CATALYTIC ANTIBODIES

Abstract: Polyclonal catalytic antibodies are provided. Polyclonal antibodies that are capable of catalyzing the hydrolsis of a carbonate are also provided. Preferred polyclonal catalytic antibodies are raised against a phosphate of general formula (I), wherein each of R and R' represents a radical which together confer immunogenicity on the phosphate of general formula (I). In addition, phosphates and precursor phosphates against which antiphosphate antibodies capable of catalyzing the hydrolysis of a carbonate can be raised are provided.

Description: CATALYTIC ANTIBODIES The present invention relates to catalytic antibodies. A catalyst accelerates a chemical reaction while itself undergoing no permanent chemical change. It achieves this by lowering the energy of activation of the reaction. Enzymes are catalysts comprised of protein and are produced by living cells. Enzymes bind substrate(s) and catalyze their chemical reaction(s). In 1948 Pauling (American Scientist; 1948; .3J, 51- 58) suggested that enzymes are able to catalyze reactions because the surface configuration of the binding site is complementary no so much to reactants or products but rather to ah unstable specie on the reaction pathway with only transient existence. He referred to this transient species as the activated complex of th reaction but this species is now generally called the transition state. In 1969 Jenc s (Catalysis in Chemistry and Enzvmoloσv; 1969; McGraw-Hill, p. 288) proposed that if the complementarity between enzyme active site and the transition state contributes t catalysis, then it should be possible to obtain a catalyst by constructing such a binding site. Moreover, since it was known (Landsteiner: "The Specificity of Seroloσical Reactions"; 1947, Harvard University Press) that specific antibodies could be raise against almost any organic molecule (hapten), he proposed that such a catalytic binding site could be obtained by raising antibodies against a hapten which resembles the transition state of a chemical reaction. Herein, such haptens are called transition state analogues. Initial attempts to obtain polyclona catalytiσ antibodies by these (Raso and Stollar, Biochemistry: 1975; .14., 591-599) and other (Royer, Advances in Catalysis: 1980; 29, p. 233) methods were unsuccessful. Antibodies have been reported (Kohen, FEBS Letters: 1980; 111, 427) that react stoichiometrically with certain haptens. These antibodies were not raised against a transition state analogue. They may be considered as having been raised against a reactant and are presumed to react through nucleophilic residues fortuitously situated close to their binding site. The production of monoclonal catalytic antibodies was described in 1986 simultaneously by two groups. One group, heade by Lerner, produced monoclonal antibodies that catalyzed ester hydrolysis by raising the antibodies against phosphonate transition state analogues (Lerner, Science; 1986; 234, 1570- 1573). Patents and applications describing this work have now been published (1987 US 4 659 567 and 1988 EP-A-0 260 939). The description of this work also refers to polyclonal antibodies, bu no actual experimental examples are recited. The other group, headed by Schultz, described catalysis of carbonate hydrolysis by a monoclonal antibody that bound a phosphate transition state (Schultz, Science: 1986; 234, 1570- 1573). In this example, the monoclonal antibody was not produced via a synthetic immunogen, but was myeloma protein (the result of a cancer) first described in 1971 (Leon, Biochemistry: 1971; 10, 1424) that happened to bind certain phosphate compounds. When Schultz set out to design and produce monoclonal antibodies to catalyze carbonate hydrolysis, he employed synthetic phosphonate transition state analogues (J. Amer. Chem. Soc: 1987; 109, 2174), as had Lerner's group. Schultz has also stated (Science: 1988; 240, p. 428) that his group has been unable to generate polyclonal catalytic antibodies. Several monoclonal antibodies have now been