COMBINATION OF ANTI-PLATELET THERAPY ANTI-GPIbα WITH IMMUNOTHERAPY FOR CANCER

Abstract

The present invention relates to the field of cancer therapy using immunotherapeutic approaches. In particular, the present invention relates to compound for use in improving immunotherapy wherein said compound is a platelet inhibiting agent. The present invention also encompasses a pharmaceutical composition comprising said compound and an immunotherapeutic agent. Also comprised by the invention is a method of assessing whether a subject suffering from hepatic cancer benefits from immunotherapy. The present invention also refers to use of the amount of platelets in a sample of a subject suffering from hepatic cancer for assessing whether said subject benefits from immunotherapy as well as a device for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy. Moreover, the present invention also envisages a kit for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy.

Description

[0001] Combination of anti-platelet therapy anti-GPIba with immunotherapy for cancer

[0002] The present invention relates to the field of cancer therapy using immunotherapeutic approaches. In particular, the present invention relates to a compound for use in improving immunotherapy wherein said compound is a platelet inhibiting agent. The present invention also encompasses a pharmaceutical composition comprising said compound and an immunotherapeutic agent. Also comprised by the invention is a method of assessing whether a subject suffering from hepatic cancer benefits from immunotherapy. The present invention also refers to use of the amount of platelets in a sample of a subject suffering from hepatic cancer for assessing whether said subject benefits from immunotherapy as well as a device for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy. Moreover, the present invention also envisages a kit for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy.

[0003] Hepatocellular carcinoma (HCC) is one of the world’s deadliest and fastest-growing tumors and is the most common type of primary liver cancer in adults, with a poor prognosis. In fact, it is the world’s fifth most frequent malignancy and the third main cause of cancer death (D.S. Mandlik et al., 2023). The development of HCC is attributed to Hepatitis B virus (HBV) infection or exposure to toxins such as alcohol, aflatoxin or pyrrolizidine alkaloids. Patients suffering from cirrhosis, fatty liver disease such as non-alcoholic steatohepatitis (NASH), metabolic syndrome or diabetes mellitus are also considered to be at high risk for HCC incidence (J.M. Llovet et al., 2021).

[0004] The treatment of HCC is assigned according to tumor stages. Patients with early-stage HCC tumors are the preferred candidates for resection, transplantation and local ablation. Unfortunately, due to late diagnosis, 70% to 80% of advanced HCC cases will not benefit from tumor resection. As a result, prognosis is poor for most patients, with an average five-year survival rate of less than 15% (C. Kole et al., 2020). Current treatment options for patients with unre- sectable HCC and patients at intermediate stages are first candidates for transcatheter arterial chemoembolization (TACE). Patients with advanced disease will first receive systemic therapies. Systemic therapies have been a mainstay of treatment for advanced HCC for more than a decade, with first-line agents, including the oral multityrosine kinase inhibitors (TKIs) sorafenib, lenvatinib, and donafmib, and second-line agents, including the antiangiogenic agents regorafenib and apatinib (J. Li et al., 2023). However, the use of systemic therapies for advanced stage HCC is still controversial due to lack of efficacy and poor patient tolerance, and thus, their clinical benefit is still limited.

[0005] Immunotherapy, including immune checkpoint inhibitors (ICIs), adoptive cell therapy (ACT), cancer vaccines and cytokines, has become a paradigm for the treatment of an increasing number of cancer types (e.g. melanoma, non-small cell lung cancer, HCC, etc.) over the past two decades. It is considered to be more successful and safer in the treatment of solid tumors, resulting in long-term survival and manageable toxicity. Immunotherapy for liver cancer most often involves checkpoint inhibitors. Immune checkpoint inhibitors are inhibitory regulatory molecules of the immune system that negate tumor-expressed extracellular ligands, e.g. cytotoxic T-lymphocyte-associated antigen 4 (CTLA-4), programmed cell death protein- 1 (PD-1) and its ligand, PD-L1, that suppress intrinsic immune response. In recent years, use of immune checkpoint inhibition with monoclonal antibodies that target PD-1, PD-L1, CTLA-4, TIM-3 and LAG-3 have shown surprising safety and efficacy in the treatment of HCC (F. Foerster et al., 2022).

[0006] So far, the combination of bevacizumab (anti-VEGFa) and atezolizumab (anti-PD-Ll) is the only approved first-line immunotherapy in unresectable HCC, along with several single-agent immune checkpoint inhibitors serving as second-line treatments. Despite that immunotherapeutic approaches often achieve better and long-lasting clinical outcome than the used-to-be standard of care, sorafenib, this was only observed in a limited number of patients of approximately 15-30%.

[0007] However, there is still a wide heterogeneity of response concerning the adoption of monotherapy and a considerable number of HCC patients does not respond to such immunotherapies.

[0008] Hence, there is still an unmet need of novel compounds and therapies with increased efficacy against liver cancer and improved patient tolerance.

[0009] The technical problem underlying the present invention may be seen as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

[0010] Thus, the present invention relates to a compound for use in improving immunotherapy wherein said compound is a platelet-inhibiting agent. It is to be understood that in the specification and in the claims, “a” or “an” can mean one or more of the items referred to in the following depending upon the context in which it is used. Thus, for example, reference to “an” item can mean that one item or more than one of those items can be utilized.

[0011] As used in the following, the terms “have”, “comprise” or “include” are meant to have a nonlimiting meaning or a limiting meaning. Thus, having a limiting meaning these terms may refer to a situation in which, besides the feature introduced by these terms, no other features are present in an embodiment described, i.e. the terms have a limiting meaning in the sense of “consisting of’ or “essentially consisting of’. Having a non-limiting meaning, the terms refer to a situation where besides the feature introduced by these terms, one or more other features are present in an embodiment described.

[0012] Further, as used in the following, the terms “preferably”, “more preferably”, “most preferably”, "particularly", "more particularly", “typically”, and “more typically” are used in conjunction with features in order to indicate that these features are preferred features, i.e. the terms shall indicate that alternative features may also be envisaged in accordance with the invention.

[0013] Further, it will be understood that the term “at least one” as used herein means that one or more of the items referred to following the term may be used in accordance with the invention. For example, if the term indicates that at least one item shall be used this may be understood as one item or more than one item, i.e. two, three, four, five or any other number. Depending on the item the term refers to the skilled person understands as to what upper limit the term may refer, if any.

[0014] The term "about" in the context of the present invention means + / - 20%, + / - 10%, + / - 5%, + / - 2 % or + / - 1% from the indicated parameters or values. This also takes into account usual deviations caused by measurement techniques and the like.

[0015] The term “compound” as used herein, refers to a therapeutic agent which can be used in the treatment or prevention of a disorder or one or more symptoms thereof. In the context of the present invention, the term “compound” refers to a therapeutic agent which can be used for improving immunotherapy. Said compound according to the present invention is a plateletinhibiting agent as specified elsewhere herein.

[0016] Platelets are small, discoid blood cells formed as non-nuclear cytoplasmic vesicles from megakaryocytes in the bone marrow. Their key role in hemostasis is the initiation of the coagulation cascade in response to vascular injury, where they adhere to the extracellular matrix to form a blood clot. However, they are also implicated in tumor progression and contribution to a more aggressive and metastatic tumor phenotype in hepatocellular carcinoma. For example, it has been reported that platelets support tumor cell migration by adhering to them through adhesion receptors GPIIb / IIIa, GPIb-IX-V and P-selectin, thereby protecting them from im- muno-surveillance. It has also been reported that platelets activate the coagulation cascade by secreting thrombin and tissue factor, which results in a meshwork of platelets and fibrin shielding tumor cells and allows them to escape immune-surveillance and successfully invade distant sites (N. Pavlovic et al., 2019). In contrast thereto, platelet inhibition therapy has been shown to ameliorate liver injury and improve the disease outcome.

[0017] The term “platelet-inhibiting agent” refers to any drug capable of preventing and / or reversing platelet aggregation and or inhibiting platelet activation, and thus, inhibiting thrombus formation. Preferably, the platelet-inhibiting agent shall act as inhibitor of platelet activation.

[0018] Platelet-inhibiting agents can be divided into oral and parenteral agents, with oral agents subdividing further based on their mechanism of action. Oral agents may include for example clopidogrel, ticagrelor, prasugrel, pentoxifylline, cilostazol and dipyridamole. Glycoprotein Ilb / IIIa inhibitors such as tirofiban and eptifibatide are only available as parenteral agents (J.W. Eikelboom et al. 2012). The platelet-inhibiting agent may reversibly or irreversibly inhibit the process involved in platelet activation, and thus, resulting in decreased tendency of platelets to adhere to one another and to damaged blood vessels’ endothelium.

[0019] Preferably, the platelet-inhibiting agent is selected from the group consisting of an inhibitor of platelet glycoprotein lb (GPIb), an inhibitor of platelet glycoprotein VI (GPVI), an inhibitor of platelet glycoprotein Ilb / IIIa (GP2B / 3A), an inhibitor of thrombin or an inhibitor of P2Y12. More preferably, the platelet inhibiting agent is an inhibitor of platelet glycoprotein lb (GPIb).

[0020] The term “platelet glycoprotein lb (GPIb)” as used herein, refers to a membrane located glycoprotein found on platelets. It is a component of the GpIb-V-IX complex on platelets that binds von Willebrand factor, allowing platelet adhesion and platelet plug formation at sites of vascular injury. Preferred is platelet glycoprotein lb (GPIb) alpha chain (GPIba), and most preferably human GPIba. Human GPIba is a protein that is expressed by the gene glycoprotein lb platelet subunit alpha chromosomally located on 17pl3.2. The gene can be found under the accession number HGNG4439 (The HUGO Gene Nomenclature Committee at the European Bioinformatics Institute).

