Production of immunoproximity catalysts

Abstract: There are disclosed and claimed a process for pro­ ducing an immunoproximity catalyst for a chemical reaction and chemical reactions in which the immuno­ proximity catalyst can be used.

Description: This invention involves novel immunoproximity catalysts. The catalysts are derived from antibody molecules. The process includes selecting a hapten based on its relationship to the structure of a transition state complex of the selected reaction so that the hapten, upon attachment to a carrier protein and injection (with or without adjuvant) into an animal, elicits an immune response in which the desired antibody is produced. This antibody is modified by the specific, covalent attachment of the catalytic group involved in the selected reaction, yielding an immunoproximity catalyst which is a substrate-specific, antibody catalyst. Catalysts are widely used in the chemical, pharmaceutical, and food processing industries. There is a continuing demand for new and/or improved catalysts. Improvements in specificity, selectivity, rate enhancement, and stability are desirable. Among known catalysts, enzymes -naturally occurring amino acid or RNA polymers - excel in terms of their ability to generate the highest possible reaction rates while maintaining high specificity. Simpler catalysts -based on organics or metals, for instance lack the specificity of enzymes but are often very stable. These simpler catalysts generally lack the stereo- and regiochemical control of reactions inherent in enzymes. The success of enzymes as catalysts is attributed to their ability to fold (i.e. assume a spatial structure) so as to create specific binding pockets for the reactant and to place catalytically active groups in this pocket in close proximity to the scissle bond. These pockets are referred to as active sites. A major obstacle to creating new enzymes - specific for a predetermined reactant and able to catalyze the desired reaction - is the rudimentary understanding of how proteins fold to form active sites. Minor alterations to an enzyme (and its catalytic properties) are possible via site directed mutagenesis. However, substitution of a single amino acid for another in a protein sequence can affect the folding and/or function of the molecule in large and unpredictable ways. Description of the Prior Art International Patent PCT/US84/01951; G.P. Royer (Advances In Catalysis 29 , 197, 1980; R.A. Lerner (Advances in Immunology 36 , 1, 1984); A. Tramontano et al (Proc. Nat. Acad. Sci., USA 83 , 6736, 1986); Kohen et al (FEBS Letters 100 , 137, 1979; 104 , 201, 1979; 111 , 427, 1980; and Biochim. Biophys. Acta 629, 328, 1980); L. Slobin (Biochemistry 5 , 2836, 1966); copending U.S. Patent Application U.S.S.N. 877, 273, filed June 23, 1986; V. Raso and B.D. Stollar (Biochemistry 14 , 585, 591, 1975); A. Tramontano et al. (Science 234 , 1566, 1986); and S.J. Pollack et al. (Science 234 , 1570, 1986) are cited as representative of the state of the art. The present invention comprises a process for producing an immunoproximity catalyst for a chemical reaction comprising the following steps: (1) identifying a reactant and a catalytic group for said reaction; (2) selecting a hapten which corresponds to the transition state complex of said reactant and said catalytic group; (3) stimulating an immune response for production of antibodies by said hapten; (4) isolating purified antibodies from said immune response which are specific for said hapten; (5) selecting a converting hapten which corresponds to the transition state complex of said reactant and said catalytic group; (6) modifying said purified antibodies by reacting them with the converting hapten thereby covalently attaching the catalytic group to said antibodies while said catalytic group is specifically bound in binding sites of said purified antibodies as a part of said converting hapten yielding antibody-catalytic group complexes which are active for said chemical reaction; and breaking the bond between the Y group and the Q cat group thereby leaving the catalytic group bound to the antibody; and (7) isolating the antibody-catalytic group complexes which are active for said chemical reaction from said modification; wherein the reactant and the catalytic group complex are represented by the formula the hapten is represented by the formula and the converting hapten is represented by the formula wherein X represents a reaction site which comprises the portion of the reactant altered in the reaction, K represents said catalytic group, and Y represents the reaction site which comprises the analogous portion of the hapten molecule and the converting hapten molecule, and X and Y are related to each other in that Y has a higher valence state and one or more bonds than X, and wherein Q and Q' represent one or more substituents bonded to Y in the hapten and converting hapten, respectively, and wherein Q cat represents the catalytic group and linker group bound to Y which is represented by the formula K - L wherein K represents the catalytic group and L represents the linker group which is the remainder of the molecule which is used to form a permanent bond between