Therapeutic bacteriophages displaying cancer cell-targeting moieties along with cytokines for the treatment of cancer

Abstract

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

Description

[Technical Field]

[0001] The present technology relates generally to bacteriophages engineered to display cancer cell targeting moieties and cytokines. The technology also relates generally to the use of such bacteriophages in the treatment of cancer. [Background technology]

[0002] Despite significant advances in cancer treatment, cancer remains the second most common cause of death in industrialized countries (Siegel R. et al. ACS Journal, Cancer Statistics 2021, incorporated herein by reference). Cancer is a complex and challenging disease, often requiring simultaneous action on several therapeutic targets to maximize the chances of treatment success. This strategy, called combination therapy, involves clinicians treating patients by combining two or more therapeutic agents (Mokhtari et al. Oncotarget 2017 Jun 6;8(23):38022-38043, incorporated herein by reference). By targeting different pathways to inhibit or eliminate cancer cells, combination therapy provides superior results over monotherapy through synergistic effects and has become the cornerstone of cancer treatment. However, combination therapy has important limitations. By increasing the number of treatments, combination therapy also increases the cost of care and the risk of side effects.

[0003] A solution to this problem would be a therapeutic modality capable of multitasking therapeutic activity, enabling combination therapy with a single drug. To solve this problem, bacteriophages capable of multitasking therapeutic activity have been developed to provide next-generation immunotherapy for cancer treatment (WO2022073127, incorporated herein by reference). These bacteriophages function as therapeutic scaffolds displaying combinations of immunomodulatory agents. Because bacteriophages can multitask therapeutic activity, their antitumor activity is coupled to the combination of therapeutic molecules carried by them and the therapeutic synergy that may result from these combinations.

[0004] In view of the above, finding combinations of therapeutic molecules that act synergistically when displayed on bacteriophages is key to enhancing the antitumor activity of the treatment. Summary of the Invention

[0005] According to various aspects, the present technology relates to a bacteriophage that simultaneously displays at least one cytokine and at least one cancer cell targeting moiety. In some cases, the bacteriophage of the present technology is a synthetic bacteriophage. In some cases, the bacteriophage of the present technology is a therapeutic bacteriophage. In some cases, the bacteriophage of the present technology is a synthetic therapeutic bacteriophage. In some cases, the cytokines include 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- In some cases, the cancer cell targeting moiety is selected from Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD59, CD24, E-cadherin, cMet, MUC1, or CD133, or a combination thereof.

[0006] According to various aspects, the present technology relates to a method for reducing tumor size in a subject, the method comprising administering to the subject a therapeutically effective amount of a bacteriophage as defined herein.

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

[0008] According to various aspects, the present technology relates to pharmaceutical compositions comprising a bacteriophage that simultaneously displays a cytokine and a cancer cell targeting moiety, along with a suitable pharmaceutical carrier.

[0009] According to various aspects, the present technology relates to a method for reducing tumor size in a subject, the method comprising administering to the subject a pharmaceutical composition as defined herein.

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

[0011] According to various aspects, the present technology relates to compositions comprising a cytokine and a bacteriophage that displays a moiety that targets cancer cells.

[0012] According to various aspects, the present technology relates to bacteriophages that display one or more therapeutic agents that target cancer cell markers on one or more of their coating proteins, and bacteriophages that display one or more cytokines on one or more of their coating proteins.

[0013] According to various aspects, the present technology 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 bacteriophage as defined herein.

[0014] According to various aspects, the present technology relates to the use of an effective amount of a bacteriophage as defined herein for the treatment of cancer in a subject in need thereof.

[0015] According to various aspects, the present technology relates to the use of an effective amount of a bacteriophage as defined herein in the manufacture of a medicament for the treatment of cancer in a subject.

[0016] According to various aspects, the present technology relates to a kit comprising a bacteriophage as defined herein together with instructions for administering the drug to a subject. [Brief explanation of the drawings]

[0017] [Figure 1] 1A, 1B, and 1C are schematic diagrams of a bacteriophage production and vector overview according to one embodiment of the present technology. FIG. 1A is a schematic diagram of an exemplary configuration of a bacterial production strain secreting bacteriophage. A bacteriophage secretion system can be composed of a bacteriophage machinery vector combined with a bacteriophage scaffold vector. FIG. 1B shows examples of bacteriophage machinery vectors: M13K07, pTAT004, and pTAT025. FIG. 1C shows examples of bacteriophage scaffold vectors: pTAT002, pTAT013 (including derivatives pTAT044 and pTAT070), and pTAT014 (including derivatives pTAT017, pTAT060, and pTAT071). [Figure 2] Figure 1 shows a schematic diagram of the mode of action of a bacteriophage displaying an anti-PD-L1 checkpoint inhibitor scFv as a cancer cell-targeting moiety and IL-2 as an immunomodulatory cytokine. The bacteriophage binds to cancer cells via the anti-PD-L1 scFv displayed on pIII, which localizes the bacteriophage to the cancer cell and inhibits the PD-L1 checkpoint, enhancing T cell activation. The presence of IL-2 cytokine bound to the bacteriophage results in a synergistic effect, further enhancing the antitumor immune response and improving tumor clearance. In parallel, filamentous bacteriophages can be internalized by cancer cells via endocytosis, providing additional natural immunogenic activity through (1) activation of TLR9, which elicits an immune response, and (2) bacteriophage-derived antigens that can be presented by MHC molecules, leading to the activation of cytotoxic T cells and subsequent elimination of cells displaying these antigens. [Figure 3]Figures 3A and 3B are graphs demonstrating that bacteriophage particles can dually display a functional cancer cell targeting moiety and a cytokine. Figure 3A: Assessment of bacteriophage binding to PD-L1 by indirect ELISA. The graph shows the HRP signal of an ELISA quantifying the binding of different phage preparations to immobilized human PD-L1 protein. PEG-NaCl purified phage were diluted to 1 x 10 phage / mL in TBS. The signal was measured using an anti-pVIII-HRP antibody to detect the presence of bacteriophage particles. Figure 3B: Assessment of bacteriophage displaying recombinant cytokines on pIX by sandwich ELISA. The graph shows the HRP signal of an ELISA quantifying the binding of different phage preparations to immobilized anti-mouse IL2 antibody or anti-human IL15 antibody. PEG-NaCl purified phage were diluted to 1 x 10 phage / mL in TBS. The signal was measured using an anti-pVIII-HRP antibody to detect the presence of phage particles. [Figure 4] Figure 10 is a graph demonstrating the ability of bacteriophage to display biologically active cytokines. The graph shows the signal measured at 630 nm from a HEK-Blue™ IL-2 reporter cell assay, where activation of the IL-2 receptor is quantified by either: PBS (vehicle), 10 bacteriophage displaying anti-PD-L1 scFv (phage-PD-L1), 10 bacteriophage displaying anti-PD-L1 scFv and murine IL-2 (mIL-2) cytokine (mIL-2-phage-PD-L1), or 0.2 ng mIL-2. [Figure 5]Figure 1 shows microscopy images demonstrating that bacteriophage displaying a cytokine and a cancer cell targeting moiety can bind to cancer cells. PD-L1+ A20 cancer cells were exposed to phage (not displaying a cancer targeting moiety) or bacteriophage displaying the mIL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1). Binding of the phage and mIL-2-phage-PD-L1 was then revealed using an anti-M13 antibody conjugated to FITC. [Figure 6] Figure 1 shows the physical coupling of IL-2 cytokine to cancer cell-targeting bacteriophage to provide a synergistic anti-tumor effect. Mice were engrafted with tumors by injecting 10 CT26 cancer cells into the right flank. Treatment was administered when tumor volumes were contained within 50-80 mm . Individual tumor volumes were then measured for each mouse. Treatment was administered on days 0, 4, and 7 using intratumoral injections of either PBS, 5 x 10 molecules of murine IL-2 (mIL-2), 10 particles of bacteriophage displaying anti-PD-L1 scFv (phage-PD-L1), 5 x 10 molecules of mIL-2 in conjunction with 10 particles of phage-PD-L1, or 10 particles of bacteriophage displaying anti-PD-L1 scFv (mIL-2-phage-PD-L1). Tumor volume was calculated by multiplying the maximum measurement by the square of the perpendicular measurement and dividing by 2 (solid line = ablated mice, dotted line = non-ablated mice). [Figure 7]Figure 1 shows the systemic antitumor activity of bacteriophage displaying cytokines and cancer cell-targeting moieties. Tumors were engrafted into both flanks of mice. 5 x 106 A20 cancer cells were injected into the right flank, followed four days later by 5 x 106 A20 cancer cells injected into the left flank. Right tumors were treated when tumor volumes reached 50-100 mm3, while left tumors were untreated. Treatments were administered on days 0, 4, and 7 using intratumoral injections of either PBS or 1012 particles of bacteriophage displaying mIL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1). Tumor volumes for injected and uninjected tumors were calculated by multiplying the maximum measurement by the square of the perpendicular measurement and dividing by 2. For injected and uninjected tumors, tumor removal is shown for both treatments. [Figure 8] This heat map shows that the antitumor activity of bacteriophage displaying cytokines and cancer cell-targeting moieties is mediated by the immune response. Bacteriophage displaying the mIL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1) activates all major immune pathways. A20 tumors measuring 75-150 mm3 were extracted from BALB / c mice, microdissected, and cultured ex vivo on chips. Microdissected tumors were then treated with PBS (control), mIL-2-phage-PD-L1, or the anti-PD-L1 checkpoint inhibitor atezolizumab (benchmark reference). Treatment-induced fold-change cytokine levels were assessed. Cytokine fold-change relative to the corresponding PBS condition is shown on a log-scale heat map. [Figure 9]Figures 9A and 9B show graphs and histological images, respectively, demonstrating that the antitumor activity of bacteriophages displaying cytokines and cancer cell-targeting moieties is mediated by extensive immune infiltration of tumors. BALB / c mice bearing A20 tumors were intratumorally injected with either PBS or bacteriophages displaying 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, mice were sacrificed, and tumors were removed and processed for histological examination. Tumor images were analyzed using Qpath to identify and differentiate cells within the tissue. Using a machine learning approach, tumor and immune cells were detected within tumors and automatically quantified across different samples (Figure 9A). Representative images of tumors treated with PBS or mIL-2-phage-PD-L1 are shown (Figure 9B). Both images share the same scale, and tissue damage and tumor shrinkage can be observed. Pale sections of tissue visible in most of the mIL-2-phage-PD-L1 treated tumors indicate the presence of necrosis. [Figure 10] This graph shows that the therapeutic activity of bacteriophage displaying cytokines and cancer cell-targeting moieties is mediated by a long-term, adaptive, and systemic antitumor immune response. Tumors were engrafted by injecting 5 x 10 A20 cancer cells into the right flank of mice. Tumors were then treated when their volume reached 80-100 mm. Treatment was performed on days 0, 4, and 7 using intratumoral injections of either mIL-2 cytokine or 10 particles of bacteriophage displaying anti-PD-L1 scFv (mIL-2-phage-PD-L1). Mice with complete tumor clearance were considered cured and maintained for 160 days. On day 160, new tumors were formed by injecting 5 x 10 A20 cancer cells, but this time into the left flank of the mice. Naive mice that had not been exposed to A20 cancer cells or received mIL-2-phage-PD-L1 treatment were also treated. Tumor volume was then measured for each mouse, calculated by multiplying the maximum measurement by the square of the perpendicular measurement and dividing by two. DETAILED DESCRIPTION OF THE INVENTION

[0018] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0019] The recitation herein of numerical ranges by endpoints is intended to include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 4.32, and 5).

[0020] Whether or not the term "about" is explicitly used herein, all quantities given herein are meant to refer to the actual given value, and also to refer to approximations to such a given value that are reasonably inferred based on ordinary skill in the art, including equivalents and approximations resulting from experimental and / or measurement conditions for such a given value. For example, the term "about" in the context of a given value or range refers to a value or range that is within 20%, preferably within 15%, more preferably within 10%, more preferably within 9%, more preferably within 8%, more preferably within 7%, more preferably within 6%, and more preferably within 5% of the given value or range.

[0021] The term "and / or," as used herein, should be considered as a specific disclosure of each of the two specified features or components with or without the other. For example, "A and / or B" should be considered as a specific disclosure of (i) A, (ii) B, and (iii) each of A and B, as if each were individually set forth herein.

