Therapeutic bacteriophages exhibiting cancer cell targeting moieties and cytokines for treatment of cancer

Abstract

The present technology relates generally to a bacteriophage that simultaneously exhibits at least one cytokine and at least one cancer cell targeting moiety, and methods of using the bacteriophage to treat cancer.

Description

Technical Field

[0001] The technology of the present invention generally relates to a phage engineered to display a cancer cell targeting moiety and a cytokine. The technology of the present invention also generally relates to the use of such phages in the treatment of cancer. Background Art

[0002] Despite significant progress in cancer treatment, cancer remains the second leading 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 disease to treat, often requiring simultaneous action on several therapeutic targets to maximize the chance of treatment success. This strategy is called combination therapy, where clinicians treat patients by combining two or more therapeutic agents (Mokhtari et al., Oncotarget, June 6, 2017; 8(23):38022 - 38043; incorporated herein by reference). Combination therapy inhibits or eliminates cancer cells by targeting different pathways, provides better results than monotherapy via synergistic effects, and has become the cornerstone of cancer treatment. However, combination therapy has an important limitation: due to the increased treatment, the cost of care and the risk of side effects of combination therapy also increase.

[0003] A solution to this problem is to adopt a treatment modality capable of multitasking therapeutic activities, allowing the use of a single agent to achieve combination therapy. To address this problem, a phage capable of multitasking therapeutic activities has been developed to provide a next - generation immunotherapy for the treatment of cancer (WO2022073127; incorporated herein by reference). Such phages are used as therapeutic scaffolds to display combinations of immunomodulators. Since phages can multitask therapeutic activities, their anti - tumor activity is combined with the combination of therapeutic molecules they carry and the therapeutic synergies that these combinations can produce.

[0004] In view of the above, finding a combination of therapeutic molecules that exhibit synergistic effects when displayed on phages is the key to enhancing the anti - tumor activity of treatment. Summary of the Invention

[0005] In accordance with various aspects, the technology of the present invention relates to a phage that simultaneously displays at least one cytokine and at least one cancer cell targeting moiety. In some cases, the phage of the technology of the present invention is a synthetic phage. In some cases, the phage of the technology of the present invention is a therapeutic phage. In some cases, the phage of the technology of the present invention is a synthetic therapeutic phage. In some cases, the cytokine is selected from: IL-1α, IL-1b, IL-1ra, 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, TNFα, LTα, LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, FLT3 ligand, G-CSF, GM-CSF, IFNα, IFNβ, IFNω, IFNγ, LIF, M-CSF, MIF, OSM, SCF, TGFβ1, TGFβ2, TGFβ3, and TSLP ligand. In some cases, the cancer cell targeting moiety targets Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD59, CD24, E-cadherin, cMet, MUC1, or CD133, or a combination thereof.

[0006] In accordance with various aspects, the technology of the present invention relates to a method for reducing the tumor size of a subject, the method comprising administering to the subject a therapeutically effective amount of a phage as defined herein.

[0007] In accordance with various aspects, the technology of the present invention relates to a method for treating cancer in a subject, the method comprising administering to the subject a therapeutically effective amount of a phage as defined herein.

[0008] In accordance with various aspects, the technology of the present invention relates to a pharmaceutical composition comprising a phage that simultaneously displays a cytokine and a cancer cell targeting moiety and a suitable pharmaceutical carrier.

[0009] In accordance with various aspects, the technology of the present invention relates to a method for reducing the tumor size of a subject, the method comprising administering to the subject a pharmaceutical composition as defined herein.

[0010] In various aspects, the technology of the present invention relates to a method for treating cancer in a subject, the method comprising administering to the subject a pharmaceutical composition as defined herein.

[0011] In various aspects, the technology of the present invention relates to a composition comprising a phage displaying a cytokine and a moiety targeting cancer cells.

[0012] In various aspects, the technology of the present invention relates to a phage, wherein the phage displays on one or more of its coat proteins one or more therapeutic agents targeting cancer cell markers, and wherein the phage displays on one or more of its coat proteins one or more cytokines.

[0013] In various aspects, the technology of the present invention relates to a method for treating cancer in a subject in need thereof, the method comprising administering to the subject in need thereof an effective amount of a phage as defined herein.

[0014] In various aspects, the technology of the present invention relates to the use of an effective amount of a phage as defined herein for treating cancer in a subject in need thereof.

[0015] In various aspects, the technology of the present invention relates to the use of an effective amount of a phage as defined herein in the manufacture of a medicament for treating cancer in a subject.

[0016] In various aspects, the technology of the present invention relates to a kit comprising a phage as defined herein and instructions for administering the medicament to a subject. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 A, 1B, and 1C are schematic representations of an overview of phage production and vectors according to one embodiment of the technology of the present invention. Figure 1 A is a schematic representation of an exemplary configuration of a bacterial production strain secreting phage. The phage secretion system can be composed of a combination of a phage machinery vector and a phage scaffold vector. Figure 1 B shows examples of phage machinery vectors: M13K07, pTAT004, and pTAT025. Figure 1 C shows examples of phage scaffold vectors: pTAT002, pTAT013 (including derivatives pTAT044 and pTAT070), and pTAT014 (including derivatives pTAT017, pTAT060, and pTAT071).

[0018] Figure 2Schematic representation of the mode of action of a phage that displays an anti-PD-L1 checkpoint inhibitor scFv as a cancer cell targeting moiety and IL-2 as an immunomodulatory cytokine. The phage binds to cancer cells via the anti-PD-L1 scFv displayed on pIII, which localizes the phage to cancer cells and inhibits the PD-L1 checkpoint to enhance T cell activation. The presence of the IL-2 cytokine conjugated to the phage acts synergistically to further enhance the anti-tumor immune response and improve tumor clearance. Meanwhile, filamentous phage can be internalized by cancer cells via endocytosis, thereby providing additional innate immunogenic activity through: (1) activation of TLR9, which initiates an immune response; and (2) antigens from the phage can be presented via MHC molecules, which activates cytotoxic T cells and subsequently eliminates the cells presenting these antigens.

[0019] Figure 3 A and 3B are diagrams showing that phage particles can dual display functional cancer cell targeting moieties and cytokines. Figure 3 A: Binding of phage to PD-L1 was evaluated by indirect ELISA. The figure shows the HRP signal of the ELISA, which quantifies the binding of different phage preparations to immobilized human PD-L1 protein. PEG-NaCl purified phage was diluted at 1×10 11 phage / ml in TBS. The signal was measured using an anti-pVIII-HRP antibody to detect the presence of phage particles. Figure 3 B: Phage displaying recombinant cytokines on pIX was evaluated by sandwich ELISA. The figure shows the HRP signal of the ELISA, which quantifies the binding of different phage preparations to immobilized anti-mouse IL2 antibody or anti-human IL15 antibody. PEG-NaCl purified phage was diluted at 1×10 11 phage / ml in TBS. The signal was measured using an anti-pVIII-HRP antibody to detect the presence of phage particles.

[0020] Figure 4 is a diagram showing that phage can display bioactive cytokines. The figure shows the signal measured at 630 nm by HEK-Blue TM IL-2 reporter cell assay, which quantifies the activation of the IL-2 receptor by: PBS (vehicle), 10 11 phage displaying anti-PD-L1 scFv (phage-PD-L1), 10 11 phage displaying anti-PD-L1 scFv and murine IL-2 (mIL-2) cytokine (mIL-2-phage-PD-L1) or 0.2 ng mIL-2.

[0021] Figure 5 is a microscope image that shows that phages displaying cytokines and cancer cell targeting moieties can bind to cancer cells. PD-L1 + A20 cancer cells were exposed to phages (not displaying cancer targeting moieties) or to phages displaying the mIL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1). Binding of phages and mIL-2-phage-PD-L1 was then revealed using an anti-M13 antibody conjugated to FITC.

[0022] Figure 6 is a figure showing the physical conjugation of the IL-2 cytokine with phages targeting cancer cells to provide a synergistic anti-tumor effect. Tumors were implanted into mice by injecting 10 6 CT26 cancer cells into the right flank of the mice. Treatments were administered when the tumor volume was between 50 - 80 mm 3 . Then the individual tumor volumes of each mouse were measured. Treatments were performed by intratumoral injection of the following on days 0, 4, and 7: PBS, 5×10 11 mouse IL-2 (mIL-2) molecules, 10 11 phage particles displaying anti-PD-L1 scFv (phage-PD-L1), 5×10 11 mIL-2 molecules and 10 11 phage-PD-L1 particles, or 10 11 phage particles displaying the mIL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1). Tumor volume was calculated by multiplying the largest measured value by the square of the perpendicular measurement and dividing by two (solid line = mice with cleared tumors, dashed line = mice without cleared tumors).

[0023] Figure 7 is a figure showing that phages displaying cytokines and cancer cell targeting moieties have systemic anti-tumor activity. Tumors were implanted into both flanks of mice. 5×10 6 A20 cancer cells were injected into the right flank, and 4 days later, 5×10 6 A20 cancer cells were injected into the left flank. The right tumor was treated when the tumor volume was between 50 - 100 mm 3 , and the left tumor was left untreated. Treatments were performed by intratumoral injection of the following on days 0, 4, and 7: PBS or 10 12 phage particles displaying the mIL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1). Tumor volumes of the injected and non-injected tumors were calculated by multiplying the largest measured value by the square of the perpendicular measurement and dividing by two. Tumor clearance of the injected and non-injected tumors under the two treatments was indicated.

[0024] Figure 8 This is a heat map showing that the anti-tumor activity of phages displaying cytokine and cancer cell targeting moieties is mediated by the immune response. The phage displaying the mIL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1) activates all major immune pathways. A20 tumors of 75 - 150 mm 3 were excised from BALB / c mice, microdissected, and cultured ex vivo on a chip. The microdissected tumors were then treated with PBS (control), mIL-2-phage-PD-L1, or the anti-PD-L1 checkpoint inhibitor atezolizumab (benchmark reference). The fold changes in cytokine levels induced by the treatment were evaluated. The cytokine fold changes relative to the corresponding PBS condition were shown as a heat map on a logarithmic scale.

[0025] Figure 9 Figures 9A and 9B are a graph and a histological image respectively showing that the anti-tumor activity of phages displaying cytokine and cancer cell targeting moieties is mediated by massive immune infiltration of the tumor. BALB / c mice bearing A20 tumors received intratumoral administration of PBS or the phage displaying the mIL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1) on days 0, 4, and 7. On day 8 of the experiment, the mice were sacrificed, the tumors were excised and processed histologically. Images of the tumors were analyzed using Qpath to identify and distinguish the cells within the tissue. Using machine learning methods, tumor cells and immune cells within the tumors were detected and automatically quantified in different samples ( Figure 9 A). Representative images of tumors treated with PBS or mIL-2-phage-PD-L1 are presented ( Figure 9 B). The two images have the same scale, and tissue damage as well as tumor shrinkage can be observed. The pale portion of the tissue that can be seen in most areas of the tumors treated with mIL-2-phage-PD-L1 indicates the presence of necrosis.

[0026] Figure 10 This is a graph showing that the therapeutic activity of phages displaying cytokine and cancer cell targeting moieties is mediated by long-term adaptive and systemic anti-tumor immune responses. Tumors were transplanted by injecting 5×10 6 A20 cancer cells into the right flank of mice. Then, when the tumor volume was between 80 - 100 mm 3 the tumors were treated. On days 0, 4, and 7, 10 11treated with phage particles presenting the mIL-2 cytokine and the anti-PD-L1 scFv (mIL-2-phage-PD-L1). Mice in which the tumors were completely cleared were considered cured and were maintained for 160 days. On day 160, 5 × 10 6 A20 cancer cells were injected to form new tumors, but this time on the left flank of the mice. The same was done with untreated mice ( mice) that had never been exposed to A20 cancer cells and had never been treated with mIL-2-phage-PD-L1. Then the tumor volume of each mouse was measured. The tumor volume was calculated by multiplying the largest measured value by the square of the perpendicular measurement and dividing by two. DETAILED DESCRIPTION

[0027] As used herein, unless the context clearly dictates otherwise, the singular forms “a,” “an,” and “the” include plural referents.

[0028] Numeric ranges are inclusive of the numbers defining the range (e.g., the recitation of 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0029] Whether or not the term “about” is explicitly used herein, each quantity given herein is meant to refer to the actual given value, and is also meant to refer to an approximation of this given value that would be reasonably inferred based on ordinary skill in the art, including equivalents and approximations due to the experimental and / or measurement conditions for this given value. By way of example, in the context of a given value or range, the term “about” refers to a value or range within 20%, preferably 15%, more preferably 10%, more preferably 9%, more preferably 8%, more preferably 7%, more preferably 6%, and still more preferably 5% of the given value or range.

[0030] The phrase “and / or” as used herein is to be understood as specifically disclosing each of the two specified features or components, whether or not in conjunction with the other. For example, “A and / or B” is to be understood as specifically disclosing (i) A, (ii) B, and (iii) each of A and B, as if each were set forth individually herein.

