VIRAL VECTOR

Abstract

The invention concerns a novel viral vector with modified viral capsid or viral envelope; a pharmaceutical composition or immunogenic agent or vaccine comprising same; a target cell transformed or transfected with same; a combination therapeutic comprising same; use of same in treatment of cancer, and a method of treating cancer using same.

Claims

1. A viral vector having attached to its viral capsid or envelope, polypeptides that have not been genetically encoded by said viral vector but have been attached to the capsid or envelope covalently or non-covalently wherein: i) at least one of said polypeptides comprises an antigen from, or of, a pathogen that a subject has been prior immunised against; and ii) at least one other of said polypeptides is an anti-tumor or anti-cancer specific polypeptide and so stimulates an anti-tumor or anti-cancer immune response in a subject exposed to said vector.

2. The viral vector according to claim 1 wherein said polypeptides comprise fusion polypeptides, a part of which comprises an antigen from, or of, a pathogen that a subject has been prior immunised against; and another part of which is an anti-tumor or anti-cancer specific polypeptide and so stimulates an anti-tumor or anti-cancer immune response in a subject exposed to said vector.

3. The viral vector according to claim 1, wherein said polypeptides are polylysine-modified or polyarginine-modified for attaching same to said capsid.

4. The viral vector according to claim 1, wherein said polypeptides are attached to the capsid or envelope by a cell penetrating peptide; a cholesterol moiety; or an electrostatic, disulfide or amide bond linkage.

5. The viral vector according to claim 1, wherein said polypeptides are selected from the group consisting of Major Histocompatibility Complex of class I (MHC-I)-specific polypeptides, Major Histocompatibility Complex of class II (MHC-II)-specific polypeptides and DC specific polypeptides.

6. The viral vector according to claim 1, wherein at least one or a plurality of said polypeptides are MHC-I-specific polypeptides and at least one or a plurality of said polypeptides are MHC-II-specific polypeptides.

7. The viral vector according to claim 1, wherein said viral vector is a member of a family selected from the group consisting of: Adenoviruses, Reoviruses, Papillomaviruses, Picornaviruses, Caliciviruses, Herpesviruses, Poxviruses, Hepadnaviruses, Flavivirus, Togavirus, Coronavirus, Hepatitis D, Orthomyxovirus, Paramyxovirus, Rhabdovirus, Bunyavirus, Filovirus and Retroviruses.

8. The viral vector according to claim 1 wherein said virus is selected from the group comprising: Adenovirus, Herpes Simplex Virus 1 (HSV-1), Herpes Simplex Virus 2 (HSV-2), Vaccinia, Vesicular stomatitis Indiana virus (VSV), Measles Virus (MeV), Maraba virus and New Castle Disease (NDV) virus.

9. (canceled)

10. The viral vector according to claim 1, wherein said viral vector is oncolytic.

11. The viral vector according to claim 1, wherein said polypeptide of i) is: TABLE-US-00005 (SEQ ID NO: 1) QYIKANSKFIGITEL (Tetanus toxin); (SEQ ID NO: 2) ARYVSQQTRANPNPY (Pertussis); (SEQ ID NO: 3) IQSKRFAPLYAVEAK (Polio Mahoney); (SEQ ID NO: 4) SPVYVGNGVHANLHV (Diphtheria); (SEQ ID NO: 5) PVFAGANYAAWAVNVAQVI (Diphtheria); (SEQ ID NO: 6) ARYVSQQTRANPNPY (Pertussis); (SEQ ID NO: 7) IQSKRFAPLYAVEAK (Polio Mahoney); or (SEQ ID NO: 8) SPVYVGNGVHANLHV (Diphtheria).

12. The viral vector according to claim 1, wherein said polypeptide of ii) is: TABLE-US-00006 (SEQ ID NO: 9) KVPRNQDWL (gp100); (SEQ ID NO: 10) SLLMWITQC (NY-ESO-1); (SEQ ID NO: 11) RGPESRLLEFYLAMPFATPM (NY-ESO-1); (SEQ ID NO: 12) YLAMPFATPMEAELARRSLA (NY-ESO-1); (SEQ ID NO: 13) RGPESRLLEFYLAMPFATPMEAELARRSLA (NY-ESO-1; (SEQ ID NO: 14) PGVLLKEFTVSGNILTIRLTAADHR (NY-ESO-1); (SEQ ID NO: 15) YLAMPFATPMEAELARRSLA (NY-ESO-1); (SEQ ID NO: 16) YLAMPFATPMEAELARRSLAEE (NY-ESO-1); (SEQ ID NO: 17) VFGIELMEVDPIGHLYIFAT (MAGE-A3); or (SEQ ID NO: 19) VFGIELMEVDPIGHLY (MAGE-A3).

13. A pharmaceutical composition or immunogenic agent or vaccine comprising said viral vector according to claim 1 and a suitable carrier.

