COMBINATIONS INCLUDING ABX196 FOR THE TREATMENT OF CANCER

Abstract

An antitumor pharmaceutical combination includes (i) a compound ABX196 and (ii) at least one chemotherapeutic agent and/or at least one immunotherapeutic agent, for use in the treatment of cancer.

Claims

1. A method of treating cancer, comprising administration, in a patient in need thereof, of a therapeutically effective amount of an antitumor pharmaceutical combination comprising: (i) a compound ABX196 of formula (I) ##STR00018## and, (ii) at least one chemotherapeutic agent and/or at least one immunotherapeutic agent, wherein said at least one immunotherapeutic agent is not an antigen.

2. The method according to claim 1, wherein the compound ABX196 of formula (I) ##STR00019## is comprised in a vaccine composition (iii) further comprising a tumor antigen.

3. The method according to claim 2, wherein the compound ABX196 of formula (I) (i) ##STR00020## or the vaccine composition (iii) and the at least one chemotherapeutic agent and/or the at least one immunotherapeutic agent (ii) are administered separately.

4. The method according to claim 2, wherein the compound ABX196 of formula (I) (i) ##STR00021## or the vaccine composition (iii) and the at least one chemotherapeutic agent and/or the at least one immunotherapeutic agent (ii) are administered semi-simultaneously.

5. The method according to claim 2, wherein the administrations of the compound ABX196 of formula (I) (i) ##STR00022## or of the vaccine composition (iii) and of the at least one chemotherapeutic agent and/or the at least one immunotherapeutic agent (ii) are spaced out over a period of time so as to obtain maximum efficacy of the combination.

6. A method for treating cancer, comprising the administration, in a patient in need thereof, of a therapeutically effective amount of a vaccine composition comprising a compound ABX196 of formula (I) ##STR00023## and a tumor antigen, in combination with a therapeutically effective amount of at least one chemotherapeutic agent and/or a therapeutically effective amount of at least one immunotherapeutic agent, wherein said at least one immunotherapeutic agent is not an antigen.

7. A method for treating cancer, comprising the administration, in a patient in need thereof, of a therapeutically effective amount of a compound ABX196 of formula (I) ##STR00024## in combination with a therapeutically effective amount of at least one chemotherapeutic agent and/or a therapeutically effective amount of at least one immunotherapeutic agent, wherein said at least one immunotherapeutic agent is not an antigen.

8. The method according to claim 7, wherein said combination further comprises an antitumor antigen.

9. A combined preparation comprising: (i) one or more dosage units of a compound ABX196 of formula (I) ##STR00025## and (ii) one or more dosage units of at least one chemotherapeutic agent and/or one or more dosage units of an immunotherapeutic agent, wherein said at least one immunotherapeutic agent is not an antigen, for treatment of a cancer.

10. The combined preparation for its use according to claim 9, wherein the compound ABX196 of formula (I) ##STR00026## is comprised in a vaccine composition further comprising a tumor antigen.

11. The method according to claim 1, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide, epirubicin, idarubicin, mitoxantrone and oxaliplatin.

12. The method according to claim 1, wherein the chemotherapeutic agent is doxorubicin or sorafenib.

13. The method according to claim 1, wherein the immunotherapeutic agent is an antibody specific of a tumor antigen selected from the group consisting of Her2/neu, EGFR, VEGF, CD20, CD52, CD33, TACE, cathepsin S, uPA, uPAR, PD-1, Glypican-3, claudin-3, claudin-4, BMCA and CTLA4.

14. The method according to claim 1, wherein the immunotherapeutic agent is a monoclonal anti-PD1 antibody.

15. The method according to claim 1, wherein the cancer is selected from the group consisting of melanoma, hepatocarcinoma, colorectal cancer and bladder cancer.

16. The method according to claim 6, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide, epirubicin, idarubicin, mitoxantrone and oxaliplatin.

17. The method according to claim 6, wherein the chemotherapeutic agent is doxorubicin or sorafenib.

18. The method according to claim 6, wherein the immunotherapeutic agent is an antibody specific of a tumor antigen selected from the group consisting of Her2/neu, EGFR, VEGF, CD20, CD52, CD33, TACE, cathepsin S, uPA, uPAR, PD-1, Glypican-3, claudin-3, claudin-4, BMCA and CTLA4.

19. The method according to claim 6, wherein the immunotherapeutic agent is a monoclonal anti-PD1 antibody.

20. The method according to claim 6, wherein the cancer is selected from the group consisting of melanoma, hepatocarcinoma, colorectal cancer and bladder cancer.

21. The method according to claim 7, wherein the chemotherapeutic agent is selected from the group consisting of doxorubicin, cyclophosphamide, epirubicin, idarubicin, mitoxantrone and oxaliplatin.

22. The method according to claim 7, wherein the chemotherapeutic agent is doxorubicin or sorafenib.

23. The method according to claim 7, wherein the immunotherapeutic agent is an antibody specific of a tumor antigen selected from the group consisting of Her2/neu, EGFR, VEGF, CD20, CD52, CD33, TACE, cathepsin S, uPA, uPAR, PD-1, Glypican-3, claudin-3, claudin-4, BMCA and CTLA4.

24. The method according to claim 7, wherein the immunotherapeutic agent is a monoclonal anti-PD1 antibody.

25. The method according to claim 7, wherein the cancer is selected from the group consisting of melanoma, hepatocarcinoma, colorectal cancer and bladder cancer.

Description

BRIEF DESCRIPTION OF THE FIGURES

[0146] FIG. 1: Mean and median volume of B16-F10 tumors engrafted in C57BL/6 mice treated, in Example 1, with TRP2-ABX196 on D3, anti-PD-1 antibodies on D10, D14, D17 and D21 or a combination of TRP2-ABX196 and anti-PD-1 antibodies.

[0147] FIG. 2: Survival of C57BL/6 mice bearing B16-F10 tumors and treated, in Example 1, with TRP2-ABX196, anti-PD-1 antibodies or a combination of TRP2-ABX196 and anti-PD-1 antibodies. ***: p<0.001; ****: p<0.0001; n.s.: not significant.

[0148] FIG. 3: Mean and median volume of B16-F10 tumors engrafted in C57BL/6 mice treated, in Example 1, with TRP2-ABX196 on D3, Doxorubicin on D7 or a combination of TRP2-ABX196 and Doxorubicin.

[0149] FIG. 4: Survival of C57BL/6 mice bearing B16-F10 tumors and treated, in Example 1, with TRP2-ABX196, Doxorubicin or a combination of TRP2-ABX196 and Doxorubicin. **: p<0.01 ***: p<0.001; ****: p<0.0001; n.s.: not significant.

[0150] FIG. 5: Mean tumor volume (mm.sup.3) of B16F10 bearing mice of example 5 exposed to vehicle, anti-PD-1 monoclonal antibodies, ABX196 administered i.v, or to the combination of anti-PD-1 monoclonal antibodies and ABX196 administered i.v. Mean+/−SEM.

[0151] FIG. 6: Mean tumor volume (mm.sup.3) of B16F10 bearing mice of Example 5 exposed to vehicle, anti-PD-1 monoclonal antibodies, ABX196 administered i.t, or to the combination of anti-PD-1 monoclonal antibodies and ABX196 administered i.t. Mean+/−SEM.

[0152] FIG. 7: Survival of B16F10 bearing mice of Example 5 exposed to vehicle, anti-PD-1 monoclonal antibodies, ABX196 administered i.v, or to the combination of anti-PD-1 monoclonal antibodies and ABX196 administered i.v. Mean+/−SEM.

[0153] FIG. 8: Survival of B16F10 bearing mice of Example 5 exposed to vehicle, anti-PD-1 monoclonal antibodies, ABX196 administered i.t or to the combination of anti-PD-1 monoclonal antibodies and ABX196 administered i.t. Mean+/−SEM.

[0154] FIG. 9: Mean tumor volume (mm.sup.3) of CT-26 bearing mice of Example 6 exposed to vehicle, anti-PD-1 monoclonal antibodies, ABX196 administered i.v, or to the combination of anti-PD-1 monoclonal antibodies and ABX196 administered i.v. Mean+/−SEM.

[0155] FIG. 10: Mean tumor volume (mm.sup.3) of MBT-2 bearing mice of Example 6 exposed to vehicle, anti-PD-1 monoclonal antibodies, ABX196 administered i.v, or to the combination of anti-PD-1 monoclonal antibodies and ABX196. Mean+/−SEM

[0156] FIG. 11: Survival of MBT-2 bearing mice of Example 6 exposed to vehicle (2), anti-PD-1 monoclonal antibodies (4), ABX196 administered i.v (3), or to the combination of anti-PD-1 monoclonal antibodies and ABX196 (1).

[0157] FIG. 12: % of tumor invasion of the liver from each groups of mice of Example 7 at day 20. G1: Vehicle; G2: Sorafenib; G3: Sorafenib+ABX196; G4: Doxorubicin; G5 Doxorubicin+ABX196; G6: Anti-PD-1; G7 Anti-PD-1+ABX196

DESCRIPTION OF THE SEQUENCES

[0158]

TABLE-US-00001 SEQ ID NO: Sequence Description 1 KFGWTGPDCNRKKPA Amino acid sequence of GGNCSVYDFFVWLHYY peptide CI II-TRP2.sub.180-188 2 KFGWTGPDCNRKKPA Amino acid sequence of Class II epitope 3 SVYDFFVWL Amino acid sequence of an immunodominant peptide from the tumor associated antigen tyrosinase-related protein-2 4 GGGSVYDFFVWLGGGS Amino acid sequence of SKFGWTGPDCNRKKPA a peptide comprising the sequence of an  immunodominant peptide from TRP2

EXAMPLES

Example 1

[0159] This example demonstrates that a vaccine composition comprising the compound ABX196 and a tumor antigen increases the anticancerous effects of chemotherapeutic agents such as doxorubicin or immunotherapeutic agents such as anti-PD-1 antibodies.

1. Materials and Methods

[0160] 1.1. Compounds

[0161] The vaccine composition used comprises the peptide CI II-TRP2.sub.180-188 and the ABX196 adjuvant.

[0162] The peptide CI II-TRP2180188 of sequence KFGWTGPDCNRKKPA GG NCSVYDFFVWLHYY (SEQ ID NO: 1), is composed of a Class II epitope (KFGWTGPDCNRKKPA (SEQ ID NO: 2)) fused with an immunodominant peptide from the tumor associated antigen tyrosinase-related protein-2 which is over-expressed in melanocyte (SVYDFFVWL (SEQ ID NO: 3)).

[0163] The adjuvant ABX196 has the property to activate NKT cells.

[0164] The chemotherapeutic agent used was doxorubicin (DOXO CELL, Cell Pharm).

[0165] The immunotherapeutic agent used was anti-PD-1 antibody (ref.: BE0146, BioXcell; clone: RMP1-14, reactivity: mouse; isotype: Rat IgG2a; storage conditions: +4° C.).

[0166] 1.2. Compounds Vehicles

[0167] Doxorubicin was diluted in NaCl 0.9%.

