An Antibody Delivery System for Regulated Expression of Therapeutic Levels of Monoclonal Antibodies In Vivo

Abstract

Monoclonal antibody (mAb) delivery by gene transfer in vivo may be an attractive alternative to current mAb therapies for applications that require long-term therapy. This article describes a transfer system that allows inducible high-level expression of unmodified mAbs in vivo. A recombinant adeno-associated viral (rAAV) vector is used that comprises an expression cassette consisting of a dimerizer-regulated promoter that drives expression of the antibody heavy and light chains linked by a 2A self-processing peptide and a furin cleavage site. Following intravenous injection of the rAAV vector, serum mAb levels >1 mg/ml were attained by administration of the inducer, rapamycin. Antibody expression could be rapidly shut off by discontinuing treatment with rapamycin. By optimizing the furin cleavage sequence, this system generated native antibody in vivo, decreasing the likelihood of a host immune response to foreign sequences. In summary, this optimized mAb expression system allows regulated high-level expression of native full-length mAbs in vivo and may offer a new opportunity for delivery of therapeutic mAbs in the clinic.

Growth inhibition of an established A431 xenograft tumor by a full-length anti-EGFR antibody following gene delivery by AAV

“…21,32,33 Serum levels of 14E1 peaked at over 1000 mg ml À1 at day 56 postinjection and persisted at levels greater than 600 mg ml À1 for over 4 months. Administration of AAV2/1-14E1 prior to tumor challenge resulted in significant inhibition of tumor growth, prolonged survival time and tumor regression.…”

“…Many different smallmolecule-regulated gene-expression systems have been developed and incorporated into AAV delivery systems including those induced by tetracycline, Rapamycin and mifepristone. 40,41 Recently, Fang et al 32 demonstrated regulated expression of a full-length antibody following AAV delivery by incorporating the Furin/2A technology with a dual vector Rapamycin system. Further improvements for a gene-regulated AAV system for antibodies could include the incorporation of a one-vector system versus a dual-vector system.…”

Translating 2A Research into Practice

“…, resulted in the expression of antibodies with no residual amino acids (Fang et al, 2007). Proteins expressed in plants could have their 2A extensions removed by endogenous proteinases acting on similar hybrid linker peptides.…”

2A - The “go-to” Technology for Transgene Co-expression

“…, which removes the 2A "tag" [21,38]. The presence of 2A, however, can be used for detection of protein expression and localization using anti-2A antibodies [2,3].…”

“…Importantly, the co-translational nature of this cleavage means that by including various signal sequences within 2A polyproteins, either up-or downstream of 2A, proteins can be either targeted co-translationally to the exocytic pathway or post-translationally to different cellular compartments [34][35][36]. Of the many 2A peptides identified to date [10,27,37], four have been widely used in biotechnology and biomedicine: F2A, equine rhinitis A virus ("E2A"), porcine teschovirus-1 ("P2A") and Thosea asigna virus ("T2A") ( Table 1) [2][3][4]36,[38][39][40][41][42][43].…”