Use of monoclonal antibodies is emerging as a highly promising and fast-developing scenario for innovative treatment of viral, autoimmune and tumour diseases. The search for diagnostic and therapeutic antibodies currently depends on in vitro screening approaches, such as phage and yeast display technologies. Antibody production still represents a critical step for preclinical and clinical evaluations. Accordingly, improving production of monoclonal antibodies represents an opportunity, to facilitate downstream target validations. SINEUP RNAs are long non-coding transcripts, possessing the ability to enhance translation of selected mRNAs. We applied SINEUP technology to semi-stable production of monoclonal antibodies in HEK293E cells, which allows for episomal propagation of the expression vectors encoding the heavy and light chains of IgGs. Co-expression of SINEUP RNA with mRNAs encoding heavy and light chains of IgG4s was able to increase the production of different anti-CLDN1 antibodies up to three-fold. Improved production of monoclonal antibodies was achieved both in transiently transfected HEK293E cells and in cellular clones with stable expression of the SINEUP. Compared to antibody preparations obtained under standard conditions, the anti-CLDN1 IgG4s produced in the presence of the SINEUP transcript showed unaltered post-translational modifications, and retained the ability to recognize their target. We thus propose SINEUP technology as a valuable tool to enhance semi-stable antibody production in human cell lines.
Bicyclic peptides assembled around small organic scaffolds are gaining an increasing interest as new potent, stable and highly selective therapeutics because of their uncommon ability to specifically recognize protein targets, of their small size that favor tissue penetration and of the versatility and easiness of the synthesis. We have here rationally designed bicyclic peptides assembled around a common tri-bromo-methylbenzene moiety in order to mimic the structure of the CFC domain of the oncogene Cripto-1 and, more specifically, to orient in the most fruitful way the hot spot residues H120 and W123. Through the CFC domain, Cripto-1 binds the ALK4 receptor and other protein partners supporting uncontrolled cell growth and proliferation. Soluble variants of CFC have the potential to inhibit these interactions suppressing the protein activity. A CFC analog named B3 binds ALK4 in vitro with an affinity in the nanomolar range. Structural analyses in solution via NMR and CD show that B3 has rather flexible conformations, like the parent CFC domain. The functional effects of B3 on the Cripto-1-positive NTERA cancer cell line have been evaluated showing that both CFC and B3 are cytotoxic for the cells and block the Cripto-1 intracellular signaling. Altogether, the data suggest that the administration of the soluble CFC and of the structurally related analog has the potential to inhibit tumor growth.
Background: Monoclonal antibodies (mAbs) against cancer biomarkers are key reagents in diagnosis and therapy. One such relevant biomarker is a preferentially expressed antigen in melanoma (PRAME) that is selectively expressed in many tumors. Knowing mAb’s epitope is of utmost importance for understanding the potential activity and therapeutic prospective of the reagents. Methods: We generated a mAb against PRAME immunizing mice with PRAME fragment 161–415; the affinity of the antibody for the protein was evaluated by ELISA and SPR, and its ability to detect the protein in cells was probed by cytofluorimetry and Western blotting experiments. The antibody epitope was identified immobilizing the mAb on bio-layer interferometry (BLI) sensor chip, capturing protein fragments obtained following trypsin digestion and performing mass spectrometry analyses. Results: A mAb against PRAME with an affinity of 35 pM was obtained and characterized. Its epitope on PRAME was localized on residues 202–212, taking advantage of the low volumes and lack of fluidics underlying the BLI settings. Conclusions: The new anti-PRAME mAb recognizes the folded protein on the surface of cell membranes suggesting that the antibody’s epitope is well exposed. BLI sensor chips can be used to identify antibody epitopes.
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