Trail receptor binding molecules

Abstract

The invention relates to a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor (TRAIL-R) binding molecule comprising an extracellular domain of TRAIL, or a TRAIL-R binding fragment thereof, and an extracellular domain of NKp46, or a fragment thereof. The TRAIL-R binding molecule can be used to induce death receptor and FADD mediated apoptosis in cancer cells and activate NK and / or T cells to target cancer cells. The invention further relates to a chimeric antigen receptor comprising a TRAIL-R binding molecules.

Description

[0001] TRAIL RECEPTOR BINDING MOLECULES

[0002] TECHNICAL FIELD

[0003] The present invention generally relates to TRAIL receptor binding molecules, and to uses thereof.

[0004] BACKGROUND

[0005] Natural killer (NK) cells belong to the innate immune system and are important effector cells for our defense against viral infections and malignancies. NK cells use germline-encoded receptors that are capable of recognizing proteins or structures on aberrant cells that differentiate them from healthy cells. An integration of signals from activating receptors and inhibitory receptors ultimately decides the action of the NK cell, either to kill or leave the encountered cell unharmed (see Fig. 5). Many of the inhibitory receptors, such as the killer immunoglobulin receptors (KIRs) and NKG2A / cluster of differentiation 94 (CD94), recognize major histocompatibility complex (MHC) class l-molecules, expressed by all nucleated cells, forging a safety system to protect healthy cells. The natural cytotoxicity receptors (NCRs) together with NKG2D are considered the major activating receptors that, when triggered, provide activating signals in the NK cell. NKG2D recognizes stress-induced ligands, such as MHC class I polypeptide-related sequence A / B (MICA / B) and UL 16 binding proteins (ULBPs). Several viral ligands have been defined for the NCRs but cellular ligands were long a mystery before characterization of B7-H6 and its binding to NKp30 as well as the binding to heparan sulphate sequences by all three NCRs, i.e., NKp30, NKp44 and NKp46. Antibody-mediated blockade of NKp46 has shown that NKp46 plays a key role in NK cell recognition of cancer cells of multiple different histotypes. Several NKp46 ligands have been proposed in recent years, including vimentin, complement factor P, Siglec 6-7, CD4 and ER-stress-induced calreticulin. However, it remains unclear to what extent these ligands account for NKp46-dependent antitumor immune responses that have been reported across cancer cell lines.

[0006] NK cell activation leads to release of cytotoxic granules that contain perforin and different granzymes that contribute to apoptosis induction or lysis of the target cells. NK cells can also kill target cells by the use of death ligands. Death ligands are expressed on NK cells and through interaction with their cognate receptors on the target cell, they can induce apoptosis via the extrinsic apoptosis pathway. These include the Fas-ligand and tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL), which activates CD95 / Fas and the TRAIL receptors (TRAIL-R1 / death receptor 4 (DR4) and TRAIL-R2 / DR5), respectively. By activation of the death receptors, the death inducing signalling complex is formed, comprising activated death receptors, the Fas-associated death domain (FADD) adaptor protein and procaspase 8, which leads to caspase activation and ultimately to cell death. TRAIL-R3 and RAIL-R4 also bind TRAIL but lack complete intracellular signaling domains and are considered decoy receptors that protect against TRAIL-mediated cytotoxicity.

[0007] Dulanermin is a recombinant soluble human TRAIL that activates apoptotic pathways in cancer cells by binding to TRAIL-R1 and TRAIL-R2. Dulanermin has been shown to enhance progression-free survival in patients with non-small-cell lung cancer (NSCLC). However, Dulanermin has in other studies been shown to have limited efficacy, potentially due to insufficient binding to TRAIL-R1 / R2.

[0008] WO 2016 / 207278 discloses multispecific NK-engage proteins that bind and specifically redirect NK cells to lyse target cells of interest without non-specific activation of NK cells in absence of target cells. The multispecific NK-engage proteins comprise a first antigen binding domain binding to human NKp46, a second antigen-binding domain binding to an antigen expressed by a tumor cell or an infectious cell and a CD16A binding polypeptide.

[0009] CN109970869 discloses a chimeric receptor ligand targeting a TRAIL receptor. The chimeric receptor ligand comprises i) a death receptor-binding domain, ii) an intracellular second conduction domain, preferably DAP12, ill) T2A, iv) an extracellular signal peptide, preferably CD8a signal peptide, and v) a intracellular first conduction domain, preferably an intracellular domain of NKp44.

[0010] There is still a need for new molecules useful in treatment of cancers.

[0011] SUMMARY

[0012] It is a general objective to provide a multifunctional molecule useful in cancer treatment.

[0013] This and other objectives are met by embodiments as disclosed herein.

[0014] The present invention is defined in the independent claims. Further embodiments of the present invention are defined in the dependent claims.

[0015] An aspect of the invention relates to a tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor (TRAIL-R) binding molecule comprising an extracellular domain of TRAIL, or a TRAIL-R binding fragment thereof, and an extracellular domain of NKp46, or a fragment thereof. Another aspect of the invention relates to a chimeric antigen receptor (CAR) comprising an antigen recognition domain comprising a TRAIL-R binding molecule according to above, a transmembrane domain and an intracellular domain.

[0016] Related aspects define a nucleic acid molecule encoding a TRAIL-R binding molecule or a CAR according to above, an expression vector comprising a promoter and a nucleic acid molecule according to above operatively controlled by the promoter and a host cell comprising an expression vector according to above, wherein the promoter is constitutively active or inducible active in the host cell.

[0017] Other aspects of the invention also relate to a NK or T cell comprising a CAR according to above or a nucleotide sequence encoding a CAR according to above, and a TRAIL-R binding molecule according to above or a NK or T cell according to above for use as a medicament and for use in treatment of cancer, or in the case of the TRAIL-R binding molecule according to the above for use in treatment of a viral infection.

[0018] The TRAIL-R binding molecules of the invention are bifunctional molecules comprising two different domains capable of interacting with TRAIL receptors. The TRAIL-R binding molecules can thereby interact with TRAIL receptors on cancer cells to induce death receptor and FADD mediated apoptosis and, in a trifunctional setting, also activate NK and T cells to target the cancer cells.

[0019] BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The embodiments, together with further objects and advantages thereof, may best be understood by making reference to the following description taken together with the accompanying drawings, in which:

[0021] Fig. 1. NKp46-dependent CRISPR screen identifies death receptors but no other extrinsic pathwayproteins. (1A) Cytotoxicity assay with WT K562 cells and IL-2 / IL-15-stimulated NK cells at E:T ratio 1 :1 (n=7). (1 B) Cytotoxicity assay with K526 tKO and polyclonally activated NK cells at E:T ratio 8:1 (n=7). (1 C) Genome-wide CRISPR screen design overview. (1 D) Genome-wide CRISPR screen results from one replicate using K562 tKO cell line. (1 E) Genome-wide CRISPR screen results from one replicate using K562 tKO cell line, including blockade against NKG2D receptor. (1 F) Scheme displaying screen rankings of selected genes from tKO and tKO / block screen. Error bars represent SEM and one-way ANOVA analysis with Sidak’s multiple comparisons test was used for statistics. Fig. 2. TRAIL-receptor depletion but not FADD depletion reduced NK susceptibility. (2A), (2B) and (2C) Cytotoxicity assays with seven days IL-2 / IL-15-stimulated NK cells at E:T ratio 1 :1 , combined with blocking antibodies towards NK cell receptors, against B7H6ne9 K562 cells (2A), B7H6ne9FADDne9 K562 cells clone P1 B2 (dotted) (2B), and B7H6ne9FADDne9 K562 cells clone P1 C10 (striped) (2C), (n=5). (2D), (2E) and (2F) Functional assays with three days IL-2 / IL-15-stimulated NK cells, combined with blocking antibodies towards NK cell receptors, against B7H6ne9 K562 cells and B7H6ne9TRAIL-R1 / R2ne9 K562 cells clone P1 C11 ; (2D) Cytotoxicity assay at E:T ration 1 :1 (n=6), (2E) Degranulation assay (n=11). (2F) Intracellular IFNy production assay (n=10). (2G) Degranulation assay with polyclonal TRAIL-R1 / R2ne9 PC3 cells with five days IL-2 / IL-15-stimulated NK cells (n=6). Error bars represent SEM and one-way ANOVA analysis with Sidak’s multiple comparisons test was used for statistic testing of (2D), (2E), (2F) and (2G).

[0022] Fig. 3. (3A) Degranulation assay using three days IL-2 / IL-15-stimulated NK cells, combined with blocking antibodies towards NK cell receptors, against B7-H6ne9 K562 cells and B7-H6ne9TRAIL-R1 / R2ne9 K562 cells, clone P1 F6. (3B) Functional screen of various cell lines using cytotoxicity (E:T ratios 1 :1 except 3:1 for HL60 and 4:1 for KG1) and degranulation assays. (3C) TRAIL-R1 / R2 staining of promising cell lines. (3D) TRAIL-R1 / R2 staining of PC3 TRAIL-R1 / R2 dKO cells.

[0023] Fig. 4. TRAIL|OWNK cells are less NKp46-dependent compared to TRAILS. (4A) TRAIL staining of resting (solid) and IL-2 / IL-15 stimulated (striped) NK cells. (4B) and (40) Cytotoxicity assays with TRAILS IL- 2 / IL-15-stimulated NK cells at E:T ratio 1 :1 and TRAIL|OWresting NK cells at E:T ratio 4:1 , combined with blocking antibodies towards indicated NK cell receptors, towards B7H6ne9 K562 cells (4B) or B7H6ne9TRAIL-R1ne9TRAIL-R2ne9 K562 cells, clone P1 C11 (4C) (n=7). (4D) NKp46 staining of WT and NKp46-overexpressing NK92. (4E) and (4F) Cytotoxicity assays with WT-NK92 (light grey) or NKp46- NK92 (dark grey) against B7-H6ne9PVRne9Nectin-2ne9 tKO K562 cells at indicated E:T ratios (4E) and with addition of indicated blocking antibodies at E:T ratio 5:1 (4F) (n=1). (4G) Luminescence measurement using displayed NanoBiT constructs. (4H) TRAIL staining of TRAIL-overexpressing and TRAIL+NKp46- overexpressing HEK293 cells with or without NKp46 blockade. (4I) TRAIL-R1-Fc binding of WT and NKp46-overexpressing HEK293 cells. (4J) TRAIL-R1-Fc binding of TRAIL-overexpressing and TRAIL+NKp46-overexpressing HEK293 with or without NKp46 blockade. Error bars represent SEM and one-way ANOVA analysis with Sidak’s multiple comparisons test was used for statistics.

[0024] Fig. 5. A schematic illustration of an integration of signals from activating receptors and inhibitory receptors ultimately decides the action of the NK cell, either to kill or leave the encountered cell unharmed. Fig. 6. Illustrates NKp46 and TRAIL interaction with TRAIL-R in target cells triggering NK cell degranulation and release of granzymes and perforins.

[0025] Figs. 7 to 9. Illustrations of TRAIL-R binding molecules according to various embodiments.

