Isolation and engineering of a high affinity antibody against P-selectin glycoprotein ligand-1 (PSGL-1)

We aim to develop novel protein antagonists of P-selectin adhesion as anti- inflammatory therapeutics. Blocking P-selectin adhesion is particularly attractive because this adhesion mediates leukocyte rolling which occurs early in the inflammatory cascade before extensive tissue damage caused by the amplification of inflammation by proinflammatory cytokines. Currently, no subnanomolar antagonists of selectin adhesion are available. The low affinity of current antagonists results in the need for frequent administration and large doses in order to obtain inhibition. High affinity antagonists are desirable because they can be administered in smaller amounts thus reducing the risk of harmful side effects and reducing production costs. Our approach for developing high affinity antagonists is to combine error prone PCR and in vivo homologous recombination to mimic in yeast the broad spectrum of mutagenic strategies exploited by B cells such as somatic hypermutation, receptor revision (... CDR replacement), receptor editing (chain shuffling), and amino acid insertions and deletions. Together with yeast surface display and flow cytometric screening (FACS), this approach has been used to effect at least a five order of magnitude affinity improvement in a single chain antibody (scFv) directed against the N-terminal 19 amino acids of P-selectin glycoprotein ligand- 1 (PSGL- 1). Three rounds of engineering were performed after an initial pool of binders was isolated from a non-immune scFv library. Chain shuffling was found to be important for generating an improved mutant in the first round of engineering.

Department dc:contributor.department

Massachusetts Institute of Technology. Dept. of Chemical Engineering.

Grantor dc:publisher

Massachusetts Institute of Technology

Year dc:date.issued

2005

Author dc:creator

• Swers, Jeffrey Seth

Advisor dc:contributor.advisor

• K. Dane Wittrup.

• 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/1721.1/32323

OAI identifier oai:identifier

oai:dspace.mit.edu:1721.1/32323