title: The efficacy of CD3 x CD19 bispecific monoclonal antibody (BsAb) in a clonogenic assay: the effect of repeated addition of BsAb and interleukin-2.
abstract: To evaluate the potency by which human T cells are targeted and activated by bispecific monoclonal antibodies (BsAbs) to lyse tumor cells, a clonogenic assay was developed. The efficacy of a CD3 x CD19 BsAb binding to both the CD3 T-cell antigen and the CD19 B-cell antigen was already proven in 51Cr-release assays and in 3-day activation cultures. To achieve more quantitative results, a 14-day clonogenic assay, based on limiting-dilution, was performed for the determination of the initial and residual number of clonogenic units obtained with a CD19+ pre-pre-B acute lymphoblastic leukemia (ALL-B) cell line. Elimination of up to 5 logs of ALL-B cells by freshly isolated peripheral blood mononuclear cells (PBMCs) cultured with BsAb plus interleukin-2 (IL-2) could be detected. The presence of human IgG did not abolish the effect. Repeated addition of each of the two agents was necessary, because a single treatment produced only a 1- to 2-log kill. CD3 monoclonal antibody and IL-2 stimulation ("lymphokine-activated killer cell" conditions) resulted in only a 2-log kill. The number of T cells proved critical in lysis of ALL-B cells, with a 5-log kill using a T-cell:B-cell ratio of 3:1 but with only a 1-log kill using a ratio of 1:1. PBMCs isolated from patients with non-Hodgkin's lymphoma, both in relapse or remission, proved to be as competent as those from healthy donors in removing ALL-B cells. This clonogenic assay shows the importance of repeated administration of CD3 x CD19 BsAb and IL-2 and offers the possibility to compare it with other therapies in B-cell malignancy.
authors.affiliation: Department of Immunology and Hematology, University Hospital of Utrecht, The Netherlands.
authors.forename: I A
authors.initials: IA
authors.lastname: Haagen
authors.forename: A J
authors.initials: AJ
authors.lastname: Geerars
authors.forename: W B
authors.initials: WB
authors.lastname: de Lau
authors.forename: B J
authors.initials: BJ
authors.lastname: Bast
authors.forename: B C
authors.initials: BC
authors.lastname: de Gast
mesh_terms: D018033:Antibodies, Bispecific / Q000276:immunology / Q000494:pharmacology* / Q000627:therapeutic use; D015703:Antigens, CD / Q000276:immunology*; D018941:Antigens, CD19; D000944:Antigens, Differentiation, B-Lymphocyte / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D003602:Cytotoxicity, Immunologic / Q000187:drug effects; D006801:Humans; D007376:Interleukin-2 / Q000494:pharmacology*; D015979:Killer Cells, Lymphokine-Activated / Q000187:drug effects* / Q000276:immunology; D008213:Lymphocyte Activation / Q000187:drug effects; D008228:Lymphoma, Non-Hodgkin / Q000276:immunology / Q000473:pathology / Q000628:therapy; D015452:Precursor B-Cell Lymphoblastic Leukemia-Lymphoma / Q000473:pathology / Q000628:therapy*; D011994:Recombinant Proteins / Q000494:pharmacology; D016176:T-Lymphocyte Subsets / Q000187:drug effects* / Q000276:immunology; D013997:Time Factors; D014407:Tumor Cells, Cultured / Q000187:drug effects; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology; D014410:Tumor Stem Cell Assay / Q000379:methods*
chemical_list: D018033:Antibodies, Bispecific; D015703:Antigens, CD; D018941:Antigens, CD19; D000944:Antigens, Differentiation, B-Lymphocyte; D017252:CD3 Complex; D007376:Interleukin-2; D011994:Recombinant Proteins; D014409:Tumor Necrosis Factor-alpha