Novel prognostic biomarkers in patients with cancer

Abstract

The present disclosure relates to methods and systems for predicting a subject's responsiveness to treatment or therapy. Specifically, the methods and systems provided herein may predict whether a subject suffering from disease or cancer may respond to immune therapy, including treatment with an immune checkpoint inhibitor such as agonist anti-CD137 antibodies, PD-1 antagonists, anti-CTL4 antibodies, or any combination thereof.

Description

NOVEL PROGNOSTIC BIOMARKERS IN PATIENTS WITH CANCER TECHNICAL FIELD

[0001] The present disclosure relates to methods and systems for predicting the likelihood that a subject suffering from a disease, e.g., cancer, may experience a complete response, partial response, stable disease, or any other efficacy outcome when treated with an anti-CD137 and / or anti -PD-1 antibody.BACKGROUND

[0002] In the past decades, advances in molecular biomarkers and the progress in treatment options have together contributed to improvements in cancer diagnosis, classification, and individualized therapy. Such advances have resulted in an increase in patient overall survival. Despite progress, however, in many cases tumors do not respond to the currently available treatments or relapse after initial response. Further, the toxicity of the treatment itself may be associated with a high risk of severe side effects as well as therapy induced autoimmune inflammation in various tissues. Thus, the identification of biomarkers to predict patient populations that may respond favorably to treatment is essential in effectively treating the disease or cancer.

[0003] An increasing body of evidence suggests that the immune system operates as a significant barrier to tumor formation and progression. The principle that naturally-occurring T cells with anti-tumor potential or activity exist in a patient with cancer has rationalized the development of diverse immunotherapeutic approaches in oncology. Immune cells, such as T cells, macrophages, and natural killer (NK) cells, can exhibit anti-tumor activity and effectively control the occurrence and growth of malignant tumors. Tumor-specific or tumor-associated antigens can induce immune cells to recognize and eliminate malignancies (Chen & Mellman, (2013) Immunity 39(1): 1-10). In spite of the existence of tumor-specific immune responses, malignant tumors often evade or avoid immune attack through a variety of immunomodulatory mechanisms resulting in the failure to control tumor occurrence and progression (Motz & Coukos, (2013) Immunity 39(l):61-730). An emerging hallmark of cancer is the exploitation of these immunomodulatory mechanisms and the disablement of anti-tumor immune responses, resulting in tumor evasion and escape from immunological killing (Hanahan and Weinberg (2011) Cell 144(5):646-674).

[0004] Approaches in the immunotherapy of cancer involve counteracting these immune evasion and escape mechanisms and inducing the endogenous immune system to reject tumors. Antibody blockade of T cell co-inhibitory molecules, also known as immune checkpoints, have emerged as a frontline treatment for cancer. ntibodies targeting CTLA-4, PD-1, and PD-L1 have shown therapeutic benefit in human clinical trials. The anti-CTLA-4 antibody ipilimumab (Yervoy®) and the anti-PD-1 antibodies nivolumab (OPDIV O®) and pembrolizumab (KEYTRUDA®), are currently approved for treating cancer patients.Moreover, therapies targeting CD 137 have recently been explored. CD 137 (alternatively known as “tumor necrosis factor receptor superfamily member 9” (TNFRSF9), 4-1 BB, and “induced by lymphocyte activation” (ILA)) is a transmembrane co-stimulatory receptor protein belonging to the tumor necrosis factor superfamily. CD 137 is a T cell co-stimulatory receptor induced upon T-cell receptor (TCR) activation (Nam et al., (2005) Curr Cancer Drug Targets 5:357-363; Watts et al., (2005) Annu Rev Immunol 23:23-68). In addition to its expression on activated CD4+ and CD8+ T cells, CD 137 is also expressed on CD4+CD25+ regulatory T cells, activated NK and NK-T cells, monocytes, neutrophils, and dendritic cells.

[0005] A report on the clinical evaluation of an agonistic CD137 antibody (Urelumab, BMS-663513; Bristol-Myers Squibb) documented the observation of treatment-related adverse events in human subjects, including indications of severe hepatotoxicity (transaminitis) correlating with antibody dose (Segal et al., (2016) Clin Cancer Res 23(8): 1929-1936). In contrast, a different agonistic CD137 antibody (Utomilumab, PF-05082566; Pfizer) tested in combination with an anti-PD-1 antibody (pembrolizumab), though not resulting in any dose-limiting toxicities, showed comparable results to anti-PD-1 antibody therapy alone (Tolcher, A. et al., (2017) Clin Cancer Res 23(18): 5349-5357).Moreover, not all subjects may respond to treatment with CD137 agonists, anti-PD-1 antibody, or combinations thereof. Given that treatment may prove ineffective for some subjects and may additionally result in adverse side effects, it is imperative to identify predictive biomarkers in order to determine prior to therapy which subjects and tumors might respond to treatments including CD1 7 agonists and / or anti-PD-1 antibodies, and which patients might develop severe side effects.

[0006] Neural cell adhesion molecule (NCAM, also interchangeably referred to as “CD56”) is a cell adhesion molecule belonging to the immunoglobulin superfamily. A ceil surface molecule, NCAM is expressed in the nervous system as well as in various cell types,such as NK cells, cardiomyocytes, and neuroendocrine cells. There are three main isoforms of NCAM (NCAM-120, NCAM-140, and NCAM-180), all generated by alternative splicing from a single gene, differing in their intracellular domain length. A hallmark of NCAM expression on cells is that its presence allows for binding to other molecules. For example, molecules that express NCAM can bind to each other by homotypic adhesion, i.e., adhesion mediated by adhesion molecules between identical cell types.

[0007] In terms of the immune system, CD56 (i.e., NCAM) is the archetypal phenotypic marker of NK cells, however, CD56 may also be expressed by many more immune cells, including a T cells, y8 T cells, DCs, and monocytes. Natural killer cells are generally characterized in humans by expression of the phenotypic marker CD56 in the absence of CD3 (CD56+CD3-). Typically, NK cells are further subdivided into two subsets, CD56dimand CD56bn8htcells. Whereas most NK cells in peripheral blood are CD56di™, CD56brlghtNK cells are more abundant in tissues. (Van Acker, H H, t al., “CD56 in the immune system: more than a marker for cytotoxicity?” Front. Immunol. (2017) 8: 892). Characterization of the CD56br!ghtpopulation shows that these NK cells produce immunoregulatory cytokines, including interferon-y (IFNy), tumor necrosis factor-beta (TNF-P), tumor necrosis factor-a (TNF-a), granulocyte macrophage-colony stimulating factor (GMCSF), IL-10, and 11,-13. On the other hand, the CD56dimsubset is the terminally differentiated successor of the CD56bnghtpopulation and is primarily responsible for exerting cytolytic functions. It has also been shown, however, that CD56dimNK cells can produce cytokines, specifically IFN-y, after cell triggering via NKp46 or NKp30 activating receptors or after stimulation with combinations of IL-2, IL-12, and IL-15.

[0008] The relationship between the immune system and cancer is complex. Effector cells of the immune system have the capacity to recognize and kill malignant cells whereas immune system-mediated inflammation may promote tumor growth and regulatory cells may suppress the anti-tumor responses. An important aspect of all anti-tumor responses is the ability of the immune cells (e.g., NK cells) to migrate to the tumor site and to interact with each other and with malignant cells. As such, cell adhesion molecules including receptors of the immunoglobulin superfamily (e.g., NCAM or CD56) are important in mediating these processes (Harjunpaa H, et al., (2019) Cell Adhesion Molecules and Their Roles and Regulation in the Immune and Tumor Microenvironment. Front. Immunol. 10:1078.).

[0009] The tumor microenvironment (TME), i.e., the frequency, location and functional orientation of different immune cell subsets, varies substantially between tumor types and also between individuals. Given the complexity, there remains a need to identify biomarkers that may predict patient response, for example, to anticipate which patients will respond to varying forms of treatment, thus optimizing treatment strategy and reducing toxicities and costs. As such, new treatment strategies are still needed to improve clinical outcomes in patients.BRIEF DESCRIPTION OF DRAWINGS

[0010] The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.

[0011] FIG. 1 shows the number of CD56+ cells per mm2in patients in complete response, partial response, stable disease or progressive disease following treatment for head and neck squamous cell carcinoma (“HNSCC”), melanoma, or non-small cell lung cancer (“NSCLC”). Each dot represents a baseline tumor sample. Results were obtained via mul tiplex i mm unofl uorescence;

[0012] FIG. 2 illustrates that CTX-471, a novel antibody agonist of CD137, monotherapy shows pharmacodynamic correlates of tumor immunity. Paired pre- and post-CTX-471 treatment tumor and blood samples were analyzed using multiplexed immunofluorescence and by flow cytometry. FIG. 2A shows a volcano plot including all pharmacodynamic correlates measured, with those showing a nominal p value of lower than p=0.05 indicated with the horizontal dashed line. FIGs 2B-2E, show the plotted raw and foldchange values of the indicated correlates. FIG. 2F shows levels of PD-L1 on tumor tissue that were observed to increase following treatment. The lower two panels of FIG. 2F show the respective H& E images from panels immediately above. Asterisks indicate differences significant at either p=0.05 (*) or p=0.01(**);

[0013] FIG. 3 shows tumor cell cobblestone staining. Each sample was taken from a HNSCC, melanoma, or NSCLC tumor. CR, PR, SD, and PD represents disease state. The staining indicates that CD56+ are more numerous in disease control samples;

[0014] FIG. 4 provides a summary of all pharmacodynamic and response biomarkers measured;

[0015] FIG. 5 shows neural crest-derived tumor response to monotherapy and CD137 expression. FIG. 5A is a waterfall plot that shows the change in tumor size within tumors derived from the neural crest: small cell lung cancer and melanoma. Within tliis subset an overall response rate of 31% was achieved. FIG. 5B is a series of graphs comparing the densities of activated (GDI 37+) for various lymphocyte subsets within all baseline tumor samples classified as liigh or low NCAM based upon median CD56 cell density. Asterisks indicate differences significant at p=0.001(***);

[0016] FIG. 6 shows a proposed mechanism by which NCAM may render tumors sensitive to agonistic CD 137 antibody treatment;

[0017] FIG. 7 illustrates biomarker correlates linked with disease control on CTX-471 monotherapy. Tumor and blood samples were taken at baseline and compared between patients with tumors showing disease control (CR, PR, and SD) or progressive disease (PD). FIG. 7A shows all response biomarkers measured as a volcano plot with those showing a nominal p value of lower than p=0.05 indicated by a hori zontal dashed line. FIG. 7B shows an antibody to CD56 / NCAM was included within the multiplexed immunofluorescent panel in order to detect NK cells. However, in addition to NK cell staining, a surprisingly liigh staining in tumor cells was observed associated with disease control. FIGs. 7C-7F show other baseline phenotypes of interest including blood cytokines TNFa (FIG. 7D) and 11-6 (FIG. 7E), monocytes per ml of blood (FIG. 7F) and regulatory T cells (FIG. 7C). Asterisks indicate differences significant at either p=0.05 (*) or p=0.01(**);

[0018] FIG. 8 illustrates NK cell correlates and disease control by CTX-471 monotherapy. A focused survey of NK subsets was made within tumor and blood samples taken at baseline and compared between patients with tumors showing disease control (CR, PR, and SD) or progressive disease (PD). Subsets measured in tissue included activated NK (FIGs. 8A and 8B), as well as NKT cells (FIG. 8C). Circulating NK cells (FIGs. 8D-8F) also showed significant differences and trends with disease control in baseline samples. Asterisks indicate differences significant at either p=0.05 (*) or p=0.01(**);

[0019] FIG. 9 illustrates a novel baseline circulating biomarker of CTX-471 response. An NK Cell phenotype in the baseline samples predicted response to CTX-471.Unbiased analysis of NK cell-focused flow cytometry panel was conducted using the Ozette Discovery platform for full annotation of all robust cellular phenotypes. Ozette Discovery-identified an NK Cell phenotype: CD16+CD1 lb+CD56+CD3-CD19-CD314-CD337-TIGIT-CD137- which clearly distinguished melanoma patients with disease control from those showing progressive disease. For example, a threshold of 1% as indicated by the dotted lines would classify PR patient samples as distinct from PD and most SD samples. This unique phenotype went undetected using conventional analytical approaches and highlights the importance of leveraging the full extent of the data and an unbiased discovery approach;

[0020] FIG. 10 illustrates a possible optimal cutoff for CD56+ tumor cell density. FIG. 10A show's the different CD56 density thresholds that were evaluated. FIG 10B show's that the patient samples were ranked based upon their CD 6 density scores using multiplex CD56 immunofluorescence. The median CD56 value was used in the given ROC analysis, where samples with CD56 intensity values at or above the median CD56 intensity score were considered “high” and those below the median were considered “low”. Calculations were made using the “pROC” package with the R statistical software;

[0021] FIG. 11 shows that CTX-471 increases levels of CD137+ circulating NK cells. CTX-471 exposure caused a significant increase in levels of circulating NK cells that express CD137 / 4-1BB.;

[0022] FIG. 12 demonstrates that CD 137+ NK cells are enriched in NCAM “high” tumors. Baseline tissue samples were analyzed by multiplex immunofluorescence (mlF) and classified by median CD56 levels as high or low CD56 / NCAM samples. Levels of tumor enrichment (“within” vs. “overall”) are shown for CD 137+ lymphocytes in high vs low' NCAM samples. Only GDI 37+ NK cells are significantly (p<0.05) enriched within NCAM “high” tumors;

[0023] FIG. 13 illustrates that C TX-471 disease control is associated with certain genetic signatures. FIG. 13A shows 31 genes that were differentially (pcO. Ol) expressed with disease control rate (DCR) in patient samples (n=9). FIG. 13B presents a gene set enrichment analysis across 7 databases. The analyses for 31 differentially expressed genes (DEGs) is shown and ranked by combined odds ratio / p-value score. Results were filtered for > 2 genes matched per pathway with p-value < 0.05. Red boxes indicate neuronal-linked genetic signatures associated with DCR; and

[0024] FIG. 14 shows baseline features of disease control enriched in NCAM tumors. Baseline tissue samples evaluable for disease control (CR, PR, or SD vs PD) were analyzed by mIF. Features significantly (p<0.05) enriched within disease control tumor samples compared with overall levels are shown. Activated lymphocytes (T and NK cells) were enriched within tumors of patients showing disease control. Activation markers measured include CD137 and PD-1.DETAILED DESCRIPTIONDefinitions

[0025] As used herein, the following definitions are provided to facilitate the understanding of the present disclosure.

