Production and characterization of an active single-chain variable fragment antibody recognizing CD25.
The alpha subunit of the interleukin-2 receptor (IL-2Ralpha, CD25) is a potential target in therapeutic approaches for hematolopoietic malignancies expressing CD25 on their cell surface, such as adult T cell leukemia/lymphomas. Recent reports have demonstrated that depletion of CD4+CD25+ regulatory T cells with anti-CD25 antibodies may enhance host tumor immunity. We previously raised a mouse monoclonal antibody (mAb), Ta60b mAb (IgG1kappa), specifically recognizing CD25, and an attempt was made here to produce a single chain Fv fragment (scFv) from this mAb as an initial step to development of scFv-based therapeutics. cDNA fragments encoding for the variable regions of the light and heavy chains of the Ta60b mAb were thus isolated by polymerase chain reaction-mediated cloning, and, an expression vector constructed to express Ta60b scFv fused with the maltose binding protein (MBP) in the periplasm of Escherichia coli. The soluble form of MBP-Ta60b fused scFv could be extracted and affinity-purified with an amylose/agarose column, allowing its immunoreactivity to be analyzed by enzyme-linked immunosorbent assay (ELISA), mixed hemadsorption assay, and fluorescence activated cell sorting. In addition, binding activity was studied by competitive ELISA and surface plasmon resonance. The results showed that Ta60b scFv obtained from periplasm retains good reactivity, although its KD value was 4-fold lower than that of the whole Ta60b antibody, suggesting possible clinical use for treatment of patients with CD25-expressing tumors and also for enhancing anti-tumor immunity.
Department of Pathology, Aichi Medical University School of Medicine, Aichi 480-1195, Japan.
Hideki
H
Muramatsu
Kazuhiro
K
Yoshikawa
Takeshi
T
Hayashi
Syuntaro
S
Takasu
Yoichi
Y
Kawada
Kazuo
K
Uchida
Shigeki
S
Sato
Toshitada
T
Takahashi
Shinsuke
S
Saga
Ryuzo
R
Ueda
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D004797:Enzyme-Linked Immunosorbent Assay; D005434:Flow Cytometry; D006801:Humans; D007128:Immunoglobulin Fragments / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000276:immunology*; D051379:Mice; D008969:Molecular Sequence Data; D015375:Receptors, Interleukin-2 / Q000276:immunology*; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification; D020349:Surface Plasmon Resonance
D007128:Immunoglobulin Fragments; D007135:Immunoglobulin Variable Region; D015375:Receptors, Interleukin-2; D011994:Recombinant Proteins