Compositions and methods comprising fusion polypeptides for use in treating cancer

Abstract

A composition of matter comprising at least one fusion polypeptide which comprises a targeting moiety which binds a marker of a myeloid-derived suppressor cell (MDSC) attached to an immune cell-activating cytokine, wherein said immune cell-activating cytokine is masked by a masking agent, such that when said at least one fusion polypeptide is in a vicinity of an MSDC. said masking agent unmasks said immune cell -activating cytokine via an activity of an MSDC- specific protease so as to allow cytokine activity of said immune cell-activating cytokine. Also provided are methods and uses of such a composition for the treatment of cancer.

Description

TITLECOMPOSITIONS AND METHODS COMPRISING FUSION POLYPEPTIDES FOR USE IN TREATING CANCERCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority of U.S. Provisional Application No. 63 / 689.451, filed August 30. 2024 and U.S. Provisional Application No. 63 / 691,342, filed September 6, 2024, both of which are incorporated by reference herein in their entirety.SEQUENCE LISTING STATEMENT

[0002] The XML file, entitled IMM 24-02-PCT SL.xml, created on August 28, 2025, comprising 147,734 bytes, submitted concurrently with the filing of this application is incorporated herein by reference.FIELD AND BACKGROUND OF THE INVENTION

[0003] The present invention, in some embodiments thereof, relates to compositions comprising fusion polypeptides for use in treating cancer.

[0004] Immunotherapy has revolutionized treatment for cancer patients. Nonetheless, a large number of patients either do not benefit from these therapies or face early disease relapse. Monotherapies targeting the tumor-associated myeloid compartment have limited capability for complete disease remission. In the past decade, a plethora of drugs and compounds have emerged as potential inhibitors of myeloid-derived suppressor cells (MDSCs) in cancer. Although a few have been FDA-approved, most are stuck at the stage of clinical trials or even preclinical models due to the lack of understanding of the complex mechanisms governing MDSC generation, expansion, recruitment, activation, and immunosuppression - rendering it seemingly impossible for any single approach to fully control or eradicate MDSCs and consequently elicit robust antitumor effects. Thus, an optimal strategy appears to lie in combining MDSC-targeting treatments with other anticancer therapies. However, the implementation of such combinatorial or dual therapeutic approaches requires careful fine-tuning of factors such as dosages or treatment regimens that maximize efficacy and minimize adverse effects (Li et al., 2021).

[0005] Cytokine-based cancer therapy involves using cytokines, which are small proteins that play a crucial role in cell signaling, to treat cancer. Cytokines can modulate the immune system and have various effects on the growth, differentiation, and activity of immune cells, making them valuable tools in cancer immunotherapy. However, relatively modest response rates and clinical success diminished the interest in cytokines during the last four decades. Key reasons for initialfailure 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. 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 ligand 1 (PDL1) and activation of off-target cells, for example of immunosuppressive regulatory T cells (Treg cells). Consequently, the therapeutic efficacy of cytokines has been limited (Saxton et al., 2022).

[0006] A specific sub-population of MDSCs have recently been reported as playing a central immunosuppressive role in the tumor microenvironment (TME), these are the Receptor Expressed on Myeloid Cells 2 positive (TREM2+) regulatory macrophages. These tumor-site specific cells are considered as an optimal target for activating anti-tumor immunity with minimal side effects and toxicity (Giladi and Amit, 2018; Katzenelenbogen et al. 2020). Indeed, potent human TREM2 antagonistic antibodies as well as anti Gpnmb antibodies are disclosed in W02023 / 012802 and in WO2021152592.

[0007] Additional background art includes:

[0008] WO2021016640 disclosing masked therapeutic agents comprising one or more tumor- associated protease cleavage sites.

[0009] AU2013227477 disclosing molecules comprising a targeting moiety capable of targeting to unwanted cells and at least one further moiety that has a masked immune cell binding region so as to prevent binding of the further moiety to an immune cell, wherein the masked immune cell binding region, optionally a T cell binding region, is capable of being selectively unmasked by selective cleavage of one or more linkers that contain at least one protease cleavage site when the molecule is in the vicinity of the unwanted cells so as to allow binding of the further moiety to an immune cell.SUMMARY OF THE INVENTION

[0010] According to an aspect of some embodiments of the present invention there is provided a composition of matter comprising at least one fusion polypeptide which comprises a targeting moiety which binds a marker of a myeloid-derived suppressor cell (MDSC) attached to an immune cell-activating cytokine, wherein the immune cell-activating cytokine is masked by a masking agent, such that w hen the at least one fusion polypeptide is in a vicinity of an MSDC, the masking agent unmasks the immune cell-activating cytokine via an activity of an MSDC-specific protease so as to allow cytokine activity of the immune cell-activating cytokine.[0001 1 ] According to some embodiments of the invention, the MDSC is a tumor associated macrophage (TAM).

[0012] According to some embodiments of the invention, the targeting moiety is attached to the immune cell-activating cytokine directly.

[0013] According to some embodiments of the invention, the targeting moiety is attached to the immune cell -activating cytokine indirectly.

[0014] According to some embodiments of the invention, the targeting moiety is attached to the immune cell-activating cytokine via at least one linker.

[0015] According to some embodiments of the invention, the at least one fusion polypeptide is a homodimer or a homotrimer.

[0016] According to some embodiments of the invention, the at least one fusion polypeptide is a heterodimer or a heterotrimer.

[0017] According to some embodiments of the invention, the masking agent is attached to the at least one fusion polypeptide via a cleavable sequence of at least one protease specific to MDSC.

[0018] According to some embodiments of the invention, the protease is a matrix metalloproteinases (MMP).

[0019] According to some embodiments of the invention, the MMP is MMP 14.

[0020] According to some embodiments of the invention, the marker is myeloid marker mediates a suppressor function of MDSCs.

[0021] According to some embodiments of the invention, the targeting moiety is characterized by a binding activity and optionally an inhibitory activity of the suppressor function.

[0022] According to some embodiments of the invention, the marker is selected from the group consisting of TREM2, GPNMB, CSF1R, LILRB1 / ILT2, LILRB2 / ILT4, LILRB4, PDL1, PDL2, CLEVER1, SIRPA, SIGLEC-1, SIGLEC-7, SIGLEC-9, SIGLEC-10, SIGLEC-15, SLAMF7, MRC1 / CD206 , LGALS9 / Gal ectin-9, VSIG-4 and MARCO.

[0023] According to some embodiments of the invention, the marker is TREM2.

[0024] According to some embodiments of the invention, the cytokine is selected from the group consisting of Interleukin-2 (IL-2), Interferon-alpha (IFN-a), Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin-12 (IL-12), Tumor Necrosis Factor-alpha (TNF-a), Interferon-gamma (IFN-y), Interleukin- 15 (IL-15), Interleukin- 18 (IL-18), Interleukin- 21 (IL-21) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).

[0025] According to some embodiments of the invention, the cytokine is Interleukin-2 (IL-

[0026] According to some embodiments of the invention, the cytokine is modified to have enhanced receptor selectivity to preferentially stimulate effector immune cells.

[0027] According to some embodiments of the invention, the fusion polypeptide activates lymphoid cells selected from the group consisting of T cells (e.g., CD4 T cells, NKT and CD8 T cells) and NK cells.

[0028] According to some embodiments of the invention, the targeting moiety is an antibody.

[0029] According to an aspect of some embodiments of the present invention there is provided a polynucleotide encoding the fusion polypeptide as described herein.

[0030] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition comprising as an active ingredient the fusion polypeptide or polynucleotide as described herein and a pharmaceutically acceptable carrier or diluent.

[0031] 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 pharmaceutical composition as described herein, thereby treating cancer

[0032] According to an aspect of some embodiments of the present invention there is provided a pharmaceutical composition as described herein for use in treating cancer.

[0033] According to some embodiments of the invention, the cancer is PD-1, PD-L1 or CTLA-4-resistant.

[0034] According to some embodiments of the invention, the cancer is MHC-1 independent or dependent.

[0035] According to some embodiments of the invention, the cancer is MHC-1 dependent.

[0036] According to some embodiments of the invention, the cancer is overexpressing TREM2 and optionally MMP14.

[0037] According to some embodiments of the invention, the cancer is selected from the group consisting of brain cancer, breast cancer, colon cancer, esophagus cancer, kidney cancer , liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testical cancer and cancer of the uterus.

[0038] According to some embodiments of the invention, the invention comprises a composition of matter, method, or composition for use, wherein the immunocytokine is a fusion polypeptide, wherein the fusion polypeptide comprises:

[0039] (i) an anti-TREM2 antibody, or a TREM2-binding portion thereof; and

[0040] (ii) one or more cytokines; and, optionally,

[0041] (iii) a cytokine mask, preferably, an IL2RB polypeptide sequence; preferably IL2RB(6-208) or IL2RB(6-97);

[0042] wherein (i) and (ii) are covalently linked, directly or through a linker and / or a Sushi Domain of IL15Ra (SD); and (ii) and (iii) are linked by a cleavable or non-cleavable linker, preferably a protease-cleavable linker, preferably an MMP14, MMP12, or MMP11 protease- cleavable linker, or a combination thereof; and wherein, optionally, (i) and (iii) are covalently linked, directly or through a linker, and (ii) is linked by a cleavable or non-cleavable linker, and hence (ii) can be released.

[0043]

[0044] In some embodiments, the anti-TREM2 antibody comprises at least one immunoglobulin heavy chain and at least one immunoglobulin light chain, wherein the at least one heavy chain comprises

[0045] HC-CDR1: GYTFTNFW

[0046] HC-CDR2: IYPGTGNT

[0047] HC-CDR3: AREAYYTNPGFAY; and

[0048] the at least one light chain comprises

[0049] LC-CDR1: QNIVHSNGNTY

[0050] LC-CDR2: KVS

[0051] LC-CDR3 : FQGSH VP YT;

[0052] wherein the antibody is optionally humanized.

[0053] In some of these embodiments, the fusion polypeptide is E3C7-IL15-MV1 or MiTE144.

[0054] In some embodiments, the method or use of treating cancer with these fusion peptides or immunocytokines further comprises administering an additional therapeutic agent or other therapy prior to, consecutively, and / or concurrently with the fusion polypeptide, wherein the additional therapeutic agent and the fusion polypeptide are in the same composition or in separate compositions.

[0055] In some of these embodiments, the additional therapeutic agent or other therapy are selected from immune check point blockage therapy, an anti-cancer therapy, a chemotherapy, an anti-inflammatory agent, a cytokine, adoptive cell therapy, and radiotherapy.

[0056] 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 / ormaterials 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.

[0057]

[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 show n 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. l is a schematic illustration of a “Myeloid-targeted immunocytokine and NK / T cell Enhancer” (MiTE), which is an embodiment of the fusion polypeptide of the invention.

[0062] FIG. 2 shows average tumor volumes (mm3 +SEM) in transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 on day 0 and were randomized on day 7 when the average tumor size reached 50 mm3. Mice were then treated intraperitoneally with isotype aRSV (lOOpg / dose, n=7), aRSV-IL2SK (lOOpg / dose, n=7) and aTREM2-IL2SK (lOOpg / dose. n=7) weekly for a total of two injections. Myeloid- targeted immunocytokine effectively abolishes MC38 tumor growth in vivo. Of note, aTREM2-IL2SK has no masking agent.

[0063] FIG. 3 shows an ELISA analysis of aTREM2-IL2SK antibody concentration in serum from WT C57BL / 6J mice overtime upon intraperitoneal administration (200pg / dose), using recombinant human TREM2 and an HRP-conjugated anti-human IgG antibody. The myeloid- targeted immuncytokine exhibits reduced half-life (compared to a naked antibody, anti-Trem2).

[0064] FIG. 4 shows probability of survival across time of transgenic hTREM2 C57BL / 6J mice, which were inoculated s.c. with MC38 on day 0 and were randomized on day 7 when the average tumor size reached 50 mm3. Mice were then treated intraperitoneally with isotype control aRSV (lOOpg / dose, n=7), aRSV-IL2SK (lOOpg / dose, n=7) and aTREM2-IL2SK (lOOpg / dose, n=7) every 3 days for a total of two injections. After the second injection, all mice in the treatment groups aRSV-IL2SK and aTREM2-IL2SK died due to cytokine release syndrome. The first generation of the immunocytokine causes dramatic systemic toxicity.

[0065] FIG. 5 shows an ELISA analysis of IFNy, IL-2 and IL-6 levels in serum of WT C57BL / 6J mice which was collected 24 hours after intravenous administration of anti-humanTREM2 (aTREM2; 200pg / dose, n=5), aTREM2-IL2SK (200pg / dose. n=5, aRSV (200pg / dose, n=5) or aRSV-IL2SK (200pg / dose, n=5), demonstrating systemic toxicity.

[0066] FIG. 6 shows single-cell RNA-sequencing analysis of macrophages in vivo represented as a dotplot. These macrophages orignate in spleen, tumor and peritoneum from WT C57BL / 6J mice (n=5), which were inoculated s.c. with MCA205 on day 0. and were collected on day 10 when tumors reached an average tumor volume of 500mm3. This led to the identification of matrix metalloproteinases (MMPs) specific to tumor-associated macrophages.

[0067] FIG. 7 shows bulk RNA-sequencing analysis of healthy (blue) versus tumor (red) tissues across 14 different human tumor types from TCGA and GTEX RNA-seq data (brain (n=1842), breast (n=1390), colon (n=637). esophagus (n=847), kidney (n=1043), liver(n=531), lung (n=1410), ovary (n=515), pancreas (n=350), prostate (n=646), skin (n=1281), stomach (n=623), testis (n=319), uterus (n=135)) as barplots, demonstrating that MMP14 and TREM2 are highly expressed together in all major solid tumors types and are only minimally expressed in the matched healthy tissue.

[0068] FIG. 8 is a schematic illustration of the design of fusion polypeptides. Upper panel shows homodimeric and heterdimeric embodiments. Lower panel shows exemplary polypeptides: “Myeloid-targeted immunocytokine and NK / T cell Enhancers (MiTEs)”, according to some embodiments of the invention. MiTE-76 is also referred to as “variant 1”, MiTE-95 is also referred to as “variant 2”. MiTE-144 is also referred to as “variant 3” and MiTE-208 is also referred to as “variant 4”. In these configurations the antibody (e.g., anti-Trem2) is fused by a linker element to a native or mutant type of a cytokine (e.g., IL 2 “superkine” with engineered enhanced receptor selectivity' that preferentially stimulates effector immune cells (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. The amino acid sequences of these configurations are as set forth in SEQ ID Nos. 89, 98, 102, 110, 114120, 127, 131, 137 and 144.

[0069] FIG. 9 shows OD at 630nm measuring SEAP secretion of HEK Blue CD122 / CD132 reporter cells. Using this reporter cells, it is possible to quantify the bioactivity of IL2 / IL2SK of different fusion proteins after incubation with or without recombinant human TAM- specific MMP14. IL2 / IL2SK activity is dependent on TAM-specific protease cleavage.

[0070] FIG. 10 shows OD at 630nm measuring SEAP secretion of HEK Blue CD 122 / CD 132 reporter cells. First, monocytes were isolated from blood and differentiated to tumor-associated macrophages across 7 days using 30ng / mL hM-CSF cytokine and starting on day 5, additionally with lOng / ml hIL-4. The supernatant was then concentrated via ultra centrifugalcolumns. Constructs encoding the indicated fusions were incubated in the presence of the concentrated supernatant overnight at 37°C degrees, similar to the control condition using recombinant MMP14. IL2SK actiti vit\ was measured via HEK Blue CD122 / CD132 reporter assay. The sectreome of human macrophages efficiently cleaves the indicated fusions (denoted MiTEs), demonstrating TAM-specificity.

[0071] FIG. 11 shows mean fluorescence intensity (MFI) measured via FACS using PE- conjugated anti-human IgG stainings. Prior, either HEK parental or HEK cells overexpressing hTREM2 were treated for 4h with the indicated fusions (denoted MiTEs) (20nM). MiTEs bind hTREM2 overexpressed in HEK cells similar to unmodified aTREM2 mAb in vitro.

[0072] FIG. 12 shows OD (450nm-570nm) after a direct ELISA assay. Plates were coated with 0.5pg / ml recombinant human TREM2 overnight and binding was assessed using anti-human IgG conjugated with HRP the next day. The indicated fusions (denoted MiTEs) with and without cleavage bind recombinant human TREM2.

[0073] FIG 13 shows Surface Plasmon Resonance (SPR) analysis of the interactions of two biomolecules with respect to binding kinetics and affinity as well as binding specificity. The indicated fusions (denoted MiTEs) bind almost covalently to recombinant human TREM2.

[0074] FIG 14 shows Surface Plasmon Resonance (SPR) analysis of the interactions of soluble recombinant IL2RB with respect to its binding kinetics and affinity as well as binding specificity. The indicated fusions (denoted MiTEs) were first bound to recombinant human TREM2 to the chip and then, the binding kinetics of IL2 / IL2SK of either hetero- or homo-MiTE molecules, which were or were not digested with recombinant human MMP14 beforehand, was determined using soluble IL2RB. Without MMP14 digestion, soluble IL2RB was not able to bind IL2SK. Upon digestion with MMP14, soluble IL2RB was able to bind to both hetero- and homo- MiTEs in a similar matter.