described that catalyze hydrolysis or other acyl transfer reactions (Science: 1988; 241, 1188 and references therein), all of which were raised against phosphonate transition state analogues. No group has yet demonstrated the successful production of the polyclonal catalytic antibodies. Attempts by Schultz and others are discussed above. One group (Green et al, J.C.S. Che . Comm♦ : 1988, 106) has described a polyclonal antibody preparation that acts as a template in a photochemical reaction, influencing the product distribution. However, those antibodies were not raised against transition state analogue but were raised against a reaction product. Moreover catalysis was not claimed and the reaction was not analyzed kinetically. Another group (Igen Inc., 1985, WO 85/ 02414) claimed that monoclonal catalytic antibodies were inducibl by: a reactant, a reaction intermediate, or an analogue of the reactant, product or reaction intermediate. However, the data provided in this patent related to polyclonal antisera which were not raised against transition state analogues. Instead, these antisera were obtained by immunization with a reactant and an analogue of a product. Very concentrated antibody preparations were employed to bring about the chemical reaction, and although catalysis was claimed it was not demonstrated by kinetic analysis In particular the data presented does not distinguish between catalysis or stoichiometric reaction similar to that previously described by Kohen (above). The use of monoclonal catalytic antibodies has the disadvantage that production of monoclonal antibodies involves the use of cancer cells such as mouse myeloma cells, with the concomitant safety doubts associated with the use of cancer cells. It would be advantageous to use polyclonal catalytic antibodies raised against transition state analogues since these do not have the same doubts as to safety associated with them as does the use of monoclonal antibodies. The production of polyclonal antibodies is also simpler and less expensive than the production of monoclonal antibodies. According to a first aspect of the present invention there are provided polyclonal catalytic antibodies. These polyclonal antibodies preferrably are raised against a phosphate of general Formula Is R 0- P—0 R 1 OH wherein each of R and R' represents a radical which together confer immunogenicity on the phosphate of general Formula I. More preferably, these polyclonal antibodies are capable of catalyzing the hydrolysis of a carbonate. R and R' may be selected to be analogous to a carbonate which it is desired to hydrolyze. Either or both of R and R* separately or together comprise a immunogenic moiety. When one of R and R is an immunogenic moiety, it may be that the other is not an immunogenic moiety. A immunogenic moiety is a moiety which confers immogenicity on the phosphate of general Formula I. R and/or R may comprise a hydrocarbyl radical, which is optionally substituted and which may be the immunogenic moiety itself, may be a linkage between the phosphate group and the immunogenic moiety, or either one of R and R λ may comprise such a hydrocarbyl radical which is non-immunogenic. The optional substituent(s) may for example be chosen from halogen atoms (particularly fluorine, chlorine and bromine atoms) and C. - C . alkoxy, C_ - C g alkanoyl, nitro, amino, mono- or di- ( c ι _ ) alkyla ino, hydroxy or trifluoromethyl groups. The hydrocarbyl radical is preferably a C. - ~ Q alkyl group more preferably a C. - C g alkyl group; or an aryl or alkyl aryl group more preferably a CO - C. alkyl C g or C. Q aryl group, particularly a phenyl group, a benzyl group or a napthyl group. In some embodiments it is preferred that the hydrocarbyl radical comprises a nitrophenol group or other effective hapten as this may increase the immunogenicity of phosphates of general Formula I. One or both of R and/or R' may comprise a medium sized ring, such as aryl, arylalkyl (such as benzyl), cycloalkyl (such as cyclohexyl) or a heterocyclic ring (any of