IMMUNOTOXINS FOR USE IN TREATING CANCER

Abstract

The present invention relates generally to immunotoxins. More particularly, the invention relates to immunotoxins (e.g. immunotoxins that comprise an antibody that binds to EpCAM or binds to MUC-1) for use in the treatment of cancer. The invention also relates to a method for treating cancer, said method comprising administering to a subject in need thereof a therapeutically effective amount of an immunotoxin. The invention also relates to the use an immunotoxin in the manufacture of a medicament for treating cancer.

Claims

1-29. (canceled)

30. A method of treating a carcinoma in humans, said method comprising systemically administering to a human in need thereof a therapeutically effective amount of an immunotoxin that induces immunogenic cell death of cancer cells, wherein the immunotoxin comprises a murine antibody conjugated to a full-length Pseudomonas exotoxin A (PE), wherein said murine antibody binds to EpCAM or to MUC-1 and comprises an Fc domain, and wherein said immunotoxin (i) is used in combination with an immunostimulant; or (ii) is the sole active agent used in the treatment regimen.

31. The method of claim 30, wherein said carcinoma is colon carcinoma.

32. The method of claim 30, wherein said carcinoma is a metastatic and/or advanced carcinoma.

33. The method of claim 30, wherein said carcinoma is an EpCAM-positive carcinoma.

34. The method of claim 30, wherein said carcinoma is a MUC-1-positive carcinoma.

35. The method of claim 30, wherein said murine antibody is a full-length antibody, preferably an IgG antibody, more preferably an IgG.sub.1 antibody.

36. The method of claim 30, wherein said full-length Pseudomonas exotoxin A (PE) has the amino acid sequence set forth in SEQ ID NO: 3.

37. The method of claim 30, wherein said murine antibody that binds to EpCAM is the antibody MOC31.

38. The method of claim 30, wherein said immunotoxin is MOC31PE.

39. The method of claim 30, wherein said murine antibody that binds to MUC-1 is the antibody BM7.

40. The method of claim 30, wherein said immunotoxin is BM7PE.

41. The method of claim 30, wherein said immunostimulant is a checkpoint inhibitor.

42. The method of claim 41, wherein said checkpoint inhibitor is a PD-1 antibody, a PD-L1 antibody or a CTLA-4 antibody.

43. The method of claim 41, wherein said checkpoint inhibitor is selected from the group consisting of Nivolumab, Pembrolizumab, Ipilimumab, Atezolizumab, Durvalumab, Avelumab and Cemiplimab.

44. The method of claim 30, wherein said immunotoxin is administered intravenously.

45. A method for stimulating an immune response in a human subject, said method comprising administering an immunotoxin to said subject, wherein the immunotoxin comprises a murine antibody conjugated to a full-length Pseudomonas exotoxin A (PE), wherein said murine antibody binds to EpCAM or to MUC-1 and comprises an Fc domain, wherein said immunotoxin is systemically administered, and wherein said immunotoxin (i) is used in combination with an immunostimulant; or (ii) is the sole active agent used in the treatment regimen.

46. A method for inducing immunogenic cell death in a human subject, said method comprising administering an immunotoxin to said subject, wherein the immunotoxin comprises a murine antibody conjugated to a full-length Pseudomonas exotoxin A (PE), wherein said murine antibody binds to EpCAM or to MUC-1 and comprises an Fc domain, wherein said immunotoxin is systemically administered, and wherein said immunotoxin (i) is used in combination with an immunostimulant; or (ii) is the sole active agent used in the treatment regimen.

Description

[0341] The invention will be further described with reference to the following non-limiting Examples with reference to the following drawings in which:

[0342] FIG. 1: Overall survival: Kaplan-Meier plot.

[0343] (a) Overall survival of patients with EpCAM-positive metastatic cancers treated with MOC31PE (n=34) or with MOC31PE plus Cyclosporin A (CsA, n=23), p<0.066 (b) Overall survival of patients with EpCAM-positive metastatic colorectal cancer treated with MOC31PE (n=15) or with MOC31PE plus CsA (n=18). The MOC31PE treated group and MOC31 PE plus CsA treated group are indicated by arrows, p<0.001. The significance of differences in survival between MOC31 PE and MOC31 PE plus CsA patients was determined by the log-rank test. Cum survival=cumulative survival.

[0344] FIG. 2: Box plot showing the cytokine level in patient serum

[0345] The levels (pg/ml) of indicated cytokines in serum of metastatic colorectal cancer patients; pre-treatment (pre, n=20), two weeks after MOC31 PE (post MOC31PE, n=9) and MOC31PE plus CsA (post MOC31PE+CsA, n=12) treatment measured by multiplex cytokine ELISA assay. The box plots show median values (horizontal lines), interquartile ranges (the box lengths), extreme values (*) and outliers (o)

[0346] FIG. 3: MOC31PE induced cytotoxicity and HMGB1 release in colorectal cancer cell lines.

[0347] (a) HCT116, SW480 and HT29 cells were incubated with MOC31 PE (1-1000 ng/ml) for 24 h. Cell viability is expressed as a percentage (mean) of the value obtained in vehicle treated cells. Results are representative of 2 independent experiments, each plated in at least triplicate. (b) Western blot analysis of high mobility group box 1 protein (HMGB1) in supernatant from HCT116 cells, untreated (vehicle control), treated with MOC31 PE immunotoxin (10 and 100 ng/ml) and MOC31 monoclonal antibody (100 ng/ml) for 24 h. Equal volumes of supernatants were run on SDS-PAGE gel and stained with anti-HMGB1 antibody. Results are representative of 2 independent experiments. FIG. 3 (b) also shows western blot analysis of high mobility group box 1 protein (HMGB1) in supernatant from SW480 cells, untreated (vehicle control) and treated with MOC31PE immunotoxin (100 and 1000 ng/ml). Recombinant HMGB1 (with a tag) was included as a positive control in the western blot.

[0348] FIG. 4: Ex vivo maturation of immature dendritic cells (DCs) induced by MOC31PE.

[0349] Immature DCs treated with conditioned medium from MOC31PE treated colorectal cancer cells (HCT116 or SW480) show a significant decrease in CD14 expression compared to immature DCs+conditioned medium from non-treated cells (HCT116 or SW480). Mean fluorescence intensity (MFI) decrease in CD14 expression analyzed by flow cytometry. Concentrations of MOC31PE were as indicated, 100 or 1000 ng/ml. The result is representative of triplicates. **** p<0.0001, *** p<0.0010, ** p<0.0025.

