FIELD

[0001] The present disclosure relates to combination prime:boost therapies for inducing an immune response where bacteria are used as priming vaccine.

BACKGROUND

[0002] Oncolytic viruses (OVs) specifically infect, replicate in and kill malignant cells, leaving normal tissues unaffected. Several OVs have reached advanced stages of clinical evaluation for the treatment of various neoplasms. Viral agents have the potential to be substituted or combined with standard cancer therapies with the goal of achieving reduced toxicity and improved therapeutic efficacy. Several oncolytic viruses are known. The Anti-tumour efficacy of oncolytic viruses not only depends on their oncolytic activity, but may also depend on their ability to evade anti-tumour immunity. Ideally, an OV not only evades, but stimulates the anti-tumour immune response. This immune-mediated tumour response may to play a critical role in the overall efficacy of OV therapy. Indeed, tumour-specific adaptive immune cells can patrol the tissues and destroy tumour cells that have been missed by the OV’s oncolytic activity. Moreover, their activation of the immune system may prevent tumour recurrence.

[0003] Various strategies have been developed to improve OV-induced antitumour immunity. One such strategy involves the administration of antigen through a priming administration, and a subsequent administration of an oncolytic vector also expressing an antigen— a heterologous prime:boost strategy. When applied in a heterologous prime:boost setting to treat a murine melanoma model, a VSV-human dopachrome tautomerase (hDCT) oncolytic vaccine using adenovirus as the priming vector not only induced an increased tumour-specific immunity to DCT but also a concomitant reduction in antiviral adaptive immunity. Although VSV was shown to be effective using hDCT as a tumour associated antigen, it has been postulated that it is difficult to predict what tumour associated antigens will be effective in a heterologous prime:boost setting.

SUMMARY OF THE INVENTION

[0004] It is an object of the present disclosure to improve at least aspect of anticancer vaccines, specifically the efficacy of oncolytic viral prime:boost therapeutic methods. [0005] The authors of the present disclosure have identified a combination prime:boost therapy that induces an immune response in a mammal. The combination prime:boost therapy according to the present disclosure uses Listeria monocytogenes (LM) expressing the antigen as the priming vaccine. A recombinant oncolytic virus expressing the antigen is used as the oncolytic boosting virus. In various embodiments, the oncolytic virus is a Maraba MG1 virus. Exemplary combination therapies according to the present disclosure include use ofMAGE-A3, Human Papilloma Virus E6/E7 fusion protein, human Six-Transmembrane Epithelial Antigen of the Prostate protein, or Cancer Testis Antigen 1 as the tumour associated antigen (“TAA”). In particular examples, the antigenic protein is MAGEA3. Examples, the therapeutic benefits provided by a heterologous prime:boost combination therapy using LM as the priming vector are superior to benefits associated with the corresponding combination therapy using adenovirus as the priming vector. Adenovirus is only effective at priming the immune response when administered intramuscularly, and has no direct oncolytic effects. In contrast, and without wishing to be bound by theory, the authors of the present disclosure believe that improved efficacy associated with using LM as the prime may be the result of direct killing of tumour cells by the LM, of local inflammation in the tumour

microenvironment may be triggered upon injection of the LM, of induction of both cellular and humoral immunity by the LM, or a combination thereof.

[0006] The results discussed herein show that a recombinant Listeria

monocytogenes bacteria provides at least one advantage over the corresponding recombinant adenovirus disclosed in the‘478 publication. The authors of the present disclosure found that, in mice, the therapeutic benefits provided by the LM:Maraba MG1 therapy, using Ova as a model TAA, were improved compared to the corresponding Ad:Maraba OVA MG1 therapy, also using Ova as a model antigenic protein, since the mice treated with the LM:Maraba MG1 therapy were observed to have smaller tumours and prolonged survival. These results are unexpected and were not predicted, because there is no way to predict if or how efficacy will be affected if the priming vector is changed. One would not have be able to predict which, if any, priming vector would provide a beneficial effect on the immune response, survival, and/or tumour size in a prime:boost combination therapy.

[0007] In various embodiments, a method of treating a cancer in a mammal is provided, said cancer being a tumour associated antigen, said method comprising (1) administering Listeria monocytogenes (LM) bacteria, said bacteria comprising a nucleic acid that expresses a tumour associated antigen, wherein said LM virus is formulated for use in generating an immunity to said tumour associated antigen; and administering an oncolytic virus comprising a nucleic acid that expresses a tumour associated antigen, wherein said oncolytic virus is formulated for use in providing a therapeutic oncolytic effect in said mammal.

[0008] In various embodiments, the LM bacteria is an attenuated LM bacteria. In various embodiments the LM bacteria has an increased immunogenicity over wild-type LM bacterial. In various embodiments the attenuated LM bacteria lacks the

transcriptional activator PfrA or has the transcriptional activator PfrA with reduced activity. In various embodiments, the attenuated LM bacteria expresses a fusion protein of PrfA and the antigenic protein. In various embodiments the Lm bacteria expresses the antigenic protein and Listeriolysin O (LLO) as a fusion protein.

[0009] In various embodiments, a combination prime:boost therapy is provided for use in inducing an immune response in a mammal, wherein the combination comprises: a Listeria monocytogenes (LM) bacteria that is capable of expressing an antigenic protein, and that is formulated to generate an immunity to the protein in the mammal; and a Maraba MG1 virus that is capable of expressing an antigenic protein, and that is formulated to induce the immune response in the mammal; wherein the antigenic protein capable of being expressed by the LM bacteria and the antigenic protein capable of being expressed by the Maraba MG1 virus are both based on the same tumour associated antigen.

