WO2018056824A1

WO2018056824A1 - Manipulation of immune activity by modulation of expression - stub1

Info

Publication number: WO2018056824A1
Application number: PCT/NL2017/050639
Authority: WO
Inventors: Antonius Nicolaas Maria Schumacher; Thijn Reinout BRUMMELKAMP; Chong SUN; Lucas Tilmann JAE; Riccardo Ernesto MEZZADRA
Original assignee: Stichting Het Nederlands Kanker
Priority date: 2016-09-23
Filing date: 2017-09-25
Publication date: 2018-03-29
Also published as: WO2018056825A1; EP3516390A1; WO2018056824A8; WO2018056825A8

Abstract

The present invention relates to the field of immunity, immune activity and more particularly to the field of "immune check points" including the PD-1/PD-L1 axis, and conditions or diseases involving PD-1/PD-L1 axis signaling. Provided are modulators of immune activity, which modulators influence the activity and/or expression of STUB1.

Description

Title: Manipulation of immune activity by modulation of expression - STUB1. FIELD OF THE INVENTION

The present invention relates to the field of immunity, immune activity and more particularly to the field of "immune check points" including the PD-1/PD-L1 axis, and conditions or diseases involving PD-1/PD-L1 axis signaling. Provided are modulators of immune activity, which modulators influence the activity and/or expression of STUB1. The modulators may modulate immune activity, e.g. T-cell activity (towards its target), PD-1/PD-L1 axis signaling and/or PD- L1 expression. Also provided for are methods for screening compounds capable of modulating immune activity, PD-1/PD-L1 axis signaling and/or PD-L1 expression as well as methods for treating conditions or diseases involving aberrant immune activity, functioning of PD-1/PD-L1 axis signaling and/or altered PD-L1 levels, such as cancer and autoimmune diseases.

BACKGROUND OF THE INVENTION

The immune system is a host defense system comprising many biological structures, molecules, and processes within an organism that protects against disease. To function properly, an immune system must detect a wide variety of agents, known as pathogens, from viruses to parasitic worms, and distinguish them from the organism's own healthy tissue.

The immune system can be classified into several subsystems, such as the humoral immune system and the cell-based immune system (also referred to as cell-mediated immunity). While the humoral immune system is concerned with aspects of immunity that is mediated by antibodies, cell-mediated immunity is an immune response that does not involve antibodies. Rather, cell-mediated immunity involves, for example, the activation of phagocytes, T-cells such as antigen-specific cytotoxic T-lymphocytes or helper T-lymphocytes, and the release of various cytokines, for example by such T-cells in response to an antigen (upon binding of the TCR of the T cell to a peptide:MHC complex on the target cell). Cell-mediated immunity plays an important role in mediating immune responses in diseases or conditions such as cancer, infections, and autoimmune diseases.

An important component of cell-mediated immunity is the so-called "T-cell mediated immunity" (or T-cell immune activity). T cell or T lymphocyte is a type of lymphocyte that plays a central role in cell-mediated immunity (Williams et al (2007), Annual Review of Immunology, Vol. 25: 171-192; Wei F et al (2013) PNAS; VOL: 1 10, E2480-2489). T cells can be distinguished from other lymphocytes, such as B cells and natural killer cells, by the presence of a T-cell receptor on the cell surface. They are called T-cells because they mature in the thymus from thymocytes.

Once they have completed their development in the thymus, T-cells enter the bloodstream and lymphoid system and are carried by the circulation. To participate in an adaptive immune response, a naive T cell must first encounter antigen in the form of a peptide: MHC complex on the surface of an activated antigen-presenting cell (APC), and is thereby induced to proliferate and differentiate into "effector T cells" (Immunobiology, 5th edition, The Immune System in Health and Disease (2001) by Charles A Janeway, Jr, Paul Travers, Mark Walport, and Mark J Shlomchik, New York: Garland Science; ISBN-10: 0-8153-3642-X).

Effector T cells encompass a broad variety of T cells including T helper cells and T killer cells. Effector T cells are capable of killing or destroying pathogens, infected cells, or aberrant cells (e.g. cancer cells displaying tumor antigens) due to their ability to induce apoptosis and to secrete cytokines such as IFN gamma (IFNg, also referred to as INFg) and TNF alpha (TNFa), as well as chemokines including CXCL9 and CXCL10, and others. Effector T cells can also secrete perforin-granzymes (Immunobiology, 5th edition, The Immune System in Health and Disease (2001) by Charles A Janeway, Jr, Paul Travers, Mark Walport, and Mark J Shlomchik, New York: Garland Science; ISBN-10: 0-8153-3642-X).

Effector T cells have also been shown to play an important role in anti-tumor immunity (e.g. against tumor cells displaying tumor antigens). However, tumor microenvironments can pose particular challenges for effector T cells. Specifically, multiple studies have shown that tumors have the ability to suppress immune responses mediated by effector T cells by inhibiting effector T cell function or activity (e.g. secretion of cytokines as mentioned above) and/or reducing or blocking proliferation of effector T cells. One way by which tumors achieve these effects is through expression of so-called inhibitory "immune check points" (Romano and Romero (2015), Journal for immunotherapy, Vol 3: 15). Immune checkpoints are molecules in the immune system that either turn up or turn down a signal from immune cells (e.g. secretion of cytokines from effector T cells), for example, so as to reduce immune responses to mitigate collateral tissue damage. One such immune check point consists of the programmed death-ligand 1 (PD-L1) and its receptor, the programmed death-1 receptor (PD-1). PD-L1 and PD-1 are often referred to as the "PD-1/PD-L1 axis" or "PD-1/PD-L1 pathway" The PD-1/PD-L1 axis is also referred to as a "negative immune checkpoint" or 'inhibitory immune checkpoint' because it reduces or turns down immune signals (e.g. secretion of cytokines by effector T cells). Normally, inhibitory immune check points, such as for instance the "PD-1/PD-L1 axis, serve as safeguard mechanisms aimed at keeping the immune system from overreacting to a stimulus or mistaking a component of the body for a dangerous invader. In the context of cancer, tumor cells protect themselves from the host immune system or escape host immune surveillance (e.g. cancer cells displaying tumor antigens should normally be recognized and destroyed by effector T cells) by inhibiting or interfering with effector T cell function or activity (e.g. cytokine production or effector T cell proliferation) by signaling through the PD-1/PD-L1 axis, as further discussed below (Taku Okazaki and Tasuku Honjo (2007), International Immunology, Vol: 19, pages 813-824; Iwai Y et al (2002), PNAS Vol: 99, pages 12293).

PD-L1 is a transmembrane glycoprotein also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1). In human, PD-L1 is encoded by the CD274 gene. PD-L1 can be expressed on a variety of cell types, including placenta, vascular endothelium, pancreatic islet cells, muscle, hepatocytes, epithelium, and mesenchymal stem cells, as well as on B cells, T cells, dendritic cells, macrophages, mast cells, and others (Suya Dai et al (2014) Cellular Immunology, Vol:290, pages 72-79). The expression of PD-L1 is further up-regulated (i.e. increased compared to resting conditions) on cells by various immune stimuli including for instance anti-lgM antibody, LPS and anti-CD40 antibody for B cells, anti-CD3 antibody for T cells, anti-CD40 antibody, LPS, IFN gamma and granulocyte macrophage colony stimulating factor for macrophages and anti-CD40 antibody, IFN gamma, IL-4, IL-12 and GM-CSF for dendritic cells (Taku Okazaki and Tasuku Honjo (2007), International Immunology, Vol: 19, pages 813-824).

PD-1 (also known as CD279 or cluster of differentiation 279) is a cell surface receptor that belongs to the immunoglobulin superfamily. More specifically, PD-1 is a member of the CD28 family of receptors, which includes CD28, CTLA-4, ICOS, PD-1 , and BTLA. In human, PD-1 is encoded by the PDCD1 gene. PD-1 is expressed at the surface of immune cells such as activated T cells, including effector T cells (e.g. killer T cells), B cells, NKT cells, and myeloid cells (Suya Dai et al (2014) Cellular Immunology, Vol:290, pages 72-79; Gianchecchi et al (2013), Autoimmun. Rev. 12 (2013) 1091-1100).

Under normal situation, the PD-1/PD-L1 axis plays a major role in suppressing the immune system during particular events such as pregnancy, tissue allografts, autoimmune diseases and other disease states such as hepatitis. Normally, the immune system reacts to foreign antigens that have accumulated in the lymph nodes or spleen by triggering the proliferation of antigen-specific CD8+ effector T cells (also known as killer T cells). The binding of PD-L1 to its receptor PD-1 transmits an inhibitory signal which reduces the proliferation of these CD8+ T cells within the lymphoid organs. More specifically, binding of PD-L1 to its receptor PD-1 on T cells delivers a signal that inhibits T cell receptor (TCR)-mediated activation of the T cell, as for instance reflected by cytokine (e.g. IL-2 and others) production and T cell proliferation, thus effectively dampening or suppressing the immune response (Wei F et al (2013) PNAS; VOL: 1 10, E2480-2489). Also in non-lymphoid tissues, binding of PD-L1 to PD-1 on T cells inhibits T cell activation.

Under normal situations, the suppression of the immune system by the PD-1/PD-L1 axis is meant to minimize or avoid the death of bystander host cells (e.g. healthy cells) and to prevent the development of autoimmune diseases. Indeed, it was shown that PD-L1 deficiency in mouse or PD-L1 dysregulation in human due to the occurrence of SNP(s) in the gene encoding the PD-L1 protein or the PD-1 receptor was associated with autoimmunity. These results show the importance of the PD-1/PD-L1 axis in preventing overshooting of the immune system against host cells.

Under pathological conditions, such as cancers, the suppression of the immune system by the PD-1/PD-L1 axis is maladaptive and detrimental to the host, because it allows the tumor cells to escape immune surveillance and continue growing. For this reason, the PD-1/PD-L1 axis has become a main center of interest for the treatment of various cancers such as melanoma, breast, lung, kidney, ovary, bladder, colon, hepatocellular, gastrointestinal tract (Gl) cancer, Hodgkin's lymphoma, and colorectal cancers, and others It was shown that in the cancer disease state, the expression of PD-L1 is often up-regulated (i.e. a higher expression of the protein, e.g. in the cell surface) at the external surface (cell surface) of cancer cells (Taku Okazaki and Tasuku Honjo (2007), International Immunology, Vol: 19, pages 813-824). In this context, the interaction between the PD-L1 on the cancer cell surface and the PD-1 receptor on an immune cell (e.g. T-cell) is promoted. This leads to decreased or reduced immune cell (e.g. effector T cell) function or activity, e.g. decreased or reduced secretion of cytokines and/or decreased or reduced proliferation of T cells, which in turn prevents or hinders the immune system from attacking the tumor cells. PD-L1 can also be expressed by non-cancerous cells within the tumor micro-environment, with the same deleterious effects on immune cell function.

Globally, this allows cancer cells to escape detection by the host immune system. These results prompted the development of new cancer therapies aimed at inhibiting or blocking the PD-1/PD-L1 axis. For instance, PD-1/PD-L1 axis inhibitors that block the interaction of PD-L1 with the PD-1 receptor are currently being used to prevent the cancer from evading the immune system (Brahmer et al. (2010) J Clin Oncol 28:3167-75; Brahmer et al. (2012) N. Engl J Med 366:2455-65; Flies et al. (2011 ) Yale J Biol Med 84:409-21.; Topalian et al. (2012b) N Engl J Med 366:2443-54.). Examples of PD-1/PD-L1 axis inhibitors include anti-PD-L1 antibodies (e.g. BMS-936559), as well as anti-PD-1 antibodies (e.g. nivolumab (BMS-936558), and combination thereof. Although cancer therapies relying on the use of such compounds have shown promising clinical results in humans, such treatment is still not optimal.

For instance, one of the drawbacks associated with the use of antibodies includes their large size (limits diffusion into solid tumors) and their ability to activate antibody dependent cell- mediated cytotoxicity, through their Fc-region. While Fc-mediated effects are an important part of the efficacy of many antibody therapeutics, in the case of PD-1/PD-L1 axis inhibition this may be counterproductive. A further drawback of anti-PD-L1 or anti-PD-1 antibodies is their lack of specificity for cancer cells or lack of specific effects on cancer cells (i.e. they target healthy cells or has effects on healthy cells as well). Others adverse effects experienced by patients treated with such compounds include fatigue, infusion reactions, diarrhea, arthralgia, rash, nausea, pruritus, headache, rash, hypothyroidism, hepatitis, endophtalmitis, diabetes mellitus, myasthenia gravis, pneumonitis, vitiligo, colitis, hypophysitis, thyroditis, and others. Further, not all subjects respond to anti-PD-L1 antibody- or anti-PD-1 antibody based therapy.

Alternative (non-antibody) PD-L1 or PD-1 or PD-1/PD-L1 axis inhibitors are being developed such as engineered affinity proteins (e.g. engineered Affimer protein scaffold), which are smaller in size than antibodies, and thus have the potential to better diffuse within solid tumors. However, such inhibitor compounds also lack cell specificity, i.e. target healthy cells in addition to cancer cells.

