Tumor blood vessel-targeted aav therapy for cancer treatment

Abstract

The present disclosure relates to novel adeno-associated adenovirus (AAV) vectors comprising targeting peptides. More particularly, the present disclosure relates to an adeno- associated serotype 2 viral vector, AAV2, comprising a transgene encoding LIGHT, wherein a viral capsid of the AAV2 vector comprises a targeting peptide that changes its tropism to target tumor endothelial cells, and uses thereof. The present disclosure also relates to use of the vectors in therapy, in particular in the treatment of cancers.

Description

[0001] TUMOR BLOOD VESSEL-TARGETED AAV THERAPY FOR CANCER TREATMENT

[0002] TECHNICAL FIELD

[0003] The present disclosure relates to novel adeno-associated virus (AAV) vectors comprising targeting peptides. Particularly, the present disclosure relates to AAV vectors comprising a transgene encoding the immune-activating protein LIGHT, or a transgene encoding a lymphotoxin beta receptor (LTpR) agonist, such as LIGHT and / or lymphotoxins, and uses thereof. In some embodiments the vectors are used in therapy, in particular in the treatment of cancers.

[0004] BACKGROUND

[0005] Cancer is one of the most prevalent deadly diseases, which despite recent advances in diagnosis and treatment still accounts for a substantial number of deaths each year. In spite of the current developments, there is still a need for new and better medical treatments of cancer.

[0006] SUMMARY

[0007] An object of the present disclosure is to provide novel AAV vectors that may be used in medical treatments. Another object is to provide novel ways to increase T cell infiltration into tumors, especially via tumor blood vessels. This object is obtained by an adeno-associated serotype 2 viral vector, (AAV2) comprising a transgene encoding LIGHT, wherein a viral capsid of the AAV2 vector comprises a targeting peptide targeting tumor blood vessels.

[0008] According to some aspects, a pharmaceutical composition is provided. The pharmaceutical composition contains an AAV2 vector as described above, and a pharmaceutically acceptable carrier or excipient. The AAV2 vector or the pharmaceutical composition as described above may be for use in therapy. In some embodiments, the therapy is cancer therapy or immunotherapy.

[0009] According to some aspects, the disclosure proposes a method of targeting LIGHT to cells in the tumor microenvironment in a subject in need thereof, wherein the cells in the tumor microenvironment comprise tumor endothelial cells of a tumor blood vessel, the method comprising: providing an AAV2 viral vector encoding LIGHT as described above, and administering the AAV2 viral vector to the subject, or a pharmaceutical composition thereof, whereby the AAV2 viral vector transduces the targeted cells, thereby inducing the targeted cells to express LIGHT. Thus, it is a general objective of the present disclosure to use recombinant AAV2 vectors encoding LIGHT (AAV2-TEC(LIGHT)) to target either endothelial cells within tumors, or other cells in the tumor microenvironment. It is a particular objective to provide AAV2-TEC(LIGHT) for cancer immunotherapy.

[0010] This and other objectives are met by embodiments as disclosed herein. The present invention is defined in the independent claims. Further embodiments of the present invention are defined in the dependent claims.

[0011] Other objects and advantages will become apparent to those skilled in the art from a review of the ensuing detailed description, which proceeds with reference to the following illustrative drawings, and the attendant claims.

[0012] BRIEF DESCRIPTION OF THE DRAWINGS

[0013] The above, as well as additional objects, features and advantages of the present inventive concept, will be better understood through the following illustrative and non-limiting detailed description of different embodiments of the present inventive concept, with reference to the appended drawings, wherein:

[0014] Figure 1 illustrates a proposed mode of action for AAV2-lntpi (LIGHT) therapy.

[0015] Figure 2 shows prolonged survival in glioma-bearing mice after AAV-BR1 -LIGHT therapy, in association with induction of T cell-rich TLS and tumor-associated high endothelial venules (TA- HEVs) that recruit T cells. AAV-BR1-GFP is also referred to as BR1-GFP, and AAV-BR1-LIGHT is also referred to as BR1 -LIGHT. Figure 2A illustrates the experimental layout used to obtain data shown in Figures 2B-2H. Figure 2B shows a Kaplan-Meier survival curve of CT-2A tumor-bearing mice treated with AAV-BR1-GFP or AAV-BR1-LIGHT. Figure 2C shows a Kaplan-Meier survival curve of mGC1 tumor-bearing mice treated with AAV-BR1-GFP or AAV-BR1-LIGHT. Figure 2D shows representative RNAscope images illustrating colocalization of LIGHT mRNA with Pecaml mRNA in CT-2A tumor-bearing brains that received AAV-BR1-LIGHT treatment. Figure 2E shows quantification of the number of TLS per brain after AAV-BR1-GFP or AAV-BR1 -LIGHT treatment. Figure 2F shows the T cell to B cell ratio within the TLS. Figure 2G shows the proportion of the tumor blood vessel area which was HEV positive. Figure 2H shows quantification of CD3+T cells touching TA-HEVs vs other tumor blood vessels.

[0016] Figure 3 shows that AAV-BR1-LIGHT therapy promotes T cell functionality, which correlates with tumor regression. AAV-BR1-GFP is also referred to as BR1-GFP, and AAV-BR1 -LIGHT is also referred to as BR1 -LIGHT. Figure 3A illustrates the experimental layout used to obtain data shown in Figures 3B-3E. Figure 3B shows the quantification of granzyme (GZM)-B expression on isolated CD8+T cells. Figure 30 shows the quantification of interferon (IFN)-y and tumor necrosis factor (TNF)-a expression on isolated CD8+T cells. Figure 3D shows the quantification of CD69 expression on isolated CD8+T cells. Figure 3E shows the quantification of CD107a expression on isolated CD8+T cells. Figure 3F shows the proportion of brains presenting with a macroscopic tumor (“tumor”) or not (“no macroscopic tumor”) in each treatment group at day 14 and day 21.

[0017] Figure 4 shows that AAV-BR1 -LIGHT therapy promotes stem-like CD8+T cells. Figure 4A illustrates the experimental layout used to obtain data shown in Figures 4B-4C. Figure 4B shows the quantification of stem-like T cells (% of tumor-targeted CD8+T cells) in AAV-BR1-GFP- or AAV- BR1-LIGHT-treated groups. Figure 40 shows the ratio of stem-like T cells / exhausted T cells in AAV- GFP- or AAV-LIGHT-treated groups.

[0018] Figure 5 shows the development of novel tumor blood vessel-targeted AAV2 vectors by rational design. Figure 5A shows the cloning strategy followed for the development of tumor blood vessel-targeted AAV2 vectors by cyclic peptide insertion. Figure 5B shows the VP3 structures of AAV2-lntpi and AAV2-iRGD vectors after peptide insertion, compared to AAV2-WT.

[0019] Figure 6 shows the vector biodistribution properties of AAV2-lntpi and AAV2-iRGD vectors in healthy mice following systemic administration. Figure 6A illustrates the experimental layout used to obtain data shown in Figures 6B-D. Figure 6B shows the quantification of AAV2-WT vector genomes (vg) in various organs of healthy mice following systemic administration. Figure 60 shows the quantification of AAV2-lntpi vector genomes (vg) in various organs of healthy mice following systemic administration. Figure 6D shows the quantification of AAV2-iRGD vector genomes (vg) in various organs of healthy mice following systemic administration.

[0020] Figure 7 shows the tumor blood vessel-targeting ability of AAV2-lntpi and AAV2-iRGD vectors in glioma-bearing mice. Figure 7A illustrates the experimental layout used to obtain data shown in Figures 7B-F. Figure 7B shows representative immunofluorescence images of vector- encoded GFP in the blood vessels within the tumor area, following systemic administration of AAV2- WT, AAV2-lntpi or AAV2-iRGD. Figure 70 shows representative immunofluorescence images of vector-encoded GFP in the blood vessels within the non-tumor area in each treatment group. Figure 7D shows the quantification of the proportion of GFP-transfected blood vessels within the tumor area in each treatment group. Figure 7E shows the quantification of the proportion of GFP- transfected activated blood vessels within the tumor area in each treatment group. Figure 7F shows the quantification of the proportion of GFP-transfected blood vessels within the non-tumor area in each treatment group. Figure 8 shows the ability of AAV2-lntpi and AAV2-iRGD vectors to transduce human brain microvascular endothelial cells (HBMVECs) in vitro. Figure 8A shows the quantification of transduced HBMVECs when treated with mock, AAV2-WT, AAV2-lntpi or AAV2-iRGD vectors. Figure 8B shows a comparison of the transduction efficiency of AAV2-lntpi vector compared to AAV2-BR1 and AAV2-WT vectors at various multiplicity of infection (MOI). Figure 8C shows the quantification of the relative transduction of HBMVECs when treated with AAV2-WT or AAV2-lntpi in conjunction with an antibody (12G10) blocking active a5pi integrin. Figure 8D shows the quantification of the relative transduction of HBMVECs when treated with AAV2-WT or AAV2-lntpi in conjunction with an isotype control antibody. Figure 8E shows the quantification of the relative transduction of HBMVECs when treated with AAV2-WT or AAV2-lntpi in conjunction with various concentrations of heparin. Figure 8F shows the quantification of the relative expression levels of hLIGHT by HBMVECs following treatment with AAV2-lntpi(hLIGHT) at various MOIs. Figure 8G shows the quantification of the relative expression levels of VCAM-1 by HBMVECs following treatment with AAV2-lntpi(hLIGHT) at various MOIs. Figure 8H shows the quantification of the relative expression levels of SELE by HBMVECs following treatment with AAV2-lntpi(hLIGHT) at various MOIs. Figure 8I shows the quantification of the relative expression levels of MADCAM-1 by HBMVECs following treatment with AAV2-lntpi-(hLIGHT) at various MOIs.

[0021] Figure 9 shows the ability of AAV2-lntpi to transduce the human glioblastoma vasculature ex vivo. Figure 9A illustrates the experimental layout used to obtain data shown in Figure 9B. Figure 9B shows representative immunofluorescence images of vector-encoded GFP in the blood vessels within the tumor area in brain slices from two patients following treatment with AAV2-lntpi compared to AAV2-WT and AAV2-iRGD.

[0022] Figure 10 shows prolonged survival in glioma-bearing mice following treatment with AAV2- Intpi-LIGHT. AAV2-lntpi-GFP is here referred to as AAV-lntpi-GFP, and AAV2-lntpi-LIGHT is here referred to as AAV-lntpi-LIGHT. Figure 10A illustrates the experimental layout used to obtain data shown in Figures 10B-E. Figure 10B shows a Kaplan-Meier survival curve of CT-2A tumorbearing mice treated with AAV2-lntpi-GFP or AAV2-lntpi-LIGHT, and a Kaplan-Meier survival curve of CT-2A tumor-bearing mice treated with AAV2-BR1-GFP or AAV2-BR1-LIGHT. Figure 10C shows representative immunofluorescence images of fibrinogen leakage in each treatment group. Figure 10D shows the quantification of fibrinogen positive area within the tumor area in each treatment group. Figure 10E shows the quantification of fibrinogen positive area within the nontumor area in each treatment group. Figure 11 shows the inducible expression of LIGHT using the mini-Xon AAV system. Figure 11A illustrates the encoding of LIGHT on a self-complementary mini-Xon AAV system, in which expression is regulated using a splice modulator drug (LMI070). Figure 11B shows the quantification of normalized LIGHT levels expressed by HEK293 cells following treatment with AAV-mini-Xon- LIGHT in conjunction with various concentrations of the drug LMI070.

[0023] Figure 12 shows the development of exclusively soluble or exclusively membrane-bound LIGHT. Figure 12A illustrates the strategy used to develop exclusively soluble or exclusively membrane-bound LIGHT. Figure 12B shows the detection of soluble LIGHT (SLIGHT) following transfection of HEK293 cells with plasmids encoding exclusively soluble LIGHT or exclusively membrane-bound LIGHT. Figure 12C shows the detection of membrane-bound LIGHT (mLIGHT) following transfection of HEK293 cells with plasmids encoding exclusively soluble LIGHT or exclusively membrane-bound LIGHT.

[0024] DETAILED DESCRIPTION

[0025] The present disclosure relates to new vectors, which may encode said protein LIGHT, or a lymphotoxin beta receptor (LTpR) agonist, including LIGHT and lymphotoxins, wherein the vectors comprise targeting peptides. The vectors, and the pharmaceutical compositions comprising said vectors, may be also used in medical treatments, such as cancer therapies.

[0026] The aim of the present disclosure is to provide new ways to express LIGHT and / or lymphotoxins in the tumor microenvironment, and thereby increase T cell recruitment and infiltration into tumors, which may be used in therapy.

[0027] Aspects of the present disclosure will be described more fully hereinafter with reference to the accompanying drawings. The targeting peptides, vectors and methods disclosed herein can, however, be realized in many different forms and should not be construed as being limited to the aspects set forth herein. Like numbers in the drawings refer to like elements throughout.

[0028] The terminology used herein is for the purpose of describing particular aspects of the disclosure only and is not intended to limit the disclosure. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which the present invention belongs.

[0029] As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0030] AAV stands for Adeno-Associated Virus, which are small viruses with a single-stranded DNA (ssDNA) genome of about 4.8 kbp. AAVs are small viruses that infect humans and some other primate species. AAV vectors can infect both dividing and quiescent cells and persist in an extrachromosomal state without integrating into the genome of the host cell. Several natural AAV serotypes exist, and a multitude of recombinant AAV serotypes have been developed. The ssDNA AAV genome consists of two open reading frames (ORFs), Rep and Cap, flanked by two 145-base inverted terminal repeats (ITRs). Thus, the genome comprises ITRs of 145 bases at both ends of the DNA strand and two ORFs. The ITRs pair to allow for synthesis of the complementary DNA strand. Rep and Cap are translated to produce multiple distinct proteins (Rep78, Rep68, Rep52, Rep40 - required for the AAV life cycle; VP1 , VP2, VP3 - capsid proteins), where Rep is composed of four overlapping genes encoding Rep proteins required for the AAV life cycle, and Cap contains overlapping nucleotide sequences encoding the capsid virion proteins (VPs): VP1 (90 kDa), VP2 (72kD) and VP3 (60 kD), which interact with each other to form a capsid with icosahedral symmetry. The capsid of an AAV is an icosahedron assembled from 60 VP monomers with approximately 50 copies of VP3, 5 copies of VP2, and 5 copies of VP1. The capsid protrusions harbor variable regions which are important for binding to certain receptors for cell attachment (primary receptor binding) and internalization (secondary receptor binding) which determines the virus tropism and thus results in different serotypes.

