Patent ID: 12239686

In summary, the Examples below show:

Example 1—that LTX-315 is the most potent of the 5 tested compounds in an in vitro cytotoxic activity study against a panel of 37 human cancer cell lines.

Example 2—that LTX-315 is the most potent of the 5 tested compounds in an in vitro cytotoxic activity study against a panel of 10 lymphoma cell lines.

Example 3—that LTX 315 has a mean EC50value greater than 1200 μg/ml (833 μM) against human red blood cells.

Example 4—that the anti-tumour activity of LTX-315 resulted in a complete tumour response in 3 of 7 treated mice for the Group receiving the optimal dose (Group 1) in an investigation into the effect of LTX-315 at different dose levels on a murine A20 B-cell lymphoma in mice.

Example 5—that four different LTX-315 treatment regimes demonstrated a strong anti tumour effect against murine CT26WT (multidrug resistant) tumours.

Example 6—that LTX-315 has a broad spectrum of activity against various multidrug resistant cancer cell lines and, significantly, a much weaker cytotoxic effect on normal human cells.

Example 7—that complete tumour regression following initial treatment of solid murine tumours with LTX-315 resulted in a form of endogenous long-term protection against growth of the same tumours following re-inoculation.

Example 8—that treatment with LTX-315 may confer long term protection against specific tumours by eliciting an immune response.

Example 9—that an anti A20 cell immune response have been induced by the injection of the cocktail of LTX-315 and lysed A20 cells.

Example 10—that treatment with LTX-315 induces hallmarks of immunogenic cell death by mitochondria distortion in human melanoma cells.

Example 11—that treatment with LTX-315 in combination with an anti-CTLA-4 antibody caused a complete and long-lasting tumor regression in a high proportion of test subjects and induced an adaptive immune response. Anti-PD-1 antibody also showed an ability to act in combination with LTX-315 to inhibit tumour growth.

Example 12—that treatment with LTX-315 in combination with an anti-PD-L1 antibody caused tumour regression in a high proportion of test subjects and induced an adaptive immune response.

Example 1

In Vitro Cytotoxic Activity Study of 5 Test Compounds Against a Panel of 37 Human Cancer Cell Lines

1. Study Aim

To determine the concentrations of five novel compounds to obtain a 50% inhibition of proliferation (IC50) against a panel of 37 human cancer cell lines.

2. Materials and Methods

2.1. Test Substances

2.1.1. Test Substances

Test substances, LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1) provided in powder form.
2.1.2. Positive ControlTriton X-100 was used as positive control, supplied by Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier, France).
2.1.3. Drug Vehicle and Storage ConditionsCompounds were stored at 4° C. Powder was first dissolved in serum free culture medium (RPMI 1640, Lonza, Verviers, Belgium) and further diluted using serum-free culture medium to reach appropriate dilutions. Stock solution was not stored and was prepared fresh the day of experiment.1% (final concentration) Triton X-100 was obtained by dilution using culture medium.
2.2. Tumor Cell Lines and Culture Conditions
2.2.1. Tumor Cell Lines

Cancer cell lines and culture media were purchased and provided by Oncodesign. The details of the cell lines is presented in Table 1 below.

TABLE 1Cell linesOriginSourceBLOODCCRF-CEMacute lymphoblastic leukemia, T cellsPharmacellaCCRF-acute lymphoblastic leukemia, T cellsPharmacellCEM/VLBHL-60acute promyelocytic leukemia, AML,ATCCbpluripotent differentiationHL-60/ADRacute promyelocytic leukemia, AMLPharmacellK-562chronic myeloid leukemia, pleuralATCCeffusion metastasisK-562/Gleevecchronic myeloid leukemia, pleuralOncodesigneffusion metastasisRPMI 8226myeloma, B cells, Igl-typePharmacellBRAINSH-SY5Yneuroblastoma, bone marrowATCCmetastasisSK-N-ASneuroblastoma, bone marrowATCCmetastasisU-87 MGglioblastoma, astrocytomaATCCBREASTMCF-7invasive ductal carcinoma, pleuralPharmacelleffusion metastasisMCF7/mdradenocarcinoma, pleural effusionPharmacellmetastasisMDA-MB-231invasive ductal carcinoma, pleuralPharmacelleffusion metastasisMDA-MB-invasive ductal carcinoma, pleuralATCC435Seffusion metastasisT-47Dinvasive ductal carcinoma, pleuralATCCeffusion metastasisCOLONCOLO 205colorectal adenocarcinoma, ascitesATCCmetastasisHCT 116colorectal carcinomaATCCHCT-15colorectal adenocarcinomaATCCHT-29colorectal adenocarcinomaATCCENDOTHELIUMHUV-EC-CnormalATCCKIDNEY786-Orenal cell adenocarcinomaATCCA-498carcinomaATCCLIVERHep G2hepatocellular carcinomaATCCSK-HEP-1adenocarcinoma, ascites metastasisATCCLUNGA549carcinomaPharmacellCalu-6anaplastic carcinomaATCCNCI-H460carcinoma, pleural effusionATCCmetastasisOVARYIGROV-1carcinomaPharmacellIGROV-carcinomaPharmacell1/CDDPNIH:OVCAR-3adenocarcinoma, ascites metastasisPharmacellSK-OV-3adenocarcinoma, ascites metastasisPharmacellPANCREASBxPC-3adenocarcinomaATCCPANC-1carcinomaATCCPROSTATEDU 145carcinoma, brain metastasisPharmacellPC-3adenocarcinoma, bone metastasisATCCSKINA-431epidermoid carcinomaATCCMalme-3MMalignant melanomaATCCSK-MEL-2malignant melanoma, skin metastasisATCCa- Pharmacell, Parisb- ATCC, Manassas, Virginia, USA
2.2.2. Culture Conditions

Tumor cells were grown as adherent monolayers or as suspensions at 37° C. in a humidified atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640 containing 2 mM L-glutamine (Lonza, Belgium) and supplemented with 10% fetal bovine serum (FBS, Lonza). For experimental use, the adherent cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (Lonza), diluted in Hanks' medium without calcium or magnesium (Lonza) and neutralized by addition of complete culture medium. Cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.

Mycoplasmadetection was performed using the MycoAlert®MycoplasmaDetection Kit (Lonza) in accordance with the manufacturer's instructions. All tested cells were found to be negative formycoplasmacontamination.

3. Experimental Design and Treatments

3.1. Cell Lines Amplification and Plating

Tumor cells were plated in 96-well flat-bottom microtitration plates (Nunc, Dutscher, Brumath, France) and incubated at 37° C. for 24 hours before treatment in 190 μl of drug-free culture medium supplemented or not with 10% FBS for adherent or suspension growing cell lines, respectively.

Implantation densities for each cell lines are summarized in Table 2 below:

TABLE 2ImplantationImplantationdensitiesdensitiesCell lines(cells/well)Cell lines(cells/well)CCRF-CEM25,000HUV-EC-C20,000CCRF-CEM/VLB25,000786-O15,000HL-6020,000A-49815,000HL-60/ADR20,000Hep G215,000K-56220,000SK-HEP-115,000K-562/IMR20,000A54915,000RPMI 822620,000Calu-615,000SH-SY5Y20,000NCI-H46015,000SK-N-AS15,000IGROV-115,000U-87 MG15,000IGROV-1/CDDP15,000MCF-720,000NIH:OVCAR-315,000MCF7/mdr20,000SK-OV-315,000MDA-MB-23115,000BxPC-315,000MDA-MB-435S20,000PANC-115,000T-47D15,000DU 14515,000COLO 20515,000PC-315,000HCT 11615,000A-43115,000HCT-1515,000Malme-3M15,000HT-2920,000SK-MEL-215,000
3.2. IC50Determination

The adherent cell lines were washed once with 200 μl FBS-free culture medium before treatment. Tumor cells were incubated for 4 hours with 10 concentrations of compounds in ¼ dilution step with a top dose of 400 μM (range 4×10−4to 4×10−10M), with 1% (final concentration) Triton X-100 as positive control and FBS-free culture medium as negative control. The cells (190 μl) were incubated in a 200 μl final volume of FBS-free culture medium containing test substances at 37° C. under 5% CO2.

Three independent experiments were performed, each concentration being tested in quadruplicate. Control cells were treated with vehicle alone. At the end of treatments, the cytotoxic activity was evaluated by a MTS assay (see § 3.3).

Dilutions of tested compound as well as distribution to plates containing cells were performed using a Sciclone ALH 3000 liquid handling system (Caliper Life Sciences S.A.). According to automate use, a single range of concentrations was tested whatever the cell lines to be tested. The range was not adapted for each cell line.

3.3. MTS Assay

The in vitro cytotoxic activity of the test substance was revealed by a MTS assay (BALTROP J. A. et al., Bioorg. Med. Chem. Lett. 1991, 1:611-614) using a novel tetrazolium compound (MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling reagent named PMS (phenazine methosulfate). Like MTT, MTS is bioreduced by cells into a formazan product that is directly soluble in culture medium without processing, unlike MTT.

