1. Abatacept (hCTLA4-Ig) For the Treatment of Rheumatoid Arthritis: Product Attributes and Mechanism of Action Joy Williams, Ph.D. Arthritis Advisory Committee September 6, 2005

2. Continuous Marketing Application (Rolling submission of BLA) Unit:Date of submission: Pharmacology and Toxicology reviewable unit11/15/04 Clinical pharmacology and biopharmaceutics Full clinical reviewable unit12/20/04 Chemistry, Manufacturing and Controls (CMC) reviewable unit3/31/05 Abatacept BLA PDUFA action date for BLA 125118 (Abatacept): 12/31/05

3. Review team Regulatory Project manager: Erik Laughner Product reviewers: Joy Williams, chair Elizabeth Shores Susan Kirshner Ennan Guan Edward Max Barbara Rellahan Clinical reviewers: Keith Hull – Chair, clinical reviewer Jeffrey Siegel –Team leader Mark Walton-Director Pharm/Tox: Anita O'Connor-toxicology reviewer Anil Rajpal-Pharm reviewer Hannan Guan Statisticians: Kyung Lee-stat reviewer Boguang Zhen-stat team leader Facilities reviewer: Ann deMarco DSI/BiMo: Dianne Tesch DDMAC: Catherine Gray DDRE: Hyon Kwon

4. Abatacept (CTLA4-Ig) homodimer CTLA-4 domain: Extracellular sequence of human CTLA-4 IgG1 Fc domain: Hinge, CH2 and CH3 regions of human IgG1 Fc sequence

5. Final formulation Abatacept for Injection, 250 mg/vial sterile, non-pyrogenic lyophile for intravenous administration following reconstitution with Sterile Water for Injection; supplied with non-siliconized syringe Excipients: maltose monohydrate, sodium phosphate monobasic, Sodium chloride, hydrochloric acid, sodium hydroxide Abatacept manufacturing process Abatacept is produced as a secreted protein in large scale culture of Chinese hamster ovary cell line Series of purification/viral clearance steps

6. activated T cell Antigen presenting cell TCR B7 MHC + peptide CD28 T cell activation requires signals from TCR and costimulatory molecules

7. T cell Antigen presenting cell CTLA4 – a CD28 homologue that binds to B7 with higher affinity than CD28 TCR B7 MHC + peptide CD28 CTLA4

8. CTLA4 extracellular domain (B7-binding domain) is fused to IgG1 Fc to create soluble CTLA4-Ig CTLA4-Ig CTLA4 IgG1 Fc B7 CTLA4 extracellular domain

9. T cell Antigen presenting cell TCR B7 MHC + peptide CD28 CTLA4-Ig binds to B7 molecules and inhibits CD28-B7 interactions; suppresses T cell response

10. Additional possible mechanisms of action to consider: B7 IDO tryptophan kynurenine T cell proliferation is inhibited B7 engaged by CTLA4-Ig directly signals to dendritic cells Suppresses T cell response INF-  Grohmann et al. Nat Immunol. 3:1097-101 Mellor et al. JI 171:1652-5 Boasso et al. Blood 105: 1574-81 Munn et al. JI 172: 4100-10

11. ? Additional possible mechanisms of action to consider: Potential functions of the IgG1 Fc portion of abatacept CTLA4 IgG1 Fc Binding to Fc receptors Antibody-dependent Cellular cytoxicity (ADCC) Phagocytosis/clearance of opsonized cells ? Resulting in elimination of B7-expressing cells and suppression of T cell response B7 expressing cell

12. The paradox: CTLA4-Ig may disrupt critical pathways required for maintenance of immune tolerance

13. T regulatory cell Antigen presenting cell Effector T cell B7-CTLA4 T regulatory cells are critical components in the maintenance of peripheral tolerance to tissue-specific self-antigens In both humans and mice, absence of T regulatory cells is associated with aggressive autoimmunity Self-antigen

14. CD28-B7 interactions are essential for both thymic development of Tregs and for their peripheral homeostasis Decrease in Tregs (CD4+CD25+) cells in lymph nodes of wildtype mice treated with CTLA4Ig for 10 days Tang et al. JI 171: 3348-52 0 1 2 3 4 % CD4+CD25+ thymocytes wildtype B7 knockout Decreased development of Tregs In the thymus in B7 knockout mice In certain inbred mouse strains, autoimmune disease is exacerbated in the absence of B7 or CD28 due to loss of Tregs Williams et al. (not published) Implications for development of fetal immune system during gestation?

15. T cell Antigen presenting cell Activated T cells express CTLA4: CTLA4 transduces a negative signal to T cells and functions to attenuate the immune response TCR B7 MHC + peptide CD28 CTLA4

16. T cell Antigen presenting cell TCR B7 MHC + peptide CD28 CTLA4 Activated T cells express CTLA4: CTLA4 transduces a negative signal to T cells and functions to attenuate the immune response; CTLA4-Ig may interfere with this process

17. Mechanisms of action that may function to ameliorate RA: Blocking CD28 costimulation Induction of suppressor antigen-presenting cells (via IDO) Depletion of B7-expressing cells Mechanisms of action that may exacerbate autoimmunity: Inhibition of T regulatory (CD4+CD25+) cell maintenance/development Inhibition of endogenous CTLA4 signals Abatacept (CTLA4-Ig) Mechanism of Action Summary