The use of hybrid hybridomas to target human cytotoxic T lymphocytes

Abstract

This study describes a general strategy to produce hybrid monoclonal antibodies that are capable of targeting human cytotoxic T lymphocytes (CTL) against any cell carrying the appropriate target antigen. This is done by fusing a HAT-sensitive, G418-resistant anti-T3 hybridoma with immune spleen cells (or with other hybridomas) that produce antibodies against the desired target antigen. In the hybrid hybridomas the reassortment of Ig heavy and light chains results in the production of bifunctional antibody molecules. Because of their double specificity, these antibodies are able to bridge human CTL to target cells and trigger cytotoxic function. We have isolated several stable hybrid hybridomas in which one specificity is against T3 and one either against HLA antigens (class II, DC-1, A3), human Ig (IgM, IgE, kappa), Toxoplasma gondii or an ovary carcinoma-associated antigen. In all of these cases we show that culture supernatants can efficiently and specifically target any CTL clone against any target cell that possesses the relevant surface antigen recognized by the antibody. Furthermore, the killing requires as little as 0.1 ng/ml of antibody, occurs at effector to target ratios comparable to those used in conventional cytotoxic assays and does not affect bystander cells. Nonspecific killing of Fc receptor-positive cells can be avoided using F(ab')2 fragments. As an example, we show that it is possible to change the major histocompatibility complex class and allospecificity of a CTL clone and target CTL against non-major histocompatibility complex antigens such as Ig, parasites and tumor-associated antigens.

Induction of Human T Lymphocyte Cytotoxicity and Inhibition of Tumor Growth by Tumor-Specific Diabody-Based Molecules Secreted from Gene-Modified Bystander Cells

“…Although various bispecific Ab fragments (tandem single-chain variable fragment (scFv) and single-chain diabodies) have already been shown to be expressed and secreted by eukaryotic cells (37,38), our results demonstrate for the first time that human cells can be engineered to simultaneously produce a bispecific, two-chain dAb and a chimeric B7 dAb fusion protein. The use of diabodies would have some advantages over the use of other formats of bispecific Abs, including the lack of an Fc portion, thus avoiding the killing of FcR-positive bystander cells (39) and their small size for better penetration of tumors (40). The anti-CEA Ab used in this study has shown excellent tumor localization in colon carcinoma patients in an scFv format (41) and in colon-carcinoma xenografts in nude mice, both as a bispecific dAb ␣CEA ϫ ␣CD3 (data not shown) as well as a bivalent ␣CEA dAb (42).…”

New simplified molecular design for functional T cell receptor

“…4A, B) and IL-3 (data not shown) in a manner which was dependent on the number of each of the two types of stimulator cells. Furthermore, this stimulation could be inhibited specifically by TR66 (data not shown), the CD3&-specific mAb [8] from which the FvCD3 domain of our chimeric TcR was derived. We tested eight BW transfectants in total and all showed similar stimulation patterns to those showed in Fig.…”

“…1B); TR66 is specific for the human C D~E chain [8], which was already successfully modified to bispecific single-chain Fv molecules by Traunecker et al [lo]. The final construction (Fig.…”

Bispecific Minibodies Targeting HER2/neu and CD16 Exhibit Improved Tumor Lysis When Placed in a Divalent Tumor Antigen Binding Format

“…For example, BsAbs can target tumor antigens and human effector cell trigger molecules on T-cells via CD3/TcR (24,25) and CD28 (26). BsAbs directed against Fc␥RI (27) trigger tumor cytotoxicity by neutrophils, monocytes, and macrophages.…”