At the end of the cells treatment, 40 μl of a 0.22 μm filtered freshly combined solution of MTS (20 ml at 2 mg/ml, Ref G1111, Batch 235897, Exp 03/2009, Promega, Charbonnières, France) and PMS (1 ml at 0.92 mg/ml, Ref P9625, Batch 065K0961, Sigma) in Dulbecco's Phosphate Buffered Saline (DPBS, Ref 17-513Q, Batch 6MB0152, Cambrex), were added in each well. Culture plates were incubated for 2 h at 37° C. Absorbency (OD) were measured at 490 nm in each well using VICTOR3TM1420 multilabeled counter (Wallac, PerkinElmer, Courtaboeuf, France).

4. Data Presentation

4.1. IC50Determination

The dose response inhibition of proliferation (IC) was expressed as follows:

IC=ODdrug-exposed⁢wellsODdrug-free⁢wells×100The OD values are the mean of 4 experimental measurements.IC50: drug concentration to obtain a 50% inhibition of cell proliferation.

The dose-response curves were plotted using XLFit 3 (IDBS, United Kingdom). The IC50determination values were calculated using the XLFit 3 software from semi-log curves. Individual IC50determination values as well as mean and SD values were generated.

4.2. Resistance Index (RI)

Resistance index was calculated using the following formula:

RIcompound⁢A=IC50⁢compoundA(Resistant⁢cell⁢líne)IC50⁢compoundA(Sensitive⁢cell⁢line)

Resistance index was calculated for each compound for each couple of sensitive and resistant cell lines. Individual resistance index was calculated when IC50values of both sensitive and corresponding resistant cell lines were determined within same experiment. In addition, resistance index was also calculated ratio of mean IC50values obtained during three independent experiments.

5. Results

5.1. LTX-302

All thirty seven human tumor cell lines tested were sensitive to LTX-302 compound with IC50values ranging from 4.83±0.96 μM to 20.09±4.07 μM for T-47D and Hep G2 cell lines, respectively.

Mean IC50value for LTX-302 compound obtained on the 37 tumor cell lines was 12.05+4.27 μM with a median value of 11.70 μM. Mean IC50value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.

Hematological and lung cancer cell lines were the most sensitive to LTX-302 compound (median IC50values 7.96 μM (n=7) and 9.02 μM (n=3) for hematological and lung cancer cell lines, respectively) whereas liver cancer cell lines were the most resistant (median IC50value 17.84 μM, n=2).

Activity of LTX-302 compound seemed to be slightly decreased by acquired resistance towards doxorubicin as exhibited by the RI values of both HL-60/ADR and MCF-7/mdr cell lines (1.31 and 1.23 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-302 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.33 for IGROV-1/CDDP cell line.

5.2. LTX-313

All thirty seven (37) human tumor cell lines tested were sensitive to LTX-313 compound with IC50values ranging from 4.01±0.39 μM to 18.49±4.86 μM for RPMI 8226 and U-87 MG cell lines, respectively.

Mean IC50value for LTX-313 compound obtained on the 37 tumor cell lines was 9.60±3.73 μM with a median value of 8.83 μM. Mean IC50value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.

Hematological cancer cell lines were the most sensitive to LTX-313 compound (median IC50value 7.04 μM, n=7) whereas liver cancer cell lines were the most resistant (median IC50value 13.71 μM, n=2).

Activity of LTX-313 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 1.16 and 1.24 for CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-313 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.49 for IGROV-1/CDDP cell line.

5.3. LTX-315

All thirty seven human tumor cell lines tested were sensitive to LTX-315 compound with IC50values ranging from 1.18±0.25 μM to 7.16±0.99 μM for T-47D and SK-OV-3 cell lines, respectively.

Mean IC50value for LTX-315 compound obtained on the 37 tumor cell lines was 3.63±1.45 μM with a median value of 3.27 μM. Mean IC50value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.

Breast, hematological and lung cancer cell lines were the most sensitive to LTX-315 compound (median IC50values 2.45 μM (n=5), 2.60 μM (n=7) and 2.83 μM (n=3) for breast, hematological and lung cancer cell lines respectively) whereas liver cancer cell lines were the most resistant (median IC50value 5.86 μM, n=2).

Activity of LTX-315 compound seemed to be slightly decreased by acquired resistance towards doxorubicin as exhibited by the RI values of HL-60/ADR and MCF-7/mdr cell lines (1.45 and 1.12 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-315 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.50 for IGROV-1/CDDP cell line.

5.4. LTX-320

All thirty seven human tumor cell lines tested were sensitive to LTX-320 compound with IC50values ranging from 3.46±0.22 μM to 16.64±3.15 μM for T-47D and Hep G2 cell lines, respectively.

Mean IC50value for LTX-320 compound obtained on the 37 tumor cell lines was 7.58±2.79 μM with a median value of 6.92 μM. Mean IC50value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.

Hematological, breast, kidney and brain cancer cell lines were the most sensitive to LTX-320 compound (median IC50values 6.04 μM (n=7), 6.60 μM (n=5), 6.60 μM (n=2) and 6.92 μM (n=3) for hematological, breast, kidney and brain cancer cell lines respectively) whereas liver cancer cell lines were the most resistant (median IC50value 11.46 μM, n=2).

Activity of LTX-320 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of HL-60/ADR and MCF-7/mdr cell lines (0.90 and 1.19 for HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-320 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.49 for IGROV-1/CDDP cell line.

5.5. LTX-329

All thirty seven human tumor cell lines tested were sensitive to LTX-329 compound with IC50values ranging from 2.43±0.34 μM to 16.90±1.18 μM for T-47D and U-87 MG cell lines, respectively.

Mean IC50value for LTX-329 compound obtained on the 37 tumor cell lines was 8.17±3.20 μM with a median value of 7.89 μM. Mean IC50value obtained for the normal cell line (HUV-EC-C) was higher than for any of the tumor cell lines.

Breast and hematological cancer cell lines were the most sensitive to LTX-329 compound (median IC50values 4.92 μM (n=5) and 5.26 μM (n=7) for breast and hematological cancer cell lines respectively) whereas ovarian cancer cell lines were the most resistant (median IC50value 13.37 μM, n=4).

Activity of LTX-329 compound seemed not to be modified by acquired resistance towards doxorubicin as exhibited by the RI values of CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines (0.76, 0.80 and 1.07 for CCRF-CEM/VLB, HL-60/ADR and MCF-7/mdr cell lines, respectively). On the contrary, activity of LTX-329 compound seemed to be increased by acquired resistance towards cisplatin as exhibited by a RI value of 0.46 for IGROV-1/CDDP cell line.

5.6. General Comments

T-47D breast cancer cell line is the most sensitive cell line whatever the LTX compound tested.

Hematological cancer cell lines are the most sensitive histological type for all five compounds tested, liver and ovarian cancer cell lines being within the most resistant cell lines. All five compounds tested exhibited highest activity on IGROV-1/CDDP cell line (resistant to cisplatin) than on parental IGROV-1 ovarian cancer cell line. Doxorubicin resistance seemed to slightly decrease activity of LTX compounds.

LTX-315 compound is the most potent compound from the five compounds tested.

6. Conclusions

All five compounds tested (i.e. LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329) exhibited cytolytic activity against 37 human cancer cell lines tested with IC50values in micromolar to ten micromolar range.LTX-315 compound is the most potent compound tested with IC50values between 1 and 5 micromolar on all 37 human cancer cell lines tested.

Example 2

In Vitro Cytotoxic Activity Study of 5 Test Compounds Against a Panel of 10 Lymphoma Cell Lines

1. Study Aim

To determine the concentrations of five novel compounds to obtain a 50% inhibition of proliferation (IC50) against a panel of 10 lymphoma cell lines.

2. Materials and Methods

2.1. Test Substances

2.1.1. Test Substances

Test substances, LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329 (see Table 1) provided in powder form.
2.1.2. Positive Control

Triton X-100 was used as positive control and supplied by Oncodesign (Dijon, France) from Sigma (Saint Quentin Fallavier, France).

2.1.3. Drug Vehicle and Storage Condition

Compounds were stored at 4° C. Powder was first dissolved in serum free culture medium (RPMI 1640, Lonza, Verviers, Belgium) and further diluted using serum-free culture medium to reach appropriate dilutions. Stock solution was not stored and was prepared fresh the day of experiment.1% (final concentration) Triton X-100 was obtained by dilution using culture medium.
2.2. Tumor Cell Lines and Culture Conditions
2.2.1. Tumor Cell Lines

Cancer cell lines and culture media were purchased and provided by Oncodesign. The details of the cells lines are presented in Table 3 below.

TABLE 3N°Cell linesOriginSourceBLOOD1DaudiBurkitt’s lymphoma, B cells,ATCCaperipheral blood2Hs 445Hodgkin’s lymphoma, lymph nodeATCC3KARPAS-Anaplastic large cell lymphoma, T cells,DSMZb299peripheral blood4MinoMantle cell lymphoma, peripheral bloodATCC5NAMALWABurkitt’s lymphoma, B cells, peripheralATCCblood6RajiBurkitt’s lymphoma, B cells, peripheralDSMZblood7RamosBurkitt’s lymphoma, B cells, peripheralATCCblood8SU-DHL-1Anaplastic large cell lymphoma,DSMZpleural effusion9ToledoNon-Hodgkin’s B cell lymphoma,ATCCperipheral blood10U-937Lymphoma, histiocytic, macrophageATCCdifferentiation, pleural effusionaAmerican Type Culture Collection, Manassas, Virginia, USAbDeutsche Sammlung von Mikroorganismen und Zellkuturen Gmbh, Braunschweig, Germany
2.2.2. Culture Conditions

Tumor cells were grown as suspensions at 37° C. in a humidified atmosphere (5% CO2, 95% air). The culture medium for each cell line is described in Table 4 below. For experimental use, cells were counted in a hemocytometer and their viability was assessed by 0.25% trypan blue exclusion.

