WO2025062128A1 - Modified adenovirus - Google Patents

Abstract

The invention concerns a modified adenoviral vector of serotype Ad5 comprising cancer selective targeting motif; a pharmaceutical composition comprising same; a combination therapeutic comprising same, the use of said adenoviral vector or pharmaceutical composition or combination therapeutic as a medicament and, in particular to treat cancer; and a method of treating cancer using said modified adenoviral vector or pharmaceutical composition or combination therapeutic.

Description

Modified Adenovirus

Field of the Invention

The invention concerns a modified adenoviral vector of serotype Ad5 comprising cancer selective targeting motif; a pharmaceutical composition comprising same; a combination therapeutic comprising same; the use of said adenoviral vector or pharmaceutical composition or combination therapeutic as a medicament and, in particular to treat cancer; and a method of treating cancer using said modified adenoviral vector or pharmaceutical composition or combination therapeutic.

Background of the Invention

Brain cancers remain amongst the most devastating and lethal of cancer types. Not only are they fast growing but they are also difficult to treat and often reoccur after removal. In the UK, only -40% of people diagnosed with a malignant brain tumour will survive more than 1 year and five-year survival rates are devastatingly low at approximately 5%. Generally, the term brain cancer encompasses over 130 different tumour types and refers to any tumour starting in the brain or the spinal cord.

Glioblastoma (GBM) is the most common high-grade type of brain tumour in adults. Clinical presentation of GBM can be non-specific and can often result in patients presenting to multiple primary care facilities before receiving a specialist referral. Early symptoms include headaches, intracranial pressure, fatigue and weakness, vomiting, visual disturbances, personality changes and seizures. Current treatment strategies involve maximal safe surgical resection of the tumour followed by radiotherapy and temozolomide chemotherapy. This is, in part, due to the nature of this disease as well as the efficacy of available treatments. Brain tumours often invade healthy brain tissue rendering surgical resection or radiotherapy dangerous or ineffective due to inability to incomplete removal of the tumour. Consequently, novel and more effective therapeutic approaches are urgently required for treatment of GBM.

Adenoviruses have demonstrated feasibility and safety as oncolytic virotherapies in several cancer backgrounds. A number of oncolytic human adenoviruses (HAdV) virotherapies have entered clinical trials and have demonstrated safety and feasibility, although delivery and efficacy require optimization before oncolytic adenovirus can be used as an effective cancer therapy. However, the brain is protected from pathogens and toxic substances by the blood brain barrier (BBB), which also provides a formidable barrier to the systemic delivery of virusbased therapies to GBM via the intravenous route. Local delivery of targeted adenoviral therapies with enhanced tumour specificity at cell entry level therefore provides a potential solution to overcome these issues by limiting infection to tumour cells and enabling more of the healthy tissue to remain unaffected.

Previously, we developed a highly tumour selective oncolytic adenovirus termed Ad5NULL-A20 that engages avp6 integrin solely for cell entry. The Ad5NULL platform vector is a modified Ad5 vector containing modifications to prevent off target interactions. The KO1 mutation in the fiber knob protein (S408E, P409A) prevents binding to the native Ad5 receptor, CAR, whilst mutations in the hypervariable region 7 (HVR7) of the hexon ablate binding to FX in the blood which limits this interaction and prevents off target sequestration in the liver. Finally, the Ad5NULL platform also contains a mutation of the RGD motif in the penton-based protein to prevent secondary interactions with cellular integrins. However, since the expression of avp6 integrin is limited to epithelial carcinomas, this therapy is unlikely to be effective in GBM which normally arises from glial cells, and therefore do not express this integrin.

Accordingly, we therefore looked to investigate whether the favourably Ad5NULL platform could be retargeted to utilise for treatment of GBM and other brain cancers.

It has been suggested that brain cancers including glioma upregulate avp3 and avp5 integrins which are involved in angiogenesis and invasive growth. We therefore looked to utilise this surface marker expression to selectively target Ad5NULL. Incorporation of an OvPs/avPs integrin binding RGD cell targetting peptide into Ad5NULL results in a retargeted vector, highly selective for avp3 and avp5 integrin binding. In totality, ablating the natural adenovirus tropisms and replacing with a singular tropism generated a modified vector with a high affinity route of infection, which not only improved selectivity, but also the activity of the virus. We have therefore developed a novel avp3 and avp5 integrin targeted vector based on the Ad5NULL background termed Ad5_NuLLRGD, that exclusively transduces cells expressing avp3/5 integrin. These novel vectors will likely benefit from a proven clinical safety profile of adenoviral vectors in glioma whilst possessing higher specificity, fewer off target effects and lower seroprevalence to improve efficacy and clinical outcomes.

Statements of the Invention

According to a first aspect of the invention there is provided a viral vector of Ad5 serotype adenovirus, herein referred to as Ad5NULL-RGD, modified to: a) comprise at least one of 1421 G, T423N, E424S, E450Q or L426Y point mutation(s) in the hexon hypervariable region 7 (HVR7 mutation) wherein said mutation prevents virus binding with coagulation factor 10 (FX); b) comprise at least one of S408E or P409A point mutation(s) in the fiber knob region AB loop (KO1 mutation) wherein said mutation prevents virus binding with the coxsackie and adenovirus receptor (CAR); c) comprise at least one of D342E or D342A point mutation(s) in the penton integrin binding motif Arg-Gly-Asp (RGD) wherein said mutation prevents virus binding with OvPs/avPs integrin; and d) present or express an OvPs/avPs integrin binding RGD cell targetting peptide.

