EP0454778A4 - Molecules with antibody combining sites that exhibit stereospecific catalysis - Google Patents

Abstract

A stereoisomeric analog-ligand having a conformation that substantially corresponds to the conformation of a hydrolytic transition state of an amide or ester reactant ligand and the stereochemistry of the nearby atoms is used to produce receptor molecules of predetermined specificity. The receptor molecules include an antibody combining site that binds stereospecifically to one stereoisomer of the analog-ligand and also to one stereoisomer of the reactant ligand and thereby stabilizes the tetrahedral carbon atom of the amide or ester hydrolysis transition state of that reactant ligand to catalytically hydrolyze the reactant ligand at a predetermined site.

Description

MO ECOLES WITH ANTIBODY COMBINING SITES THAT EXHIBIT STEREOSPECIFIC CATALYSIS

Description Cross-Reference to Related Application This application is a continuation-in-part of copending application Serial No. 083,681 filed August 7, 1987, which is a continuation-in-part of copending application Serial No. 055,177, filed May 28, 1987, disclosures of which are incorporated herein by reference. Technical Field

The present invention relates to antibodies, antigens and immunogens, and more particularly to molecules that contain &n antibody combining site that binds stereospecfically to and stabilizes the tetrahedral carbon atom of an amide or ester hydrolysis transition state and stereoselectively catalyzes hydrolysis of such bonds. Background of the Invention Binding phenomena between ligands and receptors play many crucial roles in biological systems. Exemplary of such phenomena are the binding of oxygen molecules to deoxyhe oglobin to form oxyhemoglobin, and the binding of a substrate to an enzyme that acts upon it such as between a protein and a protease like trypsin. Still further examples of biological binding phenomena include the binding of an antigen to an antibody, and the binding of complement component C3 to the so-called CRl receptor. Many drugs and other therapeutic agents are also believed to be dependent upon binding phenomena. For example, opiates such as morphine are reported to bind to specific receptors in the brain. Opiate agonists and antagonists are reported to compete with drugs like morphine for those binding sites. Ligands such as man-made drugs, like morphine and its derivatives, and those that are naturally present in biological systems such as^' endorphins and hormones bind to receptors that are naturally present in biological systems, and will be treated together herein. Such binding may lead to a number of the phenomena of biology, including particularly the hydrolysis of amide and ester bonds as where proteins are hydrolyzed into constituent polypeptides by an enzyme such as trypsin or papain, or where a fat is cleaved into glycerine and three carboxylic acids, respectively.

Slobin, Biochemistry, 12836-2844 (1966) reported preparing antibodies to a js-nitrocarbobenzoxy conjugate of bovine serum albumin. Those antibodies were thereafter used to hydrolyze £-nitrophenyl acetate and epsilon-a inocaproate esters. The reaction of the acetate ester was described by a second-order rate constant and was said to appear to be nonspecific.

The second-order rate constant obtained using normal gamma globulin was said to be about equal to that of the specially prepared antibodies. The presence of the specially prepared antibodies was said to inhibit the hydrolysis of the aminocaproate ester.

Kohnen and co-workers also reported attempts using antibodies to catalyze esterolysis. The antibodies utilized by this group were, in each instance, raised to a portion of the ultimately utilized substrate molecule that did not contain the bond to be hydrolyzed.

In their initial work [FEBS Letters, 100:137-140 (1979) and Biochim. Biophys. Aeta, 629:328-337 (1980)] anti-steroid antibodies were used to hydrolyze 7-umbelliferone (7-hydroxycoumerin) esters of a carboxyethyl thioether of a steroid. I each instance, an increase in hydroly ^' : rate was observed as compared to background or to a rate^' obtained with normal IgG. In both instances, turn over numbers were low (about one mole of substrates per mole of antibody per minute, or less), a the reaction rates declined with time, reaching a plateau with saturation of the antibody. That slow down in rate was attributed to an irreversible binding of the steroidal acid product to the antibody.

Kohen et al. also reported hydrolyses of 7-[-N-(2,4-dinitrophenyl)-6-aminohexanoyl]-coumerin using monoclonal antibodies raised to the dinitrophenyl portions of that substrate molecule [FEBS Letters, 13,1:427-431 (1980)1. Here, a rate increase over background was also reported, but the reaction was said to be stoichiometric rather than catalytic. A decrease in rate that approached zero was reported as saturation of the antibody was reached. Again, the decrease was attributed to product inhibition caused by binding of the product acid to the antibody since some of the initial hydrolysis activity could be regenerated by chromatography of an antibody-substrate-product mixture.

When strong antibody binding is directed to stable states of substrate molecules, the slow rate of dissociation of the complex will impede catalysis. Such is thought to be the situation for the results reported by Kohnen and co-workers.

The above constructs, though interesting, are severely limited by the failure to address the mechanism of binding energy utilization which is essential to enzymes [W. P. Jencks, Adv. Enzymol. , 4 , 219 (1975)]. Those deficiencies aay be redressed by using a transition state analog as the hapten to elicit the desired antibodies. This hapten (also referred to herein as an "analog-ligand*) can assume the cole of an inhibitor in the catalytic system.

Thus, immunologieal binding aay be used to experimentally divert binding interactions to catalytic processes. For example, it was suggested that use of an antibody to a haptenic group that resembles the transition state of a given reaction should cause an acceleration in substrate reaction by forcing substrates to resemble the transition state. Jencks, W.P., Catalysis in Chemistry and Enzymology, page 288 (McGraw-Hill, New York 1969). Notwithstanding that broad suggestion, specific transition state haptens were not suggested, nor were specific reactions suggested in which the concept might be tested.

Hydrolysis of amide and ester bonds is thought by presently accepted chemical theory to proceed in aqueous media by a reaction at the carbonyl carbon atom to form a transition state that contains a tetrahedral carbon atom bonded to (a) a carbon atom of the acid portion of the amide or ester, (b) two oxygen atoms, one being from the carbonyl group and the other from a hydroxyl ion or water molecule of the medium, and (c) the oxygen atom of the alcohol portion of an ester or the nitrogen atom of the amine portion of an amide. Transition states of such reactions are useful mental constructs that by definition, cannot be isolated, as compared to intermediates, which are isolatable.

Although the above hydrolytic transition states can not be isolated, a large amount of scientific literature has been devoted to the subject. Some of that literature is discussed hereinafter.

Whereas the before-described transition state for amide and ester hydrolyses is believed to be well understood, the parameters of the topology, e.g_*, size, shape and charge, of receptor binding sites in which particular amides, such as proteins, or esters, such as fats, react through those transition states is not as well understood. It would therefore be beneficial if the topology of a plurality of binding sites were known so that the interactions of the ligands that bind in those sites could be studied, unfortunately, the topology of receptor binding sites in biological hydrolyses is generally unknown, except for a relatively small number of enzymes whose X-ray crystal structures have been determined.

This lack of knowledge of binding site topology stems in part from a lack of knowledge of even the location in cells of many binding sites of receptors. In addition, for those receptor binding sites whose location is known, the chemical identity; i.e., protein and carbohydrate composition, of the binding site is generally unknown. Thus, the investigator is generally stymied in seeking to understand the topological requirements of receptor binding sites and therefore in seeking to construct therapeutic agents that can fulfill those requirements. Investigators must therefore screen potential therapeutic agents in animal or cell culture studies to ascertain whether a potential therapeutic agent may be useful. Such systems, while useful, are expensive and time-consuming to use. Even where the topology and chemical reactivity of a hydrolytic receptor such as an enzyme are know , enzymes such as hydrolytic proteases ^* typically cleave their substrates, polypeptide chains, adjacent to a particular amino acid residue that aay occur several times in the polypeptide chain of the protein. While such relatively random cleavage can be useful in obtaining a polypeptide map of the protein, that relatively random cleavage is not as useful where particular amino acid residue sequences are desired to be produced.

For example, modern genetic engineering techniques have been useful in preparing fusion proteins that contain a desired protein or polypeptide fused to the transcription product of a vector gene such as the lac z gene. The use of such fusion proteins is, however, hindered by the presence of fragments of the vector gene product. It would also therefore be beneficial if proteolytic enzyme-like molecules could be developed that would cleave such fusion products between the wanted and unwanted fusion polypeptide or protein portions. Recently, Lerner, Tramontane and Janda [Science, 234, 1566 (1986)] reported monoclonal antibodies that catalytically hydrolyzed an ester.

Tramontane and Lerner, also describe using monoclonal antibodies to hydrolyze esters in U.S. Patent No. 4,656,567. Pollack, Jacobs and Schultz [Science, 234, 1570 (1986) ] reported a myeloma protein denominated MOPC167 [Leon et al.. Blochem., 10, 1424 (1971)] that catalyzes the hydrolysis of a carbonate.

In the two Lerner and Tramontane disclosures, the antibodies were raised to a phosphonate that was synthesized to represent a stable analog of the tetrahedral hydrolytic transition state of the carboxylic acid ester or carbonate ester. The Pollack et al. antibody principally discussed was a myeloma protein that happened to bind to a phosphonate that was structurally analogous to the carbonate analog hydrolyzed. Thus, in the Lerner and Tramontane et al. work, the substrate to be hydrolyzed was preselected, with the immunizing analog and hydrolytic antibodies being synthesized in accordance with the desired product. Pollack et al. designed the substrate to be hydrolyzed once they knew the specificity of the myeloma protein. Pollack et al. also reported (above) the existence of a catalytic antibody, substrated and analog substrate system for carbonate hydrolysis similar in concept to that of Lerner et al. Work relating to that system is reported in Jacobs et al., J. Am. Chem Soc. , 109, 2174 (1987) . Published patent application WO 85/02414 discusses the possible use of antibodies as catalysts, and presents data relating to the use of polyclonal serum in hydrolyzing o-nitrophenyl- beta-D-galactoside. The antibodies useful in that application are said to be inducible by a reactant, a reaction intermediate or to an analog of the reactant, product or reaction intermediate. The term "analog" is there defined to encompass isomers, homologs or other compounds sufficiently resembling the reactant in' terms of chemical structure that an antibody raised to an analog can participate in an immunological reaction with the reactant but will not necessarily catalyze a reaction of the analog.

The data provided in that specification only indicate that some cleavage of the substrate (reactant) galactoside occurred over an eighteen hour time period using a relatively concentrated antibody preparation (1:10 and Is 0 dilutions). Although catalysis was alleged, catalytic activity was not shown since no turn over of the allegedly catalytic antibody was shown, nor was there an indication of the percentage of substrate galactoside cleaved. That application did indicate that beta-D- galactosidase cleaved about ten times as auch substrate as did the polyclonal antibodies, presuming linearity of absorbance at the unnamed concentration of substrate studied.