[0350] FIG. 5: FIG. 1: BM7PE induced cytotoxicity in breast cancer cell line.

[0351] (A) Inhibition of protein synthesis in Muc-1 expressing T47D breast cancer cells. BM7PE inhibits protein synthesis effectively in a dose-dependent way. (B) Cell viability in Muc-1 expressing T47D breast cancer cells. The cytotoxicity was measured after 24 hr treatment.

[0352] FIG. 6: BM7PE induced cytotoxicity in colorectal cancer cell line.

[0353] Cell viability in Muc-1 expressing HCT116, SW480 and HT29 colorectal cancer cells. Cells were incubated with BM7PE (100 ng/ml) for 48 h. Cell viability is expressed as a percentage (mean) of the value obtained in vehicle treated cells.

[0354] FIG. 7: Ex vivo maturation of immature dendritic cells (DCs) induced by BM7PE.

[0355] Immature DCs treated with conditioned medium from BM7PE treated colorectal cancer cells (HCT116 or SW480) show a significant decrease in CD14 expression compared to immature DCs+conditioned medium from non-treated cells (HCT116 or SW480). Mean fluorescence intensity (MFI) decrease in CD14 expression analyzed by flow cytometry. Concentrations of BM7PE were as indicated, 100 or 1000 ng/ml. The result is representative of triplicates. **** p<0.0001, ** p<0.0025.

[0356] FIG. 8: Ex vivo maturation of immature dendritic cells (DCs) induced by MOC31PE.

[0357] Immature DCs treated with conditioned medium from MOC31PE treated colorectal cancer cells (HCT116) show a decrease in CD14 expression compared to immature DCs+conditioned medium from non-treated cells or cells treated with oxaliplatin (A) and show an increase in CD86 expression compared to immature DCs+conditioned medium from non-treated cells or cells treated with oxaliplatin (B). Mean fluorescence intensity (MFI) of CD14 expression and CD86 expression was analyzed by flow cytometry. Concentrations of MOC31PE were as indicated, 0.05 or 0.1 μg/ml. The data is from three biological triplicates, each in three technical triplicates.

[0358] FIG. 9: Ex vivo maturation of immature dendritic cells (DCs) induced by BM7PE.

[0359] Immature DCs treated with conditioned medium from BM7PE treated colorectal cancer cells (HCT116) show a decrease in CD14 expression compared to immature DCs+conditioned medium from non-treated cells or cells treated with oxaliplatin (A) and show an increase in CD86 expression compared to immature DCs+conditioned medium from non-treated cells or cells treated with oxaliplatin (B). Mean fluorescence intensity (MFI) of CD14 expression and CD86 expression was analyzed by flow cytometry. Concentrations of BM7PE were as indicated, 0.05 or 0.1 μg/ml. The data is from three biological triplicates, each in three technical triplicates.

[0360] FIG. 10: MOC31PE-treated cancer cells release ATP ATP assay demonstrating that MOC31PE-treated HCT116 cells release ATP. For comparison, data is also shown for oxaliplatin, MOC31 antibody (MOC31-ab) and the PE toxin (PE).

[0361] FIG. 11: MOC31PE promotes activation of killer T cells (CD8+) ex vivo

[0362] Bone marrow-derived monocytes were thawed and cultured with the cytokines IL-4+GM-CSF for 2 days to generate immature DCs. HCT116 cells were treated with immunotoxin (MOC31PE) for 24 hrs and then the isolated conditioning medium (i.e. the supernatant with the HCT116 cells removed) was co-incubated with immature DCs for 24 hrs. DCs were stained for CD14-FITC, CD86-PE and analysed by flow cytometry. A portion of the treated DCs were transferred to a T-cell culture. The T cells become activated by the mature DCs, and the activation was measured by expression of the degranulation marker CD107a (−Cy5) and IFNγ (−FITC), and TNFα (−PE) in a flow cytometry analysis.

EXAMPLE 1

[0363] Abstract:

[0364] BACKGROUND: We recently performed a first-in-man phase I clinical trial of the anti-EpCAM immunotoxin MOC31 PE in patients with metastatic cancer. Patients were treated intravenously with MOC31 PE alone and in combination with the immunosuppressor Cyclosporin A (CsA), once every 2 weeks up to 4 times. MOC31PE was well tolerated, and only a rapidly reversible dose-limiting liver toxicity was noticed. No complete or partial response was observed, as assessed by CT 8 weeks after start of therapy. In the present study, however, we reveal an unexpected long survival time of MOC31 PE monotherapy treated patients.

[0365] METHODS: We performed a retrospective analysis of overall survival (OS) in the two study arms, MOC31 PE administered alone (34 patients) and in combination with CsA (23 patients). Hazard ratio (HR) was estimated from Cox models, survival curves by the Kaplan-Meier method, patient sera were analyzed by multiplex biometric ELISA-based immunoassay. The putative immunogenic effect of MOC31PE was analyzed in vitro by release of HMGB1, and ex vivo in a dendritic cell maturation assay.

[0366] FINDINGS: The median OS time for all patients treated with MOC31 PE alone was 12.7 months (95% CI=5.6-19.8 months) as compared to 6.2 months (95% CI=5.6-6.8 months) (p=0.066) for the MOC31 PE+CsA group. This finding was unexpected, as preclinical studies had indicated that CsA would improve efficacy and block generation of neutralizing anti-MOC31 PE antibodies. To avoid possible bias related to the heterogeneity of cancers in our study population, we analyzed specifically the results for patients with the most frequent tumor type; colorectal cancer (CRC). The Kaplan-Meier survival plot shows that CRC-patients treated with MOC31PE alone (n=15) had a median OS of 16.3 months (95% CI=5.6-27.0 months) compared to 6.0 months (CI=5.8-6.2 months) for CRC-patients in the combination group (n=18) (HR=0.248, 95% credible interval: 0.109-0.564). The cytokine Th1 profile obtained in patient sera indicates that MOC31PE induces a novel and previously unknown immunogenic cell death mechanism. This was further supported by data demonstrating that MOC31PE-induced cell death of colorectal cancer cells released immune stimulating factors that caused maturation of patient-derived immature dendritic cells ex vivo.

