Anti-NKG2a immunocytokines and uses thereof in treatment

Abstract

Anti-NKG2A immunocytokines are provided. Accordingly, there is provided a composition of matter comprising at least one fusion polypeptide comprising an anti-NKG2A antibody; and an immune cell-activating cytokine. Also provided are anti-NKG2A antibodies. Also provided are polynucleotides encoding the composition of matter or antibody, host cells comprising the composition of matter or antibody and methods of producing and using same in treatment.

Description

[0001] ANTI-NKG2A IMMUNOCYTOKINES AND USES THEREOF IN TREATMENT

[0002] RELATED APPLICATION / S

[0003] This application claims the benefit of priority of US Provisional Patent Application No. 63 / 777,013 filed on March 25, 2025, the contents of which are incorporated herein by reference in their entirety.

[0004] SEQUENCE LISTING STATEMENT

[0005] The XML file, entitled 106712.xml, created on March 24, 2026, comprising 413,696 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.

[0006] FIELD AND BACKGROUND OF THE INVENTION

[0007] The present invention, in some embodiments thereof, relates to anti-NKG2A immunocytokines and uses thereof in treatment.

[0008] Cancer remains a leading cause of death worldwide, accounting for approximately 10 million deaths annually (Bray et al., 2024, CA: A Cancer Journal for Clinicians, 74(3), pp. 229-263. Available at: https: / / doi(dot)org / 10(dot)3322 / caac(dot)21834). While immunotherapy has revolutionized cancer treatment, a significant number of patients either do not respond to these therapies or experience early disease relapse (Tan, Li and Zhu, 2020, Biomedicine & Pharmacotherapy, 124, p. 109821. Available at: https : / / doi(dot)org / 10(dot) 1016 / j (dot)biopha(dot)2020(dot) 109821). Cytokine-based cancer therapy, which utilizes small proteins crucial for cell signaling, has shown promise in modulating the immune system to combat cancer. However, the clinical success of cytokine therapies has been limited over the past four decades. FDA-approved cytokine therapies such as IFN-a and IL-2 have shown modest response rates and are associated with toxicities (Conlon, Miljkovic and Waldmann, 2019, The Official Journal of the International Society for Interferon and Cytokine Research, 39(1), pp. 6-21. Available at: https: / / doi(dot)org / 10(dot)1089 / jir(dot)2018(dot)0019). Key reasons for initial failure of cytokines for cancer immunotherapy include limited stability; side effects and toxicity which stem from the need to reach high local doses by systemic treatment; and the fact that cytokines exhibit pleiotropic effects on various cell types (Berraondo et al., 2019, British Journal of Cancer, 120(1), pp. 6-15. Available at: https: / / doi(dot)org / 10(dot)1038 / s41416-018-0328-y). While cytokines may elicit a favorable response from the intended targeted cell type, this can be accompanied by induction of inhibitory ligands such as programmed cell death ligand1 (PD-L1) and activation of off-target cells, for example of immunosuppressive regulatory T cells (Tregs). Consequently, the therapeutic efficacy of cytokines has been limited.

[0009] Recent advancements in cytokine engineering and the development of immunocytokines (cytokines coupled to antibodies) have opened up new opportunities to overcome these challenges (Shi, Liu and Lu, 2024, Acta Pharmaceutica Sinica B, 14(11), pp. 4649-4664. Available at: https: / / doi(dot)org / 10(dot)1016 / j(dot)apsb(dot)2024(dot)07(dot)024). By fusing cytokines to antibodies, their half-life can be extended, and they can be delivered to specific cell types or states expressing defined targets, avoiding off-target stimulation. Furthermore, engineering of cytokines can fine-tune their specific functions in different cell types, such as the IL-2superkine modification with increases affinity for the IL-2RP subunit of the IL-2 receptor (Levin et al., 2012, Nature, 484(7395), pp. 529-533. Available at: https : / / doi(dot)org / 10(dot)l 038 / nature 10975).

[0010] Nevertheless, the identification of optimal targets and cytokine combinations for immunocytokines that effectively stimulate anti-tumor immune responses, particularly in cytotoxic immune cells, remains an exceptionally complex challenge, demanding an intricate and comprehensive understanding of anti-cancer cytokines and their intricate dynamics within the tumor microenvironment.

[0011] NKG2A, encoded by the KLRC1 gene, is an inhibitory receptor that has been identified as an immune checkpoint in both NK and CD8 T cells (Andre et al., 2018, Cell, 175(7), pp. 1731-1743. el3. Available at: https: / / doi(dot)org / 10(dot)1016 / j(dot)cell(dot)2018(dot)10(dot)014.). This receptor interacts with HLA-E expressed on tumor cells, potentially suppressing anti-tumor immune responses. Studies have shown that blocking the NKG2A-HLA-E interaction can lead to beneficial anti-tumor responses in preclinical murine tumor models (Andre et al., 2018). This finding has translated to clinical research, with promising results observed in phase II / III clinical trials using Monalizumab (Innate Pharma), an anti-NKG2A antibody, both as a monotherapy and in combination with anti-PD-1 treatments. Given its role in modulating immune responses, NKG2A has been suggested as a potential target for immunomodulation strategies, including the development of immunocytokines (see e.g., US Patent Application Publication Nos. US20240309062, US20240092854 and US20230167164; Australian Patent Application Publication No. AU2013227477; and International Patent Application Publication No. WO2022140797 and WO2018217989).

[0012] SUMMARY OF THE INVENTION

[0013] According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising at least one fusion polypeptide comprising:(a) an anti-NKG2A antibody;

[0014] (b) an immune cell-activating cytokine; and

[0015] (c) a masking agent that masks the immune cell-activating cytokine and an amino acid sequence which is cleaved by a tumor microenvironment-associated protease, such that when the fusion polypeptide is in a vicinity of a tumor, the tumor microenvironment-associated protease cleaves the masking agent thereby exposing the immune cell-activating cytokine.

[0016] According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising at least one fusion polypeptide comprising:

[0017] (a) an anti-NKG2A antibody; and

[0018] (b) an immune cell-activating cytokine selected from the group consisting of IL-2 and IL- 12.

[0019] According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising at least one fusion polypeptide comprising:

[0020] (a) an antagonistic anti-NKG2A antibody; and

[0021] (b) an immune cell-activating cytokine.

[0022] According to some embodiments of the invention, the immune cell-activating cytokine is selected from the group consisting of IL-2, IL-12, IL-12, IL-15, IL-18, IL-21 and GM-CSF.

[0023] According to some embodiments of the invention, the immune cell-activating cytokine is selected from the group consisting of IL-2 and IL- 12.

[0024] According to some embodiments of the invention, the immune cell-activating cytokine is modified to reduce activity, alter a binding property and / or increase stability.

[0025] According to some embodiments of the invention, the immune cell-activating cytokine is in its native form.

[0026] According to some embodiments of the invention, the at least one fusion polypeptide further comprises:

[0027] (c) a masking agent that masks the immune cell-activating cytokine and an amino acid sequence which is cleaved by a tumor microenvironment-associated protease, such that when the fusion polypeptide is in a vicinity of a tumor, the tumor microenvironment-associated protease cleaves the masking agent thereby exposing the immune cell-activating cytokine.

[0028] According to some embodiments of the invention, the tumor microenvironment-associated protease is tumor microenvironment-associated immune protease.

[0029] According to some embodiments of the invention, the tumor microenvironment-associated protease is secreted by a myeloid-derived suppressor cell (MDSC).According to some embodiments of the invention, the MDSC is a tumor associated macrophage (TAM).

[0030] According to some embodiments of the invention, the protease is selected from the group consisting of matrix metalloproteinase (MMP), ADAM-9, ADAMDEC1, CathsepinL and CathepsinK.

[0031] According to some embodiments of the invention, the protease is a matrix metalloproteinase (MMP).

[0032] According to some embodiments of the invention, the MMP is selected from the group consisting of MMP2, MMP9, MMP12, MMP14 and MMP19.

[0033] According to some embodiments of the invention, the MMP is MMP14.

[0034] According to some embodiments of the invention, the tumor microenvironment-associated protease is secreted by an NK and / or cytotoxic CD 8+ T cell.

[0035] According to some embodiments of the invention, the protease is Granzyme B.

[0036] According to some embodiments of the invention, the at least one fusion polypeptide is a dimer.

[0037] According to some embodiments of the invention, the anti-NKG2A antibody comprises an antigen recognition domain comprising complementarity determining regions (CDRs) CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 or the light chain and heavy chain at least 80 % identical to those of an antibody selected from the group consisting of 30B1-1, IF7-2, 3E9-1, 5E3-2, 6B11-1, 10E2-1 and 12E2-1.

[0038] According to an aspect of some embodiments of the present invention there is provided an antibody comprising an antigen recognition domain which binds NKG2A and comprises complementarity determining regions (CDRs) CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 or the light chain and heavy chain at least 80 % identical to those of an antibody selected from the group consisting of 30B1-1, IF7-2, 3E9-1, 5E3-2, 6B11-1, 10E2-1 and 12E2-1.

[0039] According to some embodiments of the invention, the at least 80 % is at least 85 %.

[0040] According to some embodiments of the invention, the at least 80 % is at least 90 %.

[0041] According to some embodiments of the invention, the at least 80 % is at least 95 %.

[0042] According to some embodiments of the invention, the at least 80 % is 100 %.

[0043] According to an aspect of some embodiments of the present invention there is provided a polynucleotide encoding the composition of matter.

[0044] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising the polynucleotide and a cis-acting regulatory element for directing expression of the polynucleotide.According to an aspect of some embodiments of the present invention there is provided a host cell comprising the composition of matter or a polynucleotide or a nucleic acid construct encoding it.

[0045] According to an aspect of some embodiments of the present invention there is provided a method of producing at least one fusion polypeptide, the method comprising introducing into a host cell the polynucleotide or nucleic acid, or culturing the host cell.

[0046] According to some embodiments of the invention, the method comprising isolating the at least one fusion polypeptide.

[0047] According to an aspect of some embodiments of the present invention there is provided a polynucleotide encoding the antibody.

[0048] According to an aspect of some embodiments of the present invention there is provided a nucleic acid construct comprising the polynucleotide and a cis-acting regulatory element for directing expression of the polynucleotide.

[0049] According to an aspect of some embodiments of the present invention there is provided a host cell comprising the antibody or a polynucleotide or a nucleic acid construct encoding it.

[0050] According to an aspect of some embodiments of the present invention there is provided a method of producing an anti-NKG2A antibody, the method comprising introducing into a host cell the polynucleotide or nucleic acid construct, or culturing the host cell.

[0051] According to some embodiments of the invention, the method comprising isolating the antibody.

[0052] According to an aspect of some embodiments of the present invention there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter, thereby treating the cancer in the subject.

[0053] According to an aspect of some embodiments of the present invention there is provided the composition of matter, for use in treating cancer in a subject in need thereof.

[0054] According to an aspect of some embodiments of the present invention there is provided a method of treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody, thereby treating the cancer in the subject.

[0055] According to an aspect of some embodiments of the present invention there is provided the antibody, for use in treating an inflammatory disease in a subject in need thereof.

[0056] According to some embodiments of the invention, the inflammatory disease is cancer.According to some embodiments of the invention, the cancer is an NKG2A rich cancer. According to some embodiments of the invention, the cancer is selected from the group consisting of lung, melanoma, breast, ovarian, colorectal, liver, renal, head and neck and endometrial cancer.

[0057] Unless otherwise defined, all technical and / or scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention pertains. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of embodiments of the invention, exemplary methods and / or materials are described below. In case of conflict, the patent specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and are not intended to be necessarily limiting.

[0058] BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0059] Some embodiments of the invention are herein described, by way of example only, with reference to the accompanying drawings. With specific reference now to the drawings in detail, it is stressed that the particulars shown are by way of example and for purposes of illustrative discussion of embodiments of the invention. In this regard, the description taken with the drawings makes apparent to those skilled in the art how embodiments of the invention may be practiced.

[0060] In the drawings:

[0061] FIG. 1 is a schematic illustration of the immunosuppressive tumor microenvironments (TME) wherein tumor cells, TAMs and Tregs suppress NK and effector T cells and an overview of the generation of immunocytokines targeting CD8 T cells as well as y5 T cells and NK cells via NKG2A, referred to herein as “Cytotoxic-Enhancers” (CytEs) or “NK cell-targeted immunocytokines and T cell enhancers” (NiTEs), having a tumor-conditioned cytokine activity while avoiding Tregs to restore cytotoxic functions according to some embodiments of the invention.

[0062] FIG. 2 is a schematic illustration of IL-2, IL-12, IL-15, IL-18, IL-21 or GM-CSF cytokine receptors and their subunits. IL-2 and IL- 15 bind the same receptor.

[0063] FIG. 3 shows expression of individual cytokine receptor subunits and distribution of cells expressing combined cytokine receptor subunits for IL-2, IL-12, IL-15, IL-18, IL-21 or GM-CSF across 4 human cancer types (lung, breast, colon, ovarian cancer (Qian et al. 2020, Cell Res, 30, 745-762)], as assessed by scRNAseq.

[0064] FIG. 4 shows expression of individual cytokine receptor subunits and distribution of cells expressing combined cytokine receptor subunits for IL-2, IL-12, IL-15, IL-18, IL-21 or GM-CSFin the immune compartment across 4 human cancer types [lung, breast, colon, ovarian cancer (Qian et al. 2020, Cell Res, 30, 745-762)], as assessed by scRNAseq.

[0065] FIG. 5 shows expression of individual cytokine receptor subunits and distribution of cells expressing combined cytokine receptor subunits for IL-2, IL-12, IL-15, IL-18, IL-21 or GM-CSF in immune cells isolated from s.c. implanted murine 4T1 tumors, as assessed by scRNAseq.

[0066] FIG. 6 is a schematic representation of generation and mode of action of genetically modified tumor cell lines to study cytokine responses in preclinical murine tumors (4T1 model). Modified tumor cell lines are s.c. injected into murine hosts. In established tumors, cytokine expression can be induced by doxycycline administration (i.p.). In addition to the cytokines, also a barcode (Pro-Code system) is expressed allowing spatial identification of cytokine expressing tumor cells.

[0067] FIG. 7 shows cytokine expression in genetically modified mouse 4T1 tumor cells which were in-vitro stimulated with doxycycline for 72 hours. Cells were harvested and expression of genes encoding cytokines was assessed by qPCR. The figure shows that indeed IL-2s, IL- 12, IL-15, IL-18DR and GM-CSF are expressed specifically upon doxycycline stimulation, but not in control conditions.

[0068] FIG. 8 shows tumor dynamics upon cytokine treatments. Genetically modified mouse 4T1 tumor cells engineered to inducibly over-express cytokines were s.c. injected into murine recipients. In established tumors, cytokines were released in the tumor upon i.p. doxycycline injection on day 12. PBS injections were used as controls. Plots show tumor growth curves, tumor size and weight on day 19 of tumors exposed to control (no cytokine), IL-2s, IL-12, IL-15sushi, IL-18DR, IL-2 IL or GM-CSF. Normalized plots show direct comparisons of tumor volumes and weights.

[0069] FIG. 9 shows frequencies of T and NK cell subsets out of total immune cells isolated from 4T1 tumors exposed to the indicated cytokines compared to control, as assessed by scRNAseq.

[0070] FIG. 10 shows tumor dynamics upon cytokine combination treatments. Mixtures of genetically modified mouse 4T1 tumor cells engineered to inducibly over-express IL-2sk and 4T1 tumor cells engineered to inducibly over-express IL-12, IL-15sushi, IL-18DR, IL-21 or GM-CSF (in a ratio of 1 : 1) were s.c. injected into murine recipients. In established tumors, cytokine combinations were released in the tumor upon i.p. doxycycline injection on day 12. PBS injections were used as controls. Plots show tumor growth curves, tumor size and weight on day 19 of tumors exposed to control (no cytokine) and combination of IL-2sk with other indicated cytokines. Normalized plots show direct comparisons of tumor volume and weights.FIG. 11 shows frequencies of T and NK cell subsets out of total immune cells isolated from 4T1 tumors exposed to cytokine combinations, as assessed by scRNAseq.

[0071] FIG. 12 is a schematic illustration showing identification of targets enhancing CD8 T and NK cells for novel immunocytokines with a data-driven approach. Genes which are highly expressed in T cells, NK cells or gamma delta T cells in comparison to Tregs were identified in a pan-cancer dataset of scRNAseq data. Further filtering included less than 1 % expression in Tregs, and any other off-target cell type such as myeloid cells, B cells, tumor cells and stromal cells (such as epithelial and endothelial cells). The top 159 genes were then filtered for genes encoding surface proteins to obtain 44 top candidate genes.

[0072] FIG. 13 shows a heatmap depicting the expression of the top 44 target genes across different T and NK cell subsets in a pan-cancer scRNAseq dataset (Qian et al. 2020, Cell Res, 30, 745-762) including samples of lung, breast, colorectal and ovarian cancer. The genes are expressed in CD8 T, NK or y5 T cell clusters and are ordered according to the number of hits in CD8 T, NK or y5 T cell clusters from top to bottom. Gene targets for competitor immunocytokines are shown at the bottom.

[0073] FIG. 14 shows a UMAP displaying T and NK cells subsets in pan-cancer scRNAseq data (lung, breast, colorectal and ovarian cancer) and overlayed expression of the top target KLRC1 (encoding NKG2A) as well as competitor targets.

[0074] FIG. 15 shows heatmaps (top) and scatter plots (bottom) depicting NKG2A and competitor protein expression in matched blood, adjacent lung and lung cancer tissue samples of 3 human lung cancer patients, as assessed by Flow cytometry.

[0075] FIG. 16 shows scatter plots depicting NKG2A / C / E protein expression in murine B16-F10 or 4T1 tumors and spleens of naive BL6 or Balb / c mice or tumor bearing mice, as assessed by Flow cytometry using the 20D5 antibody clone.

[0076] FIG. 17 is a schematic illustration of exemplary novel designed anti-mouse immunocytokines. The immunocytokines are based on the anti-mouse 20D5 clone (targeting NKG2A / C / E) and are linked to IL-2sk, IL-2mutant with IL-2Ra bias, IL-2mutant attenuated or IL-12. Cleavable blocking units (IL2RB, IL2RA or IL12RB1) with MMP-14 or Granzyme B cleavage sites allow tumor- specific release of blocking unit and thereby restoration of cytokine activity.

[0077] FIG. 18 shows representative SDS page gel plots of the indicated anti-NKG2A / C / E immunocytokines. Ml: Protein Marker. R: Reducing condition. NR: Non-reducing condition.

[0078] FIG. 19 shows an optical density plot depicting ELISA results of the indicated immunocytokines confirming binding to recombinant murine NKG2A.FIG. 20 shows optical density plots depicting dose response of the indicated immunocytokines using IL-2 HEK blue reporter cells. HEK blue reporter cells reporting the activity of high affinity IL-2 receptor (IL2Ralpha / IL2Rbeta / IL2Rgamma) or medium affinity IL-2 receptor (IL2Rbeta / IL2RG) were used. The assay confirms that IL-2sk, IL-2 attenuated, IL-2 mutein are active and that activity of blocked IL-2sk and IL-2mutein is restored upon cleavage by MMP14. IL-2sk has similar activity in both high and medium affinity receptors cell lines in accordance with its modifications increasing its affinity to IL2RB and thereby avoiding the natural bias of wildtype IL-2 for high affinity IL-2 receptors (in which IL2RA conveys high affinity for IL-2). As intended, IL-2 attenuated has reduced activity in both cytokine receptor cell lines, whereas IL-2mutein shows increased activation in the high affinity receptor cell lines, as expected by its increased affinity for IL2RA.

[0079] FIG. 21 shows optical density plots depicting dose response of anti-RSV-IL-12 in IL-12 HEK blue reporter cells confirming activity of the heterodimeric IL- 12.

[0080] FIG. 22 shows tumor growth curves and tumor weights on day 14 post s.c. injection of the 4T1 tumors in mice intratumorally treated with three doses of 5 pg anti-RSV control, anti-RSV-IL-2sk, anti-NKG2A or anti-NKG2A-IL-2sk.

[0081] FIG. 23 is a schematic illustration of similar to figure 17, showing exemplary designed anti-human immunocytokines, based on an anti-human NKG2A.

[0082] FIG. 24 is a schematic illustration showing exemplary designed anti-human immunocytokines. Upper panel shows homodimeric, heterodimeric and homooligomeric embodiments. Lower panel shows exemplary polypeptides. In these configurations the anti-NKG2A antibody is fused by a linker element to a native or mutant type of a cytokine (round). The cytokine binds to a specific masking agent (moon-shape), thereby inactivating the cytokine activity. This masking agent is connected through a cleavable linker (e.g., MMP14, here seen as scissors) to the antibody or cytokine.

[0083] FIG. 25 is a schematic illustration of the experimental and computational workflow evaluating shared cytokine receptor landscapes in human tumors predict opposing immune programs: Cytokine activity is inferred from pan-cancer human single-cell RNA-seq data to prioritize candidate cytokines for in vivo testing. Responses to selected cytokines and cytokine combinations are profiled in murine tumors, and the resulting insights inform targeted cytokinebased therapeutic strategies.

[0084] FIG. 26 shows analyses of pan-cancer single-cell RNA-seq data [Qian, J. et al. Cell Res 30, 745-762 (2020)] from breast, lung, ovarian, and colorectal cancer (CRC) tissue including168,215 cells. The Figures shows a Circos plot depicting predicted ligand activity of selected cytokines across cell types using the downstream target genes of ligands from NicheNet.

[0085] FIGs. 27A-E demonstrate characterization of cytokine receptor expression in human pan cancer single-cell RNA-seq data. Analyses of pan-cancer single-cell RNA-seq data [Qian, J. et al. Cell Res 30, 745-762 (2020)] from breast, lung, ovarian, and colorectal cancer (CRC) tissue including 168,215 cells are shown. Figure 27A is a UMAP representation of pan-cancer T and NK cell clusters. Figure 27B is a dot plot showing the frequency (dot size) and normalized expression (color scale) of selected genes across pan-cancer T and NK cell clusters. Figure 27C is a UMAP representation of pan-cancer myeloid and B cell clusters. Figure 27D is a dot plot showing the frequency (dot size) and normalized expression (color scale) of selected genes across pan-cancer myeloid and B cell clusters. Figure 27E is a heatmap depicting predicted cytokine enrichment activity of 84 cytokines across immune cell types.

[0086] FIGs. 28A-D demonstrate tumor-restricted in vivo cytokine screening resolving cytokine-driven programs in solid tumors. Wild-type BALB / c mice were s.c. injected with 1 * 1064T1 tumor cells engineered for inducible overexpression of individual cytokines or control. For single-cell analyses, each tumor cell line was implanted at n = 4 (control n = 5 mice) and cytokine overexpression was induced on days 12 and 13. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retro-orbitally injected on day 12, and on day 13 in the morning and evening. Tumors were harvested on day 14 and obtained immune cells underwent single-cell RNA-seq, resulting in 51,073 high-quality cells. Figure 28 A shows the number of DEGs per major immune cell class for each cytokine condition (padj < 0.05, logFC > 1 or < -1) in comparison to control. Figures 28B-D show heatmaps depicting selected DEGs in conventional T cells (Tconv) (Figure 28B), NK cells (Figure 28C) or macrophages (Figure 28D) across cytokine conditions. Genes shown meet DEG criteria (padj < 0.05, log2FC > 1 or < -1) in at least one cytokine group versus control.

[0087] FIGs. 29A-C demonstrate characterization of immune cell abundances in 4T1 tumors following inducible cytokine expression. Wild-type BALB / c mice were s.c. injected with l*1064T1 tumor cells engineered for inducible overexpression of individual cytokines or control. Each tumor cell line was implanted at n = 4 mice (or

[0088] control n =5 mice respectively) and cytokine overexpression was induced by i.p injection of 0.25 mg doxycycline on days 12 and 13. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retroorbitally injected on day 12 and 13 in the morning and again on day 13 in the evening. Tumors were harvested on day 14 and obtained immune cells underwent single-cell RNA-seq, resulting in 51,073 highquality cells. CC = cell cycle. Figure 29A shows a dot plot showing thefrequency (dot size) and normalized expression (color scale) of cytokine receptor subunitencoding gene expression across immune cells (left) and heatmap depicting ModuleScores summarizing cytokine receptor co-expression within each immune cell state (right). Figure 29B shows stacked barplots showing relative abundance of immune populations among total CD45 cells, T / NK cells and myeloid cells across cytokine conditions. Figure 29C shows boxplots demonstrating frequencies of immune cell populations among total CD45 cells stratified by cytokine group. Boxes represent the interquartile range (IQR) with the median as a line, whiskers extend to the smallest and largest values within 1.5x IQR. Statistical significance was assessed by one-way ANOVA followed by Tukey’s multiple-comparisons test (*p adj < 0.05, ** p adj < 0.01, *** p adj < 0.001).

[0089] FIGs. 30A-F demonstrate cytokine-induced transcriptional reprogramming and cellular dependencies. In Figures 30A-C, wild-type BALB / c mice were s.c. injected with 1 * 1064T1 tumor cells engineered for inducible overexpression of individual cytokines or control. Each tumor cell line was implanted at n = 4 mice (or control n =5 mice respectively) and cytokine overexpression was induced by i.p. injection of 0.25 mg doxycycline on days 12 and 13. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retroorbitally injected on day 12, and on day 13 in the morning and evening. Tumors were harvested on day 14 and obtained immune cells underwent single-cell RNA-seq, resulting in 51,073 high-quality cells. Shown are heatmaps depicting selected DEGs in macrophages (Figure 30A), MDSCs (Figure 20B), or DCs (Figure 30C) across cytokine conditions. Genes shown meet DEG criteria (padj < 0.05, log2FC > 1 or < -1 in at least one cytokine group versus control). In Figures 30D-E, wild-type BALB / c mice were s.c. injected with l*1064T1 tumor cells engineered for inducible overexpression of IL-2SK. Cytokine overexpression was induced by i.p. injection of 0.25 mg doxycycline or PBS (control). Antibodies to deplete Tregs (Figure 30D), NK cells (Figure 30E) or CD8 T cells (Figure 30E) were administered one day prior to cytokine induction as follows: anti-CCR8 (200 J g, i.v., twice

[0090] weekly), anti-CTLA-4 (200 )J g, i.v., twice weekly), anti-CD8 (400 )J g, i.v., single injection) and anti-Asialo-GMl (100 J 1 first injection, 50 / 21 second injection, i.p.). n = 4-8 mice per treatment group. Tumor volume kinetics (mean ± SEM) as assessed by caliper (left) and final tumor volume on the day of harvest (right) are shown. Boxes display the interquartile range with the median as a line; whiskers extend to the minimum and maximum values. Statistical significance was assessed by two-way ANOVA (* p adj < 0.05, ** p adj < 0.01, *** p adj < 0.001).