[0026] Fig. 10. Illustration of activation of an NK cell with a TRAIL-R binding molecule according to an embodiment.

[0027] Fig. 11. Illustration of a CAR molecule comprising a TRAIL-R binding molecule according to an embodiment.

[0028] Fig. 12. (12a) Degranulation assay with WT-PC3 cells combined with blocking antibodies towards indicated NK cell receptors (n=6). (12b) Cytotoxicity assay with WT-PC3 cells combined with blocking antibodies towards indicated NK cell receptors (n=5). (12c) Degranulation assay with DR4 / 5 knock-out PC3 cells combined with blocking antibodies towards indicated NK cell receptors (n=6). (12d) Degranulation assay with WT-M14 cells combined with blocking antibodies towards indicated NK cell receptors (n=6). (12e) Cytotoxicity assay with WT-M14 cells combined with blocking antibodies towards indicated NK cell receptors (n=6). (12f) Degranulation assay with DR4 / 5 knock-out M14 cells combined with blocking antibodies towards indicated NK cell receptors (n=6). (12g) Degranulation assay with WT- SKNAS cells combined with blocking antibodies towards indicated NK cell receptors (n=6). (12h) Cytotoxicity assay with WT-SKNAS cells combined with blocking antibodies towards indicated NK cell receptors (n=6). (12i) Degranulation assay with DR4 / 5 knock-down SKNAS cells combined with blocking antibodies towards indicated NK cell receptors (n=5). (12j) Degranulation assay with negative control- treated NK cells against WT-A172 combined with blocking antibodies towards indicated NK cell receptors (n=6). (12k) Degranulation assay with TRAIL knock-down or negative control-treated NK cells against WT-A172 (n=6). (121) Degranulation assay with TRAIL knock-down NK cells against WT-A172 combined with blocking antibodies towards indicated NK cell receptors (n=6). (12m) Degranulation assay with negative control-treated NK cells against WT-A549 combined with blocking antibodies towards indicated NK cell receptors (n=6). (12n) Degranulation assay with TRAIL knock-down or negative control-treated NK cells against WT-A549 (n=6). (12o) Degranulation assay with TRAIL knock-down NK cells against WT-A549 combined with blocking antibodies towards indicated NK cell receptors (n=6). (12p) Degranulation assay with negative control-treated NK cells against WT-L482 combined with blocking antibodies towards indicated NK cell receptors (n=6). (12q) Degranulation assay with TRAIL knock-down or negative control-treated NK cells against WT-L482 (n=6). (12r) Degranulation assay with TRAIL knockdown NK cells against WT-L482 combined with blocking antibodies towards indicated NK cell receptors (n=6). (12s) Degranulation assay with negative control-treated NK cells against WT-0VCAR3 combined with blocking antibodies towards indicated NK cell receptors (n=6). (12t) Degranulation assay with TRAIL knock-down or negative control-treated NK cells against WT-0VCAR3 (n=6). (12u) Degranulation assay with TRAIL knock-down NK cells against WT-0VCAR3 combined with blocking antibodies towards indicated NK cell receptors (n=6).

[0029] DETAILED DESCRIPTION

[0030] The present invention generally relates to tumor necrosis factor (TNF) related apoptosis-inducing ligand (TRAIL) receptor (TRAIL-R) binding molecules, and to the uses thereof.

[0031] Natural killer (NK) cells possess the ability to kill cancer cells and use a set of activating and inhibitory receptors to discriminate between healthy and aberrant cells. Even though ligands for the important natural cytotoxicity receptors have been defined in the past, a cellular ligand for NKp46 (also referred to as natural cytotoxicity triggering receptor 1 (NCR1), cluster of differentiation 335 (CD335) or LY94 in the art) has been notoriously difficult to identify. Experimental data as presented herein show that the TRAIL receptors TRAIL-R1 (also referred to as death receptor 4 (DR4), and TNF receptor superfamily member 10A (TNFRSF10A)) and TRAIL-R2 (also referred to as DR5, and TNFRSF10B) areimportant for NK cell recognition through NKp46. Further, NKp46 was closely associated with TRAIL in cis in the NK cell membrane and the capacity of membrane-bound TRAIL to bind soluble TRAIL receptors was compromised by NKp46 blockade, which indicate that NKp46 and TRAIL together could bind to TRAILRIB.

[0032] The TRAIL receptors 1 and 2 have been prime targets in cancer therapies. In fact, there are several clinical trials involving the use of soluble human TRAIL as an anti-cancer agent. In such an approach, binding of the soluble human TRAIL to the TRAIL receptors 1 and 2, causes activation of the receptors and formation of the death inducing signalling complex comprising activated death receptors, the Fas- associated death domain (FADD) (also referred to as M0RT1) and procaspase 8, which leads to caspase activation and ultimately to cell death, see Fig. 6. Dulanermin is a recombinant soluble human TRAIL that activates apoptotic pathways in cancer cells by binding to the TRAIL receptors 1 and 2. Dulanermin has been shown to enhance progression-free survival (PFS) in non-small cell lung cancer (NSCLC). However, the efficacy with Dulanermin and other soluble TRAIL agents have been limited, potentially due to insufficient binding to TRAIL-R1 / R2. The present invention can solve these shortcomings with Dulanermin and other soluble TRAIL agents by potentiating the binding to the TRAIL receptors 1 and 2, which could enhance the receptor binding to activate apoptotic pathways in in cancer cells.

[0033] An aspect of the invention therefore relates to a TRAIL-R binding molecule comprising an extracellular domain of TRAIL, or a TRAIL-R binding fragment thereof, and an extracellular domain of NKp46, or a fragment thereof.

[0034] The TRAIL-R binding molecule of the invention is therefore a bifunctional or multifunctional molecule in terms of including two or more functional domains, i.e., the extracellular domain of TRAIL, or the TRAIL- R binding fragment thereof, and the extracellular domain of NKp46, or the fragment thereof. This bifunctional or multifunctional molecule can bind to TRAIL receptors, in particular TRAIL receptor 1 and / or TRAIL receptor 2. It is believed that the extracellular domain of NKp46 potentiates or enhances the binding of the extracellular domain of TRAIL to the TRAIL receptors, such as by co-binding to the TRAIL receptors and / or associating with TRAIL to enhance the binding of TRAIL to the TRAIL receptors.

[0035] The binding of the TRAIL-R binding molecule of the invention to TRAIL receptors on cancer cells can activate the death receptors (TRAIL receptor 1 and 2) to activate apoptotic pathways in the cancer cells.

[0036] Human TRAIL is 281 amino acid long transmembrane protein (SEQ ID NO: 4) comprising a cytoplasmic domain corresponding to amino acid nos. 1 -17, a transmembrane domain corresponding to amino acid nos. 18-38 and an extracellular domain corresponding to amino acid nos. 39-281 (SEQ ID NO: 5). Hence, the extracellular domain of human TRAIL as shown in SEQ ID NO: 5 corresponds to amino acid nos. 39- 281 of SEQ ID NO: 4.

[0037] In an embodiment, the TRAIL-R binding molecule could comprise the complete extracellular domain of TRAIL, preferably human TRAIL, as shown in SEQ ID NO: 5.

[0038] In another embodiment, the TRAIL-R binding molecule comprises a TRAIL-R binding fragment of the extracellular domain of TRAIL. An illustrative, but non-limiting, example of such a TRAIL-R binding fragment corresponds to amino acid nos. 114 to 281 of human TRAIL, see SEQ ID NO: 6. Other examples of TRAIL-R binding fragments correspond to amino acid nos. 102 to 281 of human TRAIL (ON 1448404), amino acid nos. 112 to 281 of human TRAIL (ON 1189481), amino acid nos. 121 to 281, amino acid nos. 122 to 281 or amino acid nos. 123 to 281 of human TRAIL (US 2015 / 125419), amino acid nos. 95 to 281 , amino acid nos. 114 to 281 , amino acid nos. 116 to 281 , amino acid nos. 119 to 281 , amino acid nos. 120 to 281 , amino acid nos. 121 to 281 or amino acid nos. 121 to 281 of human TRAIL (WO 2014 / 141094).

[0039] In an embodiment, the TRAIL-R binding fragment is selected from the group consisting of amino acid nos. 114 to 281 of SEQ ID NO: 4, amino acid nos. 102 to 281 of SEQ ID NO: 4, amino acid nos. 112 to 281 of SEQ ID NO: 4, amino acid nos. 121 to 281 of SEQ ID NO: 4, amino acid nos. 122 to 281 of SEQ ID NO: 4, amino acid nos. 123 to 281 of SEQ ID NO: 4, amino acid nos. 95 to 281 of SEQ ID NO: 4, amino acid nos. 114 to 281 of SEQ ID NO: 4, amino acid nos. 116 to 281 of SEQ ID NO: 4, amino acid nos. 119 to 281 of SEQ ID NO: 4, amino acid nos. 120 to 281 of SEQ ID NO: 4, amino acid nos. 121 to 281 of SEQ ID NO: 4, and amino acid nos. 121 to 281 of SEQ ID NO: 4, preferably amino acid nos. 114 to 281 of SEQ ID NO: 4, i.e., SEQ ID NO: 6.

[0040] Human NKp46 is a 304 amino acid long transmembrane protein (SEQ ID NO: 7) comprising an N-terminal signal peptide corresponding to amino acid nos. 1-21 , an extracellular domain corresponding to amino acid nos. 22-258 (SEQ ID NO: 8), a transmembrane domain corresponding to amino acid nos. 259-279 and a cytoplasmic domain corresponding to amino acid nos. 280-304.

[0041] In an embodiment, the TRAIL-R binding molecule could comprise the complete extracellular domain of NKp46, preferably human NKp46, as shown in SEQ ID NO: 8.

[0042] In another embodiment, the TRAIL-R binding molecule comprises a fragment of the extracellular domain of NKp46. Illustrative, but non-limiting, examples of such fragments of the extracellular domain of NKp46 include amino acid nos. 153 to 175 of human NKp46 (WO 2005 / 051973), amino acid nos. 215 to 254 of human NKp46 (WO 2005 / 000086), amino acid nos. 22-120, or amino acid nos. 121 -254 of human NKp46 (WO 2004 / 053054).

[0043] In an embodiment, the extracellular domain of NKp46 is selected from the group consisting of amino acid nos. 22-258 of SEQ ID NO: 7, amino acid nos. 153 to 175 of SEQ ID NO: 7, amino acid nos. 215 to 254 of SEQ ID NO: 7, amino acid nos. 22-120 of SEQ ID NO: 7, and amino acid nos. 121-254 of SEQ ID NO: 7, preferably amino acid nos. 22-258 of SEQ ID NO: 7, i.e., SEQ ID NO: 8. In an embodiment, the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, is fused to the extracellular domain of NKp46, or the fragment thereof. In such an embodiment, the TRAIL-R binding molecule is in the form of a TRAIL-R binding fusion protein.

[0044] In another embodiment, the TRAIL-R binding molecule comprises a linker or spacer interconnecting the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, and the extracellular domain of NKp46, or the fragment thereof.

