AU727187B2 - Treatment for insulin dependent diabetes - Google Patents

Description

AUSTRALIA

Patents Act 1990 COMPLETE SPECIFICATION STANDARD PATENT Applicant(s): BIOGEN, INC.

Invention Title: TREATMENT FOR INSULIN DEPENDENT DIABETES The following statement is a full description of this invention, including the best method of performing it known to me/us: TREATMENT FOR INSULIN DEPENDENT DIABETES FIELD OF THE INVENTION The present invention relates to a treatment for insulin dependent (type-I) diabetes. More particularly, this invention relates to the use of antibodies recognizing the integrin VLA4 (very late antigen 4) in the prevention of diabetes.

BACKGROUND OF THE INVENTION Insulin dependent diabetes (also termed type-I diabetes and formerly juvenile onset diabetes mellitus) has been classified during the past two decades as a chronic autoimmune disease. In this disorder, cells producing insulin (p cells) within the pancreatic islets are selectively targeted and destroyed by a cellular infiltrate of the pancreas. This inflammatory 20 infiltrate affecting the islets has been termed insulitis. Cells producing insulin comprise the majority of islet cells but less than 2% of the total pancreatic mass (Castano and Eisenbarth, 1990, Fujita et al., 1982 Foulis et al., 1986 The development of type I diabetes can conceptually be divided into six stages, beginning with genetic susceptibility and ending with complete p cell destruction (Eisenbarth, 1986 Stage I is genetic susceptibility, which is a necessary but insufficient 30 condition for development of the disease. A hypothetical triggering event (Stage II) leads to active autoimmunity against p cells (Stage III). In Stage III, the P cell mass is hypothesized to decline 2 and immunologic abnormalities such as autoantibodies directed against insulin and islet cytoplasmic antigens are found. Stimulated insulin secretion is still preserved at this stage. Over a period of years, however, the progressive loss of 3 cells leads to diminished insulin secretion with intravenous glucose tolerance tests (IVGTT) while the individual is still normoglycemic (Stage IV). Overt diabetes diabetes onset or clinical manifestation of disease characterized by hyperglycemia) is Stage V, and can develop years later when approximately 90% of pancreatic A cells are destroyed. In Stage V when overt diabetes is first recognized, some residual insulin production remains (as demonstrated by the presence of the connecting peptide of proinsulin,

C

peptide, in the serum) but the individual usually requires exogenous insulin for life. Finally, in Stage VI, even the remaining P cells are destroyed and C peptide can no longer be detected in the circulation.

While the initiating factor(s) and specific sequence of events leading to diabetes, including the relative importance of different cell types and cytokines, are still widely debated, a key role is generally recognized for self-antigen reactive T cells 25 (Miller et al., 1988 Harada and Makino, 1986 Koike et al., 1987 Makino et al., 1986 In addition to T lymphocytes, insulitis is characterized by macrophages, dendritic cells (Voorbij et al., 1989 and B cells, which may serve as professional antigen presenting cells (APC). Macrophages may also destroy islet p cells themselves by release of cytokines or free radicals (Nomikos et al., 1986 Thus, autoimmune diabetes relies upon both cellular migration and immune stimulation of newly resident 35 cells.

3 Cell trafficking to inflammatory sites is regulated by accessory molecules LFA-1, MAC-1 and VLA4 (Larson and Springer, 1990 Hemler et al., 1990 on the surface of lymphocytes (LFA-1, VLA4) and macrophages (Mac-1, VLA4), and by their counter-ligands ICAM (for LFA-1 and MAC-1), and VCAM (for VLA4) which are unregulated by cytokines on vascular endothelium (Larson and Springer, 1990 Lobb, 1992 [13]; Osborn, 1990,[14]). In addition, VLA4 binds to an extracellular matrix component, the CS-1 domain of fibronectin (FN) (Wayner et al., 1989 The relative importance of these pathways, for example, LFA-1 and VLA4 on lymphocytes or MAC-1 and VLA4 on monocytes, in controlling cell migration is still a subject of investigation. In vitro data suggest that the differential use of these pathways appears to depend upon the activation status of both the leukocytes and endothelial cells (Shimizu et al., 1991 Their ability to control cell migration to inflammatory sites in vivo has been directly demonstrated with monoclonal antibodies (mAbs) to ICAM, MAC-1 or VLA4 inhibiting various animal models of disease (Barton et al., 1989 phorbol esterinduced rabbit lung inflammation; Issekutz and 25 Issekutz, 1991 delayed type hypersensitivity; Issekutz, 1991 adjuvant-induced arthritis; Yednock et al., 1992 transfer of experimental allergic encephalomyelitis (EAE); Lobb, 1992 [21], asthma).

ICAM and VCAM are also found on the surface of macrophages and dendritic cells in lymphoid tissues (Dustin et al., 1986 Rice et al., 1990 Rice et al., 1991 Their distribution on these professional APC is consistent with functional data indicating a role for LFA-1 and VLA4 in T cell 4 activation (Shimuzu et al., 1990 Burkly et al., 1991 However, numerous other receptor-ligand pairs including CD4/ MHC class II and CD8/MHC class I (Rudd et al., 1989 CD2/LFA-3 (Moingeon et al., 1989 1287]), CD28/B7 (Harding et al., 1992 may also support adhesion or costimulate T cells during T/APC or T/target cell interactions. The specific contributions of these numerous pathways in the development of diabetes is unresolved. Because there are multiple molecular pathways for cell adhesion and T cell activation, it is not possible to predict whether intervention in one or more of these pathways might affect onset or severity of diabetes disease, and, in particular, which of these pathways are crucial or relevant to the disease process.

Antibodies directed to T cells have been utilized in murine and rat models for spontaneous diabetes and adoptive transfer of diabetes to deplete T cells and thus prevent disease (see, Harada and Makino, 1986 anti-Thy 1.2; Koike et al., 1987 Miller et al., 1988 and Shizuru et al., 1988 anti- CD4; Barlow and Like, 1992 anti-CD2; Like et al., 1986 anti-CDS and anti-CD8). In addition, an antibody directed to the complement receptor type 3 25 (CR3) molecule or MAC-1 on macrophages has been utilized to prevent macrophage and T cell infiltration of pancreatic tissue in a murine adoptive transfer model of disease (Hutchings et al., 1990 It is unknown whether VLA4 is relevant to insulitis or to the activity of islet-specific cells after localization in the pancreas.

Current treatment protocols suggested for type I diabetes have included certain immunomodulatory drugs summarized by Federlin and Becker [34] and references 35 cited therein. A long prediabetic period with immunologic abnormalities and progressive 3 cell destruction suggests it may be possible to halt 0 cell loss with immune intervention (Castano and Eisenbarth, 1990 Suggested agents/protocols have included certain immunomodulatory and immunosuppressive agents: levamisol, theophyllin, thymic hormones, ciamexone, anti-thymocyte globulin, interferon, nicotinamide, gamma globulin infusion, plasmapheresis or white cell transfusion. Agents such as cyclosporin A and azathioprine which impair T cell activation and T cell development, respectively, have been used in clinical trials (Zielasek et al., 1989 The most promising results have been achieved with cyclosporin A (Castano and Eisenbarth, 1990 Federlin and Becker, 1990 [34] suggest, however, that cyclosporin A may not be recommended for general or long-term use because of toxic side effects, at least when given in higher doses. Higher doses of cyclosporin, or in combination with other immunosuppressive drugs, or both, have been associated with the development of lymphoma and irreversible kidney damage (Eisenbarth, 1986 Eisenbarth, 1987 Additional studies on other suggested agents are necessary to assess 25 safety and efficacy. Even the cyclosporin A studies show that its efficacy in maintaining remission of diabetes is for one year in about 30-60% of new onset diabetes. Within 3 years, however, remissions are almost invariably lost (Castano and Eisenbarth, 1990 Treatment protocols after onset of disease are particularly problematic, since, for example, at the time diabetes is diagnosed in humans, insulitis has typically progressed already to a loss of more than of the P cells. Thus, it is possible that cyclosporin 35 A may be preventing further 3 cell destruction, but so 6 few P cells may be present at the onset of the diabetes that they cannot maintain a non-diabetic state over time (Castano and Eisenbarth, 1990 Suppression of insulitis and/or prevention of disease may be more successful if the treatment could start at an earlier phase, before disease onset.

It will be clearly understood that, although a number of prior art publications are referred to herein, this reference does not constitute an admission that any of these documents forms part of the common general knowledge in the art, in Australia or in any other country.

There are two major prerequisites in order to develop any preventive treatment for diabetes disease: (1) the ability to accurately identify the prediabetic individual and the development of safe, specific and effective preventive treatments. Significant progress has been made in identifying prediabetic individuals, however, much work remains in the development of safe, specific and effective preventive treatments as discussed and reviewed 20 by Eisenbarth and colleagues (see, Ziegler and ,Eisenbarth, 1990 Ziegler et al, 1990 Ziegler et al, 1990 It has been possible to identify certain risk factors and at-risk groups for type I diabetes and thus to predict individuals most likely to go on to clinical disease and to estimate the approximate rate of disease onset in these individuals. The ability to identify individuals with susceptibility to diabetes or to predict type I diabetes in the pre-clinical stage by the combination of genetic (HLA typing), immunological (islet 30 and insulin autoantibodies) and metabolic (first phase *o*insulin secretion to intravenous glucose preceding the development of hyperglycemia) markers makes the identification and use of prophylactic immunotherapeutic drugs and protocols possible during the evolution of the autoimmune disease process when P cell destruction is only partial. To date, there has been little success, however, in treating human diabetes. Generally, because human \\melb-files\homes\cintae\Keep\pei\69846.98.doc 13/09/00 7 treatment has been used only after onset of the disease, treatment was followed by a temporary complete or partial remission only in a certain number of patients. Since immunosuppresive mechanisms may prevent insulitis and/or diabetes, there is a need for immunosuppresive components for use in the prediabetic stage. In particular, there is a need for safer and more specifically acting compounds, monoclonal antibodies, which inhibit entry of effector cells into the pancreas or function of those cells which may have already entered the islets of Langerhans.

It has now been surprisingly discovered that administering an anti-VLA4 antibody significantly reduced the incidence of diabetes, in a rodent model of diabetes disease. The NOD mouse model of diabetes is a well established model directly comparable to human type-I diabetes. Using an adoptively transferred disease experimental protocol, irradiated non-diabetic NOD mice were administered splenocytes from spontaneously diabetic NOD mice for the acute transfer of the disease. These 20 splenocytes were treated with anti-VLA4 antibody before administration and the recipients were also treated for various periods of time after the transfer with anti-VLA4 antibody.

*c0 \\melf iles\homeS\cintae\Keep\speci\69846.98.doc 13/09/00 8 SUMMARY OF THE INVENTION Accordingly, the present invention provides novel methods for the treatment of insulin dependent (type-I) diabetes in a prediabetic. In particular, the present invention provides a method for the prevention of insulin dependent diabetes comprising the step of administering to a prediabetic individual an anti-VLA4 antibody, such as antibody HP1/2 or a humanised anti-VLA4 antibody derived from HP1/2. Also contemplated is the use of analogous antibodies, antibody fragments, soluble proteins and small molecules that mimic the action of anti-VLA4 antibodies in the treatment of diabetes. In addition, the present invention provides a method for the treatment of diabetes by administering to a mammal, including a human, with a susceptibility to diabetes an antibody capable of binding to the a4 subunit of VLA4 in an amount effective to provide inhibition of the onset of diabetes. Also contemplated is the use of recombinant and chimeric antibodies, fragments of such antibodies, polypeptides or small molecules capable 20 of binding a4/VLA4. Also contemplated are soluble forms of the natural binding proteins for VLA4, including soluble :VCAM-1, VCAM-1 peptides or VCAM-1 fusion proteins as well :as fibronectin, fibronectin having an alternatively spliced non-type III connecting segment and fibronectin peptides containing the amino acid sequence EILDV or a similar conservatively substituted amino acid sequence. These agents will act by competing with the cell-surface binding protein for VLA4.

SFor the purposes of this specification it will be 30 clearly understood that the word "comprising" means "including but not limited to", and that the word "comprises" has a corresponding meaning.

\\melb_files\homeS\cintae\Keep\speci\69846.98.doc 13/09/00 9 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 is a graph depicting the effect of anti- VLA4 antibody (Rl-2) and controls on prevention of diabetes after adoptive transfer of spleen cells; the frequency of recipients which became diabetic and day of disease onset are shown for transfer of 2x10 7 splenocytes from diabetic NOD donors without treatment (closed circles), with a non-specific rat IgG2b treatment (closed triangles), and with R1-2 anti- VLA4 treatment (closed diamonds), as well as for transfer of splenocytes from nondiabetic NOD donors (open squares); the splenocytes were transferred with R1-2 or rat IgG2b or without mAb, and then R1-2 or rat IgG2b was injected every other day through day 12 post transfer (n=8-10 for all groups).

