title: 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 (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.
authors.affiliation: Department of Medical Oncology and Cancer Research, University of Essen, D-45122 Essen, Germany. juergen.krauss@uni-essen.de
authors.forename: Jürgen
authors.initials: J
authors.lastname: Krauss
authors.forename: Michaela A E
authors.initials: MA
authors.lastname: Arndt
authors.forename: Bang K
authors.initials: BK
authors.lastname: Vu
authors.forename: Dianne L
authors.initials: DL
authors.lastname: Newton
authors.forename: Siegfried
authors.initials: S
authors.lastname: Seeber
authors.forename: Susanna M
authors.initials: SM
authors.lastname: Rybak
mesh_terms: D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry / Q000235:genetics / Q000276:immunology* / Q000302:isolation & purification; D000918:Antibody Specificity; D015703:Antigens, CD / Q000276:immunology* / Q000378:metabolism; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology* / Q000378:metabolism; D015815:Cell Adhesion Molecules / Q000276:immunology* / Q000378:metabolism; D016923:Cell Death; D045744:Cell Line, Tumor; D002470:Cell Survival; D003602:Cytotoxicity, Immunologic; D019281:Dimerization; D006801:Humans; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics / Q000276:immunology; D020128:Inhibitory Concentration 50; D037102:Lectins / Q000276:immunology* / Q000378:metabolism; D051379:Mice; D009369:Neoplasms / Q000276:immunology* / Q000473:pathology*; D020836:Protein Structure, Quaternary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D012680:Sensitivity and Specificity; D051918:Sialic Acid Binding Ig-like Lectin 2
chemical_list: D000911:Antibodies, Monoclonal; D015703:Antigens, CD; D000944:Antigens, Differentiation, B-Lymphocyte; C497867:CD22 protein, human; C497868:Cd22 protein, mouse; D015815:Cell Adhesion Molecules; D007135:Immunoglobulin Variable Region; D037102:Lectins; D011993:Recombinant Fusion Proteins; D051918:Sialic Acid Binding Ig-like Lectin 2; D012260:Ribonucleases