[0022] An "inducible promoter" refers to a regulatory region operably linked to one or more genes, such that expression of the gene(s) is increased in the presence of an inducer of the regulatory region or increased in the absence of a repressor of the regulatory region. An inducible promoter can be induced by exogenous environmental condition(s), which refers to the setting(s) or situation(s) in which the promoter described herein is induced. Exogenous environmental conditions refer to environmental conditions external to an intact (non-lysed) engineered microorganism, endogenous or natural environmental conditions relative to the tumor environment or host subject environment, or exogenously introduced perturbations to the environment. 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). Corresponding Fnr-, Anr (anaerobic nitrate respiration)-, and Dnr (dissimilatory nitrate respiration regulator)-responsive promoters are known in the art (see, e.g., Castiglione et al., 2009; Eiglmeier et al., 1989; Galimand et al., 1991; Hasegawa et al., 1998; Hoeren et al., 1993; Salmon et al., 2003, incorporated herein by reference). Examples of pH-dependent transcription factors include, but are not limited to, the phoBR-responsive promoter. Examples of thermoregulatory promoters include, but are not limited to, the pL and / or pR phage promoters and the use of a mutant cI857 repressor. Examples of ROS-level-dependent transcription factors include, but are not limited to, OxyR. Corresponding OxyR-responsive promoters include, but are not limited to, TrxCp, HemHp, sufA, AhpCp1000, AhpCp2D1, AhpCp2, AhpCpD1, AhpCp1, and DsbGp. Inducible promoters can also be induced by one or more exogenous molecules.An exogenous molecule refers to a molecule that does not naturally occur in an intact (non-lytic) engineered microorganism. Examples of exogenous molecules and their respective inducible promoters include, but are not limited to, the L-arabinose and ParaBAD promoters, the rhamnose and rhaP BAD promoters, the IPTG and Lac promoters, and the tetracycline induction system (Tet on-Tet off). An inducible promoter may also be induced by one or more endogenous molecules. An endogenous molecule refers to a molecule that is naturally produced in an intact (non-lytic) engineered microorganism. Examples of endogenous molecules and their respective inducible promoters include, but are not limited to, the diaminopimelic acid and PdapA promoters, N-acyl-homoserine lactone, and PluxI.

[0023] An inducible promoter can comprise one or more regulatory elements including, but not limited to, an enhancer sequence, a response element, a protein recognition site, an inducible element, a promoter control element, a protein binding sequence, 5' and 3' untranslated regions, a transcription initiation site, a termination sequence, a polyadenylation sequence, a riboswitch, and an intron.

[0024] The present technology is described in more detail below. This description is not intended to be a detailed list of all the different ways in which the present technology can be implemented or all the features that can be added to the present technology. For example, features shown with respect to one embodiment can be incorporated into other embodiments, and features shown with respect to a particular embodiment can be omitted from that embodiment. Furthermore, numerous modifications and additions to the various embodiments suggested herein will be apparent to those skilled in the art in light of this disclosure, and these modifications and additions do not depart from the present technology. Therefore, the following description is intended to illustrate some specific embodiments of the present technology, but does not exhaustively identify all permutations, combinations, and modifications thereof.

[0025] Components of a bacteriophage A solution for treating cancer is to use molecular scaffolds that can bind several immunomodulatory molecules to mount a potent antitumor immune response by acting simultaneously on several therapeutic targets. Filamentous bacteriophages are large immunogenic biological structures on which therapeutic proteins or peptides can be displayed to create synthetic therapeutic bacteriophages. The natural immunogenicity of filamentous bacteriophages is mediated by (1) their genomes containing CpGs, which act as TLR9 agonists and can induce innate immune responses (Sartorius et al., NPJ Vaccines, 2021 Oct 28;6(1):127, incorporated herein by reference), and (2) the presence of immunogenic antigens in their coating proteins, which can stimulate cytotoxic T cell elimination of cells displaying these antigens on MHC-1 after endocytosis of the bacteriophage (Gaubin et al., DNA And Cell Biology, Volume 22, Number 1, 2003, incorporated herein by reference). Therefore, the natural immunogenic activity of filamentous bacteriophages can be combined with specific immunomodulatory proteins or peptides to enhance their antitumor activity.

[0026] According to various embodiments, the present technology relates to operable bacteriophages in the treatment of cancer, such as those described in WO2022073127 (incorporated herein by reference). In some cases, the bacteriophage is immunogenic. In some further cases, the bacteriophage can display single or multispecific therapeutic proteins.

[0027] In some embodiments, the technology relates to bacteriophages that display one or more cancer cell targeting moieties along with one or more cytokines, the combination of these therapeutic molecules acting synergistically to enhance tumor elimination.

[0028] In some embodiments, the technology relates to bacteriophages that display one or more cancer cell targeting moieties along with one or more cytokines, the combination of these therapeutic molecules acting synergistically to enhance tumor elimination when physically bound to the bacteriophage.

[0029] Bacteriophage production In some embodiments, the bacteriophage of the present technology is produced as described in WO2022073127 (hereby incorporated by reference) and in FIG. 1.

[0030] Therapeutic activity of bacteriophages Synthetic phages displaying cancer cell targeting moieties: In some embodiments, the bacteriophage of the present technology displays one or more cancer cell targeting moieties capable of recognizing one or more cancer cell markers. The types of targeting moieties displayed by the bacteriophage 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, one or more targeting moieties can be displayed on pIII, pVI, pVII, pVIII, and / or pIX (as shown in Figure 2 of WO2022073127; incorporated herein by reference).

[0031] In some embodiments, the cancer cell marker recognized by the targeting moiety is a molecule present on cancer cells.

[0032] In another embodiment, the cancer cell marker is a molecule that is overexpressed by cancer cells.

[0033] In yet another embodiment, the cancer marker is a molecule that is specifically expressed by cancer cells.

[0034] In some embodiments, the cancer cell marker targeted by the one or more targeting moieties is a protein.

[0035] In another embodiment, the cancer cell marker targeted by the one or more targeting moieties is selected from, but not limited to, Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD59, CD24, E-cadherin, cMet, MUC1, and CD 133. In one embodiment, the cancer marker targeted by the one or more targeting moieties is PD-L1.

[0036] Cytokine-displaying synthetic phages: In some embodiments, the bacteriophage of the present technology displays a tumor-targeting moiety and one or more cytokines (Figure 2). Cytokines are molecules that can regulate immune responses by stimulating and / or inducing the differentiation of T effector cells, e.g., CD4+ and / or CD8+, and promoting the activation of B cells, macrophages, and / or dendritic cells. In some embodiments, one or more cytokines may be displayed on pIII, pVI, pVII, pVIII, and / or pIX (WO2022073127, as shown in Figure 2; incorporated herein by reference).

[0037] In some embodiments, the one or more cytokines that stimulate an immune response can be any known cytokine that stimulates and / or induces the differentiation and / or activation of T cells, B cells, macrophages, and dendritic cells.

[0038] In some embodiments, the cytokines displayed on the bacteriophage are 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-34, IL-35, IL-36, IL-37, IL-38, IL-39, IL-40, IL-41, IL-42, IL-43, IL-44, IL-45, IL-46, IL-47, IL-48, IL-49, IL-50, IL-51, IL-52, IL-53, IL-54, IL-55, IL-56, IL-57, IL-58, IL-59, IL-60, IL-61, IL-62, IL-63, IL-64, IL-65, IL-66, IL-67, IL-68, IL-69, IL-70, IL-71, IL-72, IL-73, IL-74, IL-75, IL-76, IL-77, IL-78, IL-79, IL In some cases, the cytokine displayed on the bacteriophage may be selected from, but not limited to, IL-31, IL-32, IL-33, IL-35, TNF-alpha, LT-alpha, LT-beta, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, FLT3 ligand, G-CSF, GM-CSF, IFN-alpha, IFN-beta, IFN-omega, IFN-gamma, LIF, M-CSF, MIF, OSM, SCF, TGF-beta1, TGF-beta2, TGF-beta3, and TSLP ligand. In other cases, the cytokine displayed on the bacteriophage may be selected from, but not limited to, IL-2, IL-7, IL-12, IL-15, IL-18, IL-21, TNF, GM-CSF, FLT3 ligand, and interferon gamma (IFN-gamma). In another embodiment, the cytokine displayed on the bacteriophage is IL-2. In yet another embodiment, the cytokine displayed on the bacteriophage is IL-15.

[0039] Therapeutic uses of synthetic phages and compositions containing same Treatment method In some embodiments, the synthetic therapeutic phages of the present technology can be used to treat, but are not limited to, cancer and / or tumors. Tumors can be malignant or benign. Types of cancer include adrenal gland cancer, adrenocortical cancer, anal cancer, appendix cancer, bile duct cancer, bladder cancer, bone cancer (e.g., Ewing's sarcoma tumor, osteosarcoma, malignant fibrous histiocytoma), brain cancer (e.g., astrocytoma, brainstem glioma, craniopharyngioma, ependymoma), bronchial tumors, central nervous system tumors, breast cancer, Castleman's 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, cardiac cancer, Kaposi's sarcoma, kidney cancer, and laryngeal cancer. cancer), hypopharyngeal cancer, leukemia (e.g., acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia), liver cancer, lung cancer, lymphoma (e.g., AIDS-related lymphoma, Burkitt's lymphoma, cutaneous T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, primary central nervous system lymphoma), malignant mesothelioma, multiple myeloma, myelodysplastic syndrome, nasal cavity cancer, paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, oral cancer, oral These include, but are not limited to, pharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary tumor, prostate cancer, retinoblastoma, rhabdomyosarcoma, rhabdomyosarcoma, salivary gland cancer, sarcoma, skin cancer (e.g., basal cell carcinoma, melanoma), small intestine cancer, stomach cancer, teratoma, testicular cancer, throat cancer, thymus cancer, thyroid cancer, unusual childhood cancers, urethral cancer, uterine cancer, uterine sarcoma, vaginal cancer, vulvar cancer, Waldenstrom's macroglobulinemia, and Wilms' tumor. In some embodiments, the associated symptom(s) include, but are not limited to, anemia, loss of appetite, bladder lining irritation, bleeding and bruising (thrombocytopenia), changes in taste or smell, constipation, diarrhea, dry mouth, difficulty swallowing, swelling, fatigue, hair loss (alopecia), infections, infertility, lymphedema, mouth sores, nausea, pain, peripheral neuropathy, tooth decay, urinary tract infections, and / or memory and concentration problems.

[0040] In some embodiments, a method may include preparing a pharmaceutical composition having at least one bacteriophage described herein and administering a therapeutically effective amount of the pharmaceutical composition to a subject. The bacteriophage may be administered locally, e.g., intratumorally or peritumor into tissue or a blood vessel supplying the tumor, intramuscularly, intraperitoneally, orally, or topically. The bacteriophage may also be administered systemically, e.g., intravenously or intraarterially, by infusion or injection.

[0041] In certain embodiments, administering a 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 may reduce cell proliferation, tumor growth, and / or tumor volume by at least about 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% or more compared to levels in an untreated or control subject. In some embodiments, the reduction is measured by comparing cell proliferation, tumor growth, and / or tumor volume in the subject before and after administration of the pharmaceutical composition. In some embodiments, the methods of treating or ameliorating cancer in a subject allow for an improvement in one or more symptoms of cancer by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more.

[0042] In certain embodiments, administering the pharmaceutical compositions of the present technology to a subject reduces cell proliferation, tumor growth, and / or tumor volume in treated and untreated tumors via long-range effects and a systemic immune response.

[0043] In some cases, the disclosed methods reduce cell proliferation, tumor growth, and / or tumor volume in both treated 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 levels in untreated or control subjects. In some embodiments, the reduction is measured by comparing cell proliferation, tumor growth, and / or tumor volume in the subject before and after administration of the pharmaceutical composition.

[0044] In some embodiments, the methods of treating or ameliorating cancer of the present technology allow for at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or 95% or more improvement in one or more symptoms of cancer.

[0045] In certain embodiments, the methods of the present technology induce a long-term systemic anti-tumor immune response in the subject that provides protection against tumor recurrence.

[0046] Before, during, and after administration of the pharmaceutical composition of the present technology, cancer cells and / or biomarkers in 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.

[0047] In some embodiments, the methods may comprise administering a composition of the present technology to reduce the subject's tumor volume to an undetectable size or to less than about 1%, 2%, 5%, 10%, 20%, 25%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, or 90% of the subject's tumor volume before treatment. In other embodiments, the methods may comprise administering a composition of the present technology to reduce the rate of cell proliferation or tumor growth in 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 before treatment.

[0048] Therapy using synthetic therapeutic bacteriophages with immune-based anti-cancer activity may exhibit response patterns different from those observed with conventional cytotoxic therapies. For example, tumors treated with immune-based therapies may expand 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 heavy infiltration by lymphocytes and macrophages, which are not normally present in tumor tissue. In addition, response times may be slower than those associated with standard therapies, e.g., cytotoxic therapies. In some embodiments, delivery of anti-cancer molecules may modulate tumor growth and / or ameliorate cancer symptoms in a subject, while temporarily increasing tumor volume and / or size.

[0049] Bacteriophages can be destroyed by protective factors in tissues or serum, for example, hours or days after administration (Sonnenborn et al., Microbioal Ecology in Health and Diseases, 2019, 21:3, incorporated herein by reference). Thus, pharmaceutical compositions containing bacteriophages can be readministered at therapeutically effective doses and frequencies.

