable region and at least one constant region.
Lhe IgG antibody is considered a typical antibody structure and has two intrachain disulfide bonds in the light chain (one in variable region and one in the constant region), with four in the heavy chain, and such bond encompassing a peptide loop of about 60-70 amino acids comprising a "domain" of about 110 amino acids in the chain.
IgG antibodies can be characterized into four classes, IgGl, IgG2, IgG3 and IgG4. Each immunoglobulin class has a different set of functions. Lhe following table summarizes the Physicochemical properties of each of the immunoglobulin classes and subclasses.
the type of antibody or fragment thereof can be selected for use according to the present invention based on the desired characteristics and functions that are desired for a particular therapeutic or diagnostic use, such as but not limited to serum half life, intravascular distribution, complement fixation, etc.
Antibody diversity is generated by at least 5 mechanisms, including (1) the use of multiple genes encoding parts of the antibody; (2) somatic mutation, e.g., primordial V gene mutation during B-cell ontogeny to produce different V genes in different B-cell clones; (3) somatic recombination, e.g., gene segments Jl-Jn recombine to join the main part of the V-region gene during B-cell ontogeny; (4) gene conversion where sections of DNA from a number of pseudo V region can be copied into the V region to alter the DNA sequence; and (5) nucleotide addition, e.g., when V and J regions are cut, before joining, and extra nucleotides may be inserted to code for additional amino acids.
somatic mutation e.g., primordial V gene mutation during B-cell ontogeny to produce different V genes in different B-cell clones
somatic recombination e.g., gene segments Jl-Jn recombine to join the main part of the V-region gene during
Non-limiting examples include, but are not limited to, (i) the selection/recombination of VK, J, and CK regions from germ line to B-cell clones to generate kappa chains; (ii) selection/recombination of V ⁇ , J, and C ⁇ regions from germ line to B-cell clones to generate lambda chains; (iii) selection/recombination of V H , D1-D30 and J H 1-J H 6 genes to form a functional VDJ gene encoding a heavy chain variable region.
the above mechanisms work in a coordinated fashion to generate antibody diversity and specificity.
antibody is further intended to encompass antibodies, digestion fragments, specified portions and variants thereof, including antibody mimetics or comprising portions of antibodies that mimic the structure and/or function of an antibody or specified fragment or portion thereof, including single chain antibodies and fragments thereof.
Functional fragments include
antibody fragments capable of binding to IL-17 or portions thereof including, but not limited to Fab (e.g., by papain digestion), Fab' (e.g., by pepsin digestion and partial reduction) and F(ab') 2 (e.g., by pepsin digestion), facb (e.g., by plasmin digestion), pFc' (e.g., by pepsin or plasmin digestion), Fd (e.g., by pepsin digestion, partial reduction and reaggregation), Fv or scFv (e.g., by molecular biology techniques) fragments, are encompassed by the invention (see, e.g., Colligan, Immunology, supra).
Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein.
Antibodies 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.
a combination gene encoding a F(ab') 2 heavy chain portion can be designed to include DNA sequences encoding the CHi domain and/or hinge region of the heavy chain.
the various portions of antibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques.
human antibody refers to an antibody in which substantially every part of the protein (e.g., CDR, framework, C L , C H domains (e.g., C H 1, C H 2, C H 3), hinge, (V L , V H )) is substantially non-immunogenic in humans, with only minor sequence changes or variations.
antibodies designated primate monkey, baboon, chimpanzee, etc.
rodent mouse, rat, rabbit, guinea pig, hamster, and the like
other mammals designate such species, sub-genus, genus, sub-family, family specific antibodies.
chimeric antibodies include any combination of the above.
a human antibody is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody can be produced by a non-human animal or prokaryotic or eukaryotic cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes. Further, when a human antibody is a single chain antibody, it can comprise a linker peptide that is not found in native human antibodies.
an Fv can comprise a linker peptide, such as two to about eight glycine or other amino acid residues, which connects the variable region of the heavy chain and the variable region of the light chain.
linker peptides are considered to be of human origin.
Bispecific, heterospecific, heteroconjugate or similar antibodies can also be used that are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens. In the present case, one of the binding specificities is for at least one IL-17 protein, the other one is for any other antigen. Methods for making bispecific antibodies are known in the art.
bispecific antibodies are based on the co- expression of two immunoglobulin heavy chain-light chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, Nature 305:537 (1983)). Because of the random
Such antibodies optionally further affect a specific ligand, such as but not limited to where such antibody modulates, decreases, increases, antagonizes, agonizes, mitigates, alleviates, blocks, inhibits, abrogates and/or interferes with at least one IL- 17 activity or binding, or with IL-17 receptor activity or binding, in vitro, in situ and/or in vivo.
a suitable IL- 17 antibody, specified portion or variant of the present invention can bind at least one IL- 17, or specified portions, variants or domains thereof .
a suitable IL- 17 antibody, specified portion, or variant can also optionally affect at least one of IL- 17 activity or function, such as but not limited to, RNA, DNA or protein synthesis, IL- 17 release, IL-17 receptor signaling, membrane IL- 17 cleavage, IL-17 activity, IL-17 production and/or synthesis.
IL- 17 activity or function such as but not limited to, RNA, DNA or protein synthesis, IL- 17 release, IL-17 receptor signaling, membrane IL- 17 cleavage, IL-17 activity, IL-17 production and/or synthesis.
IL- 17 antibodies (also termed IL- 17 antibodies) useful in the methods and compositions of the present invention can optionally be characterized by high affinity binding to IL- 17 and optionally and preferably having low toxicity.
an antibody, specified fragment or variant of the invention, where the individual components, such as the variable region, constant region and framework, individually and/or collectively, optionally and preferably possess low immunogenicity is useful in the present invention.
the antibodies that can be used in the invention are optionally characterized by their ability to treat patients for extended periods with measurable alleviation of symptoms and low and/or acceptable toxicity. Low or acceptable immunogenicity and/or high affinity, as well as other suitable properties, can contribute to the therapeutic results achieved.
Low immunogenicity is defined herein as raising significant HAHA, HACA or HAMA responses in less than about 75%, or preferably less than about 50% of the patients treated and/or raising low titers in the patient treated (less than about 300, preferably less than about 100 measured with a double antigen enzyme immunoassay) (Elliott et al., Lancet 344:1125-1127 (1994), entirely incorporated herein by reference).
the isolated nucleic acids of the present invention can be used for production of at least one IL- 17 antibody or specified variant thereof, which can be used to measure or effect in an cell, tissue, organ or animal (including mammals and humans), to diagnose, monitor, modulate, treat, alleviate,
CEN05189 PCT 11 help prevent the incidence of, or reduce the symptoms of, at least one IL- 17 condition, selected from, but not limited to, at least one of an immune disorder or disease, a cardiovascular disorder or disease, an infectious, malignant, and/or neurologic disorder or disease, or other known or specified IL- 17 related condition.
a method can comprise administering an effective amount of a composition or a pharmaceutical composition comprising at least one IL- 17 antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment, alleviation, prevention, or reduction in symptoms, effects or mechanisms.
the effective amount can comprise an amount of about 0.001 to 500 mg/kg per single (e.g., bolus), multiple or continuous administration, or to achieve a serum concentration of 0.01-5000 ⁇ g/ml serum concentration per single, multiple, or continuous administration, or any effective range or value therein, as done and determined using known methods, as described herein or known in the relevant arts.
At least one IL-17 antibody of the present invention can be optionally produced by a cell line, a mixed cell line, an immortalized cell or clonal population of immortalized cells, as well known in the art. See, e.g., Ausubel, et al., ed., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2001); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2 nd Edition, Cold Spring Harbor, NY (1989); Harlow and Lane, antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2001); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), each entirely incorporated herein by reference.
Human antibodies that are specific for human IL- 17 proteins or fragments thereof can be raised against an appropriate immunogenic antigen, such as isolated and/or IL- 17 protein or a portion thereof (including synthetic molecules, such as synthetic peptides).
an appropriate immunogenic antigen such as isolated and/or IL- 17 protein or a portion thereof (including synthetic molecules, such as synthetic peptides).
CEN05189 PCT 12 mammalian antibodies can be similarly raised.
Preparation of immunogenic antigens, and monoclonal antibody production can be performed using any suitable technique.
a hybridoma is produced by fusing a suitable immortal cell line (e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NSl, NS2, AE-I, L.5, >243, P3X63Ag8.653, Sp2 SA3, Sp2 MAI, Sp2 SSl, Sp2 SA5, U937, MLA 144, ACT IV, MOLT4, DA-I, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA 144, NAMAIWA, NEURO 2A, or the like, or heteromylomas, fusion products thereof, or any cell or fusion cell derived there from, or any other suitable cell line as known in the art.
a suitable immortal cell line e.g., a myeloma cell line such as, but not limited to, Sp2/0, Sp2/0-AG14, NSO, NS
antibody producing cells such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsil, or other immune or B cell containing cells, or any other cells expressing heavy or light chain constant or variable or framework or CDR sequences, either as endogenous or heterologous nucleic acid, as recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptilian, fish, mammalian, rodent, equine, ovine, goat, sheep, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single, double or triple stranded, hybridized, and the like or any combination thereof. See, e.g., Ausubel, supra, and Colligan, Immunology, supra
Antibody producing cells can also be obtained from the peripheral blood or, preferably the spleen or lymph nodes, of humans or other suitable animals that have been immunized with the antigen of interest. Any other suitable host cell can also be used for expressing heterologous or endogenous nucleic acid encoding an antibody, specified fragment or variant thereof, of the present invention.
the fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other suitable known methods, and cloned by limiting dilution or cell sorting, or other known methods. Cells which produce antibodies with the desired specificity can be selected by a suitable assay (e.g., ELISA).
Suitable methods of producing or isolating antibodies of the requisite specificity can be used, including, but not limited to, methods that select recombinant antibody from a peptide or protein library (e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK; Biolnvent, Lund,
a peptide or protein library e.g., but not limited to, a bacteriophage, ribosome, oligonucleotide, RNA, cDNA, or the like, display library; e.g., as available from Cambridge antibody Technologies, Cambridgeshire, UK; MorphoSys, Martinsreid/Planegg, DE; Biovation, Aberdeen, Scotland, UK; Biolnvent
AME Molecular Evolution
transgenic animals e.g., SCID mice, Nguyen et al., Microbiol. Immunol. 41 :901-907 (1997); Sandhu et al., Crit. Rev. Biotechnol. 16:95-118 (1996); Eren et al., Immunol. 93: 154-161
Such techniques include, but are not limited to, ribosome display (Hanes et al., Proc. Natl. Acad. Sci.
a humanized or engineered antibody has one or more amino acid residues from a source which is non-human, e.g., but not limited to mouse, rat, rabbit, non-human primate or other mammal. These human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable, constant or other domain of a known human sequence.
Such imported sequences can be used to reduce immunogenicity or reduce, enhance or modify binding, affinity, on-rate, off-rate, avidity, specificity, half-life, or any other suitable characteristic, as known in the art.
antibodies can also optionally be humanized with retention of high affinity for the antigen and other favorable biological properties.
humanized antibodies can be optionally prepared by a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art.
Humanization or engineering of antibodies of the present invention can be performed using any known method, such as but not limited to those described in, Winter (Jones et al., Nature 321 :522 (1986); Riechmann et al., Nature 332:323 (1988); Verhoeyen et al., Science 239: 1534 (1988)), Sims et al., J. Immunol. 151 : 2296 (1993); Chothia and Lesk, J. MoI. Biol. 196:901 (1987), Carter et al., Proc. Natl. Acad. Sci. U.S.A. 89:4285 (1992); Presta et al., J. Immunol.
the IL-17 antibody can also be optionally generated by immunization of a transgenic animal (e.g., mouse, rat, hamster, non-human primate, and the like) capable of producing a repertoire of human antibodies, as described herein and/or as known in the art.
a transgenic animal e.g., mouse, rat, hamster, non-human primate, and the like
Cells that produce a human IL- 17 antibody can be isolated from such animals and immortalized using suitable methods, such as the methods described herein.
Transgenic mice that can produce a repertoire of human antibodies that bind to human antigens can be produced by known methods (e.g., but not limited to, U.S. Pat. Nos: 5,770,428, 5,569,825, 5,545,806, 5,625,126, 5,625,825, 5,633,425, 5,661,016 and 5,789,650 issued to Lonberg et al; Jakobovits et al WO 98/50433, Jakobovits et al WO 98/24893, Lonberg et al WO 98/24884, Lonberg et al. WO 97/13852, Lonberg et al. WO 94/25585, Kucherlapate et al. WO 96/34096,
mice comprise at least one transgene comprising DNA from at least one human immunoglobulin locus that is functionally rearranged, or which can undergo functional rearrangement.
the endogenous immunoglobulin loci in such mice can be disrupted or deleted to eliminate the capacity of the animal to produce antibodies encoded by endogenous genes.
peptide display libraries Screening antibodies for specific binding to similar proteins or fragments can be conveniently achieved using peptide display libraries.
This method involves the screening of large collections of peptides for individual members having the desired function or structure, antibody screening of peptide display libraries is well known in the art.
the displayed peptide sequences can be from 3 to 5000 or more amino acids in length, frequently from 5-100 amino acids long, and often from about 8 to 25 amino acids long.
several recombinant DNA methods have been described.
