Viral Vectors

Abstract

The present invention relates to viral vectors having modified viral capsids or viral envelopes; pharmaceutical compositions or immunogenic agents or vaccines comprising said viral vectors; target cells transformed or transfected with said viral vectors; combination therapeutics comprising said viral vectors; the use of said viral vectors in the treatment of cancer, and methods of treating cancer using said viral vectors.

Description

[Technical field]

[0001] The present invention relates to novel viral vectors having modified viral capsids or viral envelopes; pharmaceutical compositions or immunogenic agents or vaccines comprising said viral vectors; target cells transformed or transfected with said viral vectors; combination therapeutic agents comprising said viral vectors; use of said viral vectors in the treatment of cancer, and methods of treating cancer using said viral vectors. [Background technology]

[0002] Prophylactic vaccination is one of the most effective forms of medical intervention with direct clinical and medico-economic benefits, the eradication of common deadly infectious diseases being the most obvious example. Most vaccines rely on the use of attenuated pathogens or parts of them to elicit a robust T cell memory repertoire directed against the pathogen, with CD4+ T cells dominating the memory response. After immunization, naive T cells evolve into T effector memory (TEM) that is rapidly recalled upon encountering the stimulating antigen. EM ) cells, and to T central memory (T central memory) cells, which are found primarily in lymphoid organs and are not immediately triggered in response to pathogens. CM ) cells. A robust repertoire of memory T cells exists in populations worldwide against pathogens that are included in national vaccination programs.

[0003] In contrast, the efficacy of cancer immunotherapy relies on the generation of specific antitumor CD8+ T cells that recognize peptides presented by major histocompatibility complex I (MHC-I). Rapid T cell-mediated responses are necessary for effective antitumor activity. Importantly, it has been shown that cooperation of CD4+ and CD8+ T cells is necessary for efficient antitumor immunity to occur. Indeed, it has been shown that CD4+ T cells provide signals that improve the function of CD8+ T cells within the tumor microenvironment (TME), and depletion of CD4+ cells prior to tumor challenge leads to a complete loss of tumor rejection in mouse tumor models.

[0004] However, although the central role of CD4+ T cells in T cell-mediated immunity is well recognized, how to optimally exploit the interactions between CD4+ and CD8+ T cell populations in cancer treatment strategies remains unclear.

[0005] Thus, the challenge remains how to harness pathogen-specific T cell memory reservoirs, primarily CD4 + T cells, to enhance antitumor CD8 + CTL responses.

[0006] To address this, we used our peptide-coated conditionally replicating adenovirus (PeptiCRAd) platform, based on oncolytic adenovirus coated with MHC-specific peptides (11), to evaluate the impact of re-engagement of pathogen-specific CD4+ memory T cells on antitumor CD8+ T cell responses in mice pre-immunized with a vaccine specific for a human pathogen. Without wishing to be bound by theory, it is believed that antigen-presenting cells (APCs) process the virus and tumor- and pathogen-specific peptides bound to the viral surface to present tumor-specific epitopes to CD8+ T cells and pathogen-specific epitopes to memory CD4+ T cells, which then subsequently maintains the CD8+ T cell-mediated immune response as a bystander effect.

[0007] As a proof of concept, we used naive or tetanus-pre-immunized immune-competent mice implanted with melanoma (B16.OVA) tumors. Mice were treated with intratumoral injections of PeptiCRAd coated with antigens SIINFEKL (i.e., CD8+ T cell epitope of chicken ovalbumin) and tetanus toxoid (CD4+ T cell epitope) peptides. As hypothesized, we observed a superior antitumor response in mice pre-immunized with tetanus vaccine and treated with virus coated with tetanus toxin and melanoma tumor antigens (TT-OVA-PeptiCRAd). Interestingly, in naive mice, the superiority of TT-OVA-PeptiCRAd over control treatment was lost, highlighting the prerequisite of pre-existing immunity to pathogenic antigens to exploit CD4+ T cell memory. We validated this strategy by targeting various pathogens (e.g., diphtheria and pertussis) and further combining it with checkpoint inhibitor treatment (e.g., anti-PD-1 antibody). Engagement of CD4+ T cells with a diphtheria-pertussis specific MHC-II restricted peptide delayed tumor growth in pre-immunized mice. Furthermore, a more robust effector memory CD4+ T cell infiltration was observed in the tumor microenvironment (TME) of treated animals compared to control animals. These results indicated that the proposed mechanism of action is not limited to tetanus, and the principle can be applied to other vaccine formulations.

[0008] Thus, the proposed viral vector can be used to re-engage existing CD4+ memory T cell repertoire to support and enhance antitumor CD8+ CTL responses, paving the way towards the next major improvement in cancer immunotherapy. Summary of the Invention [Means for solving the problem]

[0009] According to a first aspect of the invention there is provided a viral vector having a polypeptide attached to its viral capsid or envelope, said polypeptide being not genetically encoded by said viral vector but being covalently or non-covalently attached to said capsid or envelope, i) at least one of the polypeptides comprises an antigen derived from or of a pathogen, such as a virus or bacterium, against which the subject has previously been immunized; ii) at least one other of the polypeptides is an anti-tumor or anti-cancer specific polypeptide and stimulates an anti-tumor or anti-cancer immune response in a subject exposed to the vector; Viral vectors are provided.

[0010] The "capsid" of an adenovirus or vector refers to the protein shell of the virus. The capsid is composed of several oligomeric structural subunits made of proteins called protomers. In addition, as will be understood by those skilled in the art, certain viruses (e.g., HIV and many animal viruses) have a viral "envelope" that covers their protective capsid. The envelope is usually derived from a portion of the host cell membrane (phospholipids and proteins), but contains some viral glycoproteins. Thus, depending on the nature of the virus, the polypeptide can be attached to the capsid or the envelope.

[0011] References herein to a pathogen most typically include a virus or bacteria, but may also include any other pathogen, most suitably one against which an individual has been or may be immunized, as such pathogens typically, though not always, form part of national, international or other vaccination programmes.

[0012] The present invention relates to the reactivation of pathogen-specific T cell memory reservoirs, primarily CD4+ T cells, using antigens of or derived from a pathogen, such as a virus or bacteria, against which the subject to be treated has previously been immunized, said CD4+ T cells cooperating with cancer-specific CD8+ cytotoxic T cells to enhance the efficacy of the cancer-specific CD8+ cytotoxic T cells.

[0013] Thus, in a preferred embodiment of the invention, the antigen according to part i) may be any antigen derived from or of a pathogen, such as a virus or a bacterium, against which the subject has previously been immunised. Ideally, but not exclusively, the selected antigens would be those from the following families: measles morbillivirus, mumps rupulavirus, rubella virus, rotavirus, varicella zoster virus, flavivirus (yellow fever), hepatovirus, human papillomavirus, Streptococcus pneumoniae, Neisseria meningitidis, Corynebacterium diphtheriae, Clostridium tetani, Mycobacterium tuberculosis, poliovirus, Bordetella pertussis, Vibrio cholerae, and others. The vaccine may be of, or be derived from, a virus or bacterium that forms part of a widely implemented and / or routinely used vaccination program, such as, but not limited to, those selected from the group including, for example, B. cholerae, B. cholerae, and the like.

