ME02086B - METHOD AND COMPOSITIONS FOR THE TREATMENT AND PREVENTION OF DISEASES ASSOCIATED WITH ALPHA V BETA 5-INTEGRIN - Google Patents

Description

Description

STATE OF THE ART

Pulmonary edema ("PE") affects millions of people each year, causing significant morbidity and mortality. In patients with PE, the alveoli are filled with fluid from the pulmonary capillaries, which compromises the transfer of oxygen to the systemic circulation (Hali et al. in CURRENT THERAPY IN RESPIRATORY MEDICINE (R. Chemiack, Ed., 1986), pp. 222-227). This sequence of events leads to hypoxemia, hypercapnia, and death unless corrective measures are taken.

Any condition or agent that disrupts fluid homeostasis in the lung can lead to PE, which can be broadly divided into cardiogenic and non-cardiogenic PE (see, e.g., Kakouros and Kakouros, Hellenic J. Cardiol. 44: 385-391 (2003 ).For example, acute lung injury/adult (acute) respiratory distress syndrome or "ARDS," which can develop as a result of lung damage due to, eg, pneumonia, septic shock, trauma, aspiration of vomited contents, inhalation of chemicals, is often associated with non-cardiogenic PE. Non-cardiogenic PE is characterized by a change in the permeability of the vasculature of the lung tissue, which leads to an increase in the level of fluid in the lungs. Cardiogenic PE is often caused by weakening of the left side of the heart, and can be a complication of a heart attack, loosening or narrowing of the heart valves ( mitral or aortic valves), or any heart disease that leads to weakening and/or stiffening of the heart muscle (cardiomyopathy). A failing heart transmits its own increased pressure to the pulmonary veins. As the pressure in the pulmonary veins increases, fluid is forced into the air spaces (alveoli). This liquid then becomes a barrier to the normal exchange of oxygen, which results in short breaths. Cardiogenic PE is characterized by increased capillary hydrostatic pressure, which leads to increased fluid levels in the lungs.

PE is caused, for example, by altered capillary permeability; infection; inhaled or circulating toxins; vasoactive substances (eg, histamine, kinins); disseminated intravascular coagulation; immune reactions; radiation-related pneumonia; uremia; near drowning; smoke inhalation and acute respiratory distress syndrome; weakening of the left ventricle; mitral stenosis; bacterial endocarditis; pulmonary vein fibrosis; congenital stenosis originating in the pulmonary veins; pulmonary veno-occlusive disease; excessive fluid infusion; hypoalbuminemia (eg, renal, hepatic, nutritional, or protein-losing enteropathy); great height; drug overdose, CNS injury, subarachnoid hemorrhage, pulmonary embolism, pulmonary parenchymal disease, eclampsia, anesthesia, and cardiopulmonary bypass surgery.

Symptoms of PE may include, for example, shortness of breath, rapid and/or labored breathing, tachycardia, hypertension, chest tightness, cold extremities with or without accompanying cyanosis, cough with frothy or pink sputum, extensive use of accessory respiratory muscles, moisture accumulation with or without gurgling and their combination. Tests to diagnose PE include: blood tests, such as complete blood count (CBC), blood urea nitrogen (BUN), creatinine and serum proteins, urinalysis, arterial blood gases (ABGs), chest X-ray, and electrocardiograms (ECG), and everything used helps the doctor narrow down the diagnosis to PE.

Treatment of cardiogenic PE usually includes placing the patient on 100% oxygen, morphine to relieve anxiety and provide some beneficial effects on the heart, furosemide for diuresis, vasodilators to reduce the workload under which the myocardium must pump, and inotropic drugs such as gioputamine to the contractility of the heart would increase. Other measures that can be used are rotating clamps on three of the four limbs and reducing the blood volume by 500 ml.

Unfortunately, no specific or satisfactorily effective treatment is available for PE. Therefore, there is a need in this field for more effective and specific therapies for PE. The present invention addresses these and other problems.

BRIEF PRESENTATION OF THE ESSENCE OF THE INVENTION

The present invention provides antibodies and compositions for the treatment or prevention of pulmonary edema or acute lung injury.

The present invention provides an antibody, produced by a hybridoma, deposited as ATCC Deposit No. PTA-5817. The invention also provides an antibody that specifically binds to the αvβ5 integrin, which is a humanized form of the hybridoma-produced antibody deposited as ATCC Deposit No. PTA-5817. The invention also provides a humanized antibody, which specifically binds to the αvβ5 integrin and contains CDRs of the heavy and light chain of the antibody, produced by a hybridoma, which has been deposited as ATCC Deposit No. PTA-5817.

The invention also provides pharmaceutical compositions containing such antibodies and a pharmaceutically acceptable excipient.

The invention also provides such antibodies for use in the treatment or prevention of pulmonary edema. The invention further provides such antibodies for use in the treatment or prevention of acute lung injury. Antibodies can be used with another therapeutic agent to treat pulmonary edema or acute lung injury. Another therapeutic agent may be selected from the group consisting of: a TGFβ inhibitor

times, activated protein C, steroid, GM-CSF, platelet inhibitor, diuretic; bronchodilator agent, antibody that binds to αvβ

5 integrin, β5-binding antibody, second αvβ5 integrin antagonist, αvβ antagonist

5 integrin, (32 agonist or surfactant. Antibodies can be administered intravenously, intranasally or intrabronchially. The subject being treated is a mammal, e.g., a primate, such as a human, monkey or chimpanzee, member of the canine or feline genus.

