Article Figures & Data
Figures
- Fig. 1
A, purified HA22 (R490A) immunotoxin elution profile from Superose-12 gel filtration column chromatography. B, SDS-PAGE of HA22 (R490A) under nonreducing and reducing conditions. HA22 (R490A) was prepared as described in Materials and Methods and elution fraction 12 was analyzed on a 4-20% polyacrylamide gel. X-axis, number of fractions marked on the chromatogram. Lanes: molecular weight standards (lane M); nonreducing condition (lane 1); the purified HA22 (R490A) immunotoxin (lane 2) was reduced by boiling for 5 minutes in SDS sample buffer containing DTT. The gel was stained with Coomassie blue. The nonreduced dsFv immunotoxin migrated Mr ∼63,000 and dissociated into VL chain (Mr ∼12,000) and VH-PE38 fusion protein (Mr ∼51,000) by reduction.
- Fig. 2
Inhibition of protein synthesis (A and B) and cell viability (C) on CD22-positive cells. Inhibition of protein synthesis was determined as percentage of [3H]leucine incorporation in cells after 20 hours of treatment with indicated concentrations of immunotoxins. A, CA46 cells; B, Daudi cells. Fifty percent inhibition of protein synthesis is halfway between the level of incorporation in the absence of toxin and that in the presence of 10 μg/mL cycloheximide. C, CA46 cells were incubated with immunotoxin for 40 hours before WST-8 was added for 4 hours. Formazan production was measured at A450 nm and A650 nm. Points, mean of triplicate values; bars, SD. In A-C, •, BL22; ▿, HA22; ▪, HA22 (R490A). D, viability of HA22 (R490A) on HUVEC. HUVEC 3,000 cells/well incubated with various concentrations of immunotoxins for 72 hours. Cell viability was determined by a WST assay as described in Materials and Methods. Results are given as %viability of incubation without immunotoxin; points, mean of triplicate values; bars, SD. •, BL22; ▿, HA22; ▪, HA22 (R490A); ◊, HB21Fv-PE40; ▴, LMB-7. E, comparison of ADP-ribosylation activity between HA22 and HA22 (R490A). ADP-ribosylation assays were done as described (16) in duplicate. F, pharmacokinetics of HA22 (R490A) in mice. Normal female Swiss mice were injected i.v. with 10 μg HA22 (•) and HA22 (R490A; ▿). Blood samples were drawn at different times. The concentration of each immunotoxin in `the circulation was determined by ELISA.
- Fig. 3
Antitumor activities of HA22 and HA22 (R490A). CA46 cells were inoculated s.c. in SCID mice on day 0. A, on day 6, when tumors >100 mm3 developed, groups of 8 or 10 mice were either observed (▪) or began treatment with i.v. injections of HA22 (•) or HA22 (R490A; ▿) diluted in 0.2 mL PBS/0.2% human serum albumin. Therapy was given once every other day ×3 (on days 6, 8, and 10; arrows) at 150 μg/kg. B, the antitumor response of mice treated with HA22 (•) at 300 μg/kg every other day ×3 was contrasted with that of mice that were untreated (▪) or treated with HA22 (R490A) 300 μg/kg every other day ×3 (▿). No death was observed at these doses. Points, mean; bars, SD. C, comparisons between HA22 and HA22 (R490A) at each dose were statistically significant. *P < 0.05; **P < 0.001 (Wilcoxon's rank sum test).
Tables
- Table 1
Cytotoxicity activity (IC50) in ng/mL of BL22 and mutant immunotoxins toward various cell lines
CD22-positive cell line (Burkitt's lymphoma) CD-22-negative cell line Daudi CA46 Raji HUT-102* L540† BL22 2.78 ± 0.11 0.85 ± 0.04 2.63 ± 0.24 >2,000 >10,000 HA22 0.46 ± 0.02 0.24 ± 0.01 1.02 ± 0.09 >2,000 >10,000 HA22 (R490A) 0.18 ± 0.01 0.18 ± 0.02 0.27 ± 0.02 ∼2,000 >10,000 NOTE. Cytotoxicity data are given as IC50's, which are the concentrations of immunotoxin that cause a 50% inhibition in protein synthesis compared with controls after incubation with cells for 20 hours. Means values of three experiments ± SD are shown.
↵* CD22-negative, human T-cell lymphotrophic virus-1–positive T-cell lymphomas.
↵† CD22-negative, CD30-positive Hodgkin's lymphomas.
- Table 2
Nonspecific toxicity in mice of HA22 and HA22 (R490A)
Dose (mg/kg) BL22 HA22 (death/total) HA22 (R490A) 0.75 0/10 0/5 1.0 1/14 0/10 0/15 1.25 14/14 4/10 5/15 1.50 9/9 7/10 10/15 1.75 9/10 15/15 NOTE. On day 0, female Swiss mice (5-6 weeks, 18-22 g weight) were treated by tail vein single injection with various amounts of immunotoxins in 0.2 mL PBS containing 0.2% human serum albumin. Animal mortality was observed for 2 weeks.
- Table 3
Cytotoxicity of SS1P and SS1P (R490A)
Immunotoxin IC50 (ng/mL) A431/K5 A1847 SS1P 0.67 ± 0.01 (0.74 ± 0.07)* 3.85 ± 0.39 (4.24 ± 2.45)* SS1P (R490A) 0.48 ± 0.01 (0.46 ± 0.06)* 1.19 ± 0.18 (2.38 ± 0.61)* NOTE. Cytotoxicity assays were done by measuring incorporation of [3H]leucine in cells after 20 hours of treatment with indicated concentrations of immunotoxins. IC50 is the concentration that causes 50% inhibition of protein synthesis. Mean values of three experiments ± SD are shown.
↵* Cytotoxic effects were tested in WST-8 assay and expressed as a relative viability (%untreated control), mean ± SD.
Volume 11, Issue 4
