USE OF IMMUNOCYTOKINE COMPRISING INTERFERON-BETA OR VARIANT THEREOF FOR TREATING HUMAN EPIDERMAL GROWTH FACTOR RECEPTOR 2 POSITIVE CANCER

Abstract

The present invention relates to a use of a recombinant protein in which an interferon-beta protein and an antibody binding to a HER2 antigen are fused for the purpose of treating cancer patients of which the HER2 expression level is IHC 1+ or higher. The recombinant protein can exhibit efficacy better than conventional antibody therapeutic agents through cancer-specific anti-cancer immune responses in patients, thereby being used for more patients, and thus is effectively usable as a novel agent for treating cancer.

Claims

1. A pharmaceutical composition for preventing or treating a cancer in which a HER2 expression level is IHC 1+ or higher, comprising a recombinant protein comprising an interferon-beta; and a HER2-targeting antibody or a fragment thereof covalently linked directly or indirectly to the interferon-beta as an active component.

2. The pharmaceutical composition of claim 1, wherein the interferon-beta is a variant of the interferon-beta of SEQ ID NO:1 in which the 27.sup.th amino acid residue is substituted with threonine.

3. The pharmaceutical composition of claim 1, wherein the antibody or the fragment thereof is trastuzumab or pertuzumab.

4. The pharmaceutical composition of claim 1, wherein the recombinant protein is a protein in which the interferon-beta and the antibody or the fragment thereof are connected by a peptide linker.

5. The pharmaceutical composition of claim 1, wherein the cancer in which the HER2 expression level is IHC 1+ or higher is breast cancer or gastric cancer.

6. The pharmaceutical composition of claim 1, wherein the cancer expresses HER2 at the level of IHC 1+, or IHC 2+ in combination with negative FISH.

7. (canceled)

8. A method of treating a cancer in which a HER2 expression level is IHC 1+ or higher, comprising administering an effective amount of a composition to a subject in need thereof, the composition of claim 1.

9. A pharmaceutical composition for preventing or treating a cancer in which a HER2 expression level is IHC 1+ or higher, comprising an interferon-beta; and a HER2-targeting antibody or a fragment thereof as active components.

10. The pharmaceutical composition of claim 9, wherein the interferon-beta is a variant of the interferon-beta of SEQ ID NO:1 in which the 27.sup.th amino acid residue is substituted with threonine.

11. The pharmaceutical composition of claim 9, wherein the interferon-beta; and the HER2-targeting antibody or the fragment thereof are formulated as a single composition or separate compositions.

12. The pharmaceutical composition of claim 9, wherein the interferon-beta; and the HER2-targeting antibody or the fragment thereof are administered simultaneously, separately or sequentially.

13. (canceled)

14. A method of treating a cancer in which a HER2 expression level is IHC 1+ or higher, comprising administering an effective amount of a composition to a subject in need thereof, the composition of claim 9.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0083] FIG. 1 shows results from trastuzumab-IFNβ mutein purification experiments. SDS-PAGE was performed with proteins isolated from the first step of affinity chromatography using protein A beads, and proteins subsequently separated by ion exchange chromatography.

[0084] FIG. 2 is a graph showing direct cellular toxicity of trastuzumab and trastuzumab-mutein. NCI-N87 cells were grown in a 96-well plate, and received treatments of trastuzumab or trastuzumab-IFNβ mutein at different concentrations. After 72 hours, WST assays were performed to evaluate percentages of living cells. Although cytotoxicity was not observed in the trastuzumab-treated group, it was confirmed that the cytotoxic effect appeared only in the trastuzumab-IFNβ-treated group.

[0085] FIG. 3 is a graph showing cytotoxicity results from the indirect immune activation of trastuzumab or trastuzumab-IFNβ mutein. NCI-N87 cells were cultured in a 96-well plate, then treated with trastuzumab or trastuzumab-IFNβ mutein at different concentrations along with PBMCs. 48 hours later, percentages of viable cells were measured by WST assay.

[0086] FIGS. 4a to 4c show flow cytometry results to analyze the HER2-binding ability of trastuzumab and trastuzumab-IFNβ mutein. Three different cell lines including MCF-7 (FIG. 4a), MDA-MB-231 (FIG. 4b), and NCI-N87 (FIG. 4c) were reacted with trastuzumab or trastuzumab-IFNβ mutein and measured for their binding ability to HER2 by flow cytometry.

