Cysteine analogs of recombinant barley ribosome inactivating protein form antibody conjugates with enhanced stability and potency in vitro.
Antibody immunoconjugates were made with native and recombinant forms of the type-I ribosome inactivating protein from barley (BRIP) and with three recombinant BRIP (rBRIP) analogs engineered to contain a unique cysteine residue near the C terminus (at amino acid 256, 270, or 277). rBRIP and all three cysteine analogs (rBRIPc256, rBRIPc270, and rBRIPc277) were produced in E. coli, with yields of soluble protein as high as 1 g/L, and were as active as native BRIP in inhibiting protein synthesis in vitro. Interestingly, the position of the engineered cysteine influenced not only the efficiency of conjugation to antibody but also the efficacy and disulfide bond stability of the immunoconjugates. Anti-CD5 antibody conjugates prepared with native and rBRIP were relatively inactive against antigen-positive target cells, while the conjugate made with rBRIPc277 was 5-fold more cytotoxic. Anti-CD7 antibody conjugates made with rBRIPc277 or rBRIPc270 also exhibited improved potency and stability compared to the conjugate with native BRIP. These results indicate that engineering a cysteine residue into selected positions near the C-terminus of a type-IRIP such as BRIP can improve immunoconjugate yield, disulfide bond stability, and potency.
XOMA Corporation, Berkeley, California 94710.
S L
SL
Bernhard
M
M
Better
D M
DM
Fishwild
J A
JA
Lane
A E
AE
Orme
D A
DA
Garrison
C A
CA
Birr
S P
SP
Lei
S F
SF
Carroll
D000818:Animals; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D003545:Cysteine / Q000031:analogs & derivatives* / Q000737:chemistry / Q000276:immunology; D003601:Cytotoxicity Tests, Immunologic; D004220:Disulfides / Q000737:chemistry; D004355:Drug Stability; D004926:Escherichia coli / Q000378:metabolism; D006801:Humans; D000922:Immunotoxins / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D009699:N-Glycosyl Hydrolases; D009363:Neoplasm Proteins / Q000096:biosynthesis; D010940:Plant Proteins / Q000737:chemistry* / Q000276:immunology / Q000494:pharmacology; D010957:Plasmids; D011994:Recombinant Proteins / Q000737:chemistry / Q000276:immunology / Q000494:pharmacology; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000187:drug effects*; D013329:Structure-Activity Relationship; D014118:Toxins, Biological; D014407:Tumor Cells, Cultured
D004220:Disulfides; D000922:Immunotoxins; D009363:Neoplasm Proteins; D010940:Plant Proteins; D011994:Recombinant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D014118:Toxins, Biological; D009699:N-Glycosyl Hydrolases; C487943:ribosome-inactivating protein, Hordeum vulgare; D003545:Cysteine