oncolytic viruses

Abstract

This invention relates to gene constructs, expression cassettes comprising said gene constructs, and oncolytic viruses, and their use in methods for treating, preventing, or alleviating cancer. The gene constructs, expression cassettes, and oncolytic viruses are particularly useful (but not limited to) for treating bladder cancer, such as non-muscle-invasive bladder cancer.

Description

[0001] This invention relates to gene constructs, expression cassettes comprising said gene constructs, and oncolytic viruses, and their use in methods for treating, preventing, or alleviating cancer. The gene constructs, expression cassettes, and oncolytic viruses are particularly useful (but not limited to) for treating bladder cancer, such as non-muscle-invasive bladder cancer.

[0002] Non-muscle invasive bladder cancer (NMIBC) is a prevalent and serious health problem with a lifelong risk of recurrence. In the UK, approximately 10,000 new cases of NMIBC are diagnosed each year. Surgery is the standard treatment, but it is associated with high recurrence rates and tumor progression.

[0003] Immunotherapy with the live tuberculosis vaccine Bacillus Calmette-Guerin (BCG) injected into the bladder has been a standard treatment for 40 years, typically following surgery for high-risk diseases (intermediate and high-grade tumors). Part of the mechanism by which BCG exerts its immunotherapeutic effect is by activating key receptors that initiate the host's immune defense mechanisms, known as Toll-like receptor ligands (TLRs). While BCG can reduce the risk of recurrence, it comes at the cost of significant local and systemic toxicity, severely limiting its application. Consequently, approximately one-third of patients do not respond to BCG treatment, and another third experience serious side effects, forcing them to discontinue treatment.

[0004] Therefore, for a significant proportion (up to 60%) of bladder cancer patients who are unresponsive to or intolerant of BCG, there remains an urgent unmet need to find alternative therapies. In particular, there is a need for a novel local therapy that can utilize the immunomodulatory effects of BCG to improve efficacy while avoiding its toxic side effects.

[0005] Therefore, a first aspect of the present invention provides a gene construct comprising a first coding sequence encoding a TLR agonist and a second coding sequence encoding a cytokine.

[0006] The inventors have developed a novel anticancer virus that has shown surprising efficacy in treating BCG-unresponsive models (and ultimately, patients). Advantageously, as... Figure 2 and Figure 3 As shown, the virus enhances immunogenicity while maintaining potent tumor cytotoxicity by integrating TLR agonists (such as novel bacterial proteins) and cytokines (such as IL-15) that can promote NK cell proliferation and CD8 T cell cytotoxicity.

[0007] Preferably, the gene construct includes an expression cassette, one embodiment of which is as follows: Figure 1 As shown.

[0008] Therefore, in a second aspect of the invention, an expression cassette is provided, which includes the gene construct of the first aspect.

[0009] like Figure 1 As shown, the construct comprises a first nucleotide sequence encoding a TLR agonist (e.g., a novel bacterial protein) and a second nucleotide sequence encoding a cytokine (e.g., IL-15). However, it will be understood that, as described herein, other TLR agonists and cytokines may also be used.

[0010] In one implementation, the TLR agonist may be a bacterial TLR agonist. Alternatively, the TLR agonist may be a synthetic TLR agonist.

[0011] Preferably, the TLR agonist is a TLR2 agonist, a TLR4 agonist, or a TLR5 agonist. More preferably, the TLR agonist is a TLR2 agonist or a TLR5 agonist.

[0012] TLR agonists can be lipoproteins or lipopeptides. Examples of TLR agonists can be found in Kaczanowska S, Joseph AM, Davila E. TLR agonists: our best frenemy in cancer immunotherapy. J Leukoc Biol. 2013 Jun;93(6):847-63. doi: 10.1189 / jlb.1012501. Epub 2013 Mar8. PMID: 23475577; PMCID: PMC3656332.

[0013] In a preferred embodiment, the TLR2 agonist may be selected from the following: LpqH, LprA, LprG, LpqT, Phos1, MPB83, MPT83, LAM, AraLAM, LM, PIM2 / 6, TDM, HSP70, MymA, PE_PGRS33, EsxL, PPE18, PPE26, PPE32, PPE57, Lrp, GPLs, PILAM, PPE60, OmpA, and OmpC.

[0014] Preferably, the TLR2 agonist is Mycobacterium tuberculosis (Mycobacterium tuberculosis) Mycobacterium tuberculosis PPE60 of ) . In another preferred embodiment, the TLR2 agonist is Shigella dysenteriae ( Shigella dysenteriae The outer membrane proteins OmpA and / or OmpC.

[0015] In a preferred embodiment, the TLR4 agonist is FimH. The TLR4 agonist FimH can be derived from *Escherichia coli* (E. coli). E. coli ) and / or Salmonella enteritidis typhimurium serotype ( Salmonella enterica serovar Typhimurium ).

[0016] In a preferred embodiment, the TLR5 agonist may be selected from the following: FliC, CagL, CagY, FllA, and FlaB. CagL and CagY may be derived from Helicobacter pylori (…). Helicobacter pylori FlaA can originate from Escherichia coli. FlaB can originate from Vibrio vulnificus. Vibrio vulnificus ).

[0017] In a preferred embodiment, the TLR5 agonist is FliC. The TLR5 agonist FliC may be derived from *Escherichia coli* and / or *Burkholderia melioides* (…). Burkholderia pseudomallei Alternatively, the TLR5 agonist FliC may be derived from other Burkholderia or Salmonella strains, including but not limited to: Salmonella Typhimurium (BP). Salmonella typhimurium Salmonella enteritidis () Salmonella enteritidis Salmonella choleraesuis (Swine choleraesuis) Salmonella choleraesuis Salmonella in chickens ( Salmonella gallinarum Salmonella pullorum ( ) Salmonella pullorum ) and enteric Salmonella ( Salmonella enterica ) and its subspecies and serotypes.

[0018] As described in the examples, during the screening of TLR ligands, the inventors discovered that the bacterial flagellin FliC_BP is a particularly effective candidate ligand. Specifically, the inventors found that FliC_BP can activate TLR5 and TLR2-mediated signaling pathways, indicating that FliC_BP has a unique property of binding to the TLR2 receptor or that there is an interaction between TLR5 (the main receptor for flagellins) and TLR2. Therefore, this TLR5 / TLR2 ligand holds promise for enhancing the antitumor immune response of oncolytic viruses with innate bacterial responses.

[0019] Therefore, the most preferred TLR agonist is a TLR5 agonist.

[0020] Most preferably, the TLR5 agonist is FliC_BP.

[0021] FliC_BP is a bacterial flagellin of Burkholderia melioides. FliC_BP (GenBank: ABA48561.1) may contain the amino acid sequence of SEQ ID No: 1, which is as follows:

[0022] MVYRFSARAIRVHARSRRLAGRRTARRSHARFVRIQRWLTERSTQKIRAESRPAPSSFFGVRRNPAQGGRQRTSASARANSGIDGLIQLNLEIFMLGINSNINSLVAQQNLNGSQGALSQ AITRLSSGKRINSAADDAAGLAIATRMQTQINGLNQGVSNANDGVSILQTASSGLTSLTNSLQRIRQLAVQASNGPLSASDASALQQEVAQQISEVNRIASQTNYNGKNILDGSAGTLSFQ VGANVGQTVSVDLTQSMSAAKIGGGMVQTGQTLGTIKVAIDSSGAAWSSGSTGQETTQINVVSDGKGGFTFTDQNNQALSSTAVTAVFGSSTAGTGTAASPSFQTLALSTSATSALSATD QANATAMVAQINAVNKPQTVSNLDISTQTGAYQAMVSIDNALATVNNLQATLGAAQNRFTAIATTQQAGSNNLAQAQSQIQSADFAQETANLSRAQVLQQAGISVLAQANSLPQQVLKLLQ

[0023] [SEQ ID No: 1]

[0024] Therefore, in a preferred embodiment, FliC_BP comprises or consists of the following: an amino acid sequence substantially as shown in SEQ ID No: 1, or a fragment or variant thereof.

[0025] In one implementation, FliC_BP may be encoded by the nucleic acid sequence of SEQ ID No: 2, as shown below:

[0026]

[0027] [SEQ ID No: 2]

[0028] Therefore, in a preferred embodiment, FliC_BP is encoded by a nucleotide sequence essentially as shown in SEQ ID No: 2, or a fragment or variant thereof.

[0029] Flagella produced by *Burkholderia melioides* are typically not glycosylated. Glycosylation of the flagellar sequences in *Burkholderia melioides* can inhibit the function of TLR5 ligands in mammalian viral systems. To detect N-glycosylation sites in *Burkholderia melioides* flagella, the inventors analyzed the sequences using Net N Glyc server website software. Three N-glycosylation sites were identified (AA No. 18, 270, and 358). To avoid N-glycosylation sites, asparagine (N) at the glycosylation sites was replaced with glutamine (Q).

[0030] Therefore, in a preferred embodiment, FliC_BP may contain the optimized amino acid sequence of SEQ ID No: 3, as follows:

[0031] LGINSNINSLVAQQNLQGSQGALSQAITRLSSGKRINSAADDAAGLAIATRMQTQINGLNQGVSNANDGVSILQTASSGLTSLTNSLQRIRQLAVQASNGPLSASDASALQQEVAQQISEVNRIASQTNYNGKNILDGSAGTLSFQVGANVGQTVSVDLTQSMSAAKIGGGMVQTGQTLGTIKVAIDSSGAAW SSGSTGQETTQINVVSDGKGGFTFTDQNNQALSSTAVTAVFGSSTAGTGTAASPSFQTLALSTSATSALSATDQAQATAMVAQINAVNKPQTVSNLDISTQTGAYQAMVSIDNALATVNNLQATLGAAQNRFTAIATTQQAGSNNLAQAQSQIQSADFAQETAQLSRAQVLQQAGISVLAQANSLPQQVLKLLQ

[0032] [SEQ ID No: 3]

[0033] Therefore, in a preferred embodiment, FliC_BP comprises or consists of the following: an optimized amino acid sequence essentially as shown in SEQ ID No: 3, or a fragment or variant thereof.

[0034] In one implementation, FliC_BP may be encoded by an optimized nucleic acid sequence of SEQ ID No: 4, as follows:

[0035]

[0036] [SEQ ID No: 4]

[0037] Therefore, in a preferred embodiment, FliC_BP is encoded by an optimized nucleotide sequence or a fragment or variant thereof, as shown in SEQ ID No: 4.

[0038] To facilitate the extracellular secretion of TLR agonists (e.g., FliC_BP flagellin), the inventors further added a CD33 secretion signal. Therefore, in a preferred embodiment, the CD33 secretion signal is encoded by a first coding sequence of the gene construct described in the first aspect. Preferably, the CD33 secretion signal is located at the 5' of the sequence encoding a TLR agonist (e.g., FliC_BP).

