Oncolytic adenovirus composition with enhanced replication characteristics

Abstract

To provide oncolytic adenovirus compositions with enhanced replication properties.SOLUTION: A tumor selective recombinant adenovirus having deletion or modification in an E3 region is described. The recombinant adenovirus that expresses adenovirus death protein (ADP) but has deletion of at least three of the remaining six E3 genes exhibits enhanced virus replication. The recombinant adenovirus further includes additional modifications to allow selective replication in tumor cells and to detarget the virus from the liver. Use of the recombinant adenovirus for cancer treatment is described.SELECTED DRAWING: None

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the benefit of U.S. Provisional Application No. 62 / 655,009, filed on 9 April 2018 (which is incorporated herein by reference in its entirety).

[0002] field This disclosure relates to tumor-selective recombinant adenoviruses having deletions or other modifications in the E3 region that enhance viral replication. This disclosure further relates to the use of recombinant adenoviruses for cancer treatment. [Background technology]

[0003] background Cancer is a complex, debilitating disease that is the leading cause of death for more than 500,000 people each year. There is a great need for more effective, selective, and safer treatments for cancer. Existing treatments such as chemotherapy and surgery rarely eliminate all malignant cells and often cause adverse side effects that can outweigh the therapeutic benefits.

[0004] One approach that has the potential to address many of the shortcomings of current cancer treatments is oncolytic adenovirus therapy (Pesonen et al., Molecular Pharmaceutics 8(1):12-28, 2010). Adenoviruses (Ad) are self-replicating biological machines. They consist of a linear, double-stranded 36kb DNA genome coated with a protein coat. Adenoviruses invade, hijack, and replicate within a cell's replication mechanism, and once assembled, they induce lytic cell death and spread to surrounding cells. These remarkably similar cellular controls are targeted by mutation in cancer. This knowledge can be used to create synthetic viruses that act like guided missiles, specifically infecting, replicating, and lysing tumor cells, releasing thousands of viral progeny capable of finding and destroying distal metastases while overcoming potential resistance. Therefore, the goal of oncolytic virus design is to create viruses that specifically replicate in cancer cells but leave normal cells intact. However, designing viruses that can selectively replicate in cancer cells has presented significant challenges. Therefore, there is still a need for viruses that replicate efficiently and selectively in cancer cells. Furthermore, while many oncolytic viruses have proven safe in human cancer patients in clinical trials, most have lacked effectiveness in treating advanced cancer. As such, there is still a need to develop viruses with enhanced potency compared to current state-of-the-art technologies. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Pesonen et al., Molecular Pharmaceutics 8(1):12-28, 2010 [Overview of the Initiative] [Means for solving the problem]

[0006] Abstract Disclosed is a recombinant adenovirus exhibiting enhanced replication dynamics in tumor cells. A recombinant adenoviral genome encoding a recombinant adenovirus with enhanced replication in tumor cells is also described.

[0007] Provided herein is a recombinant adenoviral genome comprising: an E1A region encoding a modified E1A protein; an E3 region encoding adenovirus death protein (ADP) and comprising modifications in the coding sequences of at least three E3 genes selected from 12.5k, 6.7k, 19k, RIDα, RIDβ and 14.7k, wherein the modifications prevent expression of the encoded protein; and an E4 region comprising a modification (such as a deletion) of the E4orf6 / 7 coding sequence.

[0008] In some embodiments, the recombinant adenoviral genome further encodes a targeting ligand, further encodes a chimeric fiber protein, further comprises at least one modification for detargeting adenovirus from the liver, further comprises a heterologous open reading frame (ORF), further comprises a deletion of E4orf3, or any combination of the foregoing.

[0009] Further provided is an isolated cell comprising the recombinant adenoviral genome disclosed herein. Compositions comprising the recombinant adenoviral genome are also provided.

[0010] Further provided is a recombinant adenovirus encoded by the recombinant adenoviral genome, as well as compositions comprising the recombinant adenovirus disclosed herein.

[0011] Further provided is a method of inhibiting the viability of tumor cells by contacting the tumor cells with the recombinant adenoviral genome, recombinant adenovirus, or composition disclosed herein.

[0012] Also provided are methods of inhibiting tumor progression or reducing tumor volume in a subject, and methods of treating cancer in a subject, by administering to the subject a therapeutically effective amount of the recombinant adenovirus genome, recombinant adenovirus or composition disclosed herein.

[0013] Also described is a recombinant adenovirus genome having a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 and 59. Further described is a recombinant adenovirus encoded by a nucleotide sequence that is at least 95% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58 and 59. In certain embodiments, for example, there is provided: (Item 1) an E1A region encoding a modified E1a protein; an E3 region encoding adenovirus death protein (ADP) and comprising modifications in the coding sequences of at least three E3 genes selected from 12.5k, 6.7k, 19k, RIDα, RIDβ and 14.7k, wherein the modifications prevent expression of the encoded protein; an E4 region comprising a deletion of the E4orf6 / 7 coding sequence, and a recombinant adenovirus genome comprising the above. (Item 2) wherein the modified E1a protein comprises: a deletion of the LXCXE motif, a deletion of residues 2 to 11, a C124G substitution, a Y47H substitution, a Y47H substitution and a C124G substitution, or a Y47H substitution, a C124G substitution, and a deletion of residues 2 to 11 Recombinant adenovirus genomes, including those described in item 1. (Item 3) A recombinant adenovirus genome as described in item 1 or item 2, wherein at least three of the E3 genes include 12.5k, 6.7k, and 19k. (Item 4) A recombinant adenovirus genome as described in item 3, in which the 12.5k, 6.7k, and 19k genes are deleted. (Item 5) A recombinant adenovirus genome as described in item 3, wherein the 12.5k, 6.7k, and 19k genes include a start codon mutation, a mutation introducing an immature stop codon, or both. (Item 6) A recombinant adenovirus genome as described in item 1 or item 2, wherein at least three of the E3 genes include RIDα, RIDβ, and 14.7k. (Item 7) A recombinant adenovirus genome as described in item 6, lacking the RIDα, RIDβ, and 14.7k genes. (Item 8) The recombinant adenovirus genome described in item 6, wherein the RIDα, RIDβ, and 14.7k genes include a start codon mutation, a mutation introducing an immature stop codon, or both. (Item 9) A recombinant adenovirus genome according to any one of items 1 to 8, wherein at least three of the E3 genes include 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k. (Item 10) A recombinant adenovirus genome as described in item 9, lacking the 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k genes. (Item 11) A recombinant adenovirus genome as described in item 9, wherein the 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k genes include mutations in the start codon, mutations introducing immature stop codons, or both. (Item 12) A recombinant adenovirus genome as described in any one of items 1 through 11, further including a deletion of E4orf3. (Item 13) A recombinant adenovirus genome as described in any one of items 1 to 12, further encoding a targeted ligand fused to an FK506-binding protein (FKBP) and an adenovirus fiber protein fused to a wild-type FKBP-rapamycin-binding (FRB) protein or a mutant FRB protein capable of binding to rapamycin. (Item 14) The recombinant adenovirus genome described in item 13, wherein the targeted ligand is a single-domain antibody. (Item 15) The recombinant adenovirus genome described in item 14, wherein the single-domain antibody is specific to EGFR. (Item 16) A recombinant adenovirus genome as described in any one of items 1 to 15, comprising at least one modification for detargeting adenovirus from the liver. (Item 17) A recombinant adenovirus genome as described in item 16, containing mutations in the hexone protein. (Item 18) The recombinant adenovirus genome described in item 17, wherein the hexon mutation is the E451Q mutation. (Item 19) A recombinant adenovirus genome as described in any one of items 16 to 18, further comprising one or more binding sites for liver-specific microRNAs. (Item 20) The recombinant adenovirus genome described in item 19, wherein one or more binding sites for the liver-specific microRNA are located in the 3'-UTR of E1A. (Item 21) A recombinant adenovirus genome as described in item 19 or item 20, wherein the liver-specific microRNA is miR-122. (Item 22) A recombinant adenovirus genome, as described in any one of items 1 through 21, that encodes a chimeric fiber protein. (Item 23) The recombinant adenovirus genome described in item 22, wherein the chimeric fiber protein comprises a fiber shaft derived from a first adenovirus serotype and a fiber knob derived from a second adenovirus serotype. (Item 24) The recombinant adenovirus genome described in item 23, wherein the first adenovirus serotype is Ad5, and the second adenovirus serotype is Ad3, Ad9, Ad11, Ad12, Ad34, or Ad37. (Item 25) A recombinant adenovirus genome as described in item 23, wherein the first adenovirus serotype is Ad5 and the second adenovirus serotype is Ad34. (Item 26) The recombinant adenovirus genome according to item 25, comprising a modification in which the Ad34 fiber knob interferes with or inhibits binding to CD46. (Item 27) A recombinant adenovirus genome described in any one of items 1 through 25, encoding a fiber protein modified to include the RGD peptide. (Item 28) A recombinant adenovirus genome as described in any one of items 1 through 27, further including heterologous open reading frames (ORFs). (Item 29) The aforementioned heterologous ORF is a recombinant adenovirus genome as described in item 28, which includes a reporter gene. (Item 30) The aforementioned heterologous ORF is a recombinant adenovirus genome as described in item 28, containing a therapeutic gene. (Item 31) A recombinant adenovirus genome according to any one of items 28 to 30, wherein the heterologous ORF is operably linked to the self-cleaved peptide coding sequence and the ADP coding sequence within the same reading frame. (Item 32) The recombinant adenovirus genome described in item 31, wherein the self-cleaving peptide is peptide 2A. (Item 33) A recombinant adenovirus genome as described in item 32, wherein the 2A peptide comprises porcine rhinitis virus-1 (PTV1) 2A (P2A) peptide, foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, or Thosea asigna virus (TaV) 2A (T2A) peptide. (Item 34) The recombinant adenovirus genome described in item 1, wherein the nucleotide sequence of the genome is at least 95% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, or SEQ ID NO: 59. (Item 35) The recombinant adenovirus genome described in item 1, wherein the nucleotide sequence of the genome is at least 99% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, or SEQ ID NO: 59. (Item 36) The recombinant adenovirus genome described in item 1, wherein the nucleotide sequence of the genome includes SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, or SEQ ID NO: 59. (Item 37) Isolated cells containing a recombinant adenovirus genome as described in any one of items 1 through 36. (Item 38) A composition comprising a recombinant adenovirus genome as described in any one of items 1 to 36 or isolated cells as described in item 37, and a pharmaceutically acceptable carrier. (Item 39) An isolated adenovirus containing a recombinant adenovirus genome as described in any one of items 1 through 36. (Item 40) A composition comprising a recombinant adenovirus as described in item 39 and a pharmaceutically acceptable carrier. (Item 41) A method for inhibiting the viability of tumor cells, comprising contacting the tumor cells with a recombinant adenovirus genome described in any one of items 1 to 36, an adenovirus described in item 39, or a composition described in item 38 or 40. (Item 42) The method described in item 41, which is an in vitro method. (Item 43) The method according to item 41, wherein the method is an in vivo method, and the contact of the tumor cells comprises administering a therapeutically effective amount of the recombinant adenovirus genome, the adenovirus, or the composition to a subject having a tumor. (Item 44) A method for inhibiting tumor progression or reducing tumor volume in a subject, comprising administering to the subject a therapeutically effective amount of the recombinant adenovirus genome described in any one of items 1 to 36, the adenovirus described in item 39, or the composition described in item 38 or 40, thereby inhibiting tumor progression or reducing tumor volume in the subject. (Item 45) A method for treating cancer in a subject, comprising administering to the subject a therapeutically effective dose of a recombinant adenovirus genome described in any one of items 1 to 36, a recombinant adenovirus described in item 39, or a composition described in item 38 or item 40, thereby treating the cancer in the subject. (Item 46) A recombinant adenovirus genome in which the nucleotide sequence of the genome is at least 95% identical to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 58. (Item 47) The recombinant adenovirus genome according to item 46, wherein the nucleotide sequence of the genome is at least 99% identical to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 58. (Item 48) The recombinant adenovirus genome described in item 46, wherein the nucleotide sequence of the genome includes SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 58. (Item 49) An isolated adenovirus containing a recombinant adenovirus genome as described in any one of items 46 to 48.

[0014] The aforementioned and other purposes and characteristics of this disclosure will become even clearer from the following detailed description, followed by reference to the attached drawings. [Brief explanation of the drawing]

[0015] [Figure 1] Figure 1 is a pair of graphs comparing the replication of WT Ad5 reporter virus AdSyn-CO421 and E3-deleted AdSyn-CO874 in A549 lung cancer cells. The deletion of six of the seven E3 genes in AdSyn-CO874 resulted in enhanced replication compared to the WT virus.

[0016] [Figure 2]Figure 2 is a pair of graphs and tables comparing the replication dynamics of the oncolytic virus AdSyn-CO821 (lacking E3 genes RIDα, RIDβ, and 14.7k) with the corresponding virus AdSyn-CO819, which lacks six E3 genes (RIDα, RIDβ, 4.7k, 12.5k, 6.7k, and 19k). AdSyn-CO819 showed superior replication in all cancer cell lines tested.

[0017] [Figure 3] Figure 3 shows a pair of graphs illustrating the replication dynamics of the WTAd5 reporter virus AdSyn-CO421 and AdSyn-CO964 in coxsackie adenovirus receptor (CAR)-negative cells. AdSyn-CO964 is a synthetic Ad5 virus that expresses a chimeric fiber protein composed of an Ad5 shaft and an Ad34 knob. Expression of the Ad34 knob domain led to enhanced viral replication in CAR-negative cells.

[0018] [Figure 4] Figure 4 shows a pair of graphs comparing the replication of E3-deficient oncolytic Ad5 (AdSyn-CO1000) in CAR-negative cells with that of the corresponding E3-deficient oncolytic virus expressing the Ad34 knob domain (AdSyn-CO1042). AdSyn-CO1042 showed enhanced replication dynamics compared to the Ad5 knob-expressing virus in multiple cell lines.

[0019] [Figure 5]Figures 5A–5B are a pair of graphs showing the in vivo efficacy of AdSyn-CO1000 and AdSyn-CO1042 in an A549 lung tumor xenograft model. Human A549 tumor cells were inoculated into nude mice by injecting 5 × 10⁶ cells into the subcutaneous fat of the mammary gland. When the tumor reached an average volume of approximately 164 mm³ (day 0), the mice were randomized to different treatment groups (n=8 mice per group). Mice were given either a single intratumor (IT) injection of PBS (saline) or a single injection of 8 × 10⁶ PFU of the indicated virus. AdSyn-CO421 encodes the YPet fluorophore as a YPet-P2A-ADP fusion, otherwise it is wild-type Ad5. Both AdSyn-CO1000 (Figure 5A) and AdSyn-CO1042 (Figure 5B) showed enhanced antitumor activity compared to “wild-type” AdSyn-CO421.

[0020] [Figure 6] Figure 6 is a graph showing the in vivo efficacy of AdSyn-CO1042 in a normal-positioned HS578T triple-negative breast cancer model. Human HS578T tumor cells were inoculated into 7-week-old NSG mice by injecting 5 × 10⁶ cells in 100 μl of HBSS into the right mammary subcutaneous fat. When the tumor reached an average volume of approximately 168 mm³ (day 0), the mice were randomized to different treatment groups (n=8 mice per group). Mice were given three doses of either 50 μl of total volume of PBS (saline) or AdSyn-CO1042 (2 × 10⁸ PFU) on days 0, 7, and 14. All animals in the saline-treated group had to be sacrificed before day 30 due to tumor burden.

