Activity of recombinant mitogillin and mitogillin immunoconjugates.
A synthetic gene for the Aspergillus protein toxin mitogillin has been synthesized and expressed in Escherichia coli. The recombinant mitogillin is a potent inhibitor of protein synthesis in vitro with an IC50 of 9.7 pM. Immunoconjugates of recombinant mitogillin derivatized with S-acetylmercaptosuccinic anhydride and 5-methyl-2-iminothiolane modified H65 antibody kill T cell lines and peripheral blood mononuclear cells expressing the human CD5 surface antigen. Native mitogillin contains 4 cysteine residues which form two disulfide pairs (Fernandez-Luna, J. L., Lopez-Otin, C., Soriano, F., and Mendez, E. (1985) Biochemistry 24, 861-867). Three derivatives of mitogillin have been assembled which substitute alanine residues for cysteine residues 5, 147, or 5 and 147. Each of these molecules retains the ability to inhibit protein synthesis in vitro with at most a 2-fold reduction in activity. The derivative mitogillinC147A can be conjugated to 5-methyl-2-iminothiolane- modified H65 antibody directly without pretreatment with S-acetylmercaptosuccinic anhydride, and the immunoconjugate is active against HSB2 cells. Genetic manipulation of toxin genes to expose an accessible cysteine residue into a recombinant product can thus be used to generate immunotoxins without initial derivatization by nonspecific cross-linking reagents.
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XOMA Corporation, Santa Monica, California 90404.
Bernhard
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Lei
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Fishwild
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Carroll
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D000328:Adult; D000485:Allergens; D000595:Amino Acid Sequence; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology*; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D001230:Aspergillus / Q000235:genetics; D001483:Base Sequence; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D004926:Escherichia coli / Q000235:genetics; D005656:Fungal Proteins / Q000235:genetics / Q000302:isolation & purification / Q000494:pharmacology*; D005813:Genes, Synthetic; D005914:Globins / Q000235:genetics; D006801:Humans; D000922:Immunotoxins / Q000633:toxicity*; D008969:Molecular Sequence Data; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D011817:Rabbits; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000494:pharmacology; D015183:Restriction Mapping; D012156:Reticulocytes / Q000378:metabolism; D012260:Ribonucleases; D013601:T-Lymphocytes / Q000166:cytology / Q000187:drug effects*
C081947:ASPF1 protein, Aspergillus fumigatus; D000485:Allergens; D000903:Antibiotics, Antineoplastic; D000911:Antibodies, Monoclonal; D052179:Antigens, Plant; D005656:Fungal Proteins; D000922:Immunotoxins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; C036196:MITF protein, Aspergillus restrictus; D005914:Globins; D012260:Ribonucleases