CA2482042A1 - Methods for treating an autoimmune disease using a soluble ctla4 molecule and a dmard or nsaid - Google Patents

Abstract

The present invention relates to compositions and methods for treating immun e system diseases such as rheumatic disease, by administering to a subject soluble CTLA4 molecules that block endogenous B7 molecules from binding thei r ligands, alone, or in conjunction with other agents including Disease Modifying Anti-Rheumatic Drugs (DMARDs).

Description

METHODS FOR TREATING AN AUTOIMMUNE DISEASE USING A SOLUBLE

Throughout this application various publications are referenced. The disclosures of these publications in their entireties are hereby incorporated by reference into this application in order to more fully describe the state of the art to which this invention pextains.
FIELD OF THE INVENTION
The present invention relates generally to the field of immwle system diseases, e.g., rheumatic diseases. In particular, the invention relates to methods and compositions for treating immune system diseases, e.g., rheumatic diseases, such as ,rheumatoid arthritis, by administering to a subject an effective amount of soluble CTLA4 molecules alone, or in conjunction with a Disease Modifying Anti-Rheumatic Drug (DMARD).
BACKGROUND OF THE INVENTION
No cure currently exists for rheumatic diseases. Rather, therapeutic agents are used to treat the symptoms. Typically, the therapeutic agents are administered over long periods of time and the therapeutic value is often dimiilished by adverse side effects.
Rheumatic diseases encompass a group of diseases that affect the musculo-skeletal and connective tissues of the body. These diseases are characterized by chronic inflammation that often leads to permanent tissue damage, deformity, atrophy and disability.

Rheumatic diseases affect the joints, bone, soft tissue, or spinal cord (Mathies, H. 1983 Rlaeu:3aa) and are classified as inflam~ilatory rheumatism, degenerative rheumatism, e:~tra-ax-ticular rheumatism, or collagen diseases. Some rheumatic diseases are known to be autoilnmune diseases caused by a subject's altered immune response.
Rheumatoid arthritis is a progressive rheumatic disease, affecting approximately 2% of the adult population of developed countries (Utsinger, P. D., et al., 1985 Rheumatoid Ar~th~°itis, p. 140). This disease is characterized by persistent inflammatory synovitis that causes destruction of cartilage and bone erosion, leading to structural deformities in the peripheral joints. The symptoms associated with rheumatoid arthritis include joint swelling, joint tenderness, inflammation, morning stiffness, and pain, especially upon flexing. Subj ects having advanced stages of arthritis suffer from structural damage, including joint destruction with bone erosion (in: "Principals of Internal Medicine, Harrison, 13th edition, pages 1648-1655). In addition, patients can present other clinical symptoms of various organic lesions, including lesions of the skin, l~idney, heart, lung, central nervous system, and eyes due to vasculitis related to the autoimmune process.
Other symptoms that correlate with rheumatoid arthritis include elevated erythrocyte sedimentation rates, and elevated levels of serum C-reactive protein (CRP) andlor soluble IL-2 receptor (IL-2r). The erythrocyte sedimentation rate is increased in nearly all patients with active rheumatoid arthritis. The level of serum C-reactive protein is also elevated and correlates with disease activity and the likelihood of progressive joint damage. Additionally, the level of soluble IL-2r, a product of activated T-cells, is elevated in blood senun and synovial fluid of patients with active rheumatoid arthritis (see: "Principals of hiternal Medicine, Harrison, 13th edition, page 1650).
Rheumatoid arthritis is believed to be a T-cell-mediated autoimmm~.e disease, involving antigen-nonspecific intercellular interactions between T-lymphocytes and antigen-presenting cells. In general, the magnitude of the T-cell response is determined by the co-stimulatory response elicited by the interaction between T-cell surface molecules and their ligands (Mueller, et al., 1989 Ann. Rev. Imtnianol. 7:445-480). Key co-stimulatory signals are provided by the interaction between T-cell surface receptors, CD28 and CTLA4, and their ligaiids, such as B7-related molecules CD80 (i.e., B7-1) and (i.e., B7-2), on antigen presenting cells (Linsley, P. and Ledbetter, J. 1993 Afafy. Rev.
hrafhuya~l. 11:191-212).
T-cell activation in the absence of co-stimulation results in anergic T-cell response (Schwartz, R. H., 1992 G'ell 71:1065-1068) wherein the immune system becomes nonresponsive to stimulation.
I0 Since rheumatoid arthritis is thought to be a T-cell-mediated immune system disease, one strategy to develop new agents to treat rheumatoid arthritis is to identify molecules that block co-stimulatory signals between T-lymphocytes and antigen presenting cells, by blocking the interaction between endogenous CD28 or CTLA4 and B7. Potential molecules include soluble CTLA4 molecules that are modified (i.e. CTLA4 mutant molecules) to bind to B7 with higher avidity than wildtype CTLA4 (the sequence of which is shown in Figure 23) or CD28, thereby blocking the co-stimulatory signals.
Soluble forms of CD28 and CTLA4 have been constructed by fusing variable (V)-like extracellular domains of CD28 and CTLA4 to immunoglobulin (Ig) constant domains resulting in CD28Ig and CTLA4Ig. A nucleotide and amino acid sequence of CTLA4Ig is shown in Figure 24 with the protein beginning with methionine at position +1 or alanine at position -1 and ending with lysine at position +357. CTLA4Ig binds both CD80-positive and CD86-positive cells more strongly than CD28Ig (Linsley, P., et al., 1994 I~fnurzity 1:793-80). Many T-cell-dependent immune responses have been found to be blocked by CTLA4Ig both aya vitf'O afzd irz vivo. (Linsley, P., et al., 1991b, supra;
Linsley, P., et al., 1992a Scie~zce 257:792-795; Linsley, P., et al., I992b ,I. Exp. Mecl.
176:1595-1604; Lenschow, D.J., et al. 1992 Science 257:789-792; Tan, P., et al., 1992 J.
Exp. Med. 177:165-173; Turka, L.A., 1992 Proc. Natl. Acad. Sci. USA 89:11102-11105).
To alter binding affinity to natural ligands, such as B7, soluble CTLA4Ig fusion molecules were modified by mutation of amino acids in the CTLA4 portion of the molecules. Regions of CTLA4 that, when mutated, alter the binding affinity or avidity for B7 ligands include the co~nplementatzty determining region 1 (CDR-1 as descizbed i11 U.S. Patents 6,090,914, 5,773,253, 5,844,095; in copending U.S. Patent Application Serial Number 60/214,065; and by Peach et al, 1994. J. Exp. Med., 180:2049-2058) and complementarity determining region 3 (CDR-3)-like regions (CDR-3 is the conserved region of the CTLA4 extracellular domain as described in U.S. Patents 6,090,914, 5,773,253 and 5,844,095; in copending U.S. Patent Application Serial Number 60/214,065;
and by Peach, R.J., et al J Exp Mecl 1994 180:2049-2058; the CDR-3-like region encompasses the CDR-3 region and extends; by several amino acids, upstream and/or downstream of the CDR-3 motifj. The CDR-3-like region includes a hexapeptide motif MYPPPY
(SEQ
ID NO.: 20) that is highly conserved in all CD28 and CTLA4 family members.
Alanine scanning mutagenesis through the hexapeptide motif in CTLA4, and at selected residues in CD28Ig, reduced or abolished binding to CD80 (Peach, R.J., et al J Exp Med 180:2049-2058; U.S. Patent No. 5,434,131; U.S. Patent No. 6,090,914; U.S.
Patent No.
5,773,253.
Further modifications were made to soluble CTLA4Ig molecules by interchanging homologous regions of CTLA4 and CD28. These chimeric CTLA4/CD28 homologue mutant molecules identified the MYPPPY hexapeptide motif common to CTLA4 and CD28, as well as certain non-conserved amino acid residues in the CDR-1- and like regions of CTLA4, as regions responsible for increasing the binding avidity of CTLA4 with CD80 (Peach, R. J., et al., 1994 JExp Mecl 180:2049-2058).
Soluble CTLA4 molecules, such as CTLA4Ig, CTLA4 mutant molecules or chimeric CTLA4/CD28 homologue mutants as described szcp~cc, introduce a new group of therapeutic dings io treat rheumatic diseases.
Present treatments for rheumatic diseases, such as rheumatoid arthritis, include administering nonspecific cytotoxic immunosuppressive drugs known as Disease Modifying Anti-Rheumatic Drugs (D1VIARDs), such as methotrexate, il~.fliximab, cyclophosphamide, azathioprine, cyclosporin A, sulfasalazine, hydroxychloroquine, ~4 leflunomide, etanercept, and tumor necrosis factor-alpha (TNFa) or other cytokine bloclcers or antagonists. These immunosuppressive drugs suppress the entire immune system of the subject, and long-term use increases the risk of infection and oncogenesis.
Moreover, these drugs merely slow down the progress of the rheumatoid arthritis, which resumes at an accelerated pace after the therapy is discontinued.
Additionally, prolonged therapy with these nonspecific drugs produces toxic side effects, including a tendency towards development of certain malignancies, kidney failure, bone marrow suppression, pulmonary fibrosis, malignancy, diabetes, and Iiver function disorders. These drugs may also gradually cease being effective after about 2-5 yeaxs (Kelley's Textbook of Rheumatology, 6th Edition, pages 1001-1022). Newer, biologically based, DMARDs such as cytokine blockers may be more potent and may have longer lasting effects than older DMARDS such as hydrochloroquine, however, the long term safety of these newer drugs is still unknown. Reports of multiple sclerosis and lupus exist with the use of TNF
bloclcers.
Alternatively, therapeutic agents that are non-specific immunosuppressive and anti-inflammatory drugs have been used to obtain symptomatic relief. These drugs are dose-~dependent and do not protect from disease progression. These drugs include Non-Steroidal Anti-Inflammatory Drugs (NSAIDS) as well as steroid compounds (e.g., corticosteroids or glucocorticoids), such as prednisone arid methylprednisolone. Steroids also have significant toxic side effects associated with their long-term use.
(Kelley's Textbook of Rheumatology, 6th Edition, pages 829-833).
Thus, current treatments for rheumatoid arthritis are of limited efficacy, involve significant toxic side effects, and cannot be used continuously for prolonged periods of time.
Accordingly, there exists a need for treatments that are effective and more potent for treating rheumatic diseases, such as rheumatoid arthritis, and avoids the disadvantages of old conventional methods and agents, by targeting a pathophysiological mechanism of auto-inununity.
S

SUMMARY OF THE IhTVEi~TION
The present invention provides compositions and methods for treating immune system diseases, by administering to a subject soluble CTLA4 molecules, which bind to molecules on B7-positive cells, thereby inhibiting endogenous B7 molecules from binding CTLA4 and/or CD28 on T-cells. Soluble CTLA4 molecules used in the methods of the invention include CTLA4Ig and soluble C T LA4 mutant molecule L104EA29YIg.
Alternatively, the present invention provides compositions and methods for treating immune system diseases, by administering to a subject a combination of a DMARD
and a molecule that blocks B7 interaction with CTLA4 andlor CD28.
The present invention also provides methods for inhibiting T-cell function, but not causing T-cell depletion, in a human by contacting B7-positive cells in the human with soluble CTLA4. Examples of soluble CTLA4 include CTLA4Ig and soluble CTLA4 mutant molecules, such as L104EA29YIg.
The present invention also provides methods for treating (e.g. reducing symptoms of) rheumatic diseases, such as rheumatoid arthritis, by administering to a subject suffering from symptoms of arthritis, soluble CTLA4 molecules such as CTLA4Ig and/or soluble CTLA4 mutant molecule L104EA29YIg and/or a mix of any soluble CTLA molecule.
The CTLA4 mutant molecule L104EA29YIg e.g. beginning with methionine at position +1 or alanine at position -1 and ending with lysine at position +357, as shown in Figure 19, is preferred for use in the methods of the invention.
The present invention also provides methods for treating (e.g. reducing symptoms of) rheumatic diseases, such as rheumatoid arthritis, by administering to the subj ect a combination of 1) a DMARD, such as methotrexate or a molecule that bloclcs TNF
interactions, and 2) soluble CTLA4 molecules, such as CTLA4Ig.

The present invention also provides methods for reducing pathophysiological changes associated with ar~ immune system disease (e.g., rheiunatic disease), such as structural damage, by administering to the subject diagnosed with the immune system disease (e.g., rheumatoid arthritis), soluble CTLA4 molecules alone or in conjunction with other therapeutic drugs, such as a DMARD.
The present invention also provides a pharmaceutical composition for treating immune system diseases, such as rheumatic diseases, comprising a pharmaceutically acceptable carrier and a biologically effective agent, such as soluble CTLA4 molecules, alone or in conjunction with other therapeutic drugs, such as a DMARD, a NSAm, a corticosteroid and/or a glucocorticoid. .
Kits comprising pharmaceutical compositions therapeutic for immune system disease are also encompassed by the invention. In one embodiment, a kit comprising one or more of the pharmaceutical compositions of the invention is used to treat an immune system disease, e.g. rheumatoid arthritis. For example, the pharmaceutical composition comprises an effective amount of soluble CTLA4 molecules that bind to B7 molecules on B7-positive cells, thereby blocking the B7 molecules from binding CTLA4 and/or CD2~
on T-cells. Further, the kit may contain one or more immunosuppressive agents used in conjunction with the pharmaceutical compositions of the invention. Potential irnmunosuppressive agents include, but are not limited to, corticosteroids, nonsteroidal antiinflammatory drugs (e.g. Cox-2 inhibitors), prednisone, cyclosporine, cyclosporin A, azathioprine, methotrexate, TNFcc blockers or antagonists, hydroxychloroquine, sulphasalazopyrine (sulfasalazine), gold salts, infliximab, etanercept, anakinra and any biological agent targeting an inflammatory cytokine.
The present invention also provides methods for reducing the erythrocyte sedimentation rate that is associated with rheumatoid arthritis.

Additionally, the present invention provides methods for reducing the levels of certain components of blood 58111111 whlch are associated with rheumatoid arthritis, including C-reactive protein, IL-6, TNF-a, soluble ICAM-1, soluble E-selectin and/or soluble IL-2r.
BRIEF DESCRIPTION OF THE FIGURES
Figure 1A: Demographic data of patient cohorts. Demographic data including gender, race, and disease duration as described in Example 3, ifafra.
Figure 1B: Demographic data of patient cohorts. Demographic data including gender, age, weight, and disease activity, evaluated by the patient and by the physician, as described in Example 3, infiAa.
Figure 1C: Demographic data of patient cohorts as described in Example 3, infra.
Demographic data including disease activity, erythrocyte sedimentation rate (ESR), physical function (disability evaluated by health questionnaire), and C-reactive protein (CRP).
Figure 1D: Demographic data of patient cohorts as described in Example 3, infra.
Demographic data including joint swelling, joint tenderness, morning stiffness, and pain.
Figure 1E: Demographic data of patient cohorts as described in Example 3, ifzfYa.
Demographic data including prior treatments.
Figure 2: Summary of'discontinuations at day 85 by reason as described in Example 3, i:afi°a.
Figure 3A: ACR responses at Day 85 as described in Example 3, ifafr~a: ACR-20, -50, and -70 responses.

Figure 3B: ACR-20 responses at Day 85, including placebo response, as described in Example 3, it2f°a: ACR-'~0 response with 95% confidence limits.
Figure 3C: ACR-20 responses at Day 85 as described in Example 3, infra:
Difference in ACR-20 response with respect to 95% confidence intervals.
Figure 4A: Basic (20% improvement} clinical responses in swollen and tender joint count in percentage of patients at Day 85 as described in Example 3, ih~~a:
basic clinical response, ACR-20.
Figure 4B: Clinical responses (in percentage improvement) in swollen and tender joint count in percentage of patients at Day 85 as described in Example 3, infra:
change in clinical response in percentage improvement.
Figure 5A: Pain response (by Likert scale by mean unit change from baseline) in percentage of patients at Day 85 as described in Example 3, infra: pain score changes from baseline.
Figure 5B: Patient global disease changes (by Liken scale by mean unit change from baseline) in percentage of patients at Day 85 as described in Example 3, infra: patient global disease activity changes.
Figure 5C: Physician global disease changes (by Likert scale by mean unit change from baseline) in percentage of patients at Day 85 as described in Example 3, infra: physician global disease activity changes.
Figure 5D: Pain (by Likert scale by mean unit change from baseline) in percentage of patients at Day 85 as described in Example 3, infra: pain changes from baseline.
Figure 6A: Patient global assessment of disease activity change from baseline by range of 2 nits at Day 85 as described in Example 3, inf ~a; disease activity improvement.

Figure o'B: Physician global assessment of disease activity change from baseline by rmge of 2 units at Day 85 as described in Example 3, ifafra; disease activity improvement.
Figure 7A: Percent reduction in C-reactive protein (CRP) levels at Day 85 as described in Example 3, infra: percentage reduction in CRP levels from baseline.
Figure 7B: Difference in reduction in C-reactive protein (CRP) levels at Day 85 as described in Example 3, infra: percent reduction difference in CRP levels with 95%
confidence intervals.
Figure 7C: Mean reduction in C-reactive protein (CRP) levels at Day 85 as described in Example 3, inf'~a: mean change from baseline.
Figure 8: Reduction in soluble IL-2 receptor levels mean change from baseline at Day 85 as described in Example 3, ihfra.
Figure 9A: The effect of CTLA4Ig on tender joints over time as described in Example 3, infra: median difference from baseline.
Figure 9B: The effect of CTLA4Ig on tender joints over time as described in Example 3, ihfra: mean difference from baseline.
Figure 10A: The effect of CTLA4Ig on swollen joints over time as described in Example 3, inf3°a: median difference from baseline.
Figure lOB: The effect of CTLA4Ig on swollen joints over time as described in Example 3, ifafra: mean difference from baseline.
Figure 11: The effect of CTLA~.Ig on pain assessment mean difference from baseline over time as described in Example 3, infra.

Figure 12A: The effect of CTLA4Ig on patient assessment of disease activity mean difference from baseline over time as described in Example 3, irafr~a.
Figure 12B: The effect of CTLA4Ig on physician assessment of disease activity mean difference from baseline over time as described in Example 3, ifzfi°a.
Figure 13A: The effect of L104EA29YIg on tender joints over time as described in Example 3, infra: median difference from baseline.
Figure 13B: The effect of L104EA29YIg on tender joints over time as described in Example 3, infra: mean change from baseline.
Figure 14A: The effect of L104EA29YIg on swollen joints over time as described in Example 3, ihfr°a: median difference from baseline.
Figure 14B: The effect of L104EA29YIg on swollen joints over tune as described in Example 3, i~fi~a: mean change from baseline.
Figure 15: The effect of L104EA29YIg on pain assessment over time as described in Example 3, ihf~a: mean change from baseline over time.
Figure 16A: The effect of L104EA29YIg on patient assessment of disease activity mean difference from baseline over time as described in Example 3, infra.
Figure 16B: The effect of L104EA29YIg on physician assessment of disease activity mean difference from baseline over time as described in Example 3, irafi~a.
Figure 17: Percent improvement in patient disability assessed by Health Assessment Questionnaire (HAQ) compared to the baseline at Day 85 with CTLA4Ig and L104EA29YIg treatment as described in Example 3, if f-a.

Figure 18: Nucleotide and amino acid sequence of L104EIg (SEQ ID NOs: 6-7) as described irl Example 1, infra.
Figure 19: Nucleotide and amino acid sequence of L104EA29YIg (SEQ ~ NOs: 8-9) as described in Example 1, infra.
Figure 20: Nucleotide and amino acid sequence of L104EA29LIg (SEQ ID NOs: 10-11) as described in Example l, infra.
Figure 21: Nucleotide and amino acid sequence of L104EA29TIg (SEQ ID NOs: 12-13) as described in Example 1, infra.
Figure 22: Nucleotide and amino acid sequence of L104EA29WIg (SEQ 117 NOs: 14-15) as described in Example 1, infra.
Figure 23: Nucleotide and amino acid sequence of CTLA4 receptor (SEQ ID NOs:

17).
Figure 24: Nucleotide and amino acid sequence of CTLA4Ig (SEQ ID NOs: 18-19).
Figure 25: SDS gel (FIG. 25A) for CTLA4Ig (lane 1), L104EIg (lane 2), and L104EA29YIg (lane 3A); and size exclusion chromatographs of CTLA4Ig (FIG. 25B) and L104EA29YIg (FIG. 25C).
Figures 26 (left and right depictions): A ribbon diagram of the CTLA4 extracellular Ig V-like fold generated from the solution structure determined by NMR
spectroscopy.
FIG. 26 (right depiction) shows an expanded view of the CDR-1 (S25-R33) region and tile MYPPPY region indicating the location and side-chain orientation of the avidity enhancing mutations, L104 and A29.

Figures 27A & 278: FAGS assays showing binding of L104EA29YIg, L104EIg, and CTLA4ig to human CD80- or CD86-transfected CHG cells as described in Example 2, inf'a.
S Figures 28A & 28B: Graphs showing inhibition of proliferation of CD80-positive and CD86-positive CHO cells as described in Example 2, infra.
Figures 29A & 298: Graphs shoving that L104EA29YIg is more effective than CTLA4Ig at inhibiting proliferation of primary and secondary allostimulated T
cells as described in Example 2, infra.
Figures 30A-C: Graphs illustrating that L104EA29YIg is more effective than CTLA4Ig at inhibiting IL-2 (FIG. 30A), IL-4 (FIG. 30B), and gamma (y)-interferon (FIG.
30C) cytokine production of allostimulated human T cells as described in Example 2, infra.

Figure 31: A graph demonstrating that L104EA29YIg is more effective than CTLA4Ig at inhibiting proliferation of phytohemaglutinin- (PHA) stimulated monkey T cells as described in Example 2, ihfi~a.
Figure 32: A graph showing the equilibrium binding analysis of L104EA29YIg, L104EIg, and wild-type CTLA4Ig to CD86Ig.
Figixres 33A & B: Reduction in soluble ICAM-l and soluble E-selectin levels mean change from baseline at Day 8S as described in Example 3, ihfi~a.

Figure 34: A graph showing the smrlrnary of ACR20 response by visit day in response to methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, as described in Example S, iraf~a.
Figure 3S: A graph showing the summary of ACRSO response by visit day in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, as described in Example S, ihfi~a.

Figure 36: A graph showing the summary of ACR70 response by visit day in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, as described in Example 5, inf'a.
Figure 37: A graph showing the mean ACR-N over time in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy, as described in Example 5, ihf~°a.
Figure 38: A bar graph showing the ACR response in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy on day 180 with a 95%
confidence interval, as described in Example 5, iyaf~a.
Figure 39: A bar graph showing the proportion of New Active Joints in response to methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) therapy on day 180, as described in Example 5, infi°a.
Figure 40: A bar graph showing ACR response after therapy with methotrexate alone or rnethotrexate and CTLA4Ig (2 and 10 mg/kg) on day 180, as described in Example 5, itafi°a.
Figure 41: A graph showing percent improvement in tender joints after therapy with rnethotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example S, iHfra.
Figure 42: A graph showing percent improvement in swollen joints after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 rng/lcg) - mean percent improvement from baseline, as described in Example 5, irafi~a.

Figure 43: A graph showing percent improvement in pain after therapy with methotxexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, ir~a.
Figure 44: A graph showing percent improvement in regard to disease activity as reported by the subj ect after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, in~a.
Figure 45: A graph showing percent improvement in regard to disease activity as reported by the physician after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, i~fi~a.
Figure 46: A graph showing percent improvement regarding physical function after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) -mean percent improvement from baseline. as measured by HA.Q, as described in Example 5, i~ft°a.
Figure 47: A graph showing percent improvement in CRP levels function after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - mean percent improvement from baseline, as described in Example 5, ifafi-a.
Figure 48: A graph showing percent improvement in CRP levels function after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg) - median percent improvement from baseline, as described in Example 5, itift°a.
Figure 49: A graph showing the difference in ACR response rate on day 180 in two groups after therapy with CTLA4Ig (2 and 10 mg/kg) in comparison to a group treated with methotrexate (1VITY) only (95°f° Confidence Limits), as described in Example 5, it f'a.

Figure 50: A graph showing the change from baseline for SF-36 Physical health Component on day 180, in two groups after therapy with CTLA4Ig (2 and 10 mg/kg) compared to a group treated with methotrexate only (95% Confidence Limits), as described in Example 5, infra.
Figure 5I: A graph showing the change from baseline for SF-36 Mental Health Component on Day 180 in two groups after therapy with CTLA4Ig (2 and 10 mg/kg) compared to a group treated with methotrexate only (95% Confidence Limits), as described in Example 5, iyafi~a.
Figure 52: A bar graph showing CRP levels at day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, ihff°a.
Figure 53: A bar graph showing Rheumatoid Factor levels on day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, infra.
Figure 54: A bar graph showing IL-Zr levels on day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, it~fr-a.
Figure 55: A bar graph showing IL-6 levels on day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/kg), as described in Example 5, infra.
Figure 56: A bax graph showing TNFa, levels on day 180 after therapy with methotrexate alone or methotrexate and CTLA4Ig (2 and 10 mg/lcg), as described in Example 5, infra.
Figure 57: A table of the univariate methotrexate dose at screening/enrollinent for treatment group BMS 10 - treated with CTLA4Ig at 10 mg/lcg body weight as described in Example 5, if fra.

Figure 58: A table of the mivariate methotrexate dose at screening/enrolhnent for treatment group BP~iS 2 - treated with CTLA4Ig at 2 mg/lcg body weight as described in Example 5, inf-a.
Figure 59: A table of the univariate methotrexate dose at screening/enrollinent for the placebo group, as described in Example 5, inft°a.
Figure 60: A table of the univauiate methotrexate dose up to and including day 180 of the study for treatment group BMS 10 - treated with CTLA4Ig at 10 mg/kg body weight as described in Example 5, irafr°a.
Figure 61: A table of the univariate methotrexate dose up to and including day 180 of the study for treatment group BMS 2 - treated with CTLA4Ig at 2 mg/kg body weight as described in Example 5, irzfr~a.
Figure 62: A table of the univariate methotrexate dose up to and including day 180 of the study for the placebo group, as described in Example 5, ihfi~a.
Figure 63: A bar graph showing the difference in modified ACR response rates on day 180 in two groups after therapy with etanercept alone (25 mg twice weekly) or in combination with CTLA4Ig (2mg/kg), as described in Example 6, infra.
Figure 64A-C: Graphs showing percentage improvement of individual components of the modified ACR criteria as assessed on each visit day after therapy with etanercept alone (25 mg twice weekly) or in combination with CTLA4Ig (2 mg/kg) as described in Example 6, infra. A. Tender Joint Count. B. Swollen Joint Count. C. Pain Assessment.
Figure 65: A. A graph showing the change from baseline for SF-36 Physical Health Component on day 180, in two groups after therapy with etanercept (25 mg biweelcly) alone or in combination with CTLA4.Ig (2 mg/kg) (95% Confidence Limits), as described in Example 6, inf-a. B. A graph showing the change from baseline for SF-36 Mental Health Component on day 180, in two groups after therapy with etanercept (25 mg biweekly) alone or in combination with CTLA4Ig (2 mg/lcg) (95% Confidence Limits), as described in Example 6, ihfra.
Figure 66: Nucleotide sequence of a CTLA4Ig encoding a signal peptide; a wild type amino acid sequence of the extracellular domain of CTLA4 starting at methionine at position +1 to aspartic acid at position +124, or starting at alanine at position -1 to aspartic acid at position +124; and an Ig region (SEQ ID NO.: 21).
Figure 67: Amino acid sequence of a CTLA4Ig having a signal peptide; a wild type amino acid sequence of the extracellular domain of CTLA4 starting at methionine at position +1 to aspartic acid at position +124, or starting at alanine at position -1 to aspartic acid at position +124; and an Ig region (SEQ m NO.: 22).
Figure 68: A schematic diagram showing the disposition of subjects into three cohorts as described in Example 7, ircfi~a.
Figure 69: A Kaplan-Meier plot of the cumulative proportion of subjects who discontinued for any reason during the first 12 months of the study, as described in Example 7, ifzf~a.
Figure 70: A Kaplan-Meier plot of the cumulative proportion of subjects who discontinued due to lack of efficacy during the first 12 months of study, as described in Example 7, infra.
Figure 71A: A graph showing the ACR Responses on Day 180 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, ifaf~a.
~0 Figure 71B: A graph showing the 95 Percent Confidence Intervals for Differences in ACR Responses on Day 180 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
Figure 72A: A graph showing the ACR Responses on Day 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 rng/kg body weight) as described in Example 7, ifzfi~a.
Figure 72B: A graph showing the 95 Percent Confidence Intervals for Differences in AGR Responses on Day 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, i~cf~a.
Figure 73A: A graph summarizing the ACR 20 Response by Visit during a one year interval for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mglkg body weight) as described in Example 7, i~cf~a.
Figl.~re 73B: A graph summarizing the ACR 50 Response by Visit during a one year interval for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/lcg body weight) as described in Example 7, iraf -a.
Figure 73C: A graph summarizing the ACR 70 Response by Visit during a one year interval for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, ififi°a.
Figure 74: A graph showing the Mean ACR-N over a one year time interval for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infi°a.
Figure 75: A graph showilzg the Proportion of New Active Joints at Day 180 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, itafra.

Figure 76A: A graph showing the Frequency of New Tender Joints per Subject at Day 180 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, if~a.
Figure 76B: A graph showing the Frequency of New Tender Joints per Subject at Day 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/'~g body weight) as described in Example 7, ZfZf~"a.
Figure 77A: A graph showing the Frequency of New Swollen Joints per Subject at Day 180 for patients administered methotrexate alone or methotrexate and CTLA4Ig (~
or 10 mg/kg body weight) as described in Example 7, ihfra.
Figure 77B: A graph showing the Frequency of New Swollen Joints per Subject at Day 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, ihf~a.
Figure 78: A graph showing the Proportion of New Active Joints at Day 360 for patients administered rnethotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
Figure 79: Graphs showing the: A) Change from Baseline in the Physical Health Domains on Day 180, and B) Change from Baseline in the Mental Health Domains on 180, for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infi°a.
Figure 80: Graphs showing the: A) Change from Baseline in the Physical Health Domains on Day 360, and B) Change from Baseline in the Mental Health Domains on Day 360, for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/lcg body weight) as described in Example 7, inf3~a.
?0 Figure 81: A graph showing the Soluble IL-2r Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/l~g body weight) as described in Example 7, infra.
Figure 82: A graph showing the Rheumatoid Factor Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or mg/l~g body weight) as described in Example 7, inf~°a.
Figure 83: A graph showing the ICAM-1 Levels at Baseline, Days 180 and 360 for 10 patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
Figure 84: A graph showing the e-Selectin Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infra.
Figure 85: A graph showing the Serum IL-6 at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/kg body weight) as described in Example 7, infi~a.
Figure 86A: A graph showing the CRP Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/l~g body weight) as described in Example 7,~infi°a.
Figure 86B: A graph showing the TNFa Levels at Baseline, Days 180 and 360 for patients administered methotrexate alone or methotrexate and CTLA4Ig (2 or 10 mg/l~g body weight) as described in Example 7, infra.

DETAILED DESCRll'TION OF THE INVENTION
DEFINITIONS
All scientific and technical terms used in this application have meanings commonly used in the art mless otherwise specified. As used in this application, the following words or phrases have the meanings specified.
As used herein, DMARD refers to a Disease Modifying Anti-Rheumatic Drug. A
DMARD is any agent that modifies the symptoms and/or progression associated with an immune system disease, including autoimmune diseases (e.g. rheumatic diseases),, graft-related disorders and immunoproliferative diseases. DMARDs modify one or more of the symptoms and/or disease progression associated with rheumatic disease.
Symptoms of rheumatic diseases, include the following: joint swelling, pain, tenderness, morning stiffiiess, structural damage, an elevated level of serum C-reactive protein (CRP), an elevated level of soluble IL-2r, an elevated level of soluble ICAM-l, an elevated level of soluble E-selectin, an elevated level of rheumatoid factor, an elevated level of IL-6 or an elevated erythrocyte sedimentation rate (ESR). These symptoms and the reduction of these symptoms can be evaluated by any well known evaluation methods including:
Health Questionnaire Assessments; ACR 20, 50, 70; and/or Medical Outcomes Study Short Form-36.
DMARDs include, but are not limited to, dihydrofolic acid reductase inhibitors e.g., methotrexate; cyclophosphamide; cyclosporine; cyclosporin A; chloroquine;
hydroxychloroquine; sulfasalazine (sulphasalazopyrine) gold salts D-penicillamine;
leflunomide; azathioprine; anakinra; TNF blockers e.g., infliximab (REIVIICADER) or etanercept; and a biological agent that targets an inflammatory cytokine.
As used herein, NSAID refers to a Non-Steroidal Anti-Inflammatory Drug. NSAIDs reduce inflammatory reactions in a subject. NSAIDs include, but are not limited to acetyl salicylic acid, choline magnesimn salicylate, diflunisal, magnesiwn salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, lcetoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolrnetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam and tramadol.
S
As used herein, "ligand" refers to a molecule that specifically recognizes and binds another molecule, for example, a ligand for CTLA4 is a B7 molecule. In a further example, a ligand for the B7 molecule is a CTLA4 and/or CD28 molecule. The interaction of a molecule and its ligand can be regulated by compositions of the invention. For example, CTLA4 interaction with its ligand B7 can be blocked by administration of CTLA4Ig molecules. Alternatively, Tumor Necrosis Factor (TNF) interaction with its ligand, TNF receptor (TNFR), can be blocked by administration of etanercept or other TNF/TNFR blocking molecules.
1S As used herein "wild type CTLA4" or "non-mutated CTLA4" has the amino acid sequence of naturally occurring, full length CTLA4 as shown in Figure 23 (also as described in U.S. Patent Nos. 5,434,131, 5,844,095, and 5,8.51,795 herein incorporated by reference in their entirety), or any portion or derivative thereof, that recognizes and binds a B7 or interferes with a B7 so that it blocks binding to CD28 and/or CTLA4 (e.g., endogenous CD28 and/or CTLA4). In particular embodiments, the extracellular domain of wild type CTLA4 begins with methionine at position +1 and ends at aspartic acid at position +124, or the extracellular domain of wild type CTLA4 begins with alanine at position -l and ends at aspartic acid at position +124 as shown in Figure 23.
Wild type CTLA4 is a cell surface protein, having an N-terminal extracellular domain, a 2S transmembrane domain, and a C-terminal cytoplasmic domain. The extracellular domain binds to target molecules, such as a B7 molecule. In a cell, the naturally occurring, wild type CTLA4 protein is translated as an immature polypeptide, which includes a signal peptide at the N-terminal end. The immature polypeptide undergoes post-translational processing, which includes cleavage and removal of the signal peptide to generate a CTLA4 cleavage product having a newly generated N-terminal end that differs from the N-terminal end in the immature form. One skilled in the art will appreciate that additional post-translational processilig may occur, which removes one or more of the amino acids from the newly generated N-terminal end of the C1LA4 cleavage product.
Alternatively, the signal peptide may not be removed completely, generating molecules that begin before the common starting amino acid methionine. Thus, the mature protein may start at methionine at position +1 or alanine at position -1. The mature form of the CTLA4 molecule includes the extracellular domain or any portion thereof, which binds to B7.
As used herein, a "CTLA4 mutant molecule" means wildtype CTLA4 as shown in Figure 23 or any portion or derivative thereof, that has a mutation or multiple mutations (preferably in the extracellular domain of wildtype CTLA4). A CTLA4 mutant molecule has a sequence that it is similar but not identical to the sequence of wild type CTLA4 molecule, but still binds a B7. The mutations may include one or more amino acid residues substituted with an amino acid having conservative (e.g., substitute a leucine with an isoleucine) or non-conservative (e.g., subsfiitute a glycine with a tryptophan) structure or chemical properties, amino acid deletions; additions, frameshifts, or truncations. CTLA4 mutant molecules may include a non-CTLA4 molecule therein or attached thereto.
The mutant molecules may be soluble (i.e., circulating) or bound to a cell surface. Additional CTLA4 mutant molecules include those described in U.S. Patent Application Serial Numbers 09/865,321, 60/214,065 and 60/287,576; in U.S. Patent Numbers 6,090,914 5,844,095 and 5,773,253; and as described by Peach, R. J., et al., in JExp Med 180:2049-2058 (1994)). CTLA4 mutant molecules can be made synthetically or recombinantly.
"CTLA4Ig" is a soluble fusion protein comprising an extracellular domain of wildtype CTLA4 that binds B7, or a portion thereof, joined to an imrnunoglobulin constant region (Ig) , or a portion thereof . A particular embodiment comprises the extracellular domain of wild type CTLA4 (as shown in Figure 23) starting at methionine at position +1 and ending at aspartic acid at position +124 or starting at alanine at position -1 to aspartic acid at position +124; a junction amino acid residue glutamine at position +125; and an inununoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357 (DNA encoding CTLA4Ig was deposited on May 31, 1991 with the American Type Culture Collection (ATCC), 10801 University Blvd., Manassas, VA
20110-2209 under the provisions of the Budapest Treaty, and has been accorded ATCC
accession number ATCC 68629; Linsley, P., et al., 1994 Irrafnzifaity 1:793-80). CTLA4Ig-24, a Chinese Hamster Ovary (CHO) cell line expressing CTLA4Ig was deposited on May 31, 1991 with ATCC identification number CRL-10762). The soluble CTLA4Ig molecules used in the methods and/or kits of the invention may or may not include a signal (leader) peptide sequence. Typically, in the methods and/or kits of the invention, the molecules do not include a signal peptide sequence.
"L104EA29YIg" is a fusion protein that is a soluble CTLA4 mutant molecule comprising an extracellular domain of wildtype CTLA4 with amino acid changes A29Y (a tyrosine amino acid residue substituting for an alanine at position 29) and L104E (a glutamic acid amino acid residue substituting for a leucine at position +104), or a portion thereof that binds a B7 molecule, joined to an Ig tail (included in Figure 19; DNA encoding L104EA29YIg was deposited on June 20, 2000 with ATCC number PTA-2104;
copending in U.S. Patent Application Serial Numbers 09/579,927, 60/287,576 and 60/214,065, incorporated by reference herein). The soluble L104EA29YIg molecules used in the methods and/or kits of the invention may or may not include a signal (leader) peptide sequence. Typically, in the methods and/or kits of the invention, the molecules do not include a signal peptide sequence.
As used herein, "soluble" refers to any molecule, or fragments and derivatives thereof, not bound or attached to a cell, i.e., circulating. For example, CTLA4, B7 or CD28 can be made soluble by attaclung an immunoglobulin (Ig) moiety to the extracellular domain of CTLA4, B7 or CD28, respectively. Alternatively, a molecule such as CTLA4 can be rendered soluble by removing its trmsmembrane domain. Typically, the soluble molecules used in the methods, compositions and/or bits of the invention do not include a signal (or leader) sequence.
As used hereW , "soluble CTLA4 molecules" means non-cell-surface-bound (i.e.
circulating) CTLA4 molecules or any functional portion of a CTLA4 molecule that binds B7 including, but not limited to: CTLA4Ig fusion proteins (e.g. encoded by DNA
deposited with ATCC accession number 68629), wherein the extracellular domain of CTLA4 is fused to an inununoglobulin (Ig) moiety such as IgCy1 (IgCgammal), IgCy2 (IgCgamma2), IgCy3 (IgCgamma3), IgCy4 (IgCgamma4), IgC~, (IgCmu), IgCal (IgCalphal), IgCa2 (IgCalpha2), IgCB (IgCdelta) or IgCs (IgCepsilon), rendering the fusion molecule soluble, or fragments and derivatives thereof; proteins with the extracellular domain of CTLA4 fused or joined with a portion of a biologically active or chemically active protein such as the papihomavirus E7 gene product (CTLA4-E7), melanoma-associated antigen p97 (CTLA4-p97) or HIV env protein (CTLA4-env gp120) (as described in U.S. Patent No. 5,844,095, herein incorporated by reference in its entirety), or fragments and derivatives thereof; hybrid (chimeric) fusion proteins such as CD28/CTLA4Ig (as described in U.S. Patent No. 5,434,131, herein incorporated by reference in its entirety), or fragments and derivatives thereof; CTLA4 molecules with the transmembrane domain removed to render the protein soluble (Oaks, M. K., et al., 2000 Cellular Irnfraza~cology 201:144-153, herein incorporated by reference in its entirety), or fragments and derivatives thereof. "Soluble CTLA4 molecules" also include fragments, portions or derivatives thereof, and soluble CTLA4 mutant molecules, having CTLA4 binding activity. The soluble CTLA4 molecules used in the methods of the invention may or may not include a signal (leader) peptide sequence.
Typically, in the methods, compositions and/or kits of the invention, the molecules do not include a signal peptide sequence.
As used herein "the extracellular domain of CTLA4" is the portion of GTLA4 that recognizes and binds CTLA4 ligands, such as B7 molecules. For example, an extracellular domain of CTLA4 comprises methionine at position +1 to aspartic acid at position +124 (Figure 23). Alternatively, an extracellular domain of CTLA4 comprises alanine at position -1 to aspartic acid at position +124 (Figure 23). The extracellular domain includes fragments or derivatives of CTLA4 that bind a B7 molecule. The extracellular domain of CTLA4 as shown in Figure 23 znay also include mutations that change the binding avidity of the CTLA4 molecule for a B7 molecule.

As used herein, the term "mutation" means a change in the nucleotide or amino acid sequence of a wildtype molecule, for example, a change in the DNA andJor amino acid sequences of the wild-type CTLA4 extracellular domain. A mutation in DNA may change a codon leading to a change in the amino acid sequence. A DNA change may include substitutions, deletions, insertions, alternative splicing, or truncations. An amino acid change may include substitutions, deletions, insertions, additions, tntncations, or processing or cleavage errors of the protein. Alternatively, mutations in a nucleotide sequence may result in a silent mutation in the amino acid sequence as is well understood in the art. In that regard, certain nucleotide codons encode thei same amino acid.
Examples include nucleotide codons CGU, CGG, CGC, and CGA encoding the amino acid, arginine (R); or codons GAU, and GAC encoding the amino acid, aspartic acid (D).
Thus, a protein can be encoded by one or more nucleic acid molecules that differ in their specific nucleotide sequence, but still encode protein molecules having identical sequences. The amino acid coding sequence is as follows:
Amino Acid Symbol One Letter Codons Symbol Alanine Ala A GCU, GCC, GCA, GCG

Cysteine Cys G UGU, UGC .

