Methods to treat glioma in subjects according to tumor microenvironment stratification

Abstract

This disclosure relates to methods for selecting therapy for a subject with glioma, and treating said subject, according to the pretreatment tumor microenvironment, wherein when a tumor from the subject has a high level of similarity with reference samples with low tumor microenvironment, medium tumor microenvironment, or high tumor microenvironment, different therapies are selected, which can comprise an oncolytic virus, a combination of an oncolytic virus and an anti-PD-1 antibody, or a combination of an oncolytic virus, an anti-PD-1 antibody, and at least one additional immune checkpoint inhibitor, respectively.

Description

METHODS TO TREAT GLIOMA IN SUBJECTS ACCORDING TO TUMOR MICROENVIRONMENT STRATIFICATIONRelated Applications

[0001] This application claims the benefit of U.S. Provisional Application 63 / 645,542 filed on May 10, 2024, herein incorporated by reference.Incorporation of Sequence Listing

[0002] A computer readable form of the Sequence Listing “P96456515PCT00_SequenceListing.xml” (20.0 kilobytes), submitted herewith by electronic submission and created on May 4, 2025, is herein incorporated by reference.Field

[0003] The present disclosure relates to methods for selecting therapy for a subject with glioma according to the pretreatment tumor microenvironment. Also provided are methods for treating a subject with glioma according to the pretreatment tumor microenvironment.Background

[0004] The median survival of patients with recurrent glioblastoma is approximately 6-7 months. No treatment has improved survival in recurrent disease, and there is no agreed upon standard of care at disease recurrence.

[0005] DNX-2401 is a conditionally replicative adenovirus that has been investigated in patients with glioblastoma. The first in-human, single-center, phase 1 dose escalation study (Lang et al, JCO, 2018) in patients with high grade gliomas demonstrated that DNX-2401 was well tolerated, with generally mild and unrelated adverse events. The maximum tested dose was to the 10thpower. Efficacy was evaluated with intratumoral administration alone (group A) and intratumoral administration followed 14 days later by tumor resection and subsequent intramural delivery (group B) of DNX- 2401 throughout the resected tumor cavity. When administered as a single intratumoral injection, 52% of subjects (13 of 25) achieved clinical benefit defined as stable disease or better per Macdonald criteria, including 3 responses with greater than 95% reduction in enhancing tumor, with a 1-year survival rate of 32%. Examination of tumor tissue in patients treated in group B revealed infiltration by CD8+ and T-bet+ cells, as well as altered checkpoint protein expression.Summary

[0006] The present inventors have provided methods for stratifying patients with glioma for treatment according to their pretreatment tumor microenvironments and methods for treating said patients according to their pretreatment tumor microenvironments.

[0007] Accordingly, in an aspect, herein provided is a method of selecting therapy for a subject with glioma comprising: a) obtaining a tumor biopsy sample from the subject; b) determining a sample immune gene expression profile comprising markers of immune infiltration from the tumor biopsy sample, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; c) determining a tumor microenvironment (TME) of the subject from the sample immune gene expression profile, wherein the TME of the subject comprises one of a high TME (TMEhigh), a medium TME (TMEmedium) and a low TME (TMEIow); d) selecting: i. TMEhigh therapy when the TME of the subject is TMEhigh, wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor; ii. TMEmedium therapy when the TME of the subject is TMEmedium, wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and an anti-PD-1 antibody or a binding fragment thereof; or iii. TMEIow therapy when the TME of the subject is TMEIow, wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, a binding fragment of anti-PD-1 antibody, or an additional immune checkpoint inhibitor.

[0008] In another aspect, herein provided is a method of treating glioma comprising administering TMEhigh therapy to a subject that has been previously identified as having a high tumor microenvironment;wherein the high tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained pre-treatment, the immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; and wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor.

[0009] In an embodiment, the TMEhigh therapy comprises administering the oncolytic virus intratumorally prior to repeated doses of the anti-PD-1 antibody or the biding fragment thereof and repeated doses of the at least one additional immune checkpoint inhibitor.

[0010] In another embodiment, the repeated doses of the anti-PD-1 antibody or the binding fragment thereof start about 7 days to about 21 days after administration of the oncolytic virus.

[0011] In yet another embodiment, the doses of the anti-PD-1 antibody or the binding fragment thereof are administered at about 200 mg infused intravenously over about 30 minutes.

[0012] In a further embodiment, the doses of the anti-PD-1 antibody or the binding fragment thereof are administered about every 3 weeks.

[0013] In some embodiments, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises(a) an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TI GIT antibody, or a combination thereof;(b) a binding fragment of an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, or a combination thereof; or(c) a combination of (a) and (b).

[0014] In an embodiment, repeated doses of the at least one checkpoint inhibitor start about 14 days after administration of the oncolytic virus.

[0015] In an embodiment, the doses of the anti-CTLA-4 antibody or the binding fragment of the anti-CTLA-4 antibody are administered at about 3 mg / kg infused intravenously. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment of the anti-CTLA-4 antibody comprise up to 4 doses.

[0016] In an embodiment, the doses of the anti-LAG-3 antibody or the binding fragment of the anti-LAG-3 antibody are administered at about 160 mg infused intravenously. In another embodiment, the doses of the anti-LAG-3 antibody or the binding fragment of the anti-LAG-3 anti-body are administered about every 4 weeks.

[0017] In an embodiment, the doses of the anti-TI M-3 antibody or the binding fragment of the anti-TI M-3 antibody are administered at about 800 mg infused intravenously. In another embodiment, the doses of the anti-TIM-3 antibody orthe binding fragment of the anti-TIM-3 anti-body are administered about every 4 weeks.

[0018] In an embodiment, the doses of the anti-TIGIT antibody or the binding fragment of the anti-TIGIT antibody are administered at about 600 mg infused intravenously. In another embodiment, the doses of the anti-TIGIT antibody or the binding fragment of the anti-TIGIT anti-body are administered about every 3 weeks.

[0019] In yet another aspect, herein provided is method of treating glioma comprising administering TMEmedium therapy to a subject that has been previously identified as having a medium tumor microenvironment; wherein the medium tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained pre-treatment, the immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; and wherein the TMEmedium therapy comprises combined therapy of an oncolytic virus and an anti-PD-1 antibody or a binding fragment thereof.

[0020] In an embodiment, the TMEmedium therapy comprises administering the oncolytic virus intratumorally prior to repeated doses of anti-PD-1 antibody or the binding fragment thereof.

[0021] In another embodiment, the repeated doses of anti-PD-1 antibody or the binding fragment thereof start about 14 days after administration of the oncolytic virus.

[0022] In another embodiment, the doses of anti-PD-1 antibody or the binding fragment thereof are administered at about 200 mg infused intravenously over about 30 minutes.

[0023] In yet another embodiment, the doses of anti-PD-1 antibody or the binding fragment thereof are administered every 3 weeks.

[0024] In a further embodiment, the anti-PD-1 antibody or the binding fragment thereof is pembrolizumab or a fragment thereof.

[0025] In a further aspect, herein provided is a method of treating glioma comprising administering TMEIow therapy to a subject that has been previously identified as having a low tumor microenvironment; wherein the low tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained pre-treatment, the immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; and wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, binding fragment of anti-PD-1 antibody, or an additional immune checkpoint inhibitor.

[0026] In an embodiment, the TMEIow therapy comprises administering repeated doses of the oncolytic virus intratumorally.

[0027] In another embodiment, the doses of the oncolytic virus are administered about every 4 weeks.

[0028] In yet another embodiment, the repeated doses of the oncolytic virus comprise up to 6 doses.

[0029] In an embodiment, a new tumor biopsy is obtained prior to the administration of the second and subsequent doses of the oncolytic virus, and the method further comprises a step of determining a new TME of the subject from a new immunegene expression profile comprising markers of immune infiltration from the new tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores, wherein the new TME of the subject comprises one of TMEhigh, TMEmedium and TMEIow, and;I) treating the subject with a TMEhigh therapy described herein when the new TME of the subject is TMEhigh;II) treating the subject with a TMEmedium therapy described herein when the new TME of the subject is TMEmedium; orIII) continuing the TMEIow therapy when the new TME of the subject is TMEIow.

[0030] In an embodiment, the glioma is glioblastoma.

[0031] In another embodiment, the oncolytic virus is an oncolytic adenovirus.

[0032] In yet another embodiment, the oncolytic adenovirus is a conditionally replicative oncolytic adenovirus.

[0033] In yet another embodiment, the conditionally replicative oncolytic adenovirus is DNX-2401.

[0034] In a further embodiment, the oncolytic virus is administered intratumorally at about 5x1010to about 5x1011virus particles.

[0035] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments but should be given the broadest interpretation consistent with the description as a whole.Brief Description of the Drawings

[0036] For a better understanding of the various embodiments described herein, and to show more clearly how these various embodiments may be carried into effect, reference will be made, by way of example, to the accompanying drawings which show at least one example embodiment, and which are now described. The drawings are not intended to limit the scope of the teachings described herein.

[0037] Figure 1 shows mRNA expression prior to treatment in an example embodiment of the disclosure. The heatmap shows three subtypes of glioblastoma microenvironment (TME-high, left cluster; TME-medium, middle cluster; and TME-low, right cluster) in samples from a previous cohort on the basis of enrichment for immune cell types using partition around medoids clustering (prior art; reproduced from Nassiri et al, Nat Med 2023). Scores for functional orientation markers, signature scores, and expression of immune checkpoints and biomarkers are overlayed on the heatmap.

[0038] Other features and advantages of the present disclosure will become apparent from the following detailed description. It should be understood, however, that the detailed description and the specific examples, while indicating embodiments of the disclosure, are given by way of illustration only and the scope of the claims should not be limited by these embodiments but should be given the broadest interpretation consistent with the description as a whole.Detailed Description of the Disclosure

[0039] The following is a detailed description provided to aid those skilled in the art in practicing the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terminology used in the description herein is for describing particular embodiments only and is not intended to be limiting of the disclosure.

[0040] Further, the definitions and embodiments described in particular sections are intended to be applicable to other embodiments herein described for which they are suitable as would be understood by a person skilled in the art. For example, in the following passages, different aspects of the disclosure are defined in more detail. Each aspect so defined may be combined with any other aspect or aspects unless clearly indicated to the contrary. In particular, any feature described herein may be combined with any other feature or features described herein.I. Definitions

[0041] As used herein, the following terms may have meanings ascribed to them below, unless specified otherwise. However, it should be understood that other meanings that are known or understood by those having ordinary skill in the art are also possible, and within the scope of the present disclosure. In the case of conflict, the presentspecification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.

[0042] In understanding the scope of the present disclosure, the term "comprising" and its derivatives, as used herein, are intended to be open ended terms that specify the presence of the stated features, elements, components, groups, integers, and / or steps, but do not exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The foregoing also applies to words having similar meanings such as the terms, "including", "having" and their derivatives.

[0043] The term “consisting” and its derivatives, as used herein, are intended to be closed ended terms that specify the presence of stated features, elements, components, groups, integers, and / or steps, and also exclude the presence of other unstated features, elements, components, groups, integers and / or steps. The term “consisting essentially of”, as used herein, is intended to specify the presence of the stated features, elements, components, groups, integers, and / or steps as well as those that do not materially affect the basic and novel characteristic(s) of features, elements, components, groups, integers, and / or steps.

[0044] Further, terms of degree such as "substantially", "about" and "approximately" as used herein mean a reasonable amount of deviation of the modified term such that the end result is not significantly changed. These terms of degree should be construed as including a deviation of at least ±5% of the modified term if this deviation would not negate the meaning of the word it modifies.

[0045] As used in this specification and the appended claims, the singular forms “a”, “an” and “the” include plural references unless the content clearly dictates otherwise. Thus, for example, a composition containing “a compound” includes a mixture of two or more compounds.

[0046] The phrase "and / or," as used herein in the specification and in the claims, should be understood to mean "either or both" of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with "and / or" should be construed in the same fashion, i.e., "one or more" of the elements so conjoined. Other elements may optionally be present other than the elements specifically identified by the "and / or" clause, whether related or unrelated to those elements snAr.ifir.aiiv idantifiad

[0047] As used herein, "or" should be understood to have the same meaning as "and / or" as defined above. For example, when separating items in a list, "or" or "and / or" shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as "only one of or "exactly one of" or, when used in the claims, "consisting of" will refer to the inclusion of exactly one element of a number or list of elements. In general, the term "or" as used herein shall only be interpreted as indicating exclusive alternatives (i.e., "one or the other but not both") when preceded by terms of exclusivity, such as "either," "one of," "only one of," or "exactly one of."

[0048] As used herein, the phrase "at least one," in reference to a list of one or more elements, should be understood to mean at least one element selected from anyone or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements may optionally be present other than the elements specifically identified within the list of elements to which the phrase "at least one" refers, whether related or unrelated to those elements specifically identified.

[0049] It should also be understood that, in certain methods described herein that include more than one step or act, the order of the steps or acts of the method is not necessarily limited to the order in which the steps or acts of the method are recited unless the context indicates otherwise.

[0050] The term "biopsy sample" as used herein refers to a sample obtained by biopsy, for example using a surgical approach such as an open approach ora stereotactic approach. A biopsy sample is, for example, a sample specimen from a tumor.

[0051] 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 description. Ranges from any lower limit to any upper limit are contemplated. The upper and lower limits of these smaller ranges which may independently be included in the smaller ranges is also encompassed within the description, subject to any specifically excluded limit in thestated 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 description. The recitation of numerical ranges by endpoints herein includes all numbers and fractions subsumed within that range (e.g. 1 to 5 includes 1 , 1.5, 2, 2.75, 3, 3.90, 4, and 5). It is also to be understood that all numbers and fractions thereof are presumed to be modified by the term "about."

[0052] The term “nucleic acid” as used herein may refer to a biopolymer comprising monomers of nucleotides, such as deoxyribonucleic acid (DNA), ribonucleic acid (RNA) and other polynucleotides of modified nucleotides and / or nucleotide derivatives, and may be either double stranded (ds) or single stranded (ss). “Modified” bases include, for example, tritiated bases and unusual bases such as inosine. A variety of modifications can be made to DNA and RNA; thus “nucleic acid molecule”, “DNA molecule”, and “RNA molecule” embrace chemically, enzymatically, or metabolically modified forms. Examples of modified nucleotides which can be used to generate the nucleic acids disclosed herein include xanthine, hypoxanthine, 2-aminoadenine, 6- methyl, 2-propyl and other alkyl adenines, 5-halo uracil, 5-halo cytosine, 6-aza uracil, 6- aza cytosine and 6-aza thymine, pseudo uracil, 4-thiouracil, 8-halo adenine, 8- aminoadenine, 8-thiol adenine, 8-thiolalkyl adenines, 8-hydroxyl adenine and other 8- substituted adenines, 8-halo guanines, 8 amino guanine, 8-thiol guanine, 8-thiolalkyl guanines, 8-hydroxyl guanine and other 8-substituted guanines, other aza and deaza uracils, thymidines, cytosines, adenines, or guanines, 5-trifluoromethyl uracil and 5- trifluoro cytosine or fluorophore and quencher conjugated nucleotides. Alternatively, the nucleic acid molecules can be produced biologically using an expression vector. In some embodiments, modified nucleotides comprise one or more modified bases (e.g. unusual bases such as inosine, and functional modifications to the bases such as amino modifications), modified backbones (e.g. peptide nucleic acid, PNA) and / or other chemically, enzymatically, or metabolically modified forms.

[0053] The term “antibody” as used herein refers to an immunoglobulin molecule capable of specific binding to a target through at least one antigen recognition site, located in the variable region of the immunoglobulin molecule. The antibody may be from recombinant sources and / or produced in transgenic animals, and includes, without limitation, monoclonal antibodies, chimeric and humanized antibodies, and bindingfragments thereof, including for example a single chain Fab fragment, Fab’2 fragment, or single chain Fv fragment. There are five major classes of immunoglobulins: IgA, I g D, I g E , IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., lgG1 , lgG2, lgG3, lgG4, lgA1 and lgA2. Humanized or other chimeric antibodies may include sequences from one or more than one isotype, class, or species.