[0039] CD33 secretion signal may include the amino acid sequence of SEQ ID No: 5, as follows:

[0040] MPLLLLLPLLWAGALA

[0041] [SEQ ID No: 5]

[0042] Therefore, in a preferred embodiment, the CD33 secretion signal comprises or consists of the following: an amino acid sequence essentially as shown in SEQ ID No: 5, or a fragment or variant thereof.

[0043] In one embodiment, the CD33 secretion signal may be encoded by the nucleic acid sequence of SEQ ID No: 6, as follows:

[0044] atg cct ctg ctg ctg ctg ctg cct ctg ctg tgg gct ggg gct ctg gcc

[0045] [SEQ ID No: 6]

[0046] Therefore, in a preferred embodiment, the CD33 secretion signal is encoded by a nucleotide sequence, or a fragment or variant thereof, as shown in SEQ ID No: 6.

[0047] Therefore, it can be understood that FliC_BP and CD33 secretion signals can jointly contain the amino acid sequence of SEQ ID No: 7, as follows:

[0048] MPLLLLLPLLWAGALALGINSNINSLVAQQNLQGSQGALSQAITRLSSGKRINSAADDAAGLAIATRMQTQINGLNQGVSNANDGVSILQTASSGLTSLT NSLQRIRQLAVQASNGPLSASDASALQQEVAQQISEVNRIASQTNYNGKNILDGSAGTLSFQVGANVGQTVSVDLTQSMSAAKIGGGMVQTGQTLGTIKVA IDSSGAAWSSGSTGQETTQINVVSDGKGGFTFTDQNNQALSSTAVTAVFGSSTAGTGTAASPSFQTLALSTSATSALSATDQAQATAMVAQINAVNKPQTVSNLDISTQTGAYQAMVSIDNALATVNNLQATLGAAQNRFTAIATTQQAGSNNLAQAQSQIQSADFAQETAQLSRAQVLQQAGISVLAQANSLPQQVLKLLQ

[0049] [SEQ ID No: 7]

[0050] Therefore, in a preferred embodiment, the FliC_BP and CD33 secretion signal comprises or consists of the following: an amino acid sequence substantially as shown in SEQ ID No: 7, or a fragment or variant thereof.

[0051] In one implementation, the FliC_BP and CD33 secretion signals may be encoded by the nucleic acid sequence of SEQ ID No: 8, as follows:

[0052]

[0053] [SEQ ID No: 8]

[0054] Therefore, in a preferred embodiment, the FliC_BP and CD33 secretion signals are encoded by a nucleotide sequence or a fragment or variant thereof, as shown in SEQ ID No: 8.

[0055] The cytokines may be chemokines. The chemokines may be selected from the following: CXCL10, CXCL9, CXCL11, CXCL8, CXCL12, CCL2, CCL3, and CCL5.

[0056] In a preferred embodiment, the cytokines are selected from the following: IL-2, IL-6, IL-10, IL-12, non-secretory IL-12, IL-15, IL-17, IL-21, IL-33, and type I and type II interferons (IFN-α, IFN-β, IFN-γ).

[0057] However, most preferably, the cytokine is IL-15.

[0058] IL-15 may contain the amino acid sequence of SEQ ID No: 9, as follows:

[0059] MDWTWILFLVAAATRVHSNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEKKNIKEFLQSFVHIVQMFINTS

[0060] [SEQ ID No: 9]

[0061] Therefore, in a preferred embodiment, IL-15 comprises or is composed of the following: an amino acid sequence essentially as shown in SEQ ID No:9, or a fragment or variant thereof.

[0062] In one implementation, IL-15 may be encoded by the nucleic acid sequence of SEQ ID No: 10, as follows:

[0063] ATGGACTGGACCTGGATTCTGTTCCTGGTGGCAGCAGCAACAAGGGTGCACAGCAACTGGGTGAATGTGATCTCCGACCTGAAGAAGATCGAGGATCTGATCCAGAGCATGCACATCGACGCCACCCTGTACACAGAGTCCGATGTGCACCCCTCTTGCAAGGTGACCGCCATGAAGTGTTTTCTGCTGGAGCTGCAG GTCATCTCTCTGGAGAGCGGCGACGCCTCCATCCACGATACCGTGGAGAACCTGATCATCCTGGCCAACAATTCTCTGAGCTCCAACGGCAATGTGACAGAGAGCGGCTGCAAGGAGTGTGAGGAGCTGGAGAAAGAACATCAAGGAGTTCCTGCAGTCCTTTGTGCACATCGTGCAGATGTTCATCAATACATCT

[0064] [SEQ ID No: 10]

[0065] Therefore, in a preferred embodiment, IL-15 is encoded by a nucleotide sequence essentially as shown in SEQ ID No: 10, or a fragment or variant thereof.

[0066] Preferably, the first coding sequence encoding a TLR agonist and the second coding sequence encoding a cytokine are controlled by one or more promoters. Most preferably, the first coding sequence encoding a TLR agonist is controlled by a first promoter, and the second coding sequence encoding a cytokine is controlled by a second promoter.

[0067] Alternatively, in another embodiment, the gene encoding a TLR agonist and the gene encoding a cytokine are controlled by the same promoter. Therefore, in this embodiment, the gene construct according to the first aspect comprises a first promoter operatively linked to a first coding sequence encoding a TLR agonist and a second coding sequence encoding a cytokine. Preferably, when controlled by the same promoter, the first and second coding sequences are expressed as a fusion protein. The first and second coding sequences may be separated by a nucleotide sequence encoding a fusion protein linker.

[0068] In a preferred embodiment, the gene construct according to the first aspect comprises a first promoter operatively linked to a first coding sequence encoding a TLR agonist, and / or a second promoter operatively linked to a second coding sequence encoding a cytokine.

[0069] The promoter, or each promoter, can be any nucleotide sequence capable of inducing RNA polymerase to bind and transcribe the coding sequence. The promoter, or each promoter, can be a constitutive promoter or a regulatory promoter. Examples of constitutive promoters include, but are not limited to, the cytomegalovirus (CMV) promoter, the RSV promoter, and the T7 polymerase promoter.

[0070] Therefore, in a preferred embodiment, the promoter, or each promoter, may be selected from the following: CMV, RSV, T7 polymerase, EF1a, SV40, PGK1, Ac5, UBI, MP-84, and MP-135. Advantageously, the adeno-associated virus micropromoters MP-84 and MP-135 are small in size, thus providing more space for recombinant genes in small viruses.

[0071] In a preferred embodiment, the first promoter operatively linked to the first coding sequence encoding a TLR agonist (e.g., FliC_BP) is a cytomegalovirus (CMV) promoter. One embodiment of the nucleotide sequence encoding the CMV promoter is referred to herein as SEQ ID No: 12, as follows:

[0072] agctctgcttatatagacctcccaccgtacacgcctaccgcccatttgcgtcaatggggcggagttgttacgacattttggaaagtcccgttgattttggtgccaaaacaaactcccattgacgtcaatggggtggagacttggaa atccccgtgagtcaaaccgctatccacgcccattgatgtactgccaaaaccgcatcaccatggtaatagcgatgactaatacgtagatgtactgccaagtaggaaagtcccataaggtcatgtactgggcataatgccaggcgggc catttaccgtcattgacgtcaatagggggcgtacttggcatatgatacacttgatgtactgccaagtgggcagtttaccgtaaatagtccacccattgacgtcaatggaaagtccctattggcgttatactatgggaacatacgtcat tattgacgtcaatgggcgggggtcgttgggcggtcagccaggcgggccatttaccgtaagttatgtaacgcggaactccatatatgggctatgaactaatgaccccgtaattgattactattaataactagtcaataatcaatgtc

[0073] [SEQ ID No: 12]

[0074] Therefore, preferably, the first promoter comprises or consists of the following: a nucleic acid sequence essentially as shown in SEQ ID No: 12, or a fragment or variant thereof.

[0075] Preferably, the second promoter operatively linked to the second coding sequence encoding a cytokine is the RSV promoter. One embodiment of the nucleotide sequence encoding the RSV promoter is referred to herein as SEQ ID No: 13, as follows:

[0076] aatgtagtcttatgcaatacacttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccgattggtggaagtaaggtggtacgatcgtgccttat taggaaggcaacagacaggtctgacatggattggacgaaccactgaattccgcattgcagagataattgtatttaagtgcctagctcgatacaataaacgccatttgaccattcaccacattggtgtgcacc

[0077] [SEQ ID No: 13]

[0078] Therefore, preferably, the second promoter comprises or consists of the following: a nucleic acid sequence essentially as shown in SEQ ID No: 13, or a fragment or variant thereof.

[0079] like Figure 1 As shown, the gene construct (or expression cassette) contains two nucleotide sequences encoding two polyA tails.

[0080] Preferably, the gene construct or expression cassette contains a nucleotide sequence encoding a poly-A tail. More preferably, the gene construct or expression cassette contains a first nucleotide sequence and a second nucleotide sequence encoding first and second poly-A tails, respectively. Preferably, the first and second nucleotide sequences encoding the poly-A tail are located on the flanking sides of the expression cassette.

[0081] Preferably, the first poly-A tail coding sequence is located at the 3' of the first coding sequence encoding a TLR agonist (e.g., FliC_BP).

[0082] Preferably, the second poly-A tail coding sequence is located at the 3' of the second coding sequence that encodes a cytokine (e.g., IL-15).

[0083] An embodiment of the nucleotide sequence encoding the first and / or second poly-A tail (the BGH poly-A tail of CMV) is referred to herein as SEQ ID No: 14, as follows:

[0084] ccatagagcccaccgcatccccagcatgcctgctattgtcttcccaatcctcccccttgctgtcctgccccaccccaccccccagaatagaatgacacctactcagacaatg cgatgcaatttcctcattttattaggaaaggacagtgggagtggcaccttccagggtcaaggaaggcacgggggaggggcaaacaacagatggctggcaactagaaggcacag

[0085] [SEQ ID No: 14]

[0086] Therefore, preferably, the first and / or second poly-A tails contain a nucleic acid sequence or fragments or variants thereof essentially as shown in SEQ ID No: 14.

[0087] An embodiment of the nucleotide sequence encoding the first and / or second poly-A tail (SV40 poly-A tail of RSV) is referred to herein as SEQ ID No: 15, as follows:

[0088] aacttgtttattgcagcttataatggttacaaataaagcaatagcatcacaaatttcacaaataaagcatttttttcactgc

[0089] [SEQ ID No: 15]

[0090] Therefore, preferably, the first and / or second poly-A tails contain a nucleic acid sequence or fragments or variants thereof essentially as shown in SEQ ID No: 15.

[0091] Preferably, the gene construct of the first aspect or the expression cassette of the second aspect is present in the oncolytic virus.

[0092] Therefore, in a third aspect of the present invention, an oncolytic virus is provided, comprising the gene construct of the first aspect or the expression cassette of the second aspect.

[0093] like Figure 1 As shown, the oncolytic virus comprises a first nucleotide sequence encoding a TLR agonist (e.g., a novel bacterial protein, preferably FliC_BP), controlled by a first promoter (e.g., CMV); and a second nucleotide sequence encoding a cytokine (e.g., IL-15), controlled by a second promoter (e.g., RSV). Furthermore, the virus also includes two flanking polyA tails on either side of the expression cassette.

