Rearranging the domain order of a diabody-based IgG-like bispecific antibody enhances its antitumor activity and improves its degradation resistance and pharmacokinetics.

One approach to creating more beneficial therapeutic antibodies is to develop bispecific antibodies (bsAbs), particularly IgG-like formats with tetravalency, which may provide several advantages such as multivalent binding to each target antigen. Although the effects of configuration and antibody-fragment type on the function of IgG-like bsAbs have been studied, there have been only a few detailed studies of the influence of the variable fragment domain order. Here, we prepared four types of hEx3-scDb-Fc, IgG-like bsAbs, built from a single-chain hEx3-Db (humanized bispecific diabody [bsDb] that targets epidermal growth factor receptor and CD3), to investigate the influence of domain order and fusion manner on the function of a bsDb with an Fc fusion format.

Domain order of a bispecific diabody dramatically enhances its antitumor activity beyond structural format conversion: the case of the hEx3 diabody.

The domains of bispecific diabodies (BsDbs) can be ordered in four different ways; however, the influence of domain order on the cytotoxicity of BsDbs that retarget immune cells against tumor cells had not been addressed. We previously reported the marked antitumor effects of a humanized BsDb that targets epidermal growth factor receptor and CD3 (hEx3-Db). Here, we rearranged the domains of hEx3-Db to examine the influence of domain order on the function of BsDbs.

Construction and humanization of a functional bispecific EGFR × CD16 diabody using a refolding system.

We previously reported the construction and activity of a humanized, bispecific diabody (hEx3) that recruited T cells towards an epidermal growth factor receptor (EGFR) positive tumor. Herein, we describe the construction of a second functional, fully humanized, anti-EGFR bispecific diabody that recruits another subset of lymphocyte effectors, the natural killer cells, to EGFR-expressing tumor cells. After we confirmed that an anti-EGFR × anti-CD16 bispecific diabody (Ex16) consisting of a previously humanized anti-EGFR variable fragment (Fv) and a mouse anti-CD16 Fv had growth inhibitory activity, we designed a humanized anti-CD16 Fv to construct the fully humanized Ex16 (hEx16).

In vitro and in vivo antitumor effects of recombinant bispecific antibodies based on humanized anti-EGFR antibody.

We performed in vitro and in vivo experiments of the anti-epidermal growth factor receptor (EGFR) x anti-CD3 bispecific diabody (hEx3-Db) with the IgG-like bispecific antibodies (BsAbs) (hEx3-scFv-Fc and hEx3-scDb-Fc) and the anti-EGFR therapeutic antibody cetuximab to assess the effect of BsAbs on cancer growth inhibition. In vitro, efficacy of the BsAbs and cetuximab were compared by growth inhibition assays of human cell lines of bile duct (TFK-1, HuCC-T1, OCUCh-LM1), epidermoid (A431), gastric (Kato-III), colon (DLD-1, SW480), and breast (SK-BR-3, MCF-7) cancer. In vivo, in three mouse models, we evaluated the anti-tumor activity of hEx3-Db and cetuximab, assessed the effect of hEx3-Db alone, and compared the antitumor activity of hEx3-Db with the IgG-like BsAbs.

Highly enhanced cytotoxicity of a dimeric bispecific diabody, the hEx3 tetrabody.

We previously reported the utility for cancer immunotherapy of a humanized bispecific diabody (hEx3) that targets epidermal growth factor receptor and CD3. Here, we used dynamic and static light scattering measurements to show that the multimer fraction observed in hEx3 in solution is a monodisperse tetramer. The multimerization into tetramers increased the inhibition of cancer cell growth by the hEx3 diabody.

Application of the Fc fusion format to generate tag-free bi-specific diabodies.

We previously reported the use of a humanized bi-specific diabody that targets epidermal growth factor receptor and CD3 (hEx3-Db) for cancer immunotherapy. Bacterial expression can be used to express small recombinant antibodies on a large scale; however, their overexpression often results in the formation of insoluble aggregates, and in most cases artificial affinity peptide tags need to be fused to the antibodies for purification by affinity chromatography. Here, we propose a novel method for preparing refined, functional, tag-free bi-specific diabodies from IgG-like bi-specific antibodies (BsAbs) in a mammalian expression system.

Diabody-based recombinant formats of humanized IgG-like bispecific antibody with effective retargeting of lymphocytes to tumor cells.

Recently, recombinant antibodies have been dissected into antigen-binding regions and rebuilt into multivalent high-avidity formats. These new structural designs are expected to improve in vivo pharmacokinetics and efficacy in clinical use. Here, we designed effective recombinant bispecific antibody (BsAb) formats based on hEx3, a humanized bispecific diabody with epidermal growth factor receptor and CD3 retargeting.

Preferential heterodimerization of a bispecific diabody based on a humanized anti-EGFR antibody 528.

We report the utility of in vitro refolding in the preparation of monomorphous hEx3 bispecific diabodies with epidermal growth factor receptor and CD3 retargeting from insoluble aggregates in Escherichia coli. Appropriate interaction between cognate variable heavy and light chains led to the formation of functional hEx3 heterodimers in a diabody format rather than inactive homodimers. The refolded hEx3 was found to exhibit almost the equivalent activity to the hEx3 and single-chain hEx3 (hEx3-scDb) prepared in a mammalian secretion system.

Human CD4+ central and effector memory T cells produce IL-21: effect on cytokine-driven proliferation of CD4+ T cell subsets.

IL-21 regulates certain functions of T cells, B cells, NK cells and dendritic cells. Although activated CD4(+) T cells produce IL-21, data identifying the specific CD4(+) T cell subsets that produce IL-21 are conflicting. In a previous study, mouse IL-21 message was detected in T(H)2, whereas human IL-21 (hIL-21) message was found in both T(H)1 and follicular helper T cells.