[0026] It must be noted that, as used in the specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Further, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular.

[0027] As used herein, "about" will be understood by persons of ordinary skill and will vary to some extent depending on the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill given the context in which it is used, "about" will mean up to plus or minus 10% of the particular value.

[0028] As used herein, the term "agonist" refers to any molecule that partially or fully promotes, induces, increases, and / or activates a biological activity of a native polypeptide disclosed herein (e.g., CD 137). Suitable agonist molecules specifically include agonist antibodies or antibody fragments, fragments or amino acid sequence valiants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. In some embodiments, activation in the presence of the agonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% higher than the signal measured with a negative control under comparable conditions. Also disclosed herein, are methods of identifying agonists suitable for use in themethods of the disclosure. For example, these methods include, but are not limited to, binding assays such as enzyme-linked immuno-absorbent assay (ELISA), Forte Bio© systems, and radioimmunoassay (RIA). These assays determine the ability of an agonist to bind the polypeptide of interest (e.g., a receptor or ligand, e.g., CD137) and therefore indicate the ability of the agonist to promote, increase or activate the activity of the polypeptide. Efficacy of an agonist can also be determined using functional assays, such as the ability of an agonist to activate or promote the function of the polypeptide. For example, a functional assay may comprise contacting a polypeptide with a candidate agonist molecule and measuring a detectable change in one or more biological activities normally associated with the polypeptide. The potency of an agonist is usually defined by its EC50 value (concentration required to activate 50% of the agonist response). The lower the EC50 value the greater the potency of the agonist and the lower the concentration that is required to activate the maximum biological response.

[0029] As used herein, the term "antagonist" refers to any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of a native polypeptide disclosed herein (e.g., PD1 and / or PD-L1 and / or PD-L2). Suitable antagonist molecules specifically include antagonist antibodies or antibody fragments, fragments or amino acid sequence variants of native polypeptides, peptides, antisense oligonucleotides, small organic molecules, etc. In some embodiments, inhibition in the presence of the antagonist is observed in a dose-dependent manner. In some embodiments, the measured signal (e.g., biological activity) is at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35 %, at least about 40%’, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 100% lower than the signal measured with a negative control under comparable conditions.

[0030] As used herein, the term "anti-CD137 agonist antibody" (used interchangeably with the term "anti-CD137 antibody") refers to an antibody that specifically binds to CD137 and partially or fully promotes, induces, increases, and / or activates CD 137 biological activity, response, and / or downstream pathway(s) mediated by CD 137 signaling or other CD137-mediated function. In some embodiments, an anti-CD137 agonist antibody binds to CD137 and allows binding of CD137L. In some embodiments, an anti-CD137 agonistantibody binds to CD 137 and induces multimerization of CD 137. In some embodiments, an anti-CD137 agonist antibody binds to CD 137 and induces the dimerization of CD 137 trimers. In some embodiments, an anti-CD137 agonist antibody binds to CD137 and induces the multimerization of CD137 trimers. Examples of anti-CD137 agonist antibodies are provided herein. Methods for detecting formation of a trimer:trimer complex are known to those of skill in the art. For example, electron microscopy has been shown to detect such complexes, see, e.g., Won, E. The Journal of Biological Chemistry, Vol. 285 (12): 9202-9210 (2010).

[0031] As used herein, the term “antibody” refers to a whole antibody comprising two light chain polypeptides and two heavy chain polypeptides. Whole antibodies include different antibody isotypes including IgM, IgG, IgA, IgD, and IgE antibodies. The term “antibody” includes a polyclonal antibody, a monoclonal antibody, a chimerized or chimeric antibody, a humanized antibody, a primatized antibody, a deimmunized antibody, and a fully human antibody. The antibody can be made in or derived from any of a variety of species, e.g., mammals such as humans, non-human primates (e.g., orangutan, baboons, or chimpanzees), horses, cattle, pigs, sheep, goats, dogs, cats, rabbits, guinea pigs, gerbils, hamsters, rats, and mice. The antibody can be a purified or a recombinant antibody.Humanized forms of non-human antibodies are chimeric antibodies that contain minimal sequence derived from the non-human antibody. A humanized antibody is generally a human immunoglobulin (recipient antibody) in which residues from one or more CDRs are replaced by residues from one or more CDRs of a non-human antibody (donor antibody). The donor antibody can be any suitable non-human antibody, such as a mouse, rat, rabbit, chicken, or non-human primate antibody having a desired specificity, affinity, or biological effect. In some instances, selected framework region residues of the recipient antibody are replaced by the corresponding framework region residues from the donor antibody. Humanized antibodies can also comprise residues that are not found in either the recipient antibody or the donor antibody. Such modifications can be made to further refine antibody function. (See Jones et al. (1986) Nature, 321:522-525; Riechmann et al. (1988) Nature, 332:323-329; and Presta, (1992) Curr Op Struct Biol., 2:593-596).

[0032] A human antibody is one that possesses an amino acid sequence corresponding to that of an antibody produced by a human or a human cell, or derived from a non-human source that utilizes a human antibody repertoire or human antibody-encodingsequences (e.g., obtained from human sources or designed de novo). Human antibodies specifically exclude humanized antibodies.

[0033] The term “area under the curve” or “AUC” refers to the area under the curve of a receiver operating characteristic (ROC) curve, both of which are well known in the art. AUC measures are useful for comparing the accuracy of a classifier across the complete data range. Classifiers with a greater AUC have a greater capacity to classify unknowns correctly between two groups of interest (e.g., responders vs. non-responders to a treatment). ROC curves are useful for plotting the performance of a particular feature (e.g., the presence of the biomarker described herein) in distinguishing between two populations (e.g., patients that will respond to treatment and patients that will not). Typically, the feature data across the entire population (e.g., the cases and controls) are sorted in ascending order based on the value of a single feature. Then, for each value for that feature, the true positive and false positive rates for the data are calculated. The true positive rate is determined by counting the number of cases above the value for that feature and then dividing by the total number of cases. The false positive rate is determined by counting the number of controls above the value for that feature and then dividing by the total number of controls. Although this definition refers to scenarios in which a feature is elevated in cases compared to controls, this definition also applies to scenarios in which a feature is lower in cases compared to the controls (in such a scenario, samples below the value for that feature would be counted). ROC curves can be generated for a single feature as well as for other single outputs, for example, a combination of two or more features can be mathematically combined (e.g., added, subtracted, multiplied, etc.) to provide a single sum value, and this single sum value can be plotted in a ROC curve. Additionally, any combination of multiple features, in which the combination derives a single output value, can be plotted in a ROC curve. These combinations of features may comprise a test. The ROC curve is the plot of the true positive rate (sensitivity) of a test against the false positive rate (1 -specificity) of the test.

[0034] As used herein, "cancer antigen" refers to (i) tumor- specific antigens, (ii) tumor- associated antigens, (iii) cells that express tumor- specific antigens, (iv) cells that express tumor- associated antigens, (v) embryonic antigens on tumors, (vi) autologous tumor cells, (vii) tumor- specific membrane antigens, (viii) tumor- associated membrane antigens, (ix) growth factor receptors, (x) growth factor ligands, and (xi) any other type of antigen or antigen-presenting cell or material that is associated with a cancer.

[0035] As used herein, the term "cancer-specific immune response" refers to the immune response induced by the presence of tumors, cancer cells, or cancer antigens. In certain embodiments, the response includes the proliferation of cancer antigen specific lymphocytes. In certain embodiments, the response includes expression and upregulation of antibodies and T-cell receptors and the formation and release of lymphokines, chemokines, and cytokines. Both innate and acquired immune systems interact to initiate antigenic responses against the tumors, cancer cells, or cancer antigens. In certain embodiments, the cancer-specific immune response is a T cell response.

[0036] The term "carcinoma" is art recognized and refers to malignancies of epithelial or endocrine tissues, including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. The anti-CD137 antibodies and PD-1 antagonists described herein can be used to treat patients who have, who are suspected of having, or who may be at high risk for developing any type of cancer, including renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary. The term also includes carcinosarcomas, which include malignant tumors composed of carcinomatous and sarcomatous tissues. An "adenocarcinoma" refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0037] As used herein, a “clinical outcome” of treatment described herein includes a “complete response” (CR), a “partial response” (PR), “stable disease” (SD), or “progressive disease” (PD). A “complete response” to therapy means the disappearance of all detectable signs of cancer in response to treatment. A “partial response” is generally understood as meaning a decrease in tumor load in an individual, for example in terms of tumor number, size and / or growth rate. A partial response may increase the time to disease progression. “Stable Disease” is generally understood as meaning a disease state that is neither decreasing nor increasing in extent or severity i.e., absence of progression. “Progressive Disease” is generally understood as meaning a disease state that is increasing or worsening.

[0038] As used herein, the terms “comprises,” “comprising,” “containing,” “having,” and the like can have the meaning ascribed to them in U. S. Patent law and can mean “includes,” “including,” and the like; the terms “consisting essentially of’ and “consists essentially of’ likewise have the meaning ascribed in U. S. Patent law, and the terms are open-ended, allowing for the presence of more than that which is recited so long as basic or novel characteristics of that which is recited are not changed by the presence of more than that which is recited, but excludes prior art embodiments.

[0039] As used herein, the term “CD137L” or “CD 137 ligand” refers to a member of the tumor necrosis factor (TNF) family of transmembrane proteins. Alternative names and acronyms for CD137L in the art include “tumor necrosis factor superfamily member 9” (TNFSF9) and 4-1BB ligand (4-1BBL) (Alderson et al., (1994) Eur J Immunol 24(9):2219-2227.

[0040] “Diabodies,” as used herein may refer to dimeric antibody fragments. In each polypeptide of the diabody, a heavy-chain variable domain (VH) is linked to a light-chain variable domain (VL) but unlike single-chain Fv fragments, the linker between the VL and VH is too short for intramolecular pairing and as such each antigen-binding site is formed by pairing of the VH and VL of one polypeptide with the VH and VL of the other polypeptide. Diabodies thus have two antigen-binding sites, and can be monospecific or bispecific.

[0041] As used herein, the term “disease” refers to any condition or disorder that damages, interferes with, or dysregulates the normal function of a cell, tissue, or organ. In a disease such as cancer (e.g., lung cancer), the normal function of a cell, tissue, or organ can be altered to enable immune evasion and / or escape of cancer cells or tumors.

[0042] As used herein, the term “effective dose” or “effective dosage” is defined as an amount sufficient to achieve or at least partially achieve the desired effect. The term “therapeutically effective dose” is defined as an amount sufficient to cure or at least partially arrest the disease and its complications in a patient already suffering from the disease.Amounts effective for this use will depend upon the severity of the disorder being treated and the general state of the patient’s own immune system.

