News and Publications

New Vistas in Immunopharmacotherapy

In Vitro Design of Human Antibody Libraries

Phage display antibody technology is a powerful method for the design and discovery of antibodies that bind targets with high specificity and affinity. Because the use of combinatorial antibody libraries displayed on the surface of phage particles is an entirely in vitro procedure, it obviates animal-, labor-, and time-intensive immunization and hybridoma protocols required to isolate monoclonal antibodies. We extended the usefulness of phage display methods, most notably by developing fully human single-chain Fv (scFv) and Fab antibody library formats displayed on phage. These libraries allow the direct selection of antigen-specific, fully human antibodies that can be moved more expeditiously into clinical trials.

The phage coat proteins pIII and pVIII have been used extensively for the display of antibody libraries; pIII in particular has been used to select numerous highly specific human antibodies, which continue to be the focus of a great deal of research in biotechnology and medicine. We expanded the sphere of phage display antibody technology to develop antibody libraries that are combinatorial not just in composition but also in conformational space. We accomplished this step by using pVII and pIX, which are constitutively separate but physically near each other, for the simultaneous display of scFv variable heavy-chain region (V_H) and variable light-chain region (V_L) chains. This past year, we extended the usefulness of in vitro antibody library design by developing a novel phage display format in which only the pIX coat protein is used. In our model studies, this display system allowed the selection of highly specific antibodies that recognized a number of complex antigens (e.g., cholera toxin and ricin) important in both medicine and defense against bioterrorism. We are investigating the scope and advantages of this new antibody display technology as an adjunct and alternative to existing methods.

In parallel with our work on the basic science of antibody phage display technology, we continue to explore new biomedical applications for in vitro antibody libraries. For example, we used an in vitro human scFv antibody library to select for antibodies that not only specifically bound to but were internalized into cancer cells. This finding is clinically relevant, because many of the side effects of cancer chemotherapy stem from the inherent nonspecificity of the cytotoxic drugs used to kill cancer cells. By attaching such drugs to cancer cell-specific, internalizing antibodies, the deleterious side effects of many of these chemotherapeutic agents could be attenuated, improving the prognoses for cancer patients.

Although attaching a single drug molecule to a cancer cell-specific, internalizing antibody clearly is of therapeutic interest, we prepared dendritic carriers for attaching multiple drug copies to a single scFv antibody (Fig. 1). Through the use of these hybrid macromolecules, which harness the exquisite specificity and drug delivery potential of these antibodies to the potent toxicity of synthetic chemotherapeutic dendrimers, the outlook for cancer chemotherapy in the coming years could be all the brighter.

Vaccination For Septic Shock

Septic shock is 1 of the 10 leading causes of infant and adult mortality in the United States. According to the Centers for Disease Control and Prevention, this condition was directly linked to more than 30,000 deaths in 1999 alone. The prime pathogenic determinant of septic shock is lipopolysaccharide, a class of oligosaccharides liberated by lysis of bacteria that have multiple O-linked fatty acyl substituents. The Escherichia coli isoform of lipid A is the most well-studied form of lipopolysaccharide and is responsible for the toxic effects associated with most documented cases of septic shock. One promising therapeutic tactic is use of a passive vaccine, that is, monoclonal antibodies that could, in principle, be used to bind lipid A and mediate its clearance from the body during instances of infection. Although this strategy showed promise at an early stage of research, it was not effective in clinical trials, underscoring the need for alternative therapeutic strategies.

We developed an immunotherapeutic approach for treatment of septic shock that involves the use of an active vaccine, a designed synthetic variant of lipid A (compound 1 in Fig. 2), that could induce an innate immune response to future exposure to lipopolysaccharide, thereby abrogating the toxic effects. We designed a structural homolog of E coli lipid A with 2 key features. First, a bisphosphonate transition-state analog (TSA) motif, a mimic for hydroxide-catalyzed ester hydrolysis, was incorporated in place of the 2 acyloxyacyl motifs present in the native lipid A structure. This modified structure was used to elicit antibodies that not only bound lipid A but also catalytically deactivated it through catalysis of base-catalyzed fatty ester hydrolysis. Second, 2 different immunostimulatory linkers were used to attach the final TSA structure to a carrier protein for immunization. These linkers were included to circumvent the notoriously low immunogenicity of carbohydrate-based antigens, which would otherwise have severely hampered the selection of a lipid A-specific antibody binder or catalyst.

Immunization of 3 different mouse strains with compounds 2a and 2b (Fig. 2), as conjugates to the carrier protein keyhole limpet hemocyanin (KLH), led to quantitatively different immune responses, suggesting that the choice of linker directly affected the immunogenicity of the TSA structure. These differences notwithstanding, the obtained antibodies were qualitatively similar, as evinced by significant cross-reactivity of the antibodies elicited to immunogens 2a and 2b.