The invention also provides the use of the antibody of the invention for the production of a drug for the treatment of pulmonary edema. The invention further provides the use of antibodies for the production of a drug for the treatment of acute lung damage.

The invention also provides kits for the treatment or prevention of PE. The kits contain the antibodies described herein and another therapeutic agent for the treatment or prevention of PE. Another therapeutic agent can be selected from the group consisting of: TGFβ inhibitor

puta, activated protein C, steroid, GM-CSF, platelet inhibitor,

diuretic agent; a bronchodilator, an αvβ5 integrin-binding antibody, a β5-binding antibody, another αvβ5 integrin antagonist, an αvβ6 integrin antagonist, a β2 agonist, or a surfactant.

The invention also provides a hybridoma, which has been deposited as ATCC deposit number PTA-5817.

This and other embodiments of the invention are further illustrated by means of the detailed description that follows.

BRIEF DESCRIPTION OF PICTURES

Figure 1 illustratively shows the results of in vivo experiments, showing that β5-/- mice are protected against PE-induced lung injury.

Figure 2 illustrates the results of in vivo experiments, showing that an antibody that specifically binds to β5 (ie, ALULA) reduces the severity of PE induced by ischemia reperfusion.

Figure 3 illustrates the results of in vivo experiments showing that an antibody that specifically binds to β5 (ie, ALULA) reduces the severity of lung injury induced by high ventilatory volume influx.

Figure 4 illustrates the results of in vitro experiments showing that an antibody that specifically binds to β5 (ie, ALULA) blocks the adhesion of αvβ5 integrin-expressing cells to vessels coated with a range of concentrations of the αvβ5 integrin ligand, vitronectin.

DETAILED DESCRIPTION OF THE INVENTION

Introduction

This invention is based in part on the surprising finding that treatment of animals with agents that bind to αvβ5 integrins reduces the symptoms of PE. More specifically, blocking the binding of ligands to αvβ5 integrin can reduce the severity of PE.

The inventors have shown that an antibody that binds to αvβ5 integrin blocks the binding of vitronectin, a ligand of αvβ5 integrin, to αvβ5 integrin. The inventors further demonstrated that administration of an antibody that binds to αvβ5 integrin reduces the severity of PE. Consequently, the invention relates to the treatment or prevention of PE in a subject, by administering an effective amount of this antibody that binds to the worm β5 integrin.

The antibody shown was named "ALULA". The invention also provides pharmaceutical compositions containing such antibodies. As described in more detail in the Examples that follow, ALULA binds to αvβ5 integrin, and administration of ALULA to a mammalian subject reduces the severity of PE in the subject.

Definitions

An "αvβ5 antagonist" is any agent that competes with αvβ5 ligand for available ligand binding sites on αvβ5 integrins. αvβ5 antagonists include agents that specifically bind to αvβ5, β5, as well as agents that bind to αvβ5 or αvβ5. and at least one other integrin, such as, eg, αvβ3 or αvβ6.

αvβ5 integrin is a member of a family of adhesion molecules, containing non-covalently linked α/β heterodimers, which mediate, inter alia, cell-cell interactions, cell-extracellular matrix interactions, and cell-pathogen interactions. cxvβ5 is the only integrin that contains the β5 subunit. αvβ5 recognizes the peptide sequence RGD and binds vitronectin (see, e.g., Hynes, Cell 69: 11-25 (1992), and involvement in a number of disorders, including stroke, myocardial infarction, cancer (i.e., angiogenesis), and neovascularization eye disease ( see, e.g., Friedlander et al., Science 270(5241): 1500-2 (1995); Friedlander et al., PNAS USA 93(18): 9764-9 (1996); Elicieri et al., J. Cell Biol. 157(10: 149-159 (2002); Heba et al., J. Vasc. Res. 38(3): 288-300 (2001); Soeki et al., Cardiology 93(3): 168-74 (2000) and Li et al., Am. J. Physiol. 270(5 Pt 2): H1803-11 (1996). Both av and β5 have been sequenced and characterized (see, e.g., Hynes, 1992 supra, and U.S. Patent No. 5, 527, 679).

A "therapeutic dose" or "therapeutically effective amount" or "effective amount" of an αvβ5 integrin antagonist is an amount of the antagonist that prevents, alleviates, eliminates, or reduces the severity of disease symptoms associated with αvβ5 integrin, including, e.g., stroke, myocardial infarction, cancer (i.e., angiogenesis), neovascularization disease of the eye, and PE (eg, fluid accumulation in the lungs, increased hydrostatic capillary pressure in the lungs, or hypoventilation) in the patient.

The term "antibody" refers to a polypeptide encoded by an immunoglobulin gene or functional fragments thereof, which specifically binds and recognizes an antigen. Known immunoglobulin genes include the kappa, lambda, alpha, gamma, delta, epsilon, and mu constant region genes, as well as a multitude of immunoglobulin variable region genes. Light chains are classified as kappa or lambda. Heavy chains are classified as gamma, mu, alpha, delta or epsilon, which respectively define immunoglobulin classes IgG, IgM, IgA, IgD, or IgE.

