Cellular, Tissue and Gene Therapies Advisory Committee Meeting

1. Cellular, Tissue and Gene Therapies Advisory Committee Meeting Apligraf Allogeneic Cultured Keratinocytes and Fibroblasts in Bovine Collagen BLA 125400 Proposed Indication: Treatment of surgically created gingival and alveolar mucosal surface defects in adults November 17, 2011

2. FDA Presenters Mark H. Lee, PhD – Product Manufacture Division of Cellular and Gene Therapies Office of Cellular, Tissue and Gene Therapies, CBER Robert Betz, DDS – Overview of Disease and Treatments Division of Anesthesiology, General Hospital, Infection Control, and Dental Devices Office of Device Evaluation, CDRH Agnes Lim, MD – Clinical Study Design, Efficacy and Safety Division of Clinical Evaluation, Pharmacology and Toxicology Office of Cellular, Tissue and Gene Therapies, CBER

3. Product Mark H. Lee, PhD Eric Dollins, PhD Charles Durfor, PhD (CDRH) Andrew Steen, BSME (CDRH) Clinical Agnes Lim, MD Bruce Schneider, MD Robert S. Betz, DDS, Captain (Ret.) USPHS (CDRH). Diplomate, Am. Board Periodontol. Statistics John Scott, PhD Pharmacology/Toxicology Patrick Au, PhD Project Management Terrolyn Thomas, MS, MBA Labeling Loan Nguyen, PharmD Bioresearch Monitoring Janet White Epidemiology Faith Barash, MD, MPH Manufacturing and Product Quality Qiao Bobo, PhD Gang Wang, PhD Apligraf Review Team

4. Product Information

5. Product Description Apligraf is a bi-layered tissue construct consisting of an upper layer of human keratinocytes and a supporting lower layer constructed of bovine derived collagen and human neonatal foreskin-derived dermal fibroblasts. The upper and lower layers of the product make up approximately 33% and 67% of the construct. The proposed indication for Apligraf in this BLA is the treatment of surgically created gingival and alveolar mucosal surface defects in adults.

6. Regulatory History of Apligraf® • Identical to product submitted for BLA • Approved by the Center for Devices and Radiological Health in 1998 “for use with standard therapeutic compression for the treatment of non-infected partial and full-thickness skin ulcers due to venous insufficiency of greater than 1 month duration and which have not adequately responded to conventional ulcer therapy.” • Approved in 2000 by CDRH “for use with standard diabetic foot ulcer care for the treatment of full-thickness neuropathic diabetic foot ulcers of greater than three weeks duration which have not adequately responded to conventional ulcer therapy and which extend through the dermis but without tendon, muscle, capsule or bone exposure.”

7. Product Manufacture Apligraf is manufactured by combining viable allogeneic human fibroblasts and keratinocytes with type I bovine collagen over a 3-4 week process until a bi-layered structure that resembles skin dermis/epidermis forms. Apligraf does not contain Langerhans cells, melanocytes, macrophages, lymphocytes, blood vessels or hair follicles. Key manufacturing steps: • Establishment of Fibroblast and Keratinocyte Cell Banks • Production of Dermal Equivalent • Production of Epidermal Layer • Differentiation • Cornification

8. STEP QC TESTS Sterility/Mycoplasma/Virus Cell Identity & Purity Cytogenetic Stability, Tumorigenicity In vitro/In vivo Comparability Cell Banks Sterility (cellular, acellular cast mix, cell suspension residues) DE, EPI Layers Sterility (Media) Bioburden (Media - Cornification) Differentiation Cornification Mature Product Potency (Construct) Sterility (Media) Mycoplasma (Media) Bioburden (Media) Maintenance Sterility (Construct, Shipping Media) Bioburden (Construct, Ship Rinse Spent Media) Endotoxin Visual Inspection (Packaged Lots) Packaging

9. Cell Bank Qualification

10. Cell Bank Qualification and Release New cell banks of allogeneic fibroblasts and keratinocytes are introduced periodically for Apligraf manufacture. Quality and comparability of the cells are supported by: • Testing at the Master and Working Cell Bank (MCB, WCB) levels • Combination of direct testing of cells and of the cell-scaffold construct produced by the cells • Both in vitro and in vivo testing Comparability of the cells used for manufacture impacts final product quality and consistency. Scientific discussion from the Advisory Committee is requested regarding the testing approach used by the applicant.

