Markers for determining responsiveness to immune checkpoint inhibitor immunotherapy

Abstract

The present invention relates to methods for assessing the likelihood of a subject responding to immune checkpoint inhibition (ICI) treatment for cancer. Specifically, peripheral blood mononuclear cells and tumour markers can predict response to ICI treatment, thereby informing treatment. Also provide are methods of treatment of cancer with ICI immunotherapy, informed by the likelihood of treatment success.

Description

Title of InventionMARKERS FOR DETERMINING RESPONSIVENESS TO IMMUNE CHECKPOINT INHIBITOR IMMUNOTHERAPYTechnical Field

[0001] The present disclosure relates to markers and immune signatures for determining if a subject will respond to immune checkpoint inhibitor immunotherapy for cancer. Further, the present disclosure relates to methods, products and systems for assessing said markers, and methods of treatment informed by said markers and immune signatures.Priority claim

[0002] This application claims priority from Australian provisional application number 2024903613, the entire contents of which is herein incorporated.Background of Invention

[0003] Traditional treatments for cancers exploit the difference in proliferation between cancer cells, which proliferate quickly, and normal cancer cells, which proliferate at a slower rate. Such therapies, including chemotherapy and radiotherapy, have limited success for many forms of cancer and often have considerable, and use-limiting, side effects.

[0004] More recently, immunotherapies for cancers have emerged as viable and effective cancer treatments. Immunotherapies utilise the patient’s immune system by providing agents that are either directed against specific cancer-associated antigens (for example antibodies or chimeric antigen receptor T cells) or activate the patient’s immune system. The advantage of the latter is that these do not rely on knowledge of specific cancer-associated antigens and rely on the inherent mutations within a cancer. Therefore, these immune activators are useful against a variety of different cancers.

[0005] One form or immunotherapy that activates the immune system is immune checkpoint inhibition (ICI) immunotherapy. Immune checkpoints are innate negative regulators of the immune system (primarily the cellular immune system). Under homeostatic conditions these regulators are critical for “self-tolerance” and when activated down-regulate immune responses by skewing the balance immune effector cells to regulator immune cells.

[0006] Due to the immunosuppressive nature of immune checkpoints, many cancers create an immunosuppressive environment by aberrantly expressing immune checkpointactivators, thereby limiting the immune surveillance and immune response to otherwise immunogenic neo-antigens expressed by the tumour. Therefore, reversing this suppression by inhibiting immune checkpoint activation can result in activation of the immune system and antitumour immune responses.

[0007] Programmed Death-Ligand 1 (PD-L1 , also known as CD274 or B7-H1) is one of the most commonly upregulated immune checkpoint activators in tumours. Programmed Death-Ligand 1 binds to Programmed Cell Death Protein 1 (PD-1 , also known as CD279) expressed on T cells and B cells. When PD-L1 engages PD-1 it induces apoptosis on antigenspecific T cells and promotes cell survival of antigen-specific regulatory T cells. Thereby, PD- L1 expression biases the antigen-specific immune response toward tolerance.

[0008] Importantly, inhibition of PD-L1 / PD-1 signalling can disrupt the immune suppressive microenvironment by the cancer cells and result in cancer-specific immune responses leading to cancer regression.

[0009] Immune checkpoint inhibitor (I Cl) immunotherapy is used routinely for the treatment of a range of cancers. However, a significant number of patients fail to respond to immune checkpoint inhibitor therapy or respond poorly. Where a tumour is present, the efficacy of ICI immunotherapy can be gauged by assessing the size of the tumour. However, in many patients ICI immunotherapy is provided after resection of the tumour. In such cases the efficacy of the ICI immunotherapy cannot be assessed, unless a new tumour develops indicating a lack of efficacy.

[0010] Furthermore, ICI therapy can cause several and severe adverse effects and toxicities. These include the onset of autoimmune diseases (such as type 1 diabetes, thyroiditis and hepatotoxicity) as well as a range of systemic toxicities including (inter alia) diarrhoea, colitis, rashes, neurotoxicity, and nephrotoxicity.

[0011] Additionally, current ICIs are antibodies and thereby require regular intravenous administration (e.g., every 1-6 weeks) for a prolong period (e.g., 6 to 18 months), placing a significant time burden and inconvenience on the patient.

[0012] In view of the above, it would be advantageous to determine which patients are likely or unlikely to respond to ICI immunotherapy or are not responding to ICI immunotherapy,. This would allow oncologists to select more appropriate alternative treatments and would reduce the number of unnecessary adverse effects experienced by cancer patients.Summary of Invention

[0013] The present invention is predicated, at least in part, on the identification that specific markers expressed by the tumour, either alone or in combination with one or more immune cell signatures, can determine the likelihood that a subject will respond to immune checkpoint inhibition (ICI) immunotherapy and in particular PD-L1 or PD-1 ICI immunotherapy.

[0014] The present invention provides a method of determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the method comprising assessing the expression of one or more of E-selectin and / or FLIT7 within a biological sample from the subject, and assessing one or more of:• CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells;• CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;• FoxP3 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;• CD15s expression in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells;• Ki67 expression in CD8+ T cells; or• CD71 expression in CD8+ T cells, wherein an increase in the expression of one or more of E-selectin and / or FLIT7 in combination with one or more of:• an increased expression of CD15s in Ki67+ CD8+ T cells or CD71+ CD8+ T cells prior to the immunotherapy;• an increased expression of CTLA-4 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells prior to and / or after the immunotherapy;• an increased expression of FoxP3 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells prior to immunotherapy;• an increased expression of CD15s in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells prior to immunotherapy; and / or• a decreased expression of Ki67+ or CD71+ in CD8+ T cells prior to immunotherapy, determines an increased likelihood that the subject will respond to immune checkpoint inhibition immunotherapy.

[0015] E-selectin is a cell adhesion molecule involved in cell tethering and rolling, and recruitment of immune cells from the vasculature. Primarily E-selectin is expressed on vascular tissue, such as endothelial cells. Therefore, in some embodiments of the invention, the expression of E-selectin is assessed on endothelial cells from a tumour.

[0016] Endothelial cells can be identified based on a series of markers or morphology. However, in preferred embodiments, the endothelial cells express CD31 , and are identified based on CD31 expression.

[0017] There are a variety of immune checkpoint inhibitors known in the art, however in preferred embodiments the immune checkpoint inhibition immunotherapy is PD-1 immune checkpoint inhibition. PD-1 immune checkpoint inhibition may, in some embodiments, be PD- 1 receptor inhibition or, in some embodiments, PD-1 ligand (PD-L1) inhibition.

[0018] The assessment of E-selectin or FLIT7 expression may be assessment of protein expression, or gene expression. In a preferred embodiment, the expression of E-selectin is protein expression of E-selectin. In a preferred embodiment, the expression of FLIT7 is gene expression of FLIT7.

[0019] A variety of biological samples can be used in the method of the present invention. These samples can be tumour-related, in that they provide information on the phenotype or genotype of the tumour. In a preferred embodiment, the biological sample is a tumour sample. In this embodiment, E-selectin or FLIT7 are assessed in the tumour sample. In some embodiments, the expression of E-selectin and / or FLIT7 is determined in a blood sample, or a serum sample, or a plasma sample, or a lymph sample, or an isolated peripheral blood mononuclear cells (PBMCs), or in tumour infiltrating lymphocytes (TILs).

[0020] The expression of E-selectin and FLIT7 relate to the phenotype of a tumour and the tumour microenvironment (including tumour-infiltrating lymphocytes), while the makers CD15s, CTLA-4, FoxP3, Ki67 and CD71 relate to the phenotype of the subject’s immune cells and immune system. Therefore, in some embodiments, the expression of FLIT7 and / or E-selectin is assessed in a first biological sample and the expression of CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells; CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; FoxP3 marker in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; CD15s marker in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells; Ki67 marker in CD8+ T cells; or CD71 marker in CD8+ T cells, is performed in a second biological sample.

[0021] It is demonstrated herein that CD103 is associated with CD15s expression in specific peripheral T cells populations. Therefore, in some embodiments, CD103 may be used as a marker in addition to CD15s. In some embodiments, CD103 may be used as a marker in place of CD15s.

[0022] In some embodiments, the first biological sample is a tumour sample. In some embodiments, the second biological sample is a vascular sample, or a blood sample, or aserum sample, or a plasma sample, or isolated peripheral blood mononuclear cells (PBMCs), or is tumour infiltrating lymphocytes.

[0023] In some embodiments, the Ki67+ CD8+ T cells comprise Ki67+ CD8+ CD3+ T cells. In some embodiments, the Ki67+ CD8+ T cells comprise Ki67+ CD8+ CD3+ CD28+ T cells. In some embodiments, the Ki67+ CD4+ T cells comprise Ki67+ CD4+ CD8- T cells. In some embodiments, the Ki67+ CD4+ T cells comprise Ki67+ CD4+ CD3+ T cells.

[0024] Methods are known in the art to assess the expression of immune cell markers. However, in a preferred embodiment, the expression of one or more of CD15s, CD71 , CTLA4, FoxP3, Ki67, CD8, CD4, CD3, CD28 or CD103 is assessed by flow cytometry. As is known in the art, flow cytometry utilises detection antibodies. In some embodiments, CD15s expression is determined by using one or more of the antibodies FH6, HECA-452, CHO131 , or CSLEX. In some embodiments, CD71 expression is determined by using the antibody M-A712.

[0025] To determine if the expression of E-selectin in a tumour or tumour-associated endothelial cells, FLIT7 in a tumour sample, CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells; CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; FoxP3 marker in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; CD15s marker in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells; Ki67 marker in CD8+ T cells; or CD71 marker in CD8+ T cells is increased or decreased, the expression can be compared to a control standard. In some embodiments, the control standard is predetermined and based on a population of subjects that have not responded to immune checkpoint inhibition immunotherapy. In some embodiments, the control standard is based on a healthy population of healthy tissue. In such embodiments, deviations away from the control standard indicate that a subject may have a responsive phenotype.

[0026] The threshold which determines whether a subject will be within a responsive phenotype, or a non-responsive phenotype, can be determined by a person skilled in the art based on clinical studies. However, in some embodiments, a percentage of CD15s positive cells in Ki67+ CD8+ T cells of about 6.5% or greater in the biological sample from the subject determines an increased likelihood that the subject will be responsive to immune checkpoint immunotherapy. In some embodiments, a percentage of E-selectin expressing cells of about 7% or greater within endothelial cells in the biological sample from the subject determines an increased likelihood that the subject will be responsive to immune checkpoint immunotherapy. In some embodiments, a percentage of CD15s positive cells in Ki67+ CD8+ T cells of about 6.5% or greater and a percentage of E-selectin expressing cells of about 7% or greater within endothelial cells in the biological sample from the subject (preferably a tumour or tumour-associated cell sample) determines an increased likelihood that the subject will be responsive to immune checkpoint inhibition immunotherapy. In these embodiments, the expression of E- selectin may be determined using the BBA18 antibody and the expression of CD15s is determined using the HECA-452 antibody.

[0027] In some embodiments, the method of the present invention comprises using computer software executable by a processor to process data representative of the expression of one or more of E-selectin and / or FLIT7. This data may also be representative of the expression of one or more of the markers CD15s, CD71 , CTLA4, FoxP3, Ki67, CD8, CD4, CD3, or CD28, and the software comprises instructions to compare the expression of the markers to a control standard to provide a determination of the likelihood of the subject responding to immune checkpoint inhibition immunotherapy.

[0028] In some embodiments, the method comprises performing immunohistochemistry to determine the expression of E-selectin in a tumour sample or tumour endothelial cell samples.

[0029] Also provided by the present invention is a method of determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the method comprising assessing the expression of FLIT7 within a biological sample from the subject, wherein an increased expression of FLIT7 determines an increased likelihood that the subject will respond to immune checkpoint inhibition immunotherapy. In embodiments of this method, the expression of FLIT7 is gene expression of FLIT7. Preferably, the gene expression is assessed in a biological sample which is a tumour sample. However, in alternative embodiments the biological sample is a blood sample, a serum sample, a plasma sample, a lymph sample, isolated PBMCs, or tumour infiltrating lymphocytes. In embodiments, the gene expression of FLIT7 is compared to a control standard. In embodiments of this method, the control standard for FLIT7 is predetermined based on a population of subjects that have not responded to immune checkpoint inhibition immunotherapy or comparable healthy tissue. In some embodiments of this method, the method determines the likelihood of responding to immunotherapy with a single immune checkpoint inhibitor. In some embodiments of this method, the method comprises using computer software executable by a processor to process data representative of the expression of FLIT7. Methods for assessing the gene expression of FLIT7 include using PCR.

[0030] Also provided by the present invention is a method of treating or preventing cancer in a subject, the method comprising determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer as described herein. The method also comprising administering to the subject at least a single immune checkpoint inhibitor immunotherapy if thesubject is determined to have an increased likelihood of responding to immunotherapy. Preferably, the immune checkpoint immunotherapy is PD-1 receptor or PDL-1 receptor blocking immunotherapy.

[0031] The method of treatment and prevention can be performed in conjunction with other treatments such as radiotherapy, chemotherapy or surgery. In some embodiments of the method of prevention or treatment, the subject has had their cancer resected.

[0032] Alternatively, if the subject is determined to have a decreased likelihood of responding to immunotherapy, then the method of preventing or treating cancer may comprise administering to the subject two or more immune checkpoint inhibitors or increasing the dose or the number of doses of an immune checkpoint inhibitor, or performing an alternative treatment.

[0033] In embodiments of the methods of determining if a subject will respond to immune checkpoint inhibition immunotherapy and the methods of preventing or treating a cancer, the cancer is selected from one of melanoma, non-small cell lung cancer, mesothelioma, kidney cancer, head and neck cancer, Hodgkin Lymphoma, Merkel cell carcinoma, bladder cancer, breast cancer, oesophageal cancer, gastric cancer, squamous cell carcinoma of the skin, cervical cancer, a cancer with microsatellite instability and / or mismatch repair enzyme deficiency, hepatocellular carcinoma, and primary mediastinal large B-cell lymphoma, and other malignancies susceptible to therapeutic immune checkpoint inhibition. Preferably, the cancer is selected from one of melanoma, non-small cell lung cancer, or breast cancer, most preferably melanoma.

[0034] In some embodiments, the breast cancer is triple negative breast cancer, or HER2- positive breast cancer, or is hormone receptor negative breast cancer.

[0035] While many different cancers can be treated with ICI immunotherapy, tumours with a high number of mutations are most amendable to ICI immunotherapy. Accordingly, in some embodiments, the cancer is a high tumour mutational burden cancer.

[0036] The present invention also provides a kit for use in determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the kit comprising one or more of a reagent for detecting the expression of: E-selectin and / or a reagent for detecting the expression of FLIT7; and one or more reagents for detecting the expression of one or more of CD4, CD8, CD15s, CTLA-4 marker, FoxP3, Ki67 and CD71. This kit may be used in the methods of the present invention.

[0037] Preferably, the kit comprises at least one reagent for detecting the expression of E- selectin and / or a reagent for detecting the expression of FLIT7; at least one reagent for detecting the expression of Ki67 and / or CD71 ; and at least one reagent for detecting the expression of CD4, CD8, CD15s, CTLA-4 marker, or FoxP3.

