1. Field of the Invention
The present invention relates generally to methods and compositions producing novel polypeptides including antibodies capable of promoting chemical reactions. More particularly, the present invention relates to methods for modifying polypeptides to include active functionalities capable of promoting chemical reactions.
The ability to catalyze chemical reactions, such as the synthesis, modification, or cleavage of structurally complex molecules, including proteins, nucleic acids, carbohydrates, and the like, would be of great commercial and scientific benefit. To this end, antibodies have been prepared having catalytic activity resulting from the ability of the antibody combining site to selectively stabilize transition state intermediates and to overcome entropic barriers in orienting reactants in particular reactions.
Although significant catalytic activity has been observed with such antibodies, it would be desirable to provide catalytic antibodies and other polypeptides having enhanced catalytic activity and specificity. In particular, it would be desirable to be able to design catalytic antibodies and polypeptides having a combining site with a desired specificity and affinity for the reactant(s) of interest, which antibodies and polypeptides further provide catalytic or other reactive groups proximate the binding site. Such catalytic and reactive groups would be able to chemically participate in the reaction of interest.
2. Description of the Relevant Art
The preparation of catalytic antibodies against haptens that are transition state analogs is described in the following references: Pollack et al. (1986) Science 234:1570-1573; Pollack and Schultz (1987) Cold Spring Harbor Symp. Quant. Biol. 52:97-104; Jacobs et al. (1987) J. Am. Chem. Soc. 109:2174-2176; Tramontano et al. (1986) Science 234:1566-1570; Tramontano et al. (1988) J. Am. Chem. Soc. 110:2282-2286; and Janda et al. (1988) Science 241:1188-1191. The use of antibodies to overcome entropic barriers involved in orienting reaction partners is described in the following references: Napper et al. (1987) Science 237:1041-1043; Jackson et al. (1988) J. Am. Chem. Soc. 110:4841-4842; Janda et al. (1988) J. Am. Chem. Soc. 110:4835-4837; Hilvert et al. (1988) Proc. Natl. Acad. Sci. USA 85:4953-4955; and Berkovic et al. (1988) Proc. Natl. Acad. Sci. USA 85:5355-5358. Polyclonal antibodies have been generated with cofactor binding sites (Raso and Stollar (1975 ) Biochemistry 14:584-591). The generation of monoclonal antibodies having a cofactor binding site is described in Shokat et al. (1988) Angew. Chem. 100:1227-1229. The generation of affinity labels for antibody combining sites is described by Kohen et al. (1980) FEBS Lett. 111:427-431; Metzger et al. (1970) Biochemistry 9:1267-1278; and Givol et al. (1971) Biochemistry 10:3461-3466. Chemically derivatized hydrophobic and hydrophilic model systems have been shown to afford rate enhancements in hydrolytic and redox reactions by specific substrate binding (Bender et al. (1978) Cyclodextrin Chemistry, Springer-Verlag, Berlin; Tabushi (1982) Acct. Chem. Res. 15:66; Breslow (1982) Science 218:532; and Cram et al. (1978) J. Am. Chem. Soc. 106:4987). Both Cram et al. (1976) J. Am. Chem. Soc. 98:1015 and Lehn and Sirlin (1978) J.C.S. Chem. Comm. 949 have demonstrated that polyether macrocycles derivatized with thiol residues complex and accelerate acyl transfers by factors of 10.sup.3 to 10.sup.4 in thiolysis reactions of amino acid ester salts (relative to noncomplexing thiols). Furthermore, cyclodextrins derivatized with pyridoxamines have afforded 100-fold rate accelerations in transamination of pyruvic acid as well as greater than twenty-fold stereo-selectively in a-amino acid synthesis (Tabushi et al. (1985) J. Am. Chem. Soc. 107:5545; Zimmerman et al. (1983) J. Am. Chem. Soc. 105:1694; Breslow et al. (1983) J. Am. Chem. Soc. 105:1390; and Breslow et al. (1980) J. Am. Chem. Soc. 102:423). Baldwin et al. (1975) J. Am. Chem. Soc. 97:227, Collman (1977) Acct. Chem. Res. 10:265; and Traylor (1973) Proc. Natl. Acad. Sci. 78:2647 have shown that cavity containing porphyrins are also capable of mimicing the oxygen binding function of myoglobin as well as the oxidative chemistry of P-450 monoxygenase. Introduction of a free thiol into chiral macrocyclic ether has been shown to promote the transacylation of nitrophenyl glygly ester relative to uncomplexed dipeptide. Lehn and Sirlin (1978) supra. Site-directed mutagenesis has been used in conjunction with high resolution x-ray crystallography to analyze the structure of enzyme binding sites and the function of such sites in catalysis. Wilkinson et al. (1984) Nature 307:187-188; Craik et al. (1985) Science 28:291-297; Schultz et al. (1985) Biochemistry 24:6840-6848; Dalbadie-McFarland (1982) Proc. Natl. Acad. Sci. USA 79:6409-6413; and Sigal et al. (1984) J. Biol. Chem. 259:5327-5332. A cysteine in the active region of the enzyme papain has been modified with a flavin cofactor. Kaiser et al. (1984) Science 226:505-510. A thiol has been introduced into the enzymes staphylococcal nuclease and RNase S and subsequently derivatized with an oligonucleotide. Corey et al. (1987) Science 238:1401-1403. The active site serine of subtilisin has been chemically converted to a cysteine. Bender et al. (1966) J. Am. Chem. Soc. 88:3153-3154 and Koshland et al. (1966) Proc. Natl. Acad. Sci. USA 56:1606-1611. Antibodies generated against positively charged haptens contain complementary aspartate and glutamate residues (Nisonoff et al. (1975) The Antibody Molecule, Academic Press, pp. 23-27). The experimental data in Example 4 was published in Cochran et al. (1988) J. Am. Chem. Soc. 110:7888-7889.