CN116710575A - Markers responsive to PD-1/PD-L1 immunotherapy - Google Patents

Abstract

Methods of determining suitability of a subject for treatment with PD-1/PD-L1-based immunotherapy comprising receiving a sample from the subject and determining a level of HVEM in the sample, wherein an expression of HVEM above a predetermined threshold indicates that the subject is suitable for treatment by the immunotherapy, or determining suitability of a subject for non-response to PD-1/PD-L1-based immunotherapy by HVEM-based immunotherapy, comprising receiving a sample from the subject and determining a level of T cells in the sample, wherein a level of T cells above a predetermined threshold indicates that the subject is suitable for treatment by HVEM-based immunotherapy, are provided.

Description

Markers responsive to PD-1/PD-L1 immunotherapy

Cross Reference to Related Applications

The present application claims priority from U.S. provisional patent application No. 63/087,376 filed on 5 th 10 th 2020 and U.S. provisional patent application No. 63/182,968 filed on 2 th 5 th 2021, the contents of each of which are incorporated herein by reference in their entirety.

Technical Field

The present application is in the field of immunotherapy diagnostics.

Background

Immunotherapy aimed at enhancing immune response is considered as active immunotherapy and is at the front of cancer treatment. Currently, immune checkpoint blockade therapy is successfully used to treat advanced non-small cell lung cancer (NSCLC), metastatic melanoma, advanced Renal Cell Carcinoma (RCC), urinary tract cancer, tumors with high MSI, merck cell carcinoma, squamous cell carcinoma, hepatocellular carcinoma, cervical cancer, head and neck cancer, pleural mesothelioma, and the like. Some of these drugs developed and produced by different companies show tremendous promise in different cancer types and have been FDA approved, including antibodies to immune checkpoint apoptosis protein 1 receptor (PD 1), apoptosis protein 1 ligand (PDL 1), and cytotoxic T lymphocyte-associated protein 4 (CTLA-4). However, the patient response rate is still not ideal, as typically only 10% -40% of the treated patients benefit, and at the same time the patients may suffer from side effects after immunotherapy. Furthermore, treatment with these agents may induce resistance through up-regulation of other immune checkpoints. In melanoma patients, the combination of anti-PD 1 and anti-CTLA-4 therapies showed a higher response rate (60%) than single agents, however such combination therapies also involve serious treatment-related negative effects. Thus, there is a clear need for new anti-tumor immune activators.

Herpes virus invasion medium (HVEM) is a protein found on the surface of various cell types, including hematopoietic cells and non-hematopoietic cells. HVEM acts as a receptor for typical TNF-related ligands (such as LIGHT and lta), acting as a signaling receptor. However, it also acts as a ligand for immunoglobulin (Ig) superfamily molecules such as the inhibitory receptors BTLA and CD 160. Thus, for HVEM-mediated signaling networks, bi-directional signaling is possible, which may be involved in positive or negative immune responses in different situations. Deregulation of this network is involved in the pathogenesis of autoimmune and inflammatory diseases and cancer, making HVEM the target of immunotherapy. Methods of determining subjects who respond to a particular immunotherapy are now highly desirable.

Disclosure of Invention

In some embodiments, the invention provides methods of determining suitability for treating a subject with PD-1/PD-L1-based immunotherapy, comprising receiving a sample from the subject and determining a level of HVEM in the sample, wherein an expression of HVEM above a predetermined threshold indicates that the subject is suitable for treatment by the immunotherapy, and/or determining suitability for a subject that is non-responsive to treatment with PD-1/PD-L1-based immunotherapy by HVEM-based immunotherapy, comprising receiving a sample from the subject and determining a level of T cells in the sample, wherein a level of T cells above the predetermined threshold indicates that the subject is suitable for treatment by HVEM-based immunotherapy.

According to a first aspect, there is provided a method of determining suitability for treatment of a subject in need of PD-1/PD-L1-based immunotherapy, the method comprising receiving a sample from the subject and determining a level of herpes virus invasion medium (HVEM) in the sample, wherein an expression of HVEM above a predetermined threshold indicates that the subject is suitable for treatment by PD-1/PD-L1-based immunotherapy.

According to another aspect, there is provided a method of determining suitability of a subject for treatment with an HVEM-based immunotherapy for non-response to a PD-1/PD-L1-based immunotherapy, the method comprising receiving a sample from the subject and determining a T cell level in the sample, wherein a T cell level above a predetermined threshold indicates that the subject is suitable for treatment by the HVEM-based immunotherapy.

According to some embodiments, the predetermined threshold is the expression level in a healthy sample or a disease sample from a non-responder to PD-1/PD-L1 based immunotherapy.

According to some embodiments, the HVEM level is HVEM mRNA level or HVEM protein level.

According to some embodiments, the HVEM level is an HVEM protein level, and determining comprises contacting the sample with an agent that specifically binds an extracellular domain of HVEM.

According to some embodiments, the antibody comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes SEQ ID NO:1 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5 (gasssarat), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6 (QQYGSSPPYT).

According to some embodiments, the antibody or antigen binding fragment thereof comprises a detectable moiety, or wherein determining further comprises contacting the sample with a secondary antibody comprising a detectable moiety.

According to some embodiments, the sample is a disease sample.

According to some embodiments, the disease sample is a biopsy.

According to some embodiments, the sample is a body fluid.

According to some embodiments, the bodily fluid is blood or plasma.

According to some embodiments, the subject suffers from a disease or disorder treatable by PD-1/PD-L1-based immunotherapy.

According to some embodiments, the disease is cancer.

According to some embodiments, the cancer is a solid cancer.

According to some embodiments, detecting HVEM levels comprises detecting HVEM levels on the surface of cells and/or in the cytoplasm of cells in the sample.

According to some embodiments, detecting HVEM levels comprises detecting circulating HVEM levels.

According to some embodiments, PD-1/PD-L1 based immunotherapy is PD-1 and/or PD-L1 blocking therapy.

According to some embodiments, the method further comprises treating the appropriate subject with PD-1/PD-L1-based immunotherapy or HVEM-based immunotherapy.

According to some embodiments, the subject suffers from a disease or disorder treatable by PD-1/PD-L1-based immunotherapy and HVEM-based immunotherapy.

According to some embodiments, the disease is cancer.

According to some embodiments, the cancer is a solid cancer.

According to some embodiments, the sample is a disease sample.

According to some embodiments, the disease sample is a biopsy.

According to some embodiments, determining T cell levels comprises measuring CD3 expression in the sample.

According to some embodiments, determining T cell levels comprises counting T cells.

According to some embodiments, the T cell is a Tumor Infiltrating Lymphocyte (TIL).

According to some embodiments, a suitable subject is adapted to receive a combination of HVEM-based immunotherapy and PD-1/PD-L1-based immunotherapy.

According to some embodiments, the PD-1/PD-L1 based immunotherapy is PD-1 and/or PD-L1 blocking therapy, the HVEM based immunotherapy is HVEM blocking therapy, or both.

According to some embodiments, the methods of the invention further comprise treating the appropriate subject with HVEM-based immunotherapy.

According to some embodiments, the methods of the invention further comprise treating the appropriate subject with PD-1/PD-L1-based immunotherapy in combination with HVEM-based immunotherapy.

Further embodiments and full scope of applicability of the present invention will become apparent from the detailed description given hereinafter. It should be understood, however, that the detailed description and the specific examples, while indicating preferred embodiments of the invention, are given by way of illustration only, since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description.

Drawings

Fig. 1A-1g. Expression of hvem in responders and non-responders to PD-1 blocking therapy: the (1A-1D) micrograph shows immunostaining of CD3, PD-L1 and HVEM in (1A) T cells, (1B) melanoma cells, (1C) non-responders and (1D) responders. Arrows indicate specific regions of high expression. The (1E-1G) (upper panel) graph summarizes the expression levels of (1E) CD3, (1F) PD-L1 and (1G) HVEM, as well as the statistical significance of the differences in expression, in both the responders and non-responders. (lower left panel) bar graphs of (1E) CD3, (1F) PD-L1 and (1G) HVEM mean expression for all images measured by total positive pixels/field. (lower right panel) dot plots of (1E) CD3, (1F) PD-L1 and (1G) HVEM expression for each sample measured by total positive pixel/field.

FIGS. 2A-2B are graphs of the relationship between CD3 expression and PD-L1 expression (2A) in tumor samples of responders (upper panels) and non-responders (lower panels) to PD-1/PD-L1-based immunotherapy, and CD3 expression and HVEM expression (2B) in tumor samples of responders (upper panels) and non-responders (lower panels) to PD-1/PD-L1-based immunotherapy. The correlation coefficient (r) and the statistical significance (p) are given for each correlation.

FIGS. 3A-3B A plot of the relationship between CD3 expression and HVEM expression on (3A) T cells and (3B) tumor cells in samples from non-responders.