One type involves the display of a peptide sequence on the surface of a bacteriophage or cell. Each bacteriophage or cell contains the nucleotide sequence encoding the particular displayed peptide sequence.
CEN05189 PCT 16 See, e.g., U.S. Pat. Nos. 4704692, 4939666, 4946778, 5260203, 5455030, 5518889, 5534621, 5656730, 5763733, 5767260, 5856456, assigned to Enzon; 5223409, 5403484, 5571698, 5837500, assigned to Dyax, 5427908, 5580717, assigned to Affymax; 5885793, assigned to Cambridge antibody Technologies; 5750373, assigned to Genentech, 5618920, 5595898, 5576195, 5698435, 5693493, 5698417, assigned to Xoma, Colligan, supra; Ausubel, supra; or Sambrook, supra, each of the above patents and publications entirely incorporated herein by reference.
Antibodies of the present invention can also be prepared using at least one IL-17 antibody encoding nucleic acid to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such antibodies in their milk. Such animals can be provided using known methods. See, e.g., but not limited to, US patent nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; 5,304,489, and the like, each of which is entirely incorporated herein by reference.
Antibodies of the present invention can additionally be prepared using at least one IL- 17 antibody encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco and maize) that produce such antibodies, specified portions or variants in the plant parts or in cells cultured there from.
transgenic tobacco leaves expressing recombinant proteins have been successfully used to provide large amounts of recombinant proteins, e.g., using an inducible promoter. See, e.g., Cramer et al., Curr. Top. Microbol. Immunol. 240:95-118 (1999) and references cited therein.
transgenic maize have been used to express mammalian proteins at commercial production levels, with biological activities equivalent to those produced in other recombinant systems or purified from natural sources.
antibodies have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain antibodies (scFv's), including tobacco seeds and potato tubers.
scFv's single chain antibodies
tobacco seeds and potato tubers See, e.g., Conrad et al., Plant MoI. Biol. 38:101- 109 (1998) and reference cited therein.
antibodies of the present invention can also be produced using transgenic plants, according to know methods.
the antibodies of the invention can bind human IL- 17 with a wide range of affinities (K D ).
at least one human mAb of the present invention can optionally bind human IL- 17 with high affinity.
a human mAb can bind human IL- 17 with a K D equal to or less than about 10 "7 M, such as but not limited to, 0.1-9.9 (or any range or value therein) X 10 "7 , 10 "8 , 10 "9 ,10 “10 , 10 "11 , 10 42 , 10 43 or any range or value therein.
CEN05189 PCT 17 The affinity or avidity of an antibody for an antigen can be determined experimentally using any suitable method. (See, for example, Berzofsky, et ah, "Antibody-Antigen Interactions," In Fundamental Immunology, Paul, W. E., Ed., Raven Press: New York, NY (1984); Kuby, Janis Immunology, W. H. Freeman and Company: New York, NY (1992); and methods described herein).
the measured affinity of a particular antibody-antigen interaction can vary if measured under different conditions (e.g., salt concentration, pH).
measurements of affinity and other antigen- binding parameters are preferably made with standardized solutions of antibody and antigen, and a standardized buffer, such as the buffer described herein.
Nucleic Acid Molecules Using the information provided herein, such as the nucleotide sequences encoding at least 70-
nucleic acid molecules of the present invention encoding at least one IL- 17 antibody can be obtained using methods described herein or as known in the art.
Nucleic acid molecules of the present invention can be in the form of RNA, such as mRNA, hnRNA, tRNA or any other form, or in the form of DNA, including, but not limited to, cDNA and genomic DNA obtained by cloning or produced synthetically, or any combinations thereof.
the DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of the 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 anti-sense strand.
Isolated nucleic acid molecules of the present invention can include nucleic acid molecules comprising an open reading frame (ORF), optionally with one or more introns, e.g., but not limited to, at least one specified portion of at least one CDR, as CDRl, CDR2 and/or CDR3 of at least one heavy chain or light chain; nucleic acid molecules comprising the coding sequence for an IL- 17 antibody or variable region; and nucleic acid molecules which comprise a nucleotide sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode at least one IL- 17 antibody as described herein and/or as known in the art.
ORF open reading frame
introns e.g., but not limited to, at least one specified portion of at least one CDR, as CDRl, CDR2 and/or CDR3 of at least one heavy chain or light chain
nucleic acid molecules comprising the coding sequence for an IL- 17 antibody or variable region
nucleic acid variants that code for specific IL-17 antibodies of the present invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.
Non-limiting examples of isolated nucleic acid molecules of the present invention include the CDR sequences corresponding to non-limiting examples of a nucleic acid encoding, respectively, HC CDRl, HC CDR2, HC CDR3, LC CDRl, LC CDR2, LC CDR3, HC variable region and LC variable region.
nucleic acid molecules of the present invention which comprise a nucleic acid encoding an IL-17 antibody can include, but are not limited to, those encoding the amino acid
sequence encoding an antibody can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused antibody comprising an antibody fragment or portion.
a marker sequence such as a sequence encoding a peptide that facilitates purification of the fused antibody comprising an antibody fragment or portion.
the present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein.
the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides.
polynucleotides of the present invention can be used to identify, isolate, or amplify partial or full-length clones in a deposited library.
the polynucleotides are genomic or cDNA sequences isolated, or otherwise complementary to, a cDNA from a human or mammalian nucleic acid library.
the cDNA library comprises at least 80% full-length sequences, preferably at least 85% or 90% full-length sequences, and more preferably at least 95% full-length sequences.
the cDNA libraries can be normalized to increase the representation of rare sequences.
Low or moderate stringency hybridization conditions are typically, but not exclusively, employed with sequences having a reduced sequence identity relative to complementary sequences.
Moderate and high stringency conditions can optionally be employed for sequences of greater identity.
Low stringency conditions allow selective hybridization of sequences having about 70% sequence identity and can be employed to identify orthologous or paralogous sequences.
polynucleotides of this invention will encode at least a portion of an antibody encoded by the polynucleotides described herein.
the polynucleotides of this invention embrace nucleic acid sequences that can be employed for selective hybridization to a polynucleotide encoding an antibody of the present invention. See, e.g., Ausubel, supra; Colligan, supra, each entirely incorporated herein by reference.
the isolated nucleic acids of the present invention can be made using (a) recombinant methods,
the nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention.
a multi-cloning site comprising one or more endonuclease restriction sites can be inserted into the nucleic acid to aid in isolation of the polynucleotide.
translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the present invention.
a hexa-histidine marker sequence provides a convenient means to purify 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 a polynucleotide of the present invention.
Additional sequences can be added to such cloning and/or expression sequences to optimize their function in cloning and/or expression, to aid in isolation of the polynucleotide, or to improve the introduction of the polynucleotide into a cell.
Use of cloning vectors, expression vectors, adapters, and linkers is well known in the art. (See, e.g., Ausubel, supra; or Sambrook, supra) Recombinant Methods for Constructing Nucleic Acids
RNA, cDNA, genomic DNA, or any combination thereof can be obtained from biological sources using any number of cloning methodologies known to those of skill in the art.
oligonucleotide probes that selectively hybridize, under stringent conditions, to the polynucleotides of the present invention are used to identify the desired sequence in a cDNA or genomic DNA library.
the isolation of RNA, and construction of cDNA and genomic libraries, is well known to those of ordinary skill in the art. (See, e.g., Ausubel, supra; or Sambrook, supra)
a cDNA or genomic library can be screened using a probe based upon the sequence of a polynucleotide of the present invention, such as those disclosed herein.
Probes can be used to hybridize with genomic DNA or cDNA sequences to isolate homologous genes in the same or different organisms.
degrees of stringency of hybridization can be employed in the assay; and either the hybridization or the wash medium can be stringent. As the conditions for hybridization become more stringent, there must be a greater degree of complementarity between the probe and the target for duplex formation to occur.
the degree of stringency can be controlled by one or more of temperature, ionic strength, pH and the presence of a partially denaturing solvent such as formamide.
the stringency of hybridization is conveniently varied by changing the polarity of the reactant solution through, for example, manipulation of the concentration of formamide within the range of 0% to 50%.
the degree of complementarity (sequence identity) required for detectable binding will vary in accordance with the stringency of the hybridization medium and/or wash medium.
the degree of complementarity will optimally be 100%, or 70-100%, or any range or value therein.
minor sequence variations in the probes and primers can be compensated for by reducing the stringency of the hybridization and/or wash medium.
RNA amplification includes, but are not limited to, polymerase chain reaction (PCR) and related amplification processes (see, e.g., U.S. Patent Nos. 4,683,195,
PCR polymerase chain reaction
in vitro amplification methods can also be useful, for example, to clone nucleic acid sequences that code for proteins to be expressed, to make nucleic acids to use as probes for detecting the presence of the desired mRNA in samples, for nucleic acid sequencing, or for other purposes.
examples of techniques sufficient to direct persons of skill through in vitro amplification methods are found in Berger, supra, Sambrook, supra, and Ausubel, supra, as well as Mullis, et al., U.S. Patent No.
Chemical synthesis generally produces a single-stranded oligonucleotide, which can be converted into double-stranded DNA by hybridization with a complementary sequence, or by polymerization with a DNA polymerase using the single strand as a template.
a complementary sequence or by polymerization with a DNA polymerase using the single strand as a template.
One of skill in the art will recognize that while chemical synthesis of DNA can be limited to sequences of about 100 or more bases, longer sequences can be obtained by the ligation of shorter sequences.
the present invention further provides recombinant expression cassettes comprising a nucleic acid of the present invention.
a nucleic acid sequence of the present invention for example a cDNA or a genomic sequence encoding an antibody of the present invention, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell.
CEN05189 PCT 21 cassette will typically comprise a polynucleotide of the present invention operably linked to transcriptional initiation regulatory sequences that will direct the transcription of the polynucleotide in the intended host cell.
Both heterologous and non-heterologous (i.e., endogenous) promoters can be employed to direct expression of the nucleic acids of the present invention.
isolated nucleic acids that serve as promoter, enhancer, or other elements can be introduced in the appropriate position (upstream, downstream or in intron) of a non-heterologous form of a polynucleotide of the present invention so as to up or down regulate expression of a polynucleotide of the present invention.
endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution.
the present invention also relates to vectors that include isolated nucleic acid molecules of the present invention, host cells that are genetically engineered with the recombinant vectors, and the production of at least one IL- 17 antibody by recombinant techniques, as is well known in the art. See, e.g., Sambrook, et al., supra; Ausubel, et al., supra, each entirely incorporated herein by reference.
the polynucleotides can optionally be joined to a vector containing a selectable marker for propagation in a host.
a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. 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 operatively linked to an appropriate promoter.
the expression constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (e.g., UAA, UGA or UAG) appropriately positioned at the end of the mRNA to be translated, with UAA and UAG preferred for mammalian or eukaryotic cell expression.
Expression vectors will preferably but optionally include at least one selectable marker. Such markers include, e.g., but not limited to, methotrexate (MTX), dihydrofolate reductase (DHFR, US Pat.
MTX methotrexate
DHFR dihydrofolate reductase
Introduction of a vector construct into a host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other known methods. Such methods are described in the art, such as Sambrook, supra, Chapters 1 -4 and 16-18; Ausubel, supra, Chapters 1, 9, 13, 15, 16.
At least one antibody of the present invention can be expressed in a modified form, such as a fusion protein, and can include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, can be added to the N-terminus of an antibody to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to an antibody of the present invention to facilitate purification. Such regions can be removed prior to final preparation of an antibody or at least one fragment thereof. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Chapters 17.29-17.42 and 18.1-18.74; Ausubel, supra, Chapters 16, 17 and 18. Those of ordinary skill in the art are knowledgeable in the numerous expression systems available for expression of a nucleic acid encoding a protein of the present invention.
nucleic acids of the present invention can be expressed in a host cell by turning on (by manipulation) in a host cell that contains endogenous DNA encoding an antibody of the present invention.
Such methods are well known in the art, e.g., as described in US patent Nos. 5,580,734, 5,641,670, 5,733,746, and 5,733,761, entirely incorporated herein by reference.
mammalian cells useful for the production of the antibodies, specified portions or variants thereof, are mammalian cells.
Mammalian cell systems often will be in the form of monolayers 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 the COS-I (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL- 1651), HEK293, BHK21 (e.g., ATCC
CHO e.g., ATCC CRL 1610
BSC-I e.g., ATCC CRL-26
Cos-7 cells CHO cells
hep G2 cells e.g., CHO cells
P3X63Ag8.653, SP2/0-Agl4, 293 cells e.g., HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va (www.atcc.org).
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
the recombinant cell is a P3X63Ab8.653 or a SP2/0-Agl4 cell.
Expression vectors for these cells can include one or more of the following expression control sequences, such as, but not limited to an origin of replication; a promoter (e.g., late or early SV40 promoters, the CMV promoter (US Pat. Nos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk
phosphoglycerate kinase promoter an EF-I alpha promoter (US Pat.No. 5,266,491), at least one human immunoglobulin promoter; an enhancer, and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
an enhancer and/or processing information sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites (e.g., an SV40 large T Ag poly A addition site), and transcriptional terminator sequences.
ribosome binding sites e.g., RNA splice sites
polyadenylation sites e.g., an SV40 large T Ag poly A addition site
transcriptional terminator sequences e.g., Ausubel et al., supra; Sambrook, et al., supra.