[0367] INTERPRETATION: The present results reveal an unpredicted clinical benefit of anti-EpCAM immunotoxin treatment, particularly in a group of patients with advanced cancer with very limited treatment alternatives.

Introduction

[0368] One of the hallmarks of cancer is to escape anti-tumor immune response. Lately, various approaches to overcome immune resistance have been applied clinically in several tumor types. However, in spite of some cases with unforeseen durable responses only about 25% of all patients respond to immuno-oncology drugs. To improve response rates and overcome resistance new drugs and combinations are being evaluated in the clinic, and compounds with different mechanisms of immune-stimulation are investigated.

[0369] Recently, we reported the results of a phase I clinical trial with the EpCAM-targeting immunotoxin MOC31 PE, with and without parallel administration of the immunosuppressor Cyclosporin A (CsA) (1). The safety and maximum tolerated doses in patients with EpCAM-positive tumors of various types were determined, and only moderate and rapidly reversible dose-limiting liver toxicity was observed. Notably, the patients did not experience subjective side effects that could be ascribed to the treatment.

[0370] No complete or partial responses were obtained as determined by CT taken eight weeks after start of treatment. Nevertheless, in the current study we performed a detailed investigation of the data from the phase I trial and looked at the survival time of subgroups of patients. We found that the median overall survival time (OS) for all patients receiving the combination of MOC31PE and CsA was 6.2 months, as expected for this group of patients. By contrast, for the patients treated with MOC31 PE alone the median OS was more than 12 months. This difference was highly unexpected as in preclinical studies the combination was superior and even synergistic compared to the use of MOC31 PE alone (2). Based on this data, we hypothesized that MOC31PE might have induced an immunogenic response that was obliterated by the immunosuppression caused by CsA. When examining this possibility, we obtained results indicating that the MOC31PE immunotoxin represents a highly promising immunogenic stimulant, in addition to exerting its specific tumor cell killing capacity.

Material and Methods

Patients

[0371] The completed phase I trial comprised of two parts, the first with MOC31PE alone in 34 patients (modified Fibonacci dose escalation), and the second part in 23 patients with dose escalation of MOC31PE with concomitant administration of Sandimmune® (CsA) (1). In both treatment arms, MOC31PE was repeated every second week up to 4 times (day 1, 14, 28, 42). In the combination group, CsA was given one day before MOC31PE (day 0) and then for 4 subsequent days.

[0372] The inclusion criteria was EpCAM positive metastatic carcinoma, age 18 years or older, in patients with ECOG performance status 0-2. Before inclusion and study related investigations, the patients signed an approved written informed consent.

MOC31PE

[0373] The MOC31 monoclonal mouse antibody (IgG1) recognizing the CD326 antigen (EpCAM) was produced and purified to clinical grade by MCA Development, Groningen, Netherlands. Pseudomonas exotoxin A (PE) (full-length, wild-type) was isolated from the fermentation broth of Pseudomonas aeruginosa PA103, manufactured at University of Ohio, Columbus, Ohio. The MOC31 PE conjugate was produced to clinical grade at Fred Hutchinson Cancer Research Center, Biologics Production Facility, Seattle, Wash. (1).

[0374] Further details in relation to the production of Pseudomonas exotoxin A (PE) and of how it is conjugated to the MOC31 antibody are given below.

[0375] Pseudomonas exotoxin A (PE) was isolated from the fermentation broth of P. aeruginosa PA 103 (6). PE is produced by inoculation of a New Brunswick in vitro fermentor with an overnight culture of P. aeruginosa for 18-20 hrs. at 32° C. at 400 rpm. The exotoxin A was purified from 50 L of culture supernatant according to the Leppla procedure (22) with a few modifications as described by Galloway et al. (23). All procedures were performed at 5° C. The cells were harvested, and the supernatant was diluted with cold deionized water (150 litres) to bring down the ionic concentration, and DEAE cellulose (DE-52; 2 litres) slurried in water, was added. While the suspension was vigorously stirred, HCl (2M; 150 ml) diluted in cold water (4 litres) was added. After 1 h of stirring, the DE-52 resin was allowed to settle for 2 h and then was collected and washed three times with cold Tris-hydrochloride (buffer A; 0.01 M; pH 8.1). Finally, the washed DE-52 resin was transferred to a funnel and slowly eluted with cold 0.15M Tris-hydrochloride (2.5 litres; pH 8.1). The first 500 ml of eluate was discarded. The remaining 2 litres was collected, and 2-mercaptoethanol was added to a final concentration of 5 mM. Exotoxin A was precipitated by the addition of solid ammonium sulphate to 75% saturation. The precipitate was redissolved and dialyzed overnight against buffer A containing 2-mercaptoethanol (2 mM). It was then applied to a DE-52 column (200 ml) equilibrated with the same buffer. The exotoxin A bound to the column, was eluted off the column using a linear gradient of 0.01 to 0.3 M NaCl in buffer A containing 2-mercaptoethanol (2 mM). The exotoxin A fractions that were pooled were adjusted to pH 6.8 with 2M HCl.

[0376] Exotoxin A was further purified by applying to a column of hydroxylapatite equilibrated with sodium phosphate buffer (5 mM)/sodium chloride (50 mM) at pH 7.0. The bound exotoxin A was eluted off the column by a linear gradient of sodium phosphate, 5 mM to 100 mM in 50 mM sodium chloride at pH 7.0. The protein peak emerging at 40-60 mM sodium phosphate was concentrated by precipitation with ammonium sulphate, redissolved and dialyzed against buffer A containing 2-mercaptoethanol (2 mM). The exotoxin A thus purified was aliquoted and stored frozen at −70° C.

[0377] The MOC31 antibody was conjugated to the PE by a thioether bond. More specifically, MOC31 antibody that had been treated (or derivatised) using DTT (dithiothreitol) was conjugated to PE that had been derivatised using the reagent SMCC (succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate).

Ethical

[0378] The clinical phase I study was approved by the Norwegian Medicines Agency, by the Norwegian Regional Ethical Committee (REC) and by the institutional review board. The registration number is NCT01061645 with the study title “Study of MOC31-PE in antigen positive carcinomas”. All patients provided written informed consent.