[0054] The basic antibody structural unit is known in the art to comprise a tetramer composed of two identical pairs of polypeptide chains, each pair having one light (“L”) (about 25 kDa) and one heavy (“H”) chain (about 50-70 kDa). The amino-terminal portion of the light chain forms a light chain variable domain (VL) and the amino-terminal portion of the heavy chain forms a heavy chain variable domain (VH). Together, the VH and VL domains form the antibody variable region (Fv) which is primarily responsible for antigen recognition / binding. Within each of the VH and VL domains are three hypervariable regions or complementarity determining regions (CDRs, commonly denoted CDR-H1 , CDR-H2, CDR-H3, CDR-L1 , CDR-L2, and CDR-L3). The carboxy-terminal portions of the heavy and light chains together form a constant region primarily responsible for effector function. Further, these antibodies are typically produced as antigen binding fragments such as Fab, Fab' F(ab')2, Fd, Fv and single domain antibody fragments, or as single chain antibodies (e.g. scFv) in which the heavy and light chains are linked by a spacer or linker. The antibodies may include sequences from any suitable species including human. Also, the antibodies may exist in monomeric or polymeric form.

[0055] The term "antibody fragment" or “binding fragment” as used herein is intended to include without limitations Fab, Fab', F(ab')2, scFab, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and multimers thereof, and Domain Antibodies. Antibodies can be fragmented using conventional techniques. For example, F(ab')2 fragments can be generated by treating the antibody with pepsin. The resulting F(ab')2 fragment can be treated to reduce disulfide bridges to produce Fab' fragments. Papain digestion can lead to the formation of Fab fragments. Fab, Fab' and F(ab')2, scFv, dsFv, ds-scFv, dimers, minibodies, diabodies, and other fragments can also be synthesized by recombinant techniques.

[0056] As is understood in the art, the amino acid position or boundary delineating the CDR regions of an antibody can vary, depending on the context and the different definitions known in the art. Some positions within the variable regions can be viewed ashybrid CDRs in that the positions can be within a CDR region under one set of criteria while being deemed to be outside a CDR region under another set of criteria. In some embodiments, the CDRs in variable light and variable heavy chains can be delineated using the IMGT, Kabat, Chothia, AbM, Contact, or Paratome schemes, or another scheme known in art. The “Kabat” approach for defining CDRs uses sequence variability (Kabat et al. (1991 ); herein incorporated by reference). “Chothia” uses the location of structural loops (Chothia and Lesk, (1987), Chothia et al. (1992); herein incorporated by reference). The IMGT numbering scheme is an adaptation of the numbering scheme of Chothia (Lefranc et al., (1999); see also http: / / imgt.cines.fr; herein incorporated by reference). CDRs defined by “AbM” is a compromise between the Kabat and Chothia and is delineated using Oxford Molecular AbM antibody modeling software (see, Martin et al. (1989); see also www.bioinf-org.uk / abs; herein incorporated by reference). The antibody numbering scheme developed by the Chemical Computing Group (CCG) combines several antibody numbering schemes and offers a broader definition of CDR boundaries based on Martin and collaborators’ CDR definitions (see Maier et al. (2014); herein incorporated by reference). The “Contact” CDR delineations are based on analysis of known antibody-antigen crystal structures (see, e.g., MacCallum et al. (1996)). The “Paratome” approach involves computational programs based on a set of consensus regions derived from a structural alignment of a non-redundant set of known antibodyantigen complexes (Kunik et al. (2012); see also www.ofranlab.org / paratome / ; herein incorporated by reference). The “Aho” or Honegger scheme numbers the variable domains of the immunoglobulin superfamily in a homogenized format. This system is based on structural alignments of the 3D structures of the immunoglobulin variable regions covering the observed length variation. It allows to define structurally conserved Ca positions and therefore deduces appropriate framework regions and CDR lengths (see Honegger, A. and Pluckthun A. (2001 ); herein incorporated by reference). The CDRs described herein include those based on CCG, Paratome, Kabat, Chothia, or Aho definition, and it is to be understood that CDRs based on other methods are to be encompassed herein.

[0057] In some embodiments, the CDRs described herein are based on IMGT, CCG, Paratome, Kabat, Chothia, or Aho.

[0058] Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, examples of methods and materials are now described.II. Methods

[0059] The present inventors have provided methods for stratifying patients with glioma for treatment according to their pretreatment tumor microenvironments and methods for treating said patients according to their pretreatment tumor microenvironments.

[0060] Accordingly, in one aspect, the present disclosure provides a method of selecting therapy for a subject with glioma comprising: a) determining a sample immune gene expression profile comprising markers of immune infiltration from the tumor biopsy sample, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; b) determining a tumor microenvironment (TME) of the subject from the sample immune gene expression profile, wherein the TME of the subject comprises one of a high TME (TMEhigh), a medium TME (TMEmedium) and a low TME (TMEIow); c) selecting: i. TMEhigh therapy when the TME of the subject is TMEhigh, wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor; ii. TMEmedium therapy when the TME of the subject is TMEmedium, wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and an anti-PD-1 antibody or a binding fragment thereof; or iii. TMEIow therapy when the TME of the subject is TMEIow, wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, a binding fragment of anti-PD-1 antibody, or an additional immune checkpoint inhibitor.

[0061] In another aspect, herein provided is a method of treating a subject with glioma comprising: a) determining a sample immune gene expression profile comprising markers of immune infiltration from the tumor biopsy sample, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; b) determining a tumor microenvironment (TME) of the subject from the sample immune gene expression profile, wherein the TME of the subject comprises one of a high TME (TMEhigh), a medium TME (TMEmedium) and a low TME (TMEIow); c) administering: i. TMEhigh therapy when the TME of the subject is TMEhigh, wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor; ii. TMEmedium therapy when the TME of the subject is TMEmedium, wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and an anti-PD-1 antibody or a binding fragment thereof; or iii. TMEIow therapy when the TME of the subject is TMEIow, wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, a binding fragment of anti-PD-1 antibody, or an additional immune checkpoint inhibitor.

[0062] In some embodiments, the method comprises a first step of obtaining a tumor biopsy sample from the subject.

[0063] In another aspect, herein provided is a method of treating glioma comprising administering TMEhigh therapy to a subject that has been previously identified as having a high tumor microenvironment; wherein the high tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained p re-treatment, theimmune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; and wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor.

[0064] In yet another aspect, herein provided is use of a TMEhigh therapy for treating glioma in a subject that has been previously identified as having a high tumor microenvironment (TMEhigh) by a method herein disclosed, wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor. In a further aspect, herein provided is a TMEhigh therapy for use in treating glioma in a subject that has been previously identified as having a high tumor microenvironment (TMEhigh) by a method herein disclosed, wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor.

[0065] The term “TMEhigh” as used herein refers to a tumor microenvironment (TME) enriched with immune cell infiltrates and that highly expresses multiple different suppressive immune checkpoints, leading to an exhaustive immune microenvironment by complementary mechanisms. Accordingly, the term TMEhigh therapy refers to a therapy for treating TMEhigh.

[0066] The TMEhigh therapy comprising combination therapy of an oncolytic virus, a PD-1 inhibitor and at least one additional immune checkpoint inhibitor combines the initial local effects of the oncolytic virus on the tumor microenvironment with the systemic effects of innate and adaptive immune responses from virus replication, PD-1 inhibition, and blockade of at least one additional immune checkpoint. In an embodiment, the oncolytic virus is administered or for use sequentially with the anti-PD-1 antibody or the binding fragment thereof and the at least one additional immune checkpoint inhibitor. For example, a single dose of oncolytic virus is administered or for use, optionally by injection at the time of tumor biopsy, followed by administration or use of repeated doses of anti-PD-1 antibody the binding fragment thereof and repeated doses of the at least one additional immune checkpoint inhibitor.

[0067] Administration or use will depend on the pharmacokinetics of the anti-PD-1 antibody or the binding fragment thereof, the at least one additional immune checkpoint inhibitor, and the oncolytic virus in the presence of each other and can include administering the oncolytic virus about a week, about two weeks, about three weeks, or about one to about three weeks prior to administration of the anti-PD-1 antibody or the binding fragment thereof. In some embodiments, the oncolytic virus is administered intratumorally prior administration of an anti-PD-1 antibody or a binding fragment thereof and at least one additional immune checkpoint inhibitor. In other embodiments, the oncolytic virus is administered intratumorally prior to repeated doses of anti-PD-1 antibody or a binding fragment thereof and repeated doses of the at least one additional immune checkpoint inhibitor.

[0068] Administration or use of the anti-PD-1 antibody or the binding fragment thereof and the at least one additional immune checkpoint inhibitor can be on different days. For example, a dose of the at least one additional immune checkpoint inhibitor can be administered or used within 1 week or within 2 weeks of administration of a dose of the anti-PD-1 antibody or the binding fragment thereof, including either before or after administration of a dose of the anti-PD-1 antibody. When the at least one additional immune checkpoint inhibitor comprises two or more additional immune checkpoint inhibitors, each immune checkpoint inhibitor can be used or administered on different days. Administration or use of the anti-PD-1 antibody or the binding fragment thereof and the at least one additional immune checkpoint inhibitor can also be on the same day.

[0069] The term “immune checkpoint inhibitor” as used herein refers to a drug, optionally an antibody, that activates T cells to attack other cells by blocking the activity of immune checkpoint proteins involved in T cell suppression. Immune checkpoint proteins are a normal part of the immune system that prevent runaway immune responses, but they can be expressed by some cancer cells to evade being targeted by a subject’s T cells. Accordingly, immune checkpoint inhibitors can suppress immune response evasion by cancer cells, allowing T cells to attack cancer cells so that cancer cells can be eliminated. Several immune checkpoint proteins are known and include, forexample, PD-1 (PDCD1), PD-L1 (CD274), PD-L2 (PDCD1 LG2), CTLA-4, TIM-3, LAG-3, TIGIT, B7-H3, ID01 , ICOS, NOS2, ARG2 and CXCL9

[0070] In an embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises(a) an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TI GIT antibody, or a combination thereof;(b) a binding fragment of an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, or a combination thereof; or(c) a combination of (a) and (b).

[0071] In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-CTLA-4 antibody or a binding fragment thereof, an anti-LAG-3 antibody or a binding fragment thereof, and an anti-TI M-3 antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-CTLA-4 antibody or a binding fragment thereof, an anti-LAG-3 antibody or a binding fragment thereof, and an anti-TIGIT antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-CTLA-4 antibody or a binding fragment thereof, an anti-TIM-3 antibody or a binding fragment thereof, and an anti-TIGIT antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-LAG-3 antibody or a binding fragment thereof, an anti-TIM-3 antibody or a binding fragment thereof, and an anti-TIGIT antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-CTLA-4 antibody or a binding fragment thereof and an anti-LAG-3 antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-CTLA-4 antibody or a binding fragment thereof and an anti-TIM-3 antibody ora binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti- CTLA-4 antibody or a binding fragment thereof and an anti-TIGIT antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-LAG-3 antibody or a binding fragmentthereof and an anti-TIM-3 antibody ora binding fragment thereof. In anotherembodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-LAG-3 antibody or a binding fragment thereof and an anti-TIGIT antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-TIM-3 antibody or a binding fragment thereof and an anti-TIGIT antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-CTLA-4 antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-LAG-3 antibody or a binding fragment thereof. In yet another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-TIM-3 antibody or a binding fragment thereof. In a further embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-TIGIT antibody or a binding fragment thereof.

[0072] In an embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-B7-H3 antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-PD-L1 (CD274) antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-PD-L2 (PDCD1 LG2) antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-IDO1 antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-ICOS antibody or a binding fragment thereof. In another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-NOS2 antibody or a binding fragment thereof. In yet another embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-ARG2 antibody or a binding fragment thereof. In a further embodiment, the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises an anti-CXCL9 antibody or a binding fragment thereof.

[0073] In an embodiment, the at least one additional immune checkpoint inhibitor is administered or used in repeated doses.

[0074] In some embodiments, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used about 7 days after administration or use of the oncolytic virus. In some embodiment, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used about 14 days after administration or use of the oncolytic virus. In some embodiments, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used about 21 days after the administration or use of the oncolytic virus. In some embodiments, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used about 28 days after administration or use of the oncolytic virus. In some embodiments, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used about 7 days to about 28 days after administration or use of the oncolytic virus.

[0075] In some embodiments, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used by intravenous infusion. In another embodiment, the anti- CTLA-4 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes to about 120 minutes or more. In another embodiment, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes to about 90 minutes. In some embodiments, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes.

[0076] In an embodiment, about 3 mg / kg to about 9 mg / kg of the anti-CTLA-4 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, or about 9mg / kg of the anti-CTLA-4 antibody or the binding fragment thereof is used or infused intravenously. In yet another embodiment, about 3 mg / kg of the anti-CTLA-4 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used at about 3 mg / kg infused intravenously over about 30 minutes.

[0077] In some embodiments, the anti-CTLA-4 antibody or the binding fragment thereof is administered or used in repeated doses. In an embodiment, the doses of the anti-CTLA-4 antibody or the binding fragment thereof are administered about every 3 weeks.

[0078] In an embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof comprise 2 doses, 3 doses, 4 doses, 5 doses or more. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof comprise 2 doses, 3 doses, or 4 doses. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 3 months. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 6 months. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 12 months. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 18 months. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 2 years. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 3 years. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 4 years. In another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 5 years. In yet another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued for at least 10 years. In yet another embodiment, the repeated doses of the anti-CTLA-4 antibody or the binding fragment thereof are continued indefinitely.

[0079] The term “anti-CTLA-4 antibody” as used herein refers to an antibody that binds specifically to cytotoxic T-lymphocyte associated protein 4 (CTLA-4) optionally as shown in GenBank NG_011502.1 , NM_005214.5, or NM_001037631 .3. In an embodiment the anti-CTLA-4 antibody is a monoclonal antibody.

[0080] In some embodiments, the anti-CTLA-4 antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2 and CDR-H3, and the light chain variable region comprising complementarity determining region CDR-L1 , CDR-L2 and CDR-L3, wherein the CDR-L1 is CDR-L1 of amino acid sequence SEQ ID NO:1 , the CDR- L2 is CDR-L2 of amino acid sequence SEQ ID NO:1 , the CDR-L3 is CDR-L3 of amino acid sequence SEQ ID NO:1 , the CDR-H1 is CDR-H1 of amino acid sequence SEQ IDN0:2, the CDR-H2 is CDR-H2 of amino acid sequence SEQ ID NO:2, and the CDR-H3 is CDR-H3 of amino acid sequence SEQ ID NO:2.

[0081] In some embodiments, the anti-CTLA-4 antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of said light chain variable region comprises SEQ ID NO:1 and the amino acid sequence of said heavy chain variable region comprises SEQ ID NO:2.

[0082] In some embodiments, the anti-CTLA-4 antibody or the binding fragment thereof is ipilimumab (DrugBank Accession Number DB06186) or a binding fragment thereof.

[0083] In some embodiments, the anti-LAG-3 antibody or the binding fragment thereof is administered or used about 7 days after administration or use of the oncolytic virus. In some embodiment, the anti-LAG-3 antibody or the binding fragment thereof is administered or used about 14 days after administration or use of the oncolytic virus. In some embodiments, the anti-LAG-3 antibody or the binding fragment thereof is administered or used about 21 days after the administration or use of the oncolytic virus. In some embodiments, the anti-LAG-3 antibody or the binding fragment thereof is administered or used about 28 days after administration or use of the oncolytic virus. In some embodiments, the anti-LAG-3 antibody or the binding fragment thereof is administered or used about 7 days to about 28 days after administration or use of the oncolytic virus.

[0084] In some embodiments, the anti-LAG-3 antibody or the binding fragment thereof is administered or used by intravenous infusion. In another embodiment, the anti- LAG-3 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes to about 120 minutes or more. In another embodiment, the anti-LAG-3 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes to about 90 minutes. In some embodiments, the anti-LAG-3 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes.

[0085] In an embodiment, about 80 mg to about 240 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 80 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about inn ma at tho anti-LAG-3 antibody or thebinding fragment thereof is used or infused intravenously. In another embodiment, about 120 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 140 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 160 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 180 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 200 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously. In yet another embodiment, about 220 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously. In a further embodiment, about 240 mg of the anti-LAG-3 antibody or the binding fragment thereof is used or infused intravenously.

[0086] In an embodiment, the anti-LAG-3 antibody or the binding fragment thereof is administered or used at about 160 mg infused intravenously over about 30 minutes.

[0087] In some embodiments, the anti-LAG-3 antibody or the binding fragment thereof is administered or used in repeated doses. In an embodiment, the doses of the anti-LAG-3 antibody or the binding fragment thereof are administered about every 4 weeks.

