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WO1996039443A1 - Analogues de peptides et leur utilisation en tant qu'haptenes pour l'induction d'anticorps catalytiques - Google Patents

Analogues de peptides et leur utilisation en tant qu'haptenes pour l'induction d'anticorps catalytiques Download PDF

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Publication number
WO1996039443A1
WO1996039443A1 PCT/US1996/009450 US9609450W WO9639443A1 WO 1996039443 A1 WO1996039443 A1 WO 1996039443A1 US 9609450 W US9609450 W US 9609450W WO 9639443 A1 WO9639443 A1 WO 9639443A1
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Prior art keywords
same
hapten
antibodies
bond
side chain
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David E. Hansen
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IGEN Inc
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IGEN Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/44—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material not provided for elsewhere, e.g. haptens, metals, DNA, RNA, amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C229/00—Compounds containing amino and carboxyl groups bound to the same carbon skeleton
    • C07C229/46—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino or carboxyl groups bound to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton
    • C07C229/50—Compounds containing amino and carboxyl groups bound to the same carbon skeleton having amino or carboxyl groups bound to carbon atoms of rings other than six-membered aromatic rings of the same carbon skeleton with amino groups and carboxyl groups bound to carbon atoms being part of the same condensed ring system
    • 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
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C2602/00—Systems containing two condensed rings
    • C07C2602/50—Spiro compounds

Definitions

  • the present invention relates generally to antibodies, and antigens, haptens and immunogens capable of eliciting such antibodies, which include a paratope that binds to and thereby stabilizes a transition state in the cleavage or formation of a peptide bond linkage so that the cleavage or formation is catalyzed by the antibodies. Said paratope also binds to and destabilizes the substrate ground state. BACKGROUND OF THE INVENTION
  • a tetrahedral carbon atom is bonded to: a carbon atom of the acid portion of the peptide linkage or ester bond; two oxygen atoms, one corresponding to the carbonyl group and the other corresponding to a hydroxyl ion or water molecule of the medium; and either the oxygen atom of the alcohol portion of an ester or the nitrogen atom of the amine portion of the peptide linkage.
  • the transition state can be neither isolated nor detected since it exists for only about 10" 13 sec.
  • transition states reflect changes in bond lengths and bond angles as well as the formation and breakage of bonds.
  • the energy required to achieve a transition state is denoted as the activation energy which may also be considered as the difference in energy between the energy of the transition state and the energy of the reactants.
  • an enzyme preferentially binds the transition state of a reaction, thereby stabilizing it relative to the substrate and products and reducing the activation energy of the reaction, thus increasing the reaction rate.
  • aspartic proteinases are enzymes which are known to catalyze the hydrolysis of peptide linkages within a protein molecule.
  • transition state analog might be used to describe an inhibitor of this kind (1) .
  • a transition-state analog mimicking an intramolecular 6-member ring cyclization transition state was used to elicit a monoclonal antibody which acted as a stereospecific, enzyme-like catalyst (2) .
  • the monoclonal antibody so elicited accelerated, by about a factor of 170, the formation of a single enantiomer of a ⁇ -lactone from the corresponding racemic ⁇ -hydroxyester.
  • monoaryl phosphonate esters designated as analogs of the transition state in the hydrolysis of carboxylic esters, were synthesized and used as haptens to elicit specific monoclonal antibodies capable of catalyzing the hydrolysis of carboxylic esters (3) . Certain of the antibodies elicited were reportedly found to be catalytic and selective for the hydrolysis of particular aryl esters.
  • Phosphonamidates or phosphonate analog-ligands having conformations that substantially correspond to the conformation of a hydrolytic transition state of an amide or ester ligand and which have been used to produce antibodies are described in U.S. Patent 4,659,567 to
  • Antibodies so produced purportedly include a paratope that binds to and stabilizes the tetrahedral carbon atom of the amide or ester hydrolysis transition state of the ligand to hydrolyze the ligand at a predetermined site.
  • Analog-ligands which can be used to produce antibody catalysts for the hydrolysis of esters and amides are also described in European Patent Application 0,251,093 of Kollmorgen Corp. (Kollmorgen) .
  • Kollmorgen Kollmorgen
  • groups on both the substrate and the enzyme which are not involved in the chemical mechanism of bond making and breaking make an important contribution to catalysis. This is illustrated by examining the action of the enzyme succinyl-CoA acetoacetate transferase, shown below, which involves nucleophilic attack of the enzyme's glutamate carboxyl on the thioester succinyl-CoA [l] to give an anhydride intermediate.
  • the enzyme forms anhydride intermediates from "non-specific" substrates [2], as well. [2]
  • the enzyme reaction proceeds up to 3 X 10 12 fold faster with the so called specific substrate [1] (4) .
  • the non-reacting part of the substrate i.e., the CoA residue, lowers the activation energy by 72 KJmol" 1 compared with [2] .
  • the haptens disclosed in Tramontano do not provide the correct architecture to elicit antibodies that are capable of catalyzing the cleavage of a predetermined peptide sequence in a native protein.
  • These haptens do not provide the correct side-chain groups for production of antibodies that can react with predetermined sites on a protein and cause selective proteolysis in a sequence specific manner.
  • these haptens do not incorporate amino acid side-chain sub-sites on either side of the transition state analog. Without these sub-sites, the haptens cannot provide for the elicitation of catalytic antibodies capable of recognizing a specific amino acid sequence and selectively proteolyzing a peptide linkage within that sequence.
  • no general method for eliciting antibody peptidases' has been developed. OBJECTS, FEATURES AND ADVANTAGES OF THE INVENTION
  • the invention is broadly directed to antigens capable of eliciting through immunogenic methods catalytic antibodies which can catalyze the cleavage or formation of an amide, peptide, ester or glycosidic bond in a molecule.