Amongst the viruses exploited for therapeutic application, Human Adenoviruses (HAdV) have important clinical applications ranging from vectors for gene therapy and vaccines to oncolytic viruses. A number of oncolytic HAdV virotherapies have entered clinical trials and have shown safety and feasibility, although delivery and efficacy require optimization before oncolytic adenovirus can be used as an effective cancer therapy. Adenovirus 5 (Ad5) is a common vector deployed in numerous clinical trials for cancer and gene therapies (clinicaltrials.gov, 2016) because it can be genetically manipulated, and it tolerates large transgenes. However, Ad5 has a natural tropism that leads to widespread distribution of the vector. Entry of group C adenoviruses, such as Ad5, into cells is thought to involve high affinity binding of the virus to the cell through interaction of the viral fiber protein with coxsackievirus-adenovirus receptor (CAR). Therefore, for the purpose of anti-cancer therapy, targeting of adenoviral vectors to specific tissues or cell types requires modification of the normal tropism of the vector to improve specificity. We have previously reported (WO2019158914) that the Ad5 adenovirus, when modified to comprise mutations a)-c) above, ablated the native tropisms of Ad5 by mutating the main capsid proteins: hexon, fiber and penton. A triple de-targeted Ad5-based vector backbone was generated, with modifications in the hexon hypervariable region 7 (HVR7 mutation), fiber knob AB loop (KO1 mutation) and penton integrin-binding motif Arg-Gly-Asp (RGD mutation) with substitution mutations in amino acid residues responsible for binding to coagulation factor 10 (FX), coxsackie and adenovirus receptor (CAR), and avp3/5 integrins, respectively. This modified virus has reduced ability to infect off-target tissues, indeed, it is prevented/inhibited from infecting liver and spleen and also its ability to infect cells of the body in a widespread manner is also compromised. Thus, the modified adenovirus is compromised in terms of the tissue it can infect. Said modified Ad5 vector is described in detail in patent application WO2019158914. The term Ad5.3D is used in said patent disclosure but is referenced herein as Ad5Nuu.. Any sequence of Ad5 that is known in the art can be the vector according to the invention and comprise the three modifications a) - c) disclosed herein. In a preferred embodiment, the Ad5 serotype that is modified as disclosed herein comprises the accession number AC_000008.1.

As is known in the art, the hexon protein is highly conserved among the different adenovirus serotypes with the exception of nine hypervariable regions (HVRs). These HVRs reside in two distinct loops that form the exposed surface of the hexon protein, HVRs 1-6 lie within the DE1 loop and HVRs 7-9 are located within the FG1 loop. Accordingly, reference herein to at least one mutation in HVR7 refers to a mutation in the hypervariable region 7. In a preferred embodiment, hexon is as set forth in accession number AP_000211.1 Specifically, said at least one HVR7 mutation prevents interaction with coagulation Factor X thereby limiting off- target sequestration of the modified adenovirus to the liver, and improved targeting to target cancer cells. In a preferred embodiment, said at least one HVR7 mutation comprises at least one amino substitution mutation to prevent FX interaction selected from one or more of the group comprising: 1421 G, T423N, E424S, E450Q or L426Y. Most preferably, said at least one HVR7 mutation comprises additionally or alternatively at least one of 1421 G, T423N, E424S, L426Y, and E450Q point mutations. Whilst E450 is recited in these sequences, as is known in the art, this can also be known as E451 with reference to hexon. This is a known sequence alignment issue through use of an early inaccurate sequence for the hexon protein, but the two positions are regarded as analogous in the art. Thus E450 and E451 are acknowledged as being one and the same in Ad5 when considering this Hexon binding mutation, as qualified by the claim with reference to Ad5 and the specific protein

Adenoviral infection commences with recognition of host cell receptors by means of specialised proteins on the viral surface i.e. , the adenovirus fiber protein and in particular the globular carboxy-terminal domain of the adenovirus fiber protein, termed the carboxy-terminal knob domain. Accordingly, reference herein to a knob of an adenoviral fiber protein is reference to the globular carboxy-terminal domain of the adenovirus fiber protein. In a preferred embodiment, fiber is as set forth in accession number AP_000226.1. Accordingly, reference to at least one KO1 mutation refers to at least one mutation in the fiber knob region AB loop. Specifically, said at least one KO1 mutation prevents virus binding to CAR. In a preferred embodiment, said at least one KO1 mutation comprises at least one point mutation to prevent CAR binding selected from one or more of the group comprising: S408E or P409A. Most preferably said point mutation comprises S408E and P409A point mutations. Adenovirus penton base contains five Arg-Gly-Asp sequences and bind integrins alpha v beta 3 and alpha v beta 5 (avPs/ovPs) to promote viral infection by permitting virus internalization. Through prevention of this native interaction, we have found that off-site targeting to the spleen is reduced, thereby promoting tumour specific targeting and, moreover, there is a dampening release of pro-inflammatory cytokines that otherwise lead to adverse immune host responses when used in the context of anti-cancer therapy. In a preferred embodiment, penton is as set forth in accession number AAW65509.1. Accordingly, reference to at least one RGD mutation refers to at least one mutation in the penton integrin binding motif Arg-Gly-Asp (RGD mutation) wherein said mutation prevents virus binding with OvPs/avPs integrin. In a preferred embodiment, said at least one RGD mutation comprises at least one point mutation selected from the group comprising: D342E and D342A, to produce RGE or RGA, respectively. Most preferably said point mutation is D342E to produce RGE.

Reference herein to an OvPs/avPs integrin binding RGD cell targetting peptide refers to an engineered cancer cell targeting peptide that selectively targets specific host cells, in particular, specific types of cancer cells. Specifically, the present invention concerns the presentation, or expression, of an RGD peptide, ideally artificially engineered peptide, as disclosed herein targets the avp3 and avp5 integrins, which have been found to be overexpressed in brain cancers including, but not limited to, Glioblastomas. Therefore, as will be appreciated by those skilled in the art, through presentation or expression of this RGD cell targetting peptide in the viral vector, one can selectively target avp3 and avp5 integrin overexpressing cancers, in particular skin cancer, kidney cancer, ovarian cancer, prostate cancer and in particular brain cancers such as, but not limited to, chordomas, gangliocytomas, glomus jugulare, Schwannomas, astrocytomas, ependymomas, gliomas, glioblastoma, glioblastoma, multiforme mixed gliomas, hemangioblastomas, Rhabdoid tumours, meningiomas, pituitary tumours, craniopharyngiomas, germ cell tumours, pineal region tumours, medulloblastomas, CNS brain lymphomas, or metastatic forms thereof.

As will be appreciated, the virus is modified to ablate its natural tropism for OvPs/avPs integrin, which is typically used as a secondary/co-receptor for effective internalisation of the virus following CAR binding. Unexpectedly, we have found that re-introduction of the avp3 and avp5 integrins binding, through the RGD binding targeting motif, has led to superior targeting to avp3 and avp5 integrin overexpressing cells. Without wishing to be bound by theory, we believe the modified virus is utlising RGD as the primary entry receptor in place of CAR, in combination with ablation of the native penton RGD, thereby improving the on-target binding of integrins. It also may internalise through a different pathway due to the lack of co-receptor binding. In this manner, we have found a singular, high affinity route of infection not only improves selectivity, but also the activity of the virus in avp3 and avp5 integrin overexpressing cells.