[0021] The GPIb to in accordance with the present invention is, preferably GPIba, having an amino acid sequence as deposited under UniProt accession number P07359. It will be understood that the term “GPIb” also relates to variants or fragments of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned GPIb protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the GPIb protein, preferably over the entire length of the said GPIb proteins, respectively. Fragments of GPIb are preferably proteins comprising the ectodomain of GPIb, preferably of human GPIba, but lacking any transmembrane or intracellular parts of the protein. Most preferably the fragments of GPIb retain a functions to bind to thrombin, or at least comprise a binding site to thrombin, or adjacent protein sequences (10, 20, 50, 100 or 200 amino acids adjacent to such binding site). A variant of GPIb may be, preferably, a protein that is (i) a thrombin binding fragment of GPIb, and / or (ii) a protein having an amino acid sequence which is at least 80% identical to the aforementioned amino acid sequence. Inhibitors of the GPIb prevent the platelets to attach to the subendothelium. Examples of GPIb inhibitors include an- fibatide or monoclonal antibody Ab 6B4 (N. Cauwenberghs et al. 2000).

[0022] The term “platelet glycoprotein VI (GPVI)” as used herein, refers to a glycoprotein receptor for collagen which is expressed in platelets. It is encoded in humans by the GPVI gene. GPVI is involved in collagen-induced platelet adhesion and activation and plays a key role in platelet procoagulant activity and subsequent thrombin and fibrin formation. Three isoforms of GPVI are described and several orthologues have been reported in various animal species. The GPVI protein referred to in accordance with the present invention is, preferably, human GPVI having an amino acid sequence as deposited under UniProt accession number Q9HCN6. It will be understood that the term “GPVI” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned GPVI protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human GPVI protein, preferably over the entire length of the said GPVI proteins, respectively. GPVI inhibitors are capable of blocking vascular collagen sites. Examples of GPVI inhibitors include revacept, losartan or glenzocimab (P. Billiald et al. 2023). The term “platelet glycoprotein Ilb / IIIa (GP2B / 3 A)” as used herein, refers to an integrin complex found on platelets is encoded in humans by the / 7U 3 gene. It is a transmembrane receptor for fibrinogen and von Willebrand factor, and aids platelet activation. Three isoforms of GPVI are described and several orthologues have been reported in various animal species. Inhibitors of GP2B / 3A prevent platelet aggregation by blocking GP2B / 3A receptors on their platelet’s plasma membrane and inhibiting fibrinogen binding. Examples of GP2B / 3 A inhibitors include tirofiban and eptifibatide. The GP2B / 3 A protein referred to in accordance with the present invention is, preferably, human GP2B / 3A having an amino acid sequence as deposited under UniProt accession number P05106. It will be understood that the term “GP2B / 3A” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned GP2B / 3 A protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence ofthe variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human GP2B / 3A protein, preferably over the entire length of the said GP2B / 3 A proteins, respectively.

[0023] The term “P2Y12” as used herein, refers to a chemoreceptor for adenosine diphosphate (ADP) that is encoded in humans by the E2 / ? 172 gene. The P2Yn receptor is the predominant receptor involved in the ADP-stimulated activation of the glycoprotein Ilb / IIIa receptor. Activation of the glycoprotein Ilb / IIIa receptor results in enhanced platelet degranulation and thromboxane production, and prolonged platelet aggregation. Several orthologues have been reported in various animal species. P2Yn inhibitors inhibit the platelet activation and aggregation by antagonizing the platelet P2Yn receptor. This prevents the binding of ADP to the receptor which attenuates platelet aggregation and reaction of platelets to stimuli of thrombus aggregation such as thrombin. Examples of P2Yn inhibitors include clopidogrel, prasugrel, ticagrelor, cangrelor and elinogrel. The P2Yn protein referred to in accordance with the present invention is, preferably, human P2Yn having an amino acid sequence as deposited under UniProt accession number Q9H244. It will be understood that the term “P2 Y 12” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned P2Yn protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human P2Yn protein, preferably over the entire length of the said P2Yn proteins, respectively.

[0024] The compound of the present invention being a platelet inhibiting agent may be a direct or indirect inhibitor of platelet activity. A direct inhibitor as referred to in accordance with the present invention may be a compound that binds to proteins present on platelets, preferably, platelet glycoprotein lb (GPIb), platelet glycoprotein VI (GPVI), a platelet glycoprotein Ilb / IIIa (GP2B / 3A), and / or P2Y12, and inhibits the biological activity of those proteins partially or entirely. In said case, direct inhibitors may be small molecules binding to those proteins or antibodies or antibody-like binding agents, which specifically bind to those proteins. Direct inhibitors may also bind to interaction proteins for those proteins present on platelets such as thrombin. Typical direct inhibitors may, thus, be small molecules such as thrombin inhibitors or antibodies, which specifically bind to a protein.

[0025] Preferably, the compound according to the present invention is, thus, an antibody or antibodylike binding agent.

[0026] As used herein, the term “antibody” may be understood in the broadest sense as any immunoglobulin (Ig) that enables binding to its epitope. An antibody as such is a species of an antigen binding protein. Full length “antibodies” or “immunoglobulins” are generally heterotetrameric glycoproteins of about 150 kDa, composed of two identical light and two identical heavy chains. Each light chain is linked to a heavy chain by one covalent disulphide bond, while the number of disulphide linkages varies between the heavy chain of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulphide bridges. Each heavy chain has an amino terminal variable domain (VH) followed by three carboxy terminal constant domains (CH). Each light chain has a variable N-terminal domain (VL) and a single C-terminal constant domain (CL). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDR), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions of the heavy and light chains contain a binding domain that interacts with an antigen. The constant regions of the antibodies may mediate the binding of the immunoglobulin to cells or factors, including various cells of the immune system (e.g., effector cells) and the first component (Clq) of the classical complement system. Other forms of antibodies include heavy-chain antibodies, being those which consist only of two heavy chains and lack the two light chains usually found in antibod ies. Heavy-chain antibodies include the hcIgG (IgG-like) antibodies of camelids such as dromedaries, camels, llamas and alpacas, and the IgNAR antibodies of cartilaginous fishes (for example sharks). And yet other forms of antibodies include single-domain antibodies (sdAb, called Nanobody by Ablynx, the developer) being an antibody fragment consisting of a single monomeric variable antibody domain. Single-domain antibodies are typically produced from heavy-chain antibodies, but may also be derived from conventional antibodies. Antibodies can include, for instance, chimeric, humanized, (fully) human, or hybrid antibodies with dual or multiple antigen or epitope specificities, antibody fragments and antibody sub-fragments, e.g. Fab, Fab' or F(ab')2 fragments, single chain antibodies (scFv) and the like, including hybrid fragments of any immunoglobulin or any natural, synthetic or genetically engineered protein that acts like an antibody by binding to a specific antigen to form a complex.

[0027] The term “antibody-like binding agent” as used herein, refers to organic compounds that can specifically bind antigens, but are not structurally related to antibodies. Examples of antibodylike binding agents include among others affibodies, affilins, avimers, DARPins, monopodies, optimers, obodies and the like.

[0028] Preferably, the antibody or antibody-like binding agent specifically binds to the ectodomain of GPIb. The term “ectodomain of GPIb” refers to the portion of the GPIb protein that is exposed on the cytoplasmic side of a lipid bilayer of a cell, preferably of a platelet comprising such GPIb protein. Methods for determining the ectodomain of a protein are known in the art (Singer 1990; High et al. 1993). The ectodomain of GPIba is well known. In the human protein the N-terminal of the protein is extracellular, whereas the C-terminal tail is located in the cytoplasm. The transmembrane region is most likely located between amino acids 532-552 (preferably excluding the signal peptide). Hence, the ectodomain of GPIb in context of the invention preferably comprises a region located in the first 531 consecutive N-terminal amino acids of human GPIba. Said ectodomain further includes a leucine rich repeat domain, which is a preferred domain targeted in accordance with the herein disclosed invention. Hence, the compound for use in accordance with the invention specifically binds to a leucine rich repeat containing domain in the ectodomain of GPIb. Preferably, the ectodomain at least comprises at least 100, preferably at least 200 and most preferably at least 290 consecutive amino acids of the N-terminus, and preferably beginning with the N-terminus, of GPIb.

[0029] Preferably, the antibody or antibody-like binding agent specifically binds to the alpha-thrombin binding site within the ectodomain of GPIb. Such compounds preferably target GPIba directly, for example, by binding specifically to its ectodomain and thereby impairing thrombin binding to GPIba. The term “alpha-thrombin binding site of GPIb” refers to a portion of the GPIb protein that specifically binds thrombin. As used herein, the term “thrombin” refers to the serine protease that converts soluble fibrinogen into insoluble strands of fibrin and catalyses a number of other coagulation-related reactions. This term is not species-specific unless otherwise designated. The term encompasses a-thrombin, which is the native form of thrombin, as well as y- thrombin, a non-clotting derivative produced from a-thrombin that retains much of its plateletactivating capacity. Preferably, the binding of alpha-thrombin to GPIb is reduced upon binding of the antibody or antibody-like binding agent to the alpha-thrombin binding site within the ectodomain of GPIb.

[0030] The phrase “specifically binds to” as used in accordance with the present invention means that the antibody or antibody-like binding agent shall not cross-react significantly with components other than the alpha-thrombin binding site within the ectodomain of GPIb. Cross-reactivity of an antibody as mentioned herein can be tested by the skilled person by various techniques including immunological technologies such as Western blotting, ELISA or RIA based Assays or measuring of binding affinities using, e.g., Biacore technology.

[0031] The term “small molecule” as used herein, refers to a low molecular weight organic compound capable of inhibiting or reducing platelet agglutination or inhibiting platelet activity. Small molecules can act as platelet-inhibiting agents in different manners and pathways. For example, they can act by preventing the formation of secondary messengers (cyclooxygenase- 1 (COX- 1) inhibitor), by interacting with intracellular signaling pathways (phosphodiesterase (PDE) inhibitors and prostacyclin (PGL) analogue), by blocking membrane receptors (P2Yn receptor antagonists and PARI antagonist) or by inhibiting platelet aggregation (glycoprotein Ilbllla inhibitors). Other small molecules are mainly directed against platelet glycoproteins such as GPVI, GPIba and GPIIbllla or block membrane receptors such as the 2 purinergic receptors P2Yn and P2Yi as well as the receptors PARI and PAR4. Other small molecules are directed against different platelet-activation processes, such as adhesion, signaling and pro-coagulant activity (G. Jourdi et al., Int J Mol Sci. 2021).

