WO2018204677A1

WO2018204677A1 - Oncolytic adenovirus formulation

Info

Publication number: WO2018204677A1
Application number: PCT/US2018/030929
Authority: WO
Inventors: Christopher Larson; Bryan ORONSKY; Tony R. REID
Original assignee: Epicentrx, Inc.
Priority date: 2017-05-04
Filing date: 2018-05-03
Publication date: 2018-11-08

Abstract

Disclosed herein are an oncolytic adenovirus formulation for the stabilization and storage of oncolytic adenoviruses for the treatment of cancer. In some embodiments, the oncolytic adenovirus is a type 5 E1A-modified, TATA box-modified, and/or CAAT box-modified adenovirus (Ad5) optionally containing one or more therapeutic transgenes that selectively replicates in cancer cells. The formulation serves to preserve the stability, infectivity and safety of the oncolytic adenovirus during storage at refrigerator or room temperature or both.

Description

ONCOLYTIC ADENOVIRUS FORMULATION

PRIORITY

[0001] This application claims priority to U.S. provisional application no. 62/501,511, which was filed on May 4, 2017, the disclosure of which is incorporated herein by reference in its entirety.

STATEMENT REGARDING SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is provided in text format in lieu of a paper copy, and is hereby incorporated by reference into the specification. The name of the text file containing the Sequence Listing is EPRX_001_01WO_SeqList_ST25.txt. The text file is about 47 KB, was created on May 3, 2018 and is being submitted electronically via EFS-Web

FIELD

[0003] The present disclosure relates to an oncolytic adenovirus formulation for the stabilization and storage of oncolytic adenoviruses for the treatment of cancer. In some embodiments, the oncolytic adenovirus is a type 5 El A-modified, TATA box-modified, and/or CAAT box— modified adenovirus (Ad5) optionally containing one or more therapeutic transgenes that selectively replicates in cancer cells. The formulation serves to preserve the stability, infectivity and safety of the oncolytic adenovirus during storage at refrigerator or room temperature or both.

BACKGROUND

[0004] Despite extensive knowledge of the underlying molecular mechanisms that cause cancer, most advanced cancers remain incurable with current chemotherapy and radiation protocols. Oncolytic viruses have emerged as a platform technology that has the potential to significantly augment current standard treatment for a variety of malignancies (Kumar, S. et al. (2008) CURRENT OPINION IN MOLECULAR THERAPEUTICS 10(4):371-379; Kim, D. (2001) EXPERT OPINION ON BIOLOGICAL THERAPY l(3):525-538; Kim D. (2000) ONCOGENE 19(56):6660-6669). These viruses have shown promise as oncolytic agents that not only directly destroy malignant cells via an infection-to-reproduction-to-lysis chain reaction but also indirectly induce anti-tumor immunity. These immune stimulatory properties have been augmented with the insertion of therapeutic transgenes that are copied and expressed each time the virus replicates.

[0005] There remains a need for formulations to preserve the stability, infectivity and safety of the oncolytic adenovirus during storage at refrigerator or room temperature or both.

SUMMARY OF THE INVENTION

[0006] The present disclosure provides oncolytic adenovirus formulations for the stabilization and storage of oncolytic adenoviruses for the treatment of cancer.

[0007] In one aspect, this application pertains to a formulation for adenoviruses, which comprises: a) a recombinant oncolytic adenovirus; b) at least one buffer; c) at least one tonicity modifier; d) at least one sugar or at least one stabilizing agent, or both; and wherein the formulation has a pH ranging between about 7.0 and about 9.0. In one embodiment, the formulation has a pH of about 8.0.

[0008] In one aspect, this application pertains to a formulation for adenoviruses, which comprises: a) a recombinant oncolytic adenovirus; b) at least one buffer; c) at least one tonicity modifier; d) at least one sugar; e) at least one non-ionic surfactant; and f) at least one divalent cation; and wherein the formulation has a pH ranging between about 7.0 and about 9.0. In one embodiment, the formulation has a pH of about 8.0. In some embodiments, the formulation further comprises at least one inhibitor of free radical oxidation. In some embodiments, the formulation further comprises at least one cryoprotectant.

[0009] In some embodiments, the stabilizing agent is glycerol. In some embodiments, the stabilizing agent is at about 2% to about 5% (v/v).

[0010] In some embodiments, the buffer is Tris. In some embodiments, the buffer is at concentration of about ImM to about 30 mM.

[0011] In some embodiments, the tonicity modifier is NaCl. In some embodiments, the tonicity modifier is at concentration of about 10 mM to about 250 mM. In some embodiments, tonicity modifier or the divalent cation is MgCh. In some embodiments, the tonicity modifier or the divalent cation is at a concentration of about 0.1 mM to about 5 mM.

[0012] In some embodiments, the sugar is sucrose. In some embodiments, the sugar is at weight to volume percentage from about 2% to about 8%. [0013] In some embodiments, the non-ionic surfactant is polysorbate-80 or polysorbate-40. In some embodiments, the non-ionic surfactant is at a concentration of about 0.001% to about 1%.

[0014] In some embodiments, the inhibitor of free radical oxidation is EDTA. In some embodiments, the inhibitor of free radical oxidation is at a concentration of about 0.01 mM to about 5 mM.

[0015] In some embodiments, the formulation has an osmolarity of about 200 mOs/L to about 800 mOs/L. In some embodiments, the recombinant oncolytic adenovirus is at concentration from about 1 x 10⁷ vp/mL to 1 x 10¹³ vp/mL.

[0016] In some embodiments, the recombinant oncolytic adenovirus is a type 5 adenovirus (Ad5).

[0017] In some embodiments, the recombinant oncolytic adenovirus comprises an Ela promoter having one or more deletions of a functional Pea3 binding site. In some embodiments, the deletion comprises a deletion of nucleotides corresponding to about -300 to about -250 upstream of the initiation site of Ela. In some embodiments, the deletion comprises a deletion of nucleotides corresponding to about -300 to about -250 upstream of the initiation site of Ela. In some embodiments, the deletion comprises a deletion of nucleotides corresponding to -305 to -255 upstream of the initiation site of Ela. In some embodiments, the deletion comprises a deletion of nucleotides corresponding to -305 to -255 upstream of the initiation site of Ela. In some embodiments, the deletion comprises a deletion of nucleotides corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 1). In some embodiments, the Ela promoter comprises the sequence GGTGTTTTGG (SEQ ID NO: 2).

[0018] In some embodiments, the recombinant oncolytic adenovirus comprises one or more Pea3 transcription binding site deletions without one or more E2F transcription binding site deletions in the EIA region. In some embodiments, the recombinant oncolytic adenovirus comprises one or more E2F transcription binding site deletions without one or more Pea3 transcription binding site deletions in the EIA region.

[0019] In some embodiments, the recombinant oncolytic adenovirus comprises an Ela promoter having a deletion of a functional TATA box, and or having a deletion of a functional CAAT box. In some embodiments, the deletion comprises a deletion of the entire TATA box. In some embodiments, the recombinant oncolytic adenovirus may comprise a deletion of nucleotides corresponding to -29 to -26, -33 to -26, -44 to +52, or -148 to +52 upstream of the initiation site of Ela. In certain embodiments, the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 1). In certain embodiments, the TATA box deletion results in an Ela promoter that comprises the sequence CTAGGACTG (SEQ ID NO: 3).