[0088] In an embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued for at least 3 months. In another embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued for at least 6 months. In another embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued for at least 12 months. In an embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued for at least 18 months. In another embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued for at least 2 years. In another embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued for at least 3 years. In another embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued for at least 4 years. In another embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued for at least 5 years. In another embodiment, the administration or use of theanti-LAG-3 antibody or the binding fragment thereof is continued for at least 10 years. In another embodiment, the administration or use of the anti-LAG-3 antibody or the binding fragment thereof is continued indefinitely.

[0089] The term “anti-LAG-3 antibody” as used herein refers to an antibody that binds specifically to lymphocyte-activation gene 3 (LAG-3) optionally as shown in GenBank NM_002286.6, NM_001414176.1 , or NM_001414177.1 . In an embodiment the anti-LAG-3 antibody is a monoclonal antibody.

[0090] In some embodiments, the anti-LAG-3 antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2 and CDR-H3, and the light chain variable region comprising complementarity determining region CDR-L1 , CDR-L2 and CDR-L3, wherein the CDR-L1 is CDR-L1 of amino acid sequence SEQ ID NO:3, the CDR- L2 is CDR-L2 of amino acid sequence SEQ ID NO:3, the CDR-L3 is CDR-L3 of amino acid sequence SEQ ID NO:3, the CDR-H1 is CDR-H1 of amino acid sequence SEQ ID NO:4, the CDR-H2 is CDR-H2 of amino acid sequence SEQ ID NO:4, and the CDR-H3 is CDR-H3 of amino acid sequence SEQ ID NO:4.

[0091] In some embodiments, the anti-LAG-3 antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of said light chain variable region comprises SEQ ID NO: 3 and the amino acid sequence of said heavy chain variable region comprises SEQ ID NO: 4.

[0092] In some embodiments, the anti-LAG-3 antibody or the binding fragment thereof is relatlimab (DrugBank Accession Number DB14851 ) or a binding fragment thereof.

[0093] In some embodiments, the anti-TIM-3 antibody or the binding fragment thereof is administered or used about 7 days after administration or use of the oncolytic virus. In some embodiment, the anti-TIM-3 antibody or the binding fragment thereof is administered or used about 14 days after administration or use of the oncolytic virus. In some embodiments, the anti-TIM-3 antibody or the binding fragment thereof is administered or used about 21 days after the administration or use of the oncolytic virus. In some embodiments, the anti-TIM-3 antibody or the binding fragment thereof isadministered or used about 28 days after administration or use of the oncolytic virus. In some embodiments, the anti-TIM-3 antibody or the binding fragment thereof is administered or used about 7 days to about 28 days after administration or use of the oncolytic virus.

[0094] In some embodiments, the anti-TIM-3 antibody or the binding fragment thereof is administered or used by intravenous infusion. In another embodiment, the anti- TIM-3 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes to about 120 minutes or more. In another embodiment, the anti-TIM-3 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes to about 90 minutes. In some embodiments, the anti-TIM-3 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes.

[0095] In an embodiment, about 400 mg to about 1200 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, about 400 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, about 500 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, about 600 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, about 700 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment about 800 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, about 900 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, about 1000 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, about 1100 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, about 1200 mg of the anti-TIM-3 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, the anti-TIM-3 antibody or the binding fragment thereof is administered or used at about 800 mg infused intravenously over about 30 minutes.

[0096] In some embodiments, the anti-TIM-3 antibody or the binding fragment thereof is administered or used in repeated doses. In an embodiment, the doses of theanti-TIM-3 antibody or the binding fragment thereof are administered about every 4 weeks.

[0097] In an embodiment, the administration or use of the anti-TIM-3 antibody or the binding fragment thereof is continued for at least 3 months. In another embodiment, the administration or use of the anti-TIM-3 antibody or the binding fragment thereof is continued for at least 6 months. In another embodiment, the administration or use of the anti-TIM-3 antibody or the binding fragment thereof is continued for at least 12 months. In another embodiment, the administration or use of the anti-TIM-3 antibody or the binding fragment thereof is continued for at least 18 months. In another embodiment, the administration or use of the anti-TIM-3 antibody or the binding fragment thereof is continued for at least 2 years. In another embodiment, the administration or use of the anti-TIM-3 antibody or the binding fragment thereof is continued for at least 3 years. In another embodiment, the administration or use of the anti-TIM-3 antibody is continued for at least 4 years. In another embodiment, the administration or use of the anti-TIM-3 antibody or the binding fragment thereof is continued for at least 5 years. In another embodiment, the administration or use of the anti-TIM-3 antibody is continued for at least 10 years. In another embodiment, the administration or use of the anti-TIM-3 antibody or the binding fragment thereof is continued indefinitely.

[0098] The term “anti-TIM-3 antibody” as used herein refers to an antibody that binds specifically to T-cell immunoglobulin and mucin domain 3 (TIM-3) optionally as shown in GenBank NG_030444.1 , or NM_032782.5. In an embodiment the anti-TIM-3 antibody is a monoclonal antibody.

[0099] In some embodiments, the anti-TIM-3 antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2 and CDR-H3, the light chain variable region comprising complementarity determining region CDR-L1 , CDR-L2 and CDR-L3, wherein the CDR-L1 is CDR-L1 of amino acid sequence SEQ ID NO:5, the CDR- L2 is CDR-L2 of amino acid sequence SEQ ID NO:5, the CDR-L3 is CDR-L3 of amino acid sequence SEQ ID NO:5, the CDR-H1 is CDR-H1 of amino acid sequence SEQ ID NO:6, the CDR-H2 is CDR-H2 of amino acid sequence SEQ ID NO:6, and the CDR-H3 is CDR-H3 of amino acid sequence SEQ ID NO:6.

[0100] In some embodiments, the anti-TIM-3 antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of said light chain variable region comprises SEQ ID NO:5 and the amino acid sequence of said heavy chain variable region comprises SEQ ID NO:6.

[0101] In some embodiments, the anti-TIM-3 antibody or the binding fragment thereof is sabatolimab (KEGG Drug Database number D12164) or a binding fragment thereof.

[0102] In some embodiments, the anti-TIGIT antibody or the binding fragment thereof is administered or used about 7 days after administration or use of the oncolytic virus. In some embodiment, the anti-TIGIT antibody or the binding fragment thereof is administered or used about 14 days after administration or use of the oncolytic virus. In some embodiments, the anti-TIGIT antibody or the binding fragment thereof is administered or used about 21 days after the administration or use of the oncolytic virus. In some embodiments, the anti-TIGIT antibody or the binding fragment thereof is administered or used about 28 days after administration or use of the oncolytic virus. In some embodiments, the anti-TIGIT antibody or the binding fragment thereof is administered or used about 7 days to about 28 days after administration or use of the oncolytic virus.

[0103] In some embodiments, the anti-TIGIT antibody or the binding fragment thereof is administered or used by intravenous infusion. In another embodiment, the anti- TIGIT antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes to about 120 minutes or more. In another embodiment, the anti-TIGIT antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes to about 90 minutes. In some embodiments, the anti-TIGIT antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes. In another embodiment, the anti-TIGIT antibody or the binding fragment thereof is administered or used by intravenous infusion over about 60 minutes.

[0104] In an embodiment, about 600 mg of the anti-TIGIT antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, the anti-TIGIT antibody or the binding fragment thereof is administered or used at about 600 mg infused intravenously over about 60 minutes.

[0105] In some embodiments, the anti-TIGIT antibody or the binding fragment thereof is administered or used in repeated doses. In an embodiment, the doses of the anti-TIGIT antibody or the binding fragment thereof are administered about every 3 weeks.

[0106] In an embodiment, the administration or use of the anti-TIGIT antibody or the binding fragment thereof is continued for at least 3 months. In another embodiment, the administration or use of the anti-TIGIT antibody or the binding fragment thereof is continued for at least 6 months. In another embodiment, the administration or use of the anti-TIGIT antibody or the binding fragment thereof is continued for at least 1 year. In another embodiment, the administration or use of the anti-TIGIT antibody or the binding fragment thereof is continued for at least 2 years. In another embodiment, the administration or use of the anti-TIGIT antibody or the binding fragment thereof is continued for at least 3 years. In another embodiment, the administration or use of the anti-TIGIT antibody or the binding fragment thereof is continued for at least 4 years. In another embodiment, the administration or use of the anti-TIGIT antibody or the binding fragment thereof is continued for at least 5 years. In another embodiment, the administration or use of the anti-TIGIT or the binding fragment thereof antibody is continued for at least 10 years. In another embodiment, the administration or use of the anti-TIGIT antibody is continued indefinitely.

[0107] The term “anti-TIGIT antibody” as used herein refers to an antibody that binds specifically to T cell immunoreceptor with Ig and ITIM domains (TIGIT) optionally as shown in GenBank NM_173799.4. In an embodiment the anti-TIGIT antibody is a monoclonal antibody.

[0108] In some embodiments, the anti-TIGIT antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2 and CDR-H3, and the light chain variable region comprising complementarity determining region CDR-L1 , CDR-L2 and CDR-L3, wherein the CDR-L1 is CDR-L1 of amino acid sequence SEQ ID NO:7, the CDR- L2 is CDR-L2 of amino acid sequence SEQ ID NO:7, the CDR-L3 is CDR- of amino acid sequence SEQ ID NO:7, the CDR-H1 is CDR-H1 of amino acid sequence SEQ ID NO:8,the CDR-H2 is CDR-H2 of amino acid sequence SEQ ID NO:8, and the CDR-H3 is CDR- H3 of amino acid sequence SEQ ID NO:8.

[0109] In some embodiments, the anti-TIGIT antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of said light chain variable region comprises SEQ ID NO:7 and the amino acid sequence of said heavy chain variable region comprises SEQ ID NO:8.

[0110] In some embodiments, the anti-TIGIT antibody or the binding fragment thereof is tiragolumab (KEGG Drug Database number D11482) or a binding fragment thereof.

[0111] In another aspect, herein provided is a method of treating glioma comprising administering TMEmedium therapy to a subject that has been previously identified as having a medium tumor microenvironment; wherein the medium tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained p re-treatment, the immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; and wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and anti-PD-1 antibody or the binding fragment thereof.

[0112] In yet another aspect, herein provided is use of a TMEmedium therapy for treating glioma in a subject that has been previously identified as having a medium tumor microenvironment (TMEmedium) by a method herein disclosed, wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and anti-PD-1 antibody or the binding fragment thereof. In a further aspect, herein provided is a TMEmedium therapy for use in treating glioma in a subject that has been previously identified as having a medium tumor microenvironment (TMEmedium) by a method herein disclosed, wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and anti-PD-1 antibody or the binding fragment thereof.

[0113] The term “TMEmedium” as used herein refers to a TME with a moderate degree of immune cells and that expresses moderate levels of PD-1 but low levels of additional immune checkpoint genes. Accordingly, the term TMEmedium therapy refers to a therapy for treating TMEmedium.

[0114] The TMEmedium therapy comprising combination therapy of an oncolytic virus and PD-1 inhibitor combines the initial local effects of the oncolytic virus on the tumor microenvironment with the systemic effects of innate and adaptive immune responses from virus replication and PD-1 inhibition. In an embodiment, the oncolytic virus is administered sequentially with the anti-PD-1 antibody or the binding fragment thereof. For example, a single dose of oncolytic virus is administered, optionally by injection, at the time of tumor biopsy followed by administration of repeated doses of anti- PD-1 antibody or the binding fragment thereof.

[0115] Administration or use will depend on the pharmacokinetics of the anti-PD-1 antibody or the binding fragment thereof and the oncolytic virus in the presence of each other and can include administering the oncolytic virus about a week, about two weeks, about three weeks, or about one to about three weeks prior to administration of the anti- PD-1 antibody or the binding fragment thereof. In some embodiments, the oncolytic virus is administered intratumorally prior to repeated doses of anti-PD-1 antibody or the binding fragment thereof.

[0116] The term “anti-PD-1 antibody” as used in the methods and uses disclosed herein refers to an antibody that binds specifically to programmed cell death protein 1 (PD-1) optionally as shown in MW051356.1 , UMM61401 , or UMM61400. In an embodiment the anti-PD-1 antibody is a monoclonal antibody.

[0117] In some embodiments, the anti-PD-1 antibody comprises a light chain variable region and a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2 and CDR-H3, and the light chain variable region comprising complementarity determining region CDR-L1 , CDR-L2 and CDR-L3, wherein the amino acid sequences of said CDRs comprise the sequences:CDR-H1 : NYYMY SEQ ID NO: 9;CDR-H2: GINPSNGGTNFNEKFK SEQ ID NO: 10;CDR-H3: RDYRFDMGFDY SEQ ID NO: 11 ;CDR-L1 : RASKGVSTSGYSYLH SEQ ID NO: 12;CDR-L2: LASYLES SEQ ID NO: 13; andCDR-L3: QHSRDLPLT SEQ ID NO: 14.

[0118] In some embodiments, the anti-PD-1 antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of said light chain variable region comprises SEQ ID NO:17 and the amino acid sequence of said heavy chain variable region comprises SEQ ID NO:18.

[0119] In some embodiments, the anti-PD-1 antibody or the binding fragment thereof is pembrolizumab (DrugBank Accession Number: DB09037) or a binding fragment thereof.

[0120] In some embodiments, the anti-PD-1 antibody comprises a light chain variable region and / or a heavy chain variable region, the heavy chain variable region comprising complementarity determining regions CDR-H1 , CDR-H2 and CDR-H3, and / or the light chain variable region comprising complementarity determining region CDR-L1 , CDR-L2 and CDR-L3, wherein the CDR-L1 is CDR-L1 of amino acid sequence SEQ ID NO:15, the CDR- L2 is CDR-L2 of amino acid sequence SEQ ID NO: 15, the CDR-L3 is CDR-L3 of amino acid sequence SEQ ID NO:15, the CDR-H1 is CDR-H1 of amino acid sequence SEQ ID NO:16, the CDR-H2 is CDR-H2 of amino acid sequence SEQ ID NO:16, and / orthe CDR- H3 is CDR-H3 of amino acid sequence SEQ ID NO:16.

[0121] In some embodiments, the anti-PD-1 antibody comprises a light chain variable region and a heavy chain variable region, wherein the amino acid sequences of said light chain variable region comprises SEQ ID NO:15 and the amino acid sequence of said heavy chain variable region comprises SEQ ID NO:16.

[0122] In other embodiments, the anti-PD-1 antibody is nivolumab (DrugBank Accession Number DB09035) or a binding fragment thereof.

[0123] In some embodiments, the anti-PD-1 antibody or the binding fragment thereof is used or administered about 7 days after administration or use of the oncolyticvirus. In some embodiment, the anti-PD-1 antibody or the binding fragment thereof is used or administered about 14 days after administration or use of the oncolytic virus. In some embodiments, the anti-PD-1 antibody or the binding fragment thereof is used or administered about 21 days after the administration or use of the oncolytic virus. In some embodiments, the anti-PD-1 antibody or the binding fragment thereof is used or administered about 7 days to about 21 days after administration or use of the oncolytic virus. In some embodiments, the anti-PD-1 antibody or the binding fragment thereof is administered or used by intravenous infusion over about 30 minutes. In an embodiment, about 200 mg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In an embodiment, the anti-PD-1 antibody or the binding fragment thereof is administered or used at about 200 mg infused intravenously over about 30 minutes.

[0124] In an embodiment, the anti-PD-1 antibody or the binding fragment thereof is administered or used at about 200 mg infused intravenously over about 30 minutes, wherein the anti-PD-1 antibody or the binding fragment thereof is pembrolizumab or a binding fragment thereof.

[0125] In an embodiment, about 1 mg / kg to about 9 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 1 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 2 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 3 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 4 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 5 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 6 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 7 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In another embodiment, about 8 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously. In yet another embodiment, about 9 mg / kg of the anti-PD-1 antibody or the binding fragment thereof is used or infused intravenously.

[0126] In a further embodiment, the anti-PD-1 antibody or the binding fragment thereof is administered or used at about 3 mg / kg infused intravenously over about 30 minutes, wherein the anti-PD-1 antibody or the binding fragment thereof is nivolumab or a binding fragment thereof.

