A comprehensive comparison between camelid nanobodies and single chain variable fragments

Asaadi, Yasaman; Jouneghani, Fatemeh Fazlollahi; Janani, Sara; Rahbarizadeh, Fatemeh

doi:10.1186/s40364-021-00332-6

Cited by 108 publications

(102 citation statements)

References 237 publications

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“…This strategy was also selected since denosumab is a registered drug used routinely in clinical practice. The generation of Fab fragments via papain digestion is a well-described, easy-to-use method and requires minimal optimization, whereas preparing other antibody fragments (VHH, VNAR, scFv) requires extensive know-how [ 35 ]. Furthermore, it applies to multiple antibodies of different origins and subtypes [ 36 ].…”

Section: Discussionmentioning

confidence: 99%

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments

et al. 2022

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Purpose: RANKL expression in the tumor microenvironment has been identified as a biomarker of immune suppression, negating the effect of some cancer immunotherapies. Previously we had developed a radiotracer based on the FDA-approved RANKL-specific antibody denosumab, [89Zr]Zr-DFO-denosumab, enabling successful immuno-PET imaging. Radiolabeled denosumab, however, showed long blood circulation and delayed tumor uptake, potentially limiting its applications. Here we aimed to develop a smaller radiolabeled denosumab fragment, [64Cu]Cu-NOTA-denos-Fab, that would ideally show faster tumor accumulation and better diffusion into the tumor for the visualization of RANKL. Experimental design: Fab fragments were prepared from denosumab using papain and conjugated to a NOTA chelator for radiolabeling with 64Cu. The bioconjugates were characterized in vitro using SDS-PAGE analysis, and the binding affinity was assessed using a radiotracer cell binding assay. Small animal PET imaging evaluated tumor targeting and biodistribution in transduced RANKL-ME-180 xenografts. Results: The radiolabeling yield of [64Cu]Cu-NOTA-denos-Fab was 58 ± 9.2%, with a specific activity of 0.79 ± 0.11 MBq/µg (n = 3). A radiotracer binding assay proved specific targeting of RANKL in vitro. PET imaging showed fast blood clearance and high tumor accumulation as early as 1 h p.i. (2.14 ± 0.21% ID/mL), which peaked at 5 h p.i. (2.72 ± 0.61% ID/mL). In contrast, [64Cu]Cu-NOTA-denosumab reached its highest tumor uptake at 24 h p.i. (6.88 ± 1.12% ID/mL). [64Cu]Cu-NOTA-denos-Fab specifically targeted human RANKL in transduced ME-180 xenografts compared with the blocking group and negative ME-180 xenograft model. Histological analysis confirmed RANKL expression in RANKL-ME-180 xenografts. Conclusions: Here, we report on a novel RANKL PET imaging agent, [64Cu]Cu-NOTA-denos-Fab, that allows for fast tumor imaging with improved imaging contrast when compared with its antibody counterpart, showing promise as a potential PET RANKL imaging tool for future clinical applications.

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Section: Discussionmentioning

confidence: 99%

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments

et al. 2022

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“…The single-domain Ab of camelidae is fragmentary and a therapeutic Ab. Due to the small size (~ 15 kDa) and the fast metabolism (~ 30 min) in the organisms 19 – 21 , appropriate modifications are essential to improve the pharmacokinetic and pharmacodynamic performances of single domain Abs 22 , 23 that can be executed via VHH tandem (for example Cablivi: VHH-AlaAlaAla-VHH) 11 . However, human IgG Fc fusion is the general strategy for therapeutic VHH Abs 13 , 24 .…”

Section: Discussionmentioning

confidence: 99%

Development and characterization of a camelid derived antibody targeting a linear epitope in the hinge domain of human PCSK9 protein

Hong

Gao

et al. 2022

Sci Rep

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PCSK9 is an effective target for lowering LDL-c. Previously, a camelid-human chimeric heavy chain antibody VHH-B11-Fc targeting human PCSK9 was designed. It had a potent hypolipidemic effect. However, the nanobody VHH-B11 interacts with PCSK9 at low affinity, while camelid VHH exhibits some immunogenicity. Moreover, the interacting epitope is yet to be identified, although VHH-B11 was shown to have distinct hPCSK9-binding epitopes for Evolocumab. This might impede the molecule’s progress from bench to bedside. In the present study, we designed various configurations to improve the affinity of VHH-B11 with hPCSK9 (< 10 nM) that in turn enhanced the druggability of VHH-B11-Fc. Then, 17 amino acids were specifically mutated to increase the degree of humanization of the nanobody VHH-B11. Using phage display and sequencing technology, the linear epitope “STHGAGW” (amino acids 447–452) was identified in the hinge region of PCSK9 as the interacting site between VHH-B11-Fc and hPCSK9. Unlike the interaction epitope of Evolocumab, located in the catalytic region of PCSK9, the binding epitope of VHH-B11 is located in the hinge region of PCSK9, which is rarely reported. These findings indicated that a specific mechanism underlying this interaction needs to be explored.

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“…Beyond diagnostic and therapeutic applications, this characteristic is essential for the design and production of the so-called intrabodies, which recognize intracellular molecules, and therefore, should be capable of functioning in the cytoplasm [ 40 , 41 , 42 ]. This research field is gaining special relevance because of the great potential of intrabodies, for example, to characterize and modulate intracellular physiological processes and to inhibit the function of cytoplasmic targets [ 40 , 42 , 43 , 44 , 45 ]. Likewise, stability in a reducing environment is essential for expression in the cellular cytoplasm of bacteria and eukaryotic cells [ 46 ].…”

Section: Structural Bases For the Design Of Synthetic Nb Librariesmentioning

confidence: 99%

Structural Insights into the Design of Synthetic Nanobody Libraries

et al. 2022

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Single domain antibodies from camelids, or nanobodies, are a unique class of antibody fragments with several advantageous characteristics: small monomeric size, high stability and solubility and easy tailoring for multiple applications. Nanobodies are gaining increasing acceptance as diagnostic tools and promising therapeutic agents in cancer and other diseases. While most nanobodies are obtained from immunized animals of the camelid family, a few synthetic nanobody libraries constructed in recent years have shown the capability of generating high quality nanobodies in terms of affinity and stability. Since this synthetic approach has important advantages over the use of animals, the recent advances are indeed encouraging. Here we review over a dozen synthetic nanobody libraries reported so far and discuss the different approaches followed in their construction and validation, with an emphasis on framework and hypervariable loop design as critical issues defining their potential as high-class nanobody sources.

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A comprehensive comparison between camelid nanobodies and single chain variable fragments

Cited by 108 publications

References 237 publications

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments

Improved Characteristics of RANKL Immuno-PET Imaging Using Radiolabeled Antibody Fab Fragments

Development and characterization of a camelid derived antibody targeting a linear epitope in the hinge domain of human PCSK9 protein

Structural Insights into the Design of Synthetic Nanobody Libraries

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