Lloyd J. Old
Ludwig Institute for Cancer Research, New York, NY

Improving Antibodies by Evolution and Engineering

Tumor targeting put very high demands on antibodies with respect to affinity, specificity but also stability of the protein. Recent advances in the design of fully synthetic antibody libraries (9) make it now possible to obtain such antibodies. Using in vitro selection and evolution tools such as ribosome display (1-5), it has been possible to select and further improve antibodies by directed evolution totally in vitro, without the use of any cells. Picomolar affinities have been routinely obtained (2-5), and very high selectivity even to targets that are totally non-immunogenic, such as the telomeric DNA.

Using an antibody against EpCAM as a model system (6-8), the application of these technologies to tumor targeting will be discussed. An optimization of stability, valency and molecular weight has been carried out. Recently, the information gathered from directed evolution could be used in structured based engineering to complement directed evolution. Recent results on tumor targeting with these molecules will be discussed.

References:

1. J. Hanes and A. Plückthun, In vitro selection and evolution of functional proteins using ribosome display, Proc. Natl. Acad. Sci. 94, 4937-4942 (1997).

2. J. Hanes, L. Jermutus, S. Weber-Bornhauser, H. R. Bosshard and A. Plückthun, Ribosome display efficiently selects and evolves high-affinity antibodies in vitro from immune libraries, Proc. Natl. Acad. Sci 95,14130-14135 (1998).

3. J. Hanes, C. Schaffitzel, A. Knappik and A. Plückthun, Anti-insulin-antibodies from a naive library of designed fully synthetic genes were selected and evolved to picomolar affinities by using ribo-some display, Nature Biotechnology 18, 1288-1292 (2000).

4. L. Jermutus, A. Honegger, F. Schwesinger, J. Hanes and A. Plückthun, Tailoring in vitro evolution for protein activity or stability, Proc. Natl. Acad. Sci. USA 98, 75-80 (2001).

5. C. Schaffitzel, I. Berger, J. Postberg, J. Hanes, H. J. Lipps and A. Plückthun, In vitro generated antibodies specific for telomeric guanine-quadruplex DNA react with Stylonychia lemnae macronuclei, Proc. Natl. Acad. Sci. USA 98, 8572-8577 (2001).

6. R. Waibel, R. Alberto, J. Willuda, R. Finnern, R. Schibli, U. Abram, A. Egli, J. P. Mach, A. Plückthun and P. A. Schubiger, Stable One-Step Technetium-99m Labeling of His-tagged Proteins with a Novel Tc-Carbonyl Complex, Nature Biotechnology 17, 897-901.

7. J. Willuda, A. Honegger, R. Waibel, P. A. Schubiger, R. Stahel, U. Zangemeister-Wittke and A. Plückthun, High thermal stability is essential for tumor targeting of antibody fragments: engineering of a humanized anti-EGP-2 (Ep-CAM) scFv fragment, Cancer Res. 59, 5758-5767 (1999).

8. J. Willuda, S. Kubetzko, R. Waibel, P. A. Schubiger, U. Zangemeister Wittke and A. Plückthun, Tumor targeting of mono-, di- and tetravalent anti-p185HER-2 miniantibodies multimerized by self-associating peptides, J. Biol. Chem. 276, 14385-14392 (2001).

9. A. Knappik, L. Ge, A. Honegger, P. Pack, M. Fischer, G. Wellnhofer, A. Hoess, J. Wölle, A. Plückthun and B. Virnekäs, Fully synthetic Human Combinatorial Antibody libraries (HuCAL) based on modular consensus frameworks and CDRs randomized with trinucleotides, J. Mol. Biol. 296, 57-86 (2000).