Aspartic Asp D GAU, GAC
Acid Glutamic Glu E GAA, GAG, Acid PhenylalaninePhe F UUU, UUC

Glycine Gly G GGU, GGC, GGA, GGG

Histidine His H CAU, CAC

Isoleucine~Ile I AUU, AUC, AUA

Lysine Lys K AAA, AAG

Leucine Leu L UUA, UUG, CUU, CUC, CUA, CUG

Methionine Met M AUG

AsparagilzaAsn N AAU, AAC

Proline Pro P CCU, CCC, CCA, CCG

Glutarnine Ghz Q CAA, CAG

?7 Amino Acid Symbol One Letter Codons Symbol ~'g~e ~'g R CGU, CGC, CGA, CGG, AGA, AGG

Serine Ser S UCU, UCC, UCA, UCG, AGU, AGC

Threonine Thr T ACU, ACC, ACA, ACG

Valine Val V GUU, GUC, GUA, GUG

TryptophanTrp W UGG

Tyrosine Tyr ~ UAU, UAC

The mutant molecule may have one or mare mutations. As used herein, a "non-protein sequence" or "non-CTLA4 molecule" means any protein molecule that does not bind B7 and does not interfere with the binding of CTLA4 to its target. The non-CTLA4 molecule, attached to the extracellular domain of a CTLA4 molecule can alter the solubility or affinity of the CTLA4 molecule. An example includes, but is not limited to, an immunoglobulin (Ig) constant region or portion thereof. Preferably, the Ig constant region is a human or monkey Ig constant region, e.g., human C(gamma)1, including the hinge, CH2 and CH3 regions. The Ig constant region can be mutated to reduce its effector functions (IJ.S. Patents 5,637,481, 5,844,095 and 5,434,131).
As used herein, a "fragment" or "portion" is any part or segment of a molecule e.g.
CTLA4 or CD28, preferably the extracellular domain of CTLA4 or CD28 or a part or segment thereof, that recognizes and binds its target, e.g., a B7 molecule.
As used herein, 'B7" refers to the B7 family of molecules including, but not limited to, B7-1 (CD80) (Freeman et al, 1989, J Trn_ mvmol. 143:2714-2722, herein incorporated by reference in its entirety), B7-2 (CD86) (Freeman et al, 1993, Science 262:909-911 herein incorporated by reference in its entirety; Azluna et al, 1993, Nature 366:76-79 herein incorporated by reference in its entirety) that may recognize and bind CTLA4 and/or CD28. A B7 molecule can be expressed on an activated B cell.

As used herein, "CD28" refers to the molecule that recognizes and binds B7 as described in LJ.S. Serial I~o. 5,580,756 and 5,52'1,288 (hereiiz incorporated by reference in their entirety).
As used herein, "B7-positive cells" are any cells with one or more types of B7 molecules expressed on the cell surface.
As used herein, a "derivative" is a molecule that shares sequence similarity and activity of its parent molecule. For example, a derivative of CTLA4 includes a soluble C T LA4 molecule having an amino acid sequence at least 70% similar to the extracellular domain of wildtype CTLA4, and which recognizes and binds B7 e.g. CTLA4Ig or soluble CTLA4 mutant molecule L104EA29YIg. A derivative means any change to the amino acid sequence andlor chemical quality of the amino acid e.g., amino acid analogs.
As used herein, to "regulate" an immune response is to activate, stimulate, up-regulate, inhibit, block, down-regulate or modify the immune response. The auto-immune diseases described herein, may be treated by regulating an immune response e.g., by regulating functional CTLA4- and/or CD28- positive cell interactions with B7-positive cells. For example, a method for regulating an immune response comprises contacting the positive cells with a soluble CTLA4 molecule of the invention so as to form soluble CTLA4/B7 complexes, the soluble CTLA4 molecule interfering with reaction of an endogenous CTLA4 andlor CD28 molecule with said B7 molecule.
As used herein, to "block" or "inhibit" a receptor, signal or molecule means to interfere with the activation of the receptor, signal or molecule, as detected by an art-recognized test. For example, blockage of a cell-mediated immune response can be detected by determining reduction of D.heumatic Disease associated symptoms. Blockage or inhibition may be partial or total.
As used herein, "blocking B7 interaction" means to interfere with the binding of B7 to its ligands, such as CD28 and/or CTLA4, thereby obstructing T-cell and B7-positive cell interactions. Examples of agents that block B7 interactions include, but are not limited to, molecules such as an antibody (or portion or derivative thereof that recognizes and binds to the any of CTLA4, CD28 or B7 molecules (e.g. B7-l, B7-2); a soluble form (or portion or derivative thereof) of the molecules such as soluble CTLA4; a peptide fragment or other small molecule designed to interfere with the cell signal through the CTLA4/CD28/B7-mediated interaction. In a preferred embodiment, the blocking agent is a soluble CTLA4 molecule, such as CTLA4Ig (ATCC 68629) or L104EA29YIg (ATCC
PTA-2104), a soluble CD28 molecule such as CD28Ig (ATCC 68628), a soluble B7 molecule such as B7Ig (ATCC 68627), ar~ anti-B7 monoclonal antibody (e.g. ATCC
HB-253, ATCC CRL-2223, ATCC CRL-2226, ATCC HB-301, ATCC HB-11341 and monoclonal antibodies as described in by Anderson et al in U.S. Fatent 6,113,898 or Yokochi et al., 1982. J. Tmmun., 128(2)823-827), an anti-CTLA4 monoclonal antibody (e.g. ATCC HB-304, and monoclonal antibodies as described in references 82-83) and/or an anti-CD28 monoclonal antibody (e.g. ATCC HB 11944 and mAb 9.3 as described by Hansen (Hansen et al., 1980. Tmmunogenetics 10: 247-260) or Martin (Martin et al., 1984. J. Clin. Tm__m__un., 4(1):18-22)). Blocking B7 interactions can be detected by art-recognized tests such as determining reduction of immune disease (e.g., rheumatic disease) associated symptoms, by determining reduction in T-cell/B7-cell interactions or by determining reduction in B7 interaction with CTLA4 and/or CD28. Blockage may be partial or total.
As used herein, an "effective amount" of a molecule is defined as an amount that blocks the interaction of the molecule with its ligand. For example, an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 may be defined as the amount of the molecule that, when bound to B7 molecules on B7-positive cells, inhibit B7 molecules from binding endogenous ligands such as CTLA4 and CD28.
Alternatively, an effective amount of a molecule that blocks B7 interac+~ion with C T LA4 and/or CD28 may be defined as the amount of the molecule that, when bound to and/or CD28 molecules on T cells, inhibit B7 molecules from binding endogenous ligands such as CTLA4 and CD28. The inhibition or blockage may be partial or complete.

As used herein, "treating" a disease means to manage a disease by medicinal or other therapies.. Treatment of a disease may ameliorate the symptoms of a disease, reduce the severity of a disease, alter the course of disease progression and/or ameliorate or cure the basic disease problem. For example, to treat an auto-immure disease may be accomplished by regulating an immune response e.g., by regulating functional and/or CD28- positive cell interactions with B7-positive cells. Alternatively, treating an auto-immune disease may be accomplished by preventing the disease from occurnng or progressing through the use of the compositions described herein.
As used herein, "immune system disease" means any disease mediated by T-cell interactions with B7-positive cells including, but not limited to, autoimrnune diseases, graft related disorders and irnmunoproliferative diseases. Examples of immune system diseases include graft versus host disease (GVHD) (e.g., such as may result from bone marrow transplantation, or in the induction of tolerance), immune disorders associated with graft transplantation rejection, chronic rejection, and tissue or cell allo- or xenografts, including solid organs (e.g., kidney transplants), skin, islets, muscles, hepatocytes, neurons.
Examples of immunoproliferative diseases include, but are not limited to, psoriasis, T-cell lymphoma, T-cell acute lyrnphoblastic leukemia, testicular angiocentric T-cell lymphoma, benign lymphocytic angiitis, lupus (e.g. lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, diabetes (e.g. insulin dependent diabetes mellitis, type I diabetes rnellitis, type II diabetes mellitis), good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalinia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic diseases (e.g.
rheumatoid arthritis), polymyositis, scleroderna, and mixed connective tissue disease.
As used herein, "rheumatic diseases" means any disease that affects the joints, bone, soft tissue, or spinal cord (Mathies, H. 1983 Rl2eacjna) and comprises inflammatory rheumatism, degenerative rheumatism, extra-articular rhetunatism, and collagen diseases.
Additionally, rheumatic diseases include, but are not limited to,. chronic polyarthritis, J

psoriasis arthropathica, ankylosing spondylitis, rheumatoid arthritis, panarteriitis nodosa, systemic lupus erythematosus, progressive systemic scleroderma, periarthritis hiuneroscapulaizs, arthritis uratica, chondrocalcinosis, dermatomyositis, muscular rheumatism, myositis, and myogelosis. Some rheumatic diseases are known to be autoimmune diseases caused by a subject's altered immune response.
As used herein, "gene therapy" is a process to treat a disease by genetic manipulation.
Gene therapy involves introducing a nucleic acid molecule into a cell and the cell expressing a gene product encoded by the nucleic acid molecule. For example, as is well known by those skilled in the art, introducing the nucleic acid molecule into a cell may be performed by introducing an expression vector containing the nucleic acid molecule of interest into cells e,~ vivo or in vitro by a variety of methods including, for example, calcium phosphate precipitation, diethyaminoethyl dextran, polyethylene glycol (PEG), electroporation, direct injection, lipofection or viral infection (Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press 1989);
Kriegler M. Gene Transfer ad Expression: A Laboratory Manual (W. H. Freeman and Co, New York, N.Y., 1993) and Wu, Methods in Enzymology (Academic Press, New York, 1993), each of which is incorporated herein by reference). Alternatively, nucleotide sequences of interest may be introduced into a cell in vivo using a variety of vectors and by a variety of methods including, for example: direct administration of the nucleic acid into a subject (Williams et al, 1991 PNAS 88:2726-2730); or insertion of the nucleic acid molecule into a viral vector, production of the recombinant virus or viral particle, and infection of the subject with the recombinant virus (Battleman et al, 1993 JNem°osci 13:94-951; Carroll et al, 1993 J Cell Bioclaem 17E:241; Lebkowski et al, U.S. Patent 5,354,678;
Davison and Elliott, Molecular Virology: A Practical Approach (IRL Press, New York, 1993)).
Other methods used for ifi vivo transfer include encapsulation of the nucleic acid into liposomes, and direct introduction of the liposomes, or liposomes combined with a hemagglutinating Sendai virus, into a subject (IJ.S. Patent 5,824,655, incorporated by reference herein). The transfected or infected cells express the protein products encoded by the nucleic acid in order to ameliorate a disease or the symptoms of a disease.

As used herein, "Health Questionnaire Assessments (HAQs)" refers to a set of questions used to evaluate patients for symptoms of disease activity. These symptoms included:
joint swelling, joint tenderness, inflammation, morning stiffiiess, disease activity and disability evaluated by each patient in a self administered questionnaire regarding their physical well-being and function, disease activity and disability as evaluated a physician, and pain (Fries, J. F., et aL, 1982 ,I. ofRlaeumatolooy 9:789-793).
As used hereiln, "ACR" refers to clinical response studies based on criteria established by the American College of Rheumatology. A subject satisfied the "ACR20"
criterion if there was about a 20 percent improvement in tender and swollen joint counts and 20 percent improvement in three of five remaining symptoms measured, such as patient and physician global disease changes, pain, physical disability, and an acute phase reactant such as CRP or ESR (Felson, D. T., et al., 1993 Arthritis ccnd Rheunzatis3n '36:729-740;
Felson, D. T., et al., 1995 Af-tlaritis and Rheumatism 38:1-9). Similarly, a subject satisfied the "ACR50" or "ACR70" criterion if there was about a 50 or 70 percent improvement, respectively, in tender and swollen joint counts and about 50 or 70 percent improvement, respectively, in three of five remaining symptoms measured, such as patient and physician global disease changes, pain, physical disability, and an acute phase reactant such as CRP or ESR.
As used herein, the "Medical Outcomes Study Short Form-36 (SF-36)" refers to forms used to evaluate the impact of a DMARD (e.g., methotrexate or etanercept) and CTLA4Ig therapy on health-related quality of life (HRQOL). The SF-36 consists of 36 items which covers four physical and four mental domains (physical function, role-physical, bodily pain, general health, vitality, social function, role emotional, and mental health). These individual domail-~s are used to derive the physical and mental component siunmary scores which range from about 0 to 100, with higher scores indicating better quality of life. Absolute differences of 5 or more in the SF-36 scores were considered clinically meaningful.

As used hereilZ, "alleviate" refers to lessening or malting less severe, one or more of the symptoms of an immune disease (e.g., rheumatic disease) including, but not limited to, joint swelling, pain, tenderness, morning stiffiiess, struct«ral damage, an elevated level of serum C-reactive protein (CRP), an elevated level of soluble IL-2r, an elevated level of soluble ICAM-I, an elevated level of soluble E-selectin, an elevated level of rheumatoid factor, an elevated level of IL-6 or an elevated erythrocyte sedimentation rate.
h-~ order that the invention herein described may 'oe more fully understood the following description is set forth.
COMPOSITIONS AND METHODS OF THE INVENTION
The present invention provides compositions and methods for treating immune system diseases, such as rheumatic diseases, by administering to a subject an effective amount of a ligand that blocl~s B7 interactions with CTLA4 and/or CD28. For example, such ligands include: soluble CTLA4 molecules (such as CTLA4Ig, CTLA4-E7, CTLA4-p97, CTLA4-env gp 120, and mutant CTLA4 molecules such as, . CTLA4/CD28Ig, L104EA29YIg, L104EA29LIg, L104EA29TIg and/or L104EA29WIg), soluble CD28 molecules, soluble B7-I molecules, soluble B7-2 molecules, and monoclonal antibodies that recognize and bind B7, CD28 and/or CTLA4 (e.g., an anti-CTLA4 monoclonal antibody, an anti-CD28 monoclonal antibody, an anti-B7-I monoclonal antibody or an anti-B7-2 monoclonal antibody.
Further, the present invention provides compositions and methods for treating immune system diseases, such as rheumatic diseases, by administering to a subject a combination of an effective amount of 1) a DMARD (such as methotrexate or a molecule that bloclcs TNF interactions, e.g., blocl~s TNF interactions with its ligand) or other therapeutic agent, plus 2) an effective amount of a molecule that blocl~s B7 interaction with CTLA4 and/or CD28 such as soluble CTLA4 molecules (e.g., CTLA4Ig, CTLA4Ig/GD28Ig, CTLA4-E7, CTLA4-p97, CTLA4-env gp120, L104EA29YIg, L104EA2~LIg, L104EA29TIg and/or L104EA29WIg), soluble CD28 molecules, soluble B7-1 molecules, soluble molecules, and monoclonal antibodies that recognize and bind B7, CD28 and/or (e.g., an anti-CTLA4 monoclonal antibody, an anti-CD28 monoclonal antibody, an anti-B7-1 monoclonal antibody or an anti-B7-2 monoclonal antibody).
An effective amount of a molecule that blocks B7 interaction with CTLA4 and/or may be defined as the amount of anti-B7 monoclonal antibodies, soluble CTLA4 and/or soluble CD28 molecules that, when bound to B7 molecules on B7-positive cells, inhibit B7 molecules from binding endogenous ligands such as CTLA4 and CD28. The inhibition may be partial or complete.
Alternatively, an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 may be defined as the amount of anti-CTLA4 monoclonal antibody, anti-CD28 monoclonal antibody or soluble B7 (B7-1 or B7-2) molecules that, when bound to CTLA4 and/or CD28 molecules on T cells, inhibit B7 molecules from binding endogenous ligands such as CTLA4 and CD28. The inhibition may be partial or complete.
An effective amount of a molecule that blocks B7 interaction with CTLA4 and/or is an amount about O.I to 100 mg/kg weight of a subject. In another embodiment, the effective amount is an amount about 0.5 to 5 mg/kg weight of a subject, 0.1 to 5 mg/kg weight of a subject, about 5 to 10 mg/leg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mglkg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/lcg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight cf a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/lcg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/lcg weight of a subject, or about 95 to 100 mg/lcg weight of a subject.

W an embodiment, the effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28 is an amoiult about 2 mg/kg io about i0 mg/kg weight of a subject.
The preferred amount is 10 mg/kg weight of a subject. In another embodiment, the effective amount is an amount about 0.1 to 4 mg/kg weight of a subject. In another embodiment the effective amount is an amount about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subj ect, about 2.5 to 3.0 rr~g/kg weight of a subj ect, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subj ect, about 4.5 to 5.0 mg/kg weight of a subj ect, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 rng/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subj ect, about 9.5 to 10.0 mg/kg weight of a subj ect.
In another embodiment, the effective amount is an amount about 0.1 to 20 mg/kg weight of a subject. In another embodiment, the effective amount is an amount about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject or about 18 to 20 mg/kg weight of a subject.
In another embodiment, the effective amount is about 2 mg/lcg weight of a subject. In yet another embodiment, the effective amount is about 10 mg/kg weight of a subj ect.
In a specific embodiment, the molecule that blocks B7 interaction with CTLA4 and/or CD28 is soluble CTLA4 and the effective amount of a soluble CTLA4 molecule is about 2 mglkg weight of a subject. In another specific embodiment, the effective amount of a soluble CTLA4 molecule is about 10 mg/lcg weight of a subject. In another specific embodiment, an effective amount of a soluble CTLA4 is 500 mg for a subject weighing less than 60 kg, 750 ing for a subject Wezghzilg'uet~weeii 60-100 kg and 1000 mg for a subj ect weighing more than 100 kg.
An effective amount of the molecule that blocks B7 interaction with CTLA4 and/or CD28 is soluble CTLA4 may be administered to a subject daily, weekly, monthly and/or yearly, in single or multiple times per hour/day/week/month/year, depending on need. For exa~~nple, in one embodiment, the molecule may initially be administered once every two weeks for a month, and then once every month thereafter.
In a preferred embodiment, the immune disease is a rheumatic disease.
Rheumatic diseases are any diseases which are characterized by (i) inflammation or degeneration of musculo-skeletal or connective tissue structures of the body, particularly the joints, and including muscles, tendons, cartilage, synovial and fibrous tissues, (ii) accompanied by joint swelling, joint tenderness, inflammation, morning stiffness, and/or pain, or impairment of locomotion or function of those structures and, in some cases, (iii) often accompanied by serological evidence of rheumatoid factor and other inflammatory surrogate markers.
Rheumatic diseases include, but are not limited to, rheumatoid arthritis. The symptoms of rheumatoid arthritis include joint swelling, joint tenderness, inflammation, morning stiffiiess, and pain leading to physical disability. Subjects afflicted with the advanced stages of arthritis suffer from symptoms of structural damage and debilitating pain.
Other organs also can be impaired by the autoimmune mechanism.
h1 an embodiment of the invention used to treat an immune system disease, the Dr~IA,RD is methotreYate or a molecule that bloclcs TNF interactions such as etanercept, and the molecule that blocks B7 interaction with CTLA4 and/or CD28 is a soluble CTLA4.
In a fiuther embodiment, the methods of the invention comprise administering to a subject an effective amoiuzt of methotre~ate or a molecule that bloclcs TNF interactions in combination with an effective amou~.it of soluble CTLA4 in order to treat rheumatic diseases such as rheLUnatoid arthritis.
Effective amounts of methotrexate range about 0.1 to 40 mg/weelc. In one embodiment, the effective amount includes ranges of about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, or about 35 to 40 mg/ week. In one embodiment, methotrexate is administered in an amount ranging about 5 to 30 mg/week.
In one embodiment, the effective amount of a soluble CTLA4 molecule is about 2 mg/kg weight subject and the effective amount of methotrexate is about 10 to 30 mg/week. In another embodiment, the effective amount of a soluble CTLA4 molecule is about 10 mg/kg weight subject and the effective amount of methotrexate is about 10 to 30 mglweek.
In an embodiment of the invention used to treat an immune system disease, the DMARD is etanercept and the molecule that blocks B7 interaction with CTLA4 and/or CD28 is a soluble CTLA4. In a further embodiment, the methods of the invention comprise administering to a subject an effective amount of etanercept in combination with an effective amount of soluble CTLA4 in order to treat rheumatic diseases such as rheumatoid arthritis.
Effective amounts of etanercept range about 0.1 to 100 mg/week. In one embodiment, the effective amount includes ranges of about 10 to 100 mk/week, about 0.1 to 50 mg/week, about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about to 40 mg/week, about 40 to 45 mg/weelc, abcut 45 to 50 mg/weelc, about 50 tc mg/week, about 55 to 60 mg/weelc, about 60 to 65 mg/week, about 65 to 70 mg/week, about 70 to 75 mg/weelc, about 75 to 80 mg/week, about 80 to 85 mg/week, about 85 to mg/week, about 90 to 95 mg/week or about 95 to 100 mg/week. W one embodiment, 30 etanercept is administered in an amount of about 50 mg/weelc, alternatively etanercept may be administered in an amount of about 25 mg twice weeldy.

In one embodiment, the effective amount of a soluble CTLA4 molecule is about 2 mg/kg weight subject and the effective amount of etanercept is about 25 mg twice a week. Tn another embodiment, the effective amount of a soluble CTLA4 molecule is about 10 mg/kg weight subject and the effective amount of etanercept is about 25 mg twice a week.
The invention also provides compositions and methods for treating immune system diseases, such as rheumatic diseases, by administering to a subject a combination of an effective amount of an NSAID and/or other therapeutic agent plus an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28.
The invention also provides compositions and methods for treating immune system diseases, such as rheumatic diseases, by administering to a subject a an effective amount of a glucocorticoid, corticosteroid and/or other therapeutic agent plus an effective amount of a molecule that blocks B7 interaction with CTLA4 and/or CD28.
Compositiofas The present invention provides compositions for treating immune diseases, such as rheumatic diseases, comprising soluble CTLA4 molecules. Further, the present invention provides compositions comprising a biological agent that inhibits T-cell function but not T-cell depletion in a human by contacting B7-positive cells in the human with a soluble CTLA4. Examples of soluble CTLA4 include CTLA4Ig and soluble CTLA4 mutant molecules such as L104EA29YIg (Figure 19), L104EA29LIg (Figure 20), L104EA29Tig (Figure 21), and L104EA29WIg (Figure 22).
CTLA4 molecules, with mutant or wildtype sequences, may be rendered soluble by deleting the CTLA4 transmembrane segment (Oaks, M. K., et al., 2000 CellulaY
I»zfnzcjaology 201:144-153).

Alternatively, soluble CTLA4 molecules, with mutant or wildtype sequences, may be fusion proteins, wherein the CTLA4 molecules are fused to non-CTLA4 moieties such as immunoglobulin (Ig) molecules that render the CTLA4 molecules soluble. For example, a CTLA4 fusion protein may include the extracellular domain of CTLA4 fused to an immunoglobulin constant domain, resulting in the CTLA4Ig molecule (Figure 24) (Linsley, P. S., et al., 1994 Imfnufzity 1:793-80). Examples of immunoglobulin domains that may be fused to CTLA4 include, but are not limited to IgCyl (IgCgammal), IgCy2 (IgCgamma2), IgCy3 (IgCgamma3), igCy4 (IgCgamma4), IgC~, (IgCmu), IgCal (IgCalphal), IgCa2 (IgCalpha2), IgCB (IgCdelta) or IgCE (IgCepsilon).
For clinical protocols, it is preferred that the irnmunoglobulin moiety does not elicit a detrimental immune response in a subject. The preferred moiety is the immunoglobulin constant region, including the human or monkey immunoglobulin constant regions. One example of a suitable immunoglobulin region is human Cyl, including the hinge, and CH3 regions which can mediate effector functions such as binding to Fc receptors, mediating complement-dependent cytotoxicity (CDC), or mediate antibody-dependent cell-mediated cytotoxicity (ADCC). The immunoglobulin moiety may have one or more mutations therein, (e.g., in the CH2 domain, to reduce effector fiuictions such as CDC or ADCC) where the mutation modulates the binding capability of the immunoglobulin to its Iigand, by increasing or decreasing the binding capability of the immunoglobulin to Fc receptors. For example, mutations in the immunoglobulin moiety may include changes in any or alI its cysteine residues within the hinge domain, for example, the cysteines at positions +130, +136, and +139 are substituted with serine (Figure 24). The immunoglobulin moiety may also include the proline at position +148 substituted with a serine, as shown in Figure 24. Further, the mutations in the immunoglobulin moiety may include having the leucine at position +144 substituted with phenylalanine, leucine at position +145 substituted with glutarnic acid, or glycine at position +147 substituted with alanine.
Additional non-CTLA4 moieties for use in the soluble CTLA4 molecules or soluble CTLA4 mutant molecules include, but are not limited to, p97 molecule, env gp120 molecule, E7 molecule, and ova molecule (Dash, B. et al. 1994 J. Gera. Yirol. 75 (Pt 6):I389-97; Ikeda, T., et al. 1994 Gesae 138(1-2):193-6; Falk, K., et al. 1993 Cell. Ir~ana2a~aol.
150(2):447-52;
Fujisaka, K. et al. 1994 Yi3°oloby 204(2):789-93). Other molecules are also possible (Gerard, C. et al. 1994 Nezcroscieface 62(3):721; Byrn, R. et al. 1989 63(I0):4370; Smith, D.
et al. 1987 Scietzce 238:1704; Lasky, L. 1996 Science 233:209).
The soluble CTLA4 molecule of the invention can include a signal peptide sequence linked to the N-terminal end of the extracellular domain of the CTLA4 portion of the molecule. The signal peptide can be any sequence that will permit secretion of the molecule, including the signal peptide from oncostatin M (Malik, et al., (1989) Molec.
Cell. Biol. 9: 2847-2853), or CDS (Jones, N. H. et al., (1986) Nature 323:346-349), or the signal peptide from any extracellular protein. The soluble CTLA4 molecule of the invention can include the oncostatin M signal peptide linked at the N-terminal end of the extracellular domain of CTLA4, and the human immunoglobulin molecule (e.g., hinge, CHZ and GH3) linked to the C-terminal end of the extracellular domain (wildtype or mutated) of CTLA4. This molecule includes the oncostatin M signal peptide encompassing an amino acid sequence having methionine at position -26 through alanine at position -l, the CTLA4 portion encompassing an amino acid sequence having methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and the immunoglobulin portion encompassing an amino acid sequence having glutamic acid at position +126 through lysine at position +357.
Specifically, the soluble CTLA4 mutant molecules of the invention, comprising the mutated CTLA4 sequences described iyzfi~a, can be fusion molecules comprising human Ig, e.g., IgC(gamma)1 (i.e. IgCyl) moieties fused to the mutated CTLA4 fragments.
In one embodiment, the soluble CTLA4 mutant molecules comprise IgCyl (IgCgammal) fused to an extracellular domain of CTLA4 comprising a single-site mutation in the extracellular domain. The extracellulax domain of CTLA4 comprises metluonine at position +1 through aspartic acid at position +124 (e.g., Figure 23). The extracellular domain of the CTLA4 can comprise alanine at position -1 through aspartic acid at position +124 (e.g., Figure 23). Examples of single-site mutations include the following wherein the leucine at position +104 is changed to any other amino acid:
Single-site mutant: Codon change:

L104EIg Glutamic acid GAG

L104SIg Serine AGT

L104TIg Threonine ACG

L104AIg Alanine GCG

L104WIg Tryptophan TGG

L104QIg Glutamine CAG

L104KIg Lysine AAG

L 104RIg ~.
ginine CGG

L104GIg Glycine GGG

Further, the invention provides mutant molecules having the extracellular domain of CTLA4 with two mutations, fused to an Ig Cyl (IgCgammal) moiety. Examples include the following wherein the leucine at position +104 is changed to another amino acid (e.g.
glutamic acid) and the glycine at position +105, the serine at position +25, the threonine at position +30 or the alanine at position +29 is changed to any other amino acid:
Double-site mutants: Codon change:

L104EG105FIg Phenylalanine TTC

L104EG105WIg Tryptophan TGG

L104EGlO5LIg Leucine CTT

L104ES25RIg Arginine CGG

L 104ET3 OGIg Glycine GGG

L104ET30NIg Asparagine AAT

L104EA29YIg Tyrosine TAT

L104EA29LIg Leucine TTG

L104EA29TIg Threonine ACT

L104EA~9WIg Tryptophan TGG

Further still, the invention provides mutant molecules having the extracellular domain of CTLA4 comprising three mutations, fused to an IgCyl (IgCgammal ) moiety.
Examples include the following wherein the leucine at position +104 is changed to another amino acid (e.g. glutamic acid), the alanine at position +29 is changed to another amino acid (e.g. tyrosine) and the serine at position +25 is changed to another amino acid:
Triple-site Mutants: Codon changes:

L104EA29YS25KIg~ Lysine AAA

L104EA29YS25KIg Lysine AAG

L104EA29YS25NIg Asparagine AAC

L104EA29YS25RIg Arginine CGG

Soluble CTLA4 mutant molecules may have a junction amino acid residue which is located between the CTLA4 portion and the Ig portion of the molecule. The junction amino acid can be any amino acid, including glutamine. The junction amino acid can be introduced by molecular or chemical synthesis methods known in the art.
The soluble CTLA4 proteins of the invention, and fragments thereof, can be generated by cherriical synthesis methods. The principles of solid phase chemical synthesis of polypeptides are well known in the art and may be found in general texts relating to this area (Dugas, H. and Penney, C. 1951 Bioorgczraic Che~aistry, pp 54-92, Springer-Verlag, New York). The soluble CTLA4 proteins may be synthesized by solid-phase methodology utilizing an Applied Biosysterns 430A peptide synthesizer (Applied Biosystems, Foster City, Calif.) and synthesis cycles supplied by Applied Biosystems.
Protected amino acids, such as t-butoxycarbonyl-protected amino acids, and other reagents are commercially available from many chemical supply houses.

The present invention provides CTLA4 mutant molecules including a signal peptide sequence liu~ed to the N-terminal end of the extracellular domaisn of the CTLA4 portion of the mutant molecue. The signal peptide can be any sequence that will permit secretion of the mutant molecule, including the signal peptide from oncostatin M (Malik, et al., 1989 Molec. Cell. Biol. 9: 2847-2853), or CDS (Jones, N. H. et al., 1986 Nature 323:346-349), or the signal peptide from any exiracellular protein.
The irwention provides soluble CTLA4 mutant molecules comprisu-~g a single-site mutation in the extracellular domain of CTLA4 such as L104EIg (as included in Figure 18) or L104SIg, wherein L104EIg and L104SIg are mutated in their CTLA4 sequences so that leucine at position +104 is substituted with glutamic acid or serine, respectively. The single-site mutant molecules further include CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and an imm~oglobulin portion encompassing blutamic acid at position +126 through lysine at position +357. The immunoglobulin portion of the mutant molecule may also be mutated so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +148 is substituted with serine. Alternatively, the single-site soluble CTLA4 mutant molecule may have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
The invention provides soluble CTLA4 mutant molecules comprising a double-site mutation in the extracellular domain of CTLA4, such as L104EA29YIg, L104EA29LIg, L104EA29TIg or L104EA29WIg, wherein leucine at position +104 is substituted with a glutamic acid and alanine at position +29 is changed to tyrosine, leucine, threonine and tryptophan, respectively. The sequences for L104EA29YIg, L104EA29L Ig, L104EA29TIg and L104EA29WIg, starting at methionine at position +1 and ending with lysine at position +357, plus a signal (leader) peptide sequence are shown in Figures 19-2? respectively. The double-site mutant molecules fiuther comprise CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +124, a jtmction amino acid residue glutamhie at position +125, and an immunoglobulin portion encompassing glutarnic acid at position +126 through lysine at position +357.
The irnmunoglobulin portion of the mutant molecule may also be mutated, so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +148 is substituted with serine. Alternatively, these mutant molecules can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +I24.
The invention provides soluble CTLA4 mutant 11101ecules compizsmg a double-site mutation in the extracellular domain of CTLA4, such as L104EG105FIg, L104EGlO5WIg and L104EGlO5LIg, wherein leucine at position +104 is substituted with a glutamic acid and glycine at position +105 is substituted with phenylalanine, tryptophan and leucine, respectively. The double-site mutant molecules further comprise CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and an immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357.
The immunoglobulin portion of the may also be mutated, so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +14~ is substituted with serine. Alternatively, these mutant molecules can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
The invention provides L104ES25RIg which is a double-site mutant molecule comprising a CTLA4 portion encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutamine at position +125, and the immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357. The portion having the extracellular domain of CTLA4 is mutated so that serine at position +25 is substituted with arginine, and leucine at position +104 is substituted with glutamic acid.
Alternatively, L104ES25RIg can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
The invention provides soluble CTLA4 mutant molecules comprising a double-site mutation in the extracellular domain of CTLA4, such as L104ET30GIg and L104ET301~1Ig, wherein leucine at position +104 is substituted with a glutamic acid and threonine at position +30 is substituted with glycine and asparagine, respectively. The double-site mutant molecules further comprise CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +124, a junction amino acid residue glutarnine at position +125, and an immunoglobulin .portion encompassing glutamic acid at position +126 through lysine at position +357. The immunoglobulin portion of the mutant molecule may also be mutated, so that the cysteines at positions +I30, +130, and +139 are substituted with serine, and the proline at position +148 is substituted with serine. Alternatively, these mutant molecules can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
The invention provides soluble CTLA4 mutant molecules comprising a triple-site mutation in the extracellular domain of CTLA4, such as L104EA29YS25KIg, L104EA29YS25NIg, L104EA29YS25RIg, wherein leucine at position +104 is substituted with a glutamic acid, alanine at position +29 is substituted with tyrosine, and serine at position +25 is substituted with lysine, asparagine and arginine, respectively. The triple-site mutant molecules further comprise CTLA4 portions encompassing methionine at position +1 through aspartic acid at position +I24, a junction amino acid residue glutamine at position +125, and an immunoglobulin portion encompassing glutamic acid at position +126 through lysine at position +357. The immunoglobulin portion of the mutant molecule may also be mutated, so that the cysteines at positions +130, +136, and +139 are substituted with serine, and the proline at position +148 is substituted with serine. Alternatively, these mutant molecules can have a CTLA4 portion encompassing alanine at position -1 through aspartic acid at position +124.
Additional embodiments of soluble CTLA4 mutant molecules include chimeric CTLA4/CD28 homologue mutant molecules that bind a B7 (Peach, R. J., et al., Exp Med 180:2049-2058). Examples of these chimeric CTLA4/CD28 mutant molecules include HS1, HS2, HS3, HS4, HSS, HS6, HS4A, HS4B, HS7, HSB, HS9, HS10, HS11, HS12, HS13 and HS14 (U.S. patent number 5,773,253) Preferred embodiments of the invention are soluble CTLA4 molecules such as CTLA4Ig (as shown in Figure 24, starting at methionine at position +1 and ending at lysine at position +357) and soluble CTLA4 mutant L104EA29YIg (as shown in Figure 19, starting at methionine at position +1 and ending at lysine at position +357).
The invention further provides nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences corresponding to the soluble CTLA4 molecules of the invention. In one embodiment, the nucleic acid molecule is a DNA (e.g., cDNA) or a hybrid thereof. For example, a CTLA4Ig molecule can comprise a GCT or GCC
codon, encoding alanine, at nucleotide position +49 to +51 as shown in Figure 24. In another example, a CTLA4Ig molecule can comprise a GGT or GGG codon, encoding glycine, at nucleotide position +436 to +438 as shown in Figure 24. In yet another example, a CTLA4Ig molecule can comprise a CGG or CGT codon, encoding arginine, at nucleotide position +631 to +633 as shown in Figure 24. DNA encoding CTLA4Ig (Figure 24) was deposited on May 31, 1991with the American Type Culture Collection (ATCC), University Blvd., Manassas, VA 20110-2209 and has been accorded ATCC accession number ATCC 68629. DNA encoding L104EA29YIg (sequence included in Figure 19) was deposited on June 19, 2000 with ATCC and has been accorded ATCC accession number PTA-2104. Alternatively, the nucleic acid molecules are RNA or a hybrid thereof.
The nucleic acid molecules of the invention also include derivative nucleic acid molecules which differ from DNA or RNA molecules, and anti-sense molecules.
Derivative molecules include peptide nucleic acids (PNAs), and non-nucleic acid molecules including phosphorothioate, phosphotriester, phosphoramidate, and methylphosphonate molecules, that bind to single-stranded DNA or RNA in a base pair-dependent manner (Zamecnik, P. C., et al., 1978 Pr~oc. Natl. Acad. Sci.
75:280284;
Goodchild, P. C., et al., 1986 Pt-oc. Natl. <4cczel. ,Sci. 83:4143-4146).
Peptide nucleic acid molecules comprise a nucleic acid oligomer to which an amino acid residue, such as lysine, and an amino group have been added. These small molecules, also designated anti-gene agents, stop transcript elongation by binding to their complementary (template) strand of nucleic acid (Nielsen, P. E., et al., 1993 Arzticaxacef~ Df-ug Des 8:53-63).
Reviews of methods for synthesis of DNA, RNA, and their analogues can be found in:
Oligonucleotides and Analogues, eds. F. Eckstein, 1991, IRL, Press, New York;
Oligonucleotide Synthesis, ed. M. J. Gait, 1984, IRL Press, Oxford, England.
Additionally, methods for antisense RNA technology are described in U. S.
patents 5,194,428 and 5,110,802. A skilled artisan can readily obtail-~ these classes of nucleic acid molecules using the herein described soluble CTLA4 polynucleotide sequences, see for example Innovative and Pef speetives in Solid Phase Synthesis (1992) Egholm, et al. pp 325-328 or U. S. Patent No. 5,539,082.
Additionally, the invention provides a vector, which comprises the nucleotide sequences of the invention. The term vector includes, but is not limited to, plasmids, cosmids, and phagemids. In one embodiment, the vector can be an autonomously replicating vector comprising a replicon that directs the replication of the rDNA within the appropriate host cell. Alternatively, the vector can direct integration of the recombinant vector into the host cell. Various viral vectors may also be used, such as, for example, a number of well known retroviral and adenoviral vectors (Berlrner 1988 Bioteclaniques 6:616-629).
The vectors can permit expression of the soluble CTLA4 transcript or polypeptide sequences in prokaryotic or eukaryotic host cells. The vectors include expression .
vectors, comprising an expression control element, such as a promoter sequence, which enables transcription of the inserted soluble CTLA4 nucleic acid sequences and can be used for regulating the expression (e.g., transcription and/or translation) of an operably linked soluble CTLA4 sequence in an appropriate host cell. Expression control elements are lrnown in the art and include, but are not limited to, inducible promoters, constiW five promoters, secretion signals, enhancers, transcription terminators, and other transcriptional regulatory elements. Other expression control elements that are involved in translation are known in the art, and include the Shilze-Dalgamo sequence (e.g., prokaryotic host cells), and iiutiation and termination codons.

Specific initiation signals may also be required for efficient translation of a soluble CTLA4 sequence. These signals include the ATG-initiation colon and adjacent sequences. In cases where the soluble CTLA4 initiation colon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only the coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG-iiutiation colon may be provided. Furthermore, the initiation colon should be in the correct reading-frame to ensure translation of the entire insert. Exogenous transcriptional elements and initiation colons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf, D., et al, 1994 Results PYObl. Cell. Differ.
20:125-62;
Bittner, et al., 1987 Methods i~z Eizzymol. 153:516-544).
The preferred vectors for expression of the soluble CTLA4 sequences in eukaryote host cells include expression control elements, such as the baculovirus polyhedrin promoter for expression in insect cells. Other expression control elements include promoters or enhancers derived from the genomes of plant cells (e. g., heat shoclc, RUBISCO, storage protein genes), viral promoters or leader sequences or from plant viruses, and promoters or enhancers from the mammalian genes or from mammalian viruses.
The preferred vector includes at least one selectable marker gene that encodes a gene product that confers drug resistance such as resistance to ampicillin or tetracyline. The vector also comprises multiple endonuclease restriction sites that enable convenient .
insertion of exogenous DNA sequences. Methods for generating a recombinant expression vector encoding the soluble CTLA4 proteins of the invention are well known in the art, and can be found in Sambrook et al., (Nloleczdat°
Clofaisag; A Laboratory NIayaZCal, 2°d edition, Sambroolc, Fritch, and Nlaniatis 1989, Cold Spring Harbor Press) and Ausubel et al. (1989 Cury-ent Protocols in ll~Ioleciclai~ Biology, John Wiley & Sons, New York N.Y.).