[0031] Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism {in vivo) or in cell culture in vitro). Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes or other genetic material by a vector is termed transduction and the infected cells are described as transduced. Generally, a viral vector may be defined by three components including the i) protein capsid and / or envelope that encapsidates the genetic payload, and defines the vector’s tissue or cell tropism and antigen recognition, ii) the transgene of interest, which when expressed in cells, serves to confer a desired effect; and iii) the “regulatory / expression cassette,” the combined enhancer / promoter / auxiliary elements that controls stable or transient somatic expression of the transgene as an episome or as a chromosomal integrant. Thus, when referring to a “viral vector” herein, all components of the vector necessary for its function are included.

[0032] AAVs are frequently used as gene therapy vectors, by removing the Rep and Cap genes and replacing them with an expression cassette consisting of a promoter and a transgene in between the inverted terminal repeat (ITR) sequences. The expression cassette can be approximately 4.8 kbp in size. When constructing an AAV transfer plasmid, the transgene is placed between the two ITRs, and Rep and Cap are supplied in trans, i.e. the Rep and Cap genes together with viral genes needed for AAV replication are provided in trans during production of AAV vectors. Thus, ITRs are considered to be the only sequences required in cis next to the therapeutic gene: structural (Cap) and packaging (Rep) proteins can be delivered in trans. In addition to Rep and Cap, AAV requires a helper plasmid containing genes from adenovirus. These genes (E4, E2a and VA) mediate AAV replication. The transfer plasmid, Rep / Cap, and the helper plasmid are transfected into HEK293 cells, which contain the adenovirus gene E1 , to produce infectious AAV particles. Rep / Cap and the adenovirus helper genes may also be combined into a single plasmid; the separation of Rep and Cap may facilitate the viral pseudotyping. At least 13 serotypes of AAV have been identified. In the present disclosure, AAV refers to serotype 2 (AAV2).

[0033] With “AAV vectors” is referred to viral vectors that may be used for gene delivery, i.e., as gene therapy vectors. The desired gene together with a promoter to drive transcription of the gene is inserted between the ITRs that aid concatemer formation in the nucleus after the ssDNA of the vector is converted by host cell DNA polymerase complexes into double-stranded DNA. The AAV ITRs of two genomes can anneal to form head-to-tail concatemers, almost doubling the capacity of the vector. Insertion of splice sites allows for the removal of the ITRs from the transcript. An altered version of AAV has been created which is termed self-complementary adeno-associated virus (scAAV) as compared to single-stranded AAV (ssAAV). Whereas AAV packages a single strand of DNA and must wait for its second strand to be synthesized, scAAV packages two shorter strands that are complementary to each other. The genome is double-stranded and thus the expression cassette can then only be approximately 2.4 kbp, i.e. half the size of the single-stranded version of AAV. With “AAV-LIGHT” is referred to an AAV2 vector encoding LIGHT. Whereas ssAAV packages a single strand of DNA and requires the process of second-strand synthesis, scAAV packages both strands which anneal together to form double-stranded DNA. By skipping second strand synthesis scAAV allows for rapid expression in the cell.

[0034] In the present disclosure, AAV vectors, particularly of serotype 2, i.e., AAV2 vectors are used, and the capsid is modified to express a targeting peptide, such as the peptides of SEQ ID NO: 1-2, which may be incorporated as illustrated by the constructs of SEQ ID NO: 3-4. The viral vectors include an expression cassette for expressing a transgene, the transgene preferably being human LIGHT in native form, or in soluble form only or membrane-bound form only. Examples of constructs using murine LIGHT in different forms is included in SEQ ID NO: 9-14, and human LIGHT examples are included in SEQ ID NO: 30-33 and 38-40. The transduction of the AAV2 viral vector will start when the targeting peptide in the envelope of the AAV2 viral vector binds to the cell surface of the targeted cells, upon which the vector (and receptor) may be internalized in the cell using endocytosis. It may then be released from the endosomes and intracellularly trafficked to the nucleus, where it becomes uncoated and the AAV2 genome is released. If using ssAAV, the ssDNA genome is converted into dsDNA followed by transcription and nuclear export of mRNA for translation and expression of the LIGHT transgene. Any version of the capsid, i.e. any capsid based on the construct according to SEQ ID NO: 3-4, may be used in any combination with any expression cassette, i.e. comprising any variant of LIGHT and promoter constructs.

[0035] The terms “protein LIGHT” or “LIGHT” may be used interchangeably herein, and refer to the protein that in humans has the UniProt number 043557, and in mice has the UniProt number Q9QYH9, or modifications thereof. LIGHT stands for "homologous to lymphotoxin, exhibits inducible expression and competes with HSV glycoprotein D for binding to herpesvirus entry mediator, a receptor expressed on T lymphocytes". In the cluster of differentiation terminology, it is classified as CD258. LIGHT is also known under the names tumor necrosis factor superfamily member 14 (TNFSF14), a secreted protein of the TNF superfamily, herpesvirus entry mediator ligand (HVEML), sometimes also LTg, TR2, and TNLG1 D. LIGHT has been shown to interact with the receptors lymphotoxin beta receptor (LTpR or TNFRSF3), herpesvirus entry mediator (HVEM or TNFRSF14), and a soluble decoy receptor named DcR3 (TNFRSF6B).

[0036] The AAV2 vectors of the present disclosure may comprise a transgene that encodes LIGHT, preferably human LIGHT or a construct thereof of human origin, where the transgene encoding LIGHT is selected from native LIGHT, which may be both soluble and membrane-bound, exclusively soluble LIGHT, and exclusively membrane-bound LIGHT, with the two latter obtained by modifications. The native murine LIGHT nucleotide sequence is defined by SEQ ID NO:5 and the corresponding amino acid sequence by SEQ ID NO:18, and native human LIGHT nucleotide sequence by SEQ ID NO:15, and the corresponding human native LIGHT amino acid sequence by SEQ ID NO:34. Exclusively soluble murine LIGHT nucleotide sequence is defined by SEQ ID NO:6, and the corresponding amino acid sequence by SEQ ID NO: 19. Exclusively soluble human LIGHT amino acid sequence is defined by SEQ ID NO:35. The exclusively membrane-bound LIGHT having a silenced protease cleavage site, wherein the protease cleavage site may be silenced, for example by mutating amino acid 81 from L to A (L81A) as in SEQ ID NO:7 illustrating murine LIGHT having these modifications, or by deleting amino acids 78-82, referred to as ALIGHT, illustrated in SEQ ID NO:8 for murine LIGHT. It should be noted that corresponding soluble or membrane-bound human LIGHT may be used, having the same / similar modifications. The amino acid sequences for exclusively membrane bound human LIGHT is defined by SEQ ID NO:36 (hLIGHT L83A) and SEQ ID NO: 37 (hLIGHT as ALIGHT, i.e. AhLIGHT). The term “blood vessel” refers to the tubular structures that compose the circulatory system and carry the blood. The blood vessels consist of endothelial cells that line the vessel lumen, a basal lamina consisting of extracellular matrix and supporting mural cells, such as pericytes and smooth muscle cells.

[0037] “Endothelial cells” form the inner lining of a blood vessel and provide an anticoagulant barrier between the vessel wall and blood. “Pericytes” are multi-functional mural cells of the microcirculation that wrap around the endothelial cells that line the capillaries throughout the body.

[0038] A type of blood vessel that is specialized in T cell recruitment is called a high endothelial venule (HEV). With “high endothelial venules” (HEVs) is referred to endothelium specialized in recruitment of naive lymphocytes. HEVs consist of plump endothelial cells that under normal conditions are exclusively present in lymph nodes or lymphoid tissues, but by therapeutic means can be induced elsewhere in the body including in tumors. Therefore, means to increase pro- inflammatory activation of tumor endothelial cells or induce HEVs within or near tumors can increase T cell infiltration and thereby increase the efficiency of cancer immunotherapy.

[0039] With “recruitment” of lymphocytes (T and B lymphocytes) is referred to a process which attracts lymphocytes from the circulation to / into a tissue. With “infiltration” is referred to that the recruited lymphocytes enter into the tissues, such as tumor tissues. The lymphocytes are recruited from the circulation and infiltrate through the tumor blood vessels, a process which is more efficient if the vessels adopt a HEV-like phenotype. Once in the tumor, they can migrate to find and eliminate tumor cells. With T lymphocyte “activation” is referred to the process where naive T lymphocytes are activated by antigen-presenting cells, to form activated T lymphocytes. Activated CD8+T cells become cytotoxic T lymphocytes (CTLs).

[0040] The term “tertiary lymphoid structures” (TLS) refers to ectopic lymphoid aggregates / structures that develop at sites of chronic inflammation and pathological conditions, including autoimmune disease, infection, and cancer. In human cancer, spontaneous TLS formation has been documented in various tumor types and is generally associated with favorable patient outcome. TLS may serve as alternative sites for antigen presentation and T cell priming. Therefore, means to induce TLS within or near tumors is considered a novel form of cancer immunotherapy.

[0041] Intra-tumoral “antigen-presenting niches” are aggregates of immune cells found inside tumors, that are distinct from TLS. They frequently harbor antigen-presenting cells such as dendritic cells and T cells with stem-like characteristics, i.e. TCF1+stem-like CD8+T cells. Tumors that harbor these structures usually exhibit higher levels of T cell infiltration, while patients with progressive disease often lack these immune niches. Therefore, means to induce or maintain intra- tumoral antigen-presenting niches could be developed into an interesting form of cancer immunotherapy.

[0042] The terms “targeting peptide”, “vessel-targeting peptide”, or “tumor blood vessel-targeting peptide” are used to refer to peptides that are present in the capsid of the AAV2 viral vector and used to attain a certain binding specificity of the capsid, and are used to describe how a cargo or vector can be redirected to specific target molecules or specific target cells. The AAV vectors of the present disclosure comprise a targeting peptide in their capsid. The preferred targeting peptides of the present disclosure are defined in SEQ ID NO: 1-2, with their respective nucleotide sequences in SEQ ID NO:16-17.

[0043] By “therapy” as used herein is meant the treatment of any medical condition. Such treatment may be prophylactic (i.e. preventative), curative (or treatment intended to be curative), or palliative (i.e. treatment designed merely to limit, relieve or improve the symptoms of a condition). Thus, “therapy” or “treating” of a disorder, such as cancers / tumors, by means of an AAV2 viral vector as used herein, are referring to ameliorating a certain disorder or medical condition, or to cure it. In the case of cancer and tumors, the treatments may shrink or abolish the present tumors, or they may halt or prevent the further spread of the tumors. An amount adequate to accomplish this is defined as a ’’therapeutically effective amount”. Effective amounts for a given purpose will depend on the disease or condition to be treated, its severity and the size / weight and general state of the subject. Thus, the AAV2 vectors as described herein may be used in the treatment / therapy of any condition in which recruitment, infusion and activation of T lymphocytes may be beneficial, to ameliorate said conditions, and may be administered systemically or locally, and by any suitable method known in the art. The AAV2 vectors may be used to delay or reduce the extent of a cancer developing, or recurring, or for example to prevent or reduce the extent of metastasis. Most preferably the subject treated is a human being.

[0044] Blood vessels in tumors are affected by growth factors in the tumor microenvironment. Thus, they have an altered morphology and function as compared to normal blood vessels. In general, tumor blood vessels are leaky and tortuous and do not respond efficiently to inflammatory signals in the microenvironment. Therefore, tumor blood vessels are often not efficient in recruiting lymphocytes from the blood stream into the tumor.

[0045] The tumor cells reside in an environment composed of normal cells, endothelial cells, pericytes, immune cells, fibroblasts, growth factors, cytokines, chemokines and other soluble factors and extracellular matrix components. Together with the tumor cells, these cells and molecules form the “tumor microenvironment”. The tumor microenvironment in solid tumors is often immunosuppressive in nature.

[0046] In many forms of human cancer there is a strong correlation between T cell infiltration into the tumor mass and good prognosis, showing that if an immune attack is effectively mounted against the tumor, patients survive longer. Furthermore, T cell infiltration into the tumor mass is in many cases predictive of improved response to immunotherapy. Thus, therapeutic means to increase T cell infiltration into the tumor mass could improve response to immunotherapy and increase the chance of prolonging survival or possibly curing individual patients.

[0047] Priming of naive T cells (T cells also referred to as T lymphocytes) primarily occurs in lymphoid organs where professional antigen-presenting cells such as dendritic cells (DCs) present peptide epitopes from tumor-associated antigens. T lymphocytes with an appropriate T cell receptor (TCR) will interact with the peptide presented by DCs and become activated, exit the lymph nodes into the blood stream, and then migrate back to the tumor.

[0048] The migration of activated T lymphocyte across venular blood vessel walls into the tumor is a multi-step pathway including lymphocyte capture, rolling, arrest, migration, firm adhesion and transmigration through or in between blood endothelial cells lining the vessel walls. The endothelial cells need to be properly activated with expression of adhesion molecules and chemokines that match integrins and chemokine receptors expressed on the T lymphocytes. Endothelial cells in cancer tissue are in many cases dysfunctional and do not always allow for this multistep process to take place. Although pro-inflammatory cytokines are often produced in tumors, the tumor blood vessels generally do not express proteins that are necessary for lymphocyte recruitment. One important reason for this deficiency is that tumors produce pro-angiogenic growth factors. These factors induce formation of new blood vessels, and they also inhibit pro-inflammatory signaling pathways and reduce production of adhesion molecules and cytokines involved in T cell recruitment. Therefore, reducing pro-angiogenic signaling in endothelial cells in tumors may lead to increased infiltration of T lymphocytes (T lymphocyte infiltration).