[0014] In a preferred embodiment only, said polypeptide of part i) QYIKANSKFIGITEL (tetanus toxin) (SEQ ID NO:1); ARYVSQQTRANPNPY (Pertussis) (SEQ ID NO:2); IQSKRFAPLYAVEAK (Polyomahony) (SEQ ID NO:3); SPVYVGNGVHANLHV (Diphtheria) (SEQ ID NO: 4); PVFAGANYAAWAVNVAQVI (Diphtheria) (SEQ ID NO:5); ARYVSQQTRANPNPY (Pertussis) (SEQ ID NO: 6); IQSKRFAPLYAVEAK (Polyomahony) (SEQ ID NO: 7); and SPVYVGNGVHANLHV (Diphtheria) (SEQ ID NO: 8) The compound is selected from the group consisting of:

[0015] Additionally or alternatively, said polypeptides of part i) comprise a plurality of polypeptides from at least one pathogen, such as a virus or bacterium, against which the subject has previously been immunized, and in a preferred embodiment said plurality of polypeptides are from several different pathogens.

[0016] In a further preferred embodiment of the invention, the antigen according to part ii) is an anti-tumor or anti-cancer antigen and can therefore be any antigen that stimulates an anti-tumor or anti-cancer immune response in a subject exposed to said antigen. Examples of anti-cancer antigens include: These include, but are not limited to, fragments derived from the following peptides: gp100, NY-ESO-1 and Mage-A3, and more ideally, the antigen comprises a fragment of: KVPRNQDWL(gp100) (SEQ ID NO:9); SLLMWITQC (NY-ESO-1) (SEQ ID NO: 10) RGPESRLLEFYLAMPFATPM (NY-ESO-1) (SEQ ID NO: 11) YLAMPFATPMEAELARRSLA (NY-ESO-1) (SEQ ID NO: 12); RGPESRLLEFYLAMPFATPMEAELARRSLA(NY-ESO-1 (SEQ ID NO: 13); PGVLLKEFTVSGNILTIRLTAADHR (NY-ESO-1) (SEQ ID NO: 14); YLAMPFATPMEAELARRSLA (NY-ESO-1) (SEQ ID NO: 15); YLAMPFATPMEAELARRSLAEE (NY-ESO-1) (SEQ ID NO: 16); VFGIELMEVDPIGHLYIFAT (MAGE-A3) (SEQ ID NO: 17); and VFGIELMEVDPIGHLY (MAGE-A3) (SEQ ID NO: 18); Includes:

[0017] Additionally or alternatively, said polypeptide of portion ii) comprises a plurality of polypeptides which, at least collectively, if not individually, stimulate an anti-tumor or anti-cancer immune response.

[0018] Advantageously, said polypeptide stimulates both pathogen-specific (part i) and peptide-specific (part ii) immune responses in subjects who have already been pre-immunized against said pathogenic antigen.Thus, said polypeptide induces both CD4+ and CD8+ T cell responses, the former enhancing the effectiveness of the latter.Furthermore, and advantageously, since said polypeptide is not genetically encoded by said viral vector, but is covalently or non-covalently attached to capsid or envelope, said polypeptide can be attached to said virus quickly and efficiently, i.e. without the need to wait for viral replication to occur in host cells.

[0019] In a further preferred embodiment, furthermore, said polypeptide is selected from the group comprising major histocompatibility complex class I (MHC-I) specific polypeptide, major histocompatibility complex class II (MHC-II) specific polypeptide and dendritic cell (DC) specific polypeptide. Thus, said polypeptide may be a major histocompatibility complex class I (MHC-I) specific polypeptide and / or a major histocompatibility complex class II (MHC-II) specific polypeptide and / or a dendritic cell (DC) specific polypeptide.

[0020] Ideally, said viral vector comprises a plurality of MHC-I specific polypeptides and / or further a plurality of MHC-II specific polypeptides. Most ideally, the polypeptides according to part i) are MHC-II specific and / or the polypeptides according to part ii) are MHC-I specific, or vice versa.

[0021] In a further preferred embodiment of the invention, the polypeptide comprises a fusion polypeptide, one part of the fusion polypeptide comprises an antigen derived from or of a pathogen, such as a virus or a bacterium against which the subject has previously been immunized, and another part of the fusion polypeptide is an anti-tumor or anti-cancer specific polypeptide, thus stimulating an anti-tumor or anti-cancer immune response in a subject exposed to the vector.

[0022] In a further preferred embodiment, the polypeptide is polylysine or polyarginine modified, and thus includes a polylysine or polyarginine linker for attaching the polypeptide to the capsid or envelope. Typically, the polypeptide is polylysine or polyarginine extended with at least four, ideally five, six, seven, eight or nine lysines to facilitate attachment. Most typically, six lysines are used, most preferably, but not limited to, attached to the amino terminus of the polypeptide. Even more ideally, the polypeptide has a lysine or arginine tail that includes one other amino acid, preferably five lysines or arginines, followed by one proline (or any other single amino acid), and then again four more lysines or arginines.

[0023] Alternatively, other means known to those skilled in the art can be used to attach the polypeptide to the capsid or envelope, such as, but not limited to, the use of a cell penetrating peptide as a linker between the polypeptide and the virus, which is particularly useful for enveloped viruses, and the use of cholesterol moieties, electrostatic, disulfide or amide bond linkages can also be used to attach any of the polypeptides to the viral capsid or viral envelope.

[0024] In a further preferred embodiment, the viral vector is an enveloped or non-enveloped virus.

[0025] As will be appreciated by those of skill in the art, examples of enveloped viruses include families selected from the group including herpesviruses, poxviruses, hepadnaviruses, flaviviruses, togaviruses, coronaviruses, hepatitis D, orthomyxoviruses, paramyxoviruses, rhabdoviruses, bunyaviruses, filoviruses and retroviruses.

[0026] Alternatively, examples of non-enveloped viruses include a family selected from the group including adenovirus, reovirus, papillomavirus, picornavirus, calicivirus.

[0027] In a preferred embodiment, the virus is selected from the group comprising herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), vaccinia, vesicular stomatitis virus Indiana strain (VSV), measles virus (MeV), Maraba virus and Newcastle disease virus (NDV). Most ideally, the viral vector is vaccinia virus or HSV-1 or adenovirus or oncolytic adenovirus.

[0028] As will be appreciated, preferably said viral vector is oncolytic, ie, capable of infecting and killing cancer cells by selective replication in tumor cells compared to normal cells.

[0029] The viral vectors used in the present invention may contain other modifications in addition to those described above. Any further modifications may be used, but are not essential to the practice of the present invention.

