WO2003084477A2 - Mammalian cdr mimetibodies, compositions, methods and uses - Google Patents

Mammalian cdr mimetibodies, compositions, methods and uses Download PDF

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Publication number
WO2003084477A2
WO2003084477A2 PCT/US2003/009139 US0309139W WO03084477A2 WO 2003084477 A2 WO2003084477 A2 WO 2003084477A2 US 0309139 W US0309139 W US 0309139W WO 03084477 A2 WO03084477 A2 WO 03084477A2
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Prior art keywords
cdr
cdr mimetibody
according
mimetibody
variant
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PCT/US2003/009139
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French (fr)
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WO2003084477A3 (en
Inventor
George A. Heavner
David M. Knight
Bernard J. Scallon
John Ghrayeb
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Centocor, Inc.
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Priority to US60/368,791 priority
Application filed by Centocor, Inc. filed Critical Centocor, Inc.
Publication of WO2003084477A2 publication Critical patent/WO2003084477A2/en
Publication of WO2003084477A3 publication Critical patent/WO2003084477A3/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K16/00Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475Growth factors; Growth regulators
    • C07K14/505Erythropoietin [EPO]
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52Cytokines; Lymphokines; Interferons
    • C07K14/524Thrombopoietin, i.e. C-MPL ligand
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2318/00Antibody mimetics or scaffolds
    • C07K2318/10Immunoglobulin or domain(s) thereof as scaffolds for inserted non-Ig peptide sequences, e.g. for vaccination purposes

Abstract

The present invention relates to at least one novel EPO human CDR mimetibody or specified portion or variant, including isolated nucleic acids that encode at least one CDR mimetibody or specified portion or variant, CDR mimetibody or specified portion or variants, vectors, host cells, transgenic animals or plants, and methods of making and using thereof, including therapeutic compositions, methods and devices.

Description

MAMMALIAN CDR MIMETEBODIES, COMPOSITIONS, METHODS AND USES

BACKGROUND OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to mammalian CDR mimetibodies, specified portions and variants specific for bologically active proteins, fragment or ligands, CDR mimetibody encoding and complementary nucleic acids, host cells, and methods of making and using thereof, including therapeutic formulations, administration and devices.

RELATED ART

Recombinant proteins are an emerging class of therapeutic agents. Such recombinant therapeutics have engendered advances in protein formulation and chemical modification. Such modifications can potentially enhance the therapeutic utility of therapeutic proteins, such as by increaseing half lives (e.g., by blocking their exposure to proteolytic enzymes), enhancing biological activity, or reducing unwanted side effects. One such modification is the use of immunoglobulin fragments fused to receptor proteins, such as enteracept. Therapeutic proteins have also been constructed using the Fc domain to attempt to provide a longer half-life or to incorporate functions such as Fc receptor binding, protein A binding, and complement fixation.

One specific and vital role of the mammalian hematopoietic system is the production of erythrocytes, or red blood cells, which transport oxygen to the various tissues of the animal's body.

The process of producing erythrocytes ("erythropoiesis") occurs continuously throughout an animal's life span to offset erythrocyte destruction. The typical red blood cell has a relatively short life-span, usually 100 to 120 days. Erythropoiesis is a precisely controlled physiological mechanism whereby sufficient numbers of erythrocytes are produced to enable proper tissue oxygenation, but not so many as to impede circulation.

Erythropoiesis is now known to be primarily controlled by the polypeptide erythropoietin (EPO), an acidic glycoprotein. Erythropoietin is produced as the result of the expression of a single copy gene located in a chromosome of a mammal. The amino acid sequence for recombinant human EPO ("rHuEPO") is substantially identical to the amino acid sequence for EPO obtained from human urinary sources. However, the glycosylation of rHuEPO differs from that of urinary EPO and human serum EPO. In a healthy mammal, EPO is present in the blood plasma in very low concentrations, as the tissues are being sufficiently oxygenated by the existing number of circulating erythrocytes. The EPO present stimulates the production of new erythrocytes to replace those lost to the aging process. Additionally, EPO production is stimulated under conditions of hypoxia, wherein the oxygen supply to the body's tissues is reduced below normal physiological levels despite adequate perfiision of the tissue by blood. Hypoxia may be caused by hemorrhaging, radiation-induced erythrocyte destruction, various anemias, high altitude, or long periods of unconsciousness. In contrast, should the number of red blood cells in circulation exceed what is needed for normal tissue oxygenation, EPO production is reduced.

However, certain disease states involve abnormal erythropoiesis. Recombinant human EPO (rHuEPO) is being used therapeutically in a number of countries. In the United States, the U.S. Food and Drug Administration (FDA) has approved rHuEPO 's use in treating anemia associated with end- stage renal disease. Patients undergoing hemodialysis to treat this disorder typically suffer severe anemia, caused by the rupture and premature death of erythrocytes as a result of the dialysis treatment. EPO is also useful in the treatment of other types of anemia. For instance, chemotherapy-induced anemia, anemia associated with myelodysplasia, those associated with various congenital disorders, AIDS-related anemia, and prematurity-associated anemia, may be treated with EPO. Additionally, EPO may play a role in other areas, such as helping to more quickly restore a normal hematocrit in bone marrow transplantation patients, in patients preparing for autologous blood transfusions, and in patients suffering from iron overload disorders. Erythropoietin (EPO) is a glycoprotein hormone composed of 165 amino acids and four carbohydrate chains that functions as the primary regulator of erythropoiesis by binding to a specific receptor on the surface of erythrocyte precursor cells. This binding signals their proliferation and differentiation into mature red blood cells. The erythropoietin receptor is a 484-amino acid glycoprotein with high affinity for erythropoietin. For the erythropoietin receptor, ligand-induced homodimerization is the key event that governs activation .

Erythropoietin has a relatively short half-life. Intravenously administered erythropoietin is eliminated at a rate consistent with first order kinetics with a circulating half-life ranging from approximately 3 to 4 hours in patients with CRF. Within the therapeutic dose range, detectable levels of plasma erythropoietin are maintained for at least 24 hours. After subcutaneous administration of erythropoietin, peak serum levels are achieved within 5-24 hours and decline slowly thereafter. The C max and t lA after administration of erythropoietin were 1.80 ± 0.7 U/mL and 19.0 ± 5.9 hours, respectively.

Starting doses of erythropoietin range from 50-150 U/kg three times weekly. The dosage of erythropoietin must be individualized to maintain the hematocrit within the suggested target range. For surgery patients the recommended dose of erythropoietin is 300 U/kg/day s.c. for 10 days before surgery, on the day of surgery, and for 4 days after surgery or alternatively 600 U/kg s.c. in once weekly doses (21, 14 and 7 days before surgery) plus a fourth dose on the day of surgery.

Small peptidomimetics of erythropoietin were identified by several groups through screening of random phage display peptide libraries for affinity to the erythropoietin receptor. These sequences have no homology with erythropoietin. In functional assays several of these peptides showed activity, but only 1/100,000* that of recombinant erythropoietin. Although several attempts have been made to increase the potency of these peptides by preparing covalent dimers or multimers of peptidomimetics, these compounds are still 1,000 - 10,000 fold less active than erythropoietin on a molar basis. Peptide sequences from erythropoietin have also been claimed as agonistic. Increased activity of dimerized sequences comprising any or all of the native erythropoietin sequence has also been reported. These compounds have little or no oral bioavailability and their activity does not make them economically viable at this time.

Accordingly, there is a need to provide improved and/or modified versions of therapeutic proteins, which overcome one more of these and other problems known in the art.

SUMMARY OF THE INVENTION

The present invention provides isolated human mimetibodies, including modified immunoglobulins, cleavage products and other specified portions and variants thereof, as well as CDR mimetibody compositions, encoding or complementary nucleic acids, vectors, host cells, compositions, formulations, devices, transgenic animals, transgenic plants, and methods of making and using thereof, as described and/or enabled herein, in combination with what is known in the art.

The present invention also provides at least one isolated CDR mimetibody or specified portion or variant as described herein and/or as known in the art. The present invention provides, in one aspect, isolated nucleic acid molecules comprising, complementary, or hybridizing to, a polynucleotide encoding specific mimetibodies or specified portions or variants thereof, comprising at least one specified sequence, domain, portion or variant thereof. The present invention further provides recombinant vectors comprising at least one of said isolated CDR mimetibody nucleic acid molecules, host cells containing such nucleic acids and/or recombinant vectors, as well as methods of making and/or using such CDR mimetibody nucleic acids, vectors and/or host cells.

At least one CDR mimetibody or specified portion or variant of the invention mimics the binding to at least one ligand, or has at least one biological activity of, at least one protein, subunit, fragment, portion or any combination thereof. The at least one CDR mimetibody or specified portion' or variant can comprise at least one protein ligand binding' region ("LBR") that is preferably comprised of at least one extracellular, soluble, hydrophillic, external or cytoplasmic portion of said protein, which further comprises at least one amino acid from at least one complementarity determing region (CDR) of a heavy or light chain variable region.. The at least one CDR mimetibody or specified portion or variant can optionally comprise at least one LBR comprising at least a specified portion of at least one modified or replaced CDR (e.g., CDR1, CDR2 or CDR3 of the heavy or light chain variable region) and/or at least one framework region. The at least one CDR mimetibody or specified portion or variant amino acid sequence can further optionally comprise at least one specified substitution, insertion or deletion. The present invention also provides at least one isolated CDR mimetibody or specified portion or variant as described herein and/or as known in the art, wherein the CDR mimetibody or specified portion or variant has at least one activity, such as, but not limited to known biological activities. A CDR mimetibody can thus be screened for a corresponding activity according to known methods, such as at least one neutralizing activity towards a protein or fragment thereof. The present invention also provides at least one composition comprising (a) an isolated CDR mimetibody or specified portion or variant encoding nucleic acid and/or CDR mimetibody as described herein; and (b) a suitable carrier or diluent. The carrier or diluent can optionally be pharmaceutically acceptable, according to known methods. The composition can optionally further comprise at least one further compound, protein or composition. The present invention also provides at least one method for expressing at least one CDR mimetibody or specified portion or variant in a host cell, comprising culturing a host cell as described herein and/or as known in the art under conditions wherein at least one CDR mimetibody or specified portion or variant is expressed in detectable and/or recoverable amounts.

The present invention further provides at least one CDR mimetibody, specified portion or variant in a method or composition, when administered in a therapeutically effective amount, for modulation, for treating or reducing the symptoms of a(n) anemia; a(n) immune/autoimmune; and/or a(n) cancer/infecteous, as needed in many different conditions, such as but not limited to, prior to, subsequent to, or during a related disease or treatment condition, as known in the art.

The present invention further provides at least one CDR mimetibody, specified portion or variant in a method or composition, when administered in a therapeutically effective amount, for modulation, for treating or reducing the symptoms of, at least one immune, cardiovascular, infectious, malignant, and/or neurologic disease in a cell, tissue, organ, animal or patient and/or, as needed in many different conditions, such as but not limited to, prior to, subsequent to, or during a related disease or treatment 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 of a therapeutically or prophylactically effective amount of at least one CDR mimetibody or specified portion or variant, according to the present invention.

The present invention further provides at least one anti-idiotype antibody to at least one CDR mimetibody 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 complimetarity determing 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 a CDR mimetibody of the present invention. A CDR mimetibody 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 CDR mimetibody anti- idiotype antibody, comprising at least one specified sequence, domain, portion or variant thereof. The present invention further provides recombinant vectors comprising said CDR mimetibody 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 antiobody nucleic acids, vectors and/or host cells. The present invention also provides at least one method for expressing at least one CDR mimetibody, or CDR mimetibody anti-idiotype antibody, in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one CDR mimetibody or anti-idiotype antibody is expressed in detectable and/or recoverable amounts.

The present invention also provides at least one composition comprising (a) an isolated CDR mimetibody encoding nucleic acid and/or CDR mimetibody as described herein; and (b) a suitable carrier or diluent. The carrier or diluent can optionally be pharmaceutically acceptable, according to known carriers or diluents. The composition can optionally further comprise at least one further compound, protein or composition.

The present invention further provides at least one CDR mimetibody method or composition, for administering a therapeutically effective amount to modulate or treat at least one protein 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 of a therapeutically or prophylactically effective amount of at least one CDR mimetibody, according to the present invention. The present invention further provides at least one CDR mimetibody method or composition, for diagnosing at least one protein 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 CDR mimetibody, according to the present invention.

In one aspect, the present invention provides at least one isolated mammalian CDR mimetibody, comprising at least one variable region comprising at least a portion of at least one CDR that further comprises at least one of SEQID NOS: 1-1109.

In another aspect, the present invention provides at least one isolated mammalian CDR mimetibody, comprising either (i) all of the heavy chain complimentarity determining regions (CDR); or (ii) all of the light chain CDR amino acids sequences.

In another aspect, the present invention provides at least one isolated mammalian CDR mimetibody, comprising at least a portion of at least one heavy chain or light chain CDR having inserted therein at least one amino acid sequence of SEQID NOS: 1-1110 In other aspect the present invention provides at least one isolated mammalian CDR mimetibody, comprising at least one human CDR, wherein the CDR mimetibody specifically binds at least one epitope comprising at least 1-3 of at least one ligand or binding region which ligand binds to at least a portion of at least one of SEQID NOS: 1-1109.

The at least one CDR mimetibody can optionally further at least one of: bind protein 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 neutralize at least one activity of at least one protein or portion thereof. Also provided is an isolated nucleic acid encoding at least one isolated mammalian CDR mimetibody; 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 COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative, immortalized or transformed cell thereof. Also provided is a method for producing at least one CDR mimetibody, comprising translating the CDR mimetibody encoding nucleic acid under conditions in vitro, in vivo or in situ, such that the CDR mimetibody is expressed in detectable or recoverable amounts. Also provided is a composition comprising at least one isolated mammalian CDR mimetibody 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 antirheu atic, a muscle relaxant, a narcotic, a non- steroid anti-inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local anethetic, 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 epinephrine or analog, a cytokine, or a cytokine antagonist.

The present invention further provides an anti-idiotype antibody or fragment that specifically binds at least one isolated mammalian protein antibody of the present invention.

Also provided is a method for diagnosing or treating a protein 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 CDR mimetibody of the invention with, or to, the cell, tissue, organ or animal. The method can optionally further comprise using an effective amount of 0.001-50 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, 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. 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, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, 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 epinephrine or analog, a cytokine, or a cytokine antagonist.

Also provided is a medical device, comprising at least one isolated mammalian CDR mimetibody of the invention, wherein the device is suitable to contacting or administerting the at least one CDR mimetibody by at least one mode selected from parenteral, subcutaneous, intramuscular, intravenous, intrarticular, intrabronchial, intraabdominal, mtracapsular, 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.

Also provided is 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 CDR mimetibody of the present invention. The article of manufacture can optionally comprise having the container as a component of a 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 delivery device or system.

Also provided is a method for producing at least one isolated mammalian CDR mimetibody 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 CDR mimetibody. Further provided in the present invention is at least one CDR mimetibody produced by the above method.

The present invention also provides at least one method for expressing at least one CDR mimetibody, or anti-idiotype antibody, in a host cell, comprising culturing a host cell as described herein under conditions wherein at least one CDR mimetibody is expressed in detectable and/or recoverable amounts.

The present invention further provides any invention described herein.

DESCRIPTION OF THE INVENTION

The present invention provides isolated, recombinant and/or synthetic mimetibodies or specified portions or variants, as well as compositions and encoding nucleic acid molecules comprising at least one polynucleotide encoding at least one CDR mimetibody. Such mimetibodies or specified portions or variants of the present invention comprise specific CDR mimetibody sequences, domains, fragments and specified variants thereof, and methods of making and using said nucleic acids and mimetibodies or specified portions or variants, including therapeutic compositions, methods and devices.

As used herein, a "CDR mimetibody," "CDR mimetibody portion," or "CDR mimetibody fragment" and/or "CDR mimetibody variant" and the like mimics, has or simulates at least one ligand binding or at least one biological activity of at least one protein, such as ligand binding or activity in vitro, in situ and/or preferably in vivo. For example, a suitable CDR mimetibody, specified portion or variant of the present invention can bind at least one protein ligand and includes at least one protein ligand, receptor, soluble receptor, and the like. A suitable CDR mimetibody, specified portion, or variant can also modulate, increase, modify, activate, at least one protein receptor signaling or other measurable or detectable activity. Mimetibodies useful in the methods and compositions of the present invention are characterized by suitable affinity binding to protein ligands or receptors and optionally and preferably having low toxicity. In particular, a CDR mimetibody, where the individual components, such as the variable region, constant region and framework, or any portion thereof (e.g., a portion of the J, D or V rgions of the variable heavy or light chain; the hinge region, the constant heavy chain or light chain, and the like) individually and/or collectively optionally and preferably possess low immunogenicity, is useful in the present invention. The mimetibodies that can be used in the invention are optionally characterized by their ability to treat patients for extended periods with good to excellent alleviation of symptoms and low toxicity. Low immunogenicity and/or high affinity, as well as other undefined properties, may contribute to the therapeutic results achieved. "Low immunogenicity" is defined herein as raising significant HAMA, HACA or HAHA responses in less than about 75%, or preferably less than about 50, 45, 40, 35, 30, 35, 20, 15, 10, 9, 8, 7, 6, 5, 4, >,' 2, and/or 1 % of the patients treated and/or raising low titres in the patient treated (less than about 300, preferably less than about 100 measured with a double antigen enzyme immunoassay) (see, e.g., Elliott et ah, Lancet 344: 1125-1127 (1994)).

1 Utility

The isolated nucleic acids of the present invention can be used for production of at least one CDR mimetibody, fragment or specified variant thereof, which can be used to effect in an cell, tissue, organ or animal (including mammals and humans), to modulate, treat, alleviate, help prevent the incidence of, or reduce the symptoms of, at least one protein related 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, a(n) anemia; a(n) immune/autoimmune; and/or a(n) cancer/infecteous, as well as other known or specified protein related conditions.