which rings may optionally be substituted as above), wherein the ring: (i) confers a degree of rigidity in the molecule; and (ii) provides one or more significant features for antibody:substrate recognition and binding in the antibody:substrate interaction. Although it is not possible to quantify either rigidity or the significance of features for antibody:substrate recognition and interaction, those skilled in the art will recognize such groups, which will often (but not necessarily) have molecule weights in the range of from 50 to 400. As those skilled in the art will recognize, the smaller a radical is, the more rigid and/or electron-rich it needs to be to provide significant features for antibody:substrate recognition and interaction. In some embodiments, the immunogenic moiety is linked by an amide group, a diazo group, a disulphide group or an i ine group. Particularly preferred phosphates are represented by the Formula II: O OH p and Formula III: 0 2 N where KLH represents keyhole limpet haemocyanin. In some embodiments, the immunogenic moiety comprises a protein carrier such as, for example, keyhole limpet haemocyanin (KLH) , edestin, thyroglobulin, albumins such as bovine serum albumin (BSA) or human serum albumin (HSA) . It is particularly preferred that the immunogenic moiety comprises keyhole limpet haemocyanin (KLH) . According to a second aspect of the present invention there are provided phosphates of general Formula I: 0 R o—P —0 R 1 I OH wherein each of R and R' represents a radical which together confer immunogenicity on the phosphates of general Formula I, against which antiphosphate antibodies capable of catalyzing the hydrolysis of a carbonate can be raised. Preferred features of the second aspect are as for the first aspect. According to a third aspect of the present invention there are provided precursors to phosphates of general Formula I, which precursors are represented by general Formula II: R 2 0 —P 0 R 3 I OH wherein R2 and R 3 each represent a hydrocarbyl radical, which is optionally substituted such that, when the precursor is linked to an immunogenic moiety, a phosphate of Formula I may be formed. In R2 and R3 the hydrocarbyl radical are independently preferably a C. -C 20 alkyl group, more preferably a C. „ alkyl group; or an aryl or alkylaryl group more preferably a C Q -C. alkyl Cg or C. Q aryl group, particularly a phenyl group, a benzyl group, or a naphthyl group. In some embodiments it is preferred that the hydrocarbyl radical comprises .a nitrophenol group, or other effective hapten. The optional substituent(s) may for example be chosen from halogen atoms (particularly fluorine, chlorine and bromine atoms) and C, - C . alkoxy, C 2 -C- alkanoyl, carboxy, alkyl carboxy, nitro, amino, mono- or di- (C._ 4 ) alkylamino, hydroxy or trifluoromethyl groups. Either of or both substituents R 3 and R4 may include a linke moiety which facilitates linking a phosphate of general Formula I to an immunogenic moiety. Such a linker moiety may, for example, be capable of forming a direct covalent or other bond with a protein or other immunogenic moiety or may be capable of reacting with one or other species to form an appropriate linkage. particularly preferred precursor is 4-nitrophenyl 4'- carboxymethylphenyl phosphate: Methods for the production of polyclonal antibodies against haptens are well known to those skilled in the art. For example, sheep, rabbits, goat or any other suitable animals may be immunized subcutaneously with the immunogenic phosphate over a period of time, and the animals later bled, the serum separated, and the antibodies identified. The precursors according to the third aspect of the present invention can be labelled with a label to enable detection of antibody binding. This enables the specificity of antibodies in accordance with first aspect to be checked. Thus, according to a fourth aspect of the present invention there is provided a phosphate of general Formula III: R 4 0- •0 R- OH wherein R 4 and R-5 each comprises a hydrocarbyl radical