Monoclonal antibodies selectively bind tumor cell differentiation antigens in vitro and in vivo. We have devised methods of linking extremely toxic proteins to the antibodies to selectively kill tumor cells by cloning the toxins and mutating them to decrease non-target cell toxicity. Some designs using a point mutant of diphtheria toxin allow 200,000 times more toxicity to target tumor cells than nontarget cells. The transferrin receptor is particularly attractive as a target on brain tumors because it is very low or absent on neurons and glial cells and because it is highly expressed on many different types of brain tumor. In phase I and phase II clinical trials of transferrin linked to a mutant diphtheria toxin many patients showed a significant decrease in brain tumor size in response to the treatment. The dose limiting toxicity in patients appeared to be vascular damage. In animal models we have explored a new strategy to prevent this vascular toxicity by systemic delivery of chloroquine to protect the brain vasculature but not protect the brain tumor cells for the immunotoxin. We have also developed immunotoxins for treatment of dystonia and other muscle spasm diseases by targeting ricin to muscle cells. One limitation of these approaches for systemic therapy and perhaps also for brain cancer is that patients can develop an immune response to the toxin. Therefore we have explored ways to use human proteins that may be nonimmunogenic to initiate tumor cell death. Human cytotoxic proteins such as the eosinophil derived neurotoxin have been engineered and linked to antibodies and found to specifically kill tumor cells. Toxic proteins in the Bcl-2 family, Bax and Bad, that induce apoptosis, have also been engineered to bind cells and specifically kill them. As Bax and Bad are members of the Bcl-2 family of proteins that also includes anti-apoptotic members we sought to develop a new strategy to prevent neuron loss by targeting Bcl-xl to cells. Recent results show that Bcl-xl can be delivered to cells in vitro and in vivo to prevent neuron apoptosis and may have potential for inhibiting neuron loss during spinal cord injury and stroke.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Intramural Research (Z01)
Project #
1Z01NS002674-16
Application #
6432897
Study Section
(SNB)
Project Start
Project End
Budget Start
Budget End
Support Year
16
Fiscal Year
2000
Total Cost
Indirect Cost
City
State
Country
United States
Zip Code
Antignani, Antonella; Youle, Richard J (2007) The cytokine, granulocyte-macrophage colony-stimulating factor (GM-CSF), can deliver Bcl-XL as an extracellular fusion protein to protect cells from apoptosis and retain differentiation induction. J Biol Chem 282:11246-54
Youle, Richard J (2007) Cell biology. Cellular demolition and the rules of engagement. Science 315:776-7
Antignani, Antonella; Youle, Richard J (2005) A chimeric protein induces tumor cell apoptosis by delivering the human Bcl-2 family BH3-only protein Bad. Biochemistry 44:4074-82
Christiansen, Stephen P; Peterson, Daniel; To, Thu et al. (2002) Long-term effects of ricin-mAb 35 on extraocular muscles of rabbits: potential treatment for strabismus. Invest Ophthalmol Vis Sci 43:679-85
Ichinose, Makoto; Liu, Xiu-Huai; Hagihara, Naoshi et al. (2002) Extracellular Bad fused to toxin transport domains induces apoptosis. Cancer Res 62:1433-8
Nechushtan, A; Smith, C L; Lamensdorf, I et al. (2001) Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol 153:1265-76
Leonidas, D D; Boix, E; Prill, R et al. (2001) Mapping the ribonucleolytic active site of eosinophil-derived neurotoxin (EDN). High resolution crystal structures of EDN complexes with adenylic nucleotide inhibitors. J Biol Chem 276:15009-17
Liu, X H; Collier, R J; Youle, R J (2001) Inhibition of axotomy-induced neuronal apoptosis by extracellular delivery of a Bcl-XL fusion protein. J Biol Chem 276:46326-32
Richter, B W; Mir, S S; Eiben, L J et al. (2001) Molecular cloning of ILP-2, a novel member of the inhibitor of apoptosis protein family. Mol Cell Biol 21:4292-301
Frank, S; Gaume, B; Bergmann-Leitner, E S et al. (2001) The role of dynamin-related protein 1, a mediator of mitochondrial fission, in apoptosis. Dev Cell 1:515-25

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