MXPA01002670A - Methods of treating multiple myeloma and myeloma-induced bone resorption using integrin antagonists - Google Patents

Abstract

Antagonists of alpha4 integrin/alpha4 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 alpha4 integrin-dependent release of bone-resorbing factors, resulting in bone destruction in patients with multiple myeloma, is inhibited.

Description

* > - - METHODS FOR TREATING MYELOMA. MULTIPLE AND OSTEOUS OSTEOUS OSTEOUS REASSEMBLY USING INTEGRINE ANTAGONISTS FIELD OF THE INVENTION The present invention relates to a treatment for multiple myelone, and the release of factors that reabsorb bone by myeloma cells, resulting in severe bone loss, which is the most important side effect of myeloma in men. . More particularly, this invention relates to integrin antagonists, such as alpha 4-containing integrin antagonists, which inhibit the biological effects of this adhesion, associated with the lodging of multiple myeloma cells in the bone marrow; its subsequent integrin-dependent survival; and its integrin-dependent release of factors that reabsorb bone, resulting in bone destruction in patients with multiple myeloma. BACKGROUND OF THE INVENTION 0 Multiple myeloma is the hematologic malignancy, the second most common, with 15,000 new cases diagnosed each year and 30,000 to 40,000 myeloma patients in the United States of America annually (Mundy and Bertolini 1986). Eighty percent of patients suffer 5 devastating osteolytic bone destruction caused by increased osteoclast (OCL) formations and activity (Mundy and Bertolini 1986). This bone destruction can cause increased bone pain, pathological fractures, spinal cord compression, and life-threatening hypercalcemia. Because myeloma can not be cured by normal chemotherapy or stem cell transplantation (Attal et al., 1996), and due to the severe morbidity and potential mortality associated with myeloma bone disease, the treatment strategies that they control the same growth of myeloma, and in particular the osteolytic bone destruction that occurs in these patients, are vitally important. However, the pathological mechanisms responsible for increased osteoclastic activity in patients with multiple myeloma are unknown (Mundy, 1998). Bone lesions occur in severe patterns. Occasionally, patients develop discrete osteolytic lesions that are associated with solitary plasmacytomas. Some patients have diffuse osteopenia, which mimics the appearance of osteoporosis, and is due to the fact that the myeloma cells spread diffusely throughout the axial skeleton. In most patients there are multiple discrete lithic lesions that occur adjacent to myeloma cell nests. Hypercalcemia occurs as a consequence of bone destruction in approximately one third of patients with advanced disease. Rarely, patients with myeloma do not have lithic lesions or bone loss, but have an increase in the formation of new bone around the myeloma cells. This rare situation is known as osteosclerotic myeloma. Osteolytic bone lesions are by far the most common skeletal manifestations in patients with myeloma (Mundy, 1998). Although precise molecular mechanisms remain unclear, observations over 15 years have shown that: 1) the mechanism by which bone is destroyed in myeloma is via the osteoclast, the normal bone resorption cell; 2) osteoclasts accumulate on the surfaces that reabsorb bone in the myeloma adjacent to collections of myeloma cells and it seems that the mechanism by which osteoclasts are stimulated in myeloma is a local mechanism; 3) it has been known for many years that cultures of human myeloma cells in vi tro produce several osteoclast activating factors, including lymphotoxin-alpha (LT-a), interleukin-1 (IL-1), protein related to parathyroid hormone (PTHrP) and interleukin-6 (IL-6); 4) Hypercalcemia occurs in approximately one third of patients with myeloma sometime during the course of the disease. Hypercalcemia is always associated with markedly increased bone resorption and often with glomerular filtration defects; 5) the increase in osteoclastic bone resorption in myeloma is usually associated with a marked defect in osteoblast function. The activity of alkaline phosphatase in serum is decreased or in the normal range, although patients with other types of osteolytic bone disease, and radionuclide scans show no evidence of increased absorption, indicating defective osteoblast responses to increased reabsorption that is. Although several mediators listed above have been implicated in the stimulation of osteoclast activity in patients with multiple myeloma, reports of factors produced by myeloma cells have not been consistent, and some studies have been inconclusive due to the presence of other contaminating cell types, including stromal cells and macrophages, in the cell population of multiple myeloma. Interleukin-6 is an important myeloma growth factor that enhances the growth of several myeloma cell lines and newly isolated myeloma cells of patients (Bataille et al., 1989). The production of interleukin 6 can be detected in approximately 40 percent of newly isolated myeloma cells by polymerase chain reaction, but only one of 150 patients studied shows interleukin 6 production detectable by immunocytochemistry or ELISA tests (Epstein 1992 ). Interleukin 6 receptors were only detected in 6 out of 13 samples from patients with multiple myeloma (Bataille et al., 1992). In addition, mature myeloma cells have been reported to have a minimal proliferative response to interleukin 6. Interleukin 11 (IL-11) has an activity known to interleukin 6 on plasmacytomas, but to date no one has demonstrated that myeloma cells produce interleukin 11. Bataille et al., (1995) have shown perfusion of 5 patients with refractory myeloma with an antibody to interleukin 6 decreased the size of myeloma cell burden in only 2 of these patients. Interleukin 1 is an extremely potent bone resorption agent that induces hypercalcemia in animal models in the absence of renal failure (Boyce et al., 1989). In contrast, hypercalcemia rarely occurs in myeloma patients without renal failure. More importantly, in highly purified myeloma cells, interleukin 1 and only a poor production of TNF-α can not be detected, suggesting that other contaminating cell types such as macrophages may be the source of interleukin 1 and TNF-α (Epstein 1992). Similarly, LT-a is produced by most human myeloma cell lines (Bataille et al., 1995) but does not appear to be produced by myeloma cells in vivo (Alsina et al., 1996). In addition to interleukin 1, TNF-a, LT-1, and interleukin 6, myeloma cells produce a truncated form of M-CSF that is biologically active, but M-CSF does not cause hypercalcemia or induce osteoclast formation itself same in cultures of human marrow (MacDonald et al., 1986). Thus, the function of any of these factors in osteolytic bone disease in patients with myeloma has not been clearly demonstrated in vivo, so that known cytokines clearly do not fully participate in the bone resorption seen in these patients. Role of adhesive molecule interactions in myeloma bone disease Anderson and colleagues were the first group to demonstrate the importance of adhesive interactions between myeloma cells and cells in the marrow microenvironment in both cell growth and myeloma as in the development of osteolytic bone disease. Multiple myeloma cells express cell surface adhesion molecules, CD29 (VLA-4), LFA-1 and CD44 (Chauhan et al., nineteen ninety five) . These researchers suggested that the myeloma cells located in the bone marrow via the specific adhesion interactions between the extracellular matrix proteins and the bone marrow stromal cells. They also showed that the adhesion of multiple myeloma cells to the stromal cells triggered the secretion of interleukin 6 by both normal cells and multiple stromal cells derived from the bone marrow with myeloma and interleukin 6 mediated cell growth. However, the antibodies to CD29, LFA-1 or CD44 did not decrease the production of interleukin 6 by marrow stromal cells in response to myeloma cells, suggesting that another ligand-receptor interaction triggered the secretion of interleukin 6 by the stromal cells of the bone marrow attached to the myeloma cells. The mere identification of the possible path of adherence does not necessarily mean that the path is important. In this case none of the pathways involved represents a role in the production of interleukin 6. Vanderkerken et al. (1997) also examined the phenotypic adhesion profile of murine 5T2 cells and 5T33 myeloma cells in a murine myeloma model. These researchers showed that these cell lines expressed VLA-4, VLA-5, LFA-1, and CD44, and suggested that these adhesive interactions could be important for myeloma cells to bind with marrow stromal cells. However, despite many laboratory advances, the underlying mechanisms underlying increased osteoclastic bone destruction in myeloma in vivo are poorly understood. This is reflected in the inability to easily translate the data on adhesive interactions obtained in vi tro to the live stage. For example, many in vi tro studies involve both the integrin VLA-4 and the integrin LFA-1 in the adhesion of hematopoietic stem cells to the stroma of the bone marrow (reviewed in Papayannopoulou and Nakamoto, 1993). These in vitro data predict that any pathway, if blocked in vivo, will result in the peripheralization of hematopoietic stem cells from the medulla to the peripheral blood. Still, in a primate study, while a monoclonal antibody (mAb) for VLA-4 effectively periferalized stem cells, a monoclonal antibody to the beta 2 integrin chain of LFA-1 had no effect, despite increased neutrophil counts, thus demonstrating the effectiveness of the monoclonal antibody (Papayannopoulou and Nakamoto, 1993). These data show that the in vi tro results were in fact unable to accurately predict the importance in vivo. It should be noted that the role of integrin VLA-4 has been studied in metastasis of multiple tumors, including leukemias such as lymphoma, with contradictory results. Thus, the transfection of the human alpha 4 chain in Chinese hamster ovary (CHO) cells resulted in the expression of VLA-4, and rendered these cells capable of migrating to the bone cell in vivo, a phenomenon inhibited by the monoclonal antibodies for VLA-4 (Matsuura et al., 1996). In contrast, the transfection of lmfoma cells with VLA-4 strongly inhibited metastasis to the liver, lung and kidney, and had no effect on lodging and proliferation in the marrow (Gosslar et al., 1996). In addition, the expression of VLA-4 in highly metastatic murine melanoma cells strongly inhibited the formation of lung metastases in vivo (Qian et al., 1994), and did not predispose to melanoma to bone marrow metastasis. In summary, it is not clear on the basis of the m vi tro studies, how to reliably predict the importance in vivo of the accession paths. In addition, even when live studies have been conducted, the resulting data are inconsistent. A major reason for the perplexing inconsistencies in the field of multiple myeloma is that currently available animal models are not good at predicting human disease. In the case of multiple myeloma, human and murine myeloma cell lines that can be cultured rarely are associated with bone destruction in vivo (Mundy 1998). It would be very desirable to identify compounds or antagonism that inhibit the production of these bone resorption factors, thereby stopping progressive bone destruction and improving the quality of life of patients with myeloma. COMPENDIUM OF THE INVENTION We have recently used a murine model developed from multiple myeloma in which the mouse develops severe osteolysis with all the characteristics of the human disease (Garrett 1997). Using this cell line and animal model we have established that the inhibition of the integrin alpha 4 / integrin alpha 4 ligand pathway in vivo leads to the reduced ability for multiple myeloma cells to proliferate and / or survive. We have shown that cell-cell binding between myeloma cells and spinal cord cells via the VLA-4 / VCAM-1 interaction is required for an increase in the production of an activity that stimulates osteoclastic bone resorption in the bone microenvironment in vi tro. We have proposed that this interaction is critical for the accommodation of myeloma cells to the marrow compartment, to their subsequent survival and growth, to finally the progression of myeloma-induced osteolysis. We have tested this in the animal model and found that, in vivo, an integrin antagonist VLA-4 containing the alpha 4 subunit strongly inhibits the production of IgG2b subtype antibody. This isotype is the same as that produced by the 5TGM1 cell line, and is an accurate substitute for the number of myeloma cells in the marrow compartment at any time. Thus, blockade of the VLA-4 pathway strongly inhibits the production of IgG2b, and by implication, the level of myeloma burden. One aspect of the invention is a method for the treatment of multiple myeloma which comprises administering to an individual a therapeutically effective amount of a composition comprising an antagonism of an interaction between an integrin with an alpha 4 subunit (eg, VLA-4). and a ligand for this integrin (e.g., VCAM-1). This antagonist may be an alpha 4 integrin binding agent or an alpha 4 integrin ligand binding agent. Preferred agents are anti-VLA4 or anti-alpha4beta7 antibody homologs (human antibody, 'chimeric antibody, a humanized antibody and fragments thereof); homologs of the anti-VCAM-1 antibody (a human antibody, a chimeric antibody, a humanized antibody and fragments thereof); and a small inhibitor of molecules, of integrin interactions that contain the alpha 4 subunit with its ligands. The composition can be administered at a dose to provide from about 0.1 to about 20 milligrams / kilogram of body weight. In particular, 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 ligand 4. Another aspect of the invention is a method for inhibiting bone resorption associated with tumors of the bone marrow, the method comprising administering to a mammal with said tumors an antagonism of an interaction between the integrin containing the alpha 4 subunit such as VLA-4 and a ligand for this integrin containing the alpha 4 subunit, such as VCAM-1, in an amount effective to provide inhibition of bone resorption. This antagonist can be an alpha 4 integrin binding agent such as the VLA-4 binding agent or an alpha 4 integrin ligand binding agent such as a VCAM-1 binding agent. Preferred agents are the homologues of anti-VLA4 or anti-alpha 4 beta 7 antibodies (human antibody, chimeric antibody, a humanized antibody and fragments thereof); homologs of the anti-VCAM-1 antibody (a human antibody, a chimeric antibody, a humanized antibody and fragments thereof); and a small molecule inhibitor of the interaction of integrins containing the alpha 4 subunit with their respective alpha 4 integrin ligands (eg, the VCAM-I / VLA-4 interaction). The antagonist can be administered at a dose to provide from about 0.1 to about 20 milligrams / kilogram of body weight. Yet another aspect of the invention is a method for treating a subject having a disorder characterized by the presence of osteoclastogenesis, the method comprising administering to the subject an antagonist of the interaction between an integrin carrying alpha 4 subunit and a ligand for an integrin. which carries the alpha 4 subunit, in an amount sufficient to suppress osteoclastogenesis. Similarly, the antagonist can be an alpha 4 binding agent or an alpha 4 ligand binding agent. Preferred agents are anti-VLA4 antibody or antialpha 4 beta 7 (human antibody, chimeric antibody, humanized antibody and fragments thereof); homologues of anti-VCAM-1 antibodies (a human antibody, a chimeric antibody, a humanized antibody and fragments thereof); and a small molecule inhibitor of the interaction of integrins containing the alpha 4 subunit with its respective alpha 4 integrin ligands (eg, the VCAM-I / VLA-4 interaction). The composition can be administered at a dose to provide from about 0.1 to about 20 milligrams / kilogram of body weight. Unless otherwise stipulated, all references are incorporated herein by reference. BRIEF DESCRIPTION OF THE FIGURES Figure 1. Effect of neutralizing antibodies on the formation of TRAP-positive multinucleated OC-like cells in the co-cultures of 5TGM1 cells and bone marrow cells. A mixture of 5TGM1 cells (1 and 3) and marrow cells (1 and 6) in suspension was plated in 48-well culture plates and cultured with or without 10 ug / ml of anti-VCAM-1 antibody (VCAM). l Ab), anti-alpha 4 beta 1 (a4ßllAb) antibody, anti-ICAM-1 antibody (ICAM-1 Ab) or rat IgG as a control. After 6 days of culture, the cultures were fixed and the number of TRAP-positive multinucleated OC-like cells (TRAPÍ +) MNC) was determined. Both the VCAM-l antibody and the alpha 4 beta 1 antibody inhibited the formation of TRAP (+) MNC, while the ICAM-1 antibody had no effect. The data were expressed as the mean ± S.E. (n = 3). * = Significantly different from the IgG control. Figure 2. Effect of 5TGM1 and ST2 of conditioned medium on bone resorption in cultures of long-bone organs from fetal rat. The conditioned media (48 hours) obtained from ST2 alone, is 5TGM1 alone, and cocultures of ST2 and T5GM1 were tested to determine bone resorption activity in cultures of long bone organs from fetal rat labeled with calcium. Fetal rat tags were cultured in the presence of conditioned media (40 percent volume / volume) or control medium for 120 hours.The data were expressed as an increase in the percentage of calcium release over that of the control medium. release of conditioned medium from stromal cells ST2 is shown as the open bar.The release of 5TGM1 is the grated bar.The release of the conditioned medium harvested from the co-culture of 5TGM1 and ST2 is the closed bar.The data are expressed as the mean ± SE (n = 4). * = Significantly different from ST2 alone. * + + = significantly different from 5TGM1 alone Figure 3 Effect of recombinant soluble VCAM-1 (sVCAM-1) on the production of osteoclastogenic activity by 5TGM1 cells. The conditioned medium was harvested from 5TGM1 cells cultured in the presence or absence of sVCAM-1 (1 x 10"8 to 1 x 10" molar) for 24 hours. The osteoclastogenic activity of these conditioned media was tested in mouse marrow cultures. Bone marrow cells (le6 / well) were plated in 48-well plates, and cultured in the presence of conditioned media (grated bars) or control medium (IMDM) containing the same concentrations of sVCAM-1 (open bars) . After 6 days, the cultures were fixed and the number of TRAP-positive multmucleated OC-like cells was determined.