Highly effective recombinant format of a humanized IgG-like bispecific antibody for cancer immunotherapy with retargeting of lymphocytes to tumor cells.

We previously reported the marked in vitro and in vivo antitumor activity of hEx3, a humanized diabody (small recombinant bispecific antibody) with epidermal growth factor receptor (EGFR) and CD3 retargeting. Here, we fabricated a tetravalent IgG-like bispecific antibody with two kinds of single-chain Fv (scFv), i.e.

Humanization of the bispecific epidermal growth factor receptor x CD3 diabody and its efficacy as a potential clinical reagent.

Purpose: Bispecific antibodies (BsAb) have been exploited as both cancer immunodiagnostics and cancer therapeutics and show promise in clinical trials of cancer imaging and therapy. For development of BsAbs as clinical reagents, we have focused on construction of small recombinant BsAbs, called bispecific diabodies. Here, we constructed and characterized a humanized bispecific diabody.

Tumor-directed lymphocyte-activating cytokines: refolding-based preparation of recombinant human interleukin-12 and an antibody variable domain-fused protein by additive-introduced stepwise dialysis.

Integration of lymphocyte-activating cytokines (e.g., interleukin-12: IL-12) to tumor cells offers promise for cancer immunotherapy, but the preparation of such heterodimeric proteins by refolding is difficult because of subunit instability.

A highly effective and stable bispecific diabody for cancer immunotherapy: cure of xenografted tumors by bispecific diabody and T-LAK cells.

Purpose: In the field of cancer immunotherapy research, the targeting of effector cells with specific antibodies is a very promising approach. Recent advances in genetic engineering have made it possible to prepare immunoglobulin fragments consisting of variable domains using bacterial expression systems.

Methods: We have produced an anti-epidermal growth-factor receptor (EGFR) x anti-CD3 bispecific diabody (Ex3 diabody) in an Escherichia coli (E.

Evaluation of the diagnostic accuracy of the stop codon (SC) assay for identifying protein-truncating mutations in the BRCA1and BRCA2genes in familial breast cancer.

Screening for protein-truncating mutations of the BRCA1 and BRCA2 genes is useful in genetic testing for familial breast cancer because, first, the methods are usually simple and not expensive, and second, the detected mutations indicate pathogenic mutations in general. We evaluated the diagnostic accuracy of the stop codon (SC) assay for detecting protein-truncating mutations in the BRCA1 and BRCA2 genes by comparing the results with DNA sequencing in samples from 29 patients with breast cancer from 24 Japanese families with a history of breast cancer. Protein-truncating mutations were detected in 5 of the 24 families (20.

A novel adenovirus expressing human 4-1BB ligand enhances antitumor immunity.

4-1BB ligand (4-1BBL), a member of the tumor necrosis factor (TNF) superfamily, interacts with 4-1BB (CDw137) expressed on activated T cells and delivers a costimulatory signal for T cell activation and growth. Various studies have demonstrated a role for murine 4-1BB in immune function, but relatively few investigations of human 4-1BB have been conducted. Here we report on the construction of a recombinant E1/E3-deleted adenovirus encoding human 4-1BBL (Ad4-1BBL) and its stimulation of antitumor immunity.

Efficient construction of a diabody using a refolding system: anti-carcinoembryonic antigen recombinant antibody fragment.

Recombinant fragments of the variable region of antibodies are useful in many experimental and clinical applications. However, it can be difficult to obtain these materials in soluble form after their expression in bacteria. Here, we report an efficient procedure for preparing several variable-domain fragments (Fv), single-chain Fv (scFv), and a diabody (the smallest functional bispecific antibody) of anti-carcinoembryonic antigen (CEA) antibody by overexpression in Escherichia coli in inclusion bodies, using a refolding system to obtain renatured proteins.

Interleukin-17 promotes angiogenesis and tumor growth.

Interleukin-17 (IL-17) is a CD4 T-cell-derived proinflammatory cytokine. We investigated the effects of locally produced IL-17 by tumors as a means to evaluate its biologic function. Although recombinant IL-17 protein or retroviral transduction of IL-17 gene into tumors did not affect in vitro proliferation, IL-17 transfectants grew more rapidly in vivo when compared with controls.

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 gamma 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.

T-cell immunotherapy for human MK-1-expressing tumors using a fusion protein of the superantigen SEA and anti-MK-1 scFv antibody.

Background: The bacterial superantigen staphylococcal enterotoxin A (SEA) is an extremely potent activator of T lymphocytes when presented on major histocompatibility complex (MHC) class II molecules. To develop a tumor-specific superantigen for cancer therapy, we constructed a recombinant fusion protein of SEA and the single-chain variable fragment (scFv) of the FU-MK-1 antibody, which recognizes a glycoprotein antigen (termed MK-1 antigen) present on most carcinomas.

Materials And Methods: We employed recombinant DNA techniques to fuse recombinant mutant SEA to an scFv antibody derived from FU-MK-1 and the resulting fusion protein (SEA/FUscFv) was produced by a bacterial expression system, purified with a metal-affinity column, and characterized for its MK-1-binding specificity and its antitumor activity.

Novel vaccination protocol consisting of injecting MUC1 DNA and nonprimed dendritic cells at the same region greatly enhanced MUC1-specific antitumor immunity in a murine model.

In order to induce specific antitumor immunity in mice, we attempted to immunize C57BL/6 mice with DNA vaccine encoding MUC1 polypeptide. When the mice immunized with MUC1 DNA were challenged with EL4-muc, MUC1-transfected syngeneic lymphoma cells, they completely rejected tumors. When DNA vaccine was given to the EL4-muc tumor-bearing mice, this vaccination was insufficient to suppress tumor growth in the mice.

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.

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

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.