Therefore, there is a need for alternative or improved therapies, including cancer therapies or treatments, which do not suffer from one or more of the limitations above. More specifically, there is a need for alternative or new compounds and/or methods which can be used to block or inhibit or reduce the function or activity of the PD-1/PD-L1 axis in cancer cells and/or immune cells. Further, there is also a need for alternative or new compounds and/or methods which can be used to activate or enhance or increase the function or activity of the PD-1/PD- L1 axis in cells in the context of autoimmune disorders. SUMMARY OF THE INVENTION

The present invention relates to the finding of cellular proteins that may modulate immune activity, in particular modulate cell-mediated immunity, PD-1/PD-L1 axis signaling, PD-L1 expression and/or PD-L1 protein levels. It was found that modulating expression or activity of these proteins alters (e.g. up-regulates or down-regulates) the expression or amount of PD- L1 protein in a cell (e.g. at the cell surface). With respect to PD-L1 expression, within the context of the current invention, this refers to both non-stimulated PD-L1 expression and to PD-L1 expression as the consequence of (the presence of) stimuli. The skilled person knows that, for example, the expression of PD-L1 is further up-regulated (i.e. increased compared to resting conditions) on cells by various immune stimuli including for instance anti-lgM antibody, LPS and anti-CD40 antibody for B cells, anti-CD3 antibody for T cells, anti-CD40 antibody, LPS, IFN gamma and granulocyte macrophage colony stimulating factor for macrophages and anti-CD40 antibody, IFN gamma (INFg), IL-4, IL-12 and GM-CSF for dendritic cells (see, e.g. Taku Okazaki and Tasuku Honjo (2007), International Immunology, Vol: 19, pages 813- 824).

Specifically, the present inventors found that blocking the expression or down-regulating the expression of STUB1 , in a cell (e.g. cancer cell), increases the expression of PD-L1 or increases the amount of PD-L1 protein (e.g. cancer cell, pancreatic cells, etc.) in said cell.

As will be understood by the skilled person, and without being bound to any theories, it is believed that these finding may be applied, for instance in clinic or in treatment of a patient suffering from cancer or autoimmune disease, according to the following non-limiting scenarios:

Scenario 1 ) Increasing the levels of PD-L1 (e.g. at the cell surface), as a consequence of blocking, inhibiting or down-regulating STUB1 in a cell (e.g. pancreatic cell) of a subject, will enhance or increase PD-1/PD-L1 signaling or facilitate or increase binding of PD-L1 to its receptor PD-1 (as a consequence of increased availability of PD-L1). This will ultimately decrease host immune activity (e.g. decreased T-cell function such as cytokine and chemokine secretion). Such situation would be advantageous, for instance, for the treatment of an autoimmune disease (e.g. diabetes type 1 , systemic lupus erythematosus, rheumatoid arthritis, and others), where decreased host immune activity against cells (e.g. pancreatic cells) is desired.

Scenario 2) Decreasing the levels of PD-L1 (e.g. at the cell surface), as a consequence of up-regulating or increasing the expression of STUB1 in a cell (e.g. cancer cell) of a subject, will impair or decrease PD-1/PD-L1 signaling or impair or decrease binding of PD-L1 to its receptor PD-1. This will ultimately increase host immune activity (e.g. increased T-cell function such as cytokine and chemokine secretion). Such situation would be advantageous, for instance, for the treatment of cancer (e.g. bladder, lung, melanoma, colon, Gl tract, Hodgkin's lymphoma, and others), where increased host immune activity against cancer cells is desired.

The present findings (including scenarios above) have important implications for the field of immunity, in particular cell-mediated immunity, particularly for diseases or conditions involving aberrant PD-1/PD-L1 axis signaling or altered levels of PD-L1 expression or amount of PD-L1 proteins (e.g. at the cell surface), such as immunotherapy of cancer or treatment of autoimmune diseases. Specifically, the present findings may be used as follows:

1) to develop screening assays to uncover new compounds capable of modulating immune activity, in particular PD-1/PD-L1 axis signaling and/or expression and/or amount of PD-L1 protein levels (e.g. at the cell surface), e.g. by selecting compounds capable of modulating the expression or amount of STUB1.

2) to develop methods of treating diseases or conditions involving aberrant immune activity, in particular aberrant PD-1/PD-L1 axis signaling and/or altered levels of PD-L1 expression and/or amount of PD-L1 proteins (e.g. at the cell surface), such as immunotherapy of cancer and autoimmune diseases, by treating a subject with compounds capable of modulating the expression or amount of STUB1. 3) to use STUB1 , and modulators thereof, for the treatment of diseases or conditions involving aberrant PD-1/PD-L1 axis signaling or altered levels of PD-L1 expression or amount of PD-L1 proteins (e.g. at the cell surface), such as immunotherapy cancer or treatment of autoimmune diseases. These and other advantages of the invention will become obvious in the present disclosure. BRIEF DESCRIPTION OF THE FIGURES

Figure 1. Haploid genetic screen for PD-L1 in HAP1 cells. Mutagenized HAP1 cells were stained for PD-L1 , sorted by flow cytometry for high or low PD-L1 staining intensities, and gene-trap insertion sites were mapped to the human genome. For visualization, per gene the normalized coefficient of disruptive gene-trap integrations (mutational index, Ml) within the PD-L1 high and low populations is plotted on the y-axis against the combined number of total insertions on the x-axis. Genes for which insertions could only be mapped in either one of the two populations, were assigned 1 insertion in the other population to allow plotting. Genes were considered enriched in the PD-L1 high population or significantly enriched in the PD-L1 low population when P<=10E-6.

Figure 2. 8505c, A375, colo679 cell lines were transduced with a lentivirus expressing the hCas9 together with a sgRNA targeting STUB1 and selected with puromycin. Next, the selected 8505c, A375, colo679 cells were exposed to different concentrations of IFN gamma for 48h in order to induce or increase PD-L1 expression. Surface levels of PD-L1 were analyzed by flow cytometry. The results show that sgRNA targeting of STUB1 STUBIcaused an increase in PD-L1 expression in all cell lines tested (i.e. 8505c, A375, and colo679 cell lines) in response to IFN gamma stimulation, i.e. enhanced the effect of IFN gamma on PD- L1 expression. DETAILED DESCRIPTION OF THE INVENTION

Definitions

Various terms relating to the methods, compositions, uses and other aspects of the present invention are used throughout the specification and claims. Such terms are to be given their ordinary meaning in the art to which the invention pertains, unless otherwise indicated. Other specifically defined terms are to be construed in a manner consistent with the definition provided herein. Although any methods and materials similar or equivalent to those described herein can be used in the practice for testing of the present invention, the preferred materials and methods are described herein. The "Programmed Death-1 (PD-1)" receptor as used herein refers to an immune-inhibitory receptor belonging to the CD28 family. PD-1 is expressed predominantly on previously activated T 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. U64863. PD-1 is expressed on immune cells such as activated T cells (including effector T cells), B cells, myeloid cells, thymocytes, and natural killer (NK) cells (Suya Dai et al (2014) Cellular Immunology, Vol:290, pages 72-79; Gianchecchi et al (2013), Autoimmun. Rev. 12 (2013) 1091-1 100). "Programmed Death Ligand-1 (PD-L1)" as used herein refers to one of two cell surface glycoprotein ligands for PD-1 (the other being PD-L2) that down-regulates immune cell activation and cytokine secretion upon binding to PD-1. The term "PD-L1" as used herein includes human PD-L1 (hPD-L1), variants, isoforms, and species homologs of hPD-L1 , and analogs having at least one common epitope with hPD-L1. The complete hPD-L1 sequence can be found under GEN BANK Accession No. Q9NZQ7. PD-L1 is expressed on a variety of cells including cells of hematopoietic lineage such as activated T cells, B cells, monocytes, dendritic cells (DCs), mast cells, and macrophages. PD-L1 is also expressed on peripheral non-hematopoietic tissue such as heart cells, skeletal muscle cells, pancreatic islet cells, placenta cells, lung cells, hepatocytes, epithelium cells, kidney cells, mesenchymal stem cells, liver cells, and others (Suya Dai et al (2014) Cellular Immunology, Vol:290, pages 72- 79).

In more detail, PD-L1 is expressed on T and B cells, myeloid cells (e.g. dendritic cells, macrophages, neutrophils), mesenchymal stem cells, and bone marrow-derived mast cells. PD-L1 is also expressed on a wide range of nonhematopoietic cells (e.g., cornea, lung, vascular epithelium, liver nonparenchymal cells, mesenchymal stem cells, pancreatic islets, placental synctiotrophoblasts, keratinocytes, brown adipose tissue, etc.), and is upregulated on a number of cell types after activation. Both type I and type II interferons (IFNs) and hypoxia upregulate PD-L1. PD-L1 is expressed in many cancers. Any cell that expresses or can express PD-L1 , including those wherein PD-L1 is activated or introduced using a vector, is consider a suitable cell within the context of the current invention.

The term "PD-1/PD-L1 axis" as used herein consists of the PD-1 receptor and its ligand PD- L1. The term "PD-1/PD-L1 axis signaling" is a way of communication between cells (cell signaling), for instance between a first cell expressing PD-1 and a second cell expressing PD- L1 , and which involves the release of a biochemical signal (e.g. release of proteins, lipids, ions, neurotransmitters, enzymes, gases, etc), which in turn causes an effect (e.g. inhibition, activation, blockade, etc) on one or both cells. The term "cell signaling" in general refers to the biochemical relationship between a variety of signal transduction molecules that play a role in the transmission of a signal from one portion of a cell to another portion of the cell. A "cell surface receptor" includes, for example, molecules and complexes of molecules that are located on the surface of a cell and are capable of receiving a signal and transmitting such a signal across the plasma membrane of a cell. An example of a cell surface receptor of the present invention is the PD-1 receptor, which is, for example, located on the surface of activated B cells, activated T cells and myeloid cells. In the context of the present invention, an example of "PD-1/PD-L1 axis signaling" is when PD-L1 expressed at the cell surface of a first cell (e.g. cancer cells or a cancer-infiltrating immune cells) binds to its receptor PD-1 expressed at the cell surface of a second cell (e.g. a T cell, such as an effector T cell). The binding of PD-L1 to its receptor PD-1 transmits an inhibitory signal to the T-cell which results in a decrease in T cell proliferation (e.g. effector T cells) as well as T cell activity (e.g. secretion of cytokines and chemokines as discussed herein; Wei F et al (2013) PNAS; Vol: 1 10, E2480-2489). Thus, one possible end result of PD-1/PD-L1 axis signaling is the dampening or inhibition of immune activity or function mediated by T cells (e.g. effector T cells). Such situation may be detrimental in the context of cancer (e.g. lung cancer, bladder cancer, Gl tract cancer, melanoma, etc), as discussed herein. Another example of "PD-1/PD- L1 axis signaling" is when PD-L1 expressed at the cell surface of a first cell (e.g. pancreatic cells) binds to its receptor PD-1 expressed at the cell surface of a second cell (e.g. a T cell, such as an effector T cell). The binding of PD-L1 to its receptor PD-1 transmits an inhibitory signal to the T-cell which ultimately causes a reduction or inhibition of T-mediated secretion of cytokines (e.g. Interferon gamma, TNF alpha, and others) and chemokines (e.g. CXCL9, CXCL10) as well as reduced T cell (e.g. effector T cell) proliferation (Wei F et al (2013) PNAS; Vol: 1 10, E2480-2489). Thus, one possible end result of PD-1/PD-L1 axis signaling is the dampening or inhibition of immune activity or function mediated by T cells (E.g. effector T cells). Such situation may be advantageous in the context of autoimmune diseases (e.g. diabetes type 1 , rheumatoid arthritis, systemic lupus erythematosus, etc.), where dampening of an overly active immune system (e.g. T-cell mediated effects) is desired, as discussed herein. Other examples of end results of PD-1/PD-L1 axis signaling are described in the scenarios above.

The term "cancer-infiltrating (immune) cells" as used herein is known in the art and refers to white blood cells that have left the bloodstream and migrated into a tumor or cancer. They are mononuclear immune cells, which may be a mixture of different types of cells, for instance T cells, B cells, NK cells, macrophages, and others in variable proportions, T cells often being abundant cancer-infiltrating immune cells. Thus, it is understood that cancer-infiltrating immune cells, such as T-cells (e.g. effector T-cells) may express PD-L1 and/or PD-1 , as explained herein. It was shown that cancer-infiltrating immune cells are implicated in killing tumor cells, and that the presence of such cancer-infiltrating immune cells (e.g. cytotoxic T cells) in tumors is often associated with better clinical outcomes.

The term "STUB1 homology and U-Box containing protein 1 " (abbreviated as "STUB1") as used herein refers to a human gene and protein also known as "C terminus of HSC70- Interacting Protein" (also known as CHIP; UBOX1 ; SCAR16; HSPABP2; NY-CO-7; SDCCAG7). This gene encodes a protein containing tetratrico peptide repeat and a U-box that functions as an E3 ubiquitin ligase/co-chaperone and promotes ubiquitination (also known as ubiquitylation). The encoded protein binds to and ubiquitinates Heat shock cognate 71 kDa protein (Hspa8) and DNA polymerase beta (Polb), among other targets. Further, the protein encoded by this gene binds to and inhibits the ATPase activity of the chaperone proteins HSC70 and HSP70 and blocks the forward reaction of the HSC70-HSP70 substrate-binding cycle. The STUB1 protein enhances HSP70 induction during acute stress and also mediates its turnover during the stress recovery process. Hence, amongst other things, the STUB1 protein plays a role in maintaining protein homeostasis by controlling chaperone levels during stress and recovery (HGNC:1 1427; Ensembl:ENSG00000103266; Scanlan MJ et al. Int. J. Cancer 1998 May;76(5):652-658; Ballinger CA et al. Mol. Cell. Biol. 1999 Jun;19(6):4535- 4545). The term "immune activity" as used herein refers to the action or interaction, including the end results, of one or more cell of the immune system (for example, T lymphocytes (e.g. effector T cells), B lymphocytes, natural killer (NK) cells, macrophages, eosinophils, mast cells, dendritic cells, neutrophils, and others) and soluble macromolecules produced by any of these cells or the liver (including antibodies, cytokines (e.g. IFN gamma, TNF alpha), chemokines (e.g. CXCL9, CXCL10), and complement) that results in selective targeting, binding to, damage to, destruction of, and/or elimination from a vertebrate's body of invading pathogens, cells or tissues infected with pathogens, cancerous or other abnormal cells, or, in cases of autoimmunity or pathological inflammation, normal human cells or tissues. The term "immune activity" encompasses the activity or function of T cells, such as effector T cells as described herein, that is expressed towards a target cell (e.g. cancer cells or pancreatic cells) under both basal condition (non-immune challenge) and immune challenge or stimulation condition. In an embodiment, immune activity or immune response includes T cell mediated and/or B cell mediated immune responses that are influenced by modulation of T cell costimulation/ co- inhibition. Exemplary immune responses include T cell responses, e.g., cytokine production, and cellular cytotoxicity. In addition, the term immune response includes immune responses that are indirectly affected by T cell activation, e.g., antibody production (humoral responses) and activation of cytokine responsive cells, e.g. macrophages.