The preferred vectors for generating soluble CTLA4 transcripts and/or the encoded soluble CTLA=1 polypeptides are expression vectors which are compatible with prolcaryotic host cells. Prokaryotic cell expression vectors are well known in the art and are available from several commercial sources. For example, pET vectors (e.g., pET-21, Novagen Corp.), BLLTESCRIl'T phagemid (Stratagene, LaJolla, CA), pSPORT (Gibco BRL, Roclcville, MD), or ptrp-lac hybrids may be used to express soluble CTLA4 polypeptides in bacterial host cells.
Alternatively, the preferred expression vectors for generating soluble CTLA4 transcripts and/or the encoded soluble CTLA4 polypeptides are expression vectors which are compatible with eukaryotic host cells. The more preferred vectors are those compatible with vertebrate cells. Eukaryotic cell expression vectors are well known in the art and are available from several commercial sources. Typically, such vectors are provided containing convenient restriction sites for insertion of the desired DNA segment. Typical of such vectors are PSVL and pKSV-10 (Pharmacia), pBPV-1/pML2d (International Biotechnologies, Inc.), pTDTI (ATCC, #31255), and similar eukaryotic expression vectors.
Examples of expression vectors for include, but axe not limited to, vectors for mammalian host cells (e.g., BPV-1, pHyg, pRSV, pSV2, pTK2 (Maniatis); pIRES (Clontech);
pRc/CMV2, pRc/RSV, pSFV1 (Life Technologies); pVPakc Vectors, pCMV vectors, pSGS vectors (Stratagene)), retroviral vectors (e.g., pFB vectors (Stratagene)), pCDNA-3 (Invitrogen) or modified forms thereof, adenoviral vectors; adeno-associated virus vectors, baculovirus vectors, yeast vectors (e.g., pESC vectors (Stratagene)).
A host vector system is also provided. The host vector system comprises the vector of the invention in a slutable host cell. Examples of suitable host ceh_s include, but are not limited to, prokaryotic and eukaryotic cells. In accordance with the practice of the invention, eukaryotic cells are also suitable host cells. Examples of eukaryotic cells include any animal cell, whether primary or immortalized, yeast (e.g., Saccharomyces cerevisiae, Schizosaccharom~es ornbe, and Pichia pastoris), and plant cells.
Exemplary animal cells include cells from bovine, ovine, porcine, marine, equine, monkey and ape. Myeloma, COS and CHO cells are examples of animal cells that may be used as hosts. Particular CHO cells include, but are not limited to, DG44 (Chasm, et la., 1986 Som. Cell. Molec: Genet. I2:555-556; Kollcekar 1997 Biochemistry 36:10901-10909), CHO-K1 (ATCC No. CCL-61), CHO-KI Tet-On cell line (Clontech), CHO
designated ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO~ clone 13 (GEIMG, Genova, IT), CHO clone B (GEI1VIG, Genova, IT), CHO-Kl/SF designated ECACC 93061607 (CAMR, Salisbtuy, Wiltshire, UK), and RR-CHOKI designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK). Exemplary plant cells include whole plants, cell culture, or callus, from tobacco, corn, soybean, and rice cells. Corn, I O soybean, and rice seeds are also acceptable.
The CTLA4 mutant molecules of the invention may be isolated as naturally-occturing polypeptides, or from any source whether natural, synthetic, semi-synthetic or recombinant.
Accordingly, the CTLA4 mutant polypeptide molecules may be isolated as naturally-occurring proteins from any species, particularly mammalian, including bovine, ovine, porcine, marine, equine, and preferably human. Alternatively, the CTLA4 mutant polypeptide molecules may be isolated as recombinant polypeptides that are expressed in prokaryote or ettkaryote host cells,.or isolated as a chemically synthesized polypeptide.
A skilled artisan can readily employ standard isolation methods to obtain isolated CTLA4 mutant molecules. The nature and degree of isolation will depend on the source and the intended use of the isolated molecules.
CTLA4 mutant molecules and fragments or derivatives thereof, can be produced by recombinant methods. Accordingly, an isolated nucleotide sequence encoding wild-type CTLA4 molecules may be manipulated to introduce mutations, resulting in nucleotide sequences that encode the CTLA4 mutant polypeptide molecules. For example, the nucleotide sequences encoding the CTLA4 mutant molecules may be generated by site-directed mutagenesis methods, using primers and PCR amplification. The primers can include specific sequences designed to introduce desired mutations.
Alternatively, the primers can be designed to include randomized or semi-randomized sequences to introduce random mutations. Standard recombinant methods (Molecular' Cloraifag; A
Laboratory MafaZaal, 2nd edition, Saznbrooli, Fritch, and Maniatis 1959, Cold Spril-~g Harbor Press) and PCR technology (U. S. Patent No. 4,603,102) can be employed for generating and isolating CTLA4 mutant polynucleotides encoding CTLA4 mutant polypeptides.
The invention includes pharmaceutical compositions comprising pharmaceutically effective amounts of a molecule that blocks B7 interaction with CTLA4 and/or such as soluble CTLA4 molecules, CD2.8 molecules, B7 (B7-1 or B7-2,) molecules, anti-CTLA4 monoclonal antibodies, anti-CD28 monoclonal antibodies or anti-B7 (B7-I
or B7-2) monoclonal antibodies. The pharmaceutical compositions of the invention are useful for treatment of immune system diseases. In certain embodiments, immune system diseases are mediated by CD2~/CTLA4/B7 interactions. The soluble CTLA4 molecules are preferably soluble CTLA4 molecules with wildtype sequence and/or soluble molecules having one or more mutations in the extracellular domain of CTLA4.
The pharmaceutical composition can include soluble CTLA4 protein molecules and/or nucleic acid molecules, and/or vectors encoding the molecules. In preferred embodiments, the soluble CTLA4 molecules have the amino acid sequence of the extracellular domain of CTLA4 as shown in either Figures 24 or 19 (CTLA4Ig or LI04EA29Y, respectively).
Even more preferably, the soluble CTLA4 mutant molecule is L104EA29YIg as disclosed herein. The compositions may additionally include other therapeutic agents, including, but not limited to, DMAR.Ds, NSAIDs, corticosteroids, glucocorticoids, drug toxins, alkylating agents, anti-neoplastic drugs, enzymes, antibodies, or conjugates.
Am embodiment of the pharmaceutical composition of the invention comprises an effective amount of a molecule that bloclcs B7 interaction with CTLA4 and/or CD?S, such as the molecules and the suitable amounts of the molecules described supra, and an effective amount of a DMA.RD.
The amount of D1VIARDS administered to a subject varies depending on several factors including the efficacy of the drug on a specific subject and the toxicity (i.e. the tolerability) of a drug to a specific subject (Guidelines for the Management of Rhemnatoid Arthritis, Arthritis and Rheumatism ZToI. 39, No. 5, May 1996, pages 713-71 l; Physician's Desk Reference 2002, Medical Economics Company, Inc.
Montvale, NJ
07645). The following provides a range of drug dosages for each DMARD. An attending physician will determine specific dosages for each subject.
Depending on the DMARD, an effective amount can be in a range of about 1 to about 5000 mg/day. This range can be modified to an amount of about 1 to 10 mg/day, about to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 10 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to mg/day, about 700 to 750 mg/day,, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 rng/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mglday, about 1900 to 2000 rng/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day. It would be clear to one skilled in the art that dosage will vary depending on the particular DMA.RD being used. Specific examples of appropriate dosages, depending on the DMARD, are described below.
In another embodiment, an effective amount of a DMARD can be in a range of about 0.1 mg/week to 40 mg/weelc; 0.1 mg/week to 5 mg/week; 5 mg/weelc to 10 mg/weelc;
IO
mg/weelc to 30 mg/week; 30 mg/week to 35 mg/weelc; 0.1 mg/week to 100 mg/week;
or mg/weelc to 50 mg/weelc. In another embodiment, a DMARD can be administered in an amount of about 50 mg/week or 25 mg twice weeldy. It would be clear to one skilled in the ai-t that dosage range will vary depending on the particular DMARD
being used, 30 for example see below.

Methotrexate is an antimetabolite molecule that interferes with DNA synthesis, repair and cellular replication. Methotrexate fimctions as an inhibitor of dihydrofolic acid reductase i.e. it is a folic acid antagonist. Methotrexate is commonly administered in an amount about 0.1 to 40 mg per week with a common dosage ranging about 5 to 30 mg per week.
Metllotrexate may be administered to a subject in various increments: about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, or about 35 to 40 mg/ week. In one embodiment, an effective amount of a DMARD, including methotrexate, is an amount about 10 to 30 mg/week.
Cyclophosphamide, an alkylating agent, may be administered in dosages ranging about 1 to 10 mg/kg body weight per day.
Cyclosporine (e.g. NEORALR) also known as Cyclosporin A, is commonly administered in dosages ranging from about 1 to 10 mg/kg body weight per day. Dosages ranging about 2.5 to 4 mg per body weight per day are commonly used.
Chloroquine or hydroxychloroquine (e.g. PLAQUENILR), is commonly administered in dosages rangilig about 100 to 1000 mg daily. Preferred dosages range about 200-600 mg administered daily.
Sulfasalazine (e.g., AZULFIDM EN-tabsR) is commonly administered in amounts ranging about 50 to 5000 mg per day, with a common dosage of about 2000 to 3000 mg per day for adults. Dosages for children axe commonly about 5 to 100 mg/kg of body weight, up to 2 grams per day.
Gold salts are formulated for two types of administration: injection or oral.
Injectable gold salts are commonly prescribed in dosages about 5 to 100 mg doses every two to four weeks. Orally adW nistered gold salts are commonly prescribed in doses ranging about 1 to 10 mg per day.

D-penicillamine or penicillamine (CUPRIM1NE~) is commonly administered in dosages about 50 to 2000 mg per day, with preferred dosages about 125 mg per day up to mg per day.
Azathioprine is commonly administered in dosages of about 10 to 250 mg per day.
Preferred dosages range about 25 to 200 mg per day.
Analcinra (e.g. I~INERETR) is an interleukin-I receptor antagoiust. A common dosage range for anakinra is about 10 to 250 mg per day, with a recommended dosage of about 100 mg per day.
Infliximab (R.EMICADER) is a chimeric monoclonal antibody that binds to tumor necrosis factor alpha (TNFa) and inhibits the activity of TNFa. Infliximab is commonly administered in dosages about I to 20 mg/kg body weight every four to eight weeks.
Dosages of about 3 to 10 mg/kg body weight may be administered every four to eight weelcs depending on the subject.
Etanercept (e.g. ENBRELR) is a dimeric fusion protein that binds the tumor necrosis factor (TNF) and blocks its interactions with TNF receptors. Commonly administered dosages of etanercept are about 10 to 100 mg per week for adults with a preferred dosage of about 50 mg per week. Dosages for juvenile subjects range about 0.1 to 50 mg/kg body weight per week with a maximum of about 50 mg per week. For adult patients, etanercept is commonly administered e.g., injected, in 25 mg doses twice weekly e.g., 72-96 hours apart in time.
Leflunomide (AR.AVAR) is commonly administered at dosages about I and 100 mg per day. A common daily dosage is about 10 to 20 mg per day.
A further embodiment of the invention is a pharmaceutical composition comprising an effective amoiuit of a soluble CTLA4, such as CTLA4Ig, and an effective amount of a DMARD, such as methotr exate or etanercept.

A pharmaceutical composition comprising soluble CTLA4 can be used for methods for blocking B7 interaction with CTLA4 and/or CD28; or for treating immune system diseases. Effective amounts of soluble CTLA4 in the pharmaceutical composition range about 0.1 to 100 rng/kg weight of the subject. In another embodiment, the effective amount is an amount about 0.5 to 5 mglkg weight of a subj ect, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/lcg weight of a subject, about 15 to 20 111g/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 rnglkg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, or about 95 to I00 mg/kg weight of a subj ect.
In an embodiment, the effective amount of soluble CTLA4 is an amount about 2 mg/kg to about 10 mg/kg weight of a subject. In another embodiment, the effective amount is an amount about 0.1 to 4 rng/kg weight of a subject. In another embodiment the effective amount is an amount about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 m~kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject or about 3.5 to 4.0 mg/kg weight of a subject. In another embodiment, the effective amount is an amount about 0.1 to 20 mg/kg weight of a subject. In another embodiment, the effective amount is an amount about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 rng/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/lcg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/lcg weight of a subject or about 18 to 20 mg/kg weight of a subject. In an embodiment, the effective amount is 2 mg/kg weight of a subject. In another embodiment, the effective amount is about 10 ing/kg weight of a subject.
In a specific embodiment, an effective amount of soluble CTLA4 is 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg and mg for a subj ect weighing more than 100 kg.
Effective amounts of methotrexate in the pharmaceutical composition range about 0.1 to 40 mg/week. In one embodiment, the effective amount is an amount about 0.1 to mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, or about 35 to 40 mg/ week. In one embodiment, an effective amount of a DMARD, including methotrexate, is an amount about 10 to 30 mg/week.
In one embodiment, the effective amount of a soluble CTLA4 molecule is about 2 mg/kg weight subject and the effective amount of methotrexate is about 10 to 30 mg/week. In another embodiment, the effective amount of a soluble CTLA4 molecule is about 10 mg/kg weight subject and the effective amount o~methotrexate is about 10 to 30 mg/week..
Effective amounts of etanercept in the pharmaceutical composition range about 0.1 to 100 mglweelc. In one embodiment, the effective amount is ranges about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to mg/week, about 25 to 30 mg/week, about 30 to 35 mg/weelc, about 35 to 40 mg/week, about 40 to 45 mg/week, about 45 to 50 mg/week, about 50 to 55 mg/week, about 55 to mg/week, about 60 to 65 mg/week, about 65 to 70 mg/week, about 70 to 75 mg/weelc, about 75 to 80 mg/weehc, about 80 to 85 mg/week, about 85 to 90 mg/week, about 90 to mg/week or about 95 to 100 mg/week. In one embodiment, etanercept is administered in an amount ranging about 50 rng/weelc e.g., 25 mg administered twice weeldy.
In one embodiment, the effective amount of a soluble CTLA4 molecule is about 2 mg/lcg weight subject and the effective amount of etanercept is about 25 mg twice a week. W

another embodiment, the effective amount of a soluble CTLA4 molecule is about mg/kg weight subject and the effective amount of etanercept is about 25 mg twice a week.
The compositions of the invention further encompass a pharmaceutical composition comprising soluble CTLA4 in combination with other treatments for rheumatic disease including, but not limited to: collagen, dnaJ, molecules that block TNF
function (e.g., pegsunercept), molecules that block cytokine function (e.g., AMG719), molecules that block LFA-1 function (e.g., efalizumab) and stem cell transplants. These other treatments are currently being studied in clinical trials (www.clinicaltrials.~ov) to determine their effect on rheumatoid arthritis.
Collagen, for example in the form of bovine II collagen, may be orally administered to a patient suffering from rheumatoid arthritis in order to alleviate one or more symptoms of rheumatoid arthritis.
DnaJ is a small peptide which mimics a protein contained in a gene in many patients with rheumatoid arthritis. The peptide is derived from E. coli bacteria heat shock protein.
DnaJ may be orally administered to a patient suffering from rheumatoid arthritis in order to alleviate one or more symptoms of rheumatoid arthritis.
TNF is a molecule involved in the inflammatory response of patients with rheumatoid arthritis. Conceivably, any molecule that blocks TNF function e.g., by bloclcing TNF
binding to the TNF receptor (TNFR), may help modify the progression of rheumatoid arthritis and alleviate some of its symptoms. Several TNF blockers such as infliximab and etanercept, have been shown to be efficacious m treating rhetunatoid arthritis. Other TNF blockers such as pegsunercept are being developed and tested (Phase II
clinical trial) for their efficacy in treating rheumatoid arthritis.
Cytol~ines e.g., Interleukin-1 (IL-1), are cell secreted molecules involved in mediating immune responses. Conceivably, any molecule that blocks cytokine function e.g., by 5~

blocking IL-1 interaction with its receptor, may help modify the progression of rheumatoid arthritis and alleviate one or more of its symptoms. Anakulra, a recombinant protein that bloclcs IL-1 interaction with its receptor (IL-1R) has been shown to be efficacious in treating rheumatoid arthritis. An IL-1 inhibitor, AMG719, is being developed and tested (Phase II clinical trial) for its efficacy in treating rheumatoid arthritis.
Lymphocyte function associated molecule 1 (LFA-1) is a molecule composed of two subunits, CD 11 a and CD 18, which functions by mediating lymphocyte adhesion to various cell types such as endothelium. Conceivably, interference of LFA-1 function may help modify the progression of rheumatoid arthritis and alleviate one or more of its symptoms. An anti-LFA-1 antibcdy, efalizumab, is being developed and tested (Phase II
clinical trial) for its efficacy in treating rheumatoid arthritis.
Blockage of TNF, cytokine or LFA-1 interaction to their ligands by a potentially therapeutic molecule can be determined by any number of assays known to those skilled in the art. For example, competition assays may be used to test blockage by the molecule of interest e.g., a molecule can be exposed to a TNF/TNFR binding pair in order to compete with TNF to bind to TNFR. Alternatively, functional assays can be performed to test blockage e.g., a molecule can be tested for its ability to inhibit an inflammatory cascade, or any part of an inflammatory reaction such as swelling, redness or pain, caused by a cytokine.
The present invention also provides pharmaceutical compositions comprising the molecules of the inventon e.g., CTLA4Ig and an acceptable carrier or adjuvant which is known to those cf skill of the art. The pharmaceutical compositions preferably include suitable carriers and adjuvants which include any material which when combined with the molecules of the invention (e.g., a soluble CTLA4 molecule, such as, CTLA4Ig or L104EA29Y) retain the molecule's activity, and is non-reactive with the subject's immune system. These carriers and adjuvants include, but are not limited to, ion exchangers, alu.mina, aluminum stearate, lecithin, serum proteins, such as lmman serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, phosphate buffered saline solution, water, emulsions (e.g. oil/water emulsion), salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances and polyethylene glycol. Other earners may also include sterile solutions; tablets, including coated tablets and capsules. Typically such carriers contain excipients such as starch, milk, sugar (e.g. sucrose, glucose, maltose), certain types of clay, gelatin, stearic acid or salts thereof, magnesium or calcium stearate, talc, vegetable fats or oils, gums, glycols, or other known excipients. Such carriers may also include flavor and color additives or other ingredients. Compositions comprising such cariers are formulated by well known coimen tional methods. Such compositions may also be formulated within various lipid compositions, such as, for example, liposomes as well as in various polymeric compositions, such as polymer microspheres.
In a further embodiment of the invention, the present invention provides kits (i.e., a packaged combination of reagents with instructions) containing the molecules of the invention useful for blocking B7 interactions with its ligands and/or for treating an immune system disease.
The kit can contain a pharmaceutical composition that includes one or more agents, for example, a soluble CTLA4 molecule alone, or with a second agent, and an acceptable carrier or adjuvant, e.g., pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use. The agents may be provided as dry powders, usually lyophilized, including excipients that upon dissolving will provide a reagent solution having the appropriate concentration.
Second agents can include the following: steroids, glucocoi-ticoids, chug toxins, alkylating agents, anti-neoplastic cliligs, enzymes, antibodies, conjugates, GO

immunosuppressive agents, corticosteroids, DMARDs, nonsteroidal antiinflammatory dnzgs (NSAIDs), prednisone, azathioprine, methotrexate, TNFcc blockers or antagonists, infliximab, any biological agent targeting an inflammatory cytokine, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopryine), gold salts, etanercept, anakinra, cyclophosphamide, leflunomide, collagen, dnaJ, a molecule that blocks TNF
receptors (e.g., pegsunercept), a molecule that blocks cytokine function{e.g., AMG719), a molecule that blocks LFA-1 function (e.g., efalizumab), acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tohnetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate, tramadol, dihydrofolic acid reductase inhibitor, cyclosporine, cyclosporin A or D-penicillamine.
The kit comprises a container with a label and/or instructions. Suitable containers include, for example, bottles, vials, and test tubes. The containers can be formed from a variety of materials such as glass or plastic. The container can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a needle such as a hypodermic injection needle). The container can hold a pharmaceutical composition such as a pharmaceutical composition having an agent that is effective for blocking B7 interactions with its ligand andlor treating an immune system disease.
The kit can also comprise a second container comprising one or more second agents as described herein (e.g., any of the DNIARDS or NSAII7S) andlor a pharmaceutically acceptable buffer, such as phosphate-buffered saline, Ringer's solution and dextrose solution. It may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instl-uctions for use.

The lcit may also suitably include a Label and/or instmtctions on, or associated with the contaisler, the Label can provide directions for carrying out the preparation of the agents for example, dissolving of the dry powders, a.nd/or treatment for a specific immune system disease.
The label and/or the instructions can indicate directions for either in vivo or in vitro use of the pharmaceutical composition. The label and/or the instructions can indicate that the pharmaceutical camposition is used alone, or in combination with a second agent.
The Label can indicate appropriate dosages for the molecules of the invention.
For example, the label can indicate that dosages for a molecule that is effective for blocking B7 interactions with its Iigand and/or treatil~.g an im~~nune system disease is about O.I to 100 mg/kg weight of the subject, about 0.5 to 5 mglkg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about I5 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mglkg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to IO mg/lcg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to I.5 mg/kg weight of a subject, about I.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/lcg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.S mg/1cg weight of a subject, about 5.5 to 6.0 mg/lcg weight of a SLIbJeCt, about 6.0 to 6.5 mg/kg weight of a. subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/lcg weight of a subject, about 7.5 to 8.0 mg/hg weight of a.

subject, about 8,0 to 8.5 mg/kg weight o_f a subject, about 8.S to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 28 mg/kg weight of a subject, about 18 to 20 mg/Icg weight of a subject, about 0.5 mg/k~
weight of the subject, 2 mg/kg weight of the subject, 10 mg/kg weight of the subject, about 0.5 mg/kg to 100 weight of the subject, about 0.5 to IO mg/kg weight of a subject, about 0.1 to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg fox a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 1 OC hg The label and/or instructions can also indicate dosages for a second agent, such as a DMARD, is about I to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 4S0 to 500 mg/day, about 500 to 5S0 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about ?00 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to mg/day, about 1400 to 1500 ing/day, about 1500 to 1600 mg/day, about 1600 to mg/day, about 1740 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to mg/day.
The label and/or the instmctions can also indicate that the pharmaceutical composition can be used alone, or in combination, dvith a second agent to treat a condition of choice e.g., immune system diseases, autoirrunune diseases, immunoproliferative diseases,. graft-related disorders, graft versus host disease (GVHD) (e.g., suCh as may result ftom bone marrow transplantation, or in the induction of tolerance), immune disorders associated with graft transplantation rejection, immune disorders associated with chronic rejection, im~.nune disorders associated with tissue or cell allo- or xenografts (e.g., kidneys, shin, S islets, muscles, hepatocytes, neurons, solid organs and the Iike), psoriasis, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, as lupus (e.g., lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, diabetes (e.g. insulin dependent diabetes mellitus, type I diabetes mellitus, type II diabetes mellitus), good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmic, autoW unune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulcerative colitis, Sjogren's ,syndrome, rheumatic diseases (e.g., rheumatoid arthritis), polymyositis, I S scleroderma, mixed connective tissue disease, and the like.
In a specific embodiment of the invention, the kit comprises a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a first agent, wherein the first agent is a molecule that blocks B7 interaction with CTLA4 and/or CD28 such as soluble CTLA4 molecules, CD28 molecules, B7 (B7-I or B7-2) molecules, anti-CTLA4 monoclonal antibodies, anti-CD28 monoclonal antibodies or anti-B7 (B7-1 ~ or B7-2) monoclonal antibodies. In preferred embodiments, the soluble CTLA4 molecules have the amino acid sequence of the extracellulax domain of as shown in either Figures 24 or I9 (CTLA4Ig or L1041;A29Y, respectively).
Methods The invention provides methods for regulating functional CTLA4- and CD28-positive cell interactions with B i-positive cells. The methods comprise contacting the positive cells with a soluble CTLA~. molecule ofthe invention so as to regulate hulCtlollal CTLA~.- a.nd CD2~- positive cell interactions with B7-positive cells, e.g., by interfering G~.

with reaction of an endogenous CTLA4 andlor CD2S molecule with a B7 molecule.
Suitable amounts of soluble CTLA4 for use in the methods of the invention are described supra.
The present invention also provides methods for inhibiting T-cell function belt not T-cell depletion in a l2uman by contacting B7-positive cells in the human with a soluble CTLA4. Examples of soluble CTLA4 include CTLA4Ig and soluble GTLA4 mutant molecule e.g. L104EA~9YIg. The present invention further provides methods for treating immune system diseases and auto-immune diseases such as rheumatic diseases.
The methods comprise administering a therapeutic composition, comprising soluble CTLA4 molecules of the invention, to a subject in an amount effective to relieve at least one of the symptoms associated with immune system diseases. Additionally, the invention may provide long-term therapy for immune system diseases by blocking the T-celllB7-positive cell interactions, thereby blocking T-cell activation/stimulation by co-stimulatory signals such as B7 binding to CD28, leading to induction of T-cell anergy or tolerance. Immune system diseases include, but are not limited to, autoimmune diseases, immunoproliferative diseases, and graft-related disorders. Examples of graft-related diseases include graft versus host disease (GVII~) (e.g., such as may result from bone marrow transplantation, or in the induction of tolerance), , immune disorders associated 2~ with graft transplantation rejection, chronic rejection, and tissue or cell allo- or xenografts, including allo- or xenografts solid organs (e.g., kidneys), skin, islets, muscles, hepatocytes, neurons. Examples of immunoproliferative diseases include, but are not limited to, psoriasis; T cell lymphoma; T cell acute lymphoblastic leulcemia;
testicular angiocentric T cell lymphoma; benign lymphocytic angiitis; and autoimmune diseases ZS such as Iupus (e.g., lupus erythematosus, lupus nephritis), Hashimoto's thyroiditis, pnmary myxederna, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, diabetes (e.g. insulin dependent diabetes mellitus, type I
diabetes melliW s, type II diabetes melliW s), good pastlue's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoiimnune uveitis, multiple 30 sclerosis, autoirninune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis. chronic action hepatitis, ulcerative colitis, Sjogren's syndrome, rheumatic bJ

diseases (e.g., rheumatoid arthritis), polymyositis, scleroderma, and mixed connective tissue disease.
The soluble CTLA4 molecules of the invention exhibit inhibitory properties ira vivo.
Under conditions where T-celI/B7-positive cell interactions, for example T
celI/B cell interactions, are occurring as a result of contact between T cells and B7-positive cells, binding of introduced CTLA4 molecules to react to B7-positive cells, for example B
cells, may interfere, i.e., inhibit, t1'~e T cell/ B7-positive cell interactions resulting in regldation of immune responses.
The invention provides methods for regulating immune responses. Lmmzme responses downregulated (reduced) by the soluble CTLA4 molecules of the invention may be by way of inhibiting or blocking an immune response already in progress or may involve preventing the induction of an immune response. The soluble CTLA4 molecules of the invention may inhibit the functions of activated T cells, such as T lymphocyte proliferation, cytokine secretion and/or cytokine production, by suppressing T
cell responses or by inducing specific tolerance in ~T cells, or both. Further, the soluble CTLA4 molecules of this invention, interfering with the CTLA4/CD28/B7 pathway may inhibit T-cell proliferation and/or cytokine secretion, and thus result in reduced tissue destruction and induction of T-cell unresponsiveness or anergy.
A preferred embodiment of the invention comprises use of the soluble CTLA4 mutant molecule L104EA?9YIg to regulate functional CTLA4- and CD?8- positive cell interactions with B7-positive cells, to treat inunune system diseases such as rheumatic diseases and/or to downremdate immune responses. The L104EA29YIg of the invention is a soluble CTL A4 mutant molecule comprising at least the two amino acid changes, the leucine (L) to glutamie acid (E) at position +104 and the alanine (A) to tyrosine (Y) change at position +29 (Figure 19). The L104EA?9YIg molecule may encompass further mutations beyond the i~vo specif ed herein.

A preferred elmbodiment of the 111Ventloil C0111pr1seS 115e Of a molecule to blocli the interaction of B7 with CTLA4 and/or CD28 in conjunction with a DMAR.D to regulate an immune response in order to treat an immune system disease such as, a rheumatic disease.
Suitable amounts of the molecule used to block the B7 interaction with CTLA4 andlor CD28 are described supra. The molecule used to block the B7/CTLA4 interaction may be a soluble CTLA4 such as CTLA4Ig, CTLA4Ig/GD28Ig or L104EA29YIg, a soluble GD28 such as CD28Ig, a soluble B7 (B7-1 or B7-2) such as B7Ig, anti-CTLA4 monoclonal antibodies, anti-CD28 monoclonal antibodies or anti-B7 monoclonal antibodies. The DMARD may be a dihydrofolic acid reductase inhibitor such as Z O methotrexate, cyclophosphamide, cyclosporine, cyclosporin A, chloroquine, hydroxychloroquine, sulfa.salazine, sulphasalazopyrine, leflunomide, gold salts, D-penicillanline, azathioprine, anakinra, iniqiximab, etanercept, TNFa 'dlockers or a biological agent that targets an inflammatory cytokine.
A preferred embodiment includes methods for treating a rheumatic disease, such as rheumatoid arthritis, by administering an effective amount of soluble CTLA4 molecules alone, or in conjunction with an effective amount of methotrexate or a molecule that blocks TNF interactions, to a subject. Administration of an effective amount of the therapeutic composition(s), thereby relieving the subject of at least one of the symptoms associated with the disease, including reducing: joint swelling, joint tenderness, inflammation, morning ' stiffiiess, and pain, and structural damage subsequently decreasing the physical disability.
The methods of the invention also may be used to reduce at Ieast one symptom associated with rheumatoid arthritis, including reducing erythrocyte sedimentation rates, serum levels of C-reactive protein, soluble ICAM-l, soluble E-selectin and/or soluble IL-2r.
The amount of symptom relief provided by the present invention can be measured using any of the accepted criteria established to measure and document symptom relief in a clinical setting. Acceptable criteria for measuring symptom relief may include scores based on the criteria established by the American College of Rhetunatology (e.g., ACR
20}, the four measures of symptom relief (in: "CDER Guideline for the Clinical Evaluation of Anti-Inflammatory and Antirhetunatic Dings-FDA 188}, and the Health Assessmer_t Questionnaire (HAQ) (Fries, J. F., et al., 1982 J. of Rlzez~matolo~ 9:789-793). For a general description of these criteria, see "Guidance for Industry:
Clinical Development Programs for Drugs, Devices, and Biological products for the Treatment of Rheumatoid Arthritis (RA)", February 1999.
The present invention provides improving ACR response rates using the methods of the invention. The embodiments of the invention include improving ACR response rates of ACR 20, 50, and/or 70, using the methods of the invention.
The subjects treated by the present invention include mammalian subjects, including, human, monkey, ape, dog, cat, cow, horse, goat, pig, rabbit, mouse and rat.
The present invention provides various methods, local or systemic, for administering the therapeutic compositions of the invention such as soluble CTLA4. molecule alone or in conjunction with a DMARD, such as methotrexate, a molecule t$at bloclcs TNF
interactions andlor other therapeutic drug. The methods include intravenous, intramusculax, intraperitoneal, oral, inhalation and subcutaneous methods, as well as implantable pump, continuous infusion, gene therapy, liposomes, suppositories, topical contact, vesicles, capsules, biodegradable polymers, hydrogels, controlled release patch.
and injection methods. The therapeutic agent, compounded with a carrier, is commonly lyophilized for storage and is reconstituted with water or a buffered solution with a neutral pH (about pH 7-8, e.g., pH 7.5) prior to administration.
As is standard practice in the art, the compositions of the invention may be administered to the subject in any pharmaceutically acceptable form.
In accordance with the practice of the invention, the methods comprise administering to a subject the soluble CTLA4 molecules of the invention to regulate CD28- andlor positive cell interactions with B7-positive cells. The B7-positive cells are contacted with an affective amo~mt of the soluble CTLA~. molecules of the invention, or fragments or derivatives thereof, so as to form soluble CTL.A~.lI37 complexes. Suitable amounts of soluble CTLA4. are described supra. The complexes interfere with interaction between endogenous CTLA4 and CD2U mOieCUieS with B7 a,ii.i.lly mvleCuieS.
The soluble CTLA4 molecules may be administered to a subject in an amount and for a time (e.g. length of time and/or multiple times) sufficient to block endogenous B7 molecules from binding their respective Iigands, in the subject. Blockage of endogenous B7/ligand binding thereby inhibiting uiteractions between B7-positive cells with CD2S-and/or CTLA4-positive cells. In an embodiment, soluble CTLA4 may be administered to a subject daily, weeldy, monthly and/or yearly, in single or multiple times per day/weel~/month/year, depending on need. For example, in one embodiment, the molecule may initially be administered once every two weeks for a month, and then once every month thereafter.
Dosage of a therapeutic agent is dependant upon many factors including, but not limited I5 to; the type of tissue affected, the type of autoimmune disease being treated, the severity of the disease, a subject's health and response to the treatment with the agents.
Accordingly, dosages of the agents can vary depending on each subject and the mode of administration. The soluble CTLA4 molecules may be administered in an amount from about 0.1 to 100 mg/lcg weight of the patient/day. Suitable amoiuzts of soluble CTLA4 are described supra. Methotrexate may be administered to a subject in an amount from about 0.1 to 100 mg/week. Suitable amounts of soluble methotrexate are described supra.
A molecule that blocks TNF interactions e.g., etanercept, may be administered to a subject in an amount from about 0.1 to 100 mg/iveek. Suitable amounts of TNF
blockers are described supra.
The invention also encompasses the use of the compositions of the inveniion together with other pharmaceutical agents to treat immune system diseases. For example, rhemnaiic diseases may be treated with molecules of the invention in conjunction with, but not limited to, immtmosuppressants such as corticosteroids, cyclosporin ('Vlathiesen 1989 C~znzcen Lest. 44(2):151-156), prednisone, azathioprine, {R. Handsch mnacher, in:
"Drugs Used for Tinrntmosuppression" pages 126x-1276), TNFcx Mockers or antagonists fib (New England Joiur~.al of lVledicine, vol. 340: 253-259, 1999; The Lancet vol.
354: 1932-39, 1999, Annals of Internal Nledicine, vol. 130: 478-486), or any other biological agent targeting any inflammatory cytokine, nonsteroidal antiinflammatory drugs/Cox-2 inhibitors, hydroxychloroquine, sulphasalazopryine, gold salts, etanercept, infliximab, rapamycin, mycophenolate mofetil, azathioprine, tacrolismus, basiliximab, cytoxan, interferon beta-1a, interferon beta-lb, glatiramer acetate, mitoxantrone hydrochloride, anal~inra and/or other biologics.
The soluble CTLA4 molecules (preferably, L104EA29YIg) can also be used in combination with one or more of the following agents to regulate an immune response:
soluble gp39 (also known as CD40 ligand (CD40L), CD154, T-BAM, TRAP), soluble CD29, soluble CD40, soluble CD80 (e.g. ATCC 68627), soluble CD86, soluble CD28 (e.g. ATCC accession number 68628), soluble GD56, soluble Thy-l, soluble CD3, soluble TCR, soluble VLA-4, soluble VCAM-1, soluble LECAM-l, soluble ELAM-1, soluble CD44, antibodies reactive with gp39 (e.g. ATCC HB-10916, ATCC HB-12055 and ATCC IiB-12056), antibodies reactive with CD40 (e.g. ATCC HB-9I10), antibodies reactive with B7 (e.g. ATCC HB-253, ATCC CRL-2223, ATCG CRL-2226, ATCC HB-301, ATCC HB-11341, ete), antibodies reactive with CD28 (e.g. ATCC HB-11944 or mAb 9.3 as described by Martin et al (J. Clin. Tmmun. 4(1):18-22, 1980), antibodies reactive with LFA-1 (e.g. ATCC HB-9579 and ATCC TIB-213), antibodies reactive with LFA-2, antibodies reactive with IL-2, antibodies reactive with IL-12, antibodies reactive with IFN-gamma, antibodies reactive with CD2, antibodies reactive with CD48, antibodies reactive with any ICAM (e.g., IGAM-1 (ATCC CRL-2252), ICAM-2 and ICAM-3), antibodies reactive with CTLA4 (e.g. ATCC HB-304)" antibodies reactive with Thy-1, antibodies reactive with CD56, antibodies reactive with CD3, antibodies reactive with CD29, antibodies reactive with TCR, antibodies reactive with VLA-4, antibodies reactive with VCA.1VI-l, antibodies reactive with LECAM-1, antibodies reactive with ~ ELAM-1, antibodies reactive with CD44. In certalll emhnc~;mPnt.~
monoclonal antibodies are preferred. In other embodiments, antibody fragments are preferred. As persons slcilled i1z the art will readily understand, the combination can include: the soluble CTLA4 molecules of the invention and one other immtmosuppressive agent; the soluble CTLA4 molecules with two other immunosuppressive agents; the soluble CTLA4 molecules with three other irnmunosuppressive agents; and the lilce. The determination of the optimal combination and dosages can be determined and optimized using methods well known in the art.
Some specific combinations include the following: LI04EA29YIg and CD80 monoclonal antibodies (mAbs); L104EA29YIg and CD86 mAbs; L104EA29YIg, CD80 mAbs, and CD86 mAbs; L104EA29YIg and gp39 mAbs; LI04EA29YIg and CD40 mAbs; L104EA29YIg and CD28 mAbs; L104EA29YIg, CD80 and CD86 mAbs, and gp39 mAbs; L104EA29YIg, CD80 and CD86 mAbs and CD40 mAbs; and L104EA29YIg, anti-LFAl mAb, and anti-gp39 mAb. A specific example of a gp39 mAb is l~ZRi. Other com'ninations will 'ue readily appreciated and understood by persons skilled in the art.
The soluble CTLA4 molecules of the invention, for example L104EA29YIg, may be administered as the sole active ingredient or together with other drugs in immunomodulating regimens or other anti-inflammatory agents such as DMARDs e.g.
for the treatment or prevention of allo- or xenograft acute or chronic rejection or inflarrnnatory or autoimmune disorders, or to induce tolerance. For example, it may be ?0 used in combination with a calcineurin inhibitor, e.g. cyclosporin A or FI~506; an immttnosuppressive macrolide, e.g. rapamycine or a derivative thereof (e.g. 40-O-(2-hydroxy)ethyl-rapamycin); a~ lymphocyte homing agent, e.g. FTY720 or an analog thereof; corticosteroids; cyelophosphamide; azathioprene; a dihydrofolic acid reductase inhibitor such as methotrexate; leflunomide or an analog thereof; mizoribine;
mycophenolic acid; mycophenolate mofetil; 15-deoxyspergualine or an analog thereof;
immunosuppressive rn_onoclonal antibodies, e.g., monoclonal antibodies to leukocyte receptors, e.g., MHC, CD2, CD3, CD4, CD llalCDlB, CD7, CD25, CD 27, B7, CD40, GD45, CD58, CD 137, ICOS, CD150 (SLANI), 0140, ~.-1BB or their ligands; or other immunomodulatory compounds, e.g. CTLA4/CD28-Ig, or other adhesion molecule iuubitors, e.g. mAbs or Iow molecular weight inhibitors including LFA-I
antagonists, Selectin antagonists and VLA--I antagonists. The compotmd is particularly useful in combination with a compound that interferes with CD40 and its 1_ig~ld, e.g.
antibodies to CD40 and antibodies to CD40-L.
Where the soluble CTLA4 mutant molecules of the invention are administered in conjunction with other imlnunosuppressive/irnmtmomodulatory or anti-inflammatory therapy, e.g. as hereinabove specified, dosages of the co-administered mununosuppressant, immunomodulatory or anti-inflammatory compound will of course vary depending on the type of co-drug employed, e.g. whether it is a steroid or a cyclosporin, on the specific drug employed, on the condition being treated and so forth.
In accordance with the foregoing the present invention provides in a yet further aspect methods as defined above comprising co-adnunis~ation, e.g. conconutantly or in sequence, of a therapeutically effective amount of soluble CTLA4 molecules of the invention, e.g. CTLA4Ig and/or L104EA29YIg, in free form or in pharmaceutically acceptable salt form, and a second drug substance, said second drug substance being an immunosuppressant, immunomodulatory or anti-inflammatory drug, e.g. as indicated above.
Further provided are therapeutic combinations, e.g, a kit, comprising a soluble CTLA4 molecule, in free form or in pharmaceutically acceptable salt form, to be used concomitantly or in sequence with at least one pharmaceutical composition comprising an immunosuppressant, immunomodulatory or anti-inflammatory drug e.g., a DMARD, NSAm,~ glucocorticoid or corticosteroid. The kit may comprise instnlctions for its administration. The kits of the invention can be used in any method of the present invention.
The invention also provides methods for producing the soluble CTLA4 mutant molecules of the invention. Expression of soluble CTLA4 mutant molecules can be in prokaryotic cells or eukaryotic cells.
JO

Prokaryotes mast frequently are represented by various strains of bacteria.
The bacteria may be a gram positive or a gram negative. Typically, gram-negative bacteria such as E.
coli are preferred. Other microbial strains may also be used. Sequences encoding soluble CTL A4 mutant molecules can be inserted into a vector designed for expressing foreign sequences in prokaryotic cells such as E. coli. These vectors can include commonlyused prokaryotic control sequences which are defined herein to include promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, including such commonly used promoters as the beta-lactamase (penicillinase) and lactose (Iac) promoter systems (Chang, et al., (1977) Nature 198:1056), the tryptophan (trp) promoter system (Goeddel, et al., (1980) Nucleic Acids Res. 8:4057) ' and the lambda derived PL promoter and N-gene ribosome binding site (Shimatake, et aL, (1981) Nature 292:128).
Such expression vectors will also include origins of replication and selectable marlcers, such as a beta-Iactamase or neomycin phosphotransferase gene conferring resistance to antibiotics, so that the vectors can replicate in bacteria and cells carrying the plasmids can be selected for when grown in the presence of antibiotics, such as ampicillin or kanamycin.
The expression plasmid can be introduced into prokaryotic cells via a variety of standard methods, including but not limited to CaCh-shock (Cohen, (1972) Proc. Natl.
Acad. Sci.
USA 69:2110, and Sambroolc et aI. (eds.), "Molecular Clonina~ A Laboratory Manual", 2nd Edition, Cold Spring Harbor Press, (1989)) and electroporation. , In accordance with the practice of the invention, eulcaryotic cells are also suitable host cells. Examples of eukaryotic cells include any animal cell, whether primary or immortalized, yeast (e.g., Saecharomyces cerevisiae Schizosaccharomyces ombe and Piclua pastoris), and plant cells. lblyeloma, COS and CHO cells are examples of animal cells that may be used as hosts. Particular CHO cells include, but are not limited to, DG4~. (Chasm, et la., 1986 Sum. Cell. Nlolec. Genet. 12:555-556; Kolkeliar Biochemistry 36:10901-10909), CHO-ill {ATCC No. CCL-61), CHO-I~l Tet-On cell line (Clontech), CHO designated ECACC 85050302 (CAlVIR, Salisbury, Wiltshire, IJK), CHO clone 13 (GEIlVIG, Genova, IT), CHO clone B (GEINIG, Genova, IT), CHO-designated ECACC 93061607 (GAMR, Salisbury, Wiltshire, UK), and RR-CHOKI
designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK). Exemplary plant cells include tobacco (whole plants, cell culture, or callus), corn, soybean, and rice cells.
Corn, soybean, and rice seeds are also acceptable.
Nucleic acid sequences encoding the CTLA4 mutant molecules can also be inserted into a vector designed for expressing foreign sequences in an eukaryotic host. The regulatory I O elements of the vector can vary according to the particular et~l~aryotic host.
Com~~only used eukaryotic control sequences for use ir~ expression vectcrs include promoters and control sequences compatible with mammalian cells such as, for example, CMV promoter (CDM8 vector) and avian sarcoma virus (ASV) (~LN vector). Other commonly used promoters include the early and late promoters from Simian Virus (SV40) (Hers, et al., (1973) Nature 273:113), or other viral promoters such as those derived from polyoma, Adenovirus 2, and bovine papilloma virus. An inducible promoter, such as hMTII (Karin, et al., (1982) Nat«re 299:797-802) may also be used.
Vectors for expressing CTLA4 mutant molecules in eukaryotes may also carry sequences called enhancer regions. These are important in optimizing gene expression and are found either upstream or downstream of the promoter region.
Examples of expression vectors for eukaryotic host cells include, but are not limited to, vectors for mammalian host cells {e.g., BPV-1, pHyg, pRSV, pSV2, pTK2 (IVIaniatis);
AIRES (Clontech); pRc/CMV2, pRc/RSV, pSFVI (Life Technologies); pVPakc Vectors, pCIVIV vectors, pSGS vectors (Stratagene)), retroviral vectors (e.g., AFB
vectors (Stratagene)), pCDNA-3 (Invitrogen) or modified forms thereof, adenoviral vectors;
Adeno-associated virus vectors, baculovinis vectors, yeast vectors {e.g., pESC
vectors (Stratagene)). ' Nucleic acid sequences encoding CTL A4 mutant molecules can integrate into the genome of the eulcaryotie host cell and replicate as the host genome replicates.
Alternatively, the vector carrying CTLA~ mutant molecules can contain origins of replication allowing for extrachromosomal replication.
S
For expressing the nucleic acid sequences in SaccharomYces cerevisiae, the origin of replication from the endogenous yeast plasmid, the 2~, circle can be used.
(Broach, (1983) Meth. Enz. 101:307). Alternatively, sequences from the yeast genome capable of promoting autonomous replication can be used (see, for example, Stinchcomb et al., (1979) Nature 282:39); Tschemper et al., (1980) Gene 10:157; and Clarke et al., (1983) Meth. Enz. 101:300).
Transcriptional control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess et aL, (1968) J. Adv. Enzyme Rep 7:149; Holland et aL, (1978) Biochemistry 17:4900). Additional promoters known in the art include the CMV
promoter provided in the CDMS vector (Toyama and Okayama, (1990) FEBS 268:217-221); the promoter for 3-phosphoglycerate kinase (Hitzeman et al., (1980) J.
Biol. Chem.
255:2073), and those for other glycolytic enzymes.
Other promoters are inducible because they can be regulated by environmental stimuli or by the growth medium of the cells. These inducible promoters include those from the genes for heat shock proteins, alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, enzymes, associated with nitrogen catabolism, and enzymes responsible for maltose and galactose utilization.
Regulatory sequences may also be placed at the 3' end of the coding sequences.
These sequences may act to stabilize messenger RNA. Such terminators are found in the 3' untranslated region following the coding sequences in several yeast-derived and manunalian genes.