In a preferred embodiment of the invention said OvPs/avPs integrin binding RGD cell targetting peptide comprises, or consists, of a sequence selected from the group comprising:

ACDCRGDCFCG (RGD4C) (SEQ ID NO: 1);

DGARYCRGDCFDG (RGD10) (SEQ ID NO: 2);

VTGRGDSPASS (FN-RGD2) (SEQ ID NO: 3);

GCTIGRGDWAPSECKQDSDCLAGCVCGPNGFCG (1.5B) (SEQ ID NO: 4); GCPQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG (2.5D) (SEQ ID NO: 5); GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (2.5F) (SEQ ID NO: 6); or a sequence with at least 75% identity thereto and which retains the biological activity or targeting of OvPs/avPs integrin. It is preferred that the peptide has at least 75% identity with one of the recited peptide sequences, 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 therewith.

More preferably, said RGD cell targeting peptide is selected from: ACDCRGDCFCG (RGD4C) (SEQ ID NO: 1); DGARYCRGDCFDG (RGD10) (SEQ ID NO: 2); or a sequence with at least 75% identity thereto.

The skilled person will appreciate that homologues, orthologues or functional derivatives of the peptide 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. More preferably, said RGD cell targeting peptide as disclosed herein is inserted into the HI loop of HAdV fiber knob protein, preferably at a position between amino acids 304-305 of the fibre knob protein, as set forth in NCBI accession number AB724351.1 , specifically accession BAM66698. Preferably, said RGD cell targeting peptide is inserted between amino acid 542- 543 of the Ad5 vector (Uniprot P11818). In a preferred embodiment, RGD cell targeting peptide is incorporated between flanking residues TQET and GDTT. This therefore tends to indicate a preferred insertion loop for the binding epitope. In one embodiment of the invention insertion into said H1 involves the use of a short peptide sequence, or linker, such as SGG and therefore in a preferred embodiment comprises, or consists of the sequence GGSACDCRGDCFCGSGG (SEQ ID NO: 7) or GGSDGARYCRGDCFDGSGG (SEQ ID NO: 8). However, as the skilled person will appreciate, other amino acids or peptide linkers may be used with equal effect.

We show herein that Ad5NULL-RGD can engage and utilise avp3/5 integrin as a tumour selective cell entry receptor and this function is mediated by the RGD cell targetting peptide, which integrins we have shown to be upregulated in brain cancers, and notably at lower levels in healthy tissue. Therefore, as will be appreciated by those skilled in the art, through expression and incorporation of this sequence in the modified virus, the modified virus can selectively target avp3/5 integrin overexpression in cancers such as, but not limited to, glioblastoma. We also, unexpectedly, show that this effect is preferentially observed in Ad5NULL-RGD compared to Ad5 and Ad5.RGD controls, with improved transduction observed. Accordingly, despite the removal of the natural tropism of avp3/5 integrin in Ad5NULL, the introduction of the engineered RGD cell targeting peptide into the fibre knob leads to an improved “on target” transduction of several cancer types including glioma cells that express the targets, avp3 and avp5 integrins. This was even unexpectedly the case when compared to Ad5RGD, which retains the natural avp3/5 tropism and the engineered RGD4C peptide, which one would expect to have similar or greater effect, suggesting Ad5NULL to be the superior vector. Further still, advantageously, the Ad5_NuLLRGD vector was found to transduce non tumour cells less efficiently than that of Ad5.RGD, pointing towards a highly selective avp3/5 integrin tumour targeting vector.

Interestingly, Ad5 based vectors can be used as a therapy alone with oncolytic activity or for highly targeted delivery of therapeutics. Alternatively, in certain applications, such as vaccine applications, the Ad5 vectors can be made replication deficient through incorporation of further, routine modifications as known by the person skilled in the art. In a further preferred embodiment of the invention said viral vector is further modified to include at least one growth factor antibodies. Preferably the growth factor antibody is linked to the vector using a chemical linkage and then the antibody is used as a targeting moiety, e.g., bFGF, EGFR, antibodies (e.g., Cetuximab, Herceptin, Avastin or the like). The chemical linkage may comprise the use of an avidin/biotin linkage.

In a yet further preferred embodiment of the invention said viral vector is further modified to include at least one matrix degrading enzyme. Ideally, the one or more such enzymes are attached (typically, chemically linked e.g., linkage via hyaluronidase) to the outside of the virus, in this way, the matrix degrading enzyme can degrade extracellular matrix and so enable the virus to more efficiently permeate into the tumour microenvironment.

The skilled person will appreciate that homologues, orthologues or functional derivatives of the recited modified viral vector mutations will also find use in the context of the present invention. Thus, for instance mutations 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” or biochemical property (e.g., charge, hydrophobicity and/or size). Such replacements are well known in the art and are typically referred to as “conservative replacement”, “conservative mutation” or “conservative substitution”. 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.

More preferably still said a viral vector (Ad5NULL-RGD): a) comprises I421G, T423N, E424S, L426Y, and E450Q point mutations in the hexon hypervariable region 7 (HVR7 mutation) wherein said mutations prevent virus binding with coagulation factor 10 (FX); b) comprises S408E and P409A point mutations in the fiber knob region AB loop (KO1 mutation) wherein said mutations prevent virus binding with the coxsackie and adenovirus receptor (CAR); c) comprises D342E point mutation in the penton integrin binding motif Arg-Gly-Asp (to produce RGE mutation) wherein said mutation prevents virus binding with aVp3/aVp5 integrin; and d) presents or expresses an OvPs/avPs integrin binding RGD cell targetting peptide.

In yet a further preferred embodiment still, said adenovirus is further modified to include a molecule which is a transgene encoding an agent such as, but not limited to, a therapeutic agent. This embodiment therefore concerns the delivery of an agent, intracellularly, to exert a therapeutic action on the targeted cancer cell. Examples of an agent may include an agent that directly stimulates an immune responses, for example GM-CSF, IL-12; an agent to indirectly stimulate the immune system, e.g. an antibody (or fragments of an antibody), an immune checkpoint inhibitor to inhibit a co-repressor such as CTLA-4, PD-L1 , PD1 , or Lag3; a Bi-specific T cell Engaging (BiTE) antibody construct; a Bi-specific natural killer cell engaging (BiKE) antibody construct; an agent that sensitises tumours to a cell based immunotherapy e.g. encoding CD19; an agent that depletes regulatory T cells within the tumour microenvironments, anti-CD25 antibodies; an agent that sensitises a tumour to radiotherapy or for imaging e.g. by encoding sodium/iodide symporter (NIS) or somatostatin receptor type 2 (SSTR2)). Alternatively, the transgene may encode a therapeutic agent that is directly toxic to a tumour cell, e.g. by encoding the transgene Reduced Expression in Immortalized Cells (REIC/DKK3), or an enzyme that sensitises a cancer cell via conversion of a non-toxic prodrug into a toxic drug e.g. cytosine deaminase including FCLI1 or FCY1 , nitroreductase, thymidine kinase. Other transgenes known in the art and useful in the treatment of cancer may be used in the working of the invention.