The term "effector T cell" as used herein refers to a naive T cell that has encountered antigen in the form of a peptide: MHC complex on the surface of an activated antigen-presenting cell (APC), and as a result, is induced to proliferate and differentiate into "effector T cells". Effector T cells fall into two functional classes that detect different types of peptide:MHC complexes (including tumor antigens). For instance, peptides from intracellular pathogens that multiply in the cytoplasm are carried to the cell surface by MHC class I molecules and presented to CD8 T cells. These differentiate into cytotoxic T cells that kill infected target cells. Peptide antigens from pathogens multiplying in intracellular vesicles, and those derived from ingested extracellular bacteria and toxins, are carried to the cell surface by MHC class II molecules and presented to CD4 T cells. CD4 T cells can differentiate into multiple types of effector T cells, including TH1 , TH2, and TH17. Pathogens that accumulate in large numbers inside macrophage and dendritic cell vesicles tend to stimulate the differentiation of TH1 cells, whereas extracellular antigens tend to stimulate the production of TH2 cells. TH1 cells activate the microbicidal properties of macrophages, and induce B cells to make IgG antibodies that are very effective at opsonizing extracellular pathogens for uptake by phagocytic cells. TH2 cells initiate the humoral immune response by activating naive antigen- specific B cells to produce IgM antibodies. These TH2 cells can subsequently stimulate the production of different isotypes, including IgA and IgE, as well as neutralizing and/or weakly opsonizing subtypes of IgG. (Immunobiology, 5th edition, The Immune System in Health and Disease (2001 ) by Charles A Janeway, Jr, Paul Travers, Mark Walport, and Mark J Shlomchik, New York: Garland Science; ISBN-10: 0-8153-3642-X).

The term "effector T cell activity" as used herein refers to immune activity mediated by effector T cells upon signaling through the T cell receptor (TCR) expressed on T cells. In the context of the present invention, "effector T cell activity" encompasses the activity described above, for instance ability to induce apoptosis in a target cell by secreting perforin-granzymes as well as ability to kill or destroy pathogens or infected cells or aberrant cells (e.g. cancer cells displaying tumor antigens) by secreting substances such as cytokines (e.g. IFN gamma, TNF alpha) and chemokines (e.g. CXCL9, CXCL10).

The term "compound capable of modulating (e.g. increasing or decreasing) immune activity (e.g. effector T cell activity) as used herein refers to a compound, substance (a test substance in the screening method as taught herein), or agent that regulates an immune activity. Such compound may also be referred to as "modulator". "Regulating," "modifying" or "modulating" an immune activity refers to any alteration in a cell of the immune system (e.g. T cells such as effector T cells, cancer infiltrating immune cells or other immune cells) or in the activity of such cell, for example as the consequence of such alteration. Such regulation includes stimulation or suppression or reduction of the immune activity which may be manifested by an increase or decrease in the number of various cell types, an increase or decrease in the activity of these cells (e.g. secretion of cytokines, chemokines, perforin- granzymes as discussed above), or increase or decrease in signaling pathway (e.g. PD-1/PD- L1 axis) between these cells, or any other changes which can occur within the immune system.

The term "cancer" as used herein refers a broad group of various diseases characterized by the uncontrolled growth of abnormal cells in the body. Unregulated cell division and growth results in the formation of malignant tumors that invade neighboring tissues and may also metastasize to distant parts of the body through the lymphatic system or bloodstream. Non- limiting examples of cancers, and that may treated within the context of the current invention, include bladder cancer, gastrointestinal (G!) tract cancers, lung cancer, melanoma, Hodgkin's lymphoma, skin cancer (melanoma), head and neck squamous cell carcinomas (HNSCC), adrenocortical tumors, adrenal cancer, anal cancer, bile duct cancer, bladder cancer, bone cancer, brain cancer, breast cancer, central nervous system cancer, cervical cancer, chest cancer, colon cancer, colorectal cancer, endometrial cancer, epidermoid carcinoma, esophageal cancer, eye cancer, glioblastoma, glioma, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumor, gestational trophoblastic disease, head and neck, Hodgkin disease, Kaposi sarcoma, kidney cancer, laryngeal and hypopharyngeal cancer, leukemia, liver cancer (such as hepatocellular carcinoma), lung cancer (including non-small cell, small cell, and lung carcinoid tumors), lymph node cancer, lymphoma, lymphoma of the skin, melanoma, mesothelioma, mouth cancer, multiple myeloma, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oral cavity and oropharyngeal cancer, osteosarcoma, ovarian cancer, pancreatic cancer, penile cancer, pituitary cancer, prostate cancer, pediatric malignancies, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland, sarcoma, skin cancer, small intestine cancer, stomach cancer, testicular cancer, thymus cancer, throat cancer, thyroid cancer, uterine cancer, vaginal cancer, Merkel cell cancer, microsatellite unstable cancers and/or vulvar cancers.

The term "autoimmune diseases" as used herein refers to a pathological state arising from an abnormal immune response of the body to substances and tissues that are normally present in the body (i.e. "self"). Autoimmunity, on the other hand, is the presence of a self-reactive immune response (e.g., auto-antibodies, self-reactive T-cells), with or without damage or pathology resulting from it. This may be restricted to certain organs (e.g. in autoimmune thyroiditis) or involve a particular tissue in different places (e.g. Goodpasture's disease which may affect the basement membrane in both the lung and the kidney). The treatment of autoimmune diseases is typically with immunosuppression— medication that decreases the immune response. Novel treatments include cytokine blockade (or the blockade of cytokine signaling pathways), removal of effector T-cells and B-cells (e.g. anti-CD20 therapy can be effective at removing instigating B-cells). Intravenous Immunoglobulin has been helpful in treating some antibody mediated autoimmune diseases as well, possibly through negative feedback mechanisms. At least 80 types of autoimmune diseases are recognized. Non- limiting examples of autoimmune diseases include type 1 diabetes, rheumatoid arthritis, lupus (e.g. systemic lupus erythematosus), and others. Examples of other autoimmune diseases which may be treated with in the context of the current invention include but are not limited to multiple sclerosis (MS), Crohn's disease, scleroderma, Sjogren's syndrome, pemphigus vulgaris, pemphigoid, addison's disease, ankylosing spondylitis, aplastic anemia, autoimmune hemolytic anemia, autoimmune hepatitis, coeliac disease, dermatomyositis, Goodpasture's syndrome, Graves' disease, Guillain-Barre syndrome, Hashimoto's disease, idiopathic leucopenia, idiopathic thrombocytopenic purpura, male infertility, mixed connective tissue disease, myasthenia gravis, pernicious anemia, phacogenic uveitis, primary biliary cirrhosis, primary myxoedema, Reiter's syndrome, stiff man syndrome, thyrotoxicosis, ulceritive colitis, and Wegener's granulomatosis.

The term infectious (viral and non-viral) diseases or infection as used herein refers to a disease or condition attributable to the presence in a host of a foreign organism or agent that reproduces within the host. Infections typically involve breach of a normal mucosal or other tissue barrier by an infectious organism or agent. A subject that has an infection is a subject having objectively measurable infectious organisms or agents present in the subject's body. Infections are broadly classified as bacterial, viral, fungal, or parasitic based on the category of infectious organism or agent involved. Other less common types of infection are also known in the art, including, e.g., infections involving rickettsiae, mycoplasmas, and agents causing scrapie, bovine spongiform encephalopthy (BSE), and prion diseases (e.g. kuru and Creutzfeldt-Jacob disease). Examples of bacteria, viruses, fungi, and parasites which cause infection are well known in the art. Exemplary viruses include, but are not limited to: Retroviridae (e.g., human immunodeficiency viruses, such as HIV-1 (also referred to as HTLV-III), HIV-2, LAV or HTLV- MI/LAV, or HIV-III, and other isolates, such as HIV-LP; Picornaviridae (e.g. , polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses); Calciviridae (e.g. , strains that cause gastroenteritis); Togaviridae (e.g., equine encephalitis viruses, rubella viruses); Flaviviridae (e.g., dengue viruses, encephalitis viruses, yellow fever viruses); Coronaviridae (e.g. , coronaviruses); Rhabdoviridae (e.g., vesicular stomatitis viruses, rabies viruses); Filoviridae (e.g. , ebola viruses); Paramyxoviridae (e.g. , parainfluenza viruses, mumps virus, measles virus, respiratory syncytial virus); adenovirus; Orthomyxoviridae (e.g. , influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses, phleboviruses and Nairo viruses); Arena viridae (hemorrhagic fever viruses); Reoviridae (e.g. , reoviruses, orbiviurses and rotaviruses, i.e., Rotavirus A, Rotavirus B. Rotavirus C); Birnaviridae; Hepadnaviridae (Hepatitis A and B viruses); Parvoviridae (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, Human herpes virus 6, Human herpes virus 7, Human herpes virus 8, varicella zoster virus, cytomegalovirus (CMV), herpes virus; Epstein-Barr virus; Rous sarcoma virus; West Nile virus; Japanese equine encephalitis, Norwalk, papilloma virus, parvovirus B 19; Poxyiridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. , African swine fever virus); Hepatitis D virus, Hepatitis E virus, and unclassified viruses (e.g. , the etiological agents of Spongiform encephalopathies, the agent of delta hepatitis (thought to be a defective satellite of hepatitis B virus), the agents of non-A, non-B hepatitis (class l=enterally transmitted; class 2=parenterally transmitted (i.e. , Hepatitis C); Norwalk and related viruses, and astro viruses). Bacteria include both Gram negative and Gram positive bacteria. Examples of Gram positive bacteria include, but are not limited to Pasteurella species, Staphylococci species, and Streptococcus species. Examples of Gram negative bacteria include, but are not limited to Escherichia coli, Pseudomonas species, and Salmonella species. Specific examples of infectious bacteria include but are not limited to: Helicobacter pyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteria spp. (e.g. , M. tuberculosis, M. avium, M. intracellular e, M. kansasii, M. gordonae, M. leprae), Staphylococcus aureus, Neisseria gonorrhoeae, Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes (Group A Streptococcus), Streptococcus agalactiae (Group B Streptococcus), Streptococcus (viridans group), Streptococcus faecalis, Streptococcus bovis, Streptococcus (anaerobic spp.), Streptococcus pneumoniae, pathogenic Campylobacter spp.,Enterococcus spp., Haemophilus influenzae {Hemophilus influenza B, and Hemophilus influenza non- typable), Bacillus anthracis, Corynebacterium diphtheriae, Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridium perfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiella pneumoniae, Pasteurella multocida, Bacteroides spp., Fusobacterium nucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponema pertenue, Leptospira, Rickettsia, Actinomyces israelii, meningococcus, pertussis, pneumococcus, shigella, tetanus, Vibrio cholerae, yersinia, Pseudomonas species, Clostridia species, Salmonella typhi, Shigella dysenteriae, Yersinia pestis, Brucella species, Legionella pneumophila, Rickettsiae, Chlamydia, Clostridium perfringens, Clostridium botulinum, Staphylococcus aureus, Pseudomonas aeruginosa, Cryptosporidium parvum, Streptococcus pneumoniae, and Bordetella pertussis. Exemplary fungi and yeast include, but are not limited to, Cryptococcus neoformans, Candida albicans, Candida tropicalis, Candida stellatoidea, Candida glabrata, Candida krusei, Candida parapsilosis, Candida guilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorula mucilaginosa, Aspergillus fumigatus, Aspergillus flavus, Blastomyces dermatitidis, Aspergillus clavatus, Cryptococcus neoformans, Chlamydia trachomatis, Coccidioides immitis, Cryptococcus laurentii, Cryptococcus albidus, Cryptococcus gattii, Nocardia spp, Histoplasma capsulatum, Pneumocystis jirovecii (or Pneumocystis carinii), Stachybotrys chartarum, and any combination thereof. Exemplary parasites include, but are not limited to: Entamoeba histolytica; Plasmodium species (Plasmodium falciparum, Plasmodium malariae, Plasmodium ovale, Plasmodium vivax), Leishmania species (Leishmania tropica, Leishmania braziliensis, Leishmania donovani), Infectious (viral and non-viral) diseases that can be subject to the current invention, e.g treated for within the context of the current invention include such a caused by the foreign organisms as listed above./pct-s

Preferably the infectious disease is a viral, bacterial, fungal, or parasitic disease, preferably a chronic infectious disease. Throughout the description, the terms "disease" and "conditions" may be used interchangeably. The term "subject" as used herein refers to any human or non-human animal. The term "non- human animal" includes, but is not limited to, vertebrates such as nonhuman primates, sheep, dogs, cats, rabbits, ferrets, rodents such as mice, rats and guinea pigs, avian species such as chickens, amphibians, and reptiles. In preferred embodiments, the subject is a mammal such as a non-human primate, sheep, dog, cat, rabbit, ferret or rodent. In more preferred embodiments, the subject is a human. The terms, "subject," "patient" and "individual" are used interchangeably herein.

