Viral-based immuno therapeutic genetic construct comprising UL40 into which a tumour-associated antigenic peptide is incorporated

Abstract

The invention concerns a genetic construct with enhanced immunogenicity that encodes a recombinant protein or peptide sequence that includes at least a part of the human cytomegalovirus (HCMV) UL40 protein; a vector, such as a viral vector, comprising same; a pharmaceutical composition or vaccine composition comprising same; the use of said genetic construct or said viral vector or said pharmaceutical composition or said vaccine composition as a medicament; and a method of treating or preventing a disease using said genetic construct, or said viral vector, or said pharmaceutical composition or said vaccine composition.

Description

[0001] Viral-based immuno therapeutic

[0002] Field of the Invention

[0003] The invention concerns a genetic construct with enhanced immunogenicity that encodes a recombinant protein or peptide sequence that includes at least a part of the human cytomegalovirus (HCMV) LIL40 protein; a vector, such as a viral vector, comprising same; a pharmaceutical composition or vaccine composition comprising same; the use of said genetic construct or said viral vector or said pharmaceutical composition or said vaccine composition as a medicament; and a method of treating or preventing a disease using said genetic construct, or said viral vector, or said pharmaceutical composition or said vaccine composition.

[0004] Background of the Invention

[0005] Clinical and pre-clinical data suggest the importance of robust and long-lived anti-tumour T- cell responses in cancers such as colorectal cancer (CRC), with T-cell infiltration in tumours associated with improved patient survival. Cancer vaccines or other therapies that boost tumour-specific T cell responses represent one potential approach to inducing protective antitumour T cell immunity in cancer patients, particularly in patients that are post ‘curative’ treatment of intermediate stage disease (25 % relapse rate over 36 months), or conditions which predispose to cancer, such as the presence of multiple colonic polyps, Lynch syndrome, presence of dysplasia in inflammatory bowel disease in the context of CRC. Thus, development of such approaches that induce T cell responses that respond to and control primary tumour development, progression of micrometastasis and / or premalignant progression will reduce cancer burden.

[0006] T cell-mediated protection from tumour development is mediated by T cells reactive to either neoantigens that are specifically expressed by tumour cells (e.g., NY-ESO), or cancer- associated antigens that are proteins that are either mutated or aberrantly up-regulated by tumour cells (e.g., DNAJB7). One such tumour-associated antigen is 5T4, an oncofoetal antigen expressed by human trophoblasts and primary and metastatic solid tumours, with highly restricted expression in normal, adult tissues. In CRC, it is estimated that >90% of colorectal cancers are enriched for 5T4 expression. The utility of 5T4 in cancer vaccines has been demonstrated by TroVax, a recombinant modified vaccinia ankara (MVA) virus expressing 5T4, which demonstrates prophylactic and therapeutic efficacy in mouse models of melanoma and colorectal cancer using cell lines engineered to express human 5T4 4. However, a shortcoming of past and current vaccine platforms, including those developed during the COVID pandemic, is that the T-cell responses they induce wane over time. There is therefore a need for a vaccine or therapeutic having enhanced immunogenicity to promote prolonged T cell memory.

[0007] We herein disclose a viral based vector engineered to express immunogenic peptides involving a viral immunoevasin protein that promotes peptide presentation to the immune system and so enhanced immunogenicity. We have found administration with these vectors induces elevated CD8 T cell responses, as compared to controls, and affords superior CD8+ T cell associated protection. Thus, overall, we have found our invention can induce sustained and protective T-cell responses, such as against endogenously expressed tumour- associated antigen, and thus provides for a novel vaccine strategy in a variety of applications.

[0008] Statements of the Invention

[0009] According to a first aspect of the invention there is provided a genetic construct comprising nucleic acid encoding: i) human cytomegalovirus (HCMV) LIL40 protein, or a part thereof; and ii) a recombinant immunogenic or antigenic peptide sequence which is positioned within a part of said LIL40 protein of part i).

[0010] Reference herein to HMCV LIL40 protein, or a genetic construct encoding same, is refence to the coding sequence for the LIL40 protein and so does not include parts thereof that are cleaved prior to production of the final protein product such as a leader sequence.

[0011] Reference to a recombinant immunogenic or antigenic sequence is to an immunogenic or antigenic sequence from a species other than HCMV and, most suitably, an immunogenic or antigenic sequence from a species to be treated with or benefit from the use of the claimed genetic construct.

[0012] Reference herein to a genetic construct is reference to a DNA or RNA construct existing within a host or without a host, for example such a construct within a host is represented by the claimed genetic material in a bacterial or viral cell, whereas such a construct without a host is represented by isolated genetic material, such as mRNA. In a preferred embodiment of the invention said genetic construct is either DNA or RNA. Ideally, when DNA the genetic construct is gDNA or cDNA and, ideally, when RNA the genetic construct is mRNA, tRNA or non-coding RNA.

[0013] Those of skill in the art will appreciate DNA is very similar to RNA, but these two compositions differ in three primary ways:

[0014] Unlike double-stranded DNA, RNA is usually a single-stranded molecule (ssRNA) and consists of much shorter chains of nucleotides. However, double-stranded RNA (dsRNA) can form and (moreover) a single RNA molecule can, by complementary base pairing, form intrastrand double helixes, as in tRNA.

[0015] The sugar-phosphate "backbone" of DNA contains deoxyribose, whereas RNA contains ribose instead. Ribose has a hydroxyl group attached to the pentose ring in the 2' position, whereas deoxyribose does not. The hydroxyl groups in the ribose backbone make RNA more chemically labile than DNA by lowering the activation energy of hydrolysis.

[0016] The complementary base to adenine in DNA is thymine, whereas the complementary base to adenine in RNA, it is uracil, which is an unmethylated form of thymine.

[0017] In a preferred embodiment of the invention, when the construct exists within a host, ideally the host is a viral vector, and more ideally still, an adenovirus or an adenoviral vector.

[0018] Adenoviruses or adenoviral vectors are medium-sized (90-100 nm), nonenveloped (without an outer lipid bilayer) viruses with an icosahedral nucleocapsid containing a double stranded DNA genome.

[0019] In terms of categorization, in humans, there are 88 accepted human adenovirus serotypes (Ad-1 to 88) classified into seven species (Human adenovirus A to G): A - 12, 18, 31 ; B - 3, 7, 11 , 14, 16, 21 , 34, 35, 50, 55; C - 1 , 2, 5, 6, 57; D - 8, 9, 10, 13, 15, 17, 19, 20, 22, 23, 24, 25, 26, 27, 28, 29, 30, 32, 33, 36, 37, 38, 39, 42, 43, 44, 45, 46, 47, 48, 49, 51 , 53, 54, 56, 58, 59, 60, 62, 62, 64, 65, 67, 69, 70, 71 , 72, 73, 74, 75; E - 4; F - 40, 41 ; and G - 52. Accordingly, in a preferred embodiment said adenoviral vector is selected from the Ad-1 to 88 serotypes, and more preferably selected from Ad1 , 2, 5, 6 and 57 serotypes, and more preferably still is Adenovirus serotype 5 belonging to the C-subclass of adenovirus and as defined by accession number AC_000008.1. Alternatively, adenoviruses or adenoviral vectors can be categorised according to their genotype, as shown in Table 4.

[0020] As is known to those skilled in the art, LIL40 refers to an immuoevasive protein expressed by, and typically derived from, the Human Cytomegalovirus (HCMV). LIL40 is derived from the gene coding sequence for this protein. An immunoevasin is a protein expressed by some viruses that enables the virus to evade immune recognition by the host immune response. One example of viral immune evasion is interfering with MHC complexes in the infected cell, thus effectively blocking the recognition of viral protein fragments by the immune system. Some viral immunoevasins, block peptide entry into the endoplasmic reticulum (ER) by targeting the TAP transporters. Immunoevasins are particularly abundant in viruses that are capable of establishing long-term infections of the host, such as HCMV. Reference herein to LIL40 is exemplified by the protein defined by accession number Q6SW92 or F5HEM7. Other examples of the LIL40 protein are provided by different viral strains and are included within the scope of the invention.

