Article Figures & Data
Figures
- Fig. 1.
Binding of phage to Daudi cells. Phage were prepared from single colonies eluted after the second round of panning and titered as described in “Materials and Methods.” Five × 105 Daudi cells were incubated with 8 × 108 phage for 60 min at room temperature; washed with DPBS + 0.5 mm EDTA + 0.5% BSA, and incubated with 5 μg of anti-M13 antibody (Amersham) for another 20 min. Successively, a FITC-conjugated goat antimouse IgG (H+L) antibody was added to the cells, and analysis was performed using a flow cytometer (Becton and Dickinson). A, cell, cells were not incubated with phage or with anti-M13 antibody but only with FITC-conjugated goat antimouse IgG (H+L) antibody; GSSY, WT phage; GSSY+IT, 63 μg of RFB4 immunotoxin were added, together with GSSY phage to Daudi cells, and processed because the sample incubated only with the phage. B, cell, cells were not incubated with phage or with anti-M13 antibody but only with FITC-conjugated goat antimouse IgG (H+L) antibody; GSSY, WT phage; line A, mutant phage A, binder; line B, mutant phage B, binder; line C, mutant phage C, binder.
- Fig. 2.
Cytotoxicicity assay on cells isolated from patient 1. Ficoll-purified mononuclear cells from the patient were incubated with immunotoxins for 3 days at 37°C and pulsed with [3H]leucine for 6–8 h. Radiolabeled material was captured on filter-mats and counted in a Betaplate scintillation counter (Wallac). The graph shows, protein synthesis as measured by cpm of [3H]leucine incorporated into protein. The horizonial dashed line indicates, 50% inhibition of protein synthesis, which is halfway between the level of incorporation in the absence of toxin and that in the presence of 10 μg/ml of cycloheximide.
- Fig. 3.
Biacore sensograms of RFB4(Fv)-PE38 mutants. Binding profile of WT immunotoxin GSSY is compared with mutants GTTW, GTHW, and GYNW. RITs were diluted to 25 μg/ml in HEPES buffered saline buffer and injected over a chip surface containing CD22. Binding kinetics were analyzed using BIA evaluation 2.1 software. Y axis, the resonance units bound. X axis, the time in seconds.
Tables
- Table 1
DNA and amino acids sequences of CDR3 heavy chain RFB4
The nucleotide and amino acid sequence of the entire CDR3 heavy chain is shown. Hot spots with the sequence Pu-G-Py-A/T are underlined. The amino acids mutagenized are in bold. The residues are numbered according to the Kabat database (21) .
95 96 97 98 99 100 100A 100B 100C 100D 100E 100F 101 102 H S G Y G S S Y G V L F A Y CAT AGT GGC TAC GGT AGT AGC TAC GGG GTT TTG TTT GCT TAC - Table 2
Sequences of mutant phage obtained after panning
Amino acid sequences of mutant phage isolated after two rounds of panning are listed. The entire Fv of each phage was sequenced. Only amino acids in the target region are shown. Each residue is numbered according to the Kabat database (21) .
99 100 100A 100B G S S Ya G T H Wb G Y N Wb G T T Wb,c G S T Yb G K N Rb,c G S T Rd G H T F G N R Y G T A Y G T N Y G L H Y G F L Y G S R Y G R N Y G V H R G A L R G V R A G T A K G R T S -
a WT.
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b Immunotoxin made.
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c Clone found three times.
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d Clone found two times.
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- Table 3
Cytotoxic activity (IC50) in ng/ml of selected RFB4 (Fv)-PE38 mutants on six different CD22-positive cell lines
Cells were seeded at 5 × 104/well in 96-well plate 24 h before the assay. Immunotoxins were added to the plate, and cells were incubated at 37°C for 20 h and then were pulsed with [3H]leucine for 2 h. [3H]leucine incorporations were determined. Each assay was done in triplicate, and, except as noted below, at least three different assays were performed with each cell line. IC50 (expressed in ng/ml) is the toxin concentration that reduced incorporation of radioactivity by 50% compared with the cells that were not treated with the toxin. Mean values of three experiments ± SD are shown. SDs are not listed when the immunotoxins were tested only once. Below each IC50 is the significance in its difference from GSSY (WT) and GTHW when differences by Student’s t test were significant (P < 0.05).