[0021] [Figure 7A]Figures 7A-7B are graphs showing the results of safety and toxicity studies of recombinant adenoviruses in C57BL / 6 mice. Four different doses of the virus were administered intravenously to different groups of mice (n=5 mice per group) in 200 μl volumes on day 0 and again on day 7. To investigate hepatotoxicity, mice were then analyzed for survival (Figure 7A) and elevated liver enzymes (Figure 7B). (Figure 7A) Mice were monitored for macroscopic signs of survival and toxicity until day 14. WT Ad5 caused lethal toxicity at a dose of 4 × 10⁹ PFU, but AdSyn-CO181 and AdSyn-CO331 were tolerated at this dose. AdSyn-CO1042 was tolerated even at a higher dose of 2 × 10¹⁰ PFU. (Figure 7B) Blood samples were collected from all mice on day 2 (pre-administration), day 2 (48 hours after administration 1), and day 9 (48 hours after administration 2), and frozen for subsequent analysis. Blood samples were investigated for the presence of various liver enzymes, such as alanine transaminase (ALT) and aspartate transaminase (AST). The results shown are the mean AST and ALT levels from bleeding on day 2 from various treatment groups. The mean pre-administration levels of AST and ALT from all mice are also shown. [Figure 7B]Figures 7A-7B are graphs showing the results of safety and toxicity studies of recombinant adenoviruses in C57BL / 6 mice. Four different doses of the virus were administered intravenously to different groups of mice (n=5 mice per group) in 200 μl volumes on day 0 and again on day 7. To investigate hepatotoxicity, mice were then analyzed for survival (Figure 7A) and elevated liver enzymes (Figure 7B). (Figure 7A) Mice were monitored for macroscopic signs of survival and toxicity until day 14. WT Ad5 caused lethal toxicity at a dose of 4 × 10⁹ PFU, but AdSyn-CO181 and AdSyn-CO331 were tolerated at this dose. AdSyn-CO1042 was tolerated even at a higher dose of 2 × 10¹⁰ PFU. (Figure 7B) Blood samples were collected from all mice on day 2 (pre-administration), day 2 (48 hours after administration 1), and day 9 (48 hours after administration 2), and frozen for subsequent analysis. Blood samples were investigated for the presence of various liver enzymes, such as alanine transaminase (ALT) and aspartate transaminase (AST). The results shown are the mean AST and ALT levels from bleeding on day 2 from various treatment groups. The mean pre-administration levels of AST and ALT from all mice are also shown.

[0022] [Figure 8] Figure 8 is a table showing the toxicity of AdSyn-CO1000 in BALB / c mice. Two different doses of the virus, each representing 200 μl, were administered intravenously to different groups of mice (n=5 mice per group) on days 0, 6, and 12. To investigate toxicity, the mice were then analyzed for survival. WT Ad5 induced lethal toxicity in 2 out of 5 mice at a dose of 0.8 × 10⁹ PFU and killed all mice at a higher dose of 3.2 × 10⁹ PFU, while AdSyn-CO1000 was safer at slightly higher doses of 1 × 10⁹ PFU and 4 × 10⁹ PFU.

[0023] [Figure 9]Figure 9 is a graph showing the in vivo efficacy of intravenously delivered AdSyn-CO1042 in an A549 lung tumor xenograft model. Human A549 tumor cells were inoculated into NSG mice by injecting 5 × 10⁶ cells into the subcutaneous fat of the mammary gland. When the tumor reached an average volume of approximately 184 mm³ (day 0), the mice were randomized to different treatment groups (n=8 mice per group). Mice were given either a single injection of PBS (saline) or a single IV injection of 2 × 10⁹ PFU of AdSyn-CO1042. All animals in the saline-treated group had to be sacrificed before day 33 due to tumor burden. Wild-type Ad5 could not be delivered at a dose of 2 × 10⁹ PFU because this dose would result in death.

[0024] [Figure 10]Figure 10 is a set of graphs showing tumor-selective replication of AdSyn-CO1000 and AdSyn-CO1042. Human A549 cells (lung tumor) or human primary small airway epithelial cells (SAEC - normal lung) were infected with four different oncolytic viruses at an MOI of 0.12 viral particles per cell. All viruses encode the YPet fluorophore as a reporter, enabling the time-course quantification of viral replication / growth. Immediately after viral infection, virus-infected cells were imaged once every hour in the IncuCyte ZOOM imaging system to quantify the number of YPet+ virus-infected cells over 6-7 days. Data are expressed as the number of YPet+ cells over time. AdSyn-CO874 and AdSyn-CO1000 are identical viruses, except that AdSyn-CO1000 has the ΔLXCXE and ΔE4orf6 / 7 mutations and the hepatic detargeting hexone mutation that confer tumor-selective replication. Similarly, AdSyn-CO1041 and AdSyn-CO1042 are identical viruses, except that AdSyn-CO1042 has ΔLXCXE and ΔE4orf6 / 7 mutations as well as a hepatic detargeting hexone mutation. In tumor cells, AdSyn-CO1000 and AdSyn-CO1042 showed similar levels of viral growth / replication compared to AdSyn-CO874 and AdSyn-CO1041, respectively. However, in normal lung cells, AdSyn-CO1000 and AdSyn-CO1042 showed significantly attenuated growth / replication compared to AdSyn-CO874 and AdSyn-CO1041.

[0025] [Figure 11]Figure 11 is a bar graph demonstrating the effects of deletion or suppression of the E3B region on adenovirus replication. Four different recombinant viruses were investigated using a fluorescence-based virological kinetics (FBVK) assay to determine the replication rate in human A549 lung tumor cells. The graph shows the ln-slope values ​​for each recombinant adenovirus encoding the Ypet fluorescent protein. In AdSyn-CO869, the RIDα, RIDβ, and 14.7k genes (E3B genes) were all deleted from the genome. In AdSyn-CO996, the expression of the RIDα, RIDβ, and 14.7k genes (E3B genes) was suppressed by mutating the start codon or by mutating the genome to encode an immature stop codon. In AdSyn-CO874, six E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k) were deleted from the genome.

[0026] [Figure 12] Figure 12 is a bar graph showing the effect of deletion or inhibition of the E3A region on adenovirus replication. Four different viruses were investigated using the FBVK assay to determine the replication rate in human A549 lung tumor cells. The graph shows the ln-slope values ​​for each recombinant adenovirus encoding the Ypet fluorescent protein. In AdSyn-CO1002, the 12.5k, 6.7k, and 19k genes (E3A genes) were all deleted from the genome. In AdSyn-CO999, the same E3A gene was deleted, and this virus further contained E1A ΔLXCXE, ΔE4orf6 / 7, and hexon [E451Q] modifications. In AdSyn-CO1000, the E3A and E3B genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k) are deleted from the genome, and this virus further includes E1A ΔLXCXE, ΔE4orf6 / 7, and hexon [E451Q] modifications.

[0027] [Figure 13]Figure 13 is a bar graph showing that the deletion of E4orf3 does not affect viral replication. Two viruses were investigated using the FBVK assay to determine the replication rate in A549 human lung tumor cells. The bar graph shows the ln-slope values ​​for each recombinant adenovirus. AdSyn-CO1042 and AdSyn-CO1347 are identical except for the deletion of E4orf3 in AdSyn-CO1347. [Modes for carrying out the invention]

[0028] Sequence List The nucleic acid and amino acid sequences listed in the attached sequence listing are presented using standard abbreviations for nucleotide bases and three-letter codes for amino acids, as defined in 37 CFR 1.822. Only one strand of each nucleic acid sequence is shown, but complementary strands are understood to be included by any reference to the presented strand. The sequence listing was submitted as a 1.36 MB ASCII text file created on April 8, 2019, and is incorporated herein by reference. The attached sequence listing is as follows: Sequence ID 1 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO335. Sequence ID 2 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO821. Sequence ID 3 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO820. Sequence ID 4 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO819. Sequence ID 5 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1020. Sequence ID 6 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO874. Sequence ID 7 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1000. Sequence ID 8 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1067. Sequence ID 9 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1068. Sequence ID 10 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1069. Sequence ID 11 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO964. Sequence ID 12 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1041. Sequence ID 13 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1042. Sequence ID 14 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1139. Sequence ID 15 is the amino acid sequence of P2A. Sequence ID 16 is the amino acid sequence of F2A. Sequence ID 17 is the amino acid sequence of E2A. Sequence ID 18 is the amino acid sequence of T2A. Sequence ID 19 is the amino acid sequence of a modified P2A containing GSG at the N-terminus. Sequence ID 20 is the amino acid sequence of a modified F2A containing GSG at the N-terminus. Sequence ID 21 is a modified E2A amino acid sequence containing GSG at the N-terminus. Sequence ID 22 is the amino acid sequence of a modified T2A containing GSG at the N-terminus. Sequence ID 23 is the amino acid sequence of Ad5 E1A. Sequence ID 24 is the amino acid sequence of Ad5 E1A ΔLXCXE. Sequence ID 25 is the amino acid sequence of Ad5 E1A C124G. Sequence ID 26 is the amino acid sequence of Ad5 E1A Δ2-11. Sequence ID 27 is the amino acid sequence Ad5 E1A Y47H C124G. Sequence ID 28 is the amino acid sequence Ad5 E1A Δ2-11 Y47H C124G. Sequence ID 29 is the amino acid sequence Ad5 E4orf6 / 7. Sequence ID 30 is the amino acid sequence of the Ad5 fiber. Sequence ID 31 is the amino acid sequence of Ad5 FRB-fiber. Sequence ID 32 is the amino acid sequence of Ad5 FRB*-fiber. Sequence ID 33 is the amino acid sequence EGFRVHH-GS-FKBP. Sequence ID 34 is the amino acid sequence of Ad5 hexone. Sequence ID 35 is the amino acid sequence of Ad5 hexone E451Q. Sequence ID 36 is the amino acid sequence of species A(Ad12)E1A. Sequence ID 37 is the amino acid sequence of species B(Ad7)E1A. Sequence ID 38 is the amino acid sequence of species C(Ad2)E1A. Sequence ID 39 is the amino acid sequence of species C(Ad5)E1A. Sequence ID 40 is the amino acid sequence of species D(Ad9)E1A. Sequence ID 41 is the amino acid sequence of species E(Ad4)E1A. Sequence ID 42 is the amino acid sequence of species F(Ad40)E1A. Sequence ID 43 is the amino acid sequence of species G(Ad52)E1A. Sequence ID 44 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO421. Sequence ID 45 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1056. Sequence ID 46 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1250. Sequence ID 47 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1089. Sequence ID 48 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1320. Sequence ID 49 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1321. Sequence ID 50 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1325. Sequence ID 51 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1342. Sequence ID 52 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1362. Sequence ID 53 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1403. Sequence ID 54 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1404. Sequence ID 55 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO869. Sequence ID 56 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO996. Sequence ID 57 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO999. Sequence ID 58 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1002. Sequence ID 59 is the nucleotide sequence of the synthetic adenovirus AdSyn-CO1347. Sequence ID 60 is the amino acid sequence of the Ad34 fiber.

[0029] I. Abbreviations Adenovirus ADP (Adenovirus Dead Protein) CAR Coxsackieadenovirus Receptor EGFR (Epidermal Growth Factor Receptor) ERAV (Equine Rhinitis A Virus) Viral dynamics based on FBVK fluorescence FKBP FK506-binding protein FMDV (Foot-and-Mouth Disease Virus) FRB FKBP-rapamycin binding IV Intravenous IT intratumoral miR microRNA MOI Multiplicity of infection mTOR rapamycin's mammalian targets ORF Open Reading Frame PSA (Prostate-Specific Antigen) PTV1 Swine Virus-1 Rb retinoblastoma RGD Arginine-Glycine-Aspartic Acid TaV Eia asigna virus UTR Untranslated Area WT wild type

[0030] II. Terminology and Methods Unless otherwise noted, technical terms are used according to their conventional usage. Definitions of general terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press in 1994 (ISBN 0-19-854287-9); The Encyclopedia of Molecular Biology, edited by Kendrew et al., published by Blackwell Science Ltd. in 1994 (ISBN 0-632-02182-9); and Molecular Biology and Biotechnology: a Comprehensive Desk Reference, edited by Robert A. Meyers, published by VCH Publishers, Inc. in 1995 (ISBN 1-56081-569-8).

[0031] To facilitate consideration of the various embodiments of this disclosure, explanations of specific terms are provided below.

[0032] 2A peptides are a type of self-cleaving peptide encoded by certain RNA viruses, such as picornaviruses. 2A peptides function by causing ribosomes to skip the synthesis of peptide bonds at the C-terminus of the 2A element, resulting in the separation of the 2A sequence end from the downstream peptide (Kim et al., PLoS One, vol. 6(4), e18556, 2011). The "cleavage" occurs between the glycine and proline residues found at the C-terminus of the 2A peptide. Exemplary 2A peptides include, but are not limited to, those encoded by Thosea asigna virus (TaV), equine rhinitis A virus (ERAV), porcine rhinitis virus-1 (PTV1), and foot-and-mouth disease virus (FMDV), as described herein as SEQ ID NOs. 15-18. In some embodiments, the 2A peptide contains Gly-Ser-Gly at its N-terminus to improve cleavage efficiency (SEQ ID NOs. 19-22).

[0033] Adenoviruses are non-enveloped viruses with a linear double-stranded DNA genome and an icosahedral capsid. At least 68 serotypes of human adenoviruses are known and classified into seven species (species A, B, C, D, E, F, and G). Different serotypes of adenoviruses are associated with different types of diseases, with some serotypes causing respiratory illnesses (primarily species B and C), conjunctivitis (species B and D), and / or gastroenteritis (species F and G).

[0034] Adenovirus death protein (ADP): A protein synthesized late in adenovirus infection that mediates cell lysis and the release of adenoviruses in order to infect other cells. ADP is an endogenous membrane glycoprotein of 101 amino acids that localizes to the nuclear membrane, endoplasmic reticulum, and Golgi apparatus. (ADP was formerly known as E3-11.6K).

[0035] Administration: This refers to providing or administering a drug, such as a therapeutic agent (e.g., a recombinant virus or recombinant viral genome), to a subject via any effective route. Exemplary routes of administration include, but are not limited to, injection (subcutaneous, intramuscular, intradermal, intraperitoneal, intratumoral, intraosseous, and intravenous), oral, intratubal, sublingual, rectal, percutaneous, intranasal, vaginal, and inhalation routes.

[0036] Antibody: A polypeptide ligand that recognizes and binds to an antigen epitope (e.g., specifically recognizes and specifically binds), and contains at least a light chain and / or heavy chain immunoglobulin variable region. Immunoglobulin molecules consist of a heavy chain and a light chain, each of which is heavy chain variable (V H ) region and light chain variable (V L Antibodies have variable regions called Fab domains, which together are involved in binding antigens recognized by antibodies. Antibodies include intact immunoglobulins and variants and fragments of antibodies, e.g., single-domain antibodies (e.g., VH domain antibodies or camelid VHH antibodies), Fab fragments, Fab' fragments, F(ab)'2 fragments, single-chain Fv proteins ("scFv"), and disulfide-stabilized Fv proteins ("dsFv"). scFv proteins are fusion proteins in which the light chain variable region and the heavy chain variable region of an immunoglobulin are linked by a linker, while in dsFv, the chain is mutated to introduce a disulfide bond to stabilize the chain association. The term "antibody" also includes genetically modified forms, e.g., chimeric antibodies (e.g., humanized mouse antibodies) and heteroconjugate antibodies (e.g., bispecific antibodies). Pierce Catalog and Handbook, 1994-1995 (Pierce Chemical Co., Rockford, IL); Kuby, J., Immunology, 3 rd See also Ed., WH Freeman & Co., New York, 1997.

[0037] Chemotherapy agents: Any chemical agent that has therapeutic utility in the treatment of diseases characterized by abnormal cell growth. Such diseases include tumors, neoplasms and cancers, as well as diseases characterized by hyperplastic growth, such as psoriasis. In one embodiment, the chemotherapeutic agent is a radioactive compound. In one embodiment, the chemotherapeutic agent is a biologic, such as a therapeutic monoclonal antibody (e.g., specific to PD-1, PDL-1, CTLA-4, EGFR, VEGF, etc.). Those skilled in the art can easily identify useful chemotherapeutic agents (e.g., Slapak and Kufe, Principles of Cancer Therapy, Chapter 86 in Harrison's Principles of Internal Medicine, 14th edition; Perry et al., Chemotherapy, Ch. 17 in Abeloff, Clinical Oncology). 2 nd See also: (C) 2000 Churchill Livingstone, Inc; Baltzer, L., Berkery, R. (eds.): Oncology Pocket Guide to Chemotherapy, 2nd ed. St. Louis, Mosby-Year Book, 1995; Fischer, DS, Knobf, MF, Durivage, HJ (eds): The Cancer Chemotherapy Handbook, 4th ed. St. Louis, Mosby-Year Book, 1993). Combination chemotherapy involves more than one drug to treat cancer. It is an administration.

