Several attempts to attack tumours in experimental systems have been made using conjugates of chemotherapeutic agents or potent toxins with antibodies (immunotoxins). In vitro studies have been highly successful, showing target specificity of a high order in some cases. However, so far, such conjugates have been inadequate in vivo, probably for two main reasons. First, conventional heteroclonal antibodies are perhaps inappropriate, because purification by biochemical methods leaves a large amount of non-antibody gamma-globulins. The use of monoclonal antibodies may overcome this problem. Second, when whole toxins have been conjugated to antibodies there has been a strong residual nonspecific cytotoxicity due to the binding capacity of a subunit, the B-piece of the toxin. (Diphtheria toxin or ricin consist of two polypeptide subunits. The A-piece is responsible for inhibition of protein synthesis on ribosomes, and the B-piece binds to galactose residues on the cell membrane and facilitates the transmembrane passage of the A-piece.) In the present work the problem of nonspecific binding by the B-piece has been circumvented by using the A-piece only as the toxin component of immunotoxins; these immunotoxins are active both in vitro and in vivo.

Solving the structure of the stable complex between a serine protease inhibitor (serpin) and its target has been a long standing goal. We describe herein the characterization of a monoclonal antibody that selectively recognizes antithrombin in complex with either thrombin, factor Xa, or a synthetic peptide corresponding to residues P 14 to P 9 of the serpin's reactive center loop (RCL, ultimately cleaved between the P 1 and P 1 residues). Accordingly, this antibody reacts with none of the monomeric conformers of antithrombin (native, latent, and RCL-cleaved) and does not recognize heparin-activated antithrombin or antithrombin bound to a non-catalytic mutant of thrombin (S195A, in which the serine of the charge stabilizing system has been swapped for alanine). The neoepitope encompasses the motif DAFHK, located in native antithrombin on strand 4 of ␤-sheet A, which becomes strand 5 of ␤-sheet A in the RCL-cleaved and latent conformers. The inferences on the structure of the antithrombin-protease stable complex are that either a major remodeling of antithrombin accompanies the final elaboration of the complex or that, within the complex, at the most residues P 14 to P 6 of the RCL are inserted into ␤-sheet A. These conclusions limit drastically the possible locations of the defeated protease within the complex. Serine protease inhibitors (serpins)1 are mainly composed of three ␤-sheets (A, B, and C) united by nine ␣-helices (A-I); indeed, many are inhibitors that neutralize their target(s) by forming a, stoichiometric, stable complex (1-3). Formation of the stable complex involves the charge stabilizing system of the target protease (4 -8) and a surface loop of the inhibitor called the reactive center loop (RCL). The RCL connects strand 4 of ␤-sheet A to strand 1 of ␤-sheet C; it is exposed to solvent in the inhibitory serpins. By analogy with protease substrates, the 20 amino acids constituting the RCL are numbered P n -. . . -P 1 -PЈ 1 -. . . -PЈ n , where P 1 -PЈ 1 is ultimately cleaved. The mechanism of protease inhibition involves multiple steps that initiate by the formation of a reversible association, converting to a stable complex, ultimately split into regenerated enzyme and RCL-cleaved (consumed) serpin (9 -12). The RCL sustains a variety of conformations (13-16). In antithrombin (AT) that is heparin-activated (17, 18) and other inhibitory serpins such as ␣1-antitrypsin (also called ␣1-proteinase inhibitor; 19 -21) or ␣1-antichymotrypsin (22), the RCL is wholly exposed, whereas in the AT monomer, residue P 14 of the RCL disrupts ␤-sheet A (23-25). In latent AT, an intact but non-inhibitory conformer (25), and in latent type-1 plasminogen activator inhibitor (16), residues P 14 to P 3 of the RCL are completely inserted into ␤-sheet A, constituting an additional, sixth, strand. The same conversion from a five-to six-stranded ␤-sheet A occurs in the inhibitory serpins, following cleavage of the RCL (14, 26).To date, no x-ray analysis of a serpin-protease complex has been reported; thus, its structure rema...

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