Humanization of the Mouse Anti-Fas Antibody HFE7A and Crystal Structure of the Humanized HFE7A Fab Fragment.

Abstract

Apoptosis, or programmed cell death, is involved in many biological processes including embryogenesis, development of the immune system, elimination of virus-infected cells, and maintenance of tissue homeostasis.1) Binding of Fas ligand (FasL) to Fas induces apoptosis. Fas is a type-I membrane protein and belongs to the tumor necrosis factor (TNF) receptor family.2,3) FasL, a member of the TNF family, is synthesized as a type-II membrane protein and digested proteolytically to a soluble form. 4,5) Mice with mutations of Fas and/or FasL develop massive lymphadenopathy and autoimmune diseases, which indicates that the Fas-FasL system plays an important role in the elimination of autoreactive lymphoid cells. 6)The mouse monoclonal antibody HFE7A (m-HFE7A), raised against human Fas, binds to both human and mouse Fas. 7,8) Administration of m-HFE7A to mice induces apoptosis in thymocytes. However, in mice, m-HFE7A shows no sign of the hepatotoxicity observed with the hamster antimurine Fas monoclonal antibody Jo2. 9) Moreover, administration of m-HFE7A to mice prevents the injury of the liver induced by Jo2. Therefore, m-HFE7A may provide a useful treatment for autoimmune diseases such as rheumatoid arthritis and fulminant hepatitis.A major limitation in the use of murine antibodies for human therapy is the human anti-mouse antibody response, resulting in potentially harmful reactions and the rapid clearance of circulating antibody. 10,11) To reduce the immunogenicity of murine antibodies for human therapy, chimeric antibodies were initially developed.12) Chimeric antibodies consist of the mouse variable domains and the human constant domains. Even chimeric antibodies are still capable of eliciting a significant immune response.13) An approach to overcome the limitation in the clinical use of murine antibodies is humanization of murine antibodies.14) This technique involves transplantation of the murine residues in the complementarity-determining regions (CDRs), which are important for antigen binding, into a relevant human antibody. However, direct grafting of only the CDR residues has sometimes decreased the binding affinity. 15,16) Therefore, in addition to the CDR residues, some murine residues at the key positions in the framework region must also be incorporated into the human framework to maintain the proper CDR conformation. For this process, information on the three-dimensional structure is essential to minimize the number of murine residues to be transferred and avoid the human antimouse antibody response.In this report, we describe humanization of m-HFE7A by the CDR grafting method for the treatment of the human diseases. Five versions of humanized HFE7A (h-HFE7A) induced the same degree of apoptosis in WR19L12a cells as chimeric HFE7A (chi-HFE7A) does. To further probe the structural basis on antibody humanization, we selected one of five h-HFE7As, h-HFE7A-d, since the d version has the smallest number of residues transferred from m-HFE7A among five h-HFE7As and determined the crystal structure of the h-H...

Phase Separation of an IgG1 Antibody Solution under a Low Ionic Strength Condition

“…The humanized monoclonal antibody A (IgG1 subclass, MAb A) used in this investigation to target Fas ligand was produced in a mouse myeloma (NS0) cell line and was highly purified at Daiichi Sankyo Co., Ltd., Tokyo, Japan (29). The theoretical isoelectric point (pI) of the antibody is 6.5.…”

How Protein Recognizes Ladder-like Polycyclic Ethers

“…In both complexes, the B-factor values of the variable regions are lower than the value for the ligand-free 10C9 Fab. In ligand-free 10C9 Fab, there are several regions whose temperature factors are particularly high; all of the regions are CDRs: CDR-H1, Leu (38). However, these temperature factors are markedly reduced in both complexes, which suggests that the CDRs are well stabilized upon complexation.…”

FR901228 induces tumor regression associated with induction of Fas ligand and activation of Fas signaling in human osteosarcoma cells

“…In addition, FR901228 may become a candidate for clinical use as a therapeutic agent for osteosarcoma in combination with agonistic anti-Fas monoclonal antibody. Recently, we prepared a novel humanized agonistic anti-Fas monoclonal antibody HFE7A which has no hepatotoxicity (Haruyama et al, 2002;Yonehara, 2002). As blockage of the Fas/FasL system failed to completely inhibit FR901228-induced apoptosis, it is also likely that other apoptotic pathways are involved in inducing apoptosis with this agent.…”