[0038] Chimera: Composed of at least two parts of different origins. In the context of this disclosure, “chimeric adenovirus” is an adenovirus having genetic material and / or proteins derived from at least two different serotypes (e.g., Ad5 and a second serotype of adenovirus). In this context, “capsid-exchange type” adenovirus refers to a chimeric adenovirus in which the capsid protein is derived from one serotype of adenovirus and the remaining proteins are derived from another adenovirus serotype. Similarly, “chimeric fiber” is a fiber protein having amino acid sequences derived from at least two different serotypes of adenovirus. For example, a chimeric fiber may consist of a fiber shaft derived from Ad5 and a fiber knob derived from a second serotype of adenovirus (e.g., Ad34).

[0039] To bring into contact: To bring into direct physical contact, including both solid and liquid forms.

[0040] Deletion: An adenovirus genome containing a "deletion" of the adenovirus protein-coding sequence (e.g., the E4 or f6 / 7 coding sequence) refers to an adenovirus that has a complete deletion of the protein-coding sequence or a partial deletion resulting in the absence of protein expression.

[0041] Detargeted: In the context of this disclosure, “detargeted” adenovirus is a recombinant or synthetic adenovirus that includes one or more modifications that alter the viral affinity such that it no longer infects, or is substantially no longer infects, a particular cell or tissue species. In some embodiments, the recombinant or synthetic adenovirus includes a capsid mutation, e.g., a mutation in the hexon protein that detargets the virus from the liver (e.g., E451Q). In some embodiments, the recombinant or synthetic adenovirus includes a native capsid derived from an adenovirus that does not naturally infect, or is substantially no longer infects, a particular cell or tissue species. In some embodiments herein, the recombinant or synthetic adenovirus is liver-detargeted and / or spleen-detargeted.

[0042] E1A: The adenovirus early region 1A (E1A) gene and the polypeptide expressed from this gene. The E1A protein plays a role in viral genome replication by introducing cells into the cell cycle. As used herein, the term “E1A protein” refers to the protein expressed from the E1A gene and includes E1A proteins produced by any adenovirus serotype. For example, the amino acid sequence of the wild-type Ad5 E1A protein is shown herein as SEQ ID NO: 23, and modified Ad5 E1A sequences are provided herein as SEQ ID NOs: 24-28. Furthermore, wild-type E1A protein sequences from various different adenovirus serotypes are shown herein as SEQ ID NOs: 36-43. In some embodiments, the modified E1A protein comprises a protein having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 24, 25, 26, 27, 28, 36, 37, 38, 39, 40, 41, 42, or 43. The modified E1A protein intended herein contributes to replication defects of recombinant adenovirus in normal cells compared to tumor cells. The modified E1A protein disclosed herein is the Ad5 E1A protein. However, corresponding modifications can be made in any desired serotype and are therefore included in this disclosure. For example, all species of human adenovirus contain the LXCXE motif, which in Ad5 corresponds to LTCHE (residues 122-126 of SEQ ID NO: 23). Similarly, deletions of residues 2–11 and Y47H and C124G substitutions, while numbered in relation to Ad5 (e.g., SEQ ID NO: 5), can be introduced into any other serotype.

[0043] E3: This refers to the adenovirus early region 3 (E3) gene and the polypeptide expressed from it. In human adenovirus, there are seven E3 proteins (encoded from 5' to 3'): 12.5k (also known as gp12.5kDa), 6.7k (also known as CR1α), 19k (also known as gp19k), ADP (also known as CR1β or 11.6k), RIDα (10.4k), RIDβ (14.9k), and 14.7k. The RIDα, RIDβ, and 14.7k proteins form a receptor internalization and degradation complex (RID), which localizes to the nuclear membrane and causes endocytosis and degradation of various receptors, including CD95 (FasL receptor) and TNFR1 and 2 (TNF / TRAIL receptors), to protect infected cells from the host's antiviral response. The 6.7k protein is known to be involved in apoptosis modulation in infected cells, and the 19k protein is known to inhibit the insertion of class I MHC proteins in the infected host cell membrane. ADP mediates the lysis of infected cells. The function of the 12.5k protein is unknown. As used herein, the E3A gene comprises the 12.5k, 6.7k, and 19k genes, and the E3B gene comprises the RIDα, RIDβ, and 14.7k genes. In some embodiments herein, the recombinant adenovirus genome comprises a deletion of the E3A gene, a deletion of the E3B gene, or both. In other embodiments, the recombinant adenovirus genome comprises a mutation in the coding sequence of the E3A gene or the E3B gene, or both, resulting in a mutation that interferes with the expression of the encoded protein.

[0044] E4: The adenovirus early region 3 (E4) gene and the polypeptide expressed by the gene. In human adenovirus, the E4 region encodes at least six proteins, including E4orf1, E4orf2, E4orf3, E4orf4, E4orf6, and E4orf6 / 7. In some embodiments herein, the recombinant adenovirus genome includes a deletion of E4orf6 / 7. In some examples, the recombinant adenovirus genome further includes a deletion of E4orf3.

[0045] E4orf6 / 7: This is a protein encoded by the adenovirus E4 gene. The term “E4orf6 / 7 protein” includes E4orf6 / 7 proteins produced by the E4 gene derived from any adenovirus serotype. For example, the amino acid sequence of the wild-type Ad5 E4orf6 / 7 protein is shown herein as SEQ ID NO: 29. In some embodiments, the E4orf6 / 7 protein includes proteins having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity to any one of SEQ ID NOs: 29. The modified E4orf6 / 7 proteins intended herein contribute to recombinant adenovirus replication defects in normal cells compared to tumor cells. In some embodiments, the modified E4orf6 / 7 protein includes mutations (e.g., deletions) that disable or impair its E2F binding site and / or impair E2F interaction. In other embodiments, modified E4orf6 / 7 proteins include modifications that eliminate or impair the nuclear localization signal required for the efficient translocation of E2F4. Exemplary modifications of E4orf6 / 7 are discussed further in the following sections.

[0046] Epidermal growth factor receptor (EGFR): A cell surface receptor for the EGF family of extracellular protein ligands. EGFR is also known as ErbB-1 and HER1. Numerous types of cancer contain mutations that lead to EGFR overexpression.

[0047] Fibers: Adenovirus fiber proteins are trimer proteins that mediate binding to cell surface receptors. The fiber protein consists of a long N-terminal shaft and a spherical C-terminal knob.

[0048] FK506-binding proteins (FKBPs) are a family of eukaryotic proteins that function as protein folding chaperones. FKBPs are known for their ability to bind to rapamycin. An exemplary FKBP sequence is shown herein as residues 132–238 of SEQ ID NO: 33.

[0049] FKBP-rapamycin binding (FRB): A domain of the mammalian target of rapamycin (mTOR) that binds to rapamycin. An exemplary sequence of FRB is shown herein as residues 547-636 of SEQ ID NO: 31. A variant form of FRB capable of binding to both rapamycin and rapalog (referred herein as "FRB*") (also known as AP21967) is shown herein as residues 547-636 of SEQ ID NO: 32. FRB* contains a threonine-to-leucine substitution (T2098L) at position 2098 of the human mTOR, corresponding to residue 620 of SEQ ID NO: 32.

[0050] Fluorescent proteins are proteins that emit light of a specific wavelength when exposed to light of a particular wavelength. Examples of fluorescent proteins include green fluorescent proteins (GFP, EGFP, AcGFP1, Emerald, Superfolder GFP, Azami Green, mWasabi, TagGFP, TurboGFP, YPet, and ZsGreen, etc.), blue fluorescent proteins (EBFP, EBFP2, Sapphire, T-Sapphire, Azurite, and mTagBFP, etc.), cyan fluorescent proteins (ECFP, mECFP, Cerulean, CyPet, AmCyan1, Midori-Ishi Cyan, mTurquoise, and mTFP1, etc.), yellow fluorescent proteins (EYFP, Topaz, Venus, mCitrine, YPet, TagYFP, PhiYFP, ZsYellow1, and mBanana), and orange fluorescent proteins (Kusabira Orange, Kusabira Examples include, but are not limited to, Orange2, mOrange, mOrange2, and mTangerine; red fluorescent proteins (mRuby, mApple, mStrawberry, AsRed2, mRFP1, JRed, mCherry, HcRed1, mRaspberry, dKeima-Tandem, HcRed-Tandem, mPlum, AQ143, tdTomato, and E2-Crimson); far-red fluorescent proteins (e.g., Katushka2S); orange / red fluorescent proteins (dTomato, dTomato-Tandem, TagRFP, TagRFP-T, DsRed, DsRed2, DsRed-Express(T1), and DsRed-Monomer); and modified versions thereof.

[0051] A fusion protein is a protein that contains amino acid sequences derived from at least two different (heterogeneous) proteins or peptides. Fusion proteins can be produced, for example, by the expression of a nucleic acid sequence manipulated from nucleic acid sequences encoding at least a portion of two different (heterogeneous) proteins. In order to produce a fusion protein, the nucleic acid sequences must be within the same reading frame and must not contain internal stop codons. Fusion proteins, especially short fusion proteins, can also be produced by chemical synthesis.

[0052] Heterogeneous: Heterogeneous proteins or polypeptides refer to proteins or polypeptides derived from different sources or species. Similarly, heterogeneous ORFs are ORFs derived from different sources or species.

[0053] Hexon: The main adenovirus capsid protein. An exemplary hexon sequence derived from Ad5 is shown herein as SEQ ID NO: 34. A mutant hexon sequence containing the E451Q substitution is shown herein as SEQ ID NO: 35.

[0054] Immunomodulatory proteins are proteins that alter the immune system (e.g., activate, strengthen, or suppress it). Immunomodulators include, but are not limited to, cytokines (such as interleukin-2 (IL-2), IL-7, IL-12, GM-CSF, FLT3 ligand, or interferon), chemokines (such as CCL3, CCL26, CXCL7, CXCL9, and CXCL10), T cell activating ligands (such as anti-CD3 antibodies or alloantigens), costimulatory molecules (such as B7.1 / B7.2, OX40L, 4-1-BBL, or CD40L), checkpoint blocker inhibitors (such as anti-PD-1 antibodies or anti-CTLA4 antibodies), and small molecule immunomodulators.

[0055] Isolated: An “isolated” biological component (such as a nucleic acid molecule, protein, virus, or cell) is one that has been substantially separated or purified from other biological components in the organism's cells or tissues, or in the organism itself, such as other chromosomes and extrachromosomal DNA and RNA, proteins, and cells, from which it naturally exists. “Isolated” nucleic acid molecules and proteins include those purified by standard purification methods. This term also includes nucleic acid molecules and proteins prepared by recombinant expression in host cells, as well as chemically synthesized nucleic acid molecules and proteins.

[0056] MicroRNAs (miRNAs or miRs) are single-stranded RNA molecules that regulate gene expression in plants, animals, and viruses. Genes encoding microRNAs are transcribed to form primary transcript microRNAs (pre-miRNAs), which are processed to form short stem-loop molecules called precursor microRNAs (pre-miRNAs), and subsequently cleaved in a nucleotide chain-cleavage manner to form mature microRNAs. Mature microRNAs are approximately 21–23 nucleotides long and are partially complementary to the 3'UTR of one or more target messenger RNAs (mRNAs). MicroRNAs modulate gene expression by promoting the cleavage of target mRNAs or by blocking the translation of cellular transcripts. In the context of this disclosure, “liver-specific microRNAs” are microRNAs that are preferentially expressed in the liver, e.g., microRNAs expressed only in the liver or microRNAs that are significantly more expressed in the liver compared to other organs or tissue types.

[0057] Modification: A change in the sequence of a nucleic acid or protein sequence. For example, modifications of an amino acid sequence include, for example, substitutions, insertions, and deletions, or combinations thereof. Insertions include the fusion of an amino terminus and / or a carboxyl terminus, as well as the insertion of one or more amino acid residues into a sequence. Deletions are characterized by the removal of one or more amino acid residues from a protein sequence. In some embodiments herein, modifications (such as substitutions, insertions, or deletions) result in changes in function, such as a reduction or enhancement of a particular activity of a protein. As used herein, "Δ" or "delta" refers to a deletion. For example, E1AΔLXCXE refers to an E1A polypeptide having a deletion of the LXCXE motif. A substitution modification is one in which at least one residue is removed and a different residue is inserted in its place. Amino acid substitutions are typically of a single residue, but may occur simultaneously at several different positions. Substitutions, deletions, insertions, or any combination thereof may be combined to arrive at the final mutant sequence. These modifications can be prepared by modifying nucleotides in protein-coding DNA, thereby producing DNA that encodes these modifications. Techniques for making insertion, deletion, and substitution mutations at predetermined sites in DNA having known sequences are well known in the art. A “modified” protein, nucleic acid, or virus is one that has one or more of the modifications outlined above.

[0058] Neoplasms, malignant diseases, cancer, and tumors: A neoplasm is an abnormal proliferation of tissue or cells resulting from excessive cell division. Neoplastic proliferation can produce tumors. The amount of tumor in an individual is called "tumor mass" and can be measured as the number, volume, or weight of tumors. Tumors that do not metastasize are called "benign." Tumors that invade surrounding tissues and / or can metastasize are called "malignant." Malignant tumors are also called "cancer."

[0059] Hematological cancers are cancers of the blood or bone marrow. Examples of hematological (or hematopoietic) cancers include leukemia, such as acute leukemia (acute lymphoblastic leukemia, acute myeloid leukemia, acute myeloid leukemia, and myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia), chronic leukemia (chronic myeloid (granulocytic) leukemia, chronic myeloid leukemia, and chronic lymphocytic leukemia), polycythemia vera, lymphoma, Hodgkin's disease, non-Hodgkin lymphoma (painless and high-grade forms), multiple myeloma, Waldenström macroglobulinemia, heavy chain disease, myelodysplastic syndrome, hairy cell leukemia, and spinal cord malformations. In some cases, lymphoma is considered a solid tumor.

[0060] A solid tumor is a mass of abnormal tissue that typically does not contain cysts or fluid-filled areas. Solid tumors can be benign or malignant. Different types of solid tumors are named according to the type of cells that form them (e.g., sarcoma, carcinoma, and lymphoma). Examples of solid tumors, such as sarcomas and carcinomas, include fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteosarcoma, and other sarcomas, synovial tumors, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon cancer, lymphoid malignancies, pancreatic cancer, breast cancer, lung cancer, ovarian cancer, prostate cancer, hepatocellular carcinoma, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, medullary thyroid carcinoma, papillary thyroid carcinoma, pheochromocytoma, sebaceous carcinoma, papillary carcinoma, human papillomavirus (HPV) neoplasia, papillary adenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renal cell carcinoma, hepatome, cholangiocarcinoma, choriocarcinoma, Wilms' tumor, and cervical cancer. Examples include cancer, testicular tumors, seminomas, bladder cancer, melanoma, and CNS tumors (gliomas such as brainstem gliomas and mixed gliomas, glioblastoma (also known as glioblastoma multiforme), astrocytoma, CNS lymphoma, germ cell tumors, medulloblastoma, schwannoma, craniopharyogioma, ependymoma, pineal glandoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, neuroblastoma, retinoblastoma, and brain metastases).

[0061] Oncolytic viruses are viruses that selectively kill cells with impaired proliferative function, such as cancer / tumor cells. Killing of cancer cells can be detected by any method established in the art, such as determining the number of viable cells, or by detecting cytopathic effects, apoptosis, or synthesis of viral proteins in cancer cells (e.g., by metabolic labeling, immunoblotting, or RT-PCR of viral genes necessary for replication), or by reducing tumor size.

[0062] Operablely linked: When a first nucleic acid sequence has a functional relationship with a second nucleic acid sequence, the first nucleic acid sequence is operably linked to the second nucleic acid sequence. For example, if a promoter affects the transcription or expression of a coding sequence, the promoter is operably linked to the coding sequence. Generally, operably linked DNA sequences are contiguous and, if two protein coding regions need to be joined, they are within the same reading frame.