Figure 2 is a graph depicting the effect of anti- VLA4 antibody (R1-2) and controls on prevention of diabetes after adoptive transfer of spleen cells; the frequency of recipients which became diabetic and day of disease onset are shown for transfer of 3x10 7 splenocytes from diabetic NOD donors without treatment (closed circles), with a non-specific rat IgG2b treatment (closed triangles), and with R1-2 anti- VLA4 treatment (closed diamonds), as well as for 25 transfer of splenocytes from nondiabetic NOD donors (open squares); the splenocytes were transferred with R1-2 or rat IgG2b or without mAb, and then R1-2 or rat IgG2b was injected every 3.5 days through day 25 post transfer (n=4-5 for all groups).

Figure 3 is a graph depicting the effect of anti- VLA4 antibody (Rl-2) and controls on prevention of diabetes after adoptive transfer of spleen cells; the frequency of recipients which became diabetic and day of disease onset are shown for transfer of 2-3x10 7 35 splenocytes from diabetic NOD donors without 10 treatment (closed circles), with a non-specific rat IgG2b treatment (closed triangles), and with R1-2 anti- VLA4 treatment (closed diamonds), as well as for transfer of splenocytes from nondiabetic NOD donors (open squares) or for PBS alone (open circles); the splenocytes were transferred with R1-2 or rat IgG2b or without mAb, and then R1-2 or rat IgG2b was injected every 3.5 days through day 25 post transfer (n=5 for all groups).

Figure 4 is a bar graph depicting the effect of anti-VLA4 antibody (Rl-2) and controls on the degree of insulitis after adoptive transfer of spleen cells; the frequency of uninfiltrated islets (Grade 0-I infiltrate, stipled bar) and infiltrated islets (Grade II-IV insulitis, solid bar) were quantitated and shown after transfer of cells treated with R1-2, rat IgG2b or without mAb, and then R1-2 or rat IgG2b injected every days through day 25 with mice sacrificed when diabetic or on day 26 post-transfer. Pancreatic sections from n=4-5 mice were scored for each experimental group, Y-Y (non-diabetic donor cells) or D-Y (diabetic donor cells) into non-diabetic recipients with no mAb treatment, treatment with rat IgG2b or treatment with R1-2.

25 Figure 5 is a bar graph depicting the effect of anti-VLA4 antibody (Rl-2) and controls on the degree of insulitis after adoptive transfer of spleen cells; the frequency of uninfiltrated islets (Grade 0-I infiltrate, stipled bar) and infiltrated islets (Grade II-IV insulitis, solid bar) were quantitated and shown after transfer of cells treated with R1-2, rat IgG2b or without mAb, and then R1-2 or rat IgG2b injected every other day through day 12 post-transfer, then maintained without further mAb injection until sacrificed when diabetic or on day 29 post-transfer. Pancreatic 11 sections from n=4-5 mice were scored for each experimental group, Y-Y (non-diabetic donor cells) or D-Y (diabetic donor cells) into non-diabetic recipients with no mAb treatment, treatment with rat IgG2b or treatment with R1-2.

Figure 6 is a graph depicting the effect of anti- VLA4 antibody (R1-2) and controls on prevention of diabetes in a spontaneous disease model for diabetes; the frequency of recipients which became diabetic and day of disease onset are shown for NOD mice without treatment (closed squares), with a non-specific rat IgG2b treatment (closed circles), and with R1-2 anti- VLA4 treatment (closed triangles); R1-2 or rat IgG2b was injected for 8 weeks in NOD mice twice weekly from week four to week twelve of age.

Figure 7 is a graph depicting the effect of VCAM 2D-IgG fusion protein and controls on prevention of diabetes after adoptive transfer of spleen cells; the frequency of recipients which became diabetic and day of disease onset are shown for transfer of 2x10 7 splenocytes from diabetic NOD donors with an irrelevant rat LFA-3Ig fusion protein treatment (closed squares), and with VCAM 2D-IgG treatment (open circles) or of recipients which recieved PBS alone without cells 25 transferred (closed triangles); the splenocytes were transferred with VCAM 2D-IgG or rat LFA-3Ig, and then VCAM 2D-IgG or rat LFA-3Ig was injected every other day through day 17 post-transfer (n 5 for all groups).

Figure 8 is a schematic depicting structure of VCAM 2DIgG fusion protein described in Example 5. VCAM 2D-IgG is a soluble form of the ligand for VLA4 (VCAM1) .and consists of the two N-terminal domains of VCAM1 fused to the human IgG1 heavy chain constant region sequences (Hinges, C0 2 and C.

3 00n 0*" 12 DETAILED DESCRIPTION OF THE INVENTIOW The invention relates to a treatment including the prevention of insulin dependent (type I) diabetes.

More particularly, the invention relates to the use of antibodies to VLA4 in the treatment of diabetes in a prediabetic individual. The term "prediabetic" is intended to mean an individual at risk for the development of diabetes disease genetically predisposed) at any stage in the disease process prior to overt diabetes or diabetes onset. The term "diabetic" is intended to mean an individual with overt hyperglycemia fasting blood glucose levels 2 250 mg/dL). The term "overt diabetes" or "diabetes onset" is intended to mean a disease state in which the pancreatic islet cells are destroyed and which is manifested clinically by overt hyperglycemia fasting blood glucose levels 250 mg/dL).

In the first aspect, the invention provides a method of treatment of diabetes comprising the step of administering a composition capable of binding to, including blocking or coating, the VLA4 antigens on the surface of VLA4-positive cells, including lymphocytes and macrophages. For purposes of the invention, the term "binding to VLA4 antigens" is intended to mean 25 reacting with VLA4 antigens on cells and thereby interfering with interactions between VLA4 antigens and either VCAM-1 or fibronectin on the surface of other cells or thereby inducing a change in the function of the VLA4-positive cells. As demonstrated herein, such binding, including blocking or coating, of VLA4 antigens results in a prevention in or protection against the incidence of diabetes. This demonstration utilized a monoclonal antibody against VLA4 as a binding agent which effectively blocked or coated the 35 VLA4 antigens. Those skilled in the art will recognize 13 that, given this demonstration, any agent that can bind to, including those that can block or coat, VLA4 antigens can be successfully used in the method of the invention. Thus, for purposes of the invention, any agent capable of binding to VLA4 antigens on the surface of VLA4-bearing cells and which may effectively block or coat VLA4 antigens, is considered to be an equivalent of the monoclonal antibody used in the examples herein. For example, the invention contemplates as binding equivalents at least peptides, peptide mimetics, carbohydrates and small molecules capable of binding VLA4 antigens on the surface of VLA4-bearing cells.

In a preferred embodiment, the agent that is used in the method of the invention to bind to, including block or coat, cell-surface VLA4 antigens is a monoclonal antibody or antibody derivative. Preferred antibody derivatives for treatment, in particular for human treatment, include humanized recombinant antibodies, chimeric recombinant antibodies, Fab, Fab', F(ab') 2 and F(v) antibody fragments, and monomers or dimers of antibody heavy or light chains or intermixtures thereof. Thus, monoclonal antibodies against VLA4 are a preferred binding agent in the 25 method according to the invention.

The technology for producing monoclonal antibodies is well known. Briefly, an immortal cell line (typically myeloma cells) is fused to lymphocytes (typically splenocytes) from a mammal immunized with whole cells expressing a given antigen, VLA4, and the culture supernatants of the resulting hybridoma cells are screened for antibodies against the antigen.

(See, generally, Kohler et al., 1975 Immunization may be accomplished using standard 35 procedures. The unit dose and immunization regimen 14 depend on the species of mammal immunized, its immune status, the body weight of the mammal, etc. Typically, the immunized mammals are bled and the serum from each blood sample is assayed for particular antibodies using appropriate screening assays. For example, anti- VLA4 antibodies may be identified by immunoprecipitation of 1 I-labeled cell lysates from VLA4-expressing cells. (See, Sanchez-Madrid et al.

1986 [41] and Hemler et al. 1987 Anti-VLA4 antibodies may also be identified by flow cytometry, by measuring fluorescent staining of Ramos cells incubated with an antibody believed to recognize VLA4 (see, Elices et al., (1990) The lymphocytes used in the production of hybridoma cells typically are isolated from immunized mammals whose sera have already tested positive for the presence of anti-VLA4 antibodies using such screening assays.

Typically, the immortal cell line a myeloma cell line) is derived from the same mammalian species as the lymphocytes. Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").

Typically, HAT-sensitive mouse myeloma cells are S. 25 fused to mouse splenocytes using 1500 molecular weight polyethylene glycol ("PEG 1500"). Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed). Hybridomas producing a desired antibody are detected by screening the hybridoma culture supernatants. For example, hybridomas prepared to produce anti-VLA4 antibodies may be screened by testing the hybridoma culture supernatant for secreted antibodies having the ability *o 15 to bind to a recombinant a4-subunit-expressing cell line, such as transfected K-562 cells (see, Elices et al. To produce anti-VLA4 antibodies, hybridoma cells that tested positive in such screening assays were cultured in a nutrient medium under conditions and for a time sufficient to allow the hybridoma cells to secrete the monoclonal antibodies into the culture medium. Tissue culture techniques and culture media suitable for hybridoma cells are well known. The conditioned hybridoma culture supernatant may be collected and the anti-VLA4 antibodies optionally further purified by well-known methods.

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

Several mouse anti-VLA4 monoclonal antibodies have been previously described (see, Sanchez-Madrid et al., 1986 Hemler et al., 1987 Pulido et 25 al., 1991 These anti-VLA4 monoclonal antibodies such as HP1/2 and other anti-VLA4 antibodies mAb .HP2/1, HP2/4, L25, P4C2, P4G9) capable of recognizing the a chain of VLA4 will be useful in the methods of treatment according to the present invention. Anti- VLA4 antibodies that will recognize the VLA-a 4 chain epitopes involved in binding to VCAM-1 and fibronectin ligands antibodies which can bind to VLA4 at a site involved in ligand recognition and block VCAM-1 and fibronectin binding) are preferred. Such 35 antibodies have been defined as B epitope-specific -16antibodies (BI or B2) (see, Pulido et al. (1991) and are preferred anti-VLA4 antibodies according to the present invention. The RI-2 antibody used as described herein is a B epitope type antilboy.

Human monoclonal antibodies against VLA4 are another prefe-red binding agent which may block or coat VLA4 antigens in the method of the invention. These may be prepared using in virro-primed human splenocyves, as described by Boemer et al.. 1991 Alternatively. they may be prepared by repertoire cloning as described by Persson et al.. 1991 [46] or by Huang and Stollar, 1991 Another preferred binding agent which mav block or coat VLA4 antigens in the method of the invention is a chimeric recombinant antibodv having anti-VLA4 specificity and a human antibody constant region. Yet another preferred binding agent which may block or coat VLA4 antigens in the method of the invention is a humanized recombinant antibody having anti-VLA4 specificity. Humanized antibodies may be prepared. as exemplified in Jones et al.. 1986 Riechmann, 1988, Queen et al., 1989 [501; and Orlandi et al., 1989 Preferred binding agents including chimeric recombinant and humanized recombinant antibodies with B epitope specificity have been prepared and are described in the commonly owned international patent application, WO 9416094. published on July 21, 1994 The starting material for the preparation of chimeric (mouse V human C) and humanized anti-VLA4 antibodies may be a murine monoclonal anti-VLA4 antibody as previously described, a monoclonal anti-VLA4 antibody commercially available HP2/1, Amac International, Inc., Westbrook, Maine), or a monoclonal anti-VLA4 antibody prepared in accordance with the teaching herein. For example, the variable regions of *ee 17 the heavy and light chains of the anti-VLA4 antibody HP1/2 have been cloned, sequenced and expressed in combination with constant regions of human immunoglobulin heavy and light chains. Such a chimeric HP1/2 antibody is similar in specificity and potency to the murine HP1/2 antibody, and may be useful in methods of treatment according to the present invention. The HP1/2 V, DNA sequence and its translated amino acid sequences are set forth in SEQ ID NO: 1 and SEQ ID NO: 2, respectively. The HP1/2 VK DNA sequence and its translated amino acid sequence are set forth in SEQ ID NO: 3 and SEQ ID NO: 4, respectively. Similarly, humanized recombinant anti-VLA4 antibodies may be useful in these methods. A preferred humanized recombinant anti-VLA4 antibody is an AS/SVMDY antibody, for example, the AS/SVMDY antibody produced by the cell line deposited with the ATCC on November 3, 1992 and given accession no. CRL 11175. The AS/SVMDY humanized antibody is at least equipotent with or perhaps more potent than the murine HP1/2 antibody. The AS V, DNA sequence and its translated amino acid sequences are set forth in SEQ ID NO: 5 and SEQ ID NO: 6, respectively. The SVMDY VK DNA sequence and its translated amino acid sequence are set forth in SEQ ID 25 NO: 7 and SEQ ID NO: 8, respectively.