Detailed Description

The present invention provides, in some embodiments, methods of determining suitability for treating a subject with PD-1/PD-L1-based immunotherapy, comprising receiving a sample from the subject and determining HVEM levels in the sample, wherein an expression of HVEM above a predetermined threshold indicates that the subject is suitable for treatment by the immunotherapy. The invention also provides in some embodiments a method of determining suitability of a subject for treatment by HVEM-based immunotherapy for treatment by PD-1/PD-L1-based immunotherapy, comprising receiving a sample from the subject and determining a level of T cells in the sample, wherein a level of T cells above a predetermined threshold indicates that the subject is suitable for treatment by HVEM-based immunotherapy.

It is well known that not all subjects respond to PD-1/PD-L1 based immunotherapy. Even some subjects with high expression of PD-1 or high expression of PD-L1 respond notoriously unresponsively. The present invention is based on the surprising finding that the expression of HVEM is surprisingly associated with a response to PD-1/PD-L1 based immunotherapy. HVEM has been proposed as a negative prognostic marker for cancer therapy, but as described below, samples of subjects found to be responsive to PD-1 blocking therapy showed a significant increase in protein expression of HVEM. This suggests that HVEM is indeed a prognostic marker of PD-1/PD-L1 reactivity. The invention is further based on the surprising discovery that infiltration of T cells into cancer in subjects that do not respond to PD-1/PD-L1 immunotherapy is indicative of HVEM levels and thus indicative of response to HVEM-based immunotherapy or a combination of PD-1/PD-L1 and HVEM-based immunotherapy treatments.

By a first aspect, there is provided a method of determining suitability for treating a subject with PD-1/PD-L1-based immunotherapy comprising receiving a sample from the subject and determining the level of HVEM in the sample, wherein an expression of HVEM above a predetermined threshold indicates that the subject is suitable for treatment by the immunotherapy.

By another aspect, a method of determining suitability of a subject for treatment with an HVEM-based immunotherapy for treatment with a PD-1/PD-L1-based immunotherapy is provided, comprising receiving a sample from the subject and determining a level of T cells in the sample, wherein a level of T cells above a predetermined threshold indicates that the subject is suitable for treatment with the HVEM-based immunotherapy.

By another aspect, there is provided a method of determining suitability for treating a subject with a combination of PD-1/PD-L1-based immunotherapy and HVEM-based immunotherapy comprising receiving a sample from the subject and determining a level of T cells in the sample, wherein a level of T cells above a predetermined threshold indicates that the subject is suitable for treatment by the combination immunotherapy.

In some embodiments, the method is a method of assessing reactivity. In some embodiments, the evaluating includes prediction. The term "assessing responsiveness (assessing responsiveness)" as used herein refers to determining the likelihood that a subject is responsive to a treatment, i.e., success or failure of a treatment. In some embodiments, assessing responsiveness comprises characterizing the subject as a responder to the treatment. In some embodiments, assessing responsiveness comprises characterizing the subject as a non-responder to the treatment.

The term "response" or "responsiveness" to treatment refers to an improvement in at least one relevant clinical parameter compared to an untreated subject diagnosed with the same pathology (e.g., same type, stage, degree, and/or pathological classification), or compared to a clinical parameter of the same subject prior to treatment. The response is not necessarily a complete recovery or eradication of the disease, but may simply be an improvement in the disease.

The term "non-responder" to treatment refers to a patient that does not experience an improvement in at least one clinical parameter and is diagnosed as having the same condition as an untreated subject diagnosed with the same pathology (e.g., same type, stage, degree, and/or pathological classification), or who experiences a clinical parameter of the same subject prior to treatment. In some embodiments, the non-responders are subjects with disease progression. In some embodiments, a non-responder is a subject that does not show improvement in the disease. In some embodiments, the disease is a post-treatment disease.

In some embodiments, the method is a method of determining that it is possible to convert a non-responder to a responder by administering HVEM-based immunotherapy. In some embodiments, the method is a method of determining the suitability of a non-responder to a responder by administration of HVEM-based immunotherapy. In some embodiments, the method is a method of determining the suitability of a non-responder to PD-1/PD-L1 based immunotherapy to receive HVEM based immunotherapy. In some embodiments, a T cell level above a predetermined threshold indicates that the subject is suitable for receiving HVEM-based immunotherapy. In some embodiments, the non-responders are non-responders to PD-1/PD-L1 based immunotherapy.

In some embodiments, the HVEM is mammalian HVEM. In some embodiments, the HVEM is human HVEM. In some embodiments, the HVEM is any one of mouse, monkey, and human HVEM. In some embodiments, the HVEM is a membrane-bound HVEM. In some embodiments, the HVEM is HVEM on a cell. In some embodiments, the HVEM is HVEM on the surface of a cell. In some embodiments, the HVEM is cytoplasmic HVEM. In some embodiments, the HVEM is a soluble HVEM. In some embodiments, the HVEM is a circulating HVEM.

In some embodiments, the HVEM is tumor necrosis factor receptor superfamily member 14 (TNFRSF 14). In some embodiments, the HVEM is CD270. In some embodiments, HVEM is a receptor for BTLA. In some embodiments, HVEM is a ligand of BTLA. In some embodiments, BTLA is CD272. In some embodiments, HVEM is a receptor for tumor necrosis factor superfamily member 14 (TNFSF 14). In some embodiments, HVEM is a ligand of TNFSF 14. In some embodiments, TNFSF14 is LIGHT. In some embodiments, TNFSF14 is CD258. In some embodiments, HVEM is a receptor for CD 160. In some embodiments, HVEM is a ligand of CD 160. In some embodiments, HVEM is a receptor for lymphotoxin alpha (lta). In some embodiments, HVEM is a ligand of lta. In some embodiments, the lta is TNF- β.

In some embodiments, the HVEM level is HVEM expression. In some embodiments, the HVEM is a HVEM protein. In some embodiments, the HVEM is HVEM mRNA. In some embodiments, HVEM expression is expressed on the cell surface. In some embodiments, the HVEM expression is circulating expression. In some embodiments, the HVEM expression is tumor expression. In some embodiments, the HVEM expression is cancer HVEM expression. In some embodiments, the HVEM expression is subject expression. In some embodiments, the HVEM expression is immune cell HVEM expression. In some embodiments, the immune cell is a tumor immune cell. In some embodiments, the tumor immune cells are tumor resident immune cells. In some embodiments, the immune cells are Tumor Infiltrating Lymphocytes (TILs).

In some embodiments, the expression is protein expression. In some embodiments, the expression is mRNA expression. In some embodiments, the expression is gene expression. In some embodiments, determining comprises contacting the sample with an agent that binds HVEM. In some embodiments, determining comprises contacting the sample with an agent that binds HVEM mRNA. In some embodiments, determining comprises contacting the sample with an agent that binds HVEM DNA. In some embodiments, binding comprises specific binding. In some embodiments, determining comprises contacting the sample with an anti-HVEM antibody. In some embodiments, contacting comprises incubating. In some embodiments, the contacting is under conditions sufficient for the agent to bind to HVEM. In some embodiments, the contacting is in vitro. In some embodiments, the contacting is in vivo. In some embodiments, the condition is a physiological condition. In some embodiments, the conditions are that there is sufficient time for bonding. In some embodiments, the binding is hybridization. In some embodiments, the bonding is adhesive. Conditions under which proteins in a sample bind to proteins (e.g., antibodies) are well known in the art, and the skilled artisan can select appropriate conditions for such detection. Also, the conditions for nucleic acid hybridization are well known in the art and can be selected by the skilled artisan.

In some embodiments, expression refers to expression above a predetermined threshold. In some embodiments, the threshold is the expression level in a healthy sample. In some embodiments, the threshold is T cell level in a healthy sample. In some embodiments, the threshold is an expression level in a healthy population. In some embodiments, the threshold is T cell level in a healthy population. In some embodiments, the expression level is average expression. In some embodiments, the T cell level is an average level. In some embodiments, the average is an average in the sample. In some embodiments, the average is an average within a defined region. In some embodiments, the average is an average in a population. In some embodiments, the threshold is the level of expression in a disease sample from a non-responder. In some embodiments, the threshold is the average T cell level in a disease sample from a non-responder. In some embodiments, the non-responders are non-responders to the combination therapy. In some embodiments, the responder is a responder to PD-1/PD-L1-based immunotherapy. In some embodiments, the non-responders are non-responders to PD-1/PD-L1 based immunotherapy. In some embodiments, the threshold is the level of expression in a non-responsive population. In some embodiments, the threshold is T cell level in a non-responsive population. In some embodiments, the T cell level is T cell number.

