Title : Engineering novel selectivity into metalloantibodies germline-encoded precursor to the murine anti-S1P metalloantibody in E.coli and baculovirus-insect cell system
The scientific aspect of this research focuses upon the use of metals by antibodies in the mammalian adaptive immune system. It is estimated that one third of all proteins are metalloproteins. However, the use of metals by antibodies to recognize and bind to antigens is still somewhat of a novelty. In recent years, antibodies that target biologically active lipids have been studied as promising therapeutic agents. Many physiological processes, such as cell growth, differentiation, survival, and pathophysiological processes, such as cancer, cardiovascular disease, multiple sclerosis, neuropathic pain, involve sphinosine-1-phosphate (S1P) signaling. LT1002 is a murine monoclonal antibody that binds to S1P with high affinity and specificity. Previously in our lab, a 1.9 Å x-ray crystal structure of humanized LT1009 Fab antibody version of the murine LT1002 anti-S1P antibody was determined. The x-ray crystal data indicates the novel finding that it employs two bridging calcium ions in binding to its lipid antigen. The two calcium interact with aspartic acid residues from the CDR-L1 and -L3 loops of the antibody variable light chain (VL). Furthermore, the amino acids involved in metal coordination are encoded in the germ-line sequences of immunoglobulin kappa light chain genes within the genomes of diverse mammalian species and are included in several antibodies that have been previously analyzed. This study is designed to identify other naturally occurring functional metalloantibodies and to test the hypothesis that the use of metal coordination chemistry by antibodies to recognize their antigens is evolutionarily conserved in the mammalian immune system.
Inductively-coupled plasma-mass spectrometry (ICP-MS) indicates that LT1002 binds to calcium, magnesium and, to much lesser extent, barium. In order to study the chemical properties of this novel metalloantibody, I first developed methods for recombinant expression and purification. I then altered the residues observed to coordinate the Ca ions and tested their ability to bind more biochemically active metals, such as Fe, Ni, Cu, and Zn. It is expected that these novel, engineered metalloantibody scaffolds might serve as substrates for further development as a class of engineered metalloantibodies with the ability to catalyze charge transfer reactions in water. In conclusion, metal binding is inherent to a class of antibodies that derive from germline light chain sequences with conserved metal coordinating residues in CDR-L1 and -L3. Also, I used Sf9 antibody expression system to test whether two additional antibodies (Q425 and 2C10) with these conserved residues function as metalloantibodies.
Audience take away:
• Introducing novel class of antibodies that binds to metals.
• Metalloantibodies as therapeutic agents
• Introducing novel high yield expression technique for antibodies in Baculovirus SF9 insect cells
• Enzymatic activity of metalloantibodies