[0045] The linker or spacer is then preferably a peptide or a polypeptide linker or spacer comprising one or more amino acids. Various such linkers or spacers traditionally used to interconnect polypeptide molecules or domains can be used according to the embodiments. Illustrative, but non-limiting, examples of such linkers or spacers include poly-G linkers, such as Gn, wherein n is a positive integer, typically n=1 -12, various GS-linkers, such as (GGGGS)m, or (GS)n, wherein m is a positive integer, typically m=1-5, EAAAK-linkers, such as (EAAAK)m.

[0046] Fig. 7 is a schematic illustration of a TRAIL-R binding molecule comprising the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, connected to the extracellular domain of NKp46, or the fragment thereof, via a linker or spacer.

[0047] The bifunctional TRAIL-R binding molecule shown in Fig. 7 can be used in treatment of cancer by binding to TRAIL receptor 1 and 2 expressed by the cancer cells and present in the cell membrane of the cancer cells. The bifunctional TRAIL-R binding molecule thereby finds therapeutic uses in applications where soluble TRAIL, such as in the form of Dulanermin, has been used in the art. Such cancers are then characterized by cancer cells that are susceptible to TRAIL-R1 and / or TRAIL-R2 mediated extrinsic apoptosis, also referred to as FADD mediated extrinsic apoptosis as shown in the left part of Fig. 6. In such a case, binding of the bifunctional TRAIL-R binding molecule to TRAIL-R1 and / or TRAIL-R2 in the cell membrane of a cancer cell causes activation of the receptors and formation of the death inducing signalling complex comprising activated death receptors, FADD and procaspase 8, which leads to caspase (caspase 3 / 7) activation and ultimately to cell death.

[0048] Illustrative, but non-limiting examples, of such cancers that could be treated with the TRAIL-R binding molecule according to the embodiments include colorectal cancer, such as metastatic colorectal cancer, non-Hodgkin’s lymphoma, cervical cancer, multiple myeloma, hepatocellular carcinoma, peritoneal malignancies, such as peritoneal carcinomatosis and peritoneal mesothelioma, lung cancer, such as lung adenocarcinoma, non-small-cell lung cancer, and non-squamous non-small-cell lung cancer, breast cancer, pancreatic cancer, and renal cancer.,

[0049] Not all cancer cells respond to TRAIL when treated with Dulanermin. TRAIL resistance may be due to different factors, including different expression levels of pro- and anti-apoptotic proteins within cancer cells or of microRNAs able to target proteins involved in TRAIL pathway. In this case it has been observed that the combination of TRAIL to traditional chemotherapy or radiotherapy may overcame the resistant phenotype. This means that the TRAIL-R binding molecule could be used in combination with such chemotherapy and / or radiotherapy. Illustrative, but non-limiting, examples of such chemotherapeutic agents that could be used in combination with the TRAIL-R binding molecule include alkylating agents, such as mechlorethamine, cyclophosphamide, melphalan, chlorambucil, ifosfamide, busulfan, N-Nitroso- N-methylurea (MNU), carmustine (BCNU), lomustine (CCNU), semustine (MeCCNU), fotemustine, streptozotocin, dacarbazine, mitozolomide, temozolomide, thiotepa, mitomycin, diaziquone (AZQ), cisplatin, carboplatin, oxaliplatin, procarbazine and hexamethylmelamine; antimetabolites, such as methotrexate, pemetrexed, fluorouracil, capecitabine, cytarabine, gemcitabine, decitabine, azacitidine, fludarabine, nelarabine, cladribine, clofarabine, pentostatin, thioguanine and mercaptopurine; antimicrotubule agents, such as vincristine, vinblastine, vinorelbine, vindesine, vinflunine, paclitaxel, docetaxel, etoposide and teniposide; and topoisomerase inhibitors, such as irinotecan, topotecan, etoposide, doxorubicin, mitoxantrone, teniposide, novobiocin, merbarone, and aclarubicin; cytotoxic antibiotics, such as doxorubicin, daunorubicin, epirubicin, idarubicin, pirarubicin, aclarubicin, mitoxantrone, actinomycin, bleomycin, and mitomycin.

[0050] The TRAIL-R binding molecule of the embodiments can be designed to be effective also for TRAIL resistant cancer cells. This is possible by designing the TRAIL-R binding molecule so that it can activate NK cells or T cells to target and kill the cancer cells. In such an approach, the anti-cancer effect of the TRAIL-R binding molecule is not dependent on inducing FADD mediated apoptotic pathway in the cancer cells. This is schematically shown in the right part of Fig. 10, in which the TRAIL-R binding molecule interconnects an NK cell with a target cell, such as a cancer cell, and induces NK cell degranulation involving granzyme and perforin release from the NK cell, which induces apoptosis of the target cell.

[0051] In an embodiment, the TRAIL-R binding molecule is a multifunctional TRAIL-R binding molecule, also referred to as multispecific TRAIL-R binding molecule. In such an embodiment, the TRAIL-R binding molecule comprises the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, the extracellular domain of NKp46, or the fragment thereof, and a Fc polypeptide or an antibody, or an antigen-binding fragment thereof.

[0052] Figs. 8 and 9 illustrate embodiments of such TRAIL-R binding molecules comprising three functional domains. Fig. 8 illustrates an embodiment of the TRAIL-R binding molecule comprising the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, the extracellular domain of NKp46, or the fragment thereof, and a Fc polypeptide. Fig. 9 illustrates an embodiment of the TRAIL-R binding molecule comprising the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, the extracellular domain of NKp46, or the fragment thereof, and an antibody, or an antigen-binding fragment thereof.

[0053] In an embodiment, the Fc polypeptide is an immunoglobulin G (IgG) Fc polypeptide binding to CD16 (also known as FcyRIII), and in particular to CD16a (also referred to as FcyRllla).

[0054] CD16 is found on the surface of NK cells, neutrophils, monocytes, macrophages, and certain T cells. CD16 has been identified as Fc receptors FcyRllla (CD16a) and FcyRlllb (CD16b), which participate in signal transduction. While FcyRllla is expressed on mast cells, macrophages, and NK cells as a transmembrane receptor, FcyRlllb is only expressed on neutrophils. FcyRllla and FcyRlllb together are able to activate degranulation, phagocytosis, and oxidative burst. These receptors bind to the Fc portion of IgG antibodies, which then activates antibody-dependent cell-mediated cytotoxicity (ADCC) in human NK cells. In humans, monocytes expressing CD16 have a variety of ADCC capabilities in the presence of specific antibodies, and can kill primary leukemic cells, cancer cell lines, and virally infected cells.

[0055] After binding to ligands, such as the conserved section of IgG antibodies, CD16 on human NK cells induce gene transcription of surface activation molecules, such as interleukin-2 receptor (IL-2R) (also referred to as CD25) and inflammatory cytokines, such as IFNy and TNF. Activation of the CD16 further induces degranulation of the NK cells as shown in the right part of Fig. 10 with release of perforin and granzymes. Perforin is a pore forming cytolytic protein found in the granules of NK cells and cytotoxic T lymphocytes (CTLs). Upon degranulation, perforin molecules translocate to the target cell. Perforin then binds to the target cell’s plasma membrane and oligomerizes to form pores on the target cell. The pore formed allows for the passive diffusion of a family of pro-apoptotic proteases, known as the granzymes, into the target cell. Granzymes are serine proteases released by cytoplasmic granules within NK cells and CTLs. They induce programmed cell death (apoptosis) in the target cell, thus eliminating cells that have become cancerous. In NK cells and CTLs, granzymes are packaged in cytotoxic granules along with perforin. Fc polypeptide as used herein correspond to the fragment crystallizable (Fc) region, which is the tail region of an antibody and that interacts with Fc receptors.

[0056] In an embodiment, the antibody, or the antigen-binding fragment thereof, binds specifically to a surface receptor on a T cell or an NK cell. In a particular embodiment, the antibody, or the antigen-binding fragment thereof, is also capable of, by binding specifically to the surface receptor, activating the surface receptor to thereby induce an activation of the T cell or the NK cell.

[0057] The specificity of an antibody, or an antigen-binding fragment thereof, can be determined based on affinity and / or avidity. The affinity, represented by the equilibrium constant for the dissociation (Kd) of an antigen with the antibody, or the antigen-binding fragment thereof, is a measure for the binding strength between an antigenic determinant and an antigen-binding site on the antibody, or the antigen-binding fragment thereof. The lesser the value of Kd, the stronger the binding strength between the antigenic determinant and the antibody, or the antigen-binding fragment thereof. Alternatively, the affinity can also be expressed as the affinity constant (Ka), which is 1 / Kd. As will be clear to the skilled person, affinity can be determined in a manner known per se, depending on the specific antigen of interest.

[0058] Avidity is the measure of the strength of binding between an antibody, or an antigen-binding fragment thereof, and the pertinent antigen. Avidity is related to both the affinity between an antigenic determinant and its antigen binding site on the antibody, or the antigen-binding fragment thereof, and the number of pertinent binding sites present on the antibody, or the antigen-binding fragment thereof.

[0059] Typically, antibodies, or antigen-binding fragments thereof, will bind to their antigen with a dissociation constant (Kd) of 105to 1012moles / liter (M) or less, and preferably 107to 1012M or less and more preferably 108to 1012M, i . e. , with an association constant (Ka) of 105to 1012M1or more, and preferably 107to 1012M'1or more and more preferably 108to 1012M1.

[0060] Generally, any Kd value greater than 104M (or any Kavalue lower than 104M1) is generally considered to indicate non-specific binding. Preferably, an antibody, or an antigen-binding fragment thereof, will bind to the antigen with an affinity less than 500 nM, preferably less than 200 nM, more preferably less than 10 nM, such as less than 5 nM or even lower, such as 1 nM or lower. Specific binding of an antibody, or an antigen-binding fragment thereof, to an antigen or antigenic determinant can be determined in any suitable manner known per se, including, for example, Scatchard analysis and / or competitive binding assays, such as radioimmunoassays (RIA), enzyme immunoassays (EIA) and sandwich competition assays, and the different variants thereof known per se in the art.

[0061] An antigen-binding fragment of an antibody as used herein can be selected from a group consisting of a single chain antibody, a Fv fragment, a scFv fragment, a Fab fragment, a F(ab’)2 fragment, a Fab’ fragment, a Fd fragment, a single-domain antibody (sdAb), a scFv-Fc fragment, and a di-scFv fragment. In a particular embodiment, the antigen-binding fragment of an antibody is a scFv fragment.

[0062] In an embodiment, the antibody is a monoclonal antibody, or the antigen-binding fragment is an antigenbinding fragment of a monoclonal antibody.

[0063] In another embodiment, the antibody is a polyclonal antibody, or the antigen-binding fragment is an antigen-binding fragment of a polyclonal antibody.

[0064] In an embodiment, the antibody, or the antigen-binding fragment thereof, binds specifically to CD3.

[0065] CD3 is a protein complex and T cell co-receptor that is involved in activating both the cytotoxic T cells, i.e., CTLs, (CD8+T cells) and T helper cells (CD4+T cells).

[0066] For instance, the antibody, or the antigen-binding fragment thereof, such as scFv, binding specifically to CD3 enables, upon binding to CD3, activation of CD3 on a T cell and thereby activation of the T cell.

