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A Bispecific Recombinant Immunotoxin, DT2219, Targeting Human CD19 and CD22 Receptors in a Mouse Xenograft Model of B-Cell Leukemia/Lymphoma

Authors' Affiliation: Department of Therapeutic Radiology-Radiation Oncology, Section on Molecular Cancer Therapeutics, University of Minnesota Cancer Center, Minneapolis, Minnesota

Requests for reprints: Daniel A. Vallera, University of Minnesota Cancer Center, MMC 367, Minneapolis, MN 55455. Phone: 612-626-6664; Fax: 612-624-3913; E-mail: valle001{at}umn.edu.

A novel bispecific single-chain fusion protein, DT2219, was assembled consisting of the catalytic and translocation domains of diphtheria toxin (DT₃₉₀) fused to two repeating sFv subunits recognizing CD19 and CD22 and expressed in Escherichia coli. Problems with yield, purity, and aggregation in the refolding step were solved by incorporating a segment of human muscle aldolase and by using a sodium N-lauroyl-sarcosine detergent-based refolding procedure. Problems with reduced efficacy were addressed by combining the anti-CD19 and anti-CD22 on the same single-chain molecule. DT2219 had greater anticancer activity than monomeric or bivalent immunotoxins made with anti-CD19 and anti-CD22 sFv alone and it showed a higher level of binding to patient leukemia cells and to CD19⁺CD22⁺ Daudi or Raji cells than did anti-CD19 and anti-CD22 parental monoclonal antibodies. The resulting DT2219, mutated to enhance its avidity, was cytotoxic to Daudi cells in vitro (IC₅₀ = 0.3 nmol/L). In vivo, DT2219 was effective in a flank tumor therapy model in which it significantly inhibited tumor growth (P < 0.05) and in a systemic model in which it significantly prolonged survival of severe combined immunodeficient mice with established Daudi (P < 0.008) compared with controls. DT2219 has broader reactivity in recognizing B-cell malignancies, has more killing power, and requires less toxin than using individual immunotoxin, which warrants further investigation as a new drug for treating B leukemia/lymphoma.

Key Words: immunotoxin • diphtheria toxin • leukemia • lymphoma • xenograft model • anti-CD19 sFv • anti-CD22 sFv

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