EP4103208A1 - Interventions thérapeutiques recombinantes contre le cancer - Google Patents

Interventions thérapeutiques recombinantes contre le cancer

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Publication number
EP4103208A1
EP4103208A1 EP21754580.5A EP21754580A EP4103208A1 EP 4103208 A1 EP4103208 A1 EP 4103208A1 EP 21754580 A EP21754580 A EP 21754580A EP 4103208 A1 EP4103208 A1 EP 4103208A1
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EP
European Patent Office
Prior art keywords
bcg
disa
cells
tumor
protein
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP21754580.5A
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German (de)
English (en)
Other versions
EP4103208A4 (fr
Inventor
William R. Bishai
Trinity J. BIVALACQUA
Alok Singh
Monali PRAHARAJ
Takahiro Yoshida
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Johns Hopkins University
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Johns Hopkins University
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Publication date
Application filed by Johns Hopkins University filed Critical Johns Hopkins University
Publication of EP4103208A1 publication Critical patent/EP4103208A1/fr
Publication of EP4103208A4 publication Critical patent/EP4103208A4/fr
Pending legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/74—Vectors or expression systems specially adapted for prokaryotic hosts other than E. coli, e.g. Lactobacillus, Micromonospora
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/66—Microorganisms or materials therefrom
    • A61K35/74—Bacteria
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/02—Bacterial antigens
    • A61K39/04—Mycobacterium, e.g. Mycobacterium tuberculosis
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/39—Medicinal preparations containing antigens or antibodies characterised by the immunostimulating additives, e.g. chemical adjuvants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/51—Medicinal preparations containing antigens or antibodies comprising whole cells, viruses or DNA/RNA
    • A61K2039/52—Bacterial cells; Fungal cells; Protozoal cells
    • A61K2039/523—Bacterial cells; Fungal cells; Protozoal cells expressing foreign proteins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/54—Medicinal preparations containing antigens or antibodies characterised by the route of administration
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/555—Medicinal preparations containing antigens or antibodies characterised by a specific combination antigen/adjuvant
    • A61K2039/55588—Adjuvants of undefined constitution
    • A61K2039/55594—Adjuvants of undefined constitution from bacteria

Definitions

  • JHU4280_2WO_Sequence_Listing.txt was created on February 11, 2021, and is 157 kb.
  • the file can be assessed using Microsoft Word on a computer that uses Windows OS.
  • Urothelial cancer of the bladder is the most common type of bladder cancer (BC) in North America, South America, Europe and Asia.
  • NMIBC Non-Muscle Invasive Bladder Cancer
  • BCG Bacillus Calmette Guerin
  • BCG has shown to be a very effective vehicle for delivery of antigens.
  • One embodiment of the present invention provides a vector including a nucleic acid sequence expressing a protein or functional part thereof that makes a STING agonist including 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, the product of the Vibrio cholerae DncV protein); 2’-3’cGAMP (also known as 2’-5’, 3’-5’ cGAMP, the product of the human cGAS protein) and a combination thereof, as examples.
  • 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, the product of the Vibrio cholerae
  • Some vectors of the present invention include a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rvl354c protein, or a functional part thereof; a second nucleic acid sequence encoding a 3’ -3’ cyclic GMP- AMP synthase (DncV) protein, or a functional part thereof; a third nucleic acid sequence encoding a 2’ -3’ cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (disA) protein, or a functional part thereof and a combination thereof.
  • a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rvl354c protein, or a functional part thereof; a second nucleic acid sequence encoding a 3’ -3’ cyclic GMP- AMP synthase (DncV) protein
  • nucleic acid sequences express proteins that make one or more of the STING agonist as described in the definition section of the specification.
  • Some vectors of the present invention include in addition to one or more of the sequences listed above a fifth nucleic acid sequence encoding a PanC protein and a PanD protein or functional part thereof.
  • Vectors including a nucleic acid sequence encoding a PanC protein and a PanD protein or functional part thereof are typically free of an antibiotic resistance gene.
  • Suitable vectors used in the present invention may include vectors that replicate episomally in multiple copies, or vectors that integrate into a bacterial chromosome in single copy or are otherwise present in the bacterial cell.
  • a vector of the present invention may stably integrate into a bacterial genome or it may stably replicate as an episomal plasmid.
  • Suitable third nucleic acid sequences include those that overexpress the cyclase domains of the cyclic GMP-AMP synthase (cGAS) protein.
  • Other suitable third nucleic acid sequence may express a cyclic GMP-AMP synthase (cGAS) protein having a regulatory DNA recognition capability that is non- functional.
  • Vectors of the present invention may also include nucleic acid sequences that encode sequences or proteins that knock out the expression of PDE genes of a strain of Mycobacteria used in the present invention.
  • Another embodiment of the present invention provides a strain of Mycobacteria including any one of the vectors of the present invention including a vector comprising a protein or functional part thereof that makes a STING agonist.
  • STING agonist include c-di-AMP (also known as 3’-5’ c-di-AMP); c-di-GMP (also known as 3’-5’ c-di- ;AD$4 ,j&,jP;5AD #NY ⁇ X[ ⁇ d[ N ⁇ ,l&.l% ,l&.lP;5AD% aUR ]_ ⁇ QbPa ⁇ S aUR Vibrio cholerae DncV protein); 2’-3’cGAMP (also known as 2’-5’, 3’-5’ cGAMP, the product of the human cGAS protein) and a combination thereof, as examples.
  • nucleic acid sequence includes a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof; a second nucleic acid sequence encoding a 3’-3’cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof; a third nucleic acid sequence encoding a 2’-3’cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (disA) protein, or a functional part thereof and a combination thereof.
  • a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof; a second nucleic acid sequence encoding a 3’-3’cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof; a third nu
  • strains of Mycobacterium used in the present invention include Mycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof, for example.
  • Another strain used in the present invention is Mycobacterium bacillus Calmette Guerin (BCG).
  • BCG Mycobacterium bacillus Calmette Guerin
  • a strain of Mycobacteria used in the present invention may be a panthothenate auxotroph of BCG lacking its panCD genetic operon.
  • panCD auxotoph strains lack genomic sequences able to encode functional PanC and/or PanD protein.
  • strains of Mycobacteria that are pantothenate auxotrophs comprise vectors of the present invention including a panCD nucleic acid encoding the PanC and PanD proteins or functional parts thereof.
  • Pantenate auxotrophs of the present invention are preferably free of a genomic antibiotic resistant gene or unable to encode functional proteins that provide antibiotic resistance.
  • Another embodiment of the present invention provides a pharmaceutical composition, including any one of the strains of Mycobacteria of the present invention, and a pharmaceutically acceptable carrier.
  • Another embodiment of the present invention provides a method of eliciting a type 1 interferon response, enhancing the expression of pro-inflammatory cytokine, and/or eliciting trained immunity in a subject including the steps of: administering a pharmaceutical composition including anyone of the strains of the present invention into a subject; and eliciting a type 1 interferon response, enhancing the expression of pro-inflammatory cytokine, and/or eliciting trained immunity in the subject.
  • the pharmaceutical composition is administered into the bladder of the subject by a catheter.
  • Another embodiment provides a method of using a strain of Mycobacteria of the present invention to treat or prevent cancer in a subject.
  • the method includes the steps of: administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein that makes a STING agonist or a functional part thereof to a subject having cancer; and treating or preventing cancer in the subject.
  • the present invention may be used to treat or prevent cancers including epithelial cancers, breast cancer, non-muscle invasive bladder cancer, as examples.
  • the cancer is a BCG-unresponsive non-muscle invasive bladder cancer (BCG-unresponsive NMIBC) and the pharmaceutical composition is administered by intravesical instillation.
  • BCG-unresponsive NMIBC BCG-unresponsive non-muscle invasive bladder cancer
  • the cancer is a BCG-na ⁇ ve non-muscle invasive bladder cancer (BCG-na ⁇ ve NMIBC) and the pharmaceutical composition is administered by intravesical instillation.
  • BCG-na ⁇ ve NMIBC BCG-na ⁇ ve non-muscle invasive bladder cancer
  • the pharmaceutical composition is administered by intravesical instillation.
  • the cancer is selected from the group consisting of colon cancer, uterine cancer, cervical cancer, vaginal cancer, esophageal cancer, nasopharyngeal cancer, endobronchial cancer, and a combination thereof and the pharmaceutical composition is administered to a luminal surface of the epithelial cancer.
  • the cancer is selected from a solid tumor or a liquid tumor and the pharmaceutical composition is administered by intratumoral injection and/or by systemic infusion.
  • the methods of the present invention may include the step of administering a checkpoint inhibitor, such as an anti-PD1 antibody, an anti- PDL1 antibody, or a combination thereof, as example.
  • a checkpoint inhibitor such as an anti-PD1 antibody, an anti- PDL1 antibody, or a combination thereof, as example.
  • the cancer is bladder cancer and the pharmaceutical composition is administered via a catheter.
  • One embodiment of the present invention provides an expression vector including a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof; a second nucleic acid sequence encoding a cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof; a third nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (disA) protein which functions as a diadenylate cyclase, or a functional part thereof, or a combination thereof.
  • DncV cyclic GMP-AMP synthase
  • cGAS cyclic GMP-AMP synthase
  • a fourth nucleic acid sequence encoding a DNA integrity scanning (disA) protein which functions as a diadenylate cyclase, or a functional part thereof, or a combination thereof.
  • Some expression vectors of the present invention include a first nucleic acid sequence that overexpresses the cyclase domains of the Rv1354c protein when compared to the expression of a native Rv1354c protein as a reference. Some expression vectors of the present invention include a second nucleic acid sequence that overexpresses the cyclic GMP-AMP synthase (DncV) protein, when compared to the expression of a native DncV protein. Some expression vectors of the present invention include a third nucleic acid sequence that overexpresses the cyclase domains of the cyclic GMP-AMP synthase (cGAS) protein when compared to the expression of a native cGAS protein.
  • DncV cyclic GMP-AMP synthase
  • Suitable Rv1354 proteins used in the present invention include a Mycobacterium tuberculosis Rv1354 protein.
  • Suitable DncV proteins used in the present invention include a Vibrio cholera DncV protein.
  • Suitable cGAS proteins used in the present invention include a Homo sapiens cGAS protein.
  • Suitable DisA proteins used in the present invention include a Mycobacterium tuberculosis disA protein.
  • Another embodiment of the present invention provides a strain of BCG including a cdnP gene, an Rv1354c gene, an Rv1357c gene, or a combination thereof, wherein the cdnP gene is unable to express a functional cyclic di-nucleotide phosphodiesterase (CdnP) protein, the Rv1354c gene is unable to express a functional Rv1345c protein, and/or the Rv1357c gene is unable to express a functional Rv1357 protein.
  • Some BCG strains of the present invention may have an Rv1354c gene that includes a non-functional EAL domain.
  • the BCG strains of the present invention may include any of the expression vectors of the present invention.
  • Another embodiment of the present invention provides a method of treating or preventing bladder cancer including the steps of: administering a pharmaceutical composition including a strain of BCG including an expression vector of the present invention into the bladder of a subject; and treating or preventing bladder cancer in the subject when compared to a reference subject who was not administered the pharmaceutical composition.
  • the pharmaceutical composition may be administered by any suitable means including by a catheter.
  • Another embodiment of the present invention provides a method of eliciting a type 1 interferon response in a subject including the steps of: administering a pharmaceutical composition including a strain of BCG including an expression vector of the present invention into the subject such as the subject’s bladder; and enhancing a type 1 interferon response in the subject compared to a reference subject not administered the pharmaceutical composition.
  • Another embodiment of the present invention provides a method of treating or preventing cancer in a subject including the steps of: administering a pharmaceutical composition comprising a strain of BCG including an expression vector of the present 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 the 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.
  • Mycobacteria used in the present invention examples include Mycobacterium tuberculosis, Mycobacterium bovis, Mycobacterium bovis Bacillus Calmette Guerin (referred to a BCG), Mycobacterium smegmatis, Mycobacterium avium complex, and other non-tuberculous mycobacteria (NTM).
  • BCG Mycobacterium bovis
  • NTM non-tuberculous mycobacteria
  • BCG strains used 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 Czechoslovakian), BCG Russia (also known as BCG Moscow), BCG Moreau (also known as BCG Brazil), BCG Japan (also known as BCG Tokyo), BCG Sweden (also known as BCG Gothenburg), BCG Birkhaug, BCG Glaxo, BCG Frappier (also known as BCG Montreal), BCG Phipps, or other available BCG strains.
  • BCG Pasteur BCG-Pasteur-Aeras
  • BCG Tice also known as BCG Chicago
  • BCG-Connaught also known as BCG Toronto
  • BCG Danish also known as BCG Toronto
  • BCG Danish also known as BCG Moscow
  • BCG Moreau also known as BCG Brazil
  • Another embodiment of the present invention provides a method of treating diabetes including the steps of: administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein or a functional part thereof that makes a STING agonist to a subject having diabetes; and treating or preventing diabetes in the subject by providing trained immunity.
  • Trained immunity refers to the ability of one antigenic stimulus to elicit more potent immune responses to a second, different antigenic stimulus introduced at a later time.
  • Trained immunity is antigen independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and is associated with epigenetic and metabolic changes. The method results in the upregulation of glycolysis mediated by the trained immunity.
  • Another embodiment of the present invention provide a method of stimulating trained immunity in a subject including the steps of: administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein or a functional part thereof that makes a STING agonist to a subject; and stimulating trained immunity in the subject. Upregulating glycolysis in the subject and/or stimulating episomal changes in histone methylation in the subject mediate trained immunity in the subject.
  • Another embodiment of the present invention provides a method of treating or preventing a viral infection in a subject including the steps of: administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein or a functional part thereof that makes a STING agonist to a subject; and treating or preventing the viral infection in the subject.
  • Stimulating trained immunity in the subject treats or prevents the viral infection in the subject. Upregulating glycolysis in the subject and/or stimulating episomal changes in histone methylation in the subject mediate trained immunity in the subject.
  • Another embodiment of the present invention provides a method of treating or preventing a bacterial infection, or a drug-resistant bacterial infection in a subject including the steps of: administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein or a functional part thereof that makes a STING agonist to a subject; and treating or preventing the bacterial infection or the drug-resistant bacterial infection in the subject.
  • Stimulating trained immunity in the subject treats or prevents the bacterial infection in the subject. Upregulating glycolysis in the subject and/or stimulating episomal changes in histone methylation in the subject mediate trained immunity in the subject.
  • the methods of the present invention may use one or more of the vectors of the present invention or one or more strain of bacteria including a vector of the present invention.
  • Another embodiment of the present invention provides a method of suppressing the expression of myeloid-derived suppressor cells (MDSCs), M2 macrophages, and Treg cells in a tumor and inducing the expression of macrophages, dendritic cells (DCs), and T effector cells in a tumor.
  • MDSCs myeloid-derived suppressor cells
  • M2 macrophages M2 macrophages
  • Treg cells in a tumor and inducing the expression of macrophages, dendritic cells (DCs), and T effector cells in a tumor.
  • DCs dendritic cells
  • the method includes the steps of administering a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein that makes a STING agonist or a functional part thereof to a subject having a tumor; suppressing the expression of MDSCs, M2 macrophages, and Treg cells in the tumor; and inducing the expression of macrophages, DCs, and T effector cells in the tumor.
  • a pharmaceutical composition including a strain of Mycobacteria including a vector expressing a protein that makes a STING agonist or a functional part thereof to a subject having a tumor; suppressing the expression of MDSCs, M2 macrophages, and Treg cells in the tumor; and inducing the expression of macrophages, DCs, and T effector cells in the tumor.
  • M1 macrophages having induced expression in a tumor includes M1 macrophages.
  • An example of T effector cells having induced expression in a tumor includes CD4+ T cells and CD8+ T cells.
  • Suppressing the expression of MDSCs, M2 macrophages, and Treg cells in the tumor of subjects administered a Mycobacteria including a vector of the present invention is observed when compared to the expression of MDSCs, M2 macrophages, and Treg cells in a tumor of a referenced subject not administered a pharmaceutical composition including the strain of Mycobacteria.
  • Inducing the expression of macrophages, DCs, and T effector cells in a tumor is observed when compared to the expression of macrophages, DCs, and T effector cells in a tumor of a referenced subject not administered a pharmaceutical composition comprising the strain of Mycobacteria.
  • STING agonist examples include 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 a combination thereof.
  • a suitable vector of the present invention may include a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof; a second nucleic acid sequence encoding a 3’-3’cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof; a third nucleic acid sequence encoding a 2’-3’ cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; a fourth nucleic acid sequence encoding a DNA integrity scanning (DisA) protein, or a functional part thereof and a combination thereof.
  • a nucleic acid sequence selected from the group consisting of a first nucleic acid sequence encoding a Rv1354c protein, or a functional part thereof; a second nucleic acid sequence encoding a 3’-3’cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof;
  • the tumor may be a epithelial cancer, a breast cancer, or a non-muscle invasive bladder cancer, and melanoma as examples.
  • the tumor may be a non-muscle invasive bladder cancer such as a BCG-unresponsive non-muscle invasive bladder cancer (BCG-unresponsive NMIBC) and the pharmaceutical composition can be administered by intravesical instillation.
  • the tumor may be a non-muscle invasive bladder cancer such as a BCG-na ⁇ ve non-muscle invasive bladder cancer (BCG-na ⁇ ve NMIBC) and the pharmaceutical composition can be administered by intravesical instillation.
  • the tumor may be an epithelial cancer selected from the group consisting of colon cancer, uterine cancer, cervical cancer, vaginal cancer, esophageal cancer, nasopharyngeal cancer, endobronchial cancer, and a combination thereof and the pharmaceutical composition can be administered to a luminal surface of the epithelial cancer.
  • the tumor is a solid tumor and the pharmaceutical composition is administered by intratumoral, intravenous, intradermal, transdermal, intravesical topical, intramuscular or subcutaneous injection.
  • the tumor is a liquid tumor and the pharmaceutical composition is administered by intravenous, intradermal, transdermal, intravesical topical, intramuscular or subcutaneous injection.