Those skilled in the art will recognize that any of the above-identified antibody or antibody derivative binding agents can also act in the method of the invention by binding to the receptor for VLA4, and may block or coat the cell-surface VLA4 antigen. Thus, antibody and antibody derivative binding agents according to the invention may include embodiments having binding specificity for VCAM-1 or fibronectin, since these molecules appear to either be important in 35 the adhesion cells or the extracellular matrix or *e* 18 interfere with traffic of cells through tissues and blood.

Alternatively, the binding agents used in the method according to the invention may not be antibodies or antibody derivatives, but rather may be soluble forms of the natural binding proteins for VLA4. These binding agents include soluble VCAM-1, VCAM-1 peptides, or VCAM-1 fusion proteins as well as fibronectin, fibronectin having an alternatively spliced non-type III connecting segment and fibronectin peptides containing the amino acid sequence EILDV or a similar conservatively substituted amino acid sequence. These binding agents will act by competing with the cellsurface binding protein for VLA4.

In this method according to the first aspect of the invention, VLA4 binding agents are preferably administered parenterally. The VLA4 binding agents are preferably administered as a sterile pharmaceutical composition containing a pharmaceutically acceptable carrier, which may be any of the numerous well known carriers, such as water, saline, phosphate buffered saline, dextrose, glycerol, ethanol, and the like, or combinations thereof. Preferably, the VLA4 binding agent, if an antibody or antibody derivative, will be administered at a dose ranging between about 0.1 mg/kg body weight/day and about 20 mg/kg body weight/day, preferably ranging between about 0.1 mg/kg body weight/day and about 10 mg/kg body weight/day and at intervals of every 1-14 days. For non-antibody or 30 antibody derivative binding agents, the dose range should preferably be between molar equivalent amounts to these amounts of antibody. Preferably, an antibody composition is administered in an amount effective to provide a plasma level of antibody of at least 1 gg/ml.

Optimization of dosages can be determined by 19 administration of the binding agents, followed by assessment of the coating of VLA4-positive cells by the agent over time after administered at a given dose in vivo. Peripheral blood mononuclear cells contained in a sample of the individual's peripheral blood should be probed for the presence of the agent in vitro (or ex vivo) using a second reagent to detect the administered agent. For example, this may be a fluorochrome labelled antibody specific for the administered agent which is then measured by standard FACS (fluorescence activated cell sorter) analysis. Alternatively, presence of the administered agent may be detected in vitro (or ex vivo) by the inability or decreased ability of the individual's cells to bind the same agent which has been itself labelled by a fluorochrome). The preferred dosage should produce detectable coating of the vast majority of VLA4positive cells. Preferably, coating is sustained in the case of a monoclonal antibody or monoclonal antibody derivative for a 1-14 day period.

In practicing this invention, treatment with VLA4 binding agents is preferrably continued for as long as the prediabetic subject maintains a stable Snormoglycemic plasma level and a stable prediabetic 25 state as reflected by a number of known markers as described above. In the Examples which follow, it has been found that anti-VLA4 mAb, R1-2 mAb, administration prevented diabetes onset during treatment and that the residual beneficial results of treatment were extended as long as two months following cessation of R1-2 treatment. To sustain the full protective effect of the VLA4 binding agent against diabetes onset, however, continuous treatment with the binding agents is preferred.

20 The method of the present invention comprises administering to a prediabetic individual a composition comprising an anti-VLA4 antibody. The examples below set forth the results observed in a rodent model of disease. These results demonstrate a protective effect of anti-VLA4 antibody in disease onset in the acute transfer model of the disease. The non-obese diabetic (NOD) mouse has become an important model of type I or insulin dependent diabetes mellitus since its introduction by Makino et al., 1980 and has been documented as a particularly relevant model for human diabetes (see, Castano and Eisenbarth Miller et al., 1988 Hutchings et al., 1990 [33] and references cited therein). That the diabetic syndromes displayed in the NOD mouse and human are similar has been shown by several lines of evidence. For example, in both the NOD mouse and human there is a strong genetic association of diabetes with loci of the major histocompatibility complex. In addition, for example, in both species, an autoimmune pathogenesis is evidenced by the presence of lymphocytic inflammation in the pancreatic islets insulitis) that appears to mediate the selective destruction of 3 cells, (ii) the presence of anti-islet cell antibodies, and (iii) the modulating effects of cyclosporin A.

Further evidence in the NOD mouse for an autoimmune etiology of diabetes disease is the ability to transfer diabetes with spleen cells (including purified splenic T cells) from diabetic donors, (ii) prevention of diabetes by in vivo treatment with antibodies specific for T cells, and (iii) failure of a thymic nude mice with NOD genetic background to develop S.moulitis or diabetes (see, Miller et al., 1988 Hutchings et al., 1990 [33] and references cited therein).

21 Although the precise events resulting in diabetes remain unclear, in the NOD mouse a progressive inflammatory response in the pancreas appears to be the initial histological lesion which begins as a periductal /perivascular mononuclear cell infiltrate at 3-4 weeks of age. At about 4-6 weeks of age, insulitis may be observed and beginning at about 12 weeks of age, overt diabetes consistent values of 1+ or higher using a Testape (Eli Lilly, Indianapolis, IN) assay for glycosuria or greater than 250 mg/dL if plasma glucose is monitored) occurs. To avoid variations in the immune status of the animals, the NOD mice are obtained from a specific pathogen-free colony and exhibit stable, high incidence of diabetes of about 80% of females and 20% of males which typically become diabetic by about 20 weeks of age. The preferred source for the NOD mice used in the experiments described herein is Taconic Farms (Germantown, NY). A large body of data, particularly from studies of the BB rat and NOD mouse has indicated that type I diabetes may be a T-cell mediated disease. Evidence to date suggests an important role for both major T cell subpopulations (CD4/L3T4 and CD8/Ly2) in the development of diabetes in man and in the NOD mouse.

25 The data supporting the essential role of T cells in diabetes do not exclude the possibility that T lymphocytes may recruit other cells macrophages) as the final effectors for P cell destruction.

Macrophages have been implicated in the disease process based on their presence in the infiltrated islet and the ability of chronic silica treatment to prevent disease (see, Hutchings et al., 1990 [33] and references cited therein).

Using the NOD strain of mice, investigators have 35 developed an acute transfer model of disease which se 22 parallels the spontaneous disease model in that transferred cells derived from diabetogenic NOD mice mediate the disease process, which is characterized by immune reactive cells that mediate insulitis and islet p cell-specific destruction. Moreover, in this model, certain monoclonal antibodies against T cells (see, Miller et al., 1988 and macrophages (see, Hutchings et al., 1990 [33] have been shown to abrogate disease onset. Such monoclonal antibodies have been used in the treatment of spontaneous disease and adoptively transferred disease, for example, anti- CD4 antibody has been shown to abrogate disease in both models (Miller et al, 1988 and Shizuru et al., 1988 Results of treatment with an agent in the adoptive transfer model or spontaneous disease model are indicative of the ability of the agent to modulate the human disease process.

S

i« o* 23 EXAMPLE 1 Effect of Anti-VLA4 Antibody Treataent on Adoptive Transfer of Diabetes For the adoptive transfer of diabetes experiments, NOD mice were obtained from Taconic Farms (Germantown, NY) or from the Joslin Diabetes Center (Boston, MA).

Spontaneously diabetic females of recent onset (13weeks of age) were used as spleen cell donors and 8 week old nondiabetic females served as recipients.

Spleen cells from 4 week old nondiabetic female donors which fail to transfer disease were used as a negative control.

Recipient mice were placed on acidified water (1:8400 dilution of concentrated HC1 in water) one week prior to sublethal irradiation (775 rad) performed in split doses (300 rad, 300 rad, and 175 rad) on each of three days (day and the day of transfer), in order to minimize any incidence of intestinal infection subsequent to high dose irradiation (Gamma Cell 1000 Cesium 137 source, Nordion International, Inc., Ontario, Canada). Spleens were harvested from diabetic donors or from nondiabetic controls, cell suspensions made and red cells lysed with Hemolytic Geys solution. Spleen cells were injected intravenously (2-3 x 107 in 0.2 ml 25 PBS) pretreated with either 75 gg R1-2 monoclonal *antibody (mAb), 75 gg rat IgG2b, or untreated. For the antibody treatment, cells were simply suspended at 1- 1.5 X 108 cells/ml with mAb at 375 Ag/ml and kept on ice until injection. The timing of injection was 30 within 3 hours after last irradiation. Some recipients received PBS alone. The anti-VLA4 mAb R1-2 and isotype-matched rat IgG2b were purchased from Pharmingen (La Jolla, CA). The R1-2 (rat anti-mouse) anti-VLA4 mAb was originally described by Holzmann et 24 al., 1989 The R1-2 anti-VLA4 mAb blocks VLA4 binding to its ligands (Hession et al., 1992 and therefore belongs by definition to the B group (Pulido et al., 1991 is equivalent to anti-human VLA4 mAbs of the B group HP1/2 or HP2/1).

The R1-2 mAb or rat IgG2b was administered at a dose of 75 gg/0.2 ml intraperitoneally every 2-3 days, a dosing regimen which was determined to maintain maximal coating of VLA4-positive cells in the peripheral blood, lymphoid organs and bone marrow as detected by staining of peripheral blood cells and single cell suspensions prepared from these organs with a fluorochrome labelled mAb specific for the R1-2 mAb and FACS analysis to measure fluorochrome positive cells (as described above). Injections were maintained through day 12 or day 24 post transfer. Mice were monitored for diabetes by testing for glycosuria with TesTape (Eli Lilly, Indianapolis, IN) and by plasma glucose levels (Glucometer, 3 Blood Glucose Meter, Miles, Inc., Elkhart, IN) and were considered diabetic after two consecutive urine positive tests [Testape values of or higher] or plasma glucose levels >250 mg/dL.

An inhibitory effect of the anti-VLA4 mAb on the 25 onset of diabetes was demonstrated when spleen cells isolated from NOD diabetic donors were treated with a saturating quantity of anti-VLA4 mAb R1-2 followed by transfer into nondiabetic irradiated hosts, as described above, and the R1-2 mAb was then administered 30 every other day for 12 days in order to maintain *maximal coating of all VLA4-positive cells in the peripheral blood and lymphoid organs for two weeks.

Figure 1 shows the frequency of recipients that became -diabetic and the day of disease onset for transfer of 2x10 7 splenocytes from diabetic NOD donor (i) 25 without treatment (closed circles); (ii) with rat IgG2b treatment (closed triangles), and (iii) with R1-2 anti- VLA4 treatment (closed diamonds) as well as for transfer of splenocytes from non-diabetic NOD donors (open squares). Injection of PBS alone gave 0% incidence. Under these conditions, only 1 of 8 individual R1-2 mAb treated recipients became diabetic, with onset on day 29 post transfer. By contrast, 6/10 and 5/9 individuals became diabetic after receiving splenocytes from diabetic donors treated with no mAb or with non-specific rat IgG2b, respectively. As shown in Figure 1, diabetes onset occurred as early as day 14 post transfer, though administration of the irrelevant rat IgG2b somewhat delayed onset.

These data demonstrate a protective effect of the R1-2 mAb which was dependent upon its specificity for VLA4. Recipients of splenocytes from nondiabetic mice or of PBS alone failed to become diabetic. Thus, treatment with anti-VLA4 antibody reduced the frequency of diabetes during 30 days post transfer.

Although the results shown in Figure 1 demonstrate that clinical diabetes occurred in only 1 of 8 anti- VLA4 treated animals, it was possible that the anti- VLA4 antibody caused only a minor delay in the onset of 25 disease. Plasma glucose levels were monitored in parallel with urine glucose in order to quantify any increase in blood sugar levels and thereby detect progression to clinical disease. In the anti-VLA4 antibody treated group shown in Figure 1, all mice were still normoglycemic on day 29 with an average plasma glucose value of 100 7 mg/dL, n=7, except for the single individual who scored as clinically diabetic by urine test and plasma glucose >500 mg/dL. Thus, disease progression was not apparent in any of the o e "35 other anti-VLA4 antibody treated recipients shown in o 26 Figure 1 on day 29 post transfer, a full 2 weeks beyond the last anti-VLA4 antibody injection. Analysis of sera from these mice confirmed that the anti-VLA4 mAb dropped to low or undetectable levels by day 18-21 post-transfer.