[0094] The inventors have discovered that FliC_BP is a particularly potent candidate factor for enhancing the antitumor immune response of oncolytic viruses with innate bacterial responses. Specifically, they found that FliC_BP can activate TLR5 and TLR2-mediated signaling pathways, suggesting that FliC_BP has a unique property of binding to the TLR2 receptor or that there is an interaction between TLR5 (the major receptor for flagellin) and TLR2. This interaction between TLR2 and TLR5 may enhance the production of pro-inflammatory cytokines, thereby promoting a stronger and more rapid immune response. The inventors believe they are the first to integrate an expression cassette encoding FliC_BP into an oncolytic virus.

[0095] Therefore, in a fourth aspect of the invention, an oncolytic virus is provided that comprises a nucleotide sequence encoding Burkholderia melioides flagellin (FliC_BP). 。

[0096] The nucleotide sequence encoding FliC_BP can be as described in the first or second aspect above. For example, the nucleotide sequence encoding FliC_BP can comprise or consist of the following: a nucleotide sequence substantially as shown in SEQ ID No: 2 or 4, or a fragment or variant thereof.

[0097] Preferably, the oncolytic virus according to the fourth aspect comprises a first promoter operatively linked to a nucleotide sequence encoding FliC_BP.

[0098] The promoter may be as described in the first or second aspect above. For example, the promoter may be selected from the cytomegalovirus (CMV) promoter, the RSV promoter, and the T7 polymerase promoter. In a preferred embodiment, the promoter operatively linked to the nucleotide sequence encoding FliC_BP is a cytomegalovirus (CMV) promoter, which preferably comprises or consists of the following: a nucleic acid sequence substantially as shown in SEQ ID No: 12, or a fragment or variant thereof.

[0099] As used in this article, the term "oncolytic virus" in connection with the third or fourth aspect should be understood as a virus that infects and lyses cancer cells but does not infect normal or healthy cells. Oncolytic viruses can be naturally occurring viruses with oncolytic activity, or they can be constructed by modifying one or more viral genes to improve their selectivity for tumors.

[0100] Preferably, the oncolytic virus is selected from the following: herpes simplex virus (HSV), Coxsackie virus, Maraba virus, measles virus (MV), Newcastle disease virus (NDV), poliovirus, reovirus, retrovirus, Seneca Valley virus (SVV), alphaviruses such as Semliki Forest virus (SFV) and Sindbis virus (SINV), vesicular stomatitis virus (VSV), Sindbis virus (SBV), adenovirus, poxvirus, parvovirus, flaviviruses such as Zika virus, paramyxovirus, picornavirus, and rhabdovirus.

[0101] Most preferably, the oncolytic virus is herpes simplex virus (HSV). The HSV virus can be HSV-1 or HSV-2. Preferably, the HSV virus is HSV-1. Most preferably, the HSV virus is a 17+ strain of HSV-1.

[0102] The inventors have discovered that oncolytic viruses (such as HSV) exert their therapeutic effects when in an active replicating state, thus exhibiting the greatest oncolytic activity against bladder tumor cells. Advantageously, actively replicating oncolytic viruses can directly destroy cancer cells and promote the release of therapeutic proteins (such as IL-15 and FliC_BP) as well as tumor antigens. This release is crucial for stimulating both innate and adaptive immune responses against tumor antigens and viral proteins, thereby achieving durable anti-tumor immunity.

[0103] Therefore, in some embodiments, the oncolytic virus is a live, replicating virus. In some embodiments, the oncolytic virus is a live, replicating HSV. In some embodiments, the oncolytic virus is a live, replicating HSV-1 or HSV-2.

[0104] HSV-1 neurotoxic protein, specifically Infecting Cell Protein 34.5 (ICP34.5), is essential for HSV-1 infection of neurons and other healthy cells because it binds to and blocks the activity of the PKR pathway, thereby promoting viral replication. ICP34.5 blocks PKR pathway activation by binding to and activating PP1α phosphatase (which dephosphorylates eIF-2α, thus preventing eIF-2's inhibition of protein translation). Therefore, for cancer cell infection, the absence of the ICP34.5 gene is preferred.

[0105] The HSV-1 genome is a single linear double-stranded DNA molecule approximately 152,000 bp in length. It is divided into two distinct regions, called the long region (UL) and the short region (US). Short repetitive sequence regions are located at both ends of the genome and between the L and S regions. ICP34.5 encodes both short repetitive sequence regions of the UL. Therefore, there are two copies of the gene in each genome. The first ICP34.5 gene encodes between 513 and 1539 bp of the genome, and the second ICP34.5 gene encodes between 124,832 and 125,858 bp of the genome.

[0106] Therefore, in a preferred embodiment, the oncolytic virus comprises a functionally lost ICP34.5 gene. The ICP34.5 gene can be functionally lost through absence, destruction, or non-function. More preferably, the oncolytic herpes simplex virus comprises a functional loss of at least two copies of the ICP34.5 gene.

[0107] Advantageously, this functional deficiency both eliminates the pathogenic mechanism of HSV-1 and enhances viral replication in cancer cells. Infection of healthy cells with ICP34.5-deficient HSV-1 activates PKR, leading to infection failure. In contrast, this HSV can replicate in cancer cells because PKR activity is not activated.

[0108] The functional loss of the ICP34.5 gene can be achieved by introducing one or more mutations into the viral DNA, rendering the expressed protein nonfunctional or with extremely low activity. Alternatively, the functional loss of the ICP34.5 gene can also be achieved by exposing the virus to an inhibitor of the encoded protein (such as an ICP34.5-specific RNA, like siRNA).

[0109] However, preferably, the functional loss of the ICP34.5 gene is achieved by integrating the gene construct of the first aspect or the expression cassette of the second aspect into the ICP34.5 gene.

[0110] Preferably, the oncolytic virus contains a functionally lost ICP47 gene. The ICP47 gene can be functionally lost through absence, destruction, or non-function.

[0111] This loss of ICP47 function produces multiple effects, thereby enhancing the lytic activity of the HSV-1 ICP34.5 / - mutant. US11 encodes UL11, which binds to PKR and prevents eIF-2α phosphorylation, thus allowing HSV-1 replication. Furthermore, US11 is normally expressed late in infection, at which point ICP34.5 inhibits PKR. However, this deletion in the HSV virus results in US11 being regulated by the α47 promoter, causing UL11 to be expressed as an early gene and blocking its activity before PKR can terminate protein synthesis. In many cancer cells, the type I IFN signaling pathway is disrupted. In the absence of ICP34.5, the virus replicates in cancer cells lacking normal IFN signaling, while normal cells have lower tolerance due to intact IFN signaling. This characteristic of the HSV-1 ICP34.5 / - ICP47- mutant adds an important safety component, as infection of normal cells would be terminated in the absence of PKR and type I IFN signaling defects.

[0112] Therefore, in a preferred embodiment, the oncolytic virus according to the third aspect may comprise, in this specific order, a 5' coding sequence encoding a cytokine (e.g., IL-15) and a 3' coding sequence encoding a TLR agonist (e.g., FliC_BP). Alternatively, the oncolytic virus according to the third aspect may comprise, in this specific order, a 5' coding sequence encoding a TLR agonist (e.g., FliC_BP) and a 3' coding sequence encoding a cytokine (e.g., IL-15). The use of 5' and 3' indicates that these features are upstream or downstream, and does not necessarily mean that these features are terminal features.

[0113] In a specific embodiment, the oncolytic virus according to the third aspect may comprise, in a specific order, a 5' coding sequence encoding a cytokine (e.g., IL-15), a first promoter (e.g., RSV), a second promoter (e.g., CMV), and a 3' coding sequence encoding a TLR agonist (e.g., FliC_BP). Alternatively, the oncolytic virus according to the third aspect may comprise, in a specific order, a 5' coding sequence encoding a TLR agonist (e.g., FliC_BP), a first promoter (e.g., CMV), a second promoter (e.g., RSV), and a 3' coding sequence encoding a cytokine (e.g., IL-15).

[0114] Alternatively, in another embodiment, the oncolytic virus according to the third aspect may comprise, in a specific order, a 5' first promoter (e.g., RSV), a coding sequence encoding a cytokine (e.g., IL-15), a second promoter (e.g., CMV), and a 3' coding sequence encoding a TLR agonist (e.g., FliC_BP). Alternatively, the oncolytic virus according to the third aspect may comprise, in a specific order, a 5' first promoter (e.g., CMV), a coding sequence encoding a TLR agonist (e.g., FliC_BP), a second promoter (e.g., RSV), and a 3' coding sequence encoding a cytokine (e.g., IL-15).

[0115] In a specific embodiment, the oncolytic virus according to the third aspect may comprise, in a specific order: a 5' poly-A tail, a coding sequence encoding a cytokine (e.g., IL-15), a first promoter (e.g., RSV), a second promoter (e.g., CMV), a coding sequence encoding a TLR agonist (e.g., FliC_BP), and a 3' poly-A tail. Alternatively, the oncolytic virus according to the third aspect may comprise, in a specific order: a 5' poly-A tail, a coding sequence encoding a TLR agonist (e.g., FliC_BP), a first promoter (e.g., CMV), a second promoter (e.g., RSV), a coding sequence encoding a cytokine (e.g., IL-15), and a 3' poly-A tail.

[0116] Alternatively, in another embodiment, the oncolytic virus according to the third aspect may comprise, in a specific order, a 5' first promoter (e.g., RSV), a coding sequence for a cytokine (e.g., IL-15), a poly-A tail, a second promoter (e.g., CMV), a coding sequence for a TLR agonist (e.g., FliC_BP), and a 3' poly-A tail. Alternatively, the oncolytic virus according to the third aspect may comprise, in a specific order, a 5' first promoter (e.g., CMV), a coding sequence for a TLR agonist (e.g., FliC_BP), a poly-A tail, a second promoter (e.g., RSV), a coding sequence for a cytokine (e.g., IL-15), and a 3' poly-A tail.

[0117] In a preferred embodiment, the oncolytic virus according to the fourth aspect may contain a 5' promoter (e.g., CMV) and a 3' coding sequence encoding a TLR agonist (e.g., FliC_BP) in this specific order.

[0118] In a particular implementation, the oncolytic virus according to the fourth aspect may comprise, in this particular order: a 5' promoter (e.g., CMV), a coding sequence encoding a TLR agonist (e.g., FliC_BP), and a 3' poly-A tail.

[0119] The inventors have demonstrated that, in the context of oncolytic viruses (such as HSV), combining TLR agonists with cytokines can enhance acquired immunity against tumors while simultaneously enhancing the oncolytic activity of the virus. Therefore, the inventors have surprisingly discovered that developing cancer therapies does not necessarily require antigens.

[0120] Therefore, in some embodiments, the gene constructs, expression cassettes, and oncolytic viruses according to the present invention do not contain antigens (e.g., tumor or cancer antigens).