preferred 2 hydrocarbyl radicals corresponding to those as defined for R and R3, and either or both of R4 and R5 comprises a label to enable detection of antibody binding. The label may, for example be a fluorescent marker, such as fluorescein, or any other label which enables selective detection of phosphates of general Formula III when bound to antibody. As described in the first aspect of the present invention, in a preferred feature, the phosphates of general Formula I comprise a nitrophenol group, or other effective hapten in order to increase the immunogenicity of phosphates of general Formula I. further purpose may be served by the use of a nitrophenol or other marker group as a marker where, in hydrolysis of the carbonate analogous to the phosphate of Formula I, the marker group is liberated enabling the progress of the catalyzed reaction to be followed. Thus, according to a fifth aspect of the present invention there are provided carbonates of general Formula IV: u II wherein R-5 and R 7 represent a hydrocarbyl radical, which is optionally substituted, and in which either or both R 6 and R7 comprise one or more marker groups which marker group is liberate in the hydrolysis of the carbonate, enabling the progress of hydrolysis to be followed. A particularly preferred marker group is a nitrophenol group. Without being bound by theory it is believed that hydrolysis of trigonyl carbonates proceeds through an unstable tetrahedral transitional state as shown in Scheme 1. Scheme 1 The tetrahedral phosphate shown in general Formula V below is a stable analogue of this tetrahedral transitional state. Moreover, the phosphate is a closer analogue of the carbonate hydrolysis transition state than other analogues such as phosphonates or phosphonamidates. General Formula V (wherein R and R' are as defined for general Formula I). Therefore, antibodies raised against a phosphate should specifically catalyze hydrolysis of the corresponding carbonate. Such antibodies are not expected to catalysis hydrolysis of totally unrelated carbonates, although catalysis of hydrolysis of analogous but not identically corresponding carbonates may be observed, at least in some cases. Specificity is, however, in general a feature of antibodies in accordance with the invention and so the carbonate which it is desired to selectively hydrolyze can be specified by choice of a phosphate analogous to the desired carbonate. Moreover, the specific catalyzed hydrolysis reaction should be inhibited by the original phosphate as the antibodies preferentially bind this compound. Thus the phosphate can be used as an inhibitor of carbonate hydrolysis to exercise control of the carbonate hydrolysis reaction. A better understanding of the present invention and its advantages will be had by referring to the following specific examples given by way of illustration. Example 1 Synthesis of Phosphate Immunoσen III (Scheme 3) (a) Preparation of 4-Nitrophenyl-4'-methoxy-carbonylmethyl) phenyl phosphate (I) A solution of methyl 4-hydroxyphenylacetate (300 mg, 1.8 mmol) in acetonitrile (2ml) was added dropwise to a stirred solution of 4-nitrophenyl phosphorodichloridate (512 mg, 2 mmol) in pyridine (4 ml) at 0°C. The mixture was stirred at this temperature for 30 minutes, water (800 microlitres) was then adde and the reaction allowed to come to room temperature over 2 hours Solvents were removed in vacuo, the residue dissolved in aqueous sodium bicarbonate (0.5M, 15 ml) and extracted with diethyl ether (2 x 15 ml). The aqueous solution was acidified with hydrochlori acid and extracted with ethyl acetate (3 x 25 ml). The combined ethyl acetate extracts were dried and concentrated in vacuo to give the crude product (550 mg, 75%) as a yellow oil (R f 0.5, developing solvent CH 2 C1 2 : MeOH 3:1). This was purified by flash chromatography (eluting solvent CH 2 C1 2 : MeOH 3:1) to give the product (I) as a colorless oil. IR nu cm _1 (neat) 3500 (b) 1725 (s) •T-H NMR 250 MHz, (dg- .DMSO) , delta 3.32 (3H,s,CH 3 ), 3.59 (2H,s,CH 2 ) 7.05 (2H,d,J 8.8Hz, H A of ABq, ArCH 2 C0 2 ) 7.12 (2H,d,J 8.8Hz, H B of ABq, ArCH 2 C0 2 ) 7.40 (2h,d,J 9.2Hz, H A of ABq, Ar-Nθ2) 8.17 (2H,d,J 9.2Hz, H B of ABq, Ar-Nθ2) (b) Preparation of 4-Nitrophenyl-4'-(carboxymethyl) phenyl phosphate (II) Aqueous sodium hydroxide (1M, 500 microlitres) was added to stirred solution of 4-nitrophenyl 4'-methoxycarbonyl methylphenyl phosphate (I) (180 mg, 0.49 mmol) in tetrahydrofuran (3ml) and water (2ml) . After five minutes a second equivalent of aqueous sodium hydroxide (1M, 500 microlitres) was added and the reaction stirred at room temperature for 1 hour. The mixture was then acidified with hydrochloric acid and extracted with ethyl acetate (3 x 20 ml). The combined ethyl acetate extracts were dried and concentrated in vacuo to give the crude product (R f 0.7 developin solvent CH 2 C1 2 : MeOH: AcOH 50:50:1) as a pale brown solid (140 mg). Recrystallization from ethyl acetate: petroleum spirit gave the pure product (50 mg, 29%) as white crystals. 1 H NMR 250 MHz (D 2 0) delta 3.54 (2H,s,CH 2 Cθ2) , 6.99 (2H,d, J 8.5Hz, H A of ABq, ArCH 2 Cθ2) 7.10 (2H,d, J 8.5Hz, H B of ABq, ArCH 2 C0 ) 7.18 (2H,d, J 9.3Hz, H A of ABq, ArNθ2) 8.08 (2H,d, J 9.3Hz, H B of ABq, ArNθ2) (c) Preparation of Immunogen (III) Ethyl dimethylaminopropylcarbodiimide (EDC) (100 mg, 0.52 mmol) was added portionwise over 5 hours to a stirred solution of 4-nitrophenyl 4'-carboxymethylphenyl phosphate (II) (45 mg, 0.13 mmol), N-hydroxyεuccinimide (100 mg, 0.87 mmol) and keyhole limpe haemocyanin (KLH) (100 mg) in water (4 ml) and pyridine (100 ml). The reaction was stirred at room temperature for a further 16 hours, dialyzed against running tap water for 3 days, and the retenate lyophylized to give the immunogen (III) as an off-white powder (50 mg ) . Scheme 3 (Λ - Ϊαtf dm Example 2 Production of Antibodies Three mature ewes (numbered 270, 271 and 272) were each immunized subcutaneously and intramuscularly with 4 mg of the above immunogen (5) in a water (1 ml) in oil emulsion of Freunds complete adjuvent (3ml). This was repeated at four weekly intervals except that 2 mg of immunogen and Freunds incomplete adjuvant were then used. The animals were bled from the external jugular vein 2 weeks after each reimmunization and the blood allowed to clot overnight, centrifuged and the serum decanted and stored at -20°C. Example 3 Synthesis of Fluorescent Label (IX) (Scheme 4) (a) Preparation of 4-aminophenyl butanol (V) Tin (II) chloride dihydrate (6.0g, 26.6 mmol, 5.2 eq) was dissolved in concentrated hydrochloric acid (10 ml) and the solution cooled to 0°C. To the cold, stirred solution was added the 4-nitrophenyl butanol (0.866 ml, l.Og, 5.13 mmol) and the mixture stirred at 0°C for 90 minutes. At the end of this time ethyl acetate (50 ml) was added to the mixture, and the aqueous phase of the resultant bisphasic mixture basified by cautious addition of solid sodium bicarbonate (to pH 8-9). The layers wer then separated, the organic phase being retained and the aqueous phase extracted twice more with ethyl acetate (2 x 50 ml aliquots). The organic layers were combined, dried over magnesiu sulphate, and the solvent removed in vacuo; the yellow-brown soli residues were crystallized from ethylacetate/petrol to afford the product 550 mg (65%) as yellow crystals. (b) Preparation of 4-(4'-Choloracetomidophenyl) butanol (VI) 4-aminophenyl butanol (V) (10 g, 6.06 mmol) was dissolved in a mixture of methylene chloride (4 ml) and pyridine (2 ml) and th solution cooled to 0°C. To the cold, stirred solution was added dropwise a solution of chloroacetyl chloride (1.5 ml, 2m73 g, 78. mmol, 3.1 eq) in methylene chloride (2 ml) which had been cooled to 0°C. The solution became orange in color and quantities of pyridinium hydrochloride precipitated. The reaction was complete (TLC EtOAc/MeOH 10:1) in 15 minutes. To the resulting cold, stirred solution from above was added a 1:1 mixture of triethylamine/water (10 ml), followed by sufficient methanol (8 ml) to keep the solution homogenous. The solution was allowed