[0010] In various embodiments the tumour associated antigen is an HPV tumour associated antigen. In various embodiments, wherein the HPV tumour associated antigen comprises the full-length HPV serotype 16 E6 and E7 oncoproteins. In various embodiments, the HPV tumour associated antigen comprises the full-length HPV serotype 18 E6 and E7 oncoproteins. In various embodiments, the Human Papilloma Virus E6/E7 fusion protein encoded by the LM bacteria has an amino acid sequence that includes HPV16 E6, HPV18 E6, HPV16 E7 and HPV18 E7 protein domains, where the protein domains are linked by proteasomally degradeable linkers. In various

embodiments, the protein domains of the protein encoded by the LM bacteria are in the order: HPV16 E6, then HPV18 E6, then HPV16 E7, then HPV18 E7. In various embodiments, the protein domains of the protein encoded by the LM bacteria are in an order other than: HPV16 E6, then HPV18 E6, then HPV16 E7, then HPV18 E7. In various embodiments, the Human Papilloma Virus E6/E7 fusion protein encoded by the Maraba MG1 virus has an amino acid sequence that includes HPV16 E6, HPV18 E6, HPV16 E7 and HPV18 E7 protein domains, where the protein domains are linked by proteasomally degradeable linkers.

[0011] In various embodiments, the tumour associated antigen is a MAGEA3 protein. In various embodiments, the MAGEA3 protein is at least 70% identical to SEQ ID NO: 1 tumour associated epitope is selected from the group consisting of:

FLWGPRALV (SEQ ID NO: 9), KVAELVHFL (SEQ ID NO: 10), EGDCAPEEK (SEQ ID NO: 17), KKLLTQHFVQENYLEY (SEQ ID NO: 18), and RKVAELVHFLLLKYR (SEQ ID NO: 19). In various embodiments, the antigenic protein expressed by the LM bacteria, the Maraba MG1 virus, or both is at least 80% identical to SEQ ID NO: 1. In various embodiments, the antigenic protein expressed by the LM bacteria, the Maraba MG1 virus, or both is at least 90% identical to SEQ ID NO: 1. In various embodiments, the antigenic protein expressed by the LM bacteria, the Maraba MG1 virus, or both is at least 95% identical to SEQ ID NO: 1.

[0012] Other aspects and features of the present disclosure will become apparent to those ordinarily skilled in the art upon review of the following description of specific embodiments in conjunction with the accompanying figures.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Embodiments of the present disclosure will now be described, by way of example only, with reference to the attached Figures.

[0014] Fig. 1 is an illustration of a treatment regimen followed in Example 1.

[0015] Fig. 2 is a graph illustrating immune response post-vaccination with priming vaccines.

[0016] Fig. 3 illustrates the results of the flow cytometry analysis of cytotoxic T cells from the vaccination of Fig. 2.

[0017] Fig. 4 is a graph illustrating immune response post-vaccination with prime- boost vaccine therapy.

[0018] Fig. 5 illustrates the results of the flow cytometry analysis of cytotoxic T cells from the vaccination of Fig. 4.

[0019] Fig. 6 is a histology image of B16F10-Ova melanoma tumours treated with

LM or control.

[0020] Fig. 7 is a histology image of B16F10-Ova melanoma tumours treated with

LM or control.

[0021] Fig. 8 shows graphs illustrating the tumour volume of tumours with different treatments. [0022] Fig. 9 shows graphs illustrating the tumour volume of tumours with different treatments.

[0023] Fig. 10 is a graph illustrating the survival curves of mice treated with different treatments.

[0024] Fig. 1 1 is a graph illustrating the percentage of mice having tumours after challenge with tumour cells to which they were naive, or re-challenge with B16F10-Ova cells of which they had been previously cured.

[0025] Fig. 12 is a graph illustrating the body weight of mice over time.

DETAILED DESCRIPTION

[0026] The present disclosure provides a combination prime:boost therapy for use in inducing an immune response in a mammal. Prime:boost immunizations can be given with unmatched vaccine delivery methods while using the same antigen, in a

‘heterologous’ prime:boost format; or with matched vaccine delivery methods, in a ‘homologous’ prime:boost. Heterologous prime:boost methods are preferable when using vectored vaccine platforms as homologous vaccination would lead to boosting of responses to both the vector and the transgene in the secondary response. In contrast, a heterologous system focuses the secondary response (that is, the boosted response) to, as the immune response to the boost (“boost” or“second”) vector is enhanced by immunity triggered by the prime (“prime” or“first”) vector.

[0027] Generally, a combination prime:boost therapy of the present disclosure includes: (1) a Listeria monocytogenes (LM) bacteria that (a) expresses an antigenic protein that includes a tumour associated antigen and (b) is formulated to generate an immunity to the TAA in the mammal; and (2) an oncolytic virus that (a) expresses a TAA that includes a tumour associated antigen (“TAA”) and (b) is formulated to induce the immune response in the mammal. The antigenic proteins expressed by the LM bacteria and the oncolytic virus (such as the Maraba MG1 virus) are the same or derived from the same tumour associated antigen, but the TAAs on the prime and the boost vectors do not need to be identical in sequence. In the context of the present disclosure, antigenic proteins from on the same tumour associated antigen would be understood to mean the two antigenic proteins include one or more sequences that result in a boosted immune response when the two vaccines are administered in a prime-boost format. That is, the antigenic protein expressed by the LM bacteria and the antigenic protein expressed by the Maraba MG1 virus may be identical, or different. If different, the antigenic proteins are sufficiently similar that the immune response to the antigenic protein expressed by the Maraba MG1 virus is increased in comparison to an immune response induced in the absence of a priming vaccine.

[0028] In one particular example, the combination prime:boost therapy of the present disclosure includes: (1) a Listeria monocytogenes (LM) bacteria that expresses a MAGEA3 protein as an antigenic protein and that is formulated to generate an immunity to the protein in the mammal; and (2) a Maraba MG1 virus that expresses a MAGEA3 protein as an antigenic protein and that is formulated to induce the immune response in the mammal. The antigenic MAGEA3 protein expressed by the LM bacteria and the antigenic MAGEA3 protein expressed by the Maraba MG1 virus may be identical, or may be variants of each other.

