IRF8-mediated reprogramming of the immune microenvironment in glioblastoma

Abstract

Provided herein is a retroviral replicating vector comprising a transgene that encodes IRF8. The vector induces killing of tumor cells within the tumor microenvironment of a brain tumor and, as such, finds use in a variety of methods of treatment, including a method for treating glioblastoma multiforma (GBM). In such a treatment, the vector may be administered intracranially or intratumorally.

Description

[0001] IRF8-MEDIATED REPROGRAMMING OF THE IMMUNE MICROENVIRONMENT

[0002] IN GLIOBLASTOMA

[0003] STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

[0004] This invention was made with government support under Grant Nos. R25 GM056847 and R35 NS 105068 awarded by the National Institutes of Health. The government has certain rights in the invention.

[0005] CROSS-REFERENCING

[0006] This application claims the benefit of U.S. provisional application serial no. 63 / 567,863, filed on March 20, 2024, which application is incorporated by reference herein.

[0007] BACKGROUND

[0008] Glioblastoma multiforma (GBM) is the most aggressive type of brain tumor and has a median overall survival of -15 months. The current standard-of-care treatments have not advanced significantly over the last 10 years. Although immunotherapy has led to breakthroughs in other tumor types, it has not seen significant success in GBM. GBM is classified as a “cold” tumor and is sparsely infiltrated with T-cells or active antigen-presenting cells (APCs), such as dendritic cells (DC).

[0009] New strategies for treating GBM and other solid tumors are of great clinical value.

[0010] SUMMARY

[0011] Provided herein is a retroviral replicating vector comprising a transgene that encodes interferon regulatory factor 8 (IRF8). The present retroviral replicating vector can be administered to a subject that has a brain tumor such as GBM, thereby improving survival and slowing tumor growth. The present retroviral replicating vector finds use in a variety of methods of treatment, including a method for treating glioblastoma multiforma (GBM).

[0012] Without wishing to be bound to any particular theory, it is thought that the present retroviral replicating vector initially infects tumor cells, which are rapidly dividing. Retroviral replicating vector produced in tumor cells then infects nearby myeloid-derived suppressor cells (MDSCs). Expression of IRF8 (which is encoded by the vector) in the infected MDSCs causes those to differentiate into antigen-presenting cells which, in turn, provides an environment that has reduced immunosuppressive signals and increased antigen-producing cells. The tumor cells can be more effectively killed by cytotoxic T cells in this environment. Based on data presented herein, reprogramming only a modest number of MDSCs (<20%) has a major impact on the immunological milieu, significantly improving survival and slowing tumor growth.

[0013] While the vector and methods have been reduced to practice using a retroviral replicating vector, glioblastoma multiforma and a transgene that encodes IRF8, the general strategy can be expanded to other types of vectors, other solid tumors and other myeloid differentiation factors. These broader embodiments will be additionally described below.

[0014] Additional embodiments and other features, advantages and variations may become apparent in view of the description that follows below.

[0015] BRIEF DESCRIPTION OF THE FIGURES

[0016] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0017] FIGS. 1A-1E. Human and murine glioblastoma contain proliferating myeloid cell populations. (FIG. 1A) Flow cytometric analysis of Ar l expression in all myeloid cells (CD45+ CD1 lb+) and M-MDSCs (CD45+ CD1 lb+ Ly6C+). (FIG. IB) Bar graph represents in vivo expression of Argl in myeloid and Ly6C+ cells. Bars represent mean of 6 biological replicates. (FIG. 1C) Expression of intracellular- Ki67 expression in immune (CD45+) and myeloid (CD45+ CD1 lb+) cells. IgG2a K isotype was used to define gates. (FIG. ID) Bar graph represents in vivo expression of Ki67 in tumor, immune, and myeloid cells. Bars show the mean of 6 biological replicate samples. (FIG. IE) Expression of proliferation markers Ki67, PCNA, Cyclin A, and Phosphorylated Histone H3 in human primary GBM samples. Flow cytometry plots are pre-gated for live CD45+ immune cells.

[0018] FIGS. 2A-2E. Transduction with IRF8 in vivo suppresses the growth of intracerebral SB28 tumors. (FIG. 2A) Schematic of in vivo studies. Pre-mixed model: SB28 cells pre-transduced at 2% with either RRV-EMPTY or RRV-IRF8 were implanted intracerebrally. Direct injection model: SB28 WT cells were implanted intracranially and concentrated 1.2e7 transducing units (TU) / mL RRV (EMPTY or IRF8) or PBS was injected at day 4 post-tumor inoculation using the same inoculation coordinates. Tumor growth kinetics were monitored using bioluminescence (BLI) twice per week until study completion. Tissues were harvested and dissociated into single cells for analysis. (FIG. 2B) Kaplan-Meier curves showing survival; Pre-mixed: SB28 WT, SB28 RRV-EMPTY, and SB28 RRV-IRF8. (FIG. 2C) Pre-mixed: BLI imaging data corresponding with tumor growth kinetics. P-values assessed on day 12 post-tumor inoculation in pre-mixed model. (FIG. 2D) Kaplan-Meier curves showing survival; Direct injection: PBS inj ., RRV-EMPTY inj RRV-IRF8 inj. (FIG. 2E) Direct Injection: averages of BLI imaging data corresponding with tumor growth kinetics (n-20 mice per group); P-values assessed on day 14 post-tumor inoculation in direct injection model.

[0019] FIGS. 3A-3E. IRF8 transduction enhances the number of glioblastoma-infiltrating cytotoxic T-cells. (FIG. 3A) Immune cell type changes between groups. Each cell type is associated with a set of genes; differential expression of gene sets is correlated with cell type abundance. (FIG. 3B) Cell type scores for each animal (n=6). Cell type profiling algorithm was previously described by Danaher et al (PMID: 28239471). (FIG. 3C) Box-and-whisker plots derived from the expression of T-cell genes. (FIG. 3D) Representative flow plots of pan T-cells (CD45+ CD3+) and CD4 (CD45+ CD3+ CD4+) or CD8 (CD45+ CD3+ CD8+) T-cells. (FIG. 3E) Left bar graph shows T cells and right bar graph shows CD8 T cells. Bars represent the mean of 9 biological replicates.

[0020] FIGS. 4A-4D. IRF8 transduction enhances the number of glioblastoma-infiltrating type 1 conventional dendritic cells. (FIG. 4A) Box-and-whisker plots derived from the expression of MHC-associated (top panel) or antigen-processing genes (bottom panel). (FIG. 4B) Representative flow plots of the pan-DC (CD45+ CD11C+ MHC 11+) and cDCl populations. Live cells were gated on CD45+ CD1 lb- CD1 lc+ MHC 11+ and CD103. The cDCl populations were further refined by selecting CD24+ XCR1+, markers for terminally differentiated cDCls capable of antigen cross-presentation. (FIG. 4C) Left bar graph shows dendritic cells and right bar graph shows migratory type 1 eDCs. Bars represent the mean of 9 biological replicates derived from day 18 tumors. (FIG. 4D) Transduction efficiency in vivo represented by expression of the RRV marker, P2A. Samples were analyzed at day 18 post-tumor inoculation. Bars represent mean of 6 biological replicates.

[0021] FIGS. 5A-5F. Infection of non-tumor cells by RRV-IRF8 is necessary for survival benefit and slowed tumor growth. (FIG. 5A) Mice were given 0.4mg / mL AZT + 2% sucrose water or 2% sucrose water-only control, with drug administration beginning two days prior to tumor inoculation and continuing until study endpoint (day 17 post-tumor inoculation). Representative flow plots of GFP+ tumor cells in mice receiving AZT (top left panel) or control water (top right panel). Bars represent the mean of 3 biological replicates (bottom panel). (FIG. 5B) BLI tumor growth kinetics plots. 6 groups; n=10 mice per group. BLI performed twice weekly until study endpoint. BLI concluded at day 60 for 2 long-term surviving animals. (FIG. 5C) Average BLI tumor growth kinetics. (FIG. 5D) Kaplan-Meier curves showing survival. (FIG. 5E) Median survival for all groups and significance comparisons for RRV-IRF8 2% vs. 100%, RRV-IRF8 2% vs 30%, and RRV-IRF8 30% vs 100%. (FIG. 5F) Box-and-whisker plots show differential expression of T-cell function- (top panel) and DC function-related genes (bottom panel) between RRV-IRF8 100% + AZT (n=6 biological replicates) and RRV-IRF8 2% groups (n=5 biological replicates).

[0022] FIGS. 6A-6D. IRF8 transduction reduces immunosuppressive myeloid cells and enhances antigen presentation. (FIG. 6A) Representative flow plots of Ar l expression in Ly6C+ cells, all plots pre-gated on live CD45+ CD1 lb-1- cells. Flow plots for RRV-EMPTY tumor (left panel) and RRV-IRF8 tumor (right panel) are shown. (FIG. 6B) Bars show Argl expression in M-MDSCs (left panel) and PMN-MDSCs (right panel), representing the mean of 6 biological replicates. (FIG. 6C) Positive and negative controls for T-cell activation (top left panel); gates set on negative control peak. T-cell / myeloid cell co-culture at 0.8 effector: 1 target ratio. Intra-tumoral myeloid cells were isolated from day 18 RRV-EMPTY or RRV-IRF8 tumors. Naive T-cells were isolated from age-matched non-tumor bearing mice. Representative flow plots show T-cell proliferation (CFSE peaks) after 4 days of co-culture (top panels). Bars (bottom panel) represent the mean of 6 biological replicates (n=3 technical replicates for each). (FIG. 6D) OT-1 T-cell / DC co-culture: CDl lc+ DCs were isolated from SB28 OVA RRV- EMPTY or RRV-IRF8 tumors and cervical lymph nodes. Representative flow plots (top two rows of panels) show T-cell proliferation (CFSE peaks) after 4 days of co-culture. Bars (bottom panel) represent the mean of 6 biological replicates (n=2 technical replicates for each biological replicate).

[0023] FIG. 7. Vector maps of RRV-IRF8 (top) and RRV-EMPTY control (bottom). Both vectors contain the P2A self-cleaving peptide linking the transgene cassette to the viral genome. The P2A sequence is also used as a marker for vector transduction following intracellular detection and flow cytometric analysis.

