WO2025199253A2 - Enzymes for depletion of glutamine - Google Patents

Abstract

An isolated, modified gamma-glutamyl transpeptidase (GOT) enzyme with nucleotide of SEQ ID NO; 1 or SEQ ID NO:2 and encoding the protein of SEQ ID NO: 3, wherein the enzyme is coupled to a polyethylene glycol (PEG). The modified GGT enzyme depletes circulating glutamine without impacting circulating asparagine in mammals, increases circulating cystine/cystine in mammals, can be used in combination with L-cyst(e)inase to treat cancers, can be used with anti-TIGIT antibodies to treat cancers, can be used as an immunosuppressive agent and induces G-CSF and GM-CSF expression in mammals.

Description

ENZYMES FOR DEPLETION OF GLUTAMINE

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This invention claims the benefit of priority to United States Provisional Application No. 63/567,251, fi led on March 19, 2024, the contents of which are incorporated, herein by reference in its entirety.

ACKNOWLEDGMENT OF GOVERNMENT SUPPORT

[0002] This invention was made with government support under Grant R01GM143243 awarded by the National Institutes of Health. The government has certain rights in the invention.

FIELD

[0003] This invention relates generally to a recombinant enzyme which can be used to deplete circulating glutamine without impacting circulating asparagine in mammals.

BACKGROUND

[0004] Amino acids are building blocks for proteins and their dysregulated metabolism is responsible for various diseases like cancers, autoimmune diseases, sepsis, aging etc. Cells rely heavily on glutamine for proliferation and depleting circulating glutamine might hold a therapeutic value in various pathologies.

[0005] Tumorigenesis requires cancer cells to adapt cellular metabolism to utilize scarce nutrients that can sustain cellular proliferation¹. One of the most frequently documented metabolic changes in proliferating cancer cells is their addiction to glutamine, the most abundant amino acid in circulation (500 μM)². Glutamine is a carbon source by directly fueling the TCA cycle, and a nitrogen source by enabling nucleotide and protein synthesis^3-5 . This metabolic adaptation of cancer cells to utilize glutamine has been extensively documented in several preclinical and clinical studies, and glutamine metabolism is an attractive therapeutic target since cancer ceils are dependent on glutamine⁶. Indeed, the extensive pathways involved in glutamine metabolism including glutamine transporters, glutaminases, and aminotransferases have all been targeted as anti-tumor therapeutics ^7-9. [0006] Despite extensive in vitro data supporting the growth arrest of cancer cell lines in the absence of glutamine, the translation of drugs targeting glutamine metabolism has been largely unsuccessful. Small molecules used to prevent glutamine uptake by inhibiting the primary glutamine transporters, ASCT2, SNAT2, and SNAT1, have suffered from poor affinity, lack of specificity, and toxicity^{10- 12} . For example, V-9302 was reported as a high affinity inhibitor of ASCT2, but other studies suggest that it preferentially inhibits SNAT2 and. LAT1 , the latter being the primary transporter for essential amino acid uptake^13,14. The naturally occurring glutamine analog 6-diazo-5-oxo- L~norleucine (DON) inhibits multiple enzymes that utilize glutamine, including mitochondrial glutaminases but has translated poorly as a therapeutic primarily due to a narrow therapeutic index leading to gastrointestinal toxicity¹⁵. Although newer, safer prodrugs of DON like JHU-083 have been developed, their efficacy derives from not only an anti-proliferative effect directly on the tumor cells but also by inducing a strong antitumor immune response^16,17 An improved, understanding of the role of the immune system and the remarkable success of immunotherapies^18,19 has renewed focus on the role of extracellular glutamine within the tumor microenvironment (TME)^20,21. Several studies have shown that targeting glutamine metabolism directly inhibits tumor growth and enhances the anti-tumor response mediated by CD8⁺ T cells^{16, 1 7}’^22,23. By contrast, other reports suggest that interfering with glutamine metabolism either directly impacts the effector function and proliferation of CD8⁺ T cells, or acts indirectly by upregulating PDL1 expression on cancer cells and functioning as a metabolic checkpoint that licenses the function of type 1 conventional dendritic cells in activating CD8⁺ T cells^24-26. A fundamental question that needs to be answered is what is the integrated impact of inhibiting glutamine metabolism of both the cancer ceils and the immune system?

[0007] Provided herein is a recombinant enzyme which can be used to deplete circulating glutamine without impacting circulating asparagine in mammals. This enzyme can further be used to increase circulating cysteine/cystine levels for an extended period, which holds a therapeutic potential as a more bioavailable alternative to N-acetylcysteine. Furthermore, this enzyme could produce synergistic results when combined with anti- cancerous drugs like cyst(e)inase and anti-TIGIT antibodies. This enzyme can further be used to mobilize myeloid precursors from bone marrows SUMMARY OF THE INVENTION

[0008] The invention relates to a pegylated γ-glutamyl transpeptidase (PEG-GGT) enzyme comprising an isolated, modified γ-glutamyl transpeptidase (GGT) enzyme with nucleotide of SEQ ID NO: 1 or SEQ ID NO:2 and encoding the protein of SEQ ID NO: 3, wherein the enzyme is coupled to a polyethylene glycol (PEG), wherein the γ-glutamyl transpeptidase (GGT) enzyme is a Helicobacter pylori y-Glutamyltranspeptidase.

[0009] In one aspect, the invention provides a pharmaceutical composition comprising the disclosed PEG-GGT enzyme with at least one pharmaceutically acceptable excipient. In one embodiment, the composition is administered to a subject in need thereof in a. therapeutically effective amount to deplete circulating glutamine without affecting asparaginase activity. In another embodiment, glutamine depletion leads to an arrest in cell proliferation and induces a state of immunosuppression.

[0010] In another embodiment, the subject has a cancer. The cancer is selected at least from breast, pancreatic, and liver cancers. In one embodiment, glutamine is depleted and induces a state of immunosuppression in a tumor microenvironment (TME) of the cancer, wherein the induced a state of immunosuppression is marked by a notable enrichment in populations of PMN-MDSCs and M2 macrophages in the TME. In another embodiment, the composition is administered, to a subject in need thereof in a therapeutically effective amount to hydrolyze glutathione (GSH) and increase the concentration of circulating cysteine and glycine in the tumor of the subject.

[0011] In yet another embodiment, the subject has an autoimmune disease including acute respiratory distress syndrome (ARDS), asthma, rheumatoid arthritis (RA) and lupus nephritis, wherein glutamine is depleted and induces a state of immunosuppression in the subject with the autoimmune disease.

[0012] Another aspect of the invention provides a method of depleting circulating glutamine comprising administering to the host or the subject a therapeutically effective amount of the pharmaceutical composition comprising the disclosed PEG-GGT enzyme with at least one pharmaceutically acceptable excipient. In one embodiment, the pharmaceutical composition is administered intravenously, intraarterially, intraperitoneally, intralesionally, intramuscularly, intravesicularlly, intranasally by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a. catheter. [0013] Another embodiment further comprises administering at least a second anticancer therapy to the subject, wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy. In some embodiments, the second anticancer therapy is an asparaginase comprising asparaginases selected from those derived from E coli, Erwinia chrysanthemi, and pegylated asparaginases.

[0014] Yet another aspect of the invention provides a glutamine depletion gene signature comprising a glutamine depletion signature of upregulated genes having one or more genes comprising MCM10, POLQ, KNTC1, BRIP1, SLFN11, PARP1, TARBP 1, , , , , , , , , , , TUBA IB, CCND3, or the combination thereof; and a glutamine depletion signature of downregulated genes having one or more genes comprising SKIT, RPS23, UAP1L1, TPT1, FJF4B, KLHL24, INPP5K, PELFI, RPS14, RND3, TMEM242, ET.F2A, KLFIO, PDK1, YPEL5, GBE1, RTN2, RPL27A, CCNG2, UBXN1, IMEM71, SAIL WIPI1, SCAPER, TOLLTP, TAFID, S()STMI, STX5, or the combination thereof

[0015] One other aspect of the invention provides a method of using the glutamine depletion gene signature of claim 17, comprising a) obtaining a subject’s cancer sample; b) obtaining transcriptomic data from the sample; c) performing single sample GSEA. (ssGSEA), d) analyzing TCGA within cancer sample, e) determining a glutamine depletion score; and f) using the glutamine depletion score to determine the effect of glutamine depletion on the subjects prognosis and clinical outcomes. In one embodiment, the highest expression of glutamine depletion signature is labeled as a high glutamine depletion score and the lowest expression of glutamine depletion are labeled as a low glutamine depletion score. In another embodiment, the high glutamine depletion score is not associated with significantly favorable prognostic outcome in most cancers. BRIEF DESCRIPTIONS OF THE DRAWINGS

[0016] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate (one) several embodiment(s) of the invention and together with the description, serve to explain the principles of the invention.

[0017] Figures 1A-1H show H. pylori GGT is a high affinity glutaminase with no asparaginase activity. Figure 1 A shows the chemical reactions catalyzed by GGTs.

Figure IB shows the crystal structure of wild-type Helibacter pylori gamma-glutamyl transferase (hp-GGT). hp-GGT exists as aββa heterodimer. hp-GGT undergoes autocatalytic cleavage into two subunits and the N terminus Threonine of the smaller subunit acts as the nucleophile. The blue surface represents the catalytic center, and the yellow surface depicts the lid-loop structure. The hp-GGT crystal structure was obtained from PDB entry 2.XQO^{: :} and visualized in PyMOL. Figure 1C shows an image of SDS- PAGE of hp-GGT (lane 2) and hp-GGT T380A mutant (lane 3) lacking autocatalytic cleavage. Figure ID is a schematic showing the conjugation of NHS-PEG-5000 chains to lysine residues of hp-GGT to produce PEG-GGT Figure 1E is an image of SDS-PAGE of PEG-GGT (lane 2) and hp-GGT (lane 3). Figure IF is a graph showing Michaelis- Menten kinetic characterization of hydrolysis reaction for hp-GGT and PEG-GGT using γ-glutamyl analog yGPNA. Figure 1G is a plot showing relative hydrolysis rate of γGPNA by PEG-GGT in presence of equimolar amounts of glutamine (black) and asparagine (blue). Figure 1H is a plot of final asparagine concentration after incubation of 2.5 mM asparagine with 0.1 and 0.2 mg/ml of PEG-GGT for 8 hours. In Figure 1H, analysis was performed using a two-tailed student’s t-test with Welsch’s correction. In Figures IF, 1G and 1H, vertical bars show mean values with error bars representing SD (Standard Deviation). Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant.

[0018] Figures 2A-2I show that PEG-GGT inhibits the growth of multiple cell lines in vitro. Figure 2A is a schematic of MTT assay to assess growth kinetics of cell lines in vitro upon PEG-GGT treatment. Figures 2A-2C show relative growth of CT26 (FIG. 2B), 4T1 (FIG. 2C), and MC38 (FIG. 2D) cells in presence of glutamine (red), presence of glutamine and PEG-GGT (blue) and absence of glutamine (black) in vitro. Figures 2E-2F show relative levels of intracellular glutamate (FIG. 2E), glutamine (FIG. 2F) and total glutathione (FIG. 2G) in CT26 cells upon PEG-GGT treatment for 48 hours. Figures 2H and 21 show merged brightfield and GFP channel image of CT26 cells at 8, 16, and 24h after treatment with MSO, PEG-GGT, and combination of PEG-GGT and MSO in vitro (FIG. 2H). Plot representing average number of sytox positive cells per field of view (2 mm x 2 mm) at different time points (FIG. 21). Scale bar, 300 uM. In Figures 2B, 2C, 2D, 2E, 2F, 2G and 21, analysis was performed using two-tailed strident’ s t-test with Welsch’s correction and vertical bars show mean values with error bars representing SD. Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant. [0019] Figures 3 A-3K show that enzymatic depletion of circulating glutamine does not inhibit tumor growth in vivo. Figures 3A-3C show a schematic of PEG-GGT administration to study pharmacokinetics and pharmacodynamics (FIG. 3 A). Plot showing glutaminase activity (FIG. 3B) and concentration of glutamine and glutamate (FIG. 3C) in mouse serum over time after PEG-GGT administration. Figures 3D and 3G show a schematic of experimental design to study tumor growth dynamics upon PEG- GGT administration in vivo in CT26 (FIG. 3D) and 4T1 (FIG. 3G) tumor model. Figures 3 E and 3 H show plots of tumor growth over time in CT26 (FIG . 3E) and 4T1 (FIG . 3 H) tumor models. (n=5). Figures 3F and 31 are plots showing concentration of glutamine and glutamate in serum during PEG-GGT treatment in the CT26 (FIG. 3F) and 4T1 (FIG. 31) tumor model. Figures 3J and 3K are bar plots showing relative levels of amino acids as measured by LC-MS in serum (FIG. 31 J) and tumors (FIG. 31 K) of PEG-GGT treated. CT26 tumors. In Figures 3E, 3F, 3H, 31, 33 and 3K analysis was performed using two tailed student’s t-test with Welsch’s correction and vertical bars show mean values with error bars representing SD. Student’s t test: ***P < 0.001 , **p < 0.01 ; *p < 0.05; ns: not significant in vitro.

