阅读说明：本技术 用于癌症的重组治疗干预 (Recombinant therapeutic intervention for cancer ) 是由 W·R·比塞 崔尼蒂·J·比瓦拉夸 阿洛克·辛格 莫纳里·普拉哈拉杰 吉田隆广 潘卡杰·普 于 2019-03-14 设计创作，主要内容包括：描述了用于治疗或预防受试者中的癌症的组合物和方法,所述治疗或预防通过将包含分枝杆菌菌株的药物组合物施用至受试者的膀胱中来进行,所述分枝杆菌菌株含有本发明的表达载体。药物组合物可以通过任何合适的手段包括通过导管施用。(Compositions and methods for treating or preventing cancer in a subject by administering a pharmaceutical composition comprising a mycobacterium strain comprising an expression vector of the invention into the bladder of a subject are described. The pharmaceutical composition may be administered by any suitable means, including by catheter.)

1. A vector comprising a nucleic acid sequence expressing a protein, or a functional part thereof, which protein, or functional part thereof, produces a STING agonist.

2. The vector of claim 1, wherein the STING agonist is selected from the group consisting of: 3'-5' c-di-AMP (also known as c-di-AMP); 3'-5' c-di-GMP (also known as c-di-GMP); 3'-3' cGAMP; 2'-3' cGAMP and combinations thereof.

3. The vector of claim 2, comprising a nucleic acid sequence selected from the group consisting of: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (dis A) protein or a functional portion thereof, and combinations thereof.

4. The vector of claim 1, further comprising a fifth nucleic acid sequence encoding a PanC and PanD protein, or functional portions thereof.

5. The vector of claim 4, wherein the vector does not contain an antibiotic resistance gene.

6. The vector of claim 1, wherein the vector is selected from the group consisting of: a vector that is stably integrated into the genome of the bacterium, a vector that replicates stably in the bacterium in more than one copy, and/or combinations thereof.

7. The vector of claim 3, further comprising a fifth nucleic acid sequence encoding a protein or nucleic acid sequence that knocks down expression of a phosphodiesterase gene or domain of a Mycobacterium (Mycobacterium).

8. The vector of claim 3, wherein the third nucleic acid sequence overexpresses the cyclase domain of the cyclic GMP-AMP synthase (cGAS) protein.

9. The vector of claim 3, wherein the third nucleic acid sequence expresses a non-functional cyclic GMP-AMP synthase (cGAS) protein having the ability to recognize regulatory DNA.

10. A mycobacterium strain comprising a vector comprising a nucleic acid sequence that expresses a protein, or functional portion thereof, that produces a STING agonist.

11. The strain of claim 10, wherein the STING agonist is selected from the group consisting of: 3'-5' c-di-AMP (also known as c-di-AMP); 3'-5' c-di-GMP (also known as c-di-GMP); 3'-3' cGAMP; 2'-3' cGAMP and combinations thereof.

12. The strain of claim 10, wherein the nucleic acid sequence is selected from the group consisting of: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (DisA) protein or a functional portion thereof, and combinations thereof.

13. The strain of claim 12, wherein the Mycobacterium strain is Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium bovis (Mycobacterium bovis), or a combination thereof.

14. The strain of claim 13, wherein the Mycobacterium strain is Mycobacterium Calmette-Guerin (BCG).

15. The strain of claim 14, wherein the strain is a pantothenate (panCD mutant) auxotroph of BCG and the vector comprises a panCD nucleic acid encoding a PanC protein or a functional portion thereof and a nucleic acid sequence encoding a PanD protein or a functional portion thereof.

16. The strain of claim 16, wherein the strain does not contain an antibiotic resistance genomic gene.

17. The strain of claim 16, wherein the vector does not contain an antibiotic resistance gene.

18. The strain of claim 10, wherein the vector is selected from the group consisting of: a vector that integrates into the chromosome of the mycobacterium, a vector that stably replicates episomally in more than one copy within the mycobacterium, and combinations thereof.

19. The strain of claim 10, wherein the strain does not contain an antibiotic resistance gene.

20. A pharmaceutical composition comprising any one of the mycobacterium strains of claims 10-19 and (ii) a pharmaceutically acceptable carrier.

21. A method of eliciting a type 1 interferon response, enhancing expression of pro-inflammatory cytokines and/or eliciting a training immunity in a subject, the method comprising the steps of:

administering a pharmaceutical composition comprising any one of the mycobacterium strains of claims 10-19; and

eliciting a type 1 interferon response, enhancing expression of pro-inflammatory cytokines, and/or eliciting a training immunity in the subject.

22. The method of claim 21, wherein the pharmaceutical composition is administered into the subject's bladder via a catheter.

23. A method of treating or preventing cancer in a subject using a mycobacterium strain comprising a vector that expresses a protein that produces a STING agonist, the method comprising the steps of:

administering to a subject having cancer a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a STING agonist-producing protein or functional portion thereof; and

treating or preventing cancer in the subject.

24. The vector of claim 23, wherein the STING agonist is selected from the group consisting of: 3'-5' c-di-AMP (also known as c-di-AMP); 3'-5' c-di-GMP (also known as c-diGMP); 3'-3' cGAMP; 2'-3' cGAMP and combinations thereof.

25. The vector of claim 24, comprising a nucleic acid sequence selected from the group consisting of: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (DisA) protein or a functional portion thereof, and combinations thereof.

26. The method of claim 23, wherein the cancer is selected from the group consisting of: epithelial cancer, breast cancer, non-muscle invasive bladder cancer, and combinations thereof.

27. The method according to claim 26, wherein the cancer is non-muscle invasive bladder cancer and BCG non-responsive non-muscle invasive bladder cancer (BCG non-responsive NMIBC) and the pharmaceutical composition is administered by intravesical instillation.

28. The method in accordance with claim 26, wherein the cancer is non-muscle invasive bladder cancer and is BCG untreated non-muscle invasive bladder cancer (BCG untreated NMIBC) and the pharmaceutical composition is administered by intravesical instillation.

29. The method of claim 26, wherein the epithelial cancer is selected from the group consisting of: colon cancer, uterine cancer, cervical cancer, vaginal cancer, esophageal cancer, nasopharyngeal cancer, bronchial cancer, and combinations thereof, and the pharmaceutical composition is administered to the luminal surface of the epithelial cancer.

30. The method of claim 23, wherein the cancer is selected from a solid tumor, a liquid tumor.

31. The method of claim 30, wherein the pharmaceutical composition is administered by intratumoral injection.

32. The method of claim 30, wherein the pharmaceutical composition is administered by systemic infusion.

33. The method of claim 23, comprising the step of administering a checkpoint inhibitor.

34. The method of claim 33, wherein the checkpoint inhibitor is selected from the group consisting of: ipilimumab (anti-CTLA-4), nivolumab (anti-PD-1), pembrolizumab (anti-PD-1), cimiraprizumab (anti-PD-1), astuzumab (anti-PD-L1), avilimumab (anti-PD-L1), de vacizumab (anti-PD-L1), and combinations thereof.

35. The method of claim 23, wherein the cancer is bladder cancer and catheter administration of the pharmaceutical composition.

Background

Urothelial cancer of the bladder is the most common type of Bladder Cancer (BC) in north america, south america, europe, and asia. Non-Muscle Invasive Bladder Cancer (NMIBC) is associated with a high recurrence rate, frequent intravesical treatment, risk of progression to late stages, and the highest lifetime treatment of all cancers. Intravesical BCG (bacillus Calmette Guerin) instillation has been the standard of care treatment for NMIBC for 30 years. Intravesical BCG instillations are effective in 60% to 70% of patients. BCG has been shown to be a very effective vehicle for antigen delivery. Many studies demonstrating that the potential immune response is biased towards the type I interferon and Th 1-induced mediated immune responses show promise. Efforts to generate recombinant bcg (rbcg) strains for NMIBC have focused on developing strains that enhance these anti-tumor immune responses. To date, such efforts have not resulted in significant improvements over traditional BCG.

Summary of The Invention

One embodiment of the invention is a vector comprising a nucleic acid sequence expressing a protein or functional part thereof that produces a STING agonist, including, by way of example, c-di-AMP (also known as 3'-5' c-di-AMP); c-di-GMP (also known as 3'-5' c-di-GMP); 3'-3' cGAMP (also known as 3'-5', 3'-5' cGAMP, a product of Vibrio cholerae (Vibrio cholerae) DncV protein); 2'-3' cGAMP (also known as 2 '-5', 3'-5' cGAMP, which is the product of human cGAS protein) and combinations thereof. Some vectors of the invention comprise a nucleic acid sequence selected from the group consisting of: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (dis A) protein or a functional portion thereof, and combinations thereof. Each of these nucleic acid sequences expresses a protein that produces one or more STING agonists as described in the definitions section of the specification. Some vectors of the invention include a fifth nucleic acid sequence encoding a PanC protein and a PanD protein, or functional portions thereof, in addition to one or more of the sequences listed above. Vectors comprising nucleic acid sequences encoding the PanC and PanD proteins or functional portions thereof typically do not contain an antibiotic resistance gene. Suitable vectors for use in the present invention may include vectors which replicate episomally in more than one copy, or vectors which integrate into the bacterial chromosome in a single copy or are otherwise present in the bacterial cell. The vectors of the invention may be stably integrated into the bacterial genome, or they may be stably replicated as episomal plasmids (episomal plasmids). Suitable third nucleic acid sequences include those that overexpress the cyclase domain of the cyclic GMP-AMP synthase (cGAS) protein. Other suitable third nucleic acid sequences may express a non-functional cyclic GMP-AMP synthase (cGAS) protein having the ability to recognize regulatory DNA. The vector of the present invention may further comprise a nucleic acid sequence encoding a sequence or protein that knocks down the expression of the PDE gene of the mycobacterium (mycobacterium) strain used in the present invention.

Another embodiment of the invention is a mycobacterium strain comprising any of the vectors of the invention, including a vector comprising a nucleic acid sequence that expresses a protein, or functional portion thereof, that produces a STING agonist. As mentioned above, examples of STING agonists include, by way of example, c-di-AMP (also known as 3'-5' c-di-AMP); c-di-GMP (also known as 3'-5' c-di-GMP); 3'-3' cGAMP (also known as 3'-5', 3'-5' cGAMP, a product of the vibrio cholerae DncV protein); 2'-3' cGAMP (also known as 2 '-5', 3'-5' cGAMP, which is the product of human cGAS protein) and combinations thereof. Examples of suitable nucleic acid sequences include nucleic acid sequences selected from the group consisting of: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a 3'-3' cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a 2'-3' cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (dis A) protein or a functional portion thereof, and combinations thereof. Examples of suitable strains of Mycobacterium for use in the present invention include, for example, Mycobacterium tuberculosis (Mycobacterium tuberculosis), Mycobacterium bovis (Mycobacterium bovis), or combinations thereof. Another strain used in the present invention is Mycobacterium Calmette-Guerin (BCG). The mycobacterium strain used in the present invention may be a pantothenate (pantonate) auxotroph of BCG lacking its panCD gene operon. The panCD auxotrophic strain lacks genomic sequences capable of encoding functional PanC and/or PanD proteins. In some embodiments, a mycobacterium strain that is pantothenate auxotrophic comprises a vector of the present invention comprising a panCD nucleic acid encoding a PanC and PanD protein, or functional portions thereof. The vectors of the invention comprising the panCD nucleic acid sequences preferably do not contain an antibiotic resistance gene or a nucleic acid sequence encoding a functional protein that provides antibiotic resistance. The mycobacterium which is pantothenate auxotrophic for the present invention preferably does not contain an antibiotic resistance genomic gene or does not encode a functional protein that provides antibiotic resistance.

Another embodiment of the invention is a pharmaceutical composition comprising any one of the mycobacterium strains of the invention and a pharmaceutically acceptable carrier.

Another embodiment of the invention is a method of eliciting a type 1 interferon response, enhancing expression of pro-inflammatory cytokines, and/or eliciting a trained immunity in a subject, the method comprising the steps of: administering a pharmaceutical composition comprising any one of the strains of the invention into a subject; and eliciting a type 1 interferon response, enhancing expression of a pro-inflammatory cytokine, and/or eliciting a training immunity in the subject. In one embodiment, the pharmaceutical composition is administered into the bladder of the subject via a catheter.

Another embodiment is a method of treating or preventing cancer in a subject using a mycobacterium strain of the invention. The method comprises the following steps: administering to a subject having cancer a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a STING agonist-producing protein or functional portion thereof; and treating or preventing cancer in a subject. By way of example, the invention may be used to treat or prevent cancer, including epithelial cancer, breast cancer, non-muscle invasive bladder cancer. In some embodiments, the cancer is BCG non-responsive non-muscle invasive bladder cancer (BCG non-responsive NMIBC), and the pharmaceutical composition is administered by intravesical instillation. In some examples, the cancer is BCG untreated non-muscle invasive bladder cancer (BCG-non-muscle innovative radiator (BCG untreated NMIBC) and the pharmaceutical composition is administered by intravesical instillation. In other examples, the cancerSelected from the group consisting of: colon cancer, uterine cancer, cervical cancer, vaginal cancer, esophageal cancer, nasopharyngeal cancer, bronchial cancer, and combinations thereof, and the pharmaceutical composition is administered to the luminal surface of the epithelial cancer. In some embodiments, the cancer is selected from a solid tumor, a liquid tumor, and the pharmaceutical composition is administered by intratumoral injection and/or by systemic infusion. The methods of the invention may include the step of administering a checkpoint inhibitor, such as, by way of example, anti-PD 1, anti-PDL 1, or a combination thereof. In another embodiment, the cancer is bladder cancer and the catheter administers the pharmaceutical composition.

One embodiment of the present invention is an expression vector comprising: a first nucleic acid sequence encoding an Rv1354c protein or a functional portion thereof; a second nucleic acid sequence encoding a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a third nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (dis a) protein or a functional part thereof that functions as a diadenosine cyclase, or a combination thereof. Some expression vectors of the invention have a first nucleic acid sequence that overexpresses the cyclase domain of the Rv1354c protein when compared to the expression of the native Rv1354c protein as a reference. Some expression vectors of the invention have a second nucleic acid sequence that overexpresses a cyclic GMP-AMP synthase (DncV) protein when compared to the expression of a native DncV protein. Some expression vectors of the invention have a third nucleic acid sequence that overexpresses the cyclase domain of the cyclic GMP-AMP synthase (cGAS) protein when compared to expression of the native cGAS protein. Suitable Rv1354 proteins for use in the present invention include mycobacterium tuberculosis Rv1354 protein. Suitable DncV proteins for use in the present invention include the Vibrio cholerae DncV protein. Suitable cGAS proteins for use in the present invention include Homo sapiens (Homo sapiens) cGAS protein. Suitable DISA proteins for use in the present invention include Mycobacterium tuberculosis DISA proteins.

Another embodiment of the invention includes BCG strains comprising a cdnP gene, an Rv1354c gene, an Rv1357c gene, or a combination thereof, wherein the cdnP gene is incapable of expressing a functional cyclic dinucleotide phosphodiesterase (cdnP) protein, the Rv1354c gene is incapable of expressing a functional Rv1345c protein, and/or the Rv1357c gene is incapable of expressing a functional Rv1357 protein. Some BCG strains of the invention may have an Rv1354c gene comprising a non-functional EAL domain. The BCG strain of the invention may comprise any expression vector of the invention.

Another embodiment of the invention is a method of treating or preventing bladder cancer, the method comprising the steps of: administering a pharmaceutical composition comprising a BCG strain comprising an expression vector of the invention into the bladder of a subject; and treating or preventing bladder cancer in the subject when compared to a reference subject not administered the pharmaceutical composition. The pharmaceutical composition may be administered by any suitable means, including by catheter.

Another embodiment of the invention is a method of eliciting a type 1 interferon response in a subject, the method comprising the steps of: administering a pharmaceutical composition comprising a BCG strain comprising an expression vector of the invention into a subject, such as the bladder of a subject; and enhancing a type 1 interferon response in the subject compared to a reference subject not administered the pharmaceutical composition.

Another embodiment of the invention is a method of treating or preventing cancer in a subject, the method comprising the steps of: administering a pharmaceutical composition comprising a BCG strain comprising an expression vector of the invention into a tumor of a subject having cancer; and treating or preventing cancer in the subject when compared to a reference subject not administered the pharmaceutical composition. The pharmaceutical composition may be administered by any suitable means including injection into a tumor. Cancers that may be treated or prevented by this method include, but are not limited to, breast cancer and/or non-muscle invasive bladder cancer.

Examples of mycobacteria for use in the present invention include Mycobacterium tuberculosis, Mycobacterium bovis BCG (known as BCG), Mycobacterium smegmatis (Mycobacterium smegmatis), Mycobacterium avium (Mycobacterium avium complex) and other non-Mycobacterium tuberculosis (NTM). Examples of BCG strains useful in the present invention, including those that overexpress STING agonists, include BCG Pasteur, BCG-Pasteur-Aeras, BCG Tice (also known as BCG Chicago), BCG-Connaught (also known as BCG Toronto), BCG Danish, BCG-Prague (also known as BCG Czechoslokian), BCG Russia (also known as BCG Moscow), BCG Moreau (also known as BCG Brazil), BCG Japan (also known as BCG GTokyo), BCG Sweden (also known as Gothenburg), Birkhaug, Glaxo, BCG (also known as BCG Montreal), BCG Phipps, or other available strains.

Another embodiment of the invention is a method of treating diabetes, comprising the steps of: administering to a subject having diabetes a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a STING agonist-producing protein or functional portion thereof; and treating or preventing diabetes in the subject by providing a training immunity. Training immunity refers to the ability of one antigenic stimulus to elicit a stronger immune response against a second, different antigenic stimulus introduced at a later time. Training immunity is antigen independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and associated with epigenetic and metabolic changes. This approach results in the upregulation of glycolysis mediated by the training immunity. The upregulation of glycolysis mentioned above is beneficial for the prevention and treatment of type 1 and type 2 diabetes.

Another embodiment of the invention is a method of stimulating a training immunity in a subject, the method comprising the steps of: administering to the subject a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a STING agonist-producing protein or functional portion thereof; and stimulating a training immunity in the subject. Wherein the method upregulates glycolysis in the subject and/or stimulates episomal changes in histone methylation in the subject that mediate a training immunity in the subject.