[0029] In the context of the present disclosure, the terms“priming LM bacteria”,

“LM priming vaccine”,“priming LM vaccine” or“priming vaccine”, should be understood to refer to a recombinant LM bacteria that is capable of expressing an antigenic protein. In the context of the present disclosure, the terms“boosting maraba virus” or“boosting vaccine” should be understood to refer to a Maraba MG1 virus that is capable of expressing an antigenic protein, respectively. In the context of the present disclosure, the priming and boosting vaccines are vectored vaccine platforms, and the present disclosure may refer to the LM bacteria and the Maraba MG1 viruses as the“priming vector” and the “boosting vector”, respectively. The terms“LM-MAGEA3”,“Listeria monocytogenes MAGEA3”, and“Listeria monocytogenes bacteria encoding MAGE-A3 protein” should all be understood to refer to a recombinant Listeria monocytogenes bacteria that is capable of expressing MAGE-A3 as an antigenic protein. The terms“MG1-MAGEA3”,“Maraba MG1 MAGEA3”, and“Maraba MG1 virus encoding MAGE-A3 protein” should all be understood to refer to a Maraba MG1 virus that is capable of expressing MAGE-A3 as an antigenic protein.

[0030] Melanoma antigen, family A, 3 (MAGEA3) is a“cancer testis antigen”. The

MAGE family of genes encoding tumour specific antigens is discussed in De Plaen et al., Immunogenetics 40:360-369 (1994), MAGEA3 is expressed in a wide variety of tumours including melanoma, colorectal and lung.

[0031] The protein“MAGEA3” may be also referred to as“MAGE-A3” and stands for melanoma-associated antigen 3. The antigenic MAGEA3 protein according to the present disclosure is a protein that includes the amino acid sequence of SEQ ID NO: 1. This amino acid sequence may be encoded by the nucleotide sequence of SEQ ID NO: 2. Alternatively, the amino acid sequence may be encoded by a codon optimized transgene that includes the nucleotide sequence of SEQ ID NO: 3. A negative sense RNA virus that expresses the protein of SEQ ID NO: 1 may include a reverse complement and RNA version of a polynucleotide of SEQ ID NO: 2 or 3. A positive sense RNA virus or a DNA virus that expresses the protein of SEQ ID NO: 1 may include a sequence that is SEQ ID NO: 2 or 3.

[0032] An example of an antigenic MAGEA3 variant protein according to the present disclosure is a protein that includes the amino acid sequence of SEQ ID NO: 4. This amino acid sequence may be encoded by the nucleotide sequence of SEQ ID NO: 5. A negative sense RNA virus that expresses the protein of SEQ ID NO: 4 may include an RNA polynucleotide which includes a sequence that is a reverse complement and RNA version of SEQ ID NO: 5. A DNA virus or RNA virus that expresses the protein of SEQ ID NO: 4 may include a sequence that is SEQ ID NO: 5. One example of such a negative sense RNA virus is a Maraba virus that includes the reverse complement and RNA version of SEQ ID NO: 6.

[0033] An LM bacteria that encodes the protein of SEQ ID NO: 4 may include a sequence that is SEQ ID NO: 7.

[0034] The above noted sequences are shown in Appendix A.

[0035] Human Papilloma Virus (HPV) oncoproteins E6/E7 are constitutively expressed in cervical cancer (Zur Hausen, H (1996) Biochem Biophys Acta 1288:F55- F78). Furthermore, HPV types 16 and 18 are the cause of 75% of cervical cancer (Walboomers JM (1999) J Pathol 189:12-19). A fusion protein of the E6/E7 oncoproteins of HPV types 16 and 18 may be used as the antigenic protein. An example of HPV E6/E7 according to the present disclosure includes sequences corresponding to the E6 and E7 transforming proteins of both the HPV16 and HPV18 serotypes, resulting in a fusion protein that includes HPV16 E6, HPV18 E6, HPV16 E7 and HPV18 E7 protein domains. The four protein domains are linked by proteasomally degradable linkers that result in the separate HPV16 E6, HPV18 E6, HPV16 E7 and HPV18 E7 proteins once the fusion protein is in the proteasome. The proteasomally degradeable linkers in a fusion protein may be the same or different. The terms“HPV E6/E7 protein”,“HPV E6/E7 fusion protein”, and“therapeutic E6E7 construct” should all be understood to be synonymous with“Human Papilloma Virus E6/E7 fusion protein”. Examples of HPV E6/E7 fusion proteins are disclosed in POT Application No: PCT/IB2017/000622, which is incorporated herein by reference. The sequence of a particular example of an HPV E6/E7 fusion protein, and the DNA sequence encoding the fusion protein, are disclosed in SEQ ID Nos: 1 and 3, respectively of the‘622 POT application. Sequences of other particular examples of HPV E6/E7 fusion proteins are disclosed in SEQ ID Nos: 5-8 of the‘622 PCT application.

[0036] Sequences of exemplary E6 and E7 proteins from HPV16 and HPV18 are disclosed in SEQ ID Nos: 9-12 of the‘622 PCT application.

• In SEQ ID NOs: 9 and 10 of the‘622 PCT application, each Xaa is independently: absent, cysteine, or a non-cystine amino acid. When the identified variable Xaa’s residules are cysteines, the sequences correspond to the wild type sequences of HPV16 E6 and HPV18 E6, respectively.

• In SEQ ID NO: 11 of the‘622 PCT application, the Xaa at position 24 is either absent, cysteine, or a non-cysteine amino acid; the Xaa at position 25 is either absent, tyrosine, or a non-tyrosine amino acid; the Xaa at position 26 is either absent, glutamic acid, or a non-glutamic acid amino acid; and the Xaa’s at positions 91 and 94 are, independently: absent, cysteine, or a non-cysteine amino acid. The sequence of SEQ ID NO: 11 corresponds to the wild type sequence of HPV16 E7 when the Xaa’s at positions 24-26 are cysteine-tyrosine-glutamic acid and the Xaa’s at positions 91 and 94 are cysteines.