[0091] FIGs. 30F and 31A-N demonstrate profiling revealing distinct cytokine-driven immune reprogramming dynamics in the TME. In Figures 30F and 31A-E, inducible IL-2SK-overexpressing and control 4T1 cells were mixed and s.c. injected into WT BALB / c mice. IL-2SK and Pro-Code barcode expression was induced by i.p. injection of 0.25 mg doxycycline on day 12, and tumors were harvested 24 hours later and OCT-embedded. A section underwent Stereo-seq and Pro-Code Immunofluorescence staining. Figure 30F is a dot plot showing the proportion of cells (dot size) and mean expression (color) of identified populations. Figure 31A is a spatial representation of major annotated cell populations. Figure 3 IB is a UMAP representation of major annotated cell populations. Figure 31C shows projection of IL-2K barcode enrichment density. Figure 3 ID shows classified tumor regions enriched for IL-2SK or control by thresholding on the staining intensity, as well as TAMs and proliferating T / NK cells. Figure 3 IE shows empirical cumulative distribution functions (CDFs) of minimum distances between selected cell populations. Kolmogorov-Smirnov (KS) test statistics and p values indicate the statistical significance in the difference between IL-2SK regions and control regions for proliferating / NKT cells (top) and TAMs (bottom). In Figures 31F-N, wild-type BALB / c mice were s.c. injected with l><1064T1 tumor cells engineered for inducible overexpression of individual cytokines (n = 4 mice / cytokine) or control (n = 5 mice). Upon tumor establishment, cytokine overexpression was induced by i.p. injection of 0.25 mg doxycycline on days 12 and 13. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retro -orbitally injected on day 12, and on day 13 in the morning and evening. Tumors were harvested on day 14 and obtained immune cells underwent single-cell RNA-seq, resulting in 51,073 high-quality cells. CC = cell cycle. Figure 3 IF shows relative abundance of immune cells among total CD45 cells at 12 hours, 24 hours and 48 hours following cytokine exposure stratified by cytokine conditions. Figure 31G shows relative abundance kinetics for selected immune cell state frequencies of total CD45 cells at 12 hours, 24 hours and 48 hours following cytokine exposure visualized using LOESS smoothing with standard error. Figure 31H is a 2D network representation of temporally -resolved cytokine conditions based on sample distance derived from MrVI model normalized to control. Hierarchical clustering was performed to obtain the six clusters as denoted by the colors. Figures 3 ILK show expression of selected average gene module scores projected on the 2D network of conventional T cells (Tconv) (Figure 311), NK cells (Figure 31 J) or macrophages (Figure 3 IK). Figures 31L-M show heatmaps demonstrating the difference in cytokine- specific module activity over area under the curve (AUC) time normalized against the control for conventional T cells (Tconv) (Figure 3 IL), NK cells (Figure 3 IM) and macrophages (Figure 3 IN).

[0092] FIGs. 32A-J demonstrate time-resolved immune cell dynamics and cytokine-regulated transcriptional programs. Wild-type BALB / c mice were s.c. injected with 1 * 1064T1 tumor cells engineered for inducible overexpression of individual cytokines or control (n = 4 mice / tumor cellline; control n =5 mice). Upon tumor establishment, cytokine overexpression was induced by i.p. injection of 0.25 mg doxycycline on days 12 and 13. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retro-orbitally injected on day 12 and 13 in the morning and on day 13 in the evening. Tumors were harvested on day 14 and obtained immune cells underwent single-cell RNA-seq, resulting in 51,073 high-quality cells. Figure 32A shows stacked barplots depicting frequencies of immune cells among total CD45 cells across timepoints (negative, 12 hours, 24 hours, 48 hours) following inducible expression of each cytokine. CC = cell cycle. Figure 32B shows relative abundance kinetics of immune cell state frequencies of total CD45 cells across timepoints following cytokine exposure visualized using LOESS smoothing with standard error. Figures 32C-F show expression of selected average gene module scores projected onto 2D network for conventional T cells (Tconv) (Figure 32C), NK cells (Figure 32D), macrophages (Figure 32E) or myeloid-derived suppressor cells (MDSCs) (Figure 32F). Figures 32G-J show heatmaps demonstrating deviation in cytokine- specific module activity over area under the curve (AUC) time normalized against the control for Tconv (Figure 32G), NK cells (Figure 32H), macrophages (Figure 321) or MDSCs (Figure 32J).

[0093] FIG. 33 shows immune cells isolated from 4T1 tumors following inducible cytokine combination expression. Wild-type BALB / c mice were s.c. injected with 1 * 106mixed 4T1 tumor cells engineered for inducible overexpression of IL-2SK and an additional cytokine overexpressing cell line (IL-12, IL-15sushi, IL-18DR, IL-21 or GM-CSF; ratio 1:1) or control (no cytokine expression). IL-2SK only 4T1 tumors were used as a reference. For single-cell analyses, tumor cell lines were implanted at n = 4 mice and cytokine overexpression was induced on days 12 and 13. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retro-orbitally injected on day 12, and on day 13 in the morning and evening. Tumors were harvested on day 14 and obtained immune cells underwent single cell RNA-seq, resulting in 37,743 high-quality cells. The Figure shows a UMAP representation of identified clusters of total sequenced cells combining individual cytokine groups (51,073 cells) and combination groups (37,743 cells). CC = cell cycle.

[0094] FIGs. 34A-B demonstrate immune cell abundance changes in 4T1 tumors upon cytokine combination treatments. Wild-type BALB / c mice were s.c. injected with 1 * 106mixed 4T1 tumor cells engineered for inducible overexpression of IL-2SK and an additional cytokine overexpressing cell line (IL-12, IL-15sushi, IL-18DR, IL-21 or GM-CSF; ratio 1:1). IL-2SK only and control (no cytokine expression) 4T1 tumor cells were used as a reference. Tumor cell lines were implanted at n = 4 mice and cytokine

[0095] overexpression was induced on days 12 and 13 by i.p. injection of 0.25 mg doxycycline. Fluorophorelabelled anti-CD45 antibodies for Zman-seq were retro-orbitally injected on day 12,and on day 13 in the morning and evening. Tumors were harvested on day 14 and obtained immune cells underwent single cell RNA-seq, resulting in 37,743 high-quality cells. CC = cell cycle. Figure 34A shows stacked barplots demonstrating relative abundance of immune populations among total CD45 cells, T / NK cells and myeloid cells across cytokine combination conditions. Figure 34B shows boxplots demonstrating frequencies of immune cell populations among total CD45 cells stratified by cytokine combination group. Boxes represent the interquartile range (IQR) with the median as a line, whiskers extend to the smallest and largest values within 1.5x IQR. Statistical significance was assessed by by one-way ANOVA followed by Tukey’s multiple-comparisons test (*p adj < 0.05, ** p adj < 0.01, *** p adj < 0.001). Statistical

[0096] significance was assessed byone-way ANOVA followed by Tukey’s multiple-comparisons test, and significant changes (p adj < 0.05) are highlighted by black dots.

[0097] FIGs. 35A-B demonstrate temporal immune cell abundance in 4T1 tumors upon cytokine and cytokine combination treatments. Wild-type BALB / c mice were s.c. injected with P IO 4T1 tumor cells engineered for inducible overexpression of individual cytokines, or mixed cells consisting of IL-2SK and an additional cytokine overexpressing cell line (IL-12, IL-15sushi, IL-18DR, IL-21 or GM-CSF; ratio 1:1). Control (no cytokine expression) 4T1 tumor cells were used as a reference. Tumor cell lines were implanted at n = 4 mice (control n =5 mice) and cytokine / cytokine combination overexpression was induced on days 12 and 13 by i.p. injection of 0.25 mg doxycycline. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retro-orbitally injected on day 12, and on day 13 in the morning and evening. Tumors were harvested on day 14 and obtained immune cells underwent single-cell RNA-seq, resulting in 37,743 high-quality cells (cytokine combination groups) and 51,073 cells (individual cytokine groups). CC = Cell cycle. Figure 35A shows stacked barplots depicting frequencies of immune cells among total CD45 cells across timepoints (negative, 12 hours, 24 hours, 48 hours) following inducible expression of cytokine combinations. Figure 35B shows relative abundance kinetics of immune cell state frequencies of total CD45 cells across timepoints following cytokine combination exposure visualized using LOESS smoothing with standard error.

[0098] FIGs. 35C-E and 36A-F demonstrate cytokine and cytokine combination driven transcriptional reprogramming. Wild-type BALB / c mice were s.c. injected with 1 * 1064T1 tumor cells engineered for inducible overexpression of individual cytokines, or mixed cells consisting of IL-2SK and an additional cytokine overexpressing cell line (IL-12, IL-15sushi, IL-18DR, IL-21 or GM-CSF; ratio 1:1). Control (no cytokine expression) 4T1 tumor cells were used as a reference. Tumor cell lines were implanted at n = 4 mice (control n =5 mice) and cytokine / cytokine combination overexpression was induced on days 12 and 13 by i.p. injection of 0.25 mgdoxycycline. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retro-orbitally injected on day 12, and on day 13 in the morning and evening. Tumors were harvested on day 14 and obtained immune cells underwent single-cell RNA-seq, resulting in 37,743 high-quality cells (cytokine combination groups) and 51,073 cells (individual cytokine groups). Figures 35C-E show heatmaps depicting selected DEGs in Tconv (Figure 35C), NK cells (Figure 35D) or DCs (Figure 35E) across individual cytokine and cytokine combination conditions. Genes shown meet DEG criteria (padj < 0.05, log2FC > 1 or < -1 in at least one cytokine group versus control). Figure 36A-B show heatmaps depicting selected DEGs in Macrophages (Figure 36A) or myeloid-derived suppressor cells (MDSCs) (Figure 36B) across individual cytokine and cytokine combination conditions. Genes shown meet DEG criteria (padj < 0.05, log2FC > 1 or < -1 in at least one cytokine group versus control. Figures 36C-F show heatmaps demonstrating the deviation in selected cytokine- specific (IL-2SK, IL- 12 and IL-2SK + IL- 12) module activity over area under the curve (AUC) time normalized against the control for conventional T cells (Tconv) (Figure 36C), NK cells (Figure 36D), macrophages (Figure 36E) or MDSCs (Figure 36F).

[0099] FIGs. 37A-E demonstrate that IL-2SK and IL- 12 synergize to elicit potent anti-tumor immunity. Wild-type BALB / c mice were s.c. injected with l*106mixed 4T1 tumor cells engineered for inducible overexpression of IL-2SK and an additional cytokine overexpressing cell line (IL-12, IL-15sushi, IL-18DR, IL-21 or GM-CSF; ratio 1:1). IL-2SK monotherapy and control (no cytokine expression) 4T1 tumor cells were used as a reference. For single-cell analyses, tumor cell lines were implanted at n = 4 mice and cytokine overexpression was induced on days 12 and 13. Fluorophore-labelled anti-CD45 antibodies for Zman-seq were retro-orbitally injected on day 12, and on day 13 in the morning and evening. Tumors were harvested on day 14 and obtained immune cells underwent single-cell RNA-seq, resulting in 37,743 high-quality cells. CC = cell cycle. Figure 37A shows relative abundance of immune cells among total CD45 cells at 12 hours, 24 hours and 48 hours following cytokine exposure stratified by cytokine conditions. Figure 37B shows relative abundance kinetics for selected immune cell state frequencies of total CD45 cells at 12 hours, 24 hours and 48 hours following cytokine exposure visualized using LOESS smoothing with standard error. Figure 37C shows the number of DEGs per major immune cell class for each cytokine condition (padj < 0.05, logFC > 1 or < -1) in comparison to control. Figure 37D shows truncated violin plots depicting expression of selected DEGs (padj < 0.05, log2FC > 1 or < -1 in at least one cytokine group versus control) in 4T1 tumors from control, IL-2SK, IL-12, and IL-2SK + IL- 12 conditions. Figure 37E shows heatmaps depicting the difference in selected cytokine- specific (IL-2SK, IL- 12 and IL-2SK + IL- 12) module activity over area under the curve(AUC) time for conventional T cells (Tconv), NK cells, macrophages and myeloid-derived suppressor cells (MDSCs).

[0100] FIGs. 38A-B demonstrates that effector-cell-biased NKG2A expression provides a target for selective cytokine delivery. Figure 38A shows boxplots depicting KLRC1 expression across healthy human tissues (GTEx) and corresponding tumor tissues from TCGA cancer entities. Boxes represent the interquartile range with the median shown as a line; whiskers extend to the smallest and largest values within 1.5x IQR. Statistical significance was assessed using the Mann-Whitney U test with Benjamini-Hochberg correction (p adj < 0.05, ** p adj < 0.01, *** p adj < 0.001). Figure 38B shows KLRC1 expression in NK cells (left) and CD8 T cells (right) from integrated single-cell RNA-seq data from blood, adjacent healthy-like tissue, and tumor across lung, breast, and colon datasets [Wu, S. Z. et al. Nat Genet 53, 1334-1347 (2021); Pelka, K. et al. Cell 184, 4734-4752.e20 (2021); Leader, A. M. et al. Cancer Cell 39, 1594-1609.el2 (2021); and Yazar, S. et al. Science 376, eabf3041 (2022)] including ~2M cells. Boxes represent the interquartile range with the median as a line, whiskers extend to 1.5x IQR, and outliers outside this range are shown as points. Statistical significance was assessed by an independent t-test (**** p < 0.0001).

[0101] FIGs. 39A-C demonstrate pan-cancer identification and validation of immunocytokine target candidates. Figures 39A shows violin plots depicting KLRC1 expression across T / NK cell states, stratified by cancer types. Each dot represents a patient. The analysis was performed on pan-cancer single-cell RNA-seq data from breast, lung, ovarian, and colorectal cancer (CRC) tissue including 168,215 cells. Figure 39B is a dot plot showing the frequency (dot size) and normalized expression (color scale) of KLRC1 across cell populations, stratified by tissue type. Figure 39C shows boxplots depicting KLRC1 expression in NK cells (left) and CD8 T cells (right) in blood, adjacent and tumor tissue, stratified by cancer type. Boxes represent the interquartile range with the median as a line, whiskers extend to 1.5x IQR, and outliers outside the range are shown as points. Statistical significance was assessed by an independent t-test (* p < 0.1, ** p < 0.05, *** p < 0.001, **** p < 0.0001).

[0102] FIG. 40 shows representative SDS page gel plots of the indicated anti-NKG2A / C / E immunocytokines. Ml: Protein Marker. R: Reducing condition. NR: Non-reducing condition.

[0103] FIG. 41 shows an optical density plot depicting ELISA results of the indicated immunocytokines confirming binding to recombinant murine NKG2A. Shown is mean ± SD.

[0104] FIG. 42 display HEK-Blue IL- 12 reporter assays readouts validating cytokine payload activity upon in vitro pre-digestion with MMP14. IL-12 signaling in IL-12RP1 / 2 reporter cells. Plots depict mean ± SD.FIGs. 43A-C demonstrates characterization and efficacy of NKG2A-targeted immunocytokines. Figure 43A is a dot plot showing the the frequency (dot size) and normalized expression (color scale) of Klrcl and selected immunocytokine-target encoding genes (currently in preclinical or clinical evaluation) across immune cell populations isolated from 4T1 tumors as assessed by single-cell RNA-seq. Figures 43B-C shows UMAP with overlayed expression density of Klrcl and selected immunocytokine-target encoding genes (currently in preclinical or clinical evaluation) across immune cell populations isolated from 4T1 tumors as assessed by single-cell RNA-seq.

[0105] FIG. 44 demonstrates the effect of anti-NKG2A-targeted immunocytokine pro-drugs on tumor growth in anti-PDl refractory tumors. Shown are tumor growth kinetics, as assessed by caliper in 4T1 tumor-bearing mice i.v. treated with anti-RSV (isotype control) antibodies (200 pg) or increasing doses of anti-NKG2A antibodies (100 pg or 200 pg; left), IL-2SK-NiTE (50 pg, 100 pg, 200 pg; middle) or IL-12-NiTE (5 pg, 10 pg, 25 pg; right) on days 8 and 10 (n = 3 mice per treatment).

[0106] FIGs. 45A-D demonstrate no systemic toxicity of anti-NKG2A-targeted immunocytokine pro-drugs. 4T1 tumor-bearing mice received 200 pg IL-2SK-based constructs or 5 pg IL-12-based constructs i.v. on days 6 and 8 post-tumor inoculation. Serum was collected 10 hours after the second dose. Plots depict serum IFN-y (n = 3-5 mice per treatment) (Figure 45A-B) and alanine transaminase (ALT) / aspartate transaminase (AST) enzyme (Figure 45C-D) levels (n = 3 per treatment). Plots depict mean ± SD. Statistical significance was assessed by one-way ANOVA followed by Dunnett’s multiple-comparisons test versus the anti-NKG2A group. * p adj < 0.05, ** p adj < 0.01, *** p adj < 0.001, **** p adj < 0.0001).

[0107] FIGs. 46A-B depict body weight upon immunocytokine treatments in percentage. 4T1 tumor-bearing mice received 200 pg IL-2SK-based constructs or 5 pg IL-12-based constructs i.v. on days 6 and 8 post-tumor inoculation (n=7 mice per treatment, except for aNKG2A-IL2SK-GZBc-IL2RB n=3). Plots depict mean ± SD.

[0108] FIGs. 47A-F demonstrate the effect of anti-NKG2A-targeted immunocytokine pro-drugs on tumor growth in anti-PDl refractory tumors. Shown tumor volume kinetics (mean ± SEM) as assessed by caliper (Figure 47A, C, E) and tumor weight (mean ± SD) on day 12 (Figure 47B, D, F) of 4T1 tumor-bearing mice i.v. treated with 200 pg IL-2SK-based constructs or 5 pg IL- 12-based constructs on days 6 and 8 post-tumor inoculation (n=7 mice per treatment, except for aNKG2A-IL2SK-GZBc-IL2RB n=3). Statistical significance was assessed by two-way ANOVA for tumor volume (Figure 47A, C, E) and by one-way ANOVA followed by Dunnett’s multiple-comparisons test for tumor weight versus anti-RSV control group (Figure 47B, D, F). * p adj < 0.05, ** p adj < 0.01, *** p adj < 0.001, **** p adj < 0.0001).

[0109] FIG. 48 demonstrates binding of the monoclonal antibody clones 30B1-1, IF7-2, 3E9-1, 5E3-2, 6B 11-1, 10E2-1 and 12E2-1 to human NKG2A, human NKG2A / CD94 and human NKG2C via ELISA. The clones have been produced through the immunization of rabbits with human NKG2A ECDs.

[0110] FIG. 49 demonstrates blocking of the interaction of human HLA-E*01:03&B2M&CMV UL40 (VMAPRTVLL, SEQ ID NO: 102) tetramer protein complex and NKG2A / CD94 heterodimer protein by the monoclonal antibody clones 30B1-1, IF7-2, 3E9-1, 5E3-2, 6B11-1, 10E2-1 and 12E2-1, herein represented as inhibition efficacy (%).

[0111] DESCRIPTION OF SPECIFIC EMBODIMENTS OF THE INVENTION

[0112] The present invention, in some embodiments thereof, relates to anti-NKG2A immunocytokines and uses thereof in treatment.

[0113] Before explaining at least one embodiment of the invention in detail, it is to be understood that the invention is not necessarily limited in its application to the details set forth in the following description or exemplified by the Examples. The invention is capable of other embodiments or of being practiced or carried out in various ways.

[0114] Immune-activating cytokines have shown limited effectiveness in cancer treatment. Finding the optimal targets and cytokine combinations for immunocytokines that boost anti-tumor responses, especially in cytotoxic cells, is a complex challenge requiring a deep understanding of anti-cancer cytokines and their interactions within the tumor microenvironment.

[0115] Whilst conceiving embodiments of the invention and in order to improve the therapeutic efficacy of cytokine therapy, the present inventors utilized data-driven approach, single cell technologies and genetical engineering and identified the combination of IL-2 and IL- 12 as extremely potent anti-tumor agent and NKG2A as a highly powerful novel target for next generation of immunocytokines. These immunocytokines work by specifically delivering cytokines to cytotoxic CD8 and NK cells while avoiding Tregs (see Examples 1-4 of the Examples section which follows). In addition, as NKG2A is an inhibitory receptor, specific embodiments suggest the immunocytokine may inhibit the suppression of the anti-tumor response by preventing the interaction between NKG2A and HLA-E expressed on tumor cells. Further, specific embodiments suggest conditional activity of the immunocytokines in the tumor microenvironment (TME) through a blocking moiety which is cleaved via tumor microenvironment-associated immune specific proteases resulting in active cytokines solely in the TME context to therebyactivate potent anti-tumor immunity, while maintaining specificity and reducing off-target effects. In addition, the present inventors generated and characterized novel anti-NKG2A antibodies exhibiting high specificity and inhibitory activity toward peptide loaded HLA-E:B2M complex interaction, which are useful as targeting moieties for the immunocytokines described herein and / or as therapeutic agents per se (see Examples 5-6 of the Examples section which follows).

[0116] Thus, according to an aspect of the present invention there is provided a composition of matter comprising at least one fusion polypeptide comprising:

[0117] (a) an anti-NKG2A antibody;

[0118] (b) an immune cell-activating cytokine; and

[0119] (c) a masking agent that masks said immune cell-activating cytokine and an amino acid sequence which is cleaved by a tumor microenvironment-associated protease, such that when said fusion polypeptide is in a vicinity of a tumor, said cancer microenvironment-associated protease cleaves said masking agent thereby exposing said immune cell-activating cytokine.

[0120] According to an additional or an alternative aspect of the present invention, there is provided a composition of matter comprising at least one fusion polypeptide comprising:

[0121] (a) an anti-NKG2A antibody; and

[0122] (b) an immune cell-activating cytokine selected from the group consisting of IL-2 and IL- 12.

[0123] According to an additional or an alternative aspect of the present invention, there is provided a composition of matter comprising at least one fusion polypeptide comprising:

[0124] (a) an antagonistic anti-NKG2A antibody; and

[0125] (b) an immune cell-activating cytokine.

[0126] As used herein “a composition of matter” comprises at least one type of a fusion polypeptide. According to a specific embodiment, the composition of matter comprises all polypeptides of the same type i.e., having the same targeting moiety, linker (if present), a masking agent (if present), cleavage sequence (if present) and immune cell-activating cytokine, or are different such as in at least one of a targeting moiety, linker (if present), masking agent (if present), cleavage sequence (if present) and immune cell-activating cytokine. Such configurations are illustrated in Figures 23-24.

[0127] Thus, according to specific embodiments, the at least one polypeptide is a dimer.

[0128] According to specific embodiments, the at least one polypeptide is an oligomer (e.g., 3, 4, 5, 6).

[0129] When different polypeptides are present in the composition, they may form a heterodimer or a heterooligomer (e.g., 3, 4, 5, 6).When the same polypeptides are present in the composition, they may form a homodimer or a homooligomer (e.g., 3, 4, 5, 6).

[0130] As used herein “fusion polypeptide” refers to a polypeptide which is not found in nature and comprises at least some (if not all) heterologous segments of the following: an anti-NKG2A antibody, an immune cell-activating moiety, a cleavable sequence, and a masking agent. As will be explained hereinbelow in more details, the fusion needs or need not include a linker or linkers. As will be further appreciated, any of the segments can be present in the at least one fusion once or more. The fusion polypeptide can be recombinantly produced such as from a single open reading frame, as plurality of open reading frames (from the same polynucleotide or different polynucleotides) or synthetically fused. When the fusion is translated from a single polynucleotide (single open reading fame) it may be referred to as “a chimeric polypeptide”.

[0131] According to a specific embodiment, the fusion polypeptide is of a single type of an amino acid sequence.

[0132] According to a specific embodiment, the at least one fusion polypeptide is of different types of amino acid sequences, e.g., when the masking agent is not on the same amino acid sequence as the cytokine.

[0133] As mentioned, the at least one fusion polypeptide comprises an anti-NKG2A antibody. As used herein, the term “NKG2A”, also known as KLRC1 and CD 159a, refers to the polypeptide expression product of the KLRC1 (corresponding to human gene ID: 3821). According to specific embodiments, the NKG2A refers to the human NKG2A, such as provided in the following Accession Numbers: NP_001291377, NP_002250, NP_015567, NP_998822, NP_998823 (SEQ ID NOs: 52-56).

[0134] NKG2A is an immune-check point inhibitor that forms a complex with CD94 that binds the MHC class I HLA-E molecule. Thus, according to specific embodiments, NKG2A activity is at least one of (or two of or all of): forming a complex with CD99, binding HLA-E, and / or delivering an inhibitory signal.

[0135] The anti-NKG2A is characterized by a binding activity to NKG2A.

[0136] As used herein “binding activity” refers to an affinity of at least KD < 10’6M, <10-7M, <10-8M, < 10’9M, IO10M, 1011M, 1012M, each possibility represents a separate embodiment of the present invention. Methods of determining binding and affinity are well known in the art and include e.g., flow cytometry, immunoprecipitation, BiaCore, HPLC, Surface Plasmon Resonance assay (SPR) and bio-layer interferometry Blitz® assay.

[0137] According to specific embodiments, the anti-NKG2A antibody is a pan-NKG2 antibody [i.e., binds several or all of the NKG2 (also known as CD 159) family i.e., NKG2A / B / C / D / E / F / H].According to other specific embodiments, the anti-NKG2A antibody specifically (or selectively) binds NKG2A.

[0138] As used herein, the term “specifically (or selectively) binds NKG2A” refers to the ability to bind NKG2A at a higher affinity compared to other polypeptides. Higher affinity can be, for examples, of at least 2, 5, 10, 100, 1000, 10000, or 100,000 or 1,000,000 fold or more using the same affinity assay, such as e.g., flow cytometry, immunoprecipitation, BiaCore, HPLC, Surface Plasmon Resonance assay (SPR) and bio-layer interferometry Blitz® assay.

[0139] According to specific embodiments, the antibody binds NKG2A with no cross reactivity with other proteins.

[0140] According to specific embodiments, the antibody binds NKG2A with no cross reactivity with other NKG2 family of proteins.

[0141] According to specific embodiments, the antibody binds NKG2A with no cross reactivity NKG2C.

[0142] According to specific embodiments, the anti-NKG2A is also characterized by an inhibitory activity i.e., an antagonistic antibody, also referred to as inhibitory antibody.

[0143] As used herein, “inhibitory activity”, “antagonistic antibody” or “inhibitory antibody” refers to a decrease of at least 10 % in NKG2A activity in comparison to an isolated NKG2A or control cell expressing same of the same origin which was not contacted with the anti-NKG2A antibody. According to a specific embodiment, the decrease is in at least 20 %, 30 %, 40 % or even higher say, 50 %, 60 %, 70 %, 80 %, 90 % or even 100 %. Determining the decrease in activity may be assessed by any assay evaluating binding to HLA-E. Alternatively, or additionally, as NKG2A forms a complex with CD94 activity may be determined by the effect on the complex formation. Alternatively, or additionally, as NKG2A delivers an inhibitory signal in an immune cell expressing same (e.g., NK cell), activity may be evaluated by determining intracellular signaling [e.g., detect phosphorylation of downstream signaling molecules (e.g., SHP-1, SHP-2) using phospho-specific antibodies], assessing expression levels of downstream signaling molecules, functional Assays (e.g., cytotoxicity assays measuring NK cell-mediated killing of target cells expressing HLA-E, assessing cytokine production, microscopy Techniques visualizing the localization and clustering of NKG2A during NK cell interactions with target cells, etc.

[0144] Inhibiting activity may be either transient or permanent.

[0145] The antibody may be a reversible or an irreversible inhibitor.

[0146] The antibody may be a competitive or non-competitive inhibitor.

[0147] According to specific embodiments, the antagonistic antibody does not provoke killing of the NKG2A-expressing cell (i.e., not a depleting antibody).The term "antibody" as used in this invention includes intact molecules as well as functional fragments thereof (that are capable of binding to an epitope of an antigen).

[0148] As used herein, the term "epitope" refers to any antigenic determinant on an antigen to which the paratope of an antibody binds. Epitopic determinants usually consist of chemically active surface groupings of molecules such as amino acids or carbohydrate side chains and usually have specific three-dimensional structural characteristics, as well as specific charge characteristics.

[0149] According to a specific embodiment, the antibody fragments include, but are not limited to, single chain, Fab, Fab’ and F(ab')2 fragments, Fd, Fcab, Fv, dsFv, scFvs, diabodies, minibodies, nanobodies, Fab expression library or single domain molecules such as VH and VE that are capable of binding to an epitope of the antigen in an HEA restricted manner.

[0150] Suitable antibody fragments for practicing some embodiments of the invention include a complementarity-determining region (CDR) of an immunoglobulin light chain (referred to herein as “light chain”), a complementarity-determining region of an immunoglobulin heavy chain (referred to herein as “heavy chain”), a variable region of a light chain, a variable region of a heavy chain, a light chain, a heavy chain, an Fd fragment, and antibody fragments comprising essentially whole variable regions of both light and heavy chains such as an Fv, a single chain Fv (scFv), a disulfide- stabilized Fv (dsFv), an Fab, an Fab’, and an F(ab’)2, or antibody fragments comprising the Fc region of an antibody.