(TRAP + MNC). The conditioned medium from 5TGM1 cells treated with 1 x 10"" M xVCAM-1 increased the formation of TRAP (+) MNC. The data were expressed as the mean ± S.E. (n = 3). * = Significantly different from the controls.

Figure 4 Effect of monoclonal antibody PS2 to VLA-4 on elevation of serum IgG2b in mice having 5TGM1 Mice were injected with le5 5TGM1 cells, which were allowed to colonize the bone marrow. The mice were divided into two groups of three, one serving as the control group, and the second treated on days 8, 11, 14 17, and 20 with 80 ug of PS / 2 antibody (approximately 4 milligrams / kilogram). The levels of IgG2b, the isotype of antibody produced by 5TGM1 myeloma cells, were measured weekly, from weeks 1 to 6. The treatment of the monoclonal antibody inhibited the production of IgG2b greatly, an indicator of the inhibition of survival of the cell. myeloma and live culture. Figure 5 Effect of monoclonal antibody M / K-2.7 to VCAM-1 on elevation of serum IgG2b in mice having T5GM1 Mice were injected with 5TGM1 cells as described in Figure 4, which were allowed to colonize the bone marrow . The mice were divided into groups of four or five, one group serving as a control group (white squares), the second / third group treated prophylactically at 80 ug (white diamonds) and 160 ug monoclonal antibody (white circles) (approximately 4 to 8 milligrams / kilogram), the fourth group was treated therapeutically at 160 ug of monoclonal antibody (triangles) The levels of IgG2b, the isotype of antibody produced by the myeloma cell 5TGM1, were measured. The treatment of the monoclonal antibody greatly inhibited the production of IgG2b, an indicator of the inhibition of the survival and growth of myeloma cells in vivo. Figure 6 Effect of anti-alpha 4 integrin antibody on the survival of mice having multiple myeloma. DETAILED DESCRIPTION OF THE INVENTION The invention relates to treatments for, among other things, preventing multiple myeloma. More particularly, the methods of the invention relate to the use of antagonists of an interaction between a mtegrin containing an alpha 4 subunit and a ligand for this method in the treatment of multiple myeloma. The term "multiple myeloma" is intended to mean a medical condition in an individual who has a neoplastic disease of plasma cells, the neoplastic clone representing cells at different stages in the plasma cell lineage of patient to patient (Mundy, 1998). Integrin alpha 4 beta 1 is a cell surface receptor for VCAM-1, fibronectin and possibly other molecules that bind to, or otherwise interact with, alpha 4 beta 1 mtegrin. With respect to this, these molecules are link to, or otherwise interact with, the alpha 4 subunit that contain mtegrma are known individually and collectively as the alpha 4 ligand (s). "The term mtegrma a4bl (" VLA-4"or" a4bl "or" a4bl " , are used interchangeably) herein refers to polypeptides that are capable of binding to VCAM-1 and members of the extracellular matrix proteins, more particularly fibronectm, or homologues or fragments thereof, although it will be appreciated by researchers with ordinary experience in the art that other ligands for VLA-4 may exist and can be analyzed using conventional methods.However, it is known that the alpha 4 subunit will associate with other beta subunits in addition to beta 1 d Thus, we can define the term "alpha 4 mtegrin" as that of integrins whose alpha 4 subunit is associated with one or other of the beta subunits. Another example of an "alpha 4" integrin is alpha 4 beta 7 (R. Lobb and M Hemler, 1994). As used herein, the term "integrin or alpha 4 integrins" means VLA-4, as well as integrins containing beta 1, beta 7 or any other beta subunit. As discussed herein, the antagonists used in the methods of the invention are not limited to a particular type or molecule structure such that, for purposes of the invention, any agent capable of binding to any mtegrin containing an alpha subunit 4 such as VLA-4 on the surface of cells carrying VLA-4 and / or alpha 4 beta 7 integrin on the surface of cells carrying alpha 4 beta 7 [see Lobb and Hemler, J. Clin. Invest., 94: 1722-1728 (1994)] and / or their respective alpha 4 ligands such as VCAM-1 and MadCAM, respectively, on the surface of VCAM-1 and cells carrying MadCAM, and which effectively block or cover VLA-4 (or alpha 4 beta 7) or VCAM-1 (or MadCAM) (ie, an "alpha 4 integrin binding agent" and an "alpha 4 integrin ligand binding agent" respectively), is considered which is equivalent to the antagonists used in the examples herein. An integrin "antagonist" includes any compound that inhibits an integrin or alpha 4 integrins binding to the ligand and / or alpha 4 integrin receptor. The anti-integrin antibody or the proteins containing antibody homologs (discussed below) as well as other molecules such as soluble forms of the ligand proteins for the integrins are useful. Soluble forms of the ligand proteins for alpha 4 integrins include soluble VCAM-1 or collagen peptides, VCAM-1 fusion proteins, or bifunctional fusion proteins VCAM-1 / Ig. For example, a soluble form of an alpha 4 mtegrin ligand or a fragment thereof can be administered to bind to a mtegrin, and preferably complete an integrin binding site in the cell, thereby leading to effects similar to 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 to alpha 4 integrin ligand but do not separate integrin-dependent signaling are included within the scope of the invention. These mutants can act as competitive inhibitors of the 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 that use an agent that antagonizes the action of more than one alpha 4 integrin, such as a single small molecule or an antibody homolog that antagonizes several alpha 4 integrins such as VLA-4 and alpha 4 beta 7 , or other combinations of alpha 4 -mictegrins. Also included within the scope of the invention are methods that use a combination of different molecules such as the combined activity that antagonizes the action of more than one alpha 4 integrin, such as methods using several molecules small or homologous antibodies that in combination antagonize the alpha 4 VLA-4 and alpha 4 beta 7 mtegrins, or another combination of mtegrins. As discussed herein, certain integrin antagonists can be fused or otherwise conjugated to, for example, 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 another molecule. Thus, for the 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 that effectively blocks or coats alpha 4 beta 7 and / or the VLA-4 mtegrin is considered to be an equivalent of the antagonist used in the examples herein. For the purposes of the invention an "integpna alpha 4 / integrin alpha 4 ligand interaction antagonist" refers to an agent, eg, a polypeptide or other molecule, which can inhibit or block the alpha 4 ligand (e.g., VCAM-1) and / or the alpha 4 integrin (eg, alpha 4 beta 7 or VLA-4) mediated binding or that can otherwise modulate the alpha 4 ligand and / or the alpha 4 integrin function, for example, by inhibiting or blocking transduction of alpha 4 integrin signal mediated by alpha 4 ligand or alpha 4 ligand signal transduction mediated by alpha 4 ligand and which is effective in the treatment of multiple myeloma, preferably of the same way as antialfa integrin antibodies are. 4. Specifically, an antagonist of the VCAM-1 / VLA-4 interaction is an agent that has one or more of the following properties: (1) coat, or bind with, VLA-4 on the surface of a cell carrying VLA-4 (e.g., a myeloma cell) with sufficient specificity to inhibit a VLA-4 / VLA-4 ligand interaction, e.g., the VCAM-1 interaction / VLA-4 between bone stromal cells and myeloma cells; (2) coat, or bind to, VLA-4 on the surface of a cell carrying VLA-4 (ie, a myeloma cell) with sufficient specificity to modify, preferably to inhibit, the transduction of a signal mediated by VLA-4, signaling mediated by VLA-4 / VCAM-l; (3) coat, or bind to, a VLA-4 ligand (e.g., VCAM-1) on bone stromal cells with sufficient specificity to inhibit the VLA-4 / VCAM interaction; (4) coat, or bind to, a VLA-4 ligand (e.g., VCAM-1) in bone stromal cells with sufficient specificity to modify, and preferably to inhibit, the interaction of VLA-4 signaling mediated by Ligand VLA-4, for example, VCAM mediated by 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, for example, a agent that binds to VLA-4 can be combined with an agent that binds to VCAM-l. For example, antibodies or antibody homologues (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 the soluble VCAM-1 peptides, the VCAM-1 fusion proteins, the bifunctional fusion proteins VCAM-1 / Ig, fibronectm, fibronectin having a split connection segment. not of type III alternatively, and fibronectin peptides containing the amino acid sequence and EILDV or a conservatively substituted amino acid sequence. Suitable forms of the natural binding proteins for VCAM-1 include soluble VLA-4 peptides, VLA-4 fusion proteins, BIF-functional fusion proteins VLA-4 / Ig and the like. As used herein, a "soluble peptide VLA-4" or a "soluble VCAM-1 peptide" is a VLA-4 or VCAM-1 polypeptide incapable of anchoring to a membrane. These soluble polypeptides include, for example, VLA-4 and VCAM polypeptides that lack a sufficient portion of their membrane extension domain to extend the polypeptide or are modified such that the membrane expansion domain is not functional. These binding agents can act by competing with cell surface binding proteins for VLA-4 or otherwise altering the function of VLA-4. For example, a soluble form of VCAM-1 (see, for example, Osborn et al., 1989, Cell, 59: 1203-1211) or a fragment thereof can be administered to bind to VLA-4, and preferably compete for the VLA-4 binding site in the myeloma cells, thereby leg to effects similar to the administration of an antagonist such as small molecules or anti-VLA-4 antibodies. In another example, VCAM-1, or a fragment thereof that is capable of binding to VLA-4 on the surface of the myeloma cells carrying VLA-4, for example, a fragment containing the two N-thermal domains of VCAM-1, can be fused with a second peptide, for example, a peptide that increases the solubility in the lifetime of the VCAM-1 fraction in vivo. The second peptide may be a fragment of a soluble peptide, preferably a human peptide, more preferably a plasma protein, or a member of a immunoglobulin superfamily. In particularly preferred embodiments the second peptide is IgG or a portion or fragment thereof, eg, the heavy chain constant region of human IgG1 and includes, at least the joint, the 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 integrin alpha 4 / l interaction by alpha 4 integrin by , for example, blocking VLA-4 by binding the VLA-4 receptors on the cell surface or blocking VCAM-1 by binding the VCAM-1 receptors on the surface of the cells. These "small molecules" by themselves can be small peptides, or organic compounds that contain larger peptides or non-peptidic organic compounds. A "small molecule", as defined herein, is not intended to encompass an antibody or an antibody homologue. Although the molecular weight of these "small molecules" is generally less than 2,000, we do not intend to apply this figure as an absolute upper limit on molecular weight. For example, small molecules such as oligosaccharides that mimic the binding domain of a VLA-4 ligand and fit into the VLA-4 receptor domain can be employed. (See, JJ Devlin et al., 1990, Science 249: 400-406 (1990), JK Scott and GP Sith, 1990, Science 249: 386-390, and U.S. Patent 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 into the VCAM-1 receptor domain can be used 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 Alternative Spliced Type III Connecting Segment Domam of Fibronectm Is Leucine-Aspartic Acid-Valme", J. Biol. Chem ., 266 (23), pp. 15075-79 (1991)). They identify the minimum active amino acid sequence necessary to bind VLA-4 and synthesize 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 identify an 8 amino acid peptide, Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr, as well as two smaller overlapping pentapeptides, Glu-Ile-Leu-Asp-Val and Leu-Asp-Val-Pro-Ser , which have inhibitory activity against cell adhesion dependent on fibronectomy. Certain major peptides containing the LDV sequence were subsequently shown to be active in vivo (TA Ferguson et al., "Two Integrm Bmdmg Peptides Open T-cell-Mediated Immune Responses In Vivo", Proc. Nati. Acad. Sci, USA, 88 , pp. 8072-76 (1991); and SM 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 (where TPro denotes 4-t? Oprol? Na), which can inhibit both the adhesion of VLA-4 as VLA-5 to fibronectin has also been described (see, for example, D.M., Nowlin et al., "A Novel Cyclic Pentapeptide Inhibits Alpha4Betal Integrin-mediated Cell Adhesion ", J. Biol. Chem., 26b (27), pp. 20352-59 (1993), and PCT publications PCT / US91 / 04862) This pentapeptide is based on the tripeptide sequence Arg-Gly-Asp of fibronectin that has been known as a common motif at the recognition site for several extracellular matrix proteins Examples of other small molecule VLA-4 inhibitors have been reported, for example, in Ada and collaborators, "Cell Adhesion Inhibitors", PCT US97 / 13013, which describes linear peptidyl compounds containing beta amino acids having 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 disclose cell adhesion inhibitory compounds containing guanidyl, urea and thiourea The United States Patent of North America No. 5,260,277 describes guanidmyl cell adhesion modulation compounds. These small molecule mimics can be produced by synthesizing a plurality of semi-peptidic or non-peptidic peptides, organic compounds, and then selecting the compounds for their ability to inhibit the interaction of the ligand mtegrma alfa 4 /? Ntegr? Na aiíc 4. See generally U.S. Patent No. 4,833,092, Scott and Smith, "Searchmg for Peptide Ligands with an Epitope Library," Science, 249, p. 386-90 (1990), and Devlm et al., "Random Peptide Librarles: A Source of Specific Protein Bmding Molecules", Science, 249, pp. 40407 (1990). In other preferred embodiments, the agent that is used in the method of the invention to bind, includes blocking or coating, the alpha 4 integrin on the cell surface and / or the alpha 4 mtegrin ligand is an anti-monoclonal antibody. VLA-4 and / or anti-alpha 4 beta 7 or an antibody homolog. Preferred antibodies and homologs for treatment, in particular for human treatment, include homologues of human antibodies, homologues of humanized antibodies, homologs of chimeric antibodies, Fab, Fab ', F (ab') 2 and f (v) fragments of antibodies, and monomers or dimers of heavy or light chains of antibodies or mixtures thereof. Monoclonal antibodies against VLA-4 are a preferred binding agent in a method of the invention. As used herein, the term "antibody homologue" includes intact antibodies consisting of light and heavy chains of immunoglobulin linked via bisulfide linkages. The term "antibody homologue" 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 the antibody homologue composed of more than one polypeptide can optionally be a bisulfide or other covalently crosslinked linkage. Accordingly, therefore, "antibody homologs" include intact immunoglobulins types IgA, IgG, IgE, IgD, IgM (as well as subtypes thereof), wherein the immunoglobulin light chains can be of layer types. or lambda. "Antibody homologs" also include intact antibody proteins that retain antigen binding specificity, eg, Fab fragments, Fab 'fragments, F (ab') 2 fragments, F (v) fragments, monomers or heavy chain dimers. , light chain monomers or dimers, dimers consisting of a heavy and a light chain, and the like. Thus the antigen binding fragments, as well as the full-length dimeric or trimeric polypeptides derived from the aforementioned antibodies are also useful. As used herein, a "humanized antibody homologue" is an antibody homologue, produced by recombinant DNA technology, in which some or all of the amino acids of a human immunoglobulin light or heavy chain that is not required for Antigen binding has been replaced by corresponding amino acids from a light or heavy chain of non-human mammalian immunoglobulin. As used herein, a "chimeric antibody homolog" is an antibody homologue, produced by recombinant DNA technology, in which all or part of the joint and the constant regions of the heavy chain immunoglobulin light chain , or both, have been replaced by the corresponding regions of another immunoglobulin light chain or heavy chain. In another aspect the invention features a variant of a chimeric molecule that includes: (1) a fraction directed to VLA-4, for example, a VCAM-1 moiety capable of binding to an antigen (ie, VLA-4) in the surface of myeloma cells carrying VLA-4; (2) optionally, a second peptide, for example, one that increases the solubility or lifetime of the fraction directed to VLA-4, for example, a member of the immunoglobulin superfamily or fragment or portion thereof , for example, a portion or fragment of IgG, e.g., the heavy chain constant region of human IgGl, eg, CH2 and CH3 of joint regions; and a toxin fraction. The fraction directed to VLA-4 can occur naturally in a lightly or VLA-4 fragment thereof, for example, a VCAM-1 peptide or a conservatively similar substituted amino acid sequence. A preferred directed fraction is a fragment of soluble VCAM-1, for example, the N 1 and 2 terminal domains of the VCAM-1 molecule. The chimeric molecule can be used to treat a subject, for example, a human, at the risk of a disease, for example, multiple myeloma, characterized by the presence of myeloma cells carrying VLA-4, and preferably activated VLA-4. . As used herein, a "human antibody homolog" is an antibody homologue produced by recombinant DNA technology, in which all amino acids of a light or heavy immunoglobulin chain are derived from a human source. Methods of making homologous antibodies Ant ± -VLA-4 The technology for producing monoclonal antibody homologs is well known. Briefly, an immortal cell line (typical myeloma cells) is fused to lymphocytes (typical splenocytes) of a mammal immunized with whole cells expressing a given antigen, for example, VLA-4, and the culture supernatants of the cells of The resulting hybridomas are selected to determine antibodies against the antigen. See, generally, Kohler et al., 1975, Nature, 265: 295-297. The immunization can be carried out using standard procedures. The unit dose and the immunization regimen depend on the immunized mammal species, its immune status, the mammalian body weight, and so on. Typically, immunized mammals are bled and the serum from each blood sample is studied for particular antibodies using appropriate screening assays. For example, anti-VLA-4 antibodies can be identified by immunoprecipitation of lysates of cells labeled with:: f'I from cells expressing VLA-4.

