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  Pharmaceutical Patents  

 

Title:  Methods of treating multiple myeloma and myeloma-induced bone resorption using integrin antagonists
United States Patent: 
7,618,630
Issued: 
November 17, 2009

Inventors:
 Mundy; Gregory R. (San Antonio, TX), Yoneda; Toshiyuki (San Antonio, TX)
Assignee:
  Board of Regents, The University of Texas System (Austin, TX)
Appl. No.:
 10/086,217
Filed:
 February 21, 2002


 

Executive MBA in Pharmaceutical Management, U. Colorado


Abstract

Antagonists of .alpha.4 integrin/.alpha.4 integrin ligand adhesion, which inhibit the biological effects of such adhesion are described and methods for their use are detailed. Such antagonists are useful in suppressing bone destruction associated with multiple myeloma. The homing of multiple myeloma cells to bone marrow and their .alpha.4 integrin-dependent release of bone-resorbing factors, resulting in bone destruction in patients with multiple myeloma, is inhibited.

Description of the Invention

SUMMARY OF THE INVENTION

We have used a recently developed murine model of multiple myeloma in which the mouse develops severe osteolysis with all the hallmarks of human disease (Garrett 1997). Using this cell line and animal model we have established that inhibition of the .alpha.4 integrin/.alpha.4 integrin ligand pathway in vivo leads to reduced capacity for multiple myeloma cells to proliferate and/or survive. We show that cell-cell attachment between myeloma cells and marrow stromal cells via the VLA-4/VCAM-1 interaction is required for an increase in the production of an activity which stimulates osteoclastic bone resorption in the bone microenvironment in vitro.

We propose that this interaction is critical to the homing of myeloma cells to the marrow compartment, to their subsequent survival and growth, to ultimately to the progression of myeloma-induced osteolysis. We tested this in the animal model and found that, in vivo, an antagonist of the alpha4 subunit-containing integrin VLA-4 strongly inhibits the production of antibody of the IgG2b subtype. This isotype is the same as that produced by the 5TGM1 cell line, and is an accurate surrogate for the number of myeloma cells in the marrow compartment at any time. Thus, blockade of the VLA-4 pathway strongly inhibits IgG2b production, and by implication, the level of myeloma burden.

One aspect of the invention is a method for treating multiple myeloma comprising administering to an individual a therapeutically effective amount of a composition comprising an antagonist of an interaction between an integrin with an .alpha.4 subunit (e.g., VLA-4) and a ligand for this integrin (e.g., VCAM-1). This antagonist can be an .alpha.4 integrin binding agent or an .alpha.4 integrin ligand binding agent. Preferred agents are anti-VLA 4 or anti-.alpha.4.beta.7 antibody homologs (human antibody, a chimeric antibody, a humanized antibody and fragments thereof); anti-VCAM-1 antibody homologs (a human antibody; a chimeric antibody, a humanized antibody and fragments thereof); and a small molecule inhibitor of interactions of .alpha.4 subunit containing integrins with their ligands. The composition can be administered at a dosage so as to provide from about 0.1. to about 20 mg/kg body weight. In particular, the preferred agents can antagonize an interaction: a) of both VLA-4 and .alpha.4.beta.7 collectively with their respective .alpha.4 ligands; or b) only of VLA-4 with its .alpha.4 ligand; or c) only of .alpha.4.beta.7 with its .alpha.4 ligand. One or more antagonists of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin could be administered in combination with one or more compounds that preferably are not an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin. Preferably, the compound to be administered in combination with an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin is a chemotherapeutic agent. Preferably, that chemotherapeutic agent is melphalan.

Another aspect of the invention is a method for inhibiting bone resorption associated with tumors of bone marrow, the method comprising administering to a mammal with said tumors an antagonist of an interaction between an .alpha.4 subunit containing integrin such as VLA-4 and a ligand for this .alpha.4 subunit containing integrin, such as VCAM-1, in an amount effective to provide inhibition of the bone resorption. This antagonist can be an .alpha.4 integrin binding agent such as a VLA-4 binding agent or an .alpha.4 integrin ligand binding agent such as a VCAM-1 binding agent. Preferred agents are anti-VLA4 or and .alpha.4.beta.7 antibody homologs (human antibody, a chimeric antibody, a humanized antibody and fragments thereof); anti-VCAM-1 antibody homologs (a human antibody, a chimeric antibody, a humanized antibody and fragments thereof); and a small molecule inhibitor of the interaction of .alpha.4 subunit-containing integrins with their respective .alpha.4 integrin ligands (e.g, the VCAM-1/VLA-4 interaction). The antagonist can be administered at a dosage so as to provide from about 0.1 to about 20 mg/kg body weight. One or more antagonists of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin could be administered in combination with one or more compounds that preferably are not an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin. Preferably, the compound to be administered in combination with an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin is a chemotherapeutic agent. Preferably, that chemotherapeutic agent is melphalan.

Yet another aspect of the invention is a method of treating a subject having a disorder characterized by the presence of osteoclastogenesis, the method comprising administering to the subject an antagonist of an interaction between an .alpha.4 subunit bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin, in an amount sufficient to suppress the osteoclastogenesis. Similarly, the antagonist can be an .alpha.4 binding agent or an .alpha.4 ligand binding agent. Preferred agents are anti-VLA4 or anti-.alpha.4.beta.7 antibody homologs (human antibody, a chimeric antibody, a humanized antibody and fragments thereof); anti-VCAM-1 antibody homologs (a human antibody, a chimeric antibody, a humanized antibody and fragments thereof); and a small molecule inhibitor of the interaction of .alpha.4 subunit-containing integrins with their respective .alpha.4 integrin ligands (e.g, the VCAM-1/VLA-4 interaction). The composition can be administered at a dosage so as to provide from about 0.1 to about 20 mg/kg body weight. One or more antagonists of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin could be administered in combination with one or more compounds that preferably are not an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin. Preferably, the compound to be administered in combination with an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin is a chemotherapeutic agent. Preferably, that chemotherapeutic agent is melphalan.

