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 (V-H) and the ligh t (V-L) chain variable domains, which, without modification, tend to d issociate, unfold, and/or nonspecifically aggregate. The fragment is u sually stabilized by producing it as a single chain recombinant molecu le 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 site s in the framework region of the Fv fragment of the monoclonal mouse a ntibody (mAb)B3. The mAb B3 binds to many human cancer cells and is be ing used in the development of a new anticancer agent. The two sites i dentified are V(H)44-V(L)105 and V(H)111-V(L)48. (V(H)44-V(L)100 and V (H)105-V(L)43 in the numbering scheme of Kabat et al., ''Sequence of P roteins of Immunological Interest,'' U.S. DHHS, NIH publication No. 91 -3242, 1991.) This design was recently tested using the chimeric prote in composed of a truncated form of Pseudomonas exotoxin and the Fv fra gment of mAb B3 with the engineered disulfide bond at V(H)44-V(L)105 ( Brinkmann et al., Proc. Natl. Acad. Sci. U.S.A. 90:7538, 1993). The ch imeric toxin was found to be just as active as the corresponding singl e chain counterpart and considerably more stable. Because these disulf ide bond sites are in the framework region, they can be located from s equence alignment alone. We expect that the disulfide bond at these si tes will stabilize the Fv fragment of most antibodies and the antigen- specific portion of the T-cell receptors, which are homologous. (C) 19 94 Wiley-Liss, Inc.