Accordingly, in a preferred embodiment of the invention said modified adenovirus comprises a molecule encoding at least one transgene as herein described, ideally said molecule is cDNA.

According to a second aspect of the invention there is provided the vector as defined herein for use as a medicament.

According to a third aspect of the invention there is provided the vector as defined herein for use in the treatment of cancer or other diseases where avp3/5 expression can be pathological, such as but not limited to: fibrosis; bacterial or viral infection; inflammation; and inflammatory disorders, such as arthritis or osteoporosis. According to a fourth aspect of the invention there is provided the vector as defined herein for use in the manufacture of a medicament to treat cancer or other diseases where avp3/5 expression can be pathological, such as but not limited to fibrosis; bacterial or viral infection; inflammation; and inflammatory disorders, such as arthritis or osteoporosis; skin cancer, kidney cancer; ovarian cancer; prostate cancer; and in particular brain cancer, such as that selected from any one or more of the following cancers: chordomas, gangliocytomas, glomus jugulare, Schwannomas, astrocytomas, ependymomas, gliomas, glioblastoma, glioblastoma, multiforme mixed gliomas, hemangioblastomas, Rhabdoid tumours, meningiomas, pituitary tumours, craniopharyngiomas, germ cell tumours, pineal region tumours, medulloblastomas, CNS brain lymphomas, or metastatic forms thereof.

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 comprising the vector as defined herein and a pharmaceutically acceptable carrier, adjuvant, diluent or excipient.

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.

The composition may be prepared by bringing into association the above defined vector with the carrier. 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.

According to a fifth aspect of the invention there is provided a combination therapeutic comprising the modified adenoviral vector described herein and at least one further therapeutic agent, including: chemotherapeutics, such as temozolomide, procarbazine, carmustine, lomustine, or vincristine; or immune checkpoint inhibitors.

According to a sixth aspect of the invention, there is provided a method for treating diseases where avp3/5 expression can be pathological, such as but not limited to fibrosis; bacterial or viral infection; inflammation; and inflammatory disorders, such as arthritis or osteoporosis; or cancer, said method comprising administering an effective amount of the viral vector or pharmaceutical composition or combination therapeutic as defined herein to a patient in need thereof. Most preferably, said cancer is to treat skin cancer, kidney cancer; ovarian cancer; prostate cancer; and in particular brain cancer, such as that selected from any one or more of the following cancers: chordomas, gangliocytomas, glomus jugulare, Schwannomas, astrocytomas, ependymomas, gliomas, glioblastoma, glioblastoma, multiforme mixed gliomas, hemangioblastomas, Rhabdoid tumours, meningiomas, pituitary tumours, craniopharyngiomas, germ cell tumours, pineal region tumours, medulloblastomas, CNS brain lymphomas, or metastatic forms thereof.

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.

In a preferred method, the viral vector according to the invention may be administered to a subject by any suitable route. Preferably this is direct intra-tumoral injection when treating malignant solid tumours, and preferably 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.

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.

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

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.

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.

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

Figure 1. (A) mRNA expression of ITGB3, ITGB5 and ITGB6 in cases of glioma (66), upper aerodigestive (33), pancreatic (46) and breast (60) cancers, obtained from TCGA dataset available at https://software.broadinstitute.org. e 1. mRNA expression of p3 integrin subunit (ITGB3), p5 integrin subunit (ITGB5) and p6 integrin subunit (ITGB6) in cases of glioma (66), upper aerodigestive (33), pancreatic (46) and breast (60) cancers, obtained from TCGA dataset available at https://software.broadinstitute.org. (B) Gene Expression Profiling Interactive Analysis (GEPIA) comparison of p3 integrin subunit (ITGB3), p5 integrin subunit (ITGB5), p3 integrin subunit (ITGB6) and av integrin subunit (ITGAV) in normal brain vs GBM tissue. RNA expression was analysed 163 tumour samples compared to 207 normal samples for each gene and presented as a boxplot on a log-scale (log2(TMP+1)) using the GEPIA software. Significance has been determined with a p-value cut-off of p=0.01. Data available at gepia.cancer-pku.cn and obtained from TGCA dataset and GTEx dataset. Figure 2. (A) Transduction of Ad5.RGD vs Ad5_NuLLRGD quantified by expression of luciferase transgene. A549 E6 cells (lung carcinoma with over-expression of avp3) and pancreatic PANC0403 cells were infected with 5000 vp/cell of luciferase expressing Ad5, Ad5.RGD and Ad5NULL.RGD. Luminescence was measured at 72 hours avp3, avp5 and avp6 in melanoma MDA-MB 435S cells (and the derivative av knock down, MDA-MB 435S av). Transduction of Ad5.RGD vs Ad5_NuLLRGD quantified by expression of GFP transgene. MDA-MB 435 S and MDA-MB 435 S av were infected with 1000 and 5000 vp/cell of GFP expressing Ad5, Ad5.RGD and Ad5NULL.RGD. GFP expression was measured at 72 hours by flow cytometry. Data is plotted as triplicate mean of an independent experiment and is representative of n=3. Error bars represent standard error of the mean. Statistical significance was determined by two-way ANOVA using T ukey’s multiple comparisons test, ns, p > 0.05; *, p < 0.05; ** p < 0.01 ; ***, p < 0.00001.

Figure 3. Percentage expression of CAR, CD46, and integrins avp3, avp5 and avp6 in LI373 and LI87-MG glioblastoma cell lines. (A) LI373 and (B) U-87 MG cells were infected with 5000 or 10,000 vp/cell respectively of luciferase expressing Ad5, Ad5.RGD and Ad5NULL.RGD. Luminescence was measured at 72 hours and normalised to total protein, quantified by BCA assay (RLU/mg protein). Data is plotted as triplicate mean (U373, n=3; U87-MG n=1). Error bars represent standard deviation of the mean. Statistical significance was determined by two- way ANOVA using Tukey’s multiple comparisons test, ns, p > 0.05; *, p < 0.05; ** p < 0.01 ; ***, p < 0.00001.

