title: Immuno-PET imaging of tumor endothelial marker 8 (TEM8).
abstract: Tumor endothelial marker 8 (TEM8) is a cell surface receptor that is highly expressed in a variety of human tumors and promotes tumor angiogenesis and cell growth. Antibodies targeting TEM8 block tumor angiogenesis in a manner distinct from the VEGF receptor pathway. Development of a TEM8 imaging agent could aid in patient selection for specific antiangiogenic therapies and for response monitoring. In these studies, L2, a therapeutic anti-TEM8 monoclonal IgG antibody (L2mAb), was labeled with (89)Zr and evaluated in vitro and in vivo in TEM8 expressing cells and mouse xenografts (NCI-H460, DLD-1) as a potential TEM8 immuno-PET imaging agent. (89)Zr-df-L2mAb was synthesized using a desferioxamine-L2mAb conjugate (df-L2mAb); (125)I-L2mAb was labeled directly. In vitro binding studies were performed using human derived cell lines with high, moderate, and low/undetectable TEM8 expression. (89)Zr-df-L2mAb in vitro autoradiography studies and CD31 IHC staining were performed with cryosections from human tumor xenografts (NCI-H460, DLD-1, MKN-45, U87-MG, T-47D, and A-431). Confirmatory TEM8 Western blots were performed with the same tumor types and cells. (89)Zr-df-L2mAb biodistribution and PET imaging studies were performed in NCI-H460 and DLD-1 xenografts in nude mice. (125)I-L2mAb and (89)Zr-df-L2mAb exhibited specific and high affinity binding to TEM8 that was consistent with TEM8 expression levels. In NCI-H460 and DLD-1 mouse xenografts nontarget tissue uptake of (89)Zr-df-L2mAb was similar; the liver and spleen exhibited the highest uptake at all time points. (89)Zr-L2mAb was highly retained in NCI-H460 tumors with <10% losses from day 1 to day 3 with the highest tumor to muscle ratios (T:M) occurring at day 3. DLD-1 tumors exhibited similar pharmacokinetics, but tumor uptake and T:M ratios were reduced ∼2-fold in comparison to NCI-H460 at all time points. NCI-H460 and DLD-1 tumors were easily visualized in PET imaging studies despite low in vitro TEM8 expression in DLD-1 cells indicating that in vivo expression might be higher in DLD-1 tumors. From in vitro autoradiography studies (89)Zr-df-L2mAb specific binding was found in 6 tumor types (U87-MG, NCI-H460, T-47D MKN-45, A-431, and DLD-1) which highly correlated to vessel density (CD31 IHC). Westerns blots confirmed the presence of TEM8 in the 6 tumor types but found undetectable TEM8 levels in DLD-1 and MKN-45 cells. This data would indicate that TEM8 is associated with the tumor vasculature rather than the tumor tissue, thus explaining the increased TEM8 expression in DLD-1 tumors compared to DLD-1 cell cultures. (89)Zr-df-L2mAb specifically targeted TEM8 in vitro and in vivo although the in vitro expression was not necessarily predictive of in vivo expression which seemed to be associated with the tumor vasculature. In mouse models, (89)Zr-df-L2mAb tumor uptakes and T:M ratios were sufficient for visualization during PET imaging. These results would suggest that a TEM8 targeted PET imaging agent, such as (89)Zr-df-L2mAb, may have potential clinical, diagnostic, and prognostic applications by providing a quantitative measure of tumor angiogenesis and patient selection for future TEM8 directed therapies.
authors.affiliation: Molecular Imaging Program, National Cancer Institute , Bethesda, Maryland 20892-1088, United States.
authors.forename: Frank
authors.initials: F
authors.lastname: Kuo
authors.forename: Stephanie
authors.initials: S
authors.lastname: Histed
authors.forename: Biying
authors.initials: B
authors.lastname: Xu
authors.forename: Veerendra
authors.initials: V
authors.lastname: Bhadrasetty
authors.forename: Lawrence P
authors.initials: LP
authors.lastname: Szajek
authors.forename: Mark R
authors.initials: MR
authors.lastname: Williams
authors.forename: Karen
authors.initials: K
authors.lastname: Wong
authors.forename: Haitao
authors.initials: H
authors.lastname: Wu
authors.forename: Kelly
authors.initials: K
authors.lastname: Lane
authors.forename: Vincent
authors.initials: V
authors.lastname: Coble
authors.forename: Olga
authors.initials: O
authors.lastname: Vasalatiy
authors.forename: Gary L
authors.initials: GL
authors.lastname: Griffiths
authors.forename: Chang H
authors.initials: CH
authors.lastname: Paik
authors.forename: Osama
authors.initials: O
authors.lastname: Elbuluk
authors.forename: Christopher
authors.initials: C
authors.lastname: Szot
authors.forename: Amit
authors.initials: A
authors.lastname: Chaudhary
authors.forename: Brad
authors.initials: B
authors.lastname: St Croix
authors.forename: Peter
authors.initials: P
authors.lastname: Choyke
authors.forename: Elaine M
authors.initials: EM
authors.lastname: Jagoda
mesh_terms: D000818:Animals; D061067:Antibodies, Monoclonal, Humanized / Q000737:chemistry / Q000493:pharmacokinetics; D015153:Blotting, Western; D003676:Deferoxamine / Q000008:administration & dosage / Q000737:chemistry; D005260:Female; D006801:Humans; D047468:Immunoprecipitation; D051379:Mice; D008819:Mice, Nude; D008840:Microfilament Proteins; D057054:Molecular Imaging; D009363:Neoplasm Proteins / Q000037:antagonists & inhibitors / Q000276:immunology*; D009369:Neoplasms / Q000000981:diagnostic imaging / Q000276:immunology / Q000378:metabolism / Q000473:pathology; D049268:Positron-Emission Tomography / Q000379:methods*; D019275:Radiopharmaceuticals / Q000493:pharmacokinetics; D011956:Receptors, Cell Surface / Q000037:antagonists & inhibitors / Q000276:immunology*; D014018:Tissue Distribution; D014407:Tumor Cells, Cultured; D023041:Xenograft Model Antitumor Assays; D015040:Zirconium / Q000493:pharmacokinetics
chemical_list: C473287:ANTXR1 protein, human; D061067:Antibodies, Monoclonal, Humanized; D008840:Microfilament Proteins; D009363:Neoplasm Proteins; D019275:Radiopharmaceuticals; D011956:Receptors, Cell Surface; D015040:Zirconium; D003676:Deferoxamine
keywords: L2; TEM8; [89Zr]-df−L2mAb; angiogenesis; anti-TEM8 antibodies; immuno-PET imaging