CEN05189 PCT 23 instance from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources.
polyadenlyation or transcription terminator sequences are typically incorporated into the vector.
An example of a terminator sequence is the polyadenlyation sequence from the bovine growth hormone gene. Sequences for accurate splicing of the transcript can also be included.
An example of a splicing sequence is the VPl intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)).
gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.
An IL- 17 antibody can be recovered and purified from recombinant cell cultures by well- known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.
High performance liquid chromatography can also be employed for purification. See, e.g., Colligan, Current Protocols in Immunology, or Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2001), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference.
Antibodies of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a eukaryotic host, including, for example, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the antibody of the present invention can be glycosylated or can be non-glycosylated, with glycosylated preferred. Such methods are described in many standard laboratory manuals, such as Sambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12, 13, 16, 18 and 20, Colligan, Protein Science, supra, Chapters 12-14, all entirely incorporated herein by reference.
the isolated proteins and antibodies of the present invention comprise at least one protein and/or antibody amino acid sequence disclosed or described herein encoded by any suitable polynucleotide, or any at least one isolated or prepared protein antibody.
the at least one protein has at least one IL-17 activity and the at least one antibody binds human IL-17 and, thereby partially or substantially modulates at least one structural or biological activity of at least one IL-17 protein.
IL-17 protein refers to a protein as described herein that has at least one IL-17-dependent activity, such as 5 - 10000%, of the activity of a known or other IL- 17 protein or active portion thereof, preferably by at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92,
the capacity of a IL-17 protein to have at least one IL-17-dependent activity is preferably assessed by at least one suitable IL-17 protein or receptor assay, as described herein and/or as known in the art.
neutralizing antibody refers to an antibody that can inhibit at least one IL-17-dependent activity by about 5-120%, preferably by at least about 10, 20, 30, 40, 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100% or more depending on the assay.
the capacity of an IL-17 antibody to inhibit an IL-17-dependent activity is preferably assessed by at least one suitable IL- 17 protein or receptor assay, as described herein and/or as known in the art.
an antibody of the invention can be of any class (IgG, IgA, IgM, IgE, IgD, etc.) or isotype and can comprise a kappa or lambda light chain.
the human antibody comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl, IgG2, IgG3 or IgG4.
Antibodies of this type can be prepared by employing a transgenic mouse or other trangenic non-human mammal comprising at least one human light chain (e.g., IgG, IgA and IgM (e.g., ⁇ l, ⁇ 2, ⁇ 3, ⁇ 4) transgenes as described herein and/or as known in the art.
the human IL-17 human antibody comprises an IgGl heavy chain and a IgGl light chain.
At least one antibody of the invention binds at least one specified epitope specific to at least one IL- 17 protein, subunit, fragment, portion or any combination thereof.
the at least one epitope can comprise at least one antibody binding region that comprises at least one portion of the protein, which epitope can optionally comprise at least one portion of at least one extracellular, soluble, hydrophillic, external or cytoplasmic portion of the protein.
the at least one specified epitope can comprise any combination of at least one amino acid sequence of at least 1-3 amino acids to the entire specified portion of contiguous amino acids of the SEQ ID NO:1.
the at least one antibody of the present invention can preferably comprise at least one antigen-binding region that comprises at least one human complementarity determining region (CDRl , CDR2 and CDR3) or variant of at least one heavy chain variable region and/or at least one human complementarity determining region (CDRl, CDR2 and CDR3) or variant of at least one light chain variable region.
the protein and antibody can have an antigen- binding region that comprises at least a portion of at least one heavy chain (HC) CDR (i.e., HC CDRl, HC CDR2 and/or HC CDR3) having the amino acid sequence of the corresponding HC CDRs 1, 2 and/or 3.
HC heavy chain
the antibody or antigen-binding portion or variant can have at least one antigen-binding region that comprises at least a portion of at least one light chain (LC) CDR (i.e., LC CDRl, LC CDR2 and/or LC CDR3).
LC light chain
the three heavy chain CDRs and the three light chain CDRs of the anitbody or antigen -binding fragment have the amino acid sequence of the corresponding CDR of at least one of mAb as described herein.
Such antibodies can be prepared by chemically joining together the various portions (e.g., CDRs, framework) of the antibody using conventional techniques, by preparing and expressing a (i.e., one or
the IL-17 antibody can comprise at least one of a heavy or light chain variable region having a defined amino acid sequence.
the IL- 17 antibody comprises at least one of at least one heavy chain variable region, and/or at least one light chain variable region.
Antibodies that bind to human IL-17 and that comprise a defined heavy or light chain variable region can be prepared using suitable methods, such as phage display (Katsube, Y., et ah, Int J MoI. Med, l(5):863-868 (1998)) or methods that employ transgenic animals, as known in the art and/or as described herein.
a transgenic mouse comprising a functionally rearranged human immunoglobulin heavy chain transgene and a transgene comprising DNA from a human immunoglobulin light chain locus that can undergo functional rearrangement, can be immunized with human IL-17 or a fragment thereof to elicit the production of antibodies.
the antibody producing cells can be isolated and hybridomas or other immortalized antibody-producing cells can be prepared as described herein and/or as known in the art.
the antibody, specified portion or variant can be expressed using the encoding nucleic acid or portion thereof in a suitable host cell.
the invention also relates to antibodies, antigen-binding fragments, immunoglobulin chains and CDRs comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein.
such antibodies or antigen-binding fragments and antibodies comprising such chains or CDRs can bind human IL-17 with high affinity (e.g., K D less than or equal to about 10 ⁇ 9 M).
Amino acid sequences that are substantially the same as the sequences described herein include sequences comprising conservative amino acid substitutions, as well as amino acid deletions and/or insertions.
a conservative amino acid substitution refers to the replacement of a first amino acid by a second amino acid that has chemical and/or physical properties (e.g, charge, structure, polarity, hydrophobicity/ hydrophilicity) that are similar to those of the first amino acid.
Conservative substitutions include replacement of one amino acid by another within the following groups: lysine (K), arginine (R) and histidine (H); aspartate (D) and glutamate (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; C, S and T.
amino acids that make up IL-17 antibodies of the present invention are often abbreviated.
amino acid designations can be indicated by designating the amino acid by its single letter code, its three letter code, name, or three nucleotide codon(s) as is well understood in the art (see Alberts, B., et al., Molecular Biology of The Cell, Third Ed., Garland Publishing, Inc.,New York, 1994):
An IL- 17 antibody of the present invention can include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation, as specified herein.
the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of amino acid substitutions, insertions or deletions for any given IL- 17 antibody, fragment or variant will not be more than 40, 30, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, such as 1-30 or any range or value therein, as specified herein.
Amino acids in an IL- 17 antibody of the present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)).
site-directed mutagenesis or alanine-scanning mutagenesis e.g., Ausubel, supra, Chapters 8, 15; Cunningham and Wells, Science 244:1081-1085 (1989)
the latter procedure introduces single alanine mutations at every residue in the molecule.
the resulting molecules are then tested for biological activity, such as, but not limited to at least one IL-17 neutralizing activity.
Sites that are critical for antibody binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. MoI. Biol. 224:899-904 (19
IL- 17 proteins of the present invention can include, but are not limited to, at least one portion, sequence or combination selected from 3-100 to all of the contiguous amino acids of at least one of SEQ ID NO: 1.
IL-17 antibodies of the present invention can include, but are not limited to, at least
CEN05189 PCT 27 one portion, sequence or combination selected from 5 to all of the contiguous amino acids of at least one IL-17 Mab, optionally including at least one of the corresponding CDRs.
Non-limiting CDRs or portions of IL- 17 proteins or antibodies of the invention that can enhance or maintain at least one of the listed activities include, but are not limited to, any of the above polypeptides, further comprising at least one mutation corresponding to at least one substitution selected from the group consisting of at least one of extracellular, intracellular, soluble, at least 10 contiguous amino acids, and the like, of SEQ ID NO: 1 .
Non-limiting variants that can enhance or maintain at least one of the listed activities include, but are not limited to, any of the above polypeptides, further comprising at least one mutation corresponding to at least one substitution selected from the group consisting of Ile48 for Val48,
Gln90 for Glu90 Leu95 for Ile95, Leu96 for Ile96, Leu99 for Ile99, PhelO3 for TyrlO3 of at least one of SEQ ID NO: 1.
A(n) IL-17 protein can further optionally comprise a polypeptide of at least one of 70-100% of the contiguous amino acids of at least one of SEQ ID NOS: 1 or any variant thereof.
the amino acid sequence of a IL- 17 protein or antibody has about 70-
proteins and antibodies of the present invention can comprise any number of contiguous amino acid residues from an antibody of the present invention, wherein that number is selected from the group of integers consisting of from 10-100% of the number of contiguous residues in an IL- 17 protein or antibody.
this subsequence of contiguous amino acids is at least about 10, 20, 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 in length, or any range or value therein.
the present invention includes at least one biologically active protein or antibody of the present invention.
Biologically active proteins or antibodies have a specific activity at least 20%, 30%, or 40%, and preferably at least 50%, 60%, or 70%, and most preferably at least 80%, 90%, or 95%-1000% of that of the native (non-synthetic), endogenous or related and known protein or antibody.
Methods of assaying and quantifying measures of enzymatic activity and substrate specificity are well known to those of skill in the art.
the invention relates to IL-17 proteins or antibodies of the invention, as described herein, which are modified by the covalent attachment of a moiety.
modification can produce a IL- 17 protein or anibody with improved pharmacokinetic properties (e.g., increased in vivo serum half-life).
the organic moiety can be a linear or branched hydrophilic polymeric group, fatty acid group, or fatty acid ester group.
the hydrophilic polymeric group can have a molecular weight of about 800 to about 120,000 Daltons and can be a polyalkane glycol (e.g., polyethylene glycol (PEG), polypropylene glycol (PPG)), carbohydrate polymer, amino acid polymer or polyvinyl pyrolidone, and the fatty acid or fatty acid ester group can comprise from about eight to about forty carbon atoms.
the modified proteins and antibodies of the invention can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the antibody or protein. Each organic moiety that is bonded to the protein or antibody of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group.
fatty acid encompasses mono-carboxylic acids and di-carboxylic acids.
poly lysine is more soluble in water than in octane.
a IL- 17 antibody or protein modified by the covalent attachment of poly lysine is encompassed by the invention.
Hydrophilic polymers suitable for modifying antibodies or proteins of the invention can be linear or branched and include, for example, polyalkane glycols (e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like), carbohydrates (e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like), polymers of hydrophilic amino acids (e.g., polylysine, polyarginine, polyaspartate and the like), polyalkane oxides (e.g., polyethylene oxide, polypropylene oxide and the like) and polyvinyl pyrolidone.
polyalkane glycols e.g., PEG, monomethoxy-polyethylene glycol (mPEG), PPG and the like
carbohydrates e.g., dextran, cellulose, oligosaccharides, polysaccharides and the like
polymers of hydrophilic amino acids e.g., polylys
the hydrophilic polymer that modifies the protein or antibody of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity.
PEG 5000 and PEG 2 o , ooo wherein the subscript is the average molecular weight of the polymer in Daltons, can be used.
the hydrophilic polymeric group can be substituted with one to about six 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 employing suitable methods.
a polymer comprising an amine group can be coupled to a carboxylate of the fatty acid or fatty acid ester, and an activated carboxylate (e.g., activated with N, N-carbonyl diimidazole) on a fatty acid or fatty acid ester can be coupled to a hydroxyl group on a polymer.
an activated carboxylate e.g., activated with N, N-carbonyl diimidazole
Fatty acids and fatty acid esters suitable for modifying antibodies of the invention can be saturated or can contain one or more units of unsaturation.
Fatty acids that are suitable for modifying antibodies of the invention include, for example, n-dodecanoate (Ci 2 , laurate), n-tetradecanoate (Ci 4 , myristate), n-octadecanoate (Ci 8 , stearate), n-eicosanoate (C 2 o, arachidate) , n-docosanoate (C 22 , behenate), n-triacontanoate (C 30 ), n-tetracontanoate (C 40 ), cz ' s- ⁇ 9-octadecanoate (Ci 8 , oleate), all cis-
CEN05189 PCT 29 ⁇ 5,8,l l,14-eicosatetraenoate (C 2 o, arachidonate), octanedioic acid, tetradecanedioic acid, octadecanedioic acid, docosanedioic acid, and the like.
Suitable fatty acid esters include mono-esters of dicarboxylic acids that comprise a linear or branched lower alkyl group.
the lower alkyl group can comprise from one to about twelve, preferably one to about six, carbon atoms.
the modified human proteins and antibodies can be prepared using suitable methods, such as by reaction with one or more modifying agents.
An "activating group” is a chemical moiety or functional group that can, under appropriate conditions, react with a second chemical group thereby forming a covalent bond between the modifying agent and the second chemical group.
amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bromo, fluoro, iodo), N-hydroxysuccinimidyl esters (NHS), and the like.
Activating groups that can react with thiols include, for example, maleimide, iodoacetyl, acrylolyl, pyridyl disulfides, 5-thiol-2-nitrobenzoic acid thiol (TNB-thiol), and the like.
An aldehyde functional group can be coupled to amine- or hydrazide-containing molecules, and an azide group can react with a trivalent phosphorous group to form phosphoramidate or phosphorimide linkages.