[0168] Anti-PD-1 antibody was prepared in phosphate buffered saline (PBS) or other suitable vehicle according to manufacturer's recommendation.

[0169] The CI II-TRP2 peptide (5 mg/tube) was resuspended in DMSO at a concentration of 50 mg/mL.

[0170] The adjuvant ABX196 was provided as a solution at 250 μg/mL The final formulation of the vaccine containing CI II-TRP2 peptide and ABX196 were prepared in phosphate buffered saline (PBS).

[0171] The vehicle solution used in group 1 at day 3 (see section 2.1.2.) contained DMSO diluted in phosphate buffered saline (PBS) at the same final concentration as for the CL II-TRP2/ABX196 vaccine.

[0172] 1.3. Treatment Doses

[0173] The peptide CI II-TRP2 was administered at the dose of 50 μg per mouse together with 1 μg of adjuvant ABX196.

[0174] The anti-PD-1 antibody was administered at the dose 10 mg/kg.

[0175] Doxorubicin was administered at the dose of 12 mg/kg.

[0176] 1.4. Routes of Administration

[0177] The vaccine composition was injected by the intra-venous route in the caudal vein of mice (IV, bolus).

[0178] Anti-PD-1 antibody was injected into the peritoneal cavity of mice (Intraperitoneally, IP).

[0179] Doxorubicin was injected intravenously in the caudal vein of mice (IV, bolus).

[0180] In all groups, the vaccine composition was administered at a dose volume of 10 mL/kg/adm (i.e. for one mouse weighing 20 g, 200 μL of vaccine composition was administered) according to the most recent body weight of mice.

[0181] 1.5. Cancer Cell Lines and Culture Conditions

[0182] 1.5.1. Cancer Cell Lines

[0183] The cell line that was used is detailed in Table 1 below.

TABLE-US-00002 TABLE 1 Cell lines used Cell line Type Specie Origin B16-F10 Melanoma mouse ATCC .sup.a .sup.a American Type Culture Collection, Manassas, Virginia, USA

[0184] The B16-F10 cell line was established from a lung metastasis arising from a spontaneously occurring melanoma in a C57BL/6J mouse.

[0185] 1.5.2. Cell Culture Conditions

[0186] Cells were grown as monolayer at 37° C. in a humidified atmosphere (5% CO.sub.2, 95% air) in their respective culture medium (see table below). The culture medium was DMEM containing 2 mM L-glutamine (ref: BE12-702F, Lonza, Verviers, Belgium) supplemented with 10% fetal bovine serum (ref: 3302, Lonza). 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 was assessed by 0.25% trypan blue exclusion assay.

[0187] 1.6. Use of Animals

[0188] 1.6.1. Animals

[0189] 95 healthy female C57BL/6 (C57BL/6J) mice, 6-7 weeks old, were obtained from JANVIER LABS (Le Genest-Saint-Isle, France). Animals were maintained in SPF health status according to the FELASA guidelines.

[0190] Animal housing and experimental procedures were realized according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals.

[0191] 1.6.2. Housing Conditions

[0192] Animals were maintained in housing rooms under controlled environmental conditions: [0193] Temperature: 22±2° C., [0194] Humidity 55±10%, [0195] Photoperiod (12h light/12h dark), [0196] HEPA filtered air, [0197] 15 air exchanges per hour with no recirculation.
Animal enclosures were provided sterile and adequate space with bedding material, food and water, environmental and social enrichment (group housing) as described: [0198] Top filter polycarbonate Eurostandard Type III or IV cages, [0199] Corn cob bedding (ref: LAB COB 12, SERLAB, France), [0200] 25 kGy Irradiated diet (Ssniff® Soest, Germany), [0201] Complete food for immunocompetent rodents—R/M-H Extrudate, [0202] Sterile, filtrated at 0.2 μm water, [0203] Environmental enrichment (SIZZLE-dri kraft—D20004 SERLAB, France).

2. Treatments

[0204] 2.1. Antitumor Activity Study of a Novel Vaccine in Combination with PD-1 Targeting Antibody or Doxorubicin in Mice Bearing Subcutaneous 816-F10 Melanoma

[0205] 2.1.1. Induction of B16-F10 Tumors in Animals

[0206] Tumor was induced by subcutaneous injection of 1×10.sup.6 of B16-F10 cells in 200 μL of PBS buffer into the right flank of 95 C57BL/6 female animals. The day of tumor cell injection in the right flank was considered as D0.

[0207] 2.1.2. Treatment Schedule

[0208] On D3, 90 out of ninety-five 95 were then randomized according to their body weight into 6 groups each of 15 animals (groups 1-6).

[0209] A statistical test (analysis of variance, ANOVA) was performed to test for homogeneity between groups using the Vivo Manager® software (Biosystemes, Couternon, France). [0210] Animals from group 1 received one IV injection of the vehicle used for the vaccine composition at day 3 and 4 IP administrations of the vehicle used for anti-PD-1 antibody on day 10, 14, 17 and 21. [0211] Animals from group 2 received one IV injection of 50 μg of CI II-TRP2 together with 1 μg of ABX196 on day 3. [0212] Animals from group 3 received 4 IP administrations of anti-PD-1 antibody on day 10, 14, 17 and 21. [0213] Animals from group 4 received one IV injection of 50 μg of CL II-TRP2 together with 1 μg of ABX196 at day 3 and 4 IP administrations of anti-PD-1 antibody on day 10, 14, 17 and 21. [0214] Animals from group 5 received one IV injection of Doxorubicin at 12 mg/kg on day 7. [0215] Animals from group 6 received one IV injection of 50 μg of CL II-TRP2 together with 1 μg of ABX196 on day 3 and one IV injection of Doxorubicin at 12 mg/kg on day 7.

[0216] The treatment schedule is summarized in Table 2 below.

TABLE-US-00003 TABLE 2 Treatment schedules No. Treatment Group Animals Treatment Dose Route Schedule 1 15 Vehicle — IV/IP D3-IV/ TW × 2 IP 2 15 CL II-TRP2/ 50 μg/1 μg IV Q1D × 1 ABX196 on D3 3 15 Anti-PD-1 Ab 10 mg/kg IP TW × 2 on D10/D14/ D17/D21 4 15 CL II-TRP2/ 50 μg/1 μg IV Q1D × 1 ABX196 on D3 Anti-PD-1 Ab 10 mg/kg IP TW × 2 on D10/D14/ D17/D21 5 15 Doxorubicin 12 mg/kg IV Q1D × 1 on D7 6 15 CL II-TRP2/ 50 μg/1 μg IV Q1D × 1 ABX196 on D3 Doxorubicin 12 mg/kg IV Q1D × 1 on D7 TOTAL 90

[0217] The monitoring of animals was performed as described in section 2.2.

[0218] 2.2. Animal Monitoring

[0219] 2.2.1. Clinical Monitoring

[0220] The viability and behavior were recorded every day. Body weights were measured twice a week until injection of tumor cells then thrice a week. The length and width of the tumor were measured thrice a week with calipers and the volume of the tumor was estimated by the formula:

[00001]$\mathrm{Tumor}\mathrm{volume}=\frac{{\mathrm{width}}^2\times \mathrm{length}}{2}$

[0221] 2.2.2. Humane Endpoints [0222] Signs of pain, suffering or distress: pain posture, pain face mask, behavior, [0223] Tumor exceeding 10% of normal body weight (individual tumor taken into account), but non-exceeding 1,500 mm.sup.3 (the sum of tumor volume on right flank/MFP and tumor volume on left flank/MFP were taken into account), [0224] Tumors interfering with ambulation or nutrition, [0225] Ulcerated tumor or tissue erosion, [0226] 20% body weight loss remaining for 2 consecutive days, [0227] Poor body condition, emaciation, cachexia, dehydration, [0228] Prolonged absence of voluntary responses to external stimuli, [0229] Rapid labored breathing, anemia, significant bleeding, [0230] Neurologic signs: circling, convulsion, paralysis, [0231] Sustained decrease in body temperature, [0232] Abdominal distension.

[0233] 2.2.3. Necropsy

[0234] Necropsy (macroscopic examination) was performed on all terminated animals in the study, and, if possible, on all euthanized moribund or found dead animals.

[0235] 2.3. Animal procedures

[0236] 2.3.1. Anesthesia

[0237] Isoflurane gas anesthesia was used for tumor inoculation and i.v. injections.

[0238] 2.3.2. Euthanasia

[0239] Euthanasia of animals was performed by gas anesthesia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.

3. Data Processing

[0240] 3.1. Health Parameters

[0241] The following evaluation criteria of health were determined using Vivo Manager® software (Biosystemes, Couternon, France): [0242] Individual and mean body weights of animals were provided. [0243] Mean body weight change (MBWC): Average weight changes of treated animals in percent (weight at day B minus weight at day A divided by weight at day A) were calculated. The intervals over which MBWC was calculated, was chosen as a function of body weight curves and the days of body weight measurement.

[0244] 3.2. Efficacy Parameters

[0245] The treatment efficacy was assessed in terms of the effects of the vaccine composition on the tumor volumes of treated animals relative to control animals. The following evaluation criteria of antitumor efficacy was determined using Vivo Manager® software (Biosystemes, Couternon, France): [0246] Individual, mean and median tumor volumes were provided, [0247] The number of tumor-free mice was provided.
Mice survival was also monitored and used as an efficacy parameter. Survival curves were drawn.
Percent treated/control (T/C (%) index) is calculated by dividing the median treated tumor volume by the median control tumor volume on day 14 and multiplying by 100. A T/C % equal or less than 42% is considered significant antitumor activity by the drug evaluation branch of the division of cancer treatment (NCI).

[00002]$T/C\%=\frac{\mathrm{Tumor}\mathrm{Volume}{\mathrm{Median}}_{treated}}{\mathrm{Tumor}\mathrm{Volume}{\mathrm{Median}}_{\mathrm{vehicle}}}\times 100$

Tumor growth inhibition is also reflective to the antitumor effectiveness and is calculated following the formula:

[00003]$\mathrm{TGI}\%=\left[\frac{1-\frac{\left(\frac{TV{M}_{tr\mathrm{eated}\mathrm{dayx}}}{TV{M}_{\mathrm{treated}\mathrm{initial}}}\right)}{\left(\frac{TV{M}_{\mathrm{vehicle}\mathrm{dayx}}}{TV{M}_{\mathrm{vehicle}\mathrm{initial}}}\right)}}{1-\left(\frac{TV{M}_{\mathrm{vehicle}\mathrm{initial}}}{TV{M}_{\mathrm{vehicle}\mathrm{dayx}}}\right)}\right]\times 100$

% TGI higher than 50% is considered as active.

[0248] 3.3. Statistical Analysis

[0249] Mean tumor volumes at defined times were analyzed with the GrapadPrism Software (Version 6.07) using the Kruskall-Wallis test for all the treatments. A significant difference between all treatments (P<0.05) was followed by pairwise comparisons using the Dunn's multiple comparison test. Survival was analyzed using the Log-rank (Mantel-Cox) test with GrapadPrism Software (Version 6.07). A significant difference between all treatments (P<0.05) was followed by pairwise comparisons using the Log-rank (Mantel-Cox) test.

