Live biopharmaceuticals that secrete synthetic bacteriophages in cancer treatment

Abstract

The present disclosure relates generally to synthetic therapeutic bacteriophages that display at least one therapeutic agent, where the at least one therapeutic agent is fused to a coating protein of the synthetic bacteriophage.

Description

Technical Field

[0001] Cross - reference to Related Applications This application claims the benefit and priority of U.S. Provisional Patent Application No. 63 / 088,643, filed on October 7, 2020; U.S. Provisional Patent Application No. 63 / 161,543, filed on March 16, 2021; and U.S. Provisional Patent Application No. 63 / 215,176, filed on June 25, 2021, the contents of which are hereby incorporated by reference in their entirety.

[0002] The present disclosure generally relates to synthetic bacteriophages presenting one or more recombinant molecules having antitumor activity, live biotherapeutics expressing and delivering such synthetic bacteriophages, and methods of using them to prevent and treat cancer.

Background Art

[0003] Despite significant progress in cancer research and treatment, cancer remains the second leading cause of death in developed countries (Siegel R. et al., ACS Journal, Cancer statistics 2021; which is hereby incorporated by reference). Cancer is a complex and difficult - to - treat disease, and in order to maximize the likelihood of treatment success, it is often necessary to act on several therapeutic targets simultaneously. This strategy is called combination therapy, and physicians treat patients by combining two or more therapeutic agents (Mokhtari et al., Oncotarget, June 2017; 8(23):38022 - 38043; which is hereby incorporated by reference). By targeting multiple different pathways to inhibit or eliminate cancerous cells, combination therapy yields better results than monotherapy and forms the basis of cancer treatment.

[0004] The need to combine multiple therapies to maximize treatment outcomes is exemplified in the field of immuno-oncology, a branch of cancer therapy that manipulates the immune system to induce tumor disappearance. In immuno-oncology, tumor disappearance is greatly improved when the tumor is considered "hot" (Duan et al., Trends in Cancer (2020), Vol. 6, No. 7, pp. 605-618; this is incorporated herein by reference). This occurs when two conditions are met: (i) immune cells are present within the tumor, and (ii) these immune cells are not suppressed by the tumor microenvironment. The current strategy for transforming cold tumors into hot tumors involves the use of two drugs: the first to promote the recruitment of immune cells and tumor invasion, and the second to ensure that the immune cells are active and not inhibited by the tumor microenvironment (Haanen J. et al., Cell (2017), vol. 170, no. 6, pp. 1055-1056, and Sevenich L., Front. Oncol. (2019), vol. 9, article 163; these are incorporated herein by reference). For example, this is achieved by combining oncolytic virus therapy that promotes tumor invasion (e.g., Talimogene Laherparepvec from AMGEN) with a checkpoint inhibitor that ensures immune cells are activated (e.g., ipilimumab from Bristol-Myers Squibb) (Puzanov I. et al., J Clin Oncol (2016), 1;34(22):26 pp. 19-26; this is incorporated herein by reference).

[0005] While combining several treatment modes offers clear therapeutic advantages compared to monotherapy, such strategies have at least two major drawbacks. Firstly, combining several treatments also combines their side effects. For example, combining a PD-L1 checkpoint inhibitor with a CTLA-4 checkpoint inhibitor may yield better results than monotherapy, but it also results in adverse events in approximately 50% of patients (Grover S. et al., Gastrointestinal and Hepatic Toxicities of Checkpoint Inhibitors: Algorithms for Management 2018 ASCO Educational book; incorporated herein by reference). This can have serious consequences, as excessive side effects sometimes lead to premature termination of the treatment, leaving patients without a therapeutic solution. Secondly, combining several treatments also combines the development costs of each treatment, resulting in increased treatment costs.

[0006] In addition, anticancer therapies typically utilize toxic mechanisms to eliminate cancer cells. Most anticancer drugs are administered systemically and spread throughout the body, so they exert toxic effects on healthy tissues and organs, resulting in side effects (Cleeland, CS et al., Nat. Rev. Clin. Oncol. (2012), Vol. 9, pp. 471-478; this is incorporated herein by reference).

[0007] Therefore, most current cancer treatments suffer from the lack of a targeted delivery approach. These treatments instead rely on high doses to reach the desired intratumoral concentration for optimal therapeutic activity at the tumor site, which increases the risk of side effects. [Prior art documents] [Patent Documents]

[0008] [Patent Document 1] PCT / US2017 / 013072 issue [Patent Document 2] WO2009101611 [Patent Document 3] WO2010027827 [Patent Document 4] WO2011066342 [Patent Document 5] U.S. Patent Application Publication No. 2015 / 0359894 [Patent Document 6] U.S. Patent Application Publication No. 2015 / 0238545 [Patent Document 7] U.S. Patent No. 5,989,463 [Non-patent literature]

[0009] [Non-Patent Document 1] Siegel R. et al., ACS Journal, Cancer statistics 2021 [Non-Patent Document 2] Mokhtari et al., Oncotarget June 2017;8(23):38022-38043 [Non-Patent Document 3] Duan et al., Trends in Cancer (2020), Vol. 6, No. 7, pp. 605-618. [Non-Patent Document 4] Haanen J. et al., Cell (2017), Vol. 170, No. 6, pp. 1055-1056. [Non-Patent Document 5] Sevenich L., Front. Oncol. (2019), Volume 9, Paper 163 [Non-Patent Document 6] Puzanov I. et al., J Clin Oncol (2016), 1;34(22):2619~26 [Non-Patent Document 7] Grover S., Gastrointestinal and Hepatic Toxicities of Checkpoint Inhibitors: Algorithms for Management 2018 ASCO Educational book [Non-licensed Document 8] Cleeland, CS, Nat. Rev. Clin. Oncol. (2012), 9, pp. 471-478 [Non-licensed Document 9] Smith and Waterman, Ad. App. Math 2:482 pages (1981) [Non-licensed Document 10] Needleman and Wunsch, J. Mol. Biol. 48:443 pages (1970) [Non-licensed Document 11] Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988) [Non-licensed Document 12] F. CORPET, "Multiple sequence alignment with hierarchical clustering", 1988, Nucl. Acids Res., 16(22), pages 10881~10890 [Non-licensed Document 13] Sambrook, Molecular Cloning. A Laboratory Manual [Non-licensed Document 14] Zapata, Protein Eng. 8(10): pages 1057~1062 (1995) [Non-licensed Document 15] Motohiro Matsuura, Front Immunol., 2013, 4:019 [Non-licensed Document 16] Steimle, Int. J. Med. Microbiol., 2016, Volume 306: 290 pages [Non-licensed Document 17] Simpson, Nat. Rev. Microbiol., 2019, 17:403 [Non-licensed Document 18] S. Sidhu, J. Mol. Biol. (2000) pp. 296, 487~495 [Non-licensed Document 19] Hecht, Nucleic Acids Research, 2017, Volume 45, No. 7, Pages 3615~3626 [Non-licensed Document 20] Chen, Adv Drug Deliv Rev. October 15, 2013; 65(10): 1357-1369 [Non-licensed Document 21] Hanら、AMB Expr(2017)7:93 pages [Non-licensed Document 22] Bharathwajら, mBio. June 29, 2021;12(3) [Non-licensed Document 23] Hobohm & Grange, Crit Rev Immunol. 2008;28(2):95~107 pages [Non-licensed Document 24] Krysko DVら, Cell Death and Disease (2013) 4, page e631 [Non-licensed Document 25] Carroll-Portillo A., Microorganisms. 2019 December; 7(12): 625 pages [Non-licensed Document 26] Takeuchi, Cell, 2010, 140: 805-820 pages [Non-licensed Document 27] "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA [Non-licensed Document 28] Greenら, 2013 [Non-licensed Document 29] McKenzieら, 2006 [Non-licensed Document 30] Vaddepally et al., Cancers (Basel) March 2020; 12(3):738. [Non-Patent Document 31] Nyati MK et al., Gene Therapy 2002; 9: pp. 844-849 [Non-Patent Document 32] Specthrie et al., J. Mol. Biol., 1992, 3:720. [Overview of the project] [Problems that the invention aims to solve]

[0010] Considering the above, there is still a need in the field of cancer treatment for therapeutic agents that can overcome at least some of the shortcomings identified above. In particular, there is a need for therapeutic agents that can act simultaneously on several therapeutic targets, while at the same time limiting side effects through localized delivery at low doses and high efficacy. [Means for solving the problem]

[0011] In various embodiments, the technology relates to a synthetic therapeutic bacteriophage that presents at least one therapeutic agent, wherein the at least one therapeutic agent is fused with a coating protein of the synthetic bacteriophage. In some implementations of these embodiments, the synthetic bacteriophage secretion system includes a synthetic bacteriophage mechanism. The synthetic bacteriophage mechanism includes a bacteriophage assembly module, a bacteriophage replication module, a bacteriophage coating module, and a therapeutic module. In some cases, the bacteriophage assembly module includes i) a bacteriophage gene gpI encoding proteins pI and pXI; or ii) a bacteriophage gene gpIV encoding protein pIV; or iii) both i) and ii). In some other cases, the bacteriophage replication module includes i) the bacteriophage gene gpII encoding proteins pII and pX; or ii) the bacteriophage gene gpV encoding protein pV; or iii) both i) and ii). In some cases, the bacteriophage coating module includes the bacteriophage genes gpIII, gpVI, gpVII, gpVIII, and gpIX, or parts thereof, encoding or encoding parts thereof the coating proteins pIII, pVI, pVII, pVIII, and pIX, respectively. In some cases, the therapeutic module includes one or more bacteriophage coating genes selected from the bacteriophage genes gpIII, gpVI, gpVII, gpVIII, and gpIX, encoding the coating proteins pIII, pVI, pVII, pVIII, and pIX, respectively. In some implementations, at least one therapeutic agent is presented in at least a portion of the coating protein.

[0012] In various embodiments, the therapeutic agent is a binding protein. In some cases, the binding protein binds to and inhibits one or more proteins, peptides, or molecules involved in carcinogenesis, cancer development, or metastasis. In some other cases, the one or more proteins, peptides, or molecules to be inhibited are selected from CSF1, CSF1R, CCR4, CCL2, CCL17, CCL22, HER2, GD2, IL-1β, IL-6, IL-10, IL-13, IL-17, IL-27, IL-35, CD20, CD27, CD30, CD33, CD70, TGF-β, M-CSF, EGFR, ERBB2, ERBB3, PGE2, VEGF, VEGFR-2, CXCR4 / CXCL12, Tie2, galectin-1, galectin-3, phosphatidylserine, and TAM and Tim phosphatidylserine receptors. In some cases, the binding protein acts as an agonist that activates a costimulatory receptor that leads to the elimination of cancer cells, one or more costimulatory receptors selected from, but not limited to, CD40, CD27, CD28, CD70, ICOS, CD357, CD226, CD137, and CD134. In some other cases, the binding protein inhibits immune checkpoint molecules, such as, but not limited to, CCR4, CTLA-4, CD80, CD86, PD-1, PD-L1, PD-L2, TIGIT, VISTA, LAG-3, TIM1, TIM3, CEACAM1, LAIR-1, HVEM, BTLA, CD47, SIRPα, CD160, CD200, CD200R, CD39, CD73, B7-H3, B7-H4, IDO, TDO, KIR, and A2aR.

[0013] In various forms, therapeutic agents stimulate an immune response.

[0014] According to various embodiments, the therapeutic agent is an antibody, an antibody mimetic, or a nanobody.

[0015] In various aspects, the therapeutic agent is cytosine deaminase.

[0016] In various embodiments, the technology relates to a living biotherapy for producing and / or delivering at least one therapeutic agent, wherein the living biotherapy comprises a recombinant bacterial organism comprising a synthetic bacteriophage secretion system capable of secreting synthetic therapeutic bacteriophages as defined herein.

[0017] In various embodiments, the technology relates to a living biotherapeutic agent for producing and / or delivering a therapeutic agent, comprising a recombinant bacterial organism including a synthetic bacteriophage secretory system capable of secreting a synthetic therapeutic bacteriophage, wherein the synthetic therapeutic bacteriophage presents the therapeutic agent. In some embodiments, the recombinant bacterial organism is selected from the Enterobacteriaceae, Pseudomonadaceae, and Vibrionaceae families. In some embodiments, the recombinant bacterial organism is a tumor-targeting bacterium, such as Escherichia coli Nissle 1917 strain and Escherichia coli MG1655 strain, but is not limited to these.

[0018] In various embodiments, the present invention relates to a method for delivering at least one therapeutic agent to a tumor site in a subject, comprising administering to a subject in need an effective amount of a synthetic therapeutic bacteriophage as defined herein or an effective amount of a live biotherapeutic agent as defined herein.

[0019] In various embodiments, the present invention relates to a method for the prevention and / or treatment of cancer in a subject requiring such treatment, comprising administering to the subject requiring such treatment an effective amount of a synthetic therapeutic bacteriophage as defined herein or an effective amount of a live biotherapy agent as defined herein.

[0020] In various embodiments, the technology relates to a method for the prevention and / or treatment of cancer in subjects requiring it, comprising administering an effective amount of a synthetic therapeutic bacteriophage to the subject requiring it, wherein the synthetic bacteriophage does not present a therapeutic agent. In some implementations, cancers include adrenal cancer, adrenocortical carcinoma, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone malignancies, brain malignancies, bronchial tumors, central nervous system tumors, breast cancer, Castleman disease, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, ocular malignancies, gallbladder cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, cardiac malignancies, Kaposi's sarcoma, kidney cancer, pharyngeal cancer, hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, and many others. The following conditions are selected: myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, gastric cancer, teratoma, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, rare childhood cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

[0021] In various embodiments, the technology relates to the use of an effective amount of a synthetic therapeutic bacteriophage as defined herein or an effective amount of a living biotherapeutic as defined herein for the prevention and / or treatment of cancer in a subject that requires it.

[0022] In various embodiments, the technology relates to the use of an effective amount of synthetic therapeutic bacteriophage for the prevention and / or treatment of cancer in a subject requiring such use, wherein the synthetic therapeutic bacteriophage does not present a therapeutic agent.

[0023] In various embodiments, the technology relates to the use of a kit comprising a synthetic therapeutic bacteriophage or a defined live biotherapy agent as described in any one of claims 1 to 33, along with instructions for administering the synthetic therapeutic bacteriophage or live biotherapy agent to a subject.

[0024] In various embodiments, the technology relates to a kit comprising a live biotherapeutic drug as defined herein, along with instructions for administering the drug to a subject. [Brief explanation of the drawing]

[0025] [Figure 1] This is a schematic diagram illustrating an exemplary configuration of a living biopharmaceutical that secretes synthetic therapeutic bacteriophages according to one embodiment of this technology. [Figure 2] This is a schematic diagram of synthetic bacteriophages for monotherapy, dual therapy, and multitherapy that present monospecific and / or bispecific therapeutic proteins. [Figure 3] This is a schematic diagram illustrating the mechanism of action of a living biopharmaceutical that secretes CD47-binding synthetic therapeutic bacteriophages. The living biopharmaceutical secretes synthetic bacteriophages that present anti-CD47 nanobodies, which are checkpoint inhibitors. The CD47 nanobodies recognize and bind to the CD47 immune checkpoint expressed on cancer cells. Therefore, the therapeutic bacteriophage binds to CD47 and prevents the CD47 immune checkpoint from inhibiting T cell activation. [Figure 4] This is a schematic diagram illustrating the immunogenic effects of synthetic bacteriophages and bacterial hosts. [Figure 5] This is a schematic diagram of an exemplary structure of a synthetic bacteriophage secretion system. Examples of constructed synthetic bacteriophage mechanism structures include M13K07(A), M13mp18-Kan(B), pTAT004(C), pTAT025(D) and their derivatives. Examples of constructed synthetic bacteriophage scaffold vectors include pTAT002(E), pTAT012(F), pTAT013(G), pTAT014(H) and their derivatives. [Figure 6] The map includes a schematic representation of pTAT001, with the sites cleaved by the selected restriction enzymes identified on the map for validation of the construct. The expected digestion products are also shown on the map, along with the experimental agarose gel of the construct after digestion with the specified enzymes. [Figure 7] Figures 7A–7C are graphs showing that live biopharmaceuticals secrete fully assembled bacteriophages presenting checkpoint inhibitors fused with pIII. (A) Infectivity of bacteriophages secreted by live biopharmaceuticals containing pTAT004 together with either pTAT002 (control) or pTAT003 (presenting anti-CD47 nanobodies), compared to infectivity of those containing M13K07. (B) Doses of synthetic bacteriophages produced by live biopharmaceuticals that present one of the following: nothing (pTAT004 + pTAT002), nanobodies on pIII (anti-CD47, pTAT004 + pTAT003; anti-PD-L1, pTAT004 + pTAT020; anti-CTLA-4, pTAT004 + pTAT019), anticharin on pIII (anti-CTLA-4, pTAT004 + pTAT030), enzyme (cytosine deaminase, pTAT004 + pTAT022), peptide on pVIII (pTAT002 + pTAT027), or anti-CD47 nanobodies on pIX (pTAT025 + pTAT002 + pTAT028), as determined by ELISA. Detection was performed using an anti-pVIII B62-FE3 (progen) antibody linked to HRP. (C)ELISA dose of bacteriophages produced by live biopharmaceuticals possessing either pTAT004 + pTAT002, pTAT004 + pTAT003, or M13K07. Detection was performed using an anti-HA antibody linked to HRP. [Figure 8]This graph shows that synthetic bacteriophages strongly bind to A20 lymphoma cancer cells. A pull-down assay was performed using bacteriophages produced by live biopharmaceuticals containing either pTAT004 and either pTAT002 (control) or pTAT003 (presenting anti-CD47 nanobodies). [Figure 9]Figures 9A-9L are graphs showing that synthetic bacteriophages presenting checkpoint inhibitors mask immune checkpoints on cancer cells. (A) Fluorescence basal signal measured on unstained A20 cells using FITC channels during flow cytometry assay. (B) Fluorescence signal of the A20 population stained with anti-CD47-FITC antibody. (C) Fluorescence signal of the A20 population first incubated with a control synthetic bacteriophage produced by a live biopharmaceutical containing pTAT004 + pTAT002, and then stained with anti-CD47-FITC antibody. (D) Fluorescence signal of the A20 population first incubated with a synthetic bacteriophage presenting anti-CD47 nanobodies on pIII, produced by a live biopharmaceutical containing pTAT004 + pTAT003, and then stained with anti-CD47-FITC antibody. (E) Fluorescence basal signal measured on unstained A20 cells using FITC channels during flow cytometry assay. (F) Fluorescence signal of the A20 population stained with anti-CD47-FITC antibody. (G) Fluorescence signal of the A20 population first incubated with a control synthetic bacteriophage produced by a live biopharmaceutical containing pTAT004 + pTAT002, and then stained with anti-CD47-FITC antibody. (H) Fluorescence signal of the A20 population first incubated with a synthetic bacteriophage presenting anti-CD47 nanobodies on pIX, produced by a live biopharmaceutical containing pTAT002 + pTAT025 + pTAT028, and then stained with anti-CD47-FITC antibody. (I) Fluorescence basal signal measured in unstained A20 cells using a PE channel during a flow cytometry assay. (J) Fluorescence signal of the A20 population stained with anti-PD-L1-PE antibody. Fluorescence signals of the A20 population, first incubated with a control synthetic bacteriophage produced by a live biopharmaceutical containing (K)pTAT004 + pTAT002, and then stained with an anti-PD-L1-PE antibody.Fluorescence signals of the A20 population, first incubated with synthetic bacteriophages presenting anti-PD-L1 nanobodies on pIII, produced by a living biopharmaceutical containing (L)pTAT004 + pTAT020, and then stained with anti-PD-L1-PE antibody. [Figure 10] This graph shows that synthetic bacteriophages displaying anti-CTLA-4 nanobodies or antikalin can bind to the CTLA-4 protein. ELISA was performed on synthetic bacteriophages displaying anti-CTLA-4 nanobodies (pTAT019), anti-CTLA-4 antikalin (pTAT030), or synthetic bacteriophages that do not display antikalin (pTAT002). [Figure 11] Figures 11A–11E are graphs showing that a functional therapeutic protein maintains its function when cloned between two protein domains. A synthetic bacteriophage presenting an anti-PD-L1 nanobody inserted in pIII can bind to the PD-L1 protein on the surface of A20 cells and compete with PE-labeled antibodies. (A) Fluorescence basal signal measured in unstained A20 cells using a PE channel during a flow cytometry assay. (B) Fluorescence signal of an A20 population stained with anti-PD-L1-PE antibody. (C) Fluorescence signal of an A20 population first incubated with a control synthetic bacteriophage (pTAT002, no presentation) and then stained with anti-PD-L1-PE antibody. (D) Fluorescence signal of an A20 population first incubated with a synthetic bacteriophage (pTAT032) presenting an anti-PD-L1 nanobody at the N-terminus of pIII and then stained with anti-PD-L1-PE antibody. (E)Fluorescence signal of the A20 population first incubated with synthetic bacteriophage (pTAT033) presenting an anti-PD-L1 nanobody inserted between the binding domain of pIII and the bacteriophage anchor domain, and then stained with an anti-PD-L1-PE antibody. [Figure 12]Figures 12A–12F are graphs showing that synthetic bacteriophages can present antigens on all of their major coat protein pVIII subunits. (A) Sanger sequencing of the pTAT027 construct. (B) Bacteriophage particles secreted by live biopharmaceuticals that present or do not present OVA epitopes on pVIII, and that present or do not present anti-CD47 nanobodies on pIII, as measured by ELISA. (C) Western blot analysis of the protein profiles of the pIII subunit in bacteriophages that present or do not present OVA on pVIII. (D–F) Flow cytometry analysis of bacteriophages binding to CD47 on the surface of A20 cells. (D), (E), (F), (F), (D), (E), (F), (F), (D), (E), (F), (F), (D), (E), (F), (D), (E), (F), (D), (E), (F), (D), (E), (F), (E), (F), (E), (D), (E), (F), (E), (F), (E), (N), (E), (F), (E), (N), (E), (F), (E), (N), (E), (F), (E), (F), (E), (F), (E), (F), (N), (F), (E), (F [Figure 13-1]Figure 13. Figures 13A-13B are graphs showing that the antitumor activity of synthetic bacteriophages is enhanced by the presentation of anti-CD47, anti-PD-L1, or anti-CTLA-4 nanobodies. (A) Mean tumor volume was measured for each mouse group treated with three intratumoral injections of bacteriophage particles ranging from 107 to 1011 particles of the control synthetic bacteriophage (pTAT002). For treatment with PBS, 107, 108, and 109 bacteriophage particles, the doses were administered on days 0, 4, and 7 (arrows); on the other hand, for treatment with 1011 bacteriophage particles, the doses were administered on days 0, 4, and 11 (gray arrows). (B) Tumor disappearance observed in mice treated with the control synthetic bacteriophage. (C) Tumor volume measured in mice treated with three intratumoral injections (arrows) of 1 × 10⁸ control synthetic bacteriophages (pTAT002) without therapeutic protein, 1 × 10⁸ synthetic bacteriophages presenting anti-PDL1 nanobodies, or 1 × 10⁸ synthetic bacteriophages presenting anti-CTLA-4 nanobodies. Individual tumor volumes are shown for each mouse (solid line = mice with disappearance, dotted line = mice without disappearance). (A~B) Data are representative of at least 5 mice per group. Tumor volume was calculated by multiplying the maximum measurement by the square of the orthogonal measurement and dividing by 2. [Figure 13-2] Continuation of Figure 13. [Figure 14-1]Figure 14. Figures 14A-14H show the synergistic effects of checkpoint inhibitors presented by synthetic bacteriophages. (A) ELISA assay showing that purified anti-PD-L1 nanobodies are functional and bind to the PD-L1 protein. (B-C) Showing the synergistic effects of checkpoint inhibitors presented by synthetic bacteriophages. Tumors were engrafted in mice by injecting 5 × 10⁶ A20 cells into the right flank. The tumors were then treated when the tumor volume reached between 100 and 200 mm³. For each mouse treated on days 0, 4, and 7, the tumor volume was measured after intratumoral injection of either PBS, 8 × 10¹⁵ anti-PD-L1 nanobodies, 1 × 10⁸ control synthetic bacteriophage particles (pTAT002), 5 × 10⁸ anti-PD-L1 nanobodies, 1 × 10⁸ control synthetic bacteriophage particles and 5 × 10⁸ anti-PD-L1 nanobodies, or 1 × 10⁸ synthetic bacteriophage particles presenting anti-PD-L1 nanobodies. Tumor disappearance data are reported in (B), and tumor volume was calculated by multiplying the maximum measurement by the square of the orthogonal measurement and dividing by 2 (solid line = mice with disappearance, dotted line = mice without disappearance) (C). [Figure 14-2] Continuation of Figure 14. [Figure 15]This graph shows that a live biopharmaceutical secreting synthetic bacteriophages that present anti-CD47 nanobodies inhibits tumor growth. Tumors were engrafted in mice by injecting 5 × 10⁶ A20 cells into the right flank. Subsequently, when the tumor volume reached between 100 and 200 mm³, the tumors were treated by injecting either 100 μL of PBS (vehicle control), 5 × 10⁸ live biopharmaceuticals (pTAT002) that secrete synthetic bacteriophages that do not present therapeutic proteins, or 5 × 10⁸ live biopharmaceuticals (pTAT003) that secrete synthetic bacteriophages that present anti-CD47 nanobodies on the pIII subunit. Tumor volume was measured at specified time points using a digital caliper. Tumor volume was calculated by multiplying the maximum measurement by the square of the quadrature measurement and dividing by 2. Only treatment with synthetic bacteriophages presenting CD47 checkpoint inhibitors induced tumor elimination. [Figure 16-1]Figure 16. Figures 16A–16B are graphs showing that live biopharmaceuticals secreting synthetic bacteriophages that present anti-PD-L1 nanobodies induce an antitumor response. Tumors were engrafted in mice by injecting 5 × 10⁶ A20 cells into the right flank. Subsequently, the tumors were treated by injecting 50 μL of PBS (vehicle control), 5 × 10⁸ live biopharmaceuticals (pTAT002) secreting a control synthetic bacteriophage that does not present the therapeutic protein, or 5 × 10⁸ live biopharmaceuticals (pTAT020) secreting a synthetic bacteriophage that presents anti-PD-L1 nanobodies on the pIII subunit, when the tumor volume was between 80 and 250 mm³. Tumor volume was measured at specified time points using digital calipers. Tumor volume was calculated by multiplying the maximum measured value by the square of the quadrature measured value and dividing by 2 (solid line = mice with disappearance, dotted line = mice with no disappearance). (B) For all mouse groups, the complete disappearance of tumors from the mice 24 days after treatment was evaluated. Mice were sacrificed and dissected to examine for metastasis and evaluate the complete disappearance of the primary tumor. Mice that did not have a primary tumor and showed no detectable metastasis at 24 days were considered to have had cancer cells eliminated. [Figure 16-2] Continuation of Figure 16. [Figure 17]Figures 17A-17C are graphs showing that both live biopharmaceuticals and synthetic therapeutic bacteriophages induce long-lasting adaptive immune responses against cancer cells. Mice with A20 tumors in the right flank were treated on days 0, 3, and 11 by intratumoral injection of either a synthetic bacteriophage presenting anti-CD47 nanobodies (A) or a live biopharmaceutical secreting a synthetic bacteriophage presenting anti-CD47 nanobodies (B). After tumor disappearance from the mice, the mice were kept for 45 days post-treatment, and then reloaded in the left flank with an injection of 5 × 10⁶ A20 cancer cells. As a control, naive mice were also reloaded with an injection of 5 × 10⁶ A20 cancer cells (C). Only mice that showed tumor disappearance after treatment acquired an adaptive immune response that prevented the formation of new tumors. (A~C) Tumor volume was calculated by multiplying the maximum measured value by the square of the quadrature measured value and dividing by 2 (solid line = mice in which disappearance was observed, dotted line = mice in which disappearance was not observed). [Figure 18] This graph shows that synthetic therapeutic bacteriophages can present functional cytosine deaminases to produce the antitumor drug 5-FU. The conversion of 5-FC to 5-FU was measured by spectrophotometer using a quartz cuvette to determine the absorbance at 255 nm and 290 nm. The concentrations of 5-FC and 5-FU were then obtained using the following formulas based on the absorbance spectra of each molecule: [5-FC] = 0.119 × A290 - 0.025 × A255, and [5-FU] = 0.185 × A255 - 0.049 × A290. [Figure 19]This graph shows that 5-FU converted by a cytosine deaminase-presenting synthetic therapeutic bacteriophage has an antiproliferative effect on cancer cells. A20 cancer cells were incubated for 42 hours with either a vehicle (PBS 12% DMSO), 200 μM 5-FC, or a control bacteriophage (pTAT002) or a cytosine deaminase-presenting bacteriophage (pTAT022) after 24 hours of incubation with 200 μM 5-FC. Cancer cell death was then monitored by trypan blue staining. Cancer cell death was observed only with 5-FU produced by the cytosine deaminase-presenting synthetic bacteriophage. [Figure 20] Figures 20A and 20B are graphs showing that the alternative start codon GTG improves the presentation and integrity of the therapeutic protein on the surface of synthetic therapeutic bacteriophages. (A) Production of synthetic bacteriophages presenting anti-PDL1 nanobodies, as measured by anti-pVIII ELISA assay, when cloned using ATG or GTG as the start codon. (B) Integrity of nbPDL1-pIII, as measured by Western blotting against phage preparations derived from expression systems where the start codon is either ATG or GTG. The complete morphology of the fusion protein is indicated by the arrow. [Figure 21]Figures 21A-21C are graphs showing that living biopharmaceuticals can be manipulated to produce bacteriophage particles that present two or more therapeutic proteins. (A) Bacteriophage production was measured by anti-PVIII sandwich ELISA after overnight growth in LB broth at 37°C for living biopharmaceuticals secreting a control bacteriophage that does not present a protein (pTAT004 + pTAT002), a bacteriophage that presents an anti-PD-L1 nanobody on pIII (pTAT032), a gpIX-deficient mutant that presents an anti-PD-L1 nanobody on pIII (pTAT032ΔgpIX), or a dual-presenter that presents an anti-PD-L1 nanobody on pIII and an anti-CTLA-4 anticharin on pIX (pTAT032ΔgpIX + pTAT035). (B) HRP signals from ELISA quantifying the binding of various phage preparations to PDL1 on the surface of A20 cells. PEG precipitation was performed on one of the following: LB (no bacteriophage), bacteriophage from pTAT002 + pTAT004 (control without presentation), bacteriophage from pTAT032 (anti-PD-L1 nanobody on pIII), and bacteriophage from pTAT032ΔgpIX + pTAT035 (anti-PD-L1 nanobody on pIII + anti-CTLA-4 anticharin on pIX). An anti-pVIII-HRP antibody was used to detect bacteriophage particles bound to A20 cells, and the signal was measured. (C) HRP signal of ELISA quantifying the binding of various phage preparations CTLA-4 immobilized in the well. PEG precipitation was performed on one of the following: LB (no bacteriophage), bacteriophage from pTAT002 + pTAT004 (control without presentation), bacteriophage from pTAT032 (anti-PD-L1 nanobody on pIII), and bacteriophage from pTAT032ΔgpIX + pTAT035 (anti-PD-L1 nanobody on pIII + anti-CTLA-4 anticharin on pIX). To detect the presence of anti-PD-L1 nanobody fused with HA on pIII or HA fused with pIII on the tail of the bacteriophage particle, the signal was measured using an anti-HA-HRP antibody. [Figure 22] Figures 22A and 22B are graphs showing that living biopharmaceuticals can secrete synthetic therapeutic bacteriophages that present a mixture of therapeutic proteins on pIII. Living biopharmaceuticals that secrete synthetic bacteriophages presenting nanobodies for PD-L1 (pTAT032), CTLA-4 (pTAT019), or both PD-L1 and CTLA-4 (pTAT032 + pTAT019) on pIII were tested by ELISA assay for binding activity to PD-L1 (A) or CTLA-4 (B). [Modes for carrying out the invention]