Exemplary vectors for plants and plant cells include, but are not linuted to, Agrobacterium T; plasmids, cauliflower mosaic virus (Cal~IV), and tomato golden mosaic vii-as (TGMV).
General aspects of mammalian cell host system transformations have been described by Axel (LT.S. Patent No. 4,399,216 issued Aug. 1_6, 1983). Mammalian cells can be transformed by methods including but not limited to, transfection in the presence of calcium phosphate, microinjection, electroporation, or via transduction with viral vectors.
Methods for introducing foreign DNA sequences into eukaryote genomes, including plant and yeast genomes include; (1) mechanical methods, such as microinjection of DNA into single cells or protoplasts, vortexing cells with glass beads in the presence of DNA, or shooting DNA-coated tungsten or gold spheres into cells or protoplasts; (2) introducing DNA by making cell membranes permeable to macromolecules through polyethylene glycol treatment or subjection to high voltage electrical pulses (electroporation); or (3) the use of liposomes (containing cDNA) which fuse to cell membranes.
Once the CTLA4 mutant molecules of the inventions axe expressed, they can be harvested by methods well known in. the art such as cell lysis (e.g.
sonication, lysozyme and/or detergents) and protein recovery performed using standard protein purification means, e.g., affinity chromatography or ion-exchange chromatography, to yield substantially pure product (R. Scopes in: "Protein Purification Principles and Practice", Third Edition, Springer-Verlag (1994); Sambrook et al. (eds.), "Molecular Cloning: A
Laboratory Manual", 2nd Edition, Cold Spring Harbor Press, (1989)). Expression of CTLA4 mutant molecules can be detected by methods known in the art. For example, the mutant molecules can be detected by Coomassie staining SDS-PAGE gels and irnmunoblotting using antibodies that bind CTLA4.
The following examples are presented to illustrate the present invention and to assist one of ordinaay skill in making and using the same. The examples are not intended in any way to otherwise limit the scope of the uivention.

E~N~1'LE
The folloZVing provides a description of the methods used to generate the nucleotide sequences encoding the CTLA4 molecules of the invention.
A CTLA4Ig encoding plasmid was first constructed, and shown to express CTLA4Ig molecules as described in U.S. Patent Nos. 5,434,131, 5,885,579 and 5,851,795.
Then single-site mutant molecules (e.g., LI04EIg) were generated from the CTLA4Ig encoding sequence, expressed and tested for binding kinetics for various B7 molecules.
The L104EIg nucleotide sequence (as included in the sequence shown in Figure 18) was used as a template to generate the double-site CTLA4 mutant sequences (as il-~cluded in the sequences shown in Figures 19-22) which were expressed as proteins and tested for binding kinetics. The double-site CTLA4 mutant sequences include: L104EA29YIg, IS LI04EA29LIg, LI04EA29TIg, and L104EA29WIg. Triple-site mutants were also generated.
CTLA4Ia Construction A genetic construct encoding CTLA4Ig comprising the extracellular domain of CTLA4 and an IgCgammal domain was constructed as described in U.S. Patents 5,434,131, 5,844,095 and 5,851,795, the contents of which are incorporated by reference herein. The extracellular domain of the CTLA4 gene was cloned by PCR using synthetic oligonucleotides corresponding to the published sequence (Dariavach et al., Em. Tome.
T_m_m__imol. 18:190I-1905 (1988)).
Because a signal peptide for CTLA4 was not identified in the CTLA4 gene, the N-terminus of the predicted sequence of CTLA4 was fused to the signal peptide of oncostatin M (MalilL et al., lVlol. and Cell. Biol. 9:2847 (1989)) in. tW0 steps using overlapping oligonucleotides. For the first step, the oligonucleotide, CTCAGTCTGGTCCTTGG ACTCCTGTTTCC..~AGCATGGCGAGCATGGCAATGCA

CGTGGCCCAGCC (SEQ ID NO:I) (which encoded the C terminal 15 amino acids from the oncostatin M signal peptide fused to the N terminal 7 amino acids of CTLA4) was used as forward primer, and TTTGGGCTCCTGATCAGAATCTGGGCACGGTTG
(SEQ ID NO: 2) (encoding amino acid residues 119-125 of the amino acid sequence encoding CTLA4 receptor and containing a Bcl I restriction enzyme site) as reverse primer. The template for this step was cDNA synthesized from I micro g of total RNA
from H38 cells (an HTLV II infected T-cell leukemic cell line provided by Drs.
Salahudin and Gallo, NCI, Bethesda, MD). A portion of the PCR product from the first step was reamplif ed, using an overlapping forward primer, encoding the N
terminal poxtion of the oncostatin IVI signal peptide and containing a Hind III
restriction endonuclease site, CTAGCCACTGAAGCTTCACCAATGGGTGTACTGCTCACACA-GAGGACGCTGCTCAGTC T GGT CC T TGCAC T C (SEQ ID Nu: 3) and the same reverse primer. The product of the PCR reaction was digested with Hind III and Bcl I
and ligated together with a Bcl 1/Xba I cleaved cDNA fragment encoding the amino acid sequences corresponding to the hinge, CH2 and CH3 regions of IgC(gamma)I into the Hind III/~ba I cleaved expression vector, CDM8 or Hind III/Xba I cleaved expression vector piLN (also known as ~LN). .
DNA encoding the amino acid sequence corresponding to CTLA4Ig has been deposited with the ATCG under the Budapest Treaty on May 31, 1991, and has been accorded ATCC
accession number 68629.
CTLA4Ia Codon Based Muta~enesis:
A mutagenesis and screening strategy was developed to identify mutant CTLA4Ig molecules that had slower rates of dissociation ("off' rates) from CD80 and/or molecules i.e. improved binding ability. W this embodiment, mutations were carried out in and/or about the residues in the CDR-I, CDR-'? (also lmown as the C' strand) and/or CDR-3 regions of the extracellular domain of CTLA4 (as described in U.S.
Patents U.S.
Patents 6,090,914, 5,773,?53 and 5,844,095; in copending U.S. Patent Application Serial Nmnber 60/?14,065; and by Peach, R.J., et al .I Evp ~Iecl 1994 180:2049-2'058.
A CDR-like region encompasses the each GDR region and extends, by several amino acids, upstream and/or downstream of the CDF~ motif). These sites were chosen based on studies of chimeric CD28/CTLA4 fusion proteins (Peach et al., J. Exp. Med..
1994, 180:2049-2058), and on a model predicting which amino acid residue side chains would be solvent exposed, and a lack of amino acid residue identity or homology at certain positions between CD28 and GTLA4. Also, any residue which is spatially in close proximity (5 to 20 Angstrom Units) to the identified residues is considered part of the present invention.
To synthesize and screen soluble CTLA4 mutant molecules with altered affinities for a B7 molecule (e.g. CD80, CD86), a two-step strategy was , adopted. The experiments entailed first generating a library of mutations at a specific colon of an extiacellular portion of CTLA4 and then screening these by BIAcore analysis to identify mutants with altered reactivity to B7. The Biacore assay system (Pharmacia, Piscataway, N.J.) uses a surface plasmon resonance detector system that essentially involves covalent binding of either CD80Ig or GD86Ig to a dextran-coated sensor chip which is located in a detector.
The test molecule can then be injected into the chamber containing the sensor chip and the amount of complementary protein that binds can be assessed based on the change in molecular mass which is physically associated with the dextran-coated side of the sensor chip; the change in molecular mass can be measured by the detector system.
Specifically, single-site mutant nucleotide sequences were generated using non-mutated (e.g., wild-type) DNA encoding CTLA4Ig (U.S. Patent Nos: 5,434,131, 5,844,095;
5,851,795; and 5,885,796; ATGC Accession No. 68629) as a template. lVlutagenic oligonucleotide PCR primers were designed for random mutagenesis of a specific colon by allowing any base at positions 1 and 2 of the colon, but only guanine or thymine at position 3 (~~G/T or also noted as NNG/T). In this manner, a specific colon encoding an amino acid could be randomly mutated to code for each of the 20 amino acids. In that regard, u~G/T mutagenesis yields 32 potential colons encoding eacla of the 20 amino acids. PCR products encoding mutations in close proximity to the CDR3-like loop of CTLA4Ig (~'~'PP~)~ were digested with SacI/~.~bal and subcioned into similarly cut i9 CTLA4Ig (as included in Figure 24) ~cLN expression vector. Tlus method was used to generate the single-site CTLA4 mutant molecule L104EIg (as hzcluded in Figure 18).
For mutagenesis in proximity to the CDR-1-Iike loop of CTLA4Ig, a silent NheI
restriction site was first introduced 5' to this loop, by PCR primer-directed mutagenesis.
PCR products were digested with NheI/XbaI and subcloned into similarly cut CTLA4Ig or LI04EIg expression vectors. This method was used to generate the double-site CTLA4 mutant molecule L104EA29YIg (as included in Figure I9). In particular, the nucleic acid molecule encoding the single-site CTLA4 mutant molecule, L104EIg, was used as a template to generate the double-site CTLA4 mutant molecule, L104EA29YIg.
The double-site mutant nucleotide sequences encoding CTLA4 mutani molecules, such as L104EA29YIg (deposited on June 19, 2000 with the American Type Culture Collection (ATGC), 1001 University Blvd., Manassas, VA 20110-2209 and accorded ATCC accession number PTA-2104), were generated by repeating the mutagenesis procedure described above using LI04EIg as a template. This method was used to generate numerous double-site mutants nucleotide sequences such as those encoding CTLA4 molecules L104EA29YIg (as included in the sequence shown in Figure 19), L104EA29LIg (as included in the sequence shown in Figure 20), L104EA29TIg (as included in the sequence shown in Figure 21), and L104EA29WIg (as included in the sequence shown in Figure 22). Triple-site mutants, such as those encoding L104EA29YS25I~Ig, LI04EA29YS25NIg and L104EA29YS25RIg, were also generated The soluble CTLA4 molecules were expressed from the nucleotide sequences and used in the phase II clinical studies described ir~ Example 3, isaf3-a.
As those spilled-in-the-art will appreciate, replication of nucleic acid sequences, especially by PCR amplification, easily introduces base changes into DNA
strands.
However, nucleotide changes do not necessarily translate into amino acid changes as some codons redmdantly encode the same amino acid. Any changes of nucleotide from the ouiginal or wildtype sequence, silent (i.e. causing no change in the translated amino ~0 acid) or otherwise, wlule not explicitly described herein, are encompassed witlun the scope of the ir_vention.
E~~a~L~ 2 The following example provides a description of the screening methods used to identify the single- and double-site mutant CTLA polypeptides, expressed from the constructs described in Example l, that exhibited a higher binding avidity for B7 molecules, compared to non-mutated CTLA4Ig molecules.
IO
Current ifa vitro and ita vivo studies indicate that CTLA4Ig by itself is unable to completely block the primil~.g of antigen specific activated T cells. Ifa viiYa studies with CTLA4Ig and either monoclonal antibody, specific for CD80 or CD86 measuring inhibition of T cell proliferation indicate that anti-CD80 monoclonal antibody did not augment GTLA4Ig inhibition. However, anti-CD86 monoclonal antibody did augment the inhibition, indicating that CTLA4Ig was not as effective at blocking CD86 interactions. These data support earlier findings by Linsley et al. (Imm~y~t , (1994), 1:793-801) showing inhibition of CD80-mediated cellular responses required approximately 100 fold lower CTLA4Ig concentrations than for CD86-mediated responses. Based on these findings, it was sturnised that soluble CTLA4 mutant molecules having a higher avidity for CD86 than wild type CTLA4 should be better able to blocl~ the priming of antigen specific activated cells than CTLA4Ig.
To tlus end, the soluble CTLA4 mutant molecules described in Example 1 above were screened using a novel screening procedure to identify several mutations in the extracellular domain of CTLA4 that improve binding avidity for CD80 and CD86.
This screening strategy provided an effective method to directly identify mutants with apparently slower "off' rates without the need for protein purification or quantitation since "ofP' rate determiliation is concentration independent (O'Shatmessy et al., (I993) Anal. I~IOGl1e111., 212:457-=168).
. s1 COS cells were transfected with individual mi_niprep purified plasmid DNA and propagated for several days. Three day conditioned culture media was applied to BIAcore biosensor chips (Pharmacia Biotech AB, Uppsala, Sweden) coated with soluble CD80Ig or CD86Ig. The specific binding and dissociation of mutant proteins was measured by surface plasmon resonance (O'Shannessy, D. J., et al., 1997 Anal.
Biochern.
212:457-468). All experiments were run on BIAcoreT~z o_r BIAcoreT~ 2000 biosensors at 25°C. Ligands were immobilized on research grade NCMS sensor chips (Phaxmacia) using standard N-ethyl-N'-{dimethylaminopropyl) carbodiimidN-hydroxysuccinimide coupling (Johnsson, B., et al. (1991) Anal. Biochem. 198: 268-277; Khilko, S.N., et al.(1993) J. Biol. Chem 268:5425-15434).
Screeiun~ Method COS cells grown in 24 well tissue culture plates were transiently transfected with mutant CTLA4Ig. Culture media containing secreted soluble mutant CTLA4Ig was collected 3 days later.
Conditioned COS cell culture media was allowed to flow over BIA.core biosensor chips derivitized with CD86Ig or CD80Ig (as described in Greene et al., 1996 J.
Biol. Chem.
271:26762-2677I), and mutant molecules were identified with off rates slower than that observed for wild type CTLA4Ig. The DNAs corresponding to selected media samples were sequenced and more DNA prepared to perform larger scale COS cell transient transfection, from which CTLA4Ig mutant protein was prepared following protein A
purification of culture media.
BIAcore analysis conditions and equilibritun binding data analysis were performed as described in J. Greene et al. 1996 .I. Biol. Claem. 271:26762-26771 and in U.S. Patent Application Serial Nos. 09/579,927, and 60/214,065 which are herein incorporated by reference.
BIAcore Data Ailalysis 8~

Senosorgram baselines were normalized to zero response units (RU) prior to analysis.
Samples were mn over mock-derivatized flow cells to determine background RU
values due to bulk refractive index differences between solutions. Equilibrium dissociation constants (Kd) were calculated from plots of Rea versus C, where Req is the steady-state response minus the response on a mock-derivatized chip, and C is the molar concentration of analyte. Binding curves were analyzed using commercial nonlinear curve-fitting software (Prism, GraphPAD Software).
Experimental data were first fit to a model for a single ligand binding to a single receptor (1-site model, i.e., a simple langmuir system, A+B~AB), and equilibrium association constants (F~=[A]~[B]\[AB]) were calculated from L~lle equation R-Rm~~CI(~.n.dTC).
Subsequently, data were fit to the simplest two-site model of ligand binding (i.e., to a receptor having two non-interacting independent binding sites as described by the equation R--Rm~i~C\(Kdl+C)+Rm~~C\(I~2+C).
The goodness-of fits of these two models were analyzed visually by comparison with experimental data and statistically by an F test of the sums-of squares. The simpler one-site model was chosen as the best fit, unless the two-site model fit significantly better (p<0.1).
Association and disassociation analyses were performed using BIA. evaluation 2.1 Software (Pharmacia). Association rate constants ko" were calculated in two ways, assuming both homogenous single-site interactions and parallel t~.vo-site interactions. For single-site interactions, kin values were calculated according to the equation Rt=Req(I-e,~ -xs(t-to , where R is a res onse at a ven time t~ R~ is the stead -state res onse- t is P ) t P ~ > > a Y P ~ o the time at the start of the injection; and lcs dR/dt=ko"~Ckott, where C is a concentration of analyte, calculated in terms of monomeric binding sites. For two-site interactions loon values were calculated according to the equation Rt=Rzq1(1-exp ksI(t-cU)+~qG(1-eYp~~s2(t-to)_ For each model, the values of lsa" were determined from the calculated slope (to about 70°'o maximal association) of plots of Ics versus C.
s~

Dissociation data were analyzed according to one site (AB=A+B) or two site (AiBj=Ai+Bj) models, and rate constants (l~°tf) were calculated from best fit curves. The binding site model was used except when the residuals were greater than machine bacl~ground (2-10RU, according to machine}, in which case the two-binding site model was employed. Half times of receptor occupancy were calculated using the relationship IO
tli2=0.693/k°~.
Flow C ometry Murine mAb L307.4 (anti-CD80) was purchased from Becton Dickinson (San Jose, California) and IT2.2 (anti-B7-0 [also known as CD86]), from Pharmingen (San Diego, California). For immunostaini.ng, CD80-positive and/or CD86-positive CHO cells were removed from their culture vessels by incubation in phosphate-buffered saline (PBS}
I S containing l OmM EDTA. CHO cells (1-10 x I05) were first incubated with mAbs or irnmunoglobulin fusion proteins in DMEM containing IO% fetal bovine serum (FBS), then washed and incubated with fluorescein isothiocyanate-conjugated goat anti-mouse or anti-human immunoglobulin second step reagents (Tago, Burlingame, California).
Cells were given a final wash and analyzed on a FACScan (Becton Dickinson).
SDS-PAGE and Size Exclusion Chromatography SDS-PAGE was performed on Tris/glycine 4-20% acrylamide gels (Novex, San Diego, CA). Analytical gels were stained with Coomassie Blue, and images of wet gels were obtained by digital scanning. CTLA4Ig (25 fig) and LI04EA29YIg (25 ~.g) were analyzed by size exclusion chromatography using a TSI~-GEL 6300 SWxL coltunn (7.8 x 300mm, Tosohaas, Montgomeryville, PA) equilibrated in phosphate buffered saline containing 0.02% NAN3 at a flow rate of 1.0 ml/min.
CTLA4Y~l~os and LI04EA29YXc1~as_ s4 Single chain CTLA4X~mos was prepared as previously described (Linsley et al., (1995) J.
Biol. Chem., 270:15417-15424}. Briefly, an oncostatin M CTLA4 (ObZCTLA4}
expression plasmid was used as a template, the forward primer, GAGGTGATAAAGCTTCACCAATGGGTGTACTGCTCACACAG (SEQ ID NO: 4) was chosen to match sequences in the vector; and the reverse primer, GTGGTGTATTGGTCTAGATCAATCAGAATCTGGGCACGGTTC (SEQ ID NO: 5}
corresponded to the last seven amino acids (i.e. amino acids 118-124) in the extracellular domain of CTLA4, and contained a restriction enzyme site, and a stop codon (TGA).
The reverse primer specified a C120S (cysteine to serine at position 120) mutation. In particular, the nucleotide sequence GCA (nucleotides 34-36) of the reverse primer shown above is replaced with one of the following nucleotide sequences: AGA, GGA, TGA, CGA, ACT, or GCT. As persons spilled in the art will understand, the nucleotide sequence .GCA is a reversed complementary sequence of the codon TGC for cysteine.
Similarly, the nucleotide sequences AGA, GGA, TGA, CGA, ACT, or GCT are the reversed complementary sequences of the codons for serine. Polymerase chain reaction products were digested with HihdIfIlX6czI and directionally subcloned into the expression vector ~LN (Bristol-Myers Squibb Company, Princeton, NJ). L104EA29YXm2os was prepared in an identical manner. Each construct was verified by DNA
sequencing.
Identification and Biochemical Characterization of High Avidity Mutants Twenty four amino acids were chosen for mutagenesis and the resulting 2300 mutant proteins assayed for CD86Ig binding by surface plasmon resonance (SPR; as described, supra). The predominant effects of mutagenesis at each site are summarized in Table II, ifzfr~cz. Random mutagenesis of some amino acids in the CDR-1 region (S25-R33) apparently did not alter Iigand binding. Mutagenesis of E3I and R33 and residues lyl9 7-Y102 apparently resulted in reduced ligand binding. llrlutagenesis of residues, 525, A29, and T30, I~93, L96, Y103, L104, and 6105, resulted in proteins with slow "on"
and/or slow "off' rates. These results confirm previous findings that residues in the (S25-R33) region, and residues in or near 1~I97-Y102 influence ligand binding (Peach et al., (I994) J. Exp. IVIed., 180:2049-2058).
ss Mutagenesis of sites 525, T30, I~.93, L96, Y103, and GI05 resulted in the identification of some mutant proteins that had slower "off' rates from CD86Ig. However, in these instances, the slow "off' rate was compromised by a slow "an" rate that resulted in S mutant proteins with an overall avidity for CD86Ig that was apparently similar to that seen with wild type CTLA4Ig. In addition, mutagenesis of K93 resulted in significant aggregation that may have been responsible for the kinetic changes observed.
Random mutagenesis of LI04 followed by COS cell transfection and screening by SPR
of culture media samples over immobilized CD86Ig yielded six media samples containing mutant proteins with approximately 2-fold slower "off' rates than wild type CTLA4Ig. VT'nen the corresponding cDNA of these mutants were sequenced, each was found to encode a Ieucine to glutamic acid mutation (LI04E). Apparently, substitution of leucine I04 to aspartic acid (L104D) did not affect CD86Ig binding.
Mutagenesis was then repeated at each site listed in Table If, this time using L104E as the PCR template instead of wild type CTLA4Ig, as described above. SPR analysis, again using immobilized CD86Ig, identified six culture media samples from mutagenesis of alanine 29 with proteins having approximately 4-fold slower "off' rates than wild type CTLA4Ig. The two slowest were tyrosine substitutions (L104EA29Y), two were leucine (L104EA29L), one was tryptophan (LI04EA29W), and one was threonine (L104EA29T). Apparently, no slow "off' rate mutants were identified when alanine 29 was randomly mutated, alone, in wild type CTLA4Ig.
The relative molecular mass and state of aggregation of piu-ified L 104E and L104EA?9YIg was assessed by SDS-PAGE and size exclusion chromatography.
L104EA29YIg (~l ~.g; lane 3) and L104EIg (~1 yg; lane 2) apparently had the same electrophoretic mobility as CTLA4Ig (~1 p.g; lane 1) order reducing (~~OkDa;
+13ME;
plus 2-mercaptoethanol) and non-reducing (~100kDa; -131VIE) conditions (FIG.
25A).
Size exclusion chromatography demonstrated that L104EA29YIg {FIG. 25C) apparently had the same mobility as dimeric CTLA4Ig (FIG. 213), The major peaks represent protein dimes while the faster eluting minor pear ilz FIO. 25B represents higher molecular weight aggregates. Approximately 5.0% of CTLA4Ig was present as higher molecular weight aggregates but there was no evidence of aggregation of L104EA29YIg or L104EIg. Therefore, the stronger binding to CD86Ig seen with L104EIg and LI04EA29YIg could not be attributed to aggregation induced by mutagenesis.
Equilibrium and Kinetic Binding Analysis Equilibrium and kinetic binding analysis was performed on protein A purified CTLA4Ig, I0 LI04EIg, and LI04EA29YIg using surface plasmon resonance (SPR). The results are shown in Table I, iraf~~. Observed equilibrium.dissociation constants (Kd;
Table I) were calculated from binding curves generated over a range of concentrations {5.0-200 nM).
L104EA29YIg binds more strongly to CD86Ig than does L104EIg or CTLA4Ig. The lower Kd of L104EA29YIg (3.2I nM) than L104EIg (6.06 nM) or CTLA4Ig (13.9 nM) IS indicates higher binding avidity of L104EA29YIg to CD86Ig. The Iower Kd of L104EA29YIg (3.66 nM) than L104EIg (4.47 nM) or CTLA4Ig (6.SI nM) indicates higher binding avidity of L104EA29YIg to CD80Ig.
Kinetic binding analysis revealed that the comparative "on" rates for CTLA4Ig, LI04EIg, 20 and L104EA29YIg binding to CD80 were similar, as were the "on" rates for CD86Ig {Table I). However, "off' rates for these molecules were not equivalent {Table I).
Compared to CTLA4Ig, L104EA29YIg had approximately 2-fold slower "off' rate from CD80Ig, and approximately 4-fold slower "off' rate from CD86Ig. L104E had "off' rates intermediate between L104EA29YIg and CTLA4Ig. Since the introduction of these 25 mutations did not significantly affect "on" rates, the increase in avidity for CD80Ig and CD86Ig observed with LI04EA~9YIg was likely primarily due to a decrease in "off' rates, To determine whether the increase in avidity of L104EA29YIg for CD86Ig and CD80Ig 30 was due to the mutations affecting the way each monomer associated as a dimes, or whether there were avidity enhancing stmctural changes introduced into each monomer, single chain consil2icts of GTLA4 and L104EA29Y extracellular domains were prepared following mutagenesis of cysteiiie 120 to serine as described supra, and by Linsley et al., (I995) J. Biol. Chem., 270:15417-15424 (84). The purified proteins CTLA4Xm?os and 4L104EA29YXm2os were shown to be monomeric by gel permeation chromatography (Linsley et al., (1995), supra), before their ligand binding properties were analyzed by SPR. Results showed that binding affinity of both monomeric proteins for CD86Ig was approximately 35-80-fold less than that seen for their respective dimers (Table I). This supports previously published data establishing that dimerization of CTLA4 was required for lugh avidity ligand binding (Greene et al., (1996) J. Biol. Chem., 271:26762-26771).
L104EA29YXc12os bound with approximately 2-fold higher affinity than CTLA4X~I2os to both CD80Ig and CD86Ig. The ilzcreased a.~i~W y was due to approximately 3-fold slower rate of dissociation from both ligands. Therefore, stronger ligand binding by L104EA29Y was most likely due to avidity enhancing structural changes that had been introduced into each monomeric chain rather than alterations in which the molecule dimerized.
Location and Structural Analysis of Avidity Enhancing Mutations The solution structure of the extracellular IgV-like domain of CTLA4 has recently been determined by NMR spectroscopy (Metzler et al., (1997) Nature Struct. Biol., 4:527-531). This allowed accurate location of leucine 104 and alanine 29 in the three dimensional fold (FIG. 26 left and right depictions). Leucine 104 is situated near the highly conserved llrrIYPPPY amino acid sequence. Alanine 29 is sit~.~ated near the C-terminal end of the CDR-I (S25-R33) region, which is spatially adjacent to the MYPPPY
region. Wlule there is significant interaction between residues at the base of these two regions, there is apparently no direct interaction bet5,veen L104 and A29 although they both comprise part of a contiguous hydrophobic core in the proteiiZ. The stnictural consequences of the two avidity enhancing mutants were assessed by modeling.
The A29Y mutaiion can be easily accommodated in the cleft between the CDR-1 (S25-R3~) region and the MYPPPY region, and may seine to stabilize the conformation of the NIYPFPY region. h~ wild type CTLA4, L104 forms extensive hydrophobic interactions with L96 and V94 near the MYPPPY region. It is lughly unlikely that the glutamic acid mutation adopts a conformation similar to that of L104 for two reasons. First, there is insuff cient space to accommodate the longer glutamic acid side chain in tile structure without significant perturbation to the CDR-1 (S25-R33 region). Second, the energetic costs of burying the negative charge of the glutamic acid side chain in the hydrophobic region would be Iarge. Instead, modeling studies predict that the glutamic acid side chain flips out on to the surface where its charge can be stabilized by solvation.
Such a conformational change can easily be accommodated by 6105, with minimal distortion to other residues in the regions.
Binding ooh Avidit<~ Mutants to CHO Cells E~pressiln~ CD80 or CD85 FACS analysis (Fig. 27) of CTLA4Ig and mutant molecules binding to stably transfected CD80+ and CD86+CHO cells was performed as described herein. CD80-positive and CD86-positive CHO cells were incubated with increasing concentrations of CTLA4Ig, L104EA29YIg, or L104EIg, and then washed. Bound immunoglobulin.fusion protein was detected using fluorescein isothiocyanate-conjugated goat anti-human immunoglobulin.
As shown in Figure 27, CD80-positive or CD86-positive CHO cells (1.5x105) were incubated with the indicated concentrations of CTLA4Ig (closed squares), L104EA29YIg (circles), or L104EIg (triangles) for 2 hr. at 23°C, washed, and incubated with fluorescein isothiocyanate-conjugated goat anti-hmnan immwzoglobulin antibody. Binding on a total of 5,000 viable cells was analyzed (single determination] on a FACScan, and mean fluorescence intensity (MFI) was determined from data histograms using PC-LYSYS.
Data were corrected for background fluorescence measured on cells incubated with second step reagent only (MFI = 7). Control L6 mAb (80 u,ghnl) gave lIrIFI <
30. These results are representative of four independent experiments.
~0 Binding of L104EA29YIg, L104EIg, and CTLA4Ig to human CD80-transfected CHO
cells is approximately equivalent (FIG. 27A). L104EA29YIg and L104EIg bind more strongly to CHO cells stably transfected with human CD86 than does CTLA4Ig (FIG.
27B).
Functional Assays:
Human CD4-positive T cells were isolated by immunomagnetic negative selection (Linsley et al., (1992) J. Exp. Med. 176:1595-1604). Isolated CD4-positive T
cells were IO stimulated with phorbal myristate acetate (PMA) plus CD80-positive or CD86-positive CHO cells in the presence of titrating concentrations of inhibitor. CD4-positive T cells (8-10 x 104/well) were cultured in the presence of 1 nl~I P11~~A with or without irradiated CHO cell stimulators. Proliferative responses were measured by the addition of p.Ci/well of [3H]thymidine during the final 7 hours of a 72 hour culture.
Inhibition of PMA plus CD80-positive CHO, or CD86-positive CHO, stimulated T cells by LI04EA29YIg and CTLA4Ig was performed. The results are shown in FIG. 28.
L104EA29YIg inhibits proliferation of CD80-positive PMA treated CHO cells more than CTLA4Ig (FIG. 28A). L104EA29YIg is also more effective than CTLA4Ig at inhibiting proliferation of CD86-positive PMA treated CHO cells (FIG. 28B). Therefore, L104EA29YIg is a more potent inhibitor of both CD80- and CD86-mediated costimulation of T cells.
Figure 29 shows inhibition by L104EA29YIg and CTLA4Ig of allostimulated human T
cells prepared above, and further allostimulated with a human B lymphoblastoid cell line (LCL) called PM that expressed CD80 and GD86 (T cells at 3.Ox10'~/well and P1VI at 8.Ox103/well). Primary allostimulation occarred for 6 days, then the cells were pulsed with 3H-thymidine for 7 hOllrS, before incorporation of radiolabel was determil~.ed.
Secondary allostimulation was performed as Follows. Seven day primary allostimulated T cells were harvested over lymphocyte separation medium (LS1VI) (ICN, Aurora, OH) and rested for 24 hours. T cells were then restimulated (secondary), in the presence of titrating amounts of CTLA4Ig or L104EA29YIg, by adding PM in the same ratio as above. Stimulation occurred for 3 days, then the cells were pulsed with radiolabel and harvested as above. The effect of L104EA29YIg on primary allostimulated T
cells is shown in FIG. 29A. The effect of L104EA29YIg on secondary allostimulated T
cells is shown in FIG. 29B. LI04EA29YIg inhibits both primary and secondary T cell proIiferative responses better than CTLA4Ig.
To measure cytokine production (Figure 30), duplicate secondary allostimulation plates were set up. After 3 days, culture media was assayed using ELISA kits (Biosource, Camaxillo, CA) using conditions recommended by the manufacturer. LI04EA29YIg vas found to be more potent than CTLA4Ig at blocking T cell IL-2, II,-4, and y-IFN
(gamma-TrrN) cytokine production following a secondary ailogeneic stimulus (FIGS. 30A-C).
The effects of L104EA29YIg and CTLA4Ig on monkey mixed lymphocyte response IS (MLR) are shown in Figure 3I. Peripheral blood mononuclear cells (PBMC'S;
3.5x104 cells/well from each monkey) from 2 monkeys were purified over lymphocyte separation medium (LSM) and mixed with 2~,g/ml phytohemaglutinin (PHA). The cells were stimulated 3 days then pulsed with radiolabel 16 hours before harvesting.
L104EA29YIg inhibited monkey T cell proliferation better than CTLA4Ig.
Table I:
Equilibrium and apparent kinetic constants are given in the following table (values are means ~ standard deviation from three different experiments):
~:nr.~obilized.A~alyte Icon (x lcoce ( ~$~
10') x I0~3) Pz-otein 1N1'1 S'' S-1 xi111 CD80Ig CTLA4Ig 3.44 ~ 0.292.21 ~ 6.51 0.18 ~ 1.08 CD80Ig L104EIg 3.02 ~- I.35 = 4.47 0.05 0,08 = 0.36 CD80Ig L104EA29YIg 2.96 ~ 0.20I.08 -!- 3.66 0.05 ~- 0.4I

CD80Ig CTLA4~ol~os 12.0 ~ 1.0 230 CIO I95 ~

CD80Ig L104EA29YYCizos 8.3 ~ 0.26 7I ~ 5 85.0 ~ 2.5 CD86Ig CTLA4Ig 5.95 y 0.57$.16 ~ 13.9 i 0.52 2.27 CD86Ig L104EIg 7.03 ~ 0.224.26 i 6.06 ~
0.11 0.05 CD86Ig L104EA29YIg 6.42 ~ 0.402.06 ~ 3.21 ~
0.03 0.23 CD86Ig CTLA4X~IZOS 16.5 ~ O.S $40 f 55 511 ~

CD86Ig L104EA29 ~'Ymzos 11.4 ~ 1.6 300 ~ 10 267 ~

9?

Table II
The effect on CD86Ig binding by mutagenesis of CTLA4Ig at the sites listed was ' determined by SPR, described supra. The predominant effect is indicated with a "+"
sign.
a Nlutagenesis SiteEffects of iYlutagenesis No Apparent Slow "on" ratel slowReduced ligand Effect "off rate binding S25 +

P26 +

G27 +

K28 +

9 +

T30 +

E31 ~ +

R33 +

I~93 +

L96 +

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The following provides a description of phase II clinical studies of human patients administered soluble CTLA4 mutant molecule L104EA29YIg (also known as LEA29Y
or LEA) or CTLA4Ig, to relieve at least one symptom associated with rheumatoid arthritis, including reducing: joint swelling, joint tenderness, inflammation, morning stiffness, and pain. The CTLA4Ig molecule used herein begins with methionine at position +1 (or alternatively with alanine at position -1) and ends with lysine at position +357 as shown in Figure 24. DNA encoding an embodiment of the CTLA4Ig molecule has been deposited as ATCC 68629. The LI04EA29YIg molecule used herein begins with methionine at position +1 (or alternatively with alanine at position-1) and ends with lysine at position +357 as shown in Figure 19. DNA encoding an embodiment of the LI04EA29YIg molecule has been deposited as ATCC PTA 2104.
Additionally, the following provides a description of human patients administered L104EA29YIg or CTLA4Ig to relieve at least one biological surrogate marker associated with rheumatoid arthritis, including reducing erythrocyte sedimentation rates, and serum levels of C-reactive protein and/or IL2 receptor.
Patient Cohorts A total of 214 patients, including 54 males and 160 females, participated in the study (Figures lA, 1B). The patients at baseline had a mean disease duration of 3.4 (+~.0) years and had failed at least one Disease IYIodifying Antirhelunatic Drug (D1VIAR.D). Stable Nonsteroidal Anti-inflammatory Drugs (NSAIDS) or steroids (<_ 10 Ing/day) were permitted and concomitant DMARDS were prohibited. The patients were randomized into groups of 25 to 32 patients per treatment group. Thirty-two patients received a placebo, 92 received L104EA29YIg, and 90 received CTLA4Ig. The patients who Collowed protocol guidelines and did not discontinue before day 57 received a total of ~.
IntraVe110LIS Inf11510115, one infusion each on days l, 15, 29, and 57. All patients were 95.

evaluated on days l, 15, 29, 43, 57, 71, and 85. The doses administered included 0.5, 2.0, or 10.0 mg/kg of L104EA29YIg (denoted as LEA., LEA2 and LEA10, respectively in Figures lA-1E) or of CTLA4Ig (denoted as CTLA.S, CTLA2 and CTLA10, respectively ixz Figures 1A-1E).
All subjects were monitored for peri-infusional adverse events and global safety by answering a questionnaire listing potential adverse events. The patients were questioned about potential adverse events that may have occurred witlin twenty-four hours post-infusion. In addition, the patients were encouraged to spontaneously report any adverse events that they experienced. The physicians routinely monitored laboratory samples from the patients for abnormalities in blood chemistry and hematology e.g.
assessed the levels of inflammatory response mediators such as cytokines (TNF, IL-5), i:~yptase and complement. The primary endpoint was the proportion of subj ects meeting the criteria on day 85.
Storage of Test Material The CTLA4Ig and L104EA29YIg were supplied in single-use glass vials containing mg/vial of CTLA4Ig or 100 mg/vial of L104EA29YIg, respectively. Prior to infusion, the CTLA4Ig and L104EA29YIg were diluted to a final concentration of 25 mg/ml with sterile water for injection (SWFI).
Administration Protocol All infusions were administered intravenously over 1 hour (Figures 1 through 17). All subjects received at least one infusion of study medication.
Group 1: 32 patients, CTLA4Ig or L104EA29YIg matching placebo.
Group ?: 26 patients; dosage 0.J mg/hg of CTL<44Ig.
~6 Group 3: 32 patients; dosage 2.0 mg/kg of CTLA4Ig.
Group 4: 32 patients; dosage 10.0 mg/lcg of CTLA4Ig.
Group 5: 32 patients; dosage 0.5 mg/kg of L104EA29YIg.
Group 6: 29 patients; dosage 2.0 mg/kg of L104EA29YIg.
Group 7: 31 patients; dosage 10.0 mg/kg of L104EA29YIg.
Clinical Monitoring Patients were evaluated for baseline symptoms of disease activity prior to receiving any infusions. These baseline evaluations included: joint swelling, joint tenderness, I5 inflammation, morning stiffiiess, disease activity evaluated by patient and physician as well as disability evaluated by Health Questionnaire Assessment (HAQ) (reported as a physical function score in Figure 1C), and pain (Figures 1A to ID).
Additionally, the baseline evaluations included erythrocyte sedimentation rates (ESR), and ser~un levels of C-reactive protein (CRP) and soluble IL-2 receptor (IL-2r) (Figures IC and 1D).
The clinical response studies were based on the criteria established by the American College of Rheumatology (ACR). A subject satisfied the ACR20 criterion if there was a 20 percent improvement in tender and swollen joint counts and 20 percent improvement in three of the five remaining symptoms measured, such as patient and physician global disease changes, pain, disability, and an acute phase reactant (Felson, D. T., et aL, 1993 Artla~itis ahd Rheumc~tisfn 36:729-740; Felson, D. T., et al., 1995 Arthritis and Rheumatism 38:1-9). Similarly, a subject satisfied the ACR50 or ACR70 criterion if there was a 50 or 70 percent improvement, respectively, in tender and swollen joint courts and 50 or 70 percent improvement, respectively, in three of the five remaining symptoms measured, such as patient and physician global disease changes, pails, physical disability, and an acute phase reactant such as CRP or ESR.