[0049] Harbouring a high number of tumor-infiltrating lymphocytes is in most cases a good prognostic factor and is associated with prolonged survival in various cancer types. Furthermore, it is also a predictive factor for positive response to immunotherapy with immune checkpoint inhibitors.

[0050] During inflammation, lymphocytes are recruited from the circulation into the affected tissue through molecular interactions between integrins and chemokine receptors expressed on the lymphocytes and adhesion molecules and chemokines expressed on endothelial cells that line the blood vessel lumen. Endothelial cells produce the proteins that are necessary for lymphocyte recruitment in response to inflammatory cytokines such as TNF. The tightly-regulated expression of these molecules is necessary to correctly guide the immune cells to the site of inflammation and prevent lymphocytes from entering into healthy, unaffected tissues.

[0051] Therefore, it is quite common that tumors are excluded from or have an insufficiently low degree of T cell infiltration.

[0052] The content of the present disclosure is aimed at overcoming the hurdles to efficient T cell trafficking into tumors. To improve recruitment of endogenous tumor antigen-reactive T cells into the tumor tissue, adeno-associated viral (AAV) vectors that are specifically targeted to the tumor microenvironment will be used to achieve a restricted production of a cytokine called LIGHT (TNFSF14, CD258). Such vectors may comprise a capsid with a targeting peptide, which may target tumor endothelial cells (TECs) in tumor blood vessels using a targeting peptide according to SEQ ID NO:1 , said capsid construct being referred to as AAV-lntpi or AAV2-lntpi, or a targeting peptide according to SEQ ID NO:2, said capsid construct being referred to as AAV-iRGD. Local expression of LIGHT will fundamentally alter the phenotype of the endothelial cells and induce expression of a set of molecules that together will enable recruitment of T cells from the circulation.

[0053] LIGHT and Lymphotoxins are key regulators of lymphoid architecture, i.e. the development and maintenance of reticular networks and vasculature in lymphoid tissues. LIGHT binds to different receptors including the lymphotoxin p receptor (LTpR or TNFRSF3), herpesvirus entry mediator (HVEM or TNFRSF14) and a soluble decoy receptor named DcR3 (TNFRSF6B). LIGHT is found in both membrane-bound and secreted forms. Binding of LIGHT to LTpR leads to various key regulatory events dependent on which cell type the LTpR is expressed by. Lymphotoxins also bind and activate LTpR, hence providing a similar effect as LIGHT in this regard. Binding of LIGHT to HVEM on T cells leads to T cell activation, while DcR3 can bind LIGHT and neutralize its activity.

[0054] LIGHT is an inducible inflammatory cytokine that effectively attracts and co-stimulates T cells and promotes vascular inflammation. By binding to LTpR on endothelial cells, LIGHT can promote vascular inflammation and provide crucial signals to initiate the formation of tumor-associated HEVs (TA-HEVs), i.e. the transformation of tumor endothelial cells into TA-HEVs, as well as the development and maintenance of TLS. Thus, targeting LIGHT to tumor endothelial cells, tumor stromal cells, or tumor cells may be a therapeutic strategy to increase T cell infiltration into tumors. However, too high a concentration of LIGHT was shown to lead to destruction of the blood vessels when administered as a recombinant protein fused with an endothelial cell targeting peptide, see Johansson-Percival et al. (2017; doi 10.1038 / ni.3836). This would imply that local production of LIGHT in endothelial cells transduced with e.g. a viral vector encoding LIGHT, would lead to autocrine signaling and a high local concentration of LIGHT, thereby destroying the vessels. Furthermore, high LIGHT expression in glioblastoma (an aggressive form of brain cancer) is associated with poor survival. Thus, targeting LIGHT to brain tumors may be a risky therapeutic strategy if not performed carefully.

[0055] However, the inventors have now shown that targeting LIGHT to tumor blood vessels using viral vectors, to achieve increased recruitment and infiltration of T lymphocytes through tumor blood vessels to the tumor microenvironment, is a promising treatment method, which overcomes the drawbacks of the prior art.

[0056] Maitituoheti M. et al. (Journal of Immunology, 2011 , Vil. 34, p 581-587) discloses LIGHT as a candidate for cancer therapy and its ability to induce anti-tumor response by improving cytotoxic T- cell infiltration, by using an AAV2 vector to deliver LIGHT into the cancer tissue via intra-tumoral injection. First of all, this reference targets the tumor directly, and not the blood vessels associated with the tumor, failing to disclose the use of modified AAV2 vectors to express LIGHT specifically in the tumor blood vessels. Instead, by specific targeting of the tumor blood vessels using a vector able to induce expression of LIGHT, the induction of high endothelial venules or tertiary lymphoid structures within the tumor microenvironment is achieved. This is a therapeutic pathway that cannot be achieved by injection of the construct of Maitituoheti M. et al. into the tumor. Thus, the same therapeutic effect would not be achieved. Further, the skilled person would not conclude that they should target the blood vessels, nor include a vessel-targeting peptide. On the contrary, there is no incentive for the skilled person to include a targeting peptide at all, as the administration route is direct injection into the tumor in this reference. Furthermore, the skilled person would not try to target the blood vessels to express LIGHT as a therapeutic approach, as it has previously been shown that this may cause deleterious destruction of the vessels themselves. Indeed, as mentioned above, targeting the LIGHT protein to the endothelial cells utilizing a vascular-targeting peptide (VTP) at high concentrations induced endothelial cell death. Furthermore, no survival benefit from this approach has been reported. Hence, the skilled person would conclude that targeting LIGHT expression to the tumor blood vessels would result in no therapeutic benefit and have a detrimental effect. Thus, targeting LIGHT to blood vessels is not an obvious choice for the skilled person.

[0057] However, it was surprisingly found by the present inventors that when targeting a construct able to express LIGHT to the blood vessels using a targeting peptide of the invention, the induction of LIGHT expression led to formation of high endothelial venules and tertiary lymphoid structures, which may activate and recruit T-cells to achieve a therapeutic effect. Use of the new vectors may thus increase pro-inflammatory activation of tumor endothelial cells or induce HEVs within or near tumors, increase T cell infiltration and thereby increase the efficiency of cancer immunotherapy.

[0058] The novel approach of targeting peptides to the blood vessels also provides new non- invasive treatment approaches for glioma and glioblastoma. The modes of administration and action of the therapeutic constructs also provide increased survival benefits with reduced treatment-related adverse events.

[0059] Use of the specific targeting peptides of the invention also achieves additional benefits. The modified vectors in the present invention were developed by capsid engineering with cyclic peptides that target tumor endothelial cells, such as SEQ ID NO: 1-2. Even though vessel-targeting peptides are previously known, they are not known to be inserted into AAV2-LIGHT vectors, nor would inserting such peptides give the same effect as the constructs of the invention. A known targeting peptide is BR1 that targets brain endothelial cells. Compared to said targeting peptide, the use of the present targeting peptides will reduce off-target effects or side effects, since the therapeutic agent (LIGHT) will be restricted to the tumor blood vessels. AAV-BR1 targets microvascular endothelial cells in the brain and can target the vasculature of the entire central nervous system after intravenous administration. This would potentially give rise to LIGHT expression in all brain endothelial cells and possibly other endothelial cells of the central nervous system, while AAV2- TEC(LIGHT) avoids LIGHT expression in healthy brain tumor blood vessels and mediates its expression selectively in brain tumor endothelial cells. Moreover, AAV-BR1 was developed to have high affinity to mouse brain endothelial cells and do not infect human endothelial cells in vitro, while the current variants of AAV2-TEC(LIGHT) was developed for human tumor-associated endothelial cells and exhibit high cross-species translatability with mouse, which then can be used as a model system.

[0060] Adeno-associated viruses (AAVs) are frequently used as gene therapy vectors, by removing the Rep and Cap genes and replacing them with an expression cassette consisting of a promoter and a transgene in between the ITR sequences.

[0061] AAV vectors are excellent tools to deliver genetic payloads to cells in vivo. AAVs are divided into at least 13 natural serotypes (AAV1-13) with tropism for different cells and tissues. Because of their relatively low immunogenicity and possibility of both systemic and local delivery, AAVs are frequently used in human gene therapy. The tropism of AAVs can be modified by creation of chimeric AAV capsids through grafting of functional motives related to receptor binding from one serotype into another. When producing a recombinant AAV vector, typically three plasmids are co-transfected into producer cells; one plasmid with the gene expression cassette in between the ITRs, one plasmid containing the Rep and Cap genes, and one plasmid with adenoviral helper genes. The AAV vector is then harvested from the cells and purified.

[0062] AAV serotype 2 (AAV2) is one of the most extensively used AAV vectors for gene therapy. AAV2 binds to heparan sulfate proteoglycans (HSPG) and presents natural tropism towards skeletal muscles, neurons, vascular smooth muscle cells and hepatocytes. AAV2-based vectors are popular because peptides can be inserted at several positions in the loops of the viral proteins building up the virus capsid in order to expand AAV2 tropism.

[0063] One position that is frequently used is the amino acid Arginine at position 588 (R588) of VP3 in the AAV2 viral capsid, where targeting peptides of e.g. 7-11 amino acids (or more) can be inserted. Insertion at this site at least partly abolishes native AAV2 binding to heparan sulfate proteoglycans (HSPG), thus modifying the vector tropism. A unique peptide known to target a specific protein can be inserted at R588, to re-target the AAV vector towards a specific receptor. Alternatively, a pool of peptides in the form of a peptide display library can be inserted at R588, followed by selection for specific binding to cells in vitro or cells / tissues in vivo. The capsid-modified AAV2-based vectors can then be used to carry a transgene that will be expressed in the targeted cells or tissues. Thus, by using capsid-modified AAV2-based vectors carrying a transgene, the transgene can be expressed in specific cells or tissues.

[0064] Even though inserts have previously been made at the R588 location in the AAV2 capsid, the utility of cyclic peptides to develop targeted / improved AAV2-vectors is not disclosed in the art, nor have the Gly and Ala amino acids been inserted during capsid engineering as spacers flanking the cyclic peptides. Genetic modification resulting in AAV2-capsid having cyclic peptides at VR-VIII will improve the specificity of the vector by enhancing its affinity towards the binding target, due to the extended protrusion of the variable region after the peptide insertion. The structure prediction as provided in Figure 5B confirms the predicted presence of cyclic conformation in VR-VIII in both proposed variants of the AAV2-TEC vectors, AAV2-lntpi and AAV2-IRGD. Furthermore, insertion of peptides will not always produce a functional vector, and hence the vector engineering of the present invention would not be obvious to the skilled person. The technical steps involved in the modified AAV2-vector development are listed below: i) Abolishment of existing Eco53kl site in AAV2 Rep protein by nucleotide substitution (gag- >gaa) of E164 amino acid. ii) Creation of new Eco53kl and Afel site near R588 with staffer sequence (AA) in between by inserting 12 nucleotides GCTCAAAGCGCT after R588 (aga) iii) DNA assembly of the nucleotide sequence encoding the cyclic peptide flanked with Gly (ggc) and Ala (get) as spacers after digestion of capsid-engineered AAV2 generated in step (iii) with Eco53kl enzyme. iv) Confirmation of the peptide insertion in the modified AAV2-vector by sequencing, followed by verification for capsid assembly by vector packaging.

[0065] Thus, the vectors of the present disclosure have beneficial therapeutic effects not achieved or possible to derive from the prior art.

[0066] When AAV2-TEC(LIGHT) is utilized to deliver the LIGHT gene (i.e. Tnfsf14) to tumor endothelial cells, the LIGHT protein is expressed by the endothelial cells of the tumor blood vessels. It can be membrane-bound or secreted, and in an autocrine loop interact with the lymphotoxin beta receptor (LTpR) on the endothelial cells, triggering downstream signaling events that will cause the endothelial cells to adopt a HEV-like phenotype, making them permissive for T cell migration across the vessel wall. LIGHT expressed by tumor endothelial cells can also interact with herpesvirus entry mediator (HVEM) on T cells, triggering downstream signaling events that lead to the activation of the T cells. Increased infiltration of activated T cells across the tumor blood vessel wall can lead to the formation of antigen-presenting niches inside brain tumors and tertiary lymphoid structures (TLS) near the brain tumors. Thus, targeting LIGHT to these tissues using an AAV2 vector provides an efficient measure of inducing the targeted cells to express LIGHT, which causes targeted endothelial cells to switch to a HEV-like phenotype, and induces increased recruitment and infiltration of T lymphocytes through tumor blood vessels to the tumor microenvironment, hence effectively killing the cancer cells.

[0067] In specific embodiments of the present disclosure, the AAV2 vector has a targeting peptide, such as a vessel-targeting peptide, inserted in the viral capsid, preferably after amino acid arginine at position 588 (R588) of the virion protein 3 (VP3) in the variable region of the AAV2 capsid. Such vector is referred to as a vessel-targeted AAV2.

[0068] In an aspect of the embodiment, the vessel-targeting peptide has the amino acid sequence ACRGDGWCG (SEQ ID NO:1) and is targeting activated integrins, e.g. integrin alpha 5 beta 1 (a5pi), which can be expressed by tumor endothelial cells. The vector with this insertion is referred to as AAV2-lntpi or AAV-lntpi . This construct comprising the AAV2 Cap gene with the peptide inserted, is defined by SEQ ID NO:3. The peptide may be inserted after R588, and forms a loop structure at the surface of the AAV2 virus particle. In a second aspect of the embodiment, the vessel-targeting peptide has the amino acid sequence CRGDKGPDC (SEQ ID NO:2) and is targeting integrins and neuropilin-1 , which can be expressed by tumor endothelial cells. The vector with this insertion is referred to as AAV2-iRGD. This construct comprising the AAV2 Cap gene with the peptide inserted, is defined by SEQ ID NO:4.

