Cancer vaccine

Abstract

To provide a nucleic acid vaccine for use in the treatment of cancer.SOLUTION: Tumor vaccines comprising nucleic acids encoding MAGED4B proteins or variants thereof are provided.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to nucleic acid-based cancer vaccines, their uses, methods for treating or preventing cancer, particularly oral cancer, and related combination therapies. The inventors have demonstrated the usefulness of nucleic acid-based cancer vaccines against several types of cancer. Specific combination therapies of interest include immunotherapy, radiation therapy, targeted therapy, and chemotherapy.

[0002] The present invention relates in particular to nucleic acid vaccines encoding at least MAGED4B protein for use in the treatment of cancer. Synergistic combinations with other anti-cancer drugs, particularly immune checkpoint inhibitors, are described. Cancer vaccines can further comprise immunologically active fragments to enhance immune responses, and additional cancer antigens, such as FJX1. [Background technology]

[0003] Although not the only cancer suitable for treatment with the vaccines described herein, one particular cancer of interest is oral cavity and oropharyngeal cancer. Oral cavity and oropharyngeal cancer (commonly referred to as head and neck cancer; HNSCC) are grouped together and are the sixth most common cancers worldwide (annual incidence rates are estimated at 275,000 cases of oral cavity cancer (OSCC) and 130,300 cases of oropharyngeal cancer (OPSCC)). In the UK, the incidence of HNSCC has increased dramatically (+92%) since the late 1970s to 7,500 cases per year; the increase in the incidence of OPSCC in the UK has been linked to human papillomavirus (HPV) infection, but the cause of the increase in OSCC incidence is unclear, and the incidence is estimated to continue to rise significantly. Patient management is often an expensive multidisciplinary approach involving surgery and / or radiation therapy followed by reconstructive surgery and rehabilitation. Treatment results in significant physical and mental morbidity and, for many patients, does not extend life. Surgery and radiation therapy remain standard treatments, but despite improvements, they are associated with significant morbidity and a relatively stable 5-year survival rate of approximately 50–60%. Immune checkpoint inhibitors against CTLA4 and PD1 / PDL1, based on enhancing preexisting antitumor immune responses, have shown efficacy across all cancer types. The recent KEYNOTE 012 trial, treating HNSCC patients with α-PD1, resulted in an 18% response rate (J. Clin. Oncol. 34, 3838–3845 (2016); British Journal of Cancer 119, 153–159 (2018)). However, most patients do not respond to checkpoint inhibitor therapy, likely due to the lack of a sufficiently strong preexisting antitumor immune response. Several companies are developing experimental approaches to treat HPV-driven HNSCC that target HPV antigens. These include peptide vaccines, mRNA vaccines, and DNA vaccines. However, most oral and oropharyngeal cancers are HPV-negative, and survival rates in this patient population are significantly lower than in patients with HPV-positive tumors. Worldwide, 6,000,000 HPV-negative HNSCC patients require treatment annually.For this disease, vaccination approaches have so far been explored only in very few cases.

[0004] Although there is an intriguing prospect for developing patient-specific vaccines based on individual tumor mutagenesis, the high cost and technical difficulties of such approaches make it unlikely that they will benefit most patients, even if successful. Identifying common tumor antigens shared among patients for the production of universal cancer vaccines would provide an inexpensive and widely available treatment for OSCC. Among different types of TAAs, cancer / testis (CT) antigens are highly promising therapeutic targets. Cellular and humoral immune responses against CT antigens are frequently observed in cancer patients, and a correlation exists between CT antigen expression and the cytolytic activity of tumor immune infiltrates. The immunogenicity and cancer specificity of CT antigens make them priority targets for cancer immunotherapy, and their therapeutic effects have been tested in various clinical settings. CT antigen vaccines are generally well tolerated, and numerous cancer vaccination trials evaluating their therapeutic efficacy are currently underway. There is a need to identify tumor antigens that can be effectively targeted for the treatment or prevention of cancers such as oral and oropharyngeal cancer.

[0005] The inventors have also identified other cancer types that may express the cancer antigens described herein or be suitable for treatment with the cancer vaccines described herein.Such cancers include head and neck cancer, oral cancer, oropharynx cancer, nasopharyngeal cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, colon cancer, kidney cancer, cholangiocarcinoma, skin melanoma, rectal cancer, thyroid cancer, bladder urothelial carcinoma, renal cancer and gastric adenocarcinoma.Those skilled in the art will further recognize the cancer types for which cancer vaccines are effective based on the mechanism of action of the vaccine.Evidence that cancer antigens are expressed in various cancer types is included in Figure 29.

[0006] The cancer antigen of interest herein is the MAGED4B antigen.It can be expressed on the surface of one or more cancer-associated cells, including tumor cells or cancer-associated cells.Another cancer antigen of interest herein is the FJX1 antigen.They are both expressed in the same cancer, for example, on the surface of one or more cancer cells or cancer-associated cells, or in the cancer microenvironment of tumors.Therefore, vaccines can contain one or both cancer antigens.

[0007] Previously, it was shown that the FJX1 protein can be broken down into small peptides by cleaving the protein so that the peptides can bind to MHC class I molecules and induce anti-cancer immune responses for MAGED4B and / or vaccination (WO2018 / 169385). These peptides are limited to those that can bind to MHC class I molecules, which imposes size restrictions, as the groove of MHC class I molecules can only tolerate peptides of 8-10 amino acids or slightly longer.

[0008] However, such peptides are thought not to activate a sufficiently sustained immune response to help patients induce a long-lasting anti-cancer response.

[0009] Because both MAGED4B and FJX1 are self-proteins, a major problem with developing a successful cancer vaccine against any one or more of these targets is that patients who require vaccination are already self-tolerized to these antigens. Furthermore, targeting only MHC class I molecules has proven insufficient to generate an immune response sufficient to confer an anti-cancer response. Such vaccine approaches do not induce the potent, balanced, and long-lasting CD4+CD8 T cell expression required for clinical efficacy. Unfortunately, despite the identification of these antigens as good targets for cancer vaccines, this technology has not, to date, produced vaccines that can be used in clinical settings.

[0010] Furthermore, by providing the vaccine as a small peptide, the vaccine functions in a very specific manner, targeting specific types of human leukocyte antigens (HLA), which can limit the usefulness of the vaccine to specific portions of the population. Thus, peptide-derived vaccines cannot provide a pan-population vaccine that is devoid of portions of the population that need it, which means that patients need to be tested to see if they are suitable.

[0011] Therapeutic cancer vaccines generally must overcome three major hurdles: low immunogenicity, pre-existing disease burden, and an immunosuppressive tumor microenvironment. Abnormally expressed autoantigens, such as MAGED4B and FJX1, face this hurdle, and high-affinity T cells that recognize these autoantigens are eliminated by central and peripheral immune tolerance mechanisms. Thus, peptide vaccines currently only sufficiently activate low-affinity T cells. However, the high doses of adjuvant drugs required to stimulate an immune response are likely to have significant side effects for patients. Therefore, there remains a long-standing unmet need for vaccines that can help eliminate tumors in patients with cancers that express MAGED4B and / or FJX1.

[0012] These problems were such that there was a significant risk that vaccines against these antigens alone would be unlikely to be effective in treating these patients. However, the inventors have unexpectedly found that it is possible to develop vaccines that produce a good immune response, generate the CD4+CD8 T cells necessary for clinical efficacy, and provide a pan-population effect, meaning that they may be of general rather than specific use. Furthermore, the inventors have noticed several additional beneficial effects when using the vaccines of the present invention, such as targeting cancer-associated cells and protecting them from the effects of other anti-cancer drugs, thereby generally making the cancer more susceptible to additional chemotherapeutic or immunotherapeutic agents. This exciting development has great benefits for the treatment of patients in need of treatment.

[0013] Furthermore, there is some evidence that the cancer vaccines described herein may act synergistically with additional anti-cancer agents, which is an exciting development in the treatment of these cancer types, as combination approaches are often clinically recommended to ensure complete cancer remission.

[0014] To obtain a successful cancer vaccine, the vaccine must enter cells and be expressed in vivo, allowing antigen presentation on major histocompatibility molecules (MHC) and T cell recognition. Co-induction of cytotoxic T cells (bearing the surface marker CD8) with helper T cells (bearing the surface marker CD4) is crucial. Both CD4+ and CD8+ T cells contain several subsets. Therefore, a DNA vaccine must fulfill many requirements before it can be considered a good clinical candidate.

[0015] Using relevant models, the present inventors have demonstrated through preclinical data that the desired immune response can be achieved using the vaccine of the present invention. In particular, the present inventors demonstrated effective tumor infiltration by CD8+ T cells after vaccination and demonstrated the presence of circulating T cells in human samples that could be stimulated by vaccine administration while also inducing the formation of newly primed T cells. The data notably demonstrated good expression and secretion of proteins from cells transfected with nucleic acid vaccines, which are appropriately processed to present their relevant epitopes to immune surveillance. Furthermore, in a mouse tumor model in which mice had palpable tumors expressing at least one human antigen, the vaccine's effect on tumor size reduction could be observed. Because tumors associated with at least MAGED4B protein are known to be particularly aggressive, these mouse model data are highly encouraging.

[0016] Thus, the vaccine of the present invention has been shown to have the ability to alter the microenvironment surrounding the tumor, making it "recognizable" to the immune system, allowing T cell infiltration and an immune response not only against the tumor itself, but also, quite unexpectedly, against cancer-associated fibroblasts, which have also been shown to express the MAGED4B protein. Thus, the vaccine according to the present invention has the ability to alter cells surrounding the tumor that act to protect such cancer cells from the immune system and anti-cancer drugs. Thus, the vaccine of the present invention has potential importance for the modulation of a wide range of cancer microenvironments, exposing tumors to the immune system and enhancing the immune response against tumors, thus greatly supporting tumor cell death.

[0017] In particular, the inventors have found that the vaccine of the present invention has minimal expression patterns in other tissues, making it highly unlikely to harm normal tissues throughout the body, thus making the vaccine clinically relevant. Thus, the present invention treats and provides protection against tumor growth.

[0018] An object of the present invention is to provide a cancer vaccine that can provide an appropriate immune response against cancer cells, particularly oral and oropharyngeal cancer cells. Summary of the Invention

[0019] The present invention provides a nucleic acid vaccine. The vaccine according to any part of the present specification can be formulated as a vaccine composition. The nucleic acid vaccine is a cancer vaccine for use in treating cancer. Various alternative vaccines are described. As used herein, any protein can be described as a cancer antigen.

[0020] Cancer vaccines can include nucleic acids encoding MAGED4B proteins or variants thereof. This can be the full-length protein, such as set forth in SEQ ID NO: 3 or SEQ ID NO: 31, but it can be truncated or modified so that sufficient amounts of protein are provided by the nucleic acid to allow the protein to be processed intracellularly and presented to the immune system. Suitable truncations are set forth in SEQ ID NOs: 35 to 37. Thus, MAGED4B proteins can be immunogenic fragments of the full-length proteins described herein. The sequence of MAGED4B proteins can be further modified to create amino acid substitutions. Such variants, modifications, and truncations are further discussed herein.

[0021] Known isoforms (variants) of MAGED4B are set forth in SEQ ID NOs: 41 to 43. In another definition, a cancer vaccine may comprise a nucleic acid encoding a MAGED4B protein, or variants and truncated versions thereof, wherein the nucleic acid sequence is as set forth in SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. Variants and truncations are as defined herein.

[0022] Optionally, the composition may also comprise a nucleic acid encoding FJX1 protein or its variant.The protein may be the full-length protein described in SEQ ID NO:4 or SEQ ID NO:33, or may be truncated or modified.Such variants, modifications and truncations are further discussed herein.

[0023] In another definition, a cancer vaccine may comprise a nucleic acid encoding an FJX1 protein, or variants and truncated versions thereof, wherein the nucleic acid sequence is as set forth in SEQ ID NO: 8, SEQ ID NO: 20, or SEQ ID NO: 21. Variants and truncations are as defined herein.

[0024] Optionally, when the vaccine comprises a nucleic acid encoding a MAGED4B protein or a variant thereof, and a nucleic acid encoding an FJX1 protein or a variant thereof, these may be provided separately, i.e., as separate nucleic acids, or on the same nucleic acid under the control of the same or different promoters, or may be present as a fusion of the two proteins.

[0025] The present invention provides a nucleic acid vaccine. The nucleic acid encodes an FJX1 protein or a variant thereof. The protein may be the full-length protein set forth in SEQ ID NO:4 or SEQ ID NO:33, or may be truncated or modified. Such variants, modifications, and truncations are further discussed herein. Optionally, the composition may also include a nucleic acid encoding a MAGED4B protein or a variant thereof. The protein may be the full-length protein set forth in SEQ ID NO:3 or SEQ ID NO:31, or may be truncated (such as those set forth in SEQ ID NOs:35-37) or modified. Such terms are as discussed herein.

[0026] The present invention provides a nucleic acid vaccine composition. The composition may comprise a nucleic acid encoding a MAGED4B protein or a variant thereof and a nucleic acid encoding an FJX1 protein or a variant thereof. The nucleic acid may encode both the MAGED4B and FJX1 proteins as a fusion protein. The composition may be two separate nucleic acid constructs encoding the MAGED4B protein or a variant thereof and the FJX1 protein or a variant thereof, respectively, for separate, simultaneous, or sequential administration to a patient in need thereof.

[0027] In another definition, a cancer vaccine may comprise a nucleic acid encoding a MAGED4B protein, or variants and truncated versions thereof, as set forth in SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26, together with a nucleic acid encoding a FJX1 protein, or variants and truncated versions thereof, as set forth in SEQ ID NO:8, SEQ ID NO:20 or SEQ ID NO:21. Variants and truncations are as defined herein. The nucleic acids may be separate or may be present on the same nucleic acid construct.

[0028] A fusion protein is a protein consisting of at least two domains encoded by separate coding sequences that are linked together so that they are transcribed and translated as a single unit to produce a single polypeptide. Thus, a nucleic acid encoding a MAGED4B protein or its variant and a nucleic acid encoding an FJX1 protein or its variant can be fused together so that they are produced as a single polypeptide upon administration. Thus, in the nucleic acid encoding the fusion protein, the fusion protein is expressed under the control of a single promoter. The order of the genes in the fusion can be in either direction, i.e., FJX1 protein or its variant followed by MAGED4B protein or its variant, or vice versa. It may be preferable for the MAGED4B protein or its variant to precede the FJX1 protein or its variant.

[0029] It may be preferable that any of the nucleic acid vaccine compositions described herein further comprise a helper motif. A helper motif can be defined as a nucleic acid sequence that directly stimulates an immune response (e.g., the DNA sequence itself can stimulate an immune response), or a nucleic acid sequence in which the motif encodes an immunogenic protein or fragment thereof. The helper motif acts to enhance the immune response to the protein encoded by the nucleic acid vaccine. Suitable helper motifs are discussed herein. One helper motif is an immunogenic fragment of tetanus toxin known as DOM.

[0030] Thus, a cancer vaccine may comprise a nucleic acid encoding a MAGED4B protein or a variant thereof together with a helper motif. The helper motif may be any suitable helper motif described herein. The helper motif may be DOM. An exemplary vaccine may comprise the sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 19, or a variant or truncation thereof. An exemplary vaccine may encode the protein sequence set forth in SEQ ID NO: 32, or a variant or truncation thereof.

[0031] Alternatively, the cancer vaccine may comprise a nucleic acid encoding the FJX1 protein or a variant thereof together with a helper motif. The helper motif may be any suitable helper motif described herein. The helper motif may be DOM. An exemplary vaccine may comprise the sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, or a variant or truncation thereof. An exemplary vaccine may encode the protein sequence set forth in SEQ ID NO: 34, or a variant or truncation thereof.

[0032] Furthermore, the cancer vaccine may comprise a nucleic acid encoding a MAGED4B protein or a variant thereof and a nucleic acid encoding an FJX1 protein or a variant thereof together with a helper motif. The helper motif may be any suitable helper motif described herein. The helper motif may be DOM. An exemplary vaccine may comprise the MAGED4B sequence set forth in SEQ ID NO: 18 or SEQ ID NO: 19, or a variant or truncation thereof. An exemplary vaccine may encode the MAGED4B protein sequence set forth in SEQ ID NO: 32, or a variant or truncation thereof. An exemplary vaccine may comprise the sequence set forth in SEQ ID NO: 22 or SEQ ID NO: 23, or a variant or truncation thereof. An exemplary vaccine may encode the protein sequence set forth in SEQ ID NO: 34, or a variant or truncation thereof.

[0033] When the cancer vaccine described herein comprises a fusion protein, a linker can be used between the coding sequences.This fusion can be between the cancer antigen MAGED4B and FJX1, or between the cancer antigen and a helper motif.Any suitable linker sequence can be used.Exemplary linker sequences are given in SEQ ID NOs: 5, 6, 10 and 11.

[0034] The nucleic acid sequence encoding the cancer antigen may contain a signal or leader sequence. Such a sequence may improve secretion of the protein from transfected cells. Exemplary leader sequences are provided in SEQ ID NOs: 1 and 27.

[0035] The nucleic acid of the cancer vaccine described herein further comprises a promoter operably linked to the coding sequence, and optionally further comprises a polyadenylation signal downstream of the coding sequence.Suitable promoters and polyadenylation signals are described herein.Suitable promoters include the sequences described in SEQ ID NO:9 and SEQ ID NO:15.

[0036] The cancer vaccines described herein can be DNA vaccines or RNA vaccines. The vaccines can also be non-naturally occurring nucleic acids as described herein.

[0037] The cancer vaccines described herein can be presented as any suitable nucleic acid construct, including a plasmid, a minicircle, a single-stranded circle, a closed linear DNA, or a single-stranded RNA. The construct can also include any other components, such as enhancers, to promote the expression of the coding sequence.

[0038] Any of these vaccines are further described below: The cancer vaccines described herein may be provided as vaccine compositions, which may contain any suitable excipients or additives, including buffers and the like, to ensure the correct pH, to stabilize the vaccine composition, to make it suitable for storage and transportation, and / or to make it suitable for administration. The cancer vaccines described herein can be used to treat cancer in animals, including humans. Optionally, the cancer vaccines are for use in treating cancer in humans. Optionally, the cancer vaccines are for use in treating cancer in non-human animals, such as domestic animals, livestock, or wild animals. Suitable animals may include cats and dogs, guinea pigs, cows, horses, sheep, rabbits, and non-human primates. Non-human primates may include chimpanzees and monkeys. Also contemplated is a method for treating cancer in animals, including humans, comprising administering the cancer vaccine described herein to a patient in need thereof. Suitable animals may include cats and dogs, guinea pigs, cows, horses, sheep, rabbits, and non-human primates. Non-human primates may include chimpanzees and monkeys. The cancer vaccines described herein may be for use in medicine, optionally for use in the treatment or prevention of cancer. The cancer vaccines described herein can be for use in exposing tumors to the immune system. The cancer vaccines described herein can be for use in sensitizing to anti-cancer drugs, optionally immunotherapies such as checkpoint inhibitors The cancer vaccines described herein may be for use in combination with anti-cancer drugs, optionally immunotherapy, such as checkpoint inhibitors, or chemotherapy. The cancer vaccines described herein target CD8 + The method may be for a method of sensitizing a tumor to infiltration by T cells, the method comprising administering a cancer vaccine to a human or animal subject. The therapeutic methods described herein may further include the use of anti-cancer drugs, and optionally immunotherapies such as checkpoint inhibitors. As used herein, a checkpoint inhibitor is an agent that can block the action of PD-1, PD-L1, PD-L2 or CTLA-4, and optionally, the agent is an antibody or an aptamer. The cancer vaccines described herein can be used in a method of increasing the effectiveness of checkpoint inhibition in a patient in need thereof, the method comprising administering a cancer vaccine described herein to a human or animal subject. The cancer vaccines described herein can be used to co-induce CD4 and CD8 T cells. The cancer vaccines, uses and methods of treatment described herein may be effective for use in the treatment of a number of cancer types, including but not limited to head and neck cancer, oral cancer, oropharyngeal cancer, nasopharyngeal cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver cancer, colon cancer, renal cancer, cholangiocarcinoma, cutaneous melanoma, rectal cancer, thyroid cancer, bladder urothelial cancer, renal cancer and gastric adenocarcinoma.

