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

Hayashi, Hiroki; Asano, Ryutaro; Tsumoto, Kouhei; Katayose, Yu; Suzuki, Masanori; Unno, Michiaki; Kodama, Hideaki; Takemura, Shin Ichi; Yoshida, Hiroshi; Makabe, Koki; Imai, Kohzoh; Matsuno, Seiki; Kumagai, Izumi; Kudo, Tetsuichi

doi:10.1007/s00262-003-0465-9

Cited by 34 publications

(24 citation statements)

References 57 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…We also confirmed the dualspecificity of hEx3 by absorption test according to previous report (data not shown; ref. 22).…”

Section: Resultsmentioning

confidence: 99%

“…Ex3 was stable and retained binding reactivity after incubation at 37jC for 48 hours. Furthermore, administration of Ex3 and T-LAK cells, without other costimulatory factors, to tumorxenografted mice resulted in pronounced inhibition of tumor growth, with complete tumor disappearance in half of the mice (22).…”

Section: Discussionmentioning

confidence: 99%

“…We previously constructed an anti-EGFR/anti-CD3 bispecific diabody, termed Ex3, which specifically targeted both LAK with T-cell phenotype (T-LAK) and resting peripheral blood mononuclear cells against EGFRpositive cell lines. Furthermore, coadministration of the Ex3 diabody with T-LAK cells in bile duct carcinoma-xenografted severe combined immunodeficient mice resulted in pronounced inhibition of tumor growth (22).…”

mentioning

confidence: 99%

See 2 more Smart Citations

Humanization of the Bispecific Epidermal Growth Factor Receptor × CD3 Diabody and Its Efficacy as a Potential Clinical Reagent

Asano

Sone

Makabe

et al. 2006

Clinical Cancer Research

Self Cite

View full text Add to dashboard Cite

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. Experimental Design: We have reported significant antitumor activity of an anti-epidermal growth factor receptor (EGFR) Â anti-CD3 bispecific diabody (Ex3) in in vitro cytotoxicity assays and in vivo. We humanized the Ex3 diabody (hEx3) by grafting the complementaritydetermining region and compared its biological properties with those of Ex3. We also tested its physiologic stability and ability to alter survival in xenografted mice. Results: The final yield of hEx3 was 10 times that of Ex3, and refolded hEx3 and Ex3 showed identical binding profiles in EGFR-positive cell lines and EGFR-transfected Chinese hamster ovary cells. hEx3 showed dose-dependent cytotoxicity to EGFR-positive cell lines, which could be specifically inhibited by parental monoclonal antibody IgGs against EGFR or CD3 antigens. The heterodimeric structure was retained in PBS for 6 months, and growth inhibition was maintained after incubation under physiologic conditions. Coadministration of hEx3 with T-LAK cells and interleukin-2 prolonged the survival of nude mice with human colon carcinoma. Conclusions:The humanized diabody hEx3 is an attractive molecule for cancer therapy and may provide important insights into the development of EGFR-based cancer-targeting reagents.

show abstract

“…We also confirmed the dualspecificity of hEx3 by absorption test according to previous report (data not shown; ref. 22).…”

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

See 1 more Smart Citation

Humanization of the Bispecific Epidermal Growth Factor Receptor × CD3 Diabody and Its Efficacy as a Potential Clinical Reagent

Asano

Sone

Makabe

et al. 2006

Clinical Cancer Research

Self Cite

View full text Add to dashboard Cite

show abstract

“…The 528 mAb is a murine IgG2a directed to the human EGFR antigen (26). The V-region genes of 528 hybridoma cells were cloned by reverse transcription (RT)-PCR as described previously (27). The heavy chain (VH) and light chain (VL) genes were then amplified by PCR using the primer mix previously reported by Krebber et al (28).…”

Section: Methodsmentioning

confidence: 99%

“…528scFv-TRAIL fusion protein was prepared from inclusion bodies expressed in E. coli as described previously (27,31). In brief, E. coli strain BL21 (DE3) harboring the bacterial expression vector pUT-528scFv-TRAIL was grown at 28˚C in Luria-Bertani broth.…”

Section: Methodsmentioning

confidence: 99%

Target cell-restricted apoptosis induction by 528scFv-TRAIL fusion protein specific for human EGFR and expressed in Escherichia coli

Badran

Asano

Nakayama³

et al. 2010

Int J Oncol

View full text Add to dashboard Cite

Abstract. We report the preparation and functional characterization of an Escherichia coli-expressed recombinant fusion protein, 528scFv-TRAIL, specific for the human epidermal growth factor receptor (EGFR) and empowered by the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). The 528scFv-TRAIL, expressed as insoluble inclusion bodies in E. coli, was solubilized and then refolded by using a modified stepwise dialysis method. Treatment with 528scFv-TRAIL resulted in the specific binding to the cell surface of EGFRpositive cells with concomitant deployment of the TRAIL moiety to DR-5 receptor in a manner comparable to a commercially available form of recombinant TRAIL (cTRAIL). 528scFv-TRAIL, prepared by either of three refolding processes described herein, showed potent cytotoxic activity against EGFR-positive TFK-1 cell line and was superior to its parental 528scFv; a recombinant variable fragment with single specificity against human EGFR. Narrow variations in the cytotoxic potential of 528scFv-TRAIL were ascribed to manipulation of redox conditions during the refolding process. Together, our findings point to the potential value of 528scFv-TRAIL for treatment of EGFR-expressing cancers. Furthermore, preparation of 528scFv-TRAIL from insoluble aggregates in a prokaryotic cell based expression system by means of in vitro refolding introduces a feasible cost-benefit, time-efficient approach for industrial-scale production.

show abstract

Bifunctional Antibodies: Preclinical and Clinical Applications

Shuptrine

Weiner

2013

Advances in Tumor Immunology and Immunotherapy

View full text Add to dashboard Cite

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

Abstract: The Ex3 diabody can be considered a highly promising reagent for study of specific targeting immunotherapy against bile duct and other EGFR-positive carcinomas.

Cited by 34 publications

References 57 publications

Humanization of the Bispecific Epidermal Growth Factor Receptor × CD3 Diabody and Its Efficacy as a Potential Clinical Reagent

Humanization of the Bispecific Epidermal Growth Factor Receptor × CD3 Diabody and Its Efficacy as a Potential Clinical Reagent

Target cell-restricted apoptosis induction by 528scFv-TRAIL fusion protein specific for human EGFR and expressed in Escherichia coli

Bifunctional Antibodies: Preclinical and Clinical Applications

Contact Info

Product

Resources

About