Additional cell transfers were performed in order to confirm that the anti-VLA4 mAb protected against transfer of diabetes. In these experiments, the anti- VLA4 antibody treatment was extended to day 25 post transfer but administered every 3.5 days thereby maintaining saturating levels of R1-2 mAb or rat IgG2b through day 26 when mice were sacrificed for pancreatic tissue. Under these conditions, an inhibitory effect of the anti-VLA4 mAb on the onset of diabetes was also demonstrated upon spleen cell transfer and R1-2 treatment. Figure 2 shows the frequency of recipients for each group) that became diabetic and the day of disease onset for transfer of 3x10 7 splenocytes from diabetic NOD donors without treatment (closed circles), (ii) with IgG2b treatment (closed triangles) and with R1-2 anti-VLA4 treatment (closed diamonds), as well as for transfer of splenocytes from nondiabetic NOD donors open squares). Injection of PBS alone gave 0% incidence. Figure 2 shows that only 25 1 out of 5 R1-2 mAb treated mice became diabetic by day 22 post transfer whereas diabetes was transferred in (o 4/4 recipients without Rl-2 mAb and 5/5 treated with rat IgG2b. Disease onset occurred as early as day 13 post transfer. These experiments, individually and collectively demonstrate that anti-VLA4 mAb reproducibly protects against development of diabetes in an acute transfer model of disease.

Further experiments were performed to determine whether the anti-VLA-4 mAb simply delayed disease onset 35 during the treatment period or if it could achieve a 27 longer-term protective effect. Figure 3 shows the onset of diabetes in mice over time after R1-2 injection (once every 3.5 days through day 25) with only 2/5 mice becoming diabetic on days 35 and 38 post transfer, 10-13 days after the last R1-2 injection. By contrast, diabetes occurred in the untreated and IgG2b treated groups as early as day 11 post transfer, with 100% incidence by days 18-21. Surprisingly, disease incidence in the R1-2 treated group did not further increase even as long as 2 months following the last R1-2 injection. Plasma glucose values monitored in parallel during this time reveal that these three individuals were consistently normoglycemic. After this point approximately 3 months posttransfer), even the negative control groups which received PBS alone or non-diabetic cells begin developing spontaneous disease. In summary, the VLA-4specific mAb reduces the incidence of diabetes transfer. Moreover, its protective effect against disease is sustained in the absence of further mAb treatment.

i.

(o* eo (4 o (4 44 .4 .e 4 4o 4 44* egO e• 28 EXAMPLE 2 Effect of Anti-VLA4 BAb on Pancreatis Insulitis For histological analysis, mice were sacrificed between 2-4 weeks post-transfer as described in this Example and pancreata harvested in 10% formalin buffered saline for paraffin-embedded sections which were stained with hematoxylin and eosin for histology. Degree of insulitis was scored as follows: Grade 0: no insulitis [islet devoid of inflammation]; Grade I: peri-insulitis [inflammatory mononuclear cells located peripheral to the islet]; Grade II: infiltrated of the islet interior contains lymphocytic inflammatory cells]; Grade III: 25-50% infiltrated [lymphocytic infiltration]; Grade IV: infiltrated. The percent of islets in each Grade was then calculated relative to the total number of islets examined. Histologic sections were examined and scored for the degree of insulitis following the adoptive transfer of NOD splenocytes with and without anti-VLA4 mAb treatment and the results tabulated. Specifically, the frequency of uninfiltrated islets (Grade 0-I infiltrate) and islets with Grade II-IV insulitis (as described above) were quantitated. For each experimental group, pancreatic sections from n= 25 mice were scored.

roes* Pancreatic tissue was recovered from recipients treated with the anti-VLA-4 mAb for various time periods in order to address its effect on the establishment of islet-specific cellular infiltrates.

30 Mice were treated with nonspecific rat IgG2b or R1-2 mAb every 3.5 days through day 14 when sacrificed.

Similarly, mice were treated through day 25 and sacrificed after diabetes was diagnosed or on day 26 S. post transfer. Mice continuously treated with the R1-2 mAb for 14 days post transfer maintain a high frequency 000 o 29 of uninfiltrated islets, with only 24% progressing to grade II-IV insulitis. By contrast those treated with nonspecific rat IgG2b show the reciprocal pattern, with 74% severe insulitis.

Likewise, in the mice treated with R1-2 though day diabetic, pancreata isolated from mice reported in Figure a high frequency of uninfiltrated islets were preserved, similar to that uninfiltrated) in nondiabetic recipients of young NOD splenocytes, as shown in Figure 4. By contrast, both the untreated or IgG2b-treated mice had only 28% uninfiltrated islets, and conversely had increased insulitis. Thus, the anti-VLA-4 mAb treatment appears to specifically inhibit or alternatively to delay the development of insulitis upon adoptive transfer of diabetogenic spleen cells.

In order to distinguish between these alternatives, the pattern of insulitis after 4 weeks post transfer was determined when mice were treated with rat IgG2b or Rl-2 mAb through day 12 and then maintained without further treatment. Mice were sacrificed upon diabetes diagnosis or on day 29 post transfer. Analysis of sera from these mice confirmed that circulating anti-VLA-4 mAb dropped to undetectable 25 levels by days 18-21 post transfer. With this protocol, the degree of insulitis in the Rl-2-treated group (69% insulitis, 25% diabetic) was similar to that in untreated recipients (73% insulitis, 60% diabetic) though still lower than that in the rat IgG2b-treated 30 mice (96% insulitis, 75% diabetic), as shown in Figure 5. Significantly, the severity of insulitis was similar between the R1-2 treated, untreated and rat IgG2b treated groups with an average of 57%, 47%, 64% Grade III/IV infiltrates, respectively. Even considering only the nondiabetic R1-2 treated o* 30 individuals, they still exhibited 59% insulitis with 52% Grade III/IV infiltrates. Recipients of nondiabetogenic NOD splenocytes had only 7% Grade III/IV infiltrates. Conversely, Figure 5 shows that the frequency of uninfiltrated islets was decreased in the R1-2 treated mice as compared to recipients of saline or nondiabetogenic spleen cells. Thus, the degree of insulitis progressed in these R1-2 treated mice (Figure 5) as compared to mice wherein R1-2 treatment was maintained (Figure 4) and approached that in the untreated and rat IgG2b treated control groups.

Taken together, these data indicate that anti-VLA-4 mAb administration can delay the progression of insulitis in an acute transfer model of disease.

o 31 EXAMPLE 3 Comparison of Different Anti-VLA4 Antibody Treatment on Adoptive Transfer of Diabetes This Example provides comparative efficacy results of PS/2, an anti-VLA4 antibody, with R1-2 using the adoptive transfer model and procedure described in Example 1. NOD mice were treated with an irrelevant control antibody (D/rat IgG2b, n 19 mice); R1-2 antibody (D/R1-2 mAb, n 24 mice); PS/2 mAb (D/PS/2 mAb, n 5 mice); or no treatment (NONE, n 26 mice). Spleen cells were injected intravenously (2-3x 107 in 0.2 ml PBS) and pretreated with either 75 gg R1-2 mAb, 75 gg PS/2 mAb, 75 Ag rat IgG2b, or untreated. Isolation and purification of PS/2 anti-VLA4 mAb was originally described by Miyake et al., 1991 The R1-2 mAb, PS/2 mAb or rat IgG2b was administered at a dose of 75 pg/0.2 ml intraperitoneally every 2-3 days, a dosing regimen which was determined to maintain maximal coating of VLA4-positive cells in the peripheral blood, lymphoid organs and bone marrow as detected by staining of peripheral blood cells and single cell suspensions prepared from these organs with a fluorochrome labelled mAb specific for the R1-2 and PS/2 mAb and FACS analysis to measure fluorochrome positive cells (as described above). Injections were maintained through days 22 to 25 post transfer. Mice were monitored for diabetes by testing for glycosuria with TesTape (Eli 30 Lilly, Indianapolis, IN) and by plasma glucose levels (Glucometer, 3 Blood Glucose Meter, Miles, Inc., Elkhart, IN) and were considered diabetic after two consecutive urine positive tests [Testape values of 1 or higher] or plasma glucose levels >250 mg/dL.

32 An inhibitory effect of the anti-VLA4 mAb on the onset of diabetes was demonstrated when spleen cells isolated from NOD diabetic donors were treated with a saturating quantity of anti-VLA4 mAb R1-2 or PS/2 followed by transfer into nondiabetic irradiated hosts, as described above, and the R1-2 mAb or PS/2 mAb was then administered every other day for 22-25 days in order to maintain maximal coating of all VLA4-positive cells in the peripheral blood and lymphoid organs for about two weeks. Table 1 shows the frequency of recipients that became diabetic and the day of disease onset for transfer of splenocytes from diabetic NOD donor without treatment (ii) with rat IgG2b treatment (D/nonspecific rat IgG2b); (iii) with R1-2 anti-VLA4 treatment (D/R1-2 mAb); (iv) with PS/2 treatment (D/PS/2 mAb) as well as for transfer of splenocytes from non-diabetic NOD donors (non-D). Nondiabetic mice receiving PBS and no splenocytes (NONE) were included as a control. Injection of PBS alone gave 4% incidence. Under these conditions, only 1 of 24 individual R1-2 mAb treated recipients became diabetic, with onset on day 22 post transfer while none of the five individual PS/2 mAb treated recipients became diabetic. By contrast, 16/19 individuals became 25 diabetic after receiving splenocytes from diabetic donors treated with no mAb or with non-specific rat IgG2b. As shown in Table 1, diabetes onset occurred as early as day 14 post transfer, though administration of the irrelevant rat IgG2b somewhat delayed onset by one 30 day.

These data demonstrate a protective effect of the R1-2 mAb and PS/2 which were dependent upon its specificity for VLA4. Recipients of splenocytes from nondiabetic mice or of PBS alone failed to become diabetic. Thus, treatment with anti-VLA4 antibody 33 reduced the frequency of diabetes during 30 days post transfer. Analysis of sera from these mice confirmed that levels of R1-2 and PS/2 anti-VLA4 mAb become undetectable between days 26 and 34 post-transfer.

TABLE I Anti-VLA-4 mAbs Inhibit Adoptive Transfer of Diabetes in NOD Mice Cells Transferred/Treatment* No. Diabetic/Total Recipients+ Day of Onset X t SEN NONE 1/26 34 Non-D 1/15 D 16/19 14 0.2 D/Nonspecific rat IgG2b 16/19 15 0.9 D/R1-2 mAb 1/24 22 D/PS/2 mAb 0/5 Spleen cells from 4 week old nondiabetic (NON-D) or from new onset diabetic NOD females were transferred, with D cells suspended in mAb or rat IgG 20 or without mAb before transfer and recipients treated twice weekly for 22-25 days. Mice were monitored for one month post transfer. Data are compiled from experiments.

"D/R1-2 and D/PS/2 mAb treated groups are 25 significantly different from D and D/rat IgG2b treated groups by Chi square test with Yates' correction as follows: R1-2 vs. IgG2b treated and D group, p<0.0001; PS/2 vs. IgG2b treated and D group, p<0.003.

*oo *o *o *o o•- 34 EXAMPLE 4 Effect of Anti-VLA4 Antibody Treatment on Spontaneous Diabetes Model This Example described efficacy results using R1-2 mAb in the spontaneous diabetes model which employs NOD mice. NOD mice were treated for 8 weeks with an irrelevant control antibody (NOD/rat IgG2b, n mice); R1-2 antibody (NOD/R1-2, n 20 mice); or no treatment (NOD, n 10 mice) starting at week four to week twelve of age. mAb was administered at a dose of 75 gg in 0.2 ml PBS iv, twice weekly. Mice were monitored for diabetic events by TesTape for glycosuria as previously described.

Figure 6 demonstrates a marked delay in diabetes onset (12-16 weeks delay) following R1-2 administration, as compared to the two control groups.

NOD mice which received irrelevant IgG2b mAb or no treatment developed diabetes as early as 13 weeks.

These spontaneous disease model results parallel the adoptive transfer results with R1-2 mAb illustrated in Figure 1 and directly demonstrate that an anti-ViA4 antibody protects against diabetes onset.

e *ooo oo 35 EXAMPW Effect of a VCAM-Ig Fusion Protein on Adoptive Transfer of Diabetes The adoptive transfer experiment described in Example 1 was repeated with a VCAM-Ig fusion protein (VCAM 2D-IgG) instead of an anti-VLA4 mAb. VCAM 2D-IgG is a soluble form of the ligand for VLA4 (VCAM1) which consists of the two N-terminal domains of VCAM1 fused to the human IgG1 heavy chain constant region sequences (Hinges,

C

2 and C.

3 The VCAM 2D-IgG DNA sequence and its translated amino acid sequence are shown in SEQ ID NO: 9. Figure 8 illustrates the fusion protein structure. The fusion protein was constructed by recombinant techniques as described below.