[0039] In a first aspect of the present invention, there is provided a cancer vaccine comprising a nucleic acid encoding the proteins MAGED4B and / or FJX1, or a variant thereof, and further encoding an immunogenic fragment of tetanus toxin.

[0040] Advantageously, two cancer-testis antigens, MAGED4B and FJX1, were found to be frequently expressed in OSCC. Overall, the two antigens were expressed at the RNA level in 96% of OSCC cases. Furthermore, this study confirmed protein expression of both antigens in oral dysplasia and OSCC cases (10 / 10 were positive; 5 cases in each condition), with no expression in nonmalignant oral mucosa. Protein expression tests in healthy tissues showed low expression. These expression data were similar to those obtained by investigating pre-existing immunity to the antigens in patients with non-HPV-associated HNSCC using HLA-A2 tetramers (currently available only for MAGED4B) and overlapping peptide pools (OPPs) against the full amino acid sequence of each antigen. These were measured in both blood and tumors using expanded tumor-infiltrating lymphocytes. Circulating MAGED4B tetramer-positive CD8+ T cells were observed in 5 / 7 HLA-A2 patients (0.04-0.1% of total CD8+ T cells), with 2 / 2 expanded TILs also showing similar frequencies of tetramer positivity. Higher levels of MAGED4B-positive CD8+ T cells (5-10-fold higher) were detected using OPP in HLA-A2-negative versus HLA-A1-positive TIL samples, demonstrating reactivity beyond HLA-A2 restriction. CD8+ T cell reactivity against FJX1 was assessed using OPP in expanded TIL samples, demonstrating CD8+ reactivity in HLA-A1 patients coexisting with MAGED4B+ CD8+ T cells. Patient data demonstrated strong immunogenicity of both antigens, most notably in the case of MAGED4B. DNA vaccines encoding full-length MAGED4B and FJX1 antigens (e.g., p.Dom-MAGED4BFL and p.Dom-FJX1FL described herein) have been developed, and preclinical data indicate that MAGED4B / FJX1-targeted DNA vaccines have great potential to suppress the growth of tumors expressing these antigens.

[0041] Even more advantageously, the provision of immunogenic fragments of tetanus toxin from Clostridium tetani can help induce potent CD4+ helper T cell responses, necessary for the induction of tumor-specific CD4+ and CD8+ T cell responses, via activation of dendritic cells (the so-called linked T cell mechanism). Such CD4 and CD8 epitopes are known to be able to bind to a wide range of mouse and human MHC class II molecules. DETAILED DESCRIPTION OF THE INVENTION

[0042] The cancer vaccines described herein induce antigen-specific T cell responses, particularly CD4 T cells, which are important for durable responses. + and CD8 + The vaccine can induce a T cell response, thereby eliciting an immune response against tumors expressing the antigen. The cancer vaccines described herein can additionally or alternatively affect the tumor microenvironment, increasing the immune visibility of tumors and promoting the infiltration of CD8 T cells into the tumor mass, thereby making tumor cells more susceptible to anticancer drugs, either alone or in combination with the aforementioned anticancer drugs. The vaccine effectively alters the tumor microenvironment, making tumors more susceptible to immune attack.

[0043] The lack of intratumoral T cells is a major barrier to the effectiveness of immune checkpoint inhibitors and other immunotherapies in cancer patients; this may be due to poor tumor immunogenicity (i.e., a lack of T cells) or the inability of T cells to infiltrate tumors (i.e., T cells in the wrong location). The inventors have clearly shown that the cancer vaccine described herein expands intratumoral T cells (Figures 10B and 10C). Thus, the present invention extends to methods for enhancing the effectiveness of immune checkpoint inhibition in patients in need thereof, comprising administering the cancer vaccine described herein to a human or animal subject. The cancer vaccine described herein may be used to sensitize tumors to the immune system or anticancer drugs. Here, data are presented showing that when the cancer vaccine described herein is used with an agent capable of causing checkpoint inhibition, a crucial synergistic effect occurs (Figure 9B).

[0044] The vaccines described herein generate an induced or elicited immune response, which may be a cellular immune response, and may include the induction or secretion of interferon gamma (IFNγ) and / or CD107a / b, a marker of cytotoxic T cells (Figures 21 and 22).

[0045] In particular, when the antigen in a cancer vaccine is a MAGED4B protein antigen, the cancer vaccine can further induce an antigen-specific response against cancer-associated fibroblasts (CAFs), which the inventors have shown to express this antigen (Figures 27 and 28). Human tumors contain many different cell types, in addition to cancer cells, including cancer-associated fibroblasts (CAFs), endothelial cells, immune cells, adipocytes, and pericytes, which form the microenvironment and promote cancer progression. Therefore, the ability to target such cells in the tumor microenvironment may be particularly useful for treating cancer in general and may not be limited to cancer subtypes that express MAGED4B per se.

[0046] All types of solid tumors contain CAF-rich subgroups, ranging from 95% in pancreatic cancer to approximately 50% in head and neck cancer. The proportion of CAFs in tumors is approximately 25–50% and may be higher in some cancers, such as pancreatic cancer, where most tumors are CAF-rich. In particular, CAF accumulation in cancer is associated with poor clinical outcomes; CAF-rich tumors are clinically aggressive, respond poorly to treatment, and are associated with poor survival rates. This is because CAFs promote many "hallmarks of malignancy," facilitating tumor growth, invasion, metastasis, and angiogenesis. A consistent finding across multiple cancer types is the inverse correlation between CAFs and CD8 T cells, suggesting a role for CAFs in tumor immune evasion. Recent studies, including those by the applicants, have shown that CAFs exclude CD8 T cells from tumors, thereby promoting resistance to anti-PD1 / PDL1 checkpoint immunotherapy (and vaccine-based immunotherapy; Ford et al., Cancer Res 80, 1846-1860 (2020)). Several clinical trials have identified CAF gene signatures in patients unresponsive to anti-PD1 / PDL1 therapy (Mariathasan, S. et al., Nature 554, 544-548 (2018)). CAF-mediated CD8 T cell exclusion is now recognized as a major contributor to checkpoint immunotherapy resistance, making CAFs an important immunotherapy target for improving checkpoint immunotherapy response rates (currently approximately 20% across cancer types). Suggested therapeutic possibilities for CAF targeting include CAF depletion, inhibition of CAF function, or CAF normalization; however, previous attempts to specifically target CAFs have been unsuccessful.

[0047] The fact that we showed that CAFs appear to consistently express MAGED4B is quite surprising. Expression was uniformly high across the CAF population and consistent across tumors, with 70% of analyzed HNSCCs containing MAGED4B-positive CAFs. This CAF MAGED4B expression was confirmed by analyzing scRNASeq HNSCC transcriptome data (Figure 28) (Puram et al., Cell. 2017;171(7):1611-1624, which also confirmed MAGED4B expression by HNSCC cells).

[0048] Generating an immune response that specifically targets CAFs is a very attractive therapeutic approach, and previous attempts have been made to target CAFs by vaccination, for example, vaccination against FAP (fibroblast activation protein). However, FAP is now known to be expressed by other cell types (such as multipotent bone marrow stromal cells) and is not CAF-specific. Although a CAF-specific antigen has not yet been identified, one may be provided by the cancer vaccine of the present invention; in this case, the antigen provided by the cancer vaccine is MAGED4B protein or a variant thereof.

[0049] The present invention relates to vaccines and methods useful for restoring responsiveness to other anti-cancer agents, particularly targeted therapies, immunotherapies and chemotherapy, in particular T cell-based immunotherapies including checkpoint inhibition with PD-1 inhibitors, PD-L1 inhibitors, CTLA-4 inhibitors, TIM3 inhibitors, LAG3 inhibitors, TIGIT inhibitors and the like, T cell agonists such as aCD40, aCD27, OX40, chimeric antigen receptor (CAR) T cells and vaccines.

[0050] T cell-mediated tumor elimination requires signals from the T cell receptor and costimulatory molecules that enable the effector function of tumor-antigen-specific T cells. There are also diverse immunosuppressive mechanisms within the tumor microenvironment that can suppress antitumor immunity. To overcome this suppression, the use of monoclonal antibodies, particularly agonistic Abs targeting costimulatory members of the tumor necrosis factor receptor (TNFR) family, has enhanced T cell function and led to promising therapeutic outcomes. TNFRs may be important targets for enhancing tumor-specific immune responses, and specific TNFRs include OX40, 4-1BB, and CD40.

[0051] The cancer vaccines described herein can be administered at therapeutically effective doses, alone or in combination with adjuvant cancer therapies such as chemotherapy, radiation therapy, immunotherapy, laser therapy, targeted therapy, and / or surgery (all of which are referred to herein as anti-cancer agents). The described cancer vaccines may result in beneficial effects, such as a reduction in tumor size, a slowing of the rate of tumor growth, inhibition or slowing of metastasis, sensitization of the tumor to an immune response or anti-cancer therapy, or an improvement in the overall clinical condition, without necessarily eradicating the tumor.

[0052] Particularly contemplated for combination therapy are cytostatic and cytotoxic agents that target tumor cells, agents that target angiogenesis (such as the angiogenesis inhibitors lenvatinib and sorafenib), agents that target markers specifically expressed by cancer cells (i.e., Herceptin, which targets HER2-positive cells), immunotherapeutics that target macrophages, immunotherapeutics that target T-cell checkpoint or agonist pathways, adoptive cell therapy (ACT) using T cells genetically modified to express chimeric antigen receptors (CAR T cells), T-cell receptors (TCRs), or in vitro expanded T cells, and vaccines.

[0053] The combinations described herein may further include immune checkpoint inhibitors, such as agents active against cytotoxic T lymphocyte-associated protein 4 (CTLA-4), PD-1, and PDL-1, because these may prevent suppression of elements of the immune system, such as MHC class presentation, T cell presentation and / or differentiation, and cytokine, chemokine, or signaling for immune cell proliferation and / or differentiation.

[0054] Thus, the cancer vaccines described herein may be combined with checkpoint inhibitors, such as antibodies against CTLA-4, PD-1, PD-L1 and PD-L2, to enhance stimulation of both cellular and humoral immune responses, although any suitable inhibitor may be used.

[0055] Thus, the cancer vaccines described herein may be combined with cytostatic and cytotoxic drugs that target tumor cells, agents that target angiogenesis, immunotherapeutics that target macrophages, immunotherapeutics that target T cell checkpoint or agonist pathways, adoptive cell therapy (ACT) using T cells genetically modified to express chimeric antigen receptors (CAR T cells), T cell receptors (TCRs) or in vitro expanded T cells, T cell agonists and vaccines.

[0056] MAGED4B antigen MAGED4B is part of a superfamily called MAGE (melanoma antigen-encoding genes). MAGE proteins share a conserved domain known as the MAGE homology domain (MHD).

[0057] Cancer vaccines can include nucleic acids encoding MAGED4B proteins or variants thereof. This can be the full-length protein, such as set forth in SEQ ID NO: 3 or SEQ ID NO: 31, but it can be truncated or modified so that sufficient amounts of protein are provided by the nucleic acid to allow the protein to be processed intracellularly and presented to the immune system. Suitable truncations are set forth in SEQ ID NOs: 35-37. Thus, MAGED4B proteins can be immunogenic fragments of the full-length proteins described herein. The sequence of MAGED4B proteins can be further modified to create amino acid substitutions. Such variants, modifications, and truncations are further discussed herein.

[0058] In another definition, a cancer vaccine may comprise a nucleic acid encoding a MAGED4B protein, or variants and truncated versions thereof, wherein the nucleic acid sequence is as set forth in SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25 or SEQ ID NO: 26. Variants and truncations are as defined herein.

[0059] The MAGED4B protein may comprise or consist of the full-length MAGED4B protein sequence. The MAGED4B protein may comprise or consist of the sequence of SEQ ID NO: 3 or SEQ ID NO: 31, or a variant thereof. In another embodiment, the MAGED4B protein may be encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NO: 7, SEQ ID NO: 16, SEQ ID NO: 17, SEQ ID NO: 24, SEQ ID NO: 25, or SEQ ID NO: 26, or a variant thereof.

[0060] Advantageously, full-length antigen design can achieve broader population coverage, where it is not focused on targeting individual HLA alleles, such as the HLA-A2 allele.

[0061] Advantageously, the MAGED4B protein may be truncated. Truncation may require the removal of all or part of the MAGE homology domain. Two potential regions of homology have been found in the MAGED4B protein, and because they are common across MAGE proteins, both are potential truncation regions. The two homology domains are identified at amino acids 412-500 and 510-682 of isoform 1, which is represented by SEQ ID NO: 3. This sequence is 741 amino acids long. If both homology domains are removed, this would reduce the length of MAGED4B by 260 amino acids while still providing a functional immunological fragment thereof. Thus, up to 40%, optionally up to 50%, of the full-length protein can be removed, and the remaining sequence will still be sufficiently immunogenic to serve as a vaccine. Possible truncations are shown in Figure 16. It may be preferable to retain known epitopes from MHD, such as the HLA-2-defined epitope RLSLLLVL. It is located between the two MHDs. As shown, each MHD in the vaccine was successfully removed, but any portion may be removed. Thus, a truncated version of MAGED4B may comprise an immunological fragment in which part or all of the MHD has been removed. Indeed, removal of the MHD may result in additional residues being removed. At a minimum, 450 amino acids of the MAGED4B sequence set forth in SEQ ID NO: 3 are included. Immunogenic fragments may be at least 400, 450, 500, 550, 600, 650, or 700 amino acids in length, or any length therebetween. Various truncations are detailed in SEQ ID NOs: 35-37. Other truncations are also possible. The inventors contemplate that removal of the MHD may enable the immune system to more clearly recognize MAGED4B-specific epitopes.

[0062] The study here is based on MAGED4B isoform 1 (SEQ ID NO: 3). However, four isoforms exist, encompassed by SEQ ID NOs: 41-43. Of these, isoform 3 (SEQ ID NO: 42) is an extremely rare alternative splice variant, with a frameshift at position 349 and a truncation at position 414. Due to these alternative splices and therefore truncations, SEQ ID NO: 42 has 85% sequence identity to SEQ ID NO: 3. Therefore, in another definition, truncations may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% sequence identity with the sequence set forth in SEQ ID NO: 3.

[0063] The cancer vaccines described herein may comprise a nucleic acid encoding any one of the MAGED4B proteins set forth in SEQ ID NOs: 41 to 43, which are variants of SEQ ID NO: 3.

[0064] Variants of MAGED4B may include modified and / or truncated variants of MAGED4B. In particular, those skilled in the art will understand that some modifications or variants of the sequence may result in the same or substantially similar immunogenic function as the unmodified sequence (i.e., the MAGED4B sequence described herein). The modifications may include amino acid residue additions, substitutions, or deletions. In one embodiment, the modifications may include or consist of the addition, substitution, or deletion of 20 or fewer amino acid residues. In another embodiment, the modifications may include or consist of the addition, substitution, or deletion of 15 or fewer amino acid residues. In another embodiment, the modifications may include or consist of the addition, substitution, or deletion of 10 or fewer amino acid residues. In another embodiment, the modifications may include or consist of the addition, substitution, or deletion of 8 or fewer amino acid residues. In another embodiment, the modifications may include or consist of the addition, substitution, or deletion of 6 or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of five or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of four or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of three or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of two or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of one or fewer amino acid residues. The addition, substitution, or deletion of amino acid residues may involve consecutive amino acid residues, multiple groups of amino acid residues, non-consecutive amino acid residues, or a combination thereof.

[0065] A variant of MAGED4B may comprise or consist of a sequence having at least 80% identity to SEQ ID NO:3. Alternatively, a variant of MAGED4B may comprise or consist of a sequence having at least 85% identity to SEQ ID NO:3. Alternatively, a variant of MAGED4B may comprise or consist of a sequence having at least 90% identity to SEQ ID NO:3. Alternatively, a variant of MAGED4B may comprise or consist of a sequence having at least 95% identity to SEQ ID NO:3. Alternatively, a variant of MAGED4B may comprise or consist of a sequence having at least 98% identity to SEQ ID NO:3. Alternatively, a variant of MAGED4B may comprise or consist of a sequence having at least 99% identity to SEQ ID NO:3. Alternatively, a variant of MAGED4B may comprise or consist of a sequence having at least 99.5% identity to SEQ ID NO:3.

[0066] Nucleic acid changes / modifications may include conservative substitutions of nucleotides using codon redundancy to encode the same MAGED4B protein as that encoded by SEQ ID NO:7, or a portion thereof.

[0067] Also included are nucleic acids or variants thereof disclosed in SEQ ID NO:7, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:24, SEQ ID NO:25 or SEQ ID NO:26. Variants may have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or 95% sequence identity with the sequence set forth in any one of these sequences. These variants therefore cover the above-mentioned truncations of the protein.

[0068] The nucleic acid of the cancer vaccine can be codon-optimized, such as the sequence set forth in SEQ ID NO: 17. The codon-optimized sequence can encode the same peptide sequence, but this is possible because most amino acids are encoded by more than one codon. This is done to facilitate nucleic acid synthesis or to prevent complete homology with the host genome. As shown in the data, the codon-optimized vaccine performed well. The coding sequence can be optimized for stability and high-level expression.

[0069] A variant of a nucleic acid encoding MAGED4B may comprise or consist of a sequence having at least 80% identity to SEQ ID NO:7. Alternatively, a variant of a nucleic acid encoding MAGED4B may comprise or consist of a sequence having at least 85% identity to SEQ ID NO:7. Alternatively, a variant of a nucleic acid encoding MAGED4B may comprise or consist of a sequence having at least 90% identity to SEQ ID NO:7. Alternatively, a variant of a nucleic acid encoding MAGED4B may comprise or consist of a sequence having at least 95% identity to SEQ ID NO:7. Alternatively, a variant of a nucleic acid encoding MAGED4B may comprise or consist of a sequence having at least 98% identity to SEQ ID NO:7. Alternatively, a variant of a nucleic acid encoding MAGED4B may comprise or consist of a sequence having at least 99% identity to SEQ ID NO:7. Alternatively, a variant of a nucleic acid encoding MAGED4B may comprise or consist of a sequence having at least 99.5% identity to SEQ ID NO:7.

[0070] The sequence identity may be over at least 600 contiguous nucleotides or amino acid residues. Alternatively, the sequence identity may be over at least 700 contiguous nucleotides or amino acid residues. Alternatively, the sequence identity may be over the entire MAGED4B sequence.

[0071] The sequence identity may be over at least 400 contiguous amino acids, at least 450 contiguous amino acids, at least 500 contiguous amino acids, or at least 550 contiguous amino acids.

[0072] In another embodiment, a variant of MAGED4B may comprise or consist of a truncated sequence of SEQ ID NO:3. For example, the sequence of SEQ ID NO:3 herein may be truncated and still provide immunogenicity. The truncated sequence may comprise at least 200 amino acids of the sequence of SEQ ID NO:3. The truncated sequence may comprise at least 300 amino acids of the sequence of SEQ ID NO:3. The truncated sequence may comprise at least 400 amino acids of the sequence of SEQ ID NO:3. The truncated sequence may comprise at least 500 amino acids of the sequence of SEQ ID NO:3. Alternatively, the truncated sequence may comprise at least 600 amino acids of the sequence of SEQ ID NO:3. Alternatively, the truncated sequence may comprise at least 700 amino acids of the sequence of SEQ ID NO:3. Suitable truncations are shown in Figure 16.

[0073] FJX1 antigen The cancer vaccine may comprise, or may additionally comprise, a nucleic acid encoding an FJX1 (Four-jointed box protein 1) protein or a variant thereof. The protein may be the full-length protein set forth in SEQ ID NO: 4 or SEQ ID NO: 33, or may be truncated or modified. Such variants, modifications and truncations are further discussed herein.

[0074] In another definition, a cancer vaccine may comprise, or additionally comprise, a nucleic acid encoding an FJX1 protein, wherein the nucleic acid sequence is as set forth in SEQ ID NO: 8, SEQ ID NO: 20, or SEQ ID NO: 21, or variants and truncations thereof, wherein variants and truncations are as defined herein.

