EP4608420A1 - Engineered bacterium and uses thereof for treating cancer - Google Patents

Abstract

The present disclosure relates to engineered gastrointestinal bacteria expressing IL-22 and/or IFN-β and uses thereof for treatment of cancer.

Description

ENGINEERED BACTERIUM AND USES THEREOF

FOR TREATING CANCER

CROSS-REFERENCE TO RELATED APPLICATIONS

This PCT application claims priority to, and the benefit of, U.S. Provisional Patent Application No. 63/419,536, filed October 26, 2022, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under grant number A1068021 awarded by the National Institutes of Health. The government has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The sequence listing submitted on October 26, 2023, as an .XML entitled “10504- 087WOl_ST26.xml’’ created on October 26, 2023, and having a file size of 26,612 bytes is hereby incorporated by reference pursuant to 37 C.F.R. § 1.52(e)(5).

FIELD

The present disclosure relates to engineered bacteria expressing IL-22 and/or IFN- and uses thereof for treatment of cancer.

BACKGROUND

Worldwide ovarian cancer (OC) has an estimated yearly incidence of 239,000 new cases and 152,000 deaths. The cost of ovarian cancer to the U.S. military is great, with respect to troop readiness, and lifetime care is estimated to be one billion dollars. Late diagnosis of OC accounts for poor prognosis, as most women have advanced disease with widespread intraperitoneal dissemination at initial presentation. OC spreads intra-abdominally, and whole abdomen irradiation (WAI) can be a logical approach to treat all sites of metastatic disease; however, an effective dose of WAI has not been used due to intestinal toxicity. Recent clinical trials of WAI have used low radiation fraction sizes and yielded suboptimal results. Increased radiation doses are clearly needed to provide a more significant therapeutic effect. Although a combination of drugs has been tested, therapeutic resistance remains a major challenge for OC. Despite optimal management with radical cytoreductive surgery, platinum/taxane-based chemotherapy, and bevacizumab which blocks blood-vessel formation by inhibiting vascular endothelial growth factor (VEGF), most patients suffer recurrence within 18 months with platinum resistant disease. WAI and chemotherapy in combination has been postulated to act synergistically and cause changes in the tumor microenvironment including decreased hypoxia, stabilized vasculature, and enhanced immune cell activation. The combinations of therapeutic WAI with chemotherapy agents used to treat widespread abdominal cancers include paclitaxel, carboplatin, and poly (ADP- ribose) polymerase (PARP) inhibitors. This combination is also limited by radiation toxicity to the intestine.

Accordingly, what are needed are compositions and methods for treating or preventing cancers. The compositions and methods disclosed herein address these and other needs.

SUMMARY

The present disclosure provides compositions and methods for treating or preventing cancers.

In some aspects, disclosed herein is a method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of an engineered gastrointestinal tract (GI) bacterium, wherein the engineered gastrointestinal tract (GI) bacterium comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN-P, or a functional fragment thereof. As disclosed herein, in some embodiments, the GI bacterium secretes IL-22 and/or IFN- , or a functional fragment thereof. In some embodiments, the cancer is an ovarian cancer. In some embodiments, the subject is a human.

In some aspects, disclosed herein is a method wherein the engineered GI bacterium is a specie of Escherichia genus or Lactobacillus genus. In some embodiments, the engineered GI bacterium is Lactobacillus reuteri. In some embodiments, the engineered GI bacterium is Escherichia coli. In some embodiments, the GI bacterium localizes in the stomach, small intestine or colon of the subject.

In some embodiments, the subject is resistant to a therapy of immune checkpoint inhibitor. In one aspect, disclosed herein the treatment results in an increased level of an immune checkpoint molecule on a tumor cell relative to a control. In some embodiments, the immune checkpoint molecule is PD-L1 or PD-L2.

In some aspects, the method further comprises administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor, or a PD-L2 inhibitor.

In some aspects, the method further comprises applying to the subject an irradiation therapy. In some embodiments, the irradiation therapy comprises total body irradiation or abdominal irradiation. As disclosed herein, in some embodiments, the irradiation therapy is administered to the subject prior to the administration of the engineered GI bacterium. In some embodiments, the engineered GI bacterium is administered to the subject via an oral administration. In some embodiments, the administration is a dosage from about IxlO⁶ to about IxlO⁹ colony forming units (CFU) of the engineered bacterium.

In some embodiments, the treatment results in an increased number of T cells in a tumor relative to a control. In certain aspects, the method further comprises administering to the subject a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a platinum-based or taxane-based chemotherapeutic agent.

DESCRIPTION OF DRAWINGS

Fig. 1 A-1B. Radiation induces PD-L1 in human Cancer Cell lines (48 h) (Fig. 1A) PD-L1 staining in OVCAR3 cell line after 48 hours of 0 and 2 Gy irradiation (Fig. IB) PDL1 qPCR in OVSAHO cells after 0, 3 and 6 Gy irradiation. P values were obtained by t-test (n=3).

Fig. 2A-2C. Intestine of mice with intra- abdominal 2F8- cis tumors is protected from WAI by LR-IL-22 treatment. Fig. 2A: No treatment; Fig. 2B: Radiation; and Fig. 2C: Radiation preceded by gavage of 10⁹ LR-IL-22 bacteria in 10 p.1 of saline. Circles show tumor nodules.

Fig. 3. LR-IL-22 administration before WAI prevents intestinal barrier breakdown. Mice received intraoral red fluorescent beads 3 hours prior to ionizing radiation. Quantitative bar graph shows that fluorescent beads leakage into the blood following 19.75 WAI at day 2 and day 5 was significantly reduced in the LR-IL-22 group compared to mice injected intra-peritoneally with IL- 22 protein of mice receiving LR control gavage. *, <0.05 calculated by t-test relative to WAI group, (n=12).

Fig. 4A-4B: LR-IL-22 gavage and WAI induce PD-L1 expression in 2F8cis tumor cells in MUC-1 mice. (Fig. 4A) Flow cytometric analysis of CD45-/CD326+/PD-L1+ 2F8cis tumor cells was performed using freshly isolated intraperitoneal 2F8cis tumors from MUC- 1 mice. Mice were given 4 fractions of whole abdomen irradiation (WAI) (6 Gy x 4) in 4 consecutive days alone or together with LR-IL-22 gavage at day 2 and day 4 (first radiation dose as day 1). Tumors were collected at day 9 and the single cell suspensions were analyzed by flow cytometry then staining. EpCAM was used to distinguish PD-L1 expressing non-tumor cells from 2F8cis tumor cells. The number of PD-L1 expressing 2F8cis tumor cells was significantly higher in WAI and WAI+LR- IL-22 groups compared to the no radiation tumor only group. WAI+ LR-IL-22 treatment further increased the number of PD-L1 expressing tumor cells compared to WAI. (n=2-3 mice; 5-7 tumor nodules per mouse; p values were calculated by t-test). The number of mice required is in the Power and Sample Size Determination section. (Fig. 4B) Representative immunofluorescent images of excised 2F8cis tumor tissue sections at day 9 showing PD-L1 expression on tumor cells in WAI treated, WAI + LRIL- 22 treated groups compared to tumor only control group. PD-L1 expression is further increased in the WAI + LRIL- 22 group compared to WAI group (n=3). Fluorescence represents PD-L1 and DAPI stain represent cell nucleus.

Fig. 5A-5B: LR- IL-22 gavage and WAI increases intra-tumoral CD8+ T-cells in 2F8cis tumors in MUC-1 mice. (Fig. 5 A) Flow cytometric analysis of tumor infiltrating CD45+/CD3+/CD8+ T-cells was performed using freshly isolated intraperitoneal 2F8cis tumors from MUC- 1 mice. Mice were given 4 fractions of whole abdomen irradiation (WAI) (6 Gy x 4) in 4 consecutive days and were given LR-IL-22 by gavage at day 2 and day 4 (first radiation dose as day 1). Tumors were collected at day 9 and the single cell suspensions were analyzed. CD45+/CD3+/CD8+ T-cells were quantified using flow cytometry. There was a significant increase in tumor infiltrating CD8+ T-cells in both WAI and WAI + LR-IL-22 groups compared to the tumor only control. WAI + LR-IL-22 treatment further increased the number of tumor- infiltrated CD8+ T-cells compared to the WAI group. (n=2-3 mice; 5-7 tumors per mice; p values were calculated by t-test. (Fig. 5B) Representative immunofluorescent images of 2F8cis excised tumor tissue sections at day 9 showing increased abundance of CD45+, CD8+ cells in the tumors from the WAI treated and WAI + LR-IL-22 treated group compared to tumor only control group. The prevalence of intra-tumoral CD45+, CD8+ cells was further increased in the WAI + LR-IL- 22 group compared to WAI group (n=3). Fluorescence represents co-expression of CD45 and CD8, and DAPI stain represents cell nucleus.

Fig. 6. Improved survival in mice with ovarian cancer by addition of PD-L1 inhibitor to WAI and LR-IL-22. Triangles designate tumor only; circles designate tumor plus 6 Gy X 4, (p=0.0001); squares designate tumor plus 6 Gy X 4 plus LR-IL-22, (/?=0.0003); Upside down triangles designate tumor plus 6 Gy X 4 plus LR-IL-22 plus PD-L1 inhibitor ( <0.0001). Significantly improved survival is achieved with LR-IL-22, and complete survival achieved with combination of PD-L1 inhibitor and LR-IL-22. P values relative to single agent group using a two-sided log rank test (n=12).

Fig 7 : A schematic showing whole abdomen Irradiation (WAI) and LR-IL-22 (LR) each induce PD-L1 expression in OC cells stimulating infiltrating T cells. These modalities are added to PD-L1 immune check point inhibitor and combination chemotherapy to improve survival.

Fig. 8. A schematic showing combination treatment of WAI (6 Gy x 4), LR-IL-22 (109 x 2) and anti-PD-Ll antibody (150 pg x 3). Mice were sacrificed, and excised tumors were quantitated for tumor burden, PD-L1 expression, infiltrating immune cells (T-cells, macrophages, neutrophils, and monocytes) and distribution of CA-MSCs. Mice were followed for survival and late effects on other organs.

Fig. 9. Significant reduction of proinflammatory tumor promoting M2 macrophages in the 2F8cis tumor took place with radiation plus LR-IL-22 gavage and radiation plus LR-IFN-beta treatment.

Fig. 10. LR-IL-22 and/or LR-IFN-0 and WAI treatment increase intra-tumoral CD8 T cells in OC.

Fig. 11. Effects on Lgr5+ cells. 23 mice were irradiated to 12 Gy TBI. Group 1) 0 Gy, 2) Irradiation only, 3) LR, 4) LR-IFN-P, 5) IFN-P protein. Mice were sacrificed at 4 days after irradiation. Mice were injected with BrdU, waited 2 hours, and then sectioned. Tissue was fixed, sections, and observed for GFP and BrdU staining.

Fig. 12A-12AG. Luminex assay for IL-IFN- . 16 C57BL/6 mice were irradiated to 12 Gy TBI. Twenty-four hours later, the mice were divided into 4 groups of 4 mice. Group 1 was irradiation only (LGR IR). Group 2 was irradiated and treated with IFN- protein (25 pg/kg) as I.P. injection (LGR IR + IFN-B). Group 3 was irradiated and gavaged with control IL (10⁹ bacteria) (LGR IR + LR). Group 4 was irradiated and gavaged with IR-IFN-P (IO⁹ bacteria). The control group was no irradiation (LGR Control). Levels of various proteins in intestine (left) and plasma (right) are shown as follows: (A) G-CSF, (B) eotaxin, (C) GM-CSF, (D) IFN-y, (E) IL-la, (F) IL- 1B, (G) IL-2, (H) IL-3, (I) IL-4, (J) IL-5, (K) IL_6, (L) IL-7, (M) IL-9, (N) IL-10, (O) IL-12 (p40), (P) IL-12 (p70), (Q) IL-13, (R) IL-15, (S) IL-17, (T) LIF), (U) LIX, (V) IP-10, (W) KC, (X) MCP- 1, (Y) MIP-la, (Z) MIP-1B, (AA) M-CSF, (AB) MIP-2, (AC) MIG, (AD) RANTES, (AE) VEGF, (AF) TNFa, (AG) TGFB.

DETAILED DESCRIPTION

Interest in immunotherapy for ovarian cancer patients has persisted. There is a direct correlation between an increase in beneficial intra-tumoral T cells and both delayed tumor recurrence and increased survival. The data herein present an approach to facilitate immunotherapy. Disclosed herein is that a genetically engineered gastrointestinal tract (GI) bacterium (e.g., Lactobacillus rente ri). which releases interleukin-22 (e.g., LR-IL-22), is an intestinal tissue radioprotector. Furthermore, both LR-IL-22 and ionizing irradiation, each induce the PD-L1 immunotherapy target in OC cells, and the combination of radiation and LR-IL-22 increases numbers of infiltrating CD8+ T-cells and other beneficial immunocytes in OC tumors. Accordingly, in some aspects, disclosed herein are compositions and methods for treating a cancer in a subject in need thereof by administering to the subject a therapeutically effective amount of an engineered gastrointestinal tract (GI) bacterium, wherein the bacterium comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN-P, or a functional fragment thereof. In some embodiments, the method further comprises applying to the subject an irradiation therapy (such as total body irradiation or abdominal irradiation). It is a surprising finding of the present invention that bacteria harboring the aforesaid vectors alone or in combination with the irradiation therapy can induce increased levels of immune checkpoint molecules on tumor cells (such as PD-L1 and/or PD-L2) and/or improve infiltration of immune cells (e.g., CD8+ T cells) into tumors. The increased levels of immune checkpoint molecules on tumors may render subjects who were resistant to immune checkpoint therapy sensitive to the therapy. Accordingly, in some embodiments, the method of any preceding aspect further comprises administrating to the subject a therapeutically effective amount of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is a PD-L1 inhibitor or a PD-L2 inhibitor.

In some embodiments, the GI bacterium is Lactobacillus subtilus, Lactobacillus reuteri, or Escherichia coli. In some embodiments, the GI bacterium secrets IL-22 and/or IFN- , or a functional fragment thereof. In some embodiments, the GI bacterium is administered to the subject via an oral administration.

