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.