[0031] "Inducible promoter" refers to a regulatory region operably linked to one or more genes, wherein the expression of the one or more genes is increased in the presence of an inducer of the regulatory region or in the absence of a repressor of the regulatory region. Inducible promoters can be induced by exogenous environmental conditions, which refer to the environment or circumstances that induce the promoters described herein. Exogenous environmental conditions refer to environmental conditions external to the intact (non-lysed) engineered microorganism, endogenous or native environmental conditions of the tumor environment, or the host subject environment, or refer to exogenously introduced environmental perturbations. Exogenous environmental conditions can be, but are not limited to, hypoxic, microaerobic or anaerobic conditions, or low intracellular and / or extracellular pH, wavelength, reactive oxygen species (ROS) levels, the 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). The corresponding Fnr-responsive promoter, Anr (anaerobic nitrate respiration)-responsive promoter, and Dnr (dissimilatory nitrate respiration regulator)-responsive promoter are known in the art (see, for example, 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, the phoBR-responsive promoter. Examples of temperature-controlled promoters include, but are not limited to, the pL and / or pR bacteriophage λ promoters and the use of the mutant cI857 repressor. Examples of ROS level-dependent transcription factors include, but are not limited to, OxyR. The corresponding OxyR-responsive promoters include, but are not limited to, TrxCp, HemHp, sufA, AhpCp1000, AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, DsbGp. Inducible promoters can also be induced by one or more exogenous molecules. Exogenous molecules refer to molecules that do not naturally occur in the intact (non-lysed) engineered microorganism. Examples of exogenous molecules and their corresponding inducible promoters 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). Inducible promoters can also be induced by one or more endogenous molecules. Endogenous molecules refer to molecules that are naturally produced in the intact (non-lysed) engineered microorganism. Examples of endogenous molecules and their corresponding inducible promoters include, but are not limited to, diaminopimelic acid and the PdapA promoter, N-acyl-homoserine lactone and the PluxI.

[0032] Inducible promoters can include one or more regulatory elements, including but not limited to enhancer sequences, response elements, protein recognition sites, induction elements, promoter control elements, protein-binding sequences, 5′ and 3′ untranslated regions, transcription start sites, termination sequences, polyadenylation sequences, riboswitches, and introns.

[0033] The technology of the present invention is explained in more detail below. This description is not intended to be a detailed catalog of all the different ways in which the technology can be implemented, or a detailed catalog of all the features that can be added to the technology of the present invention. For example, features described with respect to one embodiment can be incorporated into other embodiments, and features described with respect to a particular embodiment can be deleted from that embodiment. Additionally, many variations and additions to the various embodiments presented herein will be apparent to those skilled in the art in light of the present disclosure, and these variations and additions do not depart from the technology of the present invention. Accordingly, the following description is intended to illustrate some particular embodiments of the technology and not to specify exhaustively all of its permutations, combinations, and variations.

[0034] Components of the phage

[0035] A solution for treating cancer by acting on several therapeutic targets simultaneously is to use a molecular scaffold capable of conjugating several immunomodulatory molecules to generate a potent anti-tumor immune response. Filamentous phages are large immunogenic biological structures on which therapeutic proteins or peptides can be displayed to produce synthetic therapeutic phages. The natural immunogenicity of filamentous phages is mediated by: (1) their CpG-containing genome, which can act as a TLR9 agonist and trigger an innate immune response (Sartorius et al., NPJ Vaccines, October 28, 2021; 6(1):127; incorporated herein by reference), and (2) the presence of immunogenic antigens in their coat proteins, which can stimulate cytotoxic T cells to eliminate cells presenting these antigens on MHC-1 after endocytosis of the phage (Gaubin et al., DNA And Cell Biology, Vol. 22, No. 1, 2003; incorporated herein by reference). Thus, the natural immunogenic activities of filamentous phages can be exploited in combination with specific immunomodulatory proteins or peptides to enhance their anti-tumor activity.

[0036] According to various embodiments, the technology of the present invention relates to an operable phage for treating cancer, such as the phage described in WO2022073127 (incorporated herein by reference). In some cases, the phage is immunogenic. In some additional cases, the phage can display monospecific or multispecific therapeutic proteins.

[0037] In some embodiments, the technology of the present invention relates to a phage displaying one or more cancer cell targeting moieties and one or more cytokines, and wherein the combination of these therapeutic molecules acts synergistically to enhance tumor elimination.

[0038] In some embodiments, the technology of the present invention relates to a phage displaying one or more cancer cell targeting moieties and one or more cytokines, and wherein the combination of these therapeutic molecules can act synergistically to enhance tumor elimination only when physically conjugated to the phage.

[0039] Production of phages

[0040] In some embodiments, the phages of the technology of the present invention are produced according to that described in WO2022073127 (incorporated herein by reference) Figure 1 and as described above.

[0041] Therapeutic activity of phages

[0042] Synthetic phages displaying cancer cell targeting moieties:

[0043] In some embodiments, the phages of the technology of the present invention display one or more cancer cell targeting moieties that are capable of recognizing one or more cancer cell markers. The types of targeting moieties displayed by the phages are disclosed in WO2022073127 (incorporated herein by reference) and can be selected from, but not limited to: antibodies, antibody mimetics, natural receptors and ligands, and peptides. In some embodiments, the one or more targeting moieties can be displayed on pIII, pVI, pVII, pVIII, and / or pIX (as depicted in WO2022073127 Figure 2 ; incorporated herein by reference).

[0044] In some embodiments, the cancer cell markers recognized by the targeting moieties are molecules present on cancer cells.

[0045] In another embodiment, the cancer cell marker is a molecule overexpressed by cancer cells.

[0046] In yet another embodiment, the cancer marker is a molecule specifically expressed by cancer cells.

[0047] In some embodiments, the cancer cell markers targeted by the one or more targeting moieties are proteins.

[0048] In another embodiment, the cancer cell markers targeted by the one or more targeting moieties are selected from, but not limited to: Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD59, CD24, E-cadherin, cMet, MUC1, and CD133. In one embodiment, the cancer marker targeted by the one or more targeting moieties is PD-L1.

[0049] Phage displaying cytokines:

[0050] In some embodiments, the phages of the technology of the present invention display tumor targeting moieties and one or more cytokines ( Figure 2 ). Cytokines are molecules that can regulate the immune response by stimulating and / or inducing the differentiation of effector T cells (such as CD4+ and / or CD8+) and promoting the activation of B cells, macrophages, and / or dendritic cells. In some embodiments, the one or more cytokines can be displayed on pIII, pVI, pVII, pVIII, and / or pIX (as depicted in WO2022073127 Figure 2 ; incorporated herein by reference).

[0051] In some embodiments, the one or more cytokines that stimulate the immune response can be any cytokines known to stimulate and / or induce the differentiation and / or activation of T cells, B cells, macrophages, and dendritic cells.

[0052] In some embodiments, the cytokine displayed on the phage can be selected from, but not limited to: IL-1α, IL-1b, IL-1ra, 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, TNFα, LTα, LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, FLT3 ligand, G-CSF, GM-CSF, IFNα, IFNβ, IFNω, IFNγ, LIF, M-CSF, MIF, OSM, SCF, TGFβ1, TGFβ2, TGFβ3 and TSLP ligand. In some other cases, the cytokine displayed on the phage can be selected from IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNF, GM-CSF, FLT3 ligand and interferon γ (IFN-γ). In another embodiment, the cytokine displayed on the phage is IL-2. In yet another embodiment, the cytokine displayed on the phage is IL-15.

[0053] Therapeutic uses of synthetic phages and compositions comprising the same

[0054] Therapeutic methods

[0055] In some embodiments, the synthetic therapeutic phages of the technology of the present invention can be used for, but not limited to, treating cancer and / or tumors. The tumors can be malignant or benign. Types of cancer include, but are not limited to, adrenal cancer, adrenocortical carcinoma, anal cancer, appendiceal cancer, bile duct cancer, bladder cancer, bone cancer (e.g., Ewing sarcoma tumor, osteosarcoma, malignant fibrous histiocytoma), brain cancer (e.g., astrocytoma, brainstem glioma, craniopharyngioma, ependymoma), bronchial tumor, central nervous system tumor, breast cancer, Castleman disease, cervical cancer, colon cancer, rectal cancer, colorectal cancer, endometrial cancer, esophageal cancer, eye cancer, gallbladder cancer, gastrointestinal cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, gestational trophoblastic disease, heart cancer, Kaposi sarcoma, kidney cancer, laryngeal 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 cancer, oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdoid tumor, salivary gland cancer, sarcoma, skin cancer (e.g., basal cell carcinoma, melanoma), small intestine cancer, stomach cancer, teratoma, testicular cancer, throat cancer, thymic cancer, thyroid cancer, abnormal childhood cancer, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom macroglobulinemia, and Wilms tumor. In some embodiments, the associated symptoms include, but are not limited to, anemia, loss of appetite, irritation of the inner wall of the bladder, bleeding and bruising (thrombocytopenia), changes in taste and smell, constipation, diarrhea, dry mouth, difficulty swallowing, edema, fatigue, hair loss (alopecia), infection, infertility, lymphedema, oral ulcers, nausea, pain, peripheral neuropathy, tooth decay, urinary tract infection, and / or memory and concentration problems.

[0056] In some embodiments, the method can include preparing a pharmaceutical composition containing at least one of the phages described herein, and administering the pharmaceutical composition to a subject in a therapeutically effective amount. The phage can be administered locally, such as intratumorally, or around the tumor into the tissue or blood supply vessels, intramuscularly, intraperitoneally, orally, or topically. The phage can be administered systemically by infusion or injection, such as intravenously or intraarterially.

[0057] In certain embodiments, administering the pharmaceutical composition to a subject reduces cell proliferation, tumor growth, and / or tumor volume in the subject. In some cases, the methods of the present disclosure can reduce cell proliferation, tumor growth, and / or tumor volume by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to the levels in untreated or control subjects. In some embodiments, the reduction is measured by comparing the cell proliferation, tumor growth, and / or tumor volume in the subject before and after administering the pharmaceutical composition. In some embodiments, the method of treating or ameliorating cancer in a subject ameliorates one or more symptoms of the cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more.

[0058] In certain embodiments, administering the pharmaceutical composition of the technology of the present invention to a subject reduces cell proliferation, tumor growth, and / or tumor volume in the treated tumor and untreated tumors through abscopal effects and systemic immune responses.

[0059] In some cases, the methods of the present disclosure reduce cell proliferation, tumor growth, and / or tumor volume in the treated tumor and untreated tumors by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to the levels in untreated or control subjects. In some embodiments, the reduction is measured by comparing the cell proliferation, tumor growth, and / or tumor volume in the subject before and after administering the pharmaceutical composition.

[0060] In some embodiments, the method of treating or ameliorating cancer of the technology of the present invention ameliorates one or more symptoms of the cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more.

[0061] In certain embodiments, the method of the technology of the present invention elicits a long-term systemic anti-tumor immune response in a subject, thereby preventing tumor recurrence.

[0062] Before, during, and after administration of the pharmaceutical composition of the present invention, cancer cells and / or biomarkers of a subject can be measured in a biological sample such as blood, serum, plasma, urine, peritoneal fluid, and / or a biopsy from a tissue or organ.

[0063] In some embodiments, the method can include administering the composition of the present invention to reduce the tumor volume of the subject to an undetectable size, or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the tumor volume of the subject prior to treatment. In other embodiments, the method can include administering the composition of the present invention to reduce the cell proliferation rate or tumor growth rate of the 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.

[0064] Therapies using synthetic therapeutic bacteriophages with immune-based anti-cancer activity may exhibit a response pattern different from that observed with traditional cytotoxic therapies. For example, tumors treated with immune-based therapies may increase in size before regressing, and / or new lesions may appear (Agarwala et al., 2015; incorporated herein by reference). The increase in tumor size may be due to massive infiltration of lymphocytes and macrophages that are not normally present in tumor tissue. Additionally, the response time may be slower than that associated with standard therapies such as cytotoxic therapies. In some embodiments, delivery of the anti-cancer molecule can modulate the growth of the subject's tumor and / or improve the symptoms of cancer while temporarily increasing the volume and / or size of the tumor.

[0065] Bacteriophages may be destroyed by defense factors in tissues or serum, for example, within hours or days after administration (Sonnenborn et al., Microbioal Ecology in Health and Diseases, 2019, 21:3; incorporated herein by reference). Thus, the pharmaceutical composition containing bacteriophages can be re-administered at a therapeutically effective dose and frequency.

[0066] The pharmaceutical composition can 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 therapy, tumor-infiltrating lymphocyte therapy, natural or engineered cell therapies (e.g., TCR or CAR-T), natural killer cell therapy, Fc-mediated ADCC therapy, therapy using bispecific soluble scFv that links cytotoxic T cells to tumor cells, and soluble TCR with effector functions), stem cell therapy, and targeted therapies using antibodies or chemical compounds (e.g., BRAF or vascular endothelial growth factor inhibitors), phages.

[0067] In some embodiments, the phage can be administered sequentially, simultaneously, or subsequent to the administration of one or more chemotherapeutic agents selected from, but not limited to, methotrexate, 5-Fluorouracil, Gemcitabine (Gemzar), and

[0068] In some embodiments, the phage is administered sequentially, simultaneously, or subsequent to the administration of one or more mRNA-based drugs.