Another embodiment of the invention is a method of treating or preventing a viral infection in a subject, the method comprising the steps of: administering to the subject a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a STING agonist-producing protein or functional portion thereof; and treating or preventing a viral infection in a subject. The method stimulates a training immunity in the subject, treating or preventing a viral infection in the subject. Wherein the method upregulates glycolysis in the subject and/or stimulates episomal changes in histone methylation in the subject that mediate a training immunity in the subject.

Another embodiment of the invention is a method of treating or preventing a bacterial infection or a drug-resistant bacterial infection in a subject, the method comprising the steps of: administering to the subject a pharmaceutical composition comprising a mycobacterium strain comprising a vector that expresses a STING agonist-producing protein or functional portion thereof; and treating or preventing a bacterial infection or a drug-resistant bacterial infection in a subject. The method stimulates a training immunity in the subject, treating or preventing a bacterial infection in the subject. Wherein the method upregulates glycolysis in the subject and/or stimulates episomal changes in histone methylation in the subject that mediate a training immunity in the subject. The methods of the invention may employ one or more vectors of the invention or one or more bacterial strains comprising a vector of the invention.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references provide the skilled artisan with a general definition of many of the terms used in the present invention: singleton et al, Dictionary of Microbiology and Molecular Biology (2 nd edition. 1994); the Cambridge Dictionary of Science and Technology (Walker, 1988); the Glossary of Genetics, 5 th edition, R.Rieger et al, (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings ascribed to them below, unless otherwise specified.

By "agent" is meant any small molecule compound, antibody, nucleic acid molecule or polypeptide, or fragment thereof.

By "alteration" is meant a change (increase or decrease) in the level of expression or activity of a gene or polypeptide, as detected by standard art-known methods, such as those described herein. As used herein, alteration includes a 10% change in expression level, preferably a 25% change in expression level, more preferably a 40% change, and most preferably a 50% or greater change.

By "improving" is meant reducing, inhibiting, attenuating, reducing, arresting or stabilizing the development or progression of a disease.

By "analog" is meant a molecule that is not identical, but has similar functional or structural characteristics. For example, a polypeptide analog retains the biological activity of the corresponding naturally-occurring polypeptide, while having certain biochemical modifications that enhance the function of the analog relative to the naturally-occurring polypeptide. For example, such biochemical modifications can increase the protease resistance, membrane permeability, or half-life of the analog without altering, for example, ligand binding. In another example, the analog can include an unnatural amino acid.

"cdnP" means 1) a cdnP gene or nucleic acid sequence encoding a cyclic dinucleotide phosphodiesterase (cdnP) protein, or 2) a cyclic dinucleotide phosphodiesterase protein. Examples include the cdnP gene Rv2837c of Mycobacterium tuberculosis (M.tuberculosis) with NCBI gene ID 888920 in H37Rv, and the cdnP protein of UniProtKB/Swiss-Prot P71615.2.

"cGAs" means 1) the cGAs gene or nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein, or 2) the cyclic GMP-AMP synthase protein. Examples of cGAs include homo sapiens (H.sapiens) cGAS gene (NCBI gene ID: 115004) and the protein encoded by this gene (UniProtKB/Swiss-Prot: Q8N884.2). cGas protein is a cyclic GMP-AMP synthase from humans that produces 2'3' cGMP. 2'3' cGMP is a STING agonist in humans.

By "cyclase domain" is meant a cyclase domain of cGAS, for example, a portion of the 522 amino acid human cGAS protein described in Kranzusch et al (Cell Reports 2013; 3:1362-1368PMID 23707061). The cyclase domain can be described as having a nucleotidase (NTase) core at amino acids 160-330 and a regulatory-sensor domain at amino acids 330-522, i.e.the C-domain. Mutants of the nucleotidase core sequence, as well as mutants of the regulatory-sensor domain, can be used to produce constitutively active variants of cGAMP that are designed to produce high levels of cGAMP without the general need for activation by DNA binding. Another example of a cyclase domain includes NCBI gene ID: 887485 amino acids of Mycobacterium tuberculosis Rv1354c, and the protein encoded by this gene (UniProtKB/Swiss-Prot: P9WM13) which encodes a protein capable of both c-di-GMP (cyclic di-guanylic acid or cyclic di-GMP) synthesis (via its GGDEF domain, amino acid 201-400) and degradation (via its EAL domain, amino acid 401-623). The GAF domain (amino acids 1-200) is a regulatory domain. Mutants of the GGDEF domain and the regulatory-sensor GAF domain and polypeptides truncated to remove the EAL domain (phosphodiesterase activity) can be used to generate constitutively active variants of Rv1354c designed to produce high levels of c-di-GMP.

"DisA" or "DISA" means 1) a DisA gene or nucleic acid sequence encoding a DNA integrity Scan (DisA) protein, or 2) a DNA integrity Scan protein. Examples include NCBI gene ID: 887485, and the protein encoded by the gene is UniProtKB/Swiss-Prot: P9WNW5.1. The protein is a diadenosine cyclase such as Dey & Bishai et al Nature Medicine 2015; 21:401-6.PMID: 25730264. The DisA protein is a diaadenylate cyclase producing c-di-AMP. c-di-AMP is a STING agonist.

By "disease" is meant any condition or disorder that damages or interferes with the normal function of a cell, tissue or organ. Examples of diseases include, but are not limited to, bladder cancer.

"dncV" means a gene encoding cyclic GMP-AMP synthase that catalyzes the synthesis of the second messenger 3'3' -cyclic GMP-AMP (3'3' -cGAMP) in cellular signal transduction from GTP and ATP. It also produces c-di-AMP and c-di-GMP from ATP and GTP, respectively; however, in contrast to 2'3' -cGAMP produced by eukaryotes, 3'3' -cGAMP is the major molecule produced in vivo by DncV. It is essential for efficient intestinal colonization by vibrio cholerae (v. cholerae) and down regulates chemotactic processes that affect colonization. It is inactive against dATP, TTP, UTP and CTP. The DncV protein is a cyclic GMP-AMP synthase from Vibrio cholerae that produces 3'3' cGAMP. 3'3' cGAMP is a STING agonist.

By "EAL domain" is meant a conserved protein domain found in a variety of bacterial signaling proteins. The EAL domain may function as a diguanylate phosphodiesterase and has been shown to stimulate degradation of the second messenger cyclic di-GMP. Non-functional EAL domains will not have one or more of these functions. Examples of EAL domains include 307 amino acids of mycobacterium tuberculosis Rv1357c, the gene for which is NCBI gene ID: 886815, and the protein encoded by the gene is UniProtKB/Swiss-Prot: P9WM07, UniProtKB/Swiss-Prot: P9WM07 encodes c-di-GMP Phosphodiesterase (PDE) and comprises a single EAL domain. The activity of this enzyme is to act as a c-di-GMP phosphodiesterase, cleaving a cyclic dinucleotide (which has signaling activity) into 2GMP molecules (which lack signaling activity), such as those titled "A full-length bifunctional protein involved in c-di-GMP turn over is required for long-term subvariant circulation in Mycobacterium smegmatis", Bharati BK, Sharma IM, Kasetty S, Kumar M, Mukherjee R, Chatterji D.Microbiology.2012Jun; 158(Pt 6) 1415-27.doi:10.1099/mic.0.053892-0.Epub 2012Feb 16.PMID: 22343354. Another example of an EAL domain includes the 336 amino acid mycobacterium tuberculosis cdnP gene in H37Rv (Rv2837c), a c-di-AMP phosphodiesterase, containing an EAL domain with the ability to hydrolyze human 2'-3' cGAMP (the product of the human cGAS enzyme), as described by Jain-Dey Bishai et al Nat Chem biol.2017; 13:210 + 217PMID 28106876. The structural features of the EAL domain (cyclic dinucleotide phosphodiester activity) and GGDEF domain (cyclic dinucleotide cyclization-biosynthetic activity) are known and well described (e.g., in Schirmer T, Jenal U.S. structural and mechanical stabilizers of c-di-GMP signalling Nat Rev Microbiol. 2009; 7:724-35.PMID: 19756011).

By "effective amount" is meant the amount required to ameliorate the symptoms of the disease relative to an untreated patient. The effective amount of one or more than one active compound for use in the practice of the present invention for the therapeutic treatment of a disease will vary depending on the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. Such an amount is referred to as an "effective" amount.

"dncV" means 1) a dncV gene or nucleic acid sequence encoding a cyclic GMP-AMP synthase (dncV) protein, or 2) a cyclic GMP-AMP synthase protein. Examples include, but are not limited to NCBI gene ID: 2614190, and the protein encoded by this gene is UniProtKB/Swiss-Prot: Q9KVG7.1.

By "fragment" is meant a portion of a polypeptide or nucleic acid molecule. The portion preferably comprises at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80% or 90% of the full length of the reference nucleic acid molecule or polypeptide. A fragment may comprise, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900 or 1000 nucleotides or amino acids.

By "gene deletion" is meant deletion of the entire gene coding region of the gene of interest from the chromosome of BCG using an allelic exchange method well known to those skilled in the art. Gene replacement with a selectable marker, such as an antibiotic resistance cassette, is a form of allelic exchange and can be performed. There are also techniques to generate unlabeled deletions (no selectable marker) in which the gene is completely deleted and no selectable marker is introduced at its location.

By "gene domain deletion" is meant the use of the above allelic exchange method to remove the portion of the gene encoding a particular domain (in the present case, the EAL domain of Rv1354c encoding the CDN phosphodiesterase domain of a multifunctional polypeptide) while leaving the other portion of the polypeptide intact and in-frame (in frame).

"Homo sapiens" means Homo sapiens.

By "obtaining" or as in "obtaining an agent" is meant synthesizing, purchasing, or otherwise obtaining the agent.

By "over-expression" is meant, in a general sense, that the number of genes expressing their respective proteins is greater than the wild-type or reference gene. Examples of genes that produce overexpressed proteins in the present invention include fusion of DNA encoding the gene of interest to a strong promoter such as Phsp60 or a strong conditionally active promoter such as PtetOFF in BCG. In PtetOFF, gene expression is turned off in the presence of tetracycline, anhydrotetracycline, or doxycycline; however, when recombinant BCG is administered as an immunotherapy in humans or animal models, the gene of interest will be turned on. This conditional activity strategy has the following advantages: preventing strong overexpression of the enzymes that produce cyclic dinucleotides during BCG growth may have any detrimental effect on the viability or growth rate of the BCG organism and allow strong expression ("overexpression") only when BCG immunotherapy is administered as a therapeutic agent to a mammalian host.

"Mtb" means Mycobacterium tuberculosis.

The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. These terms apply to amino acid polymers in which one or more amino acid residues is an analogue or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers. Polypeptides may be modified, for example by the addition of carbohydrate residues to form glycoproteins. The terms "polypeptide", "peptide" and "protein" include glycoproteins as well as non-glycoproteins.

"reduce" or "decrease" means, for example, at least about 10%, 25%, 50%, 75%, or 100%, or any percentage negative change therebetween.

By "increase" is meant, for example, at least about 10%, 25%, 50%, 75%, or 100%, or any percentage positive change therebetween.

"reference" means a standard or control condition.

"reference sequence" means a defined sequence that is used as a basis for sequence comparison. The reference sequence may be a subset or the entirety of the specified sequence; for example, a segment of a full-length cDNA or gene sequence, or the entire cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence will typically be at least about 16 amino acids, preferably at least about 20 amino acids, more preferably at least about 25 amino acids, and even more preferably about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of the reference nucleic acid sequence will generally be at least about 50 nucleotides, preferably at least about 60 nucleotides, more preferably at least about 75 nucleotides, and even more preferably about 100 nucleotides or about 300 nucleotides, or any integer near or between them.

By "reference BCG strain" is meant, for example, a conventional BCG strain that does not comprise the expression vector of the invention and/or its endogenous genes and is incapable of expressing cdnP functional protein, Rv1354c functional protein, Rv1357c functional protein, or a combination thereof.

"ability to recognize regulatory DNA" means the ability of a protein to detect or bind DNA. For example, cGAS proteins are known to bind to DNA, such as cytosolic DNA, and trigger the reaction of GTP and ATP to form cyclic GMP-amp (cgamp). cGAMP binds to a stimulator of the interferon gene (STING), which triggers phosphorylation of IRF3 via TBK 1.

By "Rv 1354 c" is meant 1) an Rv1354c gene or nucleic acid sequence encoding an Rv1354c protein, or 2) an Rv1354c protein (e.g., Gupta, Kumar, and Chatterji; PLoS ONE (11 months 2010); volume 5; stage 11; and Bharati, Sharma, Kasetty, Kumar, Mukherjee, and Chatterji; microbiology (2012),158, 1415-. The Rv1354c protein is a diguanylate cyclase producing c-di-GMP. c-di-GMP is a STING agonist.

"Rv 1357 c" means 1) an Rv1357 gene or nucleic acid sequence encoding a cyclic di-GMP phosphodiesterase protein (Rv1357), or 2) a cyclic di-GMP phosphodiesterase protein (e.g., Gupta, Kumar, and Chatterji; PLoS ONE (11 months 2010); volume 5; stage 11; and Bharati, Sharma, Kasetty, Kumar, Mukherjee, and Chatterji; microbiology (2012),158, 1415-. The Rv1357c protein is a diguanylate cyclase producing c-di-GMP. c-di-GMP is a STING agonist.

By "STING agonist" is meant a molecule that binds to STING (a stimulator of interferon genes, or TMEM173), activates STING, and triggers activation of the IRF3-TBK1 pathway, resulting in increased transcription of type 1 interferons and other genes.

"CDN" means a cyclic dinucleotide, such as 3'-5' c-di-AMP, 3'-5' c-di-GMP, 3'-3' cGAMP (also known as 3'-5', 3'-5' cGAMP, which is a product of the vibrio cholerae DncV protein), or 2'-3' cGAMP (also known as 2 '-5', 3'-5' cGAMP, which is a product of the human cGAS protein).

"PAMP" means a pathogen-associated molecular pattern (pathogen associated molecular pattern). PAMPs are microbial products, including small molecules that are recognized by innate immune sensors. Examples of PAMPs are 3'-5' c-di-AMP, 3'-5' c-di-GMP, 3'-3' cGAMP.

"DAMP" means a danger-associated molecular pattern (danger associated molecular pattern). DAMPs are host-derived (i.e., human, mouse, or other mammalian disease model) molecules that are generated to signal a risk, such as infection or other normal physiological disorder. An example of DAMP is 2'-3' cGAMP, which is produced by the host sensor enzyme cGAS upon detection of double stranded DNA in the cytoplasm, as occurs during viral or certain intracellular bacterial infections.

"panCD" means a genetic operon from a bacterium or other species that encodes the biosynthetic gene panC (encoding a PanC protein having pantothenate- β -alanine ligase enzyme activity) and the biosynthetic gene panD (encoding a PanD protein having aspartate 1-decarboxylase enzyme activity). PanC and PanD proteins are involved in the protein which is also referred to as vitamin B₅The pantothenic acid or pantothenate salts (a B vitamin) are essential for the biosynthesis. Pantothenic acid, a water-soluble vitamin, is an essential nutrient for bacteria and all mycobacteria, including BCG. Pantothenic acid is required for the synthesis of coenzyme a (coa) and for the synthesis and metabolism of proteins, carbohydrates and fats.

By "specifically binds" is meant, for example, a compound, nucleic acid, peptide, protein, or antibody that recognizes and binds to a polypeptide or nucleic acid sequence, but does not substantially recognize and bind to other molecules in a sample.

By "substantially identical" is meant a polypeptide or nucleic acid molecule that exhibits at least 50% identity to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). Preferably, such sequences are at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid or nucleic acid level to the sequence used for comparison. Sequence identity is typically measured using sequence analysis software (e.g., the sequence analysis software package of Genetics Computer Group, University of Wisconsin Biotechnology Center,1710University Avenue, Madison, wis.53705, BLAST, BESTFIT, GAP, or PILEUP/prettyox programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions and/or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method of determining the degree of identity, the BLAST program can be used, where e^-3And e^-100The probability scores in between indicate closely related sequences.

By "subject" is meant a mammal, including but not limited to a human or non-human mammal, such as a bovine, equine, canine, ovine, or feline.

By "sensitivity" is meant the percentage of subjects with a particular disease.

By "specific" is meant the percentage of subjects that are correctly identified as having a particular disease, i.e., normal or healthy subjects. For example, specificity is calculated as the number of subjects with a particular disease compared to non-cancer subjects (e.g., normal, healthy subjects).

By "training immunity" is meant the ability of one antigenic stimulant to elicit a stronger immune response against a second, different antigen administered at a later time. Training immunity is antigen independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and associated with epigenetic and metabolic changes.

"Phsp 60" or "Phsp 65" means a strong mycobacterial promoter derived from the Hsp 655' UTR of Mycobacterium leprae (Mycobacterium leprae).

"5 'UTR" means the 5' untranslated region of a gene.

"3 'UTR" means the 3' untranslated region of a gene.

"WT" means a wild type.

"BCG-WT" means a wild-type strain of M.bovis BCG.

As used herein, ranges provided herein are to be understood as a shorthand representation of all values falling within the range. For example, a range of 1 to 50 is understood to include any number, combination of numbers, or sub-range from the group consisting of: 1.2, 3, 4, 5,6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

As used herein, the terms "treat", "treating" and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be understood that treating a disorder or condition does not require (although does not preclude) that the disorder, condition, or symptoms associated therewith be completely eliminated.

The term "or" as used herein is to be understood as being inclusive unless specifically stated or apparent from the context. The terms "a", "an" and "the" as used herein are to be construed as singular or plural unless specifically stated or apparent from the context.

Unless specifically stated or apparent from the context, the term "about" as used herein is understood to be within the normal tolerance of the art, e.g., within 2 standard deviations of the mean. About may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05% or 0.01% of the stated value. Unless otherwise clear from the context, all numbers provided herein are modified by the term about.

As used herein, "comprises," "comprising," "contains," "containing," and "having" and the like may have the meaning ascribed to U.S. patent law and may mean "including," "including," and the like; "consisting essentially of … …" or "consisting essentially of" likewise has the meaning ascribed to U.S. patent law, and the terms are open-ended, allowing more than the recited features, as long as the recited basic or novel features are not altered by more than the recited features, but do not include embodiments of the prior art.

Any of the compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.

As used herein, the terms "preventing", "preventive treatment" and the like refer to reducing the probability of developing a disorder or condition in a subject who does not have, but is at risk of developing, or is predisposed to developing, the disorder or condition.

