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    • br Detecting or Defining Cancer Biomarkers by Means

    2018-11-05


    Detecting or Defining Cancer Biomarkers by Means of Nanobodies Apart from therapy, nanobodies can aid in early diagnosis and cancer prevention by detecting or defining biomarkers (Table 1). Nanobodies can improve current mAb-based diagnostic techniques due to their high specificity. Furthermore, their high baicalein under extremes of temperature, pH, or ionic strength, as shown for the cancer biomarker alpha-fetoprotein (AFP) (Chen et al., 2016), ensures that the application still can occur under harsh conditions. Cell-based ELISA was successful for nanobodies against the carbonic anhydrase IX enzyme (CAIX) (Araste et al., 2014), prostate-specific membrane antigen PMSA (Zare et al., 2014), tumor-associated glycoprotein 72 (TAG-72) (Sharifzadeh et al., 2013) and HER2 (Jamnani et al., 2012). Of note, a better performance was achieved with a mixture of several nanobodies. To perform sandwich ELISA, both a capturing and detecting nanobody are used, targeting different epitopes on the antigen. Nanobodies against AFP reached a detection limit of 0.47ng/mL. One step further, a chip format sandwich ELISA with anti-HER2 nanobodies covalently bound onto a screen-printed electrode (SPE) was proposed with a detection limit of 1μg/mL (Patris et al., 2014). The most sensitive detection (0.0005ng/mL) could be achieved by means of immune-PCR with anti-AFP nanobodies (Chen et al., 2016). Due to the relative ease of single domain nanobody generation compared to conventional antibodies, an elegant and fast strategy to generate nanobodies against (unknown) cancer biomarkers is by performing immunization with patient samples. This strategy resulted in the identification of a new breast cancer-specific biomarker, cytokeratin 19 (Even-Desrumeaux et al., 2012). Similarly, the technology of nanobody-based reverse proteomics was used in glioblastoma multiforme (GBM) (Jovcevska et al., 2014). By performing mass-spectrometry analysis on nanobody-antigen pairs, the new GBM biomarkers TRIM28 and β-actin could be revealed. As all these markers are localized intracellularly, they are especially proposed for immunohistochemical-based diagnostic purposes.
    Nanobodies in Molecular Imaging Single-photon emission computed tomography (SPECT) is based on γ-rays and nanobodies are here linked to radionuclides such as 99mTc, 177Lu, 123I and 111In. On the other hand, the positron-emitting radioisotopes 68Ga, 124I or 89Zr are used for positron emission tomography (PET) purposes. Again, especially cancer-specific receptors such as EGFR (Vosjan et al., 2011) and HER2 (D\'Huyvetter et al., 2012; D\'Huyvetter et al., 2014; Xavier et al., 2013; Keyaerts et al., 2015; Massa et al., 2014; Pruszynski et al., 2013; Pruszynski et al., 2014) (Table 1) are interesting targets for tumor visualization. Recent research in this area further focuses on PMSA (Chatalic et al., 2015; Evazalipour et al., 2014), which is overexpressed in prostate cancer, and HGF (Vosjan et al., 2012), which is implicated in several cancers, though also in cardiovascular disease. Alternatively, Movahedi and coworkers raised nanobodies against the macrophage mannose receptor MMR, which is highly expressed by tumor-associated macrophages and thus serves as an alternative strategy to image the tumor stroma, especially the hypoxic areas (Movahedi et al., 2012). Prior to radiolabeling, nanobodies are conjugated with bifunctional chelating agents which possess a metal binding moiety for sequestration of the metallic radionuclide and are generally DPTA (acyclic)-, DOTA (macrocyclic), or NOTA-based. Moreover, the chelating agents are equipped with a chemically reactive functional group for attachment to the nanobody, which can occur in several ways. First of all, random conjugation can be performed via the free ε-amino-group on nanobody lysines, as shown for the anti-HER2 and the anti-PMSA nanobodies. Second, Massa and co-workers demonstrated a generic strategy for site-specific labeling of nanobodies (Massa et al., 2014). To this end, the anti-HER2 nanobody was cloned with a C-terminal unpaired cysteine following its hexahistidine tag. A reduction step was needed prior to conjugation due to spontaneous dimerization and capping of the unpaired cysteine. Subsequently, the reduced probe could be bound to maleimide-DPTA, followed by labeling with 111In. Third, the hexahistidine tag of the nanobody can be directly labeled with 99mTc(CO)3 without any chemical modification of the protein, as shown for an anti-PMSA nanobody and the anti-MMR nanobody (Evazalipour et al., 2014; Movahedi et al., 2012). Site-specific or His-mediated labeling has the advantage that antigen binding activity of the nanobody is usually unaffected. When lysine residues involved in random conjugation are located outside the antigen-binding loops, no interference with antigen binding should be expected either (Xavier et al., 2013; Keyaerts et al., 2015).