Such a method can comprise administering an effective amount of a composition or a pharmaceutical composition comprising at least one CDR mimetibody or specified portion or variant 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 0.0001 to 500 mg/kg per single or multiple administration, or to achieve a serum concentration of 0.001-5000 μg/ml serum concentration per single or multiple adminstration, or any effective range or value therein, as done and determined using known methods, as described herein or known in the relevant arts.

Citations

All publications or patents cited herein are entirely incorporated herein by reference as they show the state of the art at the time of the present invention and/or to provide description and enablement of the present invention. Publications refer to any scientific or patent publications, or any other information available in any media format, including all recorded, electronic or printed formats.

The following references are entirely incorporated herein by reference: Ausubel, et al., ed., Current

Protocols in Molecular Biology, John Wiley & Sons, Inc., NY, NY (1987-2000); Sambrook, et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor, NY (1989); Harlow and

Lane, Antibodies, a Laboratory Manual, Cold Spring Harbor, NY (1989); Colligan, et al., eds., Current

Protocols in Immunology, John Wiley & Sons, Inc., NY (1994-2000); Colligan et al., Current Protocols in Protein Science, John Wiley & Sons, NY, NY, (1997-2000).

Mimetibodies of the Present Invention

The term "CDR mimetibody "is intended to encompass any antibody fragment where a protein is inserted into, or replaces a portion of, one or more CDR regions (e.g., CDR1, CDR2 or CDR3, heavy chain or light chain), the combination of the protein and the CDR or portion thereof designated a ligand binding region (LBR) of the CDR mimetibody. Such mimetibodies include those that mimic the structure and/or function of at least one protein or fragment or analog. Functional protions of at least one CDR mimetibody of the present invention include at least one ligand- or antigen-binding fragment that bindd to at least one protein ligand or receptor. For example, CDR mimetibody fragments capable of binding to human ligands or portions thereof, including, but not limited to mimetics of Fv, Fab, Fab' and F(ab')2 fragments, and any other portion of an antibody, further comprising at least one ligand binding region (LBR) are encompassed by the present invention.

In particular, mimetibodies comprise at least one ligand binding region (LBR) that corresponds to at least one portion of at least one complementarity determining region (CDR, e.g., CDR1, CDR2 or CDR3 of HC or LC variable region) of at least one antibody or fragment or portion thereof where at least one ligand protein is inserted into or replaces at least a portion of at least one CDR of the antibody or portion thereof. Such mimetibodies of the present invention thus provide at least one suitable property as compared to known proteins, such as, but not limited to, at least one of increased half-life, increased activity, more specific activity, increased avidity, increased or descrease off rate, a selected or more suitable subset of activities, less immungenicity, increased quality or duration of at least one desired therapeutic effect, less side effects, and the like. Such fragments can be produced by enzymatic cleavage, synthetic or recombinant techniques, as known in the art and/or as described herein. For example, papain or pepsin cleavage can generate CDR mimetibody Fab or F(ab')2 fragments, respectively. Other proteases with the requisite substrate specificity can also be used to generate Fab or F(ab')2 fragments or portions thereof. Mimetibodies can also be produced in a variety of truncated forms using antibody genes in which one or more stop codons have been introduced upstream of the natural stop site. For example, a chimeric 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 mimetibodies can be joined together chemically by conventional techniques, or can be prepared as a contiguous protein using genetic engineering techniques. For example, a nucleic acid encoding the variable and constant regions of a human antibody chain can be expressed to produce a contiguous protein for use in mimetibodies of the present invention. See, e.g., Ladner et al, U.S. Patent No. 4,946,778 and Bird, R.E. et al, Science, 242: 423-426 (1988), regarding single chain mimetibodies.

As used herein, the term "human antibody" refers to an antibody in which substantially every part of the protein (e.g., LBR, framework, C , CH domains (e.g., CHI, CH2, CH3), hinge, (VL, VH)) is substantially non-immunogenic, with only minor sequence changes or variations. Such changes or variations optionally and preferably retain or reduce the immunogenicity in humans relative to non- modified human antibodies, or mimetibodies of the prsent invention. Thus, a human antibody and corresponding CDR mimetibody of the present invention is distinct from a chimeric or humanized antibody. It is pointed out that a human antibody and CDR mimetibody can be produced by a non- human animal or cell that is capable of expressing functionally rearranged human immunoglobulin (e.g., heavy chain and/or light chain) genes, and for a CDR mimetibody, wherein at least one Ig CDR is replaced by an LBR of at least one ligand protein or fragment.

Human mimetibodies that are specific for at least one protein ligand or receptor thereof can be designed against an appropriate ligand, such as isolated and/or protein receptor or ligand, or a portion thereof (including synthetic molecules, such as synthetic peptides). Preparation of such mimetibodies are performed using known techniques to identify and characterize ligand binding regions or sequences of at least one protein or portion thereof.

In a preferred embodiment, at least one CDR mimetibody or specified portion or variant of the present invention is produced by at least one cell line, mixed cell line, immortalized cell or clonal population of immortalized and/or cultured cells. Immortalized protein producing cells can be produced using suitable methods. Preferably, the at least one CDR mimetibody or specified portion or variant is generated by providing nucleic acid or vectors comprising DNA derived or having a substantially similar sequence to, at least one human immunoglobulin locus that is functionally rearranged, or which can undergo functional rearrangement, and which further comprises at least one CDR that has been replaced by at least one LBR specific for at least one protein ligand, the resulting nucleic acid encoding at least one CDR mimetibody, or specified portion or variant according to the present invention.

The term "functionally rearranged," as used herein refers to a segment of nucleic acid from an immunoglobulin locus that has undergone N(D)J recombination, thereby producing an immunoglobulin gene that encodes an immunoglobulin chain (e.g., heavy chain, light chain), or any portion thereof. A functionally rearranged immunoglobulin gene can be directly or indirectly identified using suitable methods, such as, for example, nucleotide sequencing, hybridization (e.g., Southern blotting, Northern blotting) using probes that can anneal to coding joints between gene segments or enzymatic amplification of immunoglobulin genes (e.g., polymerase chain reaction) with primers that can anneal to coding joints between gene segments. Whether a cell produces an CDR mimetibody or portion or variant comprising a particular variable region or a variable region comprising a particular sequence (e.g., at least one ligand binding region (LBR) sequence) can also be determined using suitable methods. Mimetibodies, specified portions and variants of the present invention can also be prepared using at least one CDR mimetibody or specified portion or variant encoding nucleic acid to provide transgenic animals or mammals, such as goats, cows, horses, sheep, and the like, that produce such mimetibodies or specified portions or variants in their milk. Such animals can be provided using known methods as applied for antibody encoding sequences. 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.

Mimetibodies, specified portions and variants of the present invention can additionally be prepared using at least one CDR mimetibody or specified portion or variant encoding nucleic acid to provide transgenic plants and cultured plant cells (e.g., but not limited to tobacco and maize) that produce such mimetibodies, specified portions or variants in the plant parts or in cells cultured therefrom. As a non-limiting example, 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. Also, 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. See, e.g., Hood et al., Adv. Exp. Med. Biol. 464:127-147 (1999) and references cited therein. Antibodies have also been produced in large amounts from transgenic plant seeds including antibody fragments, such as single chain mimetibodies (scFv's), including tobacco seeds and potato tubers. See, e.g., Conrad et al., Plant Mol. Biol. 38:101-109 (1998) and references cited therein. Thus, mimetibodies, specified portions and variants of the present invention can also be produced using transgenic plants, according to know methods. See also, e.g., Fischer et al., Biotechnol. Appl. Biochem. 30:99-108 (Oct., 1999), Ma et al., Trends Biotechnol. 13:522-7 (1995); Ma et al., Plant Physiol. 109:341-6 (1995); Whitelam et al., Biochem. Soc. Trans. 22:940-944 (1994); and references cited therein. The above references are entirely incorporated herein by reference. The mimetibodies of the invention can bind human protein ligands with a wide range of affinities (KD). In a preferred embodiment, at least one human CDR mimetibody of the present invention can optionally bind at least one protein ligand with high affinity. For example, at least one CDR mimetibody of the present invention can bind at least one protein ligand with a KD equal to or less than about 10"9 M or, more preferably, with a KD equal to or less than about 0.1-9.9 (or any range or value therein) X 10"10 M, 10"11, 10"12 , 10"13 or any range or value therein.

The affinity or avidity of a CDR mimetibody for at least one protein ligand can be determined experimentally using any suitable method, e.g., as used for determing antibody-antigen binding affinity or avidity. (See, for example, Berzofsky, et al, "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 CDR mimetibody-ligand interaction can vary if measured under different conditions (e.g., salt concentration, pH). Thus, measurements of affinity and other ligand-binding parameters (e.g., KD, Ka, K ) are preferably made with standardized solutions of CDR mimetibody and ligand, 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 90- 100% of the contiguous amino acids of at least one of SEQID NOS:1-1009 as well as at least one portion of an antibody, wherein the above sequences are inserted into or replace at least one CDR of at least one antibody to provide a CDR mimetibody of the present invention, further comprising specified fragments, variants or consensus sequences thereof, or a deposited vector comprising at least one of these sequences, a nucleic acid molecule of the present invention encoding at least one CDR mimetibody or specified portion or variant 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 combination thereof. The DNA can be triple-stranded, double-stranded or single-stranded, or any combination thereof. Any portion of at least one strand of 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 LBR, as LBR1, LBR2 and/or LBR3 of at least one heavy chain or light chain; nucleic acid molecules comprising the coding sequence for a CDR mimetibody or specified portion or variant; 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 CDR mimetibody as described herein and/or as known in the art. Of course, the genetic code is well known in the art. Thus, it would be routine for one skilled in the art to generate such degenerate nucleic acid variants that code for specific CDR mimetibody or specified portion or variants of the present invention. See, e.g., Ausubel, et al., supra, and such nucleic acid variants are included in the present invention.

In another aspect, the invention provides isolated nucleic acid molecules encoding a(n) CDR mimetibody or specified portion or variant having an amino acid sequence as encoded by the nucleic acid contained in the plasmid deposited as designated clone names and ATCC Deposit Nos. , respectively, deposited on

As indicated herein, nucleic acid molecules of the present invention which comprise a nucleic acid encoding a CDR mimetibody or specified portion or variant can include, but are not limited to, those encoding the amino acid sequence of a CDR mimetibody fragment, by itself; the coding sequence for the entire CDR mimetibody or a portion thereof; the coding sequence for a CDR mimetibody, fragment or portion, as well as additional sequences, such as the coding sequence of at least one signal leader or fusion peptide, with or without the aforementioned additional coding sequences, such as at least one intron, together with additional, non-coding sequences, including but not limited to, non- coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals (for example - ribosome binding and stability of mRNA); an additional coding sequence that codes for additional amino acids, such as those that provide additional functionalities. Thus, the sequence encoding a CDR mimetibody or specified portion or variant can be fused to a marker sequence, such as a sequence encoding a peptide that facilitates purification of the fused CDR mimetibody or specified portion or variant comprising a CDR mimetibody fragment or portion.

Polynucleotides Which Selectively Hybridize to a Polynucleotide as Described Herein

The present invention provides isolated nucleic acids that hybridize under selective hybridization conditions to a polynucleotide disclosed herein, or others disclosed herein, including specified variants or portions thereof. Thus, the polynucleotides of this embodiment can be used for isolating, detecting, and/or quantifying nucleic acids comprising such polynucleotides.

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 40-99% sequence identity and can be employed to identify orthologous or paralogous sequences.

Optionally, polynucleotides of this invention will encode at least a portion of a CDR mimetibody or specified portion or variant 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 a CDR mimetibody or specified portion or variant of the present invention. See, e.g., Ausubel, supra; Colligan, supra, each entirely incorporated herein by reference.

Construction of Nucleic Acids

The isolated nucleic acids of the present invention can be made using (a) recombinant methods, (b) synthetic techniques, (c) purification techniques, or combinations thereof, as well-known in the art.

The nucleic acids can conveniently comprise sequences in addition to a polynucleotide of the present invention. For example, 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. Also, translatable sequences can be inserted to aid in the isolation of the translated polynucleotide of the present invention. For example, 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

The isolated nucleic acid compositions of this invention, such as 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. In some embodiments, oligonucleotide probes that selectively hybridize, under suitable stringency 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). Synthetic Methods for Constructing Nucleic Acids

The isolated nucleic acids of the present invention can also be prepared by direct chemical synthesis by known methods (see, e.g., Ausubel, et al., supra). 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. 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.

Recombinant Expression Cassettes 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 a CDR mimetibody or specified portion or variant of the present invention, can be used to construct a recombinant expression cassette that can be introduced into at least one desired host cell. A recombinant expression 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.

In some embodiments, 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. For example, endogenous promoters can be altered in vivo or in vitro by mutation, deletion and/or substitution, as known in the art. A polynucleotide of the present invention can be expressed in either sense or anti-sense orientation as desired. It will be appreciated that control of gene expression in either sense or anti-sense orientation can have a direct impact on the observable characteristics. Another method of suppression is sense suppression. Introduction of nucleic acid configured in the sense orientation has been shown to be an effective means by which to block the transcription of target genes. Vectors And Host Cells 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 CDR mimetibody or specified portion or variant 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. Generally, a plasmid vector is introduced into a cell using suitable known methods, such as electroporation and the like, other known methods include the use of the vector as 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 optionally for at least one of 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 PatNos. 4,399,216; 4,634,665; 4,656,134; 4,956,288; 5,149,636; 5,179,017, ampicillin, neomycin (G418), mycophenolic acid, or glutamine synthetase (GS, US PatNos. 5,122,464; 5,770,359; 5,827,739) resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria or prokaryotics (the above patents are entirely incorporated hereby by reference). Appropriate culture mediums and conditions for the above-described host cells are known in the art. Suitable vectors will be readily apparent to the skilled artisan. 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 CDR mimetibody or specified portion or variant 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 a CDR mimetibody or specified portion or variant to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties can be added to a CDR mimetibody or specified portion or variant of the present invention to facilitate purification. Such regions can be removed prior to final preparation of a CDR mimetibody 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.

Illustrative of cell cultures useful for the production of the mimetibodies, 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-1 (e.g., ATCC CRL 1650), COS-7 (e.g., ATCC CRL-1651), HEK293, BHK21 (e.g., ATCC CRL-10), CHO (e.g., ATCC CRL 1610) and BSC-1 (e.g., ATCC CRL-26) cell lines, hepG2 cells, P3X63Ag8.653, SP2/0-Agl4, 293 cells, HeLa cells and the like, which are readily available from, for example, American Type Culture Collection, Manassas, Va. Preferred host cells include cells of lymphoid origin such as myeloma and lymphoma cells. Particularly preferred host cells are P3X63Ag8.653 cells (ATCC Accession Number CRL-1580) and SP2/0-Agl4 cells (ATCC Accession Number CRL-1851). In a particularly preferred embodiment, 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 PatNos. 5,168,062; 5,385,839), an HSV tk promoter, a pgk (phosphoglycerate kinase) promoter, an EF-1 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 tr.anscriptional terminator sequences. See, e.g., Ausubel et al., supra; Sambrook, et al., supra. Other cells useful for production of nucleic acids or proteins of the present invention are known and/or available, for instance, from the American Type Culture Collection Catalogue of Cell Lines and Hybridomas (www.atcc.org) or other known or commercial sources. When eukaryotic host cells are employed, 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 VP1 intron from SV40 (Sprague, et al., J. Virol. 45:773-781 (1983)). Additionally, gene sequences to control replication in the host cell can be incorporated into the vector, as known in the art.

Purification of an CDR mimetibody or specified portion or variant Thereof

A CDR mimetibody or specified portion or variant 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 ("HPLC") 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, (1997-2000), e.g., Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein by reference. Mimetibodies or specified portions or variants 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 CDR mimetibody or specified portion or variant 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.

MIMETIBODIES- SPECIFIED FRAGMENTS AND/OR VARIANTS

The isolated mimetibodies of the present invention comprise a CDR mimetibody or specified portion or variant encoded by any one of the polynucleotides of the present invention as discussed more fully herein, or any isolated or prepared CDR mimetibody or specified portion or variant thereof.

Preferably, the CDR mimetibody or ligand-binding portion or variant binds at least one protein ligand or receptor, and, thereby provides at least one biological activity of the corresponding protein or a fragment thereof. Different therapeutically or diagnostically significant proteins are well known in the art and suitable assays or biological activities of such proteins are also well known in the art. Peptides. Any number of peptides may be used in conjunction with the present invention.

Non limiting examples can be

Of particular interest are peptides that mimic the activity of EPO, TPO, growth hormone, G-CSF, GM-CSF, IL-lra, leptin, CTLA4, TRAIL, TGF- , and TGF-β. Peptide antagonists are also of interest, particularly those antagonistic to the activity of TNF, leptin, any of the interleukins (IL-1 - D -23, etc.), and proteins involved in complement activation (e.g., C3b). Targeting peptides are also of interest, including tumor-homing peptides, membrane-transporting peptides, and the like. All of these classes of peptides may be discovered by methods described in the references cited in this specification and other references. A particularly preferred group of peptides are those that bind to cytokine receptors.

Cytokines have recently been classified according to their receptor code. See Inglot (1997), Archivum Immunologiae e Therapiae Experimentalis 45: 353-7, which is hereby incorporated entirely by reference.