[0063] Pharmacoherent Vehicles: The pharmaceutically acceptable vehicles useful in this disclosure are conventional ones. (E.W. Martin, Remington's Pharmaceutical Sciences) Mack Publishing Co., Easton, PA, 15th edition (1975) contains one or more This document describes compositions and formulations suitable for the pharmaceutically effective delivery of therapeutic compounds, molecules, or drugs (e.g., recombinant viruses or recombinant viral genomes disclosed herein). Generally, the properties of the carrier will depend on the specific mode of administration used. For example, parenteral formulations typically contain an injectable fluid as a vehicle, including pharmaceutically and physiologically acceptable fluids such as water, saline, equilibrium salt solutions, aqueous dextrose, and glycerol. For solid compositions (e.g., in the form of powders, pills, tablets, or capsules), conventional non-toxic solid carriers may include, for example, pharmaceutical-grade mannitol, lactose, starch, or magnesium stearate. In addition to a biologically neutral carrier, the pharmaceutical composition to be administered may contain small amounts of non-toxic adjuncts, such as humectants or emulsifiers, preservatives, and pH buffers, for example, sodium acetate or sorbitan monolaurate.

[0064] Polypeptides, peptides, or proteins: polymers in which monomers are amino acid residues joined together through amide bonds. If the amino acid is an alpha-amino acid, either L-optical isomers or D-optical isomers may be used. The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein. These terms apply to amino acid polymers, which are artificial chemical mimics of corresponding naturally occurring amino acids, as well as naturally occurring amino acid polymers and naturally occurring amino acid polymers. The terms “residue” or “amino acid residue” include references to amino acids incorporated into proteins, polypeptides, or peptides.

[0065] A conservative substitution in a polypeptide is the use of one amino acid residue in a protein sequence in place of a different amino acid residue that has similar biological properties. Typically, conservative substitutions have little to no effect on the activity of the resulting polypeptide. For example, a protein or peptide containing one or more conservative substitutions (e.g., no more than one, no more than two, no more than three, no more than four, or no more than five substitutions) retains the structure and function of the wild-type protein or peptide. Polypeptides can be produced to contain one or more conservative substitutions by manipulating the nucleotide sequence encoding the polypeptide, for example, using standard procedures such as site-directed mutagenesis or PCR. In one example, such variants can be readily selected by testing the cross-reactivity of antibodies or the ability of antibodies to induce an immune response. Examples of conservative substitutions are given below. [Table 3]

[0066] Conservative substitutions generally maintain (a) the structure of the polypeptide backbone in the region of substitution, for example, as a sheet conformation or helix conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulkiness of the side chain.

[0067] Generally, substitutions that are expected to bring about the greatest changes in protein properties are non-conservative substitutions, such as (a) substitutions of hydrophilic residues, such as ceryl or threonyl, with hydrophobic residues, such as leucyl, isoleucyl, phenylalanyl, valyl, or alanyl; (b) substitutions of cysteine ​​or proline with any other residue; (c) substitutions of residues with an electropositive side chain, such as lysyl, arginyl, or histadyl, with an electronegative residue, such as glutamyl or aspartyl; or (d) substitutions of residues with a bulky side chain, such as phenylalanine, with a residue without a side chain, such as glycine.

[0068] To prevent, treat, or ameliorate a disease: “Preventing” a disease means preventing the complete onset of the disease. “Treating” means a therapeutic intervention that alleviates the signs or symptoms of a disease or condition after its onset. “Amoliating” means reducing the number or severity of the signs or symptoms of a disease.

[0069] Purified: The term "purified" is intended as a relative term and does not require absolute purity. Therefore, for example, a purified peptide, protein, virus, or other active compound is one that has been isolated, in whole or in part, from naturally associated proteins and other contaminants. In certain embodiments, the term "substantially purified" refers to a peptide, protein, virus, or other active compound that has been isolated from cells, cell culture media, or other crude preparations and subjected to fractionation to remove various components of the initial preparation, such as proteins, cell debris, and other components.

[0070] Rapamycin is a small molecule with known immunosuppressive and antiproliferative properties. Rapamycin is a macrolide first discovered as a product of the bacterium Streptomyces hygroscopicus, also known as sirolimus. Rapamycin binds to mTOR and inhibits its activity. Rapalog (also known as AP21967) is an analogue of rapamycin.

[0071] Recombinant: Recombinant nucleic acid molecules, proteins, or viruses are those that have sequences not found in nature, or sequences created by the artificial combination of two segments of sequences that are otherwise distinct. This artificial combination can be achieved by chemical synthesis or by artificial manipulation of segments of isolated nucleic acid molecules, for example, by genetic engineering techniques. The term “recombinant” also includes nucleic acids, proteins, and viruses that are altered only by the addition, substitution, or deletion of parts of native nucleic acid molecules, proteins, or viruses.

[0072] Replication Deficiency: Adenoviruses exhibiting "replication deficit" in non-tumor cells (compared to tumor cells) refer to adenoviruses that exhibit reduced viral replication in normal cells compared to tumor cells. Replication deficit is evident, for example, in the absence of late viral protein expression in normal cells, reduced viral DNA synthesis, reduced ability to induce E2F target genes (e.g., cyclins A and B), reduced ability to induce S phase entry, and / or reduced ability to induce cell elimination, compared to tumor cells.

[0073] RGD peptides are peptides containing the triamino acid motif arginine-glycine-aspartic acid. The RGD motif is found in numerous substrate proteins, such as fibronectin, fibrinogen, vitronectin, and osteopontin, and plays a role in cell-cell adhesion to the extracellular matrix.

[0074] Self-cleaving peptides are peptides that induce ribosomes to skip the synthesis of peptide bonds at their C-terminus, resulting in the separation of their peptide sequence from downstream polypeptides. Virus-encoded 2A peptides are a type of self-cleaving peptide. Examples of virus-encoded 2A peptides include those derived from porcine rhinitis virus-1 (PTV1), foot-and-mouth disease virus (FMDV), equine rhinitis A virus (ERAV), and Thosea asigna virus (TaV).

[0075] Sequence Identity: The identity or similarity between two or more nucleic acid sequences or two or more amino acid sequences is expressed as sequence identity or similarity. Sequence identity can be measured as a percentage of identity, with a higher percentage indicating more identical sequences. Sequence similarity can be measured as a percentage of similarity (taking into account conservative amino acid substitutions), with a higher percentage indicating more similar sequences.

[0076] Alignment methods for sequences for comparison are publicly known. Various programs and alignment algorithms have been described: Smith and Waterman, Adv. Appl. Math., Vol. 2, p. 482, 1981; Needleman and Wunsch, J. Mol. Biol., Vol. 48, p. 443, 1970; Pearson and Lipman, Proc. Natl. Acad. Sci. USA, Vol. 85, p. 2444, 1988; Higgins and Sharp, Gene, Vol. 73, pp. 237-234, 1988; Higgins and Sharp, CABIOS, Vol. 5, pp. 151-153, 1989; Corpet et al., Nuc. Acids Res., Vol. 16, pp. 10881-10890, 1988; Huang et al., Computer Appls. in the Biosciences, Vol. 8, pp. 155-165, 1992; and Pearson et al., Meth. Mol. Bio., Vol. 24, pp. 307-331, 1994. Altschul et al., J. Mol. Biol., Vol. 215, pp. 403-4010, 1990, present a detailed discussion of sequence alignment methods and homology calculations.

[0077] The NCBI's Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol., Vol. 215, pp. 403-401, 1990) is available from multiple sources, including the National Center for Biological Information (NCBI) and the Internet, for use with the sequence analysis programs blastp, blastn, blastx, tblastn, and tblastx. Additional information can be found on the NCBI website.

[0078] Serotype: A group of closely related microorganisms (such as viruses) distinguished by a characteristic set of antigens.

[0079] Subjects: Living multicellular vertebrate organisms in categories including humans and non-human mammals, e.g., veterinary subjects (e.g., mice, rats, rabbits, cats, dogs, pigs, and non-human primates).

[0080] Synthesis: This refers to substances produced by artificial means in a laboratory. For example, synthetic nucleic acids or proteins can be chemically synthesized in a laboratory.

[0081] Targeted Ligand: In the context of this disclosure, “targeted ligand” is a protein that directs recombinant adenovirus to a specific cell type that expresses a receptor or binding protein specific to the targeted ligand. In some embodiments, the targeted ligand is an antibody specific to a cell surface protein overexpressed in a tumor (e.g., EGFR).

[0082] Therapeutic agents are chemical compounds, small molecules, recombinant viruses, or other compositions, such as antisense compounds, antibodies (monoclonal antibodies (mAbs), e.g., antagonistic mAbs), peptides, or nucleic acid molecules, that, when properly administered to a subject, can induce a desired therapeutic or preventive effect. For example, therapeutic agents for cancer include drugs that prevent or inhibit the development or metastasis of cancer.

[0083] Therapeutic effective dose: The amount of a particular drug or therapeutic agent (e.g., recombinant virus) sufficient to achieve the desired effect in a subject or cell being treated with that drug. The effective dose of a drug may depend on several factors, including, but not limited to, the subject or cell being treated, and the mode of administration of the therapeutic composition.

[0084] A vector is a nucleic acid molecule that enables the insertion of foreign nucleic acids without disrupting the vector's ability to replicate in and / or be incorporated into a host cell. A vector may contain nucleic acid sequences that enable replication in a host cell, such as origins of replication. A vector may also contain one or more select marker genes and other genetic elements. An expression vector is a vector that contains the necessary regulatory sequences to enable the transcription and translation of the inserted gene(s).

[0085] Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those widely understood by those skilled in the art to which this disclosure belongs. The singular terms “a,” “an,” and “the” include plural references unless otherwise clearly indicated by the context. “A or B” means A, B, or A and B. It should be further understood that all base sizes or amino acid sizes and all molecular weight or molecular mass values ​​given with respect to nucleic acids or polypeptides are approximate and provided for illustrative purposes only. Methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In the event of any inconsistency, including the definition of terms, this specification shall prevail. In addition, materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0086] III. Overview of Some Embodiments Tumor-selective recombinant adenoviruses having modifications (such as deletions, substitutions, and / or insertions) in the E3 region are described. In particular, this disclosure describes findings that modifications (e.g., deletions) of at least three E3 genes (such as the E3A gene or the E3B gene) or modifications (e.g., deletions) of six of the seven E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k) that render E3 protein expression inactive lead to enhanced viral replication against adenoviruses having the WT E3 region (such as an increase of at least 20%, at least 50%, at least 75%, at least 90%, at least 95%, at least 100%, at least 200%, or at least 500%). Recombinant adenoviruses may further include additional modifications that enable selective replication in tumor cells and detarget the virus from the liver. The use of recombinant adenoviruses for cancer treatment is described. Additionally, a reporter virus version of the oncolytic virus is described, which can be used as a diagnostic reagent to determine whether a patient's tumor cells are likely to be reactive to the oncolytic virus.

[0087] The specific modifications disclosed herein are described in relation to the adenovirus 5 (Ad5) genome sequence. However, the same modifications and deletions can be made in any human adenovirus serotype. Adenovirus modifications for tumor selectivity, hepatic detargeting, inducible retargeting, retargeting via chimeric fiber proteins, and other modifications are described in detail in PCT publication number WO2016 / 049201 (which is incorporated herein by reference in its entirety).

[0088] A recombinant adenovirus genome is provided herein, comprising an E1A region encoding a modified E1a protein, an E3 region encoding adenovirus death protein (ADP) and containing modifications (mutations in the gene or part thereof, e.g., amino acid substitutions or deletions) in the coding sequence of at least three E3 genes selected from 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k, wherein the modifications (e.g., deletions) prevent the expression of the encoded protein, and an E4 region having modifications (e.g., deletions) in the E4 or f6 / 7 coding sequence.

[0089] In some embodiments, the modified E1A protein includes deletion of the LXCXE motif, deletion of residues 2-11, C124G substitution, Y47H substitution, Y47H substitution and C124G substitution or Y47H substitution, C124G substitution and deletion of residues 2-11 (for example, numbered relative to SEQ ID NO: 23).

[0090] In some embodiments of recombinant adenovirus genomes, at least three E3 genes include 12.5k, 6.7k, and 19k (E3A gene). In some examples, the 12.5k, 6.7k, and 19k genes are deleted. In other examples, the 12.5k, 6.7k, and 19k genes contain mutations introducing a mutated start codon, a mutation introducing an immature stop codon, or both. In certain examples, the 12.5k, 6.7k, and / or 19k genes encode an M1S amino acid substitution.

[0091] In other embodiments of recombinant adenovirus genomes, at least three E3 genes include RIDα, RIDβ, and 14.7k (E3B gene). In some examples, the RIDα, RIDβ, and 14.7k genes are deleted. In other examples, the RIDα, RIDβ, and 14.7k genes contain mutations introducing a mutated start codon, an immature stop codon, or both. In specific examples, the RIDα gene encodes an M1K substitution, the RIDβ gene encodes M1K, C30G, and M60 stop substitutions, and / or the 14.7k gene encodes M1K, M9 stop, M31 stop, and M39 stop substitutions.

[0092] In further embodiments, at least three E3 genes include 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k (all E3 genes except ADP). In some examples, the 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k genes are deleted. In other examples, the 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k genes include mutations introducing mutated start codons, immature stop codons, or both. In specific examples, the RIDα gene encodes an M1K substitution, the RIDβ gene encodes M1K, C30G, and M60 stop substitutions, and / or the 14.7k gene encodes M1K, M9 stop, M31 stop, and M39 stop substitutions.

[0093] In some embodiments, the recombinant adenovirus genome further includes a deletion of E4orf3.

[0094] In some embodiments, the genome further encodes a targeted ligand fused to FK506-binding protein (FKBP) and an adenovirus fiber protein fused to wild-type FKBP-rapamycin-binding (FRB) protein or a mutant FRB protein capable of binding to rapamycin. In some examples, the targeted ligand is a single-domain antibody, for example, a single-domain antibody specific to EGFR.

[0095] In some embodiments, the genome further includes at least one modification for detargeting adenovirus from the liver. In some examples, the modification is a mutation in the hexone protein, e.g., the E451Q mutation (e.g., numbered relative to SEQ ID NO: 34). In some examples, the modification is one or more binding sites for liver-specific microRNAs. In certain examples, one or more binding sites for liver-specific microRNAs are located in the 3'-UTR of E1A. The liver-specific microRNAs may be, for example, miR-122, miR-30, or miR-192.

[0096] In some embodiments, the genome encodes a chimeric fiber protein. In some examples, the chimeric fiber protein comprises a fiber shaft derived from a first adenovirus serotype and a fiber knob derived from a second adenovirus serotype. In certain examples, the first adenovirus serotype is Ad5, and the second adenovirus serotype is Ad3, Ad9, Ad11, Ad12, Ad34, or Ad37. In one non-limiting example, the first adenovirus serotype is Ad5, and the second adenovirus serotype is Ad34. In one example, the Ad34 fiber knob includes a modification that prevents or inhibits binding to CD46, e.g., F242S (the wild-type Ad34 fiber sequence is shown herein as SEQ ID NO: 60).

[0097] In some embodiments, the genome encodes a fiber protein that has been modified to contain the RGD peptide.

[0098] In some embodiments, the genome further comprises heterologous ORFs. In some examples, the heterologous ORFs are operably linked to the self-cleaving peptide coding sequence and the ADP coding sequence within the same reading frame. In some examples, the self-cleaving peptide is a 2A peptide. In specific examples, the 2A peptide includes porcine rhinitis virus-1 (PTV1) 2A (P2A) peptide, foot-and-mouth disease virus (FMDV) 2A (F2A) peptide, equine rhinitis A virus (ERAV) 2A (E2A) peptide, or Thosea asigna virus (TaV) 2A (T2A) peptide.

[0099] In some cases, the heterologous ORF is a reporter gene. In some cases, the reporter gene encodes a fluorescent protein, such as green fluorescent protein, yellow fluorescent protein, or red (or near-infrared) fluorescent protein. In specific cases, the fluorescent protein is YPet, mCherry, or Katsushka2S. In other cases, the reporter gene encodes a soluble factor that can be detected in serum as a biomarker of viral replication. In certain non-limiting cases, the soluble factor is PSA. In yet another case, the reporter gene encodes an imaging probe, such as luciferase, metalloprotein, sodium iodide cotransporter, or thymidine kinase, such as herpes simplex virus thymidine kinase (HSV-tk).