[0075] The FJX1 protein may comprise or consist of the full-length FJX1 protein sequence. The FJX1 protein may comprise or consist of the sequence of SEQ ID NO: 4, or a variant thereof. In another embodiment, the FJX1 protein may be encoded by a nucleic acid comprising or consisting of the sequence of SEQ ID NO: 8, or a variant thereof.

[0076] As previously discussed, full-length antigen design can achieve broader population coverage, where it is not focused on targeting individual HLA alleles, such as the HLA-A2 allele.

[0077] Variants of FJX1 may include modified and / or truncated variants of FJX1. In particular, those skilled in the art will understand that some modifications or variants of the sequence may result in the same or substantially similar immunogenic function as the unmodified sequence (i.e., the FJX1 sequence described herein). The modifications may include additions, substitutions, or deletions of amino acid residues. In one embodiment, the modifications may include or consist of additions, substitutions, or deletions of 20 or fewer amino acid residues. In another embodiment, the modifications may include or consist of additions, substitutions, or deletions of 15 or fewer amino acid residues. In another embodiment, the modifications may include or consist of additions, substitutions, or deletions of 10 or fewer amino acid residues. In another embodiment, the modifications may include or consist of additions, substitutions, or deletions of 8 or fewer amino acid residues. In another embodiment, the modifications may include or consist of additions, substitutions, or deletions of 6 or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of five or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of four or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of three or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of two or fewer amino acid residues. In another embodiment, the modification may comprise or consist of the addition, substitution, or deletion of one or fewer amino acid residues. The addition, substitution, or deletion of amino acid residues may involve consecutive amino acid residues, multiple groups of amino acid residues, non-consecutive amino acid residues, or a combination thereof.

[0078] A variant of FJX1 may comprise or consist of a sequence having at least 80% identity to SEQ ID NO:4 or SEQ ID NO:33. Alternatively, a variant of FJX1 may comprise or consist of a sequence having at least 85% identity to SEQ ID NO:4 or SEQ ID NO:33. Alternatively, a variant of FJX1 may comprise or consist of a sequence having at least 90% identity to SEQ ID NO:4 or SEQ ID NO:33. Alternatively, a variant of FJX1 may comprise or consist of a sequence having at least 95% identity to SEQ ID NO:4 or SEQ ID NO:33. Alternatively, a variant of FJX1 may comprise or consist of a sequence having at least 98% identity to SEQ ID NO:4 or SEQ ID NO:33. Alternatively, a variant of FJX1 may comprise or consist of a sequence having at least 99% identity to SEQ ID NO:4 or SEQ ID NO:33. Alternatively, a variant of FJX1 may comprise or consist of a sequence having at least 99.5% identity to SEQ ID NO:4 or SEQ ID NO:33.

[0079] Nucleic acid changes / modifications may include conservative substitutions of nucleotides using codon redundancy to encode the same FJX1 protein, or a portion thereof, as encoded by SEQ ID NO:8 or SEQ ID NO:20 or SEQ ID NO:21.

[0080] Cancer vaccine nucleic acids can be codon-optimized, such as the sequence set forth in SEQ ID NO: 21. Codon-optimized sequences can encode the same peptide sequence, but this is possible because most amino acids are coded for by more than one codon. This is done to aid in the synthesis of the nucleic acid or to prevent complete homology with the host genome.

[0081] A variant of a nucleic acid encoding FJX1 may comprise or consist of a sequence having at least 80% identity to SEQ ID NO:8. Alternatively, a variant of a nucleic acid encoding FJX1 may comprise or consist of a sequence having at least 85% identity to SEQ ID NO:8. Alternatively, a variant of a nucleic acid encoding FJX1 may comprise or consist of a sequence having at least 90% identity to SEQ ID NO:8. Alternatively, a variant of a nucleic acid encoding FJX1 may comprise or consist of a sequence having at least 95% identity to SEQ ID NO:8. Alternatively, a variant of a nucleic acid encoding FJX1 may comprise or consist of a sequence having at least 98% identity to SEQ ID NO:8. Alternatively, a variant of a nucleic acid encoding FJX1 may comprise or consist of a sequence having at least 99% identity to SEQ ID NO:8. Alternatively, a variant of a nucleic acid encoding FJX1 may comprise or consist of a sequence having at least 99.5% identity to SEQ ID NO:8. SEQ ID NO:8 in this paragraph may be replaced by SEQ ID NO:20 or SEQ ID NO:21.

[0082] The sequence identity may be over at least 300 consecutive nucleotides or amino acid residues. Alternatively, the sequence identity may be over at least 400 consecutive nucleotides or amino acid residues. Alternatively, the sequence identity may be over the entire FJX1 sequence.

[0083] In another embodiment, the variant of FJX1 may comprise or consist of a truncated sequence of SEQ ID NO: 4. For example, the sequence of SEQ ID NO: 4 herein may be truncated and still provide immunogenicity. The truncated sequence may comprise at least 200 amino acids of the sequence of SEQ ID NO: 4. The truncated sequence may comprise at least 300 amino acids of the sequence of SEQ ID NO: 4. The truncated sequence may comprise at least 400 amino acids of the sequence of SEQ ID NO: 4. Thus, immunogenic fragments of FJX1 according to any of the sequences disclosed herein are also encompassed.

[0084] Combination of MAGED4B and FJX1 In one embodiment, the nucleic acid encodes both MAGED4B and FJX1, or variants thereof. In one embodiment, the nucleic acid encodes both MAGED4B and FJX1, or variants thereof, and a linker therebetween.

[0085] The MAGED4B and FJX1 antigens may be encoded as a single fusion protein or may be encoded for separate expression. MAGED4B may be encoded N-terminal to FJX1.

[0086] Combinations can extend to a single transcription unit, such as MAGED4B-2A peptide-FJX1. 2A self-cleaving peptides, or 2A peptides, are a class of peptides 18-22 amino acids long that can cause ribosomal skipping during protein translation in cells. These peptides share the core sequence motif of DxExNPGP and are found in a wide range of virus families. They aid in polyprotein production by preventing peptide bond formation.

[0087] Helper Motif The cancer vaccines described herein can have nucleic acids that provide helper motifs. As used herein, a helper motif is a motif that enhances or improves the immunogenicity of a cancer vaccine. This can be defined as an adjuvant, a helper epitope, an immunogenic fragment, or a cytokine. Inflammatory signals upon cytosolic nucleic acid recognition can themselves enhance immunogenicity through activation of key pro-inflammatory pathways, which can be further stimulated if nucleic acid vaccines contain helper motifs, such as CpG motifs. Unmethylated CpG motifs can themselves have immunostimulatory effects through stimulation of the innate immune system via Toll-like receptor (TLR) 9. Thus, a helper motif can be a nucleic acid motif known to stimulate the immune system without the need for expression.

[0088] The helper motif can encode a protein or polypeptide, or an immunogenic fragment thereof that stimulates the immune system. Thus, the helper motif is expressed in cells. The helper motif can be present in the same construct as the cancer vaccine antigen, or can be provided as a separate construct. Advantageously, both can be provided together on the same nucleic acid construct. When both are provided together, they can be under the control of the same or different promoters. Advantageously, they can be provided as a transcriptional or translational fusion. A transcriptional fusion allows the coding sequences to be linked but does not produce a hybrid or fusion protein. A translational fusion can also be provided, in which case the product of expression is a hybrid protein. This may be preferable for vaccination. The linker molecules described herein can be used to link the cancer antigen and the helper motif together by gene fusion and polypeptide fusion.

[0089] For example, the nucleic acid sequence encoding cytokine or chemokine can also be delivered directly to the construct or a separate construct together with the nucleic acid vaccine. This allows cytokine or chemokine to appear at the same time and in the same area as the cancer vaccine. Suitable nucleic acids encode cytokines or chemokines such as interleukin (IL)-10, IL-12, dendritic cell targeting chemokine MIP3α, or IFNγ, or those further discussed below. Thus, the helper motif can be a nucleic acid encoding cytokine.

[0090] Additionally, the nucleic acid sequence can encode an immune system stimulator, which can also be delivered directly with the vaccine, including sequences containing trafficking signals (e.g., MHC class I trafficking signals (MITDs)), etc.

[0091] Alternatively, the helper motif may provide immune stimulation by other means, such as through the formation of particles. PVXCP encodes the potato virus X coat protein, which provides immune stimulation by a binding T cell mechanism that also serves DOM, allowing the formation of particles that enhance the immunogenicity of the expressed polypeptide.

[0092] As a further example, a helper motif may encode a protein, polypeptide, or immunogenic fragment thereof known to induce a strong immune response. As detailed herein, this includes the immunogenic fragment of tetanus toxin, particularly DOM. Other suitable helper epitopes may be derived from the B subunit of Escherichia coli heat-labile toxin, fragment C of tetanus toxin, diphtheria toxin B subunit, E. coli labile toxin fragment, cholera toxin fragment, OVA peptide and / or calreticulin, HIV protein NEF (negative regulatory factor), HBV surface antigen, and the promiscuous CD4 epitope PADRE.

[0093] Examples are presented herein in which various helper motifs, such as CpG motifs, MITD, PVXCP and MIP3α, are used in combination with cancer vaccines.

[0094] Suitable helper motifs include nucleic acid sequences encoding Flt3L, Flt3L-Fc fusion, CD80, CD80-Fc fusion, OX40L, IL-15, 4-1BBL, GM-CSF, CCL21a, IL-23, CCL27, CXCL10, CCL5, CCL3, LAG3, IL-15RA, CXCL10, CpG, E. coli labile toxin fragment, cholera toxin fragment, calreticulin, HIV NEF, HBV sAg, PADRE, IRF1, CCL25, IL-33, and / or IL-28B.

[0095] Particular results were obtained when the helper motif was an immunogenic fragment of tetanus toxin as described herein.

[0096] Immunogenic fragments of tetanus toxin DOM The immunogenic fragment of tetanus toxin may not have the toxic function of full-length tetanus toxin. In one embodiment, the immunogenic fragment of tetanus toxin may not include the neuron-binding domain or may not include a functional binding domain. In one embodiment, the immunogenic fragment of tetanus toxin may comprise or consist of the p30 MHC II epitope of tetanus toxin. In one embodiment, the immunogenic fragment of tetanus toxin comprises or consists of DOM. DOM may comprise or consist of the sequence of SEQ ID NO: 2 or a variant thereof.

[0097] DOM is an immunogenic fragment of tetanus toxin from Clostridium tetani (1), which is safe for human use because it does not contain the neuron-binding domain responsible for spastic paralysis. Advantageously, DOM contains the "promiscuous" p30 MHC II epitope and, potentially, other CD4 T cell epitopes. p30 can bind to a range of mouse and human MHC class II molecules and, via dendritic cell activation, induce strong CD4+ helper T cell responses necessary for the induction of tumor-specific CD4 and CD8 T cell responses (the so-called linked T cell mechanism) (2, 3). Furthermore, DOM possesses several weak CD8 epitopes that cannot compete with cancer epitopes through the process of immunodominance, providing an additional advantage for inclusion in DNA vaccines according to the present invention intended to induce strong T cell responses against cancer antigens.

[0098] In one embodiment, reference herein to DOM may alternatively be replaced by another immunogenic fragment of tetanus toxin, particularly reference herein to DOM may be replaced by the p30 or p2 epitopes of tetanus toxin, or other tetanus toxin CD4 helper epitopes, either alone or in combination.

[0099] DOM may be present as a helper motif with any of the cancer vaccines described herein. It may also be described as a helper epitope because it contains epitopes that may help induce a strong immune response. The data presented here support this (Figure 11A). The various assemblies of DNA vaccines are shown in FIG.

[0100] Combination of DOM, MAGED4B and FJX1 In one embodiment, the nucleic acid encodes DOM together with MAGED4B and / or FJX1, or variants thereof. In another embodiment, the nucleic acid encodes DOM together with MAGED4B and FJX1, or variants thereof.

[0101] In one embodiment, DOM, MAGED4B and FJX1 are encoded as a single fusion protein. In another embodiment, DOM and one of MAGED4B or FJX1 are encoded as a single fusion protein. DOM may be encoded N-terminal to MAGED4B and / or FJX1. Various assemblies of DNA vaccines are shown in Figure 7. Any suitable assembly containing a single transcription unit such as MAGED4B-2A peptide-FJX1 can be used.

[0102] Other elements The following section applies whether a cancer vaccine containing a single antigen or both antigens is used. In one embodiment, linker residues may be provided between one or more, or all, of the DOM, MAGED4B, and FJX1 antigens. The linker residues may comprise random amino acid sequences or amino acids selected to be non-immunogenic based on epitope prediction computer programs or experiments in animal models. For example, a linker may not be considered if it is not predicted or known to be an epitope (i.e., to avoid an immune response to an epitope, e.g., an artificial epitope or an epitope not found in nature). The linker may be flexible. The linker may comprise or consist of K, G, P, or S amino acid residues, or a combination thereof. In one embodiment, the linker may comprise or consist of P and / or P amino acid residues. The linker residue may be 1 to 10 amino acids in length. In another embodiment, the linker residue may be 2 to 8 amino acids in length. In another embodiment, the linker residue may be 1 to 7 amino acids in length.

[0103] The MAGED4B and FJX1 fusion protein may include a linker between the MAGED4B and FJX1 sequences. The linker between MAGED4B and FJX1 may comprise, or consist of, about 1 to about 10 amino acids. In another embodiment, the linker between MAGED4B and FJX1 may comprise, or consist of, about 1 to about 6 amino acids. In another embodiment, the linker between MAGED4B and FJX1 may comprise, or consist of, about 1 to about 5 amino acids. In another embodiment, the linker between MAGED4B and FJX1 may comprise, or consist of, about 2 to about 6 amino acids. In another embodiment, the linker between MAGED4B and FJX1 may comprise, or consist of, about 2 to about 5 amino acids. In another embodiment, the linker between MAGED4B and FJX1 may comprise, or consist of, about 3 to about 5 amino acids. In another embodiment, the linker between MAGED4B and FJX1 may comprise or consist of about 4 to about 6 amino acids, hi another embodiment, the linker between MAGED4B and FJX1 may comprise or consist of about 5 amino acids.

[0104] The linker between MAGED4B and FJX1 may comprise or consist of 3 to 5 amino acids selected from G, S, T, and A. The linker between MAGED4B and FJX1 may comprise or consist of G and / or S residues. In one embodiment, the linker between MAGED4B and FJX1 may comprise or consist of alternating G and / or S residues. In one embodiment, the linker between MAGED4B and FJX1 may comprise or consist of GSGSG (SEQ ID NO: 6 / Linker 2). Another linker may comprise or consist of GGGGG (SEQ ID NO: 10) or SSSSS (SEQ ID NO: 11). Glycine and serine amino acids, when included in the linker, are flexible, providing flexibility to the protein for efficient expression.

[0105] The advantage of such linkers is that they are not significantly immunogenic and minimize spurious epitopes (we predicted the absence of junction peptides using an MHC I prediction algorithm). The flexibility of the linker will not significantly affect the structure of the antigen, allowing for efficient translation and proteasomal processing for presentation by the MHC.

[0106] In certain embodiments encoding a DOM together with a MAGED4B and / or FJX1 fusion protein, the fusion protein may include a linker between the sequence of DOM and the sequence of MAGED4B and / or FJX1. The linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise, or consist of, about 1 to about 10 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise, or consist of, about 1 to about 8 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise, or consist of, about 2 to about 8 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise, or consist of, about 4 to about 8 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise or consist of about 5 to about 8 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise or consist of about 6 to about 8 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise or consist of about 7 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise or consist of about 8 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise or consist of about 9 amino acids. In another embodiment, the linker between the DOM and the MAGED4B and / or FJX1 sequence may comprise or consist of about 10 amino acids.

[0107] In one embodiment, the linker between DOM and MAGED4B and / or FJX1 may comprise or consist of AAAGPGP (SEQ ID NO: 5 / Linker 1). The linker between DOM and MAGED4B and / or FJX1 may comprise or consist of 3 to 10 amino acids selected from G, S, T, and A. Alternatively, the linker between DOM and MAGED4B and / or FJX1 may comprise or consist of 3 to 5 amino acids selected from G, S, T, and A. In one embodiment, the linker between DOM and MAGED4B and / or FJX1 may comprise or consist of alternating G and / or S residues. In one embodiment, the linker between DOM and MAGED4B and / or FJX1 may comprise or consist of GSGSG (SEQ ID NO: 6 / Linker 2). Alternative linkers may comprise or consist of GGGGG (SEQ ID NO: 10) or SSSSS (SEQ ID NO: 11).

[0108] Advantageously, the linker AAAGPGP (SEQ ID NO: 5 / Linker 1) minimizes the occurrence of spurious epitopes and inserts a restriction enzyme Not I site to aid cloning. In particular, an MHC I prediction algorithm was utilized to predict the absence of junction peptides. Furthermore, the linker is flexible and will not significantly affect the structure of the antigen, allowing for efficient translation and proteasomal processing for presentation by the MHC.

[0109] The nucleic acid may further encode a leader sequence, such as a signal peptide, to enhance secretion efficiency. The leader sequence may comprise or consist of an IgH signal peptide, such as the mus IgH signal peptide, or an orthologue thereof. The leader sequence may comprise or consist of the sequence MGWSCIIFFLVATATGVHS (SEQ ID NO: 1), or a functional variant thereof. The leader sequence may be N-terminal to the DOM sequence and form part of the fusion peptide therewith.

[0110] The nucleic acid may comprise one or more promoters. The promoter may comprise a eukaryotic promoter, and the nucleic acid may optionally further comprise a prokaryotic promoter, such as T7. In one embodiment, the promoter is a dual eukaryotic / prokaryotic promoter. The promoter may be a strong promoter. In one embodiment, the promoter is a viral promoter. The promoter may be selected from the group consisting of simian virus 40 early promoter (SV40), cytomegalovirus immediate early promoter (CMV), T7, human ubiquitin C promoter (UBC), human elongation factor 1 alpha promoter (EF1A), mouse phosphoglycerate kinase 1 promoter (PGK), and chicken β-actin promoter associated with a CMV early enhancer (CAGG). In one embodiment, the promoter comprises CMV. In one embodiment, the promoter comprises a CMV / T7 dual promoter, e.g., corresponding to SEQ ID NO: 9. In one embodiment, the promoter comprises a CMV / T7 dual promoter, e.g., comprising or consisting of SEQ ID NO: 9, or a functional variant thereof. A variant of SEQ ID NO:9 may have at least 80%, 85%, 90%, 95%, 98% or 99% identity to SEQ ID NO:9.

[0111] The promoter can be encoded N-terminal to the antigenic protein (e.g., DOM, MAGED4B, and / or FJX1) being expressed. This will be understood to mean that the promoter is upstream of the coding sequence. The promoter need only be operably linked to the coding sequence.

[0112] In certain embodiments, MAGED4B and FJX1 are expressed as separate polypeptides and the nucleic acid may include a promoter for each polypeptide. A single promoter may be used for one or more, or all, of the expressed antigens.

[0113] The nucleic acid may encode a polyA transcription termination sequence. In one embodiment, the polyA transcription termination sequence is a mammalian terminator containing the sequence motif AAUAAA, which promotes both polyadenylation and termination. The mammalian terminator may be any one of SV40, hGH, BGH, and rbGlob. In one embodiment, the polyA transcription termination sequence is a bovine growth hormone (BGH) polyA transcription termination sequence. The polyadenylation signal may be downstream of the cancer antigen coding sequence. The polyadenylation signal may be an LTR polyadenylation signal, a polyadenylation signal, a human growth hormone (hGH) polyadenylation signal, or a human β-globin polyadenylation signal. The SV40 polyadenylation signal may be a polyadenylation signal from a pCEP4 vector (Invitrogen, San Diego, CA).

[0114] In one embodiment, the nucleic acid is a DNA encoding: a single fusion polypeptide comprising the DOM antigen, the full-length MAGED4B antigen, and the full-length FJX1 antigen, with encoded linker residues between each antigen; the N-terminal CMV / T7 promoter; and C-terminal poly(A) sequence.

[0115] In another embodiment, the nucleic acid is a DNA encoding: a single fusion peptide comprising the DOM antigen and one of the full-length MAGED4B antigen or the full-length FJX1 antigen, with coded linker residues between each antigen; the N-terminal CMV / T7 promoter; and C-terminal poly(A) sequence.