[0069] In some embodiments, the phage is administered sequentially, concurrently, or subsequent to the administration of one or more of the following checkpoint inhibitors or other antibodies known in the art or described herein. Non-limiting examples include CTLA-4 antibodies (including but not limited to ipilimumab and tremelimumab (CP675206)), anti-4-1BB (CD137, TNFRSF9) antibodies (including but not limited to PF-05082566 and urelumab), anti-CD134 (OX40) antibodies (including but not limited to anti-OX40 antibody (Providence Health and Services)), anti-PD1 antibodies (including but not limited to nivolumab, pidilizumab, pembrolizumab (MK-3475 / SCH900475, lambrolizumab, REGN2810, PD1 (Agenus)), anti-PD-L1 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).

[0070] In some embodiments, the phage is administered sequentially, simultaneously with, or subsequent to the administration of one or more antibodies selected from anti-phosphatidylserine antibodies (including but not limited to bavituximab), TLR9 antibodies (including but not limited to MGN1703), PD1 antibodies (including but not limited to SHR-1210 (Incyte / Jiangsu Hengrui)), anti-OX40 antibodies (including but not limited to OX40 (Agenus)), anti-Tim3 antibodies (including but not limited to anti-Tim3 (Agenus / INcyte)), anti-Lag3 antibodies (including but not limited to anti-Lag3 (Agenus / INcyte)), anti-B7H3 antibodies (including but not limited to enoblituzumab (MGA-271), anti-CT-011 (hBAT, hBAT1) described in WO2009101611 (incorporated herein by reference)), anti-PDL-2 antibodies (including but not limited to AMP-224 (described in WO2010027827 and WO2011066342; incorporated herein by reference)), anti-CD40 antibodies (including but not limited to CP-870,893), anti-CD40 antibodies (including but not limited to CP-870,893).

[0071] The dosage and frequency of administration of the pharmaceutical composition can be selected based on the severity of the symptoms and the progression of the cancer. The treatment clinic can select an appropriate therapeutically effective dosage and / or frequency of administration of the pharmaceutical composition and formulation

[0072] Pharmaceutical composition and formulation

[0073] A pharmaceutical composition comprising a phage of the technology of the present invention can be used for treating, managing, ameliorating, and / or preventing cancer. There is provided a pharmaceutical composition of the technology of the present invention that comprises only one or more phages of the technology of the present invention or a combination of one or more phages of the technology of the present invention with a prophylactic agent, a therapeutic agent, and / or a pharmaceutically acceptable carrier.

[0074] In certain embodiments, the pharmaceutical composition comprises a phage of the technology of the present invention, each phage being engineered to display a recombinant protein described herein, such as one or more anti-cancer molecules.

[0075] The pharmaceutical composition of the present invention's technology can be formulated in a conventional manner using one or more physiologically acceptable carriers, which include excipients and adjuvants that assist in processing the active ingredient into a composition for pharmaceutical use. Methods for formulating pharmaceutical compositions are known in the art (see, for example, "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical composition is tableted, freeze-dried, directly compressed, conventionally mixed, dissolved, granulated, milled, emulsified, encapsulated, entrapped, or spray-dried to form tablets, granules, nanoparticles, nanocapsules, microcapsules, microtablets, pills, or powders, which may be enteric-coated or uncoated. The appropriate formulation depends on the route of administration.

[0076] The phage can be formulated into a pharmaceutical composition in any suitable dosage form (such as liquid, capsule, sachet, hard capsule, soft capsule, tablet, enteric-coated tablet, suspension powder, granule, or matrix sustained-release form for oral administration) and for any suitable type of administration (such as oral, topical, injection, intravenous, subcutaneous, intratumoral, peritumoral, immediate release, pulsed release, delayed release, or sustained release). A suitable dose of the phage can be in the range of about 10 4 to 10 16 phage particles. The composition can be administered once or multiple times per day, week, month, or year. The composition can be administered before, during, or after a meal. In one embodiment, the pharmaceutical composition is administered before the subject has a meal. In one embodiment, the pharmaceutical composition is administered with a meal. In one embodiment, the pharmaceutical composition is administered after the subject has a meal.

[0077] Phages can be formulated into pharmaceutical compositions that contain one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surfactants, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, the pharmaceutical compositions can include, but are not limited to, the addition of calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars, and various types of starches, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and surfactants, including, for example, polysorbate 20. In some embodiments, the phages of the present inventive technique can be formulated in a sodium bicarbonate solution, such as a 1 molar sodium bicarbonate solution (to buffer the acidic cellular environment, such as the stomach). Phages can be administered and formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2 - aminoethanol, histidine, procaine, etc.

[0078] Phages can be administered intravenously, for example, by infusion or injection. Alternatively, phages can be administered intratumorally and / or peritumorally. In other embodiments, phages can be administered intraarterially, intramuscularly, or intraperitoneally. In some embodiments, phages are co - administered with a polyethylene glycolylated form of rHuPH20 (PEGPH20) or other agents to disrupt the tumor stroma, thereby enhancing the penetration of the tumor capsule, collagen, and / or matrix.

[0079] The phages of the present disclosure can be administered via intratumoral injection such that the phages are directly deposited within the target tumor. Intratumoral injection of engineered phages can trigger a strong local inflammatory response as well as an immune response against tumor cells. For the injection procedure, the phages are suspended in a solution and then aspirated into a syringe. In some embodiments, a single - hole needle is used to inject the tumor. In yet another embodiment, a multi - tipped needle (Quadra - Fuse, Rex Medical) is used to inject the tumor.

[0080] Injecting the phages of the present inventive technique directly into a solid tumor intratumorally can be advantageous compared to intravenous administration. Using the intravenous injection method, only a small fraction of the phages can reach the target tumor. Particularly, in large animals and human patients with a relatively large blood volume and relatively small tumors compared to mice, intratumoral injection can be particularly beneficial. Direct injection into the tumor allows for the delivery of higher concentrations of the therapeutic agent and avoids the toxicity that can be caused by systemic administration. Additionally, intratumoral injection of phages induces a robust and local immune response within the tumor.

[0081] Depending on the location, tumor type, and tumor size, different administration techniques can be used, including but not limited to cutaneous, subcutaneous, and percutaneous injection, therapeutic endoscopic ultrasound, or intratumoral delivery via bronchoscopy. Prior to the intratumoral administration procedure, the patient is sedated in combination with local anesthesia and standard cardiac, pressure, and oxygen monitoring, or general anesthesia.

[0082] For some tumors, percutaneous injection can be employed, which is the least invasive method of administration. Ultrasound computed tomography (CT) or fluoroscopy can be used as guidance to introduce and position the needle. For example, percutaneous intratumoral injection for hepatocellular carcinoma is described in Lencioni et al., 2010 (incorporated herein by reference). Intratumoral injection for cutaneous, subcutaneous, and lymph node tumors of advanced melanoma is described, for example, in WO / 2014 / 036412 (incorporated herein by reference).

[0083] A single insertion point or multiple insertion points can be used in the percutaneous injection protocol. Using a single insertion point, the solution can be injected percutaneously along multiple tracks, provided that the radial range of the needle permits. In other embodiments, if the tumor is larger than the radial range of the needle, multiple injection points can be used. The needle can be withdrawn at will without exiting, and the direction can be changed multiple times as needed until the entire dose is injected and dispersed. To maintain sterility, a separate needle is used for each injection. The size and length of the needle vary according to the tumor type and size.

[0084] In some embodiments, an 18-gauge multi-tip needle (Quadra-Fuse, Rex Medical) is used to inject percutaneously into the tumor. The device comprises an 18-gauge puncture needle with a length of 20 cm. The needle has three retractable tips, each with four end side holes and a connector with an extension tube clamp. The tips project from the sidewall of the needle. The needle can be introduced percutaneously into the center of the tumor and positioned at the deepest margin of the tumor. The tips are deployed to the margin of the tumor. The tips are extended to their maximum length and then retracted at prescribed intervals. Optionally, one or more rotate-inject-rotate operations can be performed, where the tips are retracted, the needle is rotated 60 degrees, and then the tips are redeployed and additional injections are made.

[0085] Therapeutic endoscopic ultrasound (EUS) is employed to overcome the anatomical limitations inherent in accessing certain other tumors (Shirley et al., 2013; incorporated herein by reference). EUS-guided fine needle injection (EUS-FNI) has been successfully used in antitumor therapies for head and neck, esophageal, pancreatic, hepatic, and adrenal masses (Verna et al., 2008; incorporated herein by reference). EUS-FNI has been widely used for pancreatic cancer injection. Fine needle injection requires the use of a curved ultrasound endoscope. The cannula is carefully inserted into the esophagus, and the ultrasound endoscope is advanced into the stomach and duodenum, where the pancreas is examined and the target tumor is identified. The maximum plane is measured to estimate the tumor volume and calculate the injection volume. An appropriate volume is aspirated into a syringe. The prepared 22-gauge fine needle aspiration (FNA) needle is inserted into the working channel of the ultrasound endoscope. Under ultrasound guidance, the needle is inserted into the tumor. Depending on the size of the tumor, administration can be performed by dividing the tumor into several parts and then injecting the corresponding fractional volume into each part. An endoscopic ultrasound processor equipped with Doppler technology is used to ensure that there are no arterial or venous structures that may interfere with the entry of the needle into the tumor (Shirley et al., 2013; incorporated herein by reference). In some embodiments, the'multi-injection needle' (MIN) of EUS-FNI can be used to improve the injection distribution into the tumor compared to a straight needle (Ohara et al., 2013; incorporated herein by reference).

[0086] Intratumoral administration of lung cancer (such as non-small cell lung cancer) can be achieved by an intratracheal intratumoral delivery method as described in Celikoglu et al., 2008 (incorporated herein by reference). Bronchoscopy (transnasal or transoral) is performed to visualize the lesion to be treated. The tumor volume can be visually estimated from the length-width-height measurements visible on the bronchial surface. A needle device is then introduced through the working channel of the bronchoscope. A needle catheter comprising a metal needle attached to a plastic catheter is placed within a sheath to prevent damage to the working channel during advancement. The size and length of the needle vary and are determined according to the tumor type and the size of the tumor. Needles made of plastic are less rigid than metal needles and are therefore highly desirable as they can negotiate sharper bends in the working channel. The needle is inserted into the lesion and the phage of the present technology is injected. The needle is repeatedly inserted at several insertion points until the tumor mass is completely perfused. After each injection, the needle is completely withdrawn from the tumor and then inserted into another location. At the end of the bronchoscopic injection phase, mechanical debulking or other ablation techniques accompanied by irrigation and aspiration can be used to remove any necrotic debris caused by the treatment.

[0087] In some embodiments, phages capable of delivering immunomodulators to a target tumor are directly administered into the tumor, using methods including but not limited to percutaneous injection, EUS-FNI, or intratumoral delivery methods via bronchus. In some cases, other techniques (such as laparoscopic or open surgical techniques) are used to access the target tumor; however, these techniques are more invasive and carry a higher morbidity and longer hospital stay.

[0088] The volume injected into each lesion depends on the size of the tumor. To obtain the tumor volume, measurements of the largest plane can be made. Then, the estimated tumor volume can be used as a basis for determining the percentage of the total volume that is the injection volume. For example, an injection volume of about 20 - 40% of the total tumor volume can be used. For example, as described in, for example, WO / 2014 / 036412 (Amgen; incorporated herein by reference), for tumors with a largest dimension greater than 5 cm, up to 4 mL can be injected. For tumors with a largest dimension between 2.5 and 5 cm, up to 2 mL can be injected. For tumors with a largest dimension between 2.5 and 5 cm, up to 2 mL can be injected. For tumors with a largest dimension between 1.5 and 2.5 cm, up to 1 mL can be injected. For tumors with a largest dimension between 0.5 and 1.5 cm, up to 0.5 mL can be injected. For tumors with a largest dimension equal to or less than 0.5 cm, up to 0.1 mL can be injected. Alternatively, ultrasound scanning can be used to determine the injection volume that can be absorbed by the tumor without leaking into the surrounding tissue.

[0089] In some embodiments, the treatment regimen will include one or more intratumoral administrations. In some embodiments, the treatment regimen will include an initial dose, followed by at least one subsequent dose. One or more doses can be administered sequentially in two or more cycles. For example, the first dose can be administered on day 1, and the second dose can be administered 1, 2, 3, 4, 5, 6 days or 1, 2, 3, or 4 weeks later or at a longer interval. Additional doses can be administered 1, 2, 3, 4, 5, 6 days or 1, 2, 3, or 4 weeks or at a longer interval later. In some embodiments, the first and subsequent administrations have the same dose. 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.

[0090] The described administration routes and doses are intended only as a guide. The optimal administration route and dose can be readily determined by a skilled practitioner. The dose can be determined based on various parameters, especially based on the location of the tumor, the size of the tumor, the age, weight, and condition of the patient to be treated, and the administration route and method.

[0091] Tumor types for intratumoral delivery of the phages of the present technology include locally advanced and metastatic tumors, including but not limited to B, T, and NK cell lymphomas, colorectal cancer, melanoma (including metastatic melanoma), mycosis fungoides, Merkel carcinoma, liver cancer (including hepatocellular carcinoma and liver metastases secondary to colorectal cancer), pancreatic cancer, breast cancer, follicular lymphoma, prostate cancer, refractory liver cancer, and Merkel cell carcinoma.