DETAILED DESCRIPTION OF THE INVENTION

The invention relates to treatments for, among other things, preventing multiple myeloma. More particularly, methods of the invention relate to the use of antagonists of an interaction between an integrin containing an .alpha.4 subunit and a ligand for this integrin in the treatment of multiple myeloma. The term "multiple myeloma" is intended to mean a medical condition in an individual having a neoplastic disease of plasma cells, with the neoplastic clone representing cells at different stages in the plasma cell lineage from patient to patient (Mundy, 1998).

Alpha 4 .beta.1 integrin is a cell-surface receptor for VCAM-1, fibronectin and possibly other molecules that bind with, or otherwise interact with, alpha 4 .beta.1 integrin. In this regard, such molecules that bind with, or otherwise interact with, alpha 4 subunit containing integrin are individually and collectively referred to as ".alpha.4 ligand(s)"). The term .alpha.4.beta.1 integrin ("VLA-4" or ".alpha.4.beta.1, or ".alpha.4.beta.1 integrin", used interchangeably) herein thus refers to polypeptides which are capable of binding to VCAM-1 and members of the extracellular matrix proteins, most particularly fibronectin, or homologs or fragments thereof, although it will be appreciated by workers of ordinary skill in the art that other ligands for VLA-4 may exist and can be analyzed using conventional methods.

Nevertheless, it is known that the .alpha.4 subunit will associate with other .beta. subunits besides .beta.1 so that we may define the term ".alpha.4 integrin" as being those integrins whose .alpha.4 subunit associates with one or another of the .beta. subunits. A further example of an ".alpha.4" integrin is .alpha.4.beta.7 (R. Lobb and M. Hemler, 1994). As used herein, the term ".alpha.4 integrin(s)" means VLA-4, as well as integrins that contain .beta.1, .beta.7 or any other .beta. subunit.

As discussed herein, the antagonists used in methods of the invention are not limited to a particular type or structure of molecule so that, for purposes of the invention, any agent capable of binding to any integrin containing an .alpha.4 subunit such as VLA-4 on the surface of VLA-4 bearing cells and/or .alpha.4.beta.7 integrin on the surface of .alpha.4.beta.7-bearing cells [see Lobb and Hemler, J. Clin. Invest., 94: 1722-1728 (1994)] and/or to their respective .alpha.4 ligands such as VCAM-1 and MadCAM, respectively, on the surface of VCAM-1 and MadCAM bearing cells, and which effectively blocks or coats VLA-4 (or .alpha.4.beta.7) or VCAM-1 (or MadCAM) (i.e., a "an .alpha.4 integrin binding agent" and ".alpha.4 integrin ligand binding agent" respectively), is considered to be an equivalent of the antagonists used in the examples herein.

An integrin "antagonist" includes any compound that inhibits an .alpha.4 integrin(s) from binding with an .alpha.4 integrin ligand and/or receptor. Anti-integrin antibody or antibody homolog-containing proteins (discussed below) as well as other molecules such as soluble forms of the ligand proteins for integrins are useful. Soluble forms of the ligand proteins for .alpha.4 integrins include soluble VCAM-1 or collagen peptides, VCAM-1 fusion protein, or bifunctional VCAM-1/Ig fusion proteins. For example, a soluble form of an .alpha.4 integrin ligand or a fragment thereof may be administered to bind to integrin, and preferably compete for an integrin binding site on cells, thereby leading to effects similar to the administration of antagonists such as anti-.alpha.4 integrin (e.g., .alpha.4.beta.7 antibodies and/or VLA-4 antibodies). In particular, soluble .alpha.4 integrin mutants that bind .alpha.4 integrin ligand but do not elicit integrin-dependent signaling are included within the scope of the invention. Such mutants can act as competitive inhibitors of wild type integrin protein and are considered "antagonists". Other antagonists used in the methods of the invention are "small molecules", as defined below.

Included within the invention are methods using an agent that antagonizes the action of more than one .alpha.4 integrin, such as a single small molecule or antibody homolog that antagonizes several .alpha.4 integrins such as VLA-4 and .alpha.4.beta.7, or other combinations of .alpha.4 integrins. Also included within the scope of the invention are methods using a combination of different molecules such that the combined activity antagonizes the action of more than one .alpha.4 integrin, such as methods using several small molecules or antibody homologs that in combination antagonize the .alpha.4 integrins VLA-4 and .alpha.4.beta.7, or other combinations of integrins.

As discussed herein, certain integrin antagonists can be fused or otherwise conjugated to, for instance, an antibody homolog such as an immunoglobulin or fragment thereof and are not limited to a particular type or structure of an integrin or ligand or other molecule. Thus, for purposes of the invention, any agent capable of forming a fusion protein (as defined below) and capable of binding to .alpha.4 integrin ligands and which effectively blocks or coats .alpha.4.beta.7 and/or VLA-4 integrin is considered to be an equivalent of the antagonists used in the examples herein.

For the purposes of the invention an "antagonist of the .alpha.4 integrin ligand/.alpha.4 integrin interaction" refers to an agent, e.g., a polypeptide or other molecule, which can inhibit or block .alpha.4 ligand (e.g., VCAM-1) and/or .alpha.4 integrin (e.g., .alpha.4.beta.7 or VLA-4)-mediated binding or which can otherwise modulate .alpha.4 ligand and/or .alpha.4 integrin function, e.g., by inhibiting or blocking .alpha.4-ligand-mediated .alpha.4 integrin signal transduction or .alpha.4 ligand mediated .alpha.4 ligand signal transduction and which is effective in the treatment of multiple myeloma, preferably in the same manner as are anti-.alpha.4 integrin antibodies.