In some embodiments, the subject is a subject in need thereof. In some embodiments, the subject is a mammal. In some embodiments, the subject is a human. In some embodiments, the subject has a disease or disorder. In some embodiments, the disease or disorder is treatable by PD-1-based immunotherapy. In some embodiments, the disease or disorder is treatable by PD-L1-based immunotherapy. In some embodiments, the disease or disorder is treatable by PD-1 and/or PD-L1-based immunotherapy. In some embodiments, the disease or disorder is treatable by PD-1/PD-L1-based immunotherapy. In some embodiments, the disease is cancer. In some embodiments, the cancer is a PD-L1 expressing cancer. In some embodiments, the cancer is a PD-L1 positive cancer. In some embodiments, the disease or disorder is treatable by HVEM-based immunotherapy. In some embodiments, the cancer is a cancer that expresses HVEM. In some embodiments, the cancer is HVEM positive cancer. In some embodiments, the cancer is a cancer that expresses HVEM and PD-L1. In some embodiments, the cancer is HVEM and PD-L1 double positive cancer. In some embodiments, the cancer is a solid cancer. In some embodiments, the cancer is a hematologic cancer. In some embodiments, the cancer is skin cancer. In some embodiments, the skin cancer is melanoma. In some embodiments, the cancer is selected from skin cancer, lung cancer, kidney cancer, urinary tract cancer, liver cancer, cervical cancer, head and neck cancer, bladder cancer, and high microsatellite instability (MSI) tumor. In some embodiments, the subject is not responsive to PD-1/PD-L1-based immunotherapy. In some embodiments, the subject is not responsive to PD-1-based immunotherapy. In some embodiments, the subject is not responsive to PD-L1-based immunotherapy.

In some embodiments, the PD-1/PD-L1-based immunotherapy is PD-1-based immunotherapy and/or PD-L1-based immunotherapy. In some embodiments, the PD-1/PD-L1-based immunotherapy is PD-1-based immunotherapy or PD-L1-based immunotherapy. In some embodiments, the PD-1/PD-L1-based immunotherapy is PD-1-based immunotherapy, PD-L1-based immunotherapy, or both. In some embodiments, the PD-1/PD-L1-based immunotherapy is a combination of PD-1 and PD-L1-based immunotherapy.

In some embodiments, PD-1/PD-L1-based immunotherapy comprises PD-1 blocking therapy. In some embodiments, PD-1/PD-L1 based immunotherapy comprises PD-L1 blocking therapy. In some embodiments, PD-1/PD-L1-based immunotherapy comprises PD-1/PD-L1 blocking therapy. In some embodiments, PD-1/PD-L1-based immunotherapy comprises an anti-PD-1 blocking antibody. In some embodiments, PD-1/PD-L1-based immunotherapy comprises an anti-PD-L1 blocking antibody. In some embodiments, the immunotherapy comprises inhibiting an interaction between PD-1 and one of its ligands. In some embodiments, the PD-1 ligand is selected from PD-L1 and PD-L2. In some embodiments, immunotherapy comprises inhibiting PD-1, PD-L1, or PD-L2. In some embodiments, immunotherapy comprises inhibiting the interaction of PD-1 with PD-L1. In some embodiments, immunotherapy comprises inhibiting the interaction of PD-1 with PD-L2. In some embodiments, the method further comprises inhibiting the interaction of PD-1 with PD-L1 or PD-L2. In some embodiments, inhibiting the interaction comprises administering an agent that inhibits the interaction of PD-1 with PD-L1 or the interaction of PD-1 with PD-L2. In some embodiments, inhibiting the interaction comprises administering an agent that inhibits the interaction between PD-1 and at least one ligand thereof. In some embodiments, at least one ligand is two ligands. In some embodiments, inhibiting the interaction comprises PD-1/PD-L1 blocking therapy. In some embodiments, inhibiting the interaction comprises PD-1/PD-L1 or PD-L2 blocking therapy. In some embodiments, the agent that inhibits an interaction is an anti-PD-1 or anti-PD-L1 antibody. In some embodiments, the agent that inhibits an interaction is an anti-PD-1, anti-PD-L1, or anti-PD-L2 antibody. In some embodiments, the agent that inhibits an interaction is a PD-1/PD-L1 inhibitor. In some embodiments, the agent that inhibits an interaction is a PD-1/PD-L2 inhibitor. In some embodiments, the antibody is a blocking antibody. PD-L1/L2 and PD-1 therapies are well known in the art and include, but are not limited to, nivolumab (Opdivo), pembrolizumab (pembrolizumab) (Keytruda), alemtuzumab (atezolizumab), aviuzumab (avelumab), du Falu mab (durvalumab), cimapril Li Shan antibody (cemiplimab) (Libtayo), pidotizumab (pidirizumab), multi-tarlimab (Dostarlimab), AMP-224, AMP-514 and PDR001. In some embodiments, the anti-PD-1 immunotherapy is selected from nivolumab or pembrolizumab.

In some embodiments, the HVEM-based immunotherapy comprises HVEM blocking therapy. In some embodiments, the HVEM-based immunotherapy comprises anti-HVEM blocking antibodies. In some embodiments, the immunotherapy comprises inhibiting an interaction between HVEM and BTLA. In some embodiments, the immunotherapy comprises inhibiting HVEM. In some embodiments, the immunotherapy comprises inhibiting the interaction of HVEM and BTLA. In some embodiments, the immunotherapy comprises inhibiting the interaction of HVEM and CD 160. In some embodiments, immunotherapy does not include inhibiting the interaction of HVEM and TNFSF 14. In some embodiments, immunotherapy does not include inhibiting the interaction of HVEM and lymphotoxin alpha (lta). In some embodiments, inhibiting the interaction comprises administering an agent that inhibits the interaction. In some embodiments, the lta is TNF- β. In some embodiments, TNFSF14 is LIGHT. In some embodiments, TNFSF14 is CD258. In some embodiments, the agent that inhibits an interaction is an anti-HVEM antibody. In some embodiments, the agent that inhibits the interaction is an anti-BTLA antibody. In some embodiments, the agent that inhibits an interaction is an HVEM inhibitor. In some embodiments, the antibody is a blocking antibody. HVEM therapies are well known in the art and include, but are not limited to, blocking antibodies disclosed in international patent application WO2020222235, which is incorporated herein by reference in its entirety.

In some embodiments, the sample is a disease sample. In some embodiments, the sample is a cancer sample. In some embodiments, the sample comprises cells. In some embodiments, the cell is a disease cell. In some embodiments, the cell is a cancer cell. In some embodiments, the sample comprises tissue. In some embodiments, the sample is a biopsy. In some embodiments, the sample is a body fluid. In some embodiments, the bodily fluid is selected from at least one of the following: blood, serum, plasma, gastric fluid, intestinal fluid, saliva, bile, tumor fluid, milk, urine, interstitial fluid, cerebrospinal fluid and stool. In some embodiments, the bodily fluid is blood. In some embodiments, the bodily fluid is plasma. In some embodiments, the bodily fluid is serum.

In some embodiments, the sample comprises cancer cells. In some embodiments, the cancer cell is a circulating tumor cell. In some embodiments, the cancer cell is a melanoma cell. In some embodiments, the cancer cell is a PD-L1 positive cell. In some embodiments, the cancer cell is an HVEM positive cell.

In some embodiments, the sample comprises immune cells. In some embodiments, the cell is a hematopoietic cell. In some embodiments, the immune cell is a T cell. In some embodiments, the T cell is a CD8 positive T cell. In some embodiments, the T cell is a cytotoxic CD8 positive T cell. In some embodiments, the T cell is a CD4 positive T cell. In some embodiments, the T cell is a CD4 positive helper T cell. In some embodiments, the T cells are selected from CD8 positive and CD4 positive T cells. In some embodiments, the T cell is a CD8 positive T cell, a CD4 positive T cell, or both. In some embodiments, the immune cells are Tumor Infiltrating Lymphocytes (TILs). In some embodiments, the immune cells are not peripheral blood immune cells. In some embodiments, the immune cell is a B cell. In some embodiments, the immune cell is a Natural Killer (NK) cell. In some embodiments, the immune cell is a neutrophil. In some embodiments, the immune cell is a dendritic cell. In some embodiments, the immune cell is a macrophage. In some embodiments, the immune cell is a myeloid-derived suppressor cell (MDSC). In some embodiments, the cell is selected from the group consisting of a T cell, a B cell, an NK cell, a neutrophil, a dendritic cell, an MDSC, and a macrophage. In some embodiments, the cell is selected from the group consisting of T cells, B cells, NK cells, neutrophils, dendritic cells, and macrophages.

In some embodiments, the method comprises determining the level of T cells in the sample. In some embodiments, the T cell level is T cell number. In some embodiments, determining T cell levels comprises counting T cells. In some embodiments, determining T cell levels comprises staining T cells. T cell markers and any markers well known in the art may be used, including, for example, CD3. In some embodiments, determining the level of T cells comprises measuring CD3 expression in the sample. In some embodiments, CD3 expression is CD3 level. In some embodiments, T cell marker expression (e.g., CD3 expression) is proportional to T cell levels. In some embodiments, the expression is protein expression. In some embodiments, the expression is mRNA expression. In some embodiments, the expression is surface expression. In some embodiments, the measuring comprises flow cytometry. In some embodiments, the flow cytometry is Fluorescence Activated Cell Sorting (FACS). In some embodiments, the measurement comprises immunostaining. In some embodiments, measuring comprises amplifying. In some embodiments, measuring comprises sequencing. In some embodiments, the staining is staining for a T cell specific protein. In some embodiments, the protein is a surface protein. In some embodiments, the amplification is amplification of T cell specific mRNA. In some embodiments, the amplification is PCR. In some embodiments, the T cell is a TIL. In some embodiments, the T cell is a TIL and the determination is a determination of T cell level in the tumor.

