Method of preventing the development of rheumatoid arthritis in subjects with undifferentiated arthritis

BACKGROUND OF THE INVENTION

Rheumatoid arthritis (RA) is the most common form of arthritis that affects approximately 1% of people worldwide (Wolfe, F., "The epidemiology of drug treatment failure in rheumatoid arthritis", Baillieres Clin. Rheumatol., 9 (4) : 619-632 (November 1995)) The probability of developing RA is 2-3 times higher in women than in men, with the disease most often occurring at the age of 40-60 (Hochberg, MC et al., "Epidemiology of rheumatoid arthritis : update ", Epidemiol. Rev., 12: 247-252 (1990); Markenson, JA, "Worldwide trends in the socioeconomic impact and long-term prognosis of rheumatoid arthritis", Semin. Arthritis Rheum., 21 (2 Suppl. 1): 4-12 (October 1991); Spector, TD, "Rheumatoid arthritis ", Rheum. Dis. Clin. North Am., 16 (3): 513-537 (August 1990); and Zvaifler, NJ, "Etiology and pathogenesis of rheumatoid arthritis", in Arthritis and Allied Conditions, McCarty, DJ, Koopman, WJ, edited by Philadelphia: Lea & Febiger, 723-736 (1993)). Although RA in clinical practice is diagnosed on the basis of severe inflammation involving synovial joints, it is also a systemic disease often accompanied by extra-articular symptoms. In the natural course of RA, joint damage, physical impairment and a deterioration in the quality of life depending on the state of health occur.

More and more scientific evidence indicates that the joint structure is destroyed in the early stages of RA. In more than 90% of people, traditional radiological tests show features of joint damage within two years after the diagnosis of RA (Emery, P., "The Optimal Management of Early Rheumatoid Disease: The Key to Preventing Disability", Br. J. Rheum., 33: 765-768 (1994)). If more sensitive techniques such as MRI are used, joint damage can be detected within a few weeks of the onset of symptoms (McGonagle, D. et al., "The relationship between synovitis and bone changes in early untreated rheumatoid arthritis", Arthritis Rheum. , 42: 1706-1711 (1999) and Wakefield, RJ et al., "The value of sonography in the detection of bone erosion in patients with rheumatoid arthritis: A comparative study with conventional radiography", Arthritis Rheum., 43: 2761-2770 (2000)). These research results contributed to growth the need for treatment methods that could effectively suppress inflammatory processes that destroy bone and cartilage in the early stages of RA, and have also emphasized the growing importance of early diagnosis and treatment of RA.

However, RA diagnostic criteria developed by the American Rheumatism Association (ARA) in 1987 are less sensitive and specific when used in people with early-stage arthritis compared to those with more advanced disease. People with early arthritis tend to have fewer joints (polyarthritis) rather than many joints (polyarthritis), arthritis symptoms may persist for less than six weeks, and initially the rheumatoid factor may be undetectable in the blood and the nodules rheumatoid or erosions (bone erosions) on standard x-rays (Van Gaalen, FA et al., "Autoantibodies to Cyclic Citrullinated Peptides Predict Progression to Rheumatoid Arthritis in Patients with Undifferentiated Arthritis", Arthritis Rheum., 50 (3): 709-715 (2004). As a result, these people often at an early stage of disease meet the diagnostic criteria for RA (or any other rheumatic disease) from 1897. Thus, the method of elimination diagnoses undifferentiated arthritis. About 40% of people referred to highly specialized centers of the third reference level selected for the diagnosis and treatment of early arthritis do not meet the diagnostic criteria for RA or any other rheumatic disease (Van Gaalen, FA et al., "Autoantibodies to Cyclic Citrullinated Peptides Predict Progression to Rheumatoid Arthritis in Patients with Undifferentiated Arthritis ", Arthritis Rheum., 50 (3): 709-715 (2004)). Therefore, undifferentiated arthritis is often a recognized clinical disease entity.

The correct synovium is the tissue surrounding and separating the joint spaces. A layer of lining cells composed of macrophage-like and fibroblast-like synoviocytes covers a thin stroma of connective tissue with a small number of dendritic cells, fibroblasts, mast cells and vascular structures (Konttinen, YT et al., "Characterization of the immunocompetent cells of rheumatoid synovium from tissue sections and eluates", Arthritis Rheum., 24 (1): 71-79 (January 1981)).

In RA, the synovium is significantly thickened and swollen. As the disease progresses, proliferation and influx of synoviocytes as well as inflammatory cells in the synovium gradually increase (Konttinen, YT et al., "Characterization of the immunocompetent cells of rheumatoid synovium from tissue sections and eluates", Arthritis Rheum., 24 ( 1): 71-79 (January 1981)). Up to 50% of synovial infiltration leukocytes are T lymphocytes, mainly CD4 + with memory / activated cell phenotype (Konttinen, YT et al., "Characterization of the immunocompetent cells of rheumatoid synovium from tissue sections and eluates", Arthritis Rheum., 24 (1): 71-79 (January 1981); Forre, O. et al., "Augmented numbers of HLADR-positive T lymphocytes in the synovial fluid and synovial tissue of subjects with rheumatoid arthritis and juvenile rheumatoid arthritis: in vivo-activated T lymphocytes are potent stimulators in the mixed lymphocyte reaction", Scand. J. Immunol., 15 (2): 227-231 (February 1982); Van-Boxel, JA et al., "Predominantly T-cell infiltrate in rheumatoid synovial membranes", N. Engl. J. Med., 293 (11): 517-520 (September 1975); Kidd, BL et al., "Immunohistological features of synovitis in ankylosing spondylitis: a comparison with rheumatoid arthritis", Ann. Rheum. Dis., 48 (2): 92-98 (February 1989); Cush, JJ et al., "Phenotypic analysis of synovial tissue and peripheral blood lymphocytes isolated from subjects with rheumatoid arthritis ", Arthritis Rheum., 31 (10): 230-238 (October 1988); Laffon, A. et al., "Upregulated expression and function of VLA-4 fibronectin receptors on human activated T cells in rheumatoid arthritis", J. Clin. Invest., 88 (2): 546-552 (August 1991); and Klareskog, L. et al., "Relationship between HLA DR expressing cells and T lymphocytes of different subsets in rheumatoid synovial tissue", Scand. J. Immunol., 15 (5): 501-507 (May 1981)). derived monocytes / macrophages also begin to predominate in the rheumatically changed synovium, accounting for up to 20% of the cells. They also have the phenotype of activated cells (Firestein, GS et al., "How important are T cells in chronic rheumatoid synovitis?" Arthritis Rheum., 33 (6): 768-773 (June 1990) and Firestein, GS et al., "Quantitative analysis of cytokine gene expression in rheumatoid" , J. Immunol., 144 (9): 3347-3353 (May 1, 1990)). Monocyte-like and macrophage-like cells in the rheumatically altered synovium produce a number of pro-inflammatory molecules, including IL-1, TNF-α, IL-6, GM-CSF, as well as proteolytic enzymes, including collagenase and matrix metalloproteinase. B lymphocytes, plasma cells and neutrophils account for less than 5% of cells in the rheumatically altered synovium, although neutrophils predominate in the synovial fluid (Konttinen, YT et al., "Characterization of the immunocompetent cells of rheumatoid synovium from tissue sections and eluates" , Arthritis Rheum., 24 (1): 71-79 (January 1981); Forre, O. et al., "Augmented numbers of HLA-DR-positive T lymphocytes in the synovial fluid and synovial tissue of subjects with rheumatoid arthritis and juvenile rheumatoid arthritis: in vivo-activated T lymphocytes are potent stimulators in the mixed lymphocyte reaction", Scand. J. Immunol., 15 (2): 227-231 (February 1982); and Firestein, GS et al., "Quantitative analysis of cytokine gene expression in rheumatoid", J. Immunol., 144 (9): 3347-3353 (May 1, 1990)).

As the synovial cell proliferation increases and the inflammatory process progresses, the vascularized, inflammatory synovial tissue known as psoriasis develops. Psoriasis is responsible for infiltration of articular cartilage and destruction of bone tissue. Activated T lymphocyte products are thought to stimulate the formation and growth of psoriasis (Zvaifler, NJ et al., "Alternative models of joint destruction in rheumatoid arthritis", Arthritis Rheum., 37 (6): 783-789 (June 1994)) .

Monocytic / macrophage-like cells and dendritic cells of a rheumatically altered synovium express both class II histocompatibility complex (MHC) molecules and co-stimulatory molecules such as CD80 (B7-1) / CD86 (B72). These cells presumably act as antigen presenting cells (Balsa, A. et al., "Differential expression of the costimulatory molecules B7.1 (CD80) and B7.2 (CD86) in rheumatoid synovial tissue", Br. J. Rheumatol., 35 (1): 33-37 (January 1996); Liu, MF et al., "The presence of costimulatory molecules CD86 and CD28 in rheumatoid arthritis synovium", Arthritis Rheum., 39 (1): 110-114 (January 1996); Ranheim, EA et al., "Elevated expression of CD80 (B7 / BB1) and other accessory molecules on synovial fluid mononuclear cell subsets in rheumatoid arthritis ", Arthritis Rheum., 37 (11): 1637-1646 (November 1994); Sfikakis, PP et al., "Expression of CD28, CTLA4, CD80, and CD86 molecules in subjects with autoimmune rheumatic diseases: implications for immunotherapy", Clin. Immunol. Immunopathol., 83 (3): 195-198 (June 1997); and Thomas , R. et al., "Functional differentiation of dendritic cells in rheumatoid arthritis: role of CD86 in the synovium", J. Immunol., 156 (8): 3074-3086 (April 15, 1996)). Activated CD4 + T cells expressing CD28 are the predominant type of inflammatory infiltrate cells present in the rheumatically affected synovium. They often occur near cells expressing MHC class II molecules and co-stimulatory molecules. This demonstrates the important role of T lymphocyte activation / co-stimulation in the pathogenesis of synovitis. This corresponds to the observations from experimental studies that show that activated T lymphocytes through direct contact with synoviocytes and osteoclasts or due to the processing properties of secreted cytokines are important factors stimulating the development of synovitis and the process of bone destruction in RA. All these observations indicate the important role of activated T lymphocytes and co-stimulatory signals sent by CD28 in RA immunopathology.

The approach to RA treatment has changed and currently Disease Modifying Anti-Rheumatic Drug (DMARD) is used earlier after diagnosis and then pharmacotherapy is optimized to obtain a greater beneficial effect on the course of the disease (Redlich, K et al., "Rheumatoid Arthritis Therapy After Tumor Necrosis Factor and Interleukin-1 Blockade", Arthritis Rheum., 40 (12): 3308-3319 (2003)). However, there are no standard clinical principles for the treatment of patients with undifferentiated arthritis (Harrison, BJ et al., "Natural remission in inflammatory polyarthritis: issues of definition and prediction," Br. J. Rheumatol., 35: 1096-1100 ( 1996)). This is largely due to the inability to accurately determine the prognosis and risk of developing RA in these patients based on frequently used clinical features present at the time of going to the doctor. Undifferentiated arthritis may resolve on its own, develop into a differentiated rheumatic disease such as RA, or remain undifferentiated (Quinn, MA et al., "Evaluation and management of early inflammatory polyarthritis", Rheumatology, Third Edition, MOSBY (Elsevier Limited), 885-891 (2003)). People with undifferentiated arthritis usually do not initially use DMARD because of uncertainty Prognosis: Traditionally, non-steroidal anti-inflammatory drugs (NSAIDs) are administered instead, followed by oral corticosteroids. DMARD is usually used when the signs and symptoms of arthritis prove to be resistant to the drugs belonging to the first two treatment groups. For this reason, the sequential treatment algorithm ("pyramid method") has traditionally been used (Harrison, BJ et al., "Natural remission in inflammatory polyarthritis: issues of definition and prediction", Br. J. Rheumatol., 35: 1096-1100 (1996 )). Unfortunately, this method may not prevent damage to the joint structure before starting potentially modification therapy.

No authorized anti-rheumatic drugs have been shown to significantly change the course of undifferentiated arthritis and prevent the development of RA. The efficacy of infliximab, a drug that targets TNF alpha, has been evaluated in a clinical trial for the prevention / remission of early RA after a short course of TNF antagonist treatment. However, published data showed that 6-month TNF antagonist treatment did not prevent the transformation of undifferentiated arthritis into RA (Saleem, B. et al., Rheumatology, Academic Unit of Musculoskeletal Disease, Leeds, United Kingdom, Ann. Rheum. Dis., 66 (Suppl. II): 186 (2007)).

There is clearly a need for drugs that, through the selective activation of T lymphocytes, prevent the development of RA and allow a fundamental change in the course of the disease in people with undifferentiated arthritis who have a high risk of RA.

US 2005/196402 and US 2004/0022787 describe the use of CTLA4 molecules in the treatment of arthritis (including rheumatoid arthritis, juvenile rheumatoid arthritis, osteoarthritis, psoriatic arthritis).

SUMMARY OF THE INVENTION

The present invention relates to CTLA4 molecules intended for use in the treatment of undifferentiated arthritis characterized in that CTLA4 molecules bind to CD80 and / or CD86 to form the extracellular domain of the CTLA4 molecule as shown in the nucleotide sequence of SEQ ID NO: 2 starting at alanine at position 26 or methionine at position 27 and ending with aspartic acid at position 150.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the nucleotide sequence (SEQ ID NO: 1) of the portion of the expression cassette for the CTLA4-Ig molecule. The amino acid sequence (SEQ ID NO: 2) encoded by the nucleic acid is also provided. CTLA4-Ig molecules that can be produced from this expression cassette include molecules having the amino acid sequence having residues: (i) 26-383 of SEQ ID NO: 2, (ii) 263

382 with SEQ ID NO: 2, (iii) 27-383 with SEQ ID NO: 2 or (iv) 26-382 with SEQ ID NO: 2, or optionally (v) 25-382 with SEQ ID NO: 2 or (vi ) 25-383 of SEQ ID NO: 2. The expression cassette contains the following regions: (a) oncostatin M signal sequence (nucleotides 11-88 of SEQ ID NO: 1; amino acids 126 of SEQ ID NO: 2); (b) the extracellular domain of the human CTLA4 molecule (nucleotides 89-463 of SEQ ID NO: 1; amino acids 27-151 of SEQ ID NO: 2); (c) a modified portion of the human IgG1 constant region (nucleotides 464-1159 of SEQ ID NO: 1; amino acids 152-383 of SEQ ID NO: 2), including the modified hinge region (nucleotides 464-508 of SEQ ID NO: 1; amino acids 152-166 of SEQ ID NO: 2), the modified CH2 domain of human IgG1 (nucleotides 509-838 of SEQ ID NO: 1; amino acids 167-276 of SEQ ID NO: 2) and the CH3 domain of human IgG1 (nucleotides 839-1159) from SEQ ID NO: 1; amino acids 277-383 of SEQ ID NO: 2).

FIG. 2 shows the time to discontinuation of treatment due to the development of RA as assessed in the clinical trial described in Example III.

DETAILED DESCRIPTION OF THE INVENTION

As used here:

The terms "CTLA4-Ig" or "CTLA4-Ig molecule" or "CTLA4Ig molecule" are used interchangeably and refer to a protein molecule forming at least a polypeptide having the extracellular domain of CTLA4 or a portion thereof and an immunoglobulin constant region or portion thereof. Extracellular domain and constant region immunoglobulins may be wild-type (natural), mutated or modified, and mammalian, including human or murine, and may contain additional protein domains. The CTLA4-Ig molecule may also refer to polypeptides in multimeric form, such as dimers, tetramers and hexamers. The CTLA4-Ig molecule also has the ability to bind to CD80 and / or CD86.

The term "B7-1" refers to CD80; the term "B7-2" refers to CD86; and the term "B7" refers to both B7-1 and B7-2 (CD80 and CD86). The term "B7-1-Ig" or "B7-1Ig" refers to CD80-Ig; the term "B7-2 -Ig "or" B7-2Ig "refers to CD86-Ig.

In one embodiment, "CTLA4Ig" refers to a protein molecule having the amino acid sequence having residues: (i) 26-383 of SEQ ID NO: 2, (ii) 26-382 of SEQ ID NO: 2; (iii) 27383 of SEQ ID NO: 2, or (iv) 27-382 of SEQ ID NO: 2, or optionally (v) 25-382 of SEQ ID NO: 2 or (vi) 25-383 of SEQ ID NO: 2. In monomeric form these the proteins may be referred to as "monomers having the sequence of SEQ ID NO: 2" or monomers "having the sequence of SEQ ID NO: 2". These monomers with the sequence SEQ ID NO: 2 can dimerize such that dimer combinations can include, for example: (i) and (i); (i) and (ii); (i) and (iii); (i) and (iv); (i) and (v); (i) and (vi); (ii) and (ii); (ii) and (iii); (ii) and (iv); (ii) and (v); (ii) and (vi); (iii) and (iii); (iii) and (iv); (iii) and (v); (iii) and (vi); (iv) and (iv); (iv) and (v); (iv) and (vi); (v) and (v); (v) and (vi); and (vi) and (vi). These different combinations of dimers can also combine to form a CTLA4Ig tetramer. In monomeric form, these proteins may be referred to as "monomers having the sequence of SEQ ID NO: 2" or monomers "having the sequence of SEQ ID NO: 2". (DNA encoding CTLA4Ig, as shown in SEQ ID NO: 2, was deposited May 31, 1991. in the American Cell Culture Collection (ATCC®), 10801 University Blvd., Manassas, VA 20110-2209, in accordance with the provisions of the Budapest Treaty, and has been given ATCC® accession number 68629; a Chinese hamster ovary (CHO) cell line expressing CTLA4Ig as depicted in SEQ ID NO: 2, deposited May 31, 1991 with the identification number ATCC® CRL-10762).

"Drug substance" refers to the starting material used in the formulation of the final medicinal product. The standard composition of the drug substance CTLA4Ig is a protein with a concentration of 20 mg / ml to 60 mg / ml, a pH of 6 to 8 and a form with a higher molecular weight ( HMW) in an amount of <5%.

The term "mass of the solution to be formulated" refers to the final formulation prior to filling the container, such as the solution being formulated before filling the lyophilization vials or the solution being formulated before filling the hypodermic syringe.

The term "medicinal product" refers to a final formulation packaged in a container that can be reconstituted prior to use, as is the case with a medicinal product in lyophilized form; further dilute before use, as in the case of a liquid medicinal product; or used unchanged, as for the medicinal product as a solution for subcutaneous injection.

"Health Questionnaire Assessments (HAQs)" refers to a set of questions used to assess symptoms of disease activity in patients.

These symptoms include: joint swelling, joint tenderness, inflammation, morning stiffness, disease activity and degree of disability assessed individually by each patient in the questionnaire regarding their physical well-being and functioning, as well as disease activity and degree of disability assessed by the doctor and pain (Fries , JF et al., J. Rheumatology, 9: 789-793 (1982)).

The "MOS SF-36 Questionnaire (Medical Outcomes Study Short Form-36") refers to the forms used to assess the impact of treatment on health-related quality of life (HRQOL). The SF-36 questionnaire consists of 36 issues concerning four somatic categories and four mental categories (physical fitness, physical roles, physical pain, general health, life energy, social functioning, emotional roles and mental health). Individual categories are used to assess the physical and mental components in the range of 0 to 100, with a higher score indicating a better quality of life.

"American Society of Rheumatism (ARA) Rheumatoid Arthritis Classification Criteria (Revised Version 1987)" refers to the set of criteria described in Table 1 below. For the purposes of classification, a person is considered to be RA patient if he or she meets at least four out of seven criteria Criteria 1-4 must persist for at least six weeks.