From the data presented in that application, it is possible that a nυcleophilic replacement of the .o-nitrophenyl group occurred by a terminal amino group of a lysine residue of the antibody preparation used. Thus, the observed absorbance could have been due to formation of epsilon-amino lysinyl o-nitrophenyl aniline or to the formation of an epsilon-amino-lysinyl galactoside and o-nitrophenol, either of which occurrences would not be catalytic since the antibody was consumed, rather than turning over.

In more recent work, bimolecular amide formation catalyzed by antibody molecules has been disclosed [Benkovic et al., Proc. Natl. Acad. Sci. USA, 85:5355 (1988)], as has an antibody-catalyzed Claisen rearrangement [Jackson et al., J. Am. Chem. Soc. , 110:4841 (1988)]. None of that work, nor the previously discussed work, has contemplated the use of antibodies to catalyze any reaction in a stereospecific aanner.

Stereospecificity was shown in an antibody-catalyzed lactone-for ing reaction [Napper et al.. Science, 237:1041 (1987)] and in an antibody-catalyzed Claisen reaction [Hilvert et al., Proc. Natl. Acad. Sci. USA, 8_5:4955 (3°98)]. The use of an antibody combining site-containing aolecule to stereospecifically catalyze a hydrolysis reaction as is described hereinafter was not, however, contemplated in either of the above publications.

Brief Summary of the Invention

The present invention contemplates a receptor molecule that contains an antibody combining site or idiotype-containing polya ide that is capable of catalytically hydrolyzing a preselected, scissile carboxylic acid amide or ester bond of one stereoisomer of a reactant ligand, and not of the other stereoisomer. That antibody combinining site binds to (immunoreacts with) : (a) one stereoisomer of a reactant ligand containing that preselected scissile carboxylic acid amide or ester bond, and (b) one stereoisomer of an analog-ligand that is stereochemically analogous to the reactant ligand and that contains a tetrahedrally bonded phosphorus atom at a position analogous to that- of the sissile carbonyl carbon atom of the preselected carboxylic acid amide or ester bond of the reactant ligand. The hydrolytic transition state of the reactant ligand so bound contains a tetrahedral carbon atom bonded to (i) a carbon atom, the alpha-carbon of the acid portion of the ester or amide, (ii) two oxygen atoms, and (iii) the oxygen atom of an ester or the nitrogen atom of an amide.

Molecules containing an antibody combining site raised to the hydrolytic transition state of a reactant ligand are raised or induced by immunizing with one stereoisomer of an analog-ligand aolecule (preferably bound to a protein carrier to form a conjugate) containing an analog of a hydrolytic transition state of the ligand. The immunizing stereoisoaer of the analog-ligand hydrolytic transition state aolecule contains a tetrahedrally bonded phosphorus atom, bonded directly to (i) & carbon atom of the acid portion of the analogous ligand amide or ester, (the alpha-carbon of the acid portion) (ii) two oxygen atoms, (iii) a third oxygen atom or a nitrogen atom, the third oxygen atom or nitrogen atom being bonded to the alpha-carbon atom of an analogous ester or amide of the ligand. The alpha-carbon atom of the acid portion,

(i) above, bonded directly to the central tetrahedral phosphorus atom of the analog-ligand molecule, is included in a chain that contains at least 5 atoms, and more preferably at least 15 atoms, and preferably at least 15 atoms and including a substituted phenyl group, as is the third oxygen or nitrogen atom, (iii) above. Of the two oxygen atoms, (ii) above, bonded directly to the central atom, one oxygen atom (a) is bonded twice (doubly bonded) in an oxo group to the central atom, (b) is part of an hydroxyl group or (c) is the oxygen of an alkoxy group containing a J- J lower alkyl group. The second of those oxygen atoms bonded to the central atom is singly bonded to the central atom and is an -OR- group, wherein ₂ is selected from the group consisting of hydrogen (H) , and C-J-CJ lower alkyl. The fourth atom, (iii) above, bonded to the central atom of the analog-ligand molecule is the alcohol oxygen atom of an ester or the amine (imino) nitrogen atom of an amide of the analogous ester or amide portion of the ligand. That fourth atom is a portion of a chain that contains at least 5, more preferably at least 15 atoms, and with the remainder of the chain constitutes R,. -li¬ lt is emphasized that both the reactant ligand and analog-ligand contain at least one carbon atom that can exist in two stereoio eric foras, "and thereby provides a stereoisome ic center. That stereoisomeric center is located in each of the ligand and analog-ligand molecules at the same relative position in each molecule. The stereoiso eric center is also loeated near enough to the bond to be hydrolyzed so that the stereoisomeric center is bound by the catalytic antibody combining site-containing molecule.

The tetrahedrally bonded central atom is phosphorus so that the analog-ligand is an organophosphorus compound with an arrangement of substitutents about the phosphorus that corresponds to the tetrahedral carbon transition state. A phosphonate or phosphona idate monoacid in its ionized form also simulates the developing charge in nucleophilic attack at a carbonyl center. Moreover, phosphonamidate and phosphoramidate inhibitors of enzy ic peptide hydrolysis have been described as mimics of transition states. Galardy et al., Biochemistry, 22, 1990 ((1983); Bartlett et al., Biochemistry, 7__ , 4618 (1983); Thorsett et al., Proc. Natl. Acad. Sci. USA, 79, 2176 (1982); Jacobsen et al., J. Am. Chem. Soc. , 103, 654 (1981); Kam et al., Biochemistry, 18, 3032 (1979) and Weaver et al., J. Mol. Biol., 114, 119 (1977). In the studies described herein, phosphonate esters and phosphonamidate amides function as transition state analogs to generate antibodies that are monoclonal and that are stereospecific carboxylic esterases and amidases. In effect, these antibodies express their inherent binding energy functionally, as true enzymes, to catalytically hydrolyze esters and amides, and classically, as antibodies, to bind antigens.

Exemplary immunizing analog-ligand aolecule: that contain an analog of a hydrolytic transition state are represented by the formula:

wherein X » O or NH;

wherein R 4. _* (CH _MCO R 2 n 2 i

R₂ ■ H or C^-C^ lower alkyl; and

wherein the wavy line indicates both stereochemica isomers; and n is an integer from 1 to 8, inclusive. The analog-ligand hydrolytic transition state aolecules are themselves ligands, albeit not reactive ligands, and are also contemplated in this invention. These ligand aolecules are of relatively small molecular size and are therefore typically linked to a larger, carrier aolecule when used as immunogens to induce production of receptor molecules or are used alone as an inhibitor molecule. Such relatively small molecules are commonly referred to as haptens. These analog-ligand molecules also typically contain a linking atom or group such as a reactive aercaptan, a succinimide or other group that provides a means to attach the haptenic analog-ligand molecules to carriers for use as immunogens. Exemplary reactant ligand molecules that structurally correspond to the foregoing analog-ligand molecules are represented by the formulas

wherein X, R^ and ₃ are as before described.

The antibody combining site-containing molecules of the present invention are themselves receptors and provide information on the conformational preferences of antibody-hapten interactions through study of the intramolecular reactivity patterns of receptor-ligand complexes that are formed between the antibody combining site-containing molecules (receptors) and ligands of differing structures that contain similar or identical epitopic regions. A aethod of preparing monoclonal receptor aolecules that bind to the hydrolytic transition state of a particular amide or ester is also contemplated. Here, a before-described haptenic analog-ligand aolecule containing a hydrolytic transition state analog is provided linked to a carrier as an immunogenic conjugate. The conjugate thus provided is dissolved or dispersed in a physiologically tolerable diluent to form an inoculum. The inoculum is introduced as by injection into a suitable, non-human mammalian host in an amount sufficient to induce antibodies to the haptenic analog-ligand.

The antibodies so induced are harvested. The harvested antibodies are assayed for their ability to bind to (immunoreact with) the immunizing, haptenic ligand analog. Immunoglobulin-producing cells such as those from the spleen of an animal whose antibodies bind to the immunizing, haptenic analog-ligand are collected and are fused with myeloma cells to form hybridoma cells. The hybridoma cells are grown in a culture medium and the supernatant medium from the growing hybridoma cells is assayed for the presence-of antibodies that bind to the immunizing, haptenic analog-ligand.

Hybridoma cells whose supernatant contains such binding antibodies are then screened to determine which of those cells secreted antibodies that also hydrolyze the substrate ligand in a stereospecific manner. Hybridoma cells whose secreted antibodies bind to the immunogen, bind to a reactant ligand and hydrolyze a reactant ligand are then cloned to provide the desired monoclonal antibodies from culture medium supernatant or from the ascites of a host mammal into which the hybridoma is introduced. The described aonoclonal antibodies can be used as the receptors of this inventio Alternatively, the so-called Fc or Fc* portions of the antibodies can be removed as by enzymic cleavage to provide an antibody combining site

(idiotype-containing polya ide) that binds to the immunizing, haptenic analog-ligand such as Fab or F(ab*)2 antibody portion, respectively.

The present invention provides several benefits and advantages. One benefit is the preparation of receptors whose binding site topological requirements are tailored to a particular reactant ligand to be studied and hydrolyze a preselected bond in only one isomer of that ligand. Another benefit of the present invention is the preparation of receptors that hydrolyze the amide or ester ligand at a predetermined site of only one stereoisomer of the ligand, and that exhibit catalytic properties. An advantage of the invention is that because of the stereospecificity of the receptors that can be produced, a ligand containing a plurality of different hydrolyzable bonds such as a polypeptide or protein containing both O and L amino acid residues can be hydrolyzed at a preselected, particular hydrolyzable bond.

Yet another advantage of the present invention is the provision of receptors that can selectively remove a blocking group from only one stereoisomer in a mixture of isomers during or after synthesis, thereby facilitating recovery or use, respectively, of a desired stereoisomer.

Still further benefits and advantages of the present invention will be apparent to those skilled in the art from the discussion that follow. Detailed Description of the Invention I. Introduction

The present invention relates to aolecules collectively referred to as receptors that are antibodies and idiotype-containing poly mide

(antibody combining site or paratopic) portions induced by an analog of a reactant ligand carboxylic acid amide or ester that mimics the stereochemistry and conformation of the transition state in the reaction sequence for the hydrolysis of that reactant ligand ester or an amide. The receptor molecules (antibodies and antibody combining sites) bind to one stereoisomer of the analog-ligand and to one stereoisomer of the reactant ligand, are thought to stabilize the hydrolytic transition state of a preselected portion of the reactant ligand, and thereby exhibit catalytic properties as to only one stereoisomer of the reactant ligand.

Antibodies and enzymes are both proteins whose function depends on their ability to bind specific target molecules. Enzymatic reactions differ from immunological reactions in that in an enzymatic reaction the binding of the enzyme to its substrate typically leads to chemical catalysis, whereas a non-catalytic complex is the usual result of antibody-antigen binding.