4. Results

[0250] 4.1. Antitumor Activity Study of the Vaccine Composition of the Invention (TRP2-ABX196) in Combination with PD-1 Targeting Antibody or Doxorubicin in Mice Bearing Subcutaneous B16-F10 Melanoma

[0251] 4.1.1. Toxicity Parameters

[0252] The mean body weight curves were determined, and the effects of treatments on mice individual body weight were studied.

[0253] No deterioration in general status was observed and clinical status remained good for treated animals during the study whatever the treatment group.

[0254] For the three groups of mice that were immunized with TRP2-ABX196, an important weight loss, i.e. approximately 10%, was measured two days after immunization. The weight loss was transient in all the mice and they rapidly recovered weight at day five.

[0255] With the exception of mice treated with a combination of TRP2-ABX196 and doxorubicin, in which body weight was stabilized during the treatment period, the weight evolution was similar in all the other treatment groups.

[0256] Thus, with the exception of a transient weight loss after administration of the vaccine TRP2-ABX196 alone or in combination, all treatments were well tolerated by C57BL/6J mice bearing B16-F10 tumor.

[0257] 4.1.2. Antitumor Activity Analysis

[0258] The individual, mean and median tumor volumes curves are presented in FIGS. 1 and 3.

[0259] Mice survival curves are presented in FIGS. 2 and 4.

[0260] A summary of median survival times in days is presented in Table 3 below.

TABLE-US-00004 TABLE 3 Median survival (days) per experimental groups. Median Treatment group Survival (days) Vehicle 17 TRP2-ABX196 21 Anti-PD-1 Ab 17 TRP2-ABX196 + Anti-PD-1 Ab 28 Doxorubicin 21 TRP2-ABX196 + Doxorubicin 26

[0261] The statistical analysis of tumor volume at D17 are shown in Table 4 below.

TABLE-US-00005 TABLE 4 Statistical analysis of tumor growth between treatments at D17. Global comparison of all treatments at D17: P value < 0.0001 (Kruskal-Wallis test). Pairwise comparisons between treatments: P value according to the Dunn's multiple comparison test. TRP2- TRP2- Treatment TRP2- ABX196 + ABX196 + groups Vehic. ABX196 α-PD-1 α-PD-1 Doxo. Doxo. Vehic. — ns ns *** ns *** TRP2- ns — ns ns ns ns ABX196 α-PD-1 ns ns — *** ns **** TRP2- *** ns *** — ns ns ABX196 + α-PD-1 Doxo. ns ns ns ns — * TRP2- *** ns **** ns * — ABX196 + Doxo. Vehic.: vehicle; α-PD-1: anti-PD-1 antibody; Doxo.: doxorubicin *P < 0.05; ***P < 0.001; ****P < 0.0001

[0262] The statistical analysis of tumor volume at D19 are shown in Table 5 below.

TABLE-US-00006 TABLE 5 Statistical analysis of tumor growth between treatments at D19. Global comparison of all treatments at D19: P value < 0.0001 (Kruskal-Wallis test). Pairwise comparisons between treatments: P value according to the Dunn's multiple comparison test. TRP2- TRP2- Treatment TRP2- ABX196 + ABX196 + Groups Vehic. ABX196 α-PD-1 α-PD-1 Doxo. Doxo. Vehic. — ns ns ns ns ** TRP2- ns — ns ns ns ns ABX196 α-PD-1 ns ns — ** ns *** TRP2- ns ns ** — ns ns ABX196 + α-PD-1 Doxo. ns ns ns ns — * TRP2- ** ns *** ns * — ABX196 + Doxo. Vehic.: vehicle; α-PD-1: anti-PD-1 antibody; Doxo.: doxorubicin *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001

[0263] The statistical analysis of tumor volume at D17 and D19 in three subgroups are shown in Table 6 below.

TABLE-US-00007 TABLE 6 Statistical analysis of tumor growth between three subgroups at D17 and D19. Statistical analysis of three groups with TRP2-ABX196 as the reference group: D17 D19 P value-Kruskal-Wallis test.sup.a 0.0228 0.0121 TRP2-ABX196 + Anti-PD-1 Ab.sup.b * ns TRP2-ABX196 + Doxorubicin.sup.b * ** .sup.athree groups (TRP2-ABX196, TRP2-ABX196 + Anti-PD-1 Ab and TRP2-ABX196 + Doxorubicin are considered) .sup.bDunn's multiple comparison test with TRP2-ABX196 as the reference group *: P < 0.05; **: P < 0.01; ***: P < 0.001; ****: P < 0.0001

[0264] The statistical analysis of mice survival are shown in Table 7 below.

TABLE-US-00008 TABLE 7 Statistical analysis of mice survival between treatments. Global comparison of all survival curves: P* < 0.0001 Summary of pairwise comparisons: Treatment TRP2-ABX196 + TRP2-ABX196 + Groups TRP2-ABX196 α-PD-1 α-PD-1 Doxo. Doxo. Vehicle 0.0001** 0.279 <0.0001 0.0015 <0.0001 α-PD-1 — 0.0002 0.0710 0.624 0.22 Doxo. <0.0001 — 0.1285 α-PD-1: anti-PD-1 antibody; Doxo.: doxorubicin *Log-rank (Mantel-Cox) test **bold numbers refers to significant difference between compared groups (P < 0.05).

TABLE-US-00009 TABLE 8 Anti-tumor activity index at day 14. TRP2/ TRP2/ TRP2/ ABX196 + ABX196 + ABX196 Anti-PD-1 Doxorubicin Anti-PD-1 Doxorubicin T/C 33.5 99.8 56.3 11.8 15.4 TGI 63.1 22.5 52.7 90.5 78.5
Antitumor Activity Analysis of TRP2-ABX196 Combined with Anti-PD-1 Ab

[0265] Mice bearing B16-F10 tumors were treated at D3 with TRP2-ABX196 vaccine, anti-PD-1 Ab at D10, D14, D17 and D21 or a combination of both. The kinetics of tumor growth is shown in FIG. 1.

[0266] As expected, there was no anti-tumoral activity associated with anti-PD-1 Ab treatment in comparison to the vehicle-treated group. Those observations are supported by the T/C and TGI values showing that anti-PD-1 demonstrates any activity (Table 8; T/C>42% and TGI<50%). In contrast, mice treatment with TRP2-ABX196 resulted in a slower growth of B16-F10 tumors in comparison to that of the vehicle-treated group. T/C and TGI values reflect the anti tumor activity of TRP2/ABX196 activity (Table 8; T/C<42% and TGI>50%). The antitumor activity of TRP2/ABX196 is further improved in combination with anti-PD-1 antibody, with a complete stabilization of tumor growth up to day 14 in the latter group; as also indicated by T/C and TGI values (Table 8) which are below 15% and higher than 90%, respectively. Those values indicate a highly active treatment because following NCI guidelines, a T/C below 15% demonstrates the high potency of the treatment.

[0267] Rigorous and thorough statistical analysis of tumor growth at D17 (Table 5) indicated a significant difference of tumor volume between all groups and a highly significant decrease of tumor volume for the group of mice treated with TRP2-ABX196 vaccine and anti-PD-1 only in comparison to the vehicle-treated group.

[0268] With the aim of increasing the power of the statistical analysis and evidenced a difference in the combination group versus the vaccine-treated group, a subsequent statistical analysis was performed with three groups, i.e. TRP2-ABX196, TRP2-ABX196+Anti-PD-1 Ab and TRP2-ABX196+Doxorubicin (Table 6). This analysis indicated a significant decrease of tumor volume at D17 between TRP2-ABX196 and TRP2-ABX196 combined with anti-PD-1 antibody while this difference did not reach significance at day 19.

[0269] Mice survival is illustrated in FIG. 2. Survival of mice treated with TRP2-ABX196 and TRP2-ABX196 combined with anti-PD-1 antibody was significantly improved in comparison to that of the vehicle-treated group (Table 7) while the increase was close to significance (P=0.0710) for the combination treatment in comparison to TRP2-ABX196 vaccine alone.

Antitumor Activity Analysis of TRP2-ABX196 Combined with Doxorubicin

[0270] Mice bearing B16-F10 tumors were treated at D3 with TRP2-ABX196 vaccine, doxorubicin at D7 or a combination of both. The kinetics of tumor growth is shown in FIG. 3.

[0271] Mice treated with doxorubicin alone presented a weak and non-significant (Tables 4 and 5) decrease of tumor growth in comparison to the vehicle-treated group. Even if TGI is slightly higher than 50%, the T/C value remains superior to 42%, indicating the lack of antitumor potency of Doxorubicin. Mice treated with the combination of TRP2-ABX196 in combination with doxorubicin presented a complete stabilization of tumor growth up to day 14; the decrease in tumor volume at D17 and D19 reached significance versus the vehicle-treated group upon the complete statistical analysis of the experiment (Tables 4 and 5). The synergic effect of TRP2/ABX196 and Doxorubicin is also demonstrated by the T/C and TGI values which are <42% and >50%, respectively (Table 8).

[0272] Following a subsequent statistical analysis with three groups (Table 6), a significant decrease of tumor volume at D17 and D19 between TRP2-ABX196 and TRP2-ABX196 combined with doxorubicin was found.

[0273] Mice survival is illustrated in FIG. 4. Survival of mice treated with TRP2-ABX196 combined with doxorubicin was significantly improved in comparison to that of the vehicle-treated group (Table 7) while the survival increase did not reach significance in comparison to each treatment modality alone.

[0274] A synthesis of the median survival for all the experimental groups is shown in Table 3.

5. Conclusions

[0275] The purpose of this example was to combine the TRP2-ABX196 vaccine with either an anti-PD-1 antibody or the chemotherapeutic agent doxorubicin, known to induce immunogenic cell death.

[0276] The compounds tested, either alone or in combination were well tolerated by the animals and no drug-related severe toxicity nor death were recorded.

[0277] Transient weight loss close to 10% was observed in all the groups that received TRP2-ABX196 vaccine but all the mice recovered their normal weight rapidly, i.e. 2 to 5 days after the single vaccine injection.

[0278] The survival of mice treated with TRP2-ABX196 vaccine was significantly improved in comparison to the vehicle-treated group. For both combination groups, the anti-tumoral activity was further improved when compared to the vaccine alone with a median survival of 21 days that increased to 28 and 26 days, when combined with anti-PD-1 antibody or doxorubicin, respectively. Survival increase was close to significance with the combination of TRP2-ABX196 vaccine and anti-PD-1 antibody (P=0.071) versus vaccine alone. Even if TRP2/ABX196 and Doxorubicin treatments present an anti-tumor effect, those treatments are improved by the combination of the invention (ABX196 plus anti-PD-1 or plus Doxorubicin) as reflected by T/C ratio, which is lower than 16% when mice are treated with the combinations (see Table 8). Below 15%, treatment is considered as highly active following NCI guidelines. On the same line, the TGI (tumor growth inhibition) is up to 78% in presence of combination treatments, demonstrating that the combination treatment is highly active.