To investigate the protective effect of the TSA conjugates, we gave the immunized mice sublethal bolus doses of lipid A. We measured the efficacy of the active vaccine by using the known upregulatory effect of lipid A on the production of TNF-α. Normalizing to TNF-α production in control mice not immunized with KLH conjugates of 2a or 2b, we observed between a 4- to 20-fold reduction in TNF-α production (depending on the mouse strain) after lipid A challenge in mice protected by previous immunization with KLH conjugates of 2a or 2b.

It is unclear as yet whether this immunoprotection against the toxic effects of E coli lipid A is due to the activity of antibody catalysts or binders. Nonetheless, these results indicate the therapeutic potential of rationally designed phosphonate TSAs for harnessing the native immune system for immunotherapy of septic shock in an active vaccine program. We are isolating and characterizing the responsible antibodies for mechanistic characterization as quick-acting therapeutic entities themselves in a complementary passive vaccine approach.

Immunopharmacotherapy For Nicotine Abuse

Cigarette smoking is still the leading cause of avoidable death due to cancer worldwide; as such, it has the unique distinction of being an elective malady of pandemic proportions. Increasingly, evidence suggests that chronic use of cigarettes and other tobacco products is due in large part to the addictive psychoactive and reinforcing effects of nicotine on the brain. Although so-called nicotine replacement therapies have grown in popularity in recent years, these are not true therapies per se; rather they are merely alternative forms of nicotine delivery. Because these strategies do not attack the underlying problem, the neurostimulatory effects of nicotine and concomitant user dependence on it, they have done little to decrease tobacco dependence on the whole.

In recent years, we and other researchers have explored immunopharmacotherapy as an alternative route to tackle the problem of chronic nicotine dependence. Several different approaches have been used to elicit antibodies that bind and thereby block nicotine from exerting its neurostimulatory effects on the brain. Although the structure of nicotine does not suggest that it poses any significant difficulties in the generation of antibody binders, all synthetic nicotine haptens prepared thus far have unexplainably fallen short of their promise. This situation led us to explore the possibility of some fundamental, yet hidden, chemical foundation for this problem.

Recently, new data indicated that in aqueous solution, nicotine exists in multiple conformations arising from rotation about the pyrrolidine-pyridine linkage axis. This finding is of key relevance for our research because it suggests a possible rationale for the unvaryingly low affinity of nicotine-binding antibodies, irrespective of protein conjugation chemistry. Whereas we and others viewed nicotine as a static, torsionally constrained 2-dimensional molecule, the immune system constantly samples all accessible solution-phase conformations. In exchange for high specificity and affinity, immunization with nicotine conjugates prepared to date--by us as well as by others--inevitably leads to a heterogenous antibody response. The isolated antibodies are characterized by low conformational specificity and an affinity that is averaged over a broad sample space of nicotine conformers.

We surmised that the solution-phase conformations of nicotine might be partly responsible for our otherwise unexplainably low success in generating nicotine-binding antibodies with our first-generation nicotine hapten (compound 3 in Fig. 3). Therefore, we prepared conformationally constrained nicotine analogs 4 and 5 (Fig. 3) for immunization to elicit improved antibody binders. The haptens were attached to KLH by using the same linkage chemistry previously used for 3 to allow meaningful side-by-side comparison. We were gratified to find that the serum antibody titers resulting from immunization with haptens 4 and 5 were 7- to 8-fold higher than those obtained with the conformationally free hapten 3.

We are isolating and characterizing the affinity of monoclonal antibodies generated in response to these conformationally constrained nicotine haptens. We envision that these antibodies could be useful in improving the efficacy of immunopharmacotherapy strategies as treatments for nicotine addiction.

Publications

Ahn, J.-M., Wentworth, P., Jr., Janda, K.D. Soluble polymer-supported convergent parallel library synthesis. Chem. Commun. (Camb.) 4:480, 2003.

Cannizzaro, C.E., Ashley, J.A., Janda, K.D., Houk, K.N. Experimental determination of the absolute enantioselectivity of an antibody-catalyzed Diels-Alder reaction and theoretical explorations of the origins of stereoselectivity. J. Am. Chem. Soc. 125:2489, 2003.

Clapham, B., Lee, S.-H., Koch, G., Zimmermann, J., Janda, K.D. The preparation of polymer bound ß-ketoesters and their conversion into an array of oxazoles. Tetrahedron Lett. 43:5407, 2002.

Delgado, M., Janda, K.D. Polymeric supports for solid phase organic synthesis. Curr. Org. Chem. 6:1031, 2002.

Dickerson, T.J., Janda, K.D. A previously undescribed chemical link between smoking and metabolic disease. Proc. Natl. Acad. Sci. U. S. A. 99:15084, 2002.

