News and Publications

In Vivo and In Vitro Evolution of Proteins: From Catalysis to Gene Regulation

C. Barbas, R. Lerner, H. Almer, J. Anderson, R. Beachy,* R. Beerli, D. Burton,** B. Dreier, R. Fuller, R. Lewis, B. List, Q. Liu, C. Rader, K. Sakthivel, J. Saldana, D. Segal, D. Shabat, P. Steinberger, J. Sutton, F. Tanaka, J. Widhopf, G. Zhong

* Division of Plant Biology, TSRI
** Department of Immunology, TSRI

We are interested in the directed evolution of proteins as a route to producing therapeutic proteins and understanding the principles of molecular recognition and catalysis. To explore this area, we have developed filamentous phage display as a format for molecular evolution. The surface of the filamentous phage physically links the genotype and phenotype of the protein under study. This linkage is necessary for studies that go beyond nucleic acid libraries in which genotype and phenotype may be encoded and expressed by the same molecule. Three areas are currently being examined: catalytic antibodies, therapeutic human antibodies, and zinc finger recognition of DNA and RNA.

Using the concept of reactive immunization, we produced monoclonal antibodies to a 1,3-dicarbonyl hapten designed to act as a chemical and entropic trap. The hapten selects for antibodies with a lysine residue in the active site by forming a covalently bound enaminone. This reaction mechanism for the formation of the hapten-antibody complex was used to catalyze aldol reactions after a similar mechanism known from natural class I aldolase enzymes: formation of an enamine between the donor carbonyl compound and the /epsilonbdy/-amino group of the essential lysine residue in the binding pocket of the antibody is followed by the nucleophilic attack of this enamine on the carbonyl of the acceptor substrate and finally hydrolytic release of the aldol product.

We found antibodies that catalyze aldol as well as retro-aldol reactions of a wide variety of aliphatic open-chain and aliphatic cyclic ketones and various aromatic and aliphatic aldehydes. More than 100 different substrate combinations have been studied. We have now shown the efficient asymmetric synthesis and resolution of a variety of molecules, including tertiary and fluorinated aldols and a variety of natural products (Fig. 1). A remarkable example is the enantioselective formation of the Wieland Miescher ketone. Typical values for k_cat range from 10^-3/min to 10/min and show a ratio of k_cat/k_uncat of 10⁵ to 10⁸. Moreover, the same antibodies catalyze the decarboxylation of ß-keto acids by using the lysine residue and thus mimic the natural enzyme acetoacetate decarboxylase.

Structural and chemical characterization of these catalysts has revealed the mechanism whereby both scope and catalytic efficiency can be selected. Using one of our antibody catalysts, we created a plethora of enamine-forming antibodies and are evolving the antibodies toward ever greater efficiency. This past year we also learned how to extend the complex covalent chemistry of proteins to nucleic acids and created the first functionalized DNA enzymes. Further study of these catalysts is providing insight into the evolution and diversification of natural enzymes.

The ability to manipulate large libraries of human antibodies has immediate implications for the development of therapeutic antibodies. We have been characterizing the antibody response induced by infection with HIV type 1 (HIV-1). One of our goals is to create a potent therapeutic cocktail of antibodies specific for HIV-1 that can inhibit mother-to-child transmission of the virus and, in combination with existing HIV treatments, have a therapeutic effect on infected patients. We intend to translate our knowledge of HIV neutralization into new vaccine strategies. Our most successful antibody, IgG1-b12, can protect animals from primary challenge with HIV-1. In vitro evolution strategies have been used to increase the binding and neutralization activity of this antibody. Coupling laboratory-evolved antibodies with potent toxins, we showed that immunotoxins can effectively kill infected cells. We hope to extend this observation to new therapeutic regimens to rid the body of infected cells.

The discovery of a second class of targets for HIV-1 neutralization, a subset of the chemokine receptor family, led to a new program. We developed antibodies that bind to the HIV-1--permissive chemokine receptors and block viral entry into the cell. The strategy we used includes selection of antibodies against cells expressing one or more of the coreceptors. We hope that the design and development of antibodies to these chemokine receptors will lead to a better understanding of the process involved in viral entry.