  • Methods of the present invention may further comprise the step of administering a checkpoint inhibitor.
  • Suitable checkpoint inhibitors that may be used in the present invention include ipilimumab (anti-CTLA-4 antibody), nivolumab (anti-PD-1 antibody), pembrolizumab (anti-PD-1 antibody), cemiplimab (anti-PD-1 antibody), atezolizumab (anti-PD-L1 antibody), avelumab (anti-PD-L1 antibody), durvalumab (anti-PD-L1 antibody) and a combination thereof.
  • FIGs 1A-1B Mycobacteria overexpressing disA from the pSD5B P hsp60 ::disA plasmid construct release large amounts of c-di-AMP into the macrophage cytosol and transcribe high levels of disA mRNA.
  • FIG. 1A Mycobacteria overexpressing disA from the pSD5B P hsp60 ::disA plasmid construct release large amounts of c-di-AMP into the macrophage cytosol and transcribe high levels of disA mRNA.
  • FIG. 1A Mycobacteria overexpressing disA from the pSD5B P hsp60 ::disA plasmid construct release large amounts of c-di-AMP into the macrophage cytosol and transcribe high levels of disA mRNA.
  • FIG.1B BCG-Pasteur harboring the pSD5B Phsp60::disA plasmid or BCG-Pasteur-WT were grown to mid-exponential phase. Bacteria were lysed and mRNA was prepared. The levels of disA mRNA were determined by quantitative RT-PCR. The BCG-disA-OE strain produces ⁇ 50-fold more disA mRNA than BCG-Pasteur-WT. [0023] FIG. 2.
  • BCG overexpressing disA augments pro-inflammatory cytokines.
  • Gene Re]_R ⁇ V ⁇ [ ]_ ⁇ SVYV[T # ⁇ D7F$ ⁇ S ]_ ⁇ &V[SYNZZNa ⁇ _f Pfa ⁇ XV[R ⁇ N[Q :B&n V[ Z ⁇ b ⁇ R 6A8A ⁇ challenged with wild-type and disA overexpression strains of BCG-Pasteur.
  • FIG. 3. BCG overexpressing disA augments IRF3 signaling. Effect of disA overexpression on activation of IRF pathway measured by IRF-SEAP QUANTI Blue reporter assay. The culture supernatants of infected RAW-Blue ISG cells were assayed for IRF activation.
  • FIGs 4A-4C Increased pro-inflammatory cytokines in response to disA overexpression.
  • FIG. 4A shows differential expression of TNF-".
  • FIG. 4B shows differential expression of IL-6.
  • FIG. 5 BCG overexpressing disA induces differential immune response in human bladder cancer cells (RT4).
  • RT4 bladder cancer cells PUNYYR[TRQ dVaU dVYQraf]R 67;&DN ⁇ aRb_% dVYQ&af]R 67;&HVPR ⁇ a_NV[% N[Q 67;&DN ⁇ aRb_&disA-OE 9e]_R ⁇ V ⁇ [ YRcRY ⁇ ⁇ S ZFB5 dN ⁇ ZRN ⁇ b_RQ b ⁇ V[T N GL6F T_RR[rON ⁇ RQ ⁇ bN[aVaNaVcR _RNYraVZR D7F' [0027] FIG.
  • FIG. 8 Immune profiling of MNU-induced Fisher rat urinary bladder tumors in response to intravesical therapy using different strains of BCG. Differential gene expression in rat bladder tumor cells after therapy with wild-type and disA overexpression strains of Mycobacterium bovis BCG-Pasteur.
  • FIG. 10 Summary of relative gene expression by 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 over 8 weeks and then treated with either BCG-disA-OE or BCG-WT by intravesical instillation for 8 weeks. Bladders were removed upon necropsy at week 16, and mRNA was prepared. Quantitative RT-PCR for the cytokine or chemokine genes indicated was performed The changes shown are the fold-induction or reduction observed with BCG-disA -OE normalized to that seen with BCG-WT.
  • BCG-WT is BCG Pasteur and BCG-disA -OE was derived from BCG Pasteur.
  • FIG. 11 Diagram of two cyclic dinucleotide cyclase and phosphodiesterase proteins present in BCG: 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. 12. M. tuberculosis harboring the pSD5B P hsP60 ::disA plasmid (M.tb-disA- OE or
  • Mtb-OE is significantly attenuated for virulence in mice compared to wild type M.tb (Mtb- CDC1551).
  • Day 1 CFU counts were performed on 3 mice in each group and confirmed the implantation of 3.5 loglO CFU units. Mice were held until death.
  • the median time to death for wild-type M. tuberculosis infection was 150.5 days.
  • mice infected with the same inoculum of M.tb-disA- OE (Mtb-OE) had a median time to death of 321.5 days (p ⁇ 0.001).
  • the BCG-JAA-OE is expected to show similar loss of virulence in mice compared with BCG-WT.
  • Data are from Dey B, Dey RJ, Cheung LS, Pokkali S, Guo H, Lee JH, and Bishai WR.
  • a bacterial cyclic dinucleotide activates the cytosolic surveillance pathway and mediates innate resistance to tuberculosis. Nat Med. 2015; 21: 401-6. PMID: 25730264.
  • FIGs 13A-13B Other BCG strains are also active: BCG Tice strain overexpressing disA also shows induction of proinflammatory cytokines similar to BCG Pasteur overexpressing disA. Bone marrow derived macrophages were challenged with wild-type and disA overexpressing strains of both BCG Pasteur and BCG Tice strains at an M.O.I of 1:20 for 15 h. Culture supernatants were harvested and probed for cytokines using ELISA.
  • FIG. 13A shows Bdifferential expression pattern of TNF- ⁇ .
  • FIG. 13B shows differential expression of IL-6.
  • Mouse BMDM were challenged with the two different strains of BCG. The BCG-Tice strain was from the commercially available Onco-Tice product.
  • FIG. 14 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 mouse were challenged with wild-type and disA OE strains of BCG Pasteur for 24 h. Culture supernatants were probed for IFN- b levels using ELISA. [0036] FIG. 15 shows that intravesical instillation of BCG-disA-OE displays greatest antitumor efficacy (statistically significant improvement in pathology) in the MNU carcinogen model of non-muscle invasive bladder cancer (NIMBC).
  • NIMBC non-muscle invasive bladder cancer
  • mice received 4 intravesical treatments with MNU over the first 8 weeks (one treatment every 2 weeks) to elicit NIMBC. Over the next 8 weeks they received 4 intravesical treatments with either PBS (untreated), BCG-WT, or BCG-disA-OE (one treatment every 2 weeks).
  • rats were sacrificed, and their bladders were removed. A portion of the bladder was fixed and subjected to H&E staining and then interpreted in a blinded fashion by a Board-certified urologic pathologist.
  • the tumor involvement score and cancer stage (T2-3, T1, CIS + papillary lesions, CIS alone, or normal-dysplastic) were determined and are shown.
  • FIG. 16 shows that BCG-disA-OE reduces Tregs (CD4 + CD25 + Foxp3 + ) in murine syngeneic bladder cancer tumors.
  • mice were implanted on the flank with 5 x 10 6 BBN975 murine bladder cancer tumor cells.
  • mice received 3 intratumoral injections of either PBS (control), BCG-WT, or BCG-disA-OE (one treatment every 2 days).
  • PBS control
  • BCG-WT BCG-disA-OE
  • mice were sacrificed, and their spleens and tumors were removed.
  • the cell preparations were stained and subjected to flow cytometry.
  • BCG-disA-OE led to reduced tumor CD4 + Tregs, reduced tumor CD8 + Tregs, and reduced spleen CD4 + Tregs.
  • FIGs 17A-17B show that BCG-disA-OE is safer than BCG-WT in two mouse models.
  • FIG.17A shows that groups of BALB/c mice (immunocompetent) were exposed to 1 x 10 3 CFU (confirmed by sacrificing a group of mice and determining day 1 lung CFU counts) of either BCG- WT or BCG-disA-OE using a Glas-Col aerosolization chamber. After 4 weeks, the mice were sacrificed from each group, their lungs were removed, homogenized, and plated on 7H11 agar plates. The figure shows the mean CFU counts for the BCG-WT and BCG-disA-OE-infected mouse lungs.
  • FIG. 17B shows that groups of SCID mice (immunosuppressed) were exposed to 1 x 10 2 CFU (confirmed by sacrificing a group of mice and determining day 1 lung CFU counts) of either BCG-WT or BCG-disA-OE using a Glas-Col aerosolization chamber. A third group was uninfected. The figure shows a Kaplan-Meier survival curve for the groups of mice. As may be seen BCG-disA-OE-infected mice had a statistically significantly longer survival time than BCG-WT-infected mice. [0039] FIG.
  • BCG-disA-OE elicits statistically significantly higher levels of “Trained Immunity immunological and epigenetic marks” in CD14 + human monocytes than does BCG-WT.
  • “Trained Immunity” refers to the ability of a first immunologic stimulus to induce increased immune responses to a second antigenically different stimulus give subsequently.
  • CD14 + human monocytes were prepared from LeukoPaks collected by apheresis. On day 0 they were infected with either BCG-WT or BCG-disA-OE at a MOI of 5:1 for 3 hours. A third group of cells were not infected. After infection, cells were washed multiple times (every two days).
  • FIG. 19 shows that BCG-disA-OE elicits a greater histone activation mark (H3K4- trimethylation) in the IL6 and TNF gene promoter regions than BCG-WT.
  • Trained Immunity refers to the ability of a first immunologic stimulus to induce increased immune responses to a second antigenically different stimulus give subsequently. Trained immunity has been associated with epigenetic modifications, such as histone methylation, in the promoter region of cytokines and other immune mediators.
  • the experiment shown in Figure 19 was performed in the same set of cells and exactly the same way as that described in Figure 18 except that after the second stimulus with the TLR1/2 agonist PAM3CSK4 (abbreviated PAM3), cells were harvested fixed, chromatins were cross-linked and DNA was collected for chromatin immunoprecipitation analysis (ChIP) using an antibody specific for the H3K4-me3 histone methylation mark.
  • H3K4-me3 is known to be a gene activating mark.
  • the graph shows the relative fold change in abundance of immunoprecipitated DNA as measured by quantitative PCR using primers for the IL6 and TNF gene promoter region.
  • FIG. 20 shows the successful construction of BCG-Tice-disA-OE.
  • BCG-Tice is manufactured and sold by Merck and is the sole FDA-approved BCG available in the United States.
  • the inventors purchased BCG-Tice, prepared electrocompetent BCG-Tice, and electroporated the pSD5-hsp60-MT3692 plasmid into BCG-Tice.
  • the drawing shows the results of colony PCRs for 5 kanamycin-resistant candidate clones of transformed BCG- Tice and confirms the successful preparation of BCG-Tice-disA-OE by electroporation of the pSD5-hsp65-MT3692 plasmid into BCG-Tice. Note on nomenclature, the inventors had previously referred to this same plasmid pSD5-hsp60-MT3692.
  • FIG.21 shows that clone 2 of BCG-Tice-disA-OE from the transformation experiment shown in Figure 20 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 the late log phase cultures using log phase cultures using SYBR green based quantitative real-time PCR. The graphical data points represent the mean of 3 independent experiments ⁇ standard error mean (SEM). M.
  • FIG. 22 shows potent, statistically significantly enhanced IRF3 induction in mouse bone marrow-derived macrophages infected with BCG-Pasteur-disA-OE compared with BCG- Pasteur-WT.
  • Mouse (C57BL/6) bone marrow-derived macrophages (BMDMs), and J774.1 macrophages were infected for 3h using different strains of BCG (MOI: 20).
  • FIG. 24A shows interferon-" levels in murine BMDMs.
  • FIG. 24B shows interferon-" levels in murine BMDCs.
  • FIG. 24C shows interferon-" levels in murine J774.1 macrophages.
  • FIGs 25A-25C show that IL-6 is induced in mouse BMDMs, BMDCs and J774.1 macrophages in response to exposure to disA overexpressing BCG strains and that the IL-6 response is statistically significantly greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains.
  • Mouse (C57BL/6) bone marrow-derived macrophages (BMDMs), and J774.1 macrophages were infected for 3 h using different strains of BCG (MOI: 20). Non-internalized bacilli were washed using warm DPBS and cell were incubated for another 24 hours.
  • FIG.25A shows IL-6 levels in murine BMDMs.
  • FIG.25B shows IL-6 levels in murine BMDCs.
  • FIG.25C shows IL-6 levels in murine J774.1 macrophages.
  • FIGs 26A-26C shows that TNF is induced in mouse BMDMs, BMDCs and J774.1 macrophages in response to exposure to disA overexpressing BCG strains and that the responses are statistically significantly greater for BCG-Pasteur-disA-OE and BCG-Tice-disA-OE than for the corresponding BCG-WT strains.
  • Mouse (C57BL/6) bone marrow-derived macrophages (BMDMs), and J774.1 macrophages were infected for 3h using different strains of BCG (MOI: 20). Non-internalized bacilli were washed using warm DPBS and cell were incubated for another 24 hours.
  • FIG.26A shows TNF levels in murine BMDMs.
  • FIG.26B shows TNF levels in murine BMDCs.
  • FIG.26C shows TNF levels in murine J774.1 macrophages.
  • NBT-II cells were infected with wild-type and recombinant strains of BCG for 3h.
  • FIG. 27A shows TNF levels in NBT-II cells.
  • FIGs 28A-28D shows that of IFN-b, IFN-g, TNF and IL-Ib in are induced the in the human transitional cell papilloma RT4 bladder cancer cell line in response to exposure to disA overexpressing BCG strains and that the two responses are greater for BCG - Pasteur- dis A -OE and B C G -Tice -d is A - O E than for the corresponding BCG-WT strains.
  • RT4 cells were infected with wild-type and recombinant strains of BCG for 3h. Non-internalized bacilli were repeatedly washed using warm DPBS and cells were incubated for another 24 h.
  • FIG. 28A shows IFN-b levels in RT4 cells.
  • FIG. 28B shows IFN-g levels in RT4 cells.
  • FIG. 28C shows TNF levels in RT4 cells.
  • FIG. 28D shows IL-Ib levels in RT4 cells.
  • FIG. 29 shows that BCG-disA -OE stimulates increased IFN-b levels in multiple bladder cancer cell lines to a greater degree than BCG-WT.
  • the drawing shows the levels of IFN- ⁇ mRNA (relative expression by the 2 ⁇ C T method) following exposure to BCG-WT, BCG-disA- OE, and LPS.
  • 5637 cells are human muscle-invasive bladder cancer cells
  • RT4 cells are human transitional cell papilloma bladder cancer cells
  • NBT-II cells are rat bladder carcinoma cells induced by N-butyl-N-(-4-hydroxybutyl) nitrosamine.
  • FIGs 30A-30D shows the cytokine responses for IFN-b, IFN-g, IL-6, and TNF in BCG- WT and BCG-disA-OE-infecte d mouse lungs at different time points following aerosol infection.
  • the drawing reveals that at most time points for most cytokines, the responses are greater for BCG- Pasteur-disA -OE and BCG-Tice-disA -O E than for the corresponding BCG-WT strains.
  • BALB/c mice were infected by the aerosol route as described in Figure 19. Groups of mice were sacrificed at 2, 4, and 6 weeks after infection.
  • FIG. 30A shows IFN-b levels in BCG-WT and B C G - d is A - O E-infected mouse lungs.
  • FIG. 30B shows IFN- g levels in BCG-WT and BCG-disA-OE-infected mouse lungs.
  • FIG. 30C shows IL-6 levels in BCG-WT and BCG-disA-OE-infected mouse lungs.
  • FIGs31A-31D shows TNF levels in BCG-WT and BCG-disA-OE-infected mouse lungs.
  • FIGs31A-31D showsthecytokineresponsesforIFN-b,IFN-g,IL-6,andTNFinBCG-WT and BCG-disA-OE-infected mouse spleens at 4 weeks following aerosol infection. Thedrawingrevealsthatformostcytokines,theresponsesaregreaterforBCG-Pasteur-disA-OEandBCG-Tice-disA-OEthanforthecorrespondingBCG-WTstrains.BALB/cmicewereinfectedbythe aerosolrouteas described in Figure 17.
  • FIG.31A showsIFN-blevelsinBCG-WTandBCG-disA-OE-infectedmousespleens.
  • FIG.31B showsIFN-glevelsinBCG-WTandBCG-disA-OE-mfectedmousespleens.
  • FIG.31C showsIL-6levelsinBCG-WTandBCG-disA-OE-infectedmousespleens.
  • FIG.31D showssTNFlevelsinBCG-WTandBCG-disA-OE-infectedmousespleens.
  • FIG.32 shows the strategy used to generate"pSD5.hsp65-disA.panCD-No Kan”(SEQIDNO: 31).
  • FIG.33 showsthemolecular structure ofthepJV53, therecombineeringplasmidwhichisSEQIDNO:32
  • FIG.34 shows the molecular structure ofthe pUC-Hyg, aplasmid with difsitesflankingaHygcassettewhichisSEQIDNO:35.pUC-Hygisusedtogeneratetheplasmid“pUC-Hyg-panCD-KO”(SEQIDNO:36).
  • FIG.35 shows themolecularstructureoftheplasmid“pUC-Hyg-panCD-KO”whichisSEQIDNO:36.“pUC-Hyg-panCD-KO”isgeneratedbycloning500bpofthepanCD5’UTRononeflankoftheHygcassette,andcloning500bpofthepanCD3’UTRtheotherflank.
  • FIG.36 shows themolecularstructureoftheplasmid“pSD5.hsp65-disA.Kan”whichisSEQIDNO:30.
  • FIG.37 shows themolecularstructureoftheplasmid“pSD5.hsp65-disA.panCD—NoKan”whichisSEQIDNO:31.Thisplasmidisgeneratedusingtheschemeillustratedin Figure32.
  • FIG.38 showsthenumberofpositivespecimens.
  • FIGs 39A-39C shows confirmation of M.tb-disA overexpression phenotype of BCG- disA-OE and induction of IRF signaling. Fig.
  • FIG. 39A shows colony PCR using Kanamycin gene specific primer confirms the presence of recombinant plasmid pSD5-hsp60-MT3692 in the BCG- disA-OE (Tice) clones selected against Kanamycin (25 qg/mL).