The term ΉΑΡ1 cells" as used herein refers to a cell line commonly used for biomedical and genetic research. This cell line has a haploid karyotype except for chromosomes 8 and 15. HAP1 cells are derived from a line of cancerous cells (i.e. KBM-7), which means they are able to divide indefinitely. Due to their haploidy, HAP1 cells are useful in biomedical research and genetic experiments. When working in diploid cells, it is difficult to screen for mutations phenotypically, especially when considering recessive mutations. Because there are two copies of each gene, the effect of the mutation is often covered up by the non-mutated gene. In haploid cells, there is only one copy of most genes, so mutated phenotypes are immediately exposed. The HAP1 cell line is often used in in vitro studies as a model of leukemia (e.g. chronic myeloid leukemia). (Blomen VA et al., Science. 2015 Nov 27;350(6264):1092-6. doi: 10.1 126/science.aac7557). The term "A375 cells" as used herein refers to a human amelanotic melanoma cell line used in cytokine research, as it is not influenced by many biomolecules— e.g., prostaglandin E2; lectins; bacterial endotoxins and cytokines such as IL2, TNF; interferons or colony stimulating factors. A375 cells are extremely sensitive to growth-inhibitory effects of oncostatin M. A375 cell line is often used in in vitro studies as a model of melanoma cancer. (Prahallad A et al., Nature. 2012 Jan 26;483(7387): 100-3. doi: 10.1038/nature10868). The term "8505C cells" as used herein refers to a human thyroid carcinoma cell line used in vitro studies as a model of thyroid cancer. (Prahallad A et al., Nature. 2012 Jan 26;483(7387): 100-3. doi: 10.1038/nature10868). The term "RKO cells" as used herein refers to a colon carcinoma cell line developed by Michael Brattain. RKO cells contain wild-type p53 but lack endogenous human thyroid receptor nuclear receptor (h-TRbeta1). The RKO cell line is often used in in vitro studies as a model of colon cancer. (Corvaisier M et al., Oncotarget. 2016 Aug 4. doi: 10.18632/oncotarget.11057.).

The term "DLD1 cells" as used herein refers to a colorectal carcinoma cell line, which is often used in in vitro studies as a model of colorectal cancer. (Ahmed MA et al. , Oncotarget. 2016 Sep 17. doi: 10.18632/oncotarget.12099). The term "LOVO cells" as used herein refers to a colon cancer cell line, which is often used in in vitro studies as a model of colon cancer. (Sun C et al., Cell Rep. 2014 Apr 10;7(1 ):86-93. doi: 10.1016/j.celrep.2014.02.045).

The term Ή2030 cells" as used herein refers to a lung cancer cell line, which is often used in in vitro studies as a model of lung cancer. (Sun C et al., Cell Rep. 2014 Apr 10;7(1):86-93. doi: 10.1016/j.celrep.2014.02.045).

The term "Colo 679 cells" as used herein refers to a melanoma cell line, which is often used in in vitro studies as a model of colon colorectal cancer. (Sun et al. Nature., 2014 Apr 3;508(7494): 118-22. doi: 10.1038/nature13121.).

The skilled person is well acquainted with HAP1 cells, A375 cells, 8505C cells, RKO cells, DLD1 cells, LOVO cells, H2030 cells and colo 679 cells and variant thereof, and knows how to use and how to obtain or purchase such cells.

The singular forms "a," "an" and "the", as used herein, include plural referents unless the context clearly dictates otherwise. For example, a method for administrating a drug includes the administrating of a plurality of molecules (e.g. 10's, 100's, 1000's, 10's of thousands, 100's of thousands, millions, or more molecules). The term "and/or" as used herein indicates that one or more of the stated cases may occur, alone or in combination with at least one of the stated cases, up to with all of the stated cases. The use of the alternative (e.g., "or") should be understood to mean either one, both, or any combination thereof of the alternatives.

The term "to comprise" and its conjugations as used herein is used in its non-limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. It also encompasses the more limiting "to consist of."

The term "treatment" or "treating" as used herein comprises a treatment relieving, reducing or alleviating at least one symptom in a subject or effecting a delay of progression of a disease (e.g. cancer or autoimmune disease). Within the meaning of the present invention, the term "treat" also denotes to arrest, delay the onset (i.e., the period prior to clinical manifestation of a disease) and/or reduce the risk of developing or worsening a disease (e.g. cancer or autoimmune diseases).

The term "cytotoxic agent" as used herein refers to a substance that inhibits or prevents the function of cells and/or causes destruction of cells. The term is intended to include radioactive isotopes, chemotherapeutic agents e.g., methotrexate, adriamicin, vinca alkaloids (vincristine, vinblastine, etoposide), doxorubicin, melphalan, mitomycin C, chlorambucil, daunorubicin or other intercalating agents, enzymes and fragments thereof such as nucleolytic enzymes, antibiotics, and toxins such as small molecule toxins or enzymatically active toxins of bacterial, fungal, plant or animal origin.

A "chemotherapeutic agent" refers to a chemical compound useful in the treatment of cancer. Examples of chemotherapeutic agents include alkylating agents such as thiotepa and cyclosphosphamide (CYTOXAN®); alkyl sulfonates such as busulfan, improsulfan and piposulfan; aziridines such as benzodopa, carboquone, meturedopa, and uredopa; ethylenimines and methylamelamines including altretamine, triethylenemelamine, triethylenephosphoramide, triethylenethiophosphoramide and trimethylomelamine; acetogenins (especially bullatacin and bullatacinone); delta-9-tetrahydrocannabinol (dronabinol, MARINOL®); beta-lapachone; lapachol; colchicines; betulinic acid; a camptothecin (including the synthetic analogue topotecan (HYCAMTIN®), CPT-1 1 (irinotecan, CAMPTOSAR®), acetylcamptothecin, scopolectin, and 9-aminocamptothecin); bryostatin; callystatin; CC-1065 (including its adozelesin, carzelesin and bizelesin synthetic analogues); podophyllotoxin; podophyllinic acid; teniposide; cryptophycins (particularly cryptophycin 1 and cryptophycin 8); dolastatin; duocarmycin (including the synthetic analogues, KW-2189 and CB1-TM1); eleutherobin; pancratistatin; a sarcodictyin; spongistatin; nitrogen mustards such as chlorambucil, chlornaphazine, chlorophosphamide, estramustine, ifosfamide, mechlorethamine, mechlorethamine oxide hydrochloride, melphalan, novembichin, phenesterine, prednimustine, trofosfamide, uracil mustard; nitrosoureas such as carmustine, chlorozotocin, fotemustine, lomustine, nimustine, and ranimustine; antibiotics such as the enediyne antibiotics (e. g., calicheamicin, especially calicheamicin gammal l and calicheamicin omegaH ; CDP323, an oral alpha-4 integrin inhibitor; dynemicin, including dynemicin A; an esperamicin; as well as neocarzinostatin chromophore and related chromoprotein enediyne antibiotic chromophores), aclacinomysins, actinomycin, authramycin, azaserine, bleomycins, cactinomycin, carabicin, carminomycin, carzinophilin, chromomycins, dactinomycin, daunorubicin, detorubicin, 6-diazo-5-oxo-L- norleucine, doxorubicin (including ADRIAMYCIN®, morpholino-doxorubicin, cyanomorpholino-doxorubicin, 2-pyrrolino-doxorubicin, doxorubicin HCI liposome injection (DOXIL®), liposomal doxorubicin TLC D-99 (MYOCET®), peglylated liposomal doxorubicin (CAELYX®), and deoxydoxorubicin), epirubicin, esorubicin, idarubicin, marcellomycin, mitomycins such as mitomycin C, mycophenolic acid, nogalamycin, olivomycins, peplomycin, porfiromycin, puromycin, quelamycin, rodorubicin, streptonigrin, streptozocin, tubercidin, ubenimex, zinostatin, zorubicin; anti-metabolites such as methotrexate, gemcitabine (GEMZAR®), tegafur (UFTORAL®), capecitabine (XELODA®), an epothilone, and 5- fluorouracil (5-FU); folic acid analogues such as denopterin, methotrexate, pteropterin, trimetrexate; purine analogs such as fludarabine, 6-mercaptopurine, thiamiprine, thioguanine; pyrimidine analogs such as ancitabine, azacitidine, 6-azauridine, carmofur, cytarabine, dideoxyuridine, doxifluridine, enocitabine, floxuridine; androgens such as calusterone, dromostanolone propionate, epitiostanol, mepitiostane, testolactone; anti-adrenals such as aminoglutethimide, mitotane, trilostane; folic acid replenisher such as frolinic acid; aceglatone; aldophosphamide glycoside; aminolevulinic acid; eniluracil; amsacrine; bestrabucil; bisantrene; edatraxate; defofamine; demecolcine; diaziquone; elfornithine; elliptinium acetate; an epothilone; etoglucid; gallium nitrate; hydroxyurea; lentinan; lonidainine; maytansinoids such as maytansine and ansamitocins; mitoguazone; mitoxantrone; mopidanmol; nitraerine; pentostatin; phenamet; pirarubicin; losoxantrone; 2- ethylhydrazide; procarbazine; PSK® polysaccharide complex (JHS Natural Products, Eugene, Oreg.); razoxane; rhizoxin; sizofiran; spirogermanium; tenuazonic acid; triaziquone; 2,2',2'-trichlorotriethylamine; trichothecenes (especially T-2 toxin, verracurin A, roridin A and anguidine); urethan; vindesine (ELDISINE®, FILDESIN®); dacarbazine; mannomustine; mitobronitol; mitolactol; pipobroman; gacytosine; arabinoside ("Ara-C"); thiotepa; taxoid, e.g., paclitaxel (TAXOL®), albumin-engineered nanoparticle formulation of paclitaxel (ABRAXANE™), and docetaxel (TAXOTERE®); chloranbucil; 6-thioguanine; mercaptopurine; methotrexate; platinum agents such as cisplatin, oxaliplatin (e.g., ELOXATIN®), and carboplatin; vincas, which prevent tubulin polymerization from forming microtubules, including vinblastine (VELBAN®), vincristine (ONCOVIN®), vindesine (ELDISINE®, FILDESIN®), and vinorelbine (NAVELBINE®); etoposide (VP-16); ifosfamide; mitoxantrone; leucovorin; novantrone; edatrexate; daunomycin; aminopterin; ibandronate; topoisomerase inhibitor RFS 2000; difluoromethylornithine (DMFO); retinoids such as retinoic acid, including bexarotene (TARGRETIN®); bisphosphonates such as clodronate (for example, BONEFOS® or OSTAC®), etidronate (DIDROCAL®), NE-58095, zoledronic acid/zoledronate (ZOMETA®), alendronate (FOSAMAX®), pamidronate (AREDIA®), tiludronate (SKELID®), or risedronate (ACTONEL®); troxacitabine (a 1 ,3-dioxolane nucleoside cytosine analog); antisense oligonucleotides, particularly those that inhibit expression of genes in signaling pathways implicated in aberrant cell proliferation, such as, for example, PKC-alpha, Raf, H-Ras, and epidermal growth factor receptor (EGF-R); vaccines such as THERATOPE® vaccine and gene therapy vaccines, for example, ALLOVECTIN® vaccine, LEUVECTIN® vaccine, and VAXID® vaccine; topoisomerase 1 inhibitor (e.g., LURTOTECAN®); rmRH (e.g., ABARELIX®); BAY439006 (sorafenib; Bayer); SU-1 1248 (sunitinib, SUTENT®, Pfizer); perifosine, COX-2 inhibitor (e.g., celecoxib or etoricoxib), proteosome inhibitor (e.g., PS341 ); bortezomib (VELCADE®); CCI-779; tipifarnib (R1 1577); orafenib, ABT510; Bcl-2 inhibitor such as oblimersen sodium (GENASENSE®); pixantrone; EGFR inhibitors (see definition below); tyrosine kinase inhibitors (see definition below); serine-threonine kinase inhibitors such as rapamycin (sirolimus, RAPAMUNEC); farnesyltransferase inhibitors such as lonafarnib (SCH 6636, SARASAR™); and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above such as CHOP, an abbreviation for a combined therapy of cyclophosphamide, doxorubicin, vincristine, and prednisolone; and FOLFOX, an abbreviation for a treatment regimen with oxaliplatin (ELOXATIN™) combined with 5-FU and leucovorin. Chemotherapeutic agents as defined herein also include "anti-hormonal agents" or "endocrine therapeutics" which act to regulate, reduce, block, or inhibit the effects of hormones that can promote the growth of cancer. They may be hormones themselves, including, but not limited to: anti-estrogens with mixed agonist/antagonist profile, including, tamoxifen (NOLVADEX®), 4-hydroxytamoxifen, toremifene (FARESTON®), idoxifene, droloxifene, raloxifene (EVISTA®), trioxifene, keoxifene, and selective estrogen receptor modulators (SERMs) such as SERM3; pure anti-estrogens without agonist properties, such as fulvestrant (FASLODEX®), and EM800 (such agents may block estrogen receptor (ER) dimerization, inhibit DNA binding, increase ER turnover, and/or suppress ER levels); aromatase inhibitors, including steroidal aromatase inhibitors such as formestane and exemestane (AROMASIN®), and nonsteroidal aromatase inhibitors such as anastrazole (ARIMIDEX®), letrozole (FEMARA®) and aminoglutethimide, and other aromatase inhibitors include vorozole (RIVISOR®), megestrol acetate (MEGASE®), fadrozole, and 4(5)-imidazoles; lutenizing hormone-releaseing hormone agonists, including leuprolide (LUPRON® and ELIGARD®), goserelin, buserelin, and tripterelin; sex steroids, including progestines such as megestrol acetate and medroxyprogesterone acetate, estrogens such as diethylstilbestrol and premarin, and androgens/retinoids such as fluoxymesterone, all transretionic acid and fenretinide; onapristone; anti-progesterones; estrogen receptor down-regulators (ERDs); anti-androgens such as flutamide, nilutamide and bicalutamide; and pharmaceutically acceptable salts, acids or derivatives of any of the above; as well as combinations of two or more of the above.

