01/22/2010 – 7:45pmeSlide – P6617 – MedImmune 4 x 8 Poster Template Detection of a human anti-PDGFR  therapeutic antibody in a human GBM orthotopic rat model Schifferli, K., ¹ Barone, T., Czapiga, M., Wetzel, L., Mense, M., ¹ Plunkett, R. and Steiner, P. MedImmune, Gaithersburg, MD ¹ Roswell Park Cancer Institute, Buffalo, NY st ASCB Annual Meeting, Denver, Colorado, December 3-7, 2011: Presentation Number 1366 Abstract Glioblastoma multiforme (GBM) is the most common malignant brain tumor in adults with median survival time of one year which presents a challenge for clinical treatment. The standard of care includes tumor resection, followed by radiation and chemotherapy with temozolomide (TMZ). Despite treatment, most GBM tumors develop resistance to treatment and outcome of patients remains dismal. GBM contains several genetic alterations affecting proliferation; most commonly upregulation of autocrine signaling of platelet-derived growth factor (PDGF) and its receptor PDGFR , which in tumor cells acts as a receptor tyrosine kinase as well as a mediator of stromal support for cancer growth. MEDI-575 is a human monoclonal antibody that selectively targets PDGFRα. To determine anti-tumor activity in GBM tumors expressing PDGFR , MEDI-575 was administered at 1 mg/kg or 3 mg/kg (2x/wk) to athymic nude mice bearing three different human GBM ectopic tumors (U118, U251 and SNB-19). We found anti-tumor activity of 118%, 71%, and 78% tumor growth inhibition (dTGI), respectively, and drug exposure levels in mouse serum of 30 to 80 ug/ml of MEDI-575. The blood brain barrier (BBB), a highly specialized zone of astrocyte and endothelial cells separating circulating blood from brain tissue, is an obstacle to delivery of targeted treatment to brain tumors. To determine levels of MEDI-575 in GBM, immunofluorescence of rat brains harboring orthotopic human GBM demonstrated staining of human antibodies in GBM of rats treated with 10 mg/kg of MEDI-575 (2x/wk, 5 doses) while normal brain tissue did not show any staining. Furthermore, immunohistochemistry of rat brains with human GBM confirmed tumoral expression of PDGFR . Surprisingly, host stromal cells within the tumor mass as well as in normal brain tissue stained for PDGFR . Pharmacokinetic analysis of serum from rats with orthotopic GBM showed MEDI-575 did not drop below 100 ug/mL for 3 days after five doses of MEDI-575 at 10 mg/kg, while in the brain MEDI-575 was detected at 26.0 ug/mL 48 hours after the final dose. This indicated that the BBB of rats with implanted GBM allowed MEDI-575 to pass into the rat brain. The resulting exposure levels of MEDI-575 in the GBM tumor might be sufficient to elicit a response based on active serum levels in mouse GBM xenograft studies (30 to 80 ug/ml). These observations support ongoing Phase 2 clinical development of MEDI-575 to treat patients suffering from GBM after relapse from treatment and surgery. MEDI-575 Treatment Shows Activity in GBM Xenograft Models with Tumoral Expression of PDGFR  in Nude Mice U118-MG GBM Overexpressed PDGFR  Nude Mice U251-MG GBM Amplified PDGFR  Nude Mice SNB-19 GBM Amplified PDGFR  Nude Mice  MEDI-575 is a human IgG2 monoclonal antibody specific for human PDGFR   Analysis of PDGFR  expression in orthotopically implanted human GBM  Quantitation of MEDI-575 in orthotopically implanted human GBM and rat serum  Investigate accessibility of MEDI-575 in orthotopically implanted human GBM  PDGFR  as a key pathway in tumor stroma  Consisting of cancer-associated fibroblasts (CAFs)  Soluble factors from CAFs support tumor growth PDGFR  as an Anti-Tumor Target  PDGFR  as a kinase driver in tumor cells  Mesenchymal tissue (role in development)  GBM, sarcoma  Kinase mutations, amplifications  Validated clinically (Imatinib, dasatinib, sunitinib)  Disease linkage between PDGFR  and the pathophysiology of GBM is supported by literature references  PDGFR  widely expressed (50%) and frequently amplified (30%) in human GBM tumors  Correlates with poor prognosis  PDGF ligands (AA and CC) expression is high by mRNA  PDGF-AA expression correlates with tumor grade  GBM reported to have high frequency of PDGF autocrine stimulation of signaling pathways  MEDI-575 exhibited anti-tumor activity in three xenograft models representing human GBM  IHC analysis demonstrated clear expression of PDGFR  in GBM tumors; host stromal cells in both tumor and normal tissue were positive for PDGFR   MEDI-575 at 1 mg/kg (2x/wk) demonstrated tumor growth inhibition (dTGI) of 118% (U118-MG) and 78% (SNB-19) in GBM xenograft mouse models (71% dTGI at 3 mg/kg, 2x/wk in U251-MG)  Since MEDI-575 does not cross-react with mouse PDGFR , anti-tumor effects are mediated via tumoral inhibition of PDGFR   Exposure levels of MEDI-575 between 30  g/ml and 80  g/ml in mouse serum exhibit maximal anti-tumor activity in xenograft mouse model Pharmacokinetic Analysis of Serum and Brain Samples from Rats Treated with MEDI-575 Rats were intraperitoneally administered 5 doses of MEDI-575 (10 mg/kg); samples were collected 24, 48 and 72 hours post final dose  MEDI-575 was sustained above 100 ug/mL for 3 days in rat serum while in the brain, MEDI-575 was detected at 26.0 ug/mL 48 hours after the final dose  Results indicate that the BBB of rats allowed for the passage of MEDI-575 into the brain and GBM tumor  Immunofluorescence analysis correlated with PK analysis in serum and brain Immunofluorescence Analysis of Human IgG in Orthotopic GBM Grown in Rats Treated with MEDI-575 MEDI-575 was detected at 48 hours after the last of 5 consecutive (2x/wk) doses of MEDI-575 in orthotopic GBM tumors; MEDI-575 was absent in adjacent normal tissue [n=2]  Immunofluorescence analysis performed on rat brains demonstrated the presence of MEDI-575 clearly associated on the membrane of orthotopic GBM; peak signal was observed at 48 hours, after the last dose  Analysis of hindbrain (normal tissue, orange arrow) show the absence of MEDI-575 diffusion however in the vasculature (white arrow), human IgG could be detected  The amount of MEDI-575 detected by immunofluorescence correlated with PK in terms of signal intensity at 48 hours after the final dose  PK analysis of rat serum showed that MEDI-575 was sustained above 100 ug/ml for 3 days while in the brain, MEDI-575 could be detected 48 hours after the final dose  Data support ongoing Phase 2 clinical development of MEDI-575 to treat patients suffering from GBM after relapse from treatment and surgery Immunohistochemistry Analysis of PDGFR  in Rat Brain  PDGFR  expression was observed in GBM orthotopic tumors  Host stromal cells in orthotopic GBM tumor and in normal cells showed PDGFR  expression Introduction Objectives Results Summary and Conclusions Orthotopic GBM MEDI-575 (48 hours) MEDI-575 (72 hours) MEDI-575 (24 hours) 20x zoom4 Vehicle (48 hours) MEDI-575 (48 hours) MEDI-575 (72 hours) Hindbrain Animal 1 Animal 3 Animal 5 Animal 2 Animal 4 Animal 6 MEDI-575 (24 hours) 40x Normal Brain Tissue 40x Orthotopic Tumor Stromal 40x Orthotopic Tumor Tumoral