Isolation of cDNA of Human IgGI Heavy Chain Region and Construction of Plasmid pSAB144 In order to isolate a cDNA copy of the human IgGl heavy chain region, RNA was prepared from COS7 cells which has been transiently transfected by the plasmid VCAM1-IgG1 (also known as pSAB133). Construction of plasmid VCAM1-IgGI is described in PCT patent application WO 90/13300. The RNA was reverse transcribed to generate cDNA using reverse transcriptase and random hexamers as the primers.

25 After 30 min. at 420C, the reverse transcriptase reaction was terminated by incubation of the reaction at 95*C for 5 min. The cDNA was then amplified by PCR (Polymerase Chain Reaction, see, Sambrook et al., Molecular Cloning, Vol. 3, pp. 14.1-14.35 (Cold Spring Harbor; 1989)) using the following kinased primers: 370-31 (SEQ ID NO: 0* 5'TCGTC GAC AAA ACT CAC ACA TGC C Asp Lys Thr His Thr Cys which contains a SalI site, and 36 370-32 (SEQ ID NO: 11): GTAAATGAGT GCGGCGGCCG

CCAA,

which encodes the carboxy terminal lysine of the IgG1 heavy chain constant region, followed by a NotI site.

The PCR amplified cDNA was purified by agarose gel electrophoresis and glass bead elution for cloning in plasmid pNN03. Plasmid pNN03 was constructed by removing the synthetic polylinker sequence from the commercially available plasmid pUC8 (Pharmacia, Piscataway, New Jersey) by restriction endonuclease digestion and replacing the synthetic polylinker sequence with the following novel synthetic sequence (SEQ ID NO: 12): GCGGCCGCGG TCCAACCACC AATCTCAAAG CTTGGTACCC GGGAATTCAG ATCTGCAGCA TGCTCGAGCT CTAGATATCG ATTCCATGGA TCCTCACATC CCAATCCGCG GCCGC.

The purified PCR amplified cDNA fragment was ligated to pNN03 which had been cleaved with EcoRV, dephosphorylated, and purified by low melt agarose gel electrophoresis. The ligation reaction was used to transform E.coli JA221 and the resulting colonies were screened for a plasmid containing an insert of approximately 700 bp. The identity of the correct insert was confirmed by DNA sequence analysis, and the 25 plasmid was designated pSAB144.

Construction of Plasmid pSAB142 The plasmid pSAB142 was constructed as follows.

CDNA prepared from COS cells transfected with pSAB133 (as described in.the previous section) was subjected to 30 PCR amplification using obligonucleotides 370-01 and 370-29. Oligonucleotide 370-01 includes a NotI site and the nucleotides corresponding to amino acids 1 :through 7 of the VCAM-1 signal sequence 37 (SEQ ID NO: 13):

GAGCTCGAGGCGGCCGCACCATGCCTGGGAAGATGGTCGTG

MetProGlyLysMetValVal Oligonucleotide 370-29 corresponds to the VCAM-1 amino acids 214-219 and includes a Sail site (SEQ ID NO: 14): GTC GAC TTG CAA TTC TTT TAC The amplified DNA fragment was ligated to the vector fragment of pNN03, cleaved by EcoRV.

Construction of pSAB132 pJOD-S (Barsoum, DNA and Cell Biol., 9, pp.293-300 (1990)) was modified to insert a unique NotI site downstream from the adenovirus major late promoter so that NotI fragments could be inserted into the expression vector. pJOD-S was linearized by NotI cleavage of the plasmid DNA. The protruding 5' termini were blunt-ended using Mung Bean nuclease, and the linearized DNA fragment was purified by low melting temperature agarose gel electrophoresis. The DNA fragment was religated using T4 DNA ligase. The ligated molecules were then transformed into E.coli JA221. Colonies were screened for the absence of a NotI site. The resulting vector was designated pJOD-S delta Notl. pJOD-8 delta Notl was linearized using Sai and the 5' termini were dephosphorylated using 25 calf alkaline phosphatase. The linearized DNA fragment was purified by low melting temperature agarose gel eletrophoresis and ligated in the presence of phosphorylated oligonucleotide ACE175, which has the following sequence (SEQ ID 30 TCGACGCGGC

CGCG

The ligation mixture was transformed into E.coli JA221, and colonies were screened for the presence of a 38 plasmid having a HtI site. The desired plasmid was named pMDR901.

In order to delete the two SV40 enhancer repeats in the Sv40 promoter which controls transcription of the DHFR cDNA, pMDR901 and pJODae-tPA (Barsoum, DNA and Cell Biol., 9, pp. 293-300 (1990)), both were cleaved with AatII and DrEIII. The 2578 bp AatII-raIII fragment from pMDR901 and the 5424 bp AatII-aIII fragment from pJODAe-tPA were isolated by low melting temperature agarose gel electrophoresis and ligated together. Following transformation into E.coli JA221, the resulting plasmid, pMDR902, was isolated. pSAB132 was then formed by eliminating the EcoRI-NotI fragment of pMDR902 containing the adenovirus major late promoter and replacing it with an 839 bp EcoRI-NotI fragment from plasmid pCMV-B (Clontech, Palo Alto, California) containing the human cytomegalovirus immediate early promoter and enhancer.

Construction of pSAB146 pSAB144 was cleaved with Sall and NotI, and the 693 bp fragment isolated. pSAB142 was cleaved with NI and SalI and the 664 bp fragment was isolated.

The two fragments were ligated to pSAB132 which had been cleaved with NotI, and the 5' termini 25 dephosphorylated by calf alkaline phosphatase. The resulting plasmid, pSAB146, contained the DNA sequence encoding the VCAM-1 signal sequence, the amino terminal 219 amino acids of mature VCAM-1, ten amino acids of the hinge region of IgG1 and the C, 2 and C, 3 constant domains of IgG1.

4o 4 4.4.

39 Production of VCAM 2D-IG from a stably transformed CHO cell line A recombinant VCAM 2D-IgG expression vector was constructed as described below and transfected into CHO cells to produce a cell line continuously secreting VCAM 2D-IgG.

The 1.357 kb NotI fragment containing the VCAM 2D- IgG coding sequence of pSAB146 was purified by agarose gel electrophoresis. This fragment was ligated into the NotI cloning site of the expression vector pMDR901, which uses the adenovirus 2 major late promoter for heterologous gene expression and the selectable, amplifiable dihydrofolate reductase (dhfr) marker. The ligated DNA was used to transform E.coli DH5. Colonies containing the plasmid with the desired, correctly oriented insert were identified by the presence of 5853 and 3734 bp fragments upon digestion with Hind III; and 4301, 2555, 2293, and 438 bp fragments upon digestion with BalgI. The resultant recombinanat VCAM 2D-IgG expression vector was designated pEAG100. The identity of the correct insert was confirmed by DNA sequence analysis.

The recombinant expression plasmid pEAG100 was electroporated into dhfr-deficient CHO cells according 25 to the published protocol of J. Barsoum (DNA Cell Biol 2: 9: 293-300, 1990), with the following changes: 200 pg of PvuI-linearized pEAG100 plasmid and 200 pg of sonicated salmon sperm DNA were used in the electroporation protocol. In addition, cells were 30 selected in alpha-complete medium supplemented with 200 nM methotrexate.

To determine expression levels of secreted VCAM 2D-IgG, clones were transferred to a flat bottom 96 well microtiter plate, grown to confluency and assayed 35 by ELISA as described below.

e 40 Wells of Immulon 2 plates (Dynatech, Chantilly, Virginia) were each coated with anti-VCAM MAb 4B9 (isolated and purified on Protein A Sepharose as described by Carlos et al, 1990 with 1 00gl of anti-VCAM 4B9 MAb diluted to 10Mg/ml in 0.05 M sodium carbonate/bicarbonate buffer, pH 9.6, covered with Parafilm, and incubated overnight at 4'C. The next day, the plate contents were dumped out and blocked with 200 Ml/well of a block buffer fetal calf serum in ix PBS), which had been filtered through a 2A filter. The buffer was removed after a 1 hour incubation at room temperature and the plates were washed twice with a solution of 0.05% Tween-20 in IX PBS. Conditioned medium was added at various dilutions. As a positive control, an anti-mouse Ig was also included. Block buffer and LFA-3TIP constituted as negative controls. The samples and controls were incubated at room temperature for 2 hours.

The plates were then washed twice with a solution of 0.05% Tween-20 in IX PBS. Each well, except for the positive control well, was then filled with 501l of a 1:2000 dilution of HRP-Donkey anti-human IgG (H+L) (Jackson Immune Research Laboratories, Inc.; West Grove, Pennsylvania) in block buffer. The positive 25 control well was filled with 50 Al of a 1:2000 dilution of HRP-Goat anti-mouse lgG (H+L)(Jackson Immune Research Laboratories, Inc.; West Grove, Pennsylvania) in block buffer. The plates were then incubated for 1 hour at room temperature.

The HRP conjugated Ab solutions were removed, and the wells were washed twice with 0.05% Tween-20 in IX PBS. Then, 100 Al of HRP-substrate buffer was added to each well at room temperature. HRP-substrate buffer was prepared as follows: 0.5 ml of 42mM 35 tetramethylbenzidine (TMB), (ICN Immunobiologicals, 41 Lisle, South Carolina, Catalogue No. 980501) in DMSO (Aldrich) was slowly added to 50 ml of substrate buffer (0.1 M sodium acetate/citric acid, pH4.9); followed by addition of 7.5 Al of 30% hydrogen peroxide (Sigma, Catalogue No. H-1009).

The development of a blue color in each well was monitored at 650nm on a microtiter plate reader. After 7-10 minutes, the development was stopped by the addition of 100 Al of 2N Sulfuric acid. The resulting yellow color was read at 450nm on a microtiter plate reader. A negative control well was used to blank the machine.

Purification of VCAM 2D-IqG CHO cells expressing VCAM 2D-IgG were grown in roller bottles on collagen beads. Conditioned medium Liters) was concentrated to 500 ml using an Amicon S1Y10 spiral ultrafiltration cartridge (Amicon, Danvers, MA). The concentrate was diluted with 1 liter of Pierce Protein A binding buffer (Pierce, Rockford, IL) and gravity loaded onto a 10 ml Protein A column (Sepharose 4 Fast Flow, Pharmacia, Piscataway, NJ).

The column was washed 9 times with 10 ml of Protein A binding buffer and then 7 times with 10 ml of PBS.

VCAM 2D-IgG was eluted with twelve-5 ml steps 25 containing 25 mM H 3 P0 4 pH2.8, 100 mM NaCl. The eluted samples were neutralized by adding 0.5 M NaHP04 pH8.6 to 25 mM. Fractions were analyzed for absorbance at 280 nm and by SDS-PAGE. The three peaks fractions of highest purity were pooled, filtered, aliquoted and 30 stored at -70"C. By SDS-PAGE, the product was greater than 95% pure. The material contained less than 1 endotoxin unit per mg of protein. In some instances, it was necessary to further purify the Protein A eluate product on Q-Sepharose FF (Pharmacia). The protein A 4.

42 eluate was diluted with 3 volumes of 25 mM Tris HC1 pH and loaded onto a Q-Sepharose FF column at 10 mg VCAM 2D-IgG per ml of resin. The VCAM 2D-IgG was then eluted from the Q- Sepharose with PBS.

Evaluation of VCAM 2D-IqG Spleen cell suspensions were prepared from diabetic donors or from nondiabetic controls as described above. Spleen cells were injected intraveneously (2-3 x 107 in 0.2 ml PBS) and were pretreated with either 100 g VCAM 2D-IgG or 100g of irrelevant LFA-3Ig fusion protein control. Another group received PBS alone without cells transferred.

The fusion protein LFA-3Ig (LFA-3TIP) was isolated and purified as described in PCT US92/02050 and Miller et al., 1993 The VCAM 2D-IgG fusion protein or irrelevant LFA-3Ig protein was administered at a dose of 100 jg/0.2 ml intraperitoneally twice weekly through day 17. This concentration was sufficient to provide a serum level of fusion protein sufficient to saturate VLA4-positive cells, the serum levels determined by ELISA as described above. Diabetes onset was monitored as described above.

The results of the evaluation are shown in Figure 7. As shown in this Figure, VCAM 2D-IgG fusion protein 25 significantly inhibits the onset of diabetes in recipients of cells from diabetic donor mice (D/VCAM- Ig, open circles) with 60% incidence by day 30 posttransfer, as compared to the mice which received cells from diabetic donor (data not shown) and LFA-3Ig 30 irrelevant control Ig fusion protein (D/LFA-3 Ig) which had already achieved 60% incidence by day 15 posttransfer. Mice which received no cells (PBS only) did not develop disease. There were n 5 mice per experimental group.