[0075] FIG. 15 shows mean fluorescence intensity (MFI-1) (normalized) of prolifertation dye measurements via FACS. The proliferation of human T cells (n=3 donors) was measured after 4 days in the presence of 0. 15 nM of the indicated fusions (denoted MiTEs), which have or have not been pre-treated with MMP 14. MiTEs activate robust human CD8 and CD4 T cell proliferation in a MMP14 / TME-specific manner.

[0076] FIG. 16 shows average tumor volumes (mm3 +SEM) in transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 on day 0 and were randomized on day 9 when the average tumor size reached 50 mm3. Mice were then treated intratumorally with isotype control aRSV (5pg / dose, n=3), aTREM2-IL2SK (5pg / dose, n=3, represented by the different linesin each panel), and the indicated fusions (denoted MiTEs) individually (5pg / dose, n=3) every two days for a total of three injections. All MiTEs show strong anti-tumor effects in vivo.

[0077] FIG. 17 shows average tumor volumes (mm3 +SEM) in transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 on day 0 and were randomized on day 8 when the average tumor size reached 50 mm3. Mice were then treated intratumorally with isotype control aRSV (5pg / dose, n=3). aPDl (40pg / dose. n=3). aTREM2 (5pg / dose, n=3), MiTE-144 (5pg / dose, n=3), aTREM2 (5pg / dose) together with aPDl (40pg / dose) (n=3), and MiTE-144 (5pg / dose) together with aPDl (40pg / dose) (n=3) every two days for a total of three injections. MiTE-144 treatments outcompete combinatorial therapies of aPD-1 together with aTREM2 as w ell as their monotherapies in vivo, as seen as by more effective eradication of tumor growth.

[0078] FIG. 18A shows body weight [%] of WT C57BL / 6J mice after intraveneous administration of 50pg / dose of aRSV, aRSV-IL2SK, aTREM2, aTREM2-IL2SK and the indicated fusions (denoted MiTEs) (n=4 / group). Mice treated with MiTEs did not loose weight. FIG. 18B shows an ELISA analysis of IFNG levels in serum of WT C57BL / 6J mice which was collected 24 hours after intraveneous administration of 50pg / dose of aRSV, aRSV-IL2SK, aTREM2, aTREM2- IL2SK and indicated MiTEs individually (n=4 / group), demonstrating reduced systemic toxicity of MiTEs.

[0079] FIG. 19A shows an ELISA analysis measuring IFNy, IL6 and IL2 levels in serum of transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 and bear tumors with an average size above 100 mm3. These mice were treated intravenously with isotype control aRSV (200pg / dose, n=4-8), aTREM2 (200pg / dose, n=4-8), MiTE-76 or MiTE-144 (200pg / dose, n=4-8) on day 10 and 13 for a total of two injections. FIG. 19B shows body weight [%] over time, and FIG. 19C the spleen weight [g] of these mice on day 16. No weight loss, no elevation in serum cytokine levels and no spleen enlargement indicate no systemic toxicity and safe administration of MiTEs at high dosages.

[0080] FIG. 20 shows average tumor volumes (mm3 +SEM) in transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 on day 0 and were randomized on day 8 when the average tumor size reached 50 mm3. Mice were then treated intraveneously with isotype control aRSV (200pg / dose, n=7-12), aPDl (200pg / dose, n=7-12), aTREM2 (200pg / dose, n=7- 12), MiTE-144 (200pg / dose, n=7-12), aTREM2 (200pg / dose) together with aPDl (200pg / dose) (n=7-12), and MiTE-144 (200pg / dose) together with aPDl (200pg / dose) (n=7-12) on day 8, 10 and 13 for a total of three injections. On day 11, 5 mice per group were used for further analysis. MiTEs outcompete combinatorial therapies of aPD-1 and aTREM2 as well as their monotherapies in a systematic matter in vivo, without off-target effects and toxicity.

[0081] FIGs. 21A-F show single-cell RNA seq analyses of immune cells isolated on day 11 from MC38 tumors from mice which were intravenously treated with isotype control aRSV (200pg / dose, n=5), aPDl (200pg / dose, n=5), aTREM2 (200pg / dose, n=5), MiTE-144 (200pg / dose, n=5), aTREM2 (200pg / dose, n=5) together with aPDl (200pg / dose, n=5), and MiTE-144 (200pg / dose, n=5) together with aPDl (200pg / dose, n=5) on day 8, 10. FIG. 21 A shows a UMAP displaying identified cell clusters. FIG. 21B shows UMAPs displaying cells from each treatment group with overlay ed cell densities. Treatment with MiTE-144 and MiTE-144 together with aPDl shifts cell densities towards the T and NKT cell compartment. FIG. 21C shows frequencies of cell clusters among total immune cells in the tumors. Treatment with MiTE-144 and MiTE-144 together with aPDl reprograms frequencies of immune cell populations. FIG. 21D shows the frequencies of selected cell populations in the T / NK(T) cell compartment. Tumors of mice treated with MiTE-144 and MiTE-144 together with aPDl have increased frequencies of two clusters of proliferating NKT cells, cytotoxic CD8 T cells and proliferating cytotoxic T cells. FIG. 21E shows a Dotplot and Violinplots displaying selected differentially expressed genes in NKT cells between the aRSV control and at least one treatment group. NKT cells isolated from tumors of mice treated with MiTE-144 and MiTE-144 together with aPDl show a gene signature of increased cytotoxicity' and proliferation with reduced exhaustion / dysfunction. This includes increased expression of the genes Prfl (encoding Perforin- 1), Gzma, Gmzb (encoding Granzyme A and B) and Mki67 (encoding Ki-67). FIG. 2 IF shows a Dotplot and Violinplots displaying selected differentially expressed genes in T cells between the aRSV control and at least one of treatment group. T cells isolated from tumors of mice treated with MiTE-144 and MiTE-144 together with aPDl show a gene signature of increased cytotoxicity and proliferation with reduced exhaustion / dysfunction. This includes increased expression of the genes Prfl (encoding Perforin- 1), Gzma, Gmzb (encoding Granzyme A and B) and Mki67 (encoding Ki-67).

[0082] FIG. 22: TAM and T cells interact closely in the TME across human cancer types

[0083] (A)-(L) Data from curated spatial atlas of -1.86 million spatial single cells from publicly available MERFISH datasets of human lung, breast, colon, and ovarian cancer41,42.

[0084] (A, D, G. J) Spatial distributions of annotated cell types in a human breast (A), lung(D), colorectal (G) and ovarian (J) cancer tissue sections with individual cells color-coded by7their assigned identity7.

[0085] (B, E, H, K) Dot plots showing the expression of selected marker genes across different cell types in breast (B), lung (E), colorectal (H) and ovarian (K) cancer tissue sections. Cell type annotations were refined using ResolVI, enhancing the resolution of transcriptionalstates. Dot size represents the fraction of cells expressing each gene, and color indicates mean expression levels.

[0086] (C, F, I, L) Empirical cumulative distribution functions (CDF) of minimum distances between key immune cell populations in breast (C), lung (F). colorectal (I) and ovarian (L) cancer tissue sections, illustrating spatial proximity relationships between tumor-associated macrophages (TAMs). dendritic cells (DCs), Monocytes (Mon) and lymphocyte subsets (T cells. NK cells, and Tregs).

[0087] (M)-(N) Analyses of scRNA-seq data containing human tumor samples (n = 123) from breast, lung, and colon cancers44-46. 332,723 high quality single-cells were integrated using scVI integration followed by MultiNicheNetR analysis.

[0088] (M) Chord diagram illustrating the overall number of ligand-receptor interactions between key immune cell populations across cancer types. Interactions between TAMs, DCs, monocytes. NK cells, and T cells are shown, with connection width representing interaction frequency. Darker red hues indicate a higher number of interactions.

[0089] (N) Detailed ligand-receptor interaction network between myeloid-derived cells(TAMs, DCs, and monocytes) as ligand-expressing senders and lymphoid cells (T cells and NK cells) as receptor-expressing receivers. Each connection represents a ligand-receptor pair, with color coding corresponding to sender cell type.

[0090]

[0091] FIG. 23: Anti-TREM2-IL2SK immunocytokines induce systemic toxicity

[0092] (A) Schematic representation of aTREM2-IL2SK, a myeloid-targeted immunocytokine composed of anti-TREM2, mouse Fc-Null (N297A) IgGl and IL-2 H9 "Superkine" domains.

[0093] (B) Binding of aTREM2-IL2SK to recombinant hTrem2 protein as assessed byELISA. aRSV and aTREM2 antibodies used as control. Absorbance was measured at 450-655 nm. Data represents mean ± SD (n =3).

[0094] (C) HEK Blue CD 122 / CD 123 reporter assay demonstrating binding of aTREM2-IL2SK to IL-2R y, as well as the functional activity of IL-2SK compared to recombinant human IL-2 and aTREM2 alone. Absorbance was measured at 630 nm. Data represents mean ± SD (n=3).

[0095] (D)-(E) naive WT mice were treated with two doses (200 pg per dose) of aTREM2-IL2SK. aTREM2, or a control antibody (aRSV and aRSV-IL2SK), administered 48 hours apart (n=5). Serum was collected 10 hours after the second injection. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test, comparing eachantibody construct with our without cytokine coupling, and is denoted by *P<0.05. **P<0.01, ***P<0.001, ****P<0.0001.

[0096] (D) Bargraphs depicting protein concentration of murine IFN-y, IL-2, and IL-6 in blood serum. Data represents mean ± SD (n=5).

[0097] (E) Bargraph depicting concentration of of AST / ALT in blood serum as assessed using Roche Diagnostics cobas c 111 Chemistry Analyzer. Data represents mean ± SEM (n=5).

[0098] FIG. 24: Development of on target cytokine activation modality dependent on T AM-specific protease

