Summary In recent years, radiolabelled monoclonal antibodies have been evaluated for their use in the diagnosis and treatment of neoplastic disease. One isotope which has not been assessed for antibody targeting iS 32P, even though it has many favourable radiobiological characteristics and has been used clinically for the treatment of certain neoplastic disorders such as polycythaemia rubra vera. The main drawback so far in using 32P has been the absence of a general method for phosphorylating antibodies. We have now developed a novel process for the phosphorylation of immunoglobulins which is rapid, efficient and allows high specific activities to be achieved (>l10Ciyg-1). The Recently, however, a novel method for the covalent coupling of the strong chelating group diethylenetriaminepentaacetic acid (DTPA) to antibodies has been developed (Hnatowich et al., 1983a; Scheinberg et al., 1982;Paxton et al., 1985) which allows antibodies to be labelled with metallic radionuclides such as "'1In (Hnatowich et al., 1983a, b; Scheinberg et al., 1982;Paxton et al., 1985) and 99mTC (Lanteigne & Hnatowich, 1984).More recently another metallic radionuclide, 90Y, has been used to label DTPA-linked antibodies (Hnatowich et al., 1985) and promising radioimmunotherapeutic results have been reported (Order et al., 1986). This isotope is of considerable interest because of its pure f-ray particle emission, high emission energy, half-life and stable daughter products. However the use of 90Y does have disadvantages requiring the availability of a 90Sr-90Y generator and, in common with other chelated metallic radionuclides, there is a loss of 90Y from the antibody and large uptake by liver, kidneys, bone and bone marrow (Hnatowich et al., 1985).Another isotope with many of the ideal propeties of 90Y is 32p. Though having a longer half-life (14 days) than 90Y, this isotope has been used clinically in cancer therapy for many years (Today's drugs, 1967;Boye et al., 1984 advantageous for attacking tumours with low vascularity. Until now there has been no simple method for conjugating 32p to antibodies. In this communication we describe a simple, rapid procedure for phosphorylating antibodies which allows high specific activities to be achieved without any significant impairment of antibody function. Kemptide (Kemp et al., 1976), a heptapeptide substrate (Leu. Arg. Arg. Ala. Ser. Leu. Gly) for the cAMP-dependent protein kinase, is first covalently linked to the antibody and then phosphorylated with 32P-y-ATP and protein kinase. The antibody 32P-Kemptide conjugates show no impairment of antibody function, are stable in serum, and have a slow rate of clearance in mice (t,,2=2 days).
Materials and methods
ReagentsMonoclonal antibody secreting hybridomas were kindly provided by the following: OX7, Dr A