[0038] Further provided is a use of one or more reagents for detecting the expression of: E-selectin and / or a reagent for detecting the expression of FLIT7; and at least one reagent for detecting the expression of Ki67 and / or CD71 ; and at least one reagent for detecting the expression of CD4, CD8, CD15s, CTLA-4 marker, or FoxP3, in determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer.

[0039] Moreover, the present invention provides a system for determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the system comprising: a means for detecting the expression of E-selectin and / or FLIT7 in a blood, vascular and / or tumour sample from the subject; a processor; memory; and software resident in the memory and accessible to the processor, the software comprising a series of instructions executable by the processor to process data from the means to detect the expression of E-selectin and / or FLIT7 in the sample from the subject, to thereby determine the likelihood of the subject responding to the immune checkpoint inhibition immunotherapy.

[0040] In embodiments of the system, the means for detecting the expression of E-selectin is immunohistochemistry. Is some embodiment of the system, the means for detecting the expression FLIT7 is PCR. The system may also further comprise means for detecting the expression of one or more of: Ki67, CD71 , CD4, CD8, CD15s, CTLA-4 marker and / or FoxP3. Preferred means for detecting the expression of one or more of Ki67, CD71 , CD4, CD8, CD15s, CTLA-4 marker and / or FoxP3 is a flow cytometer.Brief Description of Drawings

[0041] Some embodiments are illustrated in the following figures in order to provide a better understanding of the present disclosure and to assist in demonstrating how some embodiments of the invention may be carried into effect. It is to be understood that the following description and figures are provided for the purpose of describing particular embodiments of the invention only and are not intended to be limiting with respect to the description or the claimed invention.

[0042] Figure 1 shows a scatter plot of the expression of E-selectin in endothelial cells within a subject’s tumour and the percentage of CD15s cells within the Ki67+CD8 T cells in theblood of each subject. Responders are indicated by triangles and non-responders are indicated by circles.

[0043] Figure 2 shows the accuracy of a combination of three immune cell signatures for predicting the response to immune checkpoint inhibitor (I Cl) immunotherapy. The three immune cell signatures are: signature 1 - CD15s expression in Ki67+CD8+ T cells (as assessed by the antibody FH6); signature 2 - CD15s expression in Ki67+CD8+ T cells (as assessed by the antibody HECA-452); and signature 3 - CTLA-4+ and FoxP3+ in CD4+ T cells.

[0044] Figure 3 shows dot plots of the expression of responders and non-responders to ICI immunotherapy for each of the three immune cell signatures of Figure 2. The solid lines represent the mean of each group and the dotted lined represent the threshold used for prediction of response.

[0045] Figure 4 shows a receiver operating characteristic (ROC) curve for the sensitivity and specificity of FLIT7 gene expression within melanoma tumour samples for predicting responsiveness of a subject to ICI immunotherapy.

[0046] Figure 5 shows box plots of the FLIT7 gene expression within a melanoma tumour of subjects that respond to, or do not respond to, PD1 ICI immunotherapy.

[0047] Figure 6 shows violin plots of the FLIT7 gene expression within a melanoma tumour from subjects that respond to, or do not respond to, PD1 ICI immunotherapy.

[0048] Figure 7A shows CD15s expression in Ki67+CD8+ T cells using anti-CD15s mAb clone CSLEX. Figure 7B shows CD15s expression in Ki67+CD8+ T cells using anti-CD15s mAb clone CHO-131.

[0049] Figure 8A shows CD15s expression on a pre-treatment sample of peripheral Ki67+ CD8 T cells (n = 16) and the capacity of CD15s to bind E-selectin (E-selectin-Fc chimeric proteins). Figure 8B shows CD15s expression on a pre-treatment sample of peripheral Ki67+ CD8 T cells (n = 16) and the capacity of CD15s to bind P-selectin.

[0050] Figure 9 shows correlation between the density of CD3+ CD8+ T cells in tumours and the matched result for CD15s expression on peripheral blood Ki67+ CD8+ T cells (assessed by flow cytometry).

[0051] Figure 10 shows a summary of the relative TCR CDR3 clonal sharing between tumour infiltrating T cells and matched peripheral T cells.Detailed Description

[0052] Aspects of the present invention, and one or more embodiments of the present invention, are directed to methods, systems and products that may be used for one or more of the following: determining the likelihood that a subject will respond to ICI immunotherapy; screening subjects for responsiveness to ICI immunotherapy; selecting subjects that are likely to respond to ICI immunotherapy; selection of personalised treatment decisions for subjects who are candidates for ICI immunotherapy; identifying patients or subjects who are not likely to respond to ICI immunotherapy; improving the response rate to ICI immunotherapy, reducing the failure rate of ICI immunotherapy; reducing the rate of subjects who experience adverse effects with minimal to no therapeutic benefit; identifying patients or subjects who should be treated with cancer treatments other than ICI immunotherapy; assessing a subject’s responsiveness to ICI immunotherapy after the commencement of treatment; and / or assessing a subject’s responsiveness to ICI immunotherapy after the completion of treatment. Other advantages of some embodiments of the present invention are also disclosed herein, or will be apparent to those skilled in the art.

[0053] The present invention is predicated, at least in part, on the identification of that specific markers expressed by a tumour, either alone or in combination with one or more immune cell signatures, can determine the likelihood that a subject will respond to immune checkpoint inhibition (ICI) immunotherapy, and in particular PD-L1 / PD-1 ICI immunotherapy.

[0054] The present invention provides a method of determining if a subject will respond to ICI immunotherapy for cancer, the method comprising assessing the expression of FLIT7 within a biological sample from the subject, wherein an increased expression of FLIT7 determines an increased likelihood that the subject will respond to ICI immunotherapy.

[0055] The present invention also provides a method of determining if a subject will respond to ICI immunotherapy for cancer, the method comprising assessing the expression of E-selectin within a biological sample from the subject, wherein an increased expression of E- selectin determines an increased likelihood that the subject will respond to ICI immunotherapy.

[0056] The present invention also provides a method of determining if a subject will respond to ICI immunotherapy for cancer, the method comprising assessing the expression of FLIT7 and E-selectin within a biological sample from the subject, wherein an increased expression of FLIT7 and E-selectin determines an increased likelihood that the subject will respond to ICI immunotherapy.

[0057] The present invention also provides a method of determining if a subject will respond to ICI immunotherapy for cancer, the method comprising assessing the expression of one or more of E-selectin and / or FLIT7 within a biological sample from the subject, and assessing one or more of:CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells;CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;FoxP3 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;CD15s expression in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells;Ki67 expression in CD8+ T cells; orCD71 expression in CD8+ T cells, wherein an increase in the expression of one or more of E-selectin and / or FLIT7 in combination with one or more of: an increased expression of CD15s in Ki67+ CD8+ T cells or CD71+ CD8+ T cells prior to ICI immunotherapy; an increased expression of CTLA-4 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells prior to and / or after the immunotherapy; an increased expression of FoxP3 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells prior to immunotherapy; an increased expression of CD15s in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells prior to immunotherapy; and / or a decreased expression of Ki67+ or CD71+ in CD8+ T cells prior to immunotherapy, determines an increased likelihood that the subject will respond to immune checkpoint inhibition immunotherapy.

[0058] MARKERS

[0059] Each of E-selectin, FUT7, CD15s, CD103, CTLA-4, FoxP3, Ki67 and CD71 within their relative population as described above, are collectively referred to for the purpose of this patent application individually as “a marker”, or collectively or as a plurality as “markers”.

[0060] In the context of the application each marker (or the markers in combination) is a molecule which is differentially present in a sample taken from a subject of one phenotypic status (e.g., i.e. being responsive to a therapy) compared with another phenotypic status (e.g., being unresponsive to a therapy). A marker is considered as differentially present between different phenotypic status groups if a quantifiable difference in the marker is present between the two or more phenotypic groups. For example, there could be a difference in the mean or median level or amount of the marker in the two or more phenotypic groups. Therefore,quantifying the markers alone, or in combination, provides an indication of which group the subject belongs to.

[0061] The aforementioned markers can be used to assess subsets of cells within a population. For example, E-selectin expressing CD31+ endothelial cells within a tumour. Of particular relevance to the present invention are immune cell signatures, namely CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells; CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; FoxP3 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; CD15s expression in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells; Ki67 expression in CD8+ T cells; or CD71 expression in CD8+ T cells. Each of these are referred to herein as immune cell signatures and quantifying the markers alone, or in the context of the immune cell signatures, provides an indication of which group the subject belongs to.

[0062] In relation to the present invention, the two phenotypic groups are those likely to respond to ICI immunotherapy, and those unlikely to respond to ICI immunotherapy, preferably PD-1 or PD-L1 ICI immunotherapy.

[0063] It is to be an understood that an increase in the expression of any one or more of the markers may be an increase in the total, median, or mean expression of the marker or may be an increase in the number of cells positive for the marker or may be an increase in the number or percentage of cells positive for the marker within a cellular subset.

[0064] It is demonstrated herein that CD103 is co-expressed with CD15s in specific peripheral T cells populations (see Example 5). Therefore, in some embodiments, CD103 may be used in addition to CD15s. In some embodiments, CD103 may used in place of CD15s. Accordingly, it is to be understood that where CD15s is utilised as a marker for a T cell population (for example CD15s expression in Ki67+ CD8+ T cells) it can be expressly substituted within this specification or the claims with CD103, or may be expressly combined with CD103 (for example CD15s expression and / or CD103 expression in Ki67+ CD8+ T cells).

[0065] E-SELECTIN

[0066] E-selectin is a cell adhesion molecule expressed on endothelial cells, which interacts with sialylated carbohydrates on the surface of immune cells to promote initial tethering and rolling of immune cells, such as leukocytes. With its expression mediated, inter alia, by cytokines, E-selectin is upregulated during inflammation and promotes recruitment of immune cells to sites of inflammation.

[0067] E-selectin (Uniprot accession number P16581 , REFSeq NP_000441 , NM_000450.2) is also known as CD62 antigen-like family member E (CD62E), Endotherlial leukocyte adhesion molecule 1 (ELAM or ELAM-1) and Leukocyte-endothelial cell adhesion molecule 2 (LECAM2). Encoded by the SELE gene, E-selectin is a 610-residue long glycoprotein in humans with homologous sequences in other specifies.

[0068] As indicated above, E-selectin is expressed by endothelial cells which comprise the inner layer of blood vessels. Accordingly, and without being bound by theory, the inventors propose that expression of E-selectin by endothelial cells within the vasculature of a tumour can assist in the adhesion and recruitment of immune cells to the tumour. As such, recruitment of immune cells to the tumour can improve the propensity of the immune system to recognize and attack a tumour. Hence, when immune-suppression is inhibited by ICI immunotherapy the increased ability conferred by E-selectin to recruit immune cells to the tumour can result in improved tumour regression and thus an improved response to ICI therapy. Accordingly, in a preferred embodiment of the invention, the expression of E-selectin is assessed on endothelial cells from a tumour. The co-expression of endothelial cell markers and E-selectin is assessed by a suitable means, such as those discussed herein.

[0069] A range of cell surface markers are known in the art for endothelial cells including CD31 (PECAM-1), CD34, CD105 (Endoglin), CD144 (VE-cad), CD146 (P1 H12, MCAM, MUC18, S-endo-1), CD202b (Tie-2), CD309 (VEGFR-2, KDR, Flk-1), VEGFR-1 (Flk-1) and Tie-1 (see Grant, D., et al. (2021). Comprehensive phenotyping of endothelial cells using flow cytometry 1 : Murine. Cytometry A, 99(3), 251-256.). Accordingly, in some embodiments, one or more of these markers are used to identify endothelial cells. In a preferred embodiment of the present invention, E-selectin is assessed on endothelial cells which express CD31.

[0070] Vascular cells, including endothelial cells, can also be identified using known histology techniques such as hematoxylin and eosin (H&E) staining, elastic staining, Factor VIII staining, Ulex europaeus I agglutinin staining, ERG, and D2-40 (podoplanin) immunohistochemical (IHC) stains among others (see Gonzalez, J., et al. (2023). The usefulness of elastin staining to detect vascular invasion in cancer. International Journal of Molecular Sciences, 24(20), 15264). One of the most widely used method for histological examination of tumours is hematoxylin-eosin (H&E) staining (see Chan, J. K. (2014). The wonderful colors of the hematoxylin-eosin stain in diagnostic surgical pathology. International journal of surgical pathology, 22(1), 12-32). Hematoxylin and eosin staining allows for distinction of many morphological characteristics of a tumour including vascularisation, vasculature structure and the quantification and distribution of tumour infiltrating lymphocyte (TILs). Advantageously, hematoxylin and eosin staining can be easily integrated into theworkflow of pathology laboratories without the need for additional staining protocols (see Chan, J. K. et al. above).

[0071] In some embodiments, the method comprises assessing the protein expression of E-selectin. In some embodiments, this protein expression is assessed in tumour samples, or in isolated cells from the tumour, such as isolated endothelial cells. In some embodiments, the protein expression is assessed in blood samples or fractions thereof, such as serum or plasma. Methods for assessing protein expression are known in the art and are described herein.

[0072] It is to be an understood that an increase in the expression of E-selectin may be an increase in the total, median, or mean expression of E-selectin or may be an increase in the number of E-selectin positive cells, or an increase in the number of E-selectin positive cells within a cellular subset, such as endothelial cells.

[0073] In a preferred embodiment, the protein expression of E-selectin is assessed via immunohistochemistry and the method comprises performing immunohistochemistry to determine the expression of E-selectin. Preferably, the immunohistochemistry is performed on a tumour sample. The immunohistochemistry can be quantification of E-selectin alone or in combination with an endothelial cell marker, such as those discussed herein.

[0074] In some embodiments, the assessment of endothelial cells in a tumour sample is based on morphology via bright-field microscopy histology, preferably using H&E. Such staining and analysis can be performed in combination with E-selectin staining and quantification.

[0075] In some embodiments, the protein expression of E-selectin is assessed via flow cytometry. Such embodiments may be performed on a liquid sample (such as a blood sample, plasma sample or serum sample) or may be performed on a disaggregated tissue sample (such as a disaggregated sample of a tumour).

[0076] Both IHC and flow cytometry allow assessment of total E-selectin in a sample and localization of E-selectin on endothelial cells.

[0077] In some embodiments, the method comprises assessing the gene expression of E- selectin. In some embodiments, this gene expression can be assessed in a biological sample comprising a tumour sample, or in a biological sample comprising isolated cells from the tumour, such as isolated endothelial cells. Methods for isolating endothelial cells are known in the art including van Beijnum, J., et al. (2008). Isolation of endothelial cells from fresh tissues. Nature Protocols, 3(6), 1085-1091. Further, methods for quantification of co-expression of proteins on endothelial cells in cancer tissue are also known in the art including Hua, Y., et al.,(2022). Cancer immunotherapies transition endothelial cells into HEVs that generate TCF1+ T lymphocyte niches through a feed-forward loop. Cancer Cell, 40(12), 1600-1618. e10, and specific methods are described herein.

[0078] In some embodiments, the gene expression is assessed in blood samples, or fractions thereof such as serum or plasma. Methods for assessing gene expression are known in the art and are described herein.

