Humanization of immunotoxins.
The construction and expression of a chimeric gene encoding a mouse/human antibody to the human transferrin receptor fused to the gene for angiogenin, a human homolog of pancreatic RNase, are described. F(ab')2-like antibody-enzyme fusions were prepared by linking the gene for human angiogenin to a chimeric anti-transferrin receptor heavy chain gene. The antibody-enzyme fusion gene was introduced into a transfectoma that secretes the chimeric light chain of the same antibody, and cell lines were cloned that synthesize and secrete the antibody-enzyme fusion protein of the expected size at a concentration of 1-5 ng/ml. Culture supernatants from clones secreting the fusion protein caused inhibition of growth and protein synthesis of K562 cells that express the human transferrin receptor but not toward a non-human-derived cell line that lacks this receptor. Whereas excess antibody to the same receptor did not itself inhibit protein synthesis, it was able to completely prevent the protein synthesis inhibition caused by the fusion protein. These results indicate that the cytotoxicity is due to a transferrin receptor-mediated mechanism involving the angiogenin portion of the fusion protein and demonstrate the feasibility of constructing recombinant antibody-RNase molecules capable of killing tumor cells bearing the transferrin receptor. The significance of the acquired cytotoxicity of a mouse/human chimeric antibody linked to a human protein may bear importantly in human therapeutic strategies that use mouse antibodies linked to toxins from plants or bacteria to target tumor cells. It is expected that the humanization of immunotoxins will lead to less toxicity and immunogenicity than currently available reagents.
Rybak
S M
SM
Biochemistry Section, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Bethesda, MD 20896.
Hoogenboom
H R
HR
Meade
H M
HM
Raus
J C
JC
Schwartz
D
D
Youle
R J
RJ
D000818:Animals; D000906:Antibodies; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D007700:Kinetics; D015464:Leukemia, Myelogenous, Chronic, BCR-ABL Positive; D051379:Mice; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010957:Plasmids; D011506:Proteins / Q000235:genetics* / Q000276:immunology; D011990:Receptors, Transferrin / Q000235:genetics* / Q000276:immunology; D011993:Recombinant Fusion Proteins / Q000494:pharmacology; D015183:Restriction Mapping; D012259:Ribonuclease, Pancreatic; D014162:Transfection
D000906:Antibodies; D000922:Immunotoxins; D009363:Neoplasm Proteins; D011506:Proteins; D011990:Receptors, Transferrin; D011993:Recombinant Fusion Proteins; C046366:angiogenin; D012259:Ribonuclease, Pancreatic