[0024] DEFINITIONS

[0025] As used herein, the terms "treatment," "treating," “treat” and the like, refer to obtaining a desired pharmacologic and / or physiologic effect and / or a response related to the treatment. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or can be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject which can be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0026] A “therapeutically effective amount” or “efficacious amount” refers to the amount of an agent (including biologic agents, such as cells), or combined amounts of two agents, that, when administered to a mammal or other subject for treating a disease, is sufficient to effect such treatment for the disease. The “therapeutically effective amount” will vary depending on the agent(s), the disease and its severity and the age, weight, etc., of the subject to be treated.

[0027] The terms “individual,” “subject,” “host,” and “patient,” used interchangeably herein, refer to a mammal, including, but not limited to, murines (e.g., rats, mice), non-human primates, humans, canines, felines, ungulates (e.g., equines, bovines, ovines, porcines, caprines), lagomorphs, etc. In some cases, the individual is a human. In some cases, the individual is a non- human primate. In some cases, the individual is a rodent, e.g., a rat or a mouse. In some cases, the individual is a lagomorph, e.g., a rabbit.

[0028] The term “operably linked” refers to a juxtaposition wherein the components so described are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression. Operably linked nucleic acid sequences may but need not necessarily be adjacent. For example, in some instances a coding sequence operably linked to a promoter may be adjacent to the promoter. In some instances, a coding sequence operably linked to a promoter may be separated by one or more intervening sequences, including coding and non-coding sequences. Also, in some instances, more than two sequences may be operably linked including but not limited to c.g., where two or more coding sequences arc operably linked to a single promoter.

[0029] DETAILED DESCRIPTION

[0030] Before the methods and compositions of the present disclosure are described in greater detail, it is to be understood that the methods and compositions are not limited to particular’ embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the methods and compositions will be limited only by the appended claims.

[0031] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the methods and compositions. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the methods and compositions, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the methods and compositions.

[0032] Certain ranges are presented herein with numerical values being preceded by the term “about.” The term “about” is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0033] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the methods and compositions belong. Although any methods and compositions similar or equivalent to those described herein can also be used in the practice or testing of the methods and compositions, representative illustrative methods and compositions are now described. All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the materials and / or methods in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present methods and compositions are not entitled to antedate such publication, as the date of publication provided may be different from the actual publication date which may need to be independently confirmed.

[0034] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0035] It is appreciated that certain features of the methods and compositions, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the methods and compositions, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present disclosure and are disclosed herein just as if each and every combination was individually and explicitly disclosed, to the extent that such combinations embrace operable processes and / or compositions. In addition, all sub-combinations listed in the embodiments describing such variables are also specifically embraced by the present methods and compositions and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

[0036] As will be apparent to those of skill in the ail upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present methods. Any recited method can be carried out in the order of events recited or in any other order that is logically possible. Vectors

[0037] As noted above, this disclosure provides a retroviral replicating vector (RRV) comprising a transgene that encodes IRF8 (interferon regulatory factor 8). IRF8 (which may be alternatively referred to as ICSBP, IRF-8, ICSBP1, IMD32A, IMD32B, H-ICSBP in the literature) is a transcription factor of the interferon (IFN) regulatory factor (IRF) family. Proteins of this family are composed of a conserved DNA-binding domain in the N-terminal region and a divergent C- terminal region that serves as the regulatory domain. The IRF family proteins bind to the IFN- stimulated response element (ISRE) and regulate expression of genes stimulated by type I IFNs, namely IFN-alpha and IFN-beta. IRF family proteins also control expression of IFN-alpha and IFN-beta-regulated genes that are induced by viral infection. See, e.g., Weisz et al (J. Biol. Chem. 1992 267 (35): 25589-96), Nehyba et al (Mol. Cell. Biol. 200222: 3942-57), Tamura et al (I. Interferon Cytokine Res. 200222: 145-52) and Yang et al (I. Immunol. 2011 187: 4426- 30).

[0038] The human IRF8 protein is encoded by Genbank Gene ID 3394. Any of the isoforms of the human protein, e.g., isoform 1 (which is 436 amino acids in length and defined by Genbank accession nos. NP_001350836.1 and GID1394533456), isoform 2 (which is 426 amino acids in length and defined by Genbank accession number P _002154.1 and GID4504567), isoform 8 (which is 222 amino acids in length and defined by Genbank accession nos. NP_001350837.1 and GID1394533468) or isoform XI (which is 436 amino acids in length and defined by Genbank accession nos. XP_047290008.1 and GID2217305806) could be used. The mouse IRF8 protein is 424 amino acids in length and is defined by Genbank accession no. NP_001288740.1 / GID685424584. A variant of a wild-type protein that has an amino acid sequence that is at least 90%, at least 95%, or at least 98%, identical to the amino acid sequence of a wild type protein may be used in any embodiment.

[0039] Retroviral replicating vectors are non-lytic replication-competent gamma retroviral vectors and are sometimes referred to as a replication-competent retrovirus (RCR). Such vectors have been reviewed in a variety of publications, including Logg et al (Methods Mol. Biol. 2004 246:499-525), Kubo et al (Cancer Gene Therapy 2019 26: 128-135), Logg et al (Methods Enzymol. 2012 507: 199-228), Lewis et al (J Virol. 1994 68:510-6) and Chen et al (Int. J. Mol. Sei. 202021: 1433). These vectors have a stringent requirement for cell division and their ability to integrate stably into the genome of cancer cells, without immediate cytolysis, contributes to long-lasting therapeutic efficacy. Thus, retroviral replicating vectors selectively infect and replicate in the tumor environment. They spread through the tumor when infected tumor cells produce infectious virus that buds off from the infected cell and spreads to neighboring replicating tumor cells. In any embodiment, the retroviral replicating vector may be a murine leukemia virus (MLV) retroviral viral vector.

[0040] Because the transgene is going to be expressed in myeloid cells, the transgene may be operably linked to a constitutive promoter or a promoter that is selectively active in myeloid cells, e.g., the CD1 lb, p47 phox or MMP14 promoters, or a promoter that is driven by promoter elements of the lysM, csflr, CD11c, CD68, macrophage SRA, and CD1 lb genes (as described in Hume Journal of Leukocyte Biology, Volume 89, Issue 4, Apr 2011, Pages 525-538).

[0041] Methods

[0042] Also provided is a method for increasing killing of tumor cells within the tumor microenvironment of a brain tumor, which method may comprise: administering the retroviral replicating vector described above to the tumor microenvironment.

[0043] A method of treatment is also provided, which method may comprise administering the retroviral replicating vector described above to a subject that has a brain tumor. In either of these embodiments, the subject may have glioblastoma multiforma (GBM).

[0044] As would be apparent, the tumor microenvironment should comprise myeloid-derived suppressor cells (MDSCs) and cytotoxic T cells, and, upon administration of the retroviral replicating vector, the myeloid-derived suppressor cells differentiate into antigen-producing cells, the number of cytotoxic cells increase, and the tumor cells are killed at a higher rate than without the vector.

[0045] In these embodiments, the retroviral replicating vector may be administered locally to the brain to the brain tumor, e.g., intracranially or intratumorally (e.g., by direct injection into the brain cavity or tumor). In other embodiments, the administering may be systemic, e.g., intravenous. In any embodiment, the retroviral replicating vector may be administered only once, by injection.

[0046] Typically the dose will be about 9xl06TU / 100 pl; however, the dose may range from about 105to about 1012TU given in one or more doses of 100 pl or scaled appropriately by blood value for larger animals and humans (roughly 2500 fold for a human compared to a mouse). Other embodiments

[0047] Also provided is a method for increasing killing of tumor cells within the tumor microenvironment of a solid tumor, where the method comprises administering a transgene that encodes a myeloid differentiation factor to the solid tumor. Also provided is a method of treatment comprising administering a transgene that encodes a myeloid differentiation factor to a subject that has a solid tumor.

[0048] In these embodiments, the cancer may be a solid tumor, e.g., a carcinoma or a sarcoma. If the solid tumor is a carcinoma, in certain embodiments, the carcinoma is a basal cell carcinoma, squamous cell carcinoma, renal cell carcinoma, ductal carcinoma in situ (DCIS), invasive ductal carcinoma, or adenocarcinoma. Carcinomas that can treated by a method disclosed herein include, but are not limited to, esophageal carcinoma, hepatocellular carcinoma, basal cell carcinoma (a form of skin cancer), squamous cell carcinoma (various tissues), bladder carcinoma, including transitional cell carcinoma (a malignant neoplasm of the bladder), bronchogenic carcinoma, colon carcinoma, colorectal carcinoma, gastric carcinoma, lung carcinoma, including small cell carcinoma and non-small cell carcinoma of the lung, adrenocortical carcinoma, thyroid carcinoma, pancreatic carcinoma, breast carcinoma, ovarian carcinoma, prostate carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinoma, cystadenocarcinoma, medullary carcinoma, renal cell carcinoma, ductal carcinoma in situ or bile duct carcinoma, choriocarcinoma, seminoma, embryonal carcinoma, Wilm's tumor, cervical carcinoma, uterine carcinoma, testicular carcinoma, osteogenic carcinoma, epithelial carcinoma, and nasopharyngeal carcinoma. Sarcomas that can be treated by a method disclosed herein include, but are not limited to, fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, chordoma, osteogenic sarcoma, osteosarcoma, angiosarcoma, endothelio sarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's sarcoma, leiomyosarcoma, rhabdomyosarcoma, and other soft tissue sarcomas. Other solid tumors that can be treated by a method disclosed herein include, but are not limited to, glioma, astrocytoma, medulloblastoma, craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma, and retinoblastoma, including primary tumors and, in certain cases, metastases. For example, the solid tumor may be colon cancer, bladder cancer, prostate cancer, breast cancer, lung cancer, or mesothelioma. In these embodiments, the transgene may administered via a retroviral vector, retroviral replicating vector, adcno-associatcd virus (AAV), adenoviral or lentiviral vector, or encapsulated RNA, for example.

