Bispecific t cell adapter (BiTE) carrying adenoviruses

Abstract

The name of the invention is an adenovirus carrying a bispecific T cell adapter (BiTE). An adenovirus comprising a sequence of formula (I): 5 'ITR-B1-BA-B2-BX-BB-BY-B3-3' ITR (I) wherein BY comprises a transgene cassette comprising four transgenes, said genes encoding FAP-BITE, CXL10, CXL9 and IFN. The disclosure also extends to pharmaceutical compositions comprising the viruses, and treatments using the viruses or formulations.

Description

[0001] This application is a divisional application. The application date of the original application is August 28, 2018, the application number is 2018800551710, and the name of the invention is “Adenovirus carrying bispecific T cell engager (BiTE)”.

[0002] The present disclosure relates to modified adenoviruses, specifically Enadenotucirev (EnAd), with FAP-BITE compositions, such as pharmaceutical formulations comprising the adenovirus, and uses of the viruses and viral formulations, particularly for therapeutic use, especially for the treatment of cancer. The present disclosure also extends to methods for preparing the viruses and DNA encoding the viruses. The present disclosure also extends to novel sequences provided in the sequence listing in combination with the technical disclosure herein, for example, wherein the exemplified viruses are replaced with alternative cassettes or alternative viruses, such as those provided in the sequence listing. Background Art

[0003] Cancer remains a huge burden on society in terms of the hardship and suffering it inflicts on patients and their loved ones, as well as the high economic costs of treating, caring for, and supporting patients.

[0004] The stroma surrounding cancer cells acts as a physical shield, potentially trapping immune cells to fight the tumor. Furthermore, the stroma shields the tumor from the hypoxic microenvironment, which is permissive and optimal for tumor growth. Some theories suggest that cells in the stroma serve as a source of energy for the tumor.

[0005] A large component of the tumor stroma is fibroblasts, which have been destroyed to play a role in initiating cancer. Other cells that infiltrate the stroma are tumor-associated macrophages (TAMs), which are type 2 (M2) macrophages that can promote tumor growth by secreting cytokines and chemokines such as IL-10 that suppress the immune response.

[0006] Targeting the tumor stroma is particularly difficult because the cells that generate the milieu are "innate" immune cells or connective tissue cells found throughout the body. Therefore, targeting these cells with therapeutic agents can result in significant off-target effects.

[0007] Therefore, there is a need for an improved method of delivering BiTEs directly to tumor cells where it can provide the greatest therapeutic benefit, specifically to tumor cells surrounded by stromal fibroblasts.

[0008] WO2018 / 041838 and WO2018 / 041827, both incorporated herein by reference, disclose certain adenoviruses encoding BITEs. However, it would be useful to enhance the activity of the BITEs embedded in the virus by incorporating activated cytokines. The integration of two cytokines co-localized with the BITEs can be easily accomplished. However, when three cytokines coexist with the BITEs, the nature of the genes begins to affect the expression of the BITEs. The present inventors created a virus NG-615 with four transgenes, such as Figure 1 As shown. However, BITE expression was reduced. Surprisingly, the virus NG-641, in which two cytokines were altered (compared to NG-615), had good activity, including good expression of BITE. Therefore, it appears that the four transgenes are compatible with the coexistence of the virus.

[0009] The presently claimed invention relates to viruses that harbor the four transgenes co-existing between the fiber, L5 and E4 regions. Summary of the Invention

[0010] The following paragraphs are an overview of the present disclosure:

[0011] 1. An adenovirus comprising a sequence of formula (I):

[0012] 5'ITR-B1-B A -B2-B X -B B -B Y -B3-3'ITR (I)

[0013] in:

[0014] B1 is a bond or includes: E1A, E1B, or E1A to E1B;

[0015] B A Includes -E2B-L1-L2-L3-E2A-L4;

[0016] B2 is a key or includes: E3;

[0017] B X is a bond or DNA sequence that includes: a restriction site, one or more transgenes, or both;

[0018] B B Including L5;

[0019] B Y Includes a transgene cassette containing four transgenes encoding FAP-BITE, CXL10, CXL9, and IFN;

[0020] B3 is a key or includes: E4.

[0021] 2. The adenovirus according to paragraph 1, wherein the encoded FAB-BITE comprises the anti-CD3 shown in SEQ ID NO: 5 or a sequence at least 95% identical thereto, such as SEQ ID NO: 5.

[0022] 3. The adenovirus according to paragraph 1 or 2, wherein the FAP-BITE comprises the anti-FAP shown in SEQ ID NO: 9 or a sequence at least 95% identical thereto, such as SEQ ID NO: 9

[0023] 4. The adenovirus of paragraph 1, wherein the encoded FAB-BITE comprises a sequence selected from SEQ ID NO: 75, SEQ ID NO: 76, or a sequence at least 95% identical to any one thereof.

[0024] 5. The adenovirus of any one of paragraphs 1 to 4, wherein the transgene cassette encodes CXL10 as shown in SEQ ID NO: 100 or a sequence at least 95% identical thereto, such as SEQ ID NO: 100.

[0025] 6. The adenovirus of any of paragraphs 1 to 5, wherein the transgene cassette encodes CXCL9 as set forth in SEQ ID NO: 99 or a sequence at least 95% identical thereto, such as SEQ ID NO: 99.

[0026] 7. The adenovirus according to any one of paragraphs 1 to 6, wherein the transgene cassette encodes the IFNα shown in SEQ ID NO: 98 or a sequence at least 95% identical thereto, such as SEQ ID NO: 98.

[0027] 8. The adenovirus of any of paragraphs 1 to 7, wherein the transgene is operably linked.

[0028] 9. The adenovirus of any one of paragraphs 1 to 8, wherein the transgene is separated by three different highly efficient self-cleaving peptides.

[0029] 10. The adenovirus of paragraph 9, wherein the self-cleaving peptide is independently selected from E2A, F2A, P2A, and T2A.

[0030] 11. The adenovirus according to any one of paragraphs 1 to 10, wherein the relative order of the transgenes from L5 to E4 is FAP-BITE, CXL10, CXL9 and IFNα, e.g. Figure 1 As shown in NG-641.

[0031] 12. The adenovirus according to any one of paragraphs 1 to 11, wherein the transgene cassette has the polynucleotide sequence shown in SEQ ID NO: 95 or a polynucleotide encoding the same amino acid sequence, specifically SEQ ID NO: 95.

[0032] 13. The adenovirus of any of paragraphs 1 to 12, wherein the adenovirus comprises SEQ ID NO: 84.

[0033] 14. The adenovirus of any one of paragraphs 1 to 13, wherein the adenovirus is replication competent.

[0034] 15. The adenovirus of any one of paragraphs 1 to 14, wherein the adenovirus is oncolytic.

[0035] 16. The adenovirus of any of paragraphs 1 to 15, wherein the virus has hexon and fiber from Ad11.

[0036] 17. A pharmaceutical composition comprising the adenovirus according to any one of paragraphs 1 to 16 and an excipient, diluent or carrier.

[0037] 18. The adenovirus according to any one of paragraphs 1 to 16 or the pharmaceutical composition according to paragraph 17 for use in therapy, such as for the treatment of cancer.

[0038] 19. A method of treating a patient comprising administering the adenovirus of any one of paragraphs 1 to 16 or the pharmaceutical composition of paragraph 17.

[0039] 20. Use of the adenovirus according to any one of paragraphs 1 to 16 or the pharmaceutical composition according to paragraph 17 for the manufacture of a medicament for the treatment of cancer.

[0040] In one embodiment, one or more BiTEs according to the present disclosure do not include a transmembrane domain and, therefore, are not expressed on the surface of cancer cells, but rather include a signal sequence that facilitates the release of the BiTE molecule from cancer cells infected by a virus.

[0041] In one embodiment, the transgene cassette is under the control of an endogenous promoter, such as a major post promoter.

[0042] Advantageously, the inventors have discovered that carrying adenovirus with BiTE molecules allows the bispecific antibody fragment molecules to "piggyback" on the adenovirus's ability to selectively infect cancer cells, thereby enabling targeted delivery of the BiTE to tumor cells.

[0043] Advantageously, BiTEs are small and can be produced in mammalian cells. Therefore, once infected with the adenovirus of the present disclosure, BiTE molecules are synthesized, secreted, and can act locally by the tumor cells, thereby spreading beyond the direct footprint of the virus. This allows the BiTEs to spread beyond the direct site of infection, but at the same time limits the spread of the virus far beyond the site of infection of the tumor cells. This minimizes the risk of undesirable off-target effects.

[0044] In one embodiment, the adenovirus is EnAd. Compared to other adenovirus platforms, such as those based on Ad5, EnAd has been shown to have enhanced oncolytic activity. EnAd has also been shown to have high selectivity for human epithelial-derived cancer cells, such as colon cancer, lung cancer, bladder cancer, and renal cancer cells. This makes it an ideal delivery vehicle for BiTE molecules, because T cells can be activated by BiTE molecules to attack target cells, while EnAd simultaneously infects and dissolves cancer cells. This results in a two-pronged attack on tumors with a synergistic oncolytic effect.

[0045] In one embodiment, the anti-CD3 component of the BITE is selective for an antigen selected from the group consisting of CD3ε, CD3γ, and CD3δ, specifically CD3ε.

[0046] FAP is a tumor stroma antigen. Advantageously, stromal cells (non-transformed cells) that express these antigens do not undergo the same level of mutation resistance selection as transformed cells. Therefore, these cells are more easily targeted for cancer therapy because they are not "moving targets." In addition, the receptor types found in stromal cells are generally common across different types of cancer. Therefore, targeting FAP may be effective for a variety of cancer types.

[0047] Advantageously, FAP is upregulated on tumor-associated fibroblasts. Fibroblasts are an important component of solid cancers, supporting growth, invasion, and recovery from intervention. They typically comprise 40% to 60% of cells in advanced cancers. Advantageously, fibroblasts are genetically stable cells that are less likely to evade treatment than cancer cells. Activated fibroblasts are also relatively similar across tumor types. Therefore, by activating T cells to target and kill FAP-expressing tumor-associated fibroblasts, the adenoviruses of the present disclosure can help reduce the spectrum of immunosuppressive pathways, such as those mediated by IL-10, TGFβ, and IDO.

[0048] In one embodiment, B X Not a key.

[0049] In one embodiment, the adenovirus is chimeric. In one embodiment, the adenovirus is oncolytic. In one embodiment, the adenovirus is chimeric and oncolytic. In one embodiment, the adenovirus is capable of replication. In one embodiment, the adenovirus is chimeric, oncolytic, and capable of replication. In one embodiment, the adenovirus is replication-competent. In another embodiment, the adenovirus is chimeric, oncolytic, and replication-competent. In one embodiment, the adenovirus is replication-defective, i.e., a vector.

[0050] In one embodiment, the BX comprises a transgene or transgene cassette, in particular, a transgene cassette encoding a BiTE. In one embodiment, the additional transgene is under the control of an exogenous promoter, such as a CMV promoter.

[0051] In some embodiments, the use of an exogenous promoter may be advantageous because the exogenous promoter can strongly and constitutively express the antibody or fragment, which may be particularly useful in certain situations, such as when the patient has severe, diffuse cancer. Advantageously, the use of a constitutive exogenous promoter results in continuous transcription of the transgene, which may be desirable in certain situations.

[0052] In one embodiment, the transgene cassette includes a Kozak sequence, for example at the beginning of the coding sequence, specifically at the L5 end of the transgene cassette.

[0053] In one embodiment, the transgene cassette further comprises a polyadenylation sequence, for example at the end of the sequence, specifically at the end of the E4 region of the transgene cassette.

[0054] In one embodiment, the transgene cassette has Figure 1 Arrangement shown, as for virus NG-641.

[0055] In one embodiment, the BiTE molecule has a short half-life, eg, 48 hours or less.

[0056] In one embodiment, the adenovirus contains only one BiTE.

[0057] In another embodiment, the adenovirus contains two BiTEs.

[0058] In one embodiment, a FAP-BiTE comprises a VH domain comprising the amino acid sequence as set forth in SEQ ID NO: 11, or an amino acid sequence that is at least 95% identical thereto.

[0059] In one embodiment, a FAP-BiTE comprises a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 10, or an amino acid sequence that is at least 95% identical thereto.

[0060] In one embodiment, the anti-CD3 portion of a FAP-BiTE comprises a VH domain comprising the amino acid sequence set forth in SEQ ID NO: 6, or an amino acid sequence that is at least 95% identical thereto.

[0061] In one embodiment, the anti-CD3 portion of a FAP-BiTE comprises a VL domain comprising the amino acid sequence set forth in SEQ ID NO: 7, or an amino acid sequence at least 95% identical thereto.

[0062] In one embodiment, the adenovirus according to the present disclosure comprises the sequence set forth in SEQ ID NO: 34 or 35, or a polynucleotide encoding the same scFv amino acid sequence, specifically SEQ ID NO: 34.

[0063] In one embodiment, the adenovirus according to the present disclosure comprises the sequence set forth in SEQ ID NO: 68, SEQ ID NO: 69, or a polynucleotide encoding the same amino acid sequence.

[0064] In one embodiment, the adenovirus according to the present disclosure comprises the sequence set forth in SEQ ID NO: 90 or a polynucleotide encoding the same amino acid sequence.

[0065] In one embodiment, the adenovirus according to the present disclosure comprises the sequence set forth in SEQ ID NO: 91 or a polynucleotide encoding the same amino acid sequence.

[0066] In one embodiment, the adenovirus according to the present disclosure comprises the sequence set forth in SEQ ID NO: 92 or a polynucleotide encoding the same amino acid sequence.

[0067] The skilled artisan recognizes that redundancy exists in the DNA code, and therefore the present disclosure extends to EnAd or Ad11 encoding a BiTE having the amino acids disclosed herein.

[0068] A C-terminal His affinity tag (such as a deca or hexa-his tag) can be used for purification of BiTEs or adenoviruses. However, it is optional and can be excluded, for example, from the final product. The skilled artisan will recognize that other affinity tags besides deca-His can be used and that these tags can also be excluded without affecting the biological function of the BiTE or adenovirus.

[0069] Thus, in one embodiment, the BiTE comprises an amino acid sequence as set forth in SEQ ID NO: 1 or 2, but does not include a His affinity tag at the C-terminal end of the sequence, such as SEQ ID NO: 61 or 62.

[0070] The exclusion of the deca-His affinity tag further extends to all other sequences disclosed herein that include a deca-His affinity tag, ie the disclosure encompasses the same amino acid or DNA sequence lacking a C-terminal His tag.

[0071] In one aspect, a composition comprising an adenovirus as described herein and a diluent or carrier is provided.

[0072] In one aspect, a method of treating a patient is provided, comprising administering a therapeutically effective amount of an adenovirus or composition as described herein.

[0073] In one embodiment, the method is used to treat cancer, such as epithelial cancer, particularly solid tumors.

[0074] In one embodiment, a method of treatment is provided, comprising administering a virus according to the present disclosure in combination with a checkpoint inhibitor (such as a PD-1 or PDL1 inhibitor), particularly wherein the checkpoint inhibitor is encoded in the virus.

[0075] In one embodiment, a method of treatment is provided comprising administering a virus according to the present disclosure, not in combination with a checkpoint inhibitor (e.g., those listed elsewhere herein, such as a PD-1 or PDL1 inhibitor), specifically wherein the checkpoint inhibitor is not encoded in the virus.

[0076] According to the present disclosure, BiTEs encoded by viruses have the ability to enhance viral cytotoxicity.

[0077] Surprisingly, the virally encoded BiTe according to the present disclosure can activate CD4+ cells and / or CD8+ cells, such as T cells even in the suppressive environment of a tumor, such as in the tumor-containing fluid environment such as ascites.

[0078] Advantageously, the virally encoded BiTe according to the present disclosure can activate cytotoxic T cells, for example, even in the suppressive environment of a tumor, such as in a tumor-containing fluid environment such as ascites.

[0079] Even more surprisingly, the virus-encoded BiTEs according to the present disclosure are able to stimulate (activate) T cell proliferation.

[0080] The BiTE-encoding viruses according to the present disclosure appear to be able to bypass, overcome, or reverse the immunosuppressive microenvironment of tumors.

[0081] In one embodiment, activation of T cells results in upregulation of T cell markers, such as CD25. DETAILED DESCRIPTION

[0082] As used herein, immune cells are cells that have a functional role in the immune system, including but not limited to macrophages, neutrophils, dendritic cells, NK cells, lymphocytes such as T lymphocytes (particularly T cells and NKT cells).

[0083] As used herein, an antigen binding site refers to the portion of a molecule that includes a pair of variable regions, particularly a cognate pair that specifically interacts with a target antigen.

[0084] Specifically, as used herein, it is intended to refer to a binding site that recognizes only its specific antigen or a binding site that has a significantly higher binding affinity for the antigen than for an antigen for which it is nonspecific, for example, a binding affinity that is 5-fold, 6-fold, 7-fold, 8-fold, 9-fold, 10-fold higher. Affinity can be measured by techniques such as BIAcore.

[0085] As used herein, a bispecific antibody molecule refers to a molecule having two antigen binding domains, which can bind to the same or different antigens. BiTEs are a subclass of bispecific antibody molecules.

[0086] As used herein, BITE refers to a bispecific T cell engager, specifically comprising an anti-CD3 binding domain and an additional binding domain, in this case an anti-FAP binding domain. Typically, the binding domain is in the form of an scFv. Figure 12 A schematic diagram of BiTE is shown in FIG.

[0087] Therefore, as used herein, bispecific T cell adapter (BiTE) refers to a class of artificial bispecific monoclonal antibodies, which include different antibodies of 2 scFv or the amino acid sequences of 4 different genes on a single peptide chain of about 55KDa. One of them is scFv specific to immune cells, such as T cell antigens expressed on the surface of T cells, such as CD3 receptors. Under the prior art, another scFv is usually combined with tumor cells by tumor-specific molecules. Therefore, BiTE can be formed between T cells and tumor cells by its specificity for the antigens on T cells and tumor cells. This causes the activation of T cells and triggers T cells to play its cytotoxic effect on tumor cells, and has nothing to do with MHC I or costimulatory molecules.

[0088] In one embodiment, the T cell engager is arranged in the format of VL1-Linker 1-VH1-Linker 2-VH2-Linker 3-VL2, for example using linkers independently selected from the linker sequences disclosed herein.

[0089] In one embodiment, the BITE linker ranges from 10 to 30 amino acids in length, such as 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30, such as the linkers disclosed herein.

[0090] As used herein, matrix or matrix antigen refers to an antigen therapeutic target in the matrix, which is contained in the molecular structure of the matrix matrix and is expressed, such as connective tissue molecules or molecules related to this matrix or antigens related to the cellular components of the matrix, such as fibroblasts, tumor-associated macrophages, dendritic cells, NK cells and / or T cells that have infiltrated the matrix. Examples of matrix antigens include but are not limited to FAP, TGFβ, TREM1, IGFBP7, FSP-1, fibroblast-associated antigens, NG2, endosialin (CD248), platelet-derived growth factor alpha receptor (PDGFR-α), platelet-derived growth factor-β receptor (PDGFR-β) and vimentin. Typically, matrix antigens are not expressed on cancer cells, that is, they are only expressed on stromal cells.

[0091] Fibroblasts can be targeted by employing the antigen fibroblast activation protein (FAP), specifically FAP-specific antibodies that do not bind CD26 (see US 2012 / 0258119, incorporated herein by reference).

[0092] FAP was originally identified as a serine protease on reactive stromal fibroblasts. Subsequent molecular cloning revealed that FAP is identical to seprase, a 170 kDa membrane-associated gelatinase expressed by melanoma cell lines. The full-length cDNA encodes a 760 amino acid (aa) H-type transmembrane protease that is highly homologous to dipeptidyl peptidase IV (DPPIV), sharing 52% identity across the entire sequence and almost 70% identity in the catalytic domain. US Pat. No. 5,587,299, incorporated herein by reference, describes nucleic acid molecules encoding FAP and their uses.

[0093] In summary, FAP is considered a multifunctional protein that carries out its biological functions in a cell-dependent manner through a combination of its protease activity and its ability to form complexes with other cell surface molecules. Overexpression of FAP in epithelial and fibroblast cell lines promotes malignant behavior, pointing to clinical scenarios in which cellular expression levels of FAP correlate with worse clinical outcomes.

[0094] Through paracrine signaling molecules, cancer cells activate stromal fibroblasts and induce FAP expression, which in turn affects cancer cell proliferation, invasion, and migration. Recent studies have demonstrated that TGF-β is a major factor promoting FAP protein expression (Chen, H et al., (2009), Exp and Molec Pathology, doi: 10.1016 / j.yexmp.2009.09.001). FAP is abundantly expressed in reactive stromal fibroblasts in 90% of human epithelial cancers, including breast, lung, colon, and ovarian cancers (Garin-Chesa, P et al., (1990), PNAS USA, 87:7236-7239). Chen et al. recently showed that FAPα affects the invasion, proliferation and migration of HO-8910PM ovarian cancer cells (Chen, H et al. (2009), Experimental and Molecular Pathology, doi: 10.1016 / j.yexmp.2009.09.001).

[0095] FAP can be targeted by binding to the antigen and sterically blocking its interaction with biologically relevant molecules. Alternatively, or in addition, FAP molecules can be cross-linked to another FAP molecule or a different molecule, for example to T cells. This cross-linking increases the visibility of FAP-bearing cells to the immune system, which can then be activated to neutralize or destroy the immune system.

[0096] The adenoviruses disclosed herein have the ability to infect tumor cells and specifically preferentially infect tumor cells. Oncolytic viral infection causes the death and lysis of cancer cells, releasing newly generated viral particles. Incorporated transgenes, such as BiTEs and cytokines, are synthesized in the cells and actively secreted by the tumor cells prior to their death. Several molecules are also released upon cell lysis.

[0097] Antibody molecules with short half-lives, such as BiTEs, may be particularly suitable for use in the present disclosure as this minimizes off-target effects since the body will quickly clear the molecule if it becomes systemically available.

[0098] Thus, adenoviruses according to the present disclosure possess at least two or three mechanisms to attack tumors, including an indirect mechanism that destroys the tumor stroma.

[0099] As used herein, a transgenic refers to a gene that has been inserted into a genomic sequence that is not natural to the virus (exogenous) or a gene that is not normally found in a specific location in the virus. Examples of transgenics are known in the art and are discussed herein. As used herein, a transgenic also includes a functional fragment of a gene, which is a portion of a gene that, when inserted, is adapted to perform the function or a substantial portion of the function of the full-length gene.

[0100] Unless the context indicates otherwise, transgenic and coding sequence are used interchangeably in the context of insertion into a viral genome. As used herein, coding sequence refers to, for example, a DNA sequence that encodes a functional RNA, peptide, polypeptide, or protein. Typically, a coding sequence is a cDNA that encodes a transgenic functional RNA, peptide, polypeptide, or protein of interest. Functional RNAs, peptides, polypeptides, and proteins of interest are described below.

[0101] Obviously, the viral genome contains coding sequences of DNA. In the context of this specification, endogenous (naturally occurring genes) in the viral genomic sequence are not considered to be transgenic unless they are subsequently modified by recombinant technology so that they are in a non-natural location or in a non-natural environment.

[0102] In one embodiment, as used herein, a transgene refers to a DNA segment containing a gene or cDNA sequence that has been isolated from one organism and introduced into a different organism, i.e., a virus of the present disclosure. In one embodiment, this non-natural DNA segment can retain the ability to produce functional RNA, peptides, polypeptides, or proteins.

[0103] Thus, in one embodiment, the inserted transgene encodes a human or humanized protein, polypeptide, or peptide.

[0104] As used herein, operably linked means that the transgene is associated with the necessary regulatory elements to render the gene functional, ie, to allow the cellular machinery to express the gene once the virus enters the cell.

[0105] In one or more embodiments, the cartridge is arranged as shown in one or more of the Figures or Examples.

[0106] As used herein, a transgene cassette refers to a DNA sequence encoding one or more transgenes in the form of one or more coding sequences and one or more regulatory elements.

[0107] The transgene cassette may encode one or more monocistronic and / or polycistronic mRNA sequences.

[0108] In one embodiment, the transgene or transgene cassette encodes a monocistronic or polycistronic mRNA and, for example, the cassette is suitable for insertion into the adenoviral genome at a position under the control of an endogenous promoter or an exogenous promoter, or a combination thereof.

[0109] As used herein, monocistronic mRNA refers to an mRNA molecule that encodes a single functional RNA, peptide, polypeptide, or protein.

[0110] In one embodiment, the transgene cassette encodes a monocistronic mRNA.

[0111] In one embodiment, a transgene cassette, in the context of a cassette encoding a monocistronic mRNA, refers to a DNA segment optionally containing an exogenous promoter (which is a regulatory sequence that will determine when and where the transgene is active) or splice sites (which are regulatory sequences that determine when the mRNA molecule will be cleaved by the spliceosome) coding sequence (i.e., the transgene), typically derived from the cDNA of the protein of interest, optionally containing a polyA signal sequence and a terminator sequence.