[0032] An indirect inhibitor in accordance with the present invention may be a nucleic acid that inhibits or reduces expression of a protein present on platelets, preferably, as specified above. Such nucleic acids can be typically designed based on a target sequence present in either the transcripts of those proteins or within the gene encoding them. Typically, those nucleic acids comprise sequences that are complementary or reverse complementary to the target sequence. Nucleic acids that can be used as indirect inhibitors of platelets in accordance with the present invention may be antisense nucleic acids, CRISPR-Cas9 like gene editing constructs, and ribozymes. Thus, preferably, the compound is selected from the group consisting of an antisense nucleic acid, CRISPR-Cas9 like gene editing construct and a ribozyme.

[0033] The term “antisense nucleic acid” as used herein, refers to a single strand DNA and / or RNA molecule that is capable of interfering with DNA and / or RNA processing. Antisense oligonucleotides comprise a nucleic acid sequence, which is complementary to a specific RNA or DNA sequence. Typically, an antisense oligonucleotide will bind, in a sequence-specific manner, to its respective complementary oligonucleotides, DNA, or RNA, thereby interfering with DNA and / or RNA processing. Preferably, said antisense nucleic acid binds to an RNA or DNA sequence, which is complementary to an RNA or DNA sequence of an enzyme or protein involved in platelet agglutination. For example, the antisense nucleic acid may bind to an RNA or DNA sequence of GPIb, GPVI, GP2B / 3A, thrombin or P2Yn, thereby inhibiting platelet function. Antisense nucleic acid shall also refer to interfering RNAs, i.e. small interfering RNAs (siRNAs) or microRNAs (miRNAs), which induce an RNA-dependent gene silencing process. Preferably, the interfering RNAs bind to an mRNA sequence

[0034] The term “CRISPR-Cas9 like gene editing construct” refers to an RNA-guided genome editing tool consisting of a Cas9 nuclease and a single-guide RNA. By base-pairing with a DNA target sequence, the single-guide RNA enables Cas9 to recognize and cut a specific target DNA sequence, generating double strand breaks that trigger cell repair mechanisms and mutations at or near the double strand break sites. Preferably, the CRISPR-Cas9 like gene editing construct targets the DNA sequence of an enzyme or protein involved in platelet agglutination, e.g. GPIb, GPVI, GP2B / 3 A, thrombin or P2Yi2.

[0035] The term "ribozyme" as used herein refers to catalytic RNA molecules possessing a well-defined tertiary structure that allows for catalyzing either the hydrolysis of one of their own phosphodiester bonds (self-cleaving ribozymes), or the hydrolysis of bonds in other RNAs, but they have also been found to catalyze the aminotransferase activity of the ribosome. Preferably, the ribozymes target the RNA sequences of an enzyme or protein involved in platelet agglutination, e g. GPIb, GPVI, GP2B / 3 A, thrombin or P2Yi2.

[0036] The term “inhibitor of thrombin” as used herein, refers to anticoagulant agents that bind to and inhibit the activity of thrombin and therefore, prevent blood clot formation. Thrombin is essential in the conversion of soluble fibrinogen to insoluble fibrin, while also stimulating platelet activation. Thrombin can be inhibited directly or indirectly by the binding of thrombin-inhibiting drugs to one or two of its three domains: (i) the active site, (ii) the fibrin-binding domain (exosite 1) and (iii) the heparin-binding domain (exosite 2). Direct thrombin inhibitors bind directly and reversibly to thrombin without the need of a cofactor. Examples of direct thrombin inhibitors can be selected from the group consisting of lepirudin, desirudin, bivalirudin, ximela- gatran, dabigatran etexilate and argatroban. Indirect thrombin inhibitors can only inhibit free thrombin and act via antithrombin and heparin cofactor II. The main representatives of indirect thrombin inhibitors are heparins, low molecular weight heparins, fondaparinux, idraparinux and danaparoid. They bind to antithrombin and potentiate the inactivation of factor Xa and other serine proteases. Preferably, the present invention encompasses any inhibitor capable of binding - directly or indirectly - to at least one of said three thrombin domains, thereby inhibiting the activity of thrombin.

[0037] The compound according to the present invention shall be used for improving immunotherapy. The term “improving” in the context of the present invention, refers, preferably, to increasing the efficacy of immunotherapy when used in treating a disease. Preferably, the term refers to improving the efficacy of existing immunotherapies for treating liver cancer. An improvement in efficacy of an immunotherapy may be characterized by, for example, an increased survival rate, long-term cancer control, increased anti-tumor immune response, etc. The term “improving” in the context of the present invention also refers to improved tolerability of immunotherapy. In particular, it means that use of the compounds of the present invention and the immunotherapeutic agents as described herein shall result in less adverse effects, such as toxicity, in the subject. It could also refer to improving responsiveness, e.g. in the form of an anti-tumor immune response, to immunotherapy where before there was none in the subject.

[0038] The term “immunotherapy” as referred to in accordance with the present invention encompasses mono-immunotherapies, i.e. immunotherapies involving administration of one immunotherapeutic drug, as well as combination immunotherapies, i.e. immunotherapies involving administration of more than one immunotherapeutic agent. Immunotherapeutic agents used for immunotherapy according to the present invention encompass, typically, therapeutically active cytokines, therapeutically active antibodies or cell-based immunotherapies.

[0039] It will be understood that the compounds referred to in accordance with the present invention may be used as a single drug therapy or it may be used in combination with other drugs or therapy. Preferably the compound of the present invention may be used in combination with an additional cancer treating agent such as an immunotherapeutic agent that is used in immunotherapy. The compounds may be administered prior to or concomitant with the immunotherapeutic agent.

[0040] Preferably, the immunotherapy in accordance with the present invention is immunotherapy of liver cancer. More preferably, the liver cancer is hepatocellular carcinoma (HCC), most preferably, unresectable HCC. As used herein the term “hepatocellular carcinoma” refers to the most common type of liver cancer, also called malignant hepatoma. HCC may have many different causes, but in instances, the underlying cause of HCC is non-alcoholic fatty liver disease (NAFLD). NAFLD is the most common liver disorder in the Western industrialized countries. It is considered to be the hepatic manifestation of the metabolic syndrome. Thus, NAFLD tends to develop in people who are overweight or obese, and / or who have diabetes, high cholesterol or high triglycerides. For most people, NAFLD cause no signs and symptoms, and no complications. But in some people with NAFLD, the fat that accumulates in the liver can cause inflammation and scarring in the liver that is believed to result in fibrosis and cirrhosis. This more serious form of NAFLD is sometimes called non-alcoholic steatohepatitis (NASH). The term “unresectable HCC” refers to a liver tumor not eligible for resection therapy given the extent of disease.

[0041] Preferably, the immunotherapy according to the present invention involves administering an immunotherapeutic agent selected from the group consisting of an anti VEGF antibody (e.g. bevacizumab), an anti-PD-Ll antibody (e.g. atezolizumab), a combination of an anti VEGF antibody and an anti-PD-Ll antibody.

[0042] The compound of the present invention, preferably, does not compete von Willebrand factor (vWF), P-sel ectin, Mac-1, coagulation factor XI and / or coagulation factor XII for GPIb binding.

[0043] The term “von Willebrand factor” (VWF) as used herein, refers to a blood glycoprotein that is encoded in humans by the VWF gene. VWF is a large multimeric glycoprotein present in blood plasma and is essential in the maintenance of hemostasis, promotes adhesion of platelets to the sites of vascular injury and acts as a chaperone for coagulation factor VIII, delivering it to the site of injury. Two isoforms of VWF are described and several orthologues have been reported in various animal species.

[0044] The VWF protein referred to in accordance with the present invention is, preferably, human VWF having an amino acid sequence as deposited under UniProt accession number P04275. It will be understood that the term “VWF” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned VWF protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human VWF protein, preferably over the entire length of the said VWF proteins, respectively.

[0045] The term “P-selectin” as used herein, refers to a type-1 transmembrane protein that is encoded in humans by the SELP gene. It functions as a cell adhesion molecule on the surface of activated endothelial cells and plays an essential role in the initial recruitment of leukocytes to the site of injury during inflammation. Several orthologues have been reported in various animal species.

[0046] The P-selectin protein referred to in accordance with the present invention is, preferably, human P-selectin having an amino acid sequence as deposited under UniProt accession number P16109. It will be understood that the term “P-selectin” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned P-selectin protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human P-selectin protein, preferably over the entire length of the said P-selectin proteins, respectively.

[0047] The term “Mac-1” as used herein, refers to the macrophage- 1 antigen (aMp2), which is a complement receptor consisting of CD1 lb (integrin aM) and CD18 (integrin P2). Mac-1 belongs to the family of integrins and is implicated in various adhesive interactions of monocytes, macrophages and granulocytes as well as in mediating the uptake of complement-coated particles and pathogens. Two isoforms are described and several orthologues have been reported in various animal species.

[0048] The Mac-1 protein referred to in accordance with the present invention is, preferably, human Mac-1 having an amino acid sequence as deposited under UniProt accession number Pl 1215. It will be understood that the term “Mac-1” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned Mac-1 protein. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid sub- stitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human Mac-1 protein, preferably over the entire length of the said Mac-1 proteins, respectively.

[0049] The term “coagulation factor XI” as used herein, refers to a zymogen form of factor Xia, one of the enzymes of the blood coagulation cascade that is encoded in humans by the Fll gene. It is produced by the liver and circulates as a homo-dimer in its inactive form. During activation of the coagulation factor XI, an internal peptide bond is cleaved by factor Xlla (or XII) in each of the two chains, resulting in activated factor Xia, a serine protease composed of two heavy and two light chains held together by disulfide bonds. This activated plasma factor XI triggers the middle phase of the intrinsic pathway of blood coagulation by activating factor IX. Two isoforms are described and several orthologues have been reported in various animal species.

[0050] The coagulation factor XI referred to in accordance with the present invention is, preferably, human coagulation factor XI having an amino acid sequence as deposited under UniProt accession number P03951. It will be understood that the term “coagulation factor XI” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned coagulation factor XI. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human coagulation factor XI, preferably over the entire length of coagulation factor XI, respectively.

