Expression of chimeric monomer and dimer proteins on the plasma membrane of mammalian cells

Chou, Wan Chih; Liao, Kuang Wen; Lo, Yu Chih; Jiang, Shu; Yeh, Ming‐Yang; Roffler, Steve R.

doi:10.1002/(sici)1097-0290(19991020)65:2<160::aid-bit5>3.0.co;2-u

Cited by 43 publications

(58 citation statements)

References 44 publications

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“…This is especially important for gene therapy since in vivo transduction efficiencies are typically low. 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.…”

Section: Discussionmentioning

confidence: 99%

“…Polyclonal rabbit serum against AFP and E. coli b-glucuronidase was produced as previously described. 19 Cell lines and tissue culture BALB/3T3 cells were obtained from the National Health Research Institutes (Hsin Chu, Taiwan). HT29 human colon carcinoma cells and B16-F1 melanoma cells were from the American Type Culture Collection (Manassas, VA).…”

Section: Reagentsmentioning

confidence: 99%

“…15,19 The XbaI DNA fragments of murine CD80 and CD86 (kindly provided by Dr Gordon Freeman, Dana-Farber Cancer Institute, Harvard Medical School, Boston) were subcloned into the pcDNA3.1/zeo(+) vector (Invitrogen, Leek, The Netherlands) to obtain pCD80/zeo and pCD86/zeo. A cDNA fragment encompassing the IgG-like C type domain, TM domain and cytoplasmic tail of murine CD80 was PCRamplified from pCD80/zeo with the primers 5 0 -aaagtcgacgctgacttctctacccccaacataact-3 0 and 5 0 -aactcgagctaaaggaagacggtctgttcagc-3 0 .…”

Section: Plasmid Constructionmentioning

confidence: 99%

“…Expression of AFP as a GPI-anchored protein (AFP-DAF) resulted in detectable shedding of AFP as previously reported. 19 Low amounts of AFP were also shed into the culture medium when the BGP linker was employed (AFP-BGP-B7), but removal of the three N-linked glycosylation sites in the linker (AFP-BGP3-B7) resulted in greatly increased shedding of AFP, demonstrating that linker glycosylation reduced cleavage from the cell surface.…”

Section: Linker Domains Can Enhance the Expression Of Chimeric Antibomentioning

confidence: 99%

“…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. Receptor shedding reduces the level of active receptor on the cell surface and soluble receptors can act as receptor antagonists.…”

mentioning

confidence: 99%

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

Liao¹,

Chen²,

Liu³

et al. 2003

Cancer Gene Ther

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Expression of CD80 or CD86 costimulatory molecules on tumor cells can produce rejection of immunogenic but not poorly immunogenic tumors. We have previously shown that anti-CD3 single-chain antibodies expressed on the surface of cells can directly activate T cells. We therefore investigated whether anti-CD3 ''receptors'' could enhance CD86-mediated rejection of poorly immunogenic tumors. Expression of anti-CD3 receptors on cells was increased by introduction of membrane-proximal ''spacer'' domains containing glycosylation sites between the single-chain antibody and the transmembrane domain of the chimeric receptors. Removal of glycosylation sites in the spacer reduced surface expression due to increased shedding of chimeric receptors from the cell surface. Induction of T-cell proliferation by anti-CD3 receptors did not correlate with the expression level of chimeric protein, but rather depended on the physical properties of the spacer. Anti-CD3 receptors effectively induced T-cell cytotoxicity, whereas coexpression with CD80 or CD86 was required for generating T-cell proliferation and IL-2 secretion. Although expression of CD86 did not significantly delay the growth of poorly immunogenic B16-F1 tumors, expression of anti-CD3 receptors with CD86 produced complete tumor rejections in 50% of mice and induced significant protection against wild-type B16-F1 tumor cells. Our results show that spacer domains can dramatically influence the surface expression and the biological activity of chimeric antibody receptors. The strong antitumor activity produced by anti-CD3 receptors and CD86 on tumor cells indicates that this strategy may be beneficial for the gene-mediated therapy of poorly immunogenic tumors.

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Section: Discussionmentioning

confidence: 99%

Section: Reagentsmentioning

confidence: 99%

Section: Plasmid Constructionmentioning

confidence: 99%

Section: Linker Domains Can Enhance the Expression Of Chimeric Antibomentioning

confidence: 99%

mentioning

confidence: 99%

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

Liao¹,

Chen²,

Liu³

et al. 2003

Cancer Gene Ther

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

Liao

Chou

et al. 2001

Biotech & Bioengineering

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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.

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CD13 (aminopeptidase N) can associate with tumor‐associated antigen L6 and enhance the motility of human lung cancer cells

Chang¹,

Chen²,

Cheng³

et al. 2005

Intl Journal of Cancer

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Cancer metastasis is a multiple-step process that involves the regulated interaction of diverse cellular proteins. We recently reported that the expression of tumor-associated antigen L6 (TAL6) promoted the invasiveness of lung cancer cells and was inversely correlated with disease-free survival of squamous lung carcinoma patients. We now report that CD13 (aminopeptidase N) can associate with TAL6 and can enhance cancer cell migration. CD13 was shown by coimmunoprecipitation to associate in vitro with TAL6 on several cancer cell lines and to associate in vivo by antibody-mediated copatching immunofluorescence. CD13 was selectively expressed on highly invasive CL1-5 lung cancer cells as compared to poorly invasive CL1-0 lung cancer cells. The role of CD13 aminopeptidase activity in regulating cell motility was investigated with chemical inhibitors, specific antibodies and a catalytically inactive CD13 protein. Inhibition of CD13 aminopeptidase activity by nontoxic concentrations of leuhistin modestly decreased the migration of CL1-5 cells. In contrast, binding of CD13 by specific antibodies significantly reduced both the migration and the invasion of CL1-5 cells. Poorly invasive CL1-0 cells that stably expressed CD13 displayed significantly (p < or = 0.0005) enhanced cell migration (300% of control). Expression of an enzymatically inactive CD13 mutant on CL1-0 cells also significantly (p < or = 0.0005) enhanced cell migration (200% of control). Our results show that TAL6 and CD13 can form a complex on lung cancer cells, that these molecules can modulate cell migration and invasion and that the influence of CD13 on cell motility did not strictly depend on its aminopeptidase activity.

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Expression of chimeric monomer and dimer proteins on the plasma membrane of mammalian cells

Cited by 43 publications

References 44 publications

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

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

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

CD13 (aminopeptidase N) can associate with tumor‐associated antigen L6 and enhance the motility of human lung cancer cells

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