  • the invention is directed to antigens capable of eliciting through immunogenic methods catalytic antibodies which can catalyze the selective cleavage or formation of a predetermined amide bond in a native polypeptide sequence.
  • the antigens can be a hapten or an immunogen comprising a hapten coupled (linked, conjugated) to a carrier molecule via a suitable coupling moiety.
  • the haptens include structural elements which are designed to (i) mimic one or more high energy intermediates or transition states in the cleavage or formation of the amide, ester or glycosidic bond and/or (ii) mimic one or more high energy conformations of the amide, ester or glycosidic bond to be cleaved.
  • the haptens according to the invention provide the correct side chain groups for production of antibodies that can react with predetermined sites on a protein and can catalyze selective proteolysis in a sequence specific manner.
  • the haptens further incorporate amino acid side-chain sub-sites surrounding the amide bond analog. These sub-sites provide for the elicitation of catalytic antibodies capable of recognizing a specific amino acid sequence and selectively proteolyzing a peptide linkage within that sequence.
  • Such catalytic antibodies are elicited with the haptens of the present invention.
  • a hapten according to the invention shown below,
  • a aa -- B aa --[CD]-- E aa -- F aa dipeptide analog incorporates not only the dipeptide analog [CD] but also sub-site amino acid residues A, B, E, F. These subsite amino acid residues can be part of a cyclic structure as well as a linear structure. The optimum number of sub ⁇ site residues is determined by the size of the antibody combining site. It is likely that the only essential criterion for effective binding of antibody to a peptide is that complementarity between the antigen combining site of the antibody and the molecular surface of the binding peptide is maintained with regard to both shape and charge.
  • the haptens according to the invention are designed in such a way such that antibodies raised against these haptens can selectively stabilize one or any of the high energy intermediates or transition states in the cleavage or formation of an amide, peptide, ester or glycosidic bond and/or destabilize the bound ground state.
  • haptens fall into three general classes: one, those in which the hybridization of the atom corresponding to the carbonyl carbon of the scissile bond of the amide or ester bond is converted from sp 2 to sp 3 hybridization; two, those in which any of the atoms corresponding to the amide, ester or glycosidic bond is replaced by a different atom; and three, those in which the atoms corresponding to the amide, ester or glycosidic bond are part of a monocyclic or bicyclic system.
  • Peptide sequences containing dipeptide analogs, according to the invention, at the bond that is required to be hydrolyzed by the catalytic antibodies of the present invention define a sequence that the catalytic antibody will hydrolyze in a native protein.
  • the binding energy of the antibody is distributed in such a way as to allow both sequence specific recognition and chemical reactivity with the native protein or peptide of interest.
  • the catalytic antibodies elicited with these haptens can then be utilized, for example, to digest epitopes on viral proteins or tumor derived growth factors or other peptides involved in life-threatening situations (e.g., tumor necrosis factor in bacterial sepsis, etc.).
  • the haptens of the invention are distinguished from prior analog-ligands in that they have been rationally designed from knowledge of mechanistic features of enzyme catalysis and provide suitable templates for generating antibody combining sites endowed with catalytic properties. Consequently, they incorporate all the necessary features to provide for antibodies capable of molecular recognition and catalytic function.
  • the invention is a method for catalyzing the cleavage or formation of a specific peptide bond within a molecule.
  • the method comprises contacting the molecule with an amount of a monoclonal antibody effective to catalyze the cleavage or formation of the peptide bond under conditions suitable for the cleavage or formation to take place; the monoclonal antibody having been prepared by a process comprising the steps of: selecting the specific peptide bond to be cleaved or formed; selecting an antigen comprising an analog of the peptide bond to be cleaved or formed, and also comprising moieties surrounding the analog of the peptide bond, which moieties substantially correspond to some or all of the moieties surrounding the peptide bond to be cleaved or formed; exposing cells capable of producing antibodies to the antigen and thereby generating antibody producing cells; hybridizing the antibody producing cells with myeloma cells and thereby generating a plurality of hybridoma cells each producing monoclonal antibodies; and screening
  • R 10 and R X1 may be the same or different and each is a side chain of a naturally occurring amino acid or an analog of said side chain;
  • R 12 is hydrogen or a second bond between T and the carbon to which T is attached provided that if R 12 is a second bond, then there is no substituent R 13 ;
  • R 13 is hydrogen;
  • L is a ligand;
  • M 3+ is Cr(III) or Co(III) ;
  • T is O or S;
  • V is N + with any negatively-charged counterion, or C;
  • X is OH, SH, NH 2 , NH 2 protected by a protecting group selected from the group consisting of terminal amino protecting groups, alkene, (C x -C 9 ) alkyl, ⁇ ⁇ C 9 )alkoxy, phenyl, phenoxy, cyclohexyl, phenylthio, phenylsulfinyl or phenylsulfonyl wherein the aforementioned phenyl groups are unsubstituted or mono-, di- or trisubstituted by halogen, (C 1 -C 4 ) alkyl, (C x - C 4 )alkoxy, ( C -C 4 ) alkoxycarbonyl, or
  • e is an integer from 1 to 10; f and g are 0 or 2 provided f and g are not both 2;
  • Y is hydrogen, COR 5 , carboxyl protected by a protecting group selected from the group consisting of terminal carboxyl protecting groups, ( ⁇ ,)alkyl, (C x - C 9 )alkoxy, phenyl, phenoxy, cyclohexyl, phenylthio, phenylsulfinyl or phenylsulfonyl wherein the aforementioned phenyl groups are unsubstituted or mono-, di- or trisubstituted by halogen, (C 1 -C 4 )alkyl, [C x - C 4 )alkoxy, or (C- L -C alkoxycarbonyl or
  • h is an integer from 1 to 10; i and j are 0 or 2 provided i and j are not both 2;
  • R 3 is hydrogen or a protecting group selected from the group consisting of amino-terminal and carboxyl- terminal protecting groups
  • R 4 being the same or not all the same when e > 1, is a side chain of a naturally occurring amino acid or an analog of said side chain provided that R 4 is CH 2 when f or g is 2; R 5 is OH, NH 2 or 0(C ⁇ C-.,,)alkyl;
  • R 6 being the same or not all the same when h > 1, is a side chain of a naturally occurring amino acid or an analog of said side chain provided that R 6 is CH 2 and when i or j is 2;
  • R 7 is OH, SH, NH 2 , OH protected by a protecting group selected from the group consisting of terminal carboxyl protecting groups, alkene, (C 1 -C 9 )alkyl, (C x - C 9 )alkoxy, phenyl, phenoxy, cyclohexyl, phenylthio, phenylsulfinyl or phenylsulfonyl wherein the aforementioned phenyl groups are unsubstituted or mono-, di- or trisubstituted by halogen, (C 1 -C 4 )alkyl, (C x - C 4 )alkoxy, ( C x -C 4 )alkoxycarbonyl; D and E are the same or different when
  • the haptens of formula XVIII may be incorporated into a peptide sequences at the bond that is required to be hydrolyzed by the catalytic antibodies of the present invention.
