A novel fully human anti-NCL immunoRNase for triple-negative breast cancer therapy

Abstract

Breast cancer is the most common cancer in women worldwide. A new promising anti-cancer therapy involves the use of monoclonal antibodies specific for target tumor-associated antigens (TAAs). A TAA of interest for immunotherapy of Triple Negative Breast Cancer (TNBC) is nucleolin (NCL), a multifunctional protein, selectively expressed on the surface of cancer cells, which regulates the biogenesis of specific microRNAs (miRNAs) involved in tumor development and drug-resistance. We previously isolated a novel human anti-NCL scFv, called 4LB5, that is endowed with selective anti-tumor effects. Here we report the construction and characterization of a novel immunoRNase constituted by 4LB5 and a human pancreatic RNase (HP-RNase) called "4LB5-HP-RNase". This immunoRNase retains both the enzymatic activity of human pancreatic RNase and the specific binding of the parental scFv to a panel of surface NCL-positive breast cancer cells. Notably, 4LB5-HP-RNase dramatically and selectively reduced the viability and proliferation of NCL-positive tumor cells in vitro and in vivo. Specifically, it induced apoptosis and reduced the levels of the tumorigenic miRNAs miR-21, -221 and -222. Thus, this novel immunoagent could be a valuable tool for the treatment of TNBC patients ineligible for currently available targeted treatments.

Keywords: cancer immunotherapy; human RNase; microRNA; nucleolin; triple negative breast cancer.

Figure 1. Schematic representation and biochemical analyses of purified 4LB5-HP-RNase

(A) Schematic representation of the human anti-Nucleolin immunoRNase 4LB5-HP-RNase. VH and VL, the Variable Heavy and Light chains of the anti-Nucleolin scFv; linker, the flexible oligopeptide conjugating VH and VL; spacer, the peptide connecting the scFv and RNase; HP-RNase, human pancreatic RNase. (B) SDS-PAGE analysis followed by Coomassie staining of the sample eluted by IMAC. Lane 1, fraction eluted from the column. (C) Western blotting analysis using an anti-His antibody of the sample as in Lane 1. (D) Zymogram of 4LB5-HP-RNase (Lane 4) using yeast RNA as a substrate; Lane 3, enzymatic activity of ERB-HP-RNase used as positive control of the native human pancreatic ribonuclease activity.

Figure 2. Binding of 4LB5-HP-RNase to NCL on cancer cells

(A) The binding of 4LB5-HP-RNase to NCL-positive MDA-MB-231, MCF-7, BT-549 or MDA-MB-436 breast cancer cells or to NCL-negative MCF-10a normal-like breast cells was tested by incubating the chimeric protein (100 nM) with the cells; 4LB5 was tested at equimolar doses as a positive control in parallel assays. (B) Binding curves of 4LB5-HP-RNase to surface NCL-positive MDA-MB-231 (rhomboids), MCF-7 (squares), BT-549 (triangles), MDA-MB-436 (circles) breast cancer cell lines performed by using increasing concentrations (5–500 nM) of the immunoRNase. All the experiments are representative of three independent experiments performed in triplicate. Mean + SD is reported. (C) ELISA assay performed by testing 4LB5-HP-RNase (50 nM) on MDA-MB-231 breast cancer cells in the absence (black) or in the presence (white) of equimolar doses of NCL-RBD recombinant protein. All the experiments are representative of three independent experiments performed in triplicate. Mean + SD is reported. (D) Pull-down assay performed on lysates of MDA-MB-231 cells using Ni-NTA resin and 4LB5 or 4LB5-HP-RNase. Ni-NTA resin incubated with total cell lysate in the absence of 4LB5 or 4LB5-HP-RNase was used as negative control. Anti-NCL antibody (upper panel) and anti-His antibody (Lower panel) were used to visualize the effective pull-down of endogenous NCL in the presence of either 4LB5 or 4LB5-HP-RNase.

