WO1990008185A1 - Molecules with antibody combining sites that exhibit stereospecific catalysis - Google Patents

Molecules with antibody combining sites that exhibit stereospecific catalysis Download PDF

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Publication number
WO1990008185A1
WO1990008185A1 PCT/US1990/000269 US9000269W WO9008185A1 WO 1990008185 A1 WO1990008185 A1 WO 1990008185A1 US 9000269 W US9000269 W US 9000269W WO 9008185 A1 WO9008185 A1 WO 9008185A1
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Prior art keywords
ligand
reactant ligand
atom
ester
reactant
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PCT/US1990/000269
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English (en)
French (fr)
Inventor
Richard Lerner
Kim Janda
Stephen Benkovic
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Scripps Clinic And Research Foundation
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Application filed by Scripps Clinic And Research Foundation filed Critical Scripps Clinic And Research Foundation
Priority to AU50382/90A priority Critical patent/AU650846B2/en
Priority to KR1019900702060A priority patent/KR910700334A/ko
Publication of WO1990008185A1 publication Critical patent/WO1990008185A1/en
Priority to NO91912786A priority patent/NO912786L/no
Priority to FI913427A priority patent/FI95928C/fi
Priority to DK911364A priority patent/DK136491D0/da

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/10—Cells modified by introduction of foreign genetic material
    • C12N5/12—Fused cells, e.g. hybridomas
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/0002—Antibodies with enzymatic activity, e.g. abzymes
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/40—Esters thereof
    • C07F9/4071—Esters thereof the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/4087—Esters with arylalkanols
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/28—Phosphorus compounds with one or more P—C bonds
    • C07F9/38—Phosphonic acids [RP(=O)(OH)2]; Thiophosphonic acids ; [RP(=X1)(X2H)2(X1, X2 are each independently O, S or Se)]
    • C07F9/42—Halides thereof
    • C07F9/425—Acid or estermonohalides thereof, e.g. RP(=X)(YR)(Hal) (X, Y = O, S; R = H, or hydrocarbon group)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies

Definitions

  • the present invention relates to antibodies, antigens and immunogens, and more particularly to molecules that contain an 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.
  • oxyhemoglobin and the binding of a substrate to an enzyme that acts upon it such as between a protein and a protease like trypsin.
  • biological binding phenomena include the binding of an antigen to an antibody, and the binding of complement component C3 to the so-called CR1 receptor.
  • 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
  • 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.
  • 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.
  • anti-steroid antibodies were used to hydrolyze 7-umbelliferone (7-hydroxycoumerin) esters of a carboxyethyl thioether of a steroid.
  • rate was observed as compared to background or to a rate obtained with normal IgG.
  • turn over numbers were low (about one mole of substrates per mole of antibody per minute, or less), and 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.
  • immunologieal binding may be used to experimentally divert binding interactions to
  • 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.
  • binding site topology stems in part from a lack of knowledge of even the location in cells of many binding sites of receptors.
  • chemical identity i.e., protein and carbohydrate composition
  • hydrolytic receptor such as an enzyme
  • hydrolytic proteases typically cleave their substrates, polypeptide chains, adjacent to a particular amino acid residue that may 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.
  • polypeptide fused to the transcription product of a vector gene such as the lac z gene.
  • 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.
  • MOPC167 Leon et al., Blochem., 10, 1424 (1971)] that catalyzes the hydrolysis of a
  • the antibodies were raised to a
  • 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) .
  • 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 present invention contemplates a receptor molecule that contains an antibody combining site or idiotype-containing polyamide that is capable of catalytically hydrolyzing a preselected, scissile carboxylic acid amide or ester bond of one
  • 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
  • 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 molecule (preferably bound to a protein carrier to form a conjugate) containing an analog of a hydrolytic transition state of the ligand.
  • an analog-ligand molecule preferably bound to a protein carrier to form a conjugate
  • transition state molecule 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.
  • oxygen atoms bonded to the central atom is singly bonded to the central atom and is an -OR 2 group, wherein R 2 is selected from the group consisting of hydrogen (H), and C 1 -C 4 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
  • both the reactant ligand and analog-ligand contain at least one carbon atom that can exist in two stereoiomeric foras, "and thereby provides a stereoisomeric center. That stereoisomeric center is located in each of the ligand and analog-ligand molecules at the same relative position in each molecule.
  • stereoisomeric 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 phosphonamidate monoacid in its ionized form also simulates the developing charge in nucleophilic attack at a carbonyl center.
  • R 2 H or C 1 -C 4 lower alkyl
  • n is an integer from 1 to 8, inclusive.
  • the analog-ligand hydrolytic transition state molecules are themselves ligands, albeit not reactive ligands, and are also contemplated in this invention. These ligand molecules are of relatively small molecular size and are therefore typically linked to a larger, carrier molecule 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 mercaptan, a succinimide or other group that provides a means to attach the haptenic analog-ligand molecules to carriers for use as immunogens.
  • the antibody combining site-containing molecules of the present invention are themselves receptors and provide information on the
  • a method of preparing monoclonal receptor molecules that bind to the hydrolytic transition state of a particular amide or ester is also provided.
  • transition state analog is provided linked to a carrier as an immunogenic conjugate.
  • the conjugate thus provided is dissolved or dispersed in a
  • 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
  • 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
  • hybridoma 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
  • the so-called Fc or Fc' portions of the antibodies can be removed as by enzymic cleavage to provide an antibody combining site
  • 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
  • topological requirements are tailored to a particular reactant ligand to be studied and hydrolyze a
  • 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
  • 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
  • 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.
  • the present invention relates to molecules collectively referred to as receptors that are antibodies and idiotype-containing polyamide
  • 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.,
  • the binding energy of the enzyme is utilized to perform the chemical reaction [Jencks, W.P., XVII International Solvay Conference (November 1983)].
  • immunological hydrolysis 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
  • 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.
  • a receptor molecule of the present invention also binds to and hydrolyzes only one of a stereoisomeric pair of otherwise identical reactant ligand molecules.
  • the reactant ligand is enantiomeric, only one of the enantiomers is hydrolyzed.
  • the reactant ligand exists in both cis and trans forms, only one of those isomers is hydrolyzed.
  • 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.
  • 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.
  • the stereoisometric center is located in a chain four atoms away from the phosphorus atom in the acid portion of the
  • 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 molecule and/or a reactant ligand molecule can contain more 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 molecule, it is of no matter herein. However, when near enough to be bound by the receptor molecule, any other
  • 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
  • stereoisomerism the stereoisomeric center
  • 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
  • the receptor molecule 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
  • 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).
  • 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.
  • stereoisomeric ester used herein, the stereoisomeric center is located in the alcohol portion of the molecule.
  • analog-ligand and reactant ligand are one of a pair of stereoisomers.
  • 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,1 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.
  • stereoisomers are an enantiomeric, R, S, pair.
  • receptor is used herein to mean a biologically active molecule that binds to a reactant ligand, inhibitor ligand, or analog-ligand.
  • the receptor molecules of the present invention are antibodies, substantially intact antibodies or idiotype-containing polyamide portions of an
  • 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.
  • 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.
  • 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.
  • idiotype-containing polyamide antibody combining site-containing receptors
  • idiotype-containing polyamide antibody combining site-containing receptors
  • 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.
  • antibodies are typically obtained from hybridoma tissue cultures or from ascites fluid obtained from mammals into which the hybridoma tissue was
  • a "ligand” is defined herein as a molecule that immunoreacts with or binds to a receptor