[0026] As used herein, the singular forms “a,” “an,” and “the” refer to multiple objects unless the context explicitly indicates otherwise.

[0027] The enumeration of numerical ranges by endpoint in this specification is intended to include all numbers contained within that range (for example, the enumeration from 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0028] The term “about” is used herein, whether expressly or otherwise, to mean that all quantities given herein refer to actual given values, and also to approximations to such given values, including equivalents and approximations to such given values ​​by experimental and / or measurement conditions, and which would be reasonably inferred on the basis of the common sense of those skilled in the art. For example, in the context of a given value or range, the term “about” refers to a value or range within 20%, preferably 15%, more preferably 10%, more preferably 9%, more preferably 8%, more preferably 7%, more preferably 6%, and more preferably 5% of the given value or range.

[0029] When used herein, the expression “and / or” should be interpreted as specifically disclosing each of the two designated features or components, either together with or without the other. For example, “A and / or B” should be interpreted as specifically disclosing (i) A, (ii) B, and (iii) A and B, as if each were described separately herein.

[0030] The expression “degree or percentage of sequence homology” in this specification refers to the degree or percentage of sequence identity between two sequences after optimal alignment. The percentage of sequence identity (or degree of identity) is determined by comparing the two aligned sequences across a comparison window, where the portion of the peptide or polynucleotide sequence in the comparison window may include additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in both sequences where the same amino acid residue or nucleic acid base is present, obtaining the number of matching positions, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0031] As used herein, the term “isolated” means that nucleic acids or polypeptides, including but not limited to viruses, proteins, glycoproteins, peptide derivatives or fragments, or polynucleotides, have been isolated from their natural environment. For example, as used herein, the expression “isolated nucleic acid molecule” means a nucleic acid that is substantially free from cell material or culture medium when prepared by recombinant DNA techniques, or from chemical precursors or other chemicals when chemically synthesized. Isolated nucleic acids also substantially free from sequences that are naturally adjacent to the nucleic acid from which they originate (i.e., sequences located at the 5' and 3' ends of the nucleic acid).

[0032] Two nucleotide sequences or amino acids are said to be "identical" if the sequences of nucleotide residues or amino acids in the two sequences are identical when aligned to obtain the greatest possible correspondence as described below. Sequence comparison between two (or more) peptides or polynucleotides is typically performed by comparing the sequences of two optimally aligned sequences across a segment or "comparison window" to identify and compare local regions with sequence similarity. Optimal alignment of sequences for comparison can be achieved by the local homology algorithm of Smith and Waterman, Ad. App. Math 2:482 (1981), the homology sorting algorithm of Needleman and Wunsch, J. Mol. Biol. 48:443 (1970), the similarity search method of Pearson and Lipman, Proc. Natl. Acad. Sci. (USA) 85:2444 (1988), by computerized implementation of these algorithms (Wisconsin Genetics Software Package, Genetics Computer Group (GCG), GAP, BESTFIT, FASTA, and TFASTA at 575 Science Dr., Madison, Wis.), or by visual inspection. Other alignment programs, such as "Multiple sequence alignment with hierarchical clustering," F. CORPET, 1988, Nucl. Acids Res., 16(22), pp. 10881-10890, can also be used.

[0033] In some embodiments, the technology relates to isolated nucleic acid molecules having sequence identity of at least about 75%, or at least about 80%, or at least about 85%, at least about 86%, or at least about 87%, or at least about 88%, or at least about 89%, or at least about 90%, or at least about 91%, or at least about 92%, or at least about 93%, or at least about 94%, or at least about 95%, or at least about 96%, or at least about 97%, or at least about 98%, or at least about 99% with respect to nucleic acid sequences described herein.

[0034] Unless otherwise defined, scientific and technical terms used in connection with the present invention shall have meanings generally understood by those skilled in the art, and furthermore, unless otherwise required by context, singular terms shall include plural forms and plural terms shall include singular forms. In general, the nomenclature and techniques used in connection with cell and tissue culture, molecular biology, and protein and oligo or polypeptide chemistry, and hybridization described herein are well known and commonly used in the art. Standard techniques are used for recombinant DNA, oligonucleotide synthesis, and tissue culture and transformation (e.g., electroporation, lipofectin). Enzymatic reactions and purification techniques are carried out according to the manufacturer's specifications or as commonly achieved in the art as described herein. The aforementioned techniques and procedures are generally carried out according to conventional methods well known in the art and as described in the various general and more specific references cited and discussed throughout this specification (see, for example, Sambrook et al., Molecular Cloning. A Laboratory Manual).

[0035] As used herein, the term “antibody” refers to immunoglobulin molecules and molecules containing the immunologically active portion of immunoglobulin molecules, i.e., antigen-binding sites that specifically bind to (“immunely react to) an antigen. Structurally, the simplest naturally occurring antibodies (e.g., IgG) consist of four polypeptide chains: two heavy (H) chains and two light (L) chains interconnected by disulfide bonds. Immunoglobulins represent a large family of molecules, including several types of molecules, such as IgD, IgG, IgA, IgM, and IgE.

[0036] As used herein, the term “bispecific antibody” refers to an artificial protein composed of fragments of two different monoclonal antibodies, which as a result bind to two different types of antigens.

[0037] The term "immunoglobulin molecule" includes, for example, hybrid antibodies or modified antibodies, and fragments thereof.

[0038] As used herein, “antigen” refers to a substance that is specifically recognized and bound by an antibody. Antigens may include, for example, peptides, proteins, glycoproteins, polysaccharides, and lipids; their equivalents and combinations. As used herein, the term “surface antigen” refers to a component of the cell’s plasma membrane and includes endogenous and superficial membrane proteins, glycoproteins, polysaccharides, and lipids that make up the cell membrane. “Endogenous membrane proteins” are transmembrane proteins that extend across the lipid bilayer of the cell membrane. Typical endogenous membrane proteins generally contain at least one “transmembrane segment” containing hydrophobic amino acid residues. Superficial membrane proteins do not extend into the hydrophobic interior of the lipid bilayer and bind to the membrane surface through non-covalent interactions with other membrane proteins.

[0039] An "antibody fragment" comprises a portion of an intact antibody and preferably has an antigen-binding region or a variable region of the intact antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, and Fv fragments; diabodies; linear antibodies (see Zapata et al., Protein Eng. 8(10): pp. 1057-1062 (1995)); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0040] Single-chain variable fragments (scFv) are typically fusion proteins of the variable regions of the heavy chain (VH) and light chain (VL) of immunoglobulins, linked by a short linker peptide of 10 to approximately 25 amino acids. The linker is usually rich in glycine for flexibility and serine or threonine for solubility. The linker can link the N-terminus of the VH to the C-terminus of the VL, or vice versa.

[0041] As used herein, “bacteriophage” refers to a virus that infects bacteria. Similarly, “archaeal phage” refers to a virus that infects archaea. The term “phage” is used herein to refer to both types of viruses, but in certain cases, as indicated by the context, it may also be used as an abbreviation to specifically refer to a bacteriophage or archaeal phage. Bacteriophages and archaeal phages are obligate intracellular parasites that infect and replicate within bacteria / archaea by utilizing some or all of the host biosynthetic mechanisms (with respect to both the process of identifying the host to infect and the process of being able to productively replicate their genome only in suitable host cells). Different bacteriophages and archaeal phages may contain different materials, but they all contain nucleic acids and proteins and can be encapsulated in lipid membranes in certain circumstances.

[0042] Depending on the phage, the nucleic acid can be either DNA or RNA (but typically not both), can exist in various forms, and the size of the nucleic acid depends on the phage. The simplest phages have genomes of only a few thousand nucleotides, while more complex phages may have more than 100,000 nucleotides in their genome, and rarely more than 1,000,000. In addition, phages may be covered by a lipid membrane and may contain different materials. The number of different types of proteins in the phage particle and the amount of each type of protein will vary depending on the phage. These proteins protect the nucleic acid from nucleases in the environment and are functional in infection.

[0043] Numerous filamentous and non-filamentous phage genomes have been sequenced, including, for example, the filamentous phages M13, f1, fd, Ifl, Ike, Xf, Pf1, and Pf3. Among the filamentous phage classes, M13 is the most well-characterized species due to its known three-dimensional structure and the well-understood function of its coat proteins. Specifically, the M13 genome encodes five coat proteins pIII, VIII, VI, VII, and IX, which are used as sites for inserting foreign DNA into the M13 vector.

[0044] As used herein, “phage genome” includes naturally occurring phage genomes and their derivatives. Generally (but not necessarily), derivatives have the ability to grow in the same host as the parent. In some embodiments, the only difference between a naturally occurring phage genome and a derivative phage genome is the addition or deletion of at least one nucleotide from at least one end of the phage genome (if the genome is linear) or along at least one location in the genome (if the genome is circular).

[0045] As used herein, “host cell,” etc., is a cell capable of forming a phage from a particular type of phage genomic DNA. In some embodiments, the phage genomic DNA is introduced into a cell by phage infection. The phage binds to a receptor molecule outside the host cell and injects its genomic DNA into the host cell. In some embodiments, the phage genomic DNA is introduced into a cell using transformation or any other suitable method. In some embodiments, the phage genomic DNA is substantially pure when introduced into a cell. The phage genomic DNA can be present in a vector when introduced into a cell. As a non-limiting example, the phage genomic DNA is present in a yeast artificial chromosome (YAC) which is introduced into a phage host cell by transformation or an equivalent method. Subsequently, the phage genomic DNA is copied and packaged as phage particles after lysis of the phage host cell.

[0046] As used herein, “surface sequence” refers to the nucleotide sequence encoding the “surface protein” of a gene package. These proteins form a proteinaceous coat that encapsulates the genome of the gene package. Typically, the surface protein instructs the gene package to assemble the polypeptide that is to be presented on the surface of the gene package, for example, a phage or bacterium.

[0047] An "inducible promoter" refers to a regulatory region operably linked to one or more genes, wherein gene expression is increased in the presence of an inducer of the regulatory region or in the absence of a repressor of the regulatory region. An inducible promoter can be induced by exogenous environmental conditions, which refers to the setting or circumstances under which the promoter described herein is induced. Exogenous environmental conditions refer to external environmental conditions for an intact (undissolved) manipulated microorganism, endogenous or native conditions for a tumor environment, or a host target environment, or an exogenously introduced disturbance for an environment. An inducible promoter may include one or more regulatory elements, including, but not limited to, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter regulatory elements, protein binding sequences, 5' and 3' untranslated regions, transcription start sites, termination sequences, polyadenylation sequences, riboswitches, and introns.

[0048] The present technology will be described in further detail below. This description is not intended to be a detailed catalog of all different ways in which the technology may be implemented, or all features that may be added to the technology. For example, features illustrated in one embodiment may be incorporated into other embodiments, and features illustrated in a particular embodiment may be omitted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be obvious to those skilled in the art in light of this disclosure, but these variations and additions do not depart from the technology. Therefore, the following description is intended to illustrate some specific embodiments of the technology and not to exhaustively identify all substitutions, combinations, and variations thereof.

[0049] A solution for treating cancer by acting simultaneously on several therapeutic targets is to use molecular scaffolds that can link several therapeutic molecules. Fibrous bacteriophages are large immunogenic biological structures on which therapeutic proteins or peptides can be presented. Therefore, the combination of the immunogenic activity of fibrous bacteriophages and therapeutic proteins or peptides can improve the effectiveness of cancer treatment. Furthermore, fibrous bacteriophages can be secreted by bacteria and provide an efficient method for locally delivering drugs to tumor sites.

[0050] According to various embodiments, the technology relates to a operable synthetic therapeutic bacteriophage-live biopharmaceutical capable of delivering synthetic therapeutic bacteriophages.

[0051] According to some embodiments, the technology relates to an operable live biopharmaceutical capable of delivering synthetic therapeutic bacteriophages for the treatment of cancer. In some implementations, the synthetic therapeutic bacteriophages are delivered by live biopharmaceutical bacteria. In some cases, the synthetic bacteriophages are immunogenic and present monospecific or multispecific therapeutic proteins.

[0052] In some embodiments, the disclosure provides live biotherapeutic agents for the delivery of synthetic therapeutic bacteriophages.

[0053] In some embodiments, the technology relates to a bacterial host manipulated by a synthetic bacteriophage secretion system comprising a synthetic bacteriophage mechanism and a synthetic bacteriophage scaffold vector (Figure 1). The synthetic bacteriophage mechanism is responsible for the replication and assembly of synthetic therapeutic bacteriophages. The synthetic bacteriophage scaffold vector serves as a template for generating nucleic acid scaffolds for the assembly of synthetic therapeutic bacteriophages.

[0054] In some embodiments, the bacterial host engineered to deliver the synthetic therapeutic bacteriophage may originate from any of the following genera: Enterobacteriaceae (Citrobacter sp., Enterobacillus sp., Enterobacter sp., Escherichia sp., Klebsiella sp., Salmonella sp., Shigella sp.), Pseudomonadaceae (Pseudomonas sp.), and Vibriaceae (Vibrio sp.). In some other embodiments, the bacteria engineered to deliver the therapeutic bacteriophage are attenuated forms derived from any of the Enterobacteriaceae (Citrobacter, Enterobacillus, Enterobacter, Escherichia, Klebsiella, Salmonella, Sigella), Pseudomonadaceae (Pseudomonas), and Vibrioaceae (Vibrio). In another embodiment, the bacterial host is a pathogenic tumor-targeting bacterium, such as Salmonella typhimurium, Salmonella choleraesuis, or Vibrio cholera. In yet another embodiment, the bacterial host is a non-pathogenic bladder colony-forming bacterium, such as Escherichia coli strain 83972 or Escherichia coli strain HU2117. In yet another embodiment, the bacterial host is a non-pathogenic tumor-targeting bacterium, such as E. coli Nissle 1917 strain or E. coli MG1655 strain, but is not limited to these.

[0055] In yet another embodiment, the bacterial host is a tumor-targeting bacterium, such as Escherichia coli, but is not limited thereto.

[0056] Bacterial hosts engineered to secrete therapeutic bacteriophages can be biologically contained to prevent their dissemination in the environment. Biological containment can be achieved by disrupting essential genes to make the bacterial host trophic. In one non-limiting example, a trophic-dependent strain bacterial host can be engineered by disrupting the gene dapA or thyA, which makes the bacterial host dependent on an exogenous source of diaminopimelic acid (DAP) or thymine, respectively. In one embodiment, the bacterial cell is biologically contained using a single biological containment strategy that disrupts only one essential gene (e.g., DAP trophicity or thymine trophicity only). In yet another embodiment, the bacterial cell is biologically contained by disrupting two or more essential genes (e.g., DAP trophicity and thymine trophicity). The essential genes that can be disrupted to create a trophication-dependent E. coli bacterial host include yhbV, yagG, hemB, secD, secF, ribD, ribE, ML, dxs, ispA, dnaX, adk, hemH, IpxH, cysS, fold, rplT, infC, thrS, nadE, gapA, yeaZ, aspS, argS, pgsA, yefM, metG, folE, yejM, gyrA, nrdA, nrdB, folC, accD, fabB, gltX, gA, zipA, dapE, dapA, der, hisS, ispG, suhB, tadA, acpS, era, rnc, ftsB, eno, pyrG, chpR, Igt, ba, pgk, and yq. gD, metK, yqgF, plsC, ygiT, pare, ribB, cca, ygjD, tdcF, yraL, yihA, ftsN, murl, murB, birA, secE, nusG, rplJ, rplL, rpoB, rpoC, ubiA, plsB, lexA, dnaB, ssb, alsK, groS, psd, orn, y jeE, rpsR, chpS, ppa, valS, yjgP, yjgQ, dnaC, ribF, IspA, ispH, dapB, folA, imp, yabQ, ftsL, ftsl, murE, murF, mraY, murD, ftsW, murG, murC, ftsQ, ftsA, ftsZ, IpxC, secM, secA, can,folK, hemL, yadR, dapD, map, rpsB, infB, nusA, ftsH, obgE, rpmA, rplU, ispB, murA, yrbB, yrbK, yhbN, rpsl, rplM, degS, mreD, mreC, mreB, accB, accC, yrdC, def, fmt, rplQ, rpoA, rpsD, rpsK, rpsM, entD, mrdB, mrdA, nadD, hlepB, rpoE, pssA, yfiO, rplS, trmD, rpsP, ffh, grpE, yfjB, csrA, ispF, ispD, rplW, rplD, rplC, rpsJ, fusA, rpsG, rpsL, trpS, yrfF, asd, rpoH, ftsX, ftsE, ftsY, frr, dxr, ispU, rfaK, kdtA, coaD, rpmB, dfp, dut, gmk, spot, gyrB, dnaN, dnaA, rpmH, rnpA, yidC, tnaB, glmS, glmU, wzyE, hemD, hemC, yigP, ubiB, ubiD, hemG, secY, rplO, rpmD, rpsE, rplR, rplF, rpsH, rpsN, rplE, rplX, rplN, rpsQ, rpmC, rplP, rpsC, rplV, rpsS, rplB, cdsA, yaeL, yaeT, IpxD, fabZ, IpxA, IpxB, dnaE, accA, tilS, proS, yafF, tsf, pyrH, olA, rlpB, leuS, Int, glnS, fldA, cydA, infA, cydC, ftsK, lolA, serS, rpsA, msbA, IpxK, kdsB, mukF, mukE, mukB, asnS, fabA, mviN, rne, yceQ, fabD, fabG, acpP, tmk, holB, lolC, lolD, lolE, purB, ymfK, minE, mind, pth, rsA, ispE, lolB, hemA, prfA, prmC, kdsA, topA, ribA, fabl, racR, dicA, ydfB, tyrS, ribC, ydiL, pheT, pheS, yhhQ, bcsB, glyQ, yibJ, gpsA, and their functional homologs, including but not limited to these.,

[0057] Bacterial hosts can be genetically engineered to be protease-deficient as a means of increasing the production and secretion of therapeutic bacteriophages. In some embodiments, the bacterial host is deficient in one or more proteases. In some other embodiments, the bacterial host is deficient in the ompT gene, which encodes protease 7 in E. coli. In another embodiment, the bacterial host is deficient in the lon gene, which encodes the Lon protease in E. coli. In yet another embodiment, the bacterial host is deficient in the ompT gene and the sulA gene, which encodes the cell division inhibitor sulA, allowing cells to divide more normally in the absence of proteases. In yet another embodiment, the bacterial host is deficient in the lon gene and the sulA gene. In yet another embodiment, the bacterial host is deficient in the lon gene, the ompT gene, and the sulA gene.

[0058] Bacterial hosts can be genetically engineered to be attenuated and evade the human immune system. Immune cells recognize and eliminate LPS presented on the outer membrane of bacteria. Strategies for manipulating the structure of LPS have been developed to evade the immune system and extend the half-life of bacteria injected into the bloodstream, and LPS modifications that enable bacteria to evade the immune system are well documented (Motohiro Matsuura, Front Immunol., 2013, Vol. 4:p. 019; Steimle et al., Int. J. Med. Microbiol., 2016, Vol. 306:p. 290; Simpson et al., Nat. Rev. Microbiol., 2019, Vol. 17:p. 403; these are incorporated herein by reference), and the inventors cite them in their entirety. Thus, it is possible to reduce the immunogenicity of modified bacteria by cleaving LPS or manipulating their biosynthetic pathways. Therefore, in some embodiments, bacterial hosts have LPS that has been modified or cleaved to evade the immune system.

[0059] In some embodiments, the synthetic bacteriophage mechanism includes a bacteriophage assembly module, a bacteriophage replication module, a bacteriophage coating module, and a therapeutic module.

[0060] In some embodiments, the bacteriophage assembly module is responsible for assembling bacteriophage-coated proteins onto a bacteriophage ssDNA scaffold. The bacteriophage assembly module may include, but is not limited to, the bacteriophage gene gpI encoding proteins pI and pXI, and the bacteriophage gene gpIV encoding protein pIV. In one embodiment, some or all of the genes encoding pI, pXI, and pIV may be derived from one or more closely related filamentous bacteriophages belonging to the Inoviridae family, such as, but is not limited to, bacteriophages M13, Fd, F1, If1, Ike, Pf1, Pf3, fs-2, and B5. In another embodiment, the genes encoding pI, pXI, and pIV may be derived from the filamentous bacteriophage M13.