[0112] In one embodiment, the transgene cassette may encode one or more polycistronic mRNA sequences.

[0113] As used herein, a polycistronic mRNA refers to an mRNA molecule that encodes two or more functional RNAs, peptides or proteins, or a combination thereof. In one embodiment, the transgene cassette encodes a polycistronic mRNA.

[0114] In one embodiment, in the context of a cassette encoding a polycistronic mRNA, a transgene cassette comprises a DNA segment optionally containing an exogenous promoter (which is a regulatory sequence that determines when and where the transgene is active) or splice sites (which are regulatory sequences that determine when the mRNA molecule will be cleaved by the spliceosome), two or more coding sequences (i.e., transgenes), typically derived from the cDNA of a protein or peptide of interest, for example, wherein each coding sequence is separated by an IRES or 2A peptide. Following the last coding sequence to be transcribed, the cassette may optionally contain a polyA sequence and a terminator sequence.

[0115] In one embodiment, the transgene cassette encodes a monocistronic mRNA followed by a polycistronic mRNA. In another embodiment, the transgene cassette encodes a polycistronic mRNA followed by a monocistronic mRNA.

[0116] In one embodiment, the adenovirus is a human adenovirus. As used herein, "adenovirus," "serotype," or "adenovirus serotype" refers to any adenovirus that can be assigned to any of the more than 50 currently known adenovirus serotypes, which are classified into subgroups A to F, and further extends to any adenovirus serotype that has not yet been identified or classified. See, for example, Strauss, "Adenovirus infections in humans," in Adenoviruses, Ginsberg, ea., Plenum Press, New York, NY, USA, pp. 451-596 (1984) and Shenk, "Adenoviridae: The Viruses and Their Replication," in Field of Virology, Vol. 2, No. 4, Knipe, 35 ea., Lippincott Williams and Wilkins, pp. 2265-2267 (2001), as shown in Table 1.

[0117] Table 1

[0118]

[0119]

[0120] In one embodiment, the adenovirus of the present disclosure is a subgroup B virus, ie, Ad11, specifically Ad11p (Slobitski strain) and its derivatives, such as EnAd.

[0121] Adenoviruses are designated to their group / serotype based on the capsid, such as hexon and / or fiber.

[0122] In one embodiment, the adenovirus of the present disclosure is not a Group A, Group C, Group D, Group E, or Group F virus. The viruses of the present disclosure do not include an adenovirus death protein.

[0123] In one embodiment, the adenovirus of the present disclosure is chimeric. When the adenovirus is chimeric, the characteristics of the outer capsid are used to determine the serotype. As used herein, a chimera refers to a virus that includes DNA from at least two different viral serotypes, including different serotypes within the same group.

[0124] In one embodiment, the oncolytic virus has fiber, hexon and penton proteins from the same serotype, such as Ad11, specifically Ad11p, for example, found at positions 30812 to 31789, 18254 to 21100 and 13682 to 15367 of the latter's genome sequence, where the nucleotide positions are relative to genbank ID 217307399 (accession number: GC689208).

[0125] In one embodiment, the adenovirus is enadenotucirev (also referred to as EnAd, and formerly known as EnAd). Enadenotucirev as used herein refers to a chimeric adenovirus of SEQ ID NO: 28. It is a replicating oncolytic chimeric adenovirus that has enhanced therapeutic properties compared to wild-type adenovirus (see WO2005 / 118825). EnAd has a chimeric E2B region that has DNA from Ad11p and Ad3, as well as a deletion in E3 / E4. The structural changes in enadenotucirev result in a genome that is approximately 3.5 kb smaller than Ad11p, thereby providing additional "space" for the insertion of transgenes. In EnAd, almost all of the E3 and E4 regions are missing. Therefore, it has significant space in the genome to accommodate additional genetic material while maintaining viability. In addition, because EnAd is a subgroup B adenovirus, pre-existing immunity in humans is not as common as, for example, Ad5. Other examples of chimeric oncolytic viruses with Ad11 fiber, penton, and hexon include OvAd1 and OvAd2 (see WO 2008 / 080003, incorporated by reference). Thus, in one embodiment, the adenovirus employed is OvAd1 or OvAd2.

[0126] EnAd appears to preferentially infect tumor cells, rapidly replicate within them, and cause cytolysis. This, in turn, can generate an inflammatory immune response, thereby stimulating the body to fight the cancer. Part of EnAd's success is hypothesized to be related to the rapid viral replication in the body.

[0127] Advantageously, carrying viruses with DNA encoding certain proteins such as BiTEs that can be expressed within cancer cells can enable the body's own defenses to be more effectively deployed against tumor cells, for example by making the cells more visible to the immune system or by preferentially delivering therapeutic genes / proteins to target tumor cells.

[0128] It is important that the expression of the transgene does not adversely affect viral replication or other beneficial properties. Therefore, the gene or genes must be inserted in a location that does not compromise the viral replication and other beneficial properties. In addition, the adenoviral genome is densely packed, making it difficult to find a suitable location for transgene insertion. This also limits the size of the transgene that can be accommodated.

[0129] As used herein, an oncolytic adenovirus is an adenovirus that preferentially kills cancer cells over non-cancerous cells. In one embodiment, the oncolytic virus is apoptotic. That is, it accelerates programmed cell death.

[0130] In one embodiment, the oncolytic virus is cytolytic. The cytolytic activity of the oncolytic adenovirus disclosed herein can be determined in representative tumor cell lines, and the data is converted into a measurement of efficacy, for example, using adenoviruses belonging to subgroup C such as Ad5 as a standard (ie, efficacy is 1). A suitable method for determining cytolytic activity is MTS determination (see Example 4, FIG2 of WO2005 / 118825, which is incorporated herein by reference).

[0131] In one embodiment, the oncolytic virus is necrotic. That is, it causes or accelerates cell necrosis or immunogenic cell death. In one embodiment, necrotic cell death is advantageous because it triggers and induces the patient's (host) immune response.

[0132] Unless the context indicates otherwise, adenovirus as used herein refers to viruses that are capable of replication (ie, replication-competent viruses) as well as replication-defective viral vectors.

[0133] Capable of replication as used herein refers to a replicative virus or a virus whose replication is dependent on factors in cancer cells, such as up-regulating factors such as p53 or the like.

[0134] In one embodiment, the virus is replicating. Functional replication in the context of this specification refers to a virus that has all the necessary machinery to replicate in cells in vitro and in vivo, i.e., without the assistance of a packaging cell line. For example, a viral vector that is deleted in the E1 region and capable of replicating in a complementing packaging cell line is not a replicating virus in this context.

[0135] Viral vectors are replication-defective and require the packaging cells to provide a complementary gene to allow replication.

[0136] As used herein, the adenoviral genome refers to the DNA sequence encoding the structural proteins and elements associated with the function / life cycle of the adenovirus.

[0137] All human adenovirus genomes checked so far have the same general organization, that is, the gene encoding a specific function is located in the same position (referred to as structural element in this article) in the viral genome. Each end of the viral genome has a short sequence called inverted terminal repeat (or ITR), which is necessary for viral replication. The viral genome contains five early transcription units (E1A, E1B, E2, E3 and E4), three delayed early units (IX, IVa2 and E2 late) and is processed to generate a late unit (main late) of five late mRNA families (L1 to L5). The protein encoded by the early genes mainly participates in the duplication and regulation of the host cell's response to infection, and the late genes encode viral structural proteins. The early genes are prefixed with the letter E, and the late genes are prefixed with the letter L.

[0138] The adenovirus genome is tightly packed, meaning that there is little non-coding sequence, and therefore finding a suitable location to insert a transgene is difficult.

[0139] In one embodiment, an oncolytic virus or a portion of an oncolytic virus according to the present disclosure may be the result of a deletion in the E4 and / or E3 regions, such as a deletion in a portion of the E4 region or a complete deletion in the E3 region, or alternatively a deletion in a portion of the E4 region (such as E4 or f4) and a complete deletion in the E3 region, such as exemplified in the sequences disclosed herein.

[0140] In one embodiment, the oncolytic virus is EnAd or an active derivative thereof that retains the essential beneficial properties of the virus. EnAd is disclosed in WO2005 / 118825 (incorporated herein by reference) and the complete sequence of the virus is provided herein as SEQ ID NO: 28. The chimeric E2B region is disclosed herein as SEQ ID NO: 60.

[0141] Advantageously, the adenoviruses of the present disclosure exhibit similar viral activities, such as replication and / or infectivity, as EnAd after infecting a variety of different colon cancer cell lines in vitro.

[0142] Structural elements of adenovirus

[0143] Because adenovirus structures are generally similar, the elements discussed below are based on structural elements and reference to common terms, which are known to those skilled in the art. When referring to an element in this article, we are referring to the DNA sequence encoding the element or the DNA sequence encoding the same structural protein of the element in adenovirus. The latter is relevant because of the redundancy of the DNA code. It may be necessary to consider the virus's preference for codon usage to achieve optimal results.

[0144] Any structural element from the adenovirus employed in the viruses of the present disclosure may include or consist of a native sequence, or may have at least 95% similarity over a given length, such as 96%, 97%, 98%, 99%, or 100%. The original sequence may be modified to omit 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the genetic material. The skilled artisan will appreciate that when making changes, the viral reading frame must not be disrupted in order to disrupt expression of the structural proteins.

[0145] In one embodiment, a given element is a full-length sequence, ie, a full-length gene.

[0146] In one embodiment, a given element is less than the full length and retains the same or corresponding function as the full length sequence.

[0147] In one embodiment, for a given element optionally in the constructs of the present disclosure, the DNA sequence may be less than full length and non-functional.

[0148] The structural genes encoding the structural or functional proteins of adenoviruses are typically linked by non-coding regions of DNA. Thus, in order to insert a transgene into the viruses of the present disclosure, there is some flexibility in where to "cut" the genomic sequence of the structural element of interest (particularly its non-coding region). Thus, for the purposes of this specification, the elements will be considered to be reference structural elements to the extent that they are appropriate for the purpose and do not encode foreign material. Thus, if appropriate, the genes will be associated with appropriate non-coding regions, such as those found in the native structure of the virus.

[0149] Thus, in one embodiment, the insert (e.g., DNA encoding restriction sites and / or transgenes) is inserted into a non-coding region of the genomic viral DNA, such as an intron or intergenic sequence. As already mentioned, some non-coding regions of adenovirus may have functions, such as in alternative splicing, transcriptional regulation, or translational regulation, and this may need to be taken into account.

[0150] The sites identified herein associated with the L5 region (eg, between the L5 and E4 regions) are suitable for accommodating various DNA sequences encoding complex entities, such as RNAi, cytokines, single-chain or multimeric proteins, such as antibodies, such as BiTEs.

[0151] As used herein, a gene refers to a coding region and optionally any non-coding sequences associated therewith, such as introns and associated exons. In one embodiment, a gene includes or consists only of the essential structural components, such as the coding region.

[0152] Following is a discussion of specific structural elements of adenovirus.

[0153] The inverted terminal repeat (ITR) sequences are common to all known adenoviruses and are so named due to their symmetry and serve as the origin of viral chromosomal replication.Another property of these sequences is their ability to form hairpins.

[0154] As used herein, a 5' ITR refers to part or all of the ITR from the 5' end of an adenovirus that retains ITR function when incorporated into an adenovirus at the appropriate position. In one embodiment, the 5' ITR comprises or consists of a sequence of about 1 bp to 138 bp of SEQ ID NO: 28, or a sequence that is 90%, 95%, 96%, 97%, 98%, or 99% identical thereto along its entire length, specifically a sequence consisting of about 1 bp to 138 bp of SEQ ID NO: 28.

[0155] As used herein, a 3' ITR refers to part or all of the ITR at the 3' end of an adenovirus that retains ITR function when incorporated into an adenovirus at the appropriate position. In one embodiment, the 3' ITR comprises or consists of a sequence from about 32189 bp to 32326 bp of SEQ ID NO: 28, or a sequence that is 90%, 95%, 96%, 97%, 98%, or 99% identical thereto along its entire length, specifically a sequence consisting of about 32189 bp to 32326 bp of SEQ ID NO: 28.

[0156] As used herein, B1 refers to a DNA sequence encoding part or all of E1A of an adenovirus, part or all of the E1B region of an adenovirus, and independently part or all of the E1A region and E1B region of an adenovirus.

[0157] When B1 is a bond, then the E1A and E1B sequences will be omitted from the virus. In one embodiment, B1 is a bond, and thus the virus is a vector.

[0158] In one embodiment, B1 further comprises a transgene. It is known in the art that the E1 region can accommodate a transgene, which can be disruptively inserted into the E1 region (i.e., in the "middle" of the sequence) or part or all of the E1 region can be deleted to provide more space to accommodate genetic material.

[0159] As used herein, E1A refers to a DNA sequence encoding part or all of the adenoviral E1A region. The latter is referred to herein as the polypeptide / protein E1A. It can be mutated such that the protein encoded by the E1A gene has conservative or non-conservative amino acid changes such that it has: the same function as the wild-type protein (i.e., the corresponding non-mutated protein); increased function compared to the wild-type protein; decreased function, such as no function compared to the wild-type protein; or a novel function compared to the wild-type protein, or a combination thereof, as appropriate.

[0160] As used herein, E1B refers to a DNA sequence encoding part or all of the adenovirus E1B region (i.e., a polypeptide or protein), which can be mutated so that the protein encoded by the E1B gene / region has conservative or non-conservative amino acid changes such that it has: the same function as the wild-type protein (i.e., the corresponding non-mutated protein); increased function compared to the wild-type protein; decreased function, such as no function compared to the wild-type protein, or a new function compared to the wild-type protein, or a combination thereof, as the case may be.

[0161] Thus, B1 may be modified or unmodified relative to a wild-type E1 region, such as wild-type E1A and / or E1B. A skilled person can easily identify whether E1A and / or E1B is present or (partially) missing or mutated.

[0162] As used herein, wild type refers to a known adenovirus. A known adenovirus is an adenovirus that has been identified and named, regardless of whether the sequence is available.

[0163] In one embodiment, B1 has a sequence of 139 bp to 3932 bp of SEQ ID NO:28.

[0164] As used in this paper, B A Refers to the DNA sequence encoding the E2B-L1-L2-L3-E2A-L4 region, including any non-coding sequences, as appropriate. Typically, this sequence will not include a transgene. In one embodiment, the sequence is substantially similar or identical to a contiguous sequence from a known adenovirus, such as the serotypes shown in Table 1, specifically a Group B virus, such as Ad3, Ad7, Ad11, Ad14, Ad16, Ad21, Ad34, Ad35, Ad51, or a combination thereof, such as Ad3, Ad11, or a combination thereof. In one embodiment, E2B-L1-L2-L3-E2A-L4 refers to a region comprising these elements and other structural elements associated with the region, such as B. A It will typically contain a sequence encoding protein IV2a, for example as follows: IV2AIV2a-E2B-L1-L2-L3-E2A-L4.

[0165] In one embodiment, the E2B region is chimeric. That is, it includes DNA sequences from two or more different adenovirus serotypes, for example, Ad3 and Ad11, such as Ad11p. In one embodiment, the E2B region comprises the sequence of 5068 bp to 10355 bp of SEQ ID NO: 28 over its entire length, or a sequence that is 95%, 96%, 97%, 98%, or 99% identical thereto.

[0166] In one embodiment, component B A The E2B in comprises the sequence shown in SEQ ID NO: 60 (which corresponds to SEQ ID NO: 3 disclosed in WO2005 / 118825).

[0167] In one embodiment, B A It has a sequence of 3933 bp to 27184 bp of SEQ ID NO: 28.

[0168] As used herein, E3 refers to a DNA sequence encoding part or all of the adenovirus E3 region (i.e., a protein or polypeptide), which can be mutated so that the protein encoded by the E3 gene has conservative or non-conservative amino acid changes, so that it has: the same function as the wild type (the corresponding non-mutated protein); increased function compared to the wild-type protein; decreased function, such as no function compared to the wild-type protein or a new function compared to the wild-type protein or a combination thereof, as the case may be.

[0169] In one embodiment, the E3 region forms an adenovirus serotype given in Table 1 or a combination thereof, in particular a group B serotype, for example Ad3, Ad7, Ad11 (in particular Ad11p), Ad14, Ad16, Ad21, Ad34, Ad35, Ad51 or a combination thereof, such as Ad3, Ad11 (in particular Ad11p) or a combination thereof.

[0170] In one embodiment, the E3 region is partially deleted, for example, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5% deleted.

[0171] In one embodiment, B2 is a bond, wherein DNA encoding the E3 region is not present.

[0172] In one embodiment, the DNA encoding the E3 region can be replaced or interrupted by a transgene. As used herein, "replacing the E3 region with a transgene as used herein" includes replacing part or all of the E3 region with a transgene.

[0173] In one embodiment, region B2 includes the sequence from 27185 bp to 28165 bp of SEQ ID NO:28.

[0174] In one embodiment, B2 consists of the sequence from 27185 bp to 28165 bp of SEQ ID NO:28.

[0175] As used in this paper, B X Refers to B B The DNA sequence near the 5' end of the L5 gene in B. As used herein, near or proximal to the 5' end of the L5 gene refers to: a non-coding region adjacent to (adjacent to) the 5' end of the L5 gene or inherently associated with it, i.e., a non-coding region that is immediately adjacent to or adjacent to the 5' major end of the L5 gene or inherently associated with it. Alternatively, near or proximal may refer to a closed L5 gene such that in B X There is no coding sequence between the 5' end of the L5 gene and the 5' end of the L5 gene.

[0176] Therefore, in one embodiment, B XDirectly linked to the base of L5, it represents the start of the coding sequence of, for example, the L5 gene.

[0177] Therefore, in one embodiment, B X A base directly linked to L5, which represents, for example, the start of a non-coding sequence, or directly linked to a non-coding region naturally associated with L5. As used herein, a non-coding region naturally associated with L5 refers to a portion of all non-coding regions that are part of the L5 gene or are adjacent to it but not part of another gene.

[0178] In one embodiment, B X The sequence comprising SEQ ID NO: 29. This sequence is an artificial non-coding sequence into which a DNA sequence can be inserted, for example, including a transgene (or transgene cassette), a restriction site, or a combination thereof. This sequence is advantageous because it acts as a buffer, allowing for some flexibility in the exact location of the transgene while minimizing disruptive effects on viral stability and viability.

[0179] One or more insertions may occur at any position of SEQ ID NO: 29 at the 5' end, the 3' end, or at any position between bp 1 and 201. In one embodiment, BX comprises SEQ ID NO: 29, wherein the DNA sequence is inserted between bp 27 and bp 28 or at a position corresponding to positions 28192 bp and 28193 bp of SEQ ID NO: 28.

[0180] In one embodiment, the insert is a restriction site insert. In one embodiment, the restriction site insert includes one or two restriction sites. In one embodiment, the restriction site is a 19bp restriction site insert including 2 restriction sites. In one embodiment, the restriction site insert is a 9bp restriction site insert including 1 restriction site. In one embodiment, the restriction site insert includes one or two restriction sites and at least one transgene, such as one or two transgenes. In one embodiment, the restriction site is a 19bp restriction site insert including 2 restriction sites and at least one transgene, such as one or two transgenes. In one embodiment, the restriction site is a 9bp restriction site insert including 1 restriction site and at least one transgene, such as one, two or three transgenes, such as one or two. In one embodiment, two restriction sites are sandwiched between one or more, such as two transgenes (e.g., in a transgenic cassette). In one embodiment, when B X When two restriction sites are included, the restriction sites are different from each other. In one embodiment, B XThe one or more restriction sites in the B are non-naturally occurring in the particular adenoviral genome into which they have been inserted. X The one or more restriction sites in the adenoviral vector are different from other restriction sites located elsewhere in the adenoviral genome, for example, from naturally occurring restriction sites and / or restriction sites introduced into other parts of the genome, such as those introduced into B Y Thus, in one embodiment, one or more restriction sites allow for specific cleavage of the DNA in the segment.

[0181] As used in this paper, B Y The related DNA sequence is B B The DNA sequence near the 3' end of the L5 gene of the present invention. As used herein, near or proximal to the 3' end of the L5 gene refers to: the non-coding region adjacent to (adjacent to) the 3' end of the L5 gene or inherently associated with it, i.e., the non-coding region that is close to or adjacent to the 3' major end of the L5 gene or inherently associated with it (i.e., all or part of the non-coding sequence endogenous to L5). Alternatively, near or proximal can refer to the closed L5 gene, such that in B Y There is no coding sequence between the L5 region and the 3' end of the L5 gene.

[0182] Therefore, in one embodiment, B Y The base directly connected to L5 represents the "end" of the coding sequence.

[0183] Therefore, in one embodiment, B Y Directly linked to the base of L5, which represents the "end" of the non-coding sequence, or directly linked to the non-coding region naturally associated with L5.

[0184] Inherently and naturally are used interchangeably herein. In one embodiment, B Y The sequence comprising SEQ ID NO: 30. This sequence is a non-coding sequence into which a DNA sequence, for example, including a transgene (or transgene cassette), a restriction site, or a combination thereof, can be inserted. This sequence is advantageous because it acts as a buffer, allowing for some flexibility in the exact location of the transgene while minimizing disruptive effects on viral stability and viability.

[0185] One or more insertions may occur in SEQ ID NO:30 from the 5' end, the 3' end, or at any point between bp 1 and bp 35, for example, between base pairs 1 / 2, 2 / 3, 3 / 4, 4 / 5, 5 / 6, 6 / 7, 7 / 8, 8 / 9, 9 / 10, 10 / 11, 11 / 12, 12 / 13, 13 / 14, 14 / 15, 15 / 16, 16 / 17, 17 / 18, 18 / 19, 19 / 20, 20 / 21, 21 / 22, 22 / 23, 23 / 24, 24 / 25, 25 / 26, 26 / 27, 27 / 28, 28 / 29, 29 / 30, 30 / 31, 31 / 32, 32 / 33, 33 / 34, or 34 / 35.

[0186] As used herein, E4 refers to a DNA sequence encoding part or all of the adenovirus E4 region (i.e., a polypeptide or protein region), which can be mutated so that the protein encoded by the E4 gene has conservative or non-conservative amino acid changes and has: the same function as the wild-type protein (corresponding non-mutated protein); increased function compared to the wild-type protein; decreased function, such as no function compared to the wild-type protein or a new function compared to the wild-type protein or a combination thereof, as appropriate. In one embodiment, the E4 region is deleted from E4 or f4.

[0187] In one embodiment, the E4 region is partially deleted, for example, 95%, 90%, 85%, 80%, 75%, 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10% or 5% deleted. In one embodiment, the E4 region has a sequence of 32188 bp to 29380 bp of SEQ ID NO: 28.

[0188] In one embodiment, B3 is a bond, ie, wherein E4 is not present.

[0189] In one embodiment, B3 has a sequence consisting of 32188 bp to 29380 bp of SEQ ID NO:28.

[0190] As used herein, numerical ranges are inclusive.

[0191] The skilled artisan will understand that the elements in the formulae herein, such as formula (I), are continuous and may include non-coding DNA sequences as well as the genes and coding DNA sequences (structural features) mentioned herein. In one or more embodiments, the formulae disclosed herein are intended to describe naturally occurring sequences in the adenovirus genome. In this context, the skilled artisan will understand that the formulae refer to the major elements that characterize the relevant segments of the genome and are not intended to be an exhaustive description of the genomic stretch of DNA.

[0192] As used herein, E1A, E1B, E3 and E4 each independently refer to wild type and equivalents thereof, such as mutant or partially deleted forms of each region as described herein, specifically wild type sequences from known adenoviruses.

[0193] As used herein, "insert" refers to a DNA sequence incorporated at the 5' end, 3' end, or within a given DNA sequence reference fragment, such that it interrupts the reference sequence. The latter is a reference sequence used as a reference point relative to where the insert is located. In the context of the present disclosure, an insert typically appears within SEQ ID NO:29 or SEQ ID NO:30. The insert can be a restriction site insertion, a transgene cassette, or both. When the sequence is interrupted, the virus will still include the original sequence, but typically it will sandwich two fragments within the insert.

[0194] In one embodiment, the transgene or transgene cassette does not include a non-biased insertion transposon, such as the TN7 transposon or a portion thereof. As used herein, the Tn7 transposon refers to a non-biased insertion transposon as described in WO2006 / 060314.

[0195] In one embodiment, B X and B Y The one or more restriction sites in the B are independently selected from restriction sites specific for the enzymes described herein, such as NotI, FseI, AsiSI, SgfI and SbfI, and the specifically inserted restriction sites are different, such as the NotI specific site and the one located at B X The FseI specific site in B Y SgfI and SbfI in.