TABLE 1

<td colspan="2">Classification criteria for RZS ARA as amended from 1987.</td>

<td>Criterion</td><td>Definition</td>

<td>1. Morning stiffness</td><td>The morning stiffness of the joints and the joint area lasts for at least 1 hour before maximum improvement occurs</td>

<td>2. Arthritis in at least three areas</td><td>Swelling of soft tissues or the presence of fluid (and not only bone tissue hypertrophy) found by the doctor in joints located in at least three areas (14 possible areas are: proximal interphalangeal joint, metacarpal joint, wrist joints, elbow, knee, ankle and metatarsophalangeal joint [on the right or left])</td>

<td>3. Arthritis of the hands</td><td>Swelling (as above) of at least one carpal joint, metatarsal or proximal inter-phalangeal joint</td>

<td>4. Symmetrical arthritis</td><td>Simultaneous joint involvement in the same area (as in criterion 2) on both sides (bilateral proximal inflammation of the interphalangeal joints, metacarpal or metatarsophalangeal joints without complete symmetry)</td>

<td>5. Rheumatoid nodules</td><td>Doctor's presence of subcutaneous nodules above the bony prominences or above the extensor surface, or in the periarticular area</td>

<td>6. Rheumatoid factor in blood serum</td><td>Demonstration by any analytical method of an abnormal amount of rheumatoid factor in blood serum that occurs in less than 5% of healthy controls</td>

<td>7. Changes in radiological tests</td><td>Presence of RA-specific changes on the radiographs of the hand and wrist in the PA projection, which must take into account the erosion or undeniable decalcification of the bones in the affected joints or directly in their vicinity (osteoarthritis of bone and joints are not included)</td>

<td colspan="2">Abbreviations: ARA (American Rheumatism Association) = American Rheumatological Society; PIP (proximal interphalangeal) = proximal interphalangeal joint; MCP (metacarpophalangeal) = metacarpal joint; mTp (metatarsophalangeal) = metatarsophalangeal joint; RA (rheumatoid arthritis) = rheumatoid arthritis, PA (posteroanterior *) = posterolateral.</td>

The term "ACR" refers to clinical response studies based on criteria established by the American College of Rheumatology. The ACR Core Data Set and definitions of response to treatment are described in Table 2 below. A person meets the "ACR20" criterion if there is a 20% improvement in the score of the number of joints with tenderness and swelling and a 20% improvement in the score of the three out of the five remaining parameters assessed, such as the overall assessment of the patient and the doctor, pain, disability physical, as well as acute phase indicators (e.g. C-reactive protein, CRP) or Biernacki's (erythrocyte sedimentation rate (ESR)) (Felson, DT et al., Arthritis Rheum., 36: 729-740 (1993); Felson, DT et al., Arthritis Rheum., 38: 1-9 (1995)). A person meets the "ACR50" or "ACR70" criterion if a 50% or 70% improvement in the score of the number of joints with tenderness and edema, respectively, and a 50% or 70% improvement in the result of the assessment of three out of the five remaining parameters, such as general assessment of the lesions by the patient and the doctor, pain, physical disability, as well as acute phase indicators such as CRP or ESR.

TABLE 2

<td colspan="2">Basic ACR data set and treatment response definitions</td>

<td>Element of the ACR Basic Data Set</td><td>Validated measurement tool</td>

<td>1. Number of joints with tenderness</td><td>68 normalized joints</td>

<td>2. Number of joints with edema</td><td>66 normalized joints</td>

<td>3. Overall assessment of the patient's pain level</td><td>Visual analog scale (VAS) 0-100 mm</td>

<td>4. Overall assessment of the patient's disease activity</td><td>Visual analog scale (VAS) 0-100 mm</td>

<td>5. Overall assessment of disease activity by a physician</td><td>Visual analog scale (VAS) 0-100 mm</td>

<td>6. General assessment of the patient's physical fitness</td><td>Health Assessment Questionnaire (HAQ)</td>

<td>7. Acute phase indicators</td><td>OB (Westergren technique) and CRP</td>

Undifferentiated arthritis is diagnosed when a person has symptomatic clinical synovitis in at least two joints and at least one, but not more than three, of the RA classification criteria established by the American Rheumatism Association (1987) listed in Table 1.

The terms "treatment" or "therapy" of undifferentiated arthritis as used herein mean drug treatment or other methods of treating the disease. Treatment of undifferentiated arthritis can weaken disease-related immune-related events, alleviate the symptoms of the disease or disorder, reduce the severity of the disease, change the course of the disease and / or cure or reduce the disease. For example, treatment of undifferentiated arthritis may involve controlling the immune response, e.g., by regulating the functional interaction of CTLA4- and / or CD28-positive cells with B7-positive cells. Optionally, treatment of undifferentiated arthritis may be to prevent the disease from occurring or inhibit its progression and the transformation of RA by using the composite products described herein. For example, treatment of undifferentiated arthritis includes inhibition of joint tissue erosion as assessed by MRI.

Serum CTLA4Ig concentration can be determined by ELISA (enzyme-linked immunosorbent assay).

CTLA4-Ig monomers and multimers

CTLA4-Ig molecules can include, e.g., CTLA4-Ig proteins in monomeric, dimeric, tetrameric, pentameric, hexameric or other multimeric form. CTLA4-Ig molecules can form a fusion protein having at least the CTLA4 extracellular domain and an immunoglobulin constant region. CTLA4-Ig molecules can have wild-type or mutated sequences, such as, for example, CTLA4 extracellular domain and immunoglobulin constant region sequences. CTLA4-Ig protein in the form of a monomer, dimer, tertamer or other multimer can be glycosylated.

In some embodiments, the invention provides populations of CTLA4-Ig molecules that have at least a certain percentage of dimeric or other multimeric molecules. For example, the invention provides CTLA4-Ig molecule populations that are greater than 90%, 95%, 96%, 97%, 98%, 99% or 99.5% CTLA4-Ig dimers. In one embodiment, the invention provides a population of CTLA4-Ig molecules that contains from about 95% to about 99.5% CTLA4-Ig dimer and from about 0.5% to about 5% CTLA4-Ig tetramer. In another embodiment, the CTLA4-Ig molecule population comprises about 98% CTLA4-Ig dimer, about 1.5% CTLA4-Ig tetramer, and about 0.5% CTLA4-Ig monomer.

In one embodiment, the invention provides a population of CTLA4-Ig molecules, wherein the population is substantially free of CTLA4-Ig monomer molecules. CTLA4-Ig molecules substantially free of monomers can be called a population of CTLA4-Ig molecules that have less than 1%, 0.5% or 0.1% monomers.

In one embodiment, the invention provides a population of CTLA4-Ig molecules, wherein the population essentially does not contain multimers larger than dimers, such as tetramers, hexamers, etc. CTLA4-Ig molecules essentially free of multimers larger than dimers can be called a population of CTLA4-Ig molecules, which have less than 6%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% CTLA4-Ig multimers larger than dimers.

In one embodiment, the CTLA4Ig molecule in monomeric form may, for example, have the amino acid sequence: (i) 26-383 of SEQ ID NO: 2, (ii) 26-382 of SeQ ID NO: 2; (iii) 27-383 with SEQ ID NO: 2, or (iv) 27-382 with SEQ ID NO: 2, or optionally (v) 25-382 with SEQ ID NO: 2 or (vi) 25-383 with SEQ ID NO: 2. When the expression cassette containing the SEQ ID NO: 1 nucleic acid sequence is expressed in CHO cells, the dominant form of the monomer has a N-terminal methionine amino acid residue (SEQ ID NO: 2 residue 27), which corresponds to the N-terminal amino acid residue of a wild type CTLA4 human molecule. However, because SEQ ID NO: 1 also includes the coding sequence for the oncostatin M signal sequence (nucleotides 11-88 SEQ ID NO: 1), the expressed protein of SEQ ID NO: 1 contains the oncostatin M signal sequence. For example, the Met-Ala variant may be present in a mixture of CTLA4-Ig molecules in about 1%, and the Ala variant may be present in a mixture of CTLA4-Ig molecules in about 8-10%. In addition, due to incomplete processing, the expressed protein of SEQ ID NO: 1 may have C-terminal variants. The dominant C-terminus is glycine at residue 382 SEQ ID NO: 2. Monomers having lysine at the C-terminus (residue 383 SEQ ID NO: 2) may be present in the mixture of CTLA4-Ig molecules, for example in an amount of about 4-5%.

The CTLA4-Ig monomer molecule may contain the extracellular domain of the human CTLA4 molecule. In one embodiment, the extracellular domain may comprise the nucleotide sequence of nucleotides 89-463 of SEQ ID NO: 1, which encodes amino acids 27-151 of SEQ ID NO: 2. In another embodiment, the extracellular domain may contain mutated sequences of the human CTLA4 molecule. In another embodiment, the extracellular domain may contain nucleotide changes in nucleotides 89-463 of SEQ ID NO: 1 of this type that cause conservative amino acid changes. In another embodiment, the extracellular domain may comprise a nucleotide sequence that is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleotide sequence 89-463 SEQ ID NO: 1.

The CTLA4-Ig monomer molecule may contain a human immunoglobulin constant region. This constant region may be part of a constant region; this constant region may have wild type or mutated sequence. The constant region may be from human IgG1, IgG2, IgG3, IgG4, IgM, IgA1, IgA2, IgD or IgE. The constant region may be from an immunoglobulin light chain or heavy chain. In case the constant region is from an IgG, IgD or IgA molecule, the constant region may contain one or more of the following constant region domains: CL, CH1, hinge, CH2 or CH3. In case the constant region is from an IgM or IgE molecule, the constant region may contain one or more of the following constant region domains: CL, CH1, CH2, CH3 or Ca4. In one embodiment, the constant region may comprise one or more IgG, IgD, IgA, IgM or IgE constant region domains.

In one embodiment, the monomer of the CTLA4-Ig molecule comprises a modified human IgG1 hinge region (nucleotides 464-508 SEQ ID NO: 1; amino acids 152-166 SEQ ID NO: 2), wherein the serine at amino acid residues 156, 162 and 165 of SEQ ID NO: 2 was constructed from cysteines present in the wild type sequence.

In one embodiment, the monomer of the CTLA4-Ig molecule comprises a modified human IgG1 CH2 region and a wild type CH3 region (modified human IgG1 CH2 domain having nucleotides 509-838 of SEQ ID NO: 1 and amino acids 167-276 of SEQ ID NO: 2; domain CH3 human IgG1 having nucleotides 839-1159 of SEQ ID NO: 1 and amino acids 277-383 of SEQ ID NO: 2).

In one embodiment, the population of CTLA4-Ig molecules comprises monomers having the sequence shown in one or more of Figures 7, 8 or 9 of US Patent No. 7,094,874, issued August 22, 2006, and in US Patent Application Publication Nos. 2003/0083246 and 2004/0022787 .

In one embodiment, the CTLA4-Ig tetramer molecule comprises two pairs or two dimers of CTLA4-Ig polypeptides, wherein each polypeptide has one of the following amino acid sequences: (i) 26-383 of SEQ ID NO: 2, (ii) 26-382 with SEQ ID NO: 2; (iii) 27-383 with SEQ ID NO: 2 or (iv) 27-382 with SEQ ID NO: 2, or optionally (v) 25-382 with SEQ ID NO: 2 or (vi) 25-383 with SEQ ID NO: 2. Each member of the polypeptide or dimer pair covalently binds to another member, and the two pairs of polypeptides are linked together by a non-covalent bond, forming a tetramer. Such tetramer molecules are capable of binding to CD80 or CD86.

In another embodiment, such tetramer molecules can bind to CD80 and CD86 with an avidity that is at least 2-fold greater than the binding avidity of CTLA4-Ig dimer (whose monomers have one of the above amino acid sequences) to CD80 or CD86. In another embodiment, such tetramer molecules can bind to CD80 and CD86 with an avidity that is at least 2-fold greater than the binding affinity or avidity of the wild type CTLA4-Ig dimer to CD80 or CD86. Such greater avidity may contribute to higher efficacy in the treatment of immune diseases and other diseases described below. In addition, greater or improved avidity can result in greater potency. For example, a therapeutic composition containing CTLA4-Ig tetramer would have higher avidity and therefore greater potency than a therapeutic composition containing the same amount of CTLA4-Ig monomer. In another embodiment, such tetramer molecules may have at least a 2-fold greater inhibition of T cell proliferation compared to CTLA4-Ig dimer (whose monomers have one of the above amino acid sequences). In another embodiment, such tetramer molecules may have at least a 2-fold higher inhibition of T cell proliferation compared to a wild type CTLA4-Ig molecule.

T lymphocyte proliferation can be measured using standard assays known in the art. For example, one of the most common ways to assess T cell proliferation is to stimulate T cells by antigen or TCR agonist antibodies and to measure, for example, incorporation of tritiated thymidine (3H-TdR) into proliferating T cells or the amount of cytokines released into the culture by proliferating T cells . Therefore, the inhibitory effect of CTLA4-Ig molecules can be measured after T cell activation or proliferation.

The affinity of the CTLA4-Ig molecule is the strength of the molecule's binding to a single ligand, including CD80, CD86 or CD8OIg or CD86Ig fusion proteins. The affinity of CTLA4-Ig for ligands can be measured by using, for example, binding interaction analysis (BIA) based on surface plasmon resonance technique. In addition to measuring binding strength, it allows the determination of binding kinetic parameters in real time, such as coupling and dissociation rate constants. The sensor chip, consisting of a slide covered with a thin metal coating, to which a surface matrix covalently attaches, is coated with one of the interacting compounds, i.e. CTLA4-Ig or one of the ligands. A solution containing another interacting compound may flow over its surface. A continuous beam of light is directed to the other side of the surface and the angle of its reflection is measured. With CTLA4-Ig binding to the ligand, the resonance angle of the light beam changes (because it depends on the refractive index of the medium near the reactive side of the sensor, which in turn correlates directly with the concentration of dissolved material in the medium). A continuous beam of light is directed to the other side of the surface and the angle of its reflection is measured. With CTLA4-Ig binding to the ligand, the resonance angle of the light beam changes (because it depends on the refractive index of the medium near the reactive side of the sensor, which in turn correlates directly with the concentration of dissolved material in the medium). This is then analyzed using a computer.

In one embodiment, CTLA4-Ig binding experiments can be performed by surface plasmon resonance (SPR) using a BIAcore apparatus (BIAcore AG, Uppsala, Sweden). The CTLA4-Ig molecule can be covalently coupled via primary amine groups to a carboxymethylated dextran matrix on a BIAcore sensor chip, thereby immobilizing CTLA4-Ig on the sensor chip. Optionally, the anti-constant region of the antibody can be used to indirectly immobilize CTLA4-Ig on the sensor surface through an Ig fragment. Ligands are then added to the chip to measure CTLA4-Ig binding to ligands. Affinity measurements can be carried out, for example, as described in van der Merwe, P. et al., J. Exp. Med. (1997) 185 (3): 393-404.

The avidity of CTLA4-Ig molecules can also be measured. Avidity can be defined as the total sum of the binding strength of two molecules or cells to each other in many places. Avidity is different from affinity, which is the strength of the molecule's binding to its ligand in one place. Without limiting the theory, higher avidity of CTLA4-Ig molecules can lead to increased inhibition of proliferation and activation of T cells by CTLA4-Ig molecules. Avidity can be measured, for example, by two types of solid phase tests: a) competitive inhibition tests and b) elution tests. In both cases, the ligand is attached to a solid support. In the competitive inhibition assay, CTLA4-Ig molecules are then added to the solution at a constant concentration together with the free ligand at various concentrations, and the amount of ligand that inhibits binding of the solid phase by 50% is determined. The less a ligand is needed, the stronger the avidity. In the elution tests, the ligand is added in solution. After equilibration, a chaotropic or denaturing agent (e.g., isothiocyanate, urea or diethylamine) is added at various concentrations to disrupt CTLA4-Ig / ligand interactions. The amount of elution-resistant CTLA4-Ig is then determined by ELISA. The higher the avidity, the more chaotropic agent is needed to elute some CTLA4-Ig. The relative avidity of a heterogeneous mixture of CTLA4-Ig molecules can be expressed as an avidity ratio (AI) equal to the concentration of the eluting agent needed to elute 50% of the bound CTLA4-Ig molecules. Improved data analysis can be performed by determining the percentage of CTLA4-Ig eluted at different concentrations of eluting agent.

Methods for producing CTLA4Ig molecules of the invention

Expression of CTLA4Ig molecules can be found in prokaryotic cells. Organ prokaryotic isms are most often different strains of bacteria. Bacteria can be gram positive or gram negative. Gram negative bacteria such as E. coli are usually preferred. Other microbial strains may also be used.

The above-coding sequences encoding CTLA4Ig molecules can be inserted into a vector for expressing foreign sequences in prokaryotic cells, such as E. coli.

These vectors may include commonly used prokaryotic control sequences, which are defined herein as including promoters for transcription initiation, optionally with an operator, along with ribosome binding site sequences, including such commonly used promoters as beta-lactamase (penicillinase) and lactose ( lac) (Chang et al., Nature, 198: 1056 (1977)), the tryptophan (trp) promoter system (Goeddel et al., Nucleic Acids Res. 8: 4057 (1980)) and the lambda PL promoter and the N gene of the ribosome binding site (Shimatake et al., Nature, 292: 128 (1981)).

Such expression vectors will also include origin of replication and selection markers such as the beta-lactamase phosphotransferase or neomycin gene conferring resistance to antibiotics in such a way that the vectors can replicate in bacteria and plasmid-bearing cells can be selected when grown in the presence of antibiotics. such as ampicillin or kanamycin.

The expression plasmid can be introduced into prokaryotic cells by a variety of standard methods, including CaCl2 shock (Cohen, Proc. Natl. Acad. Sci. USA, 69: 2110 (1972) and Sambrook et al., Eds., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Press (1989)) and electroporation method.

In accordance with the practice of the invention, eukaryotic cells are also suitable host cells. Exemplary eukaryotic cells include any animal cell, whether primary or immortalized, yeast cells (e.g., Saccharomyces cerevisiae, Schizosaccharomyces pombe and Pichia pastoris) and plant cells. Myeloma, COS and CHO cells are examples of animal cells that can be used as host cells. Specific CHO cells include, but are not limited to, DG44 (Chasin et al., Som. Cell. Molec. Genet., 12: 555-556 (1986); Kolkekar, Biochemistry, 36: 10901-10909 (1997)), CHO-K1 (ATCC ® No. CCL-61), CHO-K1 Tet-On cell line (Clontech), CHO labeled ECACC 85050302 (CAMR, Salisbury, Wiltshire, UK), CHO clone 13 (GEIMG, Genova, IT), CHO clone B (GEIMG , Genova, IT), CHO-K1 / SF designated ECACC 93061607 (CAMR, Salisbury, Wiltshire, UK) and RR-CHOK1 designated ECACC 92052129 (CAMR, Salisbury, Wiltshire, UK). Exemplary plant cells include tobacco cells (whole plants, cell cultures or calli), corn, soybean and rice. Corn, soybean and rice seeds are also allowed.

The above-described nucleic acid sequences encoding CTLA4Ig molecules can also be inserted into a vector for expressing foreign sequences in eukaryotic host cells. The regulatory elements of the vector may vary depending on the particular eukaryotic host.

Commonly used eukaryotic control sequences for use in expression vectors include mammalian cell compatible promoters and control sequences, such as the CMV promoter (CDM8 vector) and avian sarcoma virus (ASV) (hLN vector). Other commonly used promoters include the early and late promoters of simian virus 40 (SV40) (Fiers et al., Nature, 273: 113 (1973)) or other viral promoters such as those derived from polyoma, adenovirus 2 and bovine papilloma virus . An inducible promoter such as hMTII can also be used (Karin et al., Nature, 299: 797-802 (1982)).

Vectors for expressing CTLA4Ig molecules in eukaryotes can also carry sequences called enhancer regions. They are important for optimizing gene expression and are located either before or after the promoter region.

Exemplary 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 (Maniatis); pIRES (Clontech); pRc / CMV2, pRc / RSV, pSFV1 (Life Technologies ); pVPakc vectors, pCMV vectors, pSG5 (Stratagene) vectors), retroviral vectors (e.g. pFB (Stratagene) vectors), pCDNA-3 (Invitrogen) or modified forms thereof, adenoviral vectors, adenovirus dependent viral vectors, baculovirus vectors, yeast vectors (e.g. pESC (Stratagene) vectors).

Nucleic acid sequences encoding CTLA4Ig molecules can be incorporated into the genome of a eukaryotic host cell and replicate as the host genome replicates. Optionally, the vector carrying CTLA4Ig molecules may contain origin of replication allowing extrachromosomal replication.

An origin of replication from the endogenous yeast plasmid, ori 2μ can be used to express the nucleic acid sequence in Saccharomyces cerevisiae. (Broach, Meth. Enzymol., 101: 307 (1983)). Optionally, sequences from the yeast genome capable of activating autonomous replication (see, for example, Stinchcomb et al., Nature, 282: 39 (1979)) can be used; Tschemper et al., Gene, 10: 157 (1980); and Clarke et al., Meth. Enzymol., 101: 300 (1983)).