[0087] FIG. 5 is western blotting results to compare HER2 expression levels in breast cancer cell lines and gastric cancer cell lines. Based on the HER2 expression level of each cell line, they were classified as HER2 low expression, HER2 intermediate expression, and HER2 high expression.

[0088] FIGS. 6a and 6b show direct cytotoxicity results of trastuzumab and trastuzumab-IFNβ mutein in gastric cancer cell lines. Gastric cancer cell lines with different HER2 expression levels were cultured in 96-well plates, and subjected to trastuzumab or trastuzumab-IFNβ mutein treatment at different concentrations. 72 hours later, percentages of viable cells were confirmed by WST assay.

[0089] FIGS. 7a and 7b show direct cytotoxicity results of trastuzumab and trastuzumab-IFNβ mutein in breast cancer cell lines. Breast cancer cell lines with different HER2 expression levels were cultured in 96-well plates, and then treated with different concentrations of trastuzumab or trastuzumab-IFNβ mutein. Following 72 hours, percentages of viable cells were confirmed by WST assay.

[0090] FIGS. 8a and 8b are cytotoxicity results of indirect immune activation by trastuzumab and trastuzumab-IFNβ mutein in gastric cancer cell lines. Cell lines with different HER2 expression levels were cultured in 96-well plates, and were treated with different concentrations of trastuzumab or trastuzumab-IFNβ mutein together with PBMCs. 48 hours later, percentages of living cells were confirmed by WST assay.

DETAILED EXPLANATION OF THE INVENTION

[0091] Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings. However, these descriptions are provided for illustrative purposes only to help the understanding of the present invention, and the scope of the present invention is not limited by these illustrative descriptions.

[0092] 1. Materials and Methods

[0093] 1-1. Design to Prepare Fusion Proteins.

[0094] In order to establish stable cells and cell lines transiently expressing the antibody fused to the mutant protein, pCHO 1.0 (Life Technologies) was used as an expression vector. Trastuzumab was used as an antibody, and the variant of interferon-beta in which the 27th amino acid residue was substituted with threonine was used as a mutant protein (mutein).

[0095] The interferon-beta variant was fused to the heavy chain region of the antibody. A linker was cloned into the heavy chain region of the antibody, and the interferon-beta variant was cloned therein, respectively. Thereafter, a restriction enzyme AvrII cleavage site (CCTAGG) and a Bstz17I cleavage site (GTATAC) (enzymes from Thermo Scientific, USA) were inserted into the 3′ and 5′-ends of the entire gene to secure the final gene of the heavy chain. In addition, restriction enzyme EcoRV site (GATATC) and Pad site (TTAATTAA) were inserted into the 3′ and 5′-ends of the light chain of the antibody to secure the final gene of the light chain. The final genes of the heavy and light chains were inserted into the pCHO 1.0 vector.

[0096] 1-2. Expression of Fusion Protein Constructs in Mammalian Cells

[0097] Expression vectors of trastuzumab and the trastuzumab-fused interferon-beta variant (Trastuzumab-IFNβ mutein) were transformed into CHO—S cells (Thermo Scientific) using FreeStyle™ MAX reagent (Thermo Scientific). A mixture obtained by adding OptiPRO™ SFM (Thermo Scientific) to the FreeStyle™ MAX reagent-DNA complex was put into the CHO—S cells in a flask, and incubated in a humidified 8% CO.sub.2 atmosphere at atmospheric pressure. Stable transformants to express the fusion protein were selected 48 hours after transformation. Cells were sorted through secondary selection with puromycin 10-50 μg/mL and MTX 100-1,000 nM. Selected cells were incubated in the presence of glucose to express the fusion protein for 14 days at 37° C. under humidified 8% CO.sub.2 atmospheric pressure and 130 rpm.

[0098] 1-3. Purification of Fusion Proteins

[0099] Fusion proteins expressed in CHO—S cells were isolated by affinity chromatography and ion exchange chromatography. After passing the CHO—S culture medium through a column filled with protein A Mabselect sure (GE Healthcare), equilibration buffer, wash buffer, and elution buffer were sequentially eluted to obtain purified proteins. In case of the ion exchange chromatography purification method, fusion proteins obtained by affinity chromatography purification were passed through a column filled with HiTrap Q (HiTrap Q FF, GE healthcare), then elution buffer was added to obtain purified proteins.

[0100] 1-4. Cell Lines and Culture Conditions

[0101] The human gastric carcinoma (NCI-N87) cell line and the human breast cancer (MCF-7, MDA-MB-231) cell line were purchased from the Korean Cell Line Bank (KCLB).