[0069] The tumor blood vessel-targeting AAV2-TEC vectors AAV2-lntpi , and AAV2-iRGDcan bring the LIGHT transgene into endothelial cells within or near a tumor. LIGHT can be in its native form (both secreted as a soluble protein and membrane-bound), as a specifically secreted form or as a cell membrane-bound form.

[0070] The tumor blood vessel-targeted AAV2-TEC vectors, e.g. AAV2-lntpi (LIGHT) and AAV2- iRGD(LIGHT), are collectively referred to as AAV2-TEC(LIGHT) in this disclosure. An overview of the respective AAV2 vectors and their targeting peptides is shown in Figure 5.

[0071] AAV2-TEC(LIGHT) therapy can, through induction of LIGHT in transduced cells, improve the ability of tumor blood vessels to recruit T cells, or transform the tumor blood vessels into TA-HEVs, making them permissive for T cell infiltration. AAV2-TEC(LIGHT) therapy can also, through induction of LIGHT in transduced cells, induce a tumor microenvironment which favors T cell activity and thus improve T cell-mediated killing of tumor cells.

[0072] AAV2-TEC(LIGHT) therapy may also, through induction of LIGHT in transduced cells, directly activate T cells by interacting with the HVEM receptor which is expressed by T cells.

[0073] The present disclosure generally relates to peptide-modified and thereby tumor blood vesseltargeting AAV2-based vectors encoding LIGHT, denoted AAV(LIGHT), AAV-LIGHT, AAV2(LIGHT), AAV2-LIGHT, or AAV2-TEC(LIGHT) (may be used interchangeably herein), and in particular AAV2- TEC(LIGHT) for the use in cancer gene therapy and cancer immunotherapy. It also generally relates to peptide-modified and thereby tumor blood vessel-targeting AAV2-based vectors encoding LIGHT, comprising native LIGHT or modified LIGHT, where native LIGHT has been modified to either be exclusively secreted or exclusively membrane-bound. Native LIGHT can be both secreted and membrane-bound.

[0074] The transgene encoding LIGHT may be controlled by a constitutively active promoter, such as CAG, or Phosphoglycerate kinase (PGK). Furthermore the expression of LIGHT protein may be regulated using a gene expression system such as Xon or mini-Xon. Thus, the present disclosure also relates to AAV vectors where the expression system that regulates expression of LIGHT (native, secreted or membrane-bound) can be induced by a drug such as LMI070 to activate Xon or mini-Xon. In an embodiment the tumor blood vessel-targeting AAV2-TEC vectors, such as AAV2-lntpi (Figure 1) or AAV2-iRGD, can deliver the LIGHT transgene into endothelial cells within the tumor microenvironment.

[0075] The present disclosure also relates to a new form of cancer treatment, based on the concept of tailoring the tumor blood vessels by targeting tumor-associated endothelial cells and enabling them to adopt a HEV-like phenotype, allowing immune cells (primarily T cells) to infiltrate into solid tumors. Immune cell infiltration into tumors can lead to a direct attack on tumor cells as well as formation of antigen-presenting niches and tertiary lymphoid structures (TLS). As shown in the appended examples, Proof-of-Concept studies have been performed in glioblastoma, and the concept seems to be valid for not only gliomas and glioblastoma, but all solid tumors, where T lymphocyte infiltration may be impaired.

[0076] Thus, herein is proposed AAV2 vector-based gene delivery of LIGHT. When expressed, LIGHT can alter and normalize the otherwise abnormal tumor blood vessels. The vectors herein have a modification (peptide-insertion) at amino acid position 588 (R588) of VP3 in the variable region of the AAV2 capsid, which alters the tropism of the vector and leads to retargeting. One AAV vector, denoted AAV2-lntpi is able to specifically transduce tumor-associated endothelial cells (TECs) by targeting activated integrins. Another named AAV2-iRGD also targets TECs by targeting integrins and neuropilin-1.

[0077] When the LIGHT transgene is delivered to tumor endothelial cells (TECs) by a TEC-targeted AAV2 vector such as AAV2-TEC(LIGHT), the LIGHT protein is expressed by the endothelial cells and will interact in an autocrine fashion with LTpR on the same cells, allowing the blood vessel to adopt a HEV-like phenotype. Consistent with this, in murine models, formation of tumor-associated HEVs and TLS in close proximity to the tumors were observed, when using a brain endothelial cell- targeted AAV2 vector encoding LIGHT (AAV-BR1-LIGHT), as shown in Figure 2.

[0078] Figure 1 illustrates a proposed mode of action for AAV2-TEC(LIGHT) therapy using the AAV2-lntpi (LIGHT) vector. AAV2-lntpi (LIGHT) is a tumor blood vessel-targeting adeno-associated virus serotype 2 (AAV2) vector with a cyclic peptide including an RGD-motif flanking cysteine residues in the capsid for specific targeting of the AAV2 to activated integrins, e.g. integrin alpha 5 beta 1 (a5pi), on tumor endothelial cells (TECs). It carries the transgene encoding for LIGHT. Upon AAV2-lntpi (LIGHT) vector binding to and transduction of TECs, LIGHT is expressed. LIGHT transforms TECs into tumor-associated high endothelial venules (TA-HEVs), which efficiently recruit T cells to the tumor bed. The aim is to use an AAV vector as a gene therapy tool to deliver the transgene for LIGHT into blood vessels, and in particular into tumor blood vessels.

[0079] In some aspects, the present disclosure provides an adeno-associated serotype 2 viral vector (AAV2) comprising a transgene encoding LIGHT, wherein a viral capsid of the AAV2 vector comprises a targeting peptide. In a further aspect, the invention provides an adeno-associated serotype 2 viral vector (AAV2) comprising a transgene encoding a lymphotoxin beta receptor (LTpR) agonist, wherein a viral capsid of the AAV2 vector comprises a targeting peptide. The agonist is a ligand providing an agonistic effect, i.e. a ligand that activates a receptor to produce a biological response. In an embodiment, the AAV2 vector agonist is LIGHT and / or lymphotoxins.

[0080] The transgene encoding LIGHT may be selected from native LIGHT, exclusively soluble LIGHT and exclusively membrane-bound LIGHT. The transgene encoding native LIGHT gives rise to membrane-bound LIGHT, which contains a protease cleavage site and can thus be cleaved into soluble LIGHT. The protease cleavage site may be silenced to give rise to exclusively membranebound LIGHT, e.g. by mutating amino acid 81 from L to A (L81A), or by deleting five amino acids in positions 78-82 (ALIGHT). Exclusively soluble LIGHT can be developed by, e.g. deleting the transmembrane and intracellular portion (amino acid positions 1 to 71) and fusing the extracellular domain of LIGHT with a secretory signal and trimerization domain.

[0081] In an embodiment, the transgene encoding LIGHT is controlled by a constitutively active promoter, such as CAG, or an inducible gene expression system, such as Xon or mini-Xon. The vector encoding LIGHT may preferably encode human LIGHT, which may be native, exclusively soluble or exclusively membrane-bound, as discussed above.

[0082] The targeting peptide on the AAV2 capsid targets cells in the tumor microenvironment, such as cells of vessels, also referred to as endothelial cells. Endothelial cells can be tumor endothelial cells (TECs), which may be endothelial cells in the blood vessels of a tumor.

[0083] In an embodiment, the targeting peptide is a blood vessel-targeting peptide. The targeting peptide may be inserted in the viral capsid at the arginine amino acid located in position 588 (R588) of the viral capsid.

[0084] In an embodiment, the targeting peptide may target (bind) activated integrins, such as integrin alpha 5 beta 1 (a5pi) and may have an amino acid sequence defined as ACRGDGWCG (SEQ ID NO:1), or a sequence having 80 % or more, such as 85 %, 90 %, 95 % or more, identity thereto, while still having the ability to bind the target and thus the target cells. In an embodiment, the targeting peptide targets integrins and neuropilin-1 , and may have an amino acid sequence defined as CRGDKGPDC (SEQ ID NO:2), or a sequence having 80 % or more, such as 85 %, 90 %, 95 % or more, identity thereto, while still having the ability to bind the target and thus the target cells.

[0085] Sequence identity may be assessed by any convenient method. However, for determining the degree of sequence identity between sequences, computer programs that make pairwise or multiple alignments of sequences are useful, for instance EMBOSS Needle or EMBOSS stretcher may be used for pairwise sequence alignments while Clustal Omega or MUSCLE may be used for multiple sequence alignments, though any other appropriate program may be used. Whether the alignment is pairwise or multiple, it must be performed globally (i.e. across the entirety of the reference sequence) rather than locally. Sequence alignments and % identity calculations may be determined using for instance standard Clustal Omega parameters: matrix Gonnet, gap opening penalty 6, gap extension penalty 1. Alternatively, the standard EMBOSS Needle parameters may be used: matrix BLOSUM62, gap opening penalty 10, gap extension penalty 0.5. Any other suitable parameters may alternatively be used.

[0086] In an embodiment, the AAV2 vector is able to transduce the targeted cells, inducing the targeted cells to express LIGHT and / or lymphotoxins. The AAV2 vector transduction of the targeted endothelial cells may enable the endothelial cells to adopt a high endothelial venule (HEV)-like phenotype, which may induce increased recruitment and infiltration of T lymphocytes through tumor blood vessels to the tumor microenvironment in proximity to the targeted cells, and may also induce activation of the T lymphocytes. In an embodiment, AAV2 vector transduction of the targeted cells induces formation of tertiary lymphoid structures (TLS) in tissues surrounding the targeted cells, such as in the tumor microenvironment, where the tissues may be present within or near a tumor, such as in the tumor microenvironment.

[0087] In an embodiment, the invention provides the AAV2 vector as described above for use in inducing expression of LIGHT and / or lymphotoxins, such as inducing expression of LIGHT and / or lymphotoxins in / near the targeted and transduced cells. In an embodiment, the invention provides the AAV2 vector as described above for use in one or more of i) recruitment and infiltration of T lymphocytes into the tumor microenvironment, ii) activation of T lymphocytes, iii) phenotype switch of endothelial cells into TA-HEVs, iv) formation of TLS, and v) formation of antigen-presenting niches.

[0088] Typically, the process may begin with LIGHT-induced activation of the LTpR on endothelial cells, leading to transformation of the tumor-associated endothelial cells into TA-HEVs, followed by recruitment of naive lymphocytes (T and B cells) from the circulation. Formation of TLS and antigen- presenting niches through recruitment of lymphocytes and likely activation of stromal cells / pericytes takes place, followed by priming and activation of T lymphocytes targeting tumor antigens, which likely occurs in the TLS or antigen-presenting niches. The T lymphocytes that have been activated in the TLS can then kill tumor cells. T lymphocytes activated in the periphery can also contribute, since they can be recruited through the TA-HEVs.

[0089] In some aspects, the invention provides a pharmaceutical composition containing an AAV2 vector as described above, and a pharmaceutically acceptable carrier or excipient. As used herein, “pharmaceutically acceptable carrier or excipient” includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption-delaying agents and the like that are physiologically compatible. Preferably, the carrier or excipient is suitable for parenteral, e.g. intradermal, intravenous, intramuscular or subcutaneous administration (e.g. by injection or infusion). Depending on the route of administration, the vector may be coated in a material to protect them from the action of acids and other natural conditions that may inactivate or denature it. Preferred pharmaceutically-acceptable carriers comprise aqueous carriers or diluents. Examples of suitable aqueous carriers that may be employed in the pharmaceutical compositions, kits and products include water, buffered water and saline. Examples of other carriers include ethanol, polyols (such as glycerol, propylene glycol, polyethylene glycol, and the like), and suitable mixtures thereof, vegetable oils and injectable organic esters, such as ethyl oleate. Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. In many cases, it will be preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, sodium chloride and the like.

[0090] The AAV2 vectors, or the pharmaceutical compositions comprising the vectors, may be administered via one or more routes of administration using one or more of a variety of methods known in the art. As will be appreciated by the skilled artisan, the route and / or mode of administration will vary depending upon the desired results. A suitable dosage of an AAV2 vector or pharmaceutical composition thereof may be determined by a skilled medical practitioner. Actual dosage levels of the active ingredients in the pharmaceutical compositions and products of the present invention may be varied so as to obtain an amount of the activated LIGHT, which is effective to achieve the desired therapeutic response for a particular subject, i.e. patient, without being toxic to the patient. The selected dosage level will depend upon a variety of pharmacokinetic factors including the activity of the particular protein / conjugate employed, the route of administration, the time of administration, the rate of excretion of the protein, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts. Further, the use of inducible promoters may be used to control the amount of expressed LIGHT in a subject.

[0091] Dosage regimens may be adjusted to provide the optimum desired response (e.g. a therapeutic response). The vectors may be administered in a single dose or in multiple doses. The multiple doses may be administered via the same or different routes and to the same or different locations.

[0092] In some aspects, the present disclosure provides an AAV2 vector as described above, or a pharmaceutical composition, for use in therapy, such as cancer therapy. The cancer therapy may be for treating solid tumors, and can be used in cancer therapy of brain tumors. The cancer therapies may preferably include therapy of glioma, including both lower-grade tumors and glioblastoma.

[0093] In an embodiment, the present disclosure provides the AAV2 vector or the pharmaceutical composition, as described above, for use in immunotherapy, wherein the immunotherapy may induce formation of TA-HEVs, TLS, and / or antigen-presenting niches.

[0094] In some aspects, the present disclosure provides a method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV2 vector or a pharmaceutical composition as described above.