[0061] In some embodiments, the bacteriophage replication module is responsible for replicating the bacteriophage ssDNA scaffold. This module encodes a protein that recognizes the scaffold replication module located on the synthetic bacteriophage scaffold vector and induces rolling circle replication to produce a cyclized ssDNA scaffold molecule. The bacteriophage replication module may include, but is not limited to, the bacteriophage gene gpII encoding proteins pII and pX, and the bacteriophage gene gpV encoding protein pV. In one embodiment, some or all of the genes encoding pII, pX, and pV may originate from one or more closely related bacteriophages belonging to the Inoviridae family, such as, for example, bacteriophages M13, Fd, F1, If1, Ike, Pf1, Pf3, fs-2, and B5. In another embodiment, the genes encoding pII, pX, and pV may originate from the filamentous bacteriophage M13.

[0062] In some embodiments, the bacteriophage coating module includes a coating protein that is assembled onto a bacteriophage ssDNA scaffold to form a bacteriophage. The bacteriophage coating module may include, but is not limited to, the bacteriophage genes gpIII, gpVI, gpVII, gpVIII, and gpIX, or parts thereof, encoding proteins pIII, pVI, pVII, pVIII, and pIX, respectively. In some embodiments, one or more coating genes present in the bacteriophage coating module may also be present in a therapeutic module, which is fused with one or more therapeutic proteins. In some other embodiments, if one or more coating genes are present in a therapeutic module and fused with one or more therapeutic proteins, the corresponding coating genes are not present in the bacteriophage coating module. In one embodiment, some or all of the genes encoding pIII, pVI, pVII, pVIII, and pIX may be derived from one or more closely related bacteriophages belonging to the family Inoviridae, such as, but not limited to, bacteriophages M13, Fd, F1, If1, Ike, Pf1, Pf3, fs-2, and B5. In another embodiment, the genes encoding pIII, pVI, pVII, pVIII, and pIX may be derived from the filamentous bacteriophage M13.

[0063] In some embodiments, the therapeutic module includes a therapeutic protein to be presented by the therapeutic bacteriophage. The therapeutic module includes, but is not limited to, one or more bacteriophage-coated protein genes fused with one or more therapeutic proteins. The bacteriophage therapeutic module may include, but is not limited to, one or more bacteriophage-coated genes gpIII, gpVI, gpVII, gpVIII, and gpIX, which encode proteins pIII, pVI, pVII, pVIII, and pIX, respectively, fused with one or more therapeutic proteins. In one embodiment, some or all of the genes encoding pIII, pVI, pVII, pVIII, and pIX may originate from one or more closely related bacteriophages belonging to the Inoviridae family, such as, but are not limited to, bacteriophages M13, Fd, F1, If1, Ike, Pf1, Pf3, fs-2, and B5. In another embodiment, the genes encoding pIII, pVI, pVII, pVIII, and pIX may be derived from the filamentous bacteriophage M13. The therapeutic proteins presented by the bacteriophage can be fused to any of the phage-coating proteins, such as pIII, pVI, pVII, pVIII, and pIX.

[0064] In some embodiments, the therapeutic protein is fused with a mutant pVIII-coated protein that improves the presentation of large proteins on the surface of synthetic bacteriophages. A mutant pVIII protein that improves the presentation of large proteins on the surface of fibrous bacteriophages has been identified (S. Sidhu et al., J. Mol. Biol. (2000) 296, pp. 487-495; this is incorporated by reference). In some embodiments, the therapeutic protein is presented on a pVIII-coated protein identified using an approach similar to that of S. Sidhu et al. In some embodiments, the therapeutic protein is presented on a pVIII-coated protein corresponding to the pVIII(1a) mutant described by S. Sidhu et al., Mol. Biol. (2000) 296, pp. 487-495. In another embodiment, the therapeutic protein is presented on a pVIII coating corresponding to the pVIII(2e) variant described by S. Sidhu et al., Mol. Biol. (2000), pp. 296, 487-495. In yet another embodiment, the therapeutic protein is presented on a pVIII coating corresponding to the pVIII(2f) variant described by S. Sidhu et al., Mol. Biol. (2000), pp. 296, 487-495.

[0065] In some cases, overexpression of therapeutic proteins can have harmful effects on the bacterial host, potentially resulting in reduced secretion of synthetic bacteriophages. Alternative start codons rely on fluctuations in the start tRNA that initiate transcription on an incorrect set of nucleotides. This can cause ribosome arrest, improving ribosome transport on the gene and potentially producing a more complete protein product. Several codons can be used as alternative start codons (Hecht et al., Nucleic Acids Research, 2017, vol. 45, no. 7, pp. 3615–3626; this is incorporated herein by reference). In some embodiments, the start codon of the therapeutic protein is one of 64 codons. In some embodiments, translation of the therapeutic protein gene is initiated with the standard start codon ATG. In some other embodiments, the start codon of the therapeutic protein is TTG. In yet another embodiment, the start codon of the therapeutic protein is GTG.

[0066] In some embodiments, the therapeutic protein is fused to the N-terminus or C-terminus of the coating protein. In some other embodiments, the therapeutic protein is fused to the coating protein by insertion into any portion of the protein. In some other embodiments, the coating protein is fused to the therapeutic protein using one or more protein tags, for example, but not limited to, a human influenza hemagglutinin tag (HA-tag), a polyhistidine tag (His-tag), a FLAG tag, or a myc tag. In yet another embodiment, the therapeutic protein is fused to the coating protein using an amino acid linker sequence. The linker may be flexible, rigid, or cleavable, as described by Chen et al. (Adv Drug Deliv Rev. 15 October 2013; 65(10): pp. 1357-1369; this is incorporated herein by reference). In yet another embodiment, the linker may also include a tag sequence, for example, but not limited to, human influenza hemagglutinin (HA-tag), polyhistidine (His-tag), FLAG-tag, or myc-tag. One or more therapeutic proteins are fused with one or more coating proteins pIII, pVI, pVII, pVIII, and pIX in a manner that is not detrimental to the activity of the therapeutic protein and to the assembly of the bacteriophage. In some embodiments, one or more therapeutic proteins are fused with a full-length coating protein. In another embodiment, one or more therapeutic proteins may be fused with a full-length coating protein via one or more linker sequences. In yet another embodiment, one or more therapeutic proteins may be fused with a cleaved coating protein containing a domain essential for the assembly of the bacteriophage. In yet another embodiment, one or more therapeutic proteins may be fused with a cleaved coating protein containing a domain essential for the assembly of the bacteriophage via one or more linker sequences.In some embodiments, when one or more therapeutic proteins are fused to the N-terminus of a coating protein, the fusion protein further includes a leader peptide sequence at its N-terminus to ensure the transfer of the protein to the bacterial outer membrane for phage assembly. In some embodiments, the leader peptide sequence includes, but is not limited to, one or more leader peptides derived from DsbA, PelB, TorA, and PhoA signal peptides. In yet another embodiment, the leader peptide is an optimized DsbA and PelB signal peptide with improved transfer activity, as described by Han et al. (Han et al., AMB Expr (2017) 7:93; this is incorporated herein by reference). In yet another embodiment, the leader peptide is a BKC-1 derived signal peptide as described by Bharathwaj et al. (Bharathwaj et al., mBio. June 29, 2021; 12(3); this is incorporated herein by reference). In some embodiments, the leader peptide is derived from PelB. When a multidrug therapeutic bacteriophage is secreted, two or more therapeutic proteins are fused with one or more coating proteins (Figure 2). A multidrug therapeutic bacteriophage may also include a bacteriophage that presents one or more therapeutic proteins fused to each other. In some embodiments, the therapeutic proteins are fused using one or more protein tags, for example, but not limited to, human influenza hemagglutinin tags (HA-tags), polyhistidine tags (His-tags), FLAG tags, and myc tags. In other embodiments, the therapeutic proteins are fused using an amino acid linker sequence. The linker may be flexible, rigid, or cleavable, as described by Chen et al., 2013 (Adv Drug Deliv Rev. Oct. 15, 2013; 65(10): 1357-1369. Fusion Protein Linkers: Property, Design and Functionality; this is incorporated herein by reference).In yet another embodiment, the linker may also include tag sequences, for example, but not limited to, a human influenza hemagglutinin tag (HA-tag), a polyhistidine tag (His-tag), a FLAG-tag, and a myc-tag. One or more therapeutic proteins fused to one or more bacteriophage-coated proteins may be any single-specificity or multispecificity binding proteins, including, but are not limited to, antigen-binding fragments (Fab and F(ab')2), single-chain variable fragments (scFv), double-chain variable fragments (di-scFv), bispecific T cell engagers (BiTE), TCRs, soluble TCRs, single-chain T cell receptor variable regions (scTv), single-domain antibodies (nanobodies), lipocalins (anticalin), monobodies (adonectin), afibodies, affinins, affimers, afitins, alphabodies, armadillo repeat protein-based scafolds, aptamers, atrimers, avimers, DARPin, finomers, Nottin, Knitz domain peptides, and adhesins. Binding proteins also include, but are not limited to, the extracellular domains of receptors and their ligands, such as PD-1, PD-L1, CTLA-4, B7-1, B7-2, CD112, CD155, TIGIT, CD96, CD226, CD112R, CD96, CD111, CD272, B7H4, CD28, CD80, CD86, OX40, OX40-L, ICOS, ICOS-LG, CD137, CD137-L, AITR, AITR-L, CD27, CD70, TNF-α, TNFR1, TNFR2, LAG-3, TIM-3, and galectin-9.

[0067] In another embodiment, the one or more therapeutic proteins fused with one or more bacteriophage-coated proteins are peptides.

[0068] In yet another embodiment, the one or more therapeutic proteins fused with the one or more bacteriophage-coated proteins are enzymes.

[0069] In some other embodiments, the one or more therapeutic proteins fused with one or more bacteriophage-coated proteins are a combination of a binding protein and a peptide, or a combination of a binding protein and an enzyme, or a combination of an enzyme and a peptide, or a combination of a binding protein, an enzyme, and a peptide.

[0070] In some embodiments, the synthetic bacteriophage mechanism may include a regulatory module comprising optional modules, such as regulatory elements that control the activity of the synthetic bacteriophage mechanism and the synthetic bacteriophage scafold vector. As a non-limiting example, the regulatory module may turn some or all of the genes of the bacteriophage mechanism and / or the synthetic bacteriophage scafold vector on or off. Turning off the bacteriophage mechanism and / or the synthetic bacteriophage scafold vector during the large-scale production phase of living biotherapeutic drugs may be advantageous in avoiding selective pressures and evolutionary variability. The regulatory module, when present in the bacteriophage mechanism, may include one or more genes and regulatory elements encoding one or more proteins or non-coding RNAs (e.g., transcription factors, activators, repressors, riboswitches, CRISPR-Cas9, zinc finger nucleases (ZFNs), TALEs, and taRNAs) that can regulate or be used to regulate gene expression in the synthetic bacteriophage mechanism and / or synthetic bacteriophage scaffold vector.

[0071] In some embodiments, the synthetic bacteriophage mechanism may include a maintenance module that includes a replication mechanism capable of recognizing the origin (oriV) of an optional module, such as a vector containing a vegetative replication module, such as a synthetic bacteriophage scaffold vector or a vector containing a module of the synthetic bacteriophage mechanism. The maintenance module is required when the oriV of the vegetative module is incompatible with the replication mechanism of the bacterial host. The maintenance module enables the replication of any plasmid containing a vegetative replication module compatible with its replication mechanism. The maintenance module may be heterologous to the bacterial host. If vector maintenance needs to be limited to the donor bacterium, it is preferable to place the maintenance module within the chromosome of the donor bacterium (e.g., incorporate it). Alternatively, the maintenance module may be placed on one or more vectors. The maintenance module may also include one or more genes and regulatory elements involved in proper DNA distribution.

[0072] In some embodiments, some or all promoters controlling gene expression in the phage mechanism are inducible promoters. In other embodiments, some or all promoters controlling gene expression in the phage mechanism are inducible promoters induced by one or more exogenous molecules, such as, but not limited to, L-arabinose, rhamnose, IPTG, and tetracycline. In yet another embodiment, some or all promoters controlling gene expression in the phage mechanism are inducible promoters induced by one or more exogenous environmental conditions in the tumor microenvironment, such as, but not limited to, hypoxic levels (hypoxia), acidic pH (<7), and oxidative conditions (high levels of H2O2). In yet another embodiment, some or all promoters controlling gene expression in the bacteriophage mechanism are induced by one or more exogenous molecules and / or by one or more exogenous environmental conditions present in the tumor microenvironment. In another embodiment, some or all of the promoters that control the gene expression of the bacteriophage mechanism are induced by one or more molecules produced by the host bacterium, such as diaminopimelic acid and N-acylhomoserine lactone.

[0073] In some embodiments, the synthetic bacteriophage mechanism is integrated into the genome of a bacterial cell. In another embodiment, some or all of the modules of the synthetic bacteriophage mechanism are located on a synthetic bacteriophage scaffold vector. In yet another embodiment, some or all of the modules of the synthetic bacteriophage mechanism are located on one or more vectors, while some or none of the modules of the synthetic bacteriophage mechanism remain in the genome of the bacterial cell and the synthetic bacteriophage scaffold vector. When the modules of the synthetic bacteriophage mechanism are located on a vector other than the synthetic bacteriophage scaffold vector, two further modules, namely a vegetative replication module and a selection module, are present in each vector, and one further module, namely a maintenance module, is present in either one or more vectors or the genome of the bacterial host.

[0074] The vegetative replication module enables vectors containing modules of the synthetic bacteriophage mechanism to replicate within bacterial host cells. The vegetative replication module includes a replication origin oriV compatible with the bacterial host and / or an oriV compatible with the maintenance module, and includes bacteriophage M13 and / or the following families of bacterial vectors: IncA, IncB / O (Inc10), IncC, IncD, IncE, IncF1, IncF2, IncG, IncHI1, IncHI2, IncI1, IncI2, IncJ, IncK, IncL / M, IncN, IncP It may be derived from one of IncQ1, IncQ2, IncR, IncS, IncT, IncU, IncV, IncW, IncX1, IncX2, IncY, IncZ, ColE1, ColE2, ColE3, p15A, pSC101, IncP-2, IncP-5, IncP-7, IncP-8, IncP-9, Inc1, Inc4, Inc7, Inc8, Inc9, Inc11, Inc13, Inc14, and / or Inc18. In one embodiment, the vegetative replication module oriV may be derived from one of the ColE1, pSC101, F, p15A, and M13 families of bacterial vectors. For example, the vegetative replication module may be derived from the bacterial vector ColE1.

[0075] The selection module allows the vector to be stably maintained within a bacterial host. The selection module includes one or more genes that confer selectable traits for the identification of bacteria possessing one or more modules of the bacteriophage mechanism. The selection module is operably linked to one or more bacterial vectors of the synthetic bacteriophage secretion system. Selectable traits may include, but are not limited to, antibiotic resistance genes, genes encoding fluorescent proteins (including green fluorescent protein), nutrient requirement selection markers, genes encoding β-galactosidase (e.g., bacterial lacZ gene), genes encoding luciferase, genes encoding chloramphenicol acetyltransferase (e.g., bacterial cat gene), genes encoding enzymes that enable the use of nutrients that the bacterial chassis cannot process (e.g., thiA when endogenous thiA is removed from the bacterial chromosome), genes encoding β-glucuronidase, and regulatory elements involved in proper DNA distribution. In some embodiments, some or all of the promoters controlling the expression of the genes in the selection module are inductive promoters. In some embodiments, the minimally synthesized bacteriophage scaffold vector includes a vegetative replication module, a selection module, a scaffold replication module, and a packaging module.

[0076] In some embodiments, the vegetative replication module enables the synthetic bacteriophage scaffold vector to replicate in bacterial host cells. The vegetative replication module includes a replication origin oriV compatible with the bacterial host, or an oriV compatible with the maintenance module, and the following families of bacteriophage M13 and / or bacterial vectors: IncA, IncB / O (Inc10), IncC, IncD, IncE, IncF1, IncF2, IncG, IncHI1, IncHI2, IncI1, IncI2, IncJ, IncK, IncL / M, IncN, IncP, It may originate from one of IncQ1, IncQ2, IncR, IncS, IncT, IncU, IncV, IncW, IncX1, IncX2, IncY, IncZ, ColE1, ColE2, ColE3, p15A, pSC101, IncP-2, IncP-5, IncP-7, IncP-8, IncP-9, Inc1, Inc4, Inc7, Inc8, Inc9, Inc11, Inc13, Inc14 and / or Inc18. In one embodiment, the vegetative replication module oriV may originate from one of the ColE1, pSC101, F, p15A, and M13 families of bacterial vectors. For example, the vegetative replication module may originate from the bacterial vector ColE1.

[0077] In some embodiments, the scaffold replication module enables the production of ssDNA synthetic bacteriophage scaffolds. The scaffold replication module includes a replication origin (oriV) recognized by a bacteriophage replication protein, which enables rolling circle replication of the synthetic bacteriophage scaffold vector and the production of cyclized ssDNA synthetic bacteriophage scaffold molecules. The DNA sequence of bacteriophage oriV may be derived from one of closely related bacteriophages belonging to the Inoviridae family, for example, but not limited to bacteriophages M13, Fd, F1, If1, Ike, Pf1, Pf3, fs-2, and B5. In some embodiments, some or all of the promoters controlling gene expression in the scaffold replication module are inducible promoters.

[0078] In some embodiments, the bacteriophage packaging module allows the cyclized ssDNA synthetic bacteriophage scfold to be processed for assembly with the M13 bacteriophage coating protein. The packaging module includes a DNA sequence that acts as a packaging signal to initiate the assembly of the bacteriophage. The DNA sequence of the packaging signal may be derived from one of the closely related bacteriophages belonging to the Inoviridae family, for example, but not limited to, bacteriophages M13, Fd, F1, If1, Ike, Pf1, Pf3, fs-2, and B5. In some embodiments, some or all of the promoters controlling the gene expression of the bacteriophage packaging module are inducible promoters. In some embodiments, a selection module is present to allow the vector to be stably maintained within a bacterial host. The selection module includes one or more genes that confer selectable traits for identifying bacteria harboring the synthetic bacteriophage scfold vector. The selection module is operably linked to a synthetic bacteriophage scaffold vector. Selectable traits may include, but are not limited to, antibiotic resistance genes, genes encoding fluorescent proteins (including green fluorescent protein), nutrient requirement selection markers, genes encoding β-galactosidase (e.g., bacterial lacZ gene), genes encoding luciferase, genes encoding chloramphenicol acetyltransferase (e.g., bacterial cat gene), genes encoding β-glucuronidase, and genes encoding enzymes that enable the use of nutrients that the bacterial chassis cannot process (e.g., thiA when endogenous thiA is removed from the bacterial chromosome). In some embodiments, some or all of the promoters controlling the expression of the genes in the selection module are inductive promoters.

[0079] In some embodiments, a filler module is present to allow for variations in the length of the synthetic bacteriophage. The filler module contains a random sequence of DNA intended solely to alter the size of the synthetic bacteriophage scaffold vector and does not necessarily contain genes or regulatory elements. By altering the size of the scaffold vector, the filler module allows for variations in the length of the synthetic bacteriophage particle. Having shorter synthetic bacteriophages can improve the number of secreted particles because less pVIII coating protein is required per bacteriophage. On the other hand, increasing the size of the synthetic bacteriophage can increase the distance between the therapeutic proteins fused with pIII and pIX, thus improving the interaction of these therapeutic proteins with their respective targets. Therefore, in some embodiments, the filler module consists of DNA sequences with sizes ranging from 0 bp to 100,000 bp.

[0080] In some embodiments, some or all of the promoters controlling gene expression of the synthetic bacteriophage cafold vector are inducible promoters. In other embodiments, some or all of the promoters controlling gene expression of the synthetic bacteriophage cafold vector are inducible promoters induced by one or more exogenous molecules, such as, but not limited to, L-arabinose, rhamnose, IPTG, and tetracycline. In yet another embodiment, some or all of the promoters controlling gene expression of the synthetic bacteriophage cafold vector are inducible promoters induced by one or more exogenous environmental conditions of the tumor microenvironment, such as, but not limited to, hypoxic levels (hypoxia), acidic pH (<7), and oxidative conditions (high levels of H2O2). In yet another embodiment, some or all of the promoters controlling gene expression of the synthetic bacteriophage cafold vector are induced by one or more exogenous molecules and / or by one or more exogenous environmental conditions present in the tumor microenvironment and / or by one or more molecules secreted by host bacteria.

[0081] In some embodiments, synthetic therapeutic bacteriophages stimulate pattern recognition receptors (PRRs). PRRs play a crucial role in the innate immune response through activation of pro-inflammatory signaling pathways, stimulation of phagocytic responses (macrophages, neutrophils, and dendritic cells), or binding to microorganisms as secreted proteins. PRRs recognize two classes of molecules: pathogen-associated molecular patterns (PAMPs) related to microbial pathogens and viruses, and damage-associated molecular patterns (DAMPs) related to cellular components released during cell injury, death, stress, or tissue damage. PAMPs are essential molecular structures required for pathogen survival and include, for example, bacterial cell wall molecules (e.g., lipoproteins), bacterial DNA, or viral DNA. Some PRRs can be expressed by cells of the innate immune system, while others can be expressed by other cells (both immune and non-immune cells). PRRs localize on the cell surface to detect extracellular pathogens or within endosomes and the cell matrix to detect intracellular invading viruses. Examples of PRRs include, but are not limited to, Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), C-type lectin receptors (group I mannose receptors and group II asiaroglycoproteins), nucleotide oligomerization (NOD)-like receptors (NODI and NOD2), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (RIG-I, MDA5, and DDX3), collectins, pentraxin, phycolin, lipid transferases, peptidoglycan-recognizing proteins (PGRs), and leucine-rich repeat receptors (LRRs). Upon detecting pathogens, PRRs activate inflammatory and immune responses that arise against infectious pathogens. Recent evidence has shown that immune mechanisms activated by PAMP and DAMP also play a role in activating the immune response against tumor cells (Hobohm & Grange, Crit Rev Immunol. 2008;28(2):95-107, and Krysko DV et al., Cell Death and Disease (2013) 4, e631; these are incorporated herein by reference).Intratumoral injection has been shown to stimulate an immune response in certain microorganisms, such as the microorganisms of this disclosure (e.g., bacteria and bacteriophages). In some cases, these have been shown to provide therapeutic benefits in several types of cancer, including solid tumors, melanoma, basal cell carcinoma, and squamous cell carcinoma. The antitumor responses observed in these cases are thought to be partly due to the pro-inflammatory properties of the nucleic acid fraction, capsid protein, and / or cell wall fraction of the microorganisms that activate PRRs. The synthetic therapeutic bacteriophages of this disclosure can spontaneously induce an immune response in the tumor microenvironment via the presence of PAMP and DAMP, which are agonists against immune cells and PRRs found on tumor cells (Carroll-Portillo A. et al., Microorganisms. December 2019; 7(12): 625; this is incorporated herein by reference) (Figure 4). Thus, in some embodiments, the synthetic therapeutic bacteriophages of this disclosure induce an immune response at the tumor site. In these embodiments, the synthetic therapeutic bacteriophage naturally expresses a PRR agonist, such as one or more PAMPs. Examples of PAMPs are shown in Takeuchi et al. (Cell, 2010, 140:805-820; this is incorporated herein by reference). In some embodiments, the PRR is DNA derived from the synthetic bacteriophage that is recognized by immune or non-immune cells via TLR-9 and / or RIG-I.

[0082] In one embodiment, a therapeutic bacteriophage presents one or more binding proteins that inhibit immune checkpoints (Figure 3). Some anticancer drugs target and inhibit immune checkpoints to activate the immune system and initiate an immune response against autoantigens presented by cancer cells. However, when administered systemically, the altered immunomodulatory effects can lead to immunodysfunction and autoimmune disease. Immune dysfunction side effects, such as the development of undesirable autoimmune responses, can be addressed by locally delivering immune checkpoint inhibitors or inhibitors of other immunosuppressive molecules to the tumor site. The immune checkpoint molecules to be inhibited may be any known or subsequently discovered immune checkpoint molecules or other immunosuppressive molecules. In some embodiments, the immune checkpoint molecules or other immunosuppressive molecules to be inhibited are selected from, but are not limited to, CCR4, CTLA-4, CD80, CD86, PD-1, PD-L1, PD-L2, TIGIT, VISTA, LAG-3, TIM1, TIM3, CEACAM1, LAIR-1, HVEM, BTLA, CD47, SIRPα, CD160, CD200, CD200R, CD39, CD73, B7-H3, B7-H4, IDO, TDO, KIR, and A2aR. If one or more binding proteins are derived from single-chain antibodies, their sequences may be one or more of those listed in Tables 3 and 4 of PCT / US2017 / 013072, but are not limited to these; these are incorporated herein by reference.

[0083] In one embodiment, one or more binding proteins presented by a therapeutic bacteriophage bind to and inhibit one or more proteins, peptides, or molecules involved in carcinogenesis, cancer development, or metastasis. The one or more proteins, peptides, or molecules that are inhibited may be any known or subsequently discovered proteins, peptides, or molecules involved in carcinogenesis, cancer development, or metastasis. In some embodiments, one or more proteins, peptides, or molecules to be inactivated are selected from, but are not limited to, CSF1, CSF1R, CCR4, CCL2, CCL17, CCL22, HER2, GD2, IL-1β, IL-6, IL-10, IL-13, IL-17, IL-27, IL-35, CD20, CD27, CD30, CD33, CD70, TGF-β, M-CSF, EGFR, ERBB2, ERBB3, PGE2, VEGF, VEGFR-2, CXCR4 / CXCL12, Tie2, galectin-1, galectin-3, phosphatidylserine, and TAM and Tim phosphatidylserine receptors.

[0084] In one embodiment, one or more binding proteins presented by a therapeutic bacteriophage act as agonists that activate costimulatory receptors leading to the elimination of cancer cells. One or more costimulatory cell receptors activated by one or more antibody mimetics may be any known or subsequently discovered costimulatory cell receptors that lead to the elimination of cancer cells. In some embodiments, one or more cell receptors activated by one or more antibody mimetics are selected from, but are not limited to, CD40, CD28, ICOS, CD226, CD137, and CD134.

[0085] In one embodiment, one or more binding proteins presented by a therapeutic bacteriophage are antibody Fc domains that induce antibody-dependent cell-mediated cytotoxicity (ADCC). "ADCC" refers to a cell-mediated response in which nonspecific cytotoxic cells expressing Fc receptors (FcRs), such as NK cells, recognize a bound antibody on a target cell, subsequently causing lysis of the target cell. NK cells are important mediators of ADCC, inducing direct cytotoxicity through perforin and granzyme, FasL, and TRAIL interactions, as well as cytokine production. The NK cell activation required to induce ADCC can occur via Fc receptors to IgG (FcyRs) (FcyRI, FcyRIIA, and FcyRIIIA in humans; FcyRI, FcyRIII, and FcyRIV in mice) that recognize the Fc domain of IgG antibodies. Therefore, synthetic therapeutic bacteriophages that present one or more manipulated Fc domains that bind to the activating FcyR of NK can be used to recruit and activate NK to the tumor site, thereby mediating ADCC and eliminating tumor cells. Thus, in some embodiments, the synthetic therapeutic bacteriophage presents one or more Fc domains that bind to the activating FcyR of NK. One or more Fc domains may be any known or subsequently discovered Fc domains that activate NK and induce ADCC. A list of antibodies having ADCC-inducing Fc domains can be found in Table 36 by PCT / US2017 / 013072 (which is incorporated herein by reference).

[0086] In one embodiment, one or more binding proteins presented by a therapeutic bacteriophage bind to other binding proteins, for example, but not limited to, IgG antibodies, nanobodies, aphibodies, antikalin, antibody fragments, ScFV, biotin, and streptavidin.

[0087] In one embodiment, the synthetic therapeutic bacteriophage presents a combination of one or more binding proteins that inhibit immune checkpoints, and / or one or more binding proteins that inhibit proteins, peptides, or molecules involved in carcinogenesis, cancer development, or metastasis, and / or one or more binding proteins that act as agonists that activate cell receptors to prevent carcinogenesis, cancer development, or metastasis, and / or one or more Fc domains that induce ADCC and tumor cell elimination. In one embodiment, binding proteins that may be presented by a synthetic therapeutic bacteriophage include: antigen-binding fragments (Fab and F(ab')2), single-chain variable fragments (scFv), double-chain variable fragments (di-scFv), bispecific T cell engagers (BiTE), TCRs, soluble TCRs, single-chain T cell receptor variable regions (scTv), single-domain antibodies (nanobodies), lipocalins (anticalin), monobodies (adonectin), afibodies, affilins, affimers, afitins, alphabodies, armadillo repeat protein-based scaffolds, aptamers, atrimers, avimers, DARPin, finomers, notchin, Knitz domain peptides, and adhesins.

[0088] In one embodiment, a living biopharmaceutical secretes a synthetic therapeutic bacteriophage that presents one or more tumor antigen peptides. Numerous tumor antigens are known to date, including, for example, tumor-specific antigens, tumor-associated antigens (TAAs), and neoantigens, many of which are associated with specific tumors and cancer cells. These tumor antigens are typically small peptide antigens associated with specific cancer cell types that are known to stimulate an immune response. By introducing such tumor antigens, e.g., tumor-specific antigens, TAAs, and / or neoantigens, into the local tumor environment, an immune response can be induced against specific cancer cells or tumor cells of interest known to be associated with that neoantigen. In some embodiments, the one or more tumor antigen peptides presented by the synthetic therapeutic phage may be any known or subsequently discovered tumor antigens associated with cancer cells. The one or more tumor antigen peptides may be selected from, but are not limited to, Tables 26, 27, 28, 29, 30, 31, and 32 of PCT / US2017 / 013072 (which is incorporated herein by reference).

[0089] In one embodiment, one or more peptides presented by a synthetic therapeutic bacteriophage may be peptide sequences of receptor ligands or fragments of receptor ligands that activate cell receptors leading to the elimination of cancer cells. One or more peptide ligands may be any known or later discovered peptide ligands that activate cell receptors leading to the elimination of cancer cells. In some embodiments, one or more peptide ligand sequences may be derived from, but are not limited to, CD40L, CD80, CD86, ICOS ligand, CD112, CD155, CD137 ligand, and CD134 ligand.

[0090] In one embodiment, one or more peptides presented by a synthetic therapeutic bacteriophage may be soluble peptides that eliminate tumor cells.

[0091] In one embodiment, the therapeutic bacteriophage presents one or more enzymes that activate a prodrug. Examples of enzymes that activate prodrugs for cancer treatment include, but are not limited to, cytosine deaminase, purine nucleoside phosphorylase, deoxycytidine kinase, thymidylate kinase, and uridine monophosphate kinase.

[0092] In one embodiment, the therapeutic bacteriophage presents one or more enzymes that deplete metabolites essential for the growth of tumors and cancer cells. Examples of metabolites important for the growth of tumors and cancer cells include, but are not limited to, L-asparagine, L-glutamine, L-methionine, and kynurenine. The one or more enzymes that deplete metabolites essential for the growth of tumors and cancer cells presented by the synthetic therapeutic bacteriophage may be any known or subsequently discovered enzymes that deplete metabolites essential for the growth of tumors and cancer cells. Examples of enzymes that deplete metabolites essential for the growth of tumors and cancer cells include, but are not limited to, L-asparaginase, L-glutaminase, methioninase, and kynureninase.

[0093] In one embodiment, the synthetic therapeutic bacteriophage presents a combination of one or more enzymes that activate a prodrug and one or more enzymes that deplete metabolites essential for the proliferation of tumors and cancer cells.

[0094] As a means of enhancing the antitumor effects of synthetic bacteriophages, bacterial hosts secreting therapeutic bacteriophages can exert further therapeutic activity to stimulate immune responses, either naturally or after genetic engineering. Many immune cells found in the tumor microenvironment express pattern recognition receptors (PRRs), which play a crucial role in the immune response through activation of pro-inflammatory signaling pathways, stimulation of phagocytic responses (macrophages, neutrophils, and dendritic cells), or binding to microorganisms as secreted proteins. PRRs recognize two classes of molecules: pathogen-associated molecular patterns (PAMPs) related to microbial pathogens, and damage-associated molecular patterns (DAMPs) related to cellular components released during cell injury, death, stress, or tissue damage. PAMPs are essential molecular structures required for pathogen survival, such as bacterial cell wall molecules (e.g., lipoproteins) and bacterial DNA. PRRs are expressed by cells of the innate immune system, but can also be expressed by other cells (both immune and non-immune cells). PRRs (Protein Receptor Receptors) localize on the cell surface to detect extracellular pathogens, or they localize within endosomes and the cell matrix to detect invading viruses. Examples of PRRs include Toll-like receptors (TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10), C-type lectin receptors (group I mannose receptors and group II asiaroglycoproteins), nucleotide oligomerization (NOD)-like receptors (NODI and NOD2), retinoic acid-inducible gene I (RIG-I)-like receptors (RLRs) (RIG-I, MDA5, and DDX3), collectins, pentraxin, phycolin, lipid transferases, peptidoglycan-recognizing proteins (PGRs), and leucine-rich repeat receptors (LRRs). Upon detecting a pathogen, PRRs activate inflammatory and immune responses to the infectious pathogen.Recent evidence has shown that immune mechanisms activated by PAMP and DAMP also play a role in activating the immune response against tumor cells (Hobohm & Grange, Crit Rev Immunol. 2008;28(2):95-107, and Krysko DV et al., Cell Death and Disease (2013) 4, e631; these are incorporated herein by reference). The bacterial hosts of this disclosure can induce an immune response through the presence of PAMP and DAMP, which are agonists against PRRs found on immune cells and tumor cells in the tumor microenvironment. Thus, in some embodiments, the bacterial hosts of this disclosure induce an immune response at the tumor site (Figure 4). In these embodiments, the microorganisms spontaneously express PRR agonists, e.g., one or more PAMPs or DAMPs.

[0095] A bacterial host that secretes synthetic therapeutic bacteriophages can be manipulated to secrete or produce one or more immunostimulatory enzymes for the purpose of preventing the proliferation of tumor cells.

[0096] In one embodiment, the bacterial host secretes or produces one or more enzymes that deplete metabolites essential for the growth of tumors and cancer cells.

[0097] In one embodiment, a bacterial host secretes or produces 15-hydroxyprostaglandin dehydrogenase (15-PGDH), which converts prostaglandin E2 (PGE2) to 15-keto-PG. Prostaglandin E2 (PGE2) is overproduced in many tumors and contributes to cancer progression. PGE2 is a multifaceted molecule involved in numerous biological processes, including angiogenesis, apoptosis, inflammation, and immunosuppression. Local delivery of 15-PGDH to tumors has been shown to result in a significant slowing of tumor growth.

[0098] A bacterial host that secretes synthetic therapeutic bacteriophages can be manipulated to secrete one or more immunostimulatory proteins for the purpose of preventing the proliferation of tumor cells.

[0099] In one embodiment, a bacterial host secretes granulocyte-macrophage colony-stimulating factor (GM-CSF). GM-CSF is part of the immune / inflammatory cascade. Activation of GM-CSF in a small number of macrophages leads to a rapid increase in their numbers. GM-CSF has been shown to be usable as an immunostimulatory adjuvant to induce antitumor immunity.

[0100] In one embodiment, a bacterial host secretes one or more cytokines that stimulate and / or induce the differentiation of effector T cells, such as CD4+ and / or CD8+. Cytokines that stimulate and / or induce the differentiation of effector T cells include, but are not limited to, IL-2, IL-15, IL-12, IL-7, IL-21, IL-18, TNF, and interferon-gamma (IFN-gamma). One or more cytokines that stimulate and / or induce the differentiation of effector T cells secreted by a bacterial host may be any known or subsequently discovered cytokines that stimulate and / or induce the differentiation of effector T cells.

[0101] In one embodiment, the bacterial host secretes tryptophan. Catabolism of tryptophan is a central pathway for maintaining an immunosuppressive microenvironment in many types of cancer.

[0102] In one embodiment, the bacterial host secretes L-arginine. In humans, the absence of arginine in the tumor microenvironment acts as an immunosuppressive factor that inhibits the progression of the T lymphocyte cell cycle by inducing G0-G1 arrest, thereby preventing the elimination of cancer cells. Therefore, in some embodiments, the bacterial host can be engineered to contain one or more gene sequences encoding one or more enzymes of the arginine pathway. Genes involved in the arginine pathway include, but are not limited to, argA, argB, argC, argD, argE, argF, argG, argH, argl, arg.J, carA, and carB. These genes may be organized into one or more operons, either naturally or synthetically. All of the genes encoding these enzymes are repressed by arginine through the interaction of arginine with ArgR, which forms a complex that binds to the regulatory region of each gene and inhibits transcription. In some embodiments, the genetically engineered bacteria of this technology include one or more nucleic acid mutations that reduce or eliminate arginine-mediated repression of one or more operons encoding enzymes involved in converting glutamate to arginine and / or intermediate byproducts in the arginine biosynthesis pathway.

[0103] In some embodiments, a bacterial host is engineered to transfer adenosine to reduce the level of adenosine in the tumor microenvironment. Adenosine is a potent immunosuppressive molecule found in the tumor microenvironment, and therefore, reducing its levels increases the immune response against tumor cells. The adenosine transfer mechanism may be derived from any known or subsequently discovered E. coli nucleoside permease. In one embodiment, the engineered bacterial host transfers adenosine via the E. coli nucleoside permease nupG or nupC.

[0104] In some embodiments, the technology relates to the use of the live biotherapeutic and / or synthetic therapeutic bacteriophages described herein for the treatment of cancer and / or tumors, which may be malignant or benign. The types of cancer that can be treated using this technology include, but are not limited to, adrenal cancer, adrenocortical carcinoma, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone malignancies (e.g., Ewing's sarcoma, osteosarcoma, malignant fibrous histiocytoma), brain malignancies (e.g., astrocytoma, brainstem glioma, craniopharyngioma, ependymoma), bronchial tumors, central nervous system tumors, breast cancer, Castleman disease, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, ocular malignancies, gallbladder cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, cardiac malignancies, Kaposi's sarcoma, kidney cancer, pharyngeal cancer, hypopharyngeal cancer, lymphoma (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia), and liver cancer. This includes cancer, lung cancer, lymphoma (e.g., AIDS-associated lymphoma, Burkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma), malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma, skin cancer (e.g., basal cell carcinoma, melanoma), small intestine cancer, gastric cancer, teratoma, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, rare childhood cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor. In some embodiments, the symptoms associated therewith include, but are not limited to, anemia, loss of appetite, irritation of the bladder wall, bleeding and contusions (thrombocytopenia), changes in taste or smell, constipation, diarrhea, dry mouth, dysphagia, edema, fatigue, hair loss (alopecia), infections, infertility, lymphedema, stomatitis, nausea, pain, peripheral neuropathy, tooth decay, urinary tract infections, and / or problems with memory and concentration.

[0105] In some embodiments, the methods of the present technology involve administering an effective amount of at least one live biotherapeutic and / or at least one synthetic therapeutic bacteriophage described herein to a subject requiring it. Live biotherapeutic and / or synthetic therapeutic bacteriophages can be administered locally, for example, intratumorally or peritumorally, intratissue or into supplying blood vessels, intramuscularly, intraperitoneally, or by intravenous infusion into the bladder. Live biotherapeutic and / or synthetic therapeutic bacteriophages can be administered systemically, for example, intravenously or intraarterially, by infusion or injection.

[0106] In some embodiments, the Technology relates to a pharmaceutical composition comprising, for example, at least one biotherapeutic agent and / or at least one synthetic therapeutic bacteriophage described herein, and optionally one or more suitable pharmaceutically acceptable excipients, diluents, or carriers. In certain embodiments, administering at least one live biotherapeutic agent and / or at least one synthetic therapeutic bacteriophage described herein, or the composition of the Technology, to a subject requiring it reduces cell proliferation, tumor growth, and / or tumor volume in the subject. In some embodiments, the methods of the Disclosure can reduce cell proliferation, tumor growth, and / or tumor volume by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or more compared to levels in an untreated or control subject. In some embodiments, the reduction is measured by comparing cell proliferation, tumor growth, and / or tumor volume in the subject before and after administration of the pharmaceutical composition. In some embodiments, a method for treating or improving cancer in a subject enables improvement of one or more symptoms of cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more. Cancerous cells and / or biomarkers in a subject can be measured in biological samples, such as blood, serum, plasma, urine, peritoneal fluid, and / or biopsy samples from tissues or organs, before, during, and after administration of the pharmaceutical composition. In some embodiments, the method may include administering a composition of the Technology to reduce the tumor volume in a subject to an undetectable size or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the tumor volume of the subject before treatment. In other embodiments, the method may include administering the composition of the technology to reduce the rate of cell proliferation or tumor proliferation of the target to an undetectable rate, or to less than approximately 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate before treatment.

[0107] In some embodiments, the compositions of this technology may be administered alone or in combination with one or more further therapeutic agents. Non-limiting examples of therapeutic agents include conventional therapies (e.g., radiotherapy, chemotherapy), immunotherapies (e.g., vaccines, dendritic cell vaccines, or other vaccines of other antigen-presenting cells, checkpoint inhibitors, cytokine therapy, tumor-infiltrating lymphocyte therapy, naive or engineered TCR or CAR-T therapy, natural killer cell therapy, Fc-mediated ADCC therapy, bispecific soluble scFv that link cytotoxic T cells to tumor cells, and therapies using soluble TCRs with effector function), stem cell therapy, and targeted therapies using antibodies or compounds (e.g., BRAF or vascular endothelial growth factor inhibitors) and synthetic bacteriophages. In some embodiments, genetically modified bacteria are administered sequentially, simultaneously, or subsequently to a regimen of one or more chemotherapeutic agents selected from, but not limited to, methotrexate, trabectedin®, belotecan®, cisplatin®, carboplatin®, bevacizumab®, pazopanib®, 5-fluorouracil, capecitabine®, irinotecan®, gemcitabine (Gemzar), and oxaliplatin®.

[0108] In some embodiments, at least one live biotherapy agent is administered sequentially, simultaneously, or subsequently to a drug regimen using one or more of the following checkpoint inhibitors or other antibodies known in the art or described herein: non-limiting examples include CTLA-4 antibodies (including, but not limited to, ipilimumab and tremelimumab (CP675206)), anti-4-1BB (CD137, TNFRSF9) antibodies (including, but not limited to, PF-05082566 and urelumab), and anti-OX40 antibodies (Providence Health and Anti-CD134 (OX40) antibodies, including but not limited to those listed above (Services), anti-PD1 antibodies (including but not limited to nivolumab, pidilizumab, pembrolizumab (MK-3475 / SCH900475), lambrolizumab, REGN2810, PD1 (Agenus)), anti-PD1 antibodies (durvalumab (MEDI4736), avelumab (MSB0010718C), and atezolizumab (MPDL)), This includes, but is not limited to, 3280A, RG7446, and R05541267), anti-KIR antibodies (including, but not limited to, lirilumab), LAG3 antibodies (including, but not limited to, BMS-986016), anti-CCR4 antibodies (including, but not limited to, mogamulizumab), anti-CD27 antibodies (including, but not limited to, varilumab), and anti-CXCR4 antibodies (including, but not limited to, urocuplumab).

[0109] In some embodiments, at least one live biopharmaceutical and / or one synthetic therapeutic bacteriophage includes, but is not limited to, an anti-phosphatidylserine antibody (including, but not limited to, bavituximab), a TLR9 antibody (including, but not limited to, MGN1703), a PD1 antibody (including, but not limited to, SHR-1210 (Incyte / Jiangsu Hengrui)), an anti-OX40 antibody (including, but not limited to, OX40 (Agenus)), an anti-Tim3 antibody (including, but not limited to, anti-Tim3 (Agenus / INcyte)), an anti-Lag3 antibody (including, but not limited to, anti-Lag3 (Agenus / INcyte)), an anti-B7H3 antibody (including, but not limited to, enobrituzumab (MGA-271)), and WO20091. Anti-CT-011 (hBAT, hBATl) as described in 01611 (incorporated herein by reference), anti-PDL-2 antibody (including but not limited to AMP-224 (described in WO2010027827 and WO2011066342; this is incorporated herein by reference), anti-CD40 antibody (including but not limited to CP-870, 893), anti-CD40 antibody (including but not limited to CP-870, 893) The antibodies are administered sequentially, simultaneously, or subsequently to the administration of one or more antibodies selected from (not limited to) the range of antibodies described herein. Pharmaceutical compositions comprising the live biotherapeutic agents and / or synthetic therapeutic bacteriophages of the Technology can be used to treat, manage, improve, and / or prevent cancer. Pharmaceutical compositions of the Technology are provided that contain one or more live biotherapeutic agents alone or in combination with prophylactic agents, therapeutic agents, and / or pharmaceutically acceptable carriers. In certain embodiments, the pharmaceutical composition comprises one live biotherapeutic agent engineered to contain one or more genes encoding one or more anti-cancer molecules, for example, genetically modified agents described herein. In alternative embodiments, the pharmaceutical composition comprises two or more live biotherapeutic agents engineered to contain one or more genes encoding one or more anti-cancer molecules, for example, genetically modified agents described herein.In yet another embodiment, the pharmaceutical composition comprises recombinant proteins described herein, for example, synthetic therapeutic bacteriophages, each of which is engineered to present one or more anti-cancer molecules.

[0110] The pharmaceutical compositions of this technology can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants, which facilitate the processing of the active ingredient into a composition for pharmaceutical use. Methods for formulating pharmaceutical compositions are known in the art (see, for example, "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tableting, lyophilization, direct compression, conventional mixing, dissolution, granulation, centrifugation, emulsification, encapsulation, capture, or spray drying to form tablets, granules, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may or may not be enterically coated. The appropriate formulation depends on the route of administration.

[0111] Live biotherapeutic agents and / or synthetic therapeutic bacteriophages can be formulated into pharmaceutical compositions for any suitable dosage form (e.g., liquid, capsule, sachet, hard capsule, soft capsule, tablet, enteric-coated tablet, suspension powder, granules, or matrix sustained-release formulation for oral administration) and any suitable type of administration (e.g., oral, topical, injectable, intravenous, subcutaneous, intratumoral, peritumoral, immediate-release, pulsatile-release, delayed-release, or sustained-release). A suitable dosage for live biotherapeutic agents is approximately 10 bacteria 4 ~10 12 The number of doses may be in the range of one. The composition may be administered once or more times daily, weekly, monthly, or annually. The composition may be administered before, during, or after meals. In one embodiment, the pharmaceutical composition is administered before the subject ingests a meal. In one embodiment, the pharmaceutical composition is administered simultaneously with a meal. In one embodiment, the pharmaceutical composition is administered after the subject ingests a meal.

[0112] Live biotherapeutic agents and / or synthetic therapeutic bacteriophages can be formulated as pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, surfactants, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, pharmaceutical compositions may include, but are not limited to, calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and various types of starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and surfactants, including, for example, polysorbate 20. In some embodiments, the live biotherapeutic agents of the present invention can be formulated in a sodium bicarbonate solution, for example, a 1 molar sodium bicarbonate solution (for buffering an acidic cellular environment, e.g., the stomach). Live biotherapeutic agents can be administered and formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed from anions, such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed from cations, such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0113] Live biopharmaceuticals and / or synthetic therapeutic bacteriophages can be administered intravenously, for example, by infusion or injection. Alternatively, live biopharmaceuticals and / or synthetic therapeutic bacteriophages can be administered intratumorally and / or peritumorally. In other embodiments, live biopharmaceuticals and / or synthetic therapeutic bacteriophages can be administered intra-arterially, intramuscularly, or intraperitoneally. In some embodiments, the live biopharmaceuticals colonize approximately 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the tumor. In some embodiments, the live biopharmaceuticals and / or synthetic therapeutic bacteriophages are administered co-administered with PEGylated rHuPH20 (PEGPH20) or other agents to disrupt tumor septa for the purpose of enhancing the permeability of the tumor capsule, collagen, and / or stroma. In some embodiments, the live biopharmaceuticals can produce anticancer molecules, as well as one or more enzymes that degrade fibrous tissue.

[0114] In some embodiments, the treatment regimen includes one or more intratumoral doses. In some embodiments, the treatment regimen includes an initial dose and at least one subsequent dose. The one or more doses may be administered sequentially over two or more cycles. For example, the initial dose may be administered on day 1, and the second dose may be administered 1, 2, 3, 4, 5, or 6 days later, or 1, 2, 3, or 4 weeks later, or at longer intervals. Additional doses may be administered 1, 2, 3, 4, 5, or 6 days later, or 1, 2, 3, or 4 weeks later, or at longer intervals. In some embodiments, the initial dose and subsequent doses have the same dosage. In other embodiments, different doses are administered. In some embodiments, more than one dose per day is administered, for example, two, three, or more doses per day may be administered.

[0115] The live biotherapeutic and / or synthetic therapeutic bacteriophages disclosed herein can be administered topically and may be formulated in the form of ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, emulsions, or other forms well known to those skilled in the art. See, for example, "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. In one embodiment, for topical administration forms that cannot be sprayed, a viscous, semi-solid, or solid form is used, comprising one or more excipients that are compatible with the carrier or topical application and have a dynamic viscosity greater than water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, etc., which may be sterilized or mixed with adjuvants (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties (e.g., osmotic pressure). Other suitable topical drug formulations include sprayable aerosol preparations, in which the active ingredient is packaged in a mixture with a pressurized volatile substance (e.g., a gaseous propellant, e.g., Freon) in combination with a solid or liquid inert carrier, or in a squeeze bottle. Humectants or wetting agents may also be added to the pharmaceutical composition and drug formulation. Examples of such further components are well known in the art. In one embodiment, a pharmaceutical composition containing the live biotherapy agent of the art can be formulated as a sanitary product. For example, the sanitary product may be an antimicrobial preparation or a fermented product, e.g., a fermented broth. The sanitary product may be, for example, a shampoo, conditioner, cream, paste, lotion, and lip balm.

[0116] The live biotherapeutic and / or synthetic therapeutic bacteriophages disclosed herein are administered orally and can be formulated as tablets, pills, sugars, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be prepared using solid excipients, and optionally, the resulting mixture may be pulverized, and more preferably suitable adjuvants added, after which the granular mixture may be processed to obtain tablets or sugar cores. Suitable excipients include, but are not limited to, fillers, sugars including lactose, sucrose, mannitol, or sorbitol; cellulose compositions, such as corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth gum, methylcellulose, hydroxypropyl methylcellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers, such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrants, such as cross-linked polyvinylpyrrolidone, agar, alginic acid, or its salts, such as sodium alginate, may also be added.

[0117] Tablets or capsules can be prepared by conventional means using pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, rubber, kaolin, and tragacanth rubber); fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate); lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica); disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugar, cellulose derivatives, silica powder); or wetting agents (e.g., sodium lauryl sulfate). Tablets can be coated by methods well known in the art. A coating shell may be present, and common films include polylactide, polyglycolic acid, polyacid anhydride, other biodegradable polymers, polylysine alginate-alginic acid (APA), alginate-polymethylene-co-guanidine-alginic acid (A-PMCG-A), hydroxymethyl acrylate-methacrylate (HEMA-MMA), multilayer HEMA-MMAMAA, polyacrylonitrile vinyl chloride (PAN-PVC), acrylonitrile / sodium methallyl sulfonate (AN-69), polyethylene glycol / polypentamethylcyclopentasiloxane. This includes, but is not limited to, sodium dimethylsiloxane (PEG / PD5 / PDMS), poly-N,N-dimethylacrylamide (PDMAAm), silica encapsulating agent, cellulose sulfate / sodium alginate / polymethylene-co-guanidine (CS / A / PMCG), cellulose phthalate acetate, calcium alginate, k-carrageenan-carob bean gum gel beads, guerlain-xanthan gum beads, poly(lactide-co-glycolide), carrageenan, starch polyacrylate, starch polymethacrylate, polyamino acids, and enteric-coated coating polymers.

[0118] In some embodiments, live biopharmaceuticals and / or synthetic therapeutic bacteriophages are enterically coated for release into the intestinal tract or a specific region of the intestinal tract, e.g., the large intestine. Typical pH profiles from the stomach to the colon are approximately 1–4 (stomach), 5.5–6 (duodenum), 7.3–8.0 (ileum), and 5.5–6.5 (colon). In some diseases, the pH profile may be modified. In some embodiments, the coating is degraded in a specific pH environment for the purpose of specifying the site of release. In some embodiments, at least two coatings are used. In some embodiments, the outer and inner coatings are degraded at different pH levels.

[0119] In some embodiments, enteric coating materials can be used in one or more coating layers (e.g., outer, inner, and / or intermediate coating layers). Enteric-coated polymers remain deionized at low pH and therefore insoluble. However, as the pH increases in the digestive tract, the acidic functional groups can ionize, causing the polymer to swell or become soluble in intestinal fluid.

[0120] The uses and methods defined herein involve administering a therapeutically effective dose of a live biotherapy or synthetic bacteriophage as defined herein to a subject in order to achieve the effects discussed herein. Where used herein, the terms “effective dose” or “therapeutic dose” refer to the amount of a live biotherapy or synthetic bacteriophage as defined herein that is effective in producing several desired therapeutic effects as defined herein, in terms of a reasonable benefit / risk ratio applicable to any medical treatment. The therapeutically effective dose of any particular peptide in this disclosure is thought to vary from subject to subject and from patient to patient, and depends, among other things, on the effect or outcome to be achieved, the patient’s condition and the route of delivery. The terms “therapeutably acceptable,” “therapeutably suitable,” “pharmaceutically acceptable,” and “pharmaceutically suitable” are used interchangeably herein and refer to a peptide, compound, or composition that, in light of the severity of the disease and the need for treatment, is suitable to be administered to a subject in order to achieve the effects described herein, e.g., the treatment as defined herein, without undue adverse side effects.

[0121] In another embodiment, the pharmaceutical composition containing the live biotherapy of the Technology may be an edible product, such as a food product. In one embodiment, the food product is milk, concentrated milk, fermented milk (yogurt, sour milk, frozen yogurt, lactic acid fermented beverage), powdered milk, ice cream, cream cheese, dried cheese, soy milk, fermented soy milk, vegetable-fruit juice, fruit juice, sports drink, confectionery, candy, infant food (e.g., infant cake), nutritional food product, animal feed, or nutritional supplement. In one embodiment, the food product is a fermented food, such as a fermented dairy product. In one embodiment, the fermented dairy product is yogurt. In another embodiment, the fermented dairy product is cheese, milk, cream, ice cream, milkshake, or kefir. In another embodiment, the live biotherapy of the Technology is combined in a preparation containing other live bacterial cells intended to act as probiotics. In another embodiment, the food product is a beverage. In one embodiment, the beverage is a fruit juice-based beverage or a beverage containing plant or herbal extracts. In another embodiment, the food product is jelly or pudding. Other food products suitable for administering the live biopharmaceuticals of this technology are well known in the art. See, for example, U.S. Patent Application Publication No. 2015 / 0359894 and U.S. Patent Application Publication No. 2015 / 0238545. All of the contents of each of these are expressly incorporated herein by reference. In yet another embodiment, the pharmaceutical composition of this technology is injected, sprayed, or sprinkled onto food, such as bread, yogurt, or cheese.

[0122] Pharmaceutical compositions can be packaged in sealed containers indicating the amount of the drug, such as ampoules or sachets. In one embodiment, one or more pharmaceutical compositions are supplied as dry, sterile lyophilized powder or water-free concentrates in sealed containers and can be reconstituted to an appropriate concentration (e.g., with water or saline) for administration to a subject. In one embodiment, one or more prophylactic or therapeutic agents or pharmaceutical compositions are supplied as dry, sterile lyophilized powders in sealed containers stored between 2°C and 8°C and administered within 1 hour, 3 hours, 5 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, or 1 week after reconstitution. Lyophilized dosage forms may include cryoprotective agents, mainly 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotective agents include trehalose and lactose. Other suitable fillers include glycine and arginine (both can be included at concentrations of 0-0.05%), and polysorbate-80 (optimally at a concentration of 0.005-0.01%). Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ® surfactants. The pharmaceutical composition can be prepared as an injectable solution and may further contain agents useful as adjuvants, such as those used to increase absorption or dispersion, such as hyaluronidase.

[0123] In some embodiments, live biotherapeutic agents and / or synthetic therapeutic bacteriophages and their compositions are formulated for intravenous, intratumoral, or peritumoral administration. Live biotherapeutic agents and / or synthetic therapeutic bacteriophages can be formulated as depot formulations. Such long-acting formulations can be administered by implantation or injection. For example, compositions can be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or ion-exchange resin, or as a sparingly soluble derivative (e.g., as a sparingly soluble salt).

[0124] In another embodiment, the composition can be delivered by a controlled-release or sustained-release system. In one embodiment, controlled or sustained release can be achieved using a pump. In another embodiment, controlled or sustained release of the treatment of the Disclosure can be achieved using polymer materials (see, for example, U.S. Patent No. 5,989,463 incorporated herein by reference). Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyacrylamide, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. Polymers used in sustained-release formulations may be inert, free of leaching impurities, stable at storage, sterilizable, and biodegradable. In some embodiments, the controlled or sustained-release system may be positioned near the target of prevention or treatment, and therefore only a portion of the systemic dose may be required. Any suitable technique known to those skilled in the art may be used.

[0125] The live biotherapeutic and / or synthetic therapeutic bacteriophages of this technology can be administered and formulated in neutral or salt form. Pharmaceutically acceptable salts include those formed from anions, such as hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed from cations, such as sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. [Examples]

[0126] The following embodiments are provided to illustrate the implementation of various embodiments of the Disclosure. They are not intended to limit or define the entire scope of the Disclosure. It should be understood that the Disclosure is not limited to the specific embodiments described and illustrated herein and includes all modifications and variations that fall within the scope of the Disclosure as defined in the appended embodiments.

[0127] (Example 1) Manipulation of living biopharmaceuticals that secrete synthetic bacteriophages for the presentation of therapeutic proteins All bacterial strains and plasmids used in this example are listed in Table 1. Cells were typically grown in Luria broth mirror (LB) or Luria broth agar mirror medium, with antibiotics added as needed at the following concentrations: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were grown at 37°C as is customary. Cells with temperature-sensitive plasmids (pTAT00X, pTAT001) were grown at 30°C. Bacterial cultures older than 18 hours were not used in the experiment.

[0128] [Table 1A]

[0129] [Table 1B]

[0130] DNA manipulation. A detailed list of the oligonucleotide sequences used in this example is shown in Table 2. Plasmids were prepared using the EZ10-Spin Column Plasmid Miniprep Kit (BIOBASIC, #BS614) or the QIAGEN Plasmid Maxi Kit (QIAGEN) according to the manufacturer's instructions. PCR amplification was performed using TransStart FastPFU fly DNA polymerase (Civic Bioscience) for DNA partial amplification and screening. Restriction enzyme digestion was performed using NEB products and incubated at 37°C for 1 hour according to the manufacturer's recommendations. Plasmids were constructed using NEBuilder HiFi DNA Assembly Master Mix (NEB) by Gibson assembly according to the manufacturer's protocol.

[0131] [Table 2A]

[0132] [Table 2B]

[0133] [Table 2C]

[0134] DNA purification. DNA purification was performed between each step of plasmid assembly to avoid buffer incompatibility or to halt enzymatic reactions. PCR reactions were generally purified by solid-phase reversible immobilization (SPRI) using Agencourt Ampure XP DNA-binding beads (Beckman Coulter) according to the manufacturer's guidelines, or recovered and purified from agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research). If DNA samples were digested with restriction enzymes, the DNA was purified using the Monarch® PCR & DNA Cleanup Kit (NEB) according to the manufacturer's recommendations for cell suspension DNA purification protocols. After purification, DNA concentration and purity were conventionally assessed using a Nanodrop spectrophotometer as needed.

[0135] DNA transformation of E. coli by electroporation. Conventional plasmid transformation was performed by electroporation. Electrocompetent E. coli strains were prepared from 20 mL of LB broth. Subsequently, electroporation was performed at 600 nanometers (OD). 600nm The cultures, which had reached the exponential growth phase with an optical density of 0.6, were washed three times with sterile 10% glycerol solution. Subsequently, the cells were resuspended in 200 μL of water and distributed into 50 μL aliquots. Next, DNA was added to the electrocompetent cells, and the mixture was transferred to a 1 mm electroporation cuvette. Electroporation of the cells was performed using pulses of 1.8 kV, 25 μF, and 200 Ω for 5 ms. Subsequently, the cells were resuspended in 1 mL of non-selective LB medium, harvested at 37°C for 1 hour, or at 30°C for temperature-sensitive plasmids, and then plated in selective medium.

[0136] DNA transformation of E. coli by heat shock. Heat shock transformation was primarily used to clone Gibson assembly products. Chemically competent cells were prepared according to the rubidium chloride protocol as previously described (Green et al., 2013). Chemically competent cells were rapidly frozen at -80°C and stored before use. Direct transformation of EC100Dpir+ or MM294 chemically competent cells with Gibson assembly products was performed at a 1 / 10 volume ratio. Conventionally, up to 10 μL of DNA was added to 100 μL of competent cells, followed by heat shock transformation at 42°C for 45 seconds. Subsequently, the cells were resuspended in 1 mL of non-selective LB medium and harvested at 37°C for 1 hour, or at 30°C for temperature-sensitive plasmids, before being plated in selective medium.

[0137] Insertion of pTAT001 into the E. coli genome as a biological containment measure. The modified EcN::TAT001 strain is obtained by Tn7 insertion of the antibiotic resistance cassette based on the procedure described above (McKenzie et al., 2006). Integration is verified by PCR using the corresponding primers as listed in Table 2. To verify plasmid elimination, the disappearance of ampicillin resistance is confirmed. More specifically, the pTAT00X vector is purified from the E. coli DH5-Alpha+ strain and digested with SmaI + XhoI. The insert is amplified by PCR using the corresponding primers (Table 2), and the attL of the digested pTAT00X plasmid is measured. Tn7 Body part and attR Tn7 The plasmid is inserted between the sites by Gibson assembly. Subsequently, the Gibson assembly product is used to transform the electrocompetent E. coli EC100Dpir+ strain. The resulting plasmid is analyzed using restriction enzymes, and positive clones are used to transform E. coli EC100ΔdapA+pTA-MOB. The plasmid is recruited from E. coli EC100ΔdapA+pTA-MOB to MG1655 by conjugation. To mediate cassette insertion of glmS into the terminator, MG1655 is first osmothered in LB at 30°C with 1% arabinose. 600nmCulturing is performed until 0.6°C. Subsequently, the cells are subjected to heat shock at 42°C for 1 hour and incubated overnight at 37°C to allow plasmid disappearance. Then, aliquots of the bacterial culture are streaked onto LB agar plates. More than 20 colonies are analyzed, and colonies that grow only in the absence of ampicillin but contain the insert selection marker are examined by PCR using appropriate primers listed in Table 2.

[0138] Synthetic bacteriophages. We designed a living biopharmaceutical that secretes synthetic bacteriophages for the presentation of therapeutic proteins. A general description of the composition of the living biopharmaceutical is shown in Figure 1, and outlines of various constructs are shown in Figures 5 and 6.

[0139] This embodiment demonstrates various iterations of a synthetic therapeutic bacteriophage secretion system and a method for using these iterations to present therapeutic proteins at various sites on the resulting bacteriophage particles. In the first embodiment of the synthetic therapeutic bacteriophage secretion system, the system can be divided into two sets of vectors: a synthetic bacteriophage skeleton vector (e.g., pTAT002 (Figure 5E), pTAT003 (Figure 5E), pTAT012 (Figure 5F), pTAT013 (Figure 5G), pTAT014 (Figure 5H), pTAT019 (Figure 5G), pTAT20 (Figure 5G), pTAT022 (Figure 5G), or pTAT030 (Figure 5G)) and a synthetic bacteriophage mechanism vector (e.g., M13K07 (Figure 5A), pTAT004 (Figure 5C), or pTAT025 (Figure 5D)). In some cases, the synthetic therapeutic bacteriophage secretion system may be contained within a single vector (e.g., M13mp18-Kan (Figure 5B), pTAT032 (Figure 5B), or pTAT033 (Figure 5B)) or divided into three or more gene constructs, as in the system consisting of pTAT025 (Figure 5D), pTAT002 (Figure 5E), and pTAT017 (Figure 5H) or pTAT028 (Figure 5H). To improve the biological containment of the synthetic therapeutic bacteriophage secretion system, some of its genes may be incorporated into the chromosome of the host cell, either as a whole or as parts (see Figure 6, as shown for pTAT001), but an extrachromosomal DNA element containing oriM13 (e.g., pTAT012) is still required to function as a synthetic bacteriophage scaffold vector.

[0140] All the genes required for bacteriophage assembly consist of a single genetic element closely resembling the natural M13 genome. This structure has the advantage that the therapeutic fusion protein can benefit from the same level of expression as the natural protein optimized during evolution. The first step in obtaining a single vector system was to modify M13mp18 for easy selection. To this end, M13mp18 and the aph-III gene from M13K07, designed to be inserted into the lacZ gene contained in M13mp18, were amplified using primers listed in Table 2. The fragments were then assembled by Gibson, resulting in the production of M13mp18-Kan. Next, it was necessary to express the therapeutic protein from the same backbone. To achieve this, the inventors amplified the entire M13mp18-Kan backbone except for the N-terminal portion of gbIII and the nbPDL1 (anti-PD-L1 nanobody) from gBlock. The N-terminal portion of gpIII was replaced by nbPDL1 in pTAT032 (Figure 5). Another variant of this system (pTAT033) was designed to retain the entire gpIII gene, but the nbPDL1 gene was inserted centrally into pIII between the two domains constituting this coating protein (Figure 5). Both systems were successfully assembled and produced functional synthetic therapeutic bacteriophages, which will be further described in Example 3. This demonstrates that synthetic therapeutic bacteriophages can be created by directly cloning therapeutic protein fusions in the synthetic bacteriophage mechanism. Fusion with other capsid proteins, such as gpVIII and gpIX, is also possible. This will be illustrated with pTAT027 in the next section.

[0141] To construct the synthetic bacteriophage mechanism vector pTAT004, the entire M13K07 was amplified using appropriate primers shown in Table 2, excluding the gpIII gene encoding pIII. The homology tails of the primers used to amplify M13K07 were carefully designed to remove gpIII from the final construct. Next, the PCR product was purified by SPRI and assembled by Gibson's method to construct pTAT004. This assembly was used to transform MM294 chemically competent cells, and plasmid integrity was verified by digestion using NdeI. The pTAT004 synthetic bacteriophage mechanism, lacking a copy of the gpIII gene, cannot produce fully functional bacteriophage particles on its own and therefore does not produce pIII. In order to produce bacteriophage particles, gpIII must be provided trans-positively by the synthetic bacteriophage skeleton vector. Next, another synthetic bacteriophage mechanism was derived from pTAT004. To demonstrate our ability to present proteins and peptides on pVIII, we created pTAT027 by cloning an epitope derived from the chicken ovalbumin gene to the N-terminus of the gpVIII gene on pTAT004. This plasmid was assembled using primers to amplify the pTAT004 plasmid, which had several mutations introduced at the gpIX-gpVIII gene junction. First, since the start codon of gpVIII overlaps with the end of gpIX, it was mutated and reintroduced after the terminal portion of gpXI to allow for further cloning. Next, the ovalbumin epitope was encoded in the primer tail and introduced immediately after the start codon of the gpVIII gene. This construct is expected to present the OVA peptide on pVIII, although it requires an external source of pIII for the bacteriophage to be assembled correctly. The following synthetic bacteriophage skeleton vectors were designed: pTAT002, pTAT003, pTAT012, pTAT019, pTAT020, pTAT022, and pTAT030. All synthetic bacteriophage skeleton vectors are designed for high-copy plasmid replication. pMB1(Maximize DNA material for encapsulation), ori for recognition of synthetic bacteriophage backbone vectors by ssDNA rolling circle replication and phage encapsulation machinery M13 , a selection marker (here spectinomycin resistance), and the N-terminal fragment of pIII via one HA-His dual tag (pTAT002, a control without therapeutic protein), or checkpoint inhibitor binding protein (pTAT003, anti-CD47 nanobody; pTAT019, anti-CTLA-4 nanobody; pTAT020, anti-PD-L1 nanobody; pTAT022 and pTAT030, anti-CTLA-4 anticalin), or a constitutively expressed pIII C-terminal fragment linked to either a therapeutic enzyme (pTAT022, cytosine deaminase (CD)) (see Table 3 for therapeutic protein sequences).

[0142]

Table 3

[0143] In this example, anti-CD47, anti-CTLA-4, and anti-PD-L1 binding proteins were selected as therapeutic agents because they are well-characterized checkpoint inhibitors that bind to immune checkpoints expressed by cancerous cells (Vaddepally et al., Cancers (Basel) March 2020;12(3):738; which is incorporated herein by reference), while cytosine deaminase is an enzyme that converts the prodrug 5-FC to 5-FU, a chemotherapeutic agent commonly used to treat cancer (Nyati M.K. et al., Gene Therapy 2002;9:844 - 849; which is incorporated herein by reference). The first assembled synthetic bacteriophage backbone vector was pTAT003. To construct pTAT003, ori from pSB1C3 pMB1 , ori from M13K07 M13The gBlock containing the pIII N-terminal and C-terminal regions, aad7 (spectinomycin-resistant) from E. coli KN01, and anti-CD47 nanobodies with a peptide linker and constitutive promoter was amplified by PCR. The PCR products were then assembled by Gibson and transformed into chemically competent MM294 cells (Figure 5). To assemble pTAT002, the backbone was amplified from pTAT003 and the gpIII-deficient N-terminal region was amplified from M13K07. The two DNA portions were then assembled by Gibson's method. Plasmid integrity was then confirmed by digestion using ApaLI and NdeI. Plasmid pTAT012 was then constructed using the primers listed in Table 2 and assembled by Gibson assembly. The pTAT012 vector was ori M13 ,oriV pMB1It contains only the aad7 resistance gene. Therefore, this consists of a vector that complements M13K07, providing only the scaffold for bacteriophage construction and demonstrating one biological containment strategy. Sanger sequencing of pTAT002 and pTAT003 revealed a mutation at position 3 (G>T) of the P5 promoter in both pTAT003 and pTAT002. This resulting promoter, named P5mut, allows for lower levels of upstream gene expression when measured with pTAT010-P5 and pTAT010-P5mut constructs using GFP (data not shown). To facilitate construction assembly, the pTAT002 scaffold was modified and the sfGFP gene was cloned to be expressed by the P5 promoter instead of gpIII. This skeleton was assembled similarly to pTAT003, but the primers used allowed for the insertion of an additional terminator after the gene expressed by P5, and the insertion of a Gibson assembly tag (GAT) that separated different parts of the vector. Subsequently, the resulting vector, named pTAT013, was used as a template for amplifying the skeleton of subsequent constructs for the presentation of therapeutic proteins on pIII. Therefore, for the construction of pTAT019, pTAT020, pTAT022, and pTAT030, the plasmid skeleton was amplified from pTAT013, the P5mut promoter from pTAT010-P5mut, and the C-terminal portion of gpIII from M13K07. These DNA portions were then assembled by Gibson using different gBlocks encoding the therapeutic proteins to be presented. Therefore, we used a gBlock encoding an anti-CTLA-4 nanobody in pTAT019, an anti-PD-L1 nanobody in pTAT020, cytosine deaminase codA in pTAT022, and anti-CTLA-4 anticharin in pTAT030. Next, all plasmids were subjected to Illumina or Sanger sequencing after assembly, and no harmful mutations were detected. These results prepare us for efficiency testing and refinement rounds of synthetic bacteriophage secretion systems that present therapeutic proteins on pIII.The synthetic therapeutic bacteriophage secretion system can be divided into three or more DNA molecules and can maintain its function as long as sufficient protein is produced for each bacteriophage gene. To explain the plasticity of the bacteriophage genome, the inventors aimed to divide the bacteriophage mechanism into three different plasmids. As a first step, the inventors needed to delete gpIII and additional genes from M13K07. The inventors selected gpIX, another capsid gene involved in bacteriophage budding from host cells, as the second site for protein fusion. Since the coding sequence of gpIX overlaps with the coding sequence of gpVIII, deleting gpIX from M13K07 is more complex than deleting gpIII. The inventors' design used to remove gpIX required several gene refactorings to prevent disruption of the gpVIII gene. A duplication sequence between gpVIII and gpIX, where the ATG codon is the start codon of gpIX and the TGA codon is the stop codon derived from gpVIII. The duplication between the two genes was corrected by mutating the A>G at position 3, changing the ATG codon to a weaker GTG start codon, without affecting the sequence of gpVIII (both AGG and AGA encode arginine). Furthermore, the inventors introduced a stop codon by changing the third codon of gpIX from TTA to TAA, thereby inhibiting gpIX translation. Next, the resulting construct pTAT025 was obtained by amplifying pTAT004 using primers that introduced these modifications to the gpVIII / gpIX locus. Therefore, plasmid pTAT025 expresses all genes of the M13 genome except gpIII and gpIX, and these need to be provided in trans. To secrete bacteriophages, ori... M13 A bacteriophage skeleton vector encoding this is also required. The same procedure was performed on pTAT032 to obtain pTAT032ΔgpIX, a pIX-deficient bacteriophage secretion mechanism that presents anti-PDL1 nanobodies on pIII.

[0144] The gpIII deficiency of plasmid pTAT025 can be compensated for by any of the above plasmids expressing gpIII or a gpIII-therapeutic protein fusion (pTAT002, pTAT003, pTAT019, pTAT020, pTAT022, or pTAT030). However, pTAT025 also requires an exogenous supply of gpIX to produce bacteriophages. Therefore, a set of plasmids was needed to support gpIX production. For this purpose, ori from pKN23 was used. pSC101Novel skeletons were constructed by amplifying bla from pUC19 and P5-BCD1-sfGFP from pTAT010-P5, and these were assembled using GAT at the primer tails. Next, the PCR fragments were purified by SPRI, and pTAT014 was constructed by Gibson assembly, followed by transformation in MM294. The constructs were then evaluated phenotypically (GFP phenotype) and sequenced by Sanger sequencing. Next, the entire skeleton was amplified excluding the sfGFP gene and assembled with the gpIX gene amplified from M13K07. Next, both PCR products were assembled in the same manner as pTAT014 to construct a gpIX complement plasmid (pTAT017). This plasmid was further modified to amplify the entire skeleton excluding P5-BCD1 and to present anti-CD47 nanobodies (nbCD47) on pIX by adding two DNA fragments derived from gBlock. The first was a revTet expression system, and the second was pelB-nbCD47 cloned to the N-terminus of pIX. This resulted in plasmid pTAT028 after Gibson assembly and cloning in MM294. The plasmid was then confirmed by Sanger sequencing. The pTAT017 vector was further modified to present antikalin against CTLA-4 using primers to amplify the pTAT017 backbone, P5mut-BCD1 from pTAT010-P5mut, and gBlock_TAT10. These primers also changed the start codon of the antikalin fusion protein from ATG to GTG. This construct was later named pTAT035, which enables the presentation of CTLA-4 antikalin on pIX.

[0145] The first step toward the biological containment of the synthetic bacteriophage secretion system is to confine the synthetic bacteriophage mechanism to the chromosomes of the host cell. In a system where all components are extrachromosomal, bacteriophage particles can capsidize either the synthetic bacteriophage skeleton vector (90-99%) or, by random error, the synthetic bacteriophage mechanism vector (1-10%), as observed in our tests (see Figure 7A in Example II). Insertion of the synthetic bacteriophage mechanism into the host chromosome is thought to result in capsidization of only the synthetic bacteriophage skeleton vector. To mediate the chromosomal integration of the synthetic bacteriophage mechanism vector, PCR amplification of pTAT004 (without the origin of replication and antibiotic resistance genes) was cloned between the att sites of pTAT00X digested with XhoI + NdeI. Two plasmids were fused to each other by Gibson assembly, purified by SPRI, and cloned into electrocompetent EC100Dpir+ cells. Plasmid clones were then screened by digestion using EcoRI and PvuII (Figure 6). Next, a completed novel vector called pTAT001 was extracted from EC100Dpir+ and transformed into EC100Dpir+ + pTA-MOB. Subsequently, pTAT001 was recruited by conjugation from EC100Dpir+ to MG1655 on agar medium using the pTA-MOB conjugation mechanism. Next, integration of the synthetic bacteriophage mechanism was induced at 30°C for 2 hours using 1% arabinose, the plasmid backbone was removed by heat shock at 42°C for 1 hour, and then the cells were grown overnight at 37°C. The resulting cells were then prepared to be transformed with pTAT002, pTAT003, pTAT019, pTAT020, pTAT022, or pTAT030 to complete the synthetic bacteriophage secretion system. Using pTAT001, if bacteria from the environment acquire this vector, the bacteriophage particles cannot self-replicate. Therefore, this level of biological containment provides an improved measure to prevent the spread of manipulated bacteriophages into the environment.The same strategy can be used to biologically contain synthetic bacteriophage mechanisms that enable the presentation of therapeutic proteins on pIX or other bacteriophage-coated proteins.

[0146] To further improve the biological containment of the synthetic bacteriophage secretion system, therapeutic proteins fused with pIII, pIX, or any bacteriophage-coated protein can be transferred from pTAT002, pTAT003, pTAT019, pTAT020, pTAT022, pTAT028, or pTAT030 to the genome of bacterial host cells. In this way, the therapeutic module is also integrated into the genome of the bacterial host cell. In this case, the synthetic bacteriophage backbone vector is ori M13The filler module contains, optionally, only high copy number origins of replication. The length of the synthetic bacteriophage can be modified using the filler module, which is an interesting feature for the applications of bispecific phage particles (Specthrie et al., J. Mol. Biol., 1992, 3:720; this is incorporated herein by reference). For example, if the binding protein fused to the tail of the bacteriophage binds to cancer cells, and the binding protein fused to the head of the bacteriophage binds to T cells, the length of the bacteriophage affects the distance between the cancer cells and T cells. Therefore, by modifying the size of the filler module, the size of the synthetic bacteriophage can be altered, affecting the distance between cancer cells and T cells, and thus influencing the T cell response to cancer cells. Further modifications to the synthetic bacteriophage secretory system can improve the efficiency of secretion and therapeutic activity. For example, using promoters that are induceable by environmental conditions found only in the tumor microenvironment reduces potential side effects or genetic variability in live biopharmaceuticals during scaling up production. This is exemplified by the pTAT028 construct, which is suppressed by tetracycline. Dividing the synthetic bacteriophage mechanism into multiple fragments and inserting them into distant loci in the bacterial host genome also reduces the probability of recombination. Using these constructs, the synthetic bacteriophage secretion system can present several proteins and peptides on different coating proteins (Figure 2). Nevertheless, the different plasmids constructed above transform E. coli MG1655 and, when combined, demonstrate the capabilities of the synthetic bacteriophage secretion system.

[0147] (Example 2) Synthetic bacteriophages are secreted from living biopharmaceuticals and present therapeutic agents. All bacterial strains used in this example are listed in Table 1. Cells were typically grown in Luria Broth Mirror (LB) with the following antibiotic concentrations added as needed: kanamycin (Km) 50 μg / mL, spectinomycin (Sp) 100 μg / mL. All cultures were conventionally grown at 37°C with stirring (200 rpm). Bacterial cultures older than 18 hours were not used in the experiment.

[0148] Precipitation of synthetic bacteriophage particles based on polyethylene glycol. Starting from frozen stock, inoculate 5 mL of sterile LB broth containing the appropriate antibiotic at the concentration specified in the paragraph above, and incubate at 37°C overnight or within 18 hours with agitation of the culture. Transfer 1.5 mL of the overnight bacterial culture and centrifuge at 13000 g for 2 minutes. Carefully transfer 1.2 mL of the supernatant containing bacteriophage particles to a new sterile microtube, taking care not to disturb the pellet. Add 300 μL of 2.5 M NaCl / 20% PEG-8000 (w / v) to the culture supernatant (mix in a volume ratio of supernatant:PEG solution of 4:1). After thoroughly mixing by rotating the tube 15 times, incubate the mixture at 4°C for 1 hour. Next, pelletize the virions by centrifugation at 13000 g for 3 minutes. Next, remove the supernatant and resuspend the pellet in 120 μL of TBS 1× (Tris-buffered saline: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, sterile), corresponding to 1 / 10 of the initial culture volume. Keep the bacteriophage preparation on ice for another hour, vortex it, and then use immediately.

[0149] Functionality of synthetic bacteriophage particles by infection assay. To first test the integrity of the manipulated bacteriophage particles, an infection experiment was designed. A culture of *E. coli* strain ER2738 was grown overnight in LB containing appropriate antibiotics. To test the infectivity of the bacteriophage particles, 1 μL of culture supernatant was added to 1 mL of *E. coli* strain ER2738. The mixture was then incubated at 37°C for 1 hour and 30 minutes, followed by plating for CFU analysis. Infected cells could be identified by the acquisition of either a spectinomycin resistance gene (synthetic bacteriophage scaffold vector) or a kanamycin resistance gene (synthetic bacteriophage mechanism vector or M13K07).

[0150] Titer determination of synthetic bacteriophages by enzyme-linked immunosorbent assay (ELISA). Bacteriophage expression detection and quantification were performed using a commercially available Phage Titration ELISA kit (PRPHAGE, Progen) according to the manufacturer's instructions. Briefly, lyophilized M13 particles were subjected to phage titer 1,5 × 10⁶ according to the manufacturer's recommendations. 8 The bacteriophage preparations were resuspended at 1 / 1000 and 1 / 10000 and subsequently added to ELISA wells pre-coated with mouse anti-M13 (similar to the standard curve). Captured bacteriophage particles were detected by peroxidase-conjugated monoclonal anti-M13. After the addition of tetramethylbenzidine, the optical density of each well was measured at 450 nm using a Biotek plate reader instrument. To detect the modified pIII protein on the surface of the manipulated phages, the procedure was repeated using antibody HA-Tag(6E2) mouse mAb (HRP conjugate) (1:1000 Cell Signaling Technology, Danvers, MA, USA) instead of the anti-M13-HRP provided in the kit.

[0151] Evaluation of Phage Infectivity - E. coli MG1655 was transformed with plasmids pTAT002 and pTAT003. Subsequently, the resulting strains were transformed with pTAT004 to create MG1655 + pTAT002 + pTAT004 and MG1655 + pTAT003 + pTAT004 (Table 1). Strains possessing pTAT002 should produce infectious phage particles (because pTAT002 possesses a wild-type copy of the gpIII gene), while strains possessing pTAT003 should produce non-infectious phage particles. This is because wild-type gpIII is not present in pTAT004, and in pTAT003 it is fused with an anti-CD47 nanobody (Figure 3). To verify the infectivity of bacteriophage particles, phages derived from pTAT002, pTAT003, and M13K07 were purified using a PEG precipitation protocol. Next, E. coli ER2738 cells were infected with 1 μL of each phage preparation and incubated at 37°C for 1 hour and 30 minutes. Then, CFU was quantified on selected LB agar plates of host or infected cells. Bacteriophage particles derived from pTAT002 and M13K07 were infective, but phage particles derived from pTAT003 did not infect cells, confirming that a complete copy of pIII is not present in pTAT003 (Figure 7A). This result has two main implications. First, it indicates that when pIII is wild-type, the synthetic bacteriophage secretion system produces functional phage particles, and therefore all genes associated with the synthetic bacteriophage mechanism function correctly. Secondly, this indicates that the current configuration of the synthetic bacteriophage secretion system produces non-infectious phage particles when presenting therapeutic proteins on pIII. This is a crucial step toward the biological containment of this system, indicating that it should not be able to infect and replicate by infecting the native host of M13.

[0152] Secretion of synthetic bacteriophages by live biopharmaceuticals measured by ELISA - The first step in verifying the integrity of the synthetic bacteriophage secretion system is to examine the secretion by bacterial hosts of synthetic bacteriophages that present various therapeutic proteins through various fusions. To achieve this, several repeats of the synthetic bacteriophage secretion system were assembled by transforming the E. coli MG1655 strain, which produces various bacteriophages, with different vector combinations: control bacteriophage (MG1655 + pTAT004 + pTAT002), bacteriophage presenting anti-CD47 nanobodies on pIII (MG1655 + pTAT004 + pTAT003), bacteriophage presenting anti-CTLA-4 nanobodies on pIII (MG1655 + pTAT004 + pTAT019), bacteriophage presenting anti-PD-L1 nanobodies on pIII (MG1655 + pTAT004 + pTAT020), and bacteriophage presenting cytosine deaminase on pIII (MG1655 + pTAT004 + The bacteriophages were: pTAT022), a bacteriophage (MG1655 + pTAT004 + pTAT030) that presented anti-CTLA-4 anticharin on pIII, a bacteriophage (MG1655 + pTAT025 + pTAT002 + pTAT028) that presented anti-CD47 nanobody on pIX, and a bacteriophage (pTAT027 + pTAT002) that presented an epitope derived from the chicken egg albumin gene SIINFEKL on pVIII. Next, two types of ELISA assays were performed on PEG-precipitated bacteriophage particles derived from each repeat of the synthetic bacteriophage secretion system. The first ELISA assay was performed to detect the presence of pVIII, while the second ELISA assay was performed to detect the presence of the HA linker, which is present on pIII in bacteriophage particles derived from both pTAT002 and pTAT003, but not in those derived from M13K07.When the phage preparations were diluted to 1:1000, all but one strain showed a high signal in the anti-pVIII ELISA assay, confirming high levels of secretion / mL of synthetic bacteriophages (Figure 7B). Strains presenting anti-CD47 nanobodies on the pIX fusion produced bacteriophages at a lower count than the other constructs. This lower efficiency may be related to the expression system used for this construct, which differs from the others. A second ELISA confirmed the presence of linker HA in both the MG1655 + pTAT004 + pTAT002 (pIII HA) and MG1655 + pTAT004 + pTAT003 (anti-CD47 nanobodies on pIII) strains (Figure 7C). The anti-HA-HRP (Cell Signaling) antibody induced a signal only for the two modified systems expressing the HA tag, confirming the presence of fusion pIII protein derived from the bacteriophage vector scaffold in the bacteriophage particles. As expected, the MM294 + M13K07 (pIII wild-type) strain showed no signal in this assay, because the pIII protein expressed by M13K07 lacks an HA tag linker. This data supports the presentation of a therapeutic protein.

[0153] (Example 3) Synthetic bacteriophages present therapeutic binding proteins that can recognize and bind to immune checkpoints expressed on tumor cells. All bacterial strains and plasmids used in this example are listed in Table 1. Cells were typically grown in Luria Broth Mirror (LB) or Luria Broth Agar Mirror medium supplemented with the following antibiotic concentrations as needed: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were conventionally grown at 37°C for up to 18 hours before use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example II. The bacteriophage preparations were used immediately after precipitation. A20 lymphocytes (B lymphoma cells) were ordered from ATCC (TIB-208). Upon arrival, the cells were washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This culture medium was used for cell preparation for all experiments. After four passages, frozen stocks were prepared and used to initiate subsequent cultures for experiments. Throughout all experiments, cells were 2 × 10⁶ 5 pieces / mL~2×10 6 The density was maintained between particles / mL.

[0154] Synthetic bacteriophage pull-down assay. PEG-precipitated bacteriophages were resuspended in phosphate-buffered saline (PBS) + 0.2% p / v bovine serum albumin (BSA) (PBS-B) and incubated at 4°C for 1 hour. Approximately 1 × 10⁻⁶ 61 mL of cell culture at a density of cells / mL was centrifuged at 400 g for 3 minutes and resuspended in 500 μL of PBS-B. The cells were again centrifuged at 400 g for 3 minutes and then resuspended in 100 μL of bacteriophage solution. Aliquots of the cell-bacteriophage mixture were saved for further analysis. Next, the cells were washed six times with PBS-B, with 20 μL aliquots kept on ice during the first, third, and sixth washes. After washing the cells six times, 10 μL of the total aliquots was mixed with 90 μL of 5% p / v Chelex beads in a PCR tube. DNA was extracted by incubating the mixture at 50°C for 25 minutes and then at 100°C for 10 minutes. This DNA preparation was amplified by qPCR using a TransStart PFU fly DNA polymerase kit (Civic Bioscience) supplemented with EvaGreen dye (Biotium). Bacteriophage DNA was amplified using primers oTAT043 and oTAT044, as described in Table 2. The remaining 10 μL of all aliquots was diluted in 90 μL of PBS and used to assess cell count.

[0155] Evaluation of binding of synthetic bacteriophages to therapeutic targets by flow cytometry. PEG-precipitated bacteriophages were resuspended in phosphate-buffered saline (PBS) + 0.2% p / v bovine serum albumin (BSA) (PBS-B) and incubated at 4°C for 1 hour. Approximately 1 × 10⁻⁶ 61 mL of cells / mL were centrifuged at 400 g for 3 minutes and resuspended in 500 μL of PBS-B. The cells were centrifuged again at 400 g for 3 minutes and then resuspended in 100 μL of therapeutic bacteriophage solution presenting nanobodies, or in 100 μL of PBS-B for a control without bacteriophage. The cells and bacteriophages were incubated at 4°C for 1 hour. Subsequently, the mixtures were centrifuged at 400 g for 3 minutes. Subsequently, the cells were resuspended in 50 μL of PBS-B containing 1 μg of miap301 FITC anti-CD47 rat IgG2a (Biolegend) to evaluate the specificity of anti-CD47 nanobodies, or in 50 μL of PBS-B containing 0.25 μg of PE anti-CD274 (B7-H1, PD-L1) rat IgG2b (Biolegend) to evaluate the specificity of anti-PD-L1 nanobodies. The unstained control group was prepared in the same manner, except that the cells were not labeled with antibody. The cells were incubated in the dark at 4°C for 30 minutes, followed by centrifugation at 400 g for 3 minutes, and finally resuspended in 500 μL of PBS. The cells were then analyzed using a BD Accuri C6 Plus or a BD FACSJazz® Cell Sorter flow cytometer.

[0156] Evaluation of synthetic bacteriophage binding to CTLA-4 by ELISA. An ELISA assay was devised to measure the binding activity of synthetic bacteriophages to checkpoint CTLA-4. First, a 96-well plate was coated overnight at 4°C with recombinant CTLA-4 protein (R&D Systems) diluted to 10 μg / mL in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). Subsequently, the plate was washed three times with 200 μL of TBS-T. Then, to prevent nonspecific binding, the plate was incubated with 200 μL of blocking buffer (TBS-T, 3% skim milk, 1% BSA) at room temperature for 1 hour. Blocking was stopped by removing the blocking buffer and washing the plate twice with 200 μL of TBS-T. Next, 100 μL of PEG-precipitated synthetic bacteriophage, diluted with TBS 1X and displaying either anti-CTLA-4 nanobody (pTAT004 + pTAT019), anti-CTLA-4 anticharin (pTAT004 + pTAT030), or wild-type pIII (control: pTAT004 + pTAT002), was added to wells containing or not containing CTLA-4 protein and incubated at room temperature for 1 hour. Subsequently, the plates were washed three times with 200 μL of TBS-T, and 100 μL of anti-pVIII-HRP (anti-M13 / fd / F1, B62-FE2) diluted with blocking buffer (1:500) was added. The plates were incubated in the dark at room temperature for 1 hour and then washed five times with TBS-T. To measure the presence of synthetic bacteriophages, 100 μL of TMB substrate solution (ThermoFisher) was added to each well, and the plate was incubated at room temperature for 15 minutes. The reaction was stopped by adding 100 μL of stop solution (0.5 M H2SO4) to each well. Absorbance was then measured at 450 nm.

[0157] ELISA evaluation of bacteriophage binding activity to anti-PD-L1-presenting cells in A20 cells. 1 × 10⁻⁶ 5A 96-well plate was prepared by adding 100 μL of PBS containing 1 A20 cells to each well. The plate was then incubated for 30 minutes to allow the cells to settle. The plate was then tilted and the PBS was carefully removed. Next, 100 μL of 10% formalin was added to fix the cells to the plate, and the plate was incubated for 10 minutes. The fixed cells were then gently washed with 100 μL of PBS, followed by blocking with 200 μL of PBS (1% BSA, 3% milk), and then incubated for 30 minutes. The blocking buffer was removed, and then 100 μL of bacteriophage PEG preparation was added to each well. The plate was incubated for 1 hour, followed by washing three times with 200 μL of PBS. To detect bacteriophages, 100 μL of anti-HA-HRP (Cell Signaling) diluted 1 / 500 in PBS (3% milk, 1% BSA) was added, and the plate was incubated for 1 hour. The wells were washed three times with 200 μL of PBS, followed by the addition of 100 μL of tetramethylbenzidine (TMB) substrate solution to each well. The plate was incubated for 5 minutes, followed by the addition of 100 μL of stop solution. Subsequently, 100 μL from each well was transferred to a new plate, and the optical density was measured at 450 nm.

[0158] The living biopharmaceutical secretes a synthetic bacteriophage that presents anti-CD47 nanobodies that bind to the surface of A20 cells. This example aims to confirm the ability of the synthetic bacteriophage produced by the living biopharmaceutical to bind to CD47 on the surface of A20 mouse lymphoma cells. A control bacteriophage (MG1655 + pTAT004 + pTAT002) or a bacteriophage presenting anti-CD47 nanobodies on pIII (MG1655 + pTAT004 + pTAT003) was prepared as a three-repeated biological test. 10 9 A 100 μL bacteriophage preparation containing 1,5 × 10 particles is then prepared. 6The cells were mixed with A20 cells and incubated at 4°C for 1 hour. The cells were then washed six times to remove phage particles that did not specifically bind to A20 cells. Aliquots of the cell mixture were then analyzed by qPCR to quantify the number of phage particles present during the mixing stage, the first wash, the third wash, and the sixth wash (Figure 8). The results showed that bacteriophage particles produced by pTAT002 (which does not express nanobodies against CD47) were rapidly washed away from the cells, while bacteriophages from pTAT003 bound to A20 cells and were lost very little by the washing procedure, except for excess phages in the first wash. These results suggest that bacteriophage particles presenting the pIII-anti-CD47 nanobodies strongly bind to targets on cancer cells, likely via binding to the CD47 receptor.

[0159] Live biopharmaceuticals secrete synthetic bacteriophages that present anti-CD47 on pIII or pIX, which specifically bind to the CD47 receptor on the surface of tumor cells. Flow cytometry experiments were performed to confirm that synthetic bacteriophages specifically bind to CD47 on the surface of A20 cells. In this experiment, A20 cells were first incubated with either PBS-B, a control bacteriophage (MG1655 + pTAT004 + pTAT002), or a bacteriophage presenting anti-CD47 nanobodies on pIII (MG1655 + pTAT004 + pTAT003) to enable the bacteriophage to bind to CD47 on the cell surface. Subsequently, the cells were washed and incubated with anti-CD47-FITC (miap301, Biolegend) antibody. In this case, the specific binding of the synthetic bacteriophage to CD47 is thought to lead to a decrease in the binding of the anti-CD47-FITC antibody, and therefore a decrease in the FITC signal. The experiment consisted of four groups. The first group consisted of A20 cells only, which served as a negative control for measuring the background fluorescence signal (Figure 9A). The second group consisted of A20 cells incubated with anti-CD47-FITC antibody only, which served as a positive control for the fluorescence signal (Figure 9B). The third group included A20 cells that were first incubated with a synthetic bacteriophage derived from MG1655 + pTAT004 + pTAT002 (control), and then incubated with anti-CD47-FITC antibody (Figure 9C). The final group included A20 cells incubated with synthetic bacteriophage particles derived from MG1655 + pTAT004 + pTAT003 (presenting anti-CD47 nanobodies on pIII), followed by incubation with anti-CD47-FITC antibody (Figure 9D). The first two groups were used as references for untagged and tagged populations to evaluate the effect of bacteriophages on anti-CD47-FITC antibody binding. Only the bacteriophage derived from pTAT003 (presenting anti-CD47 nanobodies) was able to mask the CD47 epitope recognized by the antibody, reducing anti-CD47 antibody binding and therefore reducing FITC signaling.Next, the experiment was repeated using synthetic bacteriophage particles derived from MG1655 + pTAT025 + pTAT002 + pTAT028 (presenting anti-CD47 nanobodies on pIX) (Figures 9E-9H). The synthetic bacteriophage presenting anti-CD47 nanobodies on pIX showed a lower fluorescence shift compared to the synthetic bacteriophage presenting anti-CD47 nanobodies on pIII. The observed lower shift is associated with a lower secretion level of this construct, as discussed in Example II. These results indicate that synthetic bacteriophages derived from MG1655 + pTAT004 + pTAT003 and MG1655 + pTAT025 + pTAT002 + pTAT028 specifically bind to the CD47 receptor on the surface of A20 cells. Therefore, living biopharmaceuticals can produce functional bacteriophage particles that present anti-CD47 nanobodies on pIII or pIX, which can bind to the CD47 receptor on A20 lymphoma cells. The presented protein can be retained without distinction by both the head and tail proteins of the bacteriophage. CD47 is a key immune checkpoint, and by preventing its binding to T cell receptors, an immune response against tumor cells should be induced.

[0160] Live biopharmaceuticals secrete bacteriophages that present anti-PD-L1, specifically binding to the PD-L1 receptor on the surface of tumor cells. Synthetic bacteriophages can present various functional checkpoint inhibitors. Flow cytometry experiments were performed to demonstrate that synthetic bacteriophages presenting anti-PD-L1 nanobodies specifically bind to PD-L1 on the surface of A20 cells. In this experiment, A20 cells were first incubated with PBS-B, or phage particles derived from pTAT002 (control phage) or pTAT020 (phage presenting anti-PD-L1 nanobodies on pIII). Subsequently, the cells were washed and incubated with anti-PD-L1-PE (10F.9G2, Biolegend) antibody. In this case, it is thought that the specific binding of synthetic bacteriophages to PD-L1 reduces the binding of the anti-PD-L1-PE antibody, and therefore reduces the PE signal. The experiment consisted of four groups. The first group consisted of unstained A20 cells, which served as a negative control for measuring background fluorescence signals (Figure 9I). The second group consisted of A20 cells incubated only with anti-PD-L1-PE antibody, which served as a positive control for fluorescence signals (Figure 9J). The third group included A20 cells first incubated with synthetic bacteriophage (control) derived from MG1655 + pTAT004 + pTAT002, and then incubated with anti-PD-L1-PE antibody (Figure 9K). The final group included A20 cells incubated with synthetic bacteriophage particles (presenting anti-PD-L1 nanobodies) derived from MG1655 + pTAT004 + pTAT020, and then incubated with anti-PD-L1-PE antibody (Figure 9L). The first two groups were used as references for untagged and tagged populations to evaluate the effect of bacteriophage on anti-PD-L1-PE antibody binding. Only bacteriophages derived from pTAT020 (displaying anti-PD-L1 nanobodies) were able to mask the PD-L1 epitope recognized by the antibody, reducing the binding of anti-PD-L1 antibodies and thus decreasing the PE signal.This indicates that synthetic bacteriophages derived from MG1655 + pTAT004 + pTAT020 specifically bind to PD-L1 receptors on the surface of A20 cells. Therefore, these results support the possibility that living biopharmaceuticals can produce checkpoint inhibitors capable of binding to PD-L1 receptors on A20 lymphoma cells, such as functional bacteriophage particles presenting anti-PD-L1 nanobodies. PD-L1 is a key immune checkpoint that should trigger an immune response against tumor cells by interfering with its binding to T cell receptors.

[0161] Live biopharmaceuticals secrete synthetic bacteriophages that present anti-CTLA-4 binding proteins on pIII, specifically binding to the CTLA-4 immune checkpoint. Previous constructs have demonstrated that bacteriophages can present functional nanobodies capable of recognizing various targets on the surface of tumor cells. Synthetic bacteriophage systems can also present receptors specific to immune cells, such as proteins that recognize CTLA-4. To demonstrate that the binding proteins presented on bacteriophages can be of various types, bacteriophages were designed to present anti-CTLA-4 nanobodies (MG1655 + pTAT004 + pTAT019) or anti-CTLA4 anticarin (MG1655 + pTAT004 + pTAT030). ELISA experiments were performed to demonstrate that synthetic bacteriophages presenting anti-CTLA-4 nanobodies and anticarin proteins specifically bind to their target proteins. In this experiment, synthetic bacteriophages derived from either pTAT002 (control without presentation on pIII), pTAT019 (presentation of anti-CTLA-4 nanobody on pIII), or pTAT030 (presentation of anti-CTLA-4 antikarin on pIII) were incubated in a 96-well plate containing wells coated with mouse CTLA-4 recombinant protein. After the binding step was complete, the 96-well plate was washed with TBS-T and subsequently incubated with anti-pVIII-HRP B62-FE3 (progen). At this stage, the pVIII protein of the bacteriophage was tagged with horseradish peroxidase to reveal whether the synthetic bacteriophage was bound to the target. The presence of target-bound synthetic bacteriophages was measured by adding a TMB substrate that produces a signal at 450 nm when TMB is oxidized by the activity of the horseradish peroxidase enzyme. The signal was measured only in synthetic bacteriophages displaying anti-CTLA-4 binding proteins, demonstrating that synthetic bacteriophages can be used to target immune checkpoints using various types of binding proteins (Figure 10).Live biopharmaceuticals secrete bacteriophages containing functional therapeutic proteins inserted into fragmented functional coating proteins. To demonstrate that therapeutic proteins can be successfully presented when inserted centrally into the phage coating protein, an anti-PD-L1 nanobody was inserted between the D1 / D2 domain and the transmembrane region of pIII (see Figure 5 pTAT033). As a control, the anti-PD-L1 nanobody was also cloned to the N-terminus of pIII in the same manner as pTAT020, but directly into the bacteriophage secretion mechanism (see Figure 5 pTAT032). Subsequently, flow cytometry experiments were performed to evaluate the binding activity of the corresponding synthetic bacteriophages. In this experiment, A20 cells were first incubated with either a control synthetic bacteriophage (pTAT002, no presentation) or synthetic bacteriophages (pTAT032 and pTAT033) that presented anti-PD-L1 nanobodies on pIII, allowing the bacteriophage to bind to PD-L1 on the cell surface. Subsequently, the cells were washed and incubated with anti-PD-L1-PE (10F.9G2, Biolegend) antibody. In this case, the specific binding of the synthetic bacteriophage to PD-L1 is thought to lead to a decrease in the binding of the anti-PD-L1-PE antibody, and therefore a decrease in the PE signal. The experiment consisted of five groups. The first group consisted of A20 cells alone, which served as a negative control for measuring background fluorescence signal (Figure 11A). The second group consisted of A20 cells incubated with anti-PD-L1-PE antibody alone, which served as a positive control for fluorescence signal (Figure 11B). The third group included A20 cells that were first incubated with synthetic bacteriophages derived from MG1655 + pTAT004 + pTAT002 (control), and then incubated with anti-PD-L1-PE antibody (Figure 11C). The fourth group included A20 cells that were incubated with synthetic bacteriophage particles derived from MG1655 + pTAT032 (presenting an anti-PD-L1 nanobody inserted at the N-terminus of pIII), and then incubated with anti-PD-L1-PE antibody (Figure 11D).The final group included A20 cells incubated with synthetic bacteriophage particles derived from MG1655 + pTAT033 (displaying anti-PD-L1 nanobodies inserted into pIII) and then incubated with anti-PD-L1-PE antibody (Figure 11E). The first two groups were used as reference populations for untagged and tagged cells to evaluate the effect of bacteriophages on anti-PD-L1-PE antibody binding. Only bacteriophages derived from pTAT032 and pTAT033 were able to mask the PD-L1 epitope recognized by the antibody, which reduced anti-PD-L1 antibody binding and therefore reduced PE signaling. This indicates that synthetic bacteriophages derived from MG1655 + pTAT033 specifically bind to the PD-L1 receptor on the surface of A20 cells and can insert functional therapeutic proteins into the coating protein for proper presentation. The pTAT033 construct also suggests that proteins larger than the size of the nanobodies can be presented on the bacteriophage pIII coating protein and retain their function. The size of the pTAT033 pIII fusion protein is comparable to that of the two nanobodies, and if cloned onto pIII, both could bind to various targets. Furthermore, the phage particles derived from pTAT033 retained their infectivity, supporting the idea that both the nanobodies and the N-terminal portion of pIII retained their function and demonstrating that two binding function proteins can be cloned onto the same coat protein.

[0162] (Example 4) Synthetic bacteriophages that present peptides on PVIII All bacterial strains and plasmids used in this example are listed in Table 1. Cells were typically grown in Luria Broth Mirror (LB) or Luria Broth Agar Mirror medium supplemented with the following antibiotic concentrations as needed: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were conventionally grown at 37°C for up to 18 hours before use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example II. The bacteriophage preparations were used immediately after precipitation. A detailed list of oligonucleotide sequences used in this example can be found in Table 2. Plasmids were prepared using the EZ10-Spin Column Plasmid Miniprep Kit (BIOBASIC #BS614) or the QIAGEN Plasmid Maxi Kit (QIAGEN) according to the manufacturer's instructions. PCR amplification was performed using TransStart FastPFU fly DNA polymerase (Civic Bioscience) for DNA partial amplification and screening. Restriction enzyme digestion was performed using NEB products and incubated at 37°C for 1 hour according to the manufacturer's recommendations. Plasmids were assembled by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (NEB) according to the manufacturer's protocol. Sanger sequencing was performed using the Plateforme de sequencage de l'Universite Laval.

[0163] DNA purification. DNA purification was performed between each step of plasmid assembly to avoid buffer incompatibility or to halt enzymatic reactions. PCR reactions were generally purified by solid-phase reversible immobilization (SPRI) using Agencourt AMPure XP DNA-binding beads (Beckman Coulter) according to the manufacturer's guidelines, or recovered and purified from agarose gel using the Zymoclean Gel DNA Recovery Kit (Zymo Research). If DNA samples were digested with restriction enzymes, the DNA was purified using the Monarch® PCR & DNA Cleanup Kit (NEB) according to the manufacturer's recommendations for cell suspension DNA purification protocols. After purification, DNA concentration and purity were conventionally assessed using a Nanodrop spectrophotometer as needed.

[0164] Cell culture. A20 lymphocytes (B lymphoma cells) were ordered from ATCC (TIB-208). Upon arrival, the cells were immediately washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This culture medium was used for cell preparation for all experiments. After four passages, frozen stocks were prepared and used to initiate subsequent cultures for experiments. Throughout all experiments, the cells were 2 × 10⁶ 5 pieces / mL~2×10 6 The density was maintained between particles / mL.

[0165] Titer determination of synthetic bacteriophages by enzyme-linked immunosorbent assay (ELISA). Bacteriophage expression detection and quantification were performed using a commercially available Phage Titration ELISA kit (PRPHAGE, Progen) according to the manufacturer's instructions. Briefly, lyophilized M13 particles were subjected to phage titer 1,5 × 10⁶ according to the manufacturer's recommendations. 8The bacteriophage preparations were resuspended at 1 / 10, 1 / 100, 1 / 1000, and 1 / 10000 dilutions and added to ELISA wells pre-coated with mouse anti-M13 (similar to the standard curve). Captured bacteriophage particles were detected by peroxidase-conjugated monoclonal anti-M13. After the addition of tetramethylbenzidine, the optical density of each well was measured at 450 nm using a Biotek plate reader instrument. To detect the modified pIII protein on the surface of the manipulated phages, this procedure was repeated using antibody HA-Tag(6E2) mouse mAb (HRP conjugate) (1:1000 Cell Signaling Technology, Danvers, MA, USA) instead of the anti-M13-HRP provided in the kit.

[0166] Verification of fusion protein integrity by Western blotting. Bacteria were grown overnight at 37°C with agitation in LB broth supplemented with kanamycin and spectinomycin. The bacteria were pelleted by centrifugation, and the culture supernatant was transferred to a new tube and buffered with concentrated PBS. Phages displaying hexahistidine tags were pulled down from the supernatant by incubation at 4°C for 2 hours with agitation using Ni-NTA beads. The beads were then washed three times with PBS, and the phages were eluted by denaturation using sample buffer 4X (SB4X). The sample was denatured at 65°C for 1 hour and loaded onto a 15% acrylamide gel. The sample was transferred to the gel at 150 volts for 1 hour. The protein was then transferred to a 0.2 μm nitrocellulose membrane by applying 100 volts for 1 hour. The membrane was air-dried to evaporate trace amounts of methanol, and blocked at 4°C for 1 hour with agitation in TBS-0.1% Tween 20 - 4% dried milk. The membrane was transferred to a resealable Western blotting bag and incubated overnight at 4°C with anti-HA-HRP (Cell Signaling) diluted in blocking buffer under agitation. After three washes with TBS-0.1% Tween 20, the membrane was colorimetrically exposed by applying Immobilon ECL Ultra Western HRP substrate, and images were acquired using a Vilber Fusion FX instrument. Images were processed using Image Lab software.

[0167] Evaluation of binding of synthetic bacteriophages to therapeutic targets by flow cytometry. PEG-precipitated bacteriophages were resuspended in phosphate-buffered saline (PBS) + 0.2% p / v bovine serum albumin (BSA) (PBS-B) and incubated at 4°C for 1 hour. Approximately 1 × 10⁻⁶ 61 mL of cells / mL were centrifuged at 400 g for 3 minutes and resuspended in 500 μL of PBS-B. The cells were centrifuged again at 400 g for 3 minutes and then resuspended in 100 μL of therapeutic bacteriophage solution presenting nanobodies, or in 100 μL of PBS-B for the control without bacteriophage. The cells and bacteriophages were incubated at 4°C for 1 hour. Subsequently, the mixtures were centrifuged at 400 g for 3 minutes. Subsequently, the cells were resuspended in 50 μL of PBS-B containing 1 μg of miap301 FITC anti-CD47 rat IgG2a (Biolegend) to evaluate the specificity of anti-CD47 nanobodies, or in 50 μL of PBS-B containing 0.25 μg of PE anti-CD274 (B7-H1, PD-L1) rat IgG2b (Biolegend) to evaluate the specificity of anti-PD-L1 nanobodies. The unstained control group was prepared in the same manner, except that the cells were not labeled with antibody. The cells were incubated in the dark at 4°C for 30 minutes, followed by centrifugation at 400 g for 3 minutes, and finally resuspended in 500 μL of PBS. The cells were then analyzed using the FITC channel of a BD Accuri C6 Plus flow cytometer.

[0168] The synthetic bacteriophage secretion system produces bacteriophages that present peptides on the major coat protein pVIII. To verify that the bacteriophage secretion mechanism pTAT027 (see Example I) presents a peptide derived from the chicken ovalbumin gene (pVIII-OVA) on pVIII, Sanger sequencing was performed on the corresponding region of the construct (Figure 12A). The OVA peptide is in-frame with the pVIII protein; therefore, if the pVIII protein can be detected by antibody, the OVA peptide is necessarily present on the surface of the bacteriophage particle. The pIII-deficient pTAT027 mechanism was therefore complemented by either pTAT002 (providing HA-tagged pIII) or pTAT003 (providing anti-CD47-nanobody-HA-pIII) from E. coli MG1655. The strains were then grown overnight, and the bacteriophages were purified by PEG precipitation. Next, bacteriophage production was detected by ELISA (PRPHAGE Progene kit) using MG1655 + pTAT004 + pTAT002 and MG1655 + pTAT004 + pTAT003 as controls that do not display the OVA peptide (Figure 12B). The synthetic bacteriophage secretion system that displays the OVA peptide on pVIII produced a similar amount of bacteriophage as their counterparts with wild-type pVIII, suggesting that peptide presentation on pVIII does not hinder bacteriophage production. Regardless of the presentation of the OVA peptide on pVIII, to confirm that the bacteriophage presents either an HA-tagged pIII protein fusion (pTAT002) or an nbCD47-HA-pIII protein fusion (pTAT003), the bacteriophages were purified with Ni-NTA, analyzed by Western blotting, and the proteins were exposed using an anti-HA-HRP (Cell Signaling) antibody (Figure 12C).Bacteriophages that present the OVA peptide on pVIII exhibit a similar pattern of presentation on pIII to control bacteriophages, suggesting that bacteriophage assembly is complete in both cases and that complete bacteriophage particles can be produced.

[0169] The synthetic bacteriophage secretion system produces bacteriophages that present peptides on pVIII and functionally binding proteins on pIII that conceal immune checkpoints on the surface of cancer cells. To confirm that synthetic bacteriophages presenting OVA peptides on pVIII do not impair the integrity of the proteins presented on pIII, the function of synthetic bacteriophages presenting both OVA on pVIII and anti-CD47 nanobodies on pIII was evaluated. Flow cytometry experiments were performed to verify the binding of bacteriophages to CD47 on the surface of A20 cells. In this experiment, A20 cells were first incubated with either PBS-B, phage particles derived from MG1655 + pTAT027 + pTAT002 (control pVIII-OVA only), or phage particles derived from MG1655 + pTAT027 + pTAT003 (peptide OVA presented on pVIII and anti-CD47 nanobody presented on pIII) to enable the bacteriophage to bind to CD47 on the cell surface. The cells were then washed and incubated with anti-CD47-FITC (miap301, Biolegend) antibody. In this case, the specific binding of the synthetic bacteriophage to CD47 is thought to lead to reduced binding of the anti-CD47-FITC antibody, and therefore to a decrease in the FITC signal. The experiment consisted of three groups. The first group consisted only of A20 cells, which served as a negative control for measuring background fluorescence signaling (Figure 12D). The second group included A20 cells that were first incubated with synthetic bacteriophages derived from MG1655 + pTAT027 + pTAT002 (control, OVA on pVIII only), and then incubated with anti-CD47-FITC antibody (Figure 12E). The final group included A20 cells that were incubated with synthetic bacteriophage particles derived from MG1655 + pTAT027 + pTAT003 (displaying OVA on pVIII and anti-CD47 nanobodies on pIII), and then incubated with anti-CD47-FITC antibody (Figure 12F).The first two groups were used as references for the untagged and tagged populations to evaluate the effect of bacteriophages on anti-CD47-FITC antibody binding. As in the experiment shown in Figure 9, only bacteriophages derived from pTAT003 (presenting anti-CD47 nanobodies) were able to mask the CD47 epitope recognized by the antibody, reducing anti-CD47 antibody binding and therefore reducing FITC signaling. Thus, living biopharmaceuticals can produce functional bacteriophage particles that present anti-CD47 nanobodies capable of binding to the CD47 receptor on A20 lymphoma cells, while simultaneously presenting peptides on pVIII. CD47 is a key immune checkpoint, and by preventing its binding to T cell receptors, an immune response against tumor cells should be induced.

[0170] (Example 5) Synthetic bacteriophages that present checkpoint inhibitors have a direct antitumor effect. All bacterial strains and plasmids used in this example are listed in Table 1. Cells were typically grown in Luria Broth Mirror (LB) or Luria Broth Agar Mirror medium supplemented with the following antibiotic concentrations as needed: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were conventionally grown at 37°C for up to 18 hours before use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example 2. A20 lymphocytes (B lymphoma cells) were ordered from ATCC (TIB-208). Throughout all experiments, cells were 2 × 10⁶ cells in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. 5 pieces / mL~2×10 6The density was maintained between particles / mL.

[0171] Preparation and purification of PD-L1 nanobody proteins. The coding sequence of an anti-PD-L1 nanobody, fused with a hexahistidine tag and an HA tag at the C-terminus, was cloned into a pTrcHis vector by Gibson assembly. BL21(DE3) competent E. coli was transformed using the obtained plasmid, and transformants were selected on an ampicillin-containing LB plate. Plasmids were extracted from the transformants, and the integrity of the nanobody coding sequence was confirmed by Sanger sequencing. BL21 transformants were cultured in ampicillin-containing LB for protein expression and purification. Protein expression was induced by adding 1 mM IPTG and incubating at room temperature for 18 hours with agitation. Protein purification was performed by incubating cell lysates with Ni-NTA agarose beads (Qiagen) at 4°C for 18 hours. Protein elution was performed by incubating the Ni-NTA agarose beads with 200 mM imidazole. Next, the protein was concentrated from the eluate to a final volume of 500 μL using an Amicon UItra-15 10kDa Centrifugal Filter Unit. The concentrated protein was resuspended in sterile PBS to a final volume of 15 mL. The concentration and resuspension cycle was repeated three times. After the final concentration step, the purity of the protein was verified by spectrophotometric analysis and SDS-PAGE. The functionality of the purified and concentrated anti-PD-L1 nanobodies was confirmed by ELISA against PD-L1-expressing A20 cells.

[0172] Evaluation of anti-PD-L1 nanobody binding activity in A20 cells by ELISA. 1 × 10⁶ cells were bound to each well. 5A 96-well plate was prepared by adding 100 μL of PBS containing 100 A20 cells. The plate was then incubated for 30 minutes to allow the cells to settle. The plate was then tilted and the PBS was carefully removed. Next, 100 μL of 10% formalin was added to fix the cells to the plate, and the plate was incubated for 10 minutes. The fixed cells were then gently washed with 100 μL of PBS, followed by blocking with 200 μL of PBS (1% BSA, 3% milk), and then incubated for 30 minutes. The blocking buffer was removed, and then 100 μL of nanobodies were added to the wells at a concentration of 1 nM to 10 μM. The plate was incubated for 1 hour, followed by washing three times with 200 μL of PBS. To detect the nanobodies, 100 μL of anti-HA-HRP (Cell Signaling) diluted 1 / 500 in PBS (3% milk, 1% BSA) was added, and the plate was incubated for 1 hour. The wells were washed three times with 200 μL of PBS, followed by the addition of 100 μL of tetramethylbenzidine (TMB) substrate solution to each well. The plate was incubated for 5 minutes, followed by the addition of 100 μL of stop solution. Subsequently, 100 μL from each well was transferred to a new plate, and the optical density was measured at 450 nm. High doses of the control synthetic bacteriophage exhibit potent antitumor effects. To evaluate whether the control synthetic bacteriophage alone, i.e., those that do not display therapeutic proteins, may have antitumor effects, 5 × 10⁶ samples were placed on the right flank of mice. 6 A20 cells were subcutaneously injected, and tumor growth was monitored every two days. The mice were then divided into five treatment groups. The first group received 50 μL of PBS (vehicle control). The remaining groups received 10 μL of PEG-purified control synthetic bacteriophage (pTAT002) at increased doses. 7 pieces, 10 8 pieces, 10 9 pieces, and 10 10 50 μL of PBS containing 1 bacteriophage particle was administered. PBS, 10 7 pieces, 10 8 pieces, and 10 9For treatment with individual bacteriophages, doses were administered on days 0, 4, and 7. 10 11 In the treatment with individual bacteriophage particles, due to the presence of necrosis at the injection site, the doses were administered on days 0, 4, and 11, rather than day 7. Subsequently, the tumor was 1500 mm 3 Tumor size was monitored twice weekly using precise calipers until it exceeded a certain level or up to 24 days after the initial injection. High doses of control synthetic bacteriophages showed strong antitumor activity (Figures 13A-13B).

[0173] Synthetic bacteriophages that present anti-checkpoint nanobodies exhibit antitumor activity. To evaluate the effect of adding checkpoint inhibitors to the antitumor activity of synthetic bacteriophages, synthetic bacteriophages presenting CD47, PD-L1, and CTLA-4 checkpoint inhibitor nanobodies were developed using the process described in Example 1. 5 × 10⁶ nanobodies were placed in the right flank of mice. 6 A20 cells were subcutaneously injected, and tumor growth was monitored by observation every two days. Next, the mice were divided into two treatment groups, and all treatments were administered intratumorally on days 0, 4, and 7. The first group received 10 A20 cells that present anti-CD47 nanobodies on pIII. 9 The second group was administered an effective dose of either a synthetic bacteriophage displaying an anti-PD-L1 nanobody on pIII or a synthetic bacteriophage displaying an anti-CTLA-4 nanobody on pIII. 8 The drug was administered (Figure 13C). Subsequently, tumor size was monitored twice weekly using precise calipers until the tumor was eliminated or grew too large to continue the experiment. PBS or a control bacteriophage (10 as a control for CD47) was administered. 9 10 bacteriophage particle doses were used as controls for PD-L1 and CTLA-4. 8Compared to a control group treated with a dose of bacteriophage particles, only bacteriophages presenting anti-CD47 nanobodies, anti-PDL1 nanobodies, or anti-CTLA-4 nanobodies exhibited antitumor activity and resulted in tumor remission compared to appropriate controls. This experiment demonstrates that the presence of checkpoint inhibitors on synthetic bacteriophages enhances their antitumor effects.

[0174] When checkpoint inhibitors are presented by synthetic bacteriophages, a synergistic therapeutic effect is induced. As demonstrated in the previous section, synthetic bacteriophages presenting checkpoint inhibitors show improved antitumor efficacy, reducing the dose required to eliminate tumors by 100 times (compared to 1 / 10 of the dose required for phage alone). 10 For each individual, the anti-PD-L1 synthetic bacteriophage was 10 8 (10) Next, the inventors investigated whether checkpoint inhibitors presented by bacteriophages exhibited enhanced therapeutic activity compared to checkpoint inhibitors administered alone or in combination therapy. Enhanced activity is thought to suggest a synergistic effect between checkpoint inhibitors and bacteriophages. To test this hypothesis, the inventors measured the antitumor activity of purified anti-PD-L1 nanobodies alone or in combination with bacteriophages. As a control, the inventors first verified the functionality of the purified anti-PD-L1 nanobodies and confirmed their binding activity to A20 cells by ELISA (Figure 14A). Following the verification of the activity of the purified nanobodies, the inventors then investigated whether a synergistic effect was observed when anti-PD-L1 nanobodies were presented by bacteriophages (Figures 14B-14C). 5 × 10¹⁶ nanobodies were placed in the right flank of mice. 6 A20 cells were subcutaneously injected, and tumor growth was monitored daily. Subsequently, the mice were divided into six treatment groups, and all treatments were administered intratumorally on days 0, 4, and 7. Group 1 received only 50 μL of PBS (control vehicle), while Group 2 received 8 × 10¹⁶ cells. 1550 μL of PBS containing 1 anti-PD-L1 nanobody molecule (20 μg, equivalent to a typical treatment dose) was given to Group 3. 8 50 μL of PBS (10) containing purified control bacteriophage (pTAT002) particles 8 Group 4 includes 5 × 10¹⁶ doses (which mimic treatment with anti-PD-L1 synthetic bacteriophage particles, but do not include checkpoint inhibitors), and 5 × 10¹⁶ doses. 8 Each purified anti-PD-L1 nanobody (12.9 pg, 5 anti-PD-L1 nanobodies per bacteriophage) is presented. 8 Group 5 contains 10 (which mimics treatment with anti-PD-L1 synthetic bacteriophage particles, but does not contain bacteriophages) 8 5 × 10¹⁶ control bacteriophage (pTAT002) particles together 8 50 μL of PBS containing 10 purified anti-PD-L1 nanobodies (containing anti-PD-L1 nanobodies) 8 The last group includes 10 anti-PD-L1 nanobodies that present on pIII (which mimic treatment with anti-PD-L1 synthetic bacteriophage particles, but are not presented by the bacteriophage). 8 50 μL of PBS containing 8 × 10¹ synthetic bacteriophage (pTAT020) particles (a treatment in which a checkpoint inhibitor is presented by the synthetic bacteriophage) was administered. As expected, the group of mice treated with PBS showed no signs of antitumor activity and the experimental limits were quickly reached. 15 Mice administered with a single dose of purified anti-PD-L1 nanobodies showed a potent antitumor effect, but did not show tumor disappearance. This experimental data point demonstrates the functionality of the purified PD-L1 nanobodies. (5 × 10⁶ molecules) 8 Mice treated with 10 purified anti-PD-L1 nanobodies, and particles 10 8 The group treated with individual control bacteriophages showed moderate antitumor effects and no tumor disappearance. (5 × 10⁶ molecules) 8 Individual purified anti-PD-L1 nanobodies and 10 particles 8A group of mice treated with a combination of control bacteriophages showed improved antitumor effects, indicating that adding bacteriophages to checkpoint inhibitor treatment enhanced the effect, but no disappearance was observed. A group of mice treated with synthetic bacteriophages presenting anti-PD-L1 nanobodies showed potent antitumor activity, with four tumors disappearing in less than 10 days. (10 particles of synthetic bacteriophage presenting anti-PD-L1 nanobodies) 8 The treatment dose of 1 unit is equivalent to 8 × 10⁶ units of anti-PD-L1 nanobody alone. 15 It showed higher antitumor activity than individual molecules, which is 8 × 10⁻⁶ of the dose efficacy. 7 This corresponds to a twofold improvement. These results demonstrate that synthetic bacteriophages enhance the effects of checkpoint inhibitor molecules in unpredictable ways, regardless of whether the checkpoint inhibitor is presented directly by the bacteriophage. Furthermore, having a checkpoint inhibitor presented directly by the bacteriophage further enhances the antitumor effect in unpredictable ways compared to treatment in which the checkpoint inhibitor is administered in combination with the bacteriophage.

[0175] (Example 6) Live biopharmaceuticals secrete synthetic bacteriophages into tumors, thus exhibiting a direct antitumor effect. All bacterial strains and plasmids used in this example are listed in Table 1. Cells were typically grown in Luria Broth Mirror (LB) or Luria Broth Agar Mirror medium supplemented with the following antibiotic concentrations as needed: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were conventionally grown at 37°C for up to 18 hours before use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol detailed in Example II. A20 lymphocytes (B lymphoma cells) were ordered from ATCC (TIB-208). Throughout all experiments, cells were 2 × 10⁶ cells in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. 5 pieces / mL~2×10 6 The density was maintained between particles / mL.

[0176] Live biopharmaceuticals secreting synthetic bacteriophages that present checkpoint inhibitors can reduce the size of solid tumors. To measure the antitumor effect of live biopharmaceuticals secreting synthetic bacteriophages that present checkpoint inhibitors, developed using the process described herein, 5 × 10⁶ cells were placed in the flanks of mice. 6 Individual A20 cells were subcutaneously injected, and tumor growth was monitored daily. Next, the mice were divided into three treatment groups, and all treatments were administered when the tumor size reached 75-200 mm. 3 A single dose was administered into the tumor on day 0 of the experiment, when the target was reached. Group 1 received only PBS into the tumor (vehicle control), while Group 2 received 5 × 10⁶ doses of a control bacteriophage that does not present any checkpoint inhibitors. 8The last group was administered live biopharmaceuticals from CFUs, and the final group was given synthetic bacteriophages secreting one or more immune checkpoint inhibitors. 8 Live biopharmaceuticals from CFU were administered. Subsequently, the tumor was 1500 mm 3 Tumor size was monitored twice weekly using precise calipers until it reached or up to 24 days after treatment. Experiments were performed using various versions of synthetic bacteriophages. The first version of the system uses a synthetic bacteriophage that presents anti-CD47 nanobodies on a pIII-coated protein (as described in the previous example using pTAT003). Immediately after injection of a single dose of a live biopharmaceutical secreting anti-CD47 synthetic bacteriophage, tumor volume began to shrink. This live biopharmaceutical was able to specifically eliminate tumors within 9 days after treatment, whereas tumors treated with either PBS or a live biopharmaceutical secreting a control bacteriophage were not eliminated (Figure 15). The same experiment was performed using a live biopharmaceutical secreting a synthetic bacteriophage that presents anti-PD-L1 nanobodies on a pIII-coated protein. In this case, mice harboring A20 tumors were treated with PBS, 5 × 10⁶ 8 Unmodified CFU bacteria, 5 × 10 8 Bacteria secreting a control bacteriophage of CFU (pTAT002), or 5 × 10 8 CFUs were treated with one of the bacteria (pTAT020) secreting bacteriophages that present anti-PD-L1 nanobodies (Figures 16A-16B). Treatment with a live biopharmaceutical secreting synthetic bacteriophages presenting anti-PD-L1 nanobodies resulted in elimination in 5 out of 9 mice, demonstrating its efficacy. These data demonstrate that synthetic therapeutic bacteriophages can be delivered locally using a live biopharmaceutical approach.

[0177] Synthetic therapeutic bacteriophages can induce a complete adaptive immune response against cancer cells. To test whether treatment with synthetic bacteriophages that present anti-CD47 nanobodies, or with live biopharmaceuticals that secrete synthetic therapeutic bacteriophages that present anti-CD47 nanobodies, can induce an adaptive immune response against A20 cancer cells, mice that showed remission by these intratumoral treatments were given 5 × 10¹⁶ samples on day 46 post-treatment. 6 The mice were reloaded by injecting 5 × 10 A20 cells into their left flank (Figures 17A and 17B). As a control, naive mice were also injected with 5 × 10 A20 cells. 6 Individual A20 cells were injected into the right flank (Figure 17C). Tumor growth was monitored twice weekly in both groups to detect any tumor formation. Both treatments, either intratumoral injection of synthetic therapeutic bacteriophages or intratumoral injection of live biopharmaceuticals secreting synthetic bacteriophages, induced a complete adaptive response that prevented the formation of new tumors (Figure 17).

[0178] (Example 7) Living biopharmaceuticals secrete synthetic bacteriophages that present therapeutic enzymes with antitumor activity. Bacterial cells were typically grown in Luria broth mirror (LB) or Luria broth agar mirror (LBA) medium supplemented with the following antibiotic concentrations as needed: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were conventionally grown at 37°C for up to 18 hours before use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol described in Example 2. Bacteriophage preparations were used immediately after precipitation. A20 lymphocytes (B lymphoma cells) were ordered from ATCC (TIB-208). Throughout all experiments, cells were 2 × 10⁶ cells in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. 5 pieces / mL~2×10 6 Cells were maintained at densities between cells / mL. Cells were typically grown in Luria Broth Mirror (LB) with the following antibiotic concentrations added as needed: kanamycin (Km) 50 μg / mL, spectinomycin (Sp) 100 μg / mL. All cultures were grown conventionally at 37°C with agitation (200 rpm). Bacterial cultures older than 18 hours were not used in the experiment.

[0179] Purification of synthetic bacteriophage particles with Ni-nitrilotriacetate (Ni-NTA) beads. For the purification of synthetic bacteriophages using Ni-NTA beads, an overnight culture (20 mL) of a live biopharmaceutical secreting synthetic bacteriophage was transferred to a 50 mL tube and subsequently centrifuged at 6000 rpm for 10 minutes. Then, 18 mL of the supernatant was transferred to a new 50 mL tube and buffered with 2 mL of PBS 10X. Subsequently, 0.250 mL of Ni-NTA resin (50% slurry in PBS) was added to each tube, and all samples were incubated at 4°C for 2 hours and 30 minutes with stirring. The tubes were then centrifuged at 4000 rpm for 2 minutes, and the supernatant was removed. The beads were resuspended in 1 mL of PBS, transferred to a 1.5 mL tube, and centrifuged at 4000 rpm for 2 minutes. The supernatant was discarded, and the beads were resuspended in 1 mL of PBS. The beads, conjugated with synthetic bacteriophages, are ready for subsequent assays.

[0180] 5-fluorocytosine (5-FC) to 5-fluorouracil (5-FU) conversion assay. To measure the conversion of 5-FC to 5-FU by a synthetic bacteriophage, 250 μL of Ni-NTA beads conjugated with the synthetic bacteriophage were added to a 1.5 mL test tube, followed by 300 μL of 6 mM 5-fluorocytosine (5-FC) (12% DMSO). The tubes were then mixed and rapidly centrifuged. 50 μL of the supernatant was transferred to a spectrophotometer cuvette pre-filled with 1 mL of 0,1N HCl (used to measure the 5-FC / 5-FU ratio at T0). The sample was then resuspended by up-and-down movement and incubated at 37°C for 24 hours. In parallel, a blank solution containing 1 mL of 0.1N HCl and 50 μL of PBS / DMSO (12%) was prepared, and the OD of the blank and collected samples was read at 255 nm and 290 nm to determine the concentrations of 5-FC and 5-FU at T0. The following day, after a 24-hour incubation, the samples were rapidly centrifuged, and 50 μL of the supernatant was transferred to a spectrophotometer quartz cuvette pre-filled with 1 mL of 0.1N HCl. Subsequently, the OD of the samples was measured at 255 nm and 290 nm relative to a blank solution consisting of 1 mL of 0.1N HCl and 50 μL of PBS / DMSO (12%). Subsequently, the percentages of 5-FC and 5-FU were expressed using the formula %5-FC=[5-FC]. 24h / [5-FC] 0h =(0.119×A290 - 0.025×A255) 24h / (0.119 × A290 - 0.025 × A255) 0h , and %5-FU=[5-FU] 24h / [5-FU] 0h =(0.185×A255 - 0.049×A290) 24h / (0.185 × A255 - 0.049 × A290) 0h The calculation was performed using [this method].

[0181] 5-FU antiproliferative assay. To test the antiproliferative activity of 5-FU obtained after conversion of 5-FC by cytosine deaminase (codA), 10 units per well were used in a 96-well plate. 4A20 cells were seeded and treated three times with either a 200 μM final concentration of 5-FU conversion product, 200 μM 5-FC (control), or an equivalent volume of PBS 12% DMSO (control). The plates were then incubated at 37°C for 42 hours with 5% CO2. After the incubation period, cell viability was measured using trypan blue. Briefly, 100 μL of cell suspension was collected and mixed with 100 μL of 0.4% trypan blue. Live cells were then counted using a hemocytometer.

[0182] Live biopharmaceuticals secreting cytosine deaminase-presenting synthetic therapeutic bacteriophages convert 5-FC precursors to the chemotherapeutic agent 5-FU. Synthetic bacteriophages derived from pTAT002 (serving as a control) or pTAT022 (presenting cytosine deaminase (codA) on pIII) were purified using Ni-NTA beads and incubated with 6 mM 5-FC (12% DMSO). After 24 hours of incubation, the percentages of 5-FC and 5-FU were determined by measuring OD at 255 nm and 290 nm. Only cytosine deaminase-presenting synthetic bacteriophages were able to convert 5-FC precursors to the chemotherapeutic agent 5-FU (Figure 18), demonstrating that therapeutic enzymes can be delivered using synthetic bacteriophages produced by live biopharmaceuticals. 5-FU produced by a cytosine deaminase-presenting synthetic therapeutic bacteriophage is active and possesses antitumor activity. The antitumor activity of 5-FU produced by a cytosine deaminase-presenting synthetic bacteriophage was tested against cancer cells. A20 cancer cells were incubated for 42 hours with 200 μM 5-FU converted by a cytosine deaminase-presenting synthetic bacteriophage, an equivalent volume of the reaction mixture obtained with a control synthetic bacteriophage, an equivalent volume of the vehicle (PBS 12% DMSO), or 200 μM 5-FC. Cancer cell death was then monitored using trypan blue (Figure 19). Cancer cell death was observed only with the cytosine deaminase-presenting synthetic bacteriophage, demonstrating that this enzyme converted the 5-FC precursor to active 5-FU. Therefore, synthetic bacteriophages can be used to deliver enzymes with anticancer activity.

[0183] (Example 8) The secretion of synthetic therapeutic bacteriophages using live biopharmaceuticals can be improved by using alternative start codons. Bacterial cells were typically grown in Luria Broth Mirror (LB) or Luria Broth Agar Mirror (LBA) medium supplemented with the following antibiotic concentrations as needed: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were grown, usually at 37°C for up to 18 hours, before use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol described in Example 2. The bacteriophage preparation was used immediately after precipitation.

[0184] Titer determination of synthetic bacteriophages by enzyme-linked immunosorbent assay (ELISA). Detection and quantification of bacteriophage expression were performed using a commercially available Phage Titration ELISA kit (PRPHAGE, Progen) according to the manufacturer's instructions. In short, lyophilized M13 particles were subjected to phage titration of 1.5 × 10⁶ particles according to the manufacturer's recommendations. 8 The bacteriophage preparations were resuspended at 1 / 10, 1 / 100, 1 / 1000, and 1 / 10000 dilutions and added to ELISA wells pre-coated with mouse anti-M13 (similar to the standard curve). Captured bacteriophage particles were detected by peroxidase-conjugated monoclonal anti-M13. After the addition of tetramethylbenzidine, the optical density of each well was measured at 450 nm using a Biotek plate reader instrument. To detect the modified pIII protein on the surface of the manipulated phages, this procedure was repeated using antibody HA-Tag(6E2) mouse mAb (HRP conjugate) (1:1000 Cell Signaling Technology, Danvers, MA, USA) instead of the anti-M13-HRP provided in the kit.

[0185] Verification of fusion protein integrity by Western blotting. Bacteria were grown overnight at 37°C with agitation in LB broth supplemented with kanamycin and spectinomycin. The bacteria were pelleted by centrifugation, and the culture supernatant was transferred to a new tube and buffered with concentrated PBS. Phages displaying hexahistidine tags were pulled down from the supernatant by incubation at 4°C for 2 hours with agitation using Ni-NTA beads. The beads were then washed three times with PBS, and the phages were eluted by denaturation using sample buffer 4X (SB4X). The sample was denatured at 65°C for 1 hour and loaded onto a 15% acrylamide gel. The sample was transferred to the gel at 150 volts for 1 hour. The protein was then transferred to a 0.2 μm nitrocellulose membrane by applying 100 volts for 1 hour. The membrane was air-dried to evaporate trace amounts of methanol, and blocked at 4°C for 1 hour with agitation in TBS-0.1% Tween 20 - 4% dried milk. The membrane was transferred to a resealable Western blotting bag and incubated overnight at 4°C with anti-HA-HRP (Cell Signaling) diluted in blocking buffer under agitation. After three washes with TBS - 0.1% Tween 20, the membrane was colorimetrically exposed by applying Immobilon ECL Ultra Western HRP substrate, and images were acquired using a Vilber Fusion FX instrument. Images were processed using Image Lab software.

[0186] The secretion of synthetic bacteriophages is improved by the presence of a GTG start codon instead of an ATG start codon in the presented protein. ATG is the normal start codon for proteins and results in the highest level of translation. In some cases, overexpression of therapeutic proteins fused with bacteriophage-coating proteins can be harmful, negatively impacting the bacterial host and ultimately reducing bacteriophage secretion. GTG is an alternative start codon that results in lower levels of protein translation (Hecht et al., Nucleic Acids Research, 2017, vol. 45, no. 7, pp. 3615-3626; this is incorporated herein by reference). Furthermore, GTG as a start codon relies on fluctuations in the start tRNA, allowing translation initiation from the wrong codon. This can potentially halt ribosomes, enabling improved ribosome transport on the gene and thus producing more complete protein products. To investigate the effect of start codons on the secretion of therapeutic bacteriophages, synthetic bacteriophages presenting anti-PD-L1 nanobodies fused to pIII with either an ATG start codon (pTAT020) or a GTG start codon (pTAT020-GTG) were produced overnight, precipitated by PEG, and subsequently quantified by ELISA. The results showed that the presence of the GTG codon increased therapeutic bacteriophage secretion 100-fold (Figure 20A). Next, the integrity of the anti-PD-L1 nanobodies presented on pIII on the surface of the bacteriophages was examined by Western blotting. Both bacteriophages derived from pTAT020 and pTAT020-GTG were purified using Ni-NTA beads and released by thermal denaturation in SB4X. Subsequently, the samples were analyzed by SDS-PAGE and Western blotting using anti-HA antibodies to examine protein integrity (Figure 20B). Several bands were identified in the pTAT020 construct, with those corresponding to the complete protein product present at lower concentrations.On the one hand, the highest band (corresponding to the full-length protein product) is mostly the protein product of pTAT020-GTG, which supports that by using an alternative start codon, ribosome transport is improved, which may reduce proteolysis and maximize the secretion of therapeutic bacteriophages.

[0187] (Example 9) The synthetic bacteriophage secretion system can produce therapeutic proteins or bacteriophages presenting two or more minor coat proteins. All strains and plasmids used in this example are listed in Table 1. Cells were typically grown in Luria-Bertani (LB) broth or Luria-Bertani agar medium supplemented with the following antibiotics as needed: ampicillin (Ap) at 100 μg / mL, chloramphenicol (Cm) at 34 μg / mL, kanamycin (Km) at 50 μg / mL, nalidixic acid (Nx) at 4 μg / mL, spectinomycin (Sp) at 100 μg / mL, streptomycin (Sm) at 50 μg / mL, sulfamethoxazole (Su) at 160 μg / mL, tetracycline (Tc) at 15 μg / mL, and trimethoprim (Tm) at 32 μg / mL. All cultures were routinely grown at 37 °C for up to 18 hours before use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol shown in Example 2. A20 lymphocyte B lymphoma cells were ordered from ATCC (TIB-208). Cells were maintained at a density between 2×10 5 cells / mL and 2×10[[ID=IT]] 6 cells / mL throughout all experiments in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol.

[0188] Titer determination of synthetic bacteriophages by enzyme-linked immunosorbent assay (ELISA). Bacteriophage expression detection and quantification were performed using a commercially available Phage Titration ELISA kit (PRPHAGE, Progen) according to the manufacturer's instructions. Briefly, lyophilized M13 particles were subjected to phage titer 1,5 × 10⁶ according to the manufacturer's recommendations. 8 The bacteriophage preparations were resuspended at 1 / 10 and 1 / 100 and subsequently added to ELISA wells pre-coated with mouse anti-M13 (similar to the standard curve). Captured bacteriophage particles were detected by peroxidase-conjugated monoclonal anti-M13. After the addition of tetramethylbenzidine, the optical density of each well was measured at 450 nm using a Biotek plate reader instrument. To detect the modified pIII protein on the surface of the manipulated phages, this procedure was repeated using antibody HA-Tag(6E2) mouse mAb (HRP conjugate) (1:1000 Cell Signaling Technology, Danvers, MA, USA) instead of the anti-M13-HRP provided in the kit.

[0189] Evaluation of synthetic bacteriophage binding to PDL1 by ELISA. An ELISA assay was devised to measure the binding activity of synthetic bacteriophages to checkpoint PDL1. First, 1 × 10⁶ wells of a 96-well plate were resuspended in ice-cold PBS for 30 minutes. 5A20 cells were coated at room temperature. Next, the supernatant was gently removed by aspirate, and the cells were fixed to the plate for 10 minutes at room temperature using 10% neutral buffered formalin. Subsequently, the plate was washed once with 200 μL of PBS. Then, to prevent nonspecific binding, the plate was incubated with 200 μL of blocking buffer (PBS, 3% skim milk, 1% BSA) at room temperature for 1 hour. Blocking was stopped by removing the blocking buffer and washing the plate twice with 200 μL of TBS-T. Subsequently, 100 μL of PEG-precipitated synthetic bacteriophages, diluted with PBS 1X and presenting nanobodies (pTAT032 + pTAT035) against anti-CTLA-4 antikalin on pIX and against PDL1 on pIII, were added to the wells containing A20 cells and incubated at room temperature for 1 hour. A control without nanobodies presented against PDL1 was also performed (pTAT004 + pTAT002). Next, the plate was washed three times with 200 μL of PBS, and 100 μL of anti-FLAG-HRP (M2, Sigma-aldrich) diluted with blocking buffer (1:500) was added. Anti-FLAG-HRP (M2, Sigma-aldrich) is preferred in this case to measure only complete bacteriophages by quantifying the presence of pIX-FLAG-anticharin CTLA-4, thereby confirming that both nanobodies and anticharin are present on the same bacteriophage particle. The plate was incubated in the dark at room temperature for 1 hour, followed by five washes with TBS-T. To measure the presence of synthetic bacteriophages, 100 μL of TMB substrate solution (ThermoFisher) was added to each well, and the plate was incubated at room temperature for 15 minutes. The reaction was stopped by adding 100 μL of stop solution (0.5 M H2SO4) to each well. Absorbance was then measured at 450 nm.

[0190] Evaluation of synthetic bacteriophage binding to CTLA-4 by ELISA. An ELISA assay was devised to measure the binding activity of synthetic bacteriophages to checkpoint CTLA-4. First, a 96-well plate was coated overnight at 4°C with recombinant CTLA-4 protein (R&D Systems) diluted to 10 μg / mL in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). Subsequently, the plate was washed three times with 200 μL of TBS-T. Next, to prevent nonspecific binding, the plate was incubated with 200 μL of blocking buffer (TBS-T, 3% skim milk, 1% BSA) at room temperature for 1 hour. Blocking was stopped by removing the blocking buffer and washing the plate twice with 200 μL of TBS-T. Next, 100 μL of PEG-precipitated synthetic bacteriophage, diluted with TBS 1X and presenting nanobodies (pTAT032 + pTAT035) for anti-CTLA-4 antikalin on pIX and PDL1 on pIII, was added to wells containing CTLA-4 protein and incubated at room temperature for 1 hour. A control without antikalin presented for CTLA-4 was also performed (pTAT004 + pTAT002). Subsequently, the plate was washed three times with 200 μL of TBS-T, and 100 μL of anti-HA-HRP (Cell Signaling) diluted with blocking buffer (1:500) was added. Anti-HA-HRP (Cell Signaling) is preferred in this case to measure only complete bacteriophages by quantifying the presence of pIII-HA-nbPDL1, thereby confirming that both nanobodies and antikalin are present on the same bacteriophage particle. The plates were incubated in the dark at room temperature for 1 hour, followed by five washes with TBS-T. To measure the presence of synthetic bacteriophages, 100 μL of TMB substrate solution (ThermoFisher) was added to each well, and the plates were incubated at room temperature for 15 minutes. The reaction was stopped by adding 100 μL of stop solution (0.5 M H2SO4) to each well. Absorbance was then measured at 450 nm.

[0191] The living biopharmaceutical secretes a bispecific synthetic bacteriophage that presents anti-PDL1 nanobodies on pIII and anti-CTLA-4 on pIX, which simultaneously bind to immune checkpoints PDL1 and CTLA-4. The previous examples demonstrated that a synthetic bacteriophage secretion system can produce bacteriophage particles that bind to several molecular targets via different binding proteins. Therefore, the next step was to demonstrate that their presentations could be combined on the same bacteriophage and subsequently bound to two or more immune checkpoints. An exemplary version of the bispecific presentation system requires a bacteriophage secretion mechanism lacking both the wild-type pIII (located in the tail of the bacteriophage) and pIX (located in the head of the bacteriophage) subunits to maximize presentation efficiency. Therefore, pTAT032ΔgpIX, a plasmid lacking gpIX and presenting nanobodies to PDL1 on pIII, was used as the bacteriophage secretion mechanism. The disruption of phage secretion in pTAT032ΔgpIX due to the absence of the gpIX gene was first evaluated by an anti-pVIII ELISA assay. Plasmid pTAT032ΔgpIX produced bacteriophages at lower titers compared to the parent construct pTAT032, which supports the successful impairment of gpIX (Figure 21A). Next, we decided to test whether bacteriophage particles could present multiple functional recombinant proteins by combining pTAT032ΔgpIX (a bacteriophage mechanism lacking pIX and presenting anti-PDL1 nanobodies on pIII) with pTAT035 (where anti-CTLA-4 antikalin is presented on pIX). These plasmids were combined in MG1655 to produce bacteriophage particles, which were purified by PEG precipitation. Next, three sets of ELISAs were performed using bacteriophages that did not present proteins as controls. In the first ELISA, anti-pVIII B62-FE3 (Progen) antibody immobilized in the plate wells was used, and anti-pVIII-HRP B62-FE3 (Progen) antibody was used to determine the amount of phage produced (Figure 21A).The second ELISA was performed using A20 cells attached to the plate wells. A20 cells express PD-L1, to which bacteriophage particles derived from the dual-presentation system bind. Subsequently, the bacteriophages were exposed using an anti-pVIII-HRP B62-FE3 (Progen) antibody, which binds to pVIII and exposes bacteriophage particles attached to A20 cells (Figure 21B). The final ELISA was performed using recombinant CTLA-4 purified protein attached to the plate wells. Bacteriophages expressing CTLA-4 binding protein bind to the purified protein and are subsequently exposed using an anti-HA-HRP (Cell Signaling) antibody that binds to the pIII-HA-PD-L1 nanobody. This reveals only complete bacteriophage particles that bind to the CTLA-4 protein and also present the PD-L1 nanobody (Figure 21C). Both the control and the bacteriophage particles derived from the combination of pTAT032ΔgpIX (a bacteriophage mechanism lacking pIX and presenting an anti-PDL1 nanobody on pIII) and pTAT035 (presenting an anti-CTLA-4 anticharin on pIX) produced bacteriophage particles; however, only the latter was able to produce bacteriophages that bound to both CTLA-4 and PDL1. Taken together, these results indicate that bacteriophages can simultaneously present several functional therapeutic proteins on the same bacteriophage particle.

[0192] The living biopharmaceutical secretes a bispecific synthetic bacteriophage that presents anti-PD-L1 and anti-CTLA-4 nanobodies on pIII, simultaneously binding to the immune checkpoints PD-L1 and CTLA-4. The synthetic therapeutic bacteriophage particles can present a mixture of different therapeutic proteins on the same coating protein. To illustrate this, plasmid pTAT032 (presenting nanobodies for PD-L1 on pIII) and plasmid pTAT019 (presenting nanobodies for CTLA-4 on pIII) were combined in the same bacterium. The resulting cells secrete a synthetic therapeutic bacteriophage capable of presenting both PD-L1 and CTLA-4 nanobodies on pIII. To test this hypothesis, the initial ELISA was performed on A20 cells, as A20 cells express PD-L1, to which bacteriophage particles derived from the bispecific presentation system are thought to bind. Bacteriophages were identified using an anti-pVIII-HRP B62-FE3 (Progen) antibody, which identifies bacteriophage particles bound to pVIII-coated proteins and attached to A20 cells (Figure 22A). A second ELISA was performed on recombinant CTLA-4 protein. Bacteriophages expressing anti-CTLA-4 nanobodies bound to purified protein were subsequently identified using an anti-pVIII-HRP (Cell Signaling) antibody, which identifies complete bacteriophage particles bound to CTLA-4 protein (Figure 22B). As controls, bacteriophages presenting only nanobodies for PD-L1 (pTAT032) or only nanobodies for CTLA-4 (pTAT019) were also evaluated. As expected, these controls produced strong binding signals only when tested against their corresponding targets.In contrast, a strain (pTAT032 + pTAT019-GTG) presenting both anti-PD-L1 and anti-CTLA-4 nanobodies on pIII was able to bind to the PD-L1 and CTLA-4 targets in both ELISA tests, supporting the possibility of constructing bispecific synthetic bacteriophages using a mixture of pIII-coated proteins presenting multiple different nanobodies.

[0193] Embedding by reference All references cited herein, and such references in whole, are incorporated herein by reference where appropriate for teaching additional or alternative details, features, and / or technical background.

[0194] Equal portions While this disclosure has been described and illustrated with reference to specific embodiments, it will be understood that variations or substitutes of the features and functions disclosed above, as well as other features and functions, may preferably be combined with many other different systems or applications. Furthermore, various substitutes, modifications, changes or improvements not currently foreseen or anticipated may subsequently be made by those skilled in the art, and these too are intended to be included within the scope of the following embodiments.

Claims

1. A non-infectious synthetic therapeutic bacteriophage presenting at least a first therapeutic agent, wherein the first therapeutic agent is (a) a binding agent to one or more proteins, peptides, or molecules involved in carcinogenesis, cancer development, or metastasis; (b) an agonist that activates a costimulatory receptor leading to the elimination of cancer cells; and / or (c) a binding protein that inhibits an immune checkpoint molecule, wherein at least one therapeutic agent is fused to the C-terminal fragment of the coating protein pIII of the synthetic bacteriophage and does not present wild-type coating protein pIII.

2. The synthetic therapeutic bacteriophage according to claim 1, wherein the bacteriophage is fibrous.

3. The synthetic therapeutic bacteriophage according to claim 2, wherein the fibrous bacteriophage is M13.

4. A synthetic therapeutic bacteriophage according to any one of claims 1 to 3, further presenting a second therapeutic agent.

5. The synthetic therapeutic bacteriophage according to claim 4, wherein the second therapeutic agent is presented in a coated protein that does not present the first therapeutic agent.

6. The synthetic therapeutic bacteriophage according to claim 5, wherein the second therapeutic agent is presented in a pVIII or pIX coated protein.

7. The synthetic therapeutic bacteriophage according to claim 6, wherein the second therapeutic agent is presented in a pIX-coated protein.

8. The synthetic therapeutic bacteriophage according to any one of claims 1 to 7, wherein the first therapeutic agent is a binding protein that binds to one or more proteins, peptides, or molecules involved in carcinogenesis, cancer development, or metastasis.

9. A synthetic therapeutic bacteriophage according to claim 8, wherein one or more proteins, peptides, or molecules involved in carcinogenesis, cancer development, or metastasis are CSF1, CSF1R, CCR4, CCL2, CCL17, CCL22, HER2, GD2, IL-1β, IL-6, IL-10, IL-13, IL-17, IL-27, IL-35, CD20, CD27, CD30, CD33, CD70, TGF-β, M-CSF, EGFR, ERBB2, ERBB3, PGE2, VEGF, VEGFR-2, CXCR4 / CXCL12, Tie2, galectin-1, galectin-3, phosphatidylserine, and TAM and Tim phosphatidylserine receptors.

10. The synthetic therapeutic bacteriophage according to any one of claims 1 to 7, wherein the first therapeutic agent is a binding protein that acts as an agonist that activates a costimulatory receptor that leads to the elimination of cancer cells.

11. The synthetic therapeutic bacteriophage according to claim 10, wherein one or more costimulatory cell receptors are selected from CD40, CD27, CD28, CD70, ICOS, CD357, CD226, CD137, and CD134.

12. The synthetic therapeutic bacteriophage according to any one of claims 1 to 7, wherein the first therapeutic agent is a binding protein that inhibits an immune checkpoint molecule.

13. The synthetic therapeutic bacteriophage according to claim 12, wherein the immune checkpoint molecule is selected from CCR4, CTLA-4, CD80, CD86, PD-1, PD-L1, PD-L2, TIGIT, VISTA, LAG-3, TIM1, TIM3, CEACAM1, LAIR-1, HVEM, BTLA, CD47, SIRPα, CD160, CD200, CD200R, CD39, CD73, B7-H3, B7-H4, IDO, TDO, KIR, and A2aR.

14. The synthetic therapeutic bacteriophage according to claim 13, wherein the immune checkpoint molecule is CD47, PD-L1, or CTLA-4.

15. A synthetic therapeutic bacteriophage according to any one of claims 1 to 14, wherein the binding protein is an antibody, antibody fragment, antibody mimetic, nanobody, or antikalin.

16. The synthetic therapeutic bacteriophage according to claim 15, wherein the first therapeutic agent is an antibody, antibody fragment, or nanobody that binds to PD-L1.

17. A composition comprising a synthetic therapeutic bacteriophage according to any one of claims 1 to 16 for delivering at least a first therapeutic agent to a tumor site in a target.

18. A composition comprising a synthetic therapeutic bacteriophage according to any one of claims 1 to 16 for the prevention and / or treatment of cancer in a subject.

19. Cancers include adrenal cancer, adrenocortical carcinoma, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone malignancies, brain malignancies, bronchial tumors, central nervous system tumors, breast cancer, Castleman disease, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye malignancies, gallbladder cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, cardiac malignancies, Kaposi's sarcoma, kidney cancer, pharyngeal cancer, hypopharyngeal cancer, leukemia, liver cancer, lung cancer, lymphoma, malignant mesothelioma, multiple myeloma, and myelodysplasia. The composition according to claim 18, selected from syndromes, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma, skin cancer, small intestine cancer, gastric cancer, teratoma, testicular cancer, pharyngeal cancer, thymic cancer, thyroid cancer, rare childhood cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenström macroglobulinemia, and Wilms' tumor.

20. The composition according to claim 18 or 19, which is administered intratumorally or peritumorally.

21. The composition according to any one of claims 18 to 20, which is administered in combination with a checkpoint inhibitor.

22. The composition according to claim 21, wherein the checkpoint inhibitor is an anti-PD-1 antibody.

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