[0020] Figures 4A-4D show that transcriptomics reveals adaptation pathways upon glutamine depletion. Figure 4A is a schematic of the study design for RNA- Sequencing upon PEG-GGT treatment., (n 3). Figure 3B is a volcano plot showing differentially expressed genes in PEG-GGT -treated tumors compared to vehicle (PBS, 10% glycerol) treated tumors. Figure 3C is a plot showing differentially expressed hallmark gene sets (FDR<0.01) in PEG-GGT treated tumors compared to vehicle treated tumors. Figure 4D shows the visualization of differentially expressed (FDR Q value<0.()5) gene ontology biological processes (GOBP) clusters using cystoscape. Each dot in a cluster represents a GOBP gene set, red color indicates enriched pathways while blue indicates down regulated pathways in PEG-GGT treated group. The thickness of the line connecting two dots represents shared genes in the GOBP datasets. [0021] Figures 5A-5J show that enzymatic depletion of circulating glutamine is immunosuppressive. Figures 5 A and. 5B show enrichment plots (left) and heatmap (right) of genes involved in IFN-a (FIG- 5 A) and TNF-a (FIG 5B) response upon PEG-GGT treatment. Figures 5C and 5D are plots representing relative fraction of immune cells in PEG-GGT and vehicle treated mice calculated from the deconvolution of RNA-Seq data (FIG 5C), B B cells, T -T cells, NK--NK cells, DC-Dendritic cells, GN--- Granulocytes, MF -Macrophages. Heatmap of Ml and M2 transcripts in PEG-GGT and vehicle treated mice (FIG 5D). Figure 5E is a violin plot of percentage of CD206^high macrophages in PEG-GGT and vehicle treated group. (Vehicle: n=6, PEG-GGT: n=8). Figures 5F, 5G and 5h violin plots of percentage of PMN-MDSCs and Cd1 1 b GR- 1 cells in PEG-GGT treated tumors (FIG. 5F, FIG. 5H). Bar plot of relative mRNA expression of G-CSF and GM-CSF transcripts upon PEG-GGT treatment in CT'26 cell line in vitro (G) (n=4). Figures 51 and 5J are violin plot showing percentage of CD8 (FIG. 51) and NK (FIG. 51) cells in PEG-GGT and vehicle treated tumors. In Figures 5C, 5E, 5F, 5G, 5H, 51 and 5J, analysis was performed using two-tailed student’s t-test with Welsch’s correction. In Figures. 5C and FIG 5G, vertical bars show mean values with error bars representing SD. In Figures 5 A, 5B and 5D, the scale of the heatmaps represent Z-score. Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant.

[0022] Figures 6A-6K show that glutamine depletion is not associated with favorable outcomes in human cancers. Figure 6A shows a schematic representing the construction of gene signature associated with glutamine depletion in human cancers and workflow for TCGA analysis of this gene signature. Figures 6B, 6C, 6D are violin plots showing ssGSEA glutamine depletion score for breast cancer categorized by tumor size (FIG. 6B), molecular subtype (FIG. 6C) and stage (FIG. 6D). Figures 6E, 6H, 6J are Kaplan-Meier plots of five-year overall survival probability for glutamine depletion high and glutamine depletion low groups in breast (FIG. 6E), pancreatic (FIG. 6H), and liver (FIG. 6J) cancers. FIG. 6F shows a heatmap of PMN-MDSC gene signature in PEG-GGT treated tumors. Figures 6G, 61, 6K are violin plot of PMN-MDSC score for glutamine depletion high and. glutamine depletion low groups in breast (FIG. 6G), pancreatic (FIG. 61), and liver (FIG. 6K) cancers. In Figures 6B, 6C, 6D, 6G, 61 and 6K, analysis was performed, using two-tailed, student’s t-test with Welsch’s correction. In Figure 6F, the scale of the heatmap represents a Z-score. Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant. In Figures 6E, 6H and 6J, survival analysis was performed using the Log-Rank test. [0023] Figures 7A-7E show that. PEG-GGT is stable in FBS, and its activity is required for growth inhibitory effect in vitro. Figures 7 A is a plot showing PEG-GGT mediated γGPNA hydrolysis rate over time when incubated in serum. Figure 7B shows a relative number of CT26 (FIG. 7B), 4T1 (FIG. 7C), and MC38 (FIG. 7D) cells after 24h in presence of glutamine (red) and presence of glutamine and heat inactivated PEG-GGT (blue). (n=3). Figure 9C is a schematic showing the process for extracting metabolites from CT26 cells in vitro. Figure 7D is a bar plot showing a relative number of CT26 cells when left untreated or treated with PEG-GGT and supplemented with either GSH or NAC. Figure 7E are light microscopy images showing cells when left untreated or treated with PEG-GGT and supplemented with either GSH or NAC. Scale bar, 300 μM. In Figures 7B and 7D, analysis was performed using two-tailed student’s t-test with Welsch’s correction. In Figures 7 A and 7B, vertical bars show mean values with error bars representing SD. Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant.

[0024] Figures 8A-8F show that enzymatic depletion of circulating glutamine does not inhibit tumor growth m vivo. Figures 8 A and 8B are plots showing average percentage weight change in PEG-GGT and vehicle treated group in CT26 (FIG. 8A) and 4T1 (FIG. 8B) tumor model. (n=5). Figure 8C is a bar plot showing relative concentration of glutamine in PEG-GGT, and vehicle treated CI'26 tumors. Figure 8D is a schematic showing the procedure for extracting metabolites from tumor tissue for LC-MS. Figures 8E and 8F are heatmaps showing relative amounts of amino acids in serum and tumor upon PEG-GGT treatment. In Figures 8A, 8B and 8C analysis was performed using two- tailed student’s t-test with Welsch’s correction and vertical bars show mean values with error bars representing SD. In Figures 8E and 8F, the scale of the heatmaps represent Z- score. Student’s t test: ***p < 0.001 ; **p < 0.01; *p < 0.05; ns: not significant.

[0025] Figures 9A-9H shows that transcriptomics reveals adaptation pathways upon glutamine depletion. Figures 9A-9F are enrichment plots (left) and heatmap of genes (right) for .AIR (FIG. 9 A), E2F (FIG. 9B), mTORC1 (FIG. 9C), Myc (FIG. 9D) TFNy signaling (FIG. 9E); and protein translation (FIG. 9F). Figure 9G is a bar plot showing mRNA fold change of genes related to ceil cycle in CT26 cell line treated with PEG-GGT in vitro. (n=3). Figure 9H is a plot showing relative number of CT26 cells after combined treatment of mTORC1 inhibitor temsirolimus and PEG-GGT compared to PEG-GGT and temsirolimus alone in vitro. (n=3). In Figures 9G and 9H, analysis was performed using two-tailed student’s t-test with Welsch’s con-ection and vertical bars show mean values with error bars representing SD. In figures 9A-9F, the scale of the heatmaps represent Z-score. Student’s t test: ***p < 0.001; **p < 0.01; *p < 0.05; ns: not significant.

[0026] Figures 10A-10G show that enzymatic depletion of circulating glutamine is immunosuppressive. Figure 10A is a design of lymphoid and myeloid flow panel. Figure 10B is a violin plot showing percentage of B cells in PEG-GGT, and vehicle treated tumors. Figures 10C, 10D, 10E, and 10F are violin plots of percentage of CD11b- CDI l c+ dendritic cells (FIG. IOC), CD4+ T cells, (FIG. 10D), l^;oxP3 • Tregs (FIG. 10E) and. monocytic MDSCs (FIG. 10F) in PEG-GGT, and vehicle-treated tumors. Figure 10G is aa gating strategy for CD1 lb+GR-l+ cells. In Figures 10B, 10C, 10D, 10E and 1 OF, analysis was performed using two-tailed student’s t-test with Welsch’s correction.

Student’s t test: ***p < 0.001; **p < 0.01 ; *p < 0.05; ns: not significant.

[0027] Figures 11A-1 IF show that glutamine depletion is not associated with favorable outcomes in human cancers. Figures 11 A- 1 IF are Kaplan-Mei er plot of five- year overall survival probability for glutamine depletion high and glutamine depletion low groups in melanoma (FIG. 11 A), kidney renal cell carcinoma (FIG. 11B), head and neck squamous cell carcinoma. (FIG. 11C) sarcoma (FIG. 1 1D), cervical squamous cell carcinoma (FIG. 1 IE) and stomach adenocarcinoma (FIG. 11F). In Figures 11 A-11F, analysis was performed using the Log-Rank test.

DETAILED DESCRIPTION

[0028] The present invention may be understood more readily by reference to the following detailed description of preferred embodiments of the invention and the Examples included therein and to the Figures and their previous and following description.

I. Definitions

[0029] To facilitate an understanding of the principles and features of the various embodiments of the disclosure, various illustrative embodiments are explained herein. Although exemplary embodiments of the disclosure are explained in detail, it is to be understood that other embodiments are contemplated. Accordingly, it is not intended that the disclosure is limited in its scope to the details of construction and arrangement of components set forth in the description or examples. The disclosure is capable of other embodiments and of being practiced or carried out in various ways. [0030] In describing the exemplaiy embodiments, specific terminology will be resorted to for the sake of clarity'. As used in the specification and the appended claims, the singular forms “a,” “an” and “the” include plural references unless the context clearly dictates otherwise. For example, reference to a component is intended also to include composition of a plurality of components. References to a. composition containing “a” constituent is intended to include other constituents in addition to the one named.

[0031] Ranges may be expressed herein as from “about” or “approximately” or “ substantially” one particular value and/or to “about” or “approximately” or “substantially” another particular value. When such a range is expressed, other exemplaiy embodiments include from the one particular value and/or to the other particular value. [0032] The term “cancer” includes, but is not limited to, the following proliferative diseases: Acute Lymphoblastic Leukemia (ALL), Acute Myeloid Leukemia. (AML), Adrenocortical Carcinomas, Childhood cancers, AIDS-Related Cancers, Kaposi Sarcoma, AIDS-Related Lymphoma, Primary/ CNS Lymphoma, Anal Cancer, Astrocytomas, Atypical Teratoid/Rhabdoid Tumor, Basal Cell Carcinoma, Skin Cancer (Nonmelanoma), Bile Duct Cancer, Bladder Cancer, Bone Cancer, Ewing Sarcoma Family of Tumors, Osteosarcoma, and Malignant Fibrous Histiocytoma, Brain Stem Glioma, Atypical Teratoid/Rhabdoid Tumor, Embryonal Tumors, Germ Cell Tumors, Craniopharyngioma, Ependymoma, Breast Cancer, Bronchial Tumors, Burkitt Lymphoma, Non-Hodgkin Lymphoma, Carcinoid. Tumor, Gastrointestinal Carcinoma, Cardiac (Heart) Tumors, Primary- Lymphoma, Cervical Cancer, Cholangiocarcinoma, Chordoma, Chronic Lymphocytic Leukemia (CLL), Chronic Myelogenous Leukemia (CML), Chronic Myeloproliferative Neoplasms, Colon Cancer, Colorectal Cancer, Craniopharyngioma, Cutaneous T-Cell Lymphoma, Mycosis Fungoides and Sezary Syndrome, Ductal Carcinoma. In Situ (DCIS), Embryonal Tumors, Endometrial Cancer, Ependymoma, Esophageal Cancer, Esthesioneuroblastoma, Extracranial Germ Cell Tumor, Extragonadal Germ Cell Tumor, Eye Cancer, Intraocular Melanoma, Retinoblastoma, Fallopian Tube Cancer, Fibrous Histiocytoma of Bone, Malignant, and Osteosarcoma, Gallbladder Cancer, Gastric (Stomach) Cancer, Gastrointestinal Carcinoid Tumor, Gastrointestinal Stromal Tumors (GIST), Germ Cell Tumor, Ovarian, Testicular, Gestational Trophoblastic Disease, Glioma, Hairy Cell Leukemia, Head and