[0020] In some embodiments, the recombinant oncolytic adenovirus comprises one or more nucleotide sequences comprising a transgene. In some embodiments, the one or more nucleotide sequence is inserted into an Elb-19K insertion site, wherein the Elb-19K insertion site is located between the start site of Elb-19K and the stop codon of Elb-19K. In some embodiments, the one or more nucleotide sequences is inserted between nucleotides corresponding to 1714 and 1917 of the Ad5 genome (SEQ ID NO: 1). In some embodiments, the one or more nucleotide sequences is inserted between nucleotides corresponding to 1714 and 1917 of the Ad5 genome (SEQ ID NO: 1). In some embodiments, the one or more nucleotide sequence is inserted between CTGACCTC (SEQ ID NO: 4) and TCACCAGG (SEQ ID NO: 5).

[0021] In some embodiments, the Elb-19K insertion site comprises a deletion of about 200 nucleotides adjacent the start site of Elb-19K. In some embodiments, the Elb-19K insertion site comprises a deletion of 202 nucleotides adjacent the start site of Elb-19K. In some embodiments, the Elb-19K insertion site comprises a deletion corresponding to nucleotides 1714-1917 of the Ad5 genome (SEQ ID NO: 1). In some embodiments, the one or more nucleotide sequences is inserted between nucleotides corresponding to 1714 and 1917 of the Ad5 genome (SEQ ID NO: 1). In some embodiments, the one or more nucleotide sequences is inserted between CTGACCTC (SEQ ID NO: 4) and TCACCAGG (SEQ ID NO: 5). In some embodiments, the recombinant oncolytic adenovirus comprises, in a 5' to 3' orientation, CTGACCTC (SEQ ID NO: 4), the one or more nucleotide sequences, and TCACCAGG (SEQ ID NO: 5).

[0022] In some embodiments, the transgene encodes a polypeptide selected from the group consisting of TGF-β, a TGF-β trap, an IL-10 trap, CD80, CD19, CD20, IL-1, IL-2, IL-3, IL-4, IL- 5, IL-6, IL-7, IL-8, IL-9, IL-12A/p35, IL-12B/p40, IL-23A/pl9, IL-15, CD154, CD70, TNF-alpha, CD86, CD137, CD137L, BORIS/CTCFL, FGF, ICAM, IL24, GM-CSF, MAGE, MAGE- A3, NY- ESO-1, KRAS, tyrosinase, gplOO, melan-A, acetylcholine, interferon-gamma, DKKl/Wnt, p53, Ox40L, GM-CSF, an IL-15 IL-15 receptor fusion protein, GITRL, CD40L, CD70, secreted flagellin, IL-12, IL-10 trap, thymidine kinase, CD30, mesothelin, MUC-1, CEA, Her-2/neu, EGFRvIII, MARTI, BRAF, Podocalyxin (Podxl), p53, tyrosinase, melan-A, the androgen- receptor, an anti-PD-1 antibody heavy chain or light chain, an anti-PD-Ll antibody heavy chain or light chain, an anti-CTLA-4 antibody heavy chain or light chain, fragments thereof, and derivatives thereof.

[0023] In some embodiments, the transgene encodes a tumor antigen selected from the group consisting of gpl 00, MART -I /Melan A, gp75 (TRP-I ), tyrosinase, melanoma proteoglycan, a MACE family antigen, MAGE-I , MACE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-I 2, a BAGE family antigen, a GAGE family antigens, GAGE-I, GAGE-2, a RAGE family antigen, RAGE-I , N-acetylglucosaminyltransf erase- V, pi 5, β-catenin, MUM-I , cyclin dependent kinase-4, p21 -ras, BCR-a6/, p53, pi 85 HER2/neu, epidermal growth factor receptor, carcinoembryonic antigen (CEA), a carcinoma-associated mutated mucin, a MUC-1 gene products, an Epstein-Barr Virus EBNA gene product, papillomavirus E7, papillomavirus E6, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), an idiotypic antigens, KSA, kinesin 2, HIP-55, TGF β -1 anti-apoptotic factor, tumor protein D52, HI FT, NY-BR-I , NY-BR-62, NY-BR-75, NY-BR- 85, NY-BR-87, NY-BR-96,BCY1 , BFA4, BCY3, BCZ4, fragments thereof, and derivatives thereof.

[0024] In one aspect, this application pertains a method of treating a cancer in a subject, wherein the method comprises administering to the subject one or more of the fore-mentioned formulations. In some embodiments, the cancer is anal cancer, basal cell carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoma, cholangiocarcinoma, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, gastroesophageal cancer, gastrointestinal (GI) cancer, gastrointestinal stromal tumor, hepatocellular carcinoma, gynecologic cancer, head and neck cancer, hematologic cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, merkel cell carcinoma, mesothelioma, neuroendocrine cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pediatric cancer, prostate cancer, renal cell carcinoma, sarcoma, skin cancer, small cell lung cancer, squamous cell carcinoma of the skin, stomach cancer, testicular cancer, or thyroid cancer.

DESCRIPTION OF THE DRAWINGS

[0025] The invention can be more completely understood with reference to the following drawings. [0026] FIG. 1 is a graph depicting titers of the viruses in Formulations A, B, or C after 0, 1, 4, or 10 freeze-thaw cycles.

[0027] FIG. 2 is a graph depicting titers of the viruses in Formulations A, B, or C stored at -80°, - 20°, 4°, and room temperature ("RT"). Titers were measured at two and four weeks, and one tube in each formulation was stored at 37° and titered at two weeks.

[0028] FIG. 3 is a comparison of titers of the viruses in Formulations A, B, or C at 4° and room temperature ("RT"). Titers were measured at two and four weeks.

DETAILED DESCRIPTION I. DEFINITIONS

[0029] While the following terms are believed to be well understood by one of ordinary skill in the art, the following definitions are set forth to facilitate explanation of the presently disclosed subject matter.

[0030] Throughout the present specification, the terms "about" and/or "approximately" may be used in conjunction with numerical values and/or ranges. The term "about" is understood to mean those values near to a recited value. For example, "about 40 [units]" may mean within ± 25% of 40 (e.g., from 30 to 50), within ± 20%, ± 15%, ± 10%, ± 9%, ± 8%, ± 7%, ± 6%, ± 5%, ± 4%, ± 3%), ± 2%), ± 1%), less than ± 1%, or any other value or range of values therein or there below. Furthermore, the phrases "less than about [a value]" or "greater than about [a value]" should be understood in view of the definition of the term "about" provided herein. The terms "about" and "approximately" may be used interchangeably.

[0031] Throughout the present specification, numerical ranges are provided for certain quantities. It is to be understood that these ranges comprise all subranges therein. Thus, the range "from 50 to 80" includes all possible ranges therein (e.g., 51-79, 52-78, 53-77, 54-76, 55-75, 60-70, etc.). Furthermore, all values within a given range may be an endpoint for the range encompassed thereby (e.g., the range 50-80 includes the ranges with endpoints such as 55-80, 50-75, etc.).

[0032] The term "a" or "an" refers to one or more of that entity. As such, the terms "a" (or "an"), "one or more" and "at least one" are used interchangeably herein.

[0033] As used herein, the verb "comprise" as is used in this description and in the claims and its conjugations are used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. The present invention may suitably "comprise", "consist of, or "consist essentially of, the steps, elements, and/or reagents described in the claims.