[0121] Based on the above, those skilled in the art will understand the nucleotide and amino acid sequences of the oncolytic virus implementation schemes of the third and fourth aspects.

[0122] In some embodiments, a shuttle vector may be used to produce the oncolytic virus of the present invention. Therefore, for the avoidance of doubt, the encoding nucleotide sequence of the shuttle vector (referred to as p-34.5 DC RSV CMV) used to produce the oncolytic virus of the present invention is provided herein as SEQ ID No: 11, and is as follows:

[0123]

[0124] [SEQ ID No: 11]

[0125] Alternatively, in another embodiment, the encoding nucleotide sequence for the shuttle vector (referred to as p-34.5 DC CMV S-FLiC-M3 RSV) for producing the oncolytic virus according to the invention is provided herein as SEQ ID No: 16, and the sequence is as follows:

[0126]

[0127] [SEQ ID No: 16]

[0128] Alternatively, in another embodiment, the encoding nucleotide sequence for the shuttle vector (referred to as p-34.5 DC CMV RSV IL-15ED) for producing the oncolytic virus according to the invention is provided herein as SEQ ID No: 17, and the sequence is as follows:

[0129]

[0130] [SEQ ID No: 17]

[0131] Alternatively, in another embodiment, the encoding nucleotide sequence for the shuttle vector (referred to as p-34.5 DC RSV CMV S-FLiC-M3) for producing the oncolytic virus according to the invention is provided herein as SEQ ID No: 18, and the sequence is as follows:

[0132]

[0133] [SEQ ID No: 18]

[0134] Therefore, in a preferred embodiment, the shuttle vector for producing the oncolytic virus according to the invention is encoded by the nucleotide sequence shown in SEQ ID No: 11, 16, 17 or 18, or a fragment or variant thereof.

[0135] The oncolytic virus according to the third and fourth aspects of the invention is particularly suitable for treating cancer, preferably bladder cancer.

[0136] Therefore, according to a fifth aspect of the invention, a gene construct according to the first aspect, an expression cassette according to the second aspect, or an oncolytic virus according to the third or fourth aspect is provided for treatment.

[0137] According to the sixth aspect, gene constructs according to the first aspect, expression cassettes according to the second aspect, or oncolytic viruses according to the third or fourth aspect are provided for the treatment, prevention, or improvement of cancer.

[0138] According to the seventh aspect, a method for treating, preventing, or improving cancer in a subject is provided, the method comprising administering, or having already administered, a therapeutically effective amount of, the gene construct according to the first aspect, the expression cassette according to the second aspect, or the oncolytic virus according to the third or fourth aspect to a subject who requires such treatment.

[0139] The cancer mentioned could be bladder cancer.

[0140] In a preferred embodiment, the cancer is bladder cancer. More preferably, the cancer is non-muscle-invasive bladder cancer (NMIBC). Preferably, the bladder cancer is BCG-refractory bladder cancer.

[0141] Advantageously, the gene constructs, expression cassettes, and oncolytic viruses according to the invention facilitate the direct destruction of cancer cells and promote the release of therapeutic proteins (such as IL-15 and FliC_BP) and tumor antigens. This release is crucial for stimulating innate and adaptive immune responses against tumor antigens and viral proteins, thereby achieving durable antitumor immunity.

[0142] Therefore, in some embodiments, the gene constructs, expression cassettes, and oncolytic viruses according to the present invention can stimulate innate and adaptive immune responses.

[0143] In some embodiments, the gene constructs, expression cassettes, and oncolytic viruses of the present invention can stimulate TLR2 and TLR-5 mediated signaling pathways.

[0144] It should be understood that the oncolytic virus according to the present invention can be used as a drug, which can be used as a monotherapy (i.e., using the oncolytic virus alone) to treat cancer. Alternatively, the oncolytic virus according to the present invention can be used as an adjunct therapy or in combination with known therapies to treat, alleviate, or prevent cancer.

[0145] The oncolytic viruses of the present invention can be combined into a variety of different compositions, depending particularly on the manner of application. Thus, for example, the compositions can be powders, tablets, capsules, liquids, ointments, creams, gels, hydrogels, aerosols, sprays, micelle solutions, transdermal patches, liposome suspensions, polygons, emulsions, lipid nanoparticles (e.g., peptides, DNA, or RNA on the surface or encapsulated), or any other form suitable for administration to humans or animals requiring vaccination. Lipid nanoparticles may contain one or more components selected from: cationic lipids (preferably ionizable), phosphatidylcholine, cholesterol, and polyethylene glycol (PEG) lipids. It should be understood that the vehicle for the medicament according to the present invention should be a carrier to which the administered subject is well tolerated.

[0146] The drug comprising the oncolytic virus of the present invention can be used in a variety of ways. For example, it may require oral administration, in which case the agent may be contained in the composition and taken orally, for example, in the form of tablets, capsules, or liquids. Compositions comprising the agents and drugs of the present invention can be administered by inhalation (e.g., intranasal). The composition can also be formulated as a topical medication. For example, a cream or ointment may be applied to the skin.

[0147] The oncolytic virus of the present invention can also be introduced into sustained-release or delayed-release devices. For example, such devices can be implanted subcutaneously or epidermally, allowing for continuous release of the drug over weeks or even months. The device can be located at least near the treatment site. Such devices are particularly advantageous when long-term use of the oncolytic virus is required for treatment, and when frequent administration (e.g., at least daily injections) is typically necessary.

[0148] However, in a preferred embodiment, the drug according to the invention can be administered to the subject by injection into the bloodstream, muscle, skin, or directly into the site of treatment. The injection can be intravenous (bolus or infusion), subcutaneous (bolus or infusion), intradermal (bolus or infusion), or intramuscular (bolus or infusion). In some embodiments, the injection is intravenous. Typically, the injection is intravesical or intratumoral. For intravesical injection, it can be administered into the bladder via a catheter.

[0149] It should be understood that the required amount of oncolytic virus depends on its biological activity and bioavailability, which in turn depend on the route of administration, the physicochemical properties of the oncolytic virus, and whether it is used as monotherapy or in combination therapy. The frequency of administration is also affected by the half-life of the active ingredient in the treated subject. Those skilled in the art can determine the optimal dosage, which will vary depending on the type of oncolytic virus used, the strength of the composition, the route of administration, and the type and progression of the cancer. Furthermore, other factors, including the subject's age, weight, sex, diet, and timing of administration, may necessitate dosage adjustments depending on the specific circumstances of the treated subject.

[0150] Typically, depending on the agent used, the daily dose of the oncolytic virus of the present invention suitable for immunization is from 0.001 µg / kg body weight to 100 mg / kg body weight. More preferably, the daily dose of the agent is from 1 μg / kg body weight to 100 mg / kg body weight, more preferably from 10 μg / kg body weight to 10 mg / kg body weight, and most preferably from about 100 μg / kg body weight to 10 mg / kg body weight. In some embodiments, the dose of the oncolytic virus of the present invention suitable for immunization is from 10E4 plaque-forming units (PFU) / mL to 10E9 PFU / mL.

[0151] The daily dose can be administered as a single application (e.g., a single injection daily). Alternatively, the oncolytic virus may need to be administered two or more times a day. For example, the oncolytic virus can be administered as initial immunization and subsequent booster immunization, or as two booster immunizations administered at weekly or monthly intervals. Preferably, the oncolytic virus can be administered as initial immunization and subsequent booster immunization, with an interval of two to six weeks. Specific formulations of the oncolytic virus according to the invention and precise treatment regimens (e.g., daily doses and frequency of administration) can be prepared using known procedures, such as those commonly used in the pharmaceutical industry (e.g., in vivo experiments, clinical trials, etc.).

[0152] The inventors believe they are the first to propose an oncolytic virus containing genes encoding TLR agonists and genes encoding cytokines, or an oncolytic virus containing genes encoding FliC_BP.

[0153] According to an eighth aspect of the invention, a pharmaceutical composition is provided comprising a gene construct according to the first aspect, an expression cassette according to the second aspect or an oncolytic virus according to the third or fourth aspect, and a pharmaceutically acceptable medium.

[0154] The ninth aspect of the present invention also provides a method for preparing the pharmaceutical composition of the eighth aspect, the method comprising combining a therapeutically effective amount of a gene construct according to the first aspect, an expression cassette according to the second aspect, or an oncolytic virus according to the third or fourth aspect with a pharmaceutically acceptable mediator.

[0155] The “subject” can be a vertebrate, mammal, or domestic animal. Therefore, the compositions and medicines according to the invention can be used to treat any mammal, such as livestock (e.g., horses), pets, or for other veterinary purposes. However, most preferably, the subject is a human.

[0156] The "therapeutic effective dose" of an oncolytic virus refers to the amount described above required to alleviate, prevent, or treat any given disease (preferably as a preventative treatment) when administered to a subject.

[0157] For example, the oncolytic virus of the present invention can be used at a dose of about 0.001 µg to about 1 mg, preferably about 0.001 µg to about 500 µg. Preferably, the amount of oncolytic virus is about 0.01 µg to about 250 µg, and most preferably about 0.1 µg to about 100 µg. Preferably, the oncolytic virus according to the present invention is administered at a dose of 1-50 µg.

[0158] The oncolytic virus of the present invention may also contain a pharmaceutically acceptable medium. As used herein, a "pharmaceutically acceptable medium" is any known compound or combination of known compounds known to those skilled in the art for use in formulating pharmaceutical compositions.

[0159] In one embodiment, the pharmaceutically acceptable medium can be a solid, and the composition can be in powder or tablet form. Pharmaceutically acceptable solid mediums can include one or more substances that can also act as flavoring agents, lubricants, solubilizers, suspending agents, dyes, fillers, glidants, tableting aids, inert binders, sweeteners, preservatives, coating agents, or tablet disintegrants. The medium can also be an encapsulating material. In powders, the medium is a subdivided solid mixed with the subdivided active agent of the present invention. In tablets, the active agent (e.g., the oncolytic virus of the present invention) can be mixed with a medium having the necessary tableting properties in an appropriate proportion and compressed into the desired shape and size. Powders and tablets preferably contain up to 99% active agent. Suitable solid mediums include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, polyvinylpyrrolidone, low-melting-point waxes, and ion exchange resins. In another embodiment, the pharmaceutical medium can be a gel, and the composition can be a cream or other similar form. In another embodiment, the oncolytic virus or pharmaceutical composition can be lyophilized.

[0160] However, the drug carrier can be a liquid, and the pharmaceutical composition can be in solution form. Liquid carriers are used to prepare solutions, suspensions, emulsions, syrups, elixirs, and pressurized compositions. The oncolytic virus according to the invention can be dissolved or suspended in a pharmaceutically acceptable liquid carrier, such as water, organic solvents, mixtures of the two, or pharmaceutically acceptable oils or fats. The liquid carrier may contain other suitable pharmaceutical additives, such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavorings, suspending agents, thickeners, colorants, viscosity modifiers, stabilizers, or osmotic pressure modifiers. Suitable examples of liquid carriers for oral and parenteral administration include water (partially containing the above-mentioned additives, such as cellulose derivatives, preferably sodium carboxymethyl cellulose solution), alcohols (including monohydric and polyhydric alcohols, such as ethylene glycol) and their derivatives, and oils (such as fractionated coconut oil and peanut oil). For parenteral administration, the carrier can also be an oily ester, such as ethyl oleate and isopropyl myristate. Sterile liquid carriers can be used in sterile liquid compositions for parenteral administration. For pressurized compositions, the liquid medium may be a halocarbon or other pharmaceutically acceptable propellant.

