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Preparation of integrin alpha(v)beta3-targeting Ab 38C2 constructs
Subhash C Sinha1, Sanjib Das, Lian-Sheng Li
1The Skaggs Institute for Chemical Biology, 10550 North Torrey Pines Road, La Jolla, California 92037, USA. subhash@scripps.edu
Nature Protocols
|April 5, 2007
Summary
Antibody constructs targeting integrins alpha(v)beta3 and alpha(v)beta5 were prepared using chemical linkers. These novel antibody constructs show potential for targeted immunotherapy applications.
Area of Science:
- Bioconjugation Chemistry
- Immunotherapy
- Molecular Targeting
Background:
- Integrins alpha(v)beta3 and alpha(v)beta5 are overexpressed on various cancer cells.
- Monoclonal antibody 38C2 targets these integrins.
- Chemical modification of antibodies can enhance targeted delivery.
Purpose of the Study:
- To describe a protocol for preparing antibody constructs targeting specific integrins.
- To develop chemically programmed antibodies for enhanced targeting.
- To evaluate the potential of these constructs for immunotherapy.
Main Methods:
- Utilized diketone and vinylketone linkers for antibody conjugation.
- Conjugated targeting agents to monoclonal aldolase antibody 38C2 via reactive Lys residues.
- Employed a 38C2-catalyzed retro-aldol reaction for vinylketone linker activation.
Main Results:
- Successfully formed antibody constructs (38C2 conjugates and cp38C2) in under 1 hour (diketone) or 2-16 hours (vinylketone).
- Diketone linker formed enaminone derivatives with Lys residues.
- Pro-vinylketone linker generated vinylketone linker for Michael-type adduct formation.
Conclusions:
- Developed a robust method for creating antibody constructs targeting integrins.
- The chemically programmed 38C2 constructs are retargeted to integrin-overexpressing cells.
- These antibody constructs represent promising candidates for targeted immunotherapy.
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01:10Integrins
Animal and protozoan cells do not have cell walls to help maintain shape and provide structural stability. Instead, these eukaryotic cells secrete a sticky mass of carbohydrates and proteins into the spaces between adjacent cells. This network of proteins and molecules is called an extracellular matrix or ECM.
Some ECM proteins assemble into a basement membrane to which the remaining components adhere. Proteoglycans typically form the bulk of the ECM while fibrous proteins, like collagen,...
Some ECM proteins assemble into a basement membrane to which the remaining components adhere. Proteoglycans typically form the bulk of the ECM while fibrous proteins, like collagen,...
01:15Activation of Integrins
Integrins bind ligands and transmit information from outside the cell to inside or vice-versa through an "outside-in signaling" or "inside-out signaling."
In "outside-in signaling," external factors in the extracellular space bind to exposed ligand binding sites on integrins. This causes the inactive protein to undergo a conformational change to become active. Integrins are often clustered on the cell membrane. Repetitive and regularly spaced ligand binding events provide an effective stimulus.
In "outside-in signaling," external factors in the extracellular space bind to exposed ligand binding sites on integrins. This causes the inactive protein to undergo a conformational change to become active. Integrins are often clustered on the cell membrane. Repetitive and regularly spaced ligand binding events provide an effective stimulus.