[0095] In some aspects, the present disclosure provides a method of targeting LIGHT and / or lymphotoxins to cells in a tumor microenvironment in a subject in need thereof, wherein the cells that are targeted in the tumor microenvironment primarily comprise tumor endothelial cells, the method comprising: providing an AAV2 viral vector encoding LIGHT or lymphotoxins as described above, and administering the AAV2 viral vector to the subject, or a pharmaceutical composition thereof, whereby the AAV2 viral vector transduces the targeted cells, thereby inducing the targeted cells to express LIGHT or lymphotoxins.

[0096] In some aspects, the present disclosure provides use of an AAV2 viral vector encoding LIGHT or lymphotoxins as described above, for targeting LIGHT or lymphotoxins to cells in the tumor microenvironment in a subject.

[0097] Thus, provided herein are tumor blood vessel-targeted AAV2(LIGHT) vectors comprising an immune-stimulator gene encoding LIGHT, which may induce LIGHT expression in tumor endothelial cells, wherein LIGHT can induce HEVs, improve T cell recruitment to the tumor bed and activate the recruited T cells. Also provided herein are methods for treating cancers by promoting a robust T-cell recruitment and response, followed with transformation of tumor blood vessels to high endothelial venules (HEVs). The AAV2 vectors may specifically target tumor vasculature, such as the glioblastoma tumor vasculature. In some embodiments, and inducible system is used, and it may be possible to turn the LIGHT expression ON and OFF in the transduced tumor blood vessels to prevent any treatment-related inflammation, such as switch ON and OFF the LIGHT expression in the tumor vasculature endothelium using a Xon-AAV2-LIGHT or miniXon-AAV2-LIGHT vector. The targeting peptides (tumor endothelial anchoring peptides) may specifically target tumor endothelial cells. The use of LIGHT may improve the treatment outcome of tumors such as glioblastoma and other solid tumors, and the use of soluble LIGHT (SLIGHT) may improve the outcome of LIGHT- based immunotherapy in treating tumors. Further, the use of membrane-bound LIGHT (mLIGHT) may improve the outcome of LIGHT-based immunotherapy for tumors. Thus, provided herein are methods for treating cancers, such as glioblastoma, by delivering the immune-stimulator gene LIGHT or bioactive variants of LIGHT intravenously using the novel AAV2 vectors.

[0098] The content of this disclosure thus enables treatment of disorders where T cell recruitment and activation is of importance, such as cancers, by administering the viral vectors of the invention. In the drawings and specification, there have been disclosed exemplary aspects of the disclosure. However, many variations and modifications can be made to these aspects without substantially departing from the principles of the present disclosure. Thus, the disclosure should be regarded as illustrative rather than restrictive, and not as being limited to the particular aspects discussed above. Accordingly, although specific terms are employed, they are used in a generic and descriptive sense only and not for purposes of limitation.

[0099] The description of the example embodiments provided herein have been presented for purposes of illustration. The description is not intended to be exhaustive or to limit example embodiments to the precise form disclosed, and modifications and variations are possible in light of the above teachings or may be acquired from practice of various alternatives to the provided embodiments. The examples discussed herein were chosen and described in order to explain the principles and the nature of various example embodiments and its practical application to enable one skilled in the art to utilize the example embodiments in various manners and with various modifications as are suited to the particular use contemplated. The features of the embodiments described herein may be combined in all possible combinations of methods, products, and systems. It should be appreciated that the example embodiments presented herein may be practiced in any combination with each other. It should also be noted that the word “comprising” does not necessarily exclude the presence of other elements or steps than those listed and the words “a” or “an” preceding an element do not exclude the presence of a plurality of such elements. It should further be noted that any reference signs do not limit the scope of the claims, that the example embodiments may be realized in the broadest sense of the claims.

[0100] EXAMPLES

[0101] The Examples herein describe the development and efficacy of tumor blood vessel-targeted adeno- associated viral (AAV) serotype 2 vectors, particularly encoding LIGHT, for use in gene and immunotherapy of cancer.

[0102] Example 1 , Therapeutic targeting of LIGHT to brain endothelial cells using AAV-LIGHT prolongs survival and induces T cell-rich TLS and TA-HEVs in murine glioma

[0103] Materials and Methods

[0104] Cell culture. CT-2A murine glioma cells were cultured in RPMI 1640 medium supplemented with 10% heat-inactivated fetal bovine serum (FBS) and 1 % penicillin streptomycin (PEST). mGClGFAP.3 murine glioma cells were cultured in DMEM / F12 Glutamax medium supplemented with 1% 1M HEPES, 1 % PEST, 2% B27 and 2% insulin (Sigma-Aldrich), on culture dishes coated with growth factor-depleted extracellular matrix (Sigma-Aldrich). Cells were cultured at 37°C with 5% CO2 in a humidified cell incubator.

[0105] Orthotopic murine glioma models. 6-8 week-old female C57BL / 6 mice were used for orthotopic implantation of CT-2A glioma cells, and 6-13 week-old C57BL / 6 G / tv-a mice were used for orthotopic implantation of mGClGFAP.3 cells. CT-2A cells (5x104) or mGClGFAP.3 cells (2x104) were intracranially (i.c) injected in 2pl of Dulbecco’s phosphate-buffered saline (DPBS). Mice were monitored daily and scored for tumor-related symptoms according to the Uppsala University scoring system for animal welfare. The mice were sacrificed when the welfare score reached a maximum of 0.5.

[0106] Adeno-associated viral (AAV) vector packaging and treatments. The brain vessel-targeting AAV2-BR1 capsid (PMID 27137490) was used to package the AAV-BR1-GFP and AAV-BR1-LIGHT vectors. The transgenes were under the control of synthetic CMV early enhancer / chicken actin (GAG) promoter. AAV2-BR1 vectors were packaged in HEK293 cells by triple transfection of plasmids pAAV-CAG GFP or pAAV-CAG LIGHT, pCapsid NRGTEWD (BR1) and pHelper, purified by density gradient centrifugation and guantified by gPCR (viral genomes, vg). Mice bearing CT-2A or mGClGFAP.3 tumors were treated with 3 intravenous injections of AAV-BR1-GFP or AAV-BR1- LIGHT (dose range: 1*1011to 5*1012vg / injection in 100pl DPBS) at days 4, 7 and 10 post-tumor implantation. After virus treatment, the mice were monitored for tumor-related symptoms.

[0107] RNAScope: Cryopreserved brain tissues were sectioned with a cryostat to 14pm-thick coronal sections and stored at -80°C. RNAscope Multiplex Fluorescent V2 Assay (ACD Bio-Techne) was performed according to the manufacturer’s protocol. In brief, sections were incubated with cold 4% PFA for 20 minutes and permeabilized with protease III. The mRNA of the respective targets was probed using RNAscope® Probe- Mm-Tnfsf14 (ACD Bio-Techne) and RNAscope® Probe- Mm- Pecam1-C3 (ACD Bio-Techne), and visualized using OpalTM 570 (Akoya Biosciences) and OpalTM 620 (Akoya Biosciences) dyes at 1 :1500 dilution. Images were taken with a Leica TCS SP8 confocal microscope (Leica Microsystems).

[0108] Image analysis: To quantify the numbers of TLS in each sample, representative sections of each segment of the cerebrum were stained for CD45, B220 and CD3. TLS-like structures were defined as compact CD45+immune cell aggregates that were positive for B220+B cells and CD3+T cells. TLS area and CD3:B220 ratio within the TLS were quantified using Imaged software.

[0109] To quantify the proportion of MAdCAM-1+TA-HEVs and the number of CD3+T cells in contact with TA-HEVs as compared to other tumor blood vessels, representative sections were stained for CD31 , MAdCAM-1 and CD3. Quantifications were made using confocal images of 5 randomly selected fields of view per sample at 25x magnification. Cell Profiler (Broad Institute) was used to quantify the proportion of CD31+tumor blood vessel area that was also positive for MAdCAM-1. The cell counter in Imaged (NIH) was used to manually count the number of CD3+T cells in contact with MAdCAM-1+CD31+TA-HEVs compared to MAdCAM-1“CD31+tumor blood vessels.

[0110] Results

[0111] Figure 2 shows that AAV-BR1-LIGHT therapy prolongs survival in glioma-bearing mice and promotes the formation of T cell-rich TLS and TA-HEVs. Figure 2A illustrates the experimental layout used to obtain data shown in Figures 2B-2H. In brief, the murine glioma cell lines CT-2A or mGC1 were implanted intracranially in C57BL / 6 mice on day 0, followed by intravenous administration of either AAV-BR1-GFP or AAV-BR1 -LIGHT on days 4, 7 and 10 post-tumor implantation. Mice were monitored for glioma-related symptoms and sacrificed at the humane endpoint, i.c. , intracranial, IF, immunofluorescence. Figures 2B and 2C show Kaplan-Meier survival curves of CT-2A tumor-bearing mice (n=9-17 mice / group) or mGC1 tumor-bearing mice (n=11-12 mice / group) treated with AAV-BR1-GFP or AAV-BR1-LIGHT. Statistics: Logrank test. In both models, mice treated with AAV-LIGHT survived significantly longer than mice treated with AAV- GFP, and a proportion of the mice completely cleared their tumors. Figure 2D shows a representative RNAscope image depicting the colocalization of LIGHT mRNA with Pecaml mRNA in CT-2A tumor-bearing brains that received AAV-BR1-LIGHT treatment. Scale bars 25pm. LIGHT expression was detected in tumor endothelial cells in AAV-BR1-LIGHT-treated tumors. Figure 2E shows quantification of the number of TLS per brain (n=9-12 mice / group). Statistics: unpaired t-test. AAV-BR1-LIGHT treatment induced a significantly higher number of TLS per brain compared to the AAV-BR1-GFP control. Figure 2F shows the T cell to B cell ratio within the TLS (n=9-31 TLS / group) in the indicated groups. Statistics: Mann-Whitney test. AAV-BR1 -LIGHT therapy induced TLS with a higher T cell to B cell ratio compared to those that formed after AAV-BR1-GFP administration. Figure 2G shows the proportion of the tumor blood vessel area which was HEV positive (n=7-9 mice / group) in the indicated groups. Statistics: unpaired t-test. AAV-BR1 -LIGHT therapy resulted in a significantly higher proportion of TA-HEVs. Figure 2H shows quantification of CD3+T cells touching TA-HEVs vs tumor blood vessels (n=7-9 mice / group) in the indicated groups. Statistics: two-way ANOVA with Sidak’s correction. A higher number of CD3+T cells were in close proximity to TA-HEVs compared to tumor blood vessels, suggesting an enhanced T cell recruitment characteristic of functional HEVs. Key to statistics: *p<0.05, ***p<0.001 and ****p<0.0001. Bar graphs show mean±SEM.

[0112] Conclusion

[0113] Therapeutic targeting of LIGHT to brain endothelial cells using AAV-BR1 -LIGHT prolongs survival in murine glioma and induces T cell rich TLS and functional TA-HEVs.

[0114] Example 2, AAV-BR1 -LIGHT therapy promotes T cell functionality, which correlates with tumor regression in murine glioma

[0115] Materials and Methods

[0116] Cell culture and tumor studies were performed as described in Example 1 , with samples collected at day 14 or day 21 post-tumor implantation. AAV packaging and treatments were performed as described in Example 1.

[0117] Ex vivo T cell functionality assays. CD8+T cells were isolated from mice bearing AAV-BR1- GFP or AAV-BR1-LIGHT-treated CT-2A-Ova tumors on day 14 post-tumor implantation, using the mouse CD8a+T Cell Isolation Kit (Miltenyi Biotec) according to manufacturer’s instruction. All T cell functionality assays were performed in 96-well plates in T cell medium: RPMI 1640 added with 10% FBS, 2 mM L-glutamine, 10 mM HEPES, 20 pm p-mercaptoethanol, 1 mM sodium-pyruvate, 100 U / ml PEST and 100 lU / ml IL-2. Isolated CD8+T cells and CT-2A-Ova cells were co-cultured at a 6:1 T celktumor cell ratio. After 20 hours of co-culture, cytokine secretion was blocked by adding GolgiStop (BD Biosciences) to the culture medium along with CD107a antibody to stain degranulating CD8+T cells. After 24 hours, all cells were harvested by trypsinization and stained with a panel of antibodies and analyzed using the CytoFLEX LX (Beckman Coulter) as described in Example 17.

[0118] Flow cytometry. Cells were stained with a fixable viability dye according to the manufacturer’s instructions. Unspecific Fc receptor binding was blocked by using anti-mouse CD16 / CD32 antibodies (Biolegend). Cells were stained for the markers of interest using fluorochrome-conjugated antibodies diluted in Brilliant Stain Buffer Plus (BD Biosciences). Nuclear and intracellular proteins were stained using True-Nuclear™ Transcription Factor Buffer Set (BioLegend) according to the manufacturer’s protocol. Stained samples were acquired using an LSR Fortessa (BD Biosciences) or a CytoFLEX LX (Beckman Coulter). Data were analyzed using FlowJo software version 10.5.3 (FlowJo LLC).

[0119] Results

[0120] Figure 3 shows that AAV-BR1 -LIGHT therapy promotes T cell functionality, which results in early tumor regression. Figure 3A illustrates the experimental layout used to obtain data shown in Figures 3B-E. In brief, CT-2A tumor cells were implanted intracranially in C57BL / 6 mice on day 0, followed by intravenous administration of either AAV-BR1-GFP or AAV-BR1-LIGHT on days 4, 7 and 10. Mice were sacrificed at day 14 and CD8+T cells were isolated from tumor-bearing brains using magnetic beads (n=5-8 mice / group). The CD8+T cells were co-cultured with CT-2A cells for 24 hours followed by flow cytometry analysis, i.c. , intracranial. Figures 3B-E show quantifications of the percentage of CD8+T cells expressing granzyme (GZM)-b (Figure 3B), interferon (IFN)-y and tumor necrosis factor (TNF)-a (Figure 3C), CD69 (Figure 3D) or CD107a (Figure 3E) in the indicated groups. CD8+T cells isolated from brains bearing AAV-BR1-LIGHT-treated tumors showed an increased functionality when restimulated with tumor cells in vitro compared to those treated with AAV-BR1-GFP. Figure 3F shows the proportion of brains presenting with a macroscopic tumor (“tumor”) or not (“no macroscopic tumor”) in each treatment group at day 14 and day 21. The proportion of mice with no macroscopic tumor after AAV-BR1 -LIGHT therapy was higher than after AAV-BR1-GFP treatment, indicating that therapeutic expression of LIGHT in the tumor blood vessels led to tumor regression. Statistics in Figures 3B,D,E: unpaired t-test. Statistics in Figure 3C: two-way ANOVA with Sidak’s correction. Key to statistics: *p<0.05 and **p<0.01. Bar graphs show mean±SEM.