[0099] (A), (C) Analyses of scRNA-seq data containing human tumor samples (n = 123) and their adjacent healthy-like samples (n = 67) from breast, lung, and colon cancers44-46and PBMC dataset63(n=108).[000100] (A) Dot plot showing the expression of immune-related proteases across immune cell subsets derived from tumor tissues or PBMCs. Dot size represents the fraction of cells expressing each gene, and color intensity indicates mean expression levels.[000101 ] (B)-(C) TAMs, peritoneal tissue-resident macrophages and spleen monocytes were isolated from MC38 and MCA205 tumor-bearing mice and FACS sorted to undergo single-cell RNA-seq (n = 5, 8266 single cells).[000102] (B) Dot plot showing the expression of MMPs in TAMs, peritoneal tissue-resident macrophages, and spleen monocytes. Dot size represents the fraction of cells expressing each gene, and color intensity indicates mean expression levels.[000103] (C) Lolli-pop plot showing the co-expression scores of MMPs with TREM2 in human (top) and murine TAMs (bottom). Each lolli-pop represents an MMP gene, with significant correlations marked in pink and non-significant ones in gray.[000104] (D) Schematic representation of four MiTE constructs (MiTE76, MiTE95,MiTE144, and MiTE208) designed for MMP-14-dependent activation (scissors). Each construct consists of an anti-TREM2 antibody backbone based on human Fc-Null (N297A) IgGl which is fused to IL-2H9 "Superkine"(orange) and a blocking domain (green), which prevents IL-2 activity in its inactive state. The blocking domain is linked via an MMP- 14 cleavage site (dashed line), allowing selective activation in MMP-14-rich tumor microenvironments upon enzymatic cleavage. The knob-into-hole engineering approach for heterodimerization was employed in MiTE144 and MiTE208. Ridgway, J.B.B., Presta. L.G.. and Carter, P. (1996). ‘Knobs-into-holes ’ engineering of antibody CH3 domains for heavy chain heterodimerization. Protein Engineering 9, 617-621.[000105] (E) HEK Blue CD122 / CD123 reporter assay demonstrating binding of MiTE molecules to recombinant IL-2R0, as well as the functional activity of IL-2SK with or without MMP-14 digestion, compared to aTREM2-IL2SK which does not pocess any blocking domain (n=3). Data represents mean ± SD (n =3).[000106] (F)-(G) T cells were isolated from the blood of three human donors, activated withCD3 / CD28 beads, and incubated for 3 days with different concentrations of MiTE constructs or control conditions, including aTREM2, aTREM2-IL2SK, IL-2, and undigested / digested MiTE molecules. Proliferation was assessed using eFluor 450 proliferation dye by flow cytometry, with MFI used as readout and represented as inverse MFI (1 / MFI).[000107] (F) Heatmap showing dose-dependent T cell proliferation in response to MiTE molecules, with and without prior digestion by MMP-14 in CD4 (top) and CD8 T cells (bottom). Heatmap color intensity represents proliferation levels, with higher 1 / MFI values (darker red) indicating increased T cell proliferation.[000108] (G) Bar plot depicting T cell proliferation represented as 1 / MFI at 0. 15nM antibody treatment concentration. Statistical significance was assessed by t-tests and is indicated as *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001. Data represents mean ± SEM.[000109] FIG. 25: MiTE robustly abrogates tumor growth without inducing systemic toxicities[0001 10] (A)-(C) hTREM2 mice were s.c. injected with IM MC38 tumors (day 0) and intravenously treated with 200 pg per dose of aRSV, aTREM2, or MiTE144 on day 10 and day 13. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test, comparing each treatment condition to aRSV, and is indicated as: ns- non significant. * P>0.05, *P<0.05, **P<0.01. ***P<0.001, ***P<0.0001.[0001 1 1 ] (A) Body weight monitoring of hTREM2 mice (n=8). Treatment days are indicated with arrows.[0001 12] (B) Bargraphs depicting protein concentration of murine IFN-y, IL-2, and IL-6 in blood serum collected 10 hours after the second dose (day 13) as assessed by measured by ELISA. Data represents mean ± SD (n=4).[0001 13] (C) Bargraph depicting concentration of of AST / ALT in blood serum as assessed using Roche Diagnostics cobas c 111 Chemistry Analyzer. Data represents mean ± SD (n=4).[0001 14] (D)-(F) WT or hTREM2 mice were s.c. injected with IM MC38 tumor cells (day0) and treated with antibodies or immunocytokines (200pg / dose) on day 8, 10 and 13. Data represents mean ± SEM (n = 7). Statistical significance was assessed using one-way ANOVAfollowed by Tukey’s multiple comparisons test, comparing each treatment condition to aRSV, and is indicated as: ns- non significant, * P>0.05, *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001.[0001 15] (D) Left: Tumor volume kinetics of mice treated with aRSV, aTREM2, aRSV-IL2SK (masked), or MiTE144. Arrows indicate treatment days. Right: Bargraph displaying the final tumor weight measured on day 15.[0001 16] (E) Left: Tumor volume kinetics of mice treated with aRSV, aTREM2. anti-PD-1, aTREM2 + anti-PD-1, MiTE144, or MiTE144 + anti-PD-1. Arrows indicate treatment days. Right: Bargraph displaying the final tumor weight on day 16[0001 17] (F) Left: Tumor volume kinetics of mice treated with aRSV, aTREM2, anti-CTLA-4, aTREM2 + anti-CTLA-4, MiTE144. or MiTE144 + anti-CTLA-4. Arrows indicate treatment days. Right: Bargraph displaying the final tumor weight on day 15.[0001 18] (G) WT or hTREM2 mice were s.c. injected with 0.5M MCA205 tumor cells (day0). Data represents mean ± SEM (n = 7). Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test, comparing each treatment condition to aRSV, and is indicated as: ns- non significant, *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001. Left: Tumor volume kinetics of mice treated with 200 pg per dose aRSV, aTREM2, aRSV-IL2SK (masked), or MiTE144 on day 8, 10 and 13. Arrows indicate treatment days. Right: Bargraph displaying the final tumor weight on day 15.[0001 19] FIG. 26: MiTE-based therapies robustly reprogram the tumor microenvironment [000120] (A)-(J) hTREM2 mice were s.c. injected with IM MC38 tumor cells (day 0). Mice were intravenously treated with 200 pg per dose of anti-RSV, anti-TREM2, anti-PD-1, anti- TREM2 + anti-PD-1, anti-CTLA-4, anti-TREM2 + anti-CTLA-4, MiTE144, MiTE144 + anti-PD- 1, or MiTE144 + anti-CTLA-4 on days 8 and 10 (n = 5). Immune cells were isolated on day 11, FACS sorted (total CD45+and T / NK cell enrichment) and underwent scRNAseq employing SPID- seq resulting in a total of 45,505 high-quality cells from 45 mice.[000121 ] (A) UMAP representation depicting identified immune cell clusters.[000122] (B) Dot plot showing the expression of selected marker genes across different immune cell types. Dot size represents the fraction of cells expressing each gene, and color indicates scaled average expression levels.[000123] (C) UMAP feature plots displaying the normalized expression levels of selected genes (hTREM2 = human TREM2 gene). Higher expression is indicated by darker shading.[000124] (D) Graph representation of treatment clustering using MrVI local sample distances aggregated for each treatment condition. Each node represents a treatment in the network and each edge denotes the inverse of the distance compared to the control condition (aRSV) and the graphwas visualized by Kamada-Kawai graph layout. Hierarchical clustering was applied to the treatment distance matrix and the treatments are grouped into four distinct color-coded clusters.[000125] (E) Bubble plot showing the number of differentially expressed genes (DEGs) across immune cell subsets per treatment group in comparison to aRSV control group. DEGs were defined as genes with padj < 0.05 and LogFC > 1 or < - E The size of each bubble represents the number of DEGs (nDEGs), while colors indicate different immune cell types.[000126] (F) Bar graphs depicting the frequencies of myeloid cell subsets stratified by treatment groups and shown per individual mouse.[000127] (G) Box plots showing the frequencies of selected macrophage cell subsets per treatment group. Statistical significance was assessed using pairwise Wilcoxon tests (Mann- Whitney U), followed by Benjamini-Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. * padj < 0.05. Boxes show the median and interquartile range (IQR), while whiskers extend to E5 times the IQR. Each dot represents an individual mouse.[000128] (H) Heatmap showing the enrichment of selected MrVI modules relevant inMacrophages across treatment groups. Color intensity represents treatment-scaled module enrichment score, with darker red indicating higher enrichment.[000129] (I) Dot plot showing the expression of selected genes and DEGs across treatment conditions in macrophages. Rows represent different treatment conditions. DEGs (padj < 0.05, LogFC > 1 or < -1) between aRSV and at least one treatment group are underlined. Dot size represents the percentage of cells expressing each gene, while color intensity reflects the scaled average expression level.[000130] (J) Violin plots showing the expression levels of selected DEGs between aRSV andMiTE treatment groups in Macrophages. Each dot represents an individual mouse.[000131 ][000132] FIG. 27: MiTE limits NK and T cell exhaustion and enhances their cytotoxicity' and proliferation in the TME[000133] (A-J) hTREM2 mice were s.c. injected with IM MC38 tumor cells (day 0). Mice were intravenously treated with 200 pg per dose of anti-RSV, anti-TREM2, anti-PD-1, anti- TREM2 + anti-PD-1, anti-CTLA-4, anti-TREM2 + anti-CTLA-4, MiTE144, MiTE144 + anti-PD- 1, or MiTE144 + anti-CTLA-4 on days 8 and 10 (n = 5). Immune cells were isolated on day 11, FACS sorted (total CD45+and T / NK. cell enrichment) and underwent scRNAseq employing SPID- seq resulting in a total of 45,505 high-quality cells from 45 mice.[000134] (A) Left: UMAP representation of identified T and NK cell populations. Right:Kernel density estimates display the distribution of cells across different treatment conditions in UMAP space.[000135] (B) Bar graphs depicting the frequencies of T and NK cell subsets stratified by treatment and show n per individual mouse.[000136] (C) Box plots showing the frequencies of NK cells proliferating of total T / NK cells(left) and ratio of NK cells proliferating to the sum of NK cells int. and NK cells early subset per treatment group (right). Statistical significance was assessed using pairwise Wilcoxon tests (Mann- Whitney U), followed by Benjamini -Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. *padj < 0.05. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000137] (D) Dot plot showing the expression of selected genes and DEGs across treatment conditions in NK cells. Rows represent different treatment conditions. DEGs (padj < 0.05, LogFC > 1 or < -1) between aRSV and at least one treatment group are underlined. Dot size represents the percentage of cells expressing each gene, while color intensity reflects the scaled average expression level.[000138] (E) Violin plots showing the expression levels of selected DEGs between aRSV andMiTE treatment groups in NK cells. Each dot represents an individual mouse.[000139] (F) Box plots showing the frequencies of total Tregs cells (Tregs early activation +Tregs effector + Tregs effector proliferating) of total T / NK cells. Statistical significance was assessed using pairwise Wilcoxon tests (Mann- Whitney U), followed by Benjamini -Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. *padj < 0.05. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000140] (G) Box plots showing the frequencies of selected T cell populations of total T / NK cells. Statistical significance was assessed using pairwise Wilcoxon tests (Mann-Whitney U), followed by Benjamini -Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. *padj < 0.05. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000141 ] (H) Box plots showing total counts of T cells (left) and CD8 T cells (right) per tumor tissue across treatment conditions. Counts are represented as cells per g tissue (xl O5) and were assessed using FACS and counting beads. Statistical significance was assessed using t-tests.Comparisons were performed against the anti-RSV control group. *P<0.05, **P<0.01. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000142] (I) Dot plot showing the expression of selected genes and DEGs across treatment conditions in T cells. Rows represent different treatment conditions. DEGs (padj < 0.05, LogFC > 1 or < -1) between aRSV and at least one treatment group are underlined. Dot size represents the percentage of cells expressing each gene, while color intensity reflects the scaled average expression level[000143] (J) Violin plots showing the expression levels of selected DEGs between aRSV andMiTE treatment groups in T cells. Each dot represents an individual mouse.[000144] (K) Chord diagrams depict conserved ligand-receptor interactions between mouse and human single-cell RNA-Seq datasets (FIG. 22N,M), focusing on myeloid cells (DCs, TAMs; senders) and lymphoid subsets (NK cells, T cells, and Tregs; receivers) in the tumor microenvironment. Shown are interactions which are enriched in the anti-RSV condition in comparison with MiTE144 alone (left), MiTE144 + anti-PD-l (middle), or MiTE144 + anti- CTLA-4 (right); i.e. interactions which are disrupted with each treatment. Line thickness reflects interaction strength, and colors indicate cell ty pe identity7as shown in the legend.[000145] FIG. 28: MiTE treatment efficiently remodels the immune landscape of human patient-derived tumor fragments[000146] (A)-(H) Fresh tumor tissue from five renal cell carcinoma patients was processed into 1-2 mm3fragments, cryopreserved, and subsequently thawed and cultured for 48 hours with control IgGs (IgGl + IgG4), MiTE144 (+ IgG4), anti-PD-1 (+ IgG4), or both MiTE144 + anti-PD- 1 in combination. Following culture, fragments were digested. CD45+immune cells were enriched, stained with a CITE-seq antibody panel, sorted for viability, and processed for single-cell transcriptomic, and proteomic profiling using the lOx Genomics Chromium 5' platform, yielding 93,087 high-quality7single cells for analysis.[000147] (A) totalVI74was applied to integrate transcriptomic and proteomic modalities, followed by unsupervised clustering of the integrated latent space. Figure depicts UMAP projection of identified CD45+clusters which were annotated based on gene expression and surface protein profiles.[000148] (B) Dot plot showing the expression of selected marker genes across different immune cell types. Dot size represents the fraction of cells expressing each gene, and color indicates scaled average expression levels.[000149] (C) Protein expression of selected immune markers (TREM2, PD1, ICOS, CD56,CD25, GITR, CD122, CD132) overlaid on the UMAP, based on denoised CITE-seq surface antibody signals.[000150] (D) Treatment enrichment density plots showing the distribution of immune cells under the indicated treatment conditions.[000151 ] (E) Stacked bar plots showing the composition of CD45+immune populations across treatment conditions for each patient.[000152] (F) Log2 fold-change in abundance of immune cell populations relative to the isotype control for MiTE144 monotherapy (left) or combination therapy with anti-PD-1 (right).[000153] (G) Violin plots of patient-level cell type proportions in CD8 cycling, CD8 memory,NK intermediate, and Treg subsets across treatment conditions. Each dot indicates a patient donor. [000154] (FI) Dot plot showing the expression of selected genes and DEGs across treatment conditions in T cells (top) and NK cells (bottom). Rows represent different treatment conditions. Dot size represents the percentage of cells expressing each gene, while color intensity reflects the scaled average expression level.[000155] (I) Schematic illustration of mode of action of MiTE molecules.[000156] FIG. 29: Cell annotations and clustering of human transcriptomic datasets, related to FIG. 22[000157] (A-H) Data from curated spatial atlas of -1.86 million single cells from publicly available MERFISH datasets of human lung, breast, colon, and ovarian cancer41 42Cells are color-coded based on their assigned cell type. Tumor-associated macrophages ( =TAMs), tissueresident macrophages (=TRMs). dendritic cells (=DCs), natural killer ( = NK) cells, regulatory T cells (= Tregs). Monocyte-like (=Mon-like). mesenchymal stem cells (= MSCs)[000158] (A)-(D) UMAP representation of identified clusters in human cancers, namely (A) breast cancer, (B) lung cancer, (C) colon cancer and (D) ovarian cancer.[000159] (E)-(H) Empirical cumulative distribution functions (CDF) of minimum distances between key immune cell populations in breast (E), lung (F), colorectal (G) and ovarian (H) cancer tissue sections, illustrating spatial proximity relationships among infiltrating immune cell populations, including tumor-associated macrophages (TAMs), monocyte-derived macrophages (MoMac), monocyte-like cells (Mon-like), Monocytes (Mon), dendritic cells (DCs), Neutrophils (Neut), Granolocytes and lymphocyte subsets (T cells, NK cells, and Tregs).[000160] (I) Analyses of scRNA-seq data containing human tumor samples (n = 123) from breast, lung, and colon cancers44'46. 332,723 high quality single-cells were integrated using scVI integration48followed by MultiNicheNetR49analysis. Dot plot displaying marker gene expressionused for cell annotation. Dot size represents the fraction of cells expressing each marker gene within a given cell type, and color intensity indicates the mean expression level.[000161 ] FIG. 30: Characterization of anti-TREM2 antibody and its functional effects, related to FIG. 23[000162] A) Box plots display TREM2 expression levels in a bulk RNA-seq dataset comprising 7,275 TCGA samples and 4,294 GTEx samples, spanning 14 different human cancer types compared to corresponding healthy tissues.[000163] (B)-(D) Monocytes were isolated from the bone marrow7of either hTREM2 orTrem2-KO mice and in vitro differentiated into tumor-associated macrophages (TAMs) using M- CSF+IL-4. These macrophages were treated as indicated (control IgG, Binder control antibody (Ab210), or anti-TREM2 (Ab219 (E3C7)) and submitted for single-cell RNA-sequencing.[000164] (B) 2D Contour plots depict the density of cells from each treatment group.[000165] (C) Heatmap showing top DEGs across treatment groups.[000166] (D) Violin plots showing the expression of selected genes Mmpl2, Siglecl, Sppl, and Itg2) across different experimental conditions.[000167] (E) TREM2-ov erexpressing Jurkat cells were incubated with increasing concentrations of soluble TREM2. Subsequent anti-TREM2 binding was assessed by flow cytometry, revealing dose-dependent competition only at supraphy siological sTREM2 concentrations.[000168] (F)-(G) hTREM2 mice were s.c. injected with IM MC38 cells (day 0). The mice were treated with 200pg / dose aTREM2 or control (aRSV) on day 10 and day 13. Data represents mean ± SEM (n = 7). Statistical significance was assessed by t-test and is indicated as: * P>0.05, *P<0.05. **P<0.01, ***P<0.001, ***P<0.0001.[000169] (F) Left: Tumor volume kinetics. Arrows indicate treatment days. Right: Bargraph displaying the final tumor weight measured on day 16.[000170] (G) Tumor volumes kinetics per individual mouse.[000171 ] FIG. 31: Characterization of anti-TREM2-IL2SK immunocytokine and its functional effects, related to FIG. 23[000172] (A) Correlations between IL-2 expression level and the infiltration levels of CD8+T and NK cells across TCGA cancers. Infiltration levels of immune cells was obtained from the TIMER2.0 database. Correlation values are color-coded, with significant correlations indicated.[000173] (B) Picture displaying SDS-PAGE analysis of anti-TREM2 and anti-TREM2-IL2SK antibodies under reducing (R) and non-reducing (NR) conditions.[000174] (C) Table showing SPR analysis results of anti-TREM2 and anti-TREM2-IL2SK, measuring interaction with recombinant human Trem2 or IL-2RJ3.[000175] (D) HEK parental cells or HEK cells overexpressing hTREM2 were incubated with anti-RSV, anti-RSV-IL-2SK, anti-TREM2, or anti-TREM2-IL2SK. Upond washing, cells were stained with anti-IgG antibodies and MFI was assessed by FACS analysis to confirm binding to HEK cells overexpressing human TREM2. Data represents mean ± SD (n =3).[000176] (E) Bioactivity assay of anti-TREM2-IL2SK using HEK Blue CD122 / CD132 reporter cells (Target (T)) and HEK cells overexpressing hTREM2 (Effector(E)). Measurement of binding to hTREM2 and functional activity of IL-2SK via engagement with IL-2Rp / y. Absorbance was measured at 630-650 nm. Data represents mean ± SEM (n=3).