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Order Caudovirales
Published in Paul Pumpens, Peter Pushko, Philippe Le Mercier, Virus-Like Particles, 2022
Paul Pumpens, Peter Pushko, Philippe Le Mercier
The phage λ-based display was used for the cell targeting. Thus, the cancer cells were targeted by the display of high-affinity nanobody against HER2 (Shoae-Hassani et al. 2013). The human 293T cell line was targeted by the display of chemically coupled human holotransferrin, and delivery of the GFP-encoding gene was realized (Khalaj-Kondori et al. 2011). The mammalian cell lines were also targeted by display of the following addressing tools: the RGD motif (Dunn 1996); TAT transduction domain of HIV-1 (Eguchi et al. 2001; Wadia et al. 2013); single-chain antibodies (Gupta et al. 2003) and single-chain anti-CEA antibody (Vaccaro et al. 2006); the full-length adenoviral penton base or its central domain of aa 286–393 (Piersanti et al. 2004); ανβ3 integrin-binding peptide (Zanghi et al. 2005) together with delivery of luciferase gene (Lankes et al. 2007); ubiquitinylation and CD-40 binding motifs (Zanghi et al. 2007); FcγRI (Sapinoro et al. 2008). The Cry1Ac toxin was displayed to address the VLPs to the receptor of Bacillus thuringiensis insecticidal toxin (Vílchez et al. 2004).
Scanning Angle Interference Microscopy (SAIM)
Published in Qiu-Xing Jiang, New Techniques for Studying Biomembranes, 2020
Cristina Bertocchi, Timothy J. Rudge, Andrea Ravasio
SAIM is feasible for fixed samples as well as live-cell imaging. To avoid loss of accuracy we do recommend special attention to the choice of fluorophores. Although SAIM does not require special fluorophores, the fluorophores should have high photostability to minimize photobleaching during the imaging scanning sequence and should be bright with high quantum yield to provide a good signal-to-noise ratio. Among the fluorescent proteins and synthetic dyes compatible with SAIM, genetically encoded fluorescent proteins have the principal advantage of being small and suitable for live-cell imaging and are capable of achieving maximal labeling specificity, removing any possible problems associated with nonspecific labeling. Furthermore, they do not require fixation or permeabilization procedures that could affect cellular nanostructure. Some fluorescent proteins successfully used in SAIM include green fluorescent proteins such as EGFP and mEmerald, red fluorescent protein mCherry,33 and photoconverted tdEOS25 that has excellent brightness and photostability. In addition, chromobodies (generated by the fusion of a fluorescent protein to a nanobody34), a novel species of extremely small antibodies that are endogenously synthesized within cultured cells, could possibly be used to prepare samples for SAIM.
Role of Engineered Proteins as Therapeutic Formulations
Published in Peter Grunwald, Pharmaceutical Biocatalysis, 2019
Khushboo Gulati, Krishna Mohan Poluri
Single variable domain from antibody heavy chain (HCAbs) is known as nanobody. Nanobodies are of nanometer size and also contain mutations that prevent the interaction of heavy chain with light chain. These mutations include V42F, G49E, L50R, and W52G that keep the HCAbs more stable and soluble in aqueous environment. Nanobodies are alternatively known as VHH domains. Structurally, they are small, 15 kDa in size, and comprise two β-sheets, three variable loops, and a disulfide bond. Nanobodies are highly stable both chemically and thermally compared to antibodies. Hence, they can be produced to reside in more harsh conditions (Hassanzadeh-Ghassabeh et al., 2013). Nanobodies can be designed for the targets for which the antibodies are not accessible (Muyldermans, 2013). A Belgian company “Ablynx” is designing nanobodies for treating number of diseases that include: psoriasis, rheumatoid arthritis, inflammatory, neurological disorders, and oncology (Steeland et al., 2016).
Investigation of the therapeutic potential of recombinant bispecific bivalent anti-PD-L1/VEGF nanobody in inhibition of angiogenesis
Published in Immunopharmacology and Immunotoxicology, 2023
Ayda Hassanzadeh Eskafi, Akbar Oghalaei, Fereidoun Mahboudi, Hajarsadat Ghaderi, Mahdi Behdani, Alireza Shoari, Fatemeh Kazemi-Lomedasht
In this study, a bivalent anti-PD-L1/VEGF nanobody was developed and its activity was evaluated. The bivalent anti-PD-L1/VEGF nanobody constructed using the IgA1 hinge that features a much extended hinge due to an insertion of the duplicated stretch of amino acids [32]. The bivalent anti-PD-L1/VEGF nanobody was expressed in E. coli host system, which is considered the best for generating recombinant proteins because of its affordability, short culture time, and high protein yield. The recombinant bivalent anti-PD-L1/VEGF nanobody significantly inhibited proliferation and tube formation of HUVEC and A431 cells. In addition, bivalent anti-PD-L1/VEGF nanobody significantly inhibited neovascularization of chick chorioallantoic membrane in an ex ovo CAM assay. As a result, the obtained bivalent anti-PD-L1/VEGF nanobody might be a valid alternative to nanobodies.
Monoclonal antibodies used for the management of hemataological disorders
Published in Expert Review of Hematology, 2022
Kanjaksha Ghosh, Kinjalka Ghosh
MoAbs used for therapeutic purposes are continuously improving through molecular engineering. They are humanized, and efficiency of these antibodies is increased with Fc receptor modification, defucosylation, improved techniques for attachment of various toxic, and radioactive payloads (These are small molecules like Radioisotopes, cytotoxic drugs, cytokines, antibiotics, etc.). Minibodies and nanobodies are antibody fragments of variable and hypervariable regions suitably stitched together leading to increased affinities and effectiveness. These antibodies are produced in Camelids or are produced by engineering and polymerization of VH segment of the antibody. It is now possible to negotiate and attach to cellular receptor sites where previous whole antibody or its Fab fragments were not able to reach due to steric hindrance. Very small VH fragment of the desired antibody now called nanobody can enter the cytoplasm of a cell after crossing the cell membrane and can attach to cytoplasmic effector molecules.
Nanobodies in Human Infections: Prevention, Detection, and Treatment
Published in Immunological Investigations, 2020
Marzieh Sanaei, Neda Setayesh, Zargham Sepehrizadeh, Mehdi Mahdavi, Mohammad Hossein Yazdi
In summary, antigen-specific Nbs can be obtained in a two-step process, which is recognized as library construction and screening for the specific nanobody as displayed in Figure 4. At first, a nanobody library should be created, which contains the VHH gene fragment repertoire. In order to achieve this goal, a camel/llama is immunized with an immunogen (single antigen, combination of several antigens, cancerous tissue) in order to raise the HCAbs. Although llamas are easier to raise, camels are preferred, because of their high ratio of HCAbs in comparison with regular antibodies (Muyldermans 2001). Lymphocytes as the source of mRNA can be used to prepare cDNA after the immunization. After that, constant region-specific primers were used in order to amplify the VHHs, and also distinguish them from the VHs, due to their difference in length: the longest is VH and the shorter is VHH (Nguyen et al. 2001). Actually, two consecutive PCR reactions are accomplished, and specific VHH sequences of ̴ 400 bp (Nb) are also screened. After the amplified VHH gene fragment cloning into a phage display vector, and transforming it into E. coli TG1 cells, an Nb library will be attained. The most frequently used genes for displaying VHHs on the bacteriophage virion surface are pIII or pVIII of bacteriophage M13. It is noteworthy to say that immune library construction is not always feasible, because of the antigen low immunogenicity or high toxicity. So, nonimmune libraries (naive or synthetic) could be used as an alternative (Goldman et al. 2006).