Preparation of integrin alpha(v)beta3-targeting Ab 38C2 constructs.
This protocol describes the preparation of Ab constructs using agents that target cells expressing integrins alpha(v)beta3 and alpha(v)beta5, 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.
Sinha
Subhash C
SC
The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. subhash@scripps.edu
Das
Sanjib
S
Li
Lian-Sheng
LS
Lerner
Richard A
RA
Barbas
Carlos F
CF
D017151:Antibodies, Catalytic / Q000276:immunology* / Q000378:metabolism; D016503:Drug Delivery Systems / Q000379:methods*; D005634:Fructose-Bisphosphate Aldolase / Q000276:immunology* / Q000378:metabolism; D007140:Immunoglobulin Fab Fragments / Q000276:immunology* / Q000378:metabolism; D007158:Immunologic Techniques; D007167:Immunotherapy / Q000379:methods; D039302:Integrin alphaVbeta3 / Q000276:immunology* / Q000378:metabolism; D013058:Mass Spectrometry
D017151:Antibodies, Catalytic; D007140:Immunoglobulin Fab Fragments; D039302:Integrin alphaVbeta3; C109536:antibody aldolase; D005634:Fructose-Bisphosphate Aldolase