In some embodiments, determining expression comprises detecting expression. In some embodiments, determining the expression comprises quantifying the expression. In some embodiments, determining expression comprises detecting expression and quantifying the detected expression. Methods for measuring protein expression are well known in the art and include, for example, western blotting, immunohistochemistry, immunocytochemistry, immunofluorescence quantification, ELISA, FACS, and protein array methods. Any such method may be employed. Methods for measuring mRNA/gene expression are also well known in the art and include, for example, northern blotting, RT-PCR, qPCR, microarray hybridization, and sequencing (e.g., whole transcriptome sequencing). Any such method may be employed.

In some embodiments, detecting comprises contacting the sample with an agent that specifically binds HVEM. In some embodiments, detecting comprises contacting the sample with an agent that binds HVEM mRNA. In some embodiments, detecting comprises contacting the sample with an agent that binds HVEM DNA. In some embodiments, the agent specifically binds HVEM. In some embodiments, the agent binds to a protein other than HVEM. In some embodiments, the agent binds to an extracellular domain of HVEM. In some embodiments, the agent binds to a cytoplasmic domain of HVEM. In some embodiments, the agent binds to a transmembrane domain of HVEM. In some embodiments, the detection is detection of an extracellular domain of HVEM. In some embodiments, the detection is detection of an intracellular domain of HVEM. In some embodiments, the detection is detection of a transmembrane domain of HVEM. In some embodiments, the detection is detection of surface expression of HVEM. In some embodiments, the detection is detection of cytoplasmic expression of HVEM. In some embodiments, the detection is detecting expression of the surface and cytoplasm of HVEM.

In some embodiments, the human HVEM comprises or consists of the amino acid sequence: MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPVGSECCPKCSPGYRVKEACGELTGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMGLRASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCSWLVTKAGAGTSSSHWVWWFLSGSLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 17). In some embodiments, the signal peptide of HVEM comprises SEQ ID NO:17 from amino acids 1 to 38 or consists thereof. In some embodiments, the signal peptide of HVEM comprises SEQ ID NO:17 from amino acids 1 to 36 or a combination thereof. In some embodiments, the HVEM extracellular domain comprises SEQ ID NO:17 or consists of amino acids 39-202 of 17. In some embodiments, the HVEM extracellular domain comprises SEQ ID NO:17 or consists of amino acids 37-202 of 17. In some embodiments, the HVEM transmembrane domain comprises SEQ ID NO:17, or consists of amino acids 203-223 of seq id no. In some embodiments, the HVEM cytoplasmic domain comprises SEQ ID NO:17, amino acids 224-283 or a combination thereof. In some embodiments, SEQ ID NO:17 are transmembrane forms of HVEM. The generation of primers and/or probes that bind to these sequences is within the knowledge of the skilled artisan. Likewise, sequencing data consistent with these sequences may be used to identify the expression of HVEM mRNA. It will be appreciated that the protein sequence may include a degree of variation in the population, and thus any given human HVEM molecule may have a sequence identical to SEQ ID NO:17 are not exactly the same. In some embodiments, the HVEM comprises SEQ ID NO:17 (homolog). In some embodiments, the HVEM extracellular domain comprises SEQ ID NO:17 from amino acids 39-202. In some embodiments, the HVEM extracellular domain comprises SEQ ID NO:17 from amino acids 37-202. In some embodiments, the homolog is at least 70, 75, 80, 85, 90, 92, 93, 95, 97, 98, or 99% homolog. Each possibility represents a separate embodiment of the invention. In some embodiments, SEQ ID NO:17 are isoforms 1 of human HVEM. HVEM isoform 1 can be found in Uniprot entry Q92956-1.

In some embodiments, the human HVEM comprises or consists of the amino acid sequence: MEPPGDWGPPPWRSTPKTDVLRLVLYLTFLGAPCYAPALPSCKEDEYPVGSECCPKCSPGYRVKEACGELTGTVCEPCPPGTYIAHLNGLSKCLQCQMCDPAMGLRASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTK (SEQ ID NO: 18). In some embodiments, the signal peptide of HVEM comprises SEQ ID NO:18 from amino acids 1 to 38 or consists thereof. In some embodiments, the signal peptide of HVEM comprises SEQ ID NO:18 from amino acids 1 to 36 or consists thereof. In some embodiments, SEQ ID NO:18 are soluble forms of HVEM. In some embodiments, SEQ ID NO:18 are forms of HVEM lacking transmembrane and cytoplasmic domains. In some embodiments, the HVEM extracellular domain comprises SEQ ID NO:18 or consist of the same. The generation of primers and/or probes that bind to these sequences is within the knowledge of the skilled artisan. Likewise, sequencing data consistent with these sequences may be used to identify the expression of HVEM mRNA. It will be appreciated that the protein sequence may include a degree of variation in the population, and thus any given human HVEM molecule may have a sequence identical to SEQ ID NO:18 are not exactly the same. In some embodiments, the HVEM comprises SEQ ID NO: 18. In some embodiments, the HVEM extracellular domain comprises SEQ ID NO:18 from amino acids 37 to 202. In some embodiments, the homolog is at least 70, 75, 80, 85, 90, 92, 93, 95, 97, 98, or 99% homolog. Each possibility represents a separate embodiment of the invention. In some embodiments, SEQ ID NO:18 are isoforms 2 of human HVEM.

Alternative isoforms of HVEM are also known, in which the extracellular domain is truncated. This isoform can be found in Uniprot entry Q92956-2. In some embodiments, the human HVEM with the truncated extracellular domain comprises or consists of the amino acid sequence: MVSRPPRTPLSPSSWTPAMGLRASRNCSRTENAVCGCSPGHFCIVQDGDHCAACRAYATSSPGQRVQKGGTESQDTLCQNCPPGTFSPNGTLEECQHQTKCSWLVTKAGAGTSSSHWVWWFLSGSLVIVIVCSTVGLIICVKRRKPRGDVVKVIVSVQRKRQEAEGEATVIEALQAPPDVTTVAVEETIPSFTGRSPNH (SEQ ID NO: 19). In some embodiments, the signal peptide of HVEM comprises SEQ ID NO:19 from amino acids 1 to 16 or consists thereof. In some embodiments, the HVEM extracellular domain comprises SEQ ID NO:19 or consists of amino acids 17-118 of 19. In some embodiments, the HVEM transmembrane domain comprises SEQ ID NO:19 or consists of amino acids 119-139 thereof. In some embodiments, the HVEM cytoplasmic domain comprises SEQ ID NO:19, amino acids 140-199 or consist thereof. In some embodiments, SEQ ID NO:19 are transmembrane forms of HVEM. In some embodiments, SEQ ID NO:19 are HVEM forms comprising truncated extracellular domains. The generation of primers and/or probes that bind to these sequences is within the knowledge of the skilled artisan. Likewise, sequencing data consistent with these sequences may be used to identify the expression of HVEM mRNA. It will be appreciated that the protein sequence may include a degree of variation in the population, and thus any given human HVEM molecule may have a sequence identical to SEQ ID NO:19 are not exactly the same. In some embodiments, the HVEM comprises SEQ ID NO: 19. In some embodiments, the HVEM extracellular domain comprises SEQ ID NO:19 from amino acids 17-118. In some embodiments, the homolog is at least 70, 75, 80, 85, 90, 92, 93, 95, 97, 98, or 99% homolog. Each possibility represents a separate embodiment of the invention.

In some embodiments, the agent is an antibody or antigen-binding fragment thereof. In some embodiments, the antibody or fragment thereof is a fab fragment. In some embodiments, the antibody or fragment thereof is a single chain antibody (scFv). In some embodiments, the antibody or fragment thereof is a single domain antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a rodent antibody. In some embodiments, the antibody is a goat antibody. In some embodiments, the antibody is a rabbit antibody. In some embodiments, the antibody is a humanized antibody. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a polyclonal antibody.

As used herein, the term "antibody" refers to a polypeptide or group of polypeptides that includes at least one binding domain formed by folding a polypeptide chain having a three-dimensional binding space, the shape and charge distribution of which is complementary to the characteristics of an antigenic determinant of an antigen. Antibodies typically have a tetrameric form, comprising two identical pairs of polypeptide chains, each pair having one "light" chain and one "heavy" chain. The variable region of each light/heavy chain pair forms an antibody binding site. Antibodies may be oligoclonal, polyclonal, monoclonal, chimeric, camelized (CDR-grafted), multispecific, bispecific, catalytic, humanized, fully human, anti-idiotypic (anti-idiotypic) and antibodies and fragments, including epitope-binding fragments, variants or derivatives thereof, that may be labeled in soluble or binding form, alone or in combination with other amino acid sequences. The antibody may be from any species. The term antibody also includes binding fragments including, but not limited to Fv, fab, fab ', F (ab') 2 single chain antibodies (svFC), dimer variable regions (diabodies), and disulfide-linked variable regions (dsFv). In particular, antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site. The antibody fragment may or may not be fused to another immunoglobulin domain, including but not limited to an Fc region or fragment thereof. Those skilled in the art will further appreciate that other fusion products may be produced, including but not limited to scFv-Fc fusions, variable regions (e.g., VL and VH) to Fc fusions, and scFv-Fc fusions.

