Preliminary X-ray diffraction studies of the transcriptional inhibitory antibody Fab41.4

The binding of transcription factor ATF-1 to DNA contributes to gene expression and regulation of cell growth. Antibody Mab41.4, raised against ATF-1, and its derivatives Fab41.4 and scFv41.4 inhibit specific DNA binding in vitro and induce apoptotic death of tumor cells in vivo. Structural studies of Fab41.4 were performed to gain insight into the mechanism of action of this potentially therapeutic antibody. The optimal conditions for crystallization of Fab41.4 were determined. Crystals were needle-like in appearance, displayed C2 space-group symmetry and diffracted to a resolution of 1.6 Å. The unit-cell parameters were determined to be a = 186.64, b = 40.22, c = 55.58 Å, α = γ = 90, β = 96.93°. The data set was 97.7% complete. Molecular replacement was performed, resulting in an R value of 44.6%

Preliminary X-ray diffraction studies of the transcriptional inhibitory antibody Fab41.4

The binding of transcription factor ATF-1 to DNA contributes to gene expression and regulation of cell growth. Antibody Mab41.4, raised against ATF-1, and its derivatives Fab41.4 and scFv41.4 inhibit specific DNA binding in vitro and induce apoptotic death of tumor cells in vivo. Structural studies of Fab41.4 were performed to gain insight into the mechanism of action of this potentially therapeutic antibody. The optimal conditions for crystallization of Fab41.4 were determined. Crystals were needle-like in appearance, displayed C2 space-group symmetry and diffracted to a resolution of 1.6 Å. The unit-cell parameters were determined to be a = 186.64, b = 40.22, c = 55.58 Å, α = γ = 90, β = 96.93°. The data set was 97.7% complete. Molecular replacement was performed, resulting in an R value of 44.6%

Structural studies of an inhibitory antibody, Mab41.4, to its epitope, ATF -1

The field of oncology is increasingly utilizing antibodies for the treatment of cancer. Sarcoma, a type of cancer that arises from non-epithelial extra-skeletal tissues of the body tends to be highly malignant. Sarcomas also do not react well to conventional therapeutic regimens such as radiation and chemotherapy. Clear Cell Sarcoma of tendon sheath and aponeurosis is characterized by the t(12;22)(q13;q12) translocation resulting in a fusion protein EWS/ATF-1. A monoclonal antibody, Mab41.4, has been determined to bind to the DNA binding domain of the ATF-1 portion of EWS/ATF-1 preventing binding of the fusion protein to the CRE DNA. To fully understand the mechanism of interaction between the antibody and fusion protein, the Fab fragment of the Mab, Fab41.4, was used to perform a crystallographic study. The structure of Fab41.4 has been determined and refined to 1.6 Angstroms resolution utilizing data collected at the Stanford Synchrotron Radiation Laboratory. The unit cell parameters at 100K were determined to be a = 186.64 Å, b = 40.23 Å, c = 55.58 Å, α = γ = 90°, and β = 96.93°. The structure was solved by the molecular replacement method using EPMR software. Analysis of the structure determined the binding site to be a deep pocket. The walls of the pocket were comprised of the complementary determining regions (CDR) L1 and H1, whereas CDR H3 comprised the bottom of the pocket. CDR H3 was determined to be hydrophobic in nature, which complemented the hydrophobicity observed by an area of the antigen, Peptide C. Since Mab41.4 cannot be internalized by cells, a smaller sized portion of the antibody termed scFv41.4 was cloned. Comparison of Fab41.4 and scFv41.4 determined that the scFv had undergone a frameshift in Framework Region 4 resulting in termination of the light chain and a deletion in Framework Region 3 of the heavy chain. This resulted in a shift of CDR H3 according to Kabat numbering scheme. Molecular modeling was employed to illustrate the differences between Fab41.4 and scFv41.4. It was found that the new CDR H3 was also hydrophobic in nature