[0079] In some embodiments, a threshold of E-selection of at least 2 times a control standard, at least 2.5 times a controls standard, at least 3 times a control standard, at least 3.5 times a controls standard, at least 4 times a control standard, at least 4.5 times a controls standard, at least 5 times a control standard, at least 5.5 times a controls standard, at least 6 times a control standard, at least 6.5 times a controls standard, at least 7 times a control standard, at least 7.5 times a controls standard, at least 8 times a control standard, at least 8.5 times a controls standard, at least 9 times a control standard, at least 9.5 times a controls standard, at least 10 times a control standard, at least 10.5 times a controls standard, at least 11 times a control standard, at least 11.5 times a controls standard, at least 12 times a control standard, at least 12.5 times a controls standard, at least 13 times a control standard, at least 13.5 times a controls standard, at least 14 times a control standard, at least 14.5 times a controls standard, at least 15 times a control standard, at least 15.5 times a controls standard, at least 16 times a control standard, at least 16.5 times a controls standard, at least 17 times a control standard, at least 17.5 times a controls standard, at least 18 times a control standard, at least 18.5 times a controls standard, at least 19 times a control standard, at least 19.5 times a controls standard, at least 20 times a control standard, at least 20.5 times a controls standard, at least 21 times a control standard, at least 21.5 times a controls standard, at least 22 times a control standard, at least 22.5 times a controls standard, at least 23 times a control standard, at least 23.5 times a controls standard, at least 24 times a control standard, at least 24.5 times a controls standard, at least 25 times a control standard, at least 25.5 times a controls standard, at least 26 times a control standard, at least 26.5 times a controls standard, at least 27 times a control standard, at least 27.5 times a controls standard, at least 28 times a control standard, at least 28.5 times a controls standard, at least 29 times a control standard, at least 29.5 times a controls standard, at least 30 times a control standard, at least 30.5 times a controls standard, at least 31 times a control standard, at least 31.5 times a controls standard, at least 32 times a control standard, at least 32.5 times a controls standard, at least 33 times a control standard, at least 33.5 times a controls standard, at least 34 times a control standard, at least 34.5 times a controls standard, at least 35 times a control standard, at least 35.5 times a controls standard, at least 36 times a control standard, at least 36.5 times a controls standard, at least 37 times a control standard, at least 37.5 times a controls standard, at least 38 times a control standard,at least 38.5 times a controls standard, or at least 39 times a control standard, indicates a likelihood that a patient will respond to ICI immunotherapy. Suitable controls standards are discussed herein, but may include the mean or median of a non-responsive population.

[0080] In some embodiments, a percentage of E-selectin of about 2% or greater, or about 2.5% or greater, or about 3% or greater, or about 3.5% or greater, or about 4% or greater, or about 4.5% or greater, or about 5% or greater, or about 5.5% or greater, or about 6% or greater, or about 6.5% or greater, or about 7% or greater, or about 7.5% or greater, or about 8% or greater, or about 8.5% or greater, or about 9% or greater, or about 9.5% or greater, or about 10% or greater, or about 10.5% or greater, or about 11% or greater, or about 11.5% or greater, or about 12% or greater, or about 12.5% or greater, or about 13% or greater, or about 13.5% or greater, or about 14% or greater, or about 14.5% or greater, or about 15% or greater, or about 15.5% or greater, or about 16% or greater within endothelial cells in the biological sample from the subject determines an increased likelihood that the subject will be responsive to immune checkpoint immunotherapy.

[0081] In a preferred embodiment, a percentage of E-selectin expressing cells of about 7% or greater within endothelial cells in a biological sample from the subject determines an increased likelihood that the subject will be responsive to ICI immunotherapy.

[0082] In some embodiments, a percentage of E-selectin expressing cells of about 7% or greater within endothelial cells in the biological sample from the subject, in combination with one or more of the following immune cell signatures determines an increased likelihood that the subject will be responsive to ICI immunotherapy:• an increased expression of CD15s in Ki67+ CD8+ T cells or CD71+ CD8+ T cells;• an increased expression of CTLA-4 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;• an increased expression of FoxP3 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;• an increased expression of CD15s in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells; or• a decreased expression of Ki67+ or CD71+ in CD8+.In a preferred embodiment, a percentage of E-selectin expressing cells of about 7% or greater within endothelial cells in a biological sample from the subject and a percentage of 6.5% of CD15s+ cells within Ki67+CD8+ T cells determines an increased likelihood that the subject will be responsive to ICI immunotherapy. Preferably, E-selectin is assessed in a biological sample comprising tumour tissue or derived from tumour tissue.

[0083] FUT7

[0084] Fucosyltransferase VII (UniProt accession number Q11130, REFSeq NP_004470, NM_004479.4) is a 342 residue long fucosyltransferase in humans belonging to the a1 , 3 / 4- fucosyltransferase family and is involved in N- and O-fucosylation of a range of proteins. Fucosyltransferase VII is also known as (inter alia) FLIT7, Fucosyltransferase 7 (Alpha (1 ,3) Fucosyltransferase), Galactoside 3-L-Fucosyltransferase, Alpha-(1 ,3)-Fucosyltransferase, Fucosyltransferase VII, FucT-VII, Fuc-TVIl, Alpha (1 ,3) Fucosyltransferase, Selectin-Ligand Synthase, Selectin Ligand Synthase, EC 2.4.1.65, and EC 2.4.1.

[0085] FUT7 has been implicated in the malignancy of a range of cancers including, hepatocellular carcinoma and lung cancer where it is proposed to promote cancer cell adhesion and intravasation (see Li, D., Sun, H., Bai, G., et al. (2018). a-1 ,3-Fucosyltransferase-VII siRNA inhibits the expression of SLex and hepatocarcinoma cell proliferation. International Journal of Molecular Medicine, 42(5), 2700-2708.; and Liang, J. X., Gao, W., & Cai, L. (2017). Fucosyltransferase VII promotes proliferation via the EGFR / AKT / mTOR pathway in A549 cells. OncoTargets and Therapy, 10, 3971-3978).

[0086] CD15 (also known as Lewis X) is a myeloid antigen found in both monocytic and myeloid cell linages. CD15 is a ligand for selectins and as such is involved in transendothelial cellular migration of cells via E-selectin.

[0087] Fucosyltransferase-VII effectively utilises alpha-2, 3-sialyllactosamine to promote the biosynthesis of the sialyl Lewis x tetrasaccharide (CD15s) and directs cell surface expression of CD15s. CD15s expression by immune cells (particularly T cells) indicates an activated phenotype, with CD15s+CD4+ T cells being shown to secrete cytokines, while CD15+CD4+ T cells have a suppressive Treg phenotype. Notably, it has been observed that FUT7 expression can determine CD15s expression in mammalian cell lines (see Natsuka, S. et al. (1994). Molecular cloning of a cDNA encoding a novel human leukocyte alpha-1 , 3- fucosyltransferase capable of synthesizing the sialyl Lewis x determinant. Journal of Biological Chemistry, 269(24), 16789-16794. Erratum in: Journal of Biological Chemistry, 269(32), 20806; and Kimura, H. et al., (1997). Distinct substrate specificities of five human alpha-1 , 3- fucosyltransferases for in vivo synthesis of the sialyl Lewis x and Lewis x epitopes. Biochemical and Biophysical Research Communications, 237(1), 131-137).

[0088] Previous work by the inventors has demonstrated that CD 15s expression within a range of immune cell subsets provide an immune cell signature which is predictive of a subject’s response to ICI immunotherapy (see PCT / AU2023 / 050654 - the entire contents of which is incorporated herein). However, for the first time, the inventors of the present invention have demonstrated that FUT7 expression in tumour-related samples is also predictive of a subject’sresponse to ICI immunotherapy. This is particularly surprising as FLIT7 expression within a tumour-related sample does not exclusively represent the expression of FLIT7 within T cells, and does not determine the relative expression of FLIT7 within specific cell subsets. Further, the level of FLIT7 within a tumour-related sample may be influenced by multiple variables, such as the abundance of tumour infiltrating immune cells, which do not necessarily align with the percentage of CD15s expressing cells within T cell subsets (as discussed in PCT / AU2023 / 050654). Hence, it would not have been expected that total FLIT7 expression within a tumour-related sample (for example a tumour biopsy) would have been an accurate predictor of the immune phenotype of a subject or their likelihood of response to ICI immunotherapy.

[0089] As illustrated in Figure 4, the area under the curve (AUC) for the receiver operating characteristic curve for FLIT7 is 0.732 which is higher than current commercial multi-gene assays, such as nCounter® PanCancer IO 360™ Panel (AUC of 0.638 in the context of melanoma). Further, unlike other tests in the art which rely on multiplexing or quantification of several markers, FUT7 is a single marker thereby providing an easy, quick and low-cost test relative to other tests.

[0090] In some embodiments, the method comprises assessing the gene expression of FUT7. In some embodiments, this gene expression is assessed in a biological sample comprising a tumour sample, or a tumour-associated sample comprising isolated cells from the tumour, such as TILs. In some embodiments, the gene expression is assessed in blood samples, or some fractions thereof such as serum or plasma. Methods for assessing gene expression are known in the art and are described herein.

[0091] In some embodiments, the method comprises assessing the protein expression of FUT7. In some embodiments, this protein expression is assessed in tumour samples, or a tumour-associated sample comprising isolated cells from the tumour, such as TILs. In some embodiments, the protein expression is assessed in blood samples, or fractions thereof such as serum or plasma. Methods for assessing protein expression are known in the art and are described herein.

[0092] It is to be an understood that an increase in the expression of FUT7 may be an increase in the total, median, or mean expression of FUT7 in a sample or in a population, or may be an increase in the number of FUT7 positive cells, or an increase in the number of FUT7 positive cells within a cellular subset, such as TILs.

[0093] Methods are known in the art for determining appropriate thresholds for diagnostic and prognostic markers. Such methods can be used in clinical studies to determine appropriatethresholds and parameters for determining if a subject will be responsive or unresponsive to ICI immunotherapy based on their marker profile. For example, following quantification of the markers in relevant samples, the expression in the samples can be compared to control standards by any appropriate statistical analysis. Methods of statistical analysis are known in the art and the appropriate method will be determined based on many factors including (but not limited to) the number of samples collected, the number of markers being analysed and the nature of the samples ( / .e., paired or unpaired). Appropriate statistical analysis techniques include negative binomial models, pair-wise comparison and Bayesian approaches, and receiver operating characteristics (ROCs). Programs and statistical frameworks for performing such analysis include edgeR, DESeq, baySeq, EBSeq, limma-voom, QPROT and maSigPro.

[0094] Methods for performing statistical analysis are provided in: Yunshun Chen et al. (2021), edgeR: differential analysis of sequence read count data User’s Guide; Bergemann, T.L., and Wilson, J. (2011), Proportion statistics to detect differentially expressed genes: a comparison with log-ratio statistics. BMC Bioinformatics 12, 228; Hardcastle, T.J. and Kelly, K.A. (2010) baySeq: Empirical Bayesian methods for identifying differential expression in sequence count data. BMC Bioinformatics 11 , 422; Leng N. et al., (2013), EBSeq: an empirical Bayes hierarchical model for inference in RNA-seq experiments, Bioinformatics, 29, 8, p.1035- 1043; Law, C.W., et al. (2014), Voom: precision weights unlock linear model analysis tools for RNA-seq read counts. Genome Biol 15, R29 (2014); Conesa A. et al. (2006), maSigPro: a method to identify significantly differential expression profiles in time-course microarray experiments, Bioinformatics, 22, 9, p. 1096-1102; Parodi, S et al. (2008), Not proper ROC curves as new tool for the analysis of differentially expressed genes in microarray experiments. BMC Bioinformatics 9, 410; and Gordon S., et al. (2015) QPROT: Statistical method for testing differential expression using protein-level intensity data in label-free quantitative proteomics. J Proteomics. 3;129:121-126.

[0095] Predictive threshold can be derived based on ROC curves or other statistical analysis of populations. For example, and based on the ROC curve presented in Figure 4, a threshold of 18.5 times the mean expression of FUT7 in a population of non-responsive patients provides for a sensitivity of 67.1% and specificity of 71.4% for predicting a likelihood of responding. Different thresholds may be selected depending on the desire for higher sensitivity or specificity. Further, the threshold may vary depending on the cancer being assessed and the clinical population. Such determinations are known within the art and are part of the common general knowledge of a person of ordinary skill in the art.

[0096] In view of the above, in at least some embodiments, a threshold of FLIT7 of at least18.5 times a control standard (such as the mean expression in a non-responding population) indicates that a subject will respond to ICI immunotherapy.

[0097] In some embodiments, a threshold of FLIT7 of at least 2 times a control standard, at least 2.5 times a controls standard, at least 3 times a control standard, at least 3.5 times a controls standard, at least 4 times a control standard, at least 4.5 times a controls standard, at least 5 times a control standard, at least 5.5 times a controls standard, at least 6 times a control standard, at least 6.5 times a controls standard, at least 7 times a control standard, at least 7.5 times a controls standard, at least 8 times a control standard, at least 8.5 times a controls standard, at least 9 times a control standard, at least 9.5 times a controls standard, at least 10 times a control standard, at least 10.5 times a controls standard, at least 11 times a control standard, at least 11.5 times a controls standard, at least 12 times a control standard, at least12.5 times a controls standard, at least 13 times a control standard, at least 13.5 times a controls standard, at least 14 times a control standard, at least 14.5 times a controls standard, at least 15 times a control standard, at least 15.5 times a controls standard, at least 16 times a control standard, at least 16.5 times a controls standard, at least 17 times a control standard, at least 17.5 times a controls standard, at least 18 times a control standard, at least 18.5 times a controls standard, at least 19 times a control standard, at least 19.5 times a controls standard, at least 20 times a control standard, at least 20.5 times a controls standard, at least 21 times a control standard, at least 21.5 times a controls standard, at least 22 times a control standard, at least 22.5 times a controls standard, at least 23 times a control standard, at least 23.5 times a controls standard, at least 24 times a control standard, at least 24.5 times a controls standard, at least 25 times a control standard, at least 25.5 times a controls standard, at least 26 times a control standard, at least 26.5 times a controls standard, at least 27 times a control standard, at least 27.5 times a controls standard, at least 28 times a control standard, at least 28.5 times a controls standard, at least 29 times a control standard, at least 29.5 times a controls standard, at least 30 times a control standard, at least 30.5 times a controls standard, at least 31 times a control standard, at least 31.5 times a controls standard, at least 32 times a control standard, at least 32.5 times a controls standard, at least 33 times a control standard, at least 33.5 times a controls standard, at least 34 times a control standard, at least 34.5 times a controls standard, at least 35 times a control standard, at least 35.5 times a controls standard, at least 36 times a control standard, at least 36.5 times a controls standard, at least 37 times a control standard, at least 37.5 times a controls standard, at least 38 times a control standard, at least 38.5 times a controls standard, or at least 39 times a control standard, indicates a likelihood that a patient will respond to ICI immunotherapy. Suitable controls standards are discussed herein, but may include the mean or median of a non-responsive population.

[0098] IMMUNE CELL SIGNATURES

[0099] The present inventors have previously shown that analysis of the immune cell subsets CD15s in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; CTLA-4 in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; FoxP3 in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; CD15s in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; Ki67 in CD8+ T cells; or CD71 in CD8+ T cells within a subject can indicate the likelihood of the subject responding to ICI therapy, and in particular PD-1 or PD-L1 inhibitor immunotherapy (see PCT / AU2023 / 050654).

[0100] Specifically, as indicated above, and as disclosed in PCT / AU2023 / 050654, an increased expression of CD15s in Ki67+ CD8+ T cells or CD71+ CD8+ T cells prior to ICI immunotherapy determines an increased likelihood that the subject will respond to immune checkpoint inhibition immunotherapy.