  • Fig. 39B shows real time PCR showing differential disA expression in different clones of BCG Tice BCG-disA-OE Tice. Transcript levels were measured in total RNA isolated from the late log phase cultures using log phase culture. M. tuberculosis sigA (Rv2703) was used as a reference gene, and relative expression mm7H was calculated by 2 method.
  • FIGs 40A-40C show BC G i i di AMP as strong inducers of type I interferon in STING-dependent manner.
  • HMDMs human monocyte-derived macrophages
  • Fig. 41A shows measurements 24 h post- infection with BCG-disA-OE (Tice).
  • Fig.41B shows measurements 24 h post-infection with BCG- disA-OE (Pasteur).
  • C-D Quantitative measurement of IL-6 in culture supernatants of wild-type C57BL/6-derived BMDMs, BMDCs, J774.1 macrophages and human monocyte-derived macrophages (HMDMs).
  • Fig. 41C shows measurements 24 h post-infection with BCG-disA-OE (Tice).
  • FIGs 43A-43E show BCG-disA-OE induces significantly higher Th1 cytokines and chemokines as compared to WT BCG.
  • Fig. 43A shows relative gene expression analyses of different cytokines and chemokines in IFN-k activated macrophages at 6 h post-infection by wild- type BCG (Tice) and BCG-disA-OE (Tice) strains.
  • FIGs 44A-44C show differential apoptotic induction in murine BMDMs and J774.1 macrophage after infection with different BCG strains.
  • FIG. 44A shows measurements in murine BMDMs.
  • Fig. 44B shows measurements in J774.1 macrophages.
  • FIGs 45A-45C show internalization and differential toxicity of WT and BCG-disA-OE strains in human urothelial carcinoma cells.
  • A-C Cell viability of RT4 (Human bladder cancer cell line representing grade I carcinoma), 5637 (Human bladder cancer cell line representing grade II carcinoma) and J82 (Human bladder cancer cell line representing grade III) cells exposed to different MOIs of wild-type BCG. Cell viability was measured using CellTiter-Glo Luminescent Cell Viability assay.
  • Fig. 45A shows cell viability of RT4 cells.
  • Fig. 45B shows cell viability of 5637 cells.
  • Fig.45C shows cell viability of J82 cells.
  • FIGs 46A-46D show BCG Tice overexpressing c-di-AMP as a stronger inducer of antitumor cytokine response in urothelial carcinoma cells.
  • A-D Quantitative measurement of differential TNF-e, IL-6, IL-1f and IFN-k levels using ELISA in different urothelial carcinoma cells 24 h after infection with different wild-type BCG (Tice) and BCG-disA-OE (Tice) strains.
  • FIGs 47A-47G show BCG Pasteur overexpressing c-di-AMP as a stronger inducer of antitumor cytokine response in urothelial carcinoma cells.
  • Fig. 47A shows TNF-e levels in 5637 cells.
  • Fig. 47B shows TNF-e levels in 75 cells.
  • Fig. 47C shows Il-6 levels in UPPL1595 cells.
  • Fig. 47D shows IL-1f levels in MB49 cells.
  • Fig.47E shows IL-1f levels in UPPL1595 cells.
  • Fig. 47F shows IFN-k levels in BBN975 cells.
  • Fig.47G shows IFN-k levels in NBT-II cells.
  • FIGs 48A-48B show stronger macrophage reprogramming towards M1 phenotype after infection with BCG strains overexpressing c-di-AMP.
  • Fig. 48A shows wild-type BMDMs infected with different BCG strains and for 24 h at 1:10 MOIs. Cell surface and intracellular straining was carried out and cells were analyzed using flow-cytometry (BD LSR II flow cytometer. Bar diagram showing percentage of TNF- ⁇ positive antigen presenting mouse macrophages (MHC Class II CD lib F4/80 ) following infection with wild-type and c-di-AMP overexpressing BCG Tice and Pasteur strains. Data were processed using FlowJo software (Tree Star vlO). Fig.
  • FIGs 49A-49B show stronger reprogramming of M2 macrophages after infection with BCG strains overexpressing c-di-AMP.
  • Fig. 49A shows percentage of M2 macrophage surface markers (CD206 CD124 ) positivity on mouse BMDM macrophages (CDllb F4/80 ) after infection with wild-type.
  • Fig. 49B shows percentage of IL-10 producing macrophages of M2
  • CD206 CD 124 mouse macrophages CD206 CD 124 mouse macrophages population.
  • FIGs 50A-50B show stronger induction of monocytic MDSCs secreting IL-10 in murine BMDMs after infection with wild-type and c-di-AMP overexpressing BCG strains.
  • Fig. 50A shows the percentage of M-MDSCs of total myeloid cells (CD45+).
  • Fig. 50B shows the percentage of IL-10 producing M-MDSCs after infection of murine BMDMs with WT and BCG- disA- OE strains. Briefly, wild-type BMDM macrophages were infected with different BCG strains and for 24 h at 1:10 MOIs. Cell surface and intracellular straining was carried out and cells were analyzed using flow-cytometry (BD LSR II flow cytometer.
  • BD LSR II flow cytometer flow-cytometry
  • FIGs 51A-51B show differential induction of classical (inflammatory) monocytes after infection with wild-type and c-di-AMP overexpressing BCG Tice.
  • Fig. 51A shows a bar diagram
  • Fig. 51B shows representative flow-cytometry plots showing different percentage of monocyte populations.
  • Cell surface staining was carried out and cells were analyzed using flow- cytometry (BD LSR II flow cytometer). Data were processed using FlowJo software (Tree Star v10).
  • FIGs 52A-52B show BCG overexpressing c-di-AMP as a potent inducer of proinflammatory cytokines in human monocyte-derived macrophages.
  • Fig. 52A is a bar diagram showing percentage of MHC class II positive classical macrophages producing TNF-e (TNF- + + + - + + + - e HLA-DR /CD14 CD16) and IL-6 (IL-6 HLA-DR /CD14 CD16). Briefly, human monocytes were isolated from PBMCs drawn from different healthy blood donors.
  • FIGs 53A-53C show BCG overexpressing c-di-AMP strongly suppressing M2 macrophage phenotypes.
  • Fig. 53A shows percentage of immunosuppressive M2 (CD206+CD163+) macrophages of total transitional (CD14+CD16+) macrophages.
  • Fig. 53B shows percentage of IL-10 producing macrophages of total M2 macrophages (CD206+CD163+) after infection of HMDMs with WT and BCG-disA-OE Tice strains.
  • Fig.53C shows representative flow-cytometry plots showing M2 cell surface phenotypes and IL-10 producing cells of M2 macrophages. Briefly, human monocytes were isolated from PBMCs drawn from different healthy blood donors. Negatively selected human monocytes were differentiated into M2 macrophages in presence of M-CSF.
  • FIGs 54 shows BCG overexpressing c-di-AMP infected macrophages enhanced phagocytosis. HMDMs were infected with WT BCG and BCG-disA-OE strains for 6 h and phagocytic activity was measured by quantifying intracellular FITC-labeled IgG-opsonized latex beads.
  • FIGs 55A-55B show BCG overexpressing c-di-AMP as a potent inducer of proinflammatory cytokines in primary human monocytes.
  • BCG-disA-OE induces significantly higher gene expression of TNF-e and IL-6 in primary human monocytes as compared to WT BCG.
  • TNF-e and IL-6 expression was accessed in primary human monocytes isolated from different healthy donors using qRT-PCR.
  • RNU6A was used as reference gene, and relative mm7H expression was calculated by 2 method.
  • Fig. 55A shows TNF-e levels.
  • Fig.55B shows IL-6 levels.
  • FIGs 56A-56E show BCG overexpressing c-di-AMP as a stronger inducer of trained immunity epigenetic marks in human monocytes after training.
  • Fig. 56A shows H3K4me3 levels on TNF-e gene promoter.
  • Fig.56B shows H3K4me3 levels on IL-6 gene promoter.
  • FIGs 57A-57C shows BCG overexpressing c-di-AMP demonstrating improved antitumor activity in the MNU carcinogen model of NMIBC.
  • Fig.57A Schematic of intravesical treatment strategy of BCG in MNU carcinogen model of NMIBC.
  • Fig.57B is a graph bar showing tumor involvement index.
  • Fig. 57C is a graph showing staging of tumors.
  • FIG 58A 58C h BCG di A OE strains attenuated for virulence in vivo.
  • Fig.58A illustrates BALB/c BCG aerosol challenge model.
  • Fig. 58C shows implantation (day 01) of BCG strains following aerosol challenge in BALB/c mice.
  • FIGs 59A-59C show BCG strain overexpressing c-di-AMP attenuated for virulence in a severely immunocompromised (SCID) mouse model of aerosol infection.
  • Fig. 59A illustrates SCID mice model of BCG aerosol infection.
  • Fig. 59C shows implantation (day 01) of BCG strains following aerosol challenge in SCID mice.
  • FIGs 60A-60D show BCG overexpressing c-di-AMP as a stronger inducer of Th1 cytokines in vivo.
  • A-B Quantitative levels of IFN-f, TNF-e, IL-6 and IFN-k in lung homogenates from BALB/c mice at 4 weeks after infection with different BCG strains using ELISA.
  • Fig. 60A shows results with WT BCG (Tice) and BCD-disA-OE (Tice).
  • 60B shows results with WT BCG (Pasteur) and BCD-disA-OE (Pasteur).
  • C-D Quantitative levels of IFN-f, TNF-e, IL-6 and IFN-k in lung homogenates from BALB/c mice at 4 weeks after infection with different BCG strains using ELISA.
  • Fig.60C shows results with WT BCG (Tice) and BCD-disA-OE (Tice).
  • FIGs 61A-61B show therapeutic intratumoral injection of BCG overexpressing c-di- AMP leading to greater antitumor activities in MB49 model of bladder cancer.
  • Fig.61A shows a 5 schematic representation of intratumoral injection of tumors. Mice were implanted with 1x10 3 MB49 cells on day 0, then accessed for tumor volume until the group averaged ⁇ 40mm . At that 6 time, mice were treated with 5x10 wild-type or BCG-disA-OE strain in a total volume of 50 $L rd or with PBS alone every 3 day for a total of 3 treatments.
  • FIG. 61B shows tumor outgrowth of MB49 bearing animals treated with vehicle (PBS), wild-type and BCG-disA-OE following treatments as shown in figure 61A. Two-way ANOVA.
  • FIGs 62A-62D show BCG overexpressing c-di-AMP inducing stronger infiltration of IFN-# at tumor site following intratumoral administration.
  • A-B Dot plot showing relative abundance of percentage of CD4 or CD8 of total CD3 populations in single cells isolated from tumor.
  • Fig.62A shows percentage of CD4 cells.
  • Fig. 62B shows percentage of CD8 cells.
  • C-D Dot plot showing higher percentage of interferon-# producing CD4 T cells inside single cells isolated from tumors receiving BCG-disA-OE.
  • agent any small molecule chemical compound, antibody, nucleic acid molecule, or polypeptide, or fragments thereof.
  • alteration is meant a change (increase or decrease) in the expression level or activity of a gene or polypeptide as detected by standard methods known in the art such as those described herein. As used herein, an alteration includes a 10% change in expression level, preferably a 25% change, more preferably a 40% change, and most preferably a 50% or greater change in expression level.
  • ameliorate decrease, suppress, attenuate, diminish, arrest, or stabilize the development or progression of a disease.
  • analog is meant a molecule that is not identical but has analogous functional or structural features.
  • a polypeptide analog retains the biological activity of a corresponding naturally occurring polypeptide, while having certain biochemical modifications that enhance the analog's function relative to a naturally occurring polypeptide. Such biochemical modifications could, for example, increase the analog's protease resistance, membrane permeability, or half-life, without altering, for example, ligand binding.
  • An analog may include an unnatural amino acid, in another example.
  • cdnP is meant either 1) a cdnP gene or nucleic acid sequence that encodes a cyclic di-nucleotide phosphodiesterase (cdnP) protein or 2) the cyclic di-nucleotide phosphodiesterase protein.
  • examples include the M. tuberculosis cdnP gene in H37Rv, Rv2837c, having NCBI Gene ID 888920, and a cdnP protein of UniProtKB/Swiss-Prot P71615.2.
  • cGas is meant either 1) a cGas gene or nucleic acid sequence that encodes a cyclic GMP-AMP synthase (cGAS) protein, or 2) the cyclic GMP-AMP synthase protein.
  • cGas include the H. sapiens cGAS gene (NCBI Gene ID: 115004) and the protein encoded by this gene (UniProtKB/Swiss-Prot: Q8N884.2).
  • the cGas protein is a cyclic GMP-AMP synthase from humans that makes 2’3’cGMP. 2’3’cGMP is a STING agonist in humans.
  • Cyclase domains of cGAS for example, refers to a portion or fragment of the 522 amino acids of the human cGAS protein described in Kranzusch et al. (Cell Reports 2013; 3:1362- 1368 PMID 23707061).
  • a cyclase domain may be described as having an NTase core situated from amino acid 160-330, and a regulatory-sensor domain that is the C-domain situated from amino acids 330-522. Mutants of the NTase core sequence as well as mutants of the regulatory- sensor domain can be used to generate constitutively active variants of cGAMP designed to produce high levels of cGAMP without the normal requirement for activation by DNA binding.
  • a cyclase domain includes M. tuberculosis Rv1354c of NCBI Gene ID: 887485, and the protein encoded by this gene (UniProtKB/Swiss-Prot: P9WM13) that encodes a 623 amino acid-long protein capable of both c-di-GMP (cyclic diguanylate or cyclic di-GMP) synthesis (via its GGDEF domain, amino acids 201-400) and degradation (via its EAL domain, amino acids 401-623).
  • the GAF domain (amino acids 1-200) is a regulatory domain.
  • the GGDEF domain as well as mutants of 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” is meant either 1) a Dis A gene or nucleic acid sequence that encodes a DNA integrity scanning (DisA) protein or 2) the DNA integrity scanning protein. Examples include M. tuberculosis disA gene Rv3586 of NCBI Gene ID: 887485, and the protein encoded by this gene is UniProtKB/Swiss-Prot: P9WNW5.1.
  • the protein is a 358 amino acid-long diadenylate cyclase as described by Dey & Bishai et al. Nature Medicine 2015;21:401-6. PMID: 25730264.
  • a DisA protein is a diadenylate cyclase that makes c-di-AMP.
  • c-di-AMP is a STING agonist.
  • 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 is meant a gene that encodes a Cyclic GMP-AMP synthase that catalyzes the synthesis of 3'3'-cyclic GMP-AMP (3'3'-cGAMP) from GTP and ATP, a second messenger in cell signal transduction.
  • dncV is also able to produce c-di-AMP and c-di-GMP from ATP and GTP, respectively; however, 3'3'-cGAMP is the dominant molecule produced by DncV in vivo, contrary to the 2'3'-cGAMP produced by eukaryotes.
  • dncV isrequired for efficient V.cholerae intestinal colonization, and down-regulates the colonization-influencing process of chemotaxis.
  • dncV is not active with dATP, TTP, UTP, and CTP.
  • the DncV protein is a cyclic GMP-AMP synthase from V.cholerae that makes 3’3’cGAMP.
  • 3’3’cGAMP is a STING agonist. signaling proteins.
  • the EAL domain may function as a diguanylate phosphodiesterase and has been shown to stimulate degradation of a second messenger, cyclic di-GMP. A non-functional EAL domain will not have one or more of these functions.
  • An example of an EAL domain includes M.
  • tuberculosis Rv1357c gene of NCBI Gene ID: 886815 and the 307 amino acid-long protein encoded by this gene is UniProtKB/Swiss-Prot: P9WM07 that encodes a c-di-GMP phosphodiesterase (PDE) and is comprised of a sole EAL domain.
  • PDE c-di-GMP phosphodiesterase
  • This enzyme activity is to serve as a c-di-GMP phosphodiesterase, cleaving the cyclic dinucleotide (which has signaling activity) into 2 GMP molecules (which lack signaling activity), as described in the article titled, “A full-length bifunctional protein involved in c-di-GMP turnover is required for long-term survival under nutrient starvation in Mycobacterium smegmatis,” Bharati BK, Sharma IM, Kasetty S, Kumar M, Mukherjee R, Chatterji D. Microbiology. 2012 Jun;158(Pt 6):1415-27. doi: 10.1099/mic.0.053892-0. Epub 2012 Feb 16.PMID: 22343354.
  • EAL domain includes the 336 amino acid-long protein encoded by M. tuberculosis cdnP gene in H37Rv (Rv2837c), a c-di-AMP phosphodiesterase comprising an EAL domain with the capability of hydrolyzing human 2’-3’cGAMP (the product of the human cGAS enzyme) as shown by Jain-Dey Bishai et al. Nat Chem Biol.2017;13:210-217 PMID 28106876.
  • EAL domains cyclic dinucleotide phosphodiesterase activity
  • GGDEF domains cyclic dinucleotide cyclization-biosynthetic activity
  • dncV is meant either 1) a dncV gene or nucleic acid sequence that encodes a cyclic GMP-AMP synthase (DncV) protein, or 2) the Cyclic GMP-AMP synthase protein. Examples include, but are not limited to, the Vibrio cholerae dncV gene of NCBI Gene ID: 2614190 and the protein encoded by this gene is UniProtKB/Swiss-Prot: Q9KVG7.1 [0098] By “fragment” is meant a portion of a polypeptide or nucleic acid molecule.
  • This portion contains, preferably, at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the entire length of the reference nucleic acid molecule or polypeptide.
  • a fragment may contain, 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.
  • gene deletion is meant using allelic exchange methodologies well-known to one skilled in the art to delete the full gene coding region of the gene of interest from the chromosome of BCG.
  • Gene replacement with selectable markers such as antibiotic resistance cassettes is a form of allelic exchange and may be performed. Technologies are also available to generate unmarked deletions (no selectable marker) in which the gene is entirely deleted, and no selectable marker is introduced in its place.