Terms used throughout this application are to be construed with ordinary and typical meaning to those of ordinary skill in the art. However, Applicants desire that the following terms be given the particular definition as provided below.

Terminology

As used in the specification and claims, the singular form "a," "an," and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a cell" includes a plurality of cells, including mixtures thereof.

The term “about” as used herein when referring to a measurable value such as an amount, a percentage, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, or ±1% from the measurable value.

“Administration” to a subject or “administering” includes any route of introducing or delivering to a subject an agent. Administration can be carried out by any suitable route, including oral, intravenous, intraperitoneal, and the like. Administration includes self-administration and the administration by another. In some embodiments, the administration of a GI bacterium to a subject is done via oral administration. The term "cancer" as used herein is defined as disease characterized by the rapid and uncontrolled growth of aberrant cells. Cancer cells can spread locally or through the bloodstream and lymphatic system to other parts of the body.

The term “cancer cells” and “tumor cells” are used interchangeably to refer to cells derived from a cancer or a tumor, or from a tumor cell line or a tumor cell culture.

As used herein, the term “carrier” encompasses any excipient, diluent, filler, salt, buffer, stabilizer, solubilizer, lipid, stabilizer, or other material well known in the art for use in pharmaceutical formulations. The choice of a carrier for use in a composition will depend upon the intended route of administration for the composition. The preparation of pharmaceutically acceptable carriers and formulations containing these materials is described in, e.g., Remington's Pharmaceutical Sciences, 21st Edition, ed. University of the Sciences in Philadelphia, Lippincott, Williams & Wilkins, Philadelphia, PA, 2005. Examples of physiologically acceptable carriers include saline, glycerol, DMSO, buffers such as phosphate buffers, citrate buffer, and buffers with other organic acids; antioxidants including ascorbic acid; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such as TWEEN (ICI, Inc.; Bridgewater, New Jersey), polyethylene glycol (PEG), and PLURONICS (BASF; Florham Park, NJ). To provide for the administration of such dosages for the desired therapeutic treatment, compositions disclosed herein can advantageously comprise between about 0.1% and 99% by weight of the total of one or more of the subject compounds based on the weight of the total composition including carrier or diluent.

A “Colony Forming Unit (CFU)” is a unit of measure of the number of viable microorganisms such as bacteria, fungi, or viruses in a sample or population.

The term “comprising” and variations thereof as used herein is used synonymously with the term “including” and variations thereof and are open, non-limiting terms. Although the terms “comprising” and “including” have been used herein to describe various embodiments, the terms “consisting essentially of’ and “consisting of” can be used in place of “comprising” and “including” to provide for more specific embodiments and are also disclosed.

“Composition” refers to any agent that has a beneficial biological effect. Beneficial biological effects include both therapeutic effects, e.g., treatment of a disorder or other undesirable physiological condition, and prophylactic effects, e.g., prevention of a disorder or other undesirable physiological condition (e.g., a radiation-induced intestinal damage). The terms also encompass pharmaceutically acceptable, pharmacologically active derivatives of beneficial agents specifically mentioned herein, including, but not limited to, a bacterium, a vector, polynucleotide, cells, salts, esters, amides, proagents, active metabolites, isomers, fragments, analogs, and the like. When the terms “composition” is used, then, or when a particular composition is specifically identified, it is to be understood that the term includes the composition per se as well as pharmaceutically acceptable, pharmacologically active vector, polynucleotide, salts, esters, amides, proagents, conjugates, active metabolites, isomers, fragments, analogs, etc. In some aspects, composition disclosed herein comprises a gastrointestinal tract (GI) bacterium, wherein the bacterium comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN-P, or a functional fragment thereof.

A "control" is an alternative subject or sample used in an experiment for comparison purposes. A control can be "positive" or "negative."

A "decrease" can refer to any change that results in a smaller amount of a symptom, disease, composition, condition, or activity. A substance is also understood to decrease the genetic output of a gene when the genetic output of the gene product with the substance is less relative to the output of the gene product without the substance. Also, for example, a decrease can be a change in the symptoms of a disorder such that the symptoms are less than previously observed. A decrease can be any individual, median, or average decrease in a condition, symptom, activity, composition in a statistically significant amount. Thus, the decrease can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% decrease so long as the decrease is statistically significant.

The term "dosage" refers to a specific amount, number, and frequency of doses over a specific period of time. And as used herein, a “dose” refers to a specified amount of medication taken at one specific time.

“Effective amount” encompasses, without limitation, an amount that can ameliorate, reverse, mitigate, prevent, or diagnose a symptom or sign of a medical condition or disorder (e.g., a cancer). Unless dictated otherwise, explicitly or by context, an “effective amount” is not limited to a minimal amount sufficient to ameliorate a condition. The severity of a disease or disorder, as well as the ability of a treatment to prevent, treat, or mitigate, the disease or disorder can be measured, without implying any limitation, by a biomarker or by a clinical parameter. The term “effective amount of a GI bacterium” refers to an amount of a GI bacterium sufficient to cause some mitigation of cancer, decrease of tumor size, prevention of relapse, improvement of survival, and/or related symptoms. The term “engineered” refers to being modified from its natural state by manipulation of genetic material.

Herein, “expression” means generation of mRNA by transcription from nucleic acids such as genes, polynucleotides, and oligonucleotides, or generation of a protein or a polypeptide by transcription from mRNA. “Suppression of expression” refers to a decrease of a transcription product or a translation product in a significant amount as compared with the case of no suppression. “Increased expression” refers to an increase of a transcription product or a translation product in a significant amount as compared with the case of no increase.

The “functional fragment,” whether attached to other sequences or not, can include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acids residues, provided the activity of the fragment is not significantly altered or impaired compared to the nonmodified peptide or protein. These modifications can provide for some additional property, such as to remove or add amino acids capable of disulfide bonding, to increase its bio-longevity, to alter its secretory characteristics, etc.

The term “gastrointestinal tract” (also referred to as “GI tract” or “digestive tract”), is a series of hollow organs joined in a long, twisting tube from the mouth to the anus. The hollow organs that make up the GI tract are the mouth, esophagus, stomach, small intestine, large intestine, and anus. The small intestine has three parts - duodenum, jejunum, and ileum. The large intestine includes the appendix, cecum, colon, and rectum. However, the term “gastrointestinal tract bacterium” or “GI bacterium” is used herein to refer to a bacterium that resides in the stomach, small intestine or large intestine, such as colon, following administration (e.g., oral administration). In some embodiments the GI bacterium localizes in the stomach, small intestine or colon. In some embodiments, the GI bacterium is selected from an Escherichia genus or a Lactobacillus genus. In some embodiments, the GI bacterium is Lactobacillus sublilus. In some embodiments, the GI bacterium is Lactobacillus reuteri. In some embodiments, the GI bacterium is Escherichia coli.

The term "gene" or "gene sequence" refers to the coding sequence or control sequence, or fragments thereof. A gene may include any combination of coding sequence and control sequence, or fragments thereof. Thus, a "gene" as referred to herein may be all or part of a native gene. A polynucleotide sequence as referred to herein may be used interchangeably with the term "gene”, or may include any coding sequence, non-coding sequence or control sequence, fragments thereof, and combinations thereof. The term "gene" or "gene sequence" includes, for example, control sequences upstream of the coding sequence (for example, the ribosome binding site). An “immune checkpoint molecule” is expressed on the surface of immune cell such as a T-cell that, when bound by a binding partner protein, can reduce T-cell activity. As one example, PD1 is an immune checkpoint molecule expressed on some T cells, and when bound by PD-L1 expressed on a cancer cell, results in a decreased in the T cell’s activity.

An "increase" can refer to any change that results in a greater amount of a symptom, disease, composition, condition or activity. An increase can be any individual, median, or average increase in a condition, symptom, activity, composition in a statistically significant amount. Thus, the increase can be a 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% increase so long as the increase is statistically significant.

An “intestinal damage”, as used herein, refers to a disruption of the homeostasis of any tissue (epithelial, connective, nervous or muscle) of any organ or compartment of the gastrointestinal tract of a mammal. The intestinal damage can be a consequence of an endogenous disruption of the homeostasis of any tissue of the gastrointestinal tract, such as a cancer. Alternatively, or additionally, the intestinal damage can be a consequence of an exogenous disruption of the homeostasis of any tissue of the gastrointestinal tract.

"Inhibit," "inhibiting," and "inhibition" mean to decrease an activity, response, condition, disease, or other biological parameter. This can include but is not limited to the complete ablation of the activity, response, condition, or disease. This may also include, for example, a 10% reduction in the activity, response, condition, or disease as compared to the native or control level. Thus, the reduction can be a 10, 20, 30, 40, 50, 60, 70, 80, 90, 100%, or any amount of reduction in between as compared to native or control levels.

“Inhibitors” of expression or of activity are used to refer to inhibitory molecules, respectively, identified using in vitro and in vivo assays for expression or activity of a described target protein, e.g., ligands, antagonists, and their homologs and mimetics. Inhibitors are agents that, e.g., inhibit expression or bind to, partially or totally block stimulation or protease activity, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of the described target protein, e.g., antagonists. A control sample (untreated with inhibitors) are assigned a relative activity value of 100%. Inhibition of a described target protein is achieved when the activity value relative to the control is about 80%, optionally 50% or 25, 10%, 5% or 1%.

“Irradiation-induced intestinal damage” used herein refers to a disruption of the homeostasis of any tissue (epithelial, connective, nervous or muscle) of any organ or compartment of the gastrointestinal tract due to irradiation, for example, due to irradiation therapy, nuclear reactor accident or irradiation terrorist event. As used herein, the terms “localize” and “localizes” mean to move to, reside, or restrict to, a particular place, locality.

As used herein, the term “metastasis” is meant to refer to the process in which cancer cells originating in one organ or part of the body, with or without transit by a body fluid, and relocate to another part of the body and continue to replicate. Metastasized cells can subsequently form tumors which may further metastasize. Metastasis thus refers to the spread of cancer, from the part of the body where it originally occurred, to other parts of the body.

The term "nucleic acid" as used herein means a polymer composed of nucleotides, e.g. deoxyribonucleotides (DNA) or ribonucleotides (RNA). The terms "ribonucleic acid" and "RNA" as used herein mean a polymer composed of ribonucleotides. The terms "deoxyribonucleic acid" and "DNA" as used herein mean a polymer composed of deoxyribonucleotides.

The terms "percent identity", “identity” and “identical to” shall be construed to mean the percentage of nucleotide bases or amino acid residues in the candidate sequence that are identical with the bases or residues of a corresponding sequence to which it is compared, after aligning the sequences and introducing gaps, if necessary to achieve the maximum percent identity for the entire sequence, and not considering any conservative substitutions as part of the sequence identity. Neither N- nor C-terminal extensions nor insertions shall be construed as reducing identity. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) that has a certain percentage (for example, 80%, 85%, 90%, or 95%) of "sequence identity" to another sequence means that, when aligned over their full lengths, that percentage of bases (or amino acids) are the same in comparing the two sequences. This alignment and the percent sequence identity can be determined using software programs known in the art. In one embodiment, default parameters are used for alignment. In one embodiment a BLAST program is used with default parameters. In one embodiment, BLAST programs BLASTN and BLASTP are used with the following default parameters: Genetic code=standard; filter=none; strand=both; cutoff=60; expect=10; Matrix=BLOSUM62; Descriptions=50 sequences; sort by=HIGH SCORE; Databases=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translations+SwissProtein+SPupdate+PIR.

"Pharmaceutically acceptable carrier" (sometimes referred to as a “carrier”) means a carrier or excipient that is useful in preparing a pharmaceutical or therapeutic composition that is generally safe and non-toxic, and includes a carrier that is acceptable for veterinary and/or human pharmaceutical or therapeutic use. The terms "carrier" or "pharmaceutically acceptable carrier" can include, but are not limited to, phosphate buffered saline solution, water, emulsions (such as an oil/water or water/oil emulsion) and/or various types of wetting agents. The term "polynucleotide" refers to a single or double stranded polymer composed of nucleotide monomers.

The term "polypeptide" refers to a compound made up of a single chain of D- or L-amino acids or a mixture of D- and L-amino acids joined by peptide bonds.

The term "promoter" or "regulatory element" refers to a region or sequence determinants located upstream or downstream from the start of transcription and which are involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. Promoters need not be of bacterial origin, for example, promoters derived from viruses or from other organisms can be used in the compositions, systems, or methods described herein.

“Recombinant” used in reference to a gene refers herein to a sequence of nucleic acids that are not naturally occurring in the genome of the bacterium. The non-naturally occurring sequence may include a recombination, substitution, deletion, or addition of one or more bases with respect to the nucleic acid sequence originally present in the natural genome of the bacterium.

As used herein, “resistant or “resistance” refers to a cancer for which the effectiveness of an anti-cancer agent is reduced as compared to a control cancer.

The term “subject” is defined herein to include animals such as mammals, including, but not limited to, primates (e.g., humans), cows, sheep, goats, horses, dogs, cats, rabbits, rats, mice and the like. In some embodiments, the subject is a human.

A “T cell” refers to a type of lymphocyte that expresses a T-cell receptor (TCR) on its surface. T cells include, but are not limited to, two major subtypes: CD8⁺ T cells and CD4⁺ T cells.

“Therapeutically effective amount” refers to the amount of a compound such as a GI bacterium comprising a vector encoding IL-22 and/or INF- that will elicit the biological or medical response of a tissue, system, animal, or human that is being sought by the researcher, veterinarian, medical doctor, or other clinician over a generalized period of time. In some embodiments, a desired response is improvement of survival. In other embodiments, a desired response is reduction of tumor size and/or mitigation of metastasis. In some embodiments, a desired response is increased infiltration of immune cells (e.g., CD8+ T cell) into tumors or a decrease in number of M2 macrophages. In some embodiments, a desired response is prevention of relapse. In some instances, a desired biological or medical response is achieved following administration of multiple dosages of the composition to the subject over a period of days, weeks, or years. The therapeutically effective amount will vary depending on the compound, the disorder or conditions and its severity, the route of administration, time of administration, rate of excretion, drug combination, judgment of the treating physician, dosage form, and the age, weight, general health, sex and/or diet of the subject to be treated.