[0127] In an embodiment, administration or use of anti-PD-1 antibody or the binding fragment thereof is repeated every 2 weeks. In another embodiment, administration or use of anti-PD-1 antibody or the binding fragment thereof is repeated every 3 weeks. In an embodiment, the administration or use of the anti-PD-1 antibody or the binding fragment thereof is continued for at least 6 months. In an embodiment, the administration or use of the anti-PD-1 antibody or the binding fragment thereof is continued for at least 1 year. In another embodiment, the administration or use of the anti-PD-1 antibody or the binding fragment thereof is continued for at least 2 years. In another embodiment, the administration or use of the anti-PD-1 antibody or the binding fragment thereof is continued for at least 3 years. In another embodiment, the administration or use of the anti-PD-1 antibody or the binding fragment thereof is continued for at least 4 years. In another embodiment, the administration or use of the anti-PD-1 antibody or the binding fragment thereof is continued for at least 5 years. In another embodiment, the administration or use of the anti-PD-1 antibody or the binding fragment thereof is continued for at least 10 years. In another embodiment, the administration or use of the anti-PD-1 antibody or the binding fragment thereof is continued indefinitely.

[0128] In another aspect, herein provided is a method of treating glioma comprising administering TMEIow therapy to a subject that has been previously identified as having a low tumor microenvironment; wherein the low tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained p re-treatment, the immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; andwherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, an binding fragment of an anti-PD-1 antibody, or an additional immune checkpoint inhibitor.

[0129] In another aspect, herein provided is use of a TMEIow therapy for treating glioma in a subject that has been previously identified as having a low tumor microenvironment (TMEIow) by a method herein disclosed, wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, an binding fragment of an anti-PD-1 antibody, or an additional immune checkpoint inhibitor. In a further aspect, herein provided is a TMEIow therapy for use in treating glioma in a subject that has been previously identified as having a low tumor microenvironment (TMEIow) by a method herein disclosed, wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, an binding fragment of an anti-PD-1 antibody, or an additional immune checkpoint inhibitor.

[0130] The term “TMEIow” as used herein refers to a TME with low levels of immune cells and low expression of immune checkpoint genes. Accordingly, TMEIow therapy is a therapy to treat TMEIow.

[0131] The TMEIow therapy can comprise repeated doses of the oncolytic virus, which can be administered by injection, for example injection into the tumor. The doses of the oncolytic virus can, for example, be administered every 4 weeks. The oncolytic virus can, for example, be administered or used in 2 to 6 doses or more. The administration or use of the anti-PD-1 antibody or the binding fragment thereof can, for example, be continued indefinitely.

[0132] In some embodiment, the TMEIow therapy comprises repeated doses of the oncolytic virus. In some embodiments, the oncolytic virus is administered or used by injection. In some embodiments, the oncolytic virus is administered or used intratumorally. In further embodiments, the oncolytic virus is used by repeat doses intratumorally.

[0133] In an embodiment, the repeated doses comprise 2 doses. In another embodiment, the repeated doses comprise 3 doses. In another embodiment, the repeated doses comprise 4 doses. In yet another embodiment, the repeated doses comprise 5 doses. In a further embodiment, the repeated doses comprise 6 doses. In a further embodiment, the repeated doses r.nmnri«a mn than 6 doses.

[0134] In an embodiment, the subject is treated with an anti-PD-1 antibody herein disclosed ora binding fragment thereof following 6 doses of the oncolytic virus. In another embodiment, the subject is treated with repeated doses of an anti-PD-1 antibody herein disclosed or a binding fragment thereof following 6 doses of the oncolytic virus.

[0135] A new tumor biopsy can be obtained prior to the second and subsequent doses of the oncolytic virus, to again determine the TME. When the TME is changed from TMEIow to TME medium or TMEhigh between biopsies, the therapy of the subject can be adjusted.

[0136] Accordingly, in an embodiment, a new tumor biopsy is obtained prior to the administration of the second and subsequent doses of the oncolytic virus, and the method further comprises a step of determining a new TME of the subject from a new immune gene expression profile comprising markers of immune infiltration from the new tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores, wherein the new TME of the subject comprises one of TMEhigh, TMEmedium and TMEIow, and;I) treating the subject with a TMEhigh therapy herein disclosed;II) treating the subject with a TMEmedium therapy herein disclosed; orIII) continuing the TMEIow therapy when the new TME of the subject is TMEIow.

[0137] Further, in another aspect, herein provided is a method of selecting therapy for a subject with glioma that has previously been treated with a TMEIow therapy comprising: a) determining a sample immune gene expression profile comprising markers of immune infiltration from a new tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; b) determining a new tumor microenvironment (TME) of the subject from the sample immune gene expression profile, wherein the new TME of the subject comprises one of a high TME (TMEhigh), a medium TME (TMEmedium) and a low TME (TMEIow); c) selecting:i. TMEhigh therapy when the new TME of the subject is TMEhigh, wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor; ii. TMEmedium therapy when the new TME of the subject is TMEmedium, wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and an anti-PD-1 antibody or a binding fragment thereof; or iii. TMEIow therapy when the new TME of the subject is TMEIow, wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, a binding fragment of anti- PD-1 antibody, or an additional immune checkpoint inhibitor.

[0138] In some embodiments, the method comprises a first step of obtaining a new tumor biopsy sample from the subject.

[0139] The term “administering” or “administration” as used herein refers to the placement of a drug, an inhibitor or a virus as disclosed herein into a subject by a method or route which results in at least partial delivery to a desired site. The drugs, an inhibitors and viruses disclosed herein can be administered by any appropriate route which results in an effective treatment in the subject. Possible routes of administration include, but are not limited to, intratumoral, intravenous, intraperitoneal, intramuscular, subcutaneous, transdermal, oral, buccal, sublingual, intranasal, or rectal routes of administration, or a combination thereof.

[0140] The term “treating”, “treatment”, and the like, as used herein, and as is well understood in the art, refers to an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results include, but are not limited to alleviation or amelioration of one or more symptoms or conditions, arresting development of disease, diminishment of extent of disease, stabilized (i.e. not worsening) state of disease, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, including regression of the disease, diminishment of the reoccurrence of disease, and remission (whether partial or total), whether detectable or undetectable. “Treating” and “treatment” may also refer to prolonging survival as compared to expected survival if not receiving treatment. “Treating”and “treatment” as used herein also include prophylactic treatment. The effect may be prophylactic in terms of completely or partially preventing a symptom of a disease and / or may be therapeutic in terms of affecting a partial or complete cure for a disease and / or symptoms of the disease. For example, a subject with early cancer can be treated to prevent progression, or alternatively a subject in remission can be treated to prevent recurrence.

[0141] Treating may referto any indicia of success in the treatment or amelioration or prevention of a cancer, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms; or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating. The treatment or amelioration of symptoms is based on one or more objective or subjective parameters; including the results of an examination by a physician. Accordingly, the term "treating" includes the administration of the methods of the present disclosure to prevent, delay, alleviate, arrest or inhibit development of the symptoms or conditions associated with glioma.

[0142] The term “subject” as used in the methods and uses disclosed herein refers to a human.

[0143] The term “glioma”, as used herein refers to a WHO grouping of three classes of diffuse gliomas, including astrocytoma, oligodendroglioma, and glioblastoma. The term “glioblastoma” as used herein refers to a WHO grade 4 CNS tumor. The term “gliosarcoma” as used herein refers to a subtype of glioblastoma.

[0144] In some embodiments of the methods and uses disclosed herein, the glioma is glioblastoma.

[0145] Any oncolytic virus that is capable of reconditioning the tumor microenvironment towards a ‘hot’ phenotype is contemplated in the methods and uses disclosed herein. In some embodiments, the oncolytic virus is an oncolytic adenovirus. In some embodiments, the oncolytic adenovirus is a conditionally replicative oncolytic adenovirus. In one embodiment, the oncolytic adenovirus is DNX-2401 (tasadenoturev; Delta-24-RGD), which is a conditionally replicative oncolytic adenovirus engineered to treat high-grade malignant gliomas, which contains two stable genetic changes in the adenovirus dsDNA genome that cause it to selectively and efficiently replicate in cancerous cells.

[0146] In an embodiment, 5x108to 5x1011virus particles are administered intratumorally. In another embodiment, 5x1010to 5x1011virus particles are administered intratumorally. In yet another embodiment, 5x108to 5x1010virus particles are administered intratumorally.

[0147] Determining the TME of the subject can be achieved by comparing a sample immune gene expression profile with a control profile comprising immune gene expression values from reference samples with known annotation TMEhigh, TMEmedium and TMEIow as described in U.S. application 63 / 460,794 and / or Nassiri et al, 2023, both of which are herein incorporated by reference in their entirety.

[0148] In an embodiment, the determining the TME of the subject comprises: a) comparing values of the sample immune gene expression profile with a control profile comprising immune gene expression values from reference samples with known annotation TMEhigh, TMEmedium and TMEIow; b) determining the level of similarity of the sample immune gene expression profile to the control profile; and c) assigning to the subject the annotation: i. TMEhigh if there is a high level of similarity of the sample immune gene expression profile to TMEhigh; a low level of similarity of the sample immune gene expression profile to TMEmedium or TMEIow; and / or a higher level of similarity of the sample immune gene expression profile to TMEhigh than to TMEmedium or TMEIow; ii. TMEmedium if there is a high level of similarity of the sample immune gene expression profile to TMEmedium; a low level of similarity of the sample immune gene expression profile to TMEhigh or TMEIow; and / or a higher level of similarity of the sample immune gene expression profile to TMEmedium than to TMEhigh or TMEIow; or iii. TMEIow if there is a high level of similarity of the sample immune gene expression profile to TMEIow; a low level of similarity of the sample immune gene expression profile to TMEhigh or TMEmedium; and / or a higher level of similarity of the sampleimmune gene expression profile to TMEIow than to TMEhigh or TM Emedium.

[0149] In an embodiment, a higher level of similarity to the control profile is indicated by a higher correlation value computed between the sample immune gene expression profile and the control profile, optionally wherein the correlation value is a correlation coefficient. In another embodiment, a lower level of similarity to the control profile is indicated by a lower correlation value computed between the sample immune gene expression profile and control profile, optionally wherein the correlation value is a correlation coefficient. In one embodiment, the correlation coefficient is a linear coefficient, optionally a Pearson correlation coefficient or a Spearman correlation coefficient.

[0150] In another embodiment, a high level of similarity is indicated by a Pearson correlation coefficient between the sample profile and the control profile having an absolute value between 0.5 to 1 , optionally between 0.75 to 1 , and a low level of similarity to the control profile is indicated by a correlation coefficient between the sample profile and the control profile having an absolute value between 0 to 0.5, optionally between 0 to 0.25.

[0151] In some embodiments, the cell type markers comprise B-cells, CD45, CD8 T cells, Cytotoxic cells, Dendritic cells, exhausted CD8 cells, macrophages, neutrophils, NK CD56dim cells, NK cells, T-cells, Gh1 cells and Treg cells.

[0152] In an embodiment, the B-cell markers comprise BLK, CD19, MS4A1 and TNFRSF17.

[0153] BLK can be from any organism or source, and optionally as shown in (NM_001715.3; or NM_001330465.2). CD19 can be from any organism or source, and optionally as shown in (NM_001178098.2, NM_001770.6, or NM_001385732.1). MS4A1 can be from any organism or source, and optionally as shown in (NG_023388.1 , NM_152866.3, NM_152867.2, or NM_021950.4). TNFRSF17 can be from any organism or source, and optionally as shown in (NM_001192.3).

[0154] In an embodiment, the CD45 marker comprises PTPRC.

[0155] PTPRC can be from any organism or source, and optionally as shown in (NG_007730.2, NM_002838.5, NM_080921 .4, or NM_001267798.2).

[0156] In an embodiment, the CD8 T cell markers comprise CD8A and CD8B.

[0157] CD8A can be from any organism or source, and optionally as shown in (NG_011608.2, NM_001768.7, NM_171827.4, NM_001145873.1 , or NM_001382698.1). CD8B can be from any organism or source, and optionally as shown in (NM_172213.5, NM_172101.5, NM_172102.5, NM_004931 .5, or NM_001178100.2).

[0158] In an embodiment, the cytotoxic cell markers comprise CTSW, GNLY, GZMA, GZMB, GZMH, KLRB1 , KLRD1 , KLRK1 and PRF1.

[0159] CTSW can be from any organism or source, and optionally as shown in (NM_001335.4). GNLY can be from any organism or source, and optionally as shown in (NM_001302758.2, NM_006433.5, or NM_012483.4). GZMA can be from any organism or source, and optionally as shown in (NM_006144.4). GZMB can be from any organism or source, and optionally as shown in (NM_004131 .6, NM_001346011 .2). KLRB1 can be from any organism or source, and optionally as shown in (NM_002258.3). KLRD1 can be from any organism or source, and optionally as shown in (NM_002262.5, NM_007334.3, NM_001114396.3, NM_001351060.2, NM_001351062.2, NM_001351063.2,NM_001414224.1 , or NM_001414225.1). KLRK1 can be from any organism or source, and optionally as shown in (NG_027762.1 , or NM_007360.4). PRF1 can be from any organism or source, and optionally as shown in (NM_005041.6, or NM_001083116.3).

[0160] In an embodiment, the dendritic cell markers comprise CCL13, CD209 and HSD11 B1.

[0161] CCL13 can be from any organism or source, and optionally as shown in (NM_005408.3). CD209 can be from any organism or source, and optionally as shown in (NG_012167.1 , NM_021155.4, NM_001144896.2, NM_001144897.2,NM_001144893.2, NM_001144894.2, NM_001144895.2, or NM_001144899.2). HSD11 B1 can be from any organism or source, and optionally as shown in (NG_012081.1 , NM_005525.4, NM_181755.3, or NM_001206741 .2).

[0162] In an embodiment, the exhausted CD8 cell markers comprise CD244, EOMES and LAG-3.

[0163] CD244 can be from any organism or source, and optionally as shown in(NM_016382.4, NM_001166663.2, or NM_001166664.2). EOMES can be from any organism or source, and optionally as shown in (NG_042182.1 , NM_001278182.2,NM_005442.4, or NM_001278183.2). LAG-3 can be from any organism or source, and optionally as shown in (NM_002286.6, NM_001414176.1 , or NM_001414177.1).

[0164] In an embodiment, the macrophage markers comprise CD163, CD68 and CD84.

[0165] CD163 can be from any organism or source, and optionally as shown in(NG_029826.1 , NM_004244.6, NM_203416.4, NM_001370145.1 , or NM_001370146.1). CD68 can be from any organism or source, and optionally as shown in (NM_001251.3, or NM_001040059.2). CD84 can be from any organism or source, and optionally as shown in (NM_001184879.2, NM_003874.4, NM_001184881 .2, NM_001184882.2, or NM_001330742.2).

[0166] In an embodiment, the mast cell markers comprise MS4A2 and TPSAB1 / B2.

[0167] MS4A2 can be from any organism or source, and optionally as shown in (NM_000139.5, or NM_001256916.2). TPSAB1 / B2 can be from any organism or source, and optionally as shown in (NM_003294.4).

[0168] In an embodiment, the neutrophil cell markers comprise CSF3R, FCGR3A / B and S100A12.

[0169] CSF3R can be from any organism or source, and optionally as shown in (NM_000760.4, NM_156039.3, or NM_172313.3). FCGR3A can be from any organism or source, and optionally as shown in (NM_000569.8, NM_001127592.2, NM_001127593.1 , NM_001127595.2, NM_001127596.2, NM_001329120.2, or NM_001329122.1). FCGR3B can be from any organism or source, and optionally as shown in (NG_032926.2, NM_001244753.2, NM_000570.5, NM_001271035.2, NM_001271036.2, NM_001271037.2). S100A12 can be from any organism or source, and optionally as shown in (NG_032926.2, NM_001244753.2, NM_000570.5, NM_001271035.2, NM_001271036.2, or NM_001271037.2).

[0170] In an embodiment, the NKCD56 dim cell markers comprise IL21 R, KIR2DL3, KIR3DL1 and KIR3DL2.

[0171] IL21 R can be from any organism or source, and optionally as shown in (NG_012222.1 , NM_021798.4, NM_181078.3, or NM_181079.5). KIR2DL3 can be from any organism or source, and optionally as shown in (NG 046935.1 , or NM_015868.3).KIR3DL1 can be from any organism or source, and optionally as shown in (NG_021414.2, NM_177749.4, or NM_001310690.1). KIR3DL2 can be from any organism or source, and optionally as shown in (NM_006737.4, or NM_001242867.2).

