Therapeutic Expression of an Anti-Death Receptor 5 Single-Chain Fixed-Variable Region Prevents Tumor Growth in Mice

Abstract

The clinical use of the single-chain fixed-variable (scFv) fragments of recombinant monoclonal antibodies as credible alternatives for classic therapeutic antibodies has two limitations: rapid blood clearance and inefficient local expression of functional molecules. In attempt to address these issues, we have developed a novel gene therapy protocol in which the anti-death receptor 5 (DR5) scFv fragments were either in vitro expressed in several tumor cell lines, or in vivo expressed in mice, using recombinant adeno-associated virus (rAAV)-mediated gene transfer. Viral transduction using the rAAV-S3C construct, which encodes a scFv molecule (S3C scFv) specific to DR5, led to stable expression in tumor cell lines and showed apoptosis-inducing activity in vitro, which could be inhibited by recombinant DR5 but not by DR4. A single i.m. injection of rAAV-S3C virus in nude mice resulted in stable expression of DR5-binding S3C scFv proteins in mouse sera for at least 240 days. Moreover, the expression of S3C scFv was associated with significant suppression of tumor growth and the increase of tumor cell apoptosis in previously established s.c. human lung LTEP-sml and liver Hep3B tumor xenografts.

Specific single chain variable fragment (ScFv) antibodies to angiotensin II AT2 receptor: evaluation of the angiotensin II receptor expression in normal and tumor-bearing mouse lung

“…The benefit of ScFv antibodies is, unlike the parental antibody, they are very small allowing them to penetrate the cell membrane more efficiently (Shi et al 2006). ScFv antibodies can also be genetically produced using cDNA of the parental antibody.…”

“…They are currently the smallest labeling system available (Malecki et al 2002). The benefit of ScFv antibodies is, unlike the parental antibody, they are very small allowing them to penetrate the cell membrane more efficiently (Shi et al 2006). The objective of the present study was to gain insight into the mechanism by which AT 2 regulates carcinogen-induced lung tumorigenesis.…”

Antibody gene therapy: an attractive approach for the treatment of cancers and other chronic diseases

“…Thus, broad application of recombinant scFv antibody is limited by manufacturing difficulty and the high cost of production. To overcome these bottle-necks, Shi et al [1] developed a promising therapeutic strategy by expressing in vivo scFv fragments of a mouse monoclonal antibody (AD5-10) that is directed against the TRAIL (tumor necrosis factor-related apoptosisinducing ligand) receptor DR5 (death receptor 5, also known as TRAIL receptor 2) [2], via recombinant adeno-associated virus (rAAV) vector-mediated antibody gene therapy.…”

“…In addition, due to their large size, it is difficult for intact antibodies to penetrate into solid tumors, further limiting their applications in cancer therapy [4]. Since AAV-mediated gene therapy enables long-term transgene expression in vivo and shows low immunogenicity, Shi et al [1] adopted this system to express scFv fragments of AD5-10 in mouse models and achieved a significant therapeutic efficacy against tumor xenografts.…”

“…Shi et al [1] demonstrated that viral transduction using the rAAV encoding scFv of AD5-10 led to stable expression and apoptosis-inducing activity in lung SCLC, liver Hep 3B and colon HCT116 carcinoma cell lines in vitro, which could be inhibited by recombinant DR5 specifically. The virally expressed scFv fragments in the media of infected HEK293 cells and the sera of transduced mice maintained its affinity for its antigen (Kd = 3.3×10 -9 M in the cell culture media and Kd = 4×10 -9 M in the mouse sera, respectively) comparing with that of the parental intact an- =10 -10 M).…”