Figure 4. (A) Flow cytometry data representing expression of the receptors, avp3, avp5, avp6 integrins, CAR and EGFR in E51 and E55 glioma stem cell lines. (B) Transduction of Ad5.RGD vs Ad5NULiRGD quantified by expression of luciferase transgene. E51 and E55 patient derived glioma stem cells were infected with 1000, 5000 and 10,000 vp/cell of luciferase expressing Ad5, Ad5.RGD and Ad5NULL.RGD. Luciferase expression was measured at 72 hours. Data is plotted as triplicate mean of an independent experiment and is representative of n=2. Error bars represent standard error of the mean. Statistical significance was determined by two-way ANOVA using Tukey’s multiple comparisons test, ns, p > 0.05; *, p < 0.05; ** p < 0.01 ; ***, p < 0.00001.

Figure 5. Cell viability was measured 96 hours post infection with oncolytic Ad5 (T1), Ad5NULiA20 and Ad5_NuLLRGD as well as replication deficient Ad5NULiA20. U-87 MG and PANC0403 cells were infected at a concentration of 500 vp/cell and cell viability was determined using CellTitreGlo kit. Percentage cell viability was normalised to uninfected cells. Data is representative of triplicate mean, n=3 independent experiments and error represents SEM. Statistical significance was determined by two-way ANOVA using Tukey’s multiple comparisons test, ns, p > 0.05; *, p < 0.05; ** p < 0.01 ; ***, p < 0.00001.

Figure 6. Transduction of Ad5.GFP, Ad5.RGD vs Ad5_NuLLRGD quantified by expression of GFP transgene. Four cell lines, E13, E51 , E55 and E56, were infected with 1000 vp/cell of GFP expressing Ad5, Ad5.RGD and Ad5NULL.RGD. GFP expression was measured at 72 hours. Data is plotted as triplicate mean of an independent experiment. Error bars represent standard error of the mean. Statistical significance was determined by two-way ANOVA using Tukey’s multiple comparisons test, ns, p > 0.05; *, p < 0.05; ** p < 0.01 ; ***, p < 0.00001.

Figure 7. Replication deficient Ad5, Ad5.RGD and Ad5_NuLLRGD (1x10^A10, 1x10^A9, 1x10^A8) were added to primary IPSC derived brain organoids (non tumour). GFP expression was measured 5 days post infection. Sections were dissociated with trypsin and GFP measured by flow cytometry. Each bar represents one section of brain organoid.

Figure 8. Replication and oncolytic Ad5Nuu_RGD (1x10^A9) were added to HiSpot media and replaced every 2 days over the course of 10 days. Cell viability was measured after 10 days using Cell Titre Gio Promega kit. Oncolytic caused greater reduction in cell death.

Figure 9. Transduction of glioma stem cell lines (E13, E51 and E55), obtained from the Glioma Cellular Genetics Resource, was evaluated using low seroprevalence Ad10 based viruses. A549 E6 (lung adenocarcinoma) cells were used as a positive control as they have high avp3 expression. PANC0403 (pancreatic ductal adenocarcinoma) cells were used as a negative control as they have lower expression of avp3 and avp5 integrins. The RGD motif was inserted into the DG loop of AdlO.GFP to mediate targeting to avp3 and avp5 integrins. An additional modification termed KO1 was made to ablate the native CAR binding. Cell lines were infected with two viral doses, 5000 and 10,000 vp/cell of GFP expressing vectors, AdlO.GFP, Ad10.RGD.GFP and Ad10.KO1.RGD.GFP. GFP expression was measured at 72 hours. Data represents mean of independent experiments (n=3; E51 and E55 and n=2; A549 E6, PANC0403, E13) and error bars represent standard deviation. Insertion of the RGD motif improved transduction in Ad10. RGD. GFP. Comparatively, the KO1 motif reduced transduction of Ad10.KO1.RGD.GFP compared to Ad10.RGD.GFP. This capacity of modified adenovirus retargeted using RGD motifs, to engage avp3 and avp5 integrin for cell entry in the absence of CAR, is a phenomenon seemingly specific for Ad5 serotype. Table 1. Primers used for recombineering to insert RGD into Ad5 fiber knob.

Materials and methods

Cell lines and Adenoviral vectors

Glioma stem cell lines (E51 and E55) were obtained from the Glioma Cellular Genetics Resource (GCGR) from Professor Steve Pollard (University of Edinburgh). Glioma stem cell lines, E13, E51 , E55 and E56 were derived from GBM patients as previously and cultured as previously described¹. Cells were grown in DMEM-F12 (cat. No. D8437). Complete DMEM- F12 with 1.25% glucose (Sigma, G8644), 2% I MEM-NEAA (Gibco, 11140-035) and Penicillin and Streptomycin (Pen/Strep), 0.16% BSA solution 7.5% (Gibco, 15260-037) 0.2% beta mercaptoethanol (Gibco, 31350-010), 1 % B27 Supplement (Gibco, 17504-044) and 0.5% N2 supplement (Gibco 17502-048). Complete media was supplemented with mouse EGF (10ng/ml, peprotech, 315-09), human FGF (10ng/ml, peprotech, 100-18b) and laminin (2pg/ml, Sigma, L2020). U-87-MG glioblastoma cell line were obtained from Dr. Florian Siebzehnrubl and U373 glioblastoma cells were purchased from ATCC. Both glioblastoma cell lines were cultured in complete DM EM supplemented with 10% FBS, 2% Pen/Strep and 1% L-Glutamine. Melanoma cell line MDA-MB 435 S parental line and MDA-MB 435 3av (Reduced av expression) and A549-E6 (lung carcinoma) were kindly gifted by Andreja Am briovic- Ristov and Dragomira Majhen (Rudjer Boskovic Institute)² and cultured in DMEM (Gibco, D6429) with 2% Pen/Strep and 1%L-Glutamine. PANC0403 were obtained from ATCC and were cultured in DMEM supplemented with 2% Pen/Strep and 1% L-Glutamine. (MDA-MB435) as in previous studies³.

Ad5NULLwas generated as previously described⁴ and harbours mutations in the hexon (HVR7), penton base (RGD/RGE) and fiber knob (KO1) proteins. A 16 amino acid peptide containing the RGC4C motif flanked by glycine and serine linkers (GGSCDCRGDCFCGSGG) was inserted between aa542 and aa543 (Uniprot P11818) into both the Ad5 and Ad5NULL vectors. Recombineering techniques were used to first insert a selection cassette into the fiber knob region and then replace this with an RGD4C oligo. A table of primers used in the recombineering process has been included below (Table 1). The RGD4C peptide was incorporated within the fiber knob HI loop which has previously proven tolerant and effective for incorporation of other peptides, including the avp6 integrin interacting peptide, A20, in previous studies⁵⁶. RGD4C was incorporated between flanking residues TQET and GDTT for Ad5 based viruses. The resulting viruses were termed Ad5.RGD and Ad5_NuLLRGD. Viral vectors were expanded in 293-TRex cells and purified using two step CsCI gradient ultracentrifugation. Flow cytometry to determine receptor expression.