[0030] In conclusion, the viral vector of the present invention is designed to stimulate immune responses against cancer, particularly by exploiting the interaction between CD4+ memory T cells and CD8+ T cells and re-engaging CD4+ memory effector T cells generated against previous (immunized) pathogenic antigens to aid in the fight against cancer, aiming to initiate a rapid and reliable anti-tumor response. Specifically, it was found that when APC / DC process the virus of the present invention, particularly the peptides attached to its surface, DC not only present tumor-specific peptides to CD8+ T cells to induce anti-tumor immune responses, but importantly, DC also present pathogen-specific peptides to CD4+ T helper cells that enhance and sustain the cytotoxic immune response. This highlights the importance of the interaction between the innate and adaptive arms of the immune system, and the central role of effector memory CD4+ T cells in supporting the ongoing anti-tumor response.

[0031] Thus, according to a second aspect of the invention, the present invention extends to a pharmaceutical composition or immunogenic agent or vaccine comprising at least one viral vector of the invention and a pharma- ceutically acceptable carrier, adjuvant, diluent or excipient.

[0032] Suitable pharmaceutical excipients are well known to those skilled in the art.The pharmaceutical composition can be formulated for administration by any suitable route, for example, intratumoral, intramuscular, intraarterial, intravenous, intrapleural, intravesicular, intracavitary or intraperitoneal injection, buccal, nasal or bronchial (inhalation), transdermal or parenteral, and can be prepared by any method well known in the art of pharmacy.

[0033] The composition can be prepared by mixing the viral vector defined above with a carrier.Generally, the formulation is prepared by uniformly and intimately mixing the viral vector with a liquid carrier or a finely divided solid carrier or both, and then, if necessary, shaping the product.The present invention extends to a method for preparing pharmaceutical compositions, which comprises combining or mixing the viral vector defined herein with a pharmaceutically or veterinarily acceptable carrier or vehicle.

[0034] According to a third aspect of the present invention there is provided a target cell transformed or transfected with the viral vector defined herein.

[0035] According to a fourth aspect of the invention there is provided a combination therapy for the treatment of cancer comprising: Combination therapeutic agents are provided comprising a viral vector or pharmaceutical composition or immunogenic agent or vaccine or cells as defined herein in combination with at least one further therapeutic agent.

[0036] Advantageously, the antitumor effect induced by the viral vector of the present invention is found to be even more pronounced when said viral vector is combined with at least one other anticancer agent, thus strengthening the rationale behind the use of combination therapy when using viral vector agents. For example, the other anticancer agent can be a checkpoint inhibitor molecule, in view of the fact that tumors have evolved several immunosuppressive mechanisms to counteract immune responses against tumors. The best characterized checkpoint pathways are the cytotoxic T-lymphocyte protein 4 (CTLA-4) pathway and the programmed cell death protein 1 pathway (PD-1 / PD-L1). Thus, the viral vector of the present invention can be used in combination with at least one checkpoint regulator, such as anti-CTLA-4, anti-PD1 or anti-PD-L1 molecules, to counteract the immunosuppressive tumor environment and trigger a strong anticancer immune response.

[0037] Additionally, the at least one other anti-cancer agent most suitably includes cyclophosphamide, as cyclophosphamide down-regulates regulatory T cells.

[0038] However, as will be appreciated by one of ordinary skill in the art, the additional therapeutic agent may be any anti-cancer agent known in the art.

[0039] Additionally or alternatively, the present invention further relates to at least one viral vector or pharmaceutical composition or immunogenic agent or vaccine or cell or combination therapy according to the present invention for use in the treatment of cancer.

[0040] Additionally or alternatively, the present invention relates to the use of at least one viral vector or pharmaceutical composition or immunogenic agent or cell or vaccine or combination therapy for treating cancer.

[0041] Additionally or alternatively, the present invention relates to the use of at least one viral vector or pharmaceutical composition or immunogenic agent or vaccine or cell or combination therapy according to the present invention in the manufacture of a medicament for treating cancer.

[0042] Thus, according to yet another aspect of the present invention there is provided a method of treating cancer comprising administering to a subject in need of cancer treatment an effective amount of at least one viral vector or pharmaceutical composition or immunogenic agent or vaccine or cells or combination therapy as described herein.

[0043] The "effective amount" of a viral vector or a pharmaceutical composition or immunogenic agent or vaccine comprising a viral vector herein refers to an amount sufficient to achieve a desired biological effect, such as cancer cell death.It is understood that an effective dosage depends on the age, sex, health and weight of the recipient, the type of concurrent treatment, if any, the frequency of treatment, and the nature of the desired effect.Typically, the effective amount is determined by the person administering the treatment.

[0044] Most preferably, the cancers referred to herein include the following cancers: nasopharyngeal carcinoma, synovial carcinoma, hepatocellular carcinoma, renal carcinoma, cancer of connective tissue, melanoma, lung cancer, intestinal cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cavity cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal gland cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, carcinoma of unknown primary site, carcinoid, carcinoid of the digestive tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer. , gallbladder 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, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meningeal cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharyngeal cancer, pleura cancer, salivary gland cancer, tongue cancer, and tonsil cancer.

[0045] In the following claims and the foregoing description of the invention, unless the context otherwise requires, either by express language or by necessary implication, the word "comprise" or variations such as "comprises" or "comprising" are used in their inclusive sense, i.e., to specify the presence of stated features but not to exclude the presence or addition of further features in various embodiments of the invention.

[0046] All references cited herein, including any patents or patent applications, are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

[0047] Preferred features of each aspect of the invention may be as described in relation to any of the other aspects.

[0048] Other features of the present invention will become clear from the following examples. Generally speaking, the present invention covers any novel one or any novel combination of features disclosed in this specification (including the appended claims and drawings). Therefore, it should be understood that a feature, integer, property, compound or chemical moiety described in connection with a particular aspect, embodiment or example of the present invention is applicable to any other aspect, embodiment or example described herein, unless inconsistent therewith.

[0049] Moreover, unless stated otherwise, any feature disclosed in this specification may be replaced by an alternative feature serving the same or a similar purpose.

[0050] Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, it is to be understood that the specification contemplates the plural as well as the singular, unless the context requires otherwise.

[0051] Embodiments of the present invention will now be described, by way of example only, with reference to the following. [Brief description of the drawings]

