Immunotherapeutic targeting of membrane Hsp70-expressing tumors using recombinant human granzyme B

Background: We have previously reported that human recombinant granzyme B (grB) mediates apoptosis in membrane heat shock protein 70 (Hsp70)-positive tumor cells in a perforin-independent manner

Enzymatic activity during the purification of grB.

<p><b>A</b>–<b>F</b> grB before enterokinase digestion; H, J’ flow through during purification process; G, I, J, K grB after enterokinase digestion: <b>A.</b> cell culture supernatant of the HEK293 producing cells; <b>B.</b> after addition of imidazole; <b>C.</b> flow through of Ni column; <b>D.</b> His-tagged grB; <b>E.</b> pooled His-tagged grB; <b>F.</b> after exchange to enterokinase buffer; <b>G.</b> after enterokinase digestion; <b>H.</b> flow through of heparin column; <b>I.</b> pooled grB containing fractions after heparin column; <b>J.</b> concentrated grB; <b>J’.</b> flow through from concentration procedure; <b>K.</b> sterile filtered (0.2 µl) active grB (final product).</p

Recombinant human grB induces apoptosis in CT26 tumor spheroids.

<p>(<b>A</b>) CT26 tumor spheroids were treated either with PBS (ctrl) or grB (4, 10, 20, 40, 80 µg/ml) for 48 h. Single cell suspensions were generated from tumor spheroids by trypsinization and the cells permeabilized before being stained for active caspase-3. The percentage of active caspase-3 positive cells was determined using a BD Biosciences FACSCalibur™. The data represent mean values of 3–4 independent experiments ± S.D. All grB-treated samples significantly differed from ctrl values (p***<0.001). (<b>B</b>) CT26 tumor spheroids were incubated for up to 14 days either with PBS (ctrl; filled circles), grB (80 µg/ml; open circles) or cam (4 µg/ml; filled triangles). Diameters of spheroids were measured on days 0, 1, 3, 6, 7, 10, and 14 after treatment. The data represent mean values of 6 independent experiments ± S.D. After a slight increase in diameter size, grB initiated a significant shrinkage of spheroids from day 7 onwards (day 7, p** = 0.004; day 10, p** = 0.0063; day 14, p*** = 0.0003). A significant reduction in tumor size was observed after treatment with cam from day 6 onwards (day 6, p** = 0.0061; day 7, p** = 0.0022; day 10, p** = 0.0019; day 14, p** = 0.0012). (<b>C</b>) Effect of treatment with inactive and active grB for 24 h and 48 h on the percentage of active caspase-3 positive cells in CT26 cells isolated from tumor spheroids. (<b>D</b>) Effect of treatment with inactive and active grB for 24 h and 48 h on the percentage of membrane Hsp70-positive CT26 cells in tumor spheroids. Spheroids were incubated with activated or inactivated grB for the indicated time periods, after which single cell suspensions were prepared following trypsinization. Cells were then incubated with cmHsp70.1-FITC mAb and membrane Hsp70 expression was determined by flow cytometry.</p

Recombinant human grB induces apoptosis in monolayer CT26 tumor, but not normal cells.

<p>(<b>A</b>) Clonogenic cell survival of CT26 tumor cells after treatment with active human grB (0.04, 0.2, 0.4, 0.6, 0.8, 1, 2, 4 µg/ml) on day 6. The data represent the mean of three independent experiments ± S.D. Significant differences in the survival of grB-treated CT26 vs. untreated controls were seen from 0.6 µg/ml upwards (<i>p</i>*<0.05). (<b>B</b>) CT26 cells were treated with PBS (ctrl), granzyme B (4 µg/ml; grB) or camptothecin (4 µg/ml; cam) for 12 h (black bars), 24 h (grey bars), and 48 h (white bars). Permeabilized cells were stained with a FITC-conjugated active caspase-3 specific antibody and the percentage of active caspase-3 positive cells determined by flow cytometry. The data represent the mean of 3–10 independent experiments ± S.D. (<i>p</i>***<0.001; <i>p</i>** = 0.01). (<b>C</b>) Light microscopical views of CT26 tumor cells after treatment with PBS (ctrl), grB (4 µg/ml), cam (4 µg/ml) or inactive grB (inact grB) for 48 h (20x objective, scale bar 100 µm). Representative images are shown from at least 3 experiments using CT26 tumor cells (<b>D</b>) CD31-positive mouse endothelial cells from healthy BALB/c mice (normal) and CT26 tumor cells (tumor) were treated either with PBS (ctrl; black bars) or grB (4 µg/ml; grey bars) and the percentage of cells that positively stained for active caspase-3 was determined by flow cytometry. The data represent the mean of six independent experiments ± S.D. Asterisks represent significantly different values (<i>p</i>*** = 0.001). Inactive grB showed similar results like PBS (data not shown) (<b>E</b>) Light microscopic phase contrast analyses of adherent growing CD31-positive mouse endothelial cells (normal) treated for 24 h with PBS (ctrl, upper graph) or grB (4 µg/ml). Both untreated and treated CD31-positive mouse endothelial cells show regular cell morphology. Similar results were obtained in three independent experiments (20x objective, scale bar 100 µm).</p

Active human grB results in tumor regression without the induction of adverse effects.

