Construction of a circularly connected VHH bispecific antibody (cyclobody) for the desirable positioning of antigen-binding sites

Hemmi, Saki; Asano, Ryutaro; Kimura, Kouki; Umetsu, Mitsuo; Nakanishi, Takeshi; Kumagai, Izumi; Makabe, Koki

doi:10.1016/j.bbrc.2019.12.018

Cited by 11 publications

(10 citation statements)

References 20 publications

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“…In addition to the C-7 bispecific VHH, various other EGFR-CD16 bispecific antibodies have recently been reported. These include a similarly structured bispecific VHH, that triggered NK cell degranulation and IFN-γ release but has not yet formally demonstrated actual tumor cell lysis [65], an anti-EGFR x anti-CD16 bispecific VHH cyclobody (Ex16), where both the N and C termini of the VHHs are linked through split-intein circular ligation to protect the compound from proteolysis, but which also negatively impacts its cytotoxic potential [66], and AFM24, an IgG 1 -scFv fusion antibody [67], that, due to its relatively large size (~200 kD versus ~30 kD for the C-7 bispecific VHH), may have reduced potential to deeply penetrate tumor tissue. In this study, we focused on the development of a bispecific antibody approach to target NK cells to EGFR expressing colorectal cancer.…”

Section: Discussionmentioning

confidence: 99%

Enhancement of NK Cell Antitumor Effector Functions Using a Bispecific Single Domain Antibody Targeting CD16 and the Epidermal Growth Factor Receptor

Toffoli

Sheikhi

Lameris

et al. 2021

Cancers

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The ability to kill tumor cells while maintaining an acceptable safety profile makes Natural Killer (NK) cells promising assets for cancer therapy. Strategies to enhance the preferential accumulation and activation of NK cells in the tumor microenvironment can be expected to increase the efficacy of NK cell-based therapies. In this study, we show binding of a novel bispecific single domain antibody (VHH) to both CD16 (FcRγIII) on NK cells and the epidermal growth factor receptor (EGFR) on tumor cells of epithelial origin. The bispecific VHH triggered CD16- and EGFR-dependent activation of NK cells and subsequent lysis of tumor cells, regardless of the KRAS mutational status of the tumor. Enhancement of NK cell activation by the bispecific VHH was also observed when NK cells of colorectal cancer (CRC) patients were co-cultured with EGFR expressing tumor cells. Finally, higher levels of cytotoxicity were found against patient-derived metastatic CRC cells in the presence of the bispecific VHH and autologous peripheral blood mononuclear cells or allogeneic CD16 expressing NK cells. The anticancer activity of CD16-EGFR bispecific VHHs reported here merits further exploration to assess its potential therapeutic activity either alone or in combination with adoptive NK cell-based therapeutic approaches.

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Section: Discussionmentioning

confidence: 99%

Enhancement of NK Cell Antitumor Effector Functions Using a Bispecific Single Domain Antibody Targeting CD16 and the Epidermal Growth Factor Receptor

Toffoli

Sheikhi

Lameris

et al. 2021

Cancers

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“…Therefore, multispecific antibodies with native IgG frameworks are still considered most desirable. Recently, intein-based protein splicing methods have proven highly effective for generating multispecific antibodies from antibody fragments [ 83 , 84 ] as well as native IgG frameworks [ 85 , 86 ]. Although the full impact of this technology has yet to be realized, the ability to make multispecifics with fully native IgG Fc regions will undoubtedly have significant implications for multispecific antibody production for a variety of reasons.…”

Section: Physical and Chemical Stabilitymentioning

confidence: 99%

Toward Drug-Like Multispecific Antibodies by Design

Sawant

Streu

et al. 2020

IJMS

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The success of antibody therapeutics is strongly influenced by their multifunctional nature that couples antigen recognition mediated by their variable regions with effector functions and half-life extension mediated by a subset of their constant regions. Nevertheless, the monospecific IgG format is not optimal for many therapeutic applications, and this has led to the design of a vast number of unique multispecific antibody formats that enable targeting of multiple antigens or multiple epitopes on the same antigen. Despite the diversity of these formats, a common challenge in generating multispecific antibodies is that they display suboptimal physical and chemical properties relative to conventional IgGs and are more difficult to develop into therapeutics. Here we review advances in the design and engineering of multispecific antibodies with drug-like properties, including favorable stability, solubility, viscosity, specificity and pharmacokinetic properties. We also highlight emerging experimental and computational methods for improving the next generation of multispecific antibodies, as well as their constituent antibody fragments, with natural IgG-like properties. Finally, we identify several outstanding challenges that need to be addressed to increase the success of multispecific antibodies in the clinic.

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“…A similar approach yielded a CD3xPRLR bispecific antibody for T-cell activation and cytokine release towards PRLR expressing breast cancer cells [104]. In addition to Fc-based bispecifics, also non-Fc, circularly connected VHH fragments (cyclobody) have been developed via SICLOPPS (Split Intein Circular Ligation of Peptides and Proteins) reaction between both C-and N-termini, forming a cyclic conformation after PTS [105]. These cyclobodies are, as discussed before for other applications, protected from proteolysis due to their cyclic topology, yet they retain their dual specificity.…”

Section: Split Inteinsmentioning

confidence: 99%

“…These cyclobodies are, as discussed before for other applications, protected from proteolysis due to their cyclic topology, yet they retain their dual specificity. An anti-EGFRxCD16 cyclobody was successfully generated to show cytotoxicity against EGFR-positive cancer cells, able to bind simultaneously EGFR and CD16 on the cell surface [105]. Applicability of the aforementioned split intein Aes PolB1 intein was demonstrated by successful bioconjugation of several therapeutically relevant formats like full-length IgG, Fc, and VHH fusions [101].…”

Section: Split Inteinsmentioning

confidence: 99%

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

Hofmann

Krah

Sellmann

et al. 2020

IJMS

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Recent years have shown a tremendous increase and diversification in antibody-based therapeutics with advances in production techniques and formats. The plethora of currently investigated bi- to multi-specific antibody architectures can be harnessed to elicit a broad variety of specific modes of actions in oncology and immunology, spanning from enhanced selectivity to effector cell recruitment, all of which cannot be addressed by monospecific antibodies. Despite continuously growing efforts and methodologies, the identification of an optimal bispecific antibody as the best possible combination of two parental monospecific binders, however, remains challenging, due to tedious cloning and production, often resulting in undesired extended development times and increased expenses. Although automated high throughput screening approaches have matured for pharmaceutical small molecule development, it was only recently that protein bioconjugation technologies have been developed for the facile generation of bispecific antibodies in a ‘plug and play’ manner. In this review, we provide an overview of the most relevant methodologies for bispecific screening purposes—the DuoBody concept, paired light chain single cell production approaches, Sortase A and Transglutaminase, the SpyTag/SpyCatcher system, and inteins—and elaborate on the benefits as well as drawbacks of the different technologies.

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Construction of a circularly connected VHH bispecific antibody (cyclobody) for the desirable positioning of antigen-binding sites

Cited by 11 publications

References 20 publications

Enhancement of NK Cell Antitumor Effector Functions Using a Bispecific Single Domain Antibody Targeting CD16 and the Epidermal Growth Factor Receptor

Enhancement of NK Cell Antitumor Effector Functions Using a Bispecific Single Domain Antibody Targeting CD16 and the Epidermal Growth Factor Receptor

Toward Drug-Like Multispecific Antibodies by Design

Greatest Hits—Innovative Technologies for High Throughput Identification of Bispecific Antibodies

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