Simplified monomeric VHH-Fc antibodies provide new opportunities for passive immunization

Greve, Henri De; Virdi, Vikram; Bakshi, Shruti; Depicker, Anna

doi:10.1016/j.copbio.2019.11.006

Cited by 20 publications

(21 citation statements)

References 25 publications

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“…Although the market consists predominantly of IgG subtype Fc fusions, there has been much interest in developing an alternative IgA-based Fc scaffold for the control of mucosal pathogens in applications such as oral passive immunization Bakema and Van Egmond, 2011;Lohse et al, 2011;De Greve et al, 2020). Unlike the IgG Fc, the IgA Fc assembles with two other subunits, a 15 kDa joining chain (JC) and a 70 kDa secretory component (SC), to form a secretory complex (sIgA) (Supplementary Figure 1).…”

Section: Introductionmentioning

confidence: 99%

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A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli

2021

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We previously isolated a single domain antibody (VHH) that binds Enterohemorrhagic Escherichia coli (EHEC) with the end-goal being the enteromucosal passive immunization of cattle herds. To improve the yield of a chimeric fusion of the VHH with an IgA Fc, we employed two rational design strategies, supercharging and introducing de novo disulfide bonds, on the bovine IgA Fc component of the chimera. After mutagenizing the Fc, we screened for accumulation levels after transient transformation in Nicotiana benthamiana leaves. We identified and characterized five supercharging and one disulfide mutant, termed ‘(5 + 1)Fc’, that improve accumulation in comparison to the native Fc. Combining all these mutations is associated with a 32-fold increase of accumulation for the Fc alone, from 23.9 mg/kg fresh weight (FW) to 599.5 mg/kg FW, as well as a twenty-fold increase when fused to a VHH that binds EHEC, from 12.5 mg/kg FW tissue to 236.2 mg/kg FW. Co-expression of native or mutated VHH-Fc with bovine joining chain (JC) and bovine secretory component (SC) followed by co-immunoprecipitation suggests that the stabilizing mutations do not interfere with the capacity of VHH-Fc to assemble with JC and FC into a secretory IgA. Both the native and the mutated VHH-Fc similarly neutralized the ability of four of the seven most prevalent EHEC strains (O157:H7, O26:H11, O111:Hnm, O145:Hnm, O45:H2, O121:H19 and O103:H2), to adhere to HEp-2 cells as visualized by immunofluorescence microscopy and quantified by fluorometry. These results collectively suggest that supercharging and disulfide bond tethering on a Fc chain can effectively improve accumulation of a VHH-Fc fusion without impacting VHH functionality.

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Section: Introductionmentioning

confidence: 99%

“…We subsequently showed that these chimeric fusions neutralize a 4th EHEC serotype, O145 (Chin-Fatt, unpublished). Simplified chimeric V H H-IgA Fc, which involve replacing the Fab region with a V H H, are much easier to produce and are encoded by a single gene, rather than four required for full sized sIgA (De Greve et al, 2020).…”

Section: Introductionmentioning

confidence: 99%

A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli

2021

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“…The conventional Igs used in passive immunization include IgG for intravenous injection and IgA for intestinal tract. Their production involves heavy chain and light chain expression and correct assembly, and the genes encoding J-chain and secretory component are also required for secretory IgA (SIgA), which is very complex and challenging (De Greve et al 2020 ). Nanobodies can be grafted on to the Fc domain of Immunoglobulin (IgG or IgA) at the hinge region to make monomeric nanobody-Fc antibodies, which are easy to manufacture because they are encoded by a single gene (De Greve et al 2020 ).…”

Section: Nanobody Expression In Plantsmentioning

confidence: 99%

“…Their production involves heavy chain and light chain expression and correct assembly, and the genes encoding J-chain and secretory component are also required for secretory IgA (SIgA), which is very complex and challenging (De Greve et al 2020 ). Nanobodies can be grafted on to the Fc domain of Immunoglobulin (IgG or IgA) at the hinge region to make monomeric nanobody-Fc antibodies, which are easy to manufacture because they are encoded by a single gene (De Greve et al 2020 ). Fc domain fusion extends their circulatory half-life to avoid rapid clearance from host (Hutt et al 2012 ), and is proved to be capable of boosting the nanobody-Fc protein accumulation levels in Arabidopsis seeds (De Buck et al 2013 ).…”

Section: Nanobody Expression In Plantsmentioning

confidence: 99%

“…Fc domain fusion extends their circulatory half-life to avoid rapid clearance from host (Hutt et al 2012 ), and is proved to be capable of boosting the nanobody-Fc protein accumulation levels in Arabidopsis seeds (De Buck et al 2013 ). Additionally, Fc domain fusion results in multiple valences, which is required for antibodies to block bacterial colonization through agglutination, and Fc domain also confers effector functions to the fused antibodies (De Greve et al 2020 ; Roche et al 2015 ).…”

Section: Nanobody Expression In Plantsmentioning

confidence: 99%

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Applications of nanobodies in plant science and biotechnology

2020

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Key message Present review summarizes the current applications of nanobodies in plant science and biotechnology, including plant expression of nanobodies, plant biotechnological applications, nanobody-based immunodetection, and nanobody-mediated resistance against plant pathogens. Abstract Nanobodies (Nbs) are variable domains of heavy chain-only antibodies (HCAbs) isolated from camelids. In spite of their single domain structure, nanobodies display many unique features, such as small size, high stability, and cryptic epitopes accessibility, which make them ideal for sophisticated applications in plants and animals. In this review, we summarize the current applications of nanobodies in plant science and biotechnology, focusing on nanobody expression in plants, plant biotechnological applications, determination of plant toxins and pathogens, and nanobody-mediated resistance against plant pathogens. Prospects and challenges of nanobody applications in plants are also discussed.

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Biochemistry, structure, and cellular internalization of a four nanobody‐bearing Fc dimer

et al. 2021

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VHH stands for the variable regions of heavy chain only of camelid IgGs. The VHH family forms a set of interesting proteins derived from antibodies that maintain their capacity to recognize the antigen, despite their relatively small molecular weight (in the 12,000 Da range). Continuing our exploration of the possibilities of those molecules, we chose to design alternative molecules with maintained antigen recognition, but enhanced capacity, by fusing four VHH with one Fc, the fragment crystallizable region of antibodies. In doing so, we aimed at having a molecule with superior quantitative antigen recognition (×4) while maintaining its size below the 110 kDa. In the present paper, we described the building of those molecules that we coined VHH2‐Fc‐VHH2. The structure of VHH2‐Fc‐VHH2 in complex with HER2 antigen was determined using electronic microscopy and modeling. The molecule is shown to bind four HER2 proteins at the end of its flexible arms. VHH2‐Fc‐VHH2 also shows an internalization capacity via HER2 receptor superior to the reference anti‐HER2 monoclonal antibody, Herceptin®, and to a simple fusion of two VHH with one Fc (VHH2‐Fc). This new type of molecules, VHH2‐Fc‐VHH2, could be an interesting addition to the therapeutic arsenal with multiple applications, from diagnostic to therapy.

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Simplified monomeric VHH-Fc antibodies provide new opportunities for passive immunization

Cited by 20 publications

References 25 publications

A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli

A Rationally Designed Bovine IgA Fc Scaffold Enhances in planta Accumulation of a VHH-Fc Fusion Without Compromising Binding to Enterohemorrhagic E. coli

Applications of nanobodies in plant science and biotechnology

Biochemistry, structure, and cellular internalization of a four nanobody‐bearing Fc dimer

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