In targeting cancer, we take a multidisciplinary approach that involves gene regulation, catalytic antibodies, drug design, and combinatorial
antibody libraries. Using a combinatorial antibody strategy, this year we prepared high-affinity
antibodies that target key angiogenic receptors, and we engineered these antibodies to simultaneously
block 2 angiogenic pathways in mice to produce a strong therapeutic effect against melanoma (Fig.
Using catalytic antibodies, we are developing a strategy to activate drugs in a highly specific fashion at the site of cancer. We also advanced our chemotherapeutic approaches based on catalytic antibodies and prodrugs. Studies with chemically programmed antibodies continue to progress, providing routes to novel treatments of melanoma and breast and ovarian cancers.
Designer Transcription Factors
From the simplest to the most complex, proteins that bind nucleic acids control the expression of genes. The nucleic acids DNA and RNA are the molecules that store the recipes of all life forms. The fertilized egg of a human contains the genetic recipe for the development and differentiation of a single cell into 2 cells, 4 cells, and so on, finally yielding a complete individual. The coordinated expression or reading of the recipes for life allows cells containing the same genetic information to perform different functions and to have distinctly different physical characteristics. Proteins that bind nucleic acids enable this coordinated control of the genetic code. Lack of coordination due to genetic defects or to viral seizure of control of the cell results in disease.
In one project, we are developing methods to produce proteins that bind to specific DNA sequences to control specified genes. As we showed earlier, these proteins can be used as specific genetic switches to turn on or turn off genes on demand, creating an operating system for genomes. To this end, we selected and designed specific zinc finger domains that will constitute an alphabet of 64 domains that will allow any DNA sequence to be bound selectively. The prospects for this second genetic code are fascinating and should have a major impact on basic and applied biology.
This year we developed the CNN family of zinc finger domains. Together with the GNN and ANN domains we have already developed, billions of transcription factors can now be prepared. Our goal is to develop a new class of therapeutic proteins that inhibit or enhance the synthesis of proteins, providing a new strategy for fighting diseases of either somatic or viral origin.
Using a novel library of transcription
factors, we have developed a strategy that effectively allows us to turn on and turn off every gene
in the genome (Fig. 3).
With this powerful new strategy, we can quickly regulate a target gene or discover other genes key in disease. We are targeting a variety of other genes involved not only in cancer and HIV disease but also in genetic diseases such as sickle cell anemia. We hope to take this genetic strategy and our other molecular approaches all the way to clinical trials.
Alwin, S., Gere, M.B., Guhl, E., Effertz, K., Barbas, C.F. III, Segal, D.J., Weitzman, M.D., Cathomen, T. Custom zinc-finger nucleases for use in human cells. Mol. Ther. 12:610, 2005.
Amir, R.J., Popkov, M., Lerner, R.A., Barbas, C.F. III, Shabat, D. Prodrug activation gated by a molecular OR logic trigger. Angew. Chem. Int. Ed. 44:4378, 2005.
Blancafort, P., Chen, E.I., Gonzalez, B., Bergquist, S., Zijlstra, A., Guthy, D., Brachat, A., Brakenhoff, R.H., Quigley, J.P., Erdmann, D., Barbas, C.F. III. Genetic reprogramming of tumor cells by zinc finger transcription factors. Proc. Natl. Acad. Sci. U. S. A. 102:11716, 2005.
Blau C.A., Barbas, C.F. III, Bomhoff, A.L., Neades, R., Yan, J., Navas, P.A., Peterson. K.R. γ-Globin gene expression in chemical inducer of dimerization (CID)-dependent multipotential cells established from human β-globin locus yeast artificial chromosome (β-YAC) transgenic mice. J. Biol Chem. 280:36642, 2005.
Chen, E.I., Florens, L., Axelrod, F.T., Monosov, E., Barbas, C.F. III, Yates, J.R. III, Felding-Habermann, B., Smith, J.W. Maspin alters the carcinoma proteome. FASEB J. 19:1123, 2005.
Chowdari, N.S., Barbas. C.F. III. Total synthesis of LFA-1 antagonist BIRT-377 via organocatalytic asymmetric construction of a quaternary stereocenter. Org. Lett. 7:867, 2005.
Corte-Real, S., Collins, C., Aires da Silva, F., Simas, J.P., Barbas, C.F. III, Chang, Y., Moore, P., Goncalves, J. Intrabodies targeting the Kaposi sarcoma-associated herpesvirus latency antigen inhibit viral persistence in lymphoma cells. Blood 106:3797, 2005.
Crotty, J., Etzkorn, C., Barbas, C.F. III, Segal, D.J., Horton, N.C. Crystallographic analysis of Aart, a designed six-finger zinc finger peptide, bound to DNA. Acta Crystallogr. F61:573, 2005.