[0102] NCI-N87 cells were cultured in RPMI-1640 (HyClone, USA) culture medium containing 10% FBS (HyClone), penicillin 100 units/mL and streptomycin 100 μg/mL, while MCF-7 and MDA-MB231 cells were cultured using DMEM (HyClone, USA) culture medium containing 10% FBS (HyClone), penicillin 100 units/mL, and streptomycin 100 μg/mL at 37° C. under atmospheric pressure of 5% CO.sub.2 with humidity.

[0103] 1-5. Examination of Direct Cytotoxicity of Interferon-Beta

[0104] In a 96-well plate, 2.0×10.sup.4 (200 μl) NCI-N87 cells were incubated in each well and divided into a control group and treatment groups of IFNβ, trastuzumab, or trastuzumab-IFNβ mutein. On the following day, IFNβ (1, 5, 10 ng/ml), trastuzumab (15, 30, 60, 120 ng/ml), or trastuzumab-IFNβ mutein (20, 40, 80, 160 ng/ml) were administered at the corresponding concentrations, respectively, and incubated for 72 hours. Then, 10 μl of EZ-Cytox (Dae-il Biotech) was added to each well, and reacted for 3 hours in an incubator. Absorbance at 450 nm was measured and compared using a SpectraMax iD3 multi-mode microplate reader (Molecular Device).

[0105] 1-6. Examination of Indirect Cytotoxicity of Interferon-Beta

[0106] In a 96-well plate, 2.0×10.sup.4 (200 μl) NCI-N87 cells were incubated in each well and divided into a control group and treatment groups of IFNβ, trastuzumab, or trastuzumab-IFNβ mutein. In the following day, peripheral blood mononuclear cells (PBMC) (Zenbio) were added at a ratio of 20:1 to cancer cells, and IFNβ (100 ng/ml), trastuzumab (100, 350 ng/ml) or trastuzumab-IFNβ mutein (100, 350 ng/ml) was treated to the corresponding concentration, respectively, and cultured for 48 hours. Then, 10 μl of EZ-Cytox (Daeil Biotech) was added to each well, and the reaction was carried out in an incubator for 3 hours. Absorbance at 450 nm was measured and compared using a SpectraMax iD3 multi-mode microplate reader (Molecular Device).

[0107] 1-7. Flow Cytometry

[0108] To measure the antibody's binding ability to HER2, flow cytometry analysis was performed. NCI-N87, MDA-MB-231, and MCF-7 cells were recovered using cell dissociation buffer (Enzyme-Free, PBS-based) (Gibco) and their cellular activities were inhibited in cold PBS (containing 2% FBS) for 1 hour at 4° C. Cells were then washed with PBS three times and incubated with 1 μg of trastuzumab and trastuzumab-IFNβ mutein diluted in PBS for 30 minutes at 4° C. Subsequently cells were washed three times with PBS and then incubated with goat anti-Human IgG FITC (Jackson) at 4° C. for 30 minutes. Fluorescent antibodies were measured by flow cytometry (CytoFLEX Flow Cytometer) (Beckman Coulter).

[0109] 1-8. Analysis of Endogenous HER2 Expression in Cancer Cells

[0110] To measure HER2 expression levels in breast cancer cell lines (HCC1954, BT-474, MDA-MB-231, BT-549) and gastric cancer cell lines (NCI-N87, KATOIII, Hs746T, MKN74, HFE145, SNU1, SNU620), western blot experiments were performed.

[0111] Each cell lines were cultured for 7 days and the culture medium was collected and centrifuged to remove cells (8000 rpm, 10 minutes). A small amount of the cell-removed culture medium was collected, mixed with 5× sample buffer, and boiled for 10 minutes at 100° C. to induce sufficient protein denaturation. Subsequently prepared protein samples were loaded on a tricine SDS-PAGE gel with a marker, and electrophoresis was performed at a voltage of 130v for 1 hour and 30 minutes. Then, the gel was separated and placed on a 3M paper with a PVDF membrane placed on top of the gel, and another 3M papers were layered, immersed in 1× transfer buffer, and proteins were transferred at the voltage of 100v for 70 minutes. Tris-buffered saline-Tween 20 (TBS-T, 0.1% Tween 20) was added with 5% and the membrane was blocked at room temperature for 1 hour and 30 minutes. The protein-transferred PVDF membrane was washed twice with TBS-T, and left immersed in TBS-T. Anti-HER2 antibody was prepared by diluting in TBS-T at the ratio of 1:1000. The membrane was then immersed in the antibody-diluted solution, reacted at room temperature for 2 hours. After completing this process, the membrane was washed with TBS-T 3 times for 10 minutes. Secondary antibodies conjugated with horseradish peroxidase (HRP) were added and reacted for 1 hour at room temperature. After washing one more time, protein bands were confirmed with ECL reagent (enhanced chemiluminescence reagent, Intron). The band intensity was measured using C-DiGit (LI-COR, USA).

