Catalytic antibodies which hydrolyze primary amides and methods

Details Catalytic antibodies which hydrolyze primary amides and methods Catalytic antibodies which hydrolyze primary amides and methods

1996-05-04

1999-05-04

Patterson, Jr., Charles L.

Drug, bio-affecting and body treating compositions

Immunoglobulin, antiserum, antibody, or antibody fragment,...

Binds hapten, hapten-carrier complex, or...

424 941, 4351885, A61K 3843, A61K 39395, C12N 900

Patent

active

059002379

DESCRIPTION:

BRIEF SUMMARY
FIELD OF THE INVENTION

This invention relates to analogs of primary amide bonds and methods of using such analogs such as for eliciting catalytic antibodies for hydrolysis of primary amide bonds. The invention also relates to catalytic antibodies capable of catalyzing the hydrolysis or formation of primary amide bonds.
This invention further relates to novel methods for using such catalytic antibodies as therapeutic agents wherein said catalytic antibodies cause hydrolysis of primary amide bonds of peptides and proteins.
This invention also relates to novel methods for using such catalytic antibodies as catalysts for hydrolysis of formation of primary amide bonds in the synthesis of chemicals.
Still further, this application relates to novel methods for selecting or screening for catalytic activity by catalytic antibodies in samples containing antibodies as well as other biological molecules. Such methods for selecting or screening are rapid and efficient and thus, allow the routine screening of larger numbers of potential catalytic antibodies.


BACKGROUND OF THE INVENTION

Various documents are cited parenthetically throughout the text of this disclosure, with full citation to these documents appearing as a list immediately preceding the claims. Other documents are cited parenthetically, in full, throughout the text of this disclosure. These documents pertain to the field of this invention, and each of these documents is hereby incorporated herein by reference.
One of the most frequently cited goals of catalytic antibody research is the generation of antibodies capable of efficient, specific hydrolysis of amide bonds (1-14). This goal partly results from the scientific challenge of generating an antibody to catalyze a relatively kinetically inert reaction and partly from the potentially vast range of practical applications for proteolytic antibodies.
Until now, no haptens have been designed which have demonstrably elicited antibodies that hydrolyze unactivated amides without cofactor assistance.
The difficulty in eliciting amide-hydrolyzing antibodies stems primarily from the difficulties associated with screening the antibodies for catalysis. The uncatalyzed rate (k.sub.uncat) of amide hydrolysis (with a half-life on the order of 7 years at neutral pH (15)) is much slower than that of typical esters (16), carbonates (17), or activated amides (8, 18). Correspondingly, it is much more difficult to detect antibodies that catalyze amide hydrolysis. If two separate reactions of different uncatalyzed rates (k.sub.uncat) are catalyzed by two antibodies with identical rate enhancements (k.sub.cat /k.sub.uncat), it will be more difficult to detect the antibody that is slower in absolute terms (k.sub.cat). There are two main reasons for this. One reason is that in the slower reaction there will be fewer turnovers per unit of time and the assay system must be able to detect extremely low levels of formed product. The second reason is that reactions with slower k.sub.uncat values typically require longer periods of antibody-substrate incubation (days or weeks) than do faster reactions (minutes or hours). Long incubation periods may lead to false results because of the possible appearance of by-products, the antibody may denature, especially at non-neutral pH values, and traces of adventitious enzymes may catalyze the reaction in question.
As mentioned above, until now no catalytic antibodies have been generated that are capable of unassisted catalytic hydrolysis of an unactivated amide bond. Documentation of rationally-designed catalytic antibody transformations of amide bonds have been limited to antibodies capable of activated amide hydrolysis (8), metallo-cofactor-assisted amide hydrolysis (20), and amide bond rearrangement (21). Also, naturally-occurring peptide hydrolysis by autoantibodies have been reported (22). It should be pointed out that in the last example the reported antibody activities were discovered rather than designed. Here we describe the first successful design of a catalytic antibody capable

REFERENCES:
patent: 5126258 (1992-06-01), Lerner et al.
Janda, et. al. (1988)Science 241, 1188-1191.
Gallacher, G, et. al. (1992) Biochem. J. 284, 675-680.

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