[0067] Examples of such CD3-binding antibodies, or antigen-binding fragments thereof, that could be used include 0KT3 (ORTHOCLONE-OKT3™ (Muromonab-CD3)); Teplizumab™ (MGA031 or PRV-031, Eli Lilly); Micromet (US 2011 / 0275787); Blinatumomab™ (Blincyto); UCHT1 (Pollard et al. 1987 J Histochem Cytochem. 35(11): 1329-38); Otelixizumab™ (Abatacept); NI0401 (W02007 / 033230); visilizumab (US5,834,597), X35-3, VIT3, BMA030 (BW264 / 56), CLB-’3 / 3, GRIS' 7, YTH12.5, Fl 11-409, CLB-T3.4.2, WT31 , WT32, SPv-T3b, 1 1 D8, XIII- 141 , XIII-46, XIII-87, 12F6, T3 / RW2-8C8, T3 / RW2-4B6, 0 T3D, M- T301, SMC2, LMT-28 and F101 .01.

[0068] The extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, the extracellular domain of NKp46, or the fragment thereof, and the Fc polypeptide or the antibody, or the antigen-binding fragment thereof, could be fused to each other in any order in the TRAIL-R binding molecule. Alternatively, the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, the extracellular domain of NKp46, or the fragment thereof, and the Fc polypeptide or the antibody, or the antigen-binding fragment thereof, could be interconnected to each other in any order using linkers or spacers. In such a case, such linkers or spacer can be selected among the above-described linkers or spacers.

[0069] In an embodiment, the TRAIL-R binding molecule of the embodiments may include a multimerization or oligomerization domain, such as a dimerization domain or a trimerization domain. Such a multimerization or oligomerization domain of the TRAIL-R binding domain is believed to retard in vivo clearance from the circulation. Illustrative, but non-limiting, examples of such multimerization or oligomerization domains include a FLAG tag, leucine and isoleucine zipper domains, a tenascin-C domain. Such a multimerization or oligomerization domain is believed to increase the stability of the TRAIL-R binding molecule and enhance its binding to the TRAIL receptor 1 and / or receptor 2.

[0070] Other examples of TRAIL modifications that could be used according to the embodiments are listed in Table 1 of Stuckey and Shah, TRAIL on Trial: Preclinical advances for cancer therapy, Trends Mol Med (2013) 19(11): 685-694. These examples include addition of a leucin zipper, a FLAG tag, an isoleucine zipper, an extracellular domain of Flt3L, human serum albumin, transferrin, a PEG, doxorubicin, and / or nanoparticles.

[0071] The invention also relates to a TRAIL-R binding molecule of the embodiments for use as a medicament.

[0072] The invention further relates to a TRAIL-R binding molecule of the embodiments for use in treatment of cancer.

[0073] Illustrative, but non-limiting examples, of such cancers that could be treated with the TRAIL-R binding molecule according to the embodiments include colorectal cancer, such as metastatic colorectal cancer, non-Hodgkin's lymphoma, cervical cancer, multiple myeloma, hepatocellular carcinoma, peritoneal malignancies, such as peritoneal carcinomatosis and peritoneal mesothelioma, lung cancer, such as lung adenocarcinoma, non-small-cell lung cancer, and non-squamous non-small-cell lung cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia, ovarian cancer, pancreatic cancer, head and neck squamous cell carcinoma, and renal cancer. In an embodiment, the cancer is selected from the group consisting of colorectal cancer, non-Hodgkin's lymphoma, Hodgkin’s lymphoma, cervical cancer, multiple myeloma, hepatocellular carcinoma, peritoneal carcinomatosis, peritoneal mesothelioma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia, ovarian cancer, pancreatic cancer, head and neck squamous cell carcinoma, neuroblastoma, glioblastoma, malignant melanoma, and renal cancer.

[0074] In a particular embodiment, the cancer is selected from the group consisting of colorectal cancer, nonHodgkin's lymphoma, cervical cancer, multiple myeloma, hepatocellular carcinoma, peritoneal carcinomatosis, peritoneal mesothelioma, lung cancer, breast cancer, pancreatic cancer, and renal cancer.

[0075] In another particular embodiment, the cancer is selected from the group consisting of non-small cell lung cancer, breast cancer, colorectal cancer, prostate cancer, leukemia, ovarian cancer, pancreatic cancer and head and neck squamous cell carcinoma.

[0076] In a further particular embodiment, the cancer is selected from the group consisting of prostate cancer, malign melanoma, neuroblastoma, ovarian cancer, Hodgkin’s lymphoma, glioblastoma, non-small cell lung cancer and leukemia, preferably selected from the group consisting of prostate cancer and leukemia.

[0077] The invention further relates to a TRAIL-R binding molecule of the embodiments for use in treatment of a viral infection.

[0078] A cell infected by a virus may, as a defense response to the viral infection, cause upregulation of TRAIL receptors on the cell membrane (Sedger et al., IFN-y Mediates a Novel Antiviral Activity Through Dynamic Modulation of TRAIL and TRAIL Receptor Expression, J Immunol (1999) 163(2): 920-926). Such TRAIL receptors can then be targeted by the TRAIL-R binding molecules of the invention to thereby induce apoptosis of the virus-infected cell or NK cell or T cell mediated killing of the virus-infected cell.

[0079] Illustrative, but non-limiting, examples of viral infections that can be treated by the TRAIL-R binding molecule of the embodiments include cytomegalovirus (CMV), hepatitis B virus, hepatitis C virus, Epstein- Barr virus, influenza virus, measles virus, and herpes virus.

[0080] The embodiments also relate to a pharmaceutical composition comprising a TRAIL-R binding molecule according to the embodiments, and a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier could be any pharmaceutically acceptable carrier, vehicle and / or excipient, including combinations thereof, that is or are compatible with the other constituent(s) of the pharmaceutical composition. Non-limiting examples of such pharmaceutically acceptable carriers include injection solutions, such as saline or buffered injection solutions.

[0081] A further embodiment is directed towards a method of treating cancer in a patient. The method comprises administering an effective amount of a TRAIL-R binding molecule and / or a pharmaceutical composition according to the embodiments to the patient.

[0082] Yet another embodiment is directed towards a method of treating a viral infection in a patient. The method comprises administering an effective amount of a TRAIL-R binding molecule and / or a pharmaceutical composition according to the embodiments to the patient.

[0083] As used herein, effective amount indicates an amount effective, at dosages and for periods of time necessary to achieve a desired result. For example, in the context of killing cancer cells an effective amount is an amount that, for example, induces, remission, reduces tumor burden, and / or prevents tumor spread or growth compared to the response obtained without administration of the cells. Effective amounts may vary according to factors, such as the disease state, age, sex, weight of the patient. Treating or treatment as used herein and is well understood in the art, means an approach for obtaining beneficial or desired results, including clinical results. Beneficial or desired clinical results could include, for instance, alleviation or amelioration of one or more symptoms or conditions, diminishment of extent of disease, stabilized state of disease, i.e., prevent worsening, preventing spread of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, diminishment of the reoccurrence of disease, and remission. Treating or treatment may also prolong survival as compared to expected survival if not receiving any treatment.

[0084] Treating as used herein also encompass preventing cancer. Preventing or prophylaxis as used herein and is well understood in the art, means an approach in which a risk of developing a disease or condition is reduced or prevented, including prolonging or delaying disease development. For instance, a patient predisposed to develop a disease, such as due to genetic or hereditary predisposition, could benefit for administration of the antibody, or the antigen-binding fragment thereof, the cell, the conjugate and / or the pharmaceutical composition according to the embodiments to prevent, reduce the risk of, delaying and / or slowing development of the disease. The patient is preferably a human patient.

[0085] The TRAIL-R binding molecule and / or the pharmaceutical composition according to the embodiments may be administered to the patient according to various routes including, but not limited to, intravenous, subcutaneous, intraperitoneal, intramuscular or intratumoral administration.

[0086] A further aspect of the embodiments includes a nucleic acid molecule encoding a TRAIL-R binding molecule according to the embodiments. Nucleic acid molecule as used herein includes polynucleotide, oligonucleotide, and nucleic acid sequence, and generally means a polymer of DNA or RNA, which may be single-stranded or double-stranded, which may contain natural, non-natural or altered nucleotides, and which may contain a natural, non-natural or altered internucleotide linkage, such as a phosphoroamidate linkage or a phosphorothioate linkage, instead of the phosphodiester found between the nucleotides of an unmodified oligonucleotide.

[0087] Another aspect of the embodiments relates to an expression vector comprising a promoter and a nucleic acid molecule encoding a TRAIL-R binding molecule according to the embodiments operatively controlled by the promoter.

[0088] The expression vector comprises at least one nucleic acid molecule comprising coding sequences that can be expressed, such as transcribed and translated, in a host cell comprising the expression vector. The expression vector is in an embodiment selected among DNA molecules, RNA molecules, plasmids, episomal plasmids and virus vectors.

[0089] In an embodiment, the expression vector is a virus vector. In a particular embodiment, the virus vector is selected from a group consisting of a lentiviral vector, an adenoviral vector, an adeno-associated viral vector, a retroviral vector, a Semliki Forest virus, a polio virus and a hybrid vector.

[0090] Lentiviruses are a subclass of retroviruses. They are adapted as gene delivery vehicles (vectors) thanks to their ability to integrate into the genome of non-dividing cells, which is the unique feature of lentiviruses as other retroviruses can infect only dividing cells. The viral genome in the form of RNA is reverse- transcribed when the virus enters a cell, such as a T cell, to produce DNA, which is then inserted into the genome at a position by the viral integrase enzyme. The vector, now called a provirus, remains in the genome and is passed on to the progeny of the cell if it divides. For safety reasons lentiviral vectors typically never carry the genes required for their replication. To produce a lentivirus, several plasmids are transfected into a so-called packaging cell line, commonly HEK 293. One or more plasmids, generally referred to as packaging plasmids, encode the virion proteins, such as the capsid and the reverse transcriptase. Another plasmid contains the genetic material to be delivered by the vector. It is transcribed to produce the single-stranded RNA viral genome and is marked by the presence of the i (psi) sequence. This sequence is used to package the genome into the virion.

[0091] Retroviruses are one of the mainstays of current gene therapy approaches. The recombinant retroviruses, such as the Moloney murine leukemia virus, have the ability to integrate into the host genome in a stable fashion. They contain a reverse transcriptase that allows integration into the host genome. Retroviral vectors can either be replication-competent or replication-defective. Replicationdefective vectors are the most common choice because the viruses have had the coding regions for the genes necessary for additional rounds of virion replication and packaging replaced with other genes, or deleted. These viruses are capable of infecting T cells and delivering their viral payload, but then fail to continue the typical lytic pathway that leads to cell lysis and death. If the vector is a lentiviral or retroviral vector then the nucleic acid sequence encoding the TRAIL-R binding molecule is preferably an RNA sequence.

[0092] Adenoviral vector as used herein include adenovirus vectors and adenovirus-derived virus vectors.