[0049] In these embodiments, the transgene may encode IRF8 (interferon regulatory factor 8), as described above, BATF3 (basic leucine zipper ATF-like transcription factor 3; also known as JDP1, JUNDM1, SNFT; Genbank accession no: AAI17490.1; GID 109658644; Gene ID: 55509) or ID2 (inhibitor of DNA binding 2; also known as GIG8, ID2A, ID2H, bHLHb26; Genbank accession no: EAX01018.1, GID119621423; Gene ID: 3398), another factor that causes myeloid cells to differentiate, or any combination thereof. The transgene may encode a wild-type protein or a variant of wild-type proteins that has an amino acid sequence that is at least 90%, at least 95%, or at least 98%, identical to the wild-type protein.

[0050] Consistent with the above, the tumor microenvironment should comprise myeloid- derived suppressor cells (MDSCs) and cytotoxic T cells, and, upon administration of the transgene, the myeloid-derived suppressor cells differentiate into antigen-producing cells, the number of cytotoxic cells increase, and the tumor cells are killed at a higher rate than without the transgene.

[0051] In these embodiments, the retroviral replicating vector may be administered locally to the tumor, e.g., by direct injection into the tumor. In other embodiments, the administering may be systemic, e.g., intravenous. However the transgene may be administered, in some embodiments it may be administered only once, by injection.

[0052] Combination therapies

[0053] The present method of treatment may comprise co-administration of the vector and at least one additional therapeutic agent. By “co-administration” it is meant that both the transgene and at least one additional therapeutic agent are administered to an individual, although not necessarily at the same time, in order to achieve a therapeutic effect that is the result of having administered both the vector and the at least one additional therapeutic agent. The administration of the vector and the at least one additional therapeutic agent can be substantially simultaneous, e.g., the polypeptide can be administered to an individual within about 1 minute to about 24 hours (e.g., within about 1 minute, within about 5 minutes, within about 15 minutes, within about 30 minutes, within about 1 hour, within about 4 hours, within about 8 hours, within about 12 hours, or within about 24 hours) of administration of the at least one additional therapeutic agent. In some cases, the transgene of the present disclosure is administered to an individual who is undergoing treatment with, or who has undergone treatment with, the at least one additional therapeutic agent. The administration of the vector can occur at different times and / or at different frequencies.

[0054] In some embodiments, the patient may additionally receive a T cell therapy (e.g., a CAR T cell therapy) or cancer vaccine that targets the tumor. Exemplary T cell therapies and cancer vaccines for GBM may target, e.g., IL13RA2, EGFRvIII, HER2, EphA2, MUC1, EGFR, PD-L1, B7-H3, NKG2D and PDPN, among others (see, e.g., Shraibman el al. Mol Cell Proteomics 2019 18:1255-1268; Wu et al. J Big Data. 2022 9: 92; Wang et al. Chin Neurosurg J. 2022 8: 34; Nakagawa et al. Neurooncol Adv. 2023 5: vdacl77; Nehama et al. EBioMedicine. 2019 47:33- 43; Yang et al. J Immunother Cancer. 2019 7: 171, Razpotnik et al. Front Immunol. 2017 8: 1181 and Akhavan et al Immunol Rev. 2019 290: 60-84). Other antigens are known for other cancers.

[0055] Other therapies include immune checkpoint inhibitors. Exemplary immune checkpoint inhibitors include inhibitors that target an immune checkpoint polypeptide such as CD27, CD28, CD40, CD 122, CD96, CD73, CD47, 0X40, GITR, CSF1R, JAK, PI3K delta, PI3K gamma, TAM, arginase, CD137 (also known as 4-1BB), ICOS, A2AR, B7-H3, B7-H4, BTLA, CTLA-4, LAG3, TIM3, VISTA, CD96, TIGIT, CD122, PD-1, PD-L1 and PD-L2. In some cases, the immune checkpoint polypeptide is a stimulatory checkpoint molecule selected from CD27, CD28, CD40, ICOS, 0X40, GITR, CD122 and CD137. In some cases, the immune checkpoint polypeptide is an inhibitory checkpoint molecule selected from A2AR, B7-H3, B7-H4, BTLA, CTLA-4, IDO, KIR, LAG3, PD-1, TIM3, CD96, TIGIT and VISTA.

[0056] Further co-therapies include for example, (a) anthracycline therapy (e.g., by administering daunomycin, doxorubicin, or mitoxantrone), (b) alkylating agent therapy (e.g., by administering mechlorethane, cyclophosphamide, ifosfamide, melphalan, cisplatin, carboplatin, nitrosourea, dacarbazine, procarbazine or busulfan), (c) topoisomerase II inhibitor therapy (e.g., by administering etoposide or teniposide), (d) bleomycin therapy, (e) anti-metabolite therapy (e.g., by administering methotrexate, 5-fluorocil, cytarabine, 6-mercaptopurine or 6- thioguanine), (f) vinca alkyloid therapy (e.g., by administering vincristine or vinblastine), (g) steroid therapy (e.g., by administering prednisone or dexamethasone and (h) radiation treatment, etc. Alternative therapies include targeted therapies and non-targeted chemotherapies, where targeted therapy includes treatment with erlotinib (Tarceva), afatinib (Gilotrif), gefitinib (Iressa) or osimertinib (Tagrisso) which may be administered to patients having an activating mutation in EGFR, crizotinib (Xalkori), ccritinib (Zykadia), alcctinib (Alcccnsa) or brigatinib (Alunbrig) which may be administered to patients having an ALK fusion, crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), crizotinib (Xalkori), entrectinib (RXDX-101), lorlatinib (PF-06463922), ropotrectinib (TPX-0005), DS-6051b, ceritinib, ensartinib or cabozantinib which may be administered to patients having an ROS1 fusion, or dabrafenib (Tafinlar) or trametinib (Mekinist) which may be administered to patients having an activating mutation in BRAF. Many other actionable mutations are known. If the patient is going to be switched to a non-targeted chemotherapy, the therapy may be, for example, a platinum-based doublet chemotherapy (in which the platinum-based doublet chemotherapy may comprise a platinumbased agent selected from cisplatin (CDDP), carboplatin (CBDCA), and nedaplatin (CDGP)) and one third-generation agent (selected from docetaxel (DTX), paclitaxel (PTX), vinorelbine (VNR), gemcitabine (GEM), irinotecan (CPT-11), pemetrexed (PEM), and tegafur gimeracil oteracil (SI)).

[0057] The following examples are offered by way of illustration and not by way of limitation.

[0058] EXPERIMENTAL

[0059] Below are examples of specific embodiments for carrying out the present invention. The examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way.

[0060] Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should, of course, be allowed for.

[0061] SUMMARY

[0062] In this study, the effects of RRV-IRF8 on survival and tumor growth kinetics were examined in the SB28 murine GBM model. Immunophenotype was analyzed by flow cytometry and gene expression assays. Functional immunosuppression and antigen presentation was assayed by ex vivo T-cell-myeloid co-culture.

[0063] Intratumoral injection of RRV-IRF8 in mice bearing intracerebral SB28 glioma significantly suppressed the tumor growth and prolonged survival. RRV-IRF8 treated tumors exhibited significant enrichment of cDCls and CD8+ T-cells. Additionally, myeloid cells derived from RRV-IRF8 tumors showed decreased expression of the immunosuppressive markers Argl and ID01 and demonstrated reduced suppression of naive T-cell proliferation in ex vivo co-culturc, compared to controls. Furthermore, DCs from RRV-IRF8 tumors showed increased antigen presentation compared to those from control tumors. In vivo treatment with azidothymidine (AZT), a viral replication inhibitor, showed that IRF8 transduction in both tumor and non-tumor cells is necessary for survival benefit, associated with a reprogrammed, cDCl- and CD8 T-cell-enriched TIME.

[0064] These results indicate that in vivo expression of IRF8 alters the GBM tumor microenvironment. Particularly, engineering of glioma-infiltrating myeloid cells may allow for enhanced antigen presentation and reduced immunosuppression, resulting in improved tumor control. These results show that a modest number of reprogrammed MDSCs can have a substantial impact on the immunological milieu, significantly enriching and activating cytotoxic T-cells. In turn, this produces a highly significant survival effect.

[0065] Among other things, these results demonstrate that:

[0066] GBM intra-tumoral myeloid cells are proliferating and a target for RRV therapy.

[0067] Expression of IRF8 significantly improves survival and slows tumor growth in murine GBM.

[0068] IRF8 expression in MDSCs is sufficient to reduce immunosuppression and enrich cDCls in vivo.

[0069] MATERIALS AND METHODS

[0070] Plasmid generation: IRF8 sequence was taken from NCBI (Gene ID: 15900). Vectors were designed using SnapGene software suite. DNA fragment assembly was done using HiFi Gibson Assembly Master Mix (Invitrogen, A46628). Resulting clones were screened using PCR and sequenced for accuracy. Confirmed clones were expanded, and plasmid DNA was isolated using a Maxiprep kit (Invitrogen, K210016).

[0071] RRV production: RRVs were made using a standard calcium phosphate transfection. Vectors were titrated using SB28 cells; flow cytometry was used to measure both Gag and P2A expression. RRV was concentrated using the BioLand Retrovirus Purification Kit (cat. #RV02- 01).

[0072] SB28 glioma cell line: Details on the establishment of the SB28 cell line were previously described26,27Green fluorescence protein (GFP) was knocked out in all SB28 cells used in this study. GFP expression in the parental SB28 cell line was disrupted using CRISPR, GFP-negative cells were then sorted out of the resulting pool population, expanded, and used in all further studies.

[0073] SB28-OVA glioma cell line: Generation of SB28 cell line expressing full-length OVA peptide was previously described28. OVA sequence from Addgene (#22533) was used.

[0074] Cell doubling time assay: SB28 WT, SB28 RRV-EMPTY (100% transduced) cells, and SB28 RRV-IRF8 (100% transduced) cells were plated at IxlO5cells (n-3 per time point) and counted (Thermofisher Countess 3) at 24 and 48-hours post-seeding. Doubling times were averaged among replicates and time points.

[0075] Secreted factor assay: SB28 WT, RRV-EMPTY 100% transduced, and RRV-IRF8 100% transduced cells were seeded at 3xl04cells and cultured for 6 days. The resulting conditioned media was centrifuged, and the supernatant was filtered through a 0.7pm filter. The LEGENDplex flow cytometry-based assay (BioLegend, 740446) was used to measure secreted factor concentration according to the manufacturer’s protocol. Data were analyzed using LEGENDplex Analysis Software.

[0076] Orthotopic glioma models: 6-10-week-old female and male C57BL / 6J mice (Jackson Laboratories) were used in all animal experiments. Animals were housed and handled in the vivarium at the University of California San Francisco. All procedures followed an approved Institutional Animal Care and Use Committee (IACUC) protocol. Uninfected SB28 cells (98%) were mixed with pre-transduced SB28 cells (2%) expressing RRV-IRF8 or RRV-EMPTY. Mice were intracranially implanted with le4 cells / 2 uL HBSS at the following coordinates relative to bregma: mediolateral 2mm, dorsoventral -3mm. Mice were monitored daily and given postoperative care, as directed in the IACUC protocol. Tumor growth was measured using bioluminescent imaging twice weekly: 3mg (lOOuL) D-Luciferin was injected intraperitoneally 10 minutes prior to image acquisition.

[0077] Subcutaneous glioma model: 4xl05SB28 cells in lOOpL of cold HBSS were mixed in 1:1 ratio with Matrigel. 200pL of cell / Matrigel slurry was injected subcutaneously in the right flank. Tumor measurements were obtained via caliper and tumor area was calculated using length (mm) x width (mm).

[0078] Isolation of tumor-infiltrating cells: Tumor were dissected out and manually disassociated into ~lmm3pieces. Tumor pieces were incubated in 600-1000 mL collagenase buffer (10 mL IX PBS, 32 mg Collagenase IV, 10 mg Deoxyribonuclease I), shaking at 700 RPM at 37C for 45 minutes, mixing thoroughly every 15 minutes. Resulting disassociated tumors were filtered through a 70 pM filter and washed; red blood cells were lysed (Lonza, BP10-548E), and cells were frozen or stained fresh for flow cytometry.

[0079] Flow cytometry: Single-cell suspensions (0.5- le6 cells / sample) of SB28 tumor tissue were incubated with anti-CD16 / CD32 Fc block (BioLegend, 156603) for 10 minutes, followed by viability staining (BioLegend, 423101) in PBS for 15 minutes. After washing, a cocktail of fhiorophore-conjugated antibodies and monocyte blocker (BioLegend, 426101) was added to cells in a total volume of lOOuL staining buffer (IX PBS, 0.5% BSA, 2mM EDTA) and incubated in the dark at 4C for 30 minutes, rocking. For intracellular staining (cytosolic and nuclear), cells were subsequently fixed and permeabilized following the manufacturer’s protocol (Invitrogen, 00-5523-00). Fluorophore-conjugated intracellular antibodies were added and incubated in the dark for at least 30 minutes, rocking. Samples were washed twice and suspended in lOOuL staining buffer. Samples were analyzed using the Invitrogen Attune NxT (Thermo Fisher Scientific) flow cytometer.

[0080] RNA preparation and gene expression assay: RNA was extracted from previously frozen tumor samples using RNeasy Mini Kit (Qiagen, 74104) and normalized to lOOng / pL. For gene expression assays, the Nanostring nCounter Mouse PanCancer Immune Profiling panel was used (Nanostring, XT-CSO-MIP1-12). Data were analyzed using nSolver and Rosalind software.

[0081] 3 '-Azido-3 -deoxy thymidine (AZT) administration via drinking water: 0.4 mg / mL AZT (Sigma, A2169) with 2% sucrose was made in drinking water and protected from light. 2% sucrose was only used as control water; all water was changed weekly. To monitor water consumption, water bottles were weighed daily. Mice consumed AZT / sucrose water at the same rate as sucrose water.

[0082] Immunosuppression: Myeloid cell / T-cell co-culture: Myeloid cells from RRV-EMPTY or RRV-IRF8 tumors were isolated and co-cultured with CFSE-stained T cells from naive nontumor bearing C57BL / 6J mice. T-cells: T-cells were isolated from spleens of naive non-tumor bearing mice using a CD3 bead isolation kit (BioLegend, 480023). T-cells were stained with 0.5 mM CFSE dye and incubated in the dark for 10 minutes. Cells were washed and activated with CD3 / CD28 beads (Gibco, 11161D). 50 lU / mL IL-2 was added to all T-cells. Myeloid cells: SB28 tumors were disassociated into single cells, as described above. Myeloid cells were isolated using a CD11b bead isolation kit (BioLegend, 480109). Co-culture: Myeloid cells and T-cclls were combined at an effector: target ratio of 0.8:1. Cells were co-culturcd for 4.5 days and stained for flow cytometry.

[0083] Antigen presentation: DC / CD8 T-cell co-culture: DCs derived from tumors and cervical lymph nodes were isolated from OVA RRV-EMPTY or OVA RRV-IRF8 mice and co-cultured with naive OT-1 T cells. T-cells: T-cells were isolated from spleens of OT-1 transgenic (Jackson Laboratory, strain 003831) naive non-tumor bearing mice using a CD8 bead isolation kit (BioLegend, 480007) and stained with CFSE dye (as above). Positive control T-cells were activated with CD3 / CD28 beads, IL-2 was added to all T-cells. DCs: SB28-OVA RRV-EMPTY or IRF8 tumors were disassociated into single cells (as above). Cervical LNs from SB28-OVA tumor-bearing mice were concurrently incubated with collagenase / DNAse for 15 minutes at 37C, then mechanically disassociated through a 70 pM filter. DCs were isolated using a CD11c bead isolation kit (Milentyi, 130-100-875). Co-culture: 5xl03DCs were combined with IxlO5OT-1 T-cells in a 96-well plate and, incubated for 4 days and stained for flow cytometry.

[0084] Statistical Analyses: Mantel-Cox (Log-rank) test was used to determine the significance in Kaplan Meier curves (GraphPad Prism vlO.l.O). For experiments comparing RRV-EMPTY versus RRV-IRF8, results were analyzed using Student’s t-test. For studies with more than two groups, results were analyzed using one-way ANOVA. Significance symbols correspond to the following: *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001.

[0085] RESULTS

[0086] Intra-tumoral myeloid cells are proliferating and are a viable target for RRV-based therapies

[0087] To target the intra-tumoral myeloid compartment, this population was first characterized in a SB28 model26. The SB28 model of GBM is clinically relevant, with its low mutational burden and resistance to immune checkpoint blockade therapy27. Like human GBM, SB28 orthotopic tumors are highly infiltrated by myeloid cells, which comprise the vast majority of intra-tumoral immune cells28,29(data not shown). The myeloid compartment was further evaluated based on Ly6C and Ly6G, which are surface markers commonly used to distinguish monocytic (M-MDSCs) and polymorphonuclear MDSCs (PMN-MDSCs), respectively30. In SB28 tumors, M-MDSCs were the dominant MDSC population (data not shown), with previous studies in other models showing that M-MDSCs are more effective at suppressing T-cell functions than PMN-MDSCs31. Within the overall myeloid population, 36.6% (± 3.859 standard deviation (SD), n=6) of cells expressed Arginase 1 (Argl), an intracellular enzyme and marker of immunosuppression32,33. When analyzed further, 37.1% (±4.94 SD, n=6) of CDl lb+Ly6C+ cells also highly expressed Argl (FIGS. 1A, IB), suggesting these M-MDSCs had an immunosuppressive phenotype. Therefore, M-MDSCs in the model represent a robust population of the TIME and are a promising target for novel myeloid-targeting therapies, such as RRV- based genetic reprogramming.

[0088] Transduction with RRV requires active cell division24,25. To determine whether intra- tumoral myeloid cells could be targeted with RRV, their expression of Ki67, a proliferation marker, was examined. Within all live cells analyzed, 17.56% (± 4.92 SD, n=6) of all immune cells, and 8.83% (± 3.49 SD, n=6) of myeloid cells, were positive for Ki67 at the time of tumor harvest, day 17 post-tumor inoculation (FIGS. 1C, ID). Because these values represent Ki67+ cclls at a single time point, cumulatively a more significant number of myeloid cells will have undergone mitosis over the lifetime of the tumor. As an integrating virus, RRV is highly persistent, and intratumoral replication and cellular transduction will continue over time, so these data suggest that a portion of the myeloid compartment may be a viable target for RRV-based therapies.

[0089] To evaluate the relevance to humans, the expanded list of proliferation markers was evaluated in clinical samples obtained from patients with primary GBM (n=2). Tumorinfiltrating immune cells were isolated from surgically resected fresh tumor samples and the expressions of PCNA (expressed in G1 and S phases), cyclin A (late S, G2, and M phases), and phosphorylated histone H3 (p-histone H3; M phase) were evaluated. Interestingly, it was found that the majority of human CD45+CD1 lb-1- cells were positive for all four markers (FIG. IE and data not shown).

[0090] IRF8 transduction of SB28 tumor cells in vitro decreases CCL2 secretion but does not impact proliferation capacity

[0091] A transgene cassette encoding the murine transcription factor IRF8 was inserted into a Moloney murine leukemia virus (MMLV)-based RRV (RRV-IRF8). In this vector, the P2A sequence encoding a “self-cleaving” peptide, which can be used to identify transduced cells by intracellular staining and flow cytometry, links the transgene cassette to the virus genome. The control vector encodes the P2A sequence but not IRF8 (RRV-EMPTY) (FIG. 7).