14. A target cell transformed or transfected with said viral vector according to claim 1.

15. A combination therapeutic for the treatment of cancer comprising: a) the viral vector according to claim 1; and b) a further cancer therapeutic agent.

16-17. (canceled)

18. A method of treating cancer in a subject, comprising: administering an effective amount of said viral vector according to claim 1 to the subject, thereby treating cancer in the subject.

19. The method according to claim 18 wherein said viral vector is administered in combination with an anti-tumour agent or an anti-seasonal disorder agent.

20. The method of claim 18, wherein said cancer is nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia/lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer or tonsil cancer.

21. (canceled)

22. A method of treating cancer in a subject, comprising: administering an effective amount of the pharmaceutical composition or immunogenic agent or vaccine according to claim 13 to the subject, thereby treating cancer in the subject.

23. A method of treating cancer in a subject, comprising: administering an effective amount of the target cell according to claim 14 to the subject, thereby treating cancer in the subject.

24. A method of treating cancer in a subject, comprising: administering an effective amount of the combination therapeutic according to claim 15 to the subject, thereby treating cancer in the subject.

Description

[0053] An embodiment of the present invention will now be described by way of example only with reference to the following wherein:

[0054] FIG. 1. Effect of recalling memory repertoire on murine model of melanoma. (A) A schematic representation of the new hybrid PeptiCRAd system. A single adenovirus is loaded with pathogen-specific peptides to evoke the pre-existing memory T cell repertoire, and with tumor-specific peptides to evoke the anti-tumor T cell repertoire. (B) Treatment scheme. 3×10.sup.5 B16.OVA cells were injected into the right flank of naïve and tetanus pre-immunized C57BL/6 mice (n=7-8). Treatments were given intra-tumorally 4 times (on days 9, 11, 13 and 15) as indicated in the figure. (C-E) The B16.OVA tumor growth was followed until the end of the experiment in naïve and pre-immunized mice. The tumor size is presented as the mean for each treatment±SEM. (Statistical analysis 2 way ANOVA, * p<0.05, ** p<0.005, *** p<0,001, **** p<0,0001);

[0055] FIG. 2 Immune cell component within the Tumour Micro Environment (TME) in pre-immunized mice after treatment. Flow cytometry analysis of the tumor samples collected from mice pre-immunized with tetanus at the end of the experiment. The frequency of (A) activated DCs, (B) CD8.sup.+ and (C) CD4.sup.+ T cells, (D-E) and CD8.sup.+ and CD4.sup.+ effector memory (CD44.sup.+CD62L.sup.−) T cells within the TME is reported. The data are plotted as bar graphs and single values. (Statistical analysis Kruskal-Wallis test ANOVA). (F) Flow cytometry analysis of the activation/exhaustion profile of the CD8.sup.+ T cells in the tumors. The bar graph depicts gMFI mean of CD8.sup.+T cells that are antigen experienced (PD1+) and exhausted (TIM3+). Significance was assessed by two-tailed unpaired student's t-test, * p<0.05, ** p<0.005, *** p<0,001, **** p<0,0001;

[0056] FIG. 3 Phenotype of immune cells in lymph-nodes. Lymph-nodes collected from Tetanus Toxin (TT) pre-immunized mice were analysed by flow cytometry to assess the level of (A) TT-specific CD4.sup.+, (B) TT-specific CD4.sup.+ expressing CD40L and (C) APCs exhibiting CD40 receptor. (Statistical analysis Ordinary One-way ANOVA * p<0.05, ** p<0.005, *** p<0,001, **** p<0,0001)

[0057] FIG. 4 Synergistic effect between hybrid PeptiCRAd and aPD1. (A) Treatment scheme. 3×10.sup.5 B16.OVA cells were injected into the right flank of C57BL/6 mice (n=7-8) pre-immunized with tetanus and the treatments were initiated on established tumors with either the hybrid PeptiCRAd only (A) or a combination with anti-PD-1 antibody (C). The tumor growth curve for mice treated without (B) or with (D) anti-PD-1 is represented as mean±SEM. (E) Complete responses (i.e. the disappearance of the total tumor mass upon treatment) for each group is depicted as the percentage of responders from all treated mice in a single group as well as the ratio of responding individuals to non-responding individuals in a single group. (F) Flow cytometry analysis of CD8.sup.+T cells in the tumor from mice treated with tyrosinase related protein 2 (TRP2) TRP2-PeptiCRAd and TT-TRP2-PeptiCRAd. The result is displayed as a single dot for each individual. The control groups that received no peptide vaccine (mock and anti-PD-1 only) are pooled and indicated as “no peptide”. Statistical analysis was assessed by 2WAY ANOVA with uncorrected Fisher's LSD (B) and Tukey's multiple comparison test (D);

