Properties of Bispecific Single Chain Antibodies Expressed in Escherichia coli

Single chain bispecific antibodies, in which the genes that encode the V_H and V_L regions are linked in tandem, may offer some advantages over other methods of bispecific antibody preparation. To begin to evaluate the potential of this system, a single chain bispecific antibody (scFv₂) that binds to the T cell receptor of a cytotoxic T cell clone and to the hapten fluorescein was constructed. The individual scFv regions were joined by a 25 amino acid linker, and the scFv₂ protein was obtained from insoluble inclusion bodies after guanidine solubilization and refolding. Fluorescein-purified scFv₂ is active at concentrations of ∼10 nM (∼1 μg/ml) and above in mediating lysis of fluorescein-coupled tumor cells by the cytotoxic T lymphocytes (CTL). This system has now been used to evaluate various features of the scFv₂ approach: (a) affinities of the scFv₂ for the TCR and fluorescein, (b) yields of the scFv₂ from several different purification schemes, (c) relationship of antigen density (i.e., fluorescein density on tumor cells) to the ability to redirect lysis of the tumor cell, and (d) relationship of scFv₂ affinity for the tumor antigen to the ability to redirect lysis. This study was performed by using several analogs of fluorescein for which the scFv₂ had different affinities. Results showed that it should be possible to select antibodies with appropriate affinities such that tumor cells with typical antigen densities of 10⁴–10⁵ molecules per cell will be lysed and normal cells that have lower levels of the antigen will be spared.