Trapscript control sequences for yeast vectors include promoters for the synthesis of glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg., 7: 149 (1968) and Holland et al., Biochemistry, 17: 4900 (1978)). Additional promoters known in the art include the CMV promoter contained in the CDM8 vector (Toyama et al., FEBS, 268: 217-221 (1990)); 3-phosphoglycerate kinase promoter (Hitzeman et al., J. Biol. Chem., 255: 2073 (1980)) and promoters for other glycolytic enzymes. Other promoters are inducible because they can be regulated by environmental stimuli or cell growth medium. These inducible promoters include promoters from the genes for heat shock proteins, alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, enzymes associated with nitrogen catabolism, and enzymes responsible for the use of maltose and galactose.

Regulatory sequences may also be located at the 3 'end of the coding sequences. These sequences can stabilize messenger RNA. Such terminators are located in the 3 'untranslated region after coding sequences in several yeast and mammalian genes.

Exemplary vectors for plants and plant cells include, but are not limited to, Agrobacterium Ti plasmids, cauliflower mosaic virus (CaMV), and tomato yellow mosaic virus (TGMV).

Mammalian cells can be transformed with methods such as transfection in the presence of calcium phosphate, microinjection, electroporation or transduction with viral vectors, among others.

Methods for introducing foreign DNA sequences into plant and yeast genomes include (1) mechanical methods, such as microinjection of DNA into individual cells or protoplasts, vortexing cells with glass beads in the presence of DNA, or shooting cells or protoplasts with tungsten or gold beads coated with DNA; (2) the introduction of DNA by making cell membranes permeable to macromolecules by treating them with polyethylene glycol or subjecting them to high voltage electrical impulses (electroporation); or (3) the use of liposomes (containing cDNA) that fuse to cell membranes.

US Patent Application Publication No. 2005/0019859 and US Patent No. 7,332,303 relate to the process of producing the proteins of the invention, in particular recombinant glycoprotein products, in animal or mammalian cell cultures.

After the protein production phase of the cell culture process, CTLA4Ig molecules are recovered from the cell culture medium by methods known to those skilled in the art. In particular, the CTLA4Ig molecule is recovered from the culture medium as a secreted polypeptide.

The culture medium is initially centrifuged to remove cellular debris and solid particles. The desired protein is then purified from DNA impurities, soluble proteins and polypeptides using the following non-limiting purification procedures commonly recognized in the art: SDS-PAGE; ammonium sulfate precipitation; ethanol precipitation; fractionation on immunoaffinity or ion exchange columns; Reverse phase HPLC; chromatography on silica or an anion exchange resin such as QAE or DEAE; chromatographic focusing; gel filtration using, for example, SEPHADEX® G-75 columns; and SEPHAROSE® protein A columns to remove impurities such as IgG. The addition of a protease inhibitor such as phenyl methylsulfonyl fluoride (PMSF) or a mixture of protease inhibitors may also be useful to inhibit proteolytic degradation during purification. One skilled in the art will recognize that purification methods suitable for the protein of interest, for example glycoprotein, may need to be amended to take into account changes in the nature of the protein after expression in recombinant cell culture.

Techniques and purification methods that have been chosen for the glycoprotein carbohydrate groups are also useful in the context of the present invention. For example, such techniques include HPLC or ion exchange chromatography using cationic or anion exchange resins in which more basic or more acidic fractions are collected, depending on which carbohydrate they were selected for. By using these techniques, you can also remove impurities at the same time.

The purification method may further comprise additional steps to inactivate and / or remove viruses and / or retroviruses that may potentially be present in the mammalian cell culture medium. A significant number of viral clearance stages are available, including but not limited to chaotrope treatment such as urea or guanidine, detergents, additional ultrafiltration / diafiltration steps, conventional separation such as ion exchange or exclusion chromatography, extreme pH values, heat, proteases, organic solvents or any combination thereof.

The purified CTLA4Ig molecule requires concentration and buffer exchange before it is stored or further processed. The Pall Filtron TFF system can be used to concentrate and exchange the elution buffer from the previous purification column with the final buffer desired for a given drug substance.

In one embodiment, purified CTLA4Ig particles, which have been concentrated and subjected to a diafiltration step, can be filled into 2L BIOTAINER® bottles, a bioprocess bag with a capacity of 50l or any suitable container. CTLA4Ig particles in such vessels can be stored prior to freezing for about 60 days at 2 ° to 8 ° C. Prolonged storage of purified CTLA4Ig molecules at 2 ° to 8 ° C can lead to an increase in the percentage of HMW forms. Therefore, for long-term storage, CTLA4Ig particles can be frozen at about -70 ° C and stored at about -40 ° C before storage. Freezing temperature may range from about -50 ° C to about -90 ° C. The freezing time can vary and depends largely on the volume of the vessel containing CTLA4Ig particles and the number of vessels that have been loaded into the freezer. For example, in one embodiment, CTLA4Ig particles are contained in 2L BIOTAINER® bottles. When loading less than four BIOTAINER® bottles with capacity to the freezer. 2l a freezing time of from about 14 to at least 18 hours may be required. When loading at least four bottles into the freezer, a freezing time of about 18 to at least 24 hours may be required. The vessels with frozen CTLA4Ig particles are stored at a temperature from about -35 ° C to about -55 ° C. The storage time at a temperature of about -35 ° C to about -55 ° C may vary and is only 18 hours. The frozen drug substance can be thawed in a controlled manner for the formulation of the medicinal product.

U.S. Patent Applications 60 / 752,267 dated December 20, 2005 and PCT / US2006 / 049074 dated December 19, 2006 concern patent production processes of proteins subject to the invention, in particular recombinant glycoprotein products, in animal or mammalian cultures phones.

Pharmaceutical composition

The present invention uses pharmaceutical compositions containing CTLA4Ig molecules mixed with an acceptable carrier or adjuvant known to those skilled in the art. Preferably, the pharmaceutical compositions contain appropriate carriers and adjuvants, which include any substance that retains CTLA4Ig activity when combined with it and that does not respond to the patient's immune system. These carriers and adjuvants include, but are not limited to, ion exchangers, alumina, aluminum stearate, lecithin, blood serum proteins such as human serum albumin, buffering substances such as phosphates, glycine, sorbic acid, potassium sorbate, a mixture of partial saturated glycerides vegetable fatty acids, phosphate buffered saline, 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, polyvinylpyrrolidone, cellulose based materials and polyethylene glycol. Other carriers also include sterile solutions; tablets, including coated tablets, and capsules. Typically, such carriers contain excipients such as starch, milk, and sugar (e.g. sucrose, glucose, maltose), certain types of clay, gelatin, stearic acid or its salts, magnesium or calcium stearate, talc, vegetable oils or fats, gums, glycols or other known excipients. Such carriers may also contain flavoring and color additives or other ingredients. Compositions containing such carriers are created by well-known traditional methods. Such compositions can also be formulated within various lipid compositions, such as, for example, liposomes, as well as various polymer compositions, such as polymer microspheres.

Formulations containing soluble CTLA4 molecules have been described in US Preliminary Patent Application No. 60 / 752,150 of December 20, 2005 and PCT / US2006 / 062297 of December 19, 2006, which are simultaneously patent pending. As described in U.S. Pat. Initial Patent Application No. 60 / 752,150, soluble CTLA4 molecules can be formulated for intravenous and subcutaneous applications. In short, the appropriate subcutaneous formulation (SC) contains CTLA4Ig molecules with a protein concentration of at least 100 mg / ml in combination with sugar at a stabilizing level in the aqueous carrier.

An example of CTLA4Ig SC available in pre-filled pre-filled syringes is shown in Table 3 below.

TABLE 3

<td colspan="2">Composition of CTLA4Ig SC, 125 mg / ml (125 mg / syringe)</td>

<td>Ingredient</td><td>Quantity (mg in the syringe)</td>

<td>CTLA4Ig</td><td> 125</td>

<td>Saccharose</td><td> 170</td>

<td>Poloxamer 188</td><td> 8.0</td>

<td>Monobasic sodium phosphate monohydrate</td><td> 0.143</td>

<td>Dibasic sodium phosphate anhydrous</td><td> 0.971</td>

<td>Water for injections</td><td>make up to 1.0 ml</td>

Examples 1 and 2 of the current specification describe the preparation of intravenous (IV) and subcutaneous (SC) CTLA4Ig formulations useful in the methods of the invention. The following is an example of a lyophilized CTLA4Ig formulation used in the method of the invention described in Example III.

TABLE 4

<td colspan="2">Composition of the freeze-dried CTLA4Ig medicinal product (250mg / vial)</td>

<td>Ingredient</td><td>Quantity (mg / vial)³</td>

<td>CTLA4Ig</td><td> 262.5</td>

<td>Maltose monohydrate</td><td> 525</td>

<td>Monobasic sodium phosphate monohydrate^b</td><td> 18.1</td>

<td>Sodium chloride^b</td><td> 15.3</td>

<td>Hydrochloric acid</td><td>Adjustment to pH 7.5</td>

<td>Sodium hydroxide</td><td>Adjustment to pH 7.5</td>

<td colspan="2">^and Includes a 5% allowance for loss for the vial, needle and syringe. ^b These ingredients are found in the solution of the CTLA4Ig drug substance.</td>

The lyophilized medicinal product may be reconstituted in an aqueous carrier. The aqueous carrier of interest herein is one of the pharmaceutically acceptable carriers (safe and non-toxic when administered to humans) and is suitable for preparing a liquid formulation after lyophilization. Typically, the lyophilized medicinal product is reconstituted to a concentration of 25 mg / ml in 10 ml of sterile water for injections, USP (SWFI), or 0.9% sodium chloride solution for injection, USP. The reconstituted solution is further diluted to a medicinal product concentration in the range of 1 to 10 mg / ml in 0.9% sodium chloride solution for injection, USP. The diluted medicinal product for injection is isotonic and is suitable for intravenous infusion.

Industrial article

In addition, an industrial article containing a medicinal product has been described, preferably with instructions for its use. The industrial article includes a container. Suitable containers include, for example, bottles, vials, syringes and test tubes. The container can be made of various materials such as glass, plastic or metal.

The container contains a freeze-dried or liquid formulation. The label attached to the container or attached to the container may contain directions for reconstitution (reconstitution) and / or use. For example, the label may indicate that the 250 mg medicinal product per vial is to be reconstituted to obtain the protein concentration described above. The label may additionally indicate that the SC formulation is suitable or intended for subcutaneous administration. The container containing the formulation may be a reusable vial allowing several administrations (e.g., 2 to 6 administrations), e.g. subcutaneous formulation. Optionally, the container may be a pre-filled syringe containing, for example, a subcutaneous formulation.

The industrial article may further comprise a second container containing, for example, a suitable carrier for the lyophilized formulation.

The industrial article may further include other materials desirable from a commercial or user point of view, including other buffers, diluents, filters, needles, syringes, and package leaflets containing instructions for use.

For surfactant-free medicinal products, it is best to use silicon-free syringes, for example when reconstituting a lyophilised medicinal product and / or transferring solutions from the vial to the infusion bag. These syringes may be packaged together with the medicinal product in the vial.

methods

The present invention provides a CTLA4Ig molecule or a pharmaceutical composition containing it for use in a method of treating individuals with undifferentiated arthritis by administering to a patient in need thereof an effective amount of a CTLA4Ig molecule or a composition containing it.

The administration of effective amounts of a CTLA4Ig molecule or pharmaceutical composition is of particular importance for alleviating at least one of the symptoms associated with the disease, including joint swelling, joint tenderness, inflammation, morning stiffness and pain, as well as reducing structural damage, thereby reducing subsequent physical disability.

The methods of the invention can also be used to improve the physical fitness of people with undifferentiated arthritis as assessed by the Health Assessment Questionnaire (HAQ) and / or the health-related quality of life assessment tool (SF-36).

The methods of the invention can also be used to inhibit joint structure damage in persons with undifferentiated arthritis assessed by erosion and edema of the bone marrow and / or synovitis of the wrist and hand joints.

The degree of relief of symptoms by the present invention can be assessed on the basis of any of the accepted criteria developed to measure and document symptom relief in clinical settings. Acceptable criteria for measuring the degree of symptom relief may include point scales based on criteria set by the American College of Rheumatology (e.g. ACR 20), four parameters for assessing the degree of symptom relief (given in: "CDER Guideline for the Clinical Evaluation of Anti-Inflammatory and Antirheumatic Drugs-FDA 1988") and the Health Assessment Questionnaire (HaQ) (Fries, JF et al., J. Rheumatology, 9: 789-793 (1982)). A general description of these criteria is found in the Industry Guidelines: Clinical Development Programs for Drugs, Devices, and Biological products for the Treatment of Rheumatoid Arthritis (RA) (Clinical development programs for drugs, medical devices and biological products intended for use in the treatment of rheumatoid inflammation joints) (February 1999).

The present invention provides various methods, local or systemic, for administering CTLA4Ig alone or in combination with other medicinal products. These methods include intravenous, intramuscular, intraperitoneal, oral, inhalation and subcutaneous methods, as well as implantable pumps, continuous intravenous infusion, gene therapy, liposomes, suppositories, topical application, vesicles, capsules and injections. CTLA4Ig in combination with a carrier is often freeze-dried for storage and reconstituted with water or a buffered solution prior to administration. This is a standard practice in the industry, and therefore the compositions of the invention may be administered to patients in any pharmaceutically acceptable form.

The most effective method of administration and dosage regimen of the formulation of the present invention depend on the patient's state of health and response to treatment, and the judgment of the attending physician. In accordance with the practice of the invention, an amount effective to treat patients is from about 0.1 to 100 mg / kg body weight. In another embodiment, the effective amount is from about 0.1 to 20 mg / kg body weight, preferably from 1 to 10 mg / kg patient weight. In a particular embodiment, the effective amount of CTLA4Ig is about 2 mg / kg patient weight. In another embodiment, the effective amount of CTLA4Ig is about 10 mg / kg patient weight. In another embodiment, the effective amount of CTLA4Ig is 500 mg for individuals weighing less than 60 kg, 750 mg for individuals weighing 60-100 kg, and 1000 mg for individuals weighing over 100 kg.

The inventive CTLA4Ig molecule formulations can be administered to the patient in an amount and time (e.g., duration and / or multiple administrations) sufficient to inhibit the binding of endogenous B7 molecules (e.g., CD80 and / or CD86) to the appropriate ligands. In this way, inhibition of binding of endogenous B7 molecules to ligand inhibits the interaction of B7 positive cells (e.g., CD80 and / or CD86 positive cells) with CD28 and / or CTLA4 positive cells. Thus, the doses of these agents may vary depending on the patient and the method of administration. US Patent Application Publication Nos. 2003/0083246 and 2004/0022787 provide dosage regimens and methods for administering CTLA4I-g molecules having the amino acid sequence set forth in SeQ ID NO: 2 for the treatment of rheumatic diseases such as rheumatoid arthritis. The effective amount of CTLA4Ig can be administered to the patient daily, once a week, once a month, and / or once a year once or repeatedly for an hour / day / week / month / year as needed. For example, in one embodiment, an effective amount of a CTLA4Ig molecule can be initially administered every other week for a month, then every other month or on days 1, 15, 29, and then once a month. For earlier doses (i.e. on days 15 and 29) time limits of +/- 3 days are allowed. For doses administered once a month, time limits of +/- 7 days are allowed.

Alternatively, a person skilled in the art could modify the administration regimen based on patient risk and / or response to treatment. For example, the scheme described above can be modified by adding the dose on Day 5 to the schedule.

The terms "four weeks," month "," months "or" per month "as used herein refer to a period of 28 ± 7 days.

Typically, doses of the CTLA4Ig molecule formulation of the present invention are determined based on body weight, and administration schedules may depend on the target profile of the lowest concentration in the dosage range in the blood serum. Typically, the target lowest concentration of CTLA4Ig molecules of the invention within the dosage range in the blood serum ranging from about 3 pg / ml to about 35 pg / ml, preferably from about 5 pg / ml to about 30 pg / ml, and more better from about 10 pg / ml to about 30 pg / ml, will be sufficient to treat undifferentiated arthritis or prevent RA in people with undifferentiated arthritis. One skilled in the art could adjust the CTLA4Ig doses and / or administration regimen so as to obtain the desired lowest concentration in the dosage range in the blood serum.

The molecules or pharmaceutical compositions of the invention may be administered by intravenous infusion over a period of 30 minutes to one hour or more. Optionally, the required dose may be provided as single or multiple subcutaneous injections.

The CTLA4Ig molecules of the invention may be administered concurrently or sequentially in combination with another immunosuppressant / immunomodulatory drug, e.g. as detailed herein, and the doses of co-administered immunosuppressive or immunomodulatory compounds will, of course, depend on the type of drug administered simultaneously.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can be administered simultaneously or sequentially in combination with the CTLA4Ig molecule of the invention. NSAIDs reduce the patient's inflammation. NSAIDs include, but are not limited to, acetylsalicylic acid, magnesium / choline salicylate, diflunisal, magnesium salicylate, salsalate, sodium salicylate, diclofenac, etodolac, fenoprofen, flurbiprofen, indomethacin, ketoprofen, ketorolac, mecloxamate, naproxamate tolmetin, acetaminophen, ibuprofen, Cox-2 inhibitors, meloxicam and tramadol.

Corticosteroids may be administered simultaneously or sequentially in combination with the CTLA4Ig molecule of the invention. For example, a low-dose oral corticosteroid (equivalent to a prednisone dose &lt;10 mg daily) or a high-dose corticosteroid administered once every six months orally (daily at a dose equivalent of prednisone 20 mg daily for a maximum of two weeks), or single intramuscular (IM) or intra-articular (IA) dose.

Examples of corticosteroids include, but are not limited to, betamethasone, budesonide, cortisol, cortisone, dexamethasone, hydrocortisone, methylprednisolone, prednisolone, prednisone, and triamcinolone.

Typically, the addition of CTLA4Ig molecules of the invention to the treatment regimen does not affect the standard doses and dosing schedules of the concomitant medications described above. However, a person skilled in the art can coordinate lower doses of concomitant medications due to the inclusion of less toxic CTLA4Ig molecules of the invention in the treatment regimen. Information on the ordination of a medicinal product may be based on the package leaflet of each concomitant medication.

As it was written earlier, the destruction of the joint structure occurs in the early phase of RA. This observation emphasizes the need for therapies that can fundamentally change, and not just stop the inflammatory processes that cause debilitating symptoms and structural damage in the early stages of RA. Therefore, more and more emphasis is put on earlier diagnosis and treatment of RA. However, RA diagnostic criteria developed by ARA in 1987 are less sensitive and specific if they are used in people with early arthritis and those people are diagnosed with undifferentiated arthritis, which is a common clinical problem.

It has recently been shown that in people with undifferentiated arthritis who simultaneously have antibodies against cyclic citrullinated peptide (anti-CCP2), the risk of developing RA is already high within one year of observation (Van Gaalen, FA et al., "Autoantibodies to Cyclic Citrullinated Peptides Predict Progression to Rheumatoid Arthritis in Patients with Undifferentiated Arthritis ", Arthritis Rheum., 50 (3): 709-715 (2004)). A positive test for the presence of an anti-cyclic citrullinated peptide antibody in blood serum has a greater predictive value for the development of RA than traditional criteria such as the presence or absence of rheumatoid factor. 83% of people with undifferentiated arthritis and a positive test for anti-CCP have developed RA within one year, compared with 18% of those in the control group without anti-CCP. These results indicate that it is possible to quickly identify a subgroup of people with undifferentiated arthritis who are at high risk of developing RA and who are therefore the ideal candidates for targeted treatment focused on mechanisms that stimulate inflammatory processes and joint destruction in RA. This approach could prevent the progression of joint damage, disability and subsequent deterioration of the quality of life typical of the natural course of RA.

Examples III and IV describe a clinical trial that aims to compare the efficacy of CTLA4Ig with placebo in the prevention of RA development in people with recently discovered undifferentiated arthritis who are at high risk of developing RA.

It is a randomized, double-blind, placebo-controlled clinical trial with two parallel treatment directions, which lasts 12 months to reach the primary endpoint and 24 months to reach the secondary endpoint. Participants were randomized in a 1: 1 ratio to treatment with CTLA4Ig or placebo for the first six months of the study. Randomization was stratified into two groups depending on the presence or absence of erosion.

Participants in this study were carefully screened to ensure that undifferentiated arthritis had recently occurred (i.e. symptoms of arthritis persist for less than 18 months), the test for anti-CCP antibodies is positive, and does not meet diagnostic criteria no other rheumatic disease and they did not use more DMARDs for more than 2 weeks or any biological medicine indicated in RA. The study looked at whether relatively short-term, 6-month CTLA4Ig monotherapy prevented progression to RA within 1 or 2 years after starting treatment. In addition, the effect of CTLA4Ig on disease activity, physical performance, health-related quality of life and PD biomarker activity in subjects with undifferentiated arthritis compared to placebo was studied both during the 6-month treatment period and for a maximum of 18 months after the last administration dose of study medication. Considering the 18-month follow-up without treatment after the last dose of study medication gave an assessment of the persistence of any observed effects of CTLA4Ig treatment. Both during treatment and follow-up, NSAIDs and oral low-dose corticosteroids (- &lt;10 mg daily prednisone or other equivalent dose), and high-dose corticosteroids were allowed. Persons with RA development at any time were withdrawn from the study. These people could receive standard treatment.

