Efficient killing of CD22+ tumor cells by a humanized diabody-RNase fusion protein

Abstract We report on the generation of a dimeric immunoenzyme capable of simultaneously delivering two ribonuclease (RNase) effector domains on one molecule to CD22 + tumor cells. As targeting moiety a diabody derived from the previously humanized scFv SGIII with grafted specificity of the murine anti-CD22 mAb RFB4 was constructed. Further engineering the interface of this construct (V L 36 Leu → Tyr ) resulted in a highly robust bivalent molecule that retained the same high affinity as the murine mAb RFB4 ( K D  = 0.2 nM). A dimeric immunoenzyme comprising this diabody and Rana pipiens liver ribonuclease I ( rap LRI) was generated, expressed as soluble protein in bacteria, and purified to homogeneity. The dimeric fusion protein killed several CD22 + tumor cell lines with high efficacy (IC 50  = 3–20 nM) and exhibited 9- to 48-fold stronger cytotoxicity than a monovalent rap LRI–scFv counterpart. Our results demonstrate that engineering of dimeric antibody–ribonuclease fusion proteins can markedly enhance their biological efficacy.