TABLE 4AdditivesCultureFBSGlucoseGlutamineNaPyrHepesCell linesmedium(%)(g/l)(mM)(mM)(mM)DaudiRPMI 164010—2110Hs 445RPMI 1640204.52110KARPAS-RPMI 164020—2——299MinoRPMI 1640154.52110NAMALWARPMI 1640102.52110RajiRPMI 164010—2110RamosRPMI 164010—2110SU-DHL-1RPMI 164010—2——ToledoRPMI 1640154.52110U-937RPMI 164010—2——

Mycoplasmadetection was performed using the MycoAlert®MycoplasmaDetection Kit (Lonza) in accordance with the manufacturer's instructions. All tested cells were found to be negative formycoplasmacontamination.

3. Experimental Design and Treatments

3.1. Cell Lines Amplification and Plating

Tumor cells were plated in 96-well flat-bottom microtitration plates (Nunc, Dutscher, Brumath, France) and incubated at 37° C. for 24 hours before treatment in 190 μl of drug-free and FBS-free culture medium.

Implantation densities for each cell lines are summarized in Table 5 below:

TABLE 5ImplantationdensitiesN°Cell lines(cells/well)1Daudi25,0002Hs 44525,0003KARPAS-29925,0004Mino25,0005NAMALWA15,0006Raji20,0007Ramos20,0008SU-DHL-125,0009Toledo25,00010U-93715,000
3.2. IC50Determination

Tumor cells were incubated for 4 hours with 10 concentrations of compounds in ¼ dilution step with a top dose of 400 μM (range 4×104 to 4×10-10 M), with 1% (final concentration) Triton X-100 as positive control and FBS-free culture medium as negative control. The cells (190 μl) were incubated in a 200 μl final volume of FBS-free culture medium containing test substances at 37° C. under 5% CO2.

Three independent experiments were performed, each concentration being issued from quadruplicate. Control cells were treated with vehicle alone. At the end of treatments, the cytotoxic activity was evaluated by a MTS assay (see § 3.3 below).

Dilutions of tested compound as well as distribution to plates containing cells were performed using a Sciclone ALH 3000 liquid handling system (Caliper Life Sciences S.A.). According to automate use, a single range of concentrations was tested whatever the cell lines to be tested. The range was not adapted for each cell line.

3.3. MTS Assay

The in vitro cytotoxic activity of the test substance was revealed by a MTS assay (Baltorp et al.) using a novel tetrazolium compound (MTS, 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxy phenyl)-2-(4-sulfophenyl)-2H-tetrazolium) and an electron coupling reagent named PMS (phenazine methosulfate). Like MTT, MTS is bioreduced by cells into a formazan product that is directly soluble in culture medium without processing, unlike MTT.

At the end of the cells treatment, 40 μl of a 0.22 μm filtered freshly combined solution of MTS (20 ml at 2 mg/ml, Ref G1111, Batch 235897, Exp 03/2009, Promega, Charbonnières, France) and PMS (1 ml at 0.92 mg/ml, Ref P9625, Batch 065K0961, Sigma) in Dulbecco's Phosphate Buffered Saline (DPBS, Ref 17-513Q, Batch 6MB0152, Cambrex), were added in each well. Culture plates were incubated for 2 h at 37° C. Absorbency (OD) were measured at 490 nm in each well using VICTOR3TM1420 multilabeled counter (Wallac, PerkinElmer, Courtaboeuf, France).

4. Data Presentation

4.1. IC50Data was Determined as in Example 1

5. Results

5.1. LTX-302

All ten human lymphoma cell lines tested were sensitive to LTX-302 compound with IC50values ranging from 5.30±2.02 μM to 12.54±3.52 UM for U-937 and Raji cell lines, respectively.

Mean IC50value for LTX-302 compound obtained on 10 sensitive cell lines was 8.11±2.44 μM with a median value of 7.53 μM.

5.2. LTX-313

All ten human lymphoma cell lines tested were sensitive to LTX-313 compound with IC50values ranging from 3.21±2.81 μM to 16.08±4.86 UM for Ramos and Raji cell lines, respectively.

Mean IC50value for LTX-313 compound obtained on 10 sensitive cell lines was 7.05±3.91 μM with a median value of 5.89 μM.

5.3. LTX-315

All ten human lymphoma cell lines tested were sensitive to LTX-315 compound with IC50values ranging from 1.15±0.42 UM to 4.93±1.03 UM for U-937 and Raji cell lines, respectively.

Mean IC50value for LTX-315 compound obtained on 10 sensitive cell lines was 3.01±1.36 UM with a median value of 2.93 μM.

5.4. LTX-320

All ten human lymphoma cell lines tested were sensitive to LTX-320 compound with IC50values ranging from 2.22+NA μM to 11.26±3.42 μM for Hs 445 and Raji cell lines, respectively.

Mean IC50value for LTX-320 compound obtained on 10 sensitive cell lines was 5.03±2.82 UM with a median value of 4.84 μM.

5.5. LTX-329

All ten human lymphoma cell lines tested were sensitive to LTX-329 compound with IC50values ranging from 2.46±NA μM to 8.70±1.70 μM for Hs 445 and Raji cell lines, respectively.

Mean IC50value for LTX-329 compound obtained on 10 sensitive cell lines was 5.76±2.27 μM with a median value of 5.72 μM.

5.6. General Comments

KARPAS-299 and Raji cell lines are the most resistant cell lines whatever the LTX compound tested.

Hs 445, Ramos and U-937 cell lines are the most sensitive cell lines whatever the LTX compound tested.

LTX-315 compound is the most potent compound from the five compounds tested.

6. Conclusions

All five compounds tested (i.e. LTX-302, LTX-313, LTX-315, LTX-320 and LTX-329) exhibited cytolytic activity against the 10 human lymphoma cell lines tested with IC50values in micromolar range.LTX-315 compound is the most potent compound tested with IC50values between 1 and 5 micromolar on all 10 human lymphoma cell lines tested.

Example 3

Haemolytic Activity In Vitro

Principle of Test

The haemolytic activity of the peptide LTX-315 against human red blood cells was measured.

Materials and Methods

Freshly collected human blood was centrifuged at 1500 rpm for 10 minutes in order to isolate the red blood cells. The red blood cells (RBC) were washed three times with PBS [35 mM phosphate buffer with 150 mM NaCl, pH 7.4] by centrifugation at 1500 rpm for 10 minutes, and adjusted to 10% haematocrit with PBS. LTX-315 solutions were added to give a final concentration range of the peptide from 1200 μg/ml to 1 μg/ml and an RBC concentration of 1%. The resulting suspension was incubated with agitation for one hour at 37° C. After incubation the suspension was centrifuged at 4000 rpm for 5 minutes, and the released haemoglobin were monitored by measuring the absorbance of the supernatant at 405 nm. PBS was used as negative control and assumed to cause no haemolysis. 0.1% Triton was used as positive control and assumed to cause complete haemolysis.

Test Substance: LTX-315

Reference substances: PBS (negative control) and Triton X-100 (positive control). Components of reaction mixtures: LTX-315, 10% Triton X-100, PBS and RBC (10% haematocrit). Details regarding these substances is presented in Table 6 below.

TABLE 6PBSRBCLTX-315/TritonConcentration(μl)(μl)X-100 (μl)Neg. Control63070—Pos. Control623707120015050300 (2 mg/ml stock)100020050250 (2 mg/ml stock)50032550125 (2 mg/ml stock)1005957035 (2 mg/ml stock)50612.57017.5 (2 mg/ml stock)105607070 (0.1 mg/ml stock)1623707 (0.1 mg/ml stock)
Method of Evaluation:

Released haemoglobin was monitored by measuring the absorbance of the supernatant at 405 nm, and percent haemolysis was calculated by the equation:

%⁢Haemolysis=⁠(A4⁢0⁢5⁢LTX-315-A4⁢0⁢5⁢PBS)⁠/(A4⁢0⁢5⁢0,1⁢%⁢Triton⁢⁢X-100-A4⁢0⁢5⁢PBS)]×100

LTX-315 concentration corresponding to 50% haemolysis (ECs) was determined from a dose-response curve.

Results

Mean value of five different experiments with standard deviation are presented in Table 7 below.

TABLE 7LTX-315MeanNumberConcentrationcell deathStandardof(μg/ml)(%)Deviationparallels1,20037.78.144531,00038.29.5760550020.47.861351003.61.14025501.60.54775100.60.8944510.00.0005

The data are also represented inFIG.1.FIG.1shows that LTX-315 has a mean value of EC50higher than 1200 μg/ml (833 μM).