[0043] The term “epitope”, as used herein, means a component of an antigen capable of specific binding to an antigen binding construct or unit. Epitopes frequently consist of surface-accessible amino acid residues and / or sugar side chains and can have specific three-dimensional structural characteristics, as well as specific charge characteristics.Conformational and non-conformational epitopes are distinguished in that the binding to the former but not the latter is lost in the presence of denaturing solvents. An epitope can comprise amino acid residues that are directly involved in the binding, and other amino acidresidues, which are not directly involved in the binding. The epitope to which an antigen binding protein binds can be determined using known techniques for epitope determination such as, for example, testing for antigen binding protein binding to antigen variants with different point mutations.

[0044] As used herein, the term “high” or “elevated” may refer to a measure that is greater than normal, greater than a standard such as a predetermined measure or a subgroup measure or that is relatively greater than another subgroup measure. For example, high CD56 refers to a measure of CD56 that is greater than a normal CD56 measure. A normal CD 6 measure may be determined according to any method available to one skilled in the art. High CD56 may also refer to a measure that is equal to or greater than a predetermined measure, such as a predetermined cutoff or threshold. High CD56 may also refer to a measure of CD56 wherein a high CD56 subgroup has relatively greater levels of CD56 than another subgroup. For example, without limitation, according to the present disclosure, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a subgroup whose measure is high (i.e., higher than the median) and another subgroup whose measure is low. CD56 can be measured by any method known to one skilled in the art such as, for example, without limitation, using any standard immunohistochemical (IHC) method. In some cases, a “high” expression level may comprise a range of expression that is very high or high and a range of expression that is “moderately high” where moderately high is a level of expression that is greater than normal, but less than “very high” or “high.” As used herein, each of these uses of the word “high” can be alternatively referred to as “elevated.”

[0045] As used herein, the term “human antibody” includes antibodies having variable and constant regions (if present) of human germline immunoglobulin sequences. Human antibodies of the disclosure can include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo) (See, e.g., Lonberg et al., (1994) Nature 368(6474): 856-859); Lonberg, (1994) Handbook of Experimental Pharmacology 113:49-101; Lonberg & Huszar, (1995) Intern. Rev. Immunol. 13:65-93, and Harding & Lonberg, (1995) Ann. N. Y. Acad. Sci. 764:536-546). However, the term “human antibody” does not include antibodies in which CDR sequences derived from the germline of anothermammalian species, such as a mouse, have been grafted onto human framework sequences (i.e. humanized antibodies).

[0046] The terms “inducing an immune response” and “enhancing an immune response” are used interchangeably and refer to the stimulation of an immune response (i.e., either passive or adaptive) to a particular antigen. The term “induce” as used with respect to inducing CDC or ADCC refer to the stimulation of particular direct cell killing mechanisms.

[0047] As used herein, the terms “inhibits,” “blocks” or “reduces” (e.g., when referring to inhibition / blocking of the PD-1 / PD-L1 signaling pathway) are used interchangeably and encompass both partial and complete inhibition / blocking as well as direct and allosteric inhibition / blocking. As used herein, “inhibition,” “blocking” or “reducing” are also intended to include any measurable decrease in biological function and / or acti vity of a target (e.g., PD-1, PD-L1). For example, when an antibody or an antigenbinding fragment thereof (e.g., an anti-PD-1 or an anti-PD-Ll antibody) is in contact with the target as compared to the target not in contact with an antibody or antigen-binding fragment. In some embodiments, an antibody or antigen -binding fragment thereof, that targets PD-1 or PD-L1, inhibits or reduces PD-1 or PD-L1 function and / or activity in a given system by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35 %■, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%.

[0048] As used herein, the term “inhibits growth” (e.g., referring to cells) is intended to include any measurable decrease in the growth of a cell, e.g., the inhibition of growth of a cell by at least about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, 70%, about 80%, about 90%, about 99%, or 100%.

[0049] As used herein, a subject “in need of prevention,” “in need of treatment,” or “in need thereof,” refers to one, who by the judgment of an appropriate medical practitioner (e.g., a doctor, a nurse, or a nurse practitioner in the case of humans; a veterinarian in the case of non-human mammals), would reasonably benefit from a given treatment (such as treatment with a composition comprising an anti-CD137 antibody and / or PD-1 antagonist).

[0050] As used herein, the term "in vivo" refers to processes that occur in a living organism.

[0051] As used herein, the terms “induces,” “increases,” “enhances,” or “stimulates” (e.g., when referring to an increase in CD137 activity) are used interchangeably and encompass both increases in activity and de novo activity (e.g., inducing activity from a previously undetectable level). The enhancement of CD 137 increases the normal level or type of activity that occurs from CD 137 in a given system in the absence of an anti-CD137 antibody or fragment thereof as the enhancer. Enhancement, induction, or stimulation are also intended to include any measurable increase in CD 137 activity (or effect on a given cell type) when in contact with an anti-CD137 antibody as compared to CD 137 not in contact with an anti-CD137 antibody, e.g., enhances / increases CD 137 activity in a given system (CD137-mediated increases in T cell activity) by at least about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, or 100%, or at least about 1.5-fold, 2-fold, 3-fold, 4-fold, 5-fold, 10-fold, 20-fold, 50-fold, and the like.

[0052] As used herein, the term “isolated antibody” is intended to refer to an antibody which is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds to human CD137, PD-1, or PD-L1 is substantially free of antibodies that specifically bind antigens other than CD137, PD-1, or PD-L1). An isolated antibody that specifically binds to an epitope may, however, have cross-reactivity to other CD 137, PD-1, or PD-L1 proteins from different species. However, the antibody continues to display specific binding to human CD137, PD-1, or PD-L1 in a specific binding assay as described herein. In addition, an isolated antibody is typically substantially free of other cellular material and / or chemicals. In some embodiments, a combination of “isolated” antibodies having different CD137, PD-1, or PD-L1 specificities is combined in a well-defined composition.

[0053] As used herein, the terms “immune therapy,” “immunotherapy,” and “immunologic therapy” refer to the treatment of disease by activating or suppressing the immune system. Activation immunotherapies amplify immune responses, and suppression immunotherapies reduce or suppress immune response.

[0054] As used herein, the term “low” may refer to a measure that is less than normal, less than a standard such as a predetermined measure or a subgroup measure that is relatively less than another subgroup measure. For example, low CD56 means a measure of CD56 thatis less than a normal CD56 measure in a particular set of samples of patients. A normal CD56 measure may be determined according to any method available to one skilled in the art. Low CD56 may also mean a measure that is less than a predetermined measure, such as a predetermined cutoff or threshold. Low CD 6 may also mean a measure wherein a low CD 6 subgroup is relatively lower than another subgroup. For example, without limitation, according to the present disclosure, two distinct patient subgroups can be created by dividing samples around a mathematically determined point, such as, without limitation, a median, thus creating a group whose measure is low (i.e., less than the median) with respect to another group whose measure is high (i.e., greater than the median). CD56 can be measured by any method known to one skilled in the art such as, for example, without limitation, using any standard immunohistochemical (IHC) method.

[0055] As used herein, the term “monoclonal antibody” refers to an antibody which displays a single binding specificity and affinity for a particular epitope. Accordingly, the term “human monoclonal antibody” refers to an antibody which displays a single binding specificity and which has variable and optional constant regions derived from human germline immunoglobulin sequences. In some embodiments, human monoclonal antibodies are produced by a hybridoma which includes a B cell obtained from a transgenic non-human animal, e.g., a transgenic mouse, having a genome comprising a human heavy chain transgene and a light chain transgene fused to an immortalized cell.

[0056] As used herein, the term "PD-1 antagonist" refers to any chemical compound or biological molecule that inhibits the PD-1 signaling pathway or that otherwise inhibits PD-1 function in a cell (e.g., an immune cell). In some embodiments, a PD-1 antagonist blocks binding of PD-L1 to PD-1 and / or PD-L2 to PD-1. In some embodiments, the PD-1 antagonist specifically binds PD-1. In some embodiments, the PD-1 antagonist specifically binds PD-Ll. In some embodiments, the PD-1 antagonist is an isolated monoclonal antibody that specifically binds human PD-1, or an antigen-binding fragment thereof. Exemplary PD-1 antagonists comprising anti -PD-1 antibodies, or antigen-binding fragments thereof, are described herein. In some embodiments, the PD-1 antagonist is an isolated monoclonal antibody that specifically binds human PD-L1, or an antigen-binding fragment thereof.Exemplary PD-1 antagonists comprising anti-PD-Ll antibodies, or antigen-binding fragments thereof, are described herein.

[0057] As used herein, the term “pre-determined cutoff” may refer to the value of a predetermined measure on subjects exhibiting certain attributes that allow the best discrimination between two or more categories of an attribute. For example, a pre-determined cutoff that allows one to discriminate between two categories such as high CD56 expression and low CD56 expression for determining overall survival may be used. Pre-determined cutoffs may be used to separate the subjects with values lower than or higher than the predetermined cutoff to optimize the prediction model.

[0058] As used herein, the term “preventing” when used in relation to a condition, refers to administration of a composition which reduces the frequency of, or delays the onset of, symptoms of a medical condition in a subject relative to a subject which does not receive the composition.[0059 j As used herein, the term “prognoses,” “prognosis,” “prognosed,” “prognosticate,” “prognosticated,” or “prognosing” relates to providing a forecast or prediction of the likely outcome of a cancer after treatment with and anti-CD137 antibody and / or an anti-PD-1 antibody. Furthermore, the difference between “prognosis” and “treatment response” is a key concept in oncology. While patients can have a good prognosis, they still can have no treatment response and experience only side effects. Thus, it is essential to predict the prognosis or the overall survival of a patient and separately predict the treatment outcome and treatment response.

[0060] As used herein, the term “Programmed Cell Death Protein 1” or “PD-1” refers to the Programmed Cell Death Protein 1 polypeptide, an immune -inhibitory receptor belonging to the CD28 family and is encoded by the PDCD1 gene in humans. Alternative names or synonyms for PD-1 include: PDCD1, PD1, CD279, and SLEB2. PD-1 is expressed predominantly on previously activated T cells, B cells, and myeloid cells in vivo, and binds to two ligands, PD-L1 and PD-L2. The term "PD-1" as used herein includes human PD-1 (hPD-1), variants, isoforms, and species homologs of hPD-1, and analogs having at least one common epitope with hPD-1. The complete hPD-1 sequence can be found under GenBank Accession No. AAC51773.

[0061] As used herein, the term "Programmed Death Ligand- 1” or “PD-L1" is one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that downregulates T cell activation and cytokine secretion upon binding to PD-1. Alternative names andsynonyms for PD-L1 include: PDCD1L1, PDL1, B7H1, B7-4, CD274, and B7-H. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-Ll, and analogs having at least one common epitope with hPD-Ll. The complete hPD-Ll sequence can be found under GenBank Accession No. Q9NZQ7.

[0062] As used herein the term “paratope” includes the antigen binding site in the variable region of an antibody that binds to an epitope. "Single-chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Generally, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the desired structure for antigen binding.

[0063] As used herein, the term “subject” includes any human or non-human animal. For example, the methods and compositions of the present invention can be used to treat a subject with an immune disorder. The term “non-human animal” includes all vertebrates, e.g., mammals and non-mammals, such as non-human primates, sheep, dog, cow, chickens, amphibians, reptiles, etc.

[0064] As used herein, the term “stratifying” or “stratification” refers to sorting patients into those who may or may not benefit from cancer therapy.

[0065] As used herein, the term “tumor” or “tumor cells” refers to all neoplastic cell growth and proliferation, whether malignant or benign, and all pre-cancerous and cancerous cells and tissues. Within the context of the present disclosure, the treatment of malignant tumors, i.e. cancers, is preferred. Thus, uses and methods described herein may be used to treat tumors, including both solid and non-solid tumors.

[0066] As used herein, the term “treat” or “treatment” means to reduce, stabilize, or inhibit progression of a symptom, such as tumor size, number of metastases or other symptoms which are caused by / associated with the presence and / or progression of a tumor. A non-limiting exemplary list of cancerous diseases and tumors which can be treated with cancer therapy, is provided herein. These tumors described herein may be metastatic or non-metastatic.

[0067] As used herein, the term “tumor microenvironment” (alternatively “cancer microenvironment”; abbreviated TME) refers to the cellular environment or milieu in whichthe tumor or neoplasm exists, including surrounding blood vessels as well as non-cancerous cells including, but not limited to, immune cells, fibroblasts, bone marrow-derived inflammatory cells, and lymphocytes. Signaling molecules and the extracellular matrix also comprise the TME. The tumor and the surrounding microenvironment are closely related and interact constantly. Tumors can influence the microenvironment by releasing extracellular signals, promoting tumor angiogenesis and inducing peripheral immune tolerance, while the immune cells in the microenvironment can affect the growth and evolution of tumor cells.