II. FORMULATION

[0034] The present disclosure provides oncolytic adenovirus formulations for the stabilization and storage of oncolytic adenoviruses for the treatment of cancer.

[0035] In one aspect, this application pertains to a formulation for adenoviruses, which comprises: a) a recombinant oncolytic adenovirus; b) at least one buffer; c) at least one tonicity modifier; d) at least one sugar or at least one stabilizing agent, or both; and wherein the formulation has a pH ranging between about 7.0 and about 9.0. In one embodiment, the stabilizing agent is glycerol. In one embodiment, the stabilizing agent is at about 2% to about 5% (v/v). In one embodiment, the formulation has a pH of about 8.0. In some embodiments, the formulation further comprises at least one antioxidant. In some embodiments, the antioxidant is vitamin A, vitamin C, vitamin E, vitamin B6, vitamin B12, folic acid, or folate. In some embodiments, the recombinant oncolytic adenovirus is purified.

[0036] In one aspect, this application pertains to a formulation for adenoviruses, which comprises: a) a recombinant oncolytic adenovirus; b) at least one buffer; c) at least one tonicity modifier; d) at least one sugar; e) at least one non-ionic surfactant; and f) at least one divalent cation, and wherein the formulation has a pH ranging between about 7.0 and about 9.0. In one embodiment, the formulation has a pH of about 8.0. In some embodiments, the formulation further comprises at least one inhibitor of free radical oxidation. In some embodiments, the formulation further comprises at least one cryoprotectant. In some embodiments, the formulation further comprises at least one antioxidant. In some embodiments, the antioxidant is vitamin A, vitamin C, vitamin E, vitamin B6, vitamin B 12, folic acid, or folate. In one embodiment, the formulation has a pH of about 8.0. In some embodiments, the formulation further comprises at least one antioxidant. In some embodiments, the antioxidant is vitamin A, vitamin C, vitamin E, vitamin B6, vitamin B 12, folic acid, or folate. In some embodiments, the recombinant oncolytic adenovirus is purified.

[0037] In some embodiments, the buffer is Tris (includes Tris-HCl and/or mono-Tris), TES,

HEPES, brucine tetrahydrate, EPPS, tricine, or histidine. In one embodiment, the buffer is at concentration of about ImM to about 30 mM. In one embodiment, the buffer is Tris at concentration of about 10 mM. In one embodiment, the buffer is Tris at concentration of about 20 mM.

[0038] In some embodiments, the tonicity modifier is MgCh, MnCh, CaCh, ZnCh, NaCl, or KC1. In one embodiment, the tonicity modifier is NaCl. In one embodiment, the tonicity modifier is at concentration of about 0.1 mM to about 5 mM. In one embodiment, the tonicity modifier is at concentration of about 10 mM to about 250 mM. In one embodiment, the the tonicity modifier is at concentration of about 25 mM to about 100 mM. In one embodiment, the tonicity modifier is at concentration of about 25 mM. In one embodiment, the tonicity modifier is NaCl at about 25 mM. In one embodiment, the tonicity modifier is NaCl at about 50 mM. In one embodiment, the tonicity modifier is NaCl at about 75 mM.

[0039] In some embodiments, the sugar is sucrose or trehalose. In one embodiment, the sugar is sucrose. In one embodiment, the sugar is at weight to volume percentage from about 2% to about 8%. In one embodiment, the sugar is at weight to volume percentage from about 3% to about 5%. In one embodiment, the sugar is at weight to volume percentage of about 5%.

[0040] In some embodiments, the non-ionic surfactant is polysorbate-80 (Tween-80) or polysorbate-40 (Tween-40). In one embodiment, the non-ionic surfactant is at a concentration of about 0.001% to about 1%. In one embodiment, the non-ionic surfactant is at a concentration of about 0.02 %.

[0041] In some embodiments, the divalent cation is MgCh, MnCh, CaCh, or ZnCh. In one embodiment, the divalent cation is MgCh. In one embodiment, the divalent cation is at a concentration of about 0.1 mM to about 5 mM. In one embodiment, the divalent cation is at a concentration of about 1 mM.

[0042] In some embodiments, the inhibitor of free radical oxidation is EDTA. In one embodiment, the inhibitor of free radical oxidation is at a concentration of about 0.01 mM to about 5 mM. In one embodiment, the inhibitor of free radical oxidation is at a concentration of about 0.05 mM to about 2 mM. In one embodiment, the inhibitor of free radical oxidation is at a concentration of about 0.1 mM.

[0043] In some embodiments, the cryoprotectant is EtOH. In some embodiments, the cryoprotectant is a concentration of about 0.01% to 5%. In some embodiments, the cryoprotectant is a concentration of about 0.1% to 2%. In one embodiment, the cryoprotectant is at a concentration of about 0.5%.

[0044] In some embodiments, the formulation has an osmolarity of about 200 mOs/L to about 800 mOs/L. In some embodiments, the formulation has an osmolarity of about 300 mOs/L to about 600 mOs/L. In some embodiments, the formulation has an osmolarity of about 400 mOs/L to about 500 mOs/L.

[0045] In one embodiment, the formulation comprises about 20 mM Tris, about 25 mM NaCl, about 2.5%) glycerol, and wherein the formulation has a pH of about 8.0. In one embodiment, the formulation comprises about 20 mM Tris, about 25 mM NaCl, about 3-5% sucrose, and wherein the formulation has a pH of about 8.0. In one embodiment, the formulation comprises about 10 mM Tris, about 75 mM NaCl, about 5% sucrose, about 0.02% polysorbate-80, about 1 mM MgC12, about 0.1 mM EDTA, about 0.5% EtOH, and wherein the formulation has a pH of about 8.0.

[0046] In some embodiments, the recombinant oncolytic adenovirus is at concentration from about 1 x 10⁷ vp/mL to 1 x 10¹³ vp/mL.

[0047] In some embodiments, the formulation further comprises an immunoadjuvant. In some embodiments, the immunoadjuvant is selected from the group consisting of 1) Alum, 2) Saponins, 3) non-ionic polymer surfactants, 4) monophosphoryl lipid A, 5) muramyl dipeptides, and 6) cytokines.

[0048] In some embodiments, the formulation further comprises a dye. In one embodiment, the dye is used for tracking injectate.

[0049] In some embodiments, the formulation further comprises a reversible protease inhibitor. In one embodiment, the reversible protease inhibitor is an inhibitor of an L3/p23 cystein protease.

II. RECOMBINANT ONCOLYTIC ADENOVIRUS

[0050] The term "virus" is used herein to refer any of the obligate intracellular parasite having no protein-synthesizing or energy-generating mechanism. The viral genome may be RNA or DNA.

A recombinantly modified virus is referred to herein as a "recombinant virus." A recombinant virus may, e.g., bemodified by recombinant DNA techniques to be replication deficient, conditionally replicating, or replication competent, and/or be modified by recombinant DNA techniques to include expression of exogenous transgenes. Chimeric viral vectors which exploit advantageous elements of each of the parent vector properties (See, e.g., Feng et al. (1997) NATURE BIOTECHNOLOGY 15:866-870) may also be useful in the practice of the present invention. Although it is generally favored to employ a virus from the species to be treated, in some instances it may be advantageous to use vectors derived from different species that possess favorable pathogenic features.