Non-limiting examples of suitable peptides for this invention appear in Tables 1 through 17 below. These peptides may be prepared by methods disclosed and/or known in the art. Single letter amino acid abbreviations are used in most cases. The X in these sequences (and throughout this specification, unless specified otherwise in a particular instance) means that any of the 20 naturally occurring amino acid residues or know derivatives thereof may be present, or any know modified amino acid thereof. Any of these peptides may be linked in tandem (i.e., sequentially), with or without linkers, and a few tandemlinked examples are provided in the table. Linkers are listed as "Δ" and may be any of the linkers described herein. Tandem repeats and linkers are shown separated by dashes for clarity. Any peptide containing a cysteinyl residue may optionally be cross-linked with another Cys-containing peptide, either or both of which may be linked to a vehicle. A few crosslinked examples are provided in the table. Any peptide having more than one Cys residue may form an intrapeptide disulfide bond, as well; see, for example, EPO-mimetic peptides in Table 1. A few examples of intrapeptide disulfide-bonded peptides are specified in the table. Any of these peptides may be derivatized as described herein, and a few derivatized examples are provided in the table. For derivatives in which the carboxyl terminus may be capped with an amino group, the capping amino group is shown as -NH2. For derivatives in which amino acid residues are substituted by moieties other than amino acid residues, the substitutions are denoted by a δ, which signifies any of the moieties known in the art, e.g., as described in Bhatnagar et al. (1996), J. Med. Chem. 39: 3814-9 and Cuthbertson et al. (1997), J. Med. Chem. 40:2876-82, which are entirely incorporated by reference. The J substituent and the Z substituents (Z5, Z_, ... Z4o) are as defined in U.S. Pat. Nos. 5,608,035, 5,786,331, and 5,880,096, which are entirely incorporated herein by reference. For the EPO-mimetic sequences (Table 1), the substituents X2 through X11 and the integer "n" are as defined in WO 96/40772, which is entirely incorporated by reference. The substituents "Ψ" "Θ," and "+" are as defined in Sparks et al. (1996), Proc. Natl. Acad. Sci. 93: 1540-4, which is entirely incorporated by reference. X4, X5, X6, and X7 are as defined in U.S. Pat. No. 5,773,569, which is hereby entirely incorporated by reference, except that: for integrin-binding peptides, XI, X2, X3, X4, X5, X6, X7, and X8 (Table 6), are as defined in PCT applications WO 95/14714, published June 1, 1995 and WO 97/08203, published March 6,1997, which are also entirely incorporated by reference; and for VIP- mimetic peptides (Table 9), Xi, Xi', Xi", X2, X3, X4, X5, X6, and Z; and the integers m and n are as defined in WO 97/40070, published October 30,1997, which is also entirely incorporated herein by reference. Xaa and Yaa below are as defined in WO 98/09985, published March 12,1998, which is entirely incorporated herein by reference. AAi, AA2, ABi, AB2, and AC are as defined in International application WO 98/53842, published December 3, 1998, which is entirely incorporated by reference. X1, X2, X3, and X4 in Table 14 only are as, defined in European application EP 0 911 393, published April 28,1999, entirely incorporated herein by reference. Residues appearing in boldface are D-amino acids, but can be optionally L-amino acids. All peptides are linked through peptide bonds unless otherwise noted. Abbreviations are listed at the end of this specification. In the "SEQID NO." column, "NR" means that no sequence listing is required for the given sequence.

Table 1-EPO-mimetic peptide sequences

Sequence/structure: SEQID NO:

YXCXXGPXTWXCXP 1

YXCXXGPXTWXCXP-YXCXXGPXTWXCXP 2 YXCXXGPXTWXCXP-A-YXCXXGPXTWXCXP 3 YXCXXGPXTWXCXP-Δ-ε-amine) 4

\

K / YXCXXGPXTWXCXP-Δ- (α-amine) 4 GGTYSCHFGPLTWVCKPQGG 5 GGDYHCRMGPLTWVCKPLGG 6 GGVYACRMGPITWVCSPLGG 7 VGNYMCHFGPITWVCRPGGG 8 GGLYLCRFGPVTWDCGYKGG 9 GGTYSCHFGPLTWVCKPQGG- 10

GGTYSCHFGPLTWVCKPQGG -Δ-GGTYSCHFGPLTWVCKPQGG 11 GGTYSCHFGPLTWVCKPQGGSSK 12 GGTYSCHFGPLTWVCKPQGGSSK 13 GGTYSCHFGPLTWVCKPQGGSSK 14

GGTYSCHFGPLTWVCKPQGGSSK-Δ-GGTYSCHFGPLTWVCKPQGGSSK GGTYSCHFGPLTWVCKPQGGSS -Δ-ε-amine)

\

K

/

GGTYSCHFGPLTWVCKPQGGSS-Δ- (α-amine) 15

GGTYSCHFGPLTWVCKPQGGSSK(-Δ-biotin) 16

CX4X5GPX6TWX7C 17 GGTYSCHGPLTWNCKPQGG 18

VGΝYMAHMGPITWVCRPGG 19

GGPHHVYACRMGPLTWIC 20

GGTYSCHFGPLTWVCKPQ 21 GGLYACHMGPMTWVCQPLRG 22

TIAQYICYMGPETWECRPSPKA 23

YSCHFGPLTWVCK 24

YCHFGPLTWVC 25

X3X4X5GPX6TWX7X8 26 YX2X3X4X5GPX6TWX7Xg 27 ιY X3X X5 6 7 8 9 ιo π 28

XιYX2CX4X5GPX6TWX7CX9XιoXιι 29

GGLYLCRFGPVTWDCGYKGG 30

GGTYSCHFGPLTWVCKPQGG 31 GGDYHCRMGPLTWVCKPLGG 32

VGΝYMCHFGPITWVCRPGGG 33

GGVYACRMGPITWVCSPLGG 34

VGΝYMAHMGPITWVCRPGG 35

GGTYSCHFGPLTWVCKPQ 36 GGLYACHMGPMT%AIVCQPLRG 37

TIAQYICYMGPETWECRPSPKA 38

YSCHFGPLTWVCK 39

YCHFGPLTWVC 40

SCHFGPLTWVCK 41 (AX2)nX3X4X5GPX6TWX7X8 42

Table 2-IL-l antagonist peptide sequences

SEQUENCE/STRUCTURE SEQ ID NO:

Z„Z7ZsZQZ5YZ6Z9Zιo 43 XXQZ5YZ6XX 44

Z7XQZ5YZ6XX 45

Figure imgf000023_0001

Zi2Zi3Zi4Zi5Zi6 i7Zi8Zi9Z2oZ2iZ22ZnZ7ZsQZ5YZ6Z9ZioL 48 Z23NZ2 Z39Z25Z26Z27Z28Z 9Z3oZ o 49

TANVSSFEWTPYYWQPYALPL 50

SWTDYGYWQPYALPISGL 51

ETPFTWEESNAYYWQPYALPL 52 ENTYSP^WADSMYWQPYALPL 53

SVGEDHNFWTSEYWQPYALPL 54

DGYDRWRQSGERYWQPYALPL 55

FEWTPGYWQPY , 56

FEWTPGYWQHY 57 FEWTPGWYQJY 58

AcFEWTPGWYQJY 59

FEVffPGWpYQJY 60

FAWTPGYWQJY 61

FEWAPGYWQJY 62 FEWVPGYWQJY 63

FEWTPGYWQJY 64

AcFEWTPGYWQJY 65

FEWTPaWYQJY 66

FEWTPSarWYQJY 67 FEWTPGYYQPY 68

FEWTPGWWQPY 69

FEWTPNYWQPY 70

FEVffPvYWQJY 71

FEWTPecGYWQJY 72 FEWTPAibYWQJY 73

FEVffSarGYWQJY 74

FEWTPGYWQPY 75

FEWTPGYWQHY 76

FEWTPGWYQJY 77 AcFEWTPGWYQJY 78

FEWTPGW-pY-QJY 79

FAWTPGYWQJY 80

FEWAPGYWQJY 81

FEWVPGYWQJY 82 FEWTPGYWQJY 83 AcFEWTPGYWQJY 84

FEWTPAWYQJY 85

FEWTPSarWYQ.TY 86

FEWTPGYYQPY 87 FEWTPGWWQPY 88

FEWTPNYWQPY 89

FEWTPVYWQJY 90

FEWTPecGYWQJY 91

FEWTPAibYWQJY 92 FEWTSarGYWQJY 93

FEWTPGYWQPYALPL 94

NapEWTPGYYQJY 95

YEWTPGYYQJY 96

FEWVPGYYQJY 97 FEWTPSYYQJY 99

FEWTPNYYQJY 99

TKPR 100

RKSSK 101

RKQDK 102 NRKQDK 103

RKQDKR 104

ENRKQDKRF 105

VTKFYF 106

VTKFY 107 VTDFY 108

SHLYWQPYSVQ 109

TLVYWQPYSLQT 110

RGDYWQPYSVQS 111

VHVYWQPYSVQT 112 RLVYWQPYSVQT 113

SRVWFQPYSLQS 114

NMVYWQPYSIQT 115

SVVFWQPYSVQT 116

TFVYWQPYALPL 117 TLVYWQPYSIQR 118 RLVYWQPYSVQR 119

SPVFWQPYSIQI 120

WIEWWQPYSVQS 121

SLIYWQPYSLQM 122 TRLYWQPYSVQR 123

RCDYWQPYSVQT 124

MRVFWQPYSVQN 125

KTVYWQPYSVQT 126

RHLYWQPYSVQR 127 ALVWWQPYSEQI 128

SRVWFQPYSLQS 129

WEQPYALPLE 130

QLVWWQPYSVQR , 131

DLRYWQPYSVQV 132 ELVWWQPYSLQL 133

DLVWWQPYSVQW 134

NGNYWQPYSFQV 135

ELVYWQPYSIQR 136

ELMY)AIQPYSVQE 137 NLLYWQPYSMQD 138

GYEWYQPYSVQR 139

SRVWYQPYSVQR 140

LSEQYQPYSVQR 141

GGGWWQPYSVQR 142 VGRWYQPYSVQR 143

VHVYWQPYSVQR 144

QARWYQPYSVQR 145

VHVYWQPYSVQT 146

RSVYWQPYSVQR 147 TRVWFQPYSVQR 148

GRIWFQPYSVQR 149

GRVWFQPYSVQR 150

ARTWYQPYSVQR 151

ARVWWQPYSVQM 152 RLMFYQPYSVQR 153 ESMWYQPYSVQR 154

HFGWWQPYSVHM 155

ARFWWQPYSVQR 156

RLVYWQPYAPIY 157 RLVYWQPYSYQT 158

RLVYWQPYSLPI 159

RLVYWQPYSVQA 160

SRVWYQPYAKGL 161

SRVWYQPYAQGL 162 SRVWYQPYAMPL 163

SRVWYQPYSVQA 164

SRVWYQPYSLGL 165

SRVWYQPYAREL 166

SRVWYQPYSRQP 167 SRVWYQPYFVQP 168

EYEWYQPYALPL 169

IPEYWQPYALPL 170

SRIWWQPYALPL 171

DPLFWQPYALPL 172 SRQWVQPYALPL 173

IRSWWQPYALPL 174

RGYWQPYALPL 175

RLLWVQPYALPL 176

EYRWFQPYALPL 177 DAYWVQPYALPL 178

WSGYFQPYALPL 179

NIEFWQPYALPL 180

TRDWVQPYALPL 181

DSSWYQPYALPL 182 IGNWYQPYALPL 183

NLRWDQPYALPL 184

LPEFWQPYALPL 185

DSYWWQPYALPL 186

RSQYYQPYALPL 187 ARFWLQPYALPL 188 NSYFWQPYALPL 189

RFMYWQPYSVQR 190

AHLFWQPYSVQR 191

WWQPYALPL 192 YYQPYALPL 193

YFQPYALGL 194

YWYQPYALPL 195

RWWQPYATPL 196

GWYQPYALGF 197 YWYQPYALGL 198

IWYQPYAMPL 199

SNMQPYQRLS 200

TFVYWQPYAVGLPAAETACN 201

TFVYWQPYSVQMTITGKVTM 202 TFVYWQPYSSHXXVPXGFPL 203

TFVYWQPYYGNPQWAIHVRH 204

TFVYWQPYVLLELPEGAVRA 205

TFVYWQPYVDYVWPIPIAQV 206

GWYQPYVDGWR 207 RWEQPYVKDGWS 208

EWYQPYALGWAR 209

GWWQPYARGL 210

LFEQPYAKALGL 211

GWEQPYARGLAG 212 AWVQPYATPLDE 213

MWYQPYSSQPAE 214

GWTQPYSQQGEV 215

DWFQPYSIQSDE 216

PWIQPYARGFG 217 RPLYWQPYSVQV 218

TLIYWQPYSVQI 219

RFDYWQPYSDQT 220

WHQFVQPYALPL 221

EWDSVYWQPYSVQTLLR 223 WEQNVYWQPYSVQSFAD 224 SDVVYWQPYSVQSLEM 225

YYDGVYWQPYSVQVMPA 226

SDIWYQPYALPL 227

QRIWWQPYALPL 228 SRIWWQPYALPL 229

RSLYWQPYALPL 230

TΠWEQPYALPL 231

WETWYQPYALPL 232

SYDWEQPYALPL 233 SRIWCQPYALPL 234

EIMFWQPYALPL 235

DYVWQQPYALPL 236

MDLLVQWYQPYALPL 237

GSKVILWYQPYALPL 238 RQGANIWYQPYALPL 239

GGGDEPWYQPYALPL 240

SQLERTWYQPYALPL 241

ETWVREWYQPYALPL 242

KKGSTQWYQPYALPL 243 LQARMNWYQPYALPL 244

EPRSQKWYQPYALPL 245

VKQKWRWYQPYALPL 246

LRRHDVWYQPYALPL 247

RSTASIWYQPYALPL 248 ESKEDQWYQPYALPL 249

EGLTMKWYQPYALPL 250

EGSREGWYQPYALPL 251

VIEWWQPYALPL 252

VWYWEQPYALPL 253 ASEWWQPYALPL 254

FYEWWQPYALPL 255

EGWWVQPYALPL 256

WGEWLQPYALPL 257

DYVWEQPYALPL 258 AHTWWQPYALPL 259 FIEWFQPYALPL 260

WLAWEQPYALPL 261

VMEWWQPYALPL 262

ERMWQPYALPL 263 NXXWXXPYALPL 264

WGNWYQPYALPL 265

TLYWEQPYALPL 266 .