[0092] It was first evaluated whether infecting SB28 cells in vitro with the RRV-IRF8 or RRV- EMPTY vectors might have any effect on cell growth. Compared to untransduced SB28 WT- cells, there was no significant effect on cell doubling time after transduction with either the RRV-EMPTY or RRV-IRF8 (data not shown), implying that neither vector transduction per se, nor exogenous expression of the IRF8 transgene impacted tumor cell proliferation rate. To better understand the effect of IRF8 transduction in GBM cells, the in vitro tumor cell culture conditioned media was characterized using a flow cytometry-based secretome assay. Of note, the assay revealed that, among the tested 13 chemokines, the secretion of CCL-2 (MCP-1) was most prominently downregulated as a result of IRF8 transduction (data not shown). This phenotype is not SB28-specific, as murine GL261 and human GBM6 GBM cells also exhibit a significant decrease in CCL-2 secretion when transduced with RRV-IRF8 (data not shown). CCL-2 is a major chemoattractant in GBM that recruits effector and regulatory T-cells (Tregs) and immature myeloid cells to the tumor34. In patient samples, elevated CCL2 is correlated with worse patient outcomes, and inhibition of CCL2 in mouse models reduces intra-tumoral MDSCs and increases T-cell cytotoxicity12,35’36. Interestingly, intra-tumoral myeloid cells from RRV-IRF8 transduced SB28 tumor-bearing mice in vivo showed significantly decreased expression of the CCR2 receptor (data not shown).

[0093] Transduction with IRF8 suppresses the growth of intracerebral SB28 GBM tumors

[0094] To evaluate the effects of IRF8 expression in vivo, the RRV-EMPTY and RRV-IRF8 were tested using two methods. First, a pre-mixed tumor establishment model was used, in which 2% of the tumor cells implanted were transduced with either the RRV-IRF8 vector or RRV- EMPTY control vector. This allows for a single intracranial injection procedure, reducing inflammation and disruption of the blood-brain barrier associated with multiple injections and survival surgeries. Pre-mixing the RRV at a low percentage allows for efficient and reproducible tumor inoculation and enables the RRV to efficiently initiate replication and spread immediately following tumor engraftment. Second, a direct injection of concentrated RRV into established tumors (day 4 post-inoculation) was performed (data not shown). This method most closely mimics a clinical setting. Tumor growth kinetics were monitored with bioluminescent imaging (BLI), and tissues were harvested at a scheduled timepoint or humane endpoint (FIG. 2A).

[0095] Mice with SB28 RRV-IRF8 tumors had significantly longer overall survival (median overall survival (mOS)=28 days pre-mixed, 27 days direct inj.) than either untransduced SB28 WT (mOS=17 days, p<0.0001), PBS injection (mOS=17.5 days, p<0.0001), or RRV-EMPTY (mOS=19 days pre-mixed, 20 days direct inj., p<0.0001) groups (FIGS. 2B, 2D). BLI revealed that RRV-IRF8 mice also had slower growth kinetics than SB28 WT and RRV-EMPTY groups (pre-mixed:p<0.0001 on day 12, direct inj:p=0.0002 on day 14). RRV-EMPTY and RRV-IRF8 tumors grew at similar rates until approximately day 12-14, when the two groups began to separate (pre-mixed: median luminescence 5.3xl05vs. 3.6xl06, p=0.0003) and remained lower for the duration of the study (FIGS. 2C, 2E).

[0096] IRF8 transduction enhances the number of GBM-infiltrating T-cells and type 1 conventional dendritic cells

[0097] Because of the significant survival benefit and tumor growth delay observed in vivo, in the absence of cell proliferation rate change in vitro, overexpression of intra-tumoral IRF8 may lead to an overall change in the TIME, perhaps associated with reduced CCL2. Nanostring analysis of overall gene expression in bulk SB28 pre-mixed tumors from each treatment group showed clear segregation of RRV-EMPTY and RRV-IRF8 transduced tumors into two distinct groups (data not shown). Tumors transduced with RRV-IRF8 showed a higher abundance of CD45+ cells when compared with controls. The abundance of different immune cell types, each defined by a subset of genes, was given a score, and the scores for the control (RRV-EMPTY) and experimental (RRV-IRF8) groups were plotted as average trends (FIG. 3A), and as individual animals (FIG. 3B). IRF8 expression resulted in the overall increase in many immune populations, especially T-cells and cytotoxic cells (data not shown).

[0098] Next, the impact of IRF8 expression on the T-cell compartment of the TIME was evaluated using a T-cell- targeted Nanostring gene expression assay and flow cytometry. Nanostring analysis showed that in RRV-IRF8 tumors, 38 T-cell-associated genes were significantly differentially expressed (data not shown). Interestingly, IRF8 transduction most significantly upregulated the levels of CD3g, Ctla4, and Gzmb, suggesting that IRF8 expression enhanced the infiltration of activated and cytotoxic T-cells, with an overall enhancement of T- cell functionality (FIG. 3C). Flow cytometric analyses detected a significant (p=0.0025) increase of T-cells in RRV-IRF8 tumors compared to control tumors (Prc-mixcd: FIG. 3C, Direct inj: data not shown). Upon evaluation of the CD4 and CD8 T-cell compartments, it was observed that CD4 cells were the majority of T-cells in control tumors, comprising approximately 80% of total T-cells. However, in IRF8-RRV tumors, the CD8 T-cell population was significantly enriched (p<0.0001) with CD4 T-cells comprising only about 42% of total T-cells (FIGS. 3D, 3E).

[0099] While innate and adaptive immune mechanisms restricted RRV infection and spread in normal tissues, it is possible that the permissive tumor microenvironment would allow for infection of proliferating intratumoral myeloid cells. Thus, any immature myeloid cells transduced by RRV-IRF8 could adopt a more cDCl-like phenotype. Nanostring analysis of the intra-tumoral DC compartment showed upregulation of 6 genes associated with DC functions in RRV-IRF8 tumors compared to controls (data not shown). In support of this data, flow cytometry analyses revealed an enrichment of the pan-DC population (CD11C+ MHC 11+) in RRV-IRF8 in both pre-mixed and direct injection tumors (pre-mixed: p=0.015, FIGS. 4B, 4C, direct inj: data not shown). Gene expression analysis revealed significant upregulation of genes associated with MHC class I (H2-Aa, H2-Abl\ MHC class II (H2-Ebl'), and antigen processing and presentation (Tapi, Psmb9). among others (data not shown). Further immunophenotyping analyses showed significant enrichment of the cDCl (CDllc+, MHC II+, CD103+, CD24+, XCR1+) population in RRV-IRF8 tumors, compared to controls (p<0.0001; FIG. 4A and data not shown), suggesting that enhanced infiltration by cytotoxic T-cell and cross-presenting cDCls contribute to the survival benefit and delayed tumor growth kinetics observed in FIG. 2.

[0100] To further elucidate the effects of exogenous IRF8 expression, RRV transduction in tumor versus myeloid cells was measured in the pre-mixed model (FIG. 4D and data not shown). It was observed that approximately 59.52% (± 8.62 SD, n=6) of tumor cells were RRV-positive. More intriguingly, a lower percentage than tumor cells, about 9.11% (± 2.14 SD, n=6), of the myeloid cell populations were also RRV-positive, indicating successful spreading and transduction of RRV into proliferating myeloid cells. A review of recent literature indicated that a small number of mature DCs can provide critical anti-tumoral functions in the immunological milieu and even a modest increase in APC abundance can improve the anti-tumor immune response37. As RRV was observed to spread to over half of the tumor cells starting from the initial 2% pre-transduced SB28 cell inoculum, the contribution of IRF8 expression was not disregarded from either population and it was sought to investigate this intriguing phenotype further.

[0101] Infection of immune cells by RRV-IRF8 is necessary for decreased tumor growth rate and survival benefit

[0102] Next, the goal was to answer a vital mechanistic question: whether the transduction of tumor cells alone is sufficient to cause the observed TIME changes and survival benefit, or if the modest population of transduced myeloid cells contributes in an essential manner. To this end, a study was designed where the following experimental conditions were compared: (1) only tumor cells were infected and RRV spread is restricted or (2) all proliferating cells could be infected and the RRV is allowed to spread freely as in previous studies. To achieve this, the first group was given the anti-retroviral drug azidothymidine (AZT), a thymidine analog that inhibits reverse transcriptase and therefore precludes the ability of the virus to replicate38. AZT is used clinically and has been previously shown to inhibit RRV spread in mice when administered through drinking water39.

[0103] As a proof of concept, the efficacy of AZT water was evaluated using tumors pre-mixed with 2% green fluorescent protein (GFP)-RRV. In this model, RRV spread was quantified based on the GFP signal at day 17 from tumor inoculation. In the control group, approximately 83.90% (± 5.30 SD, n=3) of the tumor cells were GFP-positive. In contrast, in the AZT-treated group, GFP positivity was suppressed to only 1.55% (± 0.82 SD, n=3) confirming the in vivo efficacy of AZT (FIG. 5A). The same AZT- administration scheme was then used in the RRV-IRF8 model. Tumor-bearing mice were stratified into three groups (n=10) for each RRV (EMPTY and IRF8). In group 1, mice were injected with SB28 cells containing a 2% pre-mixed RRV-population with no AZT administration, recapitulating the conditions from FIGS. 3 and 4. Groups 2 and 3 were implanted with 30% and 100% pre-mixed RRV, respectively and received AZT. In these groups, RRV reverse transcription was blocked, preventing any further spread to proliferating cells, thereby restricting transgene expression solely to RRV-IRF8 already integrated into the genomes of pre-transduced tumor cells and their progeny only. AZT administration alone did not impact tumor growth (RRV-EMPTY / control water mOS= 17 days vs. RRV-EMPTY / AZT mOS=17.5 days). As shown in FIGS. 5B and 5C, pre-mixed SB28 tumors established with RRV-IRF8 30% and 100% provide a modest survival benefit, with median survival times of 20.5 and 23 days, respectively (FIG. 5E and data not shown). Strikingly, mice with 2% RRV-IRF8 pre-mixed tumors in which RRV spread was permitted to spread freely throughout the tumor, including immune cells, showed a significant survival benefit (mOS = 33.5 days, p=0.0005) (FIGS. 5D, 5E and data not shown) compared to the other groups, including the 100% RRV-IRF8 group + AZT. This study indicates that direct infection of non-tumor cells with RRV-IRF8 is crucial for the survival benefit and suggests that even a modest level of myeloid cell transduction (FIG. 4D) may be adequate to achieve this result.