[0021] In a preferred embodiment of the invention, the genetic construct encodes only a part of the LIL40 protein and ideally the N-terminal part. Most particularly the genetic construct encodes the signal sequence of the LIL40 protein and yet, more ideally still, a part of the LIL40 protein that is about 25 - 40 amino acids in length, ideally, 30 - 40 amino acids in length, more ideally still 35 - 40 amino acids, such as 37 amino acids in length.

[0022] In a further preferred embodiment, said LIL40 protein encoded by said genetic construct comprises an N-terminal LIL40 signal sequence, most preferably having the sequence:

[0023] - MNKFSNTRIGFTCAVMAPRTLILTVGLLCMRIRSLLC (SEQ ID NO: 1); or a sequence that has 75% identity or similarity therewith.

[0024] As is known in the art, the LIL40 signal sequence comprises the nonameric peptide VMAPRTLIL (SEQ ID NO: 2); and only slight variations thereof, due the conserved nature of the sequence, depending on the viral strain. HCMV circumvents host immunity through numerous mechanisms, including downregulation of MHC class I from the surface of infected cells. Although this process restricts recognition of virus-infected cells by CD8+T cells, it concurrently renders cells vulnerable to recognition and attack by NK cells. One strategy employed by HCMV to evade NK cell killing is to up-regulate the nonclassical MHC I molecule HLA-E which binds to the inhibitory NK cell receptor LIR1 and thus antagonises NK cell function. Cell surface expression of HLA-E requires loading of the conserved nonameric peptide, VMAPRTLIL (SEQ ID NO: 2), onto HLA-E in a TAP-dependent process. VMAPRTLIL (SEQ ID NO: 2) is a naturally derived peptide from the signal sequence of classical MHC I and HLA-G molecules. HCMV exploits this process through the expression of LIL40 (Fig. 3A). This LIL40 viral protein thus promotes surface presentation of HLA-E, using a nonameric HLA-E binding peptide homologous to endogenous VMAPRTLIL (SEQ ID NO: 2) and translocating this into the endoplasmic reticulum lumen in a TAP-independent process (Fig. 3A). We herein demonstrate that, by replacing the UL40 nonameric peptide VMAPRTLIL (SEQ ID NO: 2) of UL140 signal sequence, including conserved variants thereof, with an antigenic or immunogenic peptide, and presenting or expressing same in / from a genetic construct, such as in one embodiment only a viral vector, we can enhance antigenic or immunogenic peptide presentation and subsequent stimulation of antigen specific T cells (Fig. 3A).

[0025] Therefore, according to a preferred embodiment, said UL40 signal sequence encoded by said genetic construct, comprises or consists of the sequence MNKFSNTRIGFTCAVMAPRTLILTVGLLCMRIRSLLC (SEQ ID NO: 3) wherein said nonameric peptide VMAPRTLIL (SEQ ID NO: 2), or part thereof, is replaced with an immunogenic or antigenic peptide sequence, derived from a species in which, or with which, the genetic construct is to be used; hence the reference to a recombinant protein or peptide sequence.

[0026] Therefore, accordingly in yet a further preferred embodiment of the invention, said UL40 signal sequence encoded by said genetic construct comprises, or consists, of the sequence MNKFSNTRIGFTCA(SEQ ID NO: 62)-Xi-TVGLLCMRIRSLLC (SEQ ID NO: 4), or a sequence 75% identical, or similar, thereto, wherein Xi represents an immunogenic or antigenic peptide sequence.

[0027] It is preferred that the recombinant UL40 signal sequence of the invention has at least 75% identity, or similarity, with the peptide sequence MNKFSNTRIGFTCA(SEQ ID NO: 62)-Xi- TVGLLCMRIRSLLC (SEQ ID NO: 4), and in increasing order of preference, at least 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94% 95%, 96%, 97%, 98% or 99% identity or similarity.

[0028] The skilled person will appreciate that homologues, orthologues or functional derivatives of the recombinant protein or peptide sequence of the invention comprising a signal sequence of LIL40 will also find use in the context of the present invention. Thus, for instance peptides which include one or more additions, deletions, substitutions or the like are encompassed by the present invention. In addition, it may be possible to replace one amino acid with another of similar “type”. For instance, replacing one hydrophobic amino acid with another one can be achieved by using a program such as the CLUSTAL program to compare amino acid sequences. This program compares amino acid sequences and finds the optimal alignment by inserting spaces in either sequence as appropriate. It is possible to calculate amino acid identity or similarity (identity means conservation of amino acid type) for an optimal alignment. A program like BLASTx will align the longest stretch of similar sequences and assign a value to the fit. It is thus possible to obtain a comparison where several regions of similarity are found, each having a different score. Both types of analysis are contemplated in the present invention.

[0029] As is known in the art, a variant polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions or truncations that may be present in any combination. Among preferred variants are those that vary from a reference polypeptide by conservative amino acid substitutions. Such substitutions are those that substitute a given amino acid for another amino acid of like characteristics. For example, charged amino acid residues include lysine (+), arginine (+), histidine (+), aspartate (-) and glutamate (-); polar amino acids include serine, threonine, asparagine, glutamine, and tyrosine whereas the hydrophobic amino acids include alanine, valine, leucine, isoleucine, proline, phenylalanine, tryptophan, cysteine and methionine. Generally, glycine is often found at the surface of proteins, within a loop- or coil region, providing high flexibility to the polypeptide chain at these locations. This suggests that it is rather hydrophilic. Proline, on the other hand, is generally non-polar and is mostly found buried inside the protein, although similarly to glycine, it is often found in loop regions. In contrast to glycine, proline provides rigidity to the polypeptide chain by imposing certain torsion angles on the segment of the structure. Glycine and proline are often highly conserved within a protein family since they are essential for the conservation of a particular protein fold. In addition, in the following non-limiting groups of amino acids, each amino acid within each group is considered a conservative replacement for one another: a) alanine, serine, and threonine; b) glutamic acid and aspartic acid; c) asparagine and glutamine d) arginine, histidine and lysine; e) isoleucine, leucine, methionine and valine and f) phenylalanine, tyrosine and tryptophan. Most highly preferred are variants that retain or have enhanced biological function, or immunogenicity, having regard to the reference polypeptide from which it varies. Reference herein to an immunogenic or antigenic protein or peptide sequence for use in the recombinant protein or peptide sequence of the invention refers to a sequence not part of LIL40 protein, in particular the signal sequence part thereof, that can elicit an immune response when used in viva, this response may be measured or determined using known tests such as those described herein. One test which may be used typically, but not exclusively, is whether the immunogenic or antigenic protein or peptide can cause a tumour to be recognised and acted upon by components of the immune system.

[0030] Examples of an immunogenic or antigenic peptide include but are not limited to those known in the art, such as tumour associated antigens, neoantigens, viral proteins, bacterial proteins, and fungal proteins. Particularly preferred immunogenic or antigenic peptides are those comprising 5 - 30 amino acids, ideally 5 - 20 amino acids or 7 - 9 amino acids, most ideally 8 or 9 amino acids. More preferably still, preferred immunogenic or antigenic peptides are those that associate with or characterise a disease to be treated using the technology of the invention.

[0031] As will be appreciated by those skilled in the art, measurement and / or comparison of immunogenicity can be carried out by any means known to those skilled in the art such as, but not limited to: vaccination studies in mice, including transgenic mice that express human MHC molecules; in vitro experiments using human T cell clones that react to the peptide inserted into LIL40; and clinical trials in humans.