[0172] In an embodiment, the NK cell marker comprises NCR1 .

[0173] NCR1 can be from any organism or source, and optionally as shown in (KJ892768.1).

[0174] In an embodiment, the T-cell marker comprises CD3D, CD3E, CD3G, CD6 and SH2D1A.

[0175] CD3D can be from any organism or source, and optionally as shown in (NM_000732.6, or NM_001040651 .2). CD3E can be from any organism or source, and optionally as shown in (NM_000733.4). CD3G can be from any organism or source, and optionally as shown in (NM_000073.3). CD6 can be from any organism or source, and optionally as shown in (NM_006725.5, NM_001254750.2, or NM_001254751 .2). SH2D1A can be from any organism or source, and optionally as shown in (NM_002351.5, or NM_001114937.3).

[0176] In an embodiment, the Th1 cell marker comprises TBX21.

[0177] TBX21 can be from any organism or source, and optionally as shown in (NG_012166.1 , or NM_013351.2).

[0178] In an embodiment, the Treg cell marker comprises FOXP3.

[0179] FOXP3 can be from any organism or source, and optionally as shown in (NM_014009.4, or NM_001114377.2).

[0180] In an embodiment, the immune checkpoint genes comprise PD-1 (PDCD1), PD-L1 (CD274), PD-L2 (PDCD1 LG2), CTLA-4, TIM-3, LAG-3, TIGIT, B7-H3, IDO1 , ICOS, NOS2, ARG2 and CXCL9.

[0181] PD-1 , also known as PD1 or PDCD1 , can be from any organism or source, and optionally as shown in (NG_012110.1 , or NM_005018.3). PD-L1 , also know as CD274, can be from any organism or source, and optionally as shown in (NM_014143.4, NM_001267706.2, or NM_001314029.2). PD-L2, also known as PDCD1 LG2, can be from any organism or source, and optionally as shown in (NM_025239.4). CTLA-4 can be from any organism or source, and optionally as shown in (NG_011502.1 ,NM_005214.5, or NM_001037631 .3). TIM-3, also known as HAVCR2 - hepatitis A virus cellular receptor 2, can be from any organism or source, and optionally as shown in (NG_030444.1 , or NM_032782.5). LAG-3 can be from any organism or source, and optionally as shown in (NM_002286.6, NM_001414176.1 , or NM_001414177.1 ). TIGIT can be from any organism or source, and optionally as shown in (NM_173799.4). B7-H3, also known as CD276, can be from any organism or source, and optionally as shown in (NM_001024736.2, NM_025240.3, NM_001329628.2, or NM_001329629.2). IDO1 can be from any organism or source, and optionally as shown in (NM_002164.6). ICOS can be from any organism or source, and optionally as shown in (NM_012092.4). NOS2 can be from any organism or source, and optionally as shown in (NG_011470.1 , NM_000625.4). ARG2 can be from any organism or source, and optionally as shown in (NG_011964.1 , or NM_001172.4). CXCL9 can be from any organism or source, and optionally as shown in (NM_002416.3).

[0182] In some embodiments, the functional orientation markers comprise T cell activation, T cell inhibition, Class 1 MHC, regulatory ? cells, Myeloid cell chemotaxis, M1 markers, M2 markers, Tertiary lymphoid structures, angiogenic markers.

[0183] In an embodiment, the T cell activation markers comprise CD70, CD244, CD48 and CD44.

[0184] CD70 can be from any organism or source, and optionally as shown in (NM_001252.5, or NM_001330332.2). CD244 can be from any organism or source, and optionally as shown in (NG_015991.1 , NM_016382.4, NM_001166663.2, or NM_001166664.2). CD48 can be from any organism or source, and optionally as shown in (NM_001778.4, or NM_001256030.2). CD44 can be from any organism or source, and optionally as shown in (NG_008937.1 ,NM_000610.4, NM_001001389.2, NM_001001390.2, NM_001001391 .2, NM_001001392.2, NM_001202555.2,NM_001202556.2, or NM_001202557.2).

[0185] In an embodiment, the T cell inhibition markers comprise LAG-3, TNFRSF8, CTLA-4, TIGIT, PDCD1 , HAVCR2, BTLA, ADORA2A, TNFRSF25 and LAIR1.

[0186] LAG-3 can be from any organism or source, and optionally as shown in (NM_002286.6, NM_001414176.1 , or NM_001414177.1). TNFRSF8 can be from any organism or source, and optionally as shown in (NM_001243.5, or NM_001281430.3). CTLA-4 can be from any organism or source and nntinnaiiy as shown in (see above).TIGIT can be from any organism or source, and optionally as shown in (see above). PDCD1 can be from any organism or source, and optionally as shown in (see above). HAVCR2 can be from any organism or source, and optionally as shown in (see above). BTLA can be from any organism or source, and optionally as shown in (NM_181780.4, or NM_001085357.2). ADORA2A can be from any organism or source, and optionally as shown in (NG_052804.1 , NM_001278497.2, NM_001278498.2,NM_000675.6, NM_001278499.2, or NM_001278500.2). TNFRSF25 can be from any organism or source, and optionally as shown in (NG_029910.1 , NM_ 148965.2, NM_003790.3, NM_148966.2, NM_148967.2, NM_148970.2, or NM_001039664.2). LAIR1 can be from any organism or source, and optionally as shown in (NM_002287.6, NM_001289026.3, or NM-001289027.3).

[0187] In an embodiment, the regulatory T cell markers comprise FOXP3 and TNFRSF18.

[0188] FOXP3 can be from any organism or source, and optionally as shown in (see above). TNFRSF18 can be from any organism or source, and optionally as shown in (NM_004195.3, NM_148901 .2, or NM_148902.2).

[0189] In an embodiment, the Class 1 MHC markers comprise HLA-A, HLA-B, HLA-C, HLA-E, HLA-F and B2M.

[0190] HLA-A can be from any organism or source, and optionally as shown in (Z46633.1 , or D38525.1). HLA-B can be from any organism or source, and optionally as shown in (D83043.1). HLA-C can be from any organism or source, and optionally as shown in (NG_029422.3, NM_002117.6, or NM_001243042.1). HLA-E can be from any organism or source, and optionally as shown in (NM_005516.6). HLA-F can be from any organism or source, and optionally as shown in (NG_012009.2, NM_001098479.2, NM_018950.3, or NM_001098478.2). B2M can be from any organism or source, and optionally as shown in (NM_213978.1).

[0191] In an embodiment, the myeloid cell chemotaxis markers comprise CCL2, CCL5, VEGFA and CSF1.

[0192] CCL2 can be from any organism or source, and optionally as shown in (NM_002982.4). CCL5 can be from any organism or source, and optionally as shown in (NM_002985.3, or NM_001278736.2). VEGFA can be from any organism or source, andoptionally as shown in (NM_001171623.2, NM_001171624.2, NM_001171625.2, NM_001171626.2, NM_001171627.2, NM_001171628.2, NM_001171629.2, or NM_001171630.2). CSF1 can be from any organism or source, and optionally as shown in (NM_000757.6, NM_172210.3, NM_172211.4, or NM_172212.3).

[0193] In an embodiment, the M1 markers comprise CCL2, CXCL10, GBP2, IFIT3, SLAMF7, CXCL9, CCL8, IL1 B and CD38.

[0194] CCL2 can be from any organism or source, and optionally as shown in (see above). CXCL10 can be from any organism or source, and optionally as shown in (NM_001565.4). GBP2 can be from any organism or source, and optionally as shown in (NM_004120.5). IFIT3 can be from any organism or source, and optionally as shown in (NM_001549.6, NM_001031683.4, NM_001289758.2, or NM_001289759.2). SLAMF7 can be from any organism or source, and optionally as shown in (NM_021181.5, NM_001282588.2, NM_001282589.2, NM_001282590.2, NM_001282591 .2,NM_001282592.2, NM_001282593.2, NM_001282594.2, NM_001282595.1 , or NM_001282596.2). CXCL9 can be from any organism or source, and optionally as shown in (NM_002416.3). CCL8 can be from any organism or source, and optionally as shown in (NM_005623.3). IL1 B can be from any organism or source, and optionally as shown in (NM_000576.3). CD38 can be from any organism or source, and optionally as shown in (NM_001775.4).

[0195] In an embodiment, the M2 markers comprise CXCL16, CXCR4, CD14, MRC1 , ARG1 and CCL13.

[0196] CXCL16 can be from any organism or source, and optionally as shown in (NM_001386809.1 , or NM_001100812.2). CXCR4 can be from any organism or source, and optionally as shown in (NM_001008540.2, NM_003467.3, NM_001348056.2, NM_001348059.2, or NM_001348060.2). CD14 can be from any organism or source, and optionally as shown in (NM_000591.4, NM_001040021 .3, NM_001174104.2, or NM_001174105.2). MRC1 can be from any organism or source, and optionally as shown in (NM_002438.4). ARG1 can be from any organism or source, and optionally as shown in (NM_001244438.2, NM_000045.4, or NM_001369020.1). CCL13 can be from any organism or source, and optionally as shown in (NM_005408.3).

[0197] In an embodiment, the tertiary lymphoid structure markers comprise CCL2, CCL4, CCL5, CCL8, CCL18, CCL19, C I 1 CYCI Q CYCI 10, CXCL11 and CXCL13.

[0198] CCL2 can be from any organism or source, and optionally as shown in (see above). CCL4 can be from any organism or source, and optionally as shown in (NM_002984.4). CCL5 can be from any organism or source, and optionally as shown in (NM_002985.3, or NM_001278736.2). CCL8 can be from any organism or source, and optionally as shown in (NM_005623.3). CCL18 can be from any organism or source, and optionally as shown in (NM_002988.4). CCL19 can be from any organism or source, and optionally as shown in (NM_006274.3). CCL21 can be from any organism or source, and optionally as shown in (NM_002989.4). CXCL9 can be from any organism or source, and optionally as shown in (see above). CXCL10 can be from any organism or source, and optionally as shown in (see above). CXCL11 can be from any organism or source, and optionally as shown in (NM_005409.5, or NM_001302123.2). CXCL13 can be from any organism or source, and optionally as shown in (NM_006419.3, or NM_001371558.1 ).

[0199] In an embodiment, the angiogenic markers comprise VEGFA, VEGFB, KDR, CXCR2, HIF1A and ANGPT2.

[0200] VEGFA can be from any organism or source, and optionally as shown in (see above). VEGFB can be from any organism or source, and optionally as shown in (NG_029823.1 , NM_001243733.2, or NM_003377.5). KDR can be from any organism or source, and optionally as shown in (NM_002253.4). CXCR2 can be from any organism or source, and optionally as shown in (NM_001557.4, NM_001168298.2). HIF1A can be from any organism or source, and optionally as shown in (NG_029606.1). ANGPT2 can be from any organism or source, and optionally as shown in (NM_001147.3, NM_001118887.2, NM_001118888.2, N M_001386335.1 , NM_001386336.1 , or NM_001386337.1).

[0201] In some embodiments, the signature scores comprise chemokine, cytolytic, interferon gamma signalling, interferon gamma downstream signals and T cell inflamed.

[0202] In an embodiment, the chemokine markers comprise CCL2, CCL4, CCL5, CCL8, CCL18, CCL19, CCL21 , CXCL9, CXCL10, CXCL11 and CXCL13.

[0203] In an embodiment, the cytolytic markers comprise GZMA and PRF1 .

[0204] GZMA can be from any organism or source, and optionally as shown in (see above). PRF1 can be from any organism or source, and optionally as shown in (see above).

[0205] In an embodiment, the interferon signaling markers comprise IDO1 , CXCL10, CXCL9, HLA-DRA, STAT1 and IFNG.

[0206] IDO1 can be from any organism or source, and optionally as shown in (see above). CXCL10 can be from any organism or source, and optionally as shown in (see above). CXCL9 can be from any organism or source, and optionally as shown in (see above). HLA-DRA can be from any organism or source, and optionally as shown in (see above). STAT1 can be from any organism or source, and optionally as shown in (NM_001384880.1 , NM_001384881 .1 , NM_001384882.1 , NM_001384883.1 ,NM_001384885.1 , NM_001384886.1 , N M_001384887.1 , NM_001384889.1 , or NM_001384890.1). IFNG can be from any organism or source, and optionally as shown in (NM_000619.3).

[0207] In an embodiment, the interferon gamma downstream signal markers comprise CD3D, IDO1 , CD3E, CCL5, GZMK, CD2, HLA-DRA, CXCL13, IL2RG, NKG7, HLA-E, CXCR6, LAG-3, CXCL10, STAT1 and GZMB.

[0208] CD3D can be from any organism or source, and optionally as shown in (see above). IDO1 can be from any organism or source, and optionally as shown in (see above). CD3E can be from any organism or source, and optionally as shown in (see above). CCL5 can be from any organism or source, and optionally as shown in (NM_002985.3, or NM_001278736.2). GZMK can be from any organism or source, and optionally as shown in (NM_002104.3). CD2 can be from any organism or source, and optionally as shown in (NM_001328609.2, or NM_001767.5). HLA-DRA can be from any organism or source, and optionally as shown in (see above). CXCL13 can be from any organism or source, and optionally as shown in (see above). IL2RG can be from any organism or source, and optionally as shown in (NM_000206.3). NKG7 can be from any organism or source, and optionally as shown in (NM_005601.4, or NM_001363693.2). HLA-E can be from any organism or source, and optionally as shown in (NM_005516.6). CXCR6 can be from any organism or source, and optionally as shown in (see above). LAG-3 can be from any organism or source, and optionally as shown in (NM_002286.6, NM_001414176.1 , or NM_001414177.1). CXCL10 can be from any organism or source, and optionally as shown in (see above). STAT 1 can be from any organism or source, and optionally as shown in (see above). GZMB can be from any organism or source, and optionally as shown in (see above).

[0209] In an embodiment, the T cell inflamed markers comprise CCL5, CD27, CD274 [PD-L1], CD276 [B7-H3], CD8A, CMKLR1 , CXCL9, CXCR6, HLA.DQA1 , HLA.DRB1 , HLA.E, IDO1 , LAG-3, NKG7, PDCD1 LG2 [PD-L2], PSMB10, STAT1 , and TIGIT.

[0210] CCL5 can be from any organism or source, and optionally as shown in (see above). CD27 can be from any organism or source, and optionally as shown in (NM_001413263.1 , NM_001242.5, NM_001413264.1 , NM_001413265.1 ,NM_001413266.1 , NM_001413267.1 , or NM_001413268.1 ). CD274, also known as PD- L1 , can be from any organism or source, and optionally as shown in (see above). CD276, also known as B7-H3 can be from any organism or source, and optionally as shown in (see above). CD8A can be from any organism or source, and optionally as shown in (see above). CMKLR1 can be from any organism or source, and optionally as shown in (NM_001142343.2, NM_004072.3, NM_001142344.2, or NM_001142345.2). CXCL9 can be from any organism or source, and optionally as shown in (see above). CXCR6 can be from any organism or source, and optionally as shown in (see above). HLA.DQA1 can be from any organism or source, and optionally as shown in (NG_032876.1 , or NM_002122.5). HLA-DRB1 can be from any organism or source, and optionally as shown in (NM_002124.4, NM_001243965.1 , NM_001359193.1 , or NM_001359194.1). HLA.E can be from any organism or source, and optionally as shown in (NM_005516.6). IDO1 can be from any organism or source, and optionally as shown in (see above). LAG-3 can be from any organism or source, and optionally as shown in (NM_002286.6, NM_001414176.1 , or NM_001414177.1). NKG7 can be from any organism or source, and optionally as shown in (NM_005601.4, or NM_001363693.2). PDCD1 LG2, also known as PD-L2, can be from any organism or source, and optionally as shown in (see above). PSMB10 can be from any organism or source, and optionally as shown in (NM_002801 .4). STAT 1 can be from any organism or source, and optionally as shown in (see above). TIGIT can be from any organism or source, and optionally as shown in (see above).