The therapeutically effective amount of the GI bacterium comprising a vector encoding IL-22 and/or INF-0 compositions described herein can be determined by one of ordinary skill in the art and includes exemplary dosage amounts for a mammal of from about 0.5 to about 200 mg/kg of body weight of active composition per day, which can be administered in a single dose or in the form of individual divided doses, such as from 1 to 4 times per day. Alternatively, the dosage amount can be from about 0.5 to about 150 mg/kg of body weight of active composition per day, about 0.5 to 100 mg/kg of body weight of active compound per day, about 0.5 to about 75 mg/kg of body weight of active compound per day, about 0.5 to about 50 mg/kg of body weight of active composition per day, about 0.5 to about 25 mg/kg of body weight of active composition per day, about 1 to about 20 mg/kg of body weight of active composition per day, about 1 to about 10 mg/kg of body weight of active composition per day, about 20 mg/kg of body weight of active composition per day, about 10 mg/kg of body weight of active composition per day, or about 5 mg/kg of body weight of active composition per day.

The terms “treat,” “treating,” “treatment,” and grammatical variations thereof as used herein, include partially or completely delaying, alleviating, mitigating or reducing the intensity of one or more attendant symptoms of a disorder or condition and/or alleviating, mitigating or impeding one or more causes of a disorder or condition. Treatments according to the invention may be applied preventively, prophylactically, pallatively or remedially. Prophylactic treatments are administered to a subject prior to onset (e.g., before obvious signs of cancer), during early onset (e.g. , upon initial signs and symptoms of cancer), after an established development of cancer. Prophylactic administration can occur for several minutes to months prior to the manifestation of cancer.

In some instances, the terms “treat,” “treating,” “treatment,” and grammatical variations thereof, include mitigating a cancer, and/or related symptoms or restoration of intestinal function in a subject as compared with prior to treatment of the subject or as compared with incidence of such symptom in a general or study population.

"Vector" used herein means, in respect to a nucleic acid sequence, a nucleic acid sequence comprising a regulatory nucleic acid sequence that controls the replication of an expressible gene. A vector may be either a self-replicating, extrachromosomal vector or a vector which integrates into a host genome. Alternatively, a vector may also be a vehicle comprising the aforementioned nucleic acid sequence. A vector may be a plasmid, bacteriophage (isolated, attenuated, recombinant, etc.). A vector may comprise a double-stranded or single-stranded DNA, RNA, or hybrid DNA/RNA sequence comprising double-stranded and/or single- stranded nucleotides. In some embodiments, the vector is a plasmid.

Compositions

Disclosed herein are compositions for use in the treatment of a cancer in a subject comprising administering to the subject a therapeutically effective amount of a gastrointestinal tract (GI) bacterium, wherein the bacterium comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN-P, or a functional fragment thereof.

“IL-22” refers herein to a polypeptide that, in humans, is encoded by the IL22 gene. IL- 22 is a cytokine that is involved in mediating cellular inflammatory responses. In some embodiments, the IL-22 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 14900, Entrez Gene: 50616, Ensembl: ENSG00000127318, OMIM: 605330, UniProtKB: Q9GZX6. In some embodiments, the IL-22 polypeptide comprises the sequence of SEQ ID NO: 1, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 1, or a polypeptide comprising a portion of SEQ ID NO: 1. The IL-22 polypeptide of SEQ ID NO:1 may represent an immature or pre- processed form of mature IL-22, and accordingly, included herein are mature or processed portions of the IL-22 polypeptide in SEQ ID NO: 1. In some embodiments, the IL-22 polynucleotide comprises the sequence of SEQ ID NO: 2, or a polynucleotide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 2, or a polynucleotide comprising a portion of SEQ ID NO: 2. In other embodiments, the IL-22 polynucleotide or polypeptide is that described in U.S. Patent No. 10,376,563, which is incorporated by reference in its entirety, or a polynucleotide or polynucleotide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with and IL- 22 polynucleotide or polypeptide disclosed therein.

“IFN-P” refers herein to a polypeptide that, in humans, is encoded by the IFNB1 gene. IFN- is a type 1 interferon that has anti-viral, antibacterial and anticancer activities. IFN-P binds to the type 1 IFN receptor. In some embodiments, the IFN-P polypeptide is that identified in one or more publicly available databases as follows: HGNC: 5434, Entrez Gene: 3456, Ensembl: ENSG0000017185,5 OMIM: 147640, UniProtKB: P01574. In some embodiments, the IFN-P polypeptide comprises the sequence of SEQ ID NO: 3, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 3, or a polypeptide comprising a portion of SEQ ID NO: 3. The IFN-P polypeptide of SEQ ID NO: 3 may represent an immature or pre-processed form of mature IFN-P, and accordingly, included herein are mature or processed portions of the IFN-P polypeptide in SEQ ID NO: 3. In some embodiments, the IFN- polynucleotide comprises the sequence of SEQ ID NO: 4, or a polynucleotide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 4, or a polynucleotide comprising a portion of SEQ ID NO: 4.

As noted above, the vectors described herein can be a nucleic acid sequence comprising a regulatory nucleic acid sequence that controls the replication of an expressible gene. In some embodiments, the IL-22 vector comprises a sequence at least 80% (for examples, at least 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 5. In some embodiments, the IFN-P vector comprises a sequence at least 80% (for examples, at least 85%, 90%, 95%, 98%, or 99%) identical to SEQ ID NO: 6.

In some examples, the engineered GI bacterium disclosed herein can secrete a polypeptide, such as IL-22 and/or IFN-P, or a functional fragment thereof. “Secretion,” as used herein, refers to a process by which substances are produced and released from a live bacterial cell. The bacterium and/or vector may be engineered to produce and release the polypeptide. As used herein, “release” used with respect to the bacterium releasing the polypeptide refers to disposing the polypeptide outside the bacterium upon lysis of the bacterium.

In some embodiments, the engineered GI bacterium is that described in International Publication No. WO2021/087195A1, all of which is incorporated by reference in its entirety.

Accordingly, included herein are GI bacterium comprising the herein described vectors that encode IL-22 and/or IFN-P, or a functional fragment thereof. As noted above, the term “gastrointestinal tract bacterium” or “GI bacterium” refers to a bacterium that resides in the stomach, small intestine or large intestine. The present invention therefore includes a GI bacterium comprising a herein described vector that resides or localizes in the small intestine and/or the large intestine following administration (e.g., oral administration). In some embodiments, the GI bacterium resides or localizes in the duodenum, jejunum, or ileum of the small intestine. Accordingly, included herein are GI bacteria that reside or localize in the duodenum. Included herein are GI bacteria that reside or localize in the jejunum. Included herein are GI bacteria that reside or localize in the ileum. In some embodiments, the GI bacterium resides or localizes in the colon. Accordingly, included herein are GI bacteria that reside or localize in the ascending colon. Included herein are GI bacteria that reside or localize in the transverse colon. Included herein are GI bacteria that reside or localize in the descending colon. Included herein are GI bacteria that reside or localize in the sigmoid colon. Exemplary GI bacteria include species of the lactic acid bacteria group, which are an order of gram-positive, low-GC, acid-tolerant, generally nonsporulating, nonrespiring, either rodshaped (bacilli) or spherical (cocci) bacteria that share common metabolic and physiological characteristics. Representative homolactic lactic acid bacteria genera comprises Lactococcus, Enterococcus, Streptococcus, Pediococcus, and group I lactobacilli. Lactobacillus is known to localize in the jejunum and ilium of the small intestine. Therefore, in some embodiments, the GI bacterium is a species of a Lactobacillus genus. In some embodiments, the GI bacterium may include species of lactic acid bacteria other than species of a Lactococcus genus. In some embodiments, the GI bacterium is a species of a Lactococcus genus.

Exemplary species from the Lactobacillus genus include L. acetoto terans, L. acidifarinae, L. acidipiscis, L. acidophilus, L. agilis, L. algidus, L. atimentarius, L. amytolyticus, L. amylophilus, L. amylotrophicus, L. amylovorus, L. animatis, L. antri, L. apodemi, L. aviarius, L. bifermentans, L. brevis, L. buchneri, L. camelliae, L. casei, L. catenaformis, L. ceti, L. coleohominis, L. collinoides, L. composti, L. concavus, L. coryniformis, L. crispatus, L. crustorum, L. curvatus, L. delbrueckii subsp. delbrueckii, L. delbrueckii subsp. butgaricus, L. delbrueckii subsp. lactis, L. dextrinicus, L. diolivorans, L. equi, L. equigenerosi, L. farraginis, L. farciminis, L. fermentum, L. fornicalis, L. fructivorans, L. frumenti, L. fuchuensis, L. gallinarum, L. gasseri, L. gastricus, L. ghanensis, L. graminis, L. hammesii, L. hamsteri, L. harbinensis, L. hayakitensis, L. helveticus, L. hitgardii, L. homohiochii, L. iners, L. ingluviei, L. intestinalis, L. jensenii, L. johnsonii, L. katixensis, L. kefiranofaciens, L. kefiri, L. kimchii, L. kitasatonis, L. kunkeei, L. leichmannii, L. lindneri, L. malefermentans, L. mati, L. manihotivorans, L. mindensis, L. mucosae, L. murinus, L. nagelii, L. namurensis, L. nantensis, L. oligofermentans, L. oris, L. panis, L. pantheris, L. parabrevis, L. parabuchneri, L. paracollinoides, L. parafarraginis, L. parakejiri, L. paralimenlarius, L. paraplanlarum, L. pentosus, L. perolens, L. plantarum, L. pontis, L. psittaci, L. rennini, L. reuteri, L. rhamnosus, L. rimae, L. rogosae, L. rossiae, L. ruminis, L. saerimneri, L. sakei, L. salivarius, L. sanfranciscensis, L. satsumensis, L. secaliphilus, L. sharpeae, L. siliginis, L. spicheri, L. suebicus, L. subtilus, L. thailandensis, L. ultunensis, L. vaccinostercus, L. vaginalis, L. versmoldensis, L. vini, L. vitulinus, L. ~eae. and L. zyniae. In some embodiments, the GI bacterium used in the method of treating radiation- induced intestinal damage of any aspects disclosed herein is L. subtilus. In some embodiments, the GI bacterium used in the method of treating irradiation-induced intestinal damage of any aspects disclosed herein is L. reuteri.

In some embodiments, exemplary GI bacteria include species of the Escherichia genus, which is a genus of gram-negative, non-spore-forming, facultatively anaerobic, rod-shaped bacteria from the family Enterobacteriaceae . Escherichia genus comprises E. albertii, E. coli, E. fergusonii, E. hermannii, E. marmotae, and E. vulneris. E. coli is shown to localize in the colon. Therefore, in some embodiments, the GI bacterium is E. coli. Accordingly, the GI bacterium used in the method of treating irradiation-induced intestinal damage of any aspects disclosed herein can be E. coli. In some embodiments, the GI bacterium used in the method of treating irradiation- induced intestinal damage of any aspects disclosed herein can be a species of Escherichia genus other than E. coli, such as E. albertii, E. fergusonii, E. hermannii, E. marmotae, or E. vulneris.

The compositions of the present invention may take any form, and in some embodiments, take the form of tablet, pill or capsule for oral administration.

Methods of Treatment

The current disclosure demonstrates the surprising finding that an engineered GI bacterium (e.g., Lactobacillus subtilus, Lactobacillus reuteri, or E. coli) that comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN-f>, or a functional fragment thereof, alone or in combination with an immune checkpoint therapy and/or an irradiation therapy is effective to treat cancer.

Therefore, is some aspects, provided herein are methods of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of an engineered gastrointestinal tract (GI) bacterium, wherein the engineered gastrointestinal tract (GI) bacterium comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN-P, or a functional fragment thereof. The engineered GI bacterium, vectors, and IL-22 and/or IFN- compositions used in these methods can be any of those described above or below. In some embodiments, the vector comprises a polynucleotide that encodes IL-22 or a functional fragment thereof. In some embodiments, the vector comprises a polynucleotide that encodes IFN-P or a functional fragment thereof. In some embodiments, the vector comprises a polynucleotide that encodes both IL-22 and IFN-P, or a functional fragment thereof. In some embodiments, the engineered GI bacterium is a specie of Escherichia genus or Lactobacillus genus.

As noted above, the GI bacterium used herein preferably resides or localizes in the small intestine and/or large intestine, such as colon, following administration (e.g., oral administration). In some embodiments, the GI bacterium is Escherichia coli. In some embodiments, the GI bacterium can be the species of a genus selected from the group consisting of Escherichia genus, Lactobacillus genus, Roseburian genus, and/or Akkermansia genus. In some embodiments, the GI bacterium is a species of Escherichia genus or Lactobacillus genus. In some embodiments, the GI bacterium is Lactobacillus subtilus. In some embodiments, the GI bacterium is Lactobacillus reuteri. In some embodiments, the GI bacterium is E. coli. In some embodiments, the method of any preceding aspect further comprises administrating to the subject a therapeutically effective amount of an immune checkpoint inhibitor. In some embodiments, the immune checkpoint inhibitor is selected from the group consisting of a PD-L1 inhibitor, PD-L2 inhibitor, a PD-1 inhibitor, a CTLA-4 inhibitor, a TIM-3 inhibitor, a LAG3 inhibitor, a TIGIT inhibitor, a PVR inhibitor, a PVRL2 inhibitor, a CD47/SIRPa inhibitor, a CSF1 inhibitor, or a CSF1R inhibitor). It should be understood that various compositions can be introduced into a subject simultaneously or sequentially.