[0032] Preferably said immunogenic or antigenic peptide sequence is a Major Histology Complex of Class I (MHCI) class restricted peptide sequence. As used herein "Major Histocompatibility Complex of class I" molecules refer to one of two primary classes of major histocompatibility complex (MHC) molecules (the other being MHC class II) that are found on nearly every nucleated cell of the body. Their function is to display fragments of proteins from within the cell to T cells; healthy cells will be ignored, while cells containing foreign proteins will be attacked by the immune system. Class I MHC molecules bind peptides generated mainly from degradation of cytosolic proteins by the proteasome. The bound MHC I peptide complex is then inserted into the plasma membrane of the cell. The peptide is bound to the extracellular part of the class I MHC molecule. Thus, the function of the class I MHC is to display intracellular proteins to cytotoxic T cells (CTLs). However, class I MHC can also present peptides generated from exogenous proteins, in a process known as crosspresentation. As used herein "MHC-l-specific polypeptides" refer to those peptides, which are bound to MHC-I, i.e., the extracellular part of the class I MHC molecule, and displayed to CTLs. We have found that by incorporating at least a part of the LIL40 viral immunoevasin protein in our genetic construct, such as a viral vector, promotes MHC class I peptide presentation and enhances the immunogenicity of our RAd-based vector. Indeed, unexpectedly, we have found we can induce a robust and long-lasting antigen-specific, functional CD8+ T cell responses, with subsequent protection also being enhanced.

[0033] It will be apparent to those skilled in the art that the immunogenic or antigenic peptide sequence of the invention will be chosen having regard to the nature or the purpose of the genetic construct. Most typically, the genetic construct will be used as a medicament and the nature of the immunogenic or antigenic peptide sequence will be representative of the disease to be treated, and optionally the species to be treated. For example, known human cancer antigens or epitopes will be used to create a genetic construct that treats cancer in man and, in particular, cancer antigens or epitopes that are representative of particular types of cancer will be used to create a genetic construct that treats a particular type of cancer. Similarly, antigens or epitopes that are representative of particular types of infection will be used to create a genetic construct that treats a particular type of infection. In fact, any antigen or epitope that is representative of a particular disease may be used to work the invention as it can be used to create a genetic construct that treats a particular disease. Thus the invention lies in the discovery that the LIL40 protein, or at least the N-terminal part thereof, in particular the signal sequence thereof, can be made recombinant by the insertion of an immunogenic or antigenic peptide sequence therein, which peptide is derived from a species to be treated. Further, the immunogenic or antigenic peptide sequence is inserted, ideally, in the position shown herein to create an enhanced therapeutic, that is to say, one that is MHC-I presented and negates KN evasion or negates cell killing.

[0034] In one preferred embodiment, said immunogenic or antigenic peptide is 5T4, or a fragment thereof, (Accession ID: Q9Z0L0). As known in the art, 5T4 is an oncofoetal antigen expressed by human trophoblasts and primary and metastatic solid tumours, with highly restricted expression in normal, adult tissues. In CRC, it is estimated that >90% of colorectal cancers are enriched for 5T4 expression. In a preferred embodiment of the invention said 5T4 fragment comprises or consists of an amino acid sequence selected from the group comprising or consisting of: SAPSPLVEL (SEQ ID NO: 5); NSLVSLTYV (SEQ ID NO: 6); and VSFRNLTHL (SEQ ID NO: 7). Additionally, or alternatively, said 5T4 fragment is, ideally a HLA-A2 restricted epitope, selected from the group comprising or consisting of: RLARLALVL (SEQ ID NO: 8), GAFEHLPSL (SEQ ID NO: 9) and DLPAYVRNL (SEQ ID NO: 10).

[0035] In yet a further preferred embodiment, said genetic construct of the invention is in the form of, or incorporated in, a minigene construct that is adapted to express same. As is known in the art, a minigene is a minimal gene fragment that includes an exon and the control regions necessary for the gene to express itself in the vector. Typically said adaptation includes the provision of at least one transcription control sequences (e.g., at least one promoter sequence) which mediate(s) said expression. In a preferred embodiment, said minigene includes a promoter ideally, but not exclusively, one such as the major immediate-early promoter and enhancer of the human cytomegalovirus (hCMV-MIE), which is known to be one of the most potent DNA elements, driving recombinant gene expression in mammalian cells. Alternatively, the promoter is cell / tissue specific and more ideally still adapted for inducible or constitutive expression of said coding sequence.

[0036] Those skilled in the art will appreciate that the term promoter includes the following features, which are provided by way of example only, and not by way of limitation: at least one enhancer element which is a cis acting nucleic acid sequences often found 5’ to the transcription initiation site of a gene (enhancers can also be found 3’ to a gene sequence or even located in intronic sequences and is therefore position independent) that functions to increase the rate of transcription of the gene to which the enhancer is linked. Further, enhancer activity is responsive to trans acting transcription factors (such as polypeptides) which have been shown to bind specifically to enhancer elements. The binding / activity of transcription factors (please see Eukaryotic Transcription Factors, by David S Latchman, Academic Press Ltd, San Diego) is responsive to a number of environmental cues which include, by example and not by way of limitation, intermediary metabolites (e.g., glucose, lipids), environmental effectors (e.g., light, heat,).

[0037] Further adaptations are included within the scope of the invention, which facilitate the expression of the genetic construct of the invention, including transcription termination or polyadenylation sequences. This or these adaptions also include the provision of 2A selfcleaving peptides or internal ribosome entry sites (IRES) which function to maximise expression of vector encoded genes arranged in bicistronic or multi-cistronic expression cassettes. In a preferred embodiment, where the genetic construct is provided in an adenoviral viral vector, it is inserted in the E1 adenoviral region, either as an additional insertion or as a substitution. In this manner, advantageously, immune evasion of the adenoviral vector is reduced such that the immunogenic or antigenic peptide can elicit an immune response for the purpose of vaccination and / or disease clearance.

[0038] Those skilled in the art will appreciate the invention has use in a clinical and veterinary setting.

[0039] According to a second aspect of the invention there is provided the genetic construct as defined herein for use as a medicament.

[0040] According to a third aspect of the invention there is provided the genetic construct as defined herein for use in the treatment, or prevention, of a disease such as cancer and / or an infectious disease.

[0041] According to a fourth aspect of the invention there is provided the vector as defined herein for use in the manufacture of a medicament, ideally, to treat a disease such as cancer and / or an infectious disease.

[0042] Compounds for use in medicine will generally be provided in a pharmaceutical or veterinary composition and therefore according to a yet fifth aspect of the invention there is provided a pharmaceutical composition or vaccine comprising the vector as defined herein and a pharmaceutically acceptable carrier, adjuvant, diluent or excipient.

[0043] Suitable pharmaceutical excipients are well known to those of skill in the art. Pharmaceutical compositions may be formulated for administration by any suitable route, for example oral, buccal, nasal or bronchial (inhaled), transdermal or parenteral and may be prepared by any method well known in the art of pharmacy.

[0044] The composition may be prepared by bringing into association the above defined vector with the carrier, adjuvant, diluent or excipient. In general, the formulations are prepared by uniformly and intimately bringing into association the adenovirus with liquid carriers or finely divided solid carriers or both, and then, if necessary, shaping the product. The invention extends to methods for preparing a pharmaceutical composition comprising bringing a vector as defined above in conjunction or association with a pharmaceutically or veterinary acceptable carrier or vehicle. Most suitably said vaccine or said pharmaceutical composition is formulated for human or veterinary use.

[0045] According to an even further aspect of the invention, there is provided a method for treating or preventing cancer comprising administering an effective amount of the viral vector or pharmaceutical composition or vaccine as defined herein to a patient in need thereof. Most preferably, said cancer is

[0046] Reference herein to an "effective amount" of the adenovirus or a composition comprising same is one that is sufficient to achieve a desired biological effect, such as cancer cell death. It is understood that the effective dosage will be dependent upon the age, sex, health, and weight of the recipient, kind of concurrent treatment, if any, frequency of treatment, and the nature of the effect desired. Typically, the effective amount is determined by those administering the treatment.

[0047] In a preferred method, the viral vector or vaccine or pharmaceutical composition according to the invention may be administered to a subject by any suitable route. Preferably this is by direct intra-tumoral injection when treating malignant solid tumours, including through the use of imaging guidance to target the tumour or tumours. Intra-tumoral injection includes direct injection into superficial skin, subcutaneous or nodal tumours, and imaging guided (including CT, MRI or ultrasound) injection into deeper or harder to localize deposits including in visceral organs and elsewhere. In another preferred embodiment, the adenoviral vector is injected into a blood vessel, preferably a blood vessel supplying a tumour.