[0068] As used herein, the term “tumor sample” (also referred to as “tissue sample”) is preferably derived from a subject and may be obtained via biopsy such as needle biopsy, surgical biopsy, bone marrow biopsy etc. A tumor sample may therefore include a tumor, parts of a tumor, tumor cells derived from a tumor (including tumor cell lines which may be derived from a tumor and which are grown in cell culture), but also tumor cell lines as such, and ceils and / or tissue which are / is derived from a subject and which are / is suspected of being tumorigenic or even cancerous or which are / is suspected of comprising tumorigenic or cancerous cells. A tumor tissue sample also encompasses pieces or slices of tissue that have been removed from the tumor and / or the surrounding tissue, including surgical tumor resection or the collection of a tissue sample by biopsy. A tissue sample may be obtained for the purpose of in vitro evaluation. In some embodiments, the tumor sample may result from the tumor resected from the patient. In some embodiments, the tumor sample may result from a biopsy performed in the primary tumor of the patient or performed in metastatic sample distant from the primary tumor of the patient. Generally, the tumor tissue sample may be fixed in formalin and embedded in a rigid fixative, such as paraffin (wax) or epoxy, which is placed in a mold and later hardened to produce a block which is readily cut. Thin slices of material can be then prepared using a microtome, placed on a glass slide and submitted e.g. to immunohistochemistry. The tumor tissue sample can be used in microarrays, called as tissue microarrays (TMAs). TMA consists of paraffin blocks in which up to 1000 separate tissue cores are assembled in array fashion to allow multiplex histological analysis. This technology allows rapid visualization of molecular targets in tissue specimens at a time, either at the DNA, RNA or protein level.

[0069] As used herein, the term “T cell” refers to a type of white blood cell that can be distinguished from other white blood cells by the presence of a T cell receptor on the cell surface. There are several subsets of T cells, including, but not limited to, T helper cells(a.k.a. TH cells or CD4” T cells) and subtypes, including THI, TH2, TH3, TH17, TH9, and TFH cells, cytotoxic T cells (i.e., Tc cells, CD8+T cells, cytotoxic T lymphocytes, T-killer cells, killer T cells), memory T cells and subtypes, including central memory T cells (TCM cells), effector memory T cells (TEM and TEMRA cells), and resident memory T cells (TRM cells), regulatory T cells (a.k.a. Tregcells or suppressor T cells) and subtypes, including CD4+FOXP3+Tregcells, CD4+FOXP3-Tregcells, Tri cells, Th3 cells, and Treg17 cells, natural killer T cells (a.k.a. NKT cells), mucosal associated invariant T cells (MAITs), and gamma delta!’ cells (y8T cells), including Vy9 / V52 T cells. Any one or more of the aforementioned or unmentioned T cells may be the target cell type for a method of use of the invention.

[0070] As used herein, the terms “T cell activation” or “activation of T cells” refers to a cellular process in which mature T cells, which express antigen-specific T cell receptors on their surfaces, recognize their cognate antigens and respond by entering the cell cycle, secreting cytokines or lytic enzymes, and initiating or becoming competent to perform cellbased effector functions. T cell activation requires at least two signals to become fully activated. The first occurs after engagement of the T cell antigen-specific receptor (TCR) by the antigen-major histocompatibility complex (MHC), and the second by subsequent engagement of co-stimulatory molecules (e.g., CD28). These signals are transmitted to the nucleus and result in clonal expansion of T cells, upregulation of activation markers on the cell surface, differentiation into effector cells, induction of cytotoxicity or cytokine secretion, induction of apoptosis, or a combination thereof.

[0071] As used herein, the term “T cell-mediated response” refers to any response mediated by T cells, including, but not limited to, effector T cells (e.g., CD8+cells) and helper T cells (e.g., CD4+cells). T cell mediated responses include, for example, T cell cytotoxicity and proliferation.

[0072] As used herein, the terms “therapeutically effective amount” or “therapeutically effective dose,” or similar terms used herein are intended to mean an amount of an agent (e.g., an anti-CD137 antibody or an antigen-binding fragment thereof or PD-1 antagonist) that will elicit the desired biological or medical response (e.g., an improvement in one or more symptoms of a cancer).

[0073] The terms “treat,” “treating,” and “treatment,” as used herein, refer to therapeutic or preventative measures described herein. The methods of “treatment” employadministration to a subject, in need of such treatment, a human antibody of the present disclosure, for example, a subject in need of an enhanced immune response against a particular antigen or a subject who ultimately may acquire such a disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disorder or recurring disorder, or in order to prolong the survival of a subject beyond that expected in the absence of such treatment. "Treatment" as used herein covers any treatment of a disease in a mammal, and includes: (a) preventing the disease from occurring in a subject which may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., slowing or arresting its development; or (c) relieving the disease, i.e., causing regression of the disease. The therapeutic agent may be administered before, during or after the onset of disease or injury. The treatment of ongoing disease, where the treatment stabilizes or reduces the undesirable clinical symptoms of the patient, is of particular interest. The subject therapy may be administered prior to, during the symptomatic stage of the disease, and in some cases after the symptomatic stage of the disease. In some embodiments the expression of at least one immune marker is measured using mass spectrometry by time of flight (CyTOF) or Next-generation sequencing (NGS), or quantitative polymerase chain reaction (qPCR) or any other method known in the art to measure expression levels of an immune marker. In various embodiments at least one immune marker is CD56,

[0074] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure pertains. Preferred methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the presently disclosed methods and compositions. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety.Use of The Novel Biomarker to Identify Patients

[0075] The present disclosure is based, at least in part, on the discovery that there is a highly significant relationship between biological marker CD56, as expressed in various cancer cells as detected on whole slide tissue sections as well the expression of CD56 in NK cells, and circulating CD56 and the clinical outcome of treatment, encompassing complete response (CR), partial response (PR), stable disease (SD), and / or progressive disease (PD).

[0076] As described herein, the methods provided may be used to predict or determine likelihood of a CR or PR, or whether a response is likely to be a CR or a PR. As generally understood, a ‘complete response’ to therapy means the disappearance of all detectable signs of cancer in response to treatment. A ‘partial response’ is generally understood as meaning a decrease in tumor load in an individual, for example in terms of tumor number, size and / or growth rate. A partial response may increase the time to disease progression. As used herein, stable disease is generally understood as meaning a disease state that is neither decreasing nor increasing in extent or severity i.e., absence of progression.

[0077] As described herein, the expression of CD56 in tumor cells as well as expression of CD56 in circulating immune cells and immune cells infiltrating the tumor and their location in the tumor microenvironment may be a key predictor of response to treatment or therapy. The data described herein show, using samples from patients suffering from neuroendocrine tumors (NETs), melanoma, small cell lung cancer, and head and neck cancer who were treated with anti-CD137 therapy, that a certain set of conditions favors CD137 antagonism and as a result may mediate or ameliorate tumor progression. Specifically, as mentioned above, there is a significant correlation between the density of CD56+ tumor cells and CD56+ immune cells, in particular T cells, more particularly tumor infiltrating T cells (TILs), at the tumor site and response to therapy, in particular response immune therapy, for example anti-CD137 therapy. In addition, the level of circulating CD56 may also play a role in predicting whether a subject will respond to therapy or treatment. In fact, it was shown that a lasting response to anti-CD137 therapy is correlated with a high density of CD56+ cells on tumor cells themselves or near the site of the tumor.

[0078] Furthermore, the present disclosure is also based, at least in part, on the discovery that the biomarker CD56 (also referred to as “NCAM”), either alone or in combination with one or more additional biomarkers, is predictive of the responsiveness of cancer to treatment with immune therapy, for example, an CD137 agonist and / or PD-l / PD-L1 blockade.

[0079] Accordingly, provided herein are methods for predicting the efficacy of immune therapy in a subject suffering from cancer comprising: obtaining a biological sample from the subject; assessing the biological sample by measuring a density of cells that are CD56-positive; determining a level of CD56-positive cells wherein a high number of total cells expressing CD56 is predictive of the subject’s responsiveness to immune therapy. Insome aspects, responsiveness to treatment includes improved disease-free survival (DFS), an increase in progression free survival (PFS), improved disease specific survival (DSS), or an increased overall survival (OS). In some aspects, responsiveness to treatment comprises SD, CR or PR.

[0080] The present disclosure also relates to methods for identifying subjects likely to respond to treatment with a monoclonal antibody targeting CD137 comprising: obtaining a biological sample from the subject suffering from cancer; and measuring the level of CD56-positive cells inside a tumor microenvironment to obtain total amount of CD56-positive cells, wherein the total amount of CD56-positive cells is predictive of the subject’s responsiveness to treatment,[0081 j The present disclosure also relates to method s of predicting efficacy of treatment in a subject suffering from disease and may comprise: obtaining a biological sample from the subject; assessing the biological sample by measuring levels of CD56-positive cells, CD137-positive immune cells, PD-l-positive immune cells in a tumor microenvironment and / or circulating CD56; measuring the levels of CD56-positive cells, CD 137-positive immune cells, PD-l-positive immune cells in a tumor microenvironment and / or circulating levels of CD56 to determine a total number of CD56-positive cells, CD 137-positive immune cells, PD-l-positive immune cells and / or circulating CD56; and evaluating the total number of CD56-positive cells, CD137-positive immune cells, PD-l-positive immune cells and / or circulating CD56 to predict the subject’s responsiveness to treatment. In some aspect, the subject is suffering from cancer. In some aspects, the treatment may include an immune checkpoint inhibitor (ICT). In some aspects, the treatment may include an anti-CD137 antibody, an anti-PD-1 antibody, and / or an anti-CTL4 antibody. In some aspects, the disease (e.g., cancer) may be treated with agonistic CD137 antibody monotherapy. In some aspects, the circulating CD56 is measured in a subject’s blood, plasma, and / or serum.

[0082] In some aspects, the systems and methods described herein may predict or determine the likelihood of SD, CR or PR to treatment with a CD137 agonist or PD-1 / PD-L1 blockade, and / or predict whether a response is likely to be a CR or a PR. In some aspects, the methods described herein may assess baseline tumor characteristics to predict favorable clinical responses to treatment with agonist anti-CD137 antibodies, PD-1 antagonists, anti-CTL4 antibodies, or combinations thereof.

[0083] Still, in some aspects, the methods provided herein may identify subjects with a higher probability of responding to therapy with agonist anti-CD137 antibodies and / or PD-1 antagonists by measuring or quantifying the levels of CD56-positive tumor cells, measuring or quantifying levels of immune cells expressing CD56, and / or circulating CD56. In some examples, the CD56-positive immune cells may be inside the tumor microenvironment or infiltrating immune cells. In some aspects, the CD56-positive immune cells may be outside of the tumor microenvironment or circulating. In some aspects, the immune cells are NK cells. In some aspects, levels of circulating CD56 are measured and may be a predictive biomarker. Further, circulating CD56 may include soluble CD56 found in the biological sample, for example, in the blood, plasma, and / or serum. Soluble CD56 may be shed from cells and may no longer be on the cell surface, thus circulating CD56 may be free-floating in blood, plasma, and / or serum.

[0084] Also described herein are methods for determining whether a subject suffering from cancer is likely to respond to treatment. The methods include obtaining a sample from a subject, and evaluating the presence and / or level of CD56 in the sample, and comparing the presence and / or level with one or more references, e.g., a control reference that represents a normal level of CD56, e.g., a level in an unaffected subject, and / or a disease reference that represents a level of the proteins associated with CD56, e.g., a level in a subject having cancer. In some embodiments, the methods include determining a value, e.g., a normalized expression value, for each of the biomarker CD56 and calculating a test score from the sum of each of the levels. This score can then be compared to a reference score, wherein the presence of a test score above (e.g., equal to or above, or simply above) the reference score indicates a higher likelihood that the subject may respond to treatment. In some aspects, the treatment may include an ICI. In some aspects, treatment may include an anti-CD137 antibody, an anti-PD-1 antibody, an anti-CTL4 antibody or any combination thereof.

[0085] In certain embodiments, the disclosure may provide methods for predicting treatment response in a subject. The method may include obtaining a sample from the subject; detecting levels of CD56 in the sample; comparing the levels of CD56 in the sample to reference levels; and treating a subject who has levels of CD56 above the reference levels. In some aspects, the subject may be suffering from cancer. In some aspects, the treatment may include an ICI. In some aspects, treatment may include an anti-CD137 antibody, an anti-PD-1 antibody, an anti-CTL4 antibody or any combination thereof.

[0086] In certain embodiments of each of the methods and / or aspects disclosed herein, the level of CD56 may be evaluated as either high or low. Accordingly, in some aspects, if the level of CD56 is high, the subject may be more likely to respond to the targeted therapy. In certain embodiments, if the level of CD56 is low, the subject may be less likely to respond to the targeted therapy. In certain embodiments, the targeted therapy may include an ICI inhibitor. In some aspects, the targeted therapy may include an anti-CD137 antibody, an anti-PD-1 antibody, an anti-CTL4 antibody or any combination thereof.