[0101] Further, and as disclosed in PCT / AU2023 / 050654, an increased expression of CTLA-4 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells prior to and / or after ICI immunotherapy determines an increased likelihood that the subject will respond to the ICI immunotherapy.

[0102] Further, and as disclosed in PCT / AU2023 / 050654, an increased expression of FoxP3 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells prior to ICI immunotherapy determines an increased likelihood that the subject will respond to ICI immunotherapy.

[0103] Further, and as disclosed in PCT / AU2023 / 050654, an increased expression of CD15s in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells prior to ICI immunotherapy determines an increased likelihood that the subject will respond to the ICI inhibition immunotherapy.

[0104] Further, and as disclosed in PCT / AU2023 / 050654, a decreased expression of Ki67+ or CD71+ in CD8+ T cells prior to immunotherapy determines an increased likelihood that the subject will respond to the ICI immunotherapy.

[0105] Exemplary methods for analysing these markers alone and in combination are provided in PCT / AU2023 / 050654 and are incorporated herein.

[0106] In the present invention, these immune cell signatures can be assessed in combination with one or both of E-selectin expression and / or FUT7 expression.

[0107] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and one of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells.• E-selectin; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0108] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and two of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.E-selectin; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0109] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and three of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.E-selectin; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0110] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and four of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0111] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and five of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0112] In some embodiments, the methods, kits and systems of the present invention assess the expression of FLIT7 and one of the immune cell signatures. In some embodiments, these combinations are as follows:• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells.• FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• FLIT7; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• FLIT7; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• FLIT7; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0113] In some embodiments, the methods, kits and systems of the present invention assess the expression of FLIT7 and two of the immune cell signatures. In some embodiments, these combinations are as follows:• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA- 4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• FUT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FLIT7; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• FLIT7; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FLIT7; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0114] In some embodiments, the methods, kits and systems of the present invention assess the expression of FLIT7 and three of the immune cell signatures. In some embodiments, these combinations are as follows:• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA- 4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA- 4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA- 4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FUT7; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0115] In some embodiments, the methods, kits and systems of the present invention assess the expression of FUT7 and four of the immune cell signatures. In some embodiments, these combinations are as follows:• FUT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA- 4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• FUT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA- 4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FUT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA- 4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FUT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0116] In some embodiments, the methods, kits and systems of the present invention assess the expression of FLIT7 and five of the immune cell signatures. In some embodiments, these combinations are as follows:• FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA- 4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0117] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and FLIT7 and one of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells.• E-selectin and FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin and FLIT7; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71 + CD4- CD8- T cells.• E-selectin and FLIT7; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0118] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and FLIT7 and two of the immune cell signatures. In some embodiments, these combinations are as follows:E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin and FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin and FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FUT7; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin and FUT7; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FUT7; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71 + CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0119] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and FUT7 and three of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin and FUT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells.• E-selectin and FUT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin and FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FUT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FUT7; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0120] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and FUT7 and four of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin and FUT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FLIT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.• E-selectin and FLIT7; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0121] In some embodiments, the methods, kits and systems of the present invention assess the expression of E-selectin and FUT7 and five of the immune cell signatures. In some embodiments, these combinations are as follows:• E-selectin and FUT7; and CD15s expression in Ki67+ CD8+ T cells or CD71+ CD8+ T cells; and CTLA-4 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and FoxP3 expression in Ki67+ CD4+ T cells or CD71+ CD4+ T cells; and CD15s expression in Ki67+ CD4- CD8- T cells or CD71+ CD4- CD8- T cells; and Ki67+ expression in CD8+ T cells or CD71 marker in CD8+ T cells.

[0122] In some embodiments, the Ki67+ CD8+ T cells comprise Ki67+ CD8+ CD3+ T cells; the Ki67+ CD8+ T cells comprise Ki67+ CD8+ CD3+ CD28+ T cells; the Ki67+ CD4+ T cells comprise Ki67+ CD4+ CD8- T cells, or Ki67+ CD4+ CD3+ T cells.

[0123] FUT7 (protein or gene expression) and E-selectin can be assessed in biological samples described herein.

[0124] It is to be understood that when FoxP3+ is disclosed as being expressed in a cell subpopulation, this may be considered as being interchangeable with FoxP3+ and CTLA-4+. Further, where CTLA-4+ is disclosed as being expressed in a cell subpopulation, this may beconsidered as being interchangeable with CTLA-4+ and FoxP3+. The inventors have found that quantification of one of these markers is essentially equivalent to assessing both markers together.

[0125] When assessing multiple markers in combination, the specificity and sensitivity of each marker can be assessed in combination, or separately and then combined. In some embodiments, the threshold of one or more of E-selectin and FLIT7 and one or more of the immune cell signatures described here is determined separately (for example based on an ROC curve or relative to a control standard for each marker) for an individual and then a cumulative score for that individual is calculated based on the sum of each marker or immune cell signature.

[0126] As illustrated in Figure 1 , when the percentage of CD31+ endothelial cells expressing E-selectin was quantified and combined with the immune cell signature of CD15s expression in Ki67+CD8+ T cells, the overall predictive accuracy was 100%.

[0127] In comparison, and as illustrated in Figure 2, the combination of immune cell signatures comprising: signature 1 - CD15s expression in Ki67+CD8+ T cells (as assessed by the antibody FH6); signature 2 - CD15s expression in Ki67+CD8+ T cells (as assessed by the antibody HECA-452); and signature 3 - CTLA-4+FoxP3+CD4+ T cells, provided a predictive accuracy of 88.5% (with a specificity and sensitivity of 88.5%) when an individual had a percentage of cells at or above a designated threshold for one or more the these signatures.

[0128] While the figures illustrate the combination of E-selectin with one immune cell signature, it is envisaged that combinations with other immune cell signatures disclosed herein will provide for improved accuracy, or one or more of improved sensitivity or specificity.

[0129] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of about 2% or greater, 3% or greater, 4% or greater, 5% or greater, 6% or greater, 7% or greater, 8% or greater, 9% or greater, or 10% or greater determined using the FH6 mAb in a sample from the subject prior to ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 3.5% or greater, determined using the FH6 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 5.3% or greater, determined using the FH6 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 6% or greater,determined using the FH6 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy.

[0130] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of about 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, or 2% or less determined using the FH6 mAb in a sample from the subject prior to ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 5.3% or less, determined using the FH6 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 3.5% or less, determined using the FH6 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy.

[0131] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of 7% or greater, 8% or greater, 9% or greater, 10% or greater, 11% or greater, 12% or greater, 13% or greater, 14% or greater, 15% or greater, 16% or greater, 17% or greater, 18% or greater, 19% or greater, 20% or greater, or 21% or greater determined using the HECA-452 mAb in a sample from the subject prior to ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 10.3% or greater, determined using the HECA-452 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 18% or greater, determined using the HECA-452 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 19.2% or greater, determined using the HECA-452 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy.

[0132] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of 22% or less, 20% or less, 18% or less, 16% or less, 14% or less, 13% or less, 12% or less, 11 % or less, 10% or less, 9% or less, or 8% or less, determined using the HECA- 452 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 19.2% or less,determined using the HECA-452 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 10.3% or less, determined using the HECA-452 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy.

[0133] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of 17% or greater, 18% or greater, 19% or greater, 20% or greater, 21 % or greater, 22% or greater, 23% or greater, 24% or greater, 25% or greater, 26% or greater, 27% or greater, 28% or greater, 29% or greater, or 30% or greater, determined using the CHO131 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 19.6% or greater, determined using the CHO131 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 28.6% or greater, determined using the CHO131 mAb in a sample from the subject prior to immunotherapy, is indicative that the subject is responsive to ICI immunotherapy.

[0134] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of 30% or less, 29% or less, 28% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21 % or less, 20% or less, 19% or less, or 18% or less, determined using the CHO131 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 28.6% or less, determined using the CHO131 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 19.6% or less, determined using the CHO131 mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy.

[0135] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of 18% or greater, 19% or greater, 20% or greater, 21 % or greater, 22% or greater, 23% or greater, 24% or greater, 25% or greater, 26% or greater, 27% or greater, 28% or greater, 29% or greater, or 30% or greater determined using the CSLEX mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 20.2% or greater, determined using the CSLEX mAb in asample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 28.8% or greater, determined using the CSLEX mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is responsive to ICI immunotherapy.

[0136] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of 30% or less, 29% or less, 28% or less, 27% or less, 26% or less, 25% or less, 24% or less, 23% or less, 22% or less, 21% or less, 20% or less, 19% or less, or 18% or less, determined using the CSLEX mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 28.8% or less, determined using the CSLEX mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive not to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD8+ T cells of approximately 20.2% or less, determined using the CSLEX mAb in a sample from the subject prior to ICI immunotherapy, is indicative that the subject is not responsive not to ICI immunotherapy.

[0137] In some embodiments, a percentage of cells expressing the CTLA-4 marker in Ki67+ CD4+ T cells of 42% or greater in a sample from the subject prior to and / or after the immunotherapy is indicative that the subject is responsive to ICI immunotherapy.

[0138] In some embodiments, a percentage of cells expressing the CTLA-4 marker in Ki67+ CD4+ T cells of 48% or greater, 49% or greater, 50% or greater, 51% or greater, 52% or greater, or 53% or greater, or 54% or greater, or 55% or greater, or 56 or greater, 57% or greater, 58% or greater, 59% or greater, 60% or greater, 61% or greater, or 62% or greater in a sample from the subject prior to and / or after the ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CTLA-4 marker in Ki67+ CD4+ T cells of approximately 50.1% or greater, in a sample from the subject prior to and / or after the ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CTLA-4 marker in Ki67+ CD4+ T cells of approximately 60.4% or greater, in a sample from the subject prior to and / or after the ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy.

[0139] In some embodiments, a percentage of cells expressing the CTLA-4 marker in Ki67+ CD4+ T cells of 62% or less, 61% or less, 60% or less, 59% or less, 58% or less, 57%or less, 56% or less, 55% or less, 54% or less, 53% or less, 52% or less, 51 % or less, 50% or less, 49% or less, 48% or less, 49% or less, 50% or less, 51 % or less, or 52% or less, in a sample from the subject prior to and / or after the ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CTLA-4 marker in Ki67+ CD4+ T cells of approximately 50.1% or less, in a sample from the subject prior to and / or after the ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CTLA-4 marker in Ki67+ CD4+ T cells of approximately 60.4% or less, in a sample from the subject prior to and / or after the ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy.

[0140] In some embodiments, a percentage of cells expressing the FoxP3 marker in Ki67+ CD4+ T cells of 28% or greater, 29% or greater, 30% or greater, 31 % or greater, 32% or greater, 33% or greater, 34% or greater, 35% or greater, 36% or greater, 37% or greater, or 38% or greater in a sample from the subject prior to ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the FoxP3 marker in Ki67+ CD4+ T cells of approximately 29.4% or greater in a sample from the subject prior to ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the FoxP3 marker in Ki67+ CD4+ T cells of approximately 36.9% or greater in a sample from the subject prior to ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy.

[0141] In some embodiments, a percentage of cells expressing the FoxP3 marker in Ki67+ CD4+ T cells of 38% or less, 37% or less, 36% or less, 35% or less, 34% or less, 33% or less, 32% or less, 31 % or less, 30% or less, 29% or less, or 28% or less in a sample from the subject prior to ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the FoxP3 marker in Ki67+ CD4+ T cells of approximately 29.4% or less in a sample from the subject prior to ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the FoxP3 marker in Ki67+ CD4+ T cells of approximately 36.9% or less in a sample from the subject prior to ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy.

[0142] In some embodiments, a percentage of cells expressing the FoxP3 marker and the CTLA-4 marker in CD4+ T cells of 28% or greater, 29% or greater, 30% or greater, 31 % or greater, 32% or greater, 33% or greater, 34% or greater, 35% or greater, 36% or greater, 37% or greater, or 38% or greater in a sample from the subject is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing theFoxP3 marker and the CTLA-4 marker in CD4+ T cells of approximately 32% or greater in a sample from the subject is indicative that the subject is responsive to ICI immunotherapy.

[0143] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD4- CD8- T cells of 1 % or greater, 2% or greater, 3% or greater, 4% or greater, or 5% or greater, 6% or greater, 7% or greater, or 8% or greater in a sample from the subject prior to ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD4- CD8- T cells of approximately 3.2% or greater, in a sample from the subject prior to ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD4- CD8- T cells of approximately 4.4% or greater, in a sample from the subject prior to ICI immunotherapy is indicative that the subject is responsive to ICI immunotherapy.

[0144] In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD4- CD8- T cells of 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less or 1% or less, in a sample from the subject prior to ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD4- CD8- T cells of approximately 3.2% or less, in a sample from the subject prior to ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy. In some embodiments, a percentage of cells expressing the CD15s marker in Ki67+ CD4- CD8- T cells of approximately 4.4% or less, in a sample from the subject prior to ICI immunotherapy is indicative that the subject is not responsive to ICI immunotherapy.

[0145] In some embodiments, a percentage of cells expressing the Ki67+ marker in CD8+ T cells of 1.5% or greater, or 2.8% or greater, in a sample from the subject prior to immunotherapy is indicative that the subject is not responsive to ICI immunotherapy.

[0146] In some embodiments, a percentage of cells expressing the Ki67+ marker in CD8+ T cells of 0.5% or greater, 1% or greater, 2.0% or greater, 2.5% or greater, 3.0% or greater, 3.5% or greater, or 4% or greater in a sample from the subject prior to immunotherapy is indicative that the subject is not responsive to ICI immunotherapy.

[0147] In some embodiments, a percentage of cells expressing the Ki67+ marker in CD8+ T cells of 2.8% or less of 1.5% or less in a sample from the subject prior to immunotherapy is indicative that the subject is responsive to ICI immunotherapy

[0148] In some embodiments, a percentage of cells expressing the Ki67+ marker in CD8+ T cells of 4% or less, 3.5% or less, 3.0% or less, 2.5% or less, 2.0% or less, 1% or less, or 0.5% or less, in a sample from the subject prior to immunotherapy is indicative that the subject is responsive to ICI immunotherapy.

[0149] BIOLOGICAL SAMPLES

[0150] The appropriate sample for assessing one of the markers described herein can be determined by a person skilled in the art and will be determined by factors such as tumour type and location.

[0151] In some embodiments E-selectin and / or FUT7 are assessed in a first biological sample and the immune cell signatures are assessed in a second biological sample.

[0152] In some embodiments, the first biological sample is a tumour sample, or a tumour- associated sample. In some embodiments, the first biological sample is tumour infiltrating lymphocytes. In some embodiments, the second biological sample is a vascular sample, or a lymphatic sample, or a blood sample, or a serum sample, or a plasma sample, or a lymph sample, or tumour infiltrating lymphocytes (TILs).

[0153] The means of assessing expression of the markers will depend on the sample being analysed and if gene or protein expression is being assessed.

[0154] Methods for quantifying proteins and genes in solid and liquid samples are provided below. In liquid samples, such as a blood sample, or a lymphatic sample, or a serum sample, or a plasma sample, or a lymph sample it is preferrable to use rapid multiplex assessment processes such as flow cytometry. In some embodiments, the expression of one or more of CD15s, CD71 , CTLA4, FoxP3, Ki67, CD8, CD4, CD3, or CD28 is assessed by flow cytometry.