Using our increased understanding of antibody-antigen interactions, we extended our therapeutic efforts to cancer and developed rapid methods for creating human antibodies from antibodies derived from other species. In the past year, we produced human antibodies that should cause the selective starvation of a wide variety of cancers by inhibiting angiogenesis and antibodies that will be used to deliver radioisotopes to colon cancers to destroy the tumors. We hope to see some of these antibodies used in clinical trials by our collaborators at Memorial Sloan-Kettering Cancer Center in New York City in the next few years.

Our third area of investigation involves the selection of novel zinc finger DNA-binding proteins. Zinc finger proteins are particularly well suited for this purpose because of their modularity and well-defined structural features. Each finger forms an independently folded domain that typically recognizes 3 nucleotides of DNA. We showed that proteins can be selected or designed that contain zinc fingers that recognize novel DNA sequences. These studies are aiding the elucidation of rules for sequence-specific recognition within this family of proteins. To this end, we are selecting and designing specific zinc finger domains that constitute an alphabet of 64 domains that will allow any DNA sequence to be bound selectively (Fig. 2). The prospects for this "second genetic code" are fascinating, and the code should have a major impact on basic and applied biology.

Last year, we showed that selected zinc fingers can be appropriately linked to form polydactyl proteins capable of recognizing an 18-nucleotide site and thus can target a unique locus in the human genome. These proteins bind with subnanomolar affinity and are highly specific in cell culture assays. The attachment of a nuclear localization signal and effector domains allowed repression of targeted genes and, unlike the situation with other antisense or antigene targeting methods, activation of targeted genes.

These results suggest that zinc finger proteins might be useful as genetic regulators for a variety of human aliments. The goal of this work is to develop a new class of therapeutic proteins that inhibit or enhance the synthesis of proteins and provide a new strategy for fighting diseases of either somatic or viral origin. We are developing proteins that will inhibit the growth of tumors and others that will inhibit the expression of a protein known as CCR5, which is a key to infection of human cells by HIV-1. This past year, we showed that our alphabet of proteins can be used to specifically regulate the gene for CCR5; genes important in cancer, such as erbB-2 (Fig. 2); and the genes for the ß₃ integrins. In collaboration with R. Beachy, Department of Plant Biology, we are extending this work to create plants that are disease resistant and produce higher yields.

PUBLICATIONS

Barbas, C.F. III, Heine, A., Zhong, G., Hoffmann, T., Gramatikova, S., Björnestedt, R., List, B., Anderson, J., Stura, E.A., Wilson, E.A., Lerner, R.A. Immune versus natural selection: Antibody aldolases with enzymic rates but broader scope. Science 278:2085, 1997.

Barbas, C.F. III, List, B. Alchemy, enzymes, and the blind watchmaker. Nature Biotechnol. 16:423, 1998.

Bera, T.P., Kennedy, P.E., Berger, E.A., Barbas, C.F. III, Pastan, I. Specific killing of HIV infected lymphocytes by a recombinant immunotoxin directed against the HIV-1 envelope glycoprotein. Mol. Med., in press.

Finn, M.G., Lerner, R.A., Barbas, C.F. III. Cofactor induced refinement of catalytic antibody activity: A metal-specific allosteric effect. J. Am. Chem. Soc. 120:2963, 1998.

Gauduin, M.-C., Parren, P.W.H.I., Weir, R., Allaway, G.P., Maddon, P.J., Barbas, C.F. III, Burton, D.R., Koup, R.A. Passive immunization with a human monoclonal antibody protects hu-PBL-SCID mice against challenge by primary isolates of HIV-1. Nature Med. 3:1389, 1997.

Hoffmann, T., Zhong, G., List, B., Shabat, D., Anderson, J., Gramatikova, S., Lerner, R.A., Barbas, C.F. III. Aldolase antibodies of remarkable scope. J. Am. Chem. Soc. 120:2768, 1998.