Specifically, an antagonist of the VCAM-1/VLA-4 interaction is an agent which has one or more of the following properties: (1) it coats, or binds to VLA-4 on the surface of a VLA-4 bearing cell (e.g., a myeloma cell) with sufficient specificity to inhibit a VLA-4-ligand/VLA-4 interaction, e.g., the VCAM-1/VLA-4 interaction between bone stromal cells and myeloma cells; (2) it coats, or binds to, VLA-4 on the surface of a VLA-4 bearing cell (i.e., a myeloma cell) with sufficient specificity to modify, and preferably to inhibit, transduction of a VLA-4-mediated signal e.g., VLA-4/VCAM-1-mediated signaling; (3) it coats, or binds to, a VLA-4-ligand, (e.g., VCAM-1) on bone stromal cells with sufficient specificity to inhibit the VLA-4/VCAM interaction; (4) it coats, or binds to, a VLA-4-ligand (e.g., VCAM-1) on bone stromal cells with sufficient specificity to modify, and preferably to inhibit, transduction of VLA-4-ligand mediated VLA-4 signaling, e.g., VCAM-1-mediated VLA-4 signaling. In preferred embodiments the antagonist has one or both of properties 1 and 2. In other preferred embodiments the antagonist has one or both of properties 3 and 4. Moreover, more than one antagonist can be administered to a patient, e.g., an agent which binds to VLA-4 can be combined with an agent which binds to VCAM-1.

For example, antibodies or antibody homologs (discussed below) as well as soluble forms of the natural binding proteins for VLA-4 and VCAM-1 are useful. Soluble forms of the natural binding proteins for VLA-4 include soluble VCAM-1 peptides, VCAM-1 fusion proteins, bifunctional VCAM-1/Ig fusion proteins, fibronectin, fibronectin having an alternatively spliced non-type III connecting segment, and fibronectin peptides containing the amino acid sequence EILDV or a similar conservatively substituted amino acid sequence. Soluble forms of the natural binding proteins for VCAM-1 include soluble VLA-4 peptides, VLA-4 fusion proteins, bifunctional VLA-4/Ig fusion proteins and the like. As used herein, a "soluble VLA-4 peptide" or a "soluble VCAM-1 peptide" is an VLA-4 or VCAM-1 polypeptide incapable of anchoring itself in a membrane. Such soluble polypeptides include, for example, VLA-4 and VCAM polypeptides that lack a sufficient portion of their membrane spanning domain to anchor the polypeptide or are modified such that the membrane spanning domain is non-functional. These binding agents can act by competing with the cell-surface binding protein for VLA-4 or by otherwise altering VLA-4 function. For example, a soluble form of VCAM-1(see, e.g., Osborn et al. 1989, Cell, 59: 1203-1211) or a fragment thereof may be administered to bind to VLA-4, and preferably compete for a VLA-4 binding site on myeloma cells, thereby leading to effects similar to the administration of antagonists such as small molecules or anti-VLA-4 antibodies.

In another example, VCAM-1, or a fragment thereof, which is capable of binding to VLA-4 on the surface of VLA-4 bearing myeloma cells, e.g., a fragment containing the two N-terminal domains of VCAM-1, can be fused to a second peptide, e.g., a peptide which increases the solubility or the in vivo life time of the VCAM-1 moiety. The second peptide can be a fragment of a soluble peptide, preferably a human peptide, more preferably a plasma protein, or a member of the immunoglobulin superfamily. In particularly preferred embodiments the second peptide is IgG or a portion or fragment thereof, e.g., the human IgG1 heavy chain constant region and includes, at least the hinge, CH2 and CH3 domains.

Other antagonists useful in the methods of the invention include, but are not limited to, agents that mimic the action of peptides (organic molecules called "small molecules") capable of disrupting the .alpha.4 integrin/.alpha.4 integrin ligand interaction by, for instance, blocking VLA-4 by binding VLA-4 receptors on the surface of cells or blocking VCAM-1 by binding VCAM-1 receptors on the surface of cells. These "small molecules" may themselves be small peptides, or larger peptide-containing organic compounds or non-peptidic organic, compounds. A "small molecule", as defined herein, is not intended to encompass an antibody or antibody homolog. Although the molecular weight of such "small molecules" is generally less than 2000, we don't intend to apply this figure as an absolute upper limit on molecular weight.

For instance, small molecules such as oligosaccharides that mimic the binding domain of a VLA-4 ligand and fit the receptor domain of VLA-4 may be employed. (See, J. J. Devlin et al., 1990, Science 249: 400-406 (1990), J. K. Scott and G. P. Smith, 1990, Science 249: 386-390, and U.S. Pat. No. 4,833,092 (Geysen), all incorporated herein by reference.) Conversely, small molecules that mimic the binding domain of a VCAM-1 ligand and fit the receptor domain of VCAM-1 may be employed.

Examples of other small molecules useful in the invention can be found in Komoriya et al. ("The Minimal Essential Sequence for a Major Cell Type-Specific Adhesion Site (CS1) Within the Alternatively Spliced Type III Connecting Segment Domain of Fibronectin Is Leucine-Aspartic Acid-Valine", J. Biol. Chem., 266 (23), pp. 15075-79 (1991)). They identified the minimum active amino acid sequence necessary to bind VLA-4 and synthesized a variety of overlapping peptides based on the amino acid sequence of the CS-1 region (the VLA-4 binding domain) of a particular species of fibronectin. They identified an 8-amino acid peptide, Glu-Ile-Leu-Asp-val-Pro-ser-Thr (SEQ ID NO: 5), as well as two smaller overlapping pentapeptides, Glu-Ile-Leu-Asp-Val (SEQ ID NO: 6) and Leu-Asp-Val-Pro-Ser (SEQ ID NO: 7), that possessed inhibitory activity against fibronectin-dependent cell adhesion. Certain larger peptides containing the LDV sequence were subsequently shown to be active in vivo (T.A. Ferguson et al., "Two Integrin Binding Peptides Abrogate T-cell-Mediated Immune Responses In Viv", Proc. Natl. Acad. Sci. USA, 88, pp. 8072-76 (1991); and S. M. Wahl et al., "Synthetic Fibronectin Peptides Suppress Arthritis in Rats by Interrupting Leukocyte Adhesion and Recruitment", J. Clin. Invest., 94, pp. 655-62 (1994)). A cyclic pentapeptide, Arg-Cys-Asp-TPro-Cys (SEQ ID NO: 8) (wherein TPro denotes 4-thioproline), which can inhibit both VLA-4 and VLA-5 adhesion to fibronectin has also been described. (See, e.g., D.M. Nowlin et al., "A Novel Cyclic Pentapeptide Inhibits Alpha4Beta1 Integrin-mediated Cell Adhesion", J. Biol, Chem., 268(27), pp. 20352-59 (1993); and PCT publication PCT/US91/04862). This pentapeptide was based on the tripeptide sequence Arg-Gly-Asp from FN which had been known as a common motif in the recognition site for several extracellular-matrix proteins.