[0100] In other examples, heterologous ORFs are therapeutic genes encoding, for example, RNAi sequences, proteins, antibodies or their binding fragments, chemokines, cytokines, immunomodulators, or enzymes. In some cases, therapeutic genes encode immunomodulatory proteins.

[0101] In certain non-limiting examples, immunomodulatory proteins are cytokines (e.g., IL-1α, IL-1β, IL-6, IL-9, IL-12, IL-13, IL-17, IL-18, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-33, IL-2, IL-4, IL-5, IL-7, IL-10, IL-15, IL-1RA, IFNα, IFNβ, IFNγ, TNFα, TGFβ, lymphotoxin A (LTA), GM-CSF, HMGB1, and FLT3 ligands).

[0102] In other specific examples, immunomodulatory proteins are chemokines (e.g., IL-8, CCL5, CCL17, CCL20, CCL22, CXCL9, CXCL10, CXCL11, CXCL13, CXCL12, CCL19, CCL21, CXCR2, CCR2, CCR4, CCR5, CCR6, CCR7, CCR8, CXCR3, CXCR4, CXCR5, and CRTH2, but not limited to these).

[0103] In other specific examples, immunomodulatory proteins are T cell stimulating ligands (e.g., cell surface anti-CD3, bispecific T cell engagers (BiTe), T cell stimulating MHC / HLA molecules (allogeneic) or tumor antigens, but not limited to these) or costimulatory molecules (such as B7.1, B7.2, OX40L, CD40L, CD70, LIGHT, ICOS, or 4-1BBL).

[0104] In other examples, therapeutic genes encode antibodies (mAbs, e.g., antagonistic mAbs), antigen-binding fragments of antibodies, or soluble antagonists. Specific, non-limiting examples of such molecules include anti-VEGF, anti-TGF-β, soluble TGF-β receptor, anti-PD-1, PD-IL, and LAg3.

[0105] In some embodiments, the nucleotide sequence of the genome is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, or SEQ ID NO: 59.

[0106] Furthermore, isolated cells (mammalian cells, such as mammalian tumor cells) containing the recombinant adenovirus genome disclosed herein are also provided herein.

[0107] Further provided are compositions comprising a recombinant adenovirus genome disclosed herein and a pharmaceutically acceptable carrier, such as water or saline solution.

[0108] Furthermore, isolated adenoviruses comprising the recombinant adenovirus genome disclosed herein are provided. Compositions comprising the isolated adenovirus and a pharmaceutically acceptable carrier (such as water or saline) are further provided.

[0109] Further methods are provided for inhibiting the viability of tumor cells by contacting them with recombinant adenovirus genomes, adenoviruses, or compositions described herein. In some embodiments, the method is an in vitro method. In other embodiments, the method is an in vivo method, in which contacting tumor cells involves administering a therapeutically effective amount of recombinant adenovirus genomes, adenoviruses, or compositions to a subject having a tumor.

[0110] Furthermore, a method is provided for inhibiting tumor progression or reducing tumor volume in a subject. This method comprises administering to a subject a therapeutically effective dose of a recombinant adenovirus genome, adenovirus, or composition described herein.

[0111] A method for treating cancer in a subject is further provided. This method involves administering to a subject a therapeutically effective dose of a recombinant adenovirus genome, recombinant adenovirus, or composition disclosed herein.

[0112] Furthermore, recombinant adenovirus genomes are provided herein in which the nucleotide sequence of the genome is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 58. In some embodiments, the nucleotide sequence of the genome includes or consists of SEQ ID NO: 2, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 45, SEQ ID NO: 46, SEQ ID NO: 55, SEQ ID NO: 56, or SEQ ID NO: 58. Isolated adenoviruses containing recombinant adenovirus genomes are further provided.

[0113] IV. Wild-type and mutant viral sequences A recombinant adenovirus genome is disclosed that contains a nucleotide sequence that is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59. In a particular example, the recombinant nucleic acid contains or consists of any one of SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59.

[0114] Also provided are vectors (such as plasmids or viral vectors) containing recombinant adenovirus genomes. In some embodiments, vectors are provided that contain nucleic acid molecules that are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of SEQ ID NOs. 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59. In some examples, vectors are provided that contain a nucleic acid molecule containing or consisting of any one of the nucleotide sequences among SEQ ID NOs: 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, or 59.

[0115] Exemplary wild-type and mutant adenovirus protein sequences expressed by the recombinant adenoviruses disclosed herein are provided below.

[0116] E1A and E4orf6 / 7 mutants In the following E1A sequences, the LXCXE motif is indicated by an underline. This motif is located at amino acids 122-126 of Ad5 E1A (SEQ ID NO: 23). The Y47H and C124G substitutions are indicated in bold. Modifications (including deletions) of E1A and E4orf6 / 7 contribute to tumor-selective replication of the recombinant adenovirus disclosed herein.

[0117] [ka] [ka] [ka] [ka]

[0118] Recombinant adenoviruses having genomes encoding modified E4orf6 / 7 proteins that eliminate or impair E2F binding or delete or impair nuclear localization signals are contemplated herein. In some examples, the modified E4orf6 / 7 proteins include deletions of approximately 60, 50, 40, 30, 20, or 10 amino acids at the C-terminus to delete / impair the E2F binding site. In other examples, the E4orf6 / 7 proteins include deletions, frameshifts or insertions in 10 amino acids at the C-terminus or deletions of 33 amino acids from the C-terminal third of the protein to invalidate or impair E2F binding. In some embodiments, the mutations include amino acids 81–91 that impair E2F interaction.

[0119] In other examples, the modified E4orf6 / 7 protein contains an N-terminal deletion of 58 amino acids that overrides the nuclear localization sequence required for efficient translocation of E2F4. There are eight arginine residues located between amino acids 13-38 of Ad2 and Ad5 E4orf6 / 7, which is consistent with the more than 25% arginine content of this region. The overall clustering of arginine residues at the N-terminus of E4orf6 is maintained in other adenovirus serotypes. Mutations are attempted that replace arginine residues 16, 18, 21, 22, 27 and / or 29 with alanine (or other suitable residues that override nuclear localization by this region).

[0120] In other examples, modified E4orf6 / 7 proteins include one or more modifications that induce E2F bisite occupancy by eliminating or inhibiting the ability of E4orf6 / 7. Specific non-limiting examples include mutations of F125 to proline, alanine, lysine, aspartic acid, or glutamic acid, or mutations of D121 to P, A, K, R, G, or F (amino acid positions relative to SEQ ID NO: 29).

[0121] Other E4orf6 / 7 mutations include T133A, R101A, Q105P, or any mutation that interferes with single-site occupancy of E2F, M84N or P, G, K, L, H and / or E93A or K, P, G, R, L, M (amino acid positions relative to SEQ ID NO: 29). Other intended mutations include T133Q or A, K, G, P, L, H, G141L, P, KH, F, A or V149N, K, P, H, G, E, D (amino acid positions relative to SEQ ID NO: 29).

[0122] Furthermore, recombinant adenovirus genomes with partial or complete deletions of E4orf6 / 7 are also being considered.

[0123] Fiber arrangement In the following recombinant Ad5 fiber sequences, the FRB sequence is underlined. Mutations present within the FRB* are shown in bold.

[0124] [ka] [ka] [ka]

[0125] V. Self-cleaving peptide sequence Self-cleaving peptides are peptides that induce ribosomes to skip the synthesis of peptide bonds at the C-terminus, resulting in the separation of their peptide sequence from downstream polypeptides. The use of self-cleaving peptides allows for the expression of multiple proteins adjacent to the self-cleaving peptide from a single ORF. The 2A peptide encoded by viruses is a type of self-cleaving peptide.

[0126] Like other self-cleaving peptides, 2A peptides function by causing ribosomes to skip the synthesis of peptide bonds at the C-terminus of the 2A element, resulting in the separation of the 2A sequence end from the downstream peptide (Kim et al., PLoS One, vol. 6(4), e18556, 2011). The "cleavage" occurs between the glycine and proline residues found at the C-terminus of the 2A peptide. Exemplary 2A peptides include, but are not limited to, those encoded by TaV, ERAV, PTV1, and FMDV, or their modified versions.

[0127] In certain examples herein, the 2A peptide includes PTV1 2A (P2A), FMDV 2A (F2A), ERAV 2A (E2A), or TaV 2A (T2A), the sequences of which are shown below as Sequence IDs 15-18 and described herein. P2A: ATNFSLLKQAGDVEENPGP (Sequence ID 15) F2A: VKQTLNFDLLKLAGDVESNPGP(Sequence ID 16) E2A: QCTNYALLKLAGDVESNPGP (Sequence ID 17) T2A: EGRGSLLTCGDVEENPGP (Sequence ID 18)

[0128] In some examples, the 2A peptide is modified to include Gly-Ser-Gly at its N-terminus to improve cleavage efficiency. The sequences of the modified P2A, F2A, E2A, and T2A are shown below as SEQ ID NOs. 19-22 and are described herein. Modified P2A: GSGATNFSLLKQAGDVEENPGP (Sequence ID 19) Modified F2A: GSGVKQTLNFDLLKLAGDVESNPGP (SEQ ID NO: 20) Modified E2A: GSGQCTNYALLKLAGDVESNPGP (SEQ ID NO: 21) Modified T2A: GSGEGRGSLLTCGDVEENPGP (Sequence ID 22)

[0129] In some embodiments, the 2A polypeptide is a variant of the 2A polypeptide disclosed herein. The variant may contain a polypeptide sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or higher sequence identity with respect to the wild-type or modified 2A polypeptide disclosed herein. The variant may include, for example, a 2A polypeptide from any one of SEQ ID NOs. 15, 16, 17, 18, 19, 20, 21, or 22 with at least one N-terminal amino acid deleted, for example, one, two, three, four, or five amino acids deleted. The variant may also contain a 2A polypeptide from any one of SEQ ID NOs. 15, 16, 17, 18, 19, 20, 21, or 22 with at least one C-terminal amino acid deleted, for example, one, two, three, four, or five amino acids deleted. A variant may include at least one, two, three, four, or five amino acid substitutions, such as conservative amino acid substitutions.

[0130] IV. Pharmaceutical Compositions Compositions comprising recombinant adenoviruses or recombinant adenovirus genomes disclosed herein are provided herein. These compositions are suitable for formulation and administration in vitro or in vivo, as needed. Optionally, these compositions comprise one or more of the provided agents and a pharmaceutically acceptable carrier. Suitable carriers and their formulations are described in Remington: The Science and Practice of Pharmacy. This is described in the 22nd edition, edited by Loyd V. Allen et al., Pharmaceutical Press (2012). Pharmaceutically acceptable carriers include materials that are neither biologically nor otherwise undesirable. That is, the material is administered to the subject without causing undesirable biological effects or interacting in a harmful manner with other components in the pharmaceutical composition containing it. When administered to a subject, the carrier is selected, if necessary, to minimize the degradation of the active ingredient and minimize adverse side effects in the subject.

[0131] Recombinant viruses or recombinant adenovirus genomes are administered according to known methods, for example, by intramuscular, intraperitoneal, intraracerobrospinal, subcutaneous, intra-articular, synovial bursa, intrathecal, oral, topical, intratumoral, or inhalation routes, for example, by intravenous administration, e.g., bolus or prolonged continuous infusion. Administration may be topical or systemic. The composition may be administered topically, orally, parenterally, intravenously, intra-articular, intraperitoneal, intramuscular, subcutaneous, intracavitary, percutaneous, intrahepatic, intracranial, intratumoral, intraosseous, by spraying / inhalation, and It can be administered via any of several routes of administration, including by placement via bronchoscopy. Therefore, the composition is administered by several means depending on whether a local or systemic treatment is desired, and depending on the area to be treated.

[0132] In some embodiments, the administration composition comprises recombinant adenovirus (or recombinant genome) as described herein, dissolved in a pharmaceutically acceptable carrier (e.g., an aqueous carrier). Various aqueous carriers, such as buffered saline, can be used. These solutions are sterile and typically do not contain undesirable substances. These compositions can be sterilized. The compositions may optionally contain pharmaceutically acceptable auxiliary substances, such as pH adjusters and buffers, toxicity modifiers, etc., to approximate physiological conditions, such as sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the active agent in these formulations can vary widely and will be selected primarily based on the fluid volume, viscosity, body weight, etc., depending on the selected specific mode of administration and the needs of the subject.

[0133] Pharmaceutical formulations, particularly those of recombinant viruses or recombinant adenovirus genomes, can be prepared by mixing recombinant adenovirus (or recombinant adenovirus genome) of a desired degree of purity with an optional pharmaceutically acceptable carrier, excipient, or stabilizer. Such formulations may be lyophilized or aqueous solutions.

[0134] Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at the dosage and concentration used. Acceptable carriers, excipients, or stabilizers may include acetates, phosphates, citrates, and other organic acids; antioxidants (e.g., ascorbic acid), preservatives, low molecular weight polypeptides; proteins (e.g., serum albumin or gelatin), or hydrophilic polymers (e.g., polyvinylpyrrolidone); as well as amino acids, monosaccharides, disaccharides, and other carbohydrates (e.g., glucose, mannose, or dextrin); chelating agents; as well as ionic and nonionic surfactants (e.g., polysorbate); salt-forming counterions (e.g., sodium); metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants. Recombinant adenoviruses (or one or more nucleic acids encoding recombinant adenoviruses) can be formulated in any appropriate concentration of infectious units.

[0135] Formulations suitable for oral administration may consist of (a) an effective amount of recombinant adenovirus suspended in a liquid solution, e.g., a diluent, e.g., water, saline, or PEG 400; (b) capsules, sachets, or tablets each containing a predetermined amount of the active ingredient as a liquid, solid, granule, or gelatin; (c) a suspension in a suitable liquid; and (d) a suitable emulsion. Tablet forms may include lactose, sucrose, mannitol, sorbitol, calcium phosphate, corn starch, potato starch, microcrystalline cellulose, gelatin, colloidal silicon dioxide, talc, magnesium stearate, stearic acid, and other excipients, colorants, fillers, binders, diluents, buffers, moistening agents, preservatives, flavoring agents, dyes, disintegrants, and pharmaceutically acceptable loads. The body may contain one or more of the components. The lozenge form may contain the active ingredient in a flavoring agent, such as sucrose, and in addition to the active ingredient, a carrier known in the art, in an inert base, such as an emulsion or gel of gelatin and glycerin or sucrose and acacia.

[0136] Recombinant adenoviruses or recombinant adenovirus genomes, alone or in combination with other suitable components, can be made into aerosol formulations administered by inhalation (i.e., they can be "aerosolized"). The aerosol formulations can be placed in pressurized, acceptable sprays such as dichlorodifluoromethane, propane, or nitrogen.

[0137] Suitable formulations for parenteral administration, such as intra-articular, intravenous, intramuscular, intratumor, intradermal, intraperitoneal, and subcutaneous routes, include aqueous and non-aqueous isotonic sterile injection solutions (which may contain antioxidants, buffers, bacteriostatic agents, and solutes to make the formulation isotonic with the blood of the intended recipient), as well as aqueous and non-aqueous sterile suspensions which may contain suspending agents, solubilizers, thickeners, stabilizers, and preservatives. In the provided method, the composition can be administered orally, topically, intraperitoneally, intravesically, intravesically, intratumorally, or intrathecally, for example, by intravenous injection. Parenteral administration, intratumor administration, and intravenous administration are preferred methods of administration. Formulations of the compound may be presented in sealed containers of single or multi-dose quantities, such as ampoules and vials.

[0138] Injectable solutions and suspensions can be prepared from the aforementioned types of sterile powders, granules, and tablets. Cells transduced or infected with adenovirus, or, in the case of ex vivo therapy, cells transfected with nucleic acids, can also be administered intravenously or parenterally as described above.

[0139] Pharmaceutical preparations can be in unit dosage forms. In such forms, the preparation is further divided into unit doses containing appropriate amounts of active components. Thus, pharmaceutical compositions can be administered in various unit dosage forms depending on the method of administration. For example, suitable unit dosage forms for oral administration include, but are not limited to, powders, tablets, pills, capsules, and lozenges.