(See, Sánchez-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 can also be identified by flow cytometry, for example, by measuring the fluorescent staining of the Ramos cells incubated with an antibody that is believed to recognize VLA-4 (see, Elices et al., 1990 Cell, 60: 577-584). Lymphocytes used in the production of hybridoma cells are typically isolated from immunized mammals whose sera have been tested as positive for the presence of anti-VLA-4 antibodies using these 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 the culture medium containing hypoxanthine, armnopterin and thymidine ("HAT medium"). Typically, mouse myeloma cells sensitive to the HAT medium are fused with mouse splenocytes using polyethylene glycol of molecular weight 1500 ("PEG 1500"). The hybridoma cells resulting from the fusion are selected using HAT medium, which kills unfused or unproductively fused myeloma cells (the unfused splenocytes die after several days because they do not transform). Hybridomas that produce a desired antibody are detected by selecting hybridoma culture supernatants. For example, hybridomas prepared to produce anti-VLA-4 antibodies can be selected by testing the hybridoma culture supernatant to determine secreted antibodies that have the ability to bind to a cell line expressing the recombinant alpha 4 subunit (see, Elices and collaborators, supra). To produce anti-VLA-4 antibody homologues that are intact immunoglobulins, the hybridoma cells that prove to be positive in these 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 in the culture medium. The tissue culture techniques and the convenient culture medium for the hybridoma cells are well known. The conditioned hybridoma culture supernatant can be harvested and the anti-VLA-4 antibodies optionally further purified by well-known methods. Alternatively, the desired antibody can be produced by injecting the hybridoma cells into the peritoneal cavity of an immunized mouse. Hybridoma cells proliferate in the peritoneal cavity, secreting the antibody that accumulates as fluid ascites. The antibody can be harvested by removing the ascites fluid from the peritoneal cavity with a syringe. Several anti-VLA-4 monoclonal antibodies were previously described. See, for example, Sánchez-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 HA 1/2 and other anti-VLA-4 antibodies (for example, HP2 / 1, HP2 / 4, L25, P4C2, P4G9) capable of recognizing the P chain of VLA-4 they will be useful in the methods of treatment according to the present invention. Anti-VLA-4 antibodies that will recognize the alpha 4 chain epitopes of VLA-4 mixed in to bind to VCAM-1 and fibronectin ligands (ie, antibodies that can bind to VLA-4 at a site mixed in the recognition of ligand and the en bloc linkage of VCAM-1 and fibronectin) are preferred. These antibodies have been defined as antibodies specific for epitope B (Bl or B2) (Pulido et al., 1991, supra) and are also anti-VLA-4 antibodies according to the present invention. Homologs of fully human monoclonal antibodies against VLA-4 are another preferred binding agent that can block or coat the VLA-4 antigens in the method of the invention. In their intact form these can be prepared using human splenocytes primed in vi tro, as described by Boerner et al., 1991, J. Immunol., 147, 86-95. Alternatively, they can 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. Patent No. 5,798,230 (August 25, 1998, "Process for the preparation of human monoclonal antibodies and their use") which describes the preparation of human monoclonal antibodies from B cells human According to this process, B cells that produce human antibodies are immortalized by infection with an Epstein-Barr virus, or a derivative thereof, that expresses nuclear antigen of Epstein-Barr virus 2 (EBNA2). The EBNA2 function, which is required for immortalization, is subsequently closed, which results in an increase in antibody production. In yet another embodiment for producing complete human antibodies, U.S. Patent No. 5,789,650 (August 4, 1998, "Transgenic non-human animáis for producing heterologous antibodies") describes transgenic non-human animals capable of producing heterologous antibodies and transgenic non-human animals that have inactivated endogenous immunoglobulin genes. The endogenous immunoglobulin genes are suppressed by antisense polynucleotides and / or antiserum directed against endogenous immunoglobulins. Heterologous antibodies are encoded by immunoglobulin genes not normally found in the genome of those non-human animal species. One or more transgenes containing non-human heterologous human immunoglobulin heavy chain sequences are introduced into a non-human animal whereby a transgenic animal capable of functionally rearranging transgenic immunoglobulin sequences and producing a repertoire of antibodies of various isotypes encoded by human immunoglobulin genes. These heterologous human antibodies are produced in B cells that are then immortalized, for example, by fusing with an immortalizing cell line such as a myeloma or by manipulating these B cells by other techniques to perpetuate a cell line capable of producing a human antibody homologue. completely, monoclonal heterologous. Large unhumanized human phage display libraries can 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 that can block or coat VLA-4 antigens in the method of the invention is a humanized recombinant antibody homolog having an anti-VLA-4 specificity. Following the above methods for the preparation of chimeric antibodies, a new approach was described in the European publication EP 0239400 (Winter contributors) by which the antibodies are altered by substitution by their complementary determining regions (CDR) for one species with those of another . This process can be used, for example, to replace regions of complementarity determination from human and heavy chain heavy and light chain immunoglobulm variable domains with regions of complementarity determination of domains of variable regions urines. These subsequently altered immunoglobulin variable regions can be combined with constant regions of human immunoglobulin to create antibodies that are fully human in composition except for the substituted murine complementarity determining regions. These antibodies substituted by regions of complementarity determination are predicted to be less likely to elicit an immune response in humans compared to chimeric antibodies because antibodies substituted by regions of complementarity determination contain considerably fewer non-human components. The process for humanizing monoclonal antibodies via the "grafting" of regions of determination of complementarity has been determined "reformed". (Riechmann et al., 1988, Nature 332, 323-327; Verhoeyen et al., 1988, Science 239, 1534-1536). Typically, the regions of complementarity determination (CER) of a murine antibody are transplanted over the corresponding regions in a human antibody, since it is the region of determination of complementarity (three in the heavy chains of antibodies, three in the light chains) which are the regions of the mouse antibody that bind to a specific antigen. The transplantation of regions of complementarity determination is achieved by genetic engineering whereby the DNA sequence of the complementarity determining regions are determined by cloning the murine heavy and light chain variable region (V) gene segments, and then transferred to the corresponding human V regions by site-directed mutagenesis. In the final stage of the process, segments of the human constant region gene of the desired isotype (usually gamma I for CH and kappa for CL) are added and the humanized heavy and light chain genes are coexpressed in mammalian cells to produce humanized antibody soluble. The transfer of this region of determination of complementarity to a human antibody confers on this antibody the antigen binding properties of the original murine antibody. The six regions of determination of complementarity in the murine antibody are structurally assembled on a region of "structure" of region V. The reason that the graft of the region of determination of complementarity is successful is that the regions of structure between the antibodies of mouse and humans have very similar three-dimensional structures with similar binding points for regions of complementarity determination, so that regions of complementarity determination can be exchanged. These humanized antibody homologs can 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. However, certain amino acids within the framework regions are thought to interact with regions of complementarity determination and to influence the overall antigen binding affinity. Direct transfer of regions of complementarity determination of a murine antibody to produce a recombinant humanized antibody without any modification of human V region structures frequently results in a partial or complete loss of binding affinity. In several cases, it seems critical to alter residues in the structure 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 containing modified residues in the structure regions of the acceptor antibody by combining the regions of complementarity determination of a murine monoclonal antibody. (anti-Tac) with the structure regions and human immunoglobulin constants. They have demonstrated a solution to the problem of loss of binding affinity which frequently results from the transfer of region of determination of direct complementarity without any modification of the residues of the human V region structure; Your solution involves two key steps. First, the human framework V regions are chosen by computer analysts to determine the optimal protein sequence homology with the structure of the V region of the original murine antibody, in this case, the anti-Tac monoclonal antibody. In the second step, the tertiary structure of the murine V region is modeled by computer in order to visualize the amino acid residues of the structure that are more likely to interact with the murine complementarity determining regions and these murine amino acid residues are they superimpose on the homologous human structure.