Suitable methods to introduce activating groups into molecules are known in the art (see for example, Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996)).
An activating group can be bonded directly to the organic group (e.g., hydrophilic polymer, fatty acid, fatty acid ester), or through a linker moiety, for example a divalent Ci-Ci 2 group wherein one or more carbon atoms can be replaced by a heteroatom such as oxygen, nitrogen or sulfur.
Suitable linker moieties include, 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 -CH 2 -O-CH-NH-.
Modifying agents that comprise a linker moiety can be produced, for example, by reacting a mono-Boc- alkyldiamine (e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane) with a fatty acid in the presence of l-ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) to form an amide bond between the free amine and the fatty acid carboxylate.
a mono-Boc- alkyldiamine e.g., mono-Boc-ethylenediamine, mono-Boc-diaminohexane
EDC l-ethyl-3-(3-dimethylaminopropyl) carbodiimide
the Boc protecting group can be removed from the product by treatment with trifluoroacetic acid (TFA) to expose a primary amine that can be coupled to another carboxylate as described, or can be reacted with maleic anhydride and the resulting product cyclized to produce an activated maleimido derivative of the fatty acid.
TFA trifluoroacetic acid
Modified proteins or antibodies of the invention can be produced by reacting the protein or antibody with a modifying agent.
the organic moieties can be bonded to the antibody or protein in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG.
Modified IL-17 proteins or antibodies can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of the protein and antibody. The reduced protein and antibody can then be reacted with a thiol-reactive modifying agent to produce the modified
Modified proteins and antibodies comprising an organic moiety that is bonded to specific sites of an antibody of the present invention can be prepared using suitable methods, such as reverse proteolysis (Fisch et ah, Bioconjugate Chem., 3:147-153 (1992); Werlen et ah, Bioconjugate Chem., 5:411-417 (1994); Kumaran et ah, Protein ScL 6(10):2233-2241 (1997); Itoh et ah, Bioorg. Chem., 24(1): 59-68 (1996); Capellas et ah, Biotechnol. Bioeng., 56(4):456-463
an idiotypic (Id) antibody is an antibody that recognizes unique determinants generally associated with the antigen-binding region of another antibody.
the Id can be prepared by immunizing an animal of the same species and genetic type (e.g. mouse strain) as the source of the Id antibody with the antibody or a CDR containing region thereof. The immunized animal will recognize and respond to the idiotypic determinants of the immunizing antibody and produce an anti-Id antibody.
the anti-Id antibody may also be used as an "immunogen" to induce an immune response in yet another animal, producing a so-called anti-Id antibody.
the present invention also provides at least one IL-17 antibody or protein composition comprising at least one, at least two, at least three, at least four, at least five, at least six or more IL-17 antibodies or proteins thereof, as described herein and/or as known in the art that are provided in a non-naturally occurring composition, mixture or form.
Such compositions comprise non-naturally occurring compositions comprising at least one or two IL-17 antibody or protein amino acid sequences selected from the group consisting of 5-100% of the contiguous amino acids of SEQ ID
IL-17 antibody compositions include at least one or two full length, fragments, domains or variants as at least one CDR containing portions of the IL-17 antibody sequence. Further preferred compositions comprise 40-99% of at least one of 70-100% of SEQ ID NO:1 or 2, or specified fragments, domains or variants thereof. Such composition percentages are by weight, volume, concentration, molarity, or molality as liquid or dry solutions, mixtures, suspension, emulsions or colloids, as known in the art or as described herein.
IL-17 antibody or protein compositions of the present invention can further comprise at least one of any suitable and effective amount of a composition or pharmaceutical composition comprising at least one IL- 17 antibody to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy, optionally further comprising at least one selected from at least one TNF
CEN05189 PCT 31 antagonist e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist
an antirheumatic e.g., methotrexate, auranofm, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine
a muscle relaxant a narcotic, a non-steroid inflammatory drug (NSAID)
an analgesic an anesthetic, a sedative, a local anethetic, a neuromuscular blocker
an antimicrobial e.g., aminoglycoside, an antifungal, an antiparasitic, an antiviral, a carbapenem, cephalosporin, a flurorquinolone, a macro
Non-limiting examples of such cytokines include, but are not limted to, any of IL-I to IL-23. Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR
compositions can also include toxin molecules that are associated, bound, co- formulated or co-administered with at least one antibody or protein of the present invention.
the toxin can optionally act to selectively kill the pathologic cell or tissue.
the pathologic cell can be a cancer or other cell.
Such toxins can be, but are not limited to, purified or recombinant toxin or toxin fragment comprising at least one functional cytotoxic domain of toxin, e.g., selected from at least one of ricin, diphtheria toxin, a venom toxin, or a bacterial toxin.
toxin also includes both endotoxins and exotoxins produced by any naturally occurring, mutant or recombinant bacteria or viruses which may cause any pathological condition in humans and other mammals, including toxin shock, which can result in death.
toxins may include, but are not limited to, enterotoxigenic E. coli heat-labile enterotoxin (LT), heat-stable enterotoxin (ST), Shigella cytotoxin, Aeromonas enterotoxins, toxic shock syndrome toxin-1 (TSST-I), Staphylococcal enterotoxin A (SEA), B (SEB), or C (SEC), Streptococcal enterotoxins and the like.
Such bacteria include, but are not limited to, strains of a species of enterotoxigenic E. coli (ETEC), enterohemorrhagic E. coli (e.g., strains of serotype 0157:H7), Staphylococcus species (e.g., Staphylococcus aureus, Staphylococcus pyogenes), Shigella species (e.g., Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei), Salmonella species (e.g., Salmonella typhi, Salmonella cholera-suis, Salmonella enteritidis), Clostridium species (e.g., Clostridium perfringens, Clostridium perfringens, Clostridium pere, Clostridium botulinum), Camphlobacter species (e.g., C amphlobacter jejuni, Camphlobacter fetus),
Vibrios parahemolyticus Vibrios parahemolyticus
Klebsiella species Pseudomonas aeruginosa
Streptococci See, e.g., Stein, ed., INTERNAL MEDICINE, 3rd ed., pp 1-13, Little, Brown and Co., Boston, (1990); Evans et al., eds., Bacterial Infections of Humans: Epidemiology and Control, 2d. Ed., pp 239-254, Plenum Medical Book Co., New York (1991); Mandell et al, Principles and Practice of Infectious Diseases, 3d.
compositions or combinations of the present invention can further comprise at least one of any suitable auxiliary, such as, but not limited to, diluent, binder, stabilizer, buffers, salts, lipophilic solvents, preservative, adjuvant or the like.
suitable auxiliaries are preferred.
Non-limiting examples of, and methods of preparing such sterile solutions are well known in the art, such as, but limited to, Gennaro, Ed., Remington 's Pharmaceutical Sciences, 18 th Edition, Mack Publishing Co. (Easton, PA) 1990.
Pharmaceutically acceptable carriers can be routinely selected that are suitable for the mode of administration, solubility and/or stability of the IL- 17 antibody or protein composition as well known in the art or as described herein.
Pharmaceutical excipients and additives useful in the present composition include but are not limited to proteins, peptides, amino acids, lipids, and carbohydrates (e.g., sugars, including monosaccharides, di-, tri-, terra-, and oligosaccharides; derivatized sugars such as alditols, aldonic acids, esterified sugars and the like; and polysaccharides or sugar polymers), which can be present singly or in combination, comprising alone or in combination 1-99.99% by weight or volume.
sugars including monosaccharides, di-, tri-, terra-, and oligosaccharides
derivatized sugars such as alditols, aldonic acids, esterified sugars and the like
polysaccharides or sugar polymers
Exemplary but non-limiting protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like.
Representative amino acid/antibody components which can also function in a buffering capacity, include alanine, glycine, arginine, betaine, histidine, glutamic acid, aspartic acid, cysteine, lysine, leucine, isoleucine, valine, methionine, phenylalanine, aspartame, and the like.
One preferred amino acid is glycine.
Carbohydrate excipients suitable for use in the invention include, for example, monosaccharides such as fructose, maltose, galactose, glucose, D-mannose, sorbose, and the like; disaccharides, such as lactose, sucrose, trehalose, cellobiose, and the like; polysaccharides, such as raffinose, melezitose, maltodextrins, dextrans, starches, and the like; and alditols, such as mannitol, xylitol, maltitol, lactitol, xylitol sorbitol (glucitol), myoinositol and the like.
Preferred carbohydrate excipients for use in the present invention are mannitol, trehalose, and raffinose.
IL-17 antibody or protein compositions can also include a buffer or a pH adjusting agent; typically, the buffer is a salt prepared from an organic acid or base.
Representative buffers include organic acid salts such as salts of citric acid, ascorbic acid, gluconic acid, carbonic acid, tartaric acid, succinic acid, acetic acid, or phthalic acid; Tris, tromethamine hydrochloride, or phosphate buffers.
Preferred buffers for use in the present compositions are organic acid salts such as citrate.
IL- 17 antibody or protein compositions of the invention can include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl- ⁇ -cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as
lipids e.g., phospholipids, fatty acids
steroids e.g., cholesterol
chelating agents e.g., EDTA
IL-17 antibody or protein compositions according to the invention are known in the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy", 19 th ed., Williams & Williams, (1995), and in the “Physician's Desk Reference", 52 nd ed., Medical Economics, Montvale, NJ (1998), the disclosures of which are entirely incorporated herein by reference.
Preferrred carrier or excipient materials are carbohydrates (e.g., saccharides and alditols) and buffers (e.g., citrate) or polymeric agents.
the invention provides for stable formulations, which is preferably a phosphate buffer with saline or a chosen salt, as well as preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one IL- 17 antibody or protein in a pharmaceutically acceptable formulation.
Preserved formulations contain at least one known preservative or optionally selected from the group consisting of at least one phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, phenylmercuric nitrite, phenoxyethanol, formaldehyde, chlorobutanol, magnesium chloride (e.g., hexahydrate), alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof in an aqueous diluent. Any suitable concentration or mixture can be used as known in the art, such as 0.001-5%, or any range or value therein, such as, but not limited to 0.001, 0.003, 0.005, 0.009, 0.01, 0.02, 0.03,
Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3.
benzyl alcohol e.g., 0.5, 0.9, 1.1., 1.5, 1.9, 2.0, 2.5%), 0.001-0.5% thimerosal (e.g., 0.005, 0.01), 0.001-2.0% phenol (e.g., 0.05, 0.25, 0.28, 0.5, 0.9, 1.0%), 0.0005-1.0% 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.
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%),
the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one IL-17 antibody or protein with the prescribed buffers and/or preservatives, optionally in an aqueous diluent, wherein said packaging material comprises a label that indicates that such solution can be held over a period of 1, 2, 3, 4, 5, 6, 9, 12, 18, 20, 24, 30, 36, 40, 48, 54, 60, 66, 72 hours or greater.
the invention further comprises an article of manufacture, comprising packaging material, a first vial comprising lyophilized at least one IL- 17 antibody or protein, and a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a
CEN05189 PCT 35 patient to reconstitute the at least one IL- 17 antibody or protein in the aqueous diluent to form a solution that can be held over a period of twenty-four hours or greater.
the at least one IL-17antibody or protein used in accordance with the present invention can be produced by recombinant means, including from mammalian cell or transgenic preparations, or can be purified from other biological sources, as described herein or as known in the art.
the range of at least one IL- 17 antibody in at least one product of the present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 ng/ml to about 1000 mg/ml, although lower and higher concentrations are operable and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods.
the range of at least one IL- 17 antibody in at least one product of the present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 ⁇ g/ml to about 1000 mg/ml, although lower and higher concentrations are operable and are dependent on the intended delivery vehicle, e.g., solution formulations will differ from transdermal patch, pulmonary, transmucosal, or osmotic or micro pump methods.
the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative.
preservatives include those selected from the group consisting of phenol, m- cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal, or mixtures thereof.
concentration of preservative used in the formulation is a concentration sufficient to yield an microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan.
excipients e.g. isotonicity agents, buffers, antioxidants, preservative enhancers
An isotonicity agent such as glycerin, is commonly used at known concentrations.
a physiologically tolerated buffer is preferably added to provide improved pH control.
the formulations can cover a wide range of pHs, such as from about pH 4 to about pH 10, and preferred ranges from about pH 5 to about pH 9, and a most preferred range of about 6.0 to about 8.0.
the formulations of the present invention have pH between about 6.8 and about 7.8.
Preferred buffers include phosphate buffers, most preferably sodium phosphate, particularly phosphate buffered saline (PBS).
additives such as a pharmaceutically acceptable solubilizers like Tween 20 (polyoxyethylene (20) sorbitan monolaurate), Tween 40 (polyoxyethylene (20) sorbitan monopalmitate), Tween 80 (polyoxyethylene (20) sorbitan monooleate), Pluronic F68 (polyoxyethylene polyoxypropylene block copolymers), and PEG (polyethylene glycol) or non-ionic surfactants such as polysorbate 20 or 80 or poloxamer 184 or 188, Pluronic® polyls, other block copolymers, and chelators such as EDTA and EGTA can optionally be added to the formulations or
CEN05189 PCT 36 compositions to reduce aggregation are particularly useful if a pump or plastic container is used to administer the formulation.
the presence of pharmaceutically acceptable surfactant mitigates the propensity for the protein to aggregate.
the formulations of the present invention can be prepared by a process which comprises mixing at least one IL- 17 antibody or protein and a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an aqueous diluent.
a preservative selected from the group consisting of phenol, m-cresol, p-cresol, o-cresol, chlorocresol, benzyl alcohol, alkylparaben, (methyl, ethyl, propyl, butyl and the like), benzalkonium chloride, benzethonium chloride, sodium dehydroacetate and thimerosal or mixtures thereof in an a
a measured amount of at least one IL- 17 antibody or protein in buffered solution is combined with the desired preservative in a buffered solution in quantities sufficient to provide the protein and preservative at the desired concentrations.
Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
the claimed formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one IL- 17 antibody or protein that is reconstituted with a second vial containing water, a preservative and/or excipients, preferably a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent.
a preservative and/or excipients preferably a phosphate buffer and/or saline and a chosen salt, in an aqueous diluent.
Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus can provide a more convenient treatment regimen than currently available.
Formulations of the invention can optionally be safely stored at temperatures of from about 2 to about 40 0 C and retain the biologically activity of the protein for extended periods of time, thus, allowing a package label indicating that the solution can be held and/or used over a period of 6, 12, 18, 24, 36, 48, 72, or 96 hours or greater. If preserved diluent is used, such label can include use up to 1-12 months, one-half, one and a half, and/or two years.
the solutions of at least one IL-17 antibody or protein in the invention can be prepared by a process that comprises mixing at least one antibody or protein in an aqueous diluent. Mixing is carried out using conventional dissolution and mixing procedures. To prepare a suitable diluent, for example, a measured amount of at least one antibody or protein in water or buffer is combined in quantities sufficient to provide the protein and optionally a preservative or buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the
CEN05189 PCT 37 art For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
the claimed products can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one IL- 17 antibody or protein that is reconstituted with a second vial containing the aqueous diluent.
Either a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.
the claimed products can be provided indirectly to patients by providing to pharmacies, clinics, or other such institutions and facilities, clear solutions or dual vials comprising a vial of lyophilized at least one IL- 17 antibody or protein that is reconstituted with a second vial containing the aqueous diluent.
the clear solution in this case can be up to one liter or even larger in size, providing a large reservoir from which smaller portions of the at least one antibody or protein solution can be retrieved one or multiple times for transfer into smaller vials and provided by the pharmacy or clinic to their customers and/or patients.
Recognized devices comprising these single vial systems include those pen-injector devices for delivery of a solution such as BD Pens, BD Autojector ® , Humaject ® 'NovoPen ® , B-D ® Pen, AutoPen ® , and OptiPen ® , GenotropinPen ® , Genotronorm Pen ® , Humatro Pen ® , Reco-Pen ® , Roferon Pen ® , Biojector ® , iject ® , J-tip Needle-Free Injector ® , Intraject ® , Medi-Ject ® , e.g., as made or developed by Becton Dickensen (Franklin Lakes, NJ, www.bectondickenson.com), Disetronic
Recognized devices comprising a dual vial system include those pen-injector systems for reconstituting a lyophilized drug in a cartridge for delivery of the reconstituted solution such as the HumatroPen ® .
the products presently claimed include packaging material.
the packaging material provides, in addition to the information required by the regulatory agencies, the conditions under which the product can be used.
the packaging material of the present invention provides instructions to the patient to reconstitute the at least one IL- 17 antibody or protein in the aqueous diluent to form a solution and to use the solution over a period of 2-24 hours or greater for the two vial, wet/dry, product.
the label indicates that such solution can be used over a period of 2-24 hours or greater.
the presently claimed products are useful for human pharmaceutical product use.
the formulations of the present invention can be prepared by a process that comprises mixing at least one IL- 17 antibody or protein and a selected buffer, preferably a phosphate buffer containing saline or a chosen salt. Mixing the at least one antibody or protein and buffer in an
CEN05189 PCT 38 aqueous diluent is carried out using conventional dissolution and mixing procedures.
a suitable formulation for example, a measured amount of at least one antibody or protein in water or buffer is combined with the desired buffering agent in water in quantities sufficient to provide the protein and buffer at the desired concentrations. Variations of this process would be recognized by one of ordinary skill in the art. For example, the order the components are added, whether additional additives are used, the temperature and pH at which the formulation is prepared, are all factors that can be optimized for the concentration and means of administration used.
the claimed stable or preserved formulations can be provided to patients as clear solutions or as dual vials comprising a vial of lyophilized at least one IL-17 antibody or protein that is reconstituted with a second vial containing a preservative or buffer and excipients in an aqueous diluent.
a single solution vial or dual vial requiring reconstitution can be reused multiple times and can suffice for a single or multiple cycles of patient treatment and thus provides a more convenient treatment regimen than currently available.
At least one IL-17 antibody or protein in either the stable or preserved formulations or solutions described herein can be administered to a patient in accordance with the present invention via a variety of delivery methods including SC or IM injection; transdermal, pulmonary, transmucosal, implant, osmotic pump, cartridge, micro pump, or other means appreciated by the skilled artisan, as well-known in the art.
the present invention also provides a method for modulating or treating at least one
IL-17 related disease in a cell, tissue, organ, animal, or patient, as known in the art or as described herein, using at least one antibody or protein of the present invention.
the present invention also provides a method for modulating or treating at least one IL- 17 related disease, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of obesity, an immune related disease, a cardiovascular disease, an infectious disease, a malignant disease or a neurologic disease.
the present invention also provides a method for modulating or treating at least one adult or pediatric immune or inflammation related disease, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of, or at least one inflammation related to, rheumatoid arthritis, juvenile rheumatoid arthritis, systemic onset juvenile rheumatoid arthritis, psoriatic arthritis, ankylosing spondilitis, gastric ulcer, seronegative arthropathies, osteoarthritis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, systemic lupus erythematosis, antiphospholipid syndrome, iridocyclitis, uveitis, optic neuritis, idiopathic pulmonary fibrosis, systemic vasculitis, Wegener's granulomatosis, sarcoidosis, orchitis, vasectomy or vasectomy reversal procedures, allergic atopic diseases, asthma, allergic rhinitis,
CEN05189 PCT 39 hypersensitivity pneumonitis, transplants, organ transplant rejection, graft-versus-host disease, systemic inflammatory response syndrome, sepsis syndrome, gram positive sepsis, gram negative sepsis, culture negative sepsis, fungal sepsis, neutropenic fever, urosepsis, meningococcemia, trauma, hemorrhage, burns, ionizing radiation exposure, acute pancreatitis, adult respiratory distress syndrome, rheumatoid arthritis, alcohol-induced hepatitis, chronic inflammatory pathologies, sarcoidosis, Crohn's pathology, sickle cell anemia, type I or type II diabetes, nephrosis, atopic diseases, hypersensitity reactions, allergic rhinitis, hay fever, perennial rhinitis, conjunctivitis, endometriosis, asthma, urticaria, systemic anaphalaxis, dermatitis, pernicious anemia, hemo
the present invention also provides a method for modulating or treating at least one cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least
CEN05189 PCT 40 one of cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, restenosis, diabetic aterosclerotic disease, hypertension, arterial hypertension, renovascular hypertension, syncope, shock, syphilis of the cardiovascular system, heart failure, cor pulmonale, primary pulmonary hypertension, cardiac arrhythmias, atrial ectopic beats, atrial flutter, atrial fibrillation (sustained or paroxysmal), post perfusion syndrome, cardiopulmonary bypass inflammation response, chaotic or multifocal atrial tachycardia, regular narrow QRS tachycardia, specific arrythmias, ventricular fibrillation, His bundle arrythmias, atrioventricular block, bundle branch block, myocardial ischemic disorders, coronary artery disease, angina pectoris, myocardial infarction, cardiomyopathy,
Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one IL-17 antibody or protein to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
the present invention also provides a method for modulating or treating at least one infectious disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: acute or chronic bacterial infection, acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections, HIV infection, HIV neuropathy, meningitis, hepatitis (A,B or C, or the like), septic arthritis, peritonitis, pneumonia, epiglottitis, e.
Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one IL- 17 antibody or protein to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
the present invention also provides a method for modulating or treating at least one malignant disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute
Such a method can optionally comprise administering an effective amount of
the present invention also provides a method for modulating or treating at least one neurologic disease in a cell, tissue, organ, animal or patient, including, but not limited to, at least one of: neurodegenerative diseases, multiple sclerosis, migraine headache, AIDS dementia complex, demyelinating diseases, such as multiple sclerosis and acute transverse myelitis; extrapyramidal and cerebellar disorders' such as lesions of the corticospinal system; disorders of the basal ganglia or cerebellar disorders; hyperkinetic movement disorders such as Huntington's Chorea and senile chorea; drug-induced movement disorders, such as those induced by drugs which block CNS dopamine receptors; hypokinetic movement disorders, such as Parkinson's disease; Progressive supranucleo Palsy; structural lesions of the cerebellum; spinocerebellar degenerations, such as spinal ataxia, Friedreich's ataxia, cerebellar cortical degenerations, multiple systems degenerations (Mencel, Dejerine-Thomas, Shi-Drager
demyelinating core disorders such as multiple sclerosis, acute transverse myelitis
disorders of the motor unit' such as neurogenic muscular atrophies (anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy); Alzheimer's disease; Down's Syndrome in middle age; Diffuse Lewy body disease; Senile Dementia of Lewy body type; Wernicke-Korsakoff syndrome; chronic alcoholism; Creutzfeldt- Jakob disease; Subacute sclerosing panencephalitis, Hallerrorden-Spatz disease; and Dementia pugilistica, and the like.
neurogenic muscular atrophies anterior horn cell degeneration, such as amyotrophic lateral sclerosis, infantile spinal muscular atrophy and juvenile spinal muscular atrophy
Alzheimer's disease Down's Syndrome in middle age
Diffuse Lewy body disease Senile Dementia of Lewy body type
Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one IL- 17 antibody or protein to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
a composition or pharmaceutical composition comprising at least one IL- 17 antibody or protein
Any method of the present invention can comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one IL- 17 antibody or protein to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
a composition or pharmaceutical composition comprising at least one IL- 17 antibody or protein
CEN05189 PCT 42 can optionally further comprise co-administration or combination therapy for treating such diseases, wherein the administering of said at least one IL- 17 antibody or protein, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular
Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2 nd Edition, Appleton and Lange, Stamford, CT (2000); PDR Pharmacopoeia, Tarascon Pocket Pharmacopoeia 2000, Deluxe Edition, Tarascon Publishing, Loma Linda, CA (2000), each of which references are entirely incorporated herein by reference.
TNF antagonists suitable for compositions, combination therapy, co-administration, devices and/or methods of the present invention include, but are not limited to, TNF antibodies, antigen- binding fragments thereof, and receptor molecules which bind specifically to TNF; compounds which prevent and/or inhibit TNF synthesis, TNF release or its action on target cells, such as thalidomide, tenidap, phosphodiesterase inhibitors (e.g, pentoxifylline and rolipram), A2b adenosine receptor agonists and A2b adenosine receptor enhancers; compounds which prevent and/or inhibit TNF receptor signalling, such as mitogen activated protein (MAP) kinase inhibitors; compounds which block and/or inhibit membrane TNF cleavage, such as metalloproteinase inhibitors; compounds which block and/or inhibit TNF activity, such as angiotensin converting enzyme (ACE) inhibitors (e
MAP mitogen activated protein
ACE angiotensin converting enzyme
CEN05189 PCT 43 captopril compounds which block and/or inhibit TNF production and/or synthesis, such as MAP kinase inhibitors.
a "tumor necrosis factor antibody,” “TNF antibody,” “TNF ⁇ antibody,” or fragment and the like decreases, blocks, inhibits, abrogates or interferes with TNF ⁇ activity in vitro, in situ and/or preferably in vivo.
a suitable TNF human antibody of the present invention can bind TNF ⁇ and includes TNF antibodies, antigen-binding fragments thereof, and specified mutants or domains thereof that bind specifically to TNF ⁇ .
a suitable TNF anttibody or fragment can also decrease block, abrogate, interfere, prevent and/or inhibit TNF RNA, DNA or protein synthesis, TNF release, TNF receptor signaling, membrane TNF cleavage, TNF activity, TNF production and/or synthesis.
Chimeric antibody cA2 consists of the antigen binding variable region of the high-affinity neutralizing mouse human TNF ⁇ IgGl antibody, designated A2, and the constant regions of a human IgGl, kappa immunoglobulin.
the human IgGl Fc region improves allogeneic antibody effector function, increases the circulating serum half-life and decreases the immunogenicity of the antibody.
the avidity and epitope specificity of the chimeric antibody cA2 is derived from the variable region of the murine antibody A2.
a preferred source for nucleic acids encoding the variable region of the murine antibody A2 is the A2 hybridoma cell line.
Chimeric A2 (cA2) neutralizes the cytotoxic effect of both natural and recombinant human TNF ⁇ in a dose dependent manner. From binding assays of chimeric antibody cA2 and recombinant human TNF ⁇ , the affinity constant of chimeric antibody cA2 was calculated to be 1.04XlO 10 M "1 .
murine monoclonal antibody A2 is produced by a cell line designated cl34A.
Chimeric antibody cA2 is produced by a cell line designated cl68A.