An example of an immunoglobulin (antibody) structural unit includes a tetramer. Each tetramer is composed of two identical pairs of polypeptide chains, of which each pair has one "light" (about 25 kDa) and one "heavy" chain (about 50-70 kDa). The N-terminus of each chain defines a variable region of about 100 to 110 or more amino acids, which is primarily responsible for antigen recognition. Thus, the terms "variable heavy chain" "Vh" or "VH" refer to the variable region of an immunoglobulin heavy chain, including Fv, scFv, dsFv or Fab; while the terms "variable light chain" "Vl" or "VL" refer to the variable region of an immunoglobulin light chain, including Fv, scfv, dsFv or Fab.

Examples of functional antibody fragments include, but are not limited to, complete antibody molecules, antibody fragments, such as Fv, single chain Fv (scFv), complementarity determining regions (CDRs), VL (variable light chain region), VFI ( heavy chain variable region), Fab, F(ab)2' and any combination of these or any other functional parts of an immunoglobulin peptide capable of binding to a target antigen (see, e.g., FUNDAMENTAL IMMUNOLOGY (Paul ed., 4th edition, 2001).As one skilled in the art will appreciate, various antibody fragments can be obtained by a number of methods, for example, by digestion of an intact antibody with an enzyme, such as pepsin; or by de novo synthesis. Antibody fragments are often synthesized de novo either chemically or using recombinant DNA methodology. Thus, the term antibody, as used herein, includes antibody fragments that are produced by modification of the whole antibody or those that are synthesized de novo, using recombinant DNA methodologies (eg, single-chain Fv) or those that are identified using exposed phage libraries (see, eg, McCafferty et al., (1990) Nature 348: 552). The term "antibody" also includes bivalent or bispecific molecules, dibodies, tribodies and tetrabodies. Bivalent and bispecific molecules are described in, e.g., Kostelny et al. (1992) J. Immunol. 148: 1547, Pack and Pluckthun (1992) Biochemistry 31: 1579, Hollinger et al. (1993), PNAS. USA 90: 6444, Gruber et al. (1994) J Immunol.: 5368, Zhu et al. (1997) Protein Sci. 6: 781, Hu et al. (1996) Cancer Res. 56: 3055, Adams et al. (1993) Cancer Res. 53: 4026, and McCartney, et al. (1995) Protein Eng. 8: 301.

A "humanized" antibody is an antibody that retains the reactivity of a non-human antibody, while being less immunogenic in humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing the remaining parts of the antibody with their human duplicates. See, eg, Morrison et al., PNAS USA, 81: 6851-6855 (1984); Morrison and Oi, Adv. Immunol., 44: 65-92 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); Fallen, Molec. Immun., 28: 489-498 (1991); Fallen, Molec. Immun., 31(3): 169-217 (1994).

"Single-chain Fv (scFv)" or "single-chain antibodies" refers to a protein in which the VH and VL regions of the scFv antibody contain a single chain that is coiled to form an antigen-binding site similar to that found in double-chain antibodies. Procedures for making scFv antibodies are described in, e.g., Ward et al., Exp Hematol. (5): 660-4 (1993) and Vaughan et al., Nat Biotechnol. 14(3): 309-14 (1996). Single chain Fv (scFv) antibodies optionally include a peptide linker of no more than 50 amino acids, generally no more than 40 amino acids, preferably no more than 30 amino acids and even more preferably no more than 20 amino acids in length. In some embodiments, the peptide linker is a concatamer with the sequence Gly-Gly-G! y~GIy-Ser, eg, 2, 3, 4, 5 or 6 such sequences. However, it should be taken into account that some amino acid substitutions can be made within the linker. For example, glycine can be replaced by valine. Additional peptide linkers and their use are well known in the art. See, e.g., Huston et al., Proc. Nat'l Acad. Sci. USA 8: 5879 (1988); Bird et al., Science 242: 4236 (1988); Glockshuber et al., Biochemistry 29: 1362 (1990); U.S. Patent No. 4, 946, 778, U.S. Patent No. 5, 132, 405 and Stemmer et al., Biotechniques 14: 256-265 (1993).

The term "specifically (or selectively) bind to an antibody", when referring to a protein or peptide, refers to a binding reaction, which is indicative of the existence of the protein in the presence of a heterogeneous population of proteins and other biological agents. Thus, under the indicated conditions of the immunoassays, the specified antibodies bind to a certain protein (eg, αvβ5 integrin, β5 or their parts) and do not bind in a significant amount to other proteins, which are present in the sample. Specific antibody binding under such conditions may require an antibody that is selected for its specificity for a particular protein. For example, antibodies raised against αvβ5 integrins or β5 polypeptide can be selected to obtain antibodies of specific immunoreactivity with that protein and not with other proteins, with the exception of polymorphic variants, e.g., proteins that are at least 80%, 85%, 90%, 95% or 99% identical to the sequence of interest. A number of immunoassay formats can be used to select antibodies of specific immunoreactivity with a particular protein. For example, solid-phase ELISA immunoassays, Western blots, or immunohistochemistry are routinely used to select for monoclonal antibodies of specific immunoreactivity with a protein. See, Harlow and Lane Antibodies, A Laboratory Manual, Cold Spring Harbor Publications, NY (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity. Typically, the specific or selective response will be at least twice the background signal or noise, and more commonly more than 10 to 100 times the background signal.

An agent that engages in "specific competition" for binding reduces the specific binding of an antibody to a polypeptide. The first antibody is considered to competitively inhibit the binding of the second antibody, if the binding of the second antibody to the antigen is reduced by at least 30%, generally at least about 40%, 50%, 60% or 75%, and often at least about 90%, in the presence of the first antibody , using any of the competitive binding assays known in the art (see, e.g., Harlow and Lane, supra).