VWRWEQPYALPL 267

LLWTQPYALPL 268 SRIWXX PYALPL 269

SDIWYQPYALPL 270

WGYYXX PYALPL 271

TSGWYQPYALPL 272

VHPYXXPYALPL 273 EHSYFQPYALPL 274

XXIWYQPYALPL 275

AQLHSQPYALPL 276

WANWFQPYALPL 277

SRLYSQPYALPL 278 GVTFSQPYALPL 279

SIVWSQPYALPL 280

SRDLVQPYALPL 281

HWGHVYWQPYSVQDDLG 282

SWHSVYWQPYSVQSVPE 283 WRDSVYWQPYSVQPESA 284

TWDAVYWQPYSVQKWLD 285

TPPWVYWQPYSVQSLDP 286

YWSSVYWQPYSVQSVHS 287

YWYQPYALGL 288 YWYQPYALPL 289

EWIQPYATGL 290

NWEQPYAKPL 291

AFYQPYALPL 292

FLYQPYALPL 293 VCKQPYLEWC 294 ETPFTWEESNAYYWQPYALPL 295

QGWLTWQDSVDMYWQPYALPL 296

FSEAGYTWPENTYWQPYALPL 297

TESPGGLDWAKIYWQPYALPL 298 DGYDRWRQSGERYWQPYALPL 299

TANVSSFEWTPGYWQPYALPL 300 SVGEDHNFWTSE YWQPYALPL / 301

MNDQTSEVSTFPYWQPYALPL 302

SWSEAFEQPRNLYWQPYALPL 303 QYAEPSALNDWGYWQPYALPL 304

NGDWATADWSNYYWQPYALPL 305

THDEHIYWQPYALPL 306

MLEKTYTTWTPG YWQPYALPL 307

WSDPLTRDADLYWQPYALPL 308 SDAFTTQDSQAMYWQPYALPL 309

GDDAAWRTDSLTYWQPYALPL 310

AΠRQLYRWSEMYWQPYALPL 311

ENTYSPNWADSMYWQPYALPL 312

MNDQTSEVSTFPYWQPYALPL 313 SVGEDHNFWTSEYWQPYALPL 314

QTPFTWEESNAYYWQPYALPL 315

ENPFTWQESNAYYWQPYALPL 316

VTPFTWEDSNVF YWQPYALPL 317

Q FTWEQSNAYYWQPYALPL 318 QAPLTWQESAAYYWQPYALPL 319

EPTFTWEESKAT YWQPYALPL 320

TTTLTWEESNAYYWQPYALPL 321

ESPLTWEESSALYWQPYALPL 322

ETPLTWEESNAYYWQPYALPL 323 EATFTWAESNAYYWQPYALPL 324

EALFTWKESTAYYWQPYALPL 325

STP-TWEESNAYYWQPYALPL 326

ETPFTWEESNAYYWQPYALPL 327

KAPFTWEESQAYYWQPYALPL 328 STSFTWEESNAYYWQPYALPL 329 DSTFTWEESNAYYWQPYALPL 330

YIPFTWEESNAYYWQPYALPL 331

QTAFTWEESNAYYWQPYALPL 332

ETLFTWEESNAT YWQPYALPL 333 VSSFTWEESNAYYWQPYALPL 334

QPYALPL 335

Py-1-NapPYQJYALPL 336

TANVSSFEWTPG YWQPYALPL 337

FEWTPGYWQPYALPL 338 FEWTPGYWQJYALPL 339

FEWTPGYYQJYALPL 340

ETPFTWEESNAYYWQPYALPL 341

FTWEESNAYYWQJYALPL 342

ADVLYWQPYAPVTLWV 343 GDVAEYWQPYALPLTSL 344

SWTDYGYWQPYALPISGL 345

FEWTPGYWQPYALPL 346

FEWTPGYWQJYALPL 347

FEWTPGWYQPYALPL 348 FEWTPGWYQJYALPL 349

FEWTPGYYQPYALPL 350

FEWTPGYYQJYALPL 351

TANVSSFEWTPGYWQPYALPL 352

SWTDYGYWQPYALPISGL 353 ETPFTWEESNAWAIQPYALPL 354

ENTYSPNWADSMYWQPYALPL 355

SVGEDHNFWTSEYWQPYALPL 356

DGYDRWRQSGERYWQPYALPL 357

FEWTPGYWQPYALPL 358 FEWTPGYWQPY 359

FEWTPGYWQJY 360

EWTPGYWQPY 361

FEWTPGWYQJY 362

AEWTPGYWQJY 363 FAWTPGYWQJY 364 FEATPGYWQJY 365

FEWAPGYWQJY 366

FEWTAGYWQ Y 367

FEWTPAYWQJY 368 FEWTPGAWQJY 369

FEWTPGYAQJY 370

FEWTPGYWQJA 371

FEWTGGYWQJY 372

FEWTPGYWQJY 373 FEWTJGYWQJY 374

FEVffPecGYWQJY 375

FEWTPAibYWQJY 376

FEWTPSarWYQJY 377

FEWTSarGYWQJY 378 FEWTPNYWQJY 379

FEWTPVYWQJY 380

FEWTVPYWQJY 381

AcFEWTPGWYQJY 382

AcFEVffPGYWQJY 383 INap-EVff PGYYQJY 384

YEWTPGYYQJY 385

FEWVPGYYQJY 386

FEVff PGYYQJY 387

FEVff PsYYQJY 388 FEWTPnYYQJY 389

SHLY-Nap-QPYSVQM 390

TLVY-Nap-LDPYSLQT 391

RGDY-Nap-QPYSVQS 392

NMVY-Nap-QPYSIQT 393 VYWQPYSVQ 394

VY-Nap-QPYSVQ 395

TFVYWQJYALPL 396

FEWTPGYYQJ-Bpa 397

XaaFEWTPGYYQJ-Bpa ' 398 FEWTPGY-Bpa-QJY 399 AeFEWTPGY-Bpa-QJY 400

FEWTPG-Bpa-YQJY 401

AcFEWTPG-Bpa-YQJY 402

AcFE-Bpa-TPGYYQJY 403 AcFE-Bpa-TPGYYQJY 404

Bpa-EWTPGYYQJY 405

AcBpa-EWTPGYYQJY 406

VYWQPYSVQ 407

RLVYWQPYSVQR 408 RLVY-Nap-QPYSVQR 409

RLDYWQPYSVQR 410

RLVWFQPYSVQR 411

RLVYWQPYSIQR 412

DNSSWYDSFLL 413 DNTAWYESFLA 414

DNTAWYENFLL 415

PAREDNTAWYDSFLIWC 416

TSEYDNTTWYEKFLASQ 417

SQIPDNTAWYQSFLLHG 418 SPFIDNTAWYENFLLTY 419

EQIYDNTAWYDHFLLSY 420

TPFIDNTAWYENFLLTY 421

TYTYDNTAWYERFLMSY 422

TMTQDNTAWYENFLLSY 423 TIDNTAWYANLVQTYPQ 424

TIDNTAWYERFLAQYPD 425

HIDNTAWYENFLLTYTP 426

SQDNTAWYENFLLSYKA 427

QIDNTAWYERFLLQYNA 428 NQDNTAWYESFLLQYNT 429

TIDNTAWYENFLLNHNL 430

HYDNTAWYERFLQQGWH 431

ETPFTWEESNAYYWQPYALPL 432

YEPFTWEESNAYYWQPYALPL 433 DGYDRWRQSGERYWQPYALPL 434 pY-INap-pY-QJYALPL 435

TANVSSFEWTPGYWQPYALPL 436

FEWTPGYWQJYALPL 437

FEWTPGYWQPYALPLSD 438 FEWTPGYYQJYALPL 439

FEWTPGYWQJY 440

AcFEVVTPGYWQJY 441

AcFEWTPGWYQJY 442

AcFEWTPGYYQJY 443 0 AcFEWTPaYWQJY 444

AcFEWTPaWYQJY 445

AcFEWTPaYYQJY 446

FEWTPGYYQJYALPL 447

FEWTPGYWQJYALPL 448 5 FEWTPGWYQJYALPL 449

TANVSSFEWTPGYWQPYALPL 450

AcFEWTPGYWQJY 451

AcFEWTPGWYQJY 452

AcFEWTPGYYQJY 453 0 AcFEWTPAYWQJY 454

AcFEWTPAWYQJY 455

AcFEWTPAYYQJY 456 Table 3-TPO-mimetic peptide sequences

Figure imgf000035_0001
Figure imgf000036_0001

Figure imgf000037_0001

Table 4-G-CSF-mimetic peptide sequences

Sequence/structure SEQ ID NO: EEDCK 519 EEDαK 520 pGluEDαK 521 PicSDccK 522

10 EEDCK-Δ-EEDCK 523 EEDXK-Δ-EEDXK 524

Table 5-TNF-antagonist peptide sequences Sequence/structure SEQID NO:

YCFTASENHCY 525

YCFTNSENHCY 526 YCFTRSENHCY 527

FCASENHCY 528

YCASENHCY 529

FCNSENHCY 530

FCNSENRCY 531 FCNSVENRCY 532

YCSQSVSNDCF 533

FCVSNDRCY 534

YCRKELGQVCY 535

YCKEPGQCY ' 536 YCRKEMGCY 537

FCRKEMGCY 538

YCWSQNLCY 539

YCELSQYLCY 540

YCWSQNYCY 541 YCWSQYLCY 542

DFLPHYKNTSLGHRP 543

Table 6-Integrin-binding peptide sequences

Sequence/structure SEQID NO:

RX,ETX2WX3 544

RXιETX2WX3 545

RGDGX 546

CRGDGXC 547 CX1X2RLDX3X4C 548

CARRLDAPC 549

CPSRLDSPC 550

XιX2X3RGDX4X5X6 551

CX2CRGDCX5C 552 CDCRGDCFC 553

CDCRGDCLC 554

CLCRGDCIC 555

Figure imgf000039_0001
XιX2X3DDX4X5X6X7X8 557

CWDDGWL 558

CWDDLWWLC 559

CWDDGLMC 560

CWDDGWMC 561 CSWDDGWLC 562

CPDDLWWLC 563

NGR NR

GSL NR

RGD NR CGRECPRLCQSSC 564

CNGRCVSGCAGRC 565

CLSGSLSC 566

RGD NR

NGR NR GSL NR

NGRAHA ' 567

CNGRC 568

CDCRGDCFC 569

CGSLVRC 570 DLXXL 571

RTDLDSLRTYTL 572

RTDLDSLRTY 573

RTDLDSLRT 574

RTDLDSLR 575 GDLDLLKLRLTL 576

GDLHSLRQLLSR 577

RDDLHMLRLQLW 578

SSDLHALKKRYG 579

RGDLKQLSELTW 580 RGDLAALSAPPV - 581 Table 7-Selectin antagonist peptide sequences

Sequence/structure SEQ ID NO: DITWDQLWDLMK 582

DITWDELWKIMN 583

DYTWFELWDMMQ 584

QITWAQLWNMMK 585

DMTWHDLWTLMS 586 DYSWHDLWEMMS 587

EITWDQLWEVMN 588

HVSWEQLWDIMN 589

HITWDQLWRIMT 590

RNMSWLELWEHMK 591 AEWTWDQLWHVMNPAESQ 592

HRAEWLALWEQMSP 593

KKEDWLALWRIMSV 594

ITWDQLWDLMK 595

DITWDQLWDLMK 596 DITWDQLWDLMK 597

DITWDQLWDLMK 598

CQNRYTDLVAIQNKNE 599

AENWADNEPNNKRNNED 600

RKNNKTWTWVGTKKALTNE 601 KKALTNEAENWAD 602

CQXRYTDLVAIQNKXE 603

RKXNXXWTWVGTXKXLTEE 604

AENWADGEPNNKXNXED 605

CXXXYTXLVAIQNKXE 606 RKXXXXWXWVGTXKXLTXE 607

AXNWXXXEPNNXXXED 608

XKXKTXEAXNWXX 609

Table 8-Antipathogenic peptide sequences Sequence/structure SEQ ID NO: GFFALIPKΠSSPLFKTLLSAVGSALSSSGGQQ 610

GFFALIPKΠSSPLFKTLLSAVGSALSSSGGQE 611

GFFALIPKΠSSPLFKTLLSAV 6i2 GFFALIPKΠSSPLFKTLLSAV 613

KGFFALIPKΠSSPLFKTLLSAV 614

KKGFFALIPKΠSSPLFKTLLSAV 6i5

KKGFFALIPKΠSSPLFKTLLSAV 6i6

GFFAL-PKHS 617 GIGAVLKVLTTGLPALISWIKRKRQQ 618

GIGAVLKVLTTGLPALISWIKRKRQQ 619

GIGAVLKVLTTGLPALISWIKRKRQQ 620

GIGAVLKVLTTGLPALISWIKR 621

AVLKVLTTGLPALISWIKR 622 KLLLLLKLLLLK 623

KLLLKLLLKLLK 624

KLLLKLKLKLLK 625

KKLLKLKLKLKK 626

KLLLKLLLKLLK 627 KLLLKLKLKLLK 628

KLLLLK 629

KLLLKLLK 630

KLLLKLKLKLLK 631

KLLLKLKLKLLK 632 KLLLKLKLKLLK 633

KAAAKAAAKAAK 634

KVWKVVVKVVK 635

KWVKVKVKVVK 636

KWVKVKVKVK 637 KWVKVKVKVVK, 638

KLILKL 639

KVLHLL 640

LKLRLL 641

KPLHLL 642 KLE KLVR 643 KVFHLLHL ' 644

HKFRILKL 645

KPFH-LHL 646

KlilKIKIKIIK 647 KlilKIKIKIIK 648

KIΠKIKIKΠK 649

KIPIKIKIKIPK 650

KIPIKIKIKIVK 651

RIΠRE IRΠR 652 RIΠRIRIRIIR 653

RIPRIRIRIIR 654

RIVIRIRIRLIR 655

RΠVRIRLRIIR 656

RIGIRLRVRIIR 657 KIVIRIRIRLIR 658

RIAVKWRLRFIK 659

KIGWKLRVRΠR 660

KKIGWLIIRVRR 661

RIVIRIRIRLIRIR 662 RIIVRIRLRIIRVR 663

RIGIRLRVRΠRRV 664

KIVIRIRARL-RIRIR 665

- V-aRLRI-KKIRL 666

KIGIKARVRΠRVKII 667 - Vffll^RIIHHIRL 668 fflGIKAHVRIIRVHII 669

IOYVKIHLRYIKKIRL 670

KIGHKARVHIIRYKπ 671

RIYVKPHPRYIKKIRL 672 KPGHKARPHΠRYKΠ 673

K IRIRIRLIRIRIRKrV 674

- VK-IRLRIIKKIRLIKK 675

KIGWKLRVRIIRVKIGRLR 676

KINIRIRIRL iRIRKIVKVKRIR 677 -^AVKIRLRHKKI-H-IKKπiKRVIK 678 KAGWKLRVRΠRVKIGRLRKIGWKKRVRIK 679

RIYVKPHPRYIKKIRL 680 KPGHKARPHΠRYKΠ 68 I

KIVIRIRIRLIRIRIRKIV 682 R KJRLWIKKIRLIKK 683

RIYVSKISIYIKKIRL 684

KIVIFTRIRLTSIRIRSIV 685

KPIHKARPTIIRYKMI 686 cyclicCKGFFALIPKπSSPLFKTLLSAVC 687 CKKGFFALIPKIISSPLFKTLLSAVC 688

CKKKGFFALIPKΠSSPLFKTLLSAVC 689

CyclicCRIVIRIRIRLIRIRC 690

CyclicCKPGHKARPHIIRYKπC 691

CyclicCRFAVKJRLRIIKKIRLIKKIRKRVIKC 692 KLLLKLLL KLLKC 693

KLLLKLLLKLLK 694

KLLLKLKLKLLKC 695

KLLLKLLLKLLK 696

Table 9-V-P-mimetic peptide sequences

Sequence/structure SEQ ID NO:

HSDAVFYDNYTR LRKQMAVKKYLN SILN 697

Me HSDAVFYDNYTR LRKQMAVKKYLN SILN 698

Figure imgf000043_0001
X3SX4LN 700

KKYL 701

NSILN 702

KKYL 703

KKYA 704 AVKKYL 705

NSILN 706

KKYV 707

SILauN 708

KKYLNIe 709 NSYLN 710 NSIYN 711

KKYLPPNSILN 712

LauKKYL 713

CapKKYL 714 KYL NR

KKYNIe 715

VKKYL 716

LNSILN 717

YLNS-LN 718 KKYLN 719

KKYLNS 720

KKYLNSI 721

KKYLNSIL 722

KKYL 723 KKYDA 724

AVKKYL 725

NSILN 726

KKYV 727

SILauN 728 NSYLN 729

NSIYN 730

KKYLNIe 731

KKYLPPNSILN 732

KKYL 733 KKYDA 734

AVKKYL 735

NSILN 736

IKKYV 737

SILauN 738 LauKKYL 739

CapKKYL 740

KYL NR

KYL NR

KKYNIe 741 VKKYL 742 LNSILN 743

YLNSILN 744

KKYLNIe 745

KKYLN 746 KKYLNS 747

KKYLNSI 748

KKYLNSIL 749

KKKYLD 750 cyclicCKKYLC 751 CKKYLK 752

KKYA 753

WWTDTGLW 754

WWTDDGLW 755

WWDTRGLWVWTI 756 FWGNDGIWLESG 757

DWDQFGLWRGAA 758

RWDDNGLWWVL 759

SGMWSHYGIWMG 760

GGRWDQAGLWVA 761 KLWSEQGIWMGE 762

CWSMHGLWLC 763

GCWDNTGIWVPC 764

DWDTRGLWVY 765

SLWDENGAWI 766 KWDDRGLWMH 767

QAWNERGLWT 768

QWDTRGLWVA 769

WNVHGIWQE 770

SWDTRGLWVE 771 DWDTRGLWVA 772

SWGRDGLWIE 773

EWTDNGLWAL 774

SWDEKGLWSA 775

SWDSSGLWMD 776 Table 10-Mdm/hdm antagonist peptide sequences

Sequence/structure SEQID NO:

TFSDLW 777

QETFSDLWKLLP 778 QPTFSDLWKLLP 779

QETFSDYWKLLP 780

QPTFSDYWKLLP 781

MPRFMDYWEGLN 782

VQNFIDYWTQQF 783 TGPAFTHYWATF 784

IDRAPTFRDHWFALV 785

PRPALVFADYWETLY 786

PAFSRFWSDLSAGAH 787

PAFSRFWSKLSAGAH 788 PXFXDYWXXL 789

QETFSDLWKLLP 790

QPTFSDLWKLLP 791

QETFSDYWKLLP 792

QPTFSDYWKLLP 793

Table 11-Calmodulin antagonist peptide sequences

Sequence/structure SEQ ID NO:

SCVKWGKKEFCGS 794 SCWKYWGKECGS 795

SCYEWGKLRWCGS 796

SCLRWGKWSNCGS 797

SCWRWGKYQICGS 798

SCVSWGALKLCGS 799 SCIRWGQNTFCGS 800

SCWQWGNLKICGS 801

SCVRWGQLSICGS 802

LKKFNARRKLKGAILTTMLAK 803

RRWKKNFIAVSAANRFKK 804 RKWQKTGHAVRAIGRLSS 805 INLKALAALAKKIL 806

KIWSILAPLGTTLVKLVA 807

LKKLLKLLKKLLKL 808

LKWKKLLKLLKKLLKKLL 809

AEWPSLTEIKTLSHFSV 810

AEWPSPTRVISTTYFGS 811

AELAHWPPVKTVLRSFT 812

AEGS WLQLLNLMKQMNN 813

AEWPSLTEIK 814

Table 12-Mast cell antagonists/Mast cell protease inhibitor peptide sequences

Sequence/structure SEQ ID NO:

SGSGVLKRPLPILPVTR 815 RWLSSRPLPPLPLPPRT 816

GSGSYDTLALPSLPLHPMSS 817

GSGSYDTRALPSLPLHPMSS 818

GSGSSGVTMYPKLPPHWSMA 819

GSGSSGVRMYPKLPPHWSMA 820 GSGSSSMRMVPTIPGSAKHG 821

RNR NR

QT NR

RQK NR

NRQ NR RQK NR

RNRQKT 822

RNRQ 823

RNRQK 824

NRQKT 825 RQKT 826

Table 13-SH3 antagonist peptide sequences

Sequence/structure SEQ ID NO: RPLPPLP 827 RELPPLP 828

SPLPPLP 829

GPLPPLP 830

RPLPIPP 831 RPLPIPP 832

RRLPPTP 834

RQLPPTP 835

RPLPSRP 836

RPLPTRP 837 SRLPPLP 838

RALPSPP 839

RRLPRTP 840

RPVPPIT 841

ILAPPVP 842 RPLPMLP 843

RPLPILP 844

RPLPSLP 845

RPLPSLP 846

RPLPMIP , 847 RPLPLIP 848

RPLPPTP 849

RSLPPLP 850

RPQPPPP 851

RQLPIPP 852 XXXRPLPPLPXP 853

XXXRPLPPIPXX 854

XXXRPLPPLPXX 855

RXXRPLPPLPXP 856

RXXRPLPPLPPP 857 PPPYPPPPIPXX 858

PPPYPPPPVPXX 859

LXXRPLPXTP 860

ΨXXRPLPXLP 861

PPXΘXPPPΨP 862 +PPΨPXKPXWL 863

RPXΨPΨR+SXP 864

PPVPPRPXXTL 865

ΨPΨLPΨK 866 +ΘDXPLPXLP 867

Table 14-Somatostatin or cortistatin mimetic peptide sequences

Sequence/structure SEQID NO: X1X2 -Asn-Phe-Phe-Trp-Lys-Thr-Phe-X3-Ser-X4 868

Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 869

Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 870

Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 871

Asp Arg Met Pro_Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 872 Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 873