[0051] Adenoviruses are medium-sized (90-100 nm), non-enveloped (naked), icosahedral viruses composed of a nucleocapsid and a double-stranded linear DNA genome. Adenoviruses replicate in the nucleus of mammalian cells using the host's replication machinery. The term "adenovirus" refers to any virus in the genus Adenoviridiae including, but not limited to, human, bovine, ovine, equine, canine, porcine, murine, and simian adenovirus subgenera. In particular, human adenoviruses includes the A-F subgenera as well as the individual serotypes thereof, the individual serotypes and A-F subgenera including but not limited to human adenovirus types 1, 2, 3, 4, 4a, 5, 6, 7, 8, 9, 10, 11 (Adl la and Adl lp), 12, 13, 14, 15, 16, 17, 18, 19, 19a, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 34a, 35, 35p, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, and 91. Preferred are recombinant viruses derived from human adenovirus types 2 and 5. In some embodiments, the recombinant oncolytic adenovirus is a type 5 adenovirus (Ad5). Unless stated otherwise, all adenovirus type 5 nucleotide numbers are relative to the NCBI reference sequence AC 000008.1, which is depicted herein in SEQ ID NO: 1.

[0052] In certain embodiments, the virus has one or more modifications to a regulatory sequence or promoter. A modification to a regulatory sequence or promoter comprises a deletion, substitution, or addition of one or more nucleotides compared to the wild-type sequence of the regulatory sequence or promoter.

[0053] In one embodiment, the modification of a regulatory sequence or promoter comprises a modification of sequence of a transcription factor binding site to reduce affinity for the transcription factor, for example, by deleting a portion thereof, or by inserting a single point mutation into the binding site. In certain embodiments, the additional modified regulatory sequence enhances expression in neoplastic cells, but attenuates expression in normal cells.

[0054] In certain embodiments, the modified regulatory sequence is operably linked to a sequence encoding a protein. In one embodiment, at least one of the adenoviral Ela and Elb genes (coding regions) is operably linked to a modified regulatory sequence. In one embodiment, the Ela gene is operably linked to the modified regulatory sequence. [0055] The term "operably linked" refers to a linkage of polynucleotide elements in a functional relationship. A nucleic acid sequence is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For instance, a promoter or enhancer is operably linked to a gene if it affects the transcription of the gene. Operably linked nucleotide sequences are typically contiguous. However, as enhancers generally function when separated from the promoter by several kilobases and intronic sequences may be of variable lengths, some polynucleotide elements may be operably linked but not directly flanked and may even function in trans from a different allele or chromosome.

[0056] The Ela regulatory sequence contains five binding sites for the transcription factor Pea3, designated Pea3 I, Pea3 II, Pea3 III, Pea3 IV, and Pea3 V, where Pea3 I is the Pea3 binding site most proximal to the Ela start site, and Pea3 V is most distal. The Ela regulatory sequence also contains binding sites for the transcription factor E2F, hereby designated E2F I and E2F II, where E2F I is the E2F binding site most proximal to the Ela start site, and E2F II is more distal. From the Ela start site, the binding sites are arranged: Pea3 I, E2F I, Pea3 II, E2F II, Pea3 III, Pea3 IV, and Pea3 V.

[0057] In one embodiment, at least one of these seven binding sites, or a functional binding site, is deleted. As used herein, a "functional binding site" refers to a binding site that is capable of binding to a respective binding partner, e.g., a transcription factor, e.g., a binding site that has at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40%, of the binding activity of a corresponding wild-type binding site sequence. As used herein, a "nonfunctional binding site" refers to a binding site that, e.g., has less than 30%, less than 20%, less than 10%), or 0% of the binding activity of a corresponding wild-type binding site sequence.

[0058] In certain embodiments, the recombinant oncolytic adenovirus comprises an Ela promoter having a deletion of a functional Pea3 binding site, e.g., the deletion of an entire Pea3 binding site. As used herein, a "functional Pea3 binding site" refers to a Pea3 binding site that is capable of binding to its respective transcription factor (e.g., Pea3), e.g., a Pea3 binding site that has at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40%, of the Pea3 binding activity of a corresponding wild-type Pea3 binding site sequence. As used herein, a "non-functional Pea3 binding site" refers to a Pea3 binding site that, e.g., has less than 30%, less than 20%), less than 10%, or 0% of the Pea3 binding activity of a corresponding wild-type Pea3 binding site sequence. Assays for determining whether a Pea3 binding site binds to Pea3 are known in the art. Exemplary binding assays include electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and DNAse footprinting assays.

[0059] In one embodiment, at least one Pea3 binding site, or a functional Pea3 binding site, is deleted. The deleted Pea3 binding site can be Pea3 I, Pea3 II, Pea3 III, Pea3 IV, and/or Pea3 V. In one embodiment, the deleted Pea3 binding site is Pea3 II, Pea3 III, Pea3 IV, and/or Pea3 V. In another embodiment, the deleted Pea3 binding site is Pea3 IV and/or Pea3 V. In another embodiment, the deleted Pea3 binding site is Pea3 II and/or Pea3 III. In another embodiment, the deleted Pea3 binding site is both Pea3 II and Pea3 III. In another embodiment, the Pea3 I binding site, or a functional Pea3 I binding site, is retained.

[0060] In one embodiment, at least one E2F binding site, or a functional E2F binding site, is deleted. In another embodiment, at least one E2F binding site, or a functional E2F binding site, is retained. In one embodiment, the retained E2F binding site is E2F I and/or E2F II. In another embodiment, the retained E2F binding site is E2F II. In another embodiment, the total deletion consists essentially of one or more of Pea3 II, Pea3 III, Pea3 IV, and/or Pea3 V. In one embodiment, the virus has a deletion of a 50 base pair region located from -305 to -255 upstream of the Ela initiation site, e.g., corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 1), hereafter referred to as the TAV-255 deletion. In certain embodiments, the TAV-255 deletion results in an Ela promoter that comprises the sequence GGTGTTTTGG (SEQ ID NO: 2).

[0061] In one embodiment, the recombinant oncolytic adenovirus is the oncolytic serotype 5 adenovirus (Ad5) called TAV-255 described in PCT Publication No. WO2010101921 and US Publication No. 20160017294A1, each of which is incorporated by reference herein in its entirety. It is believed that the mechanism by which the TAV-255 vector achieves this tumor selectivity is through targeted deletion of three transcriptional factor (TF) binding sites for the transcription factors Pea3 and E2F, proteins that regulate adenovirus expression of Ela, the earliest gene to be transcribed after virus entry into the host cell, through binding to specific DNA sequences. These three Pea3 and E2F deletions attenuate replication in growth-arrested, normal cells but not in malignant ones, indicating that these DNA sequences are only dispensable for transcriptional regulation and growth in cancer cells. [0062] In one embodiment, the recombinant oncolytic adenovirus is AIM-001. AIM-001 has the same El a promotor modification as in TAV-225. In addition, it has a nucleotide sequence encoding a human TGFpR-IgG fusion protein inserted in an Elb-19k insertion site.

[0063] In one embodiment, the recombinant oncolytic adenovirus comprises one or more E2F transcription binding site deletions without one or more Pea3 transcription binding site deletions in the El A region. In other embodiment, the recombinant oncolytic adenovirus comprises one or more E2F transcription binding site deletions without one or more Pea3 transcription binding site deletions in the El A region.