  • the subsite residues of the peptide sequence result in the elicitation of antibodies capable of recognizing a specific amino acid sequence and cleaving the peptide bond therein.
  • haptens may be used as antigens for in vitro elicitation of catalytic antibodies.
  • the haptens must be coupled to a suitable carrier molecule in order to obtain an immunogen suitable for immunization. Therefore, the invention is also directed to immunogens capable of eliciting catalytic antibodies.
  • immunogens comprise a hapten as hereinbeforedescribed coupled to a carrier molecule by a suitable coupling moiety.
  • the invention is directed to catalytic antibodies which are elicited by antigens comprising the haptens of the invention as described above.
  • the invention is also directed to catalytic antibodies which can catalyze a chemical reaction of interest and which are elicited through in vitro or in vivo techniques by antigens comprising haptens according to the invention as described above, wherein the antibodies have been prepared by exposing cells capable of producing antibodies to the antigens and thereby generating antibody producing cells; hybridizing the antibody producing cells with myeloma cells and thereby producing a plurality of hybridoma cells each producing monoclonal antibodies; and screening the plurality of monoclonal antibodies to identify a monoclonal antibody which catalyzes the chemical reaction of interest.
  • cells capable of producing catalytic antibodies can be stimulated to grow in culture and, therefore, can be immortalized using methodologies well known in the art.
  • lymphocytes can be so stimulated using a virus, a chemical agent or a nucleic acid (e.g., an oncogene) .
  • the invention is directed to a method for producing catalytic antibodies which can catalyze a chemical reaction of interest and which are elicited through in vitro or in vivo techniques by antigens comprising the haptens according to the invention as described above.
  • the method comprises exposing cells capable of producing antibodies to the antigens and thereby generating antibody producing cells; hybridizing the antibody producing cells with myeloma cells and thereby generating a plurality of hybridoma cells each producing monoclonal antibodies; and screening the plurality of monoclonal antibodies to identify a monoclonal antibody which catalyzes the chemical reaction of interest.
  • the invention is also directed to a method for catalyzing the cleavage or formation of a peptide bond in a molecule.
  • the method comprises contacting the molecule with an effective amount of a catalytic antibody which has been elicited by antigens comprising haptens according to the invention.
  • the invention is directed to a method for catalyzing the cleavage or formation of a specific amide bond within a specific amino acid sequence of a molecule containing numerous amino acids joined by amide bonds.
  • the method comprises contacting the molecule with an effective amount of a catalytic antibody which has been elicited by antigens comprising haptens according to the invention.
  • the haptens have complimentarity with the specific amino acid sequence.
  • the invention is directed to a spiro [4.4] nonane-containing dipeptide analog useful as a hapten.
  • the catalytic antibodies elicited by antigens comprising haptens according to the invention can be used, for example, to digest epitopes on viral proteins or tumor-derived growth factors on other peptides involved in health- or life- threatening situations.
  • Fig. 1A depicts torsionally-distorted glycyl- proline.
  • Fig. IB depicts a spiro analog of glycyl- glycine or glycyl-proline.
  • Fig. 1C depicts torsionally distorted glycyl- glycine.
  • Fig. 2 is the reaction sequence for the synthesis of trans- tert- butyldimethylsilyloxyspiro[4.4]nonane-6-ene and cis- tert- butyldimethylsilyloxyspiro[4.4]nonane-6-ene.
  • Fig. 3 is the reaction sequence for the synthesis of methyl-trans-6-hydroxyspiro[4,4]nonane- trans-1-carboxylate and methyl- rans-6- hydroxyspiro[4.4]nonane-cis-1-carboxylate.
  • Fig. 4 is the reaction sequence for the synthesis of 7-oxa-6-oxotricyclo[6.3.0.O 1,5 ] undecane.
  • Fig. 5 is the reaction sequence for N- (D- tyrosyl) -trans-7-amino-trans-6-hydroxyspiro[4.4]nonane-1- carboxyl-D-phenylalanine.
  • the invention relates to antigens which are capable of eliciting through immunogenic methods antibodies which can catalyze the cleavage or formation of a peptide linkage or the cleavage or formation pf an ester in a molecule.
  • These antigens comprise a hapten or a hapten and a suitable carrier molecule.
  • the antibodies so elicited can catalyze a chemical reaction, they are defined as "catalytic antibodies.”
  • Catalytic antibodies are identified and described in U.S. Patent No. 4,888,281, referred to above in the "Background of the Invention.”
  • the reactants undergo one or more transitions through structures which are energetically less favorable than either the reactant or product.