  • gene domain deletion is meant using the above allelic exchange methodologies to remove the portion of a gene encoding a particular domain (in the case of the present invention the EAL domain of Rvl354c which encodes the CDN phosphodiesterase domain of a multifunctional polypeptide) leaving the other portions of the polypeptide intact and in frame.
  • H . sapiens is meant Homo sapiens.
  • obtaining as in “obtaining an agent” is meant synthesizing, purchasing, or otherwise acquiring the agent.
  • overexpression is meant, in a general sense, a gene expressing its corresponding protein in a greater quantity than a wild type or reference gene.
  • An example of creating a gene overexpressing a protein in the present invention includes fusing the DNA encoding the gene of interest to a strong promoter in BCG such as Phsp60 or to a strong conditionally active promoter such as PtetOFF.
  • PtetOFF gene expression is turned off in the presence of tetracycline, anhydrotetracycline, or doxycycline; however, when the recombinant BCG is administered as an immunotherapy in a human or an animal model, the gene of interest will be turned on.
  • This conditionally active strategy has the advantage of preventing any deleterious effects on viability or growth rate that strong overexpression of cyclic dinucleotide producing enzyme might have on the BCG organisms while the BCG is being grown, and it allows for strong expression (“overexpression”) only when the BCG immunotherapy is given as a therapeutic to a mammalian host.
  • Mcb Mycobacterium tuberculosis.
  • polypeptide polypeptide
  • peptide protein
  • the terms “polypeptide,” “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an analog or mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.
  • Polypeptides can be modified, e.g., by the addition of carbohydrate residues to form glycoproteins.
  • the terms “polypeptide,” “peptide” and “protein” include glycoproteins, as well as non-glycoproteins.
  • By “reduce” or “decrease” is meant a negative alteration of at least about 10%, 25%, 50%, 75%, or 100%, for example, or any percentage in between.
  • By “increase” is meant a positive alteration of at least about 10%, 25%, 50%, 75%, or 100%, for example, or any percentage in between.
  • By “reference” is meant a standard or control condition.
  • a "reference sequence” is meant a defined sequence used as a basis for sequence comparison.
  • a reference sequence may be a subset of or the entirety of a specified sequence, for example, a segment of a full-length cDNA or gene sequence, or the complete cDNA or gene sequence.
  • the length of the reference polypeptide sequence will generally 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.
  • 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 thereabout or there between.
  • reference BCG strain is meant, for example, a conventional BCG strain that does not contain the expression vectors of the present invention and/or the endogenous genes unable to express a cdnP functional protein, a Rv1354c functional protein, a Rv1357c functional protein, or a combination thereof.
  • regulatory DNA recognition capability is meant the ability of a protein to detect or bind DNA.
  • a cGAS protein is known to bind DNA, such as cytosolic DNA, and triggers the reaction of GTP and ATP to form cyclic GMP-AMP (cGAMP).
  • cGAMP binds to the Stimulator Interferon Genes (STING) which triggers phosphorylation of IRF3 via TBK1.
  • Rv1354c is meant either 1) a Rv1354c gene or nucleic acid sequence that encodes a Rv1354c protein or 2) the Rv1354c protein (e.g., Gupta, Kumar, and Chatterji; PLoS ONE (November, 2010); Vol. 5; Issue 11; and Bhariati, Sharma, Kasetty, Kumar, Mukherjee, and Chatterji; Microbiology (2012), 158, 1415-1427).
  • the Rv1354c protein is a diguanylate cyclase that mkes c-di-GMP.
  • C-di-GMP is a STING agonist.
  • Rv1357c is meant either 1) a Rv1357 gene or nucleic acid sequence that encodes a cyclic di-GMP phosphodiesterase protein (Rv1357) protein or 2) the cyclic di-GMP phosphodiesterase protein (e.g., Gupta, Kumar, and Chatterji; PLoS ONE (November, 2010); Vol. 5; Issue 11; and Bhariati, Sharma, Kasetty, Kumar, Mukherjee, and Chatterji; Microbiology(2012), 158, 1415-1427).
  • the Rv1357c protein is a diguanylate cyclase that mkes c-di-GMP.
  • C-di-GMP is a STING agonist.
  • STING agonist is meant a molecule which binds to STING (stimulator of interferon genes, or TMEM173), activates it, and triggers activation of the IRF3-TBK1 pathway leading to increased transcription of type 1 interferon and other genes.
  • CDN cyclic dinuculeotide such as 3’-5’ c-di-AMP, 3’-5’ c-di-GMP, ,j&,j P;5AD #NY ⁇ X[ ⁇ d[ N ⁇ ,l&.l% ,l&.lP;5AD% aUR ]_ ⁇ QbPa ⁇ S aUR Vibrio cholerae DncV protein), or 2’-3’ cGAMP (also known as 2’-5’, 3’-5’ cGAMP, the product of the human cGAS protein).
  • PAMP pathogen associated molecular pattern.
  • PAMPs are microbial products including small molecules which are recognized by innate immune sensors. Examples of PAMPs are 3’-5’ c-di-AMP, 3’-5’ c-di-GMP, 3’-3’ cGAMP.
  • DAMP danger associated molecular pattern. DAMPs are host-derived (that is human, mouse, or other mammalian model of disease) molecules that are produced to signal danger such as infection or other derangement of normal physiology. An example of a DAMP is 2’-3’ cGAMP which is produced by the host sensor enzyme cGAS upon detection of double- stranded DNA in the cytosol as occurs during viral or certain intracellular bacterial infections.
  • panCD is meant the genetic operon from bacteria or other species the encodes the biosynthetic gene panC (encoding the PanC protein which has pantoate-beta-alanine ligase enzymatic activity) and the biosynthetic gene panD (encoding the PanD protein which has aspartate 1-decarboxylase enzymatic activity).
  • the PanC and PanD proteins are required for the biosynthesis of pantothenic acid or pantothenate also called vitamin B5 (a B vitamin).
  • Pantothenic acid a water-soluble vitamin, is an essential nutrient for bacteria and for all mycobacteria including BCG.
  • Pantothenic acid is required in order to synthesize coenzyme-A (CoA), as well as to synthesize and metabolize proteins, carbohydrates, and fats.
  • CoA coenzyme-A
  • specifically binds is meant a compound, nucleic acid, peptide, protein, or antibody, for example, that recognizes and binds a polypeptide or nucleic acid sequence, but which does not substantially recognize and bind other molecules in a sample.
  • substantially identical is meant a polypeptide or nucleic acid molecule exhibiting at least 50% identity to a reference amino acid sequence (for example, any one of the amino acid sequences described herein) or nucleic acid sequence (for example, any one of the nucleic acid sequences described herein).
  • such a sequence is at least 60%, more preferably 80% or 85%, and more preferably 90%, 95% or even 99% identical at the amino acid level or nucleic acid to the sequence used for comparison.
  • Sequence identity is typically measured using sequence analysis software (for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs).
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs.
  • sequence analysis software for example, Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP/PRETTYBOX programs.
  • Such software matches identical or similar sequences by assign
  • 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.
  • a BLAST program may be used, with a probability score between e -3 and e -100 indicating a closely related sequence.
  • 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.
  • sensitivity is meant the percentage of subjects with a particular disease.
  • specificity is meant the percentage of subjects correctly identified as having a particular disease, i.e., normal or healthy subjects. For example, the specificity is calculated as the number of subjects with a particular disease as compared to normal healthy subjects (e.g.,non- cancer subjects ).
  • Trained immunity is meant the ability of one antigenic stimulus to elicit more potent immune responses to a second, different antigen administered at a later time. Trained immunity is antigen-independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and is associated with epigenetic and metabolic changes.
  • Phsp60 or “Phsp65” is meant a strong mycobacterial promoter derived from the Mycobacterium leprae Hsp655’UTR.
  • 5’UTR is meant the 5’ untranslated region of a gene.
  • 3’UTR is meant the 3’ untranslated region of a gene.
  • WT is meant wild type.
  • BCG-WT is meant a wild type strain of Mycobacterium bovis bacillus Calmette Guerin.
  • a range of 1 to 50 is to be 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.
  • the terms “treat,” “treating,” “treatment,” and the like refer to reducing or ameliorating a disorder and/or symptoms associated therewith. It will be appreciated that, although not precluded, treating a disorder or condition does not require that the disorder, condition or symptoms associated therewith be completely eliminated.
  • compositions or methods provided herein can be combined with one or more of any of the other compositions and methods provided herein.
  • the terms “prevent,” “preventing,” “prevention,” “prophylactic 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 or susceptible to developing a disorder or condition.
  • Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for bladder cancer or disease, disorder, or symptom thereof. Determination of those subjects "at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g . , genetic test, enzyme or protein marker, a marker (as defined herein), family history, and the like).
  • determination of subjects susceptible to or having a urothelial cancer is determined by measuring levels of at least one of the markers.
  • the present invention relates to genetic alterations of Mycobacterium bovis BCG (hereafter, “BCG”) which generate recombinant BCG (hereafter “rBCG”) strains. These strains have greater potency as (i) tuberculosis vaccines and/or (ii) immunotherapies for non-muscle invasive bladder cancer (NMIBC).
  • BCG Mycobacterium bovis BCG
  • rBCG recombinant BCG
  • NMIBC non-muscle invasive bladder cancer
  • Some embodiments of the present 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 others.
  • CDNs cyclic dinucleotides
  • Another embodiment of this invention is to combine genetic modifications of BCG to generate multivalent CDN- overexpression modifications that include addition of novel CDN-synthesizing genetic material and/or mutations of endogenous BCG phosphodiesterase genes or genetic domains that will enhance the accumulation and release of CDNs.
  • BCG Bacillus Calmette Guerin
  • BCG Bacillus Calmette Guerin
  • bovis and the various BCG strains include the deletion of at least 15 regions of difference that comprise genomic deletions in BCG compared with virulent M. tuberculosis.
  • Key regions of difference in the development of BCG were RD1 (9.5 kb deletion leading to loss of the Esx-1 secretion system and inability to release antigens ESAT-6 and CFP-10) and RD3 (9.2 kb deletion). Regions of difference RD4-RD11 are absent in all BCG strains compared with virulent M. tuberculosis.
  • BCG tuberculosis
  • BCG has also achieved wide use as a cancer immunotherapy for nonmuscle invasive bladder cancer (NMIBC). It is given intravesically weekly for six weeks and in some instances, such as high-risk disease, it is given as maintenance therapy weekly for three weeks at 3, 6, 12, 18, 24, 30, and 36 months after initial therapy. Intravesical BCG has been shown to (i) induce a mononuclear cell infiItrate comprised predominantly of CD4 T cells and macrophages, (ii) increase the expression of interferon gamma (IFN ⁇ ) in the bladder, and (iii) increase urinary cytokine levels of IL-1, IL-2, IL-6, IL-8, IL-12, IFN ⁇ , and TNF ⁇ .
  • IFN ⁇ interferon gamma
  • BCG Despite the wide global use of BCG as (i) a vaccine for TB and (ii) an immunotherapy for NMIBC, there is considerable room for improvement in its efficacy.
  • BCG gives only partial protection predominantly against childhood disseminated tuberculosis.
  • NMIBC approximately 30% of patients have BCG-resistant disease. These individuals require riskier treatments with systemic chemotherapy and have higher rates of progression to more invasive forms of bladder cancer.
  • Urothelial cancer of the bladder is the most common malignancy of the urinary tract. It is the fourth most common cancer in males and 11th most common in females. It is estimated that approximately 79,000 new cases of bladder cancer will be diagnosed in the USA in 2017, associated with 19,870 deaths. Although the estimated five-year survival for bladder cancer patients is 78%, the rates decline dramatically for patients with locally advanced or metastatic disease. Approximately 75% of patients with bladder cancer present with a disease that is confined to the mucosa (stage Ta, carcinoma in situ ) or submucosa (stage Tl), known as non-muscle invasive bladder cancer (NMIBC). Transurethral resection is the initial treatment of choice for NMIBC.
  • MIBC muscle invasive bladder cancer
  • T2 muscle invasive bladder cancer
  • the first-line treatment option is platinum-containing chemotherapy followed by bladder removal.
  • NMIBC muscle invasive bladder cancer
  • the high rates of recurrence and significant risk of progression mandate that additional therapy be implemented. Improving clinical outcomes for patients with high risk-NMIBC therefore requires the development of novel treatments.
  • BCG Bacillus-Calmette Guerin
  • CDNs are imnortant PAMPs and DAMPs that generate valuable immune responses for TB and NMIBC.
  • PAMPs Bacterial pathogen-associated molecular patterns
  • Human cells utilize an innate immune monitoring system known as the cytosolic surveillance program (CSP) to detect nucleic acid including cyclic dinucleotides in the cytosol.
  • CSP cytosolic surveillance program
  • the CSP has now been shown to be important in anti-bacterial defenses particularly against intracellular bacteria such as Mycobacterium tuberculosis, Listeria monocytogenes, Salmonella species, and others.
  • Cytosolic pattern recognition receptors (PRRs) including STING, cGAS, DDX41 and many others are capable of binding to cytosolic CDNs 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 the induction of STAT6 which induces chemokines such as CCL2 and CCL20 independently of the TBK1-IRF3 pathway.
  • STING activation is also believed to activate the transcription factor NFKB through the IKB kinase (IKK) activation.
  • Cyclic cGAMP (cGAS) synthase is a cytosolic PRR which recognized cytosolic DNA. Upon binding to DNA it undergoes a conformational change that activates its core enzymatic activity which is to catalyze the formation of 2’ 3’ cGAMP. 2’ 3’ cGAMP in turn is a potent DAMP which activates the STING-TBK1-IRF3 axis leading to increased type 1 interferon expression as well as the STAT6 activation and IKK activation.
  • Type I IFNs produced both by innate immune cells in the tumor microenvironment and by the tumor cells themselves, are known to mediate anti-tumor effects against several malignancies, due to their ability to intervene in all phases of cancer immune-editing. (Zitvogel L, Galluzzi L, Kepp O, Smyth MJ, Kroemer G. Type I interferons in anticancer immunity. Nat Rev Immunol. 2015;15:405-14. PMID: 26027717).
  • STING (stimulator of interferon genes), is a major regulator of type I IFN innate immune responses to pathogens, following recognition of cytosolic 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 regulatory factors: at the crossroads of immunity, metabolism, and disease. Biochim Biophys Acta. 2015;1852:365-78. PMID:24807060).
  • Novel anticancer immunotherapies based on recombinant type I IFNs, type I IFN-encoding vectors, type I IFN- expressing cells, and STING agonists are therefore 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 the STING-TBK1-IRF3 axis. It is produced by mycobacteria including BCG by the disA gene which encodes the DisA protein (BCG protein WP_010950916.1 in BCG, M. tuberculosis protein Rv3586 or P9WNW5.1).
  • M.tb Mycobacterium tuberculosis synthesizes and secretes c-di-AMP, which activates the interferon regulatory factor (IRF) pathway and type I IFN responses through STING-signaling and cGAS.
  • IRF interferon regulatory factor
  • c-di-AMP overexpressing M.tb strains showed attenuation of TB in a mouse model.
  • c-di-AMP As a mucosal adjuvant, c-di-AMP exerts immune stimulatory effects causing maturation of dendritic cells, up-regulation of co-stimulatory molecules and production of pro-inflammatory cytokines, and strong Th1, Th17 and CD8 T cell responses against pathogens.
  • Ahmed D Cassol E. Role of cellular metabolism in regulating type I interferon responses: Implications for tumour immunology and treatment.
  • M.tb genome encodes a di-adenylate cyclase enzyme (DisA, also called DacA, P9WNW5.1 in the UniProtKB/Swiss-Prot databases) that synthesizes c-di-AMP from ATP or ADP.
  • DisA di-adenylate cyclase enzyme
  • the BCG protein WP_010950916.1 (NCBI reference number) is 100% identical to M. tuberculosis DisA. M.tb strains overexpressing disA intoxicate macrophages by releasing excessive c-di-AMP, a unique bacterial PAMP that activates STING-dependent IFN-b production. (Ahmed D, Cassol E. Role of cellular metabolism in regulating type I interferon responses: Implications for tumour immunology and treatment. Cancer Lett. 2017;409:20-29. PMTD: 28888999.).
  • BCG Pasteur was transformed with a kanamycin- resistance (Kan-R)-conferring plasmid that harbors the disA gene (M. tuberculosis Rv3586 or MT3692) from M.tb (the M.tb and BCG disA genes are 100% identical) fused to the strong mycobacterial promoter, P hsP60 .
  • Kan-R kanamycin- resistance
  • Addition of this plasmid to BCG-Pasteur increased the level of disA mRNA by 50-fold (Fig. lb).
  • the closely related M.tb-disA-OE strain releases 15-fold more c-di-AMP into the macrophage cytosol than wild type M. tb.
  • BMDMs from C57BL/6 mice infected with BCG- Pasteur disA-OE showed significant upregulation of IFN-b, TNF-a, IL-6 and IL-2 in comparison to uninfected or wild-type BCG infected macrophages.
  • RAWBlue ISG macrophages showed increased IRF3 levels when infected with BCG-Pasteur disA-OE, as compared to parental control.
  • BCG-disA-OE elicits anti-tumor immune responses in human bladder carcinoma (RT4) cells.
  • BCG-Pasteur-disA-OE was tested to determine whether it elicits similar immune responses in bladder cancer (BC) cells, in comparison to WT strains BCG-Pasteur and OncoTICE (the current immunotherapeutic BCG strain).
  • Human RT4 BC cells derived from human NMIBC tumors, were challenged with the wild-type (both Pasteur and TICE) and recombinant BCG Pasteur disA-OE strain at 1:20 (RT4 :: BCG) for 3h, and differential gene expression profile was determined in comparison to uninfected cells.
  • N-methyl-N-nitrosourea (MNU), a carcinogenic alkylating agent, is used to induce urothelial cancer in female Fischer rats.
  • MNU N-methyl-N-nitrosourea
  • BCG-disA-OE has significant immunotherapeutic effects in the rat bladder cancer model.
  • Urothelial dysplasia develops within eight weeks of MNU instillation, and by the 16th week after the first instillation, all rats display carcinoma-in-situ, papillary Ta, or high-grade T1 urothelial carcinoma with histopathologic and immunophenotypic features similar to those observed in human urothelial cancer.