[0151] As used herein, the terms "complementarity-determining region" or "CDR" are used interchangeably to refer to the antigen binding regions found within the variable region of the heavy and light chain polypeptides. Generally, antibodies comprise three CDRs in each of the VH (CDR HI or HI; CDR H2 or H2; and CDR H3 or H3) and three in each of the VL (CDR LI or LI; CDR L2 or L2; and CDR L3 or L3).

[0152] The identity of the amino acid residues in a particular antibody that make up a variable region or a CDR can be determined using methods well known in the art and include methods such as sequence variability as defined by Kabat et al. (See, e.g., Kabat et al., 1992, Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, NIH, Washington D.C.), location of the structural loop regions as defined by Chothia et al. (see, e.g., Chothia et al., Nature 342:877-883, 1989.), a compromise between Kabat and Chothia using Oxford Molecular's AbM antibody modeling software (now Accelrys®, see, Martin et al., 1989, Proc. Natl Acad Sci USA. 86:9268; and world wide web site www(dot)bioinf-org(dot)uk / abs), available complex crystal structures as defined by the contact definition (see MacCallum et al., J. Mol. Biol. 262:732-745, 1996) and the "conformational definition" (see, e.g., Makabe et al., Journal of Biological Chemistry, 283:1156-1166, 2008).As used herein, the “variable regions” and "CDRs" may refer to variable regions and CDRs defined by any approach known in the art, including combinations of approaches.

[0153] Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:

[0154] (i) Fv, defined as a genetically engineered fragment consisting of the variable region of the light chain (VL) and the variable region of the heavy chain (VH) expressed as two chains;

[0155] (ii) single chain Fv (“scFv”), a genetically engineered single chain molecule including the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

[0156] (iii) disulfide-stabilized Fv (“dsFv”), a genetically engineered antibody including the variable region of the light chain and the variable region of the heavy chain, linked by a genetically engineered disulfide bond.

[0157] (iv) Fab, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme papain to yield the intact light chain and the Fd fragment of the heavy chain which consists of the variable and CHI domains thereof;

[0158] (v) Fab’, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin, followed by reduction (two Fab’ fragments are obtained per antibody molecule);

[0159] (vi) F(ab’)2, a fragment of an antibody molecule containing a monovalent antigen-binding portion of an antibody molecule which can be obtained by treating whole antibody with the enzyme pepsin (i.e., a dimer of Fab’ fragments held together by two disulfide bonds);

[0160] (vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen; and

[0161] (viii) Fcab, a fragment of an antibody molecule containing the Fc portion of an antibody developed as an antigen-binding domain by introducing antigen-binding ability into the Fc region of the antibody.

[0162] According to some embodiments, the antibody is a recombinant antibody.

[0163] According to specific embodiments the antibody heavy chain constant region is chosen from, e.g., IgGl, IgG2, IgG3, IgG4, IgM, IgA, IgA2, IgD, and IgE.

[0164] The antibody may be mono- specific (capable of recognizing one epitope or protein), bispecific (capable of binding two epitopes or proteins) or multi- specific (capable of recognizing multiple epitopes or proteins).

[0165] According to specific embodiments, the antibody is a mono-specific antibody.According to specific embodiments, the antibody is a multi- specific e.g., bi-specific, trispecific, tetra- specific.

[0166] The choice of antibody type will depend on the immune effector function that the antibody is designed to elicit.

[0167] According to specific embodiments, the antibody is a naked antibody.

[0168] As used herein, the tern "naked antibody" refers to an antibody which does not comprise a heterologous effector moiety e.g., therapeutic moiety, detectable moiety. According to specific embodiments, the antibody comprises a heterologous effector moiety e.g., therapeutic moiety, detectable moiety. The effector moiety may be any molecule, including small molecule chemical compounds and polypeptides. For example, the effector moiety may be a toxin, cytotoxic drug, immune modulator, cytokine, chemokine, enzyme, a receptor construct [e.g., a chimeric antigen receptor (CAR)] or a drug payload forming an antibody-drug conjugate (ADC).

[0169] According to specific embodiments, the effector moiety is a cytokine.

[0170] According to specific embodiments, the antibody comprises an Fc domain.

[0171] The Fc region of some embodiments of the invention may be derived from an immunoglobulin isotype selected from IgG, IgA, IgM, IgD and IgE, including subclasses thereof. In some embodiments, the Fc region is derived from an IgG subclass, such as IgGl, IgG2, IgG3 or IgG4.

[0172] In some embodiments, the Fc region is engineered to modulate one or more effector functions. Such modulation may include enhancement or reduction of antibody-dependent cellular cytotoxicity (ADCC), complement-dependent cytotoxicity (CDC) and / or antibody-dependent cellular phagocytosis (ADCP).

[0173] In some embodiments, the Fc region comprises one or more amino acid substitutions that alter binding affinity to Fc receptors (FcRs) or complement proteins. In other embodiments, the Fc region is modified to reduce or eliminate effector function (Fc-silenced variants).

[0174] In some embodiments, the Fc region is glycosylated and optionally engineered to modify glycosylation patterns, including reduced fucosylation, thereby altering effector function.

[0175] It will be appreciated that Fc variants suitable for modulating effector function are well known in the art, and include mutations in the CH2 and / or CH3 domains that affect Fc receptor binding.

[0176] Methods of producing monoclonal antibodies as well as fragments thereof are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).Exemplary methods for generating antibodies employ induction of in-vivo production of antibody molecules, screening of immunoglobulin libraries (Orlandi D.R. et al., 1989. Proc. Natl. Acad. Sci. U. S. A. 86:3833-3837; Winter G. et al., 1991. Nature 349:293-299) or generation of monoclonal antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the Epstein-Barr virus (EBV)-hybridoma technique (Kohler G. et al., 1975. Nature 256:495-497; Kozbor D. et al., 1985. J. Immunol. Methods 81:31-42; Cote RJ. et al., 1983. Proc. Natl. Acad. Sci. U. S. A.

[0177] 80:2026-2030; Cole SP. et al., 1984. Mol. Cell. Biol. 62:109-120).

[0178] Antibody fragments can be obtained by pepsin or papain digestion of whole antibodies by conventional methods. For example, antibody fragments can be produced by enzymatic cleavage of antibodies with pepsin to provide a 5S fragment denoted F(ab')2. This fragment can be further cleaved using a thiol reducing agent, and optionally a blocking group for the sulfhydryl groups resulting from cleavage of disulfide linkages, to produce 3.5S Fab' monovalent fragments. Alternatively, an enzymatic cleavage using pepsin produces two monovalent Fab' fragments and an Fc fragment directly. These methods are described, for example, by Goldenberg, U.S. Pat. Nos.

[0179] 4,036,945 and 4,331,647, and references contained therein, which patents are hereby incorporated by reference in their entirety. See also Porter, R. R. [Biochem. J. 73: 119-126 (1959)]. Other methods of cleaving antibodies, such as separation of heavy chains to form monovalent lightheavy chain fragments, further cleavage of fragments, or other enzymatic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.

[0180] As described hereinabove, Fv fragments comprise an association of VH and VE chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat'l Acad. Sci. USA 69:2659-62 (19720]. Alternatively, the variable chains can be linked by an intermolecular disulfide bond or cross-linked by chemicals such as glutaraldehyde. Preferably, the Fv fragments comprise VH and VL chains connected by a peptide linker. These single-chain antigen binding proteins (sFv) are prepared by constructing a structural gene comprising DNA sequences encoding the VH and VL domains connected by an oligonucleotide. The structural gene is inserted into an expression vector, which is subsequently introduced into a host cell such as E. coli. The recombinant host cells synthesize a single polypeptide chain with a linker peptide bridging the two V domains. Methods for producing sFvs are described, for example, by [Whitlow and Filpula, Methods 2: 97-105 (1991); Bird et al., Science 242:423-426 (1988); Pack et al., Bio / Technology 11:1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.Another form of an antibody fragment is a peptide coding for a single complementaritydetermining region (CDR). CDR peptides ("minimal recognition units") can be obtained by constructing genes encoding the CDR of an antibody of interest. Such genes are prepared, for example, by using the polymerase chain reaction to synthesize the variable region from RNA of antibody-producing cells. See, for example, Larrick and Fry [Methods, 2: 106-10 (1991)].

[0181] As mentioned, the antibody fragment may comprise a Fc region of an antibody termed “Fcab”. Such antibody fragments typically comprise the CH2-CH3 domains of an antibody. Fcabs are engineering to comprise at least one modification in a structural loop region of the antibody, i.e., in a CH3 region of the heavy chain. Such antibody fragments can be generated, for example, as follows: providing a nucleic acid encoding an antibody comprising at least one structural loop region (e.g., Fc region), modifying at least one nucleotide residue of the at least one structural loop regions, transferring the modified nucleic acid in an expression system, expressing the modified antibody, contacting the expressed modified antibody with an epitope, and determining whether the modified antibody binds to the epitope. See, for example, U.S. Patent Nos. 9,045,528 and 9,133,274 incorporated herein by reference in their entirety.

[0182] Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab').sub.2 or other antigen-binding subsequences of antibodies) which contain minimal sequence derived from non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues form a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also comprise residues which are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence. The humanized antibody optimally also will comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin [Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol., 2:593-596 (1992)].There are a number of mechanisms that can be used to generate a heterodimer using an Fc domain of an antibody e.g., the CH3 domain (according to kabat) as well known in the art. Such a modification ensures correct assembly of the heterodimer via the heavy chains.

[0183] A representative example, which can be used with specific embodiments of the invention is the “knob-into-hole” (“KIH”) form. Such knob and hole mutations are well known in the art and disclosed e.g., in US Patent NO. US8216805, Gunasekaran et al., J. Biol. Chem. (2010) 285(25): 19637, Shane Atwell et Al. J. Mol. Biol. (1997) 270, 26-35; Cater et al. (Protein Engineering vol.9 no.7 pp.617-621, 1996); and A. Margaret Merchant et.al. Nature Biotechnology (1998) 16 July, the contents of which are fully incorporated herein by reference. In addition, as described in Merchant et al., Nature Biotech. 16:677 (1998), these “knobs and hole” mutations can be combined with disulfide bonds to skew formation to heterodimerization.

[0184] Thus, according to specific embodiments, one of the monomers comprises an Fc domain comprising a knob mutation(s) and the other monomer comprises an Fc domain comprising a hole mutation(s).

[0185] It is within the scope of those skilled in the art to select a specific immunoglobulin Fc domain from particular immunoglobulin classes and subclasses and to select a first Fc variant for knob mutation and the other for hole mutation.

[0186] Alternatively, heterodimers described herein can be prepared by conjugating the moieties using methods known in the art. For example, each moiety of the heterodimer can be generated separately and then conjugated to one another. A variety of coupling or cross-linking agents can be used for covalent conjugation. Examples of cross-linking agents include protein A, carbodiimide, N-succinimidyl-S-acetyl-thioacetate (SATA), 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB), o-phenylenedimaleimide (oPDM), N-succinimidyl-3-(2-pyridyldithio)propionate (SPDP), and sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohaxane-1 -carboxylate (sulfo-SMCC) (see e.g., Karpovsky et al. (1984) J. Exp. Med. 160:1686; Eiu, M A et al. (1985) Proc. Natl. Acad. Sci. (USA) 82:8648). Other methods include those described in Paulus (1985) Behring Ins. Mitt. No. 78, 118-132; Brennan et al. (1985) Science 229:81-83), and Glennie et al. (1987) J. Immunol.

[0187] 139: 2367-2375). Exemplary conjugating agents are SATA and sulfo-SMCC, both available from Pierce Chemical Co. (Rockford, Ill.).

[0188] Alternatively or additionally, the conjugation of each moiety of the heterodimer can be done via sulfhydryl bonding of the C-terminus hinge regions of the two heavy chains. In a specific embodiment, the hinge region is modified to contain an odd number of sulfhydryl residues, preferably one, prior to conjugation.A non-limiting example of a screening method for identifying anti-NKG2A antibodies that can be used with specific embodiments of the invention and assessing the specific binding and antagonistic activity if provided in Examples 5-6 in the Example section which follows.

[0189] Non-limiting examples of anti-NKG2A antibodies that can be used with specific embodiments of the invention are described in e.g., US Patent Nos. US9422368, US 10329348, US8901283, US10160810, US10711063, US Application Publication No. US20240368281, International Application Publication No. WO2016062851, Japanese Patent No. JP6333556, JP7520003, EP Patent No. EP2628753, the contents of which are fully incorporated herein by reference in their entirety.

[0190] Non-limiting sequences of anti-NKG2A antibodies and / or their CDR sequences that can be used with specific embodiments of the invention are provided in SEQ ID NOs: 67-81.

[0191] According to specific embodiments, the anti-NKG2A antibody is Monalizumab (formerly IPH2201). The CDR sequences and VL and VH sequences of Monalizumab are provided in SEQ ID NOs: 73-78.

[0192] Another example of an anti-NKG2A antibody, which sequence (or CDR sequences) can be used with specific embodiments of the invention is the anti-NKG2A Neutralizing Antibody V3S-1022-YC1139, commercially available from Creative Biolabs, CAT#: V3S-1022-YC1139). Other anti-NKG2A antibodies known to bind NKG2A are commercially available from e.g., Abeam, Miltenyi Biotec, ThermoFisher etc. their sequences (or CDR sequences) may be used with specific embodiments of the invention.

[0193] According to some embodiments of the invention, the NKG2A antibody is any one of those listed by name and sequence in Table 3 hereinbelow. In the table, each row represents a specific antibody. Also contemplated are antibodies which contain the CDRs or full-length light chain and / or heavy chain of these antibodies (SEQ ID NOs: 212-267) or homologs thereof as described below. The present inventors have shown that the antibodies of Table 3 exhibit specific binding to NKG2A and inhibitory activity toward NKG2A signaling, as described in Example 6 of the Examples section which follows.

[0194] The CDRs of the light chain variable domain and the heavy chain variable domain are designated sequentially from the N-terminus to the C-terminus as CDRL1, CDRL2 and CDRL3 (light chain), and CDRH1, CDRH2 and CDRH3 (heavy chain), respectively. In Table 3 below, the CDR sequences are indicated by underlining within each variable domain sequence set forth herein. Each CDR may be characterized as an integral component of its respective light chain variable domain or heavy chain variable domain (or a homolog thereof) or alternatively as adiscrete individual sequence. The six CDRs collectively, CDRL1, CDRL2, CDRL3, CDRH1, CDRH2, and CDRH3, define the antigen-binding specificity of the antibody.

[0195] Such antibodies can be used in any research, diagnostic and clinical applications. For example, such antibodies may be part of the fusion polypeptides described herein. Alternatively or additionally, such antibodies can be used as therapeutic agents per se to modulate immune responses.

[0196] Thus, according to an aspect of the present invention, there is provided an antibody comprising an antigen recognition domain which binds NKG2A and comprises complementarity determining regions (CDRs) CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 or the light chain and heavy chain at least 80 % identical to those of an antibody selected from the group consisting of 30B1-1, IF7-2, 3E9-1, 5E3-2, 6B11-1, 10E2-1 and 12E2-1.

[0197] According to a specific embodiment, the level of identity is at least 80 % over at least one (or at least 2, 3, 4, 5, or 6) of the CDR sequences selected from those listed in Table 3, as described herein.

[0198] According to a specific embodiment, the level of identity is at least 80 % of at least one framework or the entire sequence of an antibody selected from those listed in Table 3, as described herein.

[0199] According to a specific embodiment, the level of identity is at least 80 % over the entire sequence of any of the VL and / or VH chains listed in in Table 3, as described herein.

[0200] When referring to "at least 80 % identity," the claimed invention also refers to at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 %, or 100% identity, where each represents a separate and distinct embodiment.

[0201] Table 3:

[0202]

[0203]

[0204] * The CDRs were determined by the Kabat delineation system.** SEQ ID NO: 249 is not included in the sequence listing file due to its length being below the minimum threshold for inclusion in accordance with applicable sequence listing standards. SEQ ID NO: 249 corresponds to the amino acid sequence WDL, representing the CDRH3 of the antibody 6B11-1 as shown in the Table.

[0205] *** SEQ ID NO: 265 is not included in the sequence listing file due to its length being below the minimum threshold for inclusion in accordance with applicable sequence listing standards. SEQ ID NO: 265 corresponds to the amino acid sequence WDL, representing the CDRH3 of the antibody 12E2-1 as shown in the Table.

[0206] Non-limiting examples of nucleic acid sequences encoding the antibodies of Table 3 are provided in SEQ ID NOs: 268-323, excluding SEQ ID NOs: 305 and 321, which are not included in the sequence listing file due to their length being below the minimum threshold for inclusion in accordance with applicable sequence listing standards).

[0207] SEQ ID NO: 305 corresponds to the nucleic acid sequence TGGGACTTG, which encodes the amino acid sequence WDL representing the CDRH3 of the antibody 6B 11- 1 as shown in Table 3.

[0208] SEQ ID NO: 321 corresponds to the nucleic acid sequence TGGGACTTG, which encodes the amino acid sequence WDL, representing the CDRH3 of the antibody 12E2-1 as shown in Table 3.

[0209] As mentioned, the at least one fusion polypeptide comprises an immune cell-activating cytokine.

[0210] As used herein “immune cell-activating cytokine” refers to a cytokine which activates effector immune cells such as lymphoid and myeloid cells.

[0211] Cytokines are major regulators of innate and adaptive immunity that enable cells of the immune system to communicate over short distances. Cytokine therapy to activate the immune system of cancer patients has been an important treatment modality. Interferon alpha (IFNa) is approved for adjuvant treatment of completely resected high-risk melanoma patients and several refractory malignancies. High-dose interleukin-2 (HDIL-2) is approved for treatment of metastatic renal cell cancer and melanoma. Granulocyte-macrophage colony-stimulating factor (GM-CSF), IFN gamma (IFNy), IL-7, IL-12, IL-15 and IL-21 evaluated in clinical trials. Reviewed and incorporated by reference in Conlon et al. J Interferon Cytokine Res. 2019 Jan 1; 39(1): 6-21.

[0212] The term “immune cell-activating cytokine” includes both native cytokines and variants of these cytokines modified to e.g., reduce activity, alter a binding property and / or increase stability.Thus, according to a specific embodiment, the cytokine is a human cytokine or ortholog (e.g., mouse, rat) or synthetic version thereof (e.g., at least 80 %, 85 %, 90 %, 95 %, 98 %, or 99 % identical to the human sequence) which is capable of exerting the effector function on the immune cells. Measures are taken to reduce immunogenicity by the use of closely related homologs e.g., at least 80 % identical to the native sequence, which are capable of binding the cytokine receptor and exert function, e.g., JAK / STAT activation.

[0213] According to specific embodiments, the cytokine is in its native form (i.e., an amino acid sequence of a naturally occurring cytokine as found within a living organism).

[0214] According to other specific embodiments, the cytokine is modified to reduce activity, alter a binding property and / or increase stability.

[0215] Methods of determining cytokine activity are well known in the art and include e.g., measuring proliferation of immune cells, measuring an effector function (e.g., cytotoxicity) of immune cells, reporter cell assays, measuring phosphorylation of downstream signaling molecules, measuring STAT activation etc. An exemplary reporter assay is the HEK-blue™ assay, available for testing activation of various types of cytokines, as further described in the Examples section which follows.

[0216] Non-limiting examples of cytokines that can be used with specific embodiments of the invention include Interleukin-2 (IL-2), Interleukin- 12 (IL-12), Interleukin- 15 (IL-15), Interleukin-18 (IL-18), Interleukin-21 (IL-21), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interferon- alpha (IFN-a), Tumor Necrosis Factor-alpha (TNF-a), Interferon-gamma (IFN-y), and Granulocyte Colony-Stimulating Factor (G-CSF)

[0217] According to specific embodiments, the cytokine is selected from the group consisting of IL-2, IL-12, IL-15, IL-18, IL-21 and GM-CSF.

[0218] Non-limiting examples of amino acid sequences of cytokines that can be used with specific embodiments of the invention and nucleic acid sequences encoding same are provided in SEQ ID NOs: 1-17.

[0219] According to a specific embodiment, the cytokine is IL-2.

[0220] Non-limiting examples of amino acid sequences of IL-2 that can be used with specific embodiments of the invention and nucleic acid sequences encoding same are provided in SEQ ID NOs: 1-5.

[0221] According to a specific embodiment, the cytokine is human IL-2 (SEQ ID NO: 4) or the IL2-superkine (H9) (SEQ ID NO: 1, referred to as “IL-2S or “IL-2SK”) such as taught in Levin, A., Bates, D., Ring, A. et al. Exploiting a natural conformational switch to engineer an interleukin-2 ‘superkine’. Nature 484, 529-533 (2012). https: / / doi(dot)org / 10(dot)1038 / naturel0975. Patent(US10183980B2) “Superagonists and antagonists of interleukin-2”). The IL2-superkine (H9) exhibits preferential binding to inter-mediate affinity IL-2 receptor complex consisting of IL2RB and IL2RG which is expressed by effector T cells, NK or NKT cells, while exhibiting a mutated interface of IL2RA (CD25) to reduce the interaction of the trimeric IL-2 receptor complex that is mainly expressed by regulatory T cells.

[0222] According to a specific embodiment, a mutein cytokine is contemplated (e.g., IL-2 such as provided e.g., in SEQ ID NO: 2) engineered to have selective binding to activate effector T cells and natural killer (NK) cells without activating regulatory T cells (Tregs), which is desirable in cancer immunotherapy (see e.g., Wu et al., Nature Cancer 2023; or Xue et al. Antibody Therapeutics, Volume 4, Issue 2, April 2021, Pages 123-133; As another example, see a low affinity, cis-acting immunocytokine which selectively targets tumor-infiltrating immune cells (Ren Z, Zhang A, Sun Z, et al. Selective delivery of low-affinity IL-2 to PD-1+ T cells rejuvenates antitumor immunity with reduced toxicity. J Clin Invest. 2022;132(3):el53604. doi:10.1172 / JCI153604, each of which is incorporated by reference in its entirety).

[0223] Low affinity, cis-acting IL-2 which selectively targets tumor-infiltrating immune cells (Ren Z, Zhang A, Sun Z, et al. Selective delivery of low-affinity IL-2 to PD-1+ T cells rejuvenates antitumor immunity with reduced toxicity. J Clin Invest. 2022;132(3):el53604. doi:10.1172 / JCI153604), can be used with specific embodiments.

[0224] According to a specific embodiment, an attenuate cytokine is contemplated (e.g., IL-2att such as provided e.g., in SEQ ID NO: 3) which does not bind IL2RA, binds less to IL2RB and thus exhibits reduced binding to both IL2RA / B / G and IL2RB / G (see e.g., Canadian Patent No. CA2824253C, the contents of which are incorporated herein by reference).

[0225] A two-component split version of a cytokine that are active only upon colocalization of the two disjointed fragments at the site of the tumor (see e.g., Quijano-Rubio, A., Bhuiyan, A.M., Yang, H. et al. A split, conditionally active mimetic of IL-2 reduces the toxicity of systemic cytokine therapy. Nat Biotechnol. 41, 532-540 (2023). https: / / doi(dot)org / 10(dot)1038 / s41587-022-01510-z) can also be used. This is of special significance in IL- 12, which is highly toxic and its recombinant expression as a single protein is hindered by its size. Such a configuration is illustrated in Figures 23-24. The two subunits are split between the 2 chains of the heterodimer.

[0226] Thus, according to a specific embodiment, the cytokine is IL- 12.

[0227] Non-limiting examples of amino acid sequences of IL- 12 that can be used with specific embodiments of the invention and nucleic acid sequences encoding same are provided in SEQ ID NOs: 6-11.

[0228] According to specific embodiments, the cytokine is IL- 15.IL 15 fused to IL-15RA to enhance half-life was disclosed by Zhou T, Damsky W, Weizman OE, McGeary MK, Hartmann KP, Rosen CE, Fischer S, Jackson R, Flavell RA, Wang J, Sanmamed MF, Bosenberg MW, Ring AM.

[0229] According to specific embodiments, the cytokine is human IL- 15 (SEQ ID NO: 13) or the IL-15sushi (SEQ ID NO: 12, see e.g., Han KP et al. 2011, Cytokine. 56(3):804-10. doi: 10.1016 / j.cyto.2011.09.028).

[0230] According to specific embodiments, the cytokine is IL18.

[0231] Human IL-18 (SEQ ID NO: 15); “decoy-resistant” IL18DR (e.g., SEQ ID NO: 14) which is resistant to IL- 18 Binding peptide which is secreted by tumors to inactivate its activity (Zhou T, Damsky W, Weizman OE, McGeary MK, Hartmann KP, Rosen CE, Fischer S, Jackson R, Flavell RA, Wang J, Sanmamed MF, Bosenberg MW, Ring AM); IL-18BP is a secreted immune checkpoint and barrier to IL-18 immunotherapy. Nature. 2020 Jul;583(7817):609-614. doi: 10.1038 / s41586-020-2422-6. Epub 2020 Jun 24. PMID: 32581358; PMCID: PMC7381364); and IL-18BP which is a secreted immune checkpoint and barrier to IL- 18 immunotherapy, disclosed in e.g., Nature. 2020 Jul;583(7817):609-614. doi: 10.1038 / s41586-020-2422-6. Epub 2020 Jun 24. PMID: 32581358; PMCID: PMC7381364.), are non-limiting examples of IL-18 that can be used with specific embodiments of the invention.

[0232] The composition may include a plurality of cytokines (see e.g., variants 1 and 2 of Figure 24) which can be identical or different. When different cytokines are used and a making agent is part of the composition, then typically different masking agents are used. Though a single masking agent can be for different cytokines.

[0233] According to a specific embodiment, the at least one fusion polypeptide activates lymphoid cells selected from the group consisting of T cells (e.g., CD4 T cells, CD8 T cells, gamma delta T cells and NKT) and NK cells.

[0234] Since cytokine therapy is endowed with some disadvantages, specific embodiments envisage rendering the fusion conditionally active only in the vicinity of a tumor. Thus, the composition comprises a masking agent which is conjugated to the cytokine to mask its activity.

[0235] As used herein “a masking agent” refers to a peptide having an amino acid sequence that has an affinity sufficient to bind the cytokine in the composition, sterically interfere with its activity and / or change the conformation of the cytokine.

[0236] According to a specific embodiment, the cytokine comprises an IL-2 polypeptide and the masking agent comprises at least the binding domain to IL-2 in the IL-2 receptor (see e.g., Hsu, E. J. et al 2021. Nat. Commun. 12, 2768, and SEQ ID NOs: 22-24).According to a specific embodiment, the cytokine comprises an IL- 12 polypeptide and the masking agent comprises at least the binding domain to IL- 12 in the IL- 12 receptor (see e.g., Mansurov, A. et al. 2022, Nat. Biomed. Eng. 6, 819-829, and SEQ ID NOs: 25-27).

[0237] W02021016640 describes a domain of the IL12 receptor IL12Rbl which was fused to the IL-12, to form IL-12Rbl - IL12. This fusion is inactive, but the inclusion of an MMP or thrombin cleavage site between the receptor masking agent and the cytokine yields a pro-cytokine that can be activated in the tumor microenvironment.

[0238] According to a specific embodiment, the cytokine comprises an IL- 15 polypeptide and the masking agent comprises at least the binding domain to IL-15 in the IL-15 receptor (see e.g., Guo et al. 2021 Cell Res 31, 1190-1198, and SEQ ID NO: 28).

[0239] According to a specific embodiment, the cytokine comprises an IL- 18 polypeptide and the masking agent comprises at least the binding domain to IL- 18 in the IL- 18 receptor (see e.g., SEQ ID NO: 29). Alternatively, IL-18 is expressed naturally in an inactive precursor form (Pro-IL18) which gets cleaved by Caspase-1. Such a precursor form with an MMP14 cleavage site instead of Caspase- 1 is aimed at enhancing specificity.