  • transition states reflect changes in bond lengths and bond angles as well as formation and breakage of bonds.
  • the energy required to achieve a transition state is denoted as the activation energy, which may also be considered as the difference in energy between the energy of the transition state and the energy of the reactants.
  • Catalysts increase chemical reaction rates by lowering the activation energy of a reaction.
  • Antibodies elicited to a hapten or immunogen of the invention which antigens are chosen because, inter alia, they resemble the presumed transition state structure, a strained ground state structure, or both, can catalyze reactions.
  • the antibody thus produced should stabilize the energy of the transition state relative to reactants and products and/or destabilize the energy of the bound substrate relative to unbound substrate. This approach has been successfully demonstrated in the generation of several catalytic monoclonal antibodies.
  • Catalytic antibodies elicited with haptens according to the invention are "site specific" in that they are deliberately designed only to catalyze cleavage of peptide bonds having certain structural conformations at specific sites in a protein molecule. Likewise, these catalytic antibodies are designed only to catalyze the formation of peptide linkages from the N- and C- termini of amino acids having certain structural conformations at those termini. Therefore, haptens according to the invention may be used to elicit a site specific catalytic antibody capable of cleaving peptide bonds at specific sites in a protein molecule to produce two or more cleaved protein strands. The same catalytic antibody can then catalyze the formation of peptide bonds wherein those cleaved strands having the right structural conformation are joined.
  • the haptens of the invention are designed to mimic the transition-states or strained ground states or both for a variety of chemical reactions.
  • the reactions are the cleavage or formation of a peptide bond.
  • Certain haptens of the invention may also be able to mimic the transition states of other non-peptide types of chemical reactions.
  • a method is provided for catalyzing the cleavage or formation of a specific peptide bond within a specific amino acid sequence contained in a molecule.
  • the molecule is contacted with an amount of a monoclonal antibody effective to catalyze the cleavage or formation of the peptide bond under conditions suitable for the cleavage or formation to take place.
  • the monoclonal antibody is elicited using an antigen comprising a hapten of the invention.
  • amide bond refers to a simple amide bond (e.g., an amide bond in a side chain of a naturally occurring amino acid) or an amide bond which joins two adjacent amino acid residues, i.e., a peptide bond.
  • peptide includes dipeptides and polypeptides.
  • analog of an amide bond as used herein is defined as a normal amide bond (-CO-NH-) in which one or more moieties in the normal amide bond are replaced by one or more different moieties similar in charge and/or size to the normal moieties replaced.
  • Moiety is defined as a radical (e.g., an atom, CH 3 , C 6 H 5 , OH, NH 2 , etc.).
  • an analog of an amide bond is -CO-CF 2 wherein the normal NH moiety is replaced by the CF 2 moiety.
  • the term "antigen” is defined as a molecule which induces the formation of an antibody.
  • the term "antigen” means a molecule which is inherently immunogenic, a hapten according to the invention or an immunogen which comprises a hapten according to the invention coupled to a carrier molecule by a suitable coupling moiety.
  • Carrier molecules include, for example, keyhole limpet hemocyanin (KLH) , thyroglobulin, chicken immunoglobulin, ovalbumin, bovine serum albumin (BSA) , T-helper peptides, etc.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • T-helper peptides etc.
  • Coupling moiety refers to biotechnological cross-linking reagents well known in the art (e.g., commercially available from Pierce, Rockford, Illinois) and include, for example, Trout's reagent, dissuccinyl suberate, etc.
  • transition state analog refers to an array of atoms which is designed to approximate or “mimic” the configuration of an amide bond or an ester bond as such bonds exist in a hydrolytic transition state.
  • strained ground state refers to an array of atoms that is designed to approximate or “mimic” one or more high energy conformations of an amide or ester bond.
  • dipeptide analog refers to a structure which comprises a transition state analog or strained ground state analog or elements of both having side chains of two amino acids which are in positions analogous to those of the dipeptide being mimicked. In other words, in a dipeptide analog, the normal amide bond (i.e., -CO-NH-) between the two amino acids has been replaced by an array of atoms as defined above. Additional amino acid residues may be incorporated to surround the dipeptide analog to form a polypeptide.
  • the dipeptide analog replaces the peptide bond "targeted" for cleavage in the substrate molecule.
  • the terms "some or all” refer to a portion of the target molecule including at least the bond to be cleaved or all of the target molecule.
  • haptens designed for the purpose of eliciting antibodies to catalyze the cleavage of a specific peptide bond in a protein molecule comprising a polypeptide of many amino acid residues need only be surrounded by not more than about eight amino acid residues.
  • the target molecule is a relatively short peptide, it is advantageous to surround the peptide bond analog with all the amino acid residues of the target molecule.
  • the term "substantially corresponds" refers to moieties which are similar in charge and/or size to moieties in the amide bond analog, dipeptide analog or naturally occurring amino acid side chain analog.
  • the moieties are identical in size and charge, although such identity is not necessary for the hapten of the invention.
  • hapten as used herein is defined as a molecule which can act as an epitope. Haptens according to the invention incorporate a dipeptide analog according to the invention.
  • Physiologically acceptable salts include salts of mineral acids, for example, hydrochloric acid, sulfuric acid, nitric acid and the like, salts of monobasic carboxylic acids such as, for example, acetic acid, propionic acid and the like, salts of dibasic carboxylic acids such as, for example, maleic acid, fumaric acid and the like, and salts of tribasic carboxylic acids such as, for example, citric acid and the like.
  • naturally occurring amino acid includes the twenty essential alpha-amino acids and other alpha-amino acids which may or may not be found in proteins. These amino acids include alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, valine, 4-hydroxyproline, 5-hydroxylysine, epsilon-N- methyllysine, 3-methylhistidine, beta-alanine, gamma- aminobutyric acid, homocysteine, homoserine, citrulline, ornithine, canavanine, djenkolic acid and beta- cyanoalanine.