[0064] In certain embodiments, the recombinant oncolytic adenovirus comprises a modified TATA box-based promoter operably linked to a gene, wherein the modified TATA box-based promoter lacks a functional TATA box and permits selective expression of the gene in a hyperproliferative and/or non-growth arrested cell. As used herein, a "functional TATA box" refers to a TATA box that is capable of binding to a TATA box binding protein (TBP), e.g., a TATA box that has at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%), or at least 40%, of the TBP binding activity of a corresponding wild-type TATA box sequence. As used herein, a "non-functional TATA box" refers to a TATA box that, e.g., has less than 30%), less than 20%, less than 10%>, or 0% of the TBP binding activity of a corresponding wild-type TATA box sequence. Assays for determining whether a TBP binds to a TATA box are known in the art. Exemplary binding assays include electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and DNAse footprinting assays.

[0065] As used herein, a "modified TATA box" refers to a TATA box that has a deletion, substitution, or addition of one or more nucleotides relative to a wild-type TATA box sequence.

[0066] For example, the virus may comprise a deletion of nucleotides corresponding to -29 to -26, -33 to -26, -44 to +52, or -148 to +52 upstream of the initiation site of Ela. In certain embodiments, the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 1). In certain embodiments, the TATA box deletion results in an Ela promoter that comprises the sequence CTAGGACTG (SEQ ID NO: 3).

[0067] In certain embodiments, the recombinant oncolytic adenovirus comprises a modified CAAT box-based promoter operably linked to a gene, wherein the modified CAAT box-based promoter lacks a functional CAAT box and permits selective expression of the gene in a hyperproliferative cell and/or non-growth arrested. The TATA box-based promoter and the CAAT box-based promoter may be the same promoter (e.g., the Ad5 Ela promoter), or may be different promoters.

[0068] As used herein, "CAAT box" refers to a nucleotide sequence that is capable of binding to a C/EBP or F-Y protein. A CAAT box typically comprises a consensus sequence of GG(T/C)CAATCT.

[0069] As used herein, a "modified CAAT box" refers to a CAAT box that has a deletion, substitution, or addition of one or more nucleotides relative to a wild-type CAAT box sequence.

[0070] As used herein, a "functional CAAT box" refers to a CAAT box that is capable of binding to a C/EBP or NF-Y protein, e.g., a CAAT box that has at least 100%, at least 90%, at least 80%, at least 70%, at least 60%, at least 50%, or at least 40%, of the a C/EBP or NF-Y binding activity of a corresponding wild-type CAAT box sequence. As used herein, a "non-functional CAAT box" refers to a CAAT box that, e.g., has less than 30%, less than 20%, less than 10%, or 0% of the a C/EBP or NF-Y binding activity of a corresponding wild-type CAAT box sequence. Assays for determining whether a C/EBP or NF-Y protein binds to a CAAT box are known in the art. Exemplary binding assays include electrophoretic mobility shift assays, chromatin immunoprecipitation assays, and DNAse footprinting assays.

[0071] As used herein, "CAAT box-based promoter" refers to any gene promoter that contains a CAAT box.

[0072] As used herein, a "modified CAAT box-based promoter" refers to a CAAT box-based promoter that has been modified by a deletion, substitution, or addition of one or more nucleotides. In certain embodiments, the modification included in the modified CAAT box-based promoter comprises a deletion of one or more nucleotides of the wild-type CAAT box-based promoter sequence. In certain embodiments, the modification included in the modified CAAT box-based promoter consists of a deletion of one or more nucleotides of the wild-type CAAT box-based promoter sequence. In certain embodiments, the modification included in the modified CAAT box-based promoter comprises a deletion of the entire CAAT box of the wild-type CAAT box- based promoter sequence. In certain embodiments, the modification included in the modified CAAT box-based promoter consists of a deletion of the entire CAAT box of the wild-type CAAT box-based promoter sequence. In certain embodiments, the modification included in the modified CAAT box-based promoter comprises a deletion of the entire CAAT box-based promoter. In certain embodiments, the modification included in the modified CAAT box-based promoter consists of a deletion of the entire CAAT box-based promoter. In certain embodiments, the modification included in the modified CAAT box-based promoter does not comprise an addition of or a substitution with a separate, functional promoter sequence.

[0073] The adenoviral Elb-19k gene functions primarily as an anti-apoptotic gene and is a homolog of the cellular anti-apoptotic gene, BCL-2. Since host cell death prior to maturation of the progeny viral particles would restrict viral replication, Elb-19k is expressed as part of the El cassette to prevent premature cell death thereby allowing the infection to proceed and yield mature virions. Accordingly, in certain embodiments, a recombinant virus is provided that includes an Elb-19K insertion site, e.g., the adenovirus has an exogenous nucleotide sequence inserted into an Elb-19K insertion site. In certain embodiments, the insertion site is located between the start site of Elb-19K and the stop codon of Elb-19K.

[0074] In certain embodiments, the Elb-19K insertion site is located between the start site of Elb- 19K (i.e., the nucleotide sequence encoding the start codon of Elb-19k, e.g., corresponding to nucleotides 1714-1716 of SEQ ID NO: 1) and the stop site of Elb-19K (i.e., the nucleotide sequence encoding the stop codon of Elb-19k, e.g., corresponding to nucleotides 2242-2244 of SEQ ID NO: 1).

[0075] In certain embodiments, the Elb-19K insertion site comprises a deletion of from about 100 to about 305, about 100 to about 300, about 100 to about 250, about 100 to about 200, about 100 to about 150, about 150 to about 305, about 150 to about 300, about 150 to about 250, or about 150 to about 200 nucleotides adjacent to the start site of Elb-19K. In certain embodiments, the Elb-19K insertion site comprises a deletion of about 200 nucleotides, e.g., 202 nucleotides adjacent to the start site of Elb-19K. In certain embodiments, the Elb-19K insertion site comprises a deletion corresponding to nucleotides 1714-1917 of the Ad5 genome (SEQ ID NO: 1), or, an exogenous nucleotide sequence encoding a transgene is inserted between nucleotides corresponding to 1714 and 1917 of the Ad5 genome (SEQ ID NO: 1). In certain embodiments, an exogenous nucleotide sequence encoding a transgene is inserted between CTGACCTC (SEQ ID NO: 4) and TCACCAGG (SEQ ID NO: 5), e.g., the recombinant adenovirus comprises, in a 5' to 3' orientation, CTGACCTC (SEQ ID NO: 4), an exogenous nucleotide sequence encoding a transgene, and TCACCAGG (SEQ ID NO: 5).

[0076] Nucleic acids encoding viral genes can be incorporated into plasmids and introduced into host cells through conventional transfection or transformation techniques. Specific production and purification conditions will vary depending upon the virus and the production system employed. For adenovirus, the traditional method for the generation of viral particles is co-transfection followed by subsequent in vivo recombination of a shuttle plasmid (usually containing a small subset of the adenoviral genome and optionally containing a potential transgene an expression cassette) and an adenoviral helper plasmid (containing most of the entire adenoviral genome). Alternative technologies for the generation of adenovirus include utilization of the bacterial artificial chromosome (BAC) system, in vivo bacterial recombination in a recA÷ bacterial strain utilizing two plasmids containing complementary adenoviral sequences, and the yeast artificial chromosome (YAC) system.

[0077] In some embodiments, the recombinant oncolytic adenovirus selectively replicates in cells of a cancer. In some embodiments, wherein the cancer is selected from the group consisting of cancer of colon, rectum, breast, skin, lung, brain, and blood.