Additional examples of monoclonal TNF antibodies that can be used in the present invention are described in the art (see, e.g., U.S. Patent No. 5,231,024; M ⁇ ller, A. et al., Cytokine 2(3): 162- 169 (1990); U.S. Application No. 07/943,852 (filed September 11, 1992); Rathjen et al., International Publication No. WO 91/02078 (published February 21, 1991); Rubin et al, EPO Patent Publication No.
TNF receptor molecules useful in the present invention are those that bind TNF ⁇ with high affinity (see, e.g., Feldmann et al, International Publication No. WO 92/07076 (published April 30, 1992); Schall et al, Cell 67:361-370 (1990); and Loetscher et al, Cell 67:351-359 (1990), which references are entirely incorporated herein by reference) and optionally possess low immunogenicity.
the 55 kDa (p55 TNF-R) and the 75 kDa (p75 TNF-R) TNF cell surface receptors are useful in the present invention.
Truncated forms of these receptors comprising the extracellular domains (ECD) of the receptors or functional portions thereof (see, e.g., Corcoran et al, Eur. J. Biochem. 223:831-840 (1994)), are also useful in the present invention.
Truncated forms of the TNF receptors, comprising the ECD have been detected in urine and serum as 30 kDa and 40 kDa TNF ⁇ inhibitory binding proteins (Engelmann, H. et al, J. Biol. Chem. 265:1531-1536 (1990)).
TNF receptor multimeric molecules and TNF immunoreceptor fusion molecules, and derivatives and fragments or portions thereof, are additional examples of TNF receptor molecules which are useful in the methods and compositions of the present invention.
the TNF receptor molecules which can be used in the invention are 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 properties, can contribute to the therapeutic results achieved.
TNF receptor multimeric molecules useful in the present invention comprise all or a functional portion of the ECD of two or more TNF receptors linked via one or more polypeptide linkers or other nonpeptide linkers, such as polyethylene glycol (PEG).
the multimeric molecules can further comprise a signal peptide of a secreted protein to direct expression of the multimeric molecule.
TNF immunoreceptor fusion molecules useful in the methods and compositions of the present invention comprise at least one 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 homo-multimers. The immunoreceptor fusion molecules can also be monovalent or multivalent. An example of such a TNF immunoreceptor fusion molecule is
TNF receptor/IgG fusion protein TNF immunoreceptor fusion molecules and methods for their production have been described in the art (Lesslauer et al, Eur. J. Immunol. 27:2883-2886 (1991); Ashkenazi et al, Proc. Natl. Acad. ScL USA 55:10535-10539 (1991); Peppel et al, J. Exp. Med. / 74:1483-1489 (1991); Kolls et al., Proc. Natl. Acad. ScL USA 97:215-219 (1994); Butler et al, Cytokine 6(6):616-623 (1994); Baker et al, Eur. J. Immunol. 24:2040-2048 (1994); Beutler et al,
a functional equivalent, derivative, fragment or region of TNF receptor molecule refers to the portion of the TNF receptor molecule, or the portion of the TNF receptor molecule sequence which encodes TNF receptor molecule, that is of sufficient size and sequences to functionally resemble TNF receptor molecules that can be used in the present invention (e.g., bind TNFD with high affinity and possess low immunogenicity).
a functional equivalent of TNF receptor molecule also includes modified TNF receptor molecules that functionally resemble TNF receptor molecules that can be used in the present invention (e.g., bind TNF D with high affinity and possess low immunogenicity).
a functional equivalent of TNF receptor molecule can contain a "SILENT" codon or one or more amino acid substitutions, deletions or additions (e.g., substitution of one acidic amino acid for another acidic amino acid; or substitution of one codon encoding the same or different hydrophobic amino acid for another codon encoding a hydrophobic amino acid). See Ausubel et ah, Current
Cytokines include any known cytokine. See, e.g., CopewithCytokines.com. Cytokine antagonists 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 method of the present invention can comprise a method for treating a IL- 17 mediated disorder or disease, comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one IL- 17 antibody or protein to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.
Such a method can optionally further comprise co-administration or combination therapy for treating such disorders or diseases, wherein the administering of said at least one IL-17 antibody or protein, further comprises administering, before concurrently, and/or after, at least one selected from at least one at least one selected from at least one TNF antagonist (e.g., but not limited to a TNF antibody or fragment, a soluble TNF receptor or fragment, fusion proteins thereof, or a small molecule TNF antagonist), an antirheumatic (e.g., methotrexate, auranofin, aurothioglucose, azathioprine, etanercept, gold sodium thiomalate, hydroxychloroquine sulfate, leflunomide, sulfasalzine), a muscle relaxant, a narcotic, a non-steroid inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, a neuromuscular
treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one IL- 17 protein composition that total, on average, a range from at least about 0.001 ng to 500 milligrams of at least one IL-17 protein per kilogram of patient per dose, and preferably from at least about 0.1 ng to 100 milligrams antibody /kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition.
the effective serum concentration can comprise O.OOOlng -0.05 mg/ml serum concentration per single or multiple adminstration.
Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
Preferred doses of at least one protein can optionally include 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 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 and/or 100-500 micrograms or milligrams/kg/administration,
the dosage administered can vary depending upon known factors, such as the
CEN05189 PCT 48 pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
a dosage of active ingredient can be about 0.1 ⁇ g to 100 milligrams per kilogram of body weight.
0.0001 to 50, and preferably 0.001 to 10 milligrams per kilogram per administration or in sustained release form is effective to obtain desired results.
treatment of humans or animals can be provided as a one-time or periodic dosage of at least one antibody of the present invention 0.1 to 100 ⁇ g/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 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, 45, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000 or 3000 ⁇ g/kg, per day, or 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 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, 45, 50, 60, 70, 80, 90 or 100 mg/kg, per day, on at least one of day 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20,
Dosage forms (composition) suitable for internal administration generally contain from about
treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one IL-17 antibody composition that total, on average, a range from at least about 0.00001 to
the effective serum concentration can comprise 0.0001-500 ⁇ g/ml serum concentration per single or multiple adminstration.
Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one IL-17 antibody composition that total, on average, a range from at least about 0.001 ng to 500 milligrams of at least one IL-17antibody per kilogram of patient per dose, and preferably from at least about 0.1 ng to 100 milligrams antibody /kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition.
the effective serum concentration can comprise O.OOOlng -0.05 mg/ml serum concentration per single or multiple adminstration.
Suitable dosages are known to medical practitioners and will, of course, depend upon the particular disease state, specific activity of the composition being administered, and the particular patient undergoing treatment. In some instances, to achieve the desired therapeutic amount, it can be necessary to provide for repeated administration, i.e., repeated individual administrations of a particular monitored or metered dose, where the individual administrations are repeated until the desired daily dose or effect is achieved.
Preferred doses of at least one antibody can optionally include 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, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 62, 63, 64, 65, 66, 67, 68, 69, 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 and/or 100-500 mg/kg/administration, or any range, value or fraction
the dosage administered can vary depending upon known factors, such as the pharmacodynamic characteristics of the particular agent, and its mode and route of administration; age, health, and weight of the recipient; nature and extent of symptoms, kind of concurrent treatment, frequency of treatment, and the effect desired.
a dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight.
0.1 to 50, and preferably 0.1 to 10 milligrams per kilogram per administration or in sustained release form is effective to obtain desired results.
treatment of humans or animals can be provided as a one-time or periodic dosage of at least one antibody of the present invention 0.1 to 100 mg/kg, such as 0.5, 0.9, 1.0, 1.1, 1.5, 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,
Dosage forms (composition) suitable for internal administration generally contain from about 0.1 milligram to about 500 milligrams of active ingredient per unit or container.
the active ingredient will ordinarily be present in an amount of about 0.5-99.999% by weight based on the total weight of the composition.
the antibody or protein can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle.
a pharmaceutically acceptable parenteral vehicle examples include water, saline, Ringer's solution, dextrose solution, and 1-10% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils can also be used.
the vehicle or lyophilized powder can contain additives that maintain isotonicity (e.g., sodium chloride, mannitol) and chemical stability (e.g., buffers and preservatives).
the formulation is sterilized by known or suitable techniques. Suitable pharmaceutical carriers are described in the most recent edition of Remington's
IL- 17 antibodies of the present invention can be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other modes described here within or known in the art.
a carrier as a solution, emulsion, colloid, or suspension, or as a dry powder, using any of a variety of devices and methods suitable for administration by inhalation or other modes described here within or known in the art.
Formulations for parenteral administration can contain as common excipients sterile water or saline, polyalkylene glycols such as polyethylene glycol, oils of vegetable origin, hydrogenated naphthalenes and the like.
Aqueous or oily suspensions for injection can be prepared by using an appropriate emulsifier or humidifier and a suspending agent, according to known methods.
Agents for injection can be a non-toxic, non-orally administrable diluting agent such as aquous solution or a sterile injectable solution or suspension in a solvent.
the usable vehicle or solvent water, Ringer's
CEN05189 PCT 51 solution, isotonic saline, etc. are allowed; as an ordinary solvent, or suspending solvent, sterile involatile oil can be used.
any kind of involatile oil and fatty acid can be used, including natural or synthetic or semisynthetic fatty oils or fatty acids; natural or synthetic or semisynthtetic mono- or di- or tri-glycerides.
Parental administration is known in the art and includes, but is not limited to, conventional means of injections, a gas pressured needle-less injection device as described in U.S. Pat. No. 5,851,198, and a laser perforator device as described in U.S. Pat. No. 5,839,446 entirely incorporated herein by reference.
the invention further relates to the administration of at least one IL- 17 antibody by parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, intracapsular, intracartilaginous, intracavitary, intracelial, intracelebellar, intracerebroventricular, intracolic, intracervical, intragastric, intrahepatic, intramyocardial, intraosteal, intrapelvic, intrapericardiac, intraperitoneal, intrapleural, intraprostatic, intrapulmonary, intrarectal, intrarenal, intraretinal, intraspinal, intrasynovial, intrathoracic, intrauterine, intravesical, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means.
At least one IL-17 antibody composition can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) or any other administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as, but not limited to, creams and suppositories; for buccal, or sublingual administration such as, but not limited to, in the form of tablets or capsules; or intranasally such as, but not limited to, the form of powders, nasal drops or aerosols or certain agents; or transdermally such as not limited to a gel, ointment, lotion, suspension or patch delivery system with chemical enhancers such as dimethyl sulfoxide to either modify the skin structure or to increase the drug concentration in the transdermal patch (Junginger, et al.
At least one IL- 17 antibody composition is delivered in a particle size effective for reaching the lower airways of the lung or sinuses.
at least one IL- 17 antibody can be delivered by any of a variety of inhalation or nasal devices known in the art for administration of a therapeutic agent by inhalation.
CEN05189 PCT 52 capable of depositing aerosolized formulations in the sinus cavity or alveoli of a patient include metered dose inhalers, nebulizers, dry powder generators, sprayers, and the like.
Other devices suitable for directing the pulmonary or nasal administration of antibodies are also known in the art. All such devices can use of formulations suitable for the administration for the dispensing of antibody in an aerosol.
Such aerosols can be comprised of either solutions (both aqueous and non aqueous) or solid particles.
Metered dose inhalers like the Ventolin ® metered dose inhaler, typically use a propellent gas and require actuation during inspiration (See, e.g., WO 94/16970, WO 98/35888).
Dry powder inhalers like TurbuhalerTM (Astra), Rotahaler ® (Glaxo), Diskus ® (Glaxo), SpirosTM inhaler (Dura), devices marketed by Inhale Therapeutics, and the Spinhaler ® powder inhaler (Fisons), use breath-actuation of a mixed powder (US 4668218 Astra, EP 237507 Astra, WO 97/25086 Glaxo, WO 94/08552 Dura, US 5458135 Inhale, WO 94/06498 Fisons, entirely incorporated herein by reference).
Nebulizers like AERxTM Aradigm, the Ultravent ® nebulizer (Mallinckrodt), and the Acorn II ® nebulizer (Marquest Medical Products) (US 5404871 Aradigm, WO 97/22376), the above references entirely incorporated herein by reference, produce aerosols from solutions, while metered dose inhalers, dry powder inhalers, etc. generate small particle aerosols.
These specific examples of commercially available inhalation devices are intended to be a representative of specific devices suitable for the practice of this invention, and are not intended as limiting the scope of the invention.
a composition comprising at least one IL- 17 antibody is delivered by a dry powder inhaler or a sprayer.
an inhalation device for administering at least one antibody of the present invention.
delivery by the inhalation device is advantageously reliable, reproducible, and accurate.
the inhalation device can optionally deliver small dry particles, e.g. less than about 10 ⁇ m, preferably about 1-5 ⁇ m, for good respirability.
a spray including IL-17 antibody composition can be produced by forcing a suspension or solution of at least one IL- 17 antibody through a nozzle under pressure.
the nozzle size and configuration, the applied pressure, and the liquid feed rate can be chosen to achieve the desired output and particle size.
An electrospray can be produced, for example, by an electric field in connection with a capillary or nozzle feed.
particles of at least one IL- 17 antibody composition delivered by a sprayer have a particle size less than about 10 ⁇ m, preferably in the range of about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
Formulations of at least one IL- 17 protein or antibody composition suitable for use with a sprayer typically include antibody or protein compositions in an aqueous solution at a concentration of about 0.0000001 mg to about 1000 mg of at least one IL-17 antibody or protein composition per ml of solution or mg/gm, or any range or value therein, e.g., but not lmited to, .1, .2., .3, .4, .5, .6, .7, .8, .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, 30, 40, 45, 50, 60, 70, 80, 90 or 100 ng or ⁇ g or mg/ml or ng or ⁇ g or mg/gm.
the formulation can be any range or value therein, e.g., but not lmited to, .1, .2., .3, .4, .5, .6, .7, .8, .9,
CEN05189 PCT 53 include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
the formulation can also include an excipient or agent for stabilization of the antibody composition, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
Bulk proteins useful in formulating antibody compositions include albumin, protamine, or the like.