As noted above, it is a surprising finding of the present invention that bacteria harboring the aforesaid vectors alone or in combination with the irradiation therapy can induce increased levels of immune checkpoint molecule on tumor cells and/or improve infiltration of immune cells (e.g., CD8+ T cells) into tumors. The increased levels of immune checkpoint molecules on tumors can render subjects who were resistant to an immune checkpoint therapy sensitive to the therapy. It should be understood and herein contemplated that a cancer resistant to a therapy of immune checkpoint inhibitor refers to a cancer that can evade or survive in the presence of the immune checkpoint inhibitors, which can be attributable in part to low target antigen expression on a cancer cell. The cancer may be resistant at the beginning of treatment, or it may become resistant during treatment. Accordingly, in some embodiments, the subject in need is resistant to a therapy of immune checkpoint inhibitor. In some embodiments, the treatment results in an increased level of an immune checkpoint molecule (such as PD-L1, PD-L2, CD47, colony stimulating factor 1 (CSF1), poliovirus Receptor (PVR), and/or poliovirus receptor-related 2 (PVRL2)) on a tumor cell relative to a control. In some embodiments, the treatment results in an increased level of an immune checkpoint molecule (such as PD-1, SIRPa, CTLA-4, TIM-3, TIGIT, LAG3, or CSF1R) on immune cells (e.g., T cells, NK cells, or macrophages)

“PD-L1” refers herein to a polypeptide that, in humans, is encoded by the CD274 gene. PD-L1, is a ligand that binds with the receptor PD-1, commonly found on T cells and NK cells, and acts to block T cell and NK cell activation. In some embodiments, the PD-L1 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 17635, NCBI Entrez Gene: 29126, Ensembl: ENSG00000120217, OMIM: 605402, UniProtKB/Swiss-Prot: Q9NZQ7. In some embodiments, the PD-L1 polypeptide comprises the sequence of SEQ ID NO: 7, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 7, or a polypeptide comprising a portion of SEQ ID NO: 7. The PD-L1 polypeptide of SEQ ID NO: 7 may represent an immature or pre-processed form of mature PD-L1, and accordingly, included herein are mature or processed portions of the PD-L1 polypeptide in SEQ ID NO: 7. “PD-L2” refers herein to a polypeptide that, in humans, is encoded by the PDCD1LG2 gene. PD-L2 is known as being involved in negative regulation of activated T cell proliferation; negative regulation of interferon-gamma production from immune cells, such as T cells and NK cells. In some embodiments, the PD-L2 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 18731, NCBI Entrez Gene: 80380, Ensembl: ENSG00000197646, OMIM: 605723, UniProtKB/Swiss-Prot: Q9BQ51. In some embodiments, the PD-L2 polypeptide comprises the sequence of SEQ ID NO: 8, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 8, or a polypeptide comprising a portion of SEQ ID NO: 8. The PD-L2 polypeptide of SEQ ID NO: 8 may represent an immature or pre-processed form of mature PD- L2, and accordingly, included herein are mature or processed portions of the PD-L2 polypeptide in SEQ ID NO: 8.

“PD-1” refers herein to a polypeptide that, in humans, is encoded by the PDCD1 gene. In some embodiments, the PD-1 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 8760 NCBI Entrez Gene: 5133 Ensembl: ENSG00000188389 OMIM: 600244 UniProtKB/Swiss-Prot: Q15116. In some embodiments, the PD-1 polypeptide comprises the sequence of SEQ ID NO: 9, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 9, or a polypeptide comprising a portion of SEQ ID NO: 9. The PD-1 polypeptide of SEQ ID NO: 9 may represent an immature or pre-processed form of mature PD-1, and accordingly, included herein are mature or processed portions of the PD-1 polypeptide in SEQ ID NO: 9.

“CTLA-4” refers herein to a polypeptide that, in humans, is encoded by the CTLA4 gene. In some embodiments, the CTLA-4 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 2505, NCBI Entrez Gene: 1493, Ensembl: ENSG00000163599, OMIM: 123890, UniProtKB/Swiss-Prot: P16410. In some embodiments, the CTLA-4 polypeptide comprises the sequence of SEQ ID NO: 10, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 10, or a polypeptide comprising a portion of SEQ ID NO: 10. The CTLA-4 polypeptide of SEQ ID NO: 10 may represent an immature or pre-processed form of mature CTLA-4, and accordingly, included herein are mature or processed portions of the CTLA-4 polypeptide in SEQ ID NO: 10.

“T-cell immunoglobulin and mucin-domain containing-3 (TIM-3)” refers herein to a polypeptide that, in humans, is encoded by the HAVCR2 gene. In some embodiments, the TIM- 3 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 18437, NCBI Entrez Gene: 84868, Ensembl: ENSG00000135077, OMIM: 606652, UniProtKB/Swiss-Prot: Q8TDQ0. In some embodiments, the TIM-3 polypeptide comprises the sequence of SEQ ID NO: 11, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 11, or a polypeptide comprising a portion of SEQ ID NO: 11. The TIM-3 polypeptide of SEQ ID NO: 11 may represent an immature or pre-processed form of mature TIM-3, and accordingly, included herein are mature or processed portions of the TIM-3 polypeptide in SEQ ID NO: 11.

T cell immunoreceptor with Ig and HIM domain (TIGIT) is an immune checkpoint receptor on T cells and NK cells. On cytotoxic T cells and NK cells, interaction of TIGIT with either of its two ligands, PVR (also known as CD 155) and PVRL2, also known as nectin-2 and CD112, suppresses immune activation.

“TIGIT” refers herein to a polypeptide that, in humans, is encoded by the TIGIT gene. In some embodiments, the TIGIT polypeptide is that identified in one or more publicly available databases as follows: HGNC: 26838, NCBI Entrez Gene: 201633, Ensembl: ENSG00000181847, OMIM: 612859, UniProtKB/Swiss-Prot: Q495A1. In some embodiments, the TIGIT polypeptide comprises the sequence of SEQ ID NO: 12, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 12, or a polypeptide comprising a portion of SEQ ID NO: 12. The TIGIT polypeptide of SEQ ID NO: 12 may represent an immature or pre-processed form of mature TIGIT, and accordingly, included herein are mature or processed portions of the TIGIT polypeptide in SEQ ID NO: 12.

“Poliovirus Receptor (PVR)”, also known as CD 155, refers herein to a polypeptide that, in humans, is encoded by the PVR gene. In some embodiments, the PVR polypeptide is that identified in one or more publicly available databases as follows: HGNC: 9705, NCBI Entrez Gene: 5817, Ensembl: ENSG00000073008, OMIM: 173850, UniProtKB/Swiss-Prot: P15151. In some embodiments, the PVR polypeptide comprises the sequence of SEQ ID NO: 9, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 13, or a polypeptide comprising a portion of SEQ ID NO: 13. The PVR polypeptide of SEQ ID NO: 13 may represent an immature or pre-processed form of mature PVR, and accordingly, included herein are mature or processed portions of the PVR polypeptide in SEQ ID NO: 13.

“Poliovirus receptor-related 2 (PVRL2)”, also known as nectin-2 and CD 112 (formerly herpesvirus entry mediator B, HVEB), is a human plasma membrane glycoprotein. PVRL2 refers herein to a polypeptide that, in humans, is encoded by the NECTIN2 gene. In some embodiments, the PVRL2 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 9707 NCBI Entrez Gene: 5819, Ensembl: ENSG00000130202, OMIM: 600798, UniProtKB/Swiss-Prot: Q92692. In some embodiments, the PVRL2 polypeptide comprises the sequence of SEQ ID NO: 14, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 14, or a polypeptide comprising a portion of SEQ ID NO: 14. The PVRL2 polypeptide of SEQ ID NO: 14 may represent an immature or pre-processed form of mature PVRL2, and accordingly, included herein are mature or processed portions of the PVRL2 polypeptide in SEQ ID NO: 14.

“Lymphocyte activating 3 (LAG3)” refers herein to a polypeptide that, in humans, is encoded by the LAG3 gene. In some embodiments, the LAG3 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 6476, NCBI Entrez Gene: 3902, Ensembl: ENSG00000089692, OMIM: 153337, UniProtKB/Swiss-Prot: P18627. In some embodiments, the LAG3 polypeptide comprises the sequence of SEQ ID NO: 15, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 15, or a polypeptide comprising a portion of SEQ ID NO: 15. The LAG3 polypeptide of SEQ ID NO: 15 may represent an immature or pre-processed form of mature LAG3, and accordingly, included herein are mature or processed portions of the LAG3 polypeptide in SEQ ID NO: 15.

CD47 (Cluster of Differentiation 47) also known as integrin associated protein (IAP) is a transmembrane protein that in humans is encoded by the CD47 gene. CD47, a “don't eat me” signal for phagocytic cells, is expressed on the surface of tumor cells. CD47 functions as a ligand for signal regulatory protein-a (SIRPa), a protein expressed on immune cells such as macrophages and dendritic cells. Upon binding CD47, SIRPa initiates a signaling cascade that results in the inhibition of phagocytosis. In some embodiments, the CD47 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 6476, NCBI Entrez Gene: 3902, Ensembl: ENSG00000089692, OMIM: 153337, UniProtKB/Swiss-Prot: P18627. In some embodiments, the CD47 polypeptide comprises the sequence of SEQ ID NO: 16, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 16, or a polypeptide comprising a portion of SEQ ID NO: 16. The CD47 polypeptide of SEQ ID NO: 16 may represent an immature or pre-processed form of mature CD47, and accordingly, included herein are mature or processed portions of the CD47 polypeptide in SEQ ID NO: 16.

“Signal regulatory protein-a (SIRPa)” refers herein to a polypeptide that, in humans, is encoded by the SIRPA gene. In some embodiments, the SIRPa polypeptide is that identified in one or more publicly available databases as follows: HGNC: 9662, NCBI Entrez Gene: 140885, Ensembl: ENSG00000198053, OMIM: 602461, UniProtKB/Swiss-Prot: P78324. In some embodiments, the SIRPa polypeptide comprises the sequence of SEQ ID NO: 17, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 17, or a polypeptide comprising a portion of SEQ ID NO: 17. The SIRPa polypeptide of SEQ ID NO: 17 may represent an immature or pre-processed form of mature SIRPa, and accordingly, included herein are mature or processed portions of the SIRPa polypeptide in SEQ ID NO: 17.

“Colony stimulating factor 1 (CSF1)” refers herein to a polypeptide that, in humans, is encoded by the CSF1 gene. In some embodiments, the CSF1 polypeptide is that identified in one or more publicly available databases as follows: HGNC: 2432, NCBI Entrez Gene: 1435, Ensembl: ENSG00000184371, OMIM: 120420, UniProtKB/Swiss-Prot: P09603. In some embodiments, the CSF1 polypeptide comprises the sequence of SEQ ID NO: 18, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 18, or a polypeptide comprising a portion of SEQ ID NO: 18. The CSF1 polypeptide of SEQ ID NO: 18 may represent an immature or pre-processed form of mature CSF1, and accordingly, included herein are mature or processed portions of the CSF1 polypeptide in SEQ ID NO: 18.

“Colony stimulating factor 1 receptor (CSF1R)” refers herein to a polypeptide that, in humans, is encoded by the CSF1R gene. In some embodiments, the CSF1R polypeptide is that identified in one or more publicly available databases as follows: HGNC: 2433, NCBI Entrez Gene: 1436, Ensembl: ENSG00000182578, OMIM: 164770, UniProtKB/Swiss-Prot: P07333. In some embodiments, the CSF1R polypeptide comprises the sequence of SEQ ID NO: 19, or a polypeptide sequence having at or greater than about 80%, about 85%, about 90%, about 95%, or about 98% identity with SEQ ID NO: 19, or a polypeptide comprising a portion of SEQ ID NO: 19. The CSF1R polypeptide of SEQ ID NO: 19 may represent an immature or pre-processed form of mature CSF1R, and accordingly, included herein are mature or processed portions of the CSF1R polypeptide in SEQ ID NO: 19.

The term “PD-L1 inhibitor” refers to a composition that binds to PD-L1 and reduces or inhibits the interaction between the bound PD-L1 and PD-1. In some embodiments, the PD-L1 inhibitor is a monoclonal antibody that is specific for PD-L1 and that reduces or inhibits the interaction between the bound PD-L1 and PD-1. Non-limiting examples of PD-L1 inhibitors are atezolizumab, avelumab and durvalumab. In some embodiments, the atezolizumab is TECENTRIQ or a bioequivalent. In some embodiments, the atezolizumab has the Unique Ingredient Identifier (UNII) of the U.S. Food and Drug Administration of 52CMI0WC3Y. In some embodiments, the atezolizumab is that described in U.S. Pat. No. 8217149, which is incorporated by reference in its entirety. In some embodiments, the avelumab is BAVENCIO or a bioequivalent. In some embodiments, the avelumab has the Unique Ingredient Identifier (UNII) of the U.S. Food and Drug Administration of KXG2PJ551I. In some embodiments, the avelumab is that described in U.S. Pat. App. Pub. No. 2014321917, which is incorporated by reference in its entirety. In some embodiments, the durvalumab is IMFINZI or a bioequivalent. In some embodiments, the durvalumab has the Unique Ingredient Identifier (UNII) of the U.S. Food and Drug Administration of 28X28X9OKV. In some embodiments, the durvalumab is that described in U.S. Pat. No. 8779108, which is incorporated by reference in its entirety.

The term “PD-L2 inhibitor” refers to a composition that binds to PD-L2 and reduces or inhibits the interaction between the bound PD-L2 and PD-1. In some embodiments, the PD-L2 inhibitor is a monoclonal antibody that is specific for PD-L2 and that reduces or inhibits the interaction between the bound PD-L2 and PD-1. In some embodiments, the PD-L2 inhibitor is that described in U.S. Publication No. 2020/0369772 or U.S. Patent No. 10,934,353, which are incorporated by reference in their entireties.

As used herein, the term “PD- 1 inhibitor” refers to a composition that binds to PD- 1 and reduces or inhibits the interaction between the bound PD-1 and PD-L1. In some embodiments, the PD-1 inhibitor is a monoclonal antibody that is specific for PD-1 and that reduces or inhibits the interaction between the bound PD-1 and PD-L1. Non-limiting examples of PD-1 inhibitors are pembrolizumab, nivolumab, and cemiplimab. In some embodiments, the pembrolizumab is KEYTRUDA or a bioequivalent. In some embodiments, the pembrolizumab is that described in U.S. Pat. No. 8952136, U.S. Pat. No. 8354509, or U.S. Pat. No. 8900587, all of which are incorporated by reference in their entireties. In some embodiments, the pembrolizumab has the Unique Ingredient Identifier (UNII) of the U.S. Food and Drug Administration of DPT0O3T46P. In some embodiments, the nivolumab is OPDIVO or a bioequivalent. In some embodiments, the nivolumab has the Unique Ingredient Identifier (UNII) of the U.S. Food and Drug Administration of 31YO63LBSN. In some embodiments, the nivolumab is that described in U.S. Pat. No. 7595048, U.S. Pat. No. 8738474, U.S. Pat. No. 9073994, U.S. Pat. No. 9067999, U.S. Pat. No. 8008449, or U.S. Pat. No. 8779105, all of which are incorporated by reference in their entireties. In some embodiments, the cemiplimab is LIBTAYO or a bioequivalent. In some embodiments, the cemiplimab has the Unique Ingredient Identifier (UNII) of the U.S. Food and Drug Administration of 6QVL057INT. In some embodiments, the cemiplimab is that described in U.S. Pat. No. 10844137, which is incorporated by reference in its entirety.

