Single-chain antigen-binding proteins are novel recombinant polypeptides, composed of an antibody variable light-chain amino acid sequence (VL) tethered to a variable heavy-chain sequence (VH) by a designed peptide that links the carboxyl terminus of the VL sequence to the amino terminus of the VH sequence. These proteins have the same specificities and affinities for their antigens as the monoclonal antibodies whose VL and VH sequences were used to construct the recombinant genes that were expressed in Escherichia coli. Three of these proteins, one derived from the sequence for a monoclonal antibody to growth hormone and two derived from the sequences of two different monoclonal antibodies to fluorescein, were designed, constructed, synthesized, purified, and assayed. These proteins are expected to have significant advantages over monoclonal antibodies in a number of applications.

An electron density map of concanavalin A at 2.4-A resolution has been produced by X-ray crystallographic methods with five heavy atom derivatives. The molecule is a tetramer with all subunits identical and each containing a single polypeptide chain of 231 amino acid residues. The course of the entire backbone has been traced and three different regions of /3 structure involve about 57% of the residues. One of these /3-structure regions contains six strands of polypeptide chain and is related by a crystallographic twofold rotation axis to an additional six strands of the second c \*_>oncanavalin A (Con A1) exhibits some unusual biological properties as a result of its ability to bind various carbohydrates. It agglutinates erythrocytes from certain animal species, starch granules, and some bacteria and yeasts (Sumner and Howell, 1936a), and has been shown to precipitate various glycogens, dextrans, mannans, glycoproteins, and blood group substances (Sumner and Howell, 1936b; So and Goldstein, 1968; Leon and Young, 1970; and Lloyd et al., 1969). Carbohydrates with the minimum specificity for Con A binding contain hexose residues with the D-ara6//zo-pyranoside configuration2 at C-3, C-4, and C-5 (Goldstein et al., 1965).Con A induces transformation of lymphocytes by reversibly binding to specific sites on the cell surface (Powell and Leon, 1970; Novogrodsky and Katchalski, 1971) and inhibits phagocytosis by polymorphonuclear leucocytes (Berlin, 1972). It also agglutinates embryonic tissue cells (Moscona, 1971) and various neoplastic cells in tissue cultures (Inbar and Sachs, 1969), whereas binding sites on the corresponding adult cells and normal cells appear to be masked. Studies by Inbar et al. (1971) indicate that the site for Con A on the cell surface membrane of hamster cells has two components, one which actually binds the Con A and the other which is responsible for the agglutination.The purpose of our investigation is to determine the complete three-dimensional structure of Con A by X-ray crystallographic techniques and we report here features of the molecule, mainly the course of the polypeptide chain, subunit interactions, regions of /3 structure, the carbohydrate binding site, and the Mn2J~and Ca2^sites as interpreted from our electron density map at 2.4-A resolution. Experimental ProcedureCon A, purified and crystallized as previously described (Hardman et a!., 1971a), was used to collect the lower resolut From the

The effects of linker length on binding affinity and degree of aggregation have been examined in the antifluorescein 4-4-20 and anticarcinoma CC49 single-chain Fvs. Longer linkers in the antifluorescein sFvs have higher affinities for fluorescein and aggregate less. A proteolytically susceptible site between Lys8 and Ser9, in the previously reported 212 linker has been identified. A new linker sequence, 218 (GSTSGSGKPGSGEGSTKG) was designed in which a proline was placed at the C-terminal side of the proteolytic clip site in the 212 linker. The CC49 sFv containing the 218 linker showed reduced aggregation and was found to be more stable to proteolysis in vitro, when compared to the CC49/212 sFv. The CC49 sFv with the longer 218 linker had higher affinity than CC49/212 sFv. An aggregated CC49/212 sFv sample had higher affinity than CC49/218 sFv. The CC49/218 and CC49/212 sFvs had similar blood clearances in mice, while the aggregated CC49/212 sFv remained in circulation significantly longer. In mice bearing LS-174T human colon carcinoma xenografts, the CC49/218 sFv showed higher tumor uptake than the CC49/212 sFv and lower tumor uptake than the aggregated CC49/212 sFv. The higher tumor uptake of the CC49/218 is most likely a result of its higher resistance to proteolysis. The higher affinity and higher tumor uptake of the aggregated CC49/212 sFv are most likely due to the repetitive nature of the TAG-72 antigen and the higher avidity of multivalent aggregates. When the sFvs were radiolabeled with a lutetium-chelate the CC49/218 sFv showed a lower accumulation in the liver and spleen compared to the aggregated CC49/212 sFv.

Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)

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