Cytokine Measurement

[0379] Serum samples were taken prior to dosing and stored frozen at −70° C. All samples were thawed on ice, vortexed, spun down at 14,000×g for 10 min at 4° C. and tested in a multiplex biometric ELISA-based immunoassay. The cytokines included: interleukin-1 β (IL-1β), interleukin-2 (IL-2), interleukin-6 (IL-6), interleukin-12 (IL-12), granulocyte-macrophage colony-stimulating factor (GM-CSF), tumour necrosis factor α (TNF-α), and interferon-γ (IFN-γ), according to the manufacturer's instructions (Bioplex, Bio-Rad Lab., Inc., Hercules, Calif., USA). Serum levels of all proteins were determined using the Bio-Plex array reader Luminex IS 100 instrument (Luminex, Austin, Tex., USA) that quantifies multiplex immunoassays in a 96-well plate. The analyte concentration was calculated using a standard curve, with software provided by the manufacturer.

Cell Lines

[0380] Colon cancer cell lines HCT116, SW480 and HT29 (ATCC, Rockville, Md.) were cultured in RPMI-1640 medium supplemented with Hepes, Glutamax (all from Lonza, Austria), 10% heat-inactivated FCS (PAA, GE Healthcare, UK) at 37° C. All cell lines were routinely tested and found to be free from contamination with Mycoplasma species. The cells were routinely ID tested.

Cell Viability

[0381] The Cell Titer 96 AqueousOne solution assay (MTS (Promega Madison, Wis.)) was used to determine cell viability as previously described (3). Cells (10 000) were seeded in 96-well plates. After 24 h incubation, the medium was replaced with medium containing MOC31PE (1-1000 ng/ml), and incubated further for 24 h. The MTS solution was then added to the wells, and the absorbance was measured 2 h later at a wavelength of 490 nm. The viability of MOC31PE treated cells were compared to the values for untreated control cells and recorded as the percentage cell viability of control cells. The assays were performed in triplicate.

Western Blotting for HMGB1 Detection

[0382] HCT116 cells (2×10.sup.6) were seeded in T-25 flasks and after 24 h, treated with MOC31PE (10 and 100 ng/ml) or mAb MOC31 (100 ng/ml) or vehicle (PBS, 0.1% HSA) for 24 h. Conditioned media from cells were centrifuged and supernatants were collected. Equal volumes of supernatant were separated by NuPAGE Bis-Tris gel (Invitrogen, Carlsbad, Calif.), and subsequently transferred by electrophoresis to Immobilon membrane (Millipore, Bedford, Mass.). The membrane was blocked with 5% nonfat dry milk for 1 h at room temperature followed by incubation with rabbit anti-HMGB1 (Cell Signaling Technology (Danvers, Mass.)) in 5% w/v BSA, 1×TBS, 0.1% Tween-20 at 4° C. overnight. The membranes were washed before incubation with appropriate HRP-coupled secondary antibodies. Following several washes, the peroxidase activity was visualized with enzyme-linked chemiluminescence (ECL, Amersham Pharmacia Biotech, Buckinghamshire, UK.)

Generation of Dendritic Cells

[0383] Immature dendritic cells (DCs) were generated essentially as described in Subklewe, et al. (4). Briefly, monocytes obtained from leukapheresis product (REC Project no: 2013/624-15) were cultured for 2 days with GM-CSF and Interleukin-4 (IL-4) in Ultra-low attachment cell culture flasks (Corning). Cancer cell lines were treated for 24 h or 48 h with MOC31PE, 9.2.27PE, and MOC31 at indicated concentrations.

[0384] The immature DCs were then either matured for 24 h with MOC31PE-treated colon cancer cell lines or their supernatants (sn) in 96-well plates. As a positive control, cytokines facilitating maturation were used (IL-113, IL-6, TNF-α, IFN-γ (all from PeproTech, Rocky Hill, N.J.), prostaglandin E.sub.2(PGE2), and TLR7/8 agonist R848 (MedChem Express, Sweden) (5). Immature DCs cultured with IL-4 and GM-CSF were used as negative control. The mature DC phenotype was evaluated by flow cytometry.

Flow Cytometry

[0385] Cells were washed in staining buffer consisting of phosphate buffered saline (PBS) containing 2% FCS before staining with CD9-BV510 (M-L13, BD Biosciences, San Jose, Calif.) and CD14-FITC (61D3, Thermo Fisher Scientific Inc, Waltham, Mass.). Finally, cells were resuspended in staining buffer containing 1% paraformaldehyde. Samples were acquired on a LSR II flow cytometer (BD Bioscience) and the data were analyzed using FlowJo software (Treestar Inc., Ashland, Oreg.). An analogous experiment was also performed using a CD86 antibody to analyse CD86 expression.

Statistical Analysis

[0386] Survival was estimated by the Kaplan-Meier method and survival curves compared using the Log-rank test. Univariate analysis was conducted by Cox proportional hazards regression.

[0387] The non-parametric two-tailed Mann-Whitney test was used to compare the median values of cytokine variables in the pre- and post-treatment groups. All statistical analyses were performed using the SPSS statistical package (version 21, SPSS, Chicago, Ill.). The level of statistical significance for the cytokine analysis was set at p<0.05.

[0388] All statistical analyses for DC maturation were performed using GraphPad Prism® (GraphPad Software, Inc.). Unpaired t-tests were used for comparison of DC maturation between conditions and all p-values given are two-tailed values. The level of statistical significance for the DC maturation analysis was set to p<0.05.

Results

Overall Survival

[0389] In our recent phase I trial, the toxicity of systemic therapy with MOC31 PE alone and in combination with cyclosporin A was modest and the maximum tolerated dose (MTD) was determined (1). No objective tumor responses were found as judged by CT scans eight weeks after the first MOC31 PE infusion (1).

