Immunotherapeutic vaccine and antibody combination therapy

Abstract

The present invention relates to a combination product, composition(s) and kit of parts comprising at least (i) a therapeutic vaccine and (ii) one or more immune checkpoint modulator(s). The present invention also concerns a method for treating a proliferative or an infectious disease as well as a method for eliciting or stimulating and/or re-orienting an immune response, wherein said methods comprise administering to a subject in need thereof said combination product or said composition(s).

Claims

1. A method for treating a proliferative or an infectious disease comprising administering to a subject in need thereof an effective amount of a combination product comprising (i) a therapeutic vaccine and (ii) one or more immune checkpoint modulator(s); wherein said therapeutic vaccine comprises a modified vaccinia virus Ankara (MVA) encoding membrane anchored Human papilloma Viruses 16 (HPV-16) non-oncogenic E6 and E7 antigens and encoding interleukin 2 (IL-2); wherein said one or more immune checkpoint modulator(s) comprise(s) a modulator capable of antagonizing at least partially programmed cell death protein 1 (PD-1) pathway; and wherein said administering of said therapeutic vaccine starts before said administering of said one or more immune checkpoint modulator(s) and wherein said therapeutic vaccine is administered a first time in a first administration of said therapeutic vaccine separated by a period of time from a first administration of said one or more immune checkpoint modulator(s), and the period of time between the first administration of said therapeutic vaccine and the first administration of said one or more immune checkpoint modulator(s) is one day to eight week(s).

2. The method of claim 1, wherein said one or more immune checkpoint modulator(s) comprise(s) a modulator capable of antagonizing at least partially programmed death ligand 1 (PD-L1) or PD-1.

3. The method of claim 2, wherein said one or more immune checkpoint modulator(s) comprise(s) a modulator capable of antagonizing at least partially PD-L1.

4. The method of claim 1, wherein at least one of said one or more immune checkpoint modulator(s) is an antibody that recognizes human PD-L1.

5. The method of claim 4, wherein at least one of said one or more immune checkpoint modulator(s) is a human or humanized monoclonal antibody that specifically binds to PD-L1.

6. The method according to claim 1, wherein said proliferative disease is a cancer and wherein said infectious disease results from infection with a virus selected from the group consisting of herpes virus, papillomavirus, poxvirus, retrovirus, HCV, HBV and influenza virus.

7. The method according to claim 6, wherein said cancer is selected from the group consisting of renal cancer, prostate cancer, breast cancer, colorectal cancer, lung cancer, liver cancer, gastric cancer, bile duct carcinoma, endometrial cancer, pancreatic cancer and ovarian cancer.

8. The method according to claim 7, wherein said cancer is a non-small cell lung cancer (NSCL).

9. The method according to claim 6, wherein said cancer is selected from the group consisting of carcinoma, lymphoma, blastoma, sarcoma, leukemia, bone cancer, gastrointestinal cancer, liver cancer, pancreatic cancer, gastric cancer, colorectal cancer, esophageal cancer, oro-pharyngeal cancer, laryngeal cancer, salivary gland carcinoma, thyroid cancer, lung cancer, non-small cell lung cancer, cancer of the head or neck, skin cancer, squamous cell cancer, melanoma, uterine cancer, cervical cancer, endometrial carcinoma, vulvar cancer, ovarian cancer, breast cancer, metastatic breast cancer, prostate cancer, cancer of the endocrine system, sarcoma of soft tissue, bladder cancer, kidney cancer, glioblastoma, cancer of the central nervous system, renal cancer, clear cell carcinoma, hormone refractory prostate adenocarcinoma, hepatocarcinoma, and bile duct carcinoma.

10. The method according to claim 1 which is carried out in association with one or more conventional therapeutic modalities.

11. The method according to claim 1, wherein said therapeutic vaccine and said one or more immune checkpoint modulator(s) are formulated for from 4 to 15 administrations of 10.sup.7 to 10.sup.9 pfu of said MVA at approximately 1 to 3 week intervals interspersed with 2 to 6 administrations of 3 to 10 mg/kg of said one or more immune checkpoint modulator(s) every 2 or 3 weeks.

12. The method of claim 1, wherein the period of time between the first administration of said therapeutic vaccine and the first administration of said one or more immune check point modulator(s) is one week to eight week(s).