Biomarkers Potential biomarkers of disease activity (rheumatoid factor, CRP, ESR, soluble IL-2R, soluble ICAM-I, soluble E-selectin, and MMP-3) were also assessed. Validated enzyme immunoassay (EIA) methods were used to determine the serum concentration of IL-2sRa, sICAM-l, sE-selectill and MMP-3. TNFa and IL-6 were assessed at infusion pre and 2 hours post, if necessary. -IL-2sRa, sICAM-l, and sE-selectin were measured using commercially available colorimetric EIA kits from R&D Systems, Inc. (Minneapolis, MN). The lower and upper limits of quantitation were 312-20,000 pg/mL, 40-907 nglmL and IO-206 ng/mL, respectively. The inter-assay coefficient of variation ranged from 4.48-8.4%, 3.8-5.0%
and 5.5-9.0% respectively. According to the kit manufacturer, normal serum values range from 676-2,132 pg/mL, respectively.
N.hVlP-3 was measured using a commercially available colorimetric EIA kit from Amersham Pharmacia Biotech (Piscataway, NJ). The lower and upper limits of quantitation were 30-7,680 ng/mL. The inter-assay coefficient of variation ranged from 6.3-I0.6%. According to the kit manufacturer, normal serum values range from 28-9~
ng/mL.
IL-6 and TNFa were measured using commercially available chemiluminescent EIA
kits from R&D Systems, Inc. (Minneapolis, MN). The lower and upper limits of quantitation were 0.3-3,000 pg/mL and 0.7-7,000 pg/mL, respectively. The inter-assay coefficient of variation ranged from 3.1-5.7% and 6.4-20.7%, respectively. According to the kit manufacW rer, normal serum values range from <G.3-12 pg/mL and <0.7-7.5 pg/mL.

Antibody testing Senun samples were obtained for assessment of dmg-specific antibodies prior to dosing on day 1, and approximately on days 15, 29, 57, ~S and 169. I~ue to high, preexisting titers directed to the immunoglobulin (Ig) portion of the molecule, specific antibody formation against CTLA4Ig and LEA29Y without Ig constant regions was also assessed.
Ninety-six well Tmmulon II ELISA plates (Dynex, Chantilly, Virginia) were coated with CTLA4Ig, CTLA4Ig without the Ig constant regions, LEA29Y, or LEA29Y without the Ig constant regions at 2, 4, 2, or 1 ~.g/ml in phosphate buffered saline (PBS), respectively, and incubated overnight at 2-8°C. The plates were washed with PBS
containing 0.05%
Tween 20 and blocked for 1 hour at 37°C with PBS containing 1% bovine serum albumin (BSA). The plates were then washed and serial dilutions of the test sera or quality control (QC) sera were added to the appropriate wells and incubated for 2 hours at 37°C.
Sera was diluted threefold in PBS witch 0.25% BSA and 0.05% Tween 20 starting at a 1:10 dilution. Plates were washed and an alkaline-phosphatase-conjugated goat anti-human lcappa and lambda (Southern. Biotechnology Associates, Inc., Birmingham, Alabama) antibody cocktail was added. Following a 1-hour incubation at 37°C, the plates were washed and 1 mg/ml para-nitrophenyl phosphate in diethanolamine buffer was added to each well. After 30 minutes at 25°C, the reactions were stopped with 3N
NaOH and the absorbance (dual wavelength: 405 nm and 550 nm) was recorded.
Results were expressed as endpoint titer (EPT), defined as the reciprocal of the highest dilution that resulted in an absorbance reading fivefold greater than or equal to the mean plate-background absorbance. Plate background was determined as the absorbance measurement recorded in the absence of serum. Values were considered positive for seroconversion if they were at least two serial dilutions (ninefold) or greater relative to predose EPT values. Serum QC samples positive fox either CTLA4I5 or LEA29Y-specific antibodies were generated from immmized monkeys. An aliquot of the appropriate QC sample was assayed during each analytical run. Analytical nuns were accepted only when the QC samples were within the assay acceptance criteria.

Results CTLA4Ig and L104EA29YIg were generally well-tolerated at all dose-levels. Peri-infusional adverse events were similar across all dose groups, with the exception of headaches. Headache response of patients on day 85 increased dose-dependently 23%, 44%, and 53% in CTLA4Ig-treated patients, and 34%, 45%, and 6I% in L104EA29YIb treated patients, at 0.5, 2.0, and 10.0 mg/kg respectively. In. contrast, 31%
of the patients administered placebos experienced headaches.
I O The percent of patients that discontinued from the clinical study due to arthritis flares and other adverse events is summarized in Figure 2. A much higher percentage of patients on placebo discontinued tieatment due to arthritis flare. The CTLA4Ig treated patients discontinued treatment less with increasing doses. Very few patients treated with L104EA29YIg discontinued treatment. These results indicate a good inverse dose-I5 dependent response for CTLA4Ig, and a stronger therapeutic response with L104EA29YIg therapy.
The ACR-20, -50, and -70 responses of patients treated with CTLA4Ig, LI04EA29YIg, or placebo at day 85 are summarized in Figure 3A. Similarly, Figures 3B and C
describe 20 the ACR-20 responses with 95% confidence limits. The responses appear to be dose-dependent with a clear significant response at IO mg/kg per body weight of the patient.
The percent of patients having reduced swollen and tender joint counts compared to the patients having no response to treatment with CTLA4Ig, L104EA29YIg, or placebo, is 25 shown in Figures 4A and B. The therapeutic responses appear to be dose-dependent. A
larger percentage of patients show improvement of 20, 50, 70, and even I00% in the 2 and I O mg/kg groups for both products.
The percent of patients having reduced pain, disease activity evaluated by patient and 30 physician mean score ants with CTLA4Ig, LI04EA29YTg, or placebo, is shown in Figures ~A, B, C, and D. The therapeutic responses, as monitored by the Likert scale, appear to be dose-dependent in favor of the active treatment groups as compared to placebo on day 85. The Likert scale is a validated verbal rating scale using adjectives to rank the symptoms (The American College of Rheumatology Preliminary Core Set of Disease Activity Measures for Rheumatoid Arthritis Clinical Trials: Arthritis and Rheumatism, June 1993, 36(6):729-740).
The patient and physician assessments, of disease activity change from the baseline by at least 2 units, resulting from treatment with CTLA4Ig, L104EA29YIg, or placebo, are shown in Fimtres 6A and B. The responses appear to be dose-dependent with more marked improvement for the higher doses of active drugs.
The percent reduction in C-reactive protein (CRP) levels in patients treated with CTLA4Ig, LI04EA29YIg, or placebo, is shown in Figures 7A and B. The responses appear to be dose-dependent with a clear decrease for the 2 and 10 mg/kg active treatment groups. In addition, Figure 7B showed that the difference is quite significant compared to placebo with 95% confidence intervals. Figure 7C shows the changes in senun level changes from baseline at day 85.
The amount of serum soluble IL-2 receptor in patients treated with CTLA4Ig, LI04EA29YIg, or placebo, is shown in Figure 8. The reduction in soluble IL-2 receptor levels appears to be dose-dependent.
The amount of senun soluble IGAM-l and soluble E-selectin in patients treated with CTLA4Ig, LI04EA29YIg, or placebo, is shown in Figure 33. The reduction in soluble ICAM-l and soluble E-selectin levels appears to be dose-dependent.
The median and mean tender joint counts in patients treated with CTLA4Ig or placebo over time are shown in Figures 9A and B. The change from baseline (e.g., reduction in tender joints) appears to be more important in the ? and IO mgllcg treated groups, than in 3p the placebo or 0.a mg/lcg groups.

The median and mean swollen joint counts in patients treated with CTLA4Ig or placebo over time are shown in Figures 10A amd B. The change from baseline (e.g., reduction in swollen joints) appears to be more important in the 2 and 10 mg/kg treated groups than placebo or 0.5 mg/kg groups.
The mean pain assessment scores over time in patients treated with CTLA4Ig or placebo are shown in Figure 11. The change from baseline (e.g., reduction in pain) appears to be more important in the 2 and 10 mg/kg treated groups than placebo or 0.5 mg/kg groups.
The mean disease activity assessment scores assessed by patient or physician in patients treated with CTLA4Ig or placebo over time are shown in Figures 12A and B. The change from baseline (e.g., reduction in disease acti,~ity) appears to be more important in the 2 and 10 mg/kg treated groups than placebo or 0.5 mg/kg groups.
The median and mean tender joint counts in patients treated with L104EA29YIg (denoted as LEA in the figures) or placebo over time are shown in Figures 13A and B.
The change from baseline (e.g., reduction in tender joints) appears to be dose-dependent.
The median and mean swollen joint counts in patients treated with L104EA29YIg (denoted as LEA in the figures) or placebo over time are shown in Figures 14A
and B.
The change from baseline (e.g., reduction in swollen joints) appears to be more important in the 2 and 10 mg/kg treated groups than placebo or 0.5 mg/lcg groups.
The mean pain assessment scores in patients treated with L104EA29YIg (denoted as LEA in the figures) or placebo over time are shown in Figvzre 15. The change from baseline (e.g., reduction in pain) appears to be dose-dependent.
The mean disease activity assessment scores evaluated by patient or physician in patients treated with L104EA29YIg (denoted as LEA in the f gores) or placebo over time are 3p shown in Figures 1GA and B. The change from baseline (e.g., reduction in disease activity) appears to be dose-dependent.

The percent improvement of physical disability assessed by HAQ at day 85 for patients treated with CTLA4ig, L104EA29'~'Ig, or placebo are shown in Figure 17 (Health Assessment Questionnaire (HAQ~; Fries, J. F., et al., 1982 J. of Rlzezcfnatolo~ 9:789-793). There is a clear d05e dependent improvement with tlus parameter.
The charges from baseline for soluble IL-2r and C-reactive protein levels were dose-dependent in both treatment groups. After treatment, soluble II,-2r levels were -2%, -10%, and -22% for CTLA4Ig and -4°f°, -18%, and -32% for L104EA29YIg at 0.5, 2.0, and 10.0 mg/kg respectively, compared to +3% for the placebo. C-reactive protein levels were +12%, -15%, and -32% for CTLA4Ig and +47%, -33%, and -47% for L104EA29YIg at 0.5, 2.0, and 10.0 mg/kg respectively, compared to +20% for the placebo (Figure 7A).
No clinically remarkable findings with respect to routine hematology testing, chemistry laboratory testing with the exception of slight suppressions in TgA and IgG
levels at the higher doses of both drugs, physical findings, or vital signs assessments were observed.
Notably, neither medication induced drug-specific antibodies.

The following examples describe phase II clinical studies of human patients that will be administered L104EA29YIg, to reduce or prevent structural damage, including bone or joint erosion using validated radiographic scales. This improvement in reducing or preventing str~xctural damage is parallel to the clinical improvement measured by the clinical parameters The status of the bone structure is monitored in some of the human patients prior to treatment with CTLA4Ig or L104EA29YIg. These patients are administered from 0.5 to 20 mg/kg of CTLA4Ig or L104EA29YIg chronically every two to twelve weeks (alone or in combination with other agents) to maintain their therapeutic improvement over tune. Radiographs of patients' hands and feet are taken at predefined intervals: 6 months, and then yearly, as recormnended by the FDA guidelines. These patients are monitored in long-term extension after 6 and 12 months to determine if treatment with CTLA4Ig or L104EA29YIg reduces the progression of bone deterioration, and then ~ yearly.
The patients are monitored by radiographic methods, including X-ray andlor magnetic resonance imaging (1V~I), according to standard practice in the art (Larsen, A. K. and M.
Eek 1977 Acta. Radiol. Diag. 18:481-491; Sharp, J. T., et al., 1.985 ~lrtla~itis and Rlaezc3natism 28:1326-1335). The results of the radiographic data are evaluated for prevention of structural damage, including slowing the progression of bone erosion and cartilage damage, with joint space narrowing andlor prevention of new erosions.

A St~,idy to Evaluate the Safety and Clinical Efficacy of Two Different Doses of CTLA4I~ Administered Intravenously to Subjects with Active Rheumatoid Arthritis While Receivin~lVlethotrexate Rheumatoid Arthritis BRA) treatment is rapidly changing with an increased willingness to use more aggressive therapies to achieve larger increases in efficacy and higher success rates. The ultimate goal is to improve the subject condition in a more intensive way, by raising the rate of major and complete clinical response, to treatment and maintaining this benefit with acceptable safety.
Methotrexate remains the cornerstone of the RA treatment. Tt was the first agent that demonstrated early onset of ,action, superior efficacy and tolerability compared to the ?5 classical DMARDs (e.g. gold, hydroxychloroquine, sulfasalazine) used to treat RA.
Clinical benefit may be seen as early as 3 weeks after initiating treatment, and the maximal improvement is generally achieved by 6 months. However, methotrexate has a number of limitations. For example, despite its increased tolerability, the window 'oetween efficacy and liver toxicity is quite narrow. Subjects treated with methotrexate require careful monitoring and unacceptable toxicity is often the reason for discontinuation of treatment.

Methotrexate also does not appear .to efficiently conirol disease progression or joint deterioration. For some subjects, practitioners feel compelled to add a second DMARD
with the hope of increasing efficacy despite the risk of increased toxicity.
Alternatively, co-treatment with methotrexate and a costim~lator blocker (e.g. CD80 and CD86 blockers such as CTLA4Ig) that target the auto-immune mechanism that lies upstream of the cytokine inflammatory cascade, may also increase efficacy.
As noted in Example 3, above, significant clinical responses and reductions in surrogate marlcers of disease activity were observed for CTLA4Ig at doses of 2 and 10 mg/kg with a good tolerability profile. It has also been conFrmed that the composition CTLA4Ig, used in Example 3 above, did not induce any side effects. As a result, it was decided to continue the clinical development of CTLA4Ig for rheumatoid arthritis in Phase IIB.
The following provides a description of a Phase IIB clinical study of human patients administered soluble CTLA4 molecule with methotrexate, and the results of the study after six months.
This example describes a twelve month study in which primary efficacy was assessed after all subjects completed six months of treatment or discontinue therapy.
Efficacy, safety, and disease progression were also assessed throughout the duration of the study.
The study utilized a randomized, double blind, placebo controlled, parallel dosing design.
The study was designed to evaluate the safety, clinical activity, immunogenicity and pharmacolcinetics of two doses of CTLA4Ig: 2 or 10 mg/lcg. A total of approximately 330 subjects with active RA and receiving methotrexate were randomized to 1 of 3 dosing aa~ns: CTLA4Ig at ? mg/kg (N = 110), 10 mg/lsg (N = 110) and placebo control group (N
- 110) given monthly infusions for 12 months. All groups continued on weekly methouexate treatment (IO-30 rng weeldy) (Figures 57-62).

CTLA~kIg or a placebo were also administered on Day 15. Each dose of study medication was infused intravenously over approximately 30 minutes. The primary efficacy endpoint was the ACR 20 response rate after 6 months.
For the first 6 months, subjects were not allowed to alter their doses of corticosteroids, glucocorticoids or NSAII7s. Increases in methotrexate were also not permitted during the first six months. Decreases in methotrexate were permitted only if it was felt to be causing toxicity. Subjects were treated with methotrexate for at least 6 months, and at a stable dose for 28 days prior to first treatment of CTLA4Ig or placebo. DMARDs other than methotrexate were not permitted. Low-dose stable corticosteroids use (at 10 mg daily or less) andlor use of stable non-steroidal anti-inflammatory drugs (NSAIDs), iilcluding acetyl salicylic acid (ASA), was allowed. Analgesics that did not contain ASA
or NSAlDs were permitted in subjects experiencing pain not adequately controlled by the baseline and study medications, except for 12 hours before a joint evaluation.
Decreases in NSAIDs were permitted but only if due to adverse events such as gastrointestinal toxicity.
Test Product, Dose and Mode of Administration. Duration of Treatment CTLA4Ig at 2 mg/kg or 10 mg/kg was infused every two weeps for the first month, and monthly thereafter for 12 months.
All subjects received weekly doses of methotrexate (10-30 mg) for at least six months prior to randomization and maintained at the entry dose for the first 6 months of the trial.
Doses could only be decreased for toxicity during the first six months.
Criteria for Evaluation The primary endpoint of the first stage of the study was the proportion of subjects meeting the America~.1 College of Rheumatology criteria for '?0°,°o improvement (AGR ?0) on Day 180 (month six). The ACR '?0 definition of improvement is a ?0°'o improvement from baseline in the number of tender and swollen joint counts, and a 20%
improvement from baseline in 3 of the following 5 core sat measurements: subject global assessment of pain, subject global assessment of disease activity, physician global assessment of disease activity, subject assessment of physical function and acute phase reactant value (C-reactive protein (CRP)). The evaluation for 50% improvement (ACR 50) and 70%
improvement (ACR 70) follow similarly. Subjects, who discontinued the study due to lack of efficacy (i.e. worsening RA) were considered as ACR non-responders from that time on. For all subjects who dropped out for other reasons, their ACR response at the time of discontinuation was carried forward.
Statistical Methods Two doses of CTLA4Ig (2 mg/kg and 10 mg/kg) were compared with the placebo control group. All subjects were maintained at the same stable entry doses of methotrexate. The primary analysis was the comparison of CTLA4Ig 10 mg/kg with placebo. Sample sizes were based on a 5% level (2-tailed) of significance. Based on published studies, the placebo plus methotrexate control ACR 20 response rate at 6 months is about 25%. A
sample of 107 subjects (adjusted for a possible 15% dropout) per treatment arm was determined to yield a 94% power to detect a difference of 25% at the 5% level (two-tailed). Similarly, the sample was determined to yield a power of 95% and 90%
to detect differences of 20% and 14% in ACR 50 and ACR 70, respectively. If the comparison between CTLA4Ig 10 mg/kg and placebo was significant with regards to ACR 20, then the comparison between CTLA4Tg 2 mg/lcg and placebo was carried out. This second testing should have a power of 88%. This sequentially rejective procedure based on Clu-square tests was also used to test for differences in ACR ~0 and ACR 70 responses.
All efficacy analyses were based on a data set containing all available assessments from all subjects who received at least one dose of study medication.
Percent changes from baseline were also reported for the individual components of the ACR. .For subjects who discontinued, their last observation was carried forward.

Results De~no~r a~hv .
and baseline Characteristics:

Table III: Subject Disposition and Demographics Methotrerate + IVIethotreyateMethotrexate + +

CTLA4Ig CTLA4Ig Placebo 10 mglkg 2 mgllcg Enrolled/Randomized115 105 I19 Completed 99 (86.1%) 82 (78.1%) 78 (65.5 %) Discontinued 16 (13.9%) 23 (21.9%) 4I (34.5%) - Adverse Events2 (1.7%) 7 (6.7%) 7 (5.9%) - Lack of Efficacy12 (10.4.~0) I3 (12.4%) 29 (24.4%) - Other 2 (1.7%) 3 (2.9%) 5 (4.2%) Age (yrs) - Mean ~ 55.8 (I7 54.4 (23 - 80) 54.7 (23 - 80) (Range) - 83) Weight (kg) - Mean 77.8 (40.1 78.7 (48.4 -186.8)79.9 (44 - 140) (Range) - 144) Sex . 75% females 63 % females 66% females Race 87! white 87% white 87fo white Duration of Disease (yrs) Mean SD 9.7 9.8 9.7 8.1 8.9 8.3 Demographic and baseline clinical characteristics were similar among the treatment . groups. Sixty three to 75 percent of subjects were female, 87% were Caucasian. The mean duration of the disease at entry was 9.7 ~ 9.8, 9.7 ~ 8.1, and 8.9 ~ 8.3 years respectively in the 10, 2 mg/kg and the control group. The mean weight in kg was very similar between 77.8 and 79.9 kg with a range of 40.1 to 186.8 kg (Table III).
After 6 months, more subjects had discontinued from the control group (35.5%) than from the active treatment groups; 13.9% and 21.9% for the 10 and 2 mg/kg treated groups, respectively. The main reason was lack of efficacy: with 24.3%
discontinuing in the control group, as opposed to 12.4% and 10.4°'o discontinuing in the 2 and IO mg/lcg groups, respectively. The discontinuation rate due to adverse events was Iower in the 10 mgllcg group with 1.7%, while it was 6.7% and 5.9% in the 2 mg/kg and the control groups, respectively.

During the i~rst 3-4 months, the discontinuations appeared at a faster rate in the control group compared to tile active-~ieatlment gioups. After Day 120, the discor~tinuaticr~s fcr all treatment groups stabilized for the duration of the primary treatment period (sip months).
''able ~'~: Baseline Cli~i~al Characteristics ll~Iethotrexate + blethotre~ate +
CTLA4Ig CTLA4Ig Methotre~ate +
mg/kg 2 mglkg Placebo (n =115) (n = los) (n =119) Tender Joints (mean 30.8 12.2 28.2 12,0 29.2 13.0 SD) Swollen Joints (mean 21.3 8.4 20.2 8.9 21.8 8.8 SD) Pain (VAS 100 mm) (mean62.1 21.4 64.5 22.3 65.2 22.1 SD) Physical Function 0 0.5 1.0 0.5 I .0 0.6 (l~iAQ score of 0 to ' 3) (mean SD) Subject global assessment 60.1 20.7 59.4 23.7 62.8 21.6 (VAS 100 mm) (mean SD) Physician global assessment 62,1 14.8 61.0 16.7 63.3 15.5 (VAS 100 mm) (mean SD) CRP (mg/dL) 2.9 2. 3.2 2.6 3 .2 3.2 Morning Stiffness (in 97.9 63. 104.1 63.9 106.0 64.2 min.) I

' The mean number of tender and swollen joints at baseline was comparable among the three treatment groups. The mean number of tender joints and swollen joints in the 10 mg group was 30.8 ~ 12.2 and 21.3 ~ 8.4, respectively. The mean number of tender joints 10 and swollen joints in the 2 mg group was 28.2 ~ 12.0, and 20.2 ~ 8.9, respectively. The mean number of tender joints and swollen joints in the control group was 29.2 ~ 13.0, and 21.8 ~ 8.8, respectively. These assessments and all other clinical assessments were similar among a.ll treatment groups (Table IV).

AC'I? l~eslaonses and Core CQm~onents:
Table V~ ACR Resp onse at 6 n:anths MethotreYate lYlethotrerate + + IVIethotreYate+

CTLA4Ig CTLA4Ig Placebo 10 mg/kg 2 mg/kg (n =119) (n =115) (n =105) ACR 20 60.0% 41.9% 35.3%

Difference from control24.7 6.6 -group 95% CI l I.9, 37.5 -6.2, 19.4 -p-value < O.OOI 0.3I -ACR 50 36.5% 22.9% 11.5%

Difference from control24.8 I L 1 -group 95% CI 13.8, 35.7 1.2, 20.9 -p-value < 0.001 0.027 -ACR 70 16.5% 10.5f 1.7%

Difference from control14.8 8.8 -group 95% CI 7.5, 22.2 2.7, 14.9 -p-value < 0.001 0.005 -The improvements in ACR 20, 50, and 70 response rates in the 10 mg/kg treatment group, at six months relative to the methotrexate control group, were statistically significant (Figures 34-38, 40). The improvements in ACR 50, and ACR 70 for the 2 mg/kg group were also statistically significant. The difference in ACR 20 response between the 2 mg/kg group and the control group was 6.6%. This difference was not statistically significant, p = 0.31 (Table V, Figure 49).
IO Figures 34-37 presents the ACR response rates from Day 1 to Day 180.
Figures 38 and 40 presents the ACR20, -50 and -70 response rates on day 180 for the various treatment groups. The ACR 50 and ACR 70 response rates suggest the possibility that maximal efficacy may not have been achieved at 10 mg/kg.
Figure 39 shows the proportion of new tender and swollen joints at day 180 of the study after therapy with methotrexate alone or in combination with CTLA4Ig (administered at 2 or 10 mg/kg body weight of subject).
Figure 46 shows the mean percent improvement in physical fiu~.ction from baseline as measured by HAQ.

'1'aiole VI: lndavidual ACi~ Coanponents at day 1811 '~~a~..uu Per W°.ut iuipr v'la°.iW°,W) lYIethotrexateMethotreYate + + Methotresate +

Core Components CTLA4Ig CTLA4Ig Placebo 10 mg/kg 2 mg/kg (n = 119) (n =115) (n =105) Tender Joints 59.9% 43.3% 32.1%

Swollen Joints 54.9% 45.1% 33.4.' Pain 46.4% 22.7% 8.4%

Physical Function 41.5% ~ 17.3% 14.I%
(mI~A(~) Subject global 40.8% 9.6% 17.6%
assessment Physician global 52.0% 38.6% 25.6/
assessment CRP 31.5% 16.2% -23.6%

The 2 and 10 mg/kg dose groups demonstrated some degree of efficacy among all clinical components of the ACR response criteria (Table VI; Figures 4I-45, 47-48); the subject's global assessment in the 2 mg/kg dose group being the only exception. The reduction of tender and swollen joints appears dose-dependent. The number of tender joints was decreased by 59.9%, 43.3% and 32.I% in the 10 mg/kg, 2 mg/kg and control groups, respectively. A similar pattern was observed for the swollen joint counts with a decrease of 54.9%, 45.1 % and 33.4% in the 10 mg/kg, 2 mg/kg and control groups, respectively.
The greatest differences relative to the control group were observed with the pain assessment which decreased 46.4% and 22.7% relative to baseline for 10 mg and 2 mg/kg CTLA4Ig, respectively, compared to 8.4% in the control group. The mean CRP
decreased 31.5% and 16.2% relative to baseline in the 10 and 2 mg/lcg groups compared to an increase of 23.6% in the control group.
Health-Related Quality of Life The impact of GTLA4Ig on health-related quality of life (HRQOL) was measured by the Medical Outcomes Study Short Form-36 (SF-36). The SF-3G was administered to all subjects at baseline, 90 and 180 days. The SF-36 consists of 36 items which covers eight domains (physical function, role-physical, bodily pain, general health, vitality, social iimction, role emotional, and mental health). These individual domains are used to derive the physical and mental component sununary scores which range from 0 to 100, with higher scores indicating better quality of life. Absolute differences of 5 or more in the SF-36 scores were considered clinically meaningful.
Compared to subjects treated Wlth placebo, subjects in the CTLA4Ig 10 mg/kg group also experienced statistically significantly greater improvement in all 8 domains of the SF-36 (Figure 50-51). For subjects treated with CTLA4Ig 2 mg/kg, the improvements were also greater than those treated with placebo, but the differences were not statistically si~ni.ficant (Figure 50-51).
Baseline SF-36 scores were comparable between the three treatment groups.
Improvements in quality of Iife show a clear dose-response trend after 6 months of treatment. Subjects in the CTLA4Ig 10 mg/kg treatment group demonstrated clinically and statistically significant improvements from baseline in all 8 domains of the SF-36.
The greatest effects were shown in the role-physical, bodily pain, and role-emotional domains. This positive finding was consistent with the efficacy results. For subjects treated with CTLA4Ig 2 mg/kg, improvements from baseline were also statistically significant for alI domains except mental health.
Pharmacokinetics:
~~ PHARMACOKINETIC PARAMETER VALUES
CMAx TMA.'C AUC(TAU) T-HALF CLT VSS
(~,G/ML) (H) (~.G.H/ML) (Days) (MLJH/KG) (IlKG) Z mg/kg MEAN 57.96 0.50* 10176.14 13.50 0?3 0.07 SD 16.93 (0.00,4.00) 3069.84 5.91 0.13 0.04 10 mglkg MEAN 292.09 0.50* 50102.56 13.11 0.22 0.07 SD 67.78 (0.00,4.00) 15345.95 5.32 0.09 0.03 ~' Median (n~inimum,maximum) ?0 The phannacolLinetics of CTLA~.Ig were derived from senmz concentration versus time data bete-veen dosing days 60 and 90. Samples were collected prior to dosing on day 60, at 11'' 0.5, and 4 h after dosing, on days 67, 74, 81, and prior to dosing on Day 90.
The preliminary data indicate that botr~ Cmax and AUC values increase in a proportion comparable to the dose increment. For nominal doses increasing in a I:5 proportion, both the Cmax and AUC values increased in the proportion of I:5.04 and 1:4.92, respectively.
T-HALF, CLT, and Vss values appeared to be comparable and dose independent.
Mean Vss values were 0.07 L/kg for both dose levels, which was approximately I.6-fold the plasma volume.
Pharmacodynamics:
Table VII: Mean Baseline Values for Pharmacodynamic Biomarkers Methotre~ate lYIethotre~ate + + Methotre~ate +

CTLA4Ig GTLA4Ig Placebo Biomarker 10 mg/kg Z mg/kg (n = lI9) (n =115) (n = 105 CRP (mg/dL) 2.9 3.2 3.2 RF (ITJ/L) 207 274 179 IL-2r (pg/ml)1388 1407 1398 B,_6 (pg/~) 26.7 31.7 2L4 TNFa (pg/ml)11.8 6.0 I1.9 Serum levels of pharmacodynamic biomarlcers were analyzed at various times during the study. Baseline values are shown in Table VII. The values on Day I ~0 relative to baseline are shown in the Figures 52-56.
CRP levels decreased from baseline in both CTLA4Ib treated groups more than in the control group, with greater reduction observed in the 10 mg/lcg dosing group (see Figures 47, 48 and 52).
Rhezunatoid factor levels decreased from baseline in both CTLA4Ig-treated groups more than in the control group, with greater reduction observed in the 10 mgllcg dosing group (see Figure 53).

Soluble IL-2r levels decreased from baseline in both CTLA4Ig-treated groups more than in the control group, witl? g?-eater reduction observed in the 10 m~kg dosing group (see Figure 54).
Serum IL-6 levels decreased from in both CTLA4Ig treated groups more than in the control group (see Figure 55).
The effects of CTLA4Ig on serum TNFcc levels were inconclusive. The 2 mg/kg group IO increased and the IO mg/kg group decreased relative to the control group (see Figi.~re 56).
Safe CTLA4Ig was well tolerated at all doses. There were no deaths, malignancies or opportunistic infections in any subjects receiving CTLA4Ig. Serious adverse events (SAES) and non-serious adverse events (NSAEs) were similar or less frequent in the active-treatment groups compared to the control group.
Fewer subjects in the 10 mg/kg group discontinued due to adverse events relative to the control group (1.7% vs 5.9%, respectively). The discontinuations due to adverse events in the 2 mg/kg were similar to the control group (6.7% vs 5.9%, respectively).
The SAEs followed a pattern similar to the discontinuations due to adverse events.
No serious adverse events in the IO mg/kg dose group were considered related to the study drug.
Immuno~enicity:
No anti-drug antibody responses were detected through Day 180 at both dose levels of CTLA~.Ig.
1 l~.

CTLA4Ig significantly reduced the signs and symptoms of rheumatoid aI-tlu-Itls SLlb)eCtS receiving methotrexate as assessed by ACR response criteria. The effects Of CTLA4Ig appear to increase in proportion to dose level. The improvement from baseline in all ACR core components is higher in the 10 mg/Icg group than the 2 mg/kg group.
CTLA4Ig at 10 mg/kg doses demonstrated clinically and statistically significant improvements in all 8 domains of the SF-36. All pharmacodynamic biomarkers assayed appeared to decrease in proportion to CTLA4Ig dose level except for TNFa.
CTLA4Ig was safe and well tolerated in subjects with rheumatoid arthritis receiving methotrexate.
The adverse event profile for both CTLA4Ig doses was similar to the control group.

A Study of a co-stimulation blocker CTLA4I~ Given Monthly In Combination with Etanercept to Patients with Active Rheumatoid Arthritis.
The following example provides a description of the administration of CTLA4Ig, in combination with etanercept, to treat patients with active Rheumatoid Arthritis.
Etanercept, along with infliximab, comprises a new generation of Rheumatoid Arthritis drugs which targets Tumor Necrosis Factor (TNF. Etanercept is a dimeric fusion protein having an extracellular portion of the TNF receptor linked to the Fc portion of human imrnunoglobulin (IgGl). This fusion protein binds to TNF, blocks its interactions with cell surface TNF receptors and render TNF molecules biologically inactive.
This example describes a twelve month study in which efficacy was assessed after all subjects completed six months of treatment or discontinued therapy. Efficacy, safety and disease progression were also assessed throughout the duration of the study.
The study utilized a randomized, double-blind, placebo controlled, parallel dosing design.
A total of approximately 141 subjects with active R~ and receivinb etanercept (25 mg twice weekly) were randomized to 1 of ? dosing groups: 1) a group receiving CTLA4Ig at 2 mg/kg (n = 94) plus etanercept or 2) a placebo group receiving etanercept only (n =
47).
Test Product Dose and Mode of Administration, Duration of Treatment Ali subjects received etanercept (25 mg twice weekly) for at least 3 months prior to treatment.
Infusions of CTLA4Ig were given on Days l, 15, 30, and monthly thereafter, for IO months (primary treatment phase). Each dose of study medication was infused intravenously for approximately 30 minutes.
The primary treatment phase of the study took place during the first six months of treatment. During this period, subjects were required to remain on stable doses of etanercept (25 mg twice weekly). DMARDs other than etanercept were not permitted.
Low-dose stable corticosteroid (at I0 mg daily or less) and/or stable non-steroidal anti-inflammatory drug (NSAll~), including acetyl salicylic acid (ASA), use .was allowed.
Analgesics (that do not contain ASA or NSAIDs) were permitted in subjects experiencing pain that was not adequately controlled by the baseline and study medications, except for 12 hours before a joint evaluation.
Criteria for Evaluation The primary endpoint of this study was to collect. data regarding the proportion of subjects meeting modified American College of Rheumatology (ACR) criteria for 20°f°
improvement (ACR 20) after six months. The modified ACR 20 criteria were used to accommodate the low CRP levels in tlus study's subject population. The modified ACR
criteria were defined as 1) a greater than 20% improvement in tender and swollen joint count and 2) a greater than 20°.f° improvement in 2 of the.
remaining ~. core data set ;0 measures (global pain, physician, subject, functional assessment). CRP, which is normally a part of the standard ACR core data sets, was not included in the modified 11 (i ACR criteria due to the low levels of CRP in subjects using TNF blocl~ers, such as etanercept. The standard ACR criteria, and two alternative criteria (SF-36 Physical Health and SF-36 Mental Health) were also evaluated as secondary endpoints.
Statistical Methods Treatment of a group of patients with CTLA4Ig 2 mg/kg in combination with etanercept was compared with a control group treated with placebo plus etanercept. Based on previous studies with etanercept in similar patient populations, it was assumed that the modified ACR 20 response rate (modified criteria for evaluation) at 6 months would be 35% in the control group. This is the rate of response expected among subjects who did not respond adequately to etanercept therapy. Using a 2:1 randomization, a sample of 141 (adjusted for a possible 10% dropout) subjects (47 contxol/94 CTLA4Ig) yields a 90%
power to detect a difference of 30% at the 5% level of significance (2-tailed, based on a chi-square test with no adjustment for continuity correction).
Similarly, the sample was determined to yield a power of 91% and ~3% to detect differences of 30 and 25% in ACR 50 and 70, respectively. However, due to slow enrollment, only 122 subjects were randomized and 121 treated and analyzed (one subject was randomized but never received treatment).
Demography and Baseline Characteristics Table 1: Subject Disposition at Day 180 C'rI~A4Ig + Placebo + - TOT ~L

etanercept etanercept Randomized* 85 36 121 Completed G8 (80%) 22 (61%) 90 (74,%) Discontinued 17 (20l0) 14 (39%) 31 (26%) Adverse Events6 (7.0%) 1 (2.7,'0) 7 (6%) Laclc of Efficacy6 (7.D,'o) 12 (33,'0) 18 (IS,'o) Other 5 (5.8'0,) 1 (2.7'.'~') 6 (5%) .

rExcludes one subject that did not receive treatment After siY months, the proportion of total discontinuations were higher (39%) in the placebo plus etanercept treatment group compared to the CTLA4Ig plus etanercept group (20%). The difference was driven by a higher rate of discontinuation due to lack of efficacy in the placebo plus etanercept group (Table 1).
Demographic characteristics were similar between treatment groups. The majority of subjects were female and Caucasian. The mean duration of the disease was 13 years and the mean age was 52 years (Table 2).
Table 2. Mean Baseline Demographic and Clinical Characteristics CTLA4Ig + Placebo + TOTAL
etanercept etanercept N=85 N=36 N=121 Mean Age: yrs (Range) 50 (24 - 74) 55 (28 - 52 (24 72) - 74) Mean Weight: kg (Range) 81 (45 - 154)79 (46 - 81(45 126) - 154) Gender: female: n (%) 66 (78%) 26 (72%) 92 (76%) Race: Caucasian - n (%) 80 (94%) 36 (100%) 116 (96%) Mean Duration of Disease:13.0 10.1 12.8 8.6 13.0 yrs sd 9.7 Tender Joints (out of 28.714.0 29.513.7 28.9 68) - mean sd 13.8 Swollen Joints - (out of 66) - mean ~ sd 19.6~9.4 20.3~11.0 19.8 ~ 9.9 Baseline clinical characteristics were similar between treatment groups including a mean of 29 tender joints and 20 swollen joints. With the eYCeption of CRP values, which were lo~h~er, the baseline characteristics were typical of subjects with active rheumatoid arthritis and enrolled in clinical studies (Table 2).
ACR Responses and Core Com onents The improvements in the ACR 20 and ACR 70 responses in the CTLA4Ig +
etanercept group were statistically significant compared to the CTLA4Ig + placebo group (Table 3 and Figure 63).
Table 3: lYiodifie~l ACR Response at I3ay X80 - nuanber of subjects (%)y CTLA4Ig + etanercept*** 48.2% 25.9% 10.6%

Diff. fromPlacebo+etanercept 20.5% 6.4% 10.6%

9~% CI (1.2, 39.7) (-I0.2, 23.1) (0.4, 20.8) p-Value 0.037** 0.448 0.042**

* See Criteria for Evaluation **p < 0.05 (probability for ACR response in CTLA4Ig + etanercept vs. placebo +
etanercept) *** N = 85 and N = 36 for CTLA4Ig + etanercept: and Placebo + etanercept, respectively By two months of treatment, numerically higher responses on all components of the ACR
criteria were observed for the CTLA4Ig plus etanercept group. Three of the seven ACR
components are shown in Figure 64A-C.
The mean improvements in the individual components of the ACR criteria on Day were consistently greater in the CTLA4Ig plus etanercept treatment group compared to the placebo plus etanercept group (Table 4).

Ta~'nle 4; lVlean percent (ail} $naprovemeni.
in Iudivida~ai ACS

Components ai .i~ay 18G

ACI2 Component CTI~A4Ig + Placebo +

etanercept etanercept N=85 N=36 Tender Joints 42% (5.5) 24% (8,3) Swollen Joints 37% (5.0) 21% (8.1) Pain 34% (4.3) -1% (10.8) Physical Function (i\gIA~) 3I! (5.2) -5% (13.8) Subject Global Assessment 27% (5.4) 3% (9.5) Physician Global Assessment 43% (4.3) 27% (5.8) Qualit~l of Life Compared to baseline, subjects in the CTLA4Ig plus etanercept group demonstrated statistically significant improvements at Day 180 in all 8 subscales of the SF-compared to only one (physical fimction) in subjects in. the placebo plus etanercept group.
The absolute changes in HRQOL subscales were considered clinically meaningful.
IO Compared to the placebo plus etanercept group, subjects in the CTLA4Ig plus etanercept group experienced statistically significantly greater improvement in 4 subscales of the SF-36: role-physical, bodily pain, vitality, and social function (Figure 65).
Improvements in the other 4 subscales were also greater than the placebo plus etanercept group, although they were not statistically significant.
IS
S afety No deaths or opportunistic infections occurred during the first six months of this study.
Among the most frequently reported adverse events, headache, upper respiratory ?0 infection, musculo/skeletal pain, nausea/vomiting, hypertension, and diarrhea occurred at a higher rate in the CTLA4Ig plus etanercept group compared to the placebo plus 1?0 etanercept group. Sinus abnormalities and rash were slightly higher in the CTLA~.Ig plus etanercept group, as well.
Nlore subjects in the CTLA4Ig plus etanercept group experienced serious adverse events (SAE) (7.1%) than the etanercept plus placebo group (2.8%). However, no SAES
were considered related to the study drug.
Two subjects receiving CTLA4Ig and etanercept had a dermatological malignancy.
One subject had a basal cell carcinoma that was excised after the Day 150 visit.
The other subj ect had a squamous cell carcinoma which was a pre-existing lesion that the subj ect decided to have removed after the Day I20 visit. Another subject experienced angioedema that was considered by the investigator to be a drug reaction to azithromycin.
All adverse events (AEs) leading to discontinuation were of either of mild or of moderate intensity. One discontinuation in the CTLA4Ig plus etanercept group, due to a tremor, was considered a serious adverse event.
Trn_m__uno cenicity No subjects receiving CTLA4Ig seroconverted for CTLA4Ig or CTLA4-T specific antibodies. No significant change in GIVITs for CTLA4Ig or CTLA4-T specific antibodies was observed.
1'? 1 Comparison between CTLA4I~letanercept and CTLA4I~/methotrexate ACR responses ~Tu~r'iv°. v' ~~i~a-Aa4i~ 4W °~tu'ai.e'~'W°.'~3~ 'JS.
C~LL~,4i~ +' '~Iei~u'L't'i a iu.~,y ACI2 responses (% improvement):
CTLA4Ig + CTLA4Ig +
Etanercepta Methotrerateb 101-IOI) I101-100) 2 mglkg 0 mg/kg° 10 mg/kg 2 mglkg 0 mg/kg°
N=85 N=36 ~ N= 115 N= 105 N=119.
ACR 20 48.2% a - 27.8% 60.0 %d 41.9% 35.3%
ACR 50 29.3% 19.4% 36.5% d 22.9%d 11.8 ACR 70 10.6 °/ d 0% 16.5 %d 10.5 %d 1.7 a) Modified ACR. See criteria for evaluation b) Standard ACR criteria c) Placebo + Background therapy (etanercept or methotrexate) d) p<0.05 for the difference in ACR response vs placebo + background therapy The efficacy of CTLA4Ig plus etanercept at 2 mg/kg was similar to that observed in subjects receiving the same dose of CTLA4Ig plus methotrexate therapy (Example 5).
However, the criteria for evaluation in the methotrexate (Example 5) trial was the standard ACR, that includes CRP among the core components, while in the etanercept trial (Example 6) the criteria for evaluation was the modified ACR, that excludes CRP.
Conclusion Prelinunary assessment of the study at six months found that CTLA4Ig (2 mg/kg) in combination with etanercept reduced the signs and symptoms of rheumatoid arthritis, as compared with etanercept alone. The increases in the modified ACR?0 and ACR 70 assays were. statistically significant. Efficacy of C T LA4Ig plus etanercept therapy was observed within one month of the start of treatment. CTLA4Ig was generally safe and well tolerated when administered in combination with etanercept with the safety profile similar to etanercept therapy alone. CTLA4Ig was not ilmm.ogenic during the six month trial period. Additionally, the efficacy of CTLA4Ig therapy in combination with 12?

etanercept (Example 6) was similar to the same dose of CTLA4Ig with methotrexate (Example 5).

