In order to develop reagents for the systematic structure function analysis of IL-7, as well as the genetic construction of the IL-7R targeted fusion toxin DAB389 IL-7, we have designed and synthesized a gene encoding IL-7 using Escherichia coli condon usage bias. The synthetic IL-7 gene has been cloned in an expression vector and biologically active IL-7 has been expressed. The long term goals of this segment of the NCDDG application are to use an iterative process to develop molecular models of the tertiary structure of Il-7. We have used computational analysis of the secondary structure of Il-7, and then have used this information with the X-ray crystallographic data on two related cytokines (IL-4 and G-CSF) in order to construct molecular models of Il-7. Since these models differ considerably, we have been able to make structural predictions for each model. The determination of the disulfide bond assignment in recombinant Il-7 will allow us to pick one of these models for further structural refinement. We shall conduct site-directed mutagenesis studies in order to resolve those regions of Il-7 that re involved with receptor binding and signal transduction. We anticipate the isolation nd characterization of mutant forms of Il-7 that will be receptor antagonists, and further anticipate that these mutants will serve as candidate compounds for the treatment of many of the acute leukemias. In addition to the structure function analysis of native IL-7, we have also designed, constructed, expressed and purified, a novel fusion toxin (DAB389IL-7) that is targeted toward the IL-7 receptor. This fusion toxin is highly receptor specific and potent and will serve as the prototype of an IL-7R targeted cytotoxic agent.
The specific aims of this component of the NCDDG are to produce these and other mutant forms of Il-7 and DAB389IL-7 for experimental use in experiments proposed in the subsequent sections of this application by Drs. Foss, Rich, and Waters.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Research Project (R01)
Project #
5R01CA048626-11
Application #
6347316
Study Section
Project Start
2000-09-01
Project End
2002-08-31
Budget Start
1997-10-01
Budget End
1998-09-30
Support Year
11
Fiscal Year
2000
Total Cost
$79,323
Indirect Cost
Name
Boston Medical Center
Department
Type
DUNS #
005492160
City
Boston
State
MA
Country
United States
Zip Code
02118
Cosenza, Larry; Gorgun, Gullu; Urbano, Alexander et al. (2002) Interleukin-7 receptor expression and activation in nonhaematopoietic neoplastic cell lines. Cell Signal 14:317-25
vanderSpek, Johanna C; Sutherland, John A; Gill, Brian M et al. (2002) Structure function analysis of interleukin 7: requirement for an aromatic ring at position 143 of helix D. Cytokine 17:227-33
Gorgun, Gullu; van der Spek, Johanna; Cosenza, Larry et al. (2002) Altered biological activity associated with C-terminal modifications of IL-7. Cytokine 20:17-22
Sweeney, E B; Foss, F M; Murphy, J R et al. (1998) Interleukin 7 (IL-7) receptor-specific cell killing by DAB389 IL-7: a novel agent for the elimination of IL-7 receptor positive cells. Bioconjug Chem 9:201-7
Kelley, V R; Strom, T B (1995) A paradigm 1994: the allograft response. Blood Purif 13:199-205
Kelley, V R; Singer, G G (1993) The antigen presentation function of renal tubular epithelial cells. Exp Nephrol 1:102-11
Jevnikar, A M; Singer, G G; Coffman, T et al. (1993) Transgenic tubular cell expression of class II is insufficient to initiate immune renal injury. J Am Soc Nephrol 3:1972-7
Bloom, R D; Florquin, S; Singer, G G et al. (1993) Colony stimulating factor-1 in the induction of lupus nephritis. Kidney Int 43:1000-9
Singer, G G; Yokoyama, H; Bloom, R D et al. (1993) Stimulated renal tubular epithelial cells induce anergy in CD4+ T cells. Kidney Int 44:1030-5
Diaz-Gallo, C; Kelley, V R (1993) Self-regulation of autoreactive kidney-infiltrating T cells in MRL-lpr nephritis. Kidney Int 44:692-9

Showing the most recent 10 out of 29 publications