A novel method to purify immunotoxins from free antibodies using modified recombinant toxins

• Published in: Journal of immunological methods Vol. 182; no. 2; pp. 165 - 175
• Main Authors: Dhanabal, Mohanraj, Fryxell, Daniel K., Ramakrishnan, S.
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 09-06-1995; Elsevier
• Subjects: Analysis of the immune response. Humoral and cellular immunity; Animals; Antibodies, Monoclonal - isolation & purification; Antibody; Antibody Specificity; Antigens, Neoplasm; Base Sequence; Biological and medical sciences; Breast Neoplasms - immunology; Chromatography, Affinity - methods; Chromatography, Agarose - methods; Corynebacterium diphtheriae; Diphtheria Toxin - genetics; Diphtheria Toxin - isolation & purification; Diphtheria toxin A chain; DNA Primers - genetics; Female; Fundamental and applied biological sciences. Psychology; Fundamental immunology; Humans; Immunobiology; Immunologic Techniques; Immunotoxin; Immunotoxins - isolation & purification; Lymphokines, interleukins ( function, expression); Mice; Molecular Sequence Data; Ovarian Neoplasms - immunology; Peptide Fragments - genetics; Peptide Fragments - isolation & purification; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Regulatory factors and their cellular receptors; Ricin - genetics; Ricin - isolation & purification; Ricin A chain; Tumor Cells, Cultured; Immunotoxin; Diphtheria toxin A chain; Ricin A chain; Antibody; Toxin; Purification; Method; Column chromatography
• ISSN: 0022-1759; 1872-7905
• DOI: 10.1016/0022-1759(95)00036-A

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 trace amount of free antibody. Present studies describe a method to purify immunotoxins from free antibody in conjugation mixtures. Recombinant 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 biological activity were chemically linked to monoclonal antibodies (317G5 and 454C11) reactive to breast cancer cells. The high affinity of consecutive histidine residues for nickel-based resin (Ni-NTA) was exploited to purify immunotoxins from unreacted free antibodies. SDS-PAGE analysis of conjugates eluted from nickel column contained trace amounts of detectable free antibody whereas conjugates purified by other conventional methods using phenyl Sepharose or Cibacron blue Sepharose chromatography contained significant amounts of unconjugated antibody. Furthermore, the immunotoxin fraction containing predominantly two toxin molecules linked to one antibody can be separated from stoichiometric conjugates by Ni-NTA column. Cytotoxicity experiments showed that the complex of two toxin molecules linked to an antibody was more cytotoxic to tumor cells in vitro than the fraction enriched with immunotoxin containing equimolar stoichiometry.