The terms "polypeptide," "peptide," and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical mimetic of the corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymers. As used herein, the terms include amino acid chains of any length, including full-length proteins (ie, antigens), in which the amino acid residues are linked by covalent peptide bonds.

The term "amino acid" refers to both naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs and amino acid mimetics, which function in a manner similar to naturally occurring amino acids. Amino acids that occur in nature are those encoded by the genetic code, as well as those amino acids that are later modified, eg, hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as amino acids, which since it is in nature, i.e. j., and carbon, which is bonded to hydrogen, carboxyl group, amino group and R group, eg, homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs possess modified R groups (eg, norleucine) or modified peptide backbones, but retain the same basic chemical structure as the naturally occurring amino acid. "Amino acid mimetics" refers to chemical compounds that have a structure that differs from the general chemical structure of an amino acid, but that function in a manner similar to a naturally occurring amino acid.

Amino acids can be designated here either by their commonly known three-letter symbols or by single-letter symbols, as recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides can also be labeled with their generally accepted one-letter symbols.

As used herein, the terms "nucleic acid" and "polynucleotide" are used interchangeably. Use of the term "polynucleotide" includes oligonucleotides (i.e., short polynucleotides). This term also refers to deoxyribonucleotides, ribonucleotides, and naturally occurring variants, and may also refer to synthetic nucleic acids and/or non-naturally occurring nucleic acids (ie, including nucleic acid analogs). or modified base residues or linkers), such as, for example, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral methyl phosphonates, 2-O-methyl ribonucleotides, peptide nucleic acids (PNAs), and the like. Unless otherwise indicated, a particular nucleic acid sequence also implicitly includes conservatively modified variants thereof (eg, degenerative codon substitutions) and complementary sequences, as well as sequences that are explicitly indicated.

Specifically, degenerative codon substitutions can be made by developing sequences in which the third position of one or more selected (or all) codons is substituted with mixed-base and/or deoxyinosine residues (see, e.g., Batzer et al., Nucleic Acid Res. 19 : 5081 (1991); Ohtsuka et ah, J. Bioh Chem. 260: 2605-2608 (1985) and Cassol et al. (1992); Rossolini et ah, Moh Celh Probes 8: 91-98 (1994)).

Inhibition of αvβ5

This invention relates to the treatment or prevention of PE or acute lung injury by inhibiting the binding of ligands to the αvβ5 integrin. Antibodies that specifically bind to the β5 subunit of αvβ5 integrin were used. The disease is PE (including, eg, cardiogenic and non-cardiogenic PE). In some embodiments, treatment of PE also treats or prevents secondary disorders, such as, e.g., pulmonary fibrosis.

Antibodies

In accordance with the present invention, antibodies that specifically bind to the ctv|35 integrin, and in particular to the β5 subunit of the αvβ5 integrin, are used for the treatment or prevention of PE or acute lung injury. Suitable antibodies include humanized antibodies and antibody fragments (ie, Fv, Fab, (Fab')2, or scFv).

In the invention, monoclonal antibodies ALULA (ATCC Deposit No. PTA-5817, received February 13, 2004, ATCC, 10801 University Blvd. Manassas, VA 20110-2209) that bind to the β5 subunit of the ecvβ5 integrin, are used to treat or prevent PE or acute lung injury. Without being bound by theory, ALULA is believed to act by blocking αvβ5 integrin-mediated changes in lung vasculature permeability. In some embodiments, humanized ALULA or fragments of ALULA are used to treat PE.

Monoclonal antibodies are obtained using various techniques known to those skilled in the art. Briefly, spleen cells from an animal immunized with the desired antigen are immortalized, usually by fusing with a myeloma cell (see, for example, Kohler & Milstein, Eur. J. Immunol. 6: 511-519 (1976)). Alternative methods of immortalization include transformation with Epstein Barr virus, oncogenes, or retroviruses, or other methods well known in the art. Colonies generated from a single immortalized cell are screened for the production of antibodies of the desired specificity and affinity for the antigen, and the amounts of monoclonal antibodies produced by such cells can be increased by various techniques, including injection into the peritoneal cavity of the vertebrate host. Alternatively, DNA sequences encoding the monoclonal antibody or its binding fragment can be isolated by screening a DNA library from human B cells, according to the general protocol published by Huse et al., Science 246: 1275-1281 (1989).

Monoclonal antibodies are collected and titrated against the immunogen in an immunoassay, for example, a solid phase immunoassay with the immunogen immobilized on a solid support. Monoclonal antibodies will typically bind with a Kd of at least about 0.1 mM, more often at least about 1 μM, preferably at least about 0.1 μM or better, and most preferably, 0.01 μM or better.

For example, an animal such as a rabbit or mouse is immunized with an avj35 polypeptide, or a nucleic acid construct encoding such a polypeptide. Antibodies produced as a result of immunization can be isolated using standard procedures.

Immunoglobulins, including binding fragments and other derivatives thereof, of the present invention can be readily produced using various recombinant DNA techniques, including expression in transfected cells (eg, by immortalizing eukaryotic cells, such as myeloma or hybridoma cells) or in mice, rats, rabbits, or other vertebrates capable of producing antibodies by well-known methods. Suitable sources of cells for DNA sequences and host cells for immunoglobulin expression and secretion can be obtained from a number of sources, such as the American Type Culture Collection (Catalogue of Cell Lines and Hybridomas, Fifth edition (1985) Rockville, Md.