11. Used for Comparability MCB Qualification Testing Testing performed on cells • Microbiological and viral safety testing • Isoenzyme analysis • Karyology • Senescence • Tumorigenicity • Cell purity (CD45, CD14, CD1a, CD31, HLA-DR) Testing performed on constructs made from MCB cells • Percutaneous absorption (barrier function) • Growth Factor/Cytokine profile (PDGF a, TGF-b1, IL-1a; IL-4) • Mitochondrial tetrazolium test (MTT) • Vascular Endothelial Growth Factor quantification (supernatant) • Histological analysis of construct (potency) • Functional assessment in athymic mouse model

12. WCB Qualification Testing Testing performed on cells • Microbiological and viral safety testing • Isoenzyme analysis • Cell growth • Cell viability • Collagen biosynthesis (fibroblast) • Involucrin content (keratinocyte) Testing performed on constructs made from WCB cells • Histological analysis of construct (potency)

13. Product Potency

14. Product Potency Regulatory Requirements: Potency is defined as “the specific ability or capacity of the product, as indicated by appropriate laboratory tests… to effect a given result.” (21 CFR 600.3(s)). The applicant proposes to measure Apligraf potency using histological parameters. Scientific discussion from the Advisory Committee is requested.

15. Proposed Potency Assay Hematoxylin and Eosin (H&E) staining to distinguish fibroblasts and collagen within the lower dermal layer as well as keratinocytes within the upper epidermal layer. The applicant describes the functions of the two layers to be: • Dermal – both as structural matrix for fibroblasts and substrate for development/maintenance of upper layer • Epidermal – impart structural elements and contribute to mechanical strength, handling and barrier properties

16. Proposed Potency Assay * Viability is determined indirectly by using histological assessment of a basophilic cytoplasm, absence of severe vacuolization/necrosis.

17. Proposed Potency Assay Image from BLA 125400

18. Other Testing of Relevance to Product Potency and Function used to support histology-based assay includes in vitro and in vivo non-clinical tests performed for cell bank qualification not performed on a lot-by-lot basis

19. Other Testing Relevant to Potency • Testing performed on constructs generated from MCB: • Percutaneous water absorption - barrier function • GF/Cytokine profile [PDGF a, TGF-b1, IL-1a , IL-4] • Mitochondrial Tetrazolium Testing (MTT) • Testing on culture supernatant of constructs from MCB: • Vascular Endothelial Growth Factor (VEGF) levels * VEGF concentration is believed by the applicant to be indicative of relevant biological factors produced by keratinocytes in the construct.

20. Other Testing Relevant to Potency • An athymic mouse graft model is used to assess comparability of Apligraf performance to that of positive control (construct made with FDA approved cell strains): • Pre-graft morphology • Graft take and integration • Graft contraction • Graft morphology (epidermal, dermal) • Graft remodeling • Immunohistochemistry (involucrin)

21. Product Potency CBER’s Guidance on “Potency Tests for Cellular and Gene Therapy Products” clarifies that all potency assays used for release testing of licensed biological products must have the following characteristics: • Indicate potency specific to the product • Provide test results for release of product • Provide quantitative data • Meet pre-defined acceptance and/or rejection criteria • Establish and document the accuracy, sensitivity, specificity and reproducibility of the test methods employed through validation • Measure identity and strength (activity) of all active ingredients • Provide data to establish dating period • Meet labeling requirements

22. Topics for Discussion • Two product quality issues are being posed to the Committee for discussion: • Current approach to qualify and demonstrate comparability for new cell banks used for Apligraf manufacture • Usage of histology as basis for product potency and role of other testing relevant to potency • Clinical reviewer will discuss other studies of relevance to the potency discussion • Histology, DNA Persistence, angiogenic biomarkers

24. Clinical Background Robert S. Betz, DDS, Captain (Ret.) USPHS Diplomate, American Board of Periodontology FDA/CDRH/ODE/DAGID

25. Periodontal Anatomy (Drawing adapted from Carranza’s Clinical Periodontology, 11th Edition)

26. Periodontal Probing (Drawings adapted from Carranza’s Clinical Periodontology, 11th Edition) Coronal Cementoenamel junction Apical