[0196] As discussed above in one embodiment, region B X and / or B Y No restriction sites are included. Advantageously, the viruses and constructs of the present disclosure can be prepared without restriction sites, for example, using synthetic techniques. These techniques provide great flexibility in creating viruses and constructs. Furthermore, the inventors have determined that when viruses and constructs are prepared by synthetic techniques, their properties are not degraded.

[0197] Other regulatory sequences

[0198] As used herein, "gene expression regulator" (or regulatory / control element) refers to a genetic feature such as a promoter, enhancer or splice acceptor sequence that plays a role in gene expression, typically by initiating or enhancing transcription or translation.

[0199] As used herein, "splice acceptor sequence," "splice acceptor," or "splice site" refers to a regulatory sequence that determines when an mRNA molecule will be recognized by the small nuclear ribonucleoproteins of the spliceosomal complex. Once assembled, the spliceosome catalyzes splicing between the splice acceptor site and the upstream splice donor site of the mRNA molecule, thereby producing a mature mRNA molecule that can be translated to produce a single polypeptide or protein.

[0200] Different sized splice acceptor sequences may be employed in the present invention, and these may be described as short splice acceptor (small), splice acceptor (medium) and branched splice acceptor (large).

[0201] As used herein, SSA refers to a short splice acceptor, typically including only a splice site, e.g., 4 base pairs. As used herein, SA refers to a splice acceptor, typically including a short splice acceptor and a polypyrimidine tract, e.g., 16 bp. As used herein, bSA refers to a branched splice acceptor, typically including a short splice acceptor, a polypyrimidine tract, and a branch point, e.g., 26 base pairs.

[0202] In one embodiment, the splice acceptors SA and bSA employed in the constructs of the present disclosure are shown in SEQ ID NOs: 45 and 46, respectively. In one embodiment, SSA is used in a cassette according to the present disclosure and has the nucleotide sequence CAGG.

[0203] In one embodiment, SA is used in the cassette. In one embodiment, bSA is used in the cassette.

[0204] In one embodiment, the splice site is immediately preceded by a consensus Kozak sequence (i.e., proceeding in the 5' to 3' direction). In one embodiment, the splice site and the Kozak sequence are separated by up to 100 base pairs or fewer. In one embodiment, the Kozak sequence has the nucleotide sequence of SEQ ID NO: 47.

[0205] Typically, when under the control of an endogenous or exogenous promoter (such as an endogenous promoter), the coding sequence will be immediately followed by a Kozak sequence. The start of the coding region is indicated by a start codon (AUG), for example, in the context of the sequence (gcc)gccRccAUGg [SEQ ID NO: 48], the start of the coding sequence is indicated by the bases in bold. Lowercase letters represent the common bases at this position (although they can vary) and uppercase letters indicate highly conserved bases, i.e., the "AUGG" sequence is constant or rarely varies, if at all; "R" indicates that a purine (adenine or guanine) is usually observed at this position, and the sequence in brackets (gcc) has an uncertain meaning. Therefore, in one embodiment, the start codon AUG is incorporated into the Kozak sequence.

[0206] As used herein, an internal ribosome entry DNA sequence refers to a DNA sequence encoding an internal ribosome entry sequence (IRES). As used herein, an IRES means a nucleotide sequence that allows the initiation of translation of a messenger RNA (mRNA) sequence, comprising the start of initiation within the mRNA sequence. This is particularly useful when the cassette encodes a polycistronic mRNA. IRES is used to produce polycistronic mRNAs that are translated into multiple independent proteins or peptides. In one embodiment, the internal ribosome entry DNA sequence has the nucleotide sequence of SEQ ID NO:49. In one embodiment, a specific IRES is used only once in a genome. This may be beneficial to the stability of the genome.

[0207] As used herein, "high self-cleavage efficiency 2A peptides" or "2A peptides" refer to peptides that are efficiently cleaved after translation. Suitable 2A peptides include P2A, F2A, E2A, and T2A. The inventors have noted that once a specific DNA sequence encoding a given 2A peptide has been used once, the same specific DNA sequence may not be used again. However, the redundancy in the DNA code can be used to generate a DNA sequence that translates into the same 2A peptide. The use of 2A peptides is particularly useful when the cassette encodes a polycistronic mRNA. The use of 2A peptides results in a single translated polypeptide chain being modified after translation to generate multiple separate proteins or peptides.

[0208] In one embodiment, the encoded P2A peptide employed has an amino acid sequence of SEQ ID NO: 50. In one embodiment, the encoded F2A peptide employed has an amino acid sequence of SEQ ID NO: 51. In one embodiment, the encoded E2A peptide employed has an amino acid sequence of SEQ ID NO: 52. In one embodiment, the encoded T2A peptide employed has an amino acid sequence of SEQ ID NO: 53.

[0209] In one embodiment, the or each mRNA encoded by one or more transgenes includes a polyadenylation signal sequence, as typically found at the end of the mRNA sequence, for example as shown in SEQ ID NO: 54. Thus, one embodiment of a transgene or transgene cassette includes at least one sequence encoding a polyadenylation signal sequence.

[0210] As used herein, "Poly A," "polyadenylation signal," or "polyadenylation sequence" refers to a DNA sequence, typically containing an AATAAA site, which, once transcribed, is recognized by a multiprotein complex that cleaves and polyadenylates the nascent mRNA molecule.

[0211] In one embodiment, the polyadenylation sequence has the nucleotide sequence of SEQ ID NO:54.

[0212] In one embodiment, the construct does not comprise a polyadenylation sequence.In one embodiment, the regulator of gene expression is a splice acceptor sequence.

[0213] Advantageously, the adenoviruses of the present disclosure express and release antibody forms (such as BiTEs) and other proteins such as cytokines encoded by the transgenes therein into in vitro culture supernatants or into the tumor tissue matrix in vivo. The leader sequence can help the encoded protein / polypeptide or peptide leave the cancer cell. Therefore, in one embodiment, the encoded "protein" includes a leader sequence. As used herein, a leader sequence refers to a polynucleotide sequence located between the promoter sequence and the coding region, which can regulate gene expression at the transcriptional or translational level.

[0214] In one embodiment, an adenovirus according to the present disclosure includes a transgene that is a reporter gene encoding, for example, an imaging agent comprising bioluminescence, a fluorescent imaging agent (including an activatable fluorescent imaging agent), such as luciferase, GFP or eGFP, or red fluorescent protein.

[0215] As used herein, reporter gene or reporter sequence means the gene or DNA sequence of the product produced in eukaryotic cells that is easily detected, and can be used as a marker to determine the activity of another gene tightly connected or combined with its DNA. Reporter gene gives the characteristic or selectable marker of the cell or organism expressing them that is easy to identify and measure. Reporter gene is generally used as indicating whether a certain gene has been absorbed or expressed by cell or biological colony. Examples of common reporter genes include but are not limited to: LacZ, luciferase, GFP, eGFP, neomycin phosphotransferase, chloramphenicol acetyltransferase, sodium iodide symporter (NIS), nitroreductase (such as NfsA, NfsB) intracellular metalloproteinase, HSV1-tk or estrogen receptor.

[0216] In one embodiment, the genetic material (particularly the transgene) does not encode or express a reporter gene such as an imaging agent, luciferase, GFP or eGFP.

[0217] By examining the lytic potential in a panel of tumor cells, the preference of viruses according to the present disclosure for specific tumor types can be studied, for example, colon tumor cell lines including HT-29, DLD-1, LS174T, LS1034, SW403, HCT116, SW48, and Colo320DM. Any available colon tumor cell line is also useful for this assessment.

[0218] Prostate cell lines include DU145 and PC-3 cells. Pancreatic cell lines include Panc-1 cells. Breast tumor cell lines include the MDA231 cell line, and ovarian cell lines include the OVCAR-3 cell line. Hematopoietic cell lines include, but are not limited to, Raji and Daudi B lymphocytes, K562 erythroblastoid cells, U937 bone marrow cells, and HSB2 T lymphocytes. Other available tumor cell lines are also useful.

[0219] The present disclosure also extends to novel sequences disclosed herein.In one embodiment, the virus is shown in any one of the sequences disclosed herein.

[0220] formulations

[0221] The present disclosure also relates to pharmaceutical formulations of the viruses as described herein.

[0222] In one embodiment, a liquid parenteral formulation is provided, for example, for infusion or injection of an oncolytic replica according to the present disclosure, wherein the formulation provides a dose in the range of 1×10 10 to 1×10 14 Virus particles.

[0223] Parenteral formulations refer to formulations that are not designed to be delivered through the GI tract. Typical parenteral delivery routes include injection, implantation, or infusion. In one embodiment, the formulation is provided in the form of a bolus delivery.

[0224] In one embodiment, the parenteral formulation is in the form of an injection. Injection includes intravenous, subcutaneous, intratumoral, or intramuscular injection. As used herein, injection means injecting a liquid into the body via a syringe. In one embodiment, the methods of the present disclosure do not involve intratumoral injection.

[0225] In one embodiment, the parenteral formulation is in the form of an infusion.

[0226] As used herein, infusion refers to administering a fluid at a slower rate by drip, infusion pump, syringe driver, or equivalent device. In one embodiment, the infusion is administered over a period of 1.5 minutes to 120 minutes, such as about 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, 10 minutes, 11 minutes, 12 minutes, 13 minutes, 14 minutes, 15 minutes, 16 minutes, 17 minutes, 18 minutes, 19 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 60 minutes, 65 minutes, 70 minutes, 65 minutes, 80 minutes, 85 minutes, 90 minutes, 95 minutes, 100 minutes, 105 minutes, 110 minutes, or 115 minutes.

[0227] In one embodiment, a dose of the formulation is less than 100 ml, such as 30 ml, for example, when administered via a syringe driver. In one embodiment, a dose of the formulation is less than 10 ml, such as 9 ml, 8 ml, 7 ml, 6 ml, 5 ml, 4 ml, 3 ml, 2 ml, or 1 ml. In one embodiment, a dose of the formulation is less than 1 ml, such as 0.9 ml, 0.8 ml, 0.7 ml, 0.6 ml, 0.5 ml, 0.4 ml, 0.3 ml, 0.2 ml, or 0.1 ml.

[0228] In one embodiment, the injection is administered as a slow injection, for example, over a period of 1.5 minutes to 30 minutes.

[0229] In one embodiment, the formulation is for intravenous (iv) administration. This approach is particularly effective for the delivery of oncolytic viruses because they can quickly enter most organs and tissues and are particularly suitable for the treatment of metastases, such as established metastases, especially those located in highly vascularized areas such as the liver and lungs.

[0230] The therapeutic formulations are typically sterile and stable under the conditions of manufacture and storage. The compositions can be formulated as solutions, microemulsions, liposomes or other parenteral formulations suitable for administration to humans and can be formulated into prefilled devices such as syringes or vials, particularly as single doses.

[0231] The formulations typically include a pharmaceutically acceptable diluent or carrier, such as a non-toxic, isotonic carrier that is compatible with the virus and in which the virus is stable for a desired period of time.

[0232] The carrier can be a solvent or dispersion medium, and it contains for example water, ethanol, polyol (for example glycerol, propylene glycol and liquid polyethylene glycol etc.) and suitable mixture thereof.For example, by using a dispersant or surfactant, such as lecithin or such as nonionic surfactants such as polysorbate 80 or 40, suitable fluidity can be maintained.In dispersion, the required particle size can be assisted to maintain by the presence of a surfactant.The example of an isotonic agent comprises the sugar in the composition, such as polyols such as mannitol, sorbitol or sodium chloride.

[0233] In one embodiment, the parenteral formulation employed may include, for example, one or more of the following buffers: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, phosphate buffer and / or Tris buffer, a sugar such as dextrose, mannose, sucrose or the like, a salt such as sodium chloride, magnesium chloride or potassium chloride, a detergent such as a nonionic surfactant such as briji, PS-80, PS-40 or the like. The formulation may also include a preservative such as EDTA or ethanol or a combination of EDTA and ethanol, which is believed to prevent one or more potential degradation pathways.

[0234] In one embodiment, the formulation will include a purified oncolytic virus according to the present disclosure, for example, 1×10 10 to 1×10 14 Virus particles, such as 1×10 per dose 10 to 1×10 12 In one embodiment, the concentration of the virus in the formulation ranges from 2×10 8 vp / mL to 2×10 14 vp / mL, such as 2×10 12 vp / ml.

[0235] In one embodiment, the parenteral formulation includes glycerol.

[0236] In one embodiment, the formulation includes an oncolytic adenovirus as described herein, HEPES (N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid), glycerol, and a buffer.

[0237] In one embodiment, the parenteral formulation consists of a virus of the disclosure, eg, 5 mM HEPES, eg, 5% to 20% (v / v) glycerol, hydrochloric acid, eg, to adjust the pH to a range of 7 to 8, and water for injection.

[0238] In one embodiment, the concentration is 2×10 12 0.7 mL of the disclosed virus was formulated in 5 mM HEPES, 20% glycerol at a vp / mL with a final pH of 7.8.

[0239] A thorough discussion of pharmaceutically acceptable carriers can be found in Remington's Pharmaceutical Sciences (Mack Publishing Company, NJ, 1991).

[0240] In one embodiment, the formulation is provided as a formulation for topical administration involving inhalation.

[0241] Suitable inhalable formulations comprise inhalable powders, metered aerosols containing a propellant gas or inhalable solutions without a propellant gas.Inhalable powders according to the present disclosure typically contain a virus as described herein and a physiologically acceptable excipient.

[0242] These inhalable powders may comprise monosaccharides (e.g. glucose or arabinose), disaccharides (e.g. lactose, sucrose, maltose), oligosaccharides and polysaccharides (e.g. dextran), polyols (e.g. sorbitol, mannitol, xylitol), salts (e.g. sodium chloride, calcium carbonate) or mixtures thereof. Suitable monosaccharides or disaccharides are lactose or glucose, particularly but not limited to their hydrate forms.

[0243] Particles for deposition in the lungs need to be less than 10 microns in size, such as 1 to 9 microns, for example 0.1 μm to 5 μm, specifically 1 to 5 μm. The size of the virus-carrying particle is of primary importance, and therefore, in one embodiment, the virus according to the present disclosure can be adsorbed or absorbed onto particles, such as lactose particles of a given size.

[0244] Propellant gases that can be used to prepare inhalable aerosols are known in the art. Suitable propellant gases are selected from hydrocarbons, such as n-propane, n-butane or isobutane, and halogenated hydrocarbons, such as chlorinated and / or fluorinated derivatives of methane, ethane, propane, butane, cyclopropane or cyclobutane. The above-mentioned propellant gases can be used alone or in mixtures thereof.

[0245] Particularly suitable propellant gases are halogenated alkane derivatives selected from TG 11, TG 12, TG 134a and TG 227. Of the above halogenated hydrocarbons, TG 134a (1,1,1,2-tetrafluoroethane) and TG 227 (1,1,1,2,3,3,3-heptafluoropropane) and mixtures thereof are particularly suitable.

[0246] The inhalable aerosol containing the propellant gas may also contain other ingredients such as cosolvents, stabilizers, surfactants, antioxidants, lubricants and means of adjusting pH. All of these ingredients are known in the art.

[0247] The inhalable aerosol according to the invention containing a propellant gas may contain up to 5% by weight of active substance. The aerosol according to the invention may contain, for example, 0.002% to 5% by weight, 0.01% to 3% by weight, 0.015% to 2% by weight, 0.1% to 2% by weight, 0.5% to 2% by weight or 0.5% to 1% by weight of active ingredient.

[0248] Alternatively, local administration to the lungs can also be by administering a liquid solution or suspension formulation, for example, using a device such as a nebulizer, for example, a nebulizer connected to a compressor (e.g., the Pari LC-Jet Plus(R) nebulizer connected to a Pari Master(R) compressor manufactured by Pari Respiratory Equipment, Inc. of Richmond, Virginia).

[0249] Virus of the present invention can be dispersed in a solvent and sent, for example, in the form of a solution or suspension, for example, as described above for parenteral formulations. It can be suspended in a suitable physiological solution, for example, saline or other pharmacologically acceptable solvents or buffered solutions. Buffered solutions known in the art can contain 0.05mg to 0.15mg of disodium edetate, 8.0mg to 9.0mg NaCl, 0.15mg to 0.25mg polysorbate, 0.25mg to 0.30mg anhydrous citric acid and 0.45mg to 0.55mg sodium citrate / 1ml water, to reach a pH of approximately 4.0 to 5.0.

[0250] The therapeutic suspension or solution formulation may also contain one or more excipients. Excipients are well known in the art and include buffers (e.g., citrate buffer, phosphate buffer, acetate buffer, and bicarbonate buffer), amino acids, urea, alcohol, ascorbic acid, phospholipids, proteins (e.g., serum albumin), EDTA, sodium chloride, liposomes, mannitol, sorbitol, and glycerol. The solution or suspension may be encapsulated in liposomes or biodegradable microspheres. The formulation is typically provided in a substantially sterile form using aseptic manufacturing methods.

[0251] This may involve the production and sterilization of buffer solvents / solutions for the formulation by filtration, the sterile suspension of the antibody in the sterile buffer solvent solution, and dispensing the formulation into sterile containers by methods familiar to those of ordinary skill in the art.

[0252] The nebulizable formulations according to the present disclosure can be provided, for example, as single-dose units (e.g., sealed plastic containers or vials) packaged in foil envelopes. Each vial contains a unit dose in a volume, for example, 2 mL of solvent / solution buffer.

[0253] treat

[0254] In a further aspect, the disclosure relates to a virus or formulation thereof as described herein for use in therapy, particularly for the treatment of cancer.

[0255] In one embodiment, the therapeutic method is used to treat tumors, particularly solid tumors.

[0256] As used herein, a tumor refers to an abnormal mass of tissue caused by excessive cell division, which is uncontrolled and progressive, also known as a tumor. Tumors can be benign (non-cancerous) or malignant. Tumors encompass all forms of cancer and metastasis.

[0257] In one embodiment, the tumor is a solid tumor. Solid tumors can be localized or metastatic.

[0258] In one embodiment, the tumor is of epithelial origin.

[0259] In one embodiment, the tumor is a malignant tumor, such as colorectal cancer, liver cancer, prostate cancer, pancreatic cancer, breast cancer, ovarian cancer, thyroid cancer, kidney cancer, bladder cancer, head and neck cancer, or lung cancer.

[0260] In one embodiment, the tumor is a colorectal malignancy.

[0261] As used herein, malignant tumor refers to cancerous cells.

[0262] In one embodiment, an oncolytic adenovirus is used to treat or prevent metastasis.

[0263] In one embodiment, the methods or formulations herein are used to treat drug-resistant cancers.

[0264] In one embodiment, the virus is administered in combination with administration of a further cancer treatment or therapy.

[0265] In one embodiment, provided are viruses or formulations according to the present disclosure for the manufacture of a medicament for treating cancer, such as the cancers described above.

[0266] In a further aspect, provided is a method of treating cancer comprising administering a therapeutically effective amount of a virus or formulation according to the disclosure to a patient in need thereof, such as a human patient.

[0267] In one embodiment, the oncolytic viruses or formulations herein are administered in combination with another therapy.

[0268] As used herein, "in combination" is intended to encompass where the oncolytic virus is administered prior to, concurrently with, and / or after a cancer treatment or therapy.

[0269] Cancer treatments include surgery, radiation therapy, targeted therapy, and / or chemotherapy.

[0270] As used herein, cancer treatment refers to treatment with a therapeutic compound or biologic, such as an antibody intended for the treatment of cancer and / or its maintenance therapy.

[0271] In one embodiment, the cancer treatment is selected from any other anti-cancer therapy, including chemotherapeutic agents, targeted anti-cancer agents, radiation therapy, radioisotope therapy, or any combination thereof.

[0272] In one embodiment, viruses such as oncolytic adenoviruses of the present disclosure can be used as a pre-treatment for a therapy such as surgery (neoadjuvant therapy) to shrink the tumor, treat metastasis, and / or prevent metastasis or further metastasis. Oncolytic adenoviruses can be used after a therapy such as surgery (adjuvant therapy) to treat metastasis and / or prevent metastasis or further metastasis.

[0273] As used herein, concurrently is administration of the additional cancer therapy at the same time or about the same time as the oncolytic adenoviral formulation. The treatments may be contained in the same formulation or administered as separate formulations.

[0274] In one embodiment, the virus is administered in combination with administration of a chemotherapeutic agent.

[0275] As used herein, a chemotherapeutic agent refers to a specific anti-tumor chemical agent or a drug that selectively destroys malignant cells and tissues. For example, alkylating agents, antimetabolites, anthracyclines, plant alkaloids, topoisomerase inhibitors, and other anti-tumor agents. Other examples of chemotherapy include doxorubicin, 5-fluorouracil (5-FU), paclitaxel, capecitabine, irinotecan, and platinums such as cisplatin and oxaliplatin. The preferred dosage can be selected by the physician based on the nature of the cancer being treated.

[0276] In one embodiment, the therapeutic agent is ganciclovir, which can help control the immune response and / or tumor angiogenesis.

[0277] In one embodiment, one or more of the therapies employed in the methods herein is metronomic therapy, which is continuous or frequent treatment with low doses of an anticancer drug, often accompanied by other therapeutic approaches.

[0278] Subgroup B oncolytic adenoviruses, specifically Ad11 and its derivatives such as EnAd, may be particularly synergistic with chemotherapeutic agents because they appear to have a mechanism of action that is largely independent of apoptosis, killing cancer cells through a primarily necrotic mechanism. Furthermore, the immunosuppression that occurs during chemotherapy may allow oncolytic viruses to act more effectively.

[0279] As used herein, a therapeutic dose refers to an amount of a virus such as an oncolytic adenovirus that is suitable for achieving the desired therapeutic effect when employed in an appropriate treatment regimen, such as improving symptoms or conditions of the disease. A dose can be considered a therapeutic dose for treating cancer or metastasis when the number of viral particles is sufficient to result in: a slowing or cessation of tumor or metastatic growth, or a reduction in the size of the tumor or metastasis and / or an extension of the patient's lifespan. An appropriate therapeutic dose is typically a balance between therapeutic effect and tolerable toxicity, e.g., side effects and toxicity are tolerable given the benefits obtained from the treatment.

[0280] In one embodiment, a virus or therapeutic construct according to the disclosure (including formulations thereof) is administered weekly, for example, one dose per week on day 1, day 3, day 5, and then one dose each subsequent week.

[0281] In one embodiment, a virus or therapeutic construct according to the present disclosure (including formulations consisting thereof) is administered every two or three weeks, for example, on days 1, 3, and 5 of week 1, and also on days 1, 3, and 5 of week 2 or week 3. This dosing regimen can be repeated multiple times as appropriate.

[0282] In one embodiment, a virus or therapeutic construct according to the disclosure (including formulations consisting thereof) is administered monthly.

[0283] In one embodiment, the viruses and constructs of the present disclosure are prepared by recombinant techniques. The skilled artisan will appreciate that plasmids carrying adenoviral genomes can be made by other techniques, including completely synthetic genomes or plasmids comprising a portion of all genomes. The skilled artisan will appreciate that in the case of synthetic genomes, the insertion region may not include restriction site nucleotides, as the latter are an artifact of the gene insertion process using cloning methods.

[0284] The present disclosure further extends to an adenovirus of formula (I) or subformulae thereof obtained or obtainable by insertion of a transgene or transgene cassette.

[0285] As used herein, "is" means including.

[0286] In the context of this specification, "comprising" should be interpreted as "including".

[0287] Embodiments of the present invention that include certain features / elements are also intended to extend to alternative embodiments that "consist of" or "consist essentially of" the relevant elements / features.

[0288] The embodiments of the present invention may be combined where technically appropriate.

[0289] Technical references such as patents and applications are incorporated herein by reference.

[0290] Any embodiment specifically and explicitly described herein may form the basis of a disclaimer either alone or in combination with one or more further embodiments.

[0291] This application claims priority to GB1713765.4, WO2018 / 041838 and WO2018 / 041827, which are incorporated herein by reference. These documents may be used to correct errors in this specification, particularly errors in the sequence listing.

[0292] The present invention is further described by way of the following examples only, with reference to the accompanying drawings, in which: BRIEF DESCRIPTION OF THE DRAWINGS

[0293] Figure 1 Schematic diagram showing the NG-615, NG-640 and NG-641 transgenic cassettes

[0294] Figure 2: Viral genome replication in lung, breast, and bladder cancer cell lines. A549 (A), MDA-MB-453 (B), and RT4 (C) cell lines were treated with NG-617, NG-615, NG-640, NG-641, or enadenotucirev viral particles for up to 7 days. The amount of viral genome detected by qPCR was assessed 2, 3, 4, and 7 days after treatment.

