Anti-graft-versus-host disease effect of DT₃₉₀-anti-CD3sFv, a single- chain Fv fusion immunotoxin specifically targeting the CD3ε moiety of the T- cell receptor

In a recent study, we showed that an immunotoxin (IT) made with a conventional monoclonal antibody targeting the CD3ε 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 immunohistochemistry studies. GVHD was studied in a model in which C57BL/6 (H-2^b)-purified lymph node T cells were administered to major histocompatibility complex (MHC) antigen disparate unirradiated C.B. -17 acid (H-2(d)) 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 μg 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.