In some embodiments, the antibody is a humanized antibody, i.e., an antibody that retains the reactivity of a non-human antibody while being less immunogenic for humans. This can be achieved, for example, by retaining the non-human CDR regions and replacing other parts of the antibody with their human copies. See, eg, Morrison et al., PNAS USA, 81: 6851-6855 (1984); Morrison and Oi, Adv. Immunoh, 44: 65-92 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988); Fallen, Molec. Immun., 28: 489-498 (1991); Fallen, Molec. Immun., 31(3): 169-217 (1994). Techniques for humanizing antibodies are well known in the art and are described in, e.g., U.S. Pat. 4, 816, 567; 5, 530, 101; 5, 859, 205; 5, 585, 089; 5, 693, 761; 5, 693, 762; 5, 777, 085; 6, 180, 370; 6, 210, 671; and 6, 329, 511; WO 87/02671; EP Patent Application 0173494; Jones et al. (1986) Nature 321: 522; and Verhoyen et al. (1988) Science 239: 1534. Humanized antibodies are further described in, e.g., Winter and Milstein (1991) Nature 349: 293. For example, polynucleotides comprising a first sequence encoding humanized immunoglobulin building regions and a second set of sequences encoding the desired complementarity-determining immunoglobulin regions can be produced synthetically or by combining appropriate cDNA and genomic DNA segments. Human constant region DNA sequences can be isolated according to well known procedures from various human cells. The CDRs for producing the immunoglobulins of the present invention will be similarly derived from monoclonal ALULA antibodies.

In some cases, transferring the CDRs into a human construct results in a loss of specificity for the humanized antibody. In these cases, “back” mutations can be introduced into the construct regions of the human part of the antibody. Procedures for making "back" mutations are well known in the art and are described in, e.g., Co et al., PNAS USA 88; 2269-2273 (1991) and WO 90/07861.

In some embodiments, antibodies are antibody fragments such as Fab, F(ab')2, Fv, or scFv. Antibody fragments can be generated by any known means, including chemical digestion (eg, papain or pepsin) and recombinant procedures. Procedures for the isolation and preparation of recombinant nucleic acids are known to those skilled in the art (see, Sambrook et al., Molecular Cloning. A Laboratory Manual (2d ed. 1989); Ausubel et al., Current Protocols in Molecular Biology (1995)). Antibodies can be expressed in a number of host cells, including E. coli, other bacterial hosts, yeast and various higher eukaryotic cells, such as: COS, CHO and HeLa cell lines and myeloma cell lines.

Therapeutic treatment

As previously discussed, the invention also provides compositions comprising the antibodies of the invention. The compositions of the invention can be provided for the treatment or prevention of PE or acute lung injury.

In one embodiment, compositions of the invention (compositions containing ALULA, humanized ALULA, or ALULA fragments) can be provided for the treatment or prevention of PE in individuals with PE or those at risk of developing PE. For example, a person who has been exposed to inhalation of a toxic substance will be treated after such exposure, while patients at risk of developing PE will be able to be treated prophylactically and/or therapeutically. Examples of patients at risk for PE include patients with acute aspiration, patients presenting with symptoms of bacterial sepsis, patients whose blood cultures are positive for gram-positive or gram-negative bacteria, patients with pancreatitis, or patients in hemorrhagic shock.

The compositions of the invention can be administered on a regular basis (eg, daily) over a period of time (eg, 2, 3, 4, 5, 6, days or 1-3 weeks or more).

The compositions of the invention can be administered directly to a mammalian subject to block αvβ5 binding, by any of the known routes of administration, including, e.g., by injection (e.g., intravenously, intraperitoneally, subcutaneously, intramuscularly, or intradermally), by inhalation, by transdermal administration, rectal application, or oral administration.

The pharmaceutical compositions of the invention contain a pharmaceutically acceptable carrier. Pharmaceutically acceptable carriers are determined in part by the particularity of the mixture to be administered, as well as the particularity of the procedure by which the mixture is to be administered. Accordingly, there is a wide variety of suitable formulations of the pharmaceutical compositions of this invention (see, e.g., Remington's Pharmaceutical Sciences, 17th ed., 1989).

The compositions of the invention, alone or together with other suitable ingredients, may be made into aerosol formulations (ie, may be "misted") to be administered by inhalation. Aerosol formulations can be placed in acceptable propellants under pressure, such as: dichlorodifluoromethane, propane, nitrogen and the like.

Formulations that are suitable for administration include aqueous and non-aqueous solutions, isotonic sterile solutions, which may contain antioxidants, buffers, bacteriostatic agents and solutions that provide isotonicity to the formulation, as well as aqueous and non-aqueous sterile suspensions that may contain suspending agents, solubilizers, thickeners, stabilizers and preservatives. In practicing the present invention, the compositions may be administered, for example, orally, nasally, topically, intravenously, intraperitoneally, or intrathecally. Formulations of the compounds may be presented in single-dose or multi-dose sealed containers, such as ampoules or vials. Solutions and suspensions can be made from sterile powders, granules and tablets of the previously described types. Modulators can also be given as part of prepared food or medicine.

Formulations suitable for oral administration may include: (a) liquid solutions, such as an effective amount of the nucleic acid package suspended in diluents, such as water, saline or PEG 400; (b) capsules, sachets or tablets, each containing a certain amount of active ingredient, in the form of liquids, solids, granules or gelatin; (c) suspensions in a suitable liquid; and (d) suitable emulsions. Tablet forms may contain one or more of: lactose, sucrose, mannitol, sorbitol, calcium phosphates, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid and other bases, colors, agents fillers, binders, diluents, buffering agents, wetting agents, preservatives, fragrances, dyes, disintegrants and pharmaceutically compatible carriers. Lozenge forms may contain the active ingredient in a flavoring agent, e.g., sucrose, just as lozenges contain the active ingredient in an inert base, such as gelatin and glycerin or sucrose and acacia emulsions, gels and the like which, in addition to the active ingredient, contain carriers known in the profession.

The dose to be administered to a patient, in the context of the present invention, should be sufficient to produce a beneficial response in the recipient over time, e.g., a decrease in pulmonary capillary hydrostatic pressure, a decrease in lung fluid, a decrease in the rate of pulmonary fluid accumulation, or combinations thereof. . The optimal dosage level for each patient will depend on various factors including the effectiveness of the specific modulator used, age, body weight, physical activity, as well as the patient's diet, possible combination with other drugs, and the severity of PE. The size of the dose will also be determined by the existence, nature and degree of any adverse effect accompanying the administration of the particular compound or vector in the particular patient.

In order to determine the effective amount of an antibody of the invention to be administered, the physician may examine circulating plasma levels of the antibody. In general, the dose equivalent is approximately 1 ng/kg to 10 mg/kg for a typical recipient.

Regarding administration, antibodies can be administered in an amount determined by the LD50, as well as side effects at various concentrations, if administered to a patient of average weight and health. Application can be carried out in the form of a single dose or divided doses.

Combined therapy

In some embodiments, the antibodies of the invention are administered together with another therapeutic agent to treat or prevent acute lung injury and/or PE. For example, ALULA, humanized ALULA, or fragments of ALULA can be administered in conjunction with any of the standard treatments for PE including, e.g., diuretics, bronchodilators, narcotics, oxygen, and the use of selective dressings. In addition, the antibodies of the invention can be co-administered with agents that target metabolic pathways associated with acute lung injury and/or PE. For example, the antibodies can be co-administered with TGFβ pathway inhibitors, activated protein C, steroids, GM-CSF, platelet inhibitors, β-2 agonists, surfactants, antibodies that specifically bind to αvβ5 integrin or β5, other αvβ5 integrin antagonists, antibodies that specifically bind to αvβ6 integrin, αvβ6 integrin antagonists, thrombin receptor antagonists, anti-thrombin agents, rho kinase inhibitors and, nucleic acids that inhibit otvβ5 integrin expression including, e.g., antisense oligonucleotides and siRNA described herein. Suitable inhibitors of the TGFβ pathway include, e.g., TGFβ3 antibodies (including those that specifically block TGF-β1, TGF-β2, TGFβ33, or any combination thereof) as described in, e.g., Ling et al., J. Amer. Soc. Nephrol. 14: 377-388 (2003), McCormick et al., J. Immunol. 163: 5693-5699 (1999), and Cordeiro, Curr. Opin. Mol. Ther. 5(2): 199-203 (2003); TGFβ3 receptor type II inhibitors or TGFβ3 receptor type I kinase inhibitors as described in, e.g., DaCosta Bayfield, Mol. Pharmacol. 65(3): 744-52 (2004), Laping, Curr. Opin. Pharmacol. 3 (2): 204-8 (2003), Laping, Mol. Pharmacol. 62(l): 58-64 (2002); soluble TGFβ3 receptor type Ii as described in, e.g., Pittet, J. Clin. Invest. 107: 1537-1544 (2001); Wang et al., Exp Lung Res. 28(6): 405-17 (2002) and Wang, Thorax 54(9): 805-12 (1999); soluble peptides associated with the latent state as described in, e.g., Zhang, J. Invest. Dermatol. 121(4): 713-9 (2003); thrombospondin I inhibitors as described in, e.g., Crawford et al., Cell 93: 1159-1170 (1998), Riberiro et al., J. Biol. Chem. 274: 13586-13593 (1999), and Schultz-Cherry et al., J. Biol. Chem. 269: 26775-26782 (1994). Suitable β-2 agonists include, for example, albuterol, bitolterol, formoterol, isoproterenol, levalbuterol, metaproterenol, pirbuterol, salmeterol and terbutaline. Suitable surfactants include, e.g., exosurf, infasurf, KL-4, pumactant, survant, venticut, and surfactant TA, as described in Taeusch et al., Acta Pharmacol Sin 23 Supplement: 11-15 (2002). Suitable anti-thrombin agents include, e.g., hirudin, Hirulog (Biogen), argatroban (Texas Biotechnology), and efegatran (Lilly) and compounds described in U.S. Pat. 6, 518, 244. Suitable thrombin receptor antagonists are described in, e.g., U.S. Pat. 6, 544, 982; 6, 515, 023; 6, 403, 612; 6, 399, 581; and 5, 446, 131. Suitable rho kinase inhibitors include, e.g., γ-27632 as described in, e.g., Tasaka et al., Am JRespir Cell Mol Biol. 2005 Mar 18; [Epub ahead of print], fasudil as described in, eg, Nishikimi et al., J Hypertens. 22(9): 1787-96 (2004), l-(5-isoquinolinesulfonyl)-homopiperazine (HA-1077), (S)-(+)-2-methyl-l-[(4-methyl-5-isoquinoline )sulfonyl]-homopiperazine (H-1152P) as described in, e.g., Sasaki et al., Pharmacol Ther. 93(2-3): 225-32 (2002) and other rho kinase inhibitors described in, e.g., U.S. Pat. 6, 451, 825 and 6, 218, 410 and U.S. Patent Publication No. 20050014783 and 20030134775.