[0052] [Figure 1] Figure 1. Effect of recalling memory repertoires on a mouse model of melanoma. (A) Schematic of the novel hybrid PeptiCRAd system. A single adenovirus is loaded with a pathogen-specific peptide to elicit pre-existing memory T cell repertoire and a tumor-specific peptide to elicit antitumor T cell repertoire. (B) Treatment scheme. Naïve and tetanus-preimmunized C57BL / 6 mice (n=7-8) were injected with 3x105 B16.OVA cells into the right flank. Four treatments were administered intratumorally (on days 9, 11, 13, and 15) as indicated. (C-E) B16.OVA tumor growth was followed until the end of the experiment in naïve and preimmunized mice. Tumor sizes are presented as mean ± SEM for each treatment. (Statistical analysis 2-way analysis of variance, *p<0.05, **p<0.005, ***p<0,001, ****p<0,0001); [Diagram 2] Figure 2. Immune cell components within the tumor microenvironment (TME) in pre-immunized mice after treatment. Flow cytometric analysis of tumor samples collected from tetanus pre-immunized mice at the end of the experiment. The frequencies of (A) activated DCs, (B) CD8+ and (C) CD4+ T cells, (D-E) and CD8+ and CD4+ effector memory (CD44+CD62L-) T cells within the TME are reported. Data are plotted as bar graphs and single values. (Statistical analysis Kruskal-Wallis test ANOVA). (F) Flow cytometric analysis of the activation / exhaustion profile of CD8+ T cells in the tumor. Bar graphs show the gMFI mean of antigen-experienced (PD1+) and exhausted (TIM3+) CD8+ T cells. Significance was assessed by two-tailed unpaired Student's t-test. *p<0.05, **p<0.005, ***p<0,001, ****p<0,0001; [Diagram 3]Figure 3. Phenotype of immune cells in lymph nodes. Lymph nodes collected from tetanus toxoid (TT) pre-immunized mice were analyzed by flow cytometry to assess the levels of (A) TT-specific CD4+, (B) CD40L-expressing TT-specific CD4+ and (C) APCs displaying the CD40 receptor. (Statistical analysis: Ordinary one-way ANOVA *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001) [Figure 4] Figure 4. Synergy between hybrid PeptiCRAd and aPD1. (A) Treatment scheme. Tetanus-preimmunized C57BL / 6 mice (n=7-8) were injected with 3x105 B16.OVA cells in the right flank and treatment was initiated against established tumors with either hybrid PeptiCRAd alone (A) or in combination with anti-PD-1 antibody (C). Tumor growth curves of mice treated without (B) or with (D) anti-PD-1 are presented as mean ± SEM. (E) Complete responses (i.e., loss of total tumor mass upon treatment) for each group are illustrated as the percentage of responders from all treated mice in a single group and the ratio of responders to non-responders in a single group. (F) Flow cytometry analysis of CD8+ T cells in tumors from mice treated with tyrosinase-related protein 2 (TRP2) TRP2-PeptiCRAd and TT-TRP2-PeptiCRAd. Results are shown as one point for each individual. Control groups that did not receive peptide vaccines (mock and anti-PD-1 only) were pooled and shown as "no peptide." Statistical analysis was assessed by two-way ANOVA with Fisher's LSD (B) and Tukey's multiple comparison test (D) without correction. [Diagram 5]Figure 5. Hybrid PeptiCRAd and aPD1 efficacy in the context of a tetravalent vaccine. (A) C57BL / 6 mice (n=8) pre-immunized with polioboostrix vaccine were injected with 3x105 B16.OVA cells in the right flank. Treatment was initiated on established tumors (day 9 after tumor implantation) and mice were treated four times with DP-TRP2-PeptiCRAd (days 9, 11, 13, and 15) and three times with aPD-1 (days 9, 13, and 17). (B) Tumor volumes are presented as mean ± SEM (statistical analysis 2-way ANOVA with Tukey's multiple comparison test). (C) Levels of naive CD8+ and CD4+ (CD44-CD62L+) T cells in tumor-draining lymph nodes of naive or pre-immunized mice are reported (statistical analysis unpaired Student's t-test two-tailed, *p<0.05, **p<0.005, ***p<0,001, ****p<0,0001). (D) Effector memory (CD44+CD62L-) CD4+ T cells in tumor-draining lymph nodes and tumors are shown (statistical analysis ordinary one-way ANOVA followed by Tukey's multiple comparison test. *P≤0.05, **P≤0.01, ***P≤0.001); [Figure 6] 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. Sera from C57BL / 6 naive mice and mice pre-immunized with tetanus vaccine were collected 48 hours after the last booster immunization, and IgG anti-tetanus titers were measured. Data are shown as mean anti-tetanus IgG U / ml (statistical analysis unpaired Student's t-test two-tailed, *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001). (B) Th1 intracellular staining. Splenocytes from untreated naive mice and pre-immunized mice treated with TT-PeptiCRAd were analyzed by flow cytometry to assess the levels of IFN-γ (Th1 polarization) and Foxp3 (Treg) upon TT peptide stimulation. [Figure 7]Figure 7 (A) Single tumor growth and immune cell components within the TME. Tumor growth curves for each mouse and one graph for each group are reported with the specific treatment indicated in each graph. Tumor volumes were normalized to values ​​on the day of the first treatment and are expressed as percentage means ± SEM. Percentages displayed next to each graph indicate responders (green), defined as mice with tumor volumes less 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 in green (responders) and black (non-responders) for individual mice in each group. The frequency of all cell types analyzed was significantly higher in responders compared to non-responders. Significance was assessed by two-tailed unpaired t-tests with Welch's correction for DC activation and for CD8+ and CD4+ T cell infiltration analyses, and by two-tailed unpaired t-tests for effector memory T cell infiltration. [Figure 8] Figure 8. CD40-CD40L crosstalk effect in lymphoid organs. Splenocytes from pre-immunized mice were examined by flow cytometry for CD4+ T cells expressing (A) or not expressing (B) Foxp3. In the draining lymph nodes, the total amount of CD4+ T cells was reported (C) and memory CD4+ T cells (CD44+) were analyzed by intracellular staining for IFN-γ (Th1 phenotype) (D), IL4 (Th2 phenotype) (E) and IL-17 (Th17 phenotype) (F). The ratio between Th1 and Th2 polarized CD4+ T cells is shown in G. Statistical analysis was assessed by Tukey's multiple comparison test and ordinary one-way ANOVA. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001; [Figure 9]Figure 9. Single tumor growth and area under the curve. (A) Tumor growth curves for each mouse and one graph for each group are reported with the specific treatments indicated in each graph. (B) Area under the curve of tumor growth is reported as graph bars ± SEM for the anti-PD-1 treated group (statistical analysis unpaired Student's t-test two-tailed, *p<0.05, **p<0.005, ***p<0.001, ****p<0.0001); [Figure 10] Figure 10. Immunological characterization in polioboostrix pre-immunized mice. (A) Antibody responses induced by PolioBoostrix vaccine. C57BL / 6 mice were immunized and serum was collected 5 days after the last booster immunization and anti-diphtheria toxoid antibodies were analyzed by ELISA. Data are shown as mean IgG IU / ml ± SEM. (B) IFN-γ ELISPOT assay. Splenocytes from naive mice or mice pre-immunized with PolioBoostrix vaccine were collected 1 month after the last booster immunization and cultured for 72 hours with the stimuli indicated in the figure. Results are expressed as the mean frequency of specific IFN-γ spot-forming cells per 1X106 cells. (Statistical analysis unpaired Student's t-test two-tailed, *p<0.05, **p<0.005, ***p<0,001, ****p<0,0001). (C) IFN-γ intracellular staining. Splenocytes from naive and immunized mice were incubated with the indicated stimuli for 6 h and measured by flow cytometry after intracellular staining. (D) Percentage of TRP2-specific CD8+ T cells among all CD8+ T cells in the TME. Data were normalized to tumor volume and plotted as mean ± SEM (statistical analysis: Kruskal-Wallis test, ANOVA). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0053] Methods and Materials Study design The main goal of this study is to enhance antitumor responses by targeting CD4 +The aim of this study is to recall T cell anti-pathogen memory repertoires. As a proof of principle, we confirmed our hypothesis in tetanus-immunized B16.OVA-bearing mice compared to naive mice. To demonstrate that the use of memory repertoires is more advantageous than naive, we investigated the immunological background of the mice. We then tested our hypothesis using clinically relevant tumor peptides in combination with immune checkpoint inhibitors. Finally, we repeated the experiment using different types of vaccines to confirm the general use of the underlying main or conceptual framework. Control and treatment groups are specified in the figure legends. The number of animals for each study type was determined by the investigator (each treatment group had n=8 or more mice). Animals were randomly assigned to the control and treatment groups.