[0104] To further understand the impact of IRF8 transduction in non-tumor cells, Nanostring was used to compare bulk gene expression in RRV-IRF8 2% pre-mixed tumors versus RRV- IRF8 100% pre-mixed tumors + AZT. RNA was isolated from tumor-bearing brain quadrant at day 17 post-inoculation. Differential expression of many T-cell-related genes was observed (data not shown), including some that were not seen in previous analyses (data not shown). Interestingly, expression of Tgfb2 and checkpoint molecules Cd276 (B7-h3) and Lag3, which have been identified as negative prognostic factors in GBM patients40 42were downregulated in samples from the RRV-IRF8 2% group. Importantly, GBM tumors produce high levels of TGF02, and TGF0 signaling contributes to immunosuppression and tumor progression43. Furthermore, the DC compartment showed a significant upregulation of Cd86, a marker of mature DCs capable of activating T-cells through interaction with CD28. These data further suggest that active intratumoral replication of RRV-IRF8, associated with IRF8 transduction in myeloid cells, significantly improves anti-tumor immune responses and even reduces the expression of known GBM-promoting genes.

[0105] Two mice in the RRV-IRF8 2% group survived over 60 days post-tumor inoculation without disease progression. To assess whether these mice developed immunological memory, the mice were rechallenged with a subcutaneous injection of untransduced SB28 WT cells in the right flank. Alongside them, naive, age-matched mice were injected as controls (data not shown). While the subcutaneous tumors grew in control mice, the rechallenged mice rejected the tumor. In summary, these data collectively suggest that additional IRF8 transduction in myeloid cells suppresses tumor-intrinsic immunosuppressive factors and enhances anti-tumor immunity, leading to the acquisition of long-term adaptive immune responses. RRV-IRF8 functionally reduces myeloid-derived immunosuppression and enhances antigen presentation

[0106] Next, the functions of IRF8-reprogrammed myeloid cells were characterized. Using flow cytometry, it was found that intra-tumoral myeloid cells (M-MDSCs, PMN-MDSCs, and Macrophages) in SB28 RRV-IRF8 2% tumors expressed lower levels of two immunosuppressive markers, Argl and IDO, compared to tumors transduced with RRV-Empty (FIGS. 6A, 6B and data not shown).

[0107] Next, to investigate the immunosuppressive capabilities of myeloid cells from SB28 2% pre-mixed RRV-EMPTY versus RRV-IRF8 tumors, a myeloid / T-cell co-culture assay was utilized. Animals from both groups were euthanized at day 17-post tumor inoculation and intra- tumoral myeloid cells were isolated. Concurrently, T-cells from age-matched naive animals were isolated. Myeloid cells were co-cultured with CFSE-labeled T-cells with CD3 / CD28-stimulation for 4.5 days. T-cells cultured with myeloid cells from RRV-IRF8 tumors proliferated significantly more, undergoing 3-4 proliferation cycles, while T-cells cultured with myeloid cells derived from control tumors underwent 0-1 proliferation cycles (p<0.0001) (FIG. 6C). These data suggest that IRF8 expression can functionally reprogram intra-tumoral myeloid cells to reduce their immunosuppression and to facilitate T-cell proliferation.

[0108] Finally, the ability of DCs from RRV-IRF8 treated mice to activate T-cells in an antigenspecific manner was tested. An ovalbumin (OVA) model antigen system was used, and mice were inoculated with intracerebral SB28-OVA 2% pre-mixed RRV-EMPTY or RRV-IRF8 tumors. All animals were euthanized on day 26 and isolated CD1 lc-1- DCs from tumors and cervical lymph nodes (cLN). DCs were co-cultured with CFSE-labeled naive OT-1 CD8 T-cells. Both intra-tumoral and cLN DCs from RRV-IRF8 treated mice induced high levels of OT-1 T- cell proliferation compared to DCs from RRV-EMPTY mice (p<0.0001) (FIG. 6D and data not shown). Interestingly, intratumoral DCs from RRV-IRF8 transduced tumors induced the most robust T-cell proliferation (~5 cycles), suggesting that RRV-driven reprogramming induces the development of functional APCs in situ, after which APCs migrate to cLNs and prime T-cells.

[0109] Discussion

[0110] Lack of functional APCs and the negative contribution of immunosuppressive myeloid cells are well-recognized barriers for developing effective immunotherapy approaches for patients. A productive anti-tumor immune response relies on the crosstalk between APCs and T- cclls within the tumor microenvironment44. In situ transduction of myeloid cells with IRF8, a critical transcriptional regulator of cDCls and a suppressor of MDSCs16\ were evaluated. The data indicate that RRV-mediated reprogramming of intra-tumoral myeloid cells into cDCl-like cells can lead to reduced immunosuppression and enhanced antigen presentation in the immunologically cold GBM TIME, associated with prolonged survival.

[0111] The relevance of murine GBM models remains an essential and challenging consideration when designing immune-based therapeutics. Although the SB28 orthotopic model mimics the immunosuppressive TIME of human GBM28, there are known differences in the MDSC compartment. The data concur with previous reports demonstrating that the M-MDSC population is dominant in mouse tumors, while the PMN-MDSC population is dominant in human GBM10,45. Nevertheless, the data demonstrates that reprogramming using RRV-IRF8 directly impacts both MDSC populations, in which both Argl and IDO expression were significantly reduced. Interestingly, Trovato et al.46and Groth et al.31reported that M-MDSCs are more immunosuppressive than PMN-MDSCs and have higher capacity to inhibit T-cell proliferation. This suggests that, although M-MDSCs may be present in lower absolute numbers in GBM patients, their contribution to immunosuppression can be significant, and, therefore, reprogramming this subset cells may represent a promising therapeutic modality. The approach described herein, which efficiently reverts immunosuppression in both MDSC subsets and promotes antigen presentation, would likely improve anti-tumor immunity in patients as well.

[0112] While the data implicated a critical contribution of IRF8 transduction in non-tumor cells, the effects of IRF8 expression in tumor cells remains to be fully elucidated. In 2021, Gangoso et al. demonstrated that GBM stem cells evaded immune attack by adopting a myeloid-like transcriptional signature, including expression of IRF847and a clinical study from Lei et al. reported IRF8 as a negative prognostic biomarker in bulk glioma tissues48. On the other hand, a 2023 study by Zimmermannova et al. revealed an alternative role of IRF8, demonstrating that exogenous expression of IRF8 and other DC-regulatory genes directly converted tumor cell lines into cDCl-like cells, capable of processing and presenting antigens49. As both tumor and myeloid cells were transduced with IRF8 in the RRV system, the significance of exogenous IRF8 expression in SB28 cells must be considered. SB28 cells normally show undetectable levels of IRF8 (data not shown), and RRV-IRF8 transduced SB28 cells did not demonstrate cDCl -phenotype based on expression of the DC markers MHC II, XCR1 , and CD 103 (data not shown). These observations arc consistent with the lack of gene expression changes linked to anti-tumor effects (FIG. 5F and data not shown) and the only modest improvement of overall survival (FIGS, 5D, 5E) of mice when RRV-mediated IRF8 transduction was limited to only tumor cells. On the other hand, endogenous IRF8 expression in intra-tumoral myeloid cells was varied, with IRF8 levels being inversely correlated with Argl expression (data not shown). These data suggest that IRF8 overexpression beyond its endogenous levels is required for reprogramming MDSCs into functional cDCls.

[0113] The results demonstrate a significant impact of RRV-mediated IRF8 transduction on the immune landscape and the survival of mice bearing intracerebral SB28 tumors, even despite the modest transduction efficiency in non-tumor cells. This shows that the potential impacts of paracrine effects by tumor cells transduced with RRV-IRF8 must be considered. In vitro, a significant reduction of CCL2 secretion by transduced tumor cells was shown (data not shown). The CCL2-CCR2 axis recruits immature myeloid cells to the tumor, where they subsequently develop into MDSCs50. Notably, CCR2+ M-MDSCs have been shown to inhibit CD8 T-cell infiltration to the TIME51. RRV-IRF8 transduced tumors showed reduced percentages of CCR2+ myeloid cells (p=0.0464, n=6) in vivo (data not shown). Thus, reduced CCL2 may also contribute to the observed effects, representing a paracrine role of the current RRV-mediated IRF8 transduction approach.

[0114] Another consideration is the direct transduction of myeloid cells, and whether this transduction is critical for eDC 1 enrichment. While it was functionally demonstrated that transduction of non-tumor cells is linked to survival benefit (FIG. 5), it is crucial to determine whether MDSCs are truly being infected in situ. To this end, the intracellular levels of P2A (a component of the RRV vector) was examined by flow cytometry in both intra-tumoral cDCls and their pan-DC counterparts and significantly higher levels of P2A among cDCls was observed, suggesting that these cDCls were once MDSCs that were transduced and then adopted a cDCl phenotype. Further, cLN-derived XCR1+ cDCls expressing the transduction marker P2A were detected, suggesting migration of in situ transduced cells from the tumor to the cLN. (data not shown). The importance of antigen cross-presentation by cDCls in potentiating antitumor immunity is well-reviewed in the literature, and multiple studies have shown that even a modest increase in intra-tumoral cDCl s can significantly enhance T-cell mediating tumor killing37,52’53

[0115] Although increased generation of cDCls through RRV transduction is promising, ultimately, anti-tumor immunity relies also on the contribution of effector cells, including T- cells. CD4 T-cells represent the majority of T-cells in RRV-EMPTY tumors, albeit in modest absolute numbers. Examination of RRV-IRF8 tumors showed not only an increase in T-cell abundance overall, but also a shift from CD4 to CD8 T-cell dominance (FIG. 3C). Within the CD4 T-cell compailment exist Tregs, which have known immunosuppressive functions. Interestingly, some T-cells (including Tregs) can be recruited to the brain by CCE2, independent of CCR2, revealing another important role of CCL2 reduction34,54. Furthermore, M-MDSCs can promote Treg generation by secreting TGFB2, which was significantly downregulated in RRV- IRF8 transduced tumors55(data not shown). A modest decrease of Tregs in RRV-IRF8 tumors (p=0.0498, n=6) compared to controls was observed, suggesting that the reduction of CD4 T- cells is due, in part, to reduced CCL2 leading to less recruitment of the Treg population (data not shown). cDCls efficiently cross-present intra-tumoral tumor antigens to CD8 T-cells, as demonstrated in FIG. 6F, using the 0VA / 0T-1 system. To further elucidate the importance of CD8 T-cells in this context, subsequent studies may utilize in vivo CD8 T-cell depletion. Altogether, the study described herein demonstrates a multi-faceted impact on the recruitment of both CD4 and CD8 T-cells, concurrently reducing Treg-mediated suppression and enhancing CD8 T-cell activation.