The term “CTLA-4 inhibitor” refers to refers to a composition that reduces or inhibits the interaction between CTLA-4 and CD80/CD86. In some embodiments, the CTLA-4 inhibitor binds to CTLA-4 and reduces or inhibits the interaction between the bound CTLA-4 and CD80/CD86. In some embodiments, the CTLA-4 inhibitor is a monoclonal antibody that is specific for CTLA- 4 and that reduces or inhibits the interaction between the bound CTLA-4 and CD80/CD86. Nonlimiting examples of CTLA-4 inhibitors are ipilimumab and tremelimumab. In some embodiments, the ipilimumab is YERVOY or a bioequivalent. In some embodiments, the ipilimumab has the Unique Ingredient Identifier (UNII) of the U.S. Food and Drug Administration of 6T8C155666. In some embodiments, the ipilimumab is that described in U.S. Pat. Nos. 7,605,238 and 6,984,720, which are incorporated by references in their entireties. In some embodiments, tremelimumab has the Unique Ingredient Identifier (UNII) of the U.S. Food and Drug Administration of QEN1X95CIX. In some embodiments, the tremelimumab is that described in U.S. Pat. No. 6,682,736, which is incorporated by reference in its entirety.

The term “TIM-3 inhibitor” refers to a composition that binds to TIM-3 and reduces or inhibits the interaction between the bound TIM-3 and its ligands including, for example, galectin- 9, phosphatidyl serine, and/or carcinoembryonic antigen-related cell adhesion molecule (CEACAM)-l. In some embodiments, the TIM-3 inhibitor is a monoclonal antibody that is specific for TIM-3 and that reduces or inhibits the interaction between the bound TIM-3 and a ligand thereof. In some embodiments, the TIM-3 inhibitor is MBG453, Sym023, or TSR-022.

The term “LAG3 inhibitor” refers to a composition that binds to LAG3 and reduces or inhibits the interaction between LAG3 and its ligands including, for example, galactose lectin-3 (Galectin-3), major histocompatibility complex II (MHC II), fibrinogen-like protein 1 (FGL1), and/or hepatic sinusoid endothelial cell lectin (LSECtin). In some embodiments, the LAG3 inhibitor is a monoclonal antibody that is specific for LAG3 and that reduces or inhibits the interaction between the bound LAG3 and a ligand thereof. In some embodiments, the LAG3 inhibitor is Relatlimab, Tebotelimab, RO-7247669, Favezelimab, INCAGN-2385, IBL110, Eftilagimod alpha, Sym-022, LBL-007, leramilimab, or Fianlimab.

The term “TIGIT inhibitor” refers to a composition that binds to TIGIT and reduces or inhibits the interaction between TIGIT and its ligands, including, for example, PVR and/or PVRL2. In some embodiments, the TIGIT inhibitor is a monoclonal antibody that is specific for TIGIT and that reduces or inhibits the interaction between the bound TIGIT and a ligand thereof. In some embodiments, the TIGIT inhibitor is Vibostolimab, Ociperlimab, Tiragolumab, Domvanalimab, or BMS-986207. On the other hand, a PVR inhibitor and PVRL2 inhibitor bind to PVR and PVRL2, respectively, and reduce or inhibit the interaction between TIGIT and PVR and PVRL2. The term “CD47/SIRPa inhibitor” refers to a composition that binds to CD47 or SIRPa and reduces or inhibits the interaction between CD47 and SIRPa. In some embodiments, the CD47/SIRPa inhibitor is Magrolimab (Hu5F9-G4), Ligufalimab (AK117), AO-176, CC9002, SGN-CD47M, IBI188, SHR-1603, SRF231, ZL-1201, IMC-002, Evorpacept (ALX148), TTI- 621, TTI-662, IMM01, or FSI-189.

The term “CSF1 inhibitor” refers to a composition that binds to CSF1 and reduces or inhibits the interaction between CSF1 and CSF1R. In some embodiments, the CSF1 inhibitor is a monoclonal antibody that is specific for CSF1 and that reduces or inhibits the interaction between the bound CSF1 and CSF1R. In some embodiments, the CSF1 inhibitor is Lacnotuzumab (MCS110) or PD-0360324.

The term “CSF1R inhibitor” refers to a composition that binds to CSF1R and reduces or inhibits the interaction between CSF1 and CSF1R. In some embodiments, the CSF1R inhibitor is a monoclonal antibody that is specific for CSF1R and that reduces or inhibits the interaction between the bound CSF1R and CSF1. In some embodiments, the CSF1R inhibitor is Pexidartinib, ARRY-382, BLZ945, Emactuzumab, AMG820, Cabiralizumab, or IMC-CS4 (LY3022855).

In some examples, the method disclosed herein further comprises administering to the subject irradiation, such as total body irradiation or abdominal irradiation. The instant disclosure shows that irradiation in combination with the engineered gastrointestinal tract (GI) bacterium disclosed herein, optionally in further combination with an immune checkpoint inhibitor, is effective to treat cancer.

Accordingly, in some aspects, disclosed herein is a method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of an engineered gastrointestinal tract (GI) bacterium and administering to the subject an irradiation, wherein the engineered gastrointestinal tract (GI) bacterium comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN-P, or a functional fragment thereof.

In some aspects, disclosed herein is a method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of an engineered gastrointestinal tract (GI) bacterium and a therapeutically effective amount of an immune checkpoint inhibitor, and adminstering to the subject an irradiation, wherein the engineered gastrointestinal tract (GI) bacterium comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN- , or a functional fragment thereof.

It should be understood that various compositions or therapies can be introduced into a subject simultaneously or sequentially. In some embodiments, the irradiation is administered to the subject prior to the administration of the engineered GI bacterium and/or the immune checkpoint inhibitor. In some embodiments, the subject is resistant to an immune checkpoint inhibitor.

In some embodiments, the combination of the engineered gastrointestinal tract (GI) bacterium disclosed herein and an irradiation, optionally in further combination with an immune checkpoint inhibitor, results in an increased number of T cells in a tumor in the subject. In some embodiments, the treatment results in a reduction of an irradiation- induced intestinal damage.

As noted above, “irradiation-induced intestinal damage” refers herein to a disruption of the homeostasis of any tissue (epithelial, connective, nervous or muscle) of any organ or compartment of the gastrointestinal tract due to irradiation. Ionizing irradiation damages the intestinal barrier, kills intestinal crypt cells, damages villi, and results in a systemic increase in gut bacteria, which can lead to sepsis, and, ultimately, death. Shrinkage of antimicrobial intestinal Paneth cells that, naturally, secrete defensins, lysozyme and other antimicrobial factors, loss of intestinal crypt cells, principally Lgr5+ intestinal stem cells, is also detected after irradiation doses that produce death from gastrointestinal syndrome. Accordingly, each of the damages to the intestinal barrier, crypt cells, villi, Paneth cells, and Lgr5+ intestinal stem cells, increased inflammation of GI tract, systemic increase of gut bacteria, and sepsis are included within the definition of “irradiation-induced intestinal damage.”

In some embodiments, the method disclosed herein further comprises administering to the subject a therapeutically effective amount of a chemotherapeutic agent. In some embodiments, the chemotherapeutic agent is a platinum based or taxane-based chemotherapeutic agent. The platinum-based chemotherapeutic agent can be, for example, cisplatin, carboplatin, oxaliplatin, nedaplatin, heptaplatin, or lobaplatin. The taxane-based chemotherapeutic agent can be, for example, paclitaxel or docetaxel.

The bacterium or the composition described herein can be administered to the subject via any route including oral, topical, intravenous, subcutaneous, transcutaneous, transdermal, intramuscular, intra-joint, parenteral, intra-arteriole, intradermal, intraventricular, intracranial, intraperitoneal, intralesional, intranasal, rectal, vaginal, by inhalation or via an implanted reservoir. The term “parenteral” includes subcutaneous, intravenous, intramuscular, intraarticular, intra-synovial, intrastemal, intrathecal, intrahepatic, intralesional, and intracranial injections or infusion techniques. In some embodiments, routes of administration of the bacterium or the composition includes oral administration, rectal administration, and parenteral administration (intravenous, intramuscular, or subcutaneous). In some embodiments, the route of administration of the bacterium disclosed herein is oral administration. In some embodiments, the cancer is selected from, a hematologic cancer, lymphoma, colorectal cancer, (colon cancer, rectal cancer, colorectal adenocarcinoma), lung cancer, a head and neck cancer, ovarian cancer, prostate cancer, testicular cancer, renal cancer, skin cancer, cervical cancer, pancreatic cancer, large bowel cancer, liver cancer, cancer of the stomach (gastric cancers, stomach adenocarcinoma, gastrointestinal stromal tumor), and breast cancer. In some embodiments, the cancer is ovarian cancer.

In some embodiments, the cancer is melanoma, lung cancer (including lung adenocarcinoma, basal cell carcinoma, squamous cell carcinoma, large cell carcinoma, bronchioloalveolar carcinoma, bronchogenic carcinoma, non-small-cell carcinoma, small cell carcinoma, mesothelioma); breast cancer (including ductal carcinoma, lobular carcinoma, inflammatory breast cancer, clear cell carcinoma, mucinous carcinoma, serosal cavities breast carcinoma), colorectal cancer (colon cancer, rectal cancer, colorectal adenocarcinoma), anal cancer, pancreatic cancer (including pancreatic adenocarcinoma, islet cell carcinoma, neuroendocrine tumors), prostate cancer, prostate adenocarcinoma, ovarian carcinoma (ovarian epithelial carcinoma or surface epithelial- stromal tumor including serous tumor, endometrioid tumor and mucinous cystadenocarcinoma, sex-cord-stromal tumor), liver and bile duct carcinoma (including hepatocellular carcinoma, cholangiocarcinoma, hemangioma), esophageal carcinoma (including esophageal adenocarcinoma and squamous cell carcinoma), oral and oropharyngeal squamous cell carcinoma; salivary gland adenoid cystic carcinoma, bladder cancer, bladder carcinoma, carcinoma of the uterus (including endometrial adenocarcinoma, ocular, uterine papillary serous carcinoma, uterine clear-cell carcinoma, uterine sarcomas, leiomyosarcomas, mixed mullerian tumors), glioma, glioblastoma, medulloblastoma, and other tumors of the brain; kidney cancers (including renal cell carcinoma, clear cell carcinoma, Wilm's tumor); cancer of the head and neck (including squamous cell carcinomas), cancer of the stomach (gastric cancers, stomach adenocarcinoma, gastrointestinal stromal tumor), testicular cancer, germ cell tumor; neuroendocrine tumor, cervical cancer, carcinoids of the gastrointestinal tract, breast, and other organs; signet ring cell carcinoma, mesenchymal tumors including sarcomas, fibrosarcomas, haemangioma, angiomatosis, haemangiopericytoma, pseudoangiomatous stromal hyperplasia, myofibroblastoma, fibromatosis, inflammatory myofibroblastic tumor, lipoma, angiolipoma, granular cell tumor, neurofibroma, schwannoma, angiosarcoma, liposarcoma, rhabdomyosarcoma, osteosarcoma, leiomyoma, leiomysarcoma, skin, including melanoma, cervical, retinoblastoma, head and neck cancer, myxosarcoma, chondrosarcoma, osteosarcoma, chordoma, malignant fibrous histiocytoma, lymphangiosarcoma, mesothelioma, squamous cell carcinoma, epidermoid carcinoma, malignant skin adnexal tumors, adenocarcinoma, hepatoma, hepatocellular carcinoma, renal cell carcinoma, hypernephroma, cholangiocarcinoma, transitional cell carcinoma, choriocarcinoma, seminoma, embryonal cell carcinoma, glioma anaplastic, glioblastoma multiforme, neuroblastoma, medulloblastoma, malignant meningioma, malignant schwannoma, neurofibrosarcoma, parathyroid carcinoma, medullary carcinoma of thyroid, bronchial carcinoid, pheochromocytoma, Islet cell carcinoma, malignant carcinoid, malignant paraganglioma, melanoma, Merkel cell neoplasm, cystosarcoma phylloide, salivary cancers, thymic carcinomas, or a cancers of the vagina.

In one aspect, the cancer comprises a solid tumor. In another aspect, the cancer is selected from acute myeloid leukemia, myelodysplastic syndrome, chronic myeloid leukemia, acute lymphoblastic leukemia, myelofibrosis, multiple myeloma. In another aspect, the cancer is selected from a leukemia, a lymphoma, a sarcoma, a carcinoma and may originate in the marrow, brain, lung, breast, pancreas, liver, head and neck, skin, reproductive tract, prostate, colon, liver, kidney, intraperitoneum, bone, joint, and eye.

The disclosed methods can be performed any time prior to the onset of a cancer. In one aspect, the disclosed methods can be employed 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 months; 30, 29, 28, 27, 26, 25, 24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 minutes prior to the onset of a cancer; or 30, 29, 28, 27, 26, 25, 24,

23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, or 3 days; 60, 48, 36, 30, 24,

18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 hours; 60, 48, 36, 30, 24, 18, 15, 12, 10, 9, 8, 7, 6, 5, 4, 3, or 2 minutes after onset of a cancer; or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55,

60, 75, 90, 105, 120 minutes; 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 18, 24, 30, 36, 48, 60 hours; 3, 4,

5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 45, 60, 90 or more days; 4, 5, 6, 7, 8, 9, 10, 11, 12 or more months; 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47, 46, 45, 44, 43, 42, 41, 40, 39, 38, 37, 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25,

24, 23, 22, 21, 20, 19, 18, 17, 16, 15, 14, 13, 12, 11, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 years after the onset of a cancer.