One-Year Results of a Phase IIB Multicenter, Randomized, Double-Blind, Placebo-Controlled Study to Evaluate the Safety and Clinical Efficacy of Two Different Doses of BMS-188667 Administered Intravenously to Subjects with Active Rheumatoid Arthritis While Receiving Methotrexate.
The following example provides the one-year results from a Phase IIB, mufti-center, randomized, double-blind, placebo controlled clinical study to evaluate the safety and clinical efficacy of administering two different doses of CTLA4Ig in combination with methotrexate to treat patients with active Rheumatoid Arthritis (RA). The study presented in this example is a continuance of the six-month study presented in Example 5.
Based on preliminary efficacy results from Example 3, supra, and the standard practice of adding other therapies to MTX in the treatment of RA, this study was designed to test the hypothesis that CTLA4Ig (BMS-188667) combined with MTX may have greater clinical efficacy when compared with MTX plus placebo in RA subjects who still have active disease despite MTX treatment.
The results presented in this clinical study report are based on data from an analysis performed after all subj ects completed 6 months of treatment and again after all subj ects completed 12 months of treatment.
Throughout this Example, the 10 mg/kg CTLA4Ig plus MTX group may be referred to as the 10 mg/kg group, the 2 mg/kg plus MTX group is referred to as the 2 mg/kg group, and the CTLA4Ig (BMS-188667) placebo plus MTX group is referred to as the placebo group.

STUDY METHODOLOGY
This study compared the clinical efficacy of two different doses (10 and 2 mg/lcg) of CTLA4Ig (BMS-188667) combined with methotrexate (MTX) or with MTX plus placebo in subjects with active RA as assessed by ACR at 6 month and 12 month intervals. This study enrolled adult subjects with active R.A who had had an inadequate response to MTX.
Results after one-year of monitoring subjects with active rheumatoid arthritis who were intravenously administere: 1) CTLA4Ig at a dosage of 2 mg/kg body weight with methotrexate, 2) CTLA4Ig at a dosage of 10 mg/kg body weight with methotrexate, or 3) a placebo with methotrexate (hereinafter known as placebo), are presented herein.
Subjects with active R.A, despite treatment with MTX and who met the inclusion/exclusion criteria for this study were randomized 1:1:1 to receive one of the following treatments on a background of MTX therapy: CTLA4Ig (BMS-188667) 10 mg/kg, CTLA4Ig (BMS-188667) 2 mg/kg, or placebo. Subjeots must have been treated with MTX (10 mg to 30 mg weekly) for at least 6 months, at a stable dose for 28 days prior to Day 1.
Treatment Groups: Subjects were randomized 1:1:1 to one of three treatment groups:
1) Group 1: CTLA4Ig (BMS-188667) 10 mg/kg by intravenous infusion 2) Group 2: CTLA4Ig (BMS-188667) 2 mg/kg by intravenous infusion 3) Group 3: CTLA4Ig (BMS-188667) placebo by intravenous infusion Infusion doses were based upon the subject's body weight from the pre-treatment visit immediately prior to the Day 1 visit (for a subject on MTX monotherapy, the weight was obtained at the screening visit; for a subj ect on MTX combination therapy [in combination with other DMARDs], the weight was obtained from the washout visit, Day -2). The infusion doses were not modified during Day 1 to Day 360.

Infusions were to occur at approximately the same time of day throughout the study. All doses of study medication were administered in a fixed volume of 75 mL at a constant rate over approximately 30 minutes. The intravenous bag and line were flushed with 25 mL of dextrose 5% in water (DSW) solution at the end of each infusion. All intravenous infusions were administered with the subject in the seated position. Subjects .
were observed for Adverse Events (Aes) and changes in vital signs (blood pressure, heart rate, body temperature) from the start of each infusion (pre-dose, 15, 30, 45, 60, 75, 90, 120 minutes) and for a minimum of 2 hours after the start of the infusion. The observation period could be extended, if clinically indicated.
During the primary phase (Day 1 to Day 180) of the study, concomitant administration of selected medications was allowed. The permitted medications included:
~ MTX: Continued use of current dose (no increases, and decreases only for toxicity) ~ Systemic (non-topical) corticosteroids: Provided that the dose was stable and the total dose was less than or equal to the equivalent of prednisone 10 mg/day.
Infra-articular injections were to be avoided; however, if necessary, up to two infra-articular injections were permitted. NOTE: A joint that received an infra-articular injection was counted as "active" in ALL subsequent assessments/evaluations.
~ NSAIDs, including ASA: Provided the dose was stable ~ Acetaminophen, combination products including acetaminophen and narcotic analgesics (i.e., acetaminophen with codeine phosphate, acetaminophen with propoxyphene napsylate, acetaminophen with oxycodone hydrochloride, acetaminophen with oxycodone bitartrate, etc.), or tramadol: For subjects experiencing pain not adequately controlled by baseline or study medication (except for 12 hours before a joint evaluation) Table 1 is a schedule of study procedures and evaluations.

Table 1: Schedule of Study Procedures and Evaluations Pretreatment Treatment Period a Treatment Da '~r~~~' Screen Visit Day (-28 (-2)1 15306090120150180210240270300330360 to -2) Screening assessments Informed consent X

Complete History X X' and Physical CXR X

ECG X" X

Stabilize/Withdraw prohibited medications (ifnecessary)bX
.

Enroll Subject X X"' Randomize Subjectk X

Dosing' X X X X X X X X X X X X X

Interim Assessmentsr Duration of morningX X X X X X X X X X X X
stiffness Interim History X X X X X X X X X X
and Physicai Tenderjointcount X X X X X X X X X X X X X

Swollen joint X X X X X X X X X X X X X
count Subject's assessmentX X X X X X X X X X X X
of pain Subject's global assess of disease activity X X X X X X X X X X X X

Physician's global assess of disease activity X X X X X X X X X X X X

Subject's assess of physical function X X X X X X X X X X X X

Short form-36 health questionnaire X X X X X
(SF-36) Subjects response X X X
to therapy Safety Assessments Adverse event X X X X X X X X X X X X X X
monitoring Weightb X X X

Mammogram (femalesX X
only) Vital si s X X X X X X X X X X X X X X X

Labs CBC X X X X X X X X X X X X X X X

Chemistry panel X X X X X X X X X X X X X X X

Urinalysis X X

Urine/serum pregnancyX X X X X X X X X X X X X X X
tests Hepatitis B surfaceX
antigen Hepatitis C antibodyX

Pharmacodynamics (PD) Rheumatoid factorX X X X

CRP X X X X X X X X X X X X X

Exploratory cytokines (ICAM-1 e-Selectin, IL-6 X X X X
and TNFa) Pharmacokinetics X X X X

Immunoglobulin determinations Quantitative immunoglobulins (I G,I A,I M) X X X

Immunogenicity Anti-BMS-188667Ab X X X X X X
testin Radiographic assessments"
I

X-rays (hands/wrists X X X
and feet) Period Pretreatment ( Screen ) ~ (- ) Visit Da -28 to -2 2 1 15 30 60 90 120 150 180 210 240 270 300 330 360 a Chest X-ray and ECG was performed if not performed within 6 months or not on file.
b If subject was being treated with DMARDs on top of methotrexate therapy and did not meet initial entry criteria, the DMARDs must have been washed out for at least 28 days prior to Day 1.
c This visit was required only if the subject was on MTX therapy.
d Urine or serum pregnancy test performed within 48 hours prior to dosing, for all women of child bearing potential. Serum pregnancy test was to be processed locally.
a Subjects who discontinued must have had an "early termination" visit.
Assessments at this visit were identical to assessments performed on Day 360. The assessments for this visit replaced what might have been scheduled on the day of discontinuation. Changes in current DMARD, steroid, or NSAIDs therapy were not permitted until after these assessments were performed. Subjects were to be contacted 30 days after discontinuation to capture safety data (adverse events).
f Every effort must have been made to insure the same evaluator completed the assessment for each subject.
g Most recent weight should have been used to calculate study drug dosage. All doses administered during the study were be based on this weight.
h For Day 15, a +/- 3 day visit window was permitted. For subsequent visits, a +/- 7 day visit window was permitted.
i Complete physical examination only.
j All assessments should have been performed or administered prior to study drug administration unless otherwise indicated.
k The results of all assessments must have been reviewed for eligibility requirements before contacting the Central Randomization System for randomization.
I See Section 2.1.4.3 of the protocol for mammography rationale. If not performed within 6 months (documentation must be on file) prior to signing consent. Subjects who discontinued from the study after Day 1 required a follow-up mammogram on the one year anniversary of the mammogram that was performed during the screening period.
m Subject's body weight was provided to central randomization system.
n No radiographic assessments were required at the termination visit for subjects who discontinued within the first nine months of treatment.
o Subjects who were terminated early had adverse events and concomitant medications recorded 30 and 60 days after the last dose of study medication.
Efficacy Assessments Clinical Measurements and Responses Clinical response was assessed using the American College of Rheulnatology (ACR) Core Data Set and Response Definitions. For this assessment, data were collected on seven components: 1) tender joint count (standardized 68 joint count); 2) swollen joint count (standardized 66 joint count); 3) subject global assessment of pain; 4) subject global assessment of disease activity; 5) physician global assessment of disease activity;
6) subject assessment of physical function (MHAQ); and 7) an acute phase reactant value CRP.

The ACR 20, ACR 50, and ACR 70 definition of response corresponds to a 20%, 50%, or 70% improvement, respectively, over baseline in tender and swollen joints (components 1 and 2) and a 20%, 50%, and 70% improvement, respectively, in three of the five remaining core data set measures (components 3 to 7). A Major Clinical Response is defined as maintenance of an ACR 70 response over a continuous 6-month period.
See Table 1 for the days that data for each component was collected.
The primary efficacy analysis tested for differences in ACR 20 response between the two CTLA4Ig (BMS-188667) treatment groups and the placebo group at 6 months (Day 180).
A sequential testing procedure was employed. First, a Chi-square test was used to compare the data for the 10 mg/kg CTLA4Ig group with the data for the placebo group at the 0.05 level of significance. If this was significant, the data for the 2 mg/kg CTLA4Ig group was compared with the placebo group at the 0.05 level. This testing procedure preserved the overall alpha level at 5%. Similar analyses were carried out for the ACR 50 and ACR 70 responses at 6 months. Differences in ACR 20, ACR 50 and ACR 70 responses between each CTLA4Ig (BMS-188667) treatment group and the placebo group were summarized using point estimates and 95% confidence intervals.
Subjects who discontinued the study due to lack of efficacy (i.e., worsening RA) were considered ACR non-responders at all subsequent time points. For all subjects who discontinued for other reasons, their last ACR response was carned forward.
ACR 20, ACR 50, and ACR 70 response rates on Day 360 were compared between each CTLA4Ig (BMS-188667) treatment group and placebo at the Dumlett-adjusted 0.027 (two-tailed) level of significance.
The proportion of responders achieving an ACR 20 response at each time point was also plotted over time, and the Cochran Mantel-Haenszel test (W.G. Cochran, 1954, Some Methods of Strengthening the Common Chi-Square Test, Biometrics 10:417-451; N.
Mantel and W. Haenszel, 1959, Biostatistical Aspects of the Analysis of Data from Retrospective Studies of Disease, J Nat Cancer Inst, 22:719-748) was used to compare the frequency of subjects achieving an ACR 20 response in each CTLA4Ig (BMS-188667) group versus the placebo group.
ACR 20, ACR 50, and ACR 70 responses on Days 15, 30, 60, 90, 120, 150, 180, 240, 300, and 360 were also presented for the two CTLA4Ig (BMS-188667) groups and the placebo group. The differences in ACR responses between the CTLA4Ig (BMS-188667) groups and placebo group were summarized using 95% confidence intervals. The ACR data plotted over time were used to assess onset-of action and to determine time to maximal response.
A Major Clinical Response was defined as the maintenance of an ACR 70 response over a continuous 6-month period. At the 12-month analysis, the proportion of subjects who achieved a Major Clinical Response among the three groups was summarized.
In order to assess the integrity of the planned analyses, all subjects who received study medication and discontinued the study for any reason were considered ACR non-responders at all scheduled study visits subsequent to discontinuation.
The cumulative index, ACR-N, was evaluated at each follow-up assessment, and the AUC was assessed for up to 6 months and up to 12 months. The trapezoidal rule was used to compute the AUC. The ACR-N AUC was compared between the two CTLA4Ig (BMS-188667) treatment groups and the placebo group using an analysis of variance (ANOVA) for 6- and 12-month data. This allowed for the assessment of subject response throughout the study. These analyses were carried out on the LOCF data sets.
The distributional assumptions regarding the normality of the ACR-N AUC data were checked using the Shapiro-Wilks test on standardized residuals from the ANOVA
model at the 10% level of significance.
Surrogate biomarkers were also used to assess the efficacy of the CTLA4Ig +
MTX or placebo + MTX treatment regimens. Potential biomarkers for immunomodulation or inflammation in RA include CRP, soluble IL-2r, RF, soluble ICAM-1, E-selectin, serum IL-6, and TNFa. These parameters were summarized by treatment group, using frequencies and mean change from baseline to Day 180 and Day 360.
An Adverse Event (AE) was defined as any new or worsening illness, sign symptom or clinically significant laboratory test abnormality noted by the Investigator during the course of the study, regardless of causality. A serious adverse event (SAE) was defined as an AE that met any of the following criteria: was fatal; was life-threatening; resulted in or prolonged hospitalization; resulted in persistent or significant disability or incapacity, was cancer, was a congenital anomaly/birth defect, resulted in an overdose, resulted in the development of drug dependency or drug abuse, or was an important medical event.
Vital sign measurements were obtained at screening and at each study visit during and following study drug administration. Vital sign measurements (seated blood pressure, heart rate, and body temperature) were summarized by treatment group using means.
The two CTLA4Ig (BMS-188667) treatment groups (10 and 2 mg/kg) were compared with the placebo group. The primary analysis was the comparison of 6-month ACR response rate for 10 mg/kg and placebo groups, to be followed by the comparison of 2 mg/kg with placebo only if the former was significant. Sample sizes were based on a 5% level (two-tailed) of significance. The ACR 20 response rate for a placebo group at 6 months was estimated to be about 25% (Weinblatt M, I~remer JM, Bankhurst AD
et. al.
A trial of etanercept, a recombinant TNF:Tc fusion protein in patients with RA
receiving methotrexate. NEJM 1999; 340: 253-259). A sample of 107 subjects per treatment group (adjusted for a possible 15% discontinuation rate) was determined to yield a 94% power to detect a difference of 25% at the 5% level (two-tailed). Table 2 summarizes the power needed to detect the specified treatment differences in ACR 20, ACR 50, and responses at 6 months.

Table 2: Response Rates and Power with 107a Subjects per Group Response Control Rate (%) Treatment Difference Power (%) aSample size was adjusted for a possible 15% discontinuation rate; actual sample size was 91.
If the primary comparisons of the 10 mg/kg CTLA4Ig with placebo were significant, then for the comparison of the 2 mg/kg CTLA4Ig with placebo groups, the power of the test would be at least 0.88, 0.90, and 0.81 for the comparison involving ACR 20, ACR 50, and ACR 70 responses at 6 months, respectively (Koch DD, Gansky SA.
Statistical considerations for multiplicity in confirmatory protocols. Drug Info Journal 1996; 30:
523-534).
Statistical Analyses STUDY POPULATION
Disposition of Subjects Of 524 subjects who were enrolled in this study, 339 subjects were randomized:
115 to the 10 mg/kg group; 105 to the 2 mg/kg group; and 119 to the placebo group (Figure 68).
The most frequent reason for not being randomized was failure to meet inclusion and/or exclusion criteria.
Primary Phase (Days 1-180) A total of 256 subjects (75.5% of those randomized) completed the primary phase of the study; 83 subjects discontinued during this period (Table 3). Overall, discontinuation was more than 2-fold higher with placebo compared with 10 mg/kg CTLA4Ig group.

Discontinuation due to lack of efficacy and discontinuation due to an AE were also more than 2-fold higher with placebo than with 10 mg/kg CTLA4Ig group.
Table 3: Reasons for Discontinuation: Primary Phase (Days 1-180) CTLA4Ig (BMS 188667) 10 mg/kg 2 mglkg Placebo Total No. Treated, n 115 105 119 339 No. Discontinued, n (%) 17 (14.8)25 (23.8)41 (34.5)83 (24.5) Adverse Event 3 (2.6) 7 (6.7) 9 (7.6) 19 (5.6) Lack of Efficacy 12 (10.4)16 (15.2)28 (23.5)56 (16.5) Withdrawal of Consent 2 (1.7) 2 (1.9) 4 (3.4) 8 (2.4) Completed 180 Days of 98 (85.2)80 (76.2)78 (65.5)256 (75.5) Therapy, n (%) Cumulative Discontinuations (Days 1-360) A total of 235 subjects (69.3% of those randomized) completed both phases of the study;
104 subjects discontinued by Day 360 (Table 4). The same general pattern in disontinuations noted in the primary phase (2-fold higher incidence with placebo compared with 10 mg/kg CTLA4Ig group) was also observed overall (Days 1-360).
This included the overall discontinuation rate, discontinuations due to a lack of efficacy and discontinuations due to an AE.

Table 4: Reasons for Discontinuation: Both Phases (Days 1- 360) CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo Total No. Treated, n 115 105 119 339 No. Discontinued, n 25 (21.7)31 (29.5) 48 (40.3)104 (30.7) (%) Adverse Event 5 (4.3)1'9 (8.6) 11 (9.2) 25 (7.4) Death 0 1 (1.0) 0 1 (0.3) Lost to Follow-up 1 (0.9) 2 (1.9) 0 3 (0.9) Othera 1 (0.9) 0 1 (0.8) 2 (0.6) Lack of Efficacy 13 (11.3)17 (16.2) 30 (25.2)60 (17.7) Withdrawal of Consent 5 (4.3) 2 (1.9) 6 (5.0) 13 (3.8) Completed 360 Days of 90 (78.3)74 (70.5) 71 (59.7)235 (69.3) Therapy, n (%) a Other reasons for discontinuation were related to compliance f Subject IM101100-32-5 in the 10 mg/kg CTLA4Ig group reported an AE that was recorded as having resulted in discontinuation from the study; however, this subject was not included in this table.
A Kaplan-Meier plot of the cumulative proportion of subjects who discontinued for any reason during the first 12 months is presented in Figure 69; the cumulative proportion of subjects who discontinued due to lack of efficacy in presented in Figure 70.
Note that in both graphs after approximately 30 days of therapy, discontinuation rates with placebo were consistently higher compared with both CTLA4Ig (BMS-1~~667) groups.
Additionally, after approximately 150 days of therapy, discontinuation rates for 2 mglkg CTLA4Ig group were higher than those for 10 mg/kg.
Demography and Subject Characteristics Overall, baseline demographic characteristics and baseline clinical RA
characteristics were generally comparable across the three treatment groups and were typical of relatively advanced RA encountered in clinical practice (Table 5 and Table 6).
The majority of subjects were white females approximately 55 years old with a mean duration of RA of approximately 9 to 10 years, a relatively large number of active joints (approximately 29 tender and 21 swollen joints) and visual analogue scores (VAS) approximately 59-65 mm (100 mm scale).

Table 5: Baseline Demographic Characteristics CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo No. Randomized 115 105 119 Age (yrs) Mean SD (Range) 55.812.5 (17, 54.411.3 (23, 54.712.0 (23, 83) 80) 80) Weight (kg) Mean SD (Range) 77.818.6 (40.1,78.721.4 (48.4,79.917.6 (44.0, 144.0) 186.8) 140.0) Gender Males, n (%) 29 (25) 39 (37) 40 (34) Females, n (%) 86 (75) 66 (63) 79 (66) Race White, n (%) 100 (87) 91 (87) 104 (87) Black, n (%) 6 (5) 0 3 (3) Other, n (%) 9 (8) 14 (13) 12 (10) Duration of RA n=114a n=105 n=117a (yrs) Mean SD (Range) 9,79,g (0, 38) 9.78.1 (0, g,9g,3 (0, 41) 36) Error! Bookmark Duration of not defined. RA was not reported for 3 subjects.

Although not a component of the ACR criteria, duration of morning stiffness was also assessed and was nearly 2 hours in each of the three groups. Positive results for RF at baseline were also assessed, and the CTLA4Ig (BMS-188667) treatment groups had higher percentages of subjects who tested positive for RF (86% for both the 10 mg/kg and 2 mg/lcg CTLA4Ig groups compared to 76% for the placebo group).

Table 6: Baseline Clinical Rheumatoid Arthritis Characteristics CTLA4Ig (BMS-188667) Characteristic 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) Tender Joints, n 115 105 119 Mean SD 30.8 12.2 28.2 12.0 29.2 13.0 Range 11.0, 66.0 3.0, 62.0 4.0, 68.0 Swollen Joints, n 115 105 119 MeanSD 21.38.4 20.28.9 21.88.8 Range 9.0, 54.0 4.0, 48.0 8.0, 64.0 Pain (VAS 100 mm), n 113 104 119 Mean SD 62.1 21.4 64.3 22.3 65.2 22.1 Range 0.0, 99.0 8.0, 100.0 3.0, 95.0 Physical Function (MHAQ 115 105 119 0-3), n MeanSD 1.00.5 1.00.5 1.00.6 Range 0.0, 2.5 0.0, 2.5 0.0, 2.3 Subject Global Assess 113 105 119 (VAS 100 mm), n Mean SD 60.1 20.7 59.4 23.7 62.8 21.6 Range 10.0, 100.08.0, 99.0 4.0, 94.0 MD Global Assess (VAS 113 105 119 100 mm), n Mean SD 62.1 14.8 61.0 16.7 63.3 15.5 Range 20.0, 98.0 8.0, 95.0 18.0, 93.0 CRP (mg/dL), n 112 99 115 Mean SD 2.9 2.8 3.2 2.5 3.2 3.2 Range 0.2, 19.9 0.2, 10.8 0.2, 20.9 Morning Stiffness (in 115 103 119 minutes), n Mean SD 97.9 63.1 104.1 63.9 106.0 64.2 Range 0.0, 180.0 0.0, 180.0 0.0, 180.0 Rheumatoid Factor (lU/mL),99 90 90 n Positive 86% 86% 76%

Baseline demographics and RA characteristics of the overall population of subjects who had at least one dose of study drug and discontinued due to lack of efficacy were generally comparable to the entire study population, however, a greater proportion of subjects in this subpopulation had been diagnosed with IAA for >10 years (45%) compared to the overall study population (34%).
Medical History Findings and Prior Medications Medical history findings for subj ects in this study were consistent with relatively advanced RA and were generally similar among treatment groups. The most frequently occurring findings (in >40% of the subjects) were musculoskeletal findings (not including RA symptoms; 59.3%), gastrointestinal findings (45.1%), and genitourinary findings (42.2%). Other important medical history findings included cardiovascular disease in approximatelyr39% of subjects in all treatment groups and endocrine/metabolic findings in approximately 29% of all subjects.
Overall use of MTX, systemic (non-topical) corticosteroids, DMARDs and biologic RA
medications prior to entering the study was generally comparable across the three treatment groups (Table 7). All subjects were to have received prior treatment with rheumatic medications, including MTX, to be eligible for the study. Prior treatment with MTX was not recorded for 4 subjects. Systemic (non-topical) corticosteroid use prior to randomization was comparable among the three treatment groups, with slightly more subjects in the 2 mg/kg CTLA4Ig and placebo groups taking systemic (non-topical) corticosteroids (~67-68%) compared to subjects in the 10 mg/kg CTLA4Ig group (60.0%). Use of other DMARDs and biologic R.A medications prior to entering the study varied from 0 to 12% across treatment groups with no overall predominance in any treatment group. Mean dosing of MTX and of systemic (non-topical) corticosteroids on Day 1 were comparable among all three treatment groups (~15-16 mg/wk, ~6-7 mgs/day, respectively.

Table 7: Summary of Rheumatic Medications Prior to Enrollment CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo Prior Rheumatic Medication,(n=115) (n=105) (n=119) n (%)a No. Subjects on Prior 114 (99.1)103 (98.1) 118 (99.2) Medications Methotrexate 114 (99.1)103 (98.1) 118 (99.2) Systemic (non-topical) 69 (60.0) 71 (67.6) 80 (67.2) corticosteroids Other DMARDs 19 (16.5) 19 (18.1) 25 (21.0) Sulfasalazine 9 (7.8) 2 (1.9) 10 (8.4) Hydroxychloroquine 8 (7.0) 6 (5.7) 14 (11.8) Cyclosporine 2 (1.7) 4 (3.8) 4 (3.4) Infliximab 2 (1.7) 2 (1.9) 2 (1.7) Etanercept 1 (0.9) 4 (3.8) 1 (0.8) Chloroquine 1 (0.9) 0 0 Leflunomide 0 2 (1.9) 2 (1.7) Error! Bookmark not defined.
Categories of prior rheumatic medications were not mutually exclusive.
Error! Bookmark not defined. Administration of MTX was not recorded for 4 subjects Study Therapy Of the three treatment groups, the 10 mg/kg CTLA4Ig group had the longest mean duration of exposure for both study phases and the placebo group had the shortest mean duration of exposure for both study phases (Day 180: 163 days, 156 days, 140 days;
Day 360: 286 days, 268 days, and 234 days; 10 mglkg, 2 mg/kg, and placebo, respectively).
At Day 180 (end of the primary phase), the proportion of subj ects receiving infusions was higher in the 10 mg/kg CTLA4Ig group (85%) compared with the 2 mg/kg CTLA4Ig group (79%) and the placebo group (66%) (Table 8). At Day 330 (day of last scheduled infusion in the secondary phase), the proportion of subjects receiving infusions was also higher in the 10 mg/leg CTLA4Ig group (78%) compared with the 2 mg/kg CTLA4Ig group (70%) and the placebo group (59%).

Table 8: Subjects Who Received Infusions on Given Study Days Number (%) of Subjects CTLA4Ig (BMS-188667) Day 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) .

1 115 (100) 105 (100) 119 (100) 15 114 (99) 104 (99) 117 (98) 30 113 (98) 101 (96) 111 (93) 60 108 (94) 97 (92) 103 (87) 90 106 (92) 94 (90) 94 (79) 120 100 (87) 86 (82) 83 (70) 150 98 (85) 83 (79) 81 (68) 180 98 (85) 83 (79) 78 (66) 210 94 (82) 80 (86) 78 (66) 240 95 (83) 78 (74) 76 (64) 270 93 (81) 77 (73) 73 (61) 300 90 (78) 74 (70) 72 (61) 330 90 (78) 73 (70) 70 (59) Methotrexate Subjects were to have been treated with a "stable" dose of MTX (10-30 mg weekly) for at least 6 months, for 28 days prior to Day 1. With the exception of 4 subjects , all subjects received between 10 and 30 mg of MTX weekly in addition to CTLA4Ig (BMS-188667) during the primary phase (Day 1-180). During the secondary phase (Day 181-360), the dose of MTX could have been adjusted provided it remained between 10 and 30 mg weekly.
Measurements of Treatment Compliance During the primary phase, the number of missed infusions of study drug was <_2 at any time point (Table 9). During the secondary phase, subj ects in the placebo group appeared to have missed slightly fewer infusions than subjects in the CTLA4Ig (BMS-188667) groups. However, more placebo than CTLA4Ig (BMS-188667) subjects discontinued by these later time points (see supra).

Table 9: Number of Missed Infusions of Study Drug CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) Day 1 0 0 0 Day 15 1 1 0 Day 30 0 1 1 Day 60 0 0 0 Day 90 0 0 0 Day 120 0 1 2 Day 150 1 2 0 Day 180 1 0 1 Day 210 4 0 0 Day 240 1 2 1 Day 270 0 2 0 Day 300 1 1 0 Day 330 0 0 0 Concomitant Therapy Systemic (non-topical) corticosteroid use was generally comparable among the three groups during screening/enrollment (58-67%) and during the primary phase of the study (67-71 %), Tables 10 and 11, respectively. While corticosteroid use decreased in all three treatment groups by Day 360, more subjects in the 10 mg/kg CTLA4Ig group took systemic (non-topical) corticosteroids (63.5%) compared to the other two treatment groups (53.3% and 45.4% for the 2 mg/kg CTLA4Ig and placebo groups, respectively).
Several subjects (CTLA4Ig: 0-3%, placebo: 0-10%) received DMARDs other than MTX
during screening/enrollment.

Table 10: Summary of Rheumatic Concomitant Medications During Screening/Enrollment CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo Rheumatic Medication, (n=115) (n=105) (n=119) n (%)a No. Subjects on Prior 114 (99.1)103 (98.1) 118 (99.2) Medications Methotrexate 114 (99.1)103 (98.1) 118 (99.2) Systemic (non-topical) 67 (58.3) 70 (66.7) 75 (63.0) corticosteroids Other DMAItDs 5 (4.3) 6 (5.7) 14 (11.8) Sulfasalazine 3 (2.6) 1 (1.0) 4 (3.4) Hydroxycllloroquine 2 (1.7) 3 (2.9) 12 (10.1) Cyclosporine 1 (0.9) 1 (1.0) 2 (1.7) Etanercept 0 1 (1.0) 0 Error! Bookmark not defined.
Drug categories were not mutually exclusive.

Table 11: Subjects Who Received Clinically Relevant Concomitant Medications During Soth Study Phases CTLA4Ig (BMS-188667) Medications 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) Systemic (non-topical) corticosteroids (Primary Phase) 77 (67.0) 71 (67.6) 85 (71.4) Systemic (non-topical) corticosteroids (Secondary Phase) 73 (63.5) 56 (53.3) 54 (45.4) Error! Bookmark not defined.
Drug categories were not mutually exclusive.
Note: Subject IM101100-83-3 (10 mg/kg CTLA4Ig) took mefloquine and subject (placebo) took quinine between Days 1 and 180; subject IM101100-18-11 (10 mg/kg CTLA4Ig) took quinine between Days 181 and 360 as an antimalarial, and was not considered a significant protocol violation.
EFFICACY RESULTS
The CTLA4Ig (BMS-188667) 10 mg/kg group had superior efficacy compared to the placebo group at Day 180 and Day 360. For the 2 mg/kg CTLA4Ig group, results for some efficacy parameters were significantly better compared to the placebo group, results for most other efficacy parameters were numerically higher compared to placebo.

ACR Responses at Day 180 Analysis of the primary efficacy variable for this study, ACR20 response rate at Day 180, showed that the 10 mg/kg CTLA4Ig group was significantly (p<0.001) more effective than placebo (Table 12, Figure 71A and Figure 71B).
The ACR50 and ACR70 responses at Day 180 for the 10 mg/kg CTLA4Ig group were also significantly higher compared to the placebo group (Table 12, Figure 71A
and Figure 71B). The ACR50 and the ACR70 responses at Day 180 for the 2 mg/kg CTLA4Ig group were significantly higher compared to the placebo group. The response at Day 180 for the 2 mg/kg CTLA4Ig group was slightly higher compared to the placebo group; however, no statistically significant differences were observed.
Table 12: ACR Responses at Dav 180 CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) n (%) 70 (60.9) 44 (41.9) 42 (35.3) CI 25.6 (12.8, 38.4) 6.6 (-6.2, N/A
19.4) p-value <O.OOla 0.31 N/A

n (%) 42 (36.5) 24 (22.9) 14 (11.8) CI 24.8 (13.8, 11.1 (1.2, 20.9)N/A
35.7) p-value <O.OOla 0.027a N/A

n (%) 19 (16.5) 11 (10.5) 2 (1.7) CI 14.8 (7.5, 8.8 (2.7, 14.9) N/A
22.2) p-value <O.OOla O.OOSa N/A

Error! Bookmark not .
defined Statistically comparison of BMS-188667 significant difference for the vs placebo.

ACR Responses at Day 360 At Day 360, ACR20, ACR50 and ACR70 responses for the 10 mg/kg CTLA4Ig group were significantly (p<0.001) higher compared to the placebo group (Table 13, Figure 72A and Figure 72B). Although the same response rates for the 2 mg/kg CTLA4Ig group were numerically higher compared to the placebo group, these differences were not statistically significant.
Table 13: ACR Responses at Dav 360 CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) n (%) 72 (62.6) 43 (41.0) 43 (36.1) CI 26.5 (13.7, 39.3)4.8 (-7.9, N/A
17.6) p-value <O.OOla 0.459 N/A

n (%) 48 (41.7) 23 (21.9) 24 (20.2) CI 21.6 (9.7, 33.4)1.7 (-8.9, N/A
12.4) p-value <0.001 a 0.75 N/A

n (%) 24 (20.9) 13 (12.4) 9 (7.6) CI 13.3 (4.4, 22.2)4.8 (-3.0, N/A
12.6) p-value 0.003a 0.227 N/A

l;rror! Bookmark Statistically not defined. significant difference for the comparison of 10/mg/kg CTLA4Ig group vs placebo.

ACR Responses by Visit For the comparison of the 10 mg/kg CTLA4Ig group to the placebo group, statistically significant improvements were observed for all three response rates (ACR 20, ACR 50, and ACR 70) by Day 90, and these values remained statistically significant at every time point up to and including Day 360 (p<_0.008 for all three ACR response rates) (Figure 73A, Figure 73B, and Figure 73C). In fact, statistically significant improvements in ACR 50 and ACR 70 response for the 10 mg/kg CTLA4Ig group occurred as early as Day 30 (p=0.039 and p=0.04, respectively).
For the 2 mg/kg CTLA4Ig group, statistically significant improvements compared to placebo were observed in ACR 50 and ACR 70 responses at Day 180 (p=0.027 and p=0.005, respectively). At Day 360, improvements in ACR response were slightly greater in the 2 mg/kg CTLA4Ig group compared to the placebo group; however, no statistically significant differences were observed.
After adjusting for visit using the Cochran-Mantel Haenszel test, a significant difference in ACR 20 response was observed for the 10 mg/kg CTLA4Ig group compared to the placebo group at both Day 180 and Day 360. No significant difference was observed between the 2 mg/kg CTLA4Ig and placebo groups at both timepoints. Similar results were obtained for ACR 50 response at both time points. For ACR 70 response at both time points, a significant difference was observed for both CTLA4Ig (BMS-188667) treatment groups compared to the placebo group.
Summary of Major Clinical Response Major Clinical Response was defined as maintenance of an ACR 70 response over a continuous 6-month period. The percentages of subjects who achieved a Major Cliucal Response at Day 360 were significantly higher in both the 10 mg/kg and 2 mg/kg CTLA4Ig groups (7.8% and 5.7%, respectively) when compared to the placebo group (0.8%; p=0.008 and 0.036, respectively) (Table 14).
Table 14: Summary of Major Clinical Response by Day 360 CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) No. Subjects with a Major Response 9 (7.8) 6 (5.7)1 (0.8) Diff (CI) 7.0 (1.8, 12.2) 4.9 (0.3, 9.4) N/A

p-value 0.008a 0.036a N/A

Error! Bookmark not defined. Indicates a statisticallfor the comparison significant difference of BMS-188667 vs placebo.

Mean Numeric ACR (ACR-N) and ACR-N Area Under the Curve (ACR-N-AUC) Overall, mean numeric ACR (ACR-N) for all treatment groups increased over time during the first 6 months of the study (Figure 74). During the second 6 months, mean ACR-N increased slightly with 10 mg/lcg CTLA4Ig, but remained relatively unchanged with 2 mg/kg CTLA4Ig and placebo. At each study visit, the ACR-N was consistently higher for the 10 mg/lcg CTLA4Ig group compared to the 2 mg/kg CTLA4Ig and placebo groups.
Compared to the placebo group, the differences in values for ACR-N AUC (area under the curve) for the 10 mg/kg CTLA4Ig group was significantly (p<0.001) higher by Day 360.
Percentage Improvement from Baseline at Day 180 For the 10 mg/kg CTLA4Ig group, improvements in each individual ACR component (tender and swollen joint counts, CRP, pain, subject global assessment, physician global assessment, and physical function) at Day 180 were statistically significant relative to improvements for the placebo group (Table 15).
For the 2 mg/kg CTLA4Ig group, statistically significant improvements compared to the placebo group were observed in physician global assessment and CRP at Day 180.
Furthermore, CRP levels in the placebo group actually worsened at Day 180.
Change from baseline in mean duration of morning stiffness was comparable among the three treatment groups at Day 180.

Table 15: Mean Percentage Improvement from Baseline at Day (Individual Components of ACR Criteria) CTLA4Ig (BMS-188667) Component 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) Tender Joints n=114 n=104 n=118 Mean % Change 59.78* 43.15 31.88 Swollen Joints n=114 n=104 n=118 Mean % Change 55.28* 45.34* 33.49 ' n=108 n=98 n=114 Mean % Change 31.79* 16.41* -23.43 Pain n=109 n=102 n=118 Mean % Change 46.19* 22.09* 8.20 Subject Global n=103 n=118 Assessment n=111 Mean % Change 40.76* 9.07 17.48 MD Global Assessmentn=111 n=103 n=116 Mean % Change 51.91* 38.71* 25.14 Physical Functionn=107 n=98 n=110 Mean % Change 41.21 * 21.63 13.71 Duration Morningess n=98 n=82 n=80 Stiffn Mean SD (minutes)61.9 55.4 60.8 66.1 55.9 66.2 * Indicates p<0.05 in comparison with placebo since 95% CIs did not include zero Percentage Improvement from Baseline at Day 360 For the 10 mg/kg CTLA4Ig group, improvements in each individual ACR component (tender and swollen joint counts, CRP, pain, subject global assessment, physician global assessment, and physical function) at Day 360 were statistically significant relative to improvements for the placebo group. Mean percentage improvements from baseline to Day 360 are presented in Table 16 for all clinical parameters of the ACR
criteria.
For the 2 mg/kg CTLA4Ig group, statistically significant improvements compared to the placebo group were observed in physician global assessment and CRP at Day 360.
Furthermore, CRP levels in the placebo group actually worsened at Day 360. At Day 360, the CTLA4Ig (BMS-188667) treatment groups had greater changes from baseline in duration of morning stiffness compared to the placebo group.
Table 16: Mean Percentage Improvement from Baseline at Day 360 (Individual Components of ACR Criteria) CTLA4Ig (BMS-188667) Component 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) Tender Joints n=115 n=105 n=119 Mean % Change 66.39* 43.54* 29.97 Swollen Joints n=115 n=105 n=119 Mean % Change 59.74* 46.40 36.17 Cue' n=112 n=98 n=115 Mean%Change 27.59* 10.31* -31.26 Pain n=112 n=104 n=119 Mean % Change 44.93* 26.26 12.55 Subject Global n=105 n=119 Assessment n=113 Mean % Change 41.01 * 16.08 1.99 MD Global Assessmentn=113 n=105 n=119 Mean % Change 53.48* 37.87* 24.14 Physical Functionn=109 n=100 n=111 Mean % Change 42.32* 22.94 10.25 Duration Morningess n=88 n=71 n=72 Stiffn Mean SD 66.2 59.5* 66.6 72.2 49.7 73.9 * Indicates p<p,05 in comparison with placebo since 95% CIs did not include zero New Active Joints The proportion of new active joints was determined using the validated 28 joint count (out of 68 total tender joints and out of 66 total swollen joints) proposed by Smollen et al (Smollen JS, Breedveld FC, Eberl G, Jones I et al. Validity and reliability of the twenty-eight joint count for the assessment of RA activity. Arthritis & Rheum 1993; 38:
38-43). The proportion of new active joints (both tender and swollen) at Day 180 was lowest for subjects receiving 10 mg/kg CTLA4Ig (Figure 75).

At Day 180, the percentages of subjects reporting no new tender joints and no new swollen joints was highest in the 10 mg/kg CTLA4Ig group (Figure 76A, Figure 77A).
The percentage of subjects who reported no new tender joints and no new swollen joints was approximately 59% and 52%, respectively, in the 10 mg/kg CTLA4Ig group;
38%
and 44%, respectively, in the 2 mg/kg CTLA4Ig group; and 41% and 37%, respectively, in the placebo group.
The proportion of new active joints (both tender and swollen) at Day 360 was lowest for subjects receiving 10 mg/kg CTLA4Ig (Figure 78). This pattern for the proportion of new active joints mirrored the pattern seen at Day 180.
Similarly, at Day 360, the proportion of subjects reporting no new tender joints and no new swollen joints was highest in the 10 mg/kg CTLA4Ig group (Figure 76B, and Figure 77B). The percentage of subjects who reported no new tender and no new swollen j oints was approximately 71 % and 61 %, respectively, in the 10 mg/kg CTLA4Ig group;
41% and 44%, respectively, in the 2 mg/kg CTLA4Ig group; and 42% for both counts in the placebo group.
Improvement in Clinical Parameters Among Subjects with an ACR Response Among ACR 20, ACR 50, and ACR 70 responders, improvement in the core components of the ACR criteria were slightly greater for the two CTLA4Ig (BMS-188667) treatment groups compared to placebo.
The onset of action for subjects who received the 10 mg/kg CTLA4Ig dose occurred after approximately 15 days, with significant increases in ACR 20 improvement occurring at >_Day 60 for ACR50 at >_Day 90 for ACR70 at >_Day 30 and in each instance, continuing until Day 360 (see Figure 73A, Figure 73B and Figure 73C).

Changes from Baseline for the Health Outcomes Short Form Questionnaire (SF-36) The impact of CTLA4Ig (BMS-188667) on health-related quality of life was assessed using the Health Outcomes Short Form Questionnaire SF-36 (summary scores range from 0 to 100 with higher scores indicating a better quality of life). Analyses were performed on the LOCF (last observation carried forward) data set as well as the as the observed data set.
For the 10 mg/kg CTLA4Ig group, statistically significant improvement from baseline compared to the placebo group was observed in all four mental health and all four physical health domains of the SF-36 at Day 180, using the LOCF analysis (i.e., 95% CIs did not include 0) (Figures 79A, 79B). For the 2 mg/kg CTLA4Ig group, there were numerical improvements in the mental health or physical health domains compared to placebo at Day 180, however, these improvements were not statistically significant.
Results of analyses performed on the as-observed data set were similar to those observed for the LOCF data set except that the "role emotional" domain at Day 180 was not significantly improved (but was numerically improved) for the comparison between the 10 mg/lcg CTLA4Ig and placebo groups using the as-observed data set.
The physical component and the mental health component summary measures at Day are shown in Table 17.