[0390] We reevaluated our data at a later date and noticed that the median OS time for all patients treated with MOC31PE alone was 12.7 months (95% CI=5.6-19.8 months) as compared to 6.2 months (95% CI=5.6-6.8 months) (p=0.066) for the MOC31PE+CsA group (FIG. 1a). This difference was unexpected, as in preclinical studies the combination was superior, in fact synergistic to MOC31 PE alone both in vitro and in human tumor xenograft models (2). Therefore, we hypothesized that the observed OS difference could indicate that MOC31PE had induced an immunogenic anti-tumor effect that was obliterated by the immunosuppression caused by concomitant CsA. To examine this possibility, and to avoid bias related to the heterogeneity of tumor types in our study population, we analyzed specifically the results for patients with the most frequent tumor type; colorectal cancer. Of these, 15 patients had been treated with monotherapy and 18 with the combination. The Kaplan-Meier survival plot (FIG. 1b) shows that patients treated with MOC31PE alone had a median OS of 16.3 months (95% CI=5.6-27.0 months) compared to 6.0 months (CI=5.8-6.2 months) for patients in the MOC31 PE plus CsA group, which is comparable to OS of untreated patients with progressive disease on last line of standard chemotherapy (7, 8). In a univariable analysis, hazard ratio (HR) with 95% credible interval (Crl) was calculated for monotherapy vs combination (HR=0.248, 95% Crl: 0.109-0.564). In addition, when we excluded the five patients in the MOC31 PE monotherapy group who had received additional treatment after MOC31 PE (two with only local radiotherapy for bone metastasis) the median OS remained longer than expected (12 months, data not shown).

MOC31 PE Induced Release of HMGB1 In Vitro

[0391] To further evaluate the putative immunogenic effect of MOC31PE we wanted first to demonstrate its ability to kill colon cancer cell lines as a background for studying possible immunogenic cell death (ICD) factors such as the non-histone chromatin-binding nuclear protein high-mobility group box 1 (HMGB1) (9). In a dose-dependent manner, MOC31PE effectively decreased the cell viability of the colorectal cancer cell lines HCT116, SW480 and HT29 (FIG. 3a), with ID.sub.50 values similar for all three cell lines (100 ng/ml). Of note, release of HMGB1 was found in the supernatant of the MOC31PE treated HCT116 cells (FIG. 3b), as detected by western blot, but not in the supernatant of cells treated with the naked antibody MOC31 (100 ng/ml). Release of HMGB1 also was found in the supernatant of the MOC31PE treated SW480 cells (FIG. 3b), as detected by western blot.

[0392] It was also found that HMGB1 was released from MOC31PE treated MA-11 cells (a metastatic breast cancer cell line) (data not shown).

MOC31PE-Treated Cancer Cells Secrete Factors Responsible for Ex Vivo Maturation of Dendritic Cells

[0393] The CD14 protein level, a well known marker of immature DCs, decreases during DC maturation. In the ex vivo system used here, the CD14 level was significantly reduced by the addition of conditioned medium from MOC31PE-treated colon cancer cells (HCT116 and SW480) (p<0.0025) (FIG. 4) to an extent similar to that of the positive control (FIG. 4). The effect was MOC31PE dose-dependent, with increasing maturation of DCs with increasing MOC31PE concentrations. The irrelevant immunotoxin 9.2.27PE(10) (recognizing antigen HMW-MAA a melanoma specific antigen not expressed by colorectal cancer cells) as well as the MOC31 antibody did not kill colon cancer cells, and the supernatant from 9.2.27PE and MOC31 treated cells had no maturation effect on immature DCs. The results demonstrate that MOC31PE induces release of immune stimulating factors causing maturation of immature DCs.

[0394] Further experiments were performed which further demonstrate that MOC31PE-treated colon cancer cells release factors (immunogenic cell death factors) responsible for the ex vivo maturation of dendritic cells. The results show that conditioned media from MOC31PE-treated HCT-116 cells increases the maturation of dendritic cells (DCs) (FIG. 8A and FIG. 8B). Immature (imm) DCs were isolated from human monocytes from leukapheresis products by elutriation, and differentiated with GM-CSF and IL-4 into immDCs. The CD14 protein level, a well-known marker of immature DCs, decreases during DC maturation. The CD86 protein level, a well-known marker of mature DCs, increases during DC maturation. Interestingly, the very well-known ICD-inducer oxaliplatin, did not activate (i.e. did not enhance the maturation of) the immDCs as well as MOC31 PE (FIG. 8A and FIG. 8B). The effect was MOC31 PE dose-dependent, with increasing maturation of DCs with increasing MOC31 PE concentrations (FIG. 8A and FIG. 8B). The irrelevant immunotoxin 9.2.27PE (recognizing antigen HMW-MAA a melanoma specific antigen not expressed by colorectal cancer cells) as well as the MOC31 antibody did not kill colon cancer cells, and the supernatant from 9.2.27PE and MOC31 treated cells had no maturation effect on immature DCs (data not shown). The results demonstrate that MOC31 PE induces release of immune stimulating factors causing maturation of immDCs. The data in FIGS. 8A and 8B is from three biological triplicates, each in three technical triplicates.

Cytokines in Patients' Serum

[0395] The levels of pro-inflammatory Th1-type cytokines IL-2, IL-6, IL-12, IFN-γ and TNF-α, and the Th1 related IL-1β and GM-CSF were examined in serum from the patients with colorectal cancer: pretreatment (n=20), post-MOC31PE and post-MOC31PE+cyclosporin (in both cases 2 weeks after first immunotoxin administration, n=12).

[0396] With the exception of IL-6, the levels of all cytokines increased significantly in serum from MOC31 PE treated patients relative to the corresponding pre-treatment levels (p<0.01) (FIG. 2). Also, IL-2 (p<0.05), IL-12 (p<0.01) and GM-CSF (p<0.01) levels were significantly higher in the serum from MOC31PE treated patients compared to the combination group (FIG. 2). In table 1, the median and minimum and maximum ranges in cytokine concentration are presented. A large fraction of the pre-treatment samples had cytokine levels below the detection sensitivity of the assay. One patient in the monotherapy group (11%) and three in the combination group (25%) had all cytokine levels close to zero, the reason for which is unknown.

[0397] The results demonstrate that the MOC31 PE did induce a significant Th1 cytokine response in most patients which was abolished by the addition of CsA.

TABLE-US-00003 TABLE 1 Serum cytokines levels. Cytokine(pg/ml) IL1b (pg/ml) IL2 (pg/ml) IL6 (pg/ml) IL12 (pg/ml) GMCSF(pg/ml) IFN γ (pg/ml) TNFα (pg/ml) Median Range Median Range Median Range Median Range Median Range Median Range Median Range Pre 0 0-23 0 0-322 4 0-109  4 0-278 4 0-166 0 0-393  0 0-153 (n = 20) Post 12  0-576 58 0-824 29 0-1508 118  3-2603 165  7-1495 394 9-7912 158  0-6893 MOC31PE (n = 9) Post 3 0-31 10 0-75  16 0-67  7 1-173 34 2-167 111 0-1756 17 0-242 MOC31PE + CsA (n = 12) Serum from patients, taken before (n = 20) and two weeks after MOC31PE (n = 9) or MOC31PE plus CsA (n = 12) administration, analyzed by multiplex cytokine ELISA assay. The data is presented as the median and range of the individual cytokine levels (pg/ml).