Cells were incubated in FACS buffer (PBS (Thermo Fisher, cat. no. 10010023) with 5% fetal bovine serum (Sigma, cat. no. F9665) and labelled with mouse primary antibodies: anti-av 3 LM609 (Sigma, cat. no. mab1976), anti-av 5 P1 F6 (Abeam, cat. no. ab177004), anti-avp6 10D5 (Millipore, cat no. mab20772), anti-CAR RmcB (Millipore, cat. no. 05-644), anti-CD46 258-MEM (Novus Biologicals, cat. no. LS-B5950-50). Anti-avp3 and anti-avp6 were used at a concentration of 1 :250 and all other antibodies were diluted 1 :500. Cells were subsequently stained with a secondary anti-mouse 647-conjugated antibody (Thermo Fisher) and fixed with 4% paraformaldehyde (PFA). Appropriate fluorescence gating parameters were determined using an unstained and matched isotype control IgG-stained cells. In all experiments, doublets were eliminated using pulse geometry gates (FSC-H versus FSC-A). Single-cell suspensions were analysed using the BD Accu-riTM C6 flow cytometer; FlowJo software (FlowJo LLC) was used for subsequent analyses.

Viral transduction assays

Viral transduction assays were performed in serum free media. Cells were seeded at the optimal density 96-microwell plate (Nunc, Thermo Fisher) and transduced with stated concentrations of virus particles per cell (vp/ cell) after 24 hours. For each experimental condition, triplicate wells were treated with stated virus doses and a no virus control was included. Subsequent analysis was performed to determine transgene expression. GFP expressing viruses were imaged using the fluorescence microscopy (EVOS, ThermoFischer). When GFP was observed (typically after 72 hours) the cells were harvested using trypsin, washed with PBS and fixed in 4% PFA. Fluorescent cells were quantified using BD Accuri detected in the channel FL-1. Data was analysed using FlowJo software (FlowJo LLC). For luciferase expressing virus, cells were lysed 72 hours post infection and luciferase activity measured using a BioTek microplate reader using the luciferase assay system (Promega, cat. no. E1500) according to the manufacturer’s instructions. In addition, total protein concentration per well was determined using a microplate absorbance plate reader (BioTek) using the Pierce Micro BCA Protein assay (Thermo Fisher, cat. no. 10249133) to enable correction to relative luminescence units (RLU) per mg total protein present. Cell killing was measured using CellTitreGlo kit (Promega, G7570) according to manufactures instructions. mRNA expression analysis mRNA expression of the integrin subunits ITGB3, ITGB5 and ITGB6 in cases of glioma (n=66), upper aerodigestive (n=33), pancreatic (n=46) and breast (n=60) cancers, obtained from TCGA dataset available at https://software.broadinstitute.org. Gene Expression Profiling Interactive Analysis (GEPIA) comparison of ITGB3, ITGB5, ITGB6 and the aV integrin subunit, ITGAV, in normal brain vs GBM tissue. RNA expression was analysed 163 tumour samples compared to 207 normal samples for each gene and presented as a boxplot on a log-scale (log2(TMP+1)) using the GEPIA software. Significance has been determined with a p-value cut-off of p=0.01. Data available at gepia.cancer-pku.cn and obtained from TGCA dataset and GTEx dataset.

Statistical analysis

Statistical analysis was carried out in GraphPad (version 9) and specific tests are indicated in each figure legend and statistical significance is shown as follows; ns, p > 0.05; *, p < 0.05; **, p < 0.01 ; ***, p < 0.001 ; ****, p < 0.0001.

Results

Expression of av 3 and av 5 integrins in GBM

Epithelial cancers can be targeted through an upregulation avp6 integrin however this approach requires expression of avp6 integrin. Using mRNA data, we evaluated integrin expression in four common tumour types. Figure 1A demonstrates the mRNA expression of the p3, p5, and p6 subunits that form heterodimers with integrin av subunit to form the integrin receptors. In comparison to three other tumour groups (upper areo-digestive, breast and pancreas), glioma has the highest mRNA expression of ITGP3 and ITGP5. It also possesses the lowest expression of ITGP6, highlighting the necessity to modify our platform of avp6 targeted viruses to broaden the scope of these vectors to include brain cancer. Another important consideration is whether these integrins are present in healthy brain tissue. Figure 1 B compares upregulation of ITGB3, ITGB5, ITGB6 and ITGAV in normal brain tissue and GBM. ITGB3, ITGB5 and ITGAV are expressed to significantly higher levels in GBM compared to normal tissue, providing for a selectivity of GBM cells. Meanwhile, low expression levels of ITGB6 were observed in both healthy brain and GBM.

Ad5NULLRGD demonstrates improved transduction compared to Ad5 and Ad5RGD in non-glioma cell lines

A549 E6 and PANC0403 cell lines were transduced with luciferase expressing Ad5, Ad5.RGD and Ad5NULiRGD (Figure 2A). PANC0403 also demonstrated limited infection at 5000 vp/cell and no significant effect was observed. A549 E6 demonstrated significant increased luciferase production when infected with Ad5_NuLLRGD compared to both Ad5 and Ad5.RGD at both virus concentrations. The CAR negative cell line (MDA-MB435S) was used to determine the capacity of Ad5RGD and Ad5NULi GD to engage avp3 and avp5 integrin for cell entry in the absence of CAR (Figure 2B). MDA-MB435S av cells are depleted in the av subunit. Our data demonstrates that both Ad5.RGD and Ad5_Nuu.RGD are both able to efficiently transduce the parental cell line MDA-MB435S which expressed high levels of both avp3 and avp5 integrin. Overall transduction levels were lower in the av knock down strain, MDA-MB435S av, in correlation with the receptor expression levels.

