Ryutaro Asano

Antitumor activity of interleukin-21 prepared by novel refolding procedure from inclusion bodies expressed in Escherichia coli

Interleukin‐21 (IL‐21) has recently been identified as a novel 4‐helix‐bundle type I cytokine possessing a cytokine receptor γ chain essential for the immune response. We report the preparation and functional characterization of Escherichia coli‐expressed recombinant human IL‐21 (rIL‐21). The rIL‐21, expressed as insoluble inclusion bodies in E. coli, was solubilized and then refolded by using a modified dialysis method. The introduction of redox reagents during refolding led to a dramatic increase in the refolding efficiency. Circular dichroism spectrum analysis showed that the refolded rIL‐21 had an α‐helix as a secondary structure, which is a characteristic of type I cytokines. Flow cytometry confirmed previous reports that rIL‐21 binds to CD3‐activated T cells (T‐LAK) and to cell lines Raji, HL60, and Jurkat. rIL‐21 stimulated the proliferation of T‐LAK but not peripheral blood mononuclear cells, and this effect seems to be identical to that of co‐stimulation with anti‐CD3 antibody. Growth inhibition assay indicated that enhancement of the cytotoxicity of T‐LAK to the human bile duct carcinoma TFK‐1 depended on the concentration of rIL‐21. Thus, refolded rIL‐21 had activity identical to that of authentic IL‐21 and enhanced the anti‐tumor activity of T‐LAK. These conclusions suggest the potential use of the refolded cytokine in adoptive immunotherapy using T‐LAK cells and in the discovery of other functions of the cytokine.

A mutated superantigen SEA D227A fusion diabody specific to MUC1 and CD3 in targeted cancer immunotherapy for bile duct carcinoma.

Abstract. In cancer immunotherapy research, many bispecific antibodies (BsAbs) have been developed for directing T cells toward tumor cells. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems. Therefore, recombinant BsAbs, termed diabodies, have attracted particular attention. We have previously produced an anti-MUC1 × anti-CD3 diabody (M×3 diabody) in an Escherichia coli (E. coli) expression system. In order to reinforce the antitumor effects of the M×3 diabody, mutated superantigen staphylococcal enterotoxin A (SEA) D227A was genetically fused to the M×3 diabody. The SEA D227A fusion M×3 diabody (SEA D227A-M×3 diabody) thus constructed showed remarkable MUC1-specific antitumor effects when used with effector cells (lymphokine-activated killer cells with T-cell phenotype [T-LAK] and peripheral blood mononuclear cells [PBMCs]). In the bile duct carcinoma (BDC)-xenografted severe combined immunodeficient (SCID) mouse model, it also demonstrated strong antitumor activity when administered i.v. together with T-LAK cells and interleukin-2 (IL-2). In this experiment, the complete disappearance of tumors was observed in 3 out of 6 mice, and the other 3 showed marked retardation of tumor growth. Therefore, the SEA D227A-M×3 diabody is considered to be a promising reagent in specific targeted immunotherapy for BDC and other MUC1-positive carcinomas. This is the first report on a diabody that is effective in treating human solid cancers in the xenografted SCID mouse experimental model.

Specific and effective targeting cancer immunotherapy with a combination of three bispecific antibodies.

For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we previously constructed two kinds of bispecific antibodies (bsAbs), anti-MUC1 x anti-CD3 (M x 3) and anti-MUC1 x anti-CD28 (M x 28), which activate T cells and form bridges between them and MUC1-expressing tumor cells. In our previous studies [Cancer Res. 56 (1996) 4205] specific targeting therapy (STT) consisting of i.v. administration of lymphokine activated killer cells with a T cell phenotype (T-LAK) sensitized with two kinds of bsAbs to human BDC-grafted severe combined immunodeficient (SCID) mice demonstrated remarkable inhibition of tumor growth. However, complete cures could not be obtained. In order to improve antitumor efficacy, we have paid attention to anti-CD2 monoclonal antibodies (mAbs), thought to play an important roles in signal transduction in T cell activation or control of T cell receptor (TCR)-driven activation. Therefore, we developed another bsAb, anti-MUC1 x anti-CD2 (M x 2), in order to examine if this would show synergism with the two previously described bsAbs. The combination of the three bsAbs (M x 3, M x 28 and M x 2 bsAbs) showed highest cytotoxicity against MUC1-expressing BDC cells when given simultaneously with peripheral blood mononuclear cells (PBMCs) or T-LAK cells in vitro. When 2 x 10(7) T-LAK cells sensitized with different combinations of bsAbs were administered four times i.v. to BDC-grafted SCID mice, the best therapeutic result was obtained with a combination of all three bsAbs. These results indicate usefulness of combination of three bsAbs for targeting cancer immunotherapy.