See also Protein Design Labs - Patent of the United States of North America number 5,693,762. A different approach can be used (Tempest et al., 1991, Biotechnology 9, 266-271) and use, as a standard, the structure of the V region derived from the heavy and light chains of NEWM and REI respectively for the graft with the region of determination of complementarity without the radical introduction of mouse residues. An advantage of using the approach of Tempest et al., To construct humanized antibodies based on NEWM and REI is that the 3-dimensional structures of the NEWM and REI variable regions are known from x-ray crystallography and thus can be modeled the specific interactions between regions of complementarity determination and residues of the structure of region V. Regardless of the approach taken, examples of the initial humanized antibody homologs prepared to date have shown that this is not a direct process. However, while recognizing that these changes in structure may be necessary, it is not possible to predict, based on the prior art available, which, if any, structure residues will need to be altered to obtain functional humanized recombinant antibodies with the desired specificity. The results indicate that Jt > The changes necessary to preserve specificity and / or affinity are for the most part exclusive to a given antibody and can not be predicted based on the humanization of a different antibody. Preferred antagonists useful in the present invention include homologues of recombinant humanized and chimeric recombinant antibodies (ie, intact immunoglobulins and portions thereof) with epitope B specificity that have been prepared and described in the US Pat. North America pending joint with serial number 08 / 004,798, filed on January 12, 1993, PCT publication US94 / 00266, filed on January 7, 1994. The initial material for the preparation of chimeric antibody homologs (mouse V human C ) and humanized anti-VLA-4 can be a murine anti-VLA-4 monoclonal antibody as previously described, a commercially available anti-VLA-4 monoclonal antibody (eg, HP2 / 1, Amae International, Inc., Westbrook, Maine), or an anti-VLA-4 monoclonal antibody prepared in accordance with the teachings herein. For example, the variable regions of the heavy and light chains of anti-VLA-4 HP 1/2 antibody have been cloned, sequenced and expressed in combination with constant regions of heavy and light chains of immunoglobulins. This HP 1/2 antibody is similar in specificity and potency to the HP 1/2 mupno antibody, and may be useful in methods of treatment in accordance with the present invention. Other homologues of preferred humanized anti-VLA-4 antibodies are described in Athena Neurosciences, Ine, in PCT / US95 / 01219 (July 27, 1995). These humanized anti-VLA-4 antibodies comprise a humanized light chain and a humanized heavy chain. The humanized light chain comprises three regions of complementarity determination (CDR1, CDR2 and CDR3) having amino acid sequence from the corresponding complementary determination regions of a mouse immunoglobulin 21-6 light chain and a variable region structure of a light chain variable region sequence, human kappa except at least the position of the amino acid is occupied by the same amino acid present in the equivalent position of the light chain variable region of mouse immunoglobulin 21.6. The humanized heavy chain comprises three regions of complementarity determination (CDR1, CDR2 and CDR3) having amino acid sequence from the corresponding complementarity determining regions of a mouse immunoglobulin 21-6 heavy chain., and a variable region structure from a human heavy chain variable region structure sequence except that at least one position the amino acid position is occupied by the same amino acid present in the equivalent position of the variable region structure of immunoglobulin 21-6 heavy chain mouse. Therapeutic Applications In this method according to the first aspect of the invention, the VLA-4 binding agents, in particular, the VCAM fusions and the anti-VLA-4 antibody homologs are preferably administered parenterally. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intra-articular, intrasynovial, 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 can be any of numerous well-known vehicles, such as water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, or combinations thereof. The compounds of the present invention can be used in the form of pharmaceutically acceptable salts derived from organic acids and organic or inorganic bases. Included among these acid salts are the following: acetate, adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate, butyrate, citrate, camphorrate, camphorsulfonate, cyclopentanepropionate, digluconate, dodecyl sulfate, ethanesulfonate, fumarate, glucoheptanoate, glycerophosphate, hemisulfate, heptanoate, hexanoate , hydrochloride, hydrobromide, hydroiodide, 2-hydroxyethane sulfonate, lactate, maleate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate, pamoate, pectinate, persulfate, 3-phenylpropionate, picrate, pivalate, propionate, succinate, tartrate, thiocyanate, tosylate and undecanoate. Basic 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 on. Also, basic hydrogen-containing groups can be quaternized with agents such as lower alkyl halides, such as methyl, ethyl, propyl, and butyl chloride, bromides and iodides.; dialkyl sulfate, such as dimethyl sulfate, diethyl, dibutyl and diamyl, long chain halides such as decyl, lauryl, myristyl and stearyl chlorides, bromides and iodides, aralkyl halides, such as benzyl and phenethyl bromides and others. The soluble or dispersible products in oil or water are obtained in this way. 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 "vehicle" as used herein includes adjuvants and acceptable vehicles. Pharmaceutically acceptable carriers that can be used in the pharmaceutical compositions of this invention include, but are not limited to, ion exchangers, aluminum oxide, aluminum stearate, lecithin, whey proteins, such as human serum albumin, regulatory substances such as phosphates, glycine, sorbic acid, potassium sorbate, mixtures of partial glyceride of saturated vegetable fatty acids, water, salts or electrolytes, such as pro anna sulfate, disodium acid phosphate, potassium acid phosphate, sodium chloride, zinc salts, colloidal silicon oxide, magnesium trisilicate, polyvinyl pyrrolidone, cellulose based substances, polyethylene glycol, sodium carboxymethyl cellulose, polyacrylates, waxes, polyethylene-polyoxypropylene block polymers, polyethylene glycol and wool fat. In accordance with 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 can be formulated according to techniques known in the art using suitable dispersing or wetting agents and suspending agents. The sterile injectable preparation can 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-butanedione. Among the vehicles and acceptable 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 suspension medium. For this purpose, any soft fixed oil can be employed including synthetic mono- or diglycerides. Fatty acids, such as oleic acid and its glycemic derivatives are useful in the preparation of injectables, such as 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, tolls such as Ph.Helv or a similar alcohol. The pharmaceutical compositions of this invention, in particular the small molecule antagonists of the VLA-4 / VCAM-1 interaction, can 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, suspensions or aqueous solutions. In the case of tablets for oral use, vehicles that are commonly used include lactose and corn starch. Lubricating agents, such as magnesium stearate, are also typically added. For oral administration in capsule form, useful diluents include lactose and dry corn starch. When aqueous suspensions are required for oral use, the active ingredient is combined with emulsifying and suspending agents. If desired, certain sweeteners, sabotagers or coloring agents can also be added. Topically transdermal patches can also be used. The pharmaceutical compositions of this invention can also be used by aerosol or nasal inhalation through the use of a nebulizer, a dry powder inhaler or a metered dose inhaler. These compositions are prepared according to techniques well known in the field of pharmaceutical formulations and can be prepared as solutions in saline, employing benzyl alcohol or other suitable preservatives, absorption promoters to improve bioavailability, fluorocarbons, and / or other conventional solubilizing or dispersing agents. According to another embodiment, compositions containing a compound of this invention can also comprise an additional agent selected from the group consisting of corticosteroids, anti-inflammatories, immunosuppressants, antimetabolites, and immunomodulators. The specific compounds within each of these classes can be selected from any of those listed under the headings of appropriate groups in "Comprehensive Medicinal Chemistry," Pergamon Press, Oxford, England, pp. 425-648. 970-986 (1990), the disclosure of which is incorporated herein by reference. Also included in this group are compounds such as theophylline, sulfasalazma and ammonalicylates (anti-mflamatory); ciclosporin, FK-506, and rapamicma (immunosuppressants); cyclophosphamide and methotrexate (antimetabolites); steroids (inhaled, oral or topical) and interferons (immunomodulators). The amount of active ingredient that can be combined with the carrier materials to produce a single dose form will vary depending on the host treated, in particular mode of administration. It should be understood, however, that a specific dose and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific compound employed, age, body weight, general health, sex, diet, time of administration , speed of excretion, combination of drugs, and the judgment of the attending physician and the severity of the particular case that is being treated. The amount of active ingredient may also depend on the therapeutic or prophylactic agent, if any, with which the ingredient is coadministered. The dose and rate of dosage 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 the pathology to be treated, the specific pharmaceutical composition used, and the judgment of the attending physician. Dose levels between about 0.001 and about 100 milligrams / kilogram of body weight per day, preferably between about 0.1 and about 50 milligrams / kilogram of body weight per day of the active ingredient compound are useful. More preferably, the VLA-4 binding agent, if an antibody or antibody derivative, will be administered at a dose ranging from about 0.1 milligram / kilogram of body weight / day to about 20 milligrams / kilogram body weight / day, preferably varying between approximately 0.1 milligram / kilogram of body weight / day and approximately iu milligrams / kilogram of body weight / day and at intervals of every 1-14 days. For agents that are neither antibodies nor small molecule binding, the dose range should preferably be between equivalent molar amounts of these amounts of antibodies. Preferably, an antibody composition is administered in an amount effective to provide an antibody plasma level of less than 1 milligram / milliliter. The optimization of the doses can be determined by administration of the binding agents, followed by titration of the coating of VLA-4 positive cells by the agent during the time after administration at a given dose in vivo. Myeloma cells contained in a sample of the individual's peripheral blood (or bone marrow cells) should be tested for the presence of the agent in vitro (or ex vivo) using a second reagent to detect the agent administered. For example, this may be an antibody labeled with specific fluorochrome for the administered agent which is measured by standard FACS analysis (fluorescence activated cell sorter). Alternatively, the presence of the administered agent can be detected in vitro (or ex vivo) by the inability or decreased ability of the individual's cell to bind to the same agent that has labeled itself (eg, by a fluorochrome). The preferred dose should produce detectable coating of the vast majority of cells positive for VLA-4. Preferably, the retrieval is sustained in the case of an antibody homologue for a period of 1 to 14 days. Animal models: The animal model has been described in detail (Garrett 1997). Briefly, Radl et al. (1988) have described a murmo model of myeloma that arises spontaneously in old C57BL / KaLwR? J mice. This condition occurred in approximately 1 in 200 animals as they got older, and led to a monoclonal gamopathy with some of the characteristics of the human disease (Radl 1988). To develop a better and more reproducible animal moleel we have established and subcloned a cell line of this murine myeloma called 5TGM1, and found that it causes lesions in the characteristics of human myeloma mice, such as severe osteolysis and the participation of organs that are not Bones such as liver and kidney (Garrett 1997). Mice stained with cultured cells developed disease in a very predictable and reproducible manner, which includes the formation of a monoclonal gamopathy and radiological bone lesions. In addition, some of the mice become hypercalcemic, and bone lesions are characterized by increased osteoclastic activity. Thus, based on the histological examination of affected organs in mice that have 5TGM1 and elevated serum levels of IgG2b, 5TGM1 is defined as a mupno myeloma that acutely recapitulates the marks of the human disease. The following examples are intended to further illustrate certain preferred embodiments of the invention and are not intended to be limiting in nature. In the following examples, the restriction enzymes, plasmids and other reagents and necessary materials can be obtained from commercial sources and the methodology of cloning, ligation and other recombinant DNA methodology can be performed by methods well known in the art. Example 1: MATERIALS AND METHODS 5TGM1 Myeloma Cells 5TGM1 myeloma cells micially derived from a myeloma that arose spontaneously in aged C57BL / KaLwR? J mice (Garrett 1997, Vanderkerken 1997). Cells were cultured in Isocove Modified Dulbecco's Medium (IMDM, Life Technologies Inc., Gaithersburg, MD) supplemented with 10 percent fetal bovine serum (FBS, Summit, Fort Collins, CO) and 1 percent penicillin solution. -streptomycin (GIBCO, Grand Island, NY) at 37 ° C in 5 percent C02 in atmosphere. For the vitro experimentation described below, 5TGM1 cells were used between passage 25 and 3t > . Antibodies, soluble VCAM-l Neutralizing antibodies against murine VCAM-l (M / k-2.7), integrin VLA-4 (PS / 2), and the? -tracellular adhesion molecule-1 (ICAM-1, YN1 / 1.7 ), were kindly donated by Dr. Kensuke Miyake (Saga Medical University, Saga, Japan). VCAM-1 soluble recombinant (Lobb et al., 1991), which contains the extracellular domains 7 of human VCAM-1, was donated by Dr. Roy Lobb, Biogen Inc., Cambridge, MA. Reverse Transcription Polymerase Chain Reaction (RT-PCR) Using reverse transcription polymerase chain reaction, we confirmed the expression of VCAM-1 and of alpha 4 mtegrin in stromal cells of bone marrow and 5TGM1, respectively. Total RNA was prepared from 5TGM1, a primary culture of bone marrow stromal cells and a ST2 cell stromal cell line (RIKEN Cell Bank, Tsukuba, Japan) by a one-step RNA isolation method using TRI ol reagent ( GIBCO). Three ug of RNA were incubated with 50 ng of randomized eximer at 70 ° C during minutes and chilled on ice, then they became the first cDNA strand using reverse transcriptase (Perkin-Elmer, Branchburg, NJ) according to the manufacturer's instruction. The primers used for the polymerase chain reaction were the following: VCAM-1 murine 5'-primer; 5 '-OH-GCTGCGCGTCACCATTGTTCTC-3' -OH [SEQ ID NO: 1]; VCAM-1 murine 3 '-primer; 5'-OH-ACCACCCTCTTGAAGCCTTGTG-3'OH [SEQ ID NO: 2]; mtegrin alfa 4 mupna 5 '-primer; 5'-OH- CCCCTCAACACGAACAGATAGG-3 '-OH [SEQ ID NO: 3]; mtegpna alfa 4 raunna 3 '-primer; 5 '-OH-GCCTTGTCCTTAGCAACACTGC-3' -OH [SEQ ID NO: 4]. The polymerase chain reaction was carried out for 30 cycles consisting of one minute at 94 ° C, one minute at 55 ° C and 2 minutes at 72 ° C. The polymerase chain reaction mixture (total 50 ul) contained 10 microliters. The first strand of cDNA, 50 mM KCl, 10 mM Tris-HCl (pH 8.3), 2 mM MgCl, deoxy-NTP mixture (0.2 M each), the pair of primers (0.15 micromolar each) and 2 U of Taq DNA polymerase (Perkm-Elmer, Branchburg, NJ). The polymerase chain reaction products were separated in 2.5 percent agarose gels containing ethidium bromide and visualized under ultraviolet light. The size of the fragments was confirmed by reference to molecular weight markers. Binding of 5TGM1 cells on bone cell stromal cells For cell adhesion assays with heterotypic cells, ST2 cells (5 and 4 / well) were seeded in 48-well culture plates (Costar, Cambridge, MA) and cultured 48 hours in alfaMEM supplemented with 10 percent FBS until confluence. 