the catalytic group and the antibody molecule, and wherein Q' cat represents an analog of the selected catalytic group and linker group, bound to Y, which is represented by the formula K' - L' wherein K' represents an unreactive analog of K which will form a stable bond with Y, and wherein L' is an unreactive analog of L which induces the presence of a group in the antibody binding surface which is reactive with L and wherein R₁, R₂, R'₁ and R'₂ represent the residual chemical groups of the reactant, hapten and converting hapten which do not participate in the catalytic event, and R₁ and R'₁ and R₂ and R'₂ are, respectively, substantially similar to each other. This invention comprises a new process for the production of novel immunoproximity catalysts. The immunoproximity catalysts of the invention are capable of acting both in vitro and in yivo, and in the human body itself. The catalysts of this invention are antibody molecules obtained by an immunization with a hapten that is similar to, but distinct from, the transition state complex for the reaction of the selected reactant and catalytic group and by subsequent modification with the converting hapten. The relationship between the hapten and the reactant and catalytic group is the following: at least one chemical group in the hapten is identical in both its structure and orientation with a group in both the reactant and the catalytic group. This ensures that the hapten shares antigenic determinants with both the reactant and the catalytic group; i.e., the binding site of the antibody stimulated by hapten must interact with both the reactant and the catalytic group, the hapten must also differ structurally and chemically from the reactant and catalytic group in the vicinity of the reaction site. A reaction site analog of higher valency is substituted for the reaction site in either or both the reactant and the catalytic group; further, the reaction site analog has substituents, the role of which are to position complementary catalytic groups within the antibody binding surface and to create an additional cavity or cavities capable of enclosing cofactor molecules. The presence of one or more substantially similar groups or residues ensures that the hapten shares common antigenic determinants with both the reactant and the catalytic group. This in turn, ensures that the antibody will "recognize" the reactant in a selective manner and position the bound catalytic group for efficient catalysis. On the other hand, the hapten differs structurally and chemically from the reactant and catalytic group in the vicinity of the reaction site in the reaction. Differences include substituting a reaction site analog of higher valency for one or more such reaction sites in both the reactant and the catalytic group. In addition, residues appended from these reaction site analogs are so oriented as to induce additional, complementary groups or pockets for co-factor binding in the antibody that promote catalysis. The presence of these additional groups in the antibody binding surface increases the efficiency of the catalytic group and allows larger rate enhancements to be realized. Finally, the hapten differs from the catalytic group in the vicinity of the covalent bond to be formed with the antibody. This residue is identical to or of similar bulk and orientation to the corresponding residue in the catalytic group, i.e., it is substantially similar. However, when a photoactivatable crosslinker is to be used in the catalytic group, the corresponding group in the hapten is an unreactive analog of the crosslinker and when an electrophile group is to be used in the catalytic group, the corresponding group in the hapten is a charged analog to induce a positioned, reactive nucleophile in the antibody binding surface. The purified, specific antibody obtained in an immune response to said hapten is subsequently modified by reaction with the converting hapten. The converting hapten is similar to, but distinct from, the hapten. The relationship is as follows: the converting hapten is identical to the hapten with the following exceptions - 1) The actual catalytic group is used in the converting hapten instead of the analog used in the hapten; 2) No additional residues are appended to any of nuclei in the vicinity of the bonds to be altered during the reaction - as is the case for the hapten; and 3) The nucleus at the reaction center, i.e., the nucleus in the hapten substrate analog to which the hapten catalytic group analog is bound - may be altered to suit the stability requirements of the converting hapten. Upon modification by the converting hapten, the antibody acquires catalytic power in addition to its normal and highly selective binding capacity. The immunoproximity catalysts of the process, whether obtained polyclonally or monoclonally, are well-folded, stable proteins by virtue of their derivation in an immune response. This invention thus circumvents present uncertainties concerning folding of proteins of novel sequence. The process of immunization using the haptens of this invention to produce antibodies is defined as active immunization. The said proteins of this invention, upon modification, are