[0054] Cell lines and reagents Cell line B16.OVA, a mouse melanoma cell line expressing chicken ovalbumin (OVA), was cultured according to ATCC recommendations. Cells were grown in RPMI-1640 with low glucose and supplemented with 10% FBS, 1% antibiotics and 1% L-glutamine. Cells were cultured at 37°C and 5% CO2 in a humidified atmosphere.

[0055] The following peptides, purchased from Ontores Biotechnologies Co. Ltd (Hangzhou, China), were used throughout the study.

[0056] KKKKKQYIKANSKFIGITEL (tetanus toxin) (SEQ ID NO: 1); KKKKARYVSQQTRANPNPY (pertussis) (SEQ ID NO:2); KKKKIQSKRFAPLYAVEAK (Polyomahony) (SEQ ID NO:3); KKKKKKSPVYVGNGVHANLHV (Diphtheria) (SEQ ID NO: 4); KKKKKKPVFAGANYAAWAVNVAQVI (Diphtheria) (SEQ ID NO:5); ARYVSQQTRANPNPY (Pertussis) (SEQ ID NO: 6); IQSKRFAPLYAVEAK (Polyomahony) (SEQ ID NO: 7); and SPVYVGNGVHANLHV (Diphtheria) (SEQ ID NO: 8).

[0057] The following peptides are examples of anti-tumor / anti-cancer peptides.

[0058] Protein sequence position (aa) gp100 KKKKKK-KVPRNQDWL (SEQ ID NO: 19) 25 to 35 NY-ESO-1 KKKKKK-SLLMWITQC (SEQ ID NO: 20) 157-165 (HLA A2) NY-ESO-1 KKKKKK-RGPESRLLEFYLAMPFATPM (SEQ ID NO: 21) 81 to 100 NY-ESO-1 KKKKKK-YLAMPFATPMEAELARRSLA (SEQ ID NO: 22) 91-110 NY-ESO-1 KKKKKK-RGPESRLLEFYLAMPFATPMEAELARRSLA (SEQ ID NO: 23) 81 to 110 NY-ESO-1 KKKKKK-PGVLLKEFTVSGNILTIRLTAADHR (SEQ ID NO: 24) 119-143 NY-ESO-1 KKKKKKKKK-YLAMPFATPMEAELARRSLA (SEQ ID NO: 25) 91-110 NY-ESO-1 KKKKKK-YLAMPFATPMEAELARRSLAEE (SEQ ID NO: 26) 91-110 MAGE-A3 KKKKKK-VFGIELMEVDPIGHLYIFAT (SEQ ID NO: 27) 161 to 180 MAGE-A3 KKKKKK-VFGIELMEVDPIGHLY (SEQ ID NO: 28) 161-176 Pre-immunization of mice For tetanus and diphtheria-tetanus-polio-pertussis vaccinations, 4-6 week old female C57BL / 6 mice received a primary intramuscular (im) vaccination with Anatetall (GlaxoSmithKline, Italy: 8 IU in 100 μL) or PolioBoostrix (GlaxoSmithKline, Italy) i.e. 0.4 IU diphtheria toxoid in 100 μL, 4 IU tetanus toxoid in 100 μL, Bordetella pertussis antigens: 1.6 mg pertussis toxoid in 100 μL, 1.6 mg hemagglutinin in 100 μL, 0.5 mg pertactin in 100 μL) administered bilaterally into the quadriceps (50 uL per leg), respectively. Two intramuscular booster vaccinations (50 μL) were administered: the first vaccination 2 weeks after the first vaccination and the second 2 weeks after the first booster vaccination. Mouse IgG antibody responses to tetanus toxoid and diphtheria were measured by ELISA (Xpress Bio Frederik, MD USA). Sera from immunized mice were collected 5 days after the last immunization and before animal experiments.

[0059] Animal experiments and ethical approval All animal experiments were reviewed and approved by the Laboratory Animal Committee of the University of Helsinki and the Provincial Government of Southern Finland (License number ESAVI / 9817 / 04.10.07 / 2016).

[0060] Four- to six-week-old female C57BL / 6JOlaHsd mice were obtained from Envigo Laboratory (Bar Harbor, Maine, UK). 5 100 of B16.OVA cells were injected subcutaneously in the right flank. Details on the treatment schedule are given in the figure legends. The viral load was 1x10 complexed with 20μg of a single peptide or a 10μg+10μg mixture of the two peptides. 9 vp / tumor. Ananetall vaccine administered intratumorally was dosed at 2 IU per mouse. Checkpoint inhibitors were administered intraperitoneally at a dose of 100 μg / mouse.

[0061] Flow cytometry The antibodies used were as follows: 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-A PC(eBioscience), CCR7-V450(BD), CD11c-FITC(BD), B220-PE(eBioscience), MHC-II(AI / EI)-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). Data were acquired using a BDLSRFORTESSA flow cytometer.

[0062] Data were analyzed using FlowJo software v9 (Ashland, OR, USA).

[0063] IFN-γ ELISPOT IFN-γ ELISPOT assays were performed using a commercially available mouse ELISPOT reagent set (ImmunoSpot, Bonn Germany) testing 20 ng / uL of each peptide in in vitro stimulation of splenocytes for 72 h at 37° C. Spots were counted using an ELISPOT reader system (ImmunoSpot).

[0064] PeptiCrad complex formation PeptiCRAd complexes were prepared by mixing oncolytic adenovirus with each epitope with a poly K tail (Ontores, Zhejiang, China). Prior to treatment with the PeptiCRAd complex, we mixed the poly K-extended epitopes with Ad-5-D24-CpG at room temperature for 15 min. Further details regarding the stability and formation of the complexes are described in our previous study (11).

[0065] statistical analysis Statistical analysis was performed using Graphpad Prism 6.0 software (Graphpad Software Inc., La Jolla, CA, USA). For animal studies, a two-way analysis of variance with Tukey's multiple comparison test was used, and P < 0.05 was considered statistically significant. All results are expressed as the mean ± standard error of the mean (SEM).

[0066] Details regarding the statistical tests for each experiment are given in the corresponding figure legends.