The term "antibody" (Ab) is well-known to the skilled person and includes monoclonal antibodies, polyclonal antibodies, multi-specific antibodies (e.g. bispecific antibodies), single chain antibodies, e.g., antibodies from llama and camel, antibody fragments, e.g., variable regions and/or constant region fragments, so long as they exhibit a desired biological activity, e.g., antigen-binding activity. The term "immunoglobulin" (Ig) is used interchangeably with "antibody" herein. An "isolated antibody" is one which has been identified, and/or separated, and/or recovered from its natural environment.

The term "monoclonal antibody" is well-known to the skilled person and includes an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. In contrast to polyclonal antibody preparations which include different antibodies directed against different epitopes, each monoclonal antibody is directed against a single epitope, i.e., a single antigenic determinant. In addition to their specificity, the monoclonal antibodies are advantageous in that they may be synthesized uncontaminated by other antibodies. The monoclonal antibodies useful in the present invention may be prepared by the hybridoma methodology or may be made using recombinant DNA methods in bacterial, eukaryotic animal or plant cells (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries, using the available techniques, he monoclonal antibodies herein include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain(s) is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity.

An "antibody fragment" comprises a portion of a multimeric antibody, preferably the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, dimmers and trimers of Fab conjugates, Fv, scFv, minibodies,; dia-, tria-, and tetrabodies; linear antibodies (See Hudson et al, Nature Med. 9, 129-134 (2003)).

Members of the Camelidae family, e.g., llama, camel, and dromedaries, contain a unique type of antibody, that are devoid of light chains, and further lack the CH1 domain (Muyldermans, S., Rev. Mol. Biotechnol., 74, 277-302 (2001)). The variable region of these heavy chain antibodies are termed VHH or VHH, and constitute the smallest available intact antigen binding fragment (15 kDa) derived from a functional immunoglobulin. Methods for preparing antibodies, fragments and analogs thereof are known in the art.

Methods of screening

In a first aspect, the present invention relates to a method for screening for a compound capable of modulating immune activity, the method comprising:

(a) contacting a cell expressing the STUB1 protein with a test compound;

(b) measuring the level of expression or activity of the STUB1 protein in said cell; and

(c) selecting a test compound modulating the level of expression or activity of the STUB1 protein compared to the expression level or activity measured in the absence of the test compound,

wherein the test compound is a compound capable of decreasing immune activity if the test compound decreases the level of expression or activity of the STUB1 protein and

wherein the test compound is a compound capable of increasing immune activity if the test compound increases the level of expression or activity of the STUB1 protein.

In step (a), the cell may be any cell expressing the STUB1 protein (including recombinant cells, modified to express said protein(s). For instance, the cell may be a cell line suitable for use in screening assays, preferably a cancer cell line. Non-limiting examples of suitable cells which express the STUB1 protein include HAP1 cells, A375 cells, 8505C cells, RKO cells, DLD1 cells, LOVO cells, H2030 cells and colo 679 and variant thereof as well as other suitable cell lines. Also included are those cells that are transfected to (recombinant) express the STUB1 protein. The skilled person is well acquainted with such cell lines and knows how to obtain them.

The skilled person is also acquainted with methods for determining whether a cell, e.g. a cell line, expresses the STUB1 gene or protein, for instance by using PCR, immunohistochemistry, ELISA methods, and others. These and other methodologies may be used in step (b).

It is understood that the compounds uncovered in step (c) can be used for the development of medicaments suitable for the treatment of diseases or conditions involving (aberrant function of) the PD-1/PD-L1 axis such as cancers or autoimmune diseases, for instance such as described in the scenarios above and in the definition section.

The test compound is a compound capable of increasing immune activity if the test compound increases the level of expression or activity of the STUB1 protein (i.e. an agonist or stimulator of STUB1 ). Such compound identified by the screenings methods disclosed herein may thus for example be used to enhance T-cell function to upregulate cell-mediated immune responses, for the treatment of T cell dysfunctional disorders. The term "T cell dysfunctional disorders" as used herein refers to any condition or disease wherein there is a deficiency of T cells (not enough) or wherein the T cells or T cell function (e.g. secretion of cytokines, chemokines) is deficient or insufficient so that the immune system's ability to fight diseases (e.g. infectious diseases, cancers, etc.) is compromised or entirely absent. Non- limiting examples of T cell dysfunctional disorders include infectious diseases (e.g. diseases caused by a pathogen such as a virus such as AIDS), cancers (e.g. melanoma, lung cancer, bladder cancer, Gl tract cancer, HNSCC, and Hodgkin's lymphoma and other cancers), autoimmune diseases (e.g. rheumatoid arthritis) or any other condition or disease that would benefit from upregulation or enhancement or alteration of an immune response function (e.g. T cell function. Therefore, the compounds or modulators identified by the screenings methods disclosed herein may thus for example be used for the treatment of cancer or infections or any other condition that benefits from upregulation or enhancement of an immune response function.

In a second aspect, the present invention relates to a method for screening for a compound capable of modulating the level of expression of the PD-L1 protein in a cell, the method comprising:

(a) contacting a cell expressing the STUB1 protein with a test compound; (b) measuring the level of expression or activity of the STUB1 protein in said cell; and

wherein the test compound is a compound capable of increasing the level of expression of the PD-L1 protein if the test compound decreases the level of expression or activity of the STUB1 protein and

wherein the test compound is a compound capable of decreasing the level of expression of the PD-L1 protein if the test compound increases the level of expression or activity of the STUB1 protein.

Steps (a) and (b) may be performed as described above. It is understood that the compounds uncovered in step (c) can be used for the development of medicaments suitable for the treatment of diseases or conditions involving (aberrant function of) the PD-1/PD-L1 axis such as cancers or autoimmune diseases, for instance such as described in the scenarios above.

The test compound is a compound capable of decreasing the level of expression of the PD- L1 protein if the test compound increases the level of expression or activity of the STUB1 protein. Such compound identified by the screenings methods disclosed herein may thus for example be used to enhance T-cell function to upregulate cell-mediated immune responses, for the treatment of T cell dysfunctional disorders. Such compound identified by the screenings methods disclosed herein may thus for example be used for the treatment of cancer or infections or any other condition that benefits from upregulation or enhancement of an immune response function.

In an embodiment, relating to the method for screening as taught herein, measuring the level of expression or activity of the STUB1 protein involves measuring the level of gene expression, the level of mRNA (as a measure of transcription), the level of protein, the level of cell surface protein, activity of the protein or, phosphorylation status of the STUB1 protein. The skilled person is well-acquainted with techniques for achieving this goal.

In an embodiment relating to the method for screening as taught herein, the immune activity is mediated by a T cell, preferably an effector T cell, wherein the immune activity comprises secretion of cytokines, preferably IFN gamma, and TNF alpha, secretion of chemokines, preferably CXCL9 and CXCL10, and secretion of perforin-granzymes, following binding of a T cell receptor to a peptide-MHC complex on a target cell (also referred to herein is T-cell activity). In an embodiment relating to the method for screening as taught herein, the level of expression of the PD-L1 protein is the level of cell surface expression of the PD-L1 protein. Cell surface expression of PD-L1 protein can be performed using any suitable methods in the art, for instance flow cytometry as described in the present experimental section.

In an embodiment relating to the method for screening as taught herein, the method is screening for a compound capable of modulating PD-1/PD-L1 axis signaling, and/or capable of modulating immune activity, for instance as described above in the scenarios above.

In an embodiment relating to the method for screening as taught herein, the method further comprises measuring immune activity and/or measuring the level of expression of the PD-L1 protein and/or PD-1/PD-L1 axis signaling in the presence of the compound selected in (c). For instance, immune activity or PD-1/PD-L1 axis signaling may be measured by determining the levels of cytokines (e.g. IFN gamma, TNF alpha), chemokines (CXCL9, CXCL10) and/or perforin-granzymes released by immune cells (e.g. effector T cells), using standards techniques, in an assay wherein cells, e.g. expressing PD-L1 and/or STUB1 , for example cells of step (a), are co-incubated with immune cells expressing the PD-1 receptor such as T cells (e.g. effector T-cells), and wherein both cell types are treated with the test compound, and the results are compared to the situation wherein the cells are not treated with the selected compounds. Assays to measure the effect of PD-1 - PD-L1 axis signaling on T cells are known to those skilled in the art (Kataoka et al. Aberrant PD-L1 expression through 3'- UTR disruption in multiple cancers. Nature. 2016 May 23;534(7607):402-6. doi: 10.1038/nature18294. PubMed PMID: 27281199).

In a further aspect, the present invention relates to a method for screening for a compound capable of modulating the expression and/or activity of the STUB1 protein, the method comprising:

(i) contacting a cell expressing PD-L1 protein and expressing the STUB1 protein with a test compound;

(ii) measuring the level of expression of the PD-L1 protein; and

(iii) selecting a test compound modulating the level of expression of PD-L1 protein as compared to a cell contacted with the test compound, wherein the cell is expressing PD-L1 protein but is not expressing the STUB1 protein

wherein the test compound is a compound capable of decreasing the expression and/or activity of the STUB1 protein if the test compound increases the expression of PD-L1 and wherein the test compound is a compound capable of increasing the expression and/or activity of the STUB1 protein if the test compound decreases the expression of PD-L1.

Steps (i) and (ii) may be performed as described above (for steps (a) and (b)). It is understood that the compounds uncovered in step (iii) can be used for the development of medicaments suitable for the treatment of diseases or conditions involving (aberrant function of) the PD-1/PD-L1 axis such as cancers or autoimmune diseases, for instance such as described in the scenarios above. It is also understood that the compounds uncovered may be used in other disorders that involve (aberrant function of) a STUB1 protein.

Also provided is for a method of screening for compounds that modulate the interaction, in particular molecular interaction, between PD-L1 and STUB1.

Within the context of the current invention the term "interaction", or "interacting" refer to any physical association between proteins, directly, or indirectly via other molecules such as lipids, carbohydrates, other proteins, nucleotides, and other cell metabolites. Examples of interactions include protein-protein interactions. The term preferably refers to a stable association between two molecules (e.g. PDL-1 and STUB1) due to, for example, electrostatic, hydrophobic, ionic and/or hydrogen-bond interactions under physiological conditions. The interaction between the proteins may be either direct or indirect.

By this method for screening according to the present invention, it is possible to identify compounds capable of changing (inhibiting or augmenting) the binding property of the interaction between the proteins to be subjected (e.g. PD-L1 and STUB1 ). Such a compound can become the candidate of the therapeutic agent or the preventive agent for the disease (illness) with which the interaction between the proteins to be subjected is associated, including those disclosed herein, for example cancer, and or infectious disease, in particular those diseases that benefit from reduced or increased PD-L1-PD-1 axis signaling. Such compound identified by the screenings methods disclosed and that inhibits or reduces the interaction between PD-L1 and STUB1 may thus for example be used to enhance T-cell function to upregulate cell-mediated immune responses. Such compound identified by the screenings methods disclosed herein may thus for example be used for the treatment of cancer or infections or any other condition that benefits from upregulation or enhancement of an immune response function. The skilled person is well-aware of methods for screening for compounds that change the interaction between two proteins, including methods suitable for measuring interaction and change thereof between two membrane proteins. The screening of drugs is, for example possible by conventional enzyme-linked immunosorbent assay (ELISA) or by direct observation of one molecule using NMR spectroscopy, X-ray crystal analysis or electron microscopy, fluorescence resonance energy transfer, etc. Other suitable assays include those disclosed in, for example, WO 2004/023146 A2.

Therefore, there is provided for a method for screening for a compound for treatment of a disease, preferably selected from the group consisting of cancer, infection, or any other condition that benefits from upregulation or enhancement of an immune response function, wherein the method is characterized by utilizing the interaction between PD-L1 and STUB1.

Preferably the method comprises comparing the interaction between PD-L1 and STUB1 in the absence and presence of the compound to be screened. The membrane may be any membrane comprising a bilayer of lipids, including vesicles and artificial membranes or isolated plasma membrane. The membrane may also be the membrane of a cell expressing STUB1 and PD-L1. In the screening assay the compound to be screened may be added before, during or after PD-L1 and STUB1 interact.

As mentioned, compounds that increase or promote the interaction between PD-L1 and STUB1 are candidate drugs for treatment of conditions that benefit from reduced PD-L1-PD-1 axis signaling. Compounds that increase or promote the interaction between PD-L1 and STUB1 are candidate drugs for treatment of for example cancer, infection, or any other condition that benefits from upregulation or enhancement of an immune response function.

Also provided is for a method for screening for a compound for treatment of cancer or infection, characterized by using STUB1 , preferably characterized by further using PD-L1 , and as described herein above. In a preferred embodiment, the method of screening comprises the step of (a) contacting a cell expressing STUB1 with a test compound;

(b) measuring the level of expression or activity of STUB1 ; and

(c) selecting a test compound modulating the level of expression or activity of STUB1 compared to the expression level or activity measured in the absence of the test compound, wherein the test compound is a (candidate) compound for treatment of cancer or infection if the test compound increases the level of expression or activity of STUB1. Also provided are compounds that increases the level or activity of STUB1 , preferably wherein the compound is an activator or agonist of STUB1 , for use in the treatment of a disease in combination with an immune checkpoint inhibitor. Also provided is for the use of STUB1 in identifying of or screening for compounds for use in the treatment of cancer or infection.

Also provided is for the use of STUB1 in identifying of or screening for compounds that may be used to enhance T-cell function to upregulate cell-mediated immune responses, for the treatment of T cell dysfunctional disorders.