[0050] 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 therapy (e.g., radiation therapy, chemotherapy), immunotherapy (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 therapy (e.g., TCR or CAR-T), natural killer cell therapy, Fc-mediated ADCC therapy, therapy using bispecific soluble scFvs that link cytotoxic T cells to tumor cells, and therapy using soluble TCRs with effector function), stem cell therapy, and targeted therapy using antibodies or chemical compounds (e.g., BRAF or vascular endothelial growth factor inhibitors), bacteriophages.

[0051] In some embodiments, the bacteriophage may be administered sequentially, simultaneously, or following administration of one or more chemotherapeutic agents selected from, but not limited to, methotrexate, Trabectedin®, Belotecan®, Cisplatin®, Carboplatin®, Bevacizumab®, Pazopanib®, 5-fluorouracil, Capecitabine®, Irinotecan®, Gemcitabine (Gemzar), and Oxaliplatin®.

[0052] In some embodiments, the bacteriophage is administered sequentially, simultaneously, or subsequent to the administration of one or more mRNA-based drugs.

[0053] In some embodiments, the bacteriophage is administered sequentially, simultaneously, 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-lBB (CD137, TNFRSF9) antibodies (including but not limited to PF-05082566 and urelumab), anti-CD134 (OX40) antibodies (anti-OX40 antibodies (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), and anti-CXCR4 antibodies (including but not limited to urocuplumab).

[0054] In some embodiments, the bacteriophage is an antibody that is specifically targeted to a subject, such as an anti-phosphatidylserine antibody (including but not limited to bavituxumab), a TLR9 antibody (including but not limited to MGN1703PD1 antibody (SHR-1210 (Incyte / Jiangsu Hengrui)), an anti-OX40 antibody (including but not limited to OX40 (Agenus)), an anti-Tim3 antibody (including but not limited to anti-Tim3 (Agenus / INcyte)), an anti-Lag3 antibody (including but not limited to anti-Lag3 (Agenus / INcyte)), an anti-B7H3 antibody (enoblitutuzumab (MGA-271), anti-CT-01 as described in WO2009101611 (incorporated herein by reference)), or a combination thereof. 1 (hBAT, hBATl)), 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),

[0055] The dosage and administration frequency of the pharmaceutical composition can be selected based on the severity of the symptoms and the progression of the cancer. The appropriate therapeutically effective dose and / or administration frequency can be selected depending on the pharmaceutical composition and formulation of the treatment clinic.

[0056] Pharmaceutical Compositions and Formulations Pharmaceutical compositions comprising the bacteriophages of the present technology can be used to treat, manage, ameliorate, and / or prevent cancer. Pharmaceutical compositions of the present technology are provided that comprise one or more bacteriophages of the present technology, alone or in combination with prophylactic agents, therapeutic agents, and / or pharmaceutically acceptable carriers.

[0057] In certain embodiments, the pharmaceutical composition comprises the recombinant proteins described herein, e.g., bacteriophages of the present technology, each engineered to display one or more anti-cancer molecules.

[0058] The pharmaceutical compositions of the present technology can be formulated in a conventional manner using one or more physiologically acceptable carriers, including excipients and adjuvants, which facilitate the processing of the active ingredient into a composition for pharmaceutical use. Methods for formulating pharmaceutical compositions are known in the art (see, for example, "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA). In some embodiments, the pharmaceutical composition is subjected to tableting, lyophilization, direct compression, conventional mixing, dissolution, granulation, suspension, emulsification, encapsulation, entrapment, or spray drying to form tablets, granules, nanoparticles, nanocapsules, microcapsules, microtablets, pellets, or powders, which may or may not be enterically coated. The appropriate formulation depends on the route of administration.

[0059] The bacteriophage can be formulated into a pharmaceutical composition for any suitable dosage form (e.g., liquid, capsule, sachet, hard capsule, soft capsule, tablet, enteric coated tablet, suspended powder, granule, or matrix sustained release form for oral administration) and any suitable type of administration (e.g., oral, topical, injectable, intravenous, subcutaneous, intratumoral, peritumoral, immediate release, pulsatile release, delayed release, or sustained release). A suitable dosage for the bacteriophage is about 10 4 ~10 16 The composition may range from 100 to 1000 bacteriophage particles. The composition may be administered one or more times daily, weekly, monthly, or yearly. The composition may be administered before, during, or after a meal. In one embodiment, the pharmaceutical composition is administered before the subject ingests a meal. In one embodiment, the pharmaceutical composition is administered currently with a meal. In one embodiment, the pharmaceutical composition is administered after the subject ingests a meal.

[0060] Bacteriophages can be formulated into pharmaceutical compositions containing one or more pharmaceutically acceptable carriers, thickeners, diluents, buffers, buffering agents, surface active agents, neutral or cationic lipids, lipid complexes, liposomes, penetration enhancers, carrier compounds, and other pharmaceutically acceptable carriers or agents. For example, pharmaceutical compositions can include, but are not limited to, calcium bicarbonate, sodium bicarbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycol, and surfactants, including, for example, polysorbate 20. In some embodiments, the bacteriophages of the present technology can be formulated in a solution of sodium bicarbonate, such as a 1 molar solution of sodium bicarbonate (e.g., to buffer the acidic cellular environment, such as the stomach). Bacteriophages can be administered and formulated in neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc.

[0061] The bacteriophage may be administered intravenously, for example, by infusion or injection. Alternatively, the bacteriophage may be administered intratumorally and / or peritumorally. In other embodiments, the bacteriophage may be administered intraarterially, intramuscularly, or intraperitoneally. In some embodiments, the bacteriophage is co-administered with a PEGylated form of rHuPH20 (PEGPH20) or other agents to disrupt tumor septa to enhance penetration of the tumor capsule, collagen, and / or interstitium.

[0062] The bacteriophages of the present disclosure can be administered via intratumoral injection, resulting in the bacteriophage being deposited directly within the target tumor. Intratumoral injection of engineered bacteriophages can induce a strong local inflammatory response as well as an immune response against tumor cells. In the injection procedure, the bacteriophage is suspended in a solution and then drawn into a syringe. In some embodiments, the tumor is injected with a single-bore needle. In yet another embodiment, the tumor is injected with a multi-prong needle (Quadra-Fuse, Rex Medical).

[0063] Direct intratumoral injection of the bacteriophage of the present technology into solid tumors may be advantageous compared to intravenous administration. Using intravenous injection, only a small portion of the bacteriophage may reach the target tumor. Intratumoral injection may be particularly beneficial in large animals and human patients, which have a relatively large blood volume and relatively small tumors compared to mice. Direct injection into tumors allows for the delivery of higher concentrations of therapeutic agents and avoids the toxicity that can result from systemic administration. Furthermore, intratumoral injection of bacteriophage induces a stable local immune response within the tumor.

[0064] Depending on the location, tumor type, and tumor size, different administration techniques may be used, including, but not limited to, cutaneous, subcutaneous, and percutaneous injection, therapeutic endoscopic ultrasound, or endobronchial intratumoral delivery. Prior to the intratumoral administration procedure, the patient is administered local anesthesia and standard cardiac, blood pressure, and oxygen monitoring, or sedation in combination with complete anesthesia.

[0065] For some tumors, percutaneous injection can be used, which is the least invasive administration method. Ultrasound computed tomography (CT) or fluoroscopy can be used as guidance for introducing and positioning the needle. Percutaneous intratumoral injection is described, for example, in Lencioni et al., 2010 (incorporated herein by reference). Intratumoral injection of cutaneous tumors, subcutaneous tumors, and nodular tumors is described, for example, in WO / 2014 / 036412 (incorporated herein by reference).

[0066] A single insertion point or multiple insertion points can be used in a percutaneous injection protocol. Using a single insertion point, the solution can be injected percutaneously along multiple tracks, as long as the radial extent of the needle allows. In other embodiments, multiple injection points can be used if the tumor is larger than the radial extent of the needle. The needle can be withdrawn without exiting and can be redirected as many times as necessary 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 will vary depending on the type and size of the tumor.

[0067] In some embodiments, tumors are injected percutaneously with an 18-gauge multi-prong needle (Quadra-Fuse, Rex Medical). The device comprises a 20 cm long 18-gauge puncture needle. The needle has three retractable prongs, each with four distal holes and a connector with an extension tubing clamp. The prongs are deployed from the sidewall of the needle. The needle can be percutaneously introduced into the center of the tumor and positioned at the deepest edge of the tumor. The prongs are deployed at the tumor margin. The prongs are deployed to their maximum length and then retracted for a defined interval. Optionally, one or more rotate-inject-rotate operations may be performed, in which the prongs are retracted and the needle is rotated 60 degrees, followed by repeated prong deployment and additional injections.

[0068] Therapeutic endoscopic ultrasound (EUS) is used to overcome the inherent anatomical constraints in gaining access to certain other tumors (Shirley et al., 2013, incorporated herein by reference). EUS-guided fine-needle injection (EUS-FNI) has been successfully used in antitumor therapy for the treatment of tumors of the head and neck, esophagus, pancreas, liver, and adrenal gland (Verna et al., 2008, incorporated herein by reference). EUS-FNI is widely used for pancreatic cancer injections. Fine-needle injection requires the use of a curved ultrasound endoscope. The esophagus is carefully intubated, and the ultrasound endoscope is passed into the stomach and duodenum, where it is used to examine the pancreas and identify the target tumor. The maximum plane is measured to estimate tumor volume and calculate the injection volume. The appropriate volume is drawn into the syringe. A primed 22-gauge fine-needle aspiration (FNA) needle is passed through the working channel of the ultrasound endoscope. Under ultrasound guidance, the needle is passed into the tumor. Depending on the size of the tumor, administration can be performed by dividing the tumor into sections and then injecting a corresponding fraction of the volume into each section. The use of an installed endoscopic ultrasound processor with Doppler technology ensures the absence of arterial or venous structures that may obstruct needle passage into the tumor (Shirley et al., 2013, incorporated herein by reference). In some embodiments, "multiple injectable needles" (MINs) for EUS-FNI can be used to improve injection distribution into the tumor compared to straight needles (Ohara et al., 2013, incorporated herein by reference).

[0069] Intratumoral administration of a tumor to lung cancer, such as non-small cell lung cancer, can be achieved by intrabronchial intratumoral delivery, as described in Celikoglu et al., 2008 (incorporated herein by reference). Bronchoscopy (nasal or oral) is performed to visualize the lesion to be treated. Tumor volume can be visually estimated from visible length-width-height measurements across the bronchial surface. A needle device is then introduced through the working channel of the bronchoscope. The needle catheter, which contains a metal needle attached to a plastic catheter, is placed within a sheath to prevent damage to the working channel by the needle during advancement. The size and length of the needle vary and are determined depending on the type and size of the tumor. Plastic needles are ideal because they are less rigid than metal needles and can pass through sharper bends in the working channel. The needle is inserted into the lesion, and the bacteriophage 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 implanted in another location. At the end of the bronchoscopic injection session, removal of necrotic debris caused by the treatment can be achieved using mechanical dissection or other cauterizing techniques involving irrigation and aspiration.

[0070] In some embodiments, bacteriophages capable of delivering immunomodulatory agents to target tumors are administered directly into the tumor using methods including, but not limited to, percutaneous injection, EUS-FNI, or endobronchial intratumoral delivery methods. In some cases, other techniques, such as laparoscopic or open surgical techniques, are used to access the target tumor, but these techniques are much more invasive and result in much higher morbidity and longer hospital stays.

[0071] The volume injected into each lesion is based on the size of the tumor. A measurement of the largest plane can be taken to obtain the tumor volume. The estimated tumor volume can then provide information for determining the injection volume as a percentage of the total volume. For example, an injection volume of approximately 20-40% of the total tumor volume can be used. For example, as described in WO / 2014 / 036412 (Amgen, incorporated herein by reference), for tumors greater than 5 cm in their greatest dimension, up to 4 mL can be injected. For tumors 2.5-5 cm in their greatest dimension, up to 2 mL can be injected. For tumors 2.5-5 cm in their greatest dimension, up to 2 mL can be injected. For tumors 1.5-2.5 cm in their greatest dimension, up to 1 mL can be injected. For tumors 0.5-1.5 cm in their greatest dimension, up to 0.5 mL can be injected. For tumors 0.5 cm or less in their greatest dimension, up to 0.1 mL can be injected. Alternatively, ultrasound scanning can be used to determine the injected volume that can be taken up by the tumor without leakage into surrounding tissue.

[0072] In some embodiments, the treatment regimen includes one or more intratumoral administrations. In some embodiments, the treatment regimen includes an initial dose followed by at least one subsequent dose. One or more doses may be administered consecutively in two or more cycles. For example, a first dose may be administered on day 1, and a second dose may be administered 1, 2, 3, 4, 5, 6 days later, or 1, 2, 3, or 4 weeks later, or after a longer interval. An additional dose may be administered 1, 2, 3, 4, 5, 6 days later, or 1, 2, 3, or 4 weeks later, or after a longer interval. In some embodiments, the first and subsequent administrations have the same dose. In other embodiments, different doses are administered. In some embodiments, two or more doses per day are administered, for example, two, three, or more doses per day may be administered.