Such treatment (surgery and/or chemotherapy) will suitably be administered to a subject, particularly a human, who is suffering from, susceptible to, or at risk of bladder cancer or a disease, disorder, or symptom thereof. Determining those subjects "at risk" can be done by any objective or subjective determination of the subject or health care provider's diagnostic tests or opinions (e.g., genetic tests, enzyme or protein markers, markers (as defined herein), family history, etc.). In particular embodiments, determining that the subject is susceptible to or suffering from pancreatic cancer is determined by measuring the level of at least one marker.

Brief Description of Drawings

FIG. 1A-FIG. 1B from pSD5B P_hsp60A Mycobacterium that overexpresses DISA by a DISA plasmid construct releases large amounts of c-di-AMP into the macrophage cytoplasm and transcribes high levels of DISA mRNA. A. Using a vector pSD5B P_hsp60Mycobacterium tuberculosis of disA plasmid or J774 macrophages infected with wild type Mycobacterium tuberculosis (CDC1551) at a MOI of 1: 20. The level of c-di-AMP in macrophages was determined by LC-MS/MS 24 hours after infection. As can be observed, the M.tb-disA-OE strain produced about 15 times more c-di-AMP than the wild-type Mycobacterium tuberculosis (CDC 1551). BCG-disA-OE is expected to show similarly high levels of c-di-AMP. (data from Dey B, Dey RJ, Cheung LS, Pokkali S, Guo H, Lee JH, Bishai WR. A bacterial cyclic pyridine activities the cytotoxic peptide pathway and media activity to tissue of Nature. med. 2015; 21:401-6.PMID: 25730264.). B. carrying pSD5B P_hsp60BCG-Pasteur or BCG-Pasteur-WT of a disA plasmid grows to mid-exponential phase (mid-exponentatial phase). The bacteria were lysed and mRNA was prepared. Levels of disA mRNA were determined by quantitative RT-PCR. The BCG-disA-OE strain produces about 50 times more disA mRNA than BCG-Pasteur-WT.

Fig. 2. BCG overexpressing dis a increased proinflammatory cytokines. Gene expression profiling (qPCR) of pro-inflammatory cytokines and IFN- β in BMDM of mice challenged with wild-type BCG-Pasteur strain and DISA over-expressed BCG-Pasteur strain.

Fig. 3. BCG overexpressing dis a increased IRF3 signaling. The effect of dis overexpression on the activation of the IRF pathway was measured by the IRF-SEAP QUANTI Blue reporter assay. Culture supernatants of infected RAW-Blue ISG cells were assayed for IRF activation. Images below the IRF activation map represent QUANTI Blue assay plates and sample wells; the processing parameters for a column of holes correspond to the parameters defined above for the columns aligned with the holes. The BCG-disA-OE in this figure is derived from BCG Pasteur.

Fig. 4A-4C. An increase in proinflammatory cytokines in response to overexpression of disca. Differential expression of TNF- α (A), IL-6(B) and IL-1 β (C) in mouse BMDM challenged with wild-type BCG-Pasteur strains and DISA-overexpressing BCG-Pasteur strains. Culture supernatants were assayed for different cytokines by ELISA.

Fig. 5. BCG overexpressing dis a induced a differential immune response in human bladder cancer cells (RT 4). Differential gene expression in human RT4 bladder cancer cells challenged with wild-type BCG-Pasteur, wild-type BCG-Tice strain, and BCG-Pasteur-disA-OE. Quantitative real-time PCR based on SYBR green was used to measure mRNA expression levels.

Fig. 6. Schematic workflow for testing relative therapeutic efficacy of wild type strains and BCG-disA-OE strains.

Fig. 7. Tumor involvement index (tumor involvement index) of untreated tumor bearing rats or tumor bearing rats treated with WT BCG or with rBCG overexpressing DISA (rBCG ═ BCG-Pasteur-DISA-OE; wtBCG ═ BCG-Pasteur).

Fig. 8. Analysis of the immunoprography of MNU-induced bladder tumors in Fischer rats in response to intravesical therapy with different BCG strains. Differential gene expression in rat bladder tumor cells following therapy with wild-type mycobacterium bovis BCG-Pasteur strain and mycobacterium bovis BCG-Pasteur strain with dis a overexpression. The expression level of mRNA was measured using TaqMan-based quantitative real-time PCR. BCG-WT is BCG Pasteur, and BCG-disA-OE is derived from BCG Pasteur in this figure.

Fig. 9. Gene expression profiling of bladder from untreated MNU tumor bearing rats (MNU tumor bearing rats) or MNU tumor bearing rats treated with WT or sabg overexpressing disA.

Fig. 10. Summary of relative gene expression of BCG-disA-OE versus BCG-WT in different cells or tissues. Mouse bone marrow-derived macrophages (BMDM), human immortalized bladder cancer cell lines RT4 and 5637, and rat immortalized bladder cancer cell lines were infected with BCG-disA-OE and BCG-WT for 24 hours, and mRNA was prepared from the cells. Rats were exposed to MNU by intravesical instillation for 8 weeks, and then treated with BCG-disA-OE or BCG-WT by intravesical instillation for 8 weeks. At week 16 necropsy, bladders were removed and mRNA prepared. Quantitative RT-PCR was performed on the indicated cytokine or chemokine genes. The changes shown are the fold induction or reduction of observed BCG-disA-OE normalized against the observed BCG-WT. BCG-WT is BCG Pasteur, and BCG-disA-OE is derived from BCG Pasteur in this figure.

Fig. 11. Proposed mechanism of action of BCG overexpressing DISA.

Fig. 12. Molecular genetic modifications to BCG will increase the levels of CDN PAMP and DAMP molecules as STING agonists. PAMP: pathogen-associated molecular patterns (produced by bacteria): c-di-AMP (disA), c-di-GMP (Rv1354c), 3'3' -cGAMP (dnCV). And (3) DAMP: risk-related molecular patterns (produced by the host): 2'-3' -cGAMP. OE: and (4) overexpression. KO: knock-out (gene replacement).

FIG. 13. Two cyclic dinucleotide cyclases and phosphodiesterase proteins present in BCG: diagrams of BCG _ RS07340 and BCG _ AHM07112. BCG _ RS07340 is a bifunctional protein with both CDN cyclase and CDN PDE activities. BCG _ AHM07112 is a CDN PDE. The domains are: GAF (regulatory), GGDEF (diguanylate cyclase) and EAL diguanylate phosphodiesterase.

Fig. 14. Carries pSD5B P compared with wild type Mycobacterium tuberculosis (Mtb-CDC1551)_hsp60Mycobacterium tuberculosis (M.tb-dis A-OE or Mtb-OE) of a dis plasmid is significantly attenuated in mice. Female BALB/c mice (n ═ 10/group) 6-7 weeks old were infected with-3.5 log10 CFU by aerosol infection (aerosol infection) as described above. On day 1, 3 mice in each group were counted for CFU and 3.5log10 CFU units were confirmed implanted. Mice remained housed until death. As can be observed, the median time to death for wild-type mycobacterium tuberculosis infection was 150.5 days. In contrast, mice infected with the same M.tb-disA-OE (Mtb-OE) inoculum had a median time to death (p) of 321.5 days<0.001). Compared to BCG-WT, BCG-disA-OE is expected to show a similar loss of virulence in mice. (data from Dey B, Dey RJ, Cheung LS, Pokkali S, Guo H, Lee JH, and Bishai WR. A bacterial cyclic pyridine activities the cyclosalic Survolation pathway and media existence response to tuboculosis. Nat. Med. 2015; 21:401-6.PMID: 25730264.)

Fig. 15A-15B. Other BCG strains are also active: the BCG Tice strain overexpressing DISA also showed similar proinflammatory cytokine induction as the BCG Pasteur overexpressing DISA. Bone marrow derived macrophages were challenged with wild type strains of both BCG Pasteur and BCG tie strains and with a strain overexpressing disA at 1:20 m.o.i.for 15 h. Culture supernatants were harvested and cytokines were detected using ELISA. Differential expression patterns of TNF- α (A) and IL-6(B) in BMDM of mice challenged with two different BCG strains. The BCG-Tice strain is from a commercially available Onco-Tice product.

Fig. 16. Type I interferon responses in macrophages in response to BCG-disA-OE are STING dependent. Bone marrow-derived macrophages from STING ablated (KO) and control mice were challenged with wild-type and disA OE strains of BCG Pasteur for 24 h. The culture supernatants were probed for IFN- β levels using ELISA.

Figure 17 shows that intravesical instillation of BCG-disA-OE showed maximal anti-tumor efficacy (statistically significant pathological improvement) in an MNU oncogenic model of non-muscle invasive bladder cancer (NIMBC). Within the first 8 weeks, the rat groups received 4 intravesical treatments with MNUs (one treatment every 2 weeks) to elicit NIMBC. Over the next 8 weeks, rats received 4 intravesical treatments (one treatment every 2 weeks) with PBS (untreated), BCG-WT, or BCG-DISA-OE. At the end of the 16-week experiment, the rats were sacrificed and their bladders removed. A portion of the bladder was fixed and H & E stained and then interpreted blindly by a committee-certified urologist (bound washion). Tumor involvement scores and cancer stages were determined and shown (T2-3, T1, CIS + papillary lesions, CIS alone or normal-dysplasia). As can be observed, BCG-disA-OE instillation resulted in a statistically significant and lower tumor involvement index than PBS (untreated), whereas BCG-WT was not statistically significantly superior to PBS. This 16 week experiment was performed twice. The data in fig. 7 represent the results of experiment 1. The data in this figure (fig. 17) represents the combined results of experiment 1 plus experiment 2. The qPCR data shown in figures 8 and 9 were obtained from the use of bladder tissue at necropsy at the end of experiment 1.

FIG. 18 shows BCG-disA-OE reduces Treg in syngeneic bladder cancer tumors in mice (CD 4)⁺CD25⁺Foxp3⁺). Mouse is at rib abdomen quiltImplantation of 5X 10⁶And BBN975 mouse bladder cancer tumor cells. When the tumor was 1.5cm in diameter, mice received 3 intratumoral injections of PBS (control), BCG-WT or BCG-DISA-OE (treatment every 2 days). Two days after the last intratumoral treatment, mice were sacrificed and their spleens and tumors were removed. After tumor cells were dispersed, the cell preparations were stained and flow cytometry was performed. As can be observed, BCG-disA-OE results in reduced tumor CD4⁺Treg, reduced tumor CD8⁺Treg and reduced spleen CD4⁺Treg。

FIG. 19 shows that BCG-disA-OE is safer than BCG-WT in both mouse models. In Panel A, BALB/c mouse (immunocompetent) groups were exposed to 1X 10 using a Glas-Col nebulization chamber³BCG-WT or BCG-disA-OE of CFU (confirmed by sacrifice of a group of mice and determination of lung CFU count on day 1). After 4 weeks, mice from each group were sacrificed, their lungs removed, homogenized, and plated on 7H11 agar plates. The graph shows the mean CFU counts of the lungs of BCG-WT and BCG-disA-OE infected mice. As can be observed, a statistically significantly lower lung CFU load of BCG-disA-OE compared to BCG-WT was observed. In panel B, SCID mouse (immunosuppressed) groups were exposed to 1X 10 using a Glas-Col nebulization chamber²BCG-WT or BCG-disA-OE of CFU (confirmed by sacrifice of a group of mice and determination of lung CFU count on day 1). The third group was not infected. The graph shows the Kaplan-Meier survival curves for the mouse groups. As can be observed, BCG-disA-OE infected mice have statistically significantly longer survival times than BCG-WT infected mice.

FIG. 20 shows a CD14⁺In human monocytes, BCG-disA-OE elicits statistically significantly higher levels of "Trained immunological and epigenetic markers" (Trained immunological and epigenetic marks) than BCG-WT. By "training immunity" is meant the ability of a first immunostimulatory substance to induce an enhanced immune response to a subsequently administered second, differently antigenic stimulatory substance. In this experiment, CD14⁺Human monocytes were prepared from LeukoPak collected by apheresis. On day 0, CD14 was removed⁺Human monocytes were infected with BCG-WT or BCG-disA-OE at a MOI of 5:1 for 3 hours. The third group of cells was not infected. After infection, cells were washed more than once (every two days). After a resting time (rest period) of 6 days, monocytes were restimulated with the TLR1/2 agonist PAM3CSK4 for 2 hours. The cells were washed repeatedly and then incubated for 24 h. The level of secreted IL-1. beta. in the culture supernatants was measured by ELISA. As can be observed, while BCG-WT by itself elicits a statistically significantly higher level of immune response against the second stimulus, BCG-disA-OE elicits a statistically significantly higher response than either BCG-WT or uninfected cells, as compared to uninfected cells.

FIG. 21 shows that BCG-disA-OE elicits a greater histone activation signature (H3K 4-trimethylation) in the promoter regions of IL6 and TNF genes than BCG-WT. By "training immunity" is meant the ability of a first immunostimulatory substance to induce an enhanced immune response to a subsequently administered second, differently antigenic stimulatory substance. Training immunity is associated with epigenetic modifications such as histone methylation in the promoter regions of cytokines and other immune mediators. The experiment shown in figure 21 was performed in the same cell group and in exactly the same manner as described in figure 20, except that: following a second stimulation with the TLR1/2 agonist PAM3CSK4 (abbreviated PAM3), the fixed cells were harvested, chromatin crosslinked, and DNA was collected for chromatin immunoprecipitation analysis (ChIP) using antibodies specific for the H3K4-me3 histone methylation markers. H3K4-me3 is known as a marker of gene activation. The figure shows the relative fold change in abundance of immunoprecipitated DNA as measured by quantitative PCR using primers for the IL6 and TNF gene promoter regions. As can be observed, both BCG-Pasteur-DISA-OE and BCG-Tice-DISA-OE resulted in significantly greater levels of trimethylation of the H3K4 histone in the IL6 and TNF promoter regions than their corresponding BCG-WT strains, following challenge with the second stimulus PAM3CSK 4.

FIG. 22 shows the successful construction of BCG-Tice-dis-A-OE. Previous work by the present inventors has utilized BCG-Pasteur to construct BCG-Pasteur-dis A-OE. This strain was provided by Frank Collins, doctor 1995 to one of the present inventors. This strain is the same strain known as BCG-Pasteur-Aeras. BCG-Tice is manufactured and sold by Merck and is the only FDA approved BCG available in the United states. The present inventors purchased BCG-Tice, prepared electrocompetent BCG-Tice, and electroporated the pSD5-hsp60-MT3692 plasmid into BCG-Tice. The graph shows the results of colony PCR of 5 kanamycin-resistant candidate clones of transformed BCG-Tice, and confirms that BCG-Tice-disA-OE was successfully prepared by electroporation of the pSD5-hsp65-MT3692 plasmid into BCG-Tice. Taking note of the nomenclature, the present inventors previously referred to this same plasmid, pSD5-hsp60-MT 3692. However, the actual promoter in this strain is the promoter of the hsp65 gene of mycobacterium leprae (m.leprae). Thus, the present inventors now more correctly named the plasmid pSD5-hsp65-MT 3692.

FIG. 23 shows clone 2 of BCG-Tice-DISA-OE from the transformation experiment shown in FIG. 22 strongly expresses the DISA gene. Real-time PCR was used to show differential disA expression in four different BCG-Tice-disA-OE clones. Gene expression was measured in total RNA isolated from log late cultures using log phase cultures using SYBR green based quantitative real-time PCR. The data points illustrated represent the mean ± standard error of the mean (SEM) of 3 independent experiments. Mycobacterium tuberculosis sigA (Rv2703) was used as an internal control. Use 2^-ΔΔCTThe method performs data analysis. Welch correction (. about.P) after student's t-test<0.001；**P<0.01). The inventors generated seed lots of BCG-Rice-DISA-OE clone 2 and abbreviated this clone as "BCG-Rice-DISA-OE" in all subsequent work.

FIG. 24 shows potent, statistically significantly enhanced induction of IRF3 in mouse bone marrow-derived macrophages infected with BCG-Pasteur-disA-OE compared to BCG-Pasteur-WT. Bone marrow derived macrophages from mice (C57BL/6) were infected with wild-type and dis A overexpressing strains of BCG Pasteur (20MOI) for 3 h. Cells were washed with warm DPBS to remove non-internalized bacilli (bacillus) and then incubated for another 3 hours. IRF3 expression was measured in total RNA isolated from cell lysates using SYBR green based quantitative real-time PCR. The data points illustrated represent the mean of 3 independent experiments ±. + -Standard Error of Mean (SEM). Mouse β -actin was used as an internal control. Use 2^-ΔΔCTThe method performs data analysis. Welch correction (. about.P) after student's t-test<0.001；**P<0.01)。

FIG. 25 shows that STING is required for type I IFN (IFN-. beta.) induction in response to BCG-WT and BCG-disA-OE enhancement. Mice from wild-type animals with STING ablation (STING-KO) (C57BL/6) bone marrow-derived macrophages were infected with different BCG strains (MOI ═ 1:20) for 3 h. Cells were washed with warm DPBS to remove non-internalized bacilli and then incubated for an additional 24h before harvesting the culture supernatant. ELISA for IFN- β was performed in culture supernatants as per the manufacturer's instructions. Data points represent the mean ± standard error of the mean (s.e.m.) of 3 independent biological experiments. Students't-test followed by Welch correction (. about.p < 0.01).

FIG. 26 shows that interferon- β is induced in murine BMDM, BMDC and J774.1 macrophages when exposed to a strain of BCG with DISA over-expression, and that the IFN- β response to BCG-Pasteur-DISA-OE and BCG-Tice-DISA-OE is statistically significantly greater than the corresponding BCG-WT strain. Mice (C57BL/6) were infected with bone marrow-derived macrophages (BMDM) and J774.1 macrophages for 3h using different BCG strains (MOI: 20). The non-internalized bacilli were washed with warm DPBS and the cells were incubated for an additional 24 hours. IFN- β levels in culture supernatants were quantified using ELISA according to the manufacturer's instructions. Data points represent 3 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (. about.p < 0.001;. about.p < 0.01;. about.p < 0.05).