Cys Arg Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 874

Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 875

Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 876

Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys Lys 877 Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 878

Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 879

Cys Lys Asn Phe Phe Trp Lys Thr Phe Ser Ser Cys 880

Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 881

Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 882 Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 883

Asp Arg Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 884

Met Pro Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 885

Cys Arg Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 886

Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 887 Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 889

Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys Lys 890

Asp Arg Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 891

Met Pro Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 892

Cys Lys Asn Phe Phe Trp Lys Thr Phe Thr Ser Cys 893 Table 15-UKR antagonist peptide sequences

Sequence/structure SEQID NO: AEPMPHSLNFSQYLWYT 894

AEHTYSSLWDTYSPLAF 895

AELDLWMRHYPLSFSNR 896

AESSLWTRYAWPSMPSY 897

AEWHPGLSFGSYLWSKT 898 AEPALLNWSFFFNPGLH 899

AEWSFYNLHLPEPQTIF 900

AEPLDLWSLYSLPPLAM 901

AEPTLWQLYQFPLRLSG l 902

AEISFSELMWLRSTPAF 903 AELSEADLWTTWFGMGS 904

AESSLWRIFSPSALMMS 905

AESLPTLTSILWGKESV 906

AETLFMDLWHDKHILLT 907

AEILNFPLWHEPLWSTE 908 AESQTGTLNTLFWNTLR 909

AEPVYQYELDSYLRSYY 910

AELDLSTFYDIQYLLRT 911

AEFFKLGPNGYVYLHSA 912

FKLXXXGYVYL 913 AESTYHHLSLGYMYTLN 914

YHXLXXGYMYT 915

Table 16-Macrophage and/or T-cell inhibiting peptide sequences

Sequence/structure SEQID NO:

Xaa-Yaa-Arg NR Arg-Yaa-Xaa NR

Xaa-Arg-Yaa NR

Yaa-Arg-Xaa NR

Ala-Arg NR

Arg-Arg NR Asn-Arg NR

Asp-Arg NR

Cys-Arg NR

GIn-Arg NR

Glu-Arg NR Gly-Arg NR

His-Arg NR

Ile-Arg NR

Leu-Arg NR

Lys-Arg NR Met-Arg NR

Phe-Arg NR

Ser-Arg NR

Thr-Arg NR

Trp-Arg NR Tyr-Arg NR

Val-Arg NR

Ala-Glu-Arg ' NR

Arg-Glu-Arg NR

Asn-Glu-Arg NR Asp-Glu-Arg NR

Cys-Glu-Arg NR

Gln-Glu-Arg NR

Glu-Glu-Arg NR

Gly-Glu-Arg NR His-Glu-Arg NR Ile-Glu-Arg NR

Leu-Glu-Arg NR

Lys-Glu-Arg NR

Met-Glu-Arg NR Phe-Glu-Arg NR

Pro-Glu-Arg NR

Ser-Glu-Arg NR

Thr-Glu-Arg NR

Trp-Glu-Arg NR Tyr-Glu-Arg NR

Val-Glu-Arg NR

Arg-Ala NR

Arg-Asp NR

Arg-Cys NR Arg-Gln NR

Arg-Glu NR

Arg-Gly NR

Arg-His NR

Arg-Ile NR Arg-Leu NR

Arg-Lys NR

Arg-Met NR

Arg-Phe NR

Arg-Pro NR Arg-Ser NR

Arg-Thr NR

Arg-Trp NR

Arg-Tyr NR

Arg-Val NR Arg-Glu-Ala NR

Arg-Glu-Asn NR

Arg-Glu-Asp NR

Arg-Glu-Cys NR

Arg-Glu-Gln NR Arg-Glu-Glu NR Arg-Glu-Gly NR

Arg-Glu-His NR

Arg-Glu-Ile NR

Arg-Glu-Leu NR Arg-Glu-Lys NR

Arg-Glu-Met NR

Arg-Glu-Phe NR

Arg-Glu-Pro NR

Arg-Glu-Ser NR Arg-Glu-Thr NR

Arg-Glu-Trp NR

Arg-Glu-Tyr NR

Arg-Glu-Val NR

Ala-Arg-Glu NR Arg-Arg-Glu NR

Asn-Arg-Glu NR

Asp-Arg-Glu NR

Cys-Arg-Glu NR

Gln-Arg-Glu NR Glu-Arg-Glu NR

Gly-Arg-Glu NR

His-Arg-Glu NR

Ile-Arg-Glu NR

Leu-Arg-Glu NR Lys-Arg-Glu NR

Met-Arg-Glu NR

Phe-Arg-Glu NR

Pro-Arg-Glu NR

Ser-Arg-Glu NR Thr-Arg-Glu NR

Trp-Arg-Glu NR

Tyr-Arg-Glu NR

Val-Arg-Glu NR

Glu-Arg-Ala NR Glu-Arg-Arg NR Glu-Arg-Asn NR

Glu-Arg-Asp NR

Glu-Arg-Cys NR

Glu-Arg-Gln NR

Glu-Arg-Gly NR

Glu-Arg-His NR

Glu-Arg-Ile NR

Glu-Arg-Leu NR

Glu-Arg-Lys NR

Glu-Arg-Met NR

Glu-Arg-Phe NR

Glu-Arg-Pro NR

Glu-Arg-Ser NR

Glu-Arg-Thr NR

Glu-Arg-Trp NR

Glu-Arg-Tyr NR

Glu-Arg-Val NR

Table 17-Additional Exemplary Pharmacologically Active Peptides

Sequence/Structure SEQID NO: Activity

VEPNCDIHVMWEWECFERL 916 VEGF-antagonist

GERWCFDGPLTWVCGEES 917 VEGF-antagonist

RGWVEICVADDNGMCVTEAQ 918 VEGF-antagonist

GWDECDVARMWEWECFAGV 919 VEGF- antagonist

GERWCFDGPRAWVCGWEI 920 VEGF- antagonist

EELWCFDGPRAWVCGYVK 921 VEGF- antagonist

RGWVEICAADDYGRCLTEAQ 922 VEGF- antagonist

RGWVEICESDVWGRCL 923 VEGF- antagonist

RGWVEICESDVWGRCL 924 VEGF- antagonist

GGNECDIARMWEWECFERL 925 VEGF- antagonist

RGWVEICAADDYGRCL 926 VEGF-antagonist

CTTHWGFTLC 927 MMP inhibitor

CLRSGXGC 928 MMP inhibitor CXXHWGFXXC 929 MMP inhibitor CXPXC 930 MMP inhibitor

CRRHWGFEFC 931 MMP inhibitor STTHWGFTLS 932 MMP inhibitor CSLHWGFWWC 933 CTLA4-mimetic GFVCSGIFAVGVGRC 934 CTLA4-mimetic APGVRLGCAVLGRYC 935 CTLA4-mimetic

LLGRMK 936 Antiviral (HBV)

ICWQDWGHHRCTAGHMANLTSHASAI 937 C3b antagonist ICVVQDWGHHRCT 938 C3b antagonist

CVVQDWGHHAC 939 C3b antagonist

STGGFDDVYDWARGVSSALTTTLVATR 940 Vinculin-binding STGGFDDVYDWARRVSSALTTTLVATR 941 Vinculin-binding SRGVNFSEWLYDMSAAMKEASNVFPSRRSR 942 Vinculin-binding SSQNWDMEAGVEDLTAAMLGLLSTIHSSSR 943 Vinculin-binding SSPSLYTQFLVNYESAATRIQDLL-ASRPSR 944 Vinculin-binding SUGMIDILLGAILQRAADATRTSIPIPSLQNSIR 945 Vinculin-binding DVYTKKELIECARRVSEK 946 Vinculin-binding

EKGSYYPGSGIAQFHIDYNNVS 947 C4BP-binding

SGIAQFHIDYNNVSSAEGWHVN 948 C41BP-binding

LVTVEKGSYYPGSGIAQFHIDYNNVSSAEGWHVN 949 4BP-binding

SGIAQFHJDYNNVS 950 C4BP-binding

LLGRMK 951 anti-HBV

ALLGRMKG 952 anti-HBV

LDPAFIR 953 anti-HBV

CXXRGDC 954 Inhibition of platelet aggrepation

RPLPPLP 955 Src antagonist

PPVPPR 956 Src antagonist

XFXDXWXXLXX 957 Anti-cancer

KACRRLFGPVDSEQLSRDCD 958 pi 6-mimetic

RERWNFDFVTETPLEGDFAW 959 pi 6-mimetic

KRRQTSMTDFYHSKRRLIFS 960 pi 6-mimetic

TSMTDFYHSKRRLIFSKRKP 961 pi 6-mimetic

RRLIF 962 pi 6-mimetic KRRQTSATDFYHSKRRLIFSRQIKIWFQNRRMKWKK 963 pi 6-mimetic KRRLIFSKRQIKIWFQNRRMKWKK 964 pi 6-mimetic

Asn Gin Gly Arg His Phe Cys Gly Gly Ala Leu He His Ala Arq Phe Val Met Thr Ala Ala Ser Cys Phe Gin 965 CAP37 mimetic/LPs bindin

Arg His Phe Cys Gly Gly Ala Leu He His Ala Arg Phe Val Met Thr Ala Ala Ser Cys 499 CAP37 mimetic/LPS binding Gly Thr Arg Cys Gin Val Ala Gly Trp Gly Ser Gin Arg Ser Gly Gly Arg Leu Ser Arg Phe Pro

Arg Phe Val Asn Val 966 CAP37 mimetic/LPS binding

WHWRHRIPLQLAAGR 967 carbohydrate (GID1 alpha) mimetic

LKTPRV 968 I32GPI Ab binding

NTLKTPRV 969 I32GPI Ab binding

NTLKTPRVGGC 970 02GPI Ab binding

KDKATF 971 02GPI Ab binding

KDKATFGCHD 972 P2GP1 Ab binding

KDKATFGCHDGC 973 02GPI Ab bindinq

TLRVYK 974 02GPI Ab binding

ATLRVYKG 975 02GPI Ab binding

CATLRVYKGG 976 132GPI Ab binding

INLKALAALAKKIL 977 Membrane transporting

GWT NR Membrane transporting

GWTLNSAGYLLG 978 Membrane transporting

GWTLNSAGYLLGKINLKALAALAKKIL 979 Membrane transporting

The present invention is also particularly useful with peptides having activity in treatment of: a VEGF related condition, e.g., but not limited to, cancer, wherein the peptide is a VEGF-mimetic or a VEGF receptor antagonist, a HER2 agonist or antagonist, a CD20 antagonist and the like; asthma, wherein the protein of interest is a CKR3 antagonist, an IL-5 receptor antagonist, and the like; thrombosis, wherein the protein of interest is a GPIIb antagonist, a GP-Ha. antagonist, and the like; autoimmune diseases and other conditions involving immune modulation, wherein the protein of interest is an IL-2 receptor antagonist, a CD40 agonist or antagonist, a CD40L agonist or antagonist, a thymopoietin mimetic and the like.

For example, EPO biological activities are well known in the art. See, e.g., Anagnostou A et al Erythropoietin has a mitogenic and positive chemotactic effect on endothelial cells. Proceedings of the National Academy of Science (USA) 87: 5978-82 (1990); Fandrey J and Jelkman WE Interleukin 1 and tumor necrosis factor-alpha inhibit erythropoietin production in vitro. Annals of the New York Academy of Science 628: 250-5 (1991); Geissler K et al Recombinant human erythropoietin: A multipotential hemopoietic growth factor in vivo and in vitro. Contrib. Nephrol. 87: 1-10 (1990); Gregory CJ Erythropoietin sensitivity as a differentiation marker in the hemopoietic system. Studies of three erythropoietic colony responses in culture. Journal of Cellular Physiology 89: 289-301 (1976); Jelkman W et al Monokines inhibiting erythropoietin production in human hepatoma cultures and in isolated perfused rat kidneys. Life Sci. 50: 301-8 (1992); Kimata H et al Human recombinant erythropoietin directly stimulates B cell immunoglobulin production and proliferation in serum-free medium. Clinical and Experimental Immunology 85: 151-6 (1991); Kimata H et al Erythropoietin enhances immunoglobulin production and proliferation by human plasma cells in a serum-free medium. Clin. Immunology Immunopathol. 59: 495-501 (1991); Kimata H et al Effect of recombinant human erythropoietin on human IgE production in vitro Clinical and Experimental Immunology 83: 483-7 (1991); Koury MJ and Bondurant MC Erythropoietin retards DNA breakdown and prevents programmed cell death in erythroid progenitor cells. Science 248: 378-81 (1990); Lim VS et al Effect of recombinant human erythropoietin on renal function in humans. Kidney International 37: 131-6 (1990); Mitjavila MT et al Autocrine stimulation by erythropoietin and autonomous growth of human erythroid leukemic cells in vitro. Journal of Clinical Investigation 88: 789-97 (1991); Andre M et al Performance of an immunoradiometric assay of erythropoietin and results for specimens from anemic and polycythemic patients. Clinical Chemistry 38: 758-63 (1992); Hankins WD et al Erythropoietin-dependent and erythropoietin-producing cell lines. Implications for research and for leukemia therapy. Annals of the New York Academy of Science 554: 21-8 (1989); Kendall RGT et al Storage and preparation of samples for erytliropoietin radioimmunoassay. Clin. Lab. Haematology 13: 189-96 (1991); Krumvieh D et al Comparison of relevant biological assays for the determination of biological active erythropoietin. Dev. Biol. Stand. 69: 15-22 (1988); Ma DD et al Assessment of an EIA for measuring human serum erythropoietin as compared with RIA and an in-vitro bioassay. British Journal of Haematology 80: 431-6 (1992); Noe G et al A sensitive sandwich ELISA for measuring erythropoietin in human serum British Journal of Haematology 80: 285-92 (1992); Pauly JU et al Highly specific and highly sensitive enzyme immunoassays for antibodies to human interleukin 3 (IL3) and human erythropoietin (EPO) in serum. Behring Institut Mitteilungen 90: 112-25 (1991); Sakata S and Enoki Y Improved microbioassay for plasma erythropoietin based on CFU-E colony formation. Ann. Hematology 64: 224-30 (1992); Sanengen T et al Immunoreactive erythropoietin and erythropoiesis stimulating factor(s) in plasma from hypertransfused neonatal and adult mice. Studies with a radioimmunoassay and a cell culture assay for erythropoietin. Acta Physiol. Scand. 135: 11-6 (1989); Widness JA et al A sensitive and specific erythropoietin immunoprecipitation assay: application to pharmacokinetic studies. Journal of Lab. Clin. Med. 119: 285-94 (1992); for further information see also individual cell lines used in individual bioassays. Each of the above references are entirely incorporated herein by reference. EPO can be assayed by employing cell lines such as HCD57 , NFS-60 , TF-1 and UT-7 , which respond to the factor . EPO activity can be assessed also in a Colony formation assay by determining the number of CFU-E from bone marrow cells. An alternative and entirely different detection method is RT-PCR quantitation of cytokines.

A CDR mimetibody, or specified portion or variant thereof, that partially or preferably substantially provides at least one biological activity of at least one protein or fragment, can bind the protein or fragment ligand and thereby provide at least one activity that is otherwise mediated through the binding of protein to at least one protein ligand or receptor or through other protein-dependent or mediated mechanisms. As used herein, the term "CDR mimetibody activity" refers to a CDR mimetibody that can modulate or cause at least one protein-dependent activity by about 20-10,000%, preferably by at least about 60, 70, 80, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000 % or more depending on the assay.

The capacity of a CDR mimetibody or specified portion or variant to provide at least one protein-dependent activity is preferably assessed by at least one suitable protein biological assay, as described herein and/or as known in the art. A human CDR mimetibody or specified portion or variant of the invention can be similar to any class (IgG, IgA, IgM, etc.) or isotype and can comprise at least a portion of a kappa or lambda light chain, wherein at least one of the LBRs is replaced by at least one LBR as described herein. In one embodiment, the human CDR mimetibody or specified portion or variant comprises an IgG heavy chain or defined fragment, for example, at least one of isotypes, IgGl, IgG2, IgG3 or IgG4. In another embodiment, the human protein human CDR mimetibody or specified portion or variant thereof comprises an IgGl heavy chain and a IgGl light chain.

At least one CDR mimetibody or specified portion or variant of the invention binds at least one specified ligand specific to at least one protein, subunit, fragment, portion or any combination thereof. The at least one LBR of at least one CDR mimetibody, specified portion or variant of the present invention can optionally bind at least one specified ligand epitope of the ligand. The binding 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 sequences selected from the group consisting of a protein ligand, such as a receptor or portion thereof.

Generally, the CDR mimetibody or ligand-binding fragment of the present invention can comprise a ligand binding region (LBR) (e.g., LBRl, LBR2 and LBR3) or variant provided in at least one heavy chain variable region and at least one ligand binding region (LBRl, LBR2 and LBR3) or variant provided in at least one light chain variable region. As a non-limiting example, the CDR mimetibody or ligand-binding portion or variant can comprise at least one of the heavy chain LBR3, and/or a light chain LBR3. In a particular embodiment, the CDR mimetibody or ligand-binding fragment can have an ligand-binding region that comprises at least a portion of at least one heavy chain LBR (i.e., LBRl, LBR2 and/or LBR3) having the amino acid sequence of the corresponding LBRs 1, 2 and/or 3). In another particular embodiment, the CDR mimetibody or ligand-binding portion or variant can have an ligand-binding region that comprises at least a portion of at least one light chain LBR (i.e., LBRl, LBR2 and/or LBR3) having the amino acid sequence of the corresponding LBRs 1, 2 and/or 3 (e.g., SEQID NOS: 10, 11, and/or 12). Such mimetibodies can be prepared by joining together the various portions (e.g., LBRs, framework) of the CDR mimetibody using known techniques, by preparing and expressing at least one (i.e., one or more) nucleic acid molecules that encode the CDR mimetibody, using known techniques of recombinant DNA technology or by using any other suitable method, such as chemical synthesis.