[0073] The described routes of administration and dosages are intended as a guide only. Optimal routes of administration and dosages can be easily determined by those skilled in the art. The dosage can be determined according to various parameters, in particular the location of the tumor, the size of the tumor, the age, weight, and condition of the patient being treated, and the route and method of administration.

[0074] Tumor types in which bacteriophages can be delivered intratumorally using current technology include locally advanced and metastatic tumors (including, but not limited to, B-cell, T-cell, and NK-cell lymphomas), colon and rectal cancer, melanoma, including metastatic melanoma, mycoses, 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.

[0075] The bacteriophages disclosed herein may be administered topically and may be formulated as ointments, creams, transdermal patches, lotions, gels, shampoos, sprays, aerosols, solutions, emulsions, or other forms known to those skilled in the art. See, e.g., "Remington's Pharmaceutical Sciences," Mack Publishing Co., Easton, PA. In one embodiment, for non-sprayable topical dosage forms, a viscous to semi-solid or solid form is used that includes one or more excipients compatible with carrier or topical application and that have a dynamic viscosity greater than that of water. Suitable formulations include, but are not limited to, solutions, suspensions, emulsions, creams, salves, powders, paints, ointments, etc., which may be sterilized or mixed with auxiliary agents (e.g., preservatives, stabilizers, humectants, buffers, or salts) to affect various properties, e.g., osmotic pressure. Other suitable topical dosage forms include sprayable aerosol formulations, in which the active ingredient combined with a solid or liquid inert carrier is packaged in a mixture with a pressurized volatile substance (e.g., a gaseous propellant such as a freon) or packaged in a squeeze bottle. Moisturizing agents or humectants may also be added to pharmaceutical compositions and dosage forms. Examples of such additional ingredients are well known in the art. In one embodiment, a pharmaceutical composition comprising a bacteriophage of the present technology may be formulated as a hygiene product. For example, the hygiene product may be an antibacterial preparation or a fermented product such as a fermentation broth. The hygiene product may be, for example, a shampoo, conditioner, cream, paste, lotion, or lip balm.

[0076] The bacteriophages disclosed herein may be administered orally and may be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like. Pharmaceutical formulations for oral use can be prepared using solid excipients, optionally milling the resulting mixture and optionally adding suitable excipients, followed by processing the granular mixture to obtain tablets or dragee cores. Suitable excipients include, but are not limited to, fillers, such as sugars including lactose, sucrose, mannitol, or sorbitol; corn starch, wheat starch, rice starch, potato starch; gelatin; tragacanth gum; cellulose compositions such as methylcellulose, hydroxypropylmethylcellulose, and 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 a salt thereof, such as sodium alginate, may also be added.

[0077] Tablets or capsules can be prepared by conventional means using pharmaceutically acceptable excipients, such as binders (e.g., pregelatinized corn starch, polyvinylpyrrolidone, hydroxypropylmethylcellulose, carboxymethylcellulose, polyethylene glycol, sucrose, glucose, sorbitol, starch, gum, kaolin, and tragacanth), fillers (e.g., lactose, microcrystalline cellulose, or calcium hydrogen phosphate), lubricants (e.g., calcium, aluminum, zinc, stearic acid, polyethylene glycol, sodium lauryl sulfate, starch, sodium benzoate, L-leucine, magnesium stearate, talc, or silica powder), disintegrants (e.g., starch, potato starch, sodium starch glycolate, sugars, cellulose derivatives, silica powder), or wetting agents (e.g., sodium lauryl sulfate). Tablets may also be coated by methods well known in the art. A coating shell may be present; common membranes include polylactides, polyglycolic acids, polyanhydrides, other biodegradable polymers, alginate polylysine-alginate (APA), alginate-polymethylene-co-guanidine alginate (A-PMCG-A), hydromethyl acrylate-methyl methacrylate (HEMA-MMA), multilayer HEMA-MMMAA, polyacrylonitrile vinyl chloride (PAN-PVC), acrylonitrile / sodium methallyl sulfonate (AN-69), polyethylene glycol / polypentamethylcyclopentasiloxane / These include, but are not limited to, polydimethylsiloxane (PEG / PD5 / PDMS), poly N,N-dimethylacrylamide (PDMAAm), siliceous encapsulates, cellulose sulfate / sodium alginate / polymethylene-co-guanidine (CS / A / PMCG), cellulose acetate phthalate, calcium alginate, k-carrageenan-locust bean gum gel beads, gellan-xanthan beads, poly(lactide-co-glycolide), carrageenan, starch polyanhydride, starch polymethacrylate, polyamino acids, and enteric coating polymers.

[0078] In some embodiments, bacteriophages are coated with 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 preparation), amylose starch and starch derivatives, and dextrins (e.g., maltodextrin) are also 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.

[0079] Coating polymers may also be used, for example, phthalate derivatives, CAT, HPMCAS, polyacrylic acid derivatives, copolymers comprising acrylic acid and at least one acrylic acid ester, Eudragit™ S (poly(methacrylic acid, methyl methacrylate) 1:2), Eudragit L100™ S (poly(methacrylic acid, methyl methacrylate) 1:1), Eudragit L30D™ (poly(methacrylic acid, ethyl acrylate) 1:1), and Eudragit L100-55) (poly(methacrylic acid, ethyl acrylate) 1:1) (Eudragit™ L is an anionic polymer synthesized from methacrylic acid and methacrylic acid methyl ester), polymethyl methacrylate blended with acrylic acid and acrylic acid ester copolymers, alginic acid, ammonium, sodium, potassium, magnesium, or calcium alginate, vinyl acetate copolymer, polyvinyl acetate 30D (30% dispersion in water), neutral methacrylic acid esters including poly(dimethylaminoethyl acrylate) (Eudragit™), copolymers of methyl methacrylate and ethyl acrylate with trimethylammonioethyl methacrylate chloride, copolymers of methyl methacrylate and ethyl acrylate, zein, shellac, gum, or polysaccharides, or combinations thereof.

[0080] The coating layer may also be hydroxypropyl methylcellulose (HPMC), hydroxypropyl ethyl cellulose (HPEC), hydroxypropyl cellulose (HPC), hydroxypropyl ethyl cellulose (HPEC), hydroxymethylpropyl cellulose (HMPC), ethyl hydroxyethyl cellulose (EHEC) (Ethulose), hydroxyethyl methylcellulose (HEMC), hydroxymethyl ethyl cellulose (HMEC), propyl hydroxyethyl cellulose (PHEC), methyl hydroxyethyl cellulose (MHEC), hydrophobically modified hydroxyethyl cellulose (NEXTON), carboxymethyl hydroxyethyl cellulose (CMHEC) , methyl cellulose, ethyl cellulose, water soluble vinyl acetate copolymers, gums, polysaccharides such as alginic acid and alginates, e.g., ammonium alginate, sodium alginate, potassium alginate, carbohydrate acid phthalates, amylose acetate phthalate, cellulose acetate phthalate (CAP), cellulose ester phthalates, cellulose ether phthalates, hydroxypropyl cellulose phthalate (HPCP), hydroxypropyl ethyl cellulose phthalate (HPECP), hydroxypropyl methyl cellulose phthalate (HPMCP), hydroxypropyl methyl cellulose acetate succinate (HPMCAS) containing polymers.

[0081] Liquid preparations for oral administration may take the form of solutions, syrups, suspensions, or dry products to be reconstituted with water or other suitable vehicles before use. Such liquid preparations may be prepared by conventional means with pharmaceutically acceptable agents such as suspending agents (e.g., sorbitol syrup, cellulose derivatives, or hydrogenated edible fats), emulsifying agents (e.g., lecithin or acacia), non-aqueous vehicles (e.g., almond oil, oily esters, ethyl alcohol, or fractionated vegetable oils), and preservatives (e.g., methyl or propyl-p-hydroxybenzoate or sorbic acid). Preparations may also contain buffer salts, flavoring agents, coloring agents, and sweeteners, as needed. Preparations for oral administration may be appropriately formulated for slow-release, controlled-release, or sustained-release of the bacteriophages described herein.

[0082] In one embodiment, the bacteriophages of the present disclosure may be formulated in a composition suitable for administration to pediatric subjects. As is well known in the art, children differ from adults in many aspects, including different rates of gastric emptying, pH, gastrointestinal permeability, etc. (Ivanovska et al., Pediatrics, 134(2):361-372, 2014, incorporated herein by reference). Furthermore, the acceptability and preference of pediatric formulations, e.g., route of administration and taste attributes, are important for achieving acceptable pediatric compliance. Thus, in one embodiment, compositions suitable for administration to pediatric subjects may include easy-to-swallow or dissolvable dosage forms, or more palatable compositions, e.g., compositions with added flavorings, sweeteners, or taste-blocking agents. In one embodiment, compositions suitable for administration to pediatric subjects may also be suitable for administration to adults.

[0083] In one embodiment, a composition suitable for administration to a pediatric subject may include a solution, syrup, suspension, elixir, powder for reconstitution as a suspension or solution, dispersible / effervescent tablet, chewable tablet, gummy candy, lollipop, freeze-pop, lozenge, chewing gum, oral thin strip, orally disintegrating tablet, sachet, soft gelatin capsule, oral dusting powder, or granule. In one embodiment, the composition is a gummy candy made from a gelatin base, which provides the candy's elasticity, a desirable chewy consistency, and a longer shelf life. In some embodiments, the gummy candy may also include a sweetener or flavoring.

[0084] In one embodiment, compositions suitable for administration to pediatric subjects may include a flavoring agent. As used herein, a "flavoring agent" is a substance (liquid or solid) that provides a distinct taste and aroma to a formulation. Flavoring agents also serve to improve the mouthfeel of a formulation. Flavoring agents include, but are not limited to, strawberry, vanilla, lemon, grape, bubblegum, and cherry.

[0085] In certain embodiments, the bacteriophage may be orally administered, for example, with an inert diluent or an assimilable edible carrier. The compound may also be enclosed in hard or soft shell gelatin capsules, compressed into tablets, or incorporated directly into the patient's food or drink. For oral therapeutic administration, the compound may be incorporated with excipients and used in the form of ingestible tablets, buccal tablets, troches, capsules, elixirs, suspensions, syrups, wafers, and the like. To administer the compound by other than parenteral administration, it may be necessary to coat the compound with, or co-administer the compound with, a material to prevent its inactivation.

[0086] In some embodiments, the compositions are formulated for enteral administration via enteric-coated or non-enteric-coated nanoparticles, nanocapsules, microcapsules, or microtablets, for jejunal, duodenal, ileal, gastric shunt, or colonic administration. Pharmaceutical compositions can also be formulated in rectal compositions such as suppositories or retention enemas using conventional suppository bases such as cocoa butter or other glycerides. The compositions may be suspensions, solutions, or emulsions in oily or aqueous vehicles and may contain suspending, stabilizing, and / or dispersing agents.

[0087] The bacteriophages described herein may be administered intranasally using a suitable propellant (e.g., dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide or other suitable gas), and may be formulated in aerosol form, spray, mist, or droplets, conveniently delivered as an aerosol spray from pressurized packs or nebulizers. The pressurized aerosol dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges (e.g., gelatin) for use in an inhaler or insufflator may be formulated to contain a powder mix of the compound and a suitable powder base, such as lactose or starch.

[0088] The bacteriophage may be administered and formulated as a depot preparation. Such long-acting formulations may be administered by implantation or by injection, including intravenous, subcutaneous, local, direct injection, or infusion. For example, the composition may be formulated with a suitable polymeric or hydrophobic material (e.g., as an emulsion in an acceptable oil) or ion exchange resin, or as a sparingly soluble derivative (e.g., as a sparingly soluble salt).

[0089] In some embodiments, disclosed herein are pharmaceutically acceptable compositions in a single dosage form. The single dosage form may be in liquid or solid form. The single dosage form may be administered directly to a patient without modification, or may be diluted or reconstituted before administration. In certain embodiments, the single dosage form may be administered in a bolus form, e.g., a single injection, a single oral dose, including oral doses containing multiple tablets, capsules, pills, etc. In alternative embodiments, the single dosage form may be administered over a period of time, e.g., by infusion.

[0090] Unit dosage forms of a pharmaceutical composition may 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, pellets, or powders (which may or may not be enteric coated). Single doses of solid forms can be reconstituted by adding a liquid, typically sterile water or saline, prior to administration to a patient.

[0091] In other embodiments, 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, polymeric materials can be used to achieve controlled or sustained release of the disclosed therapies (see, e.g., U.S. Pat. No. 5,989,463, incorporated herein by reference). Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. Polymers used in sustained-release formulations can be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled or sustained release system may be placed in proximity to the prophylactic or therapeutic target, thus requiring a small fraction of the systemic dose. Any suitable technique known to those skilled in the art may be used.