27. A – C = Free Gingiva A – B = Attached Gingiva B – C = Keratinized Gingiva C – A = Probing Depth Keratinized vs. Attached Gingiva (Drawing adapted from Carranza’s Clinical Periodontology, 11th Edition)

28. The Problem Mucogingival Defects are soft tissue defects that involve both the keratinized attached gingiva and nonkeratinized alveolar mucosa at the mucogingival junction • Insufficient Zone of Attached Gingiva (vs Keratinized Gingiva) • Gingival recession – most frequent occurrence • <1 mm Attached Gingiva • Muscle Pull • Inflammation • Bone Loss • Inflammatory component • Progressive lesion

29. Possible Causes And Effects • Toothbrush abrasion • Anatomy/Heredity • Shallow Vestibular depth • Tooth eruption patterns • Root prominence • Tooth crowding • Inability to keep mucogingival junction clean • Thin gingiva • Inflammatory Factors • Faulty dental restorations (over or under contoured) • Periodontitis • Poor oral hygiene • Orthodontic Treatment – movement of tooth away from a central location in alveolus to a more facial location. • Combination of the Above – multiple concurrent etiologies may be present • Effects – tooth sensitivity, cervical tooth abrasion, root caries, esthetic problems

30. Gingival Recession Gingival recession is defined as the location of the gingival margin apical to the cementoenamel junction. (AAP) Miller Recession Classification • Class I: Gingival recession does not extend to the mucogingival junction. Interproximal bone and soft tissues are unaffected. • Class II: Gingival recession extends to or beyond the mucogingival junction. Interproximal bone and soft tissues are unaffected. • Class III: Marginal tissue recession extends to or beyond the mucogingival junction. Bone or soft tissue is lost interproximally. • Class IV: Marginal tissue recession extends to or beyond the mucogingival junction.

31. Gingival Recession (Photograph from Franscesco Cairo et.al. article, 2010 Journal of Periodontology)

32. Treatment Options Autogenous Grafts Free gingival Grafts Subepithelial connective tissue graft Lateral and rotated pedicle graft procedure Double papillae pedicle graft Coronally positioned flap procedure These procedures may be performed individually or sequentially. A popular combination is the subepithelial tissue graft + CPF. Guided Tissue Regeneration Procedures Apligraf Platelet rich Fibrin Membrane Tunneling Procedures Dental Restorations Only Do Nothing

33. Apligraf vs. Soft Tissue Autograft • In the clinical studies, Apligraf was compared to soft tissue autograft (FGG) • Does not duplicate FGG biological properties. • Connective tissue of FGG survives by “ plasmatic circulation”. • Apligraf does not “take” like soft tissue autograft. • No Apligraf DNA present after 6 months. • Cells of Apligraf do not survive beyond 6 months.

34. Apligraf Placement Soft tissue incision at mucogingival junction (Semilunar or with vertical releasing incisions) Releasing incisions were used in the clinical studies. Apligraf “Z” folded and adapted to fit defect and sutured into place (Diagrams from BLA submission)

35. Treatment Notes • Autogenous palatal graft material was harvested and placed according to standard soft tissue grafting practices. • Width of all palate grafts was 5 mm (Study 05) or 4mm (Study 06) with the length dictated by the size of the mucosal defect. Graft thickness is usually 0.75 – 1.25 mm. • Incisions made to alveolar bone were used to “tack down” both Apligraf and FGG • Apligraf membranes and FGGs were free of movement as evaluated by muscle traction after placement • Root coverage was not performed (not studied in either 05 or 06 Study)

36. If probing depth is significant, there may be a measurable difference between attached and keratinized gingiva measurements. Could the change from measuring attached gingiva (05 Study) to keratinized gingiva (06 Study) be clinically significant? Keratinized vs. Attached GingivaDiscussion Issues

37. FIN Photograph from R. Betz, D.D.S.

39. ApligrafBLA 125400 Office of Cellular, Tissue and Gene Therapies Center for Biologics Evaluation and Research Advisory Committee CTGTAC Meeting #54 November 17, 2011 Agnes Lim, M.D. Bruce Schneider, M.D. (Team Leader) Clinical Evaluation Branch Center for Biologics Evaluation and Research (CBER)

