Sortase-catalyzed in vitro functionalization of a HER2-specific recombinant Fab for tumor targeting of the plant cytotoxin gelonin.
We report on the preparation of a new type of immunotoxin via in vitro ligation of the αHer2 antigen binding fragment (Fab) of the clinically-validated antibody trastuzumab to the plant toxin gelonin, employing catalysis by the bacterial enzyme sortase A (SrtA). The αHer2 Fab was fused with the extended SrtA recognition motif LPET↓GLEH 6 at the C-terminus of its heavy chain, thereby preventing interference with antigen binding, while the toxin was equipped with a Gly 2 sequence at its N-terminus, distant to the catalytically active site in the C-terminal region. Site-specific in vitro transpeptidation led to a novel antibody-toxin conjugate wherein gelonin had effectively replaced the Fc region of a conventional (monomerized) immunoglobulin. After optimization of reaction conditions and incubation time, the resulting Fab-Gelonin ligation product was purified to homogeneity in a two-step procedure by means of Strep-Tactin affinity chromatography--utilizing the Strep-tag II appended to gelonin--and size exclusion chromatography. Binding activity of the immunotoxin for the Her2 ectodomain was indistinguishable from the unligated Fab as measured by real-time surface plasmon resonance spectroscopy. Specific cytotoxic potency of Fab-Gelonin was demonstrated against two Her2-positive cell lines, resulting in EC 50 values of ~1 nM or lower, indicating a 1000-fold enhanced cell-killing activity compared with gelonin itself. Thus, our strategy provides a convenient route to the modular construction of functional immunotoxins from Fabs of established tumor-specific antibodies with gelonin or related proteotoxins, also avoiding the elevated biosafety levels that would be mandatory for the direct biotechnological preparation of corresponding fusion proteins.
Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie; Technische Universität München; Freising-Weihenstephan, Germany.
Petra
P
Kornberger
Munich Center for Integrated Protein Science (CIPS-M) and Lehrstuhl für Biologische Chemie; Technische Universität München; Freising-Weihenstephan, Germany.
Arne
A
Skerra
D000230:Adenocarcinoma / Q000276:immunology / Q000628:therapy*; D019881:Aminoacyltransferases / Q000378:metabolism*; D061067:Antibodies, Monoclonal, Humanized / Q000235:genetics / Q000378:metabolism; D000920:Antibody-Dependent Cell Cytotoxicity; D001426:Bacterial Proteins / Q000378:metabolism*; D001943:Breast Neoplasms / Q000276:immunology / Q000628:therapy*; D002384:Catalysis; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D003546:Cysteine Endopeptidases / Q000378:metabolism*; D005260:Female; D006801:Humans; D007140:Immunoglobulin Fab Fragments / Q000235:genetics / Q000378:metabolism; D016233:Immunotherapy, Active / Q000379:methods*; D058990:Molecular Targeted Therapy; D010051:Ovarian Neoplasms / Q000276:immunology / Q000628:therapy*; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism*; D054789:Ribosome Inactivating Proteins, Type 1 / Q000235:genetics / Q000378:metabolism*; D000068878:Trastuzumab
D019881:Aminoacyltransferases; D061067:Antibodies, Monoclonal, Humanized; D001426:Bacterial Proteins; D003546:Cysteine Endopeptidases; D007140:Immunoglobulin Fab Fragments; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D000068878:Trastuzumab; C025674:GEL protein, Gelonium multiflorum; C120542:sortase A; C508053:ERBB2 protein, human
antibody-drug conjugate; binding protein; immunotoxin; plant toxin; protein ligation; transpeptidation