5TGM1 cells (5 e 6) were labeled by incubation with 10 microCi [meth? L-3H] thymidine (New England Nuclear) for 24 hours at 37 ° C in the culture medium. After the ST2 monolayer was formed, it was incubated with 1 percent bovine serum albumin (BSA, Sigma, San Luis, MO) in serum-free alphaMEM for 1 hour and the tritium-labeled 5TGM1 cells were plated over the monolayer . The system was incubated in the absence or presence of antibodies for VCAM-1 or mtegrin alfa 4 beta 1 at 37 ° C for 1 hour. The non-adherent cells were removed by washing with 5 percent trichloroacetic acid twice and PBS twice, and the adherent cells were solubilized in 300 microliters of 0.25 mM NaOH, neutralized with the same volume of 0.25 mM HCl and the radioactivity was determined. in a liquid scintillation counter. Osteoclast Formation Assay in the Cocultivation of 5TGM1 and Mouse Bone Marrow Cells Mouse bone marrow cells were obtained from male mice at 5 weeks of age C57BL as previously described (Yoneda 1993). The femurs and tibias were aseptically dissected and the ends cut off. The bone marrow cells were removed, harvested and incubated in alphaMEM supplemented with 10 percent FBS (Hyclone, Logan, UT) and 1 percent penicillin-streptomycin in dishes of 100-millimeter culture (Becton Dickinson Labware, Bedford, MA) at 37 ° C for 2 hours. Non-adherent cells containing hemopoietic osteoclast precursors and stromal cells were harvested. The bone marrow cells (1 and 6) and the 5TGM1 cell (1 and 3) in 300 microliters of culture medium were plated in 48-well culture dishes (day 0 ^ .. On day 2, 300 microliters of medium new culture was added gently to each well, and on day 4, 300 microliters of the spent medium were replaced with the same volume of new medium.On day 6, the cultures were fixed and stained for tartarate acid-resistant phosphatase (TRAP) ) using commercial kits (Sigma) Multipallous cells positive for TRAP with more than 3 nuclei were defined as osteoclast-like cells (OC-like), and were counted manually under the microscope To confirm that these osteoclast-like cells have capacity of absorbing bone, 5TGM1 cells and bone marrow cells were cocultivated in slices of 5 x 5 millimeter whale dentin in the same condition, and novo resorption was formed in these slices of dentin were examined median electronic microscope as described (Yoneda 1992). In some experiments, cultures of 5TGM1 myeloma cells and spinal cells were performed using transpozo inserts (Becton Dickinson Labware) to avoid direct contact between these two cell types (2 and 6, 24-well plates, Costar). The medulla cells are plaquearon in lower chambers and myeloma cell 5TGM1 (2 e 3) were plated in the chambers either inferior (direct contact) or superior (without contact). Long-term organ cultures of fetal rat labeled with 45Ca Conditioned media grown from 5TGM1 cultures were tested for bone reabsorption activity by cultures of fetal rat long-bone organs labeled with "Ca as previously described (Mbalaviele 1995) Pregnant rats were injected with 250 μCi of "" Ca (New England Nuclear) on the 18th day of gestation.The bone ends of the radius and ulna were obtained from 19 day old fetuses by microdissection., and were pre-cultured for 24 hours in BGJ medium (Sigma) supplemented with 0.1 percent BSA between air and the liquid phase on grids with stainless steel mesh. The grids were cultivated in the presence of conditioned medium (50 volume / volume percent) or in control medium for 120 hours. The media was changed once at 48 hours. At the end of the culture, the bones were incubated in ice-cold 5 percent trichloroacetic acid for 2 hours, and the radioactivity of Ca in bones and the medium was determined in a liquid chromatography counter. The reaosorption of bones was quantified as the percentage of Ca released in the medium from the bones as calculated by: (counts in the middle of Ca) / (counts in the middle and bone of Ca) x 100. Coculture of myeloma cells 5TGM1 with mouse stromal cell line ST2 cells ST2 cells (0.5 and 6) and 5TGM1 (4 and 6) were plated together on plates of 60 millimeter culture (Beckton Dickinson) in 10 percent IMDM supplemented with FBS grown overnight, washed with serum free IMDM twice, and incubated in 5 milliliters of IMDM without serum. After 48 hours, the conditioned medium was harvested and stored at -70 ° C until use. Effect of mAb PS2 with VLA-4 on serum IgG2b elevation in mice that had 5TGM1 Mice were injected with 1 and 5 5TGM1 cells, which were allowed to colonize the bone marrow. The mice were divided into two groups of three, one serving as the control group, and the second treated every two weeks beginning on day 8 with 8C ug of mAb PS / 2 (4 milligrams / kilogram). The levels of IgGZr, the antibody isotype produced by the myeloma cell 5TGM1, were measured weekly from weeks 1 to 6. RESULTS Expression of VCAM-1, VLA-4, and effect of the antibodies against VCAM-1 and VLA -4 on the binding of 5TGM1 to the monolayers of ST2 Using RT-PCR, we confirmed the expression of VCAM-l and integrin VLA-4 in the bone marrow stromal cells and the mielomet cells, respectively. As expected, both the ST2 stromal cell line and the primary bone marrow stromal cells expressed VCAM-1, whereas 5TGM1 did not. In contrast, 5TGM1 myeloma cells expressed mtegrma VLA-4, whereas stromal cells did not (data not shown). In addition, both the anti-VCAM-1 antibody (10 ug / ililiter) and the VLA-4 antibody (10 ug / milliliter) partially (50-80 percent) inhibited the binding of 5TGM1 cells to the ST2 monolayers, showing that the VCAM-1 and the integrin VLA-4 expressed in these cells was biologically functional and that these antibodies have neutralizing activity (the data are not shown). OC-like cell formation in the co-culture of 5TGM1 myeloma cells with mouse bone marrow cells On day 6 of the co-culture of 5TGM1 cells and mouse marrow cells, numerous positive multinucleated osteoclast (OC-like) cells were formed for TRAP. These OC-like cells exhibited the formation of resorption pits in the dentin slices, demonstrating that these cells are capable of reabsorbing bone, and possessed an osteoclastic phenotype. In experiments using transpozo inserts, the formation of OC-like cells was observed when 5TGM1 cells were cultured in direct contact with bone marrow cells. In contrast, there was only a marginal number of OC-like cells formed when 5TGM1 cells were separated from the marrow cells by the transpozole membrane. Thus, 5TGM1 cells induce the formation of osteoclasts in mixed marrow cultures, and this induction requires direct cell-to-cell contact. Effect of antibodies against VCAM-1 and integrin VLA-4 on the formation of OC-like cells in the cocultivation of 5TGM1 cells and marrow cells Both the anti-VCAM-1 antibodies (VCAM-1 Ab, 10 ug / milliliter) as anti-VLA-4-mtegpna antibody (alpha 4 beta 1 Ab, 10 ug / milliliter) dramatically inhibited the formation of OC-like cells. In contrast, the monoclonal antibody against ICAM-i, another adhesion molecule in marrow stromal cells involved in stromal interactions / myeloma, had no effect on the formation of OC-like cells 1). To determine whether this inhibition by monoclonal antibodies VCAM-1 and VLA-4 mAbs was specific for the formation of OC-like cells induced by 5TGM1 and not due to cytotoxicity, the effects of these antibodies were examined in the formation of OC-like cells. induced by 1,25 (OH, D, a stimulant widely used for osteoclastogenesis in mouse bone marrow cell cultures (Takahashi 1988) Neither the antibody VCAM-1 Ab nor the monoclonal antibody VLA-4 mAb inhibited the formation of OC-like cells induced by vitamin D3, which in itself had no effect on the expression of VCAM-1 in stromal cells (data not shown) Effects of conditioned medium harvested from cocultivation of 5TGM1 and ST2 on bone resorption Conditioned medium from the coculture of 5TGM1 cells and ST2 cells showed a marked increase in bone reabsorption in the fetal mouse long bone test (Figure 2), while the conditioned medium of 5TGM1 only caused a marginal increase, compared to the control medium. The conditional medium of ST2 cells showed no increase in bone resorption. In this way direct cell-to-cell contact via VCAM-1 and VLA-4 induces both osteoclast-like cells and the production of bone resorption factors in vitro. Effect of recombinant soluble VCAM-1 (sVCAM-1) on the production of bone and osteoclastogenic reabsorption activity by 5TGM1 cells Conditioned medium of 5TGM1 treated with a soluble recombinant form of VCAM-1 (sVCAM-1) increased bone bone resorption Fetal rat lengths in a dose-dependent manner, while the conditioned medium obtained from non-treated 5TGM1 only marginally increased bone absorption. The soluble VCAM-l itself had no effect on bone resorption (data not shown). In the mouse marrow culture system, the conditioned medium harvested from 5TGM1 cells treated with sVCAM-1 showed increased activity of OC-like cellular formation, while the conditioned medium from non-treated 5TGM1 exhibited only marginal activity from the formation OC-like cellular (Figure 3). Expression of the ligand Rank mRNA in marrow stromal cells (ST2) cultured in the presence and absence of murine myelin cells Because Rank ligand is an important mediator of OCL formation and may be the final common pathway for the effects of Osteoclastogenic cytokines in the formation of OCL, we examined the expression of Rank ligand in 5TGM1 and ST2 cells both individually and when co-cultured. We found that the cocultivation of 5TGM1 and ST2 cells induces the mRNA of ae ligand Rank in ST2 cells. In addition, while 5TGM1 cells do not express the Rank ligand, they do so when treated with sVCAM-1 (not shown). Finally, the conditioned medium for 5TGM1 cells treated with sVCAM-1 induced the Rank ligand mRNA in ST2 cells, suggesting that the VCAM-1 / VLA-4 pathway produces a cytokine in myeloma cells that increase the expression of ligand Rank for marrow stromal cells (data not shown). In summary, we show that 5TGM1 cells alone produce marginal amount of activity that stimulates OC-like cellular formation and bone resorption. However, when the 5TGM1 myeloma cells were co-cultured with bone marrow cells containing haemopoietic osteoclastic precursors and stromal cells, they adhered strongly to the stromal cells and increased the formation of OC-like cells. There were no OC-like cells formed in cocultures with 5TGM1 cells that were prevented from contacting stromal cells. In addition, in cultures of long-bone organs from fetal rat conditioned medium harvested from co-cultures of 5TGM1 myeloma cells and ST2 bone marrow stromal cells increased bone resorption activity compared to conditioned medium of either ST2 or 5TGM1 alone These data are consistent with the notion that direct cell-to-cell contact of 5TGM1 cells with bone marrow stromal cells is required for the production of osteoclast stimulating activity and bone resorption. We then determined that cell adhesion molecules are involved in the direct cell-to-cell interaction between the 5TGM1 cell and the marrow stromal cells that is necessary for the production of osteoclastogenic activity. Our data indicate that VCAM-1 and VLA-4 integrin represent a function in this cell-to-cell interaction, since neutralizing the antibodies to these two adhesion molecules profoundly decreases the formation of OC-like cells in cocultures. The VCAM-1 / mtegr? Na interaction VLA-4 is responsible for cell-to-cell communication between marrow stromal cells and 5TGM1 myeloma cells leading to increased production of osteoclastogenic activity and bone resorption. Finally, this bone reabsorption activity is partly due to the induction of the Rank ligand. Example 2: LIVE EXPERIMENTS Our in vitro studies suggest that the interaction between VLA-4 on myeloma cells with VCAM-1 in marrow stromal cells may represent a key function in the induction of bone resorption activity by myeloma. We have taken the key step of testing this hypothesis in vivo in an animal model that accurately reflects the numanian disease. A. In this experiment, the mice were injected with 1 to 5 5TGM1 myeloma cells, which were allowed to colonize the bone marrow. The mice were divided into two groups of three, one served as a control group, and the second treated every two weeks beginning on day 8 with mAb PS / 2. The levels of IgG2b, the isotype of antibody produced by the myeloma cell 5TGM1, were measured weekly from weeks 1 to 6. Treatment with mAb at a dose of 80 ug per injection (approximately 4 milligrams / kilogram) biweekly strongly inhibited the production of IgG2b, indicator of significant inhibition of survival and growth of myeloma cells in vivo (Figure 4). In addition, the treated mice showed reduced incidence of paraplegia (the three untreated animals showed paraplegia on day 42), while only one of the treated animals showed paraplegia. The two animals treated without paraplegia showed a reduction in the weight of the spleen and liver, which correlates with the growth of the tumor. Finally, the treated animals showed a reduction in the tumor area histologically (from 6.71 +/- 1.74 to 0.05 +/- 0.08 square millimeter) in the tibia and femurs. There was no effect of treatment on serum calcium levels (the data were not shown). B. In a parallel experiment, treatment with 40 ug PS / 2 biweekly had no effect on IgG2b levels (not shown). These data show that the monoclonal antibody mAb PS / 2 for VLA-4 strongly inhibits the growth of established myeloma cells in a dose-dependent manner. C. In another live experiment, 18 SCID mice were injected with 5TGM1 myeloma cells on day 0. Four mice were treated with PBS; 4 mice were treated in a prophylactic protocol with monoclonal antibody M / K-2.7 reactive against mouse VCAM1 at a dose of 80 ug (approximately 4 milligrams / kilogram) every three days beginning on day 1 (ie, days 1, 2, 5, 8, and 11). In a parallel experiment using the same protocol, five mice were treated with 160 ug of monoclonal antibody M / K-2.7. In addition, five mice were treated with 160 ug of monoclonal antibody M / K-2.7 starting on day 8 (ie, days 8, 11, 14, 17, and 20) in a therapeutic protocol. Serum was taken from all mice on days 21, 28, and 35, and the animals were radiographed then sacrificed for histology on day 35. All three treatment groups showed a reduction in serum IgG2b levels, indicator of a load of reduced myeloma cells (Figure 5). A significant effect was also observed in the spleen weights and the low dose prophylactic protocol related to the control (0.23 +/- 0.14 grams for the control against 0.08 +/- 0.04 for the treated ones). In the prophylactic high dose group 4 or 5 animals showed a clear reduction in spleen weight, but the overall value was not significant because an animal with a large spleen weight (data not shown). D. It can be investigated whether a high initial bolus dose of alpha 4 mtegrin antagonist, followed by a maintenance dose, improved efficacy. The myeloma cells have already been established in the medullary compartment, and their close VLA-4-dependent interaction with VCAM-1 does not need to be inhibited. In addition, presumably the greater the number of established myeloma cells, the greater the initial dose required to draw the cells into the peripheral circulation. A larger study with anti-VLA-4 PS / 2 antibody was then carried out. Twenty-eight SCID mice were injected with 5TGM1 myeloma cells on day 0. Nine mice received no treatment; nine mice received a control IgG monoclonal antibody paired with isotype; 10 mice were treated with monoclonal treatment PS / 2 for enterogrma alfa 4. A different therapeutic regimen was given, in which the mice were given a high dose of monoclonal antibody (200 ug) on days 4, 5, and 6, then a maintenance dose of 80 ug (approximately 4 milligrams / kilogram) for three days starting on day 8. There was a statistically significant reduction in serum IgG2b when the treated group was compared with either the IgG-treated group or the untreated in weeks 3 and 4 (the data are not presented.) Importantly, when the treated group was compared with either the group treated with control IgG and the untreated group there was a clear effect on survival ( Figure 6) Example 3: OTHER LIVE EXPERIMENTS Based on the information presented here for the first time, those of ordinary skill in the art can easily confirm and extend the importance of technologies to the fa 4 and its ligands in multiple myeloma using the murmo animal model described. The following series of experiments are within the level of experience in the art based on the present description but only serve to exemplify and not limit, the types of work. 1) The dose response to the PS / 2 monoclonal antibody to determine the optimal biweekly maintenance dose. 80 ug shows good efficacy, but 40 ug had no effect. Higher doses up to 20 milligrams / kilogram are examined two or three times per week to determine optimal dosage. 2) Patients present with the disease at different stages of severity, related to increased tumor burden. The effectiveness of the PS / 2 monoclonal antibody given at different times after the establishment of the disease is examined, that is, the start of the treatment is compared at eight days (see for example Figure 4) at the beginning after two days., three, four, and five are manas after inoculation to see how late the monoclonal antibody can be given to provide some relief from the symptoms. 3j The effects of the monoclonal antibody MK-2 to VCAM-1 murmo are examined, following the same parameters indicated above (dosing, dosing time) for the monoclonal antibody for VLA-4. It is anticipated that similar dosage levels will be required to see efficacy. 4) Other markers of myeloma progression are examined, including tumor loading at both marrow and extramedullary sites, quantification of bone lesions by radiographic analysis of the skeleton by histomorphometry; measurement of bone resorption rates by evaluation of plasma collagen cross-links; measurement of plasma monoclonal protein production; hypercalcemia cuanac is present; and mortality. 5) The disease is currently treated inefficiently with standard chemotherapy. The additive or seeding effects of the monoclonal antibodies at optimal dosage together with, either before or after, dosing with suitable chemotherapeutic regimens is examined. 6) The ability of a small molecule mtegrα alpha 4 inhibitor that is selective for a particular alpha 4 mtegrin or is selective for several alpha 4 integrins at the same time or the ability of combinations for these inhibitors to mimic the effects of antibodies Monoclonal antibodies and block the progression of myeloma are examined using the protocols and described above. Small molecule inhibitors are administered parenterally or orally, in the dosage range of 0.1 to 30 milligrams / kilogram, once or twice a day, or twice three times weekly. Additional references: Alsina M, Boyce B, Devlin R, Anderson JL, Craig F, Mundy GR, Roodman GD. Development of an in vivo model of human multiple myeloma bone disease. Blood 87: 1495-1501, 1996. Attal M, Harousseau JL, Stoppa AM, Sotto JJ, Fuzibet JG, Rossi JF, Casassus P, Maisonneuve H, Facón T, Ifrah N, Payen C, Bataille R. A prospective, randomized trial of autologou = bone marrow transplantation and chemotherapy in multiple myeloma. Intergroupe Francais du Myelome. N Engl J Med 335: 91-97, 1996. Bataille R, Jourdan M, Zhang XG, Klein B. Serum levéis of interleukin-6, a potent myeloma cell growth factor, as a refiection of disease severity in plasma cell dyscrasias. J Clin Invest «4: 2008, 1989. Bataille R, Chappard D, Klein B. Mechanisms of bone lesions in multiple myeloma. Hem Onc Clin NA 6: 285-295, 1992. Bataille R, Barlogie B, Lu ZY, Rossi JF, Lavabre-Bertrand T, Beck T, Wijdenes J, Brochier J, Klein B.