Dosing frequency for the vector or the composition of any preceding aspects, includes, but is not limited to, at least once every year, once every two years, once every three years, once every four years, once every five years, once every six years, once every seven years, once every eight years, once every nine years, once every ten year, at least once every two months, once every three months, once every four months, once every five months, once every six months, once every seven months, once every eight months, once every nine months, once every ten months, once every eleven months, at least once every month, once every three weeks, once every two weeks, once a week, twice a week, three times a week, four times a week, five times a week, six times a week, daily, two times per day, three times per day, four times per day, five times per day, six times per day, eight times per day, nine times per day, ten times per day, eleven times per day, twelve times per day, once every 12 hours, once every 10 hours, once every 8 hours, once every 6 hours, once every 5 hours, once every 4 hours, once every 3 hours, once every 2 hours, once every hour, once every 40 min, once every 30 min, once every 20 min, or once every 10 min. Administration can also be continuous and adjusted to maintaining a level of the compound within any desired and specified range.

The engineered GI bacterium disclosed herein may be administered in any amount effective to introduce the polypeptide in the bloodstream of the subject. Exemplary amounts include from about IxlO³ to about IxlO¹⁵, from about IxlO⁵ to about IxlO¹³, from about IxlO⁷ to about IxlO¹¹, from about IxlO⁶ to about IxlO⁹ colony forming units (CFU). In some embodiments, the dosage is about IxlO⁹, about IxlO⁸, about IxlO⁷, or about IxlO⁶ CFUs.

Pharmaceutically acceptable excipients or carriers are well known to those of skill in the art and may include cellulose and its derivatives (e.g. sodium carboxymethylcellulose, sodium ethylcellulose, cellulose acetate, etc.), gelatin, speckstone, solid lubricating agent (e.g. stearic acid, magnesium stearate), calcium sulphate, plant oil (e.g. pea oil, sesame oil, peanut oil, olive oil, etc.), polyols (e.g. propylene glycol, glycerol, mannitol, sorbitol, etc.), emulsifier (e.g. TWEEN), wetting agent (e.g. sodium lauryl sulfate), colorant, flavoring agent, stabilizer, anti-oxidant, antiseptic, pyrogen-free water, etc.

EXAMPLES

The following examples are set forth below to illustrate the compositions, methods, and results according to the disclosed subject matter. These examples are not intended to be inclusive of all aspects of the subject matter disclosed herein, but rather to illustrate representative methods and results. These examples are not intended to exclude equivalents and variations of the present invention which are apparent to one skilled in the art.

Example 1: Results.

Expression of PD-L1 protein is significantly induced at 48 hours after 2 Gy ionizing irradiation to human ovarian adenocarcinoma cell line, OVCAR3 and is detected by immunohistochemistry (Fig. 1A). There is also significant induction as detected by qPCR of PD- L1 mRNA in a radiation dose dependent manner in another human High-grade Serous Ovarian Cancer Cell Line, OVSAHO (Fig. IB). In the transgenic mouse model of MUC-1 mice with 2F8cis cell derived OC, the effect of adding LR- IL-22 gavage after WAI significantly reduces tumor burden and protects the intestine from radiation damage (Fig. 2). MUC-1 mice were injected I.P. with 10⁶ 2F8cis cells, then on day 5 the mice received a single fraction of 19.75 Gy WAI. The gross pathologic appearance of mice with disseminated intraabdominal 2F8cis ovarian cancer is shown at 5 days post irradiation (10 days after tumor cell injection) (Fig. 2A). The tumor burden is greatly reduced by WAI (Fig. 2B- 2C).

LR-IL-22 administration by gavage - in 100 pl before WAI prevents intestinal barrier breakdown (Fig. 3). Loss of the intestinal barrier was quantitated by measuring the leakage of intra-orally administered red fluorescent microspheres from the gut lumen into the blood. Red fluorescent bead leakage into the blood following 19.75 WAI at day 2 and day 5 was elevated but was significantly reduced in LR-IL-22 gavage treated compared to LR gavage treated (control bacteria treated) mice or IL-22 protein treated mice (Fig. 3).

WAI and LR-IL-22 each induce PD-L1 expression in 2F8cis tumors in vivo in MUC-1 mice. IO⁶ 2F8-cis cells were injected into the peritoneal cavity of MUC-1 mice and mice after 7 days received WAI (6.0 Gy x 4 daily fractions) for 4 consecutive days. Subgroups of mice received gavage of LR or LR-IL-22 at 24h and 72h after the first fraction of WAI. Quantitation of PD-L1 expression in explanted tumor cells showed that with WAI there was an increase in PD-L1 positive cells at day 9. There was a further increase by adding LR-IL-22 to WAI. Furthermore, there was a significant increase in PD-L1 mRNA by qPCR, as well as, by flow analysis (Fig. 4A), and by immunohistochemistry of sections of explanted tumor (Fig. 4B).

There is a significant increase at day 9 in the number of intra-tumoral CD8+ T-cells in 2F8cis tumors explanted from WAI treated and further increased in WAI + LR-IL-22 gavaged mice compared to control untreated tumors. Flow analysis (Fig. 5A) and immunohistochemistry of tumors (Fig. 5B) both reveal a significant increase in CD8+ T-cells by adding LR-IL-22 to WAI.

The data demonstrate that the combination of PD-L1 inhibitor added to LR-IL-22 gavage and WAI (6 Gy x 4 daily fractions) in MUC- 1 transgenic mice with diffuse intraabdominal spread of 2F8cis tumors significantly increases survival compared to other tumor-bearing groups receiving irradiation alone, LR-IL-22 alone, or untreated control mice (Fig. 6). WAI and LR-IL- 22 each or in combination will induce PD-L1 expression in OC tumor cells and that each also stimulates beneficial immunocyte infiltration (specifically, CD8+ T cell infiltration) into tumors. This study shows that the combined immune stimulatory effects of WAI added to LR-IL-22 can facilitate a therapeutic benefit of administration of a PD-L1 immune checkpoint inhibitor. Finally, this study shows that adding combination chemotherapy to LR- IL-22, WAI, and anti-PD-Ll treatment will significantly increase survival (Fig. 7).

WAI and LR-IL-22 each alone, or in combination can significantly eradicate tumors and increase survival when mice are also treated with PD-L1 inhibitors. The underlying molecular mechanism is by the induction of PD-L1 on tumor cells by WAI and LR-IL-22 Further, this study shows that there is increased CD8+ T-cell infiltration into OC tumors by this protocol of combination therapy, which includes WAI and LR-IL-22. The combination of WAI, LR-IL-22, PD-L1 inhibitors, and chemotherapy can improve survival without increasing late effects of irradiation. This study shows that LR-IL-22 is a radioprotector of the intestine during the combination treatment and can also be a drug that facilitates WAI dose escalation.

Example 2: LR-IL-22 and WAI independently upregulate PD-L1 in OC cells

LR-IL-22 gavage induces PD-L1 in OC tumor cells that are derived from injection of 2F8cis cells to form mouse ovarian tumors. MUC-1 transgenic female mice have diffuse intraabdominal ovarian cancer after I.P. injection of 10⁶ 2F8cis tumor cells.

Preparation of cells and mice with OC diffuse intra-abdominal tumors: The 2F8 mouse ovarian cancer cell line is derived from Cre-encoding adenovirus-induced orthotopic ovarian tumors. The 2F8cis cells were initially obtained by exposing 2F8 cells in vitro to increasing concentrations (up to 10 //M) of cisplatin (Sigma-Aldrich). The 2F8cis cells are then maintained in cultured Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% heat-inactivated fetal bovine serum, penicillin (100 U/ml), streptomycin (100 mg/ml), 2 mM 1- glutamine (Coming Life Sciences), and 1 //M cisplatin. Cells are regularly tested to eliminate Mycoplasma contamination.

Time of treatment of experimental tumor-bearing groups: At day 7 after I.P. injection of 10⁶2F8cis cells into MUC-1+ transgenic female mice, mice are gavaged with LR, LR-IL-22 (10⁹ bacteria in 200 zl of PBS) or inject IL-22 protein 25 pg/kg intraperitonially (I.P.). 2F8cis tumors are isolated from each of the 4 experimental groups: 1) No treatment of tumor bearing control group, 2) IL-22 protein I.P. injection group, 3) control LR gavage treated group, and 4) LR-IL-22 gavage treated group.

Analysis of cells in tumors: After gavage of LR or LR-IL-22 bacteria, or I.P. administration of IL-22 protein (PeproTech, Rocky Hill, NJ), the tumors are isolated from subgroups of mice over the next 7 days (1, 3, 5, and 7). This experiment evaluates a) the expression of PD-L1 in tumor cells and b) quantitate the abundance and phenotype of tumor- infiltrated T-lymphocytes. Absolute numbers of CD45+CD3+CD8+T cells and other phenotypes are characterized using flow cytometry. Briefly, fresh tumors, mince tumors are isolated on ice, and enzymatically digested to form single-cell suspensions according to the protocol for the mouse Tumor Dissociation Kit (Miltenyi Biotec). Cell suspensions are filtered, red blood cells are removed using Ammonium-Chloride-Potassium (ACK) lysis buffer, and single-cell suspensions are produced by filtering through a 40-micron cell strainer. Cells are then stained for flow cytometry by adding fluorescently labeled antibodies. To quantify the PD- L1 expression on the 2F8-cis tumor cells, tumor cells are separated from other nucleated cells including monocytes, the CD45-, CD326+, CD90-, PD-L1 positive cells with antibodies to each (BD biosciences). An isotype control from BD bioscience is used and dead cells are excluded from analysis with LIVE/DEAD™ Fixable Aqua (Thermo Fisher Scientific, Waltham, MA). All data analysis is performed using the flow cytometry analysis program FlowJo (Tree Star).

Since the absolute number of T-lymphocytes by Flow may not reflect the number of CD8+ T-lymphocytes that have infiltrated the tumors, immunohistochemical evaluation of the spatial distribution of CD8+ T cells within the tumor tissue is carried out. Since T cells were reported to be restricted to areas of peritumoral desmoplasia, immunohistochemical analysis is performed on tumors from all experimental groups to establish that in the LR-IL-22 treatment, as well as, other groups, T-cells are inside the tumors. Masson’s trichrome staining is performed and each experimental group for the presence of cancer-associated mesenchymal stem cells (CA-MSCs) is compared. Total RNA is isolated from tumor cells, perform quantitative RT- PCR, and measure the level of PD-L1 expression, a smooth muscle actin (rr-SMA) expression is quantitated in separated CA-MSCs. The LR-IL-22 group is compared to each other group.

WAI induces PD-L1 in human and mouse ovarian cancer lines, and in fresh human OC containing ascites fluid cells in vitro and in mouse OC cell line derived tumors in vivo.

In vitro induction of PD-L1 in human OC cell lines and fresh human OC ascites cancer cells: This study uses doses of irradiation (0, 3, 6 and 9 Gy) and human (OVCAR3 and OVSAHO cell lines). Results are compared with mouse (2F8 and 2F8cis) OC cell lines. Cisplatin upregulates PD-L1 expression in vitro and in vivo in derived tumors to support the rationale for using 2F8-cis cells for studies of immune measure checkpoint blockade. The data show that the induction of PD-L1 expression in human OC cells with irradiation. At 24, 48, 72 and 96 hours cells are harvested and the levels of expression of PD- L1+ cancer cells are quantitated by flow cytometry, qPCR, and immunostaining. These studies are repeated with fresh OC human patient ascites fluid.

In vivo induction of PD-L1 in OC tumors in mice:_Mucl+ transgenic mice are injected I.P. with (1 x 10⁶) 2F8cis tumor cells and after 1 week, mice will be irradiated with daily 6 Gy x 4. This experiment compares groups: 1) 2F8cis tumor with no radiation, 2) 2F8cis tumor with radiation. Tumor cells are isolated, stained for PD-L1 and quantified for the PD-L1 expression by rt-PCR and immunohistochemistry. The levels of PD-L1 are quantitated by immunostaining, qPCR and PD-L1+ tumor cells flow-cytometry at 24, 48, 72 and 96 hours after WAI. 6 female mice per group x 2 groups x 4 time points = 48 mice are used for analysis.

Phenotyping of immune cells: This experiment measures abundance and phenotypes of each tumor-infiltrated T-cell subset of lymphocytes (CD45+CD3+CD8+), macrophages (CD45+, CD11c-, CDllb+, F4/80+) and M2 like macrophage subpopulation (CD45+, CDllc- , CDl lb+, F4/80+, CD206+) using flow cytometry. By immunostaining, this study evaluates the effects of WAI on the special distribution of these immune cells in the tumor. Moreover, tumor infiltrated B-cells (CD19), monocytes (CDl lb, CD115) neutrophils (Ly-6G and CDl lb), and cancer associated mesenchymal stem cells (CA-MSCs) (PDGFR-a and Sca-1) are also phenotyped and quantitated.

Establish that WAI and LR-IL-22 administered together further induce PD-L1 on the surface of OC tumor cells.

This experiment quantitiates level of expression of PD-L1 on tumor cells and the infiltration of CD8+ T-cells and NK cells in the tumors derived from 2F8cis cells, the following groups are compared: 1) 2F8cis with no radiation or bacteria, 2) 2F8cis with LR control, 3) 2F8cis with LR-IL-22, 4) 2F8cis with radiation, 5) 2F8cis with radiation and LR control, 6) 2F8cis with radiation and LR-IL-22.

The expression of PD-L1 on the surface of tumor cells is significantly elevated after WAI or after gavage of LR-IL-22 and is significantly further increased by WAI added to LR-IL22. It has been shown that LR-IL-22 is a potent radioprotector and improves survival after total body irradiation. If the magnitude of induction of PD-L1 by LR-IL-22 treatment is less than expected, the radioprotector function of LR-IL-22 can still allow the delivery of higher WAI doses, (Example 7 Gy x 4 daily fractions of WAI). Since the untreated 2F8cis tumors have baseline levels of intra-tumoral T-cells, infiltration of immune cells (CD8+ T-cells, B- cells, NK cells, and monocyte/macrophages) into 2F8cis tumors can be higher after WAI and LR-IL-22 treatment compared to other groups. The location of T-cells within tumors is determined using immunostaining. It has been shown that CA-MSCs prevent T-cells from entering the 2F8cis tumors when the tumors are formed by subcutaneous injections of a mixture of 2F8cis cells and CA- MSCs. The kinetics of arrival of CA-MSCs daily after WAI and LR-IL-22 is quantitated. Endogenous (bone marrow derived) MSCs in the tumors can be decreased by WAI and LR-IL-22 relative to CD8+ T-cells entering the tumors. A higher WAI dose can further disrupt the CA-MSC barrier, and that beneficial CD8+ T-cells, NK cells, and other immune cells can better enter the tumors.