Enzymes are believed to catalyze the hydrolysis of proteins by combining with the protein to stabilize the transition state of the hydrolysis reaction. It is generally believed that the rate of an enzymatic reaction is increased relative to the rate of a non-enzymatic reaction because of the ability of the enzyme to stabilize the transition state of the reaction; i.e., to reduce the free energy of the transition state, and thus, the free energy of activation, of the reaction [Jencks, W.P., Adv. Bnzymology, 43, 219 (1975) and Pauling, L., ABter. Scientist, 3_,6, 58 (1948)]. Support for this theory comes from the observation that substances that are thought to model the presumed transition states are often strongly bound to the enzymes as competitive inhibitors. Leinhard, G., Science, 180, 149 (1973) and Wolfenden, R., Ace. Chem. Res., 5_, 10 (1972). It is further thought that the enzyme accomplishes this lowering of the reaction free energy by binding the transition state geometry of the reactant more strongly than it binds to the corresponding substrate(s) or product(s).

This means that the intrinsic binding energy of the enzyme is much greater than can be measured from the binding of substrates or products. Essentially, the binding energy of the enzyme is utilized to perform the chemical reaction [Jencks, W.P., XVII International Solvay Conference (November 1983)].

The converse proposition is that an antibody that is prepared to optimally bind a suitable analog of a transition state would function as a catalyst. The demonstration of this result by Lerner and co-workers and Schultz and coworkers in the previously cited papers completes the correlation of enzyme function and antibody structure and provides a useful approach to devising artificial enzymes. The basic idea behind immunological hydrolysis described herein contemplates the use of analog-ligands in the preparation of antibodies of predetermined specificity that preferentially bind to and thereby stabilize the transition state of amide or ester bond hydrolysis upon binding to the specified reactant ligand. An analog-ligand siaulates the conformation of a high energy transition state in hydrolysis to induce the production of antibodies having the ability to bind related substrates and stabilize their hydrolyses. Such preferential binding and stabilization results in a reduction in the activation energy for the hydrolysis reaction, thus meeting a criterion for catalysis. Antibodies that display this property can be obtained by immunization with synthetic analogs that are chemically modified to resemble the bonding characteristics of a substrate reactant ligand undergoing bond hydrolysis; i.e., by immunization with transition state analogs of the particular reaction. In addition, a receptor molecule of the present invention also binds to and hydrolyzes only one of a stereoisomeric pair of otherwise identical reactant ligand molecules. Thus, where the reactant ligand is enantiomeric, only one of the enantiomers is hydrolyzed. Similarly, where the reactant ligand exists in both cis and trans forms, only one of those isomers is hydrolyzed.

Inasmuch as a receptor molecule of this invention exhibits stereospecificity, both the analog-ligand and reactant ligand contain at least one carbon atom that can exist in two stereoisomeric forms; i.e., a stereoisomeric center. The stereoisomeric center is located in each of the analog-ligand and reactant ligand molecules in the same positions relative to the other atoms in the analogous molecules. Thus, if the stereoisometric center is located in a chain four atoms away from the phosphorus atom in the acid portion of the analog-ligand, the stereoisomeric center is 'located in a chain four atoms away from the scissile carbonyl carbon of the reactant ligand. It is noted that an analog-ligand aolecule and/or a reactant ligand aolecule can contain aore than one stereoisomeric center. When a second, 'third or other such center is located at such a distance from the scissile carbonyl carbon (or phosphorus atom) that it is not bound by a receptor aolecule, it is of no aatter herein. However, when near enough to be bound by the receptor aolecule, any other stereoisomeric center produces additional stereoisomers. The number of such isomers is determined by the equation: number » 2ⁿ, where n is the number of stereoisomeric centers.

The at least one stereoisomeric center can be on either the carboxylic acid or alcohol or amine portions of the ester or amide reactant ligand and analog-ligand. If more than one such center is present in the reactant ligand and analog-ligand molecules, that plurality of stereoisomeric centers can be distributed in any way desired about the scissile carbonyl carbon atom (or central phosphorus atom). Any stereoisomerism provided by the central tetrahedral phosphorus atom is not considered herein. A receptor molecule of the present invention distinguishes, and stereoselectively catalyzes the hydrolysis of at least one of a pair of stereisomeric reactant ligand molecules that are present in a mixture of stereoisomeric pairs or as a separate pair of reactant ligand molecules. More preferably, a receptor molecule distinguishes and stereoselectively catalyzes the hydrolysis of only one of the stereoisomers of the reactant ligand molecule.

The above stereoselectivity of catalytic hydrolysis presumes that the locus of stereoisomerism, the stereoisomeric center, is present in the reactant ligand near enough to the bond to be hydrolyzed (the scissile carbonyl carbon) so that the stereoisomeric center is bound by the catalytic antibody combining site-containing aolecule_ff and thus, the receptor aolecule binds to only one stereoisomer and the same stereoisomer (R or S) of both the reactant ligand and anolog-ligand. The locus of the bond to be hydrolyzed is determined by the location of the phosphorus atom of the analog-ligand (and the analogous scissile carbonyl carbon of the reactant ligand) and the size of an antibody combining site. An antibody combining site is normally considered to be able to accomodate about five to about seven amino .acid residues.

The stereoisomeric center is within the volume occupied by one to about four amino acid residues (a chain length of about 12 atoms) , and more preferably one to about two amino acid residues (a chain length of about 6 atoms) on either side of the phosphorus atom of the analog-ligand (scissile carbonyl carbon of the reactant ligand). Thus, the stereoisomeric center can be on the carboxylic acid portion or on the amine or alcohol portion of the scissile carbonyl carbon of carboxylic acid amide or ester reactant ligand. In the exemplary stereoisomeric ester used herein, the stereoisomeric center is located in the alcohol portion of the molecule.

This distance can readily be determined by use of space-filling models, or where there is doubt, by simply determining whether a catalytic receptor can resolve or separate the stereoisomers of an analog-ligand. Of course, the ultimate assay is whether the catalytic receptor molecule hydrolyzes one isomer and not the other. It is to be understood that where geometric isomers are concerned, the stereoisomeric center is not a single atom, as is the ease for enantiomers. Rather, that center is present over a group of atoms whose number is governed by the number of atoms required to define the cis/trans isomers. However, for case of expression, a stereoisomeric center will be referred to herein as if it were a single atom such as a chiral atom where enantiomers are involved. As already noted, the analog-ligand and reactant ligand are one of a pair of stereoisomers. Those stereoisomers can be geometric isomers or optical isomers; i.e., enantiomers. Geometeric isomers are cis/trans isomers as are found in cyclic molecules or where double bonds are present. Optical isomers are d,l or R,S pairs of enantiomers in which the stereoisomeric center is referred to as a chiral center since the carbon atom of that center is a chiral carbon atom. In the exemplary catalytic reaction discussed hereinafter, the reactant ligand stereoisomers are an enantiomeric, R, S, pair. Although the analog-ligand utilized in this exemplary study contained both enantiomers and induced production of receptor molecules that stereoselectively hydrolyzed either the R or S forms of the reactant ligand, it should be understood that only one of the enantiomeric analog-ligands could have been utilized to induce production of only one of the enantiomeric reactant ligands.

The mechanism by which an antibody hydrolyzes an ester or amide bond of a bound reactant ligand can be thought of in terms of an "induced fit" model. As the loosely bound substrate distorts or rearranges to conform to the binding geometry of the antibody, stress can be relieved by chemical reorganization of a single, predetermined amide or ester bond such that this reorganization leads to the hydrolysis of the bond. The term "receptor" is used herein to aean a biologically active aolecule that binds to a reactant ligand, inhibitor ligand, or analog-ligand. The receptor aolecules of the. present invention are antibodies, substantially intact antibodies or idiotype-containing polyamide portions of an antibody.

Biological activity of a receptor molecule is evidenced by the binding of the receptor to its antigenic reactant ligand, inhibitor ligand. or analog-ligand upon their admixture in an aqueous medium, at least at physiological pH values, and ionic strengths. Preferably, the receptors also bind to an antigenic ligand within a pH value range of about 5 to about 9, and at ionic strengths such as that of distilled water to that of about one molar sodium chloride.

Idiotype-containing polyamide portions (antibody combining sites) of antibodies are those portions of antibody molecules that include the idiotype, and bind to the ligand or analog-ligand. Such portions include the Fab, Fab' and F(ab*)₂ fragments prepared from antibodies by well-known enzymatic cleavage techniques. See for example, U.S. Patent No. 4,342,566 to Theofilopoulos and Dixon, generally, and specifically. Pollack et al. [Science, 234, 1570 (1987)3 who reported accelerated hydrolytic rates for Fab fragments were the same as those of the native Ig. Inasmuch as the antibodies from which idiotype-containing polyamides are obtained are described as raised against or induced by immunogens, idiotype-containing polyamide (antibody combining site-containing) receptors are discussed as being "raised".or "induced" with the understanding that a cleavage step is typically required to obtain an idiotype-containing polyamide from an antibody. Intact antibodies are preferred, however, and are utilized as illustrative of the receptor molecules of this invention.

The receptors useful in the present invention are monoclonal antibodies. A "monoclonal antibody" is a receptor produced by clones of a single cell called a hybridoma that secretes but one kind of receptor molecule. The hybridoma cell is fused from an antibody-producing cell and a myeloma cell or other self-perpetuating cell line.

Techniques for preparing the monclonal antibodies of the present invention are well known. Such receptors were first described by Kohler and Milstein, Nature, 256, 495 (1975) , which is incorporated herein by reference. Monoclonal antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from mammals into which the hybridoma tissue was introduced. Both methods are described herein. A "ligand" is defined herein as a molecule that immunoreacts with or binds to a receptor molecule antibody combining site. Two types of ligand are contemplated herein. A first is termed an analog-ligand and is used as an immunogen to induce preparation of receptor molecules and as an inhibitor of the receptor molecule-catalyzed reaction. The analog-ligand is substantially inert to undergoing the catalyzed reaction. The second is referred to as the reactant ligand or reactant ligand substrate and is the molecule that undergoes the catalyzed reaction. As described herein, chemical analogs of amide or ester ligands are synthesized that incorporate phosphonamidate or phosphonate aoieties at specific, predetermined sites to mimic the conformation of the transition state in the hydrolysis of an amide or ester bond. Such analogs are suitable candidates for this investigation because it is known that phosphonamidates have been used as transition state analogs in the inhibition of proteolytic enzymes [Bartlett, et. al., Biochemistry, 22, 4618 (1983)3.