[000177] (F) hTREM2 mice were s.c. injected with IM MC38 cells (day 0). Mice were treated with lOOpg / dose anti-RSV-IL-2SK, anti-TREM2-IL2SK or control anti-RSV on day 7 and day 10. Arrows indicate treatment time points. Kaplan-Meier survival curves depict probablility of survival among the treatment groups (n=7).[000178] FIG. 32: Generation of four MiTE molecules based on protease screens, related to FIG. 24[000179] (A) Analysis of scRNA-seq data containing human tumor samples (n = 123) and their adjacent healthy-like samples (n = 67) from breast, lung, and colon cancers44'46. Dot plot showing the expression of immune-related proteases across immune cell subsets derived from tumor tissues or adjacent tissue. Dot size represents the fraction of cells expressing each gene, and color intensity indicates mean expression levels.[000180] (B)-(C) Pseudo-bulk analysis from a large single-cell dataset of 13,818,851 single cells, incorporating PBMCs (n=1.753), healthy tissues (n=147), tumor-adjacent samples (n=750) as well as tumors (n=2791) (Table S2).[000181 ] (B) Dot plot showing the expression of proteases across immune and non-immune populations. Dot size represents the percentage of patients with high expression, and color intensity indicates scaled expression[000182] (C) Dot plot showing the expression of selected marker genes across different immune and non-immune cell types. Dot size represents the fraction of cells expressing each gene, and color indicates scaled average expression levels.[000183] (D) Box plots display MMP14 expression levels in a bulk RNA-seq dataset comprising 7,275 TCGA samples and 4,294 GTEx samples, spanning 14 different human cancer types compared to corresponding healthy tissues.[000184] (E) Immunofluorescence staining of DAPI (grey), TREM2 (red) and MMP14(yellow ) in human lung tumor and tumor-adjacent tissue.[000185] (F) Violin plot showing quantification of increased TREM2+MMP14+coexpression in tumor vs. tumor-adjacent tissue (*p < 0.05). Each dot indicates a tile from the corresponding staining image and the area per tile was set to be identical in both tissues.[000186] (G) Pictures depicting SDS-PAGE analysis of MiTE molecules under reducing (R) and non-reducing (NR) conditions for different MiTE constructs, including MiTE-76, MiTE-95, MiTE-144, and MiTE-208.[000187] (H) Picture depicting SDS-PAGE analysis showing the cleavage of MiTE molecules by MMP-14 under non-reducing conditions (NR).[000188] (I) Western blot showing MiTE144 digestion by different recombinant MMPs incubated for 0.5, 2 or 20h.[000189] (J) Quantification of MiTE144 digestion (% cleaved) by different recombinantMMPs incubated for 0.5, 2 or 20h based on band intensities.[000190] FIG. 33: Validation of four MiTE molecules in vitro and in vivo, related to FIG. 24. [000191 ] (A) Table sho ing SPR analysis measuring the binding affinity of MiTE molecules to recombinant human Trem2.[000192] (B) Binding of MiTE molecules or Isotype and anti-TREM2 control antibodies to recombinant hTrem2 protein as assessed by ELISA. Absorbance was measured at 450-655 nm. Data represents mean ± SD (n =3).[000193] (C) HEK parental cells or HEK cells overexpressing hTREM2 w ere incubated withMiTE molecular, or anti-RSV or anti-TREM2 control antibodies. Upon washing, cells were stained with anti-IgG antibodies and MFI was assessed by FACS analysis to confirm binding to HEK cells overexpressing human TREM2. Data represents mean ± SD (n =3).[000194] (D) Table showing results of sandwich SPR analysis measuring the binding ofMiTE molecules to IL-2Rp. The table presents binding parameters for different MiTE constructs, including versions with were pre-cleaved with MMP-14.[000195] (E)-(I) T cells were isolated from the blood of three human donors, activated withCD3 / CD28 beads, and incubated for 3 days with different concentrations of MiTE constructs or control conditions, including aTREM2, aTREM2-IL2SK, IL-2, and undigest ed / digested MiTE molecules. Proliferation as well as CD25 and CD69 expression levels were assessed by flow cytometry, with MFI used as readout. IFN-y concentrations were assessed in the supernatant by ELISA.[000196] (E) Heatmap showing dose-dependent expression of CD25 induced by MiTE molecules, with and without prior digestion by MMP-14 in CD4 (top) and CD8 T cells (bottom). Heatmap color intensity represents CD25 expression levels, with higher MFI values depicted in darker red.[000197] (F) Bar plot depicting CD25 expression represented as MFI at 0.6nM antibody treatment concentration. Statistical significance was assessed by t-tests and is indicated as *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001. Data represents mean ± SEM.[000198] (G) Heatmap showing dose-dependent proliferation (top panels) and CD69 expression (bottom panels) induced by MiTE molecules, with and without prior digestion by MMP-14 in CD4+CD251ow vs CD4+CD25high T cells. The heatmap color intensity represents CD69 expression levels, with higher MFI values depicted in darker red. Proliferation was assessed using eFluor 450 proliferation dye by flow cytometry, with MFI used as readout and represented as inverse MFI (1 / MFI).[000199] (H) Bar plot depicting proliferation represented as inverse 1 / MFI at 0. 15nM treatment concentration (top panels) as well as CD69 expression represented as MFI at 2.5nM treatment concentration (bottom pantels) in CD4+CD25high vs CD4+CD25high T cells. Statistical significance was assessed by t-tests and is indicated as *P<0.05, **P<0.01, ***P<0.001, ***p<0.0001. Data represents mean ± SEM.[000200] (I) Bar graph depicting levels of human IFN-y as measured by ELISA. Data represents mean ± SD.[000201 ] (J)-(L) Naive WT mice (n=4) were intravenously treated with a single dose (50 pg) of aRSV, aTREM2, or indicated MiTE molecules (day 0).[000202] (J) Body weight monitoring. Treatment days are indicated with arrows.[000203] (K) Cytokine levels in serum collected 24 hours after a single dose. Levels of murine IFN-y were measured by ELISA. Data represents mean ± SD.[000204] (L) hTREM2 mice were s.c. injected with IM of MC38 tumor cells (day 0). Mice were intratumorally injected (5pg / dose) with aRSV, aTREM2-IL2SK or MiTE molecules (n=3) on day 9,11 and 13. Plot depicts tumor volumes kinetics per treatment group. Statistical significance was assessed using one-way ANOVA followed by Tukey’s multiple comparisons test, comparing each treatment condition to aRSV, and is indicated as: ns- non significant, *P<0.05, **P<0.01, ***P<0.001, ***P<0.0001. Data represents mean ± SEM[000205] FIG. 34: Pathology, and pharmacokinetics in vivo, related to FIG. 25[000206] (A) Boxplot depicting spleen weight measurements (day 15) of hTREM2 mice implanted with MC38 tumors on day 0 and treated intravenously with 200 pg per dose of aRSV, aTREM2, or MiTE144 on day 10 and day 13 (n=8).[000207] (B) Spleen immune cell counts of CD8 T cells (left) or NK cells (right) isolated from WT mice s.c. injected with IM MC38 cells (day 0). followed by two intravenous injections of 200 pg per dose of anti-RSV, anti-RSV-lL2SK, anti-TREM2, anti-TREM2-lL2SK or MiTE144 on day 8 and day 10, with spleens collected 10 hours after second dosage for analysis. Data represents mean ± SD. Statistical significance was assessed by Anovas.[000208] (C) Pharmacokinetic parameters of aTREM2-IL2SK and MiTE144, including elimination rate constant (K), calculated half-life, and estimated systemic clearance time in mice following 50ug intravenous injection.[000209] (D) Biodistribution of MiTE144 measured 24 hours post-injection. Concentrations were quantified by ELISA in the indicated tissues and normalized to tissue weight (ng per mg tissue). Data represent mean ± SEM.[000210] (E) Representative histological images of lung, liver, kidney, heart, and brain, fromWT mice s.c. injected with IM MC38 cells (day 0), followed by two intravenous injections of 200 pg per dose of anti-RSV, anti-TREM2 or MiTE144 on day 8 and day 10, with tissues collected 10 hours after second dosage for analysis. H&E staining was used to assess potential pathological changes.[00021 1 ] FIG. 35: Individual tumor growth curves and body weight measurements in vivo, related to FIG. 25[000212] (A)-(C) WT or hTREM2 mice were s.c. injected with IM MC38 tumor cells (day0) and treated with antibodies or immunocytokines (200pg / dose) on day 8, 10 and 13. Data represents mean ± SEM (n = 7).[000213] (A) Tumor volume and body weight kinetics of individual mice treated with aRSV, aTREM2, aRSV-IL2SK (masked), or MiTE144.[000214] (B) Tumor volume and body weight kinetics of individual mice treated with aRSV, aTREM2, anti-PD-1, aTREM2 + anti-PD-1, MiTE144, or MiTE144 + anti-PD-1.[000215] (C) Tumor volume and body weight kinetics of individual mice treated with anti-CTLA-4, anti-TREM2 + anti-CTLA-4 or MiTE144+anti-CTLA-4 (200pg / dose)[000216] (D) WT or hTREM2 mice were s.c. injected with 0.5M MCA205 tumor cells (day0). Mice were treated with aRSV. aTREM2, aRSV-IL2SK (masked), or MiTE144 (200 pg / dose) on day 8, 10 and 13. Data represents mean ± SEM (n = 7). Graphs depict tumor volume and body weight kinetics of individual mice.[000217] FIG. 36: Single-cell analysis of MiTE treatment response in MC38 tumor model, related to FIG. 26 and 6[000218] (A)-(J) hTREM2 mice were s.c. injected with IM MC38 tumor cells (day 0). Mice were intravenously treated with 200 pg per dose of anti-RSV, anti-TREM2, anti-PD-1, anti- TREM2 + anti-PD-1, anti-CTLA-4, anti-TREM2 + anti-CTLA-4. MiTE144, MiTE144 + anti-PD- 1, or MiTE144 + anti-CTLA-4 on days 8 and 10 (n = 5). Immune cells were isolated from tumors on day 11, FACS sorted (total CD45+and T / NK cell enrichment) and underwent scRNAseq employing SPID-seq resulting in a total of 45,505 high-quality cells from 45 mice. Immune cells were isolated from tdLNs on day 11 and acquired by FACS.[000219] (A) Gating strategy for FACS sorting immune cells (CD45 ) and T / NK cells(CD90.2+and / or NK1.1+) for scRNA-seq.[000220] (B) Violin plots and bargraphs depicting quality control metrics: shown are number of UMIs, number of unique genes and percentage of mitochondrial genes in each mouse as well as percentage of cells passing quality control per mouse (passing criteria: less than 10% mitochondrial RNA transcripts, more than 200 and less than 3500 unique genes).[000221 ] (C) Heatmap showing the MrVI local sample distances aggregated for each treatment condition. Row s represent different treatment conditions.[000222] (D) Box plots showing the frequencies of myeloid cell subsets of total myeloid cells per treatment group. Statistical significance was assessed using pairwise Wilcoxon tests (Mann- Whitney U), followed by Benjamini-Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. * padj < 0.05. Boxes show' the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000223] (E) UMAP depicting identified granulocytes clusters (basophils and neutrophils).[000224] (F) Dot plot of top marker genes distinguishing basophils from neutrophils. Dot size represents the fraction of cells expressing each gene, and color indicates scaled average expression levels.[000225] (G) Box plots showing the frequencies of granulocyte cell subsets of total myeloid cells per treatment group. Statistical significance was assessed using pairwise Wilcoxon tests (Mann- Whitney U), followed by Benjamini-Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. * padj < 0.05. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000226] (H) UMAP of neutrophils revealing three transcriptionally distinct subsets (Tl, T2, and T3), based on gene signatures from Ng et al.69[000227] (I) Dot plot showing expression of gene modules as defined by Ng et al69in the three identified neutrophil subsets. Dot size represents the fraction of cells expressing each gene, and color indicates scaled average expression levels.[000228] (J) Box plots showing frequencies of neutrophil subsets among myeloid cells per treatment condition. Statistical significance was assessed using pairwise Wilcoxon tests with Benjamini -Hochberg correction; comparisons are to the anti-RSV group (padj < 0.05). Boxes indicate the median and IQR; whiskers span 1.5x IQR; dots represent individual mice.[000229] FIG. 37: Single-cell analysis of MiTE treatment response in MC38 tumor model, related to FIG. 26 and 6[000230] (A) Gating strategy' used to FACS gate on cell populations in tdLNs and tumors to quantify cell numbers per gram tissue or per LN.[000231 ] (B) Box plots showing total counts of immune cells, myeloid cell populations andB cells per tdLN across treatment conditions. Counts were assessed using FACS and counting beads. Statistical significance was assessed using t-tests. Comparisons were performed against the anti-RSV control group. *P<0.05, **P<0.01. Boxes show' the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000232] (C) Dot plot showing the expression of selected genes and DEGs across treatment conditions in dendritic cells. Rows represent different treatment conditions. DEGs (padj < 0.05, LogFC > 1 or < -1) between aRSV and at least one treatment group are underlined. Dot size represents the percentage of cells expressing each gene, while color intensity reflects the scaled average expression level.[000233] (D) Violin plots showing the expression levels of selected DEGs between aRSV and MiTE treatment groups in dendritic cells. Each dot represents an individual mouse.[000234] FIG. 38: Single-cell analysis of MiTE treatment response in MC38 tumor model, related to FIG. 27[000235] (A)-(E) hTREM2 mice w'ere s.c. injected with IM MC38 tumor cells (day 0). Mice were intravenously treated with 200 pg per dose of anti-RSV, anti-TREM2, anti-PD-1, anti- TREM2 + anti-PD-1, anti-CTLA-4, anti-TREM2 + anti-CTLA-4, MiTE144, MiTE144 + anti-PD- 1, or MiTE144 + anti-CTLA-4 on days 8 and 10 (n = 5). Immune cells were isolated from tumors on day I I. FACS sorted (total CD45+ and T / NK. cell enrichment) and underwent scRNAseq employing SPID-seq resulting in a total of 45,505 high-quality cells from 45 mice. Immune cells were isolated from tdLNs on day 11 and acquired by FACS.[000236] (A). (E) Box plots showing the frequencies of T / NK cell subsets of total T / NK cells per treatment group. Statistical significance was assessed using pairwise Wilcoxon tests (Mann- Whitney U), followed by Benjamini-Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. * padj < 0.05. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000237] (B) Box plots showing the total count of NK cells per gram tumor tissue across treatment groups. Statistical significance was assessed using pairwise Wilcoxon tests (Mann- Whitney U), followed by Benjamini-Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. * padj < 0.05. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000238] (C), (F) Box plots showing total counts of NK and T cell populations per tdLN across treatment conditions. Counts were assessed using FACS and counting beads. Statistical significance was assessed using t-tests. Comparisons were performed against the anti-RSV control group. *P<0.05, **P<0.01. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000239] (D) Bar graphs depicting levels of secreted proteins in the supernatant of humanNK cells treated with 1 nM cleaved or non-cleaved MiTE144 in vitro for 48 h. Cleaved MiTE144 was generated by MMP14 digestion. Supernatants were analyzed using the Nomic platform.[000240] (G) Box plots showing the ratio of cell counts of different T cell clusters per gram tumor tissue across treatment groups. Statistical significance was assessed using pairwise Wilcoxon tests (Mann-Whitney U), followed by Benjamini-Hochberg (BH) correction for multiple testing. Comparisons were performed against the anti-RSV control group. * padj < 0.05. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000241 ] (H) Box plots showing the MFI of GITR in CD8 T cells in tumors across treatment groups. Statistical significance was assessed using t-tests. Comparisons were performed against the anti-RSV control group. *P<0.05, **P<0.01. Boxes show the median and interquartile range (IQR), while whiskers extend to 1.5 times the IQR. Each dot represents an individual mouse.[000242] FIG. 39: CiTE-Seq analysis of patient-derived tumor fragments treated with MiTE144, anti-PD-1 or their combination, related to FIG. 28.[000243] (A) Heatmap of surface protein expression (CITE-seq) used to annotate immune cell populations. Each row represents a cell cluster and each column a protein marker.[000244] (B) Distribution of patient samples across treatment groups.[000245] (C) Principal component analysis (PCA) of single-cell transcriptome and surface protein profiles colored by patient ID.[000246] (D) Boxplots depicting frequencies of annotated immune subsets among CD45+cells across treatment groups. Violin plots show the distribution per patient; boxes indicate median and 1QR.[000247] FIG. 40: CiTE-Seq Analysis of patient-derived tumor fragments treated with MiTE144, anti-PD-1 or their combination, related to FIG. 28.[000248] (A) Volcano plots showing differential gene expression in macrophages, dendritic cells (DCs), T cells. Tregs, and NK cells following MiTE144 treatment versus isotype control. Significantly upregulated (red) and downregulated (blue) genes are highlighted (adjusted p < 0.05, |log2FC| > 0.5).[000249] (B) Dot plots showing surface protein expression of checkpoint and activation markers in T cells and NK cells across treatment conditions, based on CITE-seq data.[000250] (C) Cross-species comparison of conserved differentially expressed genes (DEGs) across human and mouse immune subsets. Heatmaps display significance (-logio adjusted p-value) for a hypergeometric test of the overlapping DEGs conserved in human and mouse species in total CD45+cells (left) and for MiTE144-specific DEGs (right).[000251 ] FIG. 41: Schematic representation of the immunocytokines E3C7-IL15 Masked 1 (FIG. 41 A) and MiTE144 (FIG. 4 IB).[000252] FIG. 42: Recombinant TREM2-targeting immunocytokines can be produced with high yield and purity in two different formats, including bivalent IL15 format (FIG.42A, E3C7- IL15-MV1) and a monovalent IL2-superkine format (FIG.42B, MiTE144). SDS-PAGE analysis of final recombinant protein product for each masked E3C7 immunocytokine prepared under reducing (R) and non-reducing (NR) conditions shows high purity with bands at the expected molecular weights.[000253] FIG. 43: TREM2 targeting immunocytokines in both E3C7-IL15-MV1 and MiTE144 format retain specific antigen binding to TREM2 in both the presence or absence of MMP14-mediated demasking. E3C7-IL15-MV1 and MiTE-144 were incubated in presence or absence of recombinant MMP-14. Subsequently, TREM2 binding capacity was measured either by ELISA, using plates coated with recombinant human TREM2 (FIG. 43A and FIG. 43C), and by cell surface binding assay utilizing Jurkat cell line engineered to express human TREM2 (FIG. 43B and FIG. 43D). A non targeted control immunocytokine (Pali-IL2SK) is shown for reference (FIG. 43A and FIG. 43C).[000254] FIG. 44: M1TE144 immunocytokine is cleaved more rapidly than the E3C7-IL15- MV1 immunocytokine. MMP14-mediated demasking is evaluated by incubating immunocytokines with recombinant MMP14 under the indicated conditions and analyzing the resulting product using SDS-PAGE under reducing conditions. The percent of specific demasking is quantified by calculating ratio of de-masked vs masked heavy chain. (FIG. 44 A) 0.5 uM of each of the immunocytokines was incubated with increasing amount of MMP14 for 21h at 37°C; The data show that both immunocytokine formats are effectively de-masked by lug of recombinant MMP14. (FIG. 44B) 0.5uM of immunocytokines were incubated with 0.45uM of MMP14 for indicated periods of time and compared to the complete digestion condition of 0.89uM for 21 hours at 37°C.[000255] FIG. 45: MMP14-mediated de-masking induces a 10-15 fold increase in cytokine activity, resulting in activity comparable to the cytokine reference controls. E3C7-IL-15-MV1 (FIG. 45 A) and MiTE-144 (FIG. 45B) were incubated for 20h at 37C in presence or absence of recombinant MMP-14. then diluted and subsequently added to the HEK-Blue CD 122 / CD 132 (IL- 2Rp / y) reporter cell line. Signal was acquired after overnight incubation. Recombinant human IL15 and IL2 are included as reference controls.[000256] FIG. 46: Immunocytokines show dose dependent activity7on primary human immune cells following MMP14-mediated demasking. Cryopreserved Peripheral Bone Marrow Cells (PBMCs) responded more strongly to immunocytokine with an MMP14 cleaved mask than immunocytokine with intact mask. PBMCs from four donors were treated with masked or MMP14 de-masked immunocytokine for 72 hours and concentrations 0. IpM, IpM, lOpM, lOOpM, InM, lOnM, and lOOnM. (FIG. 46A) IFNy secretion was assessed through cell supernatant collection with values obtained from Nomic's nELISA multiplex assay. (FIG. 46B) CD69 expression was assessed through flow cytometry gated on live CD3+, CD8+ T Cells.