13. The method of claim 1, wherein said administering of a therapeutic vaccine occurs more than once separated by a time interval between each said administering of a therapeutic vaccine that is from 2 days to 8 weeks; and said administering of immune check point modulator(s) occurs more than once separated by a time interval between each administration of immune check point modulator(s) that is from 2 days to 8 weeks.

14. The method of claim 1, wherein administration of said combination product results in at least one of: i. a prolonged survival; ii. a reduced mortality; iii. a reduction of a tumor size; iv. eliciting and/or stimulating and/or redirecting an immune response; v. eliciting and/or stimulating and/or redirecting an immune response specific to said antigens; vi. eliciting and/or stimulating and/or redirecting a CD4+ and/or CD8+ T cell response; vii. an increased IFN-gamma secretion; and viii. any combination of i. to vii; in the treated subject.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIGS. 1A and 1B illustrates the effects on tumor volume (FIG. 1A) and mice survival (FIG. 1B) in a subcutaneous CT26-CL25 tumor model of the administration of the formulation buffer (vehicle), MVATG18124 alone, the anti PD-1 clone RMP1.14 alone, a combination of both MVATG18124 and anti PD-1 clone RMP1.14 and a combination of both MVATG18124 and the rat IgG2a isotype control.

(2) FIG. 2 illustrates the effects on mice survival in metastatic CT26-CL25 tumor model of the administration of an empty MVA (MVATGN33.1), MVATG18124 alone, an anti CTLA-4 antibody alone, its IgG2b isotype control, a combination of both MVATG18124 and anti CTLA-4 and a combination of both MVATG18124 and the IgG2b isotype control.

(3) FIG. 3A illustrates the percentage of gated CD8.sup.+CD3.sup.dim cells (referred as CD8.sup.dimCD3.sup.dim herein after) in dissociated lungs obtained from untreated (i.e. nave) mice or mice treated with MVATG18124, anti-CTLA-4 and both MVATG18124 and anti-CTLA-4.

(4) FIG. 3B illustrates an example of CD8.sup.dimCD3.sup.dim (referred as CD8.sup.+CD3.sup.dim in the Fig.) population in CD3/CD8 dot blot lung cells from mice treated with anti CTLA-4 and MVATG18124.

(5) FIGS. 4A and 4B represents the IFN- positive CD8.sup.dimCD3.sup.dim cell population obtained following ConA induction in lung samples obtained from untreated (i.e. nave) mice or mice treated with MVATG18124, anti-CTLA-4 and both. FIG. 4A: ConA-induced fold induction of percentage of IFN.sup.+ CD8.sup.dimCD3.sup.dim cells upon incubation of lung cells with bmDCs to facilitate activation. FIG. 4B: ConA-induced fold induction of percentage of IFN.sup.+ CD8.sup.dimCD3.sup.dim cells upon incubation of lung cells with anti-CD28 to facilitate activation.

(6) FIG. 5 illustrates ELispot analysis of splenic lymphocytes stimulated with the -gal-specific peptide T9L3 showed -gal specific response in splenic lymphocytes treated with MVATG18124. The raw data was transformed into histogram graph. Results are expressed as number of spot forming units (sfu) per 110.sup.6 splenocytes (mean) for each triplicate or quadruplicate.

(7) FIG. 6 represents the induction of IFN-, CD107a and KLRG1 positive cells in CD8.sup.dimCD3.sup.dim cell population obtained in lung samples stimulated with T9L-3 peptide (hatched) or irrelevant T8G peptide (dotted) following treatment with an empty vector MVATGN33.1 alone or in combination with anti-CTLA-4, MVATG18124 alone or in combination with anti-CTLA-4 or anti-CTLA-4 alone.

(8) FIGS. 7A and 7B illustrates the effect provided by TG4010 treatment on mice survival and tumor volume in a CT26-MUC1 tumor model. FIG. 7A: CT26-MUC1 lung tumor model. CT26-MUC1 cells were injected iv. Day 2 and 9, 5.Math.10.sup.7 pfu TG4010, empty MVA vector (MVATGN33.1) or buffer (SO8) were injected intravenously. Mice were weighed twice per week and sacrificed when loosing 10% of weight. Percent survival was monitored. FIG. 7B: CT26-MUC1 s.c. tumor model. CT26-MUC1 cells were injected s.c. Day 2 and 9, 1.Math.10.sup.7 pfu TG4010, empty MVA vector (MVATGN33.1) or buffer (SO8) were injected s.c. in the same flank. Mice were sacrificed when the tumors reached the size of 2000 mm.sup.3. Mean tumor volumes with SEM are shown.