[0161] Liquid pharmaceutical compositions (i.e., sterile solutions or suspensions) can be administered, for example, by subcutaneous, intradermal, intrathecal, epidural, intraperitoneal, intravenous, and especially intramuscular injection, or by urinary tract catheterization via a urethral catheter. The nucleic acid sequences or expression cassettes of the present invention can be prepared as sterile solid compositions and dissolved or suspended in sterile water, physiological saline, or other suitable sterile injection media upon administration.

[0162] The oncolytic virus of the present invention can be administered orally in the form of a sterile solution or suspension containing other solutes or suspending agents (e.g., sufficient physiological saline or glucose to make the solution isotonic), bile salts, gum arabic, gelatin, sorbitan monooleate, polysorbate 80 (an oleate copolymer of sorbitol and its anhydrides with ethylene oxide), etc. The oncolytic virus of the present invention can also be administered orally in the form of liquid or solid compositions. Compositions suitable for oral administration include solid forms, such as pills, capsules, granules, tablets, and powders; and liquid forms, such as solutions, syrups, elixirs, and suspensions. Forms suitable for parenteral administration include sterile solutions, emulsions, and suspensions. The pharmaceutical composition can be freeze-dried, lyophilized, or freeze-dried.

[0163] It should be understood that the present invention is applicable to any nucleic acid or peptide or its variants, derivatives or analogs that substantially comprise an amino acid or nucleic acid sequence of any sequence described herein, including its variants or fragments. The terms “substantially…amino acid / nucleotide / peptide sequence,” “variant,” and “fragment” can be sequences having at least 40% sequence identity with an amino acid / nucleotide / peptide sequence of any sequence described herein, for example, having 40% identity with the sequences shown in SEQ ID No: 1-20, and so on.

[0164] This document also covers amino acid / polynucleotide / peptide sequences having greater than 65%, more preferably greater than 70%, more preferably greater than 75%, and more preferably greater than 80% sequence identity with any of the stated sequences. Preferably, the amino acid / polynucleotide / peptide sequence has at least 85%, more preferably at least 90%, even more preferably at least 92%, even more preferably at least 95%, even more preferably at least 97%, even more preferably at least 98%, and most preferably at least 99% identity with any of the stated sequences.

[0165] Skilled technicians will know how to calculate the percentage of identity between two amino acid / polynucleotide / peptide sequences. To calculate the percentage of identity between two amino acid / polynucleotide / peptide sequences, the two sequences must first be aligned, and then the sequence identity value is calculated. The percentage of identity between two sequences may vary depending on the following factors: (i) the method used for sequence alignment, such as ClustalW, BLAST, FASTA, Smith-Waterman (implemented in different programs), or 3D comparison structural alignment; (ii) the parameters used in the alignment method, such as local versus global alignment, the pairing score matrix used (e.g., BLOSUM62, PAM250, Gonnet, etc.), and the gap penalty, such as functional form and constants.

[0166] After sequence alignment is completed, there are many ways to calculate the percentage of identity between two sequences. For example, the number of identical positions can be divided by: (i) the length of the shortest sequence; (ii) the alignment length; (iii) the average length of the sequences; (iv) the number of non-empty positions; or (v) the number of equivalent positions excluding dangling positions. Furthermore, it should be understood that the percentage of identity is also closely related to sequence length. Therefore, the shorter the sequence pair, the higher the probability of coincidental sequence identity.

[0167] Therefore, it should be understood that accurate alignment of protein or DNA sequences is a complex process. The commonly used multiplex alignment program ClustalW (Thompson et al., 1994, Nucleic Acids Research, 22, 4673-4680; Thompson et al., 1997, Nucleic Acids Research, 24, 4876-4882) is a preferred method for generating multiplex alignments of proteins or DNA according to the present invention. Suitable parameters for ClustalW can be as follows: For DNA alignment: vacancy opening penalty = 15.0, vacancy extension penalty = 6.66, matrix = Identity. For protein alignment: vacancy opening penalty = 10.0, vacancy extension penalty = 0.2, matrix = Gonnet. For both DNA and protein alignment: ENDGAP = -1, GAPDIST = 4. Those skilled in the art will understand that these and other parameters may need to be changed to obtain optimal sequence alignment.

[0168] Preferably, the percentage of identity between two amino acid / polynucleotide / peptide sequences can be calculated from the alignment results using the formula (N / T). 100, where N is the number of positions of identical residues in the sequence, and T is the total number of positions compared, including vacancies, and may or may not include dangling sites. Preferably, dangling sites are included in the calculation. Therefore, the most preferred method for calculating the percentage of identity between two sequences includes: (i) performing sequence alignment using the ClustalW program with a suitable set of parameters (e.g., the parameter set described above); and (ii) substituting the values ​​of N and T into the following formula: Sequence Identity = (N / T) 100.

[0169] Those skilled in the art will recognize that other methods exist for identifying similar sequences. For example, substantially similar nucleotide sequences can be encoded by sequences that hybridize with a DNA sequence or its complementary sequence under stringent conditions. Stringent conditions refer to the hybridization of nucleotides with DNA or RNA bound to a filter membrane in 3× sodium chloride / sodium citrate (SSC) at about 45°C, followed by washing at least once in 0.2×SSC / 0.1% SDS at about 20–65°C. Alternatively, substantially similar polypeptides may differ from amino acid sequences such as those shown in SEQ ID Nos. 1 to 20 by at least one amino acid, but less than 5, 10, 20, 50, or 100 amino acids.

[0170] Due to the degeneracy of the genetic code, it is clear that any nucleic acid sequence described herein can be altered or mutated without substantially affecting the protein sequence it encodes, thus obtaining its functional variants. Suitable nucleotide variants are changes resulting from the substitution of different codons encoding the same amino acid in the sequence, thus producing silent (synonymous) mutations. Other suitable variants are changes that have homologous nucleotide sequences but contain all or part of the sequence, resulting in conserved alterations by substituting different codons encoding amino acids with side chains having similar biophysical properties to the substituted amino acid. For example, small nonpolar hydrophobic amino acids include glycine, alanine, leucine, isoleucine, valine, proline, and methionine. Large nonpolar hydrophobic amino acids include phenylalanine, tryptophan, and tyrosine. Polar neutral amino acids include serine, threonine, cysteine, asparagine, and glutamine. Positively charged (basic) amino acids include lysine, arginine, and histidine. Negatively charged (acidic) amino acids include aspartic acid and glutamic acid. Therefore, it is important to understand which amino acids can be replaced by amino acids with similar biophysical properties, and skilled technicians should be familiar with the nucleotide sequences that encode these amino acids.

[0171] All features described herein (including any appended claims, abstracts and drawings) and / or all steps of any disclosed method or process may be combined with any of the foregoing aspects in any combination, unless at least some of such features and / or steps in the combination are mutually exclusive.

[0172] To better understand the present invention and to demonstrate how to implement its embodiments, the following description will be provided with reference to the accompanying drawings, in which:

[0173] Figure 1 A schematic diagram of one embodiment of the oncolytic virus of the present invention is shown. The oncolytic herpes simplex virus (oHSV-FliC_BP-IL-15) is derived from a wild-type (WT) HSV-1 17+ strain and has two gene deletions: the deletion of genes encoding (i) ICP34.5 and (ii) ICP47. oHSV-FliC_BP-IL-15 expresses Burkholderia melioidus flagellin (GenBank ABA48561.1, TLR5 ligand) under the CMV promoter and IL-15 (GenBank: AF031167.1) under the RSV promoter.

[0174] Figure 2The expression of TLR ligands and IL-15 in plasmids and oHSV virus is shown. TLR ligand plasmids or oHSV virus were screened using a TLR receptor / NF-κB reporter assay: a) TLR4 and 5 (Promega); b) TLR2 ligand plasmid (Invivogen); c) oHSV-FliC_BP (Promega). d) The concentration of IL-15 in the supernatant of 5637 cells infected with oHSV-IL-15 at MOI 1 for 48 hours was detected using the IL-15 cell bioassay (Promega). e) The TLR5-MyD88-NF-κB pathway activated in the supernatant of 5637 cells infected with oHSV-FliC_BP at MOI 1 for 24 hours was partially blocked using the human TLR5 soluble extracellular domain as a TLR5 inhibitor targeting the TLR5 receptor (α Hu TLR5-Fc, InvivoGen). f) Immunostaining of BP flagellated bacterial proteins (green) was performed on 5637 cells 24 hours after infection with oHSV-FliC_BP virus at MOI 1. g) THP-1 cells (+PMA) were treated with UV-inactivated oHSV or oHSV FLIC BP. THP-1 cells were incubated for 24 hours, and qPCR of innate cytokines was performed.

[0175] Figure 3 Displays: a) Mean tumor volume of subcutaneous MB49 tumors treated 4 times (n=10) with 108 pfu of oHSV, oHSV-FliC_BP, oHSV-IL-15, or oHSV-FliC_BP-IL-15 over 8 days. b) Kaplan-Meier survival curves. c) Reinfection experiments using the same bladder cancer cell line (MB49) on uninfected and cured mice. e) FACS analysis of TNF-α in spleen cells (alone or co-cultured with MB49 tumor cells or PMA / ionomycin) from cured and uninfected mice. e) CD4; f) CD8; g) NK.

[0176] Figure 4This study demonstrates the knockout of ICP47 in the HSV-1 genome. A plasmid (pUC57 delICP47) containing two HSV-1 flanking sequences on either side of ICP47 was designed and synthesized. A PCR-generated CMV EGFP pA cassette was constructed using pcDNA3.1+EGFP. This PCR product was digested with BBs-1 and then inserted between the flanking regions of ICP47 to generate pUC57 del ICP47 CMV EGFP pA. The viral shuttle vector was linearized (ssp-1) and added to the DNA of HSV-1-infected BHK cells, followed by transfection with calcium phosphate into BHK cells. The resulting homologous recombinant was purified by plaque purification to obtain the HSV-1 17+ 47- EGFP clone. The CMV EGFP pA cassette was removed from the virus (HSV-1 17+ 47- EGFP) via homologous recombination using pUC57 del ICP47, resulting in the HSV-1 17+ 47- clone.

[0177] Figure 5 The plasmid map of p34.5 DC EGFP is shown, which contains two HSV-1 flanking sequences on either side of ICP34.5. p34.5 DC EGFP contains dual expression cassettes (RSV-pA, CMV pA). Under the CMV promoter is the synthetic green fluorescent gene (EGFP).