Typical carbohydrates useful in formulating antibody compositions include sucrose, mannitol, lactose, trehalose, glucose, or the like.
the antibody composition formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the antibody or protein composition caused by atomization of the solution in forming an aerosol.
a surfactant which can reduce or prevent surface-induced aggregation of the antibody or protein composition caused by atomization of the solution in forming an aerosol.
Various conventional surfactants can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbitol fatty acid esters. Amounts will generally range between 0.001 and 14% by weight of the formulation.
Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan monooleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as IL-17 antibodies, or specified portions or variants, can also be included in the formulation.
Nebulizer antibody composition can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer.
a nebulizer such as jet nebulizer or an ultrasonic nebulizer.
a compressed air source is used to create a high- velocity air jet through an orifice.
a low-pressure region is created, which draws a solution of antibody composition through a capillary tube connected to a liquid reservoir.
the liquid stream from the capillary tube is sheared into unstable filaments and droplets as it exits the tube, creating the aerosol.
a range of configurations, flow rates, and baffle types can be employed to achieve the desired performance characteristics from a given jet nebulizer.
an ultrasonic nebulizer high-frequency electrical energy is used to create vibrational, mechanical energy, typically employing a piezoelectric transducer. This energy is transmitted to the formulation of antibody composition either directly or through a coupling fluid, creating an aerosol including the antibody composition.
particles of antibody composition delivered by a nebulizer have a particle size less than about 10 ⁇ m, preferably in the range of about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
Formulations of at least one IL- 17 antibody suitable for use with a nebulizer, either jet or ultrasonic typically include a concentration of about 0.1 mg to about 100 mg of at least one IL-17 antibody protein per ml of solution.
the formulation can include agents such as an excipient, a buffer, an isotonicity agent, a preservative, a surfactant, and, preferably, zinc.
the formulation can also include an excipient or agent for stabilization of the at least one IL-17 antibody composition, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate.
Bulk proteins useful in formulating at least one IL-17 antibody compositions include albumin, protamine, or the like.
Typical carbohydrates useful in formulating at least one IL-17 antibody include sucrose, mannitol, lactose, trehalose, glucose, or the like.
the at least one IL-17 antibody formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the at least one IL-17 antibody caused by atomization of the solution in forming an aerosol.
a surfactant can be employed, such as polyoxyethylene fatty acid esters and alcohols, and polyoxyethylene sorbital fatty acid esters. Amounts will generally range between 0.001 and 4% by weight of the formulation.
Especially preferred surfactants for purposes of this invention are polyoxyethylene sorbitan mono- oleate, polysorbate 80, polysorbate 20, or the like. Additional agents known in the art for formulation of a protein such as antibody protein can also be included in the formulation.
a propellant In a metered dose inhaler (MDI), a propellant, at least one IL-17 antibody, and any excipients or other additives are contained in a canister as a mixture including a liquefied compressed gas. Actuation of the metering valve releases the mixture as an aerosol, preferably containing particles in
CEN05189 PCT 55 the size range of less than about 10 ⁇ m, preferably about 1 ⁇ m to about 5 ⁇ m, and most preferably about 2 ⁇ m to about 3 ⁇ m.
the desired aerosol particle size can be obtained by employing a formulation of antibody composition produced by various methods known to those of skill in the art, including jet-milling, spray drying, critical point condensation, or the like.
Preferred metered dose inhalers include those manufactured by 3M or Glaxo and employing a hydrofluorocarbon propellant.
Formulations of at least one IL- 17 antibody for use with a metered-dose inhaler device will generally include a finely divided powder containing at least one IL-17 antibody as a suspension in a non-aqueous medium, for example, suspended in a propellant with the aid of a surfactant.
the propellant can be any conventional material employed for this purpose, such as chlorofluorocarbon, a hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA- 134a (hydro fluroalkane- 134a), HFA-227 (hydrofluroalkane-227), or the like.
the propellant is a hydrofluorocarbon.
the surfactant can be chosen to stabilize the at least one IL- 17 antibody as a suspension in the propellant, to protect the active agent against chemical degradation, and the like.
Suitable surfactants include sorbitan trioleate, soya lecithin, oleic acid, or the like. In some cases solution aerosols are preferred using solvents such as ethanol. Additional agents known in the art for formulation of a protein such as protein can also be included in the formulation.
Formulations for oral rely on the co-administration of adjuvants (e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether) to increase artificially the permeability of the intestinal walls, as well as the co-administration of enzymatic inhibitors (e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol) to inhibit enzymatic degradation.
adjuvants e.g., resorcinols and nonionic surfactants such as polyoxyethylene oleyl ether and n-hexadecylpolyethylene ether
enzymatic inhibitors e.g., pancreatic trypsin inhibitors, diisopropylfluorophosphate (DFF) and trasylol
the active constituent compound of the solid-type dosage form for oral administration can be mixed with at least one additive, including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride.
at least one additive including sucrose, lactose, cellulose, mannitol, trehalose, raffinose, maltitol, dextran, starches, agar, arginates, chitins, chitosans, pectins, gum tragacanth, gum arabic, gelatin, collagen, casein, albumin, synthetic or semisynthetic polymer, and glyceride.
These dosage forms can also contain other type(s) of additives, e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, .alpha.-tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.
additives e.g., inactive diluting agent, lubricant such as magnesium stearate, paraben, preserving agent such as sorbic acid, ascorbic acid, .alpha.-tocopherol, antioxidant such as cysteine, disintegrator, binder, thickener, buffering agent, sweetening agent, flavoring agent, perfuming agent, etc.
Tablets and pills can be further processed into enteric-coated preparations.
the liquid preparations for oral administration include emulsion, syrup, elixir, suspension and solution preparations allowable for medical use. These preparations can contain inactive diluting agents
CEN05189 PCT 56 ordinarily used in said field, e.g., water.
Liposomes have also been described as drug delivery systems for insulin and heparin (U.S. Pat. No. 4,239,754). More recently, microspheres of artificial polymers of mixed amino acids (proteinoids) have been used to deliver pharmaceuticals (U.S. Pat. No. 4,925,673).
carrier compounds described in U.S. Pat. No. 5,879,681 and U.S. Pat. No. 5,5,871,753 are used to deliver biologically active agents orally are known in the art. Mucosal Formulations and Administration
compositions and methods of administering at least one IL- 17 antibody include an emulsion comprising a plurality of submicron particles, a mucoadhesive macromolecule, a bioactive peptide, and an aqueous continuous phase, which promotes absorption through mucosal surfaces by achieving mucoadhesion of the emulsion particles (U.S. Pat. Nos. 5,514,670).
Mucous surfaces suitable for application of the emulsions of the present invention can include corneal, conjunctival, buccal, sublingual, nasal, vaginal, pulmonary, stomachic, intestinal, and rectal routes of administration.
Formulations for vaginal or rectal administration e.g.
suppositories can contain as excipients, for example, polyalkyleneglycols, vaseline, cocoa butter, and the like.
Formulations for intranasal administration can be solid and contain as excipients, for example, lactose or can be aqueous or oily solutions of nasal drops.
excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (U.S. Pat. Nos. 5,849,695).
the at least one IL- 17 antibody is encapsulated in a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated).
a delivery device such as a liposome or polymeric nanoparticles, microparticle, microcapsule, or microspheres (referred to collectively as microparticles unless otherwise stated).
suitable devices are known, including microparticles made of synthetic polymers such as polyhydroxy acids such as polylactic acid, polyglycolic acid and copolymers thereof, polyorthoesters, polyanhydrides, and polyphosphazenes, and natural polymers such as collagen, polyamino acids, albumin and other proteins, alginate and other polysaccharides, and combinations thereof (U.S. Pat. Nos. 5,814,599).
a dosage form can contain a pharmaceutically acceptable non-toxic salt of the compounds that has a low degree of solubility in body fluids, for example, (a) an acid addition salt with a polybasic acid such as phosphoric acid, sulfuric acid, citric acid, tartaric acid, tannic acid, pamoic acid, alginic acid, polyglutamic acid, naphthalene mono- or di-sulfonic acids, polygalacturonic acid, and the like; (b) a salt with a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum,
a polyvalent metal cation such as zinc, calcium, bismuth, barium, magnesium, aluminum
the compounds of the present invention or, preferably, a relatively insoluble salt such as those just described can be formulated in a gel, for example, an aluminum monostearate gel with, e.g. sesame oil, suitable for injection.
Particularly preferred salts are zinc salts, zinc tannate salts, pamoate salts, and the like.
slow release depot formulation for injection would contain the compound or salt dispersed for encapsulated in a slow degrading, non-toxic, non-antigenic polymer such as a polylactic acid/polyglycolic acid polymer for example as described in U.S. Pat. No. 3,773,919.
the compounds or, preferably, relatively insoluble salts such as those described above can also be formulated in cholesterol matrix silastic pellets, particularly for use in animals.
Additional slow release, depot or implant formulations, e.g. gas or liquid liposomes are known in the literature (U.S. Pat. Nos. 5,770,222 and "Sustained and Controlled Release Drug Delivery Systems", J. R. Robinson ed., Marcel Dekker, Inc., N.Y., 1978).
Example 1 Cloning and Expression of IL-17 protein or antibody in Mammalian Cells
a typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the antibody coding sequence, and signals required for the termination of transcription and polyadenylation of the transcript. 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 long terminal repeats (LTRS) from Retroviruses, e.g., RSV, HTLVI, HIVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter).
LTRS long terminal repeats
CMV cytomegalovirus
Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pIRESlneo, pRetro-Off, pRetro-On, PLXSN, or pLNCX (Clonetech Labs, Palo Alto, CA), pcDNA3.1 (+/-), pcDNA/Zeo (+/-) or pcDNA3.1 /Hygro (+/-) (Invitrogen), PSVL and
Mammalian host cells that could be used include human HeIa 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos I 5 Cos 7 and CV I 5 quail QCl-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.
the gene can be expressed in stable cell lines that contain the gene integrated into a chromosome.
the 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 protein or antibody, e.g., as a desired portion of at least one of SEQ ID NO: 1 or 5 or an antibody thereto.
the DHFR (dihydrofolate reductase) marker is useful to develop cell lines that carry several hundred or even several thousand copies of the gene of interest.
Another useful selection marker is the enzyme glutamine synthase (GS) (Murphy, et al., Biochem. J. 227:277-279 (1991); Bebbington, et al., Bio/Technology 10:169-175 (1992)). Using these markers, the mammalian cells are grown in selective medium and the cells with the highest resistance are selected. These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) and NSO cells are used for the production of antibodies or proteins of the present invention.
GS glutamine synthase
the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment of the CMV-enhancer (Boshart, et al., Cell 41 :521-530 (1985)). Multiple cloning sites, e.g., with the restriction enzyme cleavage sites BamHI, Xbal and Asp718, facilitate the cloning of the gene of interest.
the vectors contain in addition the 3' intron, the polyadenylation and termination signal of the rat preproinsulin gene.
Plasmid pC4 is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). The plasmid contains the mouse DHFR gene under control of the SV40 early promoter.
Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD) supplemented with the chemotherapeutic agent methotrexate.
a selective medium e.g., alpha minus MEM, Life Technologies, Gaithersburg, MD
methotrexate methotrexate
DHFR as a result of amplification of the DHFR gene. If a second gene is linked to the DHFR gene, it is usually co-amplified and over-expressed. It is known in the art that this approach can be used to develop cell lines carrying more than 1,000 copies of the amplified gene(s). Subsequently, when the methotrexate is withdrawn, cell lines are obtained that contain the amplified gene integrated into one or more chromosome(s) of the host cell.
Plasmid pC4 contains coding DNA for expressing the gene of interest (e.g., encoding at least one of SEQ ID NO:1) under control of the strong promoter of the long terminal repeat (LTR) of the Rous Sarcoma Virus (Cullen, et al., Molec. Cell. Biol. 5:438-447 (1985)) plus a fragment isolated from the enhancer of the immediate early gene of human cytomegalovirus (CMV) (Boshart, et al., Cell 41 :521-530 (1985)). Downstream of the promoter are BamHI, Xbal, and Asp718 restriction enzyme cleavage sites that allow integration of the genes.
LTR long terminal repeat
CMV cytomegalovirus
the plasmid contains the 3' intron and polyadenylation site of the rat preproinsulin gene.
Other high efficiency promoters can also be used for the expression, e.g., the human b-actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
Clontech's Tet-Off and Tet-On gene expression systems and similar systems can be used to express the IL- 17 in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547- 5551 (1992)).
Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It can be advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
the 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 desired IL-17 antibody or protein is used, e.g., DNA or
RNA coding for at least one of SEQ ID NO: 1 corresponding to at least one portion of at least one IL- 17 antibody or protein of the present invention, according to known method steps.
the isolated encoding DNA and the dephosphorylated vector are then ligated with T4 DNA ligase.