[0051] The term “coagulation factor XII” as used herein, refers to serum glycoprotein that is encoded in humans by the F12 gene. It participates in the initiation of blood coagulation, fibrinolysis and the generation of bradykinin and angiotensin. It circulates in blood as a zymogen and activates coagulation factors VII and XI. Several orthologues have been reported in various animal species.

[0052] The coagulation factor XII referred to in accordance with the present invention is, preferably, human coagulation factor XII having an amino acid sequence as deposited under UniProt accession number P00748. It will be understood that the term “coagulation factor XII” also relates to variants of said proteins. Such variants have at least the same essential biological and immunological properties as the aforementioned coagulation factor XI. In particular, they share the same essential biological and immunological properties if they are detectable by the same specific assays referred to in this specification. Moreover, it is to be understood that a variant as referred to in accordance with the present invention shall have an amino acid sequence which differs due to at least one amino acid substitution, deletion and / or addition wherein the amino acid sequence of the variant is still, preferably, at least 50%, 60%, 70%, 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% identical with the specific amino acid sequence of the human coagulation factor XII, preferably over the entire length of coagulation factor XII, respectively.

[0053] The term “compete” as used herein, refers to a compound according to the present invention competing for binding with a second compound, e.g. an antibody or an antigen-binding portion thereof, where binding of the first compound with its cognate epitope is detectably decreased in the presence of the second compound compared to the binding of the first compound in the absence of the second compound. Thus, a first compound can inhibit the binding of a second compound to its epitope without that second compound inhibiting the binding of the first compound to its respective epitope. Hence, by the phrase "does not compete", as used herein with, is meant that the compounds of the present invention do not compete for binding with any second compound. In particular, the compounds of the present invention do not compete in binding with von Willebrand factor, P-selectin, Mac-1, coagulation factor XI and / or coagulation factor XII for binding their epitope on GPIb. Preferably, the wording means that the compounds of the present invention shall not be involved in any mechanism by which such competition involving von Willebrand factor, P-selectin, Mac-1, coagulation factor XI and / or coagulation factor XII for GPIb binding may occur, e.g. steric hindrance, conformational change or binding to a common epitope on GPIb. Whether two compounds compete in binding can be tested by competition binding assays formats well known in the art.

[0054] It has been found in the studies underlying the present invention that platelets, as the second most abundant blood cell population, exert an immunosuppressive function in the tumor microenvironment, diminishing the anti-cancer effects induced by immunotherapy in murine models. As such, it was surmised that complementing immunotherapy with anti-platelet therapy might further hinder liver tumor development. Advantageously, it has been found that specific antagonism of the platelet surface receptor element GPIba promoted the overall immune response elicited by multiple subsets of immunotherapy-activated immune effector cells, reshaping the tumor immune landscape into a more inflammatory phenotype. This means that a combinatorial approach of anti-GPIba and immunotherapy is capable of driving a tumor landscape associated with immunotherapy resistance into a phenotype that shows responsiveness to immunotherapy. The data has further shown that the combinatorial therapeutic approach has achieved significantly better results than immunotherapy alone. In particular, the inhibition of GPIba enhanced anti-tumor response of the immune system by reverting T cell exhaustion (CD4 and CD8 T cells) and increasing their activation status (i.e. production of anti-tumor cytokines). In addition, the activity of NKT and NK cells was increased, while the Treg / T cell ratio and boosting macrophage pro-inflammatory activation was decreased. Moreover, macrophage pro-inflammatory activation was boosted and immunosuppressive myeloid cells were reduced. Consistent results from the two distinct pre-clinical experimental models, one induced by specific driver gene mutations and the other driven by chronic metabolic stress resembling NASH-HCC, suggested that this could be a well-conserved mechanism. Overall, tumor growth was strongly inhibited in mice treated with anti-GPIba in combination with anti-PD-Ll and anti-VEGF compared to single immunotherapy or combination therapy of anti-PD-Ll and anti-VEGF. This new combinatorial intervention might produce a more favorable response in HCC, which could be translated into future clinical practices.

[0055] All definitions and explanations made herein above apply mutatis mutandis for the embodiments described in the following.

[0056] The present invention also relates to a pharmaceutical composition comprising the compound and an immunotherapeutic agent as described herein elsewhere.

[0057] The term “pharmaceutical composition” as used herein, relates to compositions comprising the compound of the present invention and an immunotherapeutic agent and, preferably, one or more pharmaceutically acceptable carrier. The pharmaceutical compositions are, preferably, administered systemically or topically. Suitable routes of administration conventionally used for drug administration are oral, intravenous, subcutaneous, or parenteral administration as well as inhalation. However, depending on the nature and mode of action of a compound, the pharmaceutical compositions may be administered by other routes as well. Moreover, the compound of the present invention and the immunotherapeutic agent can be administered in combination with other therapeutics either in a common pharmaceutical composition or as separated pharmaceutical composition, wherein said separated pharmaceutical compositions may be provided in form of a kit. Preferably, said other therapeutics are compounds used for immunotherapy of cancer as defined elsewhere herein.

[0058] The pharmaceutical composition of the present invention is, preferably, administered in conventional dosage forms prepared by combining the drugs with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing, granulating and compressing or dissolving the ingredients as appropriate for the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of active ingredient with which it is to be combined, the route of administration and other well-known variables.

[0059] The carrier(s) must be acceptable in the sense of being compatible with the other ingredients of the formulation and being not deleterious to the recipient thereof. The pharmaceutical carrier employed may be, for example, a solid, a gel or a liquid. Exemplary of solid carriers are lactose, terra alba, sucrose, talc, gelatine, agar, pectin, acacia, magnesium stearate, stearic acid, degradable polymers like PLGA, and the like. Exemplary liquid carriers are phosphate buffered saline solution, syrup, oil such as peanut oil and olive oil, water, emulsions, various types of wetting agents, sterile solutions, and the like. Similarly, the carrier or diluent may include time delay material well known to the art, such as glyceryl mono-stearate or glyceryl distearate alone or with a wax. Said suitable carriers comprise those mentioned above and others well known in the art, see, e.g., Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, Pennsylvania.

[0060] The diluent(s) is / are selected so as not to affect the biological activity of the compound or compounds. Examples of such diluents are distilled water, physiological saline, Ringer's solutions, dextrose solution, and Hank's solution. In addition, the pharmaceutical composition or formulation may also include other carriers, adjuvants, or nontoxic, nontherapeutic, non-immuno- genic stabilizers, reactive oxygen scavengers, and the like.

[0061] The pharmaceutical composition is, preferably, administered together with standard pharmaceutical carriers according to conventional procedures. These procedures may involve mixing or dissolving the ingredients as appropriate to obtain the desired preparation. It will be appreciated that the form and character of the pharmaceutically acceptable carrier or diluent is dictated by the amount of the compound of the present invention and the immunotherapeutic agent as described herein elsewhere with which it is to be combined, the route of administration and other well-known variables. Similarly, the carrier or diluent may include time delay material well known in the art, such as glyceryl mono-stearate, or glyceryl distearate alone or with a wax. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population). The dose ratio between therapeutic and toxic effects is the therapeutic index, and it can be expressed as the ratio, LD50 / ED50. The dosage regimen will be determined by the attending physician and other clinical factors. As is well known in the medical arts, dosages for any one patient depend upon many factors, which may include the patient's size, body surface area, age, the particular compound to be administered, sex, time and route of administration, general health, and other drugs being administered concurrently. Progress can be monitored by periodic assessment.

[0062] Moreover, the present invention envisages a method of assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising the steps of: a) determining the amount of platelets in a sample of said subject; b) comparing said amount to a reference; and c) assessing whether a subject suffering from hepatic cancer benefits from immunotherapy.

[0063] The term “assessing” as used herein, refers to determining a treatment response (i.e. an adverse response, a non-response, a therapeutically effective response or an adverse side effect) of the subject to immunotherapy. This includes determining said treatment response in the subject’s current physiological state in a diagnostic approach. Moreover, the term also encompasses determining whether a subject will develop a treatment response in accordance with the present invention (i.e. an adverse response, a non-response, a therapeutically effective response or an adverse side effect) in the future, i.e. within a certain predictive window, in a prognostic approach. Thus, the assessment also allows for stratifying subjects with respect to being susceptible to immunotherapy, or not. Moreover, assessing may also include approaches where a subject is monitored for a treatment response over time, e.g., in case the immunotherapy is administered over a certain period of time as a therapeutic or preventive measure. As will be understood by those skilled in the art, such an assessment, although preferred to be, may usually not be correct for 100% of the investigated subjects. The term, however, requires that a statistically significant portion of subjects can be correctly assessed. Whether a portion is statistically significant can be determined without further ado by the person skilled in the art using various well known statistic evaluation tools, e.g., determination of confidence intervals, p-value determination, Student's t-test, Mann- Whitney test, and the like. Details may be found in Dowdy and Wearden, Statistics for Research, John Wiley & Sons, New York 1983. Typically envisaged confidence intervals are at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, and at least 95%. The p-values are, typically, 0.2, 0.1, 0.05.

[0064] The phrase “benefit from immunotherapy” may refer to any advantageous effects for a subject suffering from cancer that can be derived from applying immunotherapy as described herein. Said benefit may, for example, be an improvement in efficacy of an immunotherapy for a subject suffering from cancer, preferably liver cancer, more preferably hepatocellular carcinoma (HCC), even more preferably unresectable HCC. Considering that many subjects suffering from hepatic cancer do not respond to immunotherapy, said benefit may be a response to the immunotherapy where there was none before. Said benefit may also be a therapeutically effective response involving amelioration or cure of hepatic cancer or amelioration or cure of symptoms accompanying it. For example, the benefit may be increased survival rate, long-term cancer control, increased anti-tumor immune response and the like. A further benefit might also be a reduction of adverse side effects of immunotherapy, i.e. reduced toxicity of immunotherapeutic agents used during immunotherapy. In contrast thereto, a subject does not benefit from immunotherapy if there is no response or an adverse response to the immunotherapy as described herein and / or the compounds of the present invention.