[0116] The gene therapy-based reprogramming approach described herein may be a valuable tool as a primary viral-based modality or in combination with other therapies. For example, the approach presents the opportunity to combine RRV-IRF8 with CAR-T-based therapies to support the activation and persistence of engineered T-cells in vivo. Additionally, these studies open a new area of RRV-based gene therapies in which tumor cells are not the sole target, and RRVs may be further engineered to target myeloid cells or other populations using cell and receptor- specific promoters. References

[0117] 1. Mohammed S, Dincsan M, Ajayakumar T. Survival and quality of life analysis in glioblastoma multiforme with adjuvant chemoradiotherapy: a retrospective study. Rep Pract Oncol Radiother. 2022;27(6): 1026- 1036. doi:10.5603 / RPOR.a2022.0113

[0118] 2. Schaff LR, Mellinghoff IK. Glioblastoma and Other Primary Brain Malignancies in Adults: A Review. JAMA. 2023;329(7):574-587. doi:10.1001 / jama.2023.0023

[0119] 3. Chuntova P, Chow F, Watchmaker PB, et al. Unique challenges for glioblastoma immunotherapy-discussions across neuro-oncology and non-neuro-oncology experts in cancer immunology. Meeting Report from the 2019 SNO Immuno-Oncology Think Tank. Neuro-Oncol. 2021;23(3):356-375. doi:10.1093 / neuonc / noaa277

[0120] 4. Alban TJ, Alvarado AG, Sorensen MD, et al. Global immune fingerprinting in glioblastoma patient peripheral blood reveals immune- suppression signatures associated with prognosis. JCI Insight. 3(21):el22264. doi: 10.1172 / jci.insight.122264

[0121] 5. Sharma P, Aaroe A, Liang J, Puduvalli VK. Tumor microenvironment in glioblastoma: Current and emerging concepts. Neuro-Oncol Adv. 2023;5(l):vdad009. doi: 10.1093 / noajnl / vdad009

[0122] 6. Veglia F, Sanseviero E, Gabrilovich DI. Myeloid-derived suppressor cells in the era of increasing myeloid cell diversity. Nat Rev Immunol. 2021;21(8):485-498. doi:10.1038 / s41577-020-00490-y

[0123] 7. Rodrigues IC, Gonzalez GC, Zhang L, et al. Normal human monocytes exposed to glioma cells acquire myeloid-derived suppressor cell-like properties. Neuro-Oncol.

[0124] 2010; 12(4):351-365. doi:10.1093 / neuonc / nop023

[0125] 8. Gallina G, Dolcetti L, Serafini P, et al. Tumors induce a subset of inflammatory monocytes with immunosuppressive activity on CD8+ T cells. J Clin Invest. 2006;l 16(10):2777- 2790. doi:10.1172 / JCI28828

[0126] 9. Gabrilovich DI, Ostrand-Rosenberg S, Bronte V. Coordinated regulation of myeloid cells by tumours. Nat Rev Immunol. 2012;12(4):253-268. doi:10.1038 / nri3175

[0127] 10. Raychaudhuri B, Rayman P, Ireland J, et al. Myeloid-derived suppressor cell accumulation and function in patients with newly diagnosed glioblastoma. Neuro-Oncol. 2011;13(6):591-599. doi:10.1093 / neuonc / nor042 11 . Otvos B, Silver DJ, Mulkeams-Hubert EE, et al. Cancer Stem Cell-Secreted Macrophage Migration Inhibitory Factor Stimulates Myeloid Derived Suppressor Cell Function and Facilitates Glioblastoma Immune Evasion. Stem Cells Dayt Ohio. 2016;34(8):2026-2039. doi: 10.1002 / stem.2393

[0128] 12. Zhu X, Fujita M, Snyder FA, Okada H. Systemic delivery of neutralizing antibody targeting CCE2 for glioma therapy. J Neurooncol. 2011 ; 104(l):83-92. doi: 10.1007 / s 11060-010-0473-5

[0129] 13. Ostrand-Rosenberg S, Hom FA, Ciavattone NG. Radiotherapy Both Promotes and Inhibits Myeloid-Derived Suppressor Cell Function: Novel Strategies for Preventing the Tumor-Protective Effects of Radiotherapy. Front Oncol. 2019;9:215. doi: 10.3389 / fonc.2019.00215

[0130] 14. Tcyganov E, Mastio J, Chen E, Gabrilovich DE Plasticity of myeloid-derived suppressor cells in cancer. Curr Opin Immunol. 2018;51:76-82. doi:10.1016 / j.coi.2018.03.009

[0131] 15. Blanco E, Silva- Pilipich N, Bocanegra A, et al. Oleuropein-driven reprogramming of the myeloid cell compartment to sensitise tumours to PD-1 / PD-L1 blockade strategies. Br J Cancer. 2024;130(5):869-879. doi:10.1038 / s41416-023-02561-y

[0132] 16. Lanca T, Ungerback J, Da Silva C, et al. IRF8 deficiency induces the transcriptional, functional, and epigenetic reprogramming of cDCl into the cDC2 lineage. Immunity. 2022;55(8): 1431-1447. el l. doi: 10.1016 / j.immuni.2022.06.006

[0133] 17. Sichien D, Scott CE, Martens L, et al. IRF8 Transcription Factor Controls Survival and Function of Terminally Differentiated Conventional and Plasmacytoid Dendritic Cells, Respectively. Immunity. 2016;45(3):626-640. doi:10.1016 / j.immuni.2016.08.013

[0134] 18. Waight JD, Netherby C, Hensen ML, et al. Myeloid-derived suppressor cell development is regulated by a STAT / IRF-8 axis. J Clin Invest. 2013;123(10):4464-4478. doi:10.1172 / JCI68189

[0135] 19. Condamine T, Mastio J, Gabrilovich DI. Transcriptional regulation of myeloid- derived suppressor cells. J Leukoc Biol. 2015;98(6):913-922. doi:10.1189 / jlb.4RI0515-204R

[0136] 20. Stewart TJ, Greeneltch KM, Reid JE, et al. Interferon regulatory factor-8 modulates the development of tumour-induced CD1 lb+Gr-l+ myeloid cells. J Cell Mol Med. 2009;E3(9B):3939-3950. doi: 10.1 ll l / j,1582-4934.2009.00685.x 21 . Perez OD, Logg CR, Hiraoka K, et al. Design and selection of Toca 51 1 for clinical use: modified retroviral replicating vector with improved stability and gene expression. Mol Ther JAm Soc Gene Ther. 2012;20(9): 1689-1698. doi:10.1038 / mt.2012.83

[0137] 22. Collins SA, Shah AH, Ostertag D, Kasahara N, Jolly DJ. CLINICAL DEVELOPMENT OF RETROVIRAL REPLICATING VECTOR TOCA 511 FOR GENE THERAPY OF CANCER. Expert Opin Biol Ther. 2021 ;21(9): 1199-1214. doi: 10.1080 / 14712598.2021.1902982

[0138] 23. Hiraoka K, Inagaki A, Kato Y, et al. Retroviral replicating vector-mediated gene therapy achieves long-term control of tumor recurrence and leads to durable anticancer immunity. Neuro-Oncol. 2017;19(7):918-929. doi:10.1093 / neuonc / nox038

[0139] 24. Lewis PF, Emerman M. Passage through mitosis is required for oncoretroviruses but not for the human immunodeficiency virus. J Virol. 1994;68(1):510-516. doi:10.1128 / JVL68.1.510-516.1994

[0140] 25. Logg CR, Kasahara N. Retrovirus-mediated gene transfer to tumors: utilizing the replicative power of viruses to achieve highly efficient tumor transduction in vivo. Methods Mol Biol Clifton NJ. 2004;246:499-525. doi: 10.1385 / 1-59259-650-9:499

[0141] 26. Kosaka A, Ohkuri T, Okada H. Combination of an agonistic anti-CD40 monoclonal antibody and the COX-2 inhibitor celecoxib induces anti-glioma effects by promotion of type-1 immunity in myeloid cells and T-cells. Cancer Immunol Immunother CII. 2014;63(8):847-857. doi:10.1007 / s00262-014-1561-8

[0142] 27. Responsiveness to anti-PD-1 and anti-CTLA-4 immune checkpoint blockade in SB28 and GL261 mouse glioma models - PubMed. Accessed March 6, 2024. https: / / pubmed- ncbi-nlm-nih-gov.ucsf.idm.oclc.org / 30524896 /

[0143] 28. Simonds EF, Lu ED, Badillo O, et al. Deep immune profiling reveals targetable mechanisms of immune evasion in immune checkpoint inhibitor-refractory glioblastoma. J Immunother Cancer. 2021;9(6):e002181. doi:10.1136 / jitc-2020-002181

[0144] 29. Musca B, Russo MG, Tushe A, et al. The immune cell landscape of glioblastoma patients highlights a myeloid-enriched and immune suppressed microenvironment compared to metastatic brain tumors. Front Immunol. 2023; 14: 1236824. doi: 10.3389 / fimmu.2023.1236824 30. Bronte V, Brandau S, Chen SH, et al. Recommendations for myeloid-derived suppressor cell nomenclature and characterization standards. Nat Commun. 2016;7: 12150. doi: 10.1038 / ncomms 12150

[0145] 31. Groth C, Hu X, Weber R, et al. Immunosuppression mediated by myeloid-derived suppressor cells (MDSCs) during tumour progression. Br J Cancer. 2019; 120(1): 16-25. doi:10.1038 / s41416-018-0333-l