Examples of other small molecule VLA-4 inhibitors have been reported, for example, in Adams et al., "Cell Adhesion Inhibitors", PCT US97/13013, describing linear peptidyl compounds containing beta-amino acids which have cell adhesion inhibitory activity. International patent applications WO 94/15958 and WO 92/00995 describe cyclic peptide and peptidomimetic compounds with cell adhesion inhibitory activity. International patent applications WO 93/08823 and WO 92/08464 describe guanidinyl-, urea- and thiourea-containing cell adhesion inhibitory compounds. U.S. Pat. No. 5,260,277 describes guanidinyl cell adhesion modulation compounds.

Examples of small molecules that bind to or otherwise interact with VLA-4 molecules (and/or that specifically inhibit the binding of a ligand to VLA-4) and inhibit VLA-4 dependent cell adhesion are disclosed in PCT publication WO 01/12186 A1, published Feb. 22, 2001 (PCT application number PCT/US00/22285, filed Aug. 14, 2000), the disclosure of which is incorporated by reference herein. A preferred small molecule is BIO-8809, shown below -- see Original Patent.

Small molecules mimetic agents may be produced by synthesizing a plurality of peptides semi-peptidic compounds or non-peptidic, organic compounds, and then screening those compounds for their ability to inhibit the .alpha.4 integrin/.alpha.4 integrin ligand interaction. See generally U.S. Pat. No. 4,833,092, Scott and Smith, "Searching for Peptide Ligands with an Epitope Library", Science, 249, pp. 386-90 (1990), and Devlin et al., "Random Peptide Libraries: A Source of Specific Protein Binding Molecules", Science, 249, pp. 40407 (1990).

In other preferred embodiments, the agent that is used in the method of the invention to bind to, including block or coat, cell-surface .alpha.4 integrin and/or .alpha.4 integrin ligand is an anti-VLA-4 and/or anti-.alpha.4.beta.7 monoclonal antibody or antibody homolog. Preferred antibodies and homologs for treatment, in particular for human treatment, include human antibody homologs, humanized antibody homologs, chimeric antibody homologs, Fab, Fab', F(ab')2 and F(v) antibody fragments, and monomers or dimers of antibody heavy or light chains or mixtures thereof. Monoclonal antibodies against VLA-4 are a preferred binding agent in the method of the invention.

As used herein, the term "antibody homolog" includes intact antibodies consisting of immunoglobulin light and heavy chains linked via disulfide bonds. The term "antibody homolog" is also intended to encompass a protein comprising one or more polypeptides selected from immunoglobulin light chains, immunoglobulin heavy chains and antigen-binding fragments thereof which are capable of binding to one or more antigens. The component polypeptides of an antibody homolog composed of more than one polypeptide may optionally be disulfide-bound or otherwise covalently crosslinked.

Accordingly, therefore, "antibody homologs" include intact immunoglobulins of types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the light chains of the immunoglobulin may be of types kappa or lambda.

"Antibody homologs" also include portions of intact antibodies that retain antigen-binding specificity, for example, Fab fragments, Fab' fragments, F(ab')2 fragments, F(v) fragments, heavy chain monomers or dimers, light chain monomers or dimers, dimers consisting of one heavy and one light chain, and the like. Thus, antigen-binding fragments, as well as full-length dimeric or trimeric polypeptides derived from the above-described antibodies are themselves useful.

As used herein, a "humanized antibody homolog" is an antibody homolog, produced by recombinant DNA technology, in which some or all of the amino acids of a human immunoglobulin light or heavy chain that are not required for antigen binding have been substituted for the corresponding amino acids from a nonhuman mammalian immunoglobulin light or heavy chain.

As used herein, a "chimeric antibody homolog" is an antibody homolog, produced by recombinant DNA technology, in which all or part of the hinge and constant regions of an immunoglobulin light chain, heavy chain, or both, have been substituted for the corresponding regions from another immunoglobulin light chain or heavy chain. In another aspect the invention features a variant of a chimeric molecule which includes: (1) a VLA-4 targeting moiety, e.g., a VCAM-1 moiety capable of binding to antigen (i.e., VLA-4) on the surface of VLA-4 bearing myeloma cells; (2) optionally, a second peptide, e.g., one which increases solubility or in vivo life time of the VLA-4 targeting moiety, e.g., a member of the immunoglobulin superfamily or fragment or portion thereof, e.g., a portion or a fragment of IgG, e.g., the human IgGl heavy chain constant region, e.g., CH2 and CH3 hinge regions; and a toxin moiety. The VLA-4 targeting moiety can be any naturally occurring VLA-4 ligand or fragment thereof, e.g., a VCAM-1 peptide or a similar conservatively substituted amino acid sequence. A preferred targeting moiety is a soluble VCAM-1 fragment, e.g., the N-terminal domains 1 and 2 of the VCAM-1 molecule. The chimeric molecule can be used to treat a subject, e.g., a human, at risk for disorder, e.g., multiple myeloma, characterized by the presence of myeloma cells bearing VLA-4, and preferably activated VLA-4.

As used herein, a "human antibody homolog" is an antibody homolog produced by recombinant DNA technology, in which all of the amino acids of an immunoglobulin light or heavy chain that are derived from a human source.

Methods of Making Anti-VLA-4 Antibody Homologs

The technology for producing monoclonal antibody homologs is well known. Briefly, an immortal cell line (typically myeloma cells) is fused to lymphocytes (typically splenocytes) from a mammal immunized with whole cells expressing a given antigen, e.g., VLA-4, and the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen. See, generally, Kohler et al., 1975, Nature, 265: 295-297.