[0240] According to a specific embodiment, the cytokine comprises an IL-21 polypeptide and the masking agent comprises at least the binding domain to IL-21 in the IL-21 receptor, for example a truncated form of the IL-21 Receptor (described in Edo, Angel et al. Neurotherapeutics, Volume 19, Issue 5, 1617 - 1633 2022, and SEQ ID NO: 30).

[0241] According to a specific embodiment, the cytokine comprises an Interferon-alpha (IFN-a) polypeptide and the masking agent comprises at least the binding domain to IFN-a in the IFN-a receptor see e.g., Steiner, P. et al. 2021 Blood 138, 2258-2258.

[0242] According to a specific embodiment, the cytokine comprises an Interferon-alpha (IFN-y) polypeptide and the masking agent comprises at least the binding domain to IFN- y in the IFN- y receptor.

[0243] According to a specific embodiment, the cytokine comprises a TNFa polypeptide and the masking agent comprises at least the binding domain to TNFa in the TNFa receptor.

[0244] According to a specific embodiment, the cytokine comprises a Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF) polypeptide and the masking agent comprises at least the binding domain to GM-CSF in the GM-CSF receptor (CD116).

[0245] According to a specific embodiment, the cytokine comprises a Granulocyte Colony-Stimulating Factor (G-CSF) polypeptide and the masking agent comprises at least the binding domain to G-CSF in the G-CSF receptor (CD114).In the embodiments comprising the masking agent, the fusion polypeptide comprises a cleavable moiety which ensures that the masking agent is released from the cytokine (to ensure its activity) in the tumor microenvironment (TME) via an activity of TME-associated protease so as to allow exposure of cytokine and thus activity of the immune cell activating cytokine. Since the protease is one that is enriched in the tumor microenvironment, there is a reduction of the cytokine in normal tissues when administered systemically, compared to the systemic administration of the unmasked cytokine.

[0246] Thus, according to specific embodiments, the fusion polypeptide comprises an amino acid sequence which is cleaved by a TME-associated protease.

[0247] As used herein, the phrase "tumor microenvironment (TME)-associated protease" refers to a proteolytic enzyme produced and secreted by cells (e.g., immune cells, tumor cells, stroma cell) within or in close proximity to tumor tissues. These proteases play various roles in cancer progression, immune response and tissue remodeling. It may not be TME-specific (i.e., only expressed by the cells in the TME), it is TME-enriched, meaning that the protease is expressed by a cell in the TME at a level higher than normal tissues or most normal tissues.

[0248] According to specific embodiments, the TME-associated protease is a TME-associated immune protease.

[0249] As used herein, the phrase "tumor microenvironment (TME)-associated immune protease" refers to a proteolytic enzyme produced and secreted by immune cells within or in close proximity to tumor tissues.

[0250] According to specific embodiments, the protease is secreted by an activated immune cell. “An amino acid sequence which is cleaved by a TME-associated protease” refers to an amino acid sequence comprising cleavage site that is recognized by a protease that is highly upregulated or enriched in a cell present in the TME.

[0251] According to specific embodiments, the amino acid sequence which is cleaved by a TME-associated protease is an amino acid sequence which is cleaved by a TME-associated immune protease.

[0252] “An amino acid sequence which is cleaved by a TME-associated immune protease” refers to an amino acid sequence comprising cleavage site that is recognized by a protease that is highly upregulated or enriched in an immune cell present in the TME.

[0253] According to specific embodiments, the TME-associated immune protease is secreted by a myeloid-derived suppressor cell (MDSC).

[0254] As used herein “myeloid-derived suppressor cell (MDSC)” refers to cells of the myeloid lineage which are endowed with immune suppressor activityMDSCs migrate as immature cells from the bone marrow to peripheral tissues (or tumors), where they differentiate into mature macrophages, dendritic cells, and neutrophils without suppressive phenotypes under homeostatic conditions, but become polarized when exposed to pro-inflammatory compounds, chemokines, and cytokines. In the tumor microenvironment, they suppress the anti-tumor immune response.

[0255] Suppressor activity of MDSCs is determined by their ability to inhibit the effector function of lymphocytes. Inhibition can be caused by different mechanisms.

[0256] MDSCs include various types of cells e.g., M2-macrophages (marked by the expression of any of the following markers IDO1, iNOS, CD163, MRC1 / CD206 ARG1, CX3CR1, APOE, C1QA, GPNMB, LILRB1 / ILT2, LILRB2 / ILT4, LILRB4, VISIG4, PDL1, PDL2, CLEVER1, SIRPA, SIGLEC-1, SIGLEC-9, SIGLEC-10, SIGLEC-15, SLAMF7, CSF1R, EGAES9, TREM2), including subsets such as regulatory macrophages (marked by the expression of TREM2, GPNMB, IE17R, HIEPDA, HM0X1, and VEGFA) or , but also a heterogeneous group of myeloid progenitor cells with monocytic-like characteristics CD1 lb+Ly6G-Ly6Chlghand immature myeloid cells identified by CDllb+Ly6G+Ly6Clow.

[0257] According to a specific embodiment, the MDSC is a tumor associated macrophage (TAM) and specifically an M2 macrophage which is known for its immune suppressive function.

[0258] Non-limiting examples of such proteases, that can be used with specific embodiments, include matrix metalloproteinase (MMP), ADAM-9, ADAMDEC1, Cathsepin L and Cathepsin K.

[0259] According to a specific embodiment, the protease is a matrix metalloprotease (MMP). For example, the TME-associated protease cleavage site may be one that is cleaved by MMP1, MMP2, MMP3, MMP7, MMP8, MMP9, MMP10, MMP11, MMP12, MMP13, MMP14, MMP15, MMP16, MMP17, MMP18, MMP19, MMP20, MMP21, MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, or combinations thereof.

[0260] According to a specific embodiment, the MMP cleavage site is of MMP14. Non-limiting examples of MM14 cleavage sequences are provided in SEQ ID NOs: 79-85 and Table 1 hereinbelow. It will be appreciated that some are recognized by other MMPs, such as MMP 2 and MMP9 e.g., 82-85. Other MMP14 cleavage sequences are described in Amara, Neri, Martina Tholen, and Matthew Bogyo. "Chemical tools for selective activity profiling of endogenously expressed MMP-14 in multicellular models." ACS chemical biology 13.9 (2018): 2645-2654, which is hereby incorporated by reference in its entirety.

[0261] Other examples of proteases that can be used with specific embodiments of the invention include, but are not limited to aspartate proteases (e.g., renin), fibroblast activation protein (FAP), aspartic cathepsins (e.g., cathepsin D, caspase 1, caspase 2, etc.), cysteine cathepsins (e.g.,cathepsin B), cysteine proteases (e.g., legumain), disintegrin / metalloproteinases (ADAMs, e.g., ADAM8, ADAM9), disintegrin / metalloproteinases with thrombospondin motifs (AD AMTS, e.g., AD AMTS 1), integral membrane serine proteases (e.g., matriptase 2, MT-SPl / matriptase, TMPRSS2, TMPRSS3, TMPRSS4), kallikrein-related peptidases (KLKs, e.g. KLK4, KLK5), matrix metalloproteases (e.g., MMP-1, MMP-2, MMP-9), and serine proteases (e.g., cathepsin A, coagulation factor proteases such as elastase, plasmin, thrombin, PSA, uPA, Factor Vila, Factor Xa, and HCV NS3 / 4). Alternatively or additionally, the protease is fibroblast activation protein (FAP), urokinase-type plasminogen activator (uPA, urokinase), MT-SPl / matriptase, legumain, or a matrix metalloprotease (especially MMP-1, MMP-2, and MMP-9). Those skilled in the art will appreciate that the choice of the enzyme and the corresponding cleavable sequence will depend on the disease to be treated and the protease(s) expressed by the affected tissue or organ.

[0262] Table 1 below provides some examples of proteases and their cleavage sites.

[0263] Table 1:

[0264]

[0265]

[0266] According to specific embodiments, the TME- associated protease is secreted by an NK and / or cytotoxic CD8+ T cell.

[0267] Non-limiting examples of such proteases that can be used with specific embodiments of the invention are Granzyme B and Granzyme K.

[0268] A non-limiting example of Granzyme B cleavage a sequence is provided in SEQ ID NO:According to a specific embodiment, the at least one fusion includes a single cleavage site (e.g., Variant 1 of Figure 24).

[0269] According to a specific embodiment, the at least one fusion includes multiple cleavage sites (e.g., 2, 3, 4, 5, 6, e.g., Variants 2-4 of Figure 24).

[0270] The multiple cleavage sites may be identical or different (to improve the regulation on the conditional unmasking, releasing of the masking agent).

[0271] As mentioned, the fusion may comprise a single masking agent or a plurality of masking agents.

[0272] The masking agents can be identical or different.

[0273] The attachment of the anti-NKG2A to any heterologous effector moiety described herein can be directly of via a linker.

[0274] For example, the attachment of the anti-NKG2A to the cytokine can be directly or via a linker.

[0275] Likewise, the attachment of the cytokine to the masking agent can be directly or via a linker, where the cleavage site is part of the linker or consecutively attached thereto. In any case, there is a cleavage site between the cytokine and the masking agent and it may be referred to as a linker.

[0276] Thus, linker(s), such as amino acid or peptidomimetic sequences may be inserted between the aforementioned segments. In an embodiment, a cytokine domain is joined to a Heavy (H) chain or Light (L) chain immediately after the last amino acid at the amino(NH2)-terminus or the carboxy(C)-terminus of the Heavy (H) chain or the Light (L) chain. Linkers may have one or more properties that include a flexible conformation, an inability to form an ordered secondary structure or a hydrophobic or charged character which could promote or interact with either domain. Examples of amino acids typically found in flexible protein regions may include Gly, Asn and Ser. For example, a suitable peptide linker may be GGGGSGGGGS (SEQ ID NO: 86) or (GGGGS)n (SEQ ID NO: 87), wherein n = 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 (or any range derivable therein). Other near neutral amino acids, such as Thr and Ala, may also be used in the linker sequence. The length of the linker sequence may vary without significantly affecting the function or activity of the fusion protein (see, e.g., U.S. Pat. No. 6,087,329). Examples of linkers may also include chemical moieties and conjugating agents, such as sulfo- succinimidyl derivatives (sulfo-SMCC, sulfo-SMPB), disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG) and disuccinimidyl tartrate (DST). Examples of linkers further comprise a linear carbon chain, such as CN (where N=l-100 carbon atoms, e.g., N= 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15, or more). In some embodiments, the linker can be a dipeptide linker, such as a valine-citrulline (val-cit), a phenylalanine-lysine (phe-lys) linker, or maleimidocapronic-valine-citruline-p-aminobenzyloxycarbonyl (vc) linker. In some embodiments, the linker is sulfosuccinimidyl-4-[N-maleimidomethyl]cyclohexane-l- carboxylate (smcc). Sulfo-smcc conjugation occurs via a maleimide group which reacts with sulfhydryls (thiols, -SH), while its sulfo-NHS ester is reactive toward primary amines (as found in lysine and the protein or peptide N-terminus). Further, the linker may be maleimidocaproyl (me).

[0277] According to a specific embodiment, the different segments are arranged in an N>C orientation of: targeting moiety > [linker] >cytokine>[linker]cleavage sequence[linker]>masking agent or targeting moiety > [linker] > masking agent>[linker]cleavage sequence[linker]>cytokine, where [] is optional. Such a configuration may be homodimeric.

[0278] In a heterodimer form from N>C a specific embodiment, may be for one chain: targeting moiety > [linker] > cytokine and for another chain a targeting moiety > [linker] >cleavage sequence> [linker] > masking agent, where [] is optional.

[0279] Alternatively, in a heterodimer form from N>C a specific embodiment, may be for one chain: targeting moiety > [linker] > cleavage sequence > [linker] > cytokine and another chain targeting moiety > [linker] > masking agent, where [] is optional.

[0280] According to another embodiment, the cytokine module comprises a plurality of cytokines arranged in an N^C orientation of: cytokinei >[linker]> cytokine? > ... > cytokinen, wherein each cytokine may be independently selected, engineered, or split, and optionally masked via one or more masking moieties linked through cleavage sequences.

[0281] In certain embodiments, the cytokine is provided as a split cytokine distributed across two or more chains, such that: A first chain comprises:

[0282] targeting moiety > [linker] > cytokine fragment A > [linker] > [cleavage sequence] > [linker] > [masking moiety], and a second chain comprises:

[0283] targeting moiety > [linker] > cytokine fragment B > [linker] > cleavage sequence > [linker] > masking moiety, wherein [] is optional and wherein functional cytokine activity is restored upon chain association and / or proteolytic cleavage.

[0284] Also provided herein is a polynucleotide which encodes at least portions of the fusion polypeptide or the antibody. These polynucleotides can be used per se or in the recombinant production of the polypeptides disclosed herein.

[0285] A "recombinant" polypeptide refers to a polypeptide produced by recombinant DNA techniques; i.e., produced from cells transformed by an exogenous DNA construct encoding the desired polypeptide.

[0286] The translation products can then be assembled chemically.Thus, for example, the cytokine may be attached to the anti-NKG2A antibody of some embodiments of the invention, using standard chemical synthesis techniques widely practiced in the art [see e.g., hypertexttransferprotocol: / / worldwideweb (dot) chemistry (dot) org / portal / Chemistry)], such as using any suitable chemical linkage, direct or indirect, as via a peptide bond (when the functional moiety is a polypeptide), or via covalent bonding to an intervening linker element, such as a linker peptide or other chemical moiety, such as an organic polymer. Other non-covalent interactions are also contemplated (van der Waals, hydrogen, ionic bonds, and hydrophobic interaction), provided that stability of the complex is maintained in the pharmaceutical composition and in the body at least until reaching the TME). Chimeric peptides may be linked via bonding at the carboxy (C) or amino (N) termini of the peptides, or via bonding to internal chemical groups such as straight, branched or cyclic side chains, internal carbon or nitrogen atoms, and the like.

[0287] Exemplary methods for conjugating peptide moieties are described hereinabove and below:

[0288] SPDP conjugation - A non-limiting example of a method of SPDP conjugation is described in Cumber et al. (1985, Methods of Enzymology 112: 207-224). Briefly, a peptide, such as a detectable or therapeutic moiety (e.g., 1.7 mg / ml) is mixed with a 10-fold excess of SPDP (50 mM in ethanol); the antibody is mixed with a 25-fold excess of SPDP in 20 mM sodium phosphate, 0.10 M NaCl pH 7.2 and each of the reactions is incubated for about 3 hours at room temperature. The reactions are then dialyzed against PBS. The peptide is reduced, e.g., with 50 mM DTT for 1 hour at room temperature. The reduced peptide is desalted by equilibration on G-25 column (up to 5 % sample / column volume) with 50 mM KH2PO4 pH 6.5. The reduced peptide is combined with the SPDP-antibody in a molar ratio of 1: 10 antibody: peptide and incubated at 4 °C overnight to form a peptide- antibody conjugate.

[0289] Glutaraldehyde conjugation - A non-limiting example of a method of glutaraldehyde conjugation is described in G.T. Hermanson (1996, "Antibody Modification and Conjugation, in Bioconjugate Techniques, Academic Press, San Diego). Briefly, the antibody and the peptide (1.1 mg / ml) are mixed at a 10-fold excess with 0.05 % glutaraldehyde in 0.1 M phosphate, 0.15 M NaCl pH 6.8, and allowed to react for 2 hours at room temperature. 0.01 M lysine can be added to block excess sites. After-the reaction, the excess glutaraldehyde is removed using a G-25 column equilibrated with PBS (10 % v / v sample / column volumes)

[0290] Carbodiimide conjugation - Conjugation of a peptide with an antibody can be accomplished using a dehydrating agent such as a carbodiimide, e.g., in the presence of 4-dimethyl aminopyridine. Carbodiimide conjugation can be used to form a covalent bond between a carboxylgroup of a peptide and an hydroxyl group of an antibody (resulting in the formation of an ester bond), or an amino group of an antibody (resulting in the formation of an amide bond) or a sulfhydryl group of an antibody (resulting in the formation of a thioester bond). Likewise, carbodiimide coupling can be used to form analogous covalent bonds between a carbon group of an antibody and a hydroxyl, amino or sulfhydryl group of the peptide [see, J. March, Advanced Organic Chemistry: Reaction's, Mechanism, and Structure, pp. 349-50 & 372-74 (3d ed.), 1985]. For example, the peptide can be conjugated to an antibody via a covalent bond using a carbodiimide, such as dicyclohexylcarbodiimide [B. Neises et al. (1978), Angew Chem., Int. Ed. Engl. 17:522; A. Hassner et al. (1978, Tetrahedron Lett. 4475); E.P. Boden et al. (1986, J. Org. Chem. 50:2394) and L.J. Mathias (1979, Synthesis 561)].

[0291] Alternatively, the fusion polypeptide or antibody is translated from a single open reading frame as a recombinant polypeptide or a plurality of reading frames and the translation products are assembled under conditions which allow their tertiary organization.

[0292] It will be appreciated that certain measures (e.g., modifications) may be used / introduced to ensure correct assembly of the polypeptides. Thus, for example, the knobs into holes technique may be used, as for example.

[0293] Other methods also mostly adapted from antibodies engineering may include, but not limited to:

[0294] CrossMab Technology: Involves swapping the CHI and CL domains between one arm of the antibody. This prevents mismatched pairing of light and heavy chains, ensuring that each arm of the bispecific antibody binds correctly to its target.

[0295] Dual-Variable Domain (DVD) Fab: A method where two variable domains are fused in tandem on each Fab arm, preventing heavy chain mispairing.

[0296] Fab-Arm Exchange: Involves engineering two different half-antibodies that can spontaneously exchange arms when co-expressed in the same cell line, leading to the correct formation of the desired complexes.

[0297] Controlled Fab-arm Switching: Using cysteine residues or other chemical modifications to control disulfide bonding, promoting the correct pairing of heavy and light chains during assembly.

[0298] As used herein the term “polynucleotide” refers to a single or double stranded nucleic acid sequence which is isolated and provided in the form of an RNA sequence, a complementary polynucleotide sequence (cDNA), a genomic polynucleotide sequence and / or a composite polynucleotide sequence (e.g., a combination of the above). Modifications can be included to improve bioavailability especially when the product is an mRNA.To express a polynucleotide, it is preferably ligated into a nucleic acid construct suitable for bacterial, or eukaryotic e.g., mammalian cell expression. Such a nucleic acid construct includes a promoter sequence (e.g., heterologous promoter) for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.

[0299] The expression vector of some embodiments of the invention can further include additional polynucleotide sequences that allow, for example, the translation of several proteins from a single mRNA such as an internal ribosome entry site (IRES) and / or a nucleic acid sequence encoding a self-cleavable peptide e.g., a 2A peptide (e.g., P2A, T2A, E2A); and sequences for genomic integration of the promoter-chimeric polypeptide.

[0300] Thus, according to specific embodiments, both monomers comprised in the composition are expressed from a single construct.

[0301] According to other specific embodiments, each of the monomers comprised in the composition is expressed from a different construct.

[0302] The present invention also contemplates cells comprising the composition described herein and method of generating and using same.

[0303] Thus, according to an aspect of the present invention there is provided a host cell comprising the composition of matter or antibody disclosed herein or a polynucleotide or a nucleic acid construct encoding it.

[0304] According to another aspect of the present invention, there is provided a method of producing at least one fusion polypeptide, the method comprising introducing into a host cell the polynucleotide or nucleic acid construct, or culturing the host cell.

[0305] According to an additional or an alternative aspect of the present invention, there is provided a method of producing an anti-NKG2A antibody, the method comprising introducing into a host cell the polynucleotide or nucleic acid construct, or culturing the host cell.

[0306] According to specific embodiments, the method is an in-vitro or an ex-vivo method. According to specific embodiments, the method comprises isolating the at least one fusion polypeptide or the antibody.

[0307] A variety of prokaryotic or eukaryotic cells can be used as host-expression systems to express the compositions of matter or antibodies of some embodiments of the invention. These include, but are not limited to, microorganisms, such as bacteria transformed with a recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vector containing the coding sequence; yeast transformed with recombinant yeast expression vectors containing the coding sequence; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmidexpression vectors, such as Ti plasmid, containing the coding sequence. Mammalian expression systems can also be used to express the antibodies of some embodiments of the invention. Conditions of expression in culture depend on the expression system used.

[0308] According to specific embodiments the cell is a mammalian cell.

[0309] According to specific embodiment, the cell is a human cell.

[0310] According to specific embodiments, the cell is not derived from a human embryo.

[0311] According to specific embodiments, the cell is an isolated cell.

[0312] According to specific embodiments, the cell is an immune cell.

[0313] According to other specific embodiments, the cell is not an immune cell.

[0314] According to a specific embodiment, the cell is a cell line.

[0315] According to another specific embodiment, the cell is a primary cell.

[0316] The cell may be derived from a suitable tissue including but not limited to blood, muscle, nerve, brain, heart, lung, liver, pancreas, spleen, thymus, esophagus, stomach, intestine, kidney, testis, ovary, hair, skin, bone, breast, uterus, bladder, spinal cord, or various kinds of body fluids. The cells may be derived from any developmental stage including embryo, fetal and adult stages, as well as developmental origin i.e., ectodermal, mesodermal, and endodermal origin.

[0317] Non limiting examples of mammalian cells include monkey kidney CV 1 line transformed by SV40 (COS, e.g. COS-7, ATCC CRL 1651); human embryonic kidney line (HEK293 or HEK293 cells subcloned for growth in suspension culture, Graham et al., J. Gen Virol., 36:59 1977); baby hamster kidney cells (BHK, ATCC CCL 10); mouse sertoli cells (TM4, Mather, Biol. Reprod., 23:243-251 1980); monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL- 1587); human cervical carcinoma cells (HeLa, ATCC CCL 2); NIH3T3, Jurkat, canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells (Mather et al., Annals N.Y. Acad. Sci., 383:44-68 1982); MRC 5 cells; FS4 cells; and a human hepatoma line (Hep G2), PER.C6, K562, and Chinese hamster ovary cells (CHO).

[0318] According to some embodiments of the invention, the mammalian cell is selected from the group consisting of a Chinese Hamster Ovary (CHO), HEK293, PER.C6, HT1080, NS0, Sp2 / 0, BHK, Namalwa, COS, HeLa and Vero cell.

[0319] Recovery of the polypeptide from the culture is effected following an appropriate time in the culture. The phrase "recovering the polypeptide” refers to collecting the whole fermentation medium containing the polypeptide and need not imply additional steps of separation or purification. Notwithstanding the above, polypeptides of some embodiments of the invention canbe purified using a variety of standard protein purification techniques, such as, but not limited to, affinity chromatography, ion exchange chromatography, filtration, electrophoresis, hydrophobic interaction chromatography, gel filtration chromatography, reverse phase chromatography, concanavalin A chromatography, chromatofocusing and differential solubilization.

[0320] The person of skills in the art would know which vector to select, conditions for isolation and purification and / or which chemistry to employ.

[0321] Once the antibody or fusion protein is at hand its activities such as binding to NKG2A, antagonistic activity, ability to modulate immune responses, effects on tumor growth, cytokine signaling, delivery to TME and effect of same can be determined.

[0322] The in vivo effect on e.g., tumors (e.g., biopsy) can also be evaluated especially when used in therapeutic applications to customize therapy, i.e., personalized therapy.

[0323] The antibody, fusion polypeptide or polynucleotide encoding same (e.g., DNA or mRNA) can be administered to the individual per se or as part of a pharmaceutical composition which also includes a physiologically acceptable carrier. The purpose of a pharmaceutical composition is to facilitate administration of the active ingredient to an organism.

[0324] As used herein a "pharmaceutical composition" refers to a preparation of one or more of the active ingredients described herein with other chemical components such as physiologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.

[0325] Herein the term "active ingredient" refers to the antibody or the fusion polypeptide accountable for the biological effect.

[0326] Hereinafter, the phrases "physiologically acceptable carrier" and "pharmaceutically acceptable carrier" which may be interchangeably used refer to a carrier or a diluent that does not cause significant irritation to an organism and does not abrogate the biological activity and properties of the administered compound. An adjuvant is included under these phrases.

[0327] Herein the term "excipient" refers to an inert substance added to a pharmaceutical composition to further facilitate administration of an active ingredient. Examples, without limitation, of excipients include calcium carbonate, calcium phosphate, various sugars and types of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.

[0328] Techniques for formulation and administration of drugs may be found in “Remington’s Pharmaceutical Sciences,” Mack Publishing Co., Easton, PA, latest edition, which is incorporated herein by reference.

[0329] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous andintramedullary injections as well as intrathecal, direct intraventricular, intracardiac, e.g., into the right or left ventricular cavity, into the common coronary artery, intravenous, intraperitoneal, intranasal, or intraocular injections.

[0330] Conventional approaches for drug delivery to the central nervous system (CNS) include: neurosurgical strategies (e.g., intracerebral injection or intracerebroventricular infusion); molecular manipulation of the agent (e.g., production of a chimeric fusion protein that comprises a transport peptide that has an affinity for an endothelial cell surface molecule in combination with an agent that is itself incapable of crossing the BBB) in an attempt to exploit one of the endogenous transport pathways of the BBB; pharmacological strategies designed to increase the lipid solubility of an agent (e.g., conjugation of water-soluble agents to lipid or cholesterol carriers); and the transitory disruption of the integrity of the BBB by hyperosmotic disruption (resulting from the infusion of a mannitol solution into the carotid artery or the use of a biologically active agent such as an angiotensin peptide). However, each of these strategies has limitations, such as the inherent risks associated with an invasive surgical procedure, a size limitation imposed by a limitation inherent in the endogenous transport systems, potentially undesirable biological side effects associated with the systemic administration of a chimeric molecule comprised of a carrier motif that could be active outside of the CNS, and the possible risk of brain damage within regions of the brain where the BBB is disrupted, which renders it a suboptimal delivery method.

[0331] Alternately, one may administer the pharmaceutical composition in a local rather than systemic manner, for example, via injection of the pharmaceutical composition directly into a tissue region of a patient.

[0332] The term “tissue” refers to part of an organism consisting of cells designed to perform a function or functions. Examples include, but are not limited to, brain tissue, retina, skin tissue, hepatic tissue, pancreatic tissue, bone, cartilage, connective tissue, blood tissue, muscle tissue, cardiac tissue brain tissue, vascular tissue, renal tissue, pulmonary tissue, gonadal tissue, hematopoietic tissue.

[0333] Pharmaceutical compositions of some embodiments of the invention may be manufactured by processes well known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.

[0334] Pharmaceutical compositions for use in accordance with some embodiments of the invention thus may be formulated in conventional manner using one or more physiologically acceptable carriers comprising excipients and auxiliaries, which facilitate processing of the activeingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.

[0335] For injection, the active ingredients of the pharmaceutical composition may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hank’s solution, Ringer’s solution, or physiological salt buffer. For transmucosal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art.

[0336] For oral administration, the pharmaceutical composition can be formulated readily by combining the active compounds with pharmaceutically acceptable carriers well known in the art. Such carriers enable the pharmaceutical composition to be formulated as tablets, pills, dragees, capsules, liquids, gels, syrups, slurries, suspensions, and the like, for oral ingestion by a patient. Pharmacological preparations for oral use can be made using a solid excipient, optionally grinding the resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries if desired, to obtain tablets or dragee cores. Suitable excipients are, in particular, fillers such as sugars, including lactose, sucrose, mannitol, or sorbitol; cellulose preparations such as, for example, maize starch, wheat starch, rice starch, potato starch, gelatin, gum tragacanth, methyl cellulose, hydroxypropylmethyl-cellulose, sodium carbomethylcellulose; and / or physiologically acceptable polymers such as polyvinylpyrrolidone (PVP). If desired, disintegrating agents may be added, such as cross-linked polyvinyl pyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate.