[0058] FIG. 5 Hybrid PeptiCRAd and aPD1 effects in the context of tetravalent vaccine. (A) 3×10.sup.5 B16.OVA cells were injected into the right flank of C57BL/6 mice (n=8) pre-immunized with polioboostrix vaccine. Treatments were initiated on established tumors (9 days after tumor implantation) and the mice were treated four times with DP-TRP2-PeptiCRAd (on days 9, 11, 13 and 15) and three times with aPD-1 (on days 9, 13 and 17). (B) The tumor volume is depicted as mean ±SEM (statistical analysis 2way ANOVA with Tukey's multiple comparisons test). (C) The level of naïve CD8+ and CD4+ (CD44-CD62L+) T cells in tumor draining lymph nodes of naïve or pre-immunized mice is reported (Statistical analysis unpaired student t-test two tailed, * p<0.05, ** p<0.005, ***p<0,001, ****p<0,0001). (D) Effector memory (CD44.sup.+CD62L.sup.−) CD4.sup.+ T cells in tumor draining lymph nodes and tumor is shown (Statistical analysis ordinary one-way ANOVA with Tukey's multiple comparison test. *P≤0.05, **P≤0.01, ***P≤0.001);

[0059] FIG. 6 Detection of humoral and cellular immunity in response to pre-immunization with tetanus vaccine. (A) Detection of anti-tetanus IgG in mouse serum samples. Serum from C57BL/6 naïve mice and mice pre-immunized with tetanus vaccine was collected 48 h after the last boosting and the IgG anti-tetanus titer was measured. The data are shown as mean anti-tetanus IgG U/ml (statistical analysis unpaired student t-test two tailed, * p<0.05, ** p<0.005, *** p<0,001, **** p<0,0001). (B) Th1 intracellular staining. Splenocytes from untreated naïve and TT-PeptiCRAd treated pre-immunized mice were analysed by flow cytometry to assess the level of IFN-γ (Th1 polarization) and Foxp3 (Tregs) upon TT peptide stimulation;

[0060] FIG. 7 (A) Single tumor growth and immune cell component within the TME. Tumor growth curve for each mouse and one graph for each group are reported with the specific treatment indicated in each graph. Tumor volumes are normalized against the values on the day of the first treatment and presented as mean of percentage±SEM. The percentage displayed next to each graph shows the responders (green), defined as mice with a tumor volume lower than 400% (dashed line). Flow cytometry analysis of DC activation (B) and total CD8+ T cells (C) CD4+ T cells (D) and effector memory (CD44+CD62L−) CD4+T cells (E) within the TME are reported for individual mice in green (responders) and black (non-responders) among each group. The frequency of all the analysed cell types was significantly higher in the responders compared to non-responders. Significance was assessed by two tailed unpaired t-test with Welch's correction for DC activation and for CD8.sup.+ and CD4.sup.+T cell infiltration analysis, and by two tailed unpaired t-test for the effector memory T cell infiltration;

[0061] FIG. 8 CD40-CD40L crosstalk effects in lymphoid organs. Splenocytes from pre-immunized mice were investigated by flow cytometry for CD4.sup.+T cells expressing (A) or not expressing (B) Foxp3. In draining lymph node total amount of CD4.sup.+T cells is reported (C) and the memory CD4.sup.+ T cells (CD44.sup.+) were analyzed by intracellular staining for IFN-γ (Th1 phenotype) (D), for IL4 (Th2 phenotype) (E) and for IL-17 (Th17 phenotype) (F). The ratio between Th1 and Th2 polarized CD4.sup.+ T cells is depicted in G. Statistical analysis was assessed by ordinary one-way ANOVA with Tukey's multiple comparison test. *P≤0.05, **P≤0.01, ***P≤0.001;

[0062] FIG. 9 Single tumor growth and area under the curve. (A) Tumor growth curve for each mouse and one graph for each group are reported with the specific treatment indicated in each graph. (B) Area under the curve of the tumor growth is reported as graph bars±SEM for groups treated with anti-PD-1 (Statistical analysis unpaired student t-test two tailed, * p<0.05, ** p<0.005, *** p<0,001, **** p<0,0001); and

[0063] FIG. 10 Immunological characterization in polioboostrix pre-immunized mice. (A) The antibody response induced by PolioBoostrix vaccine. C57BL/6 mice were immunized, and the sera collected after 5 days following the final booster immunization; the anti-diphtheria toxoid antibodies were analysed by ELISA. The data are shown as mean IgG IU/ml±SEM. (B) IFN-γ ELISPOT assay. Splenocytes of naïve mice or mice pre-immunized with PolioBoostrix vaccine were collected one month after the final booster immunization and cultured for 72 h with the stimuli indicated in the figure. The results were expressed as the mean frequency of specific IFN-γ spot-forming cells per 1×10.sup.6 cells. (statistical analysis unpaired student t-test two tailed, * p<0.05, ** p<0.005, *** p<0,001, **** p<0,0001). (C) IFN-γ intracellular staining. Splenocytes from naïve and immunized mice were incubated for 6 h with the indicated stimuli and measured by flow cytometry after intracellular staining. (D) The percent of TRP2-specific CD8.sup.+ T cells of all CD8.sup.+ T cells within the TME. The data were normalized to the tumor volume and plotted as mean±SEM (statistical analysis Kruskal-Wallis test ANOVA).

