Production of a Soluble Recombinant Antibody Fragment against MMP9 Using Escherichia coli

Matrix metalloproteinase 9 (MMP9) is involved in several aspects of the pathology of cancer, including invasion, metastasis, and angiogenesis. In this study, we expressed a recombinant scFv-type anti-MMP9 antibody in soluble form using Escherichia coli, purified it, and confirmed its antigen-binding ability. The convenient, rapid, inexpressive system used in this study for producing recombinant antibody fragments needs only five days, and thus can be used for the efficient production of scFv against MMP9, which can be used in a range of applications and industrial fields, including diagnosis and treatment of inflammatory and cancer-related diseases.

Generation and functional characterization of a single-chain variable fragment (scFv) of the anti-FGF2 3F12E7 monoclonal antibody

Single-chain variable fragments (scFvs) are small-sized artificial constructs composed of the immunoglobulin heavy and light chain variable regions connected by a peptide linker. We have previously described an anti-fibroblast growth factor 2 (FGF2) immunoglobulin G (IgG) monoclonal antibody (mAb), named 3F12E7, with notable antitumor potential revealed by preclinical assays. FGF2 is a known angiogenesis-associated molecule implicated in tumor progression. In this report, we describe a recombinant scFv format for the 3F12E7 mAb. The results demonstrate that the generated 3F12E7 scFv, although prone to aggregation, comprises an active anti-FGF2 product that contains monomers and small oligomers. Functionally, the 3F12E7 scFv preparations specifically recognize FGF2 and inhibit tumor growth similar to the corresponding full-length IgG counterpart in an experimental model. In silico molecular analysis provided insights into the aggregation propensity and the antigen-recognition by scFv units. Antigen-binding determinants were predicted outside the most aggregation-prone hotspots. Overall, our experimental and prediction dataset describes an scFv scaffold for the 3F12E7 mAb and also provides insights to further engineer non-aggregated anti-FGF2 scFv-based tools for therapeutic and research purposes.

Engineered Recombinant Single Chain Variable Fragment of Monoclonal Antibody Provides Protection to Chickens Infected with H9N2 Avian Influenza

Passive immunisation with neutralising antibodies can be a potent therapeutic strategy if used pre- or post-exposure to a variety of pathogens. Herein, we investigated whether recombinant monoclonal antibodies (mAbs) could be used to protect chickens against avian influenza. Avian influenza viruses impose a significant economic burden on the poultry industry and pose a zoonotic infection risk for public health worldwide. Traditional control measures including vaccination do not provide rapid protection from disease, highlighting the need for alternative disease mitigation measures. In this study, previously generated neutralizing anti-H9N2 virus monoclonal antibodies were converted to single-chain variable fragment antibodies (scFvs). These recombinant scFv antibodies were produced in insect cell cultures and the preparations retained neutralization capacity against an H9N2 virus in vitro. To evaluate recombinant scFv antibody efficacy in vivo, chickens were passively immunized with scFvs one day before, and for seven days after virus challenge. Groups receiving scFv treatment showed partial virus load reductions measured by plaque assays and decreased disease manifestation. These results indicate that antibody therapy could reduce clinical disease and shedding of avian influenza virus in infected chicken flocks.

Construction and characterization of immunoliposomes targeting fibroblast growth factor receptor 3

Fibroblast growth factor receptor 3 (FGFR3) plays an important regulatory role in tumor cell proliferation and drug resistance. FGFR3 is often constitutively active in many tumors. To deliver drugs into tumor cells by targeting FGFR3 will be a promising and potential strategy for cancer therapy. In this study, a novel fusion protein, ScFv-Cys containing a single chain variable fragment (ScFv) and an additional C-terminal cysteine residue, was generated at a rate of 10 mg/L of bacterial culture and purified at 95% by Ni-NTA chromatography. Subsequently, the recombinant ScFv-Cys was coupled with malPEG2000-DSPE and incorporated into liposomes to generate the immunoliposomes. The results indicated that immunoliposomes can specifically deliver the fluorescent molecules, Dio into bladder cancer cells highly expressing FGFR3. In conclusion, we successfully generated FGFR3-specific immunoliposomes, and proved its targeting effect and delivering ability.

Expression of the recombinant single chain variable fragments recognizing blood antigen fused with thioredoxin in Escherichia coli

The technology of recombinant single chain variable fragments (scFvs) expression has been used in research, diagnosis and treatment of diseases. In the previous study, we studied the expression of a recombinant single chain variable fragment recognizing blood A antigen (antiA-scFv) in E. coli. However, the protein was insoluble form resulting in difficulty for purification, refolding and activity assesment. Here, we present the study on fused expression of the recombinant scFv -specific blood A antigen with thioredoxin (Trx) in the expression vector pET32a (+). The results showed that the Trx/antiA-scFv fusion protein was expressed with molecular weight of 49 kDa in a soluble form reaching 40% of the total recombinant protein. This result facilitates the optimal condition of soluble protein expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

Co-expression of recombinant single chain variable fragment recognizing blood antigen fused with sumo and chaperones in Escherichia coli

Single chain variable fragments (scFv) have widely been used in research, diagnosis and treatment, but the scFv is considered as difficult protein for expression in E. coli. In previous studies, we expressed a construction of recombinant single chain variable fragments again antigen specific for blood type A (antiA-scFv) individually or fused with Trx or SUMO. However, soluble fraction was low abandant and only approximately 40% when fused with Trx, the other cases were expressed in form of inclusion body. Therefore, it was difficult for purification, refolding and activity assesment. In thispaper, we demonstrated a suitable construction for soluble production of antiA-scFv fused with SUMO (SM/antiA-scFv) in presence of chaparones. Under fermentation with 0.1 mM IPTG at 20oC, the SM/antiA-scFv was entirely expressed in soluble form. Importantly, after cleavage from SUMO with SUMOprotease, antiA-scFv was still maintained in the supernatant fraction. Therefore, it can help ensure bioactivity and is useful for purification process. To the best of our knowledge, this is the first report showing soluble recombinant scFv fused with SUMO in presence of chaperone for determination of blood group antigens. Thus, this result facilitates the optimal study of soluble expression, purification and bioactivity determination of the antiA-scFv recombinant antibody.

Generation of recombinant scFv antibody against Ochratoxin A (OTA)

Ochratoxin A (OTA) is a mycotoxin commonly found in agricultural products and can accumulate in the blood and tissues after that consuming contaminated food. Recombinant single-chain antibody fragments (scFv) against OTA were selected from phage display libraries. After of one round of biopanning against BSA-conjugated OTA (OTA-BSA), 52 and 6 phage clones displaying scFv antibodies were isolated from human (Yamo I.3) and rabbit (Bozmix I.2) libraries. Two phage clones (one from each libraries, i.e., yOTA1e3 and bOTA2a9) showed binding to free toxin by competitive ELISA. The soluble scFv antibodies were produced by superinfecting phage clones into E. coli suppressor strain HB2151. The scFv genes from these two phage clones were sub-cloned into pKP300ΔIII vectors to generate scFv-AP fusions. The binding affinity (IC50) of antibody derived from human library was higher than those from rabbit library. The binding property of recombinant antibody in the form of scFv-AP was better than those of soluble scFv form. Cross-reactivity analysis indicated that the two recombinant antibodies did not cross-react with other soluble toxins, namely AFB1, DON, ZEN and FB. The ability to use the recombinant scFv-AP to detect contaminated toxins in agricultural product (corn) was demonstrated.