E. coli HBlOl or XL-I Blue cells are then transformed and bacteria are identified that contain the fragment inserted into plasmid pC4 using, for instance, restriction enzyme analysis.
Chinese hamster ovary (CHO) cells lacking an active DHFR gene are used for transfection.
5 ⁇ g of the expression plasmid pC4 is cotransfected with 0.5 ⁇ g of the plasmid pSV2-neo using lipofectin.
the plasmid pSVZneo contains a dominant selectable marker, the neo gene from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including G418.
the cells are seeded in alpha minus MEM supplemented with 1 ⁇ g /ml G418.
the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) in alpha minus MEM supplemented with 10, 25, or 50 ng/ml of methotrexate plus 1 ⁇ g /ml G418.
hybridoma cloning plates Gibcos, Germany
single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using different concentrations of methotrexate (50 nM, 100 nM, 200 nM, 400 nM, 800 nM). Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (1 mM, 2 mM, 5 mM, 10 mM, 20 mM). The same procedure is
CEN05189 PCT 60 repeated until clones are obtained that grow at a concentration of 100 - 200 mM.
Expression of the desired gene product is analyzed, for instance, by SDS-PAGE and Western blot or by reverse phase HPLC analysis.
Transgenic mice have been used that contain human heavy and light chain immunoglobulin genes to generate high affinity, completely human, monoclonal antibodies that can be used therapeutically to inhibit the action of IL- 17 for the treatment of one or more IL-17-mediated disease.
CBA/J x C57/BL6/J F 2 hybrid mice containing human variable and constant region antibody transgenes for both heavy and light chains are immunized with human recombinant IL- 17 (Taylor et al., Intl. Immunol. 6:579-591 (1993); Lonberg, et al., Nature 368:856-859 (1994); Neuberger, M., Nature Biotech.
BSA bovine serum albumin CO 2 - carbon dioxide DMSO - dimethyl sulfoxide
EIA enzyme immunoassay
FBS fetal bovine serum H 2 O 2 - hydrogen peroxide
HRP horseradish peroxidase ⁇ ID - interadermal Ig - immunoglobulin IL- 17 - Interleukin-13 muteins IP - intraperitoneal IV - intravenous Mab - monoclonal antibody OD - optical density OPD - o-Phenylenediamine dihydro chloride PEG - polyethylene glycol
PSA penicillin, streptomycin, amphotericin
CEN05189PCT 61 Transgenic mice that can express human antibodies are known in the art (and are commecially available (e.g., from GenPharm International, San Jose, CA; Abgenix, Freemont, CA, and others) that express human immunoglobulins but not mouse IgM or Ig ⁇ .
transgenic mice contain human sequence transgenes that undergo V(D)J joining, heavy-chain class switching, and somatic mutation to generate a repertoire of human sequence immunoglobulins (Lonberg, et al., Nature 368:856-859 (1994)).
the light chain transgene can be derived, e.g., in part from a yeast artificial chromosome clone that includes nearly half of the germline human VK region.
the heavy-chain transgene can encode both human ⁇ and human ⁇ l(Fishwild, et al., Nature Biotechnology 14:845-851 (1996)) and/or ⁇ 3 constant regions.
Mice derived from appropriate genotopic lineages can be used in the immunization and fusion processes to generate fully human monoclonal antibodies to IL-17. Immunization
One or more immunization schedules using at least one IL-17 protein as an immunogen as generated according to know methods can be used to generate the IL- 17 human hybridomas.
the first several fusions can be performed after the following exemplary immunization protocol, but other similar known protocols can be used.
Several 14-20 week old female and/or surgically castrated transgenic male mice are immunized IP and/or ID with 1-1000 ⁇ g of recombinant human IL-17 protein emulsified with an equal volume of TITERMAX or complete Freund's adjuvant in a final volume of 100-400 ⁇ L (e.g., 200).
Each mouse can also optionally receive 1-10 ⁇ g in 100 ⁇ L physiological saline at each of 2 SQ sites.
the mice can then be immunized 1-7, 5- 12, 10-18, 17-25 and/or 21-34 days later IP (1-400 ⁇ g) and SQ (1-400 ⁇ g x 2) with IL-17 emulsified with an equal volume of TITERMAX or incomplete Freund's adjuvant.
Mice can be bled 12-25 and 25-40 days later by retro-orbital puncture without coagulant.
the blood is then allowed to clot at RT for one hour and the serum is collected and titered using an IL-17 EIA assay according to known methods. Fusions are performed when repeated injections do not cause titers to increase.
mice can be given a final IV booster injection of 1-400 ⁇ g IL-17 diluted in 100 ⁇ L physiological saline.
the mice can be euthanized by cervical dislocation and the spleens removed aseptically and immersed in 10 mL of cold phosphate buffered saline (PBS) containing 100 U/mL penicillin, 100 ⁇ g/mL streptomycin, and 0.25 ⁇ g/mL amphotericin B (PSA).
PBS cold phosphate buffered saline
PSA amphotericin B
the splenocytes are harvested by sterilely perfusing the spleen with PSA-PBS. The cells are washed once in cold
PSA-PBS counted using Trypan blue dye exclusion and resuspended in RPMI 1640 media containing 25 mM Hepes.
Fusion can be carried out at a 1 :1 to 1 :10 ratio of murine myeloma cells to viable spleen cells according to known methods, e.g., as known in the art.
CEN05189 PCT 62 myeloma cells can be pelleted together.
the pellet can then be slowly resuspended, over 30 seconds, in 1 mL of 50% (w/v) PEG/PBS solution (PEG molecular weight 1,450, Sigma) at 37 D C.
the fusion can then be stopped by slowly adding 10.5 mL of RPMI 1640 medium containing 25 mM Hepes (37 D C) over 1 minute.
the fused cells are centrifuged for 5 minutes at 500-1500 rpm.
HAT medium RPMI 1640 medium containing 25 mM Hepes, 10% Fetal Clone I serum (Hyclone), 1 mM sodium pyruvate, 4 mM L-glutamine, 10 ⁇ g/mL gentamicin, 2.5% Origen culturing supplement (Fisher), 10% 653-conditioned RPMI 1640/Hepes media, 50 ⁇ M 2-mercaptoethanol, 100 ⁇ M hypoxanthine, 0.4 ⁇ M aminopterin, and 16 ⁇ M thymidine) and then plated at 200 ⁇ L/well in fifteen 96-well flat bottom tissue culture plates. The plates are then placed in a humidified 37 D C incubator containing 5% CO 2 and 95% air for 7-10 days. Detection of Human IgG IL-17 antibodies in Mouse Serum
Solid phase EIA' s can be used to screen mouse sera for human IgG antibodies specific for human IL-17 protein. Briefly, plates can be coated with IL-17 protein at 2 ⁇ g/mL in PBS overnight. After washing in 0.15M saline containing 0.02% (v/v) Tween 20, the wells can be blocked with 1% (w/v) BSA in PBS, 200 ⁇ L/well for 1 hour at RT. Plates are used immediately or frozen at -20 D C for future use. Mouse serum dilutions are incubated on the IL-17 coated plates at 50 ⁇ L/well at RT for 1 hour.
the plates are washed and then probed with 50 ⁇ L/well HRP-labeled goat human IgG, Fc specific diluted 1 :30,000 in 1% BSA-PBS for 1 hour at RT.
the plates can again be washed and 100 ⁇ L/well of the citrate-phosphate substrate solution (0.1M citric acid and 0.2M sodium phosphate, 0.01% H 2 O 2 and 1 mg/mL OPD) is added for 15 minutes at RT.
Stop solution (4N sulfuric acid) is then added at 25 ⁇ L/well and the OD's are read at 490 nm via an automated plate spectrophotometer. Detection of Completely Human Immunoglobulins in Hybridoma Supernates
Hybridomas as above, can be simultaneously assayed for reactivity to IL-17 using a suitable RIA or other assay.
a suitable RIA or other assay For example, supernatants are incubated on goat human IgG Fc plates as above, washed and then probed with radiolabled IL-17 with appropriate counts per well for 1 hour at RT. The wells are washed twice with PBS and bound radiolabled IL-17 is quantitated using a suitable counter.
CEN05189 PCT 63 Human IgG l ⁇ IL- 17 secreting hybridomas can be expanded in cell culture and serially subcloned by limiting dilution. The resulting clonal populations can be expanded and cryopreserved in freezing medium (95% FBS, 5% DMSO) and stored in liquid nitrogen. Isotyping Isotype determination of the antibodies can be accomplished using an EIA in a format similar to that used to screen the mouse immune sera for specific titers. IL-17 protein can be coated on 96- well plates as described above and purified antibody at 2 ⁇ g/mL can be incubated on the plate for one hour at RT.
the plate is washed and probed with HRP labeled goat human IgGi or HRP labeled goat human IgG 3 diluted at 1 :4000 in 1% BSA-PBS for one hour at RT.
the plate is again washed and incubated with substrate solution as described above.
Binding characteristics for antibodies can be suitably assessed using an IL-17 capture EIA and BIAcore technology, for example.
Graded concentrations of purified human IL-17 antibodies can be assessed for binding to EIA plates coated with 2 ⁇ g/mL of IL-17 in assays as described above. The OD' s can be then presented as semi-log plots showing relative binding efficiencies.
Quantitative binding constants can be obtained, e.g., as follows, or by any other known suitable method.
a BIAcore CM-5 (carboxymethyl) chip is placed in a BIAcore 2000 unit.
HBS buffer (0.01 M HEPES, 0.15 M NaCl, 3 mM EDTA, 0.005% v/v P20 surfactant, pH 7.4) is flowed over a flow cell of the chip at 5 ⁇ L/minute until a stable baseline is obtained.
a solution (100 ⁇ L) of 15 mg of EDC (N-ethyl-N'-(3-dimethyl-aminopropyl)-carbodiimide hydrochloride) in 200 ⁇ L water is added to 100 ⁇ L of a solution of 2.3 mg of NHS (N-hydroxysuccinimide) in 200 ⁇ L water.
Forty (40) ⁇ L of the resulting solution is injected onto the chip.
Six ⁇ L of a solution of human IL-17 (15 ⁇ g/mL in 10 mM sodium acetate, pH 4.8) is injected onto the chip, resulting in an increase of ca. 500 RU.
the buffer is changed to TBS/Ca/Mg/BSA running buffer (20 mM Tris, 0.15 M sodium chloride, 2 mM calcium chloride, 2 mM magnesium acetate, 0.5% Triton X-100, 25 ⁇ g/mL BSA, pH 8.
Antibodies are dissolved in the running buffer at 33.33, 16.67, 8.33, and 4.17 nM.
the flow rate is adjusted to 30 ⁇ L/min and the instrument temperature to 25 D C.
Two flow cells are used for the kinetic runs, one on which IL-17 protein had been immobilized (sample) and a second, underivatized flow cell (blank).
120 ⁇ L of each antibody concentration is injected over the flow cells at 30 ⁇ L/min (association phase) followed by an uninterrupted 360 seconds of buffer flow (dissociation phase).
the surface of the chip is regenerated (Interleukin-13 muteins /antibody complex dissociated) by two sequential injections of 30 ⁇ L each of 2 M guanidine thiocyanate. Analysis of the data is done using BIA evaluation 3.0 or CLAMP 2.0, as known in the art.
each fusion is seeded in 15 plates (1440 wells/fusion) that yield several dozen antibodies specific for human IL- 17 protein. Of these, some are found to consist of a combination of human and mouse Ig chains. The remaining hybridomas secret IL-17 antibodies consisting solely of human heavy and light chains. Of the human hybridomas all are expected to be IgGlK.
IL-17 nucleotide sequence of sveral species was aligned to find areas of homology.
the species utilized were bovine (NM001008412), rat (XM236985), murine (NM010552) and human (NM002190).
NM001008412 bovine
rat rat
XM236985 murine
NM010552 human
human NM002190
the insert was subcloned using the TOPO-TA cloning kit (Invitrogen), plasmid DNA isolated, and sequenced. Analysis of the sequence data (Table 1) shows that like human IL-17, cyno IL-17 is 155 amino acid residues in length with a predicted molecular weight of 15.IkDa. Human and cyno IL-17 are 96% identical at the amino acid level (Table 2).
Table 1 Nucleotide and amino acid sequence of cyno IL-17. The putative signal sequence is
Table 2 Alignment of human and cyno IL-17. Human and cyno IL- 17 share 96% identity at the amino acid level.
HuIL17A (1) MTPGKTSLVSLLLLLSLBAIVKAGITIPRNPGCPNSBDKNFPRTVMVNLN
HuIL17A (101) NADGNVDYBMNSVPIQQEILVLRRBPPHCPNSFRLBKILVSVGCTCVTPI
HUIL17A (151) VHHVA (SEQ ID NO:4)

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CN110818789A (zh) *	2018-08-07	2020-02-21	三生国健药业（上海）股份有限公司	一种高纯度食蟹猴白细胞介素17a的纯化方法

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number	Priority date	Publication date	Assignee	Title
CN110818789A (zh) *	2018-08-07	2020-02-21	三生国健药业（上海）股份有限公司	一种高纯度食蟹猴白细胞介素17a的纯化方法
CN110818789B (zh) *	2018-08-07	2023-02-28	三生国健药业（上海）股份有限公司	一种高纯度食蟹猴白细胞介素17a的纯化方法

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