  • An amino acid consists of a carbon atom to which is bonded an amino group, a carboxyl group, a hydrogen atom and a distinctive group referred to as a "side chain".
  • the side chain is also bonded to the nitrogen atom of the amino group, thus forming a cyclic structure.
  • haptens include a side chain of a naturally occurring amino acid as well as an "analog of said side chain.”
  • the term "analog of said side chain” as used herein is defined as a side chain of a naturally occurring amino acid in which one or more moieties of the naturally occurring side chain is replaced by one or more different moieties which substantially corresponds to the naturally occurring moiety.
  • Those side chains containing a hydroxy group can be glycosylated, phosphorylated, sulphonylated or protected by a hydroxy protecting group.
  • the hydroxy group of any of the side chains may be protected by any number of suitable hydroxy protecting groups well known in the art. These include, for example, a tertiary butyl group.
  • terminal amino protecting group means any group capable of protecting the terminal amino moiety of a peptide or amino acid. Therefore, terminal amino protecting groups include acetyl, succinyl, biphenylcarbonyl, benzoyl, t-butyloxycarbonyl, carbobenzyloxy, tosyl, dansyl, isovaleryl, phthalyl, 1- adamantanesulphonyl, acetimido, benzimido, amidino, carbamyl and the functional equivalents thereof.
  • terminal carboxyl protecting group means any group capable of protecting the terminal carboxyl moiety of a peptide or amino acid.
  • Terminal carboxyl protecting groups include (Cx-C,,)alkyl, phenyl, substituted methyl esters such as methoxymethyl and phenacyl esters, 2-substituted ethyl esters such as cyclohexyl and allyl, substituted benzyl esters such as para-methoxybenzyl and para-bromobenzyl, amides such as piperidinyl and hydrazide and functional equivalents thereof.
  • L is a ligand in formula XVIII, and preferably L 4 is 4H 2 0, 4NH 3 , 2 ethylene-diamine or triethylenetetramine.
  • Haptens according to the invention contain one or more asymmetric centers and therefore exist in enantiomeric and/or diastereomeric forms. In general, the corresponding haptens according to the invention are obtained in the form of racemates or mixtures of diastereomers. If desired, techniques well known in the art for the separation of the mixtures into stereochemically homogeneous constituents may be used. Preparation of the optical isomers in a pure state is also possible by using stereochemically homogeneous starting materials.
  • Preferred haptens of formula XVIII include those haptens wherein V is carbon; X is NH 2 ; Y is COR 5 ; a and c are both 2; and R 10 , R 1]L , R 12 and R 13 are all hydrogen.
  • amino moiety of X is bound to the carboxyl terminus of an amino acid, e.g. tyrosine, and the carboxyl moiety of Y is bound to the amino terminus of an amino acid, e.g. phenylalanine, by covalent bonds or linker moieties as defined above.
  • An especially preferred hapten of the present invention has the formula:
  • HIV human immunodeficiency virus
  • gpl20 protein at its surface to attach to CD4 receptors on lymphocytes.
  • the sequence for this cell attachment has been mapped to a region on the viral protein.
  • antibodies can be generated by the methodology described in this invention to bind to this peptide sequence and cleave it in a site-specific manner.
  • such antibodies preferably bind to the "native" sequence in the protein as opposed to a linear sequence (which would occur in a denatured protein) .
  • the antigenic determinants or epitopes in the "native" protein are often conformational (i.e., three- dimensional) rather than random linear arrangements.
  • knowledge of epitopes on the protein is important in the design of antibodies having paratopes that can induce modifications of such epitopes. Therefore, haptens according to the invention are designed to have the same structural features of the epitopes, rather than random conformations. These structural features can be adopted by simple linear peptides, the lowest energy conformer being the preferred structure in solution. Secondary structural features may be introduced by cross-linking of amino-acid side-chains or the use of j ⁇ -turn mimetics.
  • Conformationally constrained haptens incorporated structures which are compatible with the epitope in the native protein may be essential for inducing the correct motif within the tertiary structure of the catalytic antibody hyper- variable binding region.
  • the advantages of conformationally constrained haptens are that they mimic the native structure in the protein and tend to mimic regions of the protein which are susceptible to cleavage. Accordingly, in the structural formula XVIII shown above for the haptens of the invention, the substituents R 10 , R 11# X and Y may be bound to one or more of the remaining substituents R 10 , R u , X and Y by a covalent bond or a linker moiety as defined above.
  • the haptens of the invention can take on a configuration mimicking that of the native ⁇ -turn or "hairpin” configuration of proteins by the formation of disulphide bridges between sulfur containing amino acid side chains which are incorporated into the hapten.
  • Disulphide bridge formation also promotes hydrogen bonding interactions. Disulphide bridge formation can be achieved by chemical methodology well known in the art.
  • the utility of the antigens of this invention coupled with appropriate screening procedures and reiterative thermodynamic perturbation studies of transition-state structures and free-energies of interaction with catalytic groups provide a methodology for production of catalytic antibodies by a rational design approach.
  • the invention also is directed to catalytic antibodies which are elicited by antigens comprising haptens according to the invention.
  • antibodies may be monoclonal or polyclonal but are preferably monoclonal and may be in the form of purified immunoglobulins (IgG, IgM, IgA, IgD or IgE) or antibody fragments, such as Fab, F(ab') 2 , F v , etc., of immunoglobulins.
  • immunoglobulins IgG, IgM, IgA, IgD or IgE
  • antibody fragments such as Fab, F(ab') 2 , F v , etc.