<p>(<b>A</b>) CT26 tumor-bearing BALB/c mice were injected (i.p.) either with PBS (ctrl), inactive grB (inact grB) or enzymatically active grB (grB; 20 µg/g mouse body weight in 200 µl PBS) on days 6, 7, 13, 14 after i.p. injection of CT26 tumor spheroids. Mice were sacrificed on day 21. The weights of tumors in mice treated with grB were significantly lower than those in the control group (n = 15 per group; <i>p</i>*<0.05). The weight of tumors in mice treated with active or inactive grB were also significantly different (<i>p</i>*<0.05), whereas there was no significant difference in the weights of tumors in control mice or mice that had been treated with inactive grB (<i>p</i> = 0.49). (<b>B</b>) Comparison of CT26 tumors derived from control mice (ctrl), active and inactive grB-treated mice (see above). Representative sections of formalin-fixed paraffin-embedded tumors stained with H&E. R1 marks necrotic/apoptotic areas in the centre of the tumor of ctrl animals; R2 marks necrotic/apoptotic areas in the border area of the tumor of active grB-treated mice; vessels are marked with a circle. (<b>C</b>) Comparison of sections from formalin-fixed paraffin-embedded liver, kidney, heart, lung and spleen derived from control mice and mice treated with grB (see above) stained with H&E were analyzed using light microscopy (20x objective, indicated scale bar 100 µm). (<b>D</b>) Intraoperative detection of Cy5.5-labeled cmHsp70.1 mAb in BALB/c mice bearing CT26 tumors. Antibody (100 µg) was injected (i.v.) into the tail vein on day 6 after tumor injection. Representative false color images and views of the Cy5.5 fluorescence of the neck part of three mice were taken 24 hours after i.v. injection. The circled area indicates the histologically proven tumor tissue; arrows indicate superficial blood vessels in the normal tissue (upper arrow) and the tumor (lower arrow). (<b>E</b>) Relative binding of cmHsp70.1 mAb to isolated CT26 tumor cells (tumor) and endothelial (normal) cells. The capacity of cells to bind cmHsp70.1-FITC is presented as mean equivalents of soluble fluorochrome (MESF) divided by the antibody binding capacity (ABC) and was determined using the Quantum Simply Cellular anti-Mouse IgG according to the manufacturer’s protocol (Bangs Laboratories, Inc., Fishers, IN, USA). The Y-axis is a logarithmic scale.</p

Confocal analysis of immunofluorescence staining of grB (ATTO488, green) and Rab4, Rab5, Rab7, Rab9, Rab11, LAMP1, and LAMP2 (Cy3, red).

<p>(<b>A</b>) CT26 tumor cells were grown on MatTek Glass Bottom Culture Dishes then incubated with 4 µg/ml ATTO488-labeled grB for 0, 5, 15, 30, 60, 120 and 240 min. After fixation, cells were stained with anti-endosomal (Rab4, Rab5, Rab7, Rab9, Rab11) or lysosomal antibodies (LAMP1, LAMP2) followed by appropriate Cy3-labeled secondary antibodies. Cells were mounted in DAPI-Vectashield and imaged using a Zeiss LSM 510 Inverted microscope (63x objective). Co-localization, as determined by an overlap of the green and red fluorescence, is visible as yellow. One representative image from 2 independent experiments is shown (scale bar, 10 µm). (<b>B</b>) Semi-quantitative analysis of the co-staining intensity and kinetics of endocytosis of grB and Rab9/11 and LAMP1/LAMP2. (<b>C</b>) Schematic representation of the mode of uptake and intracellular trafficking of grB in tumor cells.</p

Comparative flow cytometric histograms of membrane Hsp70 expression on viable (7-AAD negative) cells from primary tumors and distant metastases of three patients, and on a relapse tumor and a distant metastases of another patient using FITC-labelled IgG1 isotype-matched control antibody (open histogram) and cmHsp70.1 mAb (grey histogram).

<p>The mean fluorescence intensity of Hsp70 is much higher on metastases compared to primary and relapse tumors, as indicated by a shift of the grey peak to the right.</p