Due to the strict recruitment criteria limiting the number of randomized people to 57 (of whom 56 received treatment), no official testing of the statistical hypothesis was carried out. However, the results of this study were generally consistent with numerous efficacy parameters (clinical, radiological, MRI and biomarker activity), spoke in favor of CTLA4Ig over placebo, and provided evidence of inhibition of the rate of progression of undifferentiated arthritis into RA by CTLA4Ig administered once a month for 6 months intravenously at a dose of 10 mg / kg based on body weight. The therapeutic benefits of CTLA4Ig appear to be maintained for up to 18 months after stopping treatment.

The percentage of people who developed RA within 12 months was lower in the CTLA4Ig group (12/27, 46.2%) than in the placebo group (t6 / 24, 66.7%) (difference between treatment groups 20.5%; 95% CI: ~ 47.4%, + 7.8%). By the end of 24 months, 87.5% (12/24) of patients with undifferentiated arthritis in the placebo group and 73.9% (17/23) of those who had received CTLA4Ig treatment for 6 months (13.6% difference between treatment groups) developed RA. 95% CI: -37.6%, + 10.8%). The CTLA4Ig dose of 10 mg / kg based on body weight used in this study in subjects with undifferentiated arthritis is analogous to the 10 mg / kg dose approved for use in the treatment of RA in adults.

In this population of people with undifferentiated arthritis, CTLA4Ig treatment resulted in improved physical performance and a decrease in the activity of the physician assessed. After 6 months, more than twice as many people treated with CTLA4Ig compared to those who received placebo achieved clinically significant improvement in physical fitness (HAQ-DI) (62% compared with 24%) or showed disease remission based on the DAS-28 (CRP) score of &lt;2.6 (71% compared to 35%). The greater numerical improvement in the ILAQ-DI and DAS 28 (CRP) score observed at the end of the CTLA4Ig treatment period used in the study was still apparent after 6 and 18 months of the follow-up without treatment, although the difference between the treatment group and the placebo group was smaller after 12 and 24 months. The results of the radiological evaluation based on MRI of the palm and wrist enhanced with gadolinium contrast and traditional X-ray images of the hands and feet indicated minimal disease progression during the treatment period with the study drug in people receiving CTLA4Ig.

In particular, a decrease in serum anti-CCP2 antibody titers was observed after 6 months of CTLA4Ig treatment, and this reduction was still evident after a 6-month period without study drug treatment. For comparison, an increase in anti-CCP2 antibody titers was shown in the placebo group. Anti-CCP2 antibodies have a high predictive value for the future development of RA in both healthy individuals and patients with undifferentiated arthritis.

To date, a traditional approach to the treatment of early stage undifferentiated arthritis has been recommended, limiting the use of DMARD followed by biological agents only to patients whose signs and symptoms of arthritis are resistant to NSAIDs and low-dose corticosteroids. In this study of 56 patients with undifferentiated arthritis, CTLA4Ig was well tolerated by people with very early developmental disease. During the 6-month study treatment period, adverse events were reported at a similar frequency in the CTLA4Ig and placebo groups, no adverse events were fatal, and a small number of subjects in both treatment groups reported serious adverse events (1 in each group ) or discontinued the study due to adverse events (1 in each treatment group). In addition, there were no signs that indicated a higher risk of developing infections with CTLA4Ig, and adverse events associated with intravenous infusion occurring within 1 hour of starting CTLA4Ig administration were reported in only 1 person. The data from clinical hematological and biochemical laboratory tests were generally insignificant and no safety hazards were identified. After 12 months, four (4) participants had seropositive anti-CTLA4-T antibody status, with 2 of them being positive for neutralizing antibodies. Antibody development does not appear to correlate with clinical safety results.

The inventors have found that the administration of CTLA4Ig slowed the progression rate to unequivocal RA in patients with undifferentiated arthritis. In addition, CTLA4Ig effect on disease modification persisted for 6 months after treatment discontinuation.

A more detailed understanding of the invention will be possible by reference to the following examples. However, they are not to be understood as limiting the scope of the invention as defined by the claims.

EXAMPLE

CTLA4Ig, the lyophilized medicinal product (250 mg / vial) is a sterile, non-pyrogenic lyophilisate suitable for intravenous (IV) administration. Each single-use vial contains 250 mg CTLA4Ig reconstituted with sterile water for injections, USP, and further diluted with 0.9% sodium chloride injection, USP, at the time of use.

The batch formula for a batch size of 115 liters is described in Table 5 below.

TABLE 5

<td colspan="2">Party formula</td>

<td>Ingredient</td><td>Quantity (kg)</td>

<td>Medicinal substance CTLA4Ig^and</td><td> 4.6</td>

<td>Maltose monohydrate</td><td> 9.2</td>

<td>Hydrochloric acid</td><td>Adjustment to pH 7.5</td>

<td>Sodium hydroxide</td><td>Adjustment to pH 7.5</td>

<td>Water for injections</td><td>Padding to 119.6^b</td>

^and Medicinal substance CTLA4Ig: protein concentration 50 mg / ml, 25 mM sodium phosphate, 50mM sodium chloride, pH 7.5, &lt;5% HMW forms.

^b The density of the solution mass in the formulation = approximately 1.04 g / ml.

The required amount of CTLA4Ig drug substance is added to a cleaned and sterilized stainless steel mixing vessel equipped with a stirrer. The drug substance solution is mixed at 250 650 rpm, the solution temperature being kept in the range of 5 ° -25 ° C.

The required amount of maltose monohydrate powder is added to the mixing container. The solution is mixed for at least 10 minutes at 15 ° - 25 ° C.

The pH (pH) of the solution is adjusted to 7.3-7.7, using the previously prepared 1 N sodium hydroxide solution or 1 N hydrochloric acid if necessary. Using water for injection, USP, prepare the final batch mass (final refill) and mix at for at least 8 minutes. Samples for pH evaluation are taken from the mass of the solution in the formulation.

The mass of the solution in the formulation is pre-filtered through one filter with a pore diameter of 0.45 pm. Samples are taken from the mass of the solution in the formulation after filtering through a 0.45 pm filter for bacterial colonization and bacterial endotoxin (BET) determination.

Before filling the containers, the pre-filtered mass of the solution in the formulation is sterile filtered through two filters with a pore diameter of 0.22 pm.

The sterile filtered mass of the solution in the formulation is filled into containers and partially sealed with a gray Daikyo butyl rubber stopper with 20 nm diameter particles using a fully automatic filling and closing machine. 15 cm type I lead glass glass vials³ are washed and sterilized / depirogenized.

The filled and partially closed vials with the medicinal product are lyophilized. Table 6 below summarizes the freeze-drying (cryodication) cycle used for lyophilizing CTLA4Ig.

TABLE 6

<td colspan="2">Freeze drying cycle of the lyophilized CTLA4Ig medicinal product</td>

<td>Process parameter</td><td>In-process control</td>

<td>Charging temperature</td><td>5 ± 3 ° C</td>

<td>Freezing (shelf hold)</td><td>From 5 ° C to -45 ° C in 2.5 hours</td>

<td>Freezing</td><td>Holding at -45 ± 3 ° C for 4 hours.</td>

<td>Basic drying (shelf loading)</td><td>From -45 ° C to -19 ° C in 2 hours</td>

<td>Basic drying (vacuum)</td><td>100 ± 20 microns</td>

<td>Basic drying</td><td>Holding at -19 ± 2 ° C for 84 hours.</td>

<td>Intermediate drying (shelf loading)</td><td>From -19 ° C to 0 ° C in 2 hours</td>

<td>Intermediate drying</td><td>Holding at 0 ± 3 ° C for 8 hours.</td>

<td>Additional drying (shelf loading)</td><td>From 0 ° C to 30 ° C in 2.5 hours</td>

<td>Additional drying (vacuum)</td><td>100 ± 20 microns</td>

<td>Additional drying</td><td>Holding at 30 ± 2 ° C for 12 hours.</td>

<td>Cork closing</td><td>30 ± 3 ° C</td>

<td>Cork closure (vacuum)</td><td>500 ± 100 microns</td>

<td>Process parameter</td><td>In-process control</td>

<td>Process parameter</td><td>In-process control</td>

<td>Storage before unloading</td><td>Holding at 20 ± 3 ° C for at least 4 hours.</td>

At the end of the lyophilization cycle, the chamber pressure is increased to 500 microns using sterile filtered nitrogen and the vials are closed under vacuum. The closed vials remain inside the freeze dryer for at least 4 hours. The closed vials with lyophilized content are sealed in a capping machine with a white flip off aluminum seal with a diameter of 20 mm in air filtered with a HEPA filter. The sealed vials are rinsed with deionized water in an external vial washer. The rinsed medicinal vials are stored at 2 to 8 ° C.

The composition of the lyophilized CTLA4Ig (250 mg / vial) medicinal product is given in Table 7 below.

TABLE 7

<td colspan="2">Composition of the lyophilized CTLA4Ig medicinal product (250 mg / vial)</td>

<td>Ingredient</td><td>Quantity (mg / vial)^b</td>

<td>CTLA4Ig</td><td> 262.5</td>

<td>Maltose monohydrate</td><td> 525</td>

<td>Monobasic sodium phosphate monohydrate^b</td><td> 18.1</td>

<td>Sodium chloride^b</td><td> 15.3</td>

<td>Hydrochloric acid</td><td>Adjustment to pH 7.5</td>

<td>Sodium hydroxide</td><td>Adjustment to pH 7.5</td>

<td colspan="2">^and Includes 5% loss allowance for vial, needle and syringe. ^b These ingredients are found in the solution of the CTLA4Ig drug substance.</td>

EXAMPLE II

CTLA4Ig SC 125 mg / ml (125 mg / vial) is manufactured as a sterile, non-pyrogenic, ready-to-use solution suitable for subcutaneous administration. A batch of CTLA4Ig SC, 125 mg / ml (125 mg / vial) is produced on a 5-liter scale (3500 vials). The batch formula is described in Table 8 below.

TABLE 8

<td colspan="2">Party formula</td>

<td>Ingredient</td><td>Quantity (gm)</td>

<td>Medicinal substance CTLA4Ig^and</td><td> 625</td>

<td>Saccharose</td><td> 850</td>

<td>Poloxamer 188</td><td> 40</td>

<td>Monobasic sodium phosphate anhydrous</td><td> 0.715</td>

<td>Dibasic sodium phosphate anhydrous</td><td> 4.86</td>

<td>Water for injections</td><td>Topping up to 5.0 liters</td>

<td>Total lot size (l)</td><td> 5.0</td>

<td colspan="2">^and Medicinal substance CTLA4Ig: protein concentration 50 mg / ml, 25 mM sodium phosphate, 50mM sodium chloride, pH 7.5, &lt;5% HMW forms.</td>

As outlined in Example 1 above, the production process of CTLA4Ig SC, 125 mg / mL (125 mg / vial), includes the exchange of the drug mass buffer from 25 mM sodium phosphate, 50 mM sodium chloride pH 7.5 to 10 mM sodium phosphate pH 7.8, followed by concentrating the protein from ~ 50 mg / ml to ~ 150 mg / ml by buffer removal. Then, sucrose and poloxamer 188 are dissolved in the concentrated protein solution and the final batch mass is prepared, supplemented with 10 mM sodium phosphate buffer at pH 7.8. The mass of the solution is filtered through a sterilizing filter with a pore diameter of 0.22 microns and poured into sterilized and depyrogenized type I glass vials with a capacity of 5 cm³which are closed with 20 mm diameter rubber stoppers and 20 mm diameter flip-off aluminum seals.

The composition of CTLA4Ig SC, 125 mg / ml (125 mg / vial) is given in Table 9 below.

TABLE 9

<td colspan="2">Composition of CTLA4Ig SC, 125 mg / ml (125 mg / vial)</td>

<td>Ingredient</td><td>Quantity (mg / vial)^c</td>

<td>CTLA4Ig</td><td> 175</td>

<td>Saccharose</td><td> 238</td>

<td>Poloxamer 188</td><td> 11.2</td>

<td>Monobasic sodium phosphate anhydrous</td><td> 0.20</td>

<td>Dibasic sodium phosphate anhydrous</td><td> 1.36</td>

<td>Water for injections</td><td>Make up to 1.4 ml</td>

<td colspan="2">^and Includes 40% loss allowance for vial, needle and syringe.</td>

EXAMPLE III

Rheumatoid arthritis (RA) is an autoimmune disease that can lead to progressive joint destruction, joint deformity, significant physical disability, and poor quality of life. No medications have been shown to prevent the development of RA. This study will compare the efficacy of CTLA4Ig with placebo in the prevention of RA in people with recently discovered undifferentiated arthritis who have a high risk of developing RA during the study period.

Primary goal

Assessment of the percentage of patients with undifferentiated arthritis who develop RA as defined according to the 1987 ARA criteria during the year following the start of blinded study drug.

Secondary goals

1) Assessment of the percentage of patients with undifferentiated arthritis who develop RA as defined in 1987 ARA criteria during the two years after starting blind drug.

2) Assessment of the severity of synovitis and the degree of damage to the structure of the hand joints (wrist joints, MCP and PIP) in both treatment groups after 6, 12 and 24 months from the date of the start of treatment used as part of the examination based on MRI imaging.

3) Assessment of the percentage of participants with persistent symptomatic clinical synovitis 6, 12 and 24 months after the start of the treatment used in the study in both treatment groups.

4) Assessment of the pharmacodynamic effect of CTLA4Ig on the concentration of autoantibodies [rheumatoid factor IgM class and anti-cyclic citrullinated peptide (antiCCP2) antibodies] in blood serum.

5) Assessment of disease activity over time in both treatment groups based on the average total DAS score (CRP).

6) Evaluation of the percentage of participants in whom the total DAS score was &lt;1.6 after 6, 12 and 24 months.

7) Assessment of physical fitness and quality of life depending on health by means of Disability Index of HAQ (HAQ) and SF-36, respectively.

8) Assessment of the safety of CTLA4Ig use in a given study population, including assessment of CTLA4Ig immunogenicity after completing six months of treatment.

Tertiary goal

1) Assessment of changes in the basic components of the ACR RA (American College of Rheumatology Rheumatoid Arthritis) composite over time in both treatment groups.

Study plan

It is a randomized, double-blind, placebo-controlled study with two parallel treatment directions, which duration is 12 months to reach the primary endpoint and 24 months to reach the secondary endpoint. Participants will be randomized in a 1: 1 ratio to treatment with CTLA4Ig or placebo for the first six months of the study. Randomization will be stratified into two groups depending on the presence or absence of erosion (interpretation at headquarters). Participants will be able to take nonsteroidal anti-inflammatory drugs (NSAIDs) throughout the duration of the study. Participants will be able to take oral low-dose corticosteroids (equivalent to a prednisone dose of &lt;10 mg daily) throughout the study. A maximum of two of the high-dose corticosteroids listed below can be used simultaneously once every six months of the study, at the discretion of the investigator: oral treatment (equivalent to a dose of prednisone 20 mg daily administered daily for a maximum of two weeks) or a single intramuscular (IM) or single dose delivery dose (IA). Participants with arthritis persisting after six months of study drug use who do not meet the criteria for RA will be observed without taking study drug and will be able to use the drugs described above at the discretion of the researcher. No further doses of study medication will be administered.

Participants with a primary endpoint (RA according to ARA criteria) at any time during the study will be withdrawn from the study and will be able to receive anti-rheumatic drugs, including DMARDs and / or biologicals, at the discretion of the researcher.

Initially, participants will receive a dose based on body weight determined at the screening visit. People weighing &lt;60 kg will receive 500 mg; people weighing 60 to 100 kg will receive 750 mg, and people weighing&gt; 100 kg will receive 1 gram. CTLA4Ig will be administered on days 1, 15 and 29 and every 28 days thereafter until a total of 8 doses have been administered. Participants will be able to take oral low-dose corticosteroids (equivalent to a prednisone dose of &lt;10 mg daily) throughout the study.

For participants diagnosed with undifferentiated arthritis, an assessment will be made for the presence or absence of anti-cyclic citrullinated peptide (anti-CCP2) antibodies during the screening period. Participants with positive anti-CCP2 test results will be stratified according to the presence or absence of erosion and randomized to participate in the study drug treatment period. The study medication treatment period is six months of CTLA4Ig or placebo treatment. Participants who complete the treatment period and do not meet the criteria for diagnosing RA will be followed without taking the study medication during the observation period with masking (blinded). During the observation period of 18 months, visits will be carried out every 3 months to assess safety and efficacy.

Study population

Men or women (must not be pregnant or breastfeeding) at least 18 years old but not more than 75 years old, with undifferentiated arthritis diagnosed, who have symptomatic clinical synovitis in at least two joints and who have 1 ) meet at least one, but no more than three criteria for the diagnosis of RA established by the ARA in 1987, 2) do not meet the criteria for the diagnosis of any other rheumatic disease (e.g. lupus erythematosus) and

3) positive ELISA for anti-cyclic citrullinated peptide antibodies (Immunoscan RA Mark 2, Euro-Diagnostica, Arnhem, The Netherlands). The duration of the disease [defined as the time from the onset of symptoms (arthralgia, swelling or significant joint stiffness) of undifferentiated arthritis to the date of enrollment] must be less than 18 months. The use of any DMARDs or biologics prior to the screening phase is not allowed.

Assessment criteria

The primary result of the study will be the percentage of participants diagnosed with RA based on the 1987 ARA criteria after 12 months.

Secondary efficacy results include: percentage of participants with RA diagnosed based on the 1987 ARA criteria. after 24 months, the percentage of participants with persistent symptomatic clinical synovitis after 6, 12 and 24 months, average total DAS score (CRP) after 6, 12 and 24 months, the percentage of participants with a DAS score of &lt;1.6 after 6, 12 and 24 months, titers of rheumatoid factor and anti-CCP antibodies as well as severity of inflammation and degree of joint damage (synovitis, hand erosion and bone inflammation) in MRI examinations with gadolinium contrast assessed by the main interpreter not knowing the order of treatment. Patient-evaluated results include HAQ and SF-36.

Changes to the basic components of the ACR RA composite variable will be evaluated over time in both treatment groups as the tertiary goal of the study.

Performance analysis

The goal of the primary efficacy analysis will be to assess RA development rates after 12 months of using CTLA4Ig and placebo. Point estimators and confidence intervals for the percentage of RA patients diagnosed after 12 months in both treatment groups will be given. Participants who are withdrawn from the study with the reason "ineffective" in the clinical observation card, will be considered non-responders at the primary endpoint (i.e. will be counted in the numerator and denominator when assessing the percentage of participants who developed RA according to the ARA criteria). If the participant's data is missing during the appointment at month 12 for a reason other than withdrawal from the study, and an evaluation was carried out at the next visit, the results of that evaluation will be used to analyze the 12-month data. Otherwise the participant will not be included in the analysis. Similar analyzes will be carried out to assess RA development rates after 24 months.

The severity of synovitis and the degree of joint damage will be assessed by MRI imaging. The OMERACT 6 RA MRI scoring system will be used (Ostergaard, M. et al., "OMERACT Rheumatoid Arthritis Magnetic Resonance Imaging Studies. Core Set of MRI Acquisitions, Joint Pathology Definitions, and the OMERACT RA-MRI Scoring System", J. Rheumatology , 30 (6): 1385-1386 (2003)). Changes in the severity of erosion, edema and synovitis from baseline will be summarized after 6, 12 and 24 months using descriptive statistics.

The persistence of symptomatic clinical synovitis will be assessed after 6, 12 and 24 months. Synovial persistence rates will be summarized along with point estimators and 95% confidence intervals for the CTLA4Ig and placebo groups.

The pharmacodynamic effect of CTLA4Ig on autoantibody concentration [IgM rheumatoid factor and anti-cyclic citrullinated peptide (antiCCP2)] in serum will be assessed. The distribution of pharmacodynamic variables during the baseline visit, the appointment after 12 months and the appointment after 24 months will be summarized with changes from baseline. The percentage of participants with positive / negative results will be summarized by treatment group and baseline (positive / negative).