Also provided is for the use of STUB1 in identifying of or screening for compounds for reducing PD-L1-PD-1 axis signaling. The screening methods disclosed herein are also useful for identifying compounds that can be used to increase ubiquitination of PD-L1 and/or reduce half-life of PD-L1 in a cell. Increased expression and/or activity of STUB1 may Increases ubiquitinated PD-L1. Therefore, the screening methods as disclosed herein can be used to identify compounds that increase ubiquitination of PD-L1 , e.g. by increasing expression or activity of STUB1.

The screening methods disclosed herein are useful in identifying compounds that may be used to enhance T-cell function, to upregulate cell-mediated immune responses, for the treatment of T cell dysfunctional disorders. Such compounds identified by the screening methods disclosed herein are useful in, the treatment of conditions that benefit from reduced PD-L1-PD-1 axis signaling. Such compounds identified by the screening methods disclosed herein may be used treat cancer and infections, or any other condition that benefits from upregulation or enhancement of an immune response function.

Test compounds that can be screened in the methods disclosed herein include, without limitation, small molecules, nucleic acids (e.g., siRNA, shRNA, miRNA), and polypeptides, (e.g., antibodies).

Methods of in vitro modulation

In a further aspect, the present invention relates to an in vitro method for modulating PD-L1 protein expression and/or PD-1/PD-L1 axis signaling in a cell, the method comprising modulating the expression or activity of a protein of STUB1 protein. In an embodiment relating to the in vitro method for modulating PD-L1 protein expression and/or PD-1/PD-L1 axis signaling in a cell as taught herein, PD-L1 protein expression and/or PD-1/PD-L1 axis signaling is increased when the level of expression or activity of the STUB1 protein is increased and/or wherein PD-L1 protein expression and/or PD-1/PD-L1 axis signaling is decreased when the level of expression or activity of the STUB1 protein is decreased.

In an embodiment relating to the in vitro method for modulating PD-L1 protein expression and/or PD-1/PD-L1 axis signaling in a cell as taught herein, modulating PD-L1 protein expression and/or PD-1/PD-L1 axis signaling is performed by treating the cells in the presence of a compound identified with the method for screening of any one of the previous claims and/or by treating the cells with a siRNA, shRNA, TALENs, MEGATALENs, CRISPR or Zinc finger nucleases directed to silence expression of a gene encoding the STUB1 protein.

Treatment with STUB1 modulator

In a further aspect, the present invention relates to a modulator of the expression and/or activity of the STUB1 protein for use in modulating immune activity, PD-1/PD-L1 axis signaling and/or PD-L1 protein expression in a patient in need thereof, wherein the modulator is a modulator that increases immune activity, decreases PD-1/PD-L1 axis signaling and/or decreases PD-L1 protein expression when the modulator increases the expression and/or activity of the STUB1 protein and wherein the modulator is a modulator that decreases immune activity, increases PD-1/PD-L1 axis signaling and/or increases PD-L1 protein expression when the modulator decreases the expression and/or activity of the STUB1 protein.

In a further aspect, the present invention relates to a modulator of the expression and/or activity of the STUB1 protein for use in immunotherapy, immunotherapy in a cancer patient, preferably a patient suffering from a cancer selected from melanoma, lung cancer, bladder cancer, Gl tract cancer, HNSCC, and Hodgkin's lymphoma, and other cancers involving (aberrant function of) the PD-1/PD-L1 axis, or for use in the treatment of an autoimmune disease involving (aberrant function of) the PD-1/PD-L1 axis, preferably systemic lupus erythematosus, rheumatoid arthritis, and type 1 diabetes, wherein the modulator is a modulator that increases immune activity, decreases PD-1/PD-L1 axis signaling and/or decreases PD-L1 protein expression when the modulator increases the expression and/or activity of the STUB1 protein and/or wherein the modulator is a modulator that decreases immune activity, increases PD-1/PD-L1 axis signaling and/or increases PD-L1 protein expression when the modulator decreases the expression and/or activity of the STUB1 protein.

In an embodiment relating to the modulator of the expression and/or activity of a protein of the STUB1 protein for use as taught herein, the treatment also involves the use of a PD-1/PD-L1 axis binding antagonist and/or agonist, such as those described above.

In a further aspect, the present invention relates to the use of a modulator of the expression and/or activity of a the STUB1 protein for modulating immune activity, PD-1/PD-L1 axis signaling and/or PD-L1 protein expression wherein the modulator is a modulator that increases immune activity, decreases PD-1/PD-L1 axis signaling and/or decreases PD-L1 protein expression when the modulator increases the expression and/or activity of the STUB1 protein and/or wherein the modulator is a modulator that decreases immune activity, increases PD-1/PD-L1 axis signaling and/or increases PD-L1 protein expression when the modulator decreases the expression and/or activity of the STUB1 protein.

In a further aspect, the present invention relates to an antibody (including immunoglobulin, aptamers, affimers, single-chain antibodies, nanobodies and the like) against the STUB1 protein use in the treatment of a disorder that benefits from a decrease of immune activity, increase of PD-1/PD-L1 axis signaling and/or increases of PD-L1 protein expression, preferably wherein said disorder is an auto-immune disease.

Globally, it is understood that the STUB1 modulator compounds discussed above may be uncovered or found by the screening methods as taught herein and may be advantageously used for the treatment of diseases or conditions involving (aberrant function of) the PD-1/PD- L1 axis such as cancers or autoimmune diseases, for instance such as described in scenarios above.

Some embodiment of any of the methods, uses, compositions, or compositions for use as disclosed herein, also include the combination of modulation of CMTM6, CMTM4, or CMTM6 and CMTM4, and modulation of STUB1. In a preferred embodiment, of such methods, uses, compositions or compositions for use, CMTM6, CMTM4, or CMTM6 and CMTM4 is (are) inhibited or antagonized whereas STUB1 (the expression or activity) is stimulated, activated or promoted. CKLF Like MARVEL Transmembrane Domain Containing protein 6 and 4 (CMTM6 and CMTM4) are protein known to the skilled person, preferred are human CMTM6 and/or human CMTM4. As will be understood by the skilled person, also provided is for embodiments than encompass targeting STUB1 and CMTM4 and/or CMTM 6, for example targeting a patient with STUB1 modulators and with CMTM4/ CMTM6 modulators, and preferably in addition in combination with those other drugs as presented herein throughout.

Combination treatments with immune checkpoint inhibitors or immune checkpoint modulators. According to the invention, there is also provided for combination treatments of compounds that increases the level of expression or activity of STUB1 , preferably wherein the compound is an activator or agonist of STUB1 , in combination with immune checkpoint inhibitors or modulators.

Within the context of the current invention such activator or agonist of STUB1 is a compound that, preferably specifically, increases the level of expression or activity of STUB1 , for example may increase the amount of STUB1 protein, in the cell or at the cell surface, or may increase its activity.

The term "immune checkpoint inhibitor or modulator" as used herein refers to any molecule that directly or indirectly inhibits, partially or completely, an immune checkpoint pathway. It is generally thought that immune checkpoint pathways function to turn on or off aspects of the immune system, particularly T cells, but also for instance myeloid cells, NK cells and B cells. Following activation of a T cell, a number of inhibitory receptors can be upregulated and present on the surface of the T cell in order to suppress the immune response at the appropriate time. Aspects of the disclosure are related to the observation that inhibiting such immune checkpoint pathways and administering synthetic nanocarrier compositions comprising antigens and immunostimulators, can result in the generation of enhanced immune responses to the antigen and/or a reduction in immunosuppressive immune responses against the antigen. Examples of immune checkpoint pathways include, without limitation, PD-1/PD-L1 , CTLA4/B7-1 , TIM-3, LAG 3, By-He, H4, HAVCR2, ID01 , CD276 and VTCN1 , B7-H3, B7-H4, CD47, or KIR. Aspects of the disclosure are also related to the observation that inhibition of one checkpoint pathway, such as the CTI_A4/B7-1 pathway can lead to increased activation of the PD-1/PD-L1 pathway, for instance through increased PD- L1 expression, creating a rationale for combination treatments. Immune checkpoints and modulators thereof as well as methods of using such compounds are described in the literature. For instance, non-limiting examples of immune checkpoint inhibitors or modulators include fully human monoclonal antibodies, such as BMS-936558/MDX-1 106, BMS- 936559/MDX-1 105, ipilimumab/Yervoy, tremelimumab, BMS-986016, Durvalumab, MEDI4736, Urelumab, CDX-1 127, and Avelumab; humanized antibodies, such as CT-011 , MK-3475, Hu5F9-G4, CC-90002, MBG453, TSR-022, and Atezolizumab; and fusion proteins, such as AMP-224 and TTI-621 , and others. Other non-limiting examples of immune checkpoint modulators (agonists) include antibodies directed against e.g. CD40, OX40, GITR, CD137 (4-1 BB), CD27, ICOS, and TRAIL.

In accordance with this invention, the one or more immune checkpoint modulator(s) may independently be a polypeptide or a polypeptide- encoding nucleic acid molecule; said polypeptide comprising a domain capable of binding the targeted immune checkpoint and/or inhibiting the binding of a ligand to said targeted immune checkpoint so as to exert an antagonist function (i.e. being capable of antagonizing an immune checkpoint-mediated inhibitory signal) or an agonist function (i.e. being capable of boosting an immune checkpoint- mediated stimulatory signal). Such one or more immune checkpoint modulator(s) can be independently selected from the group consisting of peptides (e.g. peptide ligands), soluble domains of natural receptors, RNAi, antisense molecules, antibodies and protein scaffolds.

In a preferred embodiment, the immune checkpoint modulator is an antibody. In the context of the invention, the immune check modulator antibody is used in the broadest sense and encompasses e.g. naturally occurring and engineered by man as well as full length antibodies or functional fragments or analogs thereof that are capable of binding the target immune checkpoint or epitope (thus retaining the target-binding portion). It can be of any origin, e.g. human, humanized, animal (e.g. rodent or camelid antibody) or chimeric. It may be of any isotype with a specific preference for an IgGI or lgG4 isotype. In addition, it may be glycosylated or non- glycosylated. The term "antibody" also includes bispecific or multispecific antibodies so long as they exhibit the binding specificity described herein. Non-limiting examples of agonistic immune checkpoint modulators are those that exert an agonist function in the sense that they are capable of stimulating or reinforcing stimulatory signals, for example those mediated by CD28 with a specific preference for any of ICOS, CD137 (or 4- 1 BB), OX40, CD27, CD40 and GITR immune checkpoints. Standard assays to evaluate the binding ability of the antibodies toward immune checkpoints are known in the art, including for example, ELISAs, Western blots, RIAs and flow cytometry. The binding kinetics (e.g., binding affinity) of the antibodies also can be assessed by standard assays known in the art, such as by Biacore analysis. Where in the application reference is made to an immune checkpoint inhibitor, also an immune checkpoint modulator may be used, except in those cases where it is apparent from the context of the wording that this is not the case.

For example, in the instance of the PD-1/PD-L1 immune checkpoint pathway, an inhibitor may bind to PD-1 or to PD-L1 and prevent interaction between the receptor and ligand. Therefore, the inhibitor may be an anti-PD-1 antibody or anti-PD-L1 antibody. Similarly, in the instance of the CTLA4/B7-1 immune checkpoint pathway, an inhibitor may bind to CTLA4 or to B7-1 and prevent interaction between the receptor and ligand. Further examples of immune checkpoint inhibitors can be found, for example, in WO2014/144885. Such immune checkpoint inhibitors are incorporated by reference herein. In some embodiments of any one of the methods, compositions or kits provided, the immune checkpoint inhibitor is a small molecule inhibitor of an immune checkpoint pathway. In some embodiments the immune checkpoint inhibitor is a polypeptide that inhibits an immune checkpoint pathway. In some embodiments the inhibitor is a fusion protein. In some embodiments the immune checkpoint inhibitor is an antibody. In some embodiments the antibody is a monoclonal antibody. Non- limiting examples of immune checkpoint inhibitors include fully human monoclonal antibodies, such as BMS-936558/MDX-1 106, BMS-936559/MDX-1 105, ipilimumab/Yervoy, tremelimumab, BMS-986016, Durvalumab, MEDI4736, Urelumab, CDX-1 127, and Avelumab; humanized antibodies, such as CT-01 1 , MK-3475, Hu5F9-G4, CC-90002, MBG453, TSR- 022, and Atezolizumab; and fusion proteins, such as AMP-224 and TTI-621. Non-limiting examples of positive immune checkpoint modulators include antibodies against CD27, CD137.

Therefore provided is for an immune checkpoint inhibitor for use in the treatment of a disease, wherein in the treatment also involves the use of a compound that increases the level of expression or activity of STUB1 , preferably wherein the compound is an activator or agonist of STUB1.

In some embodiments, the compound is an (activating) antibody against STUB1.

Preferably the disease is a disease that benefits from decreased PD-1/PD-L1 axis signaling and/or that benefits from upregulation or enhancement of an immune response function.

Preferably the disease is cancer or infection.

Preferably the immune checkpoint inhibitor or modulators is an inhibitor of PD-1 , PD-L1 , CTLA-4 or CD47

Preferably the treatment also involves the use of a cytotoxic agent or chemotherapeutic agent or other standard of care, such as radiotherapy.

Methods of treatment In a further aspect, the present invention relates to a method for the treatment of a disorder that benefits from an increase of immune activity, decrease of PD-1/PD-L1 axis signaling and/or decrease of PD-L1 protein expression, preferably wherein said disorder is a cancer, for instance as described in the scenarios above, the method comprises administering to a human in need of such treatment a therapeutically effective amount of

-a modulator of the expression and/or activity of the STUB1 protein, wherein the modulator increases the expression and/or activity of a protein of the STUB1 family.