[0178] Figure 6 The plasmid map of p34.5 CMV RSV IL-15ED is shown, which contains two ICP34.5 flanking regions and the sequence of IL-15 ED. p34.5 CMV RSV IL-15ED contains dual expression cassettes (RSV-pA, CMV pA).

[0179] Figure 7 The plasmid map of p34.5 CMV FLIC BP RSV IL-15ED is shown, which contains two ICP34.5 flanking sequences, namely IL-15 ED and FLIC BP. p34.5 CMV FLIC BP RSV IL-15ED contains dual expression cassettes (RSV-pA, CMV pA).

[0180] Figure 8 The inactivation of HSV after exposure to 300 J UV light is shown.

[0181] Figure 9 The survival rates of four TCC bladder cancer cell lines (T24, TCCSUP, Ku19-19, and VMCUB) treated with or without UV-inactivated HSV / HSV5-15 are shown.

[0182] Figure 10 Showing the use of HEK-Blue TM TLR2 stimulation identified by hTLR-2 assay includes: a) plasmid DNA containing FLiCBP ligand; and b) FLiCBP ligand expressed from oncolytic HSV (HSV5).

[0183] Example

[0184] The inventors designed and tested a novel oncolytic virus for the treatment of non-muscle-invasive bladder cancer (NMIBC). Given the problems associated with BCG (i.e., non-responders and / or toxicity), the inventors sought a single TLR agonist to provide an alternative treatment for BCG by non-toxically stimulating NK cells and NKT cells (which have been shown to play a crucial role in BCG-induced antitumor responses in NMIBC treatment). The inventors conducted extensive screening of bacterial TLR ligands to find a suitable ligand expressed in oncolytic herpes simplex virus (oHSV). The screening resulted in the use of the TLR5 ligand, FliC BP, a flagellin derived from Burkholderia melioides. In addition to the TLR ligand, the inventors also explored the role of oHSV encoding the cytokine IL-15. The genes encoding FliC BP and IL-15 are present in the HSV strain, in which two genes (ICP34.5 and ICP47) are missing.

[0185] Materials and Methods

[0186] Production of oncolytic HSV-1 virus

[0187] All oncolytic viruses constructed and used were based on human herpes simplex virus type 1 17+ strain (Genbank X14112) obtained from ECACC.

[0188] HSV-1 ICP47 gene knockout

[0189] To knock out ICP47 from the HSV-1 genome, a plasmid (pUC57 del ICP47) was designed and synthesized, containing two HSV-1 flanking sequences on either side of ICP47. 47 143671-145261bp, 145581-146461bp Genbank X14112 (see) Figure 4Using primers CGFPA F (cloning primer 5) GAGGAAGACGAGGAGACGGGCCAGATATACGCGTTG [SEQ ID No: 19] and AGCTCCTCGTCTTCCCAATCCTCCCCCTTG [SEQ ID No: 20], a PCR-generated CMV EGFP pA cassette was constructed from pcDNA3.1+ EGFP. The PCR product was cleaved with BBs-1 and then inserted between the flanking regions of ICP47 to generate pUC57 del ICP47 CMV EGFP pA. This viral shuttle vector was linearized (ssp-1) and added to the DNA of HSV-1-infected BHK cells, which were then transfected into BHK cells with calcium phosphate. The resulting homologous recombinant was purified by plaque analysis to obtain the HSV-1 17+ 47- EGFP clone. Homologous recombination was performed using pUC57 del ICP47 to remove the CMV EGFP pA box from the virus (HSV-1 17+ 47- EGFP) to obtain the HSV-1 17+ 47- clone.

[0190] HSV-1 ICP34.5 gene knockout

[0191] To knock out the ICP34.5 gene from HSV-1 17+ 47-, a plasmid (p34.5- DC) was constructed, which contains two HSV-1 flanking sequences on either side of ICP34.5. 34.5 123462-124958bp, 125713-126790bp Genbank X14112 (see) Figure 5 The p34.5-DC contains a dual expression cassette (RSV-pA, CMV pA). Under the CMV promoter, the inventors cloned (BamHI, MfeI) a synthetic green fluorescent protein (EGFP) gene (Genbank AF188479.1) from pcDNA3.1+ EGFP, thereby generating p34.5 DC EGFP. The viral shuttle vector was linearized (ssp-1) and added to the DNA of HSV-1-infected BHK cells, followed by transfection with calcium phosphate into BHK cells. The resulting homologous recombinant was purified by plaque analysis to obtain the HSV-1 17+ 34.5-EGFP 47- clone.

[0192] The recombinant flagella (Burkholderia melioides Bp) sequence was inserted into HSV-1 17+ 34.5-47-

[0193] To express flagella from *Burkholderia melioides* (Genbank ABA48561.1, a TLR5 ligand) in oncolytic HSV-1 17+ 34.5-47-, a plasmid (pcDNA3.1+S-FLIC BP-M3) was designed and synthesized. Like all bacterial proteins, flagella produced by *Burkholderia melioides* are typically not glycosylated. Glycosylation of the Bp sequence inhibits TLR5 ligand function. To attempt to detect N-glycosylation sites in the Bp flagella, the sequence was analyzed using NetN Glyc server software. The software showed no signal transduction peptides binding to the N-glycosylation mechanism, but three N-glycosylation sites were identified (AA No. 18, 270, and 358). To avoid N-glycosylation sites, asparagine (N) at the glycosylation sites was replaced with glutamine (Q). To facilitate extracellular secretion of flagellar proteins, a CD33 secretion signal was added. After synthesizing the recombinant sequence, it was cloned into pcDNA3.1+ (BamH, EcoRI) to obtain pcDNA3.1+S-FLIC BP-M3. To generate the viral shuttle vector, S-FLIC BP-M3 was subcloned from pcDNA3.1+S-FLIC BP-M3 (BamHI, MfeI) into p34.5-DC under the CMV promoter to obtain p34.5-DC S-FLIC-BP-M3. To obtain the recombinant virus, p34.5-DC S-FLIC-BP-M3 was linearized (ssp-1), added to the DNA of HSV-1-infected BHK cells, and transfected into BHK cells with calcium phosphate. The resulting homologous recombinant was purified by plaque analysis to obtain the HSV-1 17+ 34.5- S-FLICBP-M3 47- clone.

[0194] The recombinant IL-15 sequence was inserted into HSV-1 17+ 34.5-47-

[0195] To express human interleukin-15 in oncolytic HSV-1 17+ 34.5-47- (GenBank: AF031167.1), a plasmid (pcDNA3.1+IL-15ED) was designed and synthesized (see [link to plasmid description]). Figure 6The recombinant sequence was synthesized and cloned into pcDNA3.1+ (BamH, EcoRI). To construct a viral shuttle vector containing IL-15ED, the cytokine sequence was subcloned from pcDNA3.1+ IL-15ED (PmeI, Xcm-I) into the p34.5- DC plasmid under the RSV promoter. To obtain the recombinant virus, p34.5- DC IL-15ED was linearized (ssp-1) and added to the DNA of HSV-1-infected BHK cells, which were then transfected into BHK cells with calcium phosphate. The resulting homologous recombinant was purified by plaque analysis to obtain the HSV-1 17+ 34.5- IL-15ED 47- clone.

[0196] The recombinant IL-15 and flagella (Burkholderia melioides Bp) sequences were inserted into HSV-1 17+ 34.5-47-

[0197] To express human interleukin-15 and flagellar (Bp) sequences from oncolytic HSV-1 17+ 34.5-47-, the viral shuttle plasmid containing IL-15ED was cleaved with BamHI and MfeI, and the flagellar was inserted under the CMV promoter (see [link to CMV promoter]). Figure 7 To obtain the recombinant virus, p34.5-DC IL-15ED / S-FLIC-BP-M3 was linearized (ssp-1) and added to the DNA of HSV-1-infected BHK cells, followed by transfection into BHK cells with calcium phosphate. The resulting homologous recombinant was purified by plaque analysis to obtain the HSV-1 17+34.5-IL-15ED / S-FLIC-BP-M3 47- clone.

[0198] Example 1 – Screening candidate TLR ligands

[0199] The inventors first screened several TLR ligands (i.e., TLR2, 4, or 5) to determine which ligand was best suited for cancer therapy using oHSV. Each TLR ligand was inserted into the mammalian expression vector pcDNA3.1. Previous studies have shown that N-glycosylation sites inhibit ligand-receptor interaction. Therefore, each gene was mutated to remove the N-glycosylation site, thereby converting the asparagine amino acid to glutamine. Furthermore, a sequence containing the CD33 secretion signal was added to enable the release of the TLR ligand protein from mammalian cells.

[0200] To screen recombinant TLR ligands, the inventors exposed them to NF-KB reporter systems expressing their target TLR receptors. These systems tested the ability of TLR ligands to activate the NF-KB promoter via a marker gene. TLR2 ligands were tested on the human TLR2 / NF-κB-AP-1 / SEAP reporter in HEK293 cells (Invivogen). Figure 2b). TLR4 and TLR5 ligands were selected in 293 cells transiently transfected with the pNL3.2 Luc NF-KB RE / pGL4.54 Luc2 / TK plasmid (Promega). Figure 2 a). Through this TLR ligand screening, the inventors determined that FliC_BP was the best candidate ligand ( Figure 2 a) This result is consistent with the ability of FliC_BP to interact with TLR5 on HEK cells and trigger the activation of the transcription factor NF-κB. This TLR5 ligand offers the potential to enhance antitumor immune responses using oncolytic viruses with innate bacterial responses.

[0201] Example 2 – Design and generation of expression cassettes containing TLR5 ligands and / or IL-15 and oHSV

[0202] The inventors then constructed an oncolytic virus backbone based on human HSV-1 (17+ strain) that incorporated the previously described tumor selectivity and lack of immune control. For example... Figure 1 As shown in the upper part, a schematic diagram of HSV-1 wild-type (WT) illustrates the corresponding positions of the functionally lost genes ICP34.5 and ICP47, as described below. The inventors then inserted the candidate TLR ligand FliC_BP into the oncolytic HSV viral backbone (oHSV or oHSV GFP), constructing a series of vectors expressing FliC_BP (oHSV-FliC_BP) or IL-15 (oHSV_IL-15) or both (oHSV-FliC_BP-IL-15).

[0203] ICP34.5

[0204] The HSV-1 neurotoxic protein, specifically Infecting Cell Protein 34.5 (ICP34.5), is essential for HSV-1 infection of neurons and other healthy cells because it binds to and blocks PKR, thereby allowing viral replication. ICP34.5 blocks PKR pathway activation by binding to and activating PP1α phosphatase, which in turn dephosphorylates eIF-2α, thus preventing eIF-2's inhibition of protein translation. Therefore, for infection of cancer cells, such as the treatment of bladder cancer in this study, the inventors believe that deletion, disruption, or nonfunction of the ICP34.5 gene is beneficial.