Short polypeptide chains can induce the production of antibodies that recognize and bind to a homologous protein at a predetermined specific site. The present invention carries the earlier work with polypeptides a major step forward. Here, the antibodies (receptors) are induced by one stereoisomer of an immunizing haptenic first molecule (the analog-ligand) , and recognize and bind not only to that first molecule, but also to one stereoisomer of a second, related molecule (the reactant ligand). In binding that second aolecule, the receptor causes hydrolysis (which as demonstrated herein is catalytic) of a preselected, ester or amide bond that corresponds in topology to the topology of the immunizing, haptenic first molecule. The correspondence in topology; i.e., size, shape, stereochemistry and charge, provides a means for preselecting the site at which hydrolysis of the ligand occurs. Inhibitor ligands that resemble the structure of an analog-ligand or a reactant ligand are also bound by receptor molecules.

Consequently, by synthesis of a relatively small, immunizing haptenic analog-ligand, one can induce the production of receptor molecules that recognize, bind to and catalytically cleave an ester or amide bond in another aolecule that can contain a plurality of amide or ester bonds. Thus, a receptor can be prepared that stereoselectively causes hydrolysis of a selected, predetermined amide bond of a protein or polypeptide such as the before-discussed genetically engineered fusion protein. Similarly, a receptor can be prepared that stereospecifically and catalytically hydrolyzes a selected, predetermined ester bond of a aodel compound or fat molecule.

The implication of this result is that one can confer the activity of hitherto unknown proteases and Upases to immunoglobulins. Furthermore, the activity of the antibody can be directed to any predetermined site at will by designating the amide or ester bond to be cleaved with the phosphonamidate or phosphonate configuration in the haptenic analog-ligand used for immunization.

Thus, antibodies and idiotype-containing polyamide portions of antibodies are induced by a haptenic ester or amide analog-ligand hydrolytic transition state molecule. The haptenic molecule contains a tetrahedrally bonded central phosphorus or silicon atom bonded directly to (a) a carbon atom of the carboxylic acid portion of the analogous ester or amide, (b) two oxygen atoms and (c) a third oxygen atom or a nitrogen atom, the third oxygen atom or nitrogen atom being bonded to a carbon atom (the alpha-carbon) of the alcohol or amine portion of an analogous ester or amide of the ligand.

II. Transition State of Esterolysis and Hapten

(Analog-Ligand) Design

Design of the analog-ligand flows backward from the structure of the product to be formed through the transition state for bond formation to be mimicked, and then to the analog-ligand. Reactions that involve amide or ester hydrolysis provide illustrative examples of the genereal concept and are utilized herein as exemplary for an ester or amide hydrolysis reaction.

Transacylation processes are characterized by carbonyl addition-elimination mechanisms. The acyl group may, therefore, possess varying degrees of tetrahedral character in this transition state. W. P. Jencks, Catalysis in Chemistry and Enzymology, ch. 10, (McGraw-Hill, New York, 1969). The enzymes that catalyze transacylation reactions might be expected to bind well those analogs of the reactant ligand having a tetrahedral configuration about the acyl center. This is true for serine proteases, where a covalent bond between the ligand (substrate) and the enzyme is formed temporarily [Westerik et al., J. Biol. Chem., 247, 8195 (1972); R.C. Thompson, Biochemistry, 12, 47 (1973) and Imperali et al.. Biochemistry, 25, 3760 (1986)3, as well as for enzymes that catalyze the direct hydration of amides or esters. The latter category is inhibited by compounds with a tetrahedral configuration including a phosphate, phosphonate or phosphonamidate group in lieu of the scissile amide unit [Weaver et al., J. Mol. Biol., 114, 119 (1977) and Jacobsen et al., J^ Am. Chem. Soc, 103, 654 (1981)3.

Naturally occurring and synthetic substances containing phosphorus have been studied as inhibitors of metallopeptidases. In these enzymes, the transition state would appear to contain the hydrated amide in the coordination sphere of the aetal ion [W. N. Lipscomb, Ace. Chem. Res.. 15, 232 (1982)3. A complete picture of a transition state analog might then have the phosphono group of an inhibitor as a ligand to a aetal ion or some other polarizing site [Weaver et al., J. Mol. Biol., 114, 119 (1977) and Christiansen et al_e, J. Am. Chem. Soc, 108, 545 (1986)]. The role of the aetal ions in aetallopeptidases, however, is not clearly understood, ϊt aay have a multiple function in amide hydrolysis where proton transfer steps among the tetrahedral intermediates aay be rate-limiting [L. M. Sayre, J. Am. Chem. Soc, 108, 1632 (1986)3.

The hydrolysis of carboxylic acid esters is a simpler example of transacylation that should also be approximated by the phosphonate-containing analog of the transition state. The binding of the charged phosphonate group may describe a stabilizing interaction in the transition state that would lead to catalysis. Ester hydrolysis reactions generally proceed at convenient spontaneous rates under ambient conditions that are suitable for antibodies. Therefore, any small rate acceleration can be readily detected.

The structures of the analog-ligands and reactant ligands for this investigation were selected according to certain criteria. These included the availability and stability of the organophosphorus precursors, the corresponding carboxylic acid substrate, the convenience of the chemical synthesis for its preparation, and the adaptability to diverse schemes for immunological presentation. A basic molecular unit that provides the necessary features for stereoselective catalytic hydrolysis is the substituted phenylacetic acid ester analog (Compound F) that is represented by Formula I, below.

I

The compound of Formula I, Compound F, is the analog-ligand utilized herein to raise receptors of this invention. Compound F is shown in its form prior to coupling to an antigenic carrier for immunization. It should be noted that Compound F exists as a racemic modification^' with its stereoisomeric center identified by an asterisk (*) , over the methine carbon indicating that two stereoisomeric structures (R and S) are possible. By including an amino substituent in either the acid or amine or alcohol portion of the analog-ligand, as in-the acid portion of Compound F, the analog-ligand can be provided with a functional appendage for coupling to an antigenic (immunogenic) carrier protein. Such an added appendage is useful where the analog-ligand is a hapten. The appendage and accompanying linker atoms can also be present in the reactant ligand, particularly where the reactant ligand is relatively small so that the antibody combining site can be relatively filled with the ligand.

Thus, the present invention generally relates to monoclonal receptors, that are capable of catalytically hydrolyzing a preselected amide or ester bond of one stereoisomer of a reactant ligand. The receptors contain an antibody combining site that binds: (a) to one stereoisomer of a reactant ligand that can form the tetrahedral hydrolytic transition state of a preselected ester or amide bond of the reactant; i.e., contains a preselected carboxylic acid amide or ester bond, and (b) to one stereoisomer of an analog-ligand that is stereochemically analogous to the reactant ligand and has a tetrahedrally bonded phosphorus atom located at the position occupied by the scissile carbonyl group carbon atom of the preselected ester or amide bond of the reactant ligand. The tetrahedrally bonded phosphorus atom is bonded directly to:

(i) a carbon atom (the alpha-carbon) of the acid portion of the analogous reactant ligand ester or amide that is included in a chain that contains at least 5 atoms, and more preferably at least 15 atoms, and most preferably at least 15 atoms and including a substituted phenyl group;

(ii) two oxygen atoms, one of which is bonded to the phosphorus atom by a double bond whereby the oxygen is an oxo radical, and the other of the two oxygen atoms is bonded singly to the phosphorus and singly to a radical selected from the group consisting of hydrogen and C-^-C_j lower alkyl; and (iii) a third oxygen atom or a nitrogen atom that is bonded to a carbon atom of the analogous ester or amide; i.e., to alpha-carbon of the alcohol or amine portion of the ester or amide, that is a portion of a chain that contains at least 5 atoms, more preferably at least 15 atoms, and most preferably at least 15 atoms and includes a substituted phenyl group. Where a cyclic amide or ester is the reactant ligand, there are not distinct acid and amine or alcohol portions of the aolecule. However, those skilled in organic chemistry will understand that amides and esters aust by definition contain acid and amine or alcohol portions. Thus, an imaginary line of demarcation can be drawn for such aolecules that includes at least the carbonyl carbon and its directly bonded alpha-carbon in the acid portion of the molecule and includes the amino or hydroxyl group and its directly bonded alpha-carbon in the amine or hydroxyl portion of the molecule. Such cyclic compounds also, of course, include a stereoisomeric center that is included in the reactant ligand portion that is bound by the catalytic receptor aolecule.

In another embodiment, this invention relates to a stereoselective method of catalytically hydrolyzing a preselected ester or amide bond in reactant ligand molecule. The method comprises the steps of: (a) admixing a catalytically effective amount of one of the foregoing receptors with reactant ligand molecules that contain a stereoisometric center in an aqueous medium; and (b) maintaining the admixture for a period of time sufficient for the ligand molecules to bind to the receptors and for the receptor molecules to hydrolyze the preselected bond of one of the possible stereoisomers of the reactant ligand. The products of that hydrolysis can be thereafter recovered, if desired. It is to be understood that a reactant ligand is used that has the same stereoconfiguration as the analog-ligand used to induce the receptor molecules. A stereoisomeric pair of reactant ligands can be used, although one stereoisomer reacts.

A hydrolytic method of this invention utilizes an aqueous me ium as a portion of the reaction admixture. That medium typically contains water and buffer salts. In addition, the medium can contain other salts such as sodium choride, as well as water-soluble calcium and magnesium salts as are frequently found in protein-containing media. Organic solvents such as aethanol, ethanol, acetonitrile, dimethyl sulfoxide, dioxane, hexamethylphosphoramide and N,N-diaethylforamide can also be present. Surface active agents that emulsify the reactant ligand and receptor molecule can also be present. The critical feature of ingredients present in the aqueous medium is that those ingredients not substantially interfere with or inhibit the catalytic reaction as by denaturation of the receptor molecule. Additionally, the aqueous medium is substantially free from salt, proteins generally, and enzymes, specifically, that inhibit the bond-breaking reaction catalyzed by the receptor molecule.

The aqueous medium typically has a pH value of about 5 to about 9, and preferably about pH 6.0 to about 8.0. pH Values greater and less than those recited values can also be utilized so long as the catalyzed reaction is again not substantially interfered with or inhibited. The catalytic reactions are typically carried out at ambient room temperature; i.e., at about 20 to about 25 degrees C or at 37 degrees C, and at an ambient atmospheric pressure; i.e., at about one atmosphere. However, temperatures down to about the freezing point of the aqueous medium and up to about the boiling point of the medium at atmospheric pressure can also be used. As is known, proteins such as the receptor molecule tend to denature at elevated temperatures such as those at which an aqueous medium boils, e.g. at about 100 degrees C, and thus temperatures below about 40 degrees C are preferred. As is also well known, reactions that follow aultimolecular kinetic expressions decrease in rate as the temperature decreases. Thus, a minimal temperature of about 15 degrees is preferred.