[0337] Dragee cores are provided with suitable coatings. For this purpose, concentrated sugar solutions may be used which may optionally contain gum arabic, talc, polyvinyl pyrrolidone, carbopol gel, polyethylene glycol, titanium dioxide, lacquer solutions and suitable organic solvents or solvent mixtures. Dyestuffs or pigments may be added to the tablets or dragee coatings for identification or to characterize different combinations of active compound doses.

[0338] Pharmaceutical compositions which can be used orally, include push-fit capsules made of gelatin as well as soft, sealed capsules made of gelatin and a plasticizer, such as glycerol or sorbitol. The push-fit capsules may contain the active ingredients in admixture with filler such as lactose, binders such as starches, lubricants such as talc or magnesium stearate and, optionally, stabilizers. In soft capsules, the active ingredients may be dissolved or suspended in suitable liquids, such as fatty oils, liquid paraffin, or liquid polyethylene glycols. In addition, stabilizers may be added. All formulations for oral administration should be in dosages suitable for the chosen route of administration.For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.

[0339] For administration by nasal inhalation, the active ingredients for use according to some embodiments of the invention are conveniently delivered in the form of an aerosol spray presentation from a pressurized pack or a nebulizer with the use of a suitable propellant, e.g., dichlorodifluoromethane, trichlorofluoromethane, dichloro-tetrafluoroethane or carbon dioxide. In the case of a pressurized aerosol, the dosage unit may be determined by providing a valve to deliver a metered amount. Capsules and cartridges of, e.g., gelatin for use in a dispenser may be formulated containing a powder mix of the compound and a suitable powder base such as lactose or starch.

[0340] The pharmaceutical composition described herein may be formulated for parenteral administration, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form, e.g., in ampoules or in multidose containers with optionally, an added preservative. The compositions may be suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and / or dispersing agents.

[0341] Pharmaceutical compositions for parenteral administration include aqueous solutions of the active preparation in water-soluble form. Additionally, suspensions of the active ingredients may be prepared as appropriate oily or water-based injection suspensions. Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acids esters such as ethyl oleate, triglycerides or liposomes. Aqueous injection suspensions may contain substances, which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol or dextran. Optionally, the suspension may also contain suitable stabilizers or agents which increase the solubility of the active ingredients to allow for the preparation of highly concentrated solutions.

[0342] Alternatively, the active ingredient may be in powder form for constitution with a suitable vehicle, e.g., sterile, pyrogen-free water-based solution, before use.

[0343] The pharmaceutical composition of some embodiments of the invention may also be formulated in rectal compositions such as suppositories or retention enemas, using, e.g., conventional suppository bases such as cocoa butter or other glycerides.

[0344] Pharmaceutical compositions suitable for use in context of some embodiments of the invention include compositions wherein the active ingredients are contained in an amount effective to achieve the intended purpose. More specifically, a therapeutically effective amount means an amount of active ingredients (fusion polypeptide, antibody) effective to prevent, alleviate orameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.

[0345] Determination of a therapeutically effective amount is well within the capability of those skilled in the art, especially in light of the detailed disclosure provided herein.

[0346] For any preparation used in the methods of the invention, the therapeutically effective amount or dose can be estimated initially from in vitro and cell culture assays. For example, a dose can be formulated in animal models to achieve a desired concentration or titer. Such information can be used to more accurately determine useful doses in humans.

[0347] Toxicity and therapeutic efficacy of the active ingredients described herein can be determined by standard pharmaceutical procedures in vitro, in cell cultures or experimental animals. The data obtained from these in vitro and cell culture assays and animal studies can be used in formulating a range of dosage for use in human. The dosage may vary depending upon the dosage form employed and the route of administration utilized. The exact formulation, route of administration and dosage can be chosen by the individual physician in view of the patient's condition. (See e.g., Fingl, et al., 1975, in "The Pharmacological Basis of Therapeutics", Ch. 1 P-l).

[0348] Dosage amount and interval may be adjusted individually to provide TME levels of the active ingredient are sufficient to induce or suppress the biological effect (minimal effective concentration, MEC). The MEC will vary for each preparation, but can be estimated from in vitro data. Dosages necessary to achieve the MEC will depend on individual characteristics and route of administration. Detection assays can be used to determine plasma concentrations.

[0349] Depending on the severity and responsiveness of the condition to be treated, dosing can be of a single or a plurality of administrations, with course of treatment lasting from several days to several weeks or until cure is effected or diminution of the disease state is achieved.

[0350] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity of the affliction, the manner of administration, the judgment of the prescribing physician, etc.

[0351] Compositions of some embodiments of the invention may, if desired, be presented in a pack or dispenser device, such as an FDA approved kit, which may contain one or more unit dosage forms containing the active ingredient. The pack may, for example, comprise metal or plastic foil, such as a blister pack. The pack or dispenser device may be accompanied by instructions for administration. The pack or dispenser may also be accommodated by a notice associated with the container in a form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals, which notice is reflective of approval by the agencyof the form of the compositions or human or veterinary administration. Such notice, for example, may be of labeling approved by the U.S. Food and Drug Administration for prescription drugs or of an approved product insert. Compositions comprising a preparation of the invention formulated in a compatible pharmaceutical carrier may also be prepared, placed in an appropriate container, and labeled for treatment of an indicated condition, as is further detailed above.

[0352] The compositions described herein can be used in the treatment of inflammatory diseases such as cancer.

[0353] Thus, according to an aspect of the invention, there is provided a method of treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody, thereby treating the cancer in the subject.

[0354] Alternatively or additionally, there is provided the antibody for use in treating an inflammatory disease in a subject in need thereof.

[0355] According to an aspect of the invention, there is provided a method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter, a polynucleotide encoding same or a host cell expressing same, thereby treating the cancer in the subject.

[0356] Alternatively or additionally, there is provided the composition of matter, a polynucleotide encoding same or a host cell expressing same, for use in treating cancer in a subject in need thereof.

[0357] As used herein “subject” refers to a mammal, e.g., human, diagnosed with the pathology (disease, disorder or medical condition, e.g., inflammatory disease e.g., cancer).

[0358] Inflammatory diseases that can be treated according to some embodiments of the invention include, but are not limited to, chronic inflammatory diseases and acute inflammatory diseases.

[0359] Inflammatory diseases associated with hypersensitivity

[0360] Examples of hypersensitivity include, but are not limited to, Type I hypersensitivity, Type II hypersensitivity, Type III hypersensitivity, Type IV hypersensitivity, immediate hypersensitivity, antibody mediated hypersensitivity, immune complex mediated hypersensitivity, T lymphocyte mediated hypersensitivity and DTH.

[0361] Type I or immediate hypersensitivity, such as asthma.

[0362] Type II hypersensitivity include, but are not limited to, rheumatoid diseases, rheumatoid autoimmune diseases, rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul; 15 (3):791), spondylitis, ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (l-2):49), sclerosis, systemic sclerosis (Renaudineau Y. et al., Clin DiagnLab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al., Immunol Rev 1999 Jun;169:107), glandular diseases, glandular autoimmune diseases, pancreatic autoimmune diseases, diabetes, Type I diabetes (Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), thyroid diseases, autoimmune thyroid diseases, Graves’ disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339), thyroiditis, spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15; 165 (12):7262), Hashimoto’s thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug;57 (8): 1810), myxedema, idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8): 1759); autoimmune reproductive diseases, ovarian diseases, ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 Feb;37 (2): 87), autoimmune anti-sperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43 (3): 134), repeated fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9), neurodegenerative diseases, neurological diseases, neurological autoimmune diseases, multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1;112 (1-2): 1), Alzheimer’s disease (Oron L. et al., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83), motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191), Guillain-Barre syndrome, neuropathies and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenic diseases, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000 Apr;319 (4):204), paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy, non-paraneoplastic stiff man syndrome, cerebellar atrophies, progressive cerebellar atrophies, encephalitis, Rasmussen’s encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome, polyendocrinopathies, autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156 (1):23); neuropathies, dysimmune neuropathies (Nobile- Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); neuromyotonia, acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13 ;841 :482), cardiovascular diseases, cardiovascular autoimmune diseases, atherosclerosis (Matsuura E. et al., Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9), granulomatosis, Wegener’s granulomatosis, arteritis, Takayasu’s arteritis and Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25; 112 (15-16):660); anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb Hemost.2000;26 (2): 157); vasculitises, necrotizing small vessel vasculitises, microscopic polyangiitis, Churg and Strauss syndrome, glomerulonephritis, pauci-immune focal necrotizing glomerulonephritis, crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris).

[0363] 2000 May; 151 (3): 178); antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14(4): 171); heart failure, agonist-like P-adrenoceptor antibodies in heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun;14 (2): 114); hemolytic anemia, autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan;28 (3-4):285), gastrointestinal diseases, autoimmune diseases of the gastrointestinal tract, intestinal diseases, chronic inflammatory intestinal disease (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16;138 (2): 122), autoimmune diseases of the musculature, myositis, autoimmune myositis, Sjogren’ s syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep; 123 ( 1):92); smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 Jun;53 (5-6):234), hepatic diseases, hepatic autoimmune diseases, autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326) and primary biliary cirrhosis (Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 Jun;ll (6):595).

[0364] Type IV or T cell mediated hypersensitivity, include, but are not limited to, rheumatoid diseases, rheumatoid arthritis (Tisch R, McDevitt HO. Proc Natl Acad Sci U S A 1994 Jan 18;91 (2):437), systemic diseases, systemic autoimmune diseases, systemic lupus erythematosus (Datta SK., Lupus 1998;7 (9):591), glandular diseases, glandular autoimmune diseases, pancreatic diseases, pancreatic autoimmune diseases, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647); thyroid diseases, autoimmune thyroid diseases, Graves’ disease (Sakata S. et al., Mol Cell Endocrinol 1993 Mar;92 (1):77); ovarian diseases (Garza KM. et al., J Reprod Immunol 1998 Feb;37 (2):87), prostatitis, autoimmune prostatitis (Alexander RB. et al., Urology 1997 Dec;50 (6):893), polyglandular syndrome, autoimmune polyglandular syndrome, Type I autoimmune polyglandular syndrome (HaraT. etal., Blood. 1991 Mar 1;77 (5): 1127), neurological diseases, autoimmune neurological diseases, multiple sclerosis, neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544), myasthenia gravis (Oshima M. et al., Eur J Immunol 1990 Dec;20 (12):2563), stiff-man syndrome (Hiemstra HS. et al., Proc Natl Acad Sci U S A 2001 Mar 27;98 (7):3988), cardiovascular diseases, cardiac autoimmunity in Chagas’ disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15;98 (8): 1709), autoimmune thrombocytopenic purpura (Semple JW. et al., Blood 1996 May 15;87 (10):4245), anti-helper T lymphocyte autoimmunity (Caporossi AP. et al., Viral Immunol 1998;11 (1):9), hemolytic anemia (Sallah S. et al., Ann Hematol 1997 Mar;74 (3): 139), hepatic diseases, hepatic autoimmune diseases, hepatitis, chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar;54 (3):382), biliary cirrhosis, primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551), nephric diseases, nephric autoimmune diseases, nephritis, interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140), connective tissue diseases, ear diseases,autoimmune connective tissue diseases, autoimmune ear disease (Yoo TJ. et al., Cell Immunol 1994 Aug;157 (1):249), disease of the inner ear (Gloddek B. et al., Ann N Y Acad Sci 1997 Dec 29;830:266), skin diseases, cutaneous diseases, dermal diseases, bullous skin diseases, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

[0365] Examples of delayed type hypersensitivity include, but are not limited to, contact dermatitis and drug eruption.

[0366] Examples of types of T lymphocyte mediating hypersensitivity include, but are not limited to, helper T lymphocytes and cytotoxic T lymphocytes.

[0367] Examples of helper T lymphocyte-mediated hypersensitivity include, but are not limited to, Thl lymphocyte mediated hypersensitivity and Th2 lymphocyte mediated hypersensitivity.

[0368] Autoimmune diseases

[0369] Include, but are not limited to, cardiovascular diseases, rheumatoid diseases, glandular diseases, gastrointestinal diseases, cutaneous diseases, hepatic diseases, neurological diseases, muscular diseases, nephric diseases, diseases related to reproduction, connective tissue diseases and systemic diseases.

[0370] Examples of autoimmune cardiovascular diseases include, but are not limited to atherosclerosis (Matsuura E. et al., Lupus. 1998;7 Suppl 2:S135), myocardial infarction (Vaarala O. Lupus. 1998;7 Suppl 2:S132), thrombosis (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9), Wegener’s granulomatosis, Takayasu’s arteritis, Kawasaki syndrome (Praprotnik S. et al., Wien Klin Wochenschr 2000 Aug 25; 112 (15-16):660), anti-factor VIII autoimmune disease (Lacroix-Desmazes S. et al., Semin Thromb Hemost.2000;26 (2): 157), necrotizing small vessel vasculitis, microscopic polyangiitis, Churg and Strauss syndrome, pauci-immune focal necrotizing and crescentic glomerulonephritis (Noel LH. Ann Med Interne (Paris). 2000 May; 151 (3): 178), antiphospholipid syndrome (Flamholz R. et al., J Clin Apheresis 1999; 14 (4): 171), antibody-induced heart failure (Wallukat G. et al., Am J Cardiol. 1999 Jun 17;83 (12A):75H), thrombocytopenic purpura (Moccia F. Ann Ital Med Int. 1999 Apr-Jun;14 (2): 114; Semple JW. et al., Blood 1996 May 15;87 (10):4245), autoimmune hemolytic anemia (Efremov DG. et al., Leuk Lymphoma 1998 Jan;28 (3-4):285; Sallah S. et al., Ann Hematol 1997 Mar;74 (3): 139), cardiac autoimmunity in Chagas’ disease (Cunha-Neto E. et al., J Clin Invest 1996 Oct 15;98 (8): 1709) and anti-helper T lymphocyte autoimmunity (Caporossi AP. et al., Viral Immunol 1998;11 (1):9).

[0371] Examples of autoimmune rheumatoid diseases include, but are not limited to rheumatoid arthritis (Krenn V. et al., Histol Histopathol 2000 Jul;15 (3):791; Tisch R, McDevitt HO. Proc Natl Acad Sci units S A 1994 Jan 18;91 (2):437) and ankylosing spondylitis (Jan Voswinkel et al., Arthritis Res 2001; 3 (3): 189).Examples of autoimmune glandular diseases include, but are not limited to, pancreatic disease, Type I diabetes, thyroid disease, Graves’ disease, thyroiditis, spontaneous autoimmune thyroiditis, Hashimoto’s thyroiditis, idiopathic myxedema, ovarian autoimmunity, autoimmune anti-sperm infertility, autoimmune prostatitis and Type I autoimmune polyglandular syndrome, diseases include, but are not limited to autoimmune diseases of the pancreas, Type 1 diabetes (Castano L. and Eisenbarth GS. Ann. Rev. Immunol. 8:647; Zimmet P. Diabetes Res Clin Pract 1996 Oct;34 Suppl:S125), autoimmune thyroid diseases, Graves’ disease (Orgiazzi J. Endocrinol Metab Clin North Am 2000 Jun;29 (2):339; Sakata S. et al., Mol Cell Endocrinol 1993 Mar;92 (1):77), spontaneous autoimmune thyroiditis (Braley-Mullen H. and Yu S, J Immunol 2000 Dec 15;165 (12):7262), Hashimoto’s thyroiditis (Toyoda N. et al., Nippon Rinsho 1999 Aug;57 (8): 1810), idiopathic myxedema (Mitsuma T. Nippon Rinsho. 1999 Aug;57 (8): 1759), ovarian autoimmunity (Garza KM. et al., J Reprod Immunol 1998 Feb;37 (2):87), autoimmune anti-sperm infertility (Diekman AB. et al., Am J Reprod Immunol. 2000 Mar;43 (3): 134), autoimmune prostatitis (Alexander RB. et al., Urology 1997 Dec;50 (6):893) and Type I autoimmune polyglandular syndrome (HaraT. et al., Blood. 1991 Mar 1;77 (5): 1127).

[0372] Examples of autoimmune gastrointestinal diseases include, but are not limited to, chronic inflammatory intestinal diseases (Garcia Herola A. et al., Gastroenterol Hepatol. 2000 Jan;23 (1): 16), celiac disease (Landau YE. and Shoenfeld Y. Harefuah 2000 Jan 16;138 (2): 122), colitis, ileitis and Crohn’s disease.

[0373] Examples of autoimmune cutaneous diseases include, but are not limited to, autoimmune bullous skin diseases, such as, but are not limited to, pemphigus vulgaris, bullous pemphigoid and pemphigus foliaceus.

[0374] Examples of autoimmune hepatic diseases include, but are not limited to, hepatitis, autoimmune chronic active hepatitis (Franco A. et al., Clin Immunol Immunopathol 1990 Mar;54 (3):382), primary biliary cirrhosis (Jones DE. Clin Sci (Colch) 1996 Nov;91 (5):551; Strassburg CP. et al., Eur J Gastroenterol Hepatol. 1999 Jun; 11 (6):595) and autoimmune hepatitis (Manns MP. J Hepatol 2000 Aug;33 (2):326).

[0375] Examples of autoimmune neurological diseases include, but are not limited to, multiple sclerosis (Cross AH. et al., J Neuroimmunol 2001 Jan 1 ; 112 (1-2): 1), Alzheimer’s disease (Oron L. et al., J Neural Transm Suppl. 1997;49:77), myasthenia gravis (Infante AJ. And Kraig E, Int Rev Immunol 1999;18 (l-2):83; Oshima M. et al., Eur J Immunol 1990 Dec;20 (12):2563), neuropathies, motor neuropathies (Kornberg AJ. J Clin Neurosci. 2000 May;7 (3): 191); Guillain-Barre syndrome and autoimmune neuropathies (Kusunoki S. Am J Med Sci. 2000 Apr;319 (4):234), myasthenia, Lambert-Eaton myasthenic syndrome (Takamori M. Am J Med Sci. 2000Apr;319 (4):204); paraneoplastic neurological diseases, cerebellar atrophy, paraneoplastic cerebellar atrophy and stiff-man syndrome (Hiemstra HS. et al., Proc Natl Acad Sci units S A 2001 Mar 27;98 (7):3988); non-paraneoplastic stiff man syndrome, progressive cerebellar atrophies, encephalitis, Rasmussen’s encephalitis, amyotrophic lateral sclerosis, Sydeham chorea, Gilles de la Tourette syndrome and autoimmune polyendocrinopathies (Antoine JC. and Honnorat J. Rev Neurol (Paris) 2000 Jan; 156 (1):23); dysimmune neuropathies (Nobile- Orazio E. et al., Electroencephalogr Clin Neurophysiol Suppl 1999;50:419); acquired neuromyotonia, arthrogryposis multiplex congenita (Vincent A. et al., Ann N Y Acad Sci. 1998 May 13 ;841 :482), neuritis, optic neuritis (Soderstrom M. et al., J Neurol Neurosurg Psychiatry 1994 May;57 (5):544) and neurodegenerative diseases.

[0376] Examples of autoimmune muscular diseases include, but are not limited to, myositis, autoimmune myositis and primary Sjogren’s syndrome (Feist E. et al., Int Arch Allergy Immunol 2000 Sep;123 (1):92) and smooth muscle autoimmune disease (Zauli D. et al., Biomed Pharmacother 1999 Jun;53 (5-6):234).

[0377] Examples of autoimmune nephric diseases include, but are not limited to, nephritis and autoimmune interstitial nephritis (Kelly CJ. J Am Soc Nephrol 1990 Aug;l (2): 140).

[0378] Examples of autoimmune diseases related to reproduction include, but are not limited to, repeated fetal loss (Tincani A. et al., Lupus 1998;7 Suppl 2:S 107-9).

[0379] Examples of autoimmune connective tissue diseases include, but are not limited to, ear diseases, autoimmune ear diseases (Yoo TJ. et al., Cell Immunol 1994 Aug;157 (1):249) and autoimmune diseases of the inner ear (GloddekB. etal., Ann NY Acad Sci 1997 Dec 29;830:266).

[0380] Examples of autoimmune systemic diseases include, but are not limited to, systemic lupus erythematosus (Erikson J. et al., Immunol Res 1998; 17 (l-2):49) and systemic sclerosis (Renaudineau Y. et al., Clin Diagn Lab Immunol. 1999 Mar;6 (2): 156); Chan OT. et al., Immunol Rev 1999 Jun; 169: 107).

[0381] Infectious diseases

[0382] Examples of infectious diseases include, but are not limited to, chronic infectious diseases, subacute infectious diseases, acute infectious diseases, viral diseases, bacterial diseases, protozoan diseases, parasitic diseases, fungal diseases, mycoplasma diseases and prion diseases.

[0383] Graft rejection diseases

[0384] Examples of diseases associated with transplantation of a graft include, but are not limited to, graft rejection, chronic graft rejection, subacute graft rejection, hyperacute graft rejection, acute graft rejection and graft versus host disease.Allergic diseases

[0385] Examples of allergic diseases include, but are not limited to, asthma, hives, urticaria, pollen allergy, dust mite allergy, venom allergy, cosmetics allergy, latex allergy, chemical allergy, drug allergy, insect bite allergy, animal dander allergy, stinging plant allergy, poison ivy allergy and food allergy.

[0386] Cancerous diseases

[0387] According to specific embodiments, the inflammatory disease is cancer.The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated malignant cell growth.

[0388] Cancers which may be treated or prognosed by some embodiments of the invention can be any solid or non-solid tumor (including liquid cancer), cancer metastasis and / or a pre-cancer.

[0389] Examples of cancers that can be treated according to some embodiments of the invention, include, but are not limited to, tumors of the gastrointestinal tract (colon carcinoma, rectal carcinoma, colorectal carcinoma, colorectal cancer, colorectal adenoma, hereditary nonpolyposis type 1, hereditary nonpolyposis type 2, hereditary nonpolyposis type 3, hereditary nonpolyposis type 6; colorectal cancer, hereditary nonpolyposis type 7, small and / or large bowel carcinoma, esophageal carcinoma, tylosis with esophageal cancer, stomach carcinoma, pancreatic carcinoma, pancreatic endocrine tumors), endometrial carcinoma, dermatofibrosarcoma protuberans, gallbladder carcinoma, Biliary tract tumors, prostate cancer, prostate adenocarcinoma, renal cancer (e.g., Wilms’ tumor type 2 or type 1), liver cancer (e.g., hepatoblastoma, hepatocellular carcinoma, hepatocellular cancer), bladder cancer, embryonal rhabdomyosarcoma, germ cell tumor, trophoblastic tumor, testicular germ cells tumor, immature teratoma of ovary, uterine, epithelial ovarian, sacrococcygeal tumor, choriocarcinoma, placental site trophoblastic tumor, epithelial adult tumor, ovarian carcinoma, serous ovarian cancer, ovarian sex cord tumors, cervical carcinoma, uterine cervix carcinoma, small-cell and non- small cell lung carcinoma, nasopharyngeal, breast carcinoma (e.g., ductal breast cancer, invasive intraductal breast cancer, sporadic; breast cancer, susceptibility to breast cancer, type 4 breast cancer, breast cancer- 1, breast cancer-3; breast-ovarian cancer), squamous cell carcinoma (e.g., in head and neck), neurogenic tumor, astrocytoma, ganglioblastoma, neuroblastoma, lymphomas (e.g., Hodgkin's disease, nonHodgkin's lymphoma, B cell, Burkitt, cutaneous T cell, histiocytic, lymphoblastic, T cell, thymic), gliomas, adenocarcinoma, adrenal tumor, hereditary adrenocortical carcinoma, brain malignancy (tumor), various other carcinomas (e.g., bronchogenic large cell, ductal, Ehrlich-Lettre ascites, epidermoid, large cell, Lewis lung, medullary, mucoepidermoid, oat cell, small cell, spindle cell, spinocellular, transitional cell, undifferentiated, carcinosarcoma, choriocarcinoma,cystadenocarcinoma), ependimoblastoma, epithelioma, erythroleukemia (e.g., Friend, lymphoblast), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., multiforme, astrocytoma), glioma hepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma (e.g., B cell), hypernephroma, insulinoma, islet tumor, keratoma, leiomyoblastoma, leiomyosarcoma, lymphosarcoma, melanoma, mammary tumor, mastocytoma, medulloblastoma, mesothelioma, metastatic tumor, monocyte tumor, multiple myeloma, myelodysplastic syndrome, myeloma, nephroblastoma, nervous tissue glial tumor, nervous tissue neuronal tumor, neurinoma, neuroblastoma, oligodendroglioma, osteochondroma, osteomyeloma, osteosarcoma (e.g., Ewing's), papilloma, transitional cell, pheochromocytoma, pituitary tumor (invasive), plasmacytoma, retinoblastoma, rhabdomyosarcoma, sarcoma (e.g., Ewing's, histiocytic cell, Jensen, osteogenic, reticulum cell), schwannoma, subcutaneous tumor, teratocarcinoma (e.g., pluripotent), teratoma, testicular tumor, thymoma and trichoepithelioma, gastric cancer, fibrosarcoma, glioblastoma multiforme; multiple glomus tumors, Li-Fraumeni syndrome, liposarcoma, lynch cancer family syndrome II, male germ cell tumor, mast cell leukemia, medullary thyroid, multiple meningioma, endocrine neoplasia myxosarcoma, paraganglioma, familial nonchromaffin, pilomatricoma, papillary, familial and sporadic, rhabdoid predisposition syndrome, familial, rhabdoid tumors, soft tissue sarcoma, and Turcot syndrome with glioblastoma.

[0390] According to specific embodiments, the cancer is an NKG2A rich cancer (i.e., cancer significantly expressing NKG2A).

[0391] According to specific embodiments, the cancer is selected from the group consisting of lung (e.g., NSCLC), melanoma, breast, ovarian, colorectal, liver, renal, head and neck (e.g., HNSCC) and endometrial cancer.

[0392] According to a specific embodiment, the cancer is melanoma.

[0393] According to a specific embodiment, the cancer is a solid tumor (e.g., lung cancer, liver cancer, ovarian cancer, gastric cancer and breast cancer).

[0394] According to a specific embodiment, the cancer is a primary tumor.

[0395] According to a specific embodiment, the cancer is metastatic.

[0396] According to a specific embodiment, the cancer is a secondary tumor.

[0397] According to a specific embodiment, the lung cancer is non- small cell lung cancer.

[0398] According to a specific embodiment, the lung cancer is small cell lung cancer.

[0399] According to a specific embodiment, the liver cancer is Hepatocellular carcinoma.

[0400] According to a specific embodiment, the cancer is MHC- 1 independent.

[0401] As used herein, “MHC-I independent” refer to a cancer cells which either do not express MHC-1 or express a non-functional form of MHC-I (i.e., mutated MHC-1). Such a cancer isexpected not to be recognized by CD8 cells or not to present tumor antigens on MHC- 1 molecules. This cancer may also present reduced levels of MHC-1 after fist line (or second or third line) treatment which leads to resistance.

[0402] Tumor MHC-I molecules present tumor antigens to cytotoxic CD8 T cells, and these potent immune cells are the major targeting arm of most current immunotherapies (e.g. anti-PD-1). A common tumor escape mechanism from immune checkpoint blockade (ICB) treatment is through loss of major histocompatibility class (MHC-I) molecules or additional mechanism to block activity of antigen presentation.

[0403] According to a specific embodiment, the cancer is resistant or refractory to therapy targeting PD-1 (e.g., anti-PD-1 antibodies).

[0404] Also contemplated herein are combinations with other anti-cancer treatments.

[0405] Treatment can be combined with any anti-cancer treatment known in the art, including, but not limited to, chemotherapeutic agents, radio therapeutic agents, hormonal therapy, immune modulators, engineered immune cell therapy (e.g., CAR-T) and other treatment regimens (e.g., surgery, cell transplantation e.g., hematopoietic stem cell transplantation) which are well known in the art.