[0279] These results are remarkable in the context of the strong aggressivity of the B16-F10 model and its poor noteworthy immunogenicity. Of note, the experiment has been performed with a high number of injected tumor cells, i.e. one million per animal, which might potentially explain why complete regressions of tumors were not observed.

Example 2

[0280] This example describes the specific effect obtained with the vaccine composition of the invention comprising ABX196 as adjuvant, compared to other vaccine compositions comprising other α-galactosylceramide derivatives as adjuvant.

Materials and Methods

[0281] The experiment was performed as described in Example 1 except that mice were administered with 5×10.sup.5 B16-F10 cells.

[0282] The other α-galactosylceramide derivative used was α-GalCer of the following formula:

##STR00017##

[0283] The treatment schedule of the first experiment was as follows:

TABLE-US-00010 No Treatment Group Animals Treatment Dose Route schedule 1 15 Vehicle — IV Q1D × 1 on D7/D10 2 15 ABX196 100 ng IV Q1D × 1 on D10 3 15 α-GalCer 100 ng IV Q1D × 1 on D10 4 15 Doxo- 12 mg/kg IV Q1D × 1 robucin on D7 5 15 Doxo- 12 mg/kg IV Q1D × 1 robucin on D7 ABX196 100 ng IV Q1D × 1 on D10 6 15 Doxo- 12 mg/kg IV Q1D × 1 robucin on D7 α-GalCer 100 ng IV Q1D × 1 on D10 TOTAL 90

[0284] The treatment schedule of the second experiment was as follows:

TABLE-US-00011 No Route Treatment Group Animals Treatment Dose schedule 1 15 Vehicle — IV Q1D × 1 on D7/D10 2 15 ABX196 10 ng IV Q1D × 1 on D10 3 15 α-GalCer 10 ng IV Q1D × 1 on D10 4 15 ABX196 100 ng IV Q1D × 1 on D10 5 15 α-GalCer 100 ng IV Q1D × 1 on D10 6 15 Doxo- 12 mg/kg IV Q1D × 1 robucin on D7 7 15 Doxo- 12 mg/kg IV Q1D × 1 robucin on D7 ABX196 10 ng IV Q1D × 1 on D10 8 15 Doxo- 12 mg/kg IV Q1D × 1 robucin on D7 ABX196 100 ng IV Q1D × 1 on D10 9 15 Doxo- 12 mg/kg IV Q1D × 1 robucin on D7 α-GalCer 10 ng IV Q1D × 1 on D10 10 15 Doxo- 12 mg/kg IV Q1D × 1 robucin on D7 α-GalCer 100 ng IV Q1D × 1 on D10 TOTAL 150

[0285] The treatment schedule of the third experiment was as follows:

TABLE-US-00012 No. Treatment Group Animals Treatment Dose Route Schedule 1 15 Vehicle — IV/IP D3-IV/ TW × 2 IP 2 15 TRP2/ 50 μg/1 μg IV Q1D × 1 ABX196 on D3 3 15 Anti-PD- 10 mg/kg IP TW × 2 on 1 Ab D10/D14/ D17/D21 4 15 TRP2/ 50 μg/1 μg IV Q1D × 1 ABX196 on D3 Anti-PD- 10 mg/kg IP TW × 2 on 1 Ab D10/D14/ D17/D21 5 15 TRP2/ 50 μg/1 μg IV Q1D × 1 αGalCer on D3 6 15 TRP2/ 50 μg/1 μg IV Q1D × 1 αGalCer on D3 Anti- 10 mg/kg IP TW × 2 on PD-1 Ab D10/D14/ D17/D21 TOTAL 90

[0286] The treatment schedule of the fourth experiment was as follows:

TABLE-US-00013 No. Treatment Group Animals Treatment Dose Route Schedule 1 10 Vehicle — IV/IP D3-IV/ TW × 2 IP 2 10 TRP2/ABX196 50 μg/1 μg IV Q1D × 1 on D3 3 10 TRP2/ABX196 50 μg/1 μg IV Q1D × 1 on D3 Anti-PD-1 Ab 10 mg/kg IP TW × 2 on D10/D14/ D17/D21
5 mice per group were sacrificed and autopsied at D12 and D16.
Tumor was collected and flow cytometry analysis of the following populations was performed: [0287] T Cytotoxic populations: CD45.sup.+/CD3.sup.+/CD8.sup.+/TNFα/Perforin/Granzyme [0288] Treg populations: CD45.sup.+/CD3.sup.+/CD4.sup.+/CD8.sup.−/FoxP3.sup.+

Example 3

1. Study Aims

[0289] Evaluate the anti-tumoral activity of ABX196 and α-Gal-Cer as defined in example 2 in the ectopic B16-F10 melanoma model. [0290] Evaluate the anti-tumoral activity of ABX196 and α-Gal-Cer combined with doxorubicin treatment in the ectopic B16-F10 melanoma model.

2. Materials and Methods

[0291] The experiment is the same as for example 1, except for the following.

Test and Reference Substances Vehicles

[0292] Doxorubicin was diluted in NaCl 0.9%
ABX196 was provided as a solution at 250 μg/mL
α-Gal-Cer was provided as a solution at 1 mg/mL
Dilution of ABX196 or αGalCer was performed in PBS buffer
The vehicle solution used in group 1 at day 7 contained DMSO diluted in phosphate buffered saline (PBS) at the same final concentration as the ABX196 test item.

Treatment Doses

[0293] The ABX196 and α-Gal-Cer compounds were administered at the dose of 10 or 100 ng per mouse.
Doxorubicin was administered at the dose of 12 mg/kg.

Routes of Administration

[0294] Test substance was injected by the intra-venous route in the caudal vein of mice (IV, bolus). Doxorubicin was injected intravenously in the caudal vein of mice (IV, bolus)
In all groups, test substances (ABX196 and α-Gal-Cer) were administered at a dose volume of 5 mL/kg/adm (i.e. for one mouse weighing 20 g, 100 μL of test substance was administered) according to the most recent body weight of mice. Doxorubicin was administered at a dose volume of 10 mL/kg/adm.

3. Experimental Design and Treatments

[0295] 3.1.1. Induction of B16-F10 Tumors in Animals

Tumor was induced by subcutaneous injection of 5×10.sup.5 B16-F10 cells in 200 μL of PBS buffer into the right flank of one hundred and ninety-five (195) C57BL/6 female animals. The day of tumor cell injection in the right flank was considered as D0.

[0296] 3.1.2. Treatment Schedule

On D7, one hundred fifty animals (150) out of one hundred and ninety-five (195) were then randomized according to their tumor volume into 10 groups each of 15 animals (groups 1-10). Should the volume at D7 be too small, in particular with mice presenting no measurable tumors, randomization was performed according to their body weight.
A statistical test (analysis of variance) was performed to test for homogeneity between groups using the Vivo Manager® software (Biosystemes, Couternon, France).
The treatment schedule is summarized in the table below:

TABLE-US-00014 No. Treatment Group Animals Treatment Dose Route Schedule 1 15 Vehicle — IV Q1Dx1 on D7/D10 2 15 ABX196  10 ng IV Q1Dx1 on D10 3 15 a-Gal-Cer  10 ng IV Q1Dx1 on D10 4 15 ABX196 100 ng IV Q1Dx1 on D10 5 15 a-Gal-Cer 100 ng IV Q1Dx1 on D10 6 15 Doxorubicin  12 mg/kg IV Q1Dx1 on D7 7 15 Doxorubicin  12 mg/kg IV Q1Dx1 on D7 ABX196  10 ng IV Q1Dx1 on D10 8 15 Doxorubicin  12 mg/kg IV Q1Dx1 on D7 ABX196 100 ng IV Q1Dx1 on D10 9 15 Doxorubicin  12 mg/kg IV Q1Dx1 on D7 a-Gal-Cer  10 ng IV Q1Dx1 on D10 10 15 Doxorubicin  12 mg/kg IV Q1Dx1 on D7 a-Gal-Cer 100 ng IV Q1Dx1 on D10 [0297] The animals from group 1 received one IV injection of test substance vehicle at day 7 and 10. [0298] The animals from group 2 received one IV injection of 10 ng of ABX196 on day 10. [0299] The animals from group 3 received one IV injection of 10 ng of α-Gal-Cer on day [0300] 10. [0301] The animals from group 4 received one IV injection of 100 ng of ABX196 on day 10. [0302] The animals from group 5 received one IV injection of 100 ng of α-Gal-Cer on day 10. [0303] The animals from group 6 received one IV injection of Doxorubicin at 12 mg/kg on day 7. [0304] The animals from group 7 received one IV injection of Doxorubicin at 12 mg/kg on day 7 and one IV injection of 10 ng of ABX196 on day 10. [0305] The animals from group 8 received one IV injection of Doxorubicin at 12 mg/kg on day 7 and one IV injection of 100 ng of ABX196 on day 10. [0306] The animals from group 9 received one IV injection of Doxorubicin at 12 mg/kg on [0307] day 7 and one IV injection of 10 ng of α-Gal-Cer on day 10. [0308] The animals from group 10 received one IV injection of Doxorubicin at 12 mg/kg on day 7 and one IV injection of 100 ng of α-Gal-Cer on day 10.

Example 4

1. Study Aims

[0309] Part 1: Evaluate the anti-tumoral activity of combinations of the vaccine CL II-TRP2/ABX196 or CL II-TRP2/α-Gal-Cer with an anti-PD-1 antibody in the ectopic B16-F10 melanoma model [0310] Part 2: characterization of immune infiltrates in B16-F10 tumors from mice treated with CL II-TRP2/ABX196 vaccine alone or combined with anti-PD-1 antibody treatment.

2. Materials and Methods

[0311] Part 1 of the experiment is the same as for example 1, except for the following.

Test and Reference Substances Vehicles

[0312] Anti-PD-1 antibody was prepared in phosphate buffered saline (PBS) or other suitable vehicle according to manufacturer's recommendation.
The CI II-TRP2 peptide (5 mg/tube) was resuspended in DMSO at a concentration of 50 mg/mL.
The adjuvant ABX196 was provided as a solution at 250 μg/mL
The adjuvant α-Gal-Cer was provided as a solution at 1 mg/mL
The final formulation of the vaccine containing CI II-TRP2 peptide and ABX196 was prepared in phosphate buffered saline (PBS).
The vehicle solution used in group 1 at day 3 contained DMSO diluted in phosphate buffered saline (PBS) at the same final concentration as for the CL II-TRP2/ABX196 vaccine.

Treatment Doses

[0313] The peptide CI II-TRP2 was administered at the dose of 50 μg per mouse together with 100 ng of adjuvants ABX196 or α-Gal-Cer.
The anti-PD-1 antibody was administered at the dose of 10 mg/kg.