the novel class of antibodies that are chemically reactive against their target antigenic species (the reactant), in that they catalyze cleavage of bonds in these target molecules - in the manner of an enzyme - instead of passively binding antigens. Conversion of bimolecular reactions to unimolecular reactions result in very large increases in the rate of reaction. The immunoproximity catalysts made by the process of this invention will serve this function as well as aligning the reactant and catalytic group to maximize the rate of reaction. The types of reaction catalyzed are limited only by the type of catalytic group which can be attached to said antibody. These catalytic groups include general acid-base, nucleophilic, electrophilic, and metal catalysts. It is also possible to use more than one catalytic group. Two large classes of reactions to which the catalysts made by the process of this invention are applicable are hydrolysis reactions and oxidation-reduction reactions. Definitions Reactant - the molecule to be converted chemically in a particular chemical transformation to product(s). Catalytic Group - the catalytically active moiety which is covalently bound to the elicited antibody molecule while occupying its specificity site on the antibody molecule. Cofactor - an additional molecule(s) participating in the reaction, including H₂O in hydrolytic reactions. Linker Group - the moiety included in the catalytic group used to form the covalent attachment of the catalytic group to the elicited antibody. Hapten - a molecule attached covalently to a carrier protein that elicits an antibody response directed towards itself. Antigen - the combined hapten-carrier complex used to stimulate the immune response. Converting Hapten - the molecule used to attach the catalytic group to the elicited antibody while it is occupying its specificity pocket in the antibody binding site. The selected react ant and catalytic group are represented in the transition state complex as a tetrapartite molecule wherein R₁ and R₂, which do not participate in the catalytic events, are the residual chemical groups of the reactant. X is the reaction site which comprises the portion of the reactant altered. K is the catalytic group. The haptens of this invention corresponding to the reactant-catalytic group complex are molecules selected, or chemically synthesized, having the structure wherein R ' / 1 and R ' / 2 are substantially similar to R₁ and R₂ of the reactant (except for possible addition to R ' / 1 and R ' / 2 of a group, as shown in Table 1 which links the hapten to the carrier) and Y represents nuclei and associated bonds which comprise the portion of the hapten molecule corresponding to the X region of the reactant and: (1) X and Y are related to each other in that Y has a higher valence state and one or more bonds than X. Table 2 represents this relationship between X and Y. (2) Q represents one (or more) substituents as shown in Table 3 bound to Y in (1) such that: (a) Q contains a negative charge(s) when catalysis requires a positive charge(s) in the active surface of the antibody catalyst, and vice-versa. (b) Q is polar and neutral when catalysis requires a polar component in said active surface. (c) Q is non-polar (or hydrophobic) when catalysis requires a non-polar component in said active surface. (d) When one or more cofactors are involved in the reaction, Q is selected to be a substituent of substantial bulk so as to create a cavity in the active surface to allow for one or more of said co-factors to bind, including H₂O in the case of hydrolytic reaction. (e) Q can contain in addition a group as shown in Table 1 capable of linking the hapten to carrier, in case attachment at the Y region is desired. (3) is synthesized to be a molecule of the type K' - L' FORMULA III wherein K' represents a catalytic group analog as shown in Table 4 and L' represents an unreactive linker analog of L, the linker group. L' induces the presence of a group in the antibody binding surface which is reactive with L. Both L and L' are shown in Table 5. When several possible substituents are appropriate for the catalytic process of this invention, prefered choices are selected as those which minimize differences in binding affinity between the reactant and the hapten. These relative affinities are tabulated in the treatise by Pressman and Grossberg, The Structural Basis of Antibody Specificity, Benjamin, NY, 1969, for example. These data are used to ensure a sufficient degree of similarity in binding of reactant and hapten such that both molecules will be bound by the immunoproximity catalysts resulting from this invention. 