title: Novel conjugates of single-chain Fv antibody fragments specific for stem cell antigen CD123 mediate potent death of acute myeloid leukaemia cells.
abstract: Four new single-chain Fv antibody fragments (scFvs) specific for the human leucocyte surface antigen CD123 (interleukin-3 receptor alpha) were generated to achieve preferential targeting of leukaemia stem cells (LSCs) in acute myeloid leukaemia (AML). The scFvs were isolated from a phage display library generated with spleen RNA from mice, immunized with a fusion protein consisting of the extracellular domain of CD123 and the Fc domain of a human immunoglobulin G1. The scFvs displayed CD123-specific binding on tumour cells (binding constants (K(D)) 4.5-101 nmol/l). The scFv with the highest affinity was used to design two cell death-inducing molecules. First, an immunotoxin, a fusion protein with truncated Pseudomonas Exotoxin A, induced potent apoptosis of AML-derived MOLM-13 and SKNO-1 cells at nanomolar concentrations. Second, the fusion to another scFv, specific for the low affinity Fcgamma-receptor III (CD16), created a bispecific single chain Fv (bsscFv). This bsscFv [123 x ds16] mediated potent lysis of AML-derived MOLM-13, THP-1 and SKNO-1 cells in antibody-dependent cellular cytotoxicity (ADCC) reactions at picomolar concentrations. The recruitment of CD16-positive effector cells for the lysis of AML cells via CD123 represents a novel combination with attractive prospects for future clinical testing.
authors.affiliation: Chair of Genetics, University of Erlangen-Nuremberg, Erwin-Rommel-Strasse 3, Erlangen, Germany. cstein@biologie.uni-erlangen.de
authors.forename: Christoph
authors.initials: C
authors.lastname: Stein
authors.forename: Christian
authors.initials: C
authors.lastname: Kellner
authors.forename: Markus
authors.initials: M
authors.lastname: Kügler
authors.forename: Nina
authors.initials: N
authors.lastname: Reiff
authors.forename: Kristin
authors.initials: K
authors.lastname: Mentz
authors.forename: Michael
authors.initials: M
authors.lastname: Schwenkert
authors.forename: Bernhard
authors.initials: B
authors.lastname: Stockmeyer
authors.forename: Andreas
authors.initials: A
authors.lastname: Mackensen
authors.forename: Georg H
authors.initials: GH
authors.lastname: Fey
mesh_terms: D036002:ADP Ribose Transferases / Q000276:immunology; D000818:Animals; D000920:Antibody-Dependent Cell Cytotoxicity; D017209:Apoptosis / Q000276:immunology; D001427:Bacterial Toxins / Q000276:immunology; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004306:Dose-Response Relationship, Immunologic; D000939:Epitopes; D005098:Exotoxins / Q000276:immunology; D006801:Humans; D007128:Immunoglobulin Fragments / Q000276:immunology*; D000922:Immunotoxins / Q000276:immunology; D053650:Interleukin-3 Receptor alpha Subunit / Q000276:immunology*; D015470:Leukemia, Myeloid, Acute / Q000276:immunology* / Q000473:pathology; D051379:Mice; D008807:Mice, Inbred BALB C; D057127:Single-Chain Antibodies / Q000276:immunology*; D014407:Tumor Cells, Cultured; D037521:Virulence Factors / Q000276:immunology; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D001427:Bacterial Toxins; D000939:Epitopes; D005098:Exotoxins; D007128:Immunoglobulin Fragments; D000922:Immunotoxins; D053650:Interleukin-3 Receptor alpha Subunit; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A; C055966:immunoglobulin Fv