Targeted induction of apoptosis by chimeric granzyme B fusion proteins carrying antibody and growth factor domains for cell recognition.
The serine protease granzyme B (GrB) of cytotoxic lymphocytes efficiently induces apoptosis by direct activation of caspases and cleavage of central caspase substrates. We employed human GrB as an effector function in chimeric fusion proteins that also contain the EGFR ligand TGFalpha or an ErbB2-specific single-chain antibody fragment (scFv) for selective targeting to tumor cells. GrB-TGFalpha (GrB-T) and GrB-scFv(FRP5) (GrB-5) molecules expressed in the yeast Pichia pastoris were bifunctional, cleaving synthetic and natural GrB substrates, and binding specifically to cells expressing EGFR or ErbB2 target receptors. Upon cell binding the chimeric molecules were internalized into intracellular vesicles, but could be released into the cytosol by the endosomolytic reagent chloroquine. Treatment with picomolar to nanomolar concentrations of GrB-5 and GrB-T resulted in selective and rapid tumor cell killing, accompanied by clear signs of apoptosis such as chromatin condensation, membrane blebbing, formation of apoptotic bodies and activation of endogenous initiator and effector caspases.
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Chemotherapeutisches Forschungsinstitut Georg-Speyer-Haus, Paul-Ehrlich-Strasse 42-44, D-60596 Frankfurt am Main, Germany.
Giesübel
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U
Knauer
S K
SK
Wels
W S
WS
D000906:Antibodies / Q000008:administration & dosage / Q000235:genetics; D000970:Antineoplastic Agents / Q000008:administration & dosage*; D017209:Apoptosis / Q000187:drug effects; D020169:Caspases / Q000378:metabolism; D045744:Cell Line, Tumor; D002462:Cell Membrane / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D002738:Chloroquine / Q000494:pharmacology; D022162:Cytoplasmic Vesicles / Q000187:drug effects / Q000201:enzymology; D004305:Dose-Response Relationship, Drug; D016503:Drug Delivery Systems; D004705:Endocytosis; D004789:Enzyme Activation / Q000187:drug effects; D066246:ErbB Receptors / Q000378:metabolism; D053804:Granzymes; D006801:Humans; D020128:Inhibitory Concentration 50; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D018719:Erb-b2 Receptor Tyrosine Kinases / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000008:administration & dosage* / Q000096:biosynthesis; D012697:Serine Endopeptidases / Q000008:administration & dosage* / Q000235:genetics; D016211:Transforming Growth Factor alpha / Q000008:administration & dosage / Q000235:genetics
D000906:Antibodies; D000970:Antineoplastic Agents; D020169:Caspases; D002738:Chloroquine; D066246:ErbB Receptors; D053804:Granzymes; D018719:Erb-b2 Receptor Tyrosine Kinases; D011993:Recombinant Fusion Proteins; D012697:Serine Endopeptidases; D016211:Transforming Growth Factor alpha; C509213:GZMB protein, human