[0087] Thus, in certain embodiments of each of the methods and aspects of the invention as disclosed herein, the method comprises measuring in a biological sample obtained from the subject, amount or levels of CD56. Amounts or levels of CD56 may include, for example, intra-tumoral CD56, extra-tumoral CD56, and / or circulating CD56, wherein if the amount or level of CD56 is high or moderately high then the subject may be likely to respond to treatment and / or treatment may be more likely to result in CR, PR, or SD.

[0088] In certain embodiments, the amount or levels of CD56 may be above a first threshold, such that the sample is stratified as having a “high” amounts or levels of CD56. This could lead to an optimal CD56 “high” threshold such as 205 CD56+ tumor cells per mm2(see e.g., FIGs. 10A and 10B). In some aspects, the amount or levels of CD56 may be above a second threshold, such that the sample is stratified as having a “moderately high” amounts or levels of CD56. Threshold ranges may differ depending upon patient cohort and / or timing of obtaining a sample (e.g., prior to, during, or following treatment). Thus, each of the threshold values and / or threshold ranges described herein may vary.Use of the Novel Biomarker to Treat Patients

[0089] The present disclosure also relates to methods of treating a subject suffering from a cancer that will be responsive to immune therapy comprising: obtaining a biological sample from the subject; assessing the biological sample by measuring a density of cells that are CD56-positive and / or measuring circulating levels of CD56; determining a level of CD56-positive cells and / or levels of circulating CD56; wherein a high number of total cells expressing CD56 and / or in combination with the level of circulating CD56 is predictive of the subject’s responsiveness to immune therapy; and treating the subject. In some aspects, responsiveness to treatment includes improved disease-free survival (DFS), an increase in progression free survival (PFS), improved disease specific survival (DSS), or an increased overall survival (OS). In some aspects, responsiveness to treatment comprises SD, CR or PR.

[0090] The present disclosure also relates to methods for treating a subject suffering from a cancer that will be responsive to treatment with a monoclonal antibody targeting CD137 comprising: obtaining a biological sample from the subject suffering from cancer: and measuring a level of CD56-positive cells inside a tumor microenvironment, and / or a level of CD56-positive cells outside the tumor microenvironment, and / or circulating CD56 to obtain total amount of CD56, wherein the total amount of CD56 in the biological sample is predictive of the subject’s responsiveness to treatment; and treating the subject with a monoclonal antibody targeting CD 137.

[0091] The present disclosure also relates to methods for treating a subject suffering from a disease that will be responsive to treatment with an immune therapy comprising: obtaining a biological sample from the subject; assessing the biological sample by measuring levels of CD56-positive cells, CD 137-positive immune cells, PD-l-positive immune cells in a tumor microenvironment and / or circulating levels of CD56; comparing the levels of CD56-positive cells, CD137-positive immune cells, PD-l-positive immune cells in a tumor microenvironment and / or circulating levels of CD56 to determine a total number of CD56-positive cells, CD137-positive immune cells, PD-l-positive immune cells and / or circulating CD56; and evaluating the total number of CD56-positive cells, CD137-positive immune cells, PD-l-positive immune cells and / or circulating CD56 to predict the subject’s responsiveness to immune therapy treatment; and treating the subject. In some aspect, the subject is suffering from cancer. In some aspects, the disease (e.g., cancer) may be treated with an ICI such as agonistic CD 137 antibody monotherapy.

[0092] The present disclosure may also relate to methods for treating a subject suffering from cancer comprising: obtaining a biological sample from the subject, wherein the biological sample comprises at least tumor tissue or blood; assessing the biological sample by determining the level of CD56-positive cells and / or levels of circulating CD56; and administering immune therapy to subjects having a higher level of total CD56-positive cells and / or circulating CD56 as compared to a disease free control or a sample from a subject suffering from progressive disease. In some aspects, subjects may be characterized as likely responders or non -responders.

[0093] In some embodiments, the methods described herein may be used to stratify subjects as responders or non -responders to anti-CD137 and / or anti-PD-Ll therapy and to select subjects in clinical practice, in the design of a clinical trial, or in drug discovery ordevelopment, whether relating to a single-agent therapy or a combination therapy. In further embodiments, the methods provided herein may be used to guide or effect treatment decisions in clinical practice, in a clinical trial, or in drug discovery or development. In some aspects, the methods may relate to a single-agent therapy or a combination therapy. In some aspects, the methods may help identify a subject with a particular type of tumor or cancer that will respond to treatment with a particular ICI, for example, anti-CD137 and / or anti-PD-Ll therapy.

[0094] In one embodiment at least one of cancer cell division, tumor growth, tumor size, tumor density, or tumor metastasis is reduced in a subject suffering from cancer. In another aspect, the present disclosure provides a method of improving overall survival in a subject with a solid tumor, the method comprising: obtaining a tumor sample from the subject, assessing the sample for biomarker CD56 including determining the amount of intratumoral CD56+ NK cells (i.e., tumor infiltrating) combined with the amount of CD56+ tumor cells in the TME, determining the amount of circulating CD56+ NK cells (i.e., CD56+ NK cells found outside the TME), and administering an effective amount of an anti-CD137 antibody and / or an anti-PD-1 antibody to the subject if the sample comprises an elevated amount of intratumoral CD56+ NK cells (i.e., tumor infiltrating) combined with the amount of CD56+ tumor cells in the TME and the amount of circulating CD56+ NK cells. In some embodiments, the total combined amount of intratumoral CD56+ NK cells, CD56+ tumor cells in the TME and circulating CD56+ NK cell may be compared to a healthy control sample and / or the subject’s baseline levels of CD56. Alternatively, or in addition, the total combined amount of intratumoral CD56+ NK cells, CD56+ tumor cells in the TME and circulating CD56+ NK cell may be compared to a sample showing progressive disease.

[0095] In other aspects, the present disclosure provides a method of improving overall survival in a subject with a solid tumor, comprising: (a) obtaining a tumor sample from the patient; (b) assessing the sample for levels of CD56; and (c) administering an effective amount of an anti-CD137 and / or anti-PD-1 antibody to the patient if the sample comprises high levels of CD56 expression as compared to a healthy control.

[0096] In some aspects, the subject is treated with an immune checkpoint inhibitor (ICI). In some aspects the subject is treated with an anti-CD137 antibody, an anti-PD-1 antibody, and / or an anti-CTL4 antibody. Exemplary CD137 agonist may include, but are not limited to, urelumab and utolimumal. Other forms of treatment may include antibodies suchas anti-CD137 IgGl, e.g., botensilimab, and anti-IgG4 e.g., CTX-471, pembrolizumab, and nivolumab, or any other suitable GDI 37 agonist, for example, as described in Melero, I., et al., (2023) CD137 (4-lBB)-Based Cancer Immunotherapy on Its 25th Anniversary. Cancer Discov 13 (3): 552-569. Additional treatments may include chemotherapy and immunotoxin therapy, and / or NC 'AM-targeted therapy. Moreover, any treatment may be used in combination. For example, a CD137 agonist may be administered in combination with an anti-CTL4 antibody.

[0097] Moreover, antibody molecules used in the treatment described herein may comprise a diabody, and a single-chain molecule, as well as an antigen binding fragment of an antibody (e.g., Fab, F(ab')2, and Fv). The antibody molecules used in the treatment described herein include at least one binding domain specific for CD137, PD-I, and / or CTL4. For example, an antibody molecule used herein can include a heavy (H) chain variable domain sequence (abbreviated herein as VH), and a light (L) chain variable domain sequence (abbreviated herein as VL). In some embodiments, an antibody molecule used herein comprises or consists of a heavy chain and a light chain (referred to as a half antibody). In another example, an antibody molecule used herein includes two heavy (H) chain variable domain sequences and two light (L) chain variable domain sequence, thereby forming two antigen binding sites, such as Fab, Fab', F(ab')2, Fc, Fd, Fd', Fv, single chain antibodies (scFv, for example), single variable domain antibodies, diabodies (Dab) (bivalent and bispecific), and chimeric (e.g., humanized) antibodies, which may be produced by the modification of whole antibodies or those synthesized de novo using recombinant DNA technologies. These functional antibody fragments retain the ability to selectively bind with their respective antigen. Antibodies and antibody fragments used herein can be from any class of antibodies including, but not limited to, IgG, IgA, IgM, IgD, and IgE, and from any subclass (e.g., IgGl, IgG2, IgG3, and IgG4) of antibodies. The preparation of antibody molecules can be monoclonal or polyclonal. An antibody molecule used herein can also be a human, humanized, CDR-grafted, or an in vitro generated antibody. The antibody can have a heavy chain constant region chosen from, e.g., IgGl, IgG2, IgG3, or IgG4. The antibody can also have alight chain chosen from either kappa or lambda light chains. As described herein, bispecific antigen binding constructs that can be used in the methods described herein include bispecific, trispecific, tetraspecific, or multispecific antibodies or antigen binding fragments thereof.

[0098] Antigen binding fragments of an antibody molecule are well known in the ait, and include, for example, (i) a Fab fragment, a monovalent fragment consisting of the VL,VH, CL and CHI domains; (ii) aF(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHI domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a diabody (dAb) fragment, which consists of a VH domain; (vi) a camelid or camelized variable domain; (vii) a single chain Fv (scFv) (see e.g., Bird et al. (1988) Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sei. USA 85:5879-5883); (viii) a single domain antibody. These antibody fragments are obtained using conventional techniques known to those skilled in the art, and the fragments are screened for utility in the same manner as are intact antibodies.

[0099] Antibody molecules used in the methods described herein can also be single domain antibodies that include at least one binding domain specific for CD 137, PD-1, and / or CTL4. Single domain antibodies can include antibodies whose complementary determining regions are part of a single domain polypeptide. Examples include, but are not limited to, heavy chain antibodies, antibodies naturally devoid of light chains, single domain antibodies derived from conventional 4-chain antibodies, engineered antibodies and single domain scaffolds other than those derived from antibodies. Single domain antibodies may be any of the art, or any future single domain antibodies. Single domain antibodies may be derived from any species including, but not limited to mouse, human, camel, llama, fish, shark, goat, rabbit, and bovine.

[0100] In some embodiments, antibody molecules used in the treatment described herein may include an antigen binding unit that may also be or can also comprise, e.g., a nonantibody, scaffold protein. For example, antibody molecules used in the treatment described herein may comprise optimal selectivity and affinity for CD 137, PD-1, and / or CTL4. These proteins are, generally, obtained through combinatorial chemistry-based adaptation of preexisting antigen-binding proteins. For example, the binding site of human transferrin for human transferrin receptor can be diversified using the system described herein to create a diverse library of transferrin variants, some of which have acquired affinity for different antigens. See, e.g., Ali el al. (1999) J. Biol. Chem. 274:24066-24073. The portion of human transferrin not involved with binding the receptor remains unchanged and serves as a scaffold, like framework regions of antibodies, to present the variant binding sites. The libraries are then screened, as an antibody library is, and in accordance with the methods described herein, against a target antigen of interest to identify those variants having optimalselectivity and affinity for the target antigen. See, e.g., Hey et al. (2005) TRENDS Biotechnol 23(10):514-522.

[0101] In some embodiments, the bispecific antigen binding construct used in the methods described herein may comprise a bispecific antibody, having specificity for at least two antigens but optionally having more than two binding sites, where at least one of the portions of the bispecific antibody is specific for CD137, PD-1, and / or CTL4. A bispecific antibody molecule is characterized by a first immunoglobulin variable domain sequence that has binding specificity for a first antigen and a second immunoglobulin variable domain sequence that has binding specificity for a second antigen. In some embodiments, a bispecific antibody molecule comprises a scFv or fragment thereof having binding specificity for a first antigen and a scFv or fragment thereof having binding specificity for a second antigen. (See, e.g., Kontemiann and Brinkmann (2015) Drag Discovery Today 20(7): 838-47.). A bispecific antigen binding construct can be a single multifunctional polypeptide, small molecule, or aptamer, or it can be a multimeric complex of two or more molecules that are covalently or non-covalently associated with one another. Bispecific antigen binding constructs include antibodies (or antigen binding fragments thereof) that may be linked to or co-expressed with another functional molecule, e.g., another peptide, protein, and / or aptamer. For example, an antibody or fragment thereof can be functionally linked (e.g., by chemical coupling, genetic fusion, non-covalent association or otherwise) to one or more other molecular entities, such as a protein or fragment thereof to produce a bispecific antigen binding construct with a second binding specificity. In certain embodiments, an antibody or antigen binding fragment thereof is functionally linked to one or more antibody or antigen binding fragment thereof having a different binding specificity to produce a bispecific antigen binding construct. Each antibody or antigen binding portion thereof of the construct may have one or more antigen binding specificities.