[0295] Figure 3: Virus-mediated oncolysis of lung cancer cells. A549 cells were treated with NG-617, NG-615, NG-640, NG-641, or enadenotucirev viral particles for up to 4 days. Cell viability was assessed throughout the culture period using the xCelligence system (A). The time to observe 50% killing (KT50) was determined for each viral treatment (B).

[0296] Figure 4 NG-615 transgene expression in lung and bladder cancer cells. A549 (left panel) and RT4 cells (right panel) were treated with NG-615 or enadenotucirev viral particles or left uninfected for up to 7 days. Secretion of Flt3 ligand (A), MIP1α (B), and IFNα (C) in cell supernatants was assessed by ELISA. No transgene expression was detected in enadenotucirev-treated or untreated control cells (data not shown).

[0297] Figure 5 NG-641 transgene expression in lung and bladder cancer cells. A549 (left panel) and RT4 cells (right panel) were treated with NG-641 or enadenotucirev viral particles or left uninfected for up to 7 days. CXCL9 (A), CXCL10 (B), or IFNα (C) secretion in cell supernatants was assessed by ELISA. No transgene expression was detected in enadenotucirev-treated or untreated control cells (data not shown).

[0298] Figure 6: Functional transgene expression in lung cancer cells. A549 cells were treated with NG-615, NG-641, or enadenotucirev viral particles for up to 4 days. On day 4 post-treatment, the levels of functional IFNα (A) or MIP1α (B) transgene production were assessed using a cell-based reporter assay.

[0299] Figure 7Expression of functional FAP-BiTEs in lung cancer cells. A549 cells were treated with NG-615, NG-641, or enadenotucirev viral particles for up to 4 days. Functional FAP-BiTE expression levels in cell supernatants were assessed 2 (A), 3 (B), and 4 (C) days after treatment by measuring activation of Jurkat T cells co-cultured with the FAP-expressing fibroblast line MRC-5.

[0300] Figure 8A Transgenic IFNα in supernatants of NG-641-infected A549 cells induces SEAP production in Jurkat Dual reporter cells. Jurkat-Dual reporter cells were treated with supernatants of uninfected (UIC) or enadenotucirev (EnAd) or NG-641-infected A549 cancer cell lines, and the levels of secreted embryonic alkaline phosphatase (SEAP) reporter were measured.

[0301] Figure 8B : Transgenic CXCL9 / 10 in supernatants of NG-641-infected A549 cells activates the GPCR pathway in PathHunterβ-Arrestin cells. PathHunterβ-Arrestin cells were treated with supernatants from A549 cancer cell lines that were uninfected (UIC) or infected with enadenotucirev (EnAd) or NG-641, and CXCL9 / 10-specific induction of the G protein-coupled receptor (GPCR) pathway was detected by luminescence.

[0302] Figure 9 : Transgenic CXCL9 / 10-encoded supernatants of NG-641-infected A549 cells induce downregulation of CXCR3 on the surface of activated T cells. Anti-CD3 / CD28 activated human T cells were treated with supernatants of A549 cancer cell lines that were uninfected (UIC) or infected with enadenotucirev (EnAd) or NG-641, and transgenic CXCL9 / 10-induced CXCR3 downregulation was measured by flow cytometry.

[0303] Figure 10: Levels of activated viral transgene products IFNα and Flt3L of endogenous tumor-infiltrating T cells in ex vivo cultures of primary human tumor specimens inoculated with EnAd, NG-615, NG-617, NG-640 or NG-641, anti-CD3 / 28 or uninfected (UIC) are shown in A), and levels of IFNγ, TNFα, IL-17A, granzyme B and IL-13 are shown in B).

[0304] Figure 11: Surface marker expression and intracellular cytokine activation of endogenous tumor-infiltrating T cells in in vitro cultures of primary NSCLC tumor samples treated with EnAd, NG-617, NG-640, or NG-641 or uninfected (UIC). The levels of CD4 and CD8 T cells expressing CD25, CD69, and CD107a are shown in A and B, respectively. The levels of intracellular IFNγ and TNFα expressed by CD4 and CD8 T cells are shown in C and D, respectively.

[0305] Figure 12 Schematic diagram of the BiTE antibodies disclosed herein including or lacking an optional decahistidine affinity tag. Ig SP: signal peptide; 10His: decahistidine affinity tag; L: GS linker; V L : variable light domain; V H : variable heavy domain.

[0306] Figure 13 (A) Dot blot showing recombinant BiTE quantification. (B) Graph showing ELISA results for FAP.

[0307] Figure 14 shows a graph of the expression levels of CD69 (A) and CD25 (B) on T cells cultured alone or with NHDF cells using flow cytometry in the presence of FAP BiTEs and control BiTEs. Panel (C) shows the level of IFNγ expression by T cells cultured alone or with NHDF cells in the presence of FAP BiTEs and control BiTEs as measured by intracellular cytokine staining.

[0308] Figure 15 (A) Graph showing the results of an LDH assay of the cytotoxicity of NHDF cells co-cultured with T cells and FAP BiTEs or control BiTEs. (B) Graph showing the results of an LDH assay of the cytotoxicity of BTC100 cells co-cultured with T cells and FAP BiTEs or control BiTEs. (C) Graph of NHDF cells co-cultured with T cells and FAP BiTEs and control BiTEs.

[0309] Figure 16 is a graph showing the percentage of cells expressing EpCAM and FAP across various cells and cell lines.

[0310] Figure 17 (A) shows a graph of the NHDF dose response of FAP BiTEs with increasing BiTE concentrations. Panels (B) and (C) show the results of an LDH assay showing the cytotoxicity of DLD cells that have been co-cultured with T cells and EpCAM BiTEs or control BiTEs.

[0311] Figure 18 (A) shows a graph of FAP expression in CHO cells as determined by FAP or isotope control antibodies and analyzed by flow cytometry. (B) shows a graph showing the results of an LDH assay of the cytotoxicity of CHO or CHO-FAP cells co-cultured with T cells and FAP BiTEs or control BiTEs.

[0312] Figure 19 Shown is a graph analyzing T cell activation (based on CD69 and CD25 expression levels) of CHO vs. CHO-FAP cells using flow cytometry.

[0313] Figure 20 (A) Graph showing the ability of FAP BiTEs to activate CD4+ or CD8+ T cells (based on CD69 and CD25 expression levels) analyzed using flow cytometry. (B) Graph showing the results of an LDH assay showing the cytotoxicity of NHDF cells co-cultured with CD4+ or CD8+ T cells and FAP BiTEs or control BiTEs.

[0314] Figure 21 (A) Graph showing the number of CD3+ T cells in ascites cultured with control or FAP BiTE. (B) Graph showing the level of CD25 expression by T cells in ascites cultured with control or FAP BiTE. (C) Graph showing the number of FAP+ cells in ascites cultured with control or FAP BiTE.

[0315] Figure 22 (A) Graph showing quantification of the number of viral genomes detected per cell for NG-601, NG-602, NG-603, NG-604, NG-605, NG-606, and EnAd. (B) Graph showing the oncolytic activity of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606, or EnAd assessed by infection of A549 cells.

[0316] Figure 23 A graph showing T cell activation (based on CD69 and CD25 expression levels) by NG-601, NG-602, NG-605, and NG-606 when co-cultured with CHO-FAP using flow cytometry analysis.

[0317] Figure 24 Shown are graphs showing the results of experiments to determine the amount of FAP BiTE produced from NG-605 and NG-606.

[0318] Figure 25Shown are microscopic images of Ad293 cells infected with NG-607, NG-608, NG-609, and NG-610.

[0319] Figure 26 (A) is a graph showing the ability of NG-603, NG-604, NG-605, NG-606, and EnAd to kill NHDF cells as analyzed using XCELLigence. (B) is a graph showing the ability of NG-603, NG-604, NG-605, NG-606, and EnAd to kill NHDF cells as analyzed using the LDH assay.

[0320] Figure 27 Shown are graphs showing T cell activation (based on CD69 and CD25 expression levels) by NG-603, NG-604, NG-605, NG-606 co-cultured with NHDF cells, SKOV and T cells using flow cytometry analysis.

[0321] Figure 28 (A) Graph showing T cell activation (based on CD69 and CD25 expression levels) by NG-603, NG-604, NG-605, and NG-606 in co-culture with NHDF and multiple SKOV cells against a single SKOV cell using flow cytometry analysis. (B) Graph showing the cytotoxicity of NHDF cells infected with NG-605 and NG-606 using LDH assay analysis.

[0322] Figure 29 Shown are still frame images from time-lapse videos of NHDF cell lysis by recombinant FAP BiTEs, EnAd, NG-603, or NG-605.

[0323] Figure 30 Shown are still frame images from timelapse videos of NHDF cell lysis by NG-607, NG-608, NG-609, or NG-610.

[0324] Figure 31 Shown are graphs indicating the cytotoxicity of DLD cells infected with EnAd, NG-601, NG-602, NG-603, and NG-604 in the presence or absence of T cells, analyzed using the LDH assay.

[0325] Figure 32 (A) Graph indicating the expression level of CD25 on CD3+ T cells in ascites samples infected with the viruses of the present disclosure. (B) Graph indicating the number of FAP+ cells in ascites samples infected with the viruses of the present disclosure.

[0326] Figure 33Shown are graphs indicating the number of CD3+ T cells in ascites samples obtained from cancer patients and infected with the viruses of the present disclosure.

[0327] Figure 34 Shown are graphs indicating the expression levels of CD25 on CD3+ T cells in ascites samples obtained from cancer patients and infected with the viruses of the present disclosure.

[0328] Figure 35 Shown are graphs indicating the number of FAP+ cells in ascites samples obtained from cancer patients and infected with the viruses of the present disclosure.

[0329] Figure 36 Comparison of cytokine production in T cells activated by recombinant FAP BiTE protein in the presence of human fibroblasts and polyclonal activation with anti-CD3 / CD28 beads is shown. (A) IFNγ levels measured by ELISA. (B) Cytokine levels measured by cytokine bead array.

[0330] Figure 37 FAP-targeted BiTEs induce T cell degranulation and specific cytotoxicity of FAP+ cells

[0331] (A) T cells in culture were degranulated using NHDF cells (5:1) and (B) supernatants containing BiTEs. Degranulation was assessed by externalization of CD107a after 6 hours of culture with CD107a-specific antibodies and measured by flow cytometry. CD3 / CD28 magnetic beads were used as positive controls. (C) Cytotoxicity of NHDF cells after 24 hours of co-culture with T cells (1:5) and 10-fold serial dilutions of supernatants containing BiTEs. Cytotoxicity was assessed by releasing LDH into the culture supernatant. (D) After 24 hours of co-culture with T cells (1:5) derived from PBMCs from six healthy donors and supernatants containing BiTEs, LDH release (left) and CD25 induction (right) on T cells were assessed for dissolution of NHDF.

[0332] Figure 38 EnAd expressing FAP BiTE selectively kills FAP + fibroblasts and decreased TGFb in peritoneal ascites samples

[0333] (A, B) FAP after 72 h of culture of PBMC-derived T cells with EnAd or recombinant virus + Fibroblasts (A) and EpCAM +The number of tumor cells (B). First, ascites cells were isolated from three patients' ascites and expanded in vitro. Cell number was measured by flow cytometry 72 hours after infection. (C) Induction of the activation marker CD25 on CD3 cells derived from PBMCs in (A) was measured 72 hours after infection. (D) The level of TGFb was measured by ELISA using the supernatant collected from (A).

[0334] Figure 39 Figure 2: Activation of endogenous tumor-associated T cells and associated FAP+ cell killing in malignant ascites biopsies from patients by FAP BiTE protein and EnAd-FAPBiTE virus. (A) T cell activation measured by CD25 expression. (B) Residual number of FAP+ cells measured by flow cytometry.

[0335] Figure 40 Effects of PD-L1 blocking antibodies on BiTE-mediated T cell activation in patient samples

[0336] (A) Endogenous T cell and PD-L1 expression on FAP+ cells after initial isolation from peritoneal ascites was assessed by flow cytometry. (B) Unpurified total cells from peritoneal ascites were incubated in 50% fluid from the same exudate in the presence of free BiTEs, EnAds, or recombinant viruses, with or without anti-PD-L1 blocking antibodies. After 2 days, the total cell population was harvested, and the number of CD25+ T cells was quantified by flow cytometry. (C) The amount of interferon gamma in the culture supernatant from (B, D) was measured by ELISA. (D) The number of residual FAP+ cells in (B) was measured by flow cytometry.

[0337] Figure 41 BiTE-expressing EnAds activate and redirect T cells from patient biopsies to lyse NHDF fibroblasts

[0338] (A) PD-1 expression in endogenous T cells isolated from healthy donors or malignant effusion cancer biopsy samples. PD-1 expression was measured by flow cytometry. (B) CD3 + (C) Interferon-γ levels measured by ELISA in culture supernatants collected from (B) 120 hours after treatment. (D) Real-time monitoring of NHDF fibroblast viability over 130 hours using the xCELLigence cytotoxicity assay by co-culture with PBMCs or total cancer biopsy cells (1:5) and BiTE-containing supernatants.

[0339] Figure 42Shown are the effects of immunosuppressive ascites fluid samples on FAP BiTE-mediated and anti-CD3 / CD28 bead-mediated PBMC T cell activation. (A) PBMC T cells activated with anti-CD3 / CD28 magnetic beads. (B) PBMC T cells activated with control or FAP BiTEs in the presence of NHDF cells. NS: normal serum, A: peritoneal fluid.

[0340] Figure 43 FAP BiTE expressing EnAd activated CD11b in the patient's ascites + Macrophage polarization toward a more inflammatory phenotype

[0341] (A) Unpurified total cells from ascites samples were incubated in 50% ascites fluid in the presence of free BiTEs or virally expressed BiTEs. Interferon gamma treatment was used as a positive control. After 3 days, total cell populations were harvested and CD3 expression was measured by flow cytometry. + Induction of the activation marker CD25 on cells. (B) Interferon γ levels were measured in the culture supernatants from (A) by ELISA. (C) On day 3, the expression levels of CD68, CD86, CD206, and CD163 were measured by flow cytometry on CD11b+ cells from (A). Representative flow cytometry spectra from triplicates are shown along with the complete data set.

[0342] Figure 44 Characterization of the architecture and cellular composition of solid prostate tumors

[0343] Shown are the levels of IFNg in tissue slice culture medium measured by ELISA. Supernatants were collected from slice cultures of malignant and benign tissues at the indicated time points; and the levels of IL-2 in tissue culture medium of malignant and benign tissues measured by ELISA.

[0344] Figure 45 A schematic diagram of the transgene cassette is shown.

[0345] Figure 46 Shown are graphs indicating the number of viral genomes detected per cell in NG-611, NG-612, and NG-617 treated tumor cells.

[0346] Figure 47Shown are the percentages of T cells expressing CD69 (a), CD25 (b), HLA-DR (c), CD40L (d), or cell surface CD107a (e) after coculture with EpCam-expressing SKOV cells and supernatants collected from A549 cells 24 h, 48 h, or 72 h after treatment with NG-611 viral particles compared with NG-612, enadenotucirev, or untreated control supernatants.

[0347] Figure 48 Shown are the percentages of T cells expressing CD69 (a), CD25 (b), HLA-DR (c), CD40L (d), or cell surface CD107a (e) after coculture with FAP expressing MRC-5 cells and supernatants collected from A549 cells 24 h, 48 h, or 72 h after treatment with NG-612 viral particles compared with NG-611, enadenotucirev, or untreated control supernatants.

[0348] Figure 49 The percentage of MRC-5 cells expressing EpCAM and FAP is shown

[0349] Figure 50 shows IFNγ expression in supernatants of T cell co-cultures with SKOV cells (A) or MRC-5 cells (B) 24 hours, 48 ​​hours, or 72 hours after treatment with NG-611, NG-612, or enadenotucirev viral particles or untreated control supernatants, which were incubated with supernatants collected from A549 cells.

[0350] Figure 51 Figure 2 shows the antitumor efficacy and immune activation of virally expressed BiTEs in vivo. (a) Tumor volume in mice treated with saline, enadenotucirev, or NG-611. (b) Ratio of CD8 to CD4 T cells in NG-611-treated tumors compared to enadenotucirev-treated or untreated controls.

[0351] Figure 52 Shown are graphs indicating the number of viral genomes detected per cell in NG-612 and NG-615 treated tumor cells.

[0352] Figure 53 Shown are the expressions of IFNα, MIP1α, and Flt3 L in the cell supernatants of NG-615 and enadenotucirev versus untreated control tumor cells.

[0353] Figure 54Shown are the numbers of T cells expressing CD69 (a), CD25 (b), HLA-DR (c), CD40L (d), or cell surface CD107a (e) after coculture with FAP expressing MRC-5 cells and supernatants collected from A549 cells 24 h, 48 h, or 72 h after treatment with NG-615 viral particles compared with NG-612, enadenotucirev, or untreated control supernatants.

[0354] Figure 55 Shown is IFNγ expression in supernatants of T cell cocultures with MRC-5 cells 24, 48, or 72 hours after treatment with NG-612, NG-615, or enadenotucirev viral particles or untreated control supernatant, which were incubated with supernatants collected from A549 cells.

[0355] sequence

[0356] SEQ ID NO: 1 Anti-FAP BiTE DNA coding sequence with N-terminal signal sequence and C-terminal deca-His affinity tag

[0357] SEQ ID NO: 2 Anti-FAP BiTE amino acid sequence with N-terminal signal sequence and C-terminal deca-His affinity tag

[0358] SEQ ID NO: 3: Control (anti-FHA) BiTE DNA coding sequence with N-terminal signal sequence and C-terminal deca-His affinity tag

[0359] SEQ ID NO:4: Control (anti-FHA) BiTE amino acid sequence with N-terminal signal sequence and C-terminal deca-His affinity tag

[0360] SEQ ID NO: 5: Anti-CD3 ScFv amino acid sequence

[0361] SEQ ID NO: 6: Anti-CD3 VH

[0362] SEQ ID NO: 7: Anti-CD3 VL

[0363] SEQ ID NO: 8: Anti-CD3 ScFv linker sequence

[0364] SEQ ID NO: 9: Anti-FAP ScFv

[0365] SEQ ID NO: 10: Anti-FAP VL domain

[0366] SEQ ID NO: 11: Anti-FAP VH domain

[0367] SEQ ID NO: 12: Anti-FAP and anti-EpCAM linker sequence

[0368] SEQ ID NO: 13: BiTE leader sequence

[0369] SEQ ID NO: 14: Control BiTE (anti-FHA)

[0370] SEQ ID NO: 15: Control (anti-FHA) ScFv

[0371] SEQ ID NO: 16: Control (anti-FHA) VL

[0372] SEQ ID NO: 17: Control (anti-FHA) VH

[0373] SEQ ID NO: 18: Control (anti-FHA) ScFv linker sequence

[0374] SEQ ID NO: 19: Deca-His tag sequence

[0375] SEQ ID NO: 20: FAP BiTE-P2A-RFP (ITALICS=leader, BOLD=furin cleavage site, UNDERLINE =P2A sequence, lowercase =RFP)

[0376] SEQ ID NO: 21: Control (anti-FHA) BiTE-P2A-RFP (ITALICS = leader, BOLD = furin cleavage site, UNDERLINE =P2A sequence, lowercase =RFP)

[0377] SEQ ID NO: 22: Human FAP DNA coding sequence

[0378] SEQ ID NO: 23: Human FAP amino acid sequence

[0379] SEQ ID NO: 24: CMV promoter sequence

[0380] SEQ ID NO:25:SV40 late polyadenylation sequence

[0381] SEQ ID NO:26:NG-605(EnAd-CMV-FAPBiTE)

[0382] SEQ ID NO:27:NG-606(EnAd-SA-FAPBiTE)

[0383] SEQ ID NO: 28 EnAd genome

[0384] SEQ ID NO: 29 BX DNA sequence corresponding to and including bp 28166 to bp 28366 of the EnAd genome

[0385] SEQ ID NO:30 BY DNA sequence corresponding to and comprising bp 29345 to bp 29379 of the EnAd genome

[0386] SEQ ID NO:31 HIS-Tag

[0387] SEQ ID NO: 32 splice acceptor sequence

[0388] SEQ ID NO:33 SV40 polyadenylation sequence

[0389] SEQ ID NO:34 FAP BiTE nucleic acid sequence (OKT3)

[0390] SEQ ID NO:35 FAP BiTE nucleic acid sequence (aCD3)

[0391] SEQ ID NO:36 NG-611 transgene cassette

[0392] SEQ ID NO:37 NG-612 transgene cassette

[0393] SEQ ID NO:38 NG-613 transgene cassette

[0394] SEQ ID NO:39 restriction site insertion (B X )

[0395] SEQ ID NO:40 restriction site insertion (B Y )

[0396] SEQ ID NO:41 CMV promoter sequence

[0397] SEQ ID NO:42 PGK promoter sequence

[0398] SEQ ID NO:43 CBA promoter sequence

[0399] SEQ ID NO:44 Short-splicing acceptor (SSA) DNA sequence

[0400] SEQ ID NO:45 Splice acceptor (SA) DNA sequence

[0401] SEQ ID NO:46 Branched splice acceptor (bSA) DNA sequence

[0402] SEQ ID NO:47 Kozak sequence (null sequence)

[0403] SEQ ID NO: 48 Example of a start codon

[0404] SEQ ID NO:49 Internal ribosome entry sequence (IRES)

[0405] SEQ ID NO:50 P2A peptide

[0406] SEQ ID NO:51 F2A peptide

[0407] SEQ ID NO:52 E2A peptide

[0408] SEQ ID NO:53 T2A peptide

[0409] SEQ ID NO:54 polyadenylation (polyA) sequence

[0410] SEQ ID NO:55 leader sequence

[0411] SEQ ID NO:56 leader sequence

[0412] SEQ ID NO:57 IFNγ amino acid sequence

[0413] SEQ ID NO:58 IFNα amino acid sequence

[0414] SEQ ID NO:59 TNFα amino acid sequence

[0415] SEQ ID NO: 60 corresponds to the DNA sequence of the E2B region of the EnAd genome (bp 10355 to bp 5068)

[0416] SEQ ID NO:61: Anti-FAP BiTE DNA coding sequence with N-terminal signal sequence and without C-terminal deca-His affinity tag

[0417] SEQ ID NO:62: Anti-FAP BiTE amino acid sequence with N-terminal signal sequence and without C-terminal deca-His affinity tag

[0418] SEQ ID NO:63: Control (anti-FHA) BiTE DNA coding sequence with N-terminal signal sequence and without C-terminal deca-His affinity tag

[0419] SEQ ID NO:64: Control (anti-FHA) BiTE amino acid sequence with N-terminal signal sequence and without C-terminal deca-His affinity tag

[0420] SEQ ID NO: 65: Control BiTe (anti-FHA) without C-terminal deca-His affinity tag

[0421] Q ID NO:66:NG-605 (EnAd-CMV-FAPBiTE), without deca-His affinity tag

[0422] SEQ ID NO:67:NG-606 (EnAd-SA-FAPBiTE), without deca-His affinity tag

[0423] SEQ ID NO:68: FAP BiTE nucleic acid sequence (OKT3)

[0424] SEQ ID NO:69: FAP BiTE nucleic acid sequence (aCD3)

[0425] SEQ ID NO:70:NG-611 transgene cassette

[0426] SEQ ID NO:71:NG-612 transgene cassette

[0427] SEQ ID NO:72:NG-613 transgene cassette

[0428] SEQ ID NO:73:NG-614 transgene cassette

[0429] SEQ ID NO:74:NG-617 transgene cassette

[0430] SEQ ID NO:75: FAP BiTE amino acid sequence (OKT3)

[0431] SEQ ID NO:76: FAP BiTE amino acid sequence (aCD3)

[0432] SEQ ID NO:77:NG-611 genome

[0433] SEQ ID NO:78:NG-612 genome

[0434] SEQ ID NO:79:NG-613 genome

[0435] SEQ ID NO:80:NG-614 genome

[0436] SEQ ID NO:81:NG-617 genome

[0437] SEQ ID NO:82:NG-615 genome

[0438] SEQ ID NO:83:NG-640 genome

[0439] SEQ ID NO:84:NG-641 genome

[0440] SEQ ID NO:85: zero sequence

[0441] SEQ ID NO:86: Flt3L nucleic acid sequence

[0442] SEQ ID NO: 87: zero sequence

[0443] SEQ ID NO:88: MIP1α nucleic acid sequence

[0444] SEQ ID NO: 89: Flexible linker sequence

[0445] SEQ ID NO:90: ​​IFNα nucleic acid sequence

[0446] SEQ ID NO:91:CXCL10 nucleic acid sequence

[0447] SEQ ID NO:92: CXCL9 nucleic acid sequence

[0448] SEQ ID NO:93:NG-615 transgene cassette

[0449] SEQ ID NO:94:NG-640 transgene cassette

[0450] SEQ ID NO:95:NG-641 transgene cassette

[0451] SEQ ID NO:96: FLT3L amino acid sequence

[0452] SEQ ID NO:97: MIP1α amino acid sequence

[0453] SEQ ID NO:98: IFNα amino acid sequence

[0454] SEQ ID NO:99: CXCL9 amino acid sequence

[0455] SEQ ID NO: 100: CXCL10 amino acid sequence

[0456] SEQ ID NO: 101: NG-618 genome

[0457] SEQ ID NO: 102: NG-618FAP BiTE nucleic acid sequence

[0458] SEQ ID NO: 103: NG-618 transgenic cassette

[0459] SEQ ID NO: 104 to 277 are linker sequences

[0460] SEQ ID NO:278 NG-616 genome

[0461] SEQ ID NO: 279 to 281 are primers

[0462] Examples

[0463] Example 1

[0464] Recombinant BiTEs were designed and proteins were produced as described in this example.

