Recent advances in the selection and identification of antigen-specific nanobodies

Liu, Wenshuai; Song, Haipeng; Chen, Quan; Yu, Jianliang; Xian, Ming; Nian, Rui; Feng, Dongxiao

doi:10.1016/j.molimm.2018.02.012

Cited by 86 publications

(92 citation statements)

References 96 publications

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“…demonstrated that most proteins could be isolated from jumbo squid ( Dosidicus gigas ) by pH‐shift processing due to acid and alkaline resistance . Similarly, nanobodies are usually extremely robust and resistant to acid and alkaline conditions . Therefore, we exploited this phenomenon and adjusted the pH of the PP to purify 2D5.…”

Section: Resultsmentioning

confidence: 99%

“…Conventional antibodies are composed of two heavy chains and two light chains and are heterotetrameric molecules, whereas HCAbs only consist of two heavy chains lacking CH1 . The smallest antibody type is a nanobody, which is mainly derived from variable fragments of HCAbs using molecular technology . Recently, nanobodies have attracted widespread interest because of their distinctive features, including small size (12–15 kDa, 2.5 nm in diameter, and 4.2 nm in length), simple structure, hydrophilicity, thermostability, high affinity and specificity, easy to be cloned and expressed in microorganisms with high yield, and immunogenicity comparable to human antibodies .…”

Section: Introductionmentioning

confidence: 99%

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Integrated purification of a nanobody using ammonium sulfate precipitation and Capto MMC

Fan

Liang

Li³

et al. 2019

J of Chemical Tech & Biotech

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BACKGROUND: Currently, nanobodies are undergoing intensive studies due to their characteristics of nanoscale size, high affinity and specificity, and deep tissue penetration. The development of novel and simplified methods to purify nanobodies is required because high purity and high recovery are the limiting factors for the downstream preparation of nanobodies. RESULTS: In this study, ammonium sulfate precipitation (ASP) and electropositive multimodal chromatography (Capto MMC)were integrated for tag-free nanobody (2D5) purification. ASP was our first choice for protein purification because the process is simple, easy to operate, and has low cost. ASP achieved a 36% reduction in Escherichia coli host cell proteins (HCPs), a 99.1% reduction in E. coli host cell DNA, and an 85.1% reduction in endotoxins with a recovery of 81.7% and a purity enhancement from 6.2% to 73.9%. Capto MMC was seamlessly integrated for 2D5 purification. The recovery of this step was 83.0%. The entire two-step purification platform yielded 2D5 with 397 ppm of HCPs, 198 ppb of DNA, 204 EU/mL of endotoxins, and total protein recovery of 67.8%. The purity of 2D5 was 97.5%. The purification method was also used to purify a nanobody against PD-L1 (KPU) with 97.7% purity, > 95.7% recovery, 223 ppm of HCPs, 634 ppb of DNA and 154 EU/mL of endotoxins. CONCLUSION: The established method had no effect on the affinity of 2D5. A simple and efficient purification platform that integrates ASP and Capto MMC was established, providing a more promising alternative to the conventional affinity chromatography-based nanobody purification strategy. Affinity analysis of 2D5The affinity of antibodies refers to the bond strength between the antibody binding sites and the antigenic determinant; it is one of the fundamental kinetic parameters in the drug discovery J Chem Technol Biotechnol 2020; 95: 246-254

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Section: Resultsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Integrated purification of a nanobody using ammonium sulfate precipitation and Capto MMC

Fan

Liang

Li³

et al. 2019

J of Chemical Tech & Biotech

Self Cite

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“…Currently, the nanobody is evolving into versatile research and application tool kits for related biomedical and biotechnology applications [ 18 , 29 , 30 ], including targeting drug delivery and therapy [ 17 ], disease diagnosis [ 31 ], bioimaging [ 32 ], and agricultural and plant protection [ 33 ]. Nanobodies are good candidates to improve the physicochemical properties and maintain a degree of recognition when compared to conventional antibodies [ 34 ]. The low stability and affinity of antibodies can be overcome by nanobodies due to their high stability and ability to keep affinity parameters in the nanomolar range [ 35 ].…”

Section: Discussionmentioning

confidence: 99%

A Novel Nanobody Directed against Ovine Myostatin to Enhance Muscle Growth in Mouse

et al. 2020

Animals

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Myostatin (MSTN) is a member of the transforming growth factor beta superfamily and is a negative regulator of myogenesis. It has been shown to function by controlling the proliferation of myoblasts. MSTN inhibition is considered as a promising treatment for promoting animal growth in livestock. Nanobodies, a special antibody discovered in camel, have arisen as an alternative to conventional antibodies and have shown great potential when used as tools in different biotechnology fields, such as diagnostics and therapy. In this study, we examined the effect of MSTN inhibition by RMN on the muscle growth of mice. The results showed that RMN could specifically detect and bind MSTN, as well as inhibit MSTN activity. A significant increase in skeletal muscle mass was observed after intramuscular injection of RMN into mice. Enhanced muscle growth occurred because of myofiber hypertrophy. These results offer a promising approach to enhance muscle growth that warrants further investigation in domestic animals.

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“…Conventionally, nanobodies are selected from immune phage libraries [ 25 , 26 , 27 ]. Until today, a variety of VHH with specificities against different classes of molecules such as protein ligands [ 28 ], hormones [ 29 , 30 ], small molecule drugs [ 31 ], toxins [ 32 , 33 , 34 , 35 ], and other chemicals [ 36 , 37 ] have been developed.…”

Section: Introductionmentioning

confidence: 99%

A Two-Step Approach for the Design and Generation of Nanobodies

Wagner

Wehrle

Weiss

et al. 2018

IJMS

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Nanobodies, the smallest possible antibody format, have become of considerable interest for biotechnological and immunotherapeutic applications. They show excellent robustness, are non-immunogenic in humans, and can easily be engineered and produced in prokaryotic hosts. Traditionally, nanobodies are selected from camelid immune libraries involving the maintenance and treatment of animals. Recent advances have involved the generation of nanobodies from naïve or synthetic libraries. However, such approaches demand large library sizes and sophisticated selection procedures. Here, we propose an alternative, two-step approach for the design and generation of nanobodies. In a first step, complementarity-determining regions (CDRs) are grafted from conventional antibody formats onto nanobody frameworks, generating weak antigen binders. In a second step, the weak binders serve as templates to design focused synthetic phage libraries for affinity maturation. We validated this approach by grafting toxin- and hapten-specific CDRs onto frameworks derived from variable domains of camelid heavy-chain-only antibodies (VHH). We then affinity matured the hapten binder via panning of a synthetic phage library. We suggest that this strategy can complement existing immune, naïve, and synthetic library based methods, requiring neither animal experiments, nor large libraries, nor sophisticated selection protocols.

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Recent advances in the selection and identification of antigen-specific nanobodies

Cited by 86 publications

References 96 publications

Integrated purification of a nanobody using ammonium sulfate precipitation and Capto MMC

Integrated purification of a nanobody using ammonium sulfate precipitation and Capto MMC

A Novel Nanobody Directed against Ovine Myostatin to Enhance Muscle Growth in Mouse

A Two-Step Approach for the Design and Generation of Nanobodies

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