[0121] Conclusion AAV-BR1 -LIGHT therapy of glioma-bearing mice is associated the induction of a robust tumorspecific cytotoxic CD8+T cell response, which correlates with tumor regression.

[0122] Example 3, AAV-BR1 -LIGHT therapy promotes stem-like CD8+T cells

[0123] To investigate how AAV-BR1 -LIGHT treatment affects T cell activation during earlier stages of the anti-tumor immune response, phenotypes of T cells were analyzed from samples with macroscopic tumors at day 14, in which the tumor had not yet regressed.

[0124] Materials and Methods

[0125] Cell culture and tumor studies were performed as described in Example 1 , with samples collected at day 14 post-tumor implantation. AAV packaging and treatments were performed as described in Example 1. Flow cytometry was performed as described in Example 2.

[0126] Results

[0127] Figure 4 shows that AAV-BR1 -LIGHT therapy promotes stem-like CD8+T cells. Figure 4A illustrates the experimental layout used to obtain data shown in Figures 4B-C. In brief, brains with macroscopic CT-2A-Ova tumors were collected on day 14 post-tumor implantation from AAV-BR1- GFP- or AAV-BR1-LIGHT-treated mice (n=3-9 mice / group) for flow cytometry analysis. Figure 4B shows quantifications of TCF1 / 7+PD-1+Tstem-iike cells (% of Ovadex+CD8+tumor-targeted T cells) and Figure 4C shows the ratio of Ovadex+Tstem-iike / TeXhausted cells on day 14 in AAV-BR1-GFP- or AAV- BR1-LIGHT-treated groups. AAV-LIGHT-treated mice had higher percentages of Tstem-iike cells within the tumor-targeted T cell population and exhibited a higher ratio of Tstem-iike / TeXhausted tumor-targeted T cells. Statistics: unpaired t-test. Key to statistics: *p<0.05 and **p<0.01. Bar graphs show mean±SEM.

[0128] Conclusion

[0129] AAV-BR1 -LIGHT therapy promotes stem-like CD8+T cells in murine glioma.

[0130] Example 4, Development of novel tumor blood vessel-targeted AAV2 vectors by rational design Materials and Methods

[0131] Novel tumor blood vessel-targeted AAV2-based vector development. To develop tumor blood vessel-targeted AAV2-based vectors, cyclic peptides that have the potential to bind to the tumor blood vessels and form disulfide bridges were chosen for peptide insertion in the AAV2 Cap gene. The peptide sequences of I ntpi and iRGD have di-cysteine residues that aid in the formation of disulfide bridges, and hence they adopt an extended loop / cyclic-like structure. To further attain the surface exposure of the selected peptide with minimal capsid protein alteration, the VP1 position R588 in the variable region (VR)-VI 11 loop was selected as the site of insertion. The cloning strategy utilized to facilitate the insertion of the peptide sequence in the Cap gene involved deletion of the existing Eco53kl restriction site in the AAV2 Rep gene by nucleotide substitution (gag->gaa) at the E164 site. Then, a mutagenesis protocol was performed to insert a four-amino-acids stretch (AQSA) after the R588 site in the AAV2 Cap gene to create new Afel and Eco53kl restriction sites. The AAV2 vector harboring new Afel and Eco53kl restriction sites was digested with the Eco53kl restriction enzyme, and the selected cyclic peptide (I ntpi or iRGD)-encoding nucleotide sequences flanked with the spacer amino acids (G589 and A599)-encoding nucleotide sequences were inserted by DNA assembly method. The novel cyclic peptide-bearing AAV2 capsids were verified by DNA sequencing and vector packaging. The binding targets of the novel AAV2 vectors (AAV2-lntpi and AAV2-iRGD) are indicated in the table.

[0132] Modelling of AAV2-lntpi and AAV2-iRGD capsid structures. The structures of the VP3 proteins of AAV2-lntpi and AAV2-iRGD, comprising the cyclic peptides, were predicted with the Robetta server (htp: / / robeta.bakerlab.org / ) and represented by ribbon structures using Pymol software.

[0133] Results

[0134] Figure 5 shows the development of novel tumor blood vessel-targeted AAV2 vectors by rational design. Figure 5A shows the cloning strategy followed for the development of tumor blood vessel-targeted AAV2 vectors by cyclic peptide insertion. Figure 5B shows the VP3 structures of AAV2-lntpi and AAV2-iRGD vectors after peptide insertion, compared to AAV2-WT, with the variable region-VI II (VR-VIII) indicated within the dashed circle. The VR-VIII region is enlarged and shown in the inset images for each vector. The VR-VIII of the AAV2-lntpi vector adopts an extended-open conformation after peptide insertion. The dashed line in the inset image denotes the end of VR-VRI 11 , whereas the dark structure extending beyond the dashed line denotes the extended VR-VIII loop with the disulfide bridge after the I ntpi -specific cyclic peptide insertion. Therefore, the peptide insertion has increased the protrusion length of VR-VIII in AAV2-lntpi in comparison to the AAV2-WT vector. The VR-VIII region of AAV2-iRGD adopts an extended-bent conformation due to the interaction of the cyclic peptide with the neighbouring residues. The dashed line in the inset image shows the end of VR-VIII region, whereas the dark structure extending beyond the dashed line shows the extended but bent VR-VIII loop with the disulfide bridge.

[0135] Conclusion

[0136] The novel AAV2-lntpi and AAV2-iRGD vectors were generated by tumor blood vesselbinding cyclic peptide insertion at the VR-VIII region of the AAV2 Cap gene. The Intpi peptide in the VR-VIII of the AAV2-lntpi vector adopts an extended-open confirmation, while the iRGD peptide in the VR-VIII of the AAV2-iRGD vector adopts an extended-bent conformation.

[0137] Example 5, Vector biodistribution properties of the novel AAV-lntpi and AAV2-iRGD vectors following systemic administration in healthy C57BL / 6 mice

[0138] Materials and Methods

[0139] AAV vector packaging. Packaging of AAV vectors was performed by triple transfection of HEK293 cells with a plasmid encoding AAV2 capsid (pAAV2-Rep / Cap) or AAV2-lntpi capsid (pAAV2-lntpi Rep / Cap) or AAV2-iRGD capsid (pAAV2-iRGD Rep / Cap), a plasmid encoding the desired transgene under the synthetic CMV enhancer / chicken beta-actin promoter (pAAV-CAG- GFP), and a plasmid encoding the helper gene elements (phelper), followed with iodoxinol-based gradient purification. The vector genomic titre (vgs) was estimated by SyBR chemistry-based qPCR using ITR-specific primers.

[0140] Vector biodistribution study. Healthy 6-8 week old female C57BL / 6 mice were treated with intravenous injection of 5x1O10vgs of AAV2-GFP, AAV2-lntpi-GFP or AAV2-iRGD. Four weeks post-treatment, organs were collected and genomic DNA was extracted from the liver, brain, heart, kidney, lungs and spleen. The AAV vector copy number in individual organs was assessed by qPCR using GFP-specific primers.

[0141] Results

[0142] Figure 6 shows the vector biodistribution properties of AAV2-lntpi and AAV2-iRGD vectors in healthy C57BL / 6 mice following systemic administration. Figure 6A illustrates the experimental layout used to obtain data shown in Figures 6B-D. In brief, healthy C57BL / 6 mice were treated with AAV2-WT, AAV2-lntpi or AAV2-iRGD vectors encoding GFP. Organs were collected and vector copy number was assessed in the liver, brain, heart, kidney, lungs and spleen. Figure 6B-D shows the quantification of vector genomes (vgs) in various organs of healthy mice following systemic administration of (B) AAV2-WT (n=3), (C) AAV2-lntpi (n=3) or (D) AAV2-iRGD (n=3) vectors encoding GFP. AAV2-WT mainly infected cells in the liver and to a minor extent in the kidney and spleen. The tumor blood vessel-targeting AAV2 vectors (AAV2-lntpi , AAV2-iRGD) were effectively detargeted from the liver when compared to AAV2-WT.

[0143] Conclusion

[0144] Cyclic peptide insertion in the tumor blood vessel-targeted AAVs successfully detargeted them from the liver in comparison to AAV2-WT, indicating a different tropism and reduced binding to heparin sulfate proteoglycans (HSPG). Example 6, AAV2-lntpi and AAV2-iRGD vectors transduce the tumor blood vessels in gliomabearing mice

[0145] Materials and Methods

[0146] Cell culture was performed as described in Example 1 , with the murine glioma cell line GL261 being cultured in DMEM with 10% FBS and 1 % PEST. Orthotopic murine glioma implantation was performed as described in Example 1 , with mice receiving 2*104GL261 cells.

[0147] Tumor blood vessel-targeting study. 7 days post-tumor implantation, glioma-bearing mice were treated with intravenous injection of 1x1011vg of AAV2-WT, AAV2-lntpi or AAV2-iRGD vectors encoding GFP. On day 23 post-tumor implantation, mice were sacrificed by intracardiac perfusion and brains were collected for immunofluorescence analysis.

[0148] Immunofluorescence staining and image acguisition. The brains were fixed in 4% PFA overnight, cryoprotected in 30% sucrose and cut into SOpm-thick vibratome sections. Sections were blocked in PBS containing 1% bovine serum albumin (BSA) and incubated with primary antibodies specific for GFP (1 :100, ab6673), CD31 for the blood vessels (1 :100, ma3105) and active a5pi integrin (1 :100, 553715) overnight at 4°C. Sections were then incubated with fluorophore- conjugated secondary antibodies for 2 hours at room temperature. Sections were washed in PBS, counterstained with Hoechst 33342 and mounted using Fluoromount-G. For analysis, images were captured with Leica SP8 confocal microscope (Leica Microsystems).

[0149] Image analysis. The proportion of GFP+CD31+vessels in the tumor and non-tumor region were determined by guantifying the GFP+area that co-localized with CD31+vessel area (GFP+CD31+) as compared to the total CD31+vessel area (CD31+). Similarly, the proportion of GFP+lntpi+CD31+vessels in the tumor was guantified by determining the GFP+area that colocalised with tumor blood vessels that were positive for both Intpi and CD31 (GFP+lntb1+CD31+) in comparison to the total tumor blood vessel area positive for CD31 and Intpi (CD31+lntpi+). Quantifications were made using confocal images of 4 randomly selected fields of view per sample in the tumor and non-tumor regions, at 25x magnification. Cell Profiler (Broad Institute) was used to guantify the vessel areas.

[0150] Results

[0151] Figure 7 shows the tumor blood vessel-targeting ability of AAV2-lntpi and AAV2-iRGD vectors in glioma-bearing mice. Figure 7A illustrates the experimental layout used to obtain data shown in Figures 7B-F. In brief, GL261 tumor cells were implanted intracranially in C57BL / 6 mice on day 0, followed by intravenous administration of AAV2-WT, AAV2-lntpi or AAV2-iRGD vectors encoding GFP day 7. Mice were sacrificed at day 23 and brains were collected for immunofluorescence analysis. Figures 7B-C show representative immunofluorescence images of vector-encoded GFP in the blood vessels within the (B) tumor area and (C) non-tumor area, following systemic administration of AAV2-WT, AAV2-lntpi or AAV2-iRGD. Scale bars 72pM. The AAV2-lntpi and AAV2-iRGD vector-transduced vessels are visible with GFP staining. Figure 7D shows the quantification of the proportion of vector-transduced GFP-positive blood vessels within the tumor area in each treatment group (n=3 mice / group), calculated as the ratio of GFP and CD31 double-positive area (GFP+CD31+) to the total CD31 area (CD31+area). The novel AAV2 variants are specific to the tumor blood vessels. Figure 7E shows the quantification of the proportion of vector-transduced GFP-positive activated blood vessels within the tumor area in each treatment group (n=3 mice / group), calculated as the ratio of GFP, Intpi , and CD31 triple-positive area (GFP+lntpi+CD31+area) to the total Intpi and CD31 double-positive area (lntpi+CD31+area). The novel AAV2 variants are specific to Intpr tumor blood vessels. Figure 7F shows the quantification of the proportion of GFP-positive blood vessels within the non-tumor area in each treatment group (n=3 mice / group), calculated as the ratio of GFP and CD31 double-positive area (GFP+CD31+area) to the total CD31 area (CD31+area). Compared to AAV2-WT, the tumor blood vessel-targeted AAV2-TEC vectors AAV2-lntpi and AAV2-iRGD bound significantly better to tumor blood vessels overall, as well as to tumor blood vessels expressing activated Intpi . Statistics: one-way ANOVA with Dunnett’s test for multiple comparisons. Key to statistics *p<0.01 , **p<0.01. Bar graphs show mean±SD.

[0152] Conclusion

[0153] The tumor blood vessel-targeted AAV2-TEC vectors AAV2-lntpi and AAV2-iRGD show an increased ability to bind to tumor blood vessels overall as well as to tumor blood vessels that express activated Intpi , when compared to AAV2-WT. The tumor blood vessel specificity was confirmed by the absence of binding to vessels in non-tumor tissue.