[0102] Various bispecific antibody formats are known in the art, including, for example, a bispecific IgG, a bispecific antibody fragment, a bispecific fusion protein, an appended IgG, and a bispecific antibody conjugate, described herein. Exemplary bispecific formats that can be used in the context of the present disclosure include, without limitation, e.g., scFv-based or diabody bispecific formats, IgG-scFv fusions, dual variable domain (DVD)-lg, Quadroma, knobs-into-holes, common light chain (e.g., common light chain with knobs-into-holes, etc.), CrossMab, CrossFab, (SEED)body, leucine zipper, Duobody, IgGl / lgG2, dual acting Fab (DAF)-lgG, and Mab2 bispecific formats (see, e.g., Klein et al. (2012) mAbs 4:6, 1-11, and references cited therein, for a review of the foregoing formats). (See also Spiess et al. (2015) Mol Immunol 67:95-106.) Bispecific antibodies can also be constructed using peptide / nucleic acid conjugation, e.g., wherein unnatural amino acids with orthogonal chemical reactivity are used to generate site-specific antibody-oligonucleotide conjugates which then self-assemble into multimeric complexes with defined composition, valency and geometry. (See, e.g., Kazane et al. (2013) J Am Chem Soc 135(1):340-6). In some embodiments, the bispecific antigen binding construct disclosed herein comprises a common light chain. In each of the embodiments described herein, the antibody, or antibody construct, or antigen binding fragment thereof comprises antibodies or CDRs antibodies that bind to CD 137, PD-1, and / or CTL4.

[0103] In some aspects, treatment may include inducing cytokine production and antitumor immunity. In some embodiments, treatment regimens may include administering an agonist anti-CD137 antibody first in time and administering a PD-1 antagonist second in time. Thus, the methods provided herein may combine the identification of the biomarker CD56 on cancer and / or tumor cells with a treatment that may comprise CD 137 binding antibodies and / or PD-1 antagonists. As a result, the methods and systems described herein may spare subjects that may be non-responders from certain toxicities associated with CD 137 agonism and / or PD-1 / PD-L1 blockade.

[0104] The methods described herein may be used to treat or determine the efficacy of agonistic anti-CD137 antibodies or PD-1 / PD-L1 blockers in the treatment of subjects having adrenal cortical cancer, anal cancer, bile duct cancer (e.g. peripheral cancer, distal bile duct cancer, intrahepatic bile duct cancer), bladder cancer, bone cancer (e.g. osteoblastoma, osteochondroma, hemangioma, chondromyxoid fibroma, osteosarcoma, chondrosarcoma, fibrosarcoma, malignant fibrous histiocytoma, giant cell tumor of the bone, chordoma, lymphoma, multiple myeloma), brain and central nervous system cancer (e.g. meningioma, astrocytoma, oligodendrogliomas, ependymoma, gliomas, medulloblastoma, ganglioglioma, Schwannoma, germinoma, craniopharyngioma), breast cancer (e.g. ductal carcinoma in situ, infiltrating ductal carcinoma, infiltrating, lobular carcinoma, lobular carcinoma in, situ, gynecomastia), Castleman disease (e.g. giant lymph node hyperplasia, angiofollicular lymph node hyperplasia), cervical cancer, colorectal cancer, endometrial cancer (e.g. endometrial adenocarcinoma, adenoacanthoma, papillary serous adenocarcinoma, clear cell),esophagus cancer, gallbladder cancer (mucinous adenocarcinoma, small cell carcinoma), gastrointestinal carcinoid tumors (e.g. choriocarcinoma, chorioadenoma destruens), Hodgkin's disease, non-Hodgldn's lymphoma, Kaposi's sarcoma, kidney cancer (e.g. renal cell cancer), laryngeal and hypopharyngeal cancer, liver cancer (e.g. hemangioma, hepatic adenoma, focal nodular hyperplasia, hepatocellular carcinoma), lung cancer (e.g. small cell lung cancer, non-small cell lung cancer), mesothelioma, plasmacytoma, nasal cavity and paranasal sinus cancer (e.g. esthesioneuroblastoma, midline granuloma), nasopharyngeal cancer, neuroblastoma, NETs, oral cavity and oropharyngeal cancer, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, retinoblastoma, rhabdomyosarcoma (e.g. embryonal rhabdomyosarcoma, alveolar rhabdomyosarcoma, pleomorphic rhabdomyosarcoma), salivary gland cancer,skin cancer (e.g. melanoma, nonmelanoma skin cancer), stomach cancer,testicular cancer (e.g. seminoma, nonseminoma germ cell cancer), thymus cancer, thyroid cancer (e.g. follicular carcinoma, anaplastic carcinoma, poorly differentiated carcinoma, medullary thyroid carcinoma, thyroid lymphoma), vaginal cancer, vulvar cancer, and uterine cancer (e.g. uterine leiomyosarcoma). In certain aspects, the cancer may be, e.g., neuroendocrine tumors (“NETs”), melanoma, small cell lung cancer, or head and neck cancer.

[0105] In some aspects, the level of CD56-positive cells may be assessed by immunohistochemistry and / or multiplex immunofluorescence. Alternatively, or in addition, the level of CD56-positive cells in the biological sample may be obtained using a multiplex assay, for example, flow cytometry, microarrays, and bead-based multiplex assays. In some aspects, measuring the levels of CD56-positive cells includes obtaining a simple total number of CD56 positive cells in a sample, for example the total number of CD56-positive cells both inside the TME as well as outside the TME. In some aspects, measuring the levels of CD56 positive cells includes determining the density of CD56 positive cells. The density of cells may generally refer the number of cells present within a given volume (i.e., how tightly packed together the cells are in a particular area). Such density of cells may be calculated by dividing the total cell count by the volume the cells occupy, thus cell density may be expressed as "cells per unit volume" (e.g., cells per milliliter). In some aspects, the method involves measuring soluble levels of CD56, as a liquid surrogate for tumor levels of CD56 positive cells. In some aspects, the sample containing soluble CD56 can be circulating blood, plasma, and / or serum.

[0106] In still other embodiments, a biological sample taken from a subject may be assessed for a baseline level of CD56-positive cells, for example CD56-positive tumor cells and / or CD56-positive immune cells. Moreover, the baseline level of CD56-positive cells measured inside the tumor or tumor microenvironment may be predictive of a favorable response to treatment with agonist anti-CD137 antibodies and / or PD-1 antagonist. For example, a biological sample having an elevated level of CD56-positive cells inside the tumor or TME and / or elevated levels of CD56-positive cells outside the tumor or TME may indicate that they subject is more likely to respond to treatment.

[0107] In some embodiments, a biological sample is assessed by measuring the levels of CD56-positive cells and may be further analyzed to distinguish the level of CD56-positive cells outside a tumor or TME and the level of CD56-positive cells inside a tumor or TME in order to determine a total number of CD56-positive cells in the biological sample.

[0108] The methods described herein may have a range of applications. The present method can be applied in, e.g., diagnosis (e.g., identification of candidates for anti-CD137 or anii-PD-Ll therapy or combination therapy and determination of whether relapse or resistance of a tumor or cancer has occurred), monitoring (e.g., determination of whether the anti-CD137 or anti-PD-Ll therapy or combination therapy should be used), clinical practice (e.g., selection of an anti-CD137 or anti-PD-Ll therapy or combination therapy for a particular type of tumor or cancer), clinical trial design (e.g., stratification and selection of patients and selection of single-agent or combination therapies having the greatest chance of success in clinical trial), drug discovery and drug development. Further, the methods provided herein may be used to predict whether a subject will respond to treatment with antiCD 137 or anti-PD-Ll therapy prior to the administration of the therapy. In addition, the methods may be used to assess whether a subject may have an increased probability of responding to treatment, for example, with anti-CD137 or anii-PD-Ll therapy.

[0109] Multiplex tissue analysis techniques are particularly useful for quantifying several immune checkpoint proteins, including CD56, in a tumor tissue sample. Such techniques may allow for one to five, or at least ten or more biomarkers to be measured from a single tumor tissue sample. Furthermore, it is advantageous for the technique to preserve the localization of the biomarker and be capable of distinguishing the presence of biomarkers in cancerous and non-cancerous cells. Such methods include layered immunohistochemistry (L-IHC), layered expression scanning (LES) or multiplex tissue immunoblotting (MTI)taught, for example, in U. S. Pat. Nos. 6,602,661, 6,969,615, 7,214,477 and 7,838,222; U. S. Publ. No. 2011 / 030614 (incorporated herein by reference); and in Chung & Hewitt, Meth Mol Biol, Prof Blotting Detect, Kurlen & Scofield, eds. 536: 139-148, 2009.

[0110] In some embodiments, Multiplex Tissue Imprinting (MTI) technology for measuring biomarkers may be used, wherein the method conserves biopsy tissue by allowing assessment of multiple biomarkers, in some cases at least six biomarkers. In some embodiments, alternative multiplex tissue analysis systems exist that may also be employed as part of the present invention.

[0111] In some embodiments, multiplex tissue imaging can be performed when using fluorescence (e.g. fluorophore or Quantum dots) where the signal can be measured with a multispectral imagine system. Multispectral imaging is a technique in which spectroscopic information at each pixel of an image is gathered and the resulting data analyzed with spectral image-processing software. For example, the system can take a series of images at different wavelengths that are electronically and continuously selectable and then utilized with an analysis program designed for handling such data. The system can thus be able to obtain quantitative information from multiple dyes simultaneously, even when the spectra of the dyes are highly overlapping or when they are co-localized, or occurring at the same point in the sample, provided that the spectral curves are different. Many biological materials auto fluoresce, or emit lower-energy light when excited by higher-energy light. This signal can result in lower contrast images and data. High-sensitivity cameras without multispectral imaging capability only increase the autofluorescence signal along with the fluorescence signal. Multispectral imaging can unmix, or separate out, autofluorescence from tissue and, thereby, increase the achievable signal -to-noise ratio. Briefly the quantification can be performed by following steps: i) providing a tumor tissue microarray (TMA) obtained from the patient, ii) TMA samples are then stained with anti-antibodies having specificity of the immune checkpoint protein(s) of interest, iii) the TMA slide is further stained with an epithelial cell marker to assist in automated segmentation of tumor and stroma, iv) the TMA slide is then scanned using a multispectral imaging system, v) the scanned images are processed using an automated image analysis software which allows the detection, quantification and segmentation of specific tissues through powerful pattern recognition algorithms. The machine-learning algorithm was typically previously trained to segment tumor from stroma and identify cells labelled.

[0112] Determining an expression level of a gene in a tumor sample obtained from a patient can be implemented by a panel of techniques well known in the art.

[0113] In some embodiments, concentrations of one or more biomarkers (e.g., CD56) or densities of cells expressing such biomarkers can be indicative of a patient’s immune fitness, for example, the relative ability of the patient’s immune system to combat a particular disease by itself or the relative ability of the patient’s immune system to be augmented or modified to combat a particular disease, such as cancer, by treatment with an therapeutic agent such as an anti-CD137 and / or anti-PD-1 antibody.

[0114] In some embodiments of the method, detecting, determining, quantifying, assessing, or identifying the one or more biological markers may include by immunochemical techniques in the center of the tumor (CT) and in the invasive margin (IM). In some other embodiments, quantifying one or more biological markers is done by gene expression analysis in the whole tumor tissue sample. Accordingly, in some embodiments, the tumor tissue sample may include a (global) primary tumor (as a whole), a complete tissue section, containing the center of the tumor as well as tissue directly surrounding the tumor (specifically named the “invasive margin” of the tumor) in conjunction with lymphoid islets in proximity to the tumor, the lymph nodes located at the proximity of the tumor, a tumor tissue sample collected prior surgery (for follow-up of patients after treatment for example), and a sample from a distant metastasis, also encompassing the metastatic lesion as well as the adjacent normal tissue. The tumor sample may also comprise non-tumorigenic cells. For example, tumor ceils and / or (micro) metastases are frequently surrounded by healthy tissue. Thus, a tumor sample may comprise a subset of healthy (non-tumorigenic) cells and a subset of tumori enic cells. Moreover, a blood sample may be analyzed for the concentration of certain cytokines and chemokines, whereby the blood sample does not necessarily contain tumor cells. A blood sample may also be analyzed for malignant or tumor cells. As such, the analysis of non-tumorigenic cells and tumorigenic cells can yield insight into the status of the immune status of the subject. Biological samples may also be assessed for circulating CD56.Computational Sample Assessment and Analysis

[0115] In some embodiments, methods disclosed herein may comprise collecting or obtaining a biological sample; performing an analytical method to detect and measure at least one biomarker in the biological sample (e.g., CD56 or NCAM); performing any data normalization or standardization; calculating the biomarker level; and reporting the results ofthe biomarker levels. In some embodiments, biomarker levels may be combined in some way and a single value for the combined biomarker levels may be reported. In some embodiments, the reported value may be a single number determined from the sum of all the marker calculations that is compared to a preset threshold value that may provide an indication of the presence or absence of disease. Or the diagnostic score may be a series of symbols (e.g., bars, dots, lines, heatmaps, and / or any suitable symbol) that each represent a biomarker value and the pattern of the responses may be compared to a preset pattern for determination of the presence or absence of disease. Following computation and analysis of bio marker levels, the resulting report may be predictive of whether a patient will be responsive to treatments provided herein. The methods and systems disclosed herein may comprise computer-implemented methods and systems and computer programmable products configured to determine and evaluate an objective tumor response to an anti-cancer therapy. The method may also include receiving a presence / absence of metastases, determining changes in lesions metrics, and deriving an objective tumor response based on a tumor response criteria. In some aspects, the foregoing may be implemented in a computer assisted manner for assessing and post-processing digital radiologic cross-sectional images of tumors to derive one or more tumor metrics, including total tumor burden and vascular tumor burden and may further derive objective tumor response via one or more tumor response criteria.