[0116] Ideally, the CMV / T7 promoter may be replaced with a CMV promoter. The nucleic acid can be DNA encoding: a single fusion polypeptide comprising a helper motif and a MAGED4B antigen; an operably linked promoter; and Poly A signal sequence.

[0117] The nucleic acid can be DNA encoding: a single fusion polypeptide comprising the DOM antigen and the MAGED4B antigen; an operably linked promoter; and Poly A signal sequence.

[0118] The nucleic acid may comprise a sequence encoding SEQ ID NOs: 2-4 as described herein, or a variant thereof. In another embodiment, the nucleic acid may comprise a sequence encoding SEQ ID NOs: 1-4 as described herein, or a variant thereof. In another embodiment, the nucleic acid may comprise a sequence encoding SEQ ID NOs: 2-6 as described herein, or a variant thereof. In another embodiment, the nucleic acid may comprise a sequence encoding SEQ ID NOs: 1-6 as described herein, or a variant thereof.

[0119] The nucleic acid encoding MAGED4B and / or FJX1 may be provided in a backbone vector suitable for delivery and expression in vivo. In one embodiment, the backbone vector comprises the pcDNA3.0 vector.

[0120] Any suitable nucleic acid construct can be used in vaccine.For DNA vaccine, DNA can be in the form of plasmid, minicircle, single-stranded circle or closed linear DNA.Closed linear DNA is preferred because it may not contain bacterial sequence and is the minimum vector designed for use in DNA vaccine etc.

[0121] In one embodiment, the nucleic acid may comprise or consist of any one of the vectors selected from pDOM MAGED4B-FJX1; pDOM MAGED4B, and pDOM FJX1 described herein.

[0122] In one embodiment, the nucleic acid may comprise or consist of any one of the constructs selected from DB MAGED4B-FJX1; DB MAGED4B, and DB FJX1. These may further comprise a DOM. Various configurations of these closed linear DNA structures were tested; these are shown in Figure 18.

[0123] In one embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 12. In another embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 13. In another embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 14. In one embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 16. In another embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 17. In another embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 18. In one embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 19. In another embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 20. In another embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 21. In another embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 22. In another embodiment, the nucleic acid may comprise or consist of the sequence of SEQ ID NO: 23.

[0124] Nucleic acids can comprise or consist of DNA. Nucleic acids can comprise or consist of RNA, such as mRNA or self-replicating RNA. As discussed herein, artificial nucleic acids are also contemplated.

[0125] The nucleic acid can be linear or circular, for example, in a plasmid. The nucleic acid can comprise the sequence of a mammalian expression vector, such as the pcDNA3.0 vector or its equivalent. Those skilled in the art will understand that any suitable mammalian expression vector can be used to insert a nucleic acid according to the invention described herein. It may be preferable for the vector to be a closed linear DNA.

[0126] Cancer vaccine can comprise a composition.For example, cancer vaccine can be provided in the form of pharmaceutically acceptable nucleic acid.MAGED4B and / or FJX1 antigen can be encoded by separate nucleic acid, such as vector, in the same composition.

[0127] Additional definitions As used herein, "coding sequence" or "encoding" can refer to a nucleic acid (RNA or DNA molecule) comprising a nucleotide sequence that encodes a protein or fragment thereof. The coding sequence can further comprise initiation and termination signals operably linked to regulatory elements, including a promoter and polyadenylation signal, capable of directing expression in the cells of an individual or mammal to which the nucleic acid is administered.

[0128] As used herein, "fragment" or "immunological fragment" with respect to a protein can refer to a protein or polypeptide portion thereof that is capable of eliciting an immune response in a mammal that cross-reacts with an antigen disclosed herein. The fragment can be a polypeptide fragment selected from at least one of the various amino acid sequences described herein. A fragment of a protein can comprise at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, or at least 95% of the protein.

[0129] As used herein, "identity" in the context of a nucleic acid or protein / polypeptide sequence means that the sequence has a specified percentage of the same residues as a reference sequence over a specified region. The percentage can be calculated by aligning the two sequences as appropriate, comparing the two sequences over a specified region, determining the number of positions where identical residues occur in both sequences to obtain the number of matching positions, dividing the number of matching positions by the total number of positions in the specified region, and multiplying the result by 100 to obtain the percentage of sequence identity. Optionally, when a sequence is referenced herein, sequences with at least 50% identity, 55%, 60%, 65%, 70%, 75%, 80% sequence identity, 85%, 90%, 93%, 95%, 97%, 98%, or 99% sequence identity are also encompassed.

[0130] As used herein, nucleic acids can be single-stranded or double-stranded, or can contain portions of both double-stranded and single-stranded sequences. Nucleic acids can include DNA (including cDNA), RNA, or hybrids thereof. Nucleic acids can include combinations of deoxyribonucleotides and ribonucleotides, as well as combinations of bases, including natural bases (uracil, adenine, thymine, cytosine, guanine) and unnatural bases (e.g., inosine, xanthine hypoxanthine, isocytosine, and isoguanine). Nucleic acids can be composed of any nucleotide. These nucleotides can be natural, modified, or artificial. Nucleotides can be polymerized to form RNA, DNA, locked nucleic acids (LNA), peptide nucleic acids (PNA), morpholino nucleic acids, glycol nucleic acids (GNA), threose nucleic acids (TNA), hybrids, and mixtures thereof, as well as any other artificial (xeno) nucleic acids. It may be preferable for the polynucleotide to be DNA or a modified version thereof (i.e., having modifications in the backbone, sugar residues, or nucleobases). ModRNA is considered to be a suitable nucleic acid for forming a vaccine.

[0131] The nucleic acid may be in any suitable format and may include any additional sequences or elements that may be required. The nucleic acid may be a plasmid (double-stranded circular), a minicircle, a closed linear DNA, a single-stranded circular DNA, or any other nucleic acid construct suitable for delivering a vaccine to cells. The nucleic acid may be RNA, for example, mRNA (messenger RNA), a self-replicating RNA, or a non-replicating mRNA, such as one suitable for in vitro gene transfer into dendritic cells.

[0132] As used herein, "operably linked" can mean that expression of a coding sequence is under the control of a promoter to which it is spatially linked. The promoter can be positioned 5' (upstream) or 3' (downstream) of the coding sequence under its control.

[0133] As used herein, "promoter" refers to a synthetic or naturally occurring sequence capable of conferring, activating, or enhancing expression of a coding sequence in a cell. Promoters can be derived from sources including viral, bacterial, fungal, plant, insect, and animal. Promoters can constitutively or differentially regulate expression of genetic components relative to the cell, tissue, or organ in which expression occurs, or in response to external stimuli such as inducers. Examples of suitable promoters include the lac operator promoter, the SV40 late promoter, the SV40 early promoter, the RSV-LTR promoter, the CMV promoter, or the SV40 promoter (e.g., pcDNA3.1, pVAX1, pVIVO2, pCI, pCMV, and pSV2).

[0134] The terms "signal peptide" and "leader sequence" are used interchangeably herein to refer to an amino acid sequence that can be attached to the N-terminus of a protein described herein. A signal peptide / leader sequence typically directs the localization of a protein. As used herein, a signal peptide / leader sequence preferably facilitates the secretion of a protein from the cell in which it is produced.

[0135] As used herein, "immune response" refers to the activation of an immune system, e.g., a mammalian immune system, in response to the introduction of an antigen. The immune response can be in the form of a cellular or humoral response, or both. Preferably, the immune response elicited is a CD4+ and CD8+ T cell response.

[0136] As used herein, "treatment" or "treating" can mean protecting an animal (including a human) from disease by means of preventing, suppressing, repressing, or completely eliminating the disease. Preventing disease involves administering a vaccine of the present invention to an animal (e.g., a human) prior to the onset of the disease. Suppressing disease involves administering a vaccine of the present invention to an animal (human) after the induction of the disease but prior to clinical appearance. Preventing disease involves administering a vaccine of the present invention to an animal (human) after the appearance of clinical symptoms.

[0137] The cancer may be any cancer including, but not limited to, head and neck cancer, oral cancer, oropharyngeal cancer, lung cancer, breast cancer, esophageal cancer, nasopharyngeal cancer, gastric cancer, liver cancer, colon cancer, kidney cancer, cholangiocarcinoma, cutaneous melanoma, rectal cancer, thyroid cancer, bladder urothelial carcinoma, renal cancer, and gastric adenocarcinoma.

[0138] Cancer vaccines can be used to prevent malignant or cancerous cells from developing. In some cases, cellular changes can be detected before cancer develops, and cancer vaccines are administered or used to prevent cancer development. Thus, cancer vaccines can be used to treat precancerous cells. Furthermore, cells may progress through several phenotypes before acquiring a cancerous phenotype. Thus, cancer prevention can include the treatment of precancerous but aggressive cell types. For example, in oral cancer, cells can progress through several phenotypes, including, but not limited to, oral precancerous lesions (OPML): leukoplakia, erythroplakia, lichen planus, and oral epithelial dysplasia. As a further example, some lung cancers can progress through several phenotypes, including, but not limited to, squamous dysplasia, atypical adenomatous hyperplasia, and / or carcinoma in situ (CIS).

[0139] Cancer vaccines can prevent tumor growth. Cancer vaccines can reduce tumor growth. Vaccines can prevent tumor cell metastasis. Cancer vaccines can reduce immune evasion of tumor cells. The cancer antigen of the vaccine induces or elicits an immune response directed against or reactive to the cancer or tumor expressing the antigen. The induced or elicited cellular immune response can include induction or secretion of interferon gamma (IFNγ), tumor necrosis factor alpha (TNFα), and / or formation of cytolytic granules containing perforin / granzymes. In other embodiments, the induced or elicited immune response can reduce or inhibit one or more immunosuppressive factors that promote the growth of the tumor or cancer expressing that antigen, including, but not limited to, factors that downregulate MHC presentation, factors that upregulate antigen-specific regulatory T cells (Tregs), cytokines such as PD-L1, FasL, IL-10 and TFGβ, tumor-associated macrophages, cancer-associated fibroblasts, immune suppressor cells, CTLA-4, PD-1, MDSC, MCP-1, and soluble factors produced by immune checkpoint molecules.

[0140] The vaccine can enhance the cellular immune response of a subject administered the vaccine by about 50 to about 6,000 times, about 50 to about 5,500 times, about 50 to about 5,000 times, about 50 to about 4,500 times, about 100 to about 6,000 times, about 150 to about 6,000 times, about 200 to about 6,000 times, about 250 to about 6,000 times, or about 300 to about 6,000 times compared to the cellular immune response of a subject not administered the vaccine.

[0141] The vaccine can increase interferon gamma (IFNγ) levels in a subject administered the vaccine by about 50 to about 6,000 times, about 50 to about 5,500 times, about 50 to about 5,000 times, about 50 to about 4,500 times, about 100 to about 6,000 times, about 150 to about 6,000 times, about 200 to about 6,000 times, about 250 to about 6,000 times, or about 300 to about 6,000 times compared to IFNγ levels in a subject not administered the vaccine.

[0142] The cancer vaccine can be DNA. The DNA vaccine can further include elements or reagents that prevent its integration into a chromosome. The cancer vaccine can be RNA of one or more cancer antigens.

[0143] The vaccines of the present invention may possess the characteristics necessary for an effective vaccine, such as being safe so that the vaccine itself does not cause damage, disease, or death to normal cells; being protective against disease; inducing a protective T cell response; and being easy to administer, having few side effects, biological stability, and allowing for a low cost per dose, especially when closed linear DNA is used.

[0144] Cancer vaccines may further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., immune checkpoint inhibitors or "checkpoint inhibitors"). Immune checkpoint inhibitors are any nucleic acids or proteins that prevent the suppression of any component of the immune system, such as MHC class presentation, T cell presentation and / or differentiation, B cell presentation and / or differentiation, any cytokine, chemokine, or signal transduction for immune cell proliferation and / or differentiation. Cancer vaccines may further be combined with checkpoint inhibitors, such as CTLA-4, PD-1, and PDL-1, along with antibodies, to enhance stimulation of cellular and immune responses. The use of anti-PD-1 or anti-PDL-1 antibodies prevents T cell suppression and response suppression by PD-1 or PDL-1.

[0145] Combination therapy The cancer vaccine may be used as a vaccine in combination with another therapeutically or prophylactically active ingredient. The cancer vaccine may be used as a vaccine in combination with an adjuvant. The therapeutically or prophylactically active agent may be any anti-cancer agent. Suitable anti-cancer agents include chemotherapeutic agents including, but not limited to, alkylating agents, mustard gas derivatives (mechlorethamine, cyclophosphamide, chlorambucil, melphalan, and ifosfamide), ethyleneimines, alkylsulfonates, hydrazines and triazines, nitrosoureas, metal salts (such as carboplatin, cisplatin, and oxaliplatin), plant alkaloids, vinca alkaloids, taxanes, podophyllotoxins, camptothecin analogs, antitumor antibiotics, anthracyclines, chromomycins, mitomycins, bleomycins, antimetabolites, folate antagonists, pyrimidine antagonists, purine antagonists, adenosine deaminase inhibitors, and topoisomerase (I and II) inhibitors.

[0146] Suitable anti-cancer agents include, but are not limited to, immune checkpoint inhibitors, T cell transfer therapy agents, adoptive immune cell therapy agents, or immunotherapeutics, including immune cell therapy agents, antibody therapy agents, vaccines, or immune system modulators. Also contemplated are immunotherapies that target T cell checkpoint or agonist pathways, adoptive immune cell therapy (ACT) using T cells genetically modified to express chimeric antigen receptors (CAR T cells), T cell receptors (TCR), or in vitro expanded T cells. Suitable anti-cancer agents may include radiation therapy or radiomimetic agents, targeted therapy, surgery or laser therapy. Suitable anti-cancer agents include targeted therapy.This type of therapy depends on the cancer to be treated, and may require the analysis of which gene is expressed by cancer cell, or may involve the reexamination of which gene mutation is the basis of mutagenesis.Some targeted therapy is described herein, and includes the cytostatic and cytotoxic drugs that target tumor cells, the drugs that target angiogenesis (such as the angiogenesis inhibitors lenvatinib and sorafenib), the drugs that target the markers that cancer cells specifically express (i.e., Herceptin that targets HER2-positive cells), the therapeutic agents that target macrophages, the therapeutic agents that target T cell checkpoint or agonist pathway, etc.

[0147] Additionally or alternatively, the cancer vaccine according to the present invention can be used alone or in combination with a checkpoint inhibitor to prevent or treat cancer. The checkpoint inhibitor can include an anti-PD1 binding molecule. The binding molecule includes an anti-PD1 antibody or a fragment thereof. In another embodiment, the checkpoint inhibitor can include an anti-CTLA4 binding molecule.

[0148] Advantageously, preclinical data demonstrate herein that DNA vaccines targeting MAGED4B / FJX1 and having great potential to suppress the growth of tumors expressing these antigens can be further enhanced by combining them with checkpoint inhibitors such as anti-PD1 or anti-CTLA4 binding molecules.

[0149] The anti-PD1 binding molecule may comprise an antibody or antibody variant, such as an antibody fragment or antibody mimic. The antibody or variant may be monoclonal. In one embodiment, the antibody or variant may comprise or consist of nivolumab, or an antibody or variant that competes with nivolumab for binding. In one embodiment, the antibody or variant may comprise the six heavy and light chain CDRs of nivolumab. In an alternative embodiment, the antibody or variant may comprise the variable heavy and light chain sequences of nivolumab.

[0150] The anti-CTLA4 binding molecule may comprise an antibody or antibody variant, such as an antibody fragment or antibody mimic. The antibody or variant may be monoclonal. In one embodiment, the antibody or variant may comprise or consist of ipilimumab, or an antibody or variant that competes with ipilimumab for binding. In one embodiment, the antibody or variant may comprise the six heavy and light chain CDRs of ipilimumab. In an alternative embodiment, the antibody or variant may comprise the variable heavy and light chain sequences of ipilimumab.

[0151] In one embodiment, a checkpoint inhibitor, such as an anti-PD1 or anti-CTLA4 binding molecule, can be administered by providing / administering a nucleic acid encoding the checkpoint inhibitor for in vivo expression. The checkpoint inhibitor can be encoded by a plasmid. The checkpoint inhibitor can include, for example, a DNA-encoded monoclonal antibody (DMAb), as described in Perales-Puchalt et al. (Oncotarget. 2019 Jan 1;10(1):13-16. doi:10.18632 / oncotarget.26535), which is incorporated herein by reference.

[0152] DNA-encoded monoclonal antibodies (DMAbs) can help overcome the recurring challenges of antibody production, stability, frequent high-dose requirements, and prolonged intravenous administration. Artificially engineered DMAbs can simplify the design and implementation of MAb-based therapies. DMAbs delivered via plasmid DNA injection and electroporation have been used in preclinical models for the treatment and prevention of infectious diseases, cancer, and cardiovascular disease. Perales-Puchalt et al. (Oncotarget. 2019 Jan 1;10(1):13-16. doi:10.18632 / oncotarget.26535) and Duperret EK et al. (Cancer Res. 2018;78:6363-70), both incorporated herein by reference, reported that immune checkpoint blockade drugs can be optimized and delivered in vivo. Further advances in DMAb technology include the optimization, expression, and in vivo functional characterization of anti-CTLA4 and anti-PD1 antibodies.

[0153] The use may be in the form of a mixed preparation. In another embodiment, the use may be simultaneous or sequential administration (e.g., separately formulated but administered together). In one embodiment, the vaccine according to the present invention may be administered before the checkpoint inhibitor.

[0154] Another aspect of the invention provides fusion peptides encoded by the cancer vaccine nucleic acids described herein.

[0155] In another aspect of the present invention, there is provided a composition comprising a cancer vaccine according to the present invention.

[0156] The composition may be immunogenic in a mammal, such as a human. The composition may include a pharmaceutically acceptable carrier. The composition may be a pharmaceutical composition including a pharmaceutically acceptable carrier. The composition may be for use in the prevention or treatment of cancer.

[0157] In another aspect of the present invention, there is provided a kit for the treatment or prevention of cancer, comprising: - a cancer vaccine according to the invention as described herein; and -Checkpoint inhibitors such as anti-PD1 binding molecules.

[0158] In another aspect of the present invention, there is provided a kit for the treatment or prevention of cancer, comprising: - a first cancer vaccine according to the invention described herein, wherein the nucleic acid encodes MAGED4B; - a second cancer vaccine according to the invention described herein, wherein the nucleic acid encodes FJX1; and -Optionally, a checkpoint inhibitor such as an anti-PD1 binding molecule.

[0159] Thus, there may be provided a kit for the treatment or prevention of cancer comprising: - a cancer vaccine comprising a sequence encoding a MAGED4B antigen or a variant thereof; and -Checkpoint inhibitors such as anti-PD1 binding molecules.

[0160] Thus, there may be provided a kit for the treatment or prevention of cancer comprising: - a first cancer vaccine, wherein the nucleic acid encodes MAGED4B or a variant thereof; and -Checkpoint inhibitors such as anti-PD1 binding molecules.

[0161] The cancer vaccine may further comprise one or more inhibitors of one or more immune checkpoint molecules (i.e., immune checkpoint inhibitors). The immune checkpoint inhibitor may be any nucleic acid or protein that prevents the suppression of any component of the immune system, such as MHC class presentation, T cell presentation and / or differentiation, any cytokine, chemokine, or signal transduction for immune cell proliferation and / or differentiation.

[0162] The immune checkpoint inhibitor may be one or more nucleic acid sequences encoding an antibody, a variant thereof, a fragment thereof, or a combination thereof, hi other embodiments, the immune checkpoint inhibitor may be an antibody, a variant thereof, a fragment thereof, or a combination thereof. Immune checkpoint molecules can be nucleic acid sequences, amino acid sequences, small molecules, or combinations thereof.

[0163] PD-1 and PD-L1 The immune checkpoint molecule may be programmed cell death protein 1 (PD-1), programmed cell death ligand 1 (PD-L1), a fragment thereof, a variant thereof, or a combination thereof. PD-1 is a cell surface protein encoded by the PDCD1 gene. PD-1 is a member of the immunoglobulin superfamily and is expressed on T cells and pro-B cells, thereby contributing to the fate and / or differentiation of these cells. In particular, PD-1 is a type 1 membrane protein of the CD28 / CTLA-4 family of T cell regulators and negatively regulates T cell receptor (TCR) signaling, thereby negatively regulating immune responses. PD-1 can negatively regulate CD8+ T cell responses, thereby suppressing CD8-mediated cytotoxicity and enhancing tumor growth.