JD38 Ca46 Raji Daudi Namalwa Ramos GSSY (WT) vs. GTHW 2.3 ± 0.5 3.1 ± 0.2 5.1 ± 0.15 8.1 ± 2.3 10.6 ± 1.2 252 ± 3 P < 0.01 P < 0.05 P < 0.01 P < 0.01 P < 0.01 P < 0.01 GTHW vs. WT 0.2 ± 0.09 1.4 ± 0.5 1 ± 0.14 1.7 ± 0.1 2.8 ± 0.4 32 ± 3 P < 0.01 P < 0.05 P < 0.01 P < 0.01 P < 0.01 P < 0.01 GYNW vs. WT vs. GTHW 0.6 ± 0.1 0.8 0.6 ± 0.07 2.1 ± 0.5 5.6 ± 3.3 N.D.a P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.05 GTTW vs. WT vs. GTHW 0.7 ± 0.03 1.7 ± 0.4 2.75 ± 0.3 2.0 ± 1.2 5.3 ± 0.2 N.D. P < 0.01 P < 0.05 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.01 GSTY vs. WT vs. GTHW 1.1 2 4.1 8.5 ± 0.7 9.5 N.D. P < 0.01 GKNR vs. WT vs. GTHW 5 6 10 55 ± 7 25 N.D. P < 0.01 P < 0.01 -
a N.D., not determined.
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- Table 4
Cytotoxic activity (IC50) in ng/ml of mutant immunotoxins on patient cells
Patient 1 Patient 2 Patient 3 Patient 4 Patient 5 GSSY (WT) vs. GTHW >1000 490 ± 70 34 ± 5 6.7 ± 2.3 >1000 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.01 GTHW vs. GSSY (WT) 29 ± 10 22 ± 2 1.5 ± 0.4 <1 28 ± 6 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.01 GYNW vs. GSSY (WT) vs. GTHW 105 ± 48 40 ± 5 3.4 ± 0.7 N.D.a 41 ± 2 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.05 P < 0.05 GTTW vs. GSSY (WT) vs. GTHW >1000 95 ± 9 8.5 ± 3 1.5 ± 0.6 76 ± 9 P < 0.01 P < 0.01 P < 0.01 P < 0.05 P < 0.01 P < 0.01 P < 0.01 P < 0.01 P < 0.01 GSTY vs. GSSY (WT) vs. GTHW N.D. N.D. 15 ± 2 2.1 ± 0.7 129 ± 50 P < 0.01 P < 0.01 P < 0.05 P < 0.01 P < 0.01 P < 0.05 -
Ficoll-purified mononuclear cells from patients were obtained by an approved protocol at the NIH. Cells were incubated with immunotoxins for 3 days at 37°C and pulsed with [3H]leucine for 6–8 h; protein synthesis was measured. IC50s are expressed in ng/ml; SDs are shown. Each assay was done in triplicate. Diagnosis for patients 1, 2, 3, and 5 was CLL; diagnosis for patient 4 was HCL variant.
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a N.D., not determined.
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Significance in difference between IC50s as in Table 3<$REFLINK> .
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- Table 5
Summary of BIAcore analysis mutants of RFB4-PE38
Binding kinetics were analyzed on a BIAcore 2000 Biosensor (BIAcore). On and off rates were measured by injecting immunotoxins at a concentration of 380 nm over a CM5 sensor chip (BIAcore) containing immobilized CD22. Bound material was allowed to dissociate for 5 min or more. Immunotoxin Kon (M−1S−1) Koff (S−1) Kda (nm) GSSY (WT) 2.08 × 104 1.77 × 10−3 85 GTHW 3.27 × 104 2.07 × 10−4 6 GYNW 1.84 × 104 1.91 × 10−4 10 GTTW 2.62 × 104 6.5 × 10−4 24 GSTY 3.15 × 104 1.55 × 10−3 49 -
a Kd was calculated by dividing Koff by Kon.
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Volume 8, Issue 4