[0116] In some embodiments, a user may interact with a computer interface responsive to user selections and which updates a plurality of tumor metrics in response to user input and which may synthesize and display a report to the user summarizing results, including a vascular tumor burden and an objective tumor response. In some aspects, platform technology may include the ability to perform, create, deploy, maintain, and update a wide range of panels, assay, array, and / or sequence of tests for a wide range of substances. The methods and systems in various embodiments disclosed may comprise computer systems for storing and accessing biological information and for computational analysis of complex relationships among the stored concepts. Such methods and systems may comprise a computer system, e.g., any type of system that comprises stored, e.g., digitized, data and typically enabling entry, query, display and analysis of the stored data. Such computer system can be a stand-alone computer, a multi-component computer, e.g., one in which the stored data are physically remote from the user interface, networked computers, etc. Any known method or module in the art including updating, querying, displaying, and analyzing thecontents of the databases described herein will be useful, software and hardware for electronically searching fields, categories or whole databases.

[0117] Additional aspects may include computer implemented methods for determining, using a set of normalized biomarker scores (e.g., normalized CD56 biomarker score), biomarker scores for a subject indicative of a patient's response or lack thereof to a particular therapy. In some aspects, a software program may provide a user with a visual representation presenting information related to a patient's biomarkers scores (e.g., a biomarker score, and / or a therapy score, and / or an impact score), and predicted efficacy of a therapy. Software programs may execute in any suitable computing environment including, without limitation, a cloud-computing environment, a device co-located with a user (e.g., the user's laptop, desktop, smartphone, etc.), one or more devices remote from the user (e.g., one or more servers), or any other suitable software program.

[0118] Also provided herein are systems and methods for determining therapy scores for multiple therapies based on normalized biomarker scores, e.g., normalized CD56 scores. In some embodiments, such information may be output to a user in a graphical user interface (GUI).

[0119] Disclosed herein are systems and methods for identifying a subject as a member of a cohort using normalized biomarker scores e.g., normalized CD56 scores. In some embodiments, such information is output to a user in a graphical user interface (GUI). In some aspects, systems and methods for identifying a subject as a member of a cohort using normalized CD56 biomarker scores may comprise, obtaining sequencing data for a subject, accessing biomarker information indicating distribution of values for biomarkers associated with therapy or treatment, determining normalized biomarker scores for the subject using sequencing data and biomarker information, and identifying the subject as a member of a cohort using normalized biomarker scores.

[0120] In some embodiments, the methods provided herein include predicting therapy efficacy using CD56 as a biomarker (e.g., CD56 may be associated with positive therapeutic response or non-positive therapeutic response to a particular therapy or type of therapy). Using CD56 as a biomarker in a single common quantitative framework for predicting therapy efficacy, the disclosure may provide techniques for normalizing the values of the CD56 biomarker relative to their variation in reference populations, thereby placing them ona common scale. In addition, comparing biomarker scores of a patient to those of other patients may be used to compute normalized biomarker scores. Such normalized biomarker scores may be used to more accurately predict a patient's response to a therapy.

[0121] Moreover, advances in personalized genomic sequencing and cancer genomic sequencing technologies have made it possible to obtain patient-specific information about cancer cells (e.g., tumor cells) and cancer microenvironments from one or more biological samples obtained from individual patients. This information can be used to determine a large number of parameters (or biomarkers) for each patient and, potentially, use this information to identify effective therapies and / or select one or more effective therapies for the subject (e.g., the patient). This information may also be used to determine how a subject is responding over time to a treatment and, if necessary, to select a new therapy or therapies for the subject as needed. Also, tliis information may be used to determine whether the subject should be included or excluded from participating in a clinical trial.

[0122] In some embodiments, the techniques provided herein include methods to generate “thresholds” for pre-defined biomarkers, e.g., CD56, based on data obtained from large numbers of subjects (i.e., patients), thus allowing for the creation of a normalized score for each of the biomarkers. Combinations of normalized biomarker scores for the subject may be used to analyze one more defined therapies (creating therapy scores) providing information that allows for the selection of one or more therapies for each subject based on their personal parameters. In some instances, comparing the expression level of a biomarker in a sample obtained from a subject to a reference (or control), can determined whether the subject has an altered expression level (e.g., increased / high or decreased / low) as compared to the reference (or control).

[0123] Any type of analysis may be performed on a biological sample from a subject. For example, blood analysis, cytometry analysis, histological analysis, immunohistological analysis, or any combination thereof may be performed on a biological sample from a subject.

[0124] Further, any type of sequencing data may be obtained from a biological sample of a subject. Sequencing data may include, for example, DNA sequencing data, RNA sequencing data, proteome sequencing data, or any combination thereof. Sequencing data may be obtained by any known technique. For instance, without limitation, sequencing datamay be obtained from whole genome sequencing (WGS), whole exome sequencing (WES), whole transcriptome sequencing, mRNA sequencing, DNA / RNA-hybridization, microarray, DNA / RNA chip, PCR, single nucleotide polymorphism (SNP) genotyping, or any combination thereof. Expression data (e.g., indicating expression levels) for a plurality of genes may be obtained from a biological sample. There is no limit to the number of genes which may be examined (i.e., there is no limit to the number of genes for which the expression levels may be examined).

[0125] Any dataset containing information associated with a biomarker (e.g., CD56) may be used to obtain biomarker information as described herein. In some aspects, biomarker information may be obtained from one or more databases and / or any other suitable electronic repository of data. Examples of databases include, but are not limited to, CGP (Cancer Genome Project), CPTAC (Clinical Proteomic Tumor Analysis Consortium), ICGC (International Cancer Genome Consortium), and TCGA (The Cancer Genome Atlas). In some aspects, biomarker information may be obtained from data associated with a clinical trial. In some aspects, biomarker information may be predicted in association with a clinical trial based on one or more similar drugs (e.g., drugs of a similar class such as PD-1 inhibitors). In some aspects, biomarker information may be obtained from a hospital database. In still other some aspects, biomarker information may be obtained from a commercial sequencing supplier. And in some aspects, biomarker information may be obtained from a subject (e.g., a patient) and / or a subject’s (e.g., a patient's) relative, guardian, or caretaker.

[0126] Several documents are cited throughout the text of this specification. Each of the documents cited herein (including all patents, patent applications, scientific publications, manufacturer's specifications, instructions, etc.), whether supra or infra, are hereby incorporated by reference in their entirety. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.EXAMPLES

[0127] Pharmacodynamic and Response Biomarkers in the Monotherapy Arm of a Phase 1 Trial of a novel anti-CD137 Agonist Antibody.

[0128] Data were obtained from both blood and tissue. Blood samples were subjected to flow cytometry and cytokine presence was assessed, whereas multiplex immunofluorescence was used on tissue samples. Paired pre- and post- treatment samplesshowed changes in natural killer cell activation status as measured by PD-1 and CD 137 / 4-1BB following treatment with an anti-CD137 agonist e.g., CTX-471 (FIG. 2). While staining for CD56 / NCAM levels on lymphocytes, unexpectedly a tumor cell-specific pattern of CD56 / NCAM expression in baseline samples was observed. Furthermore, FIG. 3 shows that in SCLC as well as other indications, tumor samples from patients with disease control had higher baseline levels of CD56 / NCAM positive cells compared with tumors where disease progressed. Thus, CD56+ tumor cells were indicative of improved prognosis as shown by CR, PR, or SD. Further, blood and tumor pharmacodynamic observations were confirmed and explored in pre-clinical models, allowing a greater understanding of the anti-CD137 agonist mechanism of action (see e.g., FIG. 6).

[0129] As shown in FIG. 2F, tissue and blood were taken at baseline and prior to dosing on week 9 of CTX-471 alone. Samples were analyzed by multi-parameter immunofluorescence, flow cytometry, or a panel of cytokines using the Neogenomics Multi -omyx platform. Standard lab chemistry and blood cell counts were measured at each site. Tissue samples included diagnostic samples as well as a number of paired on-treatment biopsies. Blood samples were collected on week 1, days 1 (W1D1) as well as on week 9, day 1 (W9D1) prior to dosing. The 9-week timepoint was chosen to coincide with a CT scan. Correlates measured are listed in FIG. 4. For immunofluorescence, tumor vs non-tumor locations were identified using a tumor-specific counterstain of either pan-cytokeratin or SOXIO (melanoma) as appropriate. Staining of immune cells was confirmed by staining with anti-CD45.

[0130] Figure 7 shows that to measure pharmacodynamic effects, comparisons were made between pre and post CTX-471 treatment (pharmacodynamics). To survey response biomarkers, values from samples obtained from patients with tumors showing complete (CR) or partial responses (PR) as well as stable disease (SD) were compared with tumors showing progressive disease. Pharmacodynamic analyses used samples from all available indications. Response correlate analysis was restricted to indications for which at least four patients with evaluable samples were available. Using GraphPad Prism software, paired T-tests were performed for pharmacodynamics comparisons. For response biomarkers of baseline samples, an unpaired t-test was performed using Welch’s correction when various between samples could not be assumed. Asterisks indicate differences significant at either p=0.05 (*) or p=0,01(**).The novel antibody agonist ofCD13 / , CTX-4 / 1, showed pharmacodynamic biomarkers indicating immune stimulation.

[0131] As seen in FIG. 5B, examples include increases in both CD4 and CD8 T cells expressing CD137 as well as NKp30 positive NK cells. Tumor tissue showed elevated levels of both PD- 1 and PD-L1. Paired images of pre- and post-CTX-471 treated tissue showed an increase in PD-L1 staining.CTX-471 disease control is associated with measurable baseline biomarkers.

[0132] An interesting trend was observed when comparing levels of CD56 in tissue (FIGs. 8A-C) with levels of circulating CD56 (FIGs. 8D-F). For example, total tissue NCAM (CD56) positive cell density as well as activated NK cells showed significantly higher levels at baseline in disease control samples when compared with samples taken from patients eventually showing progressive disease. In contrast, circulating NK cells, whether differentiated (“CD56dim”) or not (“CD56brJght”) were significantly lower at baseline in disease control patient samples.Baseline tumor cell expression of NCAM / CD56 is associated with disease control.

[0133] A strikingly different pattern and intensity of immunofluorescent NCAM (CD56) signal was observed in Figure 3 comparing samples from patients that did (Figure 3, Top Panels) or did not (Figure 3, Bottom Panels) show disease control. This observation is consistent with previous reports for trans-homotypic interactions between NCAM on different cells and the functional impact of those interactions. Such interactions could form a mechanistic basis for superior response to CTX-471 in tumors expressing high levels of NCAM. i.e., homotypic interactions between tumor cells and NCAM positive effector cells could promote tumor localization of cells such as NK cells which have been shown to be required for killing of tumor cells by CTX-471 preclinically. Baseline tumor samples were classified as “high” or “low” using the median CD56 positive cells per mm2. High NCAM tumors were enriched for activated (CD137+) NK cells but not for lymphocytes which typically do not express NCAM such as CD8 and CD4 T cells. High NCAM tumors could be classified based on an optional threshold of 205 CD56+ tumor cells per mm2(FIG. 10A). A mechanism by which NCAM-expressing tumors might enrich for CD137-expressing NK cells and thus for CTX-471 response is proposed in Figure 6.A novel baseline circulating cell phenotype classifies partial response to CTX-471.