  • Intravesical instillation of BCG-disA-OE strain was performed in MNU-treated rats, administered sequentially every week for 6 weeks starting eight weeks after MNU induction when tumors are visible.
  • Bladder tumors were staged by a GU pathologist according to WHO-ISUP classifications with percent tumor involvement (sum of Ta, T1 and CIS) 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.
  • BCG-disA-OE induces a characteristic cytokine and chemokine signature in rat bladders undergoing immunotherapy.
  • Rat urinary bladders from rats treated with BCG-disA-OE showed a significant induction of IFN-a/b, IFN-g, IL-1 b, TNF- ⁇ , TGF- ⁇ , iNOS, IP- 10, MCP-1 and MIP-l ⁇ in comparison to untreated or BCG-Pasteur treated rats.
  • Fig. 10 shows a summary of the cytokine expression level changes observed with BCG- disA- OE versus BCG-WT in primary cells, cancer cell lines, and in rat bladder cancer tissues.
  • cytokines associated with Thl T cell and Ml macrophage expansion, two type 1 interferons, and three pro-inflammatory chemokines were significantly upregulated by BCG-disA- OE compared to BCG-WT (2-fold to 30-fold) across these cells, cell lines and tissues.
  • cytokines associated with Th2 T cell and M2 macrophage expansion were generally down- regulated by BCG-disA-OE in comparison to BCG-WT (1-fold to 10-fold).
  • BCG immunotherapy may be effective via three immune mechanisms: (i) increased generation of tumor-specific cytotoxic CD8 T cells, (ii) cytokine environment which promotes macrophage-mediated CD4 cell activation against tumor antigens, and (iii) macrophage Ml shift promoting enhanced tumoricidal activity.
  • BCG overexpressing c-di-AMP is taken up by bladder tumor cells, and myeloid cells that are either resident or recruited to the tumor microenvironment, and induces host immune responses, including activation of STING and type I IFN responses, and NF-KB signaling, that promotes secretion of cytokines and chemokines, macrophage recruitment and apoptotic mechanisms, all of which collectively reduce tumor progression.
  • tuberculosis Rvl354c protein (100% identical to each other), (ii) the Vibrio cholerae DncV protein, Q9KVG7 in Swiss- Prot, which is a 2’-5’c-GAMP synthase, and (iii) the human cGAS protein Q8N884 in Swiss-Prot which is a 2’-3’ cGAMP synthase— may be added to BCG. These added CDN cyclase genes may be added alone or in combination. Such combinations would represent multivalent CDN overexpressing BCG. Also, as shown in Fig. 11, BCG possess several CDN phosphodiesterase genes or genes which contain phosphodiesterase domains.
  • SEQ ID NO: 1 Diadenylate cyclase DisA from BCG and other related mycobacteria, amino acid sequence (358 amino acids; BCG protein AOQ92_RS18745; NCBI Reference Sequence: NZ_CUWL01000001.1). The identical sequence is present in other strains of BCG, e.g., Mycobacterium tuberculosis as protein Rv3586 or MT3692, and in Mycobacterium bovis as protein Mb3617.
  • Plasmid pSD5B-Phsp60::disA is an episomally replicating E. coli-mycobacterial shuttle plasmid that overexpresses the BCG disA gene from the P hsp60 promoter, DNA sequence. (7742 nucleotides; promoter Phsp60 DNA comprised of a portion of the M. leprae hsp65 gene nucleotides 13 to 180 is underlined; disA coding sequence nucleotides 242 to 1318; ATG start codon and TAA stop codon shown in boldface, underline).
  • mice when mice are infected with 3.5 log10 units by the aerosol route of either M. tuberculosis harboring the pSD5B Phsp60::disA plasmid (M.tb-disA-OE or Mtb-OE) or wild type M. tuberculosis (Mtb- CDC1551), there are profound differences in the median time to death (MTD) of the animals. As can be seen, wild type M. tuberculosis (Mtb-CDC1551) gave an MTD of 150.5 days, while M.
  • tuberculosis harboring the pSD5B Phsp60::disA plasmid was a significantly weaker pathogen giving an MTD of 321.5 days.
  • a similar reduction in the pathogenicity is to be expected with BCG-disA-OE compared with BCG-WT.
  • BCG-disA-OE it is likely that should BCG-disA-OE be used as a cancer immunotherapy, one would anticipate reduced rates of bloodstream dissemination, reduced dysuria, reduced urgency and reduced malaise compared with BCG-WT.
  • tuberculosis Rv1354c protein encodes a bifunctional diguanylate cyclase/diguanylate phosphodiesterase.
  • the portion that functions as a diguanylate cyclase is an endogenous CDN-producing enzyme in BCG.
  • the full-length BCG_RS07340 polypeptide is 623 amino acids in length, and its domain structure is: N-terminus- GAF-GGDEF-EAL-C-terminus.
  • the GAF domain (approximately amino acids 1-190) is a regulatory domain which influences the activity of the other domains.
  • the GGDEF domain (approximately amino acids 190-350) is a diguanylate cyclase catalyzing the reaction 2 GTP # c- di-GMP + 2 pyrophosphates.
  • the EAL domain (approximately amino acids 350-623) is a diguanylate phosphodiesterase catalyzing the reaction c-di-GMP # 2 GMP.
  • This may be accomplished by also deleting the DNA encoding the regulatory-sensor GAF domain and/or the use of mutations in the DNA encoding the GAF domain to relieve any cyclase inhibitory activity it may possess.
  • Such techniques to generate constitutively active recombinant forms of the BCG_RS07340 protein will produce high levels of c-di-GMP in 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_RS07340; NCBI Reference Sequence: NC_008769.1; Protein ID WP 003898837.1; old locus tag BCG_1416c).
  • the identical sequence is present in other strains of BCG, e.g., Mycobacterium tuberculosis as protein Rv1354c or MT1397, and in Mycobacterium bovis as protein Mb1389c.
  • the EAL domain is from amino acid 354 to 623 and is underlined.
  • EAL domain is encoded from nucleotide 1060 to 1872 and is underlined.
  • the identical sequence is present in other strains of BCG, e.g., Mycobacterium tuberculosis as a fragment of gene Rv1354c or MT1397, and in Mycobacterium bovis as a fragment of gene Mb1389c.
  • c-GAMP is a strong inducer of the 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. It is possible to generate a recombinant dncV gene which is codon-optimized for BCG.
  • the codon-optimized structural gene may be overexpressed in BCG by fusion to a strong promoter (such as Phsp60) or a conditionally active strong promoter such as PTET-off.
  • the cGAS protein is produced by the human cGAS gene to yield a 522 amino acid polypeptide which senses cytosolic DNA and functions as a 2’-3’ cGAMP synthase.
  • the synthase or cyclase domain of cGAS becomes activated when cGAS binds to DNA.
  • This recombinant gene can also be codon-optimized for BCG.
  • the codon-optimized structural gene may be overexpressed in BCG by fusion to a strong promoter (such as Phsp60) or a conditionally active strong promoter such as PTET-off.
  • Coding sequence is 1569 nucleotides [522 codons, 1 stop codon], start codon ATG [bold underlined] at nucleotide 140; Stop codon TGA (bold, underlined) at nucleotide 1706]).
  • coli-mycobacterial shuttle plasmid that overexpresses the BCG disA gene, the human cGAS gene (with mycobacterial codon optimization), and mCherry from the Phsp60 promoter, DNA sequence.
  • this plasmid integrates as a single copy in the mycobacterial chromosome (10842 nucleotides; promoter Phsp60 DNA comprised of a portion of the M.
  • leprae hsp65 gene nucleotides 901 to 1068 is underlined; disA coding sequence are from nucleotides 1069 to 2145; human cGAS with mycobacterial codon optimization sequences are from nucleotides 2158 to 3726; ATG start codons and TAA or TGA stop codons are shown in boldface, underline).
  • tuberculosis Rv2837c over the C-terminal 316 amino acids (also called CdnP, CnpB, 3'-to-5' oligoribonuclease A, bifunctional oligoribonuclease, or PAP phosphatase NrnA).
  • the M. tuberculosis Rv2837c protein is known to hydrolyze both 3’-5’ c-di-AMP (bacterial PAMP molecule) and 2’-3’cGAMP (host DAMP molecule).
  • SEQ ID NO:14 Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP (also called CnpB, 3'- to-5' oligoribonuclease A, bifunctional oligoribonuclease, PAP phosphatase NrnA) from BCG, amino acid sequence (316 amino acids; BCG protein AHM08589.1; NCBI Reference DNA Sequence: CP003494.1 from BCG strain ATCC 35743; NCBI Reference Protein Identifier WP_003414507).
  • a similar sequence is present in Mycobacterium tuberculosis as protein Rv2837c or MT2903, and in Mycobacterium bovis as protein Mb2862c.
  • Bifunctional c-di-AMP and cGAMP phosphodiesterase CdnP also called CnpB, Rv2837c, or MT2903, 3'-to-5' oligoribonuclease A, bifunctional oligoribonuclease, PAP phosphatase NmA
  • Mycobacterium tuberculosis amino acid sequence (336 amino acids; M. tuberculosis protein WP_003905944.1; NCBI/GenBank Reference Sequence: AL123456 from M. tuberculosis strain H37Rv).
  • the M. tuberculosis protein has 20 additional amino acids at its N- terminus compared with the BCG protein (SEQ ID NO: 14) which are underlined and boldfaced.
  • BCG RS07340 EAL domain of protein BCG RS07340 (previously BCG 1416c).
  • 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 M. tuberculosis Rvl354c 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-terminus-GAF-GGDEF-EAL-C-terminus as shown.
  • the GAF domain (approximately amino acids 1-190) is a regulatory domain which influences the activity of the other domains.
  • the GGDEF domain (approximately amino acids 190-350) is a diguanylate cyclase catalyzing the reaction 2 GTP # c-di-GMP + 2 pyrophosphates.
  • 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.
  • knockout of this endogenous cyclic dinucleotide phosphodiesterase domain will increase the levels of c-di- GMP produced by BCG.
  • Targeted knockout of the EAL domain may be accomplished by gene replacement of the full-length WT BCG_RS07340 gene with one which encodes only amino acids 1-353 (the GAF-GGDEF domains), that is truncating the coding sequence of the gene to exclude the sequences that encode amino acids 354-623 (shown as the underlined DNA sequence in SEQ ID NO:5) and including an appropriate stop codon and transcription termination sequence.
  • the BCG_AHM07112 protein is an endogenous diguanylate phosphodiesterase in BCG (homologous the 307 amino acid M. tuberculosis Rv1357c protein). Some strains of BCG lack BCG_AHM07112 altogether while others such as BCG Tice harbor it.
  • the protein may be 288 amino acids in length (such as in BCG ATCC 35743) or 307 amino acids in length (such as in BCG Pasteur 1173 P2).
  • the BCG_AHM07112 protein from BCG ATCC 35743 is 288 amino acids in length and is 100% identical to the M. tuberculosis Rv1357c protein over its C-terminal 287 amino acids.
  • the domain structure of BCG_AHM07112 is that of a single EAL domain .
  • the M. tuberculosis Rv1357c protein is known to cleave 3’-5’ c-di-GMP, it is highly likely that the BCG protein performs the same reaction.
  • Knockout of this endogenous cyclic dinucleotide phosphodiesterase in BCG is anticipated to increase the levels of c-di-GMP produced by BCG.
  • Targeted knockout of the EAL domain may be accomplished by gene replacement of the full-length WT BCG_AHM07112 gene and subsequent generation of an unmarked deletion.
  • SEQ ID NO:17 [0207] Diguanylate phosphodiesterase AHM07112.1 from BCG and other related mycobacteria, amino acid sequence (288 amino acids; GenBank Reference Sequence: CP003494.1; from BCG strain ATCC 35743).
  • AHM07112.1 is 100% identical to the C-terminal 287 amino acids of the diguanylate phosphodiesterase of Mycobacterium tuberculosis protein Rv1357c or MT1400 and of Mycobacterium bovis as protein Mb1392c.
  • AHM07112.1 is 100% identical to the C-terminal 287 amino acids of the diguanylate phosphodiesterase of Mycobacterium tuberculosis protein Rv1357c or MT1400 and of Mycobacterium bovis as protein Mb1392c.
  • tuberculosis strain H37Rv The 19 amino acid N- terminal extension is present in the M. tuberculosis and in BCG Pasteur strain 1173 P2 but absent in several other BCG strains. The 19 amino acid N-terminal extension is underlined and boldfaced. The C-terminal 287 amino acids of M. tuberculosis Rvl357c are 100% identical to the BCG diguanylate phosphodiesterase AHM07112.1.
  • the present invention relates to an expression cassette or expression vector including a nucleic acid sequence encoding a Rvl354c protein, or a functional part thereof; a nucleic acid sequence encoding a cyclic GMP-AMP synthase (DncV) protein, or a functional part thereof; a nucleic acid sequence encoding a cyclic GMP-AMP synthase (cGAS) protein, or a functional part thereof; or a combination thereof.
  • the expression vector or expression cassette further includes a nucleic acid sequence encoding a DNA integrity scanning (disA) protein which functions as a diadenylate cyclase, or a functional part thereof.
  • nucleic acid sequence encoding a Rv1354c protein does not contain a phosphodiesterase gene or phosphodiesterase domain.
  • expression vector or expression cassette does not contain a phosphodiesterase gene or phosphodiesterase domain.
  • an expression vector or expression cassette of the invention includes one or more sequences operably linked to a nucleic acid of the invention which direct termination of transcription, post-transcriptional cleavage, and/or polyadenylation.
  • an expression vector or expression cassette of the invention includes a variable length intervening sequence and/or a selectable marker gene operably linked to a nucleic acid of the invention.
  • the present invention relates to a strain of Mycobacterium including an expression vector or expression cassette of the invention described herein.
  • the strain of Mycobacterium is Mycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof.
  • the strain of Mycobacterium is BCG.
  • the strain includes the plasmid of SEQ ID NO:13.
  • the present invention relates to a strain of Mycobacterium that expresses or overexpresses diadenylate cyclase and/or expresses or overexpresses one or more other cyclase genes or domains (e.g., those described herein).
  • the 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).
  • the present invention relates to a strain of Mycobacterium that expresses or overexpresses diadenylate cyclase and/or does not express a phosphodiesterase (PDE) that hydrolyzes STING agonists (e.g., contains a deletion of a PDE gene that hydrolyzes STING agonists).
  • PDE phosphodiesterase
  • the strain of Mycobacterium is Mycobacterium tuberculosis, Mycobacterium bovis, or a combination thereof.
  • the strain of Mycobacterium is BCG.
  • the rat MNU bladder cancer model is a validated model of bladder cancer in which administration of intravesical BCG can be shown to be therapeutic (Fig.6 and Kates et al. PMID 28588015).
  • the inventors extended their previous findings of the therapeutic effect of BCG-disA- OE versus BCG-WT which were shown in Figure 7.
  • the inventors have now performed the 16- week rat MNU model twice.
  • Figure 7 was based on Experiment 1 and shows that BCG-disA-OE displays a trend towards a better outcome versus BCG-WT.
  • BCG-disA-OE delivers sustained STING agonist from the intracellular compartment.
  • Bow yer et al The persistence of bacilli Calmette-Guerin in the bladder after intravesical treatment for bladder cancer. Brit J Urol. 1995; 75: 188-192.
  • PMID 7850324 evaluated 125 bladder cancer patients from 1986- 1992 who received intravesical BCG. Patients were asked to provide monthly urine samples which were then sent for mycobacterial culture.90 patients survived and were compliant with the monthly urine samples. 4/90 patients (4.4%) had persistent BCG in their urine, one for up to 16.5 months. A fifth patient required a cystectomy 7 weeks after completing intravesical BCG treatments and was found to have microscopic evidence of acid-fast bacilli in the bladder by microscopy. [0224] Durek et al. (The fate of bacillus Calmette-Guerin after intravesical instillation. J Urol. 2001; 165: 1765-1768. PMID 11342972) studied 49 patients with serial urine cultures following intravesical BCG.
  • BCG was in the urine detected in 96.4% of the specimens after 2 hours and in 67.9% after 24 hours after instillation.
  • the number of positive specimens decreased, and was 27.1% on day 7 immediately before the next instillation (FIG.38).
  • the investigators also evaluated bladder biopsies by PCR for mycobacterial DNA within 1 week after the 6 th instillation (instillations were given monthly). In 14 of 44 bladder biopsies (31.8%) mycobacterial ribosomal DNA was found. Additionally, positive PCRs for mycobacterial DNA was evident up to 24 months in between 4.2% and 37.5% of the investigated biopsies.
  • BCG-disA-OE is safer than BCG-WT in two separate mouse models
  • Intra ical BCG treatment in humans is associated with dysuria, fatigue, and malaise in treated patien s.
  • BCG-disA-OE was less capable of proliferating in immunocompetent mouse lungs than BCG-WT, and it was less lethal in a time-to-death assay in immunosuppressed mice.
  • BCG has been shown to elicit trained immunity which has been associated with its therapeutic benefit in solid and liquid tumors and for diabetes. STING agonist overexpressing BCG strains elicit stronger trained immunity changes than BCG-WT
  • Trained immunity refers to the ability of one antigenic stimulus to elicit more potent immune responses to a second, different antigen.
  • Trained immunity is antigen independent, based on heterologous CD4 and CD8 memory activation, cytokine mediated, and is associated with epigenetic and metabolic changes.
  • BCG is a potent tool as the first antigenic stimulus to elicit trained immunity to subsequent antigenic stimuli such as tumors, viral infection, or drug-resistant bacterial infections (Netea et al. Trained immunity: a program of innate immune memory in health and disease. Science 2016. PMID 27102489; and Arts et al. BCG vaccination protects against experimental viral infection in humans through the induction of cytokines associated with trained immunity. Cell Host Microbe 2018. PMID 29324233). [0231] BCG for solid and liquid tumors.