(9) FIGS. 8A and 8B illustrates the effect on mice survival (FIG. 8A) and tumor volume (FIG. 8B) provided by TG4010 treatment in combination with anti PD-1 antibody in the CT26-MUC1 s.c. tumor model. BALB/c mice were injected s.c. with 2.Math.10.sup.5 CT26-MUC1 cells. On days 2 and 9 after tumor implantation, mice were treated sc with TG4010 (also called MVATG9931) or an empty control vector at the suboptimal dose of 1.Math.10.sup.6 pfu followed by i.p. administration of 250 ug anti PD-1 (RMP1.14, IgG2a, BioXCell) on days 10, 13, 15 and 17. Mice were sacrificed when the tumors reached the size of 2000 mm.sup.3. Percent survival and mean tumor volumes were monitored over time.

(10) FIG. 9 illustrates the survival of BALB/C mice injected s.c. with CT26-MUC1 tumor cells and immunized with two injections of 110.sup.7 PFU of MVATGN33.1 or MVATG9931 on days D2 and D9 followed by i.p. administrations of 250 g of anti-PD-1 Ab on days D10, D13, D15 and D17. (*:p<0.05; **::p<0.01; ***:p<0.001)

EXAMPLES

(11) We set out to combine immune checkpoint blocking approaches with therapeutic MVA vectors with the goal of inducing antigen-specific T cell immune response with MVA and release the brakes from T cell generation with immune checkpoint antibodies. Preclinical evidence for synergistic effects of immune checkpoint blockers combined with viral vectors was to be demonstrated in mouse tumor models. This implies the use of i) murine-specific anti-immune check point antibodies and ii) an antigen-expressing MVA vector.

(12) The MVA vector chosen for these studies (MVATG18124) contains the bacterial LacZ gene encoding the beta-galactosidase Ag model (SEQ ID NO: 3) under the control of poxvirus promoter pH5R (SEQ ID NO: 4). pH5R promoter was isolated by PCR amplification from Vaccinia virus Copenhagen strain using appropriate primers. The E. coli LacZ gene was obtained by PCR amplification using primers otg19678 (SEQ ID NO: 5) and otg19679 (SEQ ID NO: 6) with pCMVBeta (Clontech) as DNA template. The pH5R and LacZ genes were cloned into a shuttle plasmid between MVA sequences extending from positions 142006 to 142987 and positions 142992 to 143992 according to GenBank sequence EF675191.1. MVATG18124 was generated into chicken embryo fibroblast (CEF) cells by transfection of shuttle plasmid into previously MVA-infected CEF, resulting in homologous recombination between shuttle plasmid DNA and MVA genome and insertion of the pH5R-LacZ cassette into deletion III. Recombinant MVA clones were isolated using conventional technology (Lullo et al., 2010, J Virol Methods 163: 195-204) and the selected clones were controlled by PCR, then amplified in CEF cells. Virus stocks were titrated on DF1 cells by plaque assay. Absence of mutation into the inserted DNA and the surrounding region was checked by DNA sequencing.

(13) It was first chosen to target the immune checkpoint blocker murine PD-1 (mPD-1) with an appropriate antibody. The rat anti mPD-1 antibody RMP1-14 (BioXcell) was chosen. This antibody was shown to block the interaction of mPD1 with its ligands (Yamazaki et al., 2005, J. Immunol. 175(3): 1586-92).

(14) The combination of mPD-1 inhibitors with the antigen-expressing MVATG18124 was tested in vivo in two mice models, respectively metastatic and subcutaneous tumor models. The colon carcinoma cell line CT26.CL25 (ATCC CRL-2639), transduced with the LacZ gene and thus stably expressing beta-galactosidase, was either injected subcutaneously to generated palpable tumors (subcutaneous model) or intravenously to generate lung metastasis. Mice were then treated with MVATG18124 expressing beta-galactosidase and murine-specific immune checkpoint blockers like anti PD-1 or anti CTLA-4 antibodies.