The reactant ligand is present in a reaction mixture in an amount up to its solubility in the aqueous medium. A two phase system that includes insoluble reactant ligand can also be used, but normally is not so used. Normally used concentrations of the reactant ligand are about 0.1 mieromolar (uM) to about 10 millimolar (mM) , with that amount also being a function of the solubility of the reactant ligand in the solvent medium. Where the product is desired, per se, relatively higher concentrations are used as compared to lower concentrations where a reaction mechanism or reaction kinetics are to be studied. An effective amount of the receptor molecule is also present. That effective amount is typically a catalytic amount; i.e., the receptor is used at a molar ratio to the reactant ligand of about 1:2 to about 1:10,000, with a molar ratio of about 1:10 to about 1:100 being preferred. The ratio of receptor molecule to reactant ligand typically depends upon the specific activity of the receptor molecule toward the reactant ligand and the purpose of the user in running the reaction. Thus, where the product is desired, a relatively higher concentration of receptor and higher receptor to reactant ligand ratio are used. Where the reaction mechanism or kinetics of the reaction are being studied, a lower concentration and ratio are typically used. A stoichiometric amount of receptor or less can also be used, but since the receptor is a catalvtic aolecule, use of even a stoichiometric amount cart be wasteful. Thus, at least a catalytic amount of the receptor is utilized. The admixture formed from mixing receptor aolecues and reactant ligand in an aqueous medium is maintained for a time period sufficient for the stereospecific binding and reaction to occur. The duration of that aaintenance period is a function of several parameters including the receptor and reactant ligand selected, their concentrations pH value and temperature, as well as what is being sought from the reaction.

Thus, where kinetics studies are being carried out, maintenance times of minutes to hours are frequently encountered. Where the reaction products are desired, maintenance times of hours to days are more usual.

III. Results

The enantiomeric Compound F covalently linked to KLH was used as an immunogenic conjugate to immunize mice. Hybridomas were prepared using spleen cells from an immunized animal. Twelve hybridomas were prepared whose secreted monoclonal antibodies (receptors) bound to Compound F coupled to BSA in an ELISA assay. Each of those binding interactions was inhibited by pre-incubation of the receptor with Compound F free in solution, thereby indicating that the observed ELISA bindings were specific to the bound haptenic analog-ligand.

Of those twelve monoclonals, the eight monoclonal receptors enumerated hereinafter were capable of catalytically hydrolyzing the exemplary enantiomeric ester reactant ligand Compound H (R,S) . Of those eight catalytic receptors, two catalyzed the hydrolysis of only the S(-) reactant ligand. Compound H [S(-)1 , whereas the other six catalyzed the hydrolysis of only the R(+) reactant ligand. Compound H [R(+)3« The specific conditions used for those stereoselective hydrolyses are discussed hereinafter.

It is to be emphasized that the receptor aolecules that hydrolyzed one enantiomer did not catalyze hydrolysis of the other enantiomer. It is also to be emphasized that no receptor was found that catalyzed hydrolysis of both the R(+) and S(-) enantiomers of Compound H.

The structures of compounds F and H, as well as the intermediates in their syntheses are shown hereinafter along with a discussion of the various syntheses involved herein.

It is believed that the above-described stereoselective catalytic hydrolyses are the first such hydrolyses ever reported. It is further believed that this is the first report of the preparation of separate antibody combining site-containing receptor molecules that can stereoselectively catalyze a reaction of each of the separate members of a stereoisomeric pair; here, enantiomers. Thus, the previously noted papers by Napper et al., Science, 237:1041 (1987) and Hilvert et al., Proc. Natl. Acad. Sci. USA, 85_:4953 (1988) both reported preparation of receptor molecules that catalyzed a reaction of only one of the two stereoisomeric substrate molecules.

The results reported herein thereby complement the results of the two prior parent applications and the Napper et al. paper, above. That complimentarity is seen in the stereoselective hydrolysis as compared to synthesis reported previously, and also in obtaining individual receptor aolecules capable of catalysing a reaction of each of the stereoisomers of the reactant ligand. Studies of the kinetics of the hydrolytic reactions have begun. Initial results indicate that the hydrolysis is relatively fast and that there is some product inhibition caused by the product acid of the reactant ligand molecule.

IV. Preparation of Analog-Ligands and Ligands

It is noted that the syntheses discussed hereinbelow relate only to one carboxylic ester as reactant ligand and one phosphonate as analog ligand. However, those syntheses can be readily adapted for the preparation of different ester and phosphonate compounds by simple substitutions of reactants. Carboxylic acid amide reactant. ligands and phosphonamide analog-ligands can also be readily prepared by procedures analogous to those described using appropriate reactants in place of those utilized herein.

Compound A

To a stirred solution of diethyl 4-aminobenzyl phosphonate (0.74 g, 3.04 mM) in 5 ml aethylene chloride (freshly distilled over calcium hydride) was added (0.32 al, 4 aM) pyridine. The aixture was cooled to 4 degrees C and trifluoroacetic anhydride (0.5 al, 3.54 aM) was added dropwise over a 5 minute period to the stirring solution. Stirring was continued for 15 minutes while the solution was allowed to warm to room temperature (about 23 degrees C) . Completion of the reaction was indicated by thin layer chromatography using a 1:1 mixture of methylene chloride CH₂C1₂) and ethyl acetate (ETOAc) as eluant (R_£ 0.2) .

The solution was thereafter diluted with 50 al of ethyl acetate. The organic solution was washed twice with successive 25 ml portions of 0.5 M HC1 and was then dried over anhydrous magnesium sulfate.

Evaporation provided a yellow oil that was purified by flash chromatography on silica gel using a 1:1 mixture of methylene chloride and ethyl acetate as eluant. The phosphonate (Compound A) (0.877 g, 85 percent yield) was obtained as a colorless crystalline material.

Proton NMR in CDC1₃ at 100 MHZ (relative to TMS as internal standard): delta 10.61 (broad singlet, 1H) , 7.53 (doublet, J«8.22 Hz, 2H) , 7.17 (double doublet, J-8.67 Hz and 2.5 Hz, 2H) , 4.02 (P, J-7.18 Hz, 2(2H)), 3.10 (doublet, J-21.62, Hz, 2H) and 1.26 (triplet, J«7.05 Hz, 2(3H).

Compound B

To a round bottom flask containing 0.2 g (5.88x10 aoles) of Compound A in 2 al of dry, freshly distilled dichloroaethane (CH₂C1₂) were added 0.8 al of triaethyl silylbromide (TMSBr; 5.9xlθ^"3 aoles). The resulting admixture was stirred at a temperature of 40^eC for a period of 3 hours.

The solvent was thereafter removed to provide a white solid. That solid was treated with a solution of 5 percent water in diethyl ether (v/v) , which dissolved the solid. Upon standing, a further white solid identified as the phosphonic acid appeared that was collected, dried and weighed 0.1274 g. Analysis by thin layer chromatography (tic) on silica gel with CH₂Cl₂/ETOAc (1:1, v:v) indicated that the phosphonic acid was pure. The phosphonic acid (0.1221 g) was dissolved in methanol to which diazomethane was added. After waiting for the reaction to take place, a cation exchange resin (proton form) was added in small amounts until the yellow color of the solution disappeared. The solvent was - removed, CH₂C1₂ added to dissolve Compound B, and the resulting solution was filtered to remove the resin beads. The solvent was thereafter removed to provide 0.1284 g of Compound B (95% yield).

Thin layer chromatography as above showed a single product.

Proton NMR in CDC1₃ at 100 MHz (relative to TMS as internal standard): delta 8.78 (broad singlet, 1H), 7.42 (multiplet, 4H) , 3.7 (doublet, J«ll Hz, 6H) , 3.17 (doublet, J«20 Hz, 2H) . Co pound C

Compound B (0.50 g; 1.6x10 moles) was placed into a round bottom flask with 4 ml of dry chloroform to which one equivalent of PC1-. (0.034 g) was added. The resulting solution was heated at a temperature of 45°C with stirring. After one hour, tic (as before) showed the reaction to be about 95 percent complete. Another one-half equivalent of PCI. was added, and the reaction maintained for another hour at which time tic showed the reaction to be complete. Sulfur dioxide was thereafter bubbled into the solution. The solvent and volatiles were then removed, and the residue was washed with diethyl ether, and dried under reduced pressure. Compound C was collected in an amount of 0.0255 g. Proton NMR in CDC1₃ at 100 MHz (relative to

TMS as internal standard): delta 8.5 (broad singlet, 1H) , 7.42 (multiplet, 4H) , 3.86 (doublet, J»14 Hz, 3H) , 3.55 (doublet, J«20 Hz, 2H) . Compound D

A racemic modification of sec-phenethyl alcohol

(alpha-methylbenzyl alcohol) (0.152 ml; 12.66x1 moles) was dissolved in dry teterahydrofuran (THF) . Sodium hydride (0.038 gj 15.83xlθ^"4 moles) was added to the solution and the resulting admixture heated under reflux for a period of 2 hours. After cooling the admixture to room temperature, 0.10 g (3.165x10 —4 moles) of Compound C were added to the cooled admixture, and the resulting admixture stirred for 5 minutes. The THF was removed, and the residue was diluted in ETOAc. That solution was washed with 0.5 M aqueous HCl, and saturated sodium chloride, and then dried over sodium sulfate. Removal of the ETOAc provided a yellow oil. That oil was purified by flash column chromoatography using CH₂Cl₂/ETOAc as solvent at 10/1, 5/1 and 3/1 (v/v) to provide 0.083 g of Compound D (65% yield).

Proton NMR in CDC1₃ at 100 MHz (relative to TMS as internal standard): delta 9.72 (broad doublet, J-10 Hz), 7.28 (multiplet, 9H) , 5.45 ( ultiplet, 1H) , diastereomeric pairs: [3.65 (doublet, J-ll Hz) 3.32 (doublet, J-ll Hz), 3B] , diastereomeric pairs: [3.1 (doublet, J«21 Hz) , 2.95 (double doublet, J»21 Hz) 2H] , diasteromeric pairs [1.6 (doublet, J-7 Hz), 1.45 (doublet, J«7 Hz) 3H] .

Compound E

Compound D (0.0250 g; 6.22 xlθ^"5 moles) was dissolved in 1 ml of ethanol with stirring. Sodium borohydride (0.0161 g; 7 equivalents) was added to the ethanol solution, and the resulting solution was stirred for a period of one hour at room temperature.

A 10 percent aqueous solution of ammonium hydroxide was then added to the above solution. The resulting solution was extracted with ETOAc, and the resulting ETOAc extract was dried over sodium sulfate. The solvent was removed to provide 0.015 g of Compound E (about 80% yield) .