• In SEQ ID NO: 12 of the‘622 PCT application, the Xaa at position 27 is either absent, cysteine, or a non-cysteine amino acid; the Xaa at position 28 is either absent, histidine, or a non-histidine amino acid; the Xaa at position 29 is either absent, glutamic acid, or a non-glutamic acid amino acid; and the Xaa’s at positions 98 and 101 are, independently: absent, cysteine, or a non-cysteine amino acid. The sequence of SEQ ID NO: 12 of the‘622 PCT application corresponds to the wild type sequence of HPV18 E7 when the Xaa’s at positions 27-29 are cysteine-histidine-glutamic acid and the Xaa’s at positions 98 and 101 are cysteines.

[0037] Six-Transmembrane Epithelial Antigen of the Prostate (huSTEAP) is a recently identified protein shown to be overexpressed in prostate cancer and up-regulated in multiple cancer cell lines, including pancreas, colon, breast, testicular, cervical, bladder, ovarian, acute lyphocytic leukemia and Ewing sarcoma (Hubert RS et al., (1999) Proc Natl Acad Sci 96:14523-14528). The STEAP gene encodes a protein with six potential membrane-spanning regions flanked by hydrophilic amino- and carboxyl- terminal domains.

[0038] Cancer Testis Antigen 1 (NYES01) is a cancer/testis antigen expressed in normal adult tissues, such as testis and ovary, and in various cancers (Nicholaou T et al., (2006) Immunol Cell Biol 84:303-317). Cancer testis antigens are a unique family of antigens, which have restricted expression to testicular germ cells in a normal adult but are aberrantly expressed on a variety of solid tumours, including soft tissue sarcomas, melanoma and epithelial cancers.

[0039] The huSTEAP and NYES01 proteins according to the present disclosure are discussed in greater detail in PCT Publication No. WO 2014/127478, and exemplary sequences of these proteins are provided in SEQ ID Nos: 10-15 of the‘478 PCT publication. The sequences are incorporated herein by reference.

[0040] The huSTEAP protein is also disclosed in greater detail in PCT Application

No. PCT/IB2017/000622. An exemplary, codon-optimized sequence of the huSTEAP protein is disclosed as SEQ ID NO: 13 in the‘622 PCT application. The corresponding DNA sequence is disclosed as SEQ ID NO: 14. The sequences are incorporated herein by reference.

[0041] In the context of the present disclosure, a“variant” of a tumour associated antigenic protein refers to a protein that (a) includes at least one tumour associated antigenic epitope from the tumour associated antigenic protein and (b) is at least 70% identical to the tumour associated antigenic protein. Preferably, the variant will be at least 80% identical to the tumour associated antigenic protein. More preferably, the variant will be at least 90% identical to the tumour associated antigenic protein. Even more preferably, the variant will be at least 95% identical to the tumour associated antigenic protein. Variants with higher sequence identities have increased likelihood that the epitopes are presented in a similar 3-dimensional manner to the wild type antigenic proteins.

[0042] Generally, a tumour associated antigenic epitope may be identified by breaking up the whole antigenic protein into overlapping series of peptides, or by generating libraries of random peptides, and looking for T cell responses by stimulating PBMCs or splenocytes from animals vaccinated with the protein target using the peptide pools. Pools having a response identify that peptide as a potential antigenic epitope. This approach is discussed by Morris, GE in Encyclopedia of Life Sciences, 2007, page 1-3 (doi: 10.1002/9780470015902.a0002624.pub2).

[0043] A database summarizing well accepted antigenic epitopes is provided by

Van der Bruggen P, Stroobant V, Vigneron N, Van den Eynde B in“Database of T cell- defined human tumour antigens: the 2013 update.” Cancer Immun 2013 13:15 and at <www.cancerimmunity.org/peptide/>. [0044] Tumour associated antigenic epitopes have been already identified for

MAGEA3. Accordingly, a variant of the MAGEA3 protein may be, for example, an antigenic protein that includes at least one tumour associated antigenic epitope selected from the group consisting of: EVDPIGHLY (SEQ ID NO: 8), FLWGPRALV (SEQ ID NO: 9), KVAELVHFL (SEQ ID NO: 10), TFPDLESEF (SEQ ID NO: 1 1), VAELVHFLL (SEQ ID NO: 12), M EVDPIGHLY (SEQ ID NO: 13), REPVTKAEML (SEQ ID NO: 14), AELVHFLLL (SEQ ID NO: 15), WQYFFPVIF (SEQ ID NO: 16), EGDCAPEEK (SEQ ID NO: 17), KKLLTQHFVQENYLEY (SEQ ID NO: 18), RKVAELVHFLLLKYR (SEQ ID NO: 19), ACYEF LWG PRALVET S (SEQ ID NO: 20), VIFSKASSSLQL (SEQ ID NO: 21),

VIFSKASSSLQL (SEQ ID NO: 22), VFGIELMEVDPIGHL (SEQ ID NO: 23),

GDNQIMPKAGLLIIV (SEQ ID NO: 24), TSYVKVLHHMVKISG (SEQ ID NO: 25), RKVAELVHFLLLKYRA (SEQ ID NO: 26), and FLLLKYRAREPVTKAE (SEQ ID NO: 27); and that is at least 70% identical to the MAGEA3 protein.

[0045] It may be desirable for variants of a tumour associated antigenic protein to include only antigenic epitopes that have high allelic frequencies, such as frequencies greater than 40% of the population. Accordingly, preferred examples of variants of MAGEA3 may include proteins that include at least one antigenic epitope selected from the group consisting of: FLWGPRALV (SEQ ID NO: 9), KVAELVHFL (SEQ ID NO: 10), EGDCAPEEK (SEQ ID NO: 17), KKLLTQHFVQENYLEY (SEQ ID NO: 18), and

RKVAELVHFLLLKYR (SEQ ID NO: 19); and that is at least 70% identical to the MAGEA3 protein.