40. Presentation Outline • BLA Supported by Two Studies; Overview of study plans • Study 05-PER-001 (05) • Study 06-PER-002-CTX (06) • Efficacy Results from Studies 05 and 06 • Safety Results • Studies 05 and 06 • Additional safety experience from Apligraf for chronic cutaneous wounds

41. Study 05-PER-001 (05) Study Design

42. Study 05-PER-001 (05) Title “A pilot Clinical Trial to Assess the Safety and Efficacy of Apligraf in Establishing A Functional Zone of Attached Gingiva” • Objectives To assess safety and efficacy in establishing a functional zone of attached gingiva • Study Design Study 05 was a randomized, single-center, within-subject controlled(graft sites matched for teeth and gingival condition) study; n=25 subjects

43. Study 05Efficacy Endpoints Primary Endpoint 1) The change in the amount of attached gingiva with Apligrafcompared to control treatment at 6 months Secondary Endpoints 1) Inflammation score 2) Color and texture match of the graft to the adjacent tissue 3) Resistance to oral muscle pull 4) Probing depth 5) Clinical attachment level 6) Subject preference or satisfaction (including pain experience) 7) Change in recession depth 8) Width of keratinized tissue

44. Study 05Major Eligibility Criteria • Included • Adults with an insufficient zone of attached gingiva that required soft tissue grafting • At least two non-adjacent teeth in contralateral quadrants of the same jaw • Root coverage was not desired at the time of grafting • Excluded • Treatment of exposed root is recommended • Acute infection in the areas intended for surgery • Health conditions/medications that could compromise wound healing --- such as, diabetes, cancer; receiving systemic corticosteroids, immunosuppressive agents • Current smokers

45. Study 05Study Plan • 25 subjects; Single center • Apligraf vs. soft tissue palatal autograft (FGG) • Within-subject control • 1st three subjects participated as training subjects; excluded from efficacy analysis, included in safety analysis • Treatment site and order of treatment were randomized; following randomization, subjects received both a palatal graft and Apligraf • Neither subject nor Investigator could be blinded • Primary efficacy evaluation at Month 6, with interim visits at Week 1, Month 1, and Month 3 • 2 adjunct laboratory studies: Histology Study and DNA Persistence Study

46. Study 05Assessment Method forPrimary Endpoint (Drawing adapted from Carranza’s Clinical Periodontology, 11th Edition) • The amount of attached gingiva was evaluated using a calibrated periodontal probe, measured to the nearest 0.5 mm Attached gingiva

47. Study 05Assessment Methods for Secondary Endpoints • Color compared to surrounding tissue • More/ Less/ Equally Red; at 1 week, 3 months, and 6 months • Texture compared to surrounding tissue • More/ Less/ Equally Firm; at 4 weeks, 3 months, 6 months • Inflammation • Scored by an examiner on a scale of 0 (absence of inflammation) to 4; at Week 1, Months 1, 3, and 6 • Probing Depth and KT width measured in mm • Clinical Attachment Level calculated from probing measurements • Resistance to Muscle Pull • Free gingiva movement when cheek/lip retracted; assessed at 6 months • Patient Satisfactionwas based on responses to 2 Questionnaires • Subject Aesthetics Questionnaire – marked response on a line between “Disappointed” and “Fully Satisfied” • Subject Discomfort Questionnaire - queried subjects for perceptions of the severity of pain, bleeding, swelling, and sensitivity for the Apligraf site, control site, and palate site. These were rated as None, Mild, Moderate, or Severe.

48. Study 05Statistical Analysis Plan • Primary Efficacy Endpoint: The absolute change in the amount of attached gingiva over 6 months between Apligraf and Control using a non-inferiority comparison • 5% significance level • n=22 subjects • Non-inferiority margin was a 1.0 mm difference in change • Analysis population: Number of subjects enrolled minus those who participated as training subjects • Secondary Efficacy Endpoints: No detailed statistical analysis plan for secondary endpoints and no adjustments were made for multiplicity

49. Study 05-PER-001 (05) Efficacy Results

50. Study 05-PER-001 • Subject Disposition • 25 enrolled and treated; all 25 completed all visits • Demographics • Mean age was 49 years • 17 female (68%); 8 male (32%) • 22 Caucasian (88%); 1 Hispanic, 1 Asian, 1 Middle Eastern