Proton NMR in CDC1₃ at 100 MHz (relative to TMS as internal standard): delta 6.95 (multiplet, 9H) , 5.48 (multiplet, IH) diasteromeric pairs: [3.62 (doublet, J«ll Hz), 3.3 (doublet, J-ll Hz) 3H] diasteromeric pairs: [3.08 (doublet, J«21 Hz) , 2.88 (doublet, J»21 Hz) , 2H] diasteromeric pairs: [1.58 (doublet, J-7 Hz), 1.45 (doublet, J»7 Hz), 3H]. Compound F (Analog Ligand)

Compound E (0.032 g; 1.046xlθ^"4 moles) was dissolved in 3 ml of dry CH₂ 1₂ to which was added triethylamine (0.0145 ml; one equivaent) , and the resulting solution was stirred for 10 minutes at room temperature. Glutaric anhydride (0.0110 g; one equivalent) was thereafter added with continued stirring. The reaction was followed by tic on silica gel with CH₂Cl₂/»ethanol (5/1, v/v) as solvent. The reaction mixture was diluted with ETOAc to which 0.5 aqueous HCl was added. Four molar aqueous HCl was thereafter added until the aqueous portion was acidic. The organic solvent layer was separated, dried over sodium sulfate and then the solvent was removed under reduced pressure. The resulting product was obtained by preparative tic on silica gel using the above solvent as eluate.

Compound F was prepared by reaction of the above-prepared compound (32 mg) with 2 ml of .t-butylamine in a sealed tube at 60°C for a period of 10 days. The solvents were removed, the product purified by fast protein liquid chromatography, and racemic Compoun ,F provided by lyophilization. A total of 26.3 mg of Compound F (85% yield) was obtained. Proton NMR in DMSO-d_g at 100 MHz (relative to DMSO as internal standard): delta 9.8 (singlet, H), 7.3 (multiplet, 9Ξ) , 5.39 (multiplet, 1H), 2.98 (doublet, J-21 Hz) _ 2.30 (multiplet, 4Ξ) , 1.82 (multiplet, 2H) , 1.45 (doublet, «7 Hz, 3H) .

Compound G

Trifluoracetic anhydride (2.8 ml) was added to a solution of 4-aminoρhenyl acetic acid (1.5 g) and sodium carbonate (1.5 g) in 10 percent aqueous acetonitrile at -10 degrees C. The solution was acidified-with 6 normal HCl (0.2 ml) and was concentrated in vacuo. Filtration through silica with a 9:1 mixture of dichloromethane and methanol provided 1.4 grams (57 percent yield by weight) of p-trifluoroacetamidophenyl acetic acid. Thin layer chromatography on silica gel using a 5/1 mixture of chloroform and methanol (v/v) as eluant provided an Rf value of 0.35. Proton NMR in DMSO-dg at 100 MHz (relative to TMS as internal standard): 7.37 (doublet, J-8.7 Hz, 2H) , 7.02 (doublet, J«8.7 Hz, 2H) , 3.3 (singlet, 2H).

The foregoing acid (0.6 g) was dissolved in thionyl chloride and the solution was heated at 40 degrees C for 2 hours. The thionyl chloride was removed in vacuo to provide the corresponding acid chloride.

A solution was prepared containing 0.0461 g (3.773xl0~⁴ aoles) of S(-) βec-phenethyl alcohol and 0.0525 al of triethylamine (one equivalent) dissolved in CH₂C1₂. That solution was stirred at room temperature for one-half hour. The above-prepared acid chloride (0.10g; 3.774x10 moles) then was added followed by another equivalent of triethylamine. Addition of the acid chloride caused the solution to turn brown, and additon the amine caused a solid to start precipitating. The reaction mixture was stirred for one-half hour. The reaction mixture was thereafter diluted with ETOAc, washed with 0.5 M aqueous HCl, the organic solvent was dried over sodium sulfate, and the organic solvent was removed. The product was purified on a silica gel column using CH₂Cl₂/ETOAc (5/1, v/v) as eluate. After drying, 0.0158 g of Compound G [S(-)] were recovered.

The R(+) isomer was prepared in a generally similar manner in a yield of 48%.

A raceminc modification containing equal amounts of both of the R(+) and S(-) enantiomeric isomers was also prepared. That material is referred to as Compound G (R,S) .

Proton NMR in CDC1₃ at 100 MHz (relative to TMS as internal standard): delta 8.1 (broad singlet, IH) , 7.3 (multiplet, 9H) , 5.9 (multiplet,

IH), 3.62 (singlet, 2H) , 1.55 (doublet, J-6 Hz, 3H) . Compound H (Reactant Ligand)

Compound G [R(+)] (0.6447 g; 1.84xl0~³ moles) was dissolved in 5 ml of ethanol in a round bottom flask and 5 equivalents of sodium borohydride were added. The resulting admixture was stirred for 2.5 hours, and then poured into 20 ml of 10 volume percent aqueous ammonium hydroxide. The aqueous solution was extracted with ETOAc, and the ETOAc solution dried over sodium sulfate. The solvent was removed under reduced pressure and the residue dissolved in 5 ml of freshly distilled CH₂C1₂.

Two equivalents of triethylamine (0.51 ml) were added to the above CH₂C1₂ solution, and the resulting solution was stirred briefly. One equivalent of glutaric anhydride (0.2095 g) was then added, and the resulting reaction mixture was stirred at room temperature for 18 hours.

Volatiles were removed under reduced pressure and the residue was redissolved in ETOAc. That solution was washed with aqueous 0.5 M HCl, sodium bicarbonate (10 percent by weight) , and then saturated aqueous sodium chloride. The organic phase was dried over sodium sulfate, and then removed under reduced pressure. The residue was dissolved in a minimal amount of methanol, and mixed hexanes were added to precipitate the product. The product was washed with additional amounts of mixed hexanes and dried to provide 0.300 g (about 43% yield).

The product, Compound H [ (•*^»)], exhibited an optical rotation of 4-30.6° with the sodium D line in a polarimeter.

The other enantiomer, Compound H [S(-)], was prepared in a generally similar manner, and exhibited an optical rotation of -30.3° as above. A racemic modification was similarly prepared using Compound G (R,S) as starting material. This product was used for initial screening, and is referred to as Compound H (R,S) . Proton NMR in DMSO d_g at 100 MHz (relative to DMSO as internal standard): delta 9.9 (singlet,

IH), 7.35 (multiplet, 9H) , 5.78 (multiplet, IH) , 3.6 (singlet, 2H) , 2.25 (multiplet, 4H) , 1.75 (multiplet, 2H), 1.45 (doublet, J-6 Hz, 3H) .

Preparation of Succinimidyl Acid Chloride

Coupling Agents for Conjugate Preparation The analog-ligand (Compound F) possessed a glutaryl half amide group that was utilized to link the haptenic analog-ligand to an antigenic (immunogenic) carrier for the induction of antibodies. Additional linking groups that contain a total of 1 to 8 methylene (CH₂) groups between carboxyl groups are also useful.

Thus, diacids such as malonic acid, glutaric acid, adipic acid, through decanedioic acid are useful. Those materials can be linked to the analog-ligand by use of an appropriate anhydride, acid chloride or other suitalbe activated bond to the acid groups. A particularly useful dicarboxylic acid-derived linking group contains an O-succinimidyl group at one carboxylic acid terminus and a acid chloride at the other terminus. The procedures below discuss the specific preparation of succinimidyl adipoyl chloride as exemplary of the syntheses for other, similar linking groups.

A solution of adipic acid monomethyl ester (5.4 g, 33.3 mmol.) in thionyl chloride (15 ml) was heated at 40 degrees C for 2 hours. The mixture was then concentrated and distilled in vacuo (boiling point 119 degrees C at 20 mm Hg) to provide 3.58 g (60 percent yield by weight) of the acid chloride methyl ester. This was dissolved in 20 ml of dichloromethane and N-hydroxysuccinimide (2.75 g,

24.0 m ol) was added, followed by triethylamine (4.2 ml, 30 mmol) . The mixture was stirred for 10 minutes then diluted with ethyl acetate and washed with 0.5 M HCl and brine. The solution was dried over anhydrous magnesium sulfate, filtered and concentrated to give 4.5 g (87.5 percent yield by weight) of methyl succinimidyl adipate as a colorless oil.

Proton NMR in CDC1₃ at 100 MHz (relative to TMS as internal standard): delta 3.73 (singlet, 3H), delta 2.90 (singlet 4H) , 2.70 (multiplet, 2H) , 2.37 (multiplet, 2H) , and 1.79 (multiplet 4H) .

A solution of methyl succinimidyl adipate (4.5 g, 17.5 mmol), chlorotrimethylsilane (11.1 ml, 87.5 mmol) and sodium iodide (13.1 g, 87.5 mmol) in 10 ml of acetonitrile was heated at reflux for 12 hours. The mixture was then cooled to room temperature and diluted with ethyl acetate. The reaction mixture was washed repeatedly with 5 percent aqueous sodium bisulfite until the organic solution was colorless. Then it was washed with brine, dried over anhydrous magnesium sulfate, filtered and concentrated to provide 3.2 ς (71 percent yield by weight) of adipic acid aonosuccinimidyl ester as a white solid. Proton NMR in CDC1₃ at 100 MHz (relative to TMS as internal standard): delta 3.90 (singlet, 4H), 2.70 (multiplet,^' 2H) , 2.4 (multiplet, 2H) , 1.80 (multiplet, 4H) .

A mixture of adipic acid succinimidyl ester (1.00 g, 3.80 mmol) and thionyl chloride (5 ml) was heated at 40 degrees C for 3 hours, then cooled to room temperature and concentrated in vacuo. The residue was stirred several times with dry hexane, the oil was separated and dried in vacuo to provide 0.97 g (90 percent yield by weight) of succinimidyl adipoyl chloride. This was dissolved in dry tetrahydrofuran to make a 5 molar solution, which was used as such in the preparation of compounds suitable for coupling to protein carriers. Proton NMR in CDC1₃ at 100 MHz (relative to TMS as internal standard): 3.00 (multiplet, 2H) , 2.90 (singlet, 4H) , 2.70 (multiplet, 2H) , 1.80 (multiplet 4H) .

The reaction of a succinimidyl acid chloride with a haptenic analog-ligand is carried out in a manner substantially similar to that discussed previously for the preparation of Compound F. That reaction bonds the acid chloride-containing portion of the succinimidyl acid chloride to the amine of the hapten, and leaves the succinimidyl group free to react later with the carrier.