[0406] The chemotherapeutic agent of the present invention can be, but not limited to, cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), asprin, sulindac, curcumin, alkylating agents including: nitrogen mustards, such as mechlor-ethamine, cyclophosphamide, ifosfamide, melphalan and chlorambucil; nitrosoureas, such as carmustine (BCNU), lomustine (CCNU), and semustine (methyl-CCNU); thylenimines / methylmelamine such as thriethylenemelamine (TEM), triethylene, thiophosphoramide (thiotepa), hexamethylmelamine (HMM, altretamine ); alkyl sulfonates such as busulfan; triazines such as dacarbazine (DTIC); antimetabolites including folic acid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxyuridine, gemcitabine, cytosine arabinoside (AraC, cytarabine ), 5-azacytidine, 2,2 •difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2 '-deoxycoformycin (pento statin), erythrohydroxynonyladenine (EHNA), fludarabine phosphate, and 2-chlorodeoxy adenosine (cladribine, 2-CdA); natural products including antimitotic drugs such as paclitaxel, vinca alkaloids including vinblastine (VLB), vincristine, and vinorelbine, taxotere, estramustine, and estramustine phosphate; epipodophylotoxins such as etoposide and teniposide; antibiotics, such as actimomycin D, daunomycin (rubidomycin), doxorubicin, mitoxantrone, idarubicin, bleomycins, plicamycin (mithramycin), mitomycinC, and actinomycin; enzymes such as L-asparaginase, cytokines such as interferon (IFN)-gamma, tumor necrosis factor (TNF)-alpha, INF -beta and GM-CSF, anti- angiogenic factors, such as angiostatin and endostatin,inhibitors of FGF or VEGF such as soluble forms of receptors for angiogenic factors, including soluble VGF / VEGF receptors, platinum coordination complexes such as cisplatin and carboplatin, anthracenediones such as mitoxantrone, substituted urea such as hydroxyurea, methylhydrazine derivatives including Nmethylhydrazine (MEH) and procarbazine, adrenocortical suppressants such as mitotane (o,p' -DDD) and aminoglutethimide; hormones and antagonists including adrenocorticosteroid antagonists such as prednisone and equivalents, dexamethasone and aminoglutethimide; progestin such as hydroxyprogesterone caproate, medroxyprogesterone acetate and megestrol acetate; estrogen such as diethylstilbestrol and ethinyl estradiol equivalents; antiestrogen such as tamoxifen; androgens including testosterone propionate and fluoxymesterone / equivalents; antiandrogens such as flutamide, gonadotropin-releasing hormone analogs and leuprolide; non-steroidal antiandrogens such as flutamide; kinase inhibitors, histone deacetylase inhibitors, methylation inhibitors, proteasome inhibitors, monoclonal antibodies, oxidants, anti-oxidants, telomerase inhibitors, BIB mimetics, ubiquitin ligase inhibitors, stat inhibitors and receptor tyrosin kinase inhibitors such as imatinib mesylate (marketed as Gleevac or Glivac) and erlotinib (an EGF receptor inhibitor) now marketed as Tarveca; and anti-virals such as oseltamivir phosphate, Amphotericin B, and palivizumab.

[0407] In some embodiments, the chemotherapeutic agent of the present invention is cytarabine (cytosine arabinoside, Ara-C, Cytosar-U), quizartinib (AC220), sorafenib (BAY 43-9006), lestaurtinib (CEP-701), midostaurin (PKC412), carboplatin, carmustine, chlorambucil, dacarbazine, ifosfamide, lomustine, mechlorethamine, procarbazine, pentostatin, (2'deoxycoformycin), etoposide, teniposide, topotecan, vinblastine, vincristine, paclitaxel, dexamethasone, methylprednisolone, prednisone, all-trans retinoic acid, arsenic trioxide, interferon- alpha, rituximab (Rituxan®), gemtuzumab ozogamicin, imatinib mesylate, Cytosar-U), melphalan, busulfan (Myleran®), thiotepa, bleomycin, platinum (cisplatin), cyclophosphamide, Cytoxan®)., daunorubicin, doxorubicin, idarubicin, mitoxantrone, 5-azacytidine, cladribine, fludarabine, hydroxyurea, 6-mercaptopurine, methotrexate, 6-thioguanine, or any combination thereof.

[0408] According to a specific embodiment, the treatment is combined with immune checkpoint blockers or inhibitors other than the anti-NKG2A antibody, such as described below. Examples of such immune checkpoint inhibitors include, but are not limited to, inhibitors of cytotoxic T-lymphocyte antigen 4 (CTLA4), programmed death 1 (PD-1) or its ligands, lymphocyte activation gene-3 (LAG3), B7 homolog 3 (B7-H3), B7 homolog 4 (B7-H4), indoleamine (2,3)-dioxygenase (IDO), adenosine A2a receptor, neuritin, B- and T-lymphocyte attenuator (BTLA), killer immunoglobulin-like receptors (KIR), T cell immunoglobulin and mucin domain-containingprotein 3 (TIM-3), inducible T cell costimulator (ICOS), CD27, CD28, CD40, CD244 (2B4), CD160, GARP, 0X40, CD137 (4-1BB), CD25, VISTA, BTLA, TNFR25, CD57, CCR2, CCRS, CCR6, CD39, CD73, CD4, CD18, CD49b, CDld, CDS, CD21, TIMI, CD19, CD20, CD23, CD24, CD38, CD93, IgM, B220 (CD45R), CD317, CDUb, Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT.

[0409] Examples of clinically approved immune checkpoint inhibitors include, but are not limited to, Ipilimumab (anti-CTLA-4), Nivolimumab (anti-PD-1) and Pembrolizumab (anti-PD-1) or anti-TREM2 blockade.

[0410] According to a specific embodiment, contemplated herein is the combination of the antibody or fusion polypeptide with inhibition of Treg cells in the tumor. For example, according to this rationale, a synergistic or additive therapeutic effect with anti-CTLA4, anti-CCR8 and / or anti-TIGIT depleting molecules is expected. Thus, according to a specific embodiment, the immune checkpoint is CTLA-4, CCR8 or TIGIT and contemplated are inhibitors thereto for depletion of Treg activity.

[0411] According to another embodiment, the treatment is combined with a Brutons tyrosine kinase (Btk) inhibitor (e.g., ibrutinib, acalabrutinib or Spebrutinib).

[0412] Additionally, molecules which act to block NK / NKT dysfunction may be effective in combination with the fusion polypeptide. Examples of such molecules include but are not limited to inhibitors e.g., antibodies, to TIGIT, TIM3, NKG2A, PD1.

[0413] According to specific embodiments, any of the genes, polynucleotides, proteins, polypeptides and / or proteinaceous moieties described herein may have a sequence of a human gene, polynucleotide, protein, polypeptide and / or proteinaceous moiety or a functional fragment or homolog thereof which exhibit the desired activity as described herein.

[0414] According to specific embodiments, the gene, polynucleotide, protein, polypeptide and / or proteinaceous moiety is of a human origin.

[0415] According to other specific embodiments, the gene, polynucleotide, protein, polypeptide and / or proteinaceous moiety is a homolog of a human gene, polynucleotide, protein, polypeptide and / or proteinaceous moiety. Such homologues can be, for example, at least 70 %, at least 75 %, at least 80 %, at least 81 %, at least 82 %, at least 83 %, at least 84 %, at least 85 %, at least 86 %, at least 87 %, at least 88 %, at least 89 %, at least 90 %, at least 91 %, at least 92 %, at least 93 %, at least 94 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the human sequence.

[0416] According to specific embodiments, the sequence of any of the genes, polynucleotides, proteins, polypeptides and / or proteinaceous moieties described herein may refer to a fragment ora homolog of the amino acid sequence or nucleic acid sequence disclosed herein which exhibit the desired activity as defined herein. The homolog (naturally occurring or synthetically / recombinantly produced) can be, for example, at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical or homologous to the polypeptide sequence provided herein or a functional fragment thereof which exhibit the desired activity as defined herein; or at least 50 %, at least 55 %, at least 60 %, at least 65 %, at least 70 %, at least 75 %, at least 80 %, at least 85 %, at least 90 %, at least 95 %, at least 96 %, at least 97 %, at least 98 %, at least 99 % or 100 % identical to the polynucleotide sequence encoding same.

[0417] Sequence identity or homology (e.g., percent homology) can be determined using any protein or nucleic acid sequence alignment algorithm such as Blast, ClustalW, and MUSCLE using default parameters.

[0418] The homolog may also refer to an ortholog, a deletion, insertion, or substitution variant, including a conservative and non-conservative amino acid substitution, as further described hereinbelow.

[0419] According to specific embodiments, the amino acid sequences described herein may comprise conservative and / or non-conservative amino acid substitutions.

[0420] The term “conservative substitution” as used herein, refers to the replacement of an amino acid present in the native sequence in the peptide with a naturally or non-naturally occurring amino or a peptidomimetics having similar steric properties. Where the side-chain of the native amino acid to be replaced is either polar or hydrophobic, the conservative substitution should be with a naturally occurring amino acid, a non-naturally occurring amino acid or with a peptidomimetic moiety which is also polar or hydrophobic (in addition to having the same steric properties as the side-chain of the replaced amino acid).

[0421] As naturally occurring amino acids are typically grouped according to their properties, conservative substitutions by naturally occurring amino acids can be easily determined bearing in mind the fact that in accordance with the invention replacement of charged amino acids by sterically similar non-charged amino acids are considered as conservative substitutions.

[0422] For producing conservative substitutions by non-naturally occurring amino acids it is also possible to use amino acid analogs (synthetic amino acids) well known in the art. A peptidomimetic of the naturally occurring amino acid is well documented in the literature known to the skilled practitioner.When affecting conservative substitutions, the substituting amino acid should have the same or a similar functional group in the side chain as the original amino acid.

[0423] The phrase "non-conservative substitutions" as used herein refers to replacement of the amino acid as present in the parent sequence by another naturally or non-naturally occurring amino acid, having different electrochemical and / or steric properties. Thus, the side chain of the substituting amino acid can be significantly larger (or smaller) than the side chain of the native amino acid being substituted and / or can have functional groups with significantly different electronic properties than the amino acid being substituted. Examples of non-conservative substitutions of this type include the substitution of phenylalanine or cycohexylmethyl glycine for alanine, isoleucine for glycine, or -NH-CH[(-CH2)5-COOH]-CO- for aspartic acid.

[0424] As used herein, "sequence identity" or "identity" in the context of two nucleic acid or polypeptide sequences includes reference to the residues in the two sequences which are the same when aligned. When percentage of sequence identity is used in reference to proteins, it is recognized that residue positions which are not identical often differ by conservative amino acid substitutions, where amino acid residues are substituted for other amino acid residues with similar chemical properties (e.g., charge or hydrophobicity) and therefore do not change the functional properties of the molecule. Where sequences differ in conservative substitutions, the percent sequence identity may be adjusted upwards to correct for the conservative nature of the substitution. Sequences which differ by such conservative substitutions are considered to have "sequence similarity" or "similarity." Means for making this adjustment are well-known to those of skill in the art. Typically, this involves scoring a conservative substitution as a partial rather than a full mismatch, thereby increasing the percentage sequence identity. Thus, for example, where an identical amino acid is given a score of 1 and a non-conservative substitution is given a score of zero, a conservative substitution is given a score between zero and 1. The scoring of conservative substitutions is calculated, e.g., according to the algorithm of Henikoff S and Henikoff JG [Amino acid substitution matrices from protein blocks. Proc. Natl. Acad. Sci. U.S.A.

[0425] 1992, 89(22): 10915-10919],

[0426] As used herein the term “about” refers to ± 10 %.

[0427] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".

[0428] The term “consisting of’ means “including and limited to”.

[0429] The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and / or parts, but only if the additional ingredients, stepsand / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

[0430] As used herein, the singular form "a", "an" and "the" include plural references unless the context clearly dictates otherwise. For example, the term "a compound" or "at least one compound" may include a plurality of compounds, including mixtures thereof.

[0431] Throughout this application, various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible subranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed subranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0432] Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases “ranging / ranges between” a first indicate number and a second indicate number and “ranging / ranges from” a first indicate number “to” a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals therebetween.

[0433] As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

[0434] As used herein, the term “treating” includes abrogating, substantially inhibiting, slowing or reversing the progression of a condition, substantially ameliorating clinical or aesthetical symptoms of a condition or substantially preventing the appearance of clinical or aesthetical symptoms of a condition.

[0435] When reference is made to particular sequence listings, such reference is to be understood to also encompass sequences that substantially correspond to its complementary sequence as including minor sequence variations, resulting from, e.g., sequencing errors, cloning errors, or other alterations resulting in base substitution, base deletion or base addition, provided that the frequency of such variations is less than 1 in 50 nucleotides, alternatively, less than 1 in 100 nucleotides, alternatively, less than 1 in 200 nucleotides, alternatively, less than 1 in 500nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.

[0436] It is understood that any Sequence Identification Number (SEQ ID NO) disclosed in the instant application can refer to either a DNA sequence or a RNA sequence, depending on the context where that SEQ ID NO is mentioned, even if that SEQ ID NO is expressed only in a DNA sequence format or a RNA sequence format.

[0437] It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable subcombination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

[0438] Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

[0439] EXAMPLES

[0440] Reference is now made to the following examples, which together with the above descriptions illustrate some embodiments of the invention in a non-limiting fashion.

[0441] MATERIALS AND METHODS

[0442] Mice - For all experiments, female (8-12 weeks old) wildtype (WT) mice (C57BL / 6 or Balb / c) (Harlan) were used. The mice were housed in the Weizmann Institute animal facility under pathogen-free conditions and 12-h light / 12-h dark cycle. All experiments were conducted as approved by the Institutional Animal Care and Use Committee.

[0443] Engineering of 4T1 tumor cells - 4T1 cells (Pulaski BA and O strand-Rosenberg S. Curr Protoc Immunol. 2001 May; Chapter 20: Unit 20.2. doi: 10.1002 / 0471142735. im2002s39) were genetically modified by introducing a blasticydin resistance cassette framed by loxP recombination sites into the Rosa26 locus by nucleofection of blasticydin / loxP plasmid, Cas9 protein (IDT) and Rosa26 sgRNA (IDT). Post blasticydin selection, a single clone was selected and a clonal cell line was generated. Afterwards, Cre and cytokine-Procode plasmids encoding IL-2s, IL-12, IL-15sushi, IL-18DR, IL-21 or GM-CSFwith Procode barcodes (generated based on Pro-Code Vector Kit, Addgene #1000000177, Wroblewska et al, 2018, Cell, 175 (4): 1141-1155.el6, NA and AA sequences are provided in SEQ ID NOs: 88-101)including a puromycin resistance cassette were introduced by reverse transfection and recombined cells were selected for 2 weeks.

[0444] Quantitative real-time PCR - RNA was isolated using a Quick-RNA Microprep kit (Zymogen R1054). Reverse transcription of RNA to cDNA was performed using a High-Capacity cDNA Reverse Transcription Kit (Thermofisher 4368814). Quantitative real-time PCR was performed using SYBR Green (Thermofisher 4385612). The following primer pairs were used:

[0445]

[0446] Production of antibodies and immunocytokines - Anti-RSV (SEQ ID: NOs 48-49), anti-RSV-IL-2SK [(SEQ ID NOs: 50-51): anti-RSV: SEQ ID NOs 48-49; IL-2sk: SEQ ID NOs: 1)], anti-RSV-IL2SK-MMPc-IL2RB (SEQ ID NOs: 209-211): anti-RSV: SEQ ID NOs 48-49; IL-2sk: SEQ ID NOs: 1, MMP14 Cleavage sites: SEQ ID NOs: 79-81, IL-2RB SEQ ID NOs: 23), anti-NKG2A-IL-2sk (SEQ ID NOs: 32-33: anti-NKG2A SEQ ID NOs: 18-20, IL-2sk: SEQ ID NO: 1), anti-NKG2A-IL2att (SEQ ID NOs: 34-35: anti-NKG2A SEQ ID NOs: 18-20, IL-2att: SEQ ID NO: 3), anti-NKG2A-IL2mut-MMPc-IL2RA (SEQ ID NOs: 36-38: anti-NKG2A SEQ ID NOs: 18-20, IL-2mut SEQ ID NO: 2, MMP14 Cleavage sites: SEQ ID NOs: 79-81, IL-2RA SEQ ID NOs:22), anti-NKG2A-IL2SK-MMPc-IL2RB (SEQ ID NOs: 39-41: anti-NKG2A SEQ ID NOs: 18-20, IL-2SK SEQ ID NO: 1, MMP14 Cleavage sites SEQ ID NOs: 79-81, IL-2RB SEQ ID NOs: 23), anti-NKG2A-IL2SK-GZBc-IL2RB (SEQ ID NOs: 42-44:, anti-NKG2A SEQ ID NOs: 18-20, IL-2SK SEQ ID NO: 1, GZBc Cleavage site SEQ ID NO: 21, IL-2RB SEQ ID NOs: 23), anti-NKG2A-IL2SK-NC-IL2RB (SEQ ID NOs: 45-47: anti-NKG2A SEQ ID NOs: 18-20, IL-2SK SEQ ID NO: 1, NC linker (SEQ ID NOs: 86-87), IL-2RB SEQ ID NOs: 23), anti-RSV-IL-12 (SEQ-ID NOs: 324-326: anti-RSV SEQ ID NOs 48-49, IL-12 SEQ ID NOs: 6-7), anti-RSV-IL-12-MMPc-IL12RBl (SEQ-ID NOs: 327-329: anti-RSV SEQ ID NOs 48-49, IL-12 SEQ ID NOs: 6-7, MMP14 Cleavage sites: SEQ ID NOs: 79-81, IL-12RB1 SEQ-ID NOs: 339-340, anti-NKG2A-IL-12 (SEQ-ID NOs: 330-332: anti-NKG2A SEQ ID NOs: 18-20, IL-12 SEQ ID NOs: 6-7), anti-NKG2A-IL-12-MMPc-IL12RBl (SEQ-ID NOs: 333-335: anti-NKG2A SEQ ID NOs: 18-20, IL-12 SEQ ID NOs: 6-7, MMP14 Cleavage sites SEQ ID NOs: 79-81, IL-12RB1 SEQ-ID NOs: 339-340), anti-NKG2A-IL-12-NC-IL12RBl (SEQ-ID NOs: 336-338: anti-NKG2A SEQ ID NOs: 18-20, IL-12 SEQ ID NOs: 6-7, NC linker SEQ ID NOs: 86-87, IL-12RB1 SEQ-ID NOs: 339-340), and custom monoclonal antibodies against human NKG2A (SEQ-ID NOs: 212-323) were generated by cloning their sequences and isolating them. The target DNA sequence was first designed, optimized, and synthesized, then sub-cloned into a proprietary expression vector. A transfection-grade plasmid was prepared for use in CHO-S cell expression. CHO-S cells were cultured in Erlenmeyer flasks at 36.5 ± 0.5 °C with 5 % CO2 on an orbital shaker. One day prior to transfection, cells were seeded at an appropriate density. On the day of transfection, the recombinant plasmid encoding the target protein was transiently transfected into the suspension CHO-S cells, followed by the addition of an enhancer and feed. The culture supernatant was harvested when cell viability fell below 80 %. For purification, the cell culture broth was centrifuged and filtered. The clarified supernatant was then loaded onto an affinity chromatography column at an appropriate flow rate. After washing and elution with suitable buffers, the eluted fractions were pooled and buffer-exchanged into the final formulation buffer. The purified protein was analyzed using SDS-PAGE, Western blot, and SEC-HPLC to assess molecular weight and purity, and the final protein concentration was determined by measuring absorbance at 280 nm (A280).

[0447] Custom monoclonal antibody production in rabbits - New Zealand rabbits were immunized with the extracellular domain (ECD) of human NKG2A protein according to standard immunization protocols. Sera were collected after the fifth immunization, and antigen- specific antibody titers were determined by indirect ELISA against recombinant human NKG2A protein. Antigen- specific B cells were isolated from peripheral blood mononuclear cells (PBMCs) using antigen-based sorting strategies. Single B cells were sorted into individual wells, and subjected to V-region sequencing. Selected clones were codon-optimized for mammalian expression and cloned into eukaryotic expression vectors containing appropriate constant regions. Following transfection, cell culture supernatants were harvested and screened for antigen- specific bindingusing indirect ELISA. For small-scale purification, supernatants were loaded onto Protein A affinity chromatography columns and bound antibodies were eluted according to standard protocols. Purified antibodies were analyzed by SDS-PAGE under reducing and non-reducing conditions to assess molecular weight and purity, and concentrations were determined by measuring absorbance at 280 nm using a NanoDrop spectrophotometer.

[0448] Tumor inoculations and in-vivo treatments - 4T1 (l-2x 106) or B16-F10 (Fidler IJ and Nicolson GL. J Natl Cancer Inst. 1976 Nov;57(5): 1199-202. doi: 10.1093 / jnci / 57.5.1199) (0.5xl06) were injected s.c. (subcutaneously). Tumor volumes and body weight were monitored twice weekly and up to daily using caliper measurements and a digital scale, respectively. Once tumors were established and reached the predefined volume, mice were i.v. (retro-orbitally), or i.t. (intratumorally) treated with the indicated constructs or i.p. with doxycycline (0.25 mg per injection) in PBS. Experimental details are provided in the respective figure legends. Tumorbearing mice were sacrificed within 19 days post-tumor implantation. Specific time points are indicated in the figure legends.

[0449] In vivo depletion experiments - To assess how depletion of specific immune cell populations influences IL-2SK-mediated anti-tumor activity, targeted depletion studies were performed as follows: Cell population depletion was started on the day prior to IL-2SK induction by doxycycline injection in established tumors (day 12). CD8 T cells were depleted by a single i.v. injection of 400 pg anti-CD8 antibody (clone YTS 169.4, Bio X Cell, Cat. No. BE0061) or rat IgG2b isotype control (Bio X Cell, Cat. No. BE0090). NK cells were depleted by i.p. injection of anti-Asialo-GMl antibody (BioLegend, Cat. No. 146002, Ultra-Leaf™ purified) twice weekly (100 pL for the first injection, 50 pL for the second injection). Tregs were depleted by i.v. injection of either anti-CTLA-4 antibody (clone 9H10, Bio X Cell, Cat. No. BE0131; 200 pg, twice weekly) or anti-CCR8 antibody (clone SA214G2, Isotype: Mouse IgG2a, in house production by R.D. Lab; 200 pg, twice weekly).

[0450] ELISA - A 96-Well ELISA microplate was coated with 0.5 pg / mL, 100 pl / well recombinant murine NKG2A (Cat. No.: 50834-M07H, Sino Biologicals), recombinant human NKG2A (Cat. No.: 13905- H07H1, Sino Biologicals), recombinant human NKG2A:CD94 (Cat. No.: CT174-H08H, Sino Biologicals) or recombinant human NKG2C (Cat. No.: 29652-H07H, Sino Biologicals) diluted in PBS pH 7.4 and incubated at 4 °C overnight. The plate was rinsed three times with 0.05 % tween20 in PBS, blocked with 1 % BSA in PBS at room temperature (RT) for 1 hour, and rinsed again. The plate was incubated with the anti-NKG2A antibodies (100 pl / well) for 2 hours at indicated concentration, at RT. Following, the plate was rinsed and incubated with Peroxidase- AffiniPure Goat anti Mouse IgG (Cat. No. 115-035-166, JacksonImmunoResearch) or Peroxidase- AffiniPure Goat anti Rabbit IgG (Cat. No.: 111-035-003, Jackson ImmunoResearch) for 20 minutes at RT. Thereafter, the plate was rinsed and incubated with a TMB Reagent (TM4500, Scytek) for 20 minutes at RT, followed by the addition of Stop Solution 2N Sulfuric Acid (DY994, R&D). OD was measured using an ELISA plate reader at dual wavelengths (450 nm and 570 nm).

[0451] HEK blue assays - HEK-Blue reporter cell lines were purchased from InvivoGen (IL-2 high affinity (IL2RA / B / G), medium affinity (IL2RB / G), IL- 12). For detecting IL-2 or IL- 12 biological activity, HEK-Blue reporter cells were incubated overnight at 37 °C with the indicated antibodies, which were serially diluted tenfold. The levels of secreted embryonic alkaline phosphatase (SEAP) in the cell culture supernatants were determined using a spectrophotometer at 630 to 650 nm.

[0452] In-vitro protease digestion assay - The immunocytokines were cocultured with active rhMMP14 (human MMP-14 catalytic domain gene (residues 112-292) doi: 10.1074 / jbc.M116.756718) in an assay buffer [50 nM Tris, 150 nM NaCl, 0.01 % Brij-35, 5 mM CaC12 (pH 7.5)] at 37 °C overnight (12 hours) for in-vitro cleavage tests.

[0453] Surface Plasmon Resonance - Affinity measurements of the immunocytokines and control antibodies were performed by surface plasmon resonance (SPR) using a Biacore T200 instrument (Cytiva) equipped with a Series S CM5 sensor chip. Recombinant mNKG2A protein (Sino Biological, Cat. No. 50834-M07H) was immobilized on the chip, and anti-NKG2A antibodies were injected as analytes to assess binding.

[0454] Inhibition assay - To assess the inhibitory activity of custom monoclonal antibody clones, ELISA plates were coated with recombinant human NKG2A / CD94 (Cat. No.: CT174-H08H, Sino Biologicals) at 1 pg / mL (100 pL / well). After coating and washing, mAb antibody clones (50 pl) and PE-Labeled Human HLA-E*01:03&B2M&CMV UL40 (VMAPRTVLL, SEQ ID NO: 208) Tetramer Protein (Cat. No.: HLU-HP2H5, ACROBiosystems) (50 pl) complex were added and incubated for 1 hour at 37 °C. After removal of unbound reagent by washing, fluorescence was measured as relative fluorescence units (RFU). Inhibitory activity was determined by decreased fluorescent signal relative to control conditions.

[0455] Spatial transcriptomics (Stereo-seq) of murine tumors with Pro-Code barcode immunofluorescence readout - To assess mosaic IL-2SK-control tumors using Stereo-seq and immunofluorescence (IF) Pro-Code staining, we s.c. injected mixed 4T1 cells (1 x 106; ratio of 0.3 control cells: 0.7 IL-2SK inducible overexpression cells) into murine hosts. Upon tumor establishment (approximately day 12), cytokine and Pro-Code expression were induced by i.p. injection of doxycycline (0.25 mg). Tumors were collected 24 hours later, embedded in OCTcompound, and stored at -80 °C. For Stereo-seq processing, 10 pm cryosections were prepared and subjected to antibody staining and spatial transcriptomic workflow according to the Stereo-seq Transcriptomics Set for Chip-on-a-Slide User Manual (mIF-compatible version 2023.3). The following kits were used: T-Slide Kit (Ref 210CT114, Lot SS-24012, No. SZ000017); Stereo-seq Transcriptomics T Kit (Ref 111KT114, Lot S 1-240228, No. SZ000195); and Library Preparation Kit M(Ref 111KL114, Lot L-231218, No. SZ000641). Briefly, cryosections were mounted on a Stereo-seq Chip Slide (pre-rinsed with nuclease-free water), dried at 37 °C for 5 minutes, and fixed in pre-cooled methanol at -20 °C for 30 minutes. After air-drying in a fume hood for 5 minutes, sections were blocked for 15 minutes at room temperature in blocking buffer (lx SSC, 10 % postcentrifuged horse serum, 5 % RI, and 0.1 % Triton X-100 in nuclease-free water; final 60 % SSC). Sections were then incubated for 45 minutes at room temperature with primary antibodies (anti-HA (rabbit, C29F4, Cell Signaling; 1:200, Cat. No.: 3724S) to detect control cells, and anti-FLAG (rat, L5, BioLegend; 1:80, Cat. No.: Biolegend 637302) to detect IL-2SK-expressing cells) in blocking buffer. After three washes with wash buffer (O.lx SSC, 5 % RI), sections were incubated with secondary antibodies (anti-rabbit AF488, Thermofisher, Al 1034; anti-rat AF555, Thermofisher, Cat. No. A78945; bothl:400) in blocking buffer for 25 minutes at room temperature in the dark. Following three additional washes, DAPI staining solution (0.2 pg / mL DAPI in lx SSC containing 5 % RI and water; final 60 % SSC) was applied for 2 minutes in the dark. Slides were rinsed once, mounted in glycerol, and image acquisition was performed using a Leica DMi8 widefield microscope (Leica Microsystems). After imaging, tissues were permeabilized in lx Permeabilization Reagent Solution at 37 °C for 10 minutes and subjected to reverse transcription at 42 °C for 3 hours. Tissue removal was performed by incubation with TR buffer at 55 °C for 10 minutes, followed by cDNA release at 55 °C overnight. The next day, cDNA was collected, purified using AMPure XP beads (Agencourt, Cat. No. A63882), amplified, and purified again.

