Compositions and methods for inducing immune responses

Abstract

The present invention relates to a composition for inducing a T cell mediated immune response for use in the treatment or prevention of prostate cancer, the composition comprising a modified vaccinia virus Ankara (MVA) vector that expresses a 5T4 antigen polypeptide under the control of a poxvirus F11 promoter. Suitably, the poxvirus F11 promoter is an endogenous MVA F11 promoter. More suitably, the vector expresses a polypeptide having the amino acid sequence represented by SEQ ID NO: 1 or the vector expresses a polypeptide encoded by a polynucleotide having the nucleic acid sequence represented by SEQ ID NO: 2. The invention also relates to uses and methods.

Description

[0001] This application is a divisional application of the invention patent application with application date of May 16, 2019, application number 201980030633.8, and invention name “Compositions and methods for inducing immune responses”. Technical Field

[0002] The present invention relates to inducing an immune response, suitably a protective immune response, to a prostate cancer-associated tumour antigen. Background Art

[0003] Prostate cancer is the most common non-skin cancer and the second leading cause of cancer death in men. In 2012, approximately 1.1 million people were diagnosed with the disease worldwide, accounting for 15% of all cancer diagnoses in men. More than 70% of prostate cancer cases occur in developed countries. For example, in 2014 alone, an estimated 233,000 men were diagnosed with the disease in the United States, and approximately 30,000 deaths were predicted (Siegel et al (2014). CA Cancer J Clin 64: 9-29). Although significant progress has been made in the treatment of prostate cancer, there are few treatments available for the late stages of the disease, and these methods have proven to be less than ideal in effectiveness. Therefore, the development of effective therapies remains a high priority for the treatment of the disease.

[0004] Efforts have been intensified to develop active immunotherapy (vaccines) for cancer (including prostate cancer). Traditional vaccines have been based on the recognition of foreign "non-self" antigens, effectively inducing protective immunity to pathogens. However, the vast majority of cancer antigens characterized so far are unchanged "self" antigens expressed by tumors and normal cells. This poses a challenge to the development of effective active immunotherapy for cancer. Despite limitations in immune surveillance and cancer clearance, cancer immunity in various tumor forms has been observed clinically (Challis & Stam (1990). Acta Oncol. 29: 545-550). In addition, histopathology of tumor sections found lymphocyte infiltration around the tumor bed, and recent studies have shown that compared with patients of similar stages without lymphocyte infiltration, patients with ovarian cancer with this infiltration around the tumor have an improved prognosis (Zhang et al (2003). N Engl J Med. 348: 203-213). Therefore, the immune repertoire contains autoreactive immune cells that can reject tumors when properly activated. These autoreactive cells also have the potential to induce tissue destruction upon recognition of target molecules on normal cells, leading to toxic autoimmunity. Therefore, the development of therapies aimed at activating host antitumor immunity using appropriate immunological targets remains a promising avenue for the successful treatment of cancer, including prostate cancer.

[0005] T cells are known to be important in the immune control of cancer, and a large body of evidence has accumulated over the past two decades showing that prime-boost regimens involving the sequential administration of different vectors encoding the same antigen can generate significantly higher immune responses with protective capacity in several animal models and clinical trials. In fact, in clinical trials, a vaccination strategy based on simian adenovirus priming and MVA boosting was shown to be the most effective approach to inducing polyfunctional protective T cell responses against certain human pathogens (Ewer et al (2013). Nat Commun. 4:2836; Antrobus et al (2014). J Am Soc Gene Therapy. 22:668-674; Borthwick et al (2014). Mol Ther. 22:464-475; Swadling et al (2014). Science translational medicine. 6:261ra153; Hodgson et al (2015). J Inf Dis. 211:1076-1086; and Ewer et al (2016). New Engl J Med. 374:1635-1646).