[0092] The phages disclosed herein can be administered locally and formulated into the form of ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, emulsions, or other forms well known to those skilled in the art. See, for example, "Remington's Pharmaceutical Sciences", Mack Publishing Co., Easton, PA. In one embodiment, for non-sprayable topical dosage forms, viscous to semi-solid or solid forms are used, which contain a carrier or one or more excipients compatible with topical application and have a dynamic viscosity greater than that of water. Suitable formulations include but are not limited to solutions, suspensions, emulsions, creams, ointments, powders, liniments, pastes, etc., which can be sterilized or mixed with adjuvants (such as preservatives, stabilizers, wetting agents, buffers, or salts) to affect various properties, such as osmotic pressure. Other suitable topical dosage forms include sprayable aerosol formulations, where the active ingredient combined with a solid or liquid inert carrier is packaged in a mixture with a pressurized volatile (such as a gas propellant, such as freon) or packaged in a squeeze bottle. A moisturizer or humectant can also be added to the pharmaceutical composition and dosage form. Examples of such additional ingredients are well known in the art. In one embodiment, the pharmaceutical composition containing the phages of the present technology can be formulated into a hygiene product. By way of example, the hygiene product can be an antibacterial formulation, or a fermentation product, such as a fermentation broth. The hygiene product can be, for example, a shampoo, a conditioner, a cream, a paste, an emulsion, and a lip balm.

[0093] The phages disclosed herein can be administered orally and formulated into tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, etc. The pharmacological compositions for oral use can be prepared using solid excipients, optionally grinding the resulting mixture, and processing the granule mixture with suitable auxiliaries as needed to obtain tablet 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 corn starch, wheat starch, rice starch, potato starch, gelatin, tragacanth, methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP) or polyethylene glycol (PEG). Disintegrants such as cross-linked polyvinylpyrrolidone, agar, alginic acid or its salts such as sodium alginate can also be added.

[0094] Tablets or capsules can be prepared with pharmaceutically acceptable excipients by conventional methods, such excipients as binders (such as pre-gelatinized corn starch, polyvinylpyrrolidone, hydroxypropylmethylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin and tragacanth); fillers (such as lactose, microcrystalline cellulose or calcium hydrogen phosphate); lubricants (such as calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc or silica); disintegrants (such as starch, potato starch, sodium starch glycolate, sugar, cellulose derivatives, silica powder); or wetting agents (such as sodium lauryl sulfate). Tablets can be coated by methods well known in the art. Coating shells can be present, and common membranes include, but are not limited to, poly(lactide), poly(glycolide), poly(anhydride), other biodegradable polymers, alginate-polylysine-alginate (APA), alginate-poly(methylene-co-guanidine)-alginate (A-PMCG-A), hydroxyethyl methacrylate-methyl methacrylate (HEMA-MMA), multilayer HEMA-MMA-MAA, polyacrylonitrile-vinyl chloride (PAN-PVC), acrylonitrile / sodium methallyl sulfonate (AN-69), polyethylene glycol / polydimethylsiloxane / polydimethylsiloxane (PEG / PD5 / PDMS), poly(N,N-dimethylacrylamide) (PDMAAm), silica encapsulates, cellulose sulfate / sodium alginate / poly(methylene-co-guanidine) (CS / A / PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolide), carrageenan, starch polyanhydride, starch polymethacrylate, polyamino acids and enteric coating polymers.

[0095] In some embodiments, the phage is encapsulated in cellulose acetate phthalate (CAP), poly(methacrylic acid-co-methyl methacrylate), cellulose acetate trimellitate (CAT), poly(vinyl acetate phthalate) (PVAP), and hydroxypropyl methylcellulose phthalate (HPMCP), fatty acids, waxes, shellac (esters of aleuritic acid), plastics, and plant fibers. Additionally, Zein, Aqua-Zein (an alcohol-free aqueous zein formulation), amylose, starch derivatives, and dextrins (such as maltodextrin) can also be used. Other known enteric coatings include ethyl cellulose, methyl cellulose, hydroxypropyl methyl cellulose, amylose acetate phthalate, cellulose acetate phthalate, hydroxypropyl methyl cellulose phthalate, ethyl acrylate, and methyl methacrylate.

[0096] The coating polymer can also comprise one or more of the following: phthalate derivatives, CAT, HPMCAS, polyacrylic acid derivatives, copolymers comprising acrylic acid and at least one acrylate, Eudragit TM S (poly(methacrylic acid, methyl methacrylate) 1:2); Eudragit LI00 TM S (poly(methacrylic acid, methyl methacrylate) 1:1); Eudragit L30D TM (poly(methacrylic acid, ethyl acrylate) 1:1); and (Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate) l:l) (Eudragit TM L is an anionic polymer synthesized from methacrylic acid and methyl methacrylate), polymethyl methacrylate mixed with a copolymer of acrylic acid and acrylate, alginic acid, ammonium alginate, sodium alginate, potassium alginate, magnesium alginate, or calcium alginate, vinyl acetate copolymer, polyvinyl acetate 30D (30% dispersed in water), neutral methacrylate comprising poly(dimethylaminoethyl acrylate) (“Eudragit E TM ”), copolymer of methyl methacrylate and ethyl acrylate with trimethylaminoethyl methacrylate chloride, copolymer of methyl methacrylate and ethyl acrylate, Zein, shellac, gums, or polysaccharides, or combinations thereof.

[0097] The coating layer may also include polymers containing the following: hydroxypropyl methylcellulose (HPMC), hydroxypropyl ethylcellulose (HPEC), hydroxypropyl cellulose (HPC), hydroxypropyl ethylcellulose (HPEC), hydroxymethylpropyl cellulose (HMPC), ethylhydroxyethyl cellulose (EHEC) (Ethulose), hydroxyethyl methylcellulose (HEMC), hydroxymethyl ethylcellulose (HMEC), propylhydroxyethyl cellulose (PHEC), methylhydroxyethyl cellulose (MHEC), hydrophobically modified hydroxyethyl cellulose (NEXTON), carboxymethylhydroxyethyl cellulose (CMHEC), methylcellulose, ethylcellulose, water-soluble vinyl acetate copolymers, gums, polysaccharides such as alginic acid and alginates (such as ammonium alginate, sodium alginate, potassium alginate), acid phthalates of carbohydrates, amylose acetate phthalate, cellulose acetate phthalate (CAP), cellulose phthalate esters, cellulose phthalate ethers, hydroxypropyl cellulose phthalate (HPCP), hydroxypropyl ethyl cellulose phthalate (HPECP), hydroxypropyl methyl cellulose phthalate (HPMCP), hydroxypropyl methyl cellulose acetate succinate (HPMCAS).

[0098] Liquid formulations for oral administration may be in the form of solutions, syrups, suspensions, or in the form of dry products reconstituted with water or other suitable vehicles before use. Such liquid formulations can be prepared by conventional methods with pharmaceutically acceptable reagents such as suspending agents (such as sorbitol syrup, cellulose derivatives or hydrogenated edible fats); emulsifying agents (such as lecithin or gum acacia); non-aqueous vehicles (such as almond oil, oily esters, ethanol or fractionated vegetable oils); and preservatives (such as methyl paraben or propyl paraben or sorbic acid). If desired, the formulations may also contain buffer salts, flavoring agents, coloring agents and sweetening agents. The formulations for oral administration can be appropriately formulated to provide slow release, controlled release or sustained release of the phages described herein.

[0099] In one embodiment, the phages of the present disclosure can be formulated in a composition suitable for administration to a pediatric subject. As is well known in the art, children differ from adults in many respects, including different gastric emptying rates, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics, 134(2):361-372, 2014; incorporated herein by reference). In addition, the acceptability and preference of pediatric formulations (e.g., route of administration and taste attributes) are crucial for achieving acceptable pediatric compliance. Thus, in one embodiment, a composition suitable for administration to a pediatric subject may include a dosage form that is easy to swallow or dissolve, or a more palatable composition, such as a composition with added flavoring agents, sweetening agents, or taste blockers. In one embodiment, a composition suitable for administration to a pediatric subject can also be administered to adults.

[0100] In one embodiment, a composition suitable for administration to a pediatric subject can include a solution, syrup, suspension, elixir, powder reconstituted into a suspension or solution, dispersible / effervescent tablets, chewable tablets, gummies, lollipops, popsicles, troches, chewing gums, oral thin strips, orally disintegrating tablets, sachets, soft gelatin capsules, sprayable oral powders or granules. In one embodiment, the composition is a gummy made from a gelatin matrix that imparts elasticity, a desired chewy consistency, and a longer shelf life to the confectionery. In some embodiments, the gummy may also contain a sweetening agent or a flavoring agent.

[0101] In one embodiment, a composition suitable for administration to a pediatric subject can include a flavoring agent. As used herein, a "flavoring agent" is a substance (liquid or solid) that provides a unique taste and aroma to a formulation. Flavoring agents also help to improve the palatability of the formulation. Flavoring agents include, but are not limited to, strawberry, vanilla, lemon, grape, bubblegum, and cherry.

[0102] In certain embodiments, the phages can be administered orally, for example, with an inert diluent or an absorbable edible carrier. The compounds can also be encapsulated in hard or soft shell gelatin capsules, compressed into tablets, or directly incorporated into the diet of the subject. For oral therapeutic administration, the compounds can be mixed with excipients and used in ingestible tablets, buccal tablets, lozenges, capsules, elixirs, suspensions, syrups, cachets, etc. To administer the compounds by a route other than parenteral administration, it may be necessary to coat the compounds with a material or co-administer the compounds with a material to prevent inactivation of the compounds.

[0103] In some embodiments, the composition is formulated for enteral administration, jejunal administration, duodenal administration, ileal administration, gastrojejunal administration, or colonic administration, via enteric-coated or uncoated nanoparticles, nanocapsules, microcapsules, or microtablets. The pharmaceutical composition can also be formulated as a rectal composition, such as a suppository or a retention enema, using, for example, conventional suppository bases such as cocoa butter or other glycerides. The composition can be a suspension, solution, or emulsion in an oily or aqueous vehicle and can contain suspending, stabilizing, and / or dispersing agents.

[0104] The phages described herein can be administered intranasally, formulated in the form of an aerosol, spray, mist, or drops, and conveniently delivered in the form of an aerosol spray from a pressurized package or nebulizer using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gases). The dosage unit of the pressurized aerosol can be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., gelatin capsules and cartridges) for inhalers or insufflators can be formulated, which contain a powder mixture of the compound and a suitable powder matrix (e.g., lactose or starch).

[0105] The phages can be administered and formulated in the form of a depot preparation. Such long-acting formulations can be administered by implantation or by injection, including intravenous injection, subcutaneous injection, local injection, direct injection, or infusion. By way of example, the composition can be formulated with a suitable polymer or hydrophobic material (e.g., formulated as an emulsion in an acceptable oil), or an ion-exchange resin, or formulated as a sparingly soluble derivative (e.g., formulated as a sparingly soluble salt).

[0106] In some embodiments, there are disclosed herein pharmaceutically acceptable compositions in a single dosage form. The single dosage form can be in liquid or solid form. The single dosage form can be administered directly to the patient without modification, or can be diluted or reconstituted before administration. In certain embodiments, the single dosage form can be administered in a bolus form, such as a single injection, a single oral dose, including an oral dose comprising multiple tablets, capsules, pills, etc. In alternative embodiments, the single dosage form can be administered over a period of time, such as by infusion.

[0107] The single dosage form of the pharmaceutical composition can be prepared by dividing the pharmaceutical composition into smaller aliquots, single-dose containers, single-dose liquid forms, or single-dose solid forms, such as tablets, granules, nanoparticles, nanocapsules, microcapsules, microtablets, pills, or powders, which can be enteric-coated or uncoated. The solid form of the single dose can be reconstituted by adding a liquid (usually sterile water or saline solution) before administration to the patient.

[0108] In other embodiments, the compositions can be delivered in controlled release or sustained release systems. In one embodiment, a pump can be used to effect controlled or sustained release. In another embodiment, a polymeric material can be used to effect 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 for sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. The polymers used in sustained release formulations can be inert, free of leachable impurities, storage stable, sterile, and biodegradable. In some embodiments, the controlled or sustained release system can be placed near the prophylactic or therapeutic target, and thus only a fraction of the systemic dose is required. Any suitable technique known to those of ordinary skill in the art can be used.

[0109] The dosage regimen can be adjusted to provide a therapeutic response. The dosage can depend on a variety of factors, including the severity and responsiveness of the disease, the route of administration, the duration of treatment (from days to months to years), and the time of disease improvement. By way of example, a single large dose can be administered at one time, several divided doses can be administered over a predetermined period of time, or the dose can be decreased or increased as indicated by the exigencies of the treatment situation. The specification of the dosage is determined by the unique characteristics of the active compound and the particular therapeutic effect to be achieved. The dosage values can vary with the type and severity of the condition to be alleviated. For any particular subject, the specific dosage regimen can be adjusted over time according to individual needs and the professional judgment of the treating clinician. The toxicity and therapeutic efficacy of the compounds provided herein can be determined by standard pharmaceutical procedures in cell cultures or animal models. By way of example, the LD50, ED50, EC50, and IC50 can be determined, and the dose ratio between toxicity and therapeutic effect (LD50 / ED50) can be calculated as the therapeutic index. Compositions that exhibit toxic side effects can be used, but they need to be carefully modified to minimize potential damage and thus reduce side effects. The dosage can be initially estimated from cell culture assays and animal models. Data obtained from in vitro and in vivo assays and animal studies can be used to formulate a dosage range for human use.