[0065] The term “subject” as used herein refers to an animal, preferably to a vertebrate and, more preferably, to a mammal. Preferably, the mammal referred to herein is a pet, such as a dog, cat, or horse, or a farming animal, such as a cow, sheep, goat or pig, or a laboratory animal, such as a rodent and, preferably, a mouse or rat. Preferably, the mammal or laboratory animal referred to herein is a monkey. More preferably, the mammal referred to herein is a human. It will be understood that the said subject shall suffer from cancer and, preferably, from hepatic cancer. The subject shall be in need of an immunotherapy. This encompasses subjects which are in need of immunotherapy due to apparent diseases or disorders that are susceptible to treatment by immunotherapy, such as liver cancer. Moreover, a subject in need of immunotherapy may also be a subject suspected to be susceptible or to benefit from administration of immunotherapy. Preferably, this includes a subject which may receive immunotherapy after successful therapy of a disease or disorder in prevention of reoccurrence of the disease or disorder or a subject which receives immunotherapy as a preventive measure.

[0066] In a first step of the method of assessing whether a subject suffering from hepatic cancer benefits from immunotherapy, the amount of platelets in a sample of a subject is determined. The phrase “determining the amount of platelets” as used herein, refers to the qualitative and quantitative determination of platelets, i.e. the phrase encompasses the determination of the presence or absence or the determination of the absolute or relative amount of platelets. The phrase further encompasses measuring the amount semi-quantitatively. The term “amount” as used herein refers to the absolute amount of platelets or the relative amount platelets, as well as any value or parameter, which correlates thereto or can be derived therefrom. Such values or parameters comprise intensity signal values from all specific physical or chemical properties obtained from the platelets or a detection molecule and / or detectable label. The values or parameters can be obtained by direct or indirect measurement. Direct measuring relates to measuring the amount of platelets present in the sample. Indirect measuring includes measuring of a signal obtained from a secondary component, i.e. a component not being the platelet itself. It is to be understood that values correlating to the aforementioned amounts or parameters can also be obtained and / or modified by all standard mathematical operations. Determining the amount in the method of the present invention may be carried out by any technique, which allows for detecting the presence or absence or the amount of platelets. For example, the amount of platelets may be determined by (i) manual counting using phase contrast microscopy, (ii) impedance analysis, (iii) optical light scatter / fluorescence analysis or (iv) immunoplatelet counting by flow cytometry. Manual methods may also be replaced by automated instrumentation. The skilled artisan is well aware of said different detection agents and / or techniques.

[0067] The term “sample” as used herein refers to any biological sample material that comprises or is suspected to comprise platelets. Preferably, the sample is a tissue, cell or fluid sample obtainable from the human body. More preferably, the sample is blood or a tissue cell or fluid sample obtainable from the liver, most preferably, the sample is a liver biopsy sample.

[0068] The term “reference” as used herein, relates to an amount of platelets which by comparison to the determined amount of platelets allows for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy. Thus, the reference in step b) in the method according to the present invention is, preferably, derived from a subject or group of subjects suffering from hepatic cancer and known to benefit from immunotherapy (responder). The reference may also be obtained from a subject or group of subjects suffering from hepatic cancer and known not to benefit from immunotherapy (non-responder).

[0069] Preferably, said reference is derived from a subject or group of subjects suffering from hepatic cancer and known to benefit from immunotherapy. Preferably, the amount of platelets being increased compared to the reference is indicative for a subject that does not benefit from immunotherapy, whereas an amount being identical or reduced is indicative for a subject that benefits from immunotherapy.

[0070] Preferably, said reference is derived from a subject or group of subjects suffering from hepatic cancer and known not to benefit from immunotherapy. In such a case, preferably, the amount of platelets being reduced compared to the reference is indicative for a subject that benefits from immunotherapy, whereas an amount being identical or increased is indicative for a subject that does not benefit from immunotherapy.

[0071] The present invention also refers to the use of the amount of platelets in a sample of a subject suffering from hepatic cancer for assessing whether said subject benefits from immunotherapy. The present invention further encompasses a device for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising: a) an analyzer for determining the amount of platelets in a sample of said subject; and b) an evaluation unit comprising a processor and a database with a stored references wherein the processor is adapted for carrying out a comparison between the determined amount of platelets and the stored references and generates the assessment by applying tangibly embedded rules for making that comparison.

[0072] The term “device” as used herein, refers to a system comprising the aforementioned units operatively linked to each other as to allow for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy, and evaluation thereof according to the method of the invention such that an assessment can be provided.

[0073] The term “analyzer” refers to an analyzing unit, typically, comprising at least one reaction zone having means suitable for determining an amount of cells, preferably platelets, in a sample. Said means comprise, preferably, biosensors, optical devices, single cell analyzing devices such as FACS analyzers, and the like. The reaction zone may either allow directly for sample application or it may be connected to a loading zone where the sample is applied. In the latter case, the sample can be actively or passively transported via the connection between the loading zone and the reaction zone to the reaction zone. Moreover, the reaction zone shall be also connected to a detector. The connection shall be such that the detector can detect the counted platelets. Suitable connections depend on the techniques used for determining the presence and / or amount of the platelets in a sample. The determined amount can be subsequently transmitted to the evaluation unit.

[0074] Said evaluation unit comprises a data processing element, such as a computer, with an implemented algorithm for determining the amount of platelets present in the sample. The processing unit as referred to in accordance with the method of the present invention, typically, comprises a Central Processing Unit (CPU) and / or one or more Graphics Processing Units (GPUs) and / or one or more Application Specific Integrated Circuits (ASICs) and / or one or more Tensor Processing Units (TPUs) and / or one or more field-programmable gate arrays (FPGAs) or the like. A data processing element may be a general purpose computer or a portable computing device, for example. It should also be understood that multiple computing devices may be used together, such as over a network or other methods of transferring data, for performing one or more steps of the methods disclosed herein. Exemplary computing devices include desktop computers, laptop computers, personal data assistants (“PDA”), cellular devices, smart or mobile devices, tablet computers, servers, and the like. In general, a data processing element comprises a processor capable of executing a plurality of instructions (such as a program of software). The evaluation unit, typically comprises or has access to a memory. A memory is a computer readable medium and may comprise a single storage device or multiple storage devices, located either locally with the computing device or accessible to the computing device across a network, for example. Computer-readable media may be any available media that can be accessed by the computing device and includes both volatile and non-volatile media. Further, computer readable-media may be one or both of removable and non-removable media. By way of example, and not limitation, computer-readable media may comprise computer storage media. Exemplary computer storage media includes, but is not limited to, RAM, ROM, EEPROM, flash memory or any other memory technology, CD-ROM, Digital Versatile Disk (DVD) or other optical disk storage, magnetic cassettes, magnetic tape, magnetic disk storage or other magnetic storage devices, or any other medium which can be used for storing a plurality of instructions capable of being accessed by the computing device and executed by the processor of the computing device. The evaluation unit may also comprise or has access to an output device. Exemplary output devices include fax machines, displays, printers, and files, for example. According to some embodiments of the present disclosure, a computing device may perform one or more steps of a method disclosed herein, and thereafter provide an output, via an output device, relating to a result, indication, ratio or other factor of the method.

[0075] Finally, the present invention also relates to a kit assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising means for determining the amount of platelets in a sample of said subject and means for making the said assessment.

[0076] The term “kit” as used herein refers to collection of the aforementioned components, typically, provided in separately or within a single container. The container also typically comprises instructions for carrying out the method of the present invention. These instructions may be in the form of a manual or may be provided by a computer program code, which is capable of carrying out or supports the determination of the biomarkers referred to in the methods of the present invention when implemented on a computer or a data processing device. The computer program code may be provided on a data storage medium or device such as an optical storage medium (e.g., a Compact Disc) or directly on a computer or data processing device or may be provided in a download format such as a link to an accessible server or cloud. Moreover, the kit may usually comprise standards for reference amounts of platelets for calibration purposes. The kit according to the present invention may also comprise further components, which are necessary for carrying out the method of the invention such as solvents, buffers, washing solutions and / or reagents required for detection. Further, it may comprise the device of the invention either in parts or in its entirety. The following are particular preferred embodiments of the present invention.

[0077] Embodiment 1 : A compound for use in improving immunotherapy wherein said compound is a platelet-inhibiting agent.

[0078] Embodiment 2: The compound for use of embodiment 1, wherein said platelet-inhibiting agent is an inhibitor of platelet glycoprotein lb (GPIb), an inhibitor of platelet glycoprotein VI (GPVI), an inhibitor of platelet glycoprotein Ilb / IIIa (GP2B / 3 A), an inhibitor of thrombin or an inhibitor of P2Yn, preferably, an inhibitor of platelet glycoprotein lb (GPIb), more preferably, an inhibitor of the alpha-thrombin binding site within the ectodomain of GPIb.

[0079] Embodiment 3 : The compound for use of embodiment 1 or 2, wherein said compound is selected from the group consisting of an antisense nucleic acid, CRISPR-Cas9 like gene editing construct, a small molecule, and a ribozyme.

[0080] Embodiment 4: The compound for use of embodiment 1 or 2, wherein said compound is an antibody or antibody-like binding agent.

[0081] Embodiment 5. The compound for use of embodiment 4, wherein said antibody or antibodylike binding agent specifically binds to the ectodomain of GPIba.

[0082] Embodiment 6: The compound for use of embodiment 5, wherein said antibody or antibodylike binding agent specifically binds to the alpha-thrombin binding site within the ectodomain of GPIba.

[0083] Embodiment 7: The compound for use of embodiment 6, wherein upon binding to the alphathrombin binding site within the ectodomain of GPIb, binding of alpha-thrombin to GPIb is reduced.

[0084] Embodiment 8: The compound for use of any one of embodiments 1 to 7, preferably embodiments 5 to 7, wherein said compound does not compete with von Willebrand factor (vWF), P- selectin, Mac-1, coagulation factor XI and / or coagulation factor XII for GPIb binding.

[0085] Embodiment 9: The compound for use of any one of embodiments 1 to 8, wherein said immunotherapy is immunotherapy of liver cancer. Embodiment 10: The compound for use of embodiment 9, wherein said liver cancer is hepatocellular carcinoma (HCC), preferably, unresectable HCC.