The mean changes in total DAS score (CRP), HAQ and all SF-36 elements after 6, 12 and 24 months from baseline will be summarized at each visit along with point estimators and 95% confidence intervals for the group treated with CTLA4Ig and a placebo group. In addition, the percentages of participants with demonstrated remission (total score on the DAS &lt;1.6) will be summarized by treatment group.

Changes to the basic components of the ACR RA composite variable will be evaluated over time in both treatment groups.

Security analysis

Relevant physical examination results as well as clinical and laboratory tests will be exchanged. Summary statistics will be presented in the table. The frequency distribution will be determined and individual lists of all adverse events will be created. Changes in clinical laboratory test results will be listed. Withdrawal / withdrawal cases will be summarized by treatment group.

Immunogenicity analysis

The distribution of immunogenicity variables and their changes relative to baseline will be summarized using descriptive statistics (geometric means, standard deviations, etc.); 95% confidence intervals for changes from baseline will also be calculated. Lack of immunogenicity was defined as no positive response. A positive response was defined based on the cut-off value for the positive test result for each assay and additional confirmation of the positive response by immunodepetition. For anti-CTLA4Ig titer determinations, this value is calculated by dividing the mean optical density (OD) value of the serum sample after dosing by the mean OD value of the corresponding serum sample before dosing (on Day 1). For anti-CTLA4Ig titer determinations, the cut-off value is calculated by dividing the mean optical density (OD) value of the serum sample taken from the patient by the mean OD value of the serum sample from the negative control on the test plate. The cut-off value is determined during test validation and can be re-determined if the test population or test reagents change. If the sample is negative, it is assigned a &lt;estimated dilution value. If the sample is positive, assessments are made in a series of dilutions and a titre value corresponding to the reciprocal of the interpolated serum dilution equivalent to the specified cut-off value for the positive sample is assigned. The positive response rate (if shown) and its 95% confidence interval will also be calculated.

Criteria for the selection of participants

Fulfilling the following criteria is a NECESSARY condition for entering the study.

Criteria for inclusion in the study

A. Signing informed consent

The participant wants to participate in the study and signed the informed consent.

B. Target group of patients

The participant must be diagnosed with undifferentiated arthritis. A participant with undifferentiated arthritis has symptomatic clinical synovitis in at least two joints and at least one, but no more than three, of the RA classification criteria established by the American Rheumatism Association (1987) is met.

The participant cannot meet the criteria for the diagnosis of any other rheumatic disease (e.g. lupus erythematosus).

The duration of the disease in the study participant [defined as the time calculated from the onset of symptoms (joint pain, joint swelling or stiffness) of arthritis to the date of enrollment in the study] must be less than 18 months.

The ELISA test result (Immunoscan RA Mark 2, Euro-Diagnostica, Arnhem, The Netherlands) for the presence of autoantibodies against cyclic citrullinated peptide must be positive.

C. Age and gender

Men and women between 18 and 75 years old. Fetal men and fertile women are eligible if they use effective contraception.

D. Concurrently used drugs

Oral corticosteroids may be taken at a constant low dose throughout the study. Dosage should be limited to the equivalent of &lt;10 mg prednisone per day for 28 days and stabilized at least 25 out of 28 days before treatment (day 1).

Exclusion criteria from the study

A. Gender and reproductive potential

1) Fertile women who are able to give birth to a child who will not or will not be able to use an acceptable method of contraception throughout the study and up to a maximum of 10 weeks after the last IV infusion of CTLA4Ig;

2) Pregnant or breastfeeding women;

3) Women with a positive pregnancy test performed during the recruitment to the study or before the study drug was administered;

4) Men who do not want or will not be able to use the appropriate method of contraception for the entire duration of study medication and for a maximum of 10 weeks after the last infusion of study medication;

B. Medical history and comorbidities

5) Persons with disabilities, incapacitation or incapability of undergoing examination-related assessments.

6) Persons meeting the criteria for the diagnosis of any other rheumatic disease (e.g. lupus erythematosus).

7) Undifferentiated arthritis lasting more than 18 months.

8) People who have previously used treatment with an authorized biological drug intended for use in RA (infliximab, etanercept, anakinra, adalimumab).

9) People with active vasculitis supplying an important organ or system.

10) Present symptoms of severe, progressive or uncontrolled disease of the kidneys, liver, hematopoietic system, gastrointestinal tract, lungs, heart, nervous system or brain. Comorbid conditions that, according to the researcher, could expose the patient to an unacceptable risk in connection with participation in this study.

11) Women who have not had age-appropriate and / or risk factors for breast cancer screening (as defined in published guidelines and / or local standards approved by the national cancer or medical association and / or the Minister of Health), or who have on the basis of breast cancer screening results, malignancy may be suspected; and women in whom malignancy cannot be excluded after additional clinical or laboratory assessments or other diagnostic tests.

12) People who have been diagnosed with cancer over the past five years (other than non-melanoma skin cancer cured after local resection). Currently diagnosed non-melanoma skin cancers should be removed prior to administration.

13) People with clinically significant alcohol or drug abuse.

14) People with any serious acute bacterial infection (such as pneumonia or pyelonephritis, unless it has been cured and resolved completely after antibiotics).

15) People with severe chronic or recurrent bacterial infections (such as recurrent pneumonia, chronic bronchiectasis).

16) People with active tuberculosis (TB) who need treatment in the last 3 years. Persons who will be positive during the screening phase of tuberculin test (PPD) will not be eligible to participate in the study, unless active tuberculosis is excluded and the result of chest x-ray examination is negative at the time of recruitment. A tuberculin test of at least 10 mm should be considered positive, although a lower threshold (5 mm) can be accepted based on clinical circumstances and the investigator's assessment in accordance with published guidelines and / or local standards approved by the medical association.

17) People who had shingles less than 2 months before being recruited for the study.

18) People who at the time of recruitment have features of active or latent bacterial or viral infection (according to the researcher's assessment), including people with features of human immunodeficiency virus (HIV) infection.

C. Results of physical examination and laboratory tests

19) People who have been tested positive for hepatitis B surface antigen.

20) People who have been shown to have antibodies to the hepatitis C virus (anti HCV), as well as a positive result of a recombinant immunoblot (RIBA) or PCR test.

21) Individuals with any of the following laboratory values:

Hgb &lt;8.5 g / dl.

WBC &lt;3000 / mm³ (3 x 10⁹/ L)

Platelets &lt;100,000 / mm³ (100 x 10⁹/ L)

Serum creatinine more than 2 times the upper limit of the ALT or AST norm more than 2 times the upper limit of the serum.

Any other laboratory test results that the researcher believes could expose the patient to an unacceptable risk in connection with participation in this study.

D. Prohibited medicines and / or treatment methods

22) People who have ever used CTLA4Ig or a treatment regimen containing CTLA4Ig.

23) Individuals who used any experimental drug for a period of 28 days (or less than 5 times the terminal half-life) before taking the dose on the first day of the study.

24) People who are currently undergoing immunoadsorption using immunological affinity columns (such as Prosorba), mycophenolate mofetil (CELLCEPT®), cyclosporin, D-penicillamine or calcineurin inhibitors.

25) People using DMARD before the screening phase.

E. Other exclusion criteria

26) Prisoners or persons under involuntary hospitalization (referred to involuntary treatment) due to mental or physical illness (e.g. infectious) must not be recruited for the examination.

People included in the MRI assessment

The radiologist at the MRI examination center in your facility is responsible for determining whether you have contraindications for this procedure. The following are some common conditions that may prevent you from having an MRI of your hands / wrists. However, this list cannot replace local healthcare standards. The final decision regarding the MRI examination for a given participant is made by the radiologist, researcher and the team of the local bioethics commission.

1) people who have been claustrophobic in the past;

2) people who may not fit in the camera gantry hole (i.e. people whose body weight exceeds 250 pounds, i.e. 113.4 kilograms);

3) people with permanent eye makeup or tattoos made directly on the palm or wrist (in the area to be examined);

4) people who have had an allergic reaction to contrast agents in the past;

5) persons who received radiological contrast agent 72 hours prior to the MRI examination;

6) persons after surgery for stiffening the wrist joints or arthroplasty of the palm or wrist joints, which are to be assessed by MRI;

7) people with an implanted pacemaker, epicardial electrodes, heart valve prostheses incompatible with the MRI environment, inserted less than two months earlier, vascular clips incompatible with the MRI environment, or ever inserted aneurysm clips incompatible with the MRI environment;

8) people with cochlear implants incompatible with the MRI environment;

9) people with implanted spinal nerve stimulators;

10) people with an infusion pump;

11) people with metal elements located in the eye / orbital area or near the brain or important neurovascular structures, people who have had a welding profession in the past, or people with bullet fragments in their bodies.

Administration of CTLA4Ig or placebo

Participants will be randomized into 1 of 2 treatment groups:

Group 1: CTLA4Ig by intravenous infusion (N = 25).

Group 2: Placebo by intravenous infusion (N = 25).

Participants receiving CTLA4Ig will be given a dose of the product based on body weight determined at the screening visit. People weighing &lt;60 kg will receive 500 mg; people weighing 60 to 100 kg will receive 750 mg, and people weighing&gt; 100 kg will receive 1 gram. Persons randomized to the placebo group will receive a 5% solution of dextrose in water or saline.

Doses administered by intravenous infusion will be calculated based on the patient's weight determined during the screening visit just before the appointment on Day 1. The Central Randomization System will confirm the patient's weight and allocate the number of CTLA4Ig vials for the visit. Participants will receive a study drug dose at each appointment during the study period (days 1, 15, 29, 57, 85, 113, 141 and 169). For doses provided on days 15 and 29, time limits of +/- 3 days are allowed; time limits of +/- 7 days are allowed for subsequent doses. The infusion should be given around the same time of day throughout the study. All doses of study drug will be administered intravenously in a constant volume of 100 ml and a constant flow rate over approximately 30 minutes. At the end of the infusion, flush the infusion line with 25 ml of a 5% dextrose solution in water or saline. The infusion solution must be provided to administration workers in a container that prevents recognition of the contents to maintain masking (plugging). The clinical trial auditor cannot know the allocation to treatment groups, which is why the study drug is administered by intravenous infusion by another competent team member. Intravenous infusions will be given to patients in a sitting position. The dose or treatment regimen is not subject to any modification. Participants will be monitored for adverse events, and vital signs (blood pressure, heart rate, respiratory rate, temperature) will be monitored from the start of each infusion (before dosing and after 60 minutes). Deadlines for assessing vital signs allow time limits of +/- 5 minutes. Participants will be followed for at least 1 hour after the start of the intravenous infusion. The observation period may be extended in cases indicated for clinical reasons.

Dose modification in the absence of adverse events

If there are no adverse events considered to be at least presumably related to study medication use, participants will complete an intravenous infusion schedule according to the protocol. For the convenience of participants and / or facility staff, the patient's assigned dose may be given within 72 hours (+/- 3 days) before or after the target day 15 or 29. For later doses, time limits of +/- 7 days may be allowed.

Treatment prohibited or restricted during the study

DMARD (e.g. methotrexate or oral or parenteral gold, sulfasalazine, chloroquine, hydroxychloroquine, D-penicillamine, azathioprine, leflunomide, cyclosporine) and biological drugs (e.g. etanercept, adalimumab, anakinra) are prohibited.

Participants will be able to take nonsteroidal anti-inflammatory drugs throughout the study. Participants will be able to take oral low-dose corticosteroids (equivalent to a prednisone dose of &lt;10 mg daily) throughout the study. Up to two of the high-dose corticosteroids listed below can be used once every six months at the discretion of the investigator: oral treatment (a dose equivalent to a dose of prednisone 20 mg per day given daily for a maximum of two weeks) or a single intramuscular (IM) dose or a single dose Supply (IA).

During this period, the use of nonsteroidal anti-inflammatory drugs (NSAIDs), including acetylsalicylic acid (ASA), is prohibited.

Administration of corticosteroids by intra-articular or intramuscular injection should be avoided. In the period of one month before the most important efficacy evaluation (i.e. on day 169, month 12 and month 24), high-dose intramuscular or intra-articular steroid administration is PROHIBITED.

In addition to the 12 hours preceding the joint assessment, the following medications may be used:

acetaminophen (paracetamol) combination products containing acetaminophen and narcotic analgesics (e.g. acetaminophen with codeine phosphate, acetaminophen with propoxyphene napsylate, acetaminophen with oxycodone hydrochloride, acetaminophen with hydrocodone bitartrate, etc.) tramadol.

TABLE 10

<td colspan="9">Procedures and observations appropriate to the study A. Phase of study drug testing with double-blind method</td>

<td>Visit Day</td><td>Day 1^and</td><td>Day 15 (+/- 3 days)</td><td>Day 29 (+/- 3 days)</td><td>Day 57 (+/- 7 days)</td><td>Day 85 (+/- 7 days)</td><td>Day 113 (+ / 7 days)</td><td>Day 141 (+ / 7 days)</td><td>Day 169 (+ / 7 days)</td>

<td>Randomization and dissection of participants (please contact the Main Randomization Center)</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Performance evaluation</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>X-rays of hands and feet</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>Hand and wrist MRI with gadolinium contrast (only in European centers)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>Determining the number of tender joints^b</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Number of joints with swelling</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Patient's assessment of pain</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Assessment of disease activity by the patient</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>General assessment of the disease activity by the doctor</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Overall patient fitness assessment (HAQ)</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>SF-36</td><td>X</td><td></td><td>X</td><td></td><td>X</td><td></td><td></td><td>X</td>

<td>Patient's response to treatment</td><td></td><td></td><td></td><td></td><td>X</td><td></td><td></td><td>X</td>

<td>Security Assessment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Adverse event control</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Ad hoc physical examination</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>ECG</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>Vital signs</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Laboratory tests</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Complete blood count</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Biochemical research set</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Urine analysis</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>Pregnancy test using a urine / serum sample (only for women able to give birth)^c</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>CRP</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>IgM RF</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>Biomarkers (IL-6, TNFa, IL-1 beta, MMP-3)</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>HLA typing</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>immunogenicity</td><td>X</td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>Anti-CCP2</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td>X</td>

<td>Drug administration</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td colspan="9">^and Before contacting the Main Randomization Center, check all grades and results. All evaluations should be performed prior to drug administration.^b An assessment of the number of 68/66 joints will be carried out.^c You must show a negative pregnancy test within 48 hours of your appointment.</td>

B. Observation after double-blind treatment

<td>Day of visit (time limits are allowed +/- 7 days in the month 9, 15, 18 and 21; and +/- 30 days in the month 12 and 24)</td><td>Month 9 (Day 253)</td><td>Month 12 (Day 365)^and</td><td>Month 15 (Day 449)</td><td>Month 18 (Day 533)</td><td>Month 21 (Day 617)</td><td>Month 24 (Day 729)</td><td>Earlier resignation</td>

<td>Performance evaluation</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>X-rays of hands and feet^b</td><td></td><td>X</td><td></td><td></td><td></td><td>X</td><td></td>

<td>Hand and wrist MRI with gadolinium contrast (only in European centers)</td><td></td><td>X</td><td></td><td></td><td></td><td>X</td><td></td>

<td>Determining the number of tender joints⁰</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Number of joints with swelling</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Patient's assessment of pain</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Assessment of disease activity by the patient</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>General assessment of the disease activity by the doctor</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Overall patient fitness assessment (HAQ)</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>SF-36</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Patient's response to treatment</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Security Assessment</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Adverse event control</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Ad hoc physical examination</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Full physical examination</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Weighting</td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td>

<td>Vital signs</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Laboratory tests</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Complete blood count</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Biochemical research set</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Urine analysis</td><td></td><td></td><td></td><td></td><td></td><td>X</td><td>X</td>

<td>Pregnancy test using a urine / serum sample (only for women able to give birth)</td><td>X^d</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>Screening for breast cancer</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td>

<td>Annually (only for women)</td><td></td><td>X</td><td></td><td></td><td></td><td>X</td><td>X</td>

<td>CRP</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td><td>X</td>

<td>IgM RF</td><td></td><td>X</td><td></td><td></td><td></td><td>X</td><td></td>

<td>Biomarkers (IL-6, TNFa, IL1 Beta, MMP-3)</td><td></td><td>X</td><td></td><td></td><td></td><td>X</td><td></td>

<td>immunogenicity</td><td>X</td><td>X</td><td></td><td></td><td></td><td></td><td></td>

<td>Anti-CCP2</td><td></td><td>X</td><td></td><td></td><td></td><td>X</td><td></td>

<td colspan="8">^and If the participant does not report to the clinic for the appointment in month 12, all assessments required in month 12 will be carried out at the next visit.^b If radiological examinations are performed between 6 and 12 months and the participant resigns / is withdrawn from the examination, the results of these examinations should be forwarded to the main interpreter for assessment according to the Sharp scale in the Genant modification.^c An assessment of the number of 68/66 joints will be carried out.^d You must show a negative pregnancy test within 48 hours of your appointment.</td>

Security Assessment

All subjects who receive a study drug dose will be evaluated for safety and immunogenicity. Safety assessment parameters include adverse events, clinically significant changes in vital signs, and abnormal laboratory results. The investigator will determine the severity of each adverse event by describing it as mild, moderately severe, severe, or very severe. Laboratory test results that the investigator considers clinically significant based on laboratory guidelines should be noted as adverse events. In addition, the investigator will establish a relationship between the adverse event and administration of study medication.

A full and / or ad hoc physical examination may be performed by a medical doctor (MD), certified osteopath (DO), medical assistant (PA), or qualified nurse (NP). Although the ad hoc physical examination may not be as comprehensive as the first complete physical examination, it should include an assessment of important body systems and organs in cases indicated for clinical reasons. These systems and organs include lymph nodes, liver, spleen and mammary gland at the discretion of the researcher. An ad hoc physical examination can capture any changes in the patient's condition (body systems and organs) since the last assessment. This examination does not preclude examination of any system and organ in cases indicated for clinical reasons.

During the screening visit, a chest x-ray should be performed if it has not already been performed six months before the date of obtaining informed written consent or if there is no relevant documentation on file.

During the screening visit, an 12-lead ECG should be performed if it has not already been done within 6 months before the date of obtaining informed written consent or if there is no relevant documentation on file. The ECG test will be repeated at the end of the treatment period (day 169) or 28 days after withdrawal / resignation if the participant completes the treatment period earlier.

To identify people with latent tuberculosis (TB), a tuberculin test (skin test using a purified protein derivative of tuberculin, PPD) is required if it was not performed within six months before the screening phase or if the file does not have adequate documentation for the test during six months). Tests for the detection of latent tuberculosis should be performed on all participants, including those who have previously received the BCG vaccine.

The tuberculin test should be performed in accordance with published guidelines containing recommendations for the performance of PPD skin tests and their interpretation in people with rheumatoid arthritis as part of qualification for treatment with biological medicines ("Preliminary guidel ines for diagnosing and treating tuberculosis in subjects with rheumatoid arthritis in immunosuppressive trials or being treated with biological agents ", Ann. Rheum. Dis., 61 (Supp.): Ii62-ii63 (2002), and centers outside of the US can also use local guidelines approved by medical societies for tuberculin testing in people with RA using biological medicines), people with weakened immunity ("Targeted Tuberculin Testing and Treatment of Latent Tuberculosis Infection ", Am. J. Respir. Crit. Care. Med., 161: 5221-5247 (2000) and" Diagnostic Standards and Classification of Tuberculosis in Adults and Children ", Am. J. Respir. Crit. Care Med., 161: 1376-1395 (2000)) and persons previously vaccinated with BCG vaccine.

Persons who will be positive during the screening phase of tuberculin test (PPD) will not be eligible to participate in the study, unless active tuberculosis is excluded and the result of chest x-ray examination is negative at the time of recruitment. A tuberculin test of at least 10 mm should be considered positive, although a lower threshold (5 mm) can be accepted based on clinical circumstances and the investigator's assessment in accordance with published guidelines and / or local standards approved by the medical association.

Prior to this study, women should be screened for breast cancer according to age and / or risk factors. Screening for breast cancer should be performed in accordance with published guidelines and / or local standards of conduct approved by the national cancer association or medical association or by the Minister of Health. In addition, the guidelines for breast cancer screening used in the research center should be made available to the local bioethics committee and explained in the informed consent documents for study participants.

The documented result of breast cancer screening performed six months before the screening phase will be considered to meet this criterion. However, screening will be necessary if there is no documentation from the facility where the screening was performed or the screening was performed more than 6 months before the study.

According to the accession criteria for breast cancer screening, women are required to repeat breast cancer screening annually.

