THE ANTI-TUMOR ANTIBODY BR96: X-RAY STRUCTURES IN ANTIGEN-BOUND AND IN FREE FORM. S. Sheriff*, C. Chang*, P.D. Jeffrey*, J. Bajorath⁺. Bristol-Myers Squibb Pharmaceutical Research Institute, *P.O. Box 4000, Princeton, NJ 08543, and ⁺3005 First Avenue, Seattle, WA 98121

Selective delivery of cytotoxic agents to tumors can be accomplished by using immunoconjugates containing monoclonal antibodies with specificity for tumor-associated antigens. The murine monoclonal antibody mBR96 (IgG3, [[kappa]]) was raised against human breast carcinoma cells. For its potential application in cancer chemotherapy, a chimeric form (cBR96) has been constructed from murine variable domains and human [[kappa]] and [[gamma]]1 constant domains. BR96 recognizes the Lewis Y (Le^y) tetrasaccharide, which was used for crystallization as the nonoate methyl ester:

Gal ([[beta]] 1->4) Nag ([[beta]] 1->3) O1-(CH₂)₈-COOCH₃

_[[arrowup]] ([[alpha]] 1->2) _[[arrowup]] ([[alpha]] 1->3)

Fuc Fuc

The structures of cBR96 Fab[[minute]], cBR96 Fab[[minute]]-Le^y and mBR96 Fab-Le^y have been determined by X-ray crystallography. BR96 binds to Le^y and interacts through complementarity determining regions (CDRs): L1, L3, H1, H2 and H3. In the binding site, a number of aromatic residues interact with Le^y: His L27D, Tyr L32, Phe L96, Tyr H32, Tyr H33, Tyr H35, Tyr H50, and Trp H100A (Kabat numbering). In addition to interactions with aromatic groups, several hydrogen bonds are formed between the antibody and Le^y. BR96 also interacts with the (CH₂)₈COOCH₃ suggesting that the antibody may be capable of recognizing a larger carbohydrate antigen.

Comparison of antigen-bound and free forms of BR96 show that VL and VH do not re-orient, but that three CDR loops undergo segmental motion (L3), conformation rearrangement (H2) or both (L1) upon antigen binding. In L1, differences are observed that are greater than 10 Å between C[[alpha]] positions. However, in contrast to other antibodies the conformation of H3 does not change significantly, despite extensive main chain interactions with Le^y.