A NOVEL METHOD TO PURIFY IMMUNOTOXINS FROM FREE ANTIBODIES USING MODIFIED RECOMBINANT TOXINS

Monoclonal antibodies linked to toxin polypeptides (immunotoxins) are developed for clinical application against cancer and graft rejection. Immunotoxins prepared by many conventional methods often contain a tr ace amount of free antibody. Present studies describe a method to puri fy immunotoxins from free antibody in conjugation mixtures. Recombinan t ricin A chain and a truncated form of diphtheria toxin (385 residues ) containing ten consecutive histidine residues at the amino terminus were prepared. The modified toxin polypeptides retaining full biologic al activity were chemically linked to monoclonal antibodies (317G5 and 454C11) reactive to breast cancer cells. The high affinity of consecu tive histidine residues for nickel-based resin (Ni-NTA) was exploited to purify immunotoxins from unreacted free antibodies. SDS-PAGE analys is of conjugates eluted from nickel column contained trace amounts of detectable free antibody whereas conjugates purified by other conventi onal methods using phenyl Sepharose or Cibacron blue Sepharose chromat ography contained significant amounts of unconjugated antibody. Furthe rmore, the immunotoxin fraction containing predominantly two toxin mol ecules linked to one antibody can be separated from stoichiometric con jugates by Ni-NTA column. Cytotoxicity experiments showed that the com plex of two toxin molecules linked to an antibody was more cytotoxic t o tumor cells in vitro than the fraction enriched with immunotoxin con taining equimolar stoichiometry.