In vitro characterization of five humanized OKT3 effector function variant antibodies

Area of Science:

• Immunology
• Transplantation Medicine
• Biotechnology

Background:

• Orthoclone OKT3 (mOKT3) effectively reverses steroid-resistant renal allograft rejection.
• Wider use is limited by human anti-mouse antibody response (HAMA) and cytokine release syndrome (CRS).
• CRS involves T cell/monocyte activation and complement cascade activation, mediated by antibody Fc regions.

Purpose of the Study:

• To engineer humanized IgG1 and IgG4-based OKT3 antibodies with Fc mutations.
• To reduce HAMA and CRS while retaining immunosuppressive efficacy.
• To investigate the role of specific amino acid residues (234, 235, 318) in Fc binding and complement fixation.

Main Methods:

• Construction of five huIgG1- and huIgG4-based OKT3 wild-type and Fc mutant antibodies.
• Characterization of binding affinity to FcgammaRI and FcgammaRII.
• Assessment of T cell activation, cytokine release, and complement fixation (C1q binding).

Main Results:

• huOKT3gamma1(A(234), A(235)) and huOKT3gamma4(A(234), A(235)) showed >100-fold reduced binding to FcgammaRI/II.
• These mutants exhibited significantly reduced T cell activation and cytokine release.
• huOKT3gamma1(A(234), A(235)) demonstrated complete inactivity in complement fixation and reduced HAMA/CRS in vivo.
• All engineered antibodies retained potent in vitro immunosuppressive effects.

Conclusions:

• Fc mutations at amino acids 234 and 235 significantly reduce HAMA and CRS.
• Engineered OKT3 variants maintain immunosuppressive efficacy for renal allograft rejection.
• These modified antibodies represent a safer therapeutic option for transplant patients.