Neck Cancer, Hepatocellular (Liver) Cancer, Histiocytosis, Langerhans Cell, Hodgkin Lymphoma, Hypopharyngeal Cancer, Islet Cell Tumors, Pancreatic Neuroendocrine Tumors, Kaposi Sarcoma, Kidney, Renal Cell, Langerhans Cell Histiocytosis, Laryngeal Cancer, Leukemia, Acute Lymphoblastic (ALL), Acute Myeloid (AML), Chronic Lymphocytic (CLL), Chronic Myelogenous (CML), Hairy Cell, Lip and Oral Cavity Cancer, Liver Cancer (Primary), Lung Cancer, Non-Small Cell, Small Cell, Lymphoma, Hodgkin, Non-Hodgkin, Macroglobulinemia, Waldenstrom, Male Breast Cancer, Melanoma, Merkel Cell Carcinoma, Mesothelioma, Metastatic Squamous Neck Cancer with Occult Primary, Midline Tract Carcinoma Involving NUT Gene, Mouth Cancer, Multiple Endocrine Neoplasia. Syndromes, Multiple Myeloma/Plasma Cell Neoplasm, Mycosis Fungoides, Myelodysplastic Syndromes, Myelodysplastic/Myeloproliferative Neoplasms, Myelogenous Leukemia, Chronic (CML), Myeloid Leukemia, Acute (.AML) Myeloma, Multiple, Myeloproliferative Neoplasms, Nasal Cavity and Paranasal Sinus Cancer, Nasopharyngeal Cancer, Neuroblastoma, Non-Hodgkin Lymphoma, Non-Small Cell Lung Cancer, Oral Cancer, Oral Cavity Cancer, Lip and Oropharyngeal Cancer, Osteosarcoma and Malignant Fibrous Histiocytoma of Bone, Ovarian Cancer, Low Malignant Potential Tumor, Pancreatic Cancer, Pancreatic Neuroendocrine Tumors (Islet Cell Tumors), Papillomatosis, Paraganglioma, Paranasal Sinus and Nasal Cavity Cancer,

Parathyroid Cancer, Penile Cancer, Pharyngeal Cancer, Pheochromocytoma, Pituitary Tumor, Plasma Cell Neoplasm/Multiple Myeloma, Pleuropulmonary Blastoma, Pregnancy and Breast Cancer, Primary Central Nervous System (CNS) Lymphoma, Primary Peritoneal Cancer, Prostate Cancer, Rectal Cancer, Renal Cell (Kidney) Cancer, Renal Pelvis and Ureter, Transitional Cell Cancer, Retinoblastoma, Rhabdomyosarcoma, Salivary' Gland Cancer, Rhabdomyosarcoma, Uterine, Small Intestine Cancer, Soft Tissue Sarcoma, Squamous Cell Carcinoma, Squamous Neck Cancer with Occult Primary, Metastatic, Stomach (Gastric) Cancer, T-Cell Lymphoma, Testicular Cancer, Throat Cancer, Thymoma and Thymic Carcinoma, Thyroid Cancer, Transitional Cell Cancer of the Renal Pelvis and Ureter, Unknown Primary', Ureter and Renal Pelvis, Transitional Cell Cancer, Urethral Cancer, Uterine Cancer, Endometrial, Uterine Sarcoma, Vaginal Cancer, Vulvar Cancer, Waldenstrom Macroglobulinemia, and Wilms Tumor.

[0033] The term “treatment' ’ as used herein includes any treatment of a. condition or disease in a subject, or particularly a human, and may include: (i) preventing the disease or condition from occurring in the subject which may be predisposed to the disease but has not yet been diagnosed as having it; (ii) inhibiting the disease or condition, i.e., arresting its development; relieving the disease or condition, i.e., causing regression of the condition; or (iii) ameliorating or relieving the conditions caused by the disease, i.e., symptoms of the disease. “Treatment,” as used herein, could be used in combination with other standard therapies or alone.

[0034] The term “effective” is used to describe an amount of a compound, composition or component which, when used within the context of its intended use, effects an intended result.

[0035] In certain embodiments, the present invention relates to methods of treating or preventing cancer, such as breast cancer, colorectal cancer, endocrine cancer, melanoma, renal cancer or B cell malignancy, with the pharmarceutical composition disclosed herein, or a pharmaceutically acceptable salt thereof. In certain embodiments. [0036] The term “effective amount” refers to that amount which is sufficient to effect treatment, as defined herein, when administered to a. subject in need of such treatment. The effective amount wall vary depending on the subject and disease state being treated, the severity of the affliction and the manner of administration, and may be determined routinely by one of ordinary skill in the art.

[0037] The term “pharmaceutically acceptable salt” is used throughout the specification to describe, where applicable, a salt form of one or more of the compounds or prodmgs described herein which are presented, to increase the solubility of the compound in the gastric or gastroenteric juices of the patient's gastrointestinal tract in order to promote dissolution and the bioavailability of the compounds. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic or organic bases and acids, where applicable. Suitable salts include those derived from alkali metals such as potassium and sodium, alkaline earth metals such as calcium, magnesium and ammonium salts, among numerous other acids and bases wdl known in the pharmaceutical art. Sodium and potassium salts are particularly preferred as neutralization salts of the phosphates according to the present invention. In a preferred embodiment, the description provides pharmaceutically acceptable salts of the modified peptides as described herein, which retain the biological effectiveness and properties of the parent compounds, and which are not biologically or otherwise harmful as the dosage administered. The compounds of this invention are capable of forming both acid and base salts by virtue of the presence of amino and carboxy groups respectively.

[0038] Enzymes can be identified by their specific activities. This definition thus includes all polypeptides that have the defined specific activity also present in other organisms, more particularly in other microorganisms. Often enzymes with similar activities can be identified by their grouping to certain families defined as PFAM or COG. PFAM (protein family database of alignments and hidden Markov models; http://pfarn.sanfferac.ukl) represents a large collection of protein sequence alignments. Each PFAM makes it possible to visualize multiple alignments, see protein domains, evaluate distribution among organisms, gain access to other databases, and visualize known protein structures. COGs (Clusters of Orthologous Groups of proteins; http://w- ww.nebi.nhn.nih.gov/COG/) are obtained by comparing protein sequences from 43 fully sequenced genomes representing 30 major phylogenetic lines. Each COG is defined from at least three lines, which permits the identification of former conserved domains.

[0039] The means of identifying homologous sequences and their percentage homology and/or identity are well known to those skilled in the art and include in particular the BLAST programs. The sequences obtained can then be exploited (e.g., aligned) using, for example, the programs CLUSTALW or MULTALIN with the default parameters indicated on those websites. Using the references given on GenBank for known genes, those skilled in the art are able to determine the equivalent genes in other organisms, bacterial strains, yeasts, fungi, mammals, plants, etc. This routine work is advantageously done using consensus sequences that can be determined by carrying out sequence alignments with genes derived from other microorganisms and designing degenerate probes to clone the corresponding gene in another organism. These routine methods of molecular biology are well known to those skilled in the art, and are described, for example, in Sambrook et. AI ¹⁰² Indeed, a person skilled in the art will understand how to select and design homologous proteins retaining substantially their γ- glutamy l trail speptidase activity .

[0040] n. Compositions

A. Glutamine

[0041] Provided herein are compositions for depleting circulating glutamine in a subject in need thereof. Glutamine plays an important role as a carrier of nitrogen, carbon, and energy. It is used for hepatic urea synthesis, for renal a mm oniagenesis, for gluconeogenesis, and as respiratory fuel for many cells. Cells get. their glutamine by either synthesizing it internally via an enzyme called glutamine synthetase (GS) or exogenously from the environment. [0042] The conversion of glutamine into glutamate is initiated by the mitochondrial enzyme, glutaminase. There are two major forms of the enzyme, K-type and L-type, which are distinguished by their Km values for glutamine and response to glutamate, wherein the Km value, or Michaelis constant, is the concentration of substrate required to reach half the maximal velocity. The L-type, also known as “liver-type'’ or GLS2, has a high Km for glutamine and is glutamate resistant. The K-type, also known as “kidney- type” or GLS1 or “KGA”, has a low Km for glutamine and is inhibited by glutamate. An alternative splice form of GLS 1 , referred to as glutaminase C or “GAC”, has recently been identified. i. The Role of Glutamine in Cancer

[0043] In addition to serving as the basic building blocks of protein synthesis, amino acids have been shown to contribute to many processes critical for growing and dividing cells, and this is particularly true for cancer cells. Nearly all definitions of cancer include reference to dysregulated proliferation. Numerous studies on glutamine metabolism in cancer indicate that many tumors are avid glutamine consumers^103-106, and this includes, but not limited to breast cancer. Certain embodiments of the invention relate to the use of the compounds described herein for the treatment of breast cancer.

[0044] Glutamine supports cell survival, growth and proliferation through metabolic and non-metabolic mechanisms. In actively proliferating cells, the metabolism of glutamine to lactate, also referred to as “glutaminolysis” is a major source of energy in the form of NADPH. The first step in glutaminolysis is the deamination of glutamine to form glutamate and ammonia, which is catalyzed by the glutaminase enzyme (GLS). Thus, deamination via. glutaminase is a control point for glutamine metabolism.

[0045] Ever since Warburg's observation that ascites tumor cells exhibited high rates of glucose consumpti on and lactate secretion in the presence of oxygen ¹⁰⁷(), researchers have been exploring how cancer cells utilize metabolic pathways to be able to continue actively proliferating. Several reports have demonstrated how glutamine metabolism supports macromolecular synthesis necessary for cells to replicate¹⁰⁸’¹⁰⁹ [0046] While many cancer cells depend on exogenous glutamine for survival, the degree of glutamine dependence among tumor cell subtypes may make a population of cells more susceptible to the reduction of glutamine. As an example, gene expression analysis of breast cancers has identified five intrinsic subtypes (luminal A, luminal B, basal, HER2+, and normal-like)¹¹⁰. Although glutamine deprivation has an impact on cell growth and viability, basal-like cells appear to be more sensitive to the reduction of exogenous glutamine¹¹¹. This supports the concept that glutamine is a very important energy source in basal -like breast cancer cell lines and suggests that inhibi tion of the glutaminase enzyme would be beneficial in the treatment of breast cancers comprised of basal-like cells.

[0047] Thus, glutaminase has been theorized to be a potential therapeutic target for the treatment of diseases characterized by actively proliferating cells, such as cancer. The lack of suitable glutaminase inhibitors has made validation of this target impossible. Therefore, the creation of glutaminase inhibitors that are specific and capable of being formulated for in vivo use could lead to a new class of therapeutics. ii. Glutamine Depletion

[0048] Glutamine contributes to major anabolic pathways and is consumed at a high rate during cell growth which often leads to its depletion in the tumor core²⁵.

Disclosed herein are studies showing how the depletion of glutamine alters the tumor and immunological landscape in vivo. Disclosed herein are compositions that leverage the high affinity glutaminase activity (low K_M) of H. pylori GGT (hp-GGT) and repurpose it as a potent in vivo glutaminase (with no asparaginase activity). Furthermore, the disclosed compositions are pegylated to produce PEGylated hp-GGT (PEG-GGT) with a half-life of 67 ± 8 hours in serum, and a. dose of 20 mg/kg which can efficiently eliminate circulating glutamine to undetectable levels without affecting asparagine for at least 48 hours in BALB/c mice.

[0049] When extracellular glutamine is abundant, intracellular glutamine triggers acetylation dependent proteasomal degradation of glutamine synthetase (Glul), the only protein capable of intracellular glutamine synthesis⁵''. -As extracellular glutamine becomes limiting, Glul degradation is inhibited, and it synthesizes glutamine intracellularly by ligating ammonia and glutamate^{5 7}’³⁸. Since Glul is degraded upon glutamine accumulation, intracellular glutamine concentration can reach only a particular threshold via Glul. Hence, glutamine flux is redirected towards nucleotide and DNA synthesis while flux towards protein synthesis is reduced, by global downregulation of translation. [0050] Disclosed herein are metabolomic studies showing decreased levels of proline and GSH upon depletion of extracellular glutamine. In multiple human cancer cell lines, reducing proline biosynthesis conserved glutamate and allowed cells to proliferate in glutamine limiting conditions providing a potential mechanism to adapt to glutamine depletion⁴⁶. In pancreatic cancer models, cells adapted to grow in low glutamine concentration (100 μM) had significantly increased Glut protein although changes in mRNA levels were not different⁶⁵. Metabolomic and transcriptomic analysis showed that adapted clones indeed had a low concentration of intracellular glutamine, a significant flux of glutamine towards nucleotide synthesis, and increased mTORC1 signaling to avoid Glut degradation The disclosed metabolomics and transcriptomic results align well with this model.

[0051] As glutamine is indispensable for hexosamine biosynthesis, depletion of glutamine induces Endoplasmic reticulum (ER) stress which increases G-CSF and GM- CSF secretion from tumors via Irela-Jnk pathway⁶⁵. Consistent with increased. ER stress, the disclosed data shows genes related to unfolded protein response were upregulated in tumors upon PEG-GGT treatment. Treatment with PEG-GGT also significantly increased G-CSF and GM-CSF transcripts in CT26 cells in vitro. In mice, G-CSF and GM-CSF mobilize myeloid precursors from the bone marrow to the tumor, where they differentiate into PMN-MDSCs^65-00. Our flow cytometry data on PEG-GGT treated tumors showed pronounced infiltration of PMN-MDSCs into the tumors.