  • BCG has a long history of therapeutic benefit as an immunotherapy for both solid and liquid tumors in humans (Hersh et al. BCG as adjuvant immunotherapy for neoplasia. Annu Rev Med 1977. PMID 324372). It has been used both systemically and intratumorally for malignancies that include melanoma, non-small cell lung cancer (NSCLC), and acute lymphoblastic leukemia (ALL). Recently there have been trials of BCG together with checkpoint inhibitors for forms of bladder cancer. [0232] BCG for diabetes. BCG vaccination has recently been shown to have therapeutic benefits in glucose control for various forms of diabetes mellitus including Type 1 diabetes mellitus (Stienstra and Netea.
  • BCG-disA-OE and trained immunity.
  • STING agonist overexpressing t ins of BCG to stimulate trained immunity, the inventors tested the ability of BCG-WT versu s CG-disA-OE to elicit potentiation of second antigen stimulation in rested human monocytes following an exposure to the BCG strains six days prior.
  • the first antigen was a BCG strain on day 0, and after six days of rest, the second antigen was the unrelated TLR-1/2 antigen PAM3CSK4.
  • BCG-Tice-disA-OE expresses much higher levels of the disA gene than BCG-WT
  • the relative expression of BCG-Tice-disA-OE clone 2 was 300:1 using the 2 &mm7H method of comparison. This indicates that disA is strongly overexpressed by being on a multicopy plasmid and driven by the M. leprae hsp65 promoter in pSD5-hsp65-MT3692 plasmid. This strong overexpression leads to much higher levels of release of the STING agonist, c-di-AMP.
  • STING agonist overexpression BCG strains such as BCG-disA-OE elicit pro- inflammatory changes in signaling pathways and cytokine secretion profiles in multiple model systems.
  • the inventors tested STING agonist overexpressing strains such as BCG-disA-OE compared to BCG-WT in multiple model systems to evaluate its relative capacity to elicit proinflammatory cytokine changes.
  • BCG-disA-OE was statistically significantly superior than BCG-WT in the majority of their tests. When the comparisons were not statistically significant, BCG-disA-OE gave the stronger of the two responses.
  • Figure 23 also shows that the elevation of type 1 IFN secretion in both BCG-disA-OE and BCG-WT is STING-dependent.
  • BCG-disA-OE is a more potent stimulator of pro-inflammatory cytokine expression and proinflammatory pathway induction than BCG-WT
  • the disA-overexpressing plasmid pSD5-hsp65-MT3692 carries a Kan resistance gene cassette conferring resistance to the antibiotic kanamycin.
  • the inventors disclose a method to generate an antibiotic gene cassette-free recombinant BCG which overexpresses a STING agonist biosynthetic gene.
  • panCD The mycobacterial genetic operon panCD encodes for the biosynthetic gene panC (Pantoate— beta-alanine ligase gene) and panD (aspartate 1 -decarboxylase gene).
  • panC and PanD are required for the biosynthesis of pantothenic acid also called vitamin B5 (a B vitamin).
  • Pantothenic acid a water-soluble vitamin, is an essential nutrient for mycobacteria such as BCG. Animals require pantothenic acid in order to synthesize coenzyme-A (CoA), as well as to synthesize and metabolize proteins, carbohydrates, and fats.
  • the anion is called pantothenate.
  • panCD in mycobacteria has been shown to yield mutant strains that can only grow in the presence of added pantothenate. As such they are auxotroph for pantothenate.
  • mpanCD mutants of Mycobacterium tuberculosis have been shown to be highly attenuated in animal infection, being rapidly cleared, because of their inability to grow in mammalian tissues where pantothenate is not available to them.
  • the inventors disclose a detailed method for generating an unmarked (no antibiotic TR[R PN ⁇ RaaR ⁇ $ mpanCD deletion mutant of BCG.
  • the inventors disclose a detailed method for generating a shuttle plasmid which harbors the mycobacterial panCD gene as well as an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65::disA construct which overexpresses the disA gene and releases excess STING agonist, c-di-AMP).
  • the shuttle plasmid is capable of replication in E. coli or in mycobacteria. It harbors an antibiotic cassette that can be conveniently removed by cleavage with a rare-cutting restriction enzyme and re-ligation.
  • the shuttle plasmid may be generated by PCR amplification of the backbone of the plasmid excluding the antibiotic resistance cassette that generates unique restriction sites at the termini and ligating in a PCR product consisting of an amplified panCD operon with the same unique restriction sites at its termini.
  • the antibiotic resistance gene-free shuttle plasmid (ligation product) may be electroporated into a BCG or E. coli auxotroph and selected for on pantothenate-free agar plates.
  • the inventors show a method to introduce the antibiotic-cassette-free plasmid harboring the mycobacterial panCD gene as well as an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65::disA P ⁇ [ ⁇ a_bPa$ V[a ⁇ N[ b[ZN_XRQ 67; mpanCD mutant.
  • STING agonists such as the Phsp65::disA P ⁇ [ ⁇ a_bPa$ V[a ⁇ N[ b[ZN_XRQ 67; mpanCD mutant.
  • panCD cassette and the construct for the biosynthesis of STING agonists could be introduced into a chromosomally integrating vector such as pMH94.
  • the antibiotic cassette could be eliminated from pMH94.
  • BCG-Tice (ATCC 35743) is a natural pantothenate auxotroph.
  • the Mycobacterium bovis BCG Tice strain (ATCC 35743) is a natural pantothenate auxotroph. This strain carries a 5 bp DNA insertion in its panC gene at base pairs 739-743. This insertion mutation change leads to a frameshift mutation after the 246 th amino acid of PanC (wild type PanC is 309 amino acids in length). As a result of the 5 bp insertion mutation, the mutant PanC polypeptide in the Mycobacterium bovis BCG Tice strain (ATCC 35743) is comprised of 246 amino acids of the wild type PanC sequence at its N-terminus followed by a 478 amino acid nonsense polypeptide at its C-terminus.
  • PanC polypeptide is highly unlikely to retain any functional pantoate--beta-alanine ligase activity (the normal enzymatic function of PanC). Additionally, the PanD polypeptide in BCG Tice (ATCC 35743) is highly unlikely to be translated because the stop codon for the panC gene (which overlaps with the ATG for panD translation initiation in the wild type sequence) is out of frame. Ribosomal termination of PanC translation is coupled with ribosomal initiation of PanD translation in the wild type panCD operon. Since there is no ribosomal termination immediately upstream of the panD start codon, ribosomal initiation of translation of the panD gene is highly unlikely to occur.
  • the inventors disclose that this natural auxotrophy enables the more rapid construction of an antibiotic gene cassette-free recombinant BCG which overexpresses a STING agonist biosynthetic gene.
  • the inventors disclose a method for introducing an antibiotic-cassette-free plasmid harboring the mycobacterial panCD gene as well as an overexpression construct for the biosynthesis of STING agonists (such as the Phsp65::disA construct) directly into BCG-Tice (ATCC 35743).
  • pSD5-hsp60-MT3692 is the same as pSD5-hsp65-MT3692.
  • the present invention relates to a pharmaceutical composition including an expression vector, expression cassette, or strain of the invention described herein and a pharmaceutically acceptable carrier.
  • the present invention relates to methods and/or compositions for treating and/or preventing cancer comprising administration of an expression vector, expression cassette, strain or pharmaceutical composition described herein to a subject.
  • the cancer is bladder cancer (e.g., non-muscle invasive bladder cancer (NMIBC)), breast cancer, or a solid tumor.
  • NMIBC non-muscle invasive bladder cancer
  • Additional embodiments of the disclosure concern methods and/or compositions for treating and/or preventing a bladder cancer in which modulation of a type 1 interferon (IFN) response is directly or indirectly related.
  • individuals with a bladder cancer such as NMIBC are treated with a modulator of the type 1 interferon response, and in some aspects an individual with bladder cancer is provided a modulator of expression type 1 interferon expression, such as an inducer of its expression.
  • the level to which an inducer of type 1 interferon expression increases type 1 interferon expression may be any level so long as it provides amelioration of at least one symptom of bladder cancer, including non-muscle-invasive bladder cancer (NMIBC).
  • the level of expression of type 1 interferon may increase by at least 2, 3, 4, 5, 10, 25, 50, 100, 1000, or more fold expression compared to the level of expression in a standard, in at least some cases.
  • An individual may monitor expression levels of type 1 interferon using standard methods in the art, such as northern assays or quantitative PCR, for example.
  • An individual known to have bladder cancer, suspected of having bladder cancer, or at risk for having bladder cancer may be provided an effective amount of an inducer of type 1 interferon expression, including a BCG strain of the present invention comprising an expression vector of the present invention.
  • the expression vector expresses a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof; or a combination thereof.
  • a BCG strain of the present invention comprising an expression vector of the present invention be administered into the bladder of the subject and that the expressed protein(s) enhance type 1 interferon expression in the bladder.
  • Those at risk for bladder cancer may be those individuals having one or more genetic factors, may be of advancing age, and/or may have a family history, for example.
  • an individual is given an agent for bladder cancer therapy in addition to the one or more inducers of type 1 interferon of the present invention.
  • additional therapy may include intravesical chemotherapies such as mitomycin C, cyclophosphamide, or a combination thereof, for example.
  • a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof
  • the additional therapy may be given prior to, at the same time as, and/or subsequent to the inducer of type 1 interferon.
  • an expression vector, expression cassette, strain, pharmaceutical composition, and/or method of the invention described herein has increased safety, increased tolerability (e.g., decreased dysuria, urgency, or malaise), and/or decreased likelihood to cause infection in the bloodstream or disseminated bloodstream infection compared to non-recombinant BCG.
  • the present invention relates to a method of treating and/or preventing cancer, including administering to a subject an expression vector, expression cassette, strain, and/or pharmaceutical composition of the invention described herein, wherein the administration results in an increased safety profile, increased tolerability (e.g., decreasing dysuria, urgency, or malaise), and/or decreased likelihood of infection in the bloodstream or disseminated bloodstream infection compared to non-recombinant BCG.
  • the cancer is, for example, bladder cancer (e.g., non-muscle-invasive bladder cancer (NMIBC)), breast cancer, or a solid tumor.
  • the solid tumor is, for example, a sarcoma, carcinoma, or lymphoma.
  • the present invention relates to a method of increasing the safety, increasing the tolerability (e.g., decreasing dysuria, urgency, or malaise), and/or decreasing the likelihood to cause infection in the bloodstream or disseminated bloodstream infection compared to non-recombinant BCG, comprising administering an expression vector, expression cassette, strain, and/or pharmaceutical compositions of the invention described herein to a subject.
  • compositions of the present invention include an effective amount of one or more inducers of expression of type 1 interferon such as such as a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof, dissolved or dispersed in a pharmaceutically acceptable carrier.
  • inducers of expression of type 1 interferon such as such as a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (
  • phrases "pharmaceutical” or “pharmacologically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal, such as, for example, a human, as appropriate.
  • the preparation of a pharmaceutical composition that comprises at least one inducer of expression of type 1 interferon or additional active ingredient will be known to those of skill in the art in light of the present disclosure, as exemplified by Remington: The Science and Practice of Pharmacy, 21 st Ed. Lippincott Williams and Wilkins, 2005, incorporated herein by reference.
  • preparations should meet sterility, pyrogenicity, general safety and purity standards as required by FDA Office of Biological Standards.
  • 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, disintegration agents, lubricants, sweetening agents, flavoring agents, dyes, such like materials and combinations thereof, as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed. Mack Printing Company, 1990, pp. 1289-1329, incorporated herein by reference). Except insofar as any conventional carrier is incompatible with the active ingredient, its use in the pharmaceutical compositions is contemplated.
  • the inducer of expression of type 1 interferon (such as a BCG strain expressing one or more of the following proteins: a RV 1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof) may comprise different types of carriers depending on whether it is to be administered in solid, liquid or aerosol form, and whether it need to be sterile for such routes of administration as injection.
  • a BCG strain expressing one or more of the following proteins: a RV 1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP-AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA)
  • the present invention can be administered intravenously, intradermally, transdermally, intrathecally, intraarterially, intraperitoneally, intranasally, intravaginally, intrarectally, intravesically (e.g., administered directly into the bladder, e.g., by injection, or by intravesical instillation), intratumorally, topically, intramuscularly, subcutaneously, mucosally, orally, topically, locally, inhalation (e.g., aerosol inhalation), injection, infusion, continuous infusion, localized perfusion bathing target cells directly, via a catheter, via a lavage, in cremes, in lipid compositions (e.g., liposomes), or by other method or any combination of the foregoing as would be known to one of ordinary skill in the art (see, for example, Remington's Pharmaceutical Sciences, 18th Ed.
  • Pharmaceutically acceptable salts include the acid addition salts, e.g., those formed with the free amino groups of a proteinaceous composition, or which are formed with inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid. Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium or ferric hydroxides; or such organic bases as isopropylamine, trimethylamine, histidine or procaine. Upon formulation, solutions will be administered in a manner compatible with the dosage formulation and in such amount as is therapeutically effective.
  • inorganic acids such as for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric or mandelic acid.
  • Salts formed with the free carboxyl groups can also be derived from inorganic bases such as for example, sodium, potassium, ammonium, calcium
  • compositions of the present invention suitable for administration is provided in a pharmaceutically acceptable carrier with or without an inert diluent.
  • the carrier should be assimilable and includes liquid, semi-solid, i.e., pastes, or solid carriers. Except insofar as any conventional media, agent, diluent or carrier is detrimental to the recipient or to the therapeutic effectiveness of a composition contained therein, its use in administrable composition for use in practicing the methods of the present invention is appropriate.
  • carriers or diluents include fats, oils, water, saline solutions, lipids, liposomes, resins, binders, fillers and the like, or combinations thereof.
  • the composition may also include various antioxidants to retard oxidation of one or more component. Additionally, 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., methylparabens, propylparabens), chlorobutanol, phenol, sorbic acid, thimerosal or combinations thereof.
  • parabens e.g., methylparabens, propylparabens
  • chlorobutanol phenol
  • sorbic acid thimerosal or combinations thereof.
  • the composition is combined with the carrier in any convenient and practical manner, i.e., by solution, suspension, emulsification, admixture, encapsulation, absorption and the like. Such procedures are routine for those skilled in the art.
  • the composition is combined or mixed thoroughly with a semi-solid or solid carrier.
  • the mixing can be carried out in any convenient manner such as grinding.
  • Stabilizing agents can be also added in the mixing process in order to protect the composition from loss of therapeutic activity, i.e., denaturation in the stomach.
  • the present invention includes the use of pharmaceutical lipid vehicle compositions that include inducer of expression of type 1 interferon, one or more lipids, and an aqueous solvent.
  • lipid includes any of a broad range of substances that is characteristically insoluble in water and extractable with an organic solvent.
  • lipid is used herein, it is not limited to any particular structure. Examples include compounds which contain long-chain aliphatic hydrocarbons and their derivatives.
  • a lipid may be naturally occurring or synthetic (i.e., designed or produced by man). However, a lipid is usually a biological substance.
  • Biological lipids are well known in the art, and include for example, neutral fats, phospholipids, phosphoglycerides, steroids, terpenes, lysolipids, glycosphingolipids, glycolipids, sulphatides, lipids with ether and ester-linked fatty acids and polymerizable lipids, and combinations thereof.
  • compositions and methods of the present invention are also encompassed by the compositions and methods of the present invention.
  • One of ordinary skill in the art would be familiar with the range of techniques that can be employed for dispersing a composition in a lipid vehicle.
  • the inducer of inducer of expression of Type 1 interferon of the present invention may be dispersed in a solution containing a lipid, dissolved with a lipid, emulsified with a lipid, mixed with a lipid, combined with a lipid, covalently bonded to a lipid, contained as a suspension in a lipid, contained or complexed with a micelle or liposome, or otherwise associated with a lipid or lipid structure by any means known to those of ordinary skill in the art.
  • the dispersion may or may not result in the formation of liposomes.
  • compositions of the present invention administered to an animal patient can be determined by physical and physiological factors such as body weight, severity of condition, the type of disease being treated, previous or concurrent therapeutic interventions, idiopathy of the patient and on the route of administration. Depending upon the dosage and the route of administration, the number of administrations of a preferred dosage and/or an effective amount may vary according to the response of the subject. The practitioner responsible for administration will, in any event, determine the concentration of active ingredient(s) in a composition and appropriate dose(s) for the individual subject. [0275] In certain aspects, pharmaceutical compositions may include, for example, at least about 0.1% of an active compound.
  • the active compound may include between about 2% to about 75% of the weight of the unit, or between about 25% to about 60%, for example, and any range derivable therein.
  • the amount of active compound(s) in each therapeutically useful composition may be prepared is such a way that a suitable dosage 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, as well as other pharmacological considerations will be contemplated by one skilled in the art of preparing such pharmaceutical formulations, and as such, a variety of dosages and treatment regimens may be desirable.
  • a dose may also comprise 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.
  • a range of about 5 mg/kg/body weight to about 100 mg/kg/body weight, about 5 microgram/kg/body weight to about 500 milligram/kg/body weight, etc. can be administered, based on the numbers described above.
  • the inducers of expression of inducer of expression of type 1 interferon of the present invention are formulated to be administered via an alimentary route.
  • Alimentary routes include all possible routes of administration in which the composition is in direct contact with the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered orally, buccally, rectally, or sublingually.
  • compositions may be formulated with an inert diluent or with an assimilable edible carrier, or they may be enclosed in hard- or soft- shell gelatin capsule, or they may be compressed into tablets, or they may be incorporated directly with the food of the diet.
  • the active compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like
  • excipients used in the form of ingestible tablets, buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like
  • ingestible tablets buccal tables, troches, capsules, elixirs, suspensions, syrups, wafers, and the like
  • PMID 9121559
  • Hwang MJ Ni X
  • Waldman M Waldman M
  • Ewig CS Hagler AT. Derivation of class II force fields.
  • the tablets, troches, pills, capsules and the like may also contain the following: a binder, such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof; an excipient, such as, for example, dicalcium phosphate, mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate or combinations thereof; a disintegrating agent, such as, for example, com starch, potato starch, alginic acid or combinations thereof; a lubricant, such as, for example, magnesium stearate; a sweetening agent, such as, for example, sucrose, lactose, saccharin or combinations thereof; a flavoring agent, such as, for example peppermint, oil of wintergreen, cherry flavoring, orange flavoring, etc.
