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.

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.

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 novel therapeutic approach for thrombocytopenia by minibody agonist of the thrombopoietin receptor.

Antibodies have brought valuable therapeutics in the clinical treatment of various diseases without serious adverse effects through their intrinsic features such as specific binding to the target antigen with high affinity, clinical safety as serum proteins, and long half-life. Agonist antibodies, furthermore, could be expected to maximize the value of therapeutic antibodies. Indeed, several IgG/IgM antibodies have been reported to induce cellular growth/differentiation and apoptosis. These agonist antibodies, however, should be further improved to exert more potent biologic activities and appropriate serum half-life depending upon the disease indications. Here, we report that IgG antibodies against the thrombopoietin receptor (Mpl), which have an absence or very weak agonist activity, can be engineered to be agonist minibodies, which include diabody or sc(Fv)2 as potent as natural ligand. Through this technological development, minibodies have been successfully constructed to bind and activate 2 types of dysfunctional mutant Mpls that cause congenital amegakaryocytic thrombocytopenia (CAMT). This drastic conversion of biologic activities by designing minibodies can be widely applicable to generate agonist minibodies for clinical application, which will constitute a new paradigm in antibody-based therapeutics.

2D7 diabody bound to the alpha2 domain of HLA class I efficiently induces caspase-independent cell death against malignant and activated lymphoid cells.

A mouse monoclonal antibody (2D7 mAb), which specifically bound to the alpha2 domain of HLA class I, rapidly induces cell aggregation accompanied by weak cytotoxicity against ARH-77 cells, suggesting that 2D7 mAb had a potential for agonist antibody. In order to enhance this cytotoxicity, 2D7 mAb was engineered to be a small bivalent antibody fragment, 2D7 diabody. The resultant 2D7 diabody showed a strong cytotoxicity against ARH-77 cells. As a notable characteristic feature, the lethal effect of 2D7 diabody was quite rapid, mediated by a caspase-independent death pathway. Furthermore, 2D7 diabody also showed cytotoxicity against several leukemia and lymphoma cell lines, and mitogen-activated peripheral blood mononuclear cells (PBMC), but not for normal resting PBMC and adherent cell lines such as HUVEC. These results suggest that 2D7 diabody could be expected as a novel therapeutic antibody for hematological malignancies as well as inflammatory diseases.