[0146] 32. Gielen PR, Schulte BM, Kers-Rebel ED, et al. Elevated levels of polymorphonuclear myeloid-derived suppressor cells in patients with glioblastoma highly express S100A8 / 9 and arginase and suppress T cell function. Neuro-Oncol. 2016; 18(9): 1253- 1264. doi:10.1093 / neuonc / now034

[0147] 33. Ren W, Zhang X, Li W, et al. Circulating and tumor- infiltrating arginase 1- expressing cells in gastric adenocarcinoma patients were mainly immature and monocytic Myeloid-derived suppressor cells. Sci Rep. 2020;10(l):8056. doi:10.1038 / s41598-020-64841-4

[0148] 34. Chang AL, Miska J, Wainwright DA, et al. CCL2 Produced by the Glioma Microenvironment Is Essential for the Recruitment of Regulatory T Cells and Myeloid-Derived Suppressor Cells. Cancer Res. 2016;76(19):5671-5682. doi: 10.1158 / 0008-5472.CAN- 16-0144

[0149] 35. Fujita M, Kohanbash G, Fellows-Mayle W, et al. COX-2 blockade suppresses gliomagenesis by inhibiting myeloid-derived suppressor cells. Cancer Res. 2011;71(7):2664- 2674. doi: 10.1158 / 0008-5472.CAN-10-3055

[0150] 36. Flores-Toro IA, Luo D, Gopinath A, et al. CCR2 inhibition reduces tumor myeloid cells and unmasks a checkpoint inhibitor effect to slow progression of resistant murine gliomas. Proc Natl Acad Sci U SA. 2020; 117(2): 1129- 1138. doi:10.1073 / pnas.1910856117

[0151] 37. Spranger S, Dai D, Horton B, Gajewski TF. Tumor-Residing Batf3 Dendritic Cells Are Required for Effector T Cell Trafficking and Adoptive T Cell Therapy. Cancer Cell. 2017;31(5):711-723.e4. doi:10.1016 / j.ccell.2017.04.003

[0152] 38. Ghodke Y, Anderson PL, Sangkuhl K, Lamba J, Altman RB, Klein TE. PharmGKB summary: zidovudine pathway. Pharmacogenel Genomics. 2012;22(12):891-894. doi:10.1097 / FPC.0b013e32835879a8

[0153] 39. Hiraoka K, Kimura T, Logg CR, et al. Therapeutic efficacy of replication- competent retrovirus vector-mediated suicide gene therapy in a multifocal colorectal cancer metastasis model. Cancer Res. 2007;67(l l):5345-5353. doi:10.1158 / 0008-5472.CAN-06-4673 40. Wang Z, Wang Z, Zhang C, et al. Genetic and clinical characterization of B7-H3 (CD276) expression and epigenetic regulation in diffuse brain glioma. Cancer Sci. 2018;109(9):2697-2705. doi: 10.1 l l l / cas.13744

[0154] 41. Sauer N, Szlasa W, Jonderko L, et al. LAG-3 as a Potent Target for Novel Anticancer Therapies of a Wide Range of Tumors. Int J Mol Sci. 2022;23(17):9958. doi:10.3390 / ijms23179958

[0155] 42. Ott M, Tomaszewski KH, Marisetty A, et al. Profiling of patients with glioma reveals the dominant immunosuppressive axis is refractory to immune function restoration. JCI Insight. 2020;5(17):el34386, 134386. doi: 10.1172 / jci.insight.134386

[0156] 43. Han J, Alvarez-Breckenridge CA, Wang QE, Yu J. TGF-P signaling and its targeting for glioma treatment. Am J Cancer Res. 2015;5(3):945-955.

[0157] 44. Raskov H, Orhan A, Christensen JP, Gogenur I. Cytotoxic CD8+ T cells in cancer and cancer immunotherapy. Br J Cancer. 2021;124(2):359-367. doi:10.1038 / s41416-020-01048- 4

[0158] 45. Raychaudhuri B, Rayman P, Huang P, et al. Myeloid derived suppressor cell infiltration of murine and human gliomas is associated with reduction of tumor infiltrating lymphocytes. J Neurooncol. 2015;122(2):293-301. doi:10.1007 / sl l060-015-1720-6

[0159] 46. Trovato R, Fiore A, Sartori S, et al. Immunosuppression by monocytic myeloid- derived suppressor cells in patients with pancreatic ductal carcinoma is orchestrated by STAT3. J Immunother Cancer. 2019;7(l):255. doi:10.1186 / s40425-019-0734-6

[0160] 47. Gangoso E, Southgate B, Bradley L, et al. Glioblastomas acquire myeloid- affiliated transcriptional programs via epigenetic immunoediting to elicit immune evasion. Cell. 2021 ; 184(9) :2454-2470.e26. doi: 10.1016 / j .cell.2021.03.023

[0161] 48. Lei J, Zhou MH, Zhang FC, Wu K, Eiu SW, Niu HQ. Interferon regulatory factor transcript levels correlate with clinical outcomes in human glioma. Aging. 2021; 13(8): 12086- 12098. doi:10.18632 / aging.202915

[0162] 49. Zimmermannova O, Ferreira AG, Ascic E, et al. Restoring tumor immunogenicity with dendritic cell reprogramming. Sci Immunol. 2023;8(85):eadd4817. doi: 10.1126 / sciimmunol.add4817

[0163] 50. Takacs GP, Kreiger CJ, Euo D, et al. Glioma-derived CCE2 and CCE7 mediate migration of immune suppressive CCR2+ / CX3CR1+ M-MDSCs into the tumor microenvironment in a redundant manner. Front Immunol. 2022; 13:993444. doi: 10.3389 / fimmu.2022.993444

[0164] 51. Lesokhin AM, Hohl TM, Kitano S, et al. Monocytic CCR2(+) myeloid-derived suppressor cells promote immune escape by limiting activated CD8 T-cell infiltration into the tumor microenvironment. Cancer Res. 2012;72(4):876-886. doi:10.1158 / 0008-5472.CAN-l l- 1792

[0165] 52. Dabbaghipour R, Ahmadi E, Entezam M, et al. Concise review: The heterogenous roles of B ATF3 in cancer oncogenesis and dendritic cells and T cells differentiation and function considering the importance of BATF3-dependent dendritic cells. Immunogenetics . Published online February 15, 2024. doi:10.1007 / s00251-024-01335-x

[0166] 53. Gardam B, Gargett T, Brown MP, Ebert LM. Targeting the dendritic cell-T cell axis to develop effective immunotherapies for glioblastoma. Front Immunol. 2023; 14: 1261257. doi:10.3389 / fimmu.2023.1261257

[0167] 54. Cedile O, Wlodarczyk A, Owens T. CCL2 recruits T cells into the brain in a CCR2-independent manner. APMIS Acta Pathol Microbiol Immunol Scand. 2017; 125(11):945- 956. doi: 10.1111 / apm.12740

[0168] 55. Huang B, Pan PY, Li Q, et al. Gr-l+CDl 15+ Immature Myeloid Suppressor Cells Mediate the Development of Tumor-Induced T Regulatory Cells and T-Cell Anergy in Tumor- Bearing Host. Cancer Res. 2006;66(2): 1123-1131. doi:10.1158 / 0008-5472.CAN-05-1299

[0169] Accordingly, the preceding merely illustrates the principles of the present disclosure. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the ail and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein

Claims

CLAIMSWhat is claimed is:

1. A retroviral replicating vector (RRV) comprising a transgene that encodes IRF8 (interferon regulatory factor 8).

2. The retroviral replicating vector of claim 1, wherein the transgene is operably linked to a constitutive promoter.

3. The retroviral replicating vector of claim 1, wherein the transgene is operably linked to a promoter that is selectively active in myeloid cells.

4. A method for increasing killing of tumor cells within the tumor microenvironment of a brain tumor, comprising: administering the retroviral replicating vector of any of claims 1-3 to the brain tumor.

5. A method of treatment, comprising: administering the retroviral replicating vector of any of claims 1-3 to a subject that has a brain tumor.

6. The method of claim 5, wherein the brain tumor is glioblastoma multiforma (GBM).

7. The method of any of claims 4-6, further comprising administering a CAR T or cancer vaccine that targets the brain tumor.

8. The method of any of claims 4-7, wherein the microenvironment of the brain tumor comprises myeloid-derived suppressor cells (MDSCs) and cytotoxic T cells.

9. The method of any of claims 4-8, wherein the administering is local to the brain tumor.

10. The method of claim 9, wherein the administering is done intracranially or intratumor lly.

11. The method of any of claims 4-8, wherein the administering is systemic.

12. The method of claim 11, wherein the administering is intravenous.

13. The method of any of claims 4-12, wherein the retroviral replicating vector is administered once, by injection.

14. A method for increasing killing of tumor cells within the tumor microenvironment of a solid tumor, comprising: administering a transgene that encodes a myeloid differentiation factor to the solid tumor.

15. A method of treatment, comprising: administering a transgene that encodes a myeloid differentiation factor to a subject that has a solid tumor.

16. The method of claim 14 or 15, wherein the solid tumor is colon cancer, bladder cancer, prostate cancer, breast cancer, lung cancer, or mesothelioma.

17. The method of any of claims 14-16, wherein the transgene is administered via a retroviral vector, adeno-associated virus (AAV), adenoviral or lentiviral vector, or encapsulated RNA.

18. The method of any of claims 14-17, further comprising administering a CAR T or cancer vaccine that targets the solid tumor.

19. The method of any of claims 14-18, wherein the tumor microenvironment of the solid tumor comprises myeloid-derived suppressor cells (MDSCs) and cytotoxic T cells.

20. The method of any of claims 14-19, wherein the administering is local to the solid tumor.

21. The method of claim 20, wherein the administering is done intracranially or intratumorally.

22. The method of any of claims 14-19, wherein the administering is systemic.

23. The method of claim 22, wherein the administering is intravenous.

24. The method of any of claims 14-23, wherein the transgene is administered once, by injection.

25. The method of any of claims 14-24, wherein the myeloid differentiation factor is IRF8 (interferon regulatory factor 8), BATF3 (basic leucine zipper ATF-like transcription factor 3) or ID2 (inhibitor of DNA binding 2).

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