[0048] According to a further aspect of the invention there is provided a method of vaccinating against cancer comprising administering said viral vector or said pharmaceutical composition or said vaccine to an individual.

[0049] Most preferably the cancer referred to herein includes any one or more of the following cancers: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor,, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, non-Hodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, pancreatic cancer, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer, stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

[0050] In the claims which follow and in the preceding description of the invention, except where the context requires otherwise due to express language or necessary implication, the word “comprises”, or variations such as “comprises” or “comprising” is used in an inclusive sense i.e. to specify the presence of the stated features but not to preclude the presence or addition of further features in various embodiments of the invention.

[0051] All references, including any patent or patent application, cited in this specification are hereby incorporated by reference. No admission is made that any reference constitutes prior art. Further, no admission is made that any of the prior art constitutes part of the common general knowledge in the art.

[0052] Preferred features of each aspect of the invention may be as described in connection with any of the other aspects.

[0053] Other features of the present invention will become apparent from the following examples. Generally speaking, the invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including the accompanying claims and drawings). Thus, features, integers, characteristics, compounds, or chemical moieties described in conjunction with a particular aspect, embodiment or example of the invention are to be understood to be applicable to any other aspect, embodiment or example described herein, unless incompatible therewith. Moreover, unless stated otherwise, any feature disclosed herein may be replaced by an alternative feature serving the same or a similar purpose.

[0054] Throughout the description and claims of this specification, the singular encompasses the plural unless the context otherwise requires. In particular, where the indefinite article is used, the specification is to be understood as contemplating plurality as well as singularity, unless the context requires otherwise.

[0055] An embodiment of the present invention will now be described by way of example only with reference to the following wherein:

[0056] Figure 1 - Mapping of h5T4-derived MHC class I epitopes. (A) Splenocytes isolated from AdZ-CTRL or AdZ2-h5T4 immunized mice were incubated with 5T4 peptide pools spanning the entire protein. Phytohemagglutinin (PHA) served as a positive control (B, C). Splenocytes isolated from mice vaccinated with AdZ-CTRL (mouse 1-4), AdZ2-CTRL (mouse 5-8), AdZ-h5t4 (mouse 9-12) and AdZ2-h5T4 (mouse 13-16) were stimulated overnight with 5T4 peptides or PHA. Each response / mouse is represented as a mean of duplicate samples ± SD. IFN-y release was quantified and is presented as spot forming unit (SFU) per well.

[0057] Figure 2. h5T4-VSF induces persistent functional 5T4-specific CD8 T cell memory responses. (A) A schematic depicting the generation of 5T4 minigene vectors. Transgene of interest was inserted in the place of the E1 region (E1A), behind HCMV major immediate early promoter, followed by PolyA signal sequence. (1) RAd-Ctrl: control vector with HCMV promoter and PolyA sequence but without transgene inserted. (2-4) Minigene vectors containing individual 5T4 epitopes inserted in the E1 region. (B-D) Mice were subcutaneously immunized with control RAd vectors or vectors expressing individual epitopes SAPSPLVEL(SEQ ID NO: 5), NSLVSLTYV (SEQ ID NO: 6) or VSFRNLTHL (SEQ ID NO: 7). Blood was withdrawn from the lateral tail vein on days 8, 41 , 71 , 124 and 169. (B) Frequency of circulating CD8+CD3+T cells. (C) Frequency of 5T4-specific IFNy+CD8+T cell responses. (D) Frequency of 5T4-specific IFNy+ TNF-+ CD8+ T cell responses. Data are shown for individual mice (n=2-3 mice / group) with mean shown. Significance was assessed by two-way ANOVA with Dunnett's correction for multiple comparisons. *p<0.05, **p<0.01 , ***p<0.005 and ****p<0.0001. Figure 3. Development of UL40-minigene adenoviral 5T4-expressing vector. (A) Schematic depicting the function of LIL40 during HCMV infection (left) and the hypothesised role for LIL40 in promoting VSF peptide loading in the endoplasmic reticulum (right). (B) A schematic demonstrating how the HCMV gene LIL40 was inserted under the HCMV promoter MIE in the place of the adenovirus E1 gene. N-terminal of LIL40 gene contains a signal sequence, with a conserved HLA-E binding peptide VMAPRTLIL (SEQ ID NO: 2; UL40i5-23). VMAPRTLIL (SEQ ID NO: 2) peptide was replaced with 5T4 immunodominant MHC class I restricted epitope VSFRNLTHL (SEQ ID NO: 7; 5T4252-26o).

[0058] Figure 4. Systemic immunization with RAd-UL40-VSF induces increased frequencies of VSF-specific T cell responses overtime. (A) 5T4 protein expression in polyps, intestinal tissue and the positive control cell lines 4T 1 and CT26. (B-E) APC-Min mice were vaccinated i.v. with 2x109pfu with RAd-Ctrl, RAd-VSF or RAd-UL40-VSF. Blood was withdrawn from the lateral tail vein on days 37, 71 , 105, 149 and 189 and stimulated ex vivo for 6 hours with VSFRNLTHL (SEQ ID NO: 7) peptide (VSF). Cells were stained for IFNy- and TNF- to measure 5T4-specific responses (B). Frequency of VSF-specific IFNy-+CD8+(C) and IFNy- +TNF+CD8+ (D) T cell responses. Data for individual mice with mean. Significance was assessed by two-way ANOVA with Tukey’s multiple comparisons. *p<0.05 **p<0.01 and ****p<0.0001. (E) Representative flow cytometry plots gated on IFNy -+and TNF-+T CD8+cells d149 post-vaccination following VSF peptide stimulation.

[0059] Figure 5. RAd-VSF-UL40 vaccination restricts microadenoma formation. (A) Intestinal transformation was assessed by immunohistochemistry and p-catenin staining to identify crypts where Ape gene deletion initiated Wnt-mediated oncogenic transformation. (Top) Typical phenotype of crypts with aberrant p-cateninhicells, displaying abnormal growth and proliferation 21 days after tamoxifen induction. (Bottom) Section from mice immunised with RAd-UL40-VSF (bottom left) or control vector (bottom right) treated with tamoxifen 170 days after vaccination. (B) Transformed crypts with positive p-catenin staining were counted with >10 stretches of the first 5cm of proximal small intestine scored, totalling 250 crypts. Data are shown as individual mice + mean: RAd-Ctrl (n=6), RAd-VSF (n=11), RAd- UL40 VSF group (n=12), from 2 experiments. Significance was assessed by ordinary oneway Anova with Dunnett's correction for multiple comparisons.

[0060] Figure 6. VSF-UL40 expressing adenovirus-based vectors restrict CRC development. C57BL / 6 mice were vaccinated with control RAd5, RAd-VSF or RAd-VSF-UL40, and after 206 days mice were challenged intra-colonically with MC38 cells. After 7, 10 and 14 days tumour development was assessed by endoscopy and images were taken. ND: tumour not detected, X: Mouse died.

[0061] Figure 7. VSF-UL40 promotes stability of surface MHC class I. A) Histogram overlay and B) quantification of surface MHC expression by TAP-deficient RMA-S cells 48 hours postvirus inoculum. As positive controls, some cells were incubated with 100uM VSF peptide.

[0062] Materials and methods

[0063] Adenoviral vector construction

[0064] Replication-deficient human adenovirus serotype 5 constructs were generated using the AdZ vector system as previously described11[see also AdZ.cf.ac.uk], These vectors lack the E1 and E3 regions required for viral replication and immune evasion respectively. In some versions, a vector was used in which the whole of E4 had also been deleted, apart from E4- ORF6. For minigene constructs, complementary oligonucleotides encoding the minigene sequence, along with flanking sequences homologous to the vector, were recombineered into pAdZ-5-CV5. For LIL40 constructs, a vector expressing LIL40 was used12. A cassette encoding a selection cassette (SacB / amp7LacZa11) was amplified by PCR and inserted by recombineering into the signal peptide of LIL40, replacing the native HLA-E epitope. Then the desired epitope was inserted by recombineering in place of the cassette, using a single oligonucleotide encoding the desired sequence flanked by sequences homologous to LIL40.