In addition, the antibodies can be co-administered with an adenovirally expressed ATPase as described in U.S. Patent Publication No. 20020192186; with the β2 adrenergic receptor as described in U.S. Patent Publication No. 20020004042; with VEGFβ antagonists as described in U.S. Patent no. 6, 284, 751; with lipid peroxidation inhibitors as described in U.S. Pat. 5, 231, 114; and with small molecule inhibitors for αvβ6, αvβ5, and αvβ3 integrins as described in, e.g., US published patent application no. 2000/40019206, 2004/0019037, 2004/0019035, 2004/0018192, 2004/0010023, 2003/0181440, 2003/0171271, 2003/0139398, 2002/00378 89, 2002/0077321, 2002/0072500, U.S. Patent No. 6, 683, 051 and Goodman et al., J. Med Chem. 45(5): 1045-51 (2002).

ALULA, humanized ALULA or fragments of ALULA and another therapeutic agent can be administered simultaneously or sequentially. For example, an antibody may be administered first, followed by a second therapeutic agent. Alternatively, another therapeutic agent can be administered first, followed by the antibody. In some cases, the antibody and the other therapeutic agent are administered in the same formulation. In other cases, the antibody and the other therapeutic agent are administered in different formulations. When the antibody and other therapeutic agent are administered in different formulations, they may be administered simultaneously or sequentially.

For administration, the antibody and other therapeutic agent may be administered as often as determined by the combined LD50 of the antibody and other therapeutic agent and, the adverse effects of the antibody and other therapeutic agent at various concentrations, applied to the subject's weight and overall health. In some cases, both the antibody and the other therapeutic agent are administered at a subtherapeutic dose or a therapeutic dose.

Packaging

The present invention also provides packages for the treatment or prevention of PE. Packages contain ALULA, humanized ALULA, or fragments of ALULA and another therapeutic agent for the treatment of PE. Suitable other therapeutic agents include, e.g., a TGFβ pathway inhibitor, activated protein C, steroid, GM-CSF, platelet inhibitor, diuretic agent, bronchodilatory agent, antibodies that specifically bind to αvβ5 integrin or β5, another αvβ5 integrin antagonist, antibodies that specifically bind to αvβ6 integrin, αvβ6 integrin antagonists, β-2 agonists and surfactants. These packages may also contain written instructions (eg, a manual) for using the package.

EXAMPLES

Example 1: Materials and procedures

Model of PE: Rodent Unilateral Ischemia-Reperfusion Lung Injury: Mice or rats are subjected to lung transplantation, cardiopulmonary bypass, pulmonary thromboangioarterectomy, or severe shock. Then, in thirty minutes, or three hours, ischemia and reperfusion were induced. To induce ischemia, a left-sided thoracotomy was performed by blocking the left hilum (eg, using an umbilical tape) for 30 minutes. To induce reperfusion, the lungs were re-inflated with a tidal volume of 12 ml/kg air, and then normal ventilation was started. The animals were sacrificed after 3 hours and the permeability of each lung was examined, eg, by measuring the extravasation of labeled albumin in the lungs, which was expressed as extravascular lung equivalents (EVPE).

PE Model: Ventilation-Induced Lung Injury in Rats: Mice or rats were ventilated with normal (6 ml/kg) or high tidal volumes (20 ml/kg). Animals were injected with 125J-labeled albumins after 4 hours, and then lung sampling and EVPE determination were performed.

Extravascular Plasma Equivalents (EVPE) Measurements: EVPEs were measured as described in, eg, Frank et al., J. Biol. Chem., 278 (45): 43939-43950 (2003)). Briefly, a vasculami tracer (eg, 125J albumin) is injected intraperitoneally into rats two hours before lung collection. Blood is taken and the lungs are removed. Radioactivity in the lungs and blood is measured. Hemoglobin concentration is measured in lung homogenate and in blood. Intravascular radioactivity in the lungs is calculated by multiplying pulses of plasma radioactivity by the volume of blood in the lungs.

Antibodies: ALULA is generated as described below. W6/32, the mission monoclonal antibody W6/32, which specifically binds to HLA A, B, and C, was obtained from ATCC. CD-I WT, a monoclonal antibody that binds to CD-I, was obtained from ATCC.

Example 2: Generation of ALULA, a murine monoclonal antibody that specifically binds to αvβ5 integrin

αvβ5-deleted mice were immunized with cells expressing a polypeptide containing the αvβ5 integrin sequence. Monoclonal antibodies that specifically bind αvβ5 integrin are identified using methods known in the art. More precisely, ALULA is identified, which specifically binds to β5. ALULA was deposited with ATCC on February 13, 2004 and has the following accession number: PTA-5817.

Example 3: β5-/- mice do not develop lung damage associated with PE

β5-/- mice and wild-type mice were ventilated as described in Example 1 above, to induce PE associated with lung injury, and EVPEs were determined. In contrast to wild-type mice, β5-/- mice did not develop PE after ventilation. These results indicate that αvβ5 is involved in PE. The results are shown in Figure 1.