[0092] Dosage regimens can be adjusted to provide a therapeutic response. Dosage can depend on several factors, including disease severity and responsiveness, the route of administration, the duration of treatment (days to months to years), and the time to disease improvement. For example, a single bolus may be administered at once, several divided doses may be administered over time, or the dose may be reduced or increased as indicated by the therapeutic situation. Dosage specifications depend on the unique characteristics of the active compound and the particular therapeutic effect to be achieved. Dosage values ​​may vary depending on the type and severity of the symptoms to be alleviated. For any particular subject, a specific dosing regimen may be adjusted over time according to the individual need 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 culture or animal models. For example, the LD50, ED50, EC50, and IC50 can be determined, and the dose ratio between toxic and therapeutic effects (LD50 / ED50) can be calculated as the therapeutic index. Compositions that exhibit toxic side effects can be carefully modified to minimize potential damage and reduce side effects. Dosages 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 use in humans.

[0093] The ingredients may be supplied separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water-free concentrate in a hermetically sealed container such as an ampoule or sachet indicating the quantity of active agent. When the mode of administration is by injection, an ampoule of sterile water for injection, saline or nutrient vehicle can be provided so that the ingredients may be mixed prior to administration.

[0094] Pharmaceutical compositions may be packaged in a hermetically sealed container, such as an ampoule or sachet, indicating the quantity of agent. In one embodiment, one or more of the pharmaceutical compositions are supplied as a dry, sterile, lyophilized powder or water-free concentrate in a hermetically sealed container, which can be reconstituted (e.g., with water or saline) to the appropriate concentration for administration to a subject. In one embodiment, one or more of the prophylactic or therapeutic agents or pharmaceutical compositions are supplied as a dry, sterile, lyophilized powder in a hermetically sealed container, stored at 2°C to 8°C, and administered within 1 hour, 3 hours, 5 hours, 6 hours, 12 hours, 24 hours, 48 ​​hours, 72 hours, or 1 week after reconstitution. Lyophilized dosage forms may include a cryoprotectant, primarily 0-10% sucrose (optimally 0.5-1.0%). Other suitable cryoprotectants include trehalose and lactose. Other suitable bulking agents include glycine and arginine, which may be present at concentrations of 0-0.05%, and polysorbate-80, which is optimally present at a concentration of 0.005-0.01%. Additional surfactants include, but are not limited to, polysorbate 20 and BRIJ surfactants. The pharmaceutical composition may be prepared as an injectable solution and may further include agents useful as adjuvants, such as agents used to increase absorption or distribution, such as hyaluronidase.

[0095] In some embodiments, bacteriophages and compositions thereof are formulated for intravenous, intratumoral, or peritumoral administration. Bacteriophages may also be formulated as depot preparations. Such long-acting formulations may be administered by implantation or by injection. For example, the compositions may be formulated with a suitable polymer or hydrophobic material (e.g., as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative (e.g., as a sparingly soluble salt).

[0096] 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, polymeric materials can be used to achieve controlled or sustained release of the disclosed treatments (see, e.g., U.S. Pat. No. 5,989,463, incorporated herein by reference). Examples of polymers used in sustained-release formulations include, but are not limited to, poly(2-hydroxyethyl methacrylate), poly(methyl methacrylate), poly(acrylic acid), poly(ethylene-co-vinyl acetate), poly(methacrylic acid), polyglycolide (PLG), polyanhydrides, poly(N-vinylpyrrolidone), poly(vinyl alcohol), polyacrylamide, poly(ethylene glycol), polylactide (PLA), poly(lactide-co-glycolide) (PLGA), and polyorthoesters. Polymers used in sustained-release formulations can be inert, free of leachable impurities, stable on storage, sterile, and biodegradable. In some embodiments, a controlled-release or sustained-release system may be placed near the prophylactic or therapeutic target, thus requiring a small fraction of the systemic dose. Any suitable technique known to those skilled in the art may be used.

[0097] The bacteriophages of the present technology can be administered and formulated as neutral or salt forms. Pharmaceutically acceptable salts include those formed with anions, such as those derived from hydrochloric acid, phosphoric acid, acetic acid, oxalic acid, tartaric acid, etc., and those formed with cations, such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxide, isopropylamine, triethylamine, 2-ethylaminoethanol, histidine, procaine, etc. [Example]

[0098] The following examples are provided to illustrate the practice of various embodiments of the present disclosure. They 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 exemplified herein, but includes all modifications and variations that fall within the scope of the present disclosure as defined in the accompanying embodiments.

[0099] Example 1 - Engineering bacteriophages that display tumor-targeting moieties and cytokines Strains, Plasmids, and Growth Conditions. All strains and plasmids used in this example are listed in Table 1. All plasmids and gBlock sequences are listed in the Sequence Listing file accompanying this specification. Cells were typically grown in Luria Broth Miller (LB) or Luria Broth Agar Miller medium supplemented with the following concentrations of antibiotics 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. Bacterial cultures older than 18 hours were not used in the experiments. [Table 1]

[0100] DNA manipulations. A detailed list of oligonucleotide sequences used in this example can be found in Table 2. Plasmids were prepared using the EZ10-Spin Column Plasmid Miniprep Kit (BIOBASIC #BS614) according to the manufacturer's instructions. For DNA segment amplification and screening, PCR amplification was performed using TransStart FastPFU fly DNA polymerase (Civic Bioscience). Plasmids were assembled by Gibson assembly using NEBuilder HiFi DNA Assembly Master Mix (NEB) according to the manufacturer's protocol. DNA digestion was performed using restriction enzymes from NEB according to the manufacturer's recommendations. Digestion was performed periodically for 1 hour.

[0101] DNA purification was performed between each step of plasmid assembly to avoid buffer incompatibilities or to stop enzymatic reactions. PCR reactions were purified by solid-phase reversible immobilization (SPRI) using Agencourt Ampure XP DNA-binding beads (Beckman Coulter) according to the manufacturer's recommendations. After purification, DNA concentration and purity were periodically assessed using a Nanodrop spectrophotometer, as needed.

[0102] DNA transformation into E. coli by electroporation. Routine plasmid transformation was performed by electroporation. Electrocompetent E. coli strains were prepared from 20 mL of LB broth. The OD was 600. 600nmCultures reaching exponential growth phase with an optical density of 0.6 at 37°C were washed three times in sterile 10% glycerol solution. The cells were then resuspended in 200 μL of 10% glycerol (1% of the initial volume) and distributed into 50 μL aliquots. DNA was then added to the electrocompetent cells (approximately 50 ng of DNA in a maximum of 10 μL of water), and the mixture was transferred into a 1 mm electroporation cuvette. The cells were electroporated for 5 ms using a pulse of 1.8 kV, 25 μF, and 200 Ω. The cells were then resuspended in 1 mL of nonselective LB medium and allowed to recover for 1 hour at 37°C before being plated onto selective medium, and the plates were incubated overnight at 37°C.

[0103] DNA transformation into E. coli by heat shock. Heat shock transformation was primarily used for cloning Gibson assembly products. Chemically competent cells were prepared according to a previously described rubidium chloride protocol (Green et al., 2013). Chemically competent cells were flash-frozen and stored at -80°C until use. Gibson assembly products were directly transformed into MM294 chemically competent cells at a 1 / 10 volume ratio. Routinely, up to 10 μL of DNA was added to 100 μL of competent cells before transformation by heat shock at 42°C for 45 seconds. Cells were then resuspended in 1 mL of non-selective LB medium and allowed to recover at 37°C for 1 hour before plating on selective medium.

[0104] Engineered phage systems are widely used in phage display approaches to express antibody-phage complexes to screen for antibodies with specific affinities. In this context, antibody variable sections are amplified using universal DNA primers from immunized animals, ligated together, cloned into a plasmid called a phagemid, and fused to the pIII protein (replacing the protein's N-terminal domain) to form a library of scFv fragments. The library is then screened to select phage that display antibody-pIII fusions that recognize the target antigen. Target-bound phages are then isolated and amplified by infecting their bacterial hosts. This step is possible because the phage still display copies of wild-type pIII (necessary for infecting the bacterial host) and the antibody-pIII fusion. After several cycles, scFv fragments that recognize the target antigen are enriched, and clones are tested and sequenced for storage. In this context, only a small number of phage that attach to the target antigen are required to isolate the desired antibody clone, meaning that screening can still be successful even if the majority of phages display only the wild-type pIII protein and not the antibody-pIII fusion. [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4]

[0105] The engineered bacteriophage system described here differs from phage display systems in several ways. For example, (1) the bacteriophage must be secreted at high levels by the producing bacteria, (2) all bacteriophage must display the therapeutic protein, and (3) as little DNA as possible must be used in the assembly of phage particles to maximize the biocontainment of the system and limit dissemination of genetic material. These additional constraints are not present in traditional phage display. Therefore, most phage display systems use M13K07 as a helper phage, expressing all proteins involved in phage replication and assembly (including a wild-type copy of gpIII), as well as a phagemid, expressing an antibody-pIII fusion protein, an origin of replication, and containing an intact encapsidation signal. Although these systems will produce antibody-phage complexes, 90% of the phage produced contain only the wild-type pIII protein from M13K07 and do not contain the phagemid-derived antibody-pIII fusion protein (Ledsgaard et al. 2018, Toxin, incorporated herein by reference). For the display of therapeutic proteins, having 90% of bacteriophage that do not display the therapeutic protein is suboptimal and undesirable, as it reduces the efficacy of the treatment.

[0106] This example describes the design of a bacteriophage secretion system for the display of therapeutic proteins. Iterations of the system are presented and their advantages and disadvantages are discussed. The list of iterations is not intended to be exhaustive, but rather illustrates variations of bacteriophage secretion systems for the combined display of cytokines and binding molecules. A general description of the various constructs described in this example is shown in Figure 1.

[0107] Repetition of the bacteriophage secretion system The bacteriophage secretion system is a flexible system that can appear in different forms. In this example, we aimed to demonstrate that the bacteriophage secretion system can be modified to allow the display of one or two therapeutic proteins on the same bacteriophage particle. In a first embodiment of the bacteriophage secretion system, the system can be divided into two sets of vectors: a bacteriophage backbone vector (e.g., pTAT002, pTAT044, pTAT070) and a bacteriophage machinery vector (e.g., pTAT004) (Figure 1). In some embodiments, the bacteriophage secretion system can be divided into three or more gene constructs, such as a system consisting of pTAT025, pTAT044, or pTAT070 and pTAT017, pTAT060, or pTAT071. In another embodiment, the bacteriophage secretion system can be on a single vector. In yet another embodiment, the bacteriophage secretion may be integrated into the genome of the producer strain bacteria.

[0108] Two-vector-based bacteriophage secretion system To create the bacteriophage machinery vector pTAT004, the entire M13K07 fragment was amplified using the appropriate primers listed in Table 2, excluding the gpIII gene encoding pIII. The homology tails of the primers used to amplify M13K07 were carefully designed to remove gpIII from the final construct. The PCR product was then purified by SPRI and assembled by the Gibson method to generate pTAT004. The assembly was transformed into MM294 chemically competent cells, and the integrity of the plasmid was verified by digestion with NdeI. The pTAT004 bacteriophage machinery does not contain a copy of the gpIII gene and therefore does not produce pIII, and therefore is unable to produce fully functional bacteriophage particles on its own. To produce bacteriophage particles, gpIII must be provided in trans by the bacteriophage backbone vector.