[0052] Polarization of M0 macrophages to Ml or M2 macrophages is influenced by the ratio of a-ketoglutarate (cx-KG) to succinate⁷⁹. Increased cx-KG demethylates repressive H3K27Me3 M2 promoters via Jmjd3 while succinate inhibits Jmjd.3 and promotes Ml phenotype by stabilizing HIF1α⁸⁰. In cell culture, contradictory results have been reported. While one study reported that glutamine deprivation impaired the expression of M2 markers which was restored by treatment with cell-permeable dimethyl- α -KG⁷⁹, another study reported that depriving M0 macrophages of glutamine promoted M2 phenotype without any cytokines by increased expression of Glul, which reduced the GABA shunt mediated flux of glutamate towards succinate⁸¹. The invention discloses an increase in M2 macrophages in PEG-GGT treated tumors. While the data supports the latter observation, the possibility of the former cannot be ruled out. Since macrophages have been shown to uptake glutamate via SLC1A2^81,82 , which is abundant in circulation upon glutamine hydrolysis, these cells might be able to maintain their α -KG pool.

[0053] To investigate whether glutamine depletion could be a viable strategy in human cancers, the inventors developed a. gene signature associated with glutamine depletion in cancers. The invention discloses that high glutamine depletion signature was not associated with favorable outcomes in most cancers, rather it was significantly unfavorable for pancreatic, liver, and skin cancers. Glutamine depletion signature was positively correlated with tumor size, stage, and aggressiveness in breast cancers.

Moreover, a high glutamine depletion score was associated with increased, infiltration of PMN-MDSC in breast, pancreatic, and liver cancers. Since glutamine is often depleted in the core of the TME²⁵, attempts to reinstate glutamine levels in the core might lift the immunological barriers created by tumor cells in the TME

B. Asparagine

[0054] The compositions and methods presented herein are different from other studies within the art that have utilized highly efficient glutaminases-asparaginases of bacterial origin to systemically deplete glutamine. Provided herein are methods of depleting circulating glutamine without affecting aparagine.

[0055] Asparagine is a naturally occurring amino acid that is found in many proteins. Asparagine synthetase (ASNS) is involved in asparagine biosynthesis. L- asparaginase (L-ASP) is an enzyme that catabolizes asparagine.

[0056] Many tumor cells exhibit high rates of glutamine consumption to support macromolecular biosynthesis and cell proliferation!. Glutamine fuels the tricarboxylic acid (TCA) cycle through anaplerosis and. contributes to the synthesis of lipids, nucleotides and non-essential amino acids. Although glutamine can contribute to synthesis of several amino acids through its catabolism to glutamate, only asparagine requires glutamine for de novo synthesis, glutamine is a substrate for asparagine synthetase (ASNS). ASNS activity is unidirectional and A TP-dependent, suggesting that cells synthesize asparagine at the expense of macromolecule synthesis and cellular energy.

[0057] As de novo asparagine synthesis is dependent on glutamine, asparagine becomes an essential amino acid when glutamine is absent''⁷. Furthermore, asparagine directly binds to LCK to promote its activity and enhance T cell activation⁷⁸. Since the bacterial glutaminase-asparaginases deplete glutamine and asparagine simultaneously, the inventors hypothesized that depleting glutamine while preserving asparagine provides a specific tool to investigate the selective impact of glutamine depletion. As there have been no reports of high affinity glutaminases (K_M in the micromolar range) with no concomitant asparaginase activity, the disclosed provides the needed tool for depleting glutamine while preserving asparagine.

[0058] C. Helicobacter pylori γ-Glutamyltranspeptidase

[0059] Helicobacter pylori (H. pylori) is a Gram-negative bacterial pathogen that colonizes the gastric mucosa. Infection puts the individual at greater risk for developing gastritis, peptic ulcer disease, and gastric cancer^{1 12}. H. pylori γ-glutamyltranspeptidase (Hp-GGT) 3 is a glutathione-degrading enzyme that has been shown to be a virulence factor in infection^{1 13}. H. pylori lacking γ-glutamyltranspeptidase has been shown to grow normally in vitro but exhibits diminished, growth rates within the gut in animal model systems. Bacterial loads of the Hp-GGT-deficient strains are reduced by nearly 70% relative to the parental strain. Although not essential for colonization, Hp-GGT clearly confers a growth advantage to the bacteria, in vivo by mechanisms that remain unclear. Hp-GGT has also been shown to up-regulate COX-2 and epidermal growth factor-related peptides in human gastric mucosal cells¹¹⁴ and to induce apoptosis in human gastric epithelial cells (6). Both these activities are abolished by inactivation of the enzyme with mechanism-based inhibitors. Despite its demonstrated involvement, in H. pylori colonization, persistence, and disease progression, biochemical characterizations of Hp- GGT have been limited.

[0060] The reclamation of extracellular glutathione and its conjugates is initiated by γ-glutamyltranspeptidase (yGT). The enzyme cleaves the γ-glutamyl amide bond to liberate cysteinylglycine, and the catalytic mechanism proceeds via a γ-glutamyl -enzyme intermediate¹¹⁵. The γ-glutamyl group can be transferred to water (hydrolysis) or to an amino acid or short, peptide (transpeptidation). Whereas mammalian yGTs are embedded in the plasma membrane by a single N-terminal transmembrane anchor and are heterologously glycosylated, bacterial homologs are soluble and localized to the periplasmic space. Overall, the yGTs are highly conserved, with mammalian and bacterial homologs often sharing >25% sequence identity.

[0061] Several studies have shown that targeting glutamine metabolism directly inhibits tumor growth and enhances the anti-tumor response mediated by CD8⁺ T cells¹¹⁶' ¹¹⁹¹. By contrast, other reports suggest that interfering with glutamine metabolism either directly impacts the effector function and proliferation of CD8⁺ T cells, or acts indirectly by upregulating PDL1 expression on cancer cells and functioning as a metabolic checkpoint that licenses the function of type 1 conventional dendritic cells in activating CDS ¹ T cells¹²⁰"¹²² A fundamental question that needs to be answered is what is the integrated impact of inhibiting glutamine metabolism of both the cancer cells and the immune system?

[0062] To answer this fundamental question about the pro and anti-tumor function of glutamine in vivo, the inventors used an enzymatic approach since enzymes can facilitate the highly specific depletion of extracellular glutamine. The studies disclosed herein show that Helicobacter pylori (H. pylori) GGT (hp-GGT) as an enzyme has no asparaginase activity and that PEGylated-hp-GGT (PEG-GGT) can be used to efficiently deplete circulating glutamine while preserving asparagine in living animals. Using syngeneic models of breast and colon cancers, the data shows that depleting circulating glutamine does not inhibit tumor growth in vivo, and this is primarily due to the enrichment of myeloid derived suppressive cells (MDSC). Based on the RNA-Seq with PEG-GGT treated cancers in mice, a signature for glutamine depletion was derived. Using this glutamine depletion score, the inventors analyzed TCGA and demonstrated that glutamine depletion scores are not associated with increased survival benefit in diverse human cancers. Consistent with the animal studies, glutamine depletion is associated with a high frequency of MDSCs in human tumors. i. Helicobacter pylori γ-Glutamyltranspeptidase sequences a. Plasmid sequence for hpGGT production:

[0063] Provided herein is the nucleic acid sequence for helicobacter pylori y- Glutamyltranspeptidase: G A TA A AG C G ii. PEG-GGT

[0066] Numerous biopharmaceuticals have successfully been PEGylated and marketed for many years. In order to couple PEG to a protein, the PEG has to be activated at its OH terminus. The activation group is chosen based on the available reactive group on the protein that will be PEGylated. In the case of proteins, the most important amino acids are lysine, cysteine, glutamic acid, aspartic acid, C -terminal carboxylic acid and the N-terminal amino group. In view of the wide range of reactive groups in a protein nearly the entire peptide chemistry- has been applied to activate the PEG moiety. Examples for this activated PEG-reagent are activated carbonates, e.g., p-nitrophenyl carbonate, succinimidyl carbonate; active esters, e.g., succinimidyl ester; and for site specific coupling aldehy des and maleimides have been developed¹²³. The availability of various chemical methods for PEG modification show's that each new development of a PEGylated protein will be a case by case study. In addition to the chemistry the molecular weight of the PEG that is attached to the protein has a strong impact on the pharmaceutical properties of the PEGylated protein. In most cases it is expected that, the higher the molecular weight of the PEG, the better the improvement of the pharmaceutical properties¹²⁴. For example, Holtsberg et al. found that, when PEG was conjugated to arginine deaminase, another amino acid degrading enzyme isolated from a microbial source, pharmacokinetic and pharmacodynamic function of the enzyme increased as the size of the PEG attachment increased from a molecular weight of 5000 Da to 20,000 Da¹²³. However, in many cases, PEGylated biopharmaceuticals show significantly reduced activity compared to the unmodified biopharmaceutical¹²⁶.

[0067] Multiple studies have tried to target the glutamine metabolism of tumors for therapeutic benefit. Enzymatic depletion of circulating glutamine via Pseudomonas 7 A and Acinelobacter asparaginase glutaminase had no significant effect on the growth of syngeneic solid tumor models including non -metastatic breast cancer (EO771 ) and metastatic melanoma (B16). These studies however documented severe lymphodepletion and splenic weight loss in treated animals²⁷. It is to be noted that both these enzymes deplete both circulating glutamine and asparagine. Disclosed herein are studies utilizing PEG-GGT, the inventors did not observe splenic weight loss compared, to untreated mice and hence these differences in toxicity might be attributable to the simultaneous depletion (or lack thereof) of both amino acids. Nonetheless, consistent with the other two enzymes, PEG-GGT did not have an anti-tumor effect in the models tested. Taken together, these observations suggest that enzymatic extracellular depletion of glutamine does not have an anti-tumor effect. Contrary to the immunosuppression induced by extracellular glutamine depletion, pan-inhibition of glutamine utilizing enzymes in TME via a. prodrug of DON promoted anti-tumor immunity with decreased infiltration of MDSCs, polarization of macrophages to Ml phenotype and reduced GM-CSF secretion in the TME¹⁶’¹⁷. While a complete understanding of this complete opposite shift in immunological landscape upon DON treatment compared to PEG-GGT treatment is lacking, we acknowledge that these two modalities of targeting glutamine metabolism are fundamentally different as DON treatment inhibits glutamine metabolism and leads to accumulation of glutamine in the TME while PEG-GGT depletes glutamine in the TME but does not inhibit intracellular glutamine metabolism. Moreover, the effects of DON are concentration dependent which further complicates direct comparison. For example, treatment of 4T1 cells with 1 μM DON leads to reduced secretion of G-CSF and GM-CSF while treatment with 50 μM DON leads to significantly increased secretion of G-CSF and GM-CSF^17,65. More studies are required on the role of different glutamine utilizing pathways in different cell types in the TME to further understand the integrated impact of glutamine metabolism in tumors. [0068] Since the studies disclosed herein have shown that PEGylated H. pylori GGT is a potent glutaminase with no asparaginase activity, we envision other applications of PEG-GGT as a molecular tool to deplete glutamine in live animals. For example, bacterial asparaginases are standard treatment for acute lymphoblastic leukemia, and there is considerable debate on the importance of glutaminase activity for therapeutic efficacy^83,84, PEG-GGT could be used to independently study the roles of glutamine and asparagine metabolism in these cancers. PEG-GGT can thus be used as a selective and efficient tool for glutamine depletion in diverse disease contexts including rheumatoid arthritis⁸⁵, acute respiratory distress syndrome (ARDS)⁸⁶, cancer cachexia⁸⁷ and cystic fibrosis⁸⁸.

[0069] The studies disclosed herein show that enzymatic depletion of glutamine is immunosuppressive and does not inhibit tumor growth. in vivo. The invention discloses a glutamine depletion gene signature and shows that glutamine depletion is not associated with favorable clinical outcomes in human cancers. Rather, a glutamine replete TME is required for optimal anti-tumor immune responses.

EXAMPLES

Example 1: II. pylori GGT is a high affinity glutaminase with no asparaginase activity [0070] Materials and Methods [0071] Generation of hp-GGT Expression Constructs: We expressed hp-GGT as described in a previous study³³. The gene encoding GGT in H. pylori has been sequenced and is available on the KEGG database (entry HP1 1 18). 1.7 kb fragment corresponding to hp-GGT sequence excluding the 26 amino acid periplasmic signal peptide sequence was provided by Integrated DNA Technologies and was PCR amplified with overhang primers containing Ndel and Xhol sites. We cloned this fragment in the pET28a (Novagen) vector using Ndel and Xhol restriction sites by Gibson Assembly. The use of the Ndel site in pET-28a allowed us to include a thrombin cleavable His-tag at the N- terminal of the expressed hp-GGT. We sequenced the construct between the T7 promoter and stop codon through Sanger sequencing (Genewiz) and it was identical to the HP1118 excluding the signal peptide. To introduce the T380A mutation in the construct expressing hp-GGT, we generated two overlapping PCR fragments using appropriate primers to introduce the desired mutation in the overlapping region and assembled the fragments using Gibson Assembly.