[0065] Table 1: Minigene primers

[0066] Table 2: UL40 Primers Mice

[0067] All studies were performed under the UK Home Office-approved Project Licenses PPL 30 / 3428, PP6945193 and P7867DADD. All animals were kept in accordance with the Cardiff University and United Kingdom Home Office regulation and all experiments were conducted according to UK Home Office guidelines. Mice were injected with 5x108PFU of RAds in subcutaneous (s.c.) infections or 2x109PFU RAds intra-venously (i.v.).

[0068] Peptides

[0069] Human 5T4 (h5T4) peptide pools were generated using forty-one 20mers, overlapping by 10 aa that covered the entire length of h5T4 protein (Table 3). The peptide pools were designed by adapting a matrix system (Table 3b) whereby 5-7 peptides at a concentration of 5 pg / ml per peptide formed a single peptide pool. Peptides were ordered from GLBiocem (Shanghai, China) at a 95% purity. For flow cytometry studies, individual MHC Class I restricted h5T4 peptides were synthesised at a purity of >95% by Peptide Synthetics (Fareham, UK): peptides 5T4i70-i78 (SAPSPLVEL (SEQ ID NO: 5)), 5T4244-252 (NSLVSLTYV (SEQ ID NO: 6)) and 5T4253-26o (VSFRNLTHL(SEQ ID NO: 7)).

[0070] 5T4 Peptides:

[0071] 5T4 PEPTIDE POOLS CONCENTRATIONS:- TRIAL STOCK (GLS Biochem):

[0072] 1mg / ml / peptide = 200ug / peptide in 200ul PBS (+ 4ul)

[0073] Final concentration in culture = 5ug / ml I peptide (Add 0.5ul to 100ul) (%DMSO in culture = 0.33%)

[0074] Enzyme-Linked ImmunoSpot (ELISpot) assay

[0075] Polymer-backed 96-well filtration plates (MerckMillipore) were pre-coated with anti-mlFN-y antibody overnight at 4°C, blocked with RPMI 5% FCS prior to plating out splenocytes + / - whole 5T4 protein (10 pg / ml, Oxford BioMedica), 5T4 peptide pools (5 pg / ml per peptide) or individual 5T4 peptides (5 pg / ml / peptide) for 18-24hr. As a positive control, Phytohemagglutinin (PHA) was used at a concentration of 10 pg / ml. The cells were discarded, and the plates were handled as per the instructions supplied by the manufacturer (Mabtech). Spot forming units were quantified using an automated ELISpot plate reader (CTL Immunospot, USA).

[0076] Flow cytometric analysis of T cell responses

[0077] Lymphocytes derived from peripheral blood were stimulated with 3 pg / ml peptides, in the presence of 2 pg / mL Brefeldin A (Sigma-Aldrich) and they were incubated for 6 hours at 37°C. Cells were then stained with Live / Dead reagent (Zombie Aqua Fixable Viability Kit, BioLegend), prior to incubation with Fc block (Biolegend) and then antibodies reactive to CD3s (clone 145-2C11 , Biolegend and CD8a (clone 53-6.7). Cells were fixed, then permeabilised with saponin and stained with anti-IFNy and anti-TNF, washed in FACS buffer and data were then acquired on Attune NxT Flow Cytometer (Thermo Fisher Scientifics). Data were analysed using FlowJo software, version 10 (TreeStar inc, USA).

[0078] Microadenoma induction and quantification APC-Min mice were injected intraperitoneally at 24-hour intervals with 200 J of tamoxifen solution in corn oil (both Sigma-Aldrich) at 10mg / ml; administered on day 1 (single dose), day 0 and 1 (double dose) and day 0, 1 and 2 (triple dose). On day 189, the small intestines were excised and divided into 3 segments: proximal, medial, and distal.

[0079] Segments were flushed with 10% neutral buffered formalin (NBF), cut open longitudinally, flattened out and rolled up. ‘Gut rolls’ were fixed in NBF overnight and immersed in >70% ethanol prior to paraffin embedding. Sections were then deparaffinised, and heat-mediated antigen retrieval was performed in HI ER buffer. Endogenous peroxide and then non-specific antibody were then blocked with hydrogen peroxide and normal horse serum (Vector Labs), respectively. Microadenomas were then stained overnight with primary rabbit anti-mouse p- catenin antibody (Abeam, ab32572) or rabbit IgG isotype control (Abeam, ab37415). ImmPRESS VR Polymer HRP anti-rabbit IgG Kit (Vector Labs, MP-6401-15) and DAB substrate (Vector Labs, SK-4105) was then used to detect microadenomas. Counterstaining was performed in Mayer’s hematoxylin and slides were mounted using Lamb DPEX (ThermoFisher Scientific). Gut rolls slides were scanned at 20x magnification, using the digital Axio Scan.ZI slide scanner (Zeiss, Germany).

[0080] Scanned histological sections were analysed with QuPath version 0.2.3 To score tumour burden, 250 intestinal crypts in the small intestine were analysed. p-cateninhi9hpositive crypts were identified as crypts displaying clusters of cells with positive, nuclear and cytoplasmic p-catenin staining. Only whole crypts with villi on both sides were considered and crypt crosscuts or crypts with staining artefacts were excluded.

[0081] 5T4 Western Blot

[0082] Microadenomas were homogenised using RIPA lysis buffer (Thermo Fisher). Protein extracts were prepared for gel electrophoresis by addition of 1 :1 Novex™ Tris-Glycine SDS Sample Buffer (Thermo Fisher) and heating at 85 °C for 5 min, followed by addition of sample to 12% Novex wedgewell Tris Glycine mini gels (Thermo Fisher) for electrophoresis. Proteins were blotted onto PVDF membrane using the mini-gel wet-transfer XCell II Blot Module (Thermo Fisher) in transfer buffer (20% methanol, 25 mM Tris-base and 192 mM Glycine). Membranes were blocked with 5% milk powder in TBS-T (Sigma-Aldrich), then stained with anti-5T4 (R and R Systems).

[0083] Colonoscopy guided orthotopic implantation of MC38 cells High-resolution optical colonoscopy was performed using the TELE PACK VET X LED endoscopic video unit (model code RP100S1), TELECAM One-Chip Camera Head (model code 20212030), Fiber Optic Light Cable (model code 69495 NE) and a HOPKINS® Straight Forward Telescope 0° (model code 64301 AA). The colon was insufflated using the TELE PACK air pump and three-way tap on the Operating Sheath (model code 61029 D; Karl Storz, Tuttlingen) passed over the Telescope prior to endoscopic examination. MC-38 cells were injected into the colonic submucosa using a 100 pL removable needle glass syringe (model code 7656-01), 22-Gauge removable transfer needle (model code 7770-02) and custom made, flexible stainless steel small hub removeable hypodermic needle (12-inch, 33-Gauge, point-style 4, 45°-bevel; model code 7803-05). Glass syringe, transfer and hypodermic needles are all products of Hamilton Company, USA and supplied by ESSLAB, UK. Cells were injected cells 1.5cm from the distal rectum and endoscopic observations were made on days 8, 10 and 14 post-implant.

[0084] Measurement of TAP-independent MHC Class I stabilisation on the cell surface RMA-S cells were seeded at 105cells / well and were mock-infected or infected with either RAd-VSF (2.5x109PFU / ml), RAd-UL40-VSF (1.40x101° PFU / ml) or an empty control vector (1.06x1011PFU / ml), at MOI 100. Cells were incubated with viral inoculum for 3hrs and then incubated with complete media for 48 hours. Control wells were stimulated with the VSFRNLTHL (SEQ ID NO: 7) peptide, which was added at a concentration of 100uM for one hour. Surface MHC class I expression (H-2Kb) was determined via flow cytometry, using PE- anti-mouse H-2Kb antibody (Biolegend; Cat No. 116507). As staining controls, some virus- infected cells unstained with anti-MHC class I antibody.

