title: A recombinant bispecific single-chain fragment variable specific for HLA class II and Fc alpha RI (CD89) recruits polymorphonuclear neutrophils for efficient lysis of malignant B lymphoid cells.
abstract: Bispecific Abs offer new perspectives for cancer immunotherapy. In this study, we describe a recombinant bispecific single-chain fragment variable (bsscFv) directed against Fc alpha RI (CD89) on polymorphonuclear neutrophils (PMNs) or monocytes/macrophages and HLA class II on lymphoma target cells. Fc alpha RI and HLA class II-directed single-chain fragment variable (scFv) fragments were isolated from phage display libraries, established from the hybridomas A77 and F3.3, respectively. The two scFv molecules were connected with a 20 aa flexible linker sequence. After expression in SF21 insect cells and chromatographic purification, the bispecific molecule showed specific binding to both Ags at K(D) values of 148 +/- 42 nM and 113 +/- 25 nM for the anti-Fc alpha RI and anti-HLA class II scFv components in the bsscFv, respectively. In Ab-dependent cytotoxicity assays with PMNs as effectors and a series of lymphoma-derived cell lines (ARH-77, RAJI, REH, NALM-6, RS4;11), the bsscFv was significantly more cytotoxic than the parental murine IgG1 and its chimeric IgG1 derivative. When targeting primary tumor cell isolates from six patients with B cell malignancies, the killing capacity of the (Fc alphaRI x HLA class II) bsscFv compared favorably to conventional HLA class II mAb. Importantly, the cell lines NALM-6 and RS411, as well as two primary tumor cell isolates, were exclusively lysed by the bsscFv. To our knowledge, this is the first report of an Fc alpha RI-directed bsscFv effectively recruiting PMNs for redirected cytotoxicity against human B cell malignancies. Our data show that an (Fc alpha RI x HLA class II) bsscFv is an interesting candidate for further engineering of small, modular immunopharmaceuticals.
authors.affiliation: Division of Hematology and Oncology, Department of Internal Medicine, University of Erlangen-Nuremberg, Erlangen, Germany.
authors.forename: Yvonne
authors.initials: Y
authors.lastname: Guettinger
authors.forename: Karin
authors.initials: K
authors.lastname: Barbin
authors.forename: Matthias
authors.initials: M
authors.lastname: Peipp
authors.forename: Joerg
authors.initials: J
authors.lastname: Bruenke
authors.forename: Michael
authors.initials: M
authors.lastname: Dechant
authors.forename: Heike
authors.initials: H
authors.lastname: Horner
authors.forename: Doreen
authors.initials: D
authors.lastname: Thierschmidt
authors.forename: Thomas
authors.initials: T
authors.lastname: Valerius
authors.forename: Roland
authors.initials: R
authors.lastname: Repp
authors.forename: Georg H
authors.initials: GH
authors.lastname: Fey
authors.forename: Bernhard
authors.initials: B
authors.lastname: Stockmeyer
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000235:genetics / Q000502:physiology*; D000920:Antibody-Dependent Cell Cytotoxicity / Q000235:genetics; D015703:Antigens, CD / Q000235:genetics / Q000502:physiology*; D016175:B-Lymphocyte Subsets / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D002460:Cell Line; D045744:Cell Line, Tumor; D002465:Cell Movement / Q000235:genetics / Q000276:immunology*; D006224:Cricetinae; D006681:HLA-D Antigens / Q000235:genetics / Q000276:immunology*; D006801:Humans; D007128:Immunoglobulin Fragments / Q000235:genetics / Q000502:physiology*; D007135:Immunoglobulin Variable Region / Q000235:genetics / Q000502:physiology*; D016393:Lymphoma, B-Cell / Q000276:immunology / Q000473:pathology / Q000628:therapy; D051379:Mice; D009504:Neutrophils / Q000276:immunology* / Q000378:metabolism / Q000473:pathology; D011485:Protein Binding / Q000235:genetics / Q000276:immunology; D011961:Receptors, Fc / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000276:immunology* / Q000378:metabolism; D014407:Tumor Cells, Cultured
chemical_list: D018033:Antibodies, Bispecific; D015703:Antigens, CD; C036832:Fc(alpha) receptor; D006681:HLA-D Antigens; D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D011961:Receptors, Fc; D011993:Recombinant Fusion Proteins; C055966:immunoglobulin Fv