Method of promoting enzyme diversity

Details Method of promoting enzyme diversity Method of promoting enzyme diversity

1998-12-30

2001-06-05

Patterson, Jr., Charles L. (Department: 1652)

Chemistry: molecular biology and microbiology

Enzyme , proenzyme; compositions thereof; process for...

Hydrolase

C435S018000

Reexamination Certificate

active

06242236

ABSTRACT:

FIELD OF THE INVENTION
The present invention relates broadly to methods for creating novel catalysts, particularly high turnover rate enzymes or biocatalysts, using animals of altered germline sequence. More particularly, the invention relates to methods for promoting rapid evolution or diversification of the specificity regions of an enzyme. The method can be used to produce antibodies with catalytic activity by encoding a functional catalytic unit or a substantial portion thereof into a germline antibody gene locus. And even more particularly, the invention relates to the placement and segmentation of the enzymatic sequences within the antibody gene loci
BACKGROUND OF THE INVENTION
Over the last decade, research has been devoted to the production of useful antibodies with catalytic activity. Such antibodies are commonly known as catalytic antibodies or Abzymes. Catalytic antibodies with enhanced turnover rates as compared with uncatalyzed reaction rates have been reported by a number of research groups. (see Suzuki et al., “Recent advances in abzyme studies,” 115(4) Journal of Biochemistry 623-8(1994) which is incorporated herein by reference, Titmas et al., “Aspects of antibody catalyzed primary amide hydrolysis, 47(2-3) Applied Biochemistry & Biotechnology 277-90 (1994) which is incorporated herein by reference). See also U.S. Pat. Nos.5,037,750 and 5,156,965 to Schochetman and Massey, which are incorporated herein by reference, which teach a method for increasing the rate of chemical reactions involving the conversion of at least one reactant to at least one product.
In some instances, large rate enhancements over uncatalyzed reaction rates have been achieved (see Thom et al., “Large rate accelerations in antibody catalysis by strategic use of haptenic charge,” 373 Nature 228-30(1995) which is incorporated herein by reference). Thus far, however, a generic means has not been found to employ traditional laboratory techniques to produce highly efficient specific catalysis with catalytic antibodies.
The traditional laboratory techniques for producing a catalytic antibody is through the use of transition state analogs (TSA) (see U.S. Pat. No. 4,792,446 to Kim and Kallenbach, which is incorporated herein by reference). The TSA is a stable mimic of the unstable intermediate conformation of a reactant molecule. Animals are immunized with TSA's in the hopes of producing antibodies which by virtue of their ability to bind the TSA's may have the ability to stabilize the transition state of the reactant and to catalyze the formation of the desired product. Hybridomas are then screened by assaying for the desired catalytic activity.
The overwhelming problem that has plagued catalytic antibody development in the vast majority of cases is the lack of high turnover rates. Except in rare instances, catalytic antibodies are orders of magnitude slower catalysts than similar enzymes found in nature.
Recently, however, selection methods have been used to produce catalytic antibodies (see Smiley et al., “Selection of catalytic antibodies for a biosynthetic reaction from a combinatorial cDNA library by complementation of an auxtrophic Escherichia coli: antibodies for orotate decarboxylation,” 91(18) Proceedings of the National Academy of Sciences of the United States of America 8319-23(1994), which is incorporated herein by reference; Janda et al., “Direct selection for a catalytic mechanism from combinatorial anyiboby libraries,” 91(7) Proceedings of the National Academy of Sciences of the United States of America 2532-6), which is incorporated herein by reference. Such methods use selection pressure to isolate antibodies with desired properties instead of the more laborious screening techniques. For example, a catalytic antibody which catalyzes the formation of a cellular growth factor may be selected for in a cell auxotrophic for such growth factor.
Advances in molecular and cell biology have given researchers the ability to alter the germ line genetic constitution of a variety of animals. Pieces of genes, whole genes, and/or chromosomal regions may be selectively deleted or added. Recently these transgenic/knock-out techniques have been used to produce human proteins in other species. In particular, it has been possible to produce human antibodies in rodents(see Green et al., “Antigen-specific human monoclonal antibodies from mice engineered with human Ig heavy and light chain YAC's” 7(1) Nature Genetics 13-21(1994), which is incorporated herein by reference; Lonberg et al., “Antigen-specific human antibodies from mice comprising four distinct genetic modifications,” 368(6474) Nature 856-9(1994), which is incorporated herein by reference; Taylor et al., “A transgenic mouse that expresses a diversity of human sequence heavy and light chain immunoglobulins,” 20-23 Nucleic Acids Research 6287-95 (1992), which is incorporated herein by reference). International applications WO 91/10741; WO 94/25585; and WO 92/03918 also discuss diversity of human sequence heavy and light chain immunoglobulins and are incorporated herein by reference. The ability to produce human antibodies in rodents allows for the controlled production of therapeutic antibodies of low immunogenicity.
In an extension of the traditional method of the catalytic antibody technique for increasing antibody binding affinity to enzymatically relevant conformations and co-factors, transgenic animals have been utilized to enhance the germline antibody metal ion binding capability. Metal ions are used as co-factors for various enzymes and transgenic mice were produced to more effectively bind metal cations (see Sarvetnick et al., 1993, “Increasing the chemical potential of the germ-line antibody repertoire.” 90(9) Proceedings of the National Academy of Sciences of the United States of America 4008-11, which is incorporated herein by reference; WO 94/25586, which is incorporated herein by reference).
In the present invention, rather than promoting binding, as has been done in past studies, applicant proposes integrating functional catalytic units into the germline composition of an animal such that the sequences encoding the specificity determining regions of the enzymatic activity are diversified in an analogous fashion to immunoglobulin variable regions. Such an approach solves the problem of low turnover rate or low catalytic activity.
SUMMARY OF THE INVENTION
Accordingly, the present invention provides a novel method for utilizing transgenic/knock-out germline altering techniques to implant sequences encoding functional catalytic domains and/or structures in the germline antibody sequence of an animal.
In accordance with one aspect of the present invention, such sequences encoding functional catalytic domains and/or structures are sequences of known enzymes with the desired catalytic function (e.g., hydrolysis of a peptide bond) but with specificity different from the desired catalytic antibody.
In still another aspect of the present invention. the sequences encoding the specificity regions of the implanted functional enzyme are inserted into the germline of the animal so as to correspond to the CDR regions of the antibody so as to promote rapid evolution/diversification of the specificity regions of the enzyme. The invention is also in the transgenic implantation of functional catalytic units or enzymes in other (non-antibody) chromosomal regions of high mutation rate (e.g., T cell receptor gene sequences).
And yet another aspect of the present invention the method provides for a varying array of chimeric antibody/enzyme structures. The placement and segmentation of the enzymatic sequence allows for the production of a multitude of antibody/enzyme structures.


REFERENCES:
patent: 4792446 (1988-12-01), Kim et al.
patent: 4963355 (1990-10-01), Kim et al.
patent: 5037750 (1991-08-01), Schochetman et al.
patent: 5156965 (1992-10-01), Schochetman et al.
patent: 5318897 (1994-06-01), Paul
patent: 5401641 (1995-03-01), Kim et al.
patent: 5914256 (1999-06-01), Wohlstadter
patent: 5919681 (1999-07-01), Wohlstadter
patent: WO

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