[0465] 1.1 BiTE Engineering

[0466] BiTEs are generated by linking two single-chain antibody fragments (ScFv) with different specificities to a flexible Gly4Ser linker. H and V L Each BiTE is designed with an N-terminal signal sequence for mammalian secretion and a C-terminal 18-histidine affinity tag for detection and purification. BiTEs are engineered by standard DNA cloning techniques and inserted into protein expression vectors ( Figure 1 ).

[0467] An anti-FAP BiTE was created de novo using anti-FAP ScFv from patent WO2010037835A2 and anti-CD3 ScFv from 2005040220 (SEQ ID 63 therein), to which a signal sequence and affinity tag were added.

[0468] Control BiTEs used anti-FHA (filamentous hemagglutinin from Bordetella pertussis) ScFv from Hussein et al., 2007 (Hussein AH et al. (2007) "Construction and characterization of single-chain variable fragment antibodies against the Bordetella pertussis surface adhesins filamentous hemagglutinin and pertussis." Infect Immunity 75, 5476-5482), and anti-CD3 ScFv from patent WO 2005040220 (SEQ ID NO: 63), to which a signal sequence and affinity tag were added.

[0469] 1.2 Recombinant BiTE Production

[0470] Recombinant BiTE proteins were produced by cloning each sequence into the pSF-CMV vector using the CMV promoter (SEQ ID NO: 24) to drive protein expression ( Figure 1). The plasmid DNA concentrations of plasmids, pSF-CMV-FAPBiTE, and pSF-CMV-ControlBiTE (Table 2) were measured by NanoDrop. An empty pSF-CMV vector was included as a negative control. 54.7 μg of each was diluted with 4 mL of OptiMEM. 109.2 ug PEI (linear, MW 25000, Polysciences, USA) was diluted in 4 mL of OptiMEM medium and mixed with 4 ml of diluted DNA to generate a DNA-PEI complex (DNA: PEI ratio of 1: 2 (w / w)). After incubation at room temperature for 20 minutes, the complex mixture was made up to 18 mL with OptiMEM, and this transfection mixture was added to a T175 flask containing 90% confluent Ad293 cells. With cell and transfection mixture at 37 ℃, 5%CO After hatching 4 hours, 30mL cell culture medium (being supplemented with the DMEM high glucose of glutamine, without phenol red) is added to the cell, and by flask at 37 ℃, 5%CO Lower hatching 48 hours.By another cell flask and pSF-CMV-GFP parallel transfection to guarantee effective transfection efficiency.In order to gather secreted protein, collect the supernatant of transfected cell, and at 4 ℃ with 350g centrifugal 5 minutes to remove cellular component (Allegra X-15R, Beckman Coulter Co., Ltd. (Beckman Coulter)).Supernatant is transferred in 10k MWCO Amicon Ultra-15 centrifugal filter device (Millipore Corporation (Millipore)).After rotating at 4750rpm and 4 ℃, the volume of retentate is regulated to obtain the concentration of 50 times higher with flow-through thing.The concentrated protein of aliquot is stored at-80 ℃.

[0471] Table 2

[0472] "p" used as a prefix in the named construct indicates that the construct is a plasmid.

[0473] Plasmid ID [Plasmid DNA]ng / ml pSF-CMV-FAPBiTE 6700 pSF-CMV-ControlBiTE 5300 pSF-Lenti-FAP 659.6

[0474] 1.3 Generation of viruses expressing FAP-BiTEs conjugated to immunomodulatory proteins

[0475] Three viruses (NG-640, NG-641, and NG-615) were generated that encode FAP-targeting BiTE molecules and two or three immunomodulatory proteins (Table 1). NG-640 encodes three transgenic proteins, the FAP-BiTE molecule and the chemokines CXCL9 and CXCL10. Both NG-641 and NG-615 encode four transgenic proteins. NG-641 encodes the FAP-BiTE, the chemokines CXCL9 and CXCL10, and the cytokine IFNα, while NG-615 encodes the FAP-BiTE, the chemokine MIP1α, the cytokine FLT3 ligand, and IFNα. A virus encoding only the FAP-BiTE molecule (NG-617) was also generated.

[0476] Table 1

[0477]

[0478]

[0479] In each transgene cassette, the cDNA encoding BiTEs and other immunomodulatory proteins was flanked at the 5' end by short splice acceptor sequences (SSA, CAGG) and at the 3' end by the SV40 late poly(A) sequence (PA, SEQUENCE ID NO: 25). The cDNA sequence for each transgene was isolated using the 2A highly efficient self-cleavable peptide sequences (P2A, T2A, E2A, SEQUENCE ID NOs: 50, 53, and 52).

[0480] Virus production

[0481] Plasmid pEnAd2.4 was used to generate plasmids pNG-615, pNG-640, and pNG-641 by direct insertion of synthetic transgene cassettes (SEQ ID NOs: 93, 94, and 95, respectively). NG-615 contains four transgenes encoding FAP-targeting BiTEs (SEQ ID NO: 102), Flt3L (SEQ ID NO. 86), MIP1α (SEQ ID NO. 88), and IFNα (SEQ ID NO. 90). NG-640 and NG-641 encode BiTes targeting FAP (SEQ ID NO. 102), CXCL9 (SEQ ID NO. 92), and CXCL10 (SEQ ID NO. 91), with NG-641 also containing a fourth transgene encoding IFNα (SEQ ID NO. 90). Figure 1 A schematic diagram of the transgene cassette is shown in The construction of the plasmid DNA was confirmed by restriction analysis and DNA sequencing.

[0482] Plasmids, pNG-615, pNG-640 and pNG-641 were linearized by restriction digestion with the enzyme Asc I to generate viral genomes. Viruses were amplified and purified according to the methods given below.

[0483] The DNA of digestion was purified by phenol / chloroform extraction and precipitated at -20°C for 16 hours in 300 μl>95% molecular biology grade ethanol and 10 μl 3M sodium acetate. The DNA was precipitated by centrifugation at 14000 rpm for 5 minutes and washed in 500 μl 70% ethanol, then centrifuged at 14000 rpm for 5 minutes again. The clean DNA pellet was air-dried, resuspended in 500 μl OptiMEM containing 15 μl liposome transfection reagent, and incubated at room temperature for 30 minutes. The transfection mixture was then added dropwise to a T-25 flask containing 293 cells grown to 70% confluence. After the cells were incubated at 37°C for 2 hours with the transfection mixture, 5% CO2 4ml of cell culture medium (DMEM high glucose and glutamine supplemented with 2% FBS) was added to the cells, and the flask was incubated at 37°C, 5% CO2.

[0484] The 293 cells of per 24 hours monitoring transfection, and per 48 hours to 72 hours supplemented extra culture medium.Monitor the generation of virus by the significant cytopathic effect (CPE) in the observation cell monolayer.Once observe extensive CPE, collect virus from 293 cells by three freeze-thaw cycles.The virus collected is used for reinfecting 293 cells so that amplification virus stock.Confirm the generation of live virus during amplification by the significant CPE in the observation cell monolayer.Once observe CPE, collect virus from 293 cells by three freeze-thaw cycles.Before by double cesium chloride bands purifying virus to produce the virus stock of purification, the virus stock of amplification is used for further amplification.

[0485] Example 2: Analysis of viral replication and oncolytic activity

[0486] Viral replication

[0487] Lung (A549), breast (MDA-MB-453) or bladder (RT4) cancer cell lines that were not infected with 1 ppc NG-615, NG-640, NG-641, NG-617, enadenotucirev (EnAd) for 72 hours or were used for quantitative viral DNA by qPCR. Cell supernatants were collected and clarified by centrifugation at 1200 rpm for 5 minutes. 50 μL supernatants were used for DNA analysis.

[0488] According to the manufacturer's protocol, DNA was extracted from the supernatant samples using the Qiagen DNeasy test kit. A standard curve using EnAd virus particles (2.5e10 to 2.5e5 vp) was also prepared and extracted using the DNeasy test kit. Each extracted sample or standard was analyzed by qPCR using viral gene-specific primers-probes for the early gene E3. Quantification of the number of viral genomes detected in each cell showed that for all tested viruses (NG-617, NG-615, NG-640, and NG-641), viral replication was observed in A549, MDA-MB-453, and RT4 (Fig. 2). Viral replication for all viruses was similar and identical to the parental EnAd virus. No viral genome was detected in uninfected cells.

[0489] Oncolytic activity

[0490] 100ppc NG-615, NG-640, NG-641, NG-617, EnAd or uninfected lung (A549) cancer cells were monitored using xCELLigence real-time cell analyzer (RTCA). Cell proliferation was monitored every 60 minutes for 96 hours. The oncolytic effect of cells was assessed by calculating the killing time 50 (KT50), the time point at which 50% lysis was reached (Fig. 3). These data show the equivalent KT50 of all test viruses, including parental EnAd viruses. No oncolytic effect was observed on untreated cells.

[0491] Together, these data indicate that inclusion of a BiTE and two or three immunomodulatory transgenes does not significantly affect the replication or oncolytic activity of the EnAd virus.

[0492] Example 3: Analysis of recombinant BiTE detection for viral-mediated transgene expression

[0493] In order to detect BiTE, the 18 histidine affinity tag of C-terminus can be detected using Western blotting technique with anti-His antibody. Protein sample is adjusted to a final volume of 15 μ L with dissolution buffer, comprising 2.5 μ L 6x Laemmli SDS sample buffer containing β-mercaptoethanol and SDS. Sample is incubated at 95 ° C for 5 minutes to denature the protein and loaded onto 15 hole 10% precast polyacrylamide gels (Mini-PROTEAN TGX precast gels, British Life Sciences Company (BioRad)). In the 1x running buffer in Mini-PROTEAN Tetra system (British Life Sciences Company), gel was run at 180V for 45 minutes. Protein from SDS gel is transferred to nitrocellulose filter by wet electroblotting in the 1x transfer buffer in miniature trans-blot cell (MiniTrans-Blot Cell) (British Life Sciences Company) at 300mA and 4 ° C in 90 minutes. Transfer is performed to limit heat in the presence of ice pack. The nitrocellulose membrane was then blocked with 5% milk in PBS-T on a shaker at room temperature for 1 hour and probed with anti-His (C-terminal) antibody (mouse α-6xHis, clone 3D5, Invitrogen, UK, #46-0693), diluted 1:5000 in PBS / 5% milk. After incubation overnight at 4°C on a shaker, the membrane was washed and probed with HRP-labeled polyclonal secondary α-mouse immunoglobulin antibody (1:10.000 in PBS / 5% milk, Dako, #P0161) at room temperature for 1 hour. For visualization, SuperSignal West Dura extended duration substrate (Thermo Fisher Scientific, UK) was applied according to the manufacturer's instructions and exposed to X-ray film and developed in an automatic film processor. The results demonstrated expression and secretion of BiTE protein in Ad293 cells transfected with BiTE expression plasmids, but not the parental vector.

[0494] Recombinant BiTE quantification

[0495] To measure the amount of recombinant BiTE protein, the BiTE signal was compared to a His-tagged (C-terminal 10His) protein standard (10x His-tagged human cathepsin D, Biolegend, #556704) using a dot blot technique. Two-fold serial dilutions of the BiTE sample and protein standard were prepared, and 1.5 μL of each was applied directly to a nitrocellulose membrane and air-dried for 20 minutes. The blocking and staining protocol described above for Western blotting was then performed. The molar concentration of the protein standard was adjusted to represent a BiTE concentration of 250 μg / mL. Results ( Figure 13A) Demonstrates the expression and secretion of BiTE proteins in Ad293 cells transfected with BiTE expression plasmids.

[0496] FAP binding ELISA

[0497] The FAP binding activity of FAP BiTEs and control (anti-FHA) BiTEs (SEQ ID NOs: 2 and 4) secreted from cells transfected with pSF-CMV-FAPBiTE or pSF-CMV-control BiTE was assessed by enzyme-linked immunosorbent assay (ELISA). Empty pSF-CMV vector supernatant was included as a negative control. ELISA plates (Nunc Immuno MaxiSorp 96-well microplates) were prepared by coating with human FAP / seprase protein (100 ng / well, Sino Biological Inc., 10464-H07H-10) in PBS buffer overnight at 4°C. Plates were washed with PBS 0.05% Tween 20 between all subsequent binding steps. Plates were blocked with 5% BSA in PBS 0.05% Tween 20 for 1 hour at room temperature. BiTE protein or protein collected from the wells transfected with the empty pSF-CMV vector was diluted 10 times into PBS / 5% BSA / 0.05% Tween 20. All samples were added to the FAP coated plate and incubated at room temperature for 2 hours. The detection antibody, anti-His (C-terminal) antibody (mouse anti-6xHis, clone 3D5, Invitrogen, UK, #46-0693) was diluted with 1:1000 and applied at room temperature for 1 hour. HRP-conjugated anti-mouse Fc (1:1000 in PBS / 5% milk, Dako) was then applied at room temperature for 1 hour, followed by HRP detection with HRP substrate solution 3.3.5.5'-tetramethylethylenediamine (TMB, Thermo Fisher Scientific). The reaction was stopped using stop solution, and color development was measured at 450nm on a plate reader. The absorbance at 450 nm was plotted for FAP BiTE, control BiTE, and empty vector supernatant, demonstrating the specific binding of FAP BiTE to FAP protein. Figure 13 B) shows specific binding of FAP BiTEs, but not control BiTEs, to recombinant FAP protein.

[0498] Assessment of transgene expression by ELISA

[0499] The expression of the chemokine or cytokine transgenes IFNα, MIP1α, FLT3L, CXCL10, and CXCL9 was assessed using ELISA. A549 and RT4 cancer cell lines were inoculated with 1 ppc of NG-615, NG-640, NG-641, NG-617, EnAd, or left uninfected for up to 7 days. Cell supernatants were clarified 4 and 7 days after inoculation, and transgene expression was assessed by ELISA.

[0500] IFNα ELISA was performed using the Verikine human IFNα kit (Pbl assay science), MIP1α ELISA was performed using the human CCL3 Quantikine ELISA kit (R&D systems), Flt3L ELISA was performed using the Flt3L human ELISA kit (Abcam), CXCL9 ELISA was performed using the CXCL9 human ELISA kit (Abcam), and CXCL10 ELISA was performed using the CXCL10 human ELISA kit (Abcam). All assays were performed according to the manufacturer's protocol.

[0501] The concentrations of secreted IFNα, MIPα, Flt3L, CXCL9, and CXCL10 were determined by interpolation from the standard curve. IFNα, MIP1α, and Flt3L expression were detectable in the cell supernatants of NG-615-treated cells, IFNα, CXCL9, and CXCL10 were detectable in the supernatants of NG-641-treated cells, and CXCL9 and CXCL10 were detectable in the supernatants of NG-640-treated cells. Figure 4 and 5 No chemokine or cytokine transgene expression was detected in EnAd-treated or untreated control cells.

[0502] Assessment of functional transgene expression by cell-based reporter gene assay

[0503] The expression of functional FAP-BiTE and IFNα transgene was evaluated in an assay using the Jurkat-Dual reporter cell line (Invivogen). This is a human immortalized T lymphocyte cell line (Jurkat) transformed by the stable integration of two inducible reporter gene constructs. One of the inducible reporter gene constructs enables the study of IFN activation of the interferon regulatory factor (IRF) pathway through the secretion and activity of secreted embryonic alkaline phosphatase (SEAP), while the second is an NF-kB-responsive secreted luciferase reporter gene activated through signaling of the T cell receptor. The activity of SEAP is proportional to the level of IFN-α present in the supernatant and can be detected by the detection substrate Quanti-Blue TM Functional MIP1α expression was measured by SEAP-induced degradation of α-MIP1α. A CCR5 reporter cell line (CHO-K1 CCR5β-arrestin, Invivogen) was used to assess functional MIP1α expression. A549 cancer cell lines were inoculated with 1 ppc of NG-615, NG-640, NG-641, NG-617, EnAd, or left uninfected. Cell supernatants were collected 2, 3, or 4 days after inoculation and clarified for analysis.

[0504] To evaluate IFNα function, 20 μL of each supernatant diluted 1:10, 1:50, or 1:250 in culture medium was added to Jurkat Dual cells (2 × 10 5 Cells / well) and incubated for 16-20 hours. The supernatant was then collected from the plate and stained with 200 μL Quanti-Blue TM The plates were analyzed by measuring the absorbance (Abs) at 640 nm using a microplate reader. A response indicating the presence of functional IFNα was detected in the supernatants of NG-615 and NG-641-treated cancer cells, but not in the supernatants of NG-640, NG-617, EnAd-treated, or uninfected controls. Figure 6A Similar levels of functional IFNα were detected in the supernatants of NG-615 and NG-641 treated cells.

[0505] To evaluate MIP1α function, CCR5 reporter cells were seeded (5 × 10 3 Cells / well) and incubated for 20-24 hours. 5 μL of supernatant from the treated tumor cells was then added to each well and incubated for 90 minutes. The activity of the luciferase reporter gene was then detected using a detection solution and quantified on a luminescence plate reader. A response indicating the presence of functional MIP1α was detected in the supernatant of NG-615-treated cancer cells and in the supernatant of cells treated with a positive control virus known to express MIP1a NG-347 ( Figure 6B ).

[0506] To evaluate the function of FAP-BiTE, MRC-5 lung fibroblasts (expressing FAP on their cell membrane) were seeded (2 × 10 4 cells / well) and incubated for 4 hours to allow the cells to adhere to the plate. Jurkat-Dual cells (2×10 5 20 μL of supernatant from treated tumor cells were added to the wells. The plates were incubated for 16-20 hours. The supernatant was then harvested and treated with 50 μL of Quanti-Luc reagent, and the plate was immediately read on a plate reader to detect luciferase activity. A response indicating the presence of functional FAP-BiTEs was detected in the supernatants of NG-617, NG-615, NG-640, and NG-641-treated cancer cells, but not in EnAd-treated or untreated control supernatants. Figure 7 Surprisingly, given the similar levels of IFNα produced by NG-615 and NG-641, supernatants from cells treated with NG-615, including the other virus containing four transgenes, NG-641, had significantly lower levels of functional FAP-BiTE expression when compared to all other BiTE expressing viruses tested.

[0507] Example 2

[0508] Prior to constructing EnAd viruses carrying the BiTE transgene, the functional activity of the recombinant BiTE protein was assessed in a number of different assays.

[0509] Isolation of human peripheral blood mononuclear cells (PBMCs)

[0510] Human PBMCs were isolated by density gradient centrifugation from fresh human blood samples from healthy donors or whole blood leukocyte cones obtained from NHS Blood and Transplant UK in Oxford. In either case, the sample was diluted 1:2 with PBS, and 25 mL of this mixture was layered onto 13 mL of Ficoll (1.079 g / mL, lymphocyte separation solution (Ficoll-Paque Plus), GE Healthcare) in a 50 mL centrifuge tube. The sample was centrifuged at 1600 rpm for 30 minutes (Allegra X-15R, Beckman Coulter) at 22°C, using the lowest deceleration setting to maintain phase separation. After centrifugation, four layers were observed, including a plasma layer at the top, an interface containing PBMCs below, and a Ficoll layer and a layer of red blood cells and granulocytes at the bottom. PBMCs were collected using a Pasteur pipette and washed twice with PBS (1200 rpm, 10 minutes at room temperature) and resuspended in RPMI culture medium supplemented with 10% FBS.

[0511] Isolation of CD3-positive T cells

[0512] CD3 positive (CD3 T cell) cells were extracted from PBMCs by depletion of non-CD3 T cells using the Pan T cell isolation kit (Miltenyi Biotec, #130-096-535) according to the manufacturer's protocol. + )T cells.

[0513] Processing of primary ascites samples

[0514] Primary human ascites samples from patients with various indications were received from the oncology ward of Churchill Hospital (Oxford University Hospital), including but not limited to ovarian cancer, pancreatic cancer, breast cancer and gastric cancer. After receiving the sample, the cells and fluid were partially separated, and the fluid of the aliquot was frozen at -20 ° C for storage and future analysis. According to the manufacturer's instructions, the cell fraction was processed with red blood cell lysis buffer (Roche (Roche), #11814389001) to remove red blood cells. The cell type present in each sample was determined by staining of EpCAM, EGFR, FAP, CD45, CD11b, CD56, CD3, CD4, CD8, PD1 and CTLA4, and analyzed by flow cytometry. Fresh cells were then used for in vitro T cell activation and target cell lysis experiments. In some cases, cells were passaged in DMEM supplemented with 10% FBS for subsequent experiments.

[0515] Cell line maintenance

[0516] All cell lines were maintained in DMEM (Sigma-Aldrich, UK) or RPMI medium (Sigma-Aldrich, UK) as specified in Table 3, supplemented with 10% (v / v) fetal bovine serum (FBS, Gibco) in a humidified incubator (MCO-17AIC, Sanyo) at 37°C and 5% CO2. TM ) and 1% (v / v) penicillin / streptomycin (10 mg / mL, Sigma-Aldrich, UK), unless otherwise stated. Cells were separated every 2 to 3 days by enzymatic dissociation with trypsin / EDTA (0.05% trypsin 0.02% EDTA, Sigma-Aldrich, UK), and then reached confluence. During this process, the culture medium was aspirated and the cells were washed with 15 ml PBS and then treated with 2 mL trypsin / EDTA at 37°C for 2 to 10 minutes. Trypsin was neutralized with 10 mL of DMEM containing 10% FBS, and a portion of the cells were transferred to a new flask containing fresh culture medium. For conventional cell culture, the culture medium was supplemented with 10% FBS for infection and viral plasmid transfection with 2% FBS and for recombinant BiTE plasmid transfection without FBS supplementation.

[0517] Table 3

[0518]

[0519]

[0520] statistics

[0521] In the case of comparison of two conditions, statistical analysis was performed using t-test. In all other cases, statistical analysis was performed by using one-way ANOVA.

[0522] Characterization of human T cell activation by recombinant FAP BiTEs

[0523] The ability of FAP BiTEs to induce T cell activation in the presence or absence of normal human dermal fibroblasts (NHDF) cells was compared. Human CD3 + T cells (70,000 cells per well in a 96-well U-bottom plate) were cultured alone or co-cultured with NHDF cells (10:1 T:NHDF). Cells were co-cultured at 37°C for 24 hours and then harvested with enzyme-free cell dissociation buffer (Thermo Fisher Scientific, #13151014). CD45 expression was then analyzed by antibody staining and flow cytometry. + CD69 on T cells ( Figure 14A ) and CD25( Figure 14B ) expression levels and expressed as geometric mean fluorescence (gMFI) values. Plate-immobilized anti-CD3 antibody (7.5 μg / mL) was used as a positive control for T cell activation. FAP BiTE selectively induced the expression of activation markers CD69 and CD25 on T cells, indicating that it was able to activate T cells.

[0524] In a second similar experiment, NHDF cells (200,000 CD3 + After 6 hours of co-culture of cells with 40,000 NHDF cells in a well of a 96-well plate and 300 ng / mL FAP or control BiTE, T cells were assessed by intracellular cytokine staining. + T cells were intracellularly stained for IFNγ expression, and Brefeldin A was added to the culture medium 5 hours before harvest. As a positive control, T cells were stimulated with soluble PMA (10 ng / mL) and ionomycin (1 μg / mL). Figure 14C The results shown in indicate that FAP BiTEs in the presence of NHDF resulted in significantly higher numbers of IFNγ-expressing T cells compared to control BiTEs.

