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. 2010 Feb 15;49(8):1451-5.
doi: 10.1002/anie.200906232.

Drug-free macromolecular therapeutics: induction of apoptosis by coiled-coil-mediated cross-linking of antigens on the cell surface

Kuangshi Wu  1 , Jihua Liu, Russell N Johnson, Jiyuan Yang, Jindrich Kopecek
Affiliations

Drug-free macromolecular therapeutics: induction of apoptosis by coiled-coil-mediated cross-linking of antigens on the cell surface

Kuangshi Wu et al. Angew Chem Int Ed Engl. .

Abstract

A new paradigm was designed for apoptosis induction mediated by the biorecognition of coiled-coil motifs at the Raji B cell surface. The heterodimerization of complementary peptides, one bound to Fab’ antibody fragment, the other as grafts to HPMA copolymer, results in crosslinking of CD20 target antigens, and consequently, initiation of apoptosis.

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Figures

Figure 1
Figure 1
Induction of apoptosis in Human Burkitt's NHL Raji B cells by crosslinking its CD20 antigens, mediated by antiparallel coiled-coil formation at cell surface. The simplified scheme is not drawn to scale.
Figure 2
Figure 2
a,b) Synthetic schemes for (CCK)9-P and Fab'-(CCE)1; c) CD spectra of CCE, CCK, (CCK)9-P, equimolar mixtures of CCE and (CCK)9-P, and Fab'-(CCE)1 and (CCK)9-P. Concentrations: [CCE]=[CCK]=[Fab’-(CCE)1]=50 μM; [(CCK)9-P]=5.60 μM. Data were acquired at 25 °C in PBS (pH 7.4).
Figure 3
Figure 3
Biorecognition of Fab’-CCE and CCK-P on Raji B cell surface. (r1-r2) Reference cells under transmitted light. (a) Exposure of cells to Fab’-(CCE)1 (0.5 μM, Rhodamine Red-X labeled) resulted in decoration of cell surface with CCE; (b) Exposure of cells to (CCK)9-P ([CCK]=25 μM, FITC labeled) did not result in staining; (c1-c3) Exposure of cells to a premixture of Fab’-(CCE)1 (0.5 μM) and (CCK)9-P([CCK]=25 μM); (d1-d3) Consecutive exposure of cells to Fab’-(CCE)1 (0.5 μM) followed 1 h later by (CCK)9-P ([CCK]=25 μM). (a, c1, and d1) red channel for Rhodamine Red-X; (b, c2, and d2) green channel for FITC; (c3 and d3) Overlay of red and green channels. Images of individual cells are shown.
Figure 4
Figure 4
Time dependence of apoptosis induction in Raji B cells by caspase 3 activity assay. Untreated: Untreated cells; Fab’-(CCE)1: Single component control at 0.5 μM; (CCK)9-P: Single component control ([CCK]=25 μM); 1F5+GAM: 1F5 Ab (0.2 μM) 1 h followed by secondary antibody GAM (10 μg/mL); Premixed: Premixture of Fab’-(CCE)1 (0.5 μM) and (CCK)9-P ([CCK]=25 μM); Consecutive: Consecutively added Fab’-(CCE)1 (0.5 μM) followed 1 h later by (CCK)9-P ([CCK]=25 μM). Results are presented as means ± SD (n=3).
Figure 5
Figure 5
Coiled-coil mediated induction of apoptosis of Raji B cells as assessed by: a) Caspase 3 activity; b) Annexin V/propidium iodide assay; and c) TUNEL assay. The concentrations of 1F5 and GAM were 0.2 μM and 10 μg/mL, respectively. Concentrations of Fab’-(CCE)1 and CCK in (CCK)9-P were 0.5 and 25 μM, respectively for P1 and C1 in two experiments of the caspase 3 assay; 1 and 25 μM, respectively for all other experiments. P-ctrl1: a premixture of Fab’ (1 μM), CCE (1 μM), CCK (25 μM), and P-NH2 (2.80 μM); P-ctrl2: a premixture of Fab’-(CCE)1 (1 μM) and P-NH2 (2.80 μM); P-ctrl3: a premixture of Fab’ (1 μM) and (CCK)9-P (2.80 μM); C-ctrl1: Fab’ (1 μM) and CCE (1 μM) for 1 h, and then CCK (25 μM) and P-NH2 (2.80 μM); C-ctrl2: Fab’-(CCE)1 (1 μM) for 1 h, then P-NH2 (2.80 μM); C-ctrl3: Fab’ (1 μM) for 1 h, then (CCK)9-P ([CCK]=25 μM / [P-NH2]=2.80 μM). Results are presented as means ± SD (n=3).
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