Example 1: Combination of MVATG18124 with Anti-PD-1 Mab

(15) The combination of mPD-1 inhibitor (commercial clone RMP1-14; BioXCell) with beta-gal-expressing MVATG18124 was tested in vivo in a subcutaneous tumor model. Balb/c mice were subcutaneously injected with 210.sup.5 CT26.CL25 cells. Day 2 and 9 after cell implantation, mice were then intravenously immunized with either 110.sup.4 pfu of MVATG18124 or formulation vehicle as negative control in combination with 4 intraperitoneal (ip) administrations at days 10, 13, 15 and 17 of 250 ug of either murine anti PD-1 antibody RMP1.14 (BioXcell) or its isotype control IgG2a (clone 2A3). In other terms, 5 groups of 10 mice were tested; a first group treated with MVATG18124 receiving 2 iv injections (group 1); a second group treated with 4 ip injections of anti-PD-1 antibody (group 2); a third group treated with both MVATG18124 and anti-PD-1 antibody receiving 2 iv injections of the therapeutic MVA and 4 ip injections of anti-PD-1 antibody (group 3); a fourth group receiving 2 iv injections of the therapeutic MVATG18124 and 4 ip injections of isotype antibody (group 4) and a control group receiving the formulation buffer (group 5). Tumor growth and survival were measured over time as illustrated in FIGS. 1A and 1B.

(16) As expected, tumor volume increased very rapidly in control group receiving formulation buffer. Rapid tumour growth was also observed in the group receiving mPD-1 antibody. A slight delay in tumor volume was seen in groups receiving MVATG18124 either alone or in combination with the isotype control. Tumor growth was greatly reduced in the combination group injected with both MVATG18124 and mPD-1 antibodies (FIG. 1A). Effects on mice survival are more pronounced since about 70% of mice treated with both MVATG18124 and mPD-1 antibodies are still alive more than 100 days following tumor implantation versus 10% of MVATG18124-treated animal group (significant differences). In contrast, control, antibody-treated and isotype-treated animals died within less than 50 days (FIG. 1B).

Example 2: Combination of MVATG18124 with Anti-CTLA-4 Mab

(17) The combination of CTLA-4 inhibitor (commercial clone 9D9; BioXCell) with antigen-expressing MVATG18124 was tested in vivo in a metastatic CT26-CL25 model. 210.sup.5 CT26.CL25 cells were injected intravenously (iv) in Balb/c mice. Days 2 and 9, MVATG18124 encoding beta-galactosidase or its empty vector control MVAN33.1 was administered iv at the dose of 1.Math.10.sup.4 pfu. 250 g of the murine anti-CTLA4 antibody 9D9 (BioXCell) or its IgG2b isotype control (clone MPC-11 BioXCell) were injected intraperitoneally (ip) at days 3 and 10. The survival of mice was followed for more than 60 days. The viral dose of 1.Math.10.sup.4 pfu was identified as optimal dose to increase survival rates in this tumor model (data not shown).

(18) As illustrated in FIG. 2, treatment with anti CTLA4 antibodies or its isotype control alone showed weak effects comparable to those observed with the empty MVA vector MVATGN33.1 (35% survival at day 20 for all three groups). In contrast, treatment with MVATG18124 alone or in combination with the isotype control increased mice survival (35% survival at day 35 for both groups). The group of mice treated with the combination of MVATG18124 and anti-CTLA4 antibodies greatly increased the percentage of 35% survival to more than 60 days.

(19) Thus, we have clearly demonstrated a clear anti-tumor effect of treating MVA-based immunotherapeutic vaccine and the immune checkpoint blocker anti CTLA4.

Example 3: Lymphoid Cell Population Studies in Dissociated Lungs of Treated Mice

(20) Determination of IFN Positive CD8dim CD3dim Cells

(21) Cellular response was examined in mice treated with either the anti-CTLA-4 antibody the antigen-expressing MVA or both as well as in untreated (i.e. nave) mice. Five BALB/c mice per group were injected iv with MVATG18124 (1.Math.10.sup.4 pfu) day 1 and 8 or ip with 250 g anti CTLA-4 (clone 9D9, BioXCell) day 2 and 9 or both. Mice were sacrificed day 15, and lungs were isolated. Lungs from all 5 mice per group were pooled, cut into small pieces in C-tubes (Miltenyi) and enzymatically dissociated using a tissue dissociation kit (Miltenyi, 130-096-730) using the Gentle OctoMACS (Miltenyi) according to the manufacturer's recommendations.

