title: Engineered antibody fragments for immuno-PET imaging of endogenous CD8+ T cells in vivo.
abstract: The noninvasive detection and quantification of CD8(+) T cells in vivo are important for both the detection and staging of CD8(+) lymphomas and for the monitoring of successful cancer immunotherapies, such as adoptive cell transfer and antibody-based immunotherapeutics. Here, antibody fragments are constructed to target murine CD8 to obtain rapid, high-contrast immuno-positron emission tomography (immuno-PET) images for the detection of CD8 expression in vivo. The variable regions of two anti-murine CD8-depleting antibodies (clones 2.43 and YTS169.4.2.1) were sequenced and reformatted into minibody (Mb) fragments (scFv-CH3). After production and purification, the Mbs retained their antigen specificity and bound primary CD8(+) T cells from the thymus, spleen, lymph nodes, and peripheral blood. Importantly, engineering of the parental antibodies into Mbs abolished the ability to deplete CD8(+) T cells in vivo. The Mbs were subsequently conjugated to S-2-(4-isothiocyanatobenzyl)-1,4,7-triazacyclononane-1,4,7-triacetic acid for (64)Cu radiolabeling. The radiotracers were injected i.v. into antigen-positive, antigen-negative, immunodeficient, antigen-blocked, and antigen-depleted mice to evaluate specificity of uptake in lymphoid tissues by immuno-PET imaging and ex vivo biodistribution. Both (64)Cu-radiolabeled Mbs produced high-contrast immuno-PET images 4 h postinjection and showed specific uptake in the spleen and lymph nodes of antigen-positive mice.
authors.affiliation: Crump Institute for Molecular Imaging and Department of Molecular and Medical Pharmacology, David Geffen School of Medicine, Howard Hughes Medical Institute, Department of Microbiology, Immunology, and Molecular Genetics, and Eli and Edythe Broad Center of Regenerative Medicine and Stem Cell Research, University of California, Los Angeles, CA 90095.
authors.forename: Richard
authors.initials: R
authors.lastname: Tavaré
authors.forename: Melissa N
authors.initials: MN
authors.lastname: McCracken
authors.forename: Kirstin A
authors.initials: KA
authors.lastname: Zettlitz
authors.forename: Scott M
authors.initials: SM
authors.lastname: Knowles
authors.forename: Felix B
authors.initials: FB
authors.lastname: Salazar
authors.forename: Tove
authors.initials: T
authors.lastname: Olafsen
authors.forename: Owen N
authors.initials: ON
authors.lastname: Witte
authors.forename: Anna M
authors.initials: AM
authors.lastname: Wu
mesh_terms: D000483:Alleles; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology*; D000918:Antibody Specificity / Q000276:immunology; D000941:Antigens / Q000737:chemistry; D018414:CD8-Positive T-Lymphocytes / Q000166:cytology*; D003301:Copper Radioisotopes / Q000737:chemistry; D000939:Epitopes / Q000737:chemistry; D005434:Flow Cytometry; D007128:Immunoglobulin Fragments / Q000276:immunology*; D007167:Immunotherapy / Q000379:methods; D051379:Mice; D008809:Mice, Inbred C3H; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D049268:Positron-Emission Tomography; D051381:Rats; D014018:Tissue Distribution
chemical_list: D000911:Antibodies, Monoclonal; D000941:Antigens; D003301:Copper Radioisotopes; D000939:Epitopes; D007128:Immunoglobulin Fragments