[0211] In some embodiments, testing the sample for cell type markers, immune checkpoint genes, functional orientation markers, and signature scores comprises or consists of measuring gene expression level of ADORA2A, ANGPT2, ARG1 , B2M, BLK, BTLA, CCL13, CCL18, CCL19, CCL2, CCL21 , CCL4, CCL5, CCL8, CD14, CD163,CD19, CD2, CD209, CD244, CD38, CD3D, CD3E, CD3G, CD44, CD48, CD6, CD68, CD70, CD84, CD8A, CD8B, CSF1 , CSF3R, CTLA-4, CTSW, CXCL10, CXCL11 , CXCL13, CXCL16, CXCL9, CXCR2, CXCR4, CXCR6, EOMES, FCGR3A / B, FOXP3, GBP2, GNLY, GZMA, GZMB, GZMH, GZMK, HAVCR2, HIF1A, HLA-A, HLA-B, HLA-C, HLA-DRA, HLA-E, HLA-F, HSD11 B1 , IDO1 , IFIT3, IFNG, IL1 B, IL21 R, IL2RG, KDR, KIR2DL3, KIR3DL1 , KIR3DL2, KLRB1 , KLRD1 , KLRK1 , LAG-3, LAIR1 , MRC1 , MS4A1 , MS4A2, NCR1 , NKG7, PDCD1 , PRF1 , PTPRC, S100A12, SH2D1A, SLAMF7, STAT1 , TBX21 , TIGIT, TNFRSF17, TNFRSF18, TNFRSF25, TNFRSF8, TPSAB1 / B2, VEGFA, VEGFB.

[0212] mRNA levels of each gene can be obtained by measuring mRNA expression, for example, by qPCR, or by directly quantifying mRNA, for example, by RNA-Sequencing or Nanostring. In an embodiment, mRNA levels are determined by measuring mRNA count.

[0213] The term “sample immune gene expression profile” or “sample gene expression profile” or “sample profile” as used herein refers to the levels of mRNA of the identified genes in the biopsy sample.

[0214] The control profile may be a reference value and / or may be derived from one or more samples, optionally from historical immune gene expression data from a pool of samples with known annotation of TMEmedium, TMEhigh or TMEIow. In an embodiment, the control profile is a value that is continually updated as further samples are collected and immune gene expression levels are measured and correlated. It will be understood that the control profile represents an average of the levels for the selected genes described herein. Average values may, for example, be the mean values or median values.

[0215] For example, a “TMEmedium control profile” may be generated by measuring the mRNA levels of the specified genes for those samples with known annotation of TMEmedium.

[0216] Methods of determining the similarity between profiles are well known in the art. Methods of determining similarity may in some embodiments provide a non- quantitative measure of similarity, for example, using visual clustering. In other embodiments, similarity may be determined using methods which provide a quantitative measure of similarity.

[0217] In an embodiment, similarity may be measured by partition-around-medoid clustering. Partitioning around medoids is a method to find a series of objects (termed medoids) that are centrally located in clusters. This algorithm minimizes the average dissimilarity between objects.

[0218] In an embodiment, similarity may be measured by computing a “correlation coefficient”, which is a measure of the interdependence of random variables that ranges in value from -1 to +1 , indicating perfect negative correlation at -1 , absence of correlation at zero, and perfect positive correlation at +1. In an embodiment, the correlation coefficient may be a linear correlation coefficient, for example, a Pearson productmoment correlation coefficient.

[0219] A Pearson correlation coefficient (r) is calculated using the following formula:

[0220] In one embodiment, x and y are the expression values of the mRNA in a sample profile and a control profile, respectively.

[0221] In an embodiment, a correlation coefficient calculated between a sample immune gene expression profile and a control profile indicates a high level of similarity to the control profile when the correlation coefficient has an absolute value between 0.5 to 1 , optionally between 0.75 to 1 , and a low level of similarity to the control profile when the correlation coefficient has an absolute value between 0 to 0.5, optionally between 0 to 0.25.

[0222] It will be appreciated that any “correlation value” which provides a quantitative scaling measure of similarity between profiles may be used to measure similarity.

[0223] A sample immune gene expression profile may be identified as being indicative of a medium tumor environment (TMEmedium), where the sample profile has high similarity to the TMEmedium control profile, low similarity to the TMEhigh or TMEIow specific control profile, or higher similarity to the TMEmedium specific control profile thanto the TMEhigh or TMEIow specific control profile. A sample profile may be identified as TMEhigh, where the sample immune gene expression profile has high similarity to the TMEhigh specific control profile, low similarity to the TMEmedium or TMEIow specific control profile, or higher similarity to the TMEhigh specific control profile than to the TMEmedium or TMEIow specific control profile. A sample profile may be identified as TMEIow, where the sample immune gene expression profile has high similarity to the TMEIow specific control profile, low similarity to the TMEmedium or TMEhigh specific control profile, or higher similarity to the TMEIow specific control profile than to the TMEmedium or TMEhigh specific control profile.

[0224] For example, in an embodiment, a sample profile may be identified as indicative of a known annotation control based on calculation of a score, which generally is defined by the following formula: score(B) = r (B, sample profile) - r (B, control profile)

[0225] where r is the Pearson correlation coefficient, and B is a vector of mRNA levels across the selected genes.

[0226] For example, a sample profile with a positive score is more similar to the TMEmedium specific control profile across the selected genes, and is therefore classified as indicative of a medium tumor microenvironment, whereas a sample with a negative score is more similar to the TMEhigh or TMEIow specific control profile across the selected geens, and is classified as “not indicative of TMEmedium”.

[0227] In an embodiment, the gene expression profiles are analyzed using combinatorial or multivariate dimension reduction statistical or mathematical methods. Combinatorial or multivariate dimension reduction statistical or mathematical methods may be clustering analysis or desirability analyses to enable comparisons between gene profiles with dimension-reduced empirical values.

[0228] In an embodiment, the clustering analysis is principal component analysis. Principal component analysis is applied as unbiased tool to visualize similarity between the gene profiles of different groups (e.g. a TMEmedium specific control profile, a TMEhigh specific control profile, a TMEIow specific control profile and a sample profile). In another embodiment, the clustering analysis is canonical correlation analysis. Canonical correlation analysis is applied as a supervised technique to test whethermultivariate gene profiles of different groups are statistically distinct, and to provide information on their level of similarity.

[0229] The above disclosure generally describes the present application. A more complete understanding can be obtained by reference to the following specific examples. These examples are described solely for the purpose of illustration and are not intended to limit the scope of the application. Changes in form and substitution of equivalents are contemplated as circumstances might suggest or render expedient. Although specific terms have been employed herein, such terms are intended in a descriptive sense and not for purposes of limitation.Examples

[0230] The following non-limiting Examples are illustrative of the present disclosure:Example 1

[0231] A multicenter phase 1 / 2 study was conducted to evaluate the combination of intratumoral delivery of DNX-2401 followed by intravenous anti-PD-1 antibody pembrolizumab in recurrent glioblastoma, first in a dose-escalation and then in a doseexpansion phase (Nassiri et al, Nat Med, 2023, herein incorporated by reference in its entirety). There were no dose-limiting toxicities, and the full dose combined treatment was well tolerated with generally mild adverse events. The objective response rate of combined treatment was 10.4% (90% confidence interval (Cl) 4.2-20.7%), which approached but did not reach statistical thresholds compared to the prespecified control rate of 5%. The secondary endpoint of overall survival at 12 months was 52.7% (95% Cl 40.1-69.2%), which was statistically greater than the prespecified control rate of 20%, and results from phase 1 trial of 32%. The median overall survival was 12.5 months (10.7-13.5 months). Objective responses led to longer survival even after controlling for lead-time bias (hazard ratio 0.20, 95% Cl 0.05-0.87). A total of 56.2% (95% Cl 41.1- 70.5%) of patients had a clinical benefit defined as stable disease or better per mRANO criteria. Three patients completed treatment with durable responses and were alive at 45, 48 and 60 months after treatment. Overall, this data suggests that sequential therapy of DNX-2401 and pembrolizumab could enhance the efficacy of treatment against these aggressive brain tumors.

[0232] Biomarkers of treatment response were characterized by obtaining gene expression data on 38 patients with biopsy specimens available before treatment in the phase 1 / 2 study. Tumors from this study were divided into three tumor microenvironment subtypes (TMEhigh, TMEmedium and TMEIow) on the basis of the degree of immune cell enrichment. TMEhigh tumors demonstrated infiltration of multiple immune cell types and highly expressed multiple complementary suppressive immune checkpoints genes. By contrast, TMEIow tumors had low immune cell infiltration and low expression of immune checkpoints. TMEmedium tumors showed moderate degree of immune cell infiltration as well as expression of PDCD-1 (gene that encodes PD-1 ) but relatively low expression of other checkpoint proteins. All of the patients in this trial who had an objective response had TMEmedium tumors before treatment (ORR=29.4%, 95% Cl 10.3-55.6%, P = 0.012), which is statistically greater than the pre-specified threshold of 5% in this trial. Patients with TMEmedium tumors were more likely to derive clinical benefit from treatment (odds ratio (OR) 4.08, 95% Cl 1.02-19.4, P = 0.036), and had statistically significantly longer survival (HR 2.27, 95% Cl 1 .09-4.49, P = 0.027). Samples were obtained from a prior trial investigating anti-PD-1 monotherapy alone in recurrent glioblastoma, and similar trends were not seen, indicating that this observation was not a consequence of systemic checkpoint inhibition alone, but rather from combination therapy.

[0233] Based on this evidence and the lack of effective therapy for recurrent glioblastoma, additional molecularly-informed clinical investigation with DNX-2401 with checkpoint inhibitors is warranted.Example 2

[0234] The treatment of recurrent glioblastoma with a combined immune checkpoint inhibitor, pembrolizumab, and oncolytic virus, DNX-2401 , represents a promising approach. Firstly, the pressing need for innovation is underscored by the grim prognosis associated with recurrent glioblastoma, which offers patients a life expectancy of only 6-7 months. In the absence of a well-established standard of care that improves overall survival, novel treatments are imperative to address this unmet medical need.

[0235] Furthermore, the identification of a tumor microenvironment favorable to this treatment allows for the precise selection of patients who are most likely to benefit from this therapy. This targeted approach minimizes the risks associated with exposingpatients who may not respond favorably, optimizing the use of the treatment and improving overall efficacy.

[0236] None of the patients in Example 1 who had TMEhigh or TMEIow tumors before treatment had an objective response to combined treatment with DNX2401 and pembrolizumab. Based on this evidence and the lack of effective therapy for recurrent glioblastoma, additional molecularly informed clinical investigation with DNX-2401 with checkpoint inhibitors is warranted.Methods

[0237] Study Duration and Schedule:

[0238] The duration of the trial for each subject is a total of 12 months. The duration for each individual patient begins during the time of intra-tumoral delivery of DNX-2401 , followed by a 2-week healing period, followed by administration of pembrolizumab every 3 weeks up to 103 weeks. However, the follow up only takes place for 12 months or until disease progression is confirmed.

[0239] The overall duration of the trial is approximately 3 years, including the preparatory phase.

[0240] The study timelines are the following:- Total trial duration: 3 years- Duration for individual patient: 1 year- FSI (First Subject In): Q1 of 2024- Interim analysis: Q4 of 2024- LSI (Last Subject In): Q4 of 2025- LSO (Last Subject Out): Q1 of 2026- DBL (Data Base Lock): Q2 of 2026- Statistical Analyses Completed: Q3 of 2026- Trial Report Completed: Q1 2027

[0241] End of Study:

[0242] The end of study for a patient enrolled in this trial is defined as the visit 12 months after the first administration of DNX-2401 . The end of the study is defined as the visit 12 months after the last patient receives their first administration of DNX-2401 .

[0243] Study Design:

[0244] Stage 1:

[0245] Previous lot concentrations of DNX-2401 are reported to be to the 11thpower (1011). Up to a total of 6 eligible subjects participate in the dose-escalation process and are enrolled using a 3x3 study design in dose-escalating cohorts as follows:• Cohort 1 : Single 1 mL dose DNX-2401 (5e11 viral particles (vp)) delivered intratumorally followed by sequential intravenous pembrolizumab every 3 weeks

[0246] Three subjects are enrolled in Cohort 1 and are followed for 21 days after the initial dose of pembrolizumab for evaluation of dose-limiting toxicity (DLT). If no DLTs are observed, and following a review of clinical data (e.g., laboratory results, adverse events), the next cohort is enrolled. If, in any patient, 1 DLT is observed out of the initial 3 subjects, then 3 additional subjects are added to the same cohort. If no additional subjects experience a DLT, then the new declared dose is 5e11 vp. If 2 or more subjects have a DLT, then the previous dose, 5e10, is considered the declared Stage 2 dose.

[0247] Stage 2:

[0248] Patients with recurrent glioblastoma (GBM) or gliosarcoma (GS) are screened for eligibility of this study. All eligible patients receive intra-tumoral delivery of 5x1010or 5x1011virus particles of DNX-2401 , based on the outcome of stage 1. Prior to virus delivery, a stereotactic-guided biopsy of the tumor is obtained and analyzed via frozen section to confirm the presence of tumor cells at the planned intra-tumoral injection target. In addition to histopathological analysis, the tissue is analyzed for gene expression, and tumor samples are classified into one of three TME subtypes (). The subtypes are low (TMEIow), intermediate or medium (TMEmedium), or high (TMEhigh) (see e.g., Figure 1). This stratifies them into one of the following three arms.

[0249] TMEmedium tumors - Phase 3, multicenter, placebo controlled randomized trial (first arm):

[0250] Subjects with TMEmedium tumors receive pembrolizumab or placebo, 14 days after delivery of DNX-2401. Pembrolizumab is infused intravenously at a dose of 200 mg over 30 minutes every 3 weeks beginning on Day 14 and continuing for up to 103 weeks or until progressive disease is confirmed. T rial follow-up is for 12 months to assess primary outcome of one-year mortality. Adverse events are < Grade 1 prior to initiation of pembrolizumab infusions, or the start is delayed (+2 day window). Alopecia and peripheral neuropathy are exceptions and, in these cases, pembrolizumab begins with either or both at < Grade 2. Additionally, > Grade 2 fatigue is an exception unless deemed severe and debilitating, as determined by the Investigator.

[0251] TMEIow tumors - Phase 0 / 2, multicenter, open-label trial (second arm):

[0252] Subjects with TMEIow tumors undergo multiple dosing of DNX- 2401 (5x1010or 5x1011virus particles of DNX-2401) at intervals of 4 ± 2 weeks for up to 6 doses total (Todo et al, Nat Med, 2022). Biopsy of tissue precedes each delivery of DNX- 2401 , and gene expression of tumor is analyzed at each point to examine changes in TME subtypes. Patients that have tumors with TMEmedium microenvironment on subsequent specimens are eligible to receive systemic pembrolizumab in an open-label format. Subsequent administration of pembrolizumab occurs either when the microenvironment is transformed to TMEmedium or after 6 doses of DNX-2401 , whether transformed or not. Trial follow-up is for 12 months to assess primary outcome of one- year mortality.

[0253] TMEhigh tumors - Phase 0 / 2, multicenter, open-label trial (third arm):

[0254] Subjects with TMEhigh tumors receive a single delivery of DNX-2401 (5x1010or 5x1011virus particles of DNX-2401 ) and then receive multiple systemic immune checkpoint inhibitors, including pembrolizumab and an additional immune checkpoint inhibitor to target complementary axes of immune checkpoint inhibition that are upregulated in these tumors. Pembrolizumab is infused intravenously at a dose of 200 mg over 30 minutes every 3 weeks. The dosing of the additional checkpoint inhibitors is either ipilimumab 3mg / kg IV every 3weeks for 4 doses, relatlimab 160mg IV every 4weeks, sabatolimab 800mg IV every 4weeks, or tiragolumab 600mg IV every 3weeks, beginning on Day 14 and continuing for up to 103 weeks (except for ipilimumab which is limited to 4 doses) or until progressive disease is confirmed. Follow-up is performed through 90 days following the final dose of either pembrolizumab or the additionalcheckpoint inhibitor, whichever is later. Thereafter, follow-up is performed every 16 weeks for overall survival, objective response, clinical benefit, and adverse events for up to 12 months to assess primary outcome of one-year mortality.

[0255] Study Population:

[0256] Subjects with first or second glioblastoma or gliosarcoma recurrence for whom gross total or partial resection is not possible or planned.