Figure 3. 4LB5-HP-RNase is internalized by TNBC cells

(A, C) MDA-MB-231 cells were cultured in the presence of Cy5 (A), 4LB5-Cy5 (B) or 4LB5-HP-RNase-Cy5 (C) for 4 h at 37°C. Labeled compounds that were not cell-associated were washed away. Cells were incubated with Hoechst nuclear staining, and microscopy images were acquired. For each experimental condition, Hoechst, Cy5 image stacks are presented as a 2 dimensional projection along with a single representative DIC image (TD). A merged image is also presented for each treatment. (D) 3D-rendering of the image series through the volume of 4LB5-HP-RNase (from 3C) treated cells to help visualization of the intracellular localization of 4LB5-HP-RNase. Scale bar = 10 μm.

Figure 4. In vitro effects of the immunoRNase on cell proliferation

(A) Dose-response curves of NCL-positive MDA-MB-231 (rhomboids), BT-549 (triangles), MDA-MB-436 (circles) and MCF-7 (black squares) breast cancer cells or NCL-negative MCF10a (empty squares) normal-like breast cells treated for 72 h with increasing doses (10–100 nM) of 4LB5-HP-RNase. Colony assays on BT-549 (B) MDA-MB-436 (C) and MDA-MB-231 (D) cells performed in the absence or in the presence of 100 nM of 4LB5-HP-RNase or 4LB5, tested in parallel assays, and stained after 10 days with Crystal violet. All the experiments are representative of three independent experiments performed in triplicate.

Figure 5. Effects of 4LB5-HP-RNase on cancer cell apoptosis

(A) Dose-response curve of NCL-positive MDA-MB-231 cells (rhomboids) treated for 72 h with increasing doses (10–200 nM) of 4LB5-HP-RNase. (B) Western Blotting analysis of total lysates from MDA-MB-231 cells treated with increasing concentrations (10–200 nM) of 4LB5-HP-RNase to evaluate PARP cleavage as a marker of apoptosis activation. Actin was used as loading control.

Figure 6. Effects of 4LB5-HP-RNase on cellular and EV-associated miRNAs

(A) NCL-dependent mature miR-21, -221 and -222 levels were analyzed by Real-Time PCR after 72 h of incubation of MDA-MB-231 cells with increasing concentrations (6–100 nM) of 4LB5-HP-RNase. (B) Comparison between the effects of 4LB5 (grey bars) and 4LB5-HP-RNase (white bars) on mature miR-21, -221 and -222 levels by using an equimolar concentration (25 nM) of compounds for the treatment of MDA-MB-231 cells. Relative microRNA expression levels were normalized for control untreated sample, and relative abundance (fold change) is reported. Data are the average of three independent experiments performed in triplicate. Mean + SD is reported. (C) The mature miR-21 levels were analyzed by Real-Time PCR on RNA extracted from exosomes derived from the conditioned medium of SW620 cancer cells treated for 72 h with equimolar doses (50 nM) of 4LB5-HP-RNase (white bars) or 4LB5 (grey bars), tested in a parallel assay. Relative microRNA expression levels were normalized for control untreated sample, and relative abundance (fold change) is reported. (D) The snoRNA RNU48 was used as negative control of RNAs cellular contamination. (E) Western Blotting analysis showing NCL in both total lysates and MVs from SW620 cells, used for the experiments reported in C and D.

Figure 7. In vivo effects of 4LB5-HP-RNase on NCL-positive tumors induced in mice by using MDA-MB-231 breast cancer cells

(A) NOD-SCID (n = 5) mice were injected with 2 × 10⁶ MDA-MB-231-Luc cells into the mammary fat pad. After 2 weeks, mice were treated with control PBS solution or 2 mg/kg of 4LB5-HP-RNase twice a week. Mice were monitored by IVIS weekly. At 6 week from injection (4 weeks of treatment), mice were sacrificed. Tumors were excised and measured. **P < 0.01 (B) In a second experiment, mice were treated with equimolar doses of 4LB5 or 4LB5-HP-RNase (2 mg/kg) or with PBS, as described for Figure 7A. *P < 0.05 (C) Representative images of H&E and Ki-67 staining of tumor tissue slices obtained from different fields are shown; 20× magnification is reported. (D) Body weight of mice treated with PBS (black bars), 4LB5 (grey bars) or 4LB5-HP-RNase (white bars). Measurements were performed at tumor excision.