  • a binder such as, for example, gum tragacanth, acacia, cornstarch, gelatin or combinations thereof
  • an excipient such as,
  • the dosage unit form 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 instance, 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, carriers such as a liquid carrier. Gelatin capsules, tablets, or pills may be enterically coated. Enteric coatings prevent denaturation of the composition in the stomach or upper bowel where the pH is acidic. See, e.g., U.S. Pat. No. 5,629,001.
  • the basic pH therein dissolves the coating and permits the composition to be released and absorbed by specialized cells, e.g., epithelial enterocytes and Peyer's patch M cells.
  • a syrup of elixir may contain the active compound sucrose as a sweetening agent methyl and propylparabens as preservatives, a dye and flavoring, such as cherry or orange flavor.
  • any material used in preparing any dosage unit form should be pharmaceutically pure and substantially non-toxic in the amounts employed.
  • the active compounds may be incorporated into sustained-release preparation and formulations.
  • compositions of the present disclosure may alternatively be incorporated with one or more excipients in the form of a mouthwash, dentifrice, buccal tablet, oral spray, or sublingual orally- administered formulation.
  • a mouthwash may be prepared incorporating the active ingredient in the required amount in an appropriate solvent, such as a sodium borate solution (Dobell's Solution).
  • the active ingredient may be incorporated into an oral solution such as one 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 humectants.
  • compositions may be fashioned into a tablet or solution form that may be placed under the tongue or otherwise dissolved in the mouth.
  • Additional formulations which are suitable for other modes of alimentary administration include suppositories.
  • Suppositories are solid dosage forms of various weights and shapes, usually medicated, for insertion into the rectum. After insertion, suppositories soften, melt or dissolve in the cavity fluids.
  • traditional carriers may include, for example, polyalkylene glycols, triglycerides or combinations thereof.
  • suppositories may be formed from mixtures containing, for example, the active ingredient in the range of about 0.5% to about 10%, and preferably about 1% to about 2%.
  • inducer of expression of type 1 interferon of the present invention may be administered via a parenteral route.
  • parenteral includes routes that bypass the alimentary tract.
  • the pharmaceutical compositions disclosed herein may be administered, for example, intravenously, intradermally, intramuscularly, intraarterially, intrathecally, subcutaneous, or intraperitoneally. See, e.g., U.S. Pat. Nos. 6,7537,514; 6,613,308; 5,466,468; 5,543,158; 5,641,515; and 5,399,363 (each specifically incorporated herein by reference in its entirety).
  • Solutions of the active compounds as free base or pharmacologically acceptable salts may be prepared in water suitably mixed with a surfactant, such as hydroxypropylcellulose. Dispersions may also be prepared in glycerol, liquid polyethylene glycols, and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the 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. Patent 5,466,468, specifically incorporated herein by reference in its entirety).
  • 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.
  • a coating such as lecithin
  • surfactants for example
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • the solution should be suitably buffered if necessary and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for intravenous, intramuscular, subcutaneous, and intraperitoneal administration.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in isotonic NaCl solution and either added hypodermoclysis fluid or injected at the proposed site of infusion, (see, for example, "Remington's Pharmaceutical Sciences” 15th Edition, pages 1035-1038 and 1570-1580). Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject.
  • Sterile injectable solutions are prepared by incorporating the active compounds in the required amount in the appropriate solvent with several of the other ingredients enumerated above, as required, followed by filtered sterilization.
  • dispersions are prepared by incorporating the various sterilized active ingredients into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • sterile powders for the preparation of sterile injectable solutions
  • the preferred methods of preparation are vacuum-drying and freeze-drying techniques which yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • a powdered composition is combined with a liquid carrier such as, e.g., water or a saline solution, with or without a stabilizing agent.
  • the active compound inducer of expression of type 1 interferon of the present invention may be formulated for administration via various miscellaneous routes, for example, topical (i.e., transdermal) administration, mucosal administration (intranasal, vaginal, etc.) and/or inhalation.
  • Pharmaceutical compositions for topical administration may include the active compound formulated for a medicated application such as an ointment, paste, cream or powder.
  • Ointments include all oleaginous, adsorption, emulsion and water-soluble based compositions for topical application, while creams and lotions are those compositions that include an emulsion base only.
  • Topically administered medications may contain a penetration enhancer to facilitate adsorption of the active ingredients through the skin.
  • Suitable penetration enhancers include glycerin, alcohols, alkyl methyl sulfoxides, pyrrolidones and luarocapram.
  • Possible bases for compositions for topical application include polyethylene glycol, lanolin, cold cream and petrolatum as well as any other suitable absorption, emulsion or water-soluble ointment base.
  • Topical preparations may also include emulsifiers, gelling agents, and antimicrobial preservatives as necessary to preserve the active ingredient and provide for a homogenous mixture.
  • Transdermal administration of the present invention may also include the use of a "patch".
  • the patch may supply one or more active substances at a predetermined rate and in a continuous manner over a fixed period of time.
  • the pharmaceutical compositions may be delivered by eye drops, intranasal sprays, inhalation, and/or other aerosol delivery vehicles. Methods for delivering compositions directly to the lungs via nasal aerosol sprays has been described e.g., in U.S. Pat. Nos.5,756,353 and 5,804,212 (each specifically incorporated herein by reference in its entirety).
  • the typical aerosol of the present invention for inhalation will consist of a suspension of active ingredients in liquid propellant or a mixture of liquid propellant and a suitable solvent.
  • Suitable propellants include hydrocarbons and hydrocarbon ethers.
  • Suitable containers will vary according to the pressure requirements of the propellant.
  • Administration of the aerosol will vary according to subject’s age, weight and the severity and response of the symptoms.
  • compositions described herein may be comprised in a kit.
  • an inducer of expression of type 1 interferon of the present invention such as a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP- AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions as a denylate cyclase, or functional part thereof
  • an inducer of expression of type 1 interferon of the present invention such as a BCG strain expressing one or more of the following proteins: a RV1354c protein, or functional part thereof; a cyclic GMP-AMP synthase (DncV) protein, or functional part thereof; a cyclic GMP- AMP synthase (cGAS) protein, or functional part thereof; a DNA integrity scanning (disA) protein which functions
  • kits may comprise a suitably aliquoted inducer of expression of type 1 interferon of the present invention and, in some cases, one or more additional agents.
  • the component(s) of the kits may be packaged either in aqueous media or in lyophilized form.
  • the container means of the kits will generally include at least one vial, test tube, flask, bottle, syringe or other container means, into which a component may be placed, and preferably, suitably aliquoted. Where there is more than one components in the kit, the kit also will generally contain a second, third or other additional container into which the additional components may be separately placed. However, various combinations of components may be included in a vial.
  • kits of the present invention also will typically include a means for containing the inducer of expression of type 1 interferon of the present invention and any other reagent containers in close confinement for commercial sale.
  • Such containers may include injection or blow-molded plastic containers into which the desired vials are retained.
  • the liquid solution is an aqueous solution, with a sterile aqueous solution being particularly preferred.
  • the inducer of expression of type 1 interferon of the present invention composition(s) may be formulated into a syringeable composition.
  • the container means may itself be a syringe, pipette, and/or other such like apparatus, 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 the other components of the kit.
  • the components of the kit may be provided as dried powder(s).
  • the powder can be reconstituted by the addition of a suitable solvent. It is envisioned that the solvent may also be provided in another container means.
  • Mycobacterium bovis Bacillus Calmette- Guerin (BCG) Pasteur (BCG-WT Pasteur) (a generous gift from Dr. Frank Collins [FDA] and identical to BCG-Pasteur provided by the Pasteur Institute to the Trudeau Institute in 1967 as TMC No. 1011) and commercially available BCG-Tice (Onco-Tice®, Merck) were used for the generation of c-di-AMP overexpressing recombinant BCG strains.
  • Genomic DNA from Mycobacterium tuberculosis (M. tb) strain CDC1551 was used for PCR amplification of disA (MT3692/Rv3586).
  • coli- mycobacterial shuttle vector (pSD5.hsp60) was used to clone M.tb gene MT3692 or Rv3586 under the strong mycobacterial promoter hsp60. Clones were confirmed by gene sequencing and were used for bacterial transformation by electroporation method. Recombinant strains were confirmed using colony PCR against kanamycin cassette, subjected to whole genome sequencing and qPCR analyses. Details of all bacterial strains, plasmids and constructs are listed in Table 3.
  • IL-2 Quantikine R2000, R and D Systems [0305] Mammalian cell culture: [0306] Cell lines: For cell-based in vitro infection assays J774.1 (American Type Culture Collection-ATCC® TIB67", Manassas, VA, USA) murine macrophage cell lines were cultivated in RPMI-Glutamax (Cat.61870-036, Fischer Scientific), supplemented with 10% heat inactivated fetal bovine serum (FBS) (Cat. 10082147, Fischer Scientific) with 1% streptomycin/penicillin at 37°C with 5% CO2.
  • FBS heat inactivated fetal bovine serum
  • Urothelial carcinoma cell lines 5637 (ATCC® HTB-9"), a human high grade urothelial cancer; RT4 (ATCC ® HTB-2"), a human transitional cell low grade urothelial cancer; J82 (ATCC® HTB-1"), a human high grade urothelial cancer; and NBT II (ATCC® CRL- 1655"), N-butyl-N-(4-hydroxybutyl) nitrosamine induced tumor cell line in Rattus norvegicus Nara Bladder Tumor No.2, UPPL1595 (luminal cell line established from a spontaneous primary bladder tumor in an Uroplakin-Cre driven PTEN/P53 knockout genetically engineered mouse model and were generously provided by Dr.
  • BBN975 basic- cell line established from , 0.05% N-Butyl-N-(4-hydroxybutyl) nitrosamine (BBN) induced murine urothelial cancer model and was generously provided by Dr. William Kim (UNC Chapel Hill)
  • MB49 murine urothelial carcinoma cells, 7,12-dimethylbenz[a]anthracene (DMBA, EMD Millipore, Cat. SSC148) were maintained as monolayer in RPMI1640 medium supplemented with 10% heat inactivated fetal bovine serum (FBS) with 1% streptomycin/penicillin at 37°C with 5% C02.
  • FBS heat inactivated fetal bovine serum
  • Mouse fibroblast cell line NCTC clone 929 [L cell, L-929, derivative of Strain L] (ATCC® CCL-1TM) were routinely maintained as monolayer in DMEM media supplemented with 10% heat inactivated fetal bovine serum (FBS) with 1% streptomycin/penicillin at 37°C with 5% C02. All cell lines were not maintained more than 10 passage cycle and Mycoplasma testing was performed periodically while cells were in culture. Reporter mouse cell line, RAW-Lucia ISG (InvivoGen, CA, USA) was cultivated in custom prepared media as per manufacturer’s instructions.
  • FBS heat inactivated fetal bovine serum
  • streptomycin/penicillin 37°C with 5% C02. All cell lines were not maintained more than 10 passage cycle and Mycoplasma testing was performed periodically while cells were in culture.
  • Reporter mouse cell line, RAW-Lucia ISG InvivoGen, CA, USA was cultivated in custom prepared media as per manufacturer’s instructions.
  • BMDMs murine bone-marrow-derived macrophages
  • BMDCs dendritic cells
  • BM cells were isolated from 4-week old wild-type (WT) C57BF/6J (Charles River laboratories, North Wilmington, Mass) and STING-KO mice (C57BF/6J-Tmeml73gt/J, Jackson laboratories). Multiple vials of bone-marrow cells were preserved in cryopreservation media containing 10% DMSO (Cat. D2650; Sigma) and 90% heat inactivated FBS (Cat. 10082147, Fischer Scientific) in liquid nitrogen.
  • 10% DMSO Cat. D2650; Sigma
  • FBS heat inactivated FBS
  • BMDM-differentiation media made from DMEM containing 10% FBS, 1% MEM amino acids (Cat. 11130051, Thermo Fisher Scientific), 1% MEM non-essential amino acids (Cat. 11140050, Thermo Fisher Scientific), 1% sodium pyruvate (Cat. 11360070, Thermo Fisher Scientific), 1% MEM vitamin (Cat. 11120052, Thermo Fisher Scientific) and antibiotics (Penicillin-Streptomycin solution) supplemented with 30% sterile mouse fibroblast F929 (ATCC® CCF-1TM) conditioned media.
  • MEM amino acids Cat. 11130051, Thermo Fisher Scientific
  • MEM non-essential amino acids Cat. 11140050, Thermo Fisher Scientific
  • sodium pyruvate Cat. 11360070, Thermo Fisher Scientific
  • MEM vitamin Cat. 11120052, Thermo Fisher Scientific
  • antibiotics Penicillin-Streptomycin solution
  • BM cells into DCs were carried out in low attachment 10 mm cell culture dish in presence of bone marrow-differentiation media in presence of recombinant murine Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) (Cat. 315-03, Peprotech) for 48 h. Non-adherent cells were washed and loosely attached cells were allowed to differentiate into BMDCs for next 6 days. Cells were characterized for macrophage and DC markers using cell-surface staining and flow cytometry analyses. Human primary monocytes and human monocyte-derived macrophages (HMDMs) were used for cell-based in vitro infection assays.
  • GM-CSF Granulocyte-Macrophage Colony-Stimulating Factor
  • PBMCs Peripheral blood-derived mononuclear cells isolated from healthy male donors (leukopacks) aged between 18-30 were used for isolation of human monocytes (HM) or human monocyte-derived macrophages (HMDM).
  • HM human monocytes
  • HMDM human monocyte-derived macrophages
  • CD 14+ human monocytes were isolated from PBMCs using magnetic labeling (Monocyte Isolation Kit II, Cat. 130-091-153, Miltenyi Biotec, San Diego, CA) and magnetic columns as per manufacturer’s instructions. The purity of isolated CD14+ cells was confirmed using a fraction of cells stained with a fluorochrome-conjugated antibody against a monocyte marker as recommended by manufacturer and cells were analyzed using BD-LSR2 flow cytometer. Human monocytes were seeded (2.0 - 3.0 X 105 cells / ml in RPMI 1640 medium supplemented with 10% FBS and 1% streptomycin/penicillin at 37°C with 5% C02.
  • Monolayers of CD14+ monocytes were differentiated into Ml [GM-CSF (20 ng/ml, PeproTech, Rocky Hill, NJ) and IFN- ⁇ (20 ng/ml, PeproTech, Rocky Hill, NJ PeproTech)] or M2 [M-CSF (20 ng/ml, PeproTech, Rocky Hill, NJ) and IL-4 (20 ng/ml, PeproTech, Rocky Hill, NJ PeproTech)] for next 7 days.
  • Ml GM-CSF (20 ng/ml, PeproTech, Rocky Hill, NJ) and IFN- ⁇ (20 ng/ml, PeproTech, Rocky Hill, NJ PeproTech)
  • M2 M-CSF (20 ng/ml, PeproTech, Rocky Hill, NJ
  • IL-4 20 ng/ml, PeproTech, Rocky Hill, NJ PeproTech
  • cell lines or primary cells were seeded at required cell density in 6-well tissue culture plates or 10 mm petri dishes.
  • log-phase wild-type and BCG-disA-OE strains were harvested by centrifugation and washed twice using DPBS to remove residual detergent and BSA then suspended in antibiotic-free RPMI 1640 media supplemented with 10% FBS.
  • the bacteria were deposited at pre-calibrated multiplicity of infection (MOI). Infection was allowed for next 4 hours, followed by repeated washing of infected cells using warm DPBS to remove non-intemalized bacteria. Infected cells were incubated until endpoints in presence of RPMI- 1640 medium supplemented with 10% FBS and antibiotics.
  • RNA expression profiling was carried out using total RNA isolated from cell lines or primary cells.
  • RNA isolation from rat bladders pieces of whole bladder samples were excised, snap frozen in liquid nitrogen immediately after harvesting and stored in RNAlater (Cat. AM7021 , Ambion) at -80°C.
  • Total RNA isolation was carried out using RNeasy system (Cat. 74106, Qiagen).
  • Real-time qPCR was performed using the StepOnePlus system (Applied Biosystems).
  • SYBR Fast green double stranded DNA binding dye Cat. 4085612, Applied Biosystems
  • Gene expression analyses in rat bladder tissues were performed using TaqMan gene expression assays.
  • Gene-specific qPCR primers were purchased from Integrated DNA Technologies and all TaqMan gene expression assays were purchased from Thermo Fischer Scientific. Amplification of RNU6a, b-actin, GAPDH were used as endogenous control for RNA samples derived from human, mouse and rat cells/tissues respectively. All experiments were performed at least in triplicate and data analyses was done using 2- ⁇ CT method. Details of NCBI gene identifiers and primer sequences are given in the Table 4.
  • Multicolor confocal microscopy [0322] Multicolor laser confocal microscopy experiments were performed to determine phagocytosis, autophagy, and colocalization studies in urothelial cancer cells and primary macrophages. Cells were allowed to adhere on sterile glass cover slips placed in 6-well tissue culture plates and infections were carried at pre-calibrated MOI. Log phase bacterial cultures were labeled using FITC (Cat. F7250, Sigma).
  • IgG-FITC conjugated latex bead phagocytosis assay kit (Item No. 500290, Cayman Chemicals, USA) was used for phagocytosis studies.
  • HMDMs were placed on sterile glass cover slip for attachment.
  • Infection was carried out at 5:1 (HMDM versus BCG) ratio for 3 hours followed by addition of IgG-FITC beads in warm RPMI 1640 media at 1 : 400 dilutions for 3 hours.
  • Nuclear staining was carried out using Hoechst 33342 (Cat. 62249, Thermo Scientific) and cells were visualized for bead phagocytosis using Zeiss LSM700 single-point, laser scanning confocal microscope. Quantification of beads was measured by mean fluorescence intensity (M.F.I.) calculations using open source Fiji Software.