In a further aspect, the present invention relates to a method for the treatment of a disorder that benefits from a decrease of immune activity, increase of PD-1/PD-L1 axis signaling and/or increase of PD-L1 protein expression, preferably wherein said disorder is an autoimmune disease (for instance as described in the scenarios above), the method comprises administering to a human in need of such treatment a therapeutically effective amount of -a modulator of the expression and/or activity of the STUB1 protein, wherein the modulator decreases the expression and/or activity of a protein of the STUB1 family.

It is understood that "an effective amount" of a modulator of the expression and/or activity of the STUB1 protein refers to the amount of a compound or a modulator as taught herein required to ameliorate the symptoms of a disease (e.g. cancer or autoimmune disease), for example, but not necessarily relative to an untreated patient.

The skilled person understands that the effective amount of active agent(s) used to practice the present disclosure for therapeutic treatment of cancer or autoimmune diseases will vary depending upon the manner of administration, the age, body weight, and general health of the subject. Ultimately, the attending physician will decide the appropriate amounts and dosage regimen. Such amount is referred to as an "effective" or "acceptable" amount. Thus, in connection with the administration of a drug which, in the context of the current disclosure, is "effective against" a disease or condition indicates that administration in a clinically appropriate manner results in a beneficial effect for at least a statistically significant fraction of patients, such as an improvement of symptoms, a cure, a reduction in at least one disease sign or symptom, extension of life, improvement in quality of life, or other effect generally recognized as positive by medical doctors familiar with treating the particular type of disease or condition. In a further aspect, the present invention relates to a method of promoting health, the method comprises communicating to a target audience, the use of a modulator of the expression and/or activity of the STUB1 protein for treating an individual with a disease or disorder that benefits from the modulation of immune activity, PD-1/PD-L1 axis signaling and/or PD-L1 protein expression.

In a further aspect, the present invention relates to the use of the STUB1 protein for modulating immune activity, PD-L1 expression, PD-L1 protein expression and/or PD-1/PD-L1 axis signaling.

Also provided is a method for decreasing half-life of PD-L1 , the method comprises increasing or promoting the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1 , preferably wherein the activator or agonist is an (activating) antibody.

Also provided is a method for reducing PD-1/PD-L1 signaling, the method comprises increasing or promoting the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1 , preferably wherein the activator or agonist is an (activating) antibody.

Also provided is a method for upregulation or enhancement of an immune response function, the method comprises increasing or promoting the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1 , preferably wherein the activator or agonist of STUB1 is an antibody.

Also provided is a method for reducing expression of PD-L1 , preferably of cell-surface PD-L1 , the method comprises increasing or promoting the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1 , preferably wherein the activator or agonist of STUB 1 is an antibody.

Also provided is a method of reducing cell-surface PD-L1 expression in a subject, the method comprising administering to the subject an activator or agonist of STUB1 , preferably wherein the activator or agonist of STUB1 is an antibody.

Also provided is a method of enhancing T-cell activation in a subject, the method comprising administering to the subject an activator or agonist of STUB1 , preferably wherein the activator or agonist of STUB1 is an antibody.

Also provided is a method of treating cancer or infectious disease in a subject, the method comprising administering to the subject an activator or agonist of STUB1 , preferably wherein the activator or agonist of STUB1 is an antibody. Also provided is a method according to any one of the foregoing methods, further comprising providing or administering an immune checkpoint inhibitor or modulator and/or a cytotoxic agent and/or chemotherapeutic agent. Methods of Diagnosis/Prognostics

In a further aspect, the present invention relates to a method for predicting immune activity towards cancer cells in a patient, the method comprising measuring the level of expression and/or activity of a STUB1 protein in cancer cells and/or cancer-infiltrating cells obtained from said patient, wherein decreased expression and/or activity of the protein STUB1 is predictive for poor activity of the T-cell towards the cancer cell and wherein increased expression and/or activity of the STUB1 is predictive for strong activity of the T-cell towards the cancer cell. Expression and/or activity may be compared to a standard, for example a healthy subject.

In a further aspect, the present invention relates to a method for predicting immune activity towards cancer cells in a patient as taught herein, wherein the method is used to determining likelihood that the patient will exhibit benefit from treatment with a PD-1/PD-L1 axis binding antagonist and/or exhibit benefit from treatment with a modulator of the expression and/or activity of a STUB1 protein. In a further aspect, the present invention relates to a method for predicting immune activity towards cells in a patient suffering from an autoimmune disease, the method comprising measuring the level of expression and/or activity of a STUB1 protein in the cells obtained from said patient, wherein increased expression and/or activity of the protein of the STUB1 is predictive for strong activity of the T-cell towards the cells and wherein decreased expression and/or activity of the STUB1 is predictive for poor activity of the T-cell towards the cells. Expression and/or activity may be compared to a standard, for example a healthy subject.

In a further aspect, the present invention relates to a method for predicting immune activity towards cells in a patient suffering from an autoimmune disease as taught herein, wherein the method is used to determine the likelihood that the patient will exhibit benefit from treatment with a PD-1/PD-L1 axis binding agonist and/or exhibit benefit from treatment with a modulator of the expression and/or activity of a STUB1 protein.

It is understood that measuring the level of expression of the STUB1 protein in cancer cells and/or cancer-infiltrating cells or other cells (e.g. pancreatic cells) obtained from a patient, can be reliably used in place of or in addition to traditional methods relying on the cell surface detection of PD-L1 protein in such cells using immunohistochemistry. Such traditional methods are known to be labor-intensive and do not always work (i.e. do not always yield PD- L1 immuno-positive staining). Therefore, the present method may be an advantageous alternative to methods relying on immunohistochemistry. EXAMPLES

Example 1 : Haploid Genetic Screen for modulator of PD-L1

In order to identify novel regulators of PD-L1 , we made use of a forward genetic screening approach in haploid human HAP1 cells. We noticed that HAP1 cells upregulate PD-L1 mRNA upon stimulation with interferon gamma (IFNg) and that this results in an increase in the abundance of PD-L1 at the cell surface of these cells that can be detected with antibodies.

Next, we created a library of loss-of-function mutants in HAP1 cells using a modified version of a retroviral gene trap (Jae et al., Science, 2013), expanded these cells, treated them with IFNg and subjected them to a staining for PD-L1 at the cell surface using antibodies. This resulted in a near-normal distribution of signal intensity when analyzed by flow-cytometry. For the genetic screen, we selected those mutants that displayed the strongest and weakest anti- PD-L1 staining, sorted a total of ca. 10 million cells for each population and analyzed their gene-trap integration sites, similar as described before (Blomen et al., Science, 2015). Material and Methods

HAP1 cells were mutagenized using gene-trap retrovirus (for example described in Carette et al. (2011). Nature, 477(7364), 340-3. doi: 10.1038/nature10348; available from www.horizon- genomics.com/hap1-wildtype.html) produced in HEK293T cells using a gene trap vector similar to that described previously (Jae et al., Science 2013 340(6131 ):479-83) in which green fluorescent protein (GFP) was exchanged for blue fluorescent protein (BFP).

Cells were seeded in 12 T175 flasks at 40% confluence. The next day, the medium was replaced with DM EM supplemented with 30% fetal calf serum (FCS) prior to transfection with 6.6 microgram gene trap plasmid per T175 flask, in combination with the packaging plasmids pCMV-Gag-pol, pCMV-VSVg and pAdVAntage (Carette et al., Science 2009 326(5957): 1231- 1235). The medium was harvested 48 hours post transfection and subsequently concentrated by ultracentrifugation at 21.000 rpm for 2 hours at 4°C. The supernatant was discarded and the pellets were resuspended in 200 microliter phosphate buffered saline (PBS, Life technologies) overnight at 4°C. Retrovirus-containing medium was collected and concentrated twice daily for three days. To generate a mutagenized HAP1 cell population, ca. 40 million HAP1 cells were repeatedly transduced with gene-trap retrovirus in the presence of 8 microgram/ml protamine sulphate (Sigma). The mutant library was subsequently expanded prior to antibody staining and flow cytometric cell sorting.

For the genetic screens measuring PD-L1 (encoded by gene CD274) at the cell surface, mutagenized HAP1 libraries (starting with either parental HAP1 cells or the respective HAP1 mutants described below) were expanded to ca. 1.5x109 cells and subsequently treated with 0.5 nanogram/microliter interferon gamma (IFNg, peprotech) for 24 hours to induce expression of PD-L1. Subsequently, ca. 3x109 cells were dissociated using trypsin-EDTA (Life technologies), washed with PBS and stained with a FITC labeled antibody directed against PD-L1 (MIH1 , BD pharmingen) at 1 :20 dilution for 30' at RT in PBS containing 0.5% w/v bovine serum albumin (Sigma) and 0.2% w/v sodium azide (Sigma). Next, the cells were washed three times with PBS containing 1 % FCS and subsequently stained with a FITC labeled polyclonal goat anti-mouse Ig (BD pharmingen) at 1 : 100 dilution for 30' at RT in PBS containing 0.5% w/v bovine serum albumin (Sigma) and 0.2% w/v sodium azide (Sigma).

Following two washes with PBS containing 1 % FCS and one wash with PBS, stained cells were passed through a 40 micrometer strainer (BD FalconTM) and subsequently fixed using BD fix buffer I (BD biosciences) for 10 minutes at 37°C, followed by a wash with PBS containing 1 % FCS. Subsequently, cells were permeabilized by suspension in cold (-20°C) BD permeabilization buffer (BD biosciences) while vortexing and incubated on ice for 30 minutes prior to incubation with 100 microgram/ml RNAse A (Qiagen, Germany) and 10 microgram/milliliter propidium iodide (Cayman Chemical) at 37°C temperature for 30 minutes. Alternatively, cells were subjected to treatment with 3 micromolar 4', 6- diamidino-2- phenylindole (DAPI) for 30 minutes. In each case, the staining was concluded by a final wash in PBS 10% FCS.

Following staining, cells were sorted on a Biorad S3 Cell sorter (Biorad) or a Moflo Asterios sorter (Beckman Coulter) to collect the highest and lowest staining populations for PD-L1 (approximately 1-5%) and 1 n DNA content.

Sorted cells were pelleted by centrifugation (2500 rpm, 10 minutes) and genomic DNA was isolated using Qiagen DNA mini kit (Qiagen). To facilitate de-crosslinking pellets were resuspended in PBS (200 microliter per 10 million cells) and after the addition of Proteinase K and lysis buffer (buffer AL, both Qiagen) incubated overnight at 56°C with agitation. The following day, DNA was isolated according to manufacturer's specifications and measured by Nanodrop2000 spectrophotometer (Thermo Fisher).

Insertion sites were amplified and cloned as described in Blomen et al., Science 2015, 350(6264): 1092-6, using the pre-adenylated linker in combination with thermostable RNA ligase 1 from Thermus scotoductus bacteriophage (Blondal et al, Nucleic Acid Research 2005, 33(1 ) 135-142, patent WO 2010/094040 A1) and sequenced on an lllumina HiSeq2500 (lllumina) using sequencing primer 5'-ctagcttgccaaacctacaggtggggtctttca-3' (SEQ ID NO: 1) as single-reads with a read-length of 65 base pairs.

Following deep sequencing, gene-trap insertion sites were identified as reads aligning uniquely to the human genome (hg19) without or with a single mismatch using bowtie (Langmead et al., Genome Biol 2009, 10:R25) for both the high and low PD-L1 sorted populations. Aligned reads were intersected with hg19 RefSeq gene coordinates (for every gene the longest RefSeq region was selected) to establish intragenic insertion sites and their orientation respective to the gene using intersectBED (Quinlan and Hall, Bioinformatics 2010, 26 (6): 841-842). For the purpose of this analysis, insertion sites integrated in sense orientation relative to the directionality of the affected gene were considered disruptive. In the case of overlapping genes, only those genomic regions unique to a single gene were considered. In order to identify genes enriched for disruptive gene-trap integrations in either query population, per gene, the number of disruptive insertion sites in that gene and the total number of disruptive gene-trap integrations in the corresponding population (e.g. PD-L1 high) was compared to the corresponding values in the other population (e.g. PD-L1 low) using a two-sided Fisher's exact test.

Resulting P-values were adjusted for multiple testing using Benjamini and Hochberg FDR correction. Fishtail plots were created in Prism 6 for Mac OS X (GraphPad, version 6. Oh) by calculating the ratio of the number of disruptive integrations per gene in both populations normalized by the number of total integrations in the two populations (deemed mutational index (Ml), plotted on the y-axis) and the sum of disruptive integrations identified in both the high and low populations (plotted on the x-axis). For genes in which one or more disruptive gene-trap insertion were identified in one population but not in the other, one insertion was assigned to that gene in the population in which no insertion could be mapped to allow Ml calculation (circumventing division by 0 and allowing plotting on a logarithmic scale): Higk(x) ,_a Lowi )

Μίίχ) - - X &High (x)≥ 1 and-Low(a:}

Low(x) „ Higkii)

where High(x) and Low(x) denote the sum of disruptive gene-trap insertions mapped in gene x in the high and low population respectively. For PD-L1 (Gene CD274) it has been described that alterations of the 3' portion of the gene can stabilize the gene product and lead to higher PD-L1 proteins levels (Kataoka et al., Nature. 2016 May 23;534(7607):402-6). As this was also recapitulated by our gene-trap insertion method (gene-trap integrations into the 3' portion of the gene resulting in increased rather than decreased staining for PD-L1), we disregarded the portion of the gene that lies downstream of exon 5 (Refseq identifier NM_014143.3).