[0093] Adenoviral DNA does not integrate into the genome and is not replicated during cell division. An adeno- derived virus vector is based on an adenovirus but in which various modifications have been done, such as relating to the nucleotide sequences coding replication proteins, regulation protein, viral surface proteins, etc.

[0094] Adeno-associated virus (AAV) is a small virus that infects humans and some other primate species. AAV can infect both dividing and non-dividing cells and may incorporate its genome into that of the host cell. Moreover, AAV mostly stays as episomal, performing long and stable expression. Whereas AAV packages a single strand of DNA and requires the process of second-strand synthesis, self- complementary adeno-associated virus (scAAV) packages both strands which anneal together to form double stranded DNA. By skipping second strand synthesis, scAAV allows for rapid expression in the cell. If the vector is an adenoviral vector then the nucleic acid sequence encoding the TRAIL-R binding molecule is preferably a DNA sequence. The Semliki Forest virus is a positive-stranded RNA virus with a genome of approximately 13,000 base pairs which encodes nine proteins. The 5’ two thirds of the genome encode four non-structural proteins concerned with RNA synthesis and the structural proteins are encoded in the 3’ third. Of the structural proteins, the C proteins makes up the icosahedral capsid which is enveloped by a lipid bilayer, derived from the host cell. The outermost surface of the virus is almost entirely covered by heterodimers of glycoproteins E1 and E2, arranged in interconnective trimers, which form an outer shell. Trimers are anchored in the membrane by an E2 cytoplasmic domain that associates with the nucleocapsid. Due to its broad host range and efficient replication, it has also been developed as a vector.

[0095] A hybrid vector is a vector virus that is genetically engineered to have qualities of more than one vector. For instance, a hybrid vector may be a combination of an adenovirus and a lentivirus.

[0096] The nucleic acid molecule encoding the TRAIL-R binding molecule is operatively controlled by the promoter in the expression vector, i.e. , is under transcriptional control of the promoter. In an embodiment, the promoter is selected from the group consisting of the human EF1a promoter, the CMV promoter, the CAG promoter, the PGK promoter, the TRE promoter, the U6 promoter and the UAS promoter if the host cell is an eukaryotic cell, such as a human cell. Illustrative, but non-limiting, examples of a promoter that could be used if the host cell is a bacterial cell include the T5 promoter, the T7 promoter, the rham promoter, the phoA promoter, the Sp6 promoter, the lac promoter, the AraBad promoter, the trp promoter and the Ptac promoter. If the host cell is a yeast cell the promoter could be selected from the group consisting of the CYC1 promoter, the ADH1 promoter, the TEF2 promoter, the pCYC promoter, a PGAL promoter and the GFD promoter as illustrative, but non-limiting, examples.

[0097] A further aspect of the embodiments relates to a host cell comprising an expression vector according to the embodiments. In such an aspect, the promoter is constitutively active in the host cell or inducible active in the host cell. This means that the nucleic acid molecule encoding that the TRAIL-R binding molecule can then be transcribed in the host cell to produce the TRAIL-R binding molecule in the host cell.

[0098] In an embodiment, the host cell is selected from the group consisting of a bacterial cell, a yeast cell, and an eukaryotic cell, such as a human cell. Illustrative examples of a human host cell include the group consisting of a T cell, a NK cell, a B cell, a monocyte, and a macrophage. In a particular embodiment, the cell is a NK cell. An aspect of the embodiments relates to a chimeric antigen receptor (CAR). The CAR comprises an antigen recognition domain comprising a TRAIL-R binding molecule according to the embodiments. The CAR also comprises a transmembrane domain and an intracellular domain.

[0099] Fig. 11 is a schematic illustration of a CAR present in the cell membrane with the antigen recognition domain represented by the TRAIL-R binding molecule as shown in Fig. 7.

[0100] In general, CARs comprise an ectodomain, a transmembrane domain, and an endodomain. The ectodomain of a CAR comprises an antigen recognition region or domain. The ectodomain may also comprise a signal tag or peptide that directs the CAR into the endoplasmic reticulum.

[0101] The transmembrane domain is the portion of the CAR that traverses the cell membrane. In general, the transmembrane domain can be derived from any transmembrane protein. In an embodiment, the transmembrane domain comprises a hydrophobic alpha helix. Illustrative, but non-limiting, examples of transmembrane domains that can be included in the CAR include all, or a portion, of the transmembrane domain of CD28, all, or a portion, of the transmembrane domain of CD8a, all, or a portion, of the transmembrane domain of CD27, all, or a portion, of the transmembrane domain of CD137 (4-1 BB), all, or a portion, of the transmembrane domain of CD134 (0X40), all, or a portion, of the transmembrane domain of CD3s, all, or a portion, of the transmembrane domain of CD3^, all, or a portion, of the transmembrane domain of CD3y, all, or a portion, of the transmembrane domain of CD35, all, or a portion, of the transmembrane domain of TCRa, and all, or a portion, of the transmembrane domain of TCR0.

[0102] The endodomain of a CAR comprises at least one signaling domain. Illustrative, but non-limiting, examples of such signaling domains that can be included in the CAR include the zeta chain of CD3 (CD3Q, CD28, CD137 (4-1 BB), ICOS, CD27, CD40, 0X40 (CD134), DNAX-activating protein 10 (DAP10), DAP12 and / or Myd88.

[0103] In an embodiment, the endodomain of the CAR comprises, such as consists of, CD3^.

[0104] In another embodiment, the endodomain of the CAR comprises, such as consists of, CD3^ and at least one co-stimulatory domain selected from the group consisting of CD28, CD137 (4-1 BB), and 0X40 (CD 134). In a further embodiment, the endodomain of the CAR comprises, such as consists of, CD3^ and at least two co-stimulatory domains selected from the group consisting of CD28, CD137 (4-1 BB), and 0X40 (CD 134).

[0105] In yet another embodiment, the endodomain of the CAR comprises, such as consists of CD3^, at least one co-stimulatory domains selected from the group consisting of CD28, CD137 (4-1 BB), and 0X40 (CD134), and at least one cytokine release domain and / or at least one transcriptional regulator domain. The at least one cytokine release domain enables the CAR T cells to secrete immune-activating cytokines, such as IL-12, IL-15, or IL-18, after activation. These cytokines assist in recruiting and activating other immune cells, such as NK cells, dendritic cells and macrophages, to the tumor site, thereby boosting the immune response and improving the anti-tumor efficacy. The at least one transcriptional regulator domain may further enhance the activity of CAR T cell by, for instance, activate pathways such as NFAT and NF-KB.

[0106] In an embodiment, the CAR comprises a hinge domain or spacer interconnecting the antigen recognition domain and the transmembrane domain.

[0107] A further aspect of the embodiments includes a nucleic acid molecule encoding a CAR according to the embodiments.

[0108] Another aspect of the embodiments relates to an expression vector comprising a promoter and a nucleic acid molecule encoding the CAR according to the embodiments operatively controlled by the promoter.

[0109] The discussion above regarding suitable expression vectors and promoter discussed in connection with the TRAIL-R binding molecule also applies to the CAR.

[0110] The invention also relates to a NK cell comprising a CAR according to the embodiments or a nucleic acid sequence encoding a CAR according to the embodiments and a T cell comprising a CAR according to the embodiments or a nucleic acid sequence encoding a CAR according to the embodiments.

[0111] Related aspects define a NK cell or a T cell according to the invention for use as a medicament, and in particular for the use in treatment of cancer. A further embodiment is directed towards a method of treating cancer in a patient. The method comprises administering an effective amount of a NK cell or a T cell according to the embodiments to the patient.

[0112] The NK cell or T cell can be administered in the form a pharmaceutical composition comprising the NK cell or the T cell and a pharmaceutically acceptable carrier.

[0113] EXAMPLES

[0114] EXAMPLE 1

[0115] NK cells possess the ability to kill cancer cells and use a set of activating and inhibitory receptors to discriminate between healthy and aberrant cells. Even though ligands for the important natural cytotoxicity receptors have been defined in the past, a cellular ligand for NKp46 has been notoriously difficult to identify. In this Example, key NK receptor ligands, B7-H6, poliovirus receptor (PVR, also referred to as CD155) and Nectin-2 (also referred to as CD112, poliovirus receptor-related 2 (PVRL2)), were deleted in K562 cells to make NK cell recognition highly dependent on NKp46 and these genetically modified K562 cells were used in an unbiased genome-wide CRISPR screen with the aim to identify novel NKp46 ligand candidates. The resulting gene hits revealed three receptors to TRAIL to be strikingly important for NK cell recognition in the highly NKp46-dependent assay. However, the gene encoding the death receptor adapter protein, FADD, which has a pivotal role in the apoptosis pathway downstream of death receptors had no role in the screen. Furthermore, FADD-deficient K562 cells displayed no reduced sensitivity to NK cell cytotoxicity, suggesting that the impact of TRAIL-Rs was different to their described role as death receptors. Instead, NKp46-mediated NK cell degranulation, cytokine responses and cytotoxicity were inhibited by knock-out of TRAIL-R1 / 2 in multiple cell lines. NKp46 was closely associated with TRAIL in cis in the NK cell membrane as determined by a NanoBiT split luciferase assay. Furthermore, the capacity of membrane-bound TRAIL to bind soluble TRAIL receptors was compromised by NKp46 blockade. Notably, NK92 cells that were overexpressing NKp46 were found to be TRAIL negative and were incapable of killing target cells via NKp46. Taken together, the data suggest that TRAIL-Rs serve as the long-sought cellular ligands to NKp46.

[0116] Methods

[0117] Cell cultures

[0118] The K562 cell line was obtained from ATCC and cultured in Iscove’s modified Dulbecco’s medium (IMDM, Gibco), supplemented with 10% heat-inactivated fetal calf serum (HI FCS, Gibco), 2 mM L-glutamine (Gibco), 1 mM sodium pyruvate (Gibco) and 1 % Penicillin-streptomycin (PEST, Gibco). It was maintained at concentration 0.1-1 million cells / ml. It was authenticated directly before Cas9 transduction (MCA by Multiplexion).

[0119] 221 G cells was gifted from Moretta lab and cultured in RPMI (Gibco), supplemented with 10% HI FCS, 2 mM L-glutamine, 1 mM sodium pyruvate and 1 % PEST. It was maintained at concentration 0.1-1 million cells / ml.

[0120] HEK293A cells were maintained in T75 flasks in Dulbecco’s modified Eagle’s minimum essential medium (Gibco), supplemented with 10% HI FCS and 1% PEST.

[0121] Both the NK-92 wild-type (WT) and the NKp46-overexpressing NK-92 cell lines were gifted from Campbell’s lab and cultured in alpha Minimum essential medium eagle with ribonucleosides and deoxyribonucleosides supplemented with 2 mM L-glutamine, 2.2 g / L sodium bicarbonate (Sigma- Aldrich), 0.2 mM inositol (Sigma-Aldrich), 0.1 mM 2-mercaptoethanol (Life Technologies), 0.02 mM folic acid (Sigma-Aldrich), 200 U / ml recombinant IL-2 (Chiron), 12.5% HI horse serum (Sigma-Aldrich) and 12.5% HI FCS. All cell lines were cultured at 37°C in 5% CO2.