[0109] Next, we designed the pTAT002, pTAT044, and pTAT070 bacteriophage backbone vectors. All of the bacteriophage backbone vectors contain the ori for high-copy plasmid replication. pMB1 (maximizing DNA material for encapsidation), ori for ssDNA rolling circle replication and recognition of the bacteriophage backbone vector by the phage encapsidation machinery M13 pTAT002 contains a selectable marker (here, spectinomycin resistance), as well as a constitutively expressed pIII C-terminal fragment for either a HA-Hi dual-tagged N-terminal fragment of pIII (pTAT002, a control with no therapeutic protein) or a checkpoint inhibitor binding protein (pTAT044, anti-PD-L1 linked via a single HA-His dual tag; pTAT070, anti-PD-L1 without a tag). In this example, the anti-PD-L1 binding protein was chosen as the therapeutic protein because it binds to a surface protein expressed by cancer cells (Vaddepally et al., Cancers (Basel) 2020 Mar;12(3):738, incorporated herein by reference). The first bacteriophage backbone vector assembled was pTAT002. To construct pTAT002, the ori pMB1 pSB1C3 (a common backbone vector for iGEM libraries), ori M13 pTAT002 was amplified by PCR from the pIII N- and C-terminal portions from M13K07, aad7 (spectinomycin resistance) from E. coli KN01, and the gBlock a constitutive promoter. The PCR products were then assembled by Gibson and transformed into chemically competent MM294 cells. The integrity of the plasmid was then verified by digestion with ApaLI and NdeI (Figure 5 in WO2022073127A1, incorporated herein by reference). After Sanger sequencing of pTAT002, a mutation was found at the third position (G>T) of the P5 promoter. P5mut (5'-TT TThe resulting promoter, designated pTAT010-P5 (ACAATTAATCATCCGGCTCGTAATTTATGTGGA-3'), allows for low-level expression of upstream genes, as measured in the pTAT010-P5 and pTAT010-P5mut constructs (data not shown). To streamline construct assembly, the pTAT002 backbone was modified, and the sfGFP gene was cloned and expressed by the P5 promoter instead of gpIII. This backbone was assembled similarly to pTAT003, except that the primers used allowed for the insertion of an additional terminator after the gene expressed by P5 and a Gibson assembly tag (GAT) that allowed for the separation of different parts of the vector. The resulting vector, designated pTAT013, was then used as a template to amplify the backbone of subsequent constructs for displaying therapeutic proteins on pIII. Therefore, for the construction of pTAT044 and pTAT070, the plasmid backbone was amplified from pTAT013, the P5mut promoter was amplified from pTAT010-P5mut, and the C-terminal portion of gpIII was amplified from M13K07. Anti-PD-L1 scFv was amplified from gBlocks with either an HA-6sHis linker (pTAT044) or no linker (pTAT070). These gBlocks also showed promise in enabling better expression levels of therapeutic bacteriophage, so the start codon of the checkpoint inhibitor fusion protein was changed from ATG to GTG. After assembly, all plasmids were then submitted for Sanger sequencing, and no deleterious mutations were detected. Based on these results, the bacteriophage secretion system with therapeutic protein display on pIII was ready for efficiency testing and rounds of improvement.

[0110] Bacteriophage secretion systems based on three or more vectors The bacteriophage secretion system can be split into two or more DNA molecules to accommodate the simultaneous display of multiple therapeutic proteins and maintain functionality as long as sufficient protein is produced for each bacteriophage gene. To facilitate the display of two therapeutic proteins on the same bacteriophage particle, we aimed to split the bacteriophage machinery into three separate plasmids. As a first step, we needed to delete gpIII, the tail fiber protein, and the bacteriophage head protein. We selected gpIX, a capsid gene located in the head of the bacteriophage and involved in budding from the host cell, as the second site for protein fusion. Deletion of gpIX from M13K07 is more complex than deleting gpIII because the coding sequence for gpIX overlaps with that of gpVIII. To remove gpIX, we had to include several gene refactorings to prevent disruption of the gpVIII gene. The overlapping sequence between gpVIII and gpIX was replaced by a 5'-AGA TGA The sequence is GTGTTTTA-3', where the bolded ATG codon is the start codon for gpIX and the underlined TGA codon is the stop codon from gpVIII. The overlap between the two genes was corrected by mutating the A>G at position 3, changing the ATG codon to a weaker GTG start codon without affecting the sequence of gpVIII (both AGG and AGA encode arginine). The third codon of gpIX was also changed from TTA to TAA, introducing a stop codon and preventing translation of gpIX. The change is underlined in the resulting sequence. 5'-AG G TGAGTGTTT A The final construct, pTAT025, was then obtained by amplifying pTAT004 with primers that introduced these modifications into the gpVIII / gpIX locus. Plasmid pTAT025 therefore expresses all genes of the M13 genome except for gpIII and gpIX, which must be provided in trans. It also requires the ori for secretion of bacteriophage. M13A bacteriophage backbone vector encoding

[0111] The gpIII deletion in plasmid pTAT025 can be complemented by any of the above-mentioned plasmids expressing gpIII or gpIII-therapeutic protein fusions (pTAT002, pTAT044, and pTAT070). However, pTAT025 also requires an exogenous supply of gpIX to produce bacteriophage. Therefore, a set of plasmids was required to support gpIX production. For this purpose, the ori from pKN23 was used. pSC101 A new backbone was generated by amplifying bla from pUC19 and P5-BCD1-sfGFP from pTAT010-P5, each of which was assembled using GAT primer tails. The PCR fragments were then purified by SPRI and assembled by Gibson assembly to generate pTAT014, which was then transformed into MM294. The construct was then evaluated phenotypically (GFP phenotype) and sequenced by Sanger sequencing. Next, this entire backbone, excluding the sfGFP gene, was amplified and assembled with the gpIX gene amplified from M13K07. Both PCR products were then assembled in the same manner as pTAT014 to generate the gpIX complementation plasmid (pTAT017). This plasmid was further modified to display mouse IL-2 (mIL-2) and human IL-15 (hIL-15) on pIX by amplifying CDS and P5mut-BCD1 from the gBlock to form pTAT071 and pTAT060, respectively. These gBlocks also showed promise in enabling better expression levels of therapeutic bacteriophages, so the start codons of the cytokine fusion proteins were changed from ATG to GTG. The plasmids were then verified by Sanger sequencing.

[0112] Incorporation of a plasmid into a functional bacteriophage secretion system. To test potential synergistic effects between cytokines and cancer cell-targeting moieties displayed on synthetic therapeutic bacteriophages, different bacteriophage secretion systems were required. To obtain these systems, the plasmids were sequentially transformed into E. coli MG1655 by electroporation. The combinations listed in Table 3 allowed for the production 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), and M13 phage displaying human IL-15 and anti-PD-L1 scFv (hIL15-phage-PD-L1). These four bacteriophage variants allowed for the analysis of the contribution of each component to the anti-tumor response in mice and the exploration of potential synergistic effects. [Table 3]

[0113] Taken together, the strains generated in this example are sufficient to explore potential synergistic effects between cytokines and cancer cell targeting moieties displayed on M13 bacteriophage. These combinations are not intended to be exhaustive, and it is reasonable to assume that other cytokines or binding agents may exhibit synergistic effects when displayed on M13 bacteriophage. This example also illustrates the ability of bacteriophage to simultaneously display two or more therapeutic agents on each subunit of two different coat proteins.

[0114] Example 2 - In vitro validation of therapeutic target engagement of bacteriophages displaying tumor-targeting molecules and cytokines Strains, Plasmids, Phage Production, and Growth Conditions. All strains and plasmids used in this example are listed in Table 1. Cells were typically grown in 2xYT broth (2xYT) supplemented with the following concentrations of antibiotics as needed: 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 longer than 18 hours before use in experiments. Bacteriophage was harvested from the supernatant of confluent bacterial cultures (grown overnight). Culture supernatants containing bacteriophage were used by centrifugation immediately after bacterial sedimentation.

[0115] Polyethylene glycol-based precipitation of bacteriophage particles. Starting from a frozen stock, 10 mL of 2xYT broth containing the appropriate antibiotic at the concentration specified in the paragraph above was inoculated and the culture was incubated overnight at 30°C with agitation, or for no longer than 18 hours. The entire overnight bacterial culture was transferred to a 2 L Erlenmeyer flask containing 500 mL of 2xYT broth containing the appropriate antibiotic. The culture was incubated at 37°C with agitation until the optical density at 600 nm reached 0.5. The culture was then placed on ice for 15 minutes and then incubated overnight at room temperature with agitation. The overnight culture was transferred to a 500 mL centrifuge bottle and centrifuged at 13,000 g for 20 minutes at 4°C. The supernatant was decanted and filtered onto a 500 mL 0.45 μm filter unit to remove residual bacteria and debris. 2.5 M NaCl / 20% PEG-8000 (w / v) was added to the filtered supernatant to obtain a 4:1 supernatant:PEG solution volume ratio. After thoroughly mixing by inverting the bottle 15 times, the mixture was then incubated at 4 °C for 1 h. The virions were then pelleted by centrifugation at 13,000 g for 20 min at 4 °C. The supernatant was then removed, and the pellet was resuspended in 1–2 mL of PBS containing 0.1 mM CaCl2. The bacteriophage preparation was kept on ice for an additional 1 h, vortexed, and stored at 4 °C. The majority of contaminating LPS was removed from the phage preparation using EndoTrap® HD (LIONEX) discontinuous chromatography according to the manufacturer's instructions. Phage titers were then interpolated by an in-house sandwich ELISA against the pVIII coating protein using a standard curve generated with purified M13K07 helper phage (NEB).

[0116] Assessment of bacteriophage binding to PD-L1 by indirect ELISA. The binding activity of bacteriophage displaying scFv against the PD-L1 checkpoint was measured by ELISA assay. A 96-well Nunc MaxiSorp™ plate was first coated overnight at 4°C with recombinant human PD-L1 protein ectodomain (Sino Biological) diluted at 2 μg / mL in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). The plate was then washed three times with 200 μL of TBS-T. To prevent nonspecific binding, the plate was then incubated with 200 μL of blocking buffer (TBS-T, 3% nonfat milk, 1% BSA) at room temperature for 1 hour. Blocking was stopped by removing the blocking buffer and washing the plate twice with 200 μL of TBS-T. Next, 100μL of purified wild-type M13 phage (phage control), or bacteriophage displaying anti-PD-L1 scFv on pIII (phage-PD-L1), or bacteriophage displaying anti-PD-L1 scFv on pIII and a cytokine on pIX (either mouse IL-2 or human IL-15, referred to as mIL2-phage-PD-L1 or hIL15-phage-PD-L1, respectively) was diluted in TBS1X and added to the wells. The plate was incubated at room temperature for 1 hour. The plate was then washed three times with 200μL of TBS-T, and 100μL of anti-pVIII-HRP (anti-M13 phage, B62-FE2) diluted in blocking buffer (1:500) was added. After incubation in the dark at room temperature for 1 hour, the wells were washed five times with TBS-T. To reveal the presence of bacteriophage, 100 μL of TMB sensitive substrate solution (BioLegend) was added to each well, and the plate was incubated at room temperature for 3–10 minutes until a blue color appeared. The reaction was stopped by adding 100 μL of stop solution (0.5 M H2SO4) to each well. The absorbance at 450 nm was then measured using a plate reader.Only bacteriophage displaying anti-PD-L1 scFv on pIII were significantly detected on PD-L1-coated wells (Figure 3A), thus verifying the biological activity of the display. Dual display of recombinant interleukin on pIX did not affect the binding efficiency of scFv on pIII.

[0117] Evaluation of bacteriophage displaying recombinant cytokines on pIX by sandwich ELISA. An ELISA assay was designed to verify the expression of recombinant human interleukin-15 (hIL-15) or murine interleukin-2 (mIL-2) displayed on bacteriophage. 96-well Nunc MaxiSorp™ plates were first coated overnight at 4°C with anti-hIL-15 or anti-mIL-2 rabbit IgG antibody (Sino Biological) diluted as recommended by the manufacturer in coating buffer (0.05 M carbonate-bicarbonate, pH 9.6). The plates were then washed three times with 200 μL of TBS-T. To prevent nonspecific binding, the plates were then incubated with 200 μL of blocking buffer (TBS-T, 3% nonfat milk, 1% BSA) at room temperature for 1 hour. Blocking was stopped by removing the blocking buffer and washing the plates twice with 200 μL of TBS-T. Next, 100μL of purified wild-type M13 phage (phage control), or bacteriophage displaying anti-PD-L1 scFv on pIII (phage-PD-L1), or bacteriophage displaying anti-PD-L1 scFv on pIII and a cytokine on pIX (either mIL-2 or hIL-15, referred to as mIL2-phage-PD-L1 or hIL15-phage-PD-L1, respectively) was diluted in TBS1X and added to the wells. The plate was incubated at room temperature for 1 hour. The plate was then washed three times with 200μL of TBS-T, and 100μL of anti-pVIII-HRP (anti-M13 / fd / F1, B62-FE2) diluted in blocking buffer (1:500) was added. After 1 hour of incubation at room temperature in the dark, the wells were washed five times with TBS-T. To reveal the presence of bacteriophage, 100 μL of TMB sensitive substrate solution was added to each well, and the plate was incubated at room temperature for 3–10 minutes until a blue color appeared. The reaction was stopped by adding 100 μL of stop solution (0.5 M H2SO4) to each well. The absorbance was then measured at 450 nm using a plate reader.Bacteriophage displaying cytokines on pIX (mIL2 or hIL15) and anti-PD-L1 scFv were detected on interleukin-specific antibody-coated wells (Figure 3B), thus verifying the expression of the recombinant proteins and the binding activity of the anti-PD-L1 scFv.

[0118] Example 3 - In vitro validation of the biological activity of cytokines displayed on bacteriophages Strains, Plasmids, Phage Production, and Growth Conditions. All strains and plasmids used in this example are listed in Table 3. Cells were typically grown in Luria Broth Miller (LB) or Luria Broth Agar Miller medium supplemented with the following concentrations of antibiotics 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 use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example II.