METHODS AND MATERIALS

Study Design

[0064] The main goal of this study is revoking the CD4.sup.+ T cell anti-pathogen memory repertoire to boost the anti-tumor response. As proof of principle, our hypothesis was verified in tetanus immunized B16.OVA bearing mice compared to naïve mice. To demonstrate that the use of the memory repertoire gave an advantage over the naïve, the mice's immunological background was examined. Subsequently, we validated our hypothesis using a clinically relevant tumor peptide in combination with an immune checkpoint inhibitor. Lastly, the experiment was repeated with a different type of vaccine, thus verifying the generic use of the underlying principal or conceptual framework. The control and treatments groups are specified in the figure legends. Animal number for each study type was determined by the investigators (each treatment group had not less than n=8 mice). Animals were randomly allocated to the control and the treatment groups.

Cell Lines and Reagents

[0065] The cell line B16.OVA, a mouse melanoma cell line expressing chicken ovalbumin (OVA), was cultured according to ATCC recommendations. The cells were cultured in RPMI-1640 with low glucose and supplemented with 10% FBS, 1% antibiotics and 1% L-Glutamine. The cells were cultivated in 37° C., 5% CO.sub.2 in a humidified atmosphere.

[0066] The following peptides, purchased from Ontores Biotechnologies Co. Ltd (Hangzhou, China), were used throughout the study:

TABLE-US-00003 (SEQ ID NO: 1) KKKKKQYIKANSKFIGITEL (Tetanus toxin); (SEQ ID NO: 2) KKKKARYVSQQTRANPNPY (Pertussis); (SEQ ID NO: 3) KKKKIQSKRFAPLYAVEAK (Polio Mahoney); (SEQ ID NO: 4) KKKKKKSPVYVGNGVHANLHV (Diphtheria); (SEQ ID NO: 5) KKKKKKPVFAGANYAAWAVNVAQVI (Diphtheria); (SEQ ID NO: 6) ARYVSQQTRANPNPY (Pertussis); (SEQ ID NO: 7) IQSKRFAPLYAVEAK (Polio Mahoney); and (SEQ ID NO: 8) SPVYVGNGVHANLHV (Diphtheria).

[0067] The following peptides are examples of anti-tumour/anti-cancer peptides

TABLE-US-00004 Position Protein Sequence (aa) gp100 KKKKKK-KVPRNQDWL 25-35 (SEQ ID NO: 19) NY-ESO-1 KKKKKK-SLLMWITQC 157-165 (SEQ ID NO: 20) (HLA A2) NY-ESO-1 KKKKKK-RGPESRLLEFYLAMPFATPM  81-100 (SEQ ID NO: 21) NY-ESO-1 KKKKKK-YLAMPFATPMEAELARRSLA  91-110 (SEQ ID NO: 22) NY-ESO-1 KKKKKK-RGPESRLLEFYLAMPFATPMEAE  81-110 LARRSLA (SEQ ID NO: 23) NY-ESO-1 KKKKKK-PGVLLKEFTVSGNILTIRLTAAD 119-143 HR (SEQ ID NO: 24) NY-ESO-1 KKKKKKKKK-YLAMPFATPMEAELARRSLA  91-110 (SEQ ID NO: 25) NY-ESO-1 KKKKKK-YLAMPFATPMEAELARRSLAEE  91-110 (SEQ ID NO: 26) MAGE-A3 KKKKKK-VFGIELMEVDPIGHLYIFAT 161-180 (SEQ ID NO: 27) MAGE-A3 KKKKKK-VFGIELMEVDPIGHLY 161-176 (SEQ ID NO: 28)

Pre-Immunization of Mice

[0068] For tetanus and diphtheria-tetanus-polio-pertussis vaccination, 4-6-week-old female C57BL/6 mice received a primary intramuscular (i.m.) vaccination of Anatetall (GlaxoSmithKline, Italy: 8 IU in 100 μL) or PolioBoostrix (GlaxoSmithKline, Italy: Diphtheria Toxoid 0.4 IU in 100 μL, Tetanus Toxoid 4 IU in 100 μL, Bordetella pertussis antigens: Pertussis Toxoid 1.6 mg in 100 μL, Hemagglutinin 1.6 mg in 100 μL and Pertactin 0.5 mg in 100 μL) respectively, administered bilaterally into the quadricep muscle (50 uL per leg). An i.m. booster vaccination (50 μL) was administered twice: a first one 2 weeks after the initial vaccinaton and a second 2 weeks after the first booster. Mouse IgG antibody responses to tetanus toxoid and diphtheria were measured by ELISA (Xpress Bio Frederik, Md. USA). Serum from immunized mice was harvested 5 days after the last immunization and prior to the animal experiment.

Animal Experiments and Ethical Permits

[0069] All animal experiments were reviewed and approved by the Experimental Animal Committee of the University of Helsinki and the Provincial Government of Southern Finland (license number ESAVI/9817/04.10.07/2016).