Inhibition of hepatitis C virus NS3 protease by peptides derived from complementarity-determining regions (CDRs) of the monoclonal antibody 8D4: tolerance of a CDR peptide to conformational changes of a target

We have synthesized and characterized peptides derived from complementarity‐determining regions (CDRs) of 8D4, a mouse monoclonal antibody against NS3 protease domain of hepatitis C virus. 8D4 inhibits enzymatic activity without its cofactor, NS4A peptide. One of the synthetic peptides derived from CDRs, CDR1 of the heavy‐chain (CDR‐H1) peptide strongly inhibited NS3 protease activity competitively in the absence of NS4A and non‐competitively in the presence of NS4A. Moreover, cyclic CDR‐H1 peptides bridged by disulfide inhibited NS3 protease more potently. The chain length of the CDR‐H1 peptide is critical for strong inhibition, even when the peptide is circularized. This finding suggests the importance of peptide conformation. In contrast to a cognate antibody molecule, CDR‐derived peptides may provide good ligands for target molecules by having a tolerance to conformational changes of the targets caused by cofactor binding or mutation.

Mutated SEA-D227A-conjugated antibodies greatly enhance antitumor activity against MUC1-expressing bile duct carcinoma.

Abstract. For the purpose of establishing a new adoptive immunotherapy for bile duct carcinoma (BDC), we have directed our attention to superantigens (SAgs), the most potent known activators of T lymphocytes. In our previous study, staphylococcal enterotoxin A (SEA) was conjugated chemically with MUSE11 mAb, which recognizes the MUC1 cancer-associated antigen, and shown to enhance the specific cytotoxic activity of T-LAK cells against MUC1-expressing BDC cells (TFK-1) in vitro and in vivo. However, it is probable that SEA might cause side-effects because of nonspecific binding to class II positive cells. In order to overcome these, we generated mutated SEA (mSEA) by changing Asp at position 227 of native SEA to Ala, which has reduced affinity to MHC class II molecules, but retains the potential for T cell activation. When mSEA-D227A was administered to rabbits to examine effects on blood pressure, 500 times more mSEA-D227A was tolerated than native SEA. This prompted us to construct a mSEA-D227A-conjugated mAb, reactive with MUC1. It augmented the antitumor activity of T-LAK cells significantly, and furthermore, mSEA-D227A could be conjugated to two bispecific antibodies, BsAb (anti-MUC1×anti-CD3) and BsAb (anti-MUC1×anti-CD28), which in combination had greater enhancing effects than mSEA-D227A-conjugated anti-MUC1 mAb, and combination of unconjugated BsAbs. These findings indicate a utility of mSEA-D227A-conjugated antibodies for targeted cancer immunotherapy.

Functional Fv fragment of an antibody specific for CD28: Fv-mediated co-stimulation of T cells.

The most predominant co‐stimulation pathway, which is critical for T cell activation and proliferation, is the CD28‐B7 pathway. The anti‐CD28 monoclonal antibody (mAb) also provides a co‐stimulatory signal to T cells. In order to construct a functional Fv fragment (complex of VH and VL domains) of anti‐CD28 antibody using a bacterial expression system, cDNA encoding the variable regions of immunoglobulin from 15E8 hybridoma cells was cloned and expressed in Escherichia coli. The Fv fragment was obtained as a soluble protein from the periplasmic fraction and showed a binding pattern similar to parental IgG. The Fv fragment induced proliferation of peripheral blood mononuclear cells in the presence of anti‐CD3 or anti‐CD2 mAb and enhanced anti‐tumor activity of anti‐MUC1×anti‐CD3 bispecific antibody when tested with lymphokine‐activated killer cells with T cell phenotype. Thus, the anti‐CD28 Fv fragment will be promising not only for the study of co‐stimulation, but also for cancer immunotherapy.