[0154] Example 7, AAV2-lntpi and AAV2-iRGD vectors transduce human brain microvascular endothelial cells (HBMVECs) in vitro

[0155] Materials and Methods

[0156] Transduction assay. Human brain microvascular endothelial cells (HBMVECs) were cultured and maintained using EBMv2 media in PureCol-coated plates. 3x104or 5x104HBMVECs were seeded in a 24-well plate and transduced with AAV2-WT, AAV2-lntpi , AAV2-iRGD or AAV-BR1 vectors encoding GFP, at various multiplicities of infection (MOI: 5x103, 1x104, 2.5x104, 5x104, 1x105). The medium was replaced after 12h of incubation, and the cells were collected for flow cytometry analysis at 72h post-transduction.

[0157] Competitive inhibition assay. 5x104HBMVECs were seeded in a 24-well plate and treated with active a5pi integrin antibody (12G10, ab30394) or isotype control antibody (IgG, ab170190) at different concentrations by incubating in ice for 30 mins. After antibody incubation, the AAV2-WT or AAV2-lntpi vectors encoding GFP were added to the cells at 2.5x104MOI, and incubated overnight. The medium was replaced after overnight incubation, and the cells were collected for flow cytometry analysis at 72h post-transduction. For the heparin-based inhibition assay, the vectors (2.5x104MOI) were treated with heparin (H3149-50KU) at various concentrations for 1 hr at RT prior to treatment. Cells were incubated with the vector and the heparin mix, and the medium was replaced after 12h of incubation. The cells were collected for flow cytometry analysis at 72h post-transduction.

[0158] In vitro analysis of hLIGHT delivery in brain endothelial cells. 1x105HBMVECs were seeded in 6-well plates and transduced with the AAV2-lntpi vector encoding human LIGHT (hLIGHT) at various MOIs. 72h post-transduction, total RNA was extracted using TRI reagent and reverse- transcribed using the iScript cDNA synthesis kit (Bio-Rad). Relative expression of gene targets such as hLIGHT, VCAM-1, SELE and MADCAM-1 were quantified using PowerUp sYBR qPCR kit in CFX96 Thermal cycler (Bio-Rad). The data was normalised against HPRT, and relative levels of expression were calculated by comparative Ct method (AACt).

[0159] Flow cytometry. The cells were assessed for GFP positivity using a CytoFLEX LX (Beckman Coulter) and data were analyzed using CytExpert software.

[0160] Results

[0161] Figure 8 shows the ability of AAV2-lntpi and AAV2-iRGD vectors to transduce human brain microvascular endothelial cells (HBMVECs) in vitro. Figures 8A-E use AAV vectors encoding GFP while Figures 8F-I use the AAV2-lntpi vector encoding human LIGHT (hLIGHT), AAV-lntpi- hLIGHT. Figure 8A shows the quantification of transduced HBMVECs when treated with mock, AAV2-WT, AAV2-lntpi or AAV2-iRGD vectors. The AAV2-lntpi vector can transduce HBMVECs, while AAV2-iRGD failed to transduce HBMVECs. Figure 8B shows a comparison of the transduction efficiency of AAV2-lntpi vector compared to AAV-BR1 and AAV2-WT vectors at various multiplicity of infection (MOI). The AAV-BR1 vector failed to transduce HBMVECs in comparison to AAV2- I ntpi . Figures 8CD show the quantification of the relative transduction of HBMVECs when treated with AAV2-WT or AAV2-lntpi in conjunction with (C) an antibody (12G10) blocking active a5pi integrin or (D) an isotype control antibody. Decreased transduction in the presence of 12G10 highlights the dependency of the AAV2-lntpi vector on activated a5pi integrins present on endothelial cells during transduction process. Figure 8E shows the quantification of the relative transduction of HBMVECs when treated with AAV2-WT or AAV2-lntpi in conjunction with various concentrations of heparin. Unaltered transduction capacity is observed for AAV2-lntpi in the presence of heparin in comparison to AAV2-WT. This shows the altered receptor usage and its independency of heparan sulfate proteoglycan (HSPG) receptors after the cyclic peptide insertion. Figures 8F-I show the quantification of the relative expression levels of (F) hLIGHT, (G) VCAM-1, (H) SELE and (I) MADCAM-1 by HBMVECs following treatment with AAV2-lntpi-LIGHT at various MOIs. AAV2-lntpi efficiently delivers LIGHT to the cells, and upregulates markers of endothelial cell activation and high endothelial venule (HEV) phenotype. Statistics in B-E: two-way ANOVA with Sidak’s test for multiple comparisons. Statistics in F-l: one-way ANOVA. Key to statistics *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. comparing AAV2-WT vs AAV2-lntB1. Graphs show mean±SD

[0162] Conclusion

[0163] AAV2-lntpi is more effective than AAV-BR1 and AAV2-iRGD at transducing HBMVECs. Furthermore, the transduction capacity of AAV2-lntpi is specifically dependent on expression of activated I ntp 1 on the target cells. AAV2-lntpi can efficiently deliver hLIGHT to HBMVECs, and upregulate markers of endothelial cell activation and the high endothelial venule (HEV) phenotype in vitro.

[0164] Example 8, AAV2-lntpi is capable of transducing the human glioblastoma vasculature ex vivo Materials and Methods

[0165] Human brain slice culture. Fresh human glioblastoma tissue resected during surgery was cut into 250pm-thick vibratome sections and cultured in human brain slice growth media containing Neurobasal A and B-27 serum supplement. The cultured brain sections were incubated with 2x1010vg of AAV2-WT, AAV2-lntpi or AAV2-iRGD encoding GFP. After 48 hours of incubation, the brain slices were fixed and stained for immunofluorescence analysis. Vessels were stained with an anti- UEA1 antibody for the blood vessels, and activated I nt|31 was stained with the 12G10 antibody.

[0166] Results

[0167] Figure 9 illustrates the ability of AAV2-lntpi to transduce the human glioblastoma vasculature ex vivo. Figure 9A illustrates the experimental layout used to obtain data shown in Figure 9B. In brief, fresh human glioblastoma tissue slices were incubated with AAV2-WT, AAV2- I ntpi or AAV2-iRGD vectors encoding for GFP for 48h. Then, the slices were stained for immunofluorescence analysis. Figure 9B shows representative immunofluorescence images of vector-encoded GFP in the blood vessels within the tumor area in brain slices from two patients following treatment with AAV2-lntpi compared to AAV2-WT and AAV2-iRGD. Tumor blood vessels are indicated by UEA-1 staining, AAV-transduced cells are identified by GFP, and active lnt|31 is indicated by 12G10 staining. Scale bars 50pm. No vector-transduced GFP was observed in the vessels of brain slices treated with AAV2-WT or AAV2-iRGD, while GFP transduction was observed in the active lnt|31 -expressing vessels of brain slices treated with AAV2-lntpi .

[0168] Conclusion

[0169] The tumor blood vessel-targeting AAV2-lntpi vector demonstrates tumor blood vesselspecific binding in human glioblastoma tissue ex vivo.

[0170] Example 9, AAV2-lntpi-LIGHT therapy prolongs survival in murine glioma Here, AAV2-lntpi is also referred to as AAV-lntpi .

[0171] Materials and Methods

[0172] Cell culture and tumor studies were performed as described in Example 1 , with mice receiving 5x104CT2A-OVA-Luc cells. Mice were treated with 1x1011vgs of AAV2-lntpi-GFP, AAV2- Intpi-LIGHT, AAV2-BR1-GFP, AAV2-BR1-LIGHT on days 4, 7 and 10 post-tumor implantation. Mice were monitored for glioma-related symptoms and sacrificed at the survival endpoint. Brains collected after intracardiac perfusion were processed and cut into 80pm-thick vibratome sections. Sections were stained with antibodies specific for fibrinogen (1 :200, DAKO A0080), blood vessels (CD31 , 1 :100, ma3105) and nuclei (Hoechst 3342) to assess the vascular leakage in the tumor and the non-tumor regions in each treatment group.

[0173] Image analysis. For analysis, images were captured with Leica SP8 confocal microscope (Leica Microsystems). Imaged software (NIH) was used to quantify the fibrinogen positive areas in the tumor and the non-tumor regions in each treatment group.

[0174] Results

[0175] Figure 10 shows prolonged survival in glioma-bearing mice following treatment with AAV2- Intpi-LIGHT. Figure 10A illustrates the experimental layout used to obtain data shown in Figures 10B-E. In brief, the murine glioma cell line CT2A-OVA-Luc was implanted intracranially in C57BL / 6 mice on day 0, followed by intravenous administration of either AAV2-lntpi-GFP, AAV2-lntpi- LIGHT, AAV-BR1-GFP, AAV-BR1 -LIGHT on days 4, 7 and 10 post-tumor implantation. Mice were monitored for glioma-related symptoms and sacrificed at the humane endpoint, when brains were collected for immunofluorescence analysis. Figure 10B shows a Kaplan-Meier survival curve of CT- 2A tumor-bearing mice treated with AAV2-lntpi-GFP or AAV2-lntpi-LIGHT (n=10 mice / group), and a Kaplan-Meier survival curve of CT-2A tumor-bearing mice treated with AAV-BR1-GFP or AAV- BR1-LIGHT (n=10 mice / group). Statistics: Logrank test. In comparison to their respective control groups, both AAV-BR1-LIGHT and AAV2-lntpi-LIGHT resulted in prolonged survival in gliomabearing mice. Figure 10C shows representative immunofluorescence images of fibrinogen (FRN) leakage in each treatment group. Figures 10D-E show the quantifications of fibrinogen positive area within (D) the tumor area and (E) the non-tumor area in each treatment group. Mice treated with AAV-BR1-LIGHT exhibited significantly higher levels of fibrinogen leakage in both the tumor and non-tumor area when compared to mice treated with AAV2-lntpi-LIGHT. Importantly, in the AAV2- Intpi-LIGHT treated group, fibrinogen-positive regions were restricted to the tumor region, thereby confirming the specificity of the AAV2-lntpi vector to the tumor blood vessels as opposed to all brain vessels.

[0176] Conclusion

[0177] Tumor blood vessel-targeted AAV2-lntpi-LIGHT prolongs survival glioma-bearing mice and reduces vascular leakage when compared to AAV2-BR1 -LIGHT.

[0178] Example 10, LIGHT can be inducibly expressed using a mini-Xon-AAV system

[0179] Materials and Methods

[0180] Inducible AAV2-mini-Xon-LIGHT construct development. The inducible AAV2-mini-Xon- LIGHT system was developed by cloning a mini-Xon system upstream of the LIGHT transgene, after removing the innate start codon of LIGHT gene in the self-complementary AAV genome system (pscAAV-mini-Xon-LIGHT). The construct was verified by transfecting HEK293 cells with 300ng of plasmid DNA using PEI transfection mix. The transfected cells were treated with the LMI070 drug at various concentrations, 4h post-transfection. 48h post-drug treatment, the cells were collected for Trizol based RNA extraction. Relatively expression levels of LIGHT were quantified by qPCR using LIGHT-specific primers. The data was normalised against GAPDH, and relative expression levels of LIGHT were calculated by comparative Ct method.

[0181] Results

[0182] Figure 11 shows the inducible expression of LIGHT using the mini-Xon AAV system. Figure 11A illustrates the encoding of LIGHT on a self-complementary mini-Xon AAV system, in which expression is regulated using a splice modulator drug (LMI070). Without LMI070, splicing modulation does not occur and thus prevents start codon incorporation for LIGHT expression. Adding LMI070 drug induces the splicing event, thereby starting production of the LIGHT protein. Figure 11 B shows the quantification of normalized LIGHT levels expressed by HEK293 cells following treatment with pscAAV-mini-Xon-LIGHT in conjunction with various concentrations of the drug LMI070. Positive correlation of LIGHT expression to the increasing concentration of drug was observed, highlighting that the mini-Xon-LIGHT construct can be used during LIGHT treatment to control LIGHT expression.

[0183] Conclusion

[0184] Regulated expression of LIGHT can be achieved using a mini-Xon system.

[0185] Example 11 , Development of exclusively soluble or exclusively membrane-bound LIGHT

[0186] In its native form, LIGHT is a 29 kDa protein that can function both as a soluble, secreted protein and as a cell surface-bound type II membrane protein. LIGHT must be in its homotrimeric form to interact with its two primary functional receptors, LTpR and HVEM. It was set out to produce variants of LIGHT that were either exclusively soluble / secreted (SLIGHT) or exclusively membranebound (mLIGHT).

[0187] Materials and Methods

[0188] Development of SLIGHT and mLIGHT constructs. SLIGHT was developed by deleting the transmembrane and intracellular portion and fusing the extracellular domain of LIGHT with a secretory signal and trimerization domain. mLIGHT was produced by amino acid substitution (L->A) at position L81 for murine LIGHT (L81A) or at position L83 for human LIGHT, or by deleting the matrix metalloproteinase (MMP) cleavage site of LIGHT (ALIGHT).

[0189] Verification of SLIGHT and mLIGHT. Plasmids encoding native LIGHT, SLIGHT or mLIGHT were used to transfect HEK293 cells. The cells were cultured for 48 hours. The culture medium (where SLIGHT, but not mLIGHT, is expected to be found) was then harvested and subjected to ELISA and western blot analysis.

[0190] Results

[0191] Figure 12 shows the development of exclusively soluble or exclusively membrane-bound LIGHT. Figure 12A illustrates the strategy used to develop exclusively soluble or exclusively membrane-bound LIGHT. Figure 12B shows the detection of soluble LIGHT (SLIGHT) following transfection of HEK293 cells with plasmids encoding SLIGHT or mLIGHT. Detection in culture medium was performed either by ELISA or western blot under reducing or non-reducing conditions. When analyzed by western blot under non-reducing conditions, SLIGHT was detected in its trimeric form, while under reducing conditions it primarily migrated in its monomeric form. Significantly higher amounts of LIGHT protein (pg / ml) were detected in the culture medium when cells had been transfected with the plasmid encoding SLIGHT, compared to the plasmid encoding native LIGHT, demonstrating efficient production and secretion of SLIGHT. Figure 12C shows the detection of membrane-bound LIGHT (mLIGHT) following transfection of HEK293 cells with plasmids encoding exclusively soluble LIGHT or exclusively membrane-bound LIGHT. Detection in culture medium was performed either by ELISA or western blot under reducing conditions. When analyzed by western blot under reducing conditions, significantly less LIGHT protein (pg / ml) was detected in the culture medium when cells were transfected with plasmids encoding for mLIGHT (LIGHT L81A and ALIGHT), compared to when a plasmid encoding for the native form of LIGHT was used, suggesting that mLIGHT is not cleaved efficiently into the medium.