Discussion

[0398] The clinical use of immunotoxins in cancer treatment has been hampered by their high immunogenicity leading to generation of anti-immunotoxin antibodies (11). We have demonstrated in preclinical experiments that combination therapy with CsA could circumvent this problem by inhibiting/delaying anti-immunotoxin antibody production, and this was confirmed in our phase I study (1, 2). However, in contrast to our expectation that the combination also could lead to increased anti-tumor efficacy, similar to that observed in preclinical experiments, we found the opposite, an observed long OS in patients treated with the MOC31 PE alone, compared to OS of those also receiving CsA. The present study demonstrates the novel and exciting finding that MOC31PE induces a strong immunogenic effect and prolonged overall survival of patients, e.g. patients with advanced colorectal cancer, a group of patients with very limited treatment alternatives.

[0399] In addition to the i.v. administered MOC31 PE clinical phase I trial, we have recently completed a clinical phase I study with intraperitoneal MOC31PE treatment of patients with colorectal cancer peritoneal metastases undergoing cytoreductive surgery and hyperthermic intraperitoneal chemotherapy (12). Also in this study, the immunotoxin treatment was well tolerated, supporting the safety of MOC31 PE treatment.

[0400] Previous clinical experience with immunotoxins, commonly highly modified or recombinant, has been disappointing (11). The underlying reasons for this might include pharmacokinetic limitations with short half-lives and low levels of active plasma concentrations, resulting in frequent injections and often severe toxicity. We report here that an immunotoxin, that has a fully murine antibody and non-modified PE conjugated with a stable and strong thioether bond, exerts an immunogenic effect.

[0401] The large size of the molecule is thought to prevent penetration of MOC31 PE deep into large solid tumor lesions. However, the immunogenic effect, and the long OS, seems to depend on specific binding of the immunotoxin to tumor cells expressing its target antigen, followed by killing of accessible tumor cells. Without wishing to be bound by theory, our data suggests that MOC31PE is inducing immunogenic cell death, involving stimulating maturation of dendritic cells and activation of T cells. The presence and increase in the levels of a number of cytokines in the serum of MOC31PE only treated patients support this. We found increased levels of IL-12 and IFNγ, both inducers of Th1 cells and necessary to promote potent cytotoxic T cells that are accountable for antitumor immunity (13). IL-2, another cytokine induced by MOC31PE, has been shown to be crucial to the expansion of CD8.sup.+ T cells and is particularly important for the functional maturation of activated T cells. In support of induction of this cytokine response being caused by MOC31PE-initiated ICD, we detected the release of HMGB1, one of the hallmarks of ICD (9), from colon cancer cells treated with MOC31 PE.

[0402] The immense success of checkpoint inhibitors in the clinic has been limited by aberrant immune checkpoint inhibition and/or absence of appropriate co-stimulation. This is observed in several cancer types with only a subset of patients responding and experiencing long term disease-free or OS. Several explanations have been suggested, including the need for stimulating the CD28 receptor (14), epigenetic stability of exhausted T-cells (15, 16), and the effect of the chaperone protein HSP90 altering the effect of proteins produced by mutated tumor genes (17). The limitations of individual checkpoint inhibitors have prompted the need for combination therapies, both with other inhibitors and with chemo- and radio-therapy (18-20).

[0403] MOC31PE has a mechanism of action that is different from checkpoint inhibitors; inhibiting protein synthesis and inducing apoptosis (2, 21). This is in addition to the immunogenic cell death anti-cancer effect described in the present study. The immunotoxin has furthermore an attractive safety profile with no subjective side effects and only modest liver toxicity. This makes it an attractive candidate for combination with checkpoint inhibitors. In summary, the MOC31PE immunotoxin represents a novel and highly promising immunostimulant for use in cancer therapies, particularly in patients with colorectal cancer.