to warm to room temperature and stirred at ambient temperature overnight. All the solvents were then remove in vacuo and the residues azeotroped repeatedly with dichloro- methane. The solid residues were then partitioned between water (30 ml) and ethylacetate (30 ml). The aqueous layer was extracte twice more with ethylacetate (2 x 30 ml). The organic phases wer combined, concentrated in vacuo and azeotroped with methanol. Th residues were then crystallized from ethyl acetate to yield pale yellow crystals (770 mg, 53%) of product (VI). (c) Preparation of Protected Derivative (VII) The mono-protected material (VI) (200 mg, 0.83 mmol) was dissolved in a 2:1 mixture of pyridine and acetonitrile (2.4 ml), and the solution cooled to 0°C. To the cold, stirred, solution was added p-nitrophenyl phosphorodichloridate (600 mg, 2.34 mmol, 2.8 eq) and stirring continued at 0°C for one hour. To the cold stirred solution above was added water (2 ml) . An immediate precipitation of a yellow white solid began to occur The mixture was allowed to warm to room temperature and stirring continued at ambient temperture for 3 hours. At the end of this time all solvent was removed in vacuo and the residues partitione between aqueous sodium bicarbonate solution (25 ml) and ether (25 ml); the aqueous layer was then washed again with ether (25 ml) and the organic layers discarded. The aqueous layer was acidifie by cautious dropwise addition of concentrated HC1 (to pH 1-2) and extracted five times with ethyl acetate (5 x 30 ml aliquots). Th organic extracts were combined, concentrated in vacuo and subjected to flash column chromatography on silica (gradient elution, start EtOAc/MeOH 10:1 increasing methanol to EtOAc/MeOH 3:1) to afford the product 785 mg (50%) as an off-white solid. (d) Deprotection to give (VIII) The protected phosphate (VII) (140 mg, 0.32 mmol) and thiourea were dissolved in methanol (3 ml) and the solution refluxed for three hours. At the end of this time, TLC (CH 2 C1 2 / MeOH 3:1) showed the reaction was complete. The solvent was the removed in vacuo and the residues subjected to flash column chromatography on silica with gradient elution (starting CH 2 Cl 2 MeOH 5:1 finishing CH 2 Cl 2 /MeOH 3:1) to afford the product (VIII mg (70%) as yellow oil. (e) Preparation of Fluorescent Label (IX) To a solution of the above phosphate VIII (3.6 mg, 0.01 mmol) in methanol (2 ml) and triethylamine (200 microlitre) was added flurosceinisothiocyanate isomer 1 (3.9 mg,, 0.01 mmol) and the reaction was stirred at room temperature for 1 hour. Volatiles were removed in vacuo and the residue azetroped with methanol (5 x 5 ml). The product was isolated by tic (developing solvent dichloromethane: methanol: acetic acid 75: 25:1) to give the purified label (IX) as a yellow solid (Rf 0.75 in the above solvent system) . Scheme 4 -21- _*) He Zf ? ) \k M- (33.) Example 4 Fluorescence Polarization Antibody Dilution Curves The fluorescent label was dissolved in methanol and the concentration of the resulting solution determined spectropho- tometrically, assuming a molar extinction coefficient of 8.78 x 10 4 1 mol-1 cm-1 at 492 run in sodium bicarbonate buffer (pH 9.0, 50 mmolar) . A portion of this solution was diluted in phosphate buffer (pH 7.5, k50 mmolar) containing sodium azide (0.1% w/v) and bovine gamma globulin (0.1% w/v) to give a stock solution of label concentration 30 nmol 1~ . Antiserum (500 micro litre) was dissolved in phosphate buffer (described above, 4.5 ml) to give an initial dilution of 1:10. Doubling dilutions were then prepared from this, giving antibody dilutions ranging from 1:10 to 1:7680. Each antibody dilution (1 ml), was incubated with the above solution of label (500 micro litre) for 1 hour at room temperature and the fluorescence polarization of each tube measured. Background values were obtained for each dilution by incubating the antibody dilution (1 ml) with phosphate buffer (500 micro litre), and these readings were subtracted from the values obtained with the label. Antibody dilution