Example 4

Pharmacodynamic Effects Relative to Murine A20 B-Cell Lymphoma Tumours in Mice

Principle of Test

The aim of the study was to investigate the effect of LTX-315 at different dose levels on a murine A20 B-cell lymphoma in mice.

Materials and Methods

The administration took place by intratumoural injection of LTX-315 dissolved in sterile saline.

Female mice were inoculated subcutaneously in the abdomen with 5 million murine A20 cells (ATCC, LGC Promochem AB, Middlesex, England) in a volume of 50 μl. The mice were divided into four groups (see Table 8 below for details). The intratumoural treatment was initiated when the tumours had reached the desired size of approximately 5 mm in diameter (minimum of 20 mm2).

Three dose levels of LTX-315, 1 mg (Group 1), 0.5 mg (Group 2) and 0.25 mg (Group 3) per injection, were investigated. The volume was 50 μl for all injections. LTX-315 was dissolved in sterile 0.9% NaCl water solution. This vehicle was used as control (Group 4). All four groups received three injections.

The mice were monitored during the study by measuring the tumours and weighing the animals regularly. The mice were followed until the maximum tumour burden of 125 mm2was reached, or until serious adverse events occurred (i.e. wound formation upon repeated treatments during the follow up period), then the mice were sacrificed. A calliper was used for tumour size measurements and weighing and physical examination were used as health control.

Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old, supplied form Harlan (England, UK)

Conditioning of animals: Animals were kept on standard laboratory chow and water.

Mean body weight, dose, route and treatment schedule is given in Table 8 below.

TABLE 8NumberInitial bodyofweight (g;ScheduleGroupanimalsmean ± SE)TreatmentDoseRoute(Day*)1720.36 ± 0.56Once1 mg inIntra1, 2, 3daily50 μl (20tumourmg/ml)2719.96 ± 0.38Once0.5 mg inIntra1, 2, 3daily50 μl (10tumourmg/ml)3920.11 ± 0.33Once0.25 mgIntra1, 2, 3dailyin 50 μltumour(5 mg/ml)4719.73 ± 0.40Once50 μlIntra1, 2, 3daily0.9%tumourNaCl inH20*Day 1 is first day of treatment
Results:

The anti-tumour effect of the various treatments is presented as mean tumour size in Table 9 below.

TABLE 9MeanMeanMeanMeantumourtumourtumourtumoursize (mm2)size (mm2)size (mm2)size (mm2)Treatmentat day 1*on day 4on day 9on day 14Group 125.82 ± 0.8003.70 ± 2.4012.43 ± 7.87Group 222.03 ± 0.63011.41 ± 4.6961.08 ± 23.84Group 321.25 ± 0.6420.60 ± 5.7168.49 ± 12.7469.42 ± 17.70Group 422.79 ± 0.6845.51 ± 5.2757.79 ± 4.3984.70 ± 7.35*Tumour size prior to start of treatment at first day of treatment

The degree of tumour response in the different treatment groups is summarised in Table 10 below.

TABLE 10Free ofTumour ResponseRelapseTumour atAnimalnopartialcompleteofend ofGroupresponseresponseresponseTumourFollow-Up1042.8%57.2%25%42.8%(3/7)(4/7)(3/7)2071.42%28.57%0%28.57%(2/7)(0/2)(2/7)377.77%22.22%0%NA0(0/9)4100%NANANANA

Discussion/Conclusions

In Group 3, receiving the lowest LTX-315 dose (0.25 mg/dose), a small inhibitory effect is observed during the first days. In Group 1 and Group 2, receiving LTX-315 doses of 1.0 mg/dose and 0.5 mg/dose respectively, all animals showed partial or complete tumour response. It was found that the anti-tumour activity resulted in a complete tumour response in 3 of 7 treated mice for the Group receiving the optimal dose (Group 1).

Generally stronger necrosis and more wound formation were observed in Group 1 compared to the other two groups. Except from the wound formation no other adverse events or toxic effects were observed in either of the groups of animals.

Both 1 mg and 0.5 mg of LTX-315 demonstrated a strong and rapid anti tumour effect in the first period of the study. However, as the study progresses more animals in Group 2 relapses than in Group 1.

Example 5

The Effect of LTX-315 on Murine CT26WT Colon Carcinoma Tumours in Mice

Materials and Methods

The administration takes place by intra-tumoural injection of LTX-315 dissolved in sterile saline (0.9% NaCl in sterile water).

Each of a total of 40 female mice was inoculated with five million murine CT26WT cells (ATCC, LGC Promochem AB, Boras, Sweden) subcutaneously on the abdomen surface in a volume of 50 μl. The mice were divided into five groups, 8 mice in each group. When the tumours reached the desired size of 20 mm2the treatment by intra tumoural injection was initiated. Group one was treated solely on day 1, Group two on day 1 and 2, Group three on day 1 and 3 and Group four on day 1, 2 and 3. All daily treatments were one single injection of 1.0 mg LTX-315 dissolved in 50 μl (20 mg/ml). Group five was treated with the 50 μl of vehicle for LTX-315 (Group 5).

The mice were monitored during the study by measuring the tumours (digital calliper) and weighing the animals regularly. The mice were followed until the maximum tumour burden of 125 mm2was reached, or until serious adverse events occurred (i.e. wound formation due to repeated injections), then the mice were sacrificed. Weighing and physical examination were used as health controls.

Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old, supplied form Harlan (England, UK)

Conditioning of animals: Standard animal facility conditions. Mean body weight, dose, route and treatment schedule is given in Table 11 below.

TABLE 11Num-Initial bodyber ofweight (g;Treat-ScheduleGroupanimalsmean ± SE)mentDoseRoute(Day*)1819.00 ± 1.087Once1 mg inIntra1daily50 μl (20tumourmg/ml)2819.56 ± 1.087Once1 mg inIntra1, 2daily50 μl (20tumourmg/ml)3819.41 ± 0.8999Once1 mg inIntra1, 3daily50 μl (20tumourmg/ml)4819.00 ± 0.9396Once1 mg inIntra1, 2, 3daily50 μl (20tumourmg/ml)5818.71 ± 0.7868Once50 μlIntra1, 2, 3(con-daily0.9%tumourtrol)NaCl inH20*Day 1 is first day of treatment
Results

The anti-tumour effect of the various treatments is presented as mean tumour size in Table 12 below.

TABLE 12MeanMeanMeanMeantumourtumourtumourtumoursize (mm2)size (mm2)size (mm2)size (mm2)Treatmentat day 1*on day 6on day 10on day 17Group 122.69 ± 0.40704.343 ± 2.2957.171 ± 4.0353.712 ± 3.712Group 222.90 ± 1.1551.458 ± 1.4585.058 ± 4.0146.644 ± 3.430Group 321.43 ± 1.1412.983 ± 2.98310.85 ± 7.5530.00 ± 0.00Group 424.09 ± 1.6530.00 ± 0.000.00 ± 0.001.308 ± 1.308Group 521.39 ± 1.68333.77 ± 3.16848.37 ± 7.03540.64 ± 19.77*Tumour size prior to start of treatment at first day of treatment

Complete tumour response was observed in the vast majority of all animals treated with LTX-315. The degree of tumour response in the different treatment groups is summarised in Table 13 below.

TABLE 13Free ofTumour ResponseRelapseTumour atAnimalnopartialcompleteofend ofGroupresponseresponseresponseTumourFollow-Up1027.5%62.5%20%50%(1/5)(4/8)2012.5%87.5%71%25%(5/7)(2/8)312.5%087.5%29%62.5%(2/7)(5/8)400100%37.5%62.5%(8/8)(5/8)5100%NANANANA(8/8)

Discussion/Conclusions

The treatment was started when the tumours had reached the desired size of a minimum of 20 mm2and animals were sacrificed when the tumours reached the maximum tumour burden of 125 mm2.

End of study was defined as day 17 when six out of eight control animals (Group 5) were sacrificed.

All LTX-315 treatment regimes resulted in a strong anti CT26WT-tumour effect.

Totally 27 of the 32 treated animals were observed with a complete tumour response and four with a partial response. Only one animal (in Group 3) did not have a response to the treatment. The results presented show that all four treated groups have very similar overall tumour response, the data also indicate that the degree of relapse of tumour was higher in Group 2 than in Group 1, 3 and 4. In addition fewer animals were observed to be free of tumour at end of follow-up in Group 2 (FIG.2).

Necrosis and complete tumour response was observed in all the treated groups. In Group 1 four out of eight animals, in Group 2 two out of eight animals, in Group 3 five out of eight animals, and in Group 4 five out of eight animals showed complete tumour response. At this stage the tumour was completely necrotic and a wound crust formed at the location of the tumour.

Necrosis at the tumour site was seen in all treatment groups. Generally, animals in Group 2, 3 and 4 showed more necrosis, wound and crust formation than the animals in Group 1 that were given only one injection of LTX-315. Group 4 animals, which were given three injections, showed the most necrosis, wound and crust formation. The difference in necrosis between Group 1 and Group 4 was quite large but the animals given the highest number of treatments seemed to cope well. No toxic or other adverse effects besides local necrotic tissue and wound formation were observed in either of the treated groups of animals.