The CDR mimetibody can comprise at least one of a heavy or light chain variable region having a defined amino acid sequence. Mimetibodies that bind to human protein ligands or receptors and that comprise a defined heavy or light chain variable region can be prepared using suitable methods, such as phage display (Katsube, Y., et al, IntJMol. Med, l(5):863-868 (1998)) or methods that employ transgenic animals, as known in the art and/or as described herein. The CDR mimetibody, 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 mimetibodies, ligand-binding fragments, immunoglobulin chains and LBRs comprising amino acids in a sequence that is substantially the same as an amino acid sequence described herein. Preferably, such mimetibodies or ligand-binding fragments and mimetibodies comprising such chains or LBRs can bind human protein ligands with high affinity (e.g., KD 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 Acid Codes

The amino acids that make up mimetibodies or specified portions or variants of the present invention are often abbreviated. The 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):

Figure imgf000060_0001

A CDR mimetibody or specified portion or variant 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.

Of course, 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 at least one of a CDR mimetibody LBR, variable, constant, light or heavy chain, or Ig 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 amino acids, such as 1-30 or any range or value therein, as specified herein.

Amino acids in a CDR mimetibody or specified portion or variant 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)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity, such as, but not limited to at least one protein related activity, as specified herein or as known in the art. Sites that are critical for CDR mimetibody or specified portion or variant binding can also be identified by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith, et al., J. Mol. Biol. 224:899-904 (1992) and de Vos, et al., Science 255:306-312 (1992)).

Mimetibodies or specified portions or variants of the present invention can comprise, but are not limited to, at least one portion, sequence or combination selected from 3 to all the of at least one of SEQID NOS: 1-1109. 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 , insertion or deletion that does not significantly affect the suitable biological activtities or functions of said CDR mimetibody.

A(n) CDR mimetibody or specified portion or variant can further optionally comprise at least one functional portion of at least one polypeptide of at least one of 90-100% of SEQID NOS:1-1109.

A CDR mimetibody can further optionally comprise an amino acid sequence selected from one or more of SEQID NOS: 1-1109.

In one embodiment, the amino acid sequence of an immunoglobulin chain, or portion thereof (e.g., comprising at least one specified variable region, LBR) has about 90-100% identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein) to the corresponding amino acid sequence of the corresponding portion of at least one of SEQID NOS: 1-1109. Preferably, 90-100% amino acid identity (i.e., 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or any range or value therein) is determined using a suitable computer algorithm, as known in the art.

Mimetibodies or specified portions or variants of the present invention can comprise any number of contiguous amino acid residues from a CDR mimetibody or specified portion or variant 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 a CDR mimetibody or specified portion or variant, including specified LBR sequences. Optionally, this subsequence of contiguous amino acids is at least about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, 250 or more amino acids in length, or any range or value therein. Further, the number of such subsequences can be any integer selected from the group consisting of from 1 to 20, such as at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more. As those of skill will appreciate, the present invention includes at least one biologically active CDR mimetibody or specified portion or variant of the present invention. Biologically active mimetibodies or specified portions or variants 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 inserted or fused protein or specified portion or variant. Methods of assaying and quantifying measures of enzymatic activity and substrate specificity, are well known to those of skill in the art.

In another aspect, the invention relates to human mimetibodies and ligand-binding fragments, as described herein, which are modified by the covalent attachment of an organic moiety. Such modification can produce a CDR mimetibody or ligand-binding fragment 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. In particular embodiments, 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 mimetibodies and ligand-binding fragments of the invention can comprise one or more organic moieties that are covalently bonded, directly or indirectly, to the CDR mimetibody or specified portion or variant. Each organic moiety that is bonded to a CDR mimetibody or ligand- binding fragment of the invention can independently be a hydrophilic polymeric group, a fatty acid group or a fatty acid ester group. As used herein, the term "fatty acid" encompasses mono-carboxylic acids and di-carboxylic acids. A "hydrophilic polymeric group," as the term is used herein, refers to an organic polymer that is more soluble in water than in octane. For example, polylysine is more soluble in water than in octane. Thus, a CDR mimetibody modified by the covalent attachment of polylysine is encompassed by the invention. Hydrophilic polymers suitable for modifying mimetibodies 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. Preferably, the hydrophilic polymer that modifies the

CDR mimetibody of the invention has a molecular weight of about 800 to about 150,000 Daltons as a separate molecular entity. For example, PEG250o, PEG50oo, PEG7500, PEG9000, PEGιoooo,PEGι250o, PEG15000, and PEG2o,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. For example, 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.

Fatty acids and fatty acid esters suitable for modifying mimetibodies of the invention can be saturated or can contain one or more units of unsaturation. Fatty acids that are suitable for modifying mimetibodies of the invention include, for example, n-dodecanoate ( 2, laurate), n-tetradecanoate (Cu, myristate), n-octadecanoate ( s, stearate), n-eicosanoate (C2o, arachidate) , n-docosanoate (C 2, behenate), n-triacontanoate (C30), n-tetracontanoate (C o), cw-Δ9-octadecanoate (C\S, oleate), all cis- Δ5,8,l l,14-eicosatetraenoate (C20, 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 mimetibodies and ligand-binding fragments can be prepared using suitable methods, such as by reaction with one or more modifying agents. A "modifying agent" as the term is used herein, refers to a suitable organic group (e.g., hydrophilic polymer, a fatty acid, a fatty acid ester) that comprises an activating group. 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. For example, amine-reactive activating groups include electrophilic groups such as tosylate, mesylate, halo (chloro, bro o, 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 C1-C12 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, -(CH2)3-, -NH-(CH2)6-NH-, -(CH2)2-NH- and -CH2-0-CH2-CH2-0-CH2-CH2-0-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. 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. (See, for example, Thompson, et al., WO 92/16221 the entire teachings of which are incorporated herein by reference.)

The modified mimetibodies of the invention can be produced by reacting an human CDR mimetibody or ligand-binding fragment with a modifying agent. For example, the organic moieties can be bonded to the CDR mimetibody in a non-site specific manner by employing an amine-reactive modifying agent, for example, an NHS ester of PEG. Modified human mimetibodies or ligand-binding fragments can also be prepared by reducing disulfide bonds (e.g., intra-chain disulfide bonds) of a CDR mimetibody or ligand-binding fragment. The reduced CDR mimetibody or ligand-binding fragment can then be reacted with a thiol-reactive modifying agent to produce the modified CDR mimetibody of the invention. Modified human mimetibodies and ligand-binding fragments comprising an organic moiety that is bonded to specific sites of a CDR mimetibody or specified portion or variant 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 al, Bioconjugate Chem., 5:411-417 (1994); Kumaran et α/., Protein Sci. 6(10):2233-2241 (1997); Itoh et α/., Bioorg. Chem., 24(1): 59-68 (1996); Capellas et al., Biotechnol. Bioeng., 56(4): 456-463 (1997)), and the methods described in Hermanson, G. T., Bioconjugate Techniques, Academic Press: San Diego, CA (1996).

CDR MIMETD3ODY OR SPECIFIED PORTION OR VARIANT COMPOSITIONS

The present invention also provides at least one CDR mimetibody or specified portion or variant composition comprising at least one, at least two, at least three, at least four, at least five, at least six or more mimetibodies or specified portions or variants 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 full length, C- and/or N-terminally deleted variants, domains, fragments, or specified variants, of the CDR mimetibody amino acid sequence selected from the group consisting of 90-100% of the contiguous amino acids of SEQID NO: 1-1109 or specified fragments, domains or variants thereof. Preferred CDR mimetibody compositions include at least one or two full length, fragments, domains or variants as at least one of the C-terminal amino acids of the protein sequence of 90-100%) of SEQID NOS: 1-1109, or specified fragments, domains or variants thereof. Further preferred compositions comprise 40-99% of at least one of 90-100% of SEQID NOS: 1-1109 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. CDR mimetibody or specified portion or variant compositions 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. Pharmaceutically acceptable 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, 18th 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 CDR mimetibody 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-, tetra-, 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. Exemplary protein excipients include serum albumin such as human serum albumin (HSA), recombinant human albumin (rHA), gelatin, casein, and the like. Representative amino acid/CDR mimetibody or specified portion or variant 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.

CDR mimetibody 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.

Additionally, the CDR mimetibody or specified portion or variant compositions of the invention can include polymeric excipients/additives such as polyvinylpyrrolidones, ficolls (a polymeric sugar), dextrates (e.g., cyclodextrins, such as 2-hydroxypropyl-β-cyclodextrin), polyethylene glycols, flavoring agents, antimicrobial agents, sweeteners, antioxidants, antistatic agents, surfactants (e.g., polysorbates such as "TWEEN 20" and "TWEEN 80"), lipids (e.g., phospholipids, fatty acids), steroids (e.g., cholesterol), and chelating agents (e.g., EDTA).

These and additional known pharmaceutical excipients and/or additives suitable for use in the CDR mimetibody compositions according to the invention are known in the art, e.g., as listed in "Remington: The Science & Practice of Pharmacy", 19th ed., Williams & Williams, (1995), and in the "Physician's Desk Reference", 52nd 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. Formulations As noted above, the invention provides for stable formulations, which can preferably include a suitable buffer with saline or a chosen salt, as well as optional preserved solutions and formulations containing a preservative as well as multi-use preserved formulations suitable for pharmaceutical or veterinary use, comprising at least one CDR mimetibody or specified portion or variant 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, 0.05, 0.09, 0.1, 0.2, 0.3, O.4., 0.5, 0.6, 0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.1, 2.8, 2.9, 3.0, 3.1, 3.2, 3.3, 3.4, 3.5, 3.6, 3.7, 3.8, 3.9, 4.0, 4.3, 4.5, 4.6, 4.7, 4.8, 4.9, or any range or value therein. Non-limiting examples include, no preservative, 0.1-2% m-cresol (e.g., 0.2, 0.3. 0.4, 0.5, 0.9, 1.0%), 0.1-3% 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.

As noted above, the invention provides an article of manufacture, comprising packaging material and at least one vial comprising a solution of at least one CDR mimetibody or specified portion or variant 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 CDR mimetibody or specified portion or variant, and a second vial comprising an aqueous diluent of prescribed buffer or preservative, wherein said packaging material comprises a label that instructs a patient to reconstitute the at least one CDR mimetibody or specified portion or variant 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 CDR mimetibody or specified portion or variant 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 amounts of at least one CDR mimetibody or specified portion or variant in the product of the present invention includes amounts yielding upon reconstitution, if in a wet/dry system, concentrations from about 1.0 μg/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.

Preferably, the aqueous diluent optionally further comprises a pharmaceutically acceptable preservative. Preferred 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. The concentration of preservative used in the formulation is a concentration sufficient to yield an anti-microbial effect. Such concentrations are dependent on the preservative selected and are readily determined by the skilled artisan. Other excipients, e.g. isotonicity agents, buffers, antioxidants, preservative enhancers, can be optionally and preferably added to the diluent. 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. Preferably 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).

Other 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 co- polymers, and chelators such as EDTA and EGTA can optionally be added to the formulations or compositions to reduce aggregation. These additives 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 CDR mimetibody or specified portion or variant 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. Mixing the at least one CDR mimetibody or specified portion or variant and preservative in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one CDR mimetibody or specified portion or variant 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 may 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 CDR mimetibody or specified portion or variant 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. 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.

The present claimed articles of manufacture are useful for administration over a period of immediately to twenty-four hours or greater. Accordingly, the presently claimed articles of manufacture offer significant advantages to the patient. Formulations of the invention can optionally be safely stored at temperatures of from about 2 to about 40°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 at least one of 1-12 months, one-half, one and a half, and/or two years.

The solutions of at least one CDR mimetibody or specified portion or variant in the invention can be prepared by a process that comprises mixing at least one CDR mimetibody or specified portion or variant 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 CDR mimetibody or specified portion or variant 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 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 may 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 CDR mimetibody or specified portion or variant 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 CDR mimetibody or specified portion or variant 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 CDR mimetibody or specified portion or variant 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 Humaject®'NovoPen®, B-D®Pen, AutoPen®, and OptiPen®. 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 CDR mimetibody or specified portion or variant 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. For the single vial, solution 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 CDR mimetibody or specified portion or variant and a selected buffer, preferably a phosphate buffer containing saline or a chosen salt. Mixing the at least one CDR mimetibody or specified portion or variant and buffer in an aqueous diluent is carried out using conventional dissolution and mixing procedures. To prepare a suitable formulation, for example, a measured amount of at least one CDR mimetibody or specified portion or variant 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 CDR mimetibody or specified portion or variant that is reconstituted with a second vial containing a preservative or buffer and excipients 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 provides a more convenient treatment regimen than currently available. At least one CDR mimetibody or specified portion or variant 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.

Therapeutic Applications

The present invention for mimetibodies also provides a method for modulating or treating anemia, in a cell, tissue, organ, animal, or patient including, but not limited to, at least one of any anemia, cancer treatment related anemia, radiotherapy or chemotherapy related anemia, viral or bacterial infection treatment related anemia, renal anemia, anemia of prematurity, pediatric and/or adult cancer-associated anemia, anemia associated with lymphoma, myeloma, multple myeloma, AIDS-associated anemia, concomitant treatment for patients with or without autologous blood donation awaiting elective surgery, preoperatve and post operative for surgery, autologous blood donation or transfusion, perioperative management, cyclic neutropenia or Kostmann syndrome (congenital agranulocytosis), end-stage renal disease, anemia associated with dialysis, chronic renal insufficiency, primary hemopoietic diseases, such as congenital hypoplastic anemia, thalassemia major, or sickle cell disease, vaso-occlusive complications of sickle cell disease. Furman et al., Pediatrics 1992; 90: 716- 728, Goldberg Science. 1988;242:1412-1415; Paul et al., Exp Hematol. 1984;12:825-830; Erslev et al., Arch Intern Med. 1968;122:230-235; Ersley et al., Ann Clin Lab Sci. 1980;10:250-257; Jacobs et al., Nature. 1985;313:806-810; Lin et al., Proc Natl Acad Sci USA. 1985;82:7580-7584; Law et al., Proc Natl Acad Sci USA. 1986;83:6920-6924; Goldwasser et al., J Biol Chem. 1974;249:4202-4206; Eaves et a., Blood. 1978;52:1196-1210; Sawyer et al., Blood. 1989;74:103-109; Winearls et al, Lancet. 1986;2: 1175-1178; Eschbach et al., N Engl J Med. 1987;316:73-78; Eschbach et al., Ann Intern Med. 1989;111:992-1000, each reference entirely incoporated herein by reference. Mimetibodies of the present invention can also be used for non-renal forms of anemia induced, for example, by chronic infections, inflammatory processes, radiation therapy, and cytostatic drug treatment, and encouraging results in patients with non-renal anemia have been reported. See, e.g., Abels RI and Rudnick SA Erythropoietin: evolving clinical applications. Experimental Hematology 19: 842-50 (1991); Graber SE and Krantz SB Erythropoietin: biology and clinical use. Hematology/Oncol. Clin. North Amer. 3 : 369-400 (1989); Jelkman W and Gross AJ (eds) Erythropoietin. Springer, Berlin 1989; Koury MJ and Bondurant MC The molecular mechanism of erythropoietin action. European Journal of Biochemistry 210: 649-63 (1992); Krantz SB Erythropoietin. Blood 77: 419-34 (1991); Tabbara IA Erythropoietin. Biology and clinical applications. Archives of Internal Medicine 153: 298- 304 (1993), each entirely incorporated herein by reference. The present invention also provides a method for modulating or treating an anemia or blood cell related condition, in a cell, tissue, organ, animal, or patient, wherein said anemia or blood cell related condition is associated with at least one including, but not limited to, at least one of immune related disease, cardiovascular disease, infectious, malignant and/or neurologic disease. Such a method can optionally comprise administering an effective amount of at least one composition or pharmaceutical composition comprising at least one CDR mimetibody or specified portion or variant 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 cancer/infecteous 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, HTV infection/HIV neuropathy, meningitis, hepatitis, septic arthritis, peritonitis, pneumonia , epiglottitis, e. coli 0157:h7, hemolytic uremic syndrome/thrombolytic thrombocytopenic purpura, malaria, dengue hemorrhagic fever, leishmaniasis, leprosy, toxic shock syndrome, streptococcal myositis, gas gangrene, mycobacterium tuberculosis, mycobacterium avium intracellulare, pneumocystis carinii pneumonia, pelvic inflammatory disease, orchitis/epidydimitis, legionella, lyme disease, influenza a, epstein-barr virus, vital-associated hemaphagocytic syndrome, vital encephalitis/aseptic meningitis, and the like; (ii) leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chromic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignamt lymphoma, non-hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, Kaposi's sarcoma, colorectal carcinoma, pancreatic carcinoma, nasopharyngeal carcinoma, malignant histiocytosis, paraneoplastic syndrome/hypercalcemia of malignancy, solid tumors, adenocarcinomas, sarcomas, malignant melanoma, and the like; or (iii) 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, and Machado-Joseph); systemic disorders (Refsum's disease, abetalipoprotemia, ataxia, telangiectasia, and mitochondrial multi.system disorder); demyelinating core disorders, such as multiple sclerosis, acute transverse myelitis; and 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. Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one TNF antibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy. See, e.g., the Merck Manual, 16th Edition, Merck & Company, Rahway, NJ (1992)

Such a method can optionally comprise administering an effective amount of at least one composition or pharmaceutical composition comprising at least one CDR mimetibody or specified portion or variant 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 cardiovascular disease in a cell, tissue, organ, animal, or patient, including, but not limited to, at least one of cardiac stun syndrome, myocardial infarction, congestive heart failure, stroke, ischemic stroke, hemorrhage, arteriosclerosis, atherosclerosis, diabetic ateriosclerotic 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), 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, dilated congestive cardiomyopathy, restrictive cardiomyopathy, valvular heart diseases, endocarditis, pericardial disease, cardiac tumors, aordic and peripheral aneuryisms, aortic dissection, inflammation of the aorta, occulsion of the abdominal aorta and its branches, peripheral vascular disorders, occulsive arterial disorders, peripheral atherlosclerotic disease, thromboangitis obliterans, functional peripheral arterial disorders, Raynaud's phenomenon and disease, acrocyanosis, erythromelalgia, venous diseases, venous thrombosis, varicose veins, arteriovenous fistula, lymphederma, lipedema, unstable angina, reperfusion injury, post pump syndrome, ischemia-reperfusion injury, and the like. Such a method can optionally comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one CDR mimetibody or specified portion or variant to a cell, tissue, organ, animal or patient in need of such modulation, treatment or therapy.