REFERENCES

[0404] 1. Y. Andersson et al., Phase I trial of EpCAM-targeting immunotoxin MOC31 PE, alone and in combination with cyclosporin. British journal of cancer 113, 1548-1555 (2015). [0405] 2. Y. Andersson, O. Engebraaten, O. Fodstad, Synergistic anti-cancer effects of immunotoxin and cyclosporin in vitro and in vivo. British journal of cancer 101, 1307-1315 (2009). [0406] 3. Y. Andersson, H. Le, S. Juell, O. Fodstad, AMP-activated protein kinase protects against anti-epidermal growth factor receptor-Pseudomonas exotoxin A immunotoxin-induced MA11 breast cancer cell death. Molecular cancer therapeutics 5, 1050-1059 (2006). [0407] 4. M. Subklewe et al., Dendritic cell maturation stage determines susceptibility to the proteasome inhibitor bortezomib. Human immunology 68, 147-155 (2007). [0408] 5. S. Spranger et al., Generation of Th1-polarizing dendritic cells using the TLR7/8 agonist CL075. Journal of immunology 185, 738-747 (2010). [0409] 6. Leppla, S. H.: Large scale purification and characterization of the exotoxin A of Pseudomonas aeruginosa. Infect & Immunity. 14: 1077-1086. [0410] 7. A. Grothey et al., Regorafenib monotherapy for previously treated metastatic colorectal cancer (CORRECT): an international, multicentre, randomised, placebo-controlled, phase 3 trial. Lancet (London, England) 381, 303-312 (2013). [0411] 8. R. J. Mayer et al., Randomized trial of TAS-102 for refractory metastatic colorectal cancer. The New England journal of medicine 372, 1909-1919 (2015). [0412] 9. O. Kepp et al., Consensus guidelines for the detection of immunogenic cell death. Oncoimmunology 3, e955691 (2014). [0413] 10. K. Risberg, O. Fodstad, Y. Andersson, The melanoma specific 9.2.27PE immunotoxin efficiently kills melanoma cells in vitro. International journal of cancer 125, 23-33 (2009). [0414] 11. I. Pastan, R. Hassan, D. J. Fitzgerald, R. J. Kreitman, Immunotoxin therapy of cancer. Nature reviews. Cancer 6, 559-565 (2006). [0415] 12. A. Y. Frøysnes I S, Larsen S G, Davidson B, └ien J M T, Olsen K H, Giercksky K E, Julsrud L, Fodstad Ø, Dueland S, Flatmark K., Novel treatment with intraperitoneal MOC31PE immunotoxin in colorectal peritoneal metastasis—results from the ImmunoPeCa phase I trial Annals of Surgical Oncology (2017). [0416] 13. A. D. Garg, D. De Ruysscher, P. Agostinis, Immunological metagene signatures derived from immunogenic cancer cell death associate with improved survival of patients with lung, breast or ovarian malignancies: A large-scale meta-analysis. Oncoimmunology 5, e1069938 (2016). [0417] 14. M. Janakiram et al., The third group of the B7-CD28 immune checkpoint family: HHLA2, TMIGD2, B7x, and B7-H3. Immunological reviews 276, 26-39 (2017). [0418] 15. K. E. Pauken et al., Epigenetic stability of exhausted T cells limits durability of reinvigoration by PD-1 blockade. Science (New York, N.Y.) 354, 1160-1165 (2016). [0419] 16. R. A. Amezquita, S. M. Kaech, Immunology: The chronicles of T-cell exhaustion. Nature 543, 190-191 (2017). [0420] 17. M. L. Siegal, Molecular genetics: Chaperone protein gets personal. Nature 545, 36-37 (2017). [0421] 18. J. Lohmueller, O. J. Finn, Current modalities in cancer immunotherapy: Immunomodulatory antibodies, CARs and vaccines. Pharmacology & therapeutics, (2017). [0422] 19. T. Kuusk et al., Antiangiogenic therapy combined with immune checkpoint blockade in renal cancer. Angiogenesis, (2017). [0423] 20. A. Esposito, C. Criscitiello, G. Curigliano, Immune checkpoint inhibitors with radiotherapy and locoregional treatment: synergism and potential clinical implications. Current opinion in oncology 27, 445-451 (2015). [0424] 21. Y. Andersson, S. Juell, O. Fodstad, Downregulation of the antiapoptotic MCL-1 protein and apoptosis in MA-11 breast cancer cells induced by an anti-epidermal growth factor receptor-Pseudomonas exotoxin a immunotoxin. International journal of cancer 112, 475-483 (2004). [0425] 22. Galloway, D. R., Hedstrom, R. C., McGowan, J. L., Kessler, S. P. and Wozniak, D. J.: Biochemical analysis of CRM66: a nonfunctional Pseudomonas aeruginosa exotoxin A. J. Biol. Chem. 264: 14869-14873. [0426] 23. Morgan, A. C., Sivam, G. P., Beaumier, P., McIntyre, R. Abrams, P. G.: Immunotoxins of Pseudomonas exotoxin A (PE): Effect of linkage on conjugate yield, potency, selectivity and toxicity. Molecular Immunology. 27: 273-282, 1990.

EXAMPLE 2

[0427] Experiments were also performed with the immunotoxin BM7PE, looking at its effect on protein synthesis, cell viability and the maturation of dendritic cells.

Material and Methods

BM7PE

[0428] BM7PE is an immunotoxin in which the antibody component is the BM7 antibody and the toxic biological molecule is PE (full-length PE). The PE was produced as described in Example 1 and was conjugated to the BM7 antibody in an analogous manner to that described in Example 1 in relation to MOC31 PE.

Cell Viability

[0429] The Cell Titer 96 AqueousOne solution assay (MTS (Promega Madison, Wis.)) was used to determine cell viability as previously described. Cells (10 000) were seeded in 96-well plates. After 24 h incubation, the medium was replaced with medium containing BM7PE (0.01-1000 ng/ml), and incubated further for 24 h (T47D) or 48 h (HT116, SW480 and HT29). The MTS solution was then added to the wells, and the absorbance was measured 2 h later at a wavelength of 490 nm. The viability of BM7PE treated cells were compared to the values for untreated control cells and recorded as the percentage cell viability of control cells. The assays were performed in triplicate.

Measurement of Protein Synthesis Inhibition

[0430] Protein synthesis inhibition caused by BM7PE was measured by using the [3H]-leucine incorporation assay (Sandvig & Olsnes, 1982, J. Biol. Chem., 257. p 7495-7503). Cells (4×10.sup.4 per well) were seeded in 48-well plates and allowed to grow overnight before addition of different concentrations of BM7PE. After 20 hr incubation, the cells were washed twice with cold phosphate-buffered saline (PBS), 0.1% FCS and incubated with [3H]leucine (2 μCi/ml) in leucine-free medium for 45 min at 37° C. The cells were then washed with 5% trichloroacetic acid (TCA) for 10 and 5 min, respectively, and dissolved in 0.1 M KOH for at least 5 min. The resultant solution was transferred to the liquid scintillator Aquasafe 300 Plus (Zinsser Analytic, Frankfurt/Main, Germany). Sample counts were determined in a liquid scintillation counter (LKB Wallac). Assays were performed in duplicate, and repeated at least three times.

Generation of Dendritic Cells

[0431] Immature dendritic cells (DCs) were generated essentially as described in Subklewe, et al. (supra). Briefly, monocytes obtained from leukapheresis product (REC Project no: 2013/624-15) were cultured for 2 days with GM-CSF and Interleukin-4 (IL-4) in Ultra-low attachment cell culture flasks (Corning). Cancer cell lines were treated for 48 h with BM7PE at indicated concentrations.

[0432] The immature DCs were then either matured for 24 h with BM7PE-treated colon cancer cell lines supernatants (sn) in 96-well plates. As a positive control, cytokines facilitating maturation were used (IL-1β, IL-6, TNF-α, IFN-γ (all from PeproTech, Rocky Hill, N.J.), prostaglandin E.sub.2(PGE2), and TLR7/8 agonist R848 (MedChem Express, Sweden)). Immature DCs cultured with IL-4 and GM-CSF were used as negative control. The mature DC phenotype was evaluated by flow cytometry.