[0525] Example 4:

[0526] To further evaluate the function of IFNα produced from the transgenic NG-641, Jurkat-Dual cells were treated with supernatants from A549 tumor cells that were uninfected or infected with 10 particles per cell (ppc) of enadenotucirev (EnAd) or NG-641 for 3 days. TM To demonstrate that SEAP secretion was IFNα-specific, IFNα was blocked by incubating A549 supernatants with IFNα-specific antibodies for 30 min prior to treatment with Jurkat-Dual reporter cell line-containing isotype control antibodies as negative controls. Data ( Figure 8A) shows that the activity of NG-641 treated tumor cell supernatant in Jurkat Dual reporter gene assay is inhibited by anti-IFNα antibody, rather than isotype control, and is therefore mediated by IFNα. The function of CXCL9 and CXL10 chemokine transgenes in NG-641 is assessed using different reporter gene assay systems. This assay uses the PathHunterβ-arrestin reporter gene cell line (Eurofins) expressing CXCR3, a receptor for two chemokines. After CXCL9 / 10 binds to CXCR3 expressed by these cells, GPCR activation leads to β-arrestin recruitment to the receptor, which is measured using a signal gain assay based on enzyme fragment complementation (EFC) technology. PathHunterβ-arrestin CXCR3 reporter gene cells were treated with supernatant of A549 tumor cells that were uninfected or infected for 3 days with 10 particles (ppc) of EnAd or NG-641 per cell. The concentration of CXCL9 / 10 in the supernatant is proportional to the luminescence in the assay. To demonstrate that GPCR activation was CXCL9 / 10 specific, CXCL9 and CXCL10 were blocked by incubating A549 supernatants with CXCL9 / 10-specific antibodies for 30 minutes prior to treatment of PathHunter β-arrestin cells. Figure 8B The data presented in show that CXCR3 reporter cell activity was increased in the presence of supernatant from NG-641-treated tumor cells compared to EnAd or uninfected controls, and this increase was blocked by antibodies to CXCL9 / 10.

[0527] As an alternative method for chemokine functionality, the ability of chemokines to downregulate the cell surface expression of their specific receptors was used as the basis for determination, and the level of CXCR3 receptors on human T cells activated by anti-CD3 / CD28 was assessed. A549 tumor cells were not infected or infected with enadenotucirev (EnAd) or NG-641 with 1 viral particle (ppc) per cell for 7 days, and supernatants were collected. The activated T cells were then treated with supernatants for 30 minutes, and the levels of CXCR3 were measured by flow cytometry, and the data were plotted as mean fluorescence intensity (MFI). In order to prove that the downregulation of cell surface CXCR3 is CXCL9 / 10 specific, CXCL9 / 10 specific antibodies were incubated with A549 tumor cell supernatants for 30 minutes to block CXCL9 and CXCL10 before the treatment of activated T cells. Figure 9 The data presented in show that selective downregulation of CXCR3 expression on CD4 and CD8 T cells is induced by supernatants of NG-641-infected A549 tumor cells, and this effect is abrogated by pretreatment with anti-CXCL9 / 10 antibodies.

[0528] Example 5: Functional activity of FAP-BiTE-expressing viruses in ex vivo human tumor cell cultures

[0529] First, samples of freshly excised human tumors from planned surgical resections, provided by the biobank under full ethical approval, were minced with scissors and a scalpel, and then single-cell suspensions were generated using a GentleMAC tissue dissociator (Miltenyi Biotec). These unfractionated cell preparations were found to include tumor cells, fibroblasts, and different immune cells, including T cells, and were used to assess the ability of viruses to infect primary tumor cells, produce their encoded transgenes, and activate tumor-infiltrating T cells also present in culture. The cells were resuspended in a culture medium consisting of: Ham's F-12 nutrient mixture, GlutaMAX TM Supplement (Gibco), 1x insulin-transferrin-selenium-ethanolamine (ITS-X) (Gibco), amphotericin B 2.5 mg / mL (Gibco TM ), penicillin 100 units / mL, streptomycin 100 mg / mL, sodium pyruvate and 10% FBS, and at ∼1×10 6 Cells / ml were seeded in 96-well plates (final volume 0.25 ml) or 24-well plates (final volume 0.5 ml). They were inoculated with EnAd, NG-615, NG-617, NG-640 or NG-641 at 1000 ppc, or left untreated (UIC). As positive T cell activation controls, some wells were also stimulated with 2 μg / ml of anti-CD3 and anti-CD28 antibodies, respectively. The cells were cultured in duplicate wells for 72 hours, and then the supernatants were collected and analyzed using a multi-cytokine fluorescent bead-based kit (LEGENDplex TM ) and flow cytometry to measure the levels of different cytokines produced. Three non-small cell lung cancer (NSCLC) samples (T016, T017, T024), one renal cell carcinoma (RCC), and one colorectal cancer (CRC) liver metastasis sample were tested. Figure 4 Consistent with the transgene expression data in NG-6, IFNα was selectively produced in cultures treated with NG-615 and NG-641. Figure 10A Flt3 ligand (FLT3L) was readily detected after NG-615 treatment, but only very low levels were detected for the other viruses, and these levels were similar to those induced by activating T cells with anti-CD3 / 28, indicating that Flt3L in NG-615 cultures is a transgene product. The results for the other cytokines showed that, as in the tumor cell line inoculation studies described in Example 3 ( Figure 7), NG-615 vaccination resulted in much lower levels of T cell activation compared to other FAP-BiTEs encoding viruses NG-617, NG-640, and another virus NG-641 with four transgenes, e.g. Figure 10B As shown in the table for IFNγ, TNFα, IL-17, granzyme B and IL-13.

[0530] The activation of endogenous tumor T cells in resected NSCLC tumor cell cultures was also measured by flow cytometry, evaluating the levels of T cell activation markers CD25, CD69, and CD107a, as well as intracellular cytokine (IFNγ and TNFα) expression by both CD4 and CD8 T cells after 3 days of culture. Figure 11A As shown in Figure 3D, EnAd had little effect on activation markers or cytokine expression, whereas NG-617, NG-640, and NG-641 treatment resulted in upregulation of all of these measures of T cell activation. The similar activation levels of the FAP-BiTE-carrying viruses are consistent with the cytokine data above ( Figure 10B )

[0531] Example 6

[0532] In this example, the ability of T cells activated by recombinant FAP BiTEs to induce cell death in fibroblast targets was assessed.

[0533] FAP BiTEs induce T cell-mediated lysis of FAP-positive cell lines and primary cells

[0534] NHDF (7,000 cells) were co-cultured with 70,000 T cells in the wells of a U-bottom 96-well plate in the presence of medium alone or 300 ng / mL control or FAP BiTE. After 24 hours of co-culture, supernatants were collected and cytotoxicity was determined by LDH assay according to the manufacturer's instructions. Figure 15 The results in A show that FAP BiTE significantly increased lysis of NHDF cells.

[0535] In similar experiments, 7,000 primary lung fibroblasts (BTC100) were incubated with 70,000 CD3 + After 24 hours of co-culture, supernatants were collected and cytotoxicity was determined by LDH assay. Figure 15 The results in panels B and C show that FAP BiTEs significantly increased lysis of primary human cancer-associated fibroblast (CAF) cells. FAP expression in these and other patient-derived cell lines was as shown in Figure 5. Figure 16 shown.

[0536] 8,000 NHDF cells were co-cultured with 40,000 T cells and the BiTE concentration ranged from 2 × 10 3 to 2×10 -2 ng / mL was used to evaluate the dose-response relationship of FAP BiTE-mediated cell lysis. After 24 hours of co-culture at 37°C, LDH assay was performed on the supernatant to determine target cell cytotoxicity. The dose-response curve was fitted using a four-parameter nonlinear fitting model integrated into GraphPad Prism, resulting in an EC50 value of 3.2 ng / mL for FAP BiTE. Results ( Figure 17A ) shows the dose-dependent relationship between FAP BiTE concentration and cytotoxicity measured by LDH assay (shown as Abs 490 ).

[0537] Example 7

[0538] Stable FAP-expressing CHO and Ad293 cell lines were generated as a means to demonstrate the FAP antigen specificity of the FAP BiTEs by comparison with parental, untransfected cells.

[0539] Generation of stably transfected cell lines expressing FAP

[0540] The protein sequence of the FAP gene was obtained from the NCBI database (SEQ ID 23) and reverse transcribed to generate a DNA coding sequence synthesized by Oxford Genetics Ltd (Oxford, UK). The FAP gene was cloned into the pSF-Lenti vector using standard cloning techniques to generate the pSF-Lenti-FAP vector. HEK293T cells were transfected with the lentiviral FAP expression vector along with pSF-CMV-HIV-Gag-Pol, pSF-CMV-VSV-G, and pSF-CMV-HIV-Rev. Lipofectamine 2000 was used as a transfection reagent and added to the vector DNA at a DNA:liposome ratio of 1:2 and incubated with the cells at 37°C. After 48 hours, the supernatant containing the lentivirus was collected and mixed with polybrene (final concentration, 8 μg / mL). The lentivirus / polybrene mixture was added to the inoculated Ad293 or CHO cells and incubated at 37°C. On day 4, the supernatant was replaced with medium containing puromycin (2 μg / mL for Ad293 and 7.5 μg / mL for CHO). Stable variants were then cloned and FAP expression was determined by staining with FAP or isotope control antibodies on the parental cell line or stably transfected variants and analyzed by flow cytometry ( Figure 18A ).

[0541] FAP BiTE-mediated target cell lysis is specific for cells expressing FAP

[0542] CHO or CHO-FAP cells (7,000 cells) were cultured alone or co-cultured with human T cells (70,000 cells) in the presence of culture medium alone or 2 μg / mL of control or FAP BiTE in wells of a U-bottom 96-well plate. After 24 hours of incubation, supernatants were collected and target cell cytotoxicity was measured by LDH cytotoxicity assay as described in Example 4 ( Figure 18B T cell activation was also determined by flow cytometry analysis of CD69 and CD25 expression levels ( Figure 19 Cytotoxicity was only observed when CHO-FAP cells were cultured with T cells and FAP BiTEs. This indicates that FAP BiTE-mediated T cell activation and target cell lysis are highly specific and limited to FAP-expressing cells, rather than the FAP-negative parental cell line.

[0543] Example 8

[0544] In further experiments, the ability of recombinant FAP BiTE proteins to activate CD4 or CD8 T cells and the ability of each of these T cell subsets to lyse NHDF cells were evaluated. CD3 T cells were cultured in the presence of 300 ng / mL control or FAP BiTE in wells of a U-bottom 96-well plate. + T cells (35,000) were co-cultured with 7,000 NHDF cells and incubated at 37°C for 24 hours. Cells were harvested and stained with antibodies against CD4 or CD8 and CD69 and CD25 and analyzed by flow cytometry. Results ( Figure 20 A) Demonstration of FAP BiTE-induced CD4 + and CD8 + Increase in activation markers CD69 and CD25 in T cells.

[0545] In similar experiments, the ability of each T cell subset (CD4 and CD8) to kill target cells was assessed. CD4 T cells were extracted from CD3 purified cells by positive selection using a CD4 T cell isolation kit (Miltenyi Biotec, #130-045-101) according to the manufacturer's protocol. + T cells, including CD8 T cells in the non-separated flow-through. In a well of a U-bottom 96-well plate, 7,000 NHDF were mixed with 35,000 CD4 + or CD8 + T cells were co-cultured with 300 ng / mL control or FAP BiTE and incubated at 37°C. After 24 hours, supernatants were collected and target cell cytotoxicity was measured by LDH cytotoxicity assay. Results ( Figure 20 B) shows that FAP BiTE induces CD4+ and CD8 + T cells to kill NHDF cells.

[0546] Example 9

[0547] Characterizing FAP BiTE-mediated activation of autologous tumor-associated lymphocytes in primary malignant ascites

[0548] To evaluate the activity of BiTE proteins using cancer patient-derived cells, CD3 + T cells and FAP + Primary malignant ascites fluid samples of cells were used for testing. Unpurified ascites cells (thus unchanged from the time of receipt) were seeded at 250,000 cells per well in U-bottom 96-well plates in 100% ascites fluid or culture medium supplemented with 1% human serum in the presence of 500 ng / mL control or FAP BiTE. Untreated wells served as negative controls. After incubation at 37°C for 5 days, the total cell population was harvested and CD3 + The number of T cells ( Figure 21 A) and CD3 + The expression level of CD25 on T cells ( Figure 21 B) Total cell number per well was determined using precision counting beads. The results demonstrate that FAP BiTEs lead to a significant increase in T cell activation of tumor-associated T cells from cancer patients.

[0549] As an extension of the above experiments, duplicate wells were harvested and FAP was determined by flow cytometry. + The number of cells ( Figure 21 C). The total cell number per well was determined using precision counting beads. The results show that FAP BiTEs resulted in a significant reduction in the number of autologous FAP-expressing cells in ascites samples.

[0550] Example 10

[0551] EnAd viruses expressing recombinant BiTEs were engineered, produced, and purified using the methods described below.

[0552] Generation of BiTE-expressing Enadenotucirev

[0553] EnAd is a replication-competent chimeric group B adenovirus containing frequent nonhomologous nucleotide substitutions from Ad3 to Ad11p in the E2B region, a nearly complete E3 deletion, and a smaller E4 deletion localized to E4 or f4 (Kuhn et al. Directed evolution generates novel oncolytic viruses for the treatment of colorectal cancer. PLoS One. 2008 Jun 18;3(6):e2409).

[0554] Plasmid pEnAd2.4 was used to generate plasmids ppEnAd2.4-CMV-FAPBiTE, pEnAd2.4-SA-FAPBiTE, pEnAd2.4-CMV-ControlBiTE, pEnAd2.4-SA-ControlBiTE (Table 4) by direct insertion of a cassette encoding a FAP BiTE (SEQ ID NO: 1) or a control BiTE (SEQ ID NO: 3). The transgenic cassette contained a 5' short splice acceptor sequence CAGG or an exogenous CMV promoter (SEQ ID NO: 24), an EpCAM, FAP or control BiTE cDNA sequence, and a 3' polyadenylation sequence (SEQ ID NO: 25). Construction of the plasmid was confirmed by DNA sequencing. The exogenous CMV promoter is constitutively active and therefore results in early expression of the transgene. The splice acceptor sequence drives expression under the control of the viral major late promoter and results in subsequent transgenic expression after viral genome replication begins.

[0555] Table 4

[0556] Plasmid ID [Plasmid DNA]ng / ml pEnAd2.4-CMV-FAPBiTE 1322.8 pEnAd2.4-SA-FAPBiTE 3918.3 pEnAd2.4-CMV-ControlBiTE 189.1 pEnAd2.4-SA-ControlBiTE 236.2 pEnAd2.4-CMV-FAPBiTE-RFP 1599 pEnAd2.4-SA-FAPBiTE-RFP 1872 pEnAd2.4-CMV-ControlBiTE-RFP 1294 pEnAd2.4-SA-ControlBiTE-RFP 2082

[0557] Virus production and characterization

[0558] Plasmids EnAd2.4-CMV-EpCAMBiTE, pEnAd2.4-SA-EpCAMBiTE, pEnAd2.4-CMV-FAPBiTE, pEnAd2.4-SA-FAPBiTE, pEnAd2.4-CMV-ControlBiTE, and pEnAd2.4-SA-ControlBiTE were linearized by restriction digestion with the enzyme AscI to produce linear viral genomes. The digested DNA was purified by isopropanol extraction and precipitated at -20°C for 16 hours in 300 μl>95% molecular biology grade ethanol and 10 μl 3M sodium acetate. The DNA was precipitated by centrifugation at 14,000 rpm for 5 minutes and washed in 500 μl 70% ethanol, then centrifuged again at 14,000 rpm for 5 minutes. The clean DNA pellet was air-dried and resuspended in 100 μl water. 6.25 μg of DNA was mixed with 15.6 μl of lipofectamine transfection reagent in OptiMEM and incubated at room temperature for 20 minutes. The transfection mixture was then added to a T-25 flask containing Ad293 cells grown to 80% confluence. After the cells were incubated with the transfection mixture at 37°C for 4 hours, 4 ml of cell culture medium (DMEM high glucose and glutamine supplemented with 10% FBS) was added to the cells in 5% CO2, and the flasks were incubated at 37°C, 5% CO2. The transfected Ad293 cells were monitored every 24 hours and additional culture medium was added every 48 to 72 hours. Virus production was monitored by observing significant cytopathic effect (CPE) in the cell monolayer. Once extensive CPE was observed, virus was collected from the Ad293 cells by three freeze-thaw cycles. Individual virus clones were selected by serially diluting the collected lysate and re-infecting the Ad293 cells, and collecting wells containing single plaques. Once the infection reached complete CPE, serial infection of the Ad293 cells was performed to amplify the viral stock. The production of live virus during amplification was confirmed by observing significant CPE in the cell monolayer.

[0559] Virus purification

[0560] Once the effective virus stock was amplified, the virus was purified by double cesium chloride density gradient centrifugation (banding) to produce NG-603, NG-604, NG-605 and NG-606 virus stocks. These stocks were titrated by micoBCA assay (Life Technologies) according to the manufacturer's instructions (Table 5).

[0561] Table 5

[0562]

[0563] Example 11

[0564] The activity of NG-601, NG-602, NG-603, NG-604, NG-605, and NG-606 viruses was characterized using the methods described below.

[0565] Characterization of BiTEs encoding EnAd activity compared with EnAd in cancer cell lines

[0566] The ability of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606, or EnAd to replicate was analyzed by infecting A549 lung cancer cells and evaluated by qPCR. 5 The cells were seeded in wells of a 24-well plate at a cell density of 10 cells / well. The plates were incubated at 37°C, 5% CO2 for 18 hours, and the cells were then infected with 100 virus particles (ppc) per cell or kept uninfected. Wells were collected 24 hours, 48 ​​hours, or 72 hours after infection, and DNA was purified using the PureLink genomic DNA micro kit (Invitrogen) according to the manufacturer's protocol. The total viral genome was quantified by qPCR for each extracted sample or standard using the EnAd hexon gene-specific primer-probe set in the reaction mixture detailed in Table 6. qPCR was performed according to the procedure in Table 7.

[0567] Table 6

[0568] Reagents Volume / well (μl) 2×qPCRBIO probe mix (PCRBiosystems) 10 EnAd forward primer 0.08 EnAd reverse primer 0.08 EnAd probe 0.8 NFW 4.04 sample 5 Pore ​​volume 20

[0569] Table 7

[0570]

[0571] Quantification of the number of viral genomes detected per cell demonstrated that NG-601, NG-602, NG-603, NG-604, NG-605, NG-606, and EnAd viral replication was comparable in the A549 cell line ( Figure 22 A).

[0572] The oncolytic activity of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606, or EnAd was evaluated by infecting A549 cells ( Figure 22 B) A549 cells were cultured at 1.5×10 4 Cells were seeded at a density of 10 cells / well in 96-well plates. The plates were incubated at 37°C, 5% CO2 for 18 hours and then incubated with increasing amounts of ppc virus (5-fold serial dilutions, 4.1×10 -7 Cells were infected with up to 5000 virus ppc) or kept uninfected. AQueous One-Solution Cell Proliferation Assay (CellTiter A549 cytotoxicity was measured using the AQueous One Solution Cell Proliferation Assay (MTS) (Promega, #G3582). Dose-response curves were fitted using a four-parameter nonlinear fitting model integrated into GraphPad Prism. The IC50 values ​​generated for each virus demonstrated that the oncolytic activity of NG-601, NG-602, NG-603, NG-604, NG-605, NG-606, and EnAd was comparable for each virus.

[0573] Confirmation of functional BiTE transgene expression from NG-603, NG-604, NG-605, and NG-606

[0574] To determine whether the viruses NG-601, NG-602, NG-605, and NG-606 produced functional BiTEs, T cell activation assays were performed using CHO, CHO-EpCAM, and CHO-FAP cell lines as target cells. 10,000 target cells were incubated with 50,000 CD3 + T cells were co-cultured in wells of a U-bottom 96-well plate, in which Ad293 viral supernatant was diluted 100-fold in culture medium and incubated for 24 hours at 37°C, 5% CO2. T cells were harvested and stained with antibodies specific for CD25 and CD69 and analyzed by flow cytometry. Results ( Figure 23 ) Indicator viruses NG-601 and NG-602 express functional BiTE transgenes that activate T cells when co-cultured with CHO-EpCAM cells, and NG-605 and NG-606 express functional BiTE transgenes that activate T cells when co-cultured with CHO-FAP cells but not with CHO cells.

[0575] Quantification of BiTE expression in colon cancer cell lines

[0576] The amount of BiTE expression in the human colon cancer cell line DLD infected with NG-601, NG-602, NG-605, and NG-606 was evaluated. DLD cells were plated at 1.2 × 10 6DLD cells were seeded at a density of 100 cells / mL in 6-well culture plates. 18 hours after seeding, DLD cells were infected with 100 ppc of EnAd, NG-601, NG-602, NG-603, NG-604, NG-605, and NG-606. The cells were cultured for 72 hours, after which the supernatant was collected from the wells and centrifuged at 1200 rpm for 5 minutes to remove cell debris. The clarified supernatant was then used in a killing assay, and the cytotoxicity was compared to a standard curve generated with known concentrations of recombinant BiTEs, thereby enabling the determination of the amount of BiTEs in the viral supernatant.

[0577] To determine the amount of FAP BiTE produced from NG-605 and NG-606, a cytotoxicity assay was performed in which 8,000 NHDFs were incubated with 40,000 CD3 + T cells were co-cultured and DLD virus supernatant was diluted to 1 / 10 3 , 1 / 10 4 and 1 / 10 5 By diluting NHDF and CD3 + T cells were co-cultured with FAP or control BiTE in 10-fold serial dilutions from 3333 to 3.33 x 10 -4 ng / μL was incubated to generate a standard curve. Supernatants were collected 24 hours after treatment and cytotoxicity was measured by LDH assay. The amount of BiTE expressed was determined by comparing the cytotoxicity of the viral supernatant with the cytotoxicity of the recombinant BiTE standard curve. Results ( Figure 24 ) Indicator viruses NG-605 and NG-606 produced 9.8 μg and 49.2 μg FAP BiTE per million DLD cells, respectively.

[0578] Example 12

[0579] In addition to encoding FAP or control BiTEs, NG-607, NG-608, NG-609, and NG-610 viruses also carry a red fluorescent protein (RFP) transgene for visualization of infected cells using fluorescence microscopy (SEQ ID NOs: 20 and 21, Table 4). The functional activities of these viruses were characterized using the methods described below.

[0580] Confirmation of transgene expression from NG-607, NG-608, NG-609, and NG-610

[0581] The ability of viruses NG-607, NG-608, NG-609, and NG-610 to produce their BiTE transgenes was assessed by infecting Ad293 cells. Ad293 cells were plated at 1 × 10 6Cells were seeded per well in 6-well plates. The plates were incubated at 37°C, 5% CO₂ for 24 hours, and then the cells were infected with 100 ppc of virus or left uninfected. 48 hours after infection, plaques were illuminated with a fluorescent mercury lamp and photographed ( FIG18 ). The results showed that viruses NG-607, NG-608, NG-609, and NG-610 expressed the RFP transgene.

[0582] Example 13

[0583] In the next series of experiments, the ability of EnAd and FAP or control BiTE viruses NG-603, NG-604, NG-605, NG-606, NG-607, NG-608, NG-609, and NG-610 to kill target cells, including tumor cells and fibroblasts, was evaluated.

[0584] In the first study, the ability of EnAd to kill DLD cells was evaluated using xCELLigence technology. DLD cells were cultured at 1.2 × 10 4 Cells / well were seeded in 48-well E-plates and incubated at 37°C, 5% CO2 for 18 hours before being infected with 100 EnAd ppc or left uninfected. XCELLigence was used to measure target cell cytotoxicity every 15 minutes during the 8-day incubation period.

[0585] In similar experiments, xCELLigence was used to investigate the relationship between SKOV tumor cells and CD3 + The ability of NG-603, NG-604, NG-605, NG-606, and EnAd to kill NHDF cells was evaluated in co-culture with T cells. NHDF cells and SKOV cells were cultured at 4 × 10 3 and 1×10 3 Cells were seeded per well in a 48-well E-plate. The plates were incubated at 37°C, 5% CO2 for 18 hours, and then the cells were infected with 100 ppc of NG-603, NG-604, NG-605, or NG-606 EnAd or left uninfected. After a 2-hour incubation, 37,500 CD3 + T cells. xCELLigence was used to measure target cell cytotoxicity every 15 minutes. Results ( Figure 26A ) demonstrated that the FAP BiTE-expressing viruses NG-605 and NG606, but not EnAd or the control BiTE-expressing viruses NG-603 and NG-604, were able to induce lysis of NHDF cells, with the kinetics depending on the promoter used for BiTE expression (CMV promoter was faster).

[0586] In similar experiments, LDH cytotoxicity assay was used in the presence of SKOV and CD3 + The ability of NG-603, NG-604, NG-605, NG-606, and EnAd to kill NHDF cells was evaluated in co-culture with T cells. NHDF cells and SKOV cells were cultured at 8 × 10 3 and 2×10 3 Cells were seeded per well in a 96-well U-bottom plate and infected with 100 ppc of NG-603, NG-604, NG-605, or NG-606 EnAd or remained uninfected. After 2 hours of incubation, 75,000 CD3 + T cells were plated and incubated at 37°C, 5% CO2. Supernatants were collected at 0, 24, 48, and 96 hours after treatment, and cytotoxicity was measured by LDH cytotoxicity assay. Results ( Figure 26B ) demonstrated that viruses NG-605 and NG606 expressing the FAP BiTE, but not EnAd or viruses NG-603 and NG-604 expressing the control BiTE, were able to induce lysis of NHDF cells, with the kinetics dependent on the promoter used for BiTE expression.