4) The remaining groups R ' / 1 and R ' / 2 comprising Y are selected to be identical or of similar size and charge to corresponding groups in X, i.e., they are substantially similar. The Formulas I-III and the algorithm set forth above define the correspondence between the hapten and reactant in this invention. That is, the hapten is selected, or synthesized chemically, to correspond to the reactant according to above Formulas I-III. The identity of X in some important reactions is illustrated in Table 6. The converting haptens of this invention corresponding to the hapten are molecules selected, or synthesized chemically, having the structure wherein R₁ and R₂ are identical to R₁ and R₂ of the reactant, Y is identical to Y of the hapten, Q cat contains the active catalytic group, K as well as the linker group, L, and; (1) Q' may be the same group(s) as in the hapten or, to decrease' the binding affinity of for the elicited antibody may be hydrogen. (2) Q cat is a molecule chemically synthesized, or selected, to be a molecule of the type K - L FORMULA V wherein K is the selected catalytic group as shown in Table 4 and L is the linker group as shown in Table 5, covalently bound to the selected catalytic group, used to permanently, covalently bind the selected catalytic group to the elicited antibody. When L is selected to contain a photoactivatable crosslinking group, L' is an unreactive analog of the crosslinking group. When L is selected to contain an electrophile group, L' is a charged unreactive analog which induces a positioned, reactive nucleophile in the antibody binding surface. Formulas IV and V and the accompanying algorithm define the relationship of the reactant and hapten to the converting hapten. That is, the converting hapten is chemically synthesized or selected to correspond to the reactant and hapten according to above Formulas IV and V. The converting hapten is incubated with the elicited antibody. The converting hapten reacts with said antibody, or is caused to react, the bond between the Y group and the Q cat group is broken (for instance with mild acid or mild base) in such a way that the integrity of the antibody molecule is preserved, and the group is removed from the antibody's binding site. This results in a fully catalytic antibody. Some Substitutions of Higher Valence Useful for Nuclei In the X Region Of The Reactant Substrate: Nuclei in X Hapten: Nuclei in Y O,S (oxygen, sulfur) N or C (nitrogen or carbon) N (nitrogen) C (carbon) C (carbon) P (phosphorus) The process of this invention comprises selecting or synthesizing a hapten according to the algorithm set forth above, preferably covalently linking said hapten(s) to carrier species such as keyhole limpet hemocyanin or similar proteins in standard use for this purpose via the linking residue provided for above, and injecting the complex into an appropriate animal as antigen to stimulate the immune response. Following a time sufficient for said response to develop, the animal is bled, serum is extracted an fractionated (preferably over a column containing covalently linked hapten) stereospecificity, selectively, and/or substrate specificity. Besides overcoming these problems, immunoproximity catalysts offer significant rate enhancements and milder reaction conditions than traditional catalysts. Immunoproximity catalysts also are of considerable use when protective groups are employed in synthesis. An immunoproximity catalyst can remove a protective group without altering the reactant in any other respect. As site-specific cleavage catalysts, immunoproximity catalysts are useful from protein sequencing to anti-cancer therapy. To facilitate protein sequencing, for example, an immunoproximity catalyst an be produced to catalyze the hydrolysis of N-terminal formyl or acetyl groups, and can be produced to catalyze the cleavage of proteins at the rare amino acids such as tryptophan, methionine, or histidine. The compounds used in the production of the immunoproximity catalysts as shown in the above tables are offered by way of illustration and not by way of limitation. These compounds can be prepared using the conventional techniques of organic synthesis. It is the intent of the inventors to commercially-develop the immunoproximity catalysts described and claimed herein under the trademark Immunozyme.

Claims: A process for producing an immunoproximity catalyst for a chemical reaction comprising the following steps: (1) identifying a reactant and a catalytic group for said reaction; (2) selecting a hapten which corresponds to the transition state complex of said reactant and said catalytic group; (3) stimulating an immune response for production of antibodies by said hapten; (4) isolating purified antibodies from said immune response which are specific for said hapten; (5) selecting a converting hapten which corresponds tp the transition state complex of said reactant and said catalytic group; (6) modifying said purified antibodies by reacting them with the converting hapten thereby covalently attaching the catalytic group to said antibodies while said catalytic group is specifically bound in binding sites of said purified antibodies as a part of said converting hapten yielding antibody-catalytic group complexes which are active for said chemical reaction; and breaking the bond between the Y group and the Q cat group thereby leaving the catalytic group bound to the antibody; and (7) isolating the antibody-catalytic group complexes which are active for said chemical reaction from said modification; wherein the reactant and the catalytic group complex are represented by the formula the hapten is represented by the formula and the converting hapten is represented by the formula wherein X