[0122] Isolation of NK cells

[0123] Buffy coats from de-identified healthy donors were obtained from the blood center at Sahlgrenska University Hospital. Peripheral blood mononuclear cells (PBMCs) were isolated by density gradient centrifugation with Lymphoprep (Stemcell technologies, cat# 07861). Human NK cell isolation kit (Miltenyi Biotec, cat# 130-092-657) were used for NK cell isolation and CD56 / CD3 staining was used to confirm purity.

[0124] Generation of polyclonally activated NK cells

[0125] Isolated NK cells were cultured in the presence of irradiated allogenic PBMCs and 221 G feeder cells in IMDM, supplemented with 10% heat-inactivated fetal calf serum, 2 mM L-glutamine, 600 U / ml IL-2, 1 % PEST and 5 pg / ml phytohemagglutinin (PHA-M, Sigma). After seven days, the medium was gradually replaced with the same medium without PHA-M. The culture was maintained at concentration 1-2 million cells / ml.

[0126] CRISPR / Cas9 engineered cell lines

[0127] LentiCas9-Blast (Sanjana et al. Improved vectors and genome-wide libraries for CRISPR screening. Nat

[0128] Methods. 2014; 11 : 783-4) (Feng Zhang, Addgene, Addgene_52962) was used for a two-time transduction of K562 cells by spinoculation in 5 pg / ml polybrene (Merck, cat# TR-1003-G), followed by selection in blasticidin before clone generation by single-cell sorting.

[0129] Generation of B7-H6ne9 NCR3LG1 sKO (single-KO) and B7-H6ne9PVRne9Nectin-2ne9 tKO (triple-KO) K562 cell lines was as previously described (Hussein et al. NKG2A gene variant predicts outcome of immunotherapy in AML and modulates the repertoire and function of NK cells, J Immunother Cancer. 2023 11 (8): e007202). A ribonucleoprotein-based method was utilized for creation of the TRAIL-R1 / R2nea cell lines. Broad Institute validated spacers for the genes TNFSRF10A, TNFSRF10B and FADD were chosen and confirmed using Synthego’s guide design verification tool (sequences can be found as SEQ ID NO: 1-3 in Table 3).

[0130] The Neon electroporation system (100 pL tips, for settings see Table 1 ; Invitrogen) was used for transfection of the cell lines.

[0131] Table 1 - Neon transfection setting for cell lines.

[0132] B7H6ne9 K562 cells were used as basis for KO of TNFRSF10A / B and FADD genes. The cells were single cell-sorted for generation of KO clones two days post transfection using a 3-laser FACSAria Fusion. Double allelic KO was confirmed using flow cytometric staining and Sanger sequencing (Eurofins Genomics).

[0133] Genome-wide CRISPR screen

[0134] K562 cells were transduced with lentiCas9-Blast (Sanjana et al., Improved vectors and genome-wide libraries for CRISPR screening. Nat Methods. 2014;1 1 (8): 783-4) (Feng Zhang, Addgene, Addgene_52962) by spinoculation in 5 pg / mL polybrene (Merck, cat# TR-1003-G) and exposed to blasticidin (30 pg / mL; InVivoGen, cat# ant-bl-1) for 20 days to select transduced cells. Clones were obtained by single cell sorting using a FACSAria III (BD Biosciences, NJ, USA). Cas9 expression was confirmed by Western blot. The genome-wide Brunello single-guide RNA library (gRNA) (Doench et al., Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. Nat Biotechnol. 2016; 34(2): 184-91) was resynthesized to include Unique Molecular Identifiers (UMI) (Schmierer et al., CRISPR / Cas9 screening using unique molecular identifiers. Mol Syst Biol. 2017; 13(10): 945). Guides were cloned in pool (oligos synthesized by CustomArray) and packaged into lentivirus. The lentiviral backbone was based on lentiGuide-Puro (Addgene_52963), with AU-flip as previously described (Cross et al. Increasing the performance of pooled CRISPR-Cas9 drop-out screening. Sci Rep. 2016; 6: 31782).

[0135] Cas9-expressing K562 cells were transduced with the Brunello library (MOI of 0.4;1 ,000 cells / guide). Transduced cells were selected (4 pg / mL puromycin) and then split into two replicates and cultured independently before cryopreservation.

[0136] Before selection experiments with NK cells, transduced cells were thawed and maintained in K562 medium with puromycin (2 pg / mL) for a total of nine days. 80 million transduced K562 cells per replicate were co-cultured with activated NK cells at effectortarget (E:T) ratio 1 :2 in flat-bottom 96-well plates for 20 hours when approximately 60% of K562 cells had died. NK cells were depleted by puromycin treatment and surviving K562 cells were pelleted and frozen. Control cells were maintained under the same conditions except for the exposure to NK cells.

[0137] Genomic DNA was isolated using the QIAamp DNA Blood Maxi kit (Qiagen), and guide and UMI sequences were amplified by PCR as described elsewhere (Schmierer et al., 2017). The sequencing was performed using an Illumina Novaseq S1 flowcell with approximately 90 million reads allocated per sample. NGS data were analyzed with the MaGeCK (Model-based Analysis of Genome-wide CRISPR- Cas9 Knockout) software (Li et al., MAGeCK enables robust identification of essential genes from genome-scale CRISPR / Cas9 knockout screens. Genome Biol. 2014; 15(12): 554) and by UMI lineage dropout analysis (Schmierer et al., 2017). tKO K562 cells were transduction with the Brunello library (MOI of 0.4; 1 ,000 cells / guide). The transduced cells were selected using 4 pg / ml puromycin and separated into two replicates and cultured separately before freezing.

[0138] Transduced cells were defrosted and cultured in K562 medium with 2 pg / ml puromycin for a maximum of nine days before selection experiments with NK cells. 80 million K562 cells per replicate were harvested and washed before being seeded in flat-bottom 96-well plates for co-culture with polyclonally activated NK cells. After 17 hours, when 80-90% of K562 cells had died, the co-culture was stopped by pooling and addition of puromycin for NK cell depletion. Surviving K562 cells were then pelleted and frozen for subsequent DNA isolation and gRNA amplification. Identical conditions were used for control cells, except for the exposure to NK cells. Two different co-culture conditions were used, with two replicates per condition. The first condition used effectortarget (E:T) ratio 1 :1 , whereas the second condition used E:T ratio 4:1 and included a blocking antibody towards NKG2D (2.5 pg / ml, clone BAT221, Miltenyi).

[0139] Flow cytometry

[0140] Cells were generally washed in buffered sodium chloride, followed by stained for 30 minutes at 4°C. For intracellular staining, cells were fixed with Cytofix buffer (BD Biosciences) for 10 minutes at 37°C, followed by permeabilization using PhosflowTM Perm Buffer III (BD biosciences) for 30 minutes on ice. The cells were then washed twice in Stain Buffer (BD biosciences), stained with primary antibody for 30 minutes at room temperature (20-25°C). After washing, the cells were blocked with goat serum for 15 minutes at 4°C, and stained with conjugated secondary antibody for 30 minutes at 4°C. A 5-laser BD LSRFortessa (BD Biosciences, NJ, USA) was used for data acquisition and data was analyzed with BD FACSDIVA version 9.0 or FlowJo software version 10.9.0.

[0141] NKp46-, TRAIL- and NKp46 / TRAIL-transfected HEK293 were stained with anti-NKp46 and anti-TRAIL antibodies. To determine the capacity to bind TRAIL-R1, cells were incubated with 2 g of Recombinant Human TRAIL-R1-Fc Chimera Protein (R&D) for 30 minutes at room temperature. After washing, cells were stained with an anti-TRAIL-R1 antibody and live-dead marker Near-IR for 30 minutes at room temperature before data acquisition.

[0142] Functional assays

[0143] Details about reagents and antibodies are shown in Table 2. For indicated assays, NK cells were prestimulated with 500 lU / ml IL-2 and 10 ng / ml IL-15 for three to seven days before being used in functional assays. Target cells were labelled with Celltrace CFSE or violet (1 :2000, Invitrogen) before being cocultured together with NK cells for three hours at specified E:T ratio before staining with live / dead-Near IR or ToPro3 (1 :1000 / 1 :5000, ThermoFischer Scientific). For degranulation assays, an antibody towards CD107a was added before co-incubation. For interferon-y (IFNy) production, target and IL-2 / IL-15- stimulated NK cells were co-cultured for one hour before addition of 1 ug / ml Brefeldin A (BD GolgiPlug), followed by four additional hours of co-culture. After washing, the cells were stained with live / dead-Near I R, fixed and permeabilize using cytofix / cytoperm for 30min at 4°C, followed by I FNy staining.

[0144] To study the role of receptors involved in the interaction between the cells, blocking antibodies were used. Blockade was performed by pre-incubating either NK cells or target cells with receptor antibodies for 15 minutes at room temperature.

[0145] Table 2 - Reagents and antibodies

[0146] NanoBiT assay

[0147] For the NanoBiT assay, constructs for TRAIL, NKp46 and NKp30 were designed. HiBiT (Promega), followed by a GS-linker, was inserted into the cytoplasmic N-terminus of TRAIL and the LgBiT (Promega) was inserted to the cytoplasmic C-termini of NKp46 and NKp30, preceded by the same linker. All constructs were synthesized by ThermoFisher and subcloned into pcDNA3.1 (+).

[0148] At the day of transfection, with -90% confluence HEK293A cells were detached using trypsin (Gibco) and transfected at 35* 104cells in 1 ml with 1 g of plasmid DNA using Iipofectamine2000 (Invitrogen) according to manufacturer protocol. Salmon sperm DNA was used as a DNA carrier. Cells were plated at 3.5x104cells / well in poly-D-lysine-coated white 96-well plate and incubated 37°C 5% CO2 incubator for 48 hours before measuring luminescence in a BMG Labtech microplate reader.

[0149] The Nano-Gio® Luciferase Assay System (Promega (USA)) was followed using the provided manufacturers protocol. Briefly, the cell growth media was washed off and replaced with 90 pl Hanks’ balanced salt solution. 10 pl of Nano-Gio® Luciferase Assay Substrate, diluted in Nano-Gio® Luciferase Assay Buffer (both Promega) was added per well and the luminescence was measured after 5 minutes using a BMG Labtech microplate reader at main emission 460 nm.

[0150] Statistical analyses

[0151] GraphPad Prism version 10.1.1 was used for statistical analyses.

[0152] Results

[0153] NKp46-dependent CRISPR screen identifies death receptors but no other extrinsic pathway proteins to be involved in NK cell killing of K562 cells

[0154] NK cell killing of the prototypic leukemia cell line K562 is largely dependent on interactions between NKp30 and its ligand B7-H6. Knockout of key ligands render target cells more resistant to NK cell cytotoxicity and shifts the dependence to other receptor-ligand pairs. Thus, NK cell killing of a triple knockout K562 variant (NCR3LG17-, PVR7-, Nectin-2z; hereafter referred to as tKO-K562) was highly dependent on the receptors NKG2D and NKp46 in NK cell killing, as compared to WT-K562 cells (Figs. 1A and 1 B). The tKO cells were used in a genome-wide CRISPR screen where gRNA library-transduced K562 cells were selected based on susceptibility to NK cell cytotoxicity (Fig. 1 C). Many of the targeted genes among the depleted cells were related to MHC-I and the I FNy signalling pathway, including HLA-E, JAK1, JAK2, TAP2, IFNGR1 and IFNGR2 (Fig. 1 D). The gene encoding B7H6, NCR3LG1, did not appear in the results of the K562 tKO screen, as it has done in previous screens, as it was already knocked out in all cells, including the unselected control cells. Instead, the gene encoding the CD2 ligand, CD58, made the most prominent top hit among the enriched cells, over the two replicates. We could also confirm the key role of the TMEM30A gene for NK cell killing.