[0067] Results and Discussion [Example 1] Pre-immunization with tetanus vaccine enhances the anti-tumor response of double-coated PeptiCRAd Here, we evaluated the feasibility of engaging CD4+ T cell memory using the PeptiCRAd vaccine platform (11), where we coated an oncolytic adenovirus with both an MHC-I-restricted tumor-specific peptide and an MHC-II-restricted pathogen-specific peptide and studied the effect on mouse tumors in mice pre-immunized against the pathogen (Figure 1A). Our hypothesis was that by adding an MHC-II-restricted pathogen-specific peptide to the PeptiCRAd platform, we would provide a more rapid and stronger T helper response, thereby enhancing tumor-specific CTL responses.

[0068] We investigated the antitumor efficacy of modified or double-coated PeptiCRAd in mice bearing B16.OVA tumors, a melanoma model expressing chicken OVA as a model antigen, that were pre-immunized intramuscularly with tetanus vaccine (15). The OVA epitope was selected because it is highly immunogenic and therefore provides a suitable model for analyzing the generation of T cell responses (16). C57BL / 6 mice were immunized three times at 2-week intervals with tetanus vaccine (Figure 1B). Five weeks after priming, serum samples were collected from the mice and anti-tetanus antibody titers were measured to confirm successful vaccination (Figure 1B and Figure 6A).

[0069] Following tumor engraftment, mice were randomized and treated with PeptiCRAd coated with a tumor-specific peptide (OVA-PeptiCRAd), a tetanus-specific peptide (TT-PeptiCRAd), or both tetanus and OVA peptides (TT-OVA-PeptiCRAd). Additionally, a tetanus vaccine was used alone or in combination with OVA-PeptiCRAd to evaluate whether a commercially available vaccine administered intratumorally could affect tumor growth. All treatments were delivered by intratumoral administration following the regimen depicted in Figure 1B.

[0070] After treatment, TT-OVA-PeptiCRAd outperformed either one of the single-coated viruses in controlling tumor growth in mice pre-immunized with tetanus toxoid vaccine (Figure 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 induce a predominantly Th1-polarized CD4+ T cell response was further supported by intracellular staining (Figure 6B). Not surprisingly, this approach also worked when tetanus vaccine was reintroduced as a combination with OVA-PeptiCRAd (vaccine+OVA-PeptiCRAd), whereas tetanus vaccine alone had no therapeutic effect (Figure 1D). Notably, when comparing vaccine+OVA-PeptiCRAd to OVA-PeptiCRAd, the latter showed significantly higher antitumor efficacy (p=0.05). This suggests that the effect is not caused by an adjuvant in the vaccine itself, but by presentation of a tetanus-specific peptide on MHC-II, engaging CD4+ T cells to support the cytotoxic CD8+ T cell response.

[0071] Interestingly, when the same experiment was performed in naive mice (mice not pre-immunized with a tetanus vaccine), no statistically significant difference was observed between OVA-PeptiCRAd and TT-OVA-PeptiCRAd (Figure 1E).

[0072] These results demonstrate that the antitumor efficacy of our virus-based PeptiCRAd cancer vaccine is greatly enhanced when co-coated with a peptide specific for a pathogen against which there is pre-existing immunity.

[0073] [Example 2] Tetanus-specific memory responses shape a favorable immune environment (TME) at the tumor site To better understand the mode of action of the dual-coated PeptiCRAd, we investigated the quality of the immune responses induced by the different treatments. To this end, we analyzed by flow cytometry the frequency of various cell populations within the tumor, most importantly activated dendritic cells (DCs), CD4+ and CD8+ T cells with effector and memory phenotypes, as well as experienced and exhausted CD8+ effector T cells.

[0074] Interestingly, we found that the frequency of activated intratumoral DCs was increased in all groups treated with PeptiCRAd in the context of tetanus antigen (either coated with TT peptide or co-injected with the whole vaccine) (Figure 2A). In contrast to these combination treatments, the use of vaccine alone resulted in poor induction of DC maturation in the TME, suggesting that the inclusion of an adenoviral adjuvant may be critical for proper DC activation in this context. Furthermore, we observed increased levels of CD4+ and CD8+ T cells in tumors in all groups of mice treated with PeptiCRAd (Figure 2B-C), which is in good agreement with what has been previously observed after treatment with virus-based drugs (11). Finally, we wanted to analyze the phenotype of these T cells. The majority of tumor-infiltrating lymphocytes (TILs) displayed a T effector memory cell phenotype, with CD8+ and CD4+ TILs in the TT-PeptiCRAd and OVA-TT-PeptiCRAd-treated groups. EMThe frequency of CD8+ T cells was increased (Figure 2D-E). To further examine T cell exhaustion, we assessed the expression levels of T cell immunoglobulin and mucin domain-containing-3 (TIM3) in PD1+ TILs. 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 (Figure 2F). We concluded that pre-existing immunity to tetanus significantly improves the overall efficacy of the treatment by modifying the immune environment at the tumor site, especially when the treatment is virus-based and contains a tetanus vaccine or tetanus peptide. Of note, the serotype 5 human adenovirus used in these experiments is non-oncolytic in mouse tumors, and therefore, the impact on tumor control is based solely on anti-tumor immune responses. To further elucidate this phenomenon, we reanalyzed all data sets by stratifying mice between responders and non-responders and reassessed their immunological response. As expected, we observed significant differences between the two groups. Regardless of the type of treatment, all responders had a continuing measurable immune response, highlighting the importance of the immune system in controlling tumor growth, regardless of the type of treatment they received. Importantly, the majority of these responders were found in the group of mice treated with TT-OVA-PeptiCRAd (Figure 7A-E).

[0075] [Example 3] TT-specific Th1-polarized CD4+ T cells expressing CD40L are detected in secondary lymphoid organs after TT-OVA-PeptiCRAd treatment To analyze possible mechanisms of the observed therapeutic effect, we evaluated the levels and phenotypes of immune cells in secondary lymphoid organs of pre-immunized mice. As expected, mice treated with PeptiCRAd showed an expansion of the CD4+ T cell compartment both in the spleen and in the draining lymph nodes (Figure 3A and Figure 8C). More importantly, a significant increase in TT-specific CD4+ T cells expressing CD40 ligand (CD40L) was observed in TT-OVA-treated mice (Figure 3B). Although some TT-specific Foxp3+ T regulatory cells (Tregs) were also detected, the majority of these CD40L+ cells were polarized toward a Th1 phenotype (Figure 8A-B). Analysis of dLNs revealed that intratumoral vaccination with TT-OVA-PeptiCRAd induced mainly IFN-γ-producing Th1 memory cells at the expense of IL-4-secreting Th2 cells, whereas no differences were observed in IL-17A-producing Th17 cells (Figure 8D-G). Since CD4+ T cell-associated CD40L has been shown to be important in stimulating cytotoxic CD8+ T cell responses, we wanted to see if we could find CD40+ antigen-presenting cells. Indeed, when pre-immunized mice were treated intratumorally with TT-OVA-PeptiCRAd, a significantly higher frequency of APCs expressing CD40+ was detected (Figure 3C), further suggesting that the double-coated PeptiCRAd could stimulate TT-specific CD4+ memory T cells, which in turn licensed professional APCs via CD40-CD40L interactions.