Immunization may be accomplished using standard procedures. The unit dose and immunization regimen depend on the species of mammal immunized, its immune status, the body weight of the mammal, etc. Typically, the immunized mammals are bled and the serum from each blood sample is assayed for particular antibodies using appropriate screening assays. For example, anti-VLA-4 antibodies may be identified by immunoprecipitation of 125I-labeled cell lysates from VLA-4-expressing cells. (See, Sanchez-Madrid et al., 1986, Eur. J. Immunol., 16: 1343-1349 and Hemler et al., 1987, J. Biol. Chem., 262, 11478-11485). Anti-VLA-4 antibodies may also be identified by flow cytometry, e.g., by measuring fluorescent staining of Ramos cells incubated with an antibody believed to recognize VLA-4 (see, Elices et al., 1990 Cell, 60: 577-584). The lymphocytes used in the production of hybridoma cells typically are isolated from immunized mammals whose sera have already tested positive for the presence of anti-VLA-4 antibodies using such screening assays.

Typically, the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, arninopterin and thymidine ("HAT medium"). Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using 1500 molecular weight polyethylene glycol ("PEG 1500"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridomas producing a desired antibody are detected by screening the hybridoma culture supernatants. For example, hybridomas prepared to produce anti-VLA-4 antibodies may be screened by testing the hybridoma culture supernatant for secreted antibodies having the ability to bind to a recombinant .alpha.4-subunit-expressing cell line (see, Elices et al., supra).

To produce anti-VLA-4 antibody homologs that are intact immunoglobulins, hybridoma cells that tested positive in such screening assays were cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium. Tissue culture techniques and culture media suitable for hybridoma cells are well known. The conditioned hybridoma culture supernatant may be collected and the anti-VLA4 antibodies optionally further purified by well-known methods.

Alternatively, the desired antibody may be produced by injecting the hybridoma cells into the peritoneal cavity of an unimmunized mouse. The hybridoma cells proliferate in the peritoneal cavity, secreting the antibody which accumulates as ascites fluid. The antibody may be harvested by withdrawing the ascites fluid from the peritoneal cavity with a syringe.

Several mouse anti-VLA-4 monoclonal antibodies have been previously described. See, e.g., Sanchez-Madrid et al., 1986, supra; Hemler et al., 1987, supra; Pulido et al., 1991, J. Biol. Chem., 266 (16), 10241-10245). These anti-VLA-4 monoclonal antibodies such as HP 1/2 and other anti-VLA-4 antibodies (e.g., HP2/1, HP2/4, L25, P4C2, P4G9) capable of recognizing the P chain of VLA-4 will be useful in the methods of treatment according to the present invention. Anti VLA-4 antibodies that will recognize the VLA-4 .alpha.4 chain epitopes involved in binding to VCAM-1 and fibronectin ligands (i.e., antibodies which can bind to VLA-4 at a site involved in ligand recognition and block VCAM-1 and fibronectin binding) are preferred. Such antibodies have been defined as B epitope-specific antibodies (B1 or B2) (Pulido et al., 1991, supra) and are also anti-VLA-4 antibodies according to the present invention.

Fully human monoclonal antibody homologs against VLA-4 are another preferred binding agent which may block or coat VLA-4 antigens in the method of the invention. In their intact form these may be prepared using in vitro-primed human splenocytes, as described by Boerner et al., 1991, J. Immunol., 147, 86-95. Alternatively, they may be prepared by repertoire cloning as described by Persson et al., 1991, Proc. Nat. Acad. Sci. USA, 88: 2432-2436 or by Huang and Stollar, 1991, J. Immunol. Methods 141, 227-236. U.S. Pat. No. 5,798,230 (Aug. 25, 1998, "Process for the preparation of human monoclonal antibodies and their use") who describe preparation of human monoclonal antibodies from human B cells. According to this process, human antibody-producing B cells are immortalized by infection with an Epstein-Barr virus, or a derivative thereof, that expresses Epstein-Barr virus nuclear antigen 2 (EBNA2). EBNA2 function, which is required for immortalization, is subsequently shut off, which results in an increase in antibody production.

In yet another method for producing fully human antibodies, U.S. Pat. No. 5,789,650 (Aug. 4, 1998, "Transgenic non-human animals for producing heterologous antibodies") describes transgenic non-human animals capable of producing heterologous antibodies and transgenic non-human animals having inactivated endogenous immunoglobulin genes. Endogenous immunoglobulin genes are suppressed by antisense polynucleotides and/or by antiserum directed against endogenous immunoglobulins. Heterologous antibodies are encoded by immunoglobulin genes not normally found in the genome of that species of non-human animal. One or more transgenes containing sequences of unrearranged heterologous human immunoglobulin heavy chains are introduced into a non-human animal thereby forming a transgenic animal capable of functionally rearranging transgenic immunoglobulin sequences and producing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes. Such heterologous human antibodies are produced in B-cells which are thereafter immortalized, e.g., by fusing with an immortalizing cell line such as a myeloma or by manipulating such B-cells by other techniques to perpetuate a cell line capable of producing a monoclonal heterologous, fully human antibody homolog.

Large nonimmunized human phage display libraries may also be used to isolate high affinity antibodies that can be developed as human therapeutics using standard phage technology (Vaughan et al., 1996). Yet another preferred binding agent which may block or coat VLA-4 antigens in the method of the invention is a humanized recombinant antibody homolog having anti-VLA-4 specificity. Following the early methods for the preparation of chimeric antibodies, a new approach was described in EP 0239400 (Winter et al.) whereby antibodies are altered by substitution of their complementarity determining regions (CDRs) for one species with those from another. This process may be used, for example, to substitute the CDRs from human heavy and light chain Ig variable region domains with alternative CDRs from murine variable region domains. These altered Ig variable regions may subsequently be combined with human Ig constant regions to created antibodies which are totally human in composition except for the substituted murine CDRs. Such CDR-substituted antibodies would be predicted to be less likely to elicit an immune response in humans compared to chimeric antibodies because the CDR-substituted antibodies contain considerably less non-human components. The process for humanizing monoclonal antibodies via CDR "grafting" has been termed "reshaping". (Riechmann et al., 1988, Nature 332, 323-327; Verhoeyen et al., 1988, Science 239, 1534-1536).