Table 17: Mean Change from Baseline to Day 180 for the SF-36 (Physical and Mental Health Components) CTLA4Ig (BMS-188667) Summary Score 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) Mental Health Componentn=115 n=103 n=118 Baseline Mean 44.52 43.06 41.75 Postbaseline Mean 48.69 45.59 44.04 Mean Change from 4.17 2.53 2.30 Baseline 95% CI (2.46, 5.88) (0.39, 4.67 (0.42, 4.17) Physical Component n=115 n=103 n=118 Baseline Mean 31.13 30.80 32.33 Postbaseline Mean 39.30 35.47 35.21 Mean Change from 8.16 4.67 2.88 Baseline 95% CI (6.33, 9.99) (3.25, 6.09) (1.54, 4.22) Results of the Health Outcomes at Day 360 were similar to those seen at Day 180. For the 10 mg/kg CTLA4Ig group, statistically significant improvements from baseline compared to the placebo group were observed in all four mental and all four physical domains of the SF-36 at Day 360, using the LOCF analysis (i.e., 95% CIs did not include 0) (Figures 80A, and 80B). For the 2 mg/kg CTLA4Ig group, a statistically significant difference in three of four physical domains at Day 360 and one of four mental domains at Day 360 compared to the placebo group was observed.
Results of analyses performed on the as-observed data set were similar to those observed for the LOCF data set.
The physical component and mental health component summary measures at Day 360 is shown in Table 18.

Table 18: Mean Change from Baseline to Day 360 for the SF-36 (Summaries of PhysicalComponent and Mental Health Component) CTLA4Ig (BMS-188667) Summary Score 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) Mental Health n=103 n=118 Component n=115 Baseline Mean 44.52 43.06 44.75 Postbaseline 48.83 45.65 43.22 Mean Mean Change 2.59 1.47 from Baseline 4.31 95% CI (2.64, 5.98) (0.64, 4.55) (-0.14, 3.08) Physical Componentn=115 n=103 n=118 Baseline Mean 31.13 30.80 32.33 Postbaseline 38.93 36.49 34.93 Mean Mean Change 5.69 2.60 from Baseline ' 7.79 95% CI (5.90, 9.68) (4.10, 7.28) (1.09, 4.11) Biomarker and Pharmacodynamic Data There were significant improvements (decreases) in 5 of the 6 biomarker/
pharmacodynamic (PD) parameters with 10 mg/kg CTLA4Ig at Day 180 (soluble IL-2r, rheumatoid factor (RF), ICAM-1, E-selectin and IL-6) and a numerical decrease in TNF-oc (Table 19). There were significant improvements (decreases) in 3 of the 6 biomarker/PD parameters with 2 mg/kg CTLA4Ig at Day 180 (soluble IL-2r, RF and IL-6) and a numerical improvement in ICAM-1. There were no significant changes in any of the biomarker/PD parameters with placebo at Day 180. There appears to be a dose response relationship with the improvements (decreases) in biomarker/PD
parameters.
Table 19: Pharmacodynamic Measures at Day 180 CTLA4Ig (BMS-188667) Parameter 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) Soluble IL-2r n=95 n=84 n=76 (Normal range : 640-2543 pg/mL) Baseline Mean (SD) 1426.19 751.761396.82 1429.13 667.84 610.21 Postbaseline Mean (SD)1112.62 699.681261.31 1470.03 637.75 473.66 Mean Change -316.23 -135.51 43.59 Table 19: Pharmacodynamic Measures at Day 180 95% CI (-417.73, -214.72)(-241.48, (-71.24, -29.53) 158.43) Rheumatoid Factor n=95 n=84 n=74 (Normal Range: 0-20 IU/mL) Baseline Mean (SD) 289.71 401.95 256.19 307.92196.11 265.48 Postbaseline Mean 185.43 269.52 227.82 276.27204.36 (SD) 320.09 Mean Change -104.27 -28.12 -0.62 95% CI (-151.53, -57.01)(-52.13, (-31.67, -4.11) 30.43) ICAM-1 n=95 n=82 n=75 Baseline Mean (SD) 404.89 137.72 393.47 150.85387.33 230.93 Postbaseline Mean 364.74 109.47 387.25 142.73386.17 (SD) 163.82 Mean Change -40.42 -6.22 1.09 95% CI (-58.06, -22.78)(-27.49, (-31.88, 15.05) 34.05) E-selectin n=89 n=80 n=71 Baseline Mean (SD) 68.07 32.93 67.32 37.1368.23 43.09 Postbaseline Mean 61.01 31.53 67.86 40.20 67.37 35.66 (SD) Mean Change -8.41 0.54 -0.68 95% CI (-13.24, -3.58)(-5.95, 7.03)(-6.87, 5.51) Table 19: Pharmacodynamic Measures at Day Serum IL-6 n=86 n=74 n=69 (Normal Range 8 pg/mL) : 0.3-14.

Baseline Mean 28.47 38.28 31.75 42.2921.20 26.51 (SD) Postbaseline 9.25 15.85 16.00 22.1323.98 37.92 Mean (SD) Mean Change -20.30 -16.10 1.98 95% CI (-27.55, -13.06) (-24.20, (-7.21, 11.17) -8.00) TNFa n=84 n=74 n=69 (1.2-8.0 pg/mL) Baseline Mean 11.17 23.72 7.51 13.25 13.12 23.20 (SD) Postbaseline 7.57 7.90 6.20 4.48 9.59 11.21 Mean (SD) Mean Change -3.66 -1.21 -3.54 95% CI (-8.62, 1.30) (-4.32, 1.90)(-7.82, 0.75) Overall, the pattern in the changes in biomarker/PD data at Day 360 were similar to that seen at Day 180. There were significant improvements (decreases) in 5 of the 6 biomarker/PD parameters with 10 mg/kg CTLA4Ig at Day 360 (soluble IL-2r, RF, ICAM-1, E-selectin and IL-6) and a numerical, but not statistically significant improvement observed for TNF-a, (Table 20). There was a significant improvement (decrease) in IL-6 only with 2 mg/kg CTLA4Ig at Day 360, however, numerical improvements were seen with RF and ICAM-1. There were no significant changes in any of the biomarker/PD parameters with placebo at Day 360. As seen with Day data, it appeared that all of the improvements (decreases) in biomarker/PD
parameters occurred in a dose response manner.
A comparison of the postbaseline means for the biomarker/PD parameters at Day 180 to those at Day 360 reveals important trends. For the 10 mg/kg CTLA4Ig group, all biomarkers/PD measures continued to decrease, with the exception of soluble IL-2r which increased slightly. For the 2 mg/kg CTLA4Ig group, mean values for 3 of the biomarkers/PD parameters either decreased slightly (ICAM-l, serum IL-6) or remained relatively constant (E-selectin) and mean values for the other 3 biomarkers/PD
measures increased slightly (soluble IL-2r, RF, TNF a). For the placebo group, mean values for all of the biomarkers/PD parameters increased slightly at Day 360, with the exception of TNF a, wluch remained relatively unchanged.

Table 20: Pharmacodynamic Measures at Day 360 CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo Measure (n=115) (n=105) (n=119) Soluble IL-2r n=68 n=56 n=55 (Normal range : 640-2543 pg/mL) Baseline Mean (SD) 1372.10 770.111373.86 567.751459.93 695.07 Postbaseline Mean (SD)1185.51 638.951413.84 452.501666.59 611.97 ~

Mean Change -194.31 39.99 206.22 95% CI (-305.67, (-69.87, 149.84)(35.88, 376.56) -82.96) Rheumatoid Factor n=69 n=55 n=58 (Normal Range: 0-20 IU/mL) Baseline Mean (SD) 261.43 333.58258.42 318.65179.12 207.72 Postbaseline Mean (SD)143.13 180.80236.61 287.36206.42 256.27 Mean Change -118.30 -25.64 20.90 95% CI (-175.19, (-58.50, 7.23)(-10.72, 52.51) -61.42) ICAM-1 n=77 n=68 n=64 Baseline Mean (SD) 406.44 145.22393.41 132.97405.67 245.16 Postbaseline Mean (SD)354.90 111.40380.42 113.20405.07 194.15 Mean Change -55.15 -12.98 1.47 95% CI (-74.80, -35.49)(-35.36, 9.39)(-26.41, 29.35) E-selectin n=75 n=68 ' n=62 Baseline Mean (SD) 68.84 34.38 66.75 37.10 69.72 44.38 Postbaseline Mean (SD)58.77 26.61 67.58 31.50 71.90 47.43 Mean Change -10.89 0.83 2.34 95% CI (-15.70, -6.08)(-5.62, 7.28)(-4.53, 9.20) Serum IL-6 n=56 n=47 n=48 (Normal Range : 0.3-14.8 pg/mL) Baseline Mean (SD) 27.68 38.56 27.19 32.45 17.27 22.47 Postbaseline Mean (SD)7.64 14.21 13.93 19.00 17.72 29.76 Mean Change -20.88 -12.72 -0.19 95% CI (-31.56, -10.19)(-22.49, -2.94)(-7.55, 7.18) TNFa n=61 n=48 n=50 (1.2-8.0 pg/mL) Baseline Mean (SD) 9.71 22.80 6.27 3.62 10.81 21.24 Postbaseline Mean (SD)6.67 4.80 7.18 8.14 9.36 26.43 Mean Change -3.02 1.08 -1.41 95% CI (-8.70, 2.67)(-1.26, 3.42)(-5.14, 2.33) The data are shown graphically for these biomarker/PD measures, as well as for changes in CRP levels, in Figures 81 through 87.

In order to assess the integrity of the planned analyses, all subjects who received study medication and discontinued the study for any reason were considered ACR non-responders at all scheduled study visits subsequent to discontinuation. Results of these analyses (Table 21) were consistent with the efficacy results already presented.
The proportion of subjects who received 10 mg/kg CTLA4Ig and achieved an ACR
20, ACR 50, or ACR 70 response at Day 180 was significantly (p<0.001) higher compared to the proportion of subjects who received placebo. For the 2 mg/kg CTLA4Ig group, a significantly (p<_0.009) higher proportion of subjects achieved either an ACR
50 or ACR 70 response.
Table 21: ACR Response at Day 180 (Non-Completer Equals Non-Responder) CTLA4Ig (BMS-188667) 10 mg/kg 2 mg/kg Placebo (n=115) (n=105) (n=119) ACR 20, n 67 (58.3) 41 (39.0) 38 (31.9) (%) Diff(CI) 26.3 (13.6, 39.1) 7.1 (-5.4, N/A
19.7) p-value <O.OOla 0.266 N/A

ACR 50, n 41 (35.7) 24 (22.9) 12 (10.1) (%) Diff (CI) 25.6 (14.8, 36.3) 12.8 (3.1, N/A
22.4) p-value <O.OOla 0.009a N/A

ACR 70, n(%) 19 (16.5) 11 (10.5) 2 (1.7) Diff (CI) 14.8 (7.5, 22.2) 8.8 (2.7, 14.9)N/A

p-value <O.OOla 0.005a N/A

Error! Bookmark not defined. Indicates a statistically significant difference for the comparison of BMS-188667 vs placebo.
In addition, all primary efficacy analyses were performed on the WOCF (worst observation carried forward) data set . ACR responses based on the WOOF data set were slightly lower than those reported in Table 13 and were comparable to those presented in Table 21. These findings confirm the consistency of ACR response rates in the CTLA4Ig (BMS-188667) treatment groups.

The dosages of anti-rheumatic concomitant medications were to be collected to assess the need for these medications at 6 and 12 months; however, the available data were inadequate to perform these analyses. Only baseline values for mean dose of methotrexate and systemic (non-topical) corticosteroids are provided.
Efficacy Conclusions CTLA4Ig (BMS-188667), admiiustered at 10 mg/kg (+ MTX) had superior efficacy compared to placebo (+ MTX) at Day 180 and Day 360. For the following efficacy parameters, administration of 10 mglkg CTLA4Ig was significantly better than placebo:
~ Primary efficacy variable: ACR20 response at Day 180 (p<0.001) ~ ACR50 and ACR70 responses at Day 180 (p<0.001) ~ ACR20, ACR50 and ACR70 responses at Day 360 (p<_0.003) ~ Statistically significant differences in ACR50 and ACR70 responses observed by Day 30 (p=0.039 and p=0.04), statistically significant differences in all 3 response rates (ACR 20, ACR50 and ACR70) observed by Day 90; these values remained statistically significant at every timepoint up to and including Day 360 (p<_0.008) ~ Proportions of subjects who achieved a Major Clinical Response (maintenance of an ACR 70 response over a continuous 6-month period) at Day 360 (p=0.008) ~ Mean numeric ACR-AUC by Day 360 (p<0.001) ~ Mean percentage improvements in each individual ACR component at Day 180 and Day 360 (p<0.05, 95% CIs did not include 0) ~ Improvements in all four mental and all four physical domains of the Health Outcomes evaluation (SF-36) at both Day 180 and Day 360 (p<0.05, 95% CIs did not include 0) In addition to the above statistically significant differences, the 10 mg/kg CTLA4Ig group had a lower number of new active joints and a higher number of subjects reporting no new active tender and swollen joints compared with the placebo group at Day 180 and at Day 360.
Significant improvement with 10 mg/kg CTLA4Ig compared with placebo was seen in nearly all measured pharmacodynamic paramenters (soluble ILl2r, RF, ICAM-l, E-selectin and IL-6) and numerical improvement in TNF-a up to 1 year.
For the 2 mg/lcg CTLA4Ig group, some efficacy parameters were significantly better compared to the placebo group:
~ ACR50 response at Day 180 (p=0.027) ~ ACR70 response at Day 180 (p=0.005) ~ Statistically significant differences in ACR70 observed by Day 60 (p=0.032) and statistically significant differences in ACR 50 and ACR 70 at Day 180 (p=0.027 and p=0.005) ~ Proportions of subjects who achieved a Major Clinical Response (maintenance of an ACR 70 response over a continuous 6-month period) at Day 360 (p=0.036) ~ Mean percentage improvements in some of the individual ACR component at Day 180 and Day 360 (p<0.05, 95% CIs did not include 0) For many other efficacy parameters, 2 mg/kg CTLA4Ig was numerically better than placebo.
SAFETY RESULTS
Overall, the safety profile of CTLA4Ig (BMS-188667) was similar to placebo.
There were no major safety problems.
Clinical Laboratory Evaluation Overall, no new safety issues emerged from the evaluation of mean changes in laboratory values. Mean values for hemoglobin, WBCs, neutrophils, platelets, ALT, AST, GGT and total protein were within the normal range at baseline and remained within the normal range during the study. In general, results of the laboratory tests did not reveal consistent out-of range values or abnormal trends that could be attributed to study medication.
Vital Signs, Physical Findings, and Observations Related to Safety On each day of study drug administration, vital signs (body temperature, heart rate, and seated blood pressure) were monitored pre-dose and at 15, 30, 45, 60, 75, 90 and 120 minutes post-infusion. Overall, mean values for all vital sign parameters were within normal range and stable throughout the 360-day study period for all treatment groups.
I

SEQUENCE LISTING
<110> Bristol Myers Squibb Company Cohen, Robert Carr, Suzette Hagerty, David Peach, Robert J.
Becker, Jean-Claude <120> METHODS FOR TREATING AN AUTOIMMUNE DISEASE USING A SOLUBLE CTLA4 MOLECULE AND A DMARD OR NSAID
<130> 30436.55WOI1 <140> Not yet known <141> 2003-04-18 <150> 60/373,852 <151> 2002-04-19 <150> 60/407,246 <151> 2002-08-30 <160> 22 <170> PatentIn version 3.2 <210> 1 <211> 65 <212> DNA
<213> Homo Sapiens <400> 1 ctcagtctgg tccttgcact cctgtttcca agcatggcga gcatggcaat gcacgtggcc 60 cagcc <210> 2 <211> 33 <212> DNA.
<213> Homo Sapiens <400> 2 tttgggctcc tgatcagaat ctgggcacgg ttg 33 <210> 3 <211> 72 <212> DNA
<213> Homo Sapiens <400> 3 ctagccactg aagcttcacc aatgggtgta ctgctcacac agaggacgct gctcagtctg 60 gtccttgcac tc 72 <210> 4 <211> 41 <212> DNA
<213> Artificial <220>
<223> Oncostatin M CTLA4 (OMCTLA4) forward primer <400> 4 gaggtgataa agcttcacca atgggtgtac tgctcacaca g 41 <210> 5 <211> 42 <212> DNA
<213> Artificial <220>
<223> Oncostatin M CTLA4 (OMCTLA4) reverse primer <400> 5 gtggtgtatt ggtctagatc aatcagaatc tgggcacggt tc 42 <210> 6 <211> 1152 <212> DNA
<213> Artificial <220>
<223> L104EIg <220>

<221> CDS

<222> (1) . .
(1149) <220>

<221> mat-peptide <222> (79) . .
() <400> 6 atg gta ctcacacag aggacgctg ctcagtctg gtccttgca 48 ggt ctg Met Val LeuThrGln ArgThrLeu LeuSerLeu ValLeuAla Gly Leu -25 -20 -l5 ctc ttt agcatggcg agcatggca atgcacgtg gcccagcot 96 ctg cca Leu Phe SerMetAla SerMetAla MetHisVal AlaGlnPro Leu Pro get gta gccagcagc cgaggcatc getagcttt gtgtgtgag 144 gtg ctg Ala Val AlaSerSer ArgGlyIle AlaSerPhe ValCysGlu Val Leu tat tct ggcaaagcc actgaggtc cgggtgaca gtgcttcgg 192 gca cca Tyr Ser GlyLysAla ThrGluVal ArgValThr ValLeuArg Ala Pro cagget gacagc caggtgactgaa gtctgtgcg gcaacctac atgatg 240 GlnAla AspSer GlnValThrGlu ValCysAla AlaThrTyr MetMet gggaat gagttg accttcctagat gattccatc tgcacgggc acctcc 288 GlyAsn GluLeu ThrPheLeuAsp AspSerIle CysThrGly ThrSer agtgga aatcaa gtgaacctcact atccaagga ctgagggcc atggac 336 SerGly AsnGln ValAsnLeuThr IleGlnGly LeuArgAla MetAsp acggga ctctac atctgcaaggtg gagctcatg tacccaccg ccatac 384 ThrGly LeuTyr IleCysLysVal GluLeuMet TyrProPro ProTyr tacgag ggcata ggcaacggaacc cagatttat gtaattgat ccagaa 432 TyrGlu GlyIle GlyAsnGlyThr GlnIleTyr ValIleAsp ProGlu ccgtgc ccagat tctgatcaggag cccaaatct tctgacaaa actcac 480 ProCys ProAsp SerAspGlnGlu ProLysSer SerAspLys ThrHis acatcc ccaccg tccccagcacct gaactcctg gggggatcg tcagtc 528 ThrSer ProPro SerProAlaPro GluLeuLeu GlyGlySer SerVal ttcctc ttcccc ccaaaacccaag gacaccctc atgatctcc cggacc 576 PheLeu PhePro ProLysProLys AspThrLeu MetIleSer ArgThr cctgag gtcaca tgcgtggtggtg gacgtgagc cacgaagac cctgag 624 ProGlu ValThr CysValValVal AspValSer HisGluAsp ProGlu 170 175 l80 gtcaag ttcaac tggtacgtggac ggcgtggag gtgcataat gccaag 672 Va1Lys PheAsn TrpTyrValAsp GlyValGlu ValHisAsn AlaLys acaaag ccgcgg gaggagcagtac aacagcacg taccgtgtg gtcagc 720 ThrLys ProArg GluGluGlnTyr AsnSerThr TyrArgVal ValSer gtcctc accgtc ctgcaccaggac tggctgaat ggcaaggag tacaag 768 ValLeu ThrVal LeuHisG1nAsp TrpLeuAsn GlyLysGlu TyrLys tgcaag gtctcc aacaaagccctc ccagccccc atcgagaaa accatc 816 CysLys ValSer AsnLysAlaLeu ProAlaPro IleGluLys ThrIle tccaaa gccaaa gggcagccccga gaaccacag gtgtacacc ctgccc 864 SerLys AlaLys GlyGlnProArg GluProG1n ValTyrThr LeuPro cca tcc cgg gat gag ctg acc aag aac cag gtc agc ctg acc tgc ctg 912 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat 960 Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn gggcagccg gagaacaac tacaagacc acgcctccc gtgctggac tcc 1008 GlyGlnPro GluAsnAsn TyrLysThr ThrProPro ValLeuAsp Ser gacggctcc ttcttcctc tacagcaag ctcaccgtg gacaagagc agg 1056 AspGlySer PhePheLeu TyrSerLys LeuThrVal AspLysSer Arg tggcagcag gggaacgtc ttctcatgc tccgtgatg catgagget ctg 1104 TrpGlnGln GlyAsnVal PheSerCys SerValMet HisGluAla Leu cacaaccac tacacgcag aagagcctc tccctgtct ccgggtaaa tga 1152 HisAsnHis TyrThrGln LysSerLeu SerLeuSer ProGlyLys <210> 7 <211> 383 <212> PRT
<213> Artificial <220>
<223> L104EIg <400> 7 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met 4'0 4 5 5 0 Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro .Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 8 <211> 1152 <212> DNA
<213> Artificial <220>
<223> L104EA29YIg <220>
<221> CDS
<222> (1)..(1149) <220>
<221> mat_peptide <222> (79) . . () <400>

atgggtgtactg ctcacacag aggacgctg ctcagtctg gtccttgca 48 MetGlyValLeu LeuThrGln ArgThrLeu LeuSerLeu ValLeuAla -25~ -20 -15 ctcctgtttcca agcatggcg agcatggca atgcacgtg gcccagcct 96 LeuLeuPhePro SerMetAla SerMetAla MetHisVal AlaGlnPro getgtggtactg gccagcagc cgaggcatc getagcttt gtgtgtgag 144 AlaValValLeu AlaSerSer ArgGlyIle AlaSerPhe ValCysGlu tatgcatctcca ggcaaatat actgaggtc cgggtgaca gtgcttcgg 192 TyrAlaSerPro GlyLysTyr ThrGluVal ArgValThr ValLeuArg caggetgacagc caggtgact gaagtctgt gcggcaacc tacatgatg 240 GlnAlaAspSer GlnValThr GluValCys AlaAlaThr TyrMetMet ggg aat gag ttg acc ttc cta gat gat tcc atc tgc acg ggc acc tcc 288 gtc aaa ggc ttc tat ccc agc gac atc gcc gtg gag tgg gag agc aat 960 Gly Leu Phe AspAsp SerIleCys ThrGly Ser Asn Thr Leu Thr Glu agtggaaat caagtgaac ctcactatc caaggactg agggccatg gac 336 SerGlyAsn GlnValAsn ThrIle GlnGly ArgAla Asp Leu Leu Met 75 , 80 85 acgggactc tacatctgc aaggtggag ctcatgtac ccaccgcca tac 384 ThrGlyLeu TyrIleCys LysValGlu LeuMetTyr ProProPro Tyr tacgagggc ataggcaac ggaacccag atttatgta attgatcca gaa 432 TyrGluGly IleGlyAsn GlyThrGln IleTyrVal IleAspPro Glu ccgtgccca gattctgat caggagccc aaatcttct gacaaaact cac 480 ProCysPro AspSerAsp GlnGluPro LysSerSer AspLysThr His acatcccca ccgtcccca gcacctgaa ctcctgggg ggatcgtca gtc 528 ThrSerPro ProSerPro AlaProGlu LeuLeuGly GlySerSer Val ttcctcttc cccccaaaa cccaaggac accctcatg atctcccgg acc 576 PheLeuPhe ProProLys ProLysAsp ThrLeuMet IleSerArg Thr cctgaggtc acatgcgtg gtggtggac gtgagccac gaagaccct gag 624 ProGluVal ThrCysVal ValValAsp ValSerHis GluAspPro Glu gtcaagttc aactggtac gtggacggc gtggaggtg cataatgcc aag 672 ValLysPhe AsnTrpTyr ValAspGly ValGluVal HisAsnAla Lys acaaagccg cgggaggag cagtacaac agcacgtac cgtgtggtc agc 720 ThrLysPro ArgGluGlu GlnTyrAsn SerThrTyr ArgValVal Ser gtcctcacc gtcctgcac caggactgg ctgaatggc aaggagtac aag 768 ValLeuThr ValLeuHis GlnAspTrp LeuAsnGly LysGluTyr Lys tgcaaggtc tccaacaaa gccctccca gcccccatc gagaaaacc atc 816 CysLysVal SerAsnLys AlaLeuPro AlaProIle GluLysThr Ile tccaaagcc aaagggcag ccccgagaa ccacaggtg tacaccctg ccc 864 SerLysAla LysGlyGln ProArgGlu ProGlnVal TyrThrLeu Pro ccatcccgg gatgagctg accaagaac caggtcagc ctgacctgc ctg 912 ProSerArg AspGluLeu ThrLysAsn GlnValSer LeuThrCys Leu gtcaaaggc ttctatccc agc atc gtggag gagagc 960 gac gcc tgg aat Val Gly PheTyrPro Ser Ile Glu Ser Lys Asp Ala Trp Asn Val Glu gggcag ccggagaac aactac aagaccacgcct cccgtgctg gactcc 1008 GlyGln ProGluAsn AsnTyr LysThrThrPro ProValLeu AspSer gacggc tccttcttc ctctac agcaagctcacc gtggacaag agcagg 1056 AspGly SerPhePhe LeuTyr SerLysLeuThr ValAspLys SerArg tggcag caggggaac gtcttc tcatgctccgtg atgcatgag getctg 1104 TrpGln GlnGlyAsn ValPhe SerCysSerVal MetHisGlu AlaLeu cacaac cactacacg cagaag agcctctccctg tctccgggt aaatga 1152 HisAsn HisTyrThr GlnLys SerLeuSerLeu SerProGly Lys <210> 9 <211> 383 <212> PRT
<213> Artificial <220>
<223> L104EA29YIg <400> 9 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Tyr Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 10 <211> 1152 <212> DNA
<213> Artificial <220>
<223> L104EA29LIg <220>

<221> CDS

<222> (1).
. (1149) <220>

<221>
mat~peptide <222> (79).
.
() <400> 10 atg gtactg ctcacacag aggacgctg ctcagtctggtc cttgca 48 ggt Met ValLeu LeuThrGln ArgThrLeu LeuSerLeuVal LeuAla Gly ctc tttcca agcatggcg agcatggca atgcacgtggcc cagcct 96 ctg Leu PhePro SerMetAla SerMetAla MetHisValAla GlnPro Leu get gtactg gccagcagc cgaggcatc getagctttgtg tgtgag 144 gtg Ala ValLeu AlaSerSer ArgGlyIle AlaSerPheVal CysGlu Val 10 15 ' 20 tat tctcca ggcaaattg actgaggtc cgggtgacagtg cttcgg 192 gca Tyr SerPro GlyLysLeu ThrGluVal ArgValThrVal LeuArg Ala cag gacagc caggtgact gaagtctgt gcggcaacctac atgatg 240 get Gln AspSer GlnValThr GluValCys AlaAlaThrTyr MetMet Ala ggg gagttg accttccta gatgattcc atctgcacgggc acctcc 288 aat Gly GluLeu ThrPheLeu AspAspSer IleCysThrGly ThrSer Asn agt aatcaa gtgaacctc actatccaa ggactgagggcc atggac 336 gga Ser AsnGln ValAsnLeu ThrIleGln GlyLeuArgAla MetAsp Gly acg gag ccaccg tac 384 gga ctc cca ctc atg tac tac atc tgc aag gtg Thr Glu ProProPro Tyr Gly Leu Leu Met Tyr Tyr Ile Cys Lys Val 90 g5 100 tac gag aac cag gta attgatcca gaa 432 ggc gga att ata acc tat ggc Tyr Glu Gln IleAspPro Glu Gly Ile Ile Tyr Gly Val Asn Gly Thr ccg tgccca tctgatcag ccc tct gacaaaact cac 480 gat gag aaa tct Pro CysPro SerAspGln Pro Ser AspLysThr His Asp Glu Lys Ser aca tcccca tccccagca gaa ctcctgggg ggatcgtca gtc 528 ccg cct Thr SerProPro SerProAla Glu Leu Gly GlySerSer Val Pro Leu ttc ctcttcccc ccaaaaccc gac accctcatg atctcccgg acc 576 aag Phe LeuPhePro ProLysPro Asp ThrLeuMet IleSerArg Thr Lys cct gaggtcaca tgcgtggtg gac gtgagccac gaagaccct gag 624 gtg Pro GluValThr CysValVal Asp ValSerHis GluAspPro Glu Val gtc aagttcaac tggtacgtg ggc gtggaggtg cataatgcc aag 672 gac Val LysPheAsn TrpTyrVal Gly ValGluVal HisAsnAla Lys Asp aca aagccgcgg gaggagcag aac agcacgtac cgtgtggtc agc 720 tac Thr LysProArg GluGluGln Asn SerThrTyr ArgValVal Ser Tyr gtc ctcaccgtc ctgcaccag tgg ctgaatggc aaggagtac aag 76g gac Val LeuThrVal LeuHisGln Trp LeuAsnGly LysGluTyr Lys Asp tgc aaggtctcc aacaaagcc cca gcccccatc gagaaaacc atc 816 ctc Cys LysValSer AsnLysAla Pro AlaProIle GluLysThr Ile Leu tcc aaagccaaa gggcagccc gaa ccacaggtg tacaccctg ccc 864 cga Ser LysAlaLys GlyGlnPro Glu ProGlnVal TyrThrLeu Pro Arg cca tcccgggat gagctgacc aac caggtcagc ctgacctgc ctg 912 aag Pro SerArgAsp GluLeuThr GlnValSer ThrCys Lys Leu Leu Asn gtc aaaggcttc tatcccagc gccgtggag agc 960 gac tgg aat atc gag Val LysGlyPhe TyrProSer AlaValGlu Ser Asp Trp Asn Ile Glu ggg cagccggag aacaactac acgcctccc 1008 aag gtg acc ctg gac tcc Gly ProGlu Tyr ThrPro Gln Asn Lys Pro Asn Thr Val Leu Asp Ser gac tccttc tac ctcacc 1056 ggc ttc agc gtg ctc aag gac aag agc agg Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag get ctg 1104 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga 1152 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 11 <211> 383 <212> PRT
<213> Artificial <220>
<223> L104EA29LIg <400> 11 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala Leu Leu Phe Pro,Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu 15 20 , Tyr Ala Ser Pro Gly Lys Leu Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 12 <211> 1152 <212> DNA
<213> Artificial <220>
<223> L104EA29TIg <220>

<221> CDS

<222> (1). 49) . (11 <220>

<221> mat-pept ide <222> (79).
.
() <400> 12 atgggtgtactg ctcaca cagaggacg ctgctcagt ctggtcctt gca 48 MetGlyValLeu LeuThr GlnArgThr LeuLeuSer LeuValLeu Ala ctcctgtttcca agcatg gcgagcatg gcaatgcac gtggcccag cct 96 LeuLeuPhePro SerMet AlaSerMet AlaMetHis ValAlaGln Pro getgtggtactg gccagc agccgaggc atcgetagc tttgtgtgt gag 144 AlaValValLeu AlaSer SerArgGly IleAlaSer PheValCys Glu l5 20 tatgcatctcca ggcaaa actactgag gtccgggtg acagtgctt cgg 192 TyrAlaSerPro .GlyLys ThrThrGlu ValArgVal ThrValLeu Arg .

caggetgacagc caggtg actgaagtc tgtgcggca acctacatg atg 240 GlnAlaAspSer GlnVal ThrGluVal CysAlaAla ThrTyrMet Met gggaatgagttg accttc ctagatgat tccatctgc acgggcacc tcc 288 G1yAsnGluLeu ThrPhe LeuAspAsp SerIleCys ThrGlyThr Ser -agtggaaatcaa gtgaac ctcactatc caaggactg agggccatg gac 336 SerGlyAsnGln ValAsn LeuThrIle GlnGlyLeu ArgAlaMet Asp acgggactctac atctgc aaggtggag ctcatgtac ccaccgcca tac 384 ThrGlyLeuTyr IleCys LysValGlu LeuMetTyr ProProPro Tyr tacgagggcata ggcaac ggaacccag atttatgta attgatcca gaa 432 TyrGluGlyIle GlyAsn GlyThrGln IleTyrVal IleAspPro Glu ccg tgc gat cag gag 480 cca gat ccc aaa tct tct tct gac aaa act cac Pro Cys Ser Ser Asp Lys Thr Pro Asp His Ser Asp Gln Glu Pro Lys aCa tCC CCa gCa CCt Ctg ggg gga tcg tca 528 CCa CCg gaa ctC gtc tCC

Thr Ser Pro Ala Pro Leu Gly Gly Ser Ser Pro Pro Glu Leu Val Ser ttc ctc aaa ccc aag acc ctc atg atc tcc cgg 576 ttc ccc gac acc cca Phe Leu Pro Lys Pro Lys Thr Leu Met Ile Ser Arg ' Phe Pro Asp Thr cct gag aca gtg gtg gtg gtg agc cac gaa gac cet 624 gtc tgc gac gag Pro Glu Thr Val Val Val Val Ser His Glu Asp Pro Val Cys Asp Glu gtc aag aac tac gtg gac gtg gag gtg cat aat gcc 672 ttc tgg ggc aag Val Lys Asn Tyr Val Asp Val Glu Val His Asn Ala Phe Trp Gly Lys aca aag cgg gag cag tac agc acg tac cgt gtg gtc 720 ccg gag aac agc Thr Lys Arg Glu Gln Tyr Ser Thr Tyr Arg Val Val Pro Glu Asn Ser gtc ctc gtc cac cag gac ctg aat ggc aag gag tac 76g acc ctg tgg aag Val Leu Val His Gln Asp Leu Asn Gly Lys Glu Tyr Thr Leu Trp Lys tgc aag tcc aaa gcc ctc gcc ccc atc gag aaa acc 816 gtc aac cca atc Cys Lys Ser Lys Ala Leu Ala Pro Ile Glu Lys Thr Val Asn Pro Ile tcc aaa aaa cag ccc cga cca cag gtg tac acc ctg 864 gcc ggg gaa ccc Ser Lys Lys Gln Pro Arg Pro Gln Val Tyr Thr Leu Ala Gly Glu Pro cca 'tcc gat ctg acc aag cag gtc agc ctg acc tgc 912 cgg gag aac ctg Pro Ser Asp Leu Thr Lys Gln Val Ser Leu Thr Cys Arg Glu Asn Leu gtc aaa ttc ccc agc gac gcc gtg gag tgg gag agc 960 ggc tat atc aat Val Lys Phe Pro Ser Asp Ala Val Glu Trp Glu Ser Gly Tyr Ile Asn ggg cag gag aac tac aag acg cct ccc gtg ctg gac 1008 ccg aac acc tcc Gly Gln Glu Thr Pro Pro Val Leu Asp Pro Asn Ser Asn Tyr Lys Thr gac ggc ttc ctc 1056 tcc ttc acc ctc gtg tac gac agc aag aag agc agg Asp Gly Phe Leu Ser Phe Thr Leu Val Tyr Asp Ser Lys Lys Ser Arg 3l5 320 325 tgg cag ggg 1104 cag aac gtc ttc tca tgc tcc gtg atg cat gag get ctg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga 1152 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 13 <211> 383 <212> PRT
<213> Artificial <220>
<223> L104EA29TIg <400> 13 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro -10 -5 -1 1 5 .
Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Thr Thr Glu Val Arg Val Thr Val Leu Arg 25 30 , 35 Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp 75 80 85 °
Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys . 185 190 195 Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys 215 220 225. 230 Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 330 335 '340 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 14 <211> 1152 <212> DNA

<213> Artificial <220>
<223> L104EA29WIg <220>
<221> CDS
<222> (1)..(1149) <220>
<221> matVpeptide <222> (79) . . () <400> 14 atg ggt gta ctg ctc aca cag agg acg ctg ctc agt ctg gtc ctt gca 48 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala ctc ctg ttt cca agc atg gcg agc atg gca atg cac gtg gcc cag cct 96 Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro -10 -5 _1 1 5 getgtggtactg gccagcagc cgaggc atcgetagcttt gtgtgt gag 144 AlaValValLeu AlaSerSer ArgGly IleAlaSerPhe ValCys Glu tatgcatctcca ggcaaatgg actgag gtccgggtgaca gtgctt cgg 192 TyrAlaSerPro GlyLysTrp ThrGlu ValArgValThr ValLeu Arg caggetgacagc caggtgact gaagtc tgtgcggcaacc tacatg atg 240 GlnAlaAspSer GlnValThr GluVal CysAlaAlaThr TyrMet Met gggaatgagttg accttccta gatgat tccatctgcacg ggcacc tcc 288 GlyAsnGluLeu ThrPheLeu AspAsp SerIleCysThr GlyThr Ser agtggaaatcaa gtgaacctc actatc caaggactgagg gccatg gac 336 SerGlyAsnGln ValAsnLeu ThrIle GlnGlyLeuArg AlaMet Asp acgggactctac atctgcaag gtggag ctcatgtaccca ccgcca tac 384 ThrGlyLeuTyr IleCysLys ValGlu LeuMetTyrPro ProPro Tyr tacgagggcata ggcaacgga acccag atttatgtaatt gatcca gaa 432 TyrGluGlyIle GlyAsnGly ThrGln IleTyrValIle AspPro Glu ccgtgcccagat tctgatcag gagccc aaatcttctgac aaaact cac 480 ProCysProAsp SerAspGln GluPro LysSerSerAsp LysThr His acatccccaccg tccccagca cctgaa ctcctgggggga tcgtca gtc 52g ThrSerProPro SerProAla ProGlu LeuLeuGlyGly SerSer Val ttc ctc ttc aaa ccc aag acc ctc atg atc tcc cgg 576 ccc cca gac acc Phe Leu Phe Lys Pro Lys Thr Leu Met Ile Ser Arg Pro Pro Asp Thr cct gag gtc tgcgtg gtg gtg gtg agc cac gaa gac cct 624 aca gac gag Pro Glu Val CysVal Val Val Val Ser His Glu Asp Pro Thr Asp Glu gtc aag ttc tggtac gtg gac gtg gag gtg cat aat gcc 672 aac ggc aag Val Lys Phe TrpTyr Val Asp Val Glu Val His Asn Ala Asn Gly Lys aca aag ccg gaggag cag tac agc acg tac cgt gtg gtc 720 cgg aac agc Thr Lys Pro GluGlu Gln Tyr Ser Thr Tyr Arg Val Val Arg Asn Ser gtc ctc acc ctgcac cag gac ctg aat ggc aag gag tac 76g gtc tgg aag Val Leu Thr LeuHis Gln Asp Leu Asn Gly Lys Glu Tyr Val Trp Lys tgc aag gtc aacaaa gcc ctc gcc ccc atc gag aaa acc 816 tcc cca atc Cys Lys Val AsnLys Ala Leu Ala Pro Ile Glu Lys Thr Ser Pro Ile tcc aaa gcc gggcag ccc cga cca cag gtg tac acc ctg 864 aaa gaa ccc Ser Lys Ala GlyGln Pro Arg Pro Gln Val Tyr Thr Leu Lys Glu Pro cca tcc cgg gagctg acc aag cag gtc agc ctg acc tgc 912 gat aac ctg Pro Ser Arg GluLeu Thr Lys Gln Val Ser Leu Thr Cys Asp Asn Leu gtc aaa ggc tatccc agc gac gcc gtg gag tgg gag agc g6p ttc atc aat Val Lys Gly TyrPro Ser Asp Ala Val Glu Trp Glu Ser Phe Ile Asn ggg cag ccg aacaac tac aag acg cct ccc gtg ctg gac 1008 gag acc tcc Gly Gln Pro Asn Thr Pro Pro Val Leu Asp Glu Asn Ser Tyr Lys Thr gac ggc tcc ttcctc tac agc ctc acc gtg gac aag a 1 ttc aag c a g 056 Asp Gly Ser gg Phe Phe Leu Tyr Ser Lys Leu Thr V
l A

a sp Lys Ser Arg tgg cag cag aac 1104 ggg gtc ttc tca tgc tcc gtg atg cat gag get ctg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu cac aac cac 1152 tac acg cag aag agc ctc tcc ctg tct ccg ggtaaa tga His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 15 <211> 383 <212> PRT
<213> Artificial <220>
<223> L104EA29WIg <400> 15 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu 15 ' 20 Tyr Ala Ser Pro Gly Lys Trp Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Glu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met, Ile Ser Arg Thr l55 160 165 Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro 250 . 255 260 Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val"Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro G1y Lys <210> l6 <211> 636 <212> DNA
<213> Homo Sapiens <220>
<221> CDS
<222> (1) . . (636) <220>

<221> mat_peptide <222> (79).
.
() <400> 16 atgggt gtactgctcaca cagaggacg ctgctc agtctggtc cttgca 48 MetGly ValLeuLeuThr GlnArgThr LeuLeu SerLeuVal LeuAla ctcctg tttccaagcatg gcgagcatg gcaatg cacgtggcc cagcct 96 LeuLeu PheProSerMet AlaSerMet AlaMet HisValAla GlnPro getgtg gtactggccagc agccgaggc atcgcc agctttgtg tgtgag 144 AlaVal ValLeuAlaSer SerArgGly IleAla SerPheVal CysGlu tatgca tctccaggcaaa gccactgag gtccgg gtgacagtg cttcgg 192 TyrAla SerProGlyLys AlaThrGlu ValArg ValThrVal LeuArg cagget gacagccaggtg actgaagtc tgtgcg gcaacctac atgatg 240 GlnAla AspSerGlnVal ThrGluVal CysAla AlaThrTyr MetMet gggaat gagttgaccttc ctagatgat tccatc tgcacgggc acctcc 288 GlyAsn GluLeuThrPhe LeuAspAsp SerTle CysThrGly ThrSer agtgga aatcaagtgaac ctcactatc caagga ctgagggcc atggac 336 SerGly AsnGlnValAsn LeuThrIle GlnGly LeuArgAla MetAsp acggga ctctacatctgc aaggtggag ctcatg tacccaccg ccatac 384 ThrGly LeuTyrIleCys LysValGlu LeuMet TyrProPro ProTyr tacctg ggcataggcaac ggaaccc~agatttat gtaattgat ccagaa 432 TyrLeu GlyIleGlyAsn GlyThrGln IleTyr ValIleAsp ProGlu ccgtgc ccagattctgac ttcctcctc tggatc cttgcagca gttagt 480 ProCys ProAspSerAsp PheLeuLeu TrpIle LeuAlaAla ValSer tcgggg ttgtttttttat agctttctc ctcaca getgtttct ttgagc 528 SerGly LeuPhePheTyr SerPheLeu LeuThr AlaValSer LeuSer aaaatg ctaaagaaaaga agccctctt acaaca ggggtctat gtgaaa 576 LysMet LeuLysLysArg SerProLeu ThrThr GlyValTyr ValLys atgccc ccaacagagcca gaatgtgaa aagcaa tttcagcct tatttt 624 MetPro ProThrGluPro GluCysGlu Gln PheGlnPro TyrPhe Lys att ccc atc aat 636 Ile Pro Ile Asn <210> 17 <211> 212 <212> PRT
<213> Homo sapiens <400> 17 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Va1 Thr Glu Val Cys Ala Ala Thr Tyr Met Met 40 45 ' 50 Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Phe Leu Leu Trp Ile Leu Ala Ala Val Ser Ser Gly Leu Phe Phe Tyr Ser Phe Leu Leu Thr Ala Val Ser Leu Ser Lys Met Leu Lys Lys Arg Ser Pro Leu Thr Thr Gly Val Tyr Val Lys Met Pro Pro Thr Glu Pro Glu Cys Glu Lys Gln Phe Gln Pro Tyr Phe Ile Pro Ile Asn <210> 18 <211> 1152 <212> DNA
<213> Artificial <220>
<223> CTLA4Ig <220>