Immunoglobulin molecules may be of any type (e.g., igG, igE, igM, igD, igA and IgY), class (e.g., igGl, igG2, igG3, igG4, igAl, and IgA 2) or subclass. In some embodiments, the antibody comprises IgG2 or IgG4. In some embodiments, the antibody comprises IgG2. In some embodiments, the antibody comprises IgG4. In some embodiments, the antibody comprises IgG1. In some embodiments, the antibody comprises IgG3. In some embodiments, the antibody comprises a modified IgG1 or IgG3 with reduced toxicity.

The basic unit of a naturally occurring antibody structure is a hetero-tetrameric glycoprotein complex of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H), linked together by non-covalent association and disulfide bonds. Each heavy and light chain has regularly spaced intrachain disulfide bridges. Five classes of human antibodies exist (IgG, igA, igM, igD and IgE), and among these, various subclasses can be identified based on structural differences, such as the number of immunoglobulin units in a single antibody molecule, the disulfide bridge structure of a single unit, and differences in chain length and sequence. The class and subclass of antibodies is its isotype.

The amino terminal regions of the heavy and light chains are more diverse in sequence than the carboxy terminal regions, and are therefore referred to as variable domains. This portion of the antibody structure confers antigen binding specificity to the antibody. The heavy chain variable domain (VH) and the light chain variable domain (VL) together form a single antigen binding site, and thus, the basic immunoglobulin unit has two antigen binding sites. Specific amino acid residues are believed to form an interface between the light and heavy chain variable domains (Chothia et al, J.mol.186,651-63 (1985); novotny and Haber (1985) Proc.Natl. Acad.Sci. USA 82 4592-4596).

The carboxy-terminal portions of the heavy and light chains form constant domains, i.e., CH1, CH2, CH3, CL. Although these domains are much less diverse, there are differences between different animal species, and further, in the same individual, there are several different antibody isoforms, each with different functions.

The term "framework region" or "FR" refers to amino acid residues in the variable domain of an antibody that do not include the high mutation region amino acid residues defined herein. The term "hypervariable region (hypervariable region)" as used herein refers to the amino acid residues in the variable domain of an antibody that are responsible for antigen binding. The high variation region includes amino acid residues from the "complementarity determining region (complementarity determining region)" or "CDR". CDRs are mainly responsible for binding to epitopes of antigens. The range of FRs and CDRs has been precisely defined (see, kabat et al).

An IMGT information system (www:// IMGT. Cines. Fr /). May also be usedV-Quest) to determine variable segments including CDRs. See, for example, brochet, X. Et al, nucleic acids Res. J6:W503-508 (2008).

As used herein, the term "humanized antibody (humanized antibody)" refers to an antibody from a non-human species whose protein sequence has been modified to increase similarity to a human antibody. Humanized antibodies can be produced by producing recombinant DNA encoding CDRs of a non-human antibody, which CDRs are surrounded by sequences similar to human antibodies. In some embodiments, the humanized antibody is a chimeric antibody. In some embodiments, humanization comprises inserting CDRs of the invention into a human antibody scaffold or framework. Humanized antibodies are well known in the art and any method that retains the CDRs of the present invention can be used to produce them.

The term "monoclonal antibody (monoclonal antibody)" or "mAb" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., including the population where the individual antibodies are identical and/or bind to the same epitope, except for variants that may occur during production of the monoclonal antibody, which are typically present in minor amounts. The term "polyclonal antibody (polyclonal antibody)" as used herein refers to a mixture of heterogeneous antibodies. Unlike polyclonal antibody agents, which typically include different antibodies directed against different determinants (epitopes), monoclonal antibodies are directed against a single determinant on an antigen. In addition to their specificity, monoclonal antibodies have the advantage that they are not contaminated with other immunoglobulins. The modifier "monoclonal" means that the antibody is characterized as being obtained from a substantially homogeneous population of antibodies, and is not to be construed as being produced by any particular method of preparation. Monoclonal antibodies for use in accordance with the methods provided herein may be prepared by the hybridoma method described for the first time by Kohler et al, nature 256:495 (1975), or may be prepared by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). "monoclonal antibodies" can also be isolated from phage antibody libraries using techniques described, for example, in Clackson et al, nature 352:624-628 (1991) and Marks et al, J.mol. Biol.222:581-597 (1991).

The mAb of the invention may be any immunoglobulin class, including IgG, igM, igD, igE or IgA. The mAb-producing hybridomas can be cultured in vitro or in vivo. High titers of mAbs can be obtained in vivo production, where cells of a single hybridoma are intraperitoneally injected into a prime-sensitized (primary-primed) Balb/c mouse to produce ascites fluid containing a high concentration of the desired mAb. Mabs of isotype IgM or IgG can be purified from such ascites fluids or culture supernatants using column chromatography methods well known to those skilled in the art.

An "antibody fragment (antibody fragment)" includes a portion of an intact antibody, preferably including the antigen-binding region thereof. Examples of antibody fragments include Fab, fab ', F (ab') 2, and Fv fragments; a diabody; tandem diabodies (taDb), linear antibodies (e.g., U.S. Pat. No. 5,641,870, example 2; zapata et al, protein Eng.8 (10): 1057-1062 (1995)); single arm antibodies, single variable domain antibodies, miniantibodies, single chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including, but not limited to, db-Fc, taDb-CH3, (scFV) 4-Fc, di-scFV, or tandem (di, tri) -scFV); and bispecific T cell adaptors (bites).

Papain digestion of antibodies produces two identical antigen binding fragments, called "Fab" fragments, each with a single antigen binding site, and a residual "Fc" fragment, the name of which reflects its ability to crystallize readily. Pepsin treatment will produce F (ab') 2 fragments that have two antigen binding sites and are still capable of cross-linking the antigen.

"Fv" is the smallest antibody fragment that contains the complete antigen recognition and antigen binding site. This region consists of a dimer of one heavy and one light chain variable domain that associates tightly, non-covalently. Three surfaces of the VH-VL dimer are also this configuration. Overall, these six highly variable regions confer specificity for antibody antigen binding. However, even a single variable domain (or half of an Fv comprising only three highly variable regions specific for an antigen) is capable of recognizing and binding antigen, although with less affinity than the entire binding site.

The Fab fragment also comprises the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments in that several residues are added at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab' wherein the cysteine residue(s) of the constant domain have at least one free thiol group. F (ab ') 2 antibody fragments were initially produced as pairs of Fab' fragments with hinge cysteines between them. Chemical ligation of other antibody fragments is also known.

The "light chain" of antibodies (immunoglobulins) of any vertebrate species can be assigned to one of two distinct types, called kappa and lambda, based on the amino acid sequence of their constant domains.

Antibodies can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five main classes of intact antibodies: igA, igD, igE, igG and IgM, and several of these can be further divided into subclasses (isotypes), such as IgGl, igG2, igG3, igG4, igA, and IgA2. The heavy chain constant domains corresponding to the different classes of antibodies are referred to as a, delta, e, gamma and mu, respectively. The subunit structure and three-dimensional organization of different classes of immunoglobulins is well known.

"Single-chain Fv" or "scFv" antibody fragments include the VH and VL domains of antibodies, wherein these domains are present in a Single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains that enables the scFv to form the desired antigen binding structure. For a discussion of scFv see Pluckaphun, the Pharmacology of Monoclonal Antibodies, volume 113, prosenburg and Moore, springer-Verlag, new York, pages 269-315 (1994).

The term "diabody" refers to a small antibody fragment having two antigen binding sites, the fragment comprising a heavy chain variable domain (VH) linked to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By pairing between two domains on the same strand using a linker that is too short, the domains are forced to pair with complementary domains on the other strand and form two antigen binding sites. The production of diabodies is known in the art and is described in Natl Acad. Sci. USA,90:6444-6448 (1993).

Monoclonal antibodies of the invention may be prepared using methods well known in the art. Examples include various techniques, such as those described in Kohler, G.and Milstein, C, nature 256:495-497 (1975); kozbor et al, immunology Today4:72 (1983); cole et al, pg.77-96in MONOCLONAL ANTIBODIES AND CANCER THERAPY,Alan R.Liss,Inc (1985).