[0587] As an extension of the above LDH experiments, cells were also harvested at 0, 24, 48, and 96 hours post-treatment, stained with antibodies to CD45, CD69, and CD25, and analyzed by flow cytometry. Figure 27 ) demonstrated that the FAP BiTE-expressing viruses NG-605 and NG-606, but not EnAd or the control BiTE-expressing viruses NG-603 and NG-604, were able to induce T cell activation, with the kinetics dependent on the promoter used for BiTE expression.

[0588] In similar experiments, the dependence on FAP for induction of FAP BiTE-mediated T cell activation was assessed. SKOV cells were plated at 2 × 10 3 cells / well alone or in combination with NHDF cells at 8 × 10 3 Cells / well were seeded. Viral particles were added to each well at 100 ppc and the plate was incubated at 37°C, 5% CO2. Two hours later, 75,000 CD3 + T cells were added and the plates were further incubated. 96 hours after infection, cells were harvested and stained for CD45 and CD25 and analyzed by flow cytometry ( Figure 28 A) The results demonstrate that the FAP BiTE-expressing viruses NG-605 and NG-606 induce T cell activation only in the presence of FAP-positive NHDF cells.

[0589] In similar experiments, the specificity of BiTE expression driven by promoters (CMV or viral MLP / SA) in NG-605 and NG-606 was further investigated. 3 NHDF cells were inoculated at 100 cells / well. 100 virus particles were added to each well and the plates were incubated at 37°C, 5% CO2. Two hours later, 40,000 CD3 cells were added and the plates were further incubated. 72 hours after infection, supernatants were collected and cytotoxicity was measured by LDH cytotoxicity assay. Results ( Figure 28 B) Demonstration that the CMV-driven virus NG-605, but not the SA-driven NG-606, is able to mediate killing of NHDF cells when infected alone.

[0590] The results indicate that both NG-605 and NG-606 are able to induce T cell activation and target cell lysis, although the kinetics differ slightly depending on the promoter used.

[0591] Time-lapse videos were acquired to visualize virus- or T-cell-mediated target cell lysis by recombinant FAP BiTEs, EnAds, NG-603, or NG-605. NHDF cells were stained with CellTracker Orange CMTMR Dye (Life Technologies, #C2927) and CD3 chromatin was stained with the CellTrace Violet Cell Proliferation Kit (Life Technologies, #C34557) according to the manufacturer's protocol. + T cells. NHDF was stained at 7.5×10 3 Cells were seeded in 24-well plates at a concentration of 1.35 × 10 4 DLD or SKOV tumor cells were co-cultured. Plates were incubated at 37°C, 5% CO2 for 18 hours. Cells were then treated with 300 ng / mL FAP BiTE or infected with 100 ppc of EnAd, NG-603, and NG-605, or left untreated. After two hours of incubation, 100,000 stained CD3 + T cells were added to the necessary wells. Videos were acquired on a Nikon TE 2000-EEclipse inverted microscope, capturing images every 15 minutes for 96 hours. Figure 29 Frames from the video are shown in . The results show that recombinant FAP BiTE and NG-605, but not EnAd or NG-603, are able to induce rapid lysis of NHDF cells.

[0592] In similar experiments, NHDF cells were stained with CellTracker Green CMFDA Dye (Life Technologies, #C2925) and CD3 chromatin was stained with CellTrace Violet Cell Proliferation Kit (Life Technologies, #C34557) according to the manufacturer's protocol. + T cells. NHDF was stained at 7.5×10 3 Cells were seeded in 24-well plates at a concentration of 1.35 × 10 4 DLD or SKOV tumor cells were co-cultured. The plates were incubated at 37°C, 5% CO2 for 18 hours. The cells were then infected with 100 ppc of NG-607, NG-608, NG-609, or NG-610 or left uninfected. After two hours of incubation, 100,000 stained CD3 + T cells were added to the necessary wells. Videos were acquired on a Nikon TE 2000-EEclipse inverted microscope, capturing images every 15 minutes for 96 hours. Figure 30 Frames from the video are shown in . The results show that all viruses resulted in tumor cell infection (RFP, red fluorescence, positive), but only NG-609 and NG-610 were able to induce rapid lysis of co-cultured NHDF cells.

[0593] Example 14

[0594] In this example, EnAd, NG-603, NG-604, NG-605, and NG-606 were evaluated for their effects on FAP from cancer patients. + Activation of autologous tumor-associated lymphocytes from primary malignant ascites. Patient samples considered suitable for further analysis were those containing CD3 + T cells and FAP + Cell samples.

[0595] In the first experiment, unpurified (and therefore unchanged from the time of receipt) ascites cells from patients were seeded at 250,000 cells per well in U-bottom 96-well plates in 100% ascites fluid. Cells were infected with 100 ppc of virus, and untreated wells served as negative controls. EnAd-CMV-GFP and EnAd-SA-GFP were also included as reporter genes in the experiment, and micrographs were taken to determine infection and late viral gene expression, respectively. After incubation at 37°C for 5 days, the total cell population was harvested and CD3 + The expression level of CD25 on T cells ( Figure 32A) Total cell number per well was determined using precision counting beads. The results demonstrate that FAPBiTE viruses NG-605 and NG-606 significantly increase T cell activation in tumor-associated lymphocytes.

[0596] As an extension of the above experiments, duplicate wells were harvested and endogenous FAP was determined by flow cytometry. + The total number of cells in each well was determined using precision counting beads. Figure 40 B) shows that NG-605 and NG-606 resulted in a significant reduction in the number of autologous FAP-expressing cells in ascites samples, indicating that some FAP + The cells have been killed by activated T cells.

[0597] In a second experiment, unpurified (and therefore unchanged from the time of receipt) ascites cells from cancer patients were seeded at 250,000 cells per well in U-bottom 96-well plates in 100% ascites fluid or in culture medium supplemented with 1% human serum. Cells were infected with 100 ppc of virus, and untreated wells served as negative controls. EnAd-CMV-GFP and EnAd-SA-GFP were also included as reporter genes to determine infection and late viral gene expression, respectively, using micrographs. After incubation at 37°C for 5 days, the total cell population was harvested and CD3 + The number of T cells ( Figure 33 ) and CD3 + The expression level of CD25 on T cells ( Figure 34 The total cell number per well was determined using precision counting beads. The results demonstrated that, for this patient, recombinant FAP BiTE and NG-605, but not NG-606, significantly increased T cell activation of tumor-associated lymphocytes in culture. Neither virus caused activation in ascites fluid.

[0598] As an extension of the above experiments, duplicate wells were harvested and FAP was determined by flow cytometry. + The number of cells ( Figure 35 The total number of cells per well was determined using precision counting beads. The results demonstrated that recombinant FAP BiTE and NG-605, but not NG-606, resulted in a significant reduction in the number of autologous FAP-expressing cells in culture. Neither virus caused FAP expression in ascites fluid. + Cell reduction.

[0599] Example 15 - Discussion

[0600] Oncolytic viruses offer an interesting new strategy that combines multiple therapeutic modalities in a single targeted self-amplifier (Keller and Bell, 2016; Seymour & Fisher, 2016). Because they replicate selectively within cancer cells and spread between cells, some oncolytic viruses are thought to mediate cell death through non-apoptotic death pathways (Ingemarsdotter et al., 2010; Li et al., 2013), which is part of the process that allows viral particles to escape from dying cells. In particular, EnAd kills cells through a pro-inflammatory process called tumor or ischemic cell death (Dyer, 2017). This non-apoptotic death mechanism leads to the release of several pro-inflammatory cellular components, such as ATP, HMGB1, and exposure of calreticulin (called damage-associated molecular patterns,

[0601] DAMPs) (Weerasinghe and Buja, 2012) and may be key to the ability of viruses to promote effective anti-cancer immune responses. However, beyond the direct lytic consequences, viruses also offer the potential to encode and express other anti-cancer biologics, thereby circumventing delivery challenges and ensuring that biologics reach their highest concentrations in the tumor microenvironment. Imlygic encodes GM-CSF, but the possibilities of viral delivery are virtually endless and offer many exciting opportunities to design multimodal therapeutic strategies with additive or synergistic anti-cancer effects (de Gruijl et al., 2015; Hermiston & Kuhn, 2002).

[0602] Encoding BiTEs in oncolytic viruses provides a powerful means of activating tumor-infiltrating lymphocytes to become cytotoxic and lyse antigen-positive target cells, thereby providing a therapeutic modality that is completely separate from the direct viral lytic effect.

[0603] BiTE-targeted cytotoxicity is fully antigen-specific and can be mediated by both CD4 and CD8 T cells

[0604] (Brischwein et al., 2006), and can be incorporated into oncolytic adenoviruses and expressed only in cells that allow viral replication. In addition, the current study shows for the first time that endogenous T cells in liquid cancer biopsies can be activated by BiTEs and virally encoded BiTEs and can kill endogenous tumor cells without any additional stimulation or reversal of immunosuppression. Importantly, this can even occur in primary fluids including the microenvironment of peritoneal ascites or pleural effusions, as surrogates for the immunosuppressive microenvironment of solid tumors.

[0605] Carrying oncolytic viruses to express BiTEs combines two distinct therapeutic mechanisms, the former providing lytic death of tumor cells that allow viral infection, and the latter targeting T cell cytotoxicity through specific selected antigens. This provides considerable flexibility in the design of therapeutic methods, and it is possible to use BiTEs to deliver cytotoxicity to tumor-associated cells that are relatively resistant to direct killing of viruses. For example, although we have illustrated the technology here using BiTEs that recognize cancer-associated antigens (EpCAM), the cytotoxicity of tumor-associated fibroblasts or other stromal cells can also be targeted using BiTE methods. In fact, even when the target recognized by BiTE is not limited to expression in the tumor microenvironment, by associating BiTE production with viral replication, the expression of BiTEs is allowed to be spatially limited to the tumor, thereby minimizing systemic toxicity. This is important because intravenously administered BiTEs show relatively short circulation kinetics (Klinger et al., 2012) and are generally associated with considerable targeted tumor extracellular toxicity (Teachey et al., 2013).

[0606] The possibility of encoding BiTEs within oncolytic viruses has been previously explored using an oncolytic vaccinia virus with a BiTE targeting Ephrin A2. This reagent showed that Ephrin BiTEs can mediate activation of PBMCs and antigen-targeted tumor cell killing in vitro and in vivo. Interestingly, although BiTEs can activate T cells, they do not lead to T cell proliferation without the addition of exogenous IL-2, whereas the BiTEs used in the current study led to extensive proliferation of PBMCs in vitro and extensive proliferation of tumor-associated lymphocytes using ex vivo clinical biopsy samples.

[0607] We believe that the observed differences may reflect different BiTE designs, different oncolytic viruses used, or may depend on the antigen density to generate sufficient CD3 cross-linking on T cells.

[0608] A central goal of oncolytic virus therapy is to create anti-cancer T cell responses that recognize patient-specific neoantigens as well as “public” tumor-associated antigens. Lytic viruses can achieve this by lysing tumor cells and virus-associated pathogen-associated molecular patterns (PAMPs) in the context of DAMPs to stimulate improved antigen presentation. Following intravenous delivery of EnAd, immunohistochemical staining of resected colon tumors showed that the virus promoted a massive influx of CD8+ T cells into tumor tissue (Garcia-Carbonero, 2017). However, although this may be a very effective approach, adaptive T cell responses ultimately rely on the expression of MHC class I antigens by tumor cells to allow targeted killing. Loss of MHC expression is a well-documented tumor immune evasion strategy (Garrido et al., 2016).

[0609] Notably, the immediate cytotoxic strategy of oncolytic viruses carried by BiTEs is exerted by tumor cells independently of MHC class I and can therefore be used to kill cancer cells even when tumor cells have lost MHC expression.

[0610] Thus, this study demonstrates that encoding BiTEs within EnAds offers a particularly promising strategy for achieving targeted expression in disseminated tumors, leveraging the known blood stability and systemic bioavailability of the virus, which has already been investigated in several early clinical trials. Notably, in a study in which the virus was injected intravenously several days before resection of primary colon cancer, subsequent immunohistological evaluation of tumor sections showed that the virus had reached the tumor site and produced a strong nuclear hexon signal, indicating successful infection and selective viral replication in tumor cells. This confirms preclinical data (Di et al., 2014; Illingworth, 2017), which indicate that this virus is stable in 100% of human blood and should be able to achieve tumor-targeted infection of disseminated and metastatic malignancies in human patients.

[0611] BiAds can be encoded by EnAds without any loss of oncolytic virulence, reflecting the virus's considerable transgene packaging capacity. The presence of the transgene does not affect the physicochemical properties of the viral particle, so the modified virus should exhibit clinical pharmacokinetics identical to the parental agent and should be able to selectively express the encoded BiTE within tumors throughout the body. This provides an exciting and potentially very effective new approach to systemically targeted cancer immunotherapy, which should now be prioritized for clinical evaluation.

[0612] Example 16

[0613] Immunosuppression of human T cell activation and target cell cytotoxicity by malignant effusions from patients

[0614] Malignant effusions represent an environment with potential immune tolerance, which has the suppressed immune response typically observed in patients with advanced metastatic cancer. Human IL-10 ELISA MAX kit (Biolegend, 430604) was used to measure the amount of IL-10, which is considered to be an anti-inflammatory cytokine, in normal serum or patient malignant effusions (A, peritoneal ascites; P, pleural effusion). The IL-10 levels in the effusions (88.1 pg / mL to 633.4 pg / mL) far exceeded the levels measured in normal serum (7.2 pg / mL to 10 pg / mL).

[0615] The ability of CD3 / CD28 beads (Gibco, 11161D) to activate PBMC T cells in the presence of normal serum, ascites or pleural fluid was studied. Human PBMC T cells (100,000 cells per well in 96-well plates) were processed with CD3 / CD28 beads (following manufacturer instructions) in normal serum or patient exudates (50%). T cells not processed in each liquid were used as negative controls. After 24 hours of cultivation, cells were collected and then analyzed for the expression levels of CD69 and CD25 on CD3+ T cells by antibody staining, and flow cytometry was expressed as the percentage of double positive (CD69+CD25+ cells). In normal serum, anti-CD3 / CD28 beads produced approximately 60% of the T cells of CD25 and CD69 double positive, and the presence of ascites fluid weakened the T cell activation in 6 / 12 liquids.

[0616] In a similar experiment, 100,000 T cells were treated with CD3 / CD28 beads in the presence of normal serum, ascites or pleural fluid (50%). Anti-CD107a or isotype control antibodies were added directly to the culture medium. After 1 hour, monensin (BD Golgistop, BD Biosciences) was added according to the manufacturer's instructions. After another 5 hours, cells were collected and analyzed by flow cytometry to determine degranulation. In normal serum, anti-CD3 / CD28 beads produced approximately 22.5% T cell degranulation, while the presence of ascites fluid weakened T cell activation in 10 / 12 fluids. Degranulation levels were significantly correlated with the amount of IL-10 in each fluid (Pearson coefficient, r=-0.7645; p=0.0038).

[0617] In similar experiments, 75,000 T cells were co-cultured with 15,000 SKOV3 and EpCAM in the presence of normal serum, ascites, or pleural fluid (50%). T cells treated with a control BiTE in each fluid served as a negative control. After 24 hours of culture, cells were harvested and analyzed for CD69 and CD25 expression on CD3+ T cells by antibody staining, and flow cytometry was performed to express the percentage of double-positive (CD69+CD25+ cells). In the presence of normal serum, the EpCAM BiTE resulted in approximately 67.6% of T cells being double-positive for CD25 and CD69, while the presence of ascites fluid attenuated T cell activation in 0 of 12 fluids and slightly induced activation in 4 of 10 fluids.

[0618] In similar experiments, 75,000 T cells were co-cultured with 15,000 SKOV3 and EpCAM beads in the presence of normal serum, ascites, or pleural fluid (50%). T cells treated with a control BiTE in each fluid served as a negative control. Anti-CD107a or isotype control antibodies were added directly to the culture medium. After 1 hour, monensin (BD Golgistop, BD Biosciences) was added according to the manufacturer's instructions. After an additional 5 hours, cells were harvested and analyzed by flow cytometry to determine degranulation. In the presence of normal serum, EpCAM BiTE beads resulted in approximately 41.4% T cell degranulation, while the presence of ascites fluid attenuated T cell activation in 2 / 12 of the fluids.

[0619] The ability of EnAd-SA-EpCAM BiTE and EnAd-SA-Control BiTE to induce T cell-mediated lysis of target cells in malignant effusions was evaluated using xCELLigence technology. SKOV3 cells were seeded at 1e4 cells / well in 48-well E-plates. Plates were incubated at 37°C, 5% CO₂ for 18 hours, after which cells were infected with 100 viral particles per cell (ppc) or left uninfected. Two hours later, normal serum or PBMC T cells from patient effusions (final, 50%) were added (5:1). xCELLigence was used to measure target cell cytotoxicity every 10 minutes. Results demonstrated that T cell BiTE-mediated lysis of SKOV3 was independent of the fluid used.

[0620] Unpurified ascites cells (thus unchanged from the time of receipt) were seeded at 100,000 cells per well in a flat-bottom 96-well plate in RPMI medium or ascites fluid. Cells were treated with EpCAM or control BiTEs, and untreated wells served as negative controls. After incubation at 37°C for 24 hours, cells were harvested and the expression levels of CD25 and CD69 on CD3 cells were determined. The results demonstrated that EpCAM BiTEs resulted in a significant increase in T cell activation (CD69 / CD25 double positive) of tumor-associated lymphocytes, with a slight increase in ascites fluid.

[0621] In a similar experiment, unpurified ascites cells (therefore unchanged from the time of receipt) were seeded in a flat-bottomed 96-well plate in RPMI culture medium or ascites fluid at 100,000 cells per well. Cells were treated with EpCAM, control BiTE or recombinant BiTE virus (100 vp / cell), and untreated wells were used as negative controls. After incubation at 37°C for 5 days, the total cell population was collected, and the number of CD3+ cells and the expression level of CD25 on CD3 cells and the number of endogenous EpCaM+ cells determined by flow cytometry were determined. Precision counting beads were used to determine the total cell number per well. The results showed that EpCAM BiTE and EnAd expressing EpCAMBiTE resulted in a significant increase in T cell activation (CD3 number, CD25) of tumor-associated lymphocytes and the cytotoxicity of EpCAM+ cells in RPMI culture medium and ascites fluid.

[0622] As an extension of the above experiments, exudate samples from six additional patients (7 in total) were processed identically in ascites fluid and the number of CD3+, CD25 expression of T cells, and the number of EpCAM+ cells were determined by flow cytometry. The results showed that EpCAM BiTE and EnAd expressing EpCAM BiTE led to a reproducible and significant increase in T cell activation of tumor-associated lymphocytes (CD3 count, CD25) and cytotoxicity of EpCAM+ cells in a series of exudate biopsy samples.

[0623] Example 17

[0624] FAP BiTE mediates T cell activation and FAP+ cell killing by different donor T cells

[0625] In additional experiments, the T cell activation properties of the recombinant FAP BiTE proteins tested in co-cultures of NHDFs and T cells were further evaluated using the methods described in Example 2, compared to control BiTEs and polyclonal T cell activation using anti-CD3 / CD28 magnetic beads.

[0626] After 24 hours of culture, the supernatant was tested for IFNγ by ELISA. Figure 36 A), and a panel of cytokines ( Figure 36 B) Control BiTEs induced no significant changes in any cytokine, however, FAP-BiTEs led to a robust increase in interferon-γ, IL-2, TNFα, IL-17, and IL-10, consistent with stimulation of distinct subsets of T cells, and IFNγ production was much greater than that elicited by anti-CD3 / CD28.

[0627] Stimulation with FAP BiTEs, but not control BiTEs, in the presence of NHDF cells also induced rapid degranulation (within 6 hours) of T cells (CD4+ and CD8+ subsets), as determined by externalization of CD107a / LAMP1 on the T cell surface (assessed by flow cytometry), which correlated well with their ability to kill target cells ( Figure 36 A and B). This degranulation induction by FAP BiTEs translates into efficient fibroblast lysis ( Figure 37 C), by interacting with PBMC T cells (EC 50 ng / mL) after 24 h of co-culture, where induced T cell activation and cytotoxicity were observed using 6 / 6 donor T cells ( Figure 37 D) Control BiTEs did not induce cytotoxicity, consistent with T cells being maintained in an inactivated state.

[0628] Example 18

[0629] Effects of FAP BiTE and EnAd-FAP BiTE viruses on cells from primary malignant ascites samples of different cancer patients

[0630] As a follow-up to the studies described in Example 16, fresh primary malignant peritoneal fluid from additional cancer patients was obtained for investigation of EnAd FAP BiTE viral activity. + Tumor cells and FAP + Three patient samples of fibroblasts were expanded ex vivo, and mixed (adherent) cell populations were cultured with PBMC-derived T cells and unmodified or BiTE-expressing EnAd viruses. After 72 hours, total cells were harvested and FAP was determined by flow cytometry. + ( Figure 38 A) and EpCAM + cell( Figure 38 In addition, the activation status of T cells (by CD25 expression) was measured. Infection with EnAd-CMV-FAPBiTE and EnAd-SA-FAPBiTE induced T cell activation and FAP in all patient samples. + Cell depletion, and EpCAM + There was no significant change in the levels of tumor cells. Neither the parental EnAd nor the control virus induced observable T cell activation, FAP + The cell numbers were similar to those of the uninfected control.

[0631] Importantly, FAP + This depletion of fibroblasts consistently resulted in a robust reduction in the levels of the immunosuppressive cytokine TGFβ detected in the supernatants.

[0632] In a second series of experiments, total (and unpurified) cells from five patient biopsy samples were evaluated to assess the activity of endogenous tumor-associated T cells in the samples. Cells were seeded in 50% ascites fluid and treated with recombinant control or FAP BiTE protein or 100 vp / cell EnAd or EnAd-BiTE virus. After 5 days of incubation, T cell activation (by CD25 expression) and the number of residual FAPs were measured by flow cytometry. + cell( Figure 39 A and B). In all three patient samples, recombinant FAP-BiTE and EnAd-CMV-FAP BiTE induced robust T cell activation, with up to approximately 80% of patient-derived T cells activated, which led to FAP + Interestingly, EnAd-SA-FAP-BiTE induced CD25 expression in 2 / 3 samples, with no observable activation or FAP in patient 1. + This may be due to insufficient viral infection of tumor cells and production of BiTE proteins (EpCAM was not detected in this sample by flow cytometry). + tumor cells), which is consistent with the requirement of tumor cells for MLP(SA)-driven transgene expression (which may also explain the lack of T cell activation and FAP expression in EnAd-SA-FAP-BiTE viruses). + Cell depletion, patient ascites samples such as Figures 42 to 44 Overall, the data show that following tumor cell infection, EnAd expressing FAP-BiTE can reproducibly lead to the activation of tumor-associated T cells to kill endogenous fibroblasts.

[0633] Another experiment investigated whether FAP-BiTE activity could be improved by blocking the PD-1 checkpoint using patient biopsy samples in which T cells were 73.6% PD-1 positive and FAP + 62.9% of cells were PDL1 positive ( Figure 40 A). Co-cultures similar to those described above were established in the presence or absence of a purified blocking mouse IgG2b antibody against human PDL1 (BioLegend, clone 29E.2A3) at a final concentration of 2.5 μg / mL. After 2 days of culture, total cells were harvested and residual FAP+ cells and T cell activation were measured. Inclusion of blocking anti-PDL1 antibody resulted in a modest increase in CD25 induction ( Figure 40 B) and IFNγ production was two-fold higher ( Figure 40 C), whereas on day 2, depletion of FAP+ cells was unchanged in either case, with almost complete lysis ( Figure 40 D).

[0634] It has been reported that tumor-associated lymphocytes (TALs) isolated from ascites of ovarian cancer patients are enriched for PD-1 expression and have impaired effector functions, including cytotoxicity and IFNg production. + The PD-1 expression of the cells was 2-fold higher than that of peripheral blood mononuclear cells (PBMCs) from three healthy donors ( Figure 41 A). To evaluate T cell function in these cancer biopsies, NHDF cells and unpurified PBMC or ascites cells (in Figure 41 The % CD3+ cells in each sample are shown in B) were co-cultured with control or FAP BiTE-containing supernatants, and supernatants were harvested 5 days later and tested for IFNγ by ELISA ( Figure 41 C). The control BiTE did not induce IFNγ. Three ascites cell samples produced IFNγ at levels similar to those of the PBMC samples, while the other three samples had a reduced response to FAPBiTE. We next investigated the ability of these T cells to induce BiTE-mediated lysis of NHDF cells. NHDFs were plated and PBMCs or ascites cells were added with BiTE-containing supernatants, and the viability of the cells in culture was monitored in real time using the xCELLigence cytotoxicity assay system. Despite the variability in IFNγ production, all ascites samples induced complete cytotoxicity of NHDF cells when FAP BiTE was added, with overall similar BiTE-mediated NHDF lysis rates similar to those when PBMCs were affected ( Figure 41 D).