<221> CDS

<222> (1)..(1149) <220>

<221> mat-pept ide <222> (79).
.
() <400> 18 atg gtactg ctcaca cagaggacg ctgctcagt ctggtcctt ca 48 ggt Met ValLeu LeuThr GlnArgThr LeuLeuSer LeuValLeu g Gly Ala ctc tttcca agcatg gcgagcatg gcaatgcac gtggcccag cct 96 ctg Leu PhePro SerMet AlaSerMet AlaMetHis ValAlaGln Pro Leu get gtactg gccagc agccgaggc atcgetagc tttgtgtgt gag 144 gtg Ala ValLeu AlaSer SerArgGly IleAlaSer PheValCys Glu Val tat tctcca ggcaaa gccactgag gtccgggtg acagtgctt cgg 192 gca Tyr SerPro GlyLys AlaThrGlu ValArgVal ThrValLeu Arg Ala cag gacagc caggtg actgaagtc tgtgcggca acctacatg atg 240 get Gln AspSer GlnVal ThrGluVal CysAlaAla ThrTyrMet Met Ala ggg gagttg accttc ctagatgat tccatctgc acgggcacc tcc 288 aat Gly GluLeu ThrPhe LeuAspAsp SerIleCys ThrGlyThr Ser Asn agt aatcaa gtgaac ctcactatc caaggactg agggccatg gac 336 gga Ser AsnGln ValAsn LeuThrIle GlnGlyLeu ArgAlaMet Asp Gly acg atc aag gag cca cca 384 gga tgc gtg ctc ccg tac ctc atg tac tac Thr Ile LysValGlu Pro Pro Gly Cys Leu Pro Tyr Leu Met Tyr Tyr tacctg ataggcaac ggaacccag tat attgat cca 432 ggc att gta gaa Tyr Gly GlyThrGln Tyr IleAsp Pro Leu Asn Ile Val Glu Gly Ile ccgtgccca gattctgat caggagccc aaatcttct gacaaa actcac 480 ProCysPro AspSerAsp GlnGluPro SerSer AspLys ThrHis Lys acatcccca ccgtcccca gcacctgaa ctcctgggt ggatcg tcagtc 528 ThrSerPro ProSerPro AlaProGlu LeuLeuGly GlySer SerVal ttcctcttc cccccaaaa cccaaggac accctcatg atctcc cggacc 576 PheLeuPhe ProProLys ProLysAsp ThrLeuMet IleSer ArgThr cctgaggtc acatgcgtg gtggtggac gtgagccac gaagac cctgag 624 ProGluVal ThrCysVal ValValAsp ValSerHis GluAsp ProGlu gtcaagttc aactggtac gtggacggc gtggaggtg cataat gccaag 672 ValLysPhe AsnTrpTyr ValAspGly ValGluVal HisAsn AlaLys acaaagccg cgggaggag cagtacaac agcacgtac cgggtg gtcagc 720 ThrLysPro ArgGluGlu GlnTyrAsn SerThrTyr ArgVal ValSer gtcctcacc gtcctgcac caggactgg ctgaatggc aaggag tacaag 768 ValLeuThr ValLeuHis GlnAspTrp LeuAsnGly LysGlu TyrLys tgcaaggtc tccaacaaa gccctccca gcccccatc gagaaa accatc 816 CysLysVal SerAsnLys AlaLeuPro AlaProIle GluLys ThrIle tccaaagcc aaagggcag ccccgagaa ccacaggtg tacacc ctgccc 864 SerLysAla LysGlyGln ProArgGlu ProGlnVal TyrThr LeuPro ccatcccgg gatgagctg accaagaac caggtcagc ctgacc tgcctg 912 ProSerArg AspGluLeu ThrLys GlnValSer LeuThr CysLeu Asn gtcaaaggc ttctatccc agcgacatc gccgtggag tgggag aat 960 agc ValLysGly Phe Pro Ser Ile AlaValGlu TrpGlu Asn Tyr Asp Ser gggcagccg gag aac aag acgcctccc ctg tcc 1008 aac tac acc gtg gac GlyGlnPro Glu ThrProPro Ser Asn Val Asn Leu Tyr Asp Lys Thr gac tcc ctc gtg agg 1056 ggc ttc acc gac ttc aag ctc agc tac agc aag Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg tgg cag cag ggg aac gtc ttc tca tgc tcc gtg atg cat gag get ctg 1104 Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu cac aac cac tac acg cag aag agc ctc tcc ctg tct ccg ggt aaa tga 1152 His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 19 <211> 383 <212> PRT
<213> Artificial <220>
<223> CTLA4Ig <400> 19 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val 135 , 140 145 150 Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr'Asn Ser Thr Tyr Arg Val Val Ser 200 205 2l0 Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro A1a Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys <210> 20 <211> 6 <212> PRT
<213> Artificial <220>
<223> MYPPPY amino acid sequence <400> 20 Met Tyr Pro Pro Pro Tyr <210> 21 <211> 1152 <212> DNA
<213> Artificial <220>
<223> CTLA4Ig <220>
<221> CDS
<222> (1)..(1149) <220>
<221> mat_peptide <222> (79) .. () ' <400> 21 atg ggt gta ctg ctc aca cag agg acg ctg ctc agt ctg gtc ctt gca 48 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala ctc ctg ttt cca agc atg gcg agc atg gca atg cac gtg gcc cag cct 96 Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro get gtg gta ctg gcc agc agc cga ggc atc gcc agc ttt gtg tgt gag 144 Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu tat gca tct cca ggc aaa gcc act gag gtc cgg gtg aca gtg ctt cgg 192 Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg cag get gac agc cag gtg act gaa gtc tgt gcg gca acc tac atg atg 240 Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met ggg aat gag ttg acc ttc cta gat gat tcc atc tgc acg ggc acc tcc 2gg Gly AsnGluLeu ThrPheLeu AspAspSerIle CysThr GlyThrSer 55 60 ' 65 70 agt ggaaatcaa gtgaacctc actatccaagga ctgagg gccatggac 336 Ser GlyAsnGln ValAsnLeu ThrIleGlnGly LeuArg AlaMetAsp acg ggactctac atctgcaag gtggagctcatg taccca ccgccatac 384 Thr GlyLeuTyr IleCysLys ValGluLeuMet TyrPro ProProTyr tac ctgggcata ggcaacgga acccagatttat gtaatt gatccagaa 432 Tyr LeuGlyIle GlyAsnGly ThrGlnIleTyr ValIle AspProGlu ccg tgcccagat tctgatcag gagcccaaatct tctgac aaaactcac 480 Pro CysProAsp SerAspGln GluProLysSer SerAsp LysThrHis aca tccccaccg tccccagca cctgaactcctg ggggga tcgtcagtc 528 Thr SerProPro SerProAla ProGluLeuLeu GlyGly SerSerVal ttc ctcttcccc ccaaaaccc aaggacaccctc atgatc tcccggacc 576 Phe LeuPhePro ProLysPro LysAspThrLeu MetIle SerArgThr cct gaggtcaca tgcgtggtg gtggacgtgagc cacgaa gaccctgag 624 Pro GluValThr CysValVal ValAspValSer HisGlu AspProGlu gtc aagttcaac tggtacgtg gacggcgtggag gtgcat aatgccaag 672 Val LysPheAsn TrpTyrVal AspGlyValGlu ValHis AsnA1aLys aca aagccgcgg gaggagcag tacaacagcacg taccgt gtggtcagc 720 Thr LysProArg GluGluGln TyrAsnSerThr TyrArg ValValSer gtc ctcaccgtc ctgcaccag gactggctgaat ggcaag gagtacaag 768 Val LeuThrVal LeuHisGln AspTrpLeuAsn GlyLys GluTyrLys tgc aaggtctcc aacaaagcc ctcccagccccc atcgag aaaaccatc 816 Cys LysValSer AsnLysAla LeuProAlaPro IleGlu LysThrIle tcc aaagccaaa gggcagccc cgagaaccacag gtgtac accctgccc 864 Ser LysAlaLys GlyGlnPro ArgGluProGln ValTyr ThrLeuPro cca tcccgggat gagctgacc aagaaccaggtc agcctg acctgcctg 912 Pro SerArgAsp GluLeuThr LysAsnGlnVal SerLeu ThrCysLeu gtc aaaggcttc tatcccagc gacatcgccgtg gagtgg gagagcaat 960 Val LysGlyPhe TyrProSer AspIleAlaVal GluTrp GluSerAsn gggcagccg gag aac tacaag accacgcctccc gtgctg gactcc 1008 aac GlyGlnPro Glu Asn TyrLys ThrThrProPro ValLeu AspSer Asn gacggctcc ttc ctc tacagc aagctcaccgtg gacaag agcagg 1056 ttc AspGlySer Phe Leu TyrSer LysLeuThrVal AspLys SerArg Phe tggcagcag ggg gtc ttctca tgctccgtgatg catgag getctg 1104 aac TrpGlnGln Gly Val PheSer CysSerValMet HisGlu AlaLeu Asn cacaaccac tac cag aagagc ctctccctgtct ccgggt aaatga 1152 acg HisAsnHis Tyr Gln LysSer LeuSerLeuSer ProGly Lys Thr <210> 22 <211> 383 <212> PRT
<213> Artificial <220>
<223> CTLA4Ig <400> 22 Met Gly Val Leu Leu Thr Gln Arg Thr Leu Leu Ser Leu Val Leu Ala Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val Ala Gln Pro Ala Val Val Leu Ala Ser Ser Arg Gly Ile Ala Ser Phe Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr Val Leu Arg Gln Ala Asp Ser Gln Val Thr Glu Val Cys Ala Ala Thr Tyr Met Met 40 . 45 50 Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr Gly Thr Ser Ser Gly Asn Gln Val Asn Leu Thr Ile Gln Gly Leu Arg Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gln Ile Tyr Val Ile Asp Pro Glu Pro Cys Pro Asp Ser Asp Gln Glu Pro Lys Ser Ser Asp Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Tle Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys

Claims (115)

What is claimed:

1. A method for blocking B7 interactions with CTLA4 and/or CD28 comprising administering to a subject an effective amount of a first agent and a second agent, wherein a) the first agent is a soluble CTLA4 molecule, and b) the second agent is selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

2. A method for treating an immune system disease comprising administering to a subject an effective amount of a first agent and a second agent, wherein a) the first agent is a soluble CTLA4 molecule, and b) the second agent is selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

3. The method of claim 1 or 2, wherein cytokine production is regulated.

4. The method of claim 1 or 2, wherein ACR 20, 50 and/or 70 response rates are improved.

5. A method for blocking B7 interactions with CTLA4 and/or CD28 comprising administering to a subject an effective amount of a soluble CTLA4 molecule, wherein the effective amount of soluble CTLA4 is about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about to 40 mg/kg weight of a subject, about 40 to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about 18 to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

6. A method for treating an immune system disease comprising administering to a subject an effective amount of a soluble CTLA4 molecule, wherein the effective amount of soluble CTLA4 is about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject,about 18 to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

7. The method of claim 5 or 6, wherein cytokine production is regulated.

8. The method of claim 5 or 6, wherein ACR 20, 50 and/or 70 response rates are improved.

9. The method of claim 1, 2, 5, or 6, wherein the soluble CTLA4 molecule comprises an extracellular domain of a CTLA4 molecule, or portion thereof, which binds a B7 antigen expressed on activated B cells.

10. The method of claim 9, wherein the extracellular domain of the CTLA4 molecule comprises the amino acids shown in Figure 23 (SEQ ID NO:17) beginning with methionine at position 1 and or with alanine at position -1 and ending with aspartic acid at position 124.

11. The method of claim 1, 2, 5, or 6, wherein the soluble CTLA4 molecule is a CTLA4 fusion molecule.

12. The method of claim 11, wherein the CTLA4 fusion molecule comprises an extracellular domain of a CTLA4 molecule which binds a B7 antigen expressed on activated B cells joined to a non-CTLA4 molecule.

13. The method of 12, wherein the non- CTLA4 molecule comprises an amino acid sequence which alters the solubility or affinity of the soluble CTLA4 molecule.

14. The method of claim 13, wherein the amino acid sequence which alters the solubility or affinity comprises an immunoglobulin moiety.

15. The method of claim 14, wherein the immunoglubulin moiety comprises one or more mutations to reduce effector function.

16. The method of claim 14, wherein the immunoglubulin moiety is an immunoglubulin constant region or portion thereof.

17. The method of claim 15, wherein the immunoglubulin constant region or portion thereof is mutated to reduce effector function.

18. The method of claim 15, wherein the immunoglubulin constant region comprises a hinge, CH2 and CH3 regions of an immunoglobulin molecule.

19. The method of claim 15, wherein the immunoglubulin constant region or portion thereof is a human or monkey immunoglobulin constant region.

20. The method of claim 1, 2, 5, or 6, wherein the soluble CTLA4 further comprises an amino acid sequence which permits secretion of the soluble CTLA4 molecule.

21. The method of claim 20, wherein the amino acid sequence which permits secretion comprises an oncostatin M signal peptide.

22. The method of claim 11, wherein the CTLA4 fusion molecule is CTLA4Ig.

23. The method of claim 22, wherein the CTLA4Ig is shown in Figure 24 (SEQ ID
NO:19) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine at position +357.

24. The method of claim 1, 2, 5, or 6, wherein the soluble CTLA4 molecule is a soluble CTLA4 mutant molecule.

25. The method of claim 24, wherein the soluble CTLA4 mutant molecule binds a antigen expressed on activated B cells and comprises a mutation in the extracellular domain of a CTLA4 molecule.

26. The method of claim 24, wherein the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), and b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with any other amino acid.

27. The method of claim 24, wherein the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with glutamic acid, and c) wherein at position +29 of the extracellular domain of CTLA4, alanine is substituted with tyrosine.

28. The method of claim 24, wherein the soluble CTLA4 mutant molecule is L104EA29YIg as shown in Figure 19 (SEQ ID NO: 9) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine at position +357 as shown in Figure 19 (SEQ ID NO:9).

29. A method for treating an immune system disease comprising administering to a subject a combination of an effective amount of a soluble CTLA4 and a second agent, wherein a) the soluble CTLA4 is a CTLA4Ig beginning with methionine at position +1 or with alanine at position -1 and ending with lysine as position +357 as shown in Figure 24 (SEQ ID NO:19), and b) the second agent is selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

30. A method for treating an immune system disease comprising administering to a subject a combination of an effective amount of a soluble CTLA4 and a second agent, wherein a) the soluble CTLA4 comprises the extracellular domain of a CTLA4 molecule as shown in Figure 23 (SEQ ID NO: 17) beginning with methionine at position +1 or with alanine at position -1 and ending with aspartic acid at position +124, and b) the second agent is selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

31. A method for treating an immune system disease comprising administering to a subject a combination of an effective amount of a soluble CTLA4 and a second agent, wherein a) the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, i) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), and ii) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with any other amino acid.
b) the second agent is selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

32. The method of claim 31, wherein the. soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with glutamic acid, and c) wherein at position +29 of the extracellular domain of CTLA4, alanine is substituted with tyrosine.

33. The method of claim 31, wherein the soluble CTLA4 mutant molecule is L104EA29YIg as shown in Figure 19 (SEQ ID NO: 9) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine at position +357 as shown in Figure 19 (SEQ ID NO: 9).

34. A method for treating an immune system disease comprising administering to a subject a combination of an effective amount of a soluble CTLA4 and a second agent, wherein a) the soluble CTLA4 is L104EA29YIg beginning with methionine at position +1 or with alanine at position -1 and ending with lysine as position +357 as shown in Figure 19 (SEQ ID NO: 9), and b) the second agent is selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

35. A method for blocking B7 interactions with CTLA4 and/or CD28 comprising administering to a subject an effective amount of a first agent and a second agent, wherein a) the first agent is a soluble CTLA4 molecule, and b) the second agent is Methotrexate, wherein the effective amount of Methotrexate is about 0.1 to 40 mg per week, about 5 to 30 mg per week, about 0.1 to 5 mg/week, about 5 to 10 mg/week, about to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/ week or about 10 to 30 mg/week.

36. A method for treating an immune system disease comprising administering to a subject an effective amount of a first agent and a second agent, wherein a) the first agent is a soluble CTLA4 molecule, and b) the second agent is Methotrexate, wherein the effective amount of Methotrexate is about 0.1 to 40 mg per week, about 5 to 30 mg per week, about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/ week or about 10 to 30 mg/week.

37. A method for blocking B7 interactions with CTLA4 and/or CD28 comprising administering to a subject an effective amount of a first agent and a second agent, wherein a) the first agent is a soluble CTLA4 molecule, and b) the second agent is Etanercept, wherein the effective amount of Etanercept is about 10 to 100 mg per week, about 50 mg per week, about 0.1 to 50 mg/kg body weight per week

38. A method for treating an immune system disease comprising administering to a subject an effective amount of a first agent and a second agent, wherein a) the first agent is a soluble CTLA4 molecule, and b) the second agent is Etanercept, wherein the effective amount of Etanercept is about 10 to 100 mg per week, about 50 mg per week, about 0.1 to 50 mg/kg body weight per week

39. A method for blocking B7 interactions with CTLA4 and/or CD28 comprising administering to a subject an effective amount of a first agent and a second agent, wherein a) the first agent is a soluble CTLA4 molecule, and b) the second agent is a DMARD, wherein the effective amount of the DMARD is about 1 to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day; about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to 2000 mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day.

40. A method for treating an immune system disease administering to a subject an effective amount of a first agent and a second agent, wherein a) the first agent is a soluble CTLA4 molecule, and b) the second agent is a DMARD, wherein the effective amount of the DMARD is about 1 to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to 2000 mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day.

41. The method of claim 40, wherein the DMARD is selected from a group consisting of a dihydrofolic acid reductase inhibitor, cyclophosphamide, cyclosponine, cyclosporin A, chloroquine, hydroxychloroquine, sulfasalazine (sulphasalazopyrine), gold salts, D-penicillamine, leflunomide, azathioprine, anakinra, a TNF blocker, a biological agent that targets an inflammatory cytokine, methotrexate or etanercept.

42. The method of claim 1, 2, 29, 30, 31, 34, 35, 36, 37, 38, 39 or 40, wherein the effective amount of soluble CTLA4 is between about 0.5 through 100 mg/kg weight of the subject, about 0.1 to 100 mg/kg weight of the subject, about 0.5 to mg/kg weight of a subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, weight of a subject, about 0.1 to mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about 18 to 20 mg/kg weight of a subject, 0.5 mg/kg weight of the subject, 2 mg/kg weight of a subject, 10 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

43. The method of claim 2, 6, 29, 30, 31, 34, 36, 38 or 40, wherein the immune system disease is a rheumatic disease.

44. The method of claim 43, wherein the rheumatic disease is rheumatoid arthritis.

45. The method of claim 2, 6, 29, 30, 31, 34, 36, 38 or 40,, wherein the immune system disease is selected from autoimmune diseases, psoriasis, immune disorders associated with graft transplantation rejection, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin dependent diabetes mellitis, good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic disease, rheumatoid arthritis, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, collagen diseases, chronic polyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenile rheumatoid arthritis, periarthritis humeroscapularis, panarteriitis nodosa, systemic lupus erythematosus, progressive systemic scleroderma, arthritis uratica, dermatomyositis, muscular rheumatism, myositis, myogelosis andchondrocalcinosis.

46. The method of claim 2, 6, 29, 30, 31, 34, 36, 38 or 4,, wherein the immune system disease is selected from graft related disorders, chronic rejection, tissue or cell allo- or xenografts, skin allo- or xenografts, islet allo- or xenografts, muscle allo- or xenografts, hepatocyte allo- or xenografts and neuron allo- or xenografts.

47. The method of claim 2, 6, 29, 30, 31, 34, 36, 38 or 40,, wherein a symptom associated with the immune system disease is alleviated and wherein the symptom is selected from the group consisting of joint swelling, pain, tenderness, morning stiffness, structural damage, an elevated level of serum C-reactive protein, an elevated level of soluble IL-2r, an elevated level of soluble ICAM-1, an elevated level of soluble E-selectin and an elevated erythrocyte sedimentation rate.

48. The method of claim 2, 6, 29, 30, 31, 34, 36, 38 or 40,, wherein treating a subject suffering from an immune system disease induces a pathophysiological change.

49. The method of claim 48, wherein the pathophysiological change associated with the immune system disease is reduced structural damage.

50. A method for treating a disease selected from autoimmune diseases, psoriasis, immune disorders associated with graft transplantation rejection, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin dependent diabetes mellitis, good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic disease, rheumatoid arthritis, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, collagen diseases, chronic polyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenile rheumatoid arthritis, periarthritis humeroscapularis, panarteriitis nodosa, systemic lupus erythematosus, progressive systemic scleroderma, arthritis uratica, dermatomyositis, muscular rheumatism, myositis, myogelosis andchondrocalcinosis, by administering to a subject an effective amount of a soluble CTLA4 molecule comprising the sequence shown in Figure 19 (SEQ ID NO: 9) beginning with methionine at position +1 or with alanine at position -1 and ending with aspartic acid at position +124, and wherein the effective amount is about 0.1 to 100 mg/kg weight of the subject, about 0.5 to mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/leg weight of a subject, about 15 to 20 mg/kg weight of a subject, about to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

51. A method for treating a disease selected from autoimmune diseases, psoriasis, immune disorders associated with graft transplantation rejection, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin dependent diabetes mellitis, good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic disease, rheumatoid arthritis, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, collagen diseases, chronic polyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenile rheumatoid arthritis, periarthritis humeroscapularis, panarteriitis nodosa, systemic lupus erythematosus, progressive systemic scleroderma, arthritis uratica, dermatomyositis, muscular rheumatism, myositis, myogelosis andchondrocalcinosis, by administering to a subject an effective amount of a soluble CTLA4 molecule L104EA29YIg having the sequence shown in Figure 19 (SEQ ID NO: 9) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine as position +357, and wherein the effective amount is about 0.1 to 100 mg/kg weight of the subject, about 0.5 to mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

52. A method for treating a disease selected from graft related disorders, chronic rejection, tissue or cell alto- or xenografts, skin allo- or xenografts, islet allo- or xenografts, muscle allo- or xenografts, hepatocyte allo- or xenografts and neuron allo- or xenografts, by administering to a subject an effective amount of a soluble CTLA4 molecule comprising the sequence shown in Figure 19 (SEQ ID NO: 9) beginning with methionine at position +1 or with alanine at position -1 and ending with aspartic acid at position +124, and wherein the effective amount is about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, weight of a subject, about 0.1 to mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about to 18 mg/kg weight of a subject, about 18 to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

53. A method for treating a disease selected from graft related disorders, chronic rejection, tissue or cell allo- or xenografts, skin allo- or xenografts, islet allo- or xenografts, muscle allo- or xenografts, hepatocyte allo- or xenografts and neuron allo- or xenografts, by administering to a subject an effective amount of a soluble CTLA4 molecule L104EA29YIg having the sequence shown in Figure 19 (SEQ
ID NO: 9) beginning with methionine at position +1 or with alanine at position and ending with lysine as position +357, and wherein the effective amount is about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about 18 to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

54. A method for treating rheumatic disease comprising administering to a subject an effective amount of a soluble CTLA4 molecule L104EA29YIg having the sequence shown in Figure 19 (SEQ ID NO: 9) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine as position +357, and wherein the effective amount is about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about 18 to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

55. The method of claim 35, 37, or 39, cytokine production is regulated.

56. The method of claim 35, 37, or 39, wherein improves ACR 20, 50 and/or 70 response rates are improved.

57. The method of claim 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 50, 51, 52, 53 or 54, wherein the soluble CTLA4 molecule comprises an amino acid sequence which permits secretion of the soluble CTLA4 molecule.

58. The method of claim 57, wherein the amino acid sequence which permits secretion comprises an oncostatin M signal peptide.

59. The method of claim 1, 2, 5, 6, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 50, 51, 52, 53 or 54, wherein administration of the agents is effected locally or systemically.

60. The method of claim 59, wherein administration is selected from the group consisting of, intravenous, intramuscular, intraperitoneal, oral, inhalation, subcutaneous, implantable pump, continuous infusion, gene therapy, liposomes, suppositories, topical contact, vesicles, capsules, biodegradable polymers, hydrogels, controlled release patch and injection methods.

61. The method of claim 1, 2, 5, 6, 29, 30, 31, 34, 35, 36, 37, 38, 39, 40, 50, 51, 52, 53 or 54, wherein the subject is selected from the group consisting of human, monkey, ape, dog, cat, cow, horse, rabbit, mouse, and rat.

62. The method of claim 22 or 29, wherein the CTLA4Ig is encoded by a nucleic acid molecule designated ATCC No. 68629.

63. The method of claim 28, 32, 51, 53 or 54, wherein L104EA29YIg is encoded by a DNA sequence designated ATCC PTA-2104.

64. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an effective amount of a first agent and a second agent, wherein a) the first agent is soluble CTLA4, and b) the second agent is selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

65. The pharmaceutical composition of claim 64, wherein the soluble CTLA4 comprises the extracellular domain of a CTLA4 molecule, or portion thereof, which binds a B7 antigen expressed on activated B cells.

66. The pharmaceutical composition of claim 64, wherein the soluble CTLA4 is a CTLA4 fusion molecule.

67. The pharmaceutical composition of claim 66, wherein the CTLA4 fusion molecule comprises an extracellular domain of a CTLA4 molecule which binds a B7 antigen expressed on activated B cells joined to a non-CTLA4 molecule.

68. The pharmaceutical composition of claim 64, wherein the soluble CTLA4 is a soluble CTLA4 mutant molecule.

69. The pharmaceutical composition of claim 68, wherein the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), and b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with any other amino acid.

70. The pharmaceutical composition of claim 68, wherein the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with glutamic acid, and c) wherein at position +29 of the extracellular domain of CTLA4, alanine is substituted with tyrosine.

71. The pharmaceutical composition of claim 68, wherein the soluble CTLA4 mutant molecule is L104EA29YIg as shown in Figure 19 (SEQ ID NO: 9), beginning with methionine at position +1 or with alanine at position -1 and ending with lysine at position +357.

72. The pharmaceutical composition of claim 64, wherein the pharmaceutically acceptable carrier is selected ion exchangers, alumina, aluminum stearate, lecithin, serum proteins, such as human serum albumin, buffer substances, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, phosphate buffered saline solution, water, emulsions, salts or electrolytes, colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone, cellulose-based substances, polyethylene glycol, sterile solutions, tablets, excipients, sucrose, glucose, maltose, flavor and color additives, lipid compositions and polymeric compositions.

73. Combination of a pharmaceutical composition comprising a soluble CTLA4 with a pharmaceutical composition comprising an agent selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol, for use in therapy.

74. A kit comprising an effective amount of a first agent and a second agent, wherein a) the first agent is soluble CTLA4, and b) the second agent is selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

75. The kit of claim 74, further comprising a label indicating that the first and second agents are useful to treat an immune system disease.

76. The kit of claim 75, wherein the label indicates an effective amount for the first agent being about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

77. The kit of claim 75, wherein the label indicates an effective amount for the second agent being about 0.1 to 40 mg per week, about 5 to 30 mg per week, about 0.1 to mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/ week, about 10 to 30 mg/week, about 10 to 100 mg/week, about 50 mg/week, about 0.1 to 50 mg/kg body weight per week, about 1 to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to 2000 mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day.

78. The kit of claim 75, wherein the label indicates an effective amount for the first and second agent a. the effective amount of the first agent being about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about 18 to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg, and b. the effective amount of the second agent is about 0.1 to 40 mg per week, about 5 to 30 mg per week, about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/ week, about 10 to 30 mg/week, about 10 to 100 mg/week, about 50 mg/week, about 0.1 to 50 mg/kg body weight per week, about 1 to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to 2000 mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day.

79. The kit of claim 75, wherein the immune system disease is selected from autoimmune diseases, psoriasis, immune disorders associated with graft transplantation rejection, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin dependent diabetes mellitis, good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic disease, rheumatoid arthritis, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, collagen diseases, chronic polyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenile rheumatoid arthritis, periarthritis humeroscapularis, panarteriitis nodosa, systemic lupus erythematosus, progressive systemic scleroderma, arthritis uratica, dennatomyositis, muscular rheumatism, myositis, myogelosis andchondrocalcinosis.

80. The kit of claim 74, further comprising a container.

81. The kit of claim 74, wherein the soluble CTLA4 comprises the extracellular domain of a CTLA4 molecule, or portion thereof, which binds a B7 antigen expressed on activated B cells.

82. The kit of claim 74, wherein the soluble CTLA4 comprises the extracellular domain of a CTLA4 molecule as shown in Figure 23 (SEQ ID NO: 17) beginning with methionine at position +1 or with alanine at position -1 and ending with aspartic acid at position +124.

83. The kit of claim 74, wherein the soluble CTLA4 is a CTLA4 fusion molecule.

84. The kit of claim 83, wherein the CTLA4 fusion molecule comprises an extracellular domain of a CTLA4 molecule which binds a B7 antigen expressed on activated B cells joined to a non-CTLA4 molecule.

85. The kit of claim 84, wherein the non-CTLA4 molecule comprises an amino acid sequence which alters the solubility or affinity which comprises an immunoglobulin moiety.

86. The kit of claim 85, wherein the immunoglubulin moiety is an immunoglubulin constant region or portion thereof.

87. The kit of claim 86, wherein the immunoglubulin constant region or portion thereof is mutated to reduce effector function.

88. The kit of claim 83, wherein the CTLA4 fusion molecule is CTLA4Ig.

89. The kit of claim 88, wherein the CTLA4Ig is shown in Figure 24 (SEQ ID
NO:19) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine at position +357.

90. The kit of claim 74, wherein the soluble CTLA4 is a soluble CTLA4 mutant molecule.

91. The kit of claim 90, wherein the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), and b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with any other amino acid.

92. The kit of claim 90, wherein the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with glutamic acid, and c) wherein at position +29 of the extracellular domain of CTLA4, alanine is substituted with tyrosine.

93. The kit of claim 90, wherein the soluble CTLA4 mutant molecule is L104EA29YIg as shown in Figure 19 (SEQ ID NO: 9) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine at position +357 as shown in Figure 19 (SEQ ID NO: 9).

94. Use of a kit comprising the pharmaceutical composition of claim 68, a container and a label.

95. A kit comprising an effective amount of a first agent and a label, wherein a) the first agent is soluble CTLA4, and b) the label indicates that the first agent can be used with a second agent selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolinetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol.

96. The kit of claim 95, wherein the label further indicates an effective amount for the first agent being about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg.

97. The kit of claim 95, wherein the label indicates an effective amount for the second agent being about 0.1 to 40 mg per week, about 5 to 30 mg per week, about 0.1 to mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/ week, about 10 to 30 mg/week, about 10 to 100 mg/week, about 50 mg/week, about 0.1 to 50 mg/kg body weight per week, about 1 to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to 2000 mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day.

98. The kit of claim 95, wherein the label indicates an effective amount for the first and second agent, a. wherein the effective amount of the first agent being about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about 40 to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about 18 to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg, and b. wherein the effective amount of the second agent is about 0.1 to 40 mg per week, about 5 to 30 mg per week, about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/ week, about 10 to 30 mg/week, about 10 to 100 mg/week, about 50 mg/week, about 0.1 to 50 mg/kg body weight per week, about 1 to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/ day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to 2000 mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day.

99. The kit of claim 95, further comprising a label indicating that the first and second agents are useful to treat an immune system disease.

100. The kit of claim 99, wherein the immune system disease is selected from autoimmune diseases, psoriasis, immune disorders associated with graft transplantation rejection, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin dependent diabetes mellitis, good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic disease, rheumatoid arthritis, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-anticular rheumatism, collagen diseases, chronic polyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenile rheumatoid arthritis, periarthritis humeroscapularis, panarteriitis nodosa, systemic lupus erythematosus, progressive systemic scleroderma, arthritis uratica, dermatomyositis, muscular rheumatism, myositis, myogelosis andchondrocalcinosis.

101. A kit comprising an effective amount of a first agent and a label, wherein a) the first agent is soluble CTLA4, and b) the label indicates that the first agent can be used with a second agent selected from a group consisting of collagen, dnaJ, a molecule that blocks TNF receptors, pegsunercept, a molecule that blocks cytokine function, AMG719, a molecule that blocks LFA-1 function, efalizumab, acetyl salicylic acid, choline magnesium salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, meclofenamate, naproxen, nabumetone, phenylbutazone, piroxicam, sulindac, tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam, codeine phosphate, propoxyphene napsylate, oxycodone hydrochloride, oxycodone bitartrate and tramadol, wherein the label further indicates an effective amount for the first agent being about 0.1 to 100 mg/kg weight of the subject, about 0.5 to 5 mg/kg weight of a subject, about 5 to 10 mg/kg weight of a subject, about 10 to 15 mg/kg weight of a subject, about 15 to 20 mg/kg weight of a subject, about 20 to 25 mg/kg weight of a subject, about 25 to 30 mg/kg weight of a subject, about 30 to 35 mg/kg weight of a subject, about 35 to 40 mg/kg weight of a subject, about to 45 mg/kg weight of a subject, about 45 to 50 mg/kg weight of a subject, about 50 to 55 mg/kg weight of a subject, about 55 to 60 mg/kg weight of a subject, about 60 to 65 mg/kg weight of a subject, about 65 to 70 mg/kg weight of a subject, about 70 to 75 mg/kg weight of a subject, about 75 to 80 mg/kg weight of a subject, about 80 to 85 mg/kg weight of a subject, about 85 to 90 mg/kg weight of a subject, about 90 to 95 mg/kg weight of a subject, about 95 to 100 mg/kg weight of a subject, about 2 to 10 mg/kg weight of a subject, about 0.1 to 4 mg/kg weight of a subject, about 0.1 to 0.5 mg/kg weight of a subject, about 0.5 to 1.0 mg/kg weight of a subject, about 1.0 to 1.5 mg/kg weight of a subject, about 1.5 to 2.0 mg/kg weight of a subject, about 2.0 to 2.5 mg/kg weight of a subject, about 2.5 to 3.0 mg/kg weight of a subject, about 3.0 to 3.5 mg/kg weight of a subject, about 3.5 to 4.0 mg/kg weight of a subject, about 4.0 to 4.5 mg/kg weight of a subject, about 4.5 to 5.0 mg/kg weight of a subject, about 5.0 to 5.5 mg/kg weight of a subject, about 5.5 to 6.0 mg/kg weight of a subject, about 6.0 to 6.5 mg/kg weight of a subject, about 6.5 to 7.0 mg/kg weight of a subject, about 7.0 to 7.5 mg/kg weight of a subject, about 7.5 to 8.0 mg/kg weight of a subject, about 8.0 to 8.5 mg/kg weight of a subject, about 8.5 to 9.0 mg/kg weight of a subject, about 9.0 to 9.5 mg/kg weight of a subject, about 9.5 to 10.0 mg/kg weight of a subject, about 0.1 to 2 mg/kg weight of a subject, about 2 to 4 mg/kg weight of a subject, about 4 to 6 mg/kg weight of a subject, about 6 to 8 mg/kg weight of a subject, about 8 to 10 mg/kg weight of a subject, about 10 to 12 mg/kg weight of a subject, about 12 to 14 mg/kg weight of a subject, about 14 to 16 mg/kg weight of a subject, about 16 to 18 mg/kg weight of a subject, about to 20 mg/kg weight of a subject, about 500 mg for a subject weighing less than 60 kg, 750 mg for a subject weighing between 60-100 kg or 1000 mg for a subject weighing more than 100 kg, and wherein the label indicates an effective amount for the second agent being about 0.1 to 40 mg per week, about 5 to 30 mg per week, about 0.1 to 5 mg/week, about 5 to 10 mg/week, about 10 to 15 mg/week, about 15 to 20 mg/week, about 20 to 25 mg/week, about 25 to 30 mg/week, about 30 to 35 mg/week, about 35 to 40 mg/ week, about 10 to 30 mg/week, about 10 to 100 mg/week, about 50 mg/week, about 0.1 to 50 mg/kg body weight per week, about 1 to about 5000 mg/day, about 1 to 10 mg/day, about 10 to 50 mg/day, about 50 to 100 mg/day, about 100 to 150 mg/day, about 150 to 200 mg/day, about 200 to 250 mg/day, about 250 to 300 mg/day, about 300 to 350 mg/day, about 350 to 400 mg/day, about 400 to 450 mg/day, about 450 to 500 mg/day, about 500 to 550 mg/day, about 550 to 600 mg/day, about 600 to 650 mg/day, about 650 to 700 mg/day, about 700 to 750 mg/day, about 750 to 800 mg/day, about 800 to 850 mg/day, about 850 to 900 mg/day, about 900 to 950 mg/day, about 950 to 1000 mg/day, about 1000 to 1100 mg/day, about 1100 to 1200 mg/day, about 1200 to 1300 mg/day, about 1300 to 1400 mg/day, about 1400 to 1500 mg/day, about 1500 to 1600 mg/day, about 1600 to 1700 mg/day, about 1700 to 1800 mg/day, about 1800 to 1900 mg/day, about 1900 to 2000 mg/day, about 2000 to 2500 mg/day, about 2500 to 3000 mg/day, about 3000 to 3500 mg/day, about 3500 to 4000 mg/day, about 4000 to 4500 mg/day or about 4500 to 5000 mg/day.

102. The kit of claim 101, wherein the label further indicates that the first and second agents are useful to treat an immune system disease selected from autoimmune diseases, psoriasis, immune disorders associated with graft transplantation rejection, T cell lymphoma, T cell acute lymphoblastic leukemia, testicular angiocentric T cell lymphoma, benign lymphocytic angiitis, lupus erythematosus, Hashimoto's thyroiditis, primary myxedema, Graves' disease, pernicious anemia, autoimmune atrophic gastritis, Addison's disease, insulin dependent diabetes mellitis, good pasture's syndrome, myasthenia gravis, pemphigus, Crohn's disease, sympathetic ophthalmia, autoimmune uveitis, multiple sclerosis, autoimmune hemolytic anemia, idiopathic thrombocytopenia, primary biliary cirrhosis, chronic action hepatitis, ulceratis colitis, Sjogren's syndrome, rheumatic disease, rheumatoid arthritis, polymyositis, scleroderma, mixed connective tissue disease, inflammatory rheumatism, degenerative rheumatism, extra-articular rheumatism, collagen diseases, chronic polyarthritis, psoriasis arthropathica, ankylosing spondylitis, juvenile rheumatoid arthritis, periarthritis humeroscapularis, panarteriitis nodosa, systemic lupus erythematosus, progressive systemic scleroderma, arthritis uratica, dermatomyositis, muscular rheumatism, myositis, myogelosis and chondrocalcinosis.

103. The kit of claim 101, further comprising a container.

104. The kit of claim 101, wherein the soluble CTLA4 comprises the extracellular domain of a CTLA4 molecule, or portion thereof, which binds a B7 antigen expressed on activated B cells.

105. The kit of claim 101, wherein the soluble CTLA4 is a CTLA4 fusion molecule.

106. The kit of claim 105, wherein the CTLA4 fusion molecule comprises an extracellular domain of a CTLA4 molecule which binds a B7 antigen expressed on activated B cells joined to a non-CTLA4 molecule.

107. The kit of claim 106, wherein the non-CTLA4 molecule comprises an amino acid sequence which alters the solubility or affinity comprises an immunoglobulin moiety.

108. The kit of claim 107, wherein the immunoglubulin moiety is an immunoglubulin constant region or portion thereof.

109. The kit of claim 108, wherein the immunoglubulin constant region or portion thereof is mutated to reduce effector function.

110. The kit of claim 105, wherein the CTLA4 fusion molecule is CTLA4Ig.

111. The kit of claim 110, wherein the CTLA4Ig is shown in Figure 24 (SEQ ID
N0:19) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine at position +357.

112. The kit of claim 101, wherein the soluble CTLA4 is a soluble CTLA4 mutant molecule.

113. The kit of claim 112, wherein the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), and b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with any other amino acid.

114. The kit of claim 112, wherein the soluble CTLA4 mutant molecule comprises an extracellular domain of CTLA4, a) wherein the extracellular domain of CTLA4 begins with methionine at position +1 or with alanine at position -1 and ends with aspartic acid at position +124 as shown in Figure 23 or 24 (SEQ ID NO: 17 or 19), b) wherein at position +104 of the extracellular domain of CTLA4, leucine is substituted with glutamic acid, and c) wherein at position +29 of the extracellular domain of CTLA4, alanine is substituted with tyrosine.

115. The kit of claim 101, wherein the soluble CTLA4 mutant molecule is L104EA29YIg as shown in Figure 19 (SEQ ID NO: 9) beginning with methionine at position +1 or with alanine at position -1 and ending with lysine at position +357 as shown in Figure 19 (SEQ ID NO: 9).