  • a catalytic antibody in accordance with the invention is a substance which is capable of changing the rate of a chemical reaction, all other conditions (e.g., temperature, reactant/substrate concentration, etc.) being the same and which does not enter into the chemical reaction and therefore is not consumed in the reaction. It is also a substance which exhibits the capability of converting multiple moles of reactant/substrate per mole of catalytic antibody; which, from a mechanistic viewpoint, binds the reactant/substrate, effects the accelerated conversion of the reactant/substrate to the product and then releases the product; and which changes the rate of the chemical reaction without shifting the position of the equilibrium.
  • the aforementioned definitions are characteristics of ideal catalysts.
  • the rate enhancement factor is a dimensionless number which expresses the rate of reaction in the presence of catalyst to the rate of reaction in the absence of catalyst, all other reaction conditions (e.g., reactant concentration, temperature, etc.) being equal.
  • Catalytic antibodies according to the invention may be elicited through both m vitro and in vivo techniques.
  • the term "elicited” as used herein means elicitation of catalytic antibodies by antigens according to the invention through both jLn vitro and in vivo techniques.
  • the skilled artisan will readily appreciate that when in vitro elicitation is involved, the haptens according to the invention, by themselves, may be used to elicit the catalytic antibodies.
  • immunogens comprising haptens complexed to a suitable carrier molecule are used to elicit the catalytic antibodies.
  • Another aspect of the invention is directed to a method for producing antibodies which can catalyze a chemical reaction of interest and which are elicited through in vitro or in vivo techniques by an antigen.
  • the antigen comprises a hapten according to the invention.
  • the haptens are designed to elicit the appropriate hypervariable binding region in an antibody molecule to express intrinsic binding energy for the transition- state of a chemical reaction, particularly a hydrolytic reaction. Arrangement of amino-acid side chains generated in the combining-site will be appropriate for performing chemical modification of an epitope of interest.
  • the method comprises exposing cells capable of producing antibodies to the antigen and thereby generating antibody producing cells; hybridizing the antibody producing cells with myeloma cells and thereby producing a plurality of hybridoma cells each producing monoclonal antibodies; and screening the plurality of monoclonal antibodies to identify a monoclonal antibody which catalyzes the chemical reaction of interest.
  • the monoclonal antibody so identified may then be replicated, again by either m. vivo or in vitro techniques, to obtain a quantity sufficient to catalyze the chemical reaction of interest.
  • the detection of antibodies with the desired catalytic activity and specificity is achieved by screening the hybridomas once they have been elicited.
  • screening may be achieved by high performance liquid chromatography (HPLC) or spectrophotometric methods (ELISA) .
  • Catalytic monoclonal antibodies are elicited "in vivo" by modification of the technique disclosed by Koprowski et al. in U.S. Patent No. 4,196,265, issued April 1, 1980, which is hereby incorporated by reference. The details of that process are known in the art.
  • a series of monoclonal antibodies directed to a specific molecule are prepared under suitable conditions. This involves first immunizing BALB/C mice with an appropriate antigen.
  • the antigen comprises a hapten according to the invention bound to a peptide or other carrier molecule.
  • Antibody-producing lymphocytes are then removed from the spleens of the immunized mice and hybridized with myeloma cells such as SP2/0 cells to produce hybridoma cells. These hybridoma cells are then plated in the wells of microliter plates. The series of monoclonal antibodies being produced by the hybridoma cells is screened under appropriate conditions to identify monoclonal antibodies which catalyze the desired reaction under appropriate conditions. Alternatively, the medium may be tested for antibodies that bind to the immunogen and the hybridomas producing these antibodies then expanded in tissue culture or grown in vivo.
  • Screening may be conveniently accomplished by treating a standardized solution of the reactant with an aliquot of medium withdrawn from a microliter well and measuring the presence of the desired product by conventional instrumental methods. This measurement may be readily conducted, for example by spectrophotometric methods or by gas-liquid or high pressure liquid chromatography. By comparison with standardized samples of the desired product or reactant, rates of reaction may be quantified. In this manner, wells containing hybridoma cells producing catalytic monoclonal antibodies are identified. The selected hybridoma cells are then cultured to yield colonies.
  • mice such as syngeneic BALB/c mice are inoculated intraperitoneally with the selected hybridoma cells and produce tumors, generally within two or three weeks. These tumors are accompanied by the production of ascites fluid which contains the desired monoclonal antibodies. The monoclonal antibodies are then separately recovered from the ascites fluid by conventional methods such as ultrafiltration, ultracentrifugation, dialysis and immunoffinity chromatography.
  • the invention is also a method for catalyzing the cleavage or formation of an amide (peptide) bond in a molecule.
  • the molecule can be a natural or synthetic peptide or protein.
  • Target molecules include biomolecules which are defined as any molecule which affects a biological system in vivo or in vitro. Biomolecules may be synthesized by cells or chemically synthesized. Examples of biomolecules include proteins, glycoproteins, peptides, steroids, and maleic acid. Also included are synthetic organic analogs of peptides, steroids, maleic acid, etc. Pharmaceutically active compounds such as theophylline, caproin, cyclosporin, etc., are also considered to be biomolecules.
  • the method comprises contacting a molecule containing one or more amide (peptide) or ester bonds with an effective amount of a catalytic antibody which has been elicited by an antigen comprising a hapten of the invention.
  • the separately recovered monoclonal antibodies are contacted with a molecule under suitable conditions permitting the formation of a complex between the monoclonal antibody and the molecule.
  • the concentration of the catalytic antibodies used is less than the equivalent concentration of the target molecule and may be in the picomolar range.
  • the antibodies should function under normal physiologic conditions in vivo. The skilled artisan will appreciate that the conditions suitable for complex formation may vary depending on the particular molecule and monoclonal antibody under consideration.
  • suitable conditions for complex formation encompass solution phase and emulsion reaction systems including a protic solvent, preferably water, maintained at a pH value between about 6.0 and about 9.0, preferably between about 6.0 and about 7.5 and at a temperature from about 4°C to about 50°C, preferably from about 20°C to about 45°C.
