Structural study of human antibody fragments with specificity for Mucin-1 antigen
MUC1 is overexpressed in many adenocarcinomas, which makes it a potential target for immunotherapy. A large number of MUC1-specific, monoclonal antibodies have been produced but the stability, solubility and the size of the antibody determines their effectiveness in cancer therapy. Smaller antibody fragments are advantageous over full-size antibodies, since small antigen binding molecules can efficiently penetrate solid tumors. As a first step towards the design of a clinically desirable antibody fragment, the crystallization of a human VH fragment has been achieved. The fragment was derived from the human single chain antibody scFvM12, which recognizes the cancer-specific hypoglycosylated MUC1. The human M12-VH domain has been crystallized through limited in-vitro proteolysis of scFvM12 antibody fragment. Crystals of M12-VH domain grew in approximately 12 months up to a size of 180x60x60µm in 100mM MES, pH 6.5, 5mM zinc sulphate, 25% v/v polyethylene glycol 550 monomethylether in the presence of low concentration of subtilisin Carlsberg. The protease addition results in complete degradation of M12-VL domain, linker and purification tags. The crystal belongs to space group C2 with unit cell dimension a = 71.34Å, b = 37.97Å, c = 37.19Å and alpha = gamma = 90°, beta = 109.674°. The solvent content of the crystal was calculated as 35.94%. The structure was solved up to 1.5Å resolution with an Rcryst factor of 15.8%, an Rfree of 19.7% and possesses a good stereochemistry. Dihedral angle values comparison of first and second complementarity-determining region (CDR) of M12-VH domain with an average value, for these two hypervariable regions, shows a significant deviation and therefore, it can be speculated that the M12-VH either suggests the existence of new canonical subclass or a link among the subclasses of canonical main-chain conformation in VH3 family. The natural existence of the stabilizing mutations Glu-H6, Arg-H66 and Ser-H52 confers the intrinsic stability to M12-VH domain under reducing conditions and therefore, the framework of this antibody domain can be used for incorporation of other specificities. This result would be helpful in structure based single domain antibody designing for biotechnological and pharmaceutical applications especially against MUC1 related cancer. The isolated VH domain undergoes aggregation due to the exposure of the hydrophobic surface involved in VH/VL interface. The camelization of the position H44, H45 and H47 can increase the hydrophilicity of the VH domain. Native and camelized M12-VH domain cloned in cytoplasmic expression vector would be helpful in assessing the immunogenic effects of camelizing the human VH domain.
info:eu-repo/classification/ddc/570, Biowissenschaften, Biologie, Antibody, Mucin-1, Limited Proteolysis, Crystallization
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