Flow Cytometry

[0433] Cells were washed in staining buffer consisting of phosphate buffered saline (PBS) containing 2% FCS before staining with CD9-BV510 (M-L13, BD Biosciences, San Jose, Calif.) and CD14-FITC (6ID3, Thermo Fisher Scientific Inc, Waltham, Mass.). Finally, cells were resuspended in staining buffer containing 1% paraformaldehyde. Samples were acquired on a LSR II flow cytometer (BD Bioscience) and the data were analyzed using FlowJo software (Treestar Inc., Ashland, Oreg.). An analogous experiment was also performed using a CD86 antibody to analyse CD86 expression.

Results

In Vitro Inhibition of Protein Synthesis and Cell Viability

[0434] In a dose-dependent manner, BM7PE effectively decreased protein synthesis (FIG. 5A) and the cell viability (FIG. 5B) of the breast cancer cell line T47D. For the colorectal cancer cell lines HCT116, SW480 and HT29 the BM7PE effectively decreased cell viability in all three cell lines (FIG. 6).

BM7PE-Treated Cancer Cells Secrete Factors Responsible for Ex Vivo Maturation of Dendritic Cells

[0435] The CD14 protein level, a well known marker of immature DCs, decreases during DCs maturation. In the ex vivo system used here, the CD14 level was significantly reduced by the addition of conditioned medium from BM7PE-treated colon cancer cells (HCT116 and SW480) (p<0.0025) (FIG. 7) to an extent similar to that of the positive control (FIG. 7). The effect was BM7PE dose-dependent, with increasing maturation of DCs with increasing BM7PE concentrations. The irrelevant immunotoxin 9.2.27PE (recognizing antigen HMW-MAA a melanoma specific antigen not expressed by colorectal cancer cells) did not kill colon cancer cells, and the supernatant from 9.2.27PE treated cells had no maturation effect on immature DCs. The results demonstrate that BM7PE induces release of immune stimulating factors causing maturation of immature DCs.

[0436] Further experiments were performed which further demonstrate that BM7PE-treated colon cancer cells release factors (immunogenic cell death factors) responsible for the ex vivo maturation of dendritic cells. The results show that conditioned media from BM7PE-treated HCT-116 cells increases the maturation of dendritic cells (DCs) (FIG. 9A and FIG. 9B). Immature (imm) DCs were isolated from human monocytes from leukapheresis products by elutriation, and differentiated with GM-CSF and IL-4 into immDCs. The CD14 protein level, a well-known marker of immature DCs, decreases during DC maturation. The CD86 protein level, a well-known marker of mature DCs, increases during DC maturation. Interestingly, the very well-known ICD-inducer oxaliplatin, did not activate (i.e. did not enhance the maturation of) the immDCs as well as BM7PE (FIG. 9A and FIG. 9B). The effect was BM7PE dose-dependent, with increasing maturation of DCs with increasing BM7PE concentrations (FIG. 9A and FIG. 9B). The irrelevant immunotoxin 9.2.27PE (recognizing antigen HMW-MAA a melanoma specific antigen not expressed by colorectal cancer cells) as well as the BM7 antibody did not kill colon cancer cells, and the supernatant from 9.2.27PE and BM7 treated cells had no maturation effect on immature DCs (data not shown). The results demonstrate that BM7PE induces release of immune stimulating factors causing maturation of immDCs. The data in FIGS. 9A and 9B is from three biological triplicates, each in three technical triplicates.

EXAMPLE 3

MOC3IPE Induced Release of ATP In Vitro

Determination of the Immunogenic Cell Death Marker—ATP.

[0437] Colon cancer cell lines HCT116 and SW480 were cultured according to manufacture (ATCC, Rockville, Md.). HCT116 cells were treated for 24 h with MOC31 PE and conditioned media from cells treated with MOC31PE (100 and 1000 ng/ml), mAb MOC31 (100 ng/ml) or vehicle (PBS, 0.1% HSA) were centrifuged. Equal volumes of cell supernatant were analyzed for the levels of secreted ATP in the supernatant by using the CellTiter-Glo® Luminescent Cell Viability Assay (Promega Madison).

[0438] The inventors have shown that the Damage Associated Molecular Patterns (DAMPs) HMGB1 and ATP, which are immunogenic cell death signals, are released by MOC31-PE-treated cancer cells (HCT116 cells and SW480 cells). FIG. 3(b) of Example 1 demonstrates the release of HMGB1. FIG. 10 shows, using an ATP assay, that MOC31PE-treated cancer cells release ATP. FIG. 10 also includes data for the ICD inducer oxaliplatin for comparison. The data in FIG. 10 indicates that MOC31 PE acts an immunogenic cell death ICD inducer. The monoclonal MOC31 antibody (MOC3-ab) and the toxin PE did not themselves act as ICD inducers.

EXAMPLE 4

MOC31PE Promotes Activation of Killer T Cells (CD8+) Ex Vivo

Materials and Methods

[0439] Bone marrow-derived monocytes were thawed and cultured with the cytokines IL-4+GM-CSF for 2 days to generate immature DCs. HCT116 cells were treated with immunotoxin (MOC31PE) for 24 hrs and then the isolated conditioning medium (i.e. the supernatant with the HCT116 cells removed) was co-incubated with immature DCs for 24 hrs. DCs were stained for CD14-FITC, CD86-PE and analysed by flow cytometry. A portion of the treated DCs were transferred to a T-cell culture. The T cells becomes activated by the mature DCs, and the activation was measured by expression of the degranulation marker CD107a (−Cy5) and IFNγ (−FITC), and TNFα (−PE) in a flow cytometry analysis (CD107a (−Cy5) and IFNγ (−FITC), and TNFα (−PE) were run in the same flow as a triple stain).

Results

[0440] ICD-factors released into the conditioning medium by MOC31PE-treated HCT116 cancer cells are able to activate immature DC to mature DC (see Example 1), which will activate T-cells leading to an increase in the population of the killer T cells (CD8+) ex vivo. This is illustrated by the data in FIG. 11. The CD8+ T-cell population (expressing the markers IFNγ, CD107a and TNFα) was analyzed by flow cytometry. The conditioning medium from untreated HCT116 cancer cells had no effect on DC and no effect on T-cells (as shown in FIG. 11). Without wishing to be bound by theory, it is believed that, in vivo, treatment with the MOC31 PE immunotoxin would result in an increase in the killer T cell (CD8+) population size and these T-cells will seek out and destroy tumor cells (e.g. metastatic tumour cells) and reduce tumor lesions, even solid tumors, by infiltrating it and releasing killing factors.