In addition to conventional methods of culturing antibodies in vivo, phage display techniques can also be used to produce antibodies in vitro. Such recombinant antibodies are produced much faster than conventional antibody production, and they can be produced against a large number of antigens. Furthermore, when conventional methods are used, many antigens have proven to be non-immunogenic or extremely toxic and therefore cannot be used to produce antibodies in animals. Furthermore, affinity maturation (i.e., increasing affinity and specificity) of recombinant antibodies is very simple and relatively rapid. Finally, a large number of different antibodies directed against a particular antigen may be generated in a selected procedure. To produce recombinant monoclonal antibodies, one can use various methods, all based on display libraries, to generate a large number of antibody pools with different antigen recognition sites. Such libraries may be generated in several ways: one can generate a synthetic pool by cloning synthetic CDR3 regions in a heavy chain germline gene pool and thus a large antibody pool from which recombinant antibody fragments with different specificities can be selected. One can use human lymphocyte pools as starting materials for constructing antibody libraries. It is possible to construct a naive repertoire of human IgM antibodies and thus create a repertoire of humans with a large diversity. This approach has been widely used successfully to select a large number of antibodies against different antigens. Protocols for phage library construction and recombinant antibody selection are provided in the well known references Current Protocols in Immunology, colligan et al (ed.), john Wiley & Sons, inc. (1992-2000), chapter 17, section 17.1.

The non-human antibodies may be humanized by any method known in the art. In one approach, non-human Complementarity Determining Regions (CDRs) are inserted into a human antibody or consensus antibody framework sequence. Further changes may then be introduced in the antibody framework to modulate affinity or immunogenicity.

In some embodiments, antibodies and portions thereof include, but are not limited to: antibodies, fragments of antibodies, fab and F (ab') 2, single domain antigen-binding recombinant fragments, and natural nanobodies. In some embodiments, the antigen binding fragment is selected from Fv, fab, F (ab') ₂ 、scFv、scFv ₂ Or scFv ₄ Fragments.

In some embodiments, the agent is an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes SEQ ID NO:1 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5 (gasssarat), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6 (QQYGSSPPYT). In some embodiments, the CDRs are according to the KABAT numbering system.

In some embodiments, an antibody or antigen binding fragment thereof comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes SEQ ID NO:11 (GFTFSSYA) CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:12 (ISGSGGST) CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:13 (AKAPGDYTAYFDY) the amino acid sequence set forth in SEQ ID NO:14 (QSVSSY) the amino acid sequence set forth in SEQ ID NO:15 The amino acid sequence set forth in (GAS), and CDR-L3 comprises SEQ ID NO:16 (QQYGSSPPYT) an amino acid sequence set forth in (d). In some embodiments, the CDRs are according to the IMGT numbering system.

In some embodiments, the agent is an antibody or antigen-binding fragment thereof comprising three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes SEQ ID NO:11 (GFTFSSYA) CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:12 (ISGSGGST) CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:13 (AKAPGDYTAYFDY) the amino acid sequence set forth in SEQ ID NO:14 (QSVSSY) the amino acid sequence set forth in SEQ ID NO:15 The amino acid sequence set forth in (GAS), and CDR-L3 comprises SEQ ID NO:16 (QQYGSSPPYT) an amino acid sequence set forth in (d). In some embodiments, the CDRs are according to the IMGT numbering system.

In some embodiments, the antibody comprises a heavy chain comprising the sequence: QVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSS (SEQ ID NO: 7). In some embodiments, the heavy chain consists of SEQ ID NO: 7. In some embodiments, the antibody comprises a heavy chain comprising the sequence: MGWSCIILFLVATATGVHSQVQLVQSGGGLVQPGGSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVSAISGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCAKAPGDYTAYFDYWGQGTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPPCPAPEFLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPEVQFNWYVDGVEVHNAKTKPREEQFNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLPSSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLGK (SEQ ID NO: 9). In some embodiments, the heavy chain consists of SEQ ID NO: 9.

In some embodiments, the antibody comprises a light chain comprising the sequence: ELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIK (SEQ ID NO: 8). In some embodiments, the light chain consists of SEQ ID NO:8, and a sequence composition thereof. In some embodiments, the antibody comprises a light chain comprising the sequence: MGWSCIILFLVATATGVHSELVLTQSPATLSLSPGERATLSCRASQSVSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPPYTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC (SEQ ID NO: 10). In some embodiments, the light chain consists of SEQ ID NO: 10.

In some embodiments, the method further comprises administering PD-1/PD-L1-based immunotherapy. In some embodiments, the administering is administering a therapeutically effective amount of an immunotherapeutic agent (immunotherapeutic). The term "therapeutically effective amount (therapeutically effective amount)" refers to an amount of a drug effective to treat a disease or disorder in a mammal. In some embodiments, a therapeutically effective amount refers to an amount effective to achieve the desired therapeutic or prophylactic effect over the necessary dose and period of time. The exact dosage form and regimen will be determined by the physician according to the patient's circumstances.

In some embodiments, detecting comprises contacting the sample with an agent that binds HVEM. In some embodiments, detecting comprises contacting the sample with an agent that binds HVEM mRNA. In some embodiments, detecting comprises contacting the sample with an agent that binds HVEM DNA. In some embodiments, the contacting is performed under conditions suitable for binding of the agent or antibody or antigen binding fragment thereof to HVEM. In some embodiments, the conditions are suitable for specific binding to HVEM. In some embodiments, the binding is hybridization. The conditions of antibody/agent binding will depend on the sample. The skilled artisan will appreciate that the conditions required for binding to a tissue sample (depending on the method/type of immobilization) are different from the conditions of binding in a liquid solution, such as whole cell lysate. Such binding conditions are well known in the art and the skilled person can select appropriate conditions for a given sample.

In some embodiments, the assay is an immunohistochemical assay. In some embodiments, the assay is an immunocytochemistry assay. In some embodiments, the detection is an immunofluorescence detection. In some embodiments, the detection is FACS detection. In some embodiments, detecting further comprises contacting the sample with a secondary agent that recognizes the agent or antibody. In some embodiments, detecting comprises detecting a secondary agent. In some embodiments, the secondary agent is an antibody. In some embodiments, the secondary agent comprises a detectable moiety. In some embodiments, the agent comprises a detectable moiety. In some embodiments, detecting comprises detecting a detectable moiety. In some embodiments, the detecting comprises a microscope. In some embodiments, the detecting comprises flow cytometry. In some embodiments, the detection comprises FACS. In some embodiments, the detection comprises ELISA.

In some embodiments, the subject has a disease or disorder. In some embodiments, the subject is suspected of having a disease or disorder. In some embodiments, the subject is at risk of developing a disease or disorder. In some embodiments, the subject has a disease or disorder that may include increased HVEM expression. In some embodiments, the subject has a disease or disorder characterized by increased HVEM expression. In some embodiments, the subject has cancer. In some embodiments, the cancer is a cancer that does not respond to first line therapy. In some embodiments, the cancer is cancer recurrence. In some embodiments, the cancer is a cancer that does not respond to PD-1/PD-L1-based immunotherapy.

In some embodiments, positive expression of HVEM comprises HVEM expression. In some embodiments, positive expression of HVEM includes elevated HVEM levels. In some embodiments, positive expression of HVEM includes increased HVEM levels. In some embodiments, positive expression of HVEM comprises overexpression of HVEM. In some embodiments, positive expression of HVEM is compared to a healthy sample. In some embodiments, the healthy sample is from healthy tissue and/or cells in the vicinity of the diseased tissue and/or cells. In some embodiments, the healthy sample comprises non-infected cells. In some embodiments, the health sample is a donor for health. In some embodiments, positive expression of HVEM is compared to a predetermined threshold. In some embodiments, the increase or overexpression is at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 70, 75, 80, 85, 90, 95, 100, 125, 150, 175, 200, 250, 300, 350, 400, 450, or 500%. Each possibility represents a separate embodiment of the invention.

In some embodiments, the HVEM expression is HVEM mRNA expression, and detecting comprises detecting HVEM mRNA. In some embodiments, the detection is specific for detecting HVEM mRNA. Methods for detecting mRNA are well known in the art and may include, for example, RNA printing, PCR, microarrays, and sequencing. Any such method may be employed. In some embodiments, the detection comprises PCR. In some embodiments, the PCR is quantitative PCR (qPCR). In some embodiments, the PCR is real-time PCR. In some embodiments, detecting comprises sequencing. In some embodiments, the sequencing is deep sequencing. In some embodiments, the sequencing is next generation sequencing. In some embodiments, the sequencing is whole transcriptome sequencing. Examples of HVEM primers or probes are well known in the art, and any such sequence or molecule may be employed. Information on the human HVEM gene can be found in entry 8764 of the Entrez gene. The sequence of human HVEM isoform 1mRNA is provided in RefSeq nm_ 003820. The sequence of the human HVEM isoform 1 protein is found in RefSeq np_003811 and SEQ ID NO: 17. Alternative HVEM isoforms (isoform 2) are also known. This isoform lacks the transmembrane domain and is therefore a soluble form of HVEM. The mRNA sequence of HVEM isoform 2 is provided in RefSeq nm_001297605, and its amino acid sequence is provided in RefSeq np_001284534 and SEQ ID NO: 18.