[0155] In order to make the interactions between NK cells and the cancer cells more dependent on the NKp46 receptor and its unknown ligand, we included a screen condition with a blocking antibody to NKG2D. The aforementioned genes in the tKO-screen appeared also in the tKO / block-screen (Fig. 1 E). However, additional genes gained in importance among the enriched cells as the killing became more NKp46- dependent. Surprisingly, these included TNFRSF10A, TNFRSF10B and TNFRSF10C. The genes TNFRSF10A-C encode the death receptors TRAIL-R1 / DR4, TRAIL-R2 / DR5 as well as TRAIL-R3 / decoy receptor-1. As the first two are involved in the extrinsic apoptosis pathway, it could be reasonable that depletion of the corresponding genes confer resistance to TRAIL-mediated NK cell killing. However, TRAIL-R3 is described to be involved in immune evasion and thought to protect against TRAIL-mediated apoptosis. Furthermore, the deletion of the extrinsic apoptosis pathway transducer protein Fas- associated death domain (FADD), as well as caspase-8 did not have an impact in the screen (Fig. 1 F). Taken together, the screen results suggested that the TRAIL-receptors had another role than initiators of the extrinsic apoptotic pathway.

[0156] TRAIL-receptor depletion but not FADD depletion reduced NK susceptibility

[0157] Next, we tested to what extent blockade of NKG2D, TRAIL receptors or NKp46 could protect B7H6nea K562 cells from NK cell cytotoxicity. As shown in in Fig. 2A, TRAIL-R blockade protected cells to the same extent as monoclonal antibodies (mAbs) to NKp46. To confirm that the extrinsic apoptotic pathway could not explain the susceptibility of the tKO cells, we knocked out FADD in B7H6nea K562 cells. If the extrinsic pathway was the major contributor, an increased resistance to NK cell killing would have been expected by depletion of the transducer protein and blockade of the TRAIL receptors would have no protective impact. However, when exposed to NK cells, the FADDnea cells did not display altered sensitivity (Figs. 2A-2C). Blockade of TRAIL-R1 / R2, in combination with NKG2D blockade, protected both FADD-competent cells and FADD-depleted cells to the same extent, indicating that TRAIL-Rs were still involved in the susceptibility to NK cells, regardless of the presence or absence of the extrinsic pathway transducer protein.

[0158] We further knocked out the TRAIL-receptors in B7H6ne9 K562 cells. TRAILR1ne9TRAILR2ne9 cells (hereafter referred to as TRAIL-R1 / R2ne9) were less sensitive to NK cell cytotoxicity, which was even more evident in the presence of NKG2D blockade, when killing is highly NKp46 dependent (Fig. 2D). Strikingly, the protective effect of NKp46 blockade was virtually abolished in TRAI L-R1 / R2nea cells.

[0159] As results from a cytotoxicity assay potentially converge the outcome from both the granzyme B-pathway as well as the extrinsic apoptosis pathway, we repeated the setup in NK cell degranulation and cytokine production experiments. When exposed to in TRAIL-R1 / R2ne9 K562 cells, significantly fewer NK cells degranulated or produced IFNy, as compared to NK cells exposed to control K562 cells (Figs. 2E-2F, Fig. 3A). In accordance with the data from the cytotoxicity experiments, presence of NKG2D blockade highlighted the impact of TRAIL-R depletion. Notably, the effect of NKp46 blockade was strongly reduced when the TRAIL receptors were lacking.

[0160] Next, we sought to validate the findings in other malignancies. We screened a number of cancer cell lines to identify cell lines, in which NKp46 blockade had a clear impact on NK cell degranulation or cytotoxicity (Fig. 3B). Identified cell lines were evaluated for TRAIL-R expression, the genes encoding TRAIL-R1 and -R2 were targeted by CRISPR / Cas9, and polyclonal TRAIL-R-negative cells were isolated by FACS (Figs. 30, 3D). As shown in Fig. 2G, NK cell degranulation against the prostate cancer cell line, PC3, was clearly reduced by blockade of NKp46 and / or NKG2D. TRAIL-R-deficient cells triggered a significantly lower degranulation response in NK cells. Importantly, while NKG2D blockade reduced NK cell degranulation, blocking antibodies to NKp46 failed to reduce NK cell degranulation, which lends further support to the notion that TRAIL-Rs are implicated in NKp46-dependent recognition of malignant cells.

[0161] TRAILIOWNK cells do not recognize target cells via NKp46

[0162] Freshly isolated, resting NK cells express very low levels of TRAIL. By stimulation with IL-2 in combination with IL-15, TRAIL levels increase (Fig. 4A). We investigated to what extent TRAI Llowand TRAILS used the same receptors for recognition of malignant cells. Resting NK cells were less cytotoxic than I L-2 / IL- 15-prestimulated cells. NKG2D blockade reduced killing of B7H6ne9 K562 by both resting and prestimulated NK cells. By contrast, NKp46 blockade only had an impact on stimulated NK cells that expressed TRAIL but failed to impact on cytotoxicity exerted by resting NK cells that lacked TRAIL expression. As shown above, the NKp46-dependent recognition observed in stimulated cells was completely abolished when TRAIL-R1 and TRAIL-R2 were depleted, whereas no change was seen for resting NK cells (Figs. 4B-4C).

[0163] Next, we tested the ability of TRAIL-negative wt-NK92 cells and NK92 cells overexpressing NKp46 (kindly provided by Prof K Campbell) to kill wt-K562 and B7H6ne9 K562 cells. Despite much higher expression of NKp46 (Fig. 4D), preliminary results indicate that NKp46-NK92 cells did not display stronger cytotoxicity responses than wt-NK92 cells (Fig. 4E). Notably, no reduction of NK92 cytotoxicity was observed in the presence of NKp46 blockade, suggesting that these cells were unable to recognize the target cells via NKp46 in the absence of TRAIL (Fig. 4F).

[0164] NKp46 and TRAIL interact in the NK cell plasma membrane

[0165] The findings above suggest that NKp46 and TRAIL cooperate to recognize TRAIL-R-expressing cancer cells. To investigate whether TRAIL and NKp46 interact in the plasma membrane, we used the NanoBiT split luciferase technology, in which two different parts of the luciferase are linked to two proteins of interest. A functional luciferase will only be generated in the cell if the two proteins interact closely to each other. As shown in Fig. 4G, a NKp46-luciferase construct generated significantly more signal than corresponding construct with NKp30, suggesting a close connection between NKp46 and TRAIL in the NK cell plasma membrane.

[0166] Next, we overexpressed TRAIL in HEK-293 cells and transfected TRAIL-expressing clones with NKp46. Interestingly, co-transfection with NKp46 enhanced TRAIL expression in HEK293 cells in all clones evaluated. Furthermore, a blocking antibody to NKp46 reduced TRAIL staining in TRAIL / NKp46- expressing cells but did not affect TRAIL staining of cells lacking NKp46 expression (Fig. 4H). Taken together, these data suggest that NKp46 and TRAIL interact closely in cis in the NK cell plasma membrane.

[0167] Findings indicate that NKp46 binds to TRAIL-R receptors

[0168] Traditionally, a common strategy to identify NK cell receptor ligands has been to immobilize the receptor in a resin and expose it to tumor cell lysates with the aim to identify ligands candidates by mass spectrometry. For NKp46, such attempts have failed, leaving the receptor an orphan for many years. The results above offer an explanation to this; the NK cell capacity to bind TRAIL-Rs to generate NKp46- mediated signalling may be highly dependent on TRAIL - TRAIL-R interactions. To test whether NKp46 has a role as a binding partner, we tested the capacity of wild-type and NKp46-transfected HEK293 cells to bind a soluble TRAIL-R1-Fc fusion protein. As seen in Fig. 4I, there was a small shift in transfected HEK293 cells suggesting that NKp46 can bind TRAIL-R1 also in the absence of TRAIL. Furthermore, TRAIL-R1-Fc fusion protein binding to TRAIL-R1 / NKp46-transfected cells was strongly inhibited by a blocking antibody to NKp46 (Fig. 4J).

[0169] In the present Example, we used a highly NKp46-driven model system in combination with an unbiased CRISPR screen on a genome-wide scale to identify novel NKp46 ligand candidates. By deleting genes encoding ligands and by addition of blocking antibodies, we could stepwise increase the NKp46- dependence of the assay and thus discern an augmented effect of a putative ligand, proving the reliance of the system.

[0170] The resulting gene hits revealed the TRAIL-receptors to be highly implicated in recognition of target cells by NK cells. This was seen already in the relatively NKp46-dependent tKO model but was even further augmented by concomitant blockade of NKG2D. The K562 cell line is known to be insensitive to TRAIL- mediated apoptosis. However, it could not be excluded that the death receptor pathway would turn out to be a key mechanism of killing in our model system. Nevertheless, for the extrinsic pathway to be functional, certain transducer proteins are required. These include the FADD protein as well as caspase 8, which are essential for the DISC complex assembly and apoptosis execution. Neither FADD nor caspase 8 were observed among the top hits in the screen. This implied that it was not as part of the extrinsic apoptosis pathway that the TRAIL-receptors showed up in the screen. The experiments using FADD-deficient target cells in combination with TRAIL-R1 / 2 blockade show that lacking the essential extrinsic apoptosis pathway transducer protein did indeed not impact the susceptibility to NK cell killing, suggesting that the TRAIL-R1 / 2 instead were involved in the GzmB-pathway. Interestingly, the gene encoding TRAIL-R3 was also a top-enriched hit in the screen. As this is considered a decoy receptor due to its lack of intracellular signalling domain and ability to prevent TRAIL-induced apoptosis, its appearance strengthened the argument that the TRAIL-receptors were involved in the GzmB pathway of killing. Furthermore, TRAIL-R1 / R2-deficient cells were not only less sensitive to NK cell cytotoxicity but, they also induced lower NK cell degranulation as well as I FNy-production. This is in accordance with a recent report showing that direct engagement of TRAIL with TRAIL-R1 -3 results in NK cell degranulation and I FNy-production (Hofle et al. Engagement of TRAIL triggers degranulation and I FNy production in human natural killer cells. EMBO Rep. 2022; 23: e54133). Importantly, NKp46 blockade did only protect K562 cells with intact expression of TRAIL-Rs, highlighting the interaction between NKp46 and the TRAIL-Rs. The NanoBiT experiments in our study indicated that NKp46 and TRAIL interact with each other in the NK cell membrane. Interestingly, the expression level of TRAIL was enhanced if the cells also overexpressed NKp46, suggesting a TRAIL dependence on NKp46 also in human NK cells. The findings that NKp46 can be found in close connection with TRAIL offer interesting possibilities when it comes to the interaction between NKp46, the TRAIL-Rs and TRAIL. NKp46-expressing HEK293 cells displayed slightly higher binding of the chimeric TRAIL-R1-Fc protein than non-expressing cells. The staining experiments also point in the direction that NKp46 / TRAIL co-expression enhances binding of the TRAIL- Rs.