[0072] Expression and purification of hp-GGT and, synthesis of PEG-GGT; We transformed hp-GGT containing pET28a plasmid in Escherichia, coll strain Rosetta 2(DE3) (Novagen) and allowed the bacteria to grow on kanamycin and chloramphenicol- resistant Ib-agar plates. We picked a single colony and inoculated it in 2xYT medium containing 34 μg/ml chloramphenicol (Sigma, #C0738) and 50 μg/ml kanamycin (Sigma, #K1377) and grew the culture overnight at 37°C and 250 rotations per minute (rpm). We used this starter culture to inoculate fresh 2xYT media with kanamycin and chloramphenicol concentrations same as for the starter culture. We kept the OD (Culture absorbance at 600 nm) at the start of the culture at 0.01. We allowed this culture to grow at 37°C with shaking at 250 rpm till the OD reached 0.5 - 0.6. At this point, we induced GGT expression by adding isopropyl β -D-thiogalactopyranoside (IPTG) (FisherScientific, #BP1755-10) to a final concentration of 500 uM and grew the culture further for 8 hours. We then centrifuged the culture at 6000 - g for 20 minutes, decanted the supernatant, and harvested the cells. We resuspended the bacterial pellet in lysis buffer containing (50 mM NaH₂PO4, 300 mM NaCl, 10 mM Imidazole (Sigma, #10250), 0.05% Tween-20, pH = 8), universal nuclease (Thermofisher, #88700) and protease inhibitor cocktail (Sigma, #P8849). We then lysed the cells by sonication and centrifuged the lysate at 21000 x g for 10 minutes at 4°C. We extracted GGT from the supernatant by affinity chromatography using Nickel chelating columns. We first equilibrated the column with binding buffer (20 mMNaH₂PO₄, 500 mM NaCl, 10 mM imidazole, pH= 7.4). We then passed the bacterial supernatant through the column followed by a wash with the binding buffer. To remove the endotoxins, we washed the column with 200 column volumes of sterile ice-cold 0.1% Triton X-l 14 (Sigma, #X114) in PBS overnight. We washed the column further with 20 column volumes of sterile PBS to wash the Triton X-l 14. Finally, we eluted, the protein in the elution buffer (20 mM NaH₂PO4, 500 mM NaCl, 500 mM imidazole, pH= 7.4). We then buffer-exchanged the eluted recombinant hp-GGT with PBS using 10 kDaNMWCO centrifugal filters (Sartorius) and stored the protein at 4°C. We then incubated the purified enzyme at 37°C for 6 hours for complete maturation of the enzyme. We determined the protein concentration by bicinchoninic acid (BCA) assay.

[0073] To make PEG-GGT, we concentrated hp-GGT in PBS to 5 mg/ml using 10 kDa NMWCO centrifugal filters and added Methoxy-PEG-5000 (methoxy-PEG- CH₂COO-NHS, Mw 5,000) (Sunbright, #ME-050AS) powder to the solution at a molar ratio of 100: 1 (100 molecules of PEG per molecule of hp-GGT) and mixed gently for 1 hour at 4°C. We removed excess PEG by buffer exchange with PBS using 10 kDa NMWCO centrifugal filters. We concentrated PEG-GGT to 5 mg/ml in 10% glycerol, sterile filtered using 0.22 μM filter, and stored PEG-GGT at. -80°C. We tested PEG-GGT for endotoxins using a chromogenic LAL-based detection assay (ThermoFisher, #A39552S).

[0074] Kinetic characterization of hp-GGT and PEG-GGT." We investigated the kinetic parameters for hydrolysis of γ-glutamyl compounds using substrate analog L- Glutamic acid y-(p-nitroanilide) (yGPNA) (Sigma, #G1135). The hydrolysis of GNA produces 4-nitroaniline whose release can be continuously measured at 412 nm. We made serial dilutions of γGPNA in 100 mM Tris-HCl (pH = 6.5) to obtain concentrations ranging from 1000 11M to 3.9 μM. We added 10 μL of GGT (40 μg/ml, 100 mM Tris, pH= 6.5) to 190 μL substrate γGPNA in a. flat transparent 96-well plate to get a final concentration of 2 μg/ml GGT. For each γGPNA substrate concentration, the corresponding control with no enzyme was also set up to determine the n on-enzymatic rate of hydrolysis. We continuously recorded the absorbance of the samples at 412 nm and 37°C immediately after the addition of the enzyme using a plate reader (Infinite 200 Pro-Tecan Life Sciences). We obtained a standard curve relating to 4-nitroanaline concentration and absorbance and calculated the extinction coefficient. We determined the kinetic constants V_max and K_M from the Lineweaver Burk plot and direct fit to Michaelis Menten equation. For determining the asparaginase activity of PEG-GGT, we added 0.1 and 0.2 mg/ml PEG-GGT to 2.5 mM asparagine in 100 mM Tris-HCl, pH=8, and measured asparagine concentration after 8 hours. For determining asparagine concentration after PEG-GGT incubation, we used a colorimetric method as described previously using ninhydrin reagent (Sigma, #151 173)²⁴.

[0075] Results

[0076] The glutaminases used to date for in vivo glutamine depletion have concomitant asparaginase activity² motivating our efforts to find a glutaminase with no asparaginase activity to uncouple the effects of asparagine depletion. Although bacterial glutaminases with no asparaginase activities exist, these enzymes have low⁷ catalytic efficiencies (k_eat/K_M -IO"³ s'V μM) because of their high K_M (2-30 mM) and are inhibited by the product glutamate²⁹'³⁰. To utilize more efficient enzymes that function efficiently at physiologically relevant glutamine concentrations (500 μM), we targeted the gamma-glutamyl transpeptidase (GGT) class of enzymes. GGTs are a class of enzymes that can either transfer the γ-glutamyl moiety from a compound to an acceptor substrate or hydrolyze the γ-glutamyl moiety³¹’³² (FIG. LA). In contrast to the asparaginase- glutaminases which have similar kinetics for glutamine and asparagine hydrolysis, we did not find reports of GGT mediated asparaginase activity in literature. We specifically focused on Helicobacter pylori (H. pylori) GGT (hp-GGT) to facilitate glutamine depletion (FIG. 1B). As the reported K_M of hp-GGT for glutamine hydrolysis is 12 ± 2 p.M'³ which is 40 fold lower than physiological glutamine concentration, it is a highly efficient glutaminase (k_cat/K_M= 1 .8 ± 0/2 s^-1/ μM) and this activity is comparable to that, of potent Acinetobacter glutaminasi ft cans asparaginase-glutaminase (k_cat/K_M=1.1 ± 0.2 s- ½ μM)²⁸. This glutaminase activity of hp-GGT is unlike mammalian membrane bound GGTs which have a 100-fold higher reaction rate for transpeptidation over hydrolysis and function as transferases rather than hydrolases under physiological conditions³⁴.

[0077] To produce hp-GGT recombinantly, we expressed and purified the codon optimized wild-type hp-GGT gene in E. coll Rosetta-2 cells with an N-terminal 6x-His tag as described previously (FIG. 1C)³³. Consistent with previous reports, the hp-GGT propeptide underwent autocatalytic cleavage, and we observed two distinct bands of 40 and 20 kDa^33,35 (FIG. IB). To confirm that this fragmentation was due to autoproteolysis, we cloned, expressed, and purified the hp-GGT T380A mutant which lacks the autocatalytic cleavage capability because of the absence of nucleophilic threonine³⁶. The T380A mutant appeared as a. single 60 kDa band on SDS-PAGE gel (FIG. 1C). Since our motivation was to explore hp-GGT as a glutaminase in vivo, we conjugated polyethylene glycol (NHS-PEG-5000) to lysine residues of hp-GGT to prevent renal clearance, increase circulation persistence, and mask immunogenicity³⁷ (FIG. 1D). We used the standard NHS ester conjugation chemistry and confirmed the conjugation of PEG chains to hp-GGT using a. standard SDS-PAGE gel (FIG. 1 E).

[0078] To verify that PEGylation did not significantly alter the catalytic efficiency of the enzyme for glutamine hydrolysis, we characterized kinetics using the chromogenic substrate, L-γ-glutamyl-para-nitroanilide (γGPNA). The Vmax [2.9 ± 0.2 pmol/(min.mg)] and K_M (21 ± 5 μM) of hp-GGT at 37 °C were consistent with the published values^33,35. PEG-GGT hydrolyzed γGPNA with similar kinetics with Vmax and K_M values of 2.4 ± 0.2 pmol/(min.mg) and 23 ± 8 μM respectively (FIG. IF). As glutamine is the most abundant physiologically relevant substrate for hp-GGT, the kinetics of glutamine hydrolysis by hp-GGT is well established and has been reported to be the same as γGPNA^33,35. To confirm that PEG-GGT showed similar kinetics for glutamine and γGPNA, we set up a. competitive assay in which we added equimolar amounts of glutamine and γGPNA and. monitored the release of the chromophore p-nitroaniline (PNA). At concentrations from 500 μM to 60 μM, the rate of release of PNA was approximately half when an equimolar amount of glutamine was present compared to no glutamine control (FIG. 1G).

Collectively, these results demonstrate that PEG-GGT has comparable kinetics to the unmodified GGT for the hydrolysis of glutamine. Furthermore, the glutaminase catalytic efficiency of PEG-GGT was comparable to published low⁷ K_M glutaminases²⁸ (Table I).

[0079] Table 1 : k_cat and K_M of reported bacterial glutaminase-asparaginases. PEG-

GGT had comparable glutaminase activity to Acinetobacter glutaminasificans glutaminase activity while having no asparaginase activity.

[0080] We next tested whether PEG-GGT could hydrolyze asparagine. In the competitive assay with yGPNA, the PNA release rate was not reduced when asparagine was added at equimolar concentrations implying no competition for substrate binding (FIG. 1G). To further bolster our finding that PEG-GGT had no asparaginase activity, we incubated 2.5 mM asparagine with 0.1 and 0.2 mg/ml PEG-GGT at 37°C and measured asparagine concentration after 8 hours (FIG. 1H). There was no reduction in asparagine concentration even at remarkably high concentrations of PEG-GGT and hence we concluded that PEG-GGT has the requisite enzymatic selectivity with no asparaginase activity.

Example 2: PEG-GGT inhibits the growth of multiple cell lines in vitro [0081] Materials and Methods

[0082] Cell culture and cell viability assays: We studied the effects of glutamine depletion on 4T1 , CT26, and MC38 cell lines. MC38 cell line was provided by Dr. Wei Peng (UH). We bought 4T1 and CT26 cells from ATCC. For each cell line, w e seeded 5xl0⁴ cells in 500 μl RPMI-1640 (Corning) media, supplemented with L-glutamine (2 mM) and 10% FBS (RnD Biosystems, S11150) in a 24-well plate. We incubated the cells at 37°C and 5% CO?.. After 24 hours, we removed the media and washed the cells with 500 μl PBS. In the first group, we added 750 μl RPMI-1640 medium without L-glutamine supplemented with 10% dialyzed FBS (dFBS) (Sigma, #F0392) and 2 mM L-glutamine to each well. In the second group, we added PEG-hp-GGT at 10 μg/ml in addition to the media added in group 1. In the third group, we added 750 μl RPMI-1640 media without L -"glutamine but supplemented with 10% dialyzed FBS (dFBS). After every 24 hours, we measured the cell viability of 3 wells from each group using MTT assay. For studying the combined effect of PEG-GGT and mTORC1 inhibition on cell growth, we performed a similar assay with regular RPMI-1640 media, and either PEG-GGT (10 μg/ml) or Temsirolimus (20 μg/ml) (Selleckchem, #81044) or both were added to the wells.

[0083] Cell culture metabolite assays: We cultured CT26 cells in 24 well plates to 60% confluency in RPMI-1640 media with 10% FBS. We then washed the cells and replaced the media with RPMI-1640 media, without glutamine and 10% dialyzed FBS (dFBS) supplemented with either 2 mM glutamine or 2 mM glutamine and 10 μg/ml PEG-GGT. After 48 hours, we washed the cells twice with PBS. We added 200 μl of 80% methanol solution stored at -80°C to the wells and kept the plate in -80°C for 10 minutes. We then scraped off the cells and vortexed the solution at 4°C for 5 minutes. We then sonicated (40 kHz) the cells at 4°C for 15 minutes. Finally, we centrifuged the mixture at 20000g at 40°C for 10 minutes. We vacuum evaporated the methanol-water solution and reconstituted the metabolites in 20 μl PBS. We determined the glutamine and glutamate levels in serum by enzymatic assay detection kit according to the manufacturer’s protocol (Promega Glutamine/Glutamate Glo kit). We assayed the total glutathione by an enzymatic assay kit according to manufactures protocol (Cayman total glutathione assay kit: #703002).