FIG. 27 shows that IL-6 was induced in mouse BMDM, BMDC, and J774.1 macrophages in response to exposure to a BCG strain that is over-expressed by DISA, and that the IL-6 response to BCG-Pasteur-DISA-OE and BCG-Tice-DISA-OE was statistically significantly greater than the corresponding BCG-WT strains. Mice (C57BL/6) were infected with bone marrow-derived macrophages (BMDM) and J774.1 macrophages for 3h using different BCG strains (MOI: 20). The non-internalized bacilli were washed with warm DPBS and the cells were incubated for an additional 24 hours. The IL-6 level in the culture supernatants was quantified using ELISA according to the manufacturer's instructions. Data points represent 3 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (. about.p < 0.001;. about.p < 0.01;. about.p < 0.05).

FIG. 28 shows that TNF was induced in mouse BMDM, BMDC, and J774.1 macrophages in response to exposure to strains of BCG overexpressed by DISA, and that the response to BCG-Pasteur-DISA-OE and BCG-Tice-DISA-OE was statistically significantly greater than the corresponding BCG-WT strains. Mice (C57BL/6) were infected with bone marrow-derived macrophages (BMDM) and J774.1 macrophages for 3h using different BCG strains (MOI: 20). The non-internalized bacilli were washed with warm DPBS and the cells were incubated for an additional 24 hours. TNF levels in culture supernatants were quantified using ELISA according to the manufacturer's instructions. Data points represent 3 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (. about.p < 0.001;. about.p < 0.01;. about.p < 0.05).

FIG. 29 shows that TNF and IFN- γ were induced in the rat bladder cancer NBT-II cell line in response to exposure to the BCG strain overexpressed by DISA, and that both responses to BCG-Pasteur-DISA-OE and BCG-Tice-DISA-OE were statistically significantly greater than the corresponding BCG-WT strains. NBT-II cells were infected with wild type and recombinant strains of BCG for 3 h. The non-internalized bacilli were washed repeatedly with warm DPBS and the cells were incubated for an additional 24 h. Culture supernatants were used to quantify TNF and IFN-. gamma.. Data points represent 3 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (P < 0.0001;. P < 0.001;. P < 0.05).

FIG. 30 shows that IFN- β, IFN- γ, TNF, and IL-1 β were induced in the human transitional cell papilloma RT4 bladder cancer cell line in response to exposure to a BCG strain that is overexpressed by dis A, and that the four responses to BCG-Pasteur-dis-A-OE and BCG-Tice-dis-A-OE were greater than the corresponding BCG-WT strains. RT4 cells were infected with wild type and recombinant strains of BCG for 3 h. The non-internalized bacilli were washed repeatedly with warm DPBS and the cells were incubated for an additional 24 h. Culture supernatants were used for the quantification of cytokines according to the manufacturer's instructions. Data points represent 2 independent biological experiments ± standard error of mean (s.e.m.). Data analysis was performed using unpaired t-test (. about.p < 0.001;. about.p < 0.01;. about.p < 0.05).

FIG. 31 shows that BCG-disA-OE stimulates increased IFN- β levels in more than one bladder cancer cell line to a greater extent than BCG-WT. The graph shows the level of IFN- β mRNA (by 2) after exposure to BCG-WT, BCG-disA-OE and LPS^-ΔΔCTRelative expression of the method). 5637 cells are human muscle-layer invasive bladder cancer cells, RT4 cells are human transitional cell papillomatosis bladder cancer cells, and NBT-II cells are rat bladder cancer cells induced by N-butyl-N- (-4-hydroxybutyl) nitrosamine.

FIG. 32 shows the cytokine responses of IFN- β, IFN- γ, IL-6 and TNF in the lungs of BCG-WT and BCG-disA-OE infected mice at different time points following aerosol infection. The figure reveals that the response to BCG-Pasteur-disc-OE and BCG-Tice-disc-OE is greater than for the corresponding BCG-WT strain at most time points for most cytokines. BALB/c mice were infected by aerosol route as described in FIG. 19. Groups of mice were sacrificed at 2,4 and 6 weeks post infection. Lung homogenates were prepared and cytokine levels were quantified using ELISA according to the manufacturer's protocol (n ═ 4 animals/treatment group ± s.e.m.). Data analysis was performed using paired t-tests (. about.p < 0.001;. about.p < 0.01;. about.p < 0.05).

FIG. 33 shows the cytokine response of IFN- β, IFN- γ, IL-6 and TNF in the spleen of BCG-WT and BCG-disA-OE infected mice 4 weeks after aerosol infection. The figure reveals that for most cytokines, the response to BCG-Pasteur-DISA-OE and BCG-Tice-DISA-OE is greater than for the corresponding BCG-WT strains. BALB/c mice were infected by aerosol route as described in FIG. 19. Groups of mice were sacrificed 4 weeks post infection. Spleen homogenates were prepared and cytokine levels were quantified using ELISA according to the manufacturer's protocol (n ═ 4 animals/treatment group ± s.e.m.). Data analysis was performed using paired t-tests (. about.p < 0.001;. about.p < 0.01;. about.p < 0.05).

Fig. 34 shows a method of generating recombinant BCG without antibiotic resistance genes overexpressing STING-agonists. In the given example, a genetically modified BCG without antibiotic resistance gene was created to overexpress the BCG disA gene and release excess c-di-AMP (a known STING agonist). However, the same strategy can be used to overexpress Rv1354c (diguanylate cyclase producing another known STING agonist, c-di-GMP), DncV (cyclic GMP-AMP synthase from Vibrio cholerae producing another known STING agonist, 3'-3' -cGAMP), cGAS (cyclic GMP-AMP synthase from human producing another known STING agonist, 2'-3' cGAMP), or another similar enzyme gene producing a STING agonist.

In step 1, the BCG-WT strain was transformed by electroporation of plasmid pJV53(SEQ ID NO:32) and selection on 7H11 agar plates containing kanamycin. pJV53 carries the gp60 and gp61 genes from the Mycobacterium phage Che9c, which encode homologues of RecE and RecT, respectively. The Che9c gp60 and gp61 encode exonuclease and DNA binding activity, respectively, and expression of these proteins significantly increases the homologous recombination capability of mycobacteria (homologous recombination proficiency). Step 1 produced "recombination-competent BCG (recombination proficient BCG)" as shown in the figure. After confirming the transformants, positive clones will be amplified in the presence of kanamycin and will be used for the preparation of electrocompetent cells. To prepare these electrocompetent cells, the bacteria were grown to mid-log phase, induced (up-regulated expression of the Che9c gp60 and Che9c gp61 recombinant engineered genes) with 0.2% acetamide for 24 hours, and then prepared into electrocompetent state.

In step 2, a linearized Allele Exchange Substrate (AES) construct corresponding to SEQ ID NO 33 was generated. AES (SEQ ID NO:33) contains the dif-Hyg-dif cassette (SEQ ID NO:34) sequence flanked on one side by the 5 'UTR of the 500bp panCD operon and on the other side by the 3' UTR of the 500bp panCD operon. AES (SEQ ID NO:33) was constructed by cloning the 5 'UTR of the 500bp panCD operon and the 3' UTR of the 500bp panCD operon into a pUC-Hyg plasmid (SEQ ID NO:35) to generate a plasmid pUC-Hyg-panCD-KO (SEQ ID NO: 36). pUC-Hyg-panCD-KO (SEQ ID NO:36) was cleaved by digestion with NruI and NcoI to generate linear AES corresponding to SEQ ID NO: 33. Alternatively, the primers SEQ ID NO:28 and SEQ ID NO:29 can be used to amplify linear AES (SEQ ID NO: 33). The linearized Allele Exchange Substrate (AES) construct corresponding to SEQ ID NO:33 was then electroporated into "recombinationable BCG" and clones were selected on 7H11 agar plates containing hygromycin and kanamycin. This procedure resulted in a "BCG carrying the panCD KO cassette" in which AES (SEQ ID NO:33) had been integrated by homologous recombination into the panCD operon of the chromosome.

In step 3, several hundred colonies of "BCG carrying the panCD KO cassette" were replica plated on both (i) 7H11 agar plates containing kanamycin and (ii) 7H11 agar containing kanamycin supplemented with pantothenate (24. mu.g/ml). Kan-resistant pantothenate auxotrophic clones were selected that grew only on kanamycin-containing 7H11 agar supplemented with pantothenate (24. mu.g/ml) but not on kanamycin-containing 7H11 agar plates lacking pantothenate. During this step, the natural action of the mycobacterium Xer recombinase, which recognizes and recombines at the dif site, results in the excision and deletion of the hygromycin cassette of the clone that is "pantothenate auxotrophic for pJV 53-bearing" as shown.

In step 4, "pantothenate auxotrophs carrying pJV 53" were plated on sucrose-containing 7H11 agar plates to select for deletions of pJV53 carrying the sacB gene (conferring lethality in the presence of sucrose). Sucrose tolerant clones were selected and confirmed to be kanamycin sensitive. This resulted in a clone that was "pantothenate auxotrophic without pJV 53".

In step 5, an electrocompetent "pantothenate auxotroph without pJV 53" was prepared. Plasmid "pSD 5.phsp65-dis A. panCD-Kan-free" (SEQ ID NO:31) was generated as described in FIG. 36. 31 was electroporated into "pantothenate auxotrophs without pJV 53" and clones were plated on pantothenate-free 7H11 agar to generate the desired "pantothenate auxotrophs carrying a dis-OE plasmid". Candidate clones were confirmed by PCR of the relevant gene and by whole genome sequencing.

FIG. 35 shows the molecular structure of a DNA fragment containing the panCD Allele Exchange Substrate (AES), which is SEQ ID NO: 33.

FIG. 36 shows the strategy used to generate "pSD5. hspC 65-dis A. panCD- -without Kan" (SEQ ID NO: 31). This protocol replaces the Kan cassette "pSD5.hsp65-dis A. Kan" (SEQ ID NO:30) with the panCD operon to produce "pSD5.hsp65-dis A. panCD- -without Kan" (SEQ ID NO: 31).

FIG. 37 shows the molecular structure of pJV53, pJV53 is a recombinant engineered plasmid of SEQ ID NO: 32.

FIG. 38 shows the molecular structure of pUC-Hyg, a plasmid with the Hyg cassette flanked by dif sites, as SEQ ID NO 35. pUC-Hyg was used to generate the plasmid "pUC-Hyg-panCD-KO" (SEQ ID NO: 36).

FIG. 39 shows the molecular structure of the plasmid "pUC-Hyg-panCD-KO" as SEQ ID NO: 36. "pUC-Hyg-panCD-KO" was generated by cloning 500bp of the panCD 5 'UTR on one flank of the Hyg cassette and 500bp of the panCD 3' UTR on the other flank.

FIG. 40 shows the molecular structure of plasmid "pSD5.hsp65-dis A. Kan" as SEQ ID NO: 30.

FIG. 41 shows the molecular structure of the plasmid "pSD5.hsp65-dis A. panCD-Kan-free" set forth as SEQ ID NO: 31. This plasmid was generated using the protocol illustrated in figure 36.

Figure 42 shows some nucleic acid and protein sequences for use in the present invention.

Figure 43 shows a description of nucleic acid and protein sequences for use in the present invention.

Fig. 44 shows the number of positive samples.

Detailed Description

In some embodiments, the invention relates to genetic alterations of mycobacterium bovis BCG (hereinafter, "BCG"), which result in recombinant BCG (hereinafter, "rBCG") strains. These strains have greater potential as (i) tuberculosis vaccines and/or (ii) immunotherapy for non-muscle invasive bladder cancer (NMIBC). Some embodiments of the invention relate to BCG strains that synthesize and secrete high levels of Cyclic Dinucleotides (CDNs), which are known to elicit valuable immunomodulatory responses from human phagocytic cells such as macrophages, dendritic cells, and other cells. Another embodiment of the invention is to combine genetic modifications of BCG to generate multivalent CDN-overexpression modifications, including the addition of new genetic material to synthesize CDN and/or mutations of endogenous BCG phosphodiesterase genes or genetic domains that will enhance the accumulation and release of CDN.

BCG

BCG (BCG vaccine) is a mutant form of M.bovis produced by Calmette and Guerin, French microbiologist, in 1921 by 13 years of serial passage of virulent M.bovis. Between 1921 and 1960, BCG was serially passaged in many laboratories around the world until defined seed lots were established and stored in the reference laboratory. Thus, many variations of BCG exist worldwide, such as BCG Pasteur, BCG Tice, BCG Tokyo, BCG Danish, BCG Montreal, and others. Most existing BCG strains have now been determined by whole genome sequencing. The major differences between virulent mycobacterium bovis and various BCG strains include the deletion of at least 15 distinct regions in BCG that contain genomic deletions compared to virulent mycobacterium tuberculosis. The key differential regions in BCG development were RD1 (a 9.5kb deletion leading to loss of the Esx-1 secretion system and inability to release the antigens ESAT-6 and CFP-10) and RD3 (a 9.2kb deletion). Compared with virulent Mycobacterium tuberculosis, no RD4-RD11 differential region exists in all BCG strains.

BCG has been used as a vaccine for the prevention of Tuberculosis (TB) since the 20 th 20 s of the 20 th century. In 2004, BCG was estimated to be given to about 1 million children, so since its first time put into use, BCG has been given to about 50 million people, and is therefore the most widely used vaccine historically. The most common is intradermal administration at birth and to date it is still administered in most countries except in the united states, canada and parts of europe. BCG has been shown to reduce the incidence of disseminated TB in children, but individuals vaccinated with BCG are not fully protected from the risk of TB.

BCG has also gained widespread use since 1977 as a cancer immunotherapy for non-muscle invasive bladder cancer (NMIBC). BCG is administered intravesically weekly for six weeks, and in some cases such as high risk illness, at 3 months, 6 months, 12 months, 18 months, 24 months, 30 months and 36 months after the initial therapy, BCG is administered weekly as a maintenance therapy for three weeks. Intravesical bcg has been shown to (i) induce infiltration of monocytes comprising primarily CD 4T cells and macrophages, (ii) increase expression of interferon gamma (IFN γ) in the bladder, and (iii) increase levels of urinary cytokines IL-1, IL-2, IL-6, IL-8, IL-12, IFN γ, and TNF α.

Although BCG is widely used globally as (i) a vaccine for TB and (ii) an immunotherapy for NMIBC, there is considerable room for improvement in its efficacy. For TB, BCG provides only partial protection primarily against disseminated tuberculosis in children. For NMIBC, about 30% of patients suffer from BCG-resistant disease. These individuals require higher risk treatment with systemic chemotherapy and have a higher rate of bladder cancer progressing to a more invasive form.

Urothelial cancer

Urothelial cancer of the bladder is the most common malignancy of the urethra. Urothelial cancer is the fourth most common cancer in men and the eleventh most common cancer in women. It is estimated that about 79,000 new cases of bladder cancer will be diagnosed in the us in 2017, associated with 19,870 deaths. Although the estimated five-year survival rate for patients with bladder cancer is 78%, the survival rate drops significantly for patients with locally advanced or metastatic disease. Approximately 75% of patients with bladder cancer present with a disease that is restricted to the mucosa (stage Ta, carcinoma in situ) or submucosa (stage T1), known as non-muscle invasive bladder cancer (NMIBC). Transurethral resection is the initial treatment of choice for NMIBC. For patients with muscle-invasive bladder cancer (MIBC; T2 or later), the first line treatment option was platinum-containing chemotherapy followed by bladder removal. For those patients with NMIBC who do not respond to intravesical treatment, there is a high risk of developing MIBC. Thus, a high recurrence rate and significant risk of progression require the administration of additional therapies. Therefore, improving the clinical outcome of patients with high risk NMIBC requires the development of new treatments.

Intravesical administration of Bacillus Calmette Guerin (BCG) developed for NMIBC in the 70's of the 20 th century provided the first successful immunotherapy against established solid cancers, and it is still the standard of care for patients with NMIBC. (activated EJ, Newton MR, O' Donnell MA, Luo Y. Black Cancer Immunotherapy: BCG and beyond. adv Urol.2012; 2012:181987.PMID:22778725.Morales A. BCG: A thwbacterium from the stone of cosmetics extruded the path for the blade Cancer Immunotherapy. Can J Urol.2017; 24:8788-8793.PMID: 28646932). The exact mechanism of the antitumor effect of the attenuated strain BCG of M.bovis is not known, but it is believed that BCG, after binding to the urothelium via fibronectin and integrin α 5 β 1, coordinates strong immune cellular and humoral immune responses, mainly Th1 responses (Redelman-Sidi G, Glickman MS, Bochner BH. the mechanism of action of BCG therapy for the tablet holder- -a current therapeutic. Nat Rev Urol. 2014; 11:153-62.PMID: 24492433). However, BCG treatment typically has a complete response rate of 55-65% to papillary tumors and 70-75% to Carcinoma In Situ (CIS). (activated EJ, Newton MR, O' Donnell MA, Luo Y. Black Cancer Immunotherapy: BCG and beyond. adv Urol.2012; 2012:181987.PMID:22778725.Morales A. BCG: A thwbacterium from the stone of cosmetics extruded the path for the blade Cancer Immunotherapy. Can J Urol.2017; 24:8788-8793.PMID: 28646932). Thus, patients with BCG unresponsive and relapsing disease and the burden on treatment-intolerant patients has prompted a need to further improve the efficacy of BCG against NMIBC.

CDNs are important PAMPs and DAMPs that generate valuable immune responses against TB and NMIBC.

Bacterial pathogen-associated molecular patterns (PAMPs).Human cells utilize an innate immune monitoring system called the cytoplasmic monitoring program (CSP) to detect nucleic acids, including cyclic dinucleotides, in the cytoplasm. CSP, originally characterized as a viral defense system, has now been shown to be important in the defense against bacteria, particularly against intracellular bacteria such as mycobacterium tuberculosis, Listeria monocytogenes (Listeria monocytogenes), Salmonella (Salmonella) species and other bacteria. Cytoplasmic Pattern Recognition Receptors (PRRs), including STING, cGAS, DDX41 and many others, are capable of binding to cytoplasmic CDN and nucleic acids leading to their activation. A key signaling event is STING activation, which leads to activation of TBK1 and IRF3 and subsequent upregulation of type I interferon expression. STING activation by cyclic dinucleotides also leads to induction of STAT6, STAT6 induces chemokines such as CCL2 and CCL20 independent of TBK1-IRF3 pathway. STING activation is also thought to activate the transcription factor NF κ B through I κ B kinase (IKK) activation.