[0046] The antigenic protein expressed by the LM bacteria does not need to have exactly the same sequence as the antigenic protein expressed by the Maraba MG1 virus. The antigenic protein expressed by Maraba MG1 must only induce an overlapping immune response to the antigenic protein expressed by the LM bacteria. For example, the LM bacteria may express the MAGEA3 and the Maraba MG virus may express a MAGEA3 variant, or vice versa, or both vectors may express different MAGEA3 variants. Since both MAGEA3 and the variant of MAGEA3 (or since both variants of MAGEA3) induce overlapping immune responses (as they both include at least one identical tumour associated antigenic sequence), the LM bacteria acts as the prime and the Maraba MG1 virus acts as the boost. It is sufficient that the immune response generated in the mammal to the first antigen results in an immune response primarily to the MAGEA3 or MAGEA3 variant when the Maraba MG1 virus is administered.

[0047] In the context of the present disclosure, it should be understood that all discussions of, and references to, a‘protein expressed by a virus’ more exactly refer to a protein expressed by a cell infected with the virus since viruses do not themselves have the capability to express proteins. Similarly, all discussions of, and references to, a‘virus that expresses a protein’ or‘virus capable of expressing a protein’ more exactly refer to a virus that includes the genetic information necessary for the protein to be expressed by a cell infected with the virus.

[0048] In the context of the present disclosure, a“combination prime:boost therapy” should be understood to refer to therapies where the LM bacteria and the Maraba MG1 virus discussed herein are to be administered as a prime:boost treatment. The LM bacteria and the Maraba MG1 virus need not be physically provided or packaged together since the LM bacteria is to be administered first and the Maraba MG1 virus is to be administered only after an immune response has been generated in the mammal. In some examples, the combination is provided to a medical institute, such as a hospital or doctor’s office, in the form of a plurality of packages of the priming vaccine, and a separate plurality of packages of the boosting Maraba MG1 virus. The packages of priming vaccine and the packages of boosting Maraba MG1 virus may be provided at different times. In other examples, the combination is provided to a medical institute, such as a hospital or doctor’s office, in the form of a package that includes both the priming vaccine and the boosting Maraba MG1 virus.

[0049] The therapy may additionally include an immune-potentiating compound, such as cyclophosphamide (CPA) that increases the prime immune response to the tumour associated antigenic protein generated in the mammal by administrating the LM bacteria. Cyclophosphamide is a chemotherapeutic agent that may lead to enhanced immune responses against the tumour associated antigenic protein. In a synergistic murine melanoma tumour model, CPA administered prior to the priming vaccine significantly increased survival, while CPA administered prior to the boosting vaccine did not.

[0050] In some exemplary combination therapies according to the present disclosure, the therapy may be used to activate the patient’s immune system to kill tumour cells with reduced toxicity to normal tissues, for example by activating antibodies and/or lymphocytes against a tumour associated antigen on the tumour. In particular examples, the therapy may display both oncolytic activity and an ability to boost adaptive cell immunity.

[0051] In some examples, the therapeutic approach disclosed herein combines:

(1) a bacteria-based vaccine, and (2) Maraba MG1 virus as an oncolytic viral vaccine, both expressing MAGEA3, or a variant thereof. Boosting with the oncolytic vaccine may lead to both tumour debulking by the oncolytic virus and a large increase in the number of tumour-specific CTL (cytotoxic T-lymphocytes) in animals primed by the bacteria-based vaccine. Paradoxically, this methodology actually generates larger anti-tumour responses in tumour-bearing, as compared to tumour-free, animals since the replication of oncolytic virus is amplified in the tumour-bearing animals, which leads to an increase in the number of antigen-specific Tumour Infiltrating Lymphocytes (TILs), when compared to the replication of oncolytic virus in the tumour-free animals and the associated number of antigen-specific Tumour Infiltrating Lymphocytes (TILs).

[0052] The expression products of these genes are processed into peptides, which, in turn, are expressed on cell surfaces. This can lead to lysis of the tumour cells by specific CTLs. The T cell response to foreign antigens includes both cytolytic T lymphocytes and helper T lymphocytes. CD8 ⁺ cytotoxic or cytolytic T cells (CTLs) are T cells which, when activated, lyse cells that present the appropriate antigen presented by HLA class I molecules. CD4 ⁺ T helper cells are T cells which secrete cytokines to stimulate macrophages and antigen-producing B cells which present the appropriate antigen by HLA class II molecules on their surface.

[0053] The term“mammal” refers to humans as well as non-human mammals.

The term“cancer” is used herein to encompass any cancer that expresses the tumour associated antigenic protein used in the viruses of interest. For example, when considering MAGEA3 as an antigenic protein, the term“cancer” encompases any cancer that expresses MAGEA3 as an antigen. Examples of such a cancer include, but are not limited to, melanoma, non-small cell lung cancer, head and neck cancer, colorectal cancer, and bladder cancer.

[0054] The LM bacteria, the oncolytic virus, or both may be independently administered to the mammal intratumourally, intravenously, intramuscularly,

intraperitoneally, or intranasally. The LM bacteria may be administered orally, intravenously, or intratumourally, but it is preferable to administer the LM bacteria intravenously or intratumourally since such administration may allow the LM bacteria to directly infect and kill cancer cells. Further, intratumoural administration may trigger local inflammation, which may increase efficacy of the prime-boost therapy. Following administration of the vaccines, an immune response is generated by the mammal within an immune response interval, e.g. within about 4 days, and extending for months, years, or potentially life.

[0055] To establish an immune response to the tumour associated antigenic protein or variant thereof, vaccination using the first vaccine and the Maraba MG1 virus may be conducted using well-established techniques. As one of skill in the art will appreciate, the amount of vector required to generate an immune response will vary with a number of factors, including, for example, the selected antigen, the selected vector, and the mammal to be treated, e.g. species, age, size, etc. In this regard, in one example, intratumoural administration of at least about 10 ⁸ colony forming units (CFUs) of an LM vector to a mouse is sufficient to generate an immune response. In another example, intravenous administration of about 10 ² -10 ⁴ CFUs of an LM vector to a mouse is sufficient to generate an immune response. Different strains of LM bacteria may require administration of different doses. Corresponding amounts would be sufficient for administration to a human to generate an immune response.