43 In summary, VLA4 binding agents such as anti-VLA4 antibodies were protective against diabetes disease onset (Examples 1, 3 and 4) and were effective in delaying the progression of insulitis (Example 2) using a murine model for human diabetes. Other VLA4 binding agents such as soluble VCAM derivatives (VCAM 2D-IgG) were also useful in protecting against diabetes disease onset (Example The foregoing examples are intended as an illustration of the method of the present invention and are not presented as a limitation of the invention as claimed hereinafter. From the foregoing disclosure, numerous modifications and additional embodiments of the invention will be apparent to those experienced in this art. For example, actual dosage used, the type of antibody or antibody fragment used, mode of administration, exact composition, time and manner of administration of the treatment, and many other features all may be varied without departing from the description above. All such modifications and additional embodiments are within the contemplation of this application and within the scope of the appended claims.

o o* o 'S S (o oe ee** o o e°° 44 LIST OF REFERENCEB CITED Castano and Eisenbarth, 1990, Annu. Rev.

Immunology 8: 647-79, "Type I Diabetes: A Chronic Autoimmune Disease of Human, Mouse, and Rat" Fujita et al., 1982, Biomed. Res. 3: 429-436, "Lymphocytic Insulitis in a Non-obese Diabetic (NOD) Strain of Mice: An Immunohistochemical and Electron Microscope Investigation" Foulis et al., 1986, Diabetologia 29: 267, "The histopathology of the pancreas in Type I (insulin-dependent) diabetes mellitus: a 25-year review of deaths in patients under 20 years of age in the United Kingdom" Eisenbarth, 1986, New Engl. J. Med.

314:1360-1368, "Type I Diabetes Mellitus A Chronic Autoimmune Disease" Miller et al., 1988, J. Immunol. 140: 52-58, "Both the Lyt-2+ and L3T4+ T Cell subsets are required for the transfer of diabetes in Nonobese diabetic mice" Harada and Makino, 1986, Exp. Anim. 501, "Suppression of overt diabetes in NOD mice by Anti-thymocyte serum or anti-Thy 1.2 antibody" Koike et al., 1987, Diabetes 36: 539, "Preventive effect of monoclonal anti- 30 L3T4 antibody on development of diabetes in NOD mice" Makino et al., 1986, Exp. Anim. 35: 495, "Absence of insulitis and overt diabetes in athymic nude mice with NOD genetic 35 background" Voorbij et al., 1989, Diabetes 35: 1623- 1629, "Dendritic cells and scavenger macrophages in pancreatic islets of prediabetic BB rats" [10] Nomikos et al., 1986, Diabetes 11302-1304, "Combined treatment with nicotinamide and desferrioxamine 45 prevents islet allograft destruction in NOD mice" [11] Larson and Springer, 1990, Immunol. Rev.

114: 181-217, "Structure and Function of Leukocyte Integrins" (12] Hemler et al., 1990, Immunol. Rev. 114: 45-66, "Structure of the Integrin VLA-4 and its Cell-Cell and Cell-matrix adhesion functions" [13] Lobb, RJR., 1992, Adhesion: Its Role in Inflammatory Diseases. ed. J.M. Harlan and D.Y. Liu, New York: W. H. Freeman.

1-18.

[14] Osborn, 1990, Cell 62: 3-6, "Leukocyte Adhesion to Endothelium in Inflammation" Wayner et al., 1989, J. Cell. Biol. 109: 1321-1330, "Identification and Characterization of the T Lymphocyte Adhesion Receptor for an Alternative Cell Attachment Domain (CS-1) in Plasma Fibronectin" [16] Shimizu et al., 1991, J. Cell Biol. 113: 1203, "Four molecular pathways to T cell adhesion to endothelial cells: roles of LFA-1 VCAM-1 and ELAM-1 and changes in pathway hierarchy under different activation conditions" *17] Barton et al., 1989, J. Immunol. 143: 1278, "The effect of anti-intercellular adhesion molecule-1 on phorbolesterinduced rabbit lung inflammation" [18] Issekutz, T.B. and Issekutz, 1991, -Clinical Immunol. and Immunopathol. 138: 300-312, "T lymphocyte migration to arthritis joints and dermal inflammation -in the rat: differing migration patterns and the involvement of VLA-4" [19] Issekutz, 1991, J. Immunol 147: 4178-4184, "Inhibition of In Vivo Lymphocyte Migration to Inflammation and Homing to Lymphoid Tissues by the TA-2 Lee*by 46 [21] [22] [23] [24] Monoclonal Antibody A Likely Role for VLA-4 In Vivo" Yednock, et al., 1992, Nature 356: 63- 66, "Prevention of experimental autoimmune encephalomyelitis by antibodies against a4#l integrin" Lobb, U.S. Patent Application Serial No.

07/821,768 filed January 13, 1992, "Treatment for Asthma" Dustin et al., 1986, J. Immunol. 137: 245-254 "Induction by IL-1 and Interferon-y Tissue Distribution, Biochemistry, and Function of a Natural Adherence Molecule (ICAM-1)" Rice et al., 1990, J. Exp. Med. 171: 1369, "Inducible Cell Adhesion Molecule 110 (INCAM-110) Is An Endothelial Receptor for Lymphocytes A CD11/CD18- Independent Adhesion Mechanism" Rice et al., 1991, Am. J. Path. 138: 385393, "Vascular and Nonvascular Expression of INCAM-110" Shimuzu et al., 1990, Immunol. Rev. 114: 109-144, "Roles of Adhesion Molecules in T-cell recognition: Fundamental Similarities between four integrins on resting human T cells (LFA-1, VLA-4, VLA-5, VLA-6) in expression, binding and costimulation" Burkly et al., 1991, Eur. J. Immunol.

21: 2871-2875, "Signaling by vascular cell adhesion molecule-1 (VCAM-1) through VLA4 promotes CD3-dependent

T

cell proliferation" Rudd et al., 1989, Immunol. Rev. 111: 225-266, "Molecular Interactions, T-Cell Subsets, and a Role of the CD4/CD8:p56' ck Complex in Human T-Cell Activation" Moingeon et al., 1989, Immunol. Rev.

11: 111-144, "The Structural Biology of CD2" 30 [26] 35 [27] 9* [28] *9 47 (29] Harding et al., 1992, Nature 156: 607- 609, "CD28-mediated signalling costimulates murine T cells and prevents induction of energy in T cell clones" [30] Shizuru et al., 1988, Science 240: 659- 662, "Immunotherapy of the Nonobese Diabetic Mouse: Treatment with an Antibody to T-Helper Lymphocytes" [31] Barlow and Like, 1992, Amer. J. Pathol.

li1: 1043-1051, "Anti-CD2 Monoclonal Antibodies Prevent Spontaneous and Adoptive Transfer of Diabetes in the BB/Wor Rat" [32] Like et al., 1986, J. Exp. Med. 164: 1145-1159, "Prevention of Diabetes in Biobreeding/Worchester Rats with Monoclonal Antibodies that Recognize T Lymphocytes or Natural Killer Cells" [33] Hutchings et al., 1990, Nature 348: 639- 642, "Transfer of diabetes in mice prevented by blockade of adhesionpromoting receptor on macrophages" [34] Federlin and Becker, 1990, Klin.

Wochenschr. 68: Suppl. XXI 38-43, "Specific Therapeutic Attempts in Experimental and Clinical Type-I Diabetes" [35] Zielasek et al., 1989, Clin. Immunol.

Immunopathol. 52: 347-365, "The 30 Potentially Simple Mathematics of Type I Diabetes" [36] Eisenbarth, 1987, Hosp. Prac. 22:167- 183, "Type I Diabetes: Clinical Implication of Autoimmunity" 35 [37] Ziegler and Eisenbarth, 1990, Horm. Res.

23: 144-150, "Multiple Target Antigens in Pre-Type I Diabetes: Implications for Prediction" [38] Ziegler et al., 1990, Diabetes Care 13: 762-765, "Predicting Type I Diabetes" [39] Ziegler et al., 1990, J. Autoimmun. 3 Suppl. i: 69-74, "Type I Diabetes: *see 48 polygenic inheritance, multiple autoantigens and 'dual' parameter prediction" Kohler, G. and Milstein, 1975, C. Nature 265: 295-497, "Continuous Cultures of Fused Cells Secreting Antibody of Predefined Specificity" [41] Sanchez-Madrid et al., 1986, Eur. J.

Immunol., 16: 1343-1349, "VLA-3: A novel polypeptide association within the VLA molecular complex: cell distribution and biochemical characterization" [42) Hemler et al., 1987, J. Biol. Chem. 262: 11478-11485, "Characterization of the cell surface heterodimer VLA4 and related peptides" [43] Elices et al., 1990, Cell 60: 577-584, "VCAM-1 on Activated Endothelium Interacts with the Leukocyte Integrin VLA4 at a Site Distinct from the VLA4/Fibronectin Binding Site" [44] Pulido et al., 1991, J. Biol. Chem., 266(16): 10241-10245, "Functional Evidence for Three Distinct and Independently Inhibitable Adhesion Activities Mediated by the Human Integrin VLA-4" [45] Boerner et al., 1991, J. Immunol.

30 147:86-95, "Production of Antigenspecific Human Monoclonal Antibodies from In Vitro-Primed Human Splenocytes" [46] Persson et al., 1991, Proc. Natl. Acad.

Sci. USA 88: 2432-2436, "Generation of 35 diverse high-affinity human monoclonal antibodies by repertoire cloning" [47] Huang and Stollar, 1991, J. Immunol.

Methods 141: 227-236, "Construction of representative immunoglobulin variable 40 region cDNA libraries from human peripheral blood lymphocytes without in vitro stimulation" [48] Jones et al., 1986, Nature 321: 522-525, "Replacing the complementarity- I 49.

determining regions in a human antibodv with those from a mouse"~ [49] ]Rlechrnann. 1988. Nature 32'-3, t.

"Reshaping human antibodies for theraDv" Queen et al., 1989. Proc. Nat'l. Acad.

Sci. USA L8:10029. "A humanized antibody that binds to the interleukin 2 receptor"* [51]Orlandi et al.. 1989. Proc. Natl. A4,cad.

Sci. USA M:3 833 "Cloning imiunoglobulin variable domains for expression by the polvmerase chain reaction" [52] International Patent Aoplication No.

WO 9416094, published on July 2 1. '1994, "Recombinant Anti- VLA4 Antibody Molecules" [53] Holznann et al. 1989. Cell 56: 37-46.

"Identification of a Murmie Peyer's Patch-Specific Lymphocyte Homing Receptor as an Intea-1i Molecule with a Chain Homologous to Human VLA-4a" [541 .Hession et al., 1992. Biochem. Biophys.

Res. Commun. LU1: 163-169, "Cloning- of Murine and Rat Vascular Cell Adhesion Molecule- I et al.. 199 1. J. Exp). Med. 173: 599-607.

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50 The foregoing documents are incorporated herein by reference in their entirety.

The entire disclosure in the comlete specification of our Australian Patent Application No. 62379/94 is by this crossreference incorporated into the present specification.

S

51 SEQUENCE LISTING GENERAL INFORMATION:

APPLICANT

NAME: Biogen, Inc.

STREET: Fourteen Cambridge Center CITY: Cambridge STATE: Massachusetts COUNTRY: USA POSTAL CODE (ZIP): 02142 TELEPHONE: 617-252-9200 TELEFAX: 617-252-9595 (ii) TITLE OF INVENTION: Treatment for Insulin Dependent Diabetes (iii) NUMBER OF SEQUENCES: (iv) COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: IBM PC compatible OPERATING SYSTEM: PC-DOS/MS-DOS SOFTWARE: PatentIn Release #1.0, CURRENT APPLICATION DATA: APPLICATION NUMBER: PCT/US94/01456 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 360 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear Version #1.25 (EPO) 3

**S

a.

(ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1 OTHER INFORMATION: /note= "pBAG159 insert: HP1/2 heavy chain variable region; amino acid 1 is Glu (E) but Gin may be substituted" (ix) FEATURE: NAME/KEY: CDS LOCATION: 1-360 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: 52 GTC AAA GTG GAG GAG TGT GGG GCA GAG CTT GTG AAG CGA GGG GCC Val Lys Leu Gin Gin Ser Gly Ala Giu Leu Val Lys Pro Gly Ala TGA GTG AAG TTG Ser Val Lys Leu ACC TAT ATG GAG Thr Tyr Met His TGG ATT GGA AGG Trp Ile Gly Arg GGG AAG TTG GAG Pro Lys Phe Gin AAG AGA GGG TGG Asn Thr Ala Trp CCC GTC TAG TAG Ala Val Tyr Tyr

TGG

Ser 21

TGG

Trp 36

ATT

Ile 51

GTG

Val 66

GTG

Leu 81

TOT

Gys 96 TGG ACA GGT TGT Gys Thr Ala Ser GTG AAG GAG AGG Val Lys Gin Arg GAT GGT GGG AGT Asp Pro Ala Ser AAG GGG AGT ATT Lys Ala Thr Ile GAG GTG AGG AGG Gin Leu Ser Ser GGA GAG GGA ATO Ala Asp Gly Met

GGG

G ly 26

GCT

Pro 41

GGG

G ly 56

AGA

Thr 71

CTG

Leu 86

TGG

Trp 101

AG

Thr 116 TTG AAG ATT AAA Phe Asn Ile Lys GAA GAG GGG GTG Glu Gin Gly Leu GAT AGT AAA TAT Asp Thr Lys Tyr GGG GAG AGG TGG Ala Asp Thr Ser ACA TGT GAG GAG Thr Ser Glu Asp GTA TGA AGG GGA Val Ser Thr Gly GTG AGG GTG TGG Val Thr Val Ser GAG Asp 31 GAG 135 Giu 46 GAG 180 Asp 61 TGG 225 Ser 76 AGT 270 Thr 91 TAT 315 Tyr 106 TGA 360 S er 121 GGT GTG GAG TTG TOG GGG GAA GGG AGG Ala Leu Asp Phe Trp Gly Gin.Gly Thr INFORMATION FOR SEQ ID NO:2: SEQUENGE CHARAGTERISTICS: LENGTH: 120 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESGRIPTION': SEQ ID NO:2: Val. Lys Leu Gin Gin Ser Gly Ala Glu Leu Val Lys Pro Gly Ala 2 6 11 16 Ser Val Lys Leu Ser Gys Thr Ala Ser Gly Phe Asn 21 26 Ile Lys Asp Thr Tyr Met His Trp Val Lys Gin Arg Pro Glu Gin Gly Leu Glu 36 41 .46 53 Trp Ile Gly Arg Ile 51 Asp Pro Ala Ser Pro Lys Phe Gin Val Lys Ala Thr lie 66 Asn Thr Ala Trp Leu Gin Leu Ser Ser 81 Gly 56 Thr 71 Leu 86 Trp 101 Thr 116 Asp Thr Lys Tyr Ala Asp Thr Ser Thr Ser Glu Asp Val Ser Thr Gly Val Thr Val Ser Ser 76 Thr 91 Ala Val Tyr Tyr Ala Leu Asp Phe Cys 96 Trp 111 Ala Asp Gly Met Gly Gin Gly Thr INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 318 base pairs TYPE: nucleic acid STRANDEDNESS: double TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: CDS LOCATION: 1-318 OTHER INFORMATION: /product= "HP1/2 variable region" light chain 9 9 *9 .9 9* (ix) FEATURE: NAME/KEY: miscfeature LOCATION: 1 OTHER INFORMATION: /note= "pBAG172 insert: HPI/2 light chain variable region" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: ATT GTG ATG ACC CAG ACT CCC AAA TTC CTG CTT GTT TCA GCA Ile Val Met Thr Gin Thr Pro Lys Phe Leu Leu Val Ser Ala 5 10

AGT

Ser 1 GGA GAC AGG GTT ACC ATA ACC TGC AAG GCC AGT CAG AGT GTG Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Ser Val

ACT

Thr AAT GAT GTA GCT Asn Asp Val Ala

TGG

Trp TAC CAA CAG AAG CCA GGG CAG TCT CCT AAA 135 Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys 54 CTG CTG ATA TAT TAT GCA TCC AAT CGC TAC ACT GGA GTC CCT GAT 180 Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 55 CGC TTC ACT GGC AGT GGA TAT GGG ACG GAT TTC ACT TTC ACC ATC 225 Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile 70 AGC ACT GTG CAG GCT GAA GAC CTG GCA GTT TAT TTC TGT CAG CAG 270 Ser Thr Val Gin Ala Glu Asp Leu Ala Val Tyr Phe Cys Gin Gin 85 GAT TAT AGC TCT CCG TAC ACG TTC GGA GGG GGG ACC AAG CTG GAG 315 Asp Tyr Ser Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 100 105 ATC 318 Ile INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 106 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Ser Il e Val Met Thr Gin Thr Pro Lys Phe Leu Leu Val Ser Ala 1 5 10 Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser Gin Ser Val Thr 20 25 Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly Gin Ser Pro Lys 40 Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr Gly Val Pro Asp 55 Arg Phe Thr Gly Ser Gly Tyr Gly Thr Asp Phe Thr Phe Thr Ile 70 Ser Thr Val Gin Ala Glu Asp Leu Ala Val Tyr Phe Cys Gin Gin 80 85 Asp Tyr Ser Ser Pro Tyr Thr Phe Gly Gly Gly Thr Lys Leu Glu 100 105 Ile 55 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 429 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

sig_peptide 1-57 mat_peptide 58-429 (ix) FEATURE: NAME/KEY: CDS LOCATION: 1-429 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1 OTHER INFORMATION: /note- "pBAG195 insert: AS heavy chain variable region"

C

C.

C,

a a (xi) SEQUENCE DESCRIPTION: SEQ ID ATG GAC TGG ACC Met Asp Trp Thr

TGG

Trp -15 AGG GTC TTC TGC Arg Val Phe Cys

TTG

Leu -10 CTG GCT GTA GCA Leu Ala Val Ala

CCA

Pro GGT GCC CAC TCC CAG GTC CAA CTG CAG GAG AGC GGT CCA Gly Ala His Ser Gin Val Gin Leu Gln Glu Ser Gly Pro GGT CTT Gly Leu GTG AGA CCT AGC CAG ACC CTG AGC Val Arg Pro Ser Gln Thr Leu Ser TTC AAC ATT Phe Asn Ile

AAA

Lys 30 GAC ACC TAT ATG Asp Thr Tyr Met

CTG

Leu

CAC

His 35

AGG

Arg 50 ACC TGC ACC GCG Thr Cys Thr Ala TGG GTG AGA CAG Trp Val Arg Gln ATT GAT CCT GCG Ile Asp Pro Ala TCT GGC 135 Ser Gly CCA CCT 180 Pro Pro ACT GGC 225 Ser Gly GGA CGA GGT CTT GAG TGG ATT GGA Gly Arg Gly Leu Glu Trp Ile Gly 56 TrLvs T\v'r ac'Cc C-AC CCC- ;AGC 77C CAG I.S' Pro vs GC-z *CCAA-C -a.G TT AGC Ser As C G S er r .a V\a.

-V

G-TO ACG-A GTC-AC AT- T Va 1.a. '71r Me::Le Leu Pr=- Leu Ser Ser val T CG 7 C AC G Cys Al a iSD) G I Me--Tr 100 ACC

:!CG

C-TA T G-A TA CCT CTC- C-AC TT C Ser Thr Tyr A!a Lau: Asp Phe GTC GC TCC T -a G G c- AG TC C Val TrVal Ser Ser Gly G-lu Ser 120 TGG C-CC CA-A GCGG 4 Trp G-1-v Gin Gl-v -hr (S

S

55.* S. S S

S.

INFORMATION FOR SEQ ID NO: S: SEQUE~NCE' ClLARACTERISTICS: kA; _LENGTH: 143 amino acids TYPE: amino acid TOPOLOGY: 1 -inea-r (M !O.ECU3E TYE-: protein __QUENCE DE-SCRITI'rON: SEQ 7D NO:6: Met As-,Tr Thr Tr:o A-rc Val. Phe C-vs Leau Leu C-lv Ala His Ser C-in Val Ci- Let: G-n Clu Sear Val Ar=o Pro Ser G-n Thr~' Lau Ser Leu Th r Cv s 20 P e Asn1 li Ls ASp- Th r Tyr Met Hi s Tmr Val 30 3:z GlY Ar:C- Leu G1,, Tr- Ile Arc Ie As-o Ala Val. Ala C-nv Pro C-'Leu Thr Al1a Sear GC-> c C-In Pro Pr Pro Al-a Ser GIv Asp Thr Lvs T-vr AsD Pro 7 vs Phe C-in Val. Arc Val 7Thr Mer: LT:a 57 Thr Ala Ala Asp Thr Ala Val Tyr Tyr Cys Ala Asp Gly Met Trp 95 100 Val Ser Thr Gly Tyr Ala Leu Asp Phe Trp 105 110 Gly Gin Gly Thr Thr Val Thr Val Ser 120 Ser Gly Glu Ser INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 386 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOCATION:

(ix) FEATURE:

NAME/KEY:

LOCATION:

sig_peptide 1-57 mat_peptide 58-386 9

C.

a. a (ix) FEATURE: NAME/KEY: CDS LOCATION: 1-386 (ix) FEATURE: NAME/KEY: misc feature LOCATION: 1 OTHER INFORMATION: /note- "pBAG198 insert: VK2 (SVMDY) light chain variable region" (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: ATG GGT TGG TCC TGC ATC ATC CTG TTC CTG GTT GCT ACC GCT ACC Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr -10 GGT GTC CAC TCC AGC ATC GTG ATG ACC CAG AGC CCA AGC AGC CTG Gly Val His Ser Ser Ile Val Met Thr Gin Ser Pro Ser Ser Leu AGC GCC AGC GTG GGT Ser Ala Ser Val Gly GAC AGA GTG ACC ATC Asp Arg Val Thr .Ile 20 ACC TGT AAG Thr Cys Lys GCC ACT 135 Ala Ser 58 CAG AGT GTG ACT AAT GAT GTA GCT TGG TAC CAG GAG AAG CCA GGT 180 Gin Ser Val Thr Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly 35 AAG GCT CCA AAG CTG CTG ATC TAC TAT GCA TCC AAT CGC TAC ACT 225 Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr 50 GGT GTG CCA GAT AGA TTC AGC GGT AGC GGT TAT GGT ACC GAC TTC 270 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Tyr Gly Thr Asp Phe 65 ACC TTC ACC ATC AGC AGC CTC CAG CCA GAG GAC ATC GCC ACC TAC 315 Thr Phe Thr Ile Ser Ser Leu Gin Pro Glu Asp Ile Ala Thr Tyr 80 TAC TGC CAG CAG GAT TAT AGC TCT CCG TAC ACG TTC GGC CAA GGG 360 Tyr Cys Gin Gin Asp Tyr Ser Ser Pro Tyr Thr Phe Gly Gin Gly 95 100 ACC AAG GTG GAA ATC AAA CGT AAG TG 386 Thr Lys Val Glu Ile Lys Arg Lys 105 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 128 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein i: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Met Gly Trp Ser Cys Ile Ile Leu Phe Leu Val Ala Thr Ala Thr -15 -10 Gly Val His Ser Ser Ile Val Met Thr Gin Ser Pro Ser Ser Leu 1 5 Ser Ala Ser Val Gly Asp Arg Val Thr Ile Thr Cys Lys Ala Ser 15 20 Gin Ser Val Thr Asn Asp Val Ala Trp Tyr Gin Gin Lys Pro Gly .i 30 35 Lys Ala Pro Lys Leu Leu Ile Tyr Tyr Ala Ser Asn Arg Tyr Thr 50 59 Gly Val Pro Asp Arg Phe Ser Gly Ser Gly Tyr Gly Thr Asp Phe 65 Thr Phe Thr Ile Ser Ser Leu Gin Pro Glu Asp Iie Ala Thr Tyr 80 Tyr Cys Gin Gin Asp Tyr Ser Ser Pro Tyr Thr Phe Gly Gin Gly 95 100 Thr Lys Val Glu Ile Lys Arg Lys 105 INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 1348 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE: NAME/KEY: VCAM-1 gene segment LOCATION: 1-219 OTHER INFORMATION: This portion of the sequence corresponds, in part, to Exons I, II and III .nucleotide sequence of the VCAM-1 gene of Cybulsky et al. Proc. Nat'1. Acad. Sci. USA 88:7861 (1991).

(ix) FEATURE: NAME/KEY: Hinge region LOCATION: 220-229 OTHER INFORMATION: This portion of the sequence corresponds, in part, to Fig. 12A in PCT/US92/ 0250 and represents the hinge region of Human IgGI heavy chain constant region.

(ix) FEATURE: NAME/KEY: Heavy chain constant region 2 LOCATION: 230-338 OTHER INFORMATION: This portion of the sequence corresponds, in part, to Fig. 12A in PCT/US92/ 02050 and represents the heavy chain constant region 2 of Human IgGl heavy chain constant region.

(ix) FEATURE: NAME/KEY: heavy chain constant region 3 LOCATION: 339-446 60 OTHER INFORMATION: This portion of the sequence corresponds, in part, to Fig. 12A in PCT/US92/ 02050 and represents the heavy chain constant region 3 of Human IgGl heavy chain constant region.