[0006] Despite its promise, there are many challenges to the use of therapeutic vaccination in cancer, with tumor tolerance to self-antigens and active immunosuppression mechanisms being two major factors that hinder efficacy. The two most advanced prostate cancer immunotherapies, Sipuleucel-T and ProstVac, target two well-defined prostate cancer antigens, prostatic acid phosphatase (PAP) and prostate-specific antigen (PSA), respectively. 5T4, an oncofetal glycoprotein belonging to the shared tumor antigen family, is another promising antigen candidate for prostate cancer vaccine. It was identified in 1990 by searching for surface shared molecules of human trophoblasts and cancer cells, with the rationale that they might play a role in the survival of the fetus in the form of a semi-allogeneic transplant (Southall et al (1990). Br J Cancer. 61: 89-95). 5T4 has become a potential target for cancer immunotherapy due to its high expression in a variety of human solid malignancies and has become the subject of intensive research (Southall et al. (1990). Br J Cancer. 61: 89-95; Starzynska et al (1994). Br J Cancer. 69: 899-902; and Amato & Stepankiw (2012). Future Oncol. 8: 231-237), and there is a clear correlation between its expression and disease progression (Stern et al (2014). Seminars in Cancer Biology. 29: 13-20; and Stern & Harrop (2016). Cancer Immunology, Immunotherapy. 2016: 1-12).

[0007] Clinical trials of vaccines targeting 5T4 began more than a decade ago with a modified vaccinia virus Ankara (MVA) expressing the 5T4 protein. The vaccine is administered as a homologous prime-boost vaccine to patients with advanced colorectal cancer under the trade name TroVax, and to date, more than 500 patients with colorectal cancer, breast cancer, kidney cancer, prostate cancer, and mesothelioma have been vaccinated during Phase I-III clinical trials (Kim et al (2010). Human Vaccines. 6: 784-791; and Al-Taei et al (2012). Lung Cancer. 77: 312-318). TroVax has a favorable safety profile, is well tolerated in patients with the highest 5T4-specific antibody titers, and has a trend toward improved progression-free survival (Harrop et al (2010); however, vaccine-specific cellular immune responses and clinical efficacy were modest, J immunotherapy. 33:999-1005; and Harrop et al (2012). Cancer Immunology, Immunotherapy. 61:2283-2294).

[0008] Harrop et al. (Cancer Immunology, Immunotherapy 2014, 62(9); 1511-1520) disclosed Results of clinical administration of an MVA expressing the 5T4 antigen under the control of the mH5 (modified H5) early promoter in combination with docetaxel in patients with castration-resistant prostate cancer. The study showed that all patients were vaccine tolerant and had significantly improved survival compared with patients receiving docetaxel alone. Patients treated with docetaxel had a longer median progression-free survival. However, the increase in efficacy measured was modest.

[0009] Cappuccini et al. (Oncotarget 2017, 8(29); 47474-47489) compared MVA expressing unmodified 5T4 antigen and the same antigen fused to MHC class 2-associated invariant chain (Ii) administered under the control of the p7.5 promoter as part of a heterologous prime-boost therapy in a mouse model of prostate cancer. This study demonstrated antibody responses to unmodified 5T4, but no measurable T cell responses were reported except to the modified antigen. The lack of T cell immune responses to unmodified "self" antigens suggests that MVA expressing unmodified 5T4 under the control of the p7.5 promoter is unlikely to be an effective antitumor vaccine.

[0010] Therefore, no vaccine in the prior art has been demonstrated to provide effective prostate cancer treatment and prevention, either alone or in combination with any other therapeutic agent.

[0011] The present invention seeks to overcome the problems associated with the prior art. Summary of the invention

[0012] We describe a combination comprising a modified vaccinia virus Ankara (MVA) vector expressing a 5T4 protein antigen under the control of an endogenous viral F11 promoter. The present invention is based on the unexpected discovery of the inventors that when used as part of a prime-boost regimen following initial immunization with an adenovirus construct expressing 5T4, 5T4 expression from the endogenous F11 promoter of MVA is sufficient to break tolerance and induce a 5T4-specific T cell immune response. Therefore, the composition of the present invention can be used to break tolerance to induce an antigen-specific immune response to treat prostate cancer. The following figures and examples provide data demonstrating these advantages.

[0013] In a first aspect, the present invention provides a composition for inducing a T cell-mediated immune response for treating or preventing prostate cancer, wherein the composition comprises a modified vaccinia virus Ankara (MVA) vector expressing a 5T4 antigen polypeptide under the control of a poxvirus F11 promoter.

[0014] The composition of the first aspect can be advantageously used to break immune tolerance to the 5T4 antigen and induce a T cell-mediated immune response to the 5T4 antigen, and this can achieve effective treatment or prevention of prostate cancer.

[0015] The 5T4 polypeptide expressed by the composition of the first aspect may have the amino acid sequence shown in SEQ ID NO:1, or may have the amino acid sequence encoded by the nucleic acid sequence shown in SEQ ID NO:2.