[0192] Conclusion

[0193] SLIGHT produced by removing the intracellular and transmembrane domains and fusing the extracellular domain of LIGHT with a secretory signal and trimerization domain is secreted in its bioactive form at a higher extent that native LIGHT. mLIGHT produced by amino acid substitution or by deleting the matrix metalloproteinase (MMP) cleavage site of LIGHT is found to be secreted to a lesser extent that native LIGHT.

[0194] SEQUENCES

[0195] Some of the sequences of the present application are presented in Table 1 below, where the different parts of the constructs are clearly identified. The rep and cap gene sequences provided are the coding sequences which can give several proteins by alternate splicing and alternate start codon usage during vector packaging. However, there is a vector backbone sequence in all these sequences (SEQ ID NO: 3-4) between residue 1867 and 1882. It should be noted that human LIGHT (SEQ ID NO:15) may be modified as the murine LIGHT (SEQ ID NO:5, not shown in the table) into exclusively soluble or membrane bound forms as in murine nucleotide sequences SEQ ID NO:6-8 (not shown). SEQ ID NO: 1-2 define the targeting peptide amino acid sequences, and SEQ ID NO:16-17 their corresponding nucleotide sequences. SEQ ID NO:3-4 show the AAV2 capsids with the respective targeting peptide. As may be seen in SEQ ID NO: 3-4, when cloning the sequence encoding the targeting peptides into the cap gene, three nucleotides encoding G is inserted directly before the peptide and three nucleotides encoding A is inserted directly after the peptide sequence. These amino acids are not part of the cap sequence nor the targeting peptide, although marked in capital letters, but are spacer amino acids (see Figure 5). These spacer sequences were included to attach the peptide to the AAV2 VRVIII loop. The AAV2-lntpi vector in SEQ ID NO:3 was generated using DNA oligos of SEQ ID NO: 22-27, the AAV2-iRGD vector in SEQ ID NO:4 was generated using DNA oligos SEQ ID NO:22-25, 28 and 29. SEQ ID NO:9-14 (not shown in the table) relate to AAV plasmid constructs comprising different modifications of murine LIGHT, murine soluble LIGHT (SLIGHT), and membrane bound murine LIGHT with different modifications to silence the protease cleavage site, and SEQ ID NO:30-33 and 38-40 show AAV plasmid constructs comprising different modifications of human LIGHT, where SEQ ID NO:9-13, and 30-33, 38 comprise the constitutively active CAG promoter, SEQ ID NO:9, 38, 40 relating to ssAAV, and SEQ ID NO: 10-14, 30-33 and 39 to scAAV. SEQ ID NO: 14, 39-40 comprise the inducible Xon system and phosphoglycerate kinase promoter. Constructs having any combinations of promoters and LIGHT variants may be made, the constructs can either be designed as single stranded (ssAAV) or self-complementary (scAAV), and the ones below are some examples. Table 1

[0196]

[0197] ITEMIZED EMBODIMENTS

[0198] 1. An adeno-associated serotype 2 viral vector, AAV2, comprising a transgene encoding LIGHT, wherein a viral capsid of the AAV2 vector comprises a targeting peptide.

[0199] 2. An adeno-associated serotype 2 viral vector, AAV2, comprising a transgene encoding a lymphotoxin beta receptor, LTpR, agonist, wherein a viral capsid of the AAV2 vector comprises a targeting peptide.

[0200] 3. The AAV2 vector according to item 2, wherein the AAV2 vector agonist is LIGHT and / or lymphotoxins.

[0201] 4. The AAV2 vector according to items 1-3, wherein the transgene encoding LIGHT is selected from native LIGHT, exclusively soluble LIGHT or exclusively membrane-bound LIGHT.

[0202] 5. The AAV2 vector according to item 4, wherein the transgene encoding LIGHT is membranebound LIGHT having a silenced protease cleavage site.

[0203] 6. The AAV2 vector according to item 5, wherein the protease cleavage site is silenced, for example by mutating amino acid 81 from L to A (L81A) in murine LIGHT or by mutating amino acid 83 from L to A (L83A) in human LIGHT or by deleting amino acids 78-82 W-E-K- L-l, ALIGHT in murine LIGHT or by deleting amino acids 81-84 E-Q-L-l, ALIGHT in human LIGHT. 7. The AAV2 vector according to items 1-6, wherein the transgene encoding LIGHT is controlled by a constitutively active promoter, such as CAG, or an inducible gene expression system, such as Xon.

[0204] 8. The AAV2 vector according to item 5, wherein LIGHT is human LIGHT.

[0205] 9. The AAV2 vector according to items 1-8, wherein the targeting peptide targets cells in a tumor microenvironment.

[0206] 10. The AAV2 vector according to items 1-9, wherein the targeting peptide targets cells of blood vessels.

[0207] 11. The AAV2 vector according to items 1-10, wherein the targeting peptide targets endothelial cells.

[0208] 12. The AAV2 vector according to item 11, wherein the endothelial cells are tumor endothelial cells, TECs.

[0209] 13. The AAV2 vector according to item 12, wherein the tumor endothelial cells are endothelial cells in blood vessels of a tumor.

[0210] 14. The AAV2 vector according to items 1-13, wherein the targeting peptide is a vessel-targeting peptide.

[0211] 15. The AAV2 vector according to items 1-14, wherein the targeting peptide is inserted in the viral capsid at the arginine amino acid located in position 588 (R588) of a VP3 protein of the viral capsid.

[0212] 16. The AAV2 vector according to items 1-15, wherein the targeting peptide targets activated integrins, such as integrin alpha 5 beta 1 , a5pi.

[0213] 17. The AAV2 vector according to items 1-16, wherein the targeting peptide has an amino acid sequence defined as ACRGDGWCG (SEQ ID NO:1). The AAV2 vector according to items 12-17, wherein the targeting peptide has an amino acid sequence defined as ACRGDGWCG (SEQ ID NO:1), or a sequence having 80 % or more, such as 85 %, 90 %, 95 % or more, identity thereto. The AAV2 vector according to items 1-15, wherein the targeting peptide targets integrins and neuropilin-1. The AAV2 vector according to items 1-15and 19, wherein the targeting peptide has an amino acid sequence defined as CRGDKGPDC (SEQ ID NO:2). The AAV2 vector according to items 12-15 and 19-20, wherein the targeting peptide has an amino acid sequence defined as CRGDKGPDC (SEQ ID NO:2), or a sequence having 80 % or more, such as 85 %, 90 %, 95 % or more, identity thereto. The AAV2 vector according to items 9-22, wherein the AAV2 vector is able to transduce the targeted cells, inducing the targeted cells to express LIGHT and / or lymphotoxins. The AAV2 vector according to items 10-22, wherein AAV2 vector transduction of the targeted endothelial cells enables the endothelial cells to adopt a high endothelial venule (HEV) phenotype. The AAV2 vector according to items 9-23, wherein AAV2 vector transduction of the targeted cells induces increased recruitment and infiltration of T lymphocytes through tumor blood vessels to the tumor microenvironment in proximity to the targeted cells. The AAV2 vector according to item 24, wherein the AAV2 vector transduction of the targeted cells induces activation of the T lymphocytes. The AAV2 vector according to items 9-25, wherein AAV2 vector transduction of the targeted cells induces formation of tertiary lymphoid structures, TLS, in tissues surrounding the targeted cells, such as in the tumor microenvironment. The AAV2 vector according to item 26, wherein the tissues are present within or near a tumor, such as in the tumor microenvironment. 28. The AAV2 vector according to any one of items 1-27, for use in inducing expression of LIGHT and / or lymphotoxins.

[0214] 29. The AAV2 vector according to any one of items 1-28, for use in one or more of i) recruitment and infiltration of T lymphocytes into a tumor microenvironment, ii) activation of T lymphocytes, iii) phenotype switch of endothelial cells into HEVs, iv) formation of TLS, and v) formation of antigen-presenting niches.

[0215] 30. A pharmaceutical composition containing an AAV2 vector of any one of items 1-29, and a pharmaceutically acceptable carrier or excipient.

[0216] 31. An AAV2 vector according to items 1-30, or a pharmaceutical composition according to item 37, for use in therapy.

[0217] 32. The AAV2 vector or pharmaceutical composition according to item 31 , for use in cancer therapy.

[0218] 33. The AAV2 vector or pharmaceutical composition according to item 32, for use in cancer therapy of solid tumors.

[0219] 34. The AAV2 vector or pharmaceutical composition according to items 32-33, for use in cancer therapy of brain tumors.

[0220] 35. The AAV2 vector or pharmaceutical composition according to item 32-34, for use in therapy of glioma, such as glioblastoma.

[0221] 36. The AAV2 vector or pharmaceutical composition according to item 31 , for use in immunotherapy.

[0222] 37. The AAV2 vector or pharmaceutical composition according to item 31 , for use in immunotherapy, wherein the immunotherapy induces formation of HEVs, TLS, and / or antigen-presenting niches. 38. A method of treating cancer in a subject in need thereof, comprising administering a therapeutically effective amount of an AAV2 vector according to any one of items 1-27, or a pharmaceutical composition of item 30. 39. A method of targeting LIGHT and / or lymphotoxins to cells in a tumor microenvironment in a subject in need thereof, wherein the cells in the tumor microenvironment comprises tumor endothelial cells of a tumor blood vessel, the method comprising: providing an AAV2 viral vector encoding LIGHT and / or lymphotoxins according to any one of items 1-27; administering the AAV2 viral vector to the subject, or a pharmaceutical composition thereof, whereby the AAV2 viral vector transduces the targeted cells, thereby inducing the targeted cells to express LIGHT and / or lymphotoxins.

[0223] 40. Use of an AAV2 viral vector encoding LIGHT and / or lymphotoxins according to any one of items 1-27 for targeting LIGHT and / or lymphotoxins to cells in a tumor microenvironment in a subject.

Claims

CLAIMS1 . An adeno-associated serotype 2 viral vector, AAV2, comprising a transgene encoding LIGHT, wherein a viral capsid of the AAV2 vector comprises a targeting peptide, wherein the targeting peptide targets tumor endothelial cells, TECs.

2. The AAV2 vector according to claim 1 , wherein the transgene encoding LIGHT is selected from native LIGHT, exclusively soluble LIGHT or exclusively membrane-bound LIGHT.

3. The AAV2 vector according to claims 1-2, wherein the transgene encoding LIGHT is controlled by a constitutively active promoter, such as CAG, or an inducible promoter, such as Xon.

4. The AAV2 vector according to claims 1-3, wherein the targeting peptide is inserted in the viral capsid at the arginine amino acid located in position 588, R588, of a VP3 protein of the viral capsid.

5. The AAV2 vector according to claims 1-4, wherein the targeting peptide targets one or more of activated integrins, such as integrin alpha 5 beta 1 , a5pi , and / or neuropilin-1..

6. The AAV2 vector according to claims 1-5, wherein the targeting peptide is cyclic.

7. The AAV2 vector according to claims 1-6, wherein the targeting peptide has an amino acid sequence selected from ACRGDGWCG (SEQ ID NO:1) and CRGDKGPDC (SEQ ID NO:2), or sequences having 80 % or more, such as 85 %, 90 %, 95 % or more, identity thereto.

8. The AAV2 vector according to claims 1-7, wherein AAV2 vector transduction of the targeted cells enables tumor endothelial cells to adopt a high endothelial venule, HEV, phenotype.

9. The AAV2 vector according to claims 1-8, wherein AAV2 vector transduction of the targeted cells induces one or more of i) phenotype switch of endothelial cells into HEVs ii) increased recruitment and infiltration of T lymphocytes through tumor blood vessels to the tumor microenvironment in proximity to the targeted cells, iii) activation of T lymphocytes, iv)formation of tertiary lymphoid structures, TLS, and v) formation of antigen-presenting niches, in tissues surrounding the targeted cells.

10. The AAV2 vector according to any one of claims 1-9, for use in inducing expression of LIGHT or any other factor that may alter the vascular phenotype to promote anti-tumor immunity.11 . A pharmaceutical composition containing an AAV2 vector of any one of claims 1-9, and a pharmaceutically acceptable carrier or excipient.

12. An AAV2 vector according to claims 1-9, or a pharmaceutical composition according to claim 11 , for use in therapy.

13. The AAV2 vector or pharmaceutical composition according to claim 12, for use in cancer therapy.

14. The AAV2 vector or pharmaceutical composition according to claim 12-13, for use in cancer therapy including one or more of i) therapy of solid tumors, ii) therapy of brain tumors, and iii) therapy of glioma.

15. The AAV2 vector or pharmaceutical composition according to claim 12, for use in immunotherapy, wherein the immunotherapy induces formation of HEVs, TLS and / or antigen-presenting niches.

16. A method of targeting LIGHT to cells in a tumor microenvironment in a subject in need thereof, wherein the cells in the tumor microenvironment comprise tumor endothelial cells of a tumor blood vessel, the method comprising: providing an AAV2 viral vector encoding LIGHT according to any one of claims 1-9; administering the AAV2 viral vector to the subject, or a pharmaceutical composition thereof, whereby the AAV2 viral vector transduces the targeted cells, thereby inducing the targeted cells to express LIGHT.

17. Use of an AAV2 viral vector encoding LIGHT according to any one of claims 1-9, for targeting LIGHT to cells in a tumor microenvironment in a subject.

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