[0110] The ingredients are provided separately, or are mixed together in unit dosage form, for example, in hermetically sealed containers (such as ampoules or sachets) marked with the quantity of the active agent, in the form of a dry lyophilized powder or an anhydrous concentrate. If the mode of administration is by injection, then ampoules of sterile water for injection, saline, or a nutrient excipient can be provided so that the ingredients can be mixed before administration.

[0111] The pharmaceutical composition can be packaged in an airtight container (such as an ampoule or sachet) labeled with the amount of the medicament. In one embodiment, one or more pharmaceutical compositions are provided in the airtight container in the form of a dry sterile lyophilized powder or an anhydrous concentrate and can be reconstituted (e.g., with water or saline) to an appropriate concentration for administration to a subject. In one embodiment, one or more prophylactic or therapeutic agents or pharmaceutical compositions are provided in the airtight container in the form of a dry sterile lyophilized powder, stored between 2°C and 8°C, and administered within 1 hour, 3 hours, 5 hours, 6 hours, 12 hours, 24 hours, 48 hours, 72 hours, or one week after reconstitution. For the lyophilized dosage form, a cryoprotectant can be included, mainly 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 BRIJ surfactants. The pharmaceutical composition can be prepared as an injectable solution and can further contain a reagent that can be used as an adjuvant, such as a reagent for increasing absorption or dispersion, such as hyaluronidase.

[0112] In some embodiments, the phage and its compositions are formulated for intravenous administration, intratumoral administration, or peritumoral administration. The phage can be formulated as a depot preparation. Such long-acting formulations can be administered by implantation or by injection. For example, the composition can be formulated with a suitable polymer or hydrophobic material (e.g., formulated as an emulsion in an acceptable oil) or an ion-exchange resin, or formulated as a slightly soluble derivative (e.g., formulated as a slightly soluble salt).

[0113] In another embodiment, the composition can be delivered in a controlled release or sustained release system. In one embodiment, a pump can be used to achieve controlled or sustained release. In another embodiment, a polymeric material 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 for sustained release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), poly(lactide-co-glycolide) (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), poly(lactide) (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. The polymers used in sustained release formulations can be inert, free of leachable impurities, storage stable, sterile, and biodegradable. In some embodiments, the controlled or sustained release system can be placed near the prophylactic or therapeutic target, and thus only a fraction of the systemic dose is required. Any suitable technique known to those of skill in the art can be used.

[0114] The phages of the present invention can be administered and formulated in neutral or salt form. Pharmaceutically acceptable salts include salts formed with anions such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and salts formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0115] Examples

[0116] The following examples are given to illustrate the practice of various embodiments of the present disclosure. These examples are not intended to limit or define the entire scope of the present disclosure. It should be understood that the present disclosure is not limited to the specific embodiments described and illustrated herein, but includes all modifications and variations that fall within the scope of the present disclosure as defined in the appended claims.

[0117] Example 1 - Engineering of Phages Displaying Tumor-Targeting Moieties and Cytokines

[0118] 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 attached to this document. Cells were generally grown in Luria broth Miller (LB) medium or on Luria broth agar Miller medium, which was supplemented as needed with the following concentrations of antibiotics: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were routinely grown at 37 °C. Bacterial cultures older than 18 hours were not used in the experiments.

[0119] Table 1: List of strains and plasmids used in this study

[0120]

[0121]

[0122] DNA manipulations. A detailed list of the oligonucleotide sequences used in this example is shown in Table 2. Plasmids were prepared using the EZ10-Spin Column Plasmid Miniprep kit (BIOBASIC #BS614) according to the manufacturer's instructions. PCR amplifications were performed using TransStart FastPFU fly DNA polymerase (Civic Bioscience) to amplify and screen DNA fragments. Plasmids were assembled by Gibson assembly using the NEBuilder HiFi DNA Assembly Master Mix (NEB) according to the manufacturer's protocol. DNA digestions were performed using restriction enzymes from NEB according to the manufacturer's recommendations. The digestion process generally lasted more than 1 hour.

[0123] DNA purification. DNA was purified between each step of plasmid assembly to avoid buffer incompatibilities or to stop enzymatic reactions. PCR reaction products 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, the DNA concentration and purity were routinely evaluated using a Nanodrop spectrophotometer as needed.

[0124] DNA was transformed into E. coli by electroporation. Conventional plasmid transformation was carried out by electroporation. Electrocompetent E. coli strains were prepared from 20 mL of LB broth. Cultures in the exponential growth phase with an optical density at 600 nanometers (OD 600nm ) of 0.6 were washed three times in a sterile 10% glycerol solution. The cells were then resuspended in 200 μL of 10% glycerol (1% of the initial volume) and divided into 50-μL aliquots. DNA was then added to the electrocompetent cells (about 50 ng of DNA was added to a maximum of 10 μL of water), and the mixture was transferred to a 1-mm electroporation cuvette. The cells were electroporated with a pulse of 1.8 kV, 25 μF, and 200 Ω for 5 ms. The cells were then resuspended in 1 mL of non-selective LB medium and recovered at 37 °C for 1 hour, then plated on selective medium, and the plates were incubated overnight at 37 °C.

[0125] DNA was transformed into E. coli by heat shock. Heat shock transformation was mainly used for cloning Gibson assembly products. Chemically competent cells were prepared according to the rubidium chloride protocol as previously described (Green et al., 2013). The chemically competent cells were flash-frozen and stored at -80 °C before use. The Gibson assembly products were directly transformed into MM294 chemically competent cells at a 1 / 10 volume ratio. Conventionally, up to 10 μL of DNA was added to 100 μL of competent cells, and then the transformation was carried out by a 45-second heat shock at 42 °C. The cells were then resuspended in 1 mL of non-selective LB medium and recovered at 37 °C for 1 hour, then plated on selective medium.

[0126] Engineered phage systems are widely used in phage display methods to express antibody-phage conjugates for screening antibodies with specific affinities. In this case, the variable regions of antibodies are amplified using universal DNA primers from immunized animals and ligated together to generate a library of scFv fragments, which are cloned into a plasmid called a phagemid and fused to the pIII protein (replacing the N-terminal domain of the protein). Next, the library is screened to select phages that display antibody-pIII fusions that recognize the target antigen. The phages that bind to the target are then isolated and amplified by infecting their bacterial host. This step is possible because the phage still displays wild-type pIII (required for infecting the bacterial host) as well as copies of the antibody-pIII fusion. After several cycles, the scFv fragments that recognize the target antigen are enriched, and the clones are tested and sequenced for preservation. In this case, only a few phages that adhere to the target antigen are needed to isolate the desired antibody clone, which means that screening can still be successful even if most of the phages display only wild-type pIII protein and not the antibody-pIII fusion.

[0127] Table 2: Oligonucleotide Sequence Listing

[0128]

[0129]

[0130]

[0131] a. Oligonucleotides are listed by PCR reaction and use, and the same oligonucleotide may appear more than once when used for multiple purposes.

[0132] b. Underlined nucleotides form the primer sites.

[0133] c. Bold nucleotides are the mutated DNA sequences

[0134] The engineered phage systems described herein differ from phage display systems in several respects. For example, (1) the production strain bacteria must secrete phages in large quantities; (2) each phage must display a therapeutic protein; and (3) as little DNA as possible must be used to assemble the phage particles to maximize the biosafety of the system and limit the spread of genetic material. These additional constraints do not exist in conventional phage display. Thus, most phage display systems use M13K07 as a helper phage, which expresses all the proteins responsible for phage replication and assembly (including the wild-type copy of gpIII), and use phagemids, which express antibody-pIII fusion proteins, an origin of replication, and contain a complete encapsidation signal. Although these systems will produce antibody-phage conjugates, 90% of the phages produced contain only the wild-type pIII protein from M13K07 and not the antibody-pIII fusion protein from the phagemid (Ledsgaard et al. 2018, Toxin; incorporated herein by reference). For the display of therapeutic proteins, it is undesirable and unwanted that 90% of the phages do not display the therapeutic protein, as this reduces the efficacy of the treatment.

[0135] In this example, we will describe the design of a phage secretion system for displaying therapeutic proteins. We will present iterations of the system and discuss their advantages and disadvantages. The list of iterations is not exhaustive but illustrates the variations of the phage secretion system for combining the display of cytokines and binding molecules. The general description of the various constructs described in this example is shown in Figure 1 shown.

[0136] Iterations of the Phage Secretion System

[0137] The phage secretion system is a plastic system that can be manifested in different forms. In this example, we aim to show that the phage secretion system can be modified to allow the display of one or two therapeutic proteins on the same phage particle. In the first embodiment of the phage secretion system, the system can be divided into a set of two vectors, namely a phage backbone vector (such as pTAT002, pTAT044, pTAT070) and a phage machinery vector (such as pTAT004)( Figure 1 ). In some embodiments, the phage secretion system can be divided into three or more genetic constructs, as in the systems composed of pTAT025, pTAT044 or pTAT070 and pTAT017 or pTAT060 or pTAT071. In another embodiment, phage secretion can be on a single vector. In yet another embodiment, phage secretion can be integrated into the genome of the production strain bacteria.

[0138] Two-Vector-Based Phage Secretion System

[0139] To generate the phage machinery vector pTAT004, the appropriate primer pairs listed in Table 2 were used to perform whole amplification of M13K07, except for the gpIII gene encoding pIII. The homologous tails of the primers for amplifying M13K07 were carefully designed to remove gpIII from the final construct. Next, the PCR products were purified by SPRI and assembled by the Gibson method to generate pTAT004. The assembly was transformed into MM294 chemically competent cells, and plasmid integrity was verified by using NdeI digestion. The pTAT004 phage machinery cannot produce fully functional phage particles on its own because it does not have a copy of the gpIII gene and thus cannot produce pIII. To prepare phage particles, gpIII must be provided in trans by a phage backbone vector.

[0140] Next, the pTAT002, pTAT044, and pTAT070 phage backbone vectors were designed. All phage backbone vectors contain an ori for high-copy plasmid replication (to maximize the DNA material for encapsidation), an ori for ssDNA rolling circle replication and recognition of the phage backbone vector by the phage encapsidation mechanism, a selectable marker (spectinomycin resistance here), and a constitutively expressed C-terminal fragment of pIII, which is linked to the HA-His dual-tagged N-terminal fragment of pIII (pTAT002, a control without a therapeutic protein) or a checkpoint inhibitor-binding protein (pTAT044, anti-PD-L1 linked via an HA-His dual tag; pTAT070, anti-PD-L1 without a tag). In this example, the anti-PD-L1 binding protein was selected as the therapeutic protein because it binds to a surface protein expressed by cancer cells (Vaddepally et al., Cancers (Basel) March 2020; 12(3):738; incorporated herein by reference). The first assembled phage backbone vector was pTAT002. To construct pTAT002, the ori was amplified by PCR from pSB1C3 (a common backbone vector of the iGEM library), the ori was amplified from M13K07, the N-terminal and C-terminal of pIII were amplified, aad7 (spectinomycin resistance) was amplified from Escherichia coli KN01, and the constitutive promoter was amplified from gBlock. Next, the PCR products were assembled by Gibson and transformed into chemically competent MM294 cells. Next, plasmid integrity was verified by using ApaLI and NdeI digestion (in pMB1 an ori for ssDNA rolling circle replication and recognition of the phage backbone vector by the phage encapsidation mechanism, a selectable marker (spectinomycin resistance here), and a constitutively expressed C-terminal fragment of pIII, which is linked to the HA-His dual-tagged N-terminal fragment of pIII (pTAT002, a control without a therapeutic protein) or a checkpoint inhibitor-binding protein (pTAT044, anti-PD-L1 linked via an HA-His dual tag; pTAT070, anti-PD-L1 without a tag). In this example, the anti-PD-L1 binding protein was selected as the therapeutic protein because it binds to a surface protein expressed by cancer cells (Vaddepally et al., Cancers (Basel) March 2020; 12(3):738; incorporated herein by reference). The first assembled phage backbone vector was pTAT002. To construct pTAT002, the ori was amplified by PCR from pSB1C3 (a common backbone vector of the iGEM library), the ori was amplified from M13K07, the N-terminal and C-terminal of pIII were amplified, aad7 (spectinomycin resistance) was amplified from Escherichia coli KN01, and the constitutive promoter was amplified from gBlock. Next, the PCR products were assembled by Gibson and transformed into chemically competent MM294 cells. Next, plasmid integrity was verified by using ApaLI and NdeI digestion (in M13 WO2022073127A1). pMB1 To construct pTAT002, the ori was amplified by PCR from pSB1C3 (a common backbone vector of the iGEM library), the ori was amplified from M13K07, the N-terminal and C-terminal of pIII were amplified, aad7 (spectinomycin resistance) was amplified from Escherichia coli KN01, and the constitutive promoter was amplified from gBlock. Next, the PCR products were assembled by Gibson and transformed into chemically competent MM294 cells. Next, plasmid integrity was verified by using ApaLI and NdeI digestion (in M13 WO2022073127A1). Figure 5; incorporated herein by reference). After Sanger sequencing of pTAT002, a mutation (G>T) was found at the third position of the P5 promoter. The resulting promoter was called P5mut (5'-TT T ACAATTAATCATCCGGCTCGTAATTTATGTGGA-3'), which allows for lower levels of expression of the upstream gene, as measured using GFP with the pTAT010-P5 and pTAT010-P5mut constructs (data not shown). To simplify construct assembly, the pTAT002 backbone was modified and the sfGFP gene was cloned to be expressed from the P5 promoter instead of gpIII. The backbone was assembled in a similar manner to pTAT003, but the primers used allowed for the insertion of an additional terminator after the gene expressed from P5 and the insertion of Gibson Assembly Tags (GAT) to separate different parts of the vector. The resulting vector (called pTAT013) was then used as a template to amplify the backbone of subsequent constructs for the display of therapeutic proteins on pIII. Thus, to construct pTAT044 and pTAT070, the backbone of the plasmid was amplified from pTAT013, the P5mut promoter was amplified from pTAT010-P5mut, and the C-terminal portion of gpIII was amplified from M13K07. The anti-PD-L1 scFv was amplified from gBlocks with an HA-6xHis linker (pTAT044) or without a linker (pTAT070). These gBlocks also changed the start codon of the checkpoint inhibitor fusion protein from ATG to GTG, as this has been shown to result in better expression levels of the therapeutic phage. All plasmids were then sent for Sanger sequencing after assembly, and no harmful mutations were detected. With these results, the phage secretion system for the display of therapeutic proteins on pIII was ready for efficiency testing and improvement rounds.

