Cloning and sequencing of the VH and V kappa genes of an anti-CD3 monoclonal antibody, and construction of a mouse/human chimeric antibody.
Mouse monoclonal antibodies against CD3 on human T lymphocytes have been used for therapy in organ-transplant patients as a potent immunosuppressive agent or for treatment of cancer as a potent T cell activating agent. However, an inherent problem in their in vivo application is the human anti-mouse antibody response. In this study, we cloned and sequenced the variable region genes of the heavy and light chains (VH and V kappa) of a mouse anti-human CD3 monoclonal antibody (OKT3) using the reverse transcription-polymerase chain reaction method. Then, we constructed a mouse/human chimeric antibody, designated as Ch OKT3, by fusing the OKT3 VH and V kappa genes to the human heavy and light chain constant region genes (C gamma 1 and C kappa) derived from a human plasma cell leukemia line (ARH77), respectively. The chimeric gene constructs were sequentially co-transfected into mouse non-Ig-producing hybridoma cells (Sp2/0) by electroporation. The Ch OKT3 antibody thus prepared bound to human peripheral blood mononuclear cells and competitively inhibited the binding of the parental MAb OKT3 to the blood mononuclear cells, indicating that this chimeric antibody seems to be suitable for in vivo therapeutic approaches.
First Department of Biochemistry, School of Medicine, Fukuoka University.
F
F
Arakawa
M
M
Kuroki
M
M
Kuwahara
T
T
Senba
H
H
Ozaki
Y
Y
Matsuoka
Y
Y
Misumi
H
H
Kanda
T
T
Watanabe
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D001483:Base Sequence; D017252:CD3 Complex / Q000276:immunology*; D002460:Cell Line; D003001:Cloning, Molecular; D005803:Genes, Immunoglobulin; D006801:Humans; D006825:Hybridomas; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry / Q000235:genetics; D007145:Immunoglobulin kappa-Chains / Q000737:chemistry / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009101:Multiple Myeloma; D016853:Muromonab-CD3 / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000737:chemistry; D013601:T-Lymphocytes / Q000276:immunology; D014162:Transfection
D000911:Antibodies, Monoclonal; D017252:CD3 Complex; D007143:Immunoglobulin Heavy Chains; D007135:Immunoglobulin Variable Region; D007145:Immunoglobulin kappa-Chains; D016853:Muromonab-CD3; D011993:Recombinant Fusion Proteins