Stable antibody expression at therapeutic levels using the 2A peptide

Abstract

Therapeutic monoclonal antibodies (mAbs) are currently being developed for the treatment of cancer and other diseases. Despite clinical success, widespread application of mAb therapies may be limited by manufacturing capabilities. In this paper, we describe a mAb delivery system that allows continuous production of a full-length antibody at high-concentrations in vivo after gene transfer. The mAb is expressed from a single open reading frame by linking the heavy and light chains with a 2A self-processing peptide derived from the foot-and-mouth disease virus. Using this expression system, we generated a recombinant adeno-associated virus vector encoding the VEGFR2-neutralizing mAb DC101 (rAAV8-DC101). A single dose of rAAV8-DC101 resulted in long-term expression of >1,000 microg/ml of DC101 in mice, demonstrating significant anti-tumor efficacy. This report describes the first feasible gene therapy approach for stable delivery of mAbs at therapeutic levels, which may serve as an attractive alternative to direct injection of mAbs.

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition

“…All the constructs, irrelevant to the origin of 2A peptides (T2A, F2A, P2A), functioned to produce the TCR in these two cell lines as demonstrated by fluorescence-activated cell sorting (FACS) analysis following gp100 tetramer staining ( Figure 1c). In contrast to a previous report, 29 the addition of furin to the 2A peptide inhibited TCR expression, whereas furin plus the addition of spacer sequences (GSG or SGSG) enabled highly efficient gene expression (up to 95%). While active in T-cell lines, these constructs produced significantly less TCR in primary PBL (o34% tetramer+; Supplementary Figure 1).…”

Coordinated protein co‐expression in plants by harnessing the synergy between an intein and a viral 2A peptide

“…This result, together with Western blot analysis, confirmed that fully assembled bioactive antibody complex was successfully produced from the IntF2A‐based polyprotein system. With the N‐extein linker‐free IntF2A system, the processed antibody light chain preserved its authentic C‐terminus without addition of any residual amino acids, which is superior to previously reported approaches (Fang et al ., 2005). The native N‐terminus of the processed downstream heavy chain fragment should also be preserved as the proline residue introduced by the action of F2A is removed upon signal peptide cleavage.…”

A Synthetic Mammalian Therapeutic Gene Circuit for Sensing and Suppressing Inflammation

“…To produce a stoichiometric amount of the heavy and light antibody chain, the effector construct was designed as a single mRNA transcript comprising coding sequences for three polypeptide chains, an anti-hTNF-a antibody heavy chain, an anti-hTNF-a antibody light chain, and hIL-1RA or mIL-10. The polypeptide-coding chains were linked through the coding sequence for the 2A peptide, which interrupts the translation of the polypeptide chain, similar as described previously 34 ( Figure 5A). These composite anti-inflammatory therapeutics were integrated in the final effector constructs pAS116 (TRE-UAS-P MIN -anti-hTNF-a Ab-hIL-1RA) and pAS134 (TRE-UAS-P MIN -anti-hTNF-a Ab-mIL-10, Table S1).…”