[0085] Statistics

[0086] All statistical analyses were conducted using GraphPad Prism software, version 8 and 9. Specific tests used are stated in the Figure legends. Parametric tests were used for normally distributed data, and non-parametric tests if the data were not distributed normally. Specific statistical tests used are detailed in each figure legend. Data was plotted as mean ± standard deviation (SD) and p-values < 0.05 were deemed significant and are displayed in the figures by an asterisk (*), with *p < 0.05; **p < 0.01 ; ***p< 0.001 and ****p<0.0001.

[0087] Results Immunization with RAd-minigene construct expressing a 5T4-derived MHC class / -restricted peptide induce long-lived functional CD8+T cell memory formation We hypothesised that induction of ‘inflationary’ 5T4-specific CD8 T cells may afford protection from cancer development. We first mapped antigenic peptides presented from the 5T4 antigen in C57BL / 6 mice. Mice were immunised with either the AdZ-h5T4 or the control vector, boosted after 6 weeks and a week later splenocytes were stimulated with peptide pools representing whole 5T4 antigen (Table 3a). Peptide pools 1 , 2, 5, 6, 8, 9 and 11 were identified as positive hits using ELISPOT, as defined by a higher IFNy expression than background (Fig. 1A). By using the matrix system (Table 1 B) and the peptide prediction program NetMHC 3.4, peptides VSFRNLTHL (SEQ ID NO: 7); NSLVSLTYV (SEQ ID NO: 6); SAPSPLVEL (SEQ ID NO: 5) and TSYVFLGIV (SEQ ID NO: 61) were identified as possible epitopes. To verify 5T4 candidate epitopes predicted by NetMHC 3.4, splenocytes were stimulated overnight and IFN-y release was quantified and compared for each peptide following stimulation with control (Fig. 1 B) or RAd vectors that expressed full-length 5T4 (Fig. 1C). We examined vectors that expressed (AdZ) or not (AdZ2) the viral E4 region that induces NK cell stress ligands and thus might alter Ad-induced immunity, although our data revealed no difference in immunogenicity of these vectors (Fig. 1 B&C). Overall, our data derived from mice vaccinated with 5T4-expressing RAd vectors revealed that all 5T4-derived peptides that we identified induced T cell responses, although TSYVFLGIV (SEQ ID NO: 61) exhibited inter-mouse variability in terms of immunogenicity.

[0088] Next, we generated RAd vectors that expressed these individual 5T4-derived epitopes. Variability in immunogenicity of TSYVFLGIV (SEQ ID NO: 61) (Fig. 1 B&C) led us to focus on peptides 5T4i70-i78 SAPSPLVEL (SEQ ID NO: 5); SAPS), 5T4244-252 NSLVSLTYV (SEQ ID NO: 6; NSL) and 5T4252-26o VSFRNLTHL (SEQ ID NO: 7; VSF). We generated minigeneexpressing RAd vectors (RAd-VSF, RAd-SAP, RAd-NSL, Fig. 2A) and subcutaneously immunized mice with these or control vector. Blood was withdrawn from the lateral tail vein over 4 months and cells were stimulated ex vivo for 6 hours with a mix of all three 5T4-derived peptides to identify functional 5T4-specific CD8 T cells. Immunisation with minigene vectors did not induce higher frequencies of circulating CD8+T cells in blood as compared to control RAd-vaccinated mice (Fig. 2B). However, RAd-VSF-vaccinated mice exhibited stable accumulation of IFNy+(Fig. 2C) and IFNy+TNF+(Fig. 2D) 5T4-specific CD8 T cells over time. In contrast, RAd-SAP and RAd-NSL vaccinated mice did not induce 5T4-specific T cells in this vaccination model (Fig. 2C&D). Thus, VSF-based adenoviral constructs were developed further. HCMV UL40 increases the immunogenicity of a minigene adenoviral-based vectors

[0089] We hypothesised that the immunogenicity of a RAd minigene vector could be enhanced if presentation of the peptide it expresses was increased. The beta-herpesvirus human cytomegalovirus (HCMV) circumvents host immunity through numerous mechanisms including downregulation of MHC class I from the surface of infected cells. Although this process restricts recognition of virus- infected cells by CD8+T cells, it concurrently renders cells vulnerable to recognition and attack by NK cells. One strategy employed by HCMV to evade NK cell killing is to up-regulate the nonclassical MHC I molecule HLA-E which binds to the inhibitory NK cell receptor LIR1 and thus antagonises NK cell function. Cell surface expression of HLA-E requires loading of a conserved nonameric peptide VMAPRTLIL (SEQ ID NO: 2) onto HLA-E in a TAP-dependent process. VMAPRTLIL (SEQ ID NO: 2) is naturally derived peptide from the signal sequence of classical MHC I and HLA-G molecules. HCMV exploits this process through the expression of UL40 (Fig. 3A). This viral protein thus promotes surface expression of HLA-E, incorporating a nonameric HLA-E binding peptide homologous to endogenous VMAPRTLIL (SEQ ID NO: 2) and translocating this into the endoplasmic reticulum lumen in a TAP-independent process (Fig. 3A). We hypothesised that replacing the UL40 nonameric peptide with 5T4252-260 VSFRNLTHL (SEQ ID NO: 7; VSF) would enhance VSF presentation and subsequent stimulation of VSF-specific T cells (Fig. 3A). To test this, we incorporated the VSF peptide into UL40 and expressed this in a RAd5 vector (Fig. 3B, RAd-UL40-VSF).

[0090] To examine whether UL40 enhanced VSF minigene vector immunogenicity we vaccinated ApcMin / +mice in which microadenoma formation can be induced with tamoxifen and are thus used to study the early stages of CRC development. We determined that a single dose of tamoxifen led to induction of -15-40 distinct individual microadenomas per 250 intestinal crypts in the upper duodenum (data not shown). Importantly, 5T4 protein expression by microadenomas was confirmed by Western blot (Fig. 4A), confirming ApcMin / +mice as a suitable model for studying whether RAd-induced 5T4-specific T cells could limit microadenoma formation.

[0091] Mice were vaccinated with RAd-VSF, RAd-UL40-VSF or control vector and were bled every 30-40 days and functional VSF-specific T cell responses were measured (Fig. 4B). To ensure maximal immunogenicity, mice were immunized via the intravenous route, which is required for maximal induction of long-lived memory T cells by adenoviral minigene vectors. Both RAd- VSF and RAd-UL40-VSF vectors induced robust CD8+T cell responses, with significantly higher frequencies of IFN-y-secreting VSF-specific CD8+T-cells than those observed in control vector-treated mice (Fig. 4C). Importantly, RAd-UL40-VSF vaccination significantly increased the frequencies of VSF-specific CD8+T cells as compared to RAd-VSF (Fig. 4C) and these responses were polyfunctional (Fig. 4D&E). Thus, our data demonstrate that incorporation of a peptide derived from a tumour-associated antigen into LIL40 can enhance the immunogenicity of an adenoviral minigene vector.

[0092] Immunisation with UL40-VSF expressing RAd vector impedes microadenoma growth in the small intestine

[0093] To determine whether induced 5T4-specific CD8+T cell responses translated into efficient control of microadenoma formation and thus the early stages of CRC development, vaccinated mice were challenged with tamoxifen 170 days after vaccination and microadenoma formation was assessed 3 weeks later. Histological examination of the first 5cm of the small intestines of control-vaccinated animals revealed p-cateninhi9hdysplastic changes, ranging from slightly enlarged crypts to transformations affecting the entire epithelium along the crypt-villus axis. Visible expansion of abnormal cells resulting from accelerated cell proliferation could be observed in the proximal small intestine, correlating with elevated p-catenin expression (Fig. 5A, top). When microadenoma formation was compared in vaccinated mice, we observed that, as compared to control-vaccinated mice (Fig. 5A, bottom right), RAd-UL40-VSF vaccination limited the formation and progression of intestinal microadenomas in the small intestines (Fig. 5A, bottom left). When p-cateninhi9hdysplastic crypts were counted, we observed a significant reduction in crypts in RAd-UL40- VSF vaccinated mice (Fig. 5B). In contrast, mice vaccinated with RAd-VSF exhibited variable crypt counts comparable to control vector-immunized mice (Fig. 5B). These data suggest that incorporation of HCMV LIL40 into an adenoviral minigene vector platform enhances induction of CD8 T cell immunity that limits the onset of microadenomas in mice.