Example 3. PD-L1 antibody treatment added to LR-IL-22 gavage and WAI will further reduce tumor volume in OC tumor-bearing mice.

WAI, LR-IL-22 gavage, and PD-L1 antibody treatment significantly increase T- cell infiltration in ovarian cancer tumors in mice. The 2F8cis tumors are known to be infiltrated by CD8+ T cells. There is a reported reduction in 2F8cis tumor size by anti-PD-Ll antibody treatment; however, the treatment is not curative. This study uses MUC-1 transgenic mice to evaluate the therapeutic efficacy anti-PD-Ll immunotherapy added to WAI and LR-IL-22. Tumor-bearing mice are randomly divided into the following 8 groups: 1) Control (no treatment), 2) WAI, 3) LR-IL- 22, 4) WAI + LR-IL-22, 5) PD-L1 antibody, 6) WAI + PD-L1 antibody, 7) LR-IL-22 + PD-L1 antibody, and 8) WAI + LR-IL-22 + PD-L1 antibody. The 2F8cis tumor cells are injected into MUC-1 mice and one week later the mice are given 6 Gy x 4 WAI. At 24 and 72 hours after the first fraction of WAI treatment, mice are treated with either LR or LR-IL22 (10⁹ bacteria in 200 /Ji). One day after the first radiation fraction, tumor-bearing mice are treated with intraperitoneal injection of 150 pg anti-PD-Ll antibody (10F, 9G2, Bio X Cell, Japan) followed by two more injections on day 12 and 14 after tumor injections (Fig. 8). Control mice receive isotype rat IgG2b (Bio X Cell). Mice are sacrificed 24 and 96 h after the last injection of anti- PD-Ll antibody. This study prepares single cell suspensions from the tumors and perform flow cytometry to analyze the number of T-cells other immune cells in the tumors. Immunostaining is performed to evaluate the distribution of T-cells and confirm that the T-cells are present inside the tumor and not sequestered in the peritumoral region.

WAI, LR-IL-22 gavage, and PD-L1 antibody treatment significantly improve survival. This study quantitates the survival of mice with 2F8-cis tumors comparing the following experimental groups: 1) Control (no treatment) 2) WAI, 3) LR-IL-22, 4) WAI + LR-IL-22, 5) PD- L1 antibody, 6) WAI + PD-L1 antibody, 7) LR-IL-22 +PD-L1 antibody, 8) WAI+LR-IL-22 + PD- L1 antibody. The mice are followed for development of the radiation-induced gastrointestinal (GI) syndrome at which time they are euthanized. Comparisons are based on a log rank test. Detail of the power analysis is described in the statistics and power analysis section.

The combination therapy of anti-PD-Ll, WAI and LR-IL-22 can significantly reduce tumor volume, tumor cell numbers, and improve survival more effectively than that detected in the other groups. Control animals should die of OC (or be euthanized according to Institutional IACUC protocols) within the third week after 2F8cis cancer cell injection. This study quantitates volume of tumor at earlier time points, since large numbers of tumor cells are required for analysis of the effects of each treatment modality.

Example 4. Addition of chemotherapy to the combination of WAI, LR-IL-22, and PD- LI antibody treatment will further improve survival in OC tumor-bearing mice.

The combination of WAI, LR-IL-22, PD-L1 antibody and chemotherapy further increase T-cell infiltrates in the ovarian tumors in mice.

This study establishes that addition of the optimal schedule of chemotherapy when added to the triple therapy regimen of Specific Aim 2 to form a quadruple therapy can further improve beneficial immunocyte infiltrates resulting in increased survival. For treating the 2F8cis tumor bearing MUC-1 mice with chemotherapeutic drugs (alone or in combination with anti-PD-Ll, WAI and LR-IL-22), This study administers chemotherapy protocols: 1) Paclitaxel (10 mg/kg) and Carboplatin (40 mg/kg) on day 3, or as backup alternative 2) Cisplatin (10.5 mg/kg every other day for 3 doses) will be used. This study compares the following groups: 1) 2F8cis tumor control, 2) WAI alone, 3) Chemotherapy alone, 4) Chemo-i- LR-IL-22, 5) Chemo-i- WAI, 6) Chemo+ LR-IL-22+ WAI, 7) Chemo + PD-L1 antibody, and 8) Chemo + LR-IL-22 + WAI + PD- L1 antibody. A week later, tumors will be removed for analysis of tumor survival and immunocyte infiltrate. The abundance and phenotype of tumor-infiltrated T-lymphocytes are quantitated by measuring absolute number of CD45+CD3+CD8+T cells and other phenotypes using flow cytometry. By immunostaining This study evaluates the effects of chemotherapy on the distribution of T-cells and whether or not they are inside the tumor tissue. This study uses 6 female mice per group for analysis x 8 groups for a total of 48 mice.

The combination of WAI, LR-IL-22, PD-L1 antibody and chemotherapy cures mice followed for over 6 months and that survivors display no toxic late effects of treatment on abdominal organs including: kidney, liver, intestine, and spinal cord.

In the 2F8-cis tumor bearing mice, in the groups where WAI is one of the modalities, the mice for any late effects to the organs (kidney, liver and intestines in particular) which were in the field of WAI is monitored. Tumor burden and survival is evaluated with all possible combinations of WAI, LR-IL-22, PD-L1 antibody and chemotherapy as listed above (8 groups). For the chemotherapy group, mice are treated as above, and followed for 60 days (n=15). This study confirms that the tissues in the radiation field show no late effects by histological and morphological analyses. Kidney: late effects due to radiation is quantitated by measuring by histopathology renal interstitial fibrosis and loss of nephron mass. Interlobular and arcuate arteries, tubular atrophy and glomerular fibrosis are measured, which are features of radiation nephropathy. Liver: This study quantitates loss of parenchymal cells, hepatic congestion, and liver fibrosis. Intestines: This study measures late effects of radiation in the intestine by measuring weight loss and by histopathology quantitating fibrosis.

Kidney, liver, and intestine are collected at day 60 and compare results between groups. Spinal cord: This study measures functional neurologic and histopathologic changes including gait, movement, and exercise wheel stamina and speed of movement, and loss of motor neurons in cross sections of spinal cord.

The addition of chemotherapy along with 6 x 4 Gy radiation may produce greater toxicity to liver, kidney, intestine, and spinal cord. To reduce radiation damage, radiation dose is redueced to 5.5 Gy x 4 daily fractions. The chemotherapy dose can be reduced if evidence of late effects at 180 days is found, but not to a level that decreases survival.

Cell lines and animal models: Cell lines: The 2F8 cell line was derived from Cre- encoding adenovirus-induced orthotopic ovarian tumors. The 2F8cis cells were cloned by incubating 2F8 cells in vitro to increasing concentrations of cisplatin. The 2F8 and its platinum- resistant derivative 2F8cis cell lines represent the primary and recurrent human disease, respectively. Cisplatin upregulates PD-L1 expression in vitro and in vivo supporting the rationale for the use of immune checkpoint blockade. This study uses well characterized established human OC cell lines, 0VCAR3 and OVSAHO. Animal model: A mouse model is used, since there are no in vitro models that can match an in vivo model of diffuse intraabdominal spread of cancer. The in vivo model uses assay of many immunocyte phenotypes, which cannot be modeled in vitro. Female mice transgenic for the expression of human MUC- 1 (a tumor associated antigen that is overexpressed by most ovarian tumor types) is used. Female MUC-1+ mice with syngeneic 2F8cis tumors will be used to compare and confirm the efficacy of the proposed combination therapy. The MUC-1 transgenic mice develop intraperitoneal (I.P.) tumors after I.P. injection of MUC-1+ 2F8, or 2F8cis (cis-platinum resistant) cells. This MUC-l/2F8cis mouse model is an established mouse model for our proposed experiments.

SEQUENCES

SEQ ID NO: 1 (amino acid sequence of human IL-22)

MAALQKSVSSFLMGTLATSCLLLLALLVQGGAAAPISSHCRLDKSNFQQPYITNRTFML

AKEASLADNNTDVRLIGEKLFHGVSMSERCYLMKQVLNFTLEEVLFPQSDRFQPYMQE

VVPFLARLSNRLSTCHIEGDDLHIQRNVQKLKDTVKKLGESGEIKAIGELDLLFMSLRNA CI

SEQ ID NO: 2 (DNA sequence of human IL-22)

ATGGCTGTCCTGCAGAAATCTATGAGTTTTTCCCTTATGGGGACTTTGGCCGCCAGC

TGCCTGCTTCTCATTGCCCTGTGGGCCCAGGAGGCAAATGCGCTGCCCATCAACACC

CGGTGCAAGCTTGAGGTGTCCAACTTCCAGCAGCCGTACATCGTCAACCGCACCTTT

ATGCTGGCCAAGGAGGCCAGCCTTGCAGATAACAACACAGACGTCCGGCTCATCGG

GGAGAAACTGTTCGAGGAGTCAGTGCTAAGGATCAGTGCTACCTGATGAAGCAGGT

GCTCAACTTCACCCTGGAAGACGTTCTGCTCCCCCAGTCAGACAGGTTCCAGCCCTA

CATGCAGGAGGTGGTGCCTTTCCTGACCAAACTCAGCAATCAGCTCAGCTCCTGTCA

CATCAGCGGTGACGACCAGAACATCCAGAAGAATGTCAGAAGGCTGAAGGAGACA

GTGAAAAAGCTTGGAGAGAGTGGAGAGATCAAAGCGATTGGGGAACTGGACCTGC

TGTTTATGTCTCTGAGAAATGCTTGCGTC

SEQ ID NO: 3 (amino acid sequence of human IFN-P)

MTNKCLLQIALLLCFSTTALSMS YNLLGFLQRS SNFQCQKLLWQLNGRLEYCLKDRMN

FDIPEEIKQLQQFQKEDAALTIYEMLQNIFAIFRQDSSSTGWNETIVENLLANVYHQINHL

KTVLEEKLEKEDFTRGKLMSSLHLKRYYGRILHYLKAKEYSHCAWTIVRVEILRNFYFI NRLTGYLRN

SEQ ID NO:4 (DNA sequence of human IFN- )

ATGAACAACAGGTGGATCCTCCACGCTGCGTTCCTGCTGTGCTTCTCCACCACAGCC

CTCTCCATCAACTATAAGCAGCTCCAGCTCCAAGAAAGGACGAACATTCGGAAATG

TCAGGAGCTCCTGGAGCAGCTGAATGGAAAGATCAACCTCACCTACAGGGCGGACT

TCAAGATCCCTATGGAGATGACGGAGAAGATGCAGAAGAGTTACACTGCCTTTGCC

ATCCAAGAGATGCTCCAGAATGTCTTTCTTGTCTTCAGAAACAATTTCTCCAGCACT

GGGTGGAATGAGACTATTGTTGTACGTCTCCTGGATGAACTCCACCAGCAGACAGT

GTTTCTGAAGACAGTACTAGAGGAAAAGCAAGAGGAAAGATTGACGTGGGAGATG

TCCTCAACTGCTCTCCACTTGAAGAGCTATTACTGGAGGGTGCAAAGGTACCTTAAA CTCATGAAGTACAACAGCTACGCCTGGATGGTGGTCCGAGCAGAGATCTTCAGGAA

CTTTCTCATCATTCGAAGACTTACCAGAAACTTCCAAAAC

SEQ ID NO: 5 (pRSET-EmGFP-IL22 Sequence) (DNA sequence, artificial sequence)

GGATCCATGGCTGTCCTGCAGAAATCTATGAGTTTTTCCCTTATGGGGACTTTGGCC

GCCAGCTGCCTGCTTCTCATTGCCCTGTGGGCCCAGGAGGCAAATGCGCTGCCCATC

AACACCCGGTGCAAGCTTGAGGTGTCCAACTTCCAGCAGCCGTACATCGTCAACCG

CACCTTTATGCTGGCCAAGGAGGCCAGCCTTGCAGATAACAACACAGACGTCCGGC

TCATCGGGGAGAAACTGTTCCGAGGAGTCAGTGCTAAGGATCAGTGCTACCTGATG

AAGCAGGTGCTCAACTTCACCCTGGAAGACGTTCTGCTCCCCCAGTCAGACAGGTTC

CAGCCCTACATGCAGGAGGTGGTGCCTTTCCTGACCAAACTCAGCAATCAGCTCAG

CTCCTGTCACATCAGCGGTGACGACCAGAACATCCAGAAGAATGTCAGAAGGCTGA

AGGAGACAGTGAAAAAGCTTGGAGAGAGTGGAGAGATCAAAGCGATTGGGGAACT

GGACCTGCTGTTTATGTCTCTGAGAAATGCTTGCGTCCCATGGTGAGCAAGGGCGA

GGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACG

GCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTACGGCAAGCTG

ACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTG

ACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCA

GCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTT

CTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGAC

ACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGGACGGCAACAT

CCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTATATCACCGCCG

ACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACAACATCGAGGA

CGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCC

CCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGAC

CCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCGCCGCCGGGAT

CACTCTCGGCATGGACGAGCTGTACAAGTAACTCGAG

SEQ ID NO: 6 (pRSET-EmGFP-IFN-P Sequence) (DNA sequence, artificial sequence)