[0134] As shown in FIGs. 9A-9B, a novel phenotype-agnostic and computationally-based approach to analyzing conventional flow cytometry data was used. A specific subset of cells that were CD14+CD11b+ and CD56+ prior to CTX-471 treatment was validated with a set of samples analyzed separately from a Discovery Set of samples used to identify the phenotype. This phenotype would likely have been overlooked using conventional humanbased flow cytometry analysis.Calculating the Theoretical Best NCAM / CD56 Cutoff

[0135] Figure 10 illustrates a possible optimal cutoff for CD56+ tumor cell density. As shown in FIG. 10A, different CD56 density thresholds that were evaluated. Methods to determine appropriate thresholds maximized sensitivity (catching all hue positives), specificity (avoiding false positives), the number of potential patients. Some PD patients were included in the “low” cutoffs and the “high” cutoffs missed some SD patients. A Receiver Operator Characteristic (ROC) analysis can be used to calculate the best sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and precision, accuracy, and false discovery. Seen in FIG 10B, the patient samples were ranked based upon their CD56 density scores using multiplex CD56 immunofluorescence. The median CD56 value was used in the given ROC analysis, where samples with CD56 intensity values at or above the median CD56 intensity score were considered “high” and those below the median were considered “low”. Calculations were made using the “pROC” package with the R statistical software.CTX-471 Increases Levels ofCD137+ Circulating NK Cells

[0136] As shown in FIG. 11, CTX-471 increases levels of CD137+ circulating NK cells. CTX-471 exposure caused a significant increase in levels of circulating NK cells that express CD137 / 4-1BB. Moreover, circulating CD56 / NCAM+ cells may then be enriched within CD56 / NCAM+ tumor tissue. Following enrichment within CD56 / NCAM positive tumor tissue, the circulating CD56 / NCAM+ cells are subsequently positioned to respond to CTX-471 and kill the tumor. As seen in FIG.12, CD137+ NK cells are enriched in NCAM “high” tumors. Baseline tissue samples were analyzed by multiplex immunofluorescence (mIF) and classified by median CD56 levels as high or low' CD56 / NCAM samples. Levels of tumor enrichment (“within” vs. “overall”) are shown for CD 137+ lymphocytes in high vs low'NCAM samples. Only CD 137+ NK cells are significantly (p<0.05) enriched within NCAM “high” tumors.CTX-471 Disease Control Is Associated With Neurological Signatures

[0137] Figure 13 illustrates that CTX-471 disease control is associated with certain genetic signatures. As seen in FIG. 13 A, 31 genes were differentially (pcO. Ol) expressed with disease control rate (DCR) in patient samples (n=9). A gene set enrichment analysis across 7 databases is shown in FIG. 13B. The analyses for 1 differentially expressed genes (DEGs) is shown and ranked by combined odds ratio / p-value score. Results were filtered for > 2. genes matched per pathway with p-value < 0.05. Red boxes indicate neuronal -linked genetic signatures associated with DCR.Baseline Features of Disease Control Enriched in NCAM Tumors

[0138] Baseline features of disease control enriched in NCAM tumors are shown in Figure 14. Baseline tissue samples evaluable for disease control (CR, PR, or SD vs PD) were analyzed by mIF. Features significantly (p<0.05) enriched within disease control tumor samples compared with overall levels are shown. Activated lymphocytes (T and NK cells) were enriched within tumors of patients showing disease control. Activation markers measured include CD 137 and PD-1.

[0139] To clarify the use of and to hereby provide notice to the public, the phrases "at least one of , ,.., and < N>" or "at least one of , ,... < N>, or combinations thereof" or ", ,... and / or < N>" are defined by the Applicant in the broadest sense, superseding any other implied definitions hereinbefore or hereinafter unless expressly asserted by the Applicant to the contrary, to mean one or more elements selected from the group comprising A, B,... and N. In other words, the phrases mean any combination of one or more of the elements A, B,... or N including any one element alone or the one element in combination with one or more of the other elements which may also include, in combination, additional elements not listed. Unless otherwise indicated or the context suggests otherwise, as used herein, "a" or "an" means "at least one" or "one or more."

[0140] While various embodiments have been described, it will be apparent to those of ordinary skill in the art that many more embodiments and implementations are possible. Accordingly, the embodiments described herein are examples, not the only possible embodiments and implementations.

Claims

1. CL IMS1. A method for predicting the efficacy of immune therapy in a subject suffering from cancer, the method comprising:3.obtaining a biological sample from the subject;4.assessing the biological sample by measuring at least one of a density of cells that are CD56-positive and / or a level of circulating CD56;5.determining a density of CD56-positive cells, and / or a level of circulating CD56+ cells to obtain a total number of CD56+ cells;6.wherein a higher total of CD56-positive cells and / or circulating CD56 is predictive of the subject’s responsiveness to immune therapy.

2. The method of claim 1, wherein the biological sample comprises blood, sera, urine, or other fluid biological sample, and / or a tissue sample.

3. The method of claim 1, wherein the density of CD56-positive cells comprises tumor cells and / or immune cells.

4. 'The method of claim 3, wherein the immune cells are natural killer cells (NK cells).

5. The method of claim 3, wherein the CD56-positive immune cells and tumor cells are CD137-positive and / or PD- 1-positive.

6. The method of claim 1, wherein the immune therapy comprises treatment with an immune checkpoint inhibitor (ICI).

7. The method of claim 1, wherein the immune therapy includes a monoclonal antibody targeting CD 137 (4- IBB).

8. The method of claim 1, wherein the immune therapy includes an anti-PD-1 antibody, an anti-PD-Ll antibody and / or an anti-CTLA-4 antibody.

9. The method of claim 1, wherein the immune therapy comprises a single-agent therapy or a combination therapy.

10. The method of claim 1, wherein the density of CD56-positive cells is measured by staining with an anti-CD56 antibody.

11. The method of claim 1, wherein the density of CD56-positive tumor cells is assessed by immunohistochemistry and / or multiplex immunofluorescence.

12. The method of claim 1, wherein the subject is suffering from neuroendocrine tumors, melanoma, small cell lung cancer, or head and neck cancer.

13. The method of claim 1, wherein the immune treatment improves the subject’s progression free survival (PFS) and / or overall survival (OS).

14. The method of claim 1, wherein the greater density of intra-tumoral CD56-positive cells as compared to the density of extra-tumoral CD56-positive cells is predictive of stable disease (SD), a complete response (CR), or partial response (PR) to immune therapy.

15. The method of claim 2, wherein the tissue is tumor tissue.

16. The method of claim 15, wherein the tumor tissue is obtained from a biopsy or an excised tumor.

17. 'The method of claim 15, wherein the tumor tissue comprises the center of the tumor and / or the invasive margin of the tumor.

18. The method of claim 1, wherein a baseline density of total CD56-positive cells in the biological sample is predictive of disease control.

19. The method of claim 1, wherein the biological sample is assessed using a multiplex assay that is selected from the group consisting of flow cytometry, microarrays, and bead-based multiplex assays.

20. A method for identifying subjects likely to respond to treatment with a monoclonal antibody targeting CD137, the method comprising:25.obtaining a biological sample from the subject; and26.measuring the level of CD56-positive cells and / or circulating CD56 to obtain total number of CD56-positive cells and / or circulating CD56 in the biological sample, wherein elevated levels of CD56-positive cells and / or circulating CD56 in the biological sample as compared to a predetermined baseline level of CD56-positive cells is predictive of the subject’s responsiveness to treatment.

21. The method of claim 20, wherein the biological sample comprises blood, sera, urine, or other fluid biological sample, and / or a tissue sample.

22. The method of claim 20, wherein the CD56-positive cells comprise tumor cells and / or immune cells.

23. The method of claim 22, wherein the immune ceils are NK cells.

24. The method of claim 20, wherein the CD56-positive cells are assessed by immunohistochemistry and / or multiplex immunofluorescence.

25. The method of claim 20, wherein the subject is suffering from melanoma, small cell lung cancer, neuroendocrine tumor, and / or head and neck cancer.

26. The method of claim 20, wherein the treatment with the monoclonal antibody targeting CD137 improves the subject’s progression free survival (PFS) and / or overall survival (OS).

27. 'The method of claim 20, wherein greater levels of CD56-positive cells inside the tumor microenvironment compared to the levels of CD56-positive cells outside the tumor microenvironment is predictive of stable disease (SD) a complete response (CR) or a partial response (PR) to treatment.

28. The method of claim 21, wherein the biological sample is tumor tissue and wherein the tumor tissue is obtained from a biopsy or an excised tumor.

29. The method of claim 28, wherein the tumor tissue comprises the center of the tumor and / or the invasive margin of the tumor.

30. The method of claim 20, wherein a baseline level CD56-positive cells inside the tumor microenvironment in the biological sample is predictive of disease control.

31. The method of claim 20, wherein the level CD56-positive cells in the biological sample is obtained using a multiplex assay that is selected from the group consisting of flow cytometry, microarrays, and bead-based multiplex assays.

32. A method of predicting efficacy of treatment with immune therapy in a subject suffering from disease, the method comprising:obtaining a biological sample from the subject;38.assessing the biological sample by measuring levels of CD56-positive cells, CD137-positive immune cells, PD-1 -positive immune cells in the biological sample, and / or circulating CD56;39.obtaining a total amount of CD56-positive cells, CD 137-positive immune cells, PD-1-positive immune cells, and circulating CD56 or a combination thereof, in the biological sample; and40.evaluating the total amount of CD56-posiiive cells, CD137-positive immune cells, and PD-1 -positive immune cells, and circulating CD56, or a combination thereof to predict the subject’s responsiveness to immune therapy treatment.

33. The method of claim 32, wherein the biological sample comprises blood, sera, urine, or other fluid biological sample, and / or a tissue sample.

34. The method of claim 32, wherein higher levels of CD56-positive cells is predictive of the subject’s responsiveness to ICI treatment.

35. The method of claim 32, wherein the CD56-positive cells comprise tumor cells and / or immune cells.

36. The method of claim 32, wherein the CD56-positive cells comprise tumor cells inside the tumor microenvironment, immune cells inside the tumor microenvironment, infiltrating immune cells, and / or circulating immune cells.

37. The method of claim 32, wherein the immune cells are natural killer cells (NK cells).

38. 'The method of claim 32, wherein the immune therapy includes treatment with a monoclonal antibody targeting CD137 (4-1BB).

39. The method of claim 32, wherein the immune therapy includes treatment comprising an anti-PD-1 antibody, an anti-PD-Ll antibody and / or an anti-CTLA-4 antibody.

40. The method of claim 32, wherein the immune therapy treatment comprises a single-agent therapy or a combination therapy.

41. The method of claim 32, wherein the level of CD56-positive cells is measured by staining with an anti-CD56 antibody.

42. The method of claim 32, wherein the level of CD56-positive cells is assessed by immunohistochemistry and / or multiplex immunofluorescence.

43. The method of claim 32, wherein the biological sample is assessed using a multiplex assay that is selected from the group consisting of flow cytometry, microarrays, and bead-based multiplex assays.

44. The method of claim 32, wherein the disease is cancer.

45. The method of claim 44, wherein the cancer is melanoma, small cell lung cancer, or head and neck cancer.

46. The method of claim 32, wherein the immune therapy treatment improves the subject’s progression free survival (PFS) and / or overall survival (OS).

47. 'The method of claim 32, wherein higher levels of total CD56-positive cells, CD 137-positive immune cells, and / or PD-l-positive immune cells as compared to the levels of CD56-positive cells outside the tumor microenvironment is predictive of stable disease (SD), a complete response (CR) or a partial response (PR) to immune therapy.

48. A method for treating a subject suffering from cancer, the method comprising: obtaining a biological sample from the subject, wherein the biological sample comprises at least tumor tissue or blood;56.assessing the biological sample by determining the level of CD56-positive cells and circulating CDS 6 to obtain a total number of CD56-positive cells and circulating CDS 6 in the biological sample; and57.administering immune therapy to subjects having a high level of total CD56-positive cells and circulating CD56.

49. 'The method of claim 48, wherein the immune therapy treatment comprises an anti-CD137 antibody, an anti-PD-1 antibody, and / or an anti-CTLA-4 antibody.

50. The method of claim 48, wherein the CD56-positive cells comprise tumor cells and / or immune cells.

51. The method of claim 50, wherein the CD56-positive immune cells comprise infiltrating immune cells and / or circulating immune cells.

52. The method of claim 48, wherein the immune therapy treatment comprises a single-agent therapy or a combination therapy.

53. The method of claim 48, wherein the level of CD56-positive cells is measured by staining with an anti-CD56 antibody.

54. The method of claim 48, wherein the level of CD56-positive cells is assessed by immunohistochemistry and / or multiplex immunofluorescence.

55. The method of claim 48, wherein the biological sample is assessed using a multiplex assay that is selected from the group consisting of flow cytometry, microarrays, and bead-based multiplex assays.

56. The method of claim 56, wherein the cancer is neuroendocrine tumors, melanoma, small cell lung cancer, or head and neck cancer.

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