[0155] In solid samples, such as tumour samples, techniques such as immunohistochemistry can be used to assess protein expression, and co-expression of proteins, in situ. In some embodiments, protein expression of E-selectin is assessed via immunohistochemistry. In some embodiments, protein expression of FUT7 is assessed via immunohistochemistry.

[0156] In some embodiments, where gene expression is assessed, the expression of one or more of the markers (such as E-selectin and / or FUT7) is assessed via polymerase chain reaction (PGR).

[0157] METHODS OF QUANTIFICATION PROTEIN

[0158] Specific methods of preforming protein analysis in cancer cells are known in the art, including Sanguedolce, F., & Zanelli, M. (2022). Immunohistochemistry in the diagnosis of primary and secondary cancers. In N. Rezaei (Ed.), Handbook of cancer and immunology (pp. 129-1). Springer.

[0159] Further methods for performing analysis of protein are known, with some briefly discussed below.

[0160] Immunohistochemistry / immunostaining

[0161] One of the most common techniques for protein quantification and localisation is immunohistochemistry. This technique comprises fixing and mounting tissue, which is subsequently sectioned prior to incubation with primary antibodies against the protein of interest. These primary antibodies are either directly labelled or can be detected by labelled secondary antibodies. Common labels include enzymes (such as horseradish peroxidase), fluorescent-tags, radio labels or conjugates such as biotin. The label can then be detected and used to identify the location and / or quantity of the protein of interest.

[0162] Methods for performing IHC are known in the art, and include: Schlederer M, et al. (2014) Reliable Quantification of Protein Expression and Cellular Localization in Histological Sections. PLoS ONE 9(7): e100822. and Goldstein, M. and Watkins, S. (2008), Immunohistochemistry. Current Protocols in Molecular Biology, 81 : 14.6.1-14.6.23.

[0163] In some embodiments, the markers of the present application are assessed using immunohistochemistry. In some embodiments, markers in a tumour sample are assessed using immunohistochemistry. In some embodiments, one or more of FLIT7 or E-selectin is assessed using immunohistochemistry.

[0164] Flow cytometry

[0165] One preferred technique for quantifying protein expression on samples, particularly liquid sample such as blood, serum, lymph and plasma, is flow cytometry. Flow cytometry uses fluorescent immunostaining of cells (which can be both extracellular and intracellular staining) followed by injection of stained samples into a flow cytometer. The flow cytometer utilises fluidics to align cells and progress them in single-file through a laser and detection system which quantifies characteristics of each cell including fluorescent intensity, cell size and density and number of fluorescent cells within a cell population. Flow cytometry allows multiplexing of many proteins on a single cell based on different excitation and output wavelengths of the fluorophores on the labelled detection antibodies.

[0166] General methods are known in the art for performing flow cytometry including El- Hajjar, L., Ali Ahmad, F., and Nasr, R. (2023). A guide to flow cytometry: Components, basic principles, experimental design, and cancer research applications. Current Protocols, 3(3), e721. Further methods for phenotyping endothelial cells by flow cytometry are also known including Grant, D., et al., (2021). Comprehensive phenotyping of endothelial cells using flow cytometry 1: Murine. Cytometry A, 99(3), 251-256.

[0167] Alternative methods for protein quantification include: High-Performance Liquid Chromatography (HPLC), Liquid Chromatography-mass spectrometry (LC / MS), Enzyme- Linked Immunosorbent Assay (ELISA), protein immunoprecipitation, immunoelectrophoresis, SDS-page and western blot. Protocols are known in the art for performing these techniques including: Mitulovic G. and Mechtler K (2006), HPLC techniques for proteomics analysis — a short overview of latest developments, Briefings in Functional Genomics, 5, 4, p. 249-260; Gao Z, et al. (2009) Identification and Verification of the Main Differentially Expressed Proteins in Gastric Cancer via iTRAQ Combined with Liquid Chromatography-Mass Spectrometry. Analytical Cellular Pathology (Amsterdam), 2019:5310684; Lome F et al. (2001), Whole cell ELISA for detection of tumor antigen expression in tumor samples, Journal of Immunological Methods, 258, 1-2, p. 47-53, ISSN 0022-1759; Kim, S. M., et al. (2017). Two different protein expression profiles of oral squamous cell carcinoma analyzed by immunoprecipitation high- performance liquid chromatography. World journal of surgical oncology, 15(1), 151; Osborne C, Brooks SA. (2006) SDS-PAGE and Western blotting to detect proteins and glycoproteins of interest in breast cancer research. Methods in Molecular Medicine, 120: p. 217-29.

[0168] Techniques are also known for simultaneous quantification of gene (mRNA) and protein expression, including REAP-seq and CITE-seq. Methodology in the art includes: Peterson V. et al. (2017) Multiplexed quantification of proteins and transcripts in single cells. Nature Biotechnology, 35, 936-939; and Stoeckius M, et al. (2017), Simultaneous epitope and transcriptome measurement in single cells. Nature Methods, 14(9): p. 865-868. Epub 2017 Jul 31.

[0169] Further, methods are known for multiplex analysis of proteins within a sample such as Van Gool, A. etal., (2020). Analytical techniques for multiplex analysis of protein biomarkers. Expert Review of Proteomics, 17(4), 257-273.

[0170] Antibodies for assessing the protein expression of the markers are known in the art and can be used for IHC or flow cytometry. Exemplary antibodies include: anti-E-selectin antibody BBA18; anti-CD31 antibody MA5-13188; anti-CD15s antibodies FH6, HECA-452, CHO131, and CSLEX; anti-CD71 antibody M-A712; anti-FoxP3 antibody PCH101; anti-CTLA-4 antibody 14D3; anti-Ki67 antibody B56; anti-CD8 antibody SK1 , anti-CD4 antibody SK3; and anti-CD3 antibody SK7.METH0DS OF RNA ISOLATION AND GENE QUANTIFICATION

[0171] Various methods for RNA isolation from a biological sample are well known in the field of biological sciences and the appropriate method will be selected by one skilled in the art in view of their specific requirements and constraints.

[0172] There are at least three major techniques which are extensively used in the art for RNA extraction. These are: organic extraction, such as phenol-Guanidine Isothiocyanate (GITC)-based solutions, silica-membrane based spin column technology, and paramagnetic particle technology.

[0173] Each of the above techniques may have various limitations and advantages. For example, phenol-GITC-based organic extraction results in isolated RNAs which are frequently contaminated with proteins and other cellular materials, as well as organic solvents such as phenol-chloroform, salts and ethanol. Additionally, such methods utilise toxic and potentially toxic reagents and therefore require safety precautions (i.e., the use of fume hoods). In comparison, Silica column and paramagnetic particle-based RNA isolation systems do not require the use of toxic organic solvents. Furthermore, such systems are typically quicker and less complex than organic solvent-based assays as well as more efficient, lower in cost, and typically can isolate higher yields of total intact RNA. In addition, the isolated RNA has relatively lower levels of contamination by cellular material and detritus such as protein. However, these methods can generate samples with significant levels of genomic DNA contamination.

[0174] A variety of commercially available kits are known in the art for RNA isolation such as AxyPrep Multisource Total RNA Miniprep (Axygen), RNeasy® Mini (Qiagen), EasySpin (Citomed), llustra RNAspin Mini RNA Isolation Kit (GE), TRIzol® and TRIzol plus RNA Purification System (Invitrogen) and E.Z.N.A.™ Total RNA Kit II (omega bio-tek). A comparison of the advantages, disadvantages and performance of each of these kits can be found in Tavares, L., et al. (2011), Comparison of different methods for DNA-free RNA isolation from SK-N-MC neuroblastoma, BMC Res Notes; 4, 3. Alternatively, protocols for RNA isolation are provided in Liu and Harada (2013), RNA Isolation from Mammalian Samples, Current Protocols in Molecular Biology; 103:4.16.1-4.16.16

[0175] Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)

[0176] RT-PCR is one of the most sensitive techniques for quantifying specific nucleic acid samples.

[0177] To perform RT-PCR, RNA is extracted and purified from a biological sample. This RNA is then reverse transcribed by a retroviral reverse transcriptase and converted to complementary DNA (cDNA). The cDNA is then combined in a buffer with a thermal stable DNA polymerase, deoxynucleotides and a forward and reverse primer.

[0178] The solution is then thermally cycled to allow denaturing (separation) of double stranded DNA, annealing of primers to the separated DNA strands and extension of new DNA copies via the DNA polymerase. This process is repeated to amplify the strand of sequence between the forward and reverse primer providing short DNA sequences known as amplicons.

[0179] The amplicons can then be visualised via gel electrophoresis, or the amplification can be monitored in real-time using inter-calculating dyes such as SYBR Green or by using fluorescent reporter probe systems such as taqman probes. Realtime PCR allows for the quantification of initial concentration of RNA by assessing the cycle-threshold (CT) value, with lower CT values indicating higher initial nucleic acid concentration.

[0180] Example protocols for performing RT PCR are provided in Mitchel, J. (2002) RT- PCR Protocols. Methods in Molecular Biology, Vol. 193; Karlin-Neumann, G., & Bizouarn, F. (Eds.). (2018). Digital PCR: Methods and protocols. Humana.

[0181] In Situ Hybridization

[0182] In situ hybridisation allows for identification and localisation of nucleic acids (such as RNA) within a biological sample. As such - unlike some other techniques - in situ hybridisation can indicate tissue distribution of nucleic acids within a sample, rather than just identifying the presence of, or quantifying the expression of, nucleic acids.

[0183] In situ hybridization utilises hybridisation between target nucleic acids (such as mRNA) with an oligonucleotide (e.g. cDNA) or RNA probe (riboprobe). Each probe is coupled with a detection moiety such as a radiolabel, enzyme or fluorophore. Hybridisation between the complementary probe nucleic acid sequence and the target sequence can then be detected or visualised to identify the locations and amount of the target nucleotide.

[0184] Techniques for performing in situ hybridisation are known in the art. For example: Henley S. R. et al. (2021), RNA in situ hybridization for human papillomavirus testing in oropharyngeal squamous cell carcinoma on a routine clinical diagnostic platform. Journal of Oral Pathology & Medicine; 50, 1 , p. 68-75.

[0185] Typically, tissue samples are taken, before being processed (e.g. frozen or paraffin embedded) and sectioned. The sections are mounted and treated with enzymes, such asproteinase K, to disrupt the cellular membrane and allow access to the internal nucleic acids. The labelled nucleic acid probe is then incubated with the sectioned tissue, followed by washing and detection of the label. The strength of the signal can be indicative of the quantity of the assayed nucleic acid in the tissue sample, while the specific location of the nucleic acid in the tissue sample can also be visualised, for example by light, fluorescent or electron microscopy.

[0186] Nuclease Protection Assays

[0187] Techniques for performing ribonuclease protection assays are known in the art, including: Henttu P. (2001), Quantification ofmRNA levels using ribonuclease protection assay. Methods in Molecular Biology; 169, p. 65-79.

[0188] This method relies on solution-phase hybridization between the mRNA molecule of interest and a radiolabelled complementary RNA molecule. Specifically, a single-stranded radioactive DNA or RNA probe, which includes at least some nucleotides that are complementary to the mRNA being analysed, is mixed with isolated RNA samples. The probe is annealed to the target mRNA by base-pairing, and the regions of the probe that are complementary to the target mRNA form double-stranded nucleic acids, while the noncomplementary regions of the probe remain single-stranded. The annealed mixture is then subjected to digestion with an enzyme specific for single-stranded DNA or RNA (for example S1 nuclease, RNase A, or RNase T1). The double-stranded annealed areas resist digestion, while all the single-stranded noncomplementary parts of the probe are digested away. In essence, areas in the probe that anneal to the mRNA are “protected” from digestion by the nucleases. The surviving, undigested parts of the probe can then be analysed by electrophoresis through an agarose or polyacrylamide gel. The amount of radiolabelled probe resistant to digestion is proportional to the amount of target mRNA in the sample.

[0189] Northern A nalysis

[0190] RNA samples are purified from tissue or cell samples before being separated by size via gel (e.g. agarose gel) electrophoresis under denaturing conditions (such as in the presence of formaldehyde or glyoxal / DMSO). The size separated RNA is then transferred to a membrane (such as a nitrocellulous or nylon membrane). This transfer may be done via techniques such as; capillary transfer, vacuum transfer, salt gradient or electrophoretic transfer. The RNA is then cross-linked or fixed to the membrane before being hybridized with a specific labelled probe. The probes can be labelled with radio-labels or fluorescent labels. Subsequently, the labelled membrane is visualised through processes such as autoradiography and the probe-bound RNA can be identified.

[0191] Northerner blotting allows for analysis and quantification based on the transcript size. This permits analysis of different expressed variants of a gene.

[0192] Examples of northern blot techniques are provided in Brown, T et al (2004), Analysis of RNA by Northern and Slot Blot Hybridization, Current Protocols in Molecular Biology; 4.9.1-4.9.19.

[0193] RNA microarray

[0194] Microarrays utilise a series of specific oligonucleotide probes immobilised in an array to a solid support. The probes at each specific location have a known sequence which will specifically hybridise to a complementary nucleic acid.

[0195] A nucleic acid sample for microarray analysis is typically prepared by reversetranscribing isolated mRNA from a sample to create cDNA. During the reverse transcription, a fluorescent label can be added to the generated cDNA or may be added upon completion.

[0196] The labelled cDNA, from the sample to be analysed, is then incubated with the immobilised probes on the microarray under high-stringency conditions, before unhybridized cDNA is removed. The fluorescence at each location is then quantified and indicates the amount of hybridised sample nucleic acid which is complementary to each immobilised probe. Because the specific sequences of the immobilised probes are known for each location in the array, the degree of fluorescence at each location is indicative of the level of each complementary nucleic acid sequence in the sample, thereby permitting the quantification of the level of the mRNA for each analysed gene in the original sample.

[0197] A range of commercially available microarray chips are known in the art including those manufacture by Affymetrix, Illumina, Agilent, Applied Microarrays, Eppendorf and Arrayit. Further, microarray protocols are known in the art including those provided by the National Human Genome Research Institute (https: / / research.nhgri.nih.gov / microarray / protocols. shtml), and Grant, G.R., et al. (2007), Analysis and Management of Microarray Gene Expression Data. Current Protocols in Molecular Biology, 77: 19.6.1-19.6.30. https: / / doi.org / 10.1002 / 0471142727.mb1906s77.

[0198] RNA sequencing (RNA-Seq)

[0199] RNA-Seq utilises next-generation sequencing platforms to analyse the sequence and expression of RNAs within cells at any given time. RNA-Seq can be used to analyse total RNA, micro RNA, transfer RNA and mRNA. Messenger RNAs are reverse- transcribed into cDNA, before adapters are ligated to each end of the cDNAs. Sequencing can be done eitherunidirectional (single-end sequencing) or bidirectional (paired-end sequencing) with the sequences aligned in silico to a reference genome database or assembled to obtain de novo transcripts. Quantification of RNA is performed by counting the number of reads that map to each locus of the reference genome. A range of tools can be used to quantify counts including HTSeq, FeatureCounts, Rcount, Maxcounts, FIXSEQ, Cuffquant, Sailfist and Kallisto.

[0200] Differential expression between two tissues (such as lesion and non-lesion tissue) can be calculated by known tools including DESeq, edgeR and Voom+limma.