Human risk associated molecular patterns (DAMPs).Cyclic cgamp (cgas) synthase is a cytoplasmic PRR that recognizes cytoplasmic DNA. Cyclic cGAMP (cgas) synthase undergoes a conformational change upon binding to DNA, which activates its core enzyme activity, catalyzing the formation of 2'3' cGAMP. 2'3' cGAMP followed by a potent DAMP that activates the STING-TBK1-IRF3 axis (axis), resulting in increased expression of type 1 interferons, as well as STAT6 activation and IKK activation.

Mechanism of STING-mediated CDN-triggered immune responses.Type I IFNs produced by both innate immune cells in the tumor microenvironment and by the tumor cells themselves are known to mediate anti-tumor effects against several malignancies because of their ability to intervene in all stages of cancer immune editing. (Zitvogel L, Galluzzi L, Kepp O, Smyth MJ, Kroemer G. type I interferons in anticipator immunology. Nat Rev immunol. 2015; 15:405-14.PMID: 26027717). STING (stimulator of the interferon gene), a major regulator of the type I IFN innate immune response to pathogens after recognition of solute DNA by the sensor cyclic GMP-AMP synthase (cGAS). cGAS catalyzes the synthesis of cyclic GMP-AMP (cgamp), which in turn functions as a second messenger that binds to and activates STING. (ZHao GN, Jiang DS, Li H. Interferon regulation factors: at the cross roads of immunity, metablism, and disease. Biochim Biophys acta.2015; 1852:365-78.PMID: 24807060). Therefore, novel anti-cancer immunotherapies based on recombinant type I IFNs, type I IFN-encoding vectors, type I IFN-expressing cells, and STING agonists are currently being developed as novel tumor immunotherapies.

Overexpression of the PAMP immunomodulator 3'-5' c-di-AMP.3'-5' c-di-AMP is a strong inducer of STING-TBK1-IRF3 axis. 3'-5' c-di-AMP is produced by mycobacteria including BCG by encoding the DisA protein (in BCGThe BCG protein WP _010950916.1, the Mycobacterium tuberculosis protein Rv3586 or P9WNW5.1). Mycobacterium tuberculosis (M.tb) synthesizes and secretes c-di-AMP, which activates the Interferon Regulatory Factor (IRF) pathway and type I IFN response via STING signaling and cGAS. (Ahmed D, Cassol E.role of cellular metabolism in regulating type I interference responses: antibiotics for tumor immunology and treatment. cancer Lett.2017; 409:20-29.PMID: 28888999). C-di-AMP over-expressed Mycobacterium tuberculosis strains show a reduction of TB in a mouse model. As a mucosal adjuvant, c-di-AMP exerts an immunostimulatory effect, leading to maturation of dendritic cells, up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines, and a strong Th1, Th17 and CD 8T cell response against pathogens. The BCG strain with c-di-AMP over-expression (rBCG-disA or BCG-disA-OE) has been constructed and surprisingly found to produce significantly higher IRF and IFN- β responses than BCG itself, indicating that bacterially derived c-di-AMP enters the host cell cytoplasm despite the absence of the ESX-1 protein secretion system. (Ahmed D, Cassol E.role of cellular metabolism in regulating type I interference responses: antibiotics for tumor immunology and treatment. cancer Lett.2017; 409:20-29.PMID: 28888999). These findings indicate that rBCG strains modified to overexpress c-di-AMP can induce better protective immunity against bladder tumors than BCG itself.

Induction of pro-inflammatory Th1 cytokines in mouse bone marrow-derived macrophages (BMDM) in response to BCG overexpressing mycobacterium tuberculosis disA (MT 3692): the Mycobacterium tuberculosis genome encodes the diadenosine cyclase (DisA, also known as DacA, P9WNW5.1 in the UniProtKB/Swiss-Prot database) for the synthesis of c-di-AMP from ATP or ADP. BCG protein WP _010950916.1(NCBI reference number) is 100% identical to M.tuberculosis DisA. Mycobacterium tuberculosis strains that overexpress DISA poison macrophages by releasing excess c-di-AMP, a unique bacterial PAMP that activates STING-dependent IFN- β production. (Ahmed D, Cassol E.role of cellular metabolism in regulating type I interference responses: antibiotics for tumor immunology and treatment. cancer Lett.2017; 409:20-29.PMID: 28888999). To enlarge or notAntigenic pool of pathogenic vaccine strains (antigeneic reporters), BCG Pasteur transformed with a plasmid conferring kanamycin resistance (Kan-R) carrying a strong mycobacterial promoter P_hsp60Fused dis A genes from M.tuberculosis (M.tuberculosis Rv3586 or MT3692) (the dis A genes of M.tuberculosis and BCG are 100% identical). Addition of this plasmid to BCG-Pasteur increased the levels of dis A mRNA by 50-fold (FIG. 1 b). The closely related M.tb-disA-OE strain released 15-fold more c-di-AMP into the macrophage cytoplasm compared to wild-type M.tuberculosis (FIG. 1a), and thus BCG-disA-OE was expected to release significantly more c-di-AMP into the host cytoplasm as well. These DISA overexpressing recombinants (rBCG or BCG-DISA-OE) are better STING-dependent IFN- β inducers than the parental strain. Most importantly, as reported in PCT/US2016/017248 filed 2/10 in 2016, the rBCG-inoculated guinea pigs were significantly better protected from aerosol infection by virulent mycobacterium tuberculosis, indicating an improved protective efficacy compared to existing BCG strains.

As shown in FIG. 2, the immune response elicited by BCG-Pasteur disA-OE was tested in an in vitro macrophage infection model. BMDM from C57BL/6 mice infected with BCG-Pasteur disA-OE showed significant upregulation of IFN- β, TNF- α, IL-6 and IL-2 compared to uninfected macrophages or wild type BCG infected macrophages.

As shown in FIG. 3, enhanced c-di-AMP-based STING activation in RAWBlue ISG macrophages was confirmed. RAWBlue macrophages showed increased levels of IRF3 when infected with BCG-Pasteur disA-OE compared to parental controls.

As shown in FIG. 4, a significant increase in secreted pro-inflammatory cytokines (TNF-. alpha., IL-6, and IL-1. beta.) was found in the culture supernatant of BMDM from BCG-Pasteur-DISA-OE infected mice. These findings indicate that BCG-Pasteur-disA-OE with an increased repertoire of antigens behaves like a STING agonist and is therefore a potent inducer of STING-dependent type I IFN. Furthermore, the immune response in macrophages in response to BCG-Pasteur disA-OE tended to be Th1, Th1 being a phenotype largely responsible for the control of NMIBC by BCG immunotherapy.

As shown in FIG. 5, BCG-disA-OE elicited anti-tumor immune responses in human bladder cancer (RT4) cells. BCG-Pasteur-disA-OE was tested to determine whether BCG-Pasteur-disA-OE elicited a similar immune response in Bladder Cancer (BC) cells as compared to WT strains BCG-Pasteur and Oncotice (the current immunotherapeutic BCG strain). Human RT4 BC cells derived from human NMIBC tumors were challenged with wild-type (both Pasteur and TICE) strains and recombinant BCG Pasteur dis A-OE strain at 1:20(RT4:: BCG) for 3h, and differential gene expression profiles were determined compared to uninfected cells. It was found that key immune mediators such as monocyte chemotactic protein 1(MCP-1)/CCL2, IFN- β and IL-1 β were significantly increased in bladder cancer cells exposed to BCG-Pasteur-dis A-OE compared to the response to the wild type strain.

As shown in fig. 6, an experimental system was established to test whether intravesical BCG-dis-a-OE immunotherapy resulted in an elevated Th1 response and anti-tumor efficacy in the MNU oncogenic model of NMIBC. The results from the above mentioned experiments using RT4 cells encouraged the present inventors to test the relative therapeutic efficacy of BCG-Pasteur disA OE in the rat NMIBC model in vivo, which was pioneered in the Bivalacqua laboratory. (Kates M, Nirschl T, Sopko NA, Matsui H, Kochel CM, Reis LO, Netto GJ, Hoque MO, Hahn NM, McConkey DJ, Baras AS, Drake CG, Bivalacqua TJ. intragenic BCG indexes CD4(+) T-Cell Expansion in an Immunol Module of blower cancer. cancer Immunol Res.2017; 5:594-603.PMID: 28588015). In this model, an oncogenic alkylating agent, N-methyl-N-nitrosourea (MNU), was used to induce urothelial cancer in female Fischer rats.

As can be observed in fig. 7, BCG-disA-OE had significant immunotherapeutic effects in the rat bladder cancer model. Urothelial dysplasia develops within 8 weeks of MNU instillation, and by 16 weeks after the first instillation, all rats show carcinoma in situ, papillary Ta or high grade T1 urothelial carcinoma with histopathological and immunophenotypic characteristics similar to those observed in human urothelial carcinoma. Using this model, Bivalacqua laboratories showed that intravesical BCG immunotherapy resulted in CD4 in the urothelium⁺T cell populationA large transient rise in volume. (Kates M, Nirschl T, Sopko NA, Matsui H, Kochel CM, Reis LO, Netto GJ, Hoque MO, Hahn NM, McConkey DJ, Baras AS, Drake CG, Bivalacqua TJ. intragenic BCG indexes CD4(+) T-Cell Expansion in an Immunol Module of blower cancer. cancer Immunol Res.2017; 5:594-603.PMID: 28588015). Intravesical instillation of BCG-disA-OE strain was performed in MNU-treated rats starting 8 weeks after MNU induction when tumors were visible, and was administered continuously weekly for 6 weeks. Bladder tumors were staged by GU pathologists according to WHO-ISUP classification, and percent tumor involvement (sum of Ta, T1 and CIS) was calculated for each group according to criteria as described. (Kates M, Nirschl T, Sopko NA, Matsui H, Kochel CM, Reis LO, Netto GJ, Hoque MO, Hahn NM, McConkey DJ, Baras AS, Drake CG, Bivalacqua TJ. intragenic BCG indexes CD4(+) T-Cell Expansion in an Immunol Module of blower cancer. cancer Immunol Res.2017; 5:594-603.PMID: 28588015). It was found that the tumor involvement index in rats treated with BCG-Pasteur disA-OE was significantly reduced compared to the bladder from untreated rats or BCG-Pasteur treated rats.

As can be observed in FIG. 8, BCG-disA-OE induced characteristic cytokine and chemokine signatures in rat bladders undergoing immunotherapy (pathological cytokine and chemokine signature). The urinary bladder of rats from rats treated with BCG-disA-OE showed significant induction of IFN-. alpha./β, IFN-. gamma., IL-1. beta., TNF-. alpha., TGF-. beta., iNOS, IP-10, MCP-1 and MIP-1. alpha. compared to untreated rats or BCG-Pasteur treated rats.

As shown in FIG. 9, CCL2 was found in the bladder of rats treated with BCG-Pasteur-DISA-OE⁺Macrophages, Nos2⁺And IL-1. beta⁺Evidence of increased infiltration of M1 macrophages, which is accompanied by increased IL-6 and IFN-expression. Interestingly, increased levels of IP-10 were found, which together with increased IFN- γ was known to promote strong T cell recruitment at the site of infection and inflammation.

FIG. 10 shows a summary of the observed changes in cytokine expression levels of BCG-disA-OE versus BCG-WT in primary cells, cancer cell lines, and in rat bladder cancer tissue. As can be observed, BCG-disA-OE significantly upregulated cytokines associated with Th 1T cell and M1 macrophage expansion, two type 1 interferons, and three proinflammatory chemokines (2-fold to 30-fold) in these cells, cell lines, and tissues compared to BCG-WT. In contrast, BCG-disA-OE generally down-regulated cytokines (1-fold to 10-fold) associated with Th 2T cell and M2 macrophage expansion compared to BCG-WT.

As shown in fig. 11, BCG immunotherapy may be effective via three immune mechanisms: (i) increased production of tumor-specific cytotoxic CD 8T cells, (ii) a cytokine milieu that promotes macrophage-mediated activation of CD4 cells against tumor antigens, and (iii) macrophage M1 conversion that promotes enhanced tumoricidal activity. The findings reported herein strongly suggest that BCG overexpressing c-di-AMP is taken up by bladder tumor cells and myeloid cells, these cells reside in or are recruited to the tumor microenvironment, and that BCG induces activation of host immune responses, including STING and type I IFN responses, as well as NF- κ B signaling that promotes secretion of cytokines and chemokines, macrophage recruitment, and apoptotic mechanisms, all of which collectively reduce tumor progression.

As shown in figure 12, in addition to overexpression of dis a resulting in increased levels of the PAMP molecule c-di-AMP, there are additional recombinant DNA modifications that can be made to BCG to enhance the production of other PAMPs and DAMP molecules. As shown in FIG. 12, the genes for other CDN cyclases (i) the BCG _ RS07340 protein or the GGDEF domain of the Mycobacterium tuberculosis Rv1354c protein (which are 100% identical to each other), (ii) the Vibrio cholerae DncV protein, which is Q9KVG7 in Swiss-Prot, which is 2 '-5' c-GAMP synthase, and (iii) the human cGAS protein Q8N884 in Swiss-Prot, which is 2'-3' cGAMP synthase- -can be added to BCG. These CDN cyclase genes added may be added individually or in combination. Such a combination would represent a multivalent CDN over-expressing BCG. Furthermore, as shown in fig. 13, BCG has several CDN phosphodiesterase genes or genes containing phosphodiesterase domains. The following endogenous phosphodiesterase genes and intragenic phosphodiesterase domains are removed using recombinant techniques: (i) the BCG WP _003414507 gene encoding a CDN PDE in BCG that is 100% identical to mycobacterium tuberculosis Rv2837c (also known as CdnP or CnpB), (ii) DNA encoding the EAL domain of the protein BCG RS07340 (previous BCG _1416c) that is 100% identical to the known PDE mycobacterium tuberculosis Rv1354c protein, and (iii) a gene encoding BCG AHM07112 that is homologous to the known PDE mycobacterium CDN Rv1357 c. Removal of the genes encoding these PDEs would serve to further increase the levels of CDN PAMP and DAMP molecules produced by the rBCG strains disclosed herein.

SEQ ID NO:1

The diadenosine cyclase DISA from BCG and other related Mycobacteria, amino acid sequence (358 amino acids; BCG protein AOQ92_ RS 18745; NCBI reference sequence: NZ _ CUWL 01000001.1). The same sequence is present in other BCG strains, for example in mycobacterium tuberculosis as protein Rv3586 or MT3692, and m.bovis as protein Mb 3617.

SEQ ID NO:2

The DNA sequence of the diadenosine cyclase DISA from BCG and other related mycobacteria (1077 nucleotides [358 codons, 1 stop codon ]; coding BCG gene AOQ92_ RS 18745; NCBI reference sequence: NZ _ CUWL 01000001.1). The same sequence is present in other BCG strains, for example in mycobacterium tuberculosis as gene Rv3586 or MT3692 and m.bovis as gene Mb 3617.

SEQ ID NO:3

Plasmid pSD5B-P_hsp60Disco is an episomally replicating E.coli (E.coli) -mycobacterial shuttle plasmid, derived from P_hsp60The promoter overexpresses BCG disA gene, DNA sequence (7742 nucleotides; containing Mycobacterium leprae hsp65 gene nucleotides 13 to 180, promoter P_hsp60The DNA is underlined; nucleotides 242 to 1318 of a dis coding sequence; the ATG start codon and TAA stop codon are shown in bold, underlined).

Mycobacteria that overexpress dis a are attenuated. As shown in FIG. 14, 3.5log was used when passing through the aerosol route₁₀Unit carrying pSD5B P_hsp60When mice are infected with Mycobacterium tuberculosis of dis A plasmids (M.tb-dis A-OE or Mtb-OE) or wild type Mycobacterium tuberculosis (Mtb-CDC1551), the median time to death (MTD) of the animals is obviously different. As can be observed, the wild type Mycobacterium tuberculosis (Mtb-CDC1551) gave an MTD of 150.5 days, while carrying pSD5B P_hsp60Mycobacterium tuberculosis (M) of the disA plasmid.tb-disA-OE or Mtb-OE) is a significantly weaker pathogen, giving an MTD of 321.5 days. A similar reduction in the pathogenicity of BCG-disA-OE is expected compared to BCG-WT. Thus, it is likely that if BCG-disA-OE is used as a cancer immunotherapy, one would expect a reduced rate of blood flow dissemination, reduced dysuria, reduced urgency and reduced fatigue compared to BCG-WT.

Addition of CDN cyclase genes other than DISA to rBCG

The PAMP immunomodulator 3'-5' c-di-GMP was overexpressed by overexpressing the GGDEF domain of the protein BCG _ RS07340 Expression of. 3'-5' c-di-GMP is a strong inducer of STING-TBK1-IRF3 axis. 3'-5' c-di-GMP is produced by mycobacteria (including BCG) via the GGDEF domain of the protein BCG _ RS07340 (formerly BCG _1416c) and via the Mycobacterium tuberculosis Rv1354c gene. BCG _ RS07340 protein (100% identical to mycobacterium tuberculosis Rv1354c protein) encodes a bifunctional diguanylate cyclase/diguanylate phosphodiesterase. Thus, part of functioning as diguanylate cyclase is the endogenous CDN-producing enzyme in BCG. The full-length BCG _ RS07340 polypeptide is 623 amino acids in length and has a domain structure of: N-terminal-GAF-GGDEF-EAL-C-terminal, as shown in FIG. 11. The GAF domain (about amino acids 1-190) is a regulatory domain that affects the activity of other domains. The GGDEF domain (about amino acid 190-350) is a diguanylate cyclase that catalyzes the reaction 2GTP → c-di-GMP +2 pyrophosphate. The EAL domain (about amino acids 350-. By genetically removing the DNA sequence encoding the C-terminal EAL domain, DNA encoding the GGDEF domain can be used to generate recombinant BCG that will overexpress diguanylate cyclase activity. This can be accomplished by also deleting the DNA encoding the regulator-sensor GAF domain and/or using mutations in the DNA encoding the GAF domain to mitigate any cyclase inhibitory activity it may have. Such a technique to produce a constitutively active recombinant form of the BCG RS07340 protein will produce high levels of c-di-GMP in the recombinant BCG.

SEQ ID NO:4

Bifunctional diguanylate cyclase/phosphodiesterase BCG _ RS07340 from BCG and other related mycobacteria, amino acid sequence (623 amino acids; BCG protein BCG _ RS 07340; NCBI reference sequence: NC-008769.1; protein ID WP 003898837.1; old locus tag BCG-1416 c). The same sequence is present in other BCG strains, for example in mycobacterium tuberculosis as protein Rv1354c or MT1397, and m.bovis as protein Mb1389 c. The EAL domain is amino acids 354 to 623 and is underlined.