(22) Lung-derived cells were plated at 2.Math.10.sup.6 cells/well (96 well plate) in T cell-specific medium (TexMACS, Miltenyi). Cells were activated by co-cultivation with peptide-loaded bone marrow-derived murine dendritic cells (bmDCs): bmDCs from BALB/c mice were generated from bone marrow cells matured in the presence of murine GM-CSF (Peprotech, 100 g/ml) for 10 days. Alternatively, activation was facilitated by incubation with 1 g of anti CD28 (clone PV-1). Concanavalin A (ConA, 5 g/ml) served as non-specific activator. Anti CD107a antibody (clone eBio1D4B) was added to label degranulating cells. Secretion of cytokines was blocked after one hour of incubation by adding GolgiPlug/Brefeldin (1:1000, BD Biosciences). After 5 hours total incubation time, cells were washed and stained for viability (LiveDead, Fixable violet dead cell staining kit) and surface markers CD8a (clone 53-6.7) and CD38 (clone 145-2C11). Cells were stained intracellularly for IFN- (clone XMG1.2) using the BD Cytofix/Cytoperm kit (BD Biosciences). Cells were fixed and analyzed by flow cytometry (Navios, Beckman Coulter).

(23) Combinatorial treatment of MVATG18124 and anti CTLA-4 in nave BALB/c mice leads to the appearance of a sub population of lymphocyte cells named CD8.sup.dimCD3.sup.dim in the lung. FIG. 3B represents an example of CD8.sup.dimCD3.sup.dim population in CD3/CD8 dot blot lung cells from mice treated with anti CTLA-4 and MVATG18124. Gating living lymphocytes, the percentages of gated CD8.sup.dimCD3.sup.dim cells in two independent experiments are illustrated in FIG. 3A. More specifically, the population of CD8.sup.dimCD3.sup.dim was clearly increased after treatment with MVATG18124 and even more after combinatorial treatment with MVATG18124+anti CTLA-4.

(24) Next, the percentage of intracellular IFN .sup.+ cells was assessed in the CD8.sup.dimCD3.sup.dim population. Within this CD8.sup.dimCD3.sup.dim population, highest induction of IFN-+ cells was observed in mice treated with MVATG18124+anti CTLA-4 (FIGS. 4A and 4B).

(25) In summary, combinatorial treatment of MVATG18124 and anti CTLA-4 in nave BALB/c mice leads to the appearance of a CD8.sup.dimCD3.sup.dim lymphocyte cell population in the lung. Upon stimulation with ConA and a high percentage of CD8.sup.dimCD3.sup.dim from mice treated with MVATG18124+anti CTLA-4 can be induced to secrete IFN-.

(26) The CD3.sup.dimCD8.sup.dim cell population was analyzed in greater detail. In the course of our analyses, we observed that the CD3.sup.dimCD8.sup.dim population was positive for the killer cell lectin like receptor G1 (KLRG1.sup.+) and negative or low for CD127 (IL-7R) (CD127.sup./flow). This phenotype is associated with antigen experienced short lived effector cells (SLECs) (Obar et al., 2011, J Immunol., doi: 10.4049/jimmunol.1102335; Sarkar et al., 2008, J Exp Med 205(3): 625-40). The CD3.sup.dimCD8.sup.dim KLRG1.sup.+ population infiltrating/present in the lung of mice treated with MVATG18124 and anti CTLA-4 responds to an antigen-specific stimulus with IFN- secretion and degranulation (CD107a).
Determination of IFN-, CD107a and KLRG1 Positive Cells in CD8.sup.dimCD3.sup.dim Cell Population