[0164] PD-1 has two ligands, PD-L1 and PD-L2, which are members of the B7 family. PD-L1 is upregulated on macrophages and dendritic cells (DCs) in response to LPS and GM-CSF treatment, and on T cells upon TCR receptor signaling. PD-L1 is expressed by several tumor cell lines.

[0165] Anti-immune checkpoint molecule antibodies The immune checkpoint inhibitor can be an antibody. The antibody can bind to or react with an immune checkpoint molecule. Therefore, the antibody can be considered an anti-immune checkpoint molecule antibody or an immune checkpoint molecule antibody. The antibody can be encoded by a nucleic acid sequence included in a cancer vaccine or can be provided as an antibody.

[0166] The antibody can be polyclonal or monoclonal. The antibody is chimeric, single chain, affinity matured, human, humanized, single chain, or fully human.

[0167] Cancer Vaccine Constructs Cancer vaccines can include nucleic acid constructs encoding cancer antigens. The nucleic acid constructs can incorporate or contain one or more heterologous nucleic acid sequences. The constructs can exist in cells as functional extrachromosomal molecules. The constructs are preferably closed linear DNA molecules.

[0168] Closed linear DNA is generally understood to be double-stranded DNA covalently closed at each end. The double-stranded portions of the DNA are therefore complementary. Upon denaturation, closed linear DNA can form single-stranded circles. The DNA can be closed at each end by any suitable structure, including cruciforms, hairpins, or hairpin loops, depending on preference. The ends of closed linear DNA can be composed of non-complementary sequences, thus rendering the DNA in a single-stranded configuration of cruciforms, hairpins, or hairpin loops. Alternatively, the sequences can be complementary. It may be preferable for the ends to be formed by a portion of a target sequence for the protelomerase enzyme. A protelomerase target sequence is any DNA sequence whose presence in a DNA template enables the enzymatic activity of protelomerase. Protelomerase cleaves the double-stranded portions of the DNA and religates them, leaving covalently closed ends. In general, protelomerase target sequences include any perfect palindromic sequence, i.e., any double-stranded DNA sequence with dyad symmetry or perfect inverted repeats. The closed linear DNA may have a portion of a protelomerase target sequence at one or both ends. Effectively, this portion is a single strand of the entire double-stranded recognition site. The protelomerase target sequence may have the same cognate protelomerase at each end, or may require a different protelomerase for each end. Closed linear DNA constructed through the action of various protelomerase enzymes has previously been disclosed in WO2010 / 086626, WO2012 / 017210, and WO2016 / 132129, all of which are incorporated herein by reference. Closed linear DNA constructed using in vitro DNA amplification followed by protelomerase enzyme cleavage has the advantage that the closed linear DNA is produced in a cell-free in vitro environment and can be scaled up for commercial use. These closed linear DNA vectors are known as Doggybone DNA or dbDNA™.Preferably, closed linear DNA vectors are generated in an in vitro, cell-free manner using Applicants' previous method based on polymerase-based amplification of a DNA template bearing at least one protelomerase target sequence, followed by further treatment of the amplified DNA with protelomerase to produce closed linear DNA.

[0169] Closed linear DNA can be constructed by converting a plasmid carrying the required protelomerase target sequence into a closed linear DNA vector, but this is not an efficient method of production.

[0170] Other closed linear DNA vectors have been constructed by various in vitro strategies, including capping of PCR products and MIDGE (minimalistic immunogenic defined gene expression) vectors, which are generated by digestion of both prokaryotic and eukaryotic backbones after isolation of the plasmid from bacterial cells, followed by ligation of the necessary DNA sequences for end refilling to hairpin sequences.

[0171] DNA "ministrings" produced in vivo in cell cultures based on the action of protelomerase are similarly closed linear DNA vectors that would be suitable for use in the present invention.

[0172] Other forms of closed linear DNA that may be suitable include those closed at both ends with cruciform structures, which may also be produced in cell culture.

[0173] It may be preferable for closed linear DNA to be produced in a cell-free system to ensure product purity; otherwise, rigorous purification of closed linear DNA produced by cell methods may be required by regulatory authorities.

[0174] The nucleic acid construct may contain regulatory elements for gene expression of the coding sequence of the nucleic acid. The regulatory elements may be a promoter, an enhancer, a stop codon, or a polyadenylation signal.

[0175] The cancer vaccines described herein can express cancer antigens in animal cells in amounts effective to induce an immune response in animals.Cancer vaccines can be useful for transfecting nucleic acids encoding cancer antigens into cells where the antigens are expressed.Closed linear DNA has been shown to be an effective construct for DNA vaccines.

[0176] Methods for preparing vaccines Closed linear DNA molecules can be formulated or produced using a combination of known equipment and techniques, but preferably they are produced using cell-free synthesis methods described in WO2010 / 086626, WO2012 / 017210 and WO2016 / 132129.

[0177] Standard recombinant techniques can be used to prepare the DNA constructs.

[0178] Other Aspects In another aspect of the invention, there is provided herein a cancer vaccine or composition according to the invention for use as a medicament.

[0179] In another aspect of the invention, there is provided herein a cancer vaccine or composition according to the invention for use in treating or preventing cancer in a subject.

[0180] In another aspect of the present invention, there is provided a method of treating or preventing cancer in a subject, the method comprising administering a cancer vaccine or composition according to the invention as described herein.

[0181] The cancer to be treated or prevented may be oral cancer and / or oropharyngeal cancer. The oral cancer and / or oropharyngeal cancer may be HPV-negative or HPV-positive oral cancer and / or oropharyngeal cancer. In one embodiment, the cancer is oral cancer. In another embodiment, the cancer is oropharyngeal cancer. In one embodiment, the cancer is squamous cell carcinoma. In another embodiment, the cancer to be treated may be lung cancer or nasopharyngeal cancer.

[0182] The cancer may be characterized by cancer cells expressing or overexpressing MAGED4B and / or FJX1. Cancers may be characterized by tumor-associated cells that express or overexpress MAGED4B.

[0183] Cancer antigen expression can be measured by routine methods such as detecting biopsies or samples with relevant antibodies and / or detecting RNA sequences present in cells.

[0184] Cancers may additionally or alternatively be characterized by their association with MAGED4B-overexpressing CAFs, which may protect cancer cells from the immune system and other anti-cancer agents.

[0185] Expression of cancer antigens on cancer cells and cancer-associated cells can be determined relative to normal cells from the same tissue or organ. Thus, expression or overexpression can be a comparative level of expression relative to normal cells.

[0186] In one embodiment, subjects are tested for the presence of MAGED4B and / or FJX1 antigens in their cancerous tissues or cells prior to treatment or prevention. In another embodiment, subjects are tested for the levels of MAGED4B and / or FJX1 antigens in their cancerous tissues or cells prior to treatment or prevention. Subjects can be selected for treatment or prevention based on whether they have MAGED4B and / or FJX1 antigens in their cancerous tissues or cells, or whether they have overexpression compared to corresponding non-cancerous tissues or cells. Cells surrounding the tumor can also be tested for cancer antigen expression, particularly MAGED4B expression.

[0187] Those skilled in the art will be familiar with vaccine administration routes and dosages. For example, administration can be subcutaneous, intramuscular, or intravenous. A typical dosage can be approximately 4-8 mg per patient / subject, administered in one or more doses. For example, multiple doses can be administered until a therapeutic effect is observed. In one embodiment, in vivo electroporation is used to enhance delivery into cells in vivo or ex vivo.

[0188] The subject may be a mammal. In one embodiment, the subject is a human. In another embodiment, the subject is a domestic animal or livestock.

[0189] In another aspect of the present invention, a polypeptide is provided that includes a MAGED4B protein or a variant or truncated version thereof. The polypeptide can be presented as a fusion with a helper motif. The polypeptide can be presented as a fusion with DOM. Exemplary polypeptide sequences are set forth herein as SEQ ID NOs: 32, 35, 36 and 37. The MAGED4B sequence can be fused with FJX1, as encoded by SEQ ID NO: 12 (in this example, fused with DOM).

[0190] Cancer vaccine composition The vaccine may be in the form of a composition, optionally a pharmaceutical composition. The pharmaceutical composition may contain from about 5 nanograms to about 10 mg of vaccine. In some embodiments, the pharmaceutical composition according to the present invention contains from about 25 nanograms to about 5 mg of vaccine. In some embodiments, the pharmaceutical composition contains from about 50 nanograms to about 1 mg of vaccine DNA.

[0191] The composition may further contain other additives for formulation purposes, which may vary depending on the method of administration.When the composition is injectable, it is sterile, pyrogen-free and particle-free.Suitable formulations may contain sodium chloride, dextrose, mannitol, sorbitol and lactose.In some cases, isotonic solutions such as phosphate buffered saline are preferred.Stabilizers may include gelatin and albumin.

[0192] The vaccine may further comprise an acceptable excipient, which may be a functional molecule such as a vehicle, adjuvant, carrier, or diluent.

[0193] Vaccination The cancer vaccines disclosed herein are provided for use in methods for treating or preventing cancer. The vaccines described herein can be used in administration or vaccination methods to induce therapeutic and / or prophylactic immune responses. The vaccination process can generate an immune response in an animal against one or more cancer antigens. The administration of the vaccine can be the genetic transfer of one or more cancer antigens as nucleic acid molecules that are expressed in cells and, when delivered to the cell surface, are recognized by the immune system and induce an immune response.

[0194] The vaccine can be administered to an animal, preferably a mammal, to induce an immune response. The mammal can be a human, a non-human primate (especially a chimpanzee or monkey), a cow, a pig, a sheep, a goat, a deer, a llama, an alpaca, a dog, a cat, a guinea pig, a rabbit, a mouse, a rat, and preferably a human, a dog, or a cat.

[0195] The vaccine dosage can be 1 μg to 10 mg of active ingredient / kg body weight / hour, and can be 20 μg to 10 mg of active ingredient / kg body weight / hour. The vaccine can be administered every 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or 31 days. The number of vaccine doses for effective treatment can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more.

[0196] Vaccines may be administered using a "prime-boost" regimen. Prime-boost immunization may be defined as a regimen of immunization with the same vaccine between a priming and a booster dose. There may be one or multiple booster doses. The treatment methods used in the Examples used a prime-boost regimen in some cases.

[0197] Cancer vaccines can be administered by a variety of routes, including orally, parenterally, sublingually, transdermally, rectally, transmucosally, topically, via inhalation, via buccal administration, intrapleurally, intravenously, intraarterially, intraperitoneally, subcutaneously, intramuscularly, intranasally, intrathecally, and intraarticularly, or a combination thereof. Vaccines can be administered by conventional syringes, needleless syringes, "microprojectile bombardment guns," or other physical methods such as tattooing, electroporation ("EP"), "hydrodynamic methods," or ultrasound.

[0198] Cancer vaccines are nucleic acids. They can be delivered using appropriate nucleic acid delivery methods used for cellular transduction. Naked nucleic acids can be used, especially when the nucleic acid is in a minimal form (such as a minicircle or closed linear DNA). However, nucleic acids can also be "packaged" for administration, for example, using different materials. These materials include lipids, linear and branched polymers, and peptides / proteins of natural or synthetic origin. Lipid / nucleic acid complexation products result in lipoplexes, which typically have layered or hexagonal structures. Polymers and nucleic acids can be complexed into polyplexes, in which polymer chains are entangled together without any regular internal structure. Peptides / proteins interact with nucleic acids to form disordered polyplexes or ordered artificial viruses with fibrous or spherical morphologies, depending on the primary structure of the peptides / proteins. Alternatively, the nucleic acid may be packaged into a viral coat such as a virus-like particle (VLP), or indeed any suitable viral vector such as lentivirus, retrovirus, and adeno-associated virus (AAV) adenovirus, modified vaccinia Ankara (MVA) or oncolytic Maraba MG1 rhabdovirus.

[0199] Cancer vaccines can be used ex vivo on cells. Thus, suitable cells, either autologous or allogeneic, can be obtained and transduced in vitro, after which these cells can be provided to patients in need. Suitable cells for ex vivo transduction include any type of antigen-presenting cell, such as natural killer cells or dendritic cells, autologous or allogeneic tumor cells (usually irradiated). Autologous cells can be transiently transduced with cancer vaccines in mRNA or DNA format. Cancer antigens can be codon-optimized and contain helper motifs that result in MHC-I and MHC-II presentation of cancer antigen peptides to both CD8+ and CD4+ T cells, resulting in a strong tumor-specific immune response after reintroduction into the patient.

[0200] Thus, the present application extends to the use of cancer vaccines to transfect cells in vitro or ex vivo prior to using those cells to treat or prevent cancer in a patient, such cells no longer comprising a cancer vaccine once administered, as the transfection may be transient.

[0201] The data show that the combination of a cancer vaccine and an immune checkpoint inhibitor induces the immune system more efficiently than a vaccine containing a cancer antigen alone, and in fact, the two act synergistically. This more efficient immune response provides enhanced efficacy in cancer treatment.

[0202] definition Checkpoint inhibitor therapy is a type of cancer immunotherapy. Checkpoint inhibitors target immune checkpoints, which are key regulators of the immune system that stimulate or suppress immune system actions that tumors can use to protect themselves from immune system attack. Checkpoint therapy can block inhibitory checkpoints and restore immune system function.

[0203] The term "immunogenic" as applied to a protein or composition of the invention means capable of eliciting an immune response in the human or animal body. The immune response may be protective.

[0204] The term "protective" means preventing cancer, reducing the risk of cancer infection, transmission and / or progression, reducing the severity of cancer, curing cancer, alleviating symptoms, or reducing the severity of cancer or cancer symptoms.

[0205] The term "treatment" means curing cancer, alleviating the symptoms, or reducing the severity of cancer or cancer symptoms.

[0206] The term "prevention" in the case of oral cancer means preventing the conversion of oral dysplasia to oral cancer.

[0207] By "antibody," we include substantially intact antibody molecules, as well as chimeric antibodies, human antibodies, humanized antibodies (in which at least one amino acid is mutated relative to a naturally occurring human antibody), single-chain antibodies, bispecific antibodies, antibody heavy chains, antibody light chains, homodimers and heterodimers of antibody heavy and / or light chains, and antigen-binding fragments and derivatives thereof. Specifically, as used herein, the term "antibody" refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules containing an antigen-binding site that specifically binds to an antigen, whether naturally occurring or partially or fully artificially produced. The term also encompasses any polypeptide or protein having a binding domain that is, or is homologous to, an antibody binding domain. These can be derived from natural sources or they can be partially or fully artificially produced. Examples of antibodies are immunoglobulin isotypes (e.g., IgG, IgE, IgM, IgD, and IgA) and their isotypic subclasses; fragments containing antigen-binding domains, such as Fab, scFv, Fv, dAb, Fd; and diabodies. Antibodies can be polyclonal or monoclonal. Monoclonal antibodies are sometimes referred to as "mAbs."

[0208] Monoclonal and other antibodies can be used to produce other antibodies or chimeric molecules that retain the specificity of the original antibody using recombinant DNA technology. Such techniques can involve introducing DNA encoding the immunoglobulin variable region, or CDRs, of an antibody into the constant region, or constant region plus framework regions, of a different immunoglobulin. See, for example, EP-A-184187, GB2188638A, or EP-A-239400, which are incorporated herein by reference. Hybridomas or other cells that produce antibodies can be subjected to genetic mutations or other changes that may or may not alter the binding specificity of the antibody produced.

[0209] Because antibodies can be modified in several ways, the term "antibody" should be interpreted to encompass any specific binding member or substance having a binding domain with the required specificity. Thus, the term encompasses antibodies, antibody fragments, derivatives, functional equivalents, and homologs of humanized antibodies, including any polypeptide containing an immunoglobulin binding domain, whether natural or wholly or partially synthetic. Chimeric molecules containing an immunoglobulin binding domain, or equivalent, fused to another polypeptide are therefore encompassed. Cloning and expression of chimeric antibodies are described in EP-A-0120694 and EP-A-0125023, both of which are incorporated herein by reference. Humanized antibodies may be engineered antibodies with non-human, e.g., murine, variable regions and human constant regions. Methods for producing humanized antibodies are described, for example, in U.S. Pat. No. 5,225,539, which is incorporated herein by reference.

[0210] The antibodies of the present disclosure may be unmodified or genetically modified. For example, the antibodies may be fully or partially glycosylated and / or selected to enhance or reduce binding to human effector systems, such as complement, FcR-containing effectors, such as macrophages, or to extend or shorten half-life. These modifications may be made to improve efficacy and, in some cases, to reduce toxic side effects.

[0211] It has been shown that fragments of complete antibodies can perform the function of binding antigens. Examples of binding molecules for use in the present invention are (i) Fab fragments consisting of the VL, VH, CL, and CH1 domains; (ii) Fd fragments consisting of the VH and CH1 domains; (iii) Fv fragments consisting of the VL and VH domains of a single antibody; (iv) dAb fragments consisting of the VH domain; (v) isolated CDR regions; (vi) F(ab')2 fragments, bivalent fragments containing two binding Fab fragments; (vii) single-chain Fv molecules (scFvs), in which the VH and VL domains are linked by a peptide linker that allows the two domains to combine to form an antigen-binding site; (viii) bispecific single-chain Fv dimers (PCT / US92 / 09965, incorporated herein by reference); and (ix) "diabodies," multivalent or multispecific fragments constructed by gene fusion (WO94 / 13804, incorporated herein by reference).

[0212] The determination of percent identity between two sequences can be achieved using a mathematical algorithm known to those skilled in the art. An example of a mathematical algorithm for comparing two sequences is the Karlin and Altschul algorithm (Proc Natl Acad Sci USA. 1990 March; 87(6): 2264-8), modified as described in Karlin and Altschul (Proc Natl Acad Sci USA. 1993 June 15; 90(12): 5873-7). The NBLAST and XBLAST programs of Altschul et al. incorporate such an algorithm and can be used with standard parameters.

[0213] Those skilled in the art will appreciate that any feature of one embodiment or aspect of the present invention may be applied to other embodiments or aspects of the present invention, as appropriate.

[0214] Any reference herein to any publication shall be deemed to be incorporated by reference for US patent purposes only.