[0201] Protocols for performing RNA-Seq and analysing data are known in the art, including: Kukurba K. R. and Montgomery S. B. (2015), RNA Sequencing and Analysis. Cold Spring Harbor Protocols, 11 : 951-969; and Costa-Silva J, et al. (2017), RNA-Seq differential expression analysis: An extended review and a software tool. PLoS ONE 12(12): e0190152. https: / / doi.Org / 10.1371 / journal.pone.0190152.

[0202] CONTROL STANDARDS

[0203] To determine which phenotypic group a subject belongs to, or has a higher likelihood of belonging to, the concentration or quantity of one or more of the disclosed markers is assess in a biological sample from the subject and, in at least some embodiments, compared to one or more control standards.

[0204] Accordingly, in some embodiments the expression of one or more of: E-selectin (preferably in tumour-associated endothelial cells), FLIT7 in a tumour sample, CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells; CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; FoxP3 marker in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; CD15s marker in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells; Ki67 marker in CD8+ T cells; or CD71 marker in CD8+ T cells is compared to a control standard.

[0205] Such control standards provide a benchmark which allow for the assessment of the relative quantity or concentration of a marker in the biological sample, thereby allowing the determination of the which phenotypic group a subject belongs to. Typically, the control standard is pre-determined and based on obtained information of a population or individual. In some embodiments, the control standard is based on the quantity or concentration of one or more the markers in a population of subjects which did not respond to I Cl immunotherapy. In some embodiments, the control standard is based on the quantity or concentration of one or more of the markers in a population of subjects which did respond to ICI immunotherapy. In some embodiments, the control standard is based on the quantity or concentration of one or more markers in a population of subjects which did respond to ICI immunotherapy, but had atleast one or more adverse or unwanted effects, or had severe adverse or unwanted effects. In some embodiments, the control standard is based on the quantity or concentration of one or more markers in a population of subjects which did respond to ICI immunotherapy and did not experience an adverse or unwanted effect, or had minimal adverse or unwanted effects.

[0206] In some embodiments, the control standard is the median quantity or concentration of the one or more markers in one of the above identified populations. In some embodiments, the control standard is the mean quantity or concentration of the one or more markers in one of the above identified populations.

[0207] In some embodiments the control standard may indicate the 5thpercentile, 10thpercentile, 15thpercentile, 20thpercentile, 25thpercentile, 30thpercentile, 35thpercentile, 40thpercentile, 45thpercentile, 50thpercentile, 55thpercentile, 60thpercentile, 65thpercentile, 70thpercentile, 75thpercentile, 80thpercentile, 85thpercentile, 90thpercentile, 95thpercentile in a population that does respond to ICI immunotherapy.

[0208] In some embodiments the control standard may indicate the 5thpercentile, 10thpercentile, 15thpercentile, 20thpercentile, 25thpercentile, 30thpercentile, 35thpercentile, 40thpercentile, 45thpercentile, 50thpercentile, 55thpercentile, 60thpercentile, 65thpercentile, 70thpercentile, 75thpercentile, 80thpercentile, 85thpercentile, 90thpercentile, 95thpercentile in a population that is unresponsive to ICI immunotherapy.

[0209] In some embodiments, where the one of more markers is lower in the population which responds to ICI treatment (e.g., Ki67% expression in CD8+ T cells) the control standard may indicate the 5thpercentile, 10thpercentile, 15thpercentile, 20thpercentile, 25thpercentile,30thpercentile, 35thpercentile, 40thpercentile, 45thpercentile, 50thpercentile, 55thpercentile,60thpercentile, 65thpercentile, 70thpercentile, 75thpercentile, 80thpercentile, 85thpercentile,90thpercentile, 95thpercentile, the median, or the mean of the concentration or quantity of the one or more markers in a non-responding population, and a concentration or quantity of the marker below the control standard indicates a higher likelihood of responding to ICI immunotherapy.

[0210] In some embodiments, where the one of more markers is lower in the population which responds to ICI treatment (e.g., Ki67% expression in CD8+ T cells) the control standard may indicate the 5thpercentile, 10thpercentile, 15thpercentile, 20thpercentile, 25thpercentile,30thpercentile, 35thpercentile, 40thpercentile, 45thpercentile, 50thpercentile, 55thpercentile,60thpercentile, 65thpercentile, 70thpercentile, 75thpercentile, 80thpercentile, 85thpercentile,90thpercentile, 95thpercentile, the median, or the mean of the concentration or quantity of theone or more markers in a responding population, and a concentration or quantity of the marker below the control standard indicates a higher likelihood of responding to ICI immunotherapy.

[0211] In some embodiments, where the one of more markers is higher in the population which responds to ICI treatment the control standard may indicate the 5thpercentile, 10thpercentile, 15thpercentile, 20thpercentile, 25thpercentile, 30thpercentile, 35thpercentile, 40thpercentile, 45thpercentile, 50thpercentile, 55thpercentile, 60thpercentile, 65thpercentile, 70thpercentile, 75thpercentile, 80thpercentile, 85thpercentile, 90thpercentile, 95thpercentile, the median, or the mean of the concentration or quantity of the one or more markers in a nonresponding population, and a concentration or quantity of the marker above the control standard indicates a higher likelihood of responding to ICI immunotherapy.

[0212] In some embodiments, where the one of more markers is higher in the population which responds to ICI treatment the control standard may indicate the 5thpercentile, 10thpercentile, 15thpercentile, 20thpercentile, 25thpercentile, 30thpercentile, 35thpercentile, 40thpercentile, 45thpercentile, 50thpercentile, 55thpercentile, 60thpercentile, 65thpercentile, 70thpercentile, 75thpercentile, 80thpercentile, 85thpercentile, 90thpercentile, 95thpercentile, the median, or the mean of the concentration or quantity of the one or more markers in a responding population, and a concentration or quantity of the marker above the control standard indicates a higher likelihood of responding to ICI immunotherapy.

[0213] IMMUNE CHECKPOINT INHIBITOR THERAPY

[0214] Immune checkpoint inhibitor immunotherapy relates to therapies aimed at inhibiting or blocking the function of an immunosuppressive immune checkpoint. Currently known immune checkpoints include PD-1 / PD-L1 , CTLA-4 / B7, A2A, B7-H3, B7-H4, BTLA, IDO, KIR, LAG3, NOX2, TIM-3, VISTA, SIGLEC7 and SIGLEC9.

[0215] Currently approved ICI therapies and their indications are known in the art (see for example Wang, D. et al. (2023), Immune Checkpoint Inhibitor Associated Myocarditis and Cardiomyopathy: A Translational Review. Biology. 12. 472. 10.3390 / biology12030472). These include Ipilimumab (CTLA-4), Nivolumab (PD-1), Pembrolizumab (PD-1), Atezolizumab (PD- L1), Durvalumab (PD-L1), Avelumab (PD-L1), Cemiplimab (PD-1), Dostarlimab (PD-1) and Relatlimab (LAG-3).

[0216] In preferred embodiments of the present invention, the ICI immunotherapy blocks PD-1 / PD-L1 signalling, and / or CTLA-4 / B7 signalling. In the most preferred embodiments, the ICI immunotherapy is PD-1 inhibitor immunotherapy or PD-L1 inhibitor immunotherapy either alone or in combination with anti-CTLA4 therapy. Accordingly, the methods, systems and kitsof the present invention, in preferred embodiments, determine the likelihood of a subject responding to anti-PD-1 receptor inhibition immunotherapy or anti-PD-1 ligand inhibition immunotherapy.

[0217] Accordingly, in some embodiments, the ICI immunotherapy is Nivolumab, Pembrolizumab, Atezolizumab, Durvalumab, Avelumab, Cemiplimab or Dostarlimab alone or in combination with another ICI immunotherapy including Ipilimumab or Relatlimab. In some preferred embodiments, the ICI immunotherapy is Nivolumab or Pembrolizumab

[0218] In some embodiments, the methods, systems and kits determine the likelihood of a subject responding to treatment with a single ICI immunotherapy, preferably PD-1 or PD-L1 inhibitor immunotherapy. In such embodiments, if a subject is determined to have a low likelihood of responding to ICI immunotherapy the subject may be treated with an alternative treatment, or may be treated with ICI immunotherapy comprising two different immune checkpoint inhibitors, or may be administered a higher dose of the ICI immunotherapy, or may be provided ICI immunotherapy more frequently. In some embodiments, the multiple immune checkpoint inhibitors include PD-1 or PD-L1 inhibitors and one or more of a CTLA-4 inhibitor (such as Ipilimumab) or LAG-3 inhibitor (such as Relatlimab). In some embodiments, the alternative treatment is chemotherapy and / or radiotherapy. In some embodiments, the alternative treatment is surgery, antibody therapy, adoptive cell therapy (such as CAR T cell therapy or tumour-infiltrating lymphocyte (TIL) therapy), hormonal-blocking therapy, targeted- therapy (such as antibody-conjugated drugs or kinase inhibitors) or photodynamic therapy.

[0219] Immune checkpoint immunotherapy is routinely utilised after surgical resection, or partial resection, of a tumour. Accordingly, the methods of the present invention may be performed prior to surgery. In such embodiments, performing the method may inform the extent of surgery.

[0220] Also provided by the present invention is a method of treating or preventing cancer in a subject, the method comprising determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer as described herein and administering to the subject an immune checkpoint inhibitor immunotherapy if the subject is determined to have an increased likelihood of responding to immunotherapy.

[0221] In some embodiments of the method of treating or preventing cancer, the subject is administered a single ICI immunotherapy if the subject is determined to have an increased likelihood of responding to ICI immunotherapy.

[0222] In some embodiments of the method of treating or preventing cancer, the subject is administered a combination of two or more ICI immunotherapies if the subject is determined to have a decreased likelihood of responding to ICI immunotherapy.

[0223] In some embodiments of the method of treating or preventing cancer, the subject has had cancer resected.

[0224] In some embodiments of the method of treating or preventing cancer, the subject may be treated with an alternative treatment, including one or more of those described herein, if a subject is determined to have a low likelihood of responding to ICI immunotherapy.

[0225] TYPES OF CANCERS

[0226] Because ICI immunotherapy activates the immune system, it can be used against a wide variety of cancers. Such cancers include, but are not limited to; melanoma, non-small cell lung cancer, mesothelioma, kidney cancer, head and neck cancer, Hodgkin Lymphoma, Merkel cell carcinoma, bladder cancer, breast cancer, oesophageal cancer, gastric cancer, squamous cell carcinoma of the skin, cervical cancer, a cancer with microsatellite instability and / or mismatch repair enzyme deficiency, hepatocellular carcinoma, and primary mediastinal large B-cell lymphoma and accordingly embodiments of the present invention may be used to determine the likelihood of responding to any one of these cancers.

[0227] In some preferred embodiments, the present invention may be used for assessing the likelihood of responding to ICI immunotherapy of melanoma, non-small cell lung cancer, or breast cancer. In some embodiments the breast cancer is triple negative breast cancer, or HER2 positive breast cancer, or hormone receptor negative breast cancer.

[0228] In some embodiments, the cancer is a high tumour mutational burden cancer, or a cancer susceptible to ICI immunotherapy.

[0229] KITS AND SYSTEMS

[0230] The present invention also provides a kit for use in determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the kit comprising one or more of a reagent for detecting the expression of: E-selectin and / or a reagent for detecting the expression of FLIT7; and one or more reagents for detecting the expression of one or more of CD4, CD8, CD15s, CTLA-4 marker, FoxP3, Ki67 and CD71.

[0231] In some embodiments, the kit comprises: at least one reagent for detecting the expression of E-selectin and / or a reagent for detecting the expression of FLIT7; at least onereagent for detecting the expression of Ki67 and / or CD71 ; and at least one reagent for detecting the expression of CD4, CD8, CD15s, CTLA-4 marker, or FoxP3.

[0232] In some embodiments of the kit, the kit is for use, or is used, in a method of determining the likelihood of a subject responding to an ICI immunotherapy as described herein. Accordingly, the present invention provides a use of one or more reagents for detecting the expression of E-selectin and / or a reagent for detecting the expression of FLIT7; and at least one reagent for detecting the expression of Ki67 and / or CD71 ; and at least one reagent for detecting the expression of CD4, CD8, CD15s, CTLA-4 marker, FoxP3, in determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer.

[0233] In some embodiments, the reagents for detecting E-selectin, Ki67, CD71 , CD4, CD8, CD15s, CTLA-4 or FoxP3 are antibodies, preferably monoclonal antibodies. In some embodiments, the reagent for detecting FUT7 is a nucleic acid. Envisaged nucleic acids include DNA primer(s) or probe(s).

[0234] In some embodiments, the reagents for detecting E-selectin, Ki67, CD71 , CD4, CD8, CD15s, CTLA-4, FoxP3 or FUT7 are antibodies, preferably monoclonal antibodies.

[0235] In some embodiments, the reagents for detecting E-selectin, Ki67, CD71 , CD4, CD8, CD15s, CTLA-4, FoxP3 are antibodies, preferably monoclonal antibodies.

[0236] In some embodiment, the reagents for detecting E-selectin, Ki67, CD71 , CD4, CD8, CD15s, CTLA-4, FoxP3 or FUT7 are nucleic acids. Envisaged nucleic acids include DNA primer(s) or probe(s).

[0237] In some embodiment, the reagent for FUT7 is a nucleic acid. Envisaged nucleic acids include DNA primer(s) or probe(s).

[0238] In aspects, the method of determining if a subject will respond to ICI immunotherapy can be performed on a computing system. In some embodiments of these aspects, the method comprises using computer software executable by a processor to process data representative of the expression of one or more of E-selectin and / or FUT7. In some embodiments, the processed data is also representative of the expression of one or more of the markers CD15s, CD71 , CTLA4, FoxP3, Ki67, CD8, CD4, CD3, or CD28. The software, when executed by a processor, can compare the expression of the markers to a control standard to provide a determination of the likelihood of the subject responding to an ICI immunotherapy. Suitable control standards are described herein.

[0239] Such a computer system can allow for automated comparison of input expression data of one or more of one or more of the markers to the control standards and provide a determined likelihood that a subject will respond to ICI immunotherapy.

[0240] Further provided by the present invention is a system for determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the system comprising: a means for detecting the expression of E-selectin and / or FLIT7 in a blood, vascular and / or tumour sample from the subject; a processor; memory; and software resident in the memory accessible to the processor, the software comprising a series of instructions executable by the processor to process data from the means to detect the expression of E-selectin and / or FLIT7 in the sample from the subject, to thereby determine the likelihood of the subject responding to the checkpoint inhibition immunotherapy.

[0241] In some embodiments of the system, the means for detecting the expression of E- selectin is immunohistochemistry. The system may include an automated slide scanner to capture images, and may comprise means for viewing or analysing scanned images.

[0242] In some embodiments of the system, the means for detecting the expression FLIT7 is PCR. Following quantification by PCR, the system may automatically quantify the expression of FLIT7 (for example by providing a cycle threshold (CT) value) and then compare this to a control standard.

[0243] In some embodiments, the system further comprises means for detecting the expression of one or more of: Ki67, CD71 , CD4, CD8, CD15s, CTLA-4 marker and / or FoxP3. In preferred embodiments, the means for detecting the expression of one or more of Ki67, CD71 , CD4, CD8, CD15s, CTLA-4 marker and / or FoxP3 is a flow cytometer. The flow cytometer may also include analysis software for providing a quantified level of one or more of Ki67, CD71 , CD4, CD8, CD15s, CTLA-4 marker and / or FoxP3 within one of the immune cell signatures disclosed herein and compare them to a control standard, such as those disclosed herein.