[0076] [Example 4] Combination with immune checkpoint inhibitors increases the number of responders and leads to complete tumor rejection.

[0077] We have previously shown that the combination of tumor-targeted PeptiCRAd with immune checkpoint inhibitors is synergistic in terms of improved antitumor efficacy. (8) Therefore, we wished to assess whether pre-existing immunity induced by the vaccine could further enhance this synergy, particularly by increasing the frequency of mice responding to treatment.

[0078] To test this hypothesis, we coated the virus with TT and the tyrosinase-related protein 2 (TRP2) peptide (TRP2 180-188 (23)), a naturally occurring melanoma-associated antigen and therefore a more clinically relevant epitope than OVA. Tetanus toxoid-preimmunized mice were implanted with subcutaneous tumors and treated intratumorally with PeptiCRAd coated with only the TRP2 peptide (TRP2-PeptiCRAd) or with PeptiCRAd coated with both the TRP2 and TT peptides (TT-TRP2-PeptiCRAd) (Figure 4A). Similarly, as in Figure 1, we observed significant inhibition of tumor growth in mice treated with the dual-coated virus compared to controls (Figure 4B).

[0079] Interestingly, when we combined PeptiCRAd treatment with a PD-1 blocking monoclonal antibody, we observed a significant increase in the efficacy of both TRP2-PeptiCRAd and TT-TRP2-PeptiCRAd treatments (Figure 4C-D). However, in terms of tumor growth control, the dual-coated PeptiCRAd was still more effective than the single-peptide coated virus (Figure 9B).

[0080] More importantly, inclusion of a TT-specific peptide 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 complete tumor eradication (Figure 4E and Figure 9A). One of the greatest advantages of combining oncolytic viruses with checkpoint inhibitors is that the virus in the tumor promotes and increases the recruitment of T lymphocytes, thereby unleashing the unprecedented activity of 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 (Figure 4C) when compared to control treatment (Figure 4F).

[0081] [Example 5] Pre-existing immunity is a general mechanism for enhancing antitumor responses and reshaping the immunological balance of the T cell repertoire.

[0082] Because we observed that pre-existing immunity to tetanus toxoid enhanced the antitumor response of dual-coated PeptiCRAd alone and in combination with PD-1 blockade, we sought to further explore whether our approach would also be effective in the context of multivalent vaccines, such as quadrivalent vaccines. Polioboostrix is ​​an attractive research model because it is a high-uptake quadrivalent vaccine that immunized 85% of infants (25). Using the same immunization regime as before, C57BL / 6 mice were pre-immunized with Polioboostrix vaccine (Figure 5A). To confirm the efficacy of the immunization protocol, serum samples and splenocytes were collected and analyzed. The quadrivalent vaccine was found to efficiently generate both antibodies and CD4+ T cells specific for pertussis and diphtheria (Figure 10A-C). For tumor growth analysis, B16.OVA tumors in naive or pre-immunized mice were treated with anti-PD1 antibody and PeptiCRAd coated with MHC-II-restricted diphtheria-pertussis peptide and MHC-I-restricted TRP2 peptide (DP-TRP2-PeptiCRAd). Consistent with our previous results, excellent anti-tumor responses were detected in pre-immunized mice treated with DP-TRP2-peptiCRAd and anti-PD1, whereas the efficacy of treatment was lost in naive mice (Figure 5B).

[0083] These results confirm that pathogen-specific pre-existing immunity enhances antitumor responses and that the mechanism of action is dependent on memory T cells. Moreover, this effect is not limited to tetanus but is applicable to other pathogens. To further validate the mechanism of action behind the improved efficacy of treatment using diphtheria and pertussis as pre-immunization vaccines, we analyzed the T cell repertoire in tumor-draining lymph nodes, TME and spleen. The frequency of naive CD8+ T and CD4+ T cells was lower in the draining lymph nodes of pre-immunized DP-TRP2 PeptiCRAd-treated mice compared to the control group (Figure 5C). Concomitantly, CD4+ T cells were significantly higher in the draining lymph nodes and TME of pre-immunized mice compared to naive and sham-treated mice. EM Increased levels of TRP2-specific CD8+ T cells were observed (Figure 5D). Furthermore, compared to naïve mice, tumor tissues from immunized mice tended to be more highly infiltrated with TRP2-specific CD8+ T cells (Figure S10D), as well as CD4+ T cells in tumors and draining lymph nodes. EM The levels of cells strongly correlated with the strength of the TRP2-specific TIL response. Collectively, the dual-coated PeptiCRAd vaccine platform can be used to stimulate pre-acquired pathogen-specific CD4+ T cell immunity to aid in the generation of effective antitumor CD8+ T cell responses.

[0084] summary Due to the high coverage of international vaccination programs, a large proportion of the world's population has been vaccinated against common pathogens, resulting in acquired pathogen-specific immunity with a robust memory T cell repertoire. These vaccines lead to the formation of immune memory that can deploy a much faster and more effective immune response when the pathogen is encountered again; indeed, the primary immune response is rather weak and slow, whereas the secondary immune response is faster and more effective (26). Although CD8+ antitumor cytotoxic T lymphocytes (CTLs) are the preferred effectors of cancer immunotherapy, CD4+ T cell help is also required for an optimally potent antitumor immune response to occur.

[0085] Therefore, we describe a new cancer immunotherapy approach that exploits the existing pathogen-specific immune memory present in the worldwide population of vaccinated individuals by investigating whether it is possible to re-engage the pathogen-associated CD4+ T cell memory population to support CTLs and convert a weak primary anti-tumor immune response into a stronger secondary anti-tumor immune response.To this end, we use our PeptiCRAd technology, which consists of a virus coated with an MHC-I restricted tumor-specific peptide, and further develop this technology by introducing a pathogen-specific MHC-II restricted peptide.

[0086] Proof of concept has been demonstrated and validated in melanoma using tetanus and polioboostrix vaccines available for humans, highlighting the universality of CD4+ memory in enhancing cancer-specific CTL responses.Importantly, this approach can be extended beyond surrogate OVA to naturally occurring tumor peptides and to other pathogens in place of tetanus, highlighting the utility of our technology in taking full advantage of the CD4+ memory T cell repertoire when designing immunotherapy treatment regimens.

[0087] Finally, when combined with immune checkpoint inhibitors such as anti-PD1, the antitumor effects were even more pronounced, strengthening the rationale behind combination therapy with oncolytic viruses.

[0088] References 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.

