Cytokine engineering through ligand/receptor dynamics : a study on granulocyte colony-simulating factor

Granulocyte colony-stimulating factor (GCSF) is a cytokine of great clinical importance, with application to elevate neutrophil counts in cancer patients undergoing chemotherapy. GCSF binds to its specific receptor (GCSFR) on bone marrow precursor cells and generates intracellular signals that cause these cells to proliferate and differentiate into mature neutrophils. However, for such cytokines that act as agonists for cell-surface receptors, these complexes undergo trafficking processes that often attenuate the intracellular signals through internalization and degradation. Additionally for GCSF, the bloodstream neutrophils represent a further negative feedback mechanism that limits the potency of the drug, as they also express GCSFR and degrade the drug through receptor-mediated endocytosis. Using the GCSF/GCSFR system, we have developed an effective method for deconvoluting the effects of binding affinity on ligand potency, and this analysis successfully predicts the half-life ranking of combinatorially generated GCSF analogs in culture. Protein structural studies and equilibrium denaturation experiments unexpectedly revealed that GCSF becomes more stable as the pH decreases from 7 to 4 (coincident with the pH range experienced along the endocytic trafficking pathway); however, similar experiments performed on analogs from the deconvolution analysis were consistent with a proposed correlation between decreased structural stability at endosomal pH and enhanced ligand half-life.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2002

Author dc:creator

• Sarkar, Casim Ali, 1975-

Advisor dc:contributor.advisor

• Douglas A. Lauffenburger.

• Chemical Engineering.

Statement dc:rights

• M.I.T. theses are protected by copyright. They may be viewed from this source for any purpose, but reproduction or distribution in any format is prohibited without written permission. See provided URL for inquiries about permission.

Licence dc:rights.uri

• http://dspace.mit.edu/handle/1721.1/7582

Language dc:language.iso

eng

Handle dc:identifier.uri

http://hdl.handle.net/1721.1/17561

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/17561