[0244] Standard techniques and equipment may be used for cell isolation, cell processing, immunological detection, flow cytometry, recombinant DNA technology, molecular biology and enzymatic reactions. The foregoing techniques and procedures may be generally performed according to methods known in the art and / or as commercially available, and are as described for example in Sambrook et al. Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); and Ausubel et a / Current Protocols in Molecular Biology (2012) John Wiley & Sons, both of which are hereby incorporated by reference.

[0245] Examples

[0246] The present disclosure is further described by the following examples. It is to be understood that the following examples are for the purpose of describing particular embodiments only, and they are not intended to be limiting with respect to the above description.

[0247] Example 1 - E-selectin expression on tumour vessels improves prediction of responsiveness to immune checkpoint inhibitor immunotherapy.

[0248] Archival formalin-fixed paraffin-embedded (FFPE) tissues (tumour-infiltrated lymph nodes, and metastatic tumours) from melanoma patients were interrogated for CD31 , E- Selectin, and tumour marker expression.

[0249] Formalin-fixed paraffin embedded (FFPE) tissue biopsies from melanoma patients were interrogated for CD31 , E-Selectin, and tumour marker expression (panel 1), as well as for CD3, CD8, CD31 , CD15s (HECA-452), CD15s (FH6), and tumour marker expression (panel 2) using two multispectral IF in-house panels. Primary antibody specifications included CD31 (JC / 70A, #MA5-13188, ThermoFisher Scientific), melanoma cocktail (HMB45+M2-7C10+M2- 9E3+T311 , #AB733, Abeam), E-selectin (polyclonal, #BBA18, R&D Systems), CD3 (SP7, #AB16669, Abeam), CD8 (C8 / 144B, #MA5- 13473, ThermoFisher Scientific), and CD15s (HECA-452, #555946, BD Pharmingen; FH6, #368102, BioLegend).

[0250] Slides were prepared by being baked at 60°C for 1 hour, dewaxed in BOND dewax solution three times for 1 min each, rehydrated in 100% ethanol, three times for 1 min each, and stained using the using the BOND RX® Automated Research Stainer. The staining protocol included blocking of endogenous peroxidases using 3% hydrogen peroxide for 10 min, followed by sequential 20 min rounds of heat-induced epitope retrieval (heat pad at 95°C), 20 min blocking of non-specific binding sites at room temperature (anti-CD31 and melanoma cocktail: Akoya blocking / antibody diluent, and E-selectin: 2.5% horse serum), 60 min primary antibody incubation at room temperature (CD31 : 1 :100 dilution, melanoma cocktail: 1 :100 dilution) or overnight at 4°C (E-selectin: 1:300 dilution), 10 min secondary antibody incubation at room temperature (CD31 : ImmPRESS® HRP universal anti-mouse / rabbit polymer kit, melanoma cocktail: Opal™ polymer HRP mouse and rabbit (Ms + Rb), and E-selectin: ImmPRESS® HRP anti-goat polymer kit), and 10 min fluorophore-tyramide signal amplification at room temperature using Opal™ 520 (1 : 100 dilution), 690 (1 :50 dilution), and 620 (1 :100 dilution) fluorophores (Akoya Biosciences®) to detect all target proteins respectively. Slides were counterstained with spectral DAPI, coverslipped using VECTASHIELD Vibrance mountingmedium, and scanned using the Vectra 3 Automated Quantitative Pathology Imaging System (Akoya Biosciences®).

[0251] Images were spectrally unmixed using the inForm® Cell Analysis software (Akoya Biosciences®) and analysed using the HALO® Image Analysis Platform (Indica Labs). Tissue segmentation and cell phenotyping based on total DAPI+ cells was performed on regions of interest encompassing tumour, stroma, and blood vessels to quantify the number of total CD31+cells, E-selectin+cells, CD31+E-selectin+cells, and melanoma* cells.

[0252] CD15s expression within blood samples were analysed as set out in PCT / AU2023 / 050654.

[0253] Following quantification of endothelial cells (defined as CD31+ cells) expressing E- selectin, the interaction between the expression of E-selectin in endothelial cells and the immune signature CD15s+Ki67+CD8+ T cells was analysed by plotting the expression of E- selectin+CD31+ cells against CD15s+Ki67+CD8+ T cells from subjects that responded to PD- 1 inhibitor immunotherapy (n=9), and those that did not (n=7).

[0254] As can be seen in Figure 1 , patients that responded to PD-1 ICI immunotherapy all had at least 7% of their tumour vascular endothelial cells expressing E-selectin and at least 6.5% of their Ki67+CD8+ T cells within their blood sample expressing CD15s. Using these cutoffs the combination of E-selectin expression on endothelial cells in a tumour and CD15s expression on systemic Ki67+CD8+ T cells differentiated between responders and nonresponders with 100% accuracy.

[0255] In comparison, and as illustrated in Figure 2, using one or more of three immune cell signatures could predict a subject’s likelihood of responding to PD-1 inhibitor immunotherapy with 88.5 % accuracy (88.5% sensitivity and 88.5% specificity).

[0256] The three cell signatures used are illustrated in Figure 3 and were CD3+ CD8+ Ki67+ CD15s+ (as measured by the FH6 antibody clone), CD3+ CD8+ Ki67+ CD15s+ (as measured by the HECA-452 antibody clone) and CD3+ CD4+ FoxP3+ CTLA-4+. A threshold of 6% CD3+ CD8+ Ki67+ T cells being CD15s+, when measure with FH6, and 18%, when measured with HECA-452, was chosen based on ROC curves. Further a threshold of 32% of Ki67+CD4+ T cells being both FoxP3+ and CTLA-4+ was chosen based on ROC curves.

[0257] Accordingly, these results indicate that assessing E-selectin expression within the vasculature of tumour samples, and combining this data with immune cell signatures, improvesthe ability to determine if a subject will respond to immune checkpoint inhibition immunotherapy for cancer.

[0258] Example 2 - FUT7 gene expression within a tumour indicates the likelihood of a subject responding to immune checkpoint inhibitor immunotherapy.

[0259] The FUT7 gene encodes for a key enzyme required for generation of CD15s carbohydrate. Accordingly, and in view of the hypothesis that CD15s PBMCs migrate to melanoma tumours, the level of FUT7 was analysed in tumour cells as a proxy for CD15s.

[0260]

[0261] To assess the role of FUT7 in predicting responsiveness to PD-1 ICI immunotherapy, the webtool ‘ROC Plotter’ (available at https: / / rocplot.org / immune and described in Kovacs et al, 2023; DOI 10.1038 / s41401 -023-01079-6) was accessed on 8 / 7 / 2024 and used. This permitted analysis of a larger dataset comprising pooled gene expression data from dozens of studies linked to clinical outcome following PD-1 ICI therapy (Kovacs SA, Fekete JT, Gyorffy B. Predictive biomarkers of immunotherapy response with pharmacological applications in solid tumors. Acta Pharmacol Sin. 2023;44(9): 1879- 1889). The following parameters were selected for the analysis:• Gene symbol: FLIT7• Response: any immune checkpoint inhibitor therapy• Sample acquisition filter: pretreatment• Primary tumour filter: no• Metastatic filter: yes• Tissue of origin: melanoma

[0262] The data was output as both an ROC curve (Figure 4), box plot (Figure 5) and violin plot (Figure 6) comparing patients that responded to PD-1 ICI immunotherapy and those that did not respond to PD-1 ICI immunotherapy.

[0263] As seen in Figure 4, the area under the curve (AUC) was 0.732, with Figures 5 and 6 showing that patients who responded to PD-1 ICI immunotherapy had a median difference of approximately 21.5-fold (Iog2 difference, approximately 2.025-fold) the expression of FLIT7 in tumour samples compared to those that did not respond.

[0264] Consequently, these data show that FUT7 expression within a melanoma tumour sample can provide a predictor of the likelihood of a subject responding to PD-1 ICI immunotherapy.

[0265] These results could be combined with E-selectin and / or immune cell signatures discussed herein to provide further predictive ability of a patient’s response to PD-1 ICI immunotherapy.

[0266] Example 3 - comparison of CD15s antibodies

[0267] As demonstrated in Figure 3, the anti-CD15s antibodies FH6 and HECA-452 differentiated between patients that responded to, or did not respond to, PD-1 ICI immunotherapy based on pre-treatment patient samples. However, several variants of the CD15s carbohydrate antigen exist, differing in the number of fucose and sulphate residues. While all mediate binding to selectins, the sensitivity of these variants for E-, P or L-selectin binding can vary.

[0268] The HECA-452 mAb (Figure 3 - signature 2) recognises at least two variants of CD15s: a canonical structure (sialyl-Lewisx) and a sulphated variant (sialyl 6-sulfo-Lewisx); these structures are collectively known as ‘cutaneous lymphocyte antigen’ (CLA). The FH6 clone (Figure 3 - signature 1) uniquely recognises the difucosyl variant of CD15s. As can be seen, this antibody binds to a smaller subset of CD8+Ki67+ cells than HECA-452, but still showed a striking increase amongst responders compared to non-responders. To ensure other anti-CD15s antibodies were predictive of response to ICI, the clones CSLEX and CHO-131 were utilised. As shown in Figure 7A (CSLEX) and Figure 7B (CHO-131), these antibodies also showed a strong, and statistically significant association, with clinical response to PD-1 ICI treatment. However, the percentage of Ki67+CD8+ T cells identified as CD15s positive in both the responder and non-responder groups varied depending on the antibody used, thereby indicating that the exact percentage of expression determining a likelihood of responding should be normalised to a control population (or control standard), such as non-responders or healthy subjects.

[0269] Example 4 - E-selectin binds to CD15s and is associated with T cell homing to tumours

[0270] To validate the hypothesis that E-selectin expression in tumour cells binds to CD15s-expressing T cells and recruits them to tumours, the binding of recombinant E- and P- selectin fusion proteins to CD15s was analysed by flow cytometry.

[0271] A selectin-binding assay, based on the method by Sakuma and Kannagi (Sakuma K, Kannagi R. Selectin-binding analysis of tumor cells. 2021 Sep 6 [Updated 2022 Mar 25], In: Nishihara S, Angata K, Aoki-Kinoshita KF, et al., editors. Glycoscience Protocols (GlycoPODv2) [Internet], Saitama (JP): Japan Consortium for Glycobiology andGlycotechnology; 2021) was utilised. Briefly, cells were resuspended in 1ml PBS with or without calcium (to establish background binding) and stained with viability stain FVS575V (BD Biosciences) for 10 minutes in the dark. After washing with the same PBS (+ / - calcium), cells were incubated with recombinant human E- or P-selectin chimeric Fc fusion proteins (R&D Systems) in the dark on ice for 30 minutes. This was followed by staining with anti-human IgG- AF488 (Thermo Fisher) in the dark, at room temperature (RT), and then surface and intracellular staining was performed for CD15s (clones HECA-452, CSLEX, CHO-131 and FH6), CD8 and Ki67+. Flow cytometry data was analysed using FCS Express (De Novo Software).

[0272] As illustrated in Figure 8A, after controlling for non-specific binding, a strong correlation was shown between E-selectin binding and CD15s expression within Ki67+ CD8 T cells detected using the HECA-452 mAb (r = 0.928, p < 0.0001). A less clear but still significant correlation was also observed between CD15s and P-selectin binding (r = 0.552, p < 0.05: Figure 8B). When using other CD15s mAbs (CHO-131 or FH6), a significant correlation with E- selectin binding was also observed, but there was no significant correlation with P-selectin binding (data not shown). These results support the hypothesis that E-selectin is likely the primary ligand for CD15s on CD8 T cells and its expression in tumours, in particular in tumour blood vessels, facilitates T cells homing from circulatory system to tumour sites.

[0273] Because CD15s mediates T cells adhesion, which is the first critical step in T cell entry to tissues, patients with more CD15s on their circulating T cells should have a corresponding increase in T cell density within their tumours. To test this, the density of CD8 TILs was quantified using multispectral immunofluorescence analysis of tissues and compared to CD15s expression in matched blood samples. As shown in Figure 9, high CD15s expression on Ki67+CD8 T cells in the blood positively correlated with high CD8 T cell density in tumours (r = 0.549; p < 0.05).

[0274] Although this correlation indicates a tumour-homing function for circulating CD15s+Ki67+CD8 T cells, to further confirm this T cells were analyses using droplet-based single cell RNA sequencing (scRNAseq) was performed together with TCR sequencing and detection of surface CD15s molecules via barcoded antibody. T cells were isolated from matched blood and CNS melanoma metastasis samples from 3 melanoma patients. Blood CD8 T cells were FACS- sorted according to surface CD71 expression (used as a surrogate surface marker of proliferation instead of intracellular Ki67). After filtering for quality control and to remove contaminating non-T cells, the merged dataset (containing CD71+blood (proliferating cells), CD7T blood (non-proliferating cells), and tumour T cell populations from 3 patients) contained 15,029 cells. The blood T cell populations were analysed for FUT7 expression based onCD15s+and CD15s_as determined by barcoded antibody binding. CD15s+cells had significantly higher expression of FUT7 compared to their CD15s_counterparts (Table 1 , below), thus validating the link between CD15s surface expression and FUT7 gene expression in T cells and further demonstrating FUT7 gene expression as an indicator for response to PD- 1 ICI therapy.

[0275] Table 1 - mean expression of FUT7 in populations of peripheral blood T cells

[0276] Additionally, for each blood T cell subset from each patient, the TCR CDR3 sequences were determined and compared to T cells obtained from the matched tumour T cells using scRepertoire (Borcherding N, Bormann NL, Kraus G. scRepertoire: An R-based toolkit for single-cell immune receptor analysis. FIOOORes. 2020;9:47. Published 2020 Jan 27). Clonal scatter plots for each patient (data not shown) were used to determine the number of T cell clones overlapping between T cells in tumour and matched blood. After normalising to the total number of blood T cells analysed, the CD15s+CD71+subset had significantly greater clonal overlap with tumour T cells than any other blood T cell subset (Figure 10) indicating movement from the peripheral blood to the tumour.

[0277] Accordingly, these data indicate that CD15s identifies a unique subset of proliferating blood T cells which home to tumours expressing E-selectin. As such, the combination of CD15s expression on peripheral blood cells and E-selectin in tumour tissue provide a strong predictor for responding to PD-1 ICI treatment.

[0278] Example 5 - CD103 is associated with CD15s expression

[0279] Peripheral blood mononuclear cells from the responder cohort were analysed using high-parameter flow cytometry to detect a broad range of markers associated with T cell memory phenotype and effector function, together with three anti-CD15s antibodies and an anti-CD103 antibody.

[0280] For analysis of surface and intracellular markers on T cells, two antibody panels were used (see Table 2 for antibody details and concentrations). PBMCs were thawed in RPMIcontaining 10% FCS, and dissociated metastatic tumours were thawed in the same buffer supplemented with 20U / ml DNAse to prevent cell clumping. Cells were washed and transferred to flow tubes for staining. Staining reagent cocktails for different panels were prepared in Brilliant Buffer Plus (BD Bioscience) (10 pl / test). Fc receptors on cells were blocked, then antibody cocktail for membrane markers (except for CD15s - FH6 clone) were added to cells at the concentrations indicated in Table 2. Cells were incubated at room temperature in the dark, then washed. To detect intracellular antigens, Foxp3 / Transcription Factor Staining Buffer Set (Invitrogen, Thermo Fisher) was used for fixing and permeabilising cells, according to the manufacturer’s protocol. Intracellular antibody cocktails were incubated for 30 minutes at room temperature protected from light, and the cells washed twice. Cells were analyzed on the day of staining using a BD LSRFortessa or BD FACSymphony A5. CD103 (integrin aE) was included for characterising T cells because it has previously been linked to tumour antigen specificity a may contribute to tumour homing.