[0119] Cell Culture. HEK-Blue™ IL-2 reporter cells, specifically designed to detect bioactive interleukin-2 (IL-2), were ordered from InvivoGen (hkb-il2). HEK-Blue™ cells were cultured under optimal conditions according to the manufacturer's guidelines. Briefly, cells were cultured in DMEM supplemented with 10% fetal bovine serum (FBS) and Normocin™ at 37°C in an atmosphere of 5% CO2 and 95% humidified air. Cells were cultured under selective pressure using HEK-Blue™ CLR Selection and puromycin to maintain stable expression of the human IL-2 receptor (IL-2R), including expression of the subunits CD25 (IL-2Rα), CD122 (IL-2Rβ), and CD132 (IL-2Rγ).

[0120] HEK-Blue™ IL-2 Reporter Assay. The HEK-Blue™ IL-2 reporter assay experiment was performed as follows: Upon reaching 80% confluency, HEK-Blue™ IL-2 cells were washed twice with cold D-PBS (Gibco). The D-PBS was removed, and 2 mL of cold Versene 1X (Gibco) was added to detach the cells. After a 5-minute incubation, 8 mL of test medium (DMEM supplemented with 10% heat-inactivated FBS) was added. Cell density was assessed using trypan blue and a hemocytometer. The cell suspension was centrifuged at 300 g for 10 minutes at 4°C, the supernatant was removed, and the cells were resuspended in test medium at a cell density of 280,000 cells / mL. 180 μL of the cell suspension, corresponding to approximately 50,000 cells, was seeded into a 96-well TC-treated culture plate. 20 μL of therapeutic agents were added to the wells to test their IL-2R activity. The plates were incubated at 37°C for 18 hours in an atmosphere of 5% CO2 and 95% humidified air. For each well, 20 μL of medium was collected and transferred to a new 96-well clear plate. 180 μL of QUANTI-Blue™ solution was added, and the plates were incubated at 37°C for 15 minutes to 6 hours. Optical density at 630 nm was acquired using a conventional microplate reader.

[0121] Bacteriophage displaying the murine IL-2 cytokine activates the IL-2 receptor. To verify that the cytokine maintains its biological activity when displayed on synthetic therapeutic bacteriophage, experiments were performed using HEK-Blue™ IL-2 reporter cells to monitor activation of the IL-2 receptor (IL-2R) by bacteriophage displaying murine IL-2 (mIL-2). HEK-Blue™ IL-2 cells were incubated with PBS (vehicle), 10 mL of bacteriophage displaying anti-PD-L1 scFv on pIII, or 10 mL of bacteriophage displaying anti-PD-L1 scFv on pIII. 11 (phage-PD-L1), which displays mIL-2 on pIX and anti-PD-L1 scFv on pIII. 11 bacteriophage (mIL-2-phage-PD-L1), or 0.2 ng of mIL-2 (10 11 Cells were treated with either mIL-2-phage-PD-L1 or an equimolar dose of mIL-2 for 18 hours. The level of IL-2R activation was monitored by measuring the optical density at 630 nm and is reported in Figure 4. Only bacteriophage displaying mIL-2 (mIL-2-phage-PD-L1) activated IL-2R as efficiently as mIL2, demonstrating that cytokines are biologically active when displayed on synthetic therapeutic bacteriophage.

[0122] Example 4: Bacteriophages displaying cytokines and cancer cell targeting moieties bind to cancer cells. Strains, Plasmids, Phage Production, and Growth Conditions. All strains and plasmids used in this example are listed in Table 3. Cells were typically grown in Luria Broth Miller (LB) or Luria Broth Agar Miller medium supplemented with the following concentrations of antibiotics 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 use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example II.

[0123] Cell culture. A20 lymphocytic B lymphoma cells were ordered from ATCC (TIB-208). Upon arrival, cells were washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This culture medium was used to prepare cells for all experiments. Frozen stocks were generated after four passages and used to initiate subsequent cultures for experiments. Cells were maintained at 2 × 10 throughout all experiments. 5 cells / mL~2×10 6 Cells were maintained at a density of 1000 cells / mL and grown at 37°C in an atmosphere of 5% CO2 and 95% humidified air.

[0124] Immunofluorescence experiment: PD-L1+A20 cancer cells were cultured at 10 11Cells were incubated with 1 mL of phage or mIL-2-phage-PD-L1 for 24 hours and then fixed to coverslips placed at the bottom of 6-well plates using 10% NBF solution. After washing with PBS, cells were permeabilized by adding 1 mL of PBS containing 0.1% Triton X-100 (PBS-Tx). The solution was removed, and cells were blocked by adding PBS-Tx containing 2% BSA (PBS-Tx-BSA). Cells were then treated with an Fc blocking antibody (CD16 / CD32 monoclonal antibody, Invitrogen). Cells were washed with PBS-Tx, and then phage was revealed using anti-M13-FITC (Progen) diluted 1 / 200 in PBS-Tx-BSA. Cells were washed with PBS-Tx, and nuclei were stained using 1 μg / mL Hoescht dye. Slides were washed with PBS-Tx and then analyzed by confocal microscopy.

[0125] Bacteriophage displaying the murine IL-2 cytokine and anti-PD-L1 scFv targeting moiety were used to target PD-L1 + Binding to Cancer Cells. Immunofluorescence experiments were performed to demonstrate that bacteriophage displaying murine IL-2 cytokine and anti-PD-L1 scFv (mIL-2-phage-PD-L1) binds to cancer cells via its anti-PD-L1 scFv targeting moiety. PD-L1 + A20 cells, 10 11 1 mL of either naked phage (therapeutic agent and cancer cell targeting moiety are not displayed on the naked phage) or mIL-2-phage-PD-L1 for 24 hours, and the PD-L1 concentration of each compound was measured. +Binding to cancer cells was assessed. Nuclei were revealed using Hoescht dye, while phage and mIL-2-phage-PD-L1 were revealed using an anti-M13 antibody conjugated to FITC according to the manufacturer's protocol and recommendations. The experiment demonstrates that only mIL-2-phage-PD-L1 can bind to A20 cells, as indicated by the FITC signal observed on the cancer cells after this treatment (Figure 5). This result demonstrates that the presence of the cancer cell targeting moiety, the anti-PD-L1 scFv, in mIL-2-phage-PD-L1 enables mIL-2-phage-PD-L1 to bind to and coat the surface of PD-L1+ cancer cells. mIL-2-phage-PD-L1 can then recruit and activate the immune system to induce an anti-tumor immune response.

[0126] Example 5: Bacteriophages displaying cytokines and cancer cell targeting moieties have enhanced local antitumor activity. Strains, Plasmids, Phage Production, and Growth Conditions. All strains and plasmids used in this example are listed in Table 3. Cells were typically grown in Luria Broth Miller (LB) or Luria Broth Agar Miller medium supplemented with the following concentrations of antibiotics 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 use in experiments. Bacteriophages were extracted from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example II.

[0127] Cell culture. CT26 colorectal cancer cells were ordered from ATCC (CRL-2638). Cells were cultured under optimal conditions at 37°C in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and penicillin-streptomycin (50 U / mL) in an atmosphere of 5% CO2 and 95% humidified air.

[0128] Tumor Mouse Model. All experiments involving mice were rigorously reviewed by the Animal Care Committee of our local university (Universite de Sherbrooke), and procedures exposed animals to minimal stress and pain. Mice were provided with water and regular chow ad libitum and were allowed to rest for at least two days after arrival. During the experiment, no more than five animals shared the same cage, and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were monitored daily.

[0129] As a general guideline, this paragraph details the workflow of a typical mouse experiment. To generate solid tumors, 10 cells resuspended in 50 µL of PBS were cultured. 6 CT26 cells were injected into the right flank of the mice. The mice were then observed daily to determine whether the tumor size was between 40 and 80 mm. 3 Tumor growth was measured until tumors reached 1500 mm. Mice then received 50 μL of treatment via intratumoral injection. Tumors were then monitored until clearance or when tumors reached 1500 mm. 3 Tumor size was followed twice a week until the tumor reached a volume of 100 μg / kg, after which the mice were sacrificed and tumors were collected from the mice. The presence of metastases in other organs was also assessed.

[0130] Bacteriophage displaying murine IL-2 cytokine and anti-PD-L1 scFv targeting moiety exhibit enhanced anti-tumor activity. To evaluate the effect of cytokine addition on the anti-tumor activity of bacteriophage displaying cancer cell targeting moieties, bacteriophage displaying a PD-L1 checkpoint inhibitor on pIII and murine interleukin-2 (mIL-2) on pIX were developed using the process described in Example I. Mice were injected with 10 6CT26 cells were injected subcutaneously and observed every two days to monitor tumor growth. 3 Once the tumor volume reached 100 μg / ml, the mice were divided into five treatment groups, and all treatments were administered intratumorally on days 0, 4, and 7. The first group received injections of vehicle (PBS) as a control (Figure 6). The second group received 5 × 10 mAb / ml of 5 × 10 mAb / ml from Sino Biological. 11 The third group received an effective dose of 14.4 ng of recombinant murine IL-2 (mIL-2) (Figure 6). The third group received 10 mAbs displaying anti-PD-L1 scFv on pIII. 11 The fourth group received an effective dose of 5 x 10 bacteriophage (phage-PD-L1) (Figure 6). 11 Molecules mIL-2 and mIL-10 11 The final group received an effective dose of 10 phage-PD-L1 scFvs displaying mIL-2 on pIX and anti-PD-L1 scFvs on pIII. 11 Patients were given an effective dose of mIL-2-phage-PD-L1. Tumor size was then monitored twice weekly using precision calipers until the tumors were removed or became too large to pursue the experiment. Experiments revealed that enhanced antitumor activity was observed only when mIL-2 was physically conjugated to bacteriophage displaying a cancer cell-targeting PD-L1 checkpoint inhibitor. This demonstrates that conjugating cytokines to bacteriophage that also display a cancer cell-targeting moiety results in a synergistic antitumor effect.

[0131] Example 6: Bacteriophages displaying cytokines and cancer cell targeting moieties have systemic antitumor activity. Strains, Plasmids, Phage Production, and Growth Conditions. All strains and plasmids used in this example are listed in Table 3. Cells were typically grown in Luria Broth Miller (LB) or Luria Broth Agar Miller medium supplemented with the following concentrations of antibiotics 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 use in experiments. Bacteriophage were obtained from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example II.

[0132] Cell culture. A20 lymphocytic B lymphoma cells were ordered from ATCC (TIB-208). Upon arrival, cells were washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This culture medium was used to prepare cells for all experiments. Frozen stocks were generated after four passages and used to initiate subsequent cultures for experiments. Cells were maintained at 2 × 10 throughout all experiments. 5 cells / mL~2×10 6 Cells were maintained at a density of 1000 cells / mL and grown at 37°C in an atmosphere of 5% CO2 and 95% humidified air.

[0133] Tumor Mouse Model. All experiments involving mice were rigorously reviewed by the Animal Care Committee of our local university (Universite de Sherbrooke), and procedures exposed animals to minimal stress and pain. Mice were provided with water and regular chow ad libitum and were allowed to rest for at least two days after arrival. During the experiment, no more than five animals shared the same cage, and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were monitored daily.

[0134] As a general guideline, this paragraph details the workflow of a typical mouse experiment. To generate solid tumors, 5 x 10 cells resuspended in 100 µL of PBS were cultured. 6 A20 cells were injected into the right flank of mice, and then 4 days later, 5 x 10 6 A20 cells were injected into the left flank to obtain mice bearing two tumors simultaneously. The mice were observed daily until the tumor volume on the right side reached 50-100 mm. 3 Tumor growth was measured until tumor volume reached 1500 mm, and mice then received 50 μL of treatment by intratumoral injection. 3 Tumor size was followed twice a week until it reached 100%, after which the mice were sacrificed.

[0135] Bacteriophage displaying murine IL-2 cytokine and anti-PD-L1 scFv targeting moieties have systemic anti-tumor activity. To evaluate whether bacteriophage displaying cytokine and cancer cell targeting moieties have systemic anti-tumor activity, bacteriophage 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 in both flanks. However, to evaluate whether a systemic anti-tumor immune response was elicited and to induce clearance of the uninjected tumor present in the left flank, the drug was injected only into tumors from the right flank. Mice were divided into two groups: those with tumors in the right flank measuring 50-100 mm. 3 The first group received vehicle injections (PBS) as a control (Figure 7). The second group received 10 mAbs displaying mIL-2 on pIX and anti-PD-L1 scFv on pIII. 12An effective dose of mIL-2-phage-PD-L1 (Figure 7) was administered to the tumors. Tumor size was then monitored twice weekly using precision calipers until the tumors were removed or became too large to pursue the experiment. Experiments demonstrated that bacteriophage displaying mIL-2 on pIX and anti-PD-L1 scFv induced clearance of not only the injected tumor (right flank), but also the uninjected tumor (left flank). These results demonstrated that bacteriophage displaying cytokines and cancer cell-targeting moieties have systemic antitumor activity (i.e., antitumor activity that exceeds local antitumor activity against the injected cell population).

