Design of transgenes for efficient expression of active chimeric proteins on mammalian cells

Abstract

Heterologous proteins expressed on the surface of cells may be useful for eliciting therapeutic responses and engineering new extracellular properties. We examined factors that control the membrane targeting of ␣-fetoprotein (AFP) and a single-chain antibody (scFv). Chimeric proteins were targeted to the plasma membrane by employing the transmembrane domain (TM) and cytosolic tail of murine CD8O (B7-1), the TM of the human platelet-derived growth factor receptor (PDGFR), the glycosylphosphatidylinositol anchor encoded by the C-terminal extension of decay-accelerating factor (DAF), and the TM of the H1 subunit of the human asialoglycoprotein receptor (ASGPR). AFP chimeric proteins containing the B7, DAF, ASGPR, or PDGFR targeting domains displayed half-lives of 12.2, 3.8, 2.4, and 1.6 h, respectively. The newly synthesized B7 chimera was rapidly transported and remained on the cell surface. Glycosylphosphatidylinositol-anchored chimeras reached the surface more slowly and significant amounts were released into the culture medium. PDGFR TM chimeras were rapidly degraded, whereas ASGPR chimeras were retained in the endoplasmic reticulum (ER). The surface expression of both AFP and scFv chimeric proteins followed the order (highest to lowest) of B7 > DAF >> PDGFR. Introduction of a dimerization domain (hinge-CH 2 -CH 3 region of human IgG1) between scFv and TM dramatically reduced cleavage of the chimeric protein, increased surface expression, and produced biologically active scFv. Our results indicate that transgenes designed for the expression of active scFv on cells should incorporate a TM that does not undergo endocytosis, include an intact cytoplasmic domain, and possess a spacer to reduce cleavage and retain biological activity.

Stable expression of chimeric anti-CD3 receptors on mammalian cells for stimulation of antitumor immunity

“…We previously showed that the murine B7-1 TM and intact cytoplasmic tail allowed high expression of AFP 19 and scFv 15 due to rapid transport and stable retention of the proteins on the cell surface. 18 In the present investigation we tested the hypothesis that introduction of a spacer domain containing oligosaccharides at the juxtamembrane position of chimeric receptors could further increase surface expression by reducing shedding. The rationale for this hypothesis was based on the observation that many endogenous receptors contain serine-and threonine-rich domains near the cell membrane that are potential sites of O-linked glycosylation.…”

“…It should be noted that 2C11-B7 already represented an improved surface receptor since it allowed about 5-10 times greater amounts of scFv to accumulate on cells as compared to the prototype scFv receptor (2C11-PDGFR). 15,18 The presence of oligosaccharide chains in the linker domain was mandatory for increased surface expression because removal of glycosylation acceptor sites (2C11-BGP3-B7) reduced surface expression to levels even below those observed without introduction of a linker (Fig 2b). Both O-linked (2C11-CD44-B7) and N-linked (2C11-BGP-B7, 2C11-g1-B7 and 2C11-e-B7) glycosylation effectively reduced shedding.…”

“…We previously showed that the transmembrane (TM) domain employed to anchor chimeric receptors affected the rate of receptor transport to the cell surface 18 and greatly influenced surface expression. 19 These studies indicated that chimeric surface receptors were also shed from the cell surface.…”

A membrane antibody receptor for noninvasive imaging of gene expression

“…We previously performed a series of studies to determine the optimal conditions for the expression of proteins 32 and scFv 26,28,33 on mammalian cells. The transmembrane domain and cytoplasmic tail derived from the B7-1 antigen was found to facilitate rapid intracellular transport of chimeric proteins to the cell surface.…”

Hapten-directed targeting to single-chain antibody receptors

“…5,13 In the present study, the anti-phOx scFv was fused to the transmembrane (TM) domain and cytoplasmic tail of murine CD80, which we have previously shown to direct efficiently the expression of heterologous proteins to the cell surface. 14 The utility of surface receptor targeting was investigated by covalently attaching phOx moieties to b-glucuronidase (bG) to create a ligand for specific recognition and binding to cells that display phOx scFv receptors. The present study investigated whether the accumulation of phOx-modified bG at receptor-positive cells could allow preferential activation of a glucuronide prodrug (p-hydroxy aniline mustard b-D-glucuronide, HAMG) for selective cell killing (Fig 1).…”