Ad5NULLRGD demonstrates improved transduction compared to Ad5.RGD in glioma cell lines

We next assessed the vectors in cell lines derived from glioblastoma patients (Figure 3A). U373 and U-87 MG are commercially available and derived from individual glioblastoma astrocytoma patients. In 11373 cells, Ad5NULi_RGD transduction was found to be superior to Ad5 and Ad5.RGD without additional modifications. Preliminary experiments were conducted using U-87 MG cells (Figure 3B). As U-87 MG cells grow in neurospheres, a concentration 10,000 vp/cell was used with similar observation that Ad5Nuu.RGD.GFP demonstrated significantly superior activity to Ad5.RGD.

In addition, we evaluated these vectors in patient-derived glioma stem cells. Receptor expression of E51 and E55 was determined for avp3, avp5, avp6 integrins, CAR and EGFR (Figure 4A). E51 cells express high levels (100%) of avp3, avp5, CAR and EGFR whereas E55 receptor expression profile demonstrated more variability and lower expression (except for avp5 integrin). Both cell lines were negative for avp6 integrin. Transduction in these cell lines was determined at three viral doses: 1000 vp/cell, 5000 vp/cell and 10,000 vp/cell. As observed in glioma cell lines Ad5_NuLLRGD was able to transduce both E51 and E55 cells significantly better than both Ad5 and Ad5.RGD at each concentration.

We have therefore demonstrated that the modifications in the NULL platform consistently improve “on target” transduction of several cancer types including glioma cells that express the targets, avp3 and avp5 integrins. These transduction experiments were repeated with E51 and E55 cells and two additional glioma stem cell lines, E13 and E56 using GFP expressing vectors (Figure 6). This data demonstrates the percentage of cells infected and as expected a similar effect was observed with superior transduction observed with Ad5_NuLLRGD compared to Ad5.RGD. Oncolytic ACI5NULLRGD is able to selectively kill U-87 MG glioma cells

We next sought to investigate the targeted cell killing activity of oncolytic versions of Ad5NULL- RGD in avp3/5 positive cancer cell lines. Oncolytic Ad5NULi_RGD demonstrates effective cell killing in avp3 positive U-87 MG cells in comparison to avp3 negative PANC0403 cells (Figure 5). Oncolytic efficacy was compared in U-87 MG (avp3 high) and PANC0403 (avp3/5 low) cells (Figure 7). Cells were transduced with 500 vp/cell oncolytic Ad5NULi_RGD, Ad5NULi_A20 and Ad5 with a T1 and dl24 mutation. Replication deficient Ad5NULi_A20 was also included as a control and did not affect cell viability. Oncolytic Ad5 (A24 T1) demonstrated no significant difference in cell killing between the two cell lines. Oncolytic Ad5NULi_A20 demonstrated significant cell killing in avp6 positive PANC0403 cells but not in U-87 MG, due to the lack of avp6 integrin expression. Ad5Nuu_RGD infection resulted in cytolysis of around 50% of avp3 positive U-87 MG but not PANC0403. This is significantly lower than the replication deficient controls. This data indicates Ad5_NuLLRGD is able to specifically infect and kill avp3 positive U- 87 MG glioma cells but not avp3/5 low PANC0403, whilst the converse was true of the avp6 selective precision virotherapy, Ad5NULL-A20, which was consistent with the receptor profiles for these respective cell lines.

Transduction in 3D models of glioblastoma

Preliminary data suggests Ad5_NuLLRGD transduces non tumour IPSC derived brain organoid sections less efficiently than Ad5.RGD (Figure 7). This has the advantage of less off target effects. HiSpots are primary patient derived tumour models grown with an air liquid interface. Preliminary data suggests oncolytic Ad5NULLRGD is more effective at tumour cell killing than Ad5 and Ad5.RGD in this HiSpot model (Figure 8).

Comparison ofKO1 modification in Ad10 vectors

We observed that a combination of modifications in Ad5NULL improved the transduction of Ad5NULiRGD compared to Ad5.RGD. We investigated ablating the native adenoviral CAR binding, through a mutation termed KO1 , in an alternative low seroprevalence vector, Ad10 targeted through RGD. We introduced the KO1 mutation into the Ad10.RGD.GFP vector to produce Ad10.KO1.RGD.GFP. The RGD motif is inserted in the DG loop of Ad10 in comparison to the HI loop of Ad5. The KO1 mutation is in the equivalent position to the Ad5NULL vector. Ad10.KO1.RGD was compared to AdlO.RGD (Figure 9). Interestingly, although we observed an increase in transduction upon introduction of the RGD motif into Ad10, as expected, addition of the KO1 mutation decreased viral transduction. Our data suggests that the KO1 mutation reduces viral infection for Ad10.KO1.RGD compared to AdlO.RGD which is not consistent with the observations described for Ad5_NuLL GD compared to Ad5.RGD.

Discussion

We engineered the basal Ad5NULL vector to improve the selectivity for avp3 and avp5 integrin which are overexpressed, making them promising targets in GBM. For proof of concept, we have developed Ad5Nuu_RGD through incorporation of RDG4C (CDCRGDCFC) into the HI loop of the fiber knob, with resulting viruses which efficiently target avp3/5 integrin, but without detrimental off target, dose limiting interactions that otherwise hinder selectivity.

Our data indicates Ad5_NuLLRGD demonstrates a marked improvement in transduction compared to Ad5 and Ad5.RGD. We show selective transduction in GBM cell lines expressing avp3 integrin. We have therefore generated and tested our first avp3 selective, Ad5Nuu_RGD that is well suited to GBM applications. We have further evaluated these vectors in relevant models of GBM including brain organoids and patient-derived 3D cultures. Future work includes incorporating transgenes that mediate tumour-specific replication and cell killing. These novel precision immunovirotherapies hold the potential to offer new therapeutic options for devastating brain cancers of significant unmet clinical need.

We hypothesised that introduction of the CAR ablating KO1 mutation would impact an alternative serotype, AdlO.RGD, in a similar way, either demonstrating an equivalent or improved transduction however we demonstrated this observation was specific to Ad5NULiRGD.

To conclude we have demonstrated introducing three additional modifications to the Ad5.RGD, through the Ad5NULL platform, significantly improved “on target” transduction and cell killing of cells expressing the target integrins, avp3 and avp5. We have also demonstrated this improvement can be harnessed as an oncolytic virotherapy and demonstrated on target GBM cell killing with reduced off target effects. These data are support the future clinical translation of oncolytic versions of Ad5NULL-RGD in avp3/5 positive cancer types such as GBM. The activity of these precision virotherapies can be enhanced through the additional engineering of the recombinant adenoviral genome to encode potent therapeutic transgenes to enhance clinical efficacy. References

1. Bates, E. A. et al. Engineering Adenoviral Vectors with Improved GBM Selectivity. Viruses 15, 1086 (2023).

2. Nestic, D. et al. avp3 Integrin Is Required for Efficient Infection of Epithelial Cells with Human Adenovirus Type 26. J. Virol, 93(1), e01474- 18 (2019).