[0070] 4-6 weeks old female C57BL/6JOIaHsd mice were obtained from Envigo (Laboratory, Bar Harbor, Maine UK). 3×10.sup.5 B16.OVA cells were injected subcutaneously into the right flank. Details about the schedule of the treatment can be found in the figure legends. Viral dose was 1×10.sup.9 vp/tumor complexed with 20 μg of a single peptide or with 10 μg+10 μg mixture of two peptides. Intratumorally administrated Anatetall vaccine was dosed at 2 IU per mouse. Checkpoint inhibitors were given intraperitoneally at a dose of 100 μg/mouse.

Flow Cytometry

[0071] The antibodies used are the following: TruStain Fcblock and anti-CD8-FITC (eBioscience, Affymetrix (Fisher), Foxp3-PE (eBioscience), CD4-PeCy7 (eBioscience), CD3-PerCPCy5.5 (eBioscience), IFNg-APC (eBioscience), CD40L-BV650 (BD Biosciences Bel Art Scienceware (Fisher), IFNg-FITC (BD), IL-17A-PE (BD), CD4-PerCPCy5.5 (BD), IL4-APC (BD), CD44-V450 (BD), CD44-PE (eBioscience), CD4-PeCy7 (eBioscience), CD3-PerCPCy5.5 (eBioscience), CD62L-APC (eBioscience), CCR7-V450 (BD), CD11c-FITC (BD), B220-PE (eBioscience), MHC-II(A-I/E-I)-PeCy7 (eBioscience), CD86-V450 (BD), CD40-APC (eBioscience), CD11b-PerCP-Cy5.5F4/80BV650 (BD), H-2Kb SVYDFFVWL-APC (ProImmune, Oxford Science Park UK), CD8a-FITC (ProImmune). The data were acquired using BDLSRFORTESSA flow cytometer.

[0072] Data were analyzed using FlowJo software v9 (Ashland, Oreg., USA).

IFN-γ ELISPOT

[0073] IFN-γ ELISPOT assays were performed using a commercially available mouse ELISPOT reagent set (ImmunoSpot, Bonn Germany) and 20 ng/uL of each peptide was tested in in vitro stimulations of splenocytes at 37° C. for 72 h. Spots were counted using an ELISPOT reader system (ImmunoSpot).

PeptiCrad Complex Formation

[0074] Oncolytic adenovirus and each epitope with a polyK tail (Ontores, Zhejiang, China) were mixed to prepare the PeptiCRAd complex. We mixed polyK-extended epitopes with Ad-5-D24-CpG for 15 minutes at room temperature prior to treatments with the PeptiCRAd complexes. More details about the stability and formation of the complex can be found in our previous study (11).

Statistical Analysis

[0075] Statistical analysis was performed using Graphpad Prism 6.0 software (Graphpad Software Inc., La Jolla, Calif. USA). For animal experiment, 2 way ANOVA with Tukey's multiple comparisons test was used and P<0.05 was considered statistically significant. All results are expressed as the mean±standard error of the mean (SEM).

[0076] Details about the statistical tests for each experiment can be found in the corresponding figure legend.

Results and Discussion

EXAMPLE 1

Pre-Immunization with Tetanus Vaccine Boosts the Antitumor Response of a Double-Coated PeptiCRAd

[0077] We herein assessed the potential of engaging the CD4+ T cell memory using the PeptiCRAd vaccine platform (11) where we herein coated an oncolytic adenovirus with both MHC-I-restricted tumor-specific peptides and MHC-II-restricted pathogen-specific peptides, and studied the effect on mice tumors in mice pre-immunized for the pathogen (FIG. 1A). Our hypothesis was that by adding the MHC-II-restricted pathogen-specific peptides to the PeptiCRAd platform we would provide a swifter and stronger T helper response, thus enhancing the tumor specific CTL response.

[0078] We investigated the anti-tumor effect of modified or double-coated PeptiCRAd in mice pre-immunized with tetanus vaccine intramuscularly and bearing B16.OVA tumors, a melanoma model expressing chicken OVA as a model antigen (15). The OVA-epitope was selected since it has a high immunogenicity and hence provides a suitable model to analyze the generation of T cell response (16). C57BL/6 mice were immunized with tetanus vaccine three times at 2-week intervals (FIG. 1B). 5 weeks after the priming, serum samples were collected from mice and anti-tetanus antibody titer was measured to confirm the success of the vaccination (FIG. 1B and FIG. 6A).

[0079] After tumor engraftment, mice were randomized and treated with PeptiCRAd coated with tumor specific peptides (OVA-PeptiCRAd), tetanus-specific peptides (TT-PeptiCRAd) or both tetanus and OVA peptides (TT-OVA-PeptiCRAd). In addition, tetanus vaccine alone or in combination with OVA-PeptiCRAd was used to assess whether intratumorally administrated commercial vaccine can affect tumor growth. All treatments were delivered by intratumoral administration according to the regimen depicted in the FIG. 1B.