ATG CCT GGG AAG ATG GTC GTG ATC CTT Met Pro Gly Lys Met Val Val Ile Leu GGA GCC Gly Ala 10 TCA AAT ATA CTT Ser Asn Ile Leu ATC GAG ACC ACC ile Glu Thr Thr TGG ATA ATG TTT Trp Ile Met Phe CCA GAA TCT AGA Pro Glu Ser Arg ACT TGC AGC ACC Thr Cys Ser Thr ACC CAG ATA GAT Thr Gin Ile Asp ACC ACA TCT ACG Thr Thr Ser Thr CAC TCT TAC CTG His Ser Tyr Leu AAA GGA ATC CAG Lys Gly Ile Gin

GCA

Ala

TAT

Tyr

ACA

Thr

AGT

Ser

CTG

Leu

TGC

Cys

GTG

Val 110 GCT TCT CAA GCT Ala Ser Gin Ala CTT GCT CAG ATT Leu Ala Gin Ile GGC TGT GAG TCC Gly Cys Glu Ser CCA CTG AAT GGG Pro Leu Asn Gly ACA ATG AAT CCT Thr Met Asn Pro ACA GCA ACT TGT Thr Ala Thr Cys GAG ATC TAC TCT Glu Ile Tyr Ser CCT CTG GAG GCT Pro Leu Glu Ala TTT AAA Phe Lys 25

GGT

Gly 40

CCA

Pro 55

AAG

Lys 70

GTT

Val 85

GAA

Glu 100

TTT

Phe 115

GGG

Gly 130 GAC TCC GTC TCA Asp Ser Val Ser TTT TTC TCT TGG Phe Phe Ser Trp GTG ACG AAT GAG Val Thr Asn Glu AGT TTT GGG AAC Ser Phe Gly Asn TCT AGG AAA TTG Ser Arg Lys Leu CCT AAG GAT CCA Pro Lys Asp Pro AAG CCG ATC ACA Lys Pro Ile Thr

J

J

TTG 135 Leu AGA 180 Arg GGG 225 Glv GAA 270 Glu GAA 315 Glu 105 GAG. 360 Glu 120 GTC 405 Val 135

I..

i: ATT CAT Ile His TTG AGT GGC Leu Ser Gly 125 AAG TGT TCA GTT Lys Cys Ser Val GAC TTA CTG AAA Asp Leu Leu Lys GCT GAT Ala Asp 140 GTA TAC CCA Val Tyr Pro TTT GAC AGG CTG GAG ATA Phe Asp Arg Leu Glu Ile 145 150 450 j..a

GGA

Gly 155 GAT CAT CTC ATG AAG AGT CAG GAA TTT CTG 495- Asp His Leu Met Lys Ser Gin Glu Phe Leu 160 165 61 GAG GAT GGA GAG Glu Asp Ala Asp

AGG

Arg 170 AAG TCC GTG GAA Lys Ser Leu Glu

ACC

Thr 175

GGA

Gly 190 AAO AGT TTG GAA Lys Ser Leu Giu AAA GTT OTT GTT Lys Val Leu Val

OTA

Val1 180

TG

Gys 195 AGO TTT AGT GOT GTG ATT GAO OAT ATT Thr Phe Thr Pro Val Ile Oiu Asp Ile 185 OGA GOT AAA TTA Arg Ala Lys Leu AGO GAG GOT OTA Arg 01n Ala Val OGA 000 TG OGA Pro Pro Gys Pro GTC TTO 000 GGA Leu Phe Pro Pro

GOT

Pro GAO OTO ACA Olu Val Thr

GAO

His 200

AAA

Lys 215

GA

Ala 230

AAA

Lys 245

TG

Gys 260

AAG

Asn 275 000 Pro 290

OTO

Leu 305

TG

Gy s 320

ATO

Ile 335 ATT OAT GAA ATO OAT Ile Asp.Olu Met Asp 205 GAA TTO CAA OTO GAG Giu Leu Gin Val Asp 220 COT GAA OTO OTO 000 Pro Giu Leu Leu Gly 235 000 AAO GAG AGO OTO Pro Lys Asp Thr Leu 250 OTO OTO OTO GAO OTO Vai Val Val Asp Val 265 TOO TACGOTO GACGG00 Trp Tyr Val Asp Gly 280 000 GAO GAO CG TAO Arg 01u Oiu Gin Tyr 295 AGO OTO OTO GAG CG Thr Vai Leu His Gin 310 AAO OTO TOG AAO AAA Lys Val Ser Asn Lys 325 TOO AAA 000 AAA 000 Ser Lys Ala Lys Oly 340 TOT OTO 000 ACA Ser Val Pro Thr AAA ACT GAG ACA Lys Thr His Thr OGA 000 TGA OTO Oly Pro Ser Val ATO ATO TOG 000 Met Ile Ser Arg AG GAG GAA GAG Ser His Oiu Asp OTO GAG OTO OAT Val 01u Val His AAO AGO AG TAG Asn Ser Thr Tyr GAG TOG OTO AAT Asp Trp Leu Asn 000 OTO OGA 000 Ala Leu Pro Ala CG 000 OGA GAA Gin Pro Arg Oiu GTA 630 Val 210 TOO 675 Gys 225 TTG 720 Ph e 240 AGO 765 Thr 255 GOT 810 Pro 270 AAT 855 Asn 285 000 900 Arg 300 000 945 Gly 315 000 990 Pro 330 OGA 1035 Pro 345 I. 55

S

*5 r* *5 5* (S S S

S

(S GAO OTO AAG TTG Glu.Val Lys Phe CO AAG ACA AAG Ala Lys Thr Lys GTG OTO AGO OTO Val Val Ser Val AAG GAG TAO AAO Lys Glu Tyr Lys ATO GAG AAA AGO Ile Giu Lys Thr 62 GAG GTG TAG ACC Gin Val Tyr Thr GAG GTG AGG GTG Gin Val Ser Leu ATC GGG GTG GAG Ile Ala Val Glu AAG AGG AGG GGT Lys Thr Thr Pro TAG AGG AAG GTG Tyr Ser Lys Leu GTG TTG TGA TGG Val Phe Ser Gys AGG GAG AAG AGG Thr Gin Lys Ser

GTG

Leu 350

AGG

Thr 365

TGG

Trp 380 GGG GGA TGG CGG Pro Pro Ser Arg TGG GTG GTC AAA Gys Leu Vai Lys GAG AGG AAT GGG Giu Ser Asn Giy GTG CTG GAG TGG Vai Leu Asp Ser

GAT

Asp 355 GAG GTG AGC Glu Leu Thr AAG AAG 1080 Lys Asn 360 TTG TAT GGG AGG Phe Tyr Pro Ser GGG GAG AAG AAG Pro Glu Asn Asn GAG 1125 Asp 375 TAG 1170 Tyr 390 GG TGG TTG Gly Ser Phe TTG GTG 1215 Phe Leu 405

AGG

Th r 410

TGG

Ser 425

GTG

Leu 440 GTG GAG AAG AGG Val Asp Lys Ser GTG ATG GAT GAG Val Met His Glu TGG GTG TGT CG Ser Leu Ser Pro

AGG

Arg 415

GGT

Ala 430 TGG GAG GAG GGG Trp Gin Gin Gly GTG GAG AAG GAG Leu His Asn His 1260 TAG 1305 Tyr 435 1348 GGT AAA Gly Lys 445 TGA GTG CGG 1* 1* (S I.

S.

(0 ~S

S

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[55 INFORM4ATION FOR SEQ ID NO:l0: SEQUENGE GHARAGTERISTIGS: LENGTH: 24 base pairs TYPE: nucleic' acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLEGULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOGATION:

OTHER INFORMATION: Primer 370-31.

This corresponds to Kinase 00,.

6..

(xi) SEQUENGE DESGRIPTION: SEQ ID NO:iO: TGGTG GAG Asp 1 AAA AGT GAG AGA TGG G Lys Thr His Thr Gys 63 INFORMATION FOR SEQ ID NO:11: SEQUENCE CHARACTERISTICS: LENGTH: 24 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOCATION:

OTHER INFORMATION: This corresponds to Kinase Primer 370-32.

(xi) SEQUENCE DESCRIPTION: SEQ ID NO:11: GTAAATGAGT GCGGCGGCCG CCAA 24 INFORMATION FOR SEQ ID NO:12: SEQUENCE CHARACTERISTICS: LENGTH: 115 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOCATION:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:12: GCGGCCGCGG TCCAACCACC AATCTCAAAG CTTGGTACCC GGGAATTCAG ATCTGCAGCA TGCTCGAGCT CTAGATATCG ATTCCATGGA TCCTCACATC 100 CCAATCCGCG GCCGC 115 INFORMATION FOR SEQ ID NO:13: SEQUENCE CHARACTERISTICS: LENGTH: 39 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear 64 (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOCATION:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:13: GA GCT CGA GGC GGC CGC ACC ATG CCT GGG AAG ATG GTC GTG 41 Met Pro Gly Lys Met Val Val 1 INFORMATION FOR SEQ ID NO:14: SEQUENCE CHARACTERISTICS: LENGTH: 23 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA (ix) FEATURE:

NAME/KEY:

LOCATION:

OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID NO:14: AA GTC GAC TTG CAA TTC TTT TAC 23 INFORMATION FOR SEQ ID t. 9 SEQUENCE CHARACTERISTICS: LENGTH: 14 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA 65 (ix) FEATURE:

NAME/KEY:

LOCATION:

CD) OTHER INFORMATION: (xi) SEQUENCE DESCRIPTION: SEQ ID TCCACGCGCC CCCC 14 5S* 4.

'5

S*

5. *5 S 55.* 5* 4 4* 5555 'S S 55 55 4; 5.55 5*

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55 S5 (5 .555

Claims (12)

1. A method for treating insulin dependent type I diabetes, comprising administering to a mammal having partial 0 cell destruction, one or more compositions comprising an antibody, a recombinant antibody, a humanised antibody, or an antigen binding fragment of such antibodies, a polypeptide or a small molecule, capable of binding to the a4 subunit of VLA-4, or combination of any of the foregoing, is an amount effective to treat diabetes.

2. The method according to claim 1, wherein the composition comprises an antibody or an antigen binding fragment thereof capable of binding to the a4 subunit of VLA-4.

3. The method according to claim 1 or claim 2, wherein the antibody is selected from the group consisting of HP1/2, HP2/1, HP2/4, L25, and P4C2, or a fragment of 20 such antibodies. 99* 9

4. The method according to any one of claims 1 to 3, wherein the antibody is a recombinant antibody, or a fragment thereof.

5. The method according to any one of claims 1 2 to 3, wherein the antibody is a humanised antibody, or a fragment thereof. 30 6. The method according to claim 1, wherein the composition comprises a soluble VCAM-1 or fibronectin polypeptide.

7. The method according to claim 6, wherein the soluble VCAM-1 polypeptide is a VCAM-IgG fusion protein. ST ~c 8. The method according to claim 6 or claim 7, \\melb_files\homeS\cintae\Keep\speci\69846.98.doc 13/09/00 0 67 wherein the soluble VCAM-1 polypeptide is a fusion protein comprising two N-terminal domains of VCAM-1 fused to the human IgGl heavy chain constant region.

9. The method according to claim 6, wherein the fibronectin polypeptide comprises the amino acid sequence EILDV. A method according to any one of claims 1 to 5, wherein the composition is administered at a dosage so as to provide from 0.1 to 10 mg/kg, based on the weight of the mammal.

11. A method according to claim 1, wherein the composition is administered at a dosage so as to provide from 0.1 to 10 mg/kg, based on the weight of the mammal.

12. A method according to any one of claims 1 to 5, wherein the composition is administered in an amount 20 effective to coat VLA4-positive cells in the peripheral blood for a period of 1-14 days. B

13. A method according to any one of claims 1 to wherein the composition is administered in an amount 25 effective to provide a plasma level of said antibody in the mammal of at least 1 gg/ml. S: 14. A method according to any one of claims 1 to 5, wherein the composition is administered prior to the 30 development of overt diabetes, as measured by a serum glucose level of less than about 250 mg/dL. A method according to any one of claims 1 to wherein the mammal is a human.

16. Use of one or more compositions comprising S an antibody, a recombinant antibody, a humanised antibody, \\melbfi les\home$\cintae\Keep\speci\69846 98.doc 13/09/00 68 or an antigen binding fragment of such antibodies, a polypeptide or a small molecule, capable of binding to the a4 subunit of VLA-4, or combination of any of the foregoing, for the manufacture of a medicament for treating insulin dependent type I diabetes in a mammal having partial p cell destruction.

17. A method according to claim 1, substantially as herein described with reference to the Examples. Dated this 14th day of September 2000 BIOGEN, INC. By their Patent Attorneys GRIFFITH HACK Fellows Institute of Patent and Trade Mark Attorneys of Australia *go *oo. S* o o* S \\melb files\homeS\cintae\Keep\speci\69846.98.doc 13/09/00