  • Single cell isolation was performed using animal tissues by harvesting tumors and spleens following necropsy. Briefly, tissues were manually disrupted before incubating in collagenase type I (Gibco) and DNase (Roche) in RMPI for 30 minutes at 37 °C. Tumor and spleen cells were dissociated through a 70- ⁇ m fdter and washed with PBS. RBC lysis was performed for 5 minutes using ACK lysis buffer (Cat. A1049201, Thermo Fisher Scientific) at room temperature. Cells were washed twice using ice-cold PBS and stained using Zombie AquaTM Fixable Viability Kit (Cat. 423101, Biolegend).
  • ACK lysis buffer Cat. A1049201, Thermo Fisher Scientific
  • Mouse BMDMs Anti-CD45 (clone 30-F11), anti-CD124 (clone I015F8), anti-I-A/I-E (clone 107630), anti-Ly6C (clone HK1.4), anti-CDl lb (clone Ml/70), anti-F4/80 (clone BM8), anti-Ly6G (clone 1A8), anti CD206 (clone C068C2), anti-TNF (clone MP6-XT22) all Biolegend and anti- IL-10 (clone JES5-16E3 eBiosciences).
  • Human HMDMs anti CD16 (clone 3G8), anti-CD14 (clone 63D3), anti-HLA-DR (clone L243), anti-CDllb (clone ICRF44), anti-CD206 (clone 15-2), anti-CD163 (clone GHE61), anti-TNF (clone MAbl 1), and anti-TNF (clone MAbl 1) all Biolegend.
  • Mouse macrophages (syngeneic MB49 model of urothelial carcinoma): CD45 (clone 30-F11, Biolegend), CD124 (IL-4Ra) (clone I015F8, Biolegend), I-a/I-e (clone M5/114.15.2, Biolegend), F4/80 (clone BM8, Biolegend), CD206 (clone C068C2, Biolegend), TNF (clone MP6- XT22, Thermo Fisher), IL-10 (clone JES5-16E3, Thermo Fisher)
  • Mouse T cells (syngeneic MB49 model of urothelial carcinoma): CD45 (clone PerCP, Biolegend), CD25 (clone PC61, Biolegend), CD3 (clone 17A2, Biolegend), CD4 (clone GK1.5, Biolegend), CD8a (clone 53-6.7, Biolegend), FOXP3 (clone MF-14, Biolegend), Mouse IFN-g (clone XMG1.2, Biolegend) and FOXP3 (clone MF-14 Biolegend).
  • PBMCs were isolated from healthy donors (leukopaks). Following magnetic separation, CD 14+ monocytes were seeded in 10 mm3 tissue culture dishes for 3 hours in warm RPMI 1640 media supplemented with 10% FBS at 37°C with 5% C02. Non-adherent cells were removed by washing cells using warm PBS. Monolayer culture of human monocytes was infected with BCG-WT and BCG-disA-OE strains at 5: 1 (monocyte versus BCG) MOIs for 4 hours in presence of RPMI 1640 supplemented with 10% FBS.
  • Non-intemalized bacilli were washed out using warm PBS and subsequently incubated for 24 hours. Cells were again washed using warm PBS and fresh warm RPMI 1640 media was added. For the following 5 days, cells were allowed to rest with a PBS wash and addition of fresh media every 2nd day. Cells were re-stimulated on day 6 with RPMI 1640 supplemented with 10% FBS (negative control, without training) or TLR1/2 agonist, Pam3Cys (Cat. tlrl-pms, InvivoGen). Following stimulation, for 24 h, culture supernatants were collected, filter sterilized and quickly snap-frozen (-80°C) for cytokine measurement.
  • Chromatin immunoprecipitation Human monocytes were fixed with a final concentration of 1% formaldehyde for 10 minutes at room temperature. Cell fixation was stopped using 125 mM glycine (Cat no.50046, Sigma-Aldrich, USA), followed by sonication to fragment cellular DNA to an average size between 300 to 600 bp using Qsonica Sonicator Q125 (Cat. 15338283, Thermo Fisher Scientific).
  • Sonicated cell lysates were subjected to immunoprecipitation (IP) by overnight incubation with recommended concentration of primary antibodies [(Histone H3K9me3 (H3K9 Trimethyl) Polyclonal Antibody cat. A-4036-100, epigentek); Anti-Histone H3 (tri methyl K4) antibody - ChIP Grade (ab8580), abcam)] in presence of magnetic Dynabeads (Cat no. 10004D, Thermo Fisher Scientific, USA) at 4°C. Non-bound material was removed by sequentially washing the Dynabeads with lysis buffer, chromatin IP (ChIP) wash buffer and Tris-EDTA (TE buffer). DNA elution was done using ChIP elution buffer.
  • ChIP chromatin IP
  • Targeted Metabolite analysis with LC-MS/MS was performed with liquid-chromatography tandem mass spectrometry (LC-MS/MS) as described earlier48. Metabolites from cells or snap-frozen xenograft tumor tissue were extracted with 80% (v/v) methanol solution equilibrated at –80 °C, and the metabolite-containing supernatants were dried under nitrogen gas.
  • the optimized MS parameters were: ESI voltage was +5,000V in positive ion mode and -4,500V in negative ion mode; dwell time was 3ms per SRM transition and the total cycle time was 1.57 seconds.
  • Hydrophilic interaction chromatography (HILIC) separations were performed on a Shimadzu UFLC system using an amide column (Waters XBridge BEH Amide, 2.1 x 150 mm, 2.5 ⁇ m).
  • the LC parameters were as follows: column temperature, 40 °C; flow rate, 0.30 ml/min.
  • Solvent A Water with 0.1% formic acid
  • Solvent B Acetonitrile with 0.1% formic acid
  • Peak integration for each targeted metabolite in SRM transition was processed with MultiQuant software (v2.1, AB Sciex).
  • the preprocessed data with integrated peak areas were exported from MultiQuant and re-imported into Metaboanalyst software for further data analysis including statistical and principle components analyses.
  • Sections of 5 m in thickness on glass slides were stained with hematoxylin-eosin for classification according to the World Health Organization/Intemational Society of Urological Pathological consensus as described earlier27.
  • high-temperature antigen retrieval (18-23 psi/126 °C) was performed by immersing the slides in Trilogy (Cell Marque). Endogenous peroxidase activity was blocked for 5 min in using Dual Endogenous Enzyme Block (Cat. S2003, Dako).
  • Ki67 (1:50, Cat. ab 16667; Abeam), CD68 (1:250, Cat. MCA341R; Serotec), CD86 (1:100, Cat. bs-1035R; Bioss) and CD206 (1:10K, Cat. ab64693; Abeam).
  • Ki67 slides were stained with ImmPACT DAB (Vector Labs) for 3 min and counterstained with haematoxylin (Richard-Alien).
  • T o determine the lung bacillary burden of wild-type and BCG-disA-OE strains 6-week- old female BALB/c mice were exposed using the aerosol route in a Glasscol inhalation exposure system (Glasscol).
  • the inoculum implanted in the lungs at day 1 (n 3 mice per group) in female BALB/c mice was determined by plating the whole lung homogenate on 7H11 selective plates containing carbenicillin (50 mg/ml), Trimethoprim (20 mg/ml), Polymyxin B (25 mg/ml) and Cycloheximide (10 mg/ml).
  • MB49 tumor cells are urothelial carcinoma line derived from an adult C57BL/6 mouse by exposure of primary bladder epithelial cell explant to 7,12-dimethylbenz[a]anthracene (DMBA) for 24 hours followed by a long-term culture79.
  • DMBA 7,12-dimethylbenz[a]anthracene
  • MB49 cells were cultured as monolayers in RPMI 1640 media supplemented with 10% FBS and 1% streptomycin/penicillin at 37°C with 5% C02. Cells were harvested using Trypsinization and cell viability was determined using Trypan blue dye. Live MB49 cells were resuspended in sterile PBS and adjusted at 1 x 10 5 live cells per 100 ⁇ l.
  • BCG-disA-OE is a genetically-engineered BCG strain in which an endogenous diadenylate cyclase gene, disA, is fused to a strong promoter, leading to a 300-fold overexpression of disA and a 15-fold increase in production of cyclic di-AMP (Fig. 39).
  • BCG-disA-OE significantly increased STING pathway activation in macrophages as measured by IRF3 induction (Fig. 39).
  • T o characterize the trained immunity-inducing potential of BCG-disA-OE versus BCG- WT, their capacity to induce cytokine expression in human monocyte-derived macrophages (HMDMs), primary murine bone marrow-derived macrophages (BMDM), and dendritic cells (BMDC) as well as a macrophage cell line (J774.1) were evaluated.
  • HMDMs human monocyte-derived macrophages
  • BMDM primary murine bone marrow-derived macrophages
  • BMDC dendritic cells
  • Cyclic dinucleotides including cyclic di-AMP are known to be potent inducers of several chemokines (CXCL9, CXCL10 [IP- 10], CXCL22, and MCP-1) as well as iNOS; consistent with this, IFN-y-primed BMDMs showed a more robust induction of these chemokines and iNOS when challenged with BCG-disA-OE strain than with BCG-WT (Fig. 41).
  • BCG-disA-OE elicits significantly higher levels of apoptotic cell death (Fig. 44) in the BMDM cells.
  • Trained immunity is associated with polarization of macrophages towards inflammatory phenotypes with a concomitant shift away from anti-inflammatory states.
  • flow cytometry was used to monitor phenotypic shifts of both murine and human primary macrophages following a 24 h exposure to BCG-disA-OE or BCG-WT.
  • the MHC class Il-expressing CD45 + CD1 lb + F4/80 + murine BMDM population were focused on, following in vitro BCG exposure using the gating scheme. As may be seen in Fig.
  • M-MDSCs Myeloid-derived suppressor cells
  • M-MDSCs monocytic-myeloid derived suppressor cells
  • BCG-disA-OE increases the levels of phagocytosis and autophagic processing within macrophages to a greater degree than BCG-WT, a phenomenon associated with enhanced peptide antigen presentation to MHC class-II molecules.
  • BCG-disA -OE REPROGRAMS MACROPHAGES EPIGENETICALLY AND POTENTIATES TRAINED IMMUNITY TO A GREATER DEGREE THAN BCG-WT
  • chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR) assays the activating histone methylation mark H3K4me3 present in the TNF-a and IL-6 promoters was quantified. It was observed that exposure to BCG-disA-OE led to greater enrichment of this mark than BCG-WT even without the heterologous second stimulation (i.e., adding RPMI media alone at day 6). Upon re-stimulation with Pam3CSK4 at day 6, the abundance of the activating epigenetic mark was further increased by both BCG strains, but BCG-disA-OE-pretreatment yielded notably more enrichment than BCG-WT (Fig. 56).
  • LC-MS was used to characterize key metabolites in primary human and murine macrophages exposed to the two BCG strains.
  • HMDMs or BMDMs showed increased catabolic signatures (elevated intracellular glucose and lactate) to a greater degree following a 24 h exposure to BCG-disA-OE than with BCG- WT.
  • the TCA cycle metabolites itaconate and fumarate were also more elevated with BCG-disA-OE than with BCG-WT.
  • Kynurenine levels were dramatically lower in macrophages following BCG-disA-OE exposure than those seen with BCG-WT, and as would be expected tryptophan levels were elevated by BCG-disA-OE while BCG-WT led to tryptophan levels comparable to the baseline seen with heat-killed BCG controls. Citrulline levels were also higher while putrescine levels were lower with BCG-disA-OE than BCG-WT suggesting that nitric oxide synthase-mediated conversion of arginine to NO (pro-inflammatory) and citrulline was more strongly induced by BCG-disA-OE.
  • BCG-disA-OE an isocitrate lyase inhibitor made by macrophages that has been shown to have antibacterial activity, was more potently induced by BCG-disA-OE than BCG-WT.
  • BCG-disA-OE elicited a greater pro-inflammatory metabolomic signature with reduced kynurenine accumulation and increases in glycolytic metabolites, NOS products, and itaconate production.
  • BCG-disA-OE versus BCG-WT was tested in a carcinogen-induced model of NMIBC in which intravesical therapies can be introduced into the bladder as they are in humans with non-invasive urothelial cancer.
  • the rat N -m ethyl - N -nitrosourea (MNU) model of bladder cancer (BC) is schematized in Fig.
  • urothelial dysplasia develops at week 14 after the first intravesical instillation of MNU and by week 24 rats display a different forms of urothelial cancer severity including carcinoma-m-V/n (CIS), papillary Ta (superficial), or higher-grade Tl- T2 urothelial carcinoma with histopathologic and immunophenotypic features similar to those observed in human bladder cancer.
  • CIS carcinoma-m-V/n
  • papillary Ta superficial
  • Tl- T2 urothelial carcinoma with histopathologic and immunophenotypic features similar to those observed in human bladder cancer.
  • groups of rats were treated with 6 weekly doses of intravesical BCG-disA-OE, BCG-WT, or no treatment from week 18-23.
  • rat urinary bladders were divided into halves for (i) RT-PCR analysis, and (ii) histologic analysis including tumor staging by a blinded genitourinary pathologist.
  • Transcriptional analysis of the whole excised bladders at week 24 showed that compared with BCG-WT, BCG-disA-OE elicited significantly increased levels of IFN-b, IFN-g, TNF-a, IL-Ib, CXCL10, MCP-1, MIP-la, and iNOS transcription while mRNA levels of the immunosuppressive cytokines IL-10 and TGF-b were reduced by both BCG strains (Fig. 57).
  • Immunohistochemical analyses revealed a significant reduction in Ki67 staining in BCG-disA-OE-treated MNU rat bladders when compared to untreated (p ⁇ 0.01) and BCG-WT (p ⁇ 0.05) suggesting reduced tumor proliferation.
  • CD68 staining of rat bladder showed significantly higher levels of macrophage recruitment with a trend toward elevation of the pro-inflammatory Ml -like CD86+ macrophages and a significant reduction in CD206+ M2-like macrophages that are associated with tumor promotion in the BCG-disA-OE-treated rats compared with untreated controls.
  • BCG-disA -OE The functional efficacy of BCG-disA -OE was also tested in a murine heterotopic, syngeneic bladder cancer model using MB49 urothelial cancer cells. Following flank engraftment with MB49 tumor cells, mice received four intratumoral treatments over 9 days as shown in Fig. 61. In this model BCG-disA-OE also showed more robust immunotherapeutic efficacy than BCG- WT as measured by tumor volume and weight after intratumoral injection of BCG-disA-OE when compared with BCG-WT (Fig. 61). Histopathology demonstrated extensive necrosis and congestion in MB49 tumors treated with BCG-disA-OE when compared to BCG-WT and untreated.
  • mice receiving BCG There were no significant changes in body weights of mice receiving BCG, however splenic weight was significantly increased by both BCG strains.
  • TME tumor microenvironment
  • BCG-disA-OE significantly reduced the abundance of immunosuppressive M2 macrophages when compared to untreated and BCG-WT and significantly (p ⁇ 0.01) increased proinflammatory Ml macrophages.
  • BCG-disA-OE recruited significantly more IFN-y-producing CD4 + T cells when compared to BCG-WT, and both BCG strains increased IFN-y-producing CD8 + T cells.
  • BCG-disA-OE While both BCG strains recruited more CD4 + and CD8 + cells to the tumors, BCG-disA-OE uniquely recruited more CD8 + T cells to the spleens of treated animals. BCG-disA-OE also significantly reduced tumor-associated T-regulatory (Treg) cells to a greater degree than BCG-WT in both tumor and spleen. In keeping with our earlier findings in primary cells, we also found that compared with BCG-WT, BCG-disA-OE elicited more potent cytokine responses and autophagy in human urothelial cancer cells representing various tumor stages.
  • BCG-disA-OE has superior antitumor efficacy than BCG- WT, and its efficacy correlates with shift in polarization of macrophages to M1, increased activation of both CD4 + and CD8 + T cells, and a reduction of local intratumoral and systemic Treg cell populations.
  • BCG-disA-OE versus BCG-WT were tested in a battery of in vitro assays. Cytokine release profiles, macrophage polarization, autophagy, phagocytosis, epigenetic modifications, and metabolic remodeling in human and murine primary cells were evaluated. In each assay system, BCG-disA-OE was a more potent potentiator of pro-inflammatory responses than BCG-WT. the cyclic-di-AMP expressing BCG was further tested in a functional in vivo assay of trained immunity, namely bladder cancer immunotherapy.
  • BCG-disA-OE has greater immunotherapeutic efficacy than did BCG-WT indicating that our in vitro results were predictive of functional efficacy in a relevant animal model.
  • BCG-disA-OE did not produce excess pathogenicity in two animal models of BCG infection or BCGosis.
  • BCG-WT did not uniformly elicit pro-inflammatory responses. For example, it was observed that treatment of murine macrophages with BCG-WT in fact induced a higher percentage of M-MDSCs (anti-inflammatory) compared with untreated controls (Fig.
  • BCG-disA-OE was superior to BCG-WT in reducing tumor growth with associated increase in tumor necrosis, and these effects were accompanied by significantly higher recruitment of Ml macrophages, IFN-y-producing CD4 cells, and reduced accumulation of Treg cells in the tumors. Elevated levels of pro-inflammatory cytokines and chemokines were observed in bladders from tumor-bearing animals treated with BCG-disA-OE compared to BCG-WT. Since non-immune cells have also been shown to possess immunological memory, the possibility that this cytokine response may have originated from myeloid cells in the TME and/or the tumor cells themselves was considered.
  • BCG-disA-OE elicited more potent cytokine responses in both primary macrophages and human urothelial cancer cells representing various tumor stages. This appeared to be a downstream consequence of STING activation since we found dramatically reduced expression in BMDMs from STING -/ - mice.
  • robust induction of several chemokines as has been observed in other studies with stimulation using exogenous STING agonists was found.

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Abstract

L'invention concerne des procédés pour supprimer l'expression de cellules myéloïdes suppressives (MDSC), de macrophages M2 et de lymphocytes Treg dans une tumeur et induire l'expression de macrophages, de cellules dendritiques (DC) et de lymphocytes T effecteurs dans une tumeur chez un sujet. Une composition pharmaceutique comprenant une souche de mycobactéries comprenant un vecteur d'expression de la présente invention est administrée à un sujet.
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WO2019203965A1 (fr) * 2018-04-17 2019-10-24 The Johns Hopkins University Interventions thérapeutiques recombinantes pour le cancer

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