III. TRANSGENES

[0078] A recombinant oncolytic adenovirus disclosed herein may comprise one or more exogenous nucleotide sequence that comprises a transgene. A transgene may encode a therapeutic nucleic acid, e.g., an antisense RNA or ribozyme RNA. A transgene may encode a therapeutic peptide or polypeptide, e.g., an oncoprotein, tumor suppressor peptide or polypeptide, enzyme, cytokine, immune modulating peptide or polypeptide, antibody, lytic peptide, vaccine antigen, a peptide or polypeptide which complements genetic defects in somatic cells, or a peptide or polypeptide which catalyzes processes leading to cell death.

[0079] In certain embodiments, the transgene encodes a therapeutic polypeptide selected from TGF-β, a TGF-β trap, an IL-10 trap, CD80, CD 19, CD20, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-12A/p35, IL-12B/p40, IL-23A/pl9, IL-15, CD154, CD70, TNF-alpha, CD86, CD137, CD137L, BORIS/CTCFL, FGF, ICAM, IL24, GM-CSF, MAGE, MAGE- A3, NY-ESO- 1, KRAS, tyrosinase, gplOO, melan-A, acetylcholine, interferon-gamma, DKKl/Wnt, p53, Ox40L, GM-CSF, an IL-15 IL-15 receptor fusion protein, GITRL, CD40L, CD70, secreted flagellin, IL- 12, IL-10 trap, thymidine kinase, CD30, mesothelin, MUC-1, CEA, Her-2/neu, EGFRvIII, MARTI, BRAF, Podocalyxin (Podxl), p53, tyrosinase, melan-A, the androgen-receptor, an anti- PD-1 antibody heavy chain or light chain, an anti-PD-Ll antibody heavy chain or light chain, an anti-CTLA-4 antibody heavy chain or light chain, fragments thereof, and derivatives thereof.

[0080] In certain embodiments, the transgene encodes a fusion protein that comprise, in an N- to C-terminal orientation: a soluble portion of an extracellular domain of a cytokine receptor; an amino acid linker; an immunoglobulin (Ig) hinge region; and an immunoglobulin (Ig) Fc domain. In some embodiments, the cytokine receptor is TGFP type II (TpRII) receptor.

[0081] In certain embodiments, the transgene encodes a tumor antigen selected from the group consisting of gpl OO, MART -I /Melan A, gp75 (TRP-I ), tyrosinase, melanoma proteoglycan, a MACE family antigen, MAGE-I , MACE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-I 2, a BAGE family antigen, a GAGE family antigens, GAGE-I, GAGE-2, a RAGE family antigen, RAGE-I, N-acetylglucosaminyltransf erase- V, pi 5, β-catenin, MUM-I , cyclin dependent kinase-4, p21 -ras, BCR-a6/, p53, pi 85 HER2/neu, epidermal growth factor receptor, carcinoembryonic antigen (CEA), a carcinoma-associated mutated mucin, a MUC-1 gene products, an Epstein-Barr Virus EBNA gene product, papillomavirus E7, papillomavirus E6, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), an idiotypic antigens, KSA, kinesin 2, HIP-55, TGF β -1 anti-apoptotic factor, tumor protein D52, HI FT, NY-BR-I , NY-BR-62, NY-BR-75, NY-BR- 85, NY-BR-87, NY-BR-96,BCY1 , BFA4, BCY3, BCZ4, fragments thereof, and derivatives thereof.

[0082] In some embodiments, the recombinant oncolytic adenovirus selectively expresses the transgene in cells of a cancer. In some embodiments, wherein the cancer is selected from the group consisting of cancer of colon, rectum, breast, skin, lung, brain, and blood.

IV. METHODS OF TREATMENT

[0083] In one aspect, the present disclosure provides a method of treating a cancer in a subject. The method comprises administering to the subject a least one of the fore-mentioned oncolytic adenovirus formulations, wherein the recombinant oncolytic adenovirus is present in the oncolytic adenovirus formulation in an effective amount for reducing tumor load in the subject by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, or at least 90%.

[0084] As used herein, "treat", "treating" and "treatment" mean the treatment of a disease in a subject, e.g., in a human. This includes: (a) inhibiting the disease, i.e., arresting its development; and (b) relieving the disease, i.e., causing regression of the disease state. As used herein, the terms "subject" and "patient" refer to an organism to be treated by the methods and compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murines, simians, equines, bovines, porcines, canines, felines, and the like), and more preferably includes humans.

[0085] Examples of cancers include solid tumors, soft tissue tumors, hematopoietic tumors and metastatic lesions. Examples of hematopoietic tumors include, leukemia, acute leukemia, acute lymphoblastic leukemia (ALL), B-cell, T-cell or FAB ALL, acute myeloid leukemia (AML), chronic myelocytic leukemia (CML), chronic lymphocytic leukemia (CLL), e.g., transformed CLL, diffuse large B-cell lymphomas (DLBCL), follicular lymphoma, hairy cell leukemia, myelodyplastic syndrome (MDS), a lymphoma, Hodgkin's disease, a malignant lymphoma, non- Hodgkin's lymphoma, Burkitt's lymphoma, multiple myeloma, or Richter's Syndrome (Richter's Transformation). Examples of solid tumors include malignancies, e.g., sarcomas, adenocarcinomas, and carcinomas, of the various organ systems, such as those affecting head and neck (including pharynx), thyroid, lung (small cell or non-small cell lung carcinoma (NSCLC)), breast, lymphoid, gastrointestinal (e.g., oral, esophageal, stomach, liver, pancreas, small intestine, colon and rectum, anal canal), genitals and genitourinary tract (e.g., renal, urothelial, bladder, ovarian, uterine, cervical, endometrial, prostate, testicular), CNS (e.g., neural or glial cells, e.g., neuroblastoma or glioma), or skin (e.g., melanoma).

[0086] In certain embodiments, the cancer is selected from anal cancer, basal cell carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoma, cholangiocarcinoma, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, gastroesophageal cancer, gastrointestinal (GI) cancer, gastrointestinal stromal tumor, hepatocellular carcinoma, gynecologic cancer, head and neck cancer, hematologic cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, merkel cell carcinoma, mesothelioma, neuroendocrine cancer, non- small cell lung cancer, ovarian cancer, pancreatic cancer, pediatric cancer, prostate cancer, renal cell carcinoma, sarcoma, skin cancer, small cell lung cancer, squamous cell carcinoma of the skin, stomach cancer, testicular cancer and thyroid cancer.

[0087] Additional exemplary hyperproliferative diseases include blood vessel proliferation disorders (e.g., restenosis, retinopathies, and atherosclerosis), fibrotic disorders (e.g., cirrhosis, e.g., hepatic cirrhosis (which may be secondary to a viral infection such as hepatitis)), mesangial disorders (e.g., human renal diseases, e.g., glomerulonephritis, diabetic nephropathy, malignant nephrosclerosis, thrombotic microangiopathy syndromes, transplant rejection, and glomerulopathies), autoimmune disorders, rheumatoid arthritis, psoriasis, lupus, idiopathic pulmonary fibrosis, sclerodermapulmonary hypertension, asthma, kidney fibrosis, COPD, cystic fibrosis, DIP, UTP, macular degeneration, hyperproliferative fibroblast disorders, and scleroderma.