GGATCCATGAACAACAGGTGGATCCTCCACGCTGCGTTCCTGCTGTGCTTCTCCACC

ACAGCCCTCTCCATCAACTATAAGCAGCTCCAGCTCCAAGAAAGGACGAACATTCG

GAAATGTCAGGAGCTCCTGGAGCAGCTGAATGGAAAGATCAACCTCACCTACAGGG

CGGACTTCAAGATCCCTATGGAGATGACGGAGAAGATGCAGAAGAGTTACACTGCC

TTTGCCATCCAAGAGATGCTCCAGAATGTCTTTCTTGTCTTCAGAAACAATTTCTCC AGCACTGGGTGGAATGAGACTATTGTTGTACGTCTCCTGGATGAACTCCACCAGCA

GACAGTGTTTCTGAAGACAGTACTAGAGGAAAAGCAAGAGGAAAGATTGACGTGG

GAGATGTCCTCAACTGCTCTCCACTTGAAGAGCTATTACTGGAGGGTGCAAAGGTA

CCTTAAACTCATGAAGTACAACAGCTACGCCTGGATGGTGGTCCGAGCAGAGATCT

TCAGGAACTTTCTCATCATTCGAAGACTTACCAGAAACTTCCAAAACCCATGGTGAG

CAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCG

ACGTAAACGGCCACAAGTTCAGCGTGTCCGGCGAGGGCGAGGGCGATGCCACCTAC

GGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCC

CACCCTCGTGACCACCTTGACCTACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCA

CATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGC

GCACCATCTTCTTCAAGGACGACGGCAACTACAAGACCCGCGCCGAGGTGAAGTTC

GAGGGCGACACCCTGGTGAACCGCATCGAGCTGAAGGGCATCGACTTCAAGGAGG

ACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTACAACAGCCACAAGGTCTAT

ATCACCGCCGACAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGACCCGCCACA

ACATCGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATC

GGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCT

GAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCGTGACCG

CCGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAACTCGAG

SEQ ID NO: 7 (polypeptide sequence of human PD-L1)

MRIFAVFIFMTYWHLLNAFTVTVPKDLYVVEYGSNMTIECKFPVEKQLDLAALIVYWE

MEDKNIIQFVHGEEDLKVQHSSYRQRARLLKDQLSLGNAALQITDVKLQDAGVYRCMI

SYGGADYKRITVKVNAPYNKINQRILVVDPVTSEHELTCQAEGYPKAEVIWTSSDHQVL

SGKTTTTNSKREEKLFNVTSTLRINTTTNEIFYCTFRRLDPEENHTAELVIPELPLAHPPNE

RTHLVILGAILLCLGVALTFIFRLRKGRMMDVKKCGIQDTNSKKQSDTHLEET

SEQ ID NO: 8 (polypeptide sequence of human PD-L2)

MIFLLLMLSLELQLHQIAALFTVTVPKELYIIEHGSNVTLECNFDTGSHVNLGAITASLQK

VENDTSPHRERATLLEEQLPLGKASFHIPQVQVRDEGQYQCIIIYGVAWDYKYLTLKVK

ASYRKINTHILKVPETDEVELTCQATGYPLAEVSWPNVSVPANTSHSRTPEGLYQVTSV

LRLKPPPGRNFSCVFWNTHVRELTLASIDLQSQMEPRTHPTWLLHIFIPFCIIAFIFIATVIA

LRKQLCQKLYSSKDTTKRPVTTTKREVNSAI

SEQ ID NO: 9 (polypeptide sequence of human PD-1) MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSN

TSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRN DSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVGVVGG LLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQWREKTP EPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWPL

SEQ ID NO: 10 (polypeptide sequence of human CTLA-4)

MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVCE

YASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTIQ GLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLFFY SFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN

SEQ ID NO: 11 (polypeptide sequence of human TIM-3)

MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGAC

PVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGI MNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQIS TLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKIQNLS LISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPLGCRFA

MP

SEQ ID NO: 12 (polypeptide sequence of human TIGIT)

MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVN

WEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGT YTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEG DLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLG NCSFFTETG

SEQ ID NO: 13 (polypeptide sequence of human Poliovirus Receptor (PVR))

MARAMAAAWPLLLVALLVLSWPPPGTGDVVVQAPTQVPGFLGDSVTLPCYLQVPNME

VTHVSQLTWARHGESGSMAVFHQTQGPSYSESKRLEFVAARLGAELRNASLRMFGLR

VEDEGNYTCLFVTFPQGSRSVDIWLRVLAKPQNTAEVQKVQLTGEPVPMARCVSTGGR

PPAQITWHSDLGGMPNTSQVPGFLSGTVTVTSLWILVPSSQVDGKNVTCKVEHESFEKP QLLTVNLTVYYPPEVSISGYDNNWYLGQNEATLTCDARSNPEPTGYNWSTTMGPLPPF AVAQGAQLL1RPVDKPINTTL1CNVTNALGARQAELTVQVKEGPPSEHSGISRNAI1FLVL GILVFLILLGIGIYFYWSKCSREVLWHCHLCPSSTEHASASANGHVSYSAVSRENSSSQD

PQTEGTR

SEQ ID NO: 14 (Poliovirus receptor-related 2 (PVRL2), homo sapiens)

MARAAALLPSRSPPTPLLWPLLLLLLLETGAQDVRVQVLPEVRGQLGGTVELPCHLLPP VPGLYISLVTWQRPDAPANHQNVAAFHPKMGPSFPSPKPGSERLSFVSAKQSTGQDTEA ELQDATLALHGLTVEDEGNYTCEFATFPKGSVRGMTWLRVIAKPKNQAEAQKVTFSQD PTTVALCISKEGRPPARISWLSSLDWEAKETQVSGTLAGTVTVTSRFTLVPSGRADGVT VTCKVEHESFEEPALIPVTLSVRYPPEVSISGYDDNWYLGRTDATLSCDVRSNPEPTGYD WSTTSGTFPTSAVAQGSQLVIHAVDSLFNTTFVCTVTNAVGMGRAEQVIFVRETPNTAG

AGATGGIIGGIIAAIIATAVAATGILICRQQRKEQTLQGAEEDEDLEGPPSYKPPTPKAKL EAQEMPSQLFTLGASEHSPLKTPYFDAGASCTEQEMPRYHELPTLEERSGPLHPGATSLG SPIPVPPGPPAVEDVSLDLEDEEGEEEEEYLDKINPIYDALSYSSPSDSYQGKGFVMSRA MYV

SEQ ID NO: 15 (polypeptide sequence of human LAG3)

MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRR AGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPL QPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTA SPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSP MDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRS FLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAII

TVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPW QCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLSLLLLVTGAF GFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEPEPEQ

SEQ ID NO: 16 (polypeptide sequence of human CD47)

MWPLVAALLLGSACCGSAQLLFNKTKSVEFTFCNDTVVIPCFVTNMEAQNTTEVYVK WKFKGRDIYTFDGALNKSTVPTDFSSAKIEVSQLLKGDASLKMDKSDAVSHTGNYTCE VTELTREGETIIELKYRVVSWFSPNENILIVIFPIFAILLFWGQFGIKTLKYRSGGMDEKTI ALLVAGLVITVIVIVGAILFVPGEYSLKNATGLGLIVTSTGILILLHYYVFSTAIGLTSFVIA ILVIQVIAYILAVVGLSLCIAACIPMHGPLLISGLSILALAQLLGLVYMKFVASNQKTIQPP RK A VEEPLN AFKES KGMMNDE SEQ ID NO: 17 (polypeptide sequence of human SIRPa)

MEPAGPAPGRLGPLLCLLLAASCAWSGVAGEEELQVIQPDKSVLVAAGETATLRCTAT

SLIPVGPIQWFRGAGPGRELIYNQKEGHFPRVTTVSDLTKRNNMDFSIRIGNITPADAGT

YYCVKFRKGSPDDVEFKSGAGTELSVRAKPSAPVVSGPAARATPQHTVSFTCESHGFSP

RDITLKWFKNGNELSDFQTNVDPVGESVSYSIHSTAKVVLTREDVHSQVICEVAHVTLQ

GDPLRGTANLSETIRVPPTLEVTQQPVRAENQVNVTCQVRKFYPQRLQLTWLENGNVS

RTETASTVTENKDGTYNWMSWLLVNVSAHRDDVKLTCQVEHDGQPAVSKSHDLKVS

AHPKEQGSNTAAENTGSNERNIYIVVGVVCTLLVALLMAALYLVRIRQKKAQGSTSSTR

LHEPEKNAREITQDTNDITYADLNLPKGKKPAPQAAEPNNHTEYASIQTSPQPASEDTLT

YADLDMVHLNRTPKQPAPKPEPSFSEYASVQVPRK

SEQ ID NO: 18 (polypeptide sequence of human CSF1)

MTAPGAAGRCPPTTWLGSLLLLVCLLASRSITEEVSEYCSHMIGSGHLQSLQRLIDSQME

TSCQITFEFVDQEQLKDPVCYLKKAFLLVQDIMEDTMRFRDNTPNAIAIVQLQELSLRLK

SCFTKDYEEHDKACVRTFYETPLQLLEKVKNVFNETKNLLDKDWNIFSKNCNNSFAEC

SSQDVVTKPDCNCLYPKAIPSSDPASVSPHQPLAPSMAPVAGLTWEDSEGTEGSSLLPGE

QPLHTVDPGSAKQRPPRSTCQSFEPPETPVVKDSTIGGSPQPRPSVGAFNPGMEDILDSA

MGTNWVPEEASGEASEIPVPQGTELSPSRPGGGSMQTEPARPSNFLSASSPLPASAKGQQ

PADVTGTALPRVGPVRPTGQDWNHTPQKTDHPSALLRDPPEPGSPRISSLRPQGLSNPST

LSAQPQLSRSHSSGSVLPLGELEGRRSTRDRRSPAEPEGGPASEGAARPLPRFNSVPLTDT GHERQSEGSFSPQLQESVFHLLVPSVILVLLAVGGLLFYRWRRRSHQEPQRADSPLEQPE GSPLTQDDRQVELPV

SEQ ID NO: 19 (polypeptide sequence of human CSF1R)

MGPGVLLLLLVATAWHGQGIPVIEPSVPELVVKPGATVTLRCVGNGSVEWDGPPSPHW

TLYSDGSSSILSTNNATFQNTGTYRCTEPGDPLGGSAAIHLYVKDPARPWNVLAQEVVV

FEDQDALLPCLLTDPVLEAGVSLVRVRGRPLMRHTNYSFSPWHGFTIHRAKFIQSQDYQ

CSALMGGRKVMSISIRLKVQKVIPGPPALTLVPAELVRIRGEAAQIVCSASSVDVNFDVF

LQHNNTKLAIPQQSDFHNNRYQKVLTLNLDQVDFQHAGNYSCVASNVQGKHSTSMFF

RVVESAYLNLSSEQNLIQEVTVGEGLNLKVMVEAYPGLQGFNWTYLGPFSDHQPEPKL

ANATTKDTYRHTFTLSLPRLKPSEAGRYSFLARNPGGWRALTFELTLRYPPEVSVIWTFI

NGSGTLLCAASGYPQPNVTWLQCSGHTDRCDEAQVLQVWDDPYPEVLSQEPFHKVTV

QSLLTVETLEHNQTYECRAHNSVGSGSWAFIPISAGAHTHPPDEFLFTPVVVACMSIMA

LLLLLLLLLLYKYKQKPKYQVRWKI1ESYEGNSYTF1DPTQLPYNEKWEFPRNNLQFGK TLGAGAFGKVVEATAFGLGKEDAVLKVAVKMLKSTAHADEKEALMSELKIMSHLGQH ENIVNLLGACTHGGPVLVITEYCCYGDLLNFLRRKAEAMLGPSLSPGQDPEGGVDYKNI HLEKKYVRRDSGFSSQGVDTYVEMRPVSTSSNDSFSEQDLDKEDGRPLELRDLLHFSSQ VAQGMAFLASKNCIHRDVAARNVLLTNGHVAKIGDFGLARDIMNDSNYIVKGNARLP VKWMAPESIFDCVYTVQSDVWSYGILLWEIFSLGLNPYPGILVNSKFYKLVKDGYQMA QPAFAPKNIYSIMQACWALEPTHRPTFQQICSFLQEQAQEDRRERDYTNLPSSSRSGGSG SSSSELEEESSSEHLTCCEQGDIAQPLLQPNNYQFC

Claims

CLAIMS What is claimed is:

1. A method of treating a cancer in a subject comprising administering to the subject a therapeutically effective amount of an engineered gastrointestinal tract (GI) bacterium, wherein the engineered GI bacterium comprises a vector comprising a polynucleotide that encodes IL-22 and/or IFN-P, or a functional fragment thereof.

2. The method of claim 1, wherein the subject is a human.

3. The method of claim 1 or claim 2, wherein the engineered GI bacterium is a specie of Escherichia genus or Lactobacillus genus.

4. The method of claim 3, wherein the engineered GI bacterium is Lactobacillus reuteri.

5. The method of claim 3, wherein the engineered GI bacterium is Escherichia coli.

6. The method of any one of claims 1 to 5, wherein the engineered GI bacterium localizes in the subject’s stomach, small intestine or colon.

7. The method of any one of claims 1 to 6, wherein the GI bacterium secretes IL-22 or a functional fragment thereof.

8. The method of any one of claims 1 to 7, wherein the GI bacterium secretes IFN-P or a functional fragment thereof.

9. The method of any one of claims 1 to 8, wherein the subject is resistant to an immune checkpoint inhibitor.

10. The method of any one of claims 1 to 9, wherein the treatment results in an increased level of an immune checkpoint molecule on a tumor cell compared to a control.

11. The method of claim 10, wherein the immune checkpoint molecule is PD-L1 or PD-L2. The method of any one of claims 1 to 11, further comprising administering to the subject a therapeutically effective amount of an immune checkpoint inhibitor. The method of claim 9 or claim 12, wherein the immune checkpoint inhibitor is a PD-L1 inhibitor, or a PD-L2 inhibitor. The method of any one of claims 1 to 13, further comprising administering irradiation to the subject. The method of claim 14, wherein the irradiation comprises total body irradiation or abdominal irradiation. The method of claim 14 or 15, wherein the irradiation is administered prior to the administration of the engineered GI bacterium. The method of any one of claims 1 to 16, wherein the engineered GI bacterium is administered to the subject via an oral administration. The method of any one of claims 1 to 17, wherein the dosage of the engineered GI bacterium is from about IxlO⁶ to about IxlO⁹ colony forming units (CFU). The method of any one of claims 1 to 18, wherein the treatment results in an increased number of T cells in a tumor compared to a control. The method of any one of claims 1 to 19, further administering to the subject a chemotherapeutic agent. The method of claim 20, wherein the chemotherapeutic agent is a platinum-based or taxane-based chemotherapeutic agent. The method of any one of claims 1- to 21, wherein the cancer is an ovarian cancer.