curves were constructed by plotting the resulting fluorescence polarization against antibody dilution. Antibody dilution curves were constructed for antisera obtained from each sheep (270, 271, and 272) 28 weeks into the immunization schedule. The titres of these antisera were 1:4000, 1:2000 and 1:2000 respectively. Example 5 Purification of IσG from Sheep 271 Anhydrous sodium sulphate (180 mg) was added, with vortex mixing, to serum (1 ml) from sheep 271. The resulting suspension was gently mixed for 30 min., centrifuged (10 min., 3000 rpm, 25°C), and the supernatant aspirated to waste. The precipated IgG was then washed (2 x 2 ml 18% Na^SO. w/v) and redissolved in phosphate buffer (2 ml, pH 7.5, 50 mmolar). The resulting solution (2 ml) was chromatographed on a "Protein G SEPHAROSE 4 Fast Flow" column (5 ml, Pharmacia) with spectrophotometric (280nm) monitoring of the eluate. The adsorbed IgG was eluted with glycine hydrochloride buffer (pH 2.7, 0.1M), and the eluate collected in fractions (2 ml) into tubes containing aqueous sodium bicarbonate (1M, 0.1 ml). The fractions containing IgG were pooled and the buffer exchanged by chromatography on a SEPHADEX G25 column (323 x 3.2 cm) with eluting buffer 50 mM phosphate pH 8.0. Example 6 Purification of IσG from Control Sheep IgG from a sheep which had not been immunized was purified as described above and used as a control. Example 7 Synthesis of Substrate To a solution of p-hydroxphenylacetic acid (152 mg, 1 mmol) and N-hydroxysuccinimide (130 mg, 2 mmol) in acetonitrile (2 ml) was added ethyl dimethylaminopropyl carbodiimide (190 mg, 1 mmol). The reaction was stirred at room temperature for 1 r», after which time, tic monitoring (developing solvent chloroform: methanol 6:1) showed that the reaction was incomplete. Another equivalent of carbodiimide was added and stirring was continued. After 15 min., tic monitoring showed complete formation of the activated ester and butylamine (150 micro litre, excess) was then added. The reaction was stirred at room temperature for 1 hour, diluted with ethyl acetate (50 ml), washed with aqueous sodium bicarbonate (0.5M, 20 ml), and dilute hydrochloric acid (1M, 20 ml), dried, and concentrated in vacuo. The residue was purified by flash chromatography (eluting solvent ethyl acetate: cyclohexane 9:1) to give the pure p-hydroxy-phenylacetamido butane (110 mg, 53% Rf 0.75 in ethyl acetate). A portion of this p- hydroxyphenylacetamide butane (92 mg, 0.44 mmol) was dissolved in acetonitrile (2 ml). To the resulting solution was added triethylamine (92 micro litres, 0.66 mmol) and p-nitrophenyl chloroformate (98.5 mg. 0.49 mmol), and the reaction mixture was stirred for 1 hour at room temperature. Volatiles were removed i vacuo and the residue was dissolved in ethyl acetate (5 ml), filtered, and the filtrate evaporated under reduced pressure to give the crude product as a white solid. Purification by the preparative tic (developing solvent ethyl acetate: cyclohex and 2:1, Rf 0.42) gave the pure substrate which was then recrystallized from ethyl acetate: cyclohexane 1:1 to give the substrate as white crystals (30 mg., 18%) MP 117°-119°C. Example 8 Hydrolysis Experiments - Sheep 271 IσG A stock solution of substrate, approximately 0.3 mM, in acetonitrile was prepared and its concentration determined spectrophotometrically (400 n ) by hydrolysis in bicarbonate buffer (pH 8.0, 50 nM) assuming a molar extinction coefficient of 18182 1 mol- cm " for nitrophenolate. A stock solution of purified IgG from sheep 271 in phosphate buffer (pH 8.0, 50 mM) was prepared and its concentration determined spectrophotometrically (280 nm) assuming a molar extinction coefficient of 2 x 10 5 1 mol-1 cm-1 for IgG. The concentration of this solution was then adjusted to 0.2 micro M b dilution with phosphate buffer (pH 8,- 50mM) . To the IgG solution (3 ml) at 25°C was added sufficient substrate solution to give a substrate concentration of 0.5 micro mol ±~ . The resulting solution was stirred and the initial reaction rate