Routes of Administration

[0314] Test substance was injected by the intra-venous route in the caudal vein of mice (IV, bolus).
Anti-PD-1 antibody was injected into the peritoneal cavity of mice (Intraperitoneally, IP)
In all groups, test substances were administered at a dose volume of 5 mL/kg/adm (i.e. for one mouse weighing 20 g, 100 μL of test substance was administered) according to the most recent body weight of mice.
Anti-PD-1 antibody was administered at a dose volume of 10 mL/kg/adm.

3. Experimental Design and Treatments

[0315] Part I: Antitumor Activity Study of a Vaccine in Combination with PD-1 Targeting Antibody in Mice Bearing Subcutaneous B16-F10 Melanoma

[0316] i. Induction of B16-F10 Tumors in Animals

Tumor was induced by subcutaneous injection of 5×10.sup.5 of B16-F10 cells in 200 μL of PBS buffer into the right flank of one hundred and fifty-six (156) C57BL/6 female animals. The day of tumor cell injection in the right flank was considered as D0.

[0317] ii. Treatment Schedule

On D3, one hundred and twenty animals (120) out of one hundred and fifty-six (156) were then randomized according to their body weight into 9 groups, six of 15 animals (groups 1-6 of ACT2) and 3 of 10 animals (group 1-3 of ACT3).
A statistical test (analysis of variance) was performed to test for homogeneity between groups using the Vivo Manager® software (Biosystemes, Couternon, France). [0318] The animals from group 1 received one IV injection of test substance vehicle at day 3 and 4 IP administrations of test reference vehicle (Antibody) on day 10,14,17 and 21. [0319] The animals from group 2 received one IV injection of 50 μg of CI II-TRP2 together with 100 ng of ABX196 on day 3. [0320] The animals from group 3 received 4 IP administrations of anti-PD-1 antibody on day 10, 14, 17 and 21. [0321] The animals from group 4 received one IV injection of 50 μg of CL II-TRP2 together with 100 ng of ABX196 on day 3 and 4 IP administrations of anti-PD-1 antibody on day 10, 14, 17 and 21. [0322] The animals from group 5 received one IV injection of 50 μg of CI II-TRP2 together with 100 ng of α-Gal-Cer on day 3. [0323] The animals from group 6 received one IV injection of 50 μg of CL II-TRP2 together with 100 ng of α-Gal-Cer on day 3 and 4 IP administrations of anti-PD-1 antibody on day 10, 14, 17 and 21. [0324] The treatment schedule is summarized in the table below:

TABLE-US-00015 No. Treatment Group Animals Treatment Dose Route Schedule 1 15 Vehicle — IV/IP D3-IV/TWx2 IP 2 15 CL II-TRP2/  50 μg/ IV Q1Dx1 on D3 ABX196 100 ng 3 15 Anti-PD-1 Ab  10 mg/kg IP TWx2 on D10/ D14/D17/D21 4 15 CL II-TRP2/  50 μg/ IV Q1Dx1 on D3 ABX196 100 ng Anti-PD-1 Ab  10 mg/kg IP TWx2 on D10/ D14/D17/D21 5 15 CL II-TRP2-  50 μg/ IV Q1Dx1 on D3 α-Gal-Cer 100 ng 6 15 CL II-TRP2-  50 μg/ IV Q1Dx1 on D3 α-Gal-Cer 100 ng Anti-PD-1 Ab  10 mg/kg IP TWx2 on D10/ D14/D17/D21 TOTAL 90
Part II: Characterization of Immune T Cell Infiltrates in B16-F10 Melanoma Tumors from Mice Treated with a Vaccine in Combination with PD-1 Targeting Antibody. [0325] The animals from group 1 received one IV injection of test substance vehicle at day 3 and 4 IP administrations of test reference vehicle (Antibody) on day 10,14,17 and 21. [0326] The animals from group 2 received one IV injection of 50 μg of CI II-TRP2 together with 100 ng of ABX196 on day 3. [0327] The animals from group 3 received one IV injection of 50 μg of CL II-TRP2 together with 100 ng of ABX196 on day 3 and 4 IP administrations of anti-PD-1 antibody on day 10, 14, 17 and 21.

[0328] The treatment schedule is summarized in the table below:

TABLE-US-00016 No. Treatment Group Animals Treatment Dose Route Schedule 1 10 Vehicle — IV/IP D3-IV/TWx2 IP 2 10 CL II-TRP2/ 50 μg/100 ng IV Q1Dx1 on D3 ABX196 3 10 CL II-TRP2/ 50 μg/100 ng IV Q1Dx1 on D3 ABX196 Anti-PD-1 Ab 10 mg/kg IP Q1Dx on D10/ D14
Immune T cell infiltrates in the tumor were evaluated on D13 and D17. At the time of termination, tumors from 5 mice of each group were collected. For group 2 and 3, balanced proportion between responding and non-responding mice was collected at each day of collection, i.e. D13 and D17.
After removal from the mice, each tumor was weighted and transferred to tubes with RPMI culture medium. The tumor was mechanically disrupted in small pieces of a few mm size with a scalpel and finally crushed with a 1 mL syringe plunger on a 70 μm sieve (Ref. 352350, FALCON). Cells were next counted after trypan blue staining and one million of cells were centrifuged and resuspended in staining buffer (PBS (ref: 17-516F, Lonza), 0.2% BSA (ref: A7030, Sigma, Saint-Quentin-Fallavier, France), 0.02% NaN.sub.3 (ref: S2002, Sigma)). The antibodies directed against the chosen markers were added to the tumor cell suspension, according to the conditions described by the supplier for each antibody. The markers CD45, CD3, CD4, CD8 were detected on the cell surface. The markers FoxP3, TNFalpha, Perforin, Granzyme were detected intracellularly, after cell permeabilization. Isotype control antibodies was used in each case as negative control. The panel of antibodies used to measure the two following populations are listed in the table below: [0329] Treg cell population: CD45.sup.+/CD3.sup.+/CD4.sup.+/CD8-/FoxP3+ [0330] T Cytotoxic populations: CD45.sup.+/CD3.sup.+/CD8.sup.+/TNFα/Perforin/Granzyme

TABLE-US-00017 Reference of Specificity Reference fluorochrome Provider isotype Isotype fluorochrome Provider FoxP3 130-093- PE Miltenyi A07796 IgG1 PE Beckman 014 Biotec Coulter CD8a 553036 PerCP BD 553933 IgG2a PerCP BD Biosciences Biosciences CD3 561389 V450 BD 560457 IgG2 V450 BD Biosciences Biosciences CD4 130-102- VioGreen Miltenyi 130-102-659 IgG2b VioGreen Miltenyi 444 Biotec Biotec CD45 557659 APC-Cy7 BD 552773 IgG2b APC-Cy7 BD Biosciences Biosciences TNFalpha 506308 APC biolegend 400412 IgG1 APC biolegend Perforin 12-9392- PE eBioscience 12-4321 IgG2a PE eBioscience 82 Granzyme 11-8898- FITC ebioscience 553988 IgG2b FITC BD 82 Biosciences
The mixture of cells and antibodies was incubated for 20 to 30 minutes at room temperature in the dark, washed, and resuspended in 200 μL staining buffer. All samples were stored on ice and protected from light until Flow Cytometry analysis.
The stained cells were analyzed with a CyFlow® space flow cytometer (LSR II, BD Biosciences) equipped with 3 excitation lasers at wavelengths 405, 488 and 633 nm. Flow cytometry data were acquired until either 10,000 mCD45+ events are recorded for each sample, or for a maximum duration of 2 minutes.

Example 5: Study of ABX196 Administered Intravenously or Intratumorally in Combination with Anti-PD-1 Antibodies

1. Study Aims

[0331] The study was performed on a syngeneic in vivo melanoma B16F10 tumor-bearing mouse model. B16F10 tumor cells were subcutaneously inoculated in immuno-competent C57BL/6 mice.

[0332] Melanoma B16F10 tumor cells were subcutaneously inoculated at day 0 by injecting cells in the flank of 8-10 weeks-old female C57BL/6 mice, and animals were then randomly assigned into control (vehicle PBS-treated) and treatments groups.

[0333] For the anti-PD-1 antibodies treatment, mice were intraperitoneally (IP) injected with anti-PD-1 antibodies at Day 6, 9, 12, 15 and 21. Furthermore, ABX196 was administered at 1 dose, intravenously or intratumorally, with one administration at Day 10 when mean of tumor size reached 100 mm.sup.3.

[0334] The animal pharmacological groups for anti-tumor efficacy assessment were organized as such: [0335] Control vehicle-treated group (PBS according to anti-PD-1 treatment schedule) [0336] Anti-PD-1-treated group (at 1 dose, intraperitoneal administration) [0337] ABX196-treated group (at 1 dose, Intravenous (iv) administration at Day10) [0338] Combination-treated group (ABX196 at 1 dose, Intravenous (iv) administration at Day10 in combination with anti-PD-1 antibodies at 1 dose, intraperitoneal (ip) administration) [0339] ABX196-treated group (at 1 dose, Intratumoral (it) administration at Day10) [0340] Combination-treated group (ABX196 at 1 dose, Intratumoral (it) administration at Day10 in combination with anti-PD-1 antibodies at 1 dose, intraperitoneal (ip) administration)

With:

[0341] 14-15 mice per group
90 mice in total for the in vivo efficacy assessment

2. Experimental Procedure

2.1 Animals

[0342] Mice (Mus musculus), Strain C57BL/6, Female

Provider: Charles River Laboratories—BP 0109—F 69592 L'Arbresle Cedex

[0343] The animals were used between 8 and 10 weeks. 6 weeks-old mice were placed in acclimatization for about 14 days (B16F10 tumor cells implantation at 8 weeks old). 90 animals were used for this study.

[0344] Ventilation and air treatment was performed through frequent turnover (8-20 volumes/hour depending on the density of animals housed) and temperature controlled between 22 and 25° C. Humidity was maintained between 40 and 70%. Artificial lighting was maintained 12 hours a day. Quantity and access to food (pellets) and drink (tap water) was checked daily. Mice were housed in collective cages, with 10 animals per cage (820 cm2 cage). Cages were renewed once a week by animal care taker.

2.2 Animal Monitoring

[0345] Tumor volume and body weight of the animals were measured and recorded three times per week. A tumor volume exceeding 2000 mm3 or a weight loss greater than 15% relative to the initial weight of the animal were considered as endpoints.

[0346] Similarly, if the mouse was (i) prostrate, or (ii) no longer cleaned its coat (hair bristling and not glossy), (iii) was less mobile, this was also considered as an endpoint.

[0347] When at least one of these conditions was met then the mice were sacrificed by cervical dislocation.

[0348] To minimize pain, suffering and anxiety related to the model, the animals were monitored every 2 days. The observation included reliable criteria such as weight loss (15%) and change of posture. An environmental enrichment was made to minimize anxiety (Plastic tubes).

[0349] Pain Procedures:

[0350] For operation steps (subcutaneous injection of tumor cells), anesthesia was achieved using Ketamine (0.33 mg/ml) and Xylazine (33.6 μg/ml) by intraperitoneal injection.

