rhBMP-2: origin, biology and preclinical safety Presentation G ver 020106 4/14/2017 rhBMP-2: origin, biology and preclinical safety Gerard E. Riedel, Ph.D. Wyeth-Genetics Institute, Inc. Cambridge, Massachusetts USA

rhBMP-2 = recombinant human bone morphogenetic protein-2

Origin of rhBMP-2 The gene for human BMP-2 was isolated This gene was inserted into a chromosome of a mammalian cell This cell line grows in large culture vessels and secretes rhBMP-2 into the media rhBMP-2 is purified, vialed and freeze-dried

Presentation G ver 020106 4/14/2017 Properties of rhBMP-2 Member of “growth and differentiation” protein family Endogenous BMP-2 is active in bone repair and in embryonic development Homodimeric, glycosylated protein that is highly conserved across species Osteoinductive in animals and humans

rhBMP-2 induces bone rat ectopic implant assay 14 days post-implantation

Mechanism of bone induction Chemotaxis/ proliferation Differentiation of mesenchymal cells Bone induction trabecular, woven bone remodels appropriately Increased vascularity

Mechanism of rhBMP-2 action Cell biology Responsive cell types, receptors and signal transduction pathways identified Bone induction biology Local administration is required

Implantation of rhBMP-2 requires a matrix Delivers rhBMP-2 to surgical site Augments rhBMP-2 retention at site Provides a compatible environment for bone induction

Matrix = ACS (absorbable collagen sponge) Commercially available in US since 1981 Approved PMA (Integra LifeSciences) Implantable hemostatic agent Extensive experience of safe use Bovine tendon Type I collagen Manufacturing process meets/exceeds all US guidance

ACS = Helistat®

rhBMP-2/ACS preparation Presentation G ver 020106 4/14/2017 rhBMP-2/ACS preparation WFI rhBMP-2 rhBMP-2 solution ACS

rhBMP-2/ACS & LT- CAGE™ device

ACS augments rhBMP-2 retention at implantation site Orthotopic model (rabbit ulna) Radiolabelled rhBMP-2 Gamma camera scintigraphy Validated method

rhBMP-2 retention with ACS Presentation G ver 020106 4/14/2017 rhBMP-2 retention with ACS % initial dose 1 4 7 14 days

Safety information rhBMP-2/ACS studies ADME studies rhBMP-2 studies implant safety studies ADME studies rhBMP-2 studies Tumor biology studies Systemic toxicity studies Reproductive toxicity studies

rhBMP-2/ACS safety studies Three anatomic sites craniofacial, long bone, spine Meyer et al. 1999. Spine 24: 747-754. Dosing >>> therapeutic range (species-adjusted) Results: no systemic effect local effects = rhBMP-2 bioactivity

Biodistribution studies of rhBMP-2/ACS Two species (rats, rabbits) Two implant sites ectopic or long-bone (+/- osteotomy) Results: rhBMP-2 is released slowly from implant site low systemic availability (rat long-bone model) maximum rhBMP-2 detected = 0.1% of dose

rhBMP-2 is slowly released from implantation site

ADME studies of rhBMP-2 Pharmacokinetics/biodistribution rats (adults and juveniles) and NHP IV administration Results: rhBMP-2 is cleared rapidly from the circulation, primarily through the liver, and rapidly degraded/excreted into urine

rhBMP-2 is rapidly cleared from the systemic circulation

slow release from implant site + rapid clearance from circulation low rhBMP-2 systemic exposure

Tumor biology studies Screening studies Some tumors express BMP-2, BMP receptors No evidence that BMP-2 initiates tumors No cytotoxic/mutagenic activity in vitro No evidence of abnormal cell biology in implant toxicity studies Pharmacology studies (in vitro)

Effect on tumor growth (in vitro) 51 tumor cell lines tested 3: growth promotion (in serum-free media) [pancreas (2), prostate] 48: no effect or inhibition 71 primary tumor isolates tested 71: no effect or inhibition Inhibition is not sufficient for therapy

Additional tumor studies (FDA consultation) 1. Screen tumor cells for BMP receptor messenger RNA 2. Assess effect on tumor cell growth in vitro (cell culture) 3. Assess effect on tumor cell growth in vivo (xenograft)

rhBMP-2 toxicity studies Systemic toxicity of rhBMP-2 single or repeat IV dosing, two species exposure > 1000x expected in patients Reproductive toxicity of rhBMP-2 repeat IV dosing, two species fertility and teratology Results: no systemic effect

Additional safety studies Tripartite guideline safety studies in vivo, in vitro cytotoxicity, mutagenicity, others General safety studies in vivo, IV administration behavior, locomotion, other body functions Results: no effect

New studies (FDA consultation) Focus: better understanding of an immune response to rhBMP-2 New clinical assay measure all human antibody isotypes (current assay measures human IgG) Neutralization assay ability of antisera to block rhBMP-2 activity

Preclinical safety findings Presentation G ver 020106 4/14/2017 Preclinical safety findings No systemic effects attributed to low systemic exposure local effects = osteoinduction by rhBMP-2 No toxicity detected Safety assessment supports use of InFUSE™ Bone Graft