Normal x-ray images of hands / wrists and feet should be taken for persons qualified to participate in the screening phase. X-rays performed in qualified patients (after confirming the positive result of anti-CCP2 antibody titers) will be sent to the main interpreter for assessment for the presence or absence of erosion. Information on erosion will be used to dissect participants as part of the study. Normal X-rays of the hands / wrists and feet will also be taken after 6, 12 and 24 months and evaluated by the main Sharp interpreter modified by Genant. If radiological examinations are carried out in the period between months 6 and 1, and the participant resigns / will be withdrawn from the examination, the results of these examinations should be forwarded for assessment to the main interpreter.

Prior to randomization, MRI scans of palm / wrists with gadolinium contrast (only on one side of the body) should be performed in all participants in European centers with positive anti-CCP2 antibody test results and symptomatic clinical synovitis in at least two hand joints or wrist. MRI examinations will be evaluated by the main interpreter. Control MRI examinations of the same hands / wrists will also be performed after 6, 12 and 24 months and evaluated by the main interpreter. MRI foot tests will not be performed.

Pregnancy tests using a urine or blood sample will be performed 48 hours before each visit to all women who are able to give birth. If any participant becomes pregnant, she will be withdrawn immediately.

MRI examinations of the hand and wrist with gadolinium contrast

All participants of the study in European centers with positive anti-CCP2 antibody test results and symptomatic clinical synovitis in at least two hand or wrist joints will have MRI of the hand and wrists with gadolinium contrast (only on one side) body) using an apparatus with a field strength of 1.5 T in the period of two weeks before administration of the first dose of study drug (from day -14 to day -1) and repeated after 6, 12 and 24 months. The results of MRI examinations will be interpreted by radiologists in the central institution by the OMERACT 6 method as follows:

Evaluation of erosion / swelling in the joints of the wrist and hands

Bone marrow erosion and edema will be assessed separately according to the OMERACT 6 classification system. Each wrist and palm bone (carpal bone, distal radius, distal ulnar segment, metacarpal base and metacarpal joint bone) is assessed separately on a scale from 0 up to 10 based on part of the bone with erosive changes and edema volume (assessed separately) compared to the volume of bone assessed. The assessment scale includes 10% increments, with 10 indicating damage to the bone under assessment by erosion or edema by more than 90%.

Evaluation of synovitis in the wrist and hand joints

Synovitis is evaluated in many areas around the wrist (distal elbow, radial-wrist joint, inter-wrist and carpal-hand joints, as well as II-V metacarpal joints). A score of 0 indicates severe damage when the volume of tissue enhancing after administration of gadolinium contrast is&gt; 2/3 of the synovial compartment evaluated.

All results of MRI examinations performed on all participants, where data from imaging tests performed both at the starting point and after 12 months are available, will be interpreted by a trained main interpreter not knowing the allocation to the treatment groups or the order of treatment.

HLA typing

Peripheral blood samples for HLA typing will be taken from all participants to be randomized on Day 1 to determine the presence or absence of alleles associated with RA susceptibility / severity (RA allele HLA-DR0401 and 0404 "common epitope" shared epitope, SE]). Randomized participants will be classified as individuals lacking these alleles, heterozygous, homozygous or double heterozygous. This information will allow an additional determination of the risk of developing a person with severe RA.

Evaluation of laboratory tests

At each visit before the infusion, blood and / or urine samples will be taken from each participant who entered the study. All laboratory findings that the investigator considers clinically significant should be recorded on the appropriate page of the clinical observation card for adverse events.

A. Hematological tests

Hemoglobin, hematocrit, total leukocytosis with smear and differentiation, platelets, erythrocytes

B. Blood biochemistry

Sodium, creatinine, potassium, urea nitrogen (BUN), chlorine, total bilirubin, total protein, alanine aminotransferase (ALT), albumin, aspartate aminotransferase (AST), calcium, gamma-glutamyl transferase (GGT), phosphorus, alkaline phosphatase, uric acid, glucose.

C. Urine analysis

Reaction (pH), protein, glucose, blood, pellet microscopy when blood, protein or glucose is detected in the test strip.

D. Liver screening (performed only during screening) HBV surface antigen (if positive - core protein), anti-HCV antibody (if positive - RIBA or PCR).

E. Pregnancy tests

Pregnancy tests using a urine or serum sample (minimum sensitivity 25 IU / L HCG) must be performed on all women within 48 hours prior to each appointment up to appointment at month 9. If any participant becomes pregnant, she will be withdrawn from the study . Pregnancy tests will be developed on site.

F. Pharmacodynamic studies (PD)

IgM RF, C-reactive protein (CRP), inflammatory cytokines (IL-6, TNFα, IL-1 beta), matrix type 3 metalloproteinase (MMP-3), anti-CCP2 antibodies.

G. Determination of immunogenicity

Anti-CTLA4Ig antibodies

H. Other

Human leukocyte antigens (HLA).

In addition, the researcher will collect samples for additional laboratory tests if deemed necessary to control the safety of the test participant.

immunogenicity

CTLA4Ig immunogenic potential will be assessed based on the level of antiCTLA4Ig antibodies.

Serum samples will be taken from subjects who complete the study drug treatment phase at the appointment on Day 1, 169, 253 and 365 for anti-CTLA4Ig antibodies.

Serum samples will be taken on days 1, 28, 56 and 85 after the last dose of study drug from subjects who did not complete the study drug treatment phase. Anti-CTLA4Ig titers will be measured in the samples.

Serum anti-CTLA4Ig and anti-CCP2 titers will be determined using a validated, sensitive enzyme-linked immunosorbent assay (ELISA).

Performance evaluation

A. X-ray of hands and feet

All participants with positive anti-CCP2 antibody titers will have hands and feet x-ray screening phase 169, month 12 and month 24. All centers must meet the technical requirements. Hand and foot x-rays will be standardized to provide sufficient image quality to assess the radiological progression of changes in RA. Radiological facilities and their employees will be qualified to participate in this study based on the technical capabilities of the equipment and the experience and licenses (licenses) possessed by x-ray technicians. The technique for performing radiological tests will be unified by using written instructions for radiological procedures and training of x-ray technicians. In addition, a film / screen system will be standardized to provide sufficient image resolution to assess erosion and narrowing of the joint gap. X-ray images taken as part of the study will be sent to the main interpretation center for quality check and assessment (blind method) by a radiologist trained and experienced in the assessment of rheumatoid arthritis according to Sharp's classification in Genant modification. A basic and additional interpreter will be designated. Interpreters will be accredited for testing based on the evaluation of a series of trial cases and establishing compliance between interpreters. Interpreters will not know the order of time points.

B. Hand and wrist MRI scan enhanced with gadolinium contrast

All participants with positive anti-CCP2 antibody titers and symptomatic clinical synovitis of the hands will have MRI of the hands / wrists with gadolinium contrast at the screening stage on day 169, month 12 and month 24. At first, select hands and wrists with more severe synovitis according to clinical evaluation and use them in all subsequent assessments. Baseline MRI (and follow-up MRI) tests should not be performed if the joints of the hands / wrists are asymptomatic or if clinical evaluation does not show synovitis.

MRI examinations will be standardized to provide sufficient image quality to assess the radiological progression of changes in RA. Radiological facilities and their employees will be qualified to participate in this study based on the technical capabilities of the equipment and the experience and licenses (licenses) held by technicians. The technique for performing radiological tests will be unified by using written instructions for radiological procedures and training of technicians. X-ray images taken as part of the examination will be sent to the main interpretation center for quality checking and assessment. Efficiency will only be assessed by the lead interpreter.

C. Assessing the number of joints

Treatment response parameters will be analyzed and discussed with the research team at a meeting of researchers or other forums as a method of normalizing the classification system by individual members of the research team. Training and instructions for assessing the number of joints will be discussed at the researchers' meeting or during the workshops.

The following employees will be able to assess the number of joints: Medical doctors, qualified osteopaths, medical assistants or qualified nurses. It is best to assess the number of joints before any other assessment or procedure.

Every effort should be made for each participant to be evaluated by the same person (s). The dates of visits should be set taking into account the availability of assessors. If the assessor cannot carry out the assessment, a competent person with similar experience can be appointed for this. The data of the person conducting the assessment should be entered in the source notes.

D. Clinical evaluation

Clinical assessment of treatment response should be carried out by the same person (s) at about the same time of day throughout the study. Clinical evaluation should be performed by persons other than those administering the study medication by intravenous infusion.

Clinical assessors will manually fill in the pages of the clinical observation card for general assessment and assessment of the number of joints. These pages will be the source documents in the study.

Participants will manually complete the HAQ and SF-36 questionnaires on the clinical observation card. These pages will be the source documents in the study.

The CRP concentration determined in the central laboratory will be used to calculate the total DAS score.

E. Pharmacodynamic assessment

The pharmacodynamic data collected in this study will include laboratory values ​​consisting of continuous variables. Biomarkers of immunomodulation or inflammation in rheumatoid arthritis, including IgM RF, CRP and anti-CCP2 antibodies will be determined.

The CRP value will be determined at each visit in the study drug treatment phase, double-blind, in the follow-up phase after double-blind treatment, and in the event of withdrawal / early withdrawal in both phases.

Rheumatoid factor (IgM RF) and biomarkers will be determined on day -21, day 1, day 169 and on months 12 and 24.

Anti-CCP2 antibodies will be screened at day 169, month 12 and month 24.

In addition, part of the serum collected at each visit will be stored and analysis of the following biomarkers will be performed: proinflammatory cytokines (IL-6, TNFo and IL-1 beta) and type 3 matrix metalloproteinase (MMP-3).

Assessment of test results

A. Physical fitness

Physical fitness will be assessed in the disability part of the Health Assessment Questionnaire (HAQ) (Fries, JF et al., "Measurement of Subject Outcome in Arthritis", Arthritis Rheum., 23: 137-145 (1980)). 20 questions enabling assessment of physical fitness in 8 categories: dressing, getting up, eating, walking, taking care of hygiene, stretching, grasping and frequently performed activities. The answers to the questions are evaluated on a 4-point scale: 0 = without any difficulties, 1 = with some difficulty, 2 = with considerable difficulty and 3 = impossible to do. A higher result means more disability. The failure rate will be calculated by adding the worst rating results for each category and dividing the sum by the number of categories assessed.

B. Quality of life depends on the state of health

The SF-36 questionnaire will be used to assess health-related quality of life (Birrell, FN et al., "How does the Short Form 36 Health Questionnaire (SF-36) in Rheumatoid Arthritis (RA) Relate to RA Outcome Measures and SF-36 Population Values? A Cross-Sectional Study ", Clin. Rheumatol., 19: 195-199 (2000); Keller, SD et al.," The SF-36 Arthritis-Specific Health Index (ASHI): n. Tests of validity in four clinical trials ", Med. Care, 37 (5 Suppi.): MS51-60 (May 1999); Ware, JE et al., "The SF36 Arthritis-Specific Health Index (ASHI): I. Development and cross-validation of scoring algorithms", Med. Care, 37 (5 Supl.): MS40-50 (May 1999); and Kosinski, M. et al., "The SF-36 Health Survey as a generic outcome measure in clinical trials of subjects with osteoarthritis and rheumatoid arthritis: relative validity of scales in relation to clinical measures of arthritis severity", Med. Care, 37 (5 Supl.): MS23-39 (May 1999)). The number of points obtained in individual subscales and two total scores will be calculated: (1) the summary result of the physical component summary (PCS) including physical fitness, performing roles in the physical aspect, pain and general health; (2) Mental Component Summary (MCS) result covering life energy, social functioning, emotional roles and mental health. The SF-36 questionnaire was recommended by the FDA as a proven tool for measuring health-related quality of life in people with RA (Guidance for Industry, Clinical Development Programs for Drugs, Devices, and Biological products for the Treatment of Rheumatoid Arthritis (RA) (Guidelines for Industry, Clinical development programs for drugs, medical devices and biological products intended for use in the treatment of rheumatoid arthritis), USA. Department of Health and Human Services, Food and Drug Evaluation and Research (February 1999).

Information on the medicinal product

Reconstitution for intravenous (IV) infusion will be performed by a pharmacist or competent center employee not otherwise associated with this study.

The medicine should always be reconstituted and diluted using silicon-free polypropylene syringes (Norm-Ject manufactured by Henke Sass Wolf in Germany.

NOTE: A separate syringe and needle MUST be used to reconstitute each vial.

The vials are sealed under vacuum. If no vial is found in any vial, it should be separated and must not be used. These vials should be stopped until agreed with the local Monitor of Test Drug.

NOTE: Vials should NOT be vented prior to reconstitution. To avoid foaming after adding SWFI, the vial should be gently inverted until the content is completely dissolved. After the lyophilized powder has completely dissolved, the vial should be vented with a needle to remove any foam contained in it.

The contents of each CTLA4Ig injection vial, 250 mg / vial, should be reconstituted in 10 ml SWFI (without bacteriostatic agent) to achieve a concentration of 25 mg / ml. To reduce the likelihood of foam formation, the SWFI stream should be directed onto the vial walls.

Each vial contains a sufficient allowance of CTLA4Ig taking into account losses during collection, so that 10 ml of reconstituted solution containing 250 mg of product can be withdrawn for parenteral administration. After reconstitution, the solution can be further diluted with 5% dextrose in water or 0.9% sodium chloride (saline).

The solution for continuous infusion should be filtered during administration through a built-in sterile, non-pyrogenic, low molecular weight protein binding filter with a pore diameter of

1.2 pm. The infusion should be given over a period of about 30 minutes. Any unused solution for infusion should not be stored for reuse.

There are no data available on the compatibility of CTLA4Ig with other intravenous substances. Whenever possible, CTLA4Ig should be administered via a separate infusion line and must not be mixed with other medicinal products. If other drugs are administered sequentially through the same infusion line, the line should be flushed appropriately and appropriately between administration of any other medicinal substances.

No incompatibilities with glass bottles or polyvinyl chloride bags or administration sets have been demonstrated.

Care should be taken to ensure sterility of the prepared solution as the medicinal product does NOT contain any antibacterial preservatives or bacteriostatic substances.

Vials for CTLA4Ig, 250 mg / vial, should be stored refrigerated (2-8 ° C) and protected from prolonged exposure to light. Intact vials are stable for at least a year under the above conditions. The diluted CTLA4Ig solution for injection should be used within 12 hours after reconstitution of the original vial.

Special guidelines for the stability of each dilution are as follows:

Reconstituted CTLA4Ig injection, 25 mg / ml, can be stored at 15-25 ° C in a lit room or refrigerated (2-8 ° C) for a maximum of 6 hours in the original vial.

CTLA4Ig, 10 mg / ml, diluted after reconstitution in physiological saline, can be stored in polyvinyl chloride (PVC) bags or in infusion bags made of materials other than PVC at 15-25 ° C in a lit room or under conditions refrigeration (2-8 ° C) for a maximum of 12 hours from the time of initial reconstruction.

Diluted CTLA4Ig injectable solutions are compatible with standard PVC infusion sets.

Results

Scheduled inter-period analysis was performed after all randomized participants reported for a visit scheduled at month 12, or for early resignation / withdrawal from the study, to examine the primary efficacy endpoint for RA. 51/56 randomized patients qualified for assessment (mean age: 45 years; mean duration of symptoms: 7 months [range 1-18 months]; mean CRP concentration: 1.1 mg / dl); 80% with oligoarthritis, 50% with erosions. RA developed before 1 year in 12/27 (44%) patients treated with CTLA4Ig and 16/24 (67%) patients receiving placebo (23% difference; 95% confidence interval -6 to +48). The time to discontinuation of treatment due to the development of RA is shown in Fig. 2.

Proposal

CTLA4Ig slows the progression rate of the disease to clear RA in patients with undifferentiated arthritis. The effect of CTLA4Ig causing disease modifications persisted for 6 months after treatment discontinuation.

EXAMPLE IV

The data from the period of 2 years of the study described in Example III are presented below. These data relate to the efficacy, safety and activity of pharmacodynamic markers (PD) in adults with undifferentiated arthritis who were treated with CTLA4Ig or double-blind placebo treatment for a maximum of 6 months. People with undifferentiated arthritis persisting after 6 months of double-blind treatment that did not meet the criteria for RA were observed without the drug being tested for RA development at a later time for a maximum of 18 additional months

Study population

A total of 184 people were screened, 57 of whom were recruited and randomized 1: 1 to the CTLA4Ig (N = 29) double-blind or placebo group (N = 28). The most common reason for screening failure and lack of randomization was failure to meet the eligibility criteria.

All but one of the randomized participants received at least 1 dose of study medication. One patient assigned to the CTLA4Ig treatment group was withdrawn from the study due to the presence of erosions detected after randomization but prior to administration of any study medication. Thus, at least 1 dose of study medication was randomized and given to a total of 56 patients - 28 in each treatment group.

(22 in the CTLA4Ig group, 17 in the placebo group) of the randomly assigned participants completed the 6-month study medication period and entered the 18-month follow-up period. 28 of them withdrew earlier before the end of the follow-up period (15 in the CTLA4Ig group, 13 in the placebo group). Sixteen (16) of these 28 participants (8 in each treatment group) dropped out of the study within 12 months. A total of 7 (25.0%) subjects in the CTLA4Ig group and 4 (14.3%) subjects in the placebo group completed the 24-month study.

Lack of efficacy was the most common reason for early resignation during the study drug treatment period, and for this reason more than twice as many people from the placebo group (n = 8, 28.5%) abandoned the study than from the CTLA4Ig group (n = 3, 10.7%). Lack of effectiveness was also the most common reason for withdrawal during the follow-up period. The percentage of participants entering the 18-month follow-up period who opted out of follow-up was also higher in the placebo group (12/17, 70.6%) than in the CTLA4Ig group (11/22, 50.0%). 14 (7 in the CTLA4Ig group, 7 in the placebo group) out of 23 subjects who discontinued due to lack of efficacy during the follow-up period, withdrew within 12 months.

Adverse events resulted in the earlier resignation of 1 person from the CTLA4Ig group during the study drug treatment and 1 person from the placebo group during the observation period.

The CTLA4Ig and placebo groups were balanced in terms of demographic characteristics in terms of age, body weight, gender distribution and race distribution. The average age of 56 randomized and treated participants was 44.8 years (range: 23 to 74 years). The majority of participants were Caucasian (85.7%) and female (71.4%). Although the majority of participants from both treatment groups were recruited at centers located in Europe (60.7% in the CTLA4Ig group, 75.0% in the placebo group), a higher percentage of people from the CTLA4Ig group were recruited in centers located in South America (17.9% compared with 7.1 % in the placebo group).

The CTLA4Ig group and the placebo group were essentially balanced in terms of baseline disease characteristics. Except for 5 people, all participants had at least 2 joints affected; 2 patients in each treatment group had only 1 joint affected, and one person in the CTLA4Ig group did not show synovitis at the screening stage or at the starting point (day 1). The latter person was recruited for the study because he met 3 criteria for the diagnosis of RA (ARA criteria from 1987), but was excluded from the primary efficacy analysis due to this significant deviation from the protocol. The remaining persons met 1-3 criteria for the diagnosis of RA, with the majority of participants (58.9%) meeting 3 criteria. For all participants, arthritis persisted for an average of 7.9 months and was slightly longer in the CTLA4Ig group (8.8 months) than in the placebo group (7.1 months). The mean CRP concentration was similar in the CTLA4Ig and placebo groups (11.2 mg / l and 10.7 mg / l, respectively), as was the proportion of subjects with baseline radiological erosion (53.5% and 57.1%, respectively).

General data from the medical history corresponded to an active inflammatory disease and were generally similar in 2 groups. Most people (&gt; 75%) in both treatment groups received anti-rheumatic drugs on Day 1 (78.6% in the CTLA4Ig group, 89.3% in the placebo group), with NSAIDs being the most commonly used anti-rheumatic drugs.

On day 1, fourteen (14) subjects received oral or injection corticosteroids, including 9 assigned to the CTLA4Ig treatment group and 5 assigned to the placebo group. Four subjects (14.3%) assigned to the CTLA4Ig treatment group and two subjects (7.1%) assigned to the placebo group on Day 1 were simultaneously using low dose oral corticosteroids (equivalent to a prednisone dose of &lt;10 mg daily).

Exposure Range

Despite the higher rate of early withdrawal from study drug treatment in the placebo group, most people in the CTLA4Ig and placebo group (89.3% and 75.0%, respectively) completed 6 months of treatment and received 8 scheduled intravenous infusions of the study drug (75.0% and 71.4%, respectively) ). One (1) person in the CTLA4Ig group (3.6%) and 5 people in the placebo group (17.9%) were given at most 3 intravenous infusions of the study drug. For no subject, study medication treatment lasted longer than 6 months and no more than 8 intravenous infusions of study medication were administered.