Typically, complementarity determining regions (CDRs) of a murine antibody are transplanted onto the corresponding regions in a human antibody, since it is the CDRs (three in antibody heavy chains, three in light chains) that are the regions of the mouse antibody which bind to a specific antigen. Transplantation of CDRs is achieved by genetic engineering whereby CDR DNA sequences are determined by cloning of murine heavy and light chain variable (V) region gene segments, and are then transferred to corresponding human V regions by site directed mutagenesis. In the final stage of the process, human constant region gene segments of the desired isotype (usually gamma I for CH and kappa for CL) are added and the humanized heavy and light chain genes are co-expressed in mammalian cells to produce soluble humanized antibody.

The transfer of these CDRs to a human antibody confers on this antibody the antigen binding properties of the original murine antibody. The six CDRs in the murine antibody are mounted structurally on a V region "framework" region. The reason that CDR-grafting is successful is that framework regions between mouse and human antibodies may have very similar 3-D structures with similar points of attachment for CDRs, such that CDRs can be interchanged. Such humanized antibody homologs may be prepared, as exemplified in Jones et al., 1986, Nature 321, 522-525; Riechmann, 1988, Nature 332, 323-327; Queen et al., 1989, Proc. Nat. Acad. Sci. USA 86, 10029; and Orlandi et al., 1989, Proc. Nat. Acad. Sci. USA 86, 3833.

Nonetheless, certain amino acids within framework regions are thought to interact with CDRs and to influence overall antigen binding affinity. The direct transfer of CDRs from a murine antibody to produce a recombinant humanized antibody without any modifications of the human V region frameworks often results in a partial or complete loss of binding affinity. In a number of cases, it appears to be critical to alter residues in the framework regions of the acceptor antibody in order to obtain binding activity.

Queen et al., 1989 (supra) and WO 90/07861 (Protein Design Labs) have described the preparation of a humanized antibody that contains modified residues in the framework regions of the acceptor antibody by combining the CDRs of a murine mAb (anti-Tac) with human immunoglobulin framework and constant regions. They have demonstrated one solution to the problem of the loss of binding affinity that often results from direct CDR transfer without any modifications of the human V region framework residues; their solution involves two key steps. First, the human V framework regions are chosen by computer analysts for optimal protein sequence homology to the V region framework of the original murine antibody, in this case, the anti-Tac mAb. In the second step, the tertiary structure of the murine V region is modeled by computer in order to visualize framework amino acid residues which are likely to interact with the murine CDRs and these murine amino acid residues are then superimposed on the homologous human framework. See also Protein Design Labs--U.S. Pat. No. 5,693,762.

One may use a different approach (Tempest et al., 1991, Biotechnology 9, 266-271) and utilize, as standard, the V region frameworks derived from NEWM and REI heavy and light chains respectively for CDR-grafting without radical introduction of mouse residues. An advantage of using the Tempest et al., approach to construct NEWM and REI based humanized antibodies is that the 3-dimensional structures of NEWM and REI variable regions are known from x-ray crystallography and thus specific interactions between CDRs and V region framework residues can be modeled.

Regardless of the approach taken, the examples of the initial humanized antibody homologs prepared to date have shown that it is not a straightforward process. However, even acknowledging that such framework changes may be necessary, it is not possible to predict, on the basis of the available prior art, which, if any, framework residues will need to be altered to obtain functional humanized recombinant antibodies of the desired specificity. Results thus far indicate that changes necessary to preserve specificity and/or affinity are for the most part unique to a given antibody and cannot be predicted based on the humanization of a different antibody.

Preferred antagonists useful in the present invention include chimeric recombinant and humanized recombinant antibody homologs (i.e., intact immunoglobulins and portions thereof) with B epitope specificity that have been prepared and are described in co-pending U.S. patent application Ser. No. 08/004,798, filed Jan. 12, 1993, PCT Publication US94/00266, filed Jan. 7, 1994. The starting material for the preparation of chimeric (mouse V-human C) and humanized anti-VLA-4 antibody homologs may be a murine monoclonal anti-VLA-4 antibody as previously described, a monoclonal anti-VLA-4 antibody commercially available (e.g., HP2/1, Amae International, Inc., Westbrook, Me.), or a monoclonal anti-VLA-4 antibody prepared in accordance with the teaching herein. For example, the variable regions of the heavy and light chains of the anti-VLA-4 antibody HP 1/2 have been cloned, sequenced and expressed in combination with constant regions of human immunoglobulin heavy and light chains. Such HP 1/2 antibody is similar in specificity and potency to the murine HP 1/2 antibody, and may be useful in methods of treatment according to the present invention.

Other preferred humanized anti-VLA4 antibody homologs are described by Athena Neurosciences, Inc. in PCT/US95/01219 (27 Jul. 1995). These humanized anti-VLA-4 antibodies comprise a humanized light chain and a humanized heavy chain. The humanized light chain comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of a mouse 21-6 immunoglobulin light chain, and a variable region framework from a human kappa light chain variable region framework sequence except in at least position the amino acid position is occupied by the same amino acid present in the equivalent position of the mouse 21.6 immunoglobulin light chain variable region framework. The humanized heavy chain comprises three complementarity determining regions (CDR1, CDR2 and CDR3) having amino acid sequences from the corresponding complementarity determining regions of a mouse 21-6 immunoglobulin heavy chain, and a variable region framework from a human heavy chain variable region framework sequence except in at least one position the amino acid position is occupied by the same amino acid present in the equivalent position of the mouse 21-6 immunoglobulin heavy chain variable region framework.

Therapeutic Applications

In this method according to the first aspect of the invention, VLA-4 binding agents, in particular, VCAM fusions and anti-VLA-4 antibody homologs are preferably administered parenterally. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intra-synovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques.