[0281] Notably, CD103 expression was enriched on the same subsets that showed the highest CD15s expression; namely, stem-like effectors and TPEX cells. Accordingly, these data suggest that CD103 may be used in addition to, or in place of, CD15s when determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer.

[0282] Antibodies

[0283] The following antibodies were used in the methods described herein.

[0284] Table 3 - Antibodies

[0285] Statistical analyses

[0286] Statistical analyses were performed using GraphPad Prism V10.4. Scatter and violin plots show median values as a red line. Two-way comparisons used Mann-Whitney test or Wilcoxon matched-pairs signed rank test for unpaired or paired data, respectively. Three or more groups (unpaired data) were compared using Kruskal-Wallis test for unmatched data or Friedman test for matched data; if significant, two-way comparisons were conducted using Dunn’s post-test. Non-significant values are indicated as ns; p values for significant results (p < 0.05) are as stated. The relationship between matched pairs of continuous variables was assessed using correlation analysis, reporting the Pearson correlation coefficient or Spearman correlation coefficient for normal or non-normal datasets, respectively, as determined using the Kolmogorov-Smirnov normality test (alpha=0.05). Simple linear regression was used to graphically display the strength and direction of the relationship. Kaplan-Meier survival analysis was conducted using logrank (Mantel-Cox) test to compare curves, and was restricted to the first 2 years’ of data. Cut-point for CD15s expression to define the groups was calculated using the ‘auto select best cutoff’ function of the KM Plotter tool (Gyorffy B. Integrated analysis of public datasets for the discovery and validation of survival-associated genes in solid tumors. Innovation (Camb). 2024;5(3):100625. Published 2024 Apr 9). Publicly available gene expression data was analysed using the ROC Plotter tool (Kovacs, S.A, 2023 - supra) on 8 / 7 / 2024 and the gene expression data Iog10 transformed and analysed in Prism.

[0287] DEFINITIONS AND QUALIFICATIONS

[0288] Referenced documents, publications and patents are to be included in their entirety by way of reference. The teachings and disclosures in such documents, publications and patents are therefore considered to form part of the disclosure of this specification.

[0289] All methods described herein can be performed in any suitable order unless indicated otherwise herein or clearly contradicted by context or the understanding of a skilled addressee. The use of any and all examples, or exemplary language (e.g., "such as", “i.e.”, “for example”), is intended merely to better illuminate the example embodiments and does not pose a limitation on the scope of the claimed invention, unless otherwise claimed or stated. No language in the specification should be construed as indicating any non-claimed element as essential.

[0290] The description provided herein is in relation to several embodiments which may share common characteristics and features. It is to be understood that one or more features of one embodiment may be combinable with one or more features of the other embodiments. In addition, a single feature or combination of features of the embodiments may constitute additional embodiments.

[0291] The subject headings used herein are included only for the ease of reference of the reader and should not be used to limit the subject matter found throughout the disclosure or the claims. The subject headings should not be used in construing the scope of the claims or the claim limitations.

[0292] Those skilled in the art will appreciate that the invention described herein is susceptible to variations and modifications other than those specifically described. It is to be understood that the invention includes all such variations and modifications. The invention also includes all of the steps, features, compositions and compounds referred to, or indicated in this specification, separately or collectively, and any and all combinations of any two or more of the steps or features.

[0293] Also, it is to be noted that, as used herein, the singular forms “a”, “an” and “the” include plural aspects unless the context already dictates otherwise.

[0294] It will be apparent to the person skilled in the art that while the invention is described herein in detail for the purposes of clarity and understanding, various modifications and alterations to the embodiments and methods described herein may be made without departing from the scope of the inventive concept disclosed in this specification.

[0295] Future patent applications may be filed on the basis of, or claiming priority from, the present application. It is to be understood that the following claims are not intended to limit the scope of what may be claimed in any such future application(s).

Claims

The claims defining the invention are as follows:

1. A method of determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the method comprising assessing the expression of one or more of E-selectin and / or FLIT7 within a biological sample from the subject, and assessing one or more of:CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells;CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;FoxP3 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;CD15s expression in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells;Ki67 expression in CD8+ T cells; orCD71 expression in CD8+ T cells, wherein an increase in the expression of one or more of E-selectin and / or FLIT7 in combination with one or more of: an increased expression of CD15s in Ki67+ CD8+ T cells or CD71+ CD8+ T cells prior to the immunotherapy; an increased expression of CTLA-4 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells prior to and / or after the immunotherapy; an increased expression of FoxP3 in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells prior to immunotherapy; an increased expression of CD15s in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells prior to immunotherapy; and / or a decreased expression of Ki67+ or CD71+ in CD8+ T cells prior to immunotherapy, determines an increased likelihood that the subject will respond to immune checkpoint inhibition immunotherapy.

2. The method of claim 1, wherein the expression of E-selectin is assessed on endothelial cells from a tumour.

3. The method of claim 2, wherein the endothelial cells express CD31.

4. The method of any one of claims 1 to 3, wherein the immune checkpoint inhibition immunotherapy is PD-1 immune checkpoint inhibition.

5. The method of any one of claims 1 to 4, wherein the immune checkpoint inhibition immunotherapy comprises PD-1 receptor inhibition.

6. The method of any one of claims 1 to 5, wherein the immune checkpoint inhibition immunotherapy comprises PD-1 ligand (PD-L1) inhibition.

7. The method of any one of claims 1 to 6, wherein the expression of E-selectin is protein expression of E-selectin.

8. The method of any one of claims 1 to 7, wherein the expression of FLIT7 is gene expression of FLIT7.

9. The method of any one of claims 1 to 8, wherein the biological sample is a tumour sample.

10. The method of any one of claims 1 to 8, wherein the expression of FLIT7 is determined in a blood sample, or a serum sample, or a plasma sample, or lymph sample, or isolated peripheral blood mononuclear cells (PBMCs), or tumour infiltrating lymphocytes (TILs).

11. The method of any one of claims 1 to 8, wherein the expression of FLIT7 and / or E-selectin is assessed in a first biological sample and the expression of CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells; CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; FoxP3 marker in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells;CD15s marker in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells; Ki67 marker in CD8+ T cells; or CD71 marker in CD8+ T cells, is performed in a second biological sample.

12. The method of claim 11 , wherein the first biological sample is a tumour sample.

13. The method of claim 11 or claim 12, wherein the second biological sample is a vascular sample, or a blood sample, or a serum sample, or a plasma sample, or lymph sample or isolated peripheral blood mononuclear cells, or tumour infiltrating lymphocytes (TILs).

14. The method of any one of claims 1 to 13, wherein the Ki67+ CD8+ T cells comprise Ki67+ CD8+ CD3+ T cells.

15. The method of any one of claims 1 to 13, wherein the Ki67+ CD8+ T cells comprise Ki67+ CD8+ CD3+ CD28+ T cells.

16. The method of any one of claims 1 to 15, wherein the Ki67+ CD4+ T cells comprise Ki67+ CD4+ CD8- T cells.

17. The method of any one of claims 1 to 16, wherein the Ki67+ CD4+ T cells comprise Ki67+ CD4+ CD3+ T cells.

18. The method of any one of claims 1 to 17, wherein the expression of one or more of CD15s, CD71 , CTLA4, FoxP3, Ki67, CD8, CD4, CD3, CD28 is assessed by flow cytometry.

19. The method of any one of claims 1 to 18, wherein the expression of E-selectin in a tumour sample or in tumour-associated endothelial cells, FLIT7 in a tumour sample, CD15s expression in Ki67+ CD8+ T cells or in CD71+ CD8+ T cells; CTLA-4 expression in Ki67+ CD4+ T cells or in CD71+ CD4+ T cells; FoxP3 marker in Ki67+ CD4+ T cells or in CD71 + CD4+ T cells; CD15s marker in Ki67+ CD4- CD8- T cells or in CD71+ CD4- CD8- T cells; Ki67 marker in CD8+ T cells; or CD71 marker in CD8+ T cells is compared to a control standard.

20. The method of claim 19, wherein the control standard is predetermined and based on a population of subjects that have not responded to immune checkpoint inhibition immunotherapy, or is based on a healthy population or healthy tissue.21 . The method of any one of claims 1 to 20, wherein a percentage of CD15s positive cells in Ki67+ CD8+ T cells of about 6.5% or greater in a biological sample from the subjectdetermines an increased likelihood that the subject will be responsive to immune checkpoint immunotherapy.

22. The method of any one of claims 1 to 21, wherein a percentage of E-selectin expressing cells of about 7% or greater within endothelial cells in a biological sample from the subject determines an increased likelihood that the subject will be responsive to immune checkpoint immunotherapy.

23. The method of any one of claims 1 to 20, wherein a percentage of CD15s positive cells in Ki67+ CD8+ T cells of about 6.5% or greater and wherein a percentage of E-selectin expressing cells of about 7% or greater within endothelial cells in a biological sample from the subject determines an increased likelihood that the subject will be responsive to immune checkpoint immunotherapy.

24. The method of any one of claims 1 to 23, wherein the expression of E-selectin is determined using the BBA18 antibody.

25. The method of any one of claims 1 to 24, wherein the method comprises using computer software executable by a processor to process data representative of the expression of one or more of E-selectin and / or FLIT7.

26. The method of claim 25, wherein the data is also representative of the expression of one or more of the markers CD15s, CD71, CTLA4, FoxP3, Ki67, CD8, CD4, CD3, or CD28, and the software comprises instructions to compare the expression of the markers to a control standard to provide a determination of the likelihood of the subject responding to the immune checkpoint inhibition immunotherapy.

27. The method of any one of claims 1 to 26, wherein the method comprises performing immunohistochemistry to determine the expression of E-selectin in a tumour sample.

28. A method of determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the method comprising assessing the expression of FLIT7within a biological sample from the subject, wherein an increased expression of FLIT7 determines an increased likelihood that the subject will respond to immune checkpoint inhibition immunotherapy.

29. The method of claim 28, wherein the expression of FLIT7 is gene expression of FLIT7.

30. The method of claim 28 or claim 29, wherein the biological sample is a tumour sample.

31. The method of claim 28 or claim 29, wherein the biological sample is a blood sample, a lymphatic sample, a serum sample or a plasma sample or peripheral blood mononuclear cells.

32. The method of any one of claims 28 to 31, wherein the expression of FLIT7 is compared to a control standard.

33. The method of claim 32, wherein the control standard is predetermined based on a population of subjects that have not responded to immune checkpoint inhibition immunotherapy, or is based on a healthy population or on healthy tissue.

34. The method of any one of claims 28 to 33, wherein the method determines the likelihood of responding to immunotherapy with a single immune checkpoint inhibitor.

35. The method of any one of claims 28 to 34, wherein the method comprises using computer software executable by a processor to process data representative of the expression of FUT7.

36. The method of any one of claims 1 to 35, wherein the method comprises using PCR to determine gene expression of FLIT7.

37. A method of treating or preventing cancer in a subject, the method comprising determining if a subject will respond to immune checkpoint inhibition immunotherapy forcancer in accordance with any one of claims 1 to 36, administering to the subject at least a single immune checkpoint inhibitor immunotherapy if the subject is determined to have an increased likelihood of responding to immunotherapy.

38. The method of claim 37, wherein the subject has had cancer resected.

39. The method of claim 37 or claim 38, wherein, if the subject is determined to have a decreased likelihood of responding to immunotherapy, the method comprises administering to the subject two or more immune checkpoint inhibitors or increasing the dose or number of doses of an immune checkpoint inhibitor, or performing an alternative treatment.

40. The method of any one of claims 1 to 39, wherein the cancer is selected from one of melanoma, non-small cell lung cancer, mesothelioma, kidney cancer, head and neck cancer, Hodgkin Lymphoma, Merkel cell carcinoma, bladder cancer, breast cancer, oesophageal cancer, gastric cancer, squamous cell carcinoma of the skin, cervical cancer, a cancer with microsatellite instability and / or mismatch repair enzyme deficiency, hepatocellular carcinoma, and primary mediastinal large B-cell lymphoma, and other malignancies susceptible to therapeutic immune checkpoint inhibition.

41. The method of any one of claims 1 to 39, wherein the cancer is selected from one of melanoma, non-small cell lung cancer, or breast cancer.

42. The method of claim 41, wherein the breast cancer is triple negative breast cancer.

43. The method of claim 41, wherein the breast cancer is HER2-positive breast cancer.

44. The method of claim 41, wherein the breast cancer is hormone receptor negative breast cancer.

45. The method of any one of claims 1 to 44, wherein the cancer is a high tumour mutational burden cancer.

46. A kit for use in determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the kit comprising one or more of a reagent for detecting the expression of: E-selectin and / or a reagent for detecting the expression of FLIT7; and one or more reagents for detecting the expression of one or more of CD4, CD8, CD15s, CTLA-4 marker, FoxP3, Ki67 and CD71.

47. The kit according to claim 46, for use in a method of any one of claims 1 to 45.

48. The kit according to claim 46 or claim 47, wherein the kit comprises: at least one reagent for detecting the expression of E-selectin and / or a reagent for detecting the expression of FLIT7; at least one reagent for detecting the expression of Ki67 and / or CD71 ; and at least one reagent for detecting the expression of CD4, CD8, CD15s, CTLA-4 marker, or FoxP3.

49. Use of one or more reagents for detecting the expression of E-selectin and / or a reagent for detecting the expression of FUT7; and at least one reagent for detecting the expression of Ki67 and / or CD71 ; and at least one reagent for detecting the expression of CD4, CD8, CD15s, CTLA-4 marker, or FoxP3, in determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer.

50. A system for determining if a subject will respond to immune checkpoint inhibition immunotherapy for cancer, the system comprising: a means for detecting the expression of E-selectin and / or FUT7 in a blood, vascular and / or tumour sample from the subject; a processor; memory; and software resident in the memory and accessible to the processor, the software comprising a series of instructions executable by the processor to process data from the means to detect the expression of E-selectin and / or FUT7 in the sample from the subject, to thereby determine the likelihood of the subject responding to the checkpoint inhibition immunotherapy.

51. The system of claim 50, wherein the means for detecting the expression of E-selectin is immunohistochemistry.

52. The system of claim 50 or claim 51, wherein the means for detecting the expression of FUT7 is PCR.

53. The system of any one of claims 50 to 52, further comprising means for detecting the expression of one or more of: Ki67, CD71, CD4, CD8, CD15s, CTLA-4 marker and / or FoxP3.

54. The system of claim 53, wherein the means for detecting the expression of one or more of Ki67, CD71 , CD4, CD8, CD15s, CTLA-4 marker and / or FoxP3 is a flow cytometer.

55. The method of any one of claims 37 to 45, the kit of any one of claims 46 to 48, the use of claim 49, or the system of any one of claims 50 to 54, wherein the immune checkpoint inhibition immunotherapy for cancer is PD-1 receptor inhibition or is PD-1 ligand (PD-L1) inhibition.

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