[0080] Following therapy, TT-OVA-PeptiCRAd was superior to either one of the single coated viruses in controlling the tumor growth in mice pre-immunized with tetanus toxoid vaccine (FIG. 1C), suggesting that the anti-tetanus memory response indeed enhances the primary immune response elicited against the OVA antigen. The ability of TT-coated PeptiCRAd to elicit mainly Th1-polarized CD4+T cell responses was further corroborated by intracellular staining (FIG. 6B). Less surprisingly, the approach worked also when the tetanus vaccine was re-introduced as a combination with OVA-PeptiCRAd (Vaccine+OVA-PeptiCRAd), whereas tetanus vaccine alone had no therapeutic efficacy (FIG. 1D). Notably, when comparing Vaccine+OVA-PeptiCRAd to OVA-PeptiCRAd, the latter showed a significantly higher anti-tumor efficacy (p=0.05). This suggests that the effect was not caused by the adjuvant contained in the vaccine itself but rather by the presentation of tetanus-specific peptides on MHC-II, engaging CD4+ T cells to help the cytotoxic CD8+ T cell response.

[0081] Interestingly, when the same experiment was performed in naïve mice (mice that had not been preimmunized with tetanus vaccine), no statistically significant differences were observed between OVA-PeptiCRAd and TT-OVA-PeptiCRAd. (FIG. 1E).

[0082] These results demonstrate that the anti-tumor efficacy of our virus-based PeptiCRAd cancer vaccine is significantly enhanced if it is simultaneously coated also with peptides that are specific for a pathogen for which a pre-existing immunity exists.

EXAMPLE 2

The Tetanus-Specific Memory Response Favourably Shapes the Immune Environment at the Tumor Site (TME)

[0083] In order to gain a deeper understanding of the mode of action of the double-coated PeptiCRAd, we investigated the quality of the immune response elicited by the different treatments. To this end, we analyzed the frequency of different cell populations in the tumor by flow cytometry, most importantly the activated dendritic cells (DC), CD4+ and CD8+ T cells with effector and memory phenotype and experienced and exhausted CD8+ effector T cells.

[0084] Interestingly, we found an increased frequency of activated intratumoral DCs in all of the groups that had been treated with PeptiCRAd in the context of tetanus antigens (either coated with the TT peptide or co-injected with the whole vaccine) (FIG. 2A). In contrast to these combination treatments, the use of vaccine alone led to poor induction of DC maturation in the TME, suggesting that inclusion of an adenoviral adjuvant may be critical for a proper DC activation in this setting. Moreover, we saw increased levels of CD4+ and CD8+ T cells in the tumors in all groups of mice treated with PeptiCRAd (FIG. 2B-C), which is well in line with what has previously been observed following treatments with virus-based drugs (11). Finally, we wanted to analyze the phenotype of these T cells. Majority of the Tumor-infiltrating lymphocytes (TILs) showed a T effector memory cell phenotype, with an increase in the frequency of CD8+ and CD4+ T.sub.EMs in groups treated with TT-PeptiCRAd and OVA-TT-PeptiCRAd (FIG. 2D-E). Moreover, the expression level of T-cell immunoglobulin and mucin-domain containing-3 (TIM3) on PD1+ TILs were assessed to study T cell exhaustion. Interestingly, we observed a significantly lower frequency of exhausted CD8+ T cells in the group of mice treated with TT-OVA-PeptiCRAd compared to the other groups, indicating that CD4+T cell help is required for optimal CD8+ T cell activity (FIG. 2F). We concluded that the tetanus pre-existing immunity improved the overall efficacy of the treatment substantially by modifying the immune environment at the tumor site, especially when the treatment was virus based and contained the tetanus vaccine or the tetanus peptides. Of note, the serotype 5 human adenovirus used in these experiments is non-oncolytic in murine tumors, and therefore the effect on tumor control is solely based on anti-tumor immune response. To better elucidate this phenomenon, we re-analyzed all the datasets by stratifying the mice between responders and non-responders and assessed again their immunological responses. As expected, we observed a significant difference between the two groups.

[0085] Irrespective of the type of therapy, all responders had an on-going measurable immune response, highlighting the importance of the immune system in controlling the tumour growth, regardless of what kind of treatment they had received. Importantly, the majority of these responders were found in the group of mice treated with TT-OVA-PeptiCRAd (FIG. 7A-E).