[0215] Embodiments of the present invention are hereinafter described, by way of example only, with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0216] [Figure 1] Overexpression of target antigens in head and neck squamous cell carcinoma (HNSC) and other promising cancer types: Transcripts for MAGED4B and FJX1 are expressed at significantly higher levels in head and neck tumors compared to adjacent normal tissue. As for other cancer types, MAGED4B expression is also significantly higher in lung adenocarcinoma (LUAD), squamous cell carcinoma (LUSC), and cholangiocarcinoma (CHOL), while FJX1 is significantly higher in many tumor types compared to their respective adjacent normal tissues. Transcriptional data are from The Cancer Genome Atlas (TCGA), processed by Li et al. (2016), and publicly available online at cistrome.shinyapps.io / timer. This data demonstrates that the target cancer antigens are expressed in multiple cancers, demonstrating the utility of the vaccines described herein. [Figure 2] Both MAGED4B and FJX1 antigens are strongly expressed in HPV-negative squamous cell carcinoma (SCC), non-malignant oral dysplasia, and nasopharyngeal carcinoma. MAGED4B (Novus-Bio: NBP1-89594) was stained at a dilution of 1:200. FJX1 (Novus-Bio: NBP1-59470) was stained at a dilution of 1:100. Staining was performed on a DAKO automated immunostainer. Testis served as a positive control with strong expression. Oral fibroepithelial polyp (FEP) served as a negative control, showing low levels of expression in non-dysplastic oral tissue. This data confirms that the target cancer antigens are expressed as proteins. [Figure 3]MAGED4B (D4B) is immunogenic in HPV-negative HNSCC patients. Flow cytometry revealed circulating D4B-specific CD8 T cells in 5 / 7 HLA-A2+ HNSC patients (four cases shown here), but not in HLA-A2+ healthy donors (HD1) using D4B501-509 / HLA-A2-PE tetramer staining. Staining was performed using peripheral blood mononuclear cells (PBMCs) from patients undergoing surgery at Poole, UK. 10 PBMCs were stained with anti-CD3 (FITC:OKT3), CD4 (APC:OKT4), CD8 (PE-Cy7:SK1 (BioLegend), DAPI (live / dead stain) (Miltenyi), and MAGED4B501-509 tetramer (PE). Flow cytometry was performed on a FACSCanto using UltraComp eBeads (Invitrogen) for compensation. Analysis was performed using FlowJo; gating was set on live / dead lymphocytes and CD8, followed by tetramer. Tetramer-positive CD8 cells are shown in the gate. This data confirms that in patients with confirmed HNSCC, there is a pool of T cells available for expansion by vaccination. Furthermore, this is encouraging data suggesting that the presence of such T cells alone does not cause any pathology in these individuals; they are not fully functional, but that individuals should otherwise use such cells to control their tumors. [Figure 4]CD8 T cells specific for both target antigens, MAGED4B and FJX1. Figure 4A: Using D4B501-509 / HLA-A2 tetramer staining and flow cytometry, D4B-specific CD8 T cells were detected in tumor-infiltrating lymphocytes (TILs; expanded with 6000 IU / ml recombinant human IL-2) from HLA-A2+ HNSCC patient HN337. 106 expanded TILs were stained with anti-CD3 (FITC:OKT3), CD4 (APC:OKT4), CD8 (PE-Cy7) (BioLegend), DAPI (live / dead stain) (Miltenyi), and MAGED4B501-509 tetramer (PE). Flow cytometry was performed on a FACSCanto using UltraComp eBeads (Invitrogen) for compensation. Analysis was performed using FlowJo. Gating was set on live and dead lymphocytes, followed by CD8+ tetramer. Tetramer-positive CD8 cells are indicated within the gate. Figure 4B: IFNγ CD8 T cells in proliferating TILs from an HLA-A2-negative, HLA-A1-positive HN337 tumor sample. TILs grown with anti-CD3 (clone OKT3) were stimulated for 8 hours with a control (peptide pool MAGED4B HLA-A2 peptides), a MAGED4B peptide pool consisting of 15-mer peptides with an 11-aa overlap across the entire antigen sequence (183 peptide pools), or an HLA-A1-only binding FJX1 peptide pool (predicted by NetMHC4.0, www.iedb.org) (three 15-mer peptides derived from the FJX1 aa sequence: ARFADGTRACVRYGI; DLVQWTDLILFDYLT; WTDLILFDYLTANFD; epitopes in bold). Flow cytometry was performed after intracellular IFNγ staining. The antibody panel for flow cytometry included anti-CD3 (FITC: OKT3), CD8 (PerCp-Cy5.5: RPA-T8), anti-CD56 (PE: HCD56), IFNγ (APC 4S.B3) (all from BioLegend), and Zombi Aqua (live / dead stain) (Miltenyi) applied after Fc receptor blocking with Human TrueStain (BioLegend). Gates indicate specific populations of IFNγ-positive CD8+ T cells after restimulation with the reagents indicated at the top of the plot.The data in these figures also indicate that in patients with established HNSCC, there is a pool of T cells available for expansion by vaccination, and these cells can also be found in the tumor. Furthermore, this is encouraging data suggesting that the presence of such T cells alone does not cause any pathology in these individuals, they are not fully functional, but that individuals should otherwise use such cells to control their tumors. [Figure 5] CD8+ T cells specific for both the target antigens MAGED4B and FJX1. Figure 4A: Using D4B501-509 and FJX115-25 HLA-A2 tetramer staining and flow cytometry, MAGED4B- and FJX1-specific CD8+ T cells were detected in tumor-infiltrating lymphocytes (TILs; expanded with 6000 IU / ml recombinant human IL-2) from HLA-A2+ Malaysian OSCC patient 06-0021-18. Expanded TILs were stained with anti-CD3 (FITC; SK7), CD4 (PerCp-Cy5.5; SK3), CD8 (BV510; RPA-T8) (BD Biosciences), FVS780 (viability stain) (BD Biosciences), and MAGED4B501-509 tetramer (PE). Flow cytometry was performed on a BD LSRFortessa using BD CompBeads for compensation. Analysis was performed using FACS Diva software. Gating was set on live and dead lymphocytes, followed by CD8+ tetramer. Tetramer-positive CD8 cells are shown in the gate. This data indicates that antigen-specific T cells were identified in the tumor. This further suggests that there is a pool of cells available for expansion in addition to de novo activation and expansion by the vaccine. [Figure 6]T cells specific for the target antigen MAGED4B express PD1 in HNSC patients and can therefore be targeted with anti-PD1. Specific T cells were detected in the circulation of PBMCs using MAGED4B501-509 tetramers. Half of the tetramer-positive population was also PD1+. This subpopulation was absent in HLA*A2-negative HNSC patients (control group). The panel consisted of anti-CD3 (FITC; OKT3), CD4 (APC: OKT4), CD8 (PE-Cy7: SK1), PD-1 (PerCp-Cy5.5: EH12.2H7), CD19 (Pacific Blue: HIB19.11), and CD14 (Pacific Blue: HCD14) (BioLegend), live / dead Violet (Pacific Blue) (Invitrogen), and MAGED4B501-509 tetramer (PE). Flow cytometry was performed on a FACSCanto using UltraComp eBeads (Invitrogen) for compensation. Analysis was performed using FlowJo software. The data confirm the specificity of the HLA-2 tetramer, since it is nonfunctional in the HLA-2-negative patient (HN366). In the HLA-2-positive patient (HN364), the data confirm the antigen sensitivity of T cells. [Figure 7]Assembly of a MAGED4B- and FJX1-targeted DNA vaccine for head and neck cancer. Diagram of the vaccine constructs: pDOM (control), vaccines delivering single antigens pDOM-MAGED4B and pDOM-FJX1, and pDOM-MAGED4B-FJX1 vaccine in which both antigens are simultaneously present. The DOM fragment of the tetanus toxin gene and the gene of interest (MAGED4B or FJX1) were linked using a seven-amino acid linker (AAAGPGP). The fusion gene was inserted between the CMV / T7 dual promoter and the BGH poly(A) site. A leader sequence encoding the mus IgH signal peptide (MGWSCIIFFLVATATGVHS) was inserted at the N-terminus of the construct to enhance secretion efficiency. When the gene of interest encoded the fused MAGED4B and FJX1 antigens, two five-amino acid linkers (GSGSG) were used to link these two genes. DOM1 was inserted into the pcDNA3.0 vector using the NotI and HindIII restriction enzyme sites to generate the pDOM vector. The genes for MAGED4B, FJX1, or their desired fusions were inserted into the pDOM vector at the NotI and XhoI restriction enzyme sites to generate DNA vaccines. As used herein, the "p" in the term "vector" refers to a plasmid vector or construct. [Figure 8]Three groups of five to six non-tumor-bearing HHD mice (transgenic for human HLA-A2 alleles) were vaccinated with 50 micrograms of p.Dom-MAGED4B (A), p.Dom-FJX1 (B), or p.Dom (C) on day 1, followed by a booster injection of the same DNA vaccine via electroporation on day 22. Their immunogenicity was assessed by IFNγ ELISpot assay. Lymphocytes isolated from mouse spleens on day 35 were seeded onto ELISpot culture dishes, and specific T cell responses were detected using overlapping peptide pools of each target antigen, MAGED4B and FJX1. The overlapping peptide pools consisted of 15-mer peptides with an 11-aa overlap across the entire sequence of each antigen (183 individual peptides were pooled for MAGED4B, and 107 peptides were pooled for FJX1). The P30 peptide was used as the vaccination standard. Each peptide was produced at 90% purity in JPT, Germany. For ELISPOT IFNγ, an ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to each responding T cell were imaged and counted using an AID ELISPOT plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassberg, Germany). This data confirms the immunogenicity of the antigen in HAL-A2 transgenic mice. [Figure 9]DNA vaccines are effective as therapeutic agents both alone and in combination with anti-PD-1. Figure 9A illustrates the treatment strategy. As indicated, each group of mice (6–10 mice) was challenged with B16 tumors expressing both MAGED4B and FJX1 and then treated with a combination DNA vaccine (DV; p.Dom-MAGED4B and p.Dom-FJX1; 100 μg / mouse, 50 μg injected intramuscularly in each leg in 100 μl saline), an anti-PD-1 antibody (200 μg / injection, 0.5 mL intraperitoneally), or a combination of DV and anti-PD-1. The control group received control IgG plus pDOM (vector backbone). Tumor size was measured every 2–3 days, and the tumor sizes for each group are shown in Figure 9B. Figure 9C (mean + sem): ELISPOT assay demonstrated a MAGED4B-specific immune response specifically in the DV and DV + α-PD1 groups, but not in the α-PD1 or control groups. Splenocytes isolated from vaccinated animals were able to secrete IFNγ upon restimulation with a full-antigen sequence-overlapping MAGED4B peptide library (183 peptides pooled together, overlapping 15-mer 11aa segments). Figure 9D is similar to Figure 9C, but uses a full-antigen overlapping FJX1 peptide library. This data demonstrates the clear impact of monotherapy (vaccine alone) and combination therapy (vaccine + anti-PD1) on tumor progression. Notably, Figures 9C and 9D show that vaccination expands antigen-specific T cells in tumor-bearing mice—these mice were exposed to antigen on the tumor but were unable to mount a successful immune response. This supports the claim that vaccines can improve immune responses and effectively "expose" tumors to the immune system. [Figure 10]DNA vaccines are effective in suppressing tumor growth. Figure 10A shows the treatment strategy, tumor volume reduction, and mechanism of action. As indicated, each group of mice (8–12 mice) was challenged with tumors expressing both MAGED4B and FJX1 and then treated with DNA vaccines (DV; p.Dom-MAGED4B and p.Dom-FJX1; 50 μg of DV was injected intramuscularly in each leg at 100 μg / mouse in 100 μl of saline). The control group received the pDOM vector backbone. Tumor size was measured every 2–3 days, and the tumor size for each group is shown in Figure 10A (mean + s.e.m.). Figure 10B shows a photograph of the cells. The tumor staining on the right panel shows T cell infiltration after vaccination with the DNA vaccine, while the left panel shows a poorly infiltrated pDOM control tumor (hematoxylin and eosin staining, original magnification: x10 objective). Figure 10C shows the results of flow cytometry analysis, demonstrating an increase in CD4+ and CD8+ immune cells in tumors recovered from vaccinated animals compared to control animals. Importantly, the checkpoint protein PD1 is significantly elevated in CD4+ and CD8+ immune cells recovered from vaccinated animals. This study used a high dose of tumor cells to accelerate cancer progression, particularly in an aggressive tumor model. Again, vaccination expanded T cells in tumor-bearing mice, demonstrating that despite exposure, they were unable to mount a strong immune response against the tumor. This supports the claim that vaccines can improve immune responses and effectively "expose" tumors to the immune system. [Figure 11]Figure 11A-B. Demonstration of essential components for the design of MAGED4B- and FJX1-targeting DNA vaccines. The therapeutic strategy is shown in both figures. Five non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg each of p.Dom-MAGED4B-FJX1, pSP-MAGED4B-FJX1 (without DOM), pDom-FJX1, and pnoSPDOM-FJX1 (without leader / SP) on day 1, followed by a booster injection of the same DNA vaccine on day 8. Their immunogenicity was assessed by IFNγ ELISpot. Lymphocytes isolated from mouse spleens on day 22 were seeded into ELISpot culture dishes, and responding specific T cells were detected using overlapping peptide pools of each target antigen, MAGED4B and FJX1. The overlapping peptide pools consisted of 15-mer peptides with an 11-aa overlap across the entire sequence of each antigen (183 individual peptides were pooled for MAGED4B, and 107 peptides were pooled for FJX1). The P30 and MHCII peptides derived from DOM were used as vaccination controls. Figure 11A shows data with and without DOM. The DOM sequence was shown to be essential for T cell induction, thus improving the response to MAGED4B. Figure 11B shows data with and without a leader sequence. The leader sequence, which directs expression of the encoded construct into the endoplasmic reticulum for secretion, is also essential for inducing T cell immunity. Each peptide was produced at 90% purity at JPT, Germany. For ELISPOT IFNγ, the ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. The spots corresponding to each responding T cell were imaged and counted using an AID Elispot plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassberg, Germany). The data show that, in general, FJX1 responses are improved by the inclusion of a leader sequence. [Figure 12]A DNA vaccine targeting both antigens in tandem as a fusion antigen induces T cell responses comparable to those of single-antigen-targeted DNA vaccines. A therapeutic strategy is presented. Three groups of five non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of each of p.Dom-MAGED4B, pDOM-FJX1, or p.Dom-MAGED4B-FJX1 on day 1, followed by a booster injection of the same DNA vaccine on day 8. Their immunogenicity was assessed by IFNγ ELISpot assay. Lymphocytes isolated from mouse spleens on day 22 were seeded into ELISpot culture dishes, and specific T cell responses were detected using overlapping peptide pools of each target antigen, MAGED4B and FJX1. The overlapping peptide pools consisted of 15-mer peptides with an 11-aa overlap across the entire sequence of each antigen (183 individual peptides were pooled for MAGED4B, and 107 peptides were pooled for FJX1). The DOM-derived P30 peptide and MHCII peptide were used as vaccination controls. DNA vaccines targeting both antigens demonstrated similar ability to induce specific T cell responses. P values ​​were calculated using Mann-Whitney analysis with GraphPad Prism 8.0. For ELISPOT IFNγ, the ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to each responding T cell were imaged and counted using an AID ELISPOT plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). [Figure 13]Assembly of alternative MAGED4B- and FJX1-targeted DNA vaccines for cancer. Diagram of vaccine constructs: pDOM (control), pMAGED4B / FJX1, pMAGED4B / FJX1-MITD, pPVXCP-MAGED4B / FJX1, and pMIP3α-MAGED4B / FJX1. The alternative fusion partners include MITD, PVXCP, and MIP3α. MITD (165 bp) encodes the MHCI (HLA-A2) trafficking signal. PVXCP (732 bp) encodes the potato virus X coat protein. MIP3α (252 bp) encodes macrophage inflammatory protein 3 alpha. The MITD, PVXCP, and MIP3α genes were optimized for human codon usage and ordered from GeneArt (Invitrogen). Genes (with or without fusion partners) were inserted between the CMV / T7 dual promoter and the BGH poly(A) site. A leader sequence encoding the mouse IgH signal peptide (MGWSCIIFFLVATATGVHS) was inserted at the N-terminus of the construct to enhance secretion efficiency. The fusion partner and gene of interest (MAGED4B or FJX1) were linked using a seven-amino acid linker (AAAGPGP). All other fusion partners were fused upstream of the gene of interest, except for MITD, which was added downstream of the gene of interest. Genes for MAGED4B, FJX1, or their fusion partners of interest were inserted into the pcDNA3 vector at the NotI, XhoI, and XbaI restriction enzyme sites to generate DNA vaccines. [Figure 14]MAGED4B-specific T cell responses were induced in C57BL / 6 mice by DNA vaccination. A therapeutic strategy is presented. Non-tumor-bearing C57BL / 6 mice were vaccinated on days 1 and 8 with 50 μg of pDOM vaccine (3 mice, as a negative control), 50 μg of pSP-MAGED4B (5 mice), 50 μg of pSP-MAGED4B-MITD (5 mice), 50 μg of pPVXCP-MAGED4B (5 mice), and 50 μg of pMIP3a-MAGED4B (5 mice). Lymphocytes isolated from mouse spleens on day 22 were seeded in ELISPOT culture dishes, and responding specific T cells were detected using overlapping peptide pools of MAGED4B. Overlapping peptide pools (OPPs) consisted of 15-mer peptides with an 11-aa overlap across the entire sequence (183 individual peptides were pooled for MAGED4B). The IFNγ Elispot kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to each responding T cell were imaged and counted using an AID Elispot plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). Graphs show the response to MAGED4B OPPs in each group. Values ​​are calculated by subtracting the number of spots without stimulation from the response. The cutoff was set at 2x background (Irr OPP, shown as a red dotted line). P values ​​were calculated using the Mann-Whitney test (Irr = irrelevant). These data demonstrate that MAGED4B responses can be generated from the full-length protein and can be improved by fusion with various helper motifs. [Figure 15]FJX1-specific T cell responses were induced in C57BL / 6 mice by DNA vaccination. Non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of pDOM vaccine (3 mice, as a negative control), 50 μg of pSP-FJX1 (5 mice), and 50 μg of pSP-FJX1-MITD (5 mice). Vaccination was administered intramuscularly on day 1. Lymphocytes isolated from mouse spleens on day 14 were seeded onto ELISPOT culture dishes, and responding specific T cells were detected using overlapping peptide pools of FJX1. The overlapping peptide pool consisted of 15-mer peptides with an 11-aa overlap in the overall sequence (107 individual peptides were pooled for FJX1). The IFNγ ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to each responding T cell were imaged and counted using an AID ELISpot plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). The graph shows the response to FJX1 OPP in each group. pSP-FJX1-MITD induced the strongest response among all groups. The median and interquartile values ​​and responses for each mouse are shown. Results were normalized by subtracting the number of spots without stimulation. The cutoff was set at 2x background (Irr OPP, shown as a red dotted line). P values ​​were calculated using the Mann-Whitney test. Only one experiment is shown because the lab shutdown due to the pandemic limited the replication of these experiments. [Figure 16]Figure 1 shows the complete MAGED4B amino acid sequence and amino acid sequence maps of three truncated fragments. As a member of the melanoma-associated antigen family, MAGED4B contains the MAGE consensus homology domain MAGED4B 412-682 (SEQ ID NO: 3). The defined HLA-A2 epitope RLSLLVIL (MAGED4B 501-509) is located within the homology domain. Three truncated MAGED4B sequences were designed as follows: 1) MAGED4B sequence type (v)1 does not contain the homology domain but does contain RLSLLVIL; 2) MAGED4B.sv2 retains the second half of the homology domain (MAGED4B 510-682) including RLSLLVIL; and 3) MAGED4B.sv3 retains the first half of the homology domain (MAGED4B 412-500) including RLSLLVIL. Thus, all of these are immunogenic fragments of MAGED4B and are suitable for use in the vaccines described herein. [Figure 17]The MAGED4B fragments detailed in Figure 16 are shown to be immunogenic. A treatment strategy is presented: four groups of five non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of p.Dom-MAGED4B (full-length), p.DOM-MAGED4Bsv1, p.Dom-MAGED4Bsv2, or p.Dom-MAGED4Bsv3 on day 1, followed by a booster injection of the same DNA vaccine on day 22. Their immunogenicity was assessed by IFNγ ELISpot. Lymphocytes isolated from mouse spleens on day 35 were seeded into ELISpot culture dishes, and responding specific T cells were detected using overlapping peptide pools of MAGED4B. The overlapping peptide pool consisted of 15-mer peptides with an 11-aa overlap in the overall sequence (183 individual peptides were pooled for MAGED4B). P30 peptide and MHCII peptide derived from tetanus DOM were used as vaccination controls. p.Dom-MAGED4Bsv3 induced significantly stronger specific T cell responses than p.Dom-MAGED4B (full-length), p.Dom-MAGED4Bsv1, and p.Dom-MAGED4Bsv2. Neither p.Dom-MAGED4Bsv1 nor p.Dom-MAGED4Bsv2 performed better than p.Dom-MAGED4B. P values ​​were calculated by one-way analysis of variance using GraphPad Prism 8.0. For ELISPOT IFNγ, an ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to individual responding T cells were imaged and counted using an AID ELISPOT plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). Thus, the MAGE homology domain can be removed in whole or in part without affecting the activity of the vaccine. [Figure 18]Vector map of the closed linear DNA (dbDNA™) used in vaccination experiments. Shown are the sequences of the closed ends of the dbDNA (TelR or TelL from the protelomerase target sequence TelRL) - these are parts of the target sequences that together form the entire sequence. The four construct configurations are as follows: basic 0 (minimal construct configuration - CMV promoter, DOM and antigen fusion, SV40 polyA signal sequence) on which all other constructs are based. Additional other constructs are: basic 1 (plus TE: triple enhancer); SV40 enh (plus TE and SV40 enhancer sequence); CpG (plus TE and part of sequence with CpG motifs). These are used in the vaccination experiments described herein. [Figure 19]MAGED4B-specific T cell responses were induced in C57BL / 6 mice by doggybone (DB) and plasmid DNA vaccines. Non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 25 μg of DB-MAGED4B-CO (5 mice), and 25 μg of pDOM-MAGED4B plasmid (5 mice). Vaccination was administered intramuscularly on day 1. Electroporation (EP) was performed on mice anesthetized intramuscularly with isoflurane using the TriGrid Delivery System with an Ichor EP device (TDS-IM). Lymphocytes isolated from mouse spleens on day 14 were seeded into ELISPOT culture dishes, and specific T cell responses were detected using overlapping peptide pools of MAGED4B. The overlapping peptide pools consisted of 15-mer peptides with an 11-aa overlap in the overall sequence (183 individual peptides were pooled for MAGED4B, and 107 individual peptides were pooled for FJX1). Tetanus DOM-derived p30 and MHCII peptides were used as controls for proper vaccination. In this experiment, FJX1 OPP served as the Irr peptide control. The IFNγ Elispot kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to each responding T cell were imaged and counted using an AID Elispot plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). Graphs show the responses to p30 and MAGED4B OPP in each group, respectively. Median + interquartile values ​​and responses for each mouse are shown. Values ​​are calculated by subtracting the number of spots without stimulation from the response. The cutoff was set at 2x background (Irr OPP, shown as a red dotted line). P values ​​were calculated using the Mann-Whitney test. [Figure 20]FJX1-specific T cell responses were induced by doggybone (DB) and plasmid DNA vaccines. Non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 25 μg of DB-DOM-FJX1-CO (5 mice), and 25 μg of pDOM-FJX1 plasmid (5 mice). The vaccine was administered intramuscularly on day 1 via EP. EP was administered to mice anesthetized with isoflurane using an intramuscular TriGrid Delivery System (TDS-IM; Ichor Medical Systems). Lymphocytes isolated from mouse spleens on day 14 were seeded in ELISPOT culture dishes, and specific T cell responses were detected using overlapping FJX1 peptide pools. Overlapping peptide pools (OPPs) consisted of 15-mer peptides with an 11-aa overlap in the overall sequence (183 individual peptides were pooled for MAGED4B, and 107 individual peptides were pooled for FJX1). Tetanus DOM-derived p30 and MHCII peptides were used as controls for proper vaccination. In this experiment, MAGED4B OPP served as the Irr peptide control. The IFNγ Elispot kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to each responding T cell were imaged and counted using an AID Elispot plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). Graphs show the responses to p30 peptide and FJX1 OPP in each group, respectively. Median and interquartile values ​​and responses for each mouse are shown. Values ​​are calculated by subtracting the number of spots without stimulation from the response. The cutoff was set at 2x background (Irr OPP, shown as a red dotted line). P values ​​were calculated using the Mann-Whitney test. [Figure 21]The dbDNA DOM-MAGED4B and pDOM-MAGED4B DNA vaccines induce specific CD4 and CD8 T cell responses. Treatment strategies are presented. Non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 10 μg of DB-MAGED4B-CO (5 mice), 10 μg of pDOM-MAGED4B-CO Basic 1 (5 mice), 10 μg of DB-MAGED4B-CO SV40 (5 mice), 10 μg of DB-MAGED4B-CO CpG (5 mice), and 10 μg of pDOM-MAGED4B plasmid (5 mice). Vaccines were administered intramuscularly (i.m.) on days 1 and 21. EP was performed on mice anesthetized with isoflurane using an intramuscular TriGrid Delivery System (TDS-IM; Ichor Medical Systems). FACS was performed after in vitro stimulation of 50 μl of blood samples collected on day 12. First, leukocytes from the collected blood were treated with RBC lysis buffer (BioLegend) and stimulated with 1 μM MAGED4B overlapping peptide pool (OPP; 1 μl of anti-CD107a-FITC and anti-CD107b-FITC (both BioLegend) in a 96-well plate. The OPP was then incubated overnight at 37°C in 5% CO2. The next day, cells were washed and stained with anti-CD3-PE, anti-CD4-PEcy7, anti-CD8-APCcy7, anti-CD137 (4-1BB)-APC, anti-PD1-PerCPcy5 (all from BioLegend), and live / dead-violet (Invitrogen). OPPs consist of 15-mer peptides with an 11-aa overlap across the entire sequence (183 individual peptides were pooled for MAGED4B). Flow cytometry was performed on a FACSCanto II. Analysis was performed using FlowJo software. Responses were assessed using FACS. CD107a / b+PD-1+ double-positive T cells and 4-1BB+PD-1+ double-positive T cells represent cytotoxic and MAGED4B OPP-specific activated populations, respectively.Responses to MAGED4B OPP from groups vaccinated with pDOM, DB-MAGED4B-CO, DB-MAGED4B-CO Basic 1, DB-MAGED4B-CO SV40, DB-MAGED4B-CO CpG, and pDOM-MAGED4B plasmids are summarized in this bar graph. Median and interquartile values ​​and individual mouse responses are shown. P values ​​were calculated using the Kruskal-Wallis test. This data demonstrates that the MAGED4B antigen can be delivered in different configurations and still induce antigen-experienced CD4 and CD8 T cells. CO is a codon-optimized sequence. [Figure 22]Doggybone DOM-FJX1 and pDOM-FJX1 DNA vaccines induce specific CD4 and CD8 T cell responses. Treatment strategies are presented. Non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 10 μg of DB-FJX1-CO (5 mice), 10 μg of DB-FJX1-CO Basic 1 (5 mice), 10 μg of DB-FJX1-CO SV40 (5 mice), 10 μg of DB-FJX1-CO CpG (5 mice), and 10 μg of pDOM-FJX1 plasmid (5 mice). Vaccines were administered intramuscularly on days 1 and 21. EP was performed on isoflurane-anesthetized mice using an intramuscular TriGrid Delivery System (TDS-IM; Ichor Medical Systems). FACS was performed after in vitro stimulation of 50 μl of collected blood samples collected on day 12. First, 50 μl of collected blood was treated with RBC lysis buffer (BioLegend). Leukocytes were stimulated with 1 μM FJX1 overlapping peptide pool (OPP; 1 μl of anti-CD107a-FITC and anti-CD107b-FITC (both BioLegend) was added simultaneously in a 96-well plate and incubated overnight at 37°C in 5% CO2. The next day, cells were washed and stained with anti-CD3-PE, anti-CD4-PEcy7, anti-CD8-APCcy7, anti-CD137(4-1BB)-APC, anti-PD1-PerCPcy5 (all BioLegend), and live / dead-violet (Invitrogen). The overlapping peptide pool consisted of 15-mer peptides with an 11-aa overlap in the overall sequence (107 individual peptides were pooled for FJX1). Flow cytometry was performed on a FACSCanto II. Analysis was performed using FlowJo software. Responses were assessed using FACS. CD107a / b+PD-1+ double-positive T cells and 4-1BB+PD-1+ double-positive T cells represent cytotoxic and FJX1 OPP-specific activated populations, respectively.Responses to FJX1 OPP from groups vaccinated with pDOM, DB-FJX1-CO, DB-FJX1-CO Basic 1, DB-FJX1-CO SV40, DB-FJX1-CO CpG, and pDOM-FJX1 plasmids are summarized in this bar graph. Median and interquartile values ​​and individual mouse responses are shown. P values ​​were calculated using the Kruskal-Wallis test. This data demonstrates that the FJX1 antigen can be delivered in different configurations and still induce antigen-experienced CD4 and CD8 T cells. CO is a codon-optimized sequence. [Figure 23]Doggybone DOM-MAGED4B and pDOM-MAGED4B DNA vaccines induce specific CD4 and CD8 T cell responses. Treatment strategies are presented. Non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 10 μg of DB-MAGED4B-CO (5 mice), 10 μg of pDOM-MAGED4B-CO Basic 1 (5 mice), 10 μg of DB-MAGED4B-CO SV40 (5 mice), 10 μg of DB-MAGED4B-CO CpG (5 mice), and 10 μg of pDOM-MAGED4B plasmid (5 mice). Vaccines were administered intramuscularly (i.m.) on days 1 and 21. EP was performed on mice anesthetized with isoflurane using an intramuscular TriGrid Delivery System (TDS-IM; Ichor Medical System). Lymphocytes isolated from mouse spleens on day 35 were seeded onto ELISPOT culture dishes, and specific responding T cells were stimulated using overlapping peptide pools (OPPs) of MAGED4B. The OPPs consisted of 15-mer peptides with an 11-aa overlap across the entire sequence (183 individual peptides were pooled for MAGED4B). The P30 peptide and MHCII peptides derived from tetanus DOM were used to evaluate the induction of CD4 responses against the DOM helper sequence. The IFNγ ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to individual responding T cells were imaged and counted using an AID ELISPOT plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). The graph shows the responses to p30 peptide, Irr OPP (FJX1 OPP), and MAGED4B OPP. DB-MAGED4B-CO Basic 0 induced the strongest response among all groups. Median + interquartile and response for each mouse are shown. The cutoff was set at 2x background (Irr OPP) and is shown as a dotted line.Values ​​are calculated by subtracting the number of spots without stimulation from the response. The number of spots / 106 cells is shown. P values ​​were calculated using the Mann-Whitney test. This data demonstrates that MAGED4B antigen can be delivered in different configurations and still induce antigen-experienced CD4 and CD8 T cells. CO is a codon-optimized sequence. [Figure 24]The dbDNA (DB)DOM-FJX1 and plasmid (p)DOM-FJX1 DNA vaccines induce specific CD4 and CD8 T cell responses. A therapeutic strategy is presented. Non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 10 μg of DB-FJX1-CO (5 mice), 10 μg of DB-FJX1-CO Basic 1 (5 mice), 10 μg of DB-FJX1-CO SV40 (5 mice), 10 μg of DB-FJX1-CO CpG (5 mice), and 10 μg of pDOM-FJX1 plasmid (5 mice). The vaccines were administered intramuscularly (i.m.) on days 1 and 21. EP was performed on isoflurane-anesthetized mice using an intramuscular TriGrid Delivery System (TDS-IM; Ichor Medical System). Lymphocytes isolated from mouse spleens on day 35 were seeded onto ELISPOT culture dishes, and specific responding T cells were detected using overlapping peptide pools of FJX1. The overlapping peptide pool (OPP) consisted of 15-mer peptides with an 11-aa overlap across the entire sequence (107 individual peptides were pooled for FJX1). The P30 peptide and MHCII peptides derived from tetanus DOM were used to evaluate the induction of CD4 responses against the DOM helper sequence. The IFNγ ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to individual responding T cells were imaged and counted using an AID ELISPOT plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). The graph shows the response to p30 peptide, Irr OPP (MAGED4B OPP), and FJX1 OPP. All groups had similar results. The median + interquartile value and response for each mouse are shown. The cutoff was set as 2x background (Irr OPP) and is shown as a dotted line. The cutoff was set as 2x background (Irr OPP) and is shown as a dotted line.Values ​​were calculated by subtracting the number of spots without stimulation from the response. The number of spots / 106 cells is shown. P values ​​were calculated using the Mann-Whitney test. This data demonstrates that FJX1 can be delivered in multiple configurations and can induce antigen-specific CD4 and CD8 T cells. [Figure 25] dbDNA (DB)DOM-MAGED4B and plasmid (p)DOM-MAGED4B DNA vaccines induce specific T cell responses. A therapeutic strategy is presented. Non-tumor-bearing C57BL / 6 mice were vaccinated with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 25 μg of DB-MAGED4B-CO (6 mice), 25 μg of pDOM-MAGED4B-CO Basic 1 (6 mice), 25 μg of DB-MAGED4B-CO SV40 (6 mice), and 25 μg of pDOM-MAGED4B plasmid (6 mice). Vaccines were administered intramuscularly on days 1 and 21. EP was performed on isoflurane-anesthetized mice using an intramuscular TriGrid Delivery System (TDS-IM; Ichor Medical Systems). Lymphocytes isolated from mouse spleens on day 35 were seeded onto ELISPOT culture dishes, and specific responding T cells were stimulated using overlapping peptide pools (OPPs) of MAGED4B. The OPPs consisted of 15-mer peptides with an 11-aa overlap across the entire sequence (183 individual peptides were pooled for MAGED4B). The IFNγ ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to individual responding T cells were imaged and counted using an AID ELISPOT plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). Responses to MAGED4B OPPs are shown. Median + interquartile values ​​and individual mice are shown. Values ​​represent the response minus the number of spots without stimulation. P values ​​were calculated using the Mann-Whitney test. [Figure 26]The dbDNA (DB)DOM-FJX1 and plasmid (p)DOM-FJX1 DNA vaccines induce specific CD4 and CD8 T cell responses. Treatment strategies are presented. Non-tumor-bearing C57BL / 6 mice were vaccinated on days 1 and 21 with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 10 μg of DB-FJX1-CO (5 mice), 10 μg of DB-FJX1-CO Basic 1 (5 mice), 10 μg of DB-FJX1-CO SV40 (5 mice), 10 μg of DB-FJX1-CO CpG (5 mice), and 10 μg of pDOM-FJX1 plasmid (5 mice). EP was performed on isoflurane-anesthetized mice using an intramuscular TriGrid Delivery System (TDS-IM; Ichor Medical Systems). Lymphocytes isolated from mouse spleens on day 35 were seeded onto ELISPOT culture dishes, and specific responding T cells were detected using overlapping peptide pools of FJX1. The overlapping peptide pool consisted of 15-mer peptides with an 11-aa overlap across the entire sequence (107 individual peptides were pooled for FJX1). The IFNγ ELISPOT kit from BD Bioscience was used according to the manufacturer's protocol. Spots corresponding to each responding T cell were imaged and counted using an AID ELISPOT plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). Values ​​were calculated by subtracting the number of spots without stimulation from the response. Graphs show responses to FJX1 OPP. Median + interquartile values ​​and responses for each mouse are shown. [Figure 27]Cancer-associated fibroblasts (CAFs) express MAGED4B. Immunohistochemical analysis of HNSCC cases demonstrated MAGED4B expression in cancer cells and cancer-associated fibroblasts, with the latter showing strong expression. Anti-MAGED4B monoclonal antibody (Santa Cruz, G12; sc-393059) was used on HNSCC tissue at a dilution of 1:50. Staining was performed with a DAKO automated immunostainer (K4065). Six individual HNSCC cases obtained from the Pathology lab at Southampton General Hospital (Southampton, UK) are presented. [Figure 28] Figures 28A-B. Cancer-associated fibroblasts (CAFs) express MAGED4B. Figure 28A: MAGED4B expression across different cell types was assessed using publicly available single-cell RNA sequencing (scRNA-seq) data generated from Smart-Seq analysis of HNSCC patient samples. Cell lineages were identified as described in Puram et al. Cell. 2017 Dec 14;171(7):1611-1624. Figure 28B: Fibroblast subpopulations were identified by unsupervised hierarchical clustering implemented in the Seurat R package (v3.2) (Butler A et al. Nat Biotechnol. 2018 Jun;36(5):411-420). Fibroblast subpopulation markers MCAM, ACTA2, and POSTIN were identified using Wilcoxon tests and compared to those previously identified in lung cancer fibroblast subpopulations and annotated into distinct subpopulations (CJ Hanley et al, bioRxiv 2020.06.08.134270). [Figure 29]Both MAGED4B and FJX1 antigens are strongly expressed in colon, prostate, rectal, breast, lung, and nasopharyngeal carcinomas. Target protein expression levels were detected by immunohistochemistry (IHC) using anti-MAGED4B (1:100; Sigma Aldrich, catalog number #HPA003554) and anti-FJX1 (1:200; Sigma Aldrich, catalog number #HPA059220) antibodies with the Dakocytomation Envision+ Dual Link System HRP (DAB+) kit (DAKO, catalog number #K4065) as previously described.