[0140] In some embodiments, the composition comprises at least two different recombinant adenoviruses or recombinant adenovirus genomes, for example, recombinant adenoviruses that bind to different cell receptors. For example, at least one of the recombinant adenoviruses in the composition may express a chimeric fiber protein. Alternatively, the recombinant adenoviruses may target different cell receptors by encoding different targeting ligand-FKBP fusion proteins among the viruses in the composition. In some examples, the composition comprises two, three, four, five, or six different recombinant adenoviruses or recombinant adenovirus genomes.

[0141] VII. Treatment Methods The recombinant adenoviruses and recombinant adenovirus genome compositions disclosed herein can be administered for therapeutic or prophylactic treatment. In particular, methods are provided for inhibiting tumor cell viability in a subject, inhibiting tumor progression in a subject, reducing tumor volume in a subject, reducing the number of metastases in a subject, and / or treating cancer in a subject. Thus, in some examples, the methods reduce tumor cell viability, tumor progression, tumor volume, tumor size, the number of metastases, or a combination thereof, by at least 20%, at least 50%, at least 75%, at least 80%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% compared to no treatment (e.g., before treatment with the recombinant adenoviruses and recombinant adenovirus genome compositions disclosed herein).

[0142] This method involves administering a therapeutically effective dose of recombinant adenovirus or recombinant adenovirus genome (or a composition thereof) to a subject. As described throughout, the adenovirus or pharmaceutical composition is administered by any number of methods, including but not limited to intravenous, intravascular, intrathecal, intramuscular, subcutaneous, intratumoral, intraperitoneal, or oral. If necessary, this method further involves administering one or more additional therapeutic agents to the subject. In some embodiments, the therapeutic agent is a chemotherapeutic agent. In other embodiments, the therapeutic agent is an immunomodulator. In yet another embodiment, the therapeutic agent is a CDK inhibitor, for example, a CDK4 inhibitor.

[0143] In some embodiments, cancer or tumor is cancer or tumor of the lung, prostate, colorectal, breast, thyroid, kidney, or liver, or a type of leukemia. In some cases, the cancer is metastatic. In some embodiments, tumor is tumor of the mammary gland, pituitary gland, thyroid gland, or prostate; tumor of the brain, liver, meninges, bone, ovaries, uterus, or cervix; monocytic or myeloid leukemia; adenocarcinoma, adenoma, astrocytoma, bladder tumor, brain tumor, Burkitt lymphoma, breast cancer, cervical cancer, colon cancer, kidney cancer, liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, rectal cancer, skin cancer, stomach cancer, testicular cancer, thyroid cancer, chondrosarcoma, choriocarcinoma, fibroma, fibrosarcoma, glioblastoma, glioma, hepatome, histiocytoma, leiomyoblastoma, leiomyosarcoma, Lymphoma, liposarcoma, breast tumor, medulloblastoma, myeloma, plasmacytoma, neuroblastoma, glioma, osteogenic sarcoma, pancreatic tumor, pituitary tumor, retinoblastoma, rhabdomyosarcoma, sarcoma, testicular tumor, thymoma, or Wilms' tumor. Tumors include both primary and metastatic solid tumors, and are found in the breast, colon, rectum, lung, oral pharynx, hypopharynx, esophagus, stomach, pancreas, liver, gallbladder and bile ducts, small intestine, urinary tract (including kidneys, bladder, and urothelium), female reproductive tract (including cervix, uterus, and ovaries, as well as choriocarcinoma and gestational trophoblastic disease), male reproductive tract (including prostate, seminal vesicles, testes, and germ cell tumors), endocrine glands (including thyroid, adrenal glands, and pituitary gland), and skin carcinomas, as well as hemangiomas, melanomas, sarcomas (those arising from bone and soft tissue, as well as Kaposi's sarcoma), and tumors of the brain, nerves, eyes, and meninges (including astrocytoma, glioma, glioblastoma, retinoblastoma, neuroma, neuroblastoma, schwannoma, and meningioma). In some embodiments, solid tumors arising from hematopoietic malignancies such as leukemia (i.e., plaques and tumors of green sarcoma, plasmacytoma, and mycosis fungoides, as well as cutaneous T-cell lymphoma / leukemia), as well as solid tumors arising in the treatment of lymphoma (both Hodgkin lymphoma and non-Hodgkin lymphoma), may be treated. In addition, treatment may be useful in preventing metastasis from the tumors described herein.

[0144] In therapeutic applications, recombinant adenovirus or recombinant adenovirus genome or compositions thereof are administered to the subject in a therapeutically effective dose. The effective dose for this use will depend on the severity of the disease and the patient's overall health condition. Single or multiple doses of the composition may be administered depending on the dosage and frequency required and tolerated by the patient. "Patient" or "subject" includes both humans and other animals, particularly mammals. Therefore, this method is applicable to both human therapeutic and veterinary applications.

[0145] An effective amount of an adenovirus having a modified sequence is determined for each individual and is based, at least in part, on the specific recombinant adenovirus used, the size, age and sex of the individual, and the size and other characteristics of the proliferating cells. For example, for the treatment of humans, at least 10 3 plaque forming units (PFU) of recombinant virus are used, for example, at least 10 depending on the type, size, and number of proliferating cells or neoplasms present 4 , at least 10 5 , at least 10 6 , at least 10 7 , at least 10 8 , at least 10 9 , at least 10 10 , at least 10 11 , or at least 10 12 PFU, for example, approximately 10 3 to 10 12 PFU of recombinant virus is used. The effective amount can be from about 1.0 pfu / kg body weight to about 10 15 pfu / kg body weight (for example, from about 10 2 pfu / kg body weight to about 10 13 pfu / kg body weight).

[0146] The recombinant adenovirus or recombinant adenovirus genome is administered in a single dose or in multiple doses (for example, 2, 3, 4, 6, or more doses). Multiple doses can be administered concurrently or consecutively (for example, over a period of days or weeks).

[0147] In some embodiments, the provided methods comprise administering to the subject one or more additional therapeutic agents, for example, an anti-cancer agent or other therapeutic treatment such as surgical resection of a tumor. Exemplary anti-cancer agents include chemotherapeutic agents, for example, mitotic inhibitors, alkylating agents, antimetabolites, intercalating antibiotics, growth factor inhibitors, cell cycle inh Examples of anticancer treatments include, but are not limited to, harmful agents, enzymes, topoisomerase inhibitors, anti-survival agents, biological response modifiers, anti-hormonal agents (e.g., anti-androgens), anti-angiogenic agents, and CDK inhibitors. Other anticancer treatments include radiotherapy and antibodies that specifically target cancer cells (e.g., therapeutic monoclonal antibodies).

[0148] Non-exclusive examples of alkylating agents include nitrogen mustards (such as mechloretamine, cyclophosphamide, melphalan, uracil mustard, or chlorambucil), alkyl sulfonates (such as busulfan), and nitrosoureas (such as carmustine, lomustine, semustine, streptozocin, or dacarbazine).

[0149] Non-exclusive examples of antimetabolites include folate analogs (such as methotrexate), pyrimidine analogs (such as 5-FU or cytarabine), and purine analogs, such as mercaptopurine or thioguanine.

[0150] Non-exclusive examples of natural products include vinca alkaloids (such as vinblastine, vincristine, or vindesine), epipodophyllotoxins (such as etoposide or teniposide), antibiotics (such as dactinomycin, daunorubicin, doxorubicin, bleomycin, plicamycin, or mitomycin C), and enzymes (such as L-asparaginase).

[0151] Other non-exclusive examples of drugs include platinum coordination complexes (such as cis-diamine-dichloroplatinum II, also known as cisplatin), substituted ureas (such as hydroxyurea), methylhydrazine derivatives (such as procarbazine), and adrenal cortical depressants (such as mitotane and aminoglutethimide).

[0152] Non-exclusive examples of hormones and antagonists include corticosteroids (such as prednisone), progestins (such as hydroxyprogesterone caproate, medroxyprogesterone acetate, and magestrol acetate), estrogens (such as diethylstilbestrol and ethinylestradiol), antiestrogenic drugs (such as tamoxifen), and androgens (such as testosterone propionate and fluoxymesterone).

[0153] Examples of commonly used chemotherapy drugs include Adriamycin, Alkeran, Ara-C, BiCNU, Busulfan, CCNU, Carboplatin, and Cisplatin. Cisplatinum, cytoxane, daunorubicin, DTIC, 5-FU, fludarabine Examples include hydra, idarubicin, ifosfamide, methotrexate, mitramycin, mitomycin, mitoxantrone, nitrogen mustard, taxol (or other taxanes, e.g., docetaxel), Velban, vincristine, and VP-16, but some newer drugs include gemcitabine (Gemzar), herceptin, irinotecan (Camptosar, CPT-11), leustatin, navelbine, rituxan STI-571, taxotere, topotecan (Hycamtin), Xeloda (capecitabine), Zevelin, and calcitriol.

[0154] Non-exclusive examples of immunomodulatory factors that can be used include AS-101 (Wyeth-Ayerst Labs.), bropyrimin (Upjohn), gamma interferon (Genentech), GM-CSF (granulocyte-macrophage colony-stimulating factor, Genetics Institute), IL-2 (Cetus or Hoffman-LaRoche), human immunoglobulin (Cutter Biological), IMREG (from Imreg, New Orleans, La.), SK&F 106528, and TNF (tumor necrosis factor, Genentech).

[0155] In some cases, the additional therapeutic agents administered are biologics, e.g., monoclonal antibodies, e.g., 3F8, avagovomab, adecatumumab, aftuzumab, alacizumab, alemtuzumab, altumomab pentetate, anatumomab mafenatox, etc. Polizumab, Alcitumomab, Bavituximab, Bectumomab, Belimumab, Vegillesomab, Bevacizumab, Bivatuzumab mertansine, Blinatumomab, Brentuximab vedotin, Cantuzumab mertansine, Capromab pendetide, Katumakisomab, CC49, Cetuximab, Citatuzumab bogatox, Sixtumumab, Clivatuzumab tetraxetan tetraxetan), conatumumab, dasetuzumab, detumomab, ecromeximab, eculizumab, edrecolomab, epratuzumab, ertumaxomab, etalacizumab, farlets Farletuzumab, Figitumumab, Galiximab, Gemtuzumab Buozogamicin, Girentuximab, Glembatumumab vedotin, Ibritumomab tiuxetan, Igovomab, Imciromab, Intetumumab, Inotuzumab ozogamicin, Ipilimumab Iratumumab, rabetsumab, lexatumumab, lintuzumab, lorvotuzumab mertansine, lucatumumab, Lumiliximab, mapatumumab, matsuzumab, mepolizumab, metelimumab, milatuzumab, mitumomab, morolimumab, nacoro Nacolomab tafenatox, naptumomab estafenatox, necitumumab, nimotuzumab, nofetumomab merpentan, ofatumumab Oportuzumab monatox, Oregovomab, Panitumumab, Pemtumomab, Pertuzumab, Pintumomab, Pritumumab, Ramucirumab, Rilotumumab, Rituximab, Robatumumab, Satumomab pendetide, Sibrotuzumab, Sonepcizumab, Takatuzumab tetraxetan, Taplitumomab paptox paptox), tenatumomab, TGN1412, tisilimmab (tremelimumab), Tigatuzumab, TNX-650, trastuzumab, tremelimumab, t These include tucotuzumab celmoleukin, bertuzumab, volociximab, botumumab, or saltumumab, or a combination thereof. In some cases, the therapeutic antibody is specific to PD-1 or PDL-1 (antagonistic mAbs, e.g., atezolizumab, MPDL3280A, BNS-936558 (nivolumab), pembrolizumab, pidilizumab, CT011, AMP-224, AMP-514, MEDI-0680, BMS-936559, BMS935559, MEDI-4736, MPDL-3280A, or MSB-0010718C).

[0156] In some cases, additional therapeutic agents are CTLA-4, LAG-3, or B7-H3 antagonists, such as Tremelimumab, BMS-986016, and MGA271, respectively.

[0157] In some cases, the additional treatment is a PD-1 or PDL-1 antagonist.

[0158] Another common treatment for some types of cancer is surgical intervention, such as surgical removal of the cancer or a portion thereof. Another example of treatment is radiation therapy, such as administering radioactive materials or energy (external phototherapy, for example) to the tumor site to eradicate the tumor or to help it regress before surgical removal.

[0159] CDK (cyclin-dependent kinase) inhibitors are drugs that inhibit the function of CDK. Non-exclusive examples of CDK inhibitors for use in the provided method include AG-024322, AT7519, AZD5438, flavopyridol, indislam, P1446A-05, PD-0332991, and P276-00 (see, for example, Lapenna et al., Nature Reviews, Vol. 8, pp. 547-566, 2009). Other CDK inhibitors include LY2835219, palbociclib, LEE011 (Novartis), the pan-CDK inhibitor AT7519, seliciclib, CYC065, butyrolactone I, hymenialdisine, SU9516, CINK4, PD0183812, or fascaplysin.

[0160] In some embodiments, the CDK inhibitor is a broad-spectrum inhibitor (such as flavopyridol, oromoucin, roscovitine, kaempaulon, SNS-032, AT7519, AG-024322, (S)-roscovitine, or R547). In other embodiments, the CDK inhibitor is a specific inhibitor (such as fascapricin, ryuvidine, purvalanol A, NU2058, BML-259, SU 9516, PD0332991, etc.). (For example, P-276-00).

[0161] The selection of drugs and dosages can be readily determined by those skilled in the art based on a given disease being treated. Combinations of drugs or compositions may be administered in parallel (e.g., as a single mixture), separately but simultaneously (e.g., via separate intravenous lines), or sequentially (e.g., one drug is administered first, followed by the second drug). Thus, the term "combination" is used to refer to the parallel, simultaneous, or sequential administration of two or more drugs or compositions.

[0162] According to the method disclosed herein, a subject is administered an effective dose of one or more of the agents provided herein. An effective dose is defined as any amount necessary to produce a desired physiological response (e.g., killing of cancer cells). The therapeutic agent is typically administered in an initial dose of about 0.001 mg / kg to about 1000 mg / kg per day. Dose ranges of about 0.01 mg / kg to about 500 mg / kg, or about 0.1 mg / kg to about 200 mg / kg, or about 1 mg / kg to about 100 mg / kg, or about 10 mg / kg to about 50 mg / kg may be used. The dosage, however, may vary depending on the subject's requirements, the severity of the condition being treated, and the compound being used. For example, the dosage may be determined empirically, taking into account the type and stage of cancer diagnosed in a particular subject. The dose administered to the subject shall, in the context of the method provided, be sufficient to produce a beneficial therapeutic response in the patient over time. Determining the appropriate dosage for a particular situation is within the scope of the skills of those skilled in the art. Therefore, the effective amount and schedule for administering a drug can be determined empirically by those skilled in the art. The dosage should not be so high that it causes substantial adverse side effects, such as undesirable cross-reactions or anaphylactic reactions. The dosage may be adjusted by the individual physician if any contraindications occur. Guidance on appropriate dosages for a given class of drugs can be found in the literature.

[0163] A method for inhibiting the viability or proliferation of tumor cells is provided herein by contacting the tumor cells with a recombinant adenovirus, recombinant adenovirus genome, or composition thereof disclosed herein. In some embodiments, the method is an in vitro method. In other embodiments, the method is an in vivo method, and contacting the tumor cells involves administering the recombinant adenovirus, recombinant adenovirus genome, or composition to a subject having a tumor.

[0164] A method is further provided for inhibiting tumor progression or reducing tumor volume in a subject by administering to the subject a therapeutically effective amount of a recombinant adenovirus or recombinant adenovirus genome (or a composition thereof) disclosed herein.

[0165] A method for treating cancer in a subject is also provided, by administering to the subject a therapeutically effective amount of a recombinant adenovirus or recombinant adenovirus genome (or a composition thereof) disclosed herein.