Dreier, B., Fuller, R.P., Segal, D.J., Lund, C., Blancafort, P., Huber, A., Koksch, B., Barbas, C.F. III. Development of zinc finger domains for recognition of the 5′ -CNN-3′ family DNA sequences and their use in the construction of artificial transcription factors. J. Biol. Chem. 280:35588, 2005.
Eberhardy, S.R., Goncalves, J., Coelho, S., Segal, D.J., Berkhout, B., Barbas, C.F. III. Inhibition of HIV-1 replication with artificial transcription factors targeting the highly conserved primer binding site. J. Virol., in press.
Gräslund, T., Li, X., Popkov, M., Barbas C.F. III. Exploring strategies for the design of artificial transcription factors: targeting sites proximal to known regulatory regions for the induction of γ-globin expression and the treatment of sickle cell disease. J. Biol. Chem. 280:3707, 2005.
Haba, K., Popkov, M., Shamis, M., Lerner, R.A., Barbas, C.F. III, Shabat, D. Single-triggered trimeric prodrugs. Angew. Chem. Int. Ed. 44:716, 2005.
Jendreyko, N., Popkov, M., Rader, C., Barbas, C.F. III. Phenotypic knockout of VEGF-R2 and Tie-2 with an intradiabody reduces tumor growth and angiogenesis in vivo. Proc. Natl. Acad. Sci. U. S. A. 102:8293, 2005.
Li, L.-S., Rader, C., Matsushita, M., Das, S., Barbas, C.F. III, Lerner, R.A., Sinha, S.C. Chemical adaptor immunotherapy: design, synthesis, and evaluation of novel integrin-targeting devices. J. Med. Chem. 47:5630, 2004.
Lund, C.V., Popkov, M., Magnenat, L., Barbas, C.F. III. Zinc finger transcription factors designed for bispecific coregulation of ErbB2 and ErbB3 receptors: insights into ErbB receptor biology. Mol. Cell. Biol. 25:9082, 2005.
Popkov, M., Jendreyko, N., McGavern, D., Rader, C., Barbas C.F. III. Targeting tumor angiogenesis with adenovirus-delivered anti-Tie-2 intrabody. Cancer Res. 65:972, 2005.
Popkov, M., Rader, C., Barbas C.F. III. Isolation of human prostate cancer reactive antibodies using phage display technology. J. Immunol. Methods 291:137, 2004.
Ramachary, D.B., Barbas, C.F. III. Direct amino acid-catalyzed asymmetric desymmetrization of meso-compounds: tandem aminoxylation/O-N bond heterolysis reactions. Org. Lett. 7:1577, 2005.
Steiner, D., Mase, N., Barbas. C.F. III. Direct asymmetric α-fluorination of aldehydes. Angew. Chem. Int. Ed. 44:3706, 2005.
Suri, J.T., Ramachary, D.B., Barbas. C.F. III. Mimicking dihydroxy acetone phosphate-utilizing aldolases through organocatalysis: a facile route to carbohydrates and aminosugars. Org. Lett. 7:1383, 2005.
Suri, J.T., Steiner, D.D., Barbas, C.F. III. Organocatalytic enantioselective synthesis of metabotropic glutamate receptor ligands. Org. Lett. 7:3885, 2005.
Tan, W., Dong, Z., Wilkinson, T.A., Barbas, C.F. III, Chow, S.A. Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc-finger protein E2C can catalyze site-specific integration in human cells. J. Virol., in press.
Tanaka, F., Barbas, C.F. III. Enamine-based reactions using organocatalysts: from aldolase antibodies to small amino acid and amine catalysts. J. Synth. Org. Chem. Jpn. 63:27, 2005.
Tanaka, F., Barbas, C.F. III. Organocatalytic approaches to enantioenriched β-amino acids. In: Enantioselective Synthesis of β-Amino Acids, 2nd ed. Juaristi, E. (Ed.). Wiley-VCH, New York, 2005, p. 195.
Tanaka, F., Barbas, C.F. III. Reactive immunization: a unique approach to aldolase antibodies. In: Catalytic Antibodies. Keinan, E. (Ed.). Wiley-VCH, New York, 2005, p. 305.
Tanaka, F., Fuller, R., Barbas, C.F. III. Development of small designer aldolase enzymes: catalytic activity, folding, and substrate specificity. Biochemistry 44:7583, 2005.
Tanaka, F., Fuller, R., Shim, H., Lerner, R.A., Barbas, C.F. III. Evolution of aldolase antibodies in vitro: correlation of catalytic activity and reaction-based selection. J. Mol. Biol. 335:1007, 2004.
Weinstain, R., Lerner, R.A., Barbas, C.F. III, Shabat, D. Antibody-catalyzed asymmetric intramolecular Michael addition of aldehydes and ketones to yield the disfavored cis-product. J. Am. Chem. Soc. 127:13104, 2005.