A recombinant immunotoxin targeting CD22 with low immunogenicity, low nonspecific toxicity, and high antitumor activity in mice

J Immunother. 2010 Apr;33(3):297-304. doi: 10.1097/CJI.0b013e3181cd1164.

Abstract

Recombinant immunotoxins (RITs) are genetically engineered proteins designed to kill cancer cells. The RIT HA22 contains the Fv portion of an anti-CD22 antibody fused to a 38 kDa fragment of Pseudomonas exotoxin A (PE38). As PE38 is a bacterial protein, patients frequently produce antibodies that neutralize its activity, preventing retreatment. We have earlier shown in mice that PE38 contains 7 major B-cell epitopes located in domains II and III of the protein. Here we present a new mutant RIT, HA22-LR-6X, in which we removed most B-cell epitopes by deleting domain II and mutating 6 residues in domain III. HA22-LR-6X is cytotoxic to several lymphoma cell lines, has very low nonspecific toxicity, and retains potent antitumor activity in mice with CA46 lymphomas. To assess its immunogenicity, we immunized 3 MHC-divergent strains of mice with 5 microg doses of HA22-LR-6X, and found that HA22-LR-6X elicited significantly lower antibody responses than HA22 or other mutant RITs with fewer epitopes removed. Furthermore, large (50 microg) doses of HA22-LR-6X induced markedly lower antibody responses than 5 microg of HA22, indicating that high doses can be administered with low immunogenicity. Our experiments show that we have correctly identified and removed B-cell epitopes from PE38, producing a highly active immunotoxin with low immunogenicity and low animal toxicity. Future studies will determine if these properties carry over to humans with cancer.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, N.I.H., Intramural

MeSH terms

  • ADP Ribose Transferases / genetics
  • ADP Ribose Transferases / immunology
  • Animals
  • Antibodies / blood
  • Antibodies / immunology
  • Antibodies, Bacterial / blood
  • Antibodies, Bacterial / immunology
  • Bacterial Toxins / genetics
  • Bacterial Toxins / immunology
  • Burkitt Lymphoma / immunology*
  • Burkitt Lymphoma / pathology
  • Burkitt Lymphoma / prevention & control
  • Cell Line, Tumor
  • Enzyme-Linked Immunosorbent Assay
  • Exotoxins / genetics
  • Exotoxins / immunology
  • Female
  • Humans
  • Immunization / methods
  • Immunotoxins / administration & dosage
  • Immunotoxins / genetics
  • Immunotoxins / immunology*
  • Male
  • Mice
  • Mice, Inbred BALB C
  • Mice, Inbred C57BL
  • Mice, Inbred NOD
  • Mice, Inbred Strains
  • Mice, SCID
  • Recombinant Proteins / immunology
  • Sialic Acid Binding Ig-like Lectin 2 / immunology*
  • Species Specificity
  • Virulence Factors / genetics
  • Virulence Factors / immunology
  • Xenograft Model Antitumor Assays

Substances

  • Antibodies
  • Antibodies, Bacterial
  • Bacterial Toxins
  • Exotoxins
  • Immunotoxins
  • Recombinant Proteins
  • Sialic Acid Binding Ig-like Lectin 2
  • Virulence Factors
  • ADP Ribose Transferases
  • toxA protein, Pseudomonas aeruginosa