title: Anti-graft-versus-host disease effect of DT390-anti-CD3sFv, a single-chain Fv fusion immunotoxin specifically targeting the CD3 epsilon moiety of the T-cell receptor.
abstract: In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3 epsilon moiety of the T-cell receptor (TCR) had a potent, but partial, graft-versus-host disease (GVHD) effect (Vallera et al, Blood 86:4367, 1995). Therefore, in this current study, we determined whether a fusion immunotoxin made with anti-CD3 single-chain Fv (sFv), the smallest unit of antibody recognizing antigen, would have anti-GVHD activity. A fusion protein was synthesized from a construct made by splicing sFv cDNA from the hybridoma 145-2C11 to a truncated form of the diphtheria toxin (DT390) gene. DT390 encodes a molecule that retains full enzymatic activity, but excludes the native DT binding domain. The DT390-anti-CD3sFv hybrid gene was cloned into a vector under the control of an inducible promoter. The protein was expressed in Escherichia coli and then purified from inclusion bodies. The DT390 moiety of the protein had full enzymatic activity compared with native DT and DT390-anti-CD3sFv, with an IC50 of 1 to 2 nmol/L against phytohemagglutinin-stimulated and alloantigen-stimulated T cells. Specificity was shown (1) by blocking the IT with parental anti-CD3 antibody, but not with a control antibody; (2) by failure of DT390-anti-CD3sFv to inhibit lipopolysaccharide-stimulated murine B cells; (3) by failure of an Ig control fusion protein, DT390-Fc, to inhibit T-cell responses; and (4) with in vivo immunohistochemisty studies. GVHD was studied in a model in which C57BL/6 (H-2b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B.-17 scid (H-2d) mice to assess GVHD effects in the absence of irradiation toxicity. Flow cytometry studies showed that donor T cells were expanded 57-fold and histopathologic analysis showed the hallmarks of a lethal model of GVHD. Control mice receiving phosphate-buffered saline showed 17% survival on day 80 after bone marrow transplantation, and mice receiving 2 micrograms DT390-Fc fusion toxin control administered in 2 daily doses for 6 days (days 0 through 5) had a 43% survival rate. In contrast, 86% of mice receiving the same dose of DT390-anti-CD3sFv were survivors on day 80, a significant improvement, although survivors still showed histopathologic signs of GVHD. These findings suggest that new anti-GVHD agents can be genetically engineered and warrant further investigation of fusion proteins for GVHD treatment.
authors.affiliation: Department of Therapeutic Radiology, University of Minnesota Hospital and Clinics, Minneapolis 55455, USA.
authors.forename: D A
authors.initials: DA
authors.lastname: Vallera
authors.forename: A
authors.initials: A
authors.lastname: Panoskaltsis-Mortari
authors.forename: C
authors.initials: C
authors.lastname: Jost
authors.forename: S
authors.initials: S
authors.lastname: Ramakrishnan
authors.forename: C R
authors.initials: CR
authors.lastname: Eide
authors.forename: R J
authors.initials: RJ
authors.lastname: Kreitman
authors.forename: P J
authors.initials: PJ
authors.lastname: Nicholls
authors.forename: C
authors.initials: C
authors.lastname: Pennell
authors.forename: B R
authors.initials: BR
authors.lastname: Blazar
mesh_terms: D000818:Animals; D001483:Base Sequence; D017252:CD3 Complex / Q000276:immunology*; D017931:DNA Primers / Q000737:chemistry; D004167:Diphtheria Toxin; D006086:Graft vs Host Disease / Q000628:therapy*; D006648:Histocompatibility; D007128:Immunoglobulin Fragments / Q000276:immunology; D007167:Immunotherapy; D000922:Immunotoxins / Q000737:chemistry* / Q000493:pharmacokinetics / Q000627:therapeutic use; D008213:Lymphocyte Activation; D008657:Metabolic Clearance Rate; D051379:Mice; D008810:Mice, Inbred C57BL; D016513:Mice, SCID; D008969:Molecular Sequence Data; D011948:Receptors, Antigen, T-Cell / Q000276:immunology*; D011993:Recombinant Fusion Proteins / Q000493:pharmacokinetics
chemical_list: D017252:CD3 Complex; D017931:DNA Primers; C111339:DT390-anti-CD3sFv; D004167:Diphtheria Toxin; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D011948:Receptors, Antigen, T-Cell; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv