AU2023405587A1 - Therapeutic bacteriophage displaying cancer cell targeting moieties along with cytokines for the treatment of cancer - Google Patents

Abstract

The present technology generally relates to a bacteriophage simultaneously displaying at least one cytokine and at least one cancer cell targeting moiety and to methods of treating a cancer using the bacteriophage.

Description

THERAPEUTIC BACTERIOPHAGE DISPLAYING CANCER CELL TARGETING MOIETIES ALONG WITH CYTOKINES FOR THE TREATMENT OF CANCER

FIELD OF TECHNOLOGY

[001] The present technology generally relates to a bacteriophage engineered to display cancer cell targeting moieties and cytokines. The present technology also generally relates to the use of such bacteriophage in the treatment of cancers.

BACKGROUND INFORMATION

[002] Despite important advances in cancer treatments, cancer remains the second most prevalent cause of death in industrialized countries (Siegel R. et al. ACS Journal, Cancer statistics 2021; incorporated herein by reference). Cancer is a complex and difficult to treat disease, often requiring acting on several therapeutic targets simultaneously to maximize chances of treatment success. This strategy is called combination therapy, where clinicians treat patients by combining two or more therapeutic agents (Mokhtari et al. Oncotarget 2017 Jun 6;8(23):38022-38043; incorporated herein by reference). By targeting different pathways to inhibit or eliminate cancer cells, combination therapy provides better results than monotherapy via synergistic effects and has become a cornerstone of cancer treatment. However, combination therapy suffers from an important limitation: by multiplying treatments, combination therapy also multiplies the costs of care and risks of side effects.

[003] A solution to this problem would be a therapeutic modality capable of multitasking therapeutic activities, allowing combination therapy with a single agent. To solve this issue, a bacteriophage capable of multitasking therapeutic activities has been developed to provide next-generation immunotherapies for the treatment of cancers (WO2022073127; incorporated herein by reference). This bacteriophage acts as a therapeutic scaffold to display a combination of immunomodulatory agents. Because the bacteriophage can multitask therapeutic activities, its antitumor activity is thus coupled to the combinations of therapeutic molecules it carries and the therapeutic synergies that can emerge from these combinations.

[004] In view of the above, finding combinations of therapeutic molecules that synergize, when displayed on the bacteriophage, is key to potentiating the antitumor activities of the treatment.

SUMMARY OF TECHNOLOGY

[005] According to various aspects, the present technology relates to a bacteriophage simultaneously displaying at least one cytokine and at least one cancer cell targeting moiety. In some instances, the bacteriophage of the present technology is a synthetic bacteriophage. In some instances, the bacteriophage of the present technology is a therapeutic bacteriophage. In some instances, the bacteriophage of the present technology is a synthetic therapeutic bacteriophage. In some instances, the cytokine is selected from: IL-la, IL- lb, IL-lra, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, [006] According to various aspects, the present technology relates to a method for reducing tumor size in a subject, the method comprising administering a therapeutically effective amount of the bacteriophage as defined herein to the subject.

[007] According to various aspects, the present technology relates to a method for treating cancer in a subject, the method comprising administering a therapeutically effective amount of the bacteriophage as defined herein to the subject.

[008] According to various aspects, the present technology relates to a pharmaceutical composition comprising a bacteriophage simultaneously displaying cytokines and cancer cell targeting moiety, together with a suitable pharmaceutical carrier.

[009] According to various aspects, the present technology relates to a method for reducing tumor size in a subject, the method comprising administering the pharmaceutical composition as defined herein to the subject.

[010] According to various aspects, the present technology relates to a method for treating a cancer in a subject, the method comprising administering the pharmaceutical composition as defined herein to the subject.

[Oi l] According to various aspects, the present technology relates to a composition comprising a bacteriophage displaying cytokines and moieties targeting cancer cells.

[012] According to various aspects, the present technology relates to a bacteriophage, wherein the bacteriophage displays one or more therapeutic agents targeting cancer cell markers on one or more of its coating proteins, and wherein the bacteriophage displays one or more cytokines on one or more of its coating proteins.

[013] According to various aspects, the present technology relates to a method for the treatment of cancer in a subject in need thereof, the method comprising administering an effective amount of the bacteriophage as defined herein to the subject in need thereof.

[014] According to various aspects, the present technology relates to the use of an effective amount of the bacteriophage as defined herein for treatment of a cancer in a subject in need thereof.

[015] According to various aspects, the present technology relates to the use of an effective amount of the bacteriophage as defined herein in the manufacture of a medicament for the treatment of cancer in a subject.

[016] According to various aspects, the present technology relates to a kit comprising the bacteriophage as defined herein together with instructions for administration of the drug to a subject.

BRIEF DESCRIPTION OF THE DRAWINGS

[017] Figures 1A, IB and 1C are schematic representations of bacteriophage production and vectors overview according to one embodiment of the present technology. Figure 1A is a schematic representation of an exemplified configuration of the bacterial production strain secreting a bacteriophage. The bacteriophage secretion system can be composed of a bacteriophage machinery vector combined with a bacteriophage scaffold vector. Figure IB illustrates examples of bacteriophage machinery vectors: M13K07, pTAT004, and pTAT025. Figure 1C shows examples of bacteriophage scaffold vectors: pTAT002, pTAT013 (including derivatives pTAT044 and pTAT070), and pTAT014 (including derivatives pTAT017, pTAT060, and pTAT071).

[018] Figure 2 is a schematic representation of a mode of action for a bacteriophage displaying an anti- PD-L1 checkpoint inhibitor scFv, as a cancer-cell targeting moiety, and IL-2, as immunomodulatory cytokine. The bacteriophage binds to cancer cells via the anti-PD-Ll scFv displayed on pill, which localizes the bacteriophage to the cancer cells and inhibits the PD-L1 checkpoint to potentiate T-cell activation. The presence of the IL-2 cytokine coupled to the bacteriophage synergizes to further potentiate the antitumor immune response and improve the clearance of tumors. Parallel to this, the filamentous bacteriophages can be internalized by the cancer cells via endocytosis providing additional natural immunogenic activities via (1) the activation of TLR9, which triggers the immune response, and (2) antigens from the bacteriophage can be presented by MHC molecules leading to the activation of cytotoxic T-cells and the subsequent elimination of cells presenting these antigens.

[020] Figure 4 is a graph showing that bacteriophages can display biologically active cytokines. The graph presents the signal measured at 630nm from an HEK-Blue™ IL-2 reporter cell assay quantifying the activation of the IL-2 receptor by either: PBS (vehicle), 10¹¹ bacteriophages displaying an anti-PD-Ll scFv (phage-PD-Ll), 10¹¹ bacteriophages displaying an anti-PD-Ll scFv and mouse IL-2 (mIL-2) cytokine (mlL- 2-phage-PD-Ll), or 0.2 ng of mIL-2.

[021] Figure 5 are microscopy images showing bacteriophages displaying a cytokine and a cancer cell targeting moiety can bind to cancer cells. PD-L1⁺ A20 cancer cells were exposed to phage (not displaying a cancer targeting moiety) or to the bacteriophage displaying the mIL-2 cytokine and the anti-PD-Ll scFv (mIL-2-phage-PD-Ll). The binding of the phage and mIL-2-phage-PD-Ll was then revealed using an anti- M13 antibody coupled to FITC.

[022] Figure 6 is a graph showing physical coupling of IL-2 cytokine to a cancer cell targeting bacteriophage to provides a synergistic antitumor effect. Tumors were engrafted in mice by injecting 10⁶ CT26 cancer cells in their right flank. Treatments were administered when tumor volumes were comprised between 50-80 mm³. Individual tumor volume was then measured for each mouse. Treatments were performed on days 0, 4, and 7 with intratumoral injection of either PBS, 5xl0ⁿ molecules of mouse IL-2 (mIL-2), 10¹¹ particles of bacteriophage displaying an anti-PD-Ll scFv (phage-PD-Ll), 5x 10' molecules of mIL-2 in conjunction with 10¹¹ particles of phage-PD-Ll, or 10¹¹ particles of bacteriophage displaying the mIL-2 cytokine and the anti-PD-Ll scFv (mIL-2-phage-PD-Ll). Tumor volume was calculated by multiplying the largest measure by the square of the perpendicular measure divided by two (solid lines = cleared mice, doted lines = non-cleared mice). [023] Figure 7 is a graph showing that bacteriophages displaying a cytokine and a cancer cell targeting moiety have systemic anti-tumoral activity. Tumors were engrafted in both flanks of mice. 5xl0⁶ A20 cancer cells were injected in the right flanks, and 4 days later, 5xl0⁶ A20 cancer cells were injected in the left flanks. Right tumors were treated when tumor volumes were comprised between 50-100 mm³, left tumors were not treated. Treatments were performed on days 0, 4, and 7 with intratumoral injection of either PBS or 10¹² particles of bacteriophage displaying the mIL-2 cytokine and the anti-PD-Ll scFv (mIL-2-phage- PD-L1). Tumor volumes of the injected tumor and non-injected tumors were calculated by multiplying the largest measure by the square of the perpendicular measure divided by two. Tumors cleared for injected and non-injected tumors are indicated for both treatments.

[024] Figure 8 is a heatmap showing that the antitumor activity of bacteriophages displaying a cytokine and a cancer cell targeting moiety is mediated by an immune response. Bacteriophages displaying the mlL- 2 cytokine and the anti-PD-Ll scFv (mIL-2-phage-PD-Ll) activate all major pathways of immunity. 75- 150 mm³ A20 tumors were extracted from BALB/c mice, micro-dissected, and cultured ex-vivo on a chip. Micro-dissected tumors were then treated with, PBS (control), mIL-2-phage-PD-Ll, or the anti-PD-Ll checkpoint inhibitor Atezolizumab (benchmarking reference). Fold change cytokine levels induced by the treatments were assessed. Cytokine fold changes against the corresponding PBS condition are shown on a log scale heatmap.

[025] Figure 9A and 9B are respectively a graph and histology images showing that the antitumor activity of bacteriophages displaying a cytokine and a cancer cell targeting moiety is mediated by a massive immune infiltration of the tumor. BALB/c mice bearing A20 tumors received an intratumoral administration on days 0, 4 and 7 of either PBS or the bacteriophage displaying the mIL-2 cytokine and the anti-PD-Ll scFv (mIL-2-phage-PD-Ll). On day 8 of the experiment, mice were sacrificed, and tumors were extracted and processed for histology. The images of tumors were analyzed using Qpath to identify and distinguish cells within the tissues. Using a machine learning approach, tumor and immune cells were detected within the tumor and quantified automatically across the different samples (Figure 9A). Representative images of tumors treated with PBS or mIL-2-phage-PD-Ll are presented (Figure 9B). Both images share the same scale and tissue damage, as well as tumor shrinkage, can be observed. Pale sections of the tissue, which can be seen on a large portion of the mIL-2-phage-PD-Ll treated tumor, denote the presence of necrosis.

[026] Figure 10 is a graph showing that the therapeutic activity of bacteriophages displaying a cytokine and a cancer cell targeting moiety is mediated by a long-term adaptive and systemic antitumor immune response. Tumors were engrafted by injecting 5xl0⁶ A20 cancer cells in the right flanks of mice. Tumors were then treated when their volume was comprised between 80-100 mm³. Treatments were performed on days 0, 4, and 7 with intratumoral injection of 10¹¹ particles of bacteriophage displaying the mIL-2 cytokine and the anti-PD-Ll scFv (mIL-2-phage-PD-Ll). Mice for which tumors were completely cleared were considered cured and were kept for 160 days. At day 160, 5xl0⁶ A20 cancer cells were injected to form new tumors, but this time in the left flanks of mice. The same was also done to naive mice, which were never exposed to A20 cancer cells and never received a mIL-2-phage-PD-Lltreatment. Tumor volumes were then measured for each mouse. Tumor volumes were calculated by multiplying the largest measure by the square of the perpendicular measure divided by two.

DETAILED DESCRIPTION OF EMBODIMENTS

[027] As used herein, the singular form “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise. [028] The recitation herein of numerical ranges by endpoints is intended to include all numbers subsumed within that range (e.g., a recitation of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[029] The term “about” is used herein explicitly or not, every quantity given herein is meant to refer to the actual given value, and it is also meant to refer to the approximation to such given value that would reasonably be inferred based on the ordinary skill in the art, including equivalents and approximations due to the experimental and/or measurement conditions for such given value. For example, the term “about” in the context of a given value or range refers to a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range.

[030] The expression “and/or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example, “A and/or B” is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein.

[031] An “inducible promoter” refers to a regulatory region that is operably linked to one or more genes, wherein expression of the gene(s) is increased in the presence of an inducer of said regulatory region or increased in the absence of a repressor of said regulatory region. An inducible promoter can be induced by exogenous environmental condition(s), which refers to setting(s) or circumstance(s) under which the promoter described herein is induced. Exogenous environmental conditions refer to the environmental conditions external to the intact (unlysed) engineered microorganism, endogenous or native to tumor environment, or the host subject environment, or to exogenously introduced perturbations to the environment. Exogenous environmental conditions can be, and are not limited to, low oxygen, microaerobic, or anaerobic conditions, or low intracellular and/or extracellular pH, wavelength, levels of reactive oxygen species (ROS), presence of specific molecules and temperature. Examples of oxygen level-dependent transcription factors include, but are not limited to, Fnr (fumarate and nitrate reductase), Anr (anaerobic nitrate respiration), and Dnr (dissimilatory nitrate respiration regulator). Corresponding Fnr responsive promoters, Anr (anaerobic nitrate respiration)-responsive promoters, and Dnr (dissimilatory nitrate respiration regulator)-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003; incorporated herein by reference). Examples of pH-dependent transcription factors include, but are not limited to, phoBR-responsive promoter. Examples of thermoregulated promoter include, but is not limited to, the pL and/or pR phage 1 promoters and the using the mutant cI857 repressor. Examples of ROS level-dependent transcription factor includes, but is not limited to, OxyR. Corresponding OxyR responsive promoters include, but are not limited to, TrxCp, HemHp, sufA, AhpCplOOO, AhpCp2Dl, AhpCp2, AhpCpDl, AhpCpl, DsbGp. An inducible promoter can also be induced by one, or more, exogenous molecule(s). Exogenous molecules refer to molecules which are not naturally present in the intact (unlysed) engineered microorganism. Examples of exogenous molecules and their respective inducible promoter include, but are not limited to, L-arabinose and the ParaBAD promoter, rhamnose and the rhaP BAD promoter, IPTG and the Lac promoter, the tetracycline-inducible system (Tet on-Tet off). An inducible promoter can also be induced by one, or more, endogenous molecule(s). Endogenous molecules refer to molecules which are naturally produced in the intact (unlysed) engineered microorganism. Examples of endogenous molecules and their respective inducible promoter include, but are not limited to, diaminopimelic acid and the PdapA promoter, N-acyl-homoserine lactone and the Pluxl.

[032] Inducible promoters can comprise one or more regulatory elements, which include, but are not limited to, enhancer sequences, response elements, protein recognition sites, inducible elements, promoter control elements, protein binding sequences, 5' and 3' untranslated regions, transcriptional start sites, termination sequences, polyadenylation sequences, riboswitches and introns.

[033] The present technology is explained in greater detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology may be implemented, or all the features that may be added to the instant technology. For example, features illustrated with respect to one embodiment may be incorporated into other embodiments, and features illustrated with respect to a particular embodiment may be deleted from that embodiment. In addition, numerous variations and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of the instant disclosure which variations and additions do not depart from the present technology. Hence, the following description is intended to illustrate some particular embodiments of the technology, and not to exhaustively specify all permutations, combinations and variations thereof.

Components of the bacteriophages

[034] A solution to treat cancers, by acting on several therapeutic targets simultaneously, is to use a molecular scaffold capable of coupling several immunomodulatory molecules to mount a potent antitumor immune response. Filamentous bacteriophages are large immunogenic biological structures upon which therapeutic proteins or peptides can be displayed to create synthetic therapeutic bacteriophages. The natural immunogenicity of filamentous bacteriophages is mediated by (1) their genome, which contains CpGs, can act as a TLR9 agonist and trigger the innate immune response (Sartorius et al., NPJ Vaccines, 2021 Oct 28;6(1): 127; incorporated herein by reference), and (2) by the presence of immunogenic antigens in their coating proteins, which can stimulate the elimination, by cytotoxic T-cells, of cells presenting these antigens on their MHC- 1 following endocytosis of the bacteriophage (Gaubin et al., DNA And Cell Biology, Volume 22, Number 1, 2003; incorporated herein by reference). The combination of the filamentous bacteriophage’s natural immunogenic activities with specific immunomodulatory proteins, or peptides, could thus be leveraged to potentiate its antitumor activity.

[035] According to various embodiments, the present technology relates to an operable bacteriophage in the treatment of cancers, such as the one described in W02022073127 (incorporated herein by reference). In some instances, the bacteriophage is immunogenic. In some further instances, the bacteriophage can display a mono- or multi-specific therapeutic proteins.

[036] In some embodiments, the present technology relates to a bacteriophage displaying one or more cancer cells targeting moieties along with one or more cytokines, and wherein the combination of these therapeutic molecules synergies to potentiate tumor elimination.

[037] In some embodiments, the present technology relates to a bacteriophage displaying one or more cancer cells targeting moieties along with one or more cytokines, and wherein the combination of these therapeutic molecules synergies to potentiate tumor elimination only when physically coupled to the bacteriophage.

Production of the bacteriophage

[038] In some embodiments, the bacteriophage of the present technology is produced as described in WO2022073127 (incorporated herein by reference) and on Figure 1.

Therapeutic activities of the bacteriophage Synthetic phages displaying cancer cells targeting moieties:

[039] In some embodiments, the bacteriophage of the present technology displays one or more cancer cells targeting moieties that are capable of recognizing one or more cancer cell markers. The type of targeting moieties displayed by the bacteriophage are disclosed in W02022073127 (incorporated herein by reference) and can be selected from, but not limited to: antibodies, antibody mimetics, natural receptor and ligands, and peptides. In some embodiments, the one or more targeting moieties can be displayed on pill, pVI, pVII, pVIII, and/or pIX (as depicted in WO2022073127 Figure 2; incorporated herein by reference).

[040] In some embodiments, the cancer cell marker recognized by the targeting moiety is a molecule present on cancer cells.

[041] In another embodiment the cancer cell marker is a molecule overexpressed by cancers cells.

[042] In yet another embodiment the cancer marker is a molecule specifically expressed by cancer cells.

[043] In some embodiments, the cancer cell markers targeted by the one or more targeting moieties are proteins.

[044] In another embodiment, the cancer cell marker targeted by the one or more targeting moieties is selected from, but not limited to: Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD59, CD24, E-Cadherin, cMet, MUC1, and CD133. In an embodiment, the cancer marker targeted by the one or more targeting moieties is PD-L1.

Synthetic phages displaying a cytokine:

[045] In some embodiments, the bacteriophage of the present technology displays a tumor targeting moiety and one or more cytokines (Figure 2). Cytokines are molecules that can modulate the immune response by stimulating and/or inducing the differentiation of T effector cells, e.g., CD4+ and/or CD8+, promoting the activation of B-cells, macrophages, and/or dendritic cells. In some embodiments, the one or more cytokines can be displayed on pill, pVI, pVII, pVIII, and/or pIX (as depicted in WO2022073127 Figure 2; incorporated herein by reference).

[046] In some embodiments, the one or more cytokines that stimulate the immune response can be any known cytokines that stimulate and/or induce the differentiation, and/or activation, of T-cells, B-cells, macrophages, and dendritic cells.

Therapeutic uses of synthetic phages and compositions comprising same Methods of treatment

[048] In some embodiments, the synthetic therapeutic phages of the present technology can be used for, but is not limited to, the treatment of cancer and/or tumors. A tumor may be malignant or benign. Types of cancer include, but are not limited to, adrenal cancer, adrenocortical carcinoma, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma tumors, osteosarcoma, malignant fibrous histiocytoma), brain cancer (e.g., astrocytomas, brain stem 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, eye cancer, gallbladder cancer, gastrointestinal cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors, gestational trophoblastic disease, heart cancer, Kaposi sarcoma, kidney cancer, largyngeal cancer, hypopharyngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myelogenous leukemia), liver cancer, lung cancer, lymphoma (e.g., AIDS- related 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 cavity cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumors, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma, skin cancer (e.g., basal cell carcinoma, melanoma), small intestine cancer, stomach cancer, teratoid tumor, testicular cancer, throat cancer, thymus cancer, thyroid cancer, unusual childhood cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macrogloblulinemia, and Wilms tumor. In some embodiments, the symptom(s) associated thereof include, but are not limited to, anemia, loss of appetite, irritation of bladder lining, bleeding and bruising (thrombocytopenia), changes in taste or smell, constipation, diarrhea, dry mouth, dysphagia, edema, fatigue, hair loss (alopecia), infection, infertility, lymphedema, mouth sores, nausea, pain, peripheral neuropathy, tooth decay, urinary tract infections, and/or problems with memory and concentration.

[049] In some embodiments, the method may comprise preparing a pharmaceutical composition with at least one bacteriophage described herein and administering the pharmaceutical composition to a subject in a therapeutically effective amount. The bacteriophage may be administered locally, e.g., intratumorally, or peritumorally into a tissue or supplying vessel, intramuscularly, intraperitoneally, orally, or topically. The bacteriophage may be administered systemically, e.g., intravenously, or intra-arterially, by infusion or injection.

[050] In certain embodiments, administering the pharmaceutical composition to the subject reduces cell proliferation, tumor growth, and/or tumor volume in a subject. In some instances, the methods of the present disclosure may 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%, or 95%, or more as compared to levels in an untreated or control subject. In some embodiments, reduction is measured by comparing cell proliferation, tumor growth, and/or tumor volume in a subject before and after administration of the pharmaceutical composition. In some embodiments, the method of treating or ameliorating a cancer in a subject allows one or more symptoms of the cancer to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or more.

[051] In certain embodiments, administering the pharmaceutical composition of the present technology to the subject reduces cell proliferation, tumor growth, and/or tumor volume of the treated tumor and nontreated tumor via abscopal effect and a systemic immune response. [052] In some instances, the methods of the present disclosure reduce cell proliferation, tumor growth, and/or tumor volume of both the treated and non-treated tumors by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95%, or more as compared to levels in an untreated or control subject. In some embodiments, reduction is measured by comparing cell proliferation, tumor growth, and/or tumor volume in a subject before and after administration of the pharmaceutical composition.

[053] In some embodiments, the method of treating or ameliorating a cancer of the present technology allows one or more symptoms of the cancer to improve by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95%, or more.

[054] In certain embodiments, the methods of the present technology trigger a long-term systemic antitumor immune response in a subject providing protection against tumor recurrence.

[055] Before, during, and after the administration of the pharmaceutical composition of the present technology, cancerous cells and/or biomarkers in a subject may be measured in a biological sample, such as blood, serum, plasma, urine, peritoneal fluid, and/or a biopsy from a tissue or organ.

[056] In some embodiments, the methods may include administration of the compositions of the present technology to reduce 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 subject's tumor volume prior to treatment. In other embodiments, the methods may include administration of the compositions of the present technology to reduce the cell proliferation rate or tumor growth rate in a subject to an undetectable rate, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the rate prior to treatment.

[057] Therapy with synthetic therapeutic bacteriophages, which has an immune-based anti-cancer activity, might present response patterns different from those observed with traditional cytotoxic therapies. For example, tumors treated with immune-based therapies may enlarge before they regress, and/or new lesions may appear (Agarwala et al., 2015; incorporated herein by reference). Increased tumor size may be due to heavy infiltration with lymphocytes and macrophages that are normally not present in tumor tissue. Additionally, response times may be slower than response times associated with standard therapies, e.g., cytotoxic therapies. In some embodiments, delivery of the anti-cancer molecule may modulate the growth of a subject's tumor and/or ameliorate the symptoms of a cancer while temporarily increasing the volume and/or size of the tumor.

[058] The bacteriophage may be destroyed, e.g., by defense factors in tissues or blood serum (Sonnenbom et al., Microbioal Ecology in Health and Diseases, 2019, 21:3; incorporated herein by reference) several hours or days after administration. Thus, the pharmaceutical composition comprising the bacteriophage may be re-administered at a therapeutically effective dose and frequency.

[059] The pharmaceutical composition may be administered alone or in combination with one or more additional 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 therapies, tumor infiltrating lymphocyte therapies, native or engineered cell therapies (e.g. TCR or CAR-T), natural killer cell therapies, Fc-mediated ADCC therapies, therapies using bispecific soluble scFvs linking cytotoxic T cells to tumor cells, and soluble TCRs with effector functions), stem cell therapies, and targeted therapies with antibodies or chemical compounds (e.g., BRAF or vascular endothelial growth factor inhibitors), bacteriophages. [060] In some embodiments, the bacteriophage may be administered sequentially, simultaneously, or subsequently to dosing with 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®.

[061] In some embodiments, the bacteriophage is administered sequentially, simultaneously, or subsequently to dosing with one or more mRNA-based drugs.

[062] In some embodiments, the bacteriophage is administered sequentially, simultaneously, or subsequently to dosing with one or more of the following checkpoint inhibitors or other antibodies known in the art or described herein. Nonlimiting examples include CTLA-4 antibodies (including but not limited to Ipilimumab and Tremelimumab (CP675206)), anti-4-lBB (CD 137, TNFRSF9) antibodies (including but not limited to PF-05082566, and Urelumab), anti CD134 (0X40) antibodies, including but not limited to Anti-OX40 antibody (Providence Health and Services), anti-PDl antibodies (including but not limited to Nivolumab, Pidilizumab, Pembrolizumab (MK- 3475/SCH900475, lambrolizumab, REGN2810, PD1 (Agenus)), anti-PD-Ll antibodies (including but not limited to Durvalumab (MEDI4736), Avelumab (MSB0010718C), and Atezolizumab (MPDL3280A, RG7446, R05541267)), andit-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 Varlilumab), anti- CXCR4 antibodies (including but not limited to Ulocuplumab).

[063] In some embodiments, the bacteriophage is administered sequentially, simultaneously, or subsequently to dosing with one or more antibodies selected from an antiphosphatidyl serine antibody (including but not limited to Bavituxumab), TLR9 antibody (including, but not limited to, MGN1703 PD1 antibody (including, but not limited to, SHR-1210 (Incyte/Jiangsu Hengrui)), anti -0X40 antibody (including, but not limited to, 0X40 (Agenus)), anti-Tim3 antibody (including, but not limited to, Anti- Tim3 (Agenus/INcyte)), anti-Lag3 antibody (including, but not limited to, Anti-Lag3 (Agenus/INcyte)), anti-B7H3 antibody (including, but not limited to, Enoblituzumab (MGA-271), anti- CT-011 (hBAT, hBATl) as described in W02009101611 (incorporated herein by reference), anti-PDL-2 antibody (including, but not limited to, AMP-224 (described in WO2010027827 and WO201 1066342; 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).

[064] The dosage of the pharmaceutical composition and the frequency of administration may be selected based on the severity of the symptoms and the progression of the cancer. The appropriate therapeutically effective dose and/or frequency of administration can be selected by a treating clinic Pharmaceutical Compositions and Formulations

Pharmaceutical Compositions and Formulations

[065] Pharmaceutical compositions comprising the bacteriophage of the present technology may be used to treat, manage, ameliorate, and/or prevent cancer. Pharmaceutical compositions of the present technology comprising one or more bacteriophage of the present technology alone or in combination with prophylactic agents, therapeutic agents, and/or pharmaceutically acceptable carriers are provided.

[066] In certain embodiments, the pharmaceutical composition comprises the bacteriophage of the present technology that are each engineered to display the recombinant protein described herein, e.g., one or more anti-cancer molecules. [067] The pharmaceutical compositions of the present technology may be formulated in a conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the active ingredients into compositions for pharmaceutical use. Methods of formulating pharmaceutical compositions are known in the art (see, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical compositions are subjected to tableting, lyophilizing, direct compression, conventional mixing, dissolving, granulating, levigating, emulsifying, encapsulating, entrapping, or spray drying to form tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. Appropriate formulation depends on the route of administration.

[068] The bacteriophage may be formulated into pharmaceutical compositions in any suitable dosage form (e.g., liquids, capsules, sachet, hard capsules, soft capsules, tablets, enteric coated tablets, suspension powders, granules, or matrix sustained release formations for oral administration) and for any suitable type of administration (e.g., oral, topical, injectable, intravenous, sub-cutaneous, intratumoral, peritumor, immediate release, pulsatile-release, delayed-release, or sustained release). Suitable dosage amounts for the bacteriophage may range from about 10⁴ to 10¹⁶ bacteriophage particles. The composition may be administered once or more daily, weekly, monthly, or annually. The composition may be administered before, during, or following a meal. In one embodiment, the pharmaceutical composition is administered before the subject eats a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In one embodiment, the pharmaceutical composition is administered after the subject eats a meal.

[069] The bacteriophage may be formulated into pharmaceutical compositions comprising one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical composition may include, but is not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols, and surfactants, including, for example, polysorbate 20. In some embodiments, the bacteriophage of the present technology may be formulated in a solution of sodium bicarbonate, e.g., 1 molar solution of sodium bicarbonate (to buffer an acidic cellular environment, such as the stomach, for example), the bacteriophage may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, procaine, etc.

[070] The bacteriophage may be administered intravenously, e.g., by infusion or injection. Alternatively, the bacteriophage may be administered intratumorally and/or peritumorally. In other embodiments, the bacteriophage may be administered intra-arterially, intramuscularly, or intraperitoneally. In some embodiments, the bacteriophage is co-administered with a PEGylated form of rHuPH20 (PEGPH20) or other agent in order to destroy the tumor septae in order to enhance penetration of the tumor capsule, collagen, and/or stroma.

[071] The bacteriophage of the disclosure may be administered via intratumoral injection, resulting in bacteriophage that is directly deposited within the target tumor. Intratumoral injection of the engineered bacteriophage may elicit a potent localized inflammatory response as well as an immune response against tumor cells. For the injection procedure, bacteriophages are suspended in solution before being withdrawn into a syringe. In some embodiments, the tumor is injected with a single hole needle. In yet another embodiment, the tumor is injected with a multipronged needle (Quadra- Fuse, Rex Medical).

[072] Direct intratumoral injection of the bacteriophage of the present technology into solid tumors may be advantageous as compared to intravenous administration. Using an intravenous injection method, only a small proportion of the bacteriophage may reach the target tumor. In particular, in large animals and human patients, which have relatively large blood volumes and relatively small tumors compared to mice, intratumoral injection may be especially beneficial. Injection directly into the tumor allows the delivery of a higher concentration of therapeutic agent and avoids the toxicity, which can result from systemic administration. In addition, intratumoral injection of bacteriophages induces robust and localized immune responses within the tumor.

[073] Depending on the location, tumor type, and tumor size, different administration techniques may be used, including but not limited to, cutaneous, subcutaneous, and percutaneous injection, therapeutic endoscopic ultrasonography, or endobronchial intratumor delivery. Prior to the intratumor administration procedures, sedation in combination with a local anesthetic and standard cardiac, pressure, and oxygen monitoring, or full anesthesia of the patient is performed.

[074] For some tumors, percutaneous injection can be employed, which is the least invasive administration method. Ultrasound computed tomography (CT) or fluoroscopy can be used as guidance to introduce and position the needle. Percutaneous intratumoral injection is for example described for hepatocellular carcinoma in Lencioni et al., 2010 (incorporated herein by reference). Intratumoral injection of cutaneous, subcutaneous, and nodal tumors is for example described in WO/2014/036412 (incorporated herein by reference) for late-stage melanoma.

[075] Single insertion points or multiple insertion points can be used in percutaneous injection protocols. Using a single insertion point, the solution may be injected percutaneously along multiple tracks, as far as the radial reach of the needle allows. In other embodiments, multiple injection points may be used if the tumor is larger than the radial reach of the needle. The needle can be pulled back without exiting and redirected as often as necessary until the full dose is injected and dispersed. To maintain sterility, a separate needle is used for each injection. Needle size and length varies depending on the tumor type and size.

[076] In some embodiments, the tumor is injected percutaneously with an 18-gauge multipronged needle (Quadra-Fuse, Rex Medical). The device comprises an 18-gauge puncture needle 20 cm in length. The needle has three retractable prongs, each with four terminal side holes and a connector with an extension tubing clamp. The prongs are deployed from the lateral wall of the needle. The needle can be introduced percutaneously into the center of the tumor and can be positioned at the deepest margin of the tumor. The prongs are deployed to the margins of the tumor. The prongs are deployed at maximum length and then are retracted at defined intervals. Optionally, one or more rotation-injection -rotation maneuvers can be performed, in which the prongs are retracted, the needle is rotated by 60 degrees, which is followed by repeat deployment of the prongs and additional injection.

[077] Therapeutic endoscopic ultrasonography (EUS) is employed to overcome the anatomical constraints inherent in gaining access to certain other tumors (Shirley et al., 2013; incorporated herein by reference). EUS-guided fine needle injection (EUS-FNI) has been successfully used for antitumor therapies for the treatment of head and neck, esophageal, pancreatic, hepatic, and adrenal masses (Verna et al, 2008; incorporated herein by reference). EUS-FNI has been extensively used for pancreatic cancer injections. Fine-needle injection requires the use of the curvilinear echoendoscope. The esophagus is carefully intubated and the echoendoscope is passed into the stomach and duodenum where the pancreatic examination occurs and the target tumor is identified. The largest plane is measured to estimate the tumor volume and to calculate the injection volume. The appropriate volume is drawn into a syringe. A primed 22 -gauge fine needle aspiration (FNA) needle is passed into the working channel of the echoendoscope. Under ultrasound guidance, the needle is passed into the tumor. Depending on the size of the tumor, administration can be performed by dividing the tumor into sections and then injecting the corresponding fractions of the volume into each section. Use of an installed endoscopic ultrasound processor with Doppler technology assures there are no arterial or venous structures that may interfere with the needle passage into the tumor (Shirley et al., 2013; incorporated herein by reference). In some embodiments, 'multiple injectable needle' (MIN) for EUS-FNI can be used to improve the injection distribution to the tumor in comparison with straight-type needles (Ohara et al., 2013; incorporated herein by reference).

[078] Intratumoral administration for lung cancer, such as non-small cell lung cancer, can be achieved through endobronchial intratumor delivery methods, as described in Celikoglu et al., 2008 (incorporated herein by reference). Bronchoscopy (trans-nasal or oral) is conducted to visualize the lesion to be treated. The tumor volume can be estimated visually from visible length-width height measurements over the bronchial surface. The needle device is then introduced through the working channel of the bronchoscope. The needle catheter, which comprises a metallic needle attached to a plastic catheter, is placed within a sheath to prevent damage by the needle to the working channel during advancement. The needle size and length vary and are determined according to tumor type and size of the tumor. Needles made from plastic are less rigid than metal needles and are ideal, since they can be passed around sharper bends in the working channel. The needle is inserted into the lesion and the bacteriophages ofthe present technology are injected. Needles are inserted repeatedly at several insertion points until the tumor mass is completely perfused. After each injection, the needle is withdrawn entirely from the tumor and is then embedded at another location. At the end of the bronchoscopic injection session, removal of any necrotic debris caused by the treatment may be removed using mechanical dissection, or other ablation techniques accompanied by irrigation and aspiration.

[079] In some embodiments, the bacteriophages capable of delivering an immune modulator to a target tumor are administrated directly into the tumor using methods, including but not limited to, percutaneous injection, EUS-FNI, or endobronchial intratumor delivery methods. In some cases, other techniques, such as laparoscopic or open surgical techniques are used to access the target tumor, however, these techniques are much more invasive and bring with them much greater morbidity and longer hospital stays.

[080] The volume injected into each lesion is based on the size of the tumor. To obtain the tumor volume, a measurement of the largest plane can be conducted. The estimated tumor volume can then inform the determination of the injection volume as a percentage of the total volume. For example, an injection volume of approximately 20-40% of the total tumor volume can be used. For example, as is for example described in WO/2014/036412 (Amgen; incorporated herein by reference), for tumors larger than 5 cm in their largest dimension, up to 4 mL can be injected. For tumors between 2.5 and 5 cm in their largest dimension, up to 2 mL can be injected. For tumors between 2.5 and 5 cm in their largest dimension, up to 2 mL can be injected. For tumors between 1.5 and 2.5 cm in their largest dimension, up to 1 ml can be injected. For tumors between 0.5 and 1.5 cm in their largest dimension, up to 0.5 mL can be injected. For tumors equal or small than 0.5 in their largest dimension, up to 0. 1 mL can be injected. Alternatively, an ultrasound scan can be used to determine the injection volume that can be taken up by the tumor without leakage into the surrounding tissue. [081] In some embodiments, the treatment regimen will include one or more intratumoral administrations. In some embodiments, a treatment regimen will include an initial dose, which is followed by at least one subsequent dose. One or more doses can be administered sequentially in two or more cycles. For example, a first dose may be administered at day 1, and a second dose may be administered after 1, 2, 3, 4, 5, 6, days or 1, 2, 3, or 4 weeks or after a longer interval. Additional doses may be administered after 1, 2, 3, 4, 5, 6, days or after 1, 2, 3, or 4 weeks or longer intervals. In some embodiments, the first and subsequent administrations have the same dosage. In other embodiments, different doses are administered. In some embodiments, more than one dose is administered per day, for example, two, three or more doses can be administered per day.

[082] The routes of administration and dosages described are intended only as a guide. The optimum route of administration and dosage can be readily determined by a skilled practitioner. The dosage may be determined according to various parameters, especially according to the location of the tumor, the size of the tumor, the age, weight, and condition of the patient to be treated, and the route and method of administration.

[083] Tumor types into which the bacteriophage of the current technology are intratumorally delivered include locally advanced and metastatic tumors, including but not limited to, B, T, and NK cell lymphomas, colon and rectal cancers, melanoma, including metastatic melanoma, mycosis fungoides, Merkel carcinoma, liver cancer, including hepatocellular carcinoma and liver metastasis secondary to colorectal cancer, pancreatic cancer, breast cancer, follicular lymphoma, prostate cancer, refractory liver cancer, and Merkel cell carcinoma.

[084] The bacteriophage disclosed herein may be administered topically and formulated in the form of an ointment, cream, transdermal patch, lotion, gel, shampoo, spray, aerosol, solution, emulsion, or other form well known to one of skill in the art. See, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. In an embodiment, for non-sprayable topical dosage forms, viscous to semisolid or solid forms comprising a carrier or one or more excipients compatible with topical application and having a dynamic viscosity greater than water are employed. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, ointments, powders, liniments, salves, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, wetting agents, buffers, or salts) for influencing various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol preparations wherein the active ingredient in combination with a solid or liquid inert carrier, is packaged in a mixture with a pressurized volatile (e.g., a gaseous propellant, such as freon) or in a squeeze bottle. Moisturizers or humectants can also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well-known in the art. In one embodiment, the pharmaceutical composition comprising the bacteriophage of the present technology may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial formulation, or a fermentation product such as a fermentation broth. Hygiene products may be, for example, shampoos, conditioners, creams, pastes, lotions, and lip balms.

[085] The bacteriophage disclosed herein may be administered orally and formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. Pharmacological compositions for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose compositions such as maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and/or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrating agents may also be added, such as cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate.

[086] Tablets or capsules can be prepared by conventional means with pharmaceutically acceptable excipients such as binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone, hydroxypropyl methylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth); 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, sugars, cellulose derivatives, silica powders); or wetting agents (e.g., sodium lauryl sulphate). The tablets may be coated by methods well known in the art. A coating shell may be present, and common membranes include, but are not limited to, polylactide, polyglycolic acid, polyanhydride, other biodegradable polymers, alginatepolylysine- alginate (APA), alginate-polymethylene-co-guanidine- alginate (A-PMCG-A), hydroymethylacrylate-methyl methacrylate (HEMA-MMA), multilayered HEMA- MMAMAA, polyacrylonitrilevinylchloride (PAN-PVC), acrylonitrile/sodium methallylsulfonate (AN-69), polyethylene glycol/poly pentamethylcyclopentasiloxane/polydimethylsiloxane (PEG/PD5/PDMS), poly N,N- dimethyl acrylamide (PDMAAm), siliceous encapsulates, cellulose sulphate/sodium alginate/polymethylene-co-guanidine (CS/A/PMCG), cellulose acetate phthalate, calcium alginate, k- carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolides), carrageenan, starch poly-anhydrides, starch polymethacrylates, polyamino acids, and enteric coating polymers.

[087] In some embodiments, the bacteriophage is coated in Cellulose acetate phthalate (CAP), Poly(methacrylic acid-co-methyl methacrylate), Cellulose acetate trimellitate (CAT), Poly(vinyl acetate phthalate) (PVAP) and Elydroxypropyl methylcellulose phthalate (EIPMCP), fatty acids, waxes, Shellac (esters of aleurtic acid), plastics and plant fibers. Additionally, Zein, Aqua-Zein (an aqueous zein formulation containing no alcohol), amylose starch and starch derivatives, and dextrins (e.g., maltodextrin) are also used. Other known enteric coatings include ethylcellulose, methylcellulose, hydroxypropyl methylcellulose, amylose acetate phthalate, cellulose acetate phthalate, hydroxyl propyl methyl cellulose phthalate, an ethylacrylate, and a methylmethacrylate.

[088] Coating polymers also may comprise one or more of, phthalate derivatives, CAT, EIPMCAS, polyacrylic acid derivatives, copolymers comprising acrylic acid and at least one acrylic acid ester, Eudragit™ S (poly(methacrylic acid, methyl methacrylate) 1:2); Eudragit LI00™ S (poly(methacrylic acid, methyl methacrylate) 1: 1); Eudragit L30D™, (poly(methacrylic acid, ethyl acrylate) 1: 1); and (Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate)l:l) (Eudragit™ L is an anionic polymer synthesized from methacrylic acid and methacrylic acid methyl ester), polymethyl methacrylate blended with acrylic acid and acrylic ester copolymers, alginic acid, ammonia alginate, sodium, potassium, magnesium or calcium alginate, vinyl acetate copolymers, polyvinyl acetate 30D (30% dispersion in water), a neutral methacrylic ester comprising poly(dimethylaminoethylacrylate) ("Eudragit E™), a copolymer of methylmethacrylate and ethylacrylate with trimethylammonioethyl methacrylate chloride, a copolymer of methylmethacrylate and ethylacrylate, Zein, shellac, gums, or polysaccharides, or a combination thereof.

[089] Coating layers may also include polymers which contain Elydroxypropylmethylcellulose (EIPMC), Elydroxypropylethylcellulose (EIPEC), Elydroxypropylcellulose (EIPC), hydroxypropylethylcellulose (EIPEC), hydroxymethylpropylcellulose (HMPC), ethylhydroxyethylcellulose (EHEC) (Ethulose), hydroxyethylmethylcellulose (HEMC), hydroxymethylethylcellulose (EIMEC), propylhydroxyethylcellulose (PEIEC), methylhydroxyethylcellulose (M El EC), hydrophobically modified hydroxyethylcellulose (NEXTON), carboxymethyl hydroxyethylcellulose (CMEIEC), Methylcellulose, Ethylcellulose, water soluble vinyl acetate copolymers, gums, polysaccharides such as alginic acid and alginates such as ammonia alginate, sodium alginate, potassium alginate, acid phthalate of carbohydrates, amylose acetate phthalate, cellulose acetate phthalate (CAP), cellulose ester phthalates, cellulose ether phthalates, hydroxypropylcellulose phthalate (EIPCP), hydroxypropylethylcellulose phthalate (EIPECP), hydroxyproplymethylcellulose phthalate (EIPMCP), hydroxyproplymethylcellulose acetate succinate (HPMCAS).

[090] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or a dry product for constitution with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils); and preservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbic acid). The preparations may also contain buffer salts, flavoring, coloring, and sweetening agents as appropriate. Preparations for oral administration may be suitably formulated for slow release, controlled release, or sustained release of the bacteriophage described herein.

[091] In one embodiment, the bacteriophage of the disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics, 134(2):361-372, 2014; incorporated herein by reference). Moreover, pediatric formulation acceptability and preferences, such as route of administration and taste attributes, are critical for achieving acceptable pediatric compliance. Thus, in one embodiment, the composition suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, such as compositions with added flavors, sweeteners, or taste blockers. In one embodiment, a composition suitable for administration to pediatric subjects may also be suitable for administration to adults.

[092] In one embodiment, the composition suitable for administration to pediatric subjects may include a solution, syrup, suspension, elixir, powder for reconstitution as suspension or solution, dispersible/effervescent tablet, chewable tablet, gummy candy, lollipop, freezer pop, troche, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, sprinkle oral powder, or granules. In one embodiment, the composition is a gummy candy, which is made from a gelatin base, giving the candy elasticity, desired chewy consistency, and longer shelf-life. In some embodiments, the gummy candy may also comprise sweeteners or flavors.

[093] In one embodiment, the composition suitable for administration to pediatric subjects may include a flavor. As used herein, "flavor" is a substance (liquid or solid) that provides a distinct taste and aroma to the formulation. Flavors also help to improve the palatability of the formulation. Flavors include, but are not limited to, strawberry, vanilla, lemon, grape, bubble gum, and cherry.

[094] In certain embodiments, the bacteriophage may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in a hard- or soft-shell gelatin capsule, compressed into tablets, or incorporated directly into the subject's diet. For oral therapeutic administration, the compounds may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer a compound by other than parenteral administration, it may be necessary to coat the compound with, or coadminister the compound with, a material to prevent its inactivation .

[095] In some embodiments, the composition is formulated for intraintestinal administration, intrajejunal administration, intraduodenal administration, intraileal administration, gastric shunt administration, or intracolic administration, via nanoparticles, nanocapsules, microcapsules, or microtablets, which are enterically coated or uncoated. The pharmaceutical compositions may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles, and may contain suspending, stabilizing and/or dispersing agents.

[096] The bacteriophage described herein may be administered intranasally, formulated in an aerosol form, spray, mist, or in the form of drops, and conveniently delivered in the form of an aerosol spray presentation from pressurized packs or a nebulizer, with the use of a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoro methane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas). Pressurized aerosol dosage units may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., of gelatin) for use in an inhaler or insufflator may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[097] The bacteriophage may be administered and formulated as depot preparations. Such long-acting formulations may be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[098] In some embodiments, disclosed herein are pharmaceutically acceptable compositions in single dosage forms. Single dosage forms may be in a liquid or a solid form. Single dosage forms may be administered directly to a patient without modification or may be diluted or reconstituted prior to administration. In certain embodiments, a single dosage form may be administered in bolus form, e.g., single injection, single oral dose, including an oral dose that comprises multiple tablets, capsule, pills, etc. In alternate embodiments, a single dosage form may be administered over a period of time, e.g., by infusion.

[099] Single dosage forms of the pharmaceutical composition may be prepared by portioning the pharmaceutical composition into smaller aliquots, single dose containers, single dose liquid forms, or single dose solid forms, such as tablets, granulates, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may be enterically coated or uncoated. A single dose in a solid form may be reconstituted by adding liquid, typically sterile water or saline solution, prior to administration to a patient.

[100] In other embodiments, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., 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-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of skill in the art may be used.

[101] Dosage regimens may be adjusted to provide a therapeutic response. Dosing can depend on several factors, including severity and responsiveness of the disease, route of administration, time course of treatment (days to months to years), and time to amelioration of the disease. For example, a single bolus may be administered at one time, several divided doses may be administered over a predetermined period of time, or the dose may be reduced or increased as indicated by the therapeutic situation. The specification for the dosage is dictated by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values may vary with the type and severity of the condition to be alleviated. For any particular subject, specific dosage regimens may be adjusted over time according to the individual need and the professional judgment of the treating clinician. Toxicity and therapeutic efficacy of compounds provided herein can be determined by standard pharmaceutical procedures in cell culture or animal models. For example, LD50, ED50, EC50, and IC50 may be determined, and the dose ratio between toxic and therapeutic effects (LD 50/ED 50) may be calculated as the therapeutic index. Compositions that exhibit toxic side effects may be used, with careful modifications to minimize potential damage to reduce side effects. Dosing may be estimated initially from cell culture assays and animal models. The data obtained from in vitro and in vivo assays and animal studies can be used in formulating a range of dosage for use in humans.

[102] The ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. If the mode of administration is by injection, an ampoule of sterile water for injection, saline or nutritive excipient can be provided so that the ingredients may be mixed prior to administration.

[103] The pharmaceutical compositions may be packaged in a hermetically sealed container such as an ampoule or sachet indicating the quantity of the agent. In one embodiment, one or more of the pharmaceutical compositions is supplied as a dry sterilized lyophilized powder or water-free concentrate in a hermetically sealed container and can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In an embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions is supplied as a dry sterile lyophilized powder in a hermetically sealed container stored between 2°C and 8°C and administered within 1 hour, within 3 hours, within 5 hours, within 6 hours, within 12 hours, within 24 hours, within 48 hours, within 72 hours, or within one week after being reconstituted. Cryoprotectants can be included for a lyophilized dosage form, principally 0-10% sucrose (optimally 0.5- 1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, either of which can be included at a concentration of 0-0.05%, and polysorbate-80 (optimally included at a concentration of 0.005-0.01%). Additional surfactants include but are not limited to polysorbate 20 and BRU surfactants. The pharmaceutical composition may be prepared as an injectable solution and can further comprise an agent useful as an adjuvant, such as those used to increase absorption or dispersion, e.g., hyaluronidase.

[104] In some embodiments, the bacteriophage and composition thereof is formulated for intravenous administration, intratumor administration, or peritumor administration. The bacteriophage may be formulated as depot preparations. Such long-acting formulations may be administered by implantation or by injection. For example, the compositions may be formulated with suitable polymeric or hydrophobic materials (e.g., as an emulsion in an acceptable oil) or ion exchange resins, or as sparingly soluble derivatives (e.g., as a sparingly soluble salt).

[105] In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump may be used to achieve controlled or sustained release. In another embodiment, polymeric materials can be used to achieve controlled or sustained release of the therapies of the present disclosure (see e.g., 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-hydroxy ethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolides (PLG), polyanhydrides, poly(N- vinyl pyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactides (PLA), poly(lactide-co-glycolides) (PLGA), and polyorthoesters. The polymer used in a sustained release formulation may be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system can be placed in proximity of the prophylactic or therapeutic target, thus requiring only a fraction of the systemic dose. Any suitable technique known to one of the skilled in the art may be used.

[106] The bacteriophage of the present technology may be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine, etc.

EXAMPLES

[107] The examples below are given to illustrate the practice of various embodiments of the present disclosure. They are not intended to limit or define the entire scope of this disclosure. It should be appreciated that the disclosure is not limited to the particular embodiments described and illustrated herein but includes all modifications and variations falling within the scope of the disclosure as defined in the appended embodiments.

EXAMPLE 1 - ENGINEERING OF THE BACTERIOPHAGE DISPLAYING A TUMOR TARGETING MOIETY AND A CYTOKINE

[108] Strains, plasmids, and growth conditions. All strains and plasmids used in this Example are described in Table 1. All plasmids and gBlock sequences are listed in the sequence listing file annexed to this document. Cells were typically grown in Luria broth Miller (LB) or on Luria broth agar Miller medium supplemented, when needed, with antibiotics at the following concentrations: ampicillin (Ap) 100 Lig ni l.. chloramphenicol (Cm) 34 Lig nil.. kanamycin (Km) 50 Lignil.. nalidixic acid (Nx) 4 Lignil.. spectinomycin (Sp) 100 Lig nil.. streptomycin (Sm) 50 Lig ni l.. sulfamethoxazole (Su) 160 Lig nil.. tetracycline (Tc) 15 lig nil.. and trimethoprim (Tm) 32 lig nil.. All cultures were routinely grown at 37°C. No bacterial cultures over 18 hours of age were used in the experiments.

Table 1: List of strains and plasmids used in this study

[109] DNA manipulations. A detailed list of oligonucleotide sequences used in this Example is found in Table 2. Plasmids were prepared using EZIO-Spin Column Plasmid Miniprep kit (BIOBASIC #BS614) according to the manufacturer’s instructions. PCR amplifications were performed using TransStart FastPFU fly DNA polymerase (Civic Bioscience) for DNA parts amplification and screening. Plasmids were assembled by Gibson assembly using the NEBuilder HiFi DNA Assembly Master Mix (NEB) following the manufacturer’s protocol. DNA digestions were carried using restriction enzymes from NEB following the manufacturer’s recommendations. Digestions were routinely carried over 1 hour.

[110] DNA purification. Purification of DNA was performed between each step of plasmid assembly to avoid buffer incompatibility or to stop enzymatic reactions. PCR reactions were purified by Solid Phase Reversible Immobilization (SPRI) using Agencourt Ampure XP DNA binding beads (Beckman Coulter) according to the manufacturer’s recommendations. After purification, DNA concentration and purity were routinely assessed using a Nanodrop spectrophotometer when necessary.

[H l] DNA transformation into E. coli by electroporation. Routine plasmid transformations were performed by electroporation. Electrocompetent E. coli strains were prepared from 20 mL of LB broth. Cultures reaching exponential growth phase of 0.6 optical density at 600 nanometers (ODeoomn) were washed three times in a sterile 10% of glycerol solution. Cells were then resuspended in 200 ill. of glycerol 10% (1% of the initial volume) and distributed in 50 ill. aliquots. The DNA was then added to the electrocompetent cells (approximately 50 ng of DNA in up to 10 ill. of water) and the mixture was transferred in a 1 mm electroporation cuvette. Cells were electroporated using a pulse of 1.8 kV, 25 pF and 200 fl for 5 ms. Cells were then resuspended in 1 mL of non-selective LB medium and recovered for 1 hour at 37°C before plating on selective media and incubating the plates overnight at 37°C. [112] DNA transformation into E. coli by heat-shock. Heat-shock transformation was mostly used to clone Gibson assembly products. Chemically competent cells were prepared according to the rubidium chloride protocol as described previously (Green et al. , 2013). Chemically competent cells were flash-frozen and conserved at -80°C before use. Gibson assembly products were directly transformed into MM294 chemically competent cells at a 1/10 volume ratio. Routinely, up to 10 ill. of DNA was added to 100 Lil , competent cells before transformation by a 45 second heat shock at 42°C. Cells were then resuspended in 1 niL of non-selective LB medium and let to recover for 1 hour at 37°C before plating on selective media.

[113] Engineered phage systems are widely used in phage display approaches to express antibodyphage conjugate to screen for antibodies with specific affinities. In this context, the variable section of antibodies is amplified using universal DNA primers from an immunized animal and linked together, creating an scFv fragment library that is cloned in a plasmid called phagemid and fused to the pill protein (replacing the N-terminal domains of the protein). The library is next screened to select phages that display an antibody-pill fusion which recognizes the target antigen. The phages that bound to the target are then isolated and amplified by infecting their bacterial host. This step is possible because phages still display copies of the wild-type pill (which is required to infect the bacterial host) as well as the antibody-pill fusion. After a few cycles, the scFv fragments recognizing the target antigen are enriched and clones are tested and sequenced for conservation. In this context, only few phages that adhere to the target antigen are needed to isolate the desired antibody clone, meaning that even if a large proportion of the phages only display the wildtype pill proteins, and not the antibody-pill fusion, the screen can still be successful.

a. Oligonucleotides are listed by PCR reaction and purpose, the same oligonucleotide can be found more than once when used for multiple purposes. b. Underlined nucleotides form the priming site. c. Bold nucleotides are mutated DNA sequences

[114] The engineered bacteriophage system described here differs from the phage display system in several ways. For instance, (1) the bacteriophage must be secreted at high levels by the production strain bacteria, (2) every bacteriophage must display the therapeutic proteins, and (3) as little DNA as possible must be used for the assembly of the phage particles to maximize biocontainment of the system and limit the dissemination of genetic material. These additional constraints do not exist in conventional phage display. As such, most phage display systems use M13K07 as the helper phage, expressing all the proteins responsible for phage replication and assembly (including a wildtype copy of gpHI), and a phagemid, expressing the antibody-pill fusion protein, an origin of replication, and containing an intact encapsidation signal. Although these systems would produce antibody-phage conjugates, 90% of the phages produced contain only the wildtype pill protein from M13K07 and not the antibody-pill fusion protein from the phagemid (Ledsgaard et al. 2018, Toxin,' incorporated herein by reference). For the display of therapeutic protein, having 90% of bacteriophages not displaying the therapeutic protein would be sub-optimal and unwanted, as it would decrease the efficacy of the treatment.

[115] In the present example, we will describe the design of the bacteriophage secretion systems for the display of therapeutic proteins. We will present iterations of the system and discuss their pros and cons. The list of iteration is not meant to be exhaustive, but rather illustrate variations of the bacteriophage secretion system for the combined display of cytokines and binding molecules. A general description of the diverse constructs described in the present example are illustrated in Figure 1.

Bacteriophage secretion system iterations

[116] The bacteriophage secretion system is a malleable system that can manifest in different forms. In this example, we aimed to show that the bacteriophage secretion system can be modified to allow the display of one or two therapeutic proteins on a same bacteriophage particle. In a first embodiment of the bacteriophage secretion system, the system can be divided in a set of two vectors, the bacteriophage backbone vector (e.g., pTAT002, pTAT044, pTAT070) and the bacteriophage machinery vector (e.g., pTAT004) (Figure 1). In some embodiments, the bacteriophage secretion system can be divided in three or more genetic constructs as in the system composed of pTAT025, pTAT044 or pTAT070 and pTAT017 or pTAT060 or pTAT071. In another embodiment, the bacteriophage secretion could be on a single vector. In yet another embodiment, the bacteriophage secretion could be integrated in the genome of the production strain bacteria.

Bacteriophage secretion systems based on two vectors [117] To generate the bacteriophage machinery vector pTAT004, M 13 K07 was amplified in its entirety using appropriate primers presented in Table 2, except for the gpHI gene, which codes for pill. The homology tails of the primers used for amplifying M13K07 were carefully designed to remove gpHI from the final construction. PCR products were next purified by SPRI and assembled by Gibson’s method, generating pTAT004. The assembly was transformed into MM294 chemically competent cells and plasmid integrity was verified by digestion using Ndel. The pTAT004 bacteriophage machinery cannot produce fully functional bacteriophage particles on its own, as it does not possess a copy of the gpHI gene, and hence, does not produce pill. In order to produce bacteriophage particles, gpHI must be provided in trans by the bacteriophage backbone vector.

[118] The pTAT002, pTAT044, and pTAT070 bacteriophage backbone vectors were next designed. All bacteriophage backbone vectors contain the OZ7_PMBI for high copy plasmid replication (maximising DNA material for encapsidation), OZVMIS for ssDNA rolling circle replication and recognition of the bacteriophage backbone vector by the phage encapsidation machinery, a selective marker (here spectinomycin resistance), and a constitutively expressed pill C-terminal fragment to either the HA-His dual tagged N-terminal fragment of pill (pTAT002, control with no therapeutic protein), or a checkpoint inhibitor binding protein (pTAT044, anti-PD-Ll linked via one HA-His dual tag; pTAT070, anti-PD-Ll without tags). In this example, the anti-PD-Ll binding protein was selected as therapeutic proteins because it binds to surface proteins expressed by cancerous cells (Vaddepally et al., Cancers (Basel) 2020 Mar; 12(3): 738; incorporated here by reference). The first bacteriophage backbone vector assembled was pTAT002. To build pTAT002, OZ-Z_PMBI was amplified by PCR from pSBlC3 (Common backbone vector for the iGEM library), oriw and the pill N-terminal and C-terminal parts from M13K07, aad7 (spectinomycin resistance) from E. coli KN01 and gBlock a constitutive promoter. The PCR products were next assembled by Gibson and transformed into chemically competent MM294 cells. Integrity of the plasmid was next verified by digestion using ApaLI and Ndel (Figure 5 in WO2022073127A1; incorporated here as reference). Following sanger sequencing of pTAT002, a mutation in the third position (G>T) of the P5 promoter was found. The resulting promoter, termed P5mut (5’- TTTACAATTAATCATCCGGCTCGTAATTTATGTGGA-3 ) allow lower levels of expression of the upstream gene, as measured with the pTAT010-P5 and pTAT010-P5mut constructs using GFP (data not shown). In order to streamline construction assembly, the pTAT002 backbone was modified and a sjGFP gene was cloned to be expressed by the P5 promoter instead of gpHI. This backbone was assembled similarly to pTAT003, but the primer used allowed the insertion of an additional terminator after the gene expressed by P5, and the insertion of Gibson assembly tags (GAT) that allowed to delimit the different parts of the vector. The resulting vector, termed pTAT013, was then used as a template to amplify the backbone of the subsequent constructs for display of therapeutic proteins on pill. As such, for the construction of pTAT044 and pTAT070, the backbone of the plasmid was amplified from pTAT013, the P5mut promoter from pTAT010-P5mut, and the C-terminal part of gpIII from M13K07. The anti-PD-Ll scFv was amplified from a gBlock with a HA-6sHis linker (pTAT044) or without a linker (pTAT070). Those gBlocks also changed the start codon of the checkpoint inhibitor fusion protein from ATG to GTG as it was shown to allow better expression levels of the therapeutic bacteriophages. All plasmids were next sent to Sanger sequencing after assembly, no deleterious mutations were detected. With these results, the bacteriophage secretion system with a display of the therapeutic protein on pill was ready for efficiency tests and improvement rounds.

Bacteriophage secretion systems based on three or more vectors

[119] The bacteriophage secretion system can be divided into two or more DNA molecules to accommodate the simultaneous display of multiple therapeutic proteins and remain functional as long as sufficient proteins of each bacteriophage gene are produced. To facilitate the display of two therapeutic proteins on a same bacteriophage particle, we aimed to split the bacteriophage machinery into three different plasmids. As a first step, we needed to delete gpIII, a tail fiber protein and a protein of the head of the bacteriophage. We selected gpIX, a capsid gene located at the head part of the bacteriophage and involved in budding from the host cell, as a second site for protein fusion. Deleting gpIX from M13K07 is more complex than deleting gpIII since the coding sequence of gpIX overlaps with the coding sequence of gpVIII. Too remove gpIX, we thus needed to include some gene refactoring to prevent interruption of the gpVIII gene. The overlapping sequence between gp VIII and gpIX is: 5 ’ -AGAT GAGTGTTTTA-3 ’ . where the bold ATG codon is the start codon of gpIX and the underlined TGA codon is the stop codon from gpVIII. The overlap 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 encodes for arginine). Also, we changed the third codon of gpIX from TTA to TAA to introduce a stop codon and prevent the translation of gpIX. The changes are underlined in the resulting sequence: 5’- AGGTGAGTGTTTAA-3 ’ . The resulting construct pTAT025 was next obtained by amplifying pTAT004 with primer introducing these modifications to the gpVIIUgpIX locus. Plasmid pTAT025 thus express all the genes of the M13 genome except for gpIII and gpIX, which needs to be provided in trans. It also needs a bacteriophage backbone vector encoding OAMIS to secrete bacteriophages.

[120] Plasmid pTAT025 gpIII deficiency can be complemented by any of the plasmid described above that express gpIII or gp ///-therapeutic protein fusion (pTAT002, pTAT044 and pTAT070). However, pTAT025 also needs an exogenous supply of gpIX to produce bacteriophages. A set of plasmids was thus needed to support gpIX production. To this end, a new backbone was generated by amplifying orz_pscioi from pKN23, bla from pUC19 and P5-BCD 1 -sfGFP from pTAT010-P5 each of which are assembled using GAT in primer tails. The PCR fragments were next purified by SPRI and assembled by Gibson assembly generating pTAT014 before transformation in MM294. The construct was next evaluated phenotypically (GFP phenotype) and sequenced by Sanger sequencing. This backbone was next amplified in its entirety except for the sfGFP gene and was assembled with the gpIX gene amplified from M13K07. Both PCR products were next assembled in the same way as for pTAT014, generating a gpIX complementation plasmid (pTAT017). This plasmid was further modified to allow the display of the mouse IL-2 (mIL-2) and human IL-15 (hIL-15) on pIX by amplifying their CDS and P5mut-BCD1 from agBlock creating pTAT071 and pTAT060 respectively. Those gBlocks also changed the start codon of the cytokine fusion protein from ATG to GTG as it was shown to allow better expression levels of the therapeutic bacteriophages. The plasmids were next confirmed by sanger sequencing.

Combining plasmids into functional bacteriophage secretion systems

[121] Different bacteriophage secretion systems are required to test potential synergies between cytokines and cancer cell targeting moieties displayed on the synthetic therapeutic bacteriophage. To obtain those systems, plasmids were transformed into E. coli MG1655 by electroporation sequentially. The combinations described in Table 3 allow the production of: the M13 phage (Phage), the M13 phage displaying an anti-PD-Ll scFv (Phage-PD-Ll), the M13 phage displaying the mouse IL-2 and the anti-PD- L1 scFv (mIL-2-Phage-PD-Ll), the M13 phage displaying human IL-15 and the anti-PD-Ll scFv (hIL15- Phage-PD-Ll). These four bacteriophage variants will allow us to dissect the contribution of each element to the anti -turn oral response in mice and explore potential synergies.

Table 3: List of test bacterial strains

[122] Altogether, the strains generated in the present example are sufficient to explore the potential synergies between cytokines and cancer cell targeting moieties displayed on the M13 bacteriophage. These combinations are not meant to be exhaustive, and it is within reason to think that other cytokines or binders could synergize when displayed on the M13 bacteriophage. This example also illustrates the ability of the bacteriophage to display simultaneously more than one therapeutic agent on each subunit of two different coat proteins.

EXAMPLE 2 -IN VITRO VALIDATION OF THE THERAPEUTIC TARGET ENGAGEMENT OF BACTERIOPHAGES DISPLAYING A TUMOR TARGETING MOLECULE AND CYTOKINES

[123] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this Example are described in Table 1. Cells were typically grown in 2xYT Broth (2xYT) supplemented, when needed, with antibiotics at the following concentrations: ampicillin (Ap) 100 pg/mL, kanamycin (Km) 50 pg/mL and/or spectinomycin (Sp) 100 pg/mL. All cultures were routinely grown at 30°C for no longer than 18 hours before use in the experiments. Bacteriophages were recovered from the supernatant of confluent bacterial culture (grown overnight). The culture supernatants containing the bacteriophages were used immediately after the bacteria precipitation by centrifugation.

[124] Polyethylene glycol-based precipitation of bacteriophage particles. Starting from frozen stock, inoculate 10 m of 2xYT broth containing the appropriate antibiotics at the concentrations specified in the paragraph above and incubate the culture at 30°C overnight with agitation, or for no longer than 18 hours. The whole overnight bacterial culture was transferred in a 2 L Erlenmeyer containing 500 m of 2xYT broth with the proper antibiotics. The culture was incubated at 37°C with agitation until the optical density at 600 nm reach 0.5. The culture was then put on ice for 15 minutes before being incubated overnight at room temperature with agitation. The overnight culture was transferred in a 500 mb centrifuge bottle and centrifuged at 13,000 g for 20 min at 4°C. The supernatant was decanted on a 500 mb 0.45 pm filter unit and filtered to remove any remaining bacteria and debris. 2.5 M NaCl / 20% PEG-8000 (w/v) was added to the filtered supernatant to obtain a 4: 1 supernatant: PEG solution volume ratio. After mixing thoroughly by inverting the bottle 15 times, the mixture is then incubated at 4 °C for 1 h. The virions are next pelleted by centrifugation at 13,000 g for 20 minutes at 4°C. The supernatant is then removed, and the pellet resuspended in 1 to 2 mb of PBS containing 0.1 mM CaCT. The bacteriophage preparation is kept on ice for another hour, vortexed, and stored at 4°C. The majority of contaminating EPS was removed from the phage preparation using the EndoTrap® HD (LIONEX) discontinuous chromatography according to the manufacturer instructions. The phage titer is then interpolated by an in-house sandwich ELISA against the pVIII coating protein using a standard curve generated with purified M13K07 Helper Phage (NEB).

[125] Assessment of bacteriophage binding to PD-L1 by indirect ELISA. The binding activity of bacteriophages displaying an scFv directed against the PD-L1 checkpoint was measured by ELISA assay. A 96 well Nunc MaxiSorp™ plate was first coated overnight at 4°C with recombinant human PD-L1 protein ectodomain (SinoBiological) diluted at a 2 pg/mL in coating buffer (0.05 M Carbonate -Bicarbonate at pH 9.6). The plate was then washed 3 times with 200 pL of TBS-T. To prevent unspecific binding, the plate was subsequently incubated with 200 pL of blocking buffer (TBS-T, 3% skimmed milk, 1% BSA) 1 hour at RT. Blocking was stopped by removing the blocking buffer and washing the plate two times with 200 pL of TBS-T. Then, 100 pL of purified wildtype M13 phage (Phage control), or of bacteriophages displaying an anti-PD-Ll scFv on pill (Phage-PD-Ll), or of bacteriophages displaying an anti-PD-Ll scFv on pill and a cytokine on pIX (either mouse IL-2 or human IL- 15, respectively called mIL2 -Phage-PD-Ll or hIL15-Phage-PD-Ll) were diluted in the TBS IX and added to the wells. The plate was incubated for Ih at RT. The plate was then washed 3 times with 200 pL of TBS-T and 100 pL of Anti-pVIII-HRP (anti- M13 phage, B62-FE2) diluted in blocking buffer (1:500) were added. After a 1 h incubation at RT in the dark and the wells were washed 5 times with TBS-T. To reveal the presence of the bacteriophages, 100 pL of TMB High Sensitivity Substrate Solution (BioLegend) was added to each well and the plate was incubated between 3 to 10 min at RT until a blue coloration appeared. The reaction was stopped by adding 100 pL of stop solution (0.5 M H2SO4) to each well. The absorbance at 450 nm was then measured using a plate reader. Only the bacteriophages displaying the anti-PD-Ll scFv on pill were significantly detected on PD-L1 coated wells (Figure 3 A), therefore validating the biological activity of the display. The dual display of a recombinant interleukin on pIX did not affect the binding efficiency of the scFv on pill.

[126] Assessment of bacteriophage displaying recombinant cytokines on pIX by sandwich ELISA. To validate the expression of a recombinant human Interleukin- 15 (hIL-15) or mouse Interleukin-2 (mIL-2) displayed on a bacteriophage, an ELISA assay was devised. A 96-well Nunc MaxiSorp™ plate was first coated overnight at 4°C with an anti-hIL-15 or an anti-mIL-2 rabbit IgG antibody (SinoBiological) diluted as recommended by the manufacturer in coating buffer (0.05 M Carbonate -Bicarbonate at pH 9.6). The plate was then washed 3 times with 200 pL of TBS-T. To prevent unspecific binding, the plate was subsequently incubated with 200 pL of blocking buffer (TBS-T, 3% skimmed milk, 1% BSA) 1 hour at RT. Blocking was stopped by removing the blocking buffer and washing the plate two times with 200 pL of TBS-T. Then, 100 pL of purified wildtype M13 phage (Phage control), or of bacteriophages displaying an anti-PD-Ll scFv on pill (Phage-PD-Ll), or of bacteriophages displaying an anti-PD-Ll scFv on pill and a cytokine on pIX (either mIL-2 or hIL-15, respectively called mIL2 -Phage-PD-Ll or hIL 15 -Phage- PD-Ll) were diluted in the TBS IX and added to the wells. The plate was incubated for Ih at RT. The plate was then washed 3 times with 200 pL of TBS-T and 100 pL of Anti-pVIII-HRP (anti-M13/fd/Fl, B62- FE2) diluted in blocking buffer (1:500) were added. After a 1 h our incubation at RT in the dark, the wells were washed 5 times with TBS-T. To reveal the presence of the bacteriophages, 100 pL of TMB High Sensitivity Substrate Solution was added to each well and the plate was incubated between 3 to 10 min at RT until a blue coloration appeared. The reaction was stopped by adding 100 pL of stop solution (0.5 M H2SO4) to each well. The absorbance was then measured at 450 nm using a plate reader. The bacteriophages displaying a cytokine on pIX (mIL2 or hIL15) and the anti-PD-Ll scFv were detected on interleukin- specific antibody coated wells (Figure 3B), thus validating the expression of the recombinant protein and the binding activity of the anti-PD-Ll scFv.

EXAMPLE 3 -IN VITRO VALIDATION OF THE BIOLOGICAL ACTIVITY OF A CYTOKINE DISPLAYED ON BACTERIOPHAGES

[127] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this Example are described in Table 3. Cells were typically grown in Luria broth Miller (LB) or on Luria broth agar Miller medium supplemented, when needed, with antibiotics at the following concentrations: ampicillin (Ap) 100 pg/mL, chloramphenicol (Cm) 34 pg/mL, kanamycin (Km) 50 pg/mL, nalidixic acid (Nx) 4 pg/mL, spectinomycin (Sp) 100 pg/mL, streptomycin (Sm) 50 pg/mL, sulfamethoxazole (Su) 160 pg/mL, tetracycline (Tc) 15 pg/mL, and trimethoprim (Tm) 32 pg/mL. All cultures were routinely grown at 37°C for no longer than 18 hours before use in the experiments. Bacteriophages were extracted from confluent bacterial culture (grown overnight) using the PEG precipitation protocol presented in example II.

[128] Cell culture. HEK-Blue™ IL-2 reporter cells, specifically designed to detect bioactive interleukin-2 (IL-2), were ordered from InvivoGen (hkb-il2). HEK-Blue™ cells were cultured under optimal conditions according to the manufacturer guidelines. Briefly, the cells were cultured in DMEM supplemented with 10% Fetal Bovine Serum (FBS) and Normocin™ in an atmosphere of 5% CO2 and 95% humidified air at 37°C. Cells were cultured under a selective pressure using HEK-Blue™ CLR Selection and puromycin to maintain the stable expression of human IL-2 receptor (IL-2R), which includes expression of the subunits CD25 (IL-2Ra), CD122 (IL-2R0), and CD132 (IL-2Ry).

[129] HEK-Blue™ IL-2 reporter assay. HEK-Blue™ IL-2 reporter assay experiments were performed as follow. HEK-Blue™ IL-2 Cells were washed twice with cold D-PBS (Gibco) when 80% confluency was reached. The D-PBS was removed and 2 mL of cold Versene IX (Gibco) was added to detach the cells. After 5 minutes of incubation, 8 mL of test medium (DMEM supplemented with 10% heat-inactivated FB S) was added. Cell density was assessed using trypan blue and an hemacytometer. The cell suspension was centrifuged at 300g for 10 minutes at 4 °C, the supernatant was removed, and cells were resuspended in test medium at a cell density of 280,000 cells/mL. 180 pL of the cell suspension, corresponding to approximatively 50,000 cells, were seeded in a 96-wells TC-treated culture plate. 20 pL of therapeutic agents were added to the wells to test for their IL-2R activation activity. The plate was incubated at 37°C for 18 hours in an atmosphere of 5% CO2 and 95% humidified air. For each well, 20 pL of medium were collected and transferred in a new 96-wells transparent plate. 180 pL of QUANTI-Blue™ Solution were added and the plate was incubated at 37 °C for 15 minutes to 6 hours. The optical density at 630 nm was acquired with a conventional microplate reader. [130] Bacteriophages displaying the mouse IL- 2 cytokine activate the IL-2 receptor. To validate that cytokines maintain their biological activity when displayed on synthetic therapeutic bacteriophages, an experiment was performed using HEK-Blue™ IL-2 reporter cells to monitor activation of the IL-2 receptor (IL-2R) by bacteriophages displaying the mouse IL-2 (mIL-2). HEK-Blue™ IL-2 Cells were treated for 18h with either PBS (vehicle), 10¹¹ of bacteriophages displaying an anti-PD-Ll scLv on pill (phage-PD- Ll), 10¹¹ bacteriophage displaying mIL-2 on pIX and an anti-PD-Ll scLv on pill (mIL-2-phage-PD-Ll), or 0.2ng of mIL-2 (equimolar dose of mIL-2 to 10¹¹ mIL-2-phage-PD-Ll). Levels of IL-2R activation were monitored by measuring optical density at 630 nm and are reported on figure 4. Only the bacteriophage displaying mIL-2 (mIL-2-phage-PD-Ll) activates IL-2R as efficiently as mIL2, demonstrating that cytokines are biologically active when displayed on a synthetic therapeutic bacteriophage.

EXAMPLE 4: BACTERIOPHAGES THAT DISPLAY A CYTOKINE AND A CANCER CELL TARGETING MOIETY BINDS TO CANCER CELLS.

[131] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this Example are described in Table 3. Cells were typically grown in Luria broth Miller (LB) or on Luria broth agar Miller medium supplemented, when needed, with antibiotics at the following concentrations: ampicillin (Ap) 100 pg/mL, chloramphenicol (Cm) 34 pg/mL, kanamycin (Km) 50 pg/mL, nalidixic acid (Nx) 4 pg/mL, spectinomycin (Sp) 100 pg/mL, streptomycin (Sm) 50 pg/mL, sulfamethoxazole (Su) 160 pg/mL, tetracycline (Tc) 15 pg/mL, and trimethoprim (Tm) 32 pg/mL. All cultures were routinely grown at 37°C for no longer than 18 hours before use in the experiments. Bacteriophages were extracted from confluent bacterial culture (grown overnight) using the PEG precipitation protocol presented in example II.

[132] Cell culture. A20 lymphocyte B lymphoma cells were ordered from ATCC (TIB-208). Upon arrival, 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 the preparation of cells for all experiments. A frozen stock was generated after 4 passages and was used to start subsequent cultures for experimentation. Cells were maintained at density between 2xl0⁵ cell/mL and 2xl0⁶ cell/mL throughout all the experiments and grown in an atmosphere of 5% CO2 and 95% humidified air at 37°C.

[133] Immunofluorescence experiment. PD-LU A20 cancer cells were incubated with phage or mlL- 2-phage-PD-Ll at lO’VmL for 24h and then fixed on cover glasses placed on the bottom of 6-well plate using a 10% NBF solution. After being washed with PBS, cells are permeabilized the cells by adding 1 mL of PBS containing 0.1% Triton X-100 (PBS-Tx). The solution was removed, and cells were blocked using by adding PBS-Tx containing 2% BSA (PBS-Tx-BSA). Cells were then treated with an Fc block antibody (CD16/CD32 Monoclonal Antibody, Invitrogen). The cells were washed with PBS-Tx and then phages were revealed using an anti-M13-FITC (Progen) 1/200 diluted in PBS-Tx-BSA as diluent. The cells were washed with PBS-Tx and nucleus were stained using Hoescht dye at 1 pg/mL. The slides were washed in PBS-Tx and subsequently analyzed by confocal microscopy.

[134] Bacteriophages displaying the mouse IL-2 cytokine and the anti-PD-Ll scFv targeting moiety hinds to PD-L1⁺ cancer cells. To demonstrate that the bacteriophages displaying the mouse IL-2 cytokine and the anti-PD-Ll scFv (mIL-2-phage-PD-Ll) binds to cancer cells via its anti-PD-Ll scFv targeting moiety, an immunofluorescence experiment was performed. PD-L1⁺ A20 cells were incubated exposed to 10ⁿ/mLof either the naked phage (no therapeutic agents and cancer cell targeting moiety are displayed on the naked phage) or mIL-2-phage-PD-Ll for 24h to assess the binding of each compound to PD-L1⁺ cancer cells. Nucleus were revealed using the Hoescht dye, while phages and mIL-2-phage-PD-Ll were revealed using an anti-M13 antibody coupled to FITC following manufacturers protocols and recommendations. The experiment shows that only mIL-2-phage-PD-Ll is capable of binding to A20 cells, as indicated by the FITC signal observed on cancer cells following this treatment (Figure 5). This result demonstrates that the presence of the cancer cell targeting moiety, the anti-PD-Ll scFv of mIL-2-phage-PD-Ll, allows mIL-2- phage-PD-Ll to bind to PD-L1⁺ cancer cells and cover their surfaces. mIL-2-phage-PD-Ll can then recruit and activate the immune system to induce an antitumor immune response.

EXAMPLE 5: BACTERIOPHAGES THAT DISPLAY A CYTOKINE AND A CANCER CELL TARGEHNG MOIETY HA VE AN ENHANCED LOCAL ANTI-TUMORAL ACTIVITY.

[135] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this Example are described in Table 3. Cells were typically grown in Luria broth Miller (LB) or on Luria broth agar Miller medium supplemented, when needed, with antibiotics at the following concentrations: ampicillin (Ap) 100 pg/mL, chloramphenicol (Cm) 34 pg/mL, kanamycin (Km) 50 pg/mL, nalidixic acid (Nx) 4 pg/mL, spectinomycin (Sp) 100 pg/mL, streptomycin (Sm) 50 pg/mL, sulfamethoxazole (Su) 160 pg/mL, tetracycline (Tc) 15 pg/mL, and trimethoprim (Tm) 32 pg/mL. All cultures were routinely grown at 37°C for no longer than 18 hours before use in the experiments. Bacteriophages were extracted from confluent bacterial culture (grown overnight) using the PEG precipitation protocol presented in example II.

[136] Cell culture. CT26 colorectal carcinoma cells were ordered from ATCC (CRL-2638). Cells were cultured under optimal conditions in RPMI-1640 supplemented with 10% Fetal Bovine Serum (FBS) and penicillin-streptomycin (50 U/mL) in an atmosphere of 5% CO2 and 95% humidified air at 37°C.

[137] Tumor mice model. All experiments involving mice were strictly evaluated by the animal care committee of our local university (Universite de Sherbrooke) and procedures exposed animals to minimal stress and pain. Mice were provided with water and normal chow ad libitum and allowed to rest a minimum of 2 days after arrival. No more than 5 individuals shared the same cage and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were followed daily during the experiments.

[138] As a general guideline, this paragraph details the workflow of a typical mouse experiment. To generate solid tumors, 10⁶ CT26 cells, resuspended in 50 pL of PBS, were injected in the right flanks of the mice. Then, mice were kept under daily observation to measure tumor growth until a tumor size of 40-80 mm³ was reached. Next, mice received 50 pL of the treatment by intratumoral injection. Tumor size was next followed twice a week until clearance or until the tumors reached a volume of 1500 mm³, after which the mice were sacrificed, and the tumors were collected from the mice. The presence of metastases in other organs was also evaluated.

[139] Bacteriophages displaying the mouse IL-2 cytokine and the anti — PD-L1 scFv targeting moiety exhibit enhanced anti-tumoral activities. To assess the effect of adding a cytokine on the anti-tumoral activity of bacteriophages displaying a cancer cell targeting moiety, bacteriophages displaying a PD-L1 checkpoint inhibitors on pill and the mouse interleukin-2 (mIL-2) on pIX was developed using the process described in example I. Mice were injected subcutaneously with 10⁶ CT26 cells in their right flanks and were observed every two days to monitor tumor growth. When the tumor reached a volume between 50 and 100 mm³, mice were divided into five treatment groups, and all treatments were administered intratumorally on days 0, 4, and 7. The first group received an injection of vehicle (PBS) as control (Figure 6). The second group received an effective dose of 5xl0ⁿ molecules (14.4 ng) of recombinant mouse IL-2 (mIL-2) from Sino Biological (Figure 6). The third group received an effective dose of 10¹¹ bacteriophages displaying an anti-PD-Ll scFv on pill (phage-PD-Ll) (Figure 6). The fourth group received an effective dose consisting of 5xl0ⁿ molecules of mIL-2 and 10¹¹ molecules of phage-PD-Ll. The fifth, and final group, received an effective dose of 10¹¹ bacteriophage displaying mIL-2 on pIX and an anti-PD-Ll scFv on pill (mIL-2- Phage-PD-Ll). Tumor sizes were then monitored twice a week using a precise caliper until tumors were either eliminated or are too large to pursue the experiment. The experiment revealed that an enhanced antitumor activity is only observed when mIL-2 is physically coupled to the bacteriophages displaying the cancer-cell targeting PD-L1 checkpoint inhibitor. This demonstrates that coupling a cytokine to a bacteriophage, also displaying a cancer cell targeting moiety, provides synergistic antitumor effect.

EXAMPLE 6: BACTERIOPHAGES THAT DISPLAY A CYTOKINE AND A CANCER CELL TARGETING MOIETY HA VE A SYSTEMIC ANTI-TUMORAL ACTIVITY.

[140] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this Example are described in Table 3. Cells were typically grown in Luria broth Miller (LB) or on Luria broth agar Miller medium supplemented, when needed, with antibiotics at the following concentrations: ampicillin (Ap) 100 pg/mL, chloramphenicol (Cm) 34 pg/mL, kanamycin (Km) 50 pg/mL, nalidixic acid (Nx) 4 pg/mL, spectinomycin (Sp) 100 pg/mL, streptomycin (Sm) 50 pg/mL, sulfamethoxazole (Su) 160 pg/mL, tetracycline (Tc) 15 pg/mL, and trimethoprim (Tm) 32 pg/mL. All cultures were routinely grown at 37°C for no longer than 18 hours before use in the experiments. Bacteriophages were obtained from confluent bacterial culture (grown overnight) using the PEG precipitation protocol presented in example II.

[141] Cell culture. A20 lymphocyte B lymphoma cells were ordered from ATCC (TIB-208). Upon arrival, 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 the preparation of cells for all experiments. A frozen stock was generated after 4 passages and was used to start subsequent cultures for experiments. Cells were maintained at density between 2xl0⁵ cell/mL and 2xl0⁶ cell/mL throughout all the experiments and grown in an atmosphere of 5% CO2 and 95% humidified air at 37°C.

[142] Tumor mice model. All experiments involving mice were strictly evaluated by the animal care committee of our local university (Universite de Sherbrooke) and procedures exposed animals to minimal stress and pain. Mice were provided with water and normal chow ad libitum and allowed to rest a minimum of 2 days after arrival. No more than 5 individuals shared the same cage and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were followed daily during the experiments.

[143] As a general guideline, this paragraph details the workflow of a typical mouse experiment. To generate solid tumors, 5xl0⁶ A20 cells resuspended in 100 pL of PBS were injected in the right flanks of the mice, and then, 5xl0⁶ A20 cells were injected in the left flank 4 days later, resulting in mice bearing two tumors simultaneously. Mice were kept under daily observation to measure tumor growth until the right tumor volume reached of 50-100 mm³, then mice received 50 pL of the treatment by intratumoral injection. Tumor size was next followed twice a week until clearance or until the combined tumor volumes (right +left) reached 1500 mm³, after which mice are sacrificed.

[144] Bacteriophages displaying the mouse IL-2 cytokine and the anti — PD-L1 scFv targeting moiety have a systemic anti-tumoral activity. To assess if bacteriophages displaying a cytokine and a cancer cell targeting moiety have systemic antitumoral activity, the bacteriophages displaying a PD-L1 checkpoint inhibitor on pill and the mouse interleukin-2 (mIL-2) on pIX (described in Example I) was injected in mice bearing tumors on both flanks. However, only tumors from the right flank were injected with the drug to assess if a systemic antitumor immune response was triggered and induce the clearance of non-injected tumors present on the left flanks. Mice were divided into two groups, tumors in the right flanks were injected when they reached 50-100 mm³ and all treatments were administered intratumorally on days 0, 4, and 7. The first group received an injection of vehicle (PBS) as control (Figure 7). The second group received an effective dose of 10¹² bacteriophage displaying mIL-2 on pIX and an anti-PD-Ll scFv on pill (mIL-2- Phage-PD-L 1 , Figure 7) . Tumor sizes were then monitored twice a week using a precise caliper until tumors were either eliminated or are too large to pursue the experiment. The experiment revealed that, bacteriophage displaying mIL-2 on pIX and an anti-PD-Ll scFv not only induced the clearance of the injected tumor (right flank), but also of the non-injected tumor (left flank). These results showed that bacteriophages that display a cytokine and a cancer cell targeting moiety have a systemic anti-tumoral activity (i.e., an anti-tumoral activity that goes beyond the local anti-tumoral activity on the injected cell population).

EXAMPLE 7: THE LOCAL AND SYSTEMIC ANTITUMOR ACTIVITY OF BACTERIOPHAGES THAT DISPLAY A CYTOKINE AND A CANCER CELL TARGETING MOIETY IS MEDIATED

BY AN IMMUNE RESPONSE.

[145] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this Example are described in Table 3. Cells were typically grown in Luria broth Miller (LB) or on Luria broth agar Miller medium supplemented, when needed, with antibiotics at the following concentrations: ampicillin (Ap) 100 pg/mL, chloramphenicol (Cm) 34 pg/mL, kanamycin (Km) 50 pg/mL, nalidixic acid (Nx) 4 pg/mL, spectinomycin (Sp) 100 pg/mL, streptomycin (Sm) 50 pg/mL, sulfamethoxazole (Su) 160 pg/mL, tetracycline (Tc) 15 pg/mL, and trimethoprim (Tm) 32 pg/mL. All cultures were routinely grown at 37°C for no longer than 18 hours before use in the experiments. Bacteriophages were obtained from confluent bacterial culture (grown overnight) using the PEG precipitation protocol presented in Example II.

[146] Cell culture. N2Q lymphocyte B lymphoma cells were ordered from ATCC (TIB-208). Upon arrival, 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 the preparation of cells for all experiments. A frozen stock was generated after 4 passages and was used to start subsequent cultures for experimentation. Cells were maintained at density between 2xl0⁵ cell/mL and 2xl0⁶ cell/mL throughout all the experiments and grown in an atmosphere of 5% CO2 and 95% humidified air at 37°C.

[147] Tumor mice model. All experiments involving mice were strictly evaluated by the animal care committee of our local university (Universite de Sherbrooke) and procedures exposed animals to minimal stress and pain. Mice were provided with water and regular chow ad libitum and allowed to rest a minimum of 2 days after arrival. No more than 5 individuals shared the same cage and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were followed daily during the experiments.

[148] As a general guideline, this paragraph details the workflow of a typical mouse experiment. To generate solid tumors, 5xl0⁶ A20 cells resuspended in 100 pL of PBS were injected in the right flanks of the mice, and then, 5xl0⁶ A20 cells were injected in the left flank 4 days later, resulting in mice bearing two tumors simultaneously. Mice were kept under daily observation to measure tumor growth until the right tumor volume reached of 50-100 mm³, then mice received 50 pL of the treatment by intratumoral injection. Tumor size was next followed twice a week until clearance or until the combined tumor volumes (right +left) reached 1500 mm³, after which mice are sacrificed.

[149] Ex-vivo culture of micro-dissected tumor (MDT) on a chip. Tumors derived from the A20 cell line were collected from BALB/c mice and micro-dissected. MDT were kept in RPMI-1640 10% FBS PEN/STREP before and after loading on specially formulated ex-vivo culture chips (MISO Chip Inc.).

MDTs were distributed 8 MDTs/channel, 4 channels/chip and incubated at 37°C + 5% CO2 for 24 hours.

[150] Treatment of micro-dissected tumor (MDT) cultured on a chip. Treatments, synthetic therapeutic phage and Atezolizumab (anti-PD-Ll checkpoint inhibitor used as benchmarking reference), were diluted to 2xl0¹² molecules/mL in culture medium. The treatments were then used to wash the corresponding chip three times for each channel before being applied. Chips were next incubated at 37°C + 5% CO2 for 48 hours and the supernatant was collected and then sent to Eve Technologies for analysis of cytokine concentrations by ELISA. Chips with MDT exposed to the same volume of PBS were also generated and used as a control.

[151] Immune infiltration assay. BALB/c mice received an injection of 5xl0⁶ A20 cells subcutaneously in their right flank for engraftment. Upon reaching 75-150 mm³, tumors were treated three times over 7 days with an intratumoral injection of either PBS or 10¹¹ particles of bacteriophage displaying mIL-2 and an anti-PD-Ll (mIL-2-Phage-PD-Ll). On day 8, mice were euthanized, and tumors were collected then fixed in formalin. Tumor samples were next sent to the Plateforme d’Histologie de 1’Universite de Sherbrooke for permeabilization and paraffin embedding. Slides were next generated and stained with hematoxylin-eosin and digitalized. Images were then observed and processed using Qpath to discern immune cells from tumor cells.

[152] The local and systemic antitumor activity of the bacteriophages displaying the mouse IL-2 cytokine and the anti — PD-L1 scFv targeting moiety is mediated by an immune activation. The mechanisms underlying the local and systemic anti-tumoral immune response observed with bacteriophage displaying mIL-2 and an anti-PD-Ll (mIL-2-Phage-PD-Ll) were investigated. As mIL-2-Phage-PD-Ll is designed to activate the immune system by acting on multiple targets via the IL-2 cytokine, the anti-PD-Ll as well as the TLR9 agonist DNA and antigenic antigen of the phage, we hypothesized that mIL-2-Phage-PD-Ll treatment should induce a broad range of cytokines involved in the activation of different branches of the immune response. To identify the immune pathway involved in the antitumor activity of mIL-2-Phage-PD- Ll, we used chips to cultivate and treat tumors ex-vivo, which allows to follow the secretion of key cytokines overtime. As such, A20 tumors were micro-dissected and cultivated ex-vivo on specialized chips (MISO chip Inc.) and exposed to equimolar quantities of either PBS (equal volume as the two other treatments), mIL-2-Phage-PD-Ll, or Atezolizumab (anti-PD-Ll). Cytokines were next dosed in the culture supernatant after 2 days of treatment and key cytokines fold change against the PBS conditions were calculated (Figure 8). While Atezolizumab produced very modest changes in cytokine levels, mainly limited to the Thl response pathway, mIL-2-Phage-PD-Ll activated all major immune pathways, most notably the acute, Thl and Th 17 immune response pathways, showing a clear engagement of multiple immune cell types in the elimination of tumor cells. Thus, the anti-tumor response triggered by mIL-2- Phage-PD-Ll is at least mediated by CD8⁺ T-cells, myeloid cells, and likely with support from B-cells through the production of anti-tumor IgG.

[153] The local and systemic antitumor activity of the bacteriophages displaying the mouse IL-2 cytokine and the anti — PD-L1 scFv targeting moiety is mediated by a massive immune infiltration of the tumor and recruitment of immune cells. With the cytokine profiling experiment showing clear signs of immune activity, the presence of immune cells was next investigated. As such, mice bearing A20 tumors between 75-150 mm3 of volume were treated on days 0,4, and 7 with 10¹¹ particles of mIL-2-Phage-PD- L1 or PBS. Then, mice were sacrificed on day 8, the tumors were collected, fixed in formalin, and processed for histology by the Plateforme d’histologie de 1’universite de Sherbrooke. After Hematoxilin-Eosin staining, tumor tissues were observed for differences in infiltrating immune cell density (Figure 9). Tumors treated with mIL-2-Phage-PD-Ll were found to be highly infiltrated with immune cells while very little immune cells were detectable in the PBS control group (Figure 9A). Tumor tissues in the mIL-2-Phage- PD-L1 treated group also had bigger necrosis patches, showing active destruction of the tumor tissue (Figure 9B). Altogether, treatment with mIL-2-Phage-PD-Ll resulted in the recruitment of multiple types of immune cells, leading to tumor elimination.

EXAMPLE 8: THE THERAPEUTIC ACTIVITY OF BACTERIOPHAGES THAT DISPLAY A CYTOKINE AND A CANCER CELL TARGETING MOIETY IS MEDIATED BY A SYSTEMIC

LONG-TERM ANTITUMOR IMMUNE RESPONSE.

[154] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this Example are described in Table 3. Cells were typically grown in Luria broth Miller (LB) or on Luria broth agar Miller medium supplemented, when needed, with antibiotics at the following concentrations: ampicillin (Ap) 100 pg/mL, chloramphenicol (Cm) 34 pg/mL, kanamycin (Km) 50 pg/mL, nalidixic acid (Nx) 4 pg/mL, spectinomycin (Sp) 100 pg/mL, streptomycin (Sm) 50 pg/mL, sulfamethoxazole (Su) 160 pg/mL, tetracycline (Tc) 15 pg/mL, and trimethoprim (Tm) 32 pg/mL. All cultures were routinely grown at 37°C for no longer than 18 hours before use in the experiments. Bacteriophages were obtained from confluent bacterial culture (grown overnight) using the PEG precipitation protocol presented in example II.

[155] Cell culture. A20 lymphocyte B lymphoma cells were ordered from ATCC (TIB-208). Upon arrival, 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 the preparation of cells for all experiments. A frozen stock was generated after 4 passages and was used to start subsequent cultures for experimentations. Cells were maintained at density between 2xl0⁵ cell/mL and 2xl0⁶ cell/mL throughout all the experiments and grown in an atmosphere of 5% CO2 and 95% humidified air at 37°C.

[156] Tumor mice model. All experiments involving mice were strictly evaluated by the animal care committee of our local university (Universite de Sherbrooke) and procedures exposed animals to minimal stress and pain. Mice were provided with water and normal chow ad libitum and allowed to rest a minimum of 2 days after arrival. No more than 5 individuals shared the same cage and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were followed daily during the experiments.

[157] The therapeutic activity of the bacteriophages displaying the mouse IL-2 cytokine and the anti — PD-L1 scFv targeting moiety is mediated by a systemic long-term antitumor immune response. To assess if the antitumoral activity of bacteriophages displaying a cytokine and a cancer cell targeting moiety is mediated by a long-term systemic antitumor immune response, a rechallenge experiment was performed in cured mice and naive mice (mice that were never exposed to A20 cancer cells or previously treated with the bacteriophages displaying a cytokine and a cancer cell targeting moiety). Cured mice were obtained by treating mice bearing A20 cancer cell tumors on their right flanks with an effective dose of 10¹² the bacteriophages displaying a PD-L1 checkpoint inhibitor and the mouse interleukin-2 (mIL2-phage-PD-Ll). Tumors were injected with the treatment when they reached 50-100 mm³ in volume and the treatment was administered intratum orally on days 0, 4, and 7 (Figure 10) . The eight mice that were cured by the treatment, and for which tumors were completely cleared, were kept in the animal facility for 160 days. At day 160, cured mice, as well as naive mice, received an injection of 5xl0⁶ A20 cancer cells to induce the formation of a tumor, but this time, cancer cells were injected in the left flanks instead of the right flank (Figure 10). The experiment reveals that new tumors were able to form and grow only in naive mice, and tumors were systematically eliminated in previously cured mice. These results illustrate that mIL2-phage-PD-Ll treatment induced an adaptive long-term and systemic antitumor immune response capable of preventing the engraftment of new tumors, and hence, preventing tumor recurrence.

[158] Any element of any embodiment may be used in any embodiment. Although the invention has been described with reference to specific embodiments, it will be understood by those skilled in the art that various changes may be made, and equivalents may be substituted for elements thereof without departing from the true spirit and scope of the invention. In addition, modifications may be made without departing from the essential teachings of the invention. Identification of equivalent compositions, methods and kits are well within the skill of the ordinary practitioner and would require no more than routine experimentation, in light of the teachings of the present disclosure. Practice of the disclosure will be still more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the disclosure in any way.

[159] All references cited in this specification, and their references, are incorporated by reference herein in their entirety where appropriate for teachings of additional or alternative details, features, and/or technical background. [160] While the disclosure has been particularly shown and described with reference to particular embodiments, it will be appreciated that variations of the above-disclosed and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, that various presently unforeseen or unanticipated alternatives, modifications, variations or improvements therein may be subsequently made by those skilled in the art which are also intended to be encompassed by the following claims.

Claims

CLAIMS:

1. A bacteriophage simultaneously displaying at least one cytokine and at least one cancer cell targeting moiety.

2. The bacteriophage of claim 1, wherein the at least one cytokine is selected from: IL-la, IL-lb, IL- Ira, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, IL- 17C, IL-17D, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A/B/IL29, IL- 30, IL-31, IL-32, IL-33, IL-35, TNF alpha, LT alpha, LT beta, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1 BBL, TRAIL, RANK, FLT3 Ligand, G-CSF, GM- CSF, IFNa, IFNP, IFNco, IFNy, LIF, M-CSF, MIF, OSM, SCF, TGFpi, TGFP2, TGFP3, and TSLP ligand.

3. The bacteriophage of claim 1, wherein the at least one cytokine is selected from: IL-2, IL-7, IL- 12, IL-15, IL-18, IL-21, TNF, GM-CSF, FLT3 Ligand, and interferon gamma (IFN -gamma).

4. The bacteriophage of claim 1, wherein the at least one cytokine is IL-2.

5. The bacteriophage of claim 1, wherein the at least one cytokine is IL-15.

6. The bacteriophage of any one of claims 1 to 5, wherein the at least one cytokine can be displayed on pin, pVI, pVII, pVIII, or pIX, or a combination thereof.

7. The bacteriophage of any one of claims 1 to 5, wherein one of the at least one cytokine is displayed on pIX.

8. The bacteriophage of any one of claims 1 to 5, wherein one of the at least one cytokine is displayed on pin.

9. The bacteriophage of any one of claims 1 to 8, wherein the at least one cancer cell targeting moiety binds Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD59, CD24, E-Cadherin, cMet, MUC1, or CD 133 or targets a combination thereof.

10. The bacteriophage of any one of claims 1 to 8, wherein the at least one cancer cell targeting moiety targets PD-L1.

11. The bacteriophage of claim 10, wherein the PD-L1 targeting moiety is an anti-PD-Ll scFv or a fragment thereof.

12. The bacteriophage of any one of claims 1 to 11, wherein the at least one cancer cell targeting moiety is displayed on pill, pVI, pVII, pVIII, or pIX, or a combination thereof.

13. The bacteriophage of any one of claims 1 to 11 , wherein one of the at least one cancer cell targeting moiety is displayed on pIX.

14. The bacteriophage of any one of claims 1 to 11 , wherein one of the at least one cancer cell targeting moiety is displayed on pill.

15. The bacteriophage of any one of claims 1 to 14, the bacteriophage being a synthetic bacteriophage .

16. The bacteriophage of any one of claims 1 to 15, the bacteriophage being a therapeutic bacteriophage.

17. A method for reducing tumor size in a subject, the method comprising administering a therapeutically effective amount of the bacteriophage of any one of claims 1 to 16 to the subject.

18. A method for treating cancer in a subject, the method comprising administering a therapeutically effective amount of the bacteriophage of any one of claims 1 to 16 to the subject.

19. A method for triggering an immune response against cancer cells in a subject, the method comprising administering the bacteriophage of any one of claims 1 to 16 to the subject.

20. The method of any one of claims 17 to 19, wherein the administration is performed intratumorally.

21. A pharmaceutical composition comprising the bacteriophage of any one of claims 1 to 16, together with a suitable pharmaceutical carrier.

22. The pharmaceutical composition of claim 21, wherein the at least one cytokine is selected from: IL-Ia, IL-lb, IL-lra, IL-2, IL-3, IL-4, IL-6, IL-9, IL-10, IL-11, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17A, IL-17B, IL- 17C, IL-17D, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL- 28A/B/IL29, IL-30, IL-31, IL-32, IL-33, IL-35, TNF alpha, LT alpha, LT beta, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, FLT3 Ligand, G-CSF, GM-CSF, IFNa, IFNP, IFNco, IFNy, LIF, M-CSF, MIF, OSM, SCF, TGFpi, TGFP2, TGFP3, and TSLP ligand.

23. The pharmaceutical composition of claim 21 or 22, wherein the at least one cytokine is selected from: IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNF, GM-CSF, FLT3 Ligand, and interferon gamma (IFN- gamma).

24. The pharmaceutical composition of any one of claims 21 to 23, wherein the at least one cytokine is IL-2.

25. The pharmaceutical composition of any one of claims 21 to 23, wherein the at least one cytokine is IL-15.

26. The pharmaceutical composition of any one of claims 21 to 25, wherein the at least one cytokine is displayed on pill, pVI, pVII, pVIII, or pIX or a combination thereof.

27. The pharmaceutical composition of any one of claims 21 to 26, wherein the at least one cytokine is displayed on pIX.

28. The pharmaceutical composition of any one of claims 21 to 26, wherein the at least one cytokine is displayed on pill.

29. The pharmaceutical composition of any one of claims 21 to 28, wherein the at least one cancer cell targeting moiety targets Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD24, E-Cadherin, cMet, MUC1, or CD 133 or a combination thereof.

30. The pharmaceutical composition of any one of claims 21 to 29, wherein the at least one cancer cell targeting moiety targets PD-L1.

31. The pharmaceutical composition of claim 30, wherein the PD-L1 targeting moiety is an anti-PD- L1 scFv or a fragment thereof.

32. The pharmaceutical composition of any one of claims 21 to 31, wherein the at least one cancer cell targeting moiety can be displayed on pill, pVI, pVII, pVIII, or pIX or a combination thereof.

33. The pharmaceutical composition of any one of claims 21 to 32, wherein the at least one cancer cell targeting moiety is displayed on pill.

34. A method for reducing tumor size in a subject, the method comprising administering the pharmaceutical composition of any one of claims 21 to 33 to the subject.

35. A method for treating a cancer in a subject, the method comprising administering the pharmaceutical composition of any one of claims 21 to 33 to the subject.

36. A method for triggering an immune response against cancer cells in a subject, the method comprising administering the pharmaceutical composition of any one of claims 21 to 22 to the subject.

37. The method of any one of claims 34 to 36, wherein the administration is performed intratumorally.