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 (rapLRI) 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 rapLRI-scFv counterpart. Our results demonstrate that engineering of dimeric antibody-ribonuclease fusion proteins can markedly enhance their biological efficacy.
Publication types
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Research Support, N.I.H., Extramural
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Research Support, Non-U.S. Gov't
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Research Support, U.S. Gov't, P.H.S.
MeSH terms
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Animals
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Antibodies, Monoclonal / chemistry
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Antibodies, Monoclonal / genetics
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Antibodies, Monoclonal / immunology*
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Antibodies, Monoclonal / isolation & purification
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Antibody Specificity
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Antigens, CD / immunology*
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Antigens, CD / metabolism
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Antigens, Differentiation, B-Lymphocyte / immunology*
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Antigens, Differentiation, B-Lymphocyte / metabolism
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Cell Adhesion Molecules / immunology*
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Cell Adhesion Molecules / metabolism
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Cell Death
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Cell Line, Tumor
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Cell Survival
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Cytotoxicity, Immunologic*
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Dimerization
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Humans
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Immunoglobulin Variable Region / chemistry
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Immunoglobulin Variable Region / genetics
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Immunoglobulin Variable Region / immunology
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Inhibitory Concentration 50
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Lectins / immunology*
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Lectins / metabolism
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Mice
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Neoplasms / immunology*
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Neoplasms / pathology*
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Protein Structure, Quaternary
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Recombinant Fusion Proteins / chemistry
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Recombinant Fusion Proteins / immunology
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Recombinant Fusion Proteins / isolation & purification
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Recombinant Fusion Proteins / metabolism*
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Ribonucleases / chemistry
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Ribonucleases / genetics
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Ribonucleases / isolation & purification
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Ribonucleases / metabolism*
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Sensitivity and Specificity
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Sialic Acid Binding Ig-like Lectin 2
Substances
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Antibodies, Monoclonal
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Antigens, CD
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Antigens, Differentiation, B-Lymphocyte
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CD22 protein, human
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Cd22 protein, mouse
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Cell Adhesion Molecules
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Immunoglobulin Variable Region
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Lectins
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Recombinant Fusion Proteins
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Sialic Acid Binding Ig-like Lectin 2
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Ribonucleases