V. Preparation of Conjugates and Inocula

Conjugates of haptenic analog-ligand molecules with antigenic (immunogenic) protein carriers such as keyhole limpet hemocyanin (KLH) can be prepared, for example, by activatiς- of the carrier with a coupling agent such as MBS (m-aaleiaidobenzoyl-N-hydroxy succinimide ester) , and coupling to the thiol group of the analog-ligand. See, for example, Liu et al., Bioehem. , 80, €90 (1979). As is also well known in the art, it is often beneficial to bind a compound to its carrier by means of an intermediate, linking group. Useful carriers are well known in the art and are generally proteins themselves. Exemplary of such carriers are keyhole limpet hemocyanin (KLH) , edestin, thyroglobulin, albumins such as bovine serum albumin or human serum albumin (BSA or HSA, respectively) , red blood cells such as sheep erythrocytes (SRBC) , tetanus toxoid, cholera toxoid as well as polyamino acids such as poly(D-lysine:D-glutamic acid), and the like.

The choice of carrier is more dependent upon the ultimate intended use of the antigen than upon the determinant portion of the antigen, and is based upon criteria not particularly involved in the present invention. For example, if the conjugate is to be used in laboratory animals, a carrier that does not generate an untoward reaction in the particular animal should be selected.

The carrier-hapten conjugate is dissolved or dispersed in an aqueous composition of a physiologically tolerable diluent such as normal saline, PBS, or sterile water to form an inoculum. An adjuvant such as complete or incomplete Freund's adjuvant or alum can also be included in the inoculum. The inoculum is introduced as by injection into the animal used to raise the antibodies in an amount sufficient to induce antibodies, as is well known. -49- Xn an exemplary procedure, 2.5 ag of a reaction product of haptenic analog-ligand and succinimidyl adipoyl chloride or succinimidyl glutaroyl chloride in 250 ul of dimethylforaamide is slowly added to 2 ag of KLH in 750 ul of 0.01 M sodium phosphate buffer at a pH value of 7.2. A temperature of 4 degrees C is utilized and the resulting admixture is stirred for about one hour to form the hapten-linked KLH conjugate. The conjugate reaction product so formed is thereafter purified by usual means.

In the present work, Compound F (5 mg) was admixed with KLH (2 mg) in water (2 ml) . The pH was adjusted to 4.5 with HCl and 10 equivalents of l-ethyl-3-(3-dimethylaminoproply)-carbodiimide were then added. The mixture was stirred for about 12 hours. The resultant crude product was injected into mice.

VI. Preparation of Monoclonal Receptors

The foregoing KLH conjugates (about 100 ug) were used to immunize mice (129G1X strain) , and monoclonal antibodies were obtained as described by Niman et al., Proc. Natl. Acad. Sci. USA, 77, 4524 (1980) and Niman et al., in Monoclonal Antibodies and T-Cell Products, ed., Katz, D.H., 23-51 (CRC Press, Boca Raton, FL 1982) • The lymphocytes employed to form the hybridomas .of the present invention can be derived from any mammal, such as a primate, rodent (e.g., mouse or rat), rabbit, guinea pig, cow, dog, sheep, pig or the like. As appropriate, the host can be sensitized by injection of the immunogen, in this instance a haptenic analog-ligand, followed by a booster injection, and then isolation of the spleen. It is preferred that the myeloma cell line be from the same species as the lymphocytes. Therefore, fused hybrids such as aouse-aouse hybrids [Shulman et al.. Nature, 276, 269 (1978)3 or rat-rat hybrids [Gal e et al.. Nature, 277, 131 (1979)] are typically utilized. However, some rat-mouse hybrids have also been successfully used in forming hybridomas [Goding, "Production of Monoclonal Antibodies by Cell Fusion," in Antibody as a Tool, Marchalonis et al. eds., John Wiley & Sons Ltd., p. 273 (1982)]. Suitable myeloma lines for use in the present invention include MPC-11 (ATCC CRL 167) , P3X63-Ag8.653 (ATCC CRL 1580), Sp2/0-Agl4 (ATCC CRL 1581), P3X63Ag8U.l (ATCC CRL 1597), Y3-Agl.2.3. (deposited at Collection Nationale de Cultures de Microorganisms, Paris, France, number 1-078) and P3X63Ag8 (ATCC TIB 9). The non-secreting murine myeloma line Sp2/0 or Sp2/0-Agl4 is preferred for use in the present invention. The hybridoma cells that are ultimately produced can be cultured following usual _in vitro tissue culture techniques for such cells as are well known. More preferably, the hybridoma cells are cultured in animals using similarly well known techniques with the monoclonal receptors being obtained from the ascites fluid so generated. The animals used for generation of the ascites fluid were female 129G1X⁺ mice bred in the mouse colony of the Scripps Clinic and Research Foundation, La Jolla, California, however, when animals other than mice are used for preparation of the hybridomas, mice or that animal type can be used for the production of ascites fluid.

In particular, an exemplary monoclonal receptor was produced by the standard hybridoma technology of Kohler et al. , Nature, 256, 495 (1975). Specifically, female 129G1X* aice were iaaunized by intraperitoneal injection with an . inoculum of 100 micr© rams of conjugate (e.g., Compound F bound to KLH) in 300 microliters of a 1:1 mixture of phosphate buffered saline (PBS) pH 7.4 and complete Freund's adjuvant. Two weeks later, the mice were again injected in a like manner with 50 micrograms of the foregoing conjugate in 300 microliters of a 1:1 mixture of PBS (pH 7.4) and 10 mg/ml alum. After an additional eight weeks, the mice were immunized intravenously with 50 micrograms of the conjugate in 200 microliters of PBS (pH 7.4). The spleens were removed from the mice 4 days later, and the spleen cells were fused to myeloma cells.

The spleens cells were pooled and a single cell suspension was made. Nucleated spleen cells (1.4xl0⁸) were then fused with 3xl0⁷ Sp2/0-Agl4 non-secreting myeloma cells in the presence of a cell fusion promoter (polyethylene glycol 2000) . The hybridoma that produces a particular monoclonal antibody was selected by seeding the spleen cells in 96-well plates and by growth in Dulbecco's modified Eagle medium (DMEM) containing 4500 mg/liter glucose (10 percent), 10 percent fetal calf serum (FCS) , hypoxanthine, aminopterin and thymidine (i.e., HAT medium) which does not support growth of the unfused myeloma cells.

After two to three weeks, the supernatant above the cell clone in each well was sampled and tested by an ELISA assay (enzyme linked immunosorbent assay as described hereafter) for the presence of antibodies against Compound F. Positive wells were cloned twice by limiting dilution. Those clones that continued to produce Compound F-specific antibody after two clonings were expanded to produce larger volumes of supernatant fluid. The hybridomas and the aonoclonal receptors produced therefrom and described herein are identified by the laboratory designations as shown in the Table hereinafter.

A aonoclonal receptor of the present invention can also be produced by introducing, as by injection, the hybridoma into the peritoneal cavity of a mammal such as a mouse. Preferably, as already noted, syngenic or semi-syngenic mammals are used, as in U.S. Patent 4,361,549, the disclosure of which is incorporated herein by reference. The introduction of the hybridoma causes formation of antibody-producing hybridomas after a suitable period of growth, e.g. 1-2 weeks, and results in a high concentration of the receptor being produced that can be recovered from the bloodstream and peritoneal exudate (ascites) of the host mouse.

Although the host mice also have normal receptors in their blood and ascites, the concentration of normal receptors is typically only about five percent that of the monoclonal receptor concentration.

Monoclonal receptors are precipitated from the ascitic fluids, purified by anion exchange chromatography, and dialyzed against three different buffers.

In the present studies, IgG fractions were typically obtained from mouse ascites by precipitation with 45 percent saturated ammonium sulfate followed by chromatography on DEAE-Sephacel with sodium chloride elution. The fraction that was eluted with 100 mM salt was dialyzed and concentrated. Protein concentrations were determined by the Lowry method. [J. Biol. Chem., 193:265 (1951) ] _• The resulting concentrated solutions containing isolated IgG fractions were typically prepared into stock solutions of receptor at 1-20 mg/ml using an appropriate buffer such as 50 aM Tris-HCl or sodium phosphate containing 0.01 M sodium azide.

Of the twelve monoclonal receptors that bound to the antigen in the ELISA, eight catalyzed the hydrolysis of racemic Compound H (R,S) substrate. Two of those eight catalyst-secreting hybridomas, one that catalyzed a reaction of each stereoisomer, were deposited as examples of the invention. The hybridomas were deposited at the American Type Culture Collection, 12301 Parklawn Drive, Rockville, MD as shown in the Hybridoma Deposit Table, below.

N.D. ■ not deposited. The present deposits were aade in compliance with the Budapest Treaty requirements that the duration of the deposits should be for J0 years.from the date of deposit or for 5 years after the last request for the deposit at the depository or for the enforceable life of a U.S. patent that matures from this application, whichever is longer. The hybridomas will be replenished should they become non-viable at the depository. A Fab fragment of a monoclonal monoclonal receptor can be was prepared from the purified receptor using predigested papain in a 0.1 M sodium acetate buffer, at a pH value of 5.5, at 37 degrees C, followed by reaction with iodoaceta ide. The Fab fragment is typically further purified by anion exchange chromatography, dialysis, and DΞAE anion exchange chromatography, and its homogeneity is judged by gel electrophoresis.

VII. Enzyme-linked I munosorbent Assay (ELISA) The binding of an analog-ligand by the induced monoclonal receptor molecule was assayed by ELISA with antibody at a fixed concentration in the range of its titer and varying inhibitor (free Compound F) concentration. Use of free Compound F as inhibitor helps to assure that an observed binding interaction is antigen-specific.

Assays were performed in flat-bottom polyvinyl microtiter plates (Dynatech, Alexandria, VA) . Illustratively, the wells were coated with a solution comprising Compound F bound to BSA as the antigen ligand in phosphate buffered saline (PBS) using 50 microliters of solution per well. BSA was used as a carrier to bind the hapten to the cell wall, and an analog-ligand/BSA conjugate was used in place of the immunizing KLH-containing to screen out possible anti-KLH antibodies.

The bound ligands were coated at 1 aicrogram per ailliliter. The plates were then incubated overnight at 37 degrees C in a dry oven. The dried plates were stored at 4 degrees C until use. Prior to the ELISA assay, dried plates were rehydrated by two washes of 2 minutes each with 10 aillimolar ( M) PBS, pH 7.4, containing 0.1 percent polyoxalkylene ^* (20) sorbitan monolaurate (Tween 20) and 0.02 percent Thimerosal (sodium ethylmercurithiosalicylate) , (Sigma, St. Louis, MO).