[0136] Example 7: The local and systemic antitumor activity of bacteriophages displaying cytokine and cancer cell targeting moieties is mediated by the immune response. Strains, Plasmids, Phage Production, and Growth Conditions. All strains and plasmids used in this example are listed in Table 3. Cells were typically grown in Luria Broth Miller (LB) or Luria Broth Agar Miller medium supplemented with the following concentrations of antibiotics 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 use in experiments. Bacteriophage were obtained from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example II.

[0137] Cell culture. A20 lymphocytic B lymphoma cells were ordered from ATCC (TIB-208). Upon arrival, cells were washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This culture medium was used to prepare cells for all experiments. Frozen stocks were generated after four passages and used to initiate subsequent cultures for experiments. Cells were maintained at 2 × 10 throughout all experiments. 5 cells / mL~2×10 6 Cells were maintained at a density of 1000 cells / mL and grown at 37°C in an atmosphere of 5% CO2 and 95% humidified air.

[0138] Tumor Mouse Model. All experiments involving mice were rigorously reviewed by the Animal Care Committee of our local university (Universite de Sherbrooke), and procedures exposed animals to minimal stress and pain. Mice were provided with water and standard chow ad libitum and were allowed to rest for at least two days after arrival. During the experiment, no more than five mice shared the same cage and were monitored daily for symptoms (isolation, inactivity, weight loss, tumor size, and dehydration).

[0139] As a general guideline, this paragraph details the workflow of a typical mouse experiment. To generate solid tumors, 5 x 10 cells resuspended in 100 µL of PBS were cultured. 6 A20 cells were injected into the right flank of mice, and then 4 days later, 5 x 10 6 A20 cells were injected into the left flank to obtain mice bearing two tumors simultaneously. The mice were observed daily until the tumor volume on the right side reached 50-100 mm. 3 Tumor growth was measured until tumor volume reached 1500 mm, and mice then received 50 μL of treatment by intratumoral injection. 3 Tumor size was followed twice a week until it reached 100%, after which the mice were sacrificed.

[0140] Ex vivo culture of microdissected tumors (MDT) on a chip. Tumors derived from the A20 cell line were harvested from BALB / c mice and microdissected. The MDT were maintained in RPMI-1640 10% FBS PEN / STREP before and after loading into specially designed ex vivo culture chips (MISO Chip Inc.). The MDT were dispensed at 8 MDT / channel, 4 channels / chip, and incubated at 37°C + 5% CO2 for 24 hours.

[0141] Treatment of microdissected tumors (MDT) cultured on a chip. Treatment agents, synthetic therapeutic phages and atezolizumab (an anti-PD-L1 checkpoint inhibitor used as a benchmark standard) were added to 2 × 10 cells in culture medium. 12 The treatment was diluted to 1000 molecules / mL. The treatment was then used to wash the corresponding chip three times for each channel before application. The chip was then incubated at 37°C + 5% CO2 for 48 hours, and the supernatant was collected and then sent to Eve Technologies for analysis of cytokine concentrations by ELISA. A chip with MDT exposed to the same volume of PBS was also generated and used as a control.

[0142] Immune infiltration assay. For engraftment, 5 x 10 6 A20 cells were injected subcutaneously into the right flank of BALB / c mice. 3 After reaching 10 μg / ml, tumors were cultured for 10 min to display PBS or mIL-2 and anti-PD-L1 (mIL-2-phage-PD-L1). 11 Mice were treated three times over a seven-day period by intratumoral injection of either one of the bacteriophage particles. On day 8, mice were euthanized, and tumors were harvested and fixed in formalin. Tumor samples were then sent to the Plateforme d'Histologie de l'Universite de Sherbrooke for permeabilization and paraffin embedding. Slides were then generated, stained with hematoxylin-eosin, and digitized. Images were then viewed and processed using Qpath to distinguish immune cells from tumor cells.

[0143] The local and systemic antitumor activity of bacteriophage displaying murine IL-2 cytokine and anti-PD-L1 scFv targeting moieties is mediated by immune activation. We investigated the mechanisms underlying the local and systemic antitumor immune responses observed with bacteriophage displaying mIL-2 and anti-PD-L1 (mIL-2-phage-PD-L1). Because mIL-2-phage-PD-L1 is designed to activate the immune system by acting on multiple targets via IL-2 cytokine, anti-PD-L1, and the phage's TLR9 agonist DNA and antigenic antigen, we hypothesized that mIL-2-phage-PD-L1 treatment should induce a wide range of cytokines involved in activating different branches of the immune response. To identify the immune pathways involved in the antitumor activity of mIL-2-phage-PD-L1, we used the chip to culture and treat tumors ex vivo, allowing us to track the secretion of key cytokines over time. Therefore, A20 tumors were microdissected and cultured ex vivo on specialized chips (MISO chip Inc.) and exposed to equimolar amounts of either PBS (same amount as the other two treatments), mIL-2-phage-PD-L1, or atezolizumab (anti-PD-L1). After 2 days of treatment, cytokines were added to the culture supernatant, and the fold changes of key cytokines relative to the PBS condition were calculated (Figure 8). While atezolizumab produced very small changes in cytokine levels that were primarily limited to the Th1 response pathway, mIL-2-phage-PD-L1 activated all major immune pathways, particularly the acute, Th1, and Th17 immune response pathways, demonstrating the clear involvement of multiple immune cell types in tumor cell elimination. Therefore, the antitumor response induced by mIL-2-phage-PD-L1 is likely mediated, at least, by CD8+ T cells, myeloid cells, and supported by B cells through the production of antitumor IgG.

[0144] The local and systemic antitumor activity of bacteriophage displaying murine IL-2 cytokine and anti-PD-L1 scFv targeting moieties is mediated by extensive immune infiltration of tumors and recruitment of immune cells. Because cytokine profiling experiments showed clear signs of immune activity, we next investigated the presence of immune cells. Therefore, mice bearing A20 tumors measuring 75–150 mm in volume were treated with 100 μg of mIL-2-phage-PD-L1 on days 0, 4, and 7. 11 Mice were then sacrificed on day 8, and tumors were collected, fixed in formalin, and processed for histological examination by the Plateforme d'histologie de l'universite de Sherbrooke. After hematoxylin-eosin staining, tumor tissues were examined for differences in the density of infiltrating immune cells (Figure 9). Tumors treated with mIL-2-phage-PD-L1 were found to be highly infiltrated with immune cells, whereas very few immune cells were detectable in the PBS control group (Figure 9A). Tumor tissues in the mIL-2-phage-PD-L1-treated group also had larger necrotic patches, indicating active destruction of tumor tissue (Figure 9B). Overall, treatment with mIL-2-phage-PD-L1 resulted in the recruitment of multiple types of immune cells, leading to tumor clearance.

[0145] Example 8: The therapeutic activity of bacteriophages displaying cytokine and cancer cell targeting moieties is mediated by a systemic long-term anti-tumor immune response. Strains, Plasmids, Phage Production, and Growth Conditions. All strains and plasmids used in this example are listed in Table 3. Cells were typically grown in Luria Broth Miller (LB) or Luria Broth Agar Miller medium supplemented with the following concentrations of antibiotics 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 use in experiments. Bacteriophage were obtained from confluent bacterial cultures (grown overnight) using the PEG precipitation protocol presented in Example II.

[0146] Cell culture. A20 lymphocytic B lymphoma cells were ordered from ATCC (TIB-208). Upon arrival, cells were washed and resuspended in RPMI-1640 supplemented with 10% fetal bovine serum (FBS) and 0.05 mM 2-mercaptoethanol. This culture medium was used to prepare cells for all experiments. Frozen stocks were generated after four passages and used to initiate subsequent cultures for experiments. Cells were maintained at 2 × 10 throughout all experiments. 5 cells / mL~2×10 6 Cells were maintained at a density of 1000 cells / mL and grown at 37°C in an atmosphere of 5% CO2 and 95% humidified air.

[0147] Tumor Mouse Model. All experiments involving mice were rigorously reviewed by the Animal Care Committee of our local university (Universite de Sherbrooke), and procedures exposed animals to minimal stress and pain. Mice were provided with water and regular chow ad libitum and were allowed to rest for at least two days after arrival. During the experiment, no more than five animals shared the same cage, and symptoms (isolation, inactivity, weight loss, tumor size, dehydration) were monitored daily.

[0148] The therapeutic activity of bacteriophage displaying murine IL-2 cytokine and anti-PD-L1 scFv targeting moiety is mediated by a systemic, long-term anti-tumor immune response. To assess whether the anti-tumor activity of bacteriophage displaying cytokine and cancer cell targeting moiety is mediated by a long-term systemic anti-tumor immune response, rechallenge experiments were performed in cured and naive mice (mice never exposed to A20 cancer cells or mice previously treated with bacteriophage displaying cytokine and cancer cell targeting moiety). Cured mice were mice bearing A20 cancer cell tumors on the right flank, treated with 100 μg of a PD-L1 checkpoint inhibitor and a bacteriophage displaying murine interleukin-2 (mIL2-phage-PD-L1). 12 The effective dose was obtained by treating the tumor with a volume of 50-100 mm. 3 Once tumor volume reached 0.05 μg / mL, the tumors were injected with treatment, which was administered intratumorally on days 0, 4, and 7 (Figure 10). Eight mice that were cured by treatment and had completely eliminated tumors were kept in the animal facility for 160 days. On day 160, the cured and naive mice were injected with 5 × 10 IgG to induce tumor formation. 6 In this study, mice received 100 A20 cancer cell injections, 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 were able to form and grow only in naive mice, while tumors were systematically eliminated in previously cured mice. These results indicate that mIL2-phage-PD-L1 treatment induced an adaptive, long-lasting, and systemic antitumor immune response that could prevent the engraftment of new tumors and thus tumor recurrence.

[0149] Any element of any embodiment may be used in any embodiment. While the present invention has been described with reference to several embodiments, those skilled in the art will recognize that various changes can be made and elements thereof can be substituted with equivalents without departing from the scope of the invention. Additionally, modifications can be made without departing from the essential teachings of the present invention. Identification of equivalent compositions, methods, and kits is within the skill of an ordinary practitioner and would require no more than routine experimentation in light of the teachings of the present disclosure. Practice of the present disclosure will be more fully understood from the following examples, which are presented herein for illustrative purposes only and should not be construed as limiting the disclosure in any way.

[0150] All references cited herein, and the references therein, where appropriate for teaching additional or alternative details, features, and / or technical background, are hereby incorporated by reference in their entirety.

[0151] While the present disclosure has been particularly shown and described with reference to particular embodiments, it should be understood that the above-disclosed variations and other features and functions, or alternatives thereof, may be desirably combined into many other different systems or applications. Also, various presently unforeseen or unanticipated substitutions, modifications, variations, or improvements therein may be made by those skilled in the art, which are also intended to be encompassed by the following claims.

Claims

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

2. The at least one cytokine is 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-1 7B, IL-17C, IL-17D, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-2 3, IL-24, IL-25, IL-26, IL-27, IL-28A / B / IL29, IL-30, IL-31, IL-32, 2. The bacteriophage of claim 1, wherein the bacteriophage is selected from IL-33, IL-35, TNF-alpha, LT-alpha, LT-beta, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, FLT3 ligand, G-CSF, GM-CSF, IFN-alpha, IFN-beta, IFN-omega, IFN-gamma, LIF, M-CSF, MIF, OSM, SCF, TGF-beta 1, TGF-beta 2, TGF-beta 3, and TSLP ligand.

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

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

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

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

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

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

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

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

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

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

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

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

15. A bacteriophage according to any one of claims 1 to 14, which is a synthetic bacteriophage.

16. A bacteriophage according to any one of claims 1 to 15, which is a therapeutic bacteriophage.

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

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

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

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

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

22. The at least one cytokine is 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-1 7B, IL-17C, IL-17D, IL-17F, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28A / B / IL29, IL-30, IL-31, IL-32 , IL-33, IL-35, TNF-alpha, LT-alpha, LT-beta, LIGHT, TWEAK, APRIL, BAFF, TL1A, GITRL, OX40L, CD40L, FASL, CD27L, CD30L, 4-1BBL, TRAIL, RANK, FLT3 ligand, G-CSF, GM-CSF, IFN-alpha, IFN-beta, IFN-omega, IFN-gamma, LIF, M-CSF, MIF, OSM, SCF, TGF-beta 1, TGF-beta 2, TGF-beta 3, and TSLP ligand.

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

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

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

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

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

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

29. 29. The pharmaceutical composition of any one of claims 21 to 28, wherein the at least one cancer cell targeting moiety targets Her2, EGFR, ER, PR, PD-L1, c-Kit, CD44, CD24, E-cadherin, cMet, MUC1, or CD133, or a combination thereof.

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

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

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

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

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

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

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

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

Images