The VLA-4 binding agents are preferably administered as a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier, which may be any of the numerous well known carriers, such as water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, or combinations thereof. The compounds of the present invention may be used in the form of pharmaceutically acceptable salts derived from inorganic or organic acids and bases. Included among such acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenyl-propionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Base salts include ammonium salts, alkali metal salts, such as sodium and potassium salts, alkaline earth metal salts, such as calcium and magnesium salts, salts with organic bases, such as dicyclohexylamine salts, N-methyl-D-glucamine, tris(hydroxymethyl)methylamine and salts with amino acids such as arginine, lysine, and so forth. Also, the basic nitrogen-containing groups can be quaternized with such agents as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides; dialkyl sulfates, such as dimethyl, diethyl, dibutyl and diamyl sulfates, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. Water or oil soluble or dispersible products are then obtained.

The pharmaceutical compositions of this invention comprise any of the compounds of the present invention, or pharmaceutically acceptable derivatives thereof, together with any pharmaceutically acceptable carrier. The term "carrier" as used herein includes acceptable adjuvants and vehicles. Pharmaceutically acceptable carriers that may be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salts or electrolytes, such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol and wool fat.

According to this invention, the pharmaceutical compositions may be in the form of a sterile injectable preparation, for example a sterile injectable aqueous or oleaginous suspension. This suspension may be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation may also be a sterile injectable solution or suspension in a non-toxic parenterally-acceptable diluent or solvent, for example as a solution in 1,3-butanediol. Among the acceptable vehicles and solvents that may be employed are water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose, any bland fixed oil may be employed including synthetic mono- or di-glycerides. Fatty acids, such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as do natural pharmaceutically-acceptable oils, such as olive oil or castor oil, especially in their polyoxyethylated versions. These oil solutions or suspensions may also contain a long-chain alcohol diluent or dispersant, such as Ph. Helv or similar alcohol.

The pharmaceutical compositions of this invention, in particular small molecule antagonists of the VLA-4/VCAM-1 interaction, may be given parenterally or orally. If given orally, they can be administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions or solutions. In the case of tablets for oral use, carriers which are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in a capsule form, useful diluents include lactose and dried corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweetening, flavoring or coloring agents may also be added. Topically-transdermal patches may also be used. The pharmaceutical compositions of this invention may also be administered by nasal aerosol or inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler. Such compositions are prepared according to techniques well-known in the art of pharmaceutical formulation and may be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to enhance bioavailability, fluorocarbons, and/or other conventional solubilizing or dispersing agents.

According to another embodiment compositions containing a compound of this invention may also comprise an additional agent selected from the group consisting of corticosteroids, anti-inflammatories, immunosuppressants, antimetabolites, and immunomodulators. Specific compounds within each of these classes may be selected from any of those listed under the appropriate group headings in "Comprehensive Medicinal Chemistry", Pergamon Press, Oxford, England, pp. 970-986 (1990), the disclosure of which is herein incorporated by reference. Also included within this group are compounds such as theophylline, sulfasalazine and aminosalicylates (anti-inflammatories); cyclosporin, FK-506, and rapamycin (immunosuppressants); cyclophosphamide and methotrexate (antimetabolites); steroids (inhaled, oral or topical) and interferons (immunomodulators).

The amount of active ingredient that may be combined with the carrier materials to produce a single dosage form will vary depending upon the host treated, and the particular mode of administration. It should be understood, however, that a specific dosage and treatment regimen for any particular patient will depend upon a variety of factors, including the activity of the specific compound employed, the age, body weight, general health, sex, diet, time of administration, rate of excretion, drug combination, and the judgment of the treating physician and the severity of the particular disease being treated. The amount of active ingredient may also depend upon the therapeutic or prophylactic agent, if any, with which the ingredient is co-administered.

The dosage and dose rate of the compounds of this invention effective to prevent, suppress or inhibit cell adhesion will depend on a variety of factors, such as the nature of the inhibitor, the size of the patient, the goal of the treatment, the nature of the pathology to be treated, the specific pharmaceutical composition used, and the judgment of the treating physician. Dosage levels of between about 0.001 and about 100 mg/kg body weight per day, preferably between about 0.1 and about 50 mg/kg body weight per day of the active ingredient compound are useful. Most preferably, the VLA-4 binding agent, if an antibody or antibody derivative, will be administered at a dose ranging between about 0.1 mg/kg body weight/day and about 20 mg/kg body weight/day, preferably ranging between about 0.1 mg/kg body weight/day and about 10 mg/kg body weight/day and at intervals of every 1-14 days. For non-antibody or small molecule binding agents, the dose range should preferably be between molar equivalent amounts to these amounts of antibody. Preferably, an antibody composition is administered in an amount effective to provide a plasma level of antibody of at least 1 mg/ml. Optimization of dosages can be determined by administration of the binding agents, followed by assessment of the coating of VLA-4-positive cells by the agent over time after administered at a given dose in vivo.

Myeloma cells contained in a sample of the individual's peripheral blood (or bone marrow cells) should be probed for the presence of the agent in vitro (or ex vivo) using a second reagent to detect the administered agent. For example, this may be a fluorochrome labeled antibody specific for the administered agent which is then measured by standard FACS (fluorescence activated cell sorter) analysis. Alternatively, presence of the administered agent may be detected in vitro (or ex vivo) by the inability or decreased ability of the individual's cells to bind the same agent which has been itself labeled (e.g., by a fluorochrome). The preferred dosage should produce detectable coating of the vast majority of VLA-4-positive cells. Preferably, coating is sustained in the case of an antibody homolog for a 1-14 day period.

Combination Therapy

In some embodiments of this invention, one or more antagonists of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin could be administered in combination with one or more compounds that may not be (preferably are not) an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin. Such compound could be a chemotherapeutic agent or another agent, including without limitation: melphalan, bisphosphonates (examples of which are ibandronate and pamidronate), thalidomide, erythropoietin, and antagonists, such as mAb blockers, of IL6 and IL15.