[0166] The following examples are provided to illustrate certain specific characteristics and / or embodiments. These examples are not intended to be construed as limiting the disclosure to the specific characteristics or embodiments described. [Examples]

[0167] (Example 1) Synthetic adenovirus with enhanced replication dynamics This example describes synthetic adenoviruses having various modifications. Some viruses include one or more modifications to at least three E3 genes, for example, deletions of three or six E3 genes, or modifications that block the expression of three or six E3 genes, enabling enhanced viral replication. In some cases, recombinant viruses include other modifications, such as liver detargeting mutations, tumor-selective mutations, targeted ligands, chimeric fiber proteins, and / or reporter genes.

[0168] [Table 1-1] [Table 1-2] [Table 1-3] [Table 1-4] [Table 1-5]

[0169] Virus description: AdSyn-CO335 (SEQ ID NO: 1) is a tumor-selective virus possessing the oncolytic mutations E1AΔLXCXE, ΔE4orf6 / 7, ΔE3-RIDα / β, and ΔE3-14.7k, as well as the hepatic detargeting hexone mutation E451Q. This virus targets EGFR genes EGFRVHH-FKBP and FRB, which are rapamycin-dependent or AP21967-dependent. * -Fibers are also expressed. The generation and characterization of AdSyn-CO335 are described in WO2016 / 049201, which is fully incorporated herein by reference.

[0170] AdSyn-CO821 (SEQ ID NO: 2) is a modified version of AdSyn-CO335 that encodes a fluorescent reporter protein (YPet). YPet is expressed as a fusion protein of a P2A self-cleaving peptide sequence and ADP (YPet-P2A-ADP). Because this virus expresses the reporter, it can be used to determine which patients are most likely to respond to AdSyn-CO335 therapy. For example, tumor biopsies from cancer patients could be used to determine whether the virus could replicate in tumor cells from biopsies. AdSyn-CO821 could also be used to screen a panel of human cancer cell lines to identify genetic mutations or gene expression profiles in tumor cells that support AdSyn-CO335 replication. These data could be used to identify "signs" of tumors that may respond to AdSyn-CO335 therapy.

[0171] AdSyn-CO820 (SEQ ID NO: 3) is an oncolytic virus that differs from AdSyn-CO335 by the deletion of three additional E3 genes (12.5k, 6.7k and 19k). Therefore, this virus lacks 12.5k, 6.7k, 19k, RIDα, RIDβ and 14.7K.

[0172] AdSyn-CO819 (SEQ ID NO: 4) is a modified version of AdSyn-CO820 that encodes a fluorescent reporter protein (YPet). The YPet reporter is encoded as a YPet-P2A-ADP fusion protein. Like AdSyn-CO820, this virus lacks six E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ and 14.7K). AdSyn-CO819 exhibits enhanced potency and replicates faster than AdSyn-CO821 in tumor cell lines. This virus can be used to infect various cancer cell lines and patient-derived tumor samples to define which cancer types and which patients are most likely to respond to AdSyn-CO820. Therefore, AdSyn-CO819 can be used to identify which patients are likely to respond to AdSyn-CO820 therapy before the start of treatment.

[0173] AdSyn-CO1020 (SEQ ID NO: 5) is an EGFRVHH-FKBP fusion protein from the ADP ORF as an EGFRVHH-FKBP-P2A-ADP fusion (EGFR + for retargeting to tumor cells) is a modified version of AdSyn-CO820 that expresses .

[0174] AdSyn-CO874 (SEQ ID NO: 6) is an E3-deleted version of wild-type Ad5 that lacks six of the E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ and 14.7K). AdSyn-CO874 expresses a YPet fluorescent reporter from the adenoviral ADP ORF as a YPet-P2A-ADP fusion protein. This virus exhibits enhanced potency and replicates faster than wild-type Ad5 in tumor cell lines.

[0175] AdSyn-CO1000 (SEQ ID NO: 7) is a modified version of AdSyn-CO874 that also contains the hexon E451Q mutation detargeting the virus from the liver (to enable intravenous delivery), and has E1A LXCXE and E4orf6 / 7 deletions that confer tumor-selective replication. This virus also encodes the YPet reporter as a YPet-P2A-ADP fusion protein. AdSyn-CO1000 exhibits a very high rate of replication.

[0176] AdSyn-CO1067 (SEQ ID NO: 8) is a version of AdSyn-CO1000 that does not contain the YPet reporter. This virus is a therapeutic candidate.

[0177] AdSyn-CO1068 (SEQ ID NO: 9) differs from AdSyn-CO1067 by having an RGD-containing peptide in the fiber HI loop. This modification increases tropism by allowing the virus to utilize RGD-integrin interactions as an alternative infection route.

[0178] AdSyn-CO1069 (SEQ ID NO: 10) is identical to AdSyn-CO1068 except that it encodes the YPet reporter as a YPet-P2A-ADP fusion protein.

[0179] AdSyn-CO964 (SEQ ID NO: 11) is a modified Ad5 virus in which the Ad5 fiber knob is replaced by an Ad34 fiber knob. This virus also expresses the YPet reporter as a YPet-P2A-ADP fusion protein. AdSyn-CO964 shows enhanced replication compared to WT Ad5 and AdSyn-CO335 in multiple cell lines tested, particularly cell lines that have low or no coxsackievirus adenovirus receptor (CAR). This may be due to the fact that the Ad34 knob binds to CD46, which is expressed on all nucleated cells, and therefore allows the virus to enter cells that do not express CAR.

[0180] AdSyn-CO1041 (SEQ ID NO: 12) also encodes a chimeric Ad5 / Ad34 fiber, but differs from AdSyn-CO964 due to the deletion of six E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7K). AdSyn-CO1041 also contains a YPet reporter expressed as a YPet-P2A-ADP fusion protein.

[0181] AdSyn-CO1042 (SEQ ID NO: 13) is a hepatic detargeting, tumor-selective version of AdSyn-CO1041. Specifically, AdSyn-CO1042 contains the E1AΔLXCXE and ΔE4orf6 / 7 oncolytic mutations, as well as the hepatic detargeting hexone mutation E451Q. AdSyn-CO1042 also contains a YPet reporter expressed as a YPet-P2A-ADP fusion protein. This virus exhibits robust replication in a variety of CAR-nonexpressing cell lines and is considerably more potent than WT Ad5. The combination of the Ad34 knob and a specific E3 gene deletion results in a virus with enhanced potency across a wide range of cancer cell lines.

[0182] AdSyn-CO1139 (SEQ ID NO: 14) is identical to AdSyn-CO1142 except for the absence of a reporter gene. Therefore, AdSyn-CO1139 is a therapeutic version of AdSyn-CO1142.

[0183] AdSyn-CO421 (SEQ ID NO: 44) is a WT Ad5 virus that expresses the YPet reporter as a YPet-P2A-ADP fusion protein. This virus is used as a control in the studies described herein.

[0184] AdSyn-CO1056 (SEQ ID NO: 45) is a hepatic detargeting, tumor-selective version of AdSyn-CO964. Specifically, AdSyn-CO1056 contains the E1AΔLXCXE and ΔE4orf6 / 7 oncolytic mutations, as well as the hepatic detargeting hexone mutation E451Q. AdSyn-CO1056 is a modified Ad5 virus in which the Ad5 fiber knob is replaced by an Ad34 fiber knob. It also contains a YPet reporter expressed as a YPet-P2A-ADP fusion protein.

[0185] AdSyn-CO1250 (SEQ ID NO: 46) is a non-reporter version of AdSyn-CO1056. This virus is a candidate for therapy.

[0186] AdSyn-CO1089 (SEQ ID NO: 47) differs from AdSyn-CO1042 by having an RGD-containing peptide (RGD4C) in the putative Ad34 fiber DG loop. RGD4C is inserted after fiber amino acid 512 (corresponding to knob amino acid 112). This modification increases tropism by allowing the virus to utilize RGD-integrin interactions as an alternative infection pathway.

[0187] AdSyn-CO1320 (SEQ ID NO: 48) is identical to AdSyn-CO1000 except that the YPet fluorescent marker is replaced with the Katushka2S fluorescent marker.

[0188] AdSyn-CO1321 (SEQ ID NO: 49) is identical to AdSyn-CO1042 except that the YPet fluorescent marker is replaced with the Katushka2S fluorescent marker.

[0189] AdSyn-CO1325 (SEQ ID NO: 50) is a hepatic detargeting (hexon E451Q), tumor-selective adenovirus containing chimeric Ad5 / Ad34 fibers, deletions of six E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7K), and oncolytic mutations in E1A ΔLXCXE and ΔE4orf6 / 7. AdSyn-CO1325 also expresses human prostate-specific antigen (PSA) as a PSA-P2A-ADP fusion protein. This virus is identical to AdSyn-CO1042 except that the YPet fluorophore of AdSyn-CO1042 is replaced by PSA.

[0190] AdSyn-CO1342 (SEQ ID NO: 51) is a hepatic detargeting, tumor-selective adenovirus encoding a chimeric Ad5 / Ad34 fiber and containing deletions of six E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k). AdSyn-CO1342 also encodes the oncolytic mutations E1A ΔLXCXE and ΔE4orf6 / 7, the hepatic detargeting hexone mutation E451Q, and the Luc2:YPet reporter expressed as a Luc2:YPet-P2A-ADP fusion protein. This virus is identical to AdSyn-CO1042 except that the YPet fluorophore of AdSyn-CO1042 is replaced by a firefly luciferase 2:YPet fusion protein.

[0191] AdSyn-CO1362 (SEQ ID NO: 52) is a hepatic detargeting, tumor-selective adenovirus that encodes a chimeric Ad5 / Ad34 fiber protein with the F242F mutation and contains deletions of six E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7K). AdSyn-CO1362 contains the oncolytic mutations E1A ΔLXCXE and ΔE4orf6 / 7, as well as the hepatic detargeting hexone mutation E451Q. This virus also contains a YPet reporter expressed as a YPet-P2A-ADP fusion protein. AdSyn-CO1362 is almost identical to AdSyn-CO1042, but differs in having a point mutation (F242S) in the Ad34 knob domain that inhibits binding to CD46.

[0192] AdSyn-CO1403 (SEQ ID NO: 53) differs from AdSyn-CO1042 by having an RGD-containing peptide (RGD4C) in the putative Ad34 fiber HI loop. RGD4C is inserted after fiber amino acid 547 (corresponding to knob amino acid 147). This modification increases tropism by allowing the virus to utilize RGD-integrin interactions as an alternative infection pathway.

[0193] AdSyn-CO1404 (SEQ ID NO: 54) differs from AdSyn-CO1042 by having an RGD-containing peptide (RGD4C) in the putative Ad34 fiber IJ loop. RGD4C is inserted after fiber amino acid 569 (corresponding to knob amino acid 169). This modification increases tropism by allowing the virus to utilize RGD-integrin interactions as an alternative infection pathway.

[0194] AdSyn-CO869 (SEQ ID NO: 55) differs from WT Ad5 in that it possesses a YPet-P2A-ADP reporter and has a deletion in the E3B gene (ΔRIDα, ΔRIDβ, Δ14.7k).

[0195] AdSyn-CO996 (SEQ ID NO: 56) differs from WT Ad5 by having the inhibition of expression of the YPet-P2A-ADP reporter and the E3B gene. Expression of RIDα, RIDβ and 14.7K is inhibited by mutation of the start codon of each gene and insertion of one or more premature stop codons.

[0196] AdSyn-CO999 (SEQ ID NO: 57) differs from WT Ad5 by comprising tumor-selective mutations (E1AΔLXCXE and ΔE4orf6 / 7), a liver detargeting mutation (hexon [E451Q]) and a deletion of the E3A gene (Δ12.5k, Δ6.7k, Δ19k). This virus also carries the YPet-P2A-ADP reporter.

[0197] AdSyn-CO1002 (SEQ ID NO: 58) differs from WT Ad5 by comprising the reporter (YPet-P2A-ADP) and a deletion of the E3A gene (Δ12.5k, Δ6.7k, Δ19k).

[0198] AdSyn-CO1347 (SEQ ID NO: 59) is a liver-detargeted, tumor-selective virus comprising a chimeric Ad5 / Ad34 fiber, deletions of E3 genes 12.5k, 6.7k, 19k, RIDα, RIDβ and 14.7K, a deletion of E4orf3, and a YPet reporter expressed as a YPet-P2A-ADP fusion protein. This virus is identical to AdSyn-CO1042, except that it further comprises an E4orf3 deletion. Studies described herein show that deletion of E4orf3 does not negatively affect viral replication in tumor cells.

[0199] (Example 2) Replication Kinetics of E3-Deleted Adenoviruses The replication dynamics of the recombinant adenovirus described in Example 1 were determined using a fluorescence-based viral dynamics (FBVK) assay, as disclosed in PCT Publication WO2017 / 147265 (fully incorporated herein by reference). Briefly, the FBVK assay can be used to determine the dynamics of adenoviruses encoding a fluorescent reporter (commonly YPet) expressed after infection. Measurement of fluorescence intensity provides a reading proportional to the number of infected cells over several stages of the viral life cycle. A semi-logarithmic plot of exponential growth in infected cells against time gives a straight line proportional to the exponential growth rate or replication rate. The replication rate of each virus in a particular cell line is reported as the "ln slope". A higher ln slope indicates a faster rate of replication.

[0200] The replication of AdSyn-CO421, AdSyn-CO819, AdSyn-CO821, AdSyn-CO874, AdSyn-CO1000, AdSyn-CO1041, and AdSyn-CO1042 was tested using the FBVK assay in several different types of cancer cell lines and normal small airway epithelial cells (SAEC). The replication rate was determined for each virus (unit = 1 / day). The results demonstrated that viruses lacking six E3 genes replicated faster than their counterpart viruses that did not lack these genes.

[0201] Figures 1-4 show a direct comparison of the replication dynamics of specific synthetic adenoviruses, as will be discussed below.

[0202] To test the effects of deletions of six E3 genes (all E3 genes except ADP) on replication, the replication of AdSyn-CO421 (WT Ad5) was compared with that of AdSyn-CO874 in A549 cells. As shown in Figure 1, the ln slope of AdSyn-CO874 was greater than that of AdSyn-CO421, indicating that E3 gene deletion enhances replication. Similar results were found in several different cancer cell lines.

[0203] In another study, the replication of AdSyn-CO821 was compared to that of AdSyn-CO819 in MDA-MB-453 breast cancer cells. AdSyn-CO821 is a hepatic detargeting oncolytic virus lacking three E3 genes (RIDα, RIDβ, and 14.7K). AdSyn-CO819 contains the same mutations as AdSyn-CO821 but has deletions of six E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7K). As shown in Figure 2, the deletion of three additional E3 genes led to enhanced replication. Similar results were found in several other cancer cell lines.

[0204] AdSyn-CO964 is an Ad5 reporter-expressing virus that expresses chimeric fiber proteins (Ad5 fiber shaft and Ad34 fiber knob). Replication of this virus was tested in a CAR-negative cell line (HS578T). AdSyn-CO421 was used as a control. As shown in Figure 3, AdSyn-CO964 shows enhanced replication in CAR-negative cells compared to the control virus with the Ad5 knob domain.

[0205] Next, replication of a liver detargeting tumor-selective reporter virus (AdSyn-CO1000) lacking six E3 genes was compared to that of a virus (AdSyn-CO1042) with the same mutation but further expressing chimeric fiber proteins (Ad5 shaft and Ad34 knob). Replication of these viruses was tested in the CAR-negative cell line HS578T. The results demonstrate that replication in CAR-negative cells is enhanced when the virus expresses a fiber protein containing the Ad34 knob domain (Figure 4).

[0206] (Example 3) Replication rate of recombinant oncolytic adenovirus in a group of tumor cell lines Recombinant adenoviruses AdSyn-CO421, AdSyn-CO819, AdSyn-CO821, AdSyn-CO874, AdSyn-CO964, AdSyn-CO1000, AdSyn-CO1041, AdSyn-CO1042, AdSyn-CO1056, AdSyn-CO1069, AdSyn-CO1089, AdSyn-CO1362, AdSyn-CO1320, AdSyn-CO1321, and AdSyn-CO1342 were investigated for their replication rates in various brain, breast, colon, head and neck, lung, prostate, and skin cancer cell lines using fluorescence-based viral dynamics (FBVK) assays. The data are shown in Tables 2A and 2B.