In order to reduce non-specific binding, hybridoma supernatantβ were diluted 1:2 in washing buffer containing 0.1 percent BSA as diluent. Fifty microliters of diluted hybridoma supernatants were thereafter added to each well and incubated for 1 hour at 4 degrees C on a gyroshaker to contact the monoclonal antibody-containing supernatant with the bound Compound F. Following two washes of 2 minutes each, 50 microliters of peroxidase-labeled goat anti-mouse IgG + igM (Tago, Burlingame, CA) , diluted 1:1000, were added to each well, and the reaction mixture was incubated at 4 degrees C for 1 hour to bind the labeled antibody to bound monoclonal antibody.

The substrate used to assay bound peroxidase activity was prepared just prior to use and consisted of 400 microgram/ml o-phenylenediamine (Sigma, St. Louis, MO) in 80 mM citrate-phosphate buffer, pH 6.0, containing 0.12 percent H₂o₂. After two final • washes, 50 microliters of substrate solution were added to each well, and color was allowed to develop for 15 minutes in the dark. Color development was stopped by adding 25 microliters of 4 molar H₂SO_< to each well and the optical density at 492 nanometers (nm) was aeasured with a Multiskan ELISA plate reader.

For another preparation of the receptor aolecules, the gene that encodes an antibody combining site-forming fragment can be obtained from any cell that produces an antibody molecule that immunoreacts as discussed herein. A preferred cell is a hybridoma cell. For examples of general reco binant DNA cloning methods, see Molecular Cloning, Maniatis et al.. Cold Spring Harbor Lab., N.Y., 1982; DNA Cloning, Glover, ed., IRL Press, McLean VA (1985). For the genomic cloning and expression of immunoglobulin genes in lymphoid cells, see Neuberger et al., Nature, 312:604-8 (1984); Ochi et al., Proc. Natl. Acad. Sci. USA, 80:6351-55 (1987); and Oi et al., Proc. Natl. Acad. Sci. USA, 80:825-29 (1983). For cloning of immunoglobulin genes from hybridoma cells and expression in Xenopus oocytes, see Roberts et al., Protein Engineering, 1:59-65 (1986), and see Wood et al. for expression in yeast. Nature, 314:446-9 (1985).

The foregoing is intended as illustrative of the present invention but riot limiting. Numerous variations and modifications can be effected without departing from true spirit and scope of the invention.

Claims

What Is Claiaed Is:

1. A receptor aolecule that contains an antibody combining site that is capable of catalytically hydrolyzing a preselected, scissile carboxylic acid amide or ester bond of one stereoisomer of a reactant ligand, said antibody combining site binding to:

(a) one of a pair of stereoisomers of a reactant ligand that contains said preselected scissile carboxylic acid amide or ester bond and

(b) one of a pair of stereoisomers of an analog-ligand that is stereochemically analogous to said reactant ligand and that contains a tetrahedrally bonded phosphorus atom at a position analogous to that of said scissile carbonyl carbon atom of said preselected carboxylic acid amide or ester bond of said reactant ligand.

2. The receptor molecule of claim 1 wherein said phosphorus atom is bonded directly to: (i) a carbon atom of the acid portion of the analogue reactant ligand amide or ester that is included in a chain containing at least 5 atoms;

(ii) one oxygen atom that is doubly bonded to said phosphorus atom;

(iii)one oxygen atom that is singly bonded to said phosphorus atom and singly bonded to a hydrogen atom or a C_χ-C₄ lower alkyl group; and (iv) a third oxygen atom or an imino nitrogen atom that is further bonded to the alpha-carbon of the alcohol or amine portion of the analogous ester or amide, respectively, and is a portion of a chain that contains at least 5 atoms. 3. The receptor aolecule of claim 1 wherein the stereoisomeric center of said stereoisomer bound by said antibody combining site is located in the alcohol or amine portion of said reactant ligand.

4. The receptor aolecule of claim 1 wherein the stereoisomeric center of said reactant ligand stereoisomer bound by said antibody combining site is within a volume occupied by one to about four amino acid residues from said scissile carboxylic acid amide or ester bond.

5. The receptor molecule of claim 1 wherein said stereoisomers are enantiomers.

6. A receptor molecule that contains an antibody combining site that is capable of catalytically hydrolyzing a preselected, scissile carboxylic acid amide or ester bond of one stereoisomer of a reactant ligand and not the other stereoisomer, said antibody combining site binding to: a) one of a pair of stereoisomers of an analog-ligand molecule represented by the formula:

wherein X « 0 or NH;

wherein R₄ « (c*^)_nco R

■ B o: ^ci~ 4 lower alkyl; and

wherein the wavy line indicates both stereochemical isomers; and n is an integer from 1 to 8, inclusive,

and.

one stereoisomer of a reactant ligand represented by the formula

wherein X, R and ₃ are as before described.

7. The receptor molecule according to claim 6 wherein said receptor is secreted by hybridoma 2H6 having ATCC accession number HB 9969 . 8. The receptor molecule according to claim 6 wherein said receptor is secreted by hybridoma 21H3 having ATCC accession number HB 9970 , 9. A hybridoma that secretes a monoclonal receptor molecule containing an antibody combining site that is capable of catalytically hydrolyzing a preselected, scissile carboxylic acid amide or ester bond of one stereoisoaer of a reactant ligand, said antibody combining site binding to:

(a) one of a pair of stereoisomers of a reactant ligand that contains said preselected scissile carboxylic acid amide or ester bond, and

(b) one of a pair of stereoisomers ,of an analog-ligand that is stereochemically analogous to said reactant ligand and that contains a tetrahedrally bonded phosphorus atom at a position analogous to that of said scissile carbonyl carbon atom of said preselected carboxylic acid amide or ester bond of said reactant ligand.

10. The hybridoma of claim 9 wherein said phosphorus atom is bonded directly to: (i) a carbon atom of the acid portion of the analogus reactant ligand amide or ester that is included in a chain containing at least 5 atoms;

(ii) one oxygen atom that is doubly bonded to said phosphorus atom; (iii) one oxygen atom that is singly bonded to said phosphorus atom and singly bonded to a hydrogen atom or a -_j-C^ lower alkyl group; and

(iv) a third oxygen atom or an imino nitrogen atom that is further bonded to the alpha-carbon of the alcohol or amine portion of the analogous ester or amide, respectively, and is a portion of a chain that contains at least 5 atoms.

11. The hybridoma of claim 9 designated 2H6 and having the ATCC accession number HB 9969 . 12. The hybridoma of claim 9 designated

21H3 and having the ATCC accession number HB 9970 .

13. A aethod of catalyzing the hydrolysis reaction of one of a pair of stereoisomeric carboxylic acid amide or ester molecules comprising the steps of: admixing a catalytically effective amount of a receptor aolecules with stereoisomeric reactant ligand aolecules in an aqueous medium to form an admixture, wherein said receptor aolecules contain an antibody combining site that is capable of hydrolyzing a preselected, scissile carboxylic acid amide or ester bond of one stereoisomer of said reactant ligand and not the other stereoisomer, said antibody combining site binding to: one of a pair of stereoisomers of a reactant ligand that contains said preselected scissile carboxylic acid amide or ester bond, and one of a pair of stereoisomers of an analog-ligand that is stereochemically analogous to said reactant ligand and that contains a tetrahedrally bonded phosphorus atom at a position analogous to that of said scissile carbonyl carbon atom of said preselected carboxylic acid amide or ester bond of said reactant ligand; and maintaining said admixture for a time period sufficient for said reactant ligand molecules to bind to said receptor molecules and for said receptor molecules to catalyze the hydrolysis of said preselected scissile bond. 14. The method of claim 13 wherein said antibody combining site binds to:

(i) a carbon atom of the acid portion of the analogue reactant ligand amide or ester that is included in a chain containing at least 5 atoms; (ii) one oxygen atom that is doubly bonded to said phosphorus atom;

(iii) one oxygen atom that is singly bonded to said phosphorus atom and singly bonded to a hydrogen atom or a C_χ-C₄ lower alkyl group; and (iv) a third oxygen atom or an imino nitrogen atom that is further bonded to the alpha-carbon of the alcohol or amine portion of the analogous ester or amide, respectively, and is a portion of a chain that contains at least 5 atoms.

15. The aethod of claim 13 wherein said reactant ligand is present as a pair of stereoisomers.

16. The aethod of claim 15 wherein said stereoisomeric pair are enantiomers. 17. The aethod of claim 13 wherein the stereoisomeric center of said stereoisomer bound by said antibody combining site is located in the alcohol or amine portion of said reactant ligand. 18. The method of claim 13 wherein the stereoisomeric center of said reactant ligand stereoisomer bound by said antibody combining site is within a volume occupied by one to about four amino acid residues from said scissile carboxylic acid amide or ester bond. 19. A method of catalyzing the hydrolysis of one of a pair of stereoisomeric carboxylic acid amide or ester molecules comprising the steps of: admixing a catalytically effective amount of a receptor molecules with stereoisomeric reactant ligand molecules in an aqueous medium to form an admixture, wherein said receptor molecules contain an antibody combining site that is capable of hydrolyzing a preselected, scissile carboxylic acid amide or ester bond of one stereoisomer of said reactant ligand and not the other stereoisomer, said antibody combining site binding to:

(a) one of a pair of stereoisomers of a reactant ligand that contains said preselected scissile carboxylic acid amide or ester bond located within a volume occupied by one to about four amino acid residues from a stereoisomeric center of said reactant ligand, and

(b) one of a pair of stereoisoaers of an analog-ligand that is stereochemically analogous to said reactant ligand, that contains a tetrahedrally bonded phosphorus atom at a position analogous to that of said scissile carbonyl carbon atom of said preselected carboxylic acid amide or ester bond of said reactant ligand, and contains a stereoisomeric center located within a volume occupied by one to four amino acid residues from said phosphorus atom, said phosphorus atom being bonded directly to:

(i) a carbon atom of the acid portion of the analogus reactant ligand amide or ester that is included in a chain containing at least 5 atoms;

(ii) one oxygen atom that is doubly bonded to said phosphorus atom;

(iii)one oxygen atom that is singly bonded to said phosphorus atom and singly bonded to a hydrogen atom or a C₁-C₄ lower alkyl group; and (iv) a third oxygen atom or an i ino nitrogen atom that is further bonded to the alpha-carbon of the alcohol or amine portion of the analogous ester or amide, respectively, and is a portion of a chain that contains at least 5 atoms; and maintaining said admixture for a time period sufficient for said reactant ligand molecules to bind to said receptor molecules and for said receptor molecules to catalyze the hydrolysis of said preselected scissile bond.

20. The method of claim 19 wherein said receptor molecules are secreted by hybridoms 2H6 having ATCC accession number HB 9969. 21. The aethod of claim 20 wherein said receptor aolecules are secreted by hybridoma 21Ξ3 having ATCC accession number HB 9970 _•