[0456] 20 ng of cDNA was used for library preparation and sequenced on the DNBSEQ-T7 platform (paired-end 100 bp).

[0457] In vivo time-labelling for Zman-sequencing - For Zman-labelling, fluorochrome-conjugated anti-CD45 antibodies were administered retro-orbitally at defined time points prior to tumor tissue harvest. Mice received anti-CD45 BB515 (48 hours before harvest), anti-CD45 BUV737 (24 hours before harvest), and anti-CD45 BV421 (12 hours before harvest), each in a total injection volume of 50 pL. For each injection, 20 pL of anti-CD45 BB515 (0.2 mg / mL stock) or 6 pL of anti-CD45 BUV737 or BV421 (0.2 mg / mL stock) was diluted with sterile PBS to a final volume of 50 pL per mouse.Isolation of single cells for How cytometry and sorting for RNA sequencing - Murine tumors were minced into small pieces on ice and digested using RPMI supplemented with 1 mg / ml Collagenase IV and 0.05 mg / ml Dnasel at 37 °C for three times 8 minutes at 80rpm. In between the incubation steps, the tissue was further dissociated using a gentleMACS dissociator. After digestion, the cells were filtered through a 100 pm cell strainer and washed with ice cold PBS. CD45 positive immune cells were enriched using CD45 MicroBeads (Miltenyi) according to the manufacturer’ s instruction. Spleens were minced into small pieces and digested using RPMI supplemented with 0.4 mg / ml Collagenase IV and 0.05 mg / ml Dnasel at 37 °C for 30 minutes. After digestion, the cells were filtered through a 100 pm cell strainer and washed with ice cold PBS. Red blood cell lysis was performed by adding 2 ml of ACK Lysis Buffer (A10492) to the pellet and incubating on ice for 5 minutes and was followed by another washing step. Cells were stained with the following fluorescent antibodies: Anti-CD8 BUV805 (BD 612898), anti-CDllb BUV737 (BD 612800), anti-CD 19 BUV661 (BD 565076), anti-TCRbeta BUV615 (BD 751212), anti-CD45 BUV395 (BD 564279), anti TCRgd BV750 (BD 746962), anti-CD4 BV605 (Biolegend 100548), anti NKG2A / C / E BV421 (BD 740065), anti-NKl.l PE-Dazzle594 (Biolegend 108748), anti-CD90.2 AF700 (Biolegendl40323), and ZombieNIR live / dead (Biolegend, 423106) stain for 30 minutes on ice. For FACS Aurora, the cells were fixed for 30 minutes using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher Scientific 00-5523-00) and stained overnight with anti-Foxp3 PE-Cy5 (eBioscience 15-5773-82). Cells were sorted on a BD FACS Symphony 6 flow cytometer or acquired on a Cytek FACS Aurora 5L. For single cell sorting, after excluding doublets and dead cells, CD45+immune cells were sorted into 384-well capture plates. The 384-well capture plates contained a lysis solution, mineral oil and barcoded poly(T) reverse transcription primers enabling scRNA sequencing.

[0458] Human lung tumors or adjacent lung tissue were minced in small pieces and digested in 4ml digestion mix with with 1 mg / ml Collagenase IV and 0.05 mg / ml Dnasel at 37 °C at 80 rpm for 30 minutes. Prior the start of the digestion and after 30 minutes of incubation, the tissue was pipetted up / down 10 times with a 5 ml syringe and a 16G needle. This was followed by pipetting up / down 5-7 times with a 5 ml syringe and a 18G needle. The tissue was incubated for another 15 minutes at 37 °C at 80 rpm. The cells were washed with cold PBS and filtered through a 100 pm filter into a new 15 ml tube. Red blood cell lysis was performed by adding 2 ml of ACK Lysis Buffer (A10492) to the pellet and incubate on ice for 5 minutes. PBMCs were isolated from blood by Ficoll gradient centrifugation followed by red blood cell lysis which was performed by adding 2 ml of ACK Lysis Buffer (A10492) to the pellet and incubate on ice for 5 minutes. Upon anotherwash with cold PBS cells isolated from tumor tissue, adjacent lung tissues or PBMCS, were stained with the following antibodies and acquired on the FACS Aurora (Cytek): anti-CD45 BUV805 (BD 612891), anti-TCRgd BUV737 (BD 748533), anti-CD19 BUV661 (BD 741604), anti-CD4 BUV395 (BD 564724), anti-NKG2A BV786 (BD 747917), anti-CD8 BV750, anti- anti-PD1 BV650 (BD 564104), anti-CD 11b BV605 (Biolegend 101237), anti-CD3 PE-Cy7 (Biolegend 300420), anti-TCRab PE-Cy5 (Biolegend 306710), anti-CD56 PE (Cytognos CYT-56PE) and ZombieNIR live / dead (Biolegend, 423106) stain for 30 minutes on ice. After this, the cells were fixed for 30 minutes using the eBioscience™ Foxp3 / Transcription Factor Staining Buffer Set (ThermoFisher Scientific 00-5523-00) and stained overnight with anti-Foxp-3 PE-Dazzle594 (Biolegend 320125).

[0459] Single cell library preparation - MARS-seq (as described in Yofe et al. Cancer Discov. (2023) 13 (12): 2610-2631, and also in Canadian Patent No, CA3226996A1) was used to generate scRNA-seq libraries. In short, the mRNA of each well was barcoded during reverse transcription, pooled from each plate, fragmented and amplified to generate libraries for Illumina sequencing (NovaSeq X).

[0460] Analysis of single cell RNAseq data - Single cell sequencing analyses were performed using Seurat (V5). After log normalization and scaling, the data underwent principal component analysis. Clustering and UMAP dimensionality reduction were performed based on 11-15 principal components and 1-1.5 resolution. Differentially expressed genes were identified by pseudo-bulking the gene counts for each individual patient and applying DESeq2.

[0461] Pan-cancer TCGA and GTEx gene expression analysis - Normalized log2 gene expression values and phenotype annotations were downloaded from the UCSC Xena Toil compendium using UCSCXenaTools. Healthy samples were defined as GTEx normal tissues and tumor samples as TCGA primary tumors. Ensembl identifiers were mapped to HGNC symbols with biomaRt, and KLRC1 expression was extracted from the normalized matrix. Expression data were merged with phenotype information and restricted to organs that contained at least ten healthy and ten tumor samples.

[0462] Pan-cancer single-cell RNA-seq cytokine receptor profiling and immunocytokine target identification - Analyses of cytokine receptor expression and identification of immunocytokine target genes were performed using the pan cancer dataset from Qian et al., comprising 233,591 single cells from patients with lung, colorectal, ovarian and breast cancer (n = 36; http: / / blueprint.lambrechtslab.org / ). T and NK cells or B and myeloid cell clusters were integrated across cancers using Harmony in Seurat (v5), and clusters were annotated based on marker gene expression identified with FindAllMarkers together with expression of lineage marker genes. Topredict cytokine ligand activity across the different immune cell types, ligand-target gene sets were curated from NicheNet, extracting a total of 84 cytokine ligands (including interleukins, TNF superfamily members, CSFs, VEGFs, and interferons) and their downstream target genes. Then the VIPER algorithm implemented in the decouplerpy package [Badia-i-Mompel, P. el al. Bioinformatics Advances 2, vbac016 (2022)] was applied to infer cytokine activity scores at singlecell resolution using the curated target gene sets. Activity scores were z-scaled across cells and averaged by our defined clusters for visualization. Genes enriched in effector clusters compared with Tregs (Tregs effector and Tregs naive like) were identified using FindMarkers to obtain positive markers with logFC > 0.2 and padj < 0.01, followed by an additional fdter requiring expression in less than 1 percent of Treg cells. Candidate genes were further filtered for off-target expression by calculating the percentage of expressing cells per global compartment in the whole tumor object and retaining genes expressed in less than 1 percent of myeloid, malignant, stromal and B cells. Genes were annotated for subcellular location using UniProt, and only cell membrane / surface expressed genes were retained as immunocytokine targets.

[0463] Spatial transcriptomics analysis - Stereo-seq data preprocessing and staining integration

[0464] Staining images (DAPI, IL2-SK, and control) were first processed in ImageJ and ImageStudio v3 to detect tracklines for downstream alignment with the sequencing data, quality assessment and multi-channel alignment of the 3 different channels. Raw sequencing data were then processed using the SAW pipeline (v7.1.1) and aligned to the mmlO genome. Cell segmentation was performed using a state-of-the-art deep learning-based approach optimized for dense tumor regions (implemented in SAW), combining nuclei detection with probabilistic cell boundary expansion (by Gaussian mixture model) to maximize transcript recovery.

[0465] Processed anndata were loaded into Python using scanpy for downstream analysis. Cells with <10 % mitochondrial reads, <30 % ribosomal reads, <10,000 UMI, and a minimum of >30 genes expressed were retained for analysis, with a total of 199,098 high-quality single cells. Genes expressed in < 20 cells and non-informative gene categories (e.g., Gm, Rik, mitochondrial, and ribosomal genes) were removed. Then data normalization was performed using library size normalization (sc.pp.normalize_total with 10,000 total counts per cell) followed by log Ip transformation. To integrate the staining image data for the IL2-SK and control intensities, staining intensity was aggregated within each expanded cell and normalized by cell area.

[0466] ResolVI modeling of spatial transcriptomics and downstream analysis

[0467] In order to address spatial confounding effects and accurately identify cell subpopulations in stereo-seq data, resolVI modeling was applied on the expanded cells to obtain corrected latentspace and counts. The resolVI model was trained with the following hyperparameters: spatial n_neighbors = 20, n_latent = 10, semisupervised=False, mixture_k=100, deeply _inject_covariates=True, encode_covariates=False, downsample_counts=50; training with a maximum epoch of 200 with learning rate=0.001 and lr_extra=0.01. The latent embedding was then visualized by constructing a k-nearest neighborhood graph of cells and applying UMAP for 2D visualization. Cell clustering was performed using the Leiden algorithm, clusters were annotated based on canonical markers.

[0468] To classify control and IL2-SK tumor regions, z-score barcode intensities of the control and IL-2SK were computed individually. Then, in the tumor cells, control regions were defined if they had a control z-score >0 and an IL2-SK z-score <1. The remaining tumor cells with IL2-SK enrichment were classified as IL2-SK regions. Proliferative T / NK cells were identified by scoring a list of strong proliferation gene signature (Mki67, Top2a, Ccnd.1, Ccnbl, Hells) with the score_genes function.

[0469] Subsequently, to statistically quantify immune cell infiltration patterns, pairwise nearest-neighbor distance analysis was performed between cell populations of interest with respect to the two classified tumor regions (control and IL2-SK). Spatial coordinates were first normalized by the median cell diameter (approximately 20 microns, derived from the square root of the cell area) to express distances in cell-equivalent units. Minimum pairwise Euclidean distance were computed between each cell and its four nearest neighbors of the target cell type. Empirical cumulative distribution functions (CDFs) were subsequently generated based on the pairwise minimum distance information to compare the spatial co-localization of proliferative T / NK cells and TAMs to each of control and IL2-SK tumor regions. Statistical significance of distribution differences was assessed using the two-sample Kolmogorov-Smirnov (KS) test with exact method computation.

[0470] Temporal transcriptomics (Zman-seq) analysis - Time bin assignment of immune cells

[0471] To assign a cytokine exposure time bins to each tumor-infiltrating immune cells, the fluorescence-minus-one FMO controls were used as unstained references to build a generalized linear model (GLM) for classifying the fluorescent FACS intensity at each time stamp (12 hours, 24 hours, 48 hours). The GLM framework was adapted and modified using the ZmanR package with modifications to train a second-order individual GLM with 14 staining markers (CD 11b, CD 19, CD4, CD64, CD8, CD49b, CD90.2, GM-CSFRa, GITR, IL-21R, IL-2Ra, IL-2Ry, TCRp, IL-18Ra) as predictors. Model fit was validated by assessing residual normality using residual-versus-fitted plots and Q-Q plots. Each cell was then assigned a cytokine exposure time bin based on itspositive antibody stains; for cells positive for multiple stains, the time bin was determined by the stain corresponding to the latest exposure time point in circulation.

[0472] Temporally-resolved, cytokine perturbation effects with MrVI modeling

[0473] To dissect temporal and cytokine-specific effects on the immune landscape, MrVI was employed, a variational inference-based deep learning framework, to generate a graph representation of temporally resolved cytokine perturbation effects normalized to control. Raw counts from preprocessed data were used as input, along with the top 5,000 highly variable genes identified using Scanpy's implementation of Seurat v3. The MrVI model was trained using combined time stamp and cytokine perturbation labels as the sample key, with the following: maximum 400 epochs, batch size of 256, learning rate of 0.001, KL-warmup period of 20 epochs, and early stopping triggered by an elbo_validation threshold of 30. The sample-aware latent space representation (z) was then used to calculate aggregated local sample distances for each temporalcytokine condition, which were subsequently normalized relative to control. A 2D graph layout was subsequently generated from the treatment distance matrix using the Kamada-Kawai force-directed algorithm to visualize temporal-cytokine relationships.

[0474] Quantification and downstream analysis of continuous cytokine exposure time (AUC time} To quantify and derive differentiation trajectories within major immune cell populations following cytokine exposure, an adapted approach was developed to estimate continuous cytokine exposure time at single-cell resolution from ZmanR package [Kirschenbaum, D. etal. Cell 187, 149-165. e23 (2024)]. Cells were partitioned into major immune populations (including Tconv cells, NK cells, Macrophages and MDSCs) and their respective latent space representations (z) from the MrVI model were expected. To estimate temporal positioning of individual cells, neighborhood-based inference was leveraged by examining the 200 nearest neighbors of each cell and analyzing their distribution across discrete time stamps. From this neighborhood composition, a single-cell area under the curve (AUC) time metric was derived similarly to ZmanR. This continuous cytokine exposure time estimate enables positioning of each cell along a temporal axis independent of its original discrete time bin assignment.

[0475] To visualize gene module dynamics along these trajectories, each cell was scored for defined gene modules using Scanpy's score_genes function and first aggregated scores per temporal-cytokine condition for projection onto 2D network representations. For heatmap visualization, the AUC time was utilized to divide cells into 100 equal bins across < 12 hours to 48 hours in each trajectory and computed the difference in module score along AUC time for each cytokine condition relative to the control. This approach enables visualization of the temporal dynamics of gene program activation while accounting for baseline temporal effects observed in the control.EXAMPLE 1

[0476] EXPRESSION PATTERN OF CYTOKINE RECEPTORS

[0477] Cytokines can elicit antitumor immunity, but pleiotropy and an incomplete understanding of their biology have limited clinical translation. Previous systematic, single-cell-resolved cytokine-response maps were generated either in vitro using PBMCs [Oesinghaus, L. et al. bioRxiv 2025.12.12.693897 (2025) doi: 10.64898 / 2025.12.12.693897] or in healthy tissue [Cui, A. et al. Nature 625, 377-384 (2024)], leaving unresolved how cytokine programs unfold within the immunosuppressive tumor microenvironment. To address this gap, a data-driven pipeline was developed that maps responses to cytokines and cytokine combinations in solid tumors and translates these response maps into targeted cytokine-based therapies. The framework integrates pan-cancer single-cell RNA-seq based prioritization of clinically relevant cytokines, targeted spatiotemporal in vivo single-cell response screening, and development of effector-directed immunocytokines guided by these chrono-immunology response maps (Figure 25).

[0478] First, decoupler [Badia-i-Mompel, P. et al. Bioinformatics Advances 2, vbac016 (2022)] was used to infer ligand activity for 84 cytokines and cytokine-like factors across immune cell types, based on downstream target gene expression collected from the NicheNet [Browaeys, R., Saelens, W. & Saeys, Y. Nat Methods 17, 159-162 (2020)] ligand-target database. This approach was applied to a pan-cancer single-cell RNA-seq atlas [Qian, J. et al. Cell Res 30, 745-762 (2020)] integrating 168,215 high-quality cells from four human cancer types (breast, lung, ovarian, and colorectal). After integrating immune cells across cancer types, the immune compartment was clustered into 28 subclusters (16 T and NK cell clusters and 12 myeloid and B cell clusters; Figures 27A-D). Hierarchical clustering of inferred activity scores revealed clear lineage structure: myeloid populations including dendritic cells (DCs), myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), exhibited enrichment for colony-stimulating factors (CSFs), VEGF family members and interferons, whereas lymphoid populations showed enrichment for interleukins and selected TNF superfamily members (Figure 27E). Notably, consistent with cytokine pleiotropy, most cytokines showed high inferred activity in both antitumor effector populations (CD8 T cells, y5 T cells, NK cells, DCs, and pDCs) and immunoregulatory populations (Tregs, MDSCs, and TAMs). Based on their strong predicted activity in effector populations and high clinical relevance reflected by extensive clinical evaluation [Berraondo, P. et al. Br J Cancer 120, 6-15 (2019) and Fu, Y., Tang, R. & Zhao, X. Front. Immunol. 14, (2023)], six cytokines were prioritized for mechanistic comparison: IL-2, IL-12, IL-15, IL-18, IL-21 and GM-CSF. The five interleukins were primarily associated withlymphoid populations, including suppressive Tregs, whereas GM-CSF (CSF2) was associated with myeloid populations, including TAMs and MDSCs (Figure 26).

[0479] The cytokines IL-2, IL-12, IL-15, IL-18, IL-21 and GM-CSF are among the most potent cytokines known to induce tumor eradication in murine models and most studied for clinical applications. Cytokines mediate their effects by binding to receptors expressed on the surface of various cell types. The receptors are made up of several subunits which are needed to allow the cytokines to induce downstream programs in the cells (Figure 2).

[0480] To identify which cells are equipped with the cytokine receptors for IL-2, IL-12, IL-15, IL- 18, IL-21 and GM-CSF in human cancer, the expression of the individual receptor subunits and percentage of combined expression of several subunits making up functional receptors were analyzed across broad cell categories in four human cancers types harnessing a single-cell sequencing dataset (Qian et al. 2020, Cell Res, 30, 745-762) (Figure 3). A high conservation of cytokine receptor expression patterns was found across cancer types which were exclusively expressed in the immune compartment, but not stromal or cancer cells (e.g., epithelial or endothelial cells). More specifically, receptors for IL-2, IL-12, IL-15, IL-18 and IL-21 were detected in the T / NK, myeloid and B cell compartment, whereas the receptor for GM-CSF was detected solely in the myeloid compartment (Figure 3).

[0481] A more detailed analyses of the immune compartment across the four human cancer types, revealed that also within the immune compartment a high level of conservation was found across cancer types (Figure 4). However, each cytokine receptor showed a unique expression pattern across cell types. For instance, in line with previous literature, the “IL-2 receptor intermediate affinity” comprising IL2RG and IL2RB, was distributed in various different T and NK subsets, while the “IL-2 receptor high affinity” comprising IL2RA, IL2RB and IL2RG was mostly expressed in Tregs - dominant immunosuppressor cells limiting immune responses against tumors. The receptors for IL- 12 and IL- 18 were also mostly found in different T and NK cell subsets, while IL-21 receptors were also detected in B cells / Plasma cells. All cytokine receptors except GM-CSF were expressed in Tregs, which are a potential off-target for cytokine therapies. Tumor associated macrophages (TAMs), dendritic cells (DCs) and myeloid-derived suppressor cell subsets were identified as cells which can sense GM-CSF (Figure 4).

[0482] To identify cellular subsets which can be stimulated by IL-2, IL- 12, IL- 15, IL- 18, IL-21 and GM-CSF in murine tumors, single-cell RNA sequencing of immune cells isolated from preclinical murine breast cancer tumors (4T1) was performed (Figure 5). Similar expression patterns as in human tumors were found, with high receptor expression in T and NK cell subsets for IL-2, IL-12, IL-15, IL-18 and IL-21, while GM-CSF receptors were detected in myeloid cells.In the mouse as well, Tregs also represent a potential sink for successful cytokine therapies making up a high percentage of the total cytokine receptor expressing cells (Figure 5).

[0483] EXAMPLE 2

[0484] THE IN-VIVO EFFECTS OF CYTOKINES ON CANCER

[0485] To study the immune response elicited by various cytokines in the tumor microenvironment, 4T1 tumor cells were genetically modified to allow the release of cytokines and a spatial barcode (Pro-code system) in an inducible fashion (Figure 6). The cytokines included in this model, IL-2s, IL-12, IL-15sushi, IL-18DR, IL-21 and GM-CSF, can be induced in established subcutaneously injected tumors by doxycycline injection. This approach allows a precise timing of cytokine release and investigating the dynamics of tumor eradication and induced immune reprogramming (Figure 6).

[0486] Quantitative real-time PCR was used to confirm the expression of the cytokines by the six 4T1 cell lines upon doxycycline administration in-vitro (Figure 7).

[0487] Following, to study the in-vivo effect of the 6 cytokines, the genetically modified mouse 4T1 tumor cells were subcutaneously (s.c.) injected into murine recipients (Figure 8). In established tumors, on day 12, cytokines expression was induced in the tumor by intraperitoneal (i.p.) doxycycline injection and was compared to PBS control injections. Furthermore, cells without a cytokine insert were used as negative controls. These negative control cells were not affected by PBS or doxycycline administration, as expected. Among the genetically modified cells, IL-2s, followed by IL- 12 and GM-CSF were the cytokines which induced the strongest reduction in tumor growth and weight. IL-15sushi and IL-18DR showed mild effects; whereas for IL-21 no an anti-tumor effect was observed (Figure 8). Directly comparing the tumor growth dynamics of the six cytokines using normalized tumor sizes confirmed the observation that IL-2s and IL- 12 were the cytokines with the most potent tumor eradication properties (Figure 8).

[0488] Next, single-cell RNAseq was performed on immune cells isolated from the tumors 48 hours post cytokine induction to capture cytokine-induced cellular immune reprogramming (Figure 9). In line with the drastic tumor eradication induced by IL-2s and IL- 12, a pronounced expansion of several proliferating NK cell and T cell subsets with IL-2s and early activated and proliferating T cell subsets with IL- 12 were observed (Figure 9). As expected by the patterns of the cytokine receptors, Tregs frequencies were increased by most of the cytokines (Figure 9).

[0489] Comparative analysis of cluster frequencies and transcriptional programs revealed distinct, cytokine- specific reprogramming signatures (Figures 28A and 29B-C). Consistent with efficacy, IL-2SK, followed by IL- 12 and GM-CSF, induced the most pronounced compositional shifts(Figures 29B-C). Differential gene expression analysis across major immune lineages using DESeq2 identified IL- 12 as the strongest transcriptional reprogramming factor (857 differentially expressed genes (DEGs); |LogFC| >1, padj <0.05), followed by IL-2SK (478) and GM-CSF (301) (Figures 28A-D and 30A-C).

[0490] IL-2SK drove a dominant NK cell-centered response at both compositional and transcriptional levels (Figures 28A and C). It expanded proliferating NK cell subsets (early, mid and late cell cycle (CC)) (Figures 29B-C) and upregulated proliferation-associated genes including Mki67, Top2a, and Ccnb2 (Figure 28C). In conventional T cells, IL-2SK induced proliferation modules, and modestly increased activation and effector features in both T and NK cells including Il2ra, Cd226, Klrgl and Ly6a (Figures 28B-C). These effects were accompanied by reduced hypoxia-associated suppressive TAMs, expansion of MDSCs with IFN -response features and mild pro-inflammatory remodeling of TAMs and MDSCs (Figures 28D, 29B-C and 30A-B).

[0491] By contrast, IL- 12 induced cytotoxic and activation programs in lymphocytes (for example Klrgl, Cd69, Gzmb, Prfl ; Figures 28B-C) and profoundly remodeled myeloid states (Figures 28A, 28D and 31A-C). This myeloid reprogramming occurred despite low IL- 12 receptor expression on TAMs and MDSCs (Figure 29A), suggesting secondary cytokine-driven cascades. IL- 12 reduced neutrophils and suppressive TAMs and reshaped MDSC population frequencies (Figure 29B-C). In macrophages, IL- 12 induced strong inflammatory, antigen-presentation and interferon-response programs (for example Ly6cl, H2-K1, Cd40, Batf2, Ifi47, Ifit2, Gbp9), while suppressing immunoregulatory programs (for example Mrcl, Argl, Veg fa), with induction of few counter-regulatory genes including Ill8bp and H2-T23 (Figure 28D). MDSCs showed the same overall directionality, shifting towards inflammatory, monocytic and antigen-presentation phenotypes, alongside a limited increase in feedback-associated genes (for example Ill8bp, Cd274, Zeb2 and H2rg) (Figure 30B). In DCs, IL-12 decreased suppressive, and maturation associated features (Cd200, Cell 7, Nrp2), while enhancing inflammatory and antigen-presentation programs (for example H2-T24, Cxcl9, Nlrp3, Ifi205, lfi44) (Figure 30C). Thus, IL- 12 potently induced lymphocyte cytotoxicity and reprogrammed myeloid states toward immunostimulatory and tumor-restraining phenotypes.

[0492] IL-15sushi expanded Tregs and proliferating conventional T cells and reduced neutrophil frequencies (Figure 29B-C). It induced expression of TCR inducible genes (for example Nr4a2, Dusp2) and enhanced exhaustion-associated markers such as Pdcdl and Tigit in conventional T cells (Figure 28B). IL18DR and IL-21 induced more modest compositional changes; IL-21 expanded DCs, whereas IL-18DR showed trends toward increased NK cells and, similarly to IL-15sushi, enhanced expression of Pdcdl and Tigit in conventional T cells (Figures 28B). GM-CSFprimarily remodeled myeloid populations and induced mixed phenotypes (Figures 28D and 30A-B). It expanded suppressive MDSCs and TAMs (Figures 29B-C) and induced both pro-inflammatory and immunoregulatory programs (Figures 28D and 30A-B). In macrophages, GM-CSF reduced several suppressive and remodeling genes (for example Tmeml76b, Apoe, Havcr2, Fcgrl, Vegfa), but concurrently upregulated other suppressive mediators (for example Lpl, Illr2, Cish) (Figure 28D and 30A). In MDSCs, it increased inflammatory and phagocytosis related genes (for example Irf5, Cybb, Cfp), while also upregulating neutrophilic, anti- apop to tic and suppressive programs (for example Cd.177, Bcl2alb, Mmpl9, Chil3) (Figure 30B). Notably, all cytokines except IL- 12 tended to expand Treg frequencies (Figures 29B-C).

[0493] To define which immune populations mediated IL-2SK-driven tumor control, in vivo depletion of Tregs, NK cells and CD8 T cells was performed (Figures 30D-E). Treg depletion using anti-CCR8 or anti-CTLA-4 antibodies further reduced tumor volume relative to IL-2SK alone (Figure 30D), indicating that Tregs limit IL-2SK efficacy despite enhanced IL-2RP affinity. NK cell depletion (anti-Asialo-GMl) markedly impaired IL-2SK-mediated tumor control, whereas CD8 T cell depletion (anti-CD8) had only a modest effect (Figure 30E), identifying NK cells as a dominant mediator of IL-2SK-driven tumor suppression in this model.