  • 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
  • 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.
  • antibodies are induced by one
  • the receptor causes hydrolysis (which as demonstrated herein is
  • topology i.e., size, shape, stereochemistry and charge
  • inhibitor ligands that resemble the structure of an analog-ligand or a reactant ligand are also bound by receptor molecules.
  • a receptor can be prepared that stereoselectively causes
  • a receptor can be prepared that stereospecifically and catalytically hydrolyzes a selected, predetermined ester bond of a model compound or fat molecule.
  • the implication of this result is that one can confer the activity of hitherto unknown proteases and Upases to immunoglobulins.
  • 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
  • haptenic ester or amide analog-ligand hydrolytic transition state 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.
  • 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.
  • transition state would appear to contain the hydrated amide in the coordination sphere of the metal 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 metal ion or some other polarizing site [Weaver et al., J. Mol. Biol., 114, 119 (1977) and Christiansen et al., J. Am. Chem. Soc, 108, 545 (1986)].
  • ⁇ t may have a multiple function in amide hydrolysis where proton transfer steps among the tetrahedral intermediates may be rate-limiting [L. M. Sayre, J. Am. Chem. Soc, 108, 1632 (1986)].
  • 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
  • Ester hydrolysis reactions generally proceed at convenient spontaneous rates under ambient conditions that are suitable for antibodies.
  • 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.
  • the compound of Formula I 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
  • 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.
  • a functional appendage for coupling to an antigenic (immunogenic) carrier protein.
  • 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.
  • 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
  • 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:
  • imaginary line of demarcation can be drawn for such molecules 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
  • 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
  • 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 medium as a portion of the reaction admixture.
  • That medium typically contains water and buffer salts.
  • 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 methanol, ethanol,
  • hexamethylphosphoramide and N,N-dimethylforamide 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
  • 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
  • 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 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
  • 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.
  • 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
  • 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
  • concentration and ratio are typically used.
  • a stoichiometric amount of receptor or less can also be used, but since the receptor is a catalytic molecule, 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 molecues 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 maintenance 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.
  • 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.
  • 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 (-)], whereas the other six catalyzed the
  • 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 HCl and was then dried over anhydrous magnesium sulfate.
  • 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, CH 2 Cl 2 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).
  • 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.
  • Trifluoracetic anhydride (2.8 ml) was added to a solution of 4-amino ⁇ henyl 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
  • 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
  • the R(+) isomer was prepared in a generally similar manner in a yield of 48%.
  • the analog-ligand (Compound F) possessed a glutaryl half amide group that was utilized to link the haptenic analog-ligand to an antigenic
  • Additional linking groups that contain a total of 1 to 8 methylene (CH 2 ) groups between carboxyl groups are also useful.
  • diacids such as malonic acid, glutaric acid, adipic acid, through decanedioic acid are useful.
  • Those materials can be linked to the
  • 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.
  • 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.
  • Conjugates of haptenic analog-ligand molecules with antigenic (immunogenic) protein carriers such as keyhole limpet hemocyanin (KLH) can be prepared, for example, by activatio- of the carrier with a coupling agent such as MBS
  • 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,
  • red blood cells such as sheep
  • erythrocytes SRBC
  • tetanus toxoid tetanus toxoid
  • cholera toxoid as well as polyamino acids
  • 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
  • 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.
  • 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.
  • 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.
  • fused hybrids such as mouse-mouse hybrids [Shulman et al.. Nature, 276, 269 (1978)3 or rat-rat hybrids [Galfre et al.. Nature, 277, 131 (1979)] are typically utilized.
  • some rat-mouse hybrids have also been successfully used in forming
  • Suitable myeloma lines for use in the present invention include MPC-11 (ATCC CRL 167), P3X63-Ag8.653 (ATCC CRL 1580), Sp2/0-Ag14 (ATCC CRL 1581), P3X63Ag8U.1 (ATCC CRL 1597), Y3-Ag1.2.3.
  • the non-secreting murine myeloma line Sp2/0 or Sp2/0-Ag14 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.
  • an exemplary monoclonal receptor was produced by the standard hybridoma technology of Kohler et al., Nature, 256, 495
  • mice were iamunized by intraperitoneal injection with an inoculum of 100 micrograms of conjugate (e.g.,
  • 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 (1.4 ⁇ 10 8 ) were then fused with 3 ⁇ 10 7 Sp2/0-Ag14 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.
  • DMEM Dulbecco's modified Eagle medium
  • a monoclonal 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.
  • a mammal such as a mouse.
  • 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.
  • 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.
  • IgG fractions were typically obtained from mouse ascites by
  • Tris-HCl or sodium phosphate containing 0.01 M sodium azide Tris-HCl or sodium phosphate containing 0.01 M sodium azide.
  • hybridomas one that catalyzed a reaction of each
  • the hybridomas were deposited at 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 iodoacetamide.
  • the Fab fragment is typically further purified by anion exchange chromatography, dialysis, and DEAE anion exchange chromatography, and its homogeneity is judged by gel electrophoresis.
  • ELISA Enzyme-linked Immunosorbent Assay
  • 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) .
  • 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.
  • PBS phosphate buffered saline
  • 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 microgram 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 (mM) PBS, pH 7.4, containing 0.1 percent polyoxalkylene (20) sorbitan monolaurate (Tween 20) and 0.02 percent Thimerosal (sodium ethylmercurithiosalicylate),
  • hybridoma supernatant ⁇ 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
  • 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 H 2 O 2 . 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 H 2 SO 4 to each well and the optical density at 492
  • nanometers (nm) was measured with a Multiskan ELISA plate reader.
  • the gene that encodes an antibody For another preparation of the receptor molecules, 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.
  • 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).

