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Construction and biological activity of a recombinant bispecific single-chain antibody designed for therapy of minimal residual colorectal cancer
- Source :
- Cancer Immunology, Immunotherapy; 19971124, Vol. 45 Issue: 3-4 p193-197, 5p
- Publication Year :
- 1997
-
Abstract
- 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 [10, 2]. 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: VL<subscript>17-1A</subscript>-VH<subscript>17-1A</subscript>-VH<subscript>CD3</subscript>-VL<subscript>CD3</subscript>. 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 remarkably, 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 α, interleukin-6 and interferon γ. Maximal cytotoxicity (<superscript>51</superscript>Cr release) was achieved without notable costimulation and was not further enhanced by adding costimulatory signals such as those delivered by anti-CD28 antibodies. CD8<superscript>+</superscript> as well as CD4<superscript>+</superscript> T cell subpopulations were recruited to exert cytotoxicity against tumor cells with different kinetics. CD8<superscript>+</superscript> cells induced high <superscript>51</superscript>Cr release within 4 h while CD4<superscript>+</superscript> 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 virto.
Details
- Language :
- English
- ISSN :
- 03407004 and 14320851
- Volume :
- 45
- Issue :
- 3-4
- Database :
- Supplemental Index
- Journal :
- Cancer Immunology, Immunotherapy
- Publication Type :
- Periodical
- Accession number :
- ejs476553
- Full Text :
- https://doi.org/10.1007/s002620050431