1. T-cell activation and cytokine production via a bispecific single-chain antibody fragment targeted to blood-stage malaria parasites
by Shigeto Yoshida, Tominari Kobayashi, Hiroyuki Matsuoka, Chisato Seki, William L. Gosnell, Sandra P. Chang, and Akira Ishii
Blood
Volume 101(6):
March 15, 2003
©2003 by American Society of Hematology

2. The nucleotide and deduced amino-acid sequences of 5. 2-OKT3 biscFv
The nucleotide and deduced amino-acid sequences of 5.2-OKT3 biscFv.Sequences encoding (Gly4Ser)3 are boxed.
The nucleotide and deduced amino-acid sequences of 5.2-OKT3 biscFv.Sequences encoding (Gly4Ser)3 are boxed. The FLAG and His-tag sequences are underlined and bold underlined, respectively.
Shigeto Yoshida et al. Blood 2003;101:
©2003 by American Society of Hematology

3. Expression and binding characteristics of 5. 2-OKT3 biscFv
Expression and binding characteristics of 5.2-OKT3 biscFv.(A) Samples of scFv were purified from culture supernatants of recombinant baculovirus-infected cells by a Ni-NTA column as described in “Materials and methods.” Purified 5.2 scFv (lane 1), OKT3 scFv...
Expression and binding characteristics of 5.2-OKT3 biscFv.(A) Samples of scFv were purified from culture supernatants of recombinant baculovirus-infected cells by a Ni-NTA column as described in “Materials and methods.” Purified 5.2 scFv (lane 1), OKT3 scFv (lane 2), and 5.2-OKT3 biscFv (lane 3) were separated by 10% SDS-PAGE and stained with Coomassie Brilliant Blue R-250 (Nippon Bio-Rad Laboratories, Tokyo, Japan). (B) Native PfMSP-1 (lanes 1-2) and rPfMSP-142 (lanes 3-4) were prepared for SDS-PAGE under nonreducing conditions and run on a 5% to 15% gradient gel. Proteins were transferred to Immobilon membranes (Millipore), probed with either 5.2 mAb followed by biotinylated goat anti–mouse IgG antibody (lanes 1,3) or 5.2-OKT3 biscFv followed by biotinylated mouse anti-FLAG M2 mAb (lanes 2,4).
Shigeto Yoshida et al. Blood 2003;101:
©2003 by American Society of Hematology

4. Flow cytometric analysis of the binding of human T cells by scFvs
Flow cytometric analysis of the binding of human T cells by scFvs.Human T cells (2 × 106) were incubated with OKT3 mAb (A), 5.2 mAb (B), OKT3 scFv (C), 5.2-OKT3 biscFv (D), or 5.2 scFv (E), followed by anti-FLAG M2 secondary antibody.
Flow cytometric analysis of the binding of human T cells by scFvs.Human T cells (2 × 106) were incubated with OKT3 mAb (A), 5.2 mAb (B), OKT3 scFv (C), 5.2-OKT3 biscFv (D), or 5.2 scFv (E), followed by anti-FLAG M2 secondary antibody. Cells were stained with FITC-conjugated goat anti–mouse IgG and analyzed by flow cytometry (solid line). Dotted line represents control cells incubated with the second and third step reagents alone. Data were obtained from 1 of 3 similar experiments.
Shigeto Yoshida et al. Blood 2003;101:
©2003 by American Society of Hematology

5. Bridge formation between CD8+ T cells and merozoites by 5
Bridge formation between CD8+ T cells and merozoites by 5.2-OKT3 biscFv.Schizont-stage parasites were enriched by treatment with E64 as described in “Materials and methods.” To release merozoites from its parasitophorous vacuole, E64 was removed completely ...
Bridge formation between CD8+ T cells and merozoites by 5.2-OKT3 biscFv.Schizont-stage parasites were enriched by treatment with E64 as described in “Materials and methods.” To release merozoites from its parasitophorous vacuole, E64 was removed completely from culture. The parasites were incubated for 3 to 5 hours in the presence of 50 μg/mL 5.2-OKT3 biscFv. When merozoites were detected in the culture, CD8+ T cells were added and incubated for one hour. (A) Bridge formation between CD8+ T cells and merozoites was detected by indirect immunofluorescence staining with FITC-conjugated anti-FLAG M2 mAb. (B) The same image is shown with DAPI staining and photographed through a fluorescence microscope. The nuclei of CD8+ T cells and merozoites were stained in blue. Original magnification, × 1000.
Shigeto Yoshida et al. Blood 2003;101:
©2003 by American Society of Hematology

6. Production of IL-2, TNF-α, and IFN-γ from mononuclear cells stimulated with scFvs.(A) ScFvs (100 μg/mL) were preincubated with rPfMSP-119–coated 96-well plates.
Production of IL-2, TNF-α, and IFN-γ from mononuclear cells stimulated with scFvs.(A) ScFvs (100 μg/mL) were preincubated with rPfMSP-119–coated 96-well plates. After washing, PBMCs (2 × 105/well) were added and cultured for 24 hours. Culture supernatants were harvested and assayed for production of IL-2, TNF-α, and IFN-γ by a sandwich ELISA. (B) Highly synchronous blood-stage cultures of P falciparum at an initial parasitemia of 1% were cocultured with PBMCs (2 × 105/well) and scFvs (50 μg/mL) for 24 hours in a 96-well plate. Culture supernatants were assayed as described in panel A. Results are presented as the means ± SDs from 3 separate experiments. *P < .01 compared with 5.2 scFv and OKT3 scFv. ND indicates not detectable.
Shigeto Yoshida et al. Blood 2003;101:
©2003 by American Society of Hematology

7. Phagocytosis rate in the presence of scFvs
Phagocytosis rate in the presence of scFvs.(A) Highly synchronous blood-stage cultures of P falciparumwere cocultured with PBMCs (2 × 105/well) in the presence or absence of scFvs (50 μg/mL) for 24 or 72 hours in a 96-well plate.
Phagocytosis rate in the presence of scFvs.(A) Highly synchronous blood-stage cultures of P falciparumwere cocultured with PBMCs (2 × 105/well) in the presence or absence of scFvs (50 μg/mL) for 24 or 72 hours in a 96-well plate. The rate of phagocytosis was evaluated by counting the number of cells ingesting one or more P falciparummerozoites per 500 monocytes. Results are presented as the means ± SDs of 3 separate experiments performed in triplicate. *P < .05 compared with 5.2 scFv and OKT3 scFv. (B) Also shown are examples of the phagocytosis. Photomicrographs of Giemsa-stained smears of in vitro P falciparum cocultured with PBMCs in the presence of 5.2-OKT3 biscFv. Many clusters of parasites phagocytosed by monocytes were observed when P falciparum was cultured with 5.2-OKT3 biscFv and PBMCs. (i) Monocytes actively phagocytosing parasites. (ii) Agglutinated clusters of merozoite phagocytosis. (iii) Monocytes phagocytosing parasites and crisis forms of parasites. CF indicates crisis form; P, malaria pigment. Original magnification, × 1000.
Shigeto Yoshida et al. Blood 2003;101:
©2003 by American Society of Hematology

8. Schematic representation of proposed mechanism of 5
Schematic representation of proposed mechanism of 5.2-OKT3 biscFv-mediated cellular inhibition of parasite growth.
Schematic representation of proposed mechanism of 5.2-OKT3 biscFv-mediated cellular inhibition of parasite growth.
Shigeto Yoshida et al. Blood 2003;101:
©2003 by American Society of Hematology