Biologic effects of ant? -mterleukm-6 murine monoclonal antibody m advanced multiple myeloma. Blood 86: 685-691, 1995. Boyce BF, Yates AJP, Mundy GR. Bolus mjections of recombinant human mterleukm-1 cause transient hypocalcemia in normal mice. Endocnnology 125: 2780-2783, 1989. Chauhan D, Uchiyama H, Urashima M, Yamamoto K, Anderson KC. Regulation of? Nterleukm-6 m multiple myeloma and bone marrow stromal cells. Stem Cells 13: 35-39, 1995. Epstein J. Myeloma phenotype: Clues to disease origin and manifestation. Hem Onc Clin NA 6: 249-256, 1992. Garret IR, Dallas S, Radl J, Mundy GR: A murine model of human myeloma bone disease. Bone 20: 515-520, 1997. Gosslar U, Jonas P, Light A, Lifka A, Naor D, Hamann A, Hozmann B. Predominant role of alpha 4 mtegrins for distinct steps of lymphoma metastasis. Proc. Nati Acad. Sci. USA. 93: 4821-4826, 1996. Lacey DL, Timms E, Tan HL, Kelley MJ, Dunstan CR, Burgess T, Elliott R, Colombero A, Elliott G, Scully S, Hsu H, Sullivan J, Hawkms N, Davy E, Capparelli C, Eli A, Qian YX, Kaufman S, Sarosi 1, Shalhoub V, Senaldi G, Guo J, Delaney J, Boyle WJ. Osteoprotegerm ligand ís a cytokine that regulates osteoclast differentiation and activation. Cell 93: 165-176, 1998. Lobb R, Chi-Rosso G, Leone D, Rosa M, Newman B, Luhowskyj S, Osborn L, Schiffer S, Benjamín C, Dougas I, Hession C, Chow P. Expression and functional characterization of a soluble form of vascular cell adhesion molecule 1. Biochem. Biphys. Res. Commun. 178: 1498-1504, 1991. Lobb, R. and Hemler, M. The Pathophysiologic Role of alpha4 Integpns In Vivo. J. Clin. Invest., 94: 1722-1728 1994 MacDonald BR, Mundy GR, Clark S, Wang EA, Kuehl TJ, Stanley ER, Roodman GD. Effects of human recombinant CSF-GM and highiy pupfied CSF-1 on the formation of multmucleated cells with osteoclast characteristics in long-term bone marrow cultures. J Bone Min Res 1: 227-223, 1986. Mbalaviele G, Chen H, Boyce BF, Mundy GR, Yoneda T: The role of cadherin m the generation of multmucleated osteoclasts from mononuclear precursor in murine marrow. J Clin Invest 95: 2757-2765, 1995. Matsuura N, Puzon-McLaughlm W, Ipe A, Morikawa Y, Kakudo K, Takada Y. Induction of experimental bone metastasis in mice by transfection of mtegrin alpha 4 beta 1 mtor tumor cells. Am J Pathol 149: 55-61, 1996. Matsuzak K, Udagawa N, Takahashi N, Yamaguchi K, Yasuda H, Shima N, Morinaga T, Toyama Y, Yabe Y, Higashio K, Suda T. Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peppheral blood mononuclear cell cultures. Biochem Biophys Res Commun 246: 199-204, 1996.