[0141] Phage secretion system based on three or more vectors

[0142] The phage 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 protein of each phage gene is produced. To facilitate the display of two therapeutic proteins on the same phage particle, we aimed to divide the phage machinery into three distinct plasmids. As a first step, we needed to delete the proteins of gpIII, tail fiber protein, and phage head. We selected gpIX as the second site for protein fusion. GpIX is a capsid gene located in the phage head and involved in budding from the host cell. Deleting gpIX from M13K07 is more complex than deleting gpIII because the coding sequence of gpIX overlaps with that of gpVIII. To remove gpIX, we needed to perform some gene reconstruction to prevent interruption of the gpVIII gene. The overlapping sequence between gpVIII and gpIX is: 5'-AGA TGA GTGTTTTA-3', where the bold ATG codon is the start codon of gpIX and the underlined TGA codon is the stop codon of gpVIII. By a mutation A>G at position 3, changing the ATG codon to the weaker GTG start codon without affecting the sequence of gpVIII (both AGG and AGA code for arginine), the overlap between the two genes can be corrected. Additionally, we changed the third codon of gpIX from TTA to TAA to introduce a stop codon and prevent translation of gpIX. In the resulting sequence, the changes are underlined: 5′-AG G TGAGTGTTT A A-3'. Next, the resulting construct pTAT025 was obtained by amplifying pTAT004 with primers that introduced these modifications into the gpVIII / gpIX locus. Thus, plasmid pTAT025 expresses all the genes of the M13 genome except gpIII and gpIX, which need to be provided in trans. It also requires a phage backbone vector encoding ori M13 to secrete phages.

[0143] The gpIII deficiency of plasmid pTAT025 can be complemented by any of the plasmids (pTAT002, pTAT044, and pTAT070) expressing gpIII or a gpIII-therapeutic protein fusion as described above. However, pTAT025 also requires exogenous provision of gpIX to produce phages. Therefore, a set of plasmids is needed to support gpIX production. For this purpose, ori was amplified from pKN23 pSC101, bla from pUC19 and P5-BCD1-sfGFP from pTAT010-P5 (each assembled using GAT in the primer tails) were used to generate a new backbone. Next, the PCR fragments were purified by SPRI and assembled by Gibson assembly to generate pTAT014, which was then transformed into MM294. Next, the construct was phenotypically evaluated (GFP phenotype) and sequenced by Sanger sequencing. Next, the backbone was amplified as a whole except for the sfGFP gene and assembled with the gpIX gene amplified from M13K07. Next, the two PCR products were assembled in the same manner as pTAT014 to generate the gpIX complementing plasmid (pTAT017). The plasmid was further modified to allow the display of mouse IL-2 (mIL-2) and human IL-15 (hIL-15) on pIX by amplifying their CDS and P5mut-BCD1 from gBlocks, resulting in pTAT071 and pTAT060, respectively. These gBlocks also changed the start codon of the cytokine fusion protein from ATG to GTG, as this was shown to result in better expression levels of the therapeutic phage. Next, the plasmids were confirmed by Sanger sequencing.

[0144] Combine the plasmids into a functional phage secretion system

[0145] Different phage secretion systems are required to test the potential synergy between the cytokines displayed on the synthetic therapeutic phages and the cancer cell targeting moieties. To obtain those systems, the plasmids were sequentially transformed into Escherichia coli MG1655 by electroporation. The combinations described in Table 3 allowed the generation of: M13 phage (phage), M13 phage displaying anti-PD-L1 scFv (phage-PD-L1), M13 phage displaying mouse IL-2 and anti-PD-L1 scFv (mIL-2-phage-PD-L1), M13 phage displaying human IL-15 and anti-PD-L1 scFv (hIL15-phage-PD-L1). These four phage variants will enable us to dissect the contribution of each component to the anti-tumor response in mice and explore potential synergies.

[0146] Table 3: List of bacterial strains tested

[0147] Strain name Plasmid content Produced phage Escherichia coli MG1655 ecTAT052 pTAT004 + pTAT002 Phage ecTAT286.1 pTAT025 + pTAT044 + pTAT060 hIL15 - phage - PD - L1 ecTAT309 pTAT004 + pTAT070 Phage - PD - L1 ecTAT312.4 pTAT025 + pTAT070 + pTAT071 mIL2 - phage - PD - L1

[0148] In summary, the strains generated in this example are sufficient to explore the potential synergy between cytokines displayed on M13 phage and cancer cell targeting moieties. These combinations are not meant to be exhaustive, and it is reasonable to assume that other cytokines or binding agents may produce synergy when displayed on M13 phage. This example also illustrates the ability of phage to display more than one therapeutic agent simultaneously on each subunit of two different coat proteins.

[0149] Example 2 - In Vitro Validation of Therapeutic Target Binding by Phage Displaying Tumor Targeting Molecules and Cytokines

[0150] Strains, plasmids, phage production, and growth conditions. All strains and plasmids used in this example are described in Table 1. Cells were generally grown in 2xYT broth (2xYT) supplemented as needed with the following concentrations of antibiotics: ampicillin (Ap) 100 μg / mL, kanamycin (Km) 50 μg / mL, and / or spectinomycin (Sp) 100 μg / mL. All cultures were routinely grown at 30 °C for no more than 18 hours prior to use in experiments. Phage were recovered from the supernatant of confluent bacterial cultures (grown overnight). The phage-containing culture supernatant was used immediately after pelleting the bacteria by centrifugation.

[0151] PEG-based precipitation of phage particles. Starting from a frozen stock, inoculate 10 mL of 2xYT broth containing the appropriate antibiotics at the concentrations specified in the above paragraph and incubate the culture with stirring overnight at 30 °C, or for no more than 18 hours. Transfer the entire overnight bacterial culture to a 2 L Erlenmeyer containing 500 mL of 2xYT broth with the appropriate antibiotics. Incubate the culture with stirring at 37 °C until the optical density at 600 nm reaches 0.5. Then place the culture on ice for 15 minutes and then incubate with stirring overnight at room temperature. Transfer the overnight culture to a 500 mL centrifuge bottle and centrifuge at 13,000 g for 20 minutes at 4 °C. Decant the supernatant onto a 500 mL 0.45 μm filter unit and filter to remove any remaining bacteria and debris. Add 2.5 M NaCl / 20% PEG-8000 (w / v) to the filtered supernatant to obtain a supernatant:PEG solution volume ratio of 4:1. After thoroughly mixing by inverting the bottle 15 times, incubate the mixture at 4 °C for 1 hour. Next, pellet the virus particles by centrifugation at 13,000 g for 20 minutes at 4 °C. Then remove the supernatant and resuspend the pellet in 1 to 2 mL of PBS containing 0.1 mM CaCl2. Keep the phage preparation on ice for an additional 1 hour, vortex, and store at 4 °C. Use according to the manufacturer's instructions. HD (LIONEX) discontinuous chromatography was used to remove most of the contaminating LPS from the phage preparation. Then, the phage titer of pVIII coat protein was extrapolated by an in-house sandwich ELISA using a standard curve generated with purified M13K07 helper phage (NEB).

[0152] The binding of phages to PD-L1 was evaluated by indirect ELISA. The binding activity of phages displaying scFv against the PD-L1 checkpoint was measured by ELISA assay. First, a 96-well Nunc MaxiSorp TM plate was coated overnight at 4 °C with the extracellular domain of recombinant human PD-L1 protein (SinoBiological), which was diluted at 2 μg / mL in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). Then the plate was washed 3 times with 200 μL of TBS-T. To prevent non-specific binding, the plate was then incubated with 200 μL of blocking buffer (TBS-T, 3% skim milk, 1% BSA) for 1 h at room temperature. Blocking was terminated by removing the blocking buffer and washing the plate twice with 200 μL of TBS-T. Then, 100 μL of each of the following was diluted in TBS1X and added to each well: purified wild-type M13 phage (phage control); or phages displaying anti-PD-L1 scFv on pIII (phage-PD-L1); or phages displaying anti-PD-L1 scFv on pIII and cytokine on pIX (mouse IL-2 or human IL-15, designated mIL2-phage-PD-L1 or hIL15-phage-PD-L1, respectively). The plate was incubated for 1 h at room temperature. Then the plate was washed 3 times with 200 μL of TBS-T, and 100 μL of anti-pVIII-HRP (anti-M13 phage, B62-FE2) diluted in blocking buffer (1:500) was added. After incubation for 1 h at room temperature in the dark, the wells were washed 5 times with TBS-T. To reveal the presence of phages, 100 μL of TMB high-sensitivity substrate solution (BioLegend) was added to each well, and the plate was incubated at room temperature for 3 to 10 min until a blue color appeared. The reaction was terminated by adding 100 μL of stop solution (0.5 M H2SO4) to each well. Then the absorbance at 450 nm was measured using a microplate reader. Only phages displaying anti-PD-L1 scFv on pIII could be significantly detected in the PD-L1-coated wells ( Figure 3 A), thus verifying the displayed biological activity. Dual display of recombinant interleukin on pIX did not affect the binding efficiency of the scFv on pIII.

[0153] Phages displaying recombinant cytokines on pIX were evaluated by sandwich ELISA. To verify the expression of recombinant human interleukin-15 (hIL-15) or murine interleukin-2 (mIL-2) displayed on phages, an ELISA assay was designed. First, a 96-well Nunc MaxiSorp TM plate was coated overnight at 4 °C with anti-hIL-15 or anti-mIL-2 rabbit IgG antibody (SinoBiological), which was diluted in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6) according to the manufacturer's recommendations. Then the plate was washed 3 times with 200 μL of TBS-T. To prevent non-specific binding, the plate was subsequently incubated with 200 μL of blocking buffer (TBS-T, 3% skim milk, 1% BSA) for 1 hour at room temperature. Blocking was terminated by removing the blocking buffer and washing the plate twice with 200 μL of TBS-T. Then, 100 μL of each of the following was diluted in TBS1X and added to each well: purified wild-type M13 phage (phage control); or phage displaying anti-PD-L1 scFv on pIII (phage-PD-L1); or phage displaying anti-PD-L1 scFv on pIII and cytokine on pIX (mIL-2 or hIL-15, designated mIL2-phage-PD-L1 or hIL15-phage-PD-L1, respectively). The plate was incubated for 1 hour at room temperature. Then the plate was washed 3 times with 200 μL of TBS-T, and 100 μL of anti-pVIII-HRP (anti-M13 / fd / F1, B62-FE2) diluted in blocking buffer (1:500) was added. After incubation for 1 hour at room temperature in the dark, the wells were washed 5 times with TBS-T. To reveal the presence of phages, 100 μL of TMB high-sensitivity substrate solution was added to each well, and the plate was incubated at room temperature for 3 to 10 minutes until a blue color appeared. The reaction was terminated by adding 100 μL of stop solution (0.5 M H2SO4) to each well. Then the absorbance at 450 nm was measured using a microplate reader. Phages displaying cytokine (mIL2 or hIL15) on pIX and anti-PD-L1 scFv were detected on wells coated with interleukin-specific antibody( Figure 3 B), thus verifying the expression of the recombinant protein and the binding activity of anti-PD-L1 scFv.

[0154] Example 3 - In vitro verification of the biological activity of cytokines displayed on phages

[0155] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this example are described in Table 3. Cells are generally grown in Luria-Bertani Miller medium (LB) or on Luria-Bertani agar Miller medium, which is supplemented as needed with antibiotics at the following concentrations: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures are routinely grown at 37 °C for no more than 18 h before use. Phage is extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol provided in Example II.