[0084] Results

[0085] As glutamine is an anabolic substrate, and its absence in culture has been shown to halt the growth of multiple cell lines in vitro³⁸, we wanted to investigate the effect of glutamine depletion via PEG-GGT on cell growth kinetics. First, we tested the serum stability of PEG-GGT in culture media with 10% FBS. PEG-GGT in culture media retained 60% of its glutaminase activity after 48 hours of incubation at 37 °C (FIG. 7 A). We measured the growth rate of CT26 (colorectal carcinoma), 4T1 (mammary carcinoma), and MC38 (colon adenocarcinoma) cell lines in vitro in the presence and absence of glutamine using MTT assay (FIG. 2 A). The lack of glutamine abolished the growth of all three cell lines tested in vitro. At 72 hours after media exchange, the relative number of cells in media supplemented with glutamine were significantly higher (CT26: p-value=0.02, 4Tl : p-value=0.002, MC38: p-value=0.001, FIG. 2B-2D). Adding PEG- GGT at 10 μg/rnl to culture media, containing 2mM glutamine also inhibited the growth of all the tested cell lines (CT26: p-value= 0.02, 4T1: p-value= 0.003, MC38: p- value=0.006, FIG. 2B-2D). To confirm that this arrest in growth could be ascribed to the enzymatic activity of PEG-GGT, we heat-inactivated the enzyme by incubating it at 70 °C for 20 min. When heat-inactivated PEG-GGT was added at the same concentration to culture media containing 2mM glutamine, we observed no impact on cell growth after 24 hours confirming that enzymatic activity is important for the observed cell growth arrest upon addition of PEG-GGT (FIG. 7B). Therefore, PEG-GGT abolishes the growth of multiple cell lines in vitro and its enzymatic activity is essential for this effect.

[0086] Previous reports have suggested that cell lines overexpressing Myc are particularly sensitive to glutamine deprivation and hence we tested the CT26 cell line in which Myc is overexpressed⁴⁸. To assess the impact of extracellular glutamine hydrolysis on intracellular glutamine and glutamate pools, we cultured CT26 cells with 2 mM glutamine, treated the cells with PEG-GGT for 48 hours and extracted the metabolites (FIG. 7C). When we measured the total glutamate and glutamine concentrations using an enzymatic assay, PEG-GGT treated cells had 3-fold lower glutamate pool (No treatment: 3. l±0,3, PEG-GGT: 1.0±0.1 p-value<0.0001) (FIG. 2E) and 4,7-fold lower glutamine pool (No treatment: 4.7±1.7, PEG-GGT: 1.0±0.8 p-value-0.002) (FIG. 2F) compared to non-treated cells. Glutathione (GSH), which is synthesized from intracellularly from glutamate, was also significantly reduced upon treatment with PEG-GGT (No treatment: 1.3±5=0.1, PEG-GGT: 1.0±0.2 p-value=0.03) (FIG. 2G). In summary, intracellular glutamate, glutamine, and total glutathione pools were reduced in CT26 cells upon PEG- GGT treatment.

[0087] Although PEG-GGT arrested the growth of cell lines, we wanted to investigate if it was cytotoxic. Upon depletion of glutamine, cells can adapt to low extracellular glutamine by synthesizing glutamine intracellularly via the enzyme glutamine synthetase (Glul) which is irreversibly inhibited by L -Methionine sulfoximine (MSO)³⁹. To test whether PEG-GGT was cytotoxic and if this cytotoxicity was synergistic with Glul inhibition, we treated cells in vitro with either MSO, PEG-GGT, or both, and monitored cell death as a function of time using microscopy. MSO as a single agent (500 μM) was not toxic and did not inhibit cell growth (FIG. 2H). PEG-GGT by itself at 10 μg/ml inhibited cell growth (FIG. 2.H) and was cytotoxic to 6 ± 1 % (p-value = 0.005) of cells after 24 hours. Remarkably, the combination of PEG-GGT and MSO was cytotoxic to more than 90 ± 9% (p-value = 0.0002) of cells after 24 hours (FIG. 2H, 21). These combined results suggest that while glutamine depletion via PEG-GGT inhibits the growth of cell lines, simultaneous glutamine depletion and inhibition of Glut is cytotoxic to cells.

Example 3: Enzymatic depletion of circulating glutamine does not inhibit tumor growth vivo

[0088] Materials and MethodsPEG-GGT in-vivo pharmacokinetics and pharmacodynamics; .All studies using animal experiments were reviewed and approved by the University of Houston (UH) IACUC (Institutional Animal Care and Use Committee). We purchased the female 6 to 8-week-old BALB/c mice from Charles River Laboratories. 8 mice were administered. PEG-GGT at a dose of 20 mg/kg body weight (b.w). We collected mouse serum at 8, 24, 48, and 72 hours. To determine the PEG-GGT concentration in serum, we first prepared, a standard curve in which we compared the rate of yGPNA hydrolysis by adding 5 μl of PEG-GGT at. different, concentrations to 200 μL of 0.5 mM yGPNA in PBS spiked with 5 μl of serum from untreated tumor-bearing mice. We also confirmed that serum from non-treated tumor-bearing mice had no γGPNA hydrolysis activity. To find the concentration of PEG-GGT in the serum of treated mice, we incubated 5 μl of serum in 200 μL of 0.5 mM GPNA in PBS and measured the rate of yGPNA hydrolysis. We determined the concentration of PEG-GGT from the standard curve generated as mentioned above. We determined the circulating glutamine and glutamate levels in serum by enzymatic assay detection kit. according to the manufacturer’s protocol (Promega Glutamine/ Glutamate Gio kit).

[0089] In vivo tumor For studying tumor growth kinetics in vivo, we injected 6-8- week-old. BALB/c mice subcutaneously either with a single cell suspension of 100K CT26 (right flank) cells or 50K 4T1 (fourth mammary fat. pad) cells suspended in RPMI-1640 media. We tested and confirmed that all cell lines were negative for mycoplasma contamination by qPCR. After the average tumor volumes reached 100 mm³, we administered either 20 mg/kg PEG-GGT IP to the treatment group every three days or the vehicle (PBS, 10% glycerol) to the control group. We measured tumor dimensions with Vernier calipers and tumor volume was approximated as L*H*H/2. We measured the tumor volume and mice weight every' three days.

[0090] In vivo metabolite measurements'. We injected 6-8-week-old BALB/c mice subcutaneously with a single cell suspension of 100,000 CT26 cells in the right flank. Once the tumor volume reached 100 mm³, we started treating the mice either with 20 mg/kg PEG-GGT or vehicle on a biweekly basis. On the 7^th day after treatment initiation (3 PEG-GGT doses) we euthanized the mice. We isolated and washed small sections of tumors (20 mg) in phosphate buffer saline (PBS) and then splash-froze them in liquid nitrogen. We collected the mouse blood from cardiac puncture and left the blood to coagulate at room temperature for 10 minutes. We then centrifuged the blood at 2000g for 10 minutes, isolated the serum and froze it in -80 °C. The amino acids were extracted from mouse tissue and serum using the liquid-liquid extraction method as explained earlier²⁵. Pool samples were used as quality control samples during MS acquisition. Agilent 6495 triple quadrupole MS coupled to Infinity 1290 LC were used for data acquisition vis multiple reaction monitoring (MRM) mode through Agilent Mass Hunter Data Acquisition Software (ver. 10.1) as described previously²⁶. Peak integration and data analysis were performed using Agilent Mass Hunter Quantitative Analysis Software. Peak areas were normalized with Tryptophan-¹⁵N₂ spike internal standard.

[0091] Results

[0092] We wanted to investigate whether PEG-GGT can be utilized to facilitate the enzymatic depletion of glutamine in vivo. First, we established the pharmacokinetics (PK) and pharmacodynamics (PD) of PEG-GGT in mice. We injected a single dose of 20 mg/kg PEG-GGT into mice intraperitoneally (ip) and measured glutaminase activity in serum collected at different time points for 72 hours by directly monitoring the hydrolysis rate of the substrate GPNA at saturating concentrations (500 iiM) (FIG. 3A). We detected glutaminase activity corresponding to 180 ± 20 μg/ml PEG-GGT in serum at 72 hours (FIG. 3B) Assuming first-order elimination kinetics, PEG-GGT has a half-life of 67 ± 8 hours in serum. We also measured glutamine and glutamate concentration in serum and while glutamine was not detectable at 24/48 hours, we detected 25 ± 5 μM glutamine at 72 hours. As expected by the hydrolysis of glutamine to glutamate by PEG- GGT, we observed an increase of glutamate reaching up to 2.6 ± 0.1 mM in serum at 72 hours (FIG. 3C).

[0093] As PEG-GGT persisted in circulation and efficiently depleted glutamine for at least 48 hours in mice, we set out to test the effect of PEG-GGT on the growth of tumors in syngeneic mouse models. Since w-e wanted to study the integrated impact of glutamine depletion on cancer cells and immune cells in the TME, we chose the CT26 tumor model which is well characterized for high tumor immune infiltrates and T cell responses⁴⁰. We implanted CT26 subcutaneously in BALB/c mice, and after the tumor was palpable, initiated treatment with PEG-GGT on a twice weekly schedule (FIG. 3D ). PEG-GGT w^;as well tolerated with no evident signs of toxicity and the kinetics of weight change were the same as the vehicle treated group during the treatment (FIG. 8A). There was no difference in the tumor growth rate of the PEG-GGT treated and vehicle treated group (FIG. 3E) even though we did not detect any circulating glutamine (<50 nM) in serum during treatment (FIG. 3F). To ensure that these results are generalizable across multiple tumor models, we implanted 4T1 breast cancer cells in mice and treated the animals with PEG-GGT using the same treatment schedule (FIG. 3G). Consistent with the CT26 model, treatment of 4T1 tumors with PEG-GGT did not inhibit tumor growth in mice (FIG. 3H) and changes in body weight were similar in treated and. control groups (FIG. SB). We also confirmed that there was no circulating glutamine in the serum of PEG-GGT treated tumor bearing mice (FIG. 31). Collectively, these results demonstrate that while PEG-GGT treatment inhibits growth of cancer cells in vitro, it does not significantly impact tumor growth in vivo.

[0094] To map the impact of PEG-GGT on circulating metabolites and tumor metabolism, we measured the relative amounts of amino acids in serum and tumors of CT26 tumor bearing mice treated either with PEG-GGT or vehicle using liquid chromatography - mass spectrometry (LC-MS) (FIG. 8D). Consistent with our hypothesis that PEG-GGT does not hydrolyze asparagine, there was no significant difference in asparagine levels in serum of PEG-GGT and vehicle treated mice (fold-change [FC]: 0.9, p-value = 0.45) (FIG. 3J). We observed a significantly decreased concentration of GSH (FC: ().03, p -value = 0.0008); and an increased concentration of cysteine (FC: 1.2, p- value = 0.01), cystine (FC: 3.9,p -value = 0.0007) and. glycine (FC: 1.8, p -value = 0.005) in serum (FIG. 3J). This observation is expected since PEG-GGT hydrolyses the GSH (reported concentration in serum ~25 μM) to cysteinyl-glycine which can be further hydrolyzed to cysteine and glycine by membrane bound dipeptidases^41,42. Mass- spectrometry based approaches for measuring amino acids unfortunately, do not always resolve glutamine and lysine, which have the same molecular weight, especially when they have same retention time on LC⁴³. Thus, while mass-spectrometry reported only one- third reduction in glutamine/lysine concentration (FIG. 8E), the direct enzymatic assay for quantifying glutamate/glutamine concentrations demonstrated a direct reduction in glutamine and. a concomitant increase in glutamate (FIG. 3F). In summary, PEG-GGT depletes circulating glutamine and glutathione resulting in increased glutamate, cysteine/cystine and glycine.

[0095] To map the metabolomic changes in the tumor tissue (which includes both interstitial/vascular and intracellular metabolites), we homogenized snap frozen CT26 tumor tissue and performed LC-MS (FIG. 8D). In the tumor tissue, we did not see a significant change in glutamate levels upon PEG-GGT treatment (FC: 0.9, p-value = 0.26) (FIG. 3K). Since the dominant contributor to the total glutamate is likely the intracellular glutamate⁴⁴, our results suggest that unlike in vitro treatment, PEG-GGT treatment did not significantly reduce intracellular glutamate. One hypothesis that can explain these results that the other non-tumor cells in the tumor function as reservoirs of glutamate⁴⁵. Consistent with the serum and in vitro profiling however, we observed a significant reduction in glutathione (FC: 0.5, p-value = 0.05), and increase in cysteine (FC: 2.2, p -value = 0.001), and cystine (FC: 5.3, p-value = 0.05) levels in PEG-GGT treated tumors (FIG. 3K). Unexpectedly proline concentrations were also decreased (FC: 0.7,p-value = 0.02) in PEG-GGT treated tumors (FIG. 3K). Both GSH and proline are present at millimolar concentrations (GSH 5-10 mM, Proline: 1-2 mM) inside the cell and need glutamate for their synthesis⁴⁴. Recent reports have shown that the inhibition of proline synthesis allowed proliferation of cancer cells in glutamine limiting conditions⁴⁶. Because LC-MS could not distinguish lysine and glutamine, we used the enzymatic assay to measure glutamine in PEG-GGT and vehicle treated tumors but while there was an overall reduction in the glutamine concentrations this was not significant in our small sample size (FC=0.3,p -value = 0.32) (FIG. 8C). In summary, despite the extracellular increase in glutamate concentrations, intracellular concentrations of glutamate were not altered in the tumor wherein cells maintain their glutamate pool in glutamine limited environments marked with downregulation of proline and GSH.