All four treatment regimes of LTX-315 tested demonstrated a strong anti tumour effect against murine CT26WT tumours.

The amount of necrosis, wound and crust formation was proportional to the number of LTX-315 treatments given.

Example 6

LTX-315 Activity Against Sensitive and Multidrug-Resistant Cancer Cells and Normal Human Cells

Characteristics of the cell lines tested are presented in Table 14 below.

TABLE 14DrugIC50Cell linesusceptibilityOriginμMHL-60SensitiveAcute promyelocytic leukemia2.07HL-60/ADRResistantAcute promyelocytic leukemia3.01MCF-7SensitiveBreast carcinoma1.94MCF-7/mdrResistantBreast carcinoma1.96IGROV-1SensitiveOvary carcinoma6.37IGROV-ResistantOvary carcinoma3.191/CDDPK-562SensitiveChronic myeloid leukemia3.27K5627/ResistantChronic myeloid leukemia2.98GleevecHUV-EC-C—Normal endothelial cells23RBC—Red blood cells833

The above data shows the broad spectrum of activity of LTX-315 against various cancer cell lines and, significantly, a much weaker cytotoxic effect on normal human cells.

Example 7

Re-challenge with murine A20 B-cell lymphoma and murine CT26WT colon carcinoma cells in mice with complete tumour regression.

This study sought to investigate the effects of tumour growth in animals that had previously shown complete tumour regression following treatment with LTX-315.

Methods: Female Balb-c mice (n=4), previously treated with LTX-315, 1 mg) or (n=9); previously treated with LTX-315 0.5 or 1 mg) were re-inoculated (s.c. in the abdominal area) with either murine A20 B cell lymphoma cells or CT26WT colon carcinoma cells (5 million) respectively 6 weeks following initial treatment with LTX-315. Tumour growth was monitored for up to 36 days following re-inoculation.

Significant inhibition (P<0.006) of tumour growth was observed in all 4 mice treated previously with LTX-315 (1 mg) in study R315-03 compared with control animals (FIG.2) and while relapse was seen in 1 animal, 3 weeks later, complete tumour regression was observed in the other 3 mice (FIG.3).

In 9 mice previously treated with LTX-315 (0.5 or 1 mg) inhibition (P<0.01) of tumour growth was observed in comparison with control animals (FIG.3). The sudden drop in tumour size inFIG.20, after Day 18, is explained by the death of 6 animals bearing large tumours. Inhibition was observed in 7 mice and complete regression in 2 of the animals (FIG.5).

Taken together these data suggest that complete tumour regression following initial treatment of solid murine tumours (murine A20 B cell lymphoma or CT26WT colon carcinoma) with LTX-315 resulted in a form of endogenous long-term protection against growth of the same tumours following re-inoculation. Inhibition of tumour growth was more pronounced in animals bearing A20 B cell lymphoma tumours when compared with animals bearing CT26WT colon tumours.

Example 8

Immunological effects of LTX-315 in a murine A20 B-cell lymphoma model. An in vivo adoptive spleen cell transfer pilot study.

This study was undertaken to investigate whether the long-term protection against growth of the same tumours following re-inoculation in animals observed in study R315-33 could be passively transferred to naive recipients via spleen cells taken from LTX-315-treated donor animals.

Ten female Balb/c mice (n=32) were each inoculated with A20 cells (5 million in 50 μL s.c.) on the abdominal surface. Once tumours had reached 20 mm2they were injected with LTX-315 (1 mg) injected intratumourally, once daily for 3 days, in a volume of 50 μL. Tumour size (mm2) and body weight were subsequently monitored and a further injection of LTX-315 was given if any tumour re-growth was observed. Subsequently, mice showing complete tumour regression were sacrificed and used as donors for transfer of splenocytes while naive donor mice were used as controls. Spleens from donor mice were excised and cells isolated. Naive receiver mice were irradiated and divided into 2 groups. Group 1 received isolated splenocytes from cured mice, whereas group 2 received isolated splenocytes from naive mice. Freshly prepared cells were injected (20×106 per 100 μl) via the tail vein. Twenty four hours later receiver mice were inoculated with 5 million murine A20 B-cell lymphoma cells on the abdominal surface as described above. Tumour size and body weight were monitored until the maximum tumour burden of ˜125 mm2was reached, or a serious adverse events occurred (i.e. wound formation due to tumour tissue necrosis) at which point mice were sacrificed.

Inhibition of tumour growth was observed in irradiated mice that received splenocytes isolated from animals that had shown complete tumour regression following treatment with LTX-315 when compared with control animals that received splenocytes from naive donors (FIG.6). It was also noted that there was a difference in the colour and texture of the tumours in recipients of splenocytes from LTX-315-treated mice suggesting an immediate inflammatory response.

Based on these observations, the data provides evidence for an adaptive immune response in the animals that received splenocytes from animals that previously showed complete regression of A20-B lymphoma tumours following treatment with LTX-315. This data suggests that treatment with LTX-315 may confer long term protection against specific tumours by eliciting an immune response.

Example 9

The objective of the study was to investigate the anti-cancer effect of prophylactic vaccination with A20 lymphoma cells lysed by 10 mg/ml LTX-315:(i) alone; and(ii) in combination with 20 mg/ml LTX-315 injected at the vaccination site prior to the vaccine.

In total, two different treatment regimens were used.

Administration was by subcutaneous injection of LTX-315 dissolved in growth media containing A20 lymphoma cells. The cell-LTX-315 “cocktail” was left for 30 min prior to injection in order to assure complete lysis of the cancer cells.

Group 1 (“vaccine”) mice were injected subcutaneously on the abdomen surface with 50 μl of a “cocktail” of ten million murine A20 cells (ATCC, LGC Promochem AB, Boras, Sweden) and 10 mg/ml LTX-315 (“A20 lysate”). Group 2 (“vaccine+adjuvant”) mice were treated as per Group 1, but in addition were given 25 μl of 20 mg/ml LTX-315 subcutaneously at the site of vaccination 5 minutes prior to the A20 lysate injection. Group 3 (“control”) mice received no treatment.

Six weeks after the treatment, all mice were inoculated with 5 million viable A20 B-cell lymphoma cells subcutaneously on the abdomen surface in a volume of 50 μl.

The mice were monitored during the study by measuring the tumour size and weighing the animals regularly. The mice were followed until the maximum tumour burden of ˜130 mm2was reached, at which point the mice were sacrificed.

Materials and Methods

Animals: Specific pathogen-free female Balb/c mice, 6-8 weeks old, supplied from Harlan Laboratories (England, UK; www.harlan.com)

Conditioning of animals: Standard animal facility conditions at the University of Tromsø.

Test substance: Murine A20 cells lysed by LTX-315 (Lot 1013687), and LTX-315 (Lot 1013687) alone

Test substance preparation: 10×106A20 cells were added to a 50 μl 10 mg/ml LTX-315/vehicle (“A20 lysate”). The test substance was ready for use 30 minutes after mixing. LTX-315 alone was dissolved in 0.9% NaCl in sterile H2O

Vehicle: RPMI-1640 w/2 mM L-glutamine or 0.9% NaCl in sterile H2O

Reference substances: Not applicable

Treatment of controls: Not applicable

Method of evaluation: Tumour size measurements and health control by weighing and examination

Additional data regarding method: A digital calliper was used for tumour size measurements and weighing and physical examination were used as health control

Mean body weight, dose, route and treatment schedule are shown in Table 15 (below).

TABLE 15Initial bodyNo ofweight (g;Treat-Cell numbersGroupanimalsmean ± SE)mentand doseRoute1817.31 ±Once10 × 106A20Sub-0.3815cells incutaneous50 μl LTX-315(10 mg/ml)2817.14 ±Once0.25 μl LTX-315Sub-0.463320 mg/ml) +cutaneous10 × 106A20cells in 50 μlLTX-315(10 mg/ml)3717.29 ±NotNot applicableNot0.3020treatedapplicable
Results:

The anti cancer effect of the various treatments is presented as mean tumour size in Table 16 below and a graphical presentation of the data is provided inFIG.7. In Table 16, Day 1 was the day of inoculation of viable A20 cells six weeks post-vaccination.

TABLE 16Mean tumourMean tumourMean tumourMean tumoursize (mm2)size (mm2)size (mm2)size (mm2)Treatmentat day 4on day 11on day 16on day 21Group 19.515 ± 1.52820.44 ± 6.19136.21 ± 10.3055.89 ± 15.27Group 27.315 ± 2.23117.13 ± 5.07829.13 ± 7.90347.16 ± 13.54Group 310.25 ± 3.10034.49 ± 8.29856.04 ± 8.33982.89 ± 14.06

Discussion/Conclusions

The inoculation of viable A20 B-cell lymphoma cells was accomplished 6 weeks after the treatment was given (day 1) and the animals were sacrificed when the tumours reached the maximum allowed tumour burden of ˜130 mm2.

The results show that the tumours developed more slowly in both LTX-315/A20-lysate treatment Groups as compared to the control Group. The median survival of Group 1 was 28 days, 33 days for Group 2, and 25 days for the control group (Group 3). Increase in median survival was 12% for Group 1 and 35% for Group 2 as compared to the control group (Group 3).

