A cell surface tethered enzyme improves efficiency in gene-directed enzyme prodrug therapy

Marais, Richard; Spooner, Robert A.; Stribbling, Stephen M.; Light, Yvonne; Martin, Janet L.; Springer, Caroline J.

doi:10.1038/nbt1297-1373

Cited by 79 publications

(86 citation statements)

References 19 publications

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“…Surface expression may decrease systemic side effects and more efficiently elicit the biological activities of proteins such as enzymes, single-chain antibodies (scFv) and cytokines. Several chimeric surface proteins have recently been described including chimeric scFv receptors for T-cell activation 15,[24][25][26] or deactivation, 27 membrane-bound cytokines, 28,29 prodrug-activating enzymes 30 and artificial Fc receptors. 31,32 Expression of engineered proteins on the surface of cells therefore represents a rich source for the development of novel therapeutics.…”

Section: Discussionmentioning

confidence: 99%

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%

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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“…[4][5][6][7] In an attempt to improve this situation, prokaryotic carboxypeptidase G2 displayed on the cell surface has been developed for GDEPT. 8,9 However, the use of a non-human enzyme may provoke undesired immune responses, particularly if multiple applications are required. We therefore sought to design a GDEPT system that is based on a human enzyme, functions extracellularly and can potentially be used with multiple prodrugs.…”

Section: Introductionmentioning

confidence: 99%

Cell surface display of a lysosomal enzyme for extracellular gene-directed enzyme prodrug therapy

2001

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Prodrug conversion is a promising approach to cytotoxic gene therapy if an efficient transfer of the generated drug to adjacent cells can be achieved. To maximize the efficacy of this strategy we sought to develop a system that is based on a human enzyme, acts extracellularly yet in close vicinity of the transduced cell and can be used with multiple prodrugs. Results obtained with a secreted version of human ␤-glucuronidase suggested that this enzyme could be a suitable candidate, although a more stringent retention of the

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“…12 Also, we anchored the enzyme to the extracellular membrane through a GPI anchor. This was achieved by the addition of the last exon of Thy1 to the C -terminus of CPG2( Q3 ) linked through a proline codon (Fig 1), an approach already proven to achieve GPI anchor display of secreted tissue inhibitor of metalloproteinases ( TIMP ).…”

Section: Generation Of Adenoviral Vectors Expressing Secreted and Gpimentioning

confidence: 99%

“…The active species of one of these drugs, ZD2767P or [ 4-[bis( 2 -iodoethyl )-amino]phenyl oxy carbonyl]-L -glutamic acid, has been shown to be 300-fold more potent than CMDA. 11 Marais et al 12 highlighted the potential of using CPG2 in a gene therapy approach by stably expressing the enzyme in human tumor cell lines. They achieved CPG2 expression within the extracellular space by linking the transmembrane region of the human tyrosine kinase receptor c-erb B2 to the C -terminus of CPG2.…”

mentioning

confidence: 99%

Adenovirus vector–mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane

et al. 2002

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Carboxypeptidase G2 ( CPG2 ) is a powerful prodrug -converting enzyme. Without a requirement for endogenous enzymes or cofactors, it can directly activate mustard alkylating prodrugs to cytotoxic species, killing both quiescent and dividing cells. This paper provides the first report of its use in the context of a clinically relevant delivery vehicle using adenovirus vectors. To strengthen the efficacy of the prodrug -activating system, the enzyme has been engineered to be secreted or glycosylphosphatidylinositol ( GPI ) anchored to the extracellular membrane of tumor cells, resulting in an enhanced bystander effect by facilitating diffusion of the active drug through extracellular, rather than intracellular, activation. Using the vectors, we have achieved expression of functional secreted or GPI -anchored CPG2 in a panel of tumor cell lines demonstrating no loss in efficacy as a result of GPI anchor retention. Despite variable transduction efficiencies inherent to these vectors, greater than 50% cell kill was achievable in all of the cell lines tested following only a single exposure to the prodrug ZD2767P. Even in cell lines refractive to infection with the vectors, substantial cell death was recorded, indicative of the enhanced bystander effect generated following extracellular prodrug activation. A direct evaluation of the efficacy of our system has been made against adenoviral delivery of herpes simples virus thymidine kinase plus ganciclovir ( GCV ), a suicide gene therapy approach already in the clinic. In a short -term human glioma culture ( IN1760 ) resistant to the clinical chemotherapeutic drug CCNU ( 1 -( 2 -chloroethyl ) -3 -cyclohexyl -1 -nitrosourea ), thymidine kinase / GCV effected no cell killing compared to 70% cell killing with our system.

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A cell surface tethered enzyme improves efficiency in gene-directed enzyme prodrug therapy

Cited by 79 publications

References 19 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

Cell surface display of a lysosomal enzyme for extracellular gene-directed enzyme prodrug therapy

Adenovirus vector–mediated delivery of the prodrug-converting enzyme carboxypeptidase G2 in a secreted or GPI-anchored form: High-level expression of this active conditional cytotoxic enzyme at the plasma membrane

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