[0096] Since we observed an increase in cysteine/cystine concentrations in serum and. tumor of PEG-GGT treated mice, we wanted to test whether increased cysteine/cystine generated from hydrolysis of GSH could be responsible for difference in tumor cell proliferation in vitro and in vivo. We treated cells cultured in RPMI-1640 media, containing 2 mM glutamine with 10 μg/ml PEG-GGT and supplemented the media with either 100 μM GSH or 100 μMN-acetyl cysteine (NAC). Supplementing the media with either GSH or NAC did not rescue the cell proliferation (FIG. 7D and 7E). These results confirmed that unlike increase in cysteine/cystine from GSH/GSSG hydrolysis, the primary impact of PEG-GGT is glutamine depletion leading to arrest in cell proliferation. Example 4: Transcriptomics reveal adaptation pathways upon glutamine depletion

[0097] Materials and Methods

[0098] mRNA isolation, sequencing. and analysis'.

[0099] We injected 6-8-week-old BALB/c mice subcutaneously with a single cell suspension of 100K CT26 cells in the right flank. Once the tumor volume reached 100 mm³, we started treating the mice either with 20 mg/kg PEG-GGT or vehicle on a biweekly basis. On the 7^th day after treatment initiation (3 PEG-GGT doses) we euthanized the mice. We isolated and washed small sections of tumors (20 mg) in PBS and then splash-froze them in liquid nitrogen. We lysed the tissue in RNeasy lysis buffer (RLT) and a. single stainless steel bead using tissue lyser (Qiagen, Hilden, Germany). We extracted total RNA using the RNeasy kit (Qiagen, #74104), DNAse treated the RNA (Invitrogen, # AM19006, and sent the RNA for sequencing to Novogene. Novogene processed the RNA to enrich mRNA and prepared the cDNA library'. They sequenced the cDNA on Illumina HiSeq 2500 in paired-end mode. We checked, the quality of sequencing data by FastQC, and the results showed good quality of reads and no further need for trimming. We aligned the sequencing data to the BALB/c reference genome (Ensembl vl .108) using STAR aligner and quantified the transcript abundance⁹³. We performed differential expression between treated and non-treated groups using the package DESeq2⁹⁴. The raw RNA-Seq data, and normalized gene-count data has been submitted to Gene Expression Omnibus (GEO) (Accession code: GSE247472). We performed Gene set enrichment analysis (GSEA.) using GSEA. software and visualized the enriched clusters in Cytoscape⁹⁵. To perform infiltrating immune cell deconvolution from RNA-Seq data, we first obtained the TPM counts for our RNA-Seq data using RSEM⁹⁰. We then made the signature matrix of immune infiltrates using immgen database⁶¹ in CibersortX⁶². We calculated the percentage of infiltrating immune cells using CibersortX. [00100] In vitro RT-qPCR:

[00101] We treated CT26 cells with either PEG-GGT or left them untreated for 120 hours in RPMI-1640 media in 24 well plates. We aspirated the media and lyzed the cells with RLT buffer. We extracted the RNA using the RNeasy kit (Qiagen, #74104). We further treated the extracted RNA with DNAse treatment kit to remove genomic DNA (Invitrogen, #AM1906) and synthesized cDNA using cDNA reverse transcription kit (Invitrogen, #4368813). We performed RT-qPCR reaction using SsoFastTM EvaGreen® Supermix with Low' ROX (Bio-Rad, # 1725211) on AriaMx Real-time PCR System (Agilent Technologies, Santa Clara, CA). We normalized the results to Actin or GAPDH (glyceraldehyde-3- phosphate dehydrogenase). To determine the fold change, we used the 2-AACt method by comparing PEG-GGT treated cells to non-treated controls. See Table S3 for the list of primer sequences used in this study.

[00102] Glutamine depletion signature and TCGA. Analysis: [00103] To construct the glutamine depletion signature, we downloaded the raw counts for cells adapted to grow⁷ in low glutamine from GEO series accession number GSE144883⁶⁵. We performed differential analysis on the adapted cell lines and filtered the genes with FDR<0.1. We chose the coordinatively upregulated or downregulated gene from our dataset and this data set to build the glutamine depletion signature. To perform ssGSEA⁷⁷ of our signature on human cancers, we downloaded the TCGA data for different cancers from cBioportal. We performed the ssGSEA on different human cancers and stratified the top 25% ssGSE.A scores as “Gin depletion high” and bottom 25% as “Gin depletion low”. We then performed overall survival analysis using clinical data in the TCGA database. For the PMN-MDSC score calculation for each patient, we first calculated the percentage of immune infiltrates from bulk RNA-Seq data using EPIC to remove the bias from non-immune cells in the data⁸⁵. We then divided the raw RSEM counts of genes in the PMN-MDSC signature with the fraction of immune cells predicted by EPIC. We then log transformed the RSEM counts, and Z -normalized the expression of PMN-MDSC related genes across the TCGA dataset. PMN-MDSC score was the sum of Z-scores for all the genes in the PMN-MDSC signature⁸⁶.

[00104] Quantification and statistical analysis

[00105] Statistical significance was assigned when p values were <0.05 using GraphPad Prism (v6.07). Tests, number of animals (n ), mean values, and statistical comparison groups are indicated in the Figure legends.

[00106] Results

[00107] To understand how tumors adapt to low extracellular glutamine in vivo, we sequenced mRNA isolated from CT26 tumors treated either with PEG-GGT or vehicle (FIG. 4A). 223 genes were differentially upregulated while 262 were downregulated (Log₂FC>0.3, FDR<0. 1). As glutamine is indispensable for nucleotide synthesis, and nucleotide availability has a profound impact on the cell cycle⁴⁷, it is not surprising that several candidate genes associated with the cell cycle like Brcal, Polq, Cdc6, Mem family proteins were significantly upregulated in the PEG-GGT treated tumors in comparison to the vehicle treated tumors (FIG. 4B). Since glutamine is a major substrate for amino acid synthesis, consistent with several reports of translation downregulation upon glutamine deprivation, we observed significant downregulation of eukaryotic translation initiation factors Eif4b, Eif2a and multiple ribosomal protein genes like Rpl28, Rps28, Rps12.^48,49. [00108] To analyze the differentially expressed pathways, we performed gene set enrichment analysis (GSEA)³⁰. Since glutamine depletion mediated reduction of nucleotide pool leads to accumulation of single-stranded DNA which activates ATR kinase^47,51, we observed an upregulation of genes related to ATR kinase activation upon replication stress (FIG. 9A). Consistent with the activation of the G2M checkpoint downstream of ATR signaling⁵², we observed a strong upregulation of G2M checkpoint signaling upon glutamine depletion (FIG. 4C). To adapt to increased, replication stress, cells must synthesize DNA at a higher rate and since E2F transcription factors directly regulate the synthesis of S phase proteins for DNA synthesis, we observed an increase in E2F target transcripts including genes involved in DNA replication⁵³ (FIG. 9B, FIG. 4D). Finally, since glutamine deficiency induces DNA alkylation damage via inhibition of ALKBH enzymes, GSEA analyses identified several significantly enriched gene clusters related to DNA damage response⁵⁴ (FIG. 4D). To validate if these pathways were also relevant upon glutamine depletion vitro, we used RT-qPCR to quantify transcript abundances of some of the genes that were most significantly upregulated in our RNA- Seq data. Transcripts of Brcal which is associated with DNA repair, Polq which has DNA polymerase activity, and Cdc6 which assembles pre-replicative complex were all significantly upregulated in CT26 cells treated with PEG-GGT for five days in vitro (FIG. 9G). In aggregate, these results demonstrate that glutamine makes an indispensable contribution to the nucleotide pool, and. upon depletion of glutamine, cells upregulate pathways associated with DNA replication and DNA repair pathways to compensate for the replication stress.

[00109] Many reports support that Myc expression reinforces glutamine dependency in diverse cancer models⁵⁵. Since CT26 tumors overexpress Myc⁴⁰, we used the RNA-Seq data to test the impact of glutamine depl etion on Myc overexpressing tumors. Unexpectedly, Myc target genes were upregulated upon glutamine depletion via PEG-GGT, suggesting a. potential adaptive mechanism to maintain the nucleotide pool⁵⁶ (FIG. 4B, 4C, FIG. 9D). Specifically, genes like Cad, Ppat, and Pfas which directly use glutamine’s y-nitrogen for nucleotide synthesis and have previously been demonstrated to be under the control of Myc, exhibited a significant upregulation in the PEG-GGT treated group. Furthermore, there was a pronounced, enrichment of gene sets pertaining to nucleotide biosynthesis and one-carbon metabolism (FIG. 4D). Collectively, our RNA- Seq data suggests that in vivo glutamine depletion leads to an elevation in the expression of downstream targets of Myc, including genes responsible for nucleotide synthesis. [00110] To explore the mechanisms underlying cellular adaptation to the depletion of extracellular glutamine, we investigated if cells synthesize glutamine intracellularly. Accordingly, we focused on Glut which is the only enzyme capable of intracellular glutamine synthesis⁵''. Since the abundance of Glul protein is determined by post- translational modification via glutamine dependent acetylation and subsequent proteasomal degradation⁵⁸, we did not observe an upregulation in Glul trreanscripts in PEG- GGT treated tumors in comparison to the untreated tumors (FIG. 4B). The mammalian target of rapamycin complex 1 ( mTORC1) inhibits proteasomal degradation of Glul⁵⁹. We observed an increase in mTORC l target transcripts (FIG. 4C, FIG. 9C) suggesting that mTORC1 might function to stabilize Glul. To verify if mTORC1 activity is essential for cell growth under glutamine depletion, we treated CT26 cells either with PEG-GGT, mTORC1 inhibitor temsirolimus, or both⁶⁰. While 20 μg/ml temsirolimus in culture media containing glutamine did not affect cell growth, the combination of 10 μg /ml PEG-GGT and 20 μg /ml temsirolimus inhibited cell growth to a. much greater extent than PEG-GGT alone (p-value = 0.003, FIG. 9H). Thus, upon glutamine depletion, there is an increase in mTORC1 signaling which when inhibited inhibits cell proliferation.

[00111] To investigate the impact of glutamine depletion on the immune system, we focused on immune related pathways within our RNA-Seq data. We observed a significant downregulation of interferon-alpha, interfer on -gamma, and TNF-a responses upon treatment with PEG-GGT (FIG. 5 A, SB, FIG. 9E). To investigate the changes in the immune cell composition upon glutamine depletion in vivo, we performed immune cell deconvolution using mouse immune cell gene signatures published in the Immgen database^61,62. We observed that tumors treated with PEG-GGT were significantly enriched in macrophages (F4/80⁺ICAM2⁺) and deprived of dendritic cells (Cd11c⁺ MHCII⁺F llt3⁺) (FIG. 5C). As macrophages can either have a pro-inflammatory (Ml) or anti-inflammatory (M2) phenotype, we investigated the Ml and M2 markers in our RNA-Seq data and observed enrichment of bona fide M2 markers Mrcl (Cd206), Cdl63 and F13al. We also observed a reduction in Cd86, H2- A b1(MHC-II), and Idol which are known markers of Ml macrophages^63,04 (FIG. 5D). Collectively, our RNA-Seq data, suggests that extracellular glutamine depletion compromises anti-tumor immunity which is marked by downregulation of IFN-a, IFN-γ and TNF-α responses, and an increased frequency of anti-inflammatory macrophages.

Example 5: Enzymatic depletion of circulating glutamine is immunosuppressive [00112] Materials and Methods [00113] Flow cytometry of tamor-infiitrating immune cells:

[00114] We cut the tumors from PEG-GGT treated or control groups into 1-10 mm³ pieces. To dissociate the tumor, we incubated the cut pieces with 0. 1% w/v collagenase D (Roche #11088858001) and 0.01% w7v DNAse (Sigma #DN25) at 37°C for 1 hour in RPMI-1640. We obtained a single-cell suspension by passing through a 70 μm cell strainer. After obtaining a single cell suspension, we washed the cells twice with PBS and labeled them with Live/dead aqua die (ThermoFisher, #L34957) in PBS for 20 minutes at room temperature (RT). The live/dead staining was stopped by adding 5 volumes of 4% FACS buffer. We then washed the cells twice with a 4% FACS buffer.