[000257] FIG. 47: Administration of E3C7-IL15-MV1 did not result in systemic elevation of IFNg observed with non-masked controls even at any of the doses tested (even up to 1563pmol). Masked E3C7-IL15-MV1 construct effectively limits systemic immune activation associated nonmasked immunocytokines in vivo. Female C57BL / 6 mice were injected twice at a 48 hour interval with the indicated dose of E3C7-IL15-MV1 or a matched non-masked E3C7-IL15 variant as a reference control. Serum was collected 24 hours following the second dose and assessed for IFNg levels.DETAILED DESCRIPTIONDEFINITIONS[000258] In order for the present disclosure to be more readily understood, certain terms are first defined below. Additional definitions for the following terms and other terms are set forth throughout the Specification.[000259] As used in this Specification and the appended claims, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.[000260] Unless specifically stated or obvious from context, as used herein, the term “or” is understood to be inclusive and covers both “or” and “and”.[000261 ] The term “and / or” where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. Thus, the term “and / or” as used in a phrase such as “A and / or B” herein is intended to include A and B; A or B; A (alone); and B (alone). Likewise, the term “and / or” as used in a phrase such as “A, B, and / or C” is intended to encompass each of the following aspects: A, B, and C; A. B, or C; A or C; A or B; B or C; A and C; A and B; B and C; A (alone); B (alone); and C (alone).[000262] The terms “e.g.,” and “i.e.,” as used herein, are used merely by way of example, without limitation intended, and should not be construed as referring only those items explicitly enumerated in the specification.[000263] The terms “or more,” “at least,” “more than,” and the like, e.g., “at least one” are understood to include but not be limited to at least 1, 2, 3, 4. 5, 6, 7, 8. 9, 10, 11. 12. 13. 14. 15. 16,17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85. 86, 87, 88, 89, 90, 91, 92, 93, 94,95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105. 106, 107, 108, 109, 110, 111, 112. 113, 114, 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 2000, 3000, 4000, 5000, or more than the stated value. Also included is any greater number or fraction in between.[000264] Conversely, the term “no more than” includes each value less than the stated value. For example, “no more than 100 amino acids” includes 100, 99, 98, 97, 96, 95, 94, 93, 92, 91, 90, 89, 88, 87, 86, 85, 84, 83, 82, 81, 80, 79, 78, 77, 76, 75, 74, 73, 72, 71, 70, 69, 68, 67, 66, 65, 64,63, 62, 61, 60, 59, 58, 57, 56, 55, 54, 53, 52, 51, 50, 49, 48, 47. 46. 45, 44, 43, 42, 41, 40, 39, 38,37. 36, 35, 34, 33, 32, 31, 30, 29, 28, 27, 26, 25, 24, 23, 22, 21. 20. 19. 18. 17. 16. 15. 14, 13, 12,1 1 , 1 , 9, 8, 7, 6, 5, 4, 3, 2, 1 , and 0 amino acids. Also included is any lesser number or fraction in between.[000265] The terms "plurality.” "at least two,” “two or more,” “at least second,” and the like, are understood to include but not limited to at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16,17, 18, 19 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42,43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68,69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85. 86. 87. 88, 89, 90, 91, 92, 93, 94,95. 96. 97, 98, 99, 100. 101, 102, 103. 104, 105. 106, 107. 108, 109, 110. I l l, 112. 113, 114. 115, 116, 117, 118, 119, 120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132, 133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145, 146, 147, 148, 149 or 150, 200, 300, 400, 500, 600, 700. 800, 900, 1000, 2000, 3000, 4000, 5000, or more. Also included is any greater number or fraction in between.[000266] Throughout the specification the word “comprising,” or variations such as “comprises” or “comprising,” will be understood to imply the inclusion of a stated element, integer or step, or group of elements, integers or steps, but not the exclusion of any other element, integer or step, or group of elements, integers or steps. It is understood that wherever aspects are described herein with the language “comprising,” otherwise analogous aspects described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. The term "consisting of' excludes any element, step, or ingredient not specified in the claim. In re Gray, 53 F.2d 520, 11 USPQ 255 (CCPA 1931); Ex parte Davis, 80 USPQ 448, 450 (Bd. App. 1948) ("consisting of' defined as "closing the claim to the inclusion of materials other than those recited except for impurities ordinarily associated therewith"). The term “consisting essentially of’ limits the scope of a claim to the specified materials or steps "and those that do not materially affect the basic and novel characteristic(s)" of the claimed disclosure.[000267] Unless specifically stated or evident from context, as used herein, the term “about” refers to a value or composition that is within an acceptable error range for the particular value or composition as determined by one of ordinary skill in the art, which will depend in part on how the value or composition is measured or determined, i.e., the limitations of the measurement system. For example, “about” or “approximately” may mean within one or more than one standard deviation per the practice in the art. “About” or “approximately” may mean a range of up to 10% (i.e., ±10%). Thus, “about” may be understood to be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.5%, 0.1%, 0.05%, 0.01%, or 0.001% greater or less than the stated value. For example, about 5 mg may include any amount between 4.5 mg and 5.5 mg. Furthermore, particularly with respect to biological systems or processes, the terms may mean up to an order of magnitude or up to 5-fold of a value. When particular values or compositions are provided in the instant disclosure,unless otherwise stated, the meaning of "about" or "approximately" should be assumed to be within an acceptable error range for that particular value or composition.[000268] As described herein, any concentration range, percentage range, ratio range or integer range is to be understood to be inclusive of the value of any integer within the recited range and, when appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated.[000269] Units, prefixes, and symbols used herein are provided using their Systeme International de Unites (SI) accepted form. Numeric ranges are inclusive of the numbers defining the range.[000270] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure is related. For example, Juo, “The Concise Dictionary of Biomedicine and Molecular Biology’’, 2nd ed., (2001), CRC Press; “The Dictionary of Cell & Molecular Biology”, 5th ed., (2013), Academic Press; and “The Oxford Dictionary Of Biochemistry And Molecular Biology”, Cammack et al. eds., 2nd ed, (2006), Oxford University Press, provide those of skill in the art with a general dictionary for many of the terms used in this disclosure.[000271 ] The present invention, in some embodiments thereof, relates to compositions comprising fusion polypeptides for use in treating cancer.[000272] 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.[000273] The therapeutic efficacy of immune cells activating cytokines in cancer therapy is limited.[000274] Whilst conceiving embodiments of the invention and in order to improve the therapeutic efficacy of cytokine therapy, the present inventors designed myeloid binding / modulating antibodies which are armed with engineered effector immune cell-activating cytokines to conditionally enhance the immune response in the tumor microenvironment (TME), thereby creating a dual-arm immunotherapeutic approach. These myeloid-targeted immunocytokines exhibit tumor-conditioned activity through a blocking moiety which is cleaved via tumor-associated-macrophage (TAM)-specific proteases resulting in active cytokines solely in the TME context. This strategy aims to activate both myeloid and effector immune cell compartments in the TME, offering a first-of-its-kind immunotherapy that could be transformative for treating immune checkpoint inhibition (ICI)-resistant tumors (e.g., anti PD-1 resistant tumors)by activating potent anti-tumor immunity, while maintaining specificity and reducing off-target effects.[000275] As is illustrated hereinbelow and in the Examples section which follows, the engineered immunocytokines exhibited inhibition of tumor growth in an MMP14-dependent manner, reprogramming of myeloid cells in tumors and hence the immunosuppressive TME, reduced systemic toxicity, overcoming tumor antigen escape, activation of T cells, NK and NK.T cells proliferation as well as synergy with immune checkpoint inhibition (as evidenced by anti PD- 1 antibody treatment).[000276] Thus, the present tool paves the way to the generation of a novel, effective, safe and personalized cancer treatment modality.[000277] Thus, according to an aspect of the invention there is provided a composition of matter comprising at least one fusion polypeptide which comprises a targeting moiety which binds a marker of a myeloid-derived suppressor cell (MDSC) attached to an immune cell-activating cytokine, wherein said immune cell-activating cytokine is masked by a masking agent, such that when said at least one fusion polypeptide is in a vicinity of an MSDC, said masking agent unmasks said immune cell-activating cytokine via an activity of an MSDC-specific protease so as to allow cytokine activity7of said immune cell-activating cytokine.[000278] 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 moiety7, linker (if present), a masking agent, cleavage sequence and immune cell-activating cytokine, or are different such as in at least one of a targeting moiety, linker (if present), masking agent, cleavage sequence and immune cellactivating cytokine.[000279] When different polypeptides are present in the composition they may form heterodimers or a heterooligomer (e.g., 3, 4, 5, 6).[000280] When the same polypeptides are present in the composition they may form homodimers or a homooligomer (e.g., 3, 4. 5, 6). Such configurations are illustrated in FIG. 8. Sequences are sub-sequences are provided in SEQ ID Nos: 89-147.[000281 ] 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: a targeting moiety, a linker, an immune cell-activating moiety, a linker, a cleavable sequence, a linker 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 suchas 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”.[000282] According to a specific embodiment, the fusion polypeptide is of a single type of an ammo acid sequence.[000283] According to a specific embodiment, the at least one fusion polypeptide is of different ty pes of amino acid sequences, e.g., when the masking agent is not on the same amino acid sequence as the cytokine.[000284] As mentioned, the fusion polypeptide comprises a targeting moiety which binds a marker of a myeloid-derived suppressor cell (MDSC).[000285] As used herein “myeloid-derived suppressor cell (MDSC)” refers to cells of the myeloid lineage which are endowed with immune suppressor activity[000286] MDSCs 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.[000287] Suppressor activity of MDSCs is determined by their ability to inhibit the effector function of lymphocytes. Inhibition can be caused by different mechanisms.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, LGALS9, TREM2), including subsets such as regulatory macrophages (marked by the expression of TREM2, GPNMB, IL17R, HILPDA, HM0X1, and VEGFA) or , but also a heterogeneous group of my eloid progenitor cells with monocytic-like characteristics CDl lb+Ly6G-Ly6Chlshand immature myeloid cells identified by CDl lb+Ly6G+Ly6Clow.[000288] 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. [000289] As used herein “a marker” refers to a cell surface molecule which is presented (e.g., expressed) on an MDSC.[000290] According to a specific embodiment, the molecule is a protein.[000291] According to a specific embodiment, the marker is presented exclusively on MDSCs or a sub-population thereof (e.g., TAM), as can be determined by fluorescence activated cell sorter (FACS).[000292] According to a specific embodiment, the marker mediates a suppressive function of MDSCs or a sub-population thereof (e.g., TAM). In this respect, it can also be referred to as an immune checkpoint molecule.[000293] According to a specific embodiment, the targeting moiety is characterized by a binding activity7and optionally (not necessarily) an inhibitory' activity7of the suppressor function. Thus, in reference to the latter function, the targeting moiety can also be referred to as inhibitory (e.g., inhibitory7antibody), also referred to herein as antagonistic (e.g., an antagonist antibody).As used herein “binding activity” refers to an affinity of at least KD < 1 O'6M or < 1 x 1 O’7M. In yet another embodiment, the targeting moiety7of the invention binds with a KD of < 5 x 10’7M. In another embodiment, the targeting moiety of the invention binds the marker with a KD of < 1 x 10'8M. In certain embodiments, the KD is < x 10’9M or < 5 x IO10M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 10’6M and > 5 x 10’15M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 10'8M and > 5 x !0’15M.According to a specific embodiment, the binding is characterized by a KD of < 1 O’9M and > 5 x 10’14M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 1 O'7M and > 5 x 10’15M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 10’8M and > 5 x 10’15M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 1 O'9M and > 5 x 10’12M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 10’6M and > 5 x 10’12M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 10'7M and > 5 x 10’12M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 10'8M and > 5 x 10’12M.According to a specific embodiment, the binding is characterized by a KD of < 5 x 10’9M and > 5 x 10’12M.[000294] Affinity can be determined by a surface plasmon resonance (SPR) assay (see exemplary conditions described in the Examples section).[000295] According to a specific activity the targeting moiety is a ligand of the marker (or a binding domain thereof to the marker) or an antibody.[000296] According to a specific embodiment, the targeting moiety is an antibody.[000297] 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).[000298] 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. [000299] 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 VL that are capable of binding to an epitope of the antigen in an HLA restricted manner.[000300] 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.[000301 ] 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).[000302] 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'sAbM 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).[000303] 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.[000304] Functional antibody fragments comprising whole or essentially whole variable regions of both light and heavy chains are defined as follows:(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;(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.(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.(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;(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);(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);(vii) Single domain antibodies or nanobodies are composed of a single VH or VL domains which exhibit sufficient affinity to the antigen; and(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.[000305] 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).[000306] Exemplary7methods 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. 80:2026-2030; Cole SP. et al., 1984. Mol. Cell. Biol. 62: 109-120).[000307] 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. 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 light-heavy chain fragments, further cleavage of fragments, or other enzy matic, chemical, or genetic techniques may also be used, so long as the fragments bind to the antigen that is recognized by the intact antibody.[000308] As described hereinabove, Fv fragments comprise an association of VH andVL chains. This association may be noncovalent, as described in Inbar et al. [Proc. Nat' 1 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 el al.. Bio / Technology 11 : 1271-77 (1993); and U.S. Pat. No. 4,946,778, which is hereby incorporated by reference in its entirety.[000309] Another form of an antibody fragment is a peptide coding for a single complementarity-determining 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)].[000310] 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.[00031 1 ] Humanized forms of non-human (e.g., murine) antibodies are chimeric molecules of immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab1, 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, Cun. Op. Struct. Biol., 2:593-596 (1992)]. In some embodiments, the antibody is an humanized version of the antibody 80E3C7, as described in PCT / US24 / 22120, incorporated herein by reference in its entirety.[000312] According to a specific embodiment, the marker is selected from the group consisting of TREM2. GPNMB. L1LRB1 / 1LT2, L1LRB2 / 1LT4, LILRB4. PDL1, PDL2, CLEVER1, SIRPA, SIGLEC-1, SIGLEC-7, SIGLEC-9, SIGLEC-10, SIGLEC-15, SLAMF7, MRC1 / CD206 , LGALS9 / Galectin-9, VSIG-4 and MARCO.[000313] These markers are known for their checkpoint activity in MDSCs.[000314] According to another embodiment the marker is not endowed with a checkpoint activity e.g., CSF1R.[000315] According to a specific embodiment, the marker is TREM2.[000316] Antibodies to TREM2 which can be utilized according to some embodiments of the invention are described in WO2021152592 and W02023 / 012802, each of which is incorporated herein by reference in its entirety.[000317] According to a specific embodiment, the antibody has the sequence of 80E3C7 of SEQ ID NO: 138 / 139 and 145 / 146, with or without the constant regions, respectively.[000318] Antibodies to GPNMB which can be utilized according to some embodiments of the invention are described in WO2021152592 and W02023 / 012802. each of which is incorporated herein by reference in its entirety.[000319] Antibodies to LILRB1 are described in US 2021 / 0122819, which is incorporated herein by reference in its entirety.[000320] Antibodies to LILRB2 or LILRB4 are described in TWI833724 and KR102129107, each of which is incorporated herein by reference in its entirety.[000321 ] Antibodies to PDL-1 or PDL-2 are described in JP17164217, JP2023099088 and KR102138447, AU2018202800S, each of which is incorporated herein by reference in its entirety. [000322] Antibodies to SIRPA are described in US 11319373, which is incorporated herein by reference in its entirety.[000323] Antibodies to SIGLEC proteins which can be utilized according to some embodiments of the invention are described in US20210253695, JP2022409920, CN20210253695, HK125696. EP2875051, IL264674, each of which is incorporated herein by reference in its entirety.[000324] Antibodies to SLAMF7 are described in Chu et al. Curr Oncol. 2023 Sep: 30(9): 7891-7903, which is incorporated herein by reference in its entirety.[000325] Antibodies to MRC1 are described in ES2831852T3, which is incorporated herein by reference in its entirety.[000326] Antibodies to Galectin-9 are described in Zhang et al. Asian Pac J Cancer Prev APJCP, 13 (6) (2012), pp. 2503-2509, which is incorporated herein by reference in its entirety.[000327] Various activity assays can be done to determine antagonistic activity of the targeting moiety. For example: FACS for phenotypical markers; ELISA soluble mediators as indicators for myeloid reprogramming, including shift from anti- towards pro-inflammatory cytokines; Single-cell analysis for gene expression profiling combined with machine learning analysis to define reprograming activity; Macrophage suppression assay, including co-culture with effector immune cells, such as T cells.[000328] The ligand can be naturally occurring or a soluble binding domain of a molecule which binds the marker.[000329] Ligands which can be used as the targeting moiety include the soluble form of PD- 1 for PDL1.[000330] Major histocompatibility complex (MHC) class I subunit 02-microglobulin (B2M) for LILRB1.[000331 ] HLA class I molecules, CDld, Angptls, myelin inhibitors (including Nogo66, MAG, and OMgp 23), or 0-amyloid for LILRB2.[000332] Galectin-8 for LILRB4.[000333] SPARC (secretory protein, acidic, and rich in cysteine), placental lactogen and oxidized, or acetylated low-density lipoprotein for CLEVER1.