Efficacy of an adapted granzyme B-based anti-CD30 cytolytic fusion protein against PI-9-positive classical Hodgkin lymphoma cells in a murine model

Abstract

Tumors develop when infiltrating immune cells contribute growth stimuli, and cancer cells are selected to survive within such a cytotoxic microenvironment. One possible immune-escape mechanism is the upregulation of PI-9 (Serpin B9) within cancer cells. This serine proteinase inhibitor selectively inactivates apoptosis-inducing granzyme B (GrB) from cytotoxic granules of innate immune cells. We demonstrate that most classical Hodgkin lymphoma (cHL)-derived cell lines express PI-9, which protects them against the GrB attack and thereby renders them resistant against GrB-based immunotherapeutics. To circumvent this disadvantage, we developed PI-9-insensitive human GrB mutants as fusion proteins to target the Hodgkin-selective receptor CD30. In contrast to the wild-type GrB, a R201K point-mutated GrB construct most efficiently killed PI-9-positive and -negative cHL cells. This was tested in vitro and also in vivo whereby a novel optical imaging-based tumor model with HL cell line L428 was applied. Therefore, this variant, as part of the next generation immunotherapeutics, also named cytolytic fusion proteins showing reduced immunogenicity, is a promising molecule for (targeted) therapy of patients with relapsing malignancies, such as cHL, and possibly other PI-9-positive malignancies, such as breast or lung carcinoma.

Figure 1

(a) Schematic structure of the protein EGb-Ki4(scFv). After purification, it is activated via enterokinase (‘E')-digestion site indicated by an arrow. (b) Coomassie-stained SDS-PAGE gel with purified Gb-Ki4(scFv) before and after activation with enterokinase. (c) Results of apoptosis assay (annexin V/propidium iodide staining) on PI-9⁻ L540cy cells and PI-9⁺ L428 cells after incubation with 33 nℳ Gb-Ki4(scFv). Numbers are % of cells within corresponding quadrant. (d and e) Expression of PI-9 in cHL cell lines L428, L1236 and L540cy. (d) Total soluble protein (40 μg/lane) was loaded on a gel for SDS-PAGE, and PI-9 was subsequently detected in a western blot using the antihuman PI-9 (clone 7D8) and GAM-PO. (e) 1 × 10⁶ cells, as indicated, were fixed, permeabilized and stained with antihuman PI-9 (7D8) and GAM-FITC. PI-9 expression was determined by flow cytometry. (f) Complex formation of PI-9 and Gb-Ki4(scFv) within cell lysates of L540cy and L428 cells after pre-incubation with Gb-Ki4(scFv) ‘G' or buffer ‘B'. Altogether, 40 μg total soluble protein was loaded on SDS-gel, and western blot was analyzed with antihuman GrB. Unspecific band was detected on the same height of CFP.

Figure 2

Enzymatic activity of Gb-Ki4(scFv) and its mutant GbR201K-Ki4(scFv) determined via a colorimetric assay based on the synthetic GrB substrate Ac-IETD-pNA. Absorbance at 405 nm was monitored in a 2-min interval for 60 min at 37 °C. Activities for both constructs were the same.

Figure 3

(a and b) Flow cytometric binding analysis of Gb-Ki4(scFv), GbR201K-Ki4(scFv) and GbR201K-H22(scFv) to CD30⁺ target cell line L428 (a), and to CD30⁻ cell line HL60 (AML) (b). Gray background is second antibody control. (c) Comparison of mean fluorescence intensities after binding of Gb-Ki4(scFv) to cell lines. MFI values correlate with CD30 surface expression. Shift for L540cy was set to 100%. (d) Confocal microscopy of CD30⁺ cell lines, L540cy and L428, after incubation with Ki4(scFv)-SNAP-BG-647 at 37 and 4 °C. Negative control is incubated only in buffer. Internalization was analyzed after 2-h incubation. Serum stability of the CFPs for at least 10 h was confirmed by residual binding activity to L540cy, measured via flow cytometry after different incubation times in 50% mouse serum (e), or PBS (f). Data shown for GbR201K-Ki4(scFv). (g) Overview of serum stability based on MFIs measured during flow cytometric analysis shown in % of MFI related to 0 h. Data in gray for Gb-Ki4(scFv) (triangle: PBS, rectangle: serum) and in black for GbR201K-Ki4(scFv) (triangle: serum, rectangle: PBS). Representative data from one out of three experiments are shown.

Figure 4

(a) Colorimetric cytotoxicity assays with various CFP concentrations (n=3 parallel cell cultures per dilution) showing strong dose-dependent effects of Gb-Ki4(scFv) and its mutants on PI9⁻ L540cy. IC₅₀ values are listed for all constructs. (b and c) Toxic effect of 11 nℳ Gb-Ki4(scFv) variants in 2 × 10⁵ L540cy cells (b; PI-9⁻) and L1236 and L428 (c; PI-9⁺) after 48 h of cultivation at 37 °C. (b) Apoptotic cells were determined via normalized annexin V-GFP flow cytometric analysis and converted to viability. The statistical significance between treated and untreated samples was P<0.005. (c) Viability was determined by XTT metabolization. Standard deviations are shown for three–five independent experiments. All statistical significances were determined via two-tailed t-test: (*) for P<0.05 and (***) for P<0.001. (d) Successful activation of caspase 3/7 by the mutated construct GbR201K-Ki4(scFv) in PI-9⁺ L428 and L1236 was demonstrated via a preluminescent caspase 3/7-DEVD-aminoluciferine substrate. Data are shown as normalized to the background signal of Gb-Ki4(scFv).

Figure 5

Results of mouse experiments with L428-induced tumors. (a) Treatment schedule indicating intravenous (i.v.) injection of L428 cells, protein injection and days of measurement. (b) Relative tumor size after treatment with fusion proteins, as indicated. Tumor size was estimated by imaging with the Cri Maestro System. The mutant GbR201K-Ki4(scFv) caused a decelerated tumor growth as compared with the unspecific control GbR201-H22(scFv) (**) and to the wild-type Gb-Ki4(scFv) (*). The difference between Gb-Ki4(scFv) and GbR201K-H22(scFv) was not significant (ns). (c) Confocal microscopy of Ki4(scFv)-SNAP-BG-Vista-Green (i) stained tissue sections of control-treated L428 tumors, Kat2 signal from transfected cells is visible in red (ii) and the overlay of an enlarged image.

Figure 6

Results of mouse experiments with L540cy-induced tumors. (a) Treatment schedule indicating i.v. injection of cells, protein injection and days of measurement. (b) Relative tumor size is shown at time points as indicated upon treatment with mutant GbR201K-Ki4(scFv) versus control GbR201K-H22(scFv). The difference between the curves was determined to be statistically significant with P<0.001 (***). (c) Confocal microscopy of Ki4(scFv)-SNAP-BG-Vista-Green-stained tissue sections of control-treated L540cy tumors. (d) Receptor depending targeting of L540cy with Ki4(scFv)-SNAP-BG-747 in vivo after 6 h. Protein was applied i.v. into L540cy-bearing mice. Measurement was taken with deep-red filter set ((730–950 nm) (i) background signal, (ii) image with fluorescence signal (yellow, Ki4(scFv)-SNAP-BG-747).