[0205] ICP47

[0206] ICP47 is involved in the lytic activity of HSV-1, therefore, defunctionalizing it can enhance its lytic activity. In many cancer cells, the type I IFN signaling pathway is disrupted. In the absence of ICP34.5, the virus replicates in cancer cells lacking normal IFN signaling, while normal cells, due to intact IFN signaling, have lower tolerance for the virus. Therefore, the inventors believe that this characteristic of the HSV-1 ICP47 / - mutant provides an important safety mechanism, as infection of normal cells will be halted in the absence of PKR and type I IFN signaling defects. Therefore, the inventors believe that deletion, disruption, or defunctionalization of the ICP47 gene is also advantageous for cancer cell infection, such as in the treatment of bladder cancer.

[0207] Now for reference Figure 1 The lower half of the diagram shows a schematic of an expression cassette integrated into the disrupted ICP34.5 gene site. This cassette contains a nucleotide sequence encoding a TLR5 ligand, derived from the flagella of *Burkholderia melioides* (FliC_BP), whose expression is controlled by the CMV promoter. The expression direction is 5' to 3' (as shown in the diagram, from left to right). The inventors have found that FliC_BP is a particularly potent candidate ligand capable of enhancing the antitumor immune response of oncolytic viruses with innate bacterial responses.

[0208] In addition, the expression cassette contains a nucleotide sequence encoding the cytokine IL-15, whose expression is controlled by the RSV promoter. Expression is directed from 3' to 5' (as shown in the figure, from right to left). IL-15 is a pleiotropic cytokine that plays a crucial role in inflammatory and protective immune responses against microbial invaders and parasites by modulating immune cells in both the innate and adaptive immune systems. IL-15 expression promotes NK cell proliferation and the cytotoxic function of CD8 T cells. FliC_BP is a bacterial protein that stimulates a strong innate response; IL-15 and FliC_BP work synergistically.

[0209] The expression cassette has two polyA tails on its flanks. The expression cassettes encoding IL-15 and FliC_BP were inserted into oHSVs with functional deletions of the ICP34.5 and ICP47 genes, thereby constructing the vector HSV-1 17+ 34.5- IL-15ED / S-FLIC-BP-M347-.

[0210] As described in the following examples, the efficacy of oHSV-1 17+ 34.5- IL-15ED / S-FLIC-BP-M3 47- in treating cancers (such as bladder cancer) was subsequently tested.

[0211] Example 3 – Characterization of oHSV expressing FliC_BP and / or IL-15 in vitro.

[0212] To verify the viral expression of FliC_BP, bladder cancer cell line 5637 was infected with oHSV-FliC_BP. Twenty-four hours later, immunostaining was performed using mouse polyclonal serum targeting BP bacterial flagellin production. The staining results showed that FliC_BP was expressed in the cytoplasm of infected cells and strongly expressed on the cell membrane. Figure 2 f). No staining signal was detected in 5637 bladder cancer cells infected with oHSV (data not shown) or with secondary antibody alone. Figure 2 f).

[0213] To detect whether virally expressed FliC_BP could activate the MyD88-NF-κB pathway, the inventors employed a TLR receptor NF-κB reporter assay. Supernatant from infected bladder cancer cells was added to 293 cells expressing the TLR5 receptor, which had been transiently transfected with a plasmid containing the NF-κB promoter located upstream of the luciferase gene. Compared to oHSV, the luciferase expression level of oHSV-FliC_BP virus increased 10-18 times (…). Figure 2 c). This indicates that the secreted FliC_BP produced only 4 hours after infecting bladder cancer cells with oHSV-FliC_BP can activate NF-KB in the reporter system. Figure 2 c).

[0214] The inventors utilized a soluble human TLR5 extracellular domain, namely a TLR5 inhibitor (α Hu TLR5-Fc, InvivoGen), to partially block the activation of the MyD88-NF-KB pathway, demonstrating that the activity of virally produced FliC_BP is at least partially achieved through TLR5 binding. This indicates that virally expressed proteins can interact in a manner similar to their bacterial counterparts, thus potentially stimulating an innate immune response. Figure 2 e).

[0215] To investigate the effect of virally expressed FliC_BP alone on a macrophage model, THP-1 cells (+PMA) were treated with UV-inactivated oHSV or oHSV FLIC BP. After culturing the virus-treated THP-1 cells for 24 hours, qPCR detection of innate cytokine transcripts was performed. The results showed that, compared with oHSV, oHSV FLIC BP enhanced the innate immune response. Figure 2 g).

[0216] To enhance the activation effect of the novel FLiC_BP virus on NK cells, the inventors aimed to introduce the cytokine IL-15. The inventors first constructed a virus expressing IL-15 (oHSV IL-15). IL-15 expression in oHSV IL-15 was detected using the IL-15 cell bioassay (Promega). Bladder cancer cell line 5637 was infected with oHSV-IL-15 virus at an MOI of 0.1 for 48 hours. The supernatant from these infected cells was added to IL-15 bioassay cells; these cells were engineered to express luciferase in response to IL-15 signaling. The assay results showed that the virus could produce biologically active IL-15 at a concentration of approximately 30 ng / ml. Figure 2 d). IL-15 was added to a virus containing FliC_BP to create oHSV-FliC_BP-IL-15, and its expression was demonstrated in an IL-15 cell bioassay.

[0217] Example 4 – In vivo efficacy of oHSV-FliC_BP-IL-15 in bladder cancer

[0218] Data show that the oncolytic virus of this invention can effectively cure up to 50% of mice in an in vivo subcutaneous model of bladder cancer. ( Figure 3 a, 3b). After 60 days of remission, these mice were again given the same bladder cancer cell line MB49, and remained tumor-free. Figure 2 c). When infected with oHSV-FliC_BP-IL-15, spleen cells (including CD4, CD8, and NK cells) from cured mice showed activation of tumor-killing cytokines (such as TNF-α) in the presence of MB49 tumor cells. Figure 3 (e, 3f, 3g). However, spleen cells in uninfected mice lacked this activation.

[0219] Example 5 – Live, replicating HSV enhances the lysis of bladder tumor cells

[0220] method

[0221] 4.2 x 10 6 293 cells were seeded at 25 cm 3Cells were incubated overnight at 37°C in culture flasks. Then, cells were infected for 1 hour in 2 ml of 2% FCS medium in each flask with HSV or HSV5-15 at an MOI of 1. HSV5 or HSV5-15 refers to the inventor's self-designation and the insertion of FliC_BP into HSV-1 or HSV-2. After incubation, the virus was removed, and 5 ml of 10% FCS medium was added to each flask. Cells were incubated for another 24 hours. The supernatant was collected, centrifuged at 1500 rpm for 5 minutes at room temperature, and transferred to 10 cm diameter culture dishes. The cell-free supernatant was exposed to 300 J UV light (in three separate exposures) and then stored at -80°C.

[0222] Four different transitional cell carcinoma (TCC) bladder cancer cell lines were used, as shown in the table below. KU19-19, VMCUB, and TCCSUP-G cells were used at a rate of 1 × 10⁶ cells per well. 4 T24 cells were seeded at a density of 8 × 10⁶ cells per well in 96-well plates (100 μL). 3 Cells were seeded at a density of 1,000 cells per well in 96-well plates. All cells were incubated overnight at 37°C.

[0223]

[0224] Cells were then treated in fresh 2% FCS medium with UV-inactivated or non-UV-inactivated HSV / HSV5-15 (MOI 0.5, 50 μL) for 2 hours. 100 μL of fresh 10% FCS medium was added, and incubation continued for 72 hours. The medium was removed, and 100 μL of MTS reagent (CellTiter 96® AQueous Assay, Promega) was added, followed by incubation at 37°C for 1 hour. Absorbance was measured at 490 nm using an ELISA reader.

[0225] result

[0226] UV irradiation successfully inactivated HSV, although a small amount of activity remained (see...). Figure 8 ).

[0227] Four TCC bladder cancer cell lines (T24, TCCSUP, Ku19-19, and VMCUB) were shown to be sensitive to oncolytic activity of HSV5-15 and the HSV viral backbone. UV inactivation of HSV5-15 and the HSV viral backbone strongly inhibited oncolytic activity of the virus and all tested TCC bladder cancer cell lines (see [link to relevant documentation]). Figure 9 ).

[0228] These results indicate that HSV and HSV5-15 have the greatest oncolytic effect on bladder tumor cells when they are in a live replicating state. They not only directly destroy cancer cells but also promote the release of therapeutic proteins such as IL-15 and FliC_BP, as well as tumor antigens. This release is crucial for stimulating innate and adaptive immune responses against tumor antigens and viral proteins, thereby achieving durable anti-tumor immunity.

[0229] The death of RU19-19 cells under the highest concentrations of UV-inactivated HSV or HSV5-15 is due to the residual activity of the virus after incomplete UV inactivation. Figure 8 This is due to the sensitivity of RU19-19 cells to HSV-induced lysis.

[0230] Example 6 – Expression of FliC_BP from plasmids or oncolytic HSV leads to TLR2 activation

[0231] The inventors aimed to investigate the effect of FliC_BP on the activation of Toll-like receptor 2 (TLR2) when expressed from plasmids and oncolytic HSV.

[0232] method

[0233] TLR2 NF-KB reaction SEAP assay

[0234]

[0235] FliC_BP protein expression from mammalian plasmids

[0236] 293 cells were spaced at 1.5 x 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1,000 cells per well in 96-well plates and incubated overnight until cell confluence reached 50-70%. ViaFect was then used to... TM Transfection reagent, DNA (7:1 ratio), and dilution buffer (serum-free medium) were warmed to room temperature and gently vortexed. Plasmid DNA (containing FliC_BP plasmid and pcDNA EGFP) was added to the diluted Opti-MEM® solution according to the above ratio. The mixture was gently pipetted to ensure thorough mixing. ViaFect was then used. TM Transfer the transfection reagent directly into the culture medium containing diluted DNA, avoiding contact with the plastic tube walls. Incubate the mixture at room temperature for 15–30 minutes. Next, add 5 µl of the transfection complex to the cells and gently vortex the culture plate on a rotary shaker (600 rpm) for 30 seconds. Repeat this process once for each sample. Then incubate the cells for 48 hours. After incubation, remove the cell supernatant and centrifuge at 2000g for 3 minutes.

[0237] FliC_BP protein expression from oncolytic HSV (HSV5)

[0238] 293 cells were spaced at 1.5 x 10⁻⁶ cells per well. 4 Cells were seeded at a density of 1000 g / well in 96-well plates and incubated overnight until cell confluence reached 50-70%. Then, cells were infected for 1 hour in 2% FCS medium with HSV5 or HSV-GFP at an MOI of 1. HSV5 refers to the inventor's self-designation and the insertion of FliC_BP into HSV-1 or HSV-2. After incubation, the virus was removed, and 10% FCS medium was added to the wells. Cells were then incubated for another 24 hours. After incubation, the cell supernatant was removed, and the cells were centrifuged at 2000g for 3 minutes.