[0257] Inclusion criteria:1 . > 18 years of age on the day of informed consent2. A single glioblastoma (GBM) or gliosarcoma (GS) tumor confirmed by documented historical histopathology. a. First or presenting second recurrence of glioblastoma or gliosarcoma (i.e. , relapse following prior treatment) at time of consent. Approval may be given by Medical Monitor or designee to proceed with enrollment with a prior non- GBM / GS diagnosis, in which case transition to GBM / GS may be accepted as first recurrence of tumor. b. Gross total or partial tumor resection, including tumor debulking, is not possible or not planned c. A single measurable tumor that is at least 10.0 mm longest diameter (LDi) x 10.0 mm shortest diameter (SDi) and that does not exceed 40.0 mm in LDi or SDi on the Screening MRI d. The measurable area of the tumor is solid / nodular and is not cystic e. Willing to provide a stereotactic biopsy sample from the brain tumor obtained prior to DNX- 2401 administration f. Tumor must be accessible for stereotactic injection g. Evidence of tumor recurrence (e.g., progression after last treatment) on the Screening MRI (15 days to 72 hours prior to DNX-2401 administration) h. Tumor location that will not risk delivery of DNX-2401 into the ventricular system3. Tumor recurrence or progression after previously failing surgical resection, chemotherapy or radiation4. Resolution of toxic effect(s) of the most recent prior chemotherapy to Grade 1 or less (except neuropathy and alopecia)5. Demonstrate adequate organ function as defined below: a. Hematological• Absolute neutrophil count (ANC) > 1 ,500 cells / mm3• White blood cells (WBC) > 2.5 x 103cells / mm3• Platelets > 100,000 cells / mm3• Hemoglobin > 10 g / dL or > 5.6 mmol / L• Absolute lymphocyte count (ALC) > 800 cells / mm3b. Renal• Creatinine < 1 ,5x upper limit of normal (ULN)• Blood urea nitrogen (BUN) <1 ,5x ULN c. Hepatic• Total bilirubin < 1.5x ULNNote: In the event that total bilirubin is > 1 .5 X ULN, the subject may be eligible if the direct bilirubin level is < ULN, following consultation with the DNAtrix Medical Monitor or designee.• Aspartate transaminase (AST) (SGOT) and alanine transaminase (ALT) (SGPT) < 2.5x ULN d. Coagulation• International Normalized Ratio (INR) <1.5x ULN• Prothrombin Time (PT) < 1 ,5x ULN• Activated Partial Thromboplastin Time (aPTT) < 1 ,5x ULN6. Adequate venous access7. Karnofsky performance status > 70%8. Afebrile at baseline / Day 0 prior to DNX-2401 administration (i.e., < 38.0°C)9. Prior anti-tumor therapies must have been completed within the following time periods prior to DNX-2401 injection: a. 2 weeks after vincristine b. 4 weeks after nitrosoureas c. 3 weeks after procarbazine or temozolomide d. 4 weeks after bevacizumab, other antibody therapy or other anti-angiogenic therapy to treat glioblastoma e. 5 half-lives for other anti-cancer agents or 2 weeks after the last dose when the half-life is unknown. A discussion of these agents takes place with the DNAtrix Medical Monitor or designee prior to establishing eligibility.10. For applicable screening candidates, external beam radiotherapy (> 5000 cGy) must have been completed at least 12 weeks prior to DNX-2401 administration11 . Females who are not of childbearing potential must be documented as such and will not be tested for pregnancy or required to utilize contraception if they meet one or more of the following definitions of non-childbearing potential: a. Amenorrheic for > 2 years without a hysterectomy and bilateral oophorectomy and a FSH value in the postmenopausal range upon pre-trial (screening) evaluation b. Post-hysterectomy, bilateral oophorectomy or tubal ligation. Tubal ligation must be confirmed with medical records of the actual procedure.12. Female subjects of childbearing potential must have a negative urine or serum pregnancy test within 24 hours prior to receiving DNX-2401 injection. If the urine test is positive or cannot be confirmed as negative, a serum pregnancy test is required to confirm negative results. The serum pregnancy test must be negative for the subject to be eligible.13. Female subjects of childbearing potential must be willing to use two highly effective birth control methods throughout the study, starting with provision of informedconsent through 180 days after the single dose of DNX-2401 and 120 days after the last dose of pembrolizumab. The two birth control methods can be either two barrier methods or a barrier method plus a hormonal method to prevent pregnancy. Examples of highly effective birth control methods include the following: a. Using twice the normal protection of birth control (i.e., double-barrier) by using a condom AND spermicidal jelly or foam, or a diaphragm AND spermicidal jelly or foam. A spermicidal jelly or foam must be used in addition to a barrier method (e.g., condom or diaphragm) b. Oral contraceptive control pills c. Depot or injectable birth control d. Intrauterine Device (IUD) e. Transdermal contraceptive patch f. Vaginal contraceptive ring14. Male subjects must agree to use an acceptable method of contraception throughout the study starting with provision of informed consent through 180 days after the single dose of DNX- 2401 and 120 days after the last dose of pembrolizumab.15. Willing and able to provide informed consent, undergo and comply with all study assessments and adhere to the protocol schedule16. Agree not to donate blood or gametes following DNX-2401 administration

[0258] Exclusion criteria:

[0259] Subjects who meet any of the following exclusion criteria are not enrolled:1. Recurrent GBM with multiple (> 2) separate enhancing tumors (measurable or nonmeasurable)2. T umor shape that is bi-lobular or multifocal tumor3. Tumor involvement that would require ventricular, brainstem or posterior fossa injection or access through a ventricle or risk of ventricular penetration in order to deliver DNX2401T umor involves both hemispheres or there is suspected cerebrospinal fluid (CSF) dissemination Documented extracranial metastases Requires, or based upon historical evidence, may require treatment with high-dose systemic corticosteroids defined as dexamethasone > 4 mg / day or bioequivalent for more than 3 consecutive days within 2 weeks prior to and following the first dose of pembrolizumab, or has demonstrated an inability to be tapered off of steroids Uncontrolled blood-sugar levels defined as HbA1 c > 7% Active autoimmune disease that requires, or has required, systemic treatment in the past 2 years (i.e. with use of disease modifying agents, corticosteroids or immunosuppressive drugs) Previous treatment with any checkpoint inhibitor (e.g., anti-PD-1 , anti-PD-L1 , or anti-PDL2 agent) or with an agent directed to another stimulatory or co-inhibitory T-cell receptor (e.g., CTLA-4, OX-40, CD137), including pembrolizumab History of (non-infectious) pneumonitis that required steroids or current pneumonitis History of interstitial lung disease Transfusions or medications (e.g., G-CSF) to treat pancytopenia or other hematological conditions within 4 weeks prior to DNX-2401 administration Prior gene transfer therapy or prior therapy with cytolytic virus of any type Live vaccines of any kind within 45 days prior to DNX-2401 administration and while participating in the study. Examples of live vaccines include, but are not limited to, the following: measles, mumps, rubella, varicella / zoster (chicken pox), yellow fever, rabies, BCG, and typhoid vaccine. Seasonal influenza vaccines for injection are generally killed virus vaccines and are permitted; however, intranasal influenza vaccines (e.g. Flu-Mist®) are live attenuated vaccines and are not allowed. Major surgery within 4 weeks and minor surgery within 2 weeks of DNX-2401 administration (Refer to Appendix 2 - Major and Minor Surgery Definitions)16. Participation in an investigational (invasive device, drug, or product) study or treatment with an investigational agent or device within 30 days prior to consent17. Any contraindication for undergoing MRI such as: individuals with pacemakers, epicardial pacer wires, infusion pumps, surgical and / or aneurysm clips, shrapnel, metal prosthesis, implants with potential magnetic properties, or metallic bodies in the eyes18. Is pregnant or breastfeeding, or planning to conceive or father children during the study, starting with the screening visit through 180 days after the single dose of DNX-2401 and 120 days after the last dose of pembrolizumab19. Evidence of active uncontrolled infection or an unstable or severe intercurrent medical condition that requires treatment and / or precludes surgery20. History of prior malignancy except for curatively treated basal or squamous cell carcinoma of the skin (non-melanoma skin cancer), cervical or vaginal intraepithelial neoplasia, non- invasive breast cancer in situ or localized prostate cancer with a prostate specific antigen (PSA) of < 4.0 ng / mL (mcg / L) at Screening. Subjects with other curatively treated malignancies who had no evidence of metastatic disease and a > 2 year disease-free interval may be enrolled after approval by the DNAtrix Medical Monitor or designee.21. Any medical condition that precludes intratumoral injection into the brain22. Immunocompromised subjects or those with autoimmune conditions, human immunodeficiency virus (HIV), or active hepatitis (according to diagnostic serology results that are positive for active HAV, HBV [Anti-HBc and HBsAg] or HCV infection)23. Pulmonary conditions including a known history of active tuberculosis (TB, Mycobacterium tuberculosis). TB testing is required for subjects recently exposed to persons with active TB or who have traveled recently to areas where TB is endemic.24. Evidence of bleeding diathesis, hemorrhage, or coagulopathy or use of anticoagulant medication or any medication that may increase the risk of bleeding that cannot be stopped prior to surgery. If the medication can be discontinued priorto DNX-2401 injection, then the subject may be eligible following consultation with the DNAtrix Medical Monitor or designee.25. Encephalitis, multiple sclerosis or other central nervous system (CNS) infection or primary CNS disease that would interfere with subject evaluation26. Li-Fraumeni Syndrome or with a known germ line deficit in the retinoblastoma gene or its related pathways27. Significant systemic or major illnesses including, but not limited to, congestive heart failure, ischemic heart disease, kidney disease or renal failure, organ transplantation or other conditions that may affect subject risk or protocol compliance28. Alcohol or substance abuse or alcohol dependency within 12 months prior to screening that has caused health consequences29. History or current diagnosis of any medical or psychological condition, and in particular, any unstable CNS condition such as delirium, confusion, etc., that might interfere with the subject’s ability to comply with the study requirements or the ability to obtain informed consent.

[0260] Additional Study Therapy, Dose and Administration Details:

[0261] Checkpoint inhibitors are administered as a 30-90 minute IV infusion. However, given the variability of infusion pumps from site to site, a window between -5 minutes and +10 minutes is permitted (i.e., if infusion time is 30 minutes -5 min / +10 min). Dosing interruptions are permitted in the case of medical / surgical events or logistical reasons not related to study therapy (e.g., elective surgery, unrelated medical events, subject vacation, and / or holidays). However, there are no dose reductions. Subjects are placed back on the checkpoint inhibitor therapy within 3 weeks of the scheduled interruption, unless otherwise discussed with the study team.

[0262] General Information on the Investigational Medical Product:

[0263] DNX-2401 is a conditionally replicative oncolytic adenovirus designed specifically for treating high-grade malignant gliomas. The virus was engineered with two stable genetic changes in the adenovirus dsDNA genome that cause it to (1 ) replicate selectively in retinoblastoma (Rb) pathway-deficient cells and (2) infect cells, such astumor cells and tumor vasculature that express certain RGD-binding integrins more efficiently. DNX-2401 , for intratumoral injection, is a sterile, preservative-free aqueous solution in single-use vials. Each vial is thawed and diluted with room temperature normal saline (USP) to achieve the required concentration for administration. The final product is a clear to translucent, colorless liquid with no evidence of particulate matter. DNX-2401 is shipped on dry ice with continuous temperature monitoring for destination storage in a < -60°C freezer.

[0264] Pembrolizumab is a monoclonal antibody that blocks the interaction between PD-1 and its ligands, PD-L1 and PD-L2, functioning as an immune checkpoint inhibitor. The drug is an lgG4 kappa immunoglobulin with an approximate molecular weight of 149 kDa.

[0265] Ipilimumab is a monoclonal antibody that binds to CTLA-4, thereby blocking its interaction with B7, functioning as an immune checkpoint inhibitor.

[0266] Relatlimab is a monoclonal antibody that binds to LAG-3, thereby blocking its interaction with MHC Class II molecules, functioning as an immune checkpoint inhibitor.

[0267] Sabatolimab is a monoclonal antibody that binds to Tl M-3, thereby inhibiting its interactions with its immune regulatory ligands such as galectin-9 or phosphatidylserine, functioning as an immune checkpoint inhibitor.

[0268] Tiragolumab is a monoclonal antibody that binds to Tl GIT, thereby inhibiting binding with ligands such as CD155 and CD 112, functioning as an immune checkpoint inhibitor.

[0269] Statistics

[0270] The primary endpoint, one-year overall survival rate are tested in a placebo- controlled randomized controlled trial (for the first arm, i.e., TME of TMEmed). The sample size estimation was based on a historical 12-month survival rate of 15% (Ballman et al, Neuro Oncol, 2007). The null hypothesis is that the DNX-2401 treated population has improved survival compared to the historical one-year survival rate.

[0271] For this randomized control study with a dichotomous end point, assuming two independent samples, a power of 80%, significance value of 5%, a control groupevent rate of 15%, and a treatment group event rate of approximately 50%, a sample size of 27 subjects per group (54 combined) is used (Nassiri et al, Nat Med, 2023).

[0272] The sample size for this study directed to subjects with a TME of TMEmedium was determined using the following formulae and calculations:wherein pi, p2 = proportion (incidence of groups #1 and #2); A = | P2-P1 1 = absolute difference between two proportions; ni = sample size for group #1 ; n2 = sample size for group #2; a = probability of type I error (usually 0.05); p = probability of type II error (usually 0.2); z = critical Z value for a given a and p; or K = ratio of sample size for group #2 to group #1 .

[0273] For a dichotomous end point, assuming a one sample study, as is the case in the second (TMEIow) and third arm (TMEhigh), with a power of 80%, significance value of 5%, a population event rate of 15% and a treatment group event rate of approximately 45% for TMEIow and 30% for TMEhigh, a sample size of 14 and 52 patients per group is used, respectively (Nassiri et al, Nat Med, 2023).

[0274] The sample size for the TMEIow arm was determined using the following formulae and calculations:wherein po = proportion (incidence) of population; pi = proportion (incidence) of study group; N = sample size for study group; a = probability of type I error (usually 0.05); p = probability of type II error (usually 0.2); and z = critical Z value for a given a or p.

[0275] The sample size for the TMEhigh arm was determined using the following formulae and calculations:wherein po = proportion (incidence) of population; pi = proportion (incidence) of study group; N = sample size for study group; a = probability of type I error (usually 0.05); p = probability of type II error (usually 0.2); and z = critical Z value for a given a or p.Objectives and Outcomes

[0276] Primary Objectives and Endpoints:

[0277] As indicated above, the primary objective of this clinical trial is to evaluate the one-year overall survival rate, with a preset control one-year survival rate of 15% (Ballman et al, Neuro Oncol, 2007). An interim analysis is performed upon recruitment of 27 patients (50% of estimated combined sample size for TMEmed) to evaluate both efficacy and safety. These data are summarized using Kaplan-Meier methods.

[0278] Secondary Objectives and Endpoints:

[0279] The secondary objectives of this clinical trial encompass the assessment of the objective response rate, median overall survival, 6-month progress free survival, and the modified radiographic response assessment in neuro-oncology (mRANO) and immunotherapy RANO (iRANO) clinical benefit rate.

[0280] The mRANO criteria, which are specifically designed for glioblastoma, define four principal response categories:

[0281] Complete Response (CR) signifies the complete disappearance of all enhancing lesions with stability or improvement in clinical status and no emergence of new or enlarging lesions.

[0282] Partial Response (PR) denotes a substantial reduction in tumor size and enhancement by at least 50%, with no new or enlarging lesions and clinical stability or improvement.

[0283] Stable Disease (SD) indicates no significant changes in tumor size and enhancement, with no new lesions and maintenance of clinical stability.

[0284] Disease Progression (PD) is characterized by a substantial increase in tumor size and enhancement, the appearance of new lesions, or clinical deterioration.

[0285] Objective response determined from magnetic resonance imaging (MRI) review is based upon the mRANO and iRANO criteria. Objective response rate, defined as proportion of complete or partial response, is summarized by the number and percentage of subjects. Corresponding 95% confidence intervals (Cis) are based upon the binomial exact method. Complete, partial responses and suspected progression are confirmed. Any partial or complete response noted on MRI is confirmed by repeat MRI at a minimum of 4 weeks (28 days with + 3-day window) later. In the case of suspectedtumor progression by MRI, and with the absence of deteriorating clinical status as determined by the Investigator, a repeat MRI is performed, if possible, at a minimum of 4 weeks (28 days with + 3-day window) later in an attempt to confirm progressive disease.

[0286] The clinical benefit rate is summarized as the number and proportion of subjects with clinical benefit, (defined as complete response, and partial response and stable disease) per mRANO and iRANO criteria along with 95% Cis for the proportion of subjects with clinical benefit based on the binomial exact method.