Most participants included in the analysis of all subjects did not miss the scheduled CTLA4Ig infusion (85.7%) or placebo (92.9%) during the study drug treatment period, and no participant in any of the treatment groups missed more than 1 scheduled intravenous infusion.

Low dose corticosteroid consumption during the study drug treatment period in the group selected for ITT (as planned) was low and similar in the CTLA4Ig group and in the placebo group, both in terms of the percentage of people using these drugs (n = 5, 17.9% in each group) as well as the average corticosteroid dose at specific time points. The same was true during the follow-up period when low-dose corticosteroids were taken by 5 out of 22 subjects entering this period in the CTLA4Ig group and 7 out of 17 subjects in the placebo group.

The number of high-dose corticosteroid treatments during the study drug treatment period in the group selected for ITT (according to planned treatment) was also similar in 2 groups: 3 treatments in the CTLA4Ig group (in 2 people) and 4 treatments in the placebo group (in 4 people). During the study drug treatment period, high-dose corticosteroids were taken by intramuscular (IM) or intra-articular (IA) injection; no person in any of the treatment groups received oral corticosteroids at a dose&gt; 10 mg per day during the study drug treatment period.

In the 18-month follow-up period, the number of high-dose corticosteroid treatments was 9 in the CTLA4Ig group and 6 in the placebo group. Two people in each treatment group received high dose corticosteroids orally at doses&gt; 10 mg daily. No person in any of the treatment groups received more than 2 high-dose corticosteroid treatments during the study medication period or follow-up.

During the follow-up period, all subjects in the CTLA4Ig group and most subjects (96.4%) in the placebo group received at least one concomitant at the same time. At the same time, the most commonly used drugs were painkillers, acetaminophen and diclofenac, each of which was taken by 7 (25.0%) people in the CTLA4Ig group and 9 (32.1%) people in the placebo group.

Performance results

The results of this study indicate that 6 months of CTLA4Ig treatment inhibits the rate of progression of undifferentiated arthritis to RA. Within 12 months, RA developed in 12 out of 26 (46.2%) patients with undifferentiated arthritis in the CTLA4Ig group and 16 out of 24 (66.7%) patients with undifferentiated arthritis in the placebo group. A 95% CI surrounding a 20.5% difference between treatment groups in favor of the CTLA4Ig treatment group for the primary efficacy endpoint was (-47.4, 7.8). Also the percentage of patients with undifferentiated arthritis who developed RA within 24 months was lower in the CTLA4Ig group (17/23, 73.9%) than in the placebo group (21/24, 87.5%), although the difference between the treatment groups (-13.6%; 95% CI: -37.6, 10.8) was less pronounced than in month 12.

It seems that CTLA4Ig's inhibition rate of disease progression to RA is more clearly seen in the subgroup of individuals with radiological erosion at the baseline than in the subgroup of those without erosion at the baseline.

The percentage of patients with undifferentiated arthritis who developed RA according to the 1987 ARA criteria within 1 year of starting study drug was lower in the CTLA4Ig group (12/26, 46.2%) than in the placebo group ( 16/24, 66.7%) (difference -20.5%; 95% CI: -47.4, 7.8).

The results of the predefined sensitivity analysis of the primary efficacy endpoint assessing each of the 55 randomized and treated trial subjects included in the ITT population identified for efficacy analysis were similar and supported CTLA4Ig. In the sensitivity analysis, the percentage of patients with undifferentiated arthritis who developed RA within 1 year of starting study drug was 42.9% in the CTLA4Ig (12/28 people) and 59.3% (16/27 people) group placebo group (difference between treatment groups -16.4%; 95% CI: -42.3, 11.0).

Fewer people with undifferentiated arthritis randomly assigned to the CTLA4Ig treatment group for 6 months (17/23, 73.9%) before 24 months had developed RA as defined by the 1987 ARA criteria compared to those assigned to the placebo group ( 21/24, 87.5%). The difference between treatment groups regarding RA development over a 24-month period was -13.6% (95% CI: -37.6, 10.8). Although after six months the study drugs were no longer administered, study participants or researchers did not know the treatment regimen used in the study until the end of the 18-month follow-up period (month 24).

On the basis of X-ray images of the feet and hands, the study showed a lower progression of structural changes in the CTLA4Ig group than in the placebo group after 12 months, which was indicated by a smaller change in the average results of assessment of erosion and joint space narrowing (JSN). In addition, in a subgroup of 21 people MRI examinations of the wrists and hands with gadolinium contrast showed minimal features of disease progression in the CTLA4Ig group at the end of the study drug treatment period, while the average change in the results of the assessment of erosion, edema and synovitis demonstrated in MRI examinations in the period from baseline to 6 months in the placebo group indicated worsening of the disease. The difference in MRI scores between treatment groups persisted for 6 months after the end of the study treatment

A prerequisite for enrollment in the study was the presence of synovitis at screening stage or on day 1. The percentage of people with undifferentiated arthritis who had symptomatic clinical synovitis after 6 months was lower among CTLA4Ig-treated patients (4/5 , 80%) compared with patients receiving placebo (12/12, 100%). The difference in the percentage of patients with persistent symptomatic clinical synovitis between the treatment groups was -20.0% (95% CI: -71.5, 15.21). After 12 months, the number of patients with persistent clinical synovitis was 10 out of 11 in the CTLA4Ig group and 7 out of 7 in the placebo group.

Hands and wrists were assessed in MRI examinations with gadolinium contrast in a total of 11 people from the CTLA4Ig group and 10 people from the placebo group; according to the protocol plan, MRI was performed only on people recruited in research centers in Europe. The mean change in the results of bone erosion and synovitis assessment of the hands and wrists based on MRI at the end of the study drug treatment period (month 6) relative to baseline indicated minimal disease progression in the CTLA4Ig treated group (mean change of 0.45 and 0.27, respectively) ), while in the placebo group the changes were larger and indicated worsening of the disease (mean change of 1.20 and 1.60, respectively). The mean change in edema scores from MRI after 6 months from baseline indicated improvement in the CTLA4Ig group (mean change -1.64) and worse in the placebo group (mean change 1.40).

A similar pattern of MRI findings was recorded after 12 months, with MRI performed at baseline and after 1 year of the study in 9 and 6 people in the CTLA4Ig and placebo groups, respectively. After 12 months, a slight change in bone erosion, edema and synovitis scores was observed compared to baseline in the CTLA4Ig group (mean change of 0.0, 0.22 and 0.22, respectively), while in the placebo group continuous disease progression was clearly visible (mean change 5.00, 6.67 and 2.33 respectively).

Only in 7 people (5 in the CTLA4Ig group, 2 in the placebo group) MRI examinations were performed at baseline and in month 24. In this small subgroup of people from Europe who did not show progression to RA and who therefore not withdrawn from the study, the mean baseline MRI score was low (typically &lt;1.0), and a small change was noted after 24 months.

At the end of the study drug treatment period (month 6), patients with undifferentiated arthritis in the CTLA4Ig group were found to have reduced disease activity (DAS 28 score [CRP]) and improved physical performance (HAQ-DI score) and dependent quality of life from the state of health (results of the PCS and MCS assessment in the SF-36 questionnaire) in relation to the baseline, while in the placebo group the average results of the evaluation of these efficacy variables remained unchanged. The difference between treatment groups was smaller after 12 and 24 months during the treatment-free period.

At the end of the study drug treatment period, disease activity on the DAS 28 (CRP) scale was lower compared to baseline in the CTLA4Ig group (mean change -1.13) while in the placebo group it remained unchanged (mean change 0.01). After 6 months, clinically significant improvement (reduction of the DAS 28 score by at least 1.2 compared to baseline) was observed in 8 out of 20 (40%) subjects from the CTLA4Ig group for whom baseline and month 6 results were available , and in 4 out of 20 people (20%) from the placebo group who had baseline and 6-month results available. Accordingly, higher rates of low disease activity (DAS 28 &lt;3.2) or disease remission (DAS 28 &lt;2.6) were observed after 6 months in the CTLA4Ig group (81.0% and 71.4%, respectively) compared to the placebo group (45.0%, respectively) and 35.0%).

The assessment of results obtained on the DAS 28 (CRP) scale after 12 and 24 months showed greater improvement in disease activity in the CTLA4Ig group than in the placebo group, but the difference between the treatment groups was smaller during the observation period without treatment. Among the 18 subjects in the CTLA4Ig group who had DAS 28 scores at baseline and after 12 months, the mean change in score was -0.50 at month 12, and disease activity was low in about two-thirds (68.4%) people. Of the 13 patients in the placebo group who had available DAS 28 scores at both time points, there was virtually no change in disease activity after 12 months compared with baseline (mean change -0.05) and disease activity was low 53.9% of people. Of the 11 patients who had a DAS 28 assessment after 24 months available, disease remission was found in 4 out of 7 patients treated with CTLA4Ig and in 2 out of 4 patients receiving placebo.

During the study drug treatment period, the greater improvement in disease activity demonstrated by the greater change from baseline and the greater proportion of patients who showed improvement was clearly visible in the CTLA4Ig group compared to the placebo group on day 29 of the study.

The percentage of subjects with clinically significant improvement in physical function (defined as a reduction of&gt; 0.3 points from baseline in the HAQ-DI questionnaire) was higher in the CTLA4Ig group than in the placebo group after 6, 12 and 24 months. At the end of the study drug treatment period, clinically significant improvements in the HAQ-DI questionnaire results were reported in 61.5% of randomized and treated subjects from the CTLA4Ig group and 24.0% from the placebo group (difference between 37.5% treated groups [95% CI: 9.9, 61.4] ). The percentage of people from the CTLA4Ig group who showed clinically significant improvement after 6 and 12 months of follow-up without treatment (36.0% in month 12 and 14.3% in month 24) was lower than after 6 months of treatment, but still had a higher numerical value than the percentage of people in the placebo group with clinically significant improvement after 12 and 24 months (12.0% and 4.2%, respectively).

A greater mean improvement in the cumulative somatic and psychological component scores in the SF-36 questionnaire compared to baseline was observed after 6, 12 and 24 months in the CTLA4Ig group compared to the placebo group. The mean improvement observed at the end of the study drug treatment period (month 6) relative to baseline was 10.23 for PCS and 2.54 for MCS in the CTLA4Ig group. In the placebo group, the mean change in cumulative somatic component (PCS) and cumulative mental component (MCS) scores after 6 months was small (1.95 and -0.30, respectively).

Among subjects from the CTLA4Ig group who were still in the study during the non-treatment follow-up period, the average improvement observed after 12 and 24 months was 3.83 and 2.46 for PCS, respectively, and 2.50 and 3.75 for MCS, respectively. In the placebo group, PCS and MCS values ​​deteriorated during the 18-month follow-up without treatment, as evidenced by the mean adverse change in these endpoints relative to baseline recorded after 12 and 24 months.

Baseline radiological results indicated minimal bone erosion or joint stenosis in randomized individuals, which met the criteria for qualifying for the study in patients without RA at the time of entry. In the 6-month treatment period with study drug, there was a slight change in the results of radiological assessment of erosion, narrowing of the joint gap or the total score compared to baseline, as evidenced by the mean change in results for all 3 endpoints after 6 months of &lt;0.13 in the CTLA4Ig group and &lt;0.47 in the placebo group. After 12 months, features indicating less progression of structural changes in the CTLA4Ig group compared to the placebo group were demonstrated when the mean change in total score was 0.02 in the CTLA4Ig group and 1.11 in the placebo group.

An analysis of the assigned data for radiological assessment of erosion and narrowing of the joint space showed small average changes, indicating a smaller progression of structural changes 6 and 12 months after the end of the study drug treatment period (i.e. at months 12 and 24) in the CTLA4Ig group (mean change total score of 0.29 and 0.02) compared to the placebo group (1.21 and 2.13).

In the CTLA4Ig group, the average percentage improvement in the individual components of the ACR variable was noted during the study drug treatment period. After 6 months, the average percentage improvement in the results of the assessment of tender and swollen joints and the severity of pain assessed by patients was 64.91%, 57.34% and 70.03%, respectively. For comparison, patients from the placebo group showed deterioration of individual components of the ACR variable during the study drug treatment period, with the mean percentage change in the results of the assessment of tenderness and swollen joints and the severity of pain assessed by patients was -63.3%, -10.5%, respectively and -83.4%.

The mean percentage improvement in major ACR variable elements observed in the CTLA4Ig group during the study drug treatment period did not persist after discontinuation of study treatment in subjects who were still enrolled in the study.

The primary and associated secondary efficacy endpoints, as well as demographics and baseline disease characteristics, were assessed separately in subgroups based on the presence or absence of erosion at study entry (i.e., randomization layers).

Demographic data were similar in the subgroup of 31 people with radiological features of erosion at the starting point and in the subgroup of 25 people without radiological features of erosion at the starting point. Baseline disease characteristics were also similar in these 2 subgroups, except for the mean and median erosion assessment results in radiology studies, which were higher in the subgroup with erosion at baseline than in the subgroup with no erosion at baseline.

In the subgroup of people with radiological features of erosion at the starting point, the percentage of patients with undifferentiated arthritis who developed RA according to the 1987 ARA criteria within 1 year of starting study drug was lower in the CTLA4Ig group (4 / 13, 30.8%) than in the placebo group (9/14, 64.3%). In the subgroup of people without radiological features of erosion, 61.5% of patients in the CTLA4Ig (8/13) group and 70.0% of patients in the placebo group (7/10) experienced RA within 12 months.

The percentage of people with undifferentiated arthritis who developed RA before 24 months was lower in the CTLA4Ig group than in the placebo group only among participants with baseline radiological erosion (50.0% [6/12 people] in the group treated with CTLA4Ig; 85.7% [12/14 people] in the placebo group). In the subgroup of people without radiological evidence of erosion at baseline, 100% of patients in the CTLA4Ig group (11/11) and 90% of patients in the placebo group (9/10) developed RA within 24 months. Security

Intravenous CTLA4Ig administered once a month based on 10 mg / kg body weight for a maximum of 6 months was generally well tolerated in the treatment of adults with undifferentiated arthritis. No deaths were reported during the study. During the study drug treatment period, serious adverse events were reported in 1 person from the CTLA4Ig (basal cell carcinoma) group and 1 person in the placebo group (sciatica). In both cases, the serious adverse events were considered by the investigator to be unrelated to the treatment used in the study. One (1) person from the CTLA4Ig and placebo group discontinued treatment during the study medication period due to adverse events. Acute infusion related adverse events (reported within 1 hour after the start of study drug administration by intravenous infusion) were reported in 1 person in each treatment group; the acute adverse event associated with intravenous infusion found in the CTLA4Ig (dyspnoea) group occurred during the first infusion and necessitated the discontinuation of treatment. Infections and infestations were reported during the study drug treatment period in a similar percentage in the CTLA4Ig group (35.7%) and in the placebo group (39.3%). None of the infections reported in the CTLA4Ig group were serious. During the study drug treatment period, adverse events were reported in a similar percentage in the CTLA4Ig group (64.3%) and in the placebo group (71.4%). All adverse events occurring in the CTLA4Ig group were mild to moderate in severity. Based on the evaluation of laboratory data and vital signs, no safety concerns were identified.

Overall, adverse events were reported in 18 (64.3%) subjects treated with CTLA4Ig and in 20 (71.4%) subjects receiving placebo during the study medication treatment period or 56 days after the last study medication administration by intravenous infusion. The incidence of adverse events was generally similar in the CTLA4Ig and placebo groups. Adverse events most commonly reported by system organ class (SOC) were infections and infestations (35.7% in the CTLA4Ig group; 39.3% in the placebo group), gastrointestinal disorders (21.4% in the CTLA4Ig group; 25.0% in the placebo group ) and thoracic and mediastinal respiratory disorders (17.9% in the CTLA4Ig group; 21.4% in the placebo group). Adverse events reported by at least 10% of people in each treatment group during the study drug treatment period were: diarrhea (14.3% in the CTLA4Ig group; 10.7% in the placebo group), headache (10.7% in the CTLA4Ig group; 7.1% in the placebo group), nasopharyngitis (10.7% in the CTLA4Ig group; 7.1% in the placebo group), urinary tract infections (7.1% in the CTLA4Ig group; 10.7% in the placebo group), sore throat and larynx (3.6% in the CTLA4Ig treated group; 14.3% in the placebo group) and gastroenteritis (0% in the CTLA4Ig group; 10.7% in the placebo group). No autoimmune disorders were reported in any of the treatment groups during the study drug treatment period.

The percentage of people in the placebo group who experienced serious adverse events was 10.7% (one serious adverse event in the form of sciatica, viral infection and throat edema). No subject from the CTLA4Ig treatment group experienced an adverse event considered serious or very severe by the investigator during the study drug treatment period.

The incidence of associated adverse events was 50% (n = 14) in the CTLA4Ig group and 35.7% (n = 10) in the placebo group during the study treatment period. The most common related adverse event was headache reported in 3 people (10.7%) in the CTLA4Ig group and 2 people (7.1%) in the placebo group. Eczema was the only other related adverse event reported by&gt; 1 CTLA4Ig treated person; eczema was reported in 2 people (7.1%).

Data from clinical laboratory tests were generally negligible and no safety hazards were identified in this group of people with undifferentiated arthritis.

During the study drug treatment period, the incidence of haematological and biochemical blood parameters meeting the sponsor's criteria for significantly abnormal changes was small and similar in the CTLA4Ig and placebo groups. Significantly abnormal changes in each hematological and biochemical parameter were found in at most 2 people in each treatment group.

Slight changes in hematological and biochemical parameters compared to baseline (from day 1) were noted during the study drug treatment and follow-up period, with significant fluctuations and a lack of a consistent pattern in 2 study groups.

In addition, neutrophils, ALT and AST levels remained stable after the CTLA4Ig treatment throughout the study period. No person in any of the treatment groups had ALT or AST values ​​greater than 3 times the upper limit of normal (ULN) at any time point in the measurement, or a neutrophil count of &lt;0.5 x 109 / L or&gt; 15 x 109 / L.

Two patients had abnormal laboratory findings considered adverse events during the study drug treatment period. Elevations of liver enzymes in the CTLA4Ig treatment group on day 141 were reported as an adverse event. This event persisted for 120 days before it resolved. None of the liver enzymes values ​​met these criteria for significantly abnormal changes. One person in the placebo group reported thrombocytopenia as an adverse event, which was the reason for discontinuation of the study treatment. These people had a platelet count of 284 x 10^-9 c / L at baseline and decreased gradually during the study to 86 x 10^-9 c / L on 169 and 22 x 10^-9 c / L on day 319 (this was the last value recorded).

The mean values ​​of all vital signs remained unchanged throughout the study drug treatment period in the CTLA4Ig and placebo groups.

In most patients from both treatment groups, the ECG was correct at the starting point and at the end of the study drug treatment period. The percentage of people whose ECG was correct at the starting point but abnormal on 169 (or at the time of early resignation) was similar in the CTLA4Ig group (1/24, 4.2%) and in the placebo group (2/23, 8.7%) .

Pharmacodynamic assessment results

CTLA4Ig treatment was associated with a mean reduction in IL-6 (-4.49 pg / ml), TNF-α (-1.70 pg / ml), IL-1p (-0.12 pg / ml) and MMP-3 (-2.29 ng / ml ) after 6 months compared to baseline. For comparison, in the placebo group after 6 months there was a slight increase in mean values ​​or no changes in IL-6 (1.08 pg / ml), TFN-α (0.07 pg / ml), IL-1 β (-0.09 pg / ml) and MMP-3 (14.34 ng / ml) from baseline.

After 6 months without any study medication in the CTLA4Ig group, the reduction in mean values ​​of most of these cytokines was still greater than in the placebo group. After 12 months, the mean change from baseline in the CTLA4Ig and placebo groups was -0.14 and 6.37 pg / ml for IL-6, respectively; -0.50 and -0.10 pg / ml for TFN-α, respectively; and -2.55 and 25.34 ng / ml for MMP-3, respectively. There were no differences between the 2 groups in the mean change in IL-1e concentration after 12 months from baseline (-0.09 and -0.24 pg / ml); however, the number of subjects for whom baseline data and 12 months were available was small (n = 7 in the placebo group and n = 13 in the CTLA4Ig group).

Only 5 subjects from the CTLA4Ig group and 3 subjects from the placebo group had data on baseline and 24-month cytokine levels available. The mean decrease in IL-6 and TFN-α was still evident in this small subset of CTLA4Ig-treated patients who did not develop RA (-3.44 pg / ml and -0.82 pg / ml, respectively).