2.3 Melanoma Cancer Cells Implantation Procedure

[0351] Cancer Cell Line:

[0352] Melanoma B16F10 cells were cultured in vitro according to provider's specifications, RPMI 1640 supplemented with FBS at the final concentration of 10%. Before implantation in mice, cell viability was assessed using Trypan Blue exclusion and a cell suspension was prepared according to the viable cell count.

[0353] Induction of Melanoma B16F10 Tumors in Mice:

B16F10 cells were implanted subcutaneously in the right flank of C57BL/6 immunocompetent mice (8 week-old female). The implantation procedure was as follows (all steps were carried out under sterile laminar flow conditions):
Mice (body weight around 20 g) were anesthetized with an intraperitoneal injection of 90 μL of anesthetic (ie 1.5 mg/Kg Ketamine and Xylazine 150 μg/Kg). The cells to be implanted were resuspended in sterile PBS and the volume needed to implant was loaded in 1 ml syringe with a 25G needle (1 000,000 cells/100 μL).

2.4 Pharmacological Treatments

[0354] Anti-PD-1 Antibodies Treatment

Anti-PD-1 monoclonal antibodies (α-PD-1) treatment schedule was applied, at 100 μg by i.p. injection. Treatment was repeated 4 times, at Days 6, 9, 12, 15 and 18.
27G gauge needle was used for i.p. injections.

Materials:

[0355] Anti-PD-L1 monoclonal antibody (α-PD-1)-(clone RMP1-14) [0356] Anti-PD-1 monoclonal antibody preparation for i.p administration [0357] Anti-PD-1 mAb dissolved in phosphate buffered saline (PBS) solution.
Dulbecco's PBS was used to dissolve anti-PD-1 Ab at a concentration of 1.0 mg/mL (volume of 100 uL). The stock solution was then aliquoted (amount for 1 day treatment) and stored at −20° C. [0358] ABX 196 treatment
ABX196 was administered at Day 10 either: [0359] by intraveneous (i.v) injection at the dose of 100 ng per mouse; or [0360] by intratumoral (i.t) injection at the dose of 10 ng per mouse

Materials:

ABX196 for i.v Administration

[0361] ABX196 was provided as a solution at 250 μg/mL in PBS. An ABX196 solution at 1 μg/mL was prepared and stored at 4° C. At Day 10, 100 μL of the 1 μg/mL ABX196 solution was injected in the tail vein of the mice from Groups 3 & 4.

ABX196 for i.t Administration

[0362] ABX196 was provided as a solution at 250 μg/mL in PBS. An ABX196 solution at 0.4 μg/mL was prepared and stored at 4° C. At Day 10, animals from Groups 5 & 6 was anesthetized and 25 μL of the 0.4 μg/mL ABX196 solution were injected in the tumor.

Summary of Pharmacological Treatments

[0363]

TABLE-US-00018 Tumor Group cell line Treatment n Host Dose Schedule Route 1 B16F10 Vehicle 20 C57BL/ PBS Days 6, i.p 6 9, 12, 15 and 18 2 B16F10 Anti-PD-1 20 C57BL/ 100 μg Days 6, i.p mAb 6 9, 12, 15 and 18 3 B16F10 ABX196 20 C57BL/ 100 ng Day 10 i.v 6 4 B16F10 Anti-PD-1 20 C57BL/ 100 μg Days 6, i.p mAb + 6 Anti- 9, 12, ABX196 PD- 15 and 18 1 mAb 100 ng Day 10 i.v ABX196 5 B16F10 ABX196 20 C57BL/6 10 ng Day 10 i.t 6 B16F10 Anti-PD-1 20 C57BL/6 100 μg Days 6, i.p mAb + Anti- 9, 12, ABX196 PD- 15 and 18 1 mAb 10 ng Day 10 i.t ABX196

3. Results

[0364] Starting from day 6 after B16F10 cell inoculation, all experimental animal groups were monitored 3 times per week over a 4 weeks period for the following parameters: [0365] Tumor size: measured by physical examination, 3 times per week, [0366] Body weight: monitored 3 times per week, and [0367] Survival: represented in a Kaplan-Meier plot. 3 times per week.

[0368] Anti-tumor activity index calculation was the same as for example 1 for the T/C (%) index and the TGI (%).

[0369] 1. Anti-Tumor Response Assessment

[0370] a) Tumor Volume

[0371] The results are given in FIGS. 5 and 6 showing the mean tumor volume (mm.sup.3) of B16F10 bearing mice exposed to the different treatments, post tumor challenge.

[0372] In particular, the results show that the combination of Anti-PD-1 Ab with ABX196 according to the invention is more effective in decreasing the tumor volume than ABX196 alone or Anti-PD-1 Ab alone, administered either i.v. or i.t.

[0373] The results are also shown in the Tables below.

[0374] Statistical analysis of mean tumor volume at Day 17 between the 6 tested groups.

[0375] Unpaired t test two tailed, with Welch's correction

TABLE-US-00019 Mean Tumor αPD-1 + αPD-1 + Volume at Day 17 ABX196 ABX196 ABX196 ABX196 (p value) αPD-1 i.v i.v i.t i.t Vehicle 0.4473 0.2838 0.0183 0.8942 0.0255 αPD-1 0.7081 0.012  0.2031 0.0245 ABX196 i.v 0.0054 0.0554 0.0169 αPD-1 + <0.0001   0.6891 ABX196 i.v ABX196 i.t 0.0001 αPD-1 + ABX196 i.t

TABLE-US-00020 Mean Tumor αPD-1 + αPD-1 + Volume at Day 17 ABX196 ABX196 ABX196 ABX196 (Significativity) αPD-1 i.v i.v i.t i.t Vehicle NS NS * NS * αPD-1 NS * NS * ABX196 i.v ** NS * αPD-1 + **** NS ABX196 i.v ABX196 i.t *** αPD-1 + ABX196 i.t Vehicle is the reference groups. Mean +/− SEM. Log-rank (Mantel-Cox) test was performed using Graph Pad Prism .fwdarw. Mean +/− SEM. Log-rank (Mantel-Cox) test was performSignificative; * p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001

[0376] b) Survival

[0377] The results are given in FIGS. 7 and 8 showing the survival of B16F10 bearing mice exposed to the different treatments, post tumor challenge.

[0378] In particular, the results show that the combination of Anti-PD-1 Ab with ABX196 according to the invention lead to a higher percentage of survival than ABX196 alone or than Anti-PD-1 Ab alone, administered either i.v. or i.t.

[0379] c) Antitumor Activity Index

TABLE-US-00021 ABX196 Anti-PD- ABX196 Anti-PD-1 + Anti-PD-1 + iv 1 it ABX196 iv ABX196 it T/C 51 76 53 42 37

[0380] Conclusion:

[0381] This study aimed at evaluating the benefit of ABX196 combined with PD-1 blockade in a syngeneic mouse model of melanoma.

[0382] There was no effect of anti-PD-1 antibody, even a supplemental dose has been administered at Day 18. The lack of effect was observed at both tumor volume (FIGS. 5 and 6) and survival levels (FIGS. 7 and 8).

[0383] Also, a transient effect of ABX196 was observed at early time points after its administration (either iv or it), it didn't translated into a significant effect.

[0384] However, it was interesting to observe a synergistic effect between ABX196 and the anti-PD1 Ab. Indeed, ABX196 was able to enhance the anti-PD-1 effect—which was not effective at all. This synergistic effect was observable with both iv and it administration of ABX196. In particular, it was observable at tumor volume level (FIGS. 5 and 6) with significative difference between anti-PD-1 and anti-PD-1+ABX196 (iv or it) (p=0,012 and 0,0245 respectively). Survival data clearly demonstrated a benefit of combinatory ABX196 and the anti-PD-1 Ab vs vehicle.

Example 6: Determination of the Anti-Tumoral Activity of ABX196 Administered Systemically Alone or Combined with Anti-PD-1 Antibody in Colon and Bladder Cancers

1. Materials and Methods

[0385] 1.1. Test and Reference Substances

1.1.1. Test Substances

[0386] ABX196.

1.1.2. Reference Substances

[0387] Anti-PD-1 antibody (ref.: BE0146, BioXcell; clone: RMP1-14, reactivity: mouse; isotype: Rat IgG2a; storage conditions: +4° C.).

1.1.3. Test and Reference Substances Vehicles

[0388] Anti-PD-1 antibody were prepared in phosphate buffered saline (PBS) or other suitable vehicle according to manufacturer's recommendation.
ABX196 was provided as a solution at 250 μg/mL.

[0389] 1.2. Treatment Doses

ABX196 was administered at the dose of 100 ng per mouse. The anti-PD-1 antibody was administered at the dose of 10 mg/kg.

[0390] 1.3. Routes of Administration

Test substance was injected by the intravenous route in the caudal vein of mice (IV, bolus). Anti-PD-1 antibody was injected into the peritoneal cavity of mice (Intraperitoneally, IP) In all groups, ABX196 was administered at a fixed dose volume of 100 μL (i.e. approximately 5 mL/kg/adm. for one mouse weighing 20 g).
Anti-PD-1 antibody was administered at a dose volume of 10 mL/kg/adm.

[0391] 1.4. Cancer Cell Line and Culture Conditions

1.4.1. Cancer Cell Line

[0392] The cell lines that were used are detailed in the table below:

TABLE-US-00022 Cell line Type Specie Origin CT-26 Colon adenocarcinoma mouse ATCC a MBT-2 Bladder carcinoma mouse ATCC a

a American Type Culture Collection, Manassas, Va., USA

[0393] The CT-26 cell line is an N-nitroso-N-methylurethane-(NNMU) induced, undifferentiated colon carcinoma cell line of BALB/C mice. [0394] The murine MBT-2 cell line was derived from a carcinogen-induced bladder tumor in C3H/HeJ mice. The MBT-2 cell line was obtained from Dr Cozzi, Memorial Sloan Kettering Cancer Center (New York, USA).

1.4.2. Cell Culture Conditions

[0395] Tumor cells were grown as 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). Tumor cells were adherent to plastic flasks. For experimental use, tumor cells were detached from the culture flask by a 5-minute treatment with trypsin-versene (ref: BE02-007E, Lonza), in Hanks' medium without calcium or magnesium (ref: BE10-543F, Lonza) and neutralized by addition of complete culture medium.

[0396] The cells were counted and their viability were assessed by 0.25% trypan blue exclusion assay.

[0397] 1.5. Animals

[0398] Sixty-three (63) healthy female BALB/c mice, 6-7 weeks old, were obtained from CHARLES RIVER (L'Arbresles) for each models syngeneic to this strain of mice (i.e. CT-26) and 68 (sixty-eight) healthy female C3H/HeJ (C3H/HeOuJ) mice, 6-7 weeks old, were obtained from The Jackson Laboratory (Bar Harbor, Me.) for the MBT-2 model.

[0399] Animals were maintained in SPF health status according to the FELASA guidelines. Animal housing and experimental procedures were realized according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals. The housing conditions of the animals were the same as in example 1.

