Conjugation of monoclonal antibodies to a synthetic peptide substrate for protein kinase: a method for labelling antibodies with 32P

Abstract

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

Construction of Phosphorylatable Chimeric Monoclonal Antibody CC49 with a Casein Kinase I Recognition Site

“…Re. In addition, the introduction of a kinase recognition site into the monoclonal antibody keeps the essential structure of the antibody intact in contrast to chemical conjugation of chelating agents necessary to bind heavy-metal radioisotopes (3, 4) or chem-ical linking of peptides with a cAMP-phosphorylation site (25). The attachment of peptides chemically to a protein creates new epitopes not present in any natural proteins of the body as both the chemical linkers used and the peptides attached form multiple new epitopes.…”

Introduction of Protein Kinase Recognition Sites into Proteins: A Review of Their Preparation, Advantages, and Applications

“…The details of the various constructions are given in the specific reports cited. Foxwell et al (11) used a non-genetic engineering procedure to introduce the cAMP-dependent protein kinase recognition site into monoclonal antibodies by using peptides containing the PKA sequence and then covalently linking these chemically to a monoclonal antibody. The phosphorylated monoclonal antibody was stable in serum as were the attached phosphates.…”

“…The introduction of a kinase recognition site into proteins keeps their essential structure intact in contrast to chemical conjugation of chelating agents necessary to bind heavy metal radioisotopes (9,10) or chemical linking of peptides with a cAMP-phosphorylation site (11). This is especially significant as antibodies are being used as human therapeutics of which it is important to minimize antigenicity.…”

Site-Specific 32 P-Labeling of Cytokines, Monoclonal Antibodies, and Other Protein Substrates for Quantitative Assays and Therapeutic Application

“…Another approach developed by Foxwell et al (18) introduced a kinase recognition site into a peptide that was then covalently linked to a monoclonal antibody. Whereas the phosphorylated monoclonal was stable in serum, proteins modified by this method would have limited potential in vivo as this approach appears to favor the generation of a heterogeneous product carrying multiple new epitopes, thus enhancing antigenicity.…”