According to the eligibility criteria for participation in the study, on day 1 all subjects tested positive for anti-CCP2 antibodies. In the placebo group, all subjects whose data were suitable for evaluation had positive antibody titer at months 6, 12 and 24. For comparison, the percentage of people with undifferentiated arthritis and anti-CCP2 positive antibody titres decreased in the CTLA4Ig group to 90.9% (20/22 people) after 6 months, to 86.7% (13/15 people) after 12 months and to 83.3 % (5/6 people) after 24 months.

These data are consistent with the results of anti-CCP2 antibody titers in blood serum. In the CTLA4Ig group, mean decreases in baseline anti-CCP2 antibody titres were observed after 6 months (-94.5 U / L) and 12 months (-6.46 U / L). In the placebo group, anti-CCP2 antibody titer was elevated from baseline after 6 months (mean change 16.32 U / L) and 12 months (149.5 U / L). Only 6 subjects from the CTLA4Ig group and 3 subjects from the placebo group had data on anti-CCP2 antibody titers available at baseline and after 24 months. One (1) of these CTLA4Ig-treated individuals had a negative anti-CCP2 antibody titer.

At baseline (day 1), the RF test was positive in 85.7% of the CTLA4Ig group and 71.4% of the placebo group. In the CTLA4Ig group, this percentage decreased to 59.1% at the end of the study drug treatment period (month 6). After 6 and 18 months of the non-treatment follow-up period, the number of subjects still in the study who had a positive RF test result was 11 out of 15 (73.3%) after 12 months and 3 out of 6 (50.0%) after 24 months. For comparison, the number of people in the placebo group who had a positive RF test result increased during the 24 months of the study (14 from 20 [70.0%] people after 6 months to 3 from 3 [100%] people after 24 months) ).

No subjects from the CTLA4Ig group were seroconverted to a positive RF titre after 6, 12 and 24 months. In the placebo group, 2 out of 7 subjects with a negative baseline RF titre were positive after 6 months, and 1 out of 2 subjects with a negative baseline RF titre was positive after 12 months.

At baseline (day 1), the percentage of people who were found to have the HLA-DRB10401, HLADRB10404 and HLA-DRB10101 "common epitope" alleles was similar in the CTLA4Ig group (46.4% [13/28]) and in the placebo group (39.3%) [11/28]).

immunogenicity

Immunogenicity data were available from a total of 23 out of 28 randomized and treated subjects in the CTLA4Ig group. No participant was found to have seropositive anti-CTLA4Ig antibody status (especially for the IgG portion of the molecule) at any time during the 24-month study period.

In four (4) of 23 people (17.4%), anti-CTLA4-T (Tip) antibody status was positive. In these 4 people, anti-CTLA4-T antibodies were not detected 3 months after the last dose (sample taken at month 9), but were detected 6 months after the last dose (sample taken at month 12). Antibody titers in these subjects were low (range: 65 to 98; sensitivity of analytical method 25). 2 of these 4 samples contained neutralizing antibodies and 2 did not.

Seroconversion to positive antibody titers does not appear to affect safety. No adverse event was reported in participants or close to the time of seroconversion to positive titers in study participants. One person reported tendon sheath inflammation on day 384, approximately 6 months after the last dose of study medication (CTLA4Ig) and near the time of seroconversion to positive titers. This adverse event was considered moderately severe, and its relationship to treatment used in the study was considered unlikely. According to reports, the event lasted for a long time.

General conclusions

The results of the primary evaluation and related major efficacy secondary endpoints obtained in this study indicate that CTLA4Ig administered intravenously as a single agent for six months at a 10-mg / kg dose based on body weight inhibits disease progression to RA in people with undifferentiated arthritis and disease modifying CTLA4Ig effects were observed 6 and 18 months after drug discontinuation. In people with undifferentiated arthritis, after 6 months of treatment, greater improvement in physical fitness, activity of the physician-evaluated disease and quality of life dependent on health status was observed in the CTLA4Ig group than in the placebo group. Radiological assessment of hands and feet indicated minimal disease progression during the study drug treatment period in subjects receiving CTLA4Ig; the progression of joint damage was also less than 6 months after stopping treatment in the CTLA4Ig group compared to the placebo group. The evaluation of the wrists and hands in MRI was more limited, but showed a similar trend. Compared with placebo, CTLA4Ig treatment was associated with a greater decrease in anti-CCP2 antibody titers in the blood serum and a reduction in the percentage of individuals with positive anti-CCP2 and RF titers. CTLA4Ig administered for 6 months once a month intravenously at a dose based on 10 mg / kg body weight was well tolerated by people with undifferentiated arthritis. The immunogenicity index (positive drug-induced seroconversion) was low and the presence of antibodies to CTLA4Ig or CTLA4-T did not correlate in this exploratory study with any clinical safety data.

LIST OF SEQUENCES &lt;110&gt; Bristol-Myers Squibb Company Vratsanos, George &lt;120&gt; METHOD OF PREVENTING THE DEVELOPMENT OF RHEUMATOID ARTHRITIS IN SUBJECTS WITH

UNDIFFERENTIATED ARTHRITIS &lt;130&gt; 11268 NP &lt;150&gt; 61 / 050,336 &lt;151&gt; 2008-05-05 &lt;160&gt; 2 &lt;170&gt; Patent In version 3.5 &lt;210&gt; 1 &lt;211&gt; 1223 &lt;212&gt; DNA &lt;213&gt; Homo sapiens &lt;220 &gt;

&lt;221&gt; CDS &lt;222&gt; (11) .. (1159) &lt;400&gt; 1 agcttcacca atg ggt gta ctg ctc aca cag agg acg ctg ctc agt ctg 49

Met Gly Val Leu Leu Thr Gin Arg Thr Leu Leu Ser Leu 15 10 gtc ctt gca ctc ctg ttt cca agc atg gcg agc atg gca atg cac gtg 97

Val Leu Ala Leu Leu Phe Pro Ser Met Ala Ser Met Ala Met His Val

twenty 25 gcc cag cct gct gtg gta ctg gcc agc agc ega ggc atc gcc agc ttt 145

Ala Gin Pro Ala Val Val Leu Ala Cheese Cheese Arg Gly Ile Ala Cheese Phe

35 40 45 gtg tgt gag tat gca tct cca ggc aaa gcc act gag gtc cgg gtg aca 193

Val Cys Glu Tyr Ala Ser Pro Gly Lys Ala Thr Glu Val Arg Val Thr

55 60 gtg ctt cgg cag gct gac agc cag gtg act gaa gtc tgt gcg gca acc 241

Val Leu Arg Gin Ala Asp Ser Gin Val Thr Glu Val Cys Ala Ala Thr '70 75 tac atg atg ggg aat gag ttg acc ttc eta gat gat tcc atc tgc acg 289

Tyr Met Met Gly Asn Glu Leu Thr Phe Leu Asp Asp Ser Ile Cys Thr

85 90 ggc acc tcc agt gga aat caa gtg aac ctc act atc caa gga ctg agg 337

Gly Thr Ser Ser Gly Asn Gin Val Asn Leu Thr Ile Gin Gly Leu Arg

100 105 gcc atg gac acg gga ctc tac atc tgc aag gtg gag ctc atg tac cca 385

Ala Met Asp Thr Gly Leu Tyr Ile Cys Lys Val Glu Leu Met Tyr Pro

110 115 120. 125 ccg cca tac tac ctg ggc ata ggc aac gga acc cag att tat gta att 433

481

Pro Pro Tyr Tyr Leu Gly Ile Gly Asn Gly Thr Gin Ile Tyr Val Ile 130 135 140 gat cca gaa ccg tgc cca gat tct gat cag gag ccc aaa tct tct gac Asp Pro Glu Pro Cys Pro Asp Ser Asp Gin Glu Pro Lys Ser Ser Asp

145 150 155 aaa act cac aca tcc cca ccg tcc cca gca cct gaa ctc ctg ggg gga Lys Thr His Thr Ser Pro Pro Ser Pro Ala Pro Glu Leu Leu Gly Gly

160 165 170

529 tcg tca gtc ttc ctc tcccc cca aaa ccc aag gac acc ctc atg atc Ser Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile

175 180 185

577 tcc cgg acc cct gag gtc aca tgc gtg gtg gtg gac gtg agc cac gaa Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 190 195 200 205

625 gac cct gag gtc aag ttc aac tgg tac gtg gac ggc gtg gag gtg cat Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His

210 215 220

673 aat gcc aag aca aag ccg cgg gag gag cag tac aac agc acg tac cgt Asn Ala Lys Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg

225 230 235

721 gtg gtc agc gtc ctc acc gtc ctg cac cag gac tgg ctg aat ggc aag Val Val Ser Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys

240 . 245 250

769 gag tac aag tgc aag gtc tcc aac aaa gcc ctc cca gcc ccc atc gag Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu

255 260 265

817

<td>aaa</td><td>acc</td><td>atc</td><td>tcc</td><td>aaa</td><td>gcc</td><td>aaa</td><td>ggg</td><td>cag</td><td>ccc</td><td>ega</td><td>gaa</td><td>ca</td><td>cag</td><td>gtg</td><td>trays</td><td> 865</td>

<td>lys</td><td>Thr</td><td>How much</td><td>Cheese</td><td>lys</td><td>ala</td><td>lys</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Arg</td><td>Glu</td><td>Pro</td><td>Gin</td><td>val</td><td>Tyr</td><td></td>

<td> 270</td><td></td><td></td><td></td><td></td><td> 275</td><td></td><td></td><td></td><td></td><td> 280</td><td></td><td></td><td></td><td></td><td> 285</td><td></td>

<td>acc</td><td>cOT</td><td>ccc</td><td>ca</td><td>tcc</td><td>CGG</td><td>gat</td><td>gag</td><td>cOT</td><td>acc</td><td>aag</td><td>aac</td><td>cag</td><td>gtc</td><td>agc</td><td>cOT</td><td> 913</td>

<td>Thr</td><td>Leu</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Arg</td><td>Asp</td><td>Glu</td><td>Leu</td><td>Thr</td><td>lys</td><td>own</td><td>Gin</td><td>val</td><td>Cheese</td><td>Leu</td><td></td>

<td></td><td></td><td></td><td></td><td> 290</td><td></td><td></td><td></td><td></td><td> 295</td><td></td><td></td><td></td><td></td><td> 300</td><td></td><td></td>

961 acc tgc ctg gtc aaa ggc tcc tat ccc agc gac atc gcc gtg gag tgg Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp

305 310 315

<td>gag</td><td>agc</td><td>aat</td><td>ggg</td><td>cag</td><td>ccg</td><td>gag</td><td>aac</td><td>aac</td><td>trays</td><td>aag</td><td>acc</td><td>ACG</td><td>Cct</td><td>ccc</td><td>gtg</td><td> 1009</td>

<td>Glu</td><td>Cheese</td><td>own</td><td>Gly</td><td>Gin</td><td>Pro</td><td>Glu</td><td>own</td><td>own</td><td>Tyr</td><td>lys</td><td>Thr</td><td>Thr</td><td>Pro</td><td>Pro</td><td>val</td><td></td>

<td></td><td></td><td> 320</td><td></td><td></td><td></td><td></td><td> 325</td><td></td><td></td><td></td><td></td><td> 330</td><td></td><td></td><td></td><td></td>

<td>cOT</td><td>gac</td><td>tcc</td><td>gac</td><td>GGC</td><td>tcc</td><td>ttc</td><td>ttc</td><td>ctc</td><td>trays</td><td>agc</td><td>aag</td><td>ctc</td><td>acc</td><td>gtg</td><td>gac</td><td> 1057</td>

<td>Leu</td><td>Asp</td><td>Cheese</td><td>Asp</td><td>Gly</td><td>Cheese</td><td>phe</td><td>phe</td><td>Leu</td><td>Tyr</td><td>Cheese</td><td>lys</td><td>Leu</td><td>Thr</td><td>val</td><td>Asp</td><td></td>

<td></td><td> 335</td><td></td><td></td><td></td><td></td><td> 340</td><td></td><td></td><td></td><td></td><td> 345</td><td></td><td></td><td></td><td></td><td></td>

<td>aag</td><td>agc</td><td>agg</td><td>TGG</td><td>cag</td><td>cag</td><td>ggg</td><td>aac</td><td>gtc</td><td>ttc</td><td>tCA</td><td>TGC</td><td>tcc</td><td>gtg</td><td>atg</td><td>cat</td><td> 1105</td>

<td>lys</td><td>Cheese</td><td>Arg</td><td>Trp</td><td>Gin</td><td>Gin</td><td>Gly</td><td>own</td><td>val</td><td>phe</td><td>Cheese</td><td>Cys</td><td>Cheese</td><td>val</td><td>Underworld</td><td>His</td><td></td>

<td> 350</td><td></td><td></td><td></td><td></td><td> 355</td><td></td><td></td><td></td><td></td><td> 360</td><td></td><td></td><td></td><td></td><td> 365</td><td></td>

<td>gag</td><td>GCT</td><td>cOT</td><td>cac</td><td>aac</td><td>cac</td><td>trays</td><td>ACG</td><td>cag</td><td>aag</td><td>agc</td><td>ctc</td><td>tcc</td><td>cOT</td><td>tct</td><td>ccg</td><td> 1153</td>

<td>Glu</td><td>ala</td><td>Leu</td><td>His</td><td>own</td><td>His</td><td>Tyr</td><td>Thr</td><td>Gin</td><td>lys</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Leu</td><td>Cheese</td><td>Pro</td><td></td>

370

375

380 ggt aaa tgagtgcgac ggccggcaag ccccgctccc cgggctctcg cggtcgcacg Gly Lys

1209 aggatgcttc tag &lt;210&gt; 2 &lt;211&gt; 383 &lt;212&gt; PRT

1223 &lt;213&gt; Homo sapiens

<td colspan="6"> &lt;400&gt; 2</td><td rowspan="2">Gin</td><td rowspan="2">Arg</td><td rowspan="2">Thr</td><td rowspan="2">Leu 10</td><td rowspan="2">Leu</td><td rowspan="2">Cheese</td><td rowspan="2">Leu</td><td rowspan="2">val</td><td rowspan="2">Leu 15</td><td rowspan="2">ala</td>

<td>Underworld 1</td><td>Gly</td><td>val</td><td>Leu</td><td>Leu 5</td><td>Thr</td>

<td>Leu</td><td>Leu</td><td>phe</td><td>Pro</td><td>Cheese</td><td>Underworld</td><td>ala</td><td>Cheese</td><td>Underworld</td><td>ala</td><td>Underworld</td><td>His</td><td>val</td><td>ala</td><td>Gin</td><td>Pro</td>

<td></td><td></td><td></td><td> 20</td><td></td><td></td><td></td><td></td><td> 25</td><td></td><td></td><td></td><td></td><td> 30</td><td></td><td></td>

<td>ala</td><td>val</td><td>val</td><td>Leu</td><td>ala</td><td>Cheese</td><td>Cheese</td><td>Arg</td><td>Gly</td><td>How much</td><td>ala</td><td>Cheese</td><td>phe</td><td>val</td><td>Cys</td><td>Glu</td>

<td></td><td></td><td> 35</td><td></td><td></td><td></td><td></td><td> 40</td><td></td><td></td><td></td><td></td><td> 45</td><td></td><td></td><td></td>

<td>Tyr</td><td>ala</td><td>Cheese</td><td>Pro</td><td>Gly</td><td>lys</td><td>ala</td><td>Thr</td><td>Glu</td><td>val</td><td>Arg</td><td>val</td><td>Thr</td><td>val</td><td>Leu</td><td>Arg</td>

<td></td><td> 50</td><td></td><td></td><td></td><td></td><td> 55</td><td></td><td></td><td></td><td></td><td> 60</td><td></td><td></td><td></td><td></td>

<td>Gin</td><td>ala</td><td>Asp</td><td>Cheese</td><td>Gin</td><td>val</td><td>Thr</td><td>Glu</td><td>val</td><td>Cys</td><td>ala</td><td>ala</td><td>Thr</td><td>Tyr</td><td>Underworld</td><td>Underworld</td>

<td> 65</td><td></td><td></td><td></td><td></td><td> 70</td><td></td><td></td><td></td><td></td><td> 75</td><td></td><td></td><td></td><td></td><td> 80</td>

<td>Gly</td><td>own</td><td>Glu</td><td>Leu</td><td>Thr</td><td>phe</td><td>Leu</td><td>Asp</td><td>Asp</td><td>Cheese</td><td>How much</td><td>Cys</td><td>Thr</td><td>Gly</td><td>Thr</td><td>Cheese</td>

<td></td><td></td><td></td><td></td><td> 85</td><td></td><td></td><td></td><td></td><td> 90</td><td></td><td></td><td></td><td></td><td> 95</td><td></td>

<td>Cheese</td><td>Gly</td><td>own</td><td>Gin</td><td>val</td><td>own</td><td>Leu</td><td>Thr</td><td>How much</td><td>Gin</td><td>Gly</td><td>Leu</td><td>Arg</td><td>ala</td><td>Underworld</td><td>Asp</td>

<td></td><td></td><td></td><td> 100</td><td></td><td></td><td></td><td></td><td> 105</td><td></td><td></td><td></td><td></td><td> 110</td><td></td><td></td>

<td>Thr</td><td>Gly</td><td>Leu</td><td>Tyr</td><td>How much</td><td>Cys</td><td>lys</td><td>val</td><td>Glu</td><td>Leu</td><td>Underworld</td><td>Tyr</td><td>Pro</td><td>Pro</td><td>Pro</td><td>Tyr</td>

<td></td><td></td><td> 115</td><td></td><td></td><td></td><td></td><td> 120</td><td></td><td></td><td></td><td></td><td> 125</td><td></td><td></td><td></td>

<td>Tyr</td><td>Leu</td><td>Gly</td><td>How much</td><td>Gly</td><td>own</td><td>Gly</td><td>Thr</td><td>Gin</td><td>How much</td><td>Tyr</td><td>val</td><td>How much</td><td>Asp</td><td>Pro</td><td>Glu</td>

<td></td><td> 130</td><td></td><td></td><td></td><td></td><td> 135</td><td></td><td></td><td></td><td></td><td> 140</td><td></td><td></td><td></td><td></td>

<td>Pro</td><td>Cys</td><td>Pro</td><td>Asp</td><td>Cheese</td><td>Asp</td><td>Gin</td><td>Glu</td><td>Pro</td><td>lys</td><td>Cheese</td><td>Cheese</td><td>Asp</td><td>lys</td><td>Thr</td><td>His</td>

<td> 145</td><td></td><td></td><td></td><td></td><td> 150</td><td></td><td></td><td></td><td></td><td> 155</td><td></td><td></td><td></td><td></td><td> 160</td>

<td>Thr</td><td>Cheese</td><td>Pro</td><td>Pro</td><td>Cheese</td><td>Pro</td><td>ala</td><td>Pro</td><td>Glu</td><td>Leu</td><td>Leu</td><td>Gly</td><td>Gly</td><td>Cheese</td><td>Cheese</td><td>val</td>

<td></td><td></td><td></td><td></td><td> 165</td><td></td><td></td><td></td><td></td><td> 170</td><td></td><td></td><td></td><td></td><td> 175</td><td></td>

<td>phe</td><td>Leu</td><td>phe</td><td>Pro</td><td>Pro</td><td>lys</td><td>Pro</td><td>lys</td><td>Asp</td><td>Thr</td><td>Leu</td><td>Underworld</td><td>How much</td><td>Cheese</td><td>Arg</td><td>Thr</td>

180 185 190

Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu Asp Pro Glu 195 200 205

Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala Lys 210 215 220

Thr Lys Pro Arg Glu Glu Gin Tyr Asn Ser Thr Tyr Arg Val Val Ser 225 230 235 240

Val Leu Thr Val Leu His Gin Asp Trp Leu Asn Gly Lys Glu Tyr Lys 245 250 255

Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile Glu Lys Thr Ile 260 265 270

Cheese Lys Ala Lys Gly Gin Pro Arg Glu Pro Gin Val Tyr Thr Leu Pro 275 280 285

Pro Ser Arg Asp Glu Leu Thr Lys Asn Gin Val Ser Leu Thr Cys Leu 290 295 300

Val Lys Gly Phe Tyr Pro Cheese Asp Ile Ala Val Glu Trp Glu Cheese Asn 305 310 315 320

Gly Gin Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp Ser 325 330 335

Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser Arg 340 345 350

Trp Gin Gin Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala Leu 355 360 365

His Asn His Tyr Thr Gin Lys Ser Leu Ser Leu Ser Pro Gly Lys 370 375 380

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