[0016] Advantageously, the composition further comprises an adjuvant, and the composition can be used to induce a T cell-mediated immune response to the 5T4 antigen polypeptide in a subject and to treat or prevent prostate cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] The present invention will now be described by way of example with reference to the accompanying drawings, in which:

[0018] Figure 1 The amino acid sequence of the 5T4 antigen is shown (SEQ ID NO: 1).

[0019] Figure 2 The nucleic acid sequence encoding the full-length 5T4 antigen is shown (SEQ ID NO: 2).

[0020] Figure 3 This indicates that the magnitude of 5T4-specific T cell responses in the blood was significantly higher after boosting with MVA.5T4 expressing 5T4 under the control of the F11 promoter compared to expression driven by the mH5 promoter. 10 C57BL / 6 mice were intramuscularly inoculated with ChAdOx1 vector expressing h5T4 antigen in VP at three-week intervals and then 10 7 pfu of h5T4-expressing MVA vector or 10 7 pfu expressing antigen driven by the F11 promoter for homologous MVA.h5T4 prime-boost. The figure shows representative data of ex vivo blood (A) and spleen (B) ELISPOT after prime-boost. X-axis: dosing regimen for groups 1-4. Y-axis: every 10 6 Spot forming cell (SFC) counts per PBMC. Bars represent medians. (C = ChAdOx1, M = MVA). Significant p values ​​are shown.

[0021] Figure 4 Flow cytometric analysis of 5T4-specific T cells in the blood and spleen of mice primed with ChAdOx1.5T4 and boosted with MVA.5T4_F11 is illustrated, indicating the generation of multifunctional CD4+ and CD8+ T cells that secrete multiple cytokines. After MVA.5T4 boosting with antigens expressed driven by the F11 promoter, PBMCs and splenocytes isolated from mice immunized with ChAdOx1.5T4 were subjected to intracellular cytokine staining (ICS). These figures show the percentage of CD4+ and CD8+ T cells that secrete IFN-γ (A), TNF-α (B) and IL-2 (C) in response to overnight stimulation with an in vitro h5T4 peptide pool. X-axis: CD4+ and CD8+ T cell responses in blood and spleen. Y-axis: percentage of 5T4-specific cytokine secreting T cells. Δ values ​​are calculated by subtracting the background (i.e., the percentage of T cells that spontaneously secrete cytokines without specific stimulation) from the percentage of cytokines secreted after T cells were exposed to the h5T4 peptide pool. Bars represent median values. DETAILED DESCRIPTION

[0022] In a first aspect, the present invention provides a composition for inducing a T cell-mediated immune response for treating or preventing prostate cancer, the composition comprising a modified vaccinia virus Ankara (MVA) vector expressing a 5T4 antigen polypeptide under the control of a poxvirus F11 promoter. MVA expressing a 5T4 antigen polypeptide expressed under the control of a poxvirus F11 promoter has not been disclosed previously, and therefore has novelty. This composition can be advantageously used to destroy immune tolerance to the 5T4 antigen and induce a T cell-mediated immune response to the 5T4 antigen, and this can achieve effective treatment or prevention of prostate cancer.

[0023] The prior art has raised a bias against the use of unmodified 5T4 antigen in vaccines for the treatment or prevention of prostate cancer. Cappuccini 2017 (supra) demonstrated that expression of unmodified 5T4 antigen by MVA can be used as part of a homologous or heterologous priming regimen to achieve an antibody response to 5T4. However, in a heterologous prime-boost regimen, the generation of an in vitro T cell-mediated immune response to 5T4 antigen expressed by MVA requires fusion of the antigen to the MHC class 2-associated invariant chain (Ii). The advantage of the present invention is that the unmodified 5T4 antigen expressed by MVA under the control of the endogenous F11 promoter induces a strong cellular immune response.

[0024] The prior art of prime-boost using MVA-based vaccine candidates generates strong T cell immune responses to a large number of different "non-self" antigens in a variety of indications. The advantage of the present invention is that it breaks immune tolerance and similarly generates a robust T cell-mediated immune response to "self" antigens. This response is unexpected and provides many benefits, including more effective treatment and simpler development and production protocols because no antigen modification or fusion is required.