EXAMPLE 3

CD40L Expressing TT-Specific, Th1 Polarized CD4+ T Cells are Detected in Secondary Lymphoid Organs Following TT-OVA-PeptiCRAd Therapy

[0086] To dissect the possible mechanism of the observed therapeutic efficacy, we assessed levels and phenotype of immune cells in secondary lymphoid organs of pre-immunized mice. As expected, PeptiCRAd treated mice showed expansion of CD4+ T cell compartment both in the spleen and in the draining lymph nodes (FIG. 3A and FIG. 8C). More importantly, a significant increase of TT-specific CD4+ T cells expressing CD40 ligand (CD40L) was observed in TT-OVA treated mice (FIG. 3B). The majority of these CD40L+ cells were polarized towards Th1 phenotype, albeit some TT-specific Foxp3+ T regulatory cells (Tregs) were also detected (FIG. 8A-B). Analysis of dLNs revealed that the intratumoral vaccination with TT-OVA-PeptiCRAd induced mainly IFN-gamma producing Th1 memory cells at the expense of IL-4 secreting Th2 cells, whereas no differences was observed in IL-17A producing Th17 cells (FIG. 8D-G). Since CD4+ T cell-associated CD40L has been shown to be important in stimulating cytotoxic CD8+ T cell responses, we wanted to study whether we can see CD40+ antigen presenting cells. Indeed, when pre-immunized mice were intratumorally treated with TT-OVA-PeptiCRAd, a significantly higher frequency of CD40+ expressing APCs was detected (FIG. 3C), further suggesting that double-coated PeptiCRAd stimulates TT-specific CD4+ memory T cells, that in turn could license professional APCs via CD40-CD40L interaction.

EXAMPLE 4

Combination with Immune Checkpoint Inhibitors Increases the Number of Responders and Leads to Complete Tumor Rejection

[0087] We have previously shown that a combination of tumor-targeted PeptiCRAd with immune checkpoint inhibitors is synergistic in terms of improved anti-tumor efficacy (8). Thus, we wanted to assess whether the vaccine-induced pre-existing immunity would further enhance this synergy, particularly by increasing the frequency of mice responding to the therapy.

[0088] In order to test this hypothesis, we coated the virus with TT and tyrosinase related protein 2 (TRP2) peptides (TRP2180-188 (23)), which is naturally occurring melanoma-associated antigen and hence more clinically relevant epitope than OVA. Tetanus toxoid pre-immunized mice were implanted with subcutaneous tumors and treated intratumorally with a PeptiCRAd coated with TRP2 peptides only (TRP2-PeptiCRAd) or with a PeptiCRAd coated with both TRP2 and TT peptides (TT-TRP2-PeptiCRAd) (FIG. 4A). Similarly, as in FIG. 1, we observed a significant inhibition of tumor growth in mice treated with the double-coated virus compared to controls (FIG. 4B).

[0089] Interestingly, when we combined the PeptiCRAd treatments with a PD-1 blocking monoclonal antibody, we observed a significant increase in efficacy of both TRP2-PeptiCRAd and TT-TRP2-PeptiCRAd treatments (FIG. 4C-D). However, the double-coated PeptiCRAd was still more effective than the virus coated with a single peptide in terms of tumor growth control (FIG. 9B).

[0090] More importantly, inclusion of TT-specific peptides in the cancer nanovaccine resulted in a 75% response rate to anti-PD1, whereas only 28% of mice treated with TRP2-PeptiCRAd and PD-1 blockade experienced a complete tumor eradication (FIG. 4E and FIG. 9A). One of the biggest advantages of combining oncolytic viruses with checkpoint inhibitors is that the viruses in the tumor facilitate and increase the T lymphocyte recruitment, thereby unleashing an unprecedented activity of the monoclonal antibodies. Along this line, we observed a significant increase in CD8+ TILs in mice treated with TT-TRP2-PeptiCRAd in combination with anti-PD-1 (FIG. 4C), when compared to the control treatments (FIG. 4F).

EXAMPLE 5

The Pre-Existing Immunity is a General Mechanism to Enhance the Anti-Tumor Response and Reshapes the Immunological Balance in T Cell Repertoire

[0091] Since we observed that pre-existing immunity to tetanus toxoid potentiates the anti-tumor response of a double-coated PeptiCRAd alone and in combination with PD-1 blockade, we sought to further investigate whether our approach is valid also in the context of a multivalent vaccine such as a tetravalent vaccine. Polioboostrix is a tetravalent vaccine with a high coverage of 85% of infants immunized, making it an attractive study model (25) C57BL/6 mice were pre-immunized with Polioboostrix vaccine with the same immunization regime as before (FIG. 5A). Serum samples and splenocytes were collected and analyzed in order to confirm the effectiveness in the immunization protocol. Tetravalent vaccine was found to efficiently generate both antibodies and CD4+ T cells specific for pertussis and diphtheria (FIG. 10A-C). For the tumor growth analysis, B16.OVA tumors in naïve or pre-immunized mice were treated with anti-PD1 antibodies and PeptiCRAd coated with MHC-II restricted Diphtheria-Pertussis peptides and MHC-I restricted TRP2 peptides (DP-TRP2-PeptiCRAd). Consistent with our previous results, a superior anti-tumor response was detected in pre-immunized treated with DP-TRP2-peptiCRAd and anti-PD1, whereas treatment efficacy was lost in naïve mice (FIG. 5B).