Any method of the present invention can comprise administering an effective amount of a composition or pharmaceutical composition comprising at least one CDR mimetibody or specified portion or variant 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 immune diseases, wherein the administering of said at least one CDR mimetibody, 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, a muscle relaxant, a narcotic, a non-steroid anti-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 macrolide, a penicillin, a sulfonamide, a tetracycline, another antimicrobial), an antipsoriatic, a corticosteriod, an anabolic steroid, a diabetes related agent, a mineral, a nutritional, a thyroid agent, a vitamin, a calcium related hormone, an antidiarrheal, an antitussive, an antiemetic, an antiulcer, a laxative, an anticoagulant, an erythropieitin (e.g., epoetin alpha), a filgrastim (e.g., G-CSF, Neupogen), a sargramostim (GM-CSF, Leukine), an immunization, an immunoglobulin, an immunosuppressive (e.g., basiliximab, cyclosporine, daclizumab), a growth hormone, a hormone replacement drug, an estrogen receptor modulator, a mydriatic, a cycloplegic, an alkylating agent, an antimetabolite, a mitotic inhibitor, a radiopharmaceutical, an antidepressant, antimanic agent, an antipsychotic, an anxiolytic, a hypnotic, a sympathomimetic, a stimulant, donepezil, tacrine, an asthma medication, a beta agonist, an inhaled steroid, a leukotriene inhibitor, a methylxanthine, a cromolyn, an epinephrine or analog, dornase alpha (Pulmozyme), a cytokine or a cytokine antagonist. Suitable dosages are well known in the art. See, e.g., Wells et al., eds., Pharmacotherapy Handbook, 2nd 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. Mimetibodies can also be used ex vivo, such as in autologous marrow culture. Briefly, bone marrow is removed from a patient prior to chemotherapy and treated with TPO and/or EPO, optionally in combination with mimetibodies, optionally in combination with one or more additional cytokines. The treated marrow is then returned to the patient after chemotherapy to speed the recovery of the marrow. In addition, TPO, alone and in combination with EPO mimetibodies and/or EPO, can also be used for the ex vivo expansion of marrow or peripheral blood progenitor (PBPC) cells. Prior to chemotherapy treatment, marrow can be stimulated with stem cell factor (SCF) or G-CSF to release early progenitor cells into peripheral circulation. These progenitors are optionally collected and concentrated from peripheral blood and then treated in culture with TPO and mimetibodies, optionally in combination with one or more other cytokines, including but not limited to SCF, G-CSF, IL-3, GM- CSF, IL-6 or IL-11, to differentiate and proliferate into high-density megakaryocyte cultures, which are optionally then be returned to the patient following high-dose chemotherapy. Doses of TPO for ex vivo treatment of bone marrow will be in the range of 100 pg/ml to 10 ng/ml, preferably 500 pg/ml to 3 ng/ml. Doses of mimetibodies will be equivalent in activity to EPO which can be used from 0.1 units/ml to 20 units/ml, preferably from 0.5 units/ml to 2 units/ml, or any range or value therein. TNF antagonists suitable for compositions, combination therapy, co-administration, devices and/or methods of the present invention (further comprising at least one anti body, specified portion and variant thereof, of the present invention), include, but are not limited to, anti-TNF antibodies, ligand-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.g., captopril); and compounds which block and/or inhibit TNF production and/or synthesis, such as MAP kinase inhibitors.

As used herein, a "tumor necrosis factor antibody," "TNF antibody," "TNFα antibody," or fragment and the like decreases, blocks, inhibits, abrogates or interferes with TNFα activity in vitro, in situ and/or preferably in vivo. For example, a suitable TNF human antibody of the present invention can bind TNFα and includes anti-TNF antibodies, antigen-binding fragments thereof, and specified mutants or domains thereof that bind specifically to TNFα. A suitable TNF antibody 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 anti-human TNFα 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. In a particular embodiment, 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 TNFα in a dose dependent manner. From binding assays of chimeric antibody cA2 and recombinant human TNFα, the affinity constant of chimeric antibody cA2 was calculated to be 1.04xl010M"1. Preferred methods for determining monoclonal antibody specificity and affinity by competitive inhibition can be found in Harlow, et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York, 1988; Colligan et al, eds., Current Protocols in Immunology, Greene Publishing Assoc. and Wiley Interscience, New York, (1992-2000); Kozbor et al, Immunol. Today, 4:12-19 (1983); Ausubel et al, eds. Current Protocols in Molecular Biology, Wiley Interscience, New York (1987-2000); and Muller, Meth. Enzymol, P2:589-601 (1983), which references are entirely incorporated herein by reference.

In a particular embodiment, 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 anti-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; Moller, 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 etal, EPO Patent Publication No. 0 218 868 (published April 22, 1987); Yone et al, EPO Patent Publication No. 0 288 088 (October 26, 1988); Liang, et al, Biochem. Biophys. Res. Comm. 737:847-854 (1986); Meager, et al, Hybridoma 5:305-311 (1987); Fendly et al, Hybridoma 6:359-369 (1987); Bringman, et al, Hybridoma 5:489-507 (1987); and Hirai, et al, J. Immunol. Meth. 96:57-62 (1987), which references are entirely incorporated herein by reference). TNF Receptor Molecules Preferred TNF receptor molecules useful in the present invention are those that bind TNFα with high affinity (see, e.g., Feldmann et al, International Publication No. WO 92/07076 (published April 30, 1992); Schall et al, Cell 57:361-370 (1990); and Loetscher et al, Cell 57:351-359 (1990), which references are entirely incorporated herein by reference) and optionally possess low immunogenicity. In particular, 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 TNFα inhibitory binding proteins (Engelmann, H. et al, J. Biol. Chem. 255:1531-1536 (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, may 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. These multimeric molecules and methods for their production have been described in U.S. Application No. 08/437,533 (filed May 9, 1995), the content of which is entirely incorporated herein by reference.

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. Sci. USA £5:10535-10539 (1991); Peppel et al, J. Exp. Med.

77-^:1483-1489 (1991); Rolls et al, Proc. Natl. Acad. Sci. USA 91:2\5-2\9 (1994); Butler et al, Cytokine 5(6):616-623 (1994); Baker et al, Eur. J. Immunol. 24:2040-2048 (1994); Beutler et al, U.S. Patent No. 5,447,851; and U.S. Application No. 08/442,133 (filed May 16, 1995), each of which references are entirely incorporated herein by reference). Methods for producing immunoreceptor fusion molecules can also be found in Capon et al, U.S. Patent No. 5,116,964; Capon et al, U.S. Patent No. 5,225,538; and Capon et al, Nature 337:525-531 (1989), which references are entirely incorporated herein by reference.

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 TNFα 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 TNFα with high affinity and possess low immunogenicity). For example, 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 al, Current Protocols in Molecular Biology, Greene Publishing Assoc. and Wiley-Interscience, New York (1987- 2000).

Cytokines include, but are not limited to all known cytokines. 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 protein mediated disorder, comprising administering an effective amount of a composition or pharmaceutical composition comprising at least one CDR mimetibody or specified portion or variant 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 immune diseases, wherein the administering of said at least one CDR mimetibody, specified portion or variant thereof, further comprises administering, before concurrently, and/or after, at least one selected from at least one other cytokines such as IL-3,-6 and -11; stem cell factor; G-CSF and GM-CSF. Within regimens of combination therapy, daily doses of other cytokines will in general be: GM-CSF, 5-15 .mu.g/kg; IL-3, 1-5 lg/kg; and G-CSF, 1-25 .mu.g/kg. Combination therapy with GM-CSF, for example, is indicated in patients with low neutrophil levels.

Typically, treatment of pathologic conditions is effected by administering an effective amount or dosage of at least one CDR mimetibody composition that total, on average, a range from at least about 0.01 to 500 milligrams of at least one CDR mimetibody or specified portion or variant /kilogram of patient per dose, and preferably from at least about 0.1 to 100 milligrams CDR mimetibody or specified portion or variant /kilogram of patient per single or multiple administration, depending upon the specific activity of contained in the composition. Alternatively, the effective serum concentration can comprise 0.1-5000 Dg/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 can optionally include 0.01, 0.02, 0.03, 0.04, 0.05. 0.06, 0.07, 0.08, 009, 0.1, 0.2,

0.3, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, and/or 30 mg/kg/administration, or any range, value or fraction thereof, or to achieve a serum concentration of 0.1, 0.5, 0.9, 1.0, 1.1, 1.2, 1.5, 1.9, 2.0, 2.5, 2.9, 3.0, 3.5, 3.9, 4.0, 4.5, 4.9, 5.0, 5.5, 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 20, 12.5, 12.9, 13.0, 13.5, 13.9, 14.0, 14.5, 4.9, 5.0, 5.5., 5.9, 6.0, 6.5, 6.9, 7.0, 7.5, 7.9, 8.0, 8.5, 8.9, 9.0, 9.5, 9.9, 10, 10.5, 10.9, 11, 11.5, 11.9, 12, 12.5, 12.9, 13.0, 13.5, 13.9, 14, 14.5, 15, 15.5, 15.9, 16, 16.5, 16.9, 17, 17.5, 17.9, 18, 18.5, 18.9, 19, 19.5, 19.9, 20, 20.5, 20.9, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 96, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, and/or 5000 μg/ml serum concentration per single or multiple administration, or any range, value or fraction thereof.

Alternatively, 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. Usually a dosage of active ingredient can be about 0.1 to 100 milligrams per kilogram of body weight. Ordinarily 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.

As a non-limiting example, treatment of humans or animals can be provided as a one-time or periodic dosage of at least one CDR mimetibody or specified portion or variant of the present invention 0.01 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, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40, or alternatively, at least one of week 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20, or any combination thereof, using single, infusion or repeated doses.

Dosage forms (composition) suitable for internal administration generally contain from about 0.0001 milligram to about 500 milligrams of active ingredient per unit or container. In these pharmaceutical compositions the active ingredient will ordinarily be present in an amount of about 0.5-95% by weight based on the total weight of the composition.

For parenteral administration, the CDR mimetibody or specified portion or variant can be formulated as a solution, suspension, emulsion or lyophilized powder in association, or separately provided, with a pharmaceutically acceptable parenteral vehicle. Examples of such vehicles are water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Liposomes and nonaqueous vehicles such as fixed oils may also be used. The vehicle or lyophilized powder may 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 phaπnaceutical carriers are described in the most recent edition of Remington's Pharmaceutical Sciences, A. Osol, a standard reference text in this field.

Therapeutic Administration

Many known and developed modes of can be used according to the present invention for administering pharmaceutically effective amounts of at least one CDR mimetibody or specified portion or variant according to the present invention. While pulmonary administration is used in the following description, other modes of administration can be used according to the present invention with suitable results.

A CDR mimetibody of the present invention can be delivered in a carrier, as a solution, emulsion, colloid, or suspension, or as a 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.

Parenteral Formulations and Administration

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. As the usable vehicle or solvent, water, Ringer's solution, isotonic saline, etc. are allowed; as an ordinary solvent, or suspending solvent, sterile involatile oil can be used. For these purposes, 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.

Alternative Delivery

The invention further relates to the administration of at least one CDR mimetibody or specified portion or variant by parenteral-, subcutaneous, intramuscular, intravenous, bolus, vaginal, rectal, buccal, sublingual, intranasal, or transdermal means. Protein, CDR mimetibody or specified portion or variant compositions can be prepared for use for parenteral (subcutaneous, intramuscular or intravenous) administration particularly in the form of liquid solutions or suspensions; for use in vaginal or rectal administration particularly in semisolid forms such as creams and suppositories; for buccal, or sublingual administration particularly in the form of tablets or capsules; or intranasally particularly in the form of powders, nasal drops or aerosols or certain agents; or transdermally particularly in the form of 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. In "Drug Permeation Enhancement"; Hsieh, D. S., Eds., pp. 59-90 (Marcel Dekker, Inc. New York 1994, entirely incorporated herein by reference), or with oxidizing agents that enable the application of formulations containing proteins and peptides onto the skin (WO 98/53847), or applications of electric fields to create transient transport pathways such as electroporation, or to increase the mobility of charged drugs through the skin such as iontophoresis, or application of ultrasound such as sonophoresis (U.S. Pat. Nos. 4,309,989 and 4,767,402) (the above publications and patents being entirely incorporated herein by reference).

Pulmonary/Nasal Administration

For pulmonary administration, preferably at least one CDR mimetibody or specified portion or variant composition is delivered in a particle size effective for reaching the lower airways of the lung or sinuses. According to the invention, at least one CDR mimetibody or specified portion or variant 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. These devices 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 CDR mimetibody or specified portion or variants are also known in the art. All such devices can use of formulations suitable for the administration for the dispensing of CDR mimetibody or specified portion or variant 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 Turbuhaler™ (Astra), Rotahaler® (Glaxo), Diskus® (Glaxo), Spiros™ 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 AERx™ 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. Preferably, a composition comprising at least one CDR mimetibody or specified portion or variant is delivered by a dry powder inhaler or a sprayer. There are a several desirable features of an inhalation device for administering at least one CDR mimetibody or specified portion or variant of the present invention. For example, 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 μm, preferably about 1-5 μm, for good respirability.

Administration of CDR mimetibody or specified portion or variant Compositions as a Spray

A spray including CDR mimetibody or specified portion or variant composition protein can be produced by forcing a suspension or solution of at least one CDR mimetibody or specified portion or variant 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.

Advantageously, particles of at least one CDR mimetibody or specified portion or variant composition protein delivered by a sprayer have a particle size less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm.

Formulations of at least one CDR mimetibody or specified portion or variant composition protein suitable for use with a sprayer typically include CDR mimetibody or specified portion or variant composition protein in an aqueous solution at a concentration of about 1 mg to about 20 mg of at least one CDR mimetibody or specified portion or variant composition 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 CDR mimetibody or specified portion or variant composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating CDR mimetibody or specified portion or variant composition proteins include albumin, protamine, or the like. Typical carbohydrates useful in formulating CDR mimetibody or specified portion or variant composition proteins include sucrose, mannitol, lactose, trehalose, glucose, or the like. The CDR mimetibody or specified portion or variant composition protein formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the CDR mimetibody or specified portion or variant composition protein 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 mimetibodies, or specified portions or variants, can also be included in the formulation.

Administration of CDR mimetibody or specified portion or variant compositions by a Nebulizer CDR mimetibody or specified portion or variant composition protein can be administered by a nebulizer, such as jet nebulizer or an ultrasonic nebulizer. Typically, in a jet nebulizer, a compressed air source is used to create a high-velocity air jet through an orifice. As the gas expands beyond the nozzle, a low-pressure region is created, which draws a solution of CDR mimetibody or specified portion or variant composition protein 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. In 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 CDR mimetibody or specified portion or variant composition protein either directly or through a coupling fluid, creating an aerosol including the CDR mimetibody or specified portion or variant composition protein. Advantageously, particles of CDR mimetibody or specified portion or variant composition protein delivered by a nebulizer have a particle size less than about 10 μm, preferably in the range of about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm.

Formulations of at least one CDR mimetibody or specified portion or variant suitable for use with a nebulizer, either jet or ultrasonic, typically include CDR mimetibody or specified portion or variant composition protein in an aqueous solution at a concentration of about 1 mg to about 20 mg of at least one CDR mimetibody or specified portion or variant 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 CDR mimetibody or specified portion or variant composition protein, such as a buffer, a reducing agent, a bulk protein, or a carbohydrate. Bulk proteins useful in formulating at least one CDR mimetibody or specified portion or variant composition proteins include albumin, protamine, or the like. Typical carbohydrates useful in formulating at least one CDR mimetibody or specified portion or variant include sucrose, mannitol, lactose, trehalose, glucose, or the like. The at least one CDR mimetibody or specified portion or variant formulation can also include a surfactant, which can reduce or prevent surface-induced aggregation of the at least one CDR mimetibody or specified portion or variant 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 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 CDR mimetibody or specified portion or variant protein can also be included in the formulation.

Administration of CDR mimetibody or specified portion or variant compositions By A Metered Dose Inhaler

In a metered dose inhaler (MDI), a propellant, at least one CDR mimetibody or specified portion or variant, 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 the size range of less than about 10 μm, preferably about 1 μm to about 5 μm, and most preferably about 2 μm to about 3 μm. The desired aerosol particle size can be obtained by employing a formulation of CDR mimetibody or specified portion or variant composition protein 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 3 M or Glaxo and employing a hydrofluorocarbon propellant.

Formulations of at least one CDR mimetibody or specified portion or variant for use with a metered-dose inhaler device will generally include a finely divided powder containing at least one CDR mimetibody or specified portion or variant 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

8? hydrochlorofluorocarbon, a hydrofluorocarbon, or a hydrocarbon, including trichlorofluoromethane, dichlorodifluoromethane, dichlorotetrafluoroethanol and 1,1,1,2-tetrafluoroethane, HFA-134a (hydrofluroalkane-134a), HFA-227 (hydrofluroalkane-227), or the like. Preferably the propellant is a hydrofluorocarbon. The surfactant can be chosen to stabilize the at least one CDR mimetibody or specified portion or variant 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. One of ordinary skill in the art will recognize that the methods of the current invention can be achieved by pulmonary administration of at least one CDR mimetibody or specified portion or variant compositions via devices not described herein. Mucosal Formulations and Administration

For absorption through mucosal surfaces, compositions and methods of administering at least one CDR mimetibody or specified portion or variant 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. For buccal administration excipients include sugars, calcium stearate, magnesium stearate, pregelinatined starch, and the like (U.S. Pat. Nos. 5,849,695).

