Prevention of growth of leukemia cells in mice by monoclonal antibodies directed against Thy 1.1 antigen disulfide linked to two ribosomal inhibitors:pokeweed antiviral protein or ricin A chain.

Pokeweed antiviral protein (PAP) and ricin A chain are potent inhibitors of protein synthesis that inactivate eukaryotic 60S ribosomal subunits. Immunotoxins were prepared by linking monoclonal anti-Thy 1.1 antibodies to PAP and ricin A chain through a disulfide bond. Both the conjugates were shown earlier to specifically inhibit protein synthesis of Thy 1.1-positive target leukemic cells (AKR SL3). In the present study, the efficacy of the immunotoxins to prevent the growth of AKR SL3 cell-induced tumor was checked in vivo in a model system. Injection of AKR SL3 cells s.c. into AKR/Cum (Thy 1.2-positive) mice developed into a solid tumor which was fatal. Administration of 31-E6:PAP and 31-E6:ricin A chain suppressed tumor growth. Suppression was specific, as similar treatment could not prevent the growth of a nontarget Thy 1.2-positive leukemia cell line (AKR SL1) derived from a congenic mouse. Unconjugated anti-Thy 1.1 immunoglobulin antibodies also showed significant tumor protection; however, administration of F(ab')2 fragment could not prevent the tumor growth. Injection of F(ab')2:PAP efficiently protected mice from AKR SL3-induced tumor. All the conjugate-treated mice showed antibody response against the toxin polypeptide. Anti-toxin antibody response was found as early as 26 days after the initiation of therapy and lasted as long as 179 days of observation. Further studies indicate that the presence of anti-toxin antibodies blocked completely the inhibitory ability of the respective immunotoxin in vitro. Anti-ricin antibodies neutralized the activity of 31-E6:ricin A chain conjugate but not OX-7:PAP immunotoxin, and similarly, anti-PAP antibodies inhibited the activity of the latter and not the activity of 31-E6:ricin A chain conjugate. These observations indicate that the use of alternate immunotoxins having an immunologically distinct toxin polypeptide may be necessary for tumor therapy during relapse, as exposure to the conjugates results in the formation of specific neutralizing anti-toxin antibodies. The anti-toxin antibodies did not prevent the binding of immunotoxin to target cells. Nevertheless, preincubation of conjugate with anti-toxin antibodies specifically blocked the respective conjugate-induced inhibition of polyuridylic acid translation in a cell-free assay system.