Results

The results of the genetic haploid genetic screen for PD-L1 levels at the cell surface in parental ('wild-type') HAP1 cells treated with IFNg are shown in Figure 1. Specifically, the genetic haploid genetic yielded a total of 215 significant outliers with an FDR-corrected P- value of smaller than 10E-6, 93 of which occurred in the PD-L1 high population and 122 in the PD-L1 low population. Besides the gene coding for PD-L1 itself (CD274), this included a set of genes known to mediate IFNg signaling events, including the receptor (IFNGR1 and IFNGR2), the kinases JAK1 and JAK2, as well as the transcription factors STAT1 and IRF1. Beyond these expected genes, the screen identified a strong regulator of PD-L1 levels: STIP1 Homology And U-Box Containing Protein 1 (STUB1 ) as a negative regulator of PD-L1 (Figure 1).

Example 2: Generation and analysis of clonal STUB1 knockout cells

Next, we sought to validate the involvement of STUB1 in surface PD-L1 levels by transducing cells with lentiviral vectors encoding fluorescently-tagged Cas9 and sgRNAs targeting the STUB1 gene.

Materials and Methods

CRISPR qRNA cloning

pLentiCrisprV2 vectors targeting STUB1 was generated as described on http://genome- engineering.org/gecko/wp-content/uploads/2013/12/lentiCRISPRv2-and-lentiGuide-oligo- cloning-protocol.pdf. The following gRNA were used:

STUB1 sgRNA#1 GGAGATGGAGAGCTATGATG (SEQ ID NO:2)

- STUB1 sgRNA#2 GGCCGTGTATTACACCAACC (SEQ ID NO 3) For production of lentiviral particles, the described plasmids were cotransfected into HEK293T cells along with packaging plasmids (psPAX2, pVSV-G). Two days after transfection, lentiviral supernatant was harvested and used for transduction. Two days after transduction cells were selected by exposing them to blasticidin or puromycin.

Results

Cell lines resembling different tissues/ tumor types were transduced with a lentivirus expressing the hCas9 together with a sgRNA targeting STUB1 and selected with puromycin. Puromycin selected 8505c, A375, colo679 were exposed to different concentrations of IFNg for 48h in order to induce or increase PD-L1 expression. Surface levels of PD-L1 were analyzed by flow cytometry. In all cases, we observed an increase in PD-L1 expression upon STUB1 KO, as shown in Figure 2. To assess whether STUB1 affects PD-L1 degradation, we disrupted STUB1 in either CMTM6 proficient or deficient A375 cells. Deletion of STUB1 resulted in a more profound increase in PD-L1 levels in CMTM6 deficient than in CMTM6 proficient cells, identifying STUB1 as an E3 ligase that causes destabilization of PD-L1 , either by direct modification of one of the lysines in the PD-L1 cytoplasmic domain or indirectly. Those data, additionally, would suggest a functional connection between STUB1 and CMTM6, as the effect of STUB1 depletion is of greater magnitude in a CMTM6 deficient than in a CMTM6 proficient context).

Claims

1. A method for screening for a compound capable of modulating immune activity, the method comprising:

(a) contacting a cell expressing a STUB1 protein with a test compound;

wherein the test compound is a compound capable of decreasing immune activity if the test compound increases the level of expression or activity of the STUB1 protein and

wherein the test compound is a compound capable of increasing immune activity if the test compound decreases the level of expression or activity of the STUB1 protein.

2. A method for screening for a compound capable of modulating the level of expression of the PD-L1 protein in a cell, the method comprising:

(a) contacting a cell expressing a STUB1 protein with a test compound;

wherein the test compound is a compound capable of increasing the level of expression of the PD-L1 protein if the test compound increases the level of expression or activity of a protein of the STUB1 protein and

wherein the test compound is a compound capable of decreasing the level of expression of the PD-L1 protein if the test compound decreases the level of expression or activity of the STUB1 protein.

3. The method for screening of any one of the previous claims, wherein measuring the level of expression or activity of the STUB1 protein involves measuring the level of gene expression, the level of mRNA, the level of protein, the level of cell surface protein, activity of the protein or, phosphorylation status of the STUB1 protein.

4. The method for screening of any one of the previous claims, wherein the immune activity is mediated by a T cell, preferably an effector T cell, wherein the immune activity comprises secretion of cytokines, preferably IFN gamma, and TNF alpha, secretion of chemokines, preferably CXCL9 and CXCL10, and secretion of perforin-granzymes, following binding of a T cell receptor to a peptide: MHC complex on a target cell.

5. The method for screening of any one of the previous claims, wherein the level of expression of the PD-L1 protein is the level of cell surface expression of the PD-L1 protein.

6. The method for screening of any one of the previous claims, wherein the method is screening for a compound capable of modulating PD-1/PD-L1 axis signaling, and/or capable of modulating immune activity.

7 The method for screening of any one of the previous claims wherein the method further comprises measuring immune activity and/or measuring the level of expression of the PD-L1 protein and/or PD-1/PD-L1 axis signaling in the presence of the compound selected in (c).

8. A method for screening for a compound capable of modulating the expression and/or activity of the STUB1 protein, the method comprising:

(ii) measuring the level of expression of the PD-L1 protein, optionally the level of cell-surface expression of PD-L2 protein; and

(iii) selecting a test compound modulating the level of expression of PD-L1 protein as compared to a cell contacted with the test compound, wherein the cell is expressing PD-L1 protein but is not expressing the STUB1 protein,

9. An in vitro method for modulating PD-L1 protein expression and/or PD-1/PD-L1 axis signaling in a cell, the method comprising modulating the expression or activity of a STUB1 protein.

10. The in vitro method for modulating PD-L1 protein expression and/or PD-1/PD-L1 axis signaling in a cell according to claim 9,

wherein PD-L1 protein expression and/or PD-1/PD-L1 axis signaling is increased when the level of expression or activity of the STUB1 protein is decreased and/or wherein PD-L1 protein expression and/or PD-1/PD-L1 axis signaling is decreased when the level of expression or activity of the STUB1 protein is increased.

1 1. The in vitro method for modulating PD-L1 protein expression and/or PD-1/PD-L1 axis signaling in a cell according to any one of claims 9-10, wherein modulating PD-L1 protein expression and/or PD-1/PD-L1 axis signaling is performed by treating the cells in the presence of a compound identified with the method for screening of any one of the previous claims and/or by treating the cells with a siRNA, shRNA, TALENs, MEGATALENs, CRISPR or Zinc finger nucleases directed to silence expression of a gene encoding the STUB1 protein.

12. A modulator of the expression and/or activity of the STUB1 protein for use in modulating immune activity, PD-1/PD-L1 axis signaling and/or PD-L1 protein expression in a patient in need thereof, wherein the modulator is a modulator that increases immune activity, decreases PD-1/PD-L1 axis signaling and/or decreases PD-L1 protein expression when the modulator increases the expression and/or activity of the STUB1 protein and

wherein the modulator is a modulator that decreases immune activity, increases PD-1/PD-L1 axis signaling and/or increases PD-L1 protein expression when the modulator decreases the expression and/or activity of the STUB1 protein.

13. A modulator of the expression and/or activity of the STUB1 protein for use in immunotherapy, immunotherapy in a cancer patient, preferably a patient suffering from a cancer selected from melanoma, lung cancer, bladder cancer, Gl tract cancer, HNSCC, and Hodgkin's lymphoma, or for use in the treatment of an autoimmune disease, preferably systemic lupus erythematosus, rheumatoid arthritis, and type 1 diabetes,

wherein the modulator is a modulator that increases immune activity, decreases PD-1/PD-L1 axis signaling and/or decreases PD-L1 protein expression when the modulator increases the expression and/or activity of the STUB1 protein and/or

wherein the modulator is a modulator that decreases immune activity, increases PD-1/PD-L1 axis signaling and/or increases PD-L1 protein expression when the modulator decreases the expression and/or activity of the STUB1 protein

14 A modulator of the expression and/or activity of a protein of the STUB1 protein for use according to any one of 12-13, wherein the treatment also involves the use of a PD-1/PD-L1 axis binding antagonist and/or agonist.

15. Use of a modulator of the expression and/or activity of the STUB1 protein for modulating immune activity, PD-1/PD-L1 axis signaling and/or PD-L1 protein expression wherein the modulator is a modulator that increases immune activity, decreases PD-1/PD-L1 axis signaling and/or decreases PD-L1 protein expression when the modulator increases the expression and/or activity of the STUB1 protein and/or

16. An antibody against the STUB1 protein for use in the treatment of a disorder that benefits from a decrease of immune activity, increase of PD-1/PD-L1 axis signaling and/or increases of PD-L1 protein expression, preferably wherein said disorder is an auto-immune disease.

17. Method for the treatment of a disorder that benefits from an increase of immune activity, decrease of PD-1/PD-L1 axis signaling and/or decrease of PD-L1 protein expression, preferably wherein said disorder is a cancer, the method comprises administering to a human in need of such treatment a therapeutically effective amount of

- a modulator of the expression and/or activity of the STUB1 protein, wherein the modulator increases the expression and/or activity of a protein of the STUB1 family.

18. Method for the treatment of a disorder that benefits from a decrease of immune activity, increase of PD-1/PD-L1 axis signaling and/or increase of PD-L1 protein expression, preferably wherein said disorder is an auto-immune disease, the method comprises administering to a human in need of such treatment a therapeutically effective amount of a modulator of the expression and/or activity of the STUB1 protein, wherein the modulator decreases the expression and/or activity of a protein of the STUB1 family.

19. Method of promoting health, the method comprises communicating to a target audience, the use of a modulator of the expression and/or activity of a the STUB1 protein for treating an individual with a disease or disorder that benefits from the modulation of immune activity, PD-1/PD-L1 axis signaling and/or PD-L1 protein expression.

20. Use of the STUB1 protein for modulating immune activity, PD-L1 expression, PD-L1 protein expression and/or PD-1/PD-L1 axis signaling.

21. A method for predicting immune activity towards cancer cells in a patient, the method comprising measuring the level of expression and/or activity of a STUB1 protein in cancer cells and/or cancer-infiltrating cells obtained from said patient, wherein decreased expression and/or activity of STUB1 protein is predictive for poor activity of the T-cell towards the cancer cell and wherein increased expression and/or activity of the STUB1 protein is predictive for strong immune activity towards cancer cells.

22. The method for predicting immune activity towards cancer cells in a patient according to claim 21 , wherein the method is used to determining likelihood that the patient will exhibit benefit from treatment with a PD-1/PD-L1 axis binding antagonist and/or exhibit benefit from treatment with a modulator of the expression and/or activity of the STUB1 protein.

23. A method for predicting immune activity towards cells in a patient suffering from an autoimmune disease, the method comprising measuring the level of expression and/or activity of the STUB1 protein in the cells obtained from said patient, wherein increased expression and/or activity of STUB1 is predictive for strong activity of the T-cell towards the cells and wherein decreased expression and/or activity of STUB1 is predictive for poor activity of the T-cell towards the cells.

24. The method for predicting immune activity towards cells in a patient suffering from an autoimmune disease according to any one of the previous claims wherein the method is used to determining likelihood that the patient will exhibit benefit from treatment with a PD-1/PD-L1 axis binding agonist and/or exhibit benefit from treatment with a modulator of the expression and/or activity of a protein of the STUB1 protein.

25. A method for screening for a compound for treatment of cancer or infection, characterized by using STUB1.

26. The method of claim 25, wherein the method of screening comprises the step of:

(a) contacting a cell expressing STUB1 with a test compound;

(b) measuring the level of expression or activity of STUB1 ; and

(c) selecting a test compound modulating the level of expression or activity of STUB1 compared to the expression level or activity measured in the absence of the test compound, wherein the test compound is a compound for treatment of cancer or infection if the test compound increases the level of expression or activity of STUB1.

27. A method for screening for a compound for treatment of a disease, preferably selected from the group consisting of cancer and infection, or a condition that benefits from upregulation or enhancement of an immune response, wherein the method is characterized by utilizing the interaction between PD-L1 and STUB1 , preferably wherein the method comprises comparing the interaction between PD-L1 and STUB1 in the absence and presence of the compound to be screened.

28. A immune checkpoint inhibitor for use in the treatment of a disease, wherein in the treatment also involves the use of a compound that increases the level of expression or activity of STUB1.

29. The immune checkpoint inhibitor for use in the treatment of a disease according to claim 28, wherein the compound is an activating antibody for STUB1.

30. The immune checkpoint inhibitor for use in the treatment of a disease according to any one of claims 28-29, wherein the disease is a disease that benefits from decreased PD-1/PD- L1 axis signaling and/or that benefits from upregulation or enhancement of an immune response function.

31. The immune checkpoint inhibitor for use in the treatment of a disease according to any one of claims 28-30, wherein the disease is cancer or infection.

32. The immune checkpoint inhibitor for use in the treatment of a disease according to any one of claims 28-31 , wherein the immune checkpoint inhibitor is an inhibitor of PD-1 , PD- L1 , CTLA-4 or CD47.

33. The immune checkpoint inhibitor for use in the treatment of a disease according to any one of claims 28-32, wherein the treatment also involves the use of a cytotoxic agent and/or chemotherapeutic agent.

34. A method for increasing ubiquitination of PD-L1 , the method comprises increasing the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1 , preferably wherein the activator or agonist is an antibody.

35. A method for decreasing half-life of PD-L1 , the method comprises increasing the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1.

36. A method for reducing PD-1/PD-L1 signaling, the method comprises increasing the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1.

37. A method for upregulation or enhancement of an immune response function, the method comprises increasing the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1.

38. A method for reducing expression of PD-L1 , preferably of cell-surface PD-L1 , the method comprises increasing the expression or activity of STUB1 , preferably by providing or administering an activator or agonist of STUB1.

39. A method according to any one of claims 34 - 38, further comprising providing or administering an immune checkpoint inhibitor and/or a cytotoxic agent and/or chemotherapeutic agent.