[0056] Once an immune response has been generated in the mammal by administration of the LM vaccine, Maraba MG1 virus encoding the tumour associated antigenic protein or a variant thereof is administered in an amount suitable for oncolytic viral therapy within a suitable immune response interval. A suitable immune response interval may be, for example, at least about 24 hours, preferably at least about 2-4 days or longer, e.g. at least about 1 week, or at least about 2 weeks. The amount of Maraba MG1 virus suitable for oncolytic viral therapy will vary with the mammal to be treated, as will be appreciated by one of skill in the art. For example, about 10 ⁸ PFU of Maraba MG1 virus encoding MAGEA3 administered IV to a mouse is sufficient for oncolytic therapy. A corresponding amount would be sufficient for use in a human.

[0057] Maraba MG1 virus encoding tumour associated antigenic protein or a variant thereof may be prepared by incorporating a reverse complement of a transgene encoding the tumour associated antigenic protein or a variant thereof into the Maraba MG1 virus using standard recombinant technology. For example, the reverse complement of the transgene may be incorporated into the genome of the Maraba MG1 virus, or alternatively, may be incorporated into the virus using a plasmid incorporating the transgene. The transgene encoding the tumour may be a codon optimized transgene.

[0058] LM bacteria encoding tumour associated antigenic protein or a variant thereof may be prepared by incorporating a transgene encoding the tumour associated antigenic protein or a variant thereof into the LM bacteria using standard recombinant technology, for example using a pJJD plasmid. The transgene encoding the tumour may be a codon optimized transgene. An exemplary strain of an LM bacteria which may be used is strain 10403S. EXAMPLES

[0059] Material and Methods

[0060] Listeria monocytogenes Bacteria. The LM bacteria used in the following examples was prepared as discussed in Dudani, Renu et al "Multiple Mechanisms Compensate to Enhance Tumour-Protective CD8 ⁺ T Cell Response in the Long-Term Despite Poor CD8 ⁺ T Cell Priming Initially: Comparison Between an Acute Versus a Chronic Intracellular Bacterium Expressing a Model Antigen." The Journal of

Immunology 168.1 1 (2002): 5737-5745.

[0061] Cell culture. LM was cultured in brain heart infusion media. Adenovirus and Maraba MG1 were expanded on HEK 293T and Vero cells, respectively. The MRB virus used in this study is the double mutant MG1 described in Brun, J et al.“Identification of genetically modified Maraba virus as an oncolytic rhabdovirus.” Mol. Ther. (2010) 18: 1440-9.

[0062] Flow Cytometry. Single-cell suspensions of freshly-isolated splenocytes were stimulated for 6h with the Ova peptide SIINFEKL (SEQ ID NO: 28) (Biomer Technology) and golgi-plug (BD Biosciences) was added after 1 h. Surface staining was performed on ice for 30 minutes in FACS buffer (3% FBS in PBS). The samples were then fixed using IC fixation buffer (BD Biosciences) for 20 minutes and permeabilized for 20 minutes using permeabilization buffer (BD Biosciences). Intracellular staining was performed at room temperature in permeabilization buffer for 30 minutes. All antibodies (CD4-488, CD3-PECY7, CD8 BV510, IFNy-APC and TNFa-PE) were mouse-specific and purchased from BD Biosciences. The samples were analyzed using an LSR Fortessa flow cytometer.

[0063] ELISPOT. Splenocytes (5x10 ⁵ ) were seeded into IFNy ELISPOT plates

(Mabtech) in serum-free DMEM with or without the Ova peptide SIINFEKL (SEQ ID NO: 28) and the assay was performed for 24h as per the manufacturer’s protocol.

[0064] Histological analysis. Tumours were fixed in formalin and special stainings were performed by the University of Ottawa Pathology core. For caspase-3 staining (antibody from Cell signaling technology), the samples were rehydrated through graded alcohol and heat-mediated antigen retrieval was performed in a citrate buffer (sodium citrate 10 mM, pH 6).

[0065] HMGB1 and LDH assays. Serum was collected 48h post-treatment by saphenous bleed. HMGB1 and LDH concentrations were determined using a mouse ELISA kit (Antibodies online) and an LDH assay (Abeam), respectively, following manufacturers’ protocols. [0066] In vivo work. As illustrated in Fig. 1 , B16F10-Ova cells (5x10 ⁵ ) were injected into the left flank of 8 weeks-old c57bl/6 female mice (Charles River). The B16F10-Ova cell line is a stable transfectant derived from B16F10 melanoma that expresses chicken ovalbumin and are discussed in Bellone M, et al.“Relevance of the tumour antigen in the validation of three vaccination strategies for melanoma.” J

Immunol (2000) 165(5):2651-2656. Immune priming was performed at day 7 with either 1x10 ⁸ PFU of Ad or Ad-Ova intramuscularly, or 1x10 ⁸ CFU of LM or LM-Ova

introtumourally. Immune boosting was performed at day 14 with 1x10 ⁸ MRB or MRB-Ova intraveneously. The animals were either sacrificed at day 21 for immune analysis, or the tumour progression was measured over time using electronic calipers. All experiments were performed in accordance with the institutional guidelines of animal care and veterinary services. Tumour volume was calculated as (length*width ² ) / 2.

[0067] Example 1. Listeria monocytogenes and Adenovirus have

comparable Ovalbumin immune-priming activity

[0068] LM and Adenovirus (Ad) variants encoding Ovalbumin (Ova) were used to compare the vaccination potential of both vaccines in heterologous prime-boost setting. First, the authors of the present disclosure compared the anti-Ova immune response induced by Ad and LM in an ELISPOT assay following the treatment regimen illustrated in Fig. 1. The results illustrated in Fig. 2 show the induction of an antigen-specific response 7-days post-vaccination using both priming vaccines. In contrast, the vaccination with empty LM did not induce Ova-specific immunity. A flow cytometry analysis confirmed that 10-15% of the cytotoxic T cells were responsive to Ova, and a significant proportion of these cells produced both IFNy and TNFa (Fig. 3). The data used to generate the graphs in Fig. 3 are shown in Table 1 , below.