[0086] Embodiment 11 : The compound for use of any one of embodiments 1 to 10, wherein said immunotherapy comprises administering an immunotherapeutic agent selected from the group consisting of: an anti VEGF antibody, an anti-PD-Ll antibody, a combination of an anti VEGF antibody and an anti-PD-Ll antibody.

[0087] Embodiment 12: A pharmaceutical composition comprising the compound as specified in any one of embodiments 1 to 10 and an immunotherapeutic agent a specified in embodiment 11.

[0088] Embodiment 13: A method of assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising the steps of: a) determining the amount of platelets in a sample of said subject; b) comparing said amount to reference; and c) assessing whether a subject suffering from hepatic cancer benefits from immunotherapy.

[0089] Embodiment 14: The method of embodiment 13, wherein said reference is derived from a subject or group of subjects suffering from hepatic cancer and known to benefit from immunotherapy.

[0090] Embodiment 15: The method of embodiment 14, wherein an amount of platelets being increased compared to the reference is indicative for a subject that does not benefit from immunotherapy, whereas an amount being identical or reduced is indicative for a subject that benefits from immunotherapy.

[0091] Embodiment 16: The method of embodiment 13, wherein said reference is derived from a subject or group of subjects suffering from hepatic cancer and known not to benefit from immunotherapy.

[0092] Embodiment 17: The method of embodiment 16, an amount of platelets being reduced compared to the reference is indicative for a subject that benefits from immunotherapy, whereas an amount being identical or increased is indicative for a subject that does not benefit from immunotherapy.

[0093] Embodiment 18: Use of the amount of platelets in a sample of a subject suffering from hepatic cancer for assessing whether said subject benefits from immunotherapy. Embodiment 19: A device for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising: a) an analyzer for determining the amount of platelets in a sample of said subject; and b) an evaluation unit comprising a processor and a database with a stored references wherein the processor is adapted for carrying out a comparison between the determined amount of platelets and the stored references and generates the assessment by applying tangibly embedded rules for making that comparison.

[0094] Embodiment 20: A kit for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising means for determining the amount of platelets in a sample of said subject and means for making the said assessment.

[0095] All references cited throughout this specification are herewith incorporated by reference with respect to the specifically mentioned disclosure content as well as in their entireties.

[0096] FIGURES

[0097] Figure 1. Experimental design of anti-platelet immunotherapy in Western diet NASH- HCC model. C57BL6 / J wildtype mice were fed on a high-fat, high-fructose, high-cholesterol, ‘Western-style’ diet for 10 months to induce NASH-associated HCC. MRI scan was performed to screen for tumor-bearing animals, which were subsequently divided into 3 groups receiving different treatments, negative control (IgG isotype), immunotherapy (anti-VEGF + anti-PD- Ll), and combinatorial anti-platelet immunotherapy (anti-GPIba + anti-VEGF + anti-PD-Ll). The interventions were delivered by intravenous (I V.) injection in a biweekly fashion for 4 weeks, and a follow-up MRI scan was carried out to assess the response rate. The mice were then sacrificed for analysis of the liver microenvironment by flow cytometry and histology.

[0098] Figure 2. Efficacy of anti-platelet immunotherapy in NASH-HCC model. Pre-therapy and post-therapy MRI images showed that immunotherapy induces a 50% disease control, whereas the addition of anti-platelet therapy enhanced that to 75%. Anti-platelet therapy likely enhanced efficacy of immunotherapy in NASH-HCC.

[0099] Figure 3. Impact of GPIba inhibition on effector cells. Inhibition of GPIba enhanced conventional CD4 and CD8 cells effector phenotype. (A) FACS analysis of the intrahepatic CD45+leukocyte population indicated that the addition of anti-platelet therapy significantly increased the number intrahepatic conventional CD4 T cells, as compared to the immunotherapy alone treatment group. Both CD4 and CD8 T cells presented a stronger activation (PD1+) and effector-like (CD44+CD62L ) status. The CD4 TH1 to CD4 TH2 ratio was doubled, suggesting a higher inflammatory microenvironment. The CD4 Treg count was highly induced by immunotherapy treatment, but such increase was abrogated by the combination with anti-platelet therapy. The CD4 Tregto CD8 Teff ratio was therefore greatly reduced. (B) The production capacity of anti-cancer cytokines, including granzyme A, interferon-y, and TNF-a, was promoted by anti-platelet therapy in CD4 and CD8 T cells.

[0100] Figure 4. Heatmap analysis of the NASH-HCC immune landscape. The heatmap analysis summarizes the general pro-inflammatory effect in the liver induced by anti-platelet therapy. In the myeloid compartment, monocyte-derived macrophages presented a Ml -like phenotype, and the myeloid-derived dendritic cells exhibited a more activated status, both of which expressed higher level of cell surface MHC-II complex. The CD4 TEM cells, CD8 T cells, and innate-like lymphoid cells (NK, NKT, y5 T cells) were strongly induced, whereas the CD4 TH2 and CD4 Treg amount was decreased. The anti-cancer cytokine production capacity of effector-like cells across the CD4 T, CD8 T, NK, and NKT cell populations was enhanced.

[0101] Figure 5. Immunofluorescence imaging of livers of mice after immunotherapy. Immunofluorescence imaging (fluorescence images not shown) were quantitatively evaluated showing that immunotherapy induced platelet (CD42b+) infiltration into the liver, and platelet extravasation from the vasculature (CD31+) to the parenchyma. The addition of anti-platelet therapy did not reverse platelet infiltration, but prevented platelet extravasation.

[0102] Figure 6. Experimental design of anti-platelet immunotherapy in HTVi liver cancer models. Hydrodynamic tail vein injection (HTVi) models were adopted to further investigate the functional role of platelets in liver cancer immunotherapy. A combination of plasmids was delivered by HTVi to transfect the hepatocytes in wildtype C57BL6 / J mice, and induce specific driver-gene mutations. The combination of KrasG12D mutation and Tp53 knockout induced immune-exclusion combined hepatocellular carcinoma and intrahepatic cholangiocarcinoma (cHCC-iCC) tumors. The combination of Ctnnbl mutation and c-myc overexpression induced immunotherapy-resistant HCC tumors. Upon tumor onset, the mice were divided into 4 groups, which were treated with IgG isotype, anti-GPIba, anti-VEGF + anti-PD-Ll, and GPIba + anti- VEGF + anti-PD-Ll. The interventions were administered by I.V. injections every 3 - 4 days, and a total of 3 injections was performed. The mice were then sacrificed for analysis of the liver microenvironment.

[0103] Figure 7. Efficacy of immunotherapy with GPIba antagonists in HTVi-induced liver cancer. In combination with immunotherapy, anti-platelet therapy reduced the tumor burden in both models, indicated by the decreased liver-to-body weight ratio and liver surface tumor nodules.

[0104] Figure 8. Experimental design of anti-platelet immunotherapy in hIL4r / GPIba HTVi HCC / iCC model. To identify the functional domain of the platelet GPIba protein, we adopted the hIL4r / GPIba transgenic mouse model, which the platelet GPIba ectodomain was replaced by a human IL4 receptor, resulting in a partial loss-of- function of the extracellular signaling pathway. HTVi (KrasG12D / sgTp53) was carried out to induce liver cancer, and immunotherapy (anti-VEGF + anti-PD-Ll) was introduced after tumor onset. The intervention was administered by I.V. injections every 3 - 4 days, and a total of 3 injections was performed. The mice were then sacrificed for analysis of the liver microenvironment.

[0105] Figure 9. Efficacy of immunotherapy in KrasG12D / sgTp53 HTVi-induced HCC / iCC. Loss of the platelet GPIba ectodomain promoted immunotherapy efficacy, which significantly reduced the liver-to-body weight ratio and liver surface tumor nodules. The loss of the platelet GPIba ectodomain did not inhibit tumor development without immunotherapy.

[0106] Figure 10. FACS analysis of intrahepatic CD45+leukocytes. FACS analysis of the intrahe- patic CD45+leukocyte population indicated that the loss of the platelet GPIba ectodomain significantly increased the number intrahepatic conventional CD4 and CD8 T cells. The CD4 Tregto CD8 Teff ratio was therefore greatly reduced. The production capacity of anti-cancer cytokines was enhanced.

[0107] Fig. 11. Immunofluorescence imaging after immunotherapy. Immunofluorescence imaging (fluorescence images not shown) were quantitatively evaluated showing that the loss of the platelet GPIba under immunotherapy significantly enhanced CD8 T cell accumulation in the peritumoral region, which was also accompanied by an increasing trend of peritumoral CD4 T cells, and intratumoral CD4 and CD8 T cells.

[0108] Figure 12. FACS analysis of circulating platelets. FACS analysis of the circulating platelets showed that more TGFb was retained on platelet surface, with the loss of GPIba ectodomain.

[0109] Figure 13. Experimental design of combination of immunotherapy and alternative antiplatelet therapies in HTVi model. To assess whether the immunotherapy-enhancing effect of anti-platelet therapy was attributed solely to the GPIba signaling pathway, or the general platelet activation or aggregation, we treated HTVi (KrasG12D / sgTp53) mice with immunotherapy combined with an array of different types of anti-platelet therapy. Figure 14. Impact of platelet inhibition on immunotherapy response. Treatments inhibiting platelet activation, including anti-GPIba, anti-GPVI, and ticagrelor, significantly reduced number of liver surface tumor nodules, when combined with immunotherapy, which was not observed when it was combined with anti-aggregation (anti-GPIIbllla) or anti-coagulation (hirudin) agents. However, all types of anti-platelet therapy seemingly caused a decreased trend of liver-to-body weight ratio, and an increased trend of circulating leukocytes in the periphery.

[0110] Figure 15. Histological analysis after anti-platelet therapy. Histological analysis showed that the combination of anti-platelet therapy with immunotherapy promoted both extra-tumoral and peri-tumoral CD 8 T cell accumulation, accompanied also by an increasing trend of intra- tumoral CD8 T cells.