  • a protic solvent preferably water
  • the ionic strength 1/2 ⁇ c ⁇ 2 , where c is the concentration and z is the electronic charge of an ionic solute, should be maintained at a value below about 2.0 moles/liter, preferably between 0.1 and 1.5 moles/liter.
  • the method of this invention may be carried out at reduced or elevated pressure, but preferably is practiced at ambient pressure.
  • suitable conditions also include the use of support materials to which the monoclonal antibody is attached. Such support materials are well-known to those of ordinary skill in the art as are methods for attaching monoclonal antibodies to them.
  • Catalytic antibodies elicited with the antigens of the invention may be useful in the treatment of autoimmune disease, cancer and thrombolytic disease.
  • the catalytic antibodies may also be useful for treatment of cardiovascular disease eliminating high density lipoproteins and for the detoxification of bacterial endotoxins.
  • Vaccines comprising synthetic peptides optionally linked to carrier proteins, for example, against foot and mouth disease (FMD) and E. coli enterotoxin, have been proven efficacious in recent years.
  • Oligopeptides having variable lengths with sequences from the receptor-binding regions of viruses which employ a specific cellular receptor for penetration of the host cell and having a transition state analog dipeptide isostere in a critical region of the sequence induce on immunization, optionally after coupling to a suitable carrier protein, catalytic antibodies that cleave the viral coat protein and prevent virus penetrating the cell.
  • the dimensional structure of Rhino 14 and Polio 1 virus particles has been charted by X-ray scattering. Regions have been identified which are binding sites to cellular receptors.
  • the region of the human immunodeficiency virus type 1 (HIV I) critical for interaction with the CD4 receptor on T-lymphocytes has been located and mapped to sequences in the.
  • oligopeptides are used which contain partial sequences from the envelope proteins of viruses critical for host cell attachment and a transition-state dipeptide isostere selected from the haptens according to the invention.
  • the resultant peptide analogs are used to induce catalytic antibodies that inactivate viruses by proteolysing segments (epitopes) of the viral coat protein critical for infectivity.
  • oligopeptides are used having sequences from the receptor binding region of retroviruses e.g., HIV I, HIVII and picorna viruses, e.g., Rhino 14, viral polypeptides, inflammatory proteins, anaphylactic proteins, lymphokines, cytokines and other polypeptide mediators of host infection or toxic syndromes.
  • retroviruses e.g., HIV I, HIVII and picorna viruses, e.g., Rhino 14, viral polypeptides, inflammatory proteins, anaphylactic proteins, lymphokines, cytokines and other polypeptide mediators of host infection or toxic syndromes.
  • the novel spiro[4.4]nonane-containing dipeptide analog 7-tran -amino-6-trans-hydroxyspiro- [4.4]nonane-1- carboxylic acid, as a racemic mixture, has been synthesized and incorporated into a longer peptide sequence.
  • This dipeptide analogue mimics both torsionally-strained glycyl-proline and glycyl-glycine as shown in Figure 1; in addition, the peptide bond has been replaced by a hydroxyethylene group, an effective transition state analogue.
  • a key step in the synthesis of the spiro[4.4]nonane-containing dipeptide analogue was the introduction of an amino functionality into the corresponding hydroxyester 12 via an intramolecular acyl- nitrene insertion reaction.
  • a stereoisomer of this material, 12 was first synthesized as is outlined in Fig. 2 and 3.
  • hydroboration of the alkene 4 produced the alcohol 6 in an epimeric ratio of 90:10.
  • Ruthenium tetraoxide catalyzed oxidation of the alcohol 6 afforded the carboxylic acid 8, which was methylated with trimethylsilyl diazomethane (TMSCHN 2 ) to yield the protected hydroxyester 10.
  • TMSCHN 2 trimethylsilyl diazomethane
  • Removal of the terfc- butyldimethylsilyl group with hydrogen fluoride gave the corresponding hydroxyester 12.
  • hydroboration of the alkene 5 yielded exclusively the alcohol 7.
  • Elaboration of the alcohol 7 through the acid 9 and the ester 11 yielded the lactone 20. That the hydroxyester 11 lactonized upon deprotection confirms the stereochemical assignment of this material, as well as the trans-stereochemistry of the hydroxyl group in 4 and the compounds synthesized from it.
  • the hydroxyester 12 was then treated with 1,1' -carbonyl-diimidazole (CDI) , followed by sodium azide to generate the azidocarbonate 13.
  • CDI 1,1' -carbonyl-diimidazole
  • TCE 1,1,2,2, -tetrachloroethane
  • Hydrolysis of carbamate 14 in hot aqueous sodium hydroxide afforded the hydroxyamino acid 15, which was directly coupled to the protected D-tyrosyl acid fluoride 16 to yield the protected dipeptide 17, as a mixture of diastereomers.
  • the derivative 15 was flanked with D-amino acids to generate a more immunogenic hapten.
  • Deprotection of 18 by catalytic transfer hydrogenation with palladium black and formic acid yielded the diastereomeric peptide derivatives 19a and 19b, which have been conjugated to the carrier proteins keyhole limpet hemocyanin (KLH) and bovine serum albumin (BSA) in preparation for immunization.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • High resolution mass spectra were determined at the Harvard Chemistry Department Mass Spectrometry Facility.
  • High-pressure liquid chromatography HPLC was performed on a Waters 600E instrument using a Waters 484 UV detector and a Waters 470 Scanning Fluorescence detector.
  • Analytic HPLC utilized an Alltech Econosphere C18 column (15 x 0.46 cm, 5 micron) ;
  • preparative HPLC utilized a Waters ⁇ Bondapack Phenyl Radial-pak cartridge (10 x 2.5 cm, 10 micron) . All elution solvents were CH 3 CN/water mixtures containing 0.1% TFA.