Table 1

[0069] The authors of the present disclosure next tested LM as a priming vaccine in a heterologous prime-boost therapy with Maraba MG 1 virus (MRB) and found that LM- Ova priming could efficiently be combined with Maraba MG1-Ova boosting (see Fig. 4). The authors observed that 30% of the cytotoxic T cells from the LM-MRB group were responsive to Ova upon vaccination (Fig. 5). The data used to generate the graphs in Fig. 5 are shown in Table 2, below.

Table 2

[0070] Maraba MG1 was not able to induce an antigen-specific immune response in the absence of a previous priming vaccine (LM+ / MRB-Ova group). The authors of the present disclosure also observed no significant difference in the response to priming using LM-Ova or Ad-Ova. Taken together, these results show that LM is as efficient as Ad at priming anti-tumour immunity in a heterologous prime-boost setting.

[0071] Example 2. The Listeria monocytogenes-Maraba prime-boost shows reduced tumour size and prolonged survival

[0072] The authors of the present disclosure performed a histological analysis of

B16F10-Ova melanoma tumours 24h-post treatment with LM to determine if LM could replicate in the tumours. The LM bacteria was observed in treated tumours by Gram staining (Fig. 6). Hematoxylin and Eosin (H&E) staining revealed that most of the tumour surface was necrotic and bloody upon LM treatment (Fig. 7), a result indicating that the LM bacteria was killing the tumours.

[0073] The authors of the present disclosure performed immunohistochemical staining for cleaved caspase-3 on tumour sections 48h-post treatment to assess the cytotoxic effect of intratumoural LM treatment. Consistent with the Hematoxylin and eosin staining shown in Fig. 7, most of the tumour surface stained positive for caspase-3.

Furthermore, the levels of LDH and HMGB1 (shown in Table 3, below), two markers of necrosis, were elevated in the serum of LM-injected, tumour-bearing mice. Taken together, these results indicate that the LM treatment contributes to tumour killing.

Table 3

[0074] The authors of the present disclosure treated and measured B16F10-Ova tumours as depicted in Fig.1 to determine if an LM-MRB prime-boost therapy could inhibit tumour growth. The results show that the LM-MRB prime-boost therapy inhibited growth of the tumours in most of the tested mice in comparison to single MRB or LM treatments (Fig. 8). The authors of the present disclosure compared the LM-MRB prime-boost therapy to the corresponding Ad-MRB prime-boost therapy and found that the LM-MRB prime-boost therapy provided improved therapeutic benefits with the mice showing smaller tumours (Fig. 9) and prolonged survival (Fig. 10) compared to the group receiving the corresponding Ad-MRB therapy. The authors of the present disclosure re-challenged animals which had been previously cured of B16F10-Ova tumours for 123 to 314 days to determine if the LM-MRB prime-boost could provide long-term protection. Animals were challenged with E0771 cells, to which they were naive, or B16F10-Ova cells. The results illustrated in Fig. 1 1 show that while all long-term survivors displayed E0771 tumours 14 days post-tumour challenge, all of the B16F10-Ova tumours were rejected. Taken together, the results show that a LM-MRB prime-boost therapy may control established tumours and provide long-term protection.

[0075] None of the animals displayed any sign of discomfort over the course of the experiment and no drop in body weight was observed (Fig. 12).

[0076] In the preceding description, for purposes of explanation, numerous details are set forth in order to provide a thorough understanding of the examples. However, it will be apparent to one skilled in the art that these specific details are not required.

Accordingly, what has been described is merely illustrative of the application of the described examples and numerous modifications and variations are possible in light of the above teachings.

[0077] Since the above description provides examples, it will be appreciated that modifications and variations can be effected to the particular examples by those of skill in the art. Accordingly, the scope of the claims should not be limited by the particular examples set forth herein, but should be construed in a manner consistent with the specification as a whole.

Appendix A - Protein and Nucleotide Sequences

Protein sequence of full length, wild type, human MAGEA3 (SEQ ID NO: 1):

MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAES

PDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAE L

VHFLLLKYRAREPVTKAEMLGSWGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGH LYI

FATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGR EDSIL

GDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGG P

HISYPPLHEWVLREGEE*

DNA sequence encoding full length, wild type, human MAGEA3 (SEQ ID NO: 2):

ATGCCTCTTGAGCAGAGGAGTCAGCACTGCAAGCCTGAAGAAGGCCTTGAGGCCCG AGGAGAGGCCCTGGGCCTGGTGGGTGCGCAGGCTCCTGCTACTGAGGAGCAGGAG GCTGCCTCCTCCTCTTCTACTCTAGTTGAAGTCACCCTGGGGGAGGTGCCTGCTGC CGAGTCACCAGATCCTCCCCAGAGTCCTCAGGGAGCCTCCAGCCTCCCCACTACCA T GAACTACCCT CT CT GG AGCCAAT CCTAT G AGGACT CCAGCAACCAAGAAGAGGAG GGGCCAAG CACCTTCCCTG ACCTG GAGT CCG AGTT CCAAGCAGCACT CAGT AG G AA G GTG G CCG AGTT G GTT CATTTT CT G CTCCT CAAGTAT CG AG CCAG GG AG CCG GT CA CAAAGGCAGAAATGCTGGGGAGTGTCGTCGGAAATTGGCAGTATTTCTTTCCTGTGA TCTTCAGCAAAGCTTCCAGTTCCTTGCAGCTGGTCTTTGGCATCGAGCTGATGGAAG TGGACCCCATCGGCCACTTGTACATCTTTGCCACCTGCCTGGGCCTCTCCTACGATG GCCTG CT G GGTG ACAAT CAG AT CAT G CCCAAG G CAGG CCTCCTG ATAATCGTCCTG G CCATAATCG CAAG AG AG GG CG ACTGTGCCCCTG AG G AG AAAATCTG G GAG GAGC TGAGTGTGTTAGAGGTGTTTGAGGGGAGGGAAGACAGTATCTTGGGGGATCCCAAG AAGCT GCT CACCCAACATTTCGT GCAGG AAAACTACCT GGAGTACCGGCAGGT CCC CGGCAGTGATCCTGCATGTTATGAATTCCTGTGGGGTCCAAGGGCCCTCGTTGAAA CCAG CTATGT G AAAGTCCT GCACCAT ATG GT AAAG AT C AGTG GAG G ACCT CACATTT CCT ACCCACCCCT GCAT GAGT GGGTTTT GAGAGAGGGGG AAG AGT GA