Mundy GR, Bertolim DR. Bone destruction and hypercalcemia m plasma cell myeloma. Seminar Oncol 3: 291, 1986. Mundy GR. Myeloma bone disease, Eur. J. Cancer 34: 246-251, 1998. Papayannopoulou T, Nakamoto B. Peppheralization of hemopoietic progenitors in primates treated with ant? -VLA4 integrin. Proc. Nati Acad. Sci. USA 90: 9374-9378, 1993. Qian F, Vaux DL, Weissman IL. Expression of the integrin a4bl on melanoma cells can inhibit the invasive stage or metastasis formation. Cell, 77: 335-347, 1994. Radl J, Croese JW, Zurcher C, Van den Enden-Vieveen MM, Leuuw AM. Animal model of human disease. Am. J. Pathol. 132: 593-59", I9né." Simonet WS, Lacey DL, Dunstan CR, Kelley M, Chang MS, Luthy R, Nguyen HQ, Wooden S, Bennett L, Boone T, Shimamoto G, DeRose M, Elliott R, Colombero A, Tan HL, Trail G, Sullivan J, Davy E, Bucay N, Renshaw-Gegg L, Hughes TM, Hill D, Pattison W, Campbell P, Boyle WJ, et al. Osteoprotegepn: a novel secreted protein mvolved? N the regulation of bone density. Cell 309-319, 1997. Takahashi N, Yamana H, Yoshiki S, Roodman GD, Mundy GR, Jones SH, Boyde A, Suda T: Osteoclast-like cell formation and regulation by osteotropic hormones m mouse bone marrow cultures. Endocnnology 122: 1373-1382, 1988.

