title: Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer.
abstract: Unlike monoclonal antibodies, clinical application of bispecific antibodies has so far lagged behind because of difficult, low-yield production techniques as well as considerable toxicity attributed to bispecific antibody preparations containing immunoglobulin-Fc parts and anti-CD3 homodimers. These difficulties were overcome by recombinant generation of a bispecific single-chain antibody (bscAb) joining two single-chain Fv fragments via a five-amino-acid glycine-serine linker. The anti-CD3 specificity directed against human T cells was combined with another specificity against the epithelial 17-1A antigen. The following domain arrangement was critical in this individual case to obtain a fully functional bscAb: VL17-1A-VH17-1A-VHCD3-VLCD3. The bscAb was expressed in chinese hamster ovary cells with a yield of 15 mg/l culture supernatant whereas numerous attempts to obtain a functional protein expression in Escherichia coli failed. The low-molecular-mass bispecific construct (60 kDa) could easily be purified by its C-terminal histidine tail. The antigen-binding properties are indistinguishable from those of the corresponding univalent single-chain Fv fragments as shown by enzyme immunoassay and flow cytometry. We could show that the bscAb, which lacks Fc parts and anti-CD3 homodimers is highly cytotoxic for 17-1A positive tumor cells in nanomolar concentrations and in the presence of human T cells. Most remarkable, it does not stimulate T lymphocyte proliferation in the absence of tumor cells and, moreover, does not induce CD25 up-regulation and the secretion of potentially toxic lymphokines such as tumor necrosis factor alpha, interleukin-6 and interferon gamma. Maximal cytotoxicity (51Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8+ as well as CD4+ T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8+ cells induced high 51Cr release within 4 h while CD4+ cells required a 20-h incubation. The systemic application of the 17-1A/CD3-bscAb could be a major improvement in therapy against disseminated micrometastatic tumor cells. A prospective, randomized clinical trial showing that an anti-17-1A monoclonal antibody could prolong survival of colorectal cancer patients after 5 and 7 years, warrants an assessment of the clinical efficacy of this bscAb exhibiting a 1000-fold higher specific cytotoxicity against tumor cells in vitro.
authors.affiliation: Institute of Immunology, University of Munich, Germany.
authors.forename: P
authors.initials: P
authors.lastname: Kufer
authors.forename: M
authors.initials: M
authors.lastname: Mack
authors.forename: R
authors.initials: R
authors.lastname: Gruber
authors.forename: R
authors.initials: R
authors.lastname: Lutterbüse
authors.forename: F
authors.initials: F
authors.lastname: Zettl
authors.forename: G
authors.initials: G
authors.lastname: Riethmüller
mesh_terms: D000818:Animals; D018033:Antibodies, Bispecific / Q000276:immunology / Q000627:therapeutic use*; D000951:Antigens, Neoplasm / Q000276:immunology*; D017252:CD3 Complex / Q000276:immunology*; D015496:CD4-Positive T-Lymphocytes / Q000276:immunology; D018414:CD8-Positive T-Lymphocytes / Q000276:immunology; D016466:CHO Cells; D015179:Colorectal Neoplasms / Q000628:therapy*; D006224:Cricetinae; D006801:Humans; D008213:Lymphocyte Activation / Q000276:immunology; D018365:Neoplasm, Residual; D015375:Receptors, Interleukin-2 / Q000276:immunology; D013601:T-Lymphocytes / Q000276:immunology
chemical_list: D018033:Antibodies, Bispecific; D000951:Antigens, Neoplasm; D017252:CD3 Complex; D015375:Receptors, Interleukin-2; C050928:carcinoma-associated antigen 17-1A