2. Experimental Design and Treatments

[0400] 2.1. Induction of CT-26, and MBT-2 Tumors in Animals

[0401] Tumors were induced by subcutaneous injection of 1×10.sup.6 of CT-26 cells in 200 μL of RPMI 1640 into the right flank of 63 female BALB/C mice. MBT-2 tumors were induced by subcutaneous injection of 1×10.sup.6 cells in 200 μL of RPMI 1640 into the right flank of sixty-eight (68) female animals.

[0402] 2.2. Treatment Schedule

[0403] The treatment started when the tumors reached a mean volume of 80-120 mm.sup.3. Forty-eight animals (48) out of sixty-three (63) for CT-26 model or sixty-eight (68) for the MBT-2 model were randomized according to their individual tumor volume into 4 groups each of 12 animals using Vivo Manager® software (Biosystemes, Couternon, France). A statistical test (analysis of variance, ANOVA) was performed to test for homogeneity between groups. The treatment schedule was as follows: [0404] Animals from group 1 received an IV injection of ABX196 vehicle and an IP injection of anti-PD-1 antibody vehicle, [0405] Animals from group 2 received an IV injection of 100 ng of ABX196, [0406] Animals from group 3 received twice weekly administrations of anti-PD-1 antibody, [0407] Animals from group 4 received an IV injection of 100 ng of ABX196 and twice weekly administrations of anti-PD-1 antibody.
The treatment schedule is summarized in the table below:

TABLE-US-00023 Dose No (mg/kg/ Adm. Treatment Group animals Treatment adm) Route schedule 1 12 Vehicle — IV ABX196 Vehicle Anti- — IP TWx2 PD-1 Ab 2 12 ABX196 100 ng IV Q1Dx1 3 12 Anti-PD-1 Ab 10 IP TWx2 4 12 ABX196 100 ng IV Q1Dx1 Anti-PD-1 Ab* 10 IP TWx2 *IP injection of anti-PD-1 Ab initiated after the first IV administration of ABX196 (Concomittant administration) without any delay.

[0408] 2.3. Animal Monitoring

2.3.1. Clinical Monitoring

[0409] All study data, including animal body weight measurements, tumor volume, clinical and mortality records, and treatment were scheduled and recorded on Vivo Manager® database (Biosystemes, Dijon, France).

[0410] The viability and behavior were recorded every day. Body weights were measured twice a week. The length and width of the tumor were measured twice a week with calipers and the volume of the tumor was estimated by the formula:

[00004]$\mathrm{Tumor}\mathrm{volume}=\frac{{\mathrm{width}}^2\times \mathrm{length}}{2}$

[0411] Anti-tumor activity index calculation was the same as for example 1 for the T/C (%) index and the TGI (%).

[0412] 2.4. Statistical Tests

[0413] All statistical analyses were performed using Vivo Manager® software (Biosystemes, Couternon, France). Statistical analysis of mean body weights, MBWC, mean tumor volumes at randomization, mean tumor volumes V, mean times to reach V and mean tumor doubling times were performed using ANOVA. Pairwise tests were performed using the Bonferroni/Dunn correction in case of significant ANOVA results. The log-Rank (Kaplan-Meier) test was used to compare the survival curves. A p value<0.05 was considered as significant.

[0414] 2.5. Humane Endpoints

The humane endpoints were the same as for example 1.

[0415] 2.6. Necropsy

[0416] Necropsy (macroscopic examination) was performed on all terminated animals in the study, and, if possible, on all euthanized moribund or found dead animals.

[0417] 2.7. Anesthesia

[0418] Isoflurane gas anesthesia was used for all procedures: tumor inoculation and i.v. injections.

[0419] 2.8. Analgesia

[0420] Non-pharmacological care was provided for all painful procedures.

Additionally, pharmacological care not interfering with studies (topic treatment) could be provided at the recommendation of the attending veterinarian.

[0421] 2.9. Euthanasia

[0422] Euthanasia of animals was performed by gas anesthesia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.

3. Results

[0423] 3.1. Antitumor Activity of the Tested Treatments in Mice Bearing Subcutaneous CT-26 Colorectal Cancer

[0424] The results are given in FIG. 9 showing the mean tumor volume (mm.sup.3) of CT-26 bearing mice exposed to the different treatments, post tumor challenge. In particular, the results show that the combination of the Anti-PD-1 Ab with ABX196 according to the invention is more effective in decreasing the tumor volume than ABX196 alone or the Anti-PD-1A b alone.

The antitumor activity indexes are given in the Table below:

TABLE-US-00024 ABX196 Anti-PD-1 ABX196 + Anti-PD-1 T/C 51 58 40 TGI 72 42 80

[0425] 3.2. Antitumor Activity of the Tested Treatments in Mice Bearing Subcutaneous MBT-2 Bladder Cancer

[0426] The results are given in FIG. 10 showing the mean tumor volume (mm.sup.3) of MBT-2 bearing mice exposed to the different treatments, post tumor challenge. In particular, the results show that the combination of the Anti-PD-1 Ab with ABX196 according to the invention is more effective in decreasing the tumor volume than ABX196 alone or the Anti-PD-1 Ab alone.

The antitumor activity indexes are given in the Table below:

TABLE-US-00025 ABX196 Anti-PD-1 ABX196 + Anti-PD-1 T/C 57 78 36 TGI 43 22 64

[0427] 3.3. Survival

[0428] The results are given in FIG. 11 showing the survival of MBT-2 bearing mice exposed to the different treatments, post tumor challenge and in the Table below

Median Survival (Days) Per MBT-2 Bearing Mice from Experimental Groups

[0429]

TABLE-US-00026 Treatment Median Survival (days) Vehicle 23 ABX196 27 Anti-PD-1 28 ABX196 + Anti-PD-1 34

[0430] The combination of ABX196 with the Anti-PD-1 antibodies significantly improves the survival of the mice compared to the treatments with ABX196 or Anti-PD-1 antibodies alone.

Statistical Analysis of Mice Survival Between Treatments

[0431]

TABLE-US-00027 ABX196 + Treatment Vehicle ABX196 Anti-PD-1 Anti-PD-1 Vehicle *0.02 *0.02      <0.0001****   ABX196   *0.02     NS ***0.0002    Anti-PD-1   *0.02     NS 0.01*  ABX196 + <0.0001**** ***0.0002  0.01* Anti-PD-1 Global comparison of all survival curves: p value 0.0045 (***)(Log-Rank (Mantel-cox) test) Summary of pairwise comparisons

4. Conclusion

[0432] The tested treatments, either alone or in combination were well tolerated by the animals and no drug-related severe toxicity nor death were recorded.

Antitumor Activity of ABX196 Plus Anti-PD-1 on Colorectal Cancer

[0433] The tumor growth delay of mice treated with ABX196 plus anti-PD-1 is significantly improved compared to vehicle-treated group (see FIG. 9). The T/C and TGI ratio clearly demonstrate the synergistic effect between ABX196 and anti-PD-1 antibodies. Only the group with ABX196+Anti-PD-1 treatment presents a T/C<42%.

Antitumor Effect on Bladder Cancer

[0434] Combination of ABX196+anti-PD-1 antibodies reduces significantly the tumor growth (see FIG. 10). The T/C and TGI ratio clearly demonstrate the synergy between between ABX196 and anti-PD-1 antibodies. The survival of mice treated with ABX196+anti-PD-1 is significantly improved in comparison to the vehicle-treated group (see FIG. 11). For combination groups, the anti-tumoral activity is further improved when compared to ABX196 alone or anti-PD-1 alone with a median survival of 27 or 28 days that increases to 34 days, when combined with anti-PD-1 antibody.

Example 7: Evaluation of the Anti-Tumoral Activity of ABX196 in Combination with Doxorubicin or Sorafenib or Anti-PD-1 Antibody in the Orthotopic Hepa 1-6 Hepatocarcinoma Model

[0435] 1. Materials and Methods

1.1 Test and Reference Substances

1.1.1 Test Substances

[0436] ABX196

1.1.2 Reference Substances

[0437] Doxorubicin: DOXO CELL, Cell Pharm.

[0438] Sorafenib (Nexavar®, Bayer Pharma, 200 mg/pill).

[0439] Anti-PD-1 antibody (ref.: BE0146, BioXcell; clone: RMP1-14, reactivity: mouse; isotype:

[0440] Rat IgG2a; storage conditions: +4° C.).

1.2. Test and Reference Substances Vehicles

[0441] Doxorubicin was diluted in NaCl 0.9%

[0442] Anti-PD-1 antibody was prepared in phosphate buffered saline (PBS) or other suitable vehicle according to manufacturer's recommendation.

[0443] Each day of administration to mice, Sorafenib pills were crushed and dissolved first in DMSO (ref: 41640, Fluka, Sigma, Saint Quentin Fallavier, France), then in Tween 20 (ref: P9416, Sigma) followed by NaCl (0.9%) addition (final ratio DMSO/Tween 20/NaCl (0.9%): 5/5/90 v/v) to reach the appropriate concentration of 10 mg/mL.

[0444] The ABX196 was provided as a solution at 250 μg/mL. Dilution of ABX196 was performed in PBS buffer.

[0445] The vehicle solution used in group 1 at day 5 contained DMSO diluted in phosphate buffered saline (PBS) at the same final concentration as the ABX196 test item.

1.3. Treatment Doses

[0446] The ABX196 was administered at the dose of 100 ng per mouse.
Doxorubicin was administered at the dose of 12 mg/kg.
The anti-PD-1 antibody was administered at the dose of 10 mg/kg.
Sorafenib was administered at the dose of 100 mg/kg.

1.4. Routes of Administration

[0447] ABX 196 was injected by the intra-venous route in the caudal vein of mice (IV, bolus). Doxorubicin was administered intravenously in the caudal vein of mice (IV, bolus) Anti-PD-1 antibody was injected into the peritoneal cavity of mice (Intraperitoneally, IP) Sorafenib was administered by oral gavage (per os, PO) via a cannula.

[0448] In all groups, ABX196 was administered at a fixed dose volume of 100 μL (approximately 5 mL/kg/adm for a mouse weighing 20 g). Doxorubicin, anti PD-1 antibody and Sorafenib were administered at a dose volume of 10 mL/kg/adm. according to the most recent body weight of mice.

1.5. Cancer Cell Line and Culture Conditions

1.5.1. Cancer Cell Line

[0449] The cell line that was used is detailed in the table below:

TABLE-US-00028 Cell line Type Origin Hepa 1-6 Hepatocellular carcinoma ATCCa

a: American Type Culture Collection, Manassas, Va., USA

[0450] The Hepa 1-6 cell line is a derivative of the BW7756 mouse hepatoma that arose in a C57/L mouse.

1.5.2. Cell Culture Conditions

[0451] Tumor cells were grown as monolayer at 37° C. in a humidified atmosphere (5% CO2, 95% air). The culture medium was DMEM containing 4 mM L-glutamine (ref: BE12-604F, Lonza, Verviers, Belgium), 4.5 g/l glucose and 1 mM NaPyr supplemented with 10% fetal bovine serum (ref: 3302, Lonza). The cells were adherent to plastic flasks.