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PCT/US1990/000269 1989-01-17 1990-01-12 Molecules with antibody combining sites that exhibit stereospecific catalysis WO1990008185A1 (en)

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AU50382/90A AU650846B2 (en) 1989-01-17 1990-01-12 Molecules with antibody combining sites that exhibit stereospecific catalysis
KR1019900702060A KR910700334A (ko) 1989-01-17 1990-01-12 입체특이적 촉매반응을 나타내는 항체 결합 부위를 지니는 분자
NO91912786A NO912786L (no) 1989-01-17 1991-07-16 Molekyler med antistoffkombinerende seter som utviser stereospesifikk katalyse.
FI913427A FI95928C (fi) 1989-01-17 1991-07-16 Molekyylit, joissa on vasta-ainetta liittävät kohdat ja joilla on stereospesifinen katalyysi
DK911364A DK136491D0 (da) 1989-01-17 1991-07-16 Molekyler med antistof-kombinerende steder, der udviser stereospecifik katalyse

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EP0521996A1 (en) * 1990-03-23 1993-01-13 Igen, Inc. Catalytic antibody components
EP1443963A2 (en) * 2001-10-22 2004-08-11 The Scripps Research Institute Antibody targeting compounds
CN110950960A (zh) * 2019-11-26 2020-04-03 中国农业大学 基于高通量测序和杂合杂交瘤技术的小分子化合物抗体的制备方法

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US5236825A (en) * 1989-01-17 1993-08-17 Scripps Clinic And Research Foundation Polyvalent metal ion-containing antibody combining site catalysts

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US4659567A (en) * 1984-09-07 1987-04-21 Scripps Clinic & Research Foundation Molecules with antibody combining sites that bind to hydrolytic transition states
US4792446A (en) * 1986-06-23 1988-12-20 Igen, Inc. Production of antibody catalysts

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US4963355A (en) * 1986-06-23 1990-10-16 Igen, Inc. Production of antibody catalysts
US5079152A (en) * 1987-05-28 1992-01-07 Scripps Clinic And Research Foundation Antibody combining sites that exhibit stereoselective synthase activity, and methods using the same

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US4659567A (en) * 1984-09-07 1987-04-21 Scripps Clinic & Research Foundation Molecules with antibody combining sites that bind to hydrolytic transition states
US4792446A (en) * 1986-06-23 1988-12-20 Igen, Inc. Production of antibody catalysts

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Title
Science, Volume 234, issued 19 December 1986, A. TRAMONTANO, "Catalytic Antibodies", see pages 1566-1570. *
Science, Volume 234, issued 19 December 1986, S.J. POLLACK, "Selective Chemical Catalysis By An Antibody," see pages 1570-1573. *
Science, Volume 241, issued 02 September 1988, K.D. JANDA, "Induction of An Antibody That Catalyzes the Hydrolysis of An Amide Bond," see pages 1188-1191. *
See also references of EP0454778A4 *

Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0521996A1 (en) * 1990-03-23 1993-01-13 Igen, Inc. Catalytic antibody components
EP0521996A4 (en) * 1990-03-23 1993-06-30 Igen, Inc. Catalytic antibody components
EP1443963A2 (en) * 2001-10-22 2004-08-11 The Scripps Research Institute Antibody targeting compounds
EP1443963B1 (en) * 2001-10-22 2014-05-21 The Scripps Research Institute Antibody targeting compounds
CN110950960A (zh) * 2019-11-26 2020-04-03 中国农业大学 基于高通量测序和杂合杂交瘤技术的小分子化合物抗体的制备方法
CN110950960B (zh) * 2019-11-26 2021-05-14 中国农业大学 基于高通量测序和杂合杂交瘤技术的小分子化合物抗体的制备方法

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