In some embodiments, the sample is a tissue sample. In some embodiments, the sample is a paraffin-embedded sample. In some embodiments, the sample is a perfusion sample. In some embodiments, the sample is from a subject. In some embodiments, the sample is a healthy sample. In some embodiments, the sample is a diseased sample. In some embodiments, the sample is a diagnostic sample. In some embodiments, the sample is a biological fluid. In some embodiments, the biological fluid is selected from the group consisting of blood, serum, plasma, urine, stool, bile, semen, tumor fluid, and cerebrospinal fluid. In some embodiments, the sample is a blood sample. In some embodiments, the sample is a cell sample. In some embodiments, the sample comprises cells. In some embodiments, the sample comprises a disease cell. In some embodiments, the sample is a tumor sample. In some embodiments, the sample is a biopsy.

In some embodiments, the method further comprises treating the appropriate subject. In some embodiments, the treatment is with PD-1 based immunotherapy. In some embodiments, the treatment is with PD-L1 based immunotherapy. In some embodiments, the treatment is with PD-1/PD-L1-based immunotherapy. In some embodiments, the treatment comprises administration of immunotherapy. In some embodiments, the treatment is with HVEM-based immunotherapy. In some embodiments, the treatment is with a combination PD-1/PD-L1-based immunotherapy and HVEM-based immunotherapy.

As used herein, the term "treatment" or "treatment" of a disease, disorder or condition includes alleviating at least one symptom thereof, reducing the severity thereof, or inhibiting the progression thereof. Treatment does not necessarily mean that the disease, disorder or condition is completely cured. For effective treatment, the compositions or methods useful herein need only reduce the severity of the disease, disorder, or condition, reduce the severity of symptoms associated therewith, or provide an improvement in the quality of life of the patient or subject.

As used herein, the terms "administering", "administering" and similar terms refer to any method of providing a subject with a composition containing an active agent in a manner that provides a therapeutic effect in sound medical practice. One aspect of the present subject matter provides a method of intravenously injecting a therapeutically effective amount of a composition of the subject matter into a patient in need thereof. Other suitable routes of administration may include parenteral, subcutaneous, oral, intramuscular, topical or intraperitoneal. In some embodiments, the administration is systemic administration. In some embodiments, the administration is topical.

The dose administered will depend on the age, health and weight of the recipient, the type of concurrent therapy (if any), the frequency of treatment, and the nature of the desired effect.

By another aspect, an agent for specifically detecting HVEM for use in the method of the invention is provided.

By another aspect, a kit comprising agents specifically detecting HVEM for use in the methods of the invention is provided.

By another aspect, an agent for specifically detecting T cells for use in the methods of the invention is provided.

By another aspect, a kit comprising an agent for specifically detecting T cells for use in the methods of the invention is provided.

In some embodiments, the kit further comprises a secondary detection molecule. In some embodiments, the detection molecule is for detecting an agent. In some embodiments, the secondary detection molecule is an antibody that binds to the agent. In some embodiments, the detection molecule comprises a detectable moiety. Examples of detectable moieties include, but are not limited to, fluorescent moieties, labels, radioactive labels, dyes, and chemiluminescent moieties. In some embodiments, the detectable moiety is a fluorescent moiety. Examples of fluorescent moieties include, but are not limited to GFP, RFP, YFP, APC, CY, CY7, and Pacific Blue (Pacific Blue). Secondary detection molecules are well known in the art and any such molecule that will detect the agents of the present invention may be used.

In some embodiments, the agent specifically binds to HVEM protein. In some embodiments, the agent specifically binds to HVEM mRNA. In some embodiments, the agent specifically binds to HVEM cDNA. In some embodiments, the agent is a primer. In some embodiments, the agent is a nucleic acid probe. In some embodiments, the agent is an antibody or antigen-binding fragment thereof.

As used herein, the term "about" when combined with a value refers to the reference value plus or minus 10%. For example, a length of about 1000 nanometers (nm) refers to a length of 1000 nm.+ -. 100 nm.

It is noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes a plurality of such polynucleotides, reference to "the polypeptide" includes one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. Accordingly, this statement is intended to serve as antecedent basis for use of exclusive terminology such as "only," "only," and the like, or use of a "negative" limitation in connection with recitation of claim elements.

Where a convention analogous to "at least one of A, B and C, etc." is used, in general such an explanation is to be taken in the sense one having skill in the art would understand the convention (e.g., "a system having at least one of A, B and C" would include but not be limited to systems of A only, B only, C, A and B together, A and C together, B and C together, and/or A, B, and C together, etc.). It will be further understood by those within the art that virtually any non-conjunctive word and/or phrase presenting two or more alternative terms, whether in the specification, claims, or drawings, should be understood to contemplate the possibilities of including one of the terms, either of the terms, or both terms. For example, the phrase "a or B" will be understood to include the possibilities of "a" or "B" or "a and B".

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination. All combinations of embodiments related to the invention are specifically included by the invention and disclosed herein as if each combination was individually and specifically disclosed. Furthermore, the invention specifically encompasses all subcombinations of the various embodiments and elements thereof and is disclosed herein as if each such subcombination was individually and specifically disclosed herein.

Other objects, advantages and novel features of the present invention will become apparent to those of ordinary skill in the art upon examination of the following examples, which are not intended to be limiting. Furthermore, the various embodiments and aspects of the invention described above and claimed in the claims below find experimental support in the following examples.

Various embodiments and aspects of the invention as described above and claimed in the claims section find experimental support in the following examples.

Examples

Generally, the terms used herein and laboratory procedures used in the present invention include molecular, biochemical, microbial and recombinant DNA techniques. This technique is explained in detail in the literature. See, for example, "Molecular Cloning: A laboratory Manual" Sambrook et al, (1989); "Current Protocols in Molecular Biology", volumes I-III, ausubel, R.M. (1994); ausubel et al, "Current Protocols in Molecular Biology", john Wiley and Sons, baltimore, maryland (1989); perbal, "A Practical Guide to Molecular Cloning", john Wiley & Sons, new york (1988); watson et al, "Recombinant DNA", scientific American Books, new York; birren et al (eds.) "Genome Analysis: A Laboratory Manual Series", volumes 1-4, cold Spring Harbor Laboratory Press, new York (1998); U.S. patent No. 4,666,828;4,683,202;4,801,531;5,192,659 and 5,272,057; "Cell Biology: A Laboratory Handbook", volumes I-III, cellis, J.E., eds. (1994); "Culture of Animal Cells-A Manual of Basic Technique", freshney, wiley-Lists, N.Y. (1994), 3 rd edition; "Current Protocols in Immunology", volumes I-III, coligan J.E., eds. (1994); stites et al (eds.), "Basic and Clinical Immunology" (8 th edition), appleton & Lange, norwalk, CT (1994); mishell and Shiigi (ed.) "Strategies for Protein Purification and Characterization-A Laboratory Course Manual" CSHL Press (1996); all of which are incorporated by reference. Other general references are also provided in this document.

Example 1: immunohistochemistry on tumor microarrays

HVEM and PD-L1 are both immune checkpoint proteins expressed on the plasma membrane of many cancers. It is believed (Malissen et al 2019, "HVEM has a broader expression than PD-L1 and constitutes a negative prognostic marker and potential treatment target for melanoma", onco immunology,8:12, e 1665976) that high expression of HVEM is a marker of poor prognosis. It is also believed that circulating HVEM levels are not associated with an immunotherapy response (Zhang et al, 2020, "Anti-PD-1therapy response predicted by the combination of exosomal PD-L1 and CD28" front. Oncol., 10:760). To determine the relative expression of these two markers, tumor Microarrays (TMAs) of melanoma cells were selected, containing paraffin-embedded tumor samples from responders and non-responders to PD-1 blocking therapy (nivolumab or pembrolizumab). TMA contained 20 patients characterized as responders and 16 patients characterized as non-responders. TMA contains 1-3 cores from each of these patients.

From each block, 5 μm sections were prepared, deparaffinized, and antigen retrieval was performed in alkaline solution (pH 9). Triple staining was performed using mouse monoclonal anti-CD 3 antibody (Proteintech, #60181-1-Ig; 5. Mu.g/ml), rabbit monoclonal anti-PD-L1 antibody (Cell Signaling, #13684, 10. Mu.g/ml) and goat polyclonal anti-HVEM IgG antibody (R & D Systems #AF356, 10. Mu.g/ml) as primary antibodies. Anti-mouse Cy2, anti-rabbit Cy3 and anti-goat Cy5 secondary antibodies (Jackson, USA; #715-545-151/152/147,3.125. Mu.g/ml respectively) were used and nuclei were stained with DAPI. In addition to TMA, T cells (primary TILs) and PD-L1 positive melanoma cells were stained. Imaging was performed with a Leica TCS SP5 confocal laser scanning microscope (X63 magnification) and Image analysis was performed using FIJI (Image J2) software.