determined spectrophotometrically (400 nm), by monitoring the liberation of nitrophenolate. This was repeated, in duplicate, for substrate concentrations ranging from 0.5 to 20 mM. The results are shown in Table 1. Table 1. -26- Sheep 271 IgG catalyzed hydrolysis of substrate. Substrate Initial Rate nmol 1~ S~ Concentration micro M (JJ (2) 0.5 1.11 1.18 0.75 1.45 1.37 1.0 1.58 1.52 1.5 1.86 1.83 1.75 1.86 1.80 2.0 2.13 2.12 2.5 2.22 2.24 3.0 2.24 2.30 3.5 2.58 2.42 4.0 2.57 2.68 5 2.78 2.92 6 3.12 2.95 8 3.42 3.22 10 3.51 3.55 12 3.40 4.04 14 3.48 3.51 16 4.12 3.94 20 4.83 4.17 These results are described by an equation of the form: Vmaχ[S] Initial Rate = + kχ[S] K m + [S] in which a saturable catalyzed hydrolysis reaction is superimposed upon a background hydrolysis of the substrate. Weighted non-linear least squares regression analysis, assuming a constant relative error structure, for the above data gives the following values: v max = 2.64 nmol 1~ s~ K m = 0.75 micro mol I -1 Ki = 9.3 x IO- 5 s- 1 Example 9 Hydrolysis Experiments - Control Sheep IσG To solutions of purified IgG from a non-immunized sheep (0.2 micro M IgG in 50 mM phosphate a buffer pH 8.0) was added substrate solution to give a range of substrate concentrations from 2 to 20 micro M. The initial rates of hydrolysis of substrate were determined as described above, and the results are shown in Table 2. Table 2 Substrate Concentration Initial Rate micro M nmol 1-i S--3-. 2.0 0.28 4.0 0.62 6.0 0.90 8.0 1.32 10.0 1.48 12.0 2.05 14.0 2.05 16.0 2.20 20.0 2.47 These results were described by an equation of the form: Initial Rate = K. [S] Linear least squares regression analysis for this data gives a value: K χ = 12.7 x 10 "1 S "1 in good agreement with the value of the rate constant of the background hydrolysis for the IgG from sheep 271 determined above. Other embodiments of the invention will be apparent to those skilled in the art from a consideration of the specification and practice of the invention disclosed herein. It is intended that the specification and the examples be considered as exemplary only, with a true scope of the spirit of the invention being indicated by the following claims and their equivalents.

Claims: What is claimed is: —29— 1. Polyclonal catalytic antibodies. 2. Polyclonal antibodies that are capable of catalyzing th hydrolysis of a carbonate. 3. Polyclonal antibodies raised against a phosphate of general Formula I: R 0 P—0 R 1 OH wherein each of R and R' represents a radical which together confer immunogenicity on the phosphate of general Formula I. 4. Phosphates of general Formula I: R 0— P —0 R' I OH wherein each of R and R' represents a radical which together confer immunogenicity on the phosphates of general Formula I, against which antiphosphate antibodies capable of catalyzing the hydrolysis of a carbonate can be raised. 5. Precursors to phosphates of general Formula I of claim 4, which precursors are represented by general Formula II: R 2 0 — P 0 R" OH wherein R-2 and R- each represent a hydrocarbyl radical, which is optionally substituted such that, when the precursor is linked to an immunogenic moiety, a phosphate of Formula I may be formed. 6. Phosphates of general Formula III: wherein R 4 and R-5 each comprises a hydrocarbyl radical and either or both of R 4 and R5 comprises a label to enable detection of antibody binding. 7. A phosphate as claimed in claim 6 in which R 4 and R5 ar 2 hydrocarbyl radicals corresponding to those as defined for R and R 3 . 8 . Carbonates of general Formula IV: wherein R° and R 7 represent a hydrocarbyl radical, which is optionally substituted, and in which either or both R 6 and R7 comprise one or more marker groups which marker group is liberate in the hydrolysis of the carbonate, enabling the progress of hydrolysis to be followed.

Inventors: GALLACHER GERARD, GALLACHER, GERARD
Applicants: THERAPEUTIC ANTIBODIES INC, THERAPEUTIC ANTIBODIES, INC.
Application year: 1990
Main classification: C07K15
IPC codes: B01J31, C07F9, C07K14, C07K16, C12N9, C12P9, G01N33