SEQ ID NO:5

Bifunctional diguanylate cyclase/phosphodiesterase BCG RS07340 from BCG and other related Mycobacteria, DNA sequence (1872 nucleotides [623 codons +1 stop codon ]; encoding BCG protein BCG RS 07340; NCBI reference sequence: NC-008769.1; protein ID WP 003898837.1; old locus tag BCG-1416 c; DNA from NC-008769.1: c1548390-1546519 Mycobacterium bovis BCG Pasteur 1173P 2). The same sequence is present in other BCG strains, for example in mycobacterium tuberculosis as protein Rv1354c or MT1397, and m.bovis as protein Mb1389 c. The EAL domain is encoded by nucleotides 1060 to 1872 and is underlined.

SEQ ID NO:6

Modified bifunctional diguanylate cyclase/phosphodiesterases from BCG and other related mycobacteria, whose EAL domain is deleted such that it functions as a monofunctional diguanylate cyclase, amino acid sequence (353 amino acids; fragment of BCG protein BCG _ RS 07340; NCBI reference sequence: NC _ 008769.1; protein ID WP 003898837.1; old locus tag BCG _1416 c). The same sequence fragments are present in other BCG strains, for example in mycobacterium tuberculosis as proteins Rv1354c or MT1397, and m.bovis as protein Mb1389 c.

SEQ ID NO:7

Modified bifunctional diguanylate cyclase/phosphodiesterases from BCG and other related mycobacteria, wherein the sequence encoding its EAL domain is deleted such that it encodes a monofunctional diguanylate cyclase, the DNA sequence (1059 nucleotides [353 codons +0 stop codons ]; fragment encoding BCG protein BCG _ RS 07340; NCBI reference sequence NC-008769.1; protein ID WP 003898837.1; old locus tag BCG _1416 c; DNA from NC-008769.1: c1548390-1546519 Mycobacterium bovis BCG Pasteur 1173P 2). The same sequence is present in other BCG strains, for example in mycobacterium tuberculosis as a fragment of the gene Rv1354c or MT1397, and in mycobacterium bovis as a fragment of the gene Mb1389 c.

Overexpression of the PAMP immunomodulator 2 '-5' c-GAMP synthase: q9KVG7 (Swiss-Prot).2 '-5' c-GAMP is a strong inducer of STING-TBK1-IRF3 axis. The Vibrio cholerae Q9KVG7 protein (436 amino acids) encoded by the dnCV gene is a known 2 '-5' c-GAMP synthase. Recombinant dncV genes optimized for BCG codons can be generated. Codon-optimized structural genes can be overexpressed in BCG by fusion to strong promoters (such as Phsp60) or conditionally active strong promoters such as PTET-off. Such a technique to produce a constitutively active recombinant form of the Q9KVG7 protein would produce high levels of 2 '-5' c-GAMP in recombinant BCG.

SEQ ID No:8

The cyclic GMP-AMP synthase, DncV, from Vibrio cholerae, amino acid sequence (436 amino acids; UniProtKB/Swiss-Prot protein ID Q9KVG7.1).

SEQ ID No:9

Circular GMP-AMP synthase from Vibrio cholerae, DncV, DNA sequence (1311 nucleotides [436 codons +1 stop codon ]; encoding UniProtKB/Swiss-Prot protein ID Q9KVG7.1; NCBI reference sequence: NC-002505.1: Vibrio cholerae O1 biovar Eltor str. N16961 chromosome I, complete sequence, and nucleotides 180419-

Overexpression of the DAMP immunomodulator 2'-3' cGAMP synthase: Q8N884(Swiss-Prot). 2'-3' cGAMP is a strong inducer of STING-TBK1-IRF3 axis. The cGAS protein is produced by the human cGAS gene to produce a 522 amino acid polypeptide that senses cytosolic DNA and functions as a 2'-3' cGAMP synthase. When cGAS binds to DNA, the synthase or cyclase domain of cGAS becomes activated. It is possible to produce recombinant cGAS genes which contain only the cyclase domain and are therefore constitutively active. The recombinant gene can also be codon optimized for BCG. Codon-optimized structural genes can be overexpressed in BCG by fusion to strong promoters (such as Phsp60) or conditionally active strong promoters such as PTET-off. Such a technique to generate constitutively active recombinant forms of cGAS protein would produce high levels of 2'-3' c-GAMP in recombinant BCG.

SEQ ID No:10

Cyclic 2'3' -GMP-AMP synthase cGAS from homo sapiens, amino acid sequence (522 amino acids, UniProtKB/Swiss-Prot protein ID Q8N884.2).

SEQ ID No:11

Circular 2'3' -GMP-AMP synthase cGAS from homo sapiens, DNA sequence of mRNA with nucleotide T replacing U (1802 nucleotides; encoding UniProtKB/Swiss-Prot protein ID Q8N884.2; NCBI reference sequence: NM-138441.2. encoding sequence 1569 nucleotides [522 codons, 1 stop codon ], start codon ATG [ bold, underlined ] at nucleotide 140, stop codon TGA (bold, underlined) at nucleotide 1706).

SEQ ID NO:12

Cyclic 2'3' -GMP-AMP synthase cGAS from homo sapiens, with mycobacterial codon optimization, DNA sequence. (1569 nucleotides [522 codons, 1 stop codon ]; encoding the UniProtKB/Swiss-Prot protein ID Q8N884.2).

SEQ ID NO:13

Plasmid pMH94H-P_hsp60hcGASCo mCherry is a shuttle plasmid of Escherichia coli-mycobacterium_hsp60The promoter overexpresses the BCG disA gene, the human cGAS gene (with mycobacterial codon optimization) and mCherry, DNA sequences. When introduced into BCG, Mycobacterium tuberculosis, Mycobacterium bovis or highly related strains, the plasmid integrates into the chromosome of the Mycobacterium as a single copy (10842 nucleotides; promoter P comprising a portion of nucleotides 901 to 1068 of the hsp65 gene of Mycobacterium leprae_hsp60The DNA is underlined; the sequence of the disA coding sequence is nucleotide 1069-2145; human cGAS with mycobacterial codon-optimized sequence is nucleotides 2158 to 3726; the ATG start codon and the TAA or TGA stop codon are shown in bold, underlined).

Knock-out of endogenous BCG phosphodiesterase genes and intragenic segments encoding phosphodiesterase domains to increase CDN PAMP and DAMP levels

CDN was overexpressed by knocking out the endogenous BCG phosphodiesterase WP _ 003414507.

The BCG AHM08589.1 protein encodes an endogenous bifunctional C-di-AMP of 316 amino acids and cGAMP phosphodiesterase in BCG, 100% identical to the 316 amino acids at the C-terminus of mycobacterium tuberculosis Rv2837C (also known as CdnP, CnpB, 3 '-to-5' oligoribonuclease a, bifunctional oligoribonuclease or PAP phosphatase NrnA). Mycobacterium tuberculosis Rv2837c is known to proteolyze both 3'-5' c-di-AMP (bacterial PAMP molecule) and 2'-3' cGAMP (host DAMP molecule). Since the 315 amino acids of the BCG protein are 100% identical at the C-terminus, knockout (gene replacement) of the BCG AHM08589.1 protein will result in increased CDN (3 '-5' C-di-AMP and 2'-3' cGAMP) levels in the recombinant BCG.

SEQ ID No:14

Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP from BCG (also known as CnpB, 3 '-to-5' oligoribonuclease A, bifunctional oligoribonuclease, PAP phosphatase NrnA), amino acid sequence (316 amino acids; BCG protein AHM 08589.1; NCBI reference DNA sequence: CP003494.1, from BCG strain ATCC 35743; NCBI reference protein identifier WP _ 003414507). Similar sequences are present in M.tuberculosis as protein Rv2837c or MT2903, and M.bovis as protein Mb2862 c.

SEQ ID No:15

The bifunctional c-di-AMP and cGAMP phosphodiesterase genes cdnP from BCG (also known as cnpB or the genes for 3 '-to-5' oligoribonuclease A, bifunctional oligoribonuclease or PAP phosphatase NrnA), the DNA sequence (951 nucleotides [316 codons and 1 stop codon ]; coding for the BCG protein AHM 08589.1; NCBI reference sequence: CP003494.1, from BCG strain ATCC 35743). Similar sequences are present in Mycobacterium tuberculosis, encoding the proteins Rv28 2837c or MT2903, and in Mycobacterium bovis, encoding the protein Mb2862 c.

SEQ ID No:16

Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP (also known as CnpB, Rv2837c, or MT2903, 3 '-to-5' oligoribonuclease A, bifunctional oligoribonuclease, PAP phosphatase NrnA) from M.tuberculosis, amino acid sequence (336 amino acids; M.tuberculosis protein WP-003905944.1; NCBI/GenBank reference sequence: AL123456, from M.tuberculosis strain H37 Rv). The M.tuberculosis protein has 20 additional amino acids at its N-terminus compared to the BCG protein (SEQ ID No:14), which are underlined and bolded.

Overexpression of CDN by knocking out endogenous BCG phosphodiesterase domain: the protein BCG _ RS07340 (formerly BCG _u) 1416c) The EAL domain of (a).The BCG _ RS07340 protein (SEQ ID No:4) is encoded by the DNA sequence shown in SEQ ID No: 5. The BCG _ RS07340 protein is 100% identical to the mycobacterium tuberculosis Rv1354c protein and is an endogenous CDN PDE in BCG. The full-length polypeptide is 623 amino acids in length and it encodes a bifunctional diguanylate cyclase/diguanylate phosphodiesterase. The domain structure is: N-terminal-GAF-GGDEF-EAL-C-terminal, as shown in FIG. 11. The GAF domain (about amino acids 1-190) is a regulatory domain that affects the activity of other domains. The GGDEF domain (about amino acid 190-350) is a diguanylate cyclase that catalyzes the reaction 2GTP → c-di-GMP +2 pyrophosphate. The EAL domain (amino acids 354 to 623, highlighted in SEQ ID No:4) is a diguanylate phosphodiesterase catalyzing the reaction c-di-GMP → 2 GMP. Since the EAL domain of this protein is known to cleave 3'-5' c-di-GMPP, knocking out the endogenous cyclic dinucleotide phosphodiesterase domain will increase the level of c-di-GMP produced by BCG. Targeted knockout of the EAL domain can be accomplished by gene replacement of the full-length WT BCG _ RS07340 gene with a gene encoding only amino acids 1-353(GAF-GGDEF domain), i.e., the coding sequence of the gene is truncated to remove the sequence encoding amino acids 354-623 (as shown by the underlined DNA sequence in SEQ ID No: 5) and to incorporate an appropriate stop codon and transcription termination sequence. Recombinant BCG lacking the EAL domain of BCG _ RS07340 will result in elevated CDN PAMP c-di-GMP levels.

Overexpression of CDN by knockout of endogenous BCG phosphodiesterase: BCG AHM07112.The BCG _ AHM07112 protein is the endogenous diguanylate phosphodiesterase in BCG (homologous to the 307 amino acid mycobacterium tuberculosis Rv1357c protein). Some BCG strains completely lack BCG _ AHM07112, while other BCG strains such as BCG Tice carry BCG _ AHM07112. In BCG strains with the polypeptide, the protein can be 288 amino acids in length (such as in BCG ATCC 35743) or 307 amino acids in length (such as in BCG Pasteur 1173P 2). From BCG ATCC 3574The BCG _ AHM07112 protein of 3 is 288 amino acids long and is 100% identical to m.tuberculosis Rv1357C protein over its C-terminal 287 amino acids. The domain structure of BCG _ AHM07112 is that of a single EAL domain (fig. 11). Since the Mycobacterium tuberculosis Rv1357c protein is known to cleave 3'-5' c-di-GMP, it is highly likely that the BCG protein undergoes the same reaction. Knocking out this endogenous cyclic dinucleotide phosphodiesterase in BCG is expected to increase the level of c-di-GMP produced by BCG. Targeted knockout of the EAL domain can be accomplished by gene replacement of the full-length WT BCG _ AHM07112 gene and subsequent generation of a marker-free deletion.

SEQ ID No:17

Diguanylate phosphodiesterase AHM07112.1 from BCG and other related Mycobacteria, amino acid sequence (288 amino acids; GenBank reference: CP 003494.1; from BCG strain ATCC 35743). AHM07112.1 is 100% identical to the C-terminal 287 amino acids of M.tuberculosis protein Rv1357C or MT1400 and the diguanylate phosphodiesterase of M.bovis such as protein Mb 1392C.

SEQ ID No:18

Diguanylate phosphodiesterase AHM07112.1 from BCG and other related Mycobacteria, DNA sequence (867 nucleotides [288 codons, 1 stop codon ]; GenBank reference sequence: CP 003494.1; from BCG strain ATCC 35743). AHM07112.1 is 100% identical to the C-terminal 287 amino acids of M.tuberculosis protein Rv1357C or MT1400 and the diguanylate phosphodiesterase of M.bovis such as protein Mb 1392C.

SEQ ID No:19

Diguanylate phosphodiesterases Rv1357c or MT1400 from Mycobacterium tuberculosis and BCG Pasteur 1173P2, amino acid sequence (307 amino acids, NCBI/GenBank reference sequence: AL 123456; from Mycobacterium tuberculosis strain H37 Rv). An N-terminal extension of 19 amino acids was present in M.tuberculosis and BCG Pasteur strain 1173P2, but not in several other BCG strains. The 19 amino acid N-terminal extension is underlined and bolded. The C-terminal 287 amino acids of M.tuberculosis Rv1357C are identical to BCG diguanylate phosphodiesterase AHM 07112.1100%.

The sequences cited in this application are summarized in table 1 below.

TABLE 1

In some embodiments, the present invention relates to an expression cassette or vector comprising a nucleic acid sequence encoding Rv1354c protein or a functional portion thereof; a nucleic acid sequence encoding a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; or a combination thereof. In some embodiments, the expression vector or cassette further comprises a nucleic acid sequence encoding a DNA integrity scanning (dis a) protein or functional portion thereof that functions as a diadenosine cyclase. In some embodiments, the nucleic acid sequence encoding Rv1354c protein does not comprise a phosphodiesterase gene or a phosphodiesterase domain. In some embodiments, the expression vector or cassette does not comprise a phosphodiesterase gene or a phosphodiesterase domain.

Methods for producing expression vectors and expression cassettes, transforming mycobacteria and isolating mycobacteria have been described. In some embodiments, the expression vector or expression cassette of the invention comprises one or more regulatory sequences, such as promoter and/or enhancer elements, operably linked to the nucleic acid of the invention, which control or affect transcription of the nucleic acid. In some embodiments, the expression vectors or expression cassettes of the invention comprise one or more sequences operably linked to a nucleic acid of the invention that direct the termination of transcription, post-transcriptional cleavage, and/or polyadenylation. In some embodiments, the expression vector or expression cassette of the invention comprises a variable length intervening sequence and/or selectable marker gene operably linked to a nucleic acid of the invention.

In some embodiments, the invention relates to a mycobacterium strain comprising an expression vector or expression cassette of the invention described herein. In some embodiments, the mycobacterium strain is mycobacterium tuberculosis, mycobacterium bovis, or a combination thereof. In some embodiments, the mycobacterium strain is BCG. In some embodiments, the strain comprises the plasmid of SEQ ID NO. 13.

In some embodiments, the invention relates to mycobacterium strains that express or overexpress a diadenosine cyclase and/or that express or overexpress one or more other cyclase genes or domains (e.g., those described herein). In some embodiments, expression or overexpression results in the release of one or more STING agonists (e.g., c-di-AMP, c-di-GMP, 2'-3' cGAMP, and/or 3'-3' cGAMP). In some embodiments, the invention relates to mycobacterium strains that express or overexpress a diadenosine cyclase and/or do not express a Phosphodiesterase (PDE) that hydrolyzes STING agonists (e.g., contain a deletion of the PDE gene that hydrolyzes STING agonists). See, for example, fig. 12. In some embodiments, the mycobacterium strain is mycobacterium tuberculosis, mycobacterium bovis, or a combination thereof. In some embodiments, the mycobacterium strain is BCG.

Statistically significant antitumor Effect of BCG-disA-OE in rat MNU bladder cancer model

The rat MNU bladder cancer model is a validated bladder cancer model in which intravesical administration of BCG can show therapeutic properties (figure 6 and Kates et al PMID 28588015). The inventors extended their previous findings on the therapeutic efficacy of BCG-disA-OE versus BCG-WT, shown in FIG. 7. The inventors have now performed the 16-week rat MNU model twice. FIG. 7 is based on experiment 1 and shows that BCG-disA-OE shows a trend towards better results than BCG-WT. After performing experiment 2 and combining its data with experiment 1, the inventors now showed that BCG-disA-OE was statistically significantly better than no treatment (p ═ 0.048), while BCG-WT was not statistically significantly better than no treatment (data shown in fig. 17).

Tumor suppressor Treg cells were reduced in a murine syngeneic bladder cancer tumor model by BCG-dis a-OE.

In the MNU rat bladder cancer model, the amount of bladder tissue at the end of the 16 week experiment was insufficient for flow cytometry. To investigate the cell population changes induced by BCG-disA-OE, the inventors developed a murine syngeneic bladder cancer tumor model using BBN975 cells. This model allowed the development of large tumors (diameter >1.5cm) on the flank of the mouse. Mice were treated with BCG-disA-OE and BCG-WT by intratumoral injection. As shown in figure 18, use of BCG-disA-OE resulted in reduced levels of tumor-associated CD4+ Treg cells, tumor-associated CD8+ Treg cells, and splenic CD4+ Treg cells.

Delivery of BCG-disA-OE from intracellular compartments (intracellular compartments) sustained STING kinase An animal agent.

Persistence of BCG in the bladder.

Bowyer et al (The persistence of bacillus calmeter-Guerin in The bladder after in vivo treatment for bladder cancer. Brit J Urol. 1995; 75:188-192.PMID 7850324) evaluated 125 patients with bladder cancer who received intravesical BCG from 1986 to 1992. The patient is asked to provide a monthly urine sample which is then sent to mycobacterial culture. 90 patients survived and urine samples were provided monthly for compliance. 4/90 (4.4%) had persistent BCG in their urine, one patient lasted for up to 16.5 months. The fifth patient required cystectomy 7 weeks after completion of intravesical BCG treatment and microscopic evidence of acid-fast bacilli was found in the bladder by microscopy.