[0094] RAd-UL40-VSF limits growth of colorectal cancer in mice

[0095] We next investigated whether RAd-UL40-VSF could restrict the growth of colorectal tumour cells within the gastrointestinal tract. To do this, we injected the murine colorectal cell line MC38 (which expresses 5T4, data not shown) into the large intestines by colonoscopy-guided orthotopic implantation, and we measured tumour development by endoscopy on days 8, 10 and 14. We identified that 5 / 5 mice developed tumours in control vector-immunized mice whereas only 2 of 3 mice immunized with RAd-VSF mice developed tumours (Fig. 6). In contrast however, in mice immunized with RAd-UL40-VSF 3 of 6 mice developed no tumours, and 1 of the remaining 3 mice that developed a tumour showed tumour regression (Fig. 6). Thus, these data imply that the RAd-UL40-VSF vector induces anti-tumour immunity in CRC and this protection may be superior to T cell responses induced by unmodified VSF-5T4 expressing minigene vectors.

[0096] VSF-UL40 promotes stability of surface MHC class I

[0097] We hypothesised that the inclusion of the signal peptide LIL40 in the RAd-VSF construct promotes antigen presentation to CD8 T cells by increasing transportation of MHC class I- restricted peptide epitopes from the cytosol to the endoplasmic reticulum (ER) in a manner independent of mammalian transporter associated with antigen processing (TAP). To test this, TAP-deficient murine RMA-S lymphoma mutant cells were transduced with adenoviral vectors after 48 hours we measured surface MHC class I, which would only be present if loaded with peptide in the endoplasmic reticulum, by flow cytometry. As a positive control, some cells were pre-incubated with VSF peptide which led to significant expression of MHC class I on the surface of TAP-deficient cells (Fig. 7A&B). Importantly, when virus-infected cells were analysed, only cells transduced with RAd-UL40-VSF exhibited surface MHC class I (Fig. 7A&B). Thus, these data suggest that the enhanced immunogenicity of RAd-UL40- VSF was a consequence of increased TAP-independent peptide loading onto MHC class I.

[0098] Discussion

[0099] As proof of concept using physiological relevant models of CRC, we report that an adenoviral minigene vector engineered to express immunodominant epitope derived from 5T4 can afford protection from CRC development. Anti-tumour protection was dependent upon the addition of the HCMV LIL40 protein into constructs which we hypothesise increases presentation of tumour antigen-derived peptide by promoting TAP-independent translocation of antigenic peptide into the ER lumen. Subsequently, LIL40 incorporation into an adenoviral vector associated with the induction of persistent high frequencies of functional CD8+T cells reactive to peptides derived from a tumour associated antigen.

[0100] A single intravenous immunisation with a non-replicating vector expressing the selected immunodominant epitope 5T4252-260 VSFRNLTHL (SEQ ID NO: 7) induced a large expansion of polyfunctional VSF-specific CD8+T cells that were maintained over time for several months post-immunisation. Various studies have reported a positive correlation between the frequencies of tumour-infiltrating CD8+T cells and the progression-free survival and overall survival in cancer patients and frequencies of CD8 T cells predicts therapeutic efficacy in mouse models. In the context of CRC, increased frequency of 5T4-specific T cell responses correlates with improved outcome of CRC. Thus, the observation that heightened 5T4- specific CD8 T cell responses in mice vaccinated with RAd-UL40-VSF correlated with improved protection from both microadenoma formation and CRC development strongly suggests that the ability to induce superior VSF-5T4 specific CD8+T cell responses as compared to a minigene vector that does not include LIL40 is critical in the anti-tumour function of RAd-UL40-VSF. Thus, CD8+T cell responses induced by adenoviral minigene vector strategies may be more resistant to the suppressive tumour microenvironment than those generated by other therapeutic approaches, including cancer vaccine strategies such as RNA-based vaccines.

[0101] Summary

[0102] In conclusion, single immunisation with a RAd5-based vector that expresses an MHC class I peptide from the human 5T4 antigen induced robust and long-lasting antigen-specific functional CD8+T cell responses. Immunogenicity and subsequent protection was enhanced by HCMV UL40. These data imply that LIL40 may have broad utility in promoting the immunogenicity of vaccine strategies that aim to generate functional anti-tumour CD8+T cell responses. In the clinical context, 25% patients suffer secondary recurrence of CRC following surgery over a 5-year period. Our findings suggest that vaccination of these patients after surgery using an adenoviral vector-based approach reported herein has the potential to prolong cancer-free survival in these patients.

[0103] The observation that LIL40 enhances the immunogenicity of a 5T4-derived peptide implies that this approach may be broadly applicable to a multitude of antigenic peptides in infectious and non-infectious diseases. Indeed, TAP-mediated translocation of peptides is a prerequisite for the presentation of peptides by MHC class I. Thus, the inclusion of any antigenic peptide into UL40-based constructs could potentially enhance the activation and expansion of a reactive CD8 T cell response.

[0104] Table 4

[0105]

[0106] References

[0107] 11 Stanton, R. J., McSharry, B. P., Armstrong, M., Tomasec, P. & Wilkinson, G. W. Re-engineering adenovirus vector systems to enable high-throughput analyses of gene function. Biotechniques 45, 659-662, 664-658, doi: 10.2144 / 000112993 (2008).

[0108] 12 Seirafian, S. An Analysis of Human Cytomegalovirus Gene Usage. Cardiff University (2013).

Claims

Claims1. A genetic construct comprising nucleic acid encoding: i) HCMV LIL40 protein, or a part thereof; and ii) a recombinant immunogenic or antigenic peptide sequence which is positioned within a part of said LIL40 protein of part i).

2. The genetic construct according to claim 1 wherein the genetic construct encodes an N-terminal part of the LIL40 protein, or a part thereof.

3. The genetic construct according to claim 2 wherein the genetic construct encodes the signal sequence, or a part thereof, of the LIL40 protein.

4. The genetic construct according to any one of claims 1 - 3 wherein said part of the LIL40 protein is about 25 - 40 amino acids in length, or about 30 - 40 amino acids in length, or about 35 - 40 amino acids, or 37 amino acids in length.

5. The genetic construct according to any one of claims 1 - 4 wherein said genetic construct encodes a sequence comprising: MNKFSNTRIGFTCAVMAPRTLILTVGLLCMRIRSLLC (SEQ ID NO: 3); or a sequence that has 75% identity or similarity therewith.

6. The genetic construct according to any one of claims 1 - 5 wherein said genetic construct encodes a sequence comprising:MNKFSNTRIGFTCA (SEQ ID NO: 62)-Xi-TVGLLCMRIRSLLC (SEQ ID NO: 4), or a sequence 75% identical, or similar, thereto; wherein Xi represents an immunogenic or antigenic peptide sequence.

7. The genetic construct according to claim 5 or 6 wherein said sequence identity or similarity is selected from the group comprising:76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94% 95%, 96%, 97%, 98% and 99% identity or similarity.

8. The genetic construct according to any one of claims 1 - 7 wherein said immunogenic or antigenic peptide comprises about 5 - 30 amino acids, or about 5 - 20 amino acids or about 7 - 9 amino acids, or 8 or 9 amino acids.

9. The genetic construct according to any one of claims 1 - 8 wherein said immunogenic or antigenic peptide, or a part thereof, is a Major Histology Complex of Class I (MHCI) class restricted peptide.

10. The genetic construct according to any one of claims 1 - 9 wherein said immunogenic or antigenic peptide sequence is derived from a species in which, or with which, the genetic construct is to be used.11 . The genetic construct according to any one of claims 1 - 10 wherein said immunogenic or antigenic peptide, or a part thereof, is representative of, or associates with or characterises a disease to be treated using the said genetic construct.

12. The genetic construct according to any one of claims 1 - 11 wherein said immunogenic or antigenic peptide, or a part thereof, is selected form the group comprising: a tumour associated antigen, a neoantigen, a viral protein, a bacterial protein, and a fungal protein.