[0088] In certain embodiments, the fore-mentioned oncolytic adenovirus formulations are administered to the subject in combination with one or more therapies, e.g., surgery, radiation, chemotherapy, immunotherapy, hormone therapy, or virotherapy.

[0089] Examples of routes of administration are intravenous (IV), intradermal,inhalation, transdermal, topical, transmucosal, and rectal administration. A preferred route of administration is IV infusion.

EXAMPLES

[0090] The following working examples are illustrative and are not intended to be limiting and it will be readily understood by one of skill in the art that other embodiments may be utilized.

[0091] Example 1

[0092] Three formulations were tested. In addition to the recombinant oncolytic adenovirus (AIM- 001), the formulations include:

Formulation A: 10 mM Tris, 1 mM MgCh, 3% sucrose, pH 8

Formulation B: 20 mM Tris, 25 mM NaCl, 2.5% glycerol, pH 8

Formulation C: 10 mM Tris, 75 mM NaCl, 5% sucrose, 0.02% Tween-80, 1 mM MgCk,

0.1 mM EDTA, 0.5% EtOH, pH 8

[0093] AIM-001 was purified and the batch split into thirds, and was dialyzed into the three different formulations. Yield from that purification was lower than expected and is less than the target manufacturing concentration, stability with higher concentrations may be different but qualitative comparison between the buffers was performed given the time constraints of the project. To test stability to freeze-thaw cycles, four tubes of AIM-001 in each formulation were subjected to 0, 1, 4, or 10 freeze-thaw cycles and titered >80% infectivity was maintained in all three formulations. The titers in each formulation are shown in the Table below and FIG. 1.

Raw titer (x1 e9 lU/ml)

Percent of baseline

[0094] Example 2

[0095] To test stability over time, samples were stored at -80°, -20°, 4°, and room temperature. Titers were measured at two and four weeks, and one tube in each formulation was stored at 37° and titered at two weeks. See FIG. 2 and the tables below. Each sample was comparably stable at -80° and -20° over the four week period, and had substantial loss at 37° after two weeks (plots are with logarithmic axes).

Buffer C

[0096] A comparison of the samples at 4° and room temperature is shown in FIG. 3. Buffer A was meaningfully less stable under these conditions, and buffers B and C were comparable with a trend toward increased stability of buffer B compared to buffer C at room temperature.

[0097] Example 3

[0098] Osmolarities of the three formulations were measured. Formulation A has an osmolarity of 111 mOs/L. Formulation B has an osmolarity of 432 mOs/L. Formulation C has an osmolarity of 405 mOs/L.

INCORPORATION BY REFERENCE [0099] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not, be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

Claims

CLAIMS:

1. A formulation for adenoviruses comprising:

a) a recombinant oncolytic adenovirus;

b) at least one buffer;

c) at least one tonicity modifier;

d) at least one sugar or at least one stabilizing agent, or both; and

wherein the formulation has a pH ranging between about 7.0 and about 9.0.

2. A formulation for adenoviruses comprising:

a) a recombinant oncolytic adenovirus;

b) at least one buffer;

c) at least one tonicity modifier;

d) at least one sugar;

e) at least one non-ionic surfactant;

f) at least one divalent cation;

g) at least one inhibitor of free radical oxidation;

h) at least one cryoprotectant; and

wherein the formulation has a pH ranging between about 7.0 and about 9.0.

3. The formulation of claim 1, wherein the stabilizing agent is glycerol.

4. The formulation of claim 1 or claim 3, wherein the stabilizing agent is at about 2% to about 5% (v/v).

5. The formulation of any one of claims 1-3, wherein the buffer is Tris.

6. The formulation of any one of claims 1-4, wherein the buffer is at concentration of about ImM to about 30 mM.

7. The formulation of any one of claims 1-6, wherein the tonicity modifier is NaCl.

8. The formulation of any one of claims 1-7, wherein the tonicity modifier is at concentration of about 10 mM to about 250 mM.

9. The formulation of any one of claims 1-8, wherein the sugar is sucrose.

10. The formulation of any one of claims 1-9, wherein the sugar is at weight to volume

percentage from about 2% to about 8%.

11. The formulation of any one of claims 2 and 5-10, wherein the non-ionic surfactant is

polysorbate-80 or polysorbate-40.

12. The formulation of any one of claims 2 and 5-11, wherein the non-ionic surfactant is at a concentration of about 0.001% to about 1%.

13. The formulation of any one of claims 1-12, wherein the tonicity modifier or the divalent cation is MgCh.

14. The formulation of any one of claims 1-13, wherein the tonicity modifier or the divalent cation is at a concentration of about 0.1 mM to about 5 mM.

15. The formulation of any one of claims 2 and 5-14, wherein the inhibitor of free radical

oxidation is EDTA.

16. The formulation of any one of claims 2 and 5-15, wherein the inhibitor of free radical

oxidation is at a concentration of about 0.01 mM to about 5 mM.

17. The formulation of any one of claims 1-16, wherein the formulation has an osmolality of about 200 mOs/L to about 800 mOs/L.

18. The formulation of any one of claims 1-17, wherein the recombinant oncolytic adenovirus is at concentration from about 1 x 10⁷ vp/mL to 1 x 10¹³ vp/mL.

19. The formulation of any one of claims 1-18, wherein the formulation comprises about 20 mM Tris, about 25 mM NaCl, about 2.5% glycerol, and wherein the formulation has a pH of about 8.0.

20. The formulation of any one of claims 1-18, wherein the formulation comprises about 20 mM Tris, about 25 mM NaCl, about 3-5% sucrose, and wherein the formulation has a pH of about 8.0.

21. The formulation of any one of claims 2 and 5-18, wherein the formulation comprises about 10 mM Tris, about 75 mM NaCl, about 5% sucrose, about 0.02% polysorbate-80, about 1 mM MgCh, about 0.1 mM EDTA, about 0.5% EtOH, and wherein the formulation has a pH of about 8.0.

22. The formulation of any one of claims 1-21, wherein the recombinant oncolytic adenovirus is a type 5 adenovirus (Ad5).

23. The formulation of any one of claims 1-22, wherein the recombinant oncolytic adenovirus comprises an El a promoter having one or more deletions of a functional Pea3 binding site.

24. The formulation of claim 23, wherein the deletion comprises a deletion of nucleotides

corresponding to about -300 to about -250 upstream of the initiation site of Ela.

25. The formulation of claim 23, wherein the deletion comprises a deletion of nucleotides

26. The formulation of claim 23, wherein the deletion comprises a deletion of nucleotides

corresponding to -305 to -255 upstream of the initiation site of Ela.

27. The formulation of claim 23, wherein the deletion comprises a deletion of nucleotides corresponding to 195-244 of the Ad5 genome (SEQ ID NO: 1).

28. The formulation of claim 23, wherein the El a promoter comprises the sequence

GGTGTTTTGG (SEQ ID NO: 2).

29. The formulation of any one of claims 1-22, wherein the recombinant oncolytic adenovirus comprises one or more Pea3 transcription binding site deletions without one or more E2F transcription binding site deletions in the El A region

30. The formulation of any one of claims 1-22, wherein the recombinant oncolytic adenovirus comprises one or more E2F transcription binding site deletions without one or more Pea3 transcription binding site deletions in the El A region.

31. The formulation of any one of claims 1-22, wherein the recombinant oncolytic adenovirus comprises an El a promoter having a deletion of a functional TATA box, and or having a deletion of a functional CAAT box.