[0025] Preferably, the MVA vector expresses the 5T4 antigen polypeptide under the control of the endogenous F11 promoter of MVA. Insertion of a polynucleotide encoding an antigen in the F11 locus of MVA under the control of the endogenous F11 promoter has been described in International Publication WO2011 / 128704. This vector expressing the 5T4 antigen polypeptide under the control of the endogenous F11 promoter has not been previously disclosed and is therefore novel. Advantageously, this configuration simplifies the preparation of the MVA vector. In addition, the Kozac-like sequence in the F11 flanking sequence is believed to contribute to translation initiation in eukaryotic cells and thus promote the expression of the 5T4 antigen by MVA.

[0026] The present inventors provide a vaccine for treating or preventing prostate cancer, comprising an MVA viral vector comprising a nucleic acid sequence encoding a full-length unmodified human 5T4 antigen polypeptide having an amino acid sequence as shown in SEQ ID NO: 1. The MVA construct is prepared so that no marker gene is present in the recombinant virus.

[0027] In a mouse model, the MVA vaccine construct of the invention ((F11)5T4) was compared to MVA constructs expressing the 5T4 antigen under the control of a modified H5 early promoter ((mH5)5T4) or under the control of a p7.5 early / late promoter ((p7.5)5T4) to measure T cell-mediated immune responses. When administered as part of a heterologous prime-boost regimen, the MVA(F11)5T4 construct induced a strong 5T4-specific T cell response as measured using an IFNγ ELISPOT assay in peripheral blood mononuclear cells (PBMCs) and splenocytes ( Figure 3 This response in PBMCs was more than three-fold higher than that induced by MVA(p7.5)5T4, whereas no response was detectable in PBMCs induced with MVA(mH5)5T4 ( Figure 3 A). The same MVA(F11)5T4 construct failed to induce the same 5T4-specific response when administered alone in a homologous prime-boost regimen. Advantageously, MVA(F11)5T4 is effective in breaking tolerance to induce a robust T cell response to 5T4 and is therefore expected to be effective in treating or preventing prostate cancer.

[0028] In certain embodiments, the poxvirus F11 promoter is the endogenous MVA F11 promoter. Endogenous enhancer sequences and Kozac-like sequences in the MVA F11 promoter region can enhance the transcription of 5T4 antigen in human cells.

[0029] In a specific embodiment, the 5T4 antigen polypeptide has the amino acid sequence provided in SEQ ID NO:1.

[0030] In another specific embodiment, the 5T4 antigen polypeptide has an amino acid sequence encoded by the nucleic acid sequence provided by SEQ ID NO: 2. After administration of the composition, the use of such a codon-optimized sequence encoding the 5T4 antigen polypeptide improves the expression of the antigen polypeptide in the subject.

[0031] In certain embodiments, the composition further comprises an adjuvant. The inclusion of an adjuvant can improve the immune response produced when the composition is administered to a subject.

[0032] The present invention also provides the use of the composition as defined above in inducing a T cell-mediated immune response to a 5T4 antigen polypeptide. The inventors have found that administration of the composition is effective in inducing an immune response to the "self" antigen 5T4. The composition is preferably used to induce a CD8+ T cell response.

[0033] Advantageously, the composition is effective for treating or preventing prostate cancer in a subject.

[0034] In another aspect, the present invention provides a method of inducing a T cell-mediated immune response to a 5T4 antigen polypeptide and inducing a T cell-mediated immune response for treating or preventing prostate cancer, the method comprising administering the composition of the first aspect to a subject in need of such a T cell-mediated immune response.

[0035] In a preferred embodiment, in this method, the composition of the present invention is 1×10 6 Up to 5×10 8 In the most preferred embodiment, in this method, the composition is administered at a dose of 1×10 7 This dose provides a robust immune response while minimizing unnecessary administration and waste of the composition.

[0036] In certain embodiments, the T cell-mediated immune response induced by the method comprises a CD8+ T cell response. Such a cytolytic T cell response is suitable for effectively removing cells expressing the 5T4 antigen from a subject.

[0037] In a preferred embodiment, the method is a prime-boost method, wherein the composition of the first aspect is administered to a subject to induce an initial T cell-mediated immune response or to strengthen an existing T cell-mediated immune response. In a particularly preferred embodiment, the composition of the first aspect is administered as a boost to a previously administered primary vaccination. This administration regimen has been shown to advantageously destroy tolerance and achieve induction of a strong anti-5T4 T cell response.

[0038] A preferred priming vaccination of this method is provided by administering an adenovirus expressing the 5T4 antigenic polypeptide, and in a most preferred embodiment, the adenovirus used is ChAdOx1.