[0112] 1-9. Examination of Direct Cytotoxicity in the Gastric Cancer Cell Lines

[0113] Experiments were performed in the same manner as in the above 1-5.

[0114] In a 96-well plate, each of NCI-N87, SNU1, SNU620, Hs746T, and KATOIII cells were cultured at the density of 2.0×10.sup.4 cells (200 μl) per well, and within each cell lines, treatment groups were assigned such as IFNβ-R27T treatment group, trastuzumab treatment group, trastuzumab-IFNβ mutein (trastuzumab-R27T) treatment group and T-DM1 treatment group. On the following day, IFNβ (1, 5, 10 ng/ml), trastuzumab (15, 30, 60, 120 ng/ml), or trastuzumab-IFNβ mutein (20, 40, 80, 160 ng/ml) were administered at the corresponding concentrations, respectively, and incubated for 72 hours. Then, 10 μl of EZ-Cytox (Daeil Biotech) was added to each well, and the reaction was carried out in an incubator for 3 hours. Absorbance at 450 nm was measured and compared using a SpectraMax iD3 multi-mode microplate reader (Molecular Device).

[0115] 1-10. Examination of Direct Cytotoxicity in the Breast Cancer Cell Lines

[0116] Experiments were performed in the same manner as in the above 1-5.

[0117] In a 96-well plate, each of BT-474 cells, SKBR3 cells, HCC1954 cells, MDA-MB-453 cells, MDA-MB-231 cells, and BT549 cells were cultured at the density of 2.0×10.sup.4 (200 μl) per well, and for each cell lines, treatment groups such as IFNβ treatment, trastuzumab treatment, and trastuzumab-IFNβ mutein (trastuzumab-R27T) treatment were assigned. On the following day, IFNβ (1, 5, 10 ng/ml), trastuzumab (15, 30, 60, 120 ng/ml), or trastuzumab-IFNβ mutein (20, 40, 80, 160 ng/ml) were administered at the corresponding concentrations, respectively and incubated for 72 hours. Then, 10 μl of EZ-Cytox (Daeil Biotech) was added to each well, and the reaction was carried out in an incubator for 3 hours. Absorbance at 450 nm was measured and compared using a SpectraMax iD3 multi-mode microplate reader (Molecular Device).

[0118] 1-11. Examination of Indirect Cytotoxicity in the Gastric Cancer Cell Lines

[0119] In a 96-well plate, each of N87 cells, KATOIII cells, Hs746T cells or MKN74 cells were divided into treatment groups of IFNβ, trastuzumab, or trastuzumab-IFNβ mutein (trastuzumab-R27T) and cultured at the concentration of 1.0×10.sup.4 cells (200 μl) per well. The next day, peripheral blood mononuclear cells (PBMC) (Zenbio) were added at a ratio of 1:1 or 1:2 to cancer cells, and IFNβ (100 ng/ml), trastuzumab (100, 350 ng/ml), or trastuzumab-IFNβ mutein (100, 350 ng/ml) were treated at the corresponding concentrations, respectively, and cultured for 3 days. Then, 10 μl of EZ-Cytox (Daeil Biotech) was added to each well, and the reaction was carried out in an incubator for 3 hours. Absorbance at 450 nm was measured and compared using a SpectraMax iD3 multi-mode microplate reader (Molecular Device).

[0120] 2. Results

[0121] 2-1. Expression and Purification of Trastuzumab-IFNβ Mutein Fusion Protein

[0122] Protein expression and purification experiments were performed under the same conditions using cell lines expressing trastuzumab-IFNβ mutein and trastuzumab. For expression conditions, CHO—S cell line was cultured at 37° C. and 5% CO.sub.2 for 10 days. Concentrations of the expressed fusion protein present in the cell culture media were measured using Cedex-bio (Roche), and purified by affinity chromatography employing AKTA instrument system and protein A beads. Thereafter, secondary purification was performed using ion exchange chromatography.