represents a reaction site which comprises the portion of the reactant altered in the reaction, K represents said catalytic group, and Y represents the reaction site which comprises the analogous portion of the hapten molecule and the converting hapten molecule, and X and Y are related to each other in that Y has a higher valence state and one or more bonds than X, and wherein Q and Q' represent one or more substituents bonded to Y in the hapten and converting hapten, respectively, and wherein Q cat represents the catalytic group and linker group bound to Y which is represented by the formula K - L wherein K represents the catalytic group and L represents the linker group which is the remainder of the molecule which is used to form a permanent bond between the catalytic group and the antibody molecule, and wherein Q' cat represents an analog of the selected catalytic group and linker group, bound to Y, which is represented by the formula K' - L' wherein K' represents an unreactive analog of K which will form a stable bond with Y, and wherein L' is an unreactive analog of L which induces the presence of a group in the antibody binding surface which is reactive with L and wherein R₁, R₂, R'₁ and R'₂ represent the residual chemical groups of the reactant, hapten and converting hapten which do not participate in the catalytic event, and R₁ and R'₁ and R₂ and R'₂ are, respectively, substantially similar to each other. The process of claim 1 wherein Q is selected from the group consisting of (a) a positively charged substituent, (b) a negatively charged substituent, (c) a polar substituent, (d) a non-polar substituent, and (e) a substituent having substantial bulk. The process of claim 1 wherein Q' is selected from the group consisting of (a) a positively charged substituent, (b) a negatively charged substituent, (c) a polar substituent, (d) a non-polar substituent, (e) a substituent having substantial bulk, and (f) hydrogen. The process of claim 1 wherein L contains a photo-activatable crosslinking group and L' is an unreactive analog of said crosslinking group. The process of claim 1 wherein L contains an electrophilic group and L' is a charged unreactive analog which induces the presence of a group in the antibody binding surface which is reactive with L. The process of claim 1 wherein X is a moiety within which there is an element selected from the group consisting of O or S, and Y is a moiety within which there is an element selected from the group consisting of N or C. The process of claim 1 wherein X is a moiety within which there is N, and Y is a moiety within which there is C. The process of claim 1 wherein X is a moiety within which there is C, and Y is a moiety within which there is P. The process of Claim 2, wherein said substituent Q is a positively charged substituent selected from the group consisting of -NH₃ (+) , -NRH₂ (+) , NR₂H (+) , NR₃ (+) and SR₂ (+) . The process of claim 2 wherein Q is a substituent hating a negative charge, said substituent selected from the group consisting of -CO₂ (-) , -PO₄ (-2) , -SO₄ (-2) , and -SO₃ (-2) . The process of claim 2 wherein Q is a polar substituent selected from the group consisting of -NO₂, -N-(CH₂F)₂, -Cl, -Br, -CF₃, -SH, and -OH. The process of claim 2 wherein Q is a non-polar substituent selected from the group consisting of -C(CH₃)₃, -CH₃, -CH(CH₃)₂, The process of claim 2 wherein Q is a substituent having substantial bulk selected from the group consisting of -CO₂ (-) , -NH₂, -NH₃ (+) , and -NO₂. The process of claim 1 wherein K is a catalyst selected from the group consisting of -OH, SH, -NH₂, CO₂H, -H₂PO₃, and a chelate containing an element selected from the group consisting of Fe, Co, Cu, Pb, Ti, Al, Pd, B or Mg. The process of claim 1 wherein K' is selected from the group consisting of -CH₂-, and the uncomplexed chelate. The process of claim 1 wherein the immune response is stimulated monoclonically and/or polyclonically. The process of claim 1 wherein the catalytic group includes an acid-base catalyst. The process of claim 1 wherein the catalytic group includes a nulceophilic catalyst. The process of claim 1 wherein the chemical reaction is an oxidation-reduction catalysis reaction. The process of claim 1 wherein the chemical reaction is hydrolysis of polydeoxynucleotides or polyribonucleotides containing a specified sequence of nucleic acids. The process of claim 1 wherein the chemical reaction is hydrolysis of protein molecules containing a specified sequence of amino acids. The process of claim 1 wherein the chemical reaction is hydrolysis of an acetal bond. The process of claim 22 wherein the acetal bond is in a polysaccharide containing a specific sequence of sugars. The process of any of claims 1, 17, 18 or 19 wherein the chemical reaction is hydrolysis of a drug having a bond selected from the group consisting of ester, amide and acetal bond. An immunoproximity catalyst obtainable by the process of any of the claims 1-24.

Inventors: KALLENBACH NEVILLE R, KIM PETER S, KALLENBACH, NEVILLE R., KIM, PETER S.
Applicants: IGEN INC, IGEN, INCORPORATED
Application year: 1988
Main classification: C12N9
IPC codes: G01N33, C07K1, C07B61, C07K14, A61K39, C12N9, C07K16