Medical Student Research Fellowship for Summer 2007
Mentor: Drs. Charlie Liu and Larry Pop/ Dr. Ellen Vitetta
Department: Cancer Immunobiology Center
Project title: The preparation and evaluation of immunotoxins containing short lived recombinant anti-CD22 monoclonal antibodies
To evaluate immunotoxins constructed by conjugating recombinant ricin toxin A chain (rRTA) to chimeric recombinant anti-human CD22(cRFB4) monoclonal antibody (MAb) mutants which have short half-lives in vivo.
Antibody therapeutics represents a new class of immunotherapeutic drugs which have efficacy in a variety of diseases including cancer. By targeting these molecules directly to disease- causing cells, many of the side effects traditionally associated with chemotherapy can be avoided One such therapeutic, developed in our laboratory is called an immuonotoxin, which is a a hybrid protein prepared by conjugating a tumor- targeting antibody to the ribotoxic portion of a potent plant toxin called ricin. These immunotoxins have been tested in vitro, in mice, and in over 300 patients with cancer. Because their long half life in the body, however, the toxin portion of the molecule can cause side effects such as vascular leak syndrome. To avoid this problem we have genetically engineered the toxin to eliminate this activity. However, another approach that we wish to explore is whether we can eliminate their toxicity by decreasing their half life in the body. To this end, we have developed a panel of cRFB4 MAbs that have alterations in the portion of the antibody molecule that controls its half life in vivo. Thee ones with the shortest half livers would be predicted to have the least toxicity. However, it must be determined whether their anti-tumor activity will be preserved. The objective of this project will be to prepare immunotoxins from two of these mutants and to evaluate their activity in vitro and in vivo. Once this is completed, they will be tested in SCID mice with human lymphoma xenografts.
1. To prepare and purify the chimeric antibodies (cRFB4 wild-type, cRFB4-H310A and cRFB4-P247W) by chromatography.
2. To analyze their functional affinity by measuring binding to CD22-positive human tumor cells and their ability to inactivate ribosomes in a cell free system.
3. To measure their internalization into CD22- positive human tumor cells.
4.To determine their cytotoxic activity to CD22 positive human tumor cells.
5. To test their persistence in circulation by pharmacokinetics parameters.
6. To evaluate their diffusion into the tissues.
1. Pop, L.M., Liu, X.Y., Ghetie, V., and Vitetta, E.S. The generation of immunotoxins
using chimeric anti-CD22 antibodies containing mutations which alter their serum
half-life. Int. Immunopharmacol., 5(7-8):1279-1290, 2005.
2. Ghetie, V., and Vitetta, E.S. Chemical construction of immunotoxins. Mol. Biotechnology, 18 (3):251-268, 2001.
3. Firan, M., Bawdon, R., Radu, C., Ober, R.J., Eaken, D, Antohe, F., Ghetie, V., and Ward, E.S. The MHC class I-related receptor, FcRn, plays an essential role in the maternofetal transfer of gamma-globulin in humans. Int. Immunol., 13 (8):993-1002, 2001.
4. Carter, P. Improving the efficacy of antibody-based cancer therapies. Nat. Rev. Cancer, 1(2):118-129, 2001.
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