We blocked the FC receptors by CD16/CD32 (done 2.4G2; BD Biosciences) antibody in 4% FACS buffer for 20 minutes. For myeloid panel, we incubated, the cells with an antibody cocktail of CD45-BUV395 (BD, #564279), CD11b-BV421 (Biolegend, #101235), CD11C-BV785 (Biolegend, #117335), F4/80-APC/Cy7 (Biolegend, #123117), Ly6C-PE (Biolegend, #128007), Ly6G-PerCP/Cy5.5 (Biolegend, #127615), CD206- AF488 (Biolegend, #141709) and CD86-AF700 (Biolegend, #105023). For lymphoid panel, we incubated the cells with an antibody cocktail of CD45-BUV395 (BD, #564279), CD3-APC (Biolegend, #100235), CD4-AF594 (Biolegend, #100446), CD8- PerCP/Cy5 (Biolegend, #100731), NKp45-e450 (Invitrogen, #48-3351-82), and CD19- APC-Cy7 (Biolegend, #115519). We fixed the cells with BD CytoFix/Perm and washed them thoroughly with BD Perm/Wash solution. For FoxP3 staining in the lymphoid panel, we labeled the cells with anti-mouse FOXP3-AF488 (Biolegend; #126405) prepared in BD Perm/Wash solution and then washed with FACS buffer. We acquired the data on a BD LSRFortessa X-20 flow cytometer apparatus and analyzed it using FlowJo software (BD Biosciences). [00115] Results [00116] To validate our finding that depletion of extracellular glutamine is immunosuppressive, we used flow cytometry to phenotype the tumor-infiltrating myeloid and lymphoid immune populations (FIG. 10A). We implanted CT26 tumors subcutaneously and utilized the same PEG-GGT treatment schedule as before (FIG. 3D). To ensure that we harvested enough cells for myeloid and lymphoid phenotyping, we harvested the tumor when the average volume reached 300-400 mm³ (7^th day after treatment). In accordance with our transcriptomic data, macrophages (Cd45⁺Cdl lb⁺Ly6C^loF4/80⁺) in PEG-GGT treated tumors were skewed towards M2 phenotype as Cd206^hlgh macrophages were significantly enriched in PEG-GGT (40 + 13%) vs vehicle treated mice (25 ± 8%,p-value = 0.03) (FIG. 5E). Furthermore, we observed a. significant increase in the polymorphonuclear myeloid derived suppressive population upon PEG-GGT treatment (7 + 3%) compared to vehicle (1 ± i%,p-value = 0.0003) (FIG. 5F). As PMN-MDSC accumulation in tumors is associated with G-CSF and GM-CSF secretion, we observed

15 ± 3 (p-value = 0.003) and 18 + 2 (p-value = 0.0003) fold increase in transcript levels of these genes respectively in CT26 cells treated with PEG-GGT in vitro^65,66 (FIG. 5G).

Cdl lb⁺GR~T' cells which form a major population of MDSCs were also enriched in PEG-GGT treated tumors (PEG-GGT: 16 ± 2% vs Vehicle: 6 + 1%, p-value = 0.0008) (FIG. 5H)⁶⁷. Unlike PMN-MDSCs, the frequency of monocytic MDSCs (Mo-MDSC) ) did not differ between PEG-GGT (5.3 + 1 .3%) and vehicle treated (3.7 ± 0.6%,p-value =^: 0.3) group (FIG. 10F). The frequency of infiltrating CD8

T cells (PEG-GGT: 2.5 ± 0.8% vs Vehicle: 3.2 ± 0.7%,p-value ~ 0.50) and NK cells

(PEG-GGT: 0.7 ± 0.6% vs Vehicle: 2.4 ± 1.0%,p-value = 0.09) did not change significantly after PEG-GG T treatment (FIG. 51 and 5J). In summary-, our flow cytometry' data demonstrates that depletion of glutamine through PEG-GGT induces a state of immunosuppression, marked by a notable enrichment in populations of PMN- MDSCs and M2 macrophages.

Example 6: Glutamine depletion is not associated with favorable outcomes in human cancers

[00117] Results

[00118] To determine whether glutamine depletion could be of therapeutic benefit in human cancers and whether it was associated, with immunosuppressive phenotype as seen in our tumor model, we derived a gene signature associated with glutamine depletion (FIG. 6A). To ensure that our gene signature is broadly reflective of glutamine depletion, we used the differentially expressed genes (FDR<0.1) from our study and a. recent study in which two pancreatic cancer cell lines (SUIT2 and 89388T) were adapted to grow in low glutamine media⁵⁹ (FIG. 6A). Based on the core set of genes common to all three datasets, we derived the glutamine depletion signature (Tables 2 and 3). [00119] Table 2: Gin depletion signature - Upregulated [00120] Table3 : Gin depletion signature Down regulated

[00121] To investigate if our glutamine depletion signature had translational relevance, we took advantage of human tumors within the TCGA. We investigated combined transcriptomic and clinical/pathological annotations in nine human cancers. To quantify the enrichment of our glutamine depletion gene signature in patient transcriptomic data, we performed single sample GSEA (ssGSEA)⁶⁸. Samples in the first quartil e with the highest expression of glutamine depletion signature were l abeled “Gln depletion high” and those in the last quartile were labeled “Gin depletion low” (FIG. 6A). A. high glutamine depletion ssGSEA score was not associated with significantly favorable prognostic outcome in most cancers (FIG. 6E, 6H, 6J, FIG. 11A-11F). Consistent with our data showing no therapeutic benefit of glutamine depletion in 4T 1 tumor model, the five-year overall survival (OS) did not differ for both groups in breast cancer (HR (Hazard Ratio): 1.1, 95% CEO.7 to 1.8). The five-year OS for patients in the “Gin depletion high” group was significantly lower in liver hepatocellular carcinoma (HR: 2.9, 95% CI: 1.8 to 4.9), and pancreatic adenocarcinoma (HR: 3.1, 95% 0: 1.7 to 5.6). The five-year OS for the “Gin depletion high” group was significantly better only in stomach adenocarcinoma (HR: 0.6, 95% CI: 0.4 to 1) (FIG. 11F). Thus, our data suggests that glutamine deprivation is not associated with better outcomes in multiple human cancers. [00122] We next evaluated if glutamine depletion score differed in different pathological subcategories of breast cancer like tumor size, molecular subtype, and stage. We observed that the glutamine depletion ssGSEA score w as significantly higher in larger tumors (T2) than smaller tumors (Tl) (0.71 ± 0.15 vs 0.64 ± 0.14, p-value = 10^-9) (FIG. 6B). Among the different molecular subtypes of breast cancers, glutamine depletion ssGSEA score was significantly enriched in more aggressive cancers like luminal B, HER2+, and basal compared to normal and luminal A breast cancers (0.81 + 0.11 vs 0.60 ± 0.10,p -value < 10^-16) (FIG. 6C). Furthermore, glutamine depletion ssGSEA score was higher in advanced stage II and stage III tumors compared to stage I tumors (0.70 ± 0.15 vs 0.65 ± 0.15, p-value = 10^-4) (FIG. 6D). Taken together, glutamine deprivation is associated with increased tumor size and more aggressive phenotypes in human breast cancers.

[00123] As our data showed that low glutamine in TME was associated with increased infiltration of PMN-MDSCs, we wanted to test whether this was also consistent in human cancers. Because of their low transcriptional activity and close resemblance to neutrophils, we still lack a unifying gene signature for PMN-MDSCs⁶⁹. However, there have been several reports of the differences in the expression of PMN-MDSCs compared to other MDSCs and neutrophils^70,71. We derived a seven-gene signature (S100A8, SI 00/19, LYZl, MMP8, MMP9, SLC27A2, and IL4R) for PMN-MDSCs based on several recent reports including single-cell RNA sequencing from 33 glioma patients'⁰"⁷⁴. We validated this signature with our mouse RNA-Seq data and confirmed that these genes were indeed upregulated in PEG-GGT treated tumors, consistent with our flow-cytometry data, on the same tumors (FIG. 6F). To restrict our analysis to immune cells and remove bias from tumor cells in the TCGA RNA-Seq data, we calculated the fraction of immune cells in the tumor using EPIC⁷⁵. We then normalized the expression of the signature genes based on immune cell infiltration and calculated “PMN-MDSC score” as the sum of Z- scores of all the genes in the signature⁷⁶. When we tested this signature in the TCGA. dataset, we found that the “Gin depletion high” group had significantly higher PMN- MDSC scores in breast (0.4 ± 0.2 vs -0.4 ± 0.3,p-value=0.04), pancreatic (0.5 ± 0.7 vs - 1.8 ± 0.9, p-value= 0.04) and liver cancer (1.0 + 0.5 vs -0.4 ± 0.4,/.?-value= 0.02) (FIG. 6G, 61, 6K). Taken together these results demonstrate that glutamine depletion is likely associated with the accumulation of PMN-MDSCs in the TME and is not associated with a therapeutic benefit in human cancers.

[00124] The complete disclosure of all patents, patent applications, and publications, and. electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRE, PDB, and. translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

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Claims

What is claimed is:

1. A pegylated γ-glutamyl transpeptidase (PEG-GGT) enzyme comprising an isolated, modified γ-glutamyl transpeptidase (GGT) enzyme with nucleotide of SEQ ID NO: 1 or SEQ ID NO:2 and encoding the protein of SEQ ID NO: 3, wherein the enzyme is coupled to a polyethylene glycol (PEG).

2. The PEG-GGT enzyme of claim 1, wherein the γ-glutamyl transpeptidase (GGT) enzyme is a. Helicobacter pylori y-Glutamyltranspeptidase.

3. A pharmaceutical composition comprising the PEG-GGT enzyme of claim 1 with at least one pharmaceutically acceptable excipient.

4. The pharmaceutical composition of claim 3, wherein the composition is administered to a subject in need thereof in a therapeutically effective amount to deplete circulating glutamine without affecting asparaginase activity.

5. The pharmaceutical composition of claim 4, wherein glutamine depletion leads to an arrest in cell proliferation and induces a state of immunosuppression.

6. The pharmaceutical composition of claim 3, wherein the subject has a cancer.

7. The pharmaceutical composition of claim 6, wherein glutamine is depleted and induces a. state of immunosuppression in a tumor microenvironment (TME) of the cancer.

8. The pharmaceutical composition of claim 7, wherein the induced a state of immunosuppression is marked by a notable enrichment in populations of PMN- MDSCs and M2 macrophages in the tumor microenvironment (TME).

9. The pharmaceutical composition of claim 3, wherein the composition is administered to a subject in need thereof in a therapeutically effective amount to hydrolyze glutathione (GSH) and increase the concentration of circulating cysteine and glycine in the tumor of the subject.

10. The pharmaceutical composition of claim 3, wherein the subject has an autoimmune disease including acute respiratory distress syndrome (ARDS), asthma, rheumatoid arthritis (RA) and lupus nephritis.

11. The pharmaceutical composition of claim 10, wherein glutamine is depleted and induces a state of immunosuppression in the subject with the autoimmune disease.

12. A method of depleting circulating glutamine comprising administering to the host or the subject a therapeutically effective amount of the pharmaceutical composition of claim 3.

13. The method of claim 12, wherein the pharmaceutical composition is administered intravenously, intraarterially, intraperitoneally, intralesionally, intramuscularly, intravesicularlly, intranasally by injection, by infusion, by continuous infusion, by localized perfusion bathing target cells directly, or via a catheter.

14. The method of claim 12, further comprising administering at least a second anticancer therapy to the subject.

15. The method of claim 14, wherein the second anticancer therapy is a surgical therapy, chemotherapy, radiation therapy, cryotherapy, hormone therapy, immunotherapy or cytokine therapy.

16. The method of claim 14, wherein the second anticancer therapy is an asparaginase comprising asparaginases selected from those derived from E. coli, Erwinia chrysanthemi, and pegylated asparaginases.

17. A glutamine depletion gene signature comprising a) a glutamine depletion signature of upregulated genes having one or more combination thereof; and b) a glutamine depletion signature of downregulated genes having one or more genes comprising SK1L, RPS23, UAO1L1 , TPTI, EIF4B, KLHL24,

18. /X method of using the glutamine depletion gene signature of claim 17, comprising a) obtaining a subject’s cancer sample; b) obtaining transcriptomic data from the sample; c) performing single sample GSEA (ssGSEA) d) analyzing TCGA within cancer sample; e) determining a glutamine depletion score; and f) using the glutamine depletion score to determine the effect of glutamine depletion on the subject’s prognosis and clinical outcomes.

19. The glutamine depletion gene signature of claim 18, wherein the highest expression of glutamine depletion signature is labeled as a high glutamine depletion score and the lowest expression of glutamine depletion are labeled as a low glutamine depletion score.

20. The glutamine depletion gene signature of claim 18, wherein the high glutamine depletion score is not associated with significantly favorable prognostic outcome in most cancers.