The biological effects of FGF2 are exerted through the formation of ternary FGF2-heparin-FGF receptor complexes in several cells in the tumor microenvironment, including endothelial and neoplastic cells9,11,12

The biological effects of FGF2 are exerted through the formation of ternary FGF2-heparin-FGF receptor complexes in several cells in the tumor microenvironment, including endothelial and neoplastic cells9,11,12. Our anti-FGF2 IgG mAb, named 3F12E7, showed promising results in experimental approaches. and also provides insights to further engineer non-aggregated anti-FGF2 scFv-based tools for therapeutic and research purposes. Subject terms: Proteins, Immunotherapy Introduction Improvements in recombinant antibody technology led to the development of a large variety of designed monoclonal antibody (mAb) constructions differing in pharmacokinetic and binding properties for research, diagnostic, and therapeutic applications. Among the recombinant antibody types, there is the single-chain variable fragment (scFv)1. ScFvs consist of artificial constructs composed of the variable regions of the heavy (VH) and light (VL) immunoglobulin chains connected by a flexible peptide linker in a way to retain the antigen-binding properties of the corresponding IgG antibody2. The scFvs are often produced with high yield using bacterial expression systems3, which comes Akt-l-1 as an advantage over the production of full-sized IgGs by hybridoma cells. Also, the expected small molecular size for such recombinant constructions may imply higher diffusion inside tumors and blood clearance1, 3. Several scFvs have been Akt-l-1 described to target tumors, with some of them already approved for clinical use4,5. To target tumor growth, our research group previously generated an immunoglobulin G (IgG) monoclonal antibody (mAb) directed against the fibroblast growth factor 2 (FGF2)6, based on the conventional hybridoma technology7. FGF2 is usually a key angiogenesis-related factor implicated in the development and progression of several tumors8C10. The biological effects of FGF2 are exerted through the formation of ternary FGF2-heparin-FGF receptor complexes in several cells in the tumor microenvironment, including endothelial and neoplastic cells9,11,12. Our anti-FGF2 IgG mAb, named 3F12E7, showed encouraging results in experimental approaches. It was demonstrated to successfully reduce tumor blood vessel density and to inhibit tumor growth and metastasis, as previously described6. However, despite the encouraging data, this full-length IgG mAb Akt-l-1 failed to inhibit the growth of already established tumors, which motivated us to design new small-sized brokers to target FGF2. In this report, we describe an scFv construct generated by the fusion of the VH and VL domains of the 3F12E7 mAb. The functional activity of the 3F12E7 scFv was evaluated considering its aggregation and binding properties. Our results revealed a soluble anti-FGF2 product that, although prone to aggregation, contains monomers and small oligomers. The obtained scFv succeeded in specifically realizing FGF2 in tumor extracts and inhibiting experimental tumor growth similar to the corresponding full-length IgG CR2 antibody counterpart. Also, in-silico predictions provide useful insights to understand the antigen-recognition by scFv models and to further engineer non-aggregated scFv-based tools for therapeutic and experimental applications. Results Construction and functional characterization of anti-FGF2 3F12E7 scFv To construct the scFv form of the anti-FGF2 3F12E7 mAb, the DNA segments encoding the antibody heavy (VH) and light (VL) variable chains were amplified from your cDNA of 3F12E7 hybridoma cells (Fig.?1a) and further sequenced. Blast analysis revealed high homology (>?98%) of the detected VH and VL domains with Akt-l-1 known mouse immunoglobulin sequences. The scFv was designed in the VH-linker-VL orientation13, using [(Gly4Ser)3] peptide as a linker (Fig.?1b). Open in a separate window Physique 1 Generation of 3F12E7 anti-FGF2 scFv. (a) Agarose gel electrophoresis of PCR amplified products of VL and VH genes from 3F12E7 hybridoma cells. Akt-l-1 (b) Design of the 3F12E7 scFv construct, created by VH and VL amino acid sequences attached by a flexible peptide linker [(G4S)3]. (c) Schematic representation of the pET26-based vector utilized for.