[0452] 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 was assessed by 0.25% trypan blue exclusion assay.

1.5.3. Animals

[0453] One hundred (100) healthy female C57BL/6 (C57BLI6J) mice, 5-6 weeks old, were obtained from JANVIER LABS (Le Genest-Saint-Isle).

[0454] Animals were maintained in SPF health status according to the FELASA guidelines. Animal housing and experimental procedures were realized according to the French and European Regulations and NRC Guide for the Care and Use of Laboratory Animals. The housing conditions were the same as in example 1.

1.6. Magnetic Resonance Imaging

[0455] All imaging experiments were performed on a 4.7T horizontal magnet (PharmaScan, Bruker Biospin GmbH, Germany) equipped with an actively shielded gradient system. All the MR images were acquired under ParaVision (PV5.1, Bruker Biospin).

1.6.1. Coils and Cradles

[0456] Mice were positioned prone in a dedicated mouse body cradle which was slidded in a volume coil (38 mm internal diameter) within the Pharmascan.

1.6.2. Anesthesia and Physiological Monitoring

[0457] During all the acquisitions, mice were continuously anesthetized using isoflurane (Minerve, Bondoufle, France) in a mixture of air via a nose piece. The mouse's breathing rate was continuously monitored using a pressure sensor taped on its abdomen. Physiological signals were monitored via a laptop placed next to the MRI workstation and connected to the sensors by fiber optic cables (SA Instruments, USA).

1.6.3. MR Imaging Sequences

Calibration and Positioning

[0458] After positioning the animal in the magnet, scout images were acquired for calibration purposes. Sagittal, coronal and axial slices were acquired. At the beginning of this acquisition, automated adjustments were performed to optimize shim, RF power and amplification of the MR signal.

TABLE-US-00029 TE/TR Matrix FOV No of Slice thickness/ No Type (ms) size (mm) slices spacing (mm) averages FLASH 6/100 128 × 128 60 × 60 NA 1/NA 1
The sequence used during this step has the following characteristics:
Where: TE is the time to echo, TR is the repetition time, FOV is the field of view.

1.6.4. T2-Weighted (T2w) Anatomical Imaging—Axial Orientation

[0459] Anatomical images were acquired using a T2w RARE sequence. The sequence used during this step has the following characteristics:

TABLE-US-00030 Slice TE/TR Matrix FOV No of thickness/ No Type (ms) size (mm) slices spacing (mm) averages RARE 38/2880 256 × 192 37 × 28 15 0.8/0.8 3 (Rare factor: 8)
If necessary, the FOV size and the sequence parameters were adapted to provide the best imaging results.

1.6.5. Image Processing

[0460] All the MR images were transferred to a Windows®-based workstation to be analyzed under ImageJ. Tumor invasion was evaluated semi-quantitavely by a visual evaluation of the percentage of tumor in the entire liver.

2. Experimental Design and Treatments

2.1. Induction of Hepa 1-6 Tumors in Animals by Intrasplenic Injection

[0461] One million (1×106) of tumor cells in 50 μL of RPMI 1640 medium were transplanted via intra-splenic injection into 100 C57BL/6 mice. Briefly, a small left subcostal flank incision was made. Spleen was exteriorized. The spleen was exposed on the sterile gauze pad, and injected under visual control with the cell suspension with a 27-gauge needle. After the cell inoculation, the spleen was excised. The day of tumor cell injection was considered as D0.

2.2. Treatment Schedule

[0462] The treatment started at D5. Ninety-nine animals (99) out of one hundred (100) were randomized according to their individual body weight into 3 groups of 13 animals and 5 groups of 12 animals using Vivo Manager® software (Biosystemes, Couternon, France). A statistical test (analysis of variance, ANOVA) was performed to test for homogeneity between groups.

The treatment schedule was as follows: [0463] Animals from group 1 received one IV injection of ABX196 vehicle at day 5, [0464] Animals from group 2 received one daily PO administration of sorafenib at 100 mg/kg/adm for 21 consecutive days, starting at day 5, [0465] Animals from group 3 received one IV injection of 100 ng of ABX196 on day 5 and one daily PO administration of sorafenib at 100 mg/kg/adm for 21 consecutive days, starting at day 5, [0466] Animals from group 4 received one IV injection of Doxorubicin at 12 mg/kg on day 5, [0467] Animals from group 5 received one IV injection of 100 ng of ABX196 and one IV injection of Doxorubicin at 12 mg/kg on day 5, [0468] Animals from group 6 received 4 IP administrations of anti-PD-1 antibody on day 7, 10, 14 and 17 (Twice weekly ×2), [0469] Animals from group 7 received one IV injection of 100 ng of ABX196 on day 5 and 4 IP administrations of anti-PD-1 antibody on day 7, 10, 14 and 17 (Twice weekly ×2).
The treatment schedule is summarized in the table below:

TABLE-US-00031 No Dose Adm. Group animals Treatment (mg/kg/adm) Route Treatment schedule 1 13 Vehicle NA IV Q1Dx1 on D5 2 13 Sorafenib 100 PO Q1Dx21 start on D5 3 12 ABX196 100 ng IV Q1Dx1 on D5 Sorafenib 100 PO Q1Dx21 start on D5 4 12 Doxorubicin  12 IV Q1Dx1 on D5 5 12 ABX196 100 ng IV Q1Dx1 on D5 Doxorubicin  12 IV Q1Dx1 on D5 6 12 Anti-PD-1  10 IP TWx2 on D7/D10/D14/D17 7 12 ABX196 100 ng IV Q1Dx1 on D5 Anti-PD-1  10 IP TWx2 on D7/D10/D14/D17

2.3. Blood Collection

[0470] At D7 and D22, approximately 120 μL of blood were collected by jugular vein puncture into blood collection tubes with clot activator. Tubes were centrifuged 30 minutes after sampling at 1,300 g for 10 minutes at room temperature to obtain serum. The serum samples was stored in propylene tubes at −80° C. All collected serum samples at D22 were analyzed for determination of circulating AFP level by ELISA analysis (Dosage Mouse α-Fetoprotein/AFP, ref: MAFP00, RD Systems).

2.4. MRI Imaging Time Points

[0471] At D19 and D20, 5 mice per group from group 1 to 8 were imaged (40 animals per day). A semi-quantitative analysis was then performed.

2.5. Mice Termination

[0472] At the time of final mice termination (around D60), liver was weighted. The number of metastases was evaluated macroscopically and the localization, the appearance (shape, colour, consistency) and the size of each of them was recorded. Macroscopic photography of the liver was taken.

[0473] Livers/tumors from all animals, sacrificed either for ethical reason or at final termination, were cut into slices 4 mm tick and fixed in 4% neutral buffered formalin for 24h to 48h, and then embedded in paraffin (Histosec®, Merck, Darmstadt, Germany).

2.6. Animal Monitoring

2.6.1. Clinical Monitoring

[0474] All study data, including animal body weight measurements, clinical and mortality records, and treatment were scheduled and recorded on Vivo Manager® database (Biosystemes, Dijon, France).

[0475] The viability and behavior were recorded every day. Body weights were measured thrice a week.

2.6.2. Humane Endpoints

[0476] Abdomen diameter superior to 25 mm
Signs of pain, suffering or distress: pain posture, pain face mask, behavior,
Tumors interfering with ambulation or nutrition,
20% body weight loss remaining for 3 consecutive days,
Poor body condition, emaciation, cachexia, dehydration,
Prolonged absence of voluntary responses to external stimuli,
Rapid labored breathing, anemia, significant bleeding,
Neurologic signs: circling, convulsion, paralysis,
Sustained decrease in body temperature,
Abdominal distension.

2.6.3. Necropsy

[0477] Necropsy (macroscopic examination) was performed on all terminated animals in the study, and, if possible, on all euthanized moribund or found dead animals.

2.6.4. Surgery

[0478] Surgery methods were described in Operating Procedures approved by IACUC.

2.6.5. Anesthesia

[0479] Isoflurane gas anesthesia was used for all procedures: surgery and blood collection.

2.6.6. Analgesia

[0480] Multimodal carprofen/buprenorphine or xylocaine/buprenorphine analgesia protocol was adapted to the severity of the surgical procedure. Non-pharmacological care was provided for all painful procedures. Additionally, pharmacological care no interfering with studies (topic treatment) could be provided at the recommendation of the attending veterinarian.

2.6.7. Euthanasia

[0481] Euthanasia of animals was performed by gas anesthesia over-dosage (Isoflurane) followed by cervical dislocation or exsanguination.

3. Data Presentation

[0482] 4.1. Health parameters

[0483] The following evaluation criteria of health were determined using Vivo Manager® software (Biosystemes, Couternon, France): [0484] Individual and/or mean (or median) body weights of animals, [0485] Mean body weight change (MBWC): Average weight change of treated animals in percent (weight at day B minus weight at day A divided by weight at day A) was calculated. The intervals over which MBWC was calculated was chosen as a function of body weight curves and the days of body weight measurement.

3.2. Efficacy Parameters

[0486] The treatment efficacy was assessed in terms of the effects of the test substance on the tumor volumes of treated animals relative to control animals. The following evaluation criteria of antitumor efficacy were determined: [0487] Individual and/or mean (or median) measurement at D19-20 of tumor invasion within the liver using MRI imaging, [0488] Measurement of circulating α-Fetoprotein in the plasma at D22, [0489] Liver weight measured at termination, [0490] Survival curves, [0491] Median survival times.

Results

Tumor Invasion

[0492] The results are given in FIG. 12 showing the percentage of tumor invasion of liver from each treated groups at day 20.

Statistical Analysis of Tumor Invasion Between Vehicle and Each Group at Day 20

[0493] Kruskal-Wallis test p value<0,0001 (****)
Pair Wise Comparison with Dunn's Test

TABLE-US-00032 Mean Dunn's multiple comparisons test rank diff. Significant? Summary Vehicle vs. Sorafenib 8.644 No ns Vehicle vs. ABX196/Sorafenib 29.35 Yes ** Vehicle vs. Doxorubicin 28.52 Yes ** Vehicle vs. ABX196/Doxorubicin 27.83 Yes * Vehicle vs. Anti-PD-1 28.14 Yes ** Vehicle vs. ABX196/Anti-PD-1 32.77 Yes ***

Summary of Alive Animals and Tumor Invasion at Day 61

[0494]

TABLE-US-00033 Fraction Fraction without Group alive detectable metastasis 1 Vehicle  4/13  3/13 2 Sorafenib  5/13  5/13 3 Sorafenib + ABX196 11/12 10/12 4 Doxorubicin  9/12  9/12 5 Doxorubicin + ABX196  8/12  6/12 6 Anti-PD-1 11/12 10/12 7 Anti-PD-1 + ABX196 12/12 12/12

CONCLUSION

[0495] This experiment demonstrates that combinations including a chemotherapeutic agent or an immunotherapeutic agent and ABX196 are more potent than each component of the combination taken alone.

[0496] As shown in FIG. 12, combinations treatments including ABX196 present less tumor invasion. The tumor invasion reduction is transduced into better survival.