Yasufumi Kikuchi

VH/VL interface engineering to promote selective expression and inhibit conformational isomerization of thrombopoietin receptor agonist single-chain diabody

Thrombopoietin receptor agonist humanized VB22B single-chain diabody (hVB22B (scFv)(2)) was found to be expressed as a mixture of two conformational isomers, a single-chain diabody form and a bivalent scFv form, which had different V(H)/V(L) (variable region of the heavy chain/light chain) association patterns. The single-chain diabody form showed significantly higher biological activity than the bivalent scFv form and, when incubated at elevated temperatures, exhibited novel isomerization to the inactive bivalent scFv form. Therefore, therapeutic development of hVB22B (scFv)(2) would require separation of the purified single-chain diabody form from the mixture of the two conformational isomers and also inhibition of isomerization into an inactive bivalent scFv form during storage. Novel V(H)/V(L) interface engineering in hVB22 (scFv)(2), in which hydrogen bonding between H39 and L38 was substituted with electrostatic interaction to enhance the desired V(H)/V(L) association and inhibit the undesired V(H)/V(L) association, enabled selective expression of the desired conformational isomer without any reduction in biological activity or thermal stability. Moreover, V(H)/V(L) interface-engineered hVB22 (scFv)(2) was completely resistant to isomerization. Because the hydrogen bonding interaction between H39 and L38 and the surrounding residues are highly conserved in human antibody sequences, V(H)/V(L) interface engineering could be generally applied to various (scFv)(2) molecules for selective expression and inhibition of the isomerization of conformational isomers.

A new disulfide-linked dimer of a single-chain antibody fragment against human CD47 induces apoptosis in lymphoid malignant cells via the hypoxia inducible factor-1α pathway

CD47 belongs to the immunoglobulin superfamily and is associated with β‐integrins. Recently it was reported that CD47 ligation rapidly induces apoptosis in B‐chronic lymphocytic leukemia (CLL) cells. Chronic lymphocytic leukemia is still an incurable hematological malignancy even with the novel therapeutic agents; therefore, new and effective agents for the treatment of CLL in clinical settings are urgently needed. We generated a murine monoclonal antibody against an extracellular domain of human CD47 (designated MABL). Subsequently, we created a disulfide‐stabilized dimer of a single‐chain antibody fragment of MABL (S‐S diabody) to get rid of the adverse effect of MABL such as hemagglutination. In this study, we analyzed the effects of this new antibody on cellular proliferation, and the molecular mechanism of CD47‐mediated apoptosis in human lymphoid malignant cells. Treatment with S‐S diabody alone induced apoptosis of CD47‐positive primary B‐CLL and leukemic cells (MOLT‐4 and JOK‐1). In addition, administration of S‐S diabody significantly prolonged the survival of severe combined immunodeficiency (SCID) mice inoculated with JOK‐1 cells. In gene expression profiling of the S‐S diabody‐treated MOLT‐4 cells, hypoxia inducible factor (HIF)‐1α downstream genes (RTP801 and BNIP3) were upregulated, and the mRNA expression levels of HIF‐1α, RTP801 and BNIP3 were increased. Knockdown of HIF‐1α by siRNA repressed S‐S diabody‐induced apoptosis in MOLT4 cells. In conclusion, CD47 will be a molecular target for the treatment of lymphoid malignancies, and S‐S diabody might have potential as a novel therapeutic agent for B‐CLL. (Cancer Sci 2011; 102: 1208–1215)

Structure of human factor VIIa/tissue factor in complex with a peptide-mimetic inhibitor: high selectivity against thrombin by introducing two charged groups in P2 and P4.

The crystal structure of human factor VIIa/soluble tissue factor (FVIIa/sTF) in complex with a highly selective peptide-mimetic FVIIa inhibitor which shows 1670-fold selectivity against thrombin inhibition has been solved at 2.6 A resolution. The inhibitor is bound to FVIIa/sTF at the S1, S2 and S3 sites and at the additional S1 subsite. Two charged groups, the amidino group in P2 and the carboxylate group in P4, form ionic interactions with Asp60 and Lys192 of FVIIa, respectively. Structural comparisons between factor VIIa and thrombin show that thrombin has oppositely charged residues, Lys60F and Glu192, in the S2 site and the S1 subsites, respectively. These data suggest that the utilization of the differences of charge distribution in the S2 site and the S1 subsites between FVIIa and thrombin is critical for achieving high selectivity against thrombin inhibition. These results will provide valuable information for the structure-based drug design of specific inhibitors for FVIIa/TF.

Protective Effect of Recombinant Human Leukemia Inhibitory Factor against Thrombocytopenia in Carboplatin-treated Mice

The protective effect of recombinant human leukemia inhibitory factor (rhLIF) against development of throrabocytopenia in carboplatin (CBDCA; cis‐diammine‐l, l‐cyclobutane dicarboxylate platinum II)‐treated mice was examined. The optimal dose of rhLIF was determined by administering doses of rhLIF ranging from 0.2 to 20 μg/day intraperitoneally to normal BALB/c mice, and by analyzing platelet counts. Platelet counts significantly increased at doses over 0.2 μg/day. When 20 μg/day rhLIF was injected for 7 days, platelet counts increased to 240% of control. A remarkable weight loss occurred in the 20μg/day group, but no weight loss was detected at doses of less than 10 μg/day. The rhLIF dose of 4 μg/day was therefore used to examine the protective effect against carboplatin‐induced thrombocytopenia in mice. The intravenous injection of 100 mg/kg carboplatin caused a significant thrombocytopenia with a nadir on day 8. The administration of 4 μg/day rhLIF intraperitoneally for 7 days promoted the recovery of platelet counts from day 5 after the injection of carboplatin. These results suggest that rhLIF at a suboptimal dose might be a useful therapeutic agent for chemotherapy‐induced thrombocytopenia.