Multiple myeloma is currently treated inefficiently with standard chemotherapeutic regimens. In some embodiments of this invention, an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin could be administered in combination with one or more standard agents for treatment of multiple myeloma, such as chemotherapeutic agents. Hence, the two compounds (an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin and a compound that may not be (preferably is not) an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin; said compound that may not be (preferably is not) an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin could be a chemotherapeutic agent or another agent for treatment or prevention of multiple myeloma, including, inter alia: melphalan, bisphosphonates (examples of which are ibandronate and pamidronate), thalidomide, erythropoietin, and antagonists, such as mAb blockers, of IL6 and IL15) could act to prevent or treat multiple myeloma synergistically; or lower dosage of either or both compounds (an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin and a compound that may not be (preferably is not) an antagonist of an interaction between an integrin with an .alpha.4 subunit and a ligand for this integrin, such as one or more standard agents for treatment of multiple myeloma) needed to provide the same effect as a higher dosage of either compound alone.

In some embodiments, this invention provides methods for treating or preventing multiple myeloma comprising administering to an individual a therapeutically or prophylactically effective amount of a first composition comprising an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin, wherein said first composition is administered in combination with a therapeutically or prophylactically effective amount of a second composition comprising a compound that may not be (preferably is not) an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin. In certain embodiments, this invention provides methods for treating or preventing multiple myeloma comprising administering to an individual a therapeutically or prophylactically effective amount of a first composition comprising an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin, wherein said first composition is administered in combination with a therapeutically or prophylactically effective amount of a second composition comprising a compound that may not be (preferably is not) an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin, wherein, to be therapeutically or prophylactically effective, a dosage of said antagonist is lower when administered in combination with said compound than not administered in combination with said compound; or

a dosage of said compound is lower when administered in combination with said antagonist than not administered in combination with said antagonist, or both. Such compound could be a chemotherapeutic agent or another agent for treating or preventing multiple myeloma, including, for example: melphalan, bisphosphonates (examples of which are ibandronate and pamidronate), thalidomide, erythropoietin, and antagonists, such as mAb blockers, of IL6 and IL15.

In some embodiments, this invention provides methods for inhibiting bone resorption associated with tumors of bone marrow, the methods comprising administering to a mammal with said tumors an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin, in an amount effective to provide inhibition of said bone resorption, wherein said antagonist is administered in combination with a compound, in an amount effective to provide inhibition of said bone resorption, that may not be (preferably is not) an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin. In certain embodiments, this invention provides methods for inhibiting bone resorption associated with tumors of bone marrow, the methods comprising administering to a mammal with said tumors an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin, in an amount effective to provide inhibition of said bone resorption, wherein said antagonist is administered in combination with a compound, in an amount effective to provide inhibition of said bone resorption, that may not be (preferably is not) an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin, wherein, to be therapeutically or prophylactically effective,

a dosage of said antagonist is lower when administered in combination with said compound than not administered in combination with said compound; or

a dosage of said compound is lower when administered in combination with said antagonist than not administered in combination with said antagonist, or both. Such compound could be a chemotherapeutic agent or another agent for inhibiting bone resorption associated with tumors of bone marrow, including, for example: melphalan, bisphosphonates (examples of which are ibandronate and pamidronate), thalidomide, erythropoietin, and antagonists, such as mAb blockers, of IL6 and IL15.

In some embodiments, this invention provides methods of treating or preventing a subject having a disorder characterized by the presence of osteoclastogenesis, the methods comprising administering to the subject an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit bearing integrin, in an amount sufficient to suppress or prevent the osteoclastogenesis, wherein said antagonist is administered in combination with a compound, in an amount sufficient to suppress or prevent the osteoclastogenesis, that may not be (preferably is not) an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin. In certain embodiments, this invention provides methods of treating or preventing a subject having a disorder characterized by the presence of osteoclastogenesis, the methods comprising administering to the subject an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit bearing integrin, in an amount sufficient to suppress or prevent the osteoclastogenesis, wherein said antagonist is administered in combination with a compound, in an amount sufficient to suppress or prevent the osteoclastogenesis, that may not be (preferably is not) an antagonist of an interaction between an .alpha.4 subunit-bearing integrin and a ligand for an .alpha.4 subunit-bearing integrin, wherein, to be therapeutically or prophylactically effective,

a dosage of said antagonist is lower when administered in combination with said compound than not administered in combination with said compound; or

a dosage of said compound is lower when administered in combination with said antagonist than not administered in combination with said antagonist, or both. Such compound could be a chemotherapeutic agent or another agent for treating or preventing a subject having a disorder characterized by the presence of osteoclastogenesis, including, for example: melphalan, bisphosphonates (examples of which are ibandronate and pamidronate), thalidomide, erythropoietin, and antagonists, such as mAb blockers, of IL6 and IL15.

In some embodiments, the pharmaceutical or prophylactic composition of this invention can also include a pharmaceutically or prophylactically effective amount of a chemotherapeutic agent or another agent, including without limitation: melphalan, bisphosphonates (examples of which are ibandronate and pamidronate), thalidomide, erythropoietin, and antagonists, such as mAb blockers, of IL6 and IL15. Said chemotherapeutic agent or another agent could be included in a second composition.

Animal Models

The animal model has been described in detail (Garrett 1997). Briefly, Radl et al. (1988) had described a murine model of myeloma which arose spontaneously in aged C57BL/KaLwRij mice. This condition occurred in approximately 1 in 200 animals as they aged, and led to a monoclonal gammopathy with some of the features of human disease (Radl 1988). To develop a better and more reproducible animal model we have established and subcloned a cell line from this murine myeloma called 5TGM1, and found that it causes lesions in mice characteristic of human myeloma, such as severe osteolysis and the involvement of non-bone organs including liver and kidney (Garrett 1997). Mice inoculated with cultured cells develop disease in a highly predictable and reproducible manner, which includes formation of a monoclonal gammopathy and radiologic bone lesions. Furthermore, some of the mice become hypercalcemic, and the bone lesions are characterized by increased osteoclast activity. Thus, based on histological examination of affected organs in 5TGM1-bearing mice and increased serum levels of IgG2b, 5TGM1 is defined as a murine myeloma which recapitulates accurately the hallmarks of human disease.
 

Claim 1 of 16 Claims

1. A method for treating multiple myeloma in a subject comprising administering to the subject a combination of an anti-VLA-4 antibody, or antigen-binding fragment thereof, and a chemotherapeutic agent, wherein said combination is therapeutically effective to treat multiple myeloma in the subject.

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