[0287] The proportion of subjects with 6-month progress free survival is calculated with 95% Cis.

[0288] The median overall survival of subjects is calculated with 95% Cis.

[0289] Exploratory Objectives:

[0290] Exploratory analyses will also be performed on plasma and cerebrospinal fluid biomarkers, tumor and tumor microenvironment multi-omic analysis, and radiometric assessments. Additional clinical metrics including progression free survival, time to tumor response, duration of response, RANO criteria modified to account for pseudoprogression and iRANO, change in Kamofsky Performance Score, change in neurological status are examined.

[0291] Tissue Biomarkers:

[0292] Genome-wide DNA-methylation profiles are generated on pre- and posttreatment tissue samples. Between 250 and 500 ng DNA is extracted from 50 mg of tissue, bisulfite converted (Zymo EZ DNA methylation Kit, Zymo 190 Research) and profiled using the Illumina HumanBeadChip 850K array or900K EPIC v2 array. Raw data files (*.idat) are imported, processed, and normalized to integrate data from multiple generations of Infinium methylation arrays. Genome-segmented copy number aberrations are inferred from methylation array data. Beta values of CpG sites are computed and combination of differentially methylated probes are used to generate signatures of response to treatment.

[0293] Biofluid Biomarker analyses:

[0294] Circulating free DNA (cfDNA) is extracted from plasma and cerebrospinal fluid (CSF) samples using the QIAamp Circulating Nucleic Acid Kit (Qiagen) andquantified through Qubit (Thermo Fisher Scientific). Between 1 and 10 ng of cfDNA from each plasma or CSF sample is quantified by a Qubit fluorometer (Qubit 4, Thermo Fisher Scientific) processed according to our cfMeDIP-seq protocol as has been previously described for 1-10 ng cfDNA per sample (Nassiri et al., 2020; Zuccato et al., 2023) In summary, cfDNA undergoes library preparation with Kapa HyperPrep Kits (Roche), immunoprecipitation of methylated cfDNA using the MagMeDIP Kit (Diagenode), cfDNA purification with version 2 of the I Pure Kit (Diagenode), and library polymerase chain reaction amplification and cleanup. Resulting libraries are sequenced on the Illumina Novaseq6000 with 100 bp pair ended reads and a median of 60-70 million reads per sample, similar to that used previously, after optimal fragment size selection are confirmed using 2100-Bioanalyzer traces (Agilent). cfMeDIP-seq data are processed as described previously (Nassiri et al. , 2020; Zuccato et al. , 2023). In brief, sequencing reads are first aligned with the human genome using Bowtie2. Reads are deduplicated and then indexed using SAMtools. Data is reduced to 300 bp genomic windows that map to regulatory features (covering CpG islands, shores, shelves, and FANTOM5 enhancers) with the MEDIPS package. Reads per kilobase million and count per million (CPM) values are calculated for these windows. Differentially methylated regions between responders and non-responders are computed and combination of these regions will be used to generate signatures of response to treatment.

[0295] Radiometric assessments:

[0296] Preoperative images typically consist of the following pulse sequences: precontrast T1 and T2, T2 fluid attenuated inversion recovery (FLAIR), diffusion weighted (b values, 0 and 1000 s / mm2), and gadolinium-enhanced T1 -weighted images. All images are manually segmented on preoperative three-dimensional T 1 post-contrast MR images and T2 FLAIR images using commercially available software (MIM Software Inc.) Shape, histogram, texture, multiscale and deep features are extracted and radiomic signatures of response to treatment will be computed.Additional Methods

[0297] Gene expression profiling and analyses:

[0298] RNA is extracted from formalin-fixed, paraffin-embedded pretreatment tumor biopsies and analyzed retrospectively on the NanoString nCounter system. When there is also tumor biopsy specimens vaiiahi at tha tima of disease progression, anexamination of gene expression changes before and after treatment in matched patient samples is performed.

[0299] The geometric mean of canonical marker genes are used to compute scores for immune cell types (Becht et al., Genome Biol, 2016), functional orientation markers, and signature scores reported herein, unless otherwise explicitly stated. Functional orientation markers and the chemokine and cytolytic signature scores are obtained from previous studies (White et al., Ann Oncol, 2022; Coppola et al., J Pathol, 2011 ; Rooney et al., Cell, 2015). Remaining marker genes are provided in Table 1. A T- cell-inflamed signature is computed as previously described using a weighted sum of normalized expression values of 18 inflammatory genes (CCL5, CD27, CD274 [PD-L1], CD276 [B7-H3], CD8A, CMKLR1 , CXCL9, CXCR6, HLA.DQA1 , HLA.DRB1 , HLA.E, IDO1 , LAG-3, NKG7, PDCD1 LG2 [PD-L2], PSMB10, STAT1 , and TIGIT) related to antigen presentation, chemokine expression, cytolytic activity, and adaptive immune resistance (Ayers et al., J Clin Invest, 2017). Glioblastoma microenvironment subtypes are obtained by partition-around-medoid clustering using immune cell type scores, as previously described (White et al., Ann Oncol, 2022). Differentially expressed genes between groups are identified by comparing Log2 fold change and Welch’s P-values. Genes with absolute value Log2FC > 1 and P <0.05 are considered differentially expressed, unless otherwise specified. Functional enrichment analysis is performed using gProfiler.

[0300] While the present disclosure has been described with reference to examples, it is to be understood that the scope of the claims should not be limited by the embodiments set forth in the examples, but should be given the broadest interpretation consistent with the description as a whole.

[0301] All publications, patents and patent applications are herein incorporated by reference in their entirety to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Where a term in the present description is found to be defined differently in a document incorporated herein by reference, the definition provided herein is to serve as the definition for the term.TablesSequencesSEQ ID NO: 1EIVLTQSPGTLSLSPGERATLSCRASQSVGSSYLAWYQQKPGQAPRLLIYGAFSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 2QVQLVESGGGVVQPGRSLRLSCAASGFTFSSYTMHWVRQAPGKGLEWVTFISYDGNNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAIYYCARTGWLGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQP REPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GS FFLYSKLTVDKS RWQQGNVFSCSVM H EALH N HYTQKSLS LS PG KSEQ ID NO: 3EIVLTQSPATLSLSPGERATLSCRASQSISSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQRSNWPLTFGQGTNLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSL SSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 4QVQLQQWGAGLLKPSETLSLTCAVYGGSFSDYYWNWIRQPPGKGLEWIGEINHRGSTNSNPSLKSRVTLSLDTSKNQFSLKLRSVTAADTAVYYCAFGYSDYEYNWFDPWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGSEQ ID NO: 5AIQLTQSPSSLSASVGDRVTITCRASESVEYYGTSLMQWYQQKPGKAPKLLIYAASNVESGVPSRFSGSGSGTDFTLTISSLQPEDFATYFCQQSRKDPSTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSK DSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 6QVQLVQSGAEVKKPGSSVKVSCKASGYTFTSYNMHWVRQAPGQGLEWMGDIYPGNGDTSYNQKFKGRVTITADKSTSTVYMELSSLRSEDTAVYYCARVGGAFPMDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRWSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPR EPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSD GSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGSEQ ID NO: 7DIVMTQSPDSLAVSLGERATINCKSSQTVLYSSNNKKYLAWYQQKPGQPPNLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQYYSTPFTFGPGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSP VTKSFNRGECSEQ ID NO: 8EVQLQQSGPGLVKPSQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGKTYYRFKWYSDYAVSVKGRITINPDTSKNQFSLQLNSVTPEDTAVFYCTRESTTYDLLAGPFDYWGQGTLVTVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRWSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKSEQ ID NO: 15EIVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYDASNRATGIPARFSGSGSGTDFTLTISSLEPEDFAVYYCQQSSNWPRTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO: 16QVQLVESGGGVVQPGRSLRLDCKASGITFSNSGMHWVRQAPGKGLEWVAVIWYDGSKRYYADSVKGRFTISRDNSKNTLFLQMNSLRAEDTAVYYCATNDDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSWTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVWDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKSEQ ID NO:17EIVLTQSPATLSLSPGERATLSCRASKGVSTSGYSYLHWYQQKPGQAPRLLIYLASYLESGVPARFSGSGSGTDFTLTISSLEPEDFAVYYCQHSRDLPLTFGGGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASWCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECSEQ ID NO:18QVQLVQSGVEVKKPGASVKVSCKASGYTFTNYYMYWVRQAPGQGLEWMGGINPSNGGTNFNEKFKNRVTLTTDSSTTTAYMELKSLQFDDTAVYYCARRDYRFDMGFDYWGQGTTVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCWVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGKCITATIONS FOR REFERENCES REFERRED TO IN THE SPECIFICATIONAyers, M. et al. 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Claims

Claims:

1. Use of a TMEhigh therapy for treating a glioma in a subject that has been previously identified as having a high tumor microenvironment; wherein the high tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained pre-treatment, the immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; and wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor.

2. The use of claim 1 , wherein the TMEhigh therapy comprises use of the oncolytic virus intratumorally prior to use of repeated doses of the anti-PD-1 antibody or the binding fragment thereof and repeated doses of the at least one additional immune checkpoint inhibitor.

3. The use of claim 2, wherein the use of repeated doses of the anti-PD-1 antibody or the binding fragment thereof starts about 7 days to about 21 days after use of the oncolytic virus.

4. The use of claim 2 or 3, wherein the doses of the anti-PD-1 antibody or the binding fragment thereof comprise about 200 mg for use intravenously over about 30 minutes.

5. The use of any one of claims 2 to 4, wherein the doses of the anti-PD-1 antibody or the binding fragment thereof are for use about every 3 weeks.

6. The use of any one of claims 2 to 5, wherein the at least one additional immune checkpoint inhibitor of the TMEhigh therapy comprises(a) an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TI GIT antibody, or a combination thereof;(b) a binding fragment of an anti-CTLA-4 antibody, an anti-LAG-3 antibody, an anti-TIM-3 antibody, an anti-TIGIT antibody, or a combination thereof; or(c) a combination of a) and b).

7. The use of claim 6, wherein the use of repeated doses of the at least one checkpoint inhibitor starts about 14 days after use of the oncolytic virus.

8. The use of claim 6 or 7, wherein the doses of the anti-CTLA-4 antibody or the binding fragment of the anti-CTLA-4 antibody comprise about 3 mg / kg for use intravenously.

9. The use of any one of claims 6 to 8, wherein the doses of the anti-CTLA-4 antibody or the binding fragment of the anti-CTLA-4 antibody are for use about every 3 weeks.

10. The use of any one of claims 6 to 9, wherein the repeated doses of the anti-CTLA- 4 antibody or the binding fragment of the anti-CTLA-4 antibody comprise up to 4 doses.11 . The use of any one of claims 6 to 10, wherein the doses of the anti-LAG-3 antibody or the binding fragment of the anti-LAG-3 antibody comprise about 160 mg for use intravenously.

12. The use of any one of claims 6 to 11 , wherein the doses of the anti-LAG-3 antibody or the binding fragment of the anti-LAG-3 antibody are for use about every 4 weeks.

13. The use of any one of claims 6 to 12, wherein the doses of the anti-TIM-3 antibody or the binding fragment of the anti-TIM-3 antibody comprise about 800 mg for use intravenously.

14. The use of any one of claims 6 to 13, wherein the doses of the anti-TI M-3 antibody or the binding fragment of the anti-TI M-3 antibody are for use about every 4 weeks.

15. The use of any one of claims 6 to 14, wherein the doses of the anti-TIGIT antibody or the binding fragment of the anti-TIGIT antibody comprise about 600 mg for use intravenously.

16. The use of any one of claims 6 to 15, wherein the doses of the anti-TIGIT antibody or the binding fragment of the anti-TIGIT antibody are for use about every 3 weeks.

17. Use of a TMEmedium therapy for treating a glioma in a subject that has been previously identified as having a medium tumor microenvironment; wherein the medium tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained pre-treatment, the immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; and wherein the TMEmedium therapy comprises combined therapy of an oncolytic virus and an anti-PD-1 antibody or a binding fragment thereof.

18. The use of claim 17, wherein the TMEmedium therapy comprises use of the oncolytic virus intratumorally prior to use of repeated doses of anti-PD-1 antibody or the binding fragment thereof.

19. The use of claim 18, wherein the use of repeated doses of anti-PD-1 antibody or the binding fragment thereof starts about 14 days after use of the oncolytic virus.

20. The use of claim 18 or 19, wherein the doses of anti-PD-1 antibody or the binding fragment thereof comprise about 200 mg for use intravenously over about 30 minutes.

21. The use of any one of claims 18to 20, wherein the doses of anti-PD-1 antibody or the binding fragment thereof are for use every 3 weeks.

22. The use of any one of claims 1 to 21 , wherein the anti-PD-1 antibody or the binding fragment thereof is pembrolizumab or a fragment thereof.

23. Use of a TMEIow therapy for treating a glioma in a subject that has been previously identified as having a low tumor microenvironment; wherein the low tumor microenvironment has been identified by determining a tumor microenvironment (TME) of the subject from a sample immune gene expression profile of a biopsy sample obtained pre-treatment, the immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; and wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, binding fragment of anti-PD-1 antibody, or an additional immune checkpoint inhibitor.

24. The use of claim 23, wherein the TMEIow therapy comprises use of repeated doses of the oncolytic virus intratumorally.

25. The use of claim 24, wherein the doses of the oncolytic virus are for use about every 4 weeks.

26. The method of claim 24 or 25, wherein the repeated doses of the oncolytic virus comprise up to 6 doses.

27. A method of selecting therapy for a subject with glioma comprising: a) determining a sample immune gene expression profile comprising markers of immune infiltration from a tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; b) determining a tumor microenvironment (TME) of the subject from the sample immune gene expression profile, wherein the TME of the subject comprises one of a high TME (TMEhigh), a medium TME (TMEmedium) and a low TME (TMEIow); c) selecting:i. TMEhigh therapy when the TME of the subject is TMEhigh, wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor; ii. TMEmedium therapy when the TME of the subject is TMEmedium, wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and an anti-PD-1 antibody or a binding fragment thereof; or iii. TMEIow therapy when the TME of the subject is TMEIow, wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, a binding fragment of anti-PD-1 antibody, or an additional immune checkpoint inhibitor.

28. A method of selecting therapy for a subject with glioma that has previously been treated with a TMEIow therapy comprising: a) determining a sample immune gene expression profile comprising markers of immune infiltration from a new tumor biopsy sample from the subject, by detecting the levels of mRNAs of cell type markers, immune checkpoint genes, functional orientation markers, and signature scores; b) determining a new tumor microenvironment (TME) of the subject from the sample immune gene expression profile, wherein the new TME of the subject comprises one of a high TME (TMEhigh), a medium TME (TMEmedium) and a low TME (TMEIow); c) selecting: i. TMEhigh therapy when the new TME of the subject is TMEhigh, wherein the TMEhigh therapy comprises combination therapy of an oncolytic virus, an anti-PD-1 antibody or a binding fragment thereof, and at least one additional immune checkpoint inhibitor; ii. TMEmedium therapy when the new TME of the subject is TMEmedium, wherein the TMEmedium therapy comprises combination therapy of an oncolytic virus and an anti-PD-1 antibody or a binding fragment thereof; oriii. TMEIow therapy when the new TME of the subject is TMEIow, wherein the TMEIow therapy comprises oncolytic virus therapy and does not comprise an anti-PD-1 antibody, a binding fragment of anti- PD-1 antibody, or an additional immune checkpoint inhibitor.

29. The method of claim 27 or 28, wherein the TMEhigh therapy is as defined in any one of claims 1-16 and 22, wherein the TMEmedium therapy is as defined in any one of claims 17-22, and / or wherein the TMEIow therapy is as defined in any one of claims 23-26.

30. The use of any one of claims 1-26 or the method of any one of claims 27 to 29, wherein the glioma is glioblastoma.31 . The use of any one of claims 1 -26 and 30 or the method of any one of claims 27 to 30, wherein the oncolytic virus is an oncolytic adenovirus.

32. The use or the method of claim 31 , wherein the oncolytic adenovirus is a conditionally replicative oncolytic adenovirus.

33. The use or the method of claim 32, wherein the conditionally replicative oncolytic adenovirus is DNX-2401 .

34. The use of any one of claims 1-26 and 30-33 or the method of any one of claims 27 to 33, wherein the oncolytic virus is for use intratumorally at about 5x1010to about 5x1011virus particles.

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