Vanderkerken K, De Raeve H, Goes E, Van Meirvenne S, Radl J, Van Riet 1, Thielemans K, Van Camp B. Organ mvolvement and phenotypic adhesion profile of 5T2 and 5T33 myeloma cells m the C57BL / KaLwR? J mouse. Brit J Cancer 76: 451-460, 1997. Vaughan T, Williams AJ, Pritchard K, Osbourn JK, Pope AR, Earnshaw JC, et al. Human antibodies with sub-nanomolar affinities isolated from a large non-immunized phage display library. Nature Biotechnology, 14: 309-314, 1996. Yasuda H, Shima N, Nakagawa N, Yamaguchi K, Kmosaki M, Mochizuki S, Tomoyasu A, Yano K, Goto M, Murakami A, Tsuda E, Morinaga T, Higashio K, Udagawa N, Takahashi N, Suda T. Osteoclast di f ferentiation factor a ligand for osteoprotegerm / osteoclastogenesis-inhibitory factor and the same to TRANCE / RankL. Proc Nati Acad Sci USA 95: 3597-3602, 1998. Yoneda T, Alsina MM, García JL, Mundy GR: Differentiation of HL-60 Cells mto cells with the osteoclast phenotype. Endocrinology 129: 683-689, 1992. Yoneda T, Lowe C, Lee CH, Gutierrez G, Niewolna M, Williams P, Izbicka E, Uehara Y, Mundy GR: Herbimycin A, pp60"tyrosine kmase mhibitor, inhibits osteoclastic bone resorption in vitro and hypercalcemia in vivo J Clin Invest 91: 2791-2795, 1993.

Claims (6)

* * CLAIMS

1. A method for treating multiple myeloma which comprises administering to an individual an amount Therapeutically effective of a composition comprising an antagonism of an interaction between a mtegrin having alpha 4 subunit and a ligand for mtegrin having alpha 4 subunit.

2. The method of claim 1, wherein the antagonist is an agent. 4.

The method of claim 1, wherein the antagonist is a ligand binding agent of integrin alpha 4.

4. The metoac ae Id rei indication 2, wherein the binding agent of ntegpna alfa 4 is selected from the group consisting of: a) an antibody homologue that antagonizes the interaction of both VLA-4 and alpha 4 beta 7 with their respective alpha 4 ligands; b) an antibody homologue that antagonizes the interaction of VLA-4 with its alpha 4 ligand; u and c) an antibody homologue that antagonizes the interaction of alpha 4 beta 7 with its alpha ligand 4.

The method of claim 4, wherein the antibody homolog is selected from the group consisting of a human antibody, a chimeric antibody , a humanized antibody and fragments thereof. * - »

6. The method of claim 3, wherein the mtegrin alpha 4 ligand binding agent is the (CLAIMS-CONTINUATION) 7. The method of claim 6, wherein the binding agent of mtegrin alpha4 ligand is a homologous of anti-VCAM-l antibody. 8. The method of claim 1, wherein the antagonist is a small molecule. 9. A method of claim 1, wherein the composition is administered at a dose such as to provide from about 0.1 to 20 mg / kg of body weight. 10. A method for inhibiting bone resorption associated with bone marrow tumors, the method comprising administering to a mammal with said tumors an antagonist 15 of an interaction between a mtegrin carrying alpha4 subunit and a ligand for integrin carrying alpha4 subunit, in an amount effective to provide inhibition of said bone reabsorption. The method of claim 10, wherein the antagonist is an alpha4 mtegrin binding agent. The method of claim 10, wherein the antagonist is a ligand binding agent of mtegrma alfa4. The method of claim 11, wherein the mtegrin alpha4 binding agent is a homologue of anti-VLA4 antibody or an anti-alpha4b3ta7 antibody homolog. The method of claim 12, wherein the mtegrin alpha4 ligand binding agent is an anti-αVCAM-1 antibody homolog. 16. The method of claim 15, wherein the antibody homolog is selected from the group consisting of a human antibody, a chimeric antibody, a humanized antibody and fragments thereof. 17. The method of claim 10, wherein the antagonist is a small molecule. 18. A method of claim 10, wherein the antagonist is administered at a dose to provide from about 0.1 to about 20 mg / kg, based on the weight of the individual. The method of claim 17, wherein the antagonist is administered in an amount effective to provide a small molecule dose of about 0.1 to 30 milligrams / kilogram of body weight. 20. A method for treating a subject having a condition characterized by the presence of osteoclastogenesis, the method comprising administering to the subject an antagonist of an interaction between a mtegrin carrying the alpha4 subunit and a ligand for a mtegrin carrying the alpha4 subunit , in an amount sufficient to suppress osteoclastogenesis. The method of claim 20, wherein the antagonist is an alpha4 integrin binding agent. 22. The method of claim 20, wherein the antagonist is a ligand binding agent of mtegrma alfa4. 23. The method of claim 21, wherein the mtegrin alpha4 binding agent is a homologue of anti-VLA4 antibody or an anti-alpha4beta7 binding agent. The method of claim 23, wherein the antibody homologue is selected from the group consisting of a human antibody, a chimeric antibody, a humanized antibody and fragments thereof. 25. The method of claim 22, wherein the ligand binding agent of alpha4 is a homolog of anti-VCAM-1 antibody. 26. The method of claim 25, wherein the antibody homolog is selected from the group consisting of a human antibody, a chimeric antibody, a humanized antibody and fragments thereof. 27. The method of claim 20, wherein the antagonist is a small molecule. The method of claim 20, wherein the antagonist is administered at a dose to provide from about 0.1 to about 20 mg / kg of body weight. 29. The method of claim 27, wherein the antagonist is administered in an amount effective to provide a small molecule dose of about 0.1-20 mg / kg body weight.