Preparation, characterization and crystallization of recombinant fragment of anti-CD3 antibody MEM-57

Jana Písačková, Vlastimil Král, Magdaléna Hořejší, Milan Fábry and Pavlína Řezáčová

Institute of Molecular Genetics of the ASCR, v.v.i., Flemingovo nam. 2, Prague 6,
166 37, Czech Republic

Monoclonal antibody MEM-57 recognizes CD3 surface glycoprotein, which associates with T-cell receptor and mediates activation signal transduction. CD3 antigen is expressed on peripheral blood T-lymphocytes [1]. Antibody MEM-57 can, therefore, be used as a part of the „Bispecific T-cell Engager“ (BiTE) antibodies in the cancer therapy [2].

Single‑chain variable fragment of MEM‑57 (scFv MEM-57) was produced in E. coli BL21(DE3) from a pET22b vector; the product, targeted into the periplasmic space by the pelB leader sequence, contained c‑myc and His₅ tag at the C-terminus. The purification consisted in two steps, nickel chelation affinity chromatography and ion‑exchange chromatography. The antigen binding activity was confirmed by flow cytometry.

Crystallographic studies were initiated, as the structural information on scFv MEM‑57 would be useful for humanization of the antibody. This poster focuses mainly on experiments aiming at pre-crystallization analysis and the improvement of protein crystallizability. Size‑exclusion chromatography, dynamic light scattering and differential scanning fluorimetry (DSF) [3] analyses were performed to seek for conditions optimal for protein crystallization. DSF was used to select composition of the starting buffer, optimal for protein stability and homogeneity, which highly affects protein crystallizability, as confirmed by the results of our initial crystallization trials.

1. Cantrell, D., T cell antigen receptor signal transduction pathways. Annu Rev Immunol, 1996. 14: 259-74.

2. Wolf, E., Hofmeister, R., Kufer, P., Schlereth, B., Baeuerle, P. A., BiTEs: bispecific antibody constructs with unique anti-tumor activity. Drug Discov Today, 2005. 10(18): 1237-44.

3. Ericsson, U. B., Hallberg, B. M., Detitta, G. T., Dekker, N., Nordlund, P., Thermofluor-based high-throughput stability optimization of proteins for structural studies. Anal Biochem, 2006. 357(2): 289-98.