[0092] These results confirm that the pathogen-specific pre-existing immunity enhances the anti-tumor response and that the mechanism of action is dependent on the memory T cells. Moreover, this effect is not restricted to tetanus but is adaptable to other pathogens as well. To further verify that the mechanism of action behind the enhanced treatment efficacy using diphtheria and pertussis as the pre-immunizing vaccine, we analyzed the T cell repertoire of the tumor draining lymph nodes, TME and spleen. The frequency of naïve CD8+T and CD4+ T cells was lower in the draining lymph nodes of the pre-immunized, DP-TRP2 PeptiCRAd treated mice compared to the control groups (FIG. 5C). Concomitantly, increased levels of CD4+T.sub.EM cells were observed in the draining lymph nodes and in the TME of pre-immunized mice compared to the naïve and mock treated mice (FIG. 5D). In addition, a trend towards higher infiltration of TRP2-specific CD8+ T-cells was seen in the tumour tissue of the immunized mice when compared to the naïve mice (FIG. 10D), and the level of CD4+T.sub.EM cells in the tumour and draining lymph nodes strongly correlated with the intensity of the TRP2-specific TIL response. Taken together, the double-coated PeptiCRAd vaccine platform can be used to stimulate pre-acquired, pathogen-specific CD4+ T cell immunity in order to help the generation of effective anti-tumor CD8+ T cell responses.

SUMMARY

[0093] Due to the high coverage of international vaccination programs, the majority of the worldwide population has been vaccinated against common pathogens, leading to acquired pathogen-specific immunity with a robust memory T cell repertoire. These vaccines lead to the formation of an immunological memory that is able to deploy a much faster and more effective immune response when re-encountering the pathogen; in fact, the primary immune response is rather weak and slow while the secondary immune response is faster and more effective (26). While CD8+ anti-tumor cytotoxic T lymphocytes (CTL) are the preferred effectors of cancer immunotherapy, CD4+ T cell help is also required for an optimally strong anti-tumor immune response to occur.

[0094] Hence, we describe a new cancer immunotherapy approach that takes advantage of the pre-existing pathogen-specific immunological memory present in the worldwide population of vaccinated individuals by investigating whether the pathogen-related CD4+ T cell memory populations could be re-engaged to support the CTLs, converting a weak primary anti-tumor immune response into a stronger secondary one. To this end, we used our PeptiCRAd technology that consists of a virus coated with MHC-I restricted tumor-specific peptides, and developed it further by introducing pathogen specific MHC-II-restricted peptides.

[0095] Proof of concept was demonstrated and validated in melanoma using tetanus and polioboostrix vaccines available for humans, highlighting the universal nature of the CD4+ memory in boosting cancer-specific CTL responses. Importantly, the approach can be extended to naturally occurring tumor peptides beyond the surrogate OVA, as well as to other pathogens instead of tetanus, highlighting the usefulness of our technique in taking full advantage of the CD4+ memory T cell repertoires when designing immunotherapeutic treatment regimens.

[0096] Finally, the anti-tumor effect was even more prominent when combined with an immune checkpoint inhibitor, such as anti-PD1, strengthening the rationale behind combination therapy with oncolytic viruses.

REFERENCES

[0097] 8. Ostroumov D, Fekete-Drimusz N, Saborowski M, Kuhnel F, Woller N. CD4 and CD8 T lymphocyte interplay in controlling tumor growth. Cell Mol Life Sci. 2018;75(4):689-713.

[0098] 11. Capasso C, Hirvinen M, Garofalo M, Romaniuk D, Kuryk L, Sarvela T, et al. Oncolytic adenoviruses coated with MHC-I tumor epitopes increase the antitumor immunity and efficacy against melanoma. Oncoimmunology. 2016;5(4):e1105429.

[0099] 15. Moore M W, Carbone F R, Bevan M J. Introduction of soluble protein into the class I pathway of antigen processing and presentation. Cell. 1988;54(6):777-85.

[0100] 16. Knocke S, Fleischmann-Mundt B, Saborowski M, Manns M P, Kuhnel F, Wirth T C, et al. Tailored Tumor Immunogenicity Reveals Regulation of CD4 and CD8 T Cell Responses against Cancer. Cell Rep. 2016;17(9):2234-46.

[0101] 23. Bloom M B, Perry-Lalley D, Robbins P F, Li Y, el-Gamil M, Rosenberg S A, et al. Identification of tyrosinase-related protein 2 as a tumor rejection antigen for the B16 melanoma. J Exp Med. 1997;185(3):453-9.

[0102] 25. Feldstein L R, Mariat S, Gacic-Dobo M, Diallo M S, Conklin L M, Wallace A S. Global Routine Vaccination Coverage, 2016. MMWR Morb Mortal Wkly Rep. 2017;66(45):1252-5.

[0103] 26. Laidlaw B J, Craft J E, Kaech S M. The multifaceted role of CD4(+) T cells in CD8(+) T cell memory. Nat Rev Immunol. 2016;16(2):102-11.