[0635] To investigate whether FAP BiTEs could mediate T cell activation in the presence of patient malignant effusion samples (all 50%), PBMC T cells were activated with control or FAP BiTEs in the presence of NHDF cells, or with anti-CD3 / CD28 magnetic beads in 50% normal human serum (NS) or different (cell-free) malignant effusion samples. Whereas in normal serum, 74% of T cells were activated (double positive for both CD25 and CD69) 24 hours after stimulation with anti-CD3 / CD28 beads, 3 / 5 ascites fluids tested had significantly attenuated T cell activation compared to the responses in NS ( Figure 42 A). However, when PBMCs were cultured with NHDF and stimulated with FAP BiTEs, no inhibition of T cell activation was observed in the presence of any of the exudates ( Figure 42 B), demonstrating that FAP BiTEs can overcome immunosuppressive mechanisms to activate T cells.

[0636] Example 19

[0637] EnAd-FAPBiTE-mediated oncolysis and T cell stimulation polarize CD11b+ TAMs in patient ascites to a more activated phenotype

[0638] To investigate whether FAP BiTE-mediated T cell activation produces Th1 cytokines, including IFNγ, TNFα, and IL-2, and subsequently eliminates FAP + The effect of fibroblasts (and the associated reduction in TGFβ1 is associated with other changes in the tumor) on tumor-associated macrophages (TAMs) in unfractionated ascites cell samples was evaluated from an immunosuppressive and pro-oncogene microenvironment to anti-tumor activity. All unpurified patient ascites cells were placed in 50% ascites fluid and treated with free control or FAP BiTE or infected with EnAd-SA-control BiTE or EnAd-SA-FAPBiTE virus (100 vp / cell). In parallel, some cells were treated with IFNγ to induce an activated CD11b myeloid cell phenotype. After 3 days of incubation, the activation state of T cells was first measured; CD25+ cells were measured by flow cytometry and IFNγ secretion was measured by ELISA.

[0639] Treatment with FAP BiTE and EnAd-SA-FAPBiTE resulted in approximately 60% of CD3 + T cells become CD25+( Figure 43 A) and a large amount of IFNγ in the culture supernatant ( Figure 43 B). No increase above background was observed for CD25 expression or IFNγ by control BiTEs or control virus. To assess TAM polarization, expression levels of CD64 and CD86 (M1 or “activated” macrophage markers) and CD206 and CD163 (M2 or TAM markers) were measured on CD11b+ cells by flow cytometry ( Figure 43 C) Treatment with free FAP BiTE or EnAd expressing FAP BiTE induces a more activated phenotype, as evidenced by a significant increase in CD64 expression and a strong decrease in CD206 and CD163 - similar to that observed when IFNγ is spiked into the cultures.

[0640] Although treatment with free FAP BiTE or control virus in this experiment did not cause significant changes in CD86 above background, EnAd expressing the FAP BiTE induced a substantial increase in CD86 expression, indicating that EnAd viral infection and FAP BiTE activity can synergistically activate primary myeloid cells in suppressive tumor microenvironments such as the malignant ascites samples tested here. In this study, IFNγ treatment induced a modest decrease in CD86, indicating that the robust increase in CD86 observed with EnAd-SA-FAPBiTE may be through an IFNγ-independent mechanism.

[0641] Example 20

[0642] EnAd-FAPBiTE activates tumor-infiltrating lymphocytes and induces cytotoxicity in ex vivo solid prostate tumor biopsies

[0643] Tissue slice culture provides one of the most realistic preclinical models for a variety of tissues, organs, and tumors. To evaluate the activity of viruses expressing FAP BiTEs in this highly clinically relevant setting, several paired punch biopsies of malignant and benign prostate tissue from resected human prostates were studied. Upon initial screening, the precursor tissue reproducibly displayed annular rings of EpCAM+ tumor cells interspersed between large areas of stroma containing scattered CD8 T cells. FAP staining was observed on fibroblasts adjacent to the tumor area.

[0644] The cores were sliced ​​into 300 μm thickness by a vibrating microtome and slice cultures were established in the presence of virus (1.5e9 vp / slice) or kept uninfected. After 7 days, the slices were fixed, paraffin-embedded, sliced ​​and T cell activation status was assessed by staining for CD25 expression by immunohistochemistry (IHC). Samples that received only EnAd-CMV-FAPBiTE or EnAd-SA-FAPBiTE showed activation of tumor-infiltrating T cells, as shown by strong CD25 staining. No CD25 positive cells were detected in either untreated or control virus-treated samples. Supernatants taken from these slice cultures on days 4 and 7 post-infection were tested by ELISA for IFNγ and IL-2, and the levels of IFNγ and IL-2 detected in cultures with EnAd-SA-FAPBiTE virus were significantly higher than those in cultures with FAP BiTE virus ( Figure 44 Increased IFNγ was detected in malignant but not benign prostate slice cultures infected with IL-2 ( Figure 44 Compared with the CMV-driven FAPBiTE virus, EnAd-SA-FAPBiTE induced higher amounts of IFNγ, which could be detected earlier.

[0645] Example 21 - Additional EnAd viruses expressing FAP BiTEs

[0646] Five viruses encoding individual BiTEs were generated (NG-611, NG-612, NG-613, NG-614, NG-617) (Table 8).

[0647] Table 8

[0648] Virus ID Transgenic cassette NG-612 (SEQ ID NO: 78 <![CDATA[SSA 1 -FAPBiTE 5 -His 3 -PA 4 ]]> NG-613 (SEQ ID NO: 79) <![CDATA[SA 6 -FAPBiTE 5 -His 3 -PA 4 ]]> NG-614 (SEQ ID NO: 73) <![CDATA[SA 6 -FAPBiTE 7 -His 3 -PA 4 ]]> NG-617 (SEQ ID NO: 81) <![CDATA[SSA 1 -FAPBiTE 5 -PA 4 ]]>

[0649] In each transgenic cassette, the cDNA encoding the BiTE is flanked at the 5' end by a short splice acceptor sequence (SSA, CAGG) or a longer splice acceptor sequence (SA, SEQUENCE ID NO: 45). At the 3' end of the BiTE, the SV40 late poly (A) sequence (PA, SEQUENCE ID NO: 54) is encoded, followed by a histidine tag (HIS) or no tag. In viruses NG-611, NG-612, NG-613, and NG-617, the anti-CD3 portion of the BiTE molecule uses a single-chain variant of the mouse anti-human CD3ε monoclonal antibody OKT3.

[0650] Virus production

[0651] Plasmid pEnAd2.4 was used to generate plasmids pNG-611, pNG-612, pNG-613, pNG-614, and pNG-617 by direct insertion of synthetic transgene cassettes (SEQ ID NOs: 70 to 74, respectively). The pNG-612, pNG-613, and pNG-617 transgene cassettes encode a FAP targeting the BiTE of SEQ ID NO. 75, and the pNG-614 transgene cassette encodes a FAP targeting the BiTE of SEQ ID NO. 76. Figure 45 A schematic diagram of the transgene cassette is shown in The construction of the plasmid DNA was confirmed by restriction analysis and DNA sequencing.

[0652] Plasmids, pNG-611, pNG-612, pNG-613, pNG-614 and pNG-617 were linearized by restriction digestion with the enzyme Asc I to generate viral genomes. Viruses were amplified and purified according to the methods given below.

[0653] The DNA of digestion was purified by phenol / chloroform extraction and precipitated at -20°C for 16 hours in 300 μl>95% molecular biology grade ethanol and 10 μl 3M sodium acetate. The DNA was precipitated by centrifugation at 14000 rpm for 5 minutes and washed in 500 μl 70% ethanol, then centrifuged at 14000 rpm for 5 minutes again. The clean DNA pellet was air-dried, resuspended in 500 μl OptiMEM containing 15 μl liposome transfection reagent, and incubated at room temperature for 30 minutes. The transfection mixture was then added dropwise to a T-25 flask containing 293 cells grown to 70% confluence. After the cells were incubated at 37°C for 2 hours with the transfection mixture, 5% CO2 4ml of cell culture medium (DMEM high glucose and glutamine supplemented with 2% FBS) was added to the cells, and the flask was incubated at 37°C, 5% CO2.

[0654] The 293 cells of per 24 hours monitoring transfection, and per 48 hours to 72 hours supplemented extra culture medium.Monitor the generation of virus by the significant cytopathic effect (CPE) in the observation cell monolayer.Once observe extensive CPE, collect virus from 293 cells by three freeze-thaw cycles.The virus collected is used for reinfecting 293 cells so that amplification virus stock.Confirm the generation of live virus during amplification by the significant CPE in the observation cell monolayer.Once observe CPE, collect virus from 293 cells by three freeze-thaw cycles.Before by double cesium chloride bands purifying virus to produce the virus stock of purification, the virus stock of amplification is used for further amplification.

[0655] Assessment of viral activity by qPCR

[0656] With 1ppc NG-611, NG-612, NG-617, enadenotucirev or keep uninfected A549 cells infected 72 hours, for by qPCR quantitative viral DNA.Collect cell supernatant and by clarifying with 1200rpm centrifugation 5 minutes.According to the manufacturer's scheme, use Qiagen DNeasy test kit to extract DNA from 45 μ L supernatant.Also use DNeasy test kit to prepare and extract the standard curve using enadenotucirev virus particle (2.5e10 to 2.5e5vp).Use the viral gene specific primer-probe for early gene E3, analyze the sample or standard of each extraction by qPCR.

[0657] Quantification of the number of viral genomes detected per cell demonstrated that NG-611, NG-612, and NG-617 showed significant genome replication in the A549 cell line ( Figure 46 ). This was similar for all tested viruses containing the parental virus enadenotucirev, indicating that the inclusion of the BiTE transgene did not affect viral replication activity. No viral genome was detected in uninfected cells (data not shown).

[0658] T cell activation and degranulation mediated by BiTE-expressing viruses.

[0659] Cancer cell infection

[0660] A549 cells were seeded into 24-well plates at a density of 2.5e5 cells / well. Plates were incubated at 37°C, 5% CO₂ for 4 hours, and then infected with 1 ppc of NG-611, NG-612, or enadenotucirev or left uninfected. Supernatants were harvested from cells 24, 48, or 72 hours after infection, clarified by centrifugation at 1200 rpm for 5 minutes, and flash frozen.

[0661] T cell assay

[0662] FAP-expressing lung fibroblast cell line MRC-5 or EpCam-expressing ovarian cancer cell line SKOV3 were seeded into 48-well plates at densities of 5.7e4 cells / well and 1.2e5 cells / well, respectively. The plates were incubated at 37°C, 5% CO₂ for 4 hours, after which the medium was replaced with 150 μL / well of thawed supernatant collected from A549 plates. Purified CD3 T cells isolated from human PBMC donors were then added to the plates, resulting in a 2:1 ratio of T cells to MRC-5 or SKOV3 cells. The co-cultures were incubated at 37°C, 5% CO₂ for 16 hours, after which the cell supernatant was collected for ELISA analysis and T cells were harvested for flow cytometry analysis. The medium containing non-adherent cells was removed from the co-culture wells and centrifuged (300 x g). The supernatant was carefully removed, diluted 1-fold with PBS 5% BSA, and stored for ELISA analysis. The adherent cell monolayer was washed once with PBS and then detached with trypsin. Trypsin was inactivated using 10% FBS RPMI culture medium, and the cells were added to the cell pellets collected from the culture supernatant. The cells were centrifuged (300xg), the supernatant was discarded, and the cell pellets were washed in 200 μL of PBS. The cells were centrifuged again and then resuspended in 50 μL of PBS containing Live / Dead Aqua (Life Technologies) for 15 minutes at room temperature. The cells were washed once in FAC buffer and then stained with a directly conjugated antibody panel: anti-CD3 conjugated to AF700; anti-CD25 conjugated to BV421; anti-HLA-DR conjugated to PE / CY5; anti-CD40L conjugated to BV605; anti-CD69 conjugated to PE and anti-CD107a conjugated to FITC. Cell samples from each co-culture condition were also stained with the relevant isotype control antibodies. All staining was performed in FAC buffer at a total volume of 50 μL / well at 4 ° C for 15 minutes. The cells were then washed twice with FACs buffer (200 μL), then resuspended in 200 μL of FACs buffer and analyzed by flow cytometry (Attune).

[0663] Upregulation of T cell activation markers

[0664] Flow cytometric analysis of T cell activation was performed by expressing T cell activation markers CD25, CD69, HLA-DR, and CD40L or T cell degranulation marker CD107a on live single cells. +When SKOV3 cells were co-cultured, the number of T cells expressing CD25, CD69, HLA-DR, CD40L, or cell surface CD107a was significantly increased when NG-611 supernatant was added to the cells compared with NG-612, enadenotucirev, or untreated control supernatant ( Figure 47 ). For all these markers, the supernatant from the A549 cells infected for 24 hours stimulated very little T cell activation, however, 48 hours after infection, the supernatant stimulated significant T cell activation on all markers. This was also the case 72 hours after infection.

[0665] When with FAP + When MRC-5 cells were co-cultured, the number of T cells expressing CD25, CD69, HLA-DR, CD40L, or cell surface CD107a was significantly increased when NG-612 supernatant was added to the cells compared with NG-611, enadenotucirev, or untreated control supernatant ( Figure 48 Some T cell activation was also observed using the NG-611 virus, likely due to low but detectable expression of EpCam (approximately 5%) in the MRC-5 cell line engaged by the EpCam BiTE expressed by the NG-611 virus ( Figure 49 ). For all these markers, the supernatant from A549 cells infected for 24 hours stimulated very little T cell activation, however, 48 hours after infection, the supernatant stimulated significant T cell activation on all markers. After incubation with the supernatant collected 72 hours after infection, CD25 and CD69 markers were also upregulated, however, supernatants were collected 72 hours after infection and 48 hours after infection, and lower levels of activation markers HLA-DR, CD40L and CD107a were detected. This may be due to the presence of high levels of BiTEs in the later stages of infection, leading to rapid and effective T cell activation, which means that effector function needs to be measured at a time point earlier than 16 hours of supernatant generation.

[0666] To detect IFNγ expression, co-culture supernatants were diluted into 5% BSA / PBS assay buffer (ranging from 1:10 to 1:1000) and ELISA was performed using the Human IFNγ Qingtikine ELISA Kit (R&D Systems) according to the manufacturer's protocol. The concentration of secreted IFNγ was determined by interpolation from the standard curve. IFNγ expression was only detected in SKOV3 cells ( Figure 50A ) on NG-611 or MRC-5 cells ( Figure 50B ) was detected in the supernatant of the co-culture of NG-611 and NG-612.

[0667] Example 22 Immune Activation and Antitumor Efficacy of BiTE-Expressing Viruses in Vivo

[0668] 18 weeks after transplantation, NSG mice were implanted with humanized CD34+ hematopoietic stem cells (from Jackson Labs) in the subcutaneous region of the flanks with HCT116 tumor cells. Once the tumors reached 80 mm 3 Up to 400mm 3 Mice were divided into groups of 3 so that each treatment group had an equal distribution of tumor volumes, with 7 mice per group. Mice were injected intratumorally with saline, enadenotucirev, or NG-611 at a rate of 5 × 10 9 Particles were injected twice per tumor. Tumors on both sides were treated. Tumor volume was measured 3 to 4 times per week and demonstrated that NG-611 treatment resulted in a significant anti-tumor response 20 days after dosing compared to enadenotucirev or untreated controls ( Figure 51 a) After 20 days post-dose, one tumor from 4 mice in each group was processed for flow cytometry, while the remaining tumors were frozen on dry ice.

[0669] Flow cytometry

[0670] Tumor samples were mechanically disaggregated immediately after excision in a small volume of RPMI medium. The disaggregated tumors were then passed through a 70 μm cell strainer and centrifuged at 300 g for 10 minutes. The cell pellets were resuspended in 100 μL of PBS containing Live / DeadAqua (Life Technologies) and kept on ice for 15 minutes. The cells were washed once in FAC buffer (5% BSA PBS) and then stained with a panel of directly conjugated antibodies: anti-CD8 (RPA-T8, AF700); anti-CD4 (RPA-T4, PE); anti-CD45 (2D1, APC-Fire 750); anti-CD3 (OKT3, PerCP-Cy5.5); anti-CD25 (M-A251, PE-Dazzle594); anti-CD69 (FN50, APC); anti-HLA-DR (L243, BV605); anti-CD107a (H4A3, FITC). Tumor cell suspension pools were also stained with relevant isotype control antibodies. All staining was performed in FAC buffer at a total volume of 50 μL / well at 4°C for 20 minutes. Cells were washed three times with FAC buffer (200 μL), then resuspended in 200 μL of FAC buffer and analyzed by flow cytometry (Attune). FAC analysis demonstrated a significant increase in the ratio of CD8 to CD4 T cells in tumors treated with NG-611 compared to enadenotucirev-treated or untreated controls ( Figure 51 b).

[0671] Example 23 - EnAd virus co-expressing FAP BiTEs and immunomodulatory cytokines and chemokines

[0672] Three viruses (NG-615, NG-640, and NG-641) encoding FAP BiTEs and immunomodulatory proteins were generated (Table 9).

[0673] Table 9

[0674]

[0675] Virus production

[0676] Plasmid pEnAd2.4 was used to generate plasmids pNG-615, pNG-616, pNG-640, and pNG-641 by directly inserting synthetic transgene cassettes (SEQ ID NOs: 93 to 95, respectively). NG-615 and NG-616 contain four transgenes encoding FAP-targeting BiTEs (SEQ ID NO: 75), Flt3L (SEQ ID NO. 96), MIP1α (SEQ ID NO. 97), and IFNα (SEQ ID NO. 98). NG-640 and NG-641 encode BiTEs targeting FAP (SEQ ID NO. 75), CXCL9 (SEQ ID NO. 99), and CXCL10 (SEQ ID NO. 100). NG-641 also contains a fourth transgene encoding IFNα (SEQ ID NO. 98). Construction of the plasmid DNA was confirmed by restriction analysis and DNA sequencing.

[0677] Plasmids, pNG-615, pNG-616, pNG-640 and pNG-641 were linearized by restriction digestion with the enzyme AscI to generate viral genomes. Viruses were amplified and purified according to the methods detailed in Example 33.

[0678] Evaluation of viral activity by qPCR and transgene ELISA

[0679] Cancer cell infection

[0680] With 1ppc NG-615, enadenotucirev or keep uninfected A549 cells infected 72 hours, for by qPCR quantitative viral DNA and by ELISA analysis transgene expression.Collect cell supernatant and by clarifying with 1200rpm centrifugal 5 minutes.45 μ L supernatant is used for DNA analysis, and remaining supernatant is used for ELISA.

[0681] qPCR

[0682] According to the manufacturer's protocol, DNA was extracted from the supernatant samples using the Qiagen DNeasy kit. A standard curve of enadenotucirev virus particles (2.5e10 to 2.5e5 vp) was also prepared and extracted using the DNeasy kit. Each extracted sample or standard was analyzed by qPCR using viral gene-specific primers-probes for the early gene E3. Quantification of the number of viral genomes detected per cell demonstrated that NG-615 replicated significantly at a similar level to the parent virus enadenotucirev in the A549 cell line ( Figure 52 These data indicate that the inclusion of BiTEs and three immunomodulatory transgenes does not significantly affect viral replication activity. No viral genome was detected in uninfected cells.

[0683] ELISA

[0684] IFNα ELISA was performed using the Verikine human IFNα kit (Pbl assay science), MIP1α ELISA was performed using the human CCL3 Quantikine ELISA kit (R&D systems), and Flt3L ELISA was performed using the Flt3L human ELISA kit (Abcam). All assays were performed according to the manufacturer's protocols.

[0685] The concentration of secreted IFNα, MIPα, or Flt3L was determined by interpolation from the standard curve. IFNα, MIP1α, and Flt3L expression could be detected in the cell supernatant of NG-615, but not in enadenotucirev or untreated control cells ( Figure 53 ).

[0686] T cell activation and degranulation mediated by BiTE-expressing viruses.

[0687] Cancer cell infection

[0688] A549 cells were seeded into 24-well plates at a density of 2.5e5 cells / well. Plates were incubated at 37°C, 5% CO₂ for 4 hours, and then infected with 1 ppc of NG-612, NG-615, or enadenotucirev or left uninfected. Supernatants were harvested from cells 24, 48, or 72 hours after infection, clarified by centrifugation at 1200 rpm for 5 minutes, and flash frozen.

[0689] T cell assay

[0690] FAP expressing lung fibroblast cell line MRC-5 was seeded into 48-well plates at a density of 5.7e4 cells / well. The plates were incubated at 37°C, 5% CO2 for 4 hours, after which the medium was replaced with 150 μL / well of thawed supernatant collected from A549 plates. Purified CD3 T cells isolated from human PBMC donors were then added to the plates, resulting in a 2:1 ratio of T cells to MRC-5 cells. The co-cultures were incubated at 37°C, 5% CO2 for 16 hours, after which the cell supernatants were collected for ELISA analysis, and T cells were harvested for flow cytometry analysis according to the methods detailed in Example 29.

[0691] Upregulation of T cell activation markers

[0692] By in vivo CD3 + Flow cytometric analysis of single cells expressing T cell activation markers CD25, CD69, HLA-DR, and CD40L or T cell degranulation marker CD107a was used to assess T cell activation. + When MRC-5 cells were co-cultured, the number of T cells expressing CD25, CD69, HLA-DR, CD40L, or CD107a was significantly increased when NG-615 or 612 supernatants were added to the cells compared with enadenotucirev or untreated control supernatants ( Figure 54 ).

[0693] secretion of stimulatory cytokine IFNγ

[0694] To detect IFNγ expression, co-culture supernatants were diluted into 5% BSA / PBS assay buffer (ranging from 1:10 to 1:1000) and ELISA was performed using the Human IFNγ Qingtikine Kit (RandD Systems) according to the manufacturer's protocol. The concentration of secreted IFNγ was determined by interpolation from the standard curve. IFNγ expression was only detectable in the supernatants of co-cultures with supernatants of A549 cells infected with NG-612 or NG-615 ( Figure 55 ).

[0695] SEQ ID NO:95 transgene cassette of NG-641

[0696]

Claims

1. An adenovirus comprising a sequence of formula (I): 5'ITR-B1-B A -B2-B X -B B -B Y -B3-3'ITR(I) in: B1 is a bond or includes: E1A, E1B, or E1A to E1B; B A Includes -E2B-L1-L2-L3-E2A-L4; B2 is a key or includes: E3; B X is a bond or DNA sequence that includes: a restriction site, one or more transgenes, or both; B B Including L5; B Y Includes a transgene cassette containing four transgenes encoding FAP-BITE, CXCL10, CXCL9, and IFN; B3 is a key or includes: E4.

2. The adenovirus according to claim 1, wherein the encoded FAB-BITE comprises the anti-CD3 shown in SEQ ID NO:5 or a sequence at least 95% identical thereto, such as SEQ ID NO:

5.

3. The adenovirus according to claim 1 or 2, wherein the FAP-BITE comprises the anti-FAP shown in SEQ ID NO: 9 or a sequence at least 95% identical thereto, such as SEQ ID NO:

9.

4. The adenovirus according to claim 1, wherein the encoded FAB-BITE comprises a sequence selected from SEQ ID NO: 75, 76 or a sequence at least 95% identical to any one thereof.

5. The adenovirus according to any one of claims 1 to 4, wherein the transgene cassette encodes CXCL10 as shown in SEQ ID NO: 100 or a sequence at least 95% identical thereto, such as SEQ ID NO:

100.

6. The adenovirus according to any one of claims 1 to 5, wherein the transgene cassette encodes CXCL9 as shown in SEQ ID NO: 99 or a sequence at least 95% identical thereto, such as SEQ ID NO:

99.

7. The adenovirus according to any one of claims 1 to 6, wherein the transgene cassette encodes the IFNα shown in SEQ ID NO: 98 or a sequence at least 95% identical thereto, such as SEQ ID NO:

98.

8. The adenovirus according to any one of claims 1 to 7, wherein the transgene is operably linked.

9. The adenovirus according to any one of claims 1 to 8, wherein the transgene is separated by three different highly efficient self-cleaving peptides.

10. The adenovirus according to claim 9, wherein the self-cleaving peptide is independently selected from E2A, F2A, P2A and T2A.

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