The data indicate a prolonged survival of the treated groups compared to the untreated control group. On day 34, when the last animal in the control group was sacrificed, 50% of the animals in Group 2 were still alive while 37.5% of the animals in Group 1 were still alive. End of study was defined as day 60. At this time-point, a total of 3 of the 16 treated animals had a complete regression of an initially developing tumour and were tumour free. At the end of the study 25% of animals from Group 1, and 12.5% of animals from Group 2 were observed to be tumour free.

Macroscopically there were morphological differences between the treated groups (Group 1 and 2) compared to the non-treated control group (Group 3). The developing tumours in the two treatment groups were observed to be whiter and harder than the tumours observed in the control group. This finding together with the slower growth rate of the tumours indicates that an anti-A20 cell immune response was induced by the vaccination with the cocktail of LTX-315 and lysed A20 cells.

Hence, LTX-315 may have a dual use by lysing the tumour cells and inducing release of danger signals from normal cells at the injection site.

Example 10

In this study, we investigated the tumoricidal effect of LTX-315 on human melanoma cells. The peptide internalized and was shown in association with mitochondria, ultimately leading to a lytic cell death. The LTX-315 peptide was designed to treat solid tumors with intratumoral injections through a two-stage mode of action: the first is the collapse of the tumor itself, while the second is the released damage-associated molecular pattern molecules (DAMPs) from the dying tumor cell, which can induce a subsequent immune protection against recurrences and metastastis.

Material and Methods

Reagents

LTX-315 and LTX-328 (K-A-Q-Dip-Q-K-Q-A-W-NH2) were made on request by Bachem AG (Bubendorf, Switzerland) and Innovagen (Lund, Sweden), respectively. LTX-315 Pacific Blue and LTX-328 Pacific Blue were purchased on request from Innovagen (Lund, Sweden) Norud (Tromsø, Norway), respectively.

Cell Culture

The A375 cell line A375 (ECACC, 88113005) is a human malignant melanoma derived from patient material, and was purchased from Public Health England (PHE Culture Collections, Porton Down, Salisbury, UK). Cells were maintained as monolayer cultures in high glucose 4.5% DMEM supplemented with 10% FBS and 1% L-glutamine, but not as antibiotics (complete media). The cell line was grown in a humidified 5% CO2atmosphere at 37° C., and was regularly tested for the presence ofmycoplasmawith MycoAlert (Lonza).

In Vitro Cytotoxicity, MTT-Assay

The cytotoxic effect of LTX-315 was investigated using the colorimetric MTT viability assay as described in Eliassen et al. (2002), 22(5): pp 2703-10. The A375 cells were seeded at a concentration of 1×105cells/ml in a volume of 0.1 ml in 96-well plates, and allowed to adhere in a complete growth media overnight. The media was then removed and the cells were washed twice in serum-free, RPMI-1650 media, before adding LTX-315 dissolved in serum-free RPMI at concentrations ranging from 2.5-300 μg/ml, and incubated for 5-180 minutes. Cells treated with a serum-free RPMI were used as negative control cells, while cells treated with 1% Triton X-100 in serum-free media were used as a positive control. The final results were calculated using the mean of three experiments, each with triplicate wells.

Confocal Microscopy

Live cell imaging with unlabeled cells-A375 cells were seeded at 10,000 cells/well in a complete media in Nunc Lab-Tec 8-wells chambered covered glass (Sigma) precoated with 25 μg/ml human fibronectin (Sigma) that were allowed to adhere overnight. Cells were washed twice with a serum-free RPMI, treated with peptide dissolved in RPMI and investigated using Bright on a Leica TCS SP5 confocal microscope, with a 63X/1.2W objective. The microscope was equipped with an incubation chamber with CO2and temperature control.

Fixed cells, mitotracker—Cells were seeded as for live cell imaging, and treated with Mitotracker CMH2XROS (Invitrogen) at 100 nm for 15 minutes prior to peptide treatment. Cells were treated with 17 μM LTX-315, with negative control serum-free RPMI only. After 60 min of incubation, cells were analyzed using a Zeiss microscope.

All confocal imaging experiments were subsequently conducted at least twice with similar results.

Fixed cells, fluorescence-labeled peptide-Subconfluential A375 cells were seeded at 8,000 cells/well as above, and transfected on the second day using the Lipofectamine LTX with Plus transfection reagents (Invitrogen) following the manufacturer's protocol. The mitochondria were labeled using the pDsRed2-Mito, and the nucleus was labeled using the GFP-Histon2B plasmid (Imaging Platform, University of Tromsø). A day after transfection, cells were washed twice with serum-free RPMI, and treated at different concentration and incubation periods with LTX-315 Pacific Blue or LTX-328 Pacific Blue. LTX-315 PB exhibited a similar cytotoxic profile as the unlabeled LTX-315 as determined by MTT assay.

Control cells were treated with unlabeled LTX-315 and also with serum-free RPMI only. After incubation, cells were fixed with 4% paraformaldehyde in PBS, and the wells were covered with Prolong Gold antifade (Invitrogen). Cells were further analyzed by use of a Leica TCS SP5 confocal microscope, with a 693, 1.2 W objective. Pacific Blue, GFP and Ds Red were excited using UV, with 488 and 561 lasers, and fluorescence channels were sequentially detected using the following band passes: UV: 420-480 nm (with attenuation), 488: 501-550 nm and 561: 576-676 nm.

TEM Electron Microscopy

A375 cells were seeded at 1×105cells per well in 6-well plates and allowed to grow for three days to optimize membrane structures in the culture, and the media was changed on the second day. Cells were washed twice in serum-free RPMI before being treated with LTX-315 dissolved in serum-free RPMI at 5, 10 and 25 μg/ml, with serum-free RPMI as a negative control. Cells were then washed with PBS twice before fixation for 24 hours in 4° C. with 4% formaldehyde and 1% gluteralaldehyde in a Hepes buffer at pH 7.8. Dehydration and post-fixation protocols included incubation in a 5% buffered tannic acid and incubation in a 1% osmium-reduced ferrocyanide. Ultrathin sections were prepared, and uranyl acetate (5%) and Reynolds's lead citrate were used for staining and contrasting. Samples were examined on a JEOL JEM-1010 transmission electron microscope, and images were taken with an Olympus Morada side-mounted TEM CCD camera (Olympus soft imaging solutions, GmbH, Germany).

Fluorescence Measurement of Reactive Oxygen Species (ROS)

A DCFDA cellular reactive oxygen species detection assay kit was purchased from Abcam®, and A375 cells seeded in a 96-well Costar black clear bottom plate with 20,000 cells per well incubated in 37° C. 16 hours prior to DCFDA assay. Cells were washed with a 100 μL/well of pre-warmed PBS one time, and incubated with 20 μM of DCFDA in a buffer solution supplied with the kit at 37° C. in a cell culture incubator for 45 min, and then washed again with a buffer solution of 100 μL/well. The cells were then stimulated with a 100 μL/well LTX-315 peptide dissolved in a buffer solution at concentrations of 17 μM for 30 min, and cells not treated were used as a negative control. The fluorescence intensity was determined at an excitation wavelength of 485 nm and an emission wavelength of 530 nm on a FLUOstar Galaxy plate reader.

Release of High Mobility-Group Box-1 (HMGB1)

A375 cells were seeded with 3×105cells/well in 6-well plates in a complete media, and allowed to adhere overnight. Cells were treated with LTX-315 or LTX-328 at 35 μM, and incubated at 37° C. and 5% CO2for different time points (5, 10, 15, 30, 60 min), and negative controls were serum-free RPMI-1650. Supernatants (S) were collected and centrifuged at 1,400 g for five minutes, and cell lysates (L) were harvested after washing with PBS twice and then subsequently lysed using a 4× Sample buffer (Invitrogen, number), 0.1 M DTT (Sigma number) and water. Supernatants were concentrated using Amicon Ultra 50K centrifugal filters (Millipore UFC505024), and the cell lysate was sonicated. Both supernatants and lysate were boiled and resolved in a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), and then electro transferred to a polyvindiline difluoride (PVDF) membrane (Millipore). The membrane was blocked in 5% milk and incubated with the HMGB1 antibody (rabbit, polyclonal, abcam ab 18256); the membrane was then rinsed several times with TBST, incubated with a horseradish peroxidase (HRP)-conjugated secondary antibody (abcam ab6721), rinsed again with TBST and then developed using WB Luminol Reagent (Santa Cruz Biotechnology, Heidelberg, Germany).

Release of Cytochrome-C

A375 cells were seeded as with HMGB1 studies, and treated with 35 μM for different time points (5, 15, 45). Supernatants were collected and concentrated as with HMGB1 studies, and samples from the supernatants were analyzed using a 4.5 hour solid form Cytochrome C-Elisa kit (R&D Systems, USA, #DCTC0) following the manufacturer's description. Shortly thereafter, a 50% diluted sample was analyzed and the optical density was determined using a microplate reader set at 450 nm, and this reading was then subtracted from the reading at 540 nm. A standard curve was generated for each set of samples assayed. Samples were run in four parallels, and the cytochrome-c released into the supernatant was expressed as a fold over the level of cytochrome-c in the supernatant of untreated cells.