Durek et al, (The fat of Bacillus Calmette-Guerin after ester intravenous infection. J Urol.2001; 165:1765-1768.PMID 11342972) 49 patients were studied for continuous urine culture following intravesical BCG. BCG in urine was detected in 96.4% of the samples after 2 hours post-instillation, and BCG in urine was detected in 67.9% of the samples after 24 hours post-instillation. The number of positive samples decreased and was 27.1% on day 7 immediately before the next instillation (fig. 44). Researchers also evaluated bladder biopsies for mycobacterial DNA by PCR within 1 week after the 6 th instillation (administered monthly instillation). Mycobacterial ribosomal DNA was found in 14 out of 44 bladder biopsies (31.8%). Furthermore, positive PCR of mycobacterial DNA was evident up to 24 months in between 4.2% and 37.5% of biopsies studied.

The fact that BCG is known to persist in bladder tissue represents an important advantage of the BCG-disA-OE strategy for delivering STING agonists in cancer. While many techniques focus on the generation of small molecule STING agonists, such agents have a relatively short exposure time. In contrast, BCG, as an intracellular microorganism and as demonstrated by the studies of Bowyer and Durek, persists in cells and tissues for several weeks. The persistence of BCG-disA-OE in tissues provides for sustained long-term delivery of STING agonists in the tumor microenvironment.

In two separate mouse models, BCG-disA-OE was safer than BCG-WT

In humans, intravesical BCG treatment is associated with dysuria, fatigue, and weakness in the treated patients. Additional more serious adverse effects are persistent cystitis and disseminated BCG disease (BCGosis) of BCG. The patient safety of BCG was extensively reviewed in O' Donnell et al (as of 2019). The incidence of BCG dissemination into the bloodstream following intravesical instillation was estimated to be 1/15,000 patients.

To test the safety of BCG-disA-OE compared to BCG-WT, the inventors used two mouse models of BCG infection, in which BCG strains were aerosolized into the lungs of immunocompetent BABL/c mice or immunosuppressed SCID mice. As shown in FIG. 19, BCG-disA-OE was less able to proliferate in immunocompetent mouse lungs than BCG-WT, and in immunosuppressed mice, BCG-disA-OE was less lethal in the time-of-death assay.

BCG has been shown to elicit a training immunity that is comparable to BCG in solid and liquid tumors and diabetes The therapeutic benefit of (a). BCG strains overexpressing STING agonists elicit stronger changes in the training immunity than BCG-WT

And (4) training immunity.Training immunity refers to the ability of an antigenic stimulant to elicit a stronger immune response against a second, different antigen. Training immunity is antigen independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and associated with epigenetic and metabolic changes. BCG is an effective tool as a first antigenic stimulant to elicit a Trained immunity against subsequent antigenic stimuli, such as tumors, viral infections or drug-resistant bacterial infections (Neetea et al. Trained immunity: a program of input immune in health and disease. science 2016.PMID 27102489; and Arts et al. BCG vaccination technologies against external viral infection in humans of the index of cellular associated with cultured cell 2018.PMID 29324233).

BCG for solid and liquid tumors.BCG has a long history of therapeutic benefit as an immunotherapy for both solid and liquid tumours in humans (Hersh et al BCG as adjuvant immunotherapy for neoplasma. annu Rev Med 1977.PMID 324372). BCG has been used systemically and intratumorally in malignancies including melanoma, non-small cell lung cancer (NSCLC) and Acute Lymphoblastic Leukemia (ALL). There have recently been trials of BCG together with checkpoint inhibitors for various forms of bladder cancer.

BCG for diabetes.BCG vaccination has recently been shown to treat various forms of diabetes (package)Including Type 1 diabetes) has therapeutic benefits (Stienstra and Neea. figure up diabetes: BCG vaccination effects on Type 1diabetes mellitus. trends Endoc Metab 2018.PMID: 30327169). This effect is thought to be mediated by the training immunity of BCG, which has been shown to result in epigenetic modifications that promote expression of pro-inflammatory cytokines as well as expression of metabolic enzymes such as those used for glycolysis.

BCG-disA-OE and training immunization.To investigate the ability of BCG strains overexpressing STING agonists to stimulate training immunity, the inventors tested that BCG-WT versus BCG-disA-OE elicited an increase in secondary antigen stimulation in resting human single cells six days before exposure to the BCG strain. The first antigen was the BCG strain at day 0 and after 6 days of rest the second antigen was the unrelated TLR-1/2 antigen PAM3CSK 4. As can be observed in FIG. 20, the immune response (secretion of IL-1. beta.) tested was enhanced by both BCG-WT and BCG-disA-OE after receiving the second stimulus, but the degree of stimulation by BCG-disA-OE was statistically significantly greater than the degree of stimulation without the first BCG stimulus or BCG-WT as the first stimulus. This revealed that BCG strains overexpressing STING, such as BCG-disA-OE, are more effective training immunostimulants than BCG-WT.

In related experiments, the present inventors performed the same BCG-first stimulation/6 days of rest/TLR-1, 2 antigen stimulation with PAM3CSK4 with human monocytes for a second time. At the end of the experiment, cellular DNA was collected and chromatin immunoprecipitation (ChIP) was performed using antibodies against H3K4 histone methylation markers. The H3K4 marker is a known transcriptional activation marker. After quantitative PCR amplification of the IL-6 promoter region of the immunoprecipitated DNA, the results showed that BCG-Pasteur-dis A-OE and BCG-Tice-dis A-OE were statistically significantly more effective at priming the H3K4 marker in the IL-6 promoter (IL-6 is a pro-inflammatory cytokine) than their respective BCG-WT strains. These results show that BCG strains that overexpress STING, such as BCG-disA-OE, are more potent stimulators of epigenetic changes associated with the training immunity than BCG-WT.

BCG-Tice-disA-OE expression ratio BMuch higher levels of the dis A gene for CG-WT

As can be observed in fig. 23, use 2^-ΔΔCTComparative method, the relative expression of BCG-Tice-disA-OE clone 2 (which was selected for seed lot preparation and storage) was 300: 1. This indicates that the disA is strongly overexpressed by being on a multicopy plasmid and driven by the Mycobacterium leprae hsp65 promoter in the pSD5-hsp65-MT3692 plasmid. This strong overexpression resulted in much higher levels of release of the STING agonist c-di-AMP.

STING agonist overexpression of BCG strains such as BCG-disA-OE trigger signaling in more than one model system Pro-inflammatory changes in the pathways and cytokine secretion profile.

The inventors tested strains overexpressing STING agonists, such as BCG-disA-OE, in more than one model system to assess their relative ability to trigger proinflammatory cytokine changes. In most of its tests, BCG-disA-OE was statistically significantly superior to BCG-WT. And BCG-disA-OE gave the stronger of the two responses when the comparison was statistically insignificant.

FIG. 25 also shows that the increase in type 1 IFN secretion in both BCG-disA-OE and BCG-WT is STING-dependent.

In summary, BCG-disA-OE is a more potent stimulator of proinflammatory cytokine expression and proinflammatory pathway induction than BCG-WT.

The following table summarizes the data:

a method of producing recombinant BCG without antibiotic gene cassettes overexpressing STING agonist biosynthesis genes.

The dis A overexpression plasmid pSD5-hsp65-MT3692 carries a Kan resistance gene cassette which confers resistance to the antibiotic kanamycin. The present inventors disclosed a method of generating recombinant BCG without antibiotic gene cassette overexpressing STING agonist biosynthesis gene.

The mycobacterial genetic operon panCD encodes the biosynthetic genes panC (pantothenate-. beta. -alanine ligase gene) and panD (aspartate 1-decarboxylase gene). The gene products PanC and PanD are also referred to as vitamin B₅(a B vitamin) is essential for the biosynthesis of pantothenic acid. Pantothenic acid is a water-soluble vitamin, and is an essential nutrient for mycobacteria such as BCG. Animals require pantothenic acid to synthesize coenzyme a (coa) and to synthesize and metabolize proteins, carbohydrates, and fats. The anion is known as pantothenate.

It has been shown that the genetic deletion of panCD in mycobacteria results in mutant strains that can only grow in the presence of added pantothenate. Thus, the mutant strain is auxotrophic for pantothenate. The Δ panCD mutant of mycobacterium tuberculosis has been shown to be highly attenuated in animal infections, being rapidly cleared because it cannot grow in mammalian tissues where pantothenate is not available.

The inventors disclosed a detailed method for generating a marker-free (antibiotic-free gene cassette) Δ panCD deletion mutant of BCG. The mutant will only be able to grow in the presence of pantothenate and is not expected to survive during infection or to serve as an effective delivery vehicle for STING agonist expression.

The present inventors disclose a detailed method for generating a shuttle plasmid carrying the mycobacterial panCD gene and an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65:: DISA construct, which overexpresses the DISA gene and releases the excess STING agonist c-di-AMP). The shuttle plasmid is capable of replication in E.coli or in Mycobacteria. It carries an antibiotic cassette that can be conveniently removed by cleavage with a rare-cutting restriction enzyme and religation. Alternatively, the shuttle plasmid may be generated by: PCR amplification the backbone of the plasmid without the antibiotic resistance cassette created restriction sites at the ends and ligated into the PCR product consisting of the amplified panCD operon with the same unique restriction sites at its ends. In either way, the shuttle plasmid (ligation product) without the antibiotic resistance gene can be electroporated into BCG or e.coli auxotrophs and selected on pantothenate-free agar plates.

In a final manifestation of the disclosure, the inventors show a method of introducing plasmids carrying antibiotic-free cassettes of the mycobacterial panCD gene and overexpression constructs for the biosynthesis of STING agonists (such as the Phsp65:: dis A construct) into the unmarked BCG Δ panCD mutants. The end result is a BCG strain that does not carry antibiotic resistance genes and that strongly overexpresses one or more than one STING agonist biosynthesis genes. In mammalian hosts or humans, such BCG strains will retain plasmids under strong selective pressure, as it requires panCD complementation from the plasmid.

In another manifestation of the disclosure, the panCD cassette and a construct for the biosynthesis of a STING agonist (such as a Phsp65:: disp construct) can be introduced into a chromosomal integration vector such as pMH 94. Using a similar approach, the antibiotic cassette could be eliminated from pMH 94. Introduction of this chromosomal integration plasmid into the unmarked BCG Δ panCD mutant also resulted in BCG strains that did not carry antibiotic resistance genes and strongly overexpressed one or more STING agonist biosynthesis genes. The disadvantage of this strategy is that the overexpression construct is single copy on the bacterial chromosome, rather than multiple copies on the plasmid, and this may result in lower levels of STING agonist release.

BCG-Tice (ATCC 35743) is a natural pantothenate auxotroph.

The present inventors have disclosed that the mycobacterium bovis BCG Tice strain (ATCC 35743) is a natural pantothenate auxotroph. This strain carries a 5bp DNA insertion in its panC gene at the base pairs 739-743. This insertional mutational change results in a frame shift mutation after amino acid 246 of PanC (the length of wild-type PanC is 309 amino acids). Due to the 5bp insertion mutation, the mutant PanC polypeptide in the Mycobacterium bovis BCG Tice strain (ATCC 35743) contained 246 amino acids of the wild-type PanC sequence at its N-terminus, followed by a nonsense polypeptide of 478 amino acids at its C-terminus. It is highly unlikely that the mutant PanC polypeptide will retain any functional pantothenate- β -alanine ligase activity (normal enzyme function of PanC). Furthermore, the PanD polypeptide in BCG Tice (ATCC 35743) is very unlikely to be translated because the stop codon of the panC gene, which overlaps the ATG where the translation of panD in the wild-type sequence is initiated, does not fit in frame (out of frame). In the wild-type panCD operon, ribosome termination of PanC translation is coupled with ribosome initiation of PanD translation. Since there is no ribosome termination immediately upstream of the panD start codon, ribosome initiation of panD gene translation is highly unlikely to occur.

The inventors disclose that this natural auxotrophy enables the more rapid construction of recombinant BCG without antibiotic gene cassettes that overexpress STING agonist biosynthetic genes.

The present inventors disclose a method for the direct introduction of plasmids carrying antibiotic-free cassettes of the mycobacterial panCD gene and overexpression constructs for the biosynthesis of STING agonists (such as the Phsp65:: dis A construct) into BCG-Tice (ATCC 35743).

Note that pSD5-hsp60-MT3692 is identical to pSD5-hsp65-MT 3692. The present inventors previously mentioned this same plasmid as pSD5-hsp60-MT 3692. However, the actual promoter in this strain is the promoter of the hsp65 gene of Mycobacterium leprae. Therefore, the present inventors can refer to the plasmid pSD-hsp60-MT3692 as pSD5-hsp65-MT 3692.

In some embodiments, the invention relates to a pharmaceutical composition comprising an expression vector, expression cassette or strain of the invention described herein and a pharmaceutically acceptable carrier.

In some embodiments, the present invention relates to methods and/or compositions for treating and/or preventing cancer comprising administering to a subject an expression vector, expression cassette, strain, or pharmaceutical composition described herein. In some embodiments, the cancer is bladder cancer (e.g., non-muscle invasive bladder cancer (NMIBC)), breast cancer, or a solid tumor. Additional embodiments of the present disclosure contemplate methods and/or compositions for treating and/or preventing bladder cancer, wherein modulation of a type 1 Interferon (IFN) response is directly or indirectly related. In certain embodiments, an individual having bladder cancer, such as NMIBC, is treated with a modulator of type 1 interferon response, and in particular embodiments, an individual having bladder cancer is provided with a modulator of type 1 interferon expression, such as an inducer of its expression.

In certain embodiments, the level at which the type 1 interferon expression inducer increases the expression of type 1 interferon can be any level so long as it provides an improvement in at least one symptom of bladder cancer, including non-muscle invasive bladder cancer (NMIBC). In at least some cases, the expression level of type 1 interferon can be increased at least 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 25-fold, 50-fold, 100-fold, 1000-fold, or more compared to the expression level in a standard. The individual may monitor the expression level of type 1 interferon using standard methods in the art, such as, for example, RNA assay (northern assay) or quantitative PCR.

Individuals known to have, suspected of having, or at risk of having bladder cancer may be provided with an effective amount of an inducer of type 1 interferon expression, including the BCG strain of the invention containing the expression vector of the invention. The expression vector expresses RV1354c protein or a functional part thereof; a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a DNA integrity scanning (dis a) protein or functional part thereof that functions as an adenylate cyclase; or a combination thereof. It is preferred that the BCG strain of the present invention comprising the expression vector of the present invention is administered into the bladder of a subject and that the expressed one or more than one protein enhances the expression of type 1 interferon in the bladder. For example, those individuals at risk of bladder cancer may be individuals with one or more genetic factors, may be elderly individuals, and/or may have a family history.

In certain embodiments of the disclosure, in addition to one or more type 1 interferon inducers of the invention, the individual is administered an agent for bladder cancer therapy. Such additional therapies may include intravesical chemotherapy such as, for example, mitomycin C, cyclophosphamide, or a combination thereof. When the combination therapy is used with one or more type 1 interferon inducers (such as a strain of BCG expressing one or more of RV1354c protein or a functional portion thereof, a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof, a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof, a DNA integrity scanning (dis) protein that functions as adenylate cyclase, or a functional portion thereof), additional therapy may be administered prior to the type 1 interferon inducer, simultaneously with the type 1 interferon inducer, and/or after the type 1 interferon inducer.

In some embodiments, the expression vectors, expression cassettes, strains, pharmaceutical compositions and/or methods of the invention described herein have increased safety, increased tolerance (e.g., reduced dysuria, urgency or weakness), and/or a reduced likelihood of causing or disseminating bloodstream infections as compared to non-recombinant BCG.

In some embodiments, the present invention relates to a method of treating and/or preventing cancer, comprising administering to a subject an expression vector, expression cassette, strain, and/or pharmaceutical composition of the invention described herein, wherein administration results in an increased safety profile, increased tolerance (e.g., reduced dysuria, urgency, or weakness), and/or a reduced likelihood of blood flow infection or disseminated blood flow infection, as compared to non-recombinant BCG. In some embodiments, the cancer is, e.g., bladder cancer (e.g., non-muscle invasive bladder cancer (NMIBC)), breast cancer, or a solid tumor. In some embodiments, the solid tumor is, for example, a sarcoma, carcinoma, or lymphoma.

In some embodiments, the present invention relates to a method of increasing safety, increasing tolerance (e.g., reducing dysuria, urgency, or weakness), and/or reducing the likelihood of causing or disseminating a blood stream infection as compared to non-recombinant BCG, comprising administering to a subject an expression vector, expression cassette, strain, and/or pharmaceutical composition of the invention described herein.

Pharmaceutical preparation

The pharmaceutical compositions of the invention comprise an effective amount of one or more type 1 interferon expression inducers, such as a BCG strain that expresses one or more of the following proteins, dissolved or dispersed in a pharmaceutically acceptable carrier: RV1354c protein or a functional portion thereof; a cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; a cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; a DNA integrity scanning (dis a) protein or functional part thereof that functions as an adenylate cyclase. The phrase "pharmaceutically or pharmacologically acceptable" refers to molecular entities and compositions that do not produce adverse, allergic, or other abnormal reactions (untoward reactions) when optionally administered to an animal such as, for example, a human. In light of The present disclosure, The preparation of pharmaceutical compositions comprising at least one type 1 interferon expression inducer or additional active ingredients will be known to those skilled in The art, as exemplified by Remington: The Science and Practice of Pharmacy, 21 st edition Lippincott Williams and Wilkins,2005, which is incorporated herein by reference. Further, for animal (e.g., human) administration, it will be understood that the formulations should meet sterility, pyrogenicity, general safety and purity Standards as required by the FDA Office of Biological Standards.

As used herein, "pharmaceutically acceptable carrier" includes any and all solvents, dispersion media, coatings, surfactants, antioxidants, preservatives (e.g., antibacterial agents, antifungal agents), isotonic agents, absorption delaying agents, salts, preservatives, drugs, drug stabilizers, gels, binders, excipients, disintegrants, lubricants, sweeteners, flavoring agents, dyes, such similar materials, and combinations thereof, as will be known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition. Mack Printing Company,1990, pp.1289-1329, which is incorporated herein by reference). Except to the extent that any conventional carrier is incompatible with the active ingredient, its use in pharmaceutical compositions is contemplated.