[0156] Cell culture. HEK-Blue TM IL-2 reporter cells, which are specifically designed for detecting bioactive interleukin-2 (IL-2), are purchased from InvivoGen (hkb-il2). TM HEK-Blue TM cells are cultured under optimal conditions according to the manufacturer's guidelines. Briefly, the cells are cultured at 37 °C in an atmosphere of 5% CO2 and 95% humid air in DMEM supplemented with 10% fetal bovine serum (FBS) and Normocin TM . HEK-Blue

[0157] CLR Selection and puromycin are used to culture the cells under selective pressure to maintain stable expression of the human interleukin-2 receptor (IL-2R), including the expression of subunits CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ).

[0157] HEK-Blue TM IL-2 reporter gene assay. The HEK-Blue TM IL-2 reporter gene assay is performed as follows. When 80% confluence is reached, the HEK-Blue is washed with cold D-PBS (Gibco). TMIL-2 cells were used twice. Remove D-PBS and add 2 mL of cold Versene 1X (Gibco) to dissociate the cells. After incubating for 5 minutes, add 8 mL of test medium (DMEM supplemented with 10% heat-inactivated FBS). Use trypan blue and a hemocytometer to evaluate the cell density. Centrifuge the cell suspension at 300 g for 10 minutes at 4 °C, remove the supernatant, and resuspend the cells in the test medium at a cell density of 280,000 cells / mL. Inoculate 180 μL of the cell suspension (corresponding to approximately 50,000 cells) into a TC-treated 96-well culture plate. Add 20 μL of the therapeutic agent to each well to test its IL-2R activation activity. Incubate the plate at 37 °C in an atmosphere of 5% CO2 and 95% humid air for 18 hours. For each well, collect 20 μL of the medium and transfer it to a new 96-well clear plate. Add 180 μL of QUANTI-Blue TM solution and incubate the plate at 37 °C for 15 minutes to 6 hours. Obtain the optical density at 630 nm using a conventional microplate reader.

[0158] Phage displaying murine IL-2 cytokine activates the IL-2 receptor. To verify that the cytokine maintains its biological activity when displayed on synthetic therapeutic phages, an experiment was conducted using HEK-Blue TM IL-2 reporter cells to monitor the activation of the IL-2 receptor (IL-2R) by phages displaying murine IL-2 (mIL-2). HEK-Blue TM IL-2 cells were treated with each of the following for 18 hours: PBS (vehicle); 10 11 phages displaying anti-PD-L1 scFv on pIII (phage-PD-L1); 10 11 phages displaying mIL-2 on pIX and anti-PD-L1 scFv on pIII (mIL-2-phage-PD-L1); or 0.2 ng of mIL-2 (mIL-2 at an equimolar dose to 10 11 mIL-2-phage-PD-L1). The level of IL-2R activation was monitored by measuring the optical density at 630 nm and reported on Figure 4 . Only the phage displaying mIL-2 (mIL-2-phage-PD-L1) activated the IL-2R as effectively as mIL2, demonstrating that the cytokine has biological activity when displayed on synthetic therapeutic phages.

[0159] Example 4: Phages displaying cytokines and cancer cell targeting moieties bind to cancer cells.

[0160] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this example are described in Table 3. Cells were generally grown in Luria broth Miller medium (LB) or on Luria broth agar Miller medium, which was supplemented as needed with the following concentrations of antibiotics: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were routinely grown at 37 °C for no more than 18 h before use in experiments. Phage were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol provided in Example II.

[0161] Cell culture. A20 lymphocyte B lymphoma cells were purchased from ATCC (TIB-208). Upon arrival, the cells were washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This medium was used to prepare cells for all experiments. Frozen stock solutions were generated after 4 passages and used to initiate subsequent cultures for experiments. In all experiments, the cells were maintained at a density of 2×10 5 cells / mL to 2×10 6 cells / mL and grown at 37 °C in an atmosphere of 5% CO2 and 95% humid air.

[0162] Immunofluorescence experiment. PD-L1 + A20 cancer cells were incubated with 10 11 / mL of phage or mIL-2-phage-PD-L1 for 24 h and then fixed on coverslips placed at the bottom of 6-well plates using 10% NBF solution. After washing with PBS, the cells were permeabilized by adding 1 mL of PBS containing 0.1% Triton X-100 (PBS-Tx). The solution was removed, and the cells were blocked by adding PBS-Tx containing 2% BSA (PBS-Tx-BSA). The cells were then treated with an Fc-blocking antibody (CD16 / CD32 monoclonal antibody, Invitrogen). The cells were washed with PBS-Tx and then the phage were revealed using anti-M13-FITC (Progen) diluted 1 / 200 in PBS-Tx-BSA as a diluent. The cells were washed with PBS-Tx and the nuclei were stained with 1 μg / mL of Hoescht dye. The slides were washed in PBS-Tx and subsequently analyzed by confocal microscopy.

[0163] Phages displaying murine IL-2 cytokine and the anti-PD-L1 scFv targeting moiety bind to PD-L1 + cancer cells. To demonstrate that phages displaying murine IL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1) bind to cancer cells via their anti-PD-L1 scFv targeting moiety, an immunofluorescence experiment was performed. PD-L1 + A20 cells were incubated for 24 hours in the presence of 10 11 / mL naked phages (naked phages do not display any therapeutic agent and cancer cell targeting moiety) or mIL-2-phage-PD-L1 to evaluate the binding of each compound to PD-L1 + cancer cells. Hoescht dye was used to reveal the cell nuclei, while an anti-M13 antibody conjugated to FITC was used to reveal phages and mIL-2-phage-PD-L1 according to the manufacturer's protocol and recommendations. The experiment showed that only mIL-2-phage-PD-L1 was able to bind to A20 cells, as indicated by the FITC signal observed on cancer cells after the treatment ( Figure 5 ). This result indicates that the presence of the cancer cell targeting moiety (i.e., the anti-PD-L1 scFv of mIL-2-phage-PD-L1) allows mIL-2-phage-PD-L1 to bind to PD-L1 + cancer cells and cover their surface. Then mIL-2-phage-PD-L1 can recruit and activate the immune system to induce an anti-tumor immune response.

[0164] Example 5: Phages displaying cytokines and cancer cell targeting moieties have enhanced local anti-tumor activity.

[0165] Strains, plasmids, phage production, and growth conditions. All strains and plasmids used in this example are described in Table 3. Cells were generally grown in Luria broth Miller medium (LB) or on Luria broth agar Miller medium, which was supplemented with antibiotics at the following concentrations as needed: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were routinely grown at 37 °C for no more than 18 hours before being used in experiments. Phages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol provided in Example II.

[0166] Cell culture. CT26 colorectal cancer cells were purchased from ATCC (CRL-2638). The cells were cultured under optimal conditions at 37 °C in an atmosphere of 5% CO2 and 95% humid air in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (50 U / mL).

[0167] Tumor mouse model. All experiments involving mice were rigorously evaluated by our local university's (Université de Sherbrooke) animal care committee, and the animals were exposed to minimal stress and pain during the experiments. Mice were provided with water and normal food ad libitum and allowed to rest for at least 2 days after arrival. A maximum of 5 mice were housed in the same cage, and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were tracked daily during the experiment.

[0168] As a general guideline, this section details the workflow of a typical mouse experiment. To generate solid tumors, 10 6 CT26 cells resuspended in 50 μL PBS were injected into the right flank of the mice. Then, the mice were observed daily to measure tumor growth until the tumor size reached 40 - 80 mm 3 . Next, the mice received 50 μL of the treatment by intratumoral injection. Next, the tumor size was tracked twice a week until clearance or until the tumor reached a volume of 1500 mm 3 , after which the mice were sacrificed and the tumors were collected from the mice. The presence of metastases in other organs was also evaluated.

[0169] Phages displaying murine IL-2 cytokine and the anti-PD-L1 scFv targeting moiety showed enhanced antitumor activity. To evaluate the effect of adding cytokines on the antitumor activity of phages displaying cancer cell targeting moieties, phages displaying the PD-L1 checkpoint inhibitor on pIII and murine interleukin-2 (mIL-2) on pIX were developed using the method described in Example I. 10 6 CT26 cells were subcutaneously injected into the right flank of the mice, and the mice were observed every two days to monitor tumor growth. When the tumors reached a volume of 50 to 100 mm 3 , the 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 a control ( Figure 6 ). The second group received an effective dose of 5 × 10 11 recombinant murine IL-2 (mIL-2) molecules ( Figure 6 ) from Sino Biological. The third group received an effective dose of 10 11Phages displaying anti-PD-L1 scFv on pIII (phage-PD-L1) ( Figure 6 ). The fourth group received an effective dose consisting of 5 × 10 11 mIL-2 molecules and 10 11 phage-PD-L1 molecules. The fifth and final group received an effective dose of 10 11 phages displaying mIL-2 on pIX and anti-PD-L1 scFv on pIII (mIL-2-phage-PD-L1). Tumor size was then monitored twice weekly using precision calipers until the tumor was eliminated or too large to continue the experiment. The experiment revealed that enhanced anti-tumor activity was observed only when mIL-2 was physically conjugated to phages displaying the PD-L1 checkpoint inhibitor targeting cancer cells. This indicates that conjugating cytokines to phages that simultaneously display cancer cell targeting moieties provides a synergistic anti-tumor effect.

[0170] Example 6: Phages displaying cytokines and cancer cell targeting moieties have systemic anti-tumor activity.

[0171] Strains, plasmids, phage production and growth conditions. All strains and plasmids used in this example are described in Table 3. Cells were generally grown in Luria broth Miller medium (LB) or on Luria broth agar Miller medium, which was supplemented as needed with the following concentrations of antibiotics: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were routinely grown at 37 °C for no more than 18 hours before use in experiments. Phages were obtained from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol provided in Example II.

[0172] Cell culture. A20 lymphocyte B lymphoma cells were purchased from ATCC (TIB-208). Upon arrival, the cells were washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This medium was used to prepare cells for all experiments. Frozen stock solutions were generated after 4 passages and used to initiate subsequent cultures for experiments. In all experiments, the cells were maintained at a density of 2 × 10 5 cells / mL to 2 × 10 6 cells / mL and grown at 37 °C in an atmosphere of 5% CO2 and 95% humid air.

[0173] Tumor mouse model. All experiments involving mice were rigorously evaluated by the animal care committee of our local university (University of Sherbrooke), and animals were exposed to minimal stress and pain during the experiments. Mice were provided with water and normal food ad libitum and allowed to rest for at least 2 days after arrival. Up to 5 mice were kept in the same cage, and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were tracked daily during the experiment.

[0174] As a general guideline, this section details the workflow of a typical mouse experiment. To generate solid tumors, 5×10 6 A20 cells resuspended in 100 μL PBS were injected into the right flank of the mice, and then 4 days later, 5×10 6 A20 cells were injected into the left flank, resulting in the mice carrying two tumors simultaneously. Mice were observed daily to measure tumor growth until the volume of the right tumor reached 50 - 100 mm 3 , and then the mice received 50 μL of the treatment by intratumoral injection. Next, the tumor size was tracked twice a week until clearance or until the total tumor volume (right + left) reached 1500 mm 3 , after which the mice were sacrificed.

[0175] Phages displaying murine IL-2 cytokine and the anti-PD-L1 scFv targeting moiety have systemic anti-tumor activity. To evaluate whether phages displaying cytokines and cancer cell targeting moieties have systemic anti-tumor activity, phages displaying a PD-L1 checkpoint inhibitor on pIII and murine interleukin-2 (mIL-2) on pIX (described in Example I) were injected into mice bearing tumors on both sides. However, only the tumors in the right flank were injected with the drug to evaluate whether a systemic anti-tumor immune response was triggered and to induce clearance of the non-injected tumors present in the left flank. The mice were divided into two groups, and when the tumors in the right flank reached 50 - 100 mm 3 , the tumors were injected, and all treatments were administered intratumorally on days 0, 4, and 7. The first group received an injection of vehicle (PBS) as a control ( Figure 7 ). The second group received an effective dose of 10 12 phages displaying mIL-2 on pIX and anti-PD-L1 scFv on pIII (mIL-2-phage-PD-L1, Figure 7)。Then, the tumor size was monitored twice a week using precision calipers until the tumor was eliminated or too large to continue the experiment. The experiment revealed that phages displaying mIL-2 on pIX and anti-PD-L1 scFv not only induced the clearance of the injected tumor (right flank), but also induced the clearance of the non-injected tumor (left flank). These results indicate that phages displaying cytokines and cancer cell targeting moieties have systemic anti-tumor activity (i.e., anti-tumor activity beyond the local anti-tumor activity of the injected cell population).

[0176] Example 7: The local and systemic anti-tumor activities of phages displaying cytokines and cancer cell targeting moieties are mediated by the immune response.

[0177] Strains, plasmids, phage production, and growth conditions. All strains and plasmids used in this example are described in Table 3. Cells were generally grown in Luria-Bertani Miller medium (LB) or on Luria-Bertani agar Miller medium, which was supplemented as needed with the following concentrations of antibiotics: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. All cultures were routinely grown at 37 °C for no more than 18 hours before use in experiments. Phages were obtained from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol provided in Example II.

