title: ImmunoPET Imaging of Murine CD4+T Cells Using Anti-CD4 Cys-Diabody: Effects of Protein Dose on T Cell Function and Imaging.
abstract: Molecular imaging of CD4+T cells throughout the body has implications for monitoring autoimmune disease and immunotherapy of cancer. Given the key role of these cells in regulating immunity, it is important to develop a biologically inert probe. GK1.5 cys-diabody (cDb), a previously developed anti-mouse CD4 antibody fragment, was tested at different doses to assess its effects on positron emission tomography (PET) imaging and CD4+T cell viability, proliferation, CD4 expression, and function.
  
  
  
  The effect of protein dose on image contrast (lymphoid tissue-to-muscle ratio) was assessed by administering different amounts of89Zr-labeled GK1.5 cDb to mice followed by PET imaging and ex vivo biodistribution analysis. To assess impact of GK1.5 cDb on T cell biology, GK1.5 cDb was incubated with T cells in vitro or administered intravenously to C57BL/6 mice at multiple protein doses. CD4 expression and T cell proliferation were analyzed with flow cytometry and cytokines were assayed.
  
  
  
  For immunoPET imaging, the lowest protein dose of 2 μg of89Zr-labeled GK1.5 cDb resulted in significantly higher % injected dose/g in inguinal lymph nodes (ILN) and spleen compared to the 12-μg protein dose. In vivo administration of GK1.5 cDb at the high dose of 40 μg caused a transient decrease in CD4 expression in spleen, blood, lymph nodes, and thymus, which recovered within 3 days postinjection; this effect was reduced, although not abrogated, when 2 μg was administered. Proliferation was inhibited in vivo in ILN but not the spleen by injection of 40 μg GK1.5 cDb. Concentrations of GK1.5 cDb in excess of 25 nM significantly inhibited CD4+T cell proliferation and interferon-γ production in vitro. Overall, using low-dose GK1.5 cDb minimized biological effects on CD4+T cells.
  
  
  
  Low-dose GK1.5 cDb yields high-contrast immunoPET images with minimal effects on T cell biology in vitro and in vivo and may be a useful tool for investigating CD4+T cells in the context of preclinical disease models. Future approaches to minimizing biological effects may include the creation of monovalent fragments or selecting anti-CD4 antibodies which target alternative epitopes.
authors.affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Crump Institute for Molecular Imaging, University of California, 570 Westwood Plaza, CNSI, PO Box 951770, Los Angeles, CA, 90095-1770, USA.
authors.forename: Amanda C
authors.initials: AC
authors.lastname: Freise
authors.affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Crump Institute for Molecular Imaging, University of California, 570 Westwood Plaza, CNSI, PO Box 951770, Los Angeles, CA, 90095-1770, USA.
authors.forename: Kirstin A
authors.initials: KA
authors.lastname: Zettlitz
authors.affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Crump Institute for Molecular Imaging, University of California, 570 Westwood Plaza, CNSI, PO Box 951770, Los Angeles, CA, 90095-1770, USA.
authors.forename: Felix B
authors.initials: FB
authors.lastname: Salazar
authors.affiliation: Department of Internal Medicine, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA.|David Geffen School of Medicine at UCLA, Clinical Translational Science Institute, Los Angeles, CA, USA.
authors.forename: Xiang
authors.initials: X
authors.lastname: Lu
authors.affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Crump Institute for Molecular Imaging, University of California, 570 Westwood Plaza, CNSI, PO Box 951770, Los Angeles, CA, 90095-1770, USA. richard.tavare@regeneron.com.|Regeneron Pharmaceuticals, Inc., 777 Old Saw Mill River Road, Tarrytown, NY, 10951, USA. richard.tavare@regeneron.com.
authors.forename: Richard
authors.initials: R
authors.lastname: Tavaré
authors.affiliation: Department of Molecular and Medical Pharmacology, David Geffen School of Medicine at UCLA, Crump Institute for Molecular Imaging, University of California, 570 Westwood Plaza, CNSI, PO Box 951770, Los Angeles, CA, 90095-1770, USA. awu@mednet.ucla.edu.
authors.forename: Anna M
authors.initials: AM
authors.lastname: Wu
mesh_terms: D000818:Animals; D000906:Antibodies / Q000378:metabolism*; D000915:Antibody Affinity / Q000276:immunology; D000918:Antibody Specificity / Q000276:immunology; D015704:CD4 Antigens / Q000378:metabolism; D015496:CD4-Positive T-Lymphocytes / Q000166:cytology / Q000276:immunology*; D049109:Cell Proliferation; D003545:Cysteine / Q000378:metabolism*; D016207:Cytokines / Q000378:metabolism; D005260:Female; D008221:Lymphoid Tissue / Q000000981:diagnostic imaging; D008810:Mice, Inbred C57BL; D049268:Positron-Emission Tomography; D014018:Tissue Distribution
chemical_list: D000906:Antibodies; D015704:CD4 Antigens; D016207:Cytokines; D003545:Cysteine
keywords: Antibody engineering; CD4; Diabody; ImmunoPET; Lymphocytes; Positron emission tomography; T cell function; T cells; Zirconium-89