[0039] In a preferred embodiment, in this method, the adenovirus expressing the 5T4 antigen polypeptide is expressed at 1×10 8 Up to 1×10 12 The dose of viral particles (VP) is preferably 1×10 9 Up to 1×10 11In the most preferred embodiment, in this method, the adenovirus is administered at a dose of 1×10 10 This dose provides a robust immune response while minimizing unnecessary administration and waste of the composition.

[0040] In another embodiment, the method of the present invention further comprises the combination of the composition of the first aspect of the present invention and an immune checkpoint inhibitor compound. In a preferred embodiment of this invention, the immune checkpoint inhibitor compound is an anti-PD1 monoclonal antibody.

[0041] Throughout the specification and the appended claims, the words "comprise" and "include" and variations such as "comprises," "comprising," "includes," and "including" will be understood to be inclusive. That is, these words are intended to convey that additional elements or integers not expressly recited may be included where the context permits.

[0042] Examples

[0043] MVA Construction

[0044] Codon-optimized polynucleotides encoding 5T4 antigenic polypeptides (NCBI reference sequence: NM_006670.4) were synthesized by GeneArt Gene Synthesis (Thermo Fisher Scientific). The 5T4 transgene was then cloned into a shuttle plasmid vector designed to have the upstream and downstream (flanks) of the F11L ORF as homology sequence arms. Insertion of the 5T4 transgene in these arms enables the use of the endogenous F11 promoter (which is part of the right homology arm) while deleting the native F11L ORF. This generates a shuttle vector (F11 shuttle vector) for the production of MVA.(F11)5T4.

[0045] Construction of MVA.(mH5)5T4 and MVA.(p7.5)5T4 was previously described in Harrop et al. (2010) and Cappuccini et al. (2017), respectively.

[0046] The MVA construct was prepared such that the marker gene was absent from the recombinant virus.

[0047] 5T4 immunogenicity

[0048] Six male C57BL / 6 mice (Harlan, UK) per group received a primary immunization on day 0 consisting of intramuscular (im) administration of 1×10 10VP ChAdOx1.5T4 (Groups 1 to 3) or 1×10 7 pfu MVA.(F11)5T4 (Group 4).

[0049] The same animals received a booster immunization on day 21 with 1 × 10 7 pfu MVA.(p7.5)5T4 (Group 1), 1×10 7 pfu MVA.(F11)5T4 (Group 2), 1×10 7 pfu MVA.(mH5)5T4 (Group 3) or 1×10 7 pfuMVA.(F11)5T4 (Group 4).

[0050] Three weeks after booster immunization (day 42), blood and spleen were collected from each mouse, and the presence of 5T4-specific T cells in PBMCs and spleen cells was detected by IFNg ELISPOT. The results of ELISPOT analysis are shown in Figure 2. Figure 3 shown.

[0051] Flow cytometric analysis of 5T4-specific T cells was also performed on PBMCs and splenocytes from mice primed with ChAdOx1.5T4 and boosted with MVA.(F11)5T4. Figure 4 Results of flow cytometric analysis are presented.

[0052] All animal procedures were performed under the terms of project license 30 / 2947 under the UK Animals (Scientific Procedures) Act (ASPA) and were approved by the Oxford University Animal Care and Ethical Review Committee. All mice were housed for at least 7 days to acclimate prior to any procedures at the Oxford University Animal Facility in the UK under specific pathogen-free (SPF) conditions.

[0053] Safety and immunogenicity of MVA-(F11)5T4 in human subjects

[0054] MVA-(F11)5T4 has been administered to human subjects in clinical trials for the treatment of advanced metastatic prostate cancer.

[0055] These prostate cancer patients received a primary immunization at week 0 with 2×10 10vp doses consisted of intramuscular (im) administration of a simian adenoviral vector ChAdOx1 encoding 5T4, and a booster dose of MVA-(F11)5T4 was administered IM at week 4, combined with an intravenous administration of the checkpoint inhibitor anti-PD1. The same patients received a second round of immunizations at weeks 12 and 16, and further received standard iv administration of anti-PD1 at weeks 8 and 12. Blood samples were collected at weeks 0, 2, 5, 9, 13, 17, 24, and 36 to measure immune responses, and any adverse events (AEs) were recorded and studied.

[0056] Eleven patients were administered MVA-(F11)5T4 and the safety profile was very favorable. On the second day after vaccination, 50% of patients experienced pain or tenderness at the injection site. Three (3) serious adverse events (SAEs) occurred, but the study concluded that none of them were caused by MVA-(F11)5T4.