[0178] Cell culture. A20 lymphocytic B lymphoma cells were purchased from ATCC (TIB-208). Upon arrival, the cells were washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This medium was used to prepare cells for all experiments. Frozen stock solutions were generated after 4 passages and used to initiate subsequent cultures for experiments. In all experiments, the cells were maintained at a density of 2×10 5 cells / mL to 2×10 6 cells / mL and grown at 37 °C in an atmosphere of 5% CO2 and 95% humid air.

[0179] Tumor mouse model. All experiments involving mice were strictly evaluated by our local university's (University of Sherbrooke) Animal Care Committee, and animals were exposed to minimal stress and pain during the experiments. Mice were provided with free access to water and regular food and allowed to rest for at least 2 days after arrival. There were a maximum of 5 mice in the same cage, and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were tracked daily during the experiment.

[0180] As a general guideline, this section details the workflow of a typical mouse experiment. To generate solid tumors, 5×10 6 A20 cells resuspended in 100 μL PBS were injected into the right flank of the mice, and then 4 days later, 5×10 6 A20 cells were injected into the left flank, resulting in the mice carrying two tumors simultaneously. Mice were observed daily to measure tumor growth until the volume of the right tumor reached 50 - 100 mm 3 , and then the mice received 50 μL of the treatment by intratumoral injection. Next, the tumor size was tracked twice a week until clearance or until the total tumor volume (right + left) reached 1500 mm 3 , after which the mice were sacrificed.

[0181] Ex vivo culture of microdissected tumors (MDT) on a chip. Tumors derived from the A20 cell line were collected from BALB / c mice and microdissected. The MDT were kept in RPMI - 1640 10% FBS PEN / STREP before and after loading onto a specially formulated ex vivo culture chip (MISO Chip Inc.). The MDT were distributed as 8 MDT / channel, 4 channels / chip, and incubated at 37 °C + 5% CO2 for 24 hours.

[0182] Treatment of microdissected tumors (MDT) cultured on a chip. The treatments, namely synthetic therapeutic phages and atezolizumab (an anti - PD - L1 checkpoint inhibitor used as a benchmark reference), were diluted in the medium to 2×10 12 molecules / mL. Then, before application, each corresponding chip of each channel was washed three times with the treatment. Next, the chips were incubated at 37 °C + 5% CO2 for 48 hours, and the supernatant was collected and then sent to Eve Technologies for cytokine concentration analysis by ELISA. Chips with MDT exposed to the same volume of PBS were also generated and used as controls.

[0183] Immune infiltration analysis. BALB / c mice received an injection of 5×10 6 A20 cells subcutaneously in their right flank for implantation. When the tumor reached 75 - 150 mm 3At that time, PBS or 10 11 phage particles expressing mIL-2 and anti-PD-L1 (mIL-2-phage-PD-L1) were injected into the tumor three times within 7 days. On the 8th day, the mice were euthanized, and the tumors were collected and then fixed in formalin. Next, the tumor samples were sent to the Plateforme d′Histologie de l′Universite de Sherbrooke for permeabilization and paraffin embedding. Next, slides were generated, stained with hematoxylin-eosin, and digitized. Then, the images were observed and processed using Qpath to distinguish immune cells from tumor cells.

[0184] The local and systemic anti-tumor activities of phages expressing murine IL-2 cytokine and anti-PD-L1 scFv targeting moieties are mediated by immune activation. The potential mechanisms of the local and systemic anti-tumor immune responses observed under phages expressing mIL-2 and anti-PD-L1 (mIL-2-phage-PD-L1) were investigated. Since mIL-2-phage-PD-L1 was designed to activate the immune system by acting on multiple targets through the phage's IL-2 cytokine, anti-PD-L1, as well as the TLR9 agonist DNA and antigenic antigens, we hypothesized that mIL-2-phage-PD-L1 treatment should induce multiple cytokines involved in different branches of the activated immune response. To identify the immune pathways involved in the anti-tumor activity of mIL-2-phage-PD-L1, we used ex vivo cultures and treated tumors on a chip, which allowed tracking of the secretion of key cytokines over time. Thus, A20 tumors were microdissected and cultured ex vivo on a dedicated chip (MISO chip Inc.) and exposed to equimolar amounts of PBS (equal volume to two other treatments), mIL-2-phage-PD-L1, or atezolizumab (anti-PD-L1). Next, 2 days after treatment, cytokines were added to the culture supernatant, and the fold change of key cytokines relative to the PBS condition was calculated ( Figure 8 ). While the cytokine levels changed very little with atezolizumab, mainly limited to the Th1 response pathway, mIL-2-phage-PD-L1 activated all major immune pathways, most notably the acute, Th1, and Th17 immune response pathways, showing significant involvement of multiple immune cell types in eliminating tumor cells. Thus, the anti-tumor response triggered by mIL-2-phage-PD-L1 is mediated by at least CD8 + T cells and myeloid cells and may be supported by B cells through the production of anti-tumor IgG.

[0185] Demonstration that the local and systemic anti-tumor activities of phages presenting murine IL-2 cytokine and the anti-PD-L1 scFv targeting moiety are mediated by substantial immune infiltration of the tumor and recruitment of immune cells. Cytokine analysis experiments showed clear signs of immune activity, and the presence of immune cells was then investigated. Thus, mice bearing A20 tumors with a volume of 75 - 150 mm 11 were treated on days 0, 4, and 7 with 10 3 mIL-2-phage-PD-L1 particles or PBS. Then, the mice were sacrificed on day 8, the tumors were collected, fixed in formalin, and processed histologically by the Histology Platform of the University of Sherbrooke. After hematoxylin and eosin staining, differences in the density of infiltrating immune cells in the tumor tissue were observed ( Figure 9 ). It was found that tumors treated with mIL-2-phage-PD-L1 were highly infiltrated with immune cells, while very few immune cells were detectable in the PBS control group ( Figure 9 A). There were also larger necrotic plaques in the tumor tissue of the mIL-2-phage-PD-L1 treatment group, indicating active destruction of the tumor tissue ( Figure 9 B). In summary, treatment with mIL-2-phage-PD-L1 caused the recruitment of multiple types of immune cells, thereby eliminating the tumor.

[0186] Example 8: Demonstration that the therapeutic activity of phages presenting cytokine and cancer cell targeting moieties is mediated by a systemic long-term anti-tumor immune response.

[0187] Strains, plasmids, phage production, and growth conditions. All strains and plasmids used in this example are described in Table 3. Cells were generally grown in Luria broth Miller medium (LB) or on Luria broth agar Miller medium, which was supplemented as needed with the following concentrations of antibiotics: ampicillin (Ap) 100 μg / mL, chloramphenicol (Cm) 34 μg / mL, kanamycin (Km) 50 μg / mL, nalidixic acid (Nx) 4 μg / mL, spectinomycin (Sp) 100 μg / mL, streptomycin (Sm) 50 μg / mL, sulfamethoxazole (Su) 160 μg / mL, tetracycline (Tc) 15 μg / mL, and trimethoprim (Tm) 32 μg / mL. Before use in experiments, all cultures were routinely grown at 37 °C for no more than 18 hours. Phages were obtained from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol provided in Example II.

[0188] Cell culture. A20 lymphocytic B lymphoma cells were purchased from ATCC (TIB - 208). Upon arrival, the cells were washed and resuspended in RPMI - 1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2 - mercaptoethanol. The medium was used to prepare cells for all experiments. After 4 passages, frozen stocks were generated and used to initiate subsequent cultures for experiments. In all experiments, the cells were maintained at a density of 2×10 5 cells / ml to 2×10 6 cells / ml and grown at 37 °C in an atmosphere of 5% CO2 and 95% humid air.

[0189] Tumor mouse model. All experiments involving mice were rigorously evaluated by our local university's (University of Sherbrooke) animal care committee, and animals were exposed to minimal stress and pain during the experimental procedures. Mice were provided with water and normal food ad libitum and allowed to rest for at least 2 days after arrival. There were at most 5 mice in the same cage, and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were tracked daily during the experiment.

[0190] Demonstrate that the therapeutic activity of phages displaying murine IL - 2 cytokine and anti - PD - L1 scFv targeting moieties is mediated by a systemic long - term anti - tumor immune response. To evaluate whether the anti - tumor activity of phages displaying cytokine and cancer cell - targeting moieties is mediated by a long - term systemic anti - tumor immune response, a rechallenge experiment was conducted in cured mice and untreated mice (mice that had never been exposed to A20 cancer cells or previously treated with phages displaying cytokine and cancer cell - targeting moieties). Cured mice were obtained by treating mice bearing A20 cancer cell tumors in the right flank with an effective dose of 10 12 phages displaying a PD - L1 checkpoint inhibitor and murine interleukin - 2 (mIL2 - phage - PD - L1). When the tumor volume reached 50 - 100 mm 3 , the treatment was injected into the tumor, and the treatment was administered intratumorally on days 0, 4, and 7 ( Figure 10 ). Eight mice that were cured by the treatment and had their tumors completely cleared were housed in the animal facility for 160 days. On day 160, the cured mice as well as the untreated mice received an injection of 5×10 6 A20 cancer cells to induce tumor formation, but this time, the cancer cells were injected into the left flank instead of the right flank ( Figure 10 ). The experiment revealed that new tumors formed and grew only in the untreated mice, while tumors in the previously cured mice were systematically eliminated. These results indicate that mIL2 - phage - PD - L1 treatment induces an adaptive long - term and systemic anti - tumor immune response capable of preventing the implantation of new tumors and thus preventing tumor recurrence.

[0191] Any element of any embodiment can be used in any embodiment. Although the invention has been described with reference to specific embodiments, those skilled in the art will understand that various changes can be made and elements thereof can be replaced with equivalents without departing from the true spirit and scope of the invention. In addition, modifications can be made without departing from the basic teachings of the invention. Identification of equivalent compositions, methods, and kits according to the teachings of the present disclosure is well within the skill of the ordinary practitioner and will require only routine experimentation. The practice of the present disclosure will be more fully understood from the following examples, which are presented herein for illustration only and should not be construed as limiting the present disclosure in any way.

[0192] All references cited in this specification and their references are hereby incorporated by reference in their entirety, where appropriate, to teach additional or alternative details, features, and / or technical background.

[0193] Although the present disclosure has been specifically shown and described with reference to particular embodiments, it should be understood that the variations and other features and functions or alternatives thereof disclosed above can ideally be combined into many other different systems or applications. In addition, various presently unforeseen or unanticipated substitutions, modifications, variations, or improvements may subsequently be made by those skilled in the art, which are also intended to be encompassed by the appended claims.

Claims

1. A phage that simultaneously displays at least one cytokine and at least one cancer cell targeting moiety.

2. The phage according to claim 1, wherein the at least one cytokine is selected from: IL-1α, IL-1b, IL-1ra, 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, TNFα, LTα, LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, FLT3 ligand, G-CSF, GM-CSF, IFNα, IFNβ, IFNω, IFNγ, LIF, M-CSF, MIF, OSM, SCF, TGFβ1, TGFβ2, TGFβ3, and TSLP ligand.

3. The phage according to 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 γ (IFN-γ).

4. The phage according to claim 1, wherein the at least one cytokine is IL-2.

5. The phage according to claim 1, wherein the at least one cytokine is IL-15.

6. The phage according to any one of claims 1 to 5, wherein the at least one cytokine is capable of being displayed on pIII, pVI, pVII, pVIII, or pIX, or a combination thereof.

7. The phage according to any one of claims 1 to 5, wherein one of the at least one cytokines is displayed on pIX.

8. The phage according to any one of claims 1 to 5, wherein one of the at least one cytokines is displayed on pIII.

9. The phage according to any one of claims 1 to 8, wherein the at least one cancer cell targeting moiety binds to Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD59, CD24, E-cadherin, cMet, MUC1, or CD133, or targets a combination thereof.

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

11. The phage according to claim 10, wherein the PD-L1 targeting moiety is an anti-PD-L1 scFv or a fragment thereof.

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

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

14. The phage according to any one of claims 1 to 11, wherein one of the at least one cancer cell targeting moieties is displayed on pIII.

15. The phage according to any one of claims 1 to 14, wherein the phage is a synthetic phage.

16. The phage according to any one of claims 1 to 15, wherein the phage is a therapeutic phage.

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

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

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

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

21. A pharmaceutical composition comprising the phage according to any one of claims 1 to 16 and a suitable pharmaceutical carrier.

22. The pharmaceutical composition according to claim 21, wherein the at least one cytokine is selected from: IL-1α, IL-1b, IL-1ra, 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, TNFα, LTα, LTβ, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, FLT3 ligand, G-CSF, GM-CSF, IFNα, IFNβ, IFNω, IFNγ, LIF, M-CSF, MIF, OSM, SCF, TGFβ1, TGFβ2, TGFβ3, and TSLP ligand.

23. The pharmaceutical composition according to 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 γ (IFN-γ).

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

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

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

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

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

29. The pharmaceutical composition according to 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 CD133, or a combination thereof.

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

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

32. The pharmaceutical composition according to any one of claims 21 to 31, wherein the at least one cancer cell targeting moiety is capable of being displayed on pIII, pVI, pVII, pVIII or pIX or a combination thereof.

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

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

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

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

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

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