Release of ATP

The supernatant of LTX-315-treated A375 cells was analyzed using an Enliten ATP luciferase assay kit (Promega, USA). Cells were then seeded as with an ROS assay, and treated with LTX-315 in different incubation times, from 1 to 15 minutes with two parallels, which was then conducted three times. Negative controls were untreated A375 cells exposed to serum-free media alone. Samples were diluted at 1:50 and 1:100, and analyzed with a Luminoscan RT luminometer according to the manufacturer's protocol.

Statistical Analysis

All data represent at least two independent experiments with at least two parallels, which were expressed as the mean±SD. Cytochrome-C release and ATP release data was compared using one-way ANOVA and a multiple comparison test, and we considered the P-value<0.05 to indicate statistical significance.

Results

Cytotoxic Effect of LTX-315 on Melanoma Cells

To investigate the effect of LTX-315 on A735 melanoma cells in vitro, we determined the IC-50 values for the peptide by a cell viability assay MTT at different incubation times. The IC-50 value was 30 μM after only five minutes of incubation, and progressed to 14 μM after 90 minutes. Further incubation up to 180 minutes did not offer any additional effect (FIG.8).

LTX-315 Treatment Causes Rapid Cell Lysis

We next wanted to assess the cell morphology of A735 melanoma cells treated with LTX-315. Cells were treated with LTX-315 at an IC-50 value, and investigated by bright field confocal microscopy. Treated cells displayed a rapid change from a normal epithelial morphology to a total collapse of the cells with an extrusion of cytoplasmic content, which was preceded by a rounding up of the cell (data not shown). These changes typically occurred within 15-60 minutes at an IC-50 value in the majority of the cells.

LTX-315 Internalizes and Targets the Mitochondria

To investigate the internalization and fate of the peptide within the cells, LTX-315 was labeled with Pacific Blue and incubated with cells at concentrations of 3 μM and 1.5 μM, respectively. The labeled LTX-315 rapidly penetrated the plasma membrane and at 1.5 μM, the peptide showed an accumulation around the mitochondria after 30 minutes of incubation but was not detected in the cell nucleus (FIG.9). The labeled non-lytic mock-sequence peptide LTX-328 did not demonstrate any internalization at any concentration or incubation time tested (FIG.10).

LTX-315 Induces Ultra-Structural Changes in Cells

We further evaluated the ultrastructural changes in treated cells by performing transmission electron microscopy (TEM), in which A375 cells were treated with either peptides dissolved directly in media or in media alone. A significant number of the cells treated with a low concentration (3.5 μM) of the LTX-315 peptide for 60 minutes showed vacuolization, as well as some altering of the mitochondrial morphology (FIG.11). The mitochondria appeared to be less electron-dense, also exhibiting some degree of reorganization, with the cristae lying further apart or not visible at all. The number of necrotic cells in these samples was less than 5%. In these low concentrations, vacuolization of the cytoplasm was observed. Another common finding in these samples were peripherally placed vacuoles, which were lined with a single membrane layer containing a homogenous material (FIG.11B). When cells were treated with higher concentration (17 μM) for 60 min, approximately 40% of them displayed a necrotic morphology with a loss of plasma membrane integrity (FIG.11C&E). The cells that were still intact displayed a great heterogeneity, from a normal appearance with microvilli to a round appearance, with mitochondria clearly affected. In this high concentration, only 4% of the cells investigated displayed vacuolization, and chromatin condensation was not visible in this material at any peptide concentration tested. These results demonstrate that LTX-315 kills the tumor cells with a lytic mode of action, while lower concentrations cause the cells to undergo ultrastructure changes, such as vacuolization and an altered mitochondrial morphology. Moreover, no significant morphological changes suggestive of apoptotic cell death were observed.

In a separate experiment, exposure of LTX-315 at 10 μg/ml to human A547 cells (an ovarian melanoma cell line) led to disintegration of the mitochondrial membrane (FIG.16).

LTX-315 Treatment Leads to Extracellular ATP Release

DAMPs are molecules that are released from intracellular sources during cellular damage. DAMPs can initiate and perpetuate an immune response through binding to Pattern Recognition Receptors (PRRs) on Antigen Presenting Cells (APCs). Among commonly known DAMPs are ATP, HMGB1, Calreticulin, Cytochrome C, mitochondrial DNA and Reactive oxygen species (ROS). We next wanted to investigate whether ATP was released into the supernatant from cells treated with LTX-315. Hence, the supernatant from treated and non-treated cells analyzed using luciferase detection assay. As shown inFIG.15, ATP was detected in the supernatant as early as after 5 minutes of treatment with LTX-315, and the release was concentration-dependent.

LTX-315 Treatment Induces Cytochrome-C Release in Supernatant

To assess whether LTX-315-treated cells released cytochrome-C into the medium, A375 cells were treated with LTX-315 at 35 μM at different time points (5, 15, 45 min). The supernatant was subsequently analyzed using an ELISA assay. Cells treated with 35 μM value had three times more cytochrome-C in the supernatant compared to untreated control cells. The increase in cytochrome-C was detected after only five minutes of treatment, and there was also an increase after 15 and 45 minutes of peptide treatment, respectively (FIG.13).

LTX-315 Treatment Leads to Extracellular HMGB1 Release

HMGB 1 is a non-histone, chromatin-binding nuclear protein. Once passively released from necrotic cells, HMGB1 is able to trigger the functional maturation of dendritic cells, cytokine stimulation and chemotaxis among several immunopotentiating effects.

HMGB 1 is normally found in the cell nucleus and would be expected in a cell lysate of healthy cells, though not in the culture media (supernatant). In order to assess the release of HMGB 1 from LTX-315-treated cells, we measured the translocation and free HMGB1 from the nuclear compartment within the cell lysate into the cell supernatant.

Both cell lysate and the cell supernatant of LTX-315- and LTX-328-treated A375 melanoma cells were analyzed using a Western blot. Cells were treated with 35 μM of either LTX-315 or LTX-328, with a gradual translocation from the cell lysate to the supernatant detected in the LTX-315-treated melanoma cells, but not in the cells treated with the mock sequence peptide LTX-328 or a serum-free medium only (FIG.14).

LTX-315 Treatment Causes the Production of Reactive Oxygen Species (ROS) in A375 Melanoma Cells

The ROS generation following LTX-315 treatment was measured by CH2DCFDA fluorometric assay. Significant amounts of ROS were generated after 15 minutes of incubation with LTX-315, and the ROS levels were concentration-dependent (FIG.12).

Discussion

LTX-315 labeled with the fluorescent molecule Pacific Blue was internalized within minutes after incubation with A375 melanoma cells, and was distributed in the cytoplasm (FIG.9). At low concentrations, accumulation of the peptide around the mitochondria was evident, whereas at higher concentrations the peptide was more spread within the cytoplasm and accumulated in circular structures closer to the cell membrane (FIG.10). If the peptide attacks the mitochondrial membrane, a decrease or even a total collapse of the mitochondrial membrane potential would be expected. A confocal imaging of cells with the membrane potential-dependent mitochondrial stain Mitotracker CMXh2ROS showed a loss of mitochondrial signal a short time after peptide treatment (data not shown). The loss of the signal shows that the peptide interaction with the mitochondria causes a loss of mitochondrial membrane potential, which is crucial for the mitochondria's most important cellular functions. An altered mitochondrial morphology was also demonstrated with TEM. Cells treated with LTX-315 for 60 minutes had less electron-dense mitochondria with an altered organization of the cristae, as well as vacuolization within the mitochondria compared to untreated cells (FIG.11). Furthermore, vacuolization was evident in approximately 20% of cells treated with 3.5 μM of LTX-315. When the mitochondria are dysfunctional, free oxygen radicals (ROS) may be formed, and by using fluorometric assays we demonstrated ROS formation within a few minutes after peptide treatment (FIG.12).

In this study, we demonstrate that treatment with the LTX-315 peptide causes an increase in ROS levels in A375 melanoma cells after treatment. One explanation for these higher levels of ROS following peptide treatment could be that the peptide enters the cells and targets the mitochondria, and the dysfunctional mitochondria then releases ROS. Through an ELISA assay, we detected the release of cytochrome-C in the supernatant of peptide-treated cells after only a few minutes of treatment (FIG.13). Cytochrome-C is a mitochondrial protein released from the intermembrane space and into the cytosol when the outer mitochondrial membrane is perturbed, and by binding to the apoptotic protease activating factor-1 (Apaf-1) it is also a part of the apoptotic cascade that eventually leads to cell death by apoptosis. However, if cytochrome-C is found in the extracellular space, it has been reported to act as a pro-inflammatory mediator, thus activating NF-kB and inducing cytokine and chemokine production. The transition of HMGB1 from the cellular compartment to the extracellular compartment was detected using a western blot (FIG.14). When the nuclear protein HMBG1 is released into the extracellular fluid, it functions as a DAMP, and can bind to both the PRR TLRs and to the RAGE receptors; the activation of these may lead to a number of inflammatory responses such as the transcription of pro-inflammatory cytokines. We also detected ATP released in the supernatant after peptide incubation (FIG.15), and presented extracellularly it functions as a DAMP by activating the purinerg P2RX7 receptors on the DC. This receptor not only functions as a pore that opens for small cationic and later bigger molecules after binding to ATP, its activation also causes the processing and release of the pro-inflammatory cytokine IL-1β.