Oral Formulations and Administration

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. 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. 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.

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 may contain inactive diluting agents 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). Furthermore, 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. Transdermal Formulations and Administration

For transdermal administration, the at least one CDR mimetibody or specified portion or variant 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 number of 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).

Prolonged Administration and Formulations

It can be sometimes desirable to deliver the compounds of the present invention to the subject over prolonged periods of time, for example, for periods of one week to one year from a single administration. Various slow release, depot or implant dosage forms can be utilized. For example, 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, copper, cobalt, nickel, cadmium and the like, or with an organic cation formed from e.g., N,N'- dibenzyl-ethylenediamine or ethylenediamine; or (c) combinations of (a) and (b) e.g. a zinc tannate salt. Additionally, 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. Another type of 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).

Having generally described the invention, the same will be more readily understood by reference to the following examples, which are provided by way of illustration and are not intended as limiting.

Example 1: Cloning and Expression of EPO CDR mimetibody in Mammalian Cells

A typical mammalian expression vector contains at least one promoter element, which mediates the initiation of transcription of mRNA, the CDR mimetibody or specified portion or variant 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, HTVI and the early promoter of the cytomegalovirus (CMV). However, cellular elements can also be used (e.g., the human actin promoter). 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 PMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC 67109). Mammalian host cells that could be used include human Hela 293, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7 and CV 1, quail QC1-3 cells, mouse L cells and Chinese hamster ovary (CHO) cells.

Alternatively, 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 CDR mimetibody or specified portion or variant. 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 often used for the production of CDR mimetibody or specified portion or variants.

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. Cloning and Expression in CHO Cells

The vector pC4 is used for the expression of CDR mimetibody or specified portion or variant. 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. The amplification of the DHFR genes in cells resistant to methotrexate (MTX) has been well documented (see, e.g., F. W. Alt, et al., J. Biol. Chem. 253:1357-1370 (1978); J. L. Hamlin and C. Ma, Biochem. et Biophys. Acta 1097:107-143 (1990); and M. J. Page and M. A. Sydenham, Biotechnology 9:64-68 (1991)). Cells grown in increasing concentrations of MTX develop resistance to the drug by overproducing the target enzyme, DHFR, as a result of amplification of the DHFR gene. 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 for expressing the gene of interest 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. Behind these cloning sites 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 EPO in a regulated way in mammalian cells (M. Gossen, and H. Bujard, Proc. Natl. Acad. Sci. USA 89: 5547-5551 (1992)). For the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well. 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 is 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 complete CDR mimetibody or specified portion or variant is used, e.g., as presented in SEQID NOS: 13, 14, 15, 16, 17, 18, corresponding to HC and LC variable regions of a CDR mimetibody of the present invention, according to known method steps. Isolated nucleic acid encoding a suitable human constant region (i.e., HC and LC regions) is also used in this construct (e.g., as provided in vector p 1351.

The isolated variable and constant region encoding DNA and the dephosphorylated vector are then ligated with T4 DNA ligase. E. coli HB101 or XL-1 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 μg of the expression plasmid pC4 is cotransfected with 0.5 μg of the plasmid pSV2-neo using lipofectin. The plasmid pSV2neo 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 μg /ml G418. After 2 days, 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 μg /ml G418. After about 10-14 days single clones are trypsinized and then seeded in 6-well petri dishes or 10 ml flasks using 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 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.

Example 2: Examplary CDR Mimetibodies Background. Erythropoietin has a relatively short half-life. Intravenously administered erythropoietin is eliminated at a rate consistent with first order kinetics with a circulating half-life ranging from approximately 3 to 4 hours in patients with CRF. Within the therapeutic dose range, detectable levels of plasma erythropoietin are maintained for at least 24 hours. After subcutaneous administration of erythropoietin, peak serum levels are achieved within 5-24 hours and decline slowly thereafter. The C max and t V_ after administration of erythropoietin were 1.80 ± 0.7 U/mL and 19.0 ±

5.9 hours, respectively.

Starting doses of erythropoietin range from 50-150 U/kg three times weekly. The dosage of erythropoietin must be individualized to maintain the hematocrit within the suggested target range. For surgery patients the recommended dose of erythropoietin is 300 U/kg/day s.c. for 10 days before surgery, on the day of surgery, and for 4 days after surgery or alternatively 600 U/kg s.c. in once weekly doses (21, 14 and 7 days before surgery) plus a fourth dose on the day of surgery.

The 1998 world wide market size for therapeutic proteins was $16.6B, of which the largest segment was $3.6B (21%) for erythropoietin. Erythropoietin is used to treat anemia. The market is large, partly because the disorder can result from other kinds of diseases, such as cancer or renal dialysis.

Small peptidomimetics of erythropoietin were identified by several groups through screening of random phage display peptide libraries for affinity to the erythropoietin receptor. These sequences have no homology with erythropoietin. In functional assays several of these peptides showed activity, but only 1/100,000* that of recombinant erythropoietin. The consensus sequences

YXCXXGPXTWXCXP SEQ ID NO:980) and CXXGWVGXCXXW (SEQ ID NO:981) that have no homology with erythropoietin were identified based solely on receptor binding. One group has > claimed peptides of the motif XXXGPXTWXX (SEQ ID NO:982) that are erythropoietin agonists while another group claims peptides of the motif XXXGPXTWXX (SEQ ID NO:983) that, when dimeric, activate the erythropoietin receptor but that are inactive as monomers.

Although several attempts have been made to increase the potency of these peptides by preparing covalent dimers or multimers of peptidomimetics, these compounds are still 1,000 - 10,000 fold less active than erythropoietin on a molar basis.

Peptide sequences from erythropoietin have also been claimed as agonistic. Increased activity of dimerized sequences comprising any or all of the native erythropoietin sequence has also been reported.

These compounds have little or no oral bioavailability and their activity does not make them economically viable at this time.

The invention provides for the design, production and use of erythropoietic mimetibodies, compounds that support peptidomimetids on an antibody scaffold. It is anticipated that these compounds will have erythropoietic activity similar to recombinant or native erythropoietin or analogs thereof, but will be superior because of extended half-life. This will permit sustained activity with less frequent dosing.

Using molecular modeling, a germline antibody sequence and the coordinates of the EMP- 1/EPO receptor crystal structure, six CDR mimetibody structures were designed. The germline antibody sequence is used merely to illustrate the concept. It is expected that any number of other antibodies of any isotype can be used. Likewise, although the structure of EMP-1 is used to create an erythropoietic CDR mimetibody, it is expected that any number of peptidomimetic structures can be substituted to create active mimetibodies. While the concept is designed to work with peptidomimetics of proteins that cause receptor multimerization that lead to activity, monomeric binding leading to receptor activation is envisioned as well. The six mimetibodies have the peptides attached to six different combinations of CDRs. Other constructs using different combinations of CDRs, different amino acids as attachment points for grafting the peptidomimetids onto the CDRs and different lengths and compositions of joining regions between the peptidomimetics and the antibodies can be used as well.

The sequences of the six example mimetibodies aligned with the germline antibody sequence are shown below.

Table 18: (SEQ IDNOS:984-990)

Light Chain

Figure imgf000091_0001

/0

7^ ^τχ-(r τ ~y~' γ-T-v; — p; -"" I Hv Pr- J

- - . 5 G I ϋ r i i - j L .. r> . _ Li A V v V ) R j" t I 1 L i V/ PR

7. F r "" . r n y - Q j-j p f I -EV TO 6 P . < G C I D i O O P i - tV4 PHO k r .- „ C S G T D T L ' i S - L C f E D ' A V Y f - 0 . f Si J L- W FPO

R F S C C " T π T I ' s i F T F r - v v Y f f 01 R -.T- .-riΛ. ΓPO

K I G 5 u - - I ϋ τ ι J i L P D A V ϊ l U l k N _. X\ . PI .

T F P ¥ r v -TV TO I I P s. k ' D I _. . w PΓ C 1 I T r F C v D I K _.-C\-

J' I . t i . K v 1 I t FV4 DRO

99 i r G .i P i r I P b K V I- I . L Wo &hO r4 . _ -. r F T F r . T Y V T I _-B/. TO V ,° Γ F . F p c i K v D K I -V ri PPC on o -n <jf "51 αf!' ( th __\ br q . yel l o w u -. t ft .t c f f rr 1 S'Nlh t^rorr -. i i^rrroi #/ H.x t due not < i f t [ . I Ml π PRO

Table 19: (SEQ ID NOS: 991-996)

Heavy Chain

10 20 30

Q V Q L V E S G G G V V Q P G R S L R L s C A A S G F T F S H-EM1.PR0

Q V Q L V E S G G G V V Q P G R S L R L s C A A S G F T F s H-EM2.PR0

Q V Q L V E S G G G V V Q P G R S L R L s C A A S G F T F s H-EM3.PR0

Q V Q L V E S G G G V V Q P G R S L R L s C A A S G F T F s H-EM4 PR0

Q V Q L V E s G G G V V Q P G R S L R L s C A A S G F T F s H-EM5 PRO

Q V Q L V E s G G G V V Q P G R S L R L s C A A S G F T F s H-SYNTH.PRO

40 50

31 S Y A M H W V R Q A P G K G L E W V A V 1 S - - Y - H-EM1.PR0

31 S Y A M H V R 0 A P G K G L E W V A V 1 S - Y H-EM2.PR0

31 S Y A M H W V R Q A P G K G L E W V A V s G 3 G G T Y S H H-EM3.PR0

31 S Y A M H W V R Q A P G K G L E W V A V | E S t G r Y S c H H-EM4.PR0

31 S Y A H V R Q A P G K G L E V A V 1 s _ - . Y - - H-EM5.PR0

31 S Y A H V R Q A P G K G L E W V A V 1 s - - - - Y - - - H-SYNTH.PRO

Figure imgf000092_0001
O

80 90 100

74 N s K N T L Y 1 Q M N S L R A E D T A V Y Y C A R D R G 1 Is H-EM1 PRO

74 N s K N T L Y L Q M N S L R A E D T A V Y Y C A R D R G 1 G H-EM2 PRO

91 N s K N T L Y L Q M N S L R A E D T A V Y Y C A R D R G 1 H-EM3.PR0

89 N s K N T L Y L Q M N S L R A E D T A V Y Y C A R D R G 1 G H-EM4 PRO

74 N s K N T L Y L Q M N S L R A E D T A V Y Y C A R 0 R G 1- E H-EM5.PR0

74 N s K N T L Y L Q N S L R A E D T A V Y Y C A R 0 R G 1 G H-SYNTH.PRO

110

104 TΓ G G 1 Y s C H r G P L T ¥ V C K P Q G S j Y Y Y Y G M D V H-EMi.PRO

104 G Q N Y Y Y Y G M D V H-E 2.PR0

121 r P - - - - _ -. -. - t - - Y ϊ G il D V H-E 3.PR0

119 G Q N Y Y Y Y G M D V H-E 4.PR0

103 G s G G T Y s C H F G P L T W V c K P 0 V G G s G - 5 | M D V H-E 5.PR0

104 G W o N Y Y Y Y G D V H-SYNTH.PRO

The three dimensional structures of the molecular models of the variable regions of the six structures were calculated and plotted and determined to provide suitable presentation of the exemplary therapeutic proteins and polypeptides for therapeutic activity. Immunoglobulins are known to have long half-lives as compared to both peptide and non-peptide drugs. The joining of two EMP-like peptides to the CDRs of an immunoglobulin provides the necessary structure for receptor dimerization and is expected to have a half-life that will allow for dosing only 1-2 times per month or less. Given the extended half-life smaller amounts of material may be required for similar erythropoietic activity. It will be clear that the invention can be practiced otherwise than as particularly described in the foregoing description and examples.

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

Claims

WHAT IS CLAIMED IS:
1. A CDR mimetibody, comprising at least one portion of a heavy chain or light chain variable region comprised of at least one human framework region and at least one ligand binding region (LBR) comprised of at least one biologically active polypeptide, which LBR further comprises at least one amino acid from at least one complementarity determing region (CDR) of a heavy or light chain variable region, wherein said biologically active polypeptide is selected from at least one of SEQ ID NOS-1-1109.
2. A CDR mimetibody according to claim 1, wherein said at least one CDR is CDR1,
CDR2 or CDR3 of the heavy or light chain variable region and said at least one human framework region is FR1, FR2 or FR3 of the heavy or light chain variable " region.
3. A CDR mimetibody according to claim 1, wherein said at least one CDR amino y acid sequence optionally further comprises at least one specified substitution, insertion or deletion.
4. A composition comprising at least one CDR mimetibody according to claim 1 and a suitable carrier or diluent.
5. A composition according to claim 4, wherein the carrier or diluent is y pharmaceutically acceptable.
6. A composition according to claim 4, wherein said composition further comprises at , least one further compound, protein or composition.
7. A method for providing at least one CDR mimetibody according to claim 1 in a host - cell, comprising culturing a host cell under conditions wherein at least one CDR mimetibody is expressed in detectable and/or recoverable amounts.
8. A method for treating a biologically active protein related pathology, comprising
(a) administering a therapeutically effective amount of at least one CDR mimetibody according to claim 1. J
9. A method according to claim 8, wherein the pathology is selected from at least one of a(n) anemia condition; a(n) immune/autoimmune condition; and/or a(n) cancer/infecteous condition.
10. The present invention further provides at least one CDR mimetibody, specified portion or variant in a method or composition, when administered in a therapeutically effective amount, for modulation, for treating or reducing the symptoms of, at least one immune, cardiovascular, infectious, malignant, and/or neurologic disease in a cell, tissue, organ, animal or patient and/or, as needed in many different conditions, such as but not limited to, prior to, subsequent to, or during a related disease or treatment condition, as known in the art and/or as described herein.
11. An anti-idiotype antibody that specifically binds at least one CDR mimetibody according to claim 1.
12. An isolated nucleic acid encoding an anti-idio-type antibody according to claim 11.
13. A nucleic acid vector, comprising an isolated nucleic acid according to claim 12.
14. A host cell comprising a nucleic acid according to claim 13.
15. A CDR mimetibody according to claim 1 , wherein the CDR region is derived from a mammal.
16. A CDR mimetibody according to claim 12, wherein the mammal is selected from a human, a mouse, a rabbit, a rat, a rodent, or a primate.
17. A CDR mimetibody isolated nucleic acid molecule, comprising, complementary, or hybridizing to, a polynucleotide encoding at least one CDR mimetibody according to claim 1.
18. A nucleic acid vector, comprising an isolated nucleic acid according to claim 17.
19. A host cell comprising a nucleic acid according to claim 18.
20. A CDR mimetibody according to claim 1, wherein said LBR comprises at least one selected from (i) all of the heavy chain complimentarity determining regions
(CDR); or (ii) all of the light chain CDR amino acids sequences.
21. A CDR mimetibody according to claim 1, wherein said CDR mimetibody binds at least one ligand for said ligand binding region with an affinity of at least one selected from at least 10"9 M, at least 10"10 M, at least 10"11 M, or at least 10"12 M.
22. A CDR mimetibody according to claim 21, wherein said CDR mimetibody substantially neutralizes at least one activity of at least one of said ligand.
23. A host cell according to claim 19, wherein said host cell is at least one selected from COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, Hep G2, 653, SP2/0, 293, HeLa, myeloma, or lymphoma cells, or any derivative, immortalized or transformed cell thereof.
24. A method for producing at least one CDR mimetibody, comprising translating at least one CDR mimetibody nucleic acid according to claim 17, under conditions in vitro, in vivo or in situ, such that the CDR mimetibody is expressed in detectable or recoverable amounts.
25. A composition according to claim 6, wherein at least one further compound, protein or composition is selected from at least one of a detectable label or reporter, a TNF antagonist, an antirheumatic, a muscle relaxant, a narcotic, a non-steroid anti- inflammatory drug (NTHE), an analgesic, an anesthetic, a sedative, a local anethetic, 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 epinephrine or analog, a cytokine, or a cytokine antagonist.
26. A method according to claim 8, wherein said effective amount is 0.001-50 mg/kilogram of the cells, tissue, organ or animal.
27. A method according to claim 8, wherein said administering is by at least one mode selected from 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.
28. A method according to claim 8, further comprising administering, prior, concurrently or after the (a) 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, a non-steroid anti-inflammatory drug (NSAID), an analgesic, an anesthetic, a sedative, a local anethetic, 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 epinephrine or analog, a cytokine, or a cytokine antagonist.
29. A medical device, comprising at least one isolated mammalian CDR mimetibody according to claim 1, wherein the device is suitable to contacting or administerting the at least one CDR mimetibody by at least one mode selected from 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.
30. 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 CDR mimetibody according to claim 1.
31. An article of manufacture according ot claim 30, wherein the article of manufacture further comprises at least one container as a component of a 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 delivery device or system.
32. A method for providing at least one isolated mammalian CDR mimetibody according to claim 1, comprising providing a host cell or transgenic animal or transgenic plant or plant cell capable of expressing in recoverable amounts the CDR mimetibody.
33. At least one CDR mimetibody produced by a method according to claim 32.
34. A CDR mimetibody according to claim 1, wherein said light chain variable region comprises at least one amino acid sequence selected from SEQ ID NOS:984-990.
35. A CDR mimetibody according to claim 1, wherein said heavy chain variable region comprises at least one amino acid sequence selected from SEQ ID NOS:991-996.
95
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EP1572079A4 (en) 2006-09-06
AU2003222069A1 (en) 2003-10-20
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WO2003084477A3 (en) 2005-09-09
EP1572079A2 (en) 2005-09-14

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