Claims

1. A composition for inducing a T cell-mediated immune response for treating or preventing prostate cancer, the composition comprising a modified vaccinia virus Ankara (MVA) vector expressing a 5T4 antigen polypeptide under the control of a poxvirus F11 promoter.

2. The composition according to claim 1, wherein The poxvirus F11 promoter is the endogenous MVAF11 promoter.

3. The composition according to claim 1 or claim 2, wherein The vector expresses a polypeptide having an amino acid sequence shown in SEQ ID NO:

1.

4. The composition according to claim 1 or claim 2, wherein The vector expresses a polypeptide encoded by a polynucleotide having a nucleic acid sequence shown in SEQ ID NO:

2.

5. A composition according to any one of the preceding claims, further comprising an adjuvant.

6. Use of a composition according to any one of the preceding claims in the preparation of a medicament for inducing a T cell-mediated immune response to a 5T4 antigen polypeptide.

7. For use according to claim 6, wherein: The T cell-mediated immune response includes a CD8+ T cell response.

8. Use of the composition according to any one of claims 1 to 6 in the preparation of a medicament for treating or preventing prostate cancer.

9. Use of the composition according to any one of claims 1 to 6 in the preparation of a medicament for inducing a T cell-mediated immune response to a 5T4 antigen polypeptide in a subject, wherein the inducing a T cell-mediated immune response to a 5T4 antigen polypeptide in a subject comprises administering the composition according to any one of claims 1 to 6 to the subject.

10. The use according to claim 9, wherein The composition is 1×10 6 Up to 5×10 8 Plaque forming units (pfu) were administered.

11. The use according to claim 10, wherein The composition is 1×10 7 pfu doses were administered.

12. The use according to any one of claims 9 to 11, wherein The T cell-mediated immune response includes a CD8+ T cell response.

13. The use according to any one of claims 9 to 12, wherein The administration is performed as part of a prime-boost vaccination regimen.

14. The use according to claim 13, wherein The administration is provided as a boost to a previous priming vaccination.

15. The use according to claim 14, wherein The previous priming vaccination was provided by administering an adenovirus expressing the 5T4 antigenic polypeptide.

16. The use according to claim 15, wherein The adenovirus is ChAdOx1.

17. The use according to claim 15 or claim 16, wherein The adenovirus was expressed at 1×10 8 Up to 1×10 12 Dosage administration of viral particles (VP).

18. The use according to claim 17, wherein The adenovirus was expressed at 1×10 9 Up to 1×10 11 Dosage administration of VP.

19. The use according to claim 17, wherein The adenovirus was expressed at 1×10 10 Dosage administration of VP.

20. Use of a composition according to any one of claims 1 to 6 in the preparation of a medicament for inducing a T cell-mediated immune response for treating or preventing prostate cancer in a subject, wherein the inducing a T cell-mediated immune response for treating or preventing prostate cancer in a subject comprises administering a composition according to any one of claims 1 to 6 to the subject.

21. The use according to claim 20, wherein The composition is 1×10 6 Up to 5×10 8 Plaque forming units (pfu) were administered.

22. The use according to claim 21, wherein The composition is 1×10 7 pfu doses were administered.

23. The use according to any one of claims 20 to 22, wherein The T cell-mediated immune response includes a CD8+ T cell response.

24. The use according to any one of claims 20 to 23, wherein The administration is performed as part of a prime-boost vaccination regimen.

25. The use according to claim 24, wherein The administration is provided as a boost to a previous priming vaccination.

26. The use according to claim 25, wherein The previous priming vaccination was provided by administering an adenovirus expressing the 5T4 antigenic polypeptide.

27. The use according to claim 26, wherein The adenovirus is ChAdOx1.

28. The use according to claim 26 or claim 27, wherein The adenovirus was expressed at 1×10 8 Up to 1×10 12 Dosage administration of VP.

29. The use according to claim 28, wherein The adenovirus was expressed at 1×10 9 Up to 1×10 11 Dosage administration of VP.

30. The use according to claim 29, wherein The adenovirus was expressed at 1×10 10 Dosage administration of VP.

31. The use according to any one of claims 20 to 30, further comprising the step of administering an immune checkpoint inhibitor compound.

32. The use according to claim 31, wherein The immune checkpoint inhibitor compound is an anti-PD1 monoclonal antibody.

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