Effective screening method of agonistic diabodies based on autocrine growth.
Agonistic diabodies that mimic the function of natural ligands are expected to increase the value of therapeutic antibodies. We have developed a method that detects agonistic diabodies based on their ability to transduce growth signals through receptors, thereby permitting cytokine-independent growth of BaF/3-derived cytokine-dependent cells. Retrovirus-mediated expression of the diabody in cytokine-dependent cells was followed by selection of clones for growth in the absence of cytokine. A diabody library derived from splenocytes of human Mpl immunized mice was constructed. Infection of cells with viral particles led to the isolation of over 500 autonomously growing clones whose cultured supernatants showed agonistic activities against Mpl. Genome-integrated diabodies were cloned; representative clone AB317 showed agonistic activities as potent as a natural ligand and cross-reactive against mouse Mpl.
Nakano
Kiyotaka
K
Genome Antibody Product Research Dept., Chugai Pharmaceutical Co. Ltd. 1-135 Komakado, Gotemba, Shizuoka 412-8513, Japan. nakanokyt@chugai-pharm.co.jp
Kojima
Tetsuo
T
Kasutani
Keiko
K
Senoh
Chiaki
C
Natori
Osamu
O
Ishii
Shinya
S
Tsunoda
Hiroyuki
H
Hattori
Kunihiro
K
D000595:Amino Acid Sequence; D000818:Animals; D000906:Antibodies / Q000235:genetics / Q000378:metabolism*; D019898:Autocrine Communication; D001666:Binding Sites, Antibody; D019556:COS Cells; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D003001:Cloning, Molecular; D015723:Gene Library; D006801:Humans; D007114:Immunization; D007143:Immunoglobulin Heavy Chains / Q000096:biosynthesis / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000096:biosynthesis / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000096:biosynthesis / Q000235:genetics; D007377:Interleukin-3 / Q000378:metabolism; D008024:Ligands; D051379:Mice; D008817:Mice, Mutant Strains; D008969:Molecular Sequence Data; D054448:Precursor Cells, B-Lymphoid / Q000276:immunology*; D053628:Receptors, Thrombopoietin / Q000819:agonists / Q000235:genetics / Q000276:immunology* / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015398:Signal Transduction; D013926:Thrombopoietin / Q000378:metabolism; D014162:Transfection
D000906:Antibodies; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D007377:Interleukin-3; D008024:Ligands; C481270:Mpl protein, mouse; D053628:Receptors, Thrombopoietin; D011993:Recombinant Fusion Proteins; C082218:MPL protein, human; D013926:Thrombopoietin