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

Watanabe, Yasuhiro; Asano, Ryutaro; Arai, Kyoko; Shimomura, Ippei; Ogata, Hiromi; Kawaguchi, Haruko; Hayashi, Hiroki; Ohtsuka, Hideo; Yoshida, Hiroshi; Katayose, Yu; Egawa, Shinichi; Nakanishi, Takeshi; Umetsu, Mitsuo; Ishida, Tadao; Imai, Kohzoh; Kudo, Tetsuichi; Unno, Michiaki; Kumagai, Izumi

doi:10.3892/or.2011.1382

Cited by 9 publications

(4 citation statements)

References 34 publications

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“…To verify the pharmacological activity of anti-EGFR and anti-EGFR-iRGD, in vitro cytotoxicity tests using MTT assays against BGC-823, Caco-2 and MCF-7 (EGFR lowly expressed) [38] were conducted. It was found that both anti-EGFR and cetuximab exhibited no anti-proliferative activity against BGC-823 cells even at the highest concentration of 240 µg/mL (Fig.…”

Section: Resultsmentioning

confidence: 99%

Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy

Sha

Zou

Kai

et al. 2015

Journal of Controlled Release

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Human tumors, including gastric cancer, frequently express high levels of epidermal growth factor receptors (EGFRs), which are associated with a poor prognosis. Targeted delivery of anticancer drugs to cancerous tissues shows potential in sparing unaffected tissues. However, it has been a major challenge for drug penetration in solid tumor tissues due to the complicated tumor microenvironment. We have constructed a recombinant protein named anti-EGFR-iRGD consisting of an anti-EGFR VHH (the variable domain from the heavy chain of the antibody) fused to iRGD, a tumor-specific binding peptide with high permeability. Anti-EGFR-iRGD, which targets EGFR and αvβ3, spreads extensively throughout both the multicellular spheroids and the tumor mass. The recombinant protein anti-EGFR-iRGD also exhibited antitumor activity in tumor cell lines, multicellular spheroids, and mice. Moreover, anti-EGFR-iRGD could improve anticancer drugs, such as doxorubicin (DOX), bevacizumab, nanoparticle permeability and efficacy in multicellular spheroids. This study draws attention to the importance of iRGD peptide in the therapeutic approach of anti-EGFR-iRGD. As a consequence, anti-EGFR-iRGD could be a drug candidate for cancer treatment and a useful adjunct of other anticancer drugs.

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Section: Resultsmentioning

confidence: 99%

Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy

Sha

Zou

Kai

et al. 2015

Journal of Controlled Release

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“…However, in this case the connection will be to the N-terminus of the scFv, i.e., close to its antigen-binding site. Typical linkers used for IgG-scFv fusion proteins are composed of 2 or 3 repeats of G 4 S. 216,217 Also other linkers such as a hydrophilic helical linker, e.g., with the sequence SNS(EEAKK) 3 SNS, have been used to fuse an scFv to the heavy chain C-terminus. 104 Short linkers might restrain antigen binding, although recently a three residue linker (GSS) was successfully used to generated an IgG-HC-scFv targeting HER2 and HER3.…”

Section: Bispecific Antibodies With a Symmetric Architecturementioning

confidence: 99%

The making of bispecific antibodies

Brinkmann

Kontermann

2017

mAbs

738

706

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During the past two decades we have seen a phenomenal evolution of bispecific antibodies for therapeutic applications. The 'zoo' of bispecific antibodies is populated by many different species, comprising around 100 different formats, including small molecules composed solely of the antigenbinding sites of two antibodies, molecules with an IgG structure, and large complex molecules composed of different antigen-binding moieties often combined with dimerization modules. The application of sophisticated molecular design and genetic engineering has solved many of the technical problems associated with the formation of bispecific antibodies such as stability, solubility and other parameters that confer drug properties. These parameters may be summarized under the term 'developability'. In addition, different 'target product profiles', i.e., desired features of the bispecific antibody to be generated, mandates the need for access to a diverse panel of formats. These may vary in size, arrangement, valencies, flexibility and geometry of their binding modules, as well as in their distribution and pharmacokinetic properties. There is not 'one best format' for generating bispecific antibodies, and no single format is suitable for all, or even most of, the desired applications. Instead, the bispecific formats collectively serve as a valuable source of diversity that can be applied to the development of therapeutics for various indications. Here, a comprehensive overview of the different bispecific antibody formats is provided.

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“…The functionalization of small bsAbs by the construction of Fc fusion formats has also been reported 3 . We found that hEx3-Fc was approximately ten times more effective at inhibiting cancer growth than hEx3-Db 24 , 34 .…”

Section: Discussionmentioning

confidence: 78%

Engineering the hinge region of human IgG1 Fc-fused bispecific antibodies to improve fragmentation resistance

Suzuki

Annaka

Konno

et al. 2018

Sci Rep

Self Cite

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Fc domain fusion can improve the therapeutic effects of relatively small biological molecules such as peptides, cytokines, and antibody fragments. Fc fusion proteins can also be used to enhance the cytotoxic effects of small bispecific antibodies (bsAbs). However, fragmentation of Fc fusion proteins, which mainly occurs around the hinge regions during production, storage, and circulation in the blood, is a major issue. In this study, we first investigated the mechanisms of fragmentation around the hinge region during storage using Fc-fused bsAbs with specificity for epidermal growth factor receptor and CD3 as a model. The fragmentation peaks generated by gel filtration analysis indicated that both contaminating proteases and dissolved active oxygen should be considered causes of fragmentation. We designed and constructed variants by introducing a point mutation into the upper hinge region, which reduced the cleavage caused by dissolved active oxygen, and shortened the hinge region to restrict access of proteases. These hinge modifications improved fragmentation resistance and did not affect the biological activity of the bsAbs in vitro. We confirmed the versatility of the hinge modifications using another Fc-fused bsAb. Our results show that hinge modifications to the Fc fusion protein, especially the introduction of a point mutation into the upper hinge region, can reduce fragmentation substantially, and these modifications can be used to improve the fragmentation resistance of other recombinant Fc fusion proteins.

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

Cited by 9 publications

References 34 publications

Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy

Tumor-penetrating peptide fused EGFR single-domain antibody enhances cancer drug penetration into 3D multicellular spheroids and facilitates effective gastric cancer therapy

The making of bispecific antibodies

Engineering the hinge region of human IgG1 Fc-fused bispecific antibodies to improve fragmentation resistance

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