(27) As described above, BALB/c mice were injected i.v. with MVA--gal or an empty vector MVATGN33.1 at 1.Math.10.sup.4 pfu. On days 3 and 10, mice received 250 g anti CTLA-4 i.p. Lungs were taken day 14 and enzymatically dissociated. Lung derived cells were plated at 2.Math.10.sup.6 cells/well (96 well plate) in T cell-specific medium (TexMACS, Miltenyi), activated by incubation with 1 g of anti CD28 (clone PV-1) and stimulated with a -gal specific peptide (T9L-3) or a control peptide (T8G) in the presence of anti CD107a antibody (clone eBio1D4B) to label degranulating cells. Secretion of cytokines was blocked after one hour of incubation by adding GolgiPlug/Brefeldin (1:1000, BD Biosciences). After 5h total incubation time, cells were washed and stained for viability (LiveDead, Fixable violet dead cell staining kit) and for the surface markers CD8a (clone 53-6.7), CD3 (clone 145-2C11) and KLRG1 (clone 2F1/KLRG1). Cells were stained intracellularly for IFN- (clone XMG1.2) using the BD Cytofix/Cytoperm kit (BD Biosciences). Cells were fixed and analyzed by flow cytometry (Navios, Beckman Coulter).. After 5 hours, cells and stained CD8a.

(28) Treatment with MVATG18124 or an empty control vector, and even more the combination of MVATG18124 and anti CTLA-4 in nave BALB/c mice lead to the appearance of a CD8.sup.dimCD3.sup.dimKLRG1.sup.+ lymphocyte cell population in the lung. Upon ex vivo stimulation with the -gal peptide T9L-3, this population secreted IFN and degranulated (CD107a) and, as illustrated in FIG. 6, the percentage was higher after treatment with MVATG18124 and anti CTLA-4 than in the other groups treated with the control vector (MVATGN33.1 alone or with anti CTLA-4), with MVATG18124 alone or with anti CTLA-4 alone. As expected, stimulation with an irrelevant peptide (T8G peptide) did not yield such induction.

(29) In conclusion, the treatment with MVATG18124 and anti CTLA-4 increases the b-gal specific response in a CD3.sup.dimCD8.sup.dimKLRG1.sup.+ cell population in the lung.

Example 4: Secretion of IFN-

(30) Further, the number of IFN secreting splenic lymphocytes was investigated following BALB/c mice treatment with MVATG18124 or MVAN33.1 at 1.Math.10.sup.4 pfu (D1 and 8) and anti CTLA-4 or its isotype control (D2 and D9, 250 g ip). Measurement was performed at day 14 by ELISpot (Enzyme-linked immunospot) assay.

(31) Plate Preparation

(32) The day preceding the experiments, membrane ELISpot plates (Millipore, ref. MSIPS4W10) were preweted with 15 l of 35% ethanol per well with maximum incubation time of 2 min. Plates were washed five times with 200 l per well of sterile water.

(33) ELISpot plates were coated with a rat anti-mouse IFN- monoclonal antibody (AN18 Mabtech, ref. 3321-3-1000) diluted at 15 g/mL in sterile DPBS (Sigma, ref. D8357) (100 l/well). The plates were covered and incubated overnight at 4 C. The next day, the plates were washed 3 times with sterile PBS (200 L/well) and were saturated for 1 h at 37 C. with 200 L/well of complete RPMI 1640 medium (RPMI1640 medium, (Sigma R0883); L-Glutamine 2 mM, (Sigma G6392); Gentamycin 0.01 g/L (Schering Plough U570036); Fetal Calf Serum 10% (JRH 12003-1000M) 550 l of a solution 510.sup.2 M bmercaptoethanol).

(34) Sample Preparation

(35) For ex vivo evaluation of the frequency of the specific CD8+ T cells induced by immunization, euthanized animals were splenectomized 7 days after last immunization. Spleens from the same group were pooled in a cell strainer in a well of a 6-wells culture plate containing 5 mL of complete medium. Spleens were crushed with a syringe piston and the cell strainer discarded. Splenocytes were collected with 8 mL of complete medium and transferred in a 15 ml falcon tube. The splenocytes suspension was laid over 4 mL of Lympholyte-M separation cell media (Cedarlane, ref. CL5035) and centrifuged (20 min, 1500g, room temperature). The interphase containing lymphocytes was collected and rinsed three times with 10 ml of PBS. Between each rinse step, cells were centrifuged (4 min at 400g) and the supernatants were discarded. The remaining red blood cells were lysed by addition on the lymphocyte pellet of 2 mL of RBC lysis buffer 1(10 solution: BD Pharm Lyse lysing solution, ref. 555899) diluted in sterile water. Each tube was gently vortexed immediately after adding the lysis solution and incubated at room temperature for 15 minutes. Lysis was topped by the addition of 10 mL DPBS followed by centrifugation 4 min at 400g., the cells were resuspended in 10 mL of complete RPMI 1640 medium. The cells were counted with a Z2 Cell Counter (Beckman Coulter) and the cell concentration was adjusted at 110.sup.7 cells per mL in complete RPMI 1640 medium.