[0089] 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。

[0090] 15.Moore MW,Carbone FR,Bevan MJ.Introduction of soluble protein into the class I pathway of antigen processing and presentation.Cell.1988;54(6):777-85。

[0091] 16.Knocke S,Fleischmann-Mundt B,Saborowski M,Manns MP,Kuhnel F,Wirth TC,et al.Tailored Tumor Immunogenicity Reveals Regulation of CD4 and CD8 T Cell Responses against Cancer.Cell Rep.2016;17(9):2234-46。

[0092] 23.Bloom MB,Perry-Lalley D,Robbins PF,Li Y,el-Gamil M,Rosenberg SA,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。

[0093] 25.Feldstein LR,Mariat S,Gacic-Dobo M,Diallo MS,Conklin LM,Wallace AS.Global Routine Vaccination Coverage,2016.MMWR Morb Mortal Wkly Rep.2017;66(45):1252-5。

[0094] 26.Laidlaw BJ,Craft JE,Kaech SM.The multifaceted role of CD4(+)T cells in CD8(+)T cell memory.Nat Rev Immunol.2016;16(2):102-11。

Claims

1. A viral vector having a polypeptide attached to a viral capsid or envelope, said polypeptide being not genetically encoded by said viral vector but being covalently or non-covalently attached to said capsid or envelope; i) at least one of said polypeptides comprises an antigen derived from or of a pathogen against which the subject has previously been immunized, said polypeptide comprising: QYIKANSKFIGITEL (tetanus toxin) (SEQ ID NO: 1); ARYVSQQTRANSPNPY (pertussis) (SEQ ID NO:2); IQSKRFAPLEAVEAK (Polymahony) (SEQ ID NO:3); SPVYVGNGVHANLHV (Diphtheria) (SEQ ID NO: 4); PVFAGANYAAWAVNVAQVI (Diphtheria) (SEQ ID NO:5); ARYVSQQTRANSPNPY (pertussis) (SEQ ID NO: 6); IQSKRFAPLEAVEAK (Polymahony) (SEQ ID NO: 7); and SPVYVGNGVHANLHV (Diphtheria) (SEQ ID NO: 8); is selected from the group comprising: ii) at least one other of the polypeptides is an anti-tumor or anti-cancer specific polypeptide that stimulates an anti-tumor or anti-cancer immune response in a subject exposed to the vector; Viral vector.

2. The viral vector of claim 1, wherein the polypeptide comprises a fusion polypeptide, one portion of the fusion polypeptide comprising an antigen derived from or of a pathogen against which a subject has previously been immunized, and another portion of the fusion polypeptide is an anti-tumor or anti-cancer specific polypeptide, thus stimulating an anti-tumor or anti-cancer immune response in a subject exposed to the vector.

3. The viral vector of claim 1 or 2, wherein the polypeptide is polylysine- or polyarginine-modified to attach the polypeptide to the capsid.

4. 3. The viral vector of claim 1 or 2, wherein the polypeptide is 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 any one of claims 1 to 4, wherein the polypeptide is selected from the group comprising major histocompatibility complex class I (MHC-I) specific polypeptides, major histocompatibility complex class II (MHC-II) specific polypeptides and DC specific polypeptides.

6. The viral vector of any one of claims 1 to 5, wherein at least one or more of the polypeptides is an MHC-I specific polypeptide and at least one or more of the polypeptides is an MHC-II specific polypeptide.

7. 7. The viral vector of any one of claims 1 to 6, wherein the viral vector is a member of a family selected from the group comprising adenovirus, reovirus, papillomavirus, picornavirus, calicivirus, herpesvirus, poxvirus, hepadnavirus, flavivirus, togavirus, coronavirus, hepatitis D, orthomyxovirus, paramyxovirus, rhabdovirus, bunyavirus, filovirus and retrovirus.

8. 8. The viral vector of claim 7, wherein the virus is selected from the group comprising herpes simplex virus 1 (HSV-1), herpes simplex virus 2 (HSV-2), vaccinia, vesicular stomatitis virus Indiana strain (VSV), measles virus (MeV), Maraba virus and Newcastle disease virus (NDV).

9. The viral vector according to claim 7 or 8, wherein the virus is a vaccinia virus, HSV-1 or an adenovirus.

10. The viral vector of any one of claims 1 to 9, wherein the viral vector is oncolytic.

11. The polypeptide of part ii) KVPRNQDWL (gp100) (SEQ ID NO: 9); SLLMWITQC (NY-ESO-1) (SEQ ID NO: 10); RGPESRLLEFYLAMPFATPM (NY-ESO-1) (SEQ ID NO: 11); YLAMPFATPMEAELARRSLA (NY-ESO-1) (SEQ ID NO: 12); RGPESRLLEFYLAMPFATPMEAELARRSLA (NY-ESO-1 (SEQ ID NO: 13); PGVLLKEFTVSGNILTIRLTAADHR (NY-ESO-1) (SEQ ID NO: 14); YLAMPFATPMEAELARRSLA (NY-ESO-1) (SEQ ID NO: 15); YLAMPFATPMEAELARRSLAEE (NY-ESO-1) (SEQ ID NO: 16); VFGIELMEVDPIGHLYIFAT (MAGE-A3) (SEQ ID NO: 17); and VFGIELMEVDPIGHLY (MAGE-A3) (SEQ ID NO: 18); The viral vector according to any one of claims 1 to 10, selected from the group comprising:

12. A pharmaceutical composition or an immunogenic agent or a vaccine comprising the viral vector according to any one of claims 1 to 11 and a suitable carrier.

13. A target cell transformed or transfected with the viral vector or pharmaceutical composition or immunogenic agent or vaccine according to any one of claims 1 to 12.

14. a) in combination with a further cancer therapeutic agent; b) the viral vector according to claims 1 to 11 or the pharmaceutical composition or immunogenic agent or vaccine according to claim 12 or the cell according to claim 13, 23. A combination therapy for the treatment of cancer comprising:

15. The viral vector of any one of claims 1 to 11, or the pharmaceutical composition or immunogenic agent or vaccine of claim 12, or the cell of claim 13, or the combination therapy of claim 14, for use in the treatment of cancer.

16. Use of the viral vector according to any one of claims 1 to 11 or the cell according to claim 13 or the combination therapeutic agent according to claim 14 for the manufacture of a medicament for the treatment of cancer.

17. The viral vector of any one of claims 1 to 11, or the pharmaceutical composition or immunogenic agent or vaccine of claim 12, or the cell of claim 13, for administration in combination with an anti-tumor or anti-seasonal disorder agent.

18. The cancer may be nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, cancer of connective tissue, melanoma, lung cancer, intestinal 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 gland cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal tumor, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of the digestive tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Williams' disease, or any combination thereof.

15. The combination therapy of claim 14, comprising or selected from the group consisting of Lumus 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, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymus cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, cardiac cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsillar cancer.

19. The cancer is selected from the group consisting of nasopharyngeal cancer, synovial cancer, hepatocellular carcinoma, renal cancer, cancer of connective tissue, melanoma, lung cancer, intestinal 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 gland 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 the digestive tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, esophageal cancer, gallbladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, 17. The use of claim 16, wherein the cancer is selected from the group comprising or consisting of 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, penile cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, gastric cancer, thymus cancer, thyroid cancer, choriocarcinoma, hydatidiform mole, uterine cancer, endometrial cancer, vaginal cancer, vulvar cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gingival cancer, cardiac cancer, lip cancer, meningeal cancer, oral cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneal cancer, pharyngeal cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsillar cancer.

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