In summary, our data suggests that LTX-315 induces lytic cell death in cancer cells, not only by direct attack on the plasma membrane, but also as a result of an injury to vital intracellular organelles after the internalization of the peptide at concentrations too low to cause an immediate loss of plasma membrane integrity. We demonstrate that the peptide treatment causes the release of several DAMPs such as CytC, ATP, HMGB1 and ROS. The DAMPs may affect the cellular integrity of the damaged cells in several ways, but are also associated with so-called immunogenic cell death. The release of tumor-specific antigens into the extracellular compartment, together with potent immune stimulatory molecules (DAMPs) such as ATP, CytC and HMGB1, can give a strong immune response. In turn, these factors will lead to a maturation and activation of DCs and other accessory cells of the adaptive immune system.

Example 11

The combination of LTX-315 with either an anti-programmed cell death protein 1 (PD-1) antibody or an anti-cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) antibody was assayed in a mouse MCA205 sarcoma model. Seven animals were studied per group. For each study, two independent experiments were performed yielding identical results. Anova statistical analyses were performed.

The anti-PD-1 antibody used in this study was a murine IgG isotype. The anti-CTLA-4 antibody was a rat IgG isotype. Both antibodies were purchased from eBioscience.

FIGS.17a,18a,19aand20ashow the timings and administration routes of LTX-315 and the antibodies in the various studies. Thus, at day-8 mice were inoculated with MCA205 cancer cells and palpable tumours were allowed to form. LTX-315 was then administered intratumorally on the days shown in the Figures and the anti-PD-1 or anti-CTLA-4 antibodies were administered i.p. on the days shown.

An adaptive immunity study using the combination of anti-CTLA-4 antibody and LTX-315 was carried out in a similar manner to that previously discussed.

The findings of these studies are discussed in the legend ofFIGS.17to20. As the effects of anti-PD1 antibodies occur in the local tumour microenvironment (anti-CTLA-4 works in lymphatic tissue), it is predicted that the combination of the peptidic compound with anti-PD1 antibodies would be more pronounced in a clinical setting in comparison to this single tumour model which is not representative of metastatic disease.

Example 12

The combination of LTX-315 with an anti-programmed cell death protein 1 ligand (PD-L1) antibody was assayed in a mouse EMT-6 mammary carcinoma model.

Materials and Methods

The anti-PD-L1 antibody had the following characteristics: ref: BE0101, Bioxcell; clone 10F.9G2; reactivity: mouse; isotype: Rat IgG2b. LTX-315 was prepared at a dose of 0.5 mg/50 μL in 0.9% sodium chloride solution. Anti-PD-L1 antibody was prepared at a concentration in phosphate-buffered saline and was administered at a dose of 10 mg/kg. LTX-315 was injected into the tumour grafted on the right flank of the mice, anti-PD-L1 antibody was injected into the peritoneal cavity of the mice.

The EMT-6 cell line was established from a transplantable murine mammary carcinoma that arose in a BALB/cCRGL mouse after implantation of a hyperplastic mammary alveolar nodule (VOLENEC F J., et al., J Surg Oncol. 13(1):39-44, 1980).

Cell culture conditions: EMT-6 tumor cells were grown as a monolayer at 37° C. in a humidified atmosphere (5% CO2, 95% air). The culture medium was RPMI 1640 containing 2 mM L-glutamine (ref: BE12-702F, Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (ref: 3302, Lonza). EMT-6 tumor cells are adherent to plastic flasks. For experimental use, tumor cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (ref: BE17-161E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium. The cells were counted in a hemocytometer and their viability were assessed by 0.25% trypan blue exclusion assay.

Use of mice: Healthy female Balb/C mice, 6-8 weeks old at reception, were obtained from CHARLES RIVER (L'Arbresles, France) and from Janvier (France). Mice were maintained in SPF health status according to the FELASA guidelines. Mouse housing and experimental procedures were realized according to the French and European Regulations (Principe d′éthique de l′expérimentation animale, Directive no 2010/63 CEE du 22 September 2010, Décret no 2013-118 du 01 février 2013) and the NRC Guide for the Care and Use of Laboratory Animals (NRC Guide for the Care and Use of Laboratory Animals). The animal facility was authorized by the French authorities (Agreement No B 21 231 011 EA). All procedures using mice were submitted to the Animal Care and Use Committee of Oncodesign (Oncomet) agreed by French authorities (CNREEA agreement No 91). Mice were individually identified with a RFID transponder and each cage was labeled with a specific code.

Housing conditions: Mice were maintained in housing rooms under controlled environmental conditions: Temperature: 22±2° C. Humidity 55±10%, Photoperiod (12 h light/12 h dark), HEPA filtered air, 15 air exchanges per hour with no recirculation. Mice enclosures provided sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: Top filter polycarbonate Eurostandard Type III or IV cages, Corn cob bedding (ref: LAB COB 12, SERLAB, France), 25 kGy Irradiated diet (Ssniff® Soest, Germany), Complete food for immunecompetent rodents—R/M-H Extrudate, Sterile, filtrated at 0.2 μm water, and Environmental enrichment (SIZZLE-dri kraft-D20004 SERLAB, France).

Induction of MET-6 tumours in mice: A first tumor was induced by subcutaneous injection of 1×106of EMT-6 cells in 200 μL of RPMI 1640 into the right flank of Balb/C female mice. The day of tumor cell injection in the right flank was considered as DO. A second tumor was induced by subcutaneous injection of 1×105of EMT-6 cells in 200 μL of RPMI 1640 into the left flank of Balb/C female mice. The day of tumor cell injection in the left flank was considered as D3.

Treatment schedule: Mice were randomized according to their body weight on D3 into four groups each of 5 mice (group 1) or 10 mice (groups 3, 5 and 7) using Vivo Manager® software (Biosystemes, Couternon, France). A statistical test (analysis of variance) was performed to test for homogeneity between groups. A statistical test (analysis of variance) was performed to test for homogeneity between groups.

The treatment schedule was as follows: the mice from group 1 were not treated; the mice from group 3 received a total of 3 intratumoral injections of LTX135; the mice from group 5 received a total of 6 IP injections of anti-PD-L1; and the mice from group 7 received a total of 3 intratumoral injections of LTX315 and a total of 6 IP injections of anti-PD-L1.

The treatment schedule is summarized in Table 17 below:

TABLE 17No.TreatmentGroupMiceTreatmentScheduleG15Untreated—G310LTX315Q1Dx3G510Anti-PD-L1Q2Dx6G710LTX315Q1Dx3Anti-PD-L1Q2Dx6Q1Dx3 = one injection for 3 consecutive days (total of 3 injections).Q2DX6 = one injection every 2 days (total of 6 injections).

Mouse monitoring: All study data, including mouse body weight measurements, tumour volume, clinical and mortality records, and treatment were scheduled and recorded on Vivo Manager® database (Biosystemes, Dijon, France). The viability and behaviour was recorded every day. Body weights were measured thrice a week. The length and width of the tumor was measured three times a week with calipers and the volume of the tumour was estimated by the following formula: tumour volume=(width2×length)/2 (SIMPSON-HERREN L. et al. Cancer Chemotherapy Rep., 54: 143, 1970).

Statistical Tests: All statistical analyses were performed using Vivo Manager® software (Biosystemes, Couternon, France). Statistical analyses of mean body weights, MBWC, mean tumor volumes at randomization, mean tumor volumes, mean times to reach mean tumour volumes and mean tumor doubling times were performed using ANOVA and pairwise tests were performed using the Bonferroni/Dunn correction in case of significant ANOVA results. A p value<0.05 was considered as significant.

Results

As shown inFIG.21, 50% of the test mice survived for more than 50 days after tumour induction when administered with both LTX-315 and anti-PD-L1 antibody, compared to 40% of the test mice treated with anti-PD-L1 antibody alone, 30% of the test mice treated with LTX-315 alone and none of the untreated mice. This shows that the combination therapy is more effective at both directly treating tumours (treating the tumour in the right flank of the mouse) and inducing an adaptive immunity response (treating the tumour in the left flank of the mouse).

Details regarding the percentage of mice that show total tumour regression in the right and/or left flank are shown in Table 18 below. Mice were tested at day 52 after inoculation or when sacrificed.

TABLE 18ProportionGroupFlankof MiceG1Right0%Left0%G3Right30%Left10%G5Right20%Left20%G7Right20%Left40%

The table shows that the combination of LTX-315 and anti-PD-L1 antibody is particularly effective in inducing adaptive immunity, as 40% of the mice treated with the combination (G7) showed total regression of the tumour in the left flank, compared to 10% of mice treated with LTX-315 alone (G3), 20% of mice treated with anti-PD-L1 antibody alone (G5) and none of the untreated mice (G1).

Median and mean tumour volume for each cohort was measured and while G3 and G5 always gave significantly lower volumes than G1, G7 was always clearly the best performing cohort.

It will be appreciated that it is not intended to limit the present invention to the above specific embodiments only, numerous embodiments, modifications and improvements being readily apparent to one of ordinary skill in the art without departing from the scope of the appended claims.