(36) Assay

(37) To perform the ELISpot assay, 100 L of lymphocyte suspension from each group (110.sup.6 cells) were added to each wells of a coated 96-wells plate. One given condition was tested in triplicates or in quadruplicates. One hundred microliter of different indicated peptides (2 g/ml in complete RPMI 1640 medium) was added to the cell suspension. ConA (Sigma, ref. C5275) was used as positive control (5 g/mL final concentration) MVA-specific peptide (S9L-8) was used for immunization control. The plates were then incubated at 37 C. in 5% CO.sub.2 for 16 to 20 hours. Then, plates were washed three times with DPBS (200 L). Biotinylated rat anti-mouse IFN- monoclonal antibody (Mabtech, ref. 3321-6-1000) was diluted at 1 g/mL in antibody mix buffer (PBS, 0.5% SVF) and distributed at 100 l/well. Plates were incubated 2 hours at room temperature in darkness, and then washed three times in DPBS (200 L). One hundred microliter of Extravidin-Phosphatase alkaline (SIGMA, ref. E2636) (Diluted 1/5000 in antibody mix buffer) was added to each well and the plates were incubated for 1 hour at room temperature in darkness. Plates were finally washed three times in DPBS (200 L). One hundred microliter of BCIP/NBT (Sigma, ref. B5655, one caps in 10 mL MilliQ water) was added to each well until blue spots develop and then plates were washed thoroughly in tap water and dried.

(38) Data Acquisition

(39) Spots were counted with an ELISpot reader (CTL Immunospot reader, S5 UV). A visual quality control (comparing machine scans and plates) was performed on each well to ensure that the counts provided by the ELISpot reader match the reality of the picture. Results were expressed as number of spot forming units (sfu) per 110.sup.6 splenic lymphocytes (mean) for each triplicate or quadruplicate. Specific ELISpot response was determined either with the DFR (eq) method (Moodie et al., Cancer Immunol Immunother. 2010 October; 59(10):1489-501) or with an empirical cut-off calculated as the mean number of spots from blank wells plus two times the standard deviation of this mean number of spots.

(40) As shown in FIG. 5, ELIspot analysis of splenic lymphocytes stimulated with the -gal-specific peptide T9L3 showed -gal specific response in splenic lymphocytes treated with MVATG18124. The specific responses increased further after combinatorial treatment with anti CTLA-4 but not with its isotype control. Stimulation with the non-specific peptide T8G showed no increase of IFN- secreting cells.

Example 5: Combinatorial Effect of TG4010 (MVA-MUC1-IL-2) and Anti PD-1 (RMP1.14) in a MU1-Positive CT26-Based Tumor Model

(41) TG4010 is a MVA vector encoding the full cDNA sequence of human MUC1 and human IL-2. Anti-tumoral efficacy provided by this vector was tested in a CT26-based MUC1-positive cell line which gives rise to MUC1-positive tumors after s.c. injection, as well as lung tumors after i.v. injection.

(42) Generation of CT26-MUC1 Cell Line

(43) The murine colon carcinoma cell line CT26 WT (ATCC CRL-2638) was stably transfected with the plasmid pTG5077 encoding the full cDNA sequence of human MUC1 under the control of the CMV promoter as well as a G418-resistance gene under the control of the SV40 promoter. CT26 cells were transfected by means of Lipofectamine LTX with pTG5077, and cultivated in the presence of 0.4 mg/ml G418 to select for stable transfectants. After 14 days, living cells were labeled with a monoclonal antibody against MUC1 (H23+second antibody Goat anti mouse-FITC). Positive cells were sorted (FACS ARIA), transferred in 96 well plates at 1 cell/well. Outgrowing clones were analyzed for stable MUC1 expression by flow cytometry up to day 60 after transfection. Four stably MUC1-expressing clones were then tested for their ability to induce tumor growth in BALB/c mice after sc injection and after iv injection. One clone was retained after verification that s.c.-implanted tumors and lung tumors obtained after iv injection were MUC1-positive.