[0217] array Sequences of potential DNA vaccine components, or coding sequences, are set forth below. DNA vaccines of the present invention may comprise any one of the nucleic acid sequences provided below, or variants thereof. Alternatively or additionally, DNA vaccines of the present invention may comprise a nucleic acid encoding any one of the amino acid sequences provided below, or variants thereof. Amino acid sequences of genes in the assembled DNA vaccine as shown in Figure 7: [ka] TIFF2026010064000003.tif227162TIFF2026010064000004.tif240160TIFF2026010064000005.tif241162TIFF2026010064000006.tif241161TIFF202 6010064000007.tif241161TIFF2026010064000008.tif241161TIFF2026010 064000009.tif241161TIFF2026010064000010.tif241162TIFF20260100640 00011.tif241159TIFF2026010064000012.tif241160TIFF2026010064000013.tif241160TIFF2026010064000014.tif241161TIFF2026010064000015.t if241159TIFF2026010064000016.tif242159TIFF2026010064000017.tif241162TIFF2026010064000018.tif241160TIFF2026010064000019.tif163160

[0218] The present invention provides: A. A cancer vaccine comprising a nucleic acid encoding the proteins MAGED4B and / or FJX1, or a variant thereof, and further encoding an immunogenic fragment of tetanus toxin. B. The cancer vaccine of paragraph A, wherein the MAGED4B protein comprises or consists of the sequence of SEQ ID NO: 3, or a variant thereof. C. The cancer vaccine of item A or item B, wherein the FJX1 protein comprises or consists of the sequence of SEQ ID NO: 4, or a variant thereof. D. The cancer vaccine of any one of paragraphs A-C, wherein the immunogenic fragment of tetanus toxin comprises or consists of the p30 MHC II epitope of tetanus toxin. E. The cancer vaccine of any one of paragraphs A-D, wherein the immunogenic fragment of tetanus toxin comprises or consists of DOM. F. The cancer vaccine of any one of paragraphs A-E, wherein the MAGED4B and FJX1 antigens are encoded as a single fusion protein. G. The cancer vaccine of any one of paragraphs A-F, wherein the immunogenic fragment of tetanus toxin, MAGED4B, and FJX1 are encoded as a single fusion protein. H. The cancer vaccine of any one of paragraphs A-G, wherein a linker residue is provided between one or more, or all, of the immunogenic fragment of tetanus toxin, MAGED4B and FJX1 antigens. I. The cancer vaccine of any one of paragraphs A-H, wherein the nucleic acid further encodes a signal peptide to increase secretion efficiency. J. The cancer vaccine of any one of paragraphs AI, wherein the nucleic acid further encodes one or more promoters. K. The cancer vaccine of any one of paragraphs A-J, wherein the nucleic acid further encodes a polyA transcription termination sequence. L. The cancer vaccine of any one of paragraphs A-K, wherein the nucleic acid comprises a sequence encoding SEQ ID NO: 2-4, or a variant thereof. M. The cancer vaccine of any one of paragraphs A to L, wherein the nucleic acid comprises a sequence encoding SEQ ID NO: 1-6. N. The cancer vaccine of any one of paragraphs AM, wherein the nucleic acid comprises or consists of a sequence encoding SEQ ID NO: 12, 13, or 14. O. A composition comprising the cancer vaccine described in any one of items A to N. P. A cancer vaccine according to any one of items A to M or a composition according to item O for use as a medicament. Q. A cancer vaccine according to any one of paragraphs A to M, or a composition according to paragraph O, for use in treating or preventing cancer in a subject. R. A method of treating or preventing cancer in a subject, the method comprising administering to a subject a cancer vaccine described in any one of paragraphs A-M, or a composition described in paragraph O. S. A cancer vaccine or composition for use according to item Q or a method of treatment or prevention according to item R, wherein the cancer to be treated or prevented is oral cavity cancer and / or oropharyngeal cancer. T. A cancer vaccine or composition for use according to any one of paragraphs P, Q or S, or a method of treatment or prevention according to paragraph R or S, wherein the cancer vaccine or composition is used in combination with administration of a checkpoint inhibitor to a subject. U. The cancer vaccine or composition for use of, or the method of, item T, wherein the checkpoint inhibitor comprises an anti-PD1 or anti-CTLA4 binding molecule, or a nucleic acid encoding an anti-PD1 or anti-CTLA4 binding molecule. V. A kit for treating or preventing cancer, comprising: - a cancer vaccine according to any one of items A to M; and - a kit comprising a checkpoint inhibitor, such as an anti-PD1 or anti-CTLA4 binding molecule. W. A kit for treating or preventing cancer, comprising: - the first cancer vaccine according to any one of paragraphs A to M, wherein the nucleic acid encodes MAGED4B; - a second cancer vaccine according to any one of paragraphs A to M, wherein the nucleic acid encodes FJX1; and optionally, - a kit comprising a checkpoint inhibitor, such as an anti-PD1 binding molecule.