[0111] Figure 16. Experimental design of anti-platelet immunotherapy in hIL4r / GPIba HTVi- HCC / iCC model. To determine if the platelet-mediated immunosuppression was caused by secretion of platelet alpha granule, we adopted the Nbeal2- / - transgenic mouse model. These mice produce activatable platelets, but without alpha granules. HTVi (KrasG12D / sgTp53) was carried out to induce liver cancer, and immunotherapy (anti-VEGF + anti-PD-Ll) was introduced after tumor onset. The intervention was administered by I V. injections every 3 - 4 days, and a total of 3 injections was performed. The mice were then sacrificed for analysis of the liver microenvironment.

[0112] Figure 17. Impact of anti-platelet therapy on immunotherapy outcome. The lack of platelet alpha granules caused a likely reduction of liver-to-body weight ratio and liver surface tumor nodules, under immunotherapy treatment.

[0113] Figure 18. FACS analysis of the intrahepatic CD45+leukocyte population. Results of FACS analysis indicated that the lack of platelet alpha granules depleted CD4 Tregcells and increased number of CD8 central memory T cells and interferon-y production in CD8 T cells.

[0114] Figure 19. Impact of anti-platelet therapy on intrahepatic environment. Histological analysis showed that immunotherapy in Neal2- / - mice promoted the amount of both CD4 and CD8 T cells in non-tumoral tissue. The lack of platelet alpha granules increases intrahepatic CD4 and CD8 T cells.

[0115] Figure 20. In silico correlational analysis between platelet markers and estimated immune cell infiltration. (A) In silico transcriptomic analysis of the TCGA LIHC dataset revealed that the expression of platelet marker ITGB3 negatively correlated with the number of anti-tumor- igenic cells, such as CD8 T cells, CD4 TCM cells, CD4 TH1 cells, NKT cells, and pDCs, whereas it positively correlated with pro-tumorigenic cells, like M2 macrophages and CD4 Tregcells. (B) In silico transcriptomic analysis of the TCGA LIHC dataset revealed that the expression of platelet markers ITGB3 and GP1BA positively correlated with the expression of molecular signatures related to an immunosuppressive and immune-exhaustive microenvironment.

[0116] Figure 21. Pharmaceutical inhibition of platelet GPIba amplified the efficacy of immunotherapy in cHCC-ICC murine model. (A) Representative images of the liver, H&E, and GFP staining, from each treatment group. (B) Quantification of liver tumor nodules. (IgG Ctrl n=20, Anti-GPIba n=8, Immunotherapy n=24, Anti-GPIba+Immunotherapy n=21). (C) Flow cytometry analysis of systemic circulating platelets GPIba geometric mean fluorescence intensity (MFI). (IgG Ctrl n=6, Anti-GPIba n=6, Immunotherapy n=6, Anti-GPIba+Immunotherapy n=6). Data presented as median ± quartiles. One-way ANOVA and Fisher’s LSD test were used to evaluate statistical significance. *P<0.05, **P<0.01, ***P<0.001, and ****p < 0.0001.

[0117] Figure 22. Pharmaceutical inhibition of platelet GPIba with immunotherapy reshaped the cHCC-ICC immune microenvironment. (A) Representative images of the H&E, CD4, CD8, TIM3, FOXP3, CD42b IHC staining of mouse liver from each treatment group. Tumor area is denoted as “T”. (B - K) Quantification of the immune cell markers in the extra-tumoral liver tissue and the intra-tumoral regions (T). (IgG Ctrl n=6, Anti-GPIba n=6, Immunotherapy n=6, Anti-GPIba+Immunotherapy n=6). Data presented as median ± quartiles. One-way ANOVA and Fisher’s LSD test were used to evaluate statistical significance. *P<0.05, **P<0.01, ***P<0.001, and ****P < 0.0001.

[0118] Figure 23. Pharmaceutical Inhibition of Platelet GPIba reverted therapeutic resistance in CtnnblT41A / Myc HCC. (A) Representative images of the liver, H&E, CD4, and CD8 IHC staining from each treatment group. Tumor area is denoted as “T”. (B) Quantification of liver tumor nodules per area. (IgG Ctrl n=3, Immunotherapy n=4, Anti-GPIba+Immunotherapy n=6). (C - F) Quantification of CD4 and CD8 T cells with in the extra-tumoral liver tissue and the intra-tumoral regions. (IgG Ctrl n=4, Immunotherapy n=5, Anti-GPIba+Immunotherapy n=6). Data presented as median ± quartiles. One-way ANOVA and Fisher’s LSD test were used to evaluate statistical significance. *P<0.05, **P<0.01, ***P<0.001, and ****p < 0.0001.

[0119] Figure 24. Pharmaceutical inhibition of Platelet GPIba sensitized MASH-HCC to immunotherapy and eliminated MASH-associated PD1+ auto-aggressive CD8 T cells. (A) Experimental design. Mice were fed with the western diet regimen for 15 months to induce MASH-HCC in WT male C57BL / 6J mice. Therapeutic interventions were administered I.V. in a biweekly fashion for 2 months. (IgG Ctrl n=l l, Immunotherapy n=l l, Anti-GPIba+Immunotherapy n=10). (B) Representative images of the liver, H&E, CD4, and CD8 IHC staining from each treatment group. (C) Quantification of liver tumor nodules quantity from mice in each treatment group (IgG Ctrl n=l l, Immunotherapy n=l l, Anti-GPIba+Immunotherapy n=10). (D) Quantification of nodules size of all liver tumors in each treatment group (IgG Ctrl n=12, Immunotherapy n=13, Anti-GPIba+Immunotherapy n=10). (E) Quantification of CD4 T cells within the extra-tumoral liver tissue (IgG Ctrl n=l l, Immunotherapy n=10, Anti- GPIba+Immunotherapy n=9). (F) Quantification of CD4 T cells within the intra -tumoral regions (IgG Ctrl n=5, Immunotherapy n=6, Anti-GPIba+Immunotherapy n=2). (G) Quantification of CD8 T cells within the extra-tumoral liver tissue (IgG Ctrl n=6, Immunotherapy n=8, Anti-GPIba+Immunotherapy n=6). (H) Quantification of CD8 T cells within the intra -tumoral regions (IgG Ctrl n=4, Immunotherapy n=4, Anti-GPIba+Immunotherapy n=3). (I) Quantification of PD1+ CD8 T cells within the extra-tumoral liver tissue (IgG Ctrl n=6, Immunotherapy n=8, Anti-GPIba+Immunotherapy n=6). (J) Quantification of PD1+ CD8 T cells within the intra -tumoral regions (IgG Ctrl n=4, Immunotherapy n=5, Anti-GPIba+Immunotherapy n=3). Data presented as median ± quartiles. One-way ANOVA and Fisher’s LSD test were used to evaluate statistical significance. *P<0.05.

[0120] EXAMPLES

[0121] The Examples merely illustrate the invention. They shall by no means be interpreted as limiting the scope of the invention.

[0122] Example 1: Mice, diets, and treatments

[0123] Male mice were housed at the German Cancer Research Center (DKFZ) (constant temperature of 20-24 °C and 45-65% humidity with a 12-h light-dark cycle). Mice were maintained under specific pathogen-free conditions and experiments were performed in accordance with German law and the governmental bodies, and with approval from the Regierung sprasidium Karlsruhe (G141-19, G275-18).

[0124] Knockout mice for Nbeal2 and transgenic mice, lacking functional GPIba, hIL4ra / GPIba-Tg, both on the background of C57BL / 6J were kindly provided by B. Nieswandt (University Hospital and Rudolf Virchow Center, University of Wurzburg, Wurzburg, Germany).

[0125] For western diet experiments, 8-week-old male C57BL / 6J mice were fed on a high-fat, high- fructose, high-cholesterol, “western diet” (Research Diets; D16022301i). For hydrodynamic tail vein injection (HTVi) experiments, plasmids were dissolved in 0.9% NaCl solution to a final volume of 10% of mouse body weight, which was subsequently delivered by intravenous injection to the lateral tail vein within 10 seconds to male C57BL / 6J mice of at least 8 weeks of age. The KrasG12D / sgTp53 model received 5pg KrasG12Dplasmid, lOpg sgTp53 plasmid, and Ipg sleeping beauty transposase (SB13), per mouse. The CtnnblT41A / c- myc model received lOpg CtnnblT41Aplasmid, lOpg c-myc plasmid, and 5pg sleeping beauty transposase (SB 13), per mouse.

[0126] For interventional studies, mice were treated bi-weekly by intravenous injection of 2 mg / kg anti-GPIba (B. Nieswandt, pop / B), 3 mg / kg anti-PD-Ll (Bioxcell, 10F.9G2), 5 mg / kg anti- VEGFa (Genentech, B20-4.1.1). Mice in the HTVi experiments were treated 14 days post- HTVi with 3 injections across 1.5 weeks. Mice in the WD experiments were treated 10 months post-diet with 8 injections across 4 weeks.

[0127] Example 2: Intrahepatic leukocyte isolation from murine models

[0128] Mouse livers were perfused once with Hank's Balanced Salt Solution (HBSS), mechanically digested, and then chemically digested with collagen IV (60 U final concentration (f. c.)) and DNase I (25 pg / ml fc.) for up to 45 min at 37 °C. Single-cell suspension was obtained by filtering the digested liver through a 100-pm mesh. Next, samples underwent a two-step Percoll gradient (40% / 80% Percoll / HBSS) and centrifugation for 25 min at 1,800g and 4 °C. Enriched leukocytes were then collected and washed. For re-stimulation, cells were incubated for 2 h at 37 °C under 5% CO2 with 1 :500 Biolegend's Cell Activation Cocktail (with brefeldin A) (423304) and 1 : 1,000 Monensin Solution (420701). Live / dead discrimination was done using ZombieDyeNIR according to the manufacturer’s instructions with subsequent staining of titrated antibodies.