(44) Therapeutic Efficacy of TG4010 in the CT26-MUC1 Tumor Model

(45) 2.Math.10.sup.5 CT26-MUC1 cells were injected s.c. or i.v. in BALB/c mice to generate sc tumors or lung tumors, respectively. On day 2 and 9 after tumor challenge, mice were treated s.c. or i.v., respectively, with 1.Math.10.sup.7 TG4010 or the empty control vector MVATGN33.1. Mean tumor volume or percent survival were monitored over time. TG4010 showed significant improvement of survival in the iv/iv lung tumor model (p=0.00642) and significant reduction of tumor growth in the sc/sc tumor model (see FIGS. 7A and 7B, respectively).

(46) Therapeutic Efficacy of TG4010 in Combination with Anti-PD1 in the CT26-MUC1 Tumor Model

(47) BALB/c mice were injected s.c. with 2.Math.10.sup.5 CT26.MUC1 cells. On days 2 and 9 after tumor implantation, mice were treated sc with TG4010 (also designated MVATG9931) or an empty control vector (MVATGN33.1) at the suboptimal dose of 1.Math.10.sup.6 pfu. On days 10, 13, 15 and 17, mice received 250 g anti PD-1 (RMP1.14, IgG2a, BioXCell). Mice were sacrificed when the tumors reached the size of 2,000 mm.sup.3. Tumor volume and animal survival were followed over time.

(48) As illustrated in FIG. 8B, tumors started to grow at day 16 and tumor volume increased rapidly and regularly over time in control group receiving formulation buffer, as expected. A delay in tumour growth was observed in the groups receiving mPD-1 antibody, TG4010 alone and empty MVATGN33.1 alone or with anti-PD-1. In contrast, tumor growth was greatly reduced in the combination group injected with both TG4010 and mPD-1 antibody. Effects on mice survival are also observed as shown in FIG. 8A. Indeed, about 70% of mice treated with both TG4010 and mPD-1 antibody are still alive more than 60 days following tumor implantation while approximately 10% and 20% of animals treated with the empty control (without or with the anti-PD-1 antibody) remained alive. Only 30 and 40% of animals respectively treated with TG4010 alone or with anti-PD-1 antibody survived 2 months after tumor implantation. In contrast, control group treated with formulation buffer died within less than 45 days. The increased survival provided by the combinatorial treatment was maintained overtime (120 days after tumor implantation) (data not shown).

(49) The same experiment as above was also conducted except that mice were treated with 2 injections of 1.Math.10.sup.7 pfu of MVATG9931 (i.e. TG4010) or the negative MVATGN33.1 control vector, optionally followed by four i.p. administrations of 250 g of anti-PD1. As illustrated in FIG. 9, a substantial increase in median survival of 70 days for the combinatorial treatment is however noticeable in comparison to 46 and 49.5 days for the mice treated with MVATG9931 and anti-PD-1, respectively. Note also that the survival increase is significant (p=0.008) when the combination of MVATG9931 and anti-PD-1 treatments is compared to the control empty virus MVATGN33.1 and anti-PD-1 modalities, suggesting a MUC-1 specific interaction at this dose of virus. At the highest dose of 110.sup.7 PFU for MVATG9931, the best therapeutic activity among all the conditions tested is achieved for the combination with significance reached against each treatment modality alone (p=0.014 against MVATG9931 and p<0.001 against anti-PD-1; FIG. 9). In conclusion, the combination of MVATG9931 with an anti-PD-1 antibody allowed to obtain the best therapeutic index in the ectopic CT26-MUC1 model in comparison to each treatment alone which paves the way to the clinical evaluation of this combination therapy approach.

(50) Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, numerous equivalents to the specific method and reagents described herein, including alternatives, variants, additions, deletions, modifications and substitutions. Such equivalents are considered to be within the scope of this invention and are covered by the following claims.