  • Column chromatography employed Aldrich silica gel 60 A (200-400 mesh) .
  • Sodium hydride (24 mmol as a 60% dispersion in mineral oil) was washed with 50 mL of anhydrous ether under nitrogen, and 10 mL of dry DMSO was added. The mixture was heated at 70-75 °c for approximately 1 hour until a clear dark-gray solution formed. The resulting solution was cooled to 0 oc, and methyltriphenylphosphonium bromide (9.0 g, 25,193 mmol) in 25 mL of dry DMSO was added. The resulting dark-red solution was stirred at room temperature for 15 minutes and then 3 (680 mg, 2.537 mmol) in 5 mL of DMSO was added.
  • the reaction mixture was stirred at 55 oC for 3 hours, cooled, and diluted with water.
  • the resulting mixture was extracted with EtOAc (4 x 40 mL) , and the combined extracts were washed with brine and dried over anhydrous MgSCs.
  • the solvent was removed by evaporation under reduced pressure, and the residue purified by chromatography on silica gel (hexanes, R f 4 0.39, R £ 5 0.68) to afford 4 (316 mg, 47%) and 5 (160 mg, 24%) as colorless oils.
  • the reaction medium was then acidified to approximately pH4 with concentrated HCl and stirred at room temperature overnight. 15mL of brine was added, and the aqueous layer was extracted with Et0Ac4 x 20 mL) . The combined organic layers were washed with brine (2x10 mL) , dried over anhydrous MgS0 4 , and the solvent removed by evaporation under reduced pressure to afford the azidoformate 13 as a colorless oil (156 ,mg, yield 92%) .
  • the acid fluoride 16 (35 mg, 0.086 mmol) in 0.5 mL of dry DMF was then added dropwise.
  • Diastereomer 19b was prepared from 18b exactly as described in Example 11 above.
  • Diastereomers 19a and 19b are prepared as described in Examples 12 and 13, and are then conjugated to the carrier protein keyhole limpet hemocyanin (KLH) .
  • KLH keyhole limpet hemocyanin
  • BALB/c mice are immunized with the KLH-conjugated compounds emulsified in complete Freund's adjuvant.
  • a blood sample is obtained from each mouse and the serum separated by centrifugation and stored at 4oC.
  • Sera obtained in this way are screened for binding activity to the original transition-state analog immunogen by standard ELISA procedures.
  • Antibody-producing mice immunized as described above and assayed for reactivity with the transition state analog peptide immunogens are sacrificed, their spleens are removed and hybidoma cells are prepared using myeloma cells.
  • Hybidomas secreting monoclonal antibodies are screened for hydrolytic activity against the peptide substrates D-tyrosyl-glycyl- glycyl-D-phenylalanine, D-tyrosyl-glycyl-D-prolyl-D- phenylalanine, and D-tyrosyl-glycyl-L-prolyl-D- phenylalanine.

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Abstract

L'invention concerne des haptènes capables d'induire des anticorps pouvant catalyser des réactions chimiques, constitués d'un haptène ou d'un haptène et d'une molécule de transport appropriée. Elle porte en particulier sur des analogues de dipeptides contenant spiro[4.]nonane, qui imitent l'état fondamental d'un peptide déformé en torsade et l'état de transition pour l'hydrolyse d'une liaison peptidique, ainsi que sur leurs procédés de synthèse et leur couplage avec des acides aminés de configuration D. Elle se rapporte également à des anticorps catalytiquement actifs pour les réactions chimiques, en particulier, le clivage ou la formation d'une liaison peptidique sélectionnée, et qui sont induits par lesdits antigènes, ainsi qu'à des procédés de production des anticorps et de catalyse du clivage ou de la formation d'une liaison peptidique dans une molécule.
PCT/US1996/009450 1995-06-06 1996-06-05 Analogues de peptides et leur utilisation en tant qu'haptenes pour l'induction d'anticorps catalytiques Ceased WO1996039443A1 (fr)

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Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2763071A1 (fr) * 1997-05-07 1998-11-13 Centre Nat Rech Scient Analogues peptidiques, et leurs utilisations notamment dans des compositions pharmaceutiques et pour le diagnostic
WO1999033968A1 (fr) * 1997-12-26 1999-07-08 Taizo Uda Procede de production d'anticorps catalytique et son procede d'utilisation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE MEDLINE ON STN, US NATIONAL LIBRARY OF MEDICINE, (Bethesda, MD, USA), No. 95031013, SMITH et al., "An Approach to Sequence-Specific Antibody Proteases. The Use of Haptens Mimicking Both a Transition State and a Distorted Ground State", Abstract 95031013; & APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY, May-June 1994, Volume 47, Number 2-3. *
JOURNAL ORG. CHEM., 1995, Vol. 60, No. 16, YUAN et al., "A Mimic of Both a Torsionally-Distorted Peptide Ground State and the Transition State for Peptide Bond Hydrolysis: Synthesis of a Spiro[4.4ÜNonyl Derivative", pages 5360-5364. *
TETRAHEDRON LETTERS, 1994, Vol. 35, No. 34, YUAN et al., "The Synthesis of Cyclobutanol-Containing Dipeptide Analogues", pages 6195-6198. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
FR2763071A1 (fr) * 1997-05-07 1998-11-13 Centre Nat Rech Scient Analogues peptidiques, et leurs utilisations notamment dans des compositions pharmaceutiques et pour le diagnostic
WO1998050423A3 (fr) * 1997-05-07 1999-08-19 Centre Nat Rech Scient Analogues peptidiques et leurs utilisations notamment dans des compositions pharmaceutiques et pour le diagnostic
WO1999033968A1 (fr) * 1997-12-26 1999-07-08 Taizo Uda Procede de production d'anticorps catalytique et son procede d'utilisation

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