Journal
NATURE PROTOCOLS
Volume 2, Issue 2, Pages 449-456Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/nprot.2007.3
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Funding
- NATIONAL CANCER INSTITUTE [R01CA104045, R01CA120289] Funding Source: NIH RePORTER
- NCI NIH HHS [R01CA104045, R01 CA120289] Funding Source: Medline
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This protocol describes the preparation of Ab constructs using agents that target cells expressing integrins alpha(v)beta(3) and alpha(v)beta(5), and the monoclonal aldolase Ab 38C2. The targeting agents are equipped with a diketone or vinylketone linker, and selectively react through the reactive Lys residues in the Ab binding sites to form 38C2 conjugates or chemically programmed 38C2 (i.e., cp38C2). The targeting agent possessing a diketone linker reacts with the Lys residues forming an enaminone derivative. By contrast, the vinylketone linker is used as the corresponding acetone adduct (i.e., a pro-vinylketone linker), and this pro-adapter undergoes a 38C2-catalyzed retro-aldol reaction to produce the vinylketone linker, which forms a Michael-type adduct with the Lys residues. The Ab construct formation is achieved in < 1 h for the diketone compounds at ambient temperature, and in 2-16 h using the pro-vinylketone linker at 37 degrees C. The 38C2 constructs are retargeted to cells over-expressing integrins, and are potential candidates for immunotherapy.
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