13. The genetic construct according to any one of claims 1 - 12 wherein said immunogenic or antigenic peptide, or a part thereof, is selected form the group comprising: a human cancer antigen, including a cancer antigen that is representative of a particular type of cancer; a human infection antigen, including an antigen that is representative of particular type of infection.

14. The genetic construct according to any one of claims 1 - 13 wherein said immunogenic or antigenic peptide, or a part thereof, is 5T4, or a fragment thereof, optionally comprising or consisting of one or more of: MPGGCSRGPAAGDGRLRLAR (SEQ ID NO: 20) AGDGRLRLARLALVLLGWVS (SEQ ID NO: 21) LALVLLGWVSSSSPTSSASS (SEQ ID NO: 22) SSSPTSSASSFSSSAPFLAS (SEQ ID NO: 23) FSSSAPFLASAVSAQPPLPD (SEQ ID NO: 24) AVSAQPPLPDQCPALCECSE (SEQ ID NO: 25) QCPALCECSEAARTVKCVNR (SEQ ID NO: 26) AARTVKCVNRNLTEVPTDLP (SEQ ID NO: 27) NLTEVPTDLPAYVRNLFLTG (SEQ ID NO: 28) AYVRNLFLTGNQLAVLPAGA (SEQ ID NO: 29) NQLAVLPAGAFARRPPLAEL (SEQ ID NO: 30) FARRPPLAELAALNLSGSRL (SEQ ID NO: 31) AALNLSGSRLDEVRAGAFEH (SEQ ID NO: 32) DEVRAGAFEHLPSLRQLDLS (SEQ ID NO: 33) LPSLRQLDLSHNPLADLSPF (SEQ ID NO: 34) HNPLADLSPFAFSGSNASVS (SEQ ID NO: 35) AFSGSNASVSAPSPLVELIL (SEQ ID NO: 36) APSPLVELILNHIVPPEDER (SEQ ID NO: 37) NHIVPPEDERQNRSFEGMVV (SEQ ID NO: 38) QNRSFEGMVVAALLAGRALQ (SEQ ID NO: 39) AALLAGRALQGLRRLELASN (SEQ ID NO: 40)GLRRLELASNHFLYLPRDVL (SEQ ID NO: 41)HFLYLPRDVLAQLPSLRHLD (SEQ ID NO: 42)AQLPSLRHLDLSNNSLVSLT (SEQ ID NO: 43) LSNNSLVSLTYVSFRNLTHL (SEQ ID NO: 44) YVSFRNLTHLESLHLEDNAL (SEQ ID NO: 45) ESLHLEDNALKVLHNGTLAE (SEQ ID NO: 46) KVLHNGTLAELQGLPHIRVF (SEQ ID NO: 47) LQGLPHIRVFLDNNPWVCDC (SEQ ID NO: 48) LDNNPWVCDCHMADMVTWLK (SEQ ID NO: 49) HMADMVTWLKETEVVQGKDR (SEQ ID NO: 50) ETEWQGKDRLTCAYPEKMR (SEQ ID NO: 51) LTCAYPEKMRNRVLLELNSA (SEQ ID NO: 52) NRVLLELNSADLDCDPILPP (SEQ ID NO: 53) DLDCDPILPPSLQTSYVFLG (SEQ ID NO: 54) SLQTSYVFLGIVLALIGAIF (SEQ ID NO: 55) IVLALIGAIFLLVLYLNRKG (SEQ ID NO: 56) LLVLYLNRKGIKKWMHNIRD (SEQ ID NO: 57) IKKWMHNIRDACRDHMEGYH (SEQ ID NO: 58) ACRDHMEGYHYRYEINADPR (SEQ ID NO: 59) YRYEINADPRLTNLSSNSDV (SEQ ID NO: 60)15. The genetic construct according to any one of claims 1 - 14 wherein said immunogenic or antigenic peptide, or a part thereof, is 5T4, or a fragment thereof, optionally comprising or consisting of one or more of: TSYVFLGIV (SEQ ID NO: 61), SAPSPLVEL (SEQ ID NO: 5), NSLVSLTYV (SEQ ID NO: 6), VSFRNLTHL (SEQ ID NO: 7), RLARLALVL (SEQ ID NO: 8), GAFEHLPSL (SEQ ID NO: 9) and DLPAYVRNL (SEQ ID NO: 10).

16. The genetic construct according to any one of claims 1 - 15 wherein said genetic construct is either DNA or RNA, including gDNA or cDNA and mRNA, tRNA or noncoding RNA.

17. The genetic construct according to any one of claims 1 - 16 wherein said genetic construct is in the form of a minigene.

18. A host cell comprising the genetic construct according to any one of claims 1 - 17.

19. The host cell according to claim 17 wherein said cell is a virus or a bacterium, optionally an adenovirus.

20. The host cell according to claim 19 wherein the genetic construct is inserted in E1 region of an adenovirus, either as an additional insertion or as a substitution.

21. The genetic construct according to any one of claims 1 - 17 or the host cell of claims 18 - 20 for use as a medicament.

22. The genetic construct according to any one of claims 1 - 17 or the host cell of claims 18 - 20 for use in the treatment of cancer or an infection.

23. Use of the genetic construct according to any one of claims 1 - 17 or the host cell of claims 18 - 20 in the manufacture of a medicament to prevent or treat a disease, including cancer or an infection.

24. A pharmaceutical composition comprising the genetic construct according to any one of claims 1 - 17 or the host cell of claims 18 - 20 and a suitable carrier.

25. The pharmaceutical composition according to claim 24 wherein said pharmaceutical composition is formulated for human or veterinary use.

26. A method for treating or preventing a disease comprising administering an effective amount of the genetic construct of claims 1 - 17 or the host cell of claims 18 - 20 or the pharmaceutical composition of claims 24 or 25 to a patient in need thereof.

27. The method according to claim 26 wherein said administering comprises intra-tumoral administration or systemic administration.

28. A method of vaccinating against a disease comprising administering an effective amount of the genetic construct of claims 1 - 17 of the host cell of claims 18 - 20 or the pharmaceutical composition of claims 24 or 25 to an individual.

29. The genetic construct of claim 22 or the use of claim 23 or the method of claim 26 wherein said cancer or disease is selected from the group comprising: nasopharyngeal cancer, synovial cancer, hepatocellular cancer, renal cancer, cancer of connective tissues, melanoma, lung cancer, bowel cancer, colon cancer, rectal cancer, colorectal cancer, brain cancer, throat cancer, oral cancer, liver cancer, bone cancer, pancreatic cancer, choriocarcinoma, gastrinoma, pheochromocytoma, prolactinoma, T-cell leukemia / lymphoma, neuroma, von Hippel-Lindau disease, Zollinger-Ellison syndrome, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, ureter cancer, oligodendroglioma, neuroblastoma, meningioma, spinal cord tumor,, osteochondroma, chondrosarcoma, Ewing's sarcoma, cancer of unknown primary site, carcinoid, carcinoid of gastrointestinal tract, fibrosarcoma, breast cancer, Paget's disease, cervical cancer, colorectal cancer, rectal cancer, esophagus cancer, gall bladder cancer, head cancer, eye cancer, neck cancer, kidney cancer, Wilms' tumor, Kaposi's sarcoma, prostate cancer, testicular cancer, Hodgkin's disease, nonHodgkin's lymphoma, oral cancer, skin cancer, mesothelioma, multiple myeloma, ovarian cancer, endocrine pancreatic cancer, glucagonoma, parathyroid cancer, penis cancer, pituitary cancer, soft tissue sarcoma, retinoblastoma, small intestine cancer,stomach cancer, thymus cancer, thyroid cancer, trophoblastic cancer, hydatidiform mole, uterine cancer, endometrial cancer, vagina cancer, vulva cancer, acoustic neuroma, mycosis fungoides, insulinoma, carcinoid syndrome, somatostatinoma, gum cancer, heart cancer, lip cancer, meninges cancer, mouth cancer, nerve cancer, palate cancer, parotid gland cancer, peritoneum cancer, pharynx cancer, pleural cancer, salivary gland cancer, tongue cancer and tonsil cancer.

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