Example 4: A monoclonal antibody that specifically binds to 35 reduces the severity of pulmonary edema associated with ischemia reperfusion

To determine the role of β5 in PE associated with ischemia-reperfusion, rats were subjected to the following treatments and EVPE measurements were performed:

1. No treatment.

2. Intraperitoneal (i.p.) injection of 4 μg per gram of W6/32.

3. I. p. 4 μg per gram (i. p. ) ALULA.

4. Ischemia-reperfusion was induced as described previously in Example 1

5. Ischemia-reperfusion was induced and 4 μg per gram of W6/32 was injected intraperitoneally.

6. Ischemia-reperfusion was induced and 4 μg per gram of ALULA was injected intraperitoneally.

In these experiments, antibodies are injected before induction of ischemia-reperfusion.

Rats treated with ALULA show a reduction in EVPE (ie, a decrease in lung cell permeability) compared to control rats, indicating that the monoclonal antibody, which specifically binds to β5, can reduce the severity of PE.

The results are shown in Figure 2.

Example 5: A monoclonal antibody that specifically binds to β5 reduces the severity of pulmonary edema associated with lung injury

To determine the role of β5 in PE associated with lung injury, mice were subjected to the following treatments and EVPE measurements were performed:

1. Normal respiratory volume and i. p. injection of 4 μg per gram of CD-I WT.

2. High respiratory volume and i. p. injection of 4 μg per gram of CD-I WT.

3. Normal respiratory volume and i. p. injection of 4 μg per gram of ALULA.

4. High respiratory volume and i. p. injection of 4 μg per gram of ALULA.

In these experiments, the antibodies are injected before treatment of the respiratory volume.

Mice treated with ALULA show a reduction in EVPE compared to control mice, indicating that a monoclonal antibody that specifically binds to β5 can reduce the severity of PE.

Thus, ALULA is the first monoclonal antibody, specific for αvβ5, found to block activity in vivo, in all mammals, and the first found to block increased vascular permeability and the development of alveolar flooding in models of acute lung injury (ie, PE).

The results are shown in Figure 3.

Example 6: Antibody ALULA Blocks Binding of αvβ5 Integrin Uganda Vitronectin to Cells Expressing αvβ5 Integrin

SW-480 cells expressing αvβ5 integrin were challenged with 0 μg/ml, 0.1 μg/ml, 0.3 μg/ml and 1 μg/ml vitronectin in the presence of 0 μg/ml, 0.3 μg/ml , 1 μg/ml and 10 μg/ml ALULA. As a negative control, a monoclonal antibody (ie, Y9A2) specific for the α9β1 integrin is used. ALULA blocks the binding of the αvβ5 integrin ligand, vitronectin, to cells. The results are shown in Figure 4.

Claims (16)

1. Antibody, produced by hybridoma, deposited as ATCC Deposit No. PTA-5817. 2. An antibody that specifically binds to uv|35 integrin, wherein the antibody is humanized from an antibody produced by a hybridoma deposited as ATCC Deposit No. PTA-5817. 3. A humanized antibody that specifically binds to the αvβ5 integrin, containing the complementarity determining regions of the heavy and light chain of the antibody produced by a hybridoma deposited as ATCC Deposit No. PTA-5817. 4. An antibody as in any one of claims 1 to 3, wherein the antibody is selected from the group consisting of: scFv, Fab and (Fab')2 5. A pharmaceutical composition containing a pharmaceutically acceptable carrier and an antibody, as in any one of claims 1 to 4. 6. An antibody as claimed in any one of claims 1 to 4 for use in the treatment or prevention of pulmonary oedema. 7. An antibody as claimed in any one of claims 1 to 4 for use in the treatment or prevention of acute lung injury. 8. An antibody as in any one of claims 1 to 4 for use as in claim 6, wherein the use further comprises administering another therapeutic agent to treat or prevent pulmonary edema. 9. An antibody as in any one of claims 1 to 4, for use as in claim 7, wherein the use further comprises administering another therapeutic agent to treat or prevent acute lung injury. 10. An antibody as in any one of claims 1 to 4 for use as in claim 8 or 9, wherein the second therapeutic agent is selected from the group consisting of: TGFβ pathway inhibitors, activated protein C, steroids, GM -CSF, platelet inhibitor, diuretic, bronchodilator, αvβ5 integrin binding antibody, β5 binding antibody, other αvβ5 integrin antagonist, αvβ6 integrin antagonist, β-2 agonist and surfactant. 11. An antibody as in any one of claims 1 to 4, for use as in any one of claims 6 to 10, which is administered intravenously, intranasally or intrabronchially. 12. Use of an antibody, as in any one of claims 1 to 4, for the production of a medicament for the treatment of pulmonary edema. 13. Use of an antibody, as in any of claims 1 to 4, for the manufacture of a medicament for the treatment of acute lung injury. 14. A kit for the treatment or prevention of pulmonary edema, comprising an antibody as in any one of claims 1 to 4 and a therapeutic agent for the treatment or prevention of pulmonary edema. 15. A kit as in claim 14, wherein the second therapeutic agent is selected from the group consisting of: TGFβ pathway inhibitor, activated protein C, steroid, GM-CSF, platelet inhibitor, diuretic agent, bronchodilator agent, antibody that binds to αvβ5 integrin, β5-binding antibody, αvβ5 integrin dear antagonist, αvβ6 integrin antagonist, 3-2 agonist and surfactant. 16. Hybrid deposited under: ATCC deposit number: PTA. -5817.