Design of interchain disulfide bonds in the framework region of the Fv fragment of the monoclonal antibody B3.
The Fv fragments are the smallest units of antibodies that retain the specific antigen binding characteristics of the whole molecule and are being used for the diagnosis and therapy of human diseases. These are noncovalently associated heterodimers of the heavy (VH) and the light (VL) chain variable domains, which, without modification, tend to dissociate, unfold, and/or nonspecifically aggregate. The fragment is usually stabilized by producing it as a single chain recombinant molecule in which the two chains are linked by means of a short polypeptide linker. An alternative strategy is to connect the two chains by means of an interchain disulfide bond. We used molecular graphics and other modeling tools to identify two possible interchain disulfide bond sites in the framework region of the Fv fragment of the monoclonal mouse antibody (mAb) B3. The mAb B3 binds to many human cancer cells and is being used in the development of a new anticancer agent. The two sites identified are VH44-VL105 and VH111-VL48. (VH44-VL100 and VH105-VL43 in the numbering scheme of Kabat et al., "Sequence of Proteins of Immunological Interest," U.S. DHHS, NIH publication No. 91-3242, 1991). This design was recently tested using the chimeric protein composed of a truncated form of Pseudomonas exotoxin and the Fv fragment of mAb B3 with the engineered disulfide bond at VH44-VL105 (Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A. 90:7538, 1993). The chimeric toxin was found to be just as active as the corresponding single chain counterpart and considerably more stable. Because these disulfide bond sites are in the framework region, they can be located from sequence alignment alone. We expect that the disulfide bond at these sites will stabilize the Fv fragment of most antibodies and the antigen-specific portion of the T-cell receptors, which are homologous.
Jung
S H
SH
Division of Cancer Biology, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892.
Pastan
I
I
Lee
B
B
D036002:ADP Ribose Transferases; D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry*; D000912:Antibodies, Neoplasm / Q000737:chemistry; D001427:Bacterial Toxins; D001666:Binding Sites, Antibody; D003553:Cystine / Q000737:chemistry*; D005098:Exotoxins / Q000737:chemistry; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry; D007147:Immunoglobulin Light Chains / Q000737:chemistry; D007135:Immunoglobulin Variable Region / Q000737:chemistry*; D000922:Immunotoxins / Q000737:chemistry; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013816:Thermodynamics; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A
D036002:ADP Ribose Transferases; D000911:Antibodies, Monoclonal; D000912:Antibodies, Neoplasm; D001427:Bacterial Toxins; D003553:Cystine; D005098:Exotoxins; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D000922:Immunotoxins; D011993:Recombinant Fusion Proteins; D037521:Virulence Factors; D000097668:Pseudomonas aeruginosa Exotoxin A