[0129] Example 3: Flow cytometry analysis

[0130] For flow cytometry analysis, antibodies against CD45 (30-F11, 103137), Ly6G (1A8, 127639), I-A / I-E (M5 / 114.15.2, 107625), CD206 (C068C2, 141732), CDl lb (MI / 70, 101242), F4 / 80 (BM8, 123114), CDl lc (N418, 117343), Ly6C (HK1.4, 128006), XCR1 (ZET, 148204), CD4 (RM4-5, 100536), CD8 (53-6.7, 100734), PD1 (29F.1A12, 135220), CD44 (IMT, 103012), CD62L (MEL-14, 104448), NK1.1 (PK136, 108745), CD19 (6D5, 115520), TCR y / 5 (UC7- 13D5, 107504), CD69 (H1.2F3, 104508), IFN-y (XMG1.2, 505810), TNF-a (MP6-XT22, 506346), TGF-pi (TW7-16B4, 141409), granzyme A (3G8.5, 149703), granzyme B (QA16A02, 372206), CD25 (PC61, 102006), FOXP3 (MF-14, 126407), Tbet (4B10, 644832), were purchased from BioLegend. Antibodies against TCR P (H57-597, 742483), IL17A (TC11- 18H10, 563354), RORyt (Q31-378, 562607), GATA3 (L50-823, 560406), were purchased from BD Biosciences. Antibodies against Ki-67 (Sol Al 5, 48-5698-82) was purchased from Invitro- gen. Antibody against Ki-67 was diluted 1 : 100 and all other antibodies were diluted 1 :200 in MACS buffer for staining.

[0131] Samples for flow cytometry were fixed using eBioscience IC fixation (00-8222-49) or FOXP3 Fix / Perm kit (00-5523-00) according to the manufacturer’s instructions. Intracellular staining was performed in eBioscience Perm buffer (00-8333-56). Cells were analyzed using BD FACSFortessa and data were analyzed using FlowJo (vlO.9.1).

[0132] Example 4: Immunohistochemistry, scanning and automated analysis

[0133] Mouse liver samples were fixed in ROTI Histofix solution (Carl Rothl, P087.1) and paraffin- embedded at the DKFZ, Department of Chronic Inflammation and Cancer (Heidelberg) as described. Briefly, 2 pm sections from FFPE and cryo-preserved tissues were prepared and stained with Hematoxylin / Eosin or IHC antibodies. Incubation in Ventana buffer and staining was performed on a NEXES immunohistochemistry robot (Ventana Instruments) using an IVIEW DAB Detection Kit (Ventana) or on a Bond MAX (Leica). Slides were scanned with a Nano Zoomer (Hamamatsu, Japan).

[0134] Antibodies that were used included anti-CD4, rat, 1 : 1000 (Thermo fisher, clone 4SM95, 14- 9766-82); anti-CD8a, rabbit, 1 :400 (Cell Signaling, clone D4W2Z, 60168); anti- CD31, rat, 1 :40 (Dianova, clone SZ31, DIA-310-BA); anti-CD42b, rabbit, 1 :200 (abeam, clone SP219, ab240268).

[0135] For quantification of stains, slides were scanned using a SCN400 slide scanner (Leica) and analyzed using Aperio ImageScope (Leica, version 12.4.0.05043), ImageJ (version 1.53q), or QuPath (version 0.4.3).

[0136] Example 5: Mouse blood profiling

[0137] Blood was collected from mice via the submandibular vein or the inferior vena cava into EDTA coated tubes. The blood cell count was measured with Element HT5 (Scil) according to the manufacturer’s instructions. Example 6: Immunohistochemistry, scanning and automated analysis

[0138] Mouse liver samples were fixed in ROTI Histofix solution (Carl Rothl, P087.1) for 48 hours at room temperature, and preserved in 70% ethanol at 4 °C. Specimens were paraffin-embedded at the DKFZ, Department of Chronic Inflammation and Cancer (Heidelberg). 2 pm sections from FFPE and cryo-preserved tissues were prepared and stained with Hematoxylin / Eosin or IHC antibodies (Table 1). Incubation in Ventana buffer and staining was performed on a NEXES immunohistochemistry robot (Ventana Instruments) using an IVIEW DAB Detection Kit (Ventana) or on a Bond MAX (Leica). Slides were scanned with a Nano Zoomer (Hamamatsu). For quantification of staining, slides were scanned using a SCN400 slide scanner (Leica) and analyzed using Aperio ImageScope (Leica, version 12.4.0.05043), Imaged (version 1.53q), or QuPath (version 0.4.3).

[0139] Table 1. Immunohistochemistry Antibodies

[0140] In summary, the data has demonstrated that the combinatorial therapeutic approach combining anti-GPIba with immunotherapy has achieved significantly better results than immunotherapy alone treatment in all non-responder models (e.g. Fig. 21, 23 or 24). In particular, the Kras model (Fig. 21), the Ctnnbl model (Fig. 23) and the wester-diet-based MASH-HCC model (Fig. 24), which are recognized as non-responding models showed a significantly improved response to immunotherapy after combining anti-GPIba with immunotherapy, as reflected by the reduced tumor burden (e.g. Fig. 21). CITED LITERATURE

[0141] D.S. Mandlik, S.K. Mandlik and H.B. Choudhary, “Immunotherapy for hepatocellular carcinoma: current status and future perspectives”, World J Gastroenterol. 2023 Feb 14; 29(6): 1054- 1075

[0142] J.M. Llovet et al., „Hepatocellular carcinoma“, Nat Rev Dis Primers 7, 6 (2021)

[0143] J.W. Eikelboom et al., “Antiplatelet drugs: Antithrombotic Therapy and Prevention of Thrombosis”, Chest. 2012 Feb; 141(2 Suppl):e89S-el l9S

[0144] C. Kole et al., “Immunotherapy for hepatocellular carcinoma: a 2021 update”, Cancers (Basel) 2020 Oct; 12(10): 2859

[0145] J. Li et al., “Immunotherapy of hepatocellular carcinoma: recent progress and new strategy”, Front. Immunol., 10 May 2023, vol. 14

[0146] F. Foerster et al., “Emerging immunotherapy for hepatocellular carcinoma: a guide for hepatol- ogists”, Hepatology 2022 Jun; 75(6): 1604-1626

[0147] Natasa Pavlovic, Bhavna Rani, Par Gerwins and Femke Heindry, “Platelets as Key Factors in Hepatocellular Carcinoma”, Cancers 2019, 11, 1022

[0148] G. Jourdi et al., “Current and novel antiplatelet therapies for the treatment of cardiovascular diseases”, Int J Mol Sci. 2021 Dec; 22(23): 13079

[0149] N. Cauwenberghs et al., “Antithrombotic effect of platelet glycoprotein Ib-blocking monoclonal antibody Fab fragments in nonhuman primates”, Arteriosclerosis, Thrombosis and Vascular Biology 2000; 20: 1347-1353

[0150] P. Billiald et al., “Targeting platelet GPVI with glenzocimab: a novel mechanism for inhibitions”, Blood Adv 2023, 7 (7): 1258-1268

Claims

CLAIMS1. A compound for use in improving immunotherapy wherein said compound is a platelet inhibiting agent.

2. The compound for use of claim 1, wherein said platelet inhibiting agent is an inhibitor of platelet glycoprotein lb (GPIb), an inhibitor of platelet glycoprotein VI (GPVI), an inhibitor of platelet glycoprotein Ilb / IIIa (GP2B / 3 A), an inhibitor of thrombin or an inhibitor of P2Y12, preferably, an inhibitor of platelet glycoprotein lb (GPIb), more preferably, an inhibitor of the alpha-thrombin binding site within the ectodomain of GPIb.

3. The compound for use of claim 1 or 2, wherein said compound is selected from the group consisting of an antisense nucleic acid, CRISPR-Cas9 like gene editing construct, a small molecule, and a ribozyme.

4. The compound for use of claim 1 or 2, wherein said compound is an antibody or antibodylike binding agent, preferably, an antibody or antibody-like binding agent specifically binds to the ectodomain of GPIb, more preferably, an antibody or antibody-like binding agent specifically binds to the alpha-thrombin binding site within the ectodomain of GPIb.

5. The compound for use of any of of claims 1 to 4, wherein said compound does not compete with von Willebrandt factor (vWF), P-selectin, Mac-1, coagulation factor XI and / or coagulation factor XII for GPIb binding.

6. The compound for use of any one of claims 1 to 5, wherein said immunotherapy is immunotherapy of liver cancer.

7. The compound for use of claim 6, wherein said liver cancer is hepatocellular carcinoma (HCC), preferably, unresectable HCC.

8. The compound for use of any one of claims 1 to 7, wherein said immunotherapy comprises administering an immunotherapeutic agent selected from the group consisting of an anti VEGF antibody, an anti-PD-Ll antibody, a combination of an anti VEGF antibody and an anti-PD-Ll antibody.

9. A pharmaceutical composition comprising the compound as specified in any one of claims 1 to 7 and an immunotherapeutic agent as specified in claim 8.

10. A method of assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising the steps of: a) determining the amount of platelets in a sample of said subject; b) comparing said amount to reference; and c) assessing whether a subject suffering from hepatic cancer benefits from immunotherapy.

11. The method of claim 10, wherein said reference is derived from a subject or group of subjects suffering from hepatic cancer and known to benefit from immunotherapy and wherein an amount of platelets being increased compared to the reference is indicative for a subject that does not benefit from immunotherapy, whereas an amount being identical or reduced is indicative for a subject that benefits from immunotherapy.

12. The method of claim 10, wherein said reference is derived from a subject or group of subjects suffering from hepatic cancer and known not to benefit from immunotherapy and wherein an amount of platelets being reduced compared to the reference is indicative for a subject that benefits from immunotherapy, whereas an amount being identical or increased is indicative for a subject that does not benefit from immunotherapy.

13. Use of the amount of platelets in a sample obtained from a subject suffering from hepatic cancer for assessing whether said subject benefits from immunotherapy.

14. A device for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising: a) an analyzer for determining the amount of platelets in a sample of said subject; and b) an evaluation unit comprising a processor and a database with a stored references wherein the processor is adapted for carrying out a comparison between the determined amount of platelets and the stored references and generates the assessment by applying tangibly embedded rules for making that comparison.

15. A kit for assessing whether a subject suffering from hepatic cancer benefits from immunotherapy comprising means for determining the amount of platelets in a sample of said subject and means for making the said assessment.

Images