Highly Effective Recombinant Format of a Humanized IgG-like Bispecific Antibody for Cancer Immunotherapy with Retargeting of Lymphocytes to Tumor Cells

Asano, Ryutaro; Watanabe, Yasuhiro; Kawaguchi, Haruko; Fukazawa, Hidesuke; Nakanishi, Takeshi; Umetsu, Mitsuo; Hayashi, Hiroki; Katayose, Yu; Unno, Michiaki; Kudo, Tetsuichi; Kumagai, Izumi

doi:10.1074/jbc.m704719200

Cited by 55 publications

(48 citation statements)

References 32 publications

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“…These antibodies included IgG-like bispecific antibodies that contain the human Fc region, and studies to assess their usefulness for clinical therapy are currently underway [15,20,21]. However, the recruitment of T cells by bispecific antibodies has led to concerns about side effects, including effects on the central nervous system, and this has led to the permanent discontinuation of study drug in some cases [22].…”

Section: Discussionmentioning

confidence: 99%

Multimerization of anti‐(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers

et al. 2013

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The construction of antibody fragments has the potential to reduce the high cost of therapeutic antibody production, but the structures of these fragments, with monovalency and the lack of an Fc region, can lead to reduced function. Multimerization is one strategy for recovering function that also yields better tumor-to-blood ratios than IgGs or monomeric antibody fragments because of rapid tumor uptake and clearance. Here, we constructed single-chain variable fragment (scFv) multimers by modifying the linker length and domain order of humanized anti-(epidermal growth factor receptor) IgG 528 (h528) and tested their ability to inhibit tumor growth. h528 scFv multimers, expressed using a bacterial expression system, were successfully fractionated and inhibited cancer growth in a multimerization-dependent manner, whereas the h528 scFv monomer showed no inhibition. h528 scFv trimers with variable heavy-light domain order and no linkers showed the highest in vitro and in vivo antitumor effects, which were comparable with those of the approved anti-(epidermal growth factor receptor) therapeutic IgG Cetuximab and Panitumumab. The trimers were also structurally stable in vitro and in vivo, which may be attributable to a strong interaction between the variable heavy and variable light domains of h528 Fv. Thus, h528 scFv multimers, especially trimers, are attractive as the next generation of anti-(epidermal growth factor receptor) therapeutic IgG and offer the possibility of low-cost production.

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Section: Discussionmentioning

confidence: 99%

Multimerization of anti‐(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers

et al. 2013

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“…Immune-deficient mice xenografted with human cancer can be used to evaluate ADCC induced from human IgG1 antibodies (34) and glycosylation in the Fc region of the antibody-heavy chain is indispensable for both their interactions with Fc receptors and FcR-mediated effector functions, including ADCC (35,36). We previously confirmed that the glycosylation pattern of hEx3-scDb-Fc is quite similar to that of humanized IgG1 produced by CHO cells (37) and the binding of hEx3-scFv-Fc mediates the inhibition of the phosphorylation of protein kinase (23). These facts suggest that the monotherapeutic effects of IgG-like BsAbs based on hEx3 are due to the induction of ADCC and the inhibition of receptor tyrosine kinase, similar to the mechanisms that have been demonstrated for cetuximab (38,39).…”

Section: Discussionmentioning

confidence: 57%

“…Therefore, we focused on IgG-like BsAbs to improve the Db and fabricated two IgG-like BsAbs based on hEx3-Db: hEx3-scFv-Fc and hEx3-scDb-Fc ( Fig. 1) (23,24). Both of these IgG-like BsAbs have two pairs of humanized Fv portions with specificity for EGFR and CD3, and were more cytotoxic than hEx3-Db.…”

Section: In Vitro and In Vivo Antitumor Effects Of Recombinant Bispecmentioning

confidence: 99%

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

Watanabe

Asano

Arai³

et al. 2011

Oncol Rep

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Abstract. 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. In vitro, hEx3-scFv-Fc showed nearly 100% killing activity for all cell lines. Both in vitro and in vivo, hEx3-Db needed CD3-positive phenotypes to induce a growth inhibitory effect. In contrast, IgG-like BsAbs showed monotherapeutic effects in vivo by inducing antibody-dependent cellular cytotoxicity (ADCC) similar to cetuximab. However, enhancement was not observed when lymphokine-activated killer cells with the T-cell phenotype were co-injected. Results suggest that IgG-like BsAbs could not efficiently direct T lymphocytes toward tumor cells to induce ADCC due to steric hindrance on binding to CD3-and Fc-receptor-positive phenotypes. Although hEx3-scFv-Fc showed high cytotoxicity in vitro, its high molecular weight limits its usefulness. With an in vivo effect comparable to hEx3-scFv-Fc and its realistic molecular weight, hEx3-scDb-Fc shows promise as a novel recombinant therapeutic antibody and may be modified to enhance its potency by prevention of steric hindrance.

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“…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

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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.

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Highly Effective Recombinant Format of a Humanized IgG-like Bispecific Antibody for Cancer Immunotherapy with Retargeting of Lymphocytes to Tumor Cells

Cited by 55 publications

References 32 publications

Multimerization of anti‐(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers

Multimerization of anti‐(epidermal growth factor receptor) IgG fragments induces an antitumor effect: the case for humanized 528 scFv multimers

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

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

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