[0219] The invention has been broadly described; the materials and methods for the data obtained and presented in the figures contained herein are provided below, and specific information regarding matters such as treatment strategies is included in the figures and legends. The methods and data provided herein are in support of the invention and illustratively show several alternative vaccine constructs, etc.

[0220] Example Example 1: Evaluation of the immunogenicity of a DNA vaccine targeting MAGED4B / FJX antigens in HPV-ve HNSCC Target antigen expression and pre-existing T cell responses in patients with HPV-unrelated HNSCC Although there is an intriguing prospect for developing patient-specific vaccines based on individual tumor mutagenesis, the high cost and technical difficulties of such approaches make it unlikely that they will benefit most patients, even if successful. Identifying common tumor antigens shared among patients for the production of universal cancer vaccines would provide inexpensive and widely available treatments for OSCC. Among different types of TAAs, cancer / testis (CT) antigens are highly promising therapeutic targets. Cellular and humoral immune responses against CT antigens are frequently observed in cancer patients, and a correlation exists between CT antigen expression and the cytolytic activity of tumor immune infiltrates. The immunogenicity and cancer specificity of CT antigens make them priority targets for cancer immunotherapy, and their therapeutic effects have been tested in various clinical settings. CT antigen vaccines are generally well tolerated, and numerous cancer vaccination trials evaluating their therapeutic efficacy are currently underway. We selected two cancer / testis antigens, MAGED4B and FJX1, which have been found to be highly expressed in OSCC. We then extended these data to an analysis of the Cancer Genome Atlas (CGA) dataset to confirm their expression. Overall, the two antigens were expressed at the RNA level in 96% of OSCC cases. Furthermore, we confirmed protein expression of both antigens in oral dysplasia and OSCC cases (10 / 10 were positive; 5 cases per condition), but not in nonmalignant oral mucosa (Figure 2). Protein expression data from healthy tissues were similar to investigations of pre-existing immunity to the antigens in patients with HPV-unrelated HNSCC using HLA-A2 tetramers (currently available only for MAGED4B) and overlapping peptide pools (OPPs) against the full amino acid sequence of each antigen. These were measured in both blood and tumors using expanded tumor-infiltrating lymphocytes. Circulating MAGED4B tetramer-positive CD8+ T cells were observed in 4 / 6 HLA-A2 patients (0.04–0.1% of total CD8+ T cells) (Figure 3), with 2 / 2 expanded TILs also being tetramer-positive at similar frequencies (Figure 4A and Figure 5).Higher levels of MAGED4B-positive CD8 T cells (5-10 times higher) were detected using OPP in HLA-A2-negative HLA-A1-positive TIL samples, demonstrating reactivity beyond HLA-A2 restriction (Figure 4B). Previously, CD8 T cell reactivity against FJX1 was assessed using OPP only in expanded TIL samples, demonstrating CD8 reactivity in HLA-A1-positive patients coexisting with MAGED4B CD8 T cells (Figure 4B). Patient data demonstrated strong immunogenicity of both antigens, most notably in the case of MAGED4B.

[0221] Example 2: Preclinical Data on DNA Vaccine Efficacy / Immunogenicity Vaccine Design In HPV+ cancers, DNA vaccines encoding immunogenic viral antigens have made great progress, and recent results from a randomized phase 2b clinical trial of cervical tumors demonstrate histopathological regression of disease. Our approach to DNA vaccination was to deliver tumor-specific peptides or antigens in the presence of immunogenic sequences from the tetanus toxin domain (Dom). The vaccine design aims to provide binding CD4 T cells for optimal induction of CD8+ T cells in cancer patients, a highly potent strategy for breaking immune tolerance. Phase 2 clinical data suggest that DNA vaccination can overcome peripheral immune tolerance in tumor tissue by eliciting CD8 T cell responses against tumor antigens (carcinoembryonic antigen; CEA) that are detected post-vaccination and demonstrate clinical benefit. Therefore, we applied the Dom-based design to generate DNA vaccines encoding full-length MAGED4B and FJX1 antigens (p.Dom-MAGED4BFL and p.Dom-FJX1FL). Here, we optimized the full-length antigen design to achieve broader population coverage and not focus on targeting individual HLA alleles (HLA-A2).

[0222] Mouse model For immunogenicity, HLA-A2 transgenic mice (HHD) were used without tumor challenge. B16 / F10 mice were transfected with human HLA-A2, MAGED4B, and FJX1 constructs to mimic the expression of these antigens in HNSCC, and used in the HLA-A2 transgenic humanized mouse model (B6.Cg-Tg(HLA-A / H2-D)2Enge / J). Tumors were administered subcutaneously.

[0223] The immunogenicity of each vaccine was confirmed, and subsequently, a single dose of vaccine was administered to non-tumor-bearing mice by electroporation (Figure 7). Antigen-specific T cell responses were measured using overlapping peptide pools (OPPs) covering the entire sequence of each antigen (MAGED4B 183 peptides; FJX1 107 peptides).

[0224] For tumor challenge experiments, mice were vaccinated when tumors were palpable (size) on day 3. The DNA vaccine was administered twice at 3-week intervals as a mixture with or without anti-PD1 (in vivo Mab anti-mouse PD1 (RMP1-14, BEO146 from BioXcell), which served as a control). The experiment was performed in parallel with immunogenicity measurements using OPP as described above. The DNA vaccine was able to reduce / suppress tumor growth at a similar level to anti-PD1 versus the control DNA vaccine (p.Dom backbone), and when combined together, produced significant synergistic effects (Figure 9B). The data were consistent with the demonstration of induction of MAGED4B-specific T cells and an increase in MAGED4B-specific T cells when combined with anti-PD1 (Figure 9C). Collectively, the preclinical data demonstrate that DNA vaccines targeting MAGED4B / FJX1 have great potential to suppress the growth of tumors expressing these antigens, which can be further enhanced by combining with anti-PD1.

[0225] Example 3: Alternative gene fusion partners - helper motifs - MITD, PVXCP, MIP3α It has been shown that MHC class I trafficking signals (MITDs) attached to the C-terminus of target antigens promote the presentation of both MHC1 and MHC2 epitopes, leading to polyepitopic proliferation of CD4 and CD8 T cells (Kreiter S, et al. J Immunol. 2008;180(1):309-18).

[0226] PVXCP (Potato virus X coat protein) is a helper sequence that has been shown to enhance the induction of T cell responses to fusion cancer antigens through a mechanism of binding T cell help similar to that of the DOM helper sequence (Savelyeva N et al. Nature biotechnology. 2001;19(8):760-4, and Stegantseva MV et al., Cancer immunology, immunotherapy :CII. 2020).

[0227] Antigens fused to the chemokine MIP3α have been shown to target immature DCs via the chemokine receptor CCR6 (Biragyn A et al. J Immunol. 2001;167(11):6644-53). After receptor-mediated uptake, the fused antigens are presented by both MHC class I and II and activate significant CD4+ and CD8+ T cell responses (Biragyn A et al. Blood. 2004;104(7):1961-9, Biragyn A, et al. J Immunol. 2007;179(2):1381-8).

[0228] These fusions are shown in Figure 13, and the legend to this diagram provides further information.

[0229] Assembly of fusion constructs and helper motifs MITD (165 bp) encodes the HLA-A2 trafficking signal. PVXCP (732 bp) encodes the potato virus X coat protein. MIP3α (252 bp) encodes the macrophage inflammatory protein 3 alpha. The MITD, PVXCP, and MIP3α genes were codon-optimized and synthesized by GeneArt (Invitrogen). A leader sequence encoding the mouse (mus) IgH signal peptide (MGWSCIIFFLVATATGVHS) was inserted at the N-terminus of each construct to enhance secretion efficiency, apart from the MIP3α fusion construct, which has its own signal peptide. The fusion partner and the gene of interest (MAGED4B or FJX1) were linked with a seven-amino acid linker (AAAGPGP). All fusion partners except MITD were fused upstream of the target cancer antigen (Figure 13). The MITD sequence was added downstream of the antigen sequence. The genes for MAGED4B, FJX1 or their desired fusions were inserted into the pcDNA3 vector at the NotI, XhoI and XbaI restriction enzyme sites to generate the DNA vaccine constructs.

[0230] Assessment of immunogenicity of fusion constructs containing DOM and different fusion helper motif partners: The general vaccination protocol for evaluation of the immunogenicity of DNA vaccines alone or in combination with electroporation was prime / boost (Figures 17; 21-26) (specific vaccine constructs are indicated in the legends of each figure):

[0231] Three groups of 5–6 non-tumor-bearing HHD mice (transgenic for human HLA-A2 alleles) were vaccinated with 50 μg of p.Dom-MAGED4B, p.Dom-FJX1, or p.Dom on day 1, followed by a booster injection of the same DNA vaccine by electroporation on day 22. Their immunogenicity was assessed by IFNγ ELISpot assay. Lymphocytes isolated from mouse spleens on day 35 were seeded onto ELISpot culture dishes (Figure 8). In the experiments shown in Figures 21–26, the variable doses of dbDNA vaccines are indicated in the table legends.

[0232] In the experiments in Figures 11, 12, and 14, vaccinations were administered on days 1 and 8, and spleens for ELISPOT were harvested on day 22 (specific vaccine constructs are indicated in the legends of each figure).

[0233] In Figures 15, 19, and 20, evaluation was performed after stimulation with the generic (specific vaccine constructs are indicated in the legends of each figure) protocol alone: ​​non-tumor-bearing C57B / 6 mice were vaccinated with 50 μg of pDOM plasmid vaccine (3 mice, as a negative control), 25 μg of DB-DOM-FJX1-CO (5 mice), and 25 μg of pDOM-FJX1 plasmid (5 mice). Vaccines were administered intramuscularly (i.m.) on day 1. In the experiment in Figure 15, mice received 50 μg of DNA vaccine. Lymphocytes isolated from mouse spleens on day 14 were seeded into ELISPOT culture dishes.

[0234] Generic ELISPOT protocol used herein: IFNγ ELISpot was performed according to the manufacturer's protocol (BD Bioscience). Briefly, lymphocytes were isolated from the spleens of vaccinated mice using Lymphoprep™ and 2.5 x 10 5Cells were seeded into ELISPOT culture dishes at 100 cells / well. Overlapping peptide pools (OPPs) for each target antigen, MAGED4B or FJX1, were added to a final concentration of 1 μM and incubated for 40 hours at 37°C in 5% RPMI supplemented with 10% FCS, 20 mM L-glutamine, and 10 U / ml penicillin / streptomycin. The overlapping peptide pools for the entire sequence of each antigen consisted of 15-mer peptides with an 11-aa overlap; 183 peptides were pooled for MAGED4B and 107 peptides for FJX1 (JPT, Germany). The FJX1 OPP served as a negative control for the MAGED4B-targeted vaccine, and vice versa. The spot-forming units (SFU) corresponding to each responding T cell were imaged and counted using an AID ELISPOT plate reader system ELR04 and software (AID Autoimmun Diagnostika GmbH, Strassburg, Germany). Graphs were generated using the PRISM graphPad package.

[0235] Example 4: Identification of MAGED4B antigen on CAFs Immunohistochemical analysis of an HNSCC case shows strong MAGED4B expression in cancer-associated fibroblasts as well as cancer cells. After antigen retrieval using a high-pH (Tris-EDTA, pH(9)) buffer, anti-MAGED4B monoclonal antibody (Santa Cruz, G12; sc-393059) was used at a 1:50 dilution on HNSCC tissue. Deparaffinization, rehydration, antigen retrieval, and IHC staining were performed using a Dako PT Link Autostainer (EnVision FLEX Target Retrieval Solution, High pH (Agilent Dako) and a DAKO Autostainer Link48 from the Department of Cellular Pathology of the University Hospital of Southampton NHS Trust). DAKO Envision FLEX Mouse Linker was applied to sections for 15 minutes; DAKO Envision FLEX HRP (20 minutes) and DAKO Substrate Working Solution (10 minutes) were applied, followed by counterstaining with DAKO Envision FLEX Hematoxylin for 5 minutes. Images were acquired at the WISH Lab using a ZEISS Axio scanner. Six individual HNSCC cases are presented (Figure 27).

[0236] Analysis of 10 HNSCC cases confirmed tumor cell expression of MAGED4B in 9 / 10 cases. The level of tumor cell expression was assessed using the H Score (the product of staining intensity (score 0–3) and the percentage of positive cells (score 0–100)); the maximum possible score is 300, i.e., 100% of tumor cells exhibit strong staining. Notably (and unexpectedly), high expression of MAGED4 was also observed in CAFs (7 / 10 cases), indicated as CAF+ in the table below. CAF staining was assessed as positive or negative. We confirmed CAF MAGED4B expression by analyzing scRNASeq HNSCC transcriptome data, which also confirmed MAGED4B expression by HNSCC cells (Figure 28A). Single-cell RNA sequencing (scRNASeq) has emerged as a powerful method for quantifying the transcriptome of individual cells, and suitable protocols are outlined in Andrews, T and Hemberg, M, Molecular Aspects of Medicine, Volume 59, February 2018, Pages 114-122. [Table 1] The table shows the intensity of staining of tumor cells: CAF+ indicates strong staining for MAGED4B in CAFs.

[0237] Example 5: Identification of MAGED4B expression in tumor and normal tissues These studies were designed to evaluate MAGED4B and FJX1 expression in both tumor and normal tissue samples. Tumor biopsies and normal tissue microarray samples from OSCC patients were obtained and subjected to immunohistochemistry analysis. Strong expression of both antigens was evident in all samples from OSCC patients: 5 / 5 samples with HPV-HNSCC and 5 / 5 in oral dysplastic tissue for each antigen (Southampton, UK cohort). Twenty-eight samples were evaluated for antigen expression; 28 / 28 samples were positive for MAGED4B and 27 / 28 samples were positive for FJX1 (Malaysia cohort). In addition, five samples from lung cancer patients (three LUAD and two LUSC samples) showed strong expression of MAGED4B.

[0238] The suitability of targeting either of these two antigens was suggested by analyses showing no / negligible expression in healthy tissues and confirmed by low staining in TMA samples (data not shown). Therefore, the vaccine is not expected to induce an immune response against normal tissues.

[0239] This study determined the expression of both antigens in multiple HPV-ve HNSCC samples from patients in Malaysia and the UK and confirmed favorable tissue expression patterns in a tissue microarray panel of most organs, including non-dysplastic oral tissues.

[0240] For Southampton OSCC patient samples: Samples were stained using an automated Dako immunostainer according to the manufacturer's instructions. [Table 2]

[0241] In a Malaysian cohort of OSCC patients, FFPE blocks were identified, and 4 μm sections were prepared on positively charged glass slides. Briefly, wax from sections was melted at 65°C for 10 minutes, followed by deparaffinization by two 5-minute washes in a xylene substitute. Sections were then rehydrated in graded ethanol (2x 100% ethanol, 2x 95% ethanol, 1x 70% ethanol) and washed in distilled water for at least 30 seconds. Subsequently, they underwent heat-induced antigen retrieval (citrate buffer pH 6 for MAGED4B; Tris-EDTA buffer pH 9 for FJX1) at 99°C for 20 minutes. Nonspecific binding was blocked by incubating sections in Dual Endogenous Enzyme Blocking Reagent from the Dako Cytomation Envision+ Dual Link System HRP (DAB+) kit for 10 minutes at room temperature. The sections were then incubated with anti-MAGED4B (1:100 dilution) and anti-FJX1 (1:200 dilution) antibodies for 16 hours at 4°C. After incubation, the sections were washed and incubated with peroxidase-conjugated polymers (conjugated to goat anti-mouse and goat anti-rabbit) for 30 minutes at room temperature. After hematoxylin staining and dehydration through graded ethanol, positive binding for each antibody was revealed under a microscope with DAB+ chromogenic substrate and coverslips. [Table 3]

[0242] The study confirmed the following: Strong expression of both antigens in HNSCC / OSCC patient samples from the UK and Malaysia. Lung cancer samples also showed strong expression of MAGED4B (FJX1 was not analyzed), confirming the overexpression data generated using the TCGA database (described in detail in TGL-100_001-R TCGA). These data correspond broadly with available RNAseq data showing favorable tissue expression patterns; strong expression in tumors and low / no expression in healthy tissues.

[0243] Example 6: Preclinical studies in the B16 mouse model To demonstrate the impact of therapeutic vaccination with the vaccines described herein on tumor progression, we employed the antigen-expressing B16 model to subcutaneously challenge HLA-A2 transgenic AAD mice. Tumors were allowed to establish for 5 days prior to vaccination with the dual vaccine on day 5, and tumor volume assessments could begin once they were measurable approximately 10 days after administration. The combined effect of vaccine treatment and anti-PD-1 therapy was also evaluated.

[0244] Vaccine monotherapy delayed tumor growth compared with controls, and this effect was even more significantly enhanced when combined with anti-PD-1. Tumor evaluation by immunohistochemistry demonstrated that mice vaccinated with the dual vaccine had increased T cell infiltrates compared with mice vaccinated with the pDOM control. Flow cytometry further demonstrated that these infiltrates contained increased numbers of activated CD4+ and CD8+ T cells compared with pDOM controls. Increased expression of the T cell exhaustion marker PD-1 indicated that combining PD-1 blockade with vaccination may be beneficial.

[0245] This study supports our proposed mechanism of action that vaccination targeting the novel antigens MAGED4B and / or FJX1 in combination with PD-1 blockade can effectively induce T cell-mediated tumor attack.

[0246] The efficacy of two doses of the vaccine administered intramuscularly (i.m.) was tested in AAD mice (HLA-A2+ / Kb+) in the BAM model (B16 melanoma tumors genetically modified to express MAGED4B and HLA-A2) or the BAF model (B16 melanoma tumors genetically modified to express huFJX1 and HLA-A2). BAM cells confirmed the expression of MAGED4B and murine FJX1, which is 95% homologous to human FJX1. BAF cells confirmed the expression of huFJX1.

[0247] The B16F10 melanoma cell line expressing the human HLA-A2 gene was a gift from Professor Eric Tartour (Universite Paris Descartes, Paris). This cell line was cultured in RPMI 1640 supplemented with 10% heat-inactivated fetal bovine serum, penicillin / streptomycin (100 U / ml), and 1 mg / ml G418. B16F10 / HLA-A2 was verified to endogenously express mouse FJX1 and was engineered to express human MAGED4B (B16F10 / HLA-A2 / MAGED4B, "BAM"). Concurrently, B16F10 / HLA-A2 was engineered to express human FJX1 (B16F10 / HLA-A2 / FJX1, "BAF"). Expression of HLA-A2 in these cell lines was confirmed by flow cytometry using a human HLA-A2-PE (clone BB7.2)-binding antibody, and MAGED4B and FJX1 expression levels were confirmed by Western blotting using custom-made anti-MAGED4B and anti-FJX1 antibodies, respectively.

[0248] The immunogenicity and efficacy of the DNA vaccine were tested in transgenic mice expressing the chimeric HLA-A2.1 / H2-Dd MHC class I molecule (B6.Cg-Immp2lTg(HLA-A / H2-D)2Enge / J) (also known as AAD mice) purchased from Jackson Laboratory, USA. AAD mice were bred in the animal laboratory for use in subsequent experiments.

[0249] Mice were cultured with the BAM cell line (10 6 Mice were challenged with either 50 μg of each vaccine or 50 μg of the pDOM DNA vaccine control (intramuscularly) in sterile saline on day 0, and then observed for palpable tumors on day 5 (average 25 mm 2pDOM controls expressed only the tetanus toxin DOM helper sequence. A booster injection was administered on day 12. Tumor growth was monitored until mice were sacrificed, and T cell infiltration of tumors was assessed by immunohistochemistry and flow cytometry. Tumor volume was estimated using the following formula: volume = 1 / 2(length x width) 2 The results for the plasmid-based vaccines are shown in Figures 9 and 10.

Claims

[Claim 1] A cancer vaccine comprising a nucleic acid encoding a MAGED4B protein or a variant thereof.

Images