[0171] In conclusion, the findings suggest that TRAIL-Rs have a role that goes beyond initiation of the extrinsic death pathway and that they serve as ligands to NKp46 (see Fig. 6). This finding opens up new avenues of study to develop novel immunotherapeutic strategies. The TRAIL-Rs have long been considered promising therapeutic targets due to their expression profile being rather limited to malignant cells. Developed therapies, including TRAIL-R agonistic antibodies and recombinant TRAIL have thus far lacked truly convincing clinical results. Molecules or chimeric antigen receptor constructs that combine both TRAIL and NKp46 may have the potential to induce stronger external death pathway triggering signals that can translate in improved clinical efficacy.

[0172] EXAMPLE 2

[0173] Cell cultures

[0174] PC3 prostate cancer cells was maintained in IMDM with 10-15% HI FCS and 1 % PEST. M14 melanoma cancer cells, SK-N-AS neuroblastoma cancer cells, SK-OV-3 ovarian cancer cells and L428 Hodgkin lymphoma cancer cells were cultured in RPMI, supplemented with 10% HI FCS, 2 mM L-glutamine, and 1 % PEST. OVCAR-3 ovarian cancer cells were maintained in RPMI, supplemented with 20% HI FCS, 0.01 mg / ml bovine insulin (Sigma-Aldrich) and 1 % PEST. A172 glioblastoma cancer cels were cultured in Dulbecco’s Modified Eagle Medium (DMEM, Gibco) supplemented with 10% HI FCS, 2 mM L- glutamine, and 1 % PEST. A549 non-small cell lung cancer cells were maintained in Ham’s F-12K Medium (Gibco) supplemented with 10% HI FCS, and 1 % PEST.

[0175] Addition to CRISPR / Cas9 engineered cell lines

[0176] TRAIL-R1 / R2neg PC3 and M14 cells were sorted polyclonally.

[0177] Gene expression silencing with siRNA Lipofectamine RNAiMAX (Thermo Fischer Scientific) was used to deliver TRAILR1 - / R2-targeting siRNA (Thermo Fisher Scientific) in SKNAS with a reverse-transfection method. SiRNAs and Lipofectamine were first diluted in Opti-MEM Reduced Serum Medium (Gibco), mixed and co-incubated for 15 minutes in cell-culture plates. SK-N-AS cells were then added to siRNA-lipid complex. Knock-down was analyzed after 72 h by flow cytometry staining. Negative Control DsiRNA (IDT) was used as control.

[0178] TRAIL and NKp46 expression knock-down were performed after 72 h IL-2 / IL-15 treatment with the Accell Human TNFSF10 siRNA and Accell Human NCR1 siRNA (Dharmacon Reagents). Accell Non-targeting siRNA #1 was used as control. The results are presented in Figs. 12a- 12u.

[0179] Table 3 - sequence information

[0180] The embodiments described above are to be understood as a few illustrative examples of the present invention. It will be understood by those skilled in the art that various modifications, combinations and changes may be made to the embodiments without departing from the scope of the present invention. In particular, different part solutions in the different embodiments can be combined in other configurations, where technically possible. The scope of the present invention is, however, defined by the appended claims.

Claims

CLAIMS1. A tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptor (TRAIL-R) binding molecule comprising an extracellular domain of TRAIL, or a TRAIL-R binding fragment thereof, and an extracellular domain of NKp46, or a fragment thereof.

2. The TRAIL-R binding molecule according to claim 1 , wherein the TRAIL-R binding molecule binds to TRAIL-R1 and / or TRAIL-R2.

3. The TRAIL-R binding molecule according to claim 1 or 2, wherein the extracellular domain of TRAIL comprises, preferably consists of, amino acid numbers 39 to 281 of human TRAIL as defined in SEQ ID NO: 4.

4. The TRAIL-R binding molecule according to any one of claims 1 to 3, wherein the TRAIL-R binding fragment is selected from the group consisting of amino acid nos. 1 14 to 281 of SEQ ID NO: 4, amino acid nos. 102 to 281 of SEQ ID NO: 4, amino acid nos. 112 to 281 of SEQ ID NO: 4, amino acid nos. 121 to 281 of SEQ ID NO: 4, amino acid nos. 122 to 281 of SEQ ID NO: 4, amino acid nos. 123 to 281 of SEQ ID NO: 4, amino acid nos. 95 to 281 of SEQ ID NO: 4, amino acid nos. 114 to 281 of SEQ ID NO:

4. amino acid nos. 116 to 281 of SEQ ID NO: 4, amino acid nos. 119 to 281 of SEQ ID NO: 4, amino acid nos. 120 to 281 of SEQ ID NO: 4, amino acid nos. 121 to 281 of SEQ ID NO: 4, and amino acid nos. 121 to 281 of SEQ ID NO: 4.

5. The TRAIL-R binding molecule according to claim 4, wherein the TRAIL-R binding fragment comprises, preferably consists of, amino acid numbers 114 to 281 of human TRAIL as defined in SEQ ID NO: 4.

6. The TRAIL-R binding molecule according to any one of claims 1 to 5, wherein the extracellular domain of NKp46 is selected from the group consisting of amino acid nos. 22-258 of SEQ ID NO: 7, amino acid nos. 153 to 175 of SEQ ID NO: 7, amino acid nos. 215 to 254 of SEQ ID NO: 7, amino acid nos. 22-120 of SEQ ID NO: 7, and amino acid nos. 121-254 of SEQ ID NO: 7.

7. The TRAIL-R binding molecule according to claim 6, wherein the extracellular domain of NKp46 comprises, preferably, consists of, amino acid numbers 22 to 258 of human NKp46 as defined in SEQ ID NO: 7.

8. The TRAIL-R binding molecule according to any one of claims 1 to 7, further comprising a linker interconnecting the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof, and the extracellular domain of NKp46, or the fragment thereof.

9. The TRAIL-R binding molecule according to any one of claims 1 to 8, wherein the TRAIL-R binding molecule is a multispecific TRAIL-R binding molecule comprising: the extracellular domain of TRAIL, or the TRAIL-R binding fragment thereof; the extracellular domain of NKp46, or the fragment thereof; and a Fc polypeptide or an antibody, or an antigen-binding fragment thereof.

10. The TRAIL-R binding molecule according to claim 9, wherein the Fc polypeptide is an immunoglobulin G (IgG) Fc polypeptide binding to cluster of differentiation 16 (CD16).11 . The TRAIL-R binding molecule according to claim 9, wherein the antibody, or the antigen-binding fragment thereof, binds specifically to cluster of differentiation 3 (CD3).

12. A TRAIL-R binding molecule according to any one of claims 1 to 11 for use as a medicament.

13. A TRAIL-R binding molecule according to any one of claims 1 to 11 for use in treatment of cancer.

14. The TRAIL-R binding molecule for use according to claim 13, wherein the cancer is selected from the group consisting of colorectal cancer, non-Hodgkin's lymphoma, Hodgkin’s lymphoma, cervical cancer, multiple myeloma, hepatocellular carcinoma, peritoneal carcinomatosis, peritoneal mesothelioma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia, ovarian cancer, pancreatic cancer, head and neck squamous cell carcinoma, neuroblastoma, glioblastoma, malignant melanoma, and renal cancer, preferably selected from the group consisting of prostate cancer, malignant melanoma, neuroblastoma, ovarian cancer, Hodgkin’s lymphoma, glioblastoma, non-small cell lung cancer and leukemia, and more preferably selected from the group consisting of prostate cancer and leukemia.

15. A TRAIL-R binding molecule according to any one of claims 1 to 11 for use in treatment of a viral infection.

16. A nucleic acid molecule encoding a TRAIL-R binding molecule according to any one of claims 1 to 11.

17. An expression vector comprising a promoter and a nucleic acid molecule according to claim 16 operatively controlled by the promoter.

18. A host cell comprising an expression vector according to claim 17, wherein the promoter is constitutively active or inducible active in the host cell.

19. A chimeric antigen receptor (CAR) comprising: an antigen recognition domain comprising a TRAIL-R binding molecule according to any one of claims 1 to 8; a transmembrane domain; and an intracellular domain.

20. The CAR according to claim 19, wherein the transmembrane domain is selected from the group consisting of all, or a portion, of the transmembrane domain of cluster of differentiation 28 (CD28), all, or a portion, of the transmembrane domain of CD8a, all, or a portion, of the transmembrane domain of CD27, all, or a portion, of the transmembrane domain of CD137, all, or a portion, of the transmembrane domain of CD134, all, or a portion, of the transmembrane domain of CD3s, all, or a portion, of the transmembrane domain of CD3^, all, or a portion, of the transmembrane domain of CD3y, all, or a portion, of the transmembrane domain of CD35, all, or a portion, of the transmembrane domain of TCRa, and all, or a portion, of the transmembrane domain of TCR0.21 . The CAR according to claim 19 or 20, wherein the intracellular signaling domain is selected from the group consisting of zeta chain of cluster of differentiation 3 (CD3Q, CD28, CD137, ICOS, CD27, CD40, CD134, DN AX-activating protein 10 (DAP10), DAP12 and / or Myd88.

22. The CAR according to any one of claims 19 to 21 , further comprising at least one cytokine secretion domain and / or at least one transcription factor domain.

23. A nucleic acid molecule encoding a CAR according to any one of claims 19 to 22.

24. A natural killer (NK) cell comprising a CAR according to any one of claims 19 to 22 or a nucleotide sequence encoding a CAR according to claim 19.

25. A T cell comprising a CAR according to any one of claims 19 to 22 or a nucleotide sequence encoding a CAR according to claim 23.

26. The NK cell according to claim 24 or the T cell according to claim 25 for use as a medicament.

27. The NK cell according to claim 24 or the T cell according to claim 25 for use in treatment of cancer.

28. The NK cell or T cell for use according to claim 27, wherein the cancer is selected from the group consisting of colorectal cancer, non-Hodgkin's lymphoma, Hodgkin’s lymphoma, cervical cancer, multiple myeloma, hepatocellular carcinoma, peritoneal carcinomatosis, peritoneal mesothelioma, lung cancer, breast cancer, pancreatic cancer, prostate cancer, leukemia, ovarian cancer, pancreatic cancer, head and neck squamous cell carcinoma, neuroblastoma, glioblastoma, malignant melanoma, and renal cancer, preferably selected from the group consisting of prostate cancer, malignant melanoma, neuroblastoma, ovarian cancer, Hodgkin’s lymphoma, glioblastoma, non-small cell lung cancer and leukemia, and more preferably selected from the group consisting of prostate cancer and leukemia.

Images