Dickerson, T.J., Janda, K.D. Glycation of the amyloid ß-protein by a nicotine metabolite: a fortuitous chemical dynamic between smoking and Alzheimer's disease. Proc. Natl. Acad. Sci. U. S. A. 100:8182, 2003.

Dickerson, T.J., Reed, N.N., Janda, K.D. Soluble polymers as scaffolds for recoverable catalysts and reagents. Chem. Rev. 102:3325, 2002.

Gambs, C., Dickerson, T.J., Mahajan, S., Pasternack, L.B., Janda, K.D. High-resolution diffusion-ordered spectroscopy to probe the microenvironment of JandaJel and Merrifield resins. J. Org. Chem. 68:3673, 2003.

Gao, C., Mao, S., Ditzel, H.J., Farnaes, L., Wirsching, P., Lerner, R.A., Janda, K.D. A cell-penetrating peptide from a novel pVII-pIX phage-displayed random peptide library. Bioorg. Med. Chem. 10:4057, 2002.

Gao, C., Mao, S., Kaufmann, G., Wirsching, P., Lerner, R.A., Janda, K.D. A method for the generation of combinatorial antibody libraries using pIX phage display. Proc. Natl. Acad. Sci. U. S. A. 99:12612, 2002.

Gao, C., Mao, S., Ronca, F., Zhuang, S., Quaranta, V., Wirsching, P., Janda, K.D. De novo identification of tumor-specific internalizing human antibody-receptor pairs by phage-display methods. J. Immunol. Methods 274:185, 2003.

Jones, L.H., Altobell, L.J. III, MacDonald, M.T., Boyle, N.A., Wentworth, P., Jr., Lerner, R.A., Janda, K.D. Active immunization with a glycolipid transition state analogue protects against endotoxic shock. Angew. Chem. Int. Ed. 41:4241, 2002.

Lee, K.J., Mao, S., Sun, C., Gao, C., Blixt, O., Arrues, S., Hom, L.G., Kaufmann, G.F., Hoffman, T.Z., Coyle, A.R., Paulson, J., Felding-Habermann, B., Janda, K.D. Phage-display selection of a human single-chain Fv antibody highly specific for melanoma and breast cancer cells using a chemoenzymatically synthesized G_M3-carbohydrate antigen. J. Am. Chem. Soc. 124:12439, 2002.

Lee, S.-H., Clapham, B., Koch, G., Zimmermann, J., Janda, K.D. Rhodium carbenoid N-H insertion reactions of primary ureas: solution and solid-phase synthesis of imidazolones. Org. Lett. 5:511, 2003.

Liu, C., Sun, C., Huang, H., Janda, K., Edgington, T. Overexpression of legumain in tumors is significant for invasion/metastasis and a candidate enzymatic target for prodrug therapy. Cancer Res. 63:2957, 2003.

Meijler, M.M., Matsushita, M., Altobell, L.J. III, Wirsching, P., Janda, K.D. A new strategy for improved nicotine vaccines using conformationally constrained haptens. J. Am. Chem. Soc. 125:7164, 2003.

Redwan, el-R.M., Larsen, N.A., Zhou, B., Wirsching, P., Janda, K.D., Wilson, I.A. Expression and characterization of a humanized cocaine-binding antibody. Biotechnol. Bioeng. 82:612, 2003.

Shimomura, O., Clapham, B., Spanka, C., Mahajan, S., Janda, K.D. Application of microgels as polymer supports for organic synthesis: preparation of a small phthalide library, a scavenger, and a borohydride reagent. J. Comb. Chem. 4:436, 2002.

Sun, C., Wirsching, P., Janda, K.D. Enabling scFvs as multi-drug carriers: a dendritic approach. Bioorg. Med. Chem. 11:1761, 2003.

Wang, Q., Raja, K.S., Janda, K.D., Lin, T., Finn, M.G. Blue fluorescent antibodies as reporters of steric accessibility in virus conjugates. Bioconjug. Chem. 14:38, 2003.

Wentworth, P., Jr., McDunn, J.E., Wentworth, A.D., Takeuchi, C., Nieva, J., Jones, T., Bautista, C., Ruedi, J.M., Gutierrez, A., Janda, K.D., Babior, B.M., Eschenmoser, A., Lerner, R.A. Evidence for antibody-catalyzed ozone formation in bacterial killing and inflammation. Science 298:2195, 2002.

Wentworth, P., Jr., Wentworth, A.D., Zhu, X., Wilson, I.A., Janda, K.D., Eschenmoser, A., Lerner, R.A. Evidence for the production of trioxygen species during antibody-catalyzed chemical modification of antigens. Proc. Natl. Acad. Sci. U. S. A. 100:1490, 2003.

Zhu, X.Y., Heine, A., Monnat, F., Houk, K.N., Janda, K.D., Wilson, I.A. Structural basis for antibody catalysis of a cationic cyclization reaction. J. Mol. Biol. 329:69, 2003.