Li, A., Baba, T.W., Sodroski, J., Zolla-Pazner, S., Gorny, M.K., Robinson, J., Posner, M.R., Katinger, H., Barbas, C.F. III, Burton, D.R., Chou, T.-C., Ruprecht, R.M. Synergistic neutralization of a chimeric SIV/HIV-1 virus with combinations of human anti-HIV-1 envelope monoclonal antibodies or hyperimmune globulins. AIDS Res. Hum. Retroviruses 13:647, 1997.

Lin, C.-H., Hoffmann, T.Z., Wirsching, P., Barbas, C.F. III, Janda, K.D., Lerner, R.A. On roads not taken in the evolution of protein catalysts: antibody steroid isomerases that use the enamine mechanism. Proc. Natl. Acad. Sci. U.S.A. 94:11773, 1997.

Lin, E.C.K., Ratnikov, B.I., Tsai, P.M., Carron, C.P., Myers, D.M., Barbas, C.F. III, Smith, J.W. Identification of a region in the integrin ß₃ subunit that confers ligand binding specificity. J. Biol. Chem. 272:23912, 1997.

List, B., Shabat, D., Barbas, C.F. III, Lerner, R.A. Enantioselective total synthesis of some brevicomins using aldolase antibody 38C2. Chem. Eur. J. 4:881, 1998.

Mo, H., Stamatatos, L., Ip, J.E., Barbas, C.F. III, Parren, P.W.H.I., Burton, D.R., Moore, J.P., Ho, D.D. Human immunodeficiency virus type 1 mutants that escape neutralization by human monoclonal antibody IgG1b12. J. Virol. 71:6869, 1997.

Rader, C., Barbas, C.F. III. Phage display of combinatorial antibody libraries. Curr. Opin. Biotechnol. 8:503, 1997.

Rader, C., Cheresh, D., Barbas, C.F. III. Phage display approach for rapid antibody humanization: Designed combinatorial V gene libraries. Proc. Natl. Acad. Sci. U.S.A., in press.

Sakthivel, K., Barbas, C.F. III. Expanding the potential of DNA for binding and catalysis: Delineation of a class of highly functionalized dUTP derivatives that are substrates for thermostable DNA polymerases. Angew. Chem., in press.

Segal, D.J., Dreier, B., Beerli, R.R., Barbas, C.F. III. Towards controlling gene expression at will: Selection and design of zinc finger domains specifically recognizing each of the 5´-GNN-3´ DNA target sequences. Proc. Natl. Acad. Sci. U.S.A., in press.

Shulman, A., Shabat, D., Barbas, C.F. III, Keinan, F.J. Teaching catalytic antibodies to undergraduate students: An organic chemistry lab experiment. Chem. Educ., in press.

Sillivan, N., Sun, Y., Binley, J., Lee, J., Barbas, C.F. III, Parren, P.W.H.I., Burton, D.R., Sodroski, J. Determinants of human immunodeficiency virus type 1 envelope glycoprotein activation by soluble CD4 and monoclonal antibodies. J. Virol. 72:6332, 1998.

Wong, N.C., Mueller, B.M., Barbas, C.F., Ruminski, P., Quaranta, V., Lin, E.C.K., Smith, J.W. /alphapub/_V Integrins mediate adhesion and migration of breast carcinoma cell lines. Clin. Exp. Metastasis 16:50, 1998.

Zhong, G., Hoffmann, T., Lerner, R.A., Danishefsky, S., Barbas, C.F. III. Antibody catalyzed enantioselective Robinson annulation. J. Am. Chem. Soc. 119:8131, 1997.

Zhong, G., Shabat, D., List, B., Anderson, J., Sinha, S.C., Lerner, R.A., Barbas, C.F. III. Catalytic enantioselective retro-aldol reactions: Kinetic resolution of ß-hydroxyketones using aldolase antibodies. Angew. Chem., in press.