32. The formulation of claim 31, wherein the deletion comprises a deletion of the entire TATA box.

33. The formulation of claim 31, wherein the deletion comprises a deletion of nucleotides corresponding to -29 to -26 of the El a promoter.

34. The formulation of claim 31, wherein the deletion comprises a deletion of nucleotides corresponding to -33 to -26 of the El a promoter.

35. The formulation of claim 31, wherein the deletion comprises a deletion of nucleotides corresponding to -44 to +52 of the El a promoter.

36. The formulation of claim 31, wherein the deletion comprises a deletion of nucleotides corresponding to -148 to +52 of the Ela promoter.

37. The formulation of claim 31, wherein the deletion comprises a deletion of nucleotides corresponding to 353-552 of the Ad5 genome (SEQ ID NO: 1).

38. The formulation of claim 31, wherein the Ela promoter comprises the sequence

CTAGGACTG (SEQ ID NO: 3).

39. The formulation of any one of claims 1-38, wherein the recombinant oncolytic adenovirus comprises one or more nucleotide sequences comprising a transgene.

40. The formulation of claim 39, wherein the nucleotide sequence is inserted into an Elb-19K insertion site, wherein the Elb-19K insertion site is located between the start site of Elb-19K and the stop codon of Elb-19K.

41. The formulation of claim 40, wherein the Elb-19K insertion site comprises a deletion of about 200 nucleotides adjacent the start site of Elb-19K.

42. The formulation of claim 40, wherein the Elb-19K insertion site comprises a deletion of 202 nucleotides adjacent the start site of Elb-19K.

43. The formulation of claim 40, wherein the Elb-19K insertion site comprises a deletion

corresponding to nucleotides 1714-1917 of the Ad5 genome (SEQ ID NO: 1).

44. The formulation of claim 40, wherein the one or more nucleotide sequence is inserted

between nucleotides corresponding to 1714 and 1917 of the Ad5 genome (SEQ ID NO: 1).

45. The formulation of claim 40, wherein the one or more nucleotide sequence is inserted

between CTGACCTC (SEQ ID NO: 4) and TCACCAGG (SEQ ID NO: 5).

46. The formulation of claim 40, wherein the recombinant oncolytic adenovirus comprises, in a 5' to 3 ' orientation, CTGACCTC (SEQ ID NO: 4), the one or more nucleotide sequence, and TCACCAGG (SEQ ID NO: 5).

47. The formulation of any one of claims 39-46, wherein the transgene encodes a polypeptide selected from the group consisting of TGF-β, a TGF-β trap, an IL-10 trap, CD80, CD19, CD20, IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, JL-7, IL-8, IL-9, IL-12A/p35, IL-12B/p40, IL- 23A/pl9, IL-15, CD154, CD70, TNF-alpha, CD86, CD137, CD137L, BORIS/CTCFL, FGF, ICAM, IL24, GM-CSF, MAGE, MAGE- A3, NY-ESO-1, KRAS, tyrosinase, gplOO, melan- A, acetylcholine, interferon-gamma, DKKl/Wnt, p53, Ox40L, GM-CSF, an IL-15 IL-15 receptor fusion protein, GITRL, CD40L, CD70, secreted flagellin, IL-12, IL-10 trap, thymidine kinase, CD30, mesothelin, MUC-1, CEA, Her-2/neu, EGFRvIII, MARTI, BRAF, Podocalyxin (Podxl), p53, tyrosinase, melan-A, the androgen-receptor, an anti-PD-1 antibody heavy chain or light chain, an anti-PD-Ll antibody heavy chain or light chain, an anti-CTLA- 4 antibody heavy chain or light chain, fragments thereof, and derivatives thereof.

48. The formulation of any one of claims 39-46, wherein the transgene encodes a tumor antigen selected from the group consisting of gpl OO, MART-I /Melan A, gp75 (TRP-I ), tyrosinase, melanoma proteoglycan, a MACE family antigen, MAGE-I , MACE-2, MAGE-3, MAGE-4, MAGE-6, MAGE-I 2, a BAGE family antigen, a GAGE family antigens, GAGE-I, GAGE-2, a RAGE family antigen, RAGE-I , N-acetylglucosaminyltransf erase- V, pi 5, β-catenin, MUM-I , cyclin dependent kinase-4, p21 -ras, BCR-a6/, p53, pi 85 HER2/neu, epidermal growth factor receptor, carcinoembryonic antigen (CEA), a carcinoma-associated mutated mucin, a MUC-1 gene products, an Epstein-Barr Virus EBNA gene product, papillomavirus E7, papillomavirus E6, prostate specific antigen (PSA), prostate specific membrane antigen (PSMA), an idiotypic antigens, KSA, kinesin 2, HIP-55, TGF β -1 anti-apoptotic factor, tumor protein D52, HI FT, NY-BR-I , NY-BR-62, NY-BR-75, NY-BR-85, NY-BR-87, NY- BR-96,BCY1 , BFA4, BCY3, BCZ4, fragments thereof, and derivatives thereof.

49. The formulation of any one of claims 1-48, wherein the recombinant oncolytic adenovirus selectively replicates in cells of a cancer.

50. The formulation of any one of claims 39-49, wherein the recombinant oncolytic adenovirus selectively expresses the transgene in cells of a cancer.

51. The formulation of claim 49 or claim 50, wherein the cancer is selected from the group

consisting of cancer of colon, rectum, breast, skin, lung, brain, and blood.

52. The formulation of any one of claims 1-51, wherein the formulation further comprises an immunoadjuvant.

53. The formulation of claim 52, wherein the immunoadjuvant is selected from the group

consisting of 1) Alum, 2) Saponins, 3) non-ionic polymer surfactants, 4) monophosphoryl lipid A, 5) muramyl dipeptides, and 6) cytokines.

54. The formulation of any one of claims 1-53, wherein the formulation further comprises a dye.

55. The formulation of any one of claims 1-54, wherein the formulation further comprises a reversible protease inhibitor.

56. The formulation of claim 55, wherein the reversible protease inhibitor is an inhibitor of an L3/p23 cystein protease.

57. The formulation of any one of claims 2, 5-18, and 21-56, wherein the cryoprotectant is

EtOH.

58. The formulation of any one of claims 2, 5-18, and 21-57, wherein the cryoprotectant is at a concentration of about 0.5%.

59. A method of treating a cancer in a subject, wherein the method comprises administering to the subject the formulation of any one of claims 1-58.

60. The method of claim 59, wherein the cancer is anal cancer, basal cell carcinoma, bladder cancer, bone cancer, brain cancer, breast cancer, carcinoma, cholangiocarcinoma, cervical cancer, colon cancer, colorectal cancer, endometrial cancer, gastroesophageal cancer, gastrointestinal (GI) cancer, gastrointestinal stromal tumor, hepatocellular carcinoma, gynecologic cancer, head and neck cancer, hematologic cancer, kidney cancer, leukemia, liver cancer, lung cancer, lymphoma, melanoma, merkel cell carcinoma, mesothelioma, neuroendocrine cancer, non-small cell lung cancer, ovarian cancer, pancreatic cancer, pediatric cancer, prostate cancer, renal cell carcinoma, sarcoma, skin cancer, small cell lung cancer, squamous cell carcinoma of the skin, stomach cancer, testicular cancer, or thyroid cancer.