Hapten-directed targeting to single-chain antibody receptors

Abstract

Artificial recombinant receptors may be useful for selectively targeting imaging and therapeutic agents to sites of gene expression. To evaluate this approach, we developed transgenes to express highly on cells a single-chain antibody (scFv) against the hapten 4-ethoxymethylene-2-phenyl-2-oxazoline-5-one (phOx). A phOx enzyme conjugate was created by covalently attaching phOx molecules to polyethylene glycol (PEG)-modified b-glucuronidase. Cells expressing phOx scFv but not control scFv receptors were selectively killed after exposure to -glucuronidase derivatized with phOx and PEG (phOx-bG-PEG) and a glucuronide prodrug (phydroxy aniline mustard b-D-glucuronide, HAMG) of p-hydroxyaniline mustard. Targeted activation of HAMG produced bystander killing of receptor-negative cells in mixed populations containing as few as 10% phOx-receptor-positive cells. Functional phOx scFv receptors were stably expressed on B16-F1 melanoma tumors in vivo. Treatment of mice bearing established phOx-receptorpositive tumors with phOx-bG-PEG and HAMG significantly (Pp.0005) suppressed tumor growth as compared with treatment with bG-PEG and HAMG or prodrug alone. phOx was unstable in the serum, suggesting alternative haptens may be more suitable for in vivo applications. Our results show that therapeutic agents can be targeted to artificial hapten receptors in vitro and in vivo. The expression of artificial receptors on target cells may allow preferential delivery of therapeutic or imaging molecules to sites of transgene expression.

Gene expression imaging by enzymatic catalysis of a fluorescent probe via membrane-anchored β-glucuronidase

“…Successful use of surface bG therefore requires efficient transport of bG to the cell surface. We previously performed a series of studies to determine the optimal conditions for the expression of proteins 22 and scFv 23,24 on mammalian cells. Based on those studies, we anchored bG on the surface of cells by fusion of the enzyme to the Ig-hingelike domains of the B7-1 antigen (e-B7) and the B7-1 TM.…”

Cutting Edge: Mechanical Forces Acting on T Cells Immobilized via the TCR Complex Can Trigger TCR Signaling

“…The construction of p2C11-g1-B7 (CD3L-2d), p2C11-CD44-B7 (CD3L-CD44), and pLNCX-phOx-g1-B7 (phOx-2d) has been described (12,13). A DNA fragment coding the ectodomain of CD43 was amplified by RT-PCR of RNA isolated from Jurkat T cells and inserted into the unique SalI site in p2C11-B7 to generate p2C11-CD43-B7 (CD3L-CD43).…”

A membrane antibody receptor for noninvasive imaging of gene expression

“…We subsequently discovered that phOx is unstable in serum, resulting in loss of binding to antibody. 27 (DNS) 2 -DTPA- 111 In, in contrast, could be specifically bound by DNS scFv receptors even after 24 h in the presence of 20% mouse serum. The high thermodynamic stability of 111 In for DTPA 34 argues that the (DNS) 2 -DTPA-111 In complex should be very stable for in vivo imaging.…”

“…27,28 B16-F1 murine melanoma cells were infected with recombinant retroviral particles, selected in G418 and sorted in a flow cytometer for high expression of the DNS or phOx scFv receptors to obtain stable B16/DNS and B16/phOx cell lines. The surface expression of the receptors was analyzed by immunofluorescence staining using an antibody with specificity for the c-myc epitope present in the receptors.…”