Tetsuro Orita

Anti-glypican 3 antibodies cause ADCC against human hepatocellular carcinoma cells

Glypican 3 (GPC3), a GPI-anchored heparan sulfate proteoglycan, is expressed in the majority of hepatocellular carcinoma (HCC) tissues. Using MRL/lpr mice, we successfully generated a series of anti-GPC3 monoclonal antibodies (mAbs). GPC3 was partially cleaved between Arg358 and Ser359, generating a C-terminal 30-kDa fragment and an N-terminal 40-kDa fragment. All mAbs that induced antibody-dependent cellular cytotoxicity (ADCC) and/or complement-dependent cytotoxicity (CDC) against cells expressing GPC3 recognized the 30-kDa fragment, indicating that the C-terminal region of GPC3 serves as an epitope for mAb with ADCC and/or CDC inducing activities. Chimeric mAbs with Fc replaced by human IgG1 were created from GC33, one of the mAbs that reacted with the C-terminal 30-kDa fragment. Chimeric GC33 induced not only ADCC against GPC3-positive human HCC cells but also was efficacious against the Huh-7 human HCC xenograft. Thus, mAbs against the C-terminal 30-kDa fragment such as GC33 are useful in therapy targeting HCC.

Generation of a humanized anti-glypican 3 antibody by CDR grafting and stability optimization.

Glypican 3 (GPC3), a glycosylphosphatidylinositol-anchored heparan sulfate proteoglycan, is expressed in a majority of hepatocellular carcinoma tissues. The murine monoclonal antibody GC33 that specifically binds to the COOH-terminal part of GPC3 causes strong antibody-dependent cellular cytotoxicity against hepatocellular carcinoma cells and exhibits strong antitumor activity in the xenograft models. To apply GC33 for clinical use, we generated a humanized GC33 from complementarity-determining region grafting with the aid of both the hybrid variable region and two-step design methods. The humanized antibody bound to GPC3 specifically and induced antibody-dependent cellular cytotoxicity as effectively as a chimeric GC33 antibody. To improve stability of the humanized GC33, we further optimized humanized GC33 by replacing the amino acid residues that may affect the structure of the variable region of a heavy chain. Substitution of Glu6 with Gln in the heavy chain significantly improved the stability under high temperatures. GC33 also has the risk of deamidation of the -Asn–Gly- sequence in the complementarity-determining region 1 of the light chain. As substitution of Asn diminished the antigen binding, we changed the neighboring Gly to Arg to avoid deamidation. The resulting humanized anti-GPC3 antibody was as efficacious as chimeric GC33 against the HepG2 xenograft and is now being evaluated in clinical trials.

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.

Binding of NF-Y transcription factor to one of the cis-elements in the myeloperoxidase gene promoter that responds to granulocyte colony-stimulating factor.

The expression of the myeloperoxidase (MPO) gene is restricted to cells of the myeloid cell lineage and is induced by granulocyte colony-stimulating factor (G-CSF). In this study, a series of deletion mutations was introduced in the promoter of the human MPO gene, which was then fused to the chloramphenicol acetyltransferase gene. The G-CSF-induced promoter activity was examined in mouse myeloid precursor FDC-P1 transformants that constitutively express the G-CSF receptor. A G-CSF-responsive element (GRE) in the MPO gene was found approximately 800 base pairs upstream from the transcription initiation site. When the 5′-flanking region of the human MPO gene contained this element, it yielded promoter activity in cells cultured with G-CSF but not in cells cultured with interleukin 3. Gel shift assays with the element showed that a specific nuclear factor(s) (NF/G-CSF) binds to the element. The NF/G-CSF was purified by affinity chromatography using an oligonucleotide of GRE. Protein sequence analysis of the purified NF/G-CSF indicated that NF/G-CSF is a ubiquitous transcription factor, NF-Y, which is composed of three subunits. The recombinant NF-Y was then shown to bind to GRE in a combination of the three subunits.

Thrombopoietic activity of human interleukin-6

Thrombopoietin (TPO), a regulatory factor in platelet production, was purified from the conditioned medium of TNK‐01 cells cultured in the presence of human interleukin‐1. The N‐terminal sequence of purified TPO was determined to be VPPGEDSKDVAAPHRQPLT, identical to that of the N‐terminal region of human interleukin‐6 (IL‐6). Two forms of TPO with molecular masses of 24 and 27 kDa were identified as IL‐6 by Western analysis using an anti‐IL‐6 antibody. Commercial recombinant human IL‐6 produced in Escherichia coli, stimulated megakaryocyte colony formation in the presence of mouse interleukin‐3 and increased the number of peripheral platelets in mice in a dose‐dependent manner. From these results, it is concluded that human IL‐6 has thrombopoietic activity.

Reduction of the leukemogenic potential of malignant murine leukemic cells by in vivo treatment with recombinant human granulocyte colony-stimulating factor

We have investigated the effect of recombinant human granulocyte colony-stimulating factor (rhG-CSF) administration on the leukemogenic potential of L-103 murine leukemic cells. Leukemogenic potential was assessed by comparing the regression lines drawn between the number of inoculated leukemic spleen cells and the mean survival time (MST) of the syngeneic recipients. rhG-CSF injected 2.5 micrograms daily for 14 days reduced the leukemogenic potential of spleen cells of the leukemic mice to 1/200 of the control. This phenomenon was not observed with the leukemic spleen cells treated with r-murine granulocyte-macrophage (rmGM)-CSF in vivo. Cytochemical study indicated that morphologically identifiable blast cells were fewer in the rhG-CSF-treated leukemic spleen. Furthermore, leukemic cells in the rhG-CSF-treated spleen were less proliferative than the control in spite of having more clonogenic cells in the leukemic cell preparation. Cytogenetical analysis showed that chromosome abnormalities found in the original leukemic cells were not altered by rhG-CSF administrations. It also showed that the frequency of the abnormal karyotype was reduced in rhG-CSF-treated leukemic spleen (4/17) as compared with the control (8/8), indicating that the mitotic fraction was smaller in the rhG-CSF-treated leukemic cells. These findings indicate that in addition to the reduced number of leukemic cells in the spleen cell preparation, a reduction of the proliferative capacity of the original leukemic cells is involved in the reduction of leukemogenic potential of leukemic cells treated with rhG-CSF in vivo.