[0239] Using HEK-Blue TM Detection and measurement of TLR2 stimulation

[0240] HEK-Blue TM The assay uses a cell culture medium specifically designed for detecting secretory embryonic alkaline phosphatase (SEAP), a reporter protein produced and secreted by cells. In the experimental setup, 20 µl of each sample was aliquoted into the wells of a 96-well plate, with six wells per sample. Additionally, 20 µl of 10 ng / ml Pam3CSK4 was added to designated control wells as a positive control, and 20 µl of sterile growth medium served as a negative control. HEK-Blue was prepared for the assay. TM hTLR2 cells were removed from the original culture medium and the cells in the T-75 culture flask were gently rinsed with 5-10 ml of pre-warmed PBS. Then, 2-5 ml of pre-warmed PBS was added to the culture flask, and the cells were incubated at 37°C for 1-2 minutes to promote detachment. The cells were then detached by gently tapping the culture flask or using a cell scraper, and gently aspirated to ensure complete dispersal of the cell clumps. Importantly, to maintain cell integrity, trypsin was not used on HEK-Blue cells. TM hTLR2 cells were detached. After detachment, the cells were stored at a rate of 2.8 × 10⁻⁶ cells / year. 5 Resuspended in HEK-Blue at a concentration of cells / ml. TM In the assay medium, immediately add approximately 180 µl of cells (equivalent to approximately 50,000 cells) to each sample well. Take care to avoid cell contamination in HEK-Blue. TMProlonged incubation at room temperature in the assay medium is avoided as this may lead to increased background or false positive results. Assembled culture plates are incubated overnight at 37°C with 5% CO2 to promote SEAP production and secretion. Following incubation, SEAP levels are measured at 600 nm using a microplate reader to quantitatively measure SEAP activity, which indicates the immune response elicited by the test sample. This assay setup assesses immune activation mediated by the TLR2 signaling pathway, providing valuable insights into cellular responses to various stimuli or treatments.

[0241] result

[0242] FliC_BP expressed by plasmids or oncolytic HSV in HEK-Blue TM TLR2 was successfully activated in the hTLR2 assay. This result shows that FliC_BP can trigger the TLR2-mediated signaling pathway, indicating that FliC_BP binds directly to the TLR2 receptor or that there is an interaction between TLR5 (the main receptor for flagellin) and TLR2.

[0243] Toll-like receptors (TLRs) are an important component of the innate immune system, responsible for recognizing and responding to microbial pathogens. Different TLR ligands can induce unique cytokine profiles through pathway-specific signaling, reflecting the complexity of TLR pathway interactions. The results showed that the flagellin FliC_BP from *Burkholderia melioides* can trigger TLR2-mediated signaling pathways, indicating that FliC_BP binds directly to the TLR2 receptor or that there is an interaction between TLR5 (the major flagellin receptor) and TLR2. This previously unreported activity of flagellins from *Burkholderia melioides*, and indeed any bacterial flagellin, could explain the observed enhanced ability of FliC_BP to stimulate the innate immune response. Simultaneous activation of the TLR2 and TLR5 pathways may amplify the production of pro-inflammatory cytokines, thereby promoting a stronger and more rapid immune response. The ability of FliC_BP to activate multiple TLR pathways could be a key factor in its potential application as a therapeutic agent, especially in oncolytic virus therapies requiring potent immune activation.

[0244] in conclusion

[0245] The inventors have developed a novel oncolytic virus that is effective against BCG-unresponsive models (and ultimately, patients). Intravesical delivery of this novel oncolytic virus promises to achieve highly selective tumor infection and cancer cell cytotoxicity. Advantageously, the virus retains potent tumor cytotoxicity and enhances immunogenicity through the incorporation of a novel bacterial protein and IL-15 (which promotes NK cell proliferation and CD8 T cell cytotoxicity). Figure 2and Figure 3 Therefore, this oncolytic virus can selectively kill cancer cells in the bladder while stimulating a powerful anti-cancer immune response, thereby preventing cancer recurrence.

Claims

1. A gene construct containing a first coding sequence encoding a TLR agonist and a second coding sequence encoding a cytokine.

2. The gene construct according to claim 1, wherein the TLR agonist is a TLR2 agonist, a TLR4 agonist, or a TLR5 agonist.

3. The gene construct according to claim 2, wherein: (i) The TLR2 agonist is selected from the following: LpqH, LprA, LprG, LpqT, PhoS1, MPB83, MPT83, LAM, AraLAM, LM, PIM2 / 6, TDM, HSP70, MymA, PE_PGRS33, EsxL, PPE18, PPE26, PPE32, PPE57, Lrp, GPLs, PILAM, PPE60, OmpA and OmpC; (ii) The TLR4 agonist is FimH; or (iii) The TLR5 agonist is selected from the following: FliC, CagL, CagY, FllA and FlaB.

4. The gene construct according to any one of the preceding claims, wherein the TLR agonist is Burkholderia melioides (…). Burkholderia pseudomallei )FliC_BP.

5. The gene construct according to claim 4, wherein FliC_BP comprises, or consists of, an amino acid sequence substantially as shown in SEQ ID No: 1 or 3, a fragment or variant thereof, and / or wherein FliC_BP is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 2 or 4, a fragment or variant thereof.

6. The gene construct according to any one of the preceding claims, wherein the first coding sequence encodes the CD33 secretion signal.

7. The gene construct of claim 6, wherein the CD33 secretion signal comprises, or is composed of, an amino acid sequence substantially as shown in SEQ ID No: 5, a fragment or variant thereof, and / or wherein the CD33 secretion signal is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 6, a fragment or variant thereof.

8. The gene construct according to any one of the preceding claims, wherein the cytokine is a chemokine, optionally wherein the chemokine is selected from the following: CXCL10, CXCL9, CXCL11, CXCL8, CXCL12, CCL2, CCL3 and CCL5.

9. The gene construct according to any one of the preceding claims, wherein the cytokines are selected from the following: IL-2, IL-6, IL-10, IL-12, non-secretory IL-12, IL-15, IL-17, IL-21, IL-33, and type I and type II interferons.

10. The gene construct according to any one of the preceding claims, wherein the cytokine is IL-15.

11. The gene construct of claim 10, wherein IL-15 comprises, or is composed of, an amino acid sequence substantially as shown in SEQ ID No: 9, or a fragment or variant thereof, and / or wherein IL-15 is encoded by a nucleotide sequence substantially as shown in SEQ ID No: 10, or a fragment or variant thereof.

12. The gene construct according to any one of the preceding claims, wherein the gene construct further comprises a first promoter operatively linked to a first coding sequence encoding a TLR agonist, and / or a second promoter operatively linked to a second coding sequence encoding a cytokine.

13. The gene construct according to claim 12, wherein the promoter or each promoter is selected from the following: CMV, RSV, T7 polymerase, EF1a, SV40, PGK1, Ac5, UBI, MP-84 and MP-135.

14. The gene construct according to claim 12 or 13, wherein, The first promoter operatively linked to the first coding sequence encoding a TLR agonist is the cytomegalovirus (CMV) promoter.

15. The gene construct of claim 14, wherein the first promoter comprises, or is composed of, a nucleic acid sequence substantially as shown in SEQ ID No: 12, a fragment or variant thereof, or is composed of, a nucleic acid sequence substantially as shown in SEQ ID No: 12, a fragment or variant thereof.

16. The gene construct according to any one of claims 12 to 15, wherein, The second promoter that is operatively linked to the second coding sequence encoding a cytokine is the RSV promoter.

17. The gene construct of claim 16, wherein the second promoter comprises, or is composed of, a nucleic acid sequence substantially as shown in SEQ ID No: 13, a fragment or variant thereof, or is composed of, a nucleic acid sequence substantially as shown in SEQ ID No: 13, a fragment or variant thereof.

18. The gene construct according to any one of the preceding claims, wherein the gene construct comprises a first nucleotide sequence and a second nucleotide sequence encoding first and second poly-A tails, preferably wherein the first poly-A tail coding sequence is located at the 3' of a first coding sequence encoding a TLR agonist, and / or wherein the second poly-A tail coding sequence is located at the 3' of a second coding sequence encoding a cytokine.

19. The gene construct of claim 18, wherein the first and / or second poly-A tail comprises a nucleic acid sequence substantially as shown in SEQ ID No: 14 or 15, or a fragment or variant thereof.

20. An expression cassette comprising the gene construct according to any one of claims 1 to 19.

21. An oncolytic virus comprising the gene construct of any one of claims 1 to 19 or the expression cassette of claim 20.

22. Oncolytic virus containing a nucleotide sequence encoding Burkholderia flagellin (FliC_BP).

23. The oncolytic virus of claim 22, wherein the oncolytic virus comprises a first promoter operatively linked to a nucleotide sequence encoding FliC_BP.

24. The oncolytic virus according to any one of claims 21 to 23, wherein the oncolytic virus is selected from the following: herpes simplex virus (HSV), Coxsackie virus, Malaba virus, measles virus (MV), Newcastle disease virus (NDV), poliovirus, reovirus, retrovirus, Seneca Valley virus (SVV), alphavirus, Semlikie Forest virus (SFV), Sindbis virus (SINV), vesicular stomatitis virus (VSV), Sindbis virus (SBV), adenovirus, poxvirus, parvovirus, flavivirus, Zika virus, paramyxovirus, cirrovirus, and rhabdovirus.

25. The oncolytic virus according to any one of claims 21 to 24, wherein: (i) The oncolytic virus is a herpes simplex virus (HSV), optionally, wherein the HSV is HSV-1 or HSV-2; and / or (ii) wherein the oncolytic virus is a live, replicable virus.

26. The oncolytic virus according to any one of claims 21 to 25, wherein the oncolytic virus comprises a functionally lost ICP34.5 gene, preferably wherein the oncolytic virus comprises at least two functionally lost copies of the ICP34.5 gene.

27. The oncolytic virus according to any one of claims 21 to 26, wherein the oncolytic virus comprises a functionally lost ICP47 gene.

28. The gene construct according to any one of claims 1 to 19, the expression cassette according to claim 20, or the oncolytic virus according to any one of claims 21 to 27, for therapeutic purposes.

29. The gene construct according to any one of claims 1 to 19, the expression cassette according to claim 20, or the oncolytic virus according to any one of claims 21 to 27, for the treatment, prevention, or mitigation of cancer.

30. The gene construct, expression cassette, or oncolytic virus used according to claim 29, wherein the cancer is bladder cancer.

31. The gene construct, expression cassette, or oncolytic virus used according to claim 29 or 30, wherein the cancer is non-muscle-invasive bladder cancer (NMIBC) or BCG-refractory bladder cancer.

32. A pharmaceutical composition comprising the gene construct of any one of claims 1 to 19, the expression cassette of claim 20, or the oncolytic virus of any one of claims 21 to 27, and a pharmaceutically acceptable medium.

33. A method for preparing the pharmaceutical composition of claim 32, the method comprising combining a therapeutically effective amount of the gene construct of any one of claims 1 to 19, the expression cassette of claim 20, or the oncolytic virus of any one of claims 21 to 27 with a pharmaceutically acceptable medium.

Images