Type 1 interferon expression inducers (such as BCG strains expressing one or more of the following proteins: RV1354c protein or a functional part thereof; cyclic GMP-AMP synthase (DncV) protein or a functional part thereof; cyclic GMP-AMP synthase (cGAS) protein or a functional part thereof; DNA integrity scanning (dis) protein or a functional part thereof acting as adenylate cyclase) may comprise different types of vectors depending on whether it is administered in solid, liquid or aerosol (aerosol) form and whether it needs to be sterile in such administration routes as injection. In some embodiments, the invention (e.g., expression vector, strain, or Pharmaceutical composition) can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intravesically (e.g., directly into the bladder, such as by injection or by intravesical instillation), intratumorally, locally, intramuscularly, subcutaneously, mucosally, orally, topically (locally), locally (locally), by inhalation (e.g., aerosol inhalation), by injection, infusion, continuous infusion, via a catheter, via lavage in cream, by direct local perfusion of liquid compositions (e.g., liposomes) in a localized perfusion bath (localized perfusion) of target cells, or by other methods or any combination of the foregoing as known to those of ordinary skill in the art (see, e.g., Remington's Pharmaceutical Sciences, 18 th edition STING gelatinants in non-immunogenic tumors, cancer Immunol res.2018, 2.22.p. PMID:29472271, incorporated herein by reference).

Pharmaceutically acceptable salts include acid addition salts, for example with free amino groups of the protein composition, or with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric or mandelic acid. Salts with free carboxyl groups may also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium or iron hydroxides; or an organic base such as isopropylamine, trimethylamine, histidine or procaine. After formulation, the solution will be administered in a manner compatible with the dosage formulation and in an amount such as is therapeutically effective. The formulations are readily administered in a variety of dosage forms, such as formulated for parenteral administration, such as an injectable solution or aerosol for delivery to the lung, or formulated for alimentary tract administration, such as a drug release capsule or the like.

Further, in accordance with the present disclosure, compositions of the present invention suitable for administration are provided in a pharmaceutically acceptable carrier, with or without an inert diluent. The carrier should be absorbable and include liquid, semi-solid (i.e., paste) or solid carriers. Except to the extent that any conventional vehicle, agent, diluent or carrier is deleterious to the recipient or to the therapeutic effect of the composition contained therein, it is suitable for use in an administrable composition used in practicing the methods of the present invention. Examples of carriers or diluents include fats, oils, water, salt solutions, lipids, liposomes, resins, binders, fillers, and the like or combinations thereof. The composition may also comprise a plurality of antioxidants to retard oxidation of one or more components. In addition, the prevention of the action of microorganisms can be brought about by preservatives such as various antibacterial and antifungal agents, including but not limited to parabens (e.g., methylparaben, propylparaben), chlorobutanol, phenol, sorbic acid, thimerosal, or combinations thereof.

In accordance with the present invention, the compositions are combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, mixing, encapsulation, absorption, and the like. Such procedures are conventional to those skilled in the art.

In a particular embodiment of the invention, the composition is intimately combined or admixed with a semi-solid carrier or a solid carrier. Mixing may be carried out in any convenient manner, such as milling. Stabilizers may also be added during mixing to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach. Examples of stabilizers for use in the composition include buffers, amino acids such as glycine and lysine, carbohydrates such as dextrose, mannose, galactose, fructose, lactose, sucrose, maltose, sorbitol, mannitol, and the like.

In additional embodiments, the invention encompasses the use of a pharmaceutical lipid vehicle composition comprising a type 1 interferon expression inducer, one or more lipids and an aqueous solvent. As used herein, the term "lipid" includes any of a wide range of substances that are characteristically insoluble in water and extractable with organic solvents. This broad class of compounds is well known to those skilled in the art, and the term "lipid" as used herein is not limited to any particular structure. Examples include compounds containing long chain aliphatic hydrocarbons and derivatives thereof. Lipids may be naturally occurring or synthetic (i.e., designed or produced by humans). However, lipids are generally a biological substance. Biolipids are well known in the art and include, for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids (lysolipids), glycosphingolipids, glycolipids, sulfates, lipids with ether and ester linked fatty acids, and polymerizable lipids, and combinations thereof. Of course, the compositions and methods of the present invention also encompass compounds other than those specifically described herein, which are understood by those of skill in the art to be lipids.

One of ordinary skill in the art will be familiar with the range of techniques that can be used to disperse the composition in a lipid vehicle. For example, the type 1 interferon expression inducers of the present invention may be dispersed in a solution containing a lipid, solubilized with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bound to a lipid, contained as a suspension in a lipid, contained or complexed with micelles or liposomes, or associated with a lipid or lipid structure by any means known to one of ordinary skill in the art. The dispersion may or may not result in the formation of liposomes.

The actual dosage amount (dosage amount) of the composition of the invention to be administered to an animal patient may be determined by physical and physiological factors such as body weight, severity of the condition, type of disease being treated, prior or concurrent therapeutic intervention, the patient's characteristics and the route of administration. Depending on the dosage and route of administration, the preferred dosage and/or the number of administrations of the effective amount may vary depending on the response of the subject. In any case, the physician responsible for administration will determine the concentration of the one or more active ingredients in the composition and the appropriate dose or doses for the individual subject.

In certain embodiments, the pharmaceutical composition may comprise, for example, at least about 0.1% of the active compound. In other embodiments, for example, the active compound may constitute between about 2% to about 75%, or between about 25% to about 60%, and any range derivable therein, by weight of the unit. Naturally, the amount of one or more active compounds in each therapeutically useful composition can be prepared in such a way that a suitable dose will be obtained in any given unit dose of the compound. Factors such as solubility, bioavailability, biological half-life, route of administration, product shelf-life, and other pharmacological considerations will be considered by those skilled in the art of preparing such pharmaceutical formulations, and thus, a variety of dosages and treatment regimens may be desirable.

In other non-limiting examples, the dose can further include from about 1 microgram/kg/body weight, about 5 microgram/kg/body weight, about 10 microgram/kg/body weight, about 50 microgram/kg/body weight, about 100 microgram/kg/body weight, about 200 microgram/kg/body weight, about 350 microgram/kg/body weight, about 500 microgram/kg/body weight, about 1 milligram/kg/body weight, about 5 milligram/kg/body weight, about 10 milligram/kg/body weight, about 50 milligram/kg/body weight, about 100 milligram/kg/body weight, about 200 milligram/kg/body weight, about 350 milligram/kg/body weight, about 500 milligram/kg/body weight to about 1000 mg/kg/body weight or more per administration, and any range derivable therein. In non-limiting examples of ranges derivable from the values listed herein, based on the values described above, a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 micrograms/kg/body weight to about 500 milligrams/kg/body weight, and the like, may be administered.

A. Digestive Composition (Alimentary Composition) and formulation

In one embodiment of the present disclosure, the type 1 interferon expression inducer of the present invention is formulated for administration via the digestive route. The digestive route includes all possible routes of administration for which the composition is in direct contact with the digestive tract. In particular, the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually. Thus, these compositions may be formulated with an inert diluent or an ingestible carrier, or they may be enclosed in hard or soft shell gelatin capsules, or they may be compressed into tablets, or they may be incorporated directly into the food of the diet.

In certain embodiments, the active compound may be incorporated into excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers (wafers), and the like (Mathiewitz E, Jacob JS, Jong YS, Carino GP, Chickering DE, Chaturvedi P, Santos CA, Vijayarghan K, Montgomery S, Bassett M, Mobile C. biological edible aqueous systems organic delivery systems. Nature.1997; 386:410-4.PMID: 9121559; Hwang MJ, Ni X, Waldman M, Ewig CS, Hagler AT. derivation of mixtures II form. VI. Carbodredgens and 3. composite, Ostre J. 9. Biocide, Mi J. TM., Ostre. III. E. IV. VI. carbide, Mo. 9. Biocide compositions of polymers, Mo. 11. 9. Biocide, Mo. 9. Biocide. 9. molecular compositions, Mo. 9. Biocide, Mo. 9. II. Biocide, Mo. 9. molecular compositions, Mo. E. Biocide, Mo., Mi. 9. Biocide, Mo., Mitsukuwa, park H, Park K.scientific reliable driver-delivery systems. crit Rev the driver Carrier Syst.1998; 15:243-84.PMID: 9699081; U.S. Pat. nos. 5,641,515; 5,580,579 No; and 5,792,451, each specifically incorporated by reference herein in its entirety). The tablets, troches, pills, capsules, and the like may also contain the following: binders such as, for example, gum tragacanth, acacia, corn starch, gelatin, or combinations thereof; excipients such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharin, cellulose, magnesium carbonate, or combinations thereof; a disintegrant such as, for example, corn starch, potato starch, alginic acid, or a combination thereof; lubricants, such as, for example, magnesium stearate; a sweetening agent such as, for example, sucrose, lactose, saccharin or combinations thereof; flavoring agents such as, for example, peppermint, oil of wintergreen, cherry flavoring (cherry flavoring), orange flavoring (orange flavoring), and the like. When the dosage unit form is a capsule, it may contain, in addition to materials of the above type, a liquid carrier. Various other materials may be present, as coatings or to otherwise modify the physical form of the dosage unit. For example, tablets, pills, or capsules may be coated with shellac, sugar or both. When the dosage form is a capsule, it may contain, in addition to materials of the above type, a carrier, such as a liquid carrier. Gelatin capsules, tablets or pills may be enteric coated. The enteric coating prevents denaturation of the composition in the stomach or upper bowel (upper bowel) where the pH is acidic. See, for example, U.S. patent No. 5,629,001. Upon reaching the small intestine, the alkaline pH therein dissolves the coating and allows the composition to be released and absorbed by specialized cells, such as intestinal epithelial cells (epithelial enterocytes) and Peyer's patch M cells. A syrup or elixir may contain the active compounds sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring such as cherry or orange flavor. Of course, any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed. Furthermore, the active compounds can be incorporated into sustained-release preparations (preparation) and preparations (formulation).

For oral administration, the compositions of the present disclosure may optionally incorporate one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, buccal spray, or sublingual oral formulation. For example, mouthwashes may be prepared by incorporating the required amount of active ingredient in a suitable solvent, such as a sodium borate solution (Dobell solution). Alternatively, the active ingredient may be incorporated into an oral solution, such as a solution containing sodium borate, glycerin, and potassium bicarbonate, or dispersed in a dentifrice, or added in a therapeutically effective amount to a composition that may include water, binders, abrasives, flavoring agents, foaming agents, and wetting agents. Alternatively, the composition may be formulated as a tablet or solution that can be placed under the tongue or otherwise dissolved in the mouth.

Additional formulations suitable for other modes of digestive administration include suppositories. Suppositories are solid dosage forms of varying weight and shape, usually filled with a drug, for insertion into the rectum. After insertion, the suppository softens, melts or dissolves in the luminal fluid. Generally, for suppositories, conventional carriers may include, for example, polyalkylene glycols, triglycerides, or combinations thereof. In certain embodiments, suppositories may be formed from mixtures containing the active ingredient, for example, in the range of from about 0.5% to about 10%, and preferably from about 1% to about 2%.

B. Parenteral compositions and formulations

In another embodiment, the type 1 interferon expression inducer of the present invention may be administered via a parenteral route. As used herein, the term "parenteral" includes routes that bypass the digestive tract. In particular, the pharmaceutical compositions disclosed herein may be administered, for example, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneously, or intraperitoneally. See, for example, U.S. patent nos. 6,7537,514; 6,613,308 No; U.S. Pat. No. 5,466,468; U.S. Pat. No. 5,543,158; U.S. Pat. No. 5,641,515; and 5,399,363 (each of which is specifically incorporated by reference herein in its entirety).

Solutions of the active compound as a free base or pharmacologically acceptable salt may be prepared in water suitably mixed with a surfactant such as hydroxypropylcellulose. Dispersions can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms. Pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (U.S. Pat. No. 5,466,468, specifically incorporated herein by reference in its entirety). In all cases, the form must be sterile and must be fluid to the extent that there is ease of injection. It must be stable under the conditions of manufacture and storage and must be protected from the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (i.e., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), suitable mixtures thereof, and/or vegetable oils. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions and by the use of surfactants. Prevention of the action of microorganisms can be caused by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride. Prolonged absorption of the injectable compositions can be brought about by the use of agents delaying absorption, for example, combinations of aluminum monostearate and gelatin.

For example, for parenteral administration as an aqueous solution, the solution should be suitably buffered if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose. These particular aqueous solutions are particularly suitable for intravenous, intramuscular, subcutaneous and intraperitoneal administration. In this regard, sterile aqueous media that can be used in accordance with the present disclosure will be known to those skilled in the art. For example, a dose can be dissolved in isotonic NaCl solution and added as a subcutaneous perfusion solution, or injected into the proposed infusion site (see, e.g., "Remington's Pharmaceutical Sciences" 15 th edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. In any event, the person responsible for administration will determine the appropriate dose for the individual subject. In addition, for human administration, the formulations should meet sterility, pyrogenicity, and general safety and purity standards as required by FDA office of biological standards.

Sterile injectable solutions are prepared by: the required amount of active compound is incorporated, as required, in a suitable solvent together with several of the other ingredients enumerated above, and then filter sterilized. Generally, dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains an alkaline dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preferred methods of preparation are vacuum drying and the freeze-drying technique which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof. The powdered composition is combined with a liquid carrier such as, for example, water or a salt solution, with or without a stabilizer.

C. Other pharmaceutical compositions and formulations

In other preferred embodiments of the invention, the active compounds of the invention (inducers of type 1 interferon expression) may be formulated for administration via a variety of other routes, such as topical (i.e. transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation. Pharmaceutical compositions for topical administration may comprise an active compound configured for drug-containing applications such as ointments, pastes, creams or powders. Ointments include all oily, adsorptive, emulsion and water-soluble based compositions for topical application, while creams and lotions are those containing only an emulsion base. Topically applied drugs may contain permeation enhancers to promote absorption of the active ingredient through the skin. Suitable penetration enhancers include glycerol, alcohols, alkyl methyl sulfoxides, pyrrolidones, and laurocapram. Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorbent, lotion or water-soluble ointment base. Topical formulations may also include emulsifiers, gelling agents, and antimicrobial preservatives as needed to preserve the active ingredients and provide a homogeneous mixture. Transdermal administration of the present invention may also include the use of "patches". For example, the patch may provide one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.

In certain embodiments, the pharmaceutical composition may be delivered by eye drops, intranasal sprays, inhalants, and/or other aerosol delivery vehicles. Methods of delivering compositions directly to the lungs via nasal aerosol sprays have been described, for example, in U.S. patent nos. 5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety). Similarly, drug delivery using intranasal microparticle resins (Takenaga M, Serizawa Y, Azechi Y, Ochiai A, Kosaka Y, Igarashi R, Mizushima Y. microparticle resins as a potential nasal drug delivery system for insulin. J Control Release. 1998; 52:81-7.PMID:9685938) and lysophosphatidyl-glycerol compounds (U.S. Pat. No. 5,725,871, specifically incorporated herein by reference in its entirety) is also well known in the pharmaceutical arts. Similarly, transmucosal drug delivery in the form of a polytetrafluoroethylene support matrix is described in U.S. Pat. No. 5,780,045 (specifically incorporated herein by reference in its entirety). The term aerosol refers to a colloidal system of finely divided solids of liquid particles dispersed in a liquefied or pressurized gaseous propellant. A typical aerosol formulation of the invention for inhalation will consist of a suspension of the active ingredient in a liquid propellant or a mixture of a liquid propellant and a suitable solvent. Suitable propellants include hydrocarbons and hydrocarbon ethers. Suitable containers will vary depending on the pressure requirements of the propellant. Aerosol administration will vary depending on the age, weight and severity of symptoms and response of the subject.

Kits of the present disclosure

Any of the compositions described herein can be included in a kit. In a non-limiting example, a type 1 interferon expression inducer of the present invention (such as a BCG strain expressing one or more of the following proteins: RV1354c protein or a functional portion thereof; cyclic GMP-AMP synthase (DncV) protein or a functional portion thereof; cyclic GMP-AMP synthase (cGAS) protein or a functional portion thereof; DNA integrity scanning (dis) protein functioning as adenylate cyclase or a functional portion thereof) can be included in the kit.

The kit may comprise a suitable aliquot of an inducer of type 1 interferon expression of the invention, and in some cases, one or more additional agents. One or more components of the kit may be packaged in an aqueous medium or in lyophilized form. The container means of the kit will generally comprise at least one vial, test tube, flask, bottle, syringe or other container means in which the components may be placed and preferably suitably aliquoted. Where more than one component is present in the kit, the kit will typically further comprise a second container, a third container or other additional containers in which additional components may be separately placed. However, various combinations of components may be contained in the vial. The kit of the invention will also typically contain a means for containing an inducer of type 1 interferon expression of the invention and any other reagent containers within closed confines for commercial sale. Such containers may include injection or blow-molded plastic containers in which the desired vials are stored.

When the components of the kit are provided in one and/or more liquid solutions, the liquid solutions are aqueous solutions, with sterile aqueous solutions being particularly preferred. One or more type 1 interferon expression inducers of the compositions of the invention may be formulated into injectable compositions. In this case, the container means may itself be a syringe, pipette and/or other such similar device from which the formulation may be applied to an infected area of the body, injected into an animal, and/or even applied to and/or mixed with other components of the kit.

However, the components of the kit may be provided as one or more dry powders. When the reagents and/or components are provided as dry powders, the powders may be reconstituted by the addition of a suitable solvent. It is envisaged that the solvent may also be provided in another container means.

The examples above have been included to provide guidance to those of ordinary skill in the art for practicing representative embodiments of the presently disclosed subject matter. In light of the present disclosure and the general level of skill in the art, those of skill in the art will appreciate that the above embodiments are intended to be exemplary only and that numerous changes, modifications and alterations may be used without departing from the scope of the presently disclosed subject matter. The above embodiments are provided by way of illustration and not by way of limitation.

All references, including publications, patent applications, and patents, cited herein are hereby incorporated by reference to the same extent as if each reference were individually and specifically indicated to be incorporated by reference and were set forth in its entirety herein.

Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., "such as") provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

Preferred embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those preferred embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.