Generation and functional characterization of the anti-transferrin receptor single-chain antibody-GAL4 (TfRscFv-GAL4) fusion protein

Abstract

BackgroundThe development of vectors for cell-specific gene delivery is a major goal of gene therapeutic strategies. Transferrin receptor (TfR) is an endocytic receptor and identified as tumor relative specific due to its overexpression on most tumor cells or tissues, and TfR binds and intakes of transferrin-iron complex. We have previously generated an anti-TfR single-chain variable fragments of immunoglobulin (scFv) which were cloned from hybridoma cell line producing antibody against TfR linked with a 20 aa-long linker sequence (G4S)4. In the present study, the anti-TfR single-chain antibody (TfRscFv) was fused to DNA-binding domain of the yeast transcription factor GAL4. The recombinant fusion protein, designated as TfRscFv-GAL4, is expected to mediate the entry of DNA-protein complex into targeted tumor cells.ResultsFusion protein TfRscFv-GAL4 was expressed in an E. coli bacterial expression system and was recovered from inclusion bodies with subsequent purification by metal-chelate chromatography. The resulting proteins were predominantly monomeric and, upon refolding, became a soluble biologically active bifunctional protein. In biological assays, the antigen-binding activity of the re-natured protein, TfRscFv-GAL4, was confirmed by specific binding to different cancer cells and tumor tissues. The cell binding rates, as indicated by flow cytometry (FCM) analysis, ranged from 54.11% to 8.23% in seven different human carcinoma cell lines. It showed similar affinity and binding potency as those of parent full-length mouse anti-TfR antibody. The positive binding rates to tumor tissues by tissue microarrays (TMA) assays were 75.32% and 63.25%, but it showed weakly binding with hepatic tissue in 5 cases, and normal tissues such as heart, spleen, adrenal cortex blood vessel and stomach. In addition, the re-natured fusion protein TfRscFv-GAL4 was used in an ELISA with rabbit anti-GAL4 antibody. The GAL4-DNA functional assay through the GAL4 complementary conjugation with the GAL4rec-GFP-pGes plasmid to verify the GLA4 activity and GAL4rec-recognized specificity functions. It also shows the complex, TfRscFv-GAL4-GAL4rec-GFP-pGes, could be taken into endochylema to express the green fluorescent protein (GFP) with 8 to 10-fold transfection efficiency.ConclusionsResults of our study demonstrated that the biofunctianality of genetically engineered fusion protein, TfRscFv-GAL4, was retained, as the fusion protein could both carry the plasmid of GAL4rec-pGes and bind TfR on tumour cells. This product was able to transfect target cells effectively in an immuno-specific manner, resulting in transient gene expression. This protein that can be applied as an effective therapeutic and diagnostic delivery to the tumor using endogenous membrane transport system with potential widespread utility.

Therapeutic Bispecific T-Cell Engager Antibody Targeting the Transferrin Receptor

“…Our previous studies also demonstrated that TfR Abs could recognize tumor cells with high efficiency in vitro and 131 I-TfR Ab displayed a feature of specific accumulation at tumor tissue in vivo (23–25). Also these TfR Ab-modified therapeutic agents exhibit tumor-specific cytotoxic activities (26–29).…”

Bifunctional fused polypeptide inhibits the growth and metastasis of breast cancer

“…Targeted fused polypeptides are an emerging hotspot in targeted therapy for breast cancer. 23 , 24 There are a number of studies on the anticancer effect of targeted fused polypeptides in the treatment of breast cancer, including targeting the con-served transmembrane domain of human epidermal growth factor receptors, 25 – 29 glucose-regulated protein 78, 30 transferrin receptor, 31 and p53. 32 These targeted fused polypeptides exhibited a potent cell death inducing effect on tumor cells and a considerable suppressing effect on tumor growth and metastasis, indicating that the targeted fused polypeptide represents a promising targeted therapy strategy.…”

Recent Progress in Lipid Nanoparticles for Cancer Theranostics: Opportunity and Challenges

“…He and coworkers recognized a TfR mAb (monoclonal antibody) tailored nanomedicines for improved tumor targeting. They demonstrated that drug entrapped HPPS based nanomedicines adapted with explicit ligands could muddle to receptors on the surface of tumor cells, resulting in the accretion of nanomedicines on the exterior surface of targeted cells [75][76][77][78][79][80][81][82][83][84][85][86][87][88][89][90]. It was concluded that developed HPPS based nanomedicine holds the potential to strengthen targeting to tumor cells and attains favored recognition by specific antibodies in a complex biological setting [91].…”