Codon optimized DNA sequence encoding full length, wild type, human MAGEA3 protein (SEQ ID NO: 3):

ATGCCCCTGGAGCAGCGGTCTCAGCATTGCAAGCCAGAGGAGGGCCTCGAGGCGA

GGGGCGAGGCCCTCGGCTTGGTGGGGGCGCAGGCTCCTGCAACCGAGGAGCAAG

AGGCCGCATCCAGTTCCTCTACCCTGGTTGAGGTGACCTTGGGTGAGGTGCCCGCC

GCGGAGAGCCCCGACCCGCCTCAAAGCCCCCAGGGTGCCAGCTCCCTGCCCACAA CAATGAACTACCCACTCTGGAGTCAGTCTTACGAGGACAGTAGTAACCAAGAGGAG

GAGGGACCCTCCACATTCCCAGACCTGGAGTCTGAATTCCAGGCAGCATTGTCTAG

AAAAGTGGCCGAATTGGTGCACTTCCTGCTGCTGAAGTATCGCGCCCGCGAGCCAG

TCACAAAAGCTGAAATGCTGGGTTCTGTCGTGGGAAATTGGCAGTACTTCTTCCCCG

T GAT CTT CAGT AAAGCGT CCAGCT CCTT GCAGCT GGT CTTTGGTAT CG AGCT GATGG

AGGTGGATCCCATCGGCCATCTGTATATCTTTGCCACATGCCTGGGCCTGAGCTAC

GATGGCCTGCTGGGCGACAACCAGATCATGCCAAAAGCTGGCCTGCTGATCATCGT

TCTGGCTATCATCGCTAGAGAAGGAGATTGCGCCCCTGAAGAAAAGATCTGGGAGG

AACTGAGCGTCCTGGAAGTCTTTGAGGGTCGTGAAGACAGCATTCTCGGGGATCCC

AAGAAGCTGCTGACCCAGCACTTCGTGCAGGAGAACTATCTGGAGTACCGCCAGGT

TCCCGGCAGCGACCCCGCTTGCTACGAGTTCCTGTGGGGCCCCAGGGCCCTGGTC

GAGACATCCTACGTGAAGGTCCTGCACCATATGGTTAAAATCAGCGGCGGCCCCCA

TATCTCTTATCCGCCGCTCCACGAGTGGGTGCTCCGGGAGGGAGAGGAG

Protein sequence of a variant of full length, wild type, human MAGEA3 (SEQ ID NO:

4):

MPLEQRSQHCKPEEGLEARGEALGLVGAQAPATEEQEAASSSSTLVEVTLGEVPAAES

PDPPQSPQGASSLPTTMNYPLWSQSYEDSSNQEEEGPSTFPDLESEFQAALSRKVAE L

VHFLLLKYRAREPVTKAEMLGSWGNWQYFFPVIFSKASSSLQLVFGIELMEVDPIGH LYI

FATCLGLSYDGLLGDNQIMPKAGLLIIVLAIIAREGDCAPEEKIWEELSVLEVFEGR EDSIL

GDPKKLLTQHFVQENYLEYRQVPGSDPACYEFLWGPRALVETSYVKVLHHMVKISGG P

HISYPPLHEWVLREGEEDYKDDDDK*

DNA sequence encoding a variant of full length, wild type, human MAGEA3 (SEQ ID NO: 5):

ATGCCCCTGGAACAGCGGAGCCAGCACTGCAAGCCCGAGGAAGGCCTGGAAGCCA

GAGGCGAAGCCCTGGGACTGGTGGGAGCCCAGGCCCCTGCCACAGAAGAACAGGA

AGCCGCCAGCAGCAGCTCCACCCTGGTGGAAGTGACCCTGGGCGAAGTGCCTGCC

GCCGAGAGCCCTGATCCCCCTCAGTCTCCTCAGGGCGCCAGCAGCCTGCCCACCA

CCATGAACTACCCCCTGTGGTCCCAGAGCTACGAGGACAGCAGCAACCAGGAAGAG

GAAGGCCCCAGCACCTTCCCCGACCTGGAAAGCGAGTTCCAGGCCGCCCTGAGCC

GGAAGGTGGCAGAGCTGGTGCACTTCCTGCTGCTGAAGTACAGAGCCCGCGAGCC

CGTGACCAAGGCCGAGATGCTGGGCAGCGTGGTGGGAAACTGGCAGTACTTCTTCC

CCGTGATCTTCTCCAAGGCCAGCAGCTCCCTGCAGCTGGTGTTCGGCATCGAGCTG

ATGGAAGTGGACCCCATCGGCCACCTGTACATCTTCGCCACCTGTCTGGGCCTGAG CTACGACGGCCTGCTGGGCGACAACCAGATCATGCCCAAGGCCGGCCTGCTGATC

ATCGTGCTGGCCATCATTGCCCGCGAGGGCGACTGCGCCCCTGAGGAAAAGATCTG

GGAGGAACTGAGCGTGCTGGAAGTGTTCGAGGGCAGAGAGGACAGCATCCTGGGC

GACCCCAAGAAGCTGCTGACCCAGCACTTCGTGCAGGAAAACTACCTGGAATACCG

CCAGGTGCCCGGCAGCGACCCCGCCTGTTACGAGTTCCTGTGGGGCCCCAGGGCT

CTGGTGGAAACCAGCTACGTGAAGGTGCTGCACCACATGGTGAAAATCAGCGGCGG

ACCCCACATCAGCTACCCCCCACTGCACGAGTGGGTGCTGAGAGAGGGCGAAGAG

G ACTACAAG G ACG ACG ACG ACAAATG A