[0494] Because cytokine responses are shaped by both spatial context and temporal dynamics, a spatiotemporal profiling was next applied to resolve cytokine-mediated immune remodeling in vivo. First, spatial reprogramming induced by IL-2SK within the tumor microenvironment was characterized. To this end, control and IL-2SK 4T1 tumor cells were mixed and subcutaneously co-injected to generate mosaic tumors, followed by cytokine- and Pro-Code-barcode induction after tumor establishment (Figure 31A-E and 30F). Tumors were harvested 24 hours after induction, and a tumor section was profiled by spatial transcriptomics [Stereo-seq (Chen, A. el al. Cell 185, 1777-1792.e21 (2022)] together with Pro-Code tag staining and immunofluorescence imaging on the same section to delineate control and IL-2SK-tumor regions (Figures 31A-E and 30F). 199,095 high-quality spatially resolved single cells were obtained using deep-leaming-based cell segmentation and subsequently ResolVI ]Ergen, C. & Yosef, N. ResolVI - addressing noise and bias in spatial transcriptomics. 2025.01.20.634005 Preprint at https: / / doi.org / 10.1101 / 2025.01.20.634005 (2025)] was applied to reduce noise in the misassignment of molecules for downstream analysis. Eight major cell clusters were identified spanning immune, stromal and malignant populations (Figures 31A-B and 30F). By quantifying barcode staining intensities in tumor cells, IL-2SK-enriched regions across the tissue section were mapped (Figure 31C). Applying enrichment thresholds, the tumor microenvironment was classified into IL-2SK and control niches, enabling side-by-side comparisons within the samesection (Figure 3 ID). Consistent with the single-cell RNA-seq atlas, proliferating T and NK cells were enriched in IL-2SK regions, whereas these regions were relatively depleted of TAMs (Figure 3 ID). Analysis of empirical cumulative distribution functions (CDF) of minimal cell-to-cell distances further revealed significant co-localization of proliferating T and NK cells with IL-2SK tumor cells (p < 0.001) and of TAMs with control tumor cells (p < 0.001) (Figure 3 IE). Together, these data show that tumor-confined IL-2SK generates spatially restricted immune remodeling with local enrichment of proliferating lymphocytes.

[0495] To characterize the temporal dynamics of cytokine-induced immune responses, cluster frequencies were quantified at 12-, 24- and 48-hours cytokine exposure bins (Figures 31F-G and 32A-B). IL-2SK showed strongly time-dependent effects, with NK cells displaying expansion of proliferating phenotypes already 12 hours following cytokine exposure (Figures 31F-G). By contrast, IL-2SK-driven expansion of proliferating conventional T cells and MDSCs with an interferon-response phenotype emerged later, predominantly at 24-48 hours (Figures 31F-G). IL-12-indcued compositional changes were more stable over time, with mature NK cells consistently expanded across timepoints (Figures 31F-G). GM-CSF-driven myeloid reprograming was most evident at 24-48 hours and included expansion of MDSC and TAM populations (Figures 31F-G).

[0496] To delineate cytokine-induced transcriptomic reprogramming over time, the deep generative model MrVI [Boyeau, P. et al. Nat Methods 22, 2264-2274 (2025)] was applied and a temporally resolved cytokine-specific two-dimensional distance similarity network was constructed (Figure 31H). Hierarchical clustering on the MrVI distance of treatment-timepoint combinations revealed largely treatment- specific groupings, indicating that each cytokine maintained a distinct transcriptional trajectory over time (Figure 31H). Notably, IL-2SK at 48 hours clustered with IL-18DR, and GM-CSF at 12 hours clustered with IL-21, suggesting shared late or early dynamics that differed from other timepoints of the same cytokine (Figure 31H). Projection of the gene programs described above (Figure 28B-D and 30A-C) onto this network revealed strongly cytokine- specific modules, whose directionality was generally conserved for a given cytokine, with intensities that were stable or time-varying across exposure bins (Figures 321-K and 32C-F). IL-2SK and IL- 12 showed the most divergent profiles, dominated by proliferation (IL-2SK) versus activation, cytotoxicity and pro-inflammatory programs (IL-12) (Figures 3 ILK). These analyses indicate that cytokines drive distinct transcriptional programs that persist as coherent trajectories over time.

[0497] To resolve continuous temporal dynamics beyond discrete time bins, the exposure labels were converted into a continuous cytokine exposure metric by analyzing the time-label distribution among each cell’s nearest neighbors and an area-under-the-curve (AUC) score was computed[Figures 31L-N and 32G-J; Zman-seq (Kirschenbaum, D. et al. Cell 187, 149-165. e23 (2024)]. Using cells from control tumors as the reference AUC trajectory, temporal deviations in cytokine-induced gene programs were quantified within major lineage clusters along differentiation trajectories (Tconv cells, NK cells, macrophages, MDSCs; Figures 31L-N and 32G-J). This revealed cell-type specific temporal patterns across cytokines. IL- 12 induced an early and relatively continuous increase in activation, interferon-response and cytotoxic programs in lymphocytes and enhanced antigen presentation in macrophages. By contrast, IL-2SK- induced NK cell cytotoxicity and IL-15sushi- or IL-18DR-associated T cell exhaustion programs emerged later, predominantly between 24 and 48 hours (Figures 31L-N). Notably, IL-2SK-driven proliferative transcriptional programs persisted in NK cells and appeared early in T cells (Figures 31L-M), whereas proliferating NK cell frequencies peaked at 12 hours and T cell expansion was most evident at 48 hours (Figures 31G). This pattern is consistent with sustained per-cell proliferative transcription in NK cells despite a transient population-level burst, and a lag in T cells where early transcriptional commitment precedes later numerical expansion. GM-CSF induced macrophage reprogramming was also highly dynamic, characterized by early emergence of inflammatory, hypoxia-, and glycolysis-related programs (Figures 3 IN and 321). Together, continuous -time analysis resolves distinct temporal ordering of cytokine programs that is not captured by population frequencies alone.

[0498] In summary, tumor-derived IL-2SK locally remodeled the tumor microenvironment, promoting accumulation of proliferating T and NK cells while reducing TAM proximity. Temporal delineation further revealed a sequence of IL-2SK effects, with early NK cell proliferation followed by later T cell and myeloid changes, whereas IL- 12 induced more temporally stable cytotoxic and myeloid-reprogramming. These spatiotemporal response maps establish distinct cytokine- specific trajectories in vivo and provide a quantitative foundation for prioritizing cytokines and combinations for therapeutic translation.

[0499] To test whether the use of several cytokines can even further enhance tumor eradication and immune activation, mixtures of tumor cell lines expressing different cytokines were injected and their effects on tumor growth were studied (Figure 10). Since IL-2s was identified as the cytokine with the most powerful properties, the effects of 1 : 1 mixtures of IL-2s together with each of the five other cytokines (IL-12, IL-15sushi, IL-18DR, IL-21 and GM-CSF) were compared to each other. Interestingly, all of the combinations allowed a strong reduction of tumor growth, more pronounced than what was observed with the individual cytokines. However, the combination of IL-2s and IL- 12 showed the strongest synergy, leading to an almost complete tumor eradication (Figure 10). Single-cell RNA sequencing revealed high frequencies ofproliferating NK cells in all combinations of IL-2s with other cytokines showcasing that IL-2s is needed to induce the NK cell proliferation phenotype (Figure 11). Again, in most cytokine combinations, Tregs increased in frequencies in comparison to control tumors. Strikingly, the combination of IL-2s and IL- 12 induced increased frequencies of cytotoxic CD8 T cells, a feature which was not detected by IL-2s or IL- 12 alone, indicating a synergistic effect of the two cytokines on this population (Figure 11).

[0500] To dissect the cellular and molecular basis of cytokine combinations, a Zman-seq was performed across all combinatorial treatments at 48 hours after cytokine induction (n = 4 per combination), recovering 37,743 high-quality single cells (Figure 33A). IL-2SK combined with IL- 12 expanded total NK cells similarly to IL-2SK alone, but shifted NK cell composition toward non-proliferating, cytotoxic subsets with early or mature differentiation phenotypes (Figures 34A-B). This combination also expanded CD8 cytotoxic T cells, moMACS and monocytes, while reducing neutrophils and MDSC subsets (Figures 34A-B). Temporal analysis confirmed persistent expansion of NK cells, moMACS and monocyte clusters over time, whereas neutrophils and MDSC frequencies remained low (Figures 37A-B and 35A-B).

[0501] Differential expression analysis revealed comparable numbers of gene level changes in T and NK cells across all cytokine combinations, whereas IL-2SK plus IL- 12 induced the highest differentially expressed gene numbers in MDSCs and macrophages, even exceeding the IL-2SK plus GM-CSF group (Figure 37C). In conventional T cells, IL-2SK plus IL- 12 did not recapitulate either monotherapy signature but instead induced a distinct type I interferon and IFN-y response program including Statl, Zbpl, Rsad.2, and Ifit3 (Figures 37D and 35C). In NK cells, the transcriptional profile resembled the IL- 12 condition, with several maturation, activation and interferon-response genes further amplified to the monotherapy including Ly6a, Cd69, Ccl9, Klrgl, and Gbp2b (Figures 37D and 35D). In macrophages and MDSCs, IL-12-driven programs predominated, yet the combination with IL-2SK further increased inflammatory features (for example Adgrel, Cd300c2, Aif Slamf7, Ly6d), type I interferon and IFN-y response genes (for example Tapi, Tgtpl, Ifit2) in macrophages, and strongly enhanced type I interferon and IFN-y response genes (for example. Statl, Ifit2, Ifit3, Isgl5, Ly64) in MDSCs (Figures 37D and 36A-B).

[0502] Other combinations largely mirrored IL-2SK-driven transcriptional programs, consistent with limited in vivo synergy. An exception was IL-2SK in combination with GM-CSF, which retained GM-CSF-associated programs in macrophages, MDSCs and DCs, and selectively upregulated MHC-II antigen-presentation genes in macrophages (H2-Dma, H2-DMb2, Cd74) and moDC-associated genes (Ear2, Mmpl2) in DCs (Figures 35E and 36A-B). Temporal analysis further supported altered program dynamics uniquely in the IL-2SK plus IL- 12 group comparedwith either monotherapy (Figures 37E and 36C-F), including changed dynamics in type I interferon and IFN-y response across T, NK and macrophage populations, prolonged NK cell cytotoxicity and enhanced and prolonged inflammatory, monocytic and antigen presentation programs in MDSCs (Figure 37E).

[0503] Taken together, using a cutting-edge genetically modified cytokine over-expressing preclinical tumor model, IL-2s and IL- 12 were identified as a highly potent cytokine combination. The reprogramming effects of this combination seems to depend on both cytotoxic T cells and proliferating NK cells, while also allowing an expansion of Tregs potentially limiting the full power of this treatment.

[0504] EXAMPLE 3

[0505] IDENTIFYING NKG2A AS A TARGET SPECIFIC TO NK AND CYTOTOXIC T CELLS

[0506] To allow specific stimulation of NK cells and cytotoxic T cells, but not Tregs, with IL-2s, IL- 12 or their combination, a data-driven approach was used to identify target proteins for novel immunocytokines (Figure 12). Using scRNAseq data from four human cancer types including lung, breast, colorectal and ovarian cancer, genes which are higher expressed in T cells, NK cells or gamma delta T cells in comparison to Tregs were identified. Further filtering of genes allowed the inclusion of genes with less than 1 % expression in Tregs, and in any other off-target cell type such as myeloid cells, B cells, tumor cells and stromal cells (such as epithelial and endothelial cells). The top 159 genes were then filtered for genes encoding surface proteins to obtain 44 top candidate genes (Figures 12-13). KLRC1 encoding NKG2A showed one of the most a favorable expression profiles across NK, T and y5 T cell clusters across the four cancer types, while being completely absent from Tregs (Figure 13). NKG2A was previously shown to be an immune checkpoint in both NK and CD8 T cells (Andre et al., 2018). Blockade of the interaction of NKG2A expressed by NK cells and CD8 T cells with HLA-E expressed by tumor cells, have been demonstrated to lead to beneficial anti-tumor responses in preclinical murine tumor models (Andre et al., 2018) as well as in phase II / III clinical trials as monotherapy or in combination with anti-PD1 (Monalizumab, Innate pharma). Interestingly, gene targets of competitor immunocytokines such as CD8A, PDCD1 (encoding PD-1), CTLA4, HAVCR2, LAG3 or TIGIT were either not expressed in NK cells or showed off-target expression in Tregs (Figures 13-14).

[0507] Further analysis of KLRC1 revealed consistent expression across cancer types and patients, spanning cytotoxic T, NK and y5 T cells populations, with the highest enrichment observed in NK 2 cells (Figure 39A). At the bulk-tissue level, KLRC1 expression was elevated in multiple TCGAcancer types relative to healthy tissues from the Genotype-Tissue Expression (GTEx) Project (Figure 38A). To resolve KLRC1 expression across tumor and healthy tissues at single-cell resolution, a second pan-cancer and tissue single-cell RNA-seq atlas encompassing lung, breast and colon cancer tissues with adjacent tissue controls, as well as PBMCs from healthy donors, comprising ~2M high-quality cells [von Locquenghien, M. et al. Cell (2025); Sheban, F. et al. Cancer Cell 43, 1227-1241.ell (2025); Wu, S. Z. et al. Nat Genet 53, 1334-1347 (2021); Pelka, K. et al. Cell 184, 4734-4752.e20 (2021); Eeader, A. M. et al. Cancer Cell 39, 1594-1609.el2 (2021); and Yazar, S. et al. Science 376, eabf3041 (2022)], was analyzed. Across tissues, KLRC1 expression was higher in CD8 T cells from adjacent tissues than in PBMCs and was further increased in tumors relative to adjacent tissue (Figures 38B and 39B-C). In NK cells, KLRC1 expression was higher in adjacent and cancer tissues than in PBMCs, with the exception from breast cancer (Figures 38B and 39B-C).

[0508] To confirm the findings on the protein level, the expression of NKG2A as well as PD-1 and CD8 was assessed in cells isolated from blood, lung cancer tissue and adjacent lung tissue from of 3 human lung cancer patients by Flow cytometry (Figure 15). Indeed, NKG2A was upregulated in NK cells, NKT cells, CD8 T cells and y5 T cells in cancer tissue; whereas the competitor targets showed less specific expression patterns. Either they were constitutively expressed (CD8) or also expressed in Tregs (PD-1) (Figure 15). Similar observations were made in murine 4T1 and B16-F10 (melanoma) tumors, in which a high NKG2A / C / E (for mice only antibodies detecting the three proteins are available) protein expression was detected in NK and T cell clusters, but not Tregs in the tumor, whereas expression in the spleen was in general low (Figure 16).

[0509] EXAMPLE 4

[0510] IMMUNOCYTOKINES TARGETING NKG2A

[0511] Eight different variants of murine immunocytokines were designed based on the anti-mouse 20D5 clone (targeting NKG2A / C / E) which are linked to IL-2sk, IL-2mutant with IL-2Ra bias, IL-2mutant attenuated or IL- 12 aiming to stimulate cytotoxic T and NK cells with these cytokines (Figure 17). The designs included versions in which the cytokines are covered with cleavable blocking units (IL2RB, IL2RA or IL12RB1) which will be released specifically in the tumor by MMP- 14 or Granzyme B cleavage allowing tumor- specific release of the blocking unit and thereby restoration of cytokine activity (Figure 17). Using the blocking unit is important in order to reduce systemic toxicities. This novel class of immunocytokines is referred to herein as “Cytotoxic-Enhancers (CytEs)” or “NK cell-targeted immunocytokines and T cell enhancers (NiTEs)”.Quality control assessments confirmed that the immunocytokines and blocking subunits have the right size as assessed by SDS page (Figures 18 and 40), bind to NKG2A in-vitro as assessed by ELISA (Figures 19 and 41) and SPR (Table 2 hereinbelow), and that the cytokines of the immunocytokines have activity in-vitro using cytokine reporter HEK blue cell lines (Figures 20-21 and 42). Masked cytokine activity was restored after in vitro MMP14 pre-digestion, as shown by HEK-Blue reporter assays for both IL-2SK- and IL- 12 NiTEs (Figure 20 and 42).

[0512] Table 2: Surface plasmon resonance (SPR) affinity measurements of engineered constructs binding to recombinant murine NKG2A.

[0513]

[0514] To test the activity of the immunocytokines in-vivo, 4T1 tumors were treated with intratumoral injection of anti-RSV control, anti-RSV-IL-2SK, anti-NKG2A and anti-NKG2A-IL-2SK (Figure 22). To confirm in vivo target availability in mice, Klrcl was highly and selectively expressed in CD8 and NK cell populations, but not in Tregs in the 4T1 tumor single-cell RNA-seq dataset (Figures 43A-C). While both anti-NKG2A and anti-RSV-IL-2s showed a reduction in tumor growth as monotherapies, the anti-NKG2A-IL-2s immunocytokine showed the strongest treatment efficacy and reduced both tumor growth and weight in the cold (i.e., immunosuppressed) 4T1 model. These findings highlight the potential of this novel immunocytokine class.

[0515] In the next step, a dose-escalation pilot study was performed in 4T1 tumor-bearing mice to determine effective dosing via intravenous injections of the NiTE design. Specifically, mice received intravenous injections of anti-NKG2A control antibody, IL-2SK-NiTEs (50, 100 or 200 pg) or IL-12-NiTEs (5, 10 or 25 pg) (n = 3 per group) on day 8 and day 10. Anti-NKG2A and IL-2SK-NiTEs produced modest, dose-dependent tumor reduction, with maximal effects at 200 pg in this anti-PD-1 refractory setting (Figure 44). The IL-12-NiTE induced strong, dose-dependent tumor regression at doses between 5-25 pg, reaching a plateau at 10-25 pg (Figure 44). At the selected doses, both IL-2SK- and IL-12-NiTEs were well tolerated. No significant systemictoxicity were detected, as assessed by serum IFN-y, alanine transaminase (ALT) and aspartate transaminase (AST), and body weight measured at 10 hours after two intravenous injections of 200 pg IL-2SK-construtcs or 5 pg IL-12-constructs in 4T1 tumor-bearing mice (Figures 45A-D, Figure 46A-B).

[0516] To test for synergy in vivo, 4T1 tumor-bearing mice were treated with anti-RSV (200 pg), anti-NKG2A (200 pg), IL-2SK-constructs (200 pg), IL-12-constructs (5 pg) or combination of IL-2SK- and IL-12-NiTEs (200pg plus 5 pg) (n = 3-7 per group) on day 6 and day 8 and monitored tumor growth and endpoint weight (Figures 47A-F). RSV-targeted masked-immunocytokines carrying the same protease-cleavable linker and cytokine payload served as architecture-matched controls (Figures 47A-F). IL-2SK-constructs monotherapy and low-dose IL-12-NiTE each reduced tumor volume and weight relative to controls; whereas the combination produced markedly stronger antitumor activity, significantly reducing both tumor volume and weight (Figures 47A-F). This effect was not observed with RSV-targeted masked cytokine controls, indicating that tumor control required NKG2A-directed targeting rather than nonspecific cytokine release or masking architecture alone (Figures 47A-F).

[0517] Taken together, using genetical engineering, single-cell technologies and data-driven target selection the present inventors identified the combination of IL-2s and IL- 12 as extremely potent anti-tumor agents and NKG2A as highly powerful novel target for the next generation of immunocytokines. These immunocytokines work by avoiding Tregs and specifically delivering IL-2s and IL-12 to cytotoxic CD8 and NK cells (Figure 1).

[0518] EXAMPLE 5

[0519] SCREENING FOR ANTAGONISTIC ANTI-NKG2A ANTIBODIES Anti-NKG2A antibody candidates are screened for their binding to recombinant human NKG2A as assessed by ELISA and SPR, as well as absence of binding to NKG2C (ELISA counter-screening). Furthermore, they are screened for their ability to block the interaction between NKG2A and HLA-E by a competitive ELISA. Functional screening tests are performed in co-cultures of human CD8 T cells or NK cells with HLA-E expressing tumors cell lines in-vitro comparing anti-NKG2A candidates and control antibodies. Degranulation (CD107a) and cytotoxicity (INFy) are assessed by FACS and ELISA. Top candidates are screened for efficient tumor control (reducing tumor growth) in different preclinical murine tumor models modified to over-express human HLA-E. For this, novel generated hNKG2A mice in which the murine NKG2A / CD94 gene is replaced with the respective human genes are used.EXAMPLE 6

[0520] SCREENING FOR ANTAGONISTIC ANTI-NKG2A ANTIBODIES To experimentally implement aspects of the screening strategy described herein, anti-NKG2A antibodies were generated by immunizing New Zealand rabbits with the recombinant extracellular domain (ECD) of human NKG2A.

[0521] Antigen- specific responses were confirmed by ELISA, followed by isolation of PBMCs, single B cell sorting and recombinant expression of antibody clones in mammalian cells.

[0522] A total of 54 monoclonal antibody clones were screened for their binding to recombinant human NKG2A as assessed by ELISA, as well as absence of binding to NKG2C (ELISA counterscreening). Furthermore, the antibodies were screened for their ability to block the interaction between NKG2A and HLA-E by a competitive ELISA assay.

[0523] Seven lead clones (see Table 3 hereinabove) were selected based on strong binding to NKG2A over NKG2C (Figure 48) and / or effective blockade of the NKG2A-HLA-E interaction (Figure 49).

[0524] Although the invention has been described in conjunction with specific embodiments thereof, it is evident that many alternatives, modifications and variations will be apparent to those skilled in the art. Accordingly, it is intended to embrace all such alternatives, modifications and variations that fall within the spirit and broad scope of the appended claims.

[0525] It is the intent of the Applicant(s) that all publications, patents and patent applications referred to in this specification are to be incorporated in their entirety by reference into the specification, as if each individual publication, patent or patent application was specifically and individually noted when referenced that it is to be incorporated herein by reference. In addition, citation or identification of any reference in this application shall not be construed as an admission that such reference is available as prior art to the present invention. To the extent that section headings are used, they should not be construed as necessarily limiting. In addition, any priority document(s) of this application is / are hereby incorporated herein by reference in its / their entirety.

Claims

WHAT IS CLAIMED IS:

1. A composition of matter comprising at least one fusion polypeptide comprising: (a) an anti-NKG2A antibody;(b) an immune cell-activating cytokine; and(c) a masking agent that masks said immune cell-activating cytokine and an amino acid sequence which is cleaved by a tumor microenvironment-associated protease, such that when said fusion polypeptide is in a vicinity of a tumor, said tumor microenvironment-associated protease cleaves said masking agent thereby exposing said immune cell-activating cytokine.

2. A composition of matter comprising at least one fusion polypeptide comprising: (a) an anti-NKG2A antibody; and(b) an immune cell-activating cytokine selected from the group consisting of IL-2 and IL- 12.

3. A composition of matter comprising at least one fusion polypeptide comprising: (a) an antagonistic anti-NKG2A antibody; and(b) an immune cell-activating cytokine.

4. The composition of matter of any one of claims 1 and 3, wherein said immune cellactivating cytokine is selected from the group consisting of IL-2, IL-12, IL-12, IL-15, IL-18, IL-21 and GM-CSF.

5. The composition of matter of any one of claims 1 and 3, wherein said immune cellactivating cytokine is selected from the group consisting of IL-2 and IL- 12.

6. The composition of matter of any one of claims 1-5, wherein said immune cellactivating cytokine is modified to reduce activity, alter a binding property and / or increase stability.

7. The composition of matter of any one of claims 1-5, wherein said immune cellactivating cytokine is in its native form.

8. The composition of matter of any one of claims 2-7, wherein said at least one fusion polypeptide further comprises:(c) a masking agent that masks said immune cell-activating cytokine and an amino acid sequence which is cleaved by a tumor microenvironment-associated protease, such that when said fusion polypeptide is in a vicinity of a tumor, said tumor microenvironment-associated protease cleaves said masking agent thereby exposing said immune cell-activating cytokine.

9. The composition of matter of any one of claims 1 and 8, wherein said tumor microenvironment-associated protease is secreted by a myeloid-derived suppressor cell (MDSC).

10. The composition of matter of claim 9, wherein said MDSC is a tumor associated macrophage (TAM).

11. The composition of matter of claim 10, wherein said protease is selected from the group consisting of matrix metalloproteinase (MMP), ADAM-9, ADAMDEC1, CathsepinL and CathepsinK.

12. The composition of matter of claim 10, wherein said protease is a matrix metalloproteinase (MMP).

13. The composition of matter of any one of claims 11-12, wherein said MMP is selected from the group consisting of MMP2, MMP9, MMP12, MMP14 and MMP19.

14. The composition of matter of any one of claims 11-12, wherein said MMP is MMP14.

15. The composition of matter of any one of claims 1 and 8, wherein said tumor microenvironment-associated protease is secreted by an NK and / or cytotoxic CD8+ T cell.

16. The composition of matter of claim 15, wherein said protease is Granzyme B.

17. The composition of matter of any one of claims 1-16, wherein said at least one fusion polypeptide is a dimer.

18. The composition of matter of any one of claims 1-17, wherein said anti-NKG2A antibody comprises an antigen recognition domain comprising complementarity determiningregions (CDRs) CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 or the light chain and heavy chain at least 80 % identical to those of an antibody selected from the group consisting of 30B1-1, IF7-2, 3E9-1, 5E3-2, 6B11-1, 10E2-1 and 12E2-1.

19. An antibody comprising an antigen recognition domain which binds NKG2A and comprises complementarity determining regions (CDRs) CDRL1, CDRL2, CDRL3, CDRH1, CDRH2 and CDRH3 or the light chain and heavy chain at least 80 % identical to those of an antibody selected from the group consisting of 30B1-1, IF7-2, 3E9-1, 5E3-2, 6B11-1, 10E2-1 and 12E2-1.

20. The composition of matter of claim 18 or the antibody of claim 19, wherein said at least 80 % is at least 85 %.

21. The composition of matter of claim 18 or the antibody of claim 19, wherein said at least 80 % is at least 90 %.

22. The composition of matter of claim 18 or the antibody of claim 19, wherein said at least 80 % is at least 95 %.

23. The composition of matter of claim 18 or the antibody of claim 19, wherein said at least 80 % is 100 %.

24. A polynucleotide encoding the composition of matter of any one of claims 1-18 and 20-23.

25. A nucleic acid construct comprising the polynucleotide of claim 24 and a cis-acting regulatory element for directing expression of said polynucleotide.

26. A host cell comprising the composition of matter of any of claims 1-18 and 20-23 or a polynucleotide or a nucleic acid construct encoding it.

27. A method of producing at least one fusion polypeptide, the method comprising introducing into a host cell the polynucleotide or nucleic acid construct of any one of claims 24-25, or culturing the host cell of claim 26.

28. The method of claim 27, comprising isolating the at least one fusion polypeptide.

29. A polynucleotide encoding the antibody of any one of claims 19-23.

30. A nucleic acid construct comprising the polynucleotide of claim 29 and a cis-acting regulatory element for directing expression of said polynucleotide.

31. A host cell comprising the antibody any one of claims 19-23 or a polynucleotide or a nucleic acid construct encoding it.

32. A method of producing an anti-NKG2A antibody, the method comprising introducing into a host cell the polynucleotide or nucleic acid construct of any one of claims 29-30, or culturing the host cell of claim 31.

33. The method of claim 32, comprising isolating the antibody.

34. A method of treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the composition of matter of any one of claims 1-18 and 20-23, thereby treating the cancer in the subject.

35. The composition of matter of any one of claims 1-18 and 20-23, for use in treating cancer in a subject in need thereof.

36. A method of treating an inflammatory disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the antibody of any one of claims 19-23, thereby treating the cancer in the subject.

37. The antibody of any one of claims 19-23, for use in treating an inflammatory disease in a subject in need thereof.

38. The method of claim 36 or the antibody for use of claim 37, wherein said inflammatory disease is cancer.

39. The method of claim 34, the composition of matter for use of claim 35 or the method or antibody for use of claim 38, wherein said cancer is an NKG2A rich cancer.

40. The method of claim 34, the composition of matter for use of claim 35 or the method or antibody for use of claim 38, wherein said cancer is selected from the group consisting of lung, melanoma, breast, ovarian, colorectal, liver, renal, head and neck and endometrial cancer.

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