title: Phage display-based generation of novel internalizing antibody fragments for immunotoxin-based treatment of acute myeloid leukemia.
abstract: The current standard treatment for acute myeloid leukemia (AML) is chemotherapy based on cytarabine and daunorubicine (7 + 3), but it discriminates poorly between malignant and benign cells. Dose-limiting off‑target effects and intrinsic drug resistance result in the inefficient eradication of leukemic blast cells and their survival beyond remission. This minimal residual disease is the major cause of relapse and is responsible for a 5-year survival rate of only 24%. More specific and efficient approaches are therefore required to eradicate malignant cells while leaving healthy cells unaffected. In this study, we generated scFv antibodies that bind specifically to the surface of AML blast cells and AML bone marrow biopsy specimens. We isolated the antibodies by phage display, using subtractive whole-cell panning with AML M2‑derived Kasumi‑1 cells. By selecting for internalizing scFv antibody fragments, we focused on potentially novel agents for intracellular drug delivery and tumor modulation. Two independent methods showed that 4 binders were internalized by Kasumi-1 cells. Furthermore, we observed the AML‑selective inhibition of cell proliferation and the induction of apoptosis by a recombinant immunotoxin comprising one scFv fused to a truncated form of Pseudomonas exotoxin A (ETA'). This method may therefore be useful for the selection of novel disease-specific internalizing antibody fragments, providing a novel immunotherapeutic strategy for the treatment of AML patients.
authors.affiliation: a Department of Experimental Medicine and Immunotherapy; Institute of Applied Medical Engineering; Helmholtz-Institute for Biomedical Engineering ; University Hospital RWTH Aachen ; Aachen , Germany.
authors.forename: Jenny
authors.initials: J
authors.lastname: Fitting
authors.forename: Tobias
authors.initials: T
authors.lastname: Blume
authors.forename: Andre
authors.initials: A
authors.lastname: Ten Haaf
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Blau
authors.forename: Stefan
authors.initials: S
authors.lastname: Gattenlöhner
authors.forename: Mehmet Kemal
authors.initials: MK
authors.lastname: Tur
authors.forename: Stefan
authors.initials: S
authors.lastname: Barth
mesh_terms: D036002:ADP Ribose Transferases / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D000912:Antibodies, Neoplasm / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D000918:Antibody Specificity / Q000235:genetics; D001427:Bacterial Toxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D001752:Blast Crisis / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D045744:Cell Line, Tumor; D005098:Exotoxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D006801:Humans; D000922:Immunotoxins / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D015470:Leukemia, Myeloid, Acute / Q000188:drug therapy* / Q000276:immunology / Q000473:pathology; D057127:Single-Chain Antibodies / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D037521:Virulence Factors / Q000235:genetics / Q000276:immunology / Q000494:pharmacology; D000097668:Pseudomonas aeruginosa Exotoxin A
chemical_list: D036002:ADP Ribose Transferases; D000912:Antibodies, Neoplasm; D001427:Bacterial Toxins; D005098:Exotoxins; D000922:Immunotoxins; D057127:Single-Chain Antibodies; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
keywords: AML immunohistochemistry; AML, acute myeloid leukemia; CDR, Complementarity Determining Region; ETA, Pseudomonas exotoxin A; PBMC, peripheral blood mononuclear cell; acute myeloid leukemia; anti-AML antibody fragments; in vitro efficacy; internalizing phage antibodies; phage display technology; primary AML blast cells; recombinant immunotoxin; scFv, single chain variable fragment