[0207] [Table 2A-1] [Table 2A-2] [Table 2A-3]

[0208] [Table 2B-1] [Table 2B-2] [Table 2B-3]

[0209] (Example 4) In vivo characterization of AdSyn-CO1042 and AdSyn-CO1000 Additional studies were conducted to further characterize recombinant adenoviruses that exhibited enhanced viral replication.

[0210] As demonstrated by in vitro FBVK assays, both AdSyn-CO1000 and AdSyn-CO1042 are E3 deletion viruses with enhanced potency / replication in tumor cells compared to WT Ad5 (see Tables 2A and 2B above). The in vivo efficacy of AdSyn-CO1000 and AdSyn-CO1042 was then tested in an A549 lung tumor xenograft model. 5 × 10⁶ cells were injected into the subcutaneous fat of the breast. 6 Human A549 tumor cells were inoculated into nude mice by cell injection. The tumor was approximately 164 mm. 3 When the mean volume was reached (day 0), the mice were randomized to different treatment groups (n=8 mice per group). Mice received either a single intratumor (IT) injection of PBS or 8 × 10⁶ doses of AdSyn-CO421, AdSyn-1000, or AdSyn-CO1042. 6 A single injection of PFU was administered. AdSyn-CO421 encodes the YPet fluorophore as a YPet-P2A-ADP fusion, otherwise it is wild-type Ad5. Both AdSyn-CO1000 (Figure 5A) and AdSyn-CO1042 (Figure 5B) showed enhanced antitumor activity compared to “wild-type” AdSyn-CO421.

[0211] AdSyn-CO1042 was also tested in an HS578T normal-position triple-negative breast cancer tumor model. 100 μl of HBSS was added to the subcutaneous fat of the right breast, containing 5 × 10⁴ 6 Human HS578T tumor cells were inoculated into 7-week-old NSG mice by cell injection. The tumors were approximately 168 mm in size. 3 When the mean volume was reached (day 0), the mice were randomized to different treatment groups (n=8 mice per group). On days 0, 7, and 14, the mice were given 50 μl of PBS or AdSyn-CO1042 (2 × 10⁻¹⁶). 8 Three doses of PFU were administered.

[0212] As shown in Figure 6, tumor volume in AdSyn-CO1042-treated mice remained low throughout the 40-day study period. All animals in the PBS-treated group had to be sacrificed before day 30 due to tumor burden.

[0213] The following study demonstrates that combinations of tumor-selective mutations (ΔLXCXE and ΔE4orf6 / 7) and hepatic detargeting hexone mutations (E451Q) result in better safety / toxicity profiles for AdSyn-CO1042 and AdSyn-CO1000 compared to WT Ad5, which is important for enabling safe intravenous (IV) delivery of higher doses of these viruses for the treatment of metastatic cancer. On day 0 and again on day 7, AdSyn-CO102, AdSyn-CO181, AdSyn-CO331, and AdSyn-CO1042 were administered in 200 μl volumes of 8 × 10⁶ doses. 8 , 4×10 9 or 2 × 10 10 The drug was administered intravenously to different groups of C57BL / 6 mice (n=5 mice per group) at the specified dose. To investigate hepatotoxicity, the mice were then analyzed for survival (Figure 7A) and elevated liver enzymes (Figure 7B).

[0214] Mice were monitored for macroscopic signs of survival and toxicity up to day 14 (Figure 7A). WT Ad5 was 4 × 10⁶ 9 PFU caused lethal toxicity at this dose, but AdSyn-CO181 and AdSyn-CO331 were tolerable at this dose. AdSyn-CO1042 was tolerable at 2 × 10⁻⁶ doses. 10Higher doses of PFU were also tolerated. Blood samples were collected from all mice on day 2 (pre-administration), day 2 (48 hours after administration 1), and day 9 (48 hours after administration 2) and frozen for subsequent analysis. Blood samples were investigated for the presence of various liver enzymes, such as alanine transaminase (ALT) and aspartate transaminase (AST). The results shown are the mean AST and ALT levels from bleeding on day 2 from various treatment groups (Figure 7B). Mean pre-administration levels of AST and ALT from all mice are also shown. These data indicate that both the ΔLXCXE and ΔE4orf6 / 7 mutations that limit viral replication in normal cells (AdSyn-CO181) and the hexone detargeting mutation present in AdSyn-CO331 lead to reduced toxicity compared to WT Ad5. AdSyn-CO1042 has 2 × 10⁻⁶ 10 Even at higher doses of PFU, reduced hepatotoxicity was observed, demonstrating that the combination of these different mutations present in AdSyn-CO1042 leads to further reductions in toxicity.

[0215] Figure 8 is a table showing the toxicity of AdSyn-CO1000 in BALB / c mice. Two different viruses in 200 μl volumes were intravenously administered to different groups of mice (n=5 mice per group) on days 0, 6, and 12 at the doses shown in the figure. To investigate toxicity, the mice were then analyzed for survival. WT Ad5 was 0.8 × 10⁻⁶. 9 The PFU dose induced lethal toxicity in 2 out of 5 mice, and 3.2 × 10⁻⁶ 9 While a higher dose of PFU killed all mice, AdSyn-CO1000 killed 1 × 10⁶ mice. 9 PFU and 4×10 9 PFU was safer at slightly higher doses. These data demonstrate that, despite its enhanced replication and death efficacy in tumor cells, AdSyn-CO1000 exhibits a better safety profile than WT Ad5 due to the tumor-selective mutations and hepatic detargeting hexone mutations present in AdSyn-CO1000.

[0216] Another study was conducted to evaluate the efficacy of IV delivery of AdSyn-CO1042 in an A549 lung cancer xenograft model. 5 × 10⁶ doses were administered to the subcutaneous fat of the breast. 6 Human A549 tumor cells were inoculated into NSG mice by cell injection. The tumor was approximately 184 mm. 3 When the mean volume was reached (day 0), the mice were randomized to different treatment groups (n=8 mice per group). The mice were given either a single injection of PBS or 2 × 10⁶ injections of AdSyn-CO1042. 9 A single IV injection of PFU was administered. As shown in Figure 9, in AdSyn-CO1042-treated animals, tumor volume remained low throughout the 33-day study period. In contrast, all animals in the saline-treated group had to be sacrificed before day 33 due to tumor burden. Wild-type Ad5 had a tumor volume of 2 × 10⁶. 9 The PFU could not be delivered at that dose because it would be fatal.

[0217] Next, AdSyn-CO1000 and AdSyn-CO1042 were evaluated in normal (non-tumor) cells. Human A549 cells (lung tumor) or human primary small airway epithelial cells (SAEC - normal lung) were infected with one of four different oncolytic viruses (AdSyn-CO874, AdSyn-CO1000, AdSyn-CO1041, and AdSyn-CO1042) at an MOI of 0.12 viral particles per cell. All viruses encode a YPet fluorophore as a reporter, enabling the time-course quantification of viral replication / growth. Immediately after viral infection, virus-infected cells were imaged once every hour using the IncuCyte ZOOM imaging system to quantify the number of YPet+ virus-infected cells over 6–7 days. Data are expressed as the number of YPet+ cells over time. AdSyn-CO874 and AdSyn-CO1000 are identical viruses, except that AdSyn-CO1000 possesses the ΔLXCXE and ΔE4orf6 / 7 mutations and the hepatic detargeting hexone mutation, which confer tumor-selective replication. Similarly, AdSyn-CO1041 and AdSyn-CO1042 are identical viruses, except that AdSyn-CO1042 possesses the ΔLXCXE and ΔE4orf6 / 7 mutations and the hepatic detargeting hexone mutation. In tumor cells, AdSyn-CO1000 and AdSyn-CO1042 showed similar levels of viral growth / replication compared to AdSyn-CO874 and AdSyn-CO1041, respectively (Figure 10). However, in normal lung cells, AdSyn-CO1000 and AdSyn-CO1042 showed significantly attenuated growth / replication compared to AdSyn-CO874 and AdSyn-CO1041. These data indicate that AdSyn-CO1000 and AdSyn-CO1042 demonstrate robust replication in tumor cells, but their replication is significantly attenuated in normal cells.

[0218] (Example 5) Missing or altered E3A, E3B, and / or E4orf3 Studies were conducted to evaluate the effects of suppressing only the E3A gene or only the E3B gene on adenovirus replication dynamics. To assess the role of the E3B gene, AdSyn-CO421, AdSyn-CO869, AdSyn-CO996, and AdSyn-CO874 were investigated using a fluorescence-based virological (FBVK) assay to determine replication rates in human A549 lung tumor cells (Figure 11). Data are reported as ln-slope values ​​for each recombinant adenovirus encoding the Ypet fluorescent protein. In AdSyn-CO869, the RIDα, RIDβ, and 14.7k genes (E3B gene) are deleted from the genome. In AdSyn-CO996, the expression of the RIDα, RIDβ, and 14.7k genes (E3B gene) is suppressed by mutating the start codon or by mutating the genome to encode an immature stop codon. Thus, the gene is still present in the genome, but it is not expressed during viral infection. In AdSyn-CO874, six E3 genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k) are deleted from the genome. These data indicate that deletion or suppression of expression of the RIDα, RIDβ, and 14.7k genes leads to increased viral replication. Deletion of additional E3 genes (12.5k, 6.7k, and 19k) in AdSyn-CO874 leads to a further increase in replication dynamics.

[0219] To evaluate the role of the E3A gene, AdSyn-CO421, AdSyn-CO1002, AdSyn-CO999, and AdSyn-CO1000 were investigated in an FBVK assay to determine their replication rate in human A549 lung tumor cells (Figure 12). Data are reported as ln-slope values ​​for each recombinant adenovirus encoding the Ypet fluorescent protein. In AdSyn-CO1002, the 12.5k, 6.7k, and 19k genes (E3A gene) are deleted from the genome. In AdSyn-CO999, the same E3A gene is deleted, and this virus further contains E1A ΔLXCXE, ΔE4orf6 / 7, and hexone [E451Q] modifications. In AdSyn-CO1000, the E3A and E3B genes (12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k) are deleted from the genome, and this virus further includes E1A ΔLXCXE, ΔE4orf6 / 7, and hexon [E451Q] modifications. These data indicate that deletion of the E3A gene leads to a slight increase in viral replication. Additional deletion of the E3B gene (RIDα, RIDβ, and 14.7k) in AdSyn-CO1000 leads to a further increase in replication dynamics.

[0220] Further studies were conducted to evaluate the effect of E4orf3 deletion on viral replication dynamics. AdSyn-CO1042 and AdSyn-CO1347 were investigated using the FBVK assay to determine replication rates in A549 human lung tumor cells (Figure 13). The graph shows the ln-slope values ​​for each recombinant adenovirus. AdSyn-CO1042 and AdSyn-CO1347 are identical except for the E4orf3 deletion in AdSyn-CO1347. These data demonstrate that E4orf3 can be deleted in Ad5 without negatively impacting viral replication in tumor cells.

[0221] Considering the numerous possible embodiments to which the principles of this disclosure can be applied, it should be recognized that the exemplary embodiments are merely examples of this disclosure and should not be considered as limiting the scope of this disclosure. Rather, the scope of the present invention is defined by the following claims. Accordingly, the inventors claim rights as their invention to all things within the scope and spirit of these claims.

Claims

1. Recombinant adenovirus genome, Modifications that confer tumor-selective replication, wherein the modification includes an E1A region encoding a modified E1a protein and an E4 region containing deletions or modifications of the E4 or f6 / 7 coding sequence, The E3 gene encodes adenovirus death protein (ADP) and includes modifications in the coding sequences of each of the E3 gene 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k, wherein the modifications interfere with the expression of the encoded protein in the E3 region and Recombinant adenovirus genome, including

2. The modified E1a protein described above LXCXE motif missing, Deletion of residues 2-11, C124G substitution, Y47H substitution, Y47H substitution and C124G substitution, or Y47H substitution, C124G substitution, and deletion of residues 2-11 A recombinant adenovirus genome according to claim 1, comprising:

3. The recombinant adenovirus genome according to claim 1 or 2, wherein the 12.5k, 6.7k, and 19k genes are deleted.

4. The recombinant adenovirus genome according to claim 1 or 2, wherein the 12.5k, 6.7k, and 19k genes include a start codon mutation, a mutation introducing an immature stop codon, or both.

5. The recombinant adenovirus genome according to any one of claims 1 to 4, wherein the RIDα, RIDβ, and 14.7k genes are deleted.

6. The recombinant adenovirus genome according to any one of claims 1 to 4, wherein the RIDα, RIDβ, and 14.7k genes include a mutation in the start codon, a mutation introducing an immature stop codon, or both.

7. The recombinant adenovirus genome according to claim 1 or 2, wherein the 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k genes are deleted.

8. The recombinant adenovirus genome according to claim 1 or 2, wherein the 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k genes include a start codon mutation, a mutation introducing an immature stop codon, or both.

9. The recombinant adenovirus genome according to claim 1 or 2, wherein the 12.5k, 6.7k, 19k, RIDα, RIDβ, and 14.7k genes are deleted, or include a mutation in the start codon, a mutation introducing an immature stop codon, or both.

10. A recombinant adenovirus genome according to any one of claims 1 to 9, further comprising a deletion of E4orf3.

11. A recombinant adenovirus genome according to any one of claims 1 to 10, comprising at least one modification for detargeting adenovirus from the liver.

12. The recombinant adenovirus genome according to claim 11, comprising a mutation in the hexone protein.

13. The recombinant adenovirus genome according to claim 12, wherein the hexon mutation is the E451Q mutation.

14. A recombinant adenovirus genome according to any one of claims 1 to 13, encoding a chimeric fiber protein.

15. The recombinant adenovirus genome according to claim 14, wherein the chimeric fiber protein comprises a fiber shaft derived from a first adenovirus serotype and a fiber knob derived from a second adenovirus serotype.

16. The recombinant adenovirus genome according to claim 15, wherein the first adenovirus serotype is Ad5, and the second adenovirus serotype is Ad3, Ad9, Ad11, Ad12, Ad34, or Ad37, and optionally the second adenovirus serotype is Ad34.

17. The recombinant adenovirus genome according to claim 16, wherein the second adenovirus serotype is Ad34, and the Ad34 fiber knob comprises a modification that prevents or inhibits binding to CD46.

18. A recombinant adenovirus genome according to any one of claims 1 to 17, further comprising heterogeneous open reading frames (ORFs).

19. The recombinant adenovirus genome according to claim 18, wherein the heterologous ORF is operably linked to the self-cleaving peptide coding sequence and the ADP coding sequence within the same reading frame, and the self-cleaving peptide is a 2A peptide.

20. The recombinant adenovirus genome according to claim 1, wherein the nucleotide sequence of the genome includes a sequence that is at least 96% identical to SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 10, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 47, SEQ ID NO: 48, SEQ ID NO: 49, SEQ ID NO: 50, SEQ ID NO: 51, SEQ ID NO: 52, SEQ ID NO: 53, SEQ ID NO: 54, SEQ ID NO: 57, or SEQ ID NO:

59.

21. An isolated cell or recombinant adenovirus comprising the recombinant adenovirus genome described in any one of claims 1 to 20.

22. A composition comprising a recombinant adenovirus genome according to any one of claims 1 to 20 or an isolated cell or recombinant adenovirus according to claim 21, and a pharmaceutically acceptable carrier.

23. A composition for use in a method for inhibiting the viability of tumor cells, comprising a recombinant adenovirus genome according to any one of claims 1 to 20, a recombinant adenovirus according to claim 21, or a composition according to claim 22, wherein the method comprises contacting the tumor cells with the recombinant adenovirus genome according to any one of claims 1 to 20, the recombinant adenovirus according to claim 21, or the composition according to claim 22.

24. The composition according to claim 23, wherein the method is an in vivo method, and contacting the tumor cells comprises administering a therapeutically effective amount of the recombinant adenovirus genome, the recombinant adenovirus, or the composition to a subject having a tumor.

25. A composition for use in a method of treating cancer in a subject, comprising a recombinant adenovirus genome according to any one of claims 1 to 20, a recombinant adenovirus according to claim 21, or a composition according to claim 22, wherein the method comprises administering to the subject a therapeutically effective amount of the recombinant adenovirus genome according to any one of claims 1 to 20, the recombinant adenovirus according to claim 21, or the composition according to claim 22, thereby treating the cancer in the subject.

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