3. Paradzik, M. et al. KANK2 Links aV 5 Focal Adhesions to Microtubules and Regulates Sensitivity to Microtubule Poisons and Cell Migration. Front. Cell Dev. Biol., (2020).

4. Uusi-Kerttula, H et al. Ad5NULL-A20: A Tropism-Modified, avp6 Integrin-Selective Oncolytic Adenovirus for Epithelial Ovarian Cancer Therapies. Clin Cancer Res,

24(17):4215-4224 (2018).

5. Uusi-Kerttula, H. et al. Pseudotyped avp6 integrin-targeted adenovirus vectors for ovarian cancer therapies. Oncotarget 7, 27926-27937 (2016).

6. Davies, J. A. et al. Efficient Intravenous Tumor Targeting Using the avp6 I ntegrin- Selective Precision Virotherapy Ad5NULL-A20. Viruses 13, 864 (2021).

Table 1

Claims

Claims

1. A viral vector of Ad5 serotype adenovirus (Ad5NULL-RGD) modified to comprise: a) at least one of 1421 G, T423N, E424S, E450Q or L426Y point mutation(s) in the hexon hypervariable region 7 (HVR7 mutation) wherein said mutation prevents virus binding with coagulation factor 10 (FX); b) at least one of S408E or P409A point mutation(s) in the fiber knob region AB loop (KO1 mutation) wherein said mutation prevents virus binding with the coxsackie and adenovirus receptor (CAR); c) at least one of D342E or D342A point mutation(s) in the penton integrin binding motif Arg-Gly-Asp (RGD) wherein said mutation prevents virus binding with OvPs/avPs integrin; and d) present or express an OvPs/avPs integrin binding RGD cell targetting peptide.

2. The viral vector according to claim 1 wherein said at least one HVR7 mutation comprises at least one of 1421 G, T423N, E424S, and L426Y point mutations.

3. The viral vector according to any one of claims 1-2 wherein least one KO1 mutation comprises S408E and P409A point mutations.

4. The viral vector according to any one of claims 1-3 wherein at least one RGD mutation comprises the point mutation D342E to produce RGE.

5. The viral vector according to any one of claims 1-4 wherein said OvPs/avPs integrin binding RGD cell targetting peptide comprises, or consists, of a sequence selected from the group comprising:

ACDCRGDCFCG (RGD4C) (SEQ ID NO: 1);

DGARYCRGDCFDG (RGD10) (SEQ ID NO: 2);

VTGRGDSPASS (FN-RGD2) (SEQ ID NO: 3);

GCTIGRGDWAPSECKQDSDCLAGCVCGPNGFCG (1.5B) (SEQ ID NO: 4); GCPQGRGDWAPTSCKQDSDCRAGCVCGPNGFCG (2.5D) (SEQ ID NO: 5); GCPRPRGDNPPLTCKQDSDCLAGCVCGPNGFCG (2.5F) (SEQ ID NO: 6); or a sequence with at least 75% identity thereto and which retains the biological activity or targeting of OvPs/avPs integrin.

6. The viral vector according to claim 5 wherein said RGD cell targeting peptide is selected from: ACDCRGDCFCG (RGD4C) (SEQ ID NO: 1); DGARYCRGDCFDG (RGD10) (SEQ ID NO: 2); or a sequence with at least 75% identity thereto.

7. The viral vector according to any one of claims 1-6 wherein said RGD cell targeting peptide is inserted into the HI loop of HAdV fiber knob protein.

8. The viral vector according to claim 7 wherein said RGD cell targeting peptide is inserted a position between amino acids 304-305 of the fibre knob protein.

9. The viral vector according to any one of claims 1-8 wherein presentation or expression of said RGD cell targetting peptide comprises at least one peptide sequence or linker.

10. The viral vector according to claim 9 wherein said linker comprises the sequence GGS.

11 . The viral vector according to any one of claims 1-10 wherein said viral vector (Ad5NULL- RGD) is modified to comprise: a) I421G, T423N, E424S, E450Q and L426Y point mutations in the hexon hypervariable region 7 (HVR7 mutation) wherein said mutations prevent virus binding with coagulation factor 10 (FX); b) S408E and P409A point mutations in the fiber knob region AB loop (KO1 mutation) wherein said mutations prevent virus binding with the coxsackie and adenovirus receptor (CAR); c) D342E point mutation in the penton integrin binding motif Arg-Gly-Asp (to produce RGE mutation) wherein said mutation prevents virus binding with aVp3/aVp5 integrin; and d) present or express an OvPs/avPs integrin binding RGD cell targetting peptide.

12. The viral vector according to any one of claims 1-10 wherein said vector is further modified to include at least one growth factor antibodies, and/or at least one matrix degrading enzyme.

13. The viral vector according to any one of claims 1-12 wherein said viral vector is further modified to include a molecule which is a transgene encoding a therapeutic agent.

14. The viral vector according to any one of claims 1-13 for use as a medicament.

15. The viral vector according to claim 14 for use in the treatment of cancer or other diseases where avp3/5 expression can be pathological.

16. Use of a viral vector according to any one of claims 1-13 in the manufacture of a medicament to treat cancer or other diseases where avp3/5 expression can be pathological.

17. A pharmaceutical composition comprising the vector according to any one of claims 1- 13 and a pharmaceutically acceptable carrier, adjuvant, diluent or excipient.

18. A combination therapeutic comprising the viral vector according to any one of claims 1-3 and at least one further therapeutic agent.

19. A method for treating a disease in a subject comprising administering an effective amount of the viral vector according to any one of claims 1-13 or pharmaceutical composition according to claim 17 or combination therapeutic according to claim 18 to a patient in need thereof.

20. The method according to claim 19 wherein said disease is cancer, optionally, skin cancer, kidney cancer; ovarian cancer; prostate cancer; and in particular brain cancer, such as that selected from any one or more of the following cancers: chordomas, gangliocytomas, glomus jugulare, Schwannomas, astrocytomas, ependymomas, gliomas, glioblastoma, glioblastoma, multiforme mixed gliomas, hemangioblastomas, Rhabdoid tumours, meningiomas, pituitary tumours, craniopharyngiomas, germ cell tumours, pineal region tumours, medulloblastomas, CNS brain lymphomas, or metastatic forms thereof.