[000334] CD47 functions as a ligand for signal regulatory protein-a (SIRPa).[000335] For SIGLEC proteins the following can be used:[000336] SIGLEC-1 : Sialylated glycans, specifically sialylated glycoproteins such as MUC1, PSGL-1, and various glycosylated pathogens.[000337] SIGLEC-7: a8, a3-linked disialic acids, gangliosides (e.g., GD3, GTlb), and sialylated glycoproteins.[000338] SIGLEC-9: a3, a4, S6, a.8, a.33 sialylated glycans, particularly a2,3-linked sialic acids and certain gangliosides (e.g., 9-O-acetyl GD3).[000339] SIGLEC-10: a6, 04 Sialylated glycans, CD24, and sialyl-Tn antigen.[000340] SIGLEC-15: Sialylated glycoproteins and glycolipids.[000341 ] SLAMF7 functions as the ligand for SLAMF7.[000342] CD206 have multiple ligands, e.g., sulphated N-Acetylgalactosamine ligand.[000343] Polyanions bind MARCO.[000344] When the ligand is not a protein, another proteinaceous binding moiety may be preferred (e.g., antibody), though synthetic chemistry can be used to prepare the fusion between a chemical moiety (e.g., polyanion) and a protein (e.g., cytokine).[000345] As used herein “immune activating cytokine” refers to a cytokine which activates effector immune cells such as lymphoid and myeloid cells.[000346] 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 modality7. 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.[000347] 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 immunogenicity7by 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.[000348] Methods of determining cytokine activity are provided hereinbelow in the Examples section which follows and include the CD 122 / CD 132 assay, which tests the activation of the JAK / STAT pathway controlled by IL-2 in dedicated cells. One such assay is the HEK- blue™ assay, available for testing activation of various types of cytokines.[000349] According to a specific embodiment, the cytokine is selected from the group consisting of Interleukin-2 (IL-2), Interferon-alpha (IFN-a), Granulocyte-Macrophage Colony- Stimulating Factor (GM-CSF), Interleukin- 12 (IL-12), Tumor Necrosis Factor-alpha (TNF-a), Interferon-gamma (IFN-y), Interleukin- 15 (IL-15), Interleukin- 18 (IL-18), Interleukin-21 (IL-21) and Granulocyte Colony-Stimulating Factor (G-CSF) or variants of these cytokines with enhanced receptor selectivity that preferentially stimulates effector immune cells.[000350] According to a specific embodiment, the cytokine is human Interleukin-2(IL-2) or the IL2-superkine (H9) (SEQ ID NO: 149, referred to as 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, reducing the interaction of the trimeric IL-2 receptor complex that is mainly expressed by regulator}' T cells.[000351 ] According to a specific embodiment, a mutein cytokine is contemplated(e.g., IL-2) engineered to have selective binding to activate effector T cells and natural killer (NK) cells without activating regulator}’ T cells (Tregs). which is desirable in cancer immunotherapy (see e g., 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 tumorinfiltrating 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).[000352] A “decoy-resistant” IL18DR 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.)[000353] IL15 fused to IL-15RA to enhance half-life (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.)[000354] Low affinity, cis-acting IL-2 which selectively targets tumor-infiltrating immune cells (Ren Z, Zhang A, Sun Z, et al. Selective deliver}' 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).[000355] 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)l 038 / 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 FIG. 8. The two subunits are splitbetween the 2 chains of the heterodimer and the masking agent is attached to one of them (in this case the p35).[000356] The composition may include a plurality of cytokines (see e.g., variants 1 and 2 of FIG. 8) which can be identical or different. If different cytokines are used, then ty pically different masking agents are used. Though a single masking agent can be for different cytokines.[000357] 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 and NKT) and NK cells.[000358] Since cytokine therapy is endowed with some disadvantages, the present inventors envisage rendering the fusion conditionally active only in the vicinity of the target cell (myeloid cell in the TME). Thus, the fusion comprises a masking agent which is conjugated to the cytokine to mask its activity.[000359] 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 fusion, sterically interfere with its activity and / or change the conformation of the cytokine.[000360] The fusion protein comprises a cleavable moiety which ensures that the masking agent is released from the cytokine (to ensure its activity ) in the TME via an activity' of an MSDC- specific protease so as to allow cytokine activity’ of the immune cell activating cytokine.[000361 ] Since the protease is one that is enriched in the tumor microenvironment and preferably clusters with the myeloid cells (e g., TAM, see Figures 6 and 7), there is a reduction of the cytokine in normal tissues when administered systemically, compared to the systemic administration of the unmasked cytokine. As shown in the Examples section which follows, the unmasked fusion (that does not include the masking agent) is toxic (see FIG. 5), while the variants which include the reversibly conjugated masking agent are safer (Figures 16 and 17).[000362] 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).[000363] 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 (see e.g., Mansurov, A. et al. 2022, Nat. Biomed. Eng. 6, 819-829).[000364] W02021016640 describes a domain of the IL12 receptor IL12Rbl which was fused to the IL 12, to form IL12Rbl - IL 12. 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.[000365] According to a specific embodiment, the cytokine comprises an IL 15 polypeptide and the masking agent comprises at least one binding domain to IL 15 in the IL15 receptor (see e.g., Guo et al. 2021 Cell Res 31, 1190-1198).[000366] According to a specific embodiment, the cytokine comprises an IL 15, stabilized by IL- 15 receptor alpha, and the masking agent comprises IL2RB, to form a complex.[000367] According to a specific embodiment, the cytokine comprises an IL 18 polypeptide and the masking agent comprises at least the binding domain to IL18 in the IL18 receptor. 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.[000368] According to a specific embodiment, the cytokine comprises an IL21 polypeptide and the masking agent comprises at least the binding domain to IL21 in the IL21 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).[000369] 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.[000370] 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.[000371 ] 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.[000372] 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).[000373] 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).[000374] The cytokine is (reversibly) conjugated to the masking agent via at least one cleavable sequence of at least one protease specific to an MDSC.[000375] An “MDSC-specific protease cleavage site” refers to a cleavage site (amino acid sequence) that is recognized by a protease that is highly upregulated or enriched in the tumor microenvironment (e.g., MDSC). It may not be MDSC-specific, meaning that the protease is onlyexpressed by the MDSC, it is MDSC-enriched, meaning that the protease is expressed by the MDSC at a level higher than normal tissues or most normal tissues.[000376] According to a specific embodiment, the cleavable sequence is of a matrix metalloprotease (MMP). For example, the tumor-associated protease cleavage site may be one that is cleaved by MMP1, MMP2. MMP3, MMP7, MMP8. MMP9, MMP 10, MMP11, MMP12, MMP13. MMP14. MMP15. MMP16. MMP17. MMP18. MMP19. MMP20. MMP21. MMP23A, MMP23B, MMP24, MMP25, MMP26, MMP27, MMP28, or combinations thereof.[000377] According to a specific embodiment, the MMP cleavage site is of MMP14 (e.g., SGRSENIRTA, SEQ ID NO: 107. other sequences are listed below and in Table 1).[000378] According to other specific embodiments the MMP 14 cleavage sites are as set forth in SEQ ID NO: 150-155. It will be appreciated that some are recognized by other MMPs, such as MMP 2 and MMP9 e.g., 152-155.[000379] 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.[000380] Other examples of proteases 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. KLK.4. KLK.5), 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.[000381 ] Table 1 below provides some examples of proteases and their cleavage sites.Table 1[000382] According to a specific embodiment, the fusion includes a single cleavage site (e.g.,MiTE-144 of FIG. 8).[000383] According to a specific embodiment, the fusion includes multiple cleavage sites (e g., 2, 3, 4, 5, 6, e g., variants MiTE-76, MiTE-95 and MiTE-208 of FIG. 8).[000384] The multiple cleavage sites may be identical or different (to improve the regulation on the conditional unmasking, releasing of the masking agent).[000385] As mentioned, the fusion may comprise a single masking agent or a plurality of masking agents (MiTE-76 and MiTE-95 of FIG. 8). [000386] The masking agents can be identical or different.[000387] The attachment of the targeting moiety to the cytokine can be directly or via a linker.[000388] 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.[000389] 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: 148) or (GGGGS)n (SEQ ID NO: 106), 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). According to a specific embodiment, the linker is (GGGGS)3 (SEQ ID NO: 135) and when comprises the cleavage site is GGGGSSGRSENIRTAGGGGS (SEQ ID NO: 95).[000390] According to a specific embodiment, the different segments are arranged in an N>C orientation of: targeting moiety>[linker]>cytokine>[linker]cleavage sequence>masking agent, where [] is optional. Such a configuration may be homodimeric.[000391 ] 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>masking agent.[000392] Also provided herein is a polynucleotide which encodes at least portions of the fusion polypeptide.[000393] The translation products can then be assembled chemically.[000394] Thus, for example, the cytokine may be attached to the targeting moiety (e.g., 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.[000395] Exemplary methods for conjugating peptide moieties are described herein below:[000396] SPDP conjugation - A non-limiting example of a method of SPDP conjugation is described in Cumber et al. (1985. Methods of Enzymology 1 12: 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 rnM 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.[000397] 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 Mlysine 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)[000398] 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 carboxyl group of 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)].[000399] Alternatively, the fusion polypeptide 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.[000400] 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, in MiTE-144 or MiTE-208.[000401 ] Other methods also mostly adapted from antibodies engineering may include, but not limited to:[000402] 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.[000403] Dual -Variable Domain (DVD) Fab: A method where two variable domains are fused in tandem on each Fab arm, preventing heavy chain mispairing.[000404] 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.[000405] 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.[000406] 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 sequences (e.g., a combination of the above). Modifications can be included to improve bioavailability7especially when the product is an mRNA.[000407] 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 for directing transcription of the polynucleotide sequence in the cell in a constitutive or inducible manner.[000408] The person of skills in the art would know which vector to select, conditions for isolation and purification and / or which chemistry to employ.[000409] Once the fusion protein is at hand its activity7on tumor grow th, cytokine signaling, delivery7to MDSCs and effect of same can be determined.[000410] The in vivo effect on tumors (e.g.. biopsy) can also be evaluated especially when used in therapeutic applications to customize therapy, i.e., personalized therapy.[00041 1 ] The 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.[000412] 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 phy siologically suitable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate administration of a compound to an organism.[000413] Herein the term "active ingredient" refers to the fusion polypeptide accountable for the biological effect.[000414] 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 activity7and properties of the administered compound. An adjuvant is included under these phrases.[000415] 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 ty pes of starch, cellulose derivatives, gelatin, vegetable oils and polyethylene glycols.[000416] 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.[000417] Suitable routes of administration may, for example, include oral, rectal, transmucosal, especially transnasal, intestinal or parenteral delivery, including intramuscular, subcutaneous and intramedullary 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.[000418] 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 artery7or 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.[000419] 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.[000420] 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.[000421 ] 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.[000422] 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 active ingredients into preparations which, can be used pharmaceutically. Proper formulation is dependent upon the route of administration chosen.[000423] 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.[000424] 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, fdlers 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.[000425] 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.[000426] 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.[000427] For buccal administration, the compositions may take the form of tablets or lozenges formulated in conventional manner.[000428] 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.[000429] 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.[000430] 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.[000431 ] 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.[000432] 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.[000433] 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) effective to prevent, alleviate or ameliorate symptoms of a disorder (e.g., cancer) or prolong the survival of the subject being treated.[000434] 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. [000435] 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.[000436] 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).[000437] 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.[000438] 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. [000439] The amount of a composition to be administered will, of course, be dependent on the subject being treated, the severity7of the affliction, the manner of administration, the judgment of the prescribing physician, etc.[000440] 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, comprisemetal 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 agency of 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.[000441 ] The compositions described herein can be used in the treatment of cancer.[000442] Thus, 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 pharmaceutical composition as described herein, thereby treating cancer[000443] Alternatively or additionally, there is provided the pharmaceutical composition as described herein for use in treating cancer.[000444] As used herein “subject” refers to a mammal, e.g., human, diagnosed with cancer.[000445] The terms "cancer" and "cancerous" refer to or describe the physiological condition in mammals that is typically characterized by unregulated malignant cell growth.[000446] Examples of cancers that can be analyzed and 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 breastcancer, 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, non-Hodgkin'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, ery throleukemia (e.g., Friend, lymphoblast), fibrosarcoma, giant cell tumor, glial tumor, glioblastoma (e.g., multiforme, astrocytoma), glioma hepatoma, heterohybridoma, heteromyeloma, histiocytoma, hybridoma [000447] (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, pituitary7tumor (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. [000448] According to a specific embodiment, the cancer is melanoma.[000449] According to a specific embodiment, the cancer is a solid tumor (e.g.. lung cancer, liver cancer, ovarian cancer, gastric cancer and breast cancer).[000450] According to a specific embodiment, the cancer is a primary7tumor.[000451 ] According to a specific embodiment, the cancer is metastatic.[000452] According to a specific embodiment, the cancer is a secondary tumor.[000453] According to a specific embodiment, the lung cancer is non-small cell lung cancer.[000454] According to a specific embodiment, the lung cancer is small cell lung cancer.[000455] According to a specific embodiment, the liver cancer is Hepatocellular carcinoma.[000456] According to a specific embodiment, the cancer is MHC-1 independent.[000457] As used herein| MHC-I independent” refer to a cancer cells which either do not express MHC-1 or express anon-functional form of MHC-I (i.e., mutated MHC-1). Such a cancer is expected 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.[000458] 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.[000459] The present findings as presented in Figures 19A-C shows that unlike ICB or T cell focused immunocytokines, TREM2-immunocytokine affects deeper and broader, more potent immune arms. The fusion polypeptide functions in a trans based mechanism, and shows a very dramatic effect on NK and NKT cells in the TME following TREM2-immunocytokine (30 fold more potent activity of the NK / NKT arm than standard ICB). While both TREM2- immunocytokine and ICB activate the T cell arm the present data in vivo demonstrates that NK / NKT cells are responding much more potently than CD8 and brings the largest efficacy tumor killing effects. Importantly the NK cells are not only not inhibited by MHC-I loss but rather get activated by such signals and therefore can be very effective even in PD-1 unresponsive tumors including potential patients with mutations in the MHC-I pathway.[000460] Also contemplated herein are combinations with other anti cancer treatments.[000461 ] Treatment can be combined with any anti-cancer treatment known in the art, including, but not limited to, chemotherapeutic agents, radiotherapeutic 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.[000462] 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 folicacid analogs such as methotrexate and trimetrexate, pyrimidine analogs such as 5-fluorouracil, fluorodeoxy uridine, gemcitabine, cytosine arabinoside (AraC, cytarabine ), 5-azacytidine, 2,2 •difluorodeoxycytidine, purine analogs such as 6-mercaptopurine, 6-thioguanine, azathioprine, 2 '-deoxycoformycin (pentostatin), erythrohydroxynonyladeninc (EHNA), fludarabine phosphate, and 2-chlorodeoxyadenosine (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.[000463] 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.[000464] According to a specific embodiment, the treatment is combined with an immune checkpoint blockers or inhibitors, such as described below.[000465] As used herein “immune checkpoint inhibition” refers to cancer immunotherapy. The therapy targets immune checkpoints, key regulators of the immune system that stimulate or inhibit its actions, which tumors can use to protect themselves from attacks by the immune system. Checkpoint therapy can block inhibitory checkpoints, activate stimulatory functions, thereby restoring immune system function. Currently approved checkpoint inhibitors target the molecules CTLA4, PD-1, and PD-L1. PD-1 is the transmembrane programmed cell death 1 protein (also called PDCD1 and CD279), which interacts with PD-L1 (PD-1 ligand 1, or CD274).[000466] Examples of immune checkpoint inhibitors include, but are not limited to. 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-containing protein 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, CDllb, Ly6G, ICAM-1, FAP, PDGFR, Podoplanin, and TIGIT.