As expected, control T cells were positive for CD3, to a lesser extent HVEM, and negative for PD-L1 (fig. 1A, cell surface expression). Melanoma cells were negative for CD3 and strongly positive for PD-L1 and HVEM (FIG. 1B, cell surface expression of both is prevalent, with cytoplasmic expression of some HVEM). Staining of non-responders found a high degree of heterogeneity between samples, with CD3, PD-L1 and HVEM being negative in some samples and highly expressed in others (fig. 1C). The same heterogeneity was also observed in the samples with the reactants (fig. 1D). However, quantification of the expression of each protein throughout the responders and non-responders population produced both expected and unexpected results. The expression of CD3 was comparable in both populations and the observed minor differences were not statistically significant (fig. 1E). The expression of PD-L1 was elevated in the responders population, and this increase was significant when all fields of all patients of this group were analyzed (fig. 1F). The results for such PD-L1 have also been previously reported and are readily understood, as subjects with highest target expression of a therapeutic molecule are most likely to respond to the therapeutic molecule. Finally, examination of HVEM expression showed one of the most unexpected results; although HVEM was judged in the literature to be a marker of poor prognosis, higher HVEM levels were observed in the samples of the responders group compared to non-responders (fig. 1G). This result is statistically significant when evaluating all images, and even when the average value for each subject is used in the calculation. This result suggests that HVEM expression is not a negative prognostic marker, in fact a positive prognostic marker for PD-1/PD-L1-based immunotherapy response, and can be used diagnostically to determine patient populations more likely to respond to these immunotherapy.

Another unexpected result was observed when these three markers were compared in pairs. Comparing CD3 expression and PD-L1 expression in tumor samples only found very weak correlation (responders: r=0.32, p=0.174; non-responders: r=0.32, p=0.225), which was comparable in both responders and non-responders (fig. 2A). In the group with the responders, the same weak correlation was observed between CD3 and HVEM (r=0.39, p=0.09) (fig. 2B, upper panel). However, in the non-responder group, a moderate to high, statistically significant positive correlation (r=0.72, p=0.002) between CD3 and HVEM expression was observed (fig. 2B, bottom panel). In other words, those subjects with a high degree of T cell infiltration may also have high HVEM expression in subjects who are not responsive to anti-PD-1/PD-L1 immunotherapy. Since HVEM can be expressed in cancer and T cells, the correlation of each cell population was tested in the non-responder group. Correlation with HVEM expressed on T cells (fig. 3a, r=0.66, p= 0.00.5) was significant and comparable to correlation with HVEM expressed on cancer cells (fig. 3b, r=0.66, p=0.006).

The combined anti-HVEM and anti-PD-1/PD-L1 treatment has been demonstrated to be effective against cancers that express PD-L1/HVEM (see international patent application WO2020222235, incorporated herein by reference in its entirety). It has also been shown that tumors with higher HVEM expression are most likely to respond to HVEM therapy (see Aubert et al, "TNFRSF14 (HVEM) is a novel immune checkpoint for cancer immunotherapy in humanized mice", preprint on bioxiv. Org, doi. Org/10.1101/711119). This new result suggests that there is still a way to determine which subjects are most likely to receive HVEM monotherapy or HVEM/PD-1/PD-L1 combination therapy. T cell infiltration in the PD-1/PD-L1 non-responder population indicates HVEM levels. Infiltration of immune cells into the tumor microenvironment suggests the possibility of changing tumors from altered immunosuppressive immunotumors to thermal immunotumors suitable for receiving HVEM monotherapy or combination therapy to ameliorate ineffective PD-1/PD-L1 therapy.

Example 2:

selecting TMA for other PD-1 treatable cancers containing responder and non-responder samples: including lung cancer (non-small cell lung cancer), kidney cancer (renal cell carcinoma), head and neck cancer, bladder cancer, and cervical cancer. HVEM was stained and quantified as described above. Higher HVEM expression was found to be associated with response to PD-1/PD-L1 therapy.

In addition to protein levels, HVEM mRNA levels were also examined. Sequencing data and/or PCR analysis from responders and non-responders were analyzed and HVEM expression quantified. HVEM mRNA levels in subjects and tumor samples were found to correlate with responses to PD-1/PD-L1 therapy.

An analysis was also performed in which unknown samples were evaluated for HVEM expression and classified as either reactive or non-reactive. This is done with samples from subjects who have not received treatment, and the treatment results are recorded and found to match the predictions. Furthermore, samples from responders/non-responders are known to be provided as blind samples, where the analyst is unaware of the status. Again, expression of HVEM above the cutoff value indicates responsiveness to treatment.

While the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations as fall within the spirit and broad scope of the appended claims.

Sequence listing

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Special Ha Xiumo medical research infrastructure and service company

<120> markers responsive to PD-1/PD-L1 immunotherapy

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<150> US63/087376

<151> 2020-10-05

<150> US63/182968

<151> 2021-05-02

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<170> PatentIn version 3.5

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Claims (30)

1. A method of determining suitability for treatment by PD-1/PD-L1-based immunotherapy in a subject in need thereof, the method comprising receiving a sample from the subject and determining a level of herpes virus invasion mediator (HVEM) in the sample, wherein an expression of HVEM above a predetermined threshold indicates that the subject is suitable for treatment by the PD-1/PD-L1-based immunotherapy.

2. The method of claim 1, wherein the predetermined threshold is an expression level in a healthy sample or a disease sample from a non-responder to the PD-1/PD-L1.

3. The method of claim 1 or 2, wherein the HVEM level is HVEM mRNA level or HVEM protein level.

4. The method of claim 3, wherein the HVEM level is an HVEM protein level and the determining comprises contacting the sample with an agent that specifically binds an extracellular domain of HVEM.

5. The method of claim 4, wherein the antibody comprises three heavy chain CDRs (CDR-H) and three light chain CDRs (CDR-L), wherein: CDR-H1 includes SEQ ID NO:1 (SYAMS), CDR-H2 comprises the amino acid sequence set forth in SEQ ID NO:2 (AISGSGGSTYYADSVKG), CDR-H3 comprises the amino acid sequence set forth in SEQ ID NO:3 (APGDYTAYFDY), CDR-L1 comprises the amino acid sequence set forth in SEQ ID NO:4 (RASQSVSSYLA), CDR-L2 comprises the amino acid sequence set forth in SEQ ID NO:5 (gasssarat), and CDR-L3 comprises the amino acid sequence set forth in SEQ ID NO:6 (QQYGSSPPYT).

6. The method of claim 4 or 5, wherein the antibody or antigen binding fragment thereof comprises a detectable moiety, or wherein the determining further comprises contacting the sample with a secondary antibody comprising a detectable moiety.

7. The method of any one of claims 1 to 6, wherein the sample is a disease sample.

8. The method of claim 7, wherein the disease sample is a biopsy.

9. The method of any one of claims 1 to 7, wherein the sample is a body fluid.

10. The method of claim 8, wherein the bodily fluid is blood or plasma.

11. The method of any one of claims 1 to 10, wherein the subject has a disease or disorder treatable by PD-1/PD-L1-based immunotherapy.

12. The method of claim 11, wherein the disease is cancer.

13. The method of claim 12, wherein the cancer is a solid cancer.

14. The method of any one of claims 1-13, wherein detecting HVEM levels comprises detecting HVEM levels on the surface of cells and/or in the cytoplasm of cells in the sample.

15. The method of any one of claims 1-14, wherein detecting HVEM levels comprises detecting circulating HVEM levels.

16. The method of any one of claims 1 to 15, wherein the PD-1/PD-L1-based immunotherapy is PD-1 and/or PD-L1 blocking therapy.

17. The method of any one of claims 1 to 16, further comprising treating a suitable subject with the PD-1/PD-L1-based immunotherapy.

18. A method of determining suitability of a subject who is not responsive to PD-1/PD-L1-based immunotherapy for treatment with HVEM-based immunotherapy, the method comprising receiving a sample from the subject and determining a level of T cells in the sample, wherein a level of T cells above a predetermined threshold indicates that the subject is suitable for treatment by the HVEM-based immunotherapy.

19. The method of claim 18, wherein the subject has a disease or disorder treatable by PD-1/PD-L1-based immunotherapy and HVEM-based immunotherapy.

20. The method of claim 19, wherein the disease is cancer.

21. The method of claim 20, wherein the cancer is a solid cancer.

22. The method of any one of claims 18 to 21, wherein the sample is a disease sample.

23. The method of claim 22, wherein the disease sample is a biopsy.

24. The method of any one of claims 18 to 23, wherein determining T cell levels comprises measuring CD3 expression in the sample.

25. The method of any one of claims 18 to 24, wherein determining T cell levels comprises counting T cells.

26. The method of any one of claims 18 to 25, wherein the T cells are Tumor Infiltrating Lymphocytes (TILs).

27. The method of any one of claims 18-26, wherein a suitable subject is adapted to receive a combination of HVEM-based immunotherapy and PD-1/PD-L1-based immunotherapy.

28. The method of any one of claims 18-27, wherein the PD-1/PD-L1-based immunotherapy is a PD-1 and/or PD-L1 blocking therapy, the HVEM-based immunotherapy is an HVEM blocking therapy, or both.

29. The method of any one of claims 18-28, further comprising treating the appropriate subject with HVEM-based immunotherapy.

30. The method of claim 29, further comprising treating a suitable subject with the PD-1/PD-L1-based immunotherapy in combination with the HVEM-based immunotherapy.