[0123] As shown in FIG. 1, it was confirmed that both the heavy chain and light chain proteins of trastuzumab-IFNβ mutein, which were produced through the two-step purification process, were observed at the position corresponding to the expected sizes.

[0124] 2-2. Anti-Cancer Efficacy of Trastuzumab-IFNβ Mutein Fusion Protein

[0125] WST assay was used to analyze a direct cytotoxic effect of the fusion protein and indirect anti-cancer efficacy through immune activation.

[0126] Referring to FIG. 2, trastuzumab-IFNβ mutein exhibited direct cytotoxicity against NCI-N87 cells, however, trastuzumab was not associated with cellular toxicity. This suggests that blocking of the signal transduction by trastuzumab does not cause significant damages or toxicity in the cells.

[0127] Referring to FIG. 3, trastuzumab-IFNβ mutein showed cytotoxicity in NCI-N87 cells through immune cell activation, and showed even greater cytotoxicity against cancer cells compared to trastuzumab.

[0128] 2-3. Targeting HER2 by Trastuzumab-IFNβ Mutein

[0129] To examine the binding ability to HER2, flow cytometry analysis was performed.

[0130] Referring to FIG. 4, it was confirmed that trastuzumab and trastuzumab-IFNβ mutein had the same HER2 binding ability in the cell line with a high HER2 expression level (NCI-N87). Similarly, it was observed that trastuzumab and trastuzumab-IFNβ mutein had the comparable HER2 binding ability even in cell lines with low HER2 expression levels (MCF-7, MDA-MB-231). These results showed that there was no change in the HER2-binding ability of trastuzumab even when it is fused to IFNβ mutein.

[0131] 2-4. Analysis of Endogenous HER2 Expression Levels in Cancer Cell Lines

[0132] Western blot experiments were carried out to compare HER2 expression levels in breast cancer cell lines and gastric cancer cell lines. The HER2 expression levels of breast cancer cell lines HCC1954, BT-474, MDA-MB-231, BT-549 and gastric cancer cell lines NCI-N87, KATOIII, Hs746T, MKN74, HFE145, SNU1, and SNU620 were compared and classified into three groups.

[0133] As shown in FIG. 5, HER2 low expression group include MDA-MB231 cells and BT-549 cells among breast cancer cell lines, and HS746T cells and KATOIII cells among gastric cancer cell lines. HER2 intermediate expression group include MDA-MB-453 breast cancer cells, and SNU1 and SNU620 gastric cancer cells. BT-474 and HCC1954 cells among breast cancer cell lines, and NCI-N87 cells among gastric cancer cells fall into HER2 high expression group.

[0134] 2-5. Direct Cytotoxic Efficacy Depending on HER2 Expression Levels in Breast Cancer Cell Lines and Gastric Cancer Cell Lines

[0135] Direct cytotoxic efficacy of the fusion protein was measured by WST assay method in NCI-N87 cells showing high levels of HER2 expression, SNU1 and SNU620 cells with medium levels of HER2 expression, and Hs746T and KATOIII cells with low levels of HER2 expression.

[0136] As shown in FIGS. 6a and 6b, trastuzumab-IFNβ mutein showed direct cytotoxic efficacy against cells with medium HER2 expression levels and gastric cancer cells with low expression levels, while trastuzumab did not show any cytotoxic effect.

[0137] In addition, as shown in FIGS. 7a and 7b, trastuzumab-IFNβ mutein showed direct cytotoxic efficacy against cells with medium HER2 expression levels and breast cancer cells with low expression levels.

[0138] 2-6. Indirect Cytotoxic Effect Depending on HER2 Expression Level in Gastric Cancer Cell Line

[0139] NCI-N87 cells with high HER2 expression or Hs746T cells with low HER2 expression were co-cultured with PBMCs to compare PBMC-mediated cytotoxic effects. Indirect anti-cancer efficacy through immune activation was measured.

[0140] As shown in FIGS. 8a and 8b, trastuzumab-IFNβ mutein exhibited cytotoxic efficacy through immune cell activation against NCI-N87 cells as well as Hs746T cells with a low HER2 expression level, proving that it has even better cancer cell cytotoxicity compared with trastuzumab.

[0141] Accordingly, it can be understood that trastuzumab-IFNβ mutein exerts a therapeutic effect on HER2-positive cancer even when the HER2 expression level is relatively low in contrast to trastuzumab which is effective only when the HER2 expression level is very high.