[000467] 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). [000468] According to a specific embodiment, the immune checkpoint is CTLA-4 or TIGIT and contemplated are inhibitors thereto for depletion of Treg activity.[000469] According to another embodiment, the treatment is combined with a Brutons tyrosine kinase (Btk) inhibitor (e.g. ibrutinib, acalabrutinib or Spebrutinib).[000470] According to a specific embodiment, contemplated herein is the combination of the fusion polypeptide with inhibition of Treg cells in the tumor. For example, the cytokine portion of the fusion is designed to bind less effectively to Treg cytokine receptors (e.g. Il-2superkine binds with much lower affinity to the trimeric receptor complex of IL2RA(CD25), IL2RB and IL2RG found on Tregs, as compared to effector Tcells NK / NKT cells) however Tregs will be affected bynative IL2 signaling and therefore boosted in the TME. Therefore, activated CD8 and NK. cells proliferate, utilizing the IL-2Superkine, and due to their proliferation, these cells produce wildtype IL-2 which then can bind to Tregs. Hence a synergy with anti-CTLA4 and anti-TIGIT depleting molecules is expected.[000471 ] Thus, according to a specific embodiment, the cancer is PD-1 -resistant.[000472] Additionally, molecules which act to block NK / NK.T 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.[000473] The present inventors also contemplate selecting a treatment type based on the presence of myeloid cells which express the marker and / or immune checkpoint molecules. Such methods are described in details in W02023 / 012802 and in WO2021152592.[000474] As used herein the term “about” refers to ± 10 %.[000475] The terms "comprises", "comprising", "includes", "including", “having” and their conjugates mean "including but not limited to".[000476] The term “consisting of’ means “including and limited to”.[000477] 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, steps and / or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.[000478] 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.[000479] 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 brevity7and 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.[000480] 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 / rangesbetween’7a 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.[000481 ] 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.[000482] 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.[000483] 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 500 nucleotides, alternatively, less than 1 in 1000 nucleotides, alternatively, less than 1 in 5,000 nucleotides, alternatively, less than 1 in 10,000 nucleotides.[000484] 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.[000485] In one embodiment, the invention provides a novel myeloid-targeted immunocytokine prodrug strategy with three key features: (1) effective TME reprogramming and TREM2+ macrophage antagonism. (2) cytokine activity masked through a blocking domain cleaved by TAM-specific protease within the TME, and (3) / ram-based mechanism allows to stimulate both innate and adaptive immunity.[000486] Immunocytokines direct cytokine activity to specific cell subsets or lineages. One embodiment of the invention is to couple cytokines to antibodies against tumor antigens75-77to localize cytokines, serving as scaffolds without intrinsic immunomodulatory effects. Another embodiment of the invention is to directly stimulate immune cells in cis (eg. anti-CD45-IL-1514)and enhance their anti -tumor efficacy. By engaging T cells, immunocytokines such as anti-PD-1- IL-27’19, anti-TCRb-IL-28, anti-41BB-IL-2nor anti-CD8-IL-215can enhance immune responses against cancer by promoting proliferation, activation, and persistence of cytotoxic T lymphocytes. In one embodiment, the cytokines are IL-2, IL-12, or IL-15, often fused to T cell-targeted ICIs, enabling synergy with ICI action. The disclosure provides that targeting the immunosuppressive myeloid compartment of the TME is crucial for durable anti-tumor immunity. Accordingly, the MiTE platform provided in this disclosure enables reprogramming of suppressive macrophages, disrupting pro-tumor networks and promoting a more favorable TME for immune activation. While the anti-TREM2 arm of MiTEs primarily reprograms TAMs, the strong activation of T and NK cells may also contribute to macrophage depletion, as indicated by reduced macrophage frequencies and a shift toward inflammatory phenotypes indicating earlier monocytic and inflammatory macrophages.[000487] In one embodiment, by integrating IL-2SK-mediated immune activation in trans, MiTEs offer a more comprehensive anti-tumor effect than cv.s-delivered immunocytokines. The MiTEs of this disclosure broadly reactivate intratumoral T cells, shifting dysfunctional T cell signatures towards stem-like T cell programs. Although IL-2SK was designed to enhance effector T cell expansion over Treg activation, the inventors observed increased Treg frequencies in the TME and lymph nodes, likely due to both direct and indirect effects. Treg depletion by anti-CTLA- 4 boosted CD8+ T cell responses and impacted macrophages via FcyR signaling71, suggesting additional therapeutic benefit.[000488] While the systemic activation of NK cells can be associated with toxicities98, the tumor-localized IL-2SK activity in trans in MiTEs may unlock NK cell potential without off-target effects, overcoming MHC-I-mediated immune escape mechanisms. Moreover, the inventors’ IL- 2 masking strategy may reduce anti-drug antibody (ADA) formation by keeping IL-2 in an inactive state until it reaches the TME. The low expression of TREM2 in healthy organs, primarily the brain, liver, and lungs36, may explain the absence of pathological changes in these tissues following MiTE144 treatment, highlighting the specificity of the inventors’ TAM-targeted, protease- activated approach.[000489] The disclosure also provides that single-cell transcriptomic and proteomic profiling of patient-derived tumor fragments from five renal cell carcinomas revealed that MiTE144 selectively activated and expanded effector CD8+T cells and NK cells, inducing transcriptional programs associated with cytotoxicity, proliferation, and cytokine responsiveness, while sparing Tregs. Some of these effects were further enhanced by anti-PD-1 co-treatment and were reflected at the protein level by upregulation of IL-2 receptor components and activation markers. Thedisclosure also provides that the use of PDTFs from multiple patients provides direct evidence of MiTE144 immunomodulatory activity in a human tumor context, underscoring its translational potential. Accordingly, the conserved response across patients and species supports further clinical development of MiTE144 as a rational combination partner for immune checkpoint blockade in solid tumors.[000490] In one embodiment, the disclosure provides that MiTEs represent a new class of myeloid-targeted immunocytokines, setting itself apart from previously reported immunocytokines and conditionally activated cytokines. Accordingly, in one embodiment, targeting immunosuppressive TAMs and integrating TME-restricted cytokine activity in trans resulting in dual innate and adaptive immune stimulation, MiTEs may overcome key resistance mechanisms and improve therapeutic efficacy in otherwise refractory tumors.[000491 ] 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.[000492] 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.EXAMPLES[000493] 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. The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the disclosure, and are not intended to limit the scope of what the inventors regard as their disclosure. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Centigrade, and pressure is at or near atmospheric.MATERIALS AND METHODSCell linesMCA-205 fibrosarcoma cells were kindly provided by the Sergio Quezada group (UCL Cancer Institute, London, UK) and maintained in DMEM (Thermo Fisher Scientific) supplemented with 10% heat-inactivated FBS, 1 mM sodium pyruvate and 2 mM L-glutamine. MC38 murine colon adenocarcinoma were cultured in RPMI-1640 medium (Gibco) supplemented with 10 mM HEPES, 2 mM L-glutamine, 10% heat-inactivated FBS, 1 mM sodium pyruvate and 1 x non-essential amino acids. Jurkat cells were maintained in RPMI with 10% FBS. HEK293 parental cells were cultured in DMEM supplemented with 10% FBS, while HEK-Blue CD122 / CD132 cells were maintained in DMEM supplemented with 2 mM L-glutamine, 10% heat-inactivated FBS and 100 pg / rnl Normocin. All cell lines were grown in tissue culture plates under standard conditions (37 °C, 5% CO2, humidified atmosphere). Human TREM2-expressing cell lines were established via viral infection with a pLV[Exp]-Neo-EFl A construct carrying cDNAs for TREM2 (NM_018965.4) and TYROBP / DAP12 (NM 003332.4).Mouse modelsFor all experiments, male and female (6-16 w eeks old) hTREM2 transgenic (Song WM, Joshita S, Zhou Y, Ulland TK, Gilfillan S, Colonna M. Humanized TREM2 mice reveal microglia-intrinsic and -extrinsic effects of R47H polymorphism. J Exp Med. 2018;215(3):745-760), Trem2-knockout (KO) or wildtype (WT) mice (C57BL / 6) (Harlan) were housed in the Weizmann Institute animal facility under pathogen-free conditions and 12-h light / 12-h dark cycle. Food and water were provided ad libitum. All experiments were conducted as approved by the Institutional Animal Care and Use Committee. MC38 (IxlO6) or MCA205 (5xl05) cells were subcutaneously injected. Tumor volumes were measured twice per week up to daily using a caliper gauge. Weight of mice was measured twice per week up to daily. After the tumors were established and a specific tumor volume reached, mice were treated intravenously or intratumorally with indicated constructs. Details on each experiment can be found in the figure legends. Tumor bearing mice were sacrificed <16 days post tumor implantation. Specific timepoints are indicated in the respective figure legends.Bone marrow^ derived macrophages (BMDMs)Mouse bone marrow cells of Trem2 knockout (KO) and hTREM2 transgenic (hTREM2) mice w ere harvested and isolated from femora and tibiae of two legs to generate bone marrow derived macrophage cells (BMDM). Bones were cleaned of surrounding tissue and flushed with CIO culture medium (RPMI-1640 supplemented with 15% serum, 1% non-essential amino acids, 10 mM HEPES buffer, 1 mM sodium pyruvate, 2 mM L-glutamine. and 50 pM P-mercaptoethanol) using a G21 syringe. The cell suspension was passed through a 70-pm strainer and centrifuged at 300 x g for 5 min at 4 °C. Pellets were resuspended in 250 pl RBC lysis solution (Sigma) per leg for 5 min at room temperature, washed, and resuspended in pre-warmed CIO medium. Cultures were plated on day 0 by plating 2 * 106cells in 10 ml CIO in 100-mm non-tissue culture plates under standard conditions (37 °C, 5% CO2). The culture medium was replaced on days 5 and 6 with C IO supplemented with 30 ng / ml human M-CSF (PeproTech) and 20 ng / ml murine IL-4 (PeproTech). On day 7, cells were detached using Accutase (Sigma-Aldrich) and collected for scRNA-seq.Isolation of human PBMCsPeripheral blood mononuclear cells (PBMCs) were isolated from fresh blood collected from a healthy donor (IRB approval: 2448-2) using sterile density gradient centrifugation with Ficoll- Paque.Human tissue sectionsFormalin-fixed, paraffin-embedded (FFPE) human tissue sections were obtained from the Hadassah Medical Center (IRB approval: HMO-0235-21 / All procedures were conducted in accordance with the Declaration of Helsinki, and written informed consent was obtained from all participants.Patient-derived tumor fragments (PDTFs)Fresh tumor samples were obtained from discarded surgical resection specimens collected from consented patients under a Cooperative Human Tissue Network-CHTN IRB approved protocol. Tumor samples were cut into l-2mm3Patient-Derived Tumor Fragments (PDTFs) and cryopreserved.Immunocvtokine and antibody productionsThe 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 HD 293F cell expression. HD 293F 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 HD 293F 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). Recombinant antibodies were generated by GenScript (Nanjing, China).Protease digestion assayTo assess the cleavage of blocking domain by MMP-14 and detect the MMP-14 dependent activity of IL-2SK in MiTEs in vitro, 2pM MiTEs and aTREM2-IL2SK controls were co-cultured with 0.75pM active rhMMP14 (human MMP-14 catalytic domain gene (residues 112-292). a gift from Prof. Irit Sagi) in an assay buffer [50mM Tris, 150mM NaCl, 0.01% Brij-35, 5mM CaC12 (pH 7.5)] at 37°C overnight for 12h. As control, assay buffer without rhMMP14 was used. The overnight digestion was followed by SDS page, a HEK blue reporter assays as described above or in vitro NK or T cell proliferation assay described below.Surface Plasmon Resonance (SPR)Affinity measurement of our immunocytokines and control antibodies were obtained by SPR, carried out on BIAcore T200 instrument (Cytiva), with Series S Sensor Chip CM5 (Cytiva). For one part of the experiments to assess the binding properties of anti-hTREM2 antibodies, hTrem2 protein (Sino Biological) was captured to the chip, and anti-hTREM2 antibodies were used as theanalyte. For other experiments to define the binding of IL-2SK to IL-2R0, hTrem2 protein and bound MiTEs were captured to the chip, and soluble recombinant IL2RP (Sino Biological) was the analyte. Sensograms were fit using steady-state affinity binding to provide equilibrium dissociation constant (KD) values.SDS-PAGE and Western BlottingMiTE144 (2 pM) was incubated in digestion buffer (50 mM Tris, 150 mM Nad, 5 mM Cad?, 0.01% Brij, pH 7.5) with 0.75 pM of MMP3, MMP7, MMP10, MMP11 (MMP Multipack. Enzo), or MMP14 (Enzo). A no-enzyme control was included. Samples were incubated at 37°C for 30 min, 2 h, or 20 h. At each timepoint, samples were mixed 1 : 1 with 2* Laemmli buffer containing 0.71 pM 2-mercaptoethanol. boiled for 5 min at 95°C, and analyzed by SDS-PAGE (4-20%. BioRad) in Tris / Glycine / SDS buffer. Gels were transferred to 0.2 pm nitrocellulose membranes using the Trans-Blot Turbo system and blocked in 5% milk in PBS-T for 45 min. Blots were probed with mouse anti-human IgG (1 :5,000, Jackson ImmunoResearch), followed by HRP-conjugated antimouse secondary (1: 10,000. Jackson ImmunoResearch), each for 45 min in 5% milk in PBS-T with three PBS-T washes in between. Detection was performed with chemiluminescent substrate (Thermo Fisher) and imaged on a Bio-Rad MP ChemiDoc system. Images were processed using ImageLab.Direct ELISA96-Well ELISA microplate was coated lOOpl / well (0.5pg / mL) recombinant hTrem2 (Sino Biological) 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 hr, and rinsed again. The plates was incubated with the anti-TREM2 antibodies (100 pl / well) for 2 hrs at indicated concentration at RT. Plate was rinsed and incubated with Peroxidase-AffiniPure Goat Anti-Mouse IgG, Fey Fragment Specific (Jackson ImmunoResearch) or a Peroxidase AffmiPure Goat Anti -Human IgG, Fey fragment (Jackson ImmunoResearch) for 20 min at RT. The plate was rinsed and incubated with TMB Reagent (Scytek) for 20 min at RT. followed by the addition of Stop Solution 2N Sulfuric Acid (R&D). OD was measured using SpectraMax Plus spectrophotometer (Molecular Devices) at dual wavelengths (450 nm and 570 nm).Competitive Binding Assay with Soluble TREM2Jurkat-TREM2 cells were cultured in RPMI with 10% FBS and seeded at 50,000 cells / well in a 96-well plate with stain buffer (BD). Cells were washed and incubated with serial dilutions (1 :3) of His-tagged TREM2 (Sino Biological), ranging from 0.293 M to 1.65* 10~6M.Anti-TREM2 was then added directly to the wells and incubated for 30 minutes at RT. Next, cells were washed and stained with Alexa Fluor 647 anti-human IgG (1:2000, Jackson ImmunoResearch) for 15 min, fixed with 150 pl fixation buffer (BioLegend), and analyzed on the Aurora (Cytek Bio). Binding was quantified by MFI in FlowJo.Flow cytometry analysis of anti-TREM2 antibodies in HEK cells200K HEK parental and HEK cells overexpressing human TREM2 were incubated indicated concentrations (serially diluted tenfold) with anti-RSV, anti-RSV-IL-2SK, anti-TREM2, or anti-TREM2-IL-2SK antibodies in 1 OO .1 DMEM (10% FBS) at 37 degrees for 30 min. Next, the cells were washed four times with MACS buffer (PBS pH 7.2, 0.5% BSA and 2 mM EDTA) and stained with PE-conjugated anti-human IgG antibodies (BD) or PE-conjugated anti-mouse IgG antibodies (BioLegend) in MACS buffer (30min. on ice. in the dark). After washing, cells were analyzed on a flow cytometer (S6 Symphony, BD). MFI of PE was assessed using FlowJO (version 10).HEK-Blue CD 122 / CD 132 reporter assaysHEK-Blue CD122 / CD132 reporter cell line was purchased from InvivoGen and the assay was performed according to manufacturer’s instructions. These cells were generated by stably introducing the human genes of the CD122 (IL-2RP) / CD132 (IL-2Ry) receptor and signaling cascade through tyrosine kinases of the Janus family (JAK1 and JAK3) and signal transducer and transcription activator 5 (STAT5) into HEK293 cells. For detecting IL-2SK biological activity, HEK-Blue CD122 / CD132 reporter cells were incubated overnight at 37°C with indicated antibodies (MiTEs, aTREM2-IL2SK and aTREM2) serially diluted tenfold. For the dual binding assay, HEK cells overexpressing hTREM2 (Effector(E)) were incubated with InM of each indicated antibody for 30min at 37°C, washed four times with PBS and added to HEK Blue CD122 / CD132 reporter cells (Target (T)) at various ratios. The levels of secreted embryonic alkaline phosphatase (SEAP) in cell culture supernatants were determined using SpectraMax Plus spectrophotometer (Molecular Devices) at 630 to 650 nm.Toxicity studies, pathology and serum isolationTumor-bearing mice were treated with 200pg per dose on days 8 and 10 after tumor inoculation. Tissues w ere extracted lOh after the second treatment. Tissues w ere fixed in 4% formaldehyde for 2 days before being embedded, sectioned, and processed for H&E staining. Naive WT mice were treated with either 50pg or 200pg two days apart. Blood was collected via facial vain, incubated at room temperature for 30min allowing the blood to clot and centrifuged for 10 min at 1500g. Serum w as collected from WT and tumor-bearing mice at defined time points: 24 hours after the first treatment for IFN-y and IL-2 quantification. 48 hours after the first treatment for IL-6 measurement, and 10 hours after the second treatment for AST / ALT analysis, using either the Roche Diagnostics cobas c 111 Chemistry Analyzer or ELISA. For pharmacokinetic analysis, serum was collected at the indicated time points, as detailed below.Pharmacokinetics and Biodistribution AnalysisPharmacokinetics of MiTE144 and aTREM2-IL2SK were evaluated in naive C57BL / 6 mice following a single intravenous injection of 50pg protein doses. Blood samples were collected at defined time points up to 7 days post-injection. Serum concentrations were quantified by direct ELISA using recombinant TREM2 (Sino Biological) as capture and Peroxidase AffiniPure Goat Anti-Human IgG, Fey fragment (Jackson ImmunoResearch) detection antibodies. Pharmacokinetic parameters including the elimination rate constant (K), serum half-life (ti / 2), and estimated clearance time were calculated.For biodistribution studies, tumor-bearing mice were intravenously injected with 200pg MiTE144. At defined endpoints, mice were euthanized and tissues (tumor, lung, spleen, kidney, brain, and liver) were collected, homogenized, digested via RIPA Buffer (150mM Sodium chloride, 1%Triton X-1000, Sodium deoxy cholate, 0.1% SDS, 50mM Tris (pH8.0), DDW), and analyzed by direct ELISA for MiTE144 content. Protein concentration was normalized to tissue weight and expressed as ng per mg tissue. All experiments were performed in biological triplicates.Cytokine secretion measurementCytokine concentration was measured using ELISA for mouse IFN-y (BioLegend), mouse IL-6 (BioLegend), mouse IL-2 (BioLegend), human IFN-y (BioLegend). 96-well ELISA microplates were coated with 0.2pg / well of capture antibody diluted in PBS, and incubated overnight. The plates were rinsed three times with 0.05% Tween-20 in PBS, blocked with 1% BSA in PBS at room temperature (RT) for 1 hour, and rinsed again. The coated plates were incubated with isolated serum for 2h at RT. After incubation, plates were rinsed and incubated with detection antibody, followed by streptavidin-HRP for 20 min at RT. The plates were rinsed and incubated with substrate solution F for 20 min at RT in the dark, followed by the addition of the Stop solution 2N Sulfuric Acid (R&D). OD was measured using a plate reader at dual wavelengths (450nm and 570nm). NK culture supernatants were collected for secreted protein analysis using Nomic Bio.Isolation, activation and proliferation rate assessment of human T cellsT cells were isolated from human blood donors using a Pan T cell isolation kit (Miltenyi Biotec). T cells were stained by proliferation tracking dye eFluor 450 (eBioscience) and seeded on anti- CD3 (lOpg / ml) and anti-CD28 (2pg / ml) pre-coated 96-well plates. After 2 days of pre-activation, control antibodies. MiTEs and MiTEs pre-treated with hMMP-14 (see above) were administered to T cells in different concentrations (0,0.04, 0.15, 0.6, 2.5, lOnM). Four days later, the cells were harvested and labelled with anti-CD3 BV711 (BD), anti-CD8 APC Cy7 (BD), anti-CD4 PE (BD), anti-CD25 BV605 (BD), anti-CD69 APC (BD) and live / dead dye Zombie Green (BioLegend) by incubating for 30 min at 4°C in the dark. Upon washing with MACS buffer, the cells were analysed on a flow cytometer and MFIs for CD25, CD69 and proliferation tracking dye w ere assessed using FlowJO (version 10).Isolation and activation assessment of human NK cellsNatural Killer (NK) cells were isolated with the EasySep™ Human NK Cell Isolation Kit (Stemcell Technologies). Following isolation, NK cells were seeded at 80,000 cells per well for a period of 48 hours. Cells were treated with InM recombinant human IL-2 (PeproTech), non-cleaved MITE144, or cleaved MITE144. Upon completion of the incubation period, culture supernatants were collected for secreted protein analysis using Nomic Bio.Human Tissue Sections and ImmunofluorescenceTissue samples were fixed in 4% paraformaldehyde (PF A) and sectioned at 5 pm thickness. Opal multiplex immunostaining was performed using the BOND RXm platform (Leica). Sections were deparaffinized with BOND dewaxing solution, and endogenous peroxidase activity was blocked using 3% H2O2 and 1% HC1 in methanol. Heat-induced epitope retrieval was performed in HIER2 buffer (pH 9) for 10 minutes. Blocking was carried out with 20% normal horse serum (Vector Labs) and 0.05% Tween-20. Primary antibodies included mouse anti-MMP14 (Mazor. Roei D. et al. Cell, Volume 185, Issue 7, 1208 - 1222. e21) and rabbit anti-TREM2 (1:200, Cell Signaling Technology), diluted in 2% NHS with 0.05% Tween-20. Detection was performed using secondaryHRP- or biotin-conjugated antibodies (Jackson ImmunoResearch, 1 : 100). followed by the Vectastain ABC kit (Vector Laboratories) and TSA fluorophores (Opals, Akoya Biosciences). Between staining rounds, antibody removal was performed using HIER2 buffer for 10 minutes before repeating the protocol from the blocking step. Nuclei were counterstained with Hoechst (1 : 1000, Invitrogen), and slides were imaged using the PhenoImager HT system (Akoya Biosciences).Isolation of single cells and sorting for RNA sequencing and Flow cytometryTumors were minced into small pieces on ice and digested using RPMI supplemented with Img / ml Collagenase IV (Enco) and 0.05mg / ml Dnasel (Sigma Aldrich) at 37 degrees for three times 8 min. In between the incubation steps, the tissue was further dissociated using a gentleMACS dissociator and C tubes (Miltenyi Biotec). After digestion, the cells were fdtered through a lOOum cell strainer and washed with ice cold PBS.Spleens were smashed through a lOOum cell strainer and washed with ice cold PBS. tdLNs were minced into small pieces and digested using RPMI supplemented with 0.4mg / ml Collagenase IV and 0.05 mg / ml Dnasel at 37 °C for 30mins. After digestion, the cells were filtered through a 100 pm cell strainer and washed with ice cold PBS.Cells isolated from tumors, spleens or tdLNs were stained with anti-CD8 BUV805 (BD). anti- CDl lb BUV737 (BD), anti-CD19 BUV661 (BD), anti-TCRbeta BUV615 (clBD), anti-GITR BUV563 (BD), anti-CD45 BUV395 (BD), anti-CD64 BV711 (BioLegend), anti-CD4 BV605 (BioLegend), anti-Ly6G Pacific blue (BioLegend), anti-Ly6C FITC (cBioLegend), anti-MHCII (I- A / I-E) PE-CY7 (BioLegend), anti-NKl. l PE-Dazzle594 (BioLegend), anti-CDl lc PE (BioLegend), anti-CD25 BV785 (BioLegend), anti-CD90.2 AF700 (BioLegend), anti-F4 / 80 APC (BioLegend), anti-CD16 / CD32 Trustain FcX (BioLegend) to prevent FC blocking and ZombieNIR live / dead (BioLegend) for 30min on ice. After washing them again with ice cold PBS, cells were sorted and acquired on a BD FACS Symphony 6 flow cytometer. After excluding doublets and dead cells, two 384-well plates of CD45+cells and CD90.2+and / or NK1.1+T / NKT / NK cells or CDl lb7F4 / 80+cells were sorted per sample. The 384-well plates contained a lysis solution, mineral oil and barcoded poly(T) reverse transcription primers enabling scRNA sequencing.To assess cell numbers per gram tissue (tumors) or per tdLN, tumor weight was assessed prior to digestion. Before acquiring the samples on the flow cytometer. lOul of Precision Count beads (BioLegend) were added to each sample and counts were calculated according to the manufractures instructor upon exporting cell counts gated in FlowJO (version 10).Single cell library preparationSPID-seq, an adapted version of the MARS-seq pipeline, was used to generate scRNA-seq libraries. In short, polyadenylated mRNA from individual cells that had been sorted into 384-well plates was captured, followed by a barcoding step of the mRNA during reverse transcription into cDNA. cDNA of each plate was pooled into a single tube, and was fragmented and amplified togenerate sequencing-ready libraries for NovaSeq X plus (Illumina) sequencing. Quality control was performed on each library created from a plate.Read alignmentSingle-cell RNA-seq (scRNA-seq) libraries were pooled at equimolar concentrations and sequenced on an Illumina NovaSeq X plus sequencer, achieving a sequencing depth of 10,000 to 50,000 reads per cell. Reads sharing identical unique molecular identifiers (UMIs) were collapsed to represent original RNA molecules. Batch quality was confirmed by ensuring minimal cross-cell contamination (less than 3%), as assessed by the frequency of spurious UMIs detected in empty wells. Read alignment was performed using the MARS-seq2.0 pipeline. Briefly, low-quality reads were filtered out, and the remaining reads were mapped to the mouse reference genome (mmlO) using HIS AT (version 0.1.6), discarding reads with multiple mapping locations. Gene annotations were based on the UCSC Genome Browser, and only exonic reads were assigned to genes. UMI uniqueness was verified within a 3 kb window. In cases where exons of different genes overlapped on the same strand, such reads were attributed to a merged gene identity with concatenated gene symbols.Preprocessing of single cell RNA-seq dataSingle cell sequencing pre-processing and analyses were performed using Seurat (V5). Cell quality control (QC) was performed to remove cells with more than 10% mitochondrial RNA transcripts, and cells with less than 200 and more than 3500 unique genes. 45,505 cells passed quality control. Further QC steps included the removal of genes which were expressed in less than 5 cells and the exclusion of selected non-essential genes (Gm, Rik, mitochondrial, ribosomal genes), resulting in a dataset with 20,043 genes. The data was normalized using log normalization with a scale factor of 10,000, and 2000 highly variable genes were selected. After scaling, principal component analysis was performed and clustering was conducted based on 16 principal components and 1.2 resolution. UMAP was used for dimensionality reduction and visualization. To improve resolution, T cells were subsetted and re-clustered (16 principal components. 1.2 resolution) and the resulting cluster labels w ere transferred back to the global dataset.Differential gene expression analysesPseudobulk gene expression data were generated using Seurat v5's AggregateExpression function, aggregating counts by Treatment, Mouse ID, and broad cell clusters (T cells, Tregs, NK cells, Macrophages, Dendritic Cells, Granulocytes). Prior to differential expression analysis, "Treatment-Mouse ID-cell cluster" samples with 20 or less cells were excluded. Genes with fewer than 10 counts in at least half of the samples were also removed. DEG analysis was performed using DESeq2 with the anti-RSV group set as reference. DEGs were filtered based on padj < 0.05 and log2FoldChange > 1 and < -1.Downstream analysis for treatment perturbation effects (MrVI)To further investigate the treatment effect on the immune landscape, we utilized a variational inference deep learning model (MrVI) to construct a graph representation of the effect of each treatment in relation to all other treatments. The raw counts of preprocessed data were used and the top 5000 most highly variable genes were determined using the scanpy implementation ofseurat v3. A MrVI model was then trained using the treatment condition as the sample key with the following model hyperparameters: n_laten = 10, n_latent_u = 5, leam_z_u_prior_scale = False, z_u_prior = False, u_prior_mixture = True, u_prior_mixture_k = 20. The model has been trained using a batch size of 256. a learning rate of 0.001, a kl warmup of 20 epochs with an early stopping based on the elbo_validation using a patience of 30.The sample aware z latent space from the model was extracted to compute the aggregated local sample distances for each treatment condition and further visualized as a heatmap. Using the treatment distance matrix, we applied the Kamada-Kawai algorithm to construct a 2D graph layout representing the treatment effects. The inverse of the distance matrix was then computed in relation to the control condition (aRSV) to represent the deviation from the control on the graph as edges.To further dissect the molecular effects, we also employed hotspot gene module analysis using the z MrVI latent space. A neighborhood similarity graph (k = 30) was first constructed and the top 1000 locally autocorrelated genes (FDR < 5%) were extracted. Significantly autocorrelated genes were subsequently grouped into modules based on their co-expression profiles in their neighborhood of cells. The gene module enrichment score was then averaged for each treatment condition and visualized as a heatmap using seaborn.PDTF ex vivo cultureCryopreserved PDTFs from five distinct renal cell carcinoma patient tumors were rapidly thawed in a 37°C water bath and immediately placed in RPMI 1640 medium supplemented with 5% fetal bovine serum (FBS). Following thawing, individual tumor fragments were cultured for 48 hours as previously detailed. For each donor, a total of 16 fragments per treatment condition were allocated to each of the four experimental arms: Isotype Control: anti-Respiratory Syncytial Virus (RSV) IgGl (lOnM) and anti-Hen Egg Lysozyme (HEL) IgG4 (lOpg / mL), anti-PD-1 Monotherapy: anti-PD-1 (10 pg / mL) and anti-RSV-IgGl (lOnM), MiTE144 Monotherapy: MITE144 (lOnM) and anti-HEL-IgG4 (10 pg / mL), Combination Therapy: MITE144 (10 nM) and anti-PD-1 (lOpg / mL).Upon completion of the 48-hour culture period, 90pl of supernatants were collected and frozen for secreted protein analysis. Next, all fragments from the same donor and treatment arm were pooled and digested into single cell suspensions. Immune cells w ere enriched from the digested fragments using CD45 magnetic-activated cell sorting (MACS). The resulting CD45-enriched cell fractions were subsequently stained with a custom Immunai CITE-seq antibody panel and subjected to fluorescence-activated cell sorting (FACS) to isolate viable CD45 cells.Single-cell gene expression (GEX) and feature (CITE-seq) libraries w ere prepared from the sorted cells using the lOx Genomics Chromium Next GEM Single Cell 5' Kit v2. Libraries were sequenced on a Novaseq X Plus sequencer using a 300-cycle kit.CITE-Seq & PDTF Analysis PreprocessingRaw data were processed using Cell Ranger v8.0.0 (lOx Genomics) mapping to the human GRCh38 (hg38) genome, and downstream analysis was performed using scanpy and muon. Cellquality control (QC) was performed to remove cells with more than 5% mitochondrial RNA transcripts, and cells with less than 500 and more than 20000 UMIs. A total of 93,087 immune cells passed quality control. We further removal genes which were expressed in less than 5 cells and had less than 10 UMI counts, and excluded selected non-essential genes (pseudo genes, mitochondrial, ribosomal process genes). The data was normalized using log normalization with a scale factor of 10,000, and 5000 highly variable genes were selected using scanpy. experimental. pp highly variable genesf) function with setting patient ‘donor’ variable as the batch key. The mRNA modality was combined with the protein modality (146 proteins) using muon to one mudata object for downstream analysis.We then trained a totalVI model for the integration of two modalities to enable joint analysis of the data and acquiring denoised protein levels. We used the patient donor as the batch key with the following model hyperparameters: n_latent=20, n_hidden=256, gene_likelihood="nb", gene_dispersion="gene-batch", protein_dispersion="protein-batch", empirical_protein_background_prior=True. The model was trained for 400 epochs using a learning rate of 0.001, a kl warmup of 20 epochs.We performed unsupervised Leiden clustering on the latent space of the model and computed the umap for visualization. Clusters were then annotated with canonical markers using both RNA and protein modalities. For visualization of the protein level, we obtained the denoised protein expression using the totalVI model (model, get normalized expression function with n_samples=25) and further filtered the expression by thresholding a protein foreground probability of >0.9 (obtained by model, get protein foreground probability with n_samples=25). \Downstream analy sis ofPDTF:In order to quantify how- strongly each donor’s immune composition responded to treatment, we first calculated the immune cell type abundance per donor per treatment (20 samples with 4 treatment conditions in 5 donors). These abundance profiles were projected to two dimensional space using PCA on all samples (Figure SUB). For each donor, we first computed a treatment variance (Tss) as the sum of squared Euclidean distances of all four treatment points from the donor’s centroid - measuring how much the treatment perturbs the immune profile. In parallel, we quantified a baseline global compositional effect (Css) as the sum of squared distances from each donor’s centroid to the global centroid - reflecting the inherent differences between donors. Since both sums involve 4 points, we scaled Cssby the degree of freedom (n - 1) and defined aT normalized treatment response score R = — , comparing the treatment-driven changes against s / 3 overall compositional variability. Finally for visualization, we computed the z-score of the ratio to standardize across donors, and identified ‘Patient 138’ as a potential outlier that had minimal treatment effect across all conditions, which we removed it for susbsequent patient-level compositional analysis.IL-2 infiltration scoreWe analyzed the correlation between IL-2 expression and immune cell infiltration across TCGA tumor samples using the TIMER2.0 database (http: / / timer.cistrome.org / ). IL-2 gene expression levels were extracted from TCGA bulk RNA-sequencing data and correlated with immune infiltration scores for CD4+T cells. CD8+T cells, regulatory T cells (Tregs), and natural killer (NK) cells using deconvolution-based estimates from TIMER2.0. Correlation analyses were performed using Spearman’s rank correlation, with statistical significance determined using Wilcoxon rank-sum tests for group comparisons.Analysis of human patient atlas for protease screeningThe single-cell RNA-seq immune cell atlas was constructed from a list of 183 publicly available cancer tissue studies (Table S2). starting from published count data or raw reads where available (mapped with CellRanger v8.0.0). Overall, we generated a large single-cell atlas of 13,818,851 single cells, incorporating PBMCs (n= 1,753), healthy tissues (n=147), tumor-adjacent samples (n=750) as well as tumors (n=2791). Doublets were detected using scDblFinder vl.13.14 and ambient RNA contamination was detected using decontX. Cell types were annotated using transfer learning from a proprietary tissue reference. Marker genes per cell type are provided in Table S2. Cell counts were pseudobulked per cell type and sample and embedded using scVI v 1.0.4 (parameters: n_layers=2; n_latent=20; gene_likelihood="nb") to obtain reconstructed gene expression values. Reconstructed gene expression was scaled to range between 0 and 1 and the fraction of samples with high expression per gene and cell type is denoted as the fraction of samples exceeding the 80% expression quantile per gene.Analysis of human spatial datasets (MERFISH)The processed MERFISH datasets were obtained from the Vizgen MERSCOPE FFPE Human Immuno-oncology, specifically slides designated as ‘‘Breast Cancer”, “Colon Cancer 1”, “Lung Cancer 1”, and “Ovarian Cancer 1” were utilized in this study. Squidpy and Scanpy were utilized for preprocessing of each individual dataset and cells with a total count of >2000 were removed. In order to account for incorrectly assigned transcripts via technical limitations such as cell segmentation error or transcript diffusion, we employed resolVI to each slide individually and obtained a corrected latent representation of the data and corrected counts. A resolVI model was then trained using the following model hyperparameters: spatial n neighbors = 20, njatent = 10, semisupervised=False, mixture_k=100, deeply _inject_covariates=True, encode_covariates=False, downsample counts = 150). The model has been trained with a maximum epoch of 100 and a learning rate of 0.001. The corrected latent space was subsequently visualized by constructing a neighborhood graph of cells and applying UMAP for a two-dimensional visualization. Clusters of cells were identified by applying the Leiden clustering algorithm on this neighborhood graph and annotation was done using canonical major lineage cell type markers.In order to quantify the distance between the major cell types as a proxy to their physical interaction, we first divide the coordinates of the cells by the median of the cell radius (obtained by the square root of the cell volume), ranging between roughly 8-9 microns which isapproximately the size of an average single cell. Then by looking at the nearest 10 cells, we can compute a pairwise distance from of each cell type to each other. Using the pairwise distance information, we then computed a cumulative distribution function (CDF) on the minimum distances to compare the spatial co-locahzation of cell types.Ligand receptor analysis of human tumors and mouse treatment perturbation dataUtilizing an in-house and published human single-cell tumor atlas consisting of Breast, Colon, and Lung datasets including their tumor and respective adjacent healthy -like tissues . we extracted the major immune populations (NK cells, T cells, DCs, Monocytes, and TAMs) to query the likelihood of interactions at the molecular level. MultiNicheNetR was performed on 332,723 single immune cells. Genes expressed in less than 10 cells were removed and using the raw counts as input, a pseudobulk level DEGs was performed comparing the tumor tissue to the adjacent healthy-like tissue per cell type. Potential ligands were extracted if they were expressed in at least 5% of the senders within their respective clusters, Log2FoldChange > 1 and padj < 0.05; denoting the myeloid compartment as senders and lymphoid compartment as receivers. For summarization of the putative ligand-receptor pairs, we counted the sum of the tumor-enriched pairs in the top 100 pairs, then we also showed the top 200 pairs for the putative ligand-receptor interactions.Similar settings were used for the mouse treatment perturbation data, querying the immune interactions among NK cells, T cells, Tregs, TAMs and DCs. In order to compare the downregulated interactions in the MiTE-induced conditions, we first compared the anti-RSV to each of the treatment condition to show the depleted interactions in all treatment conditions (a total of 8 treatments including MiTE144, MiTE144 + anti-PDl, and MiTE144 + anti-CTLA4). To show conservation with the human tumor, we extracted the top 1000 significant putative ligand-receptor pairs spanning all treatment conditions and plotted the conserved mouse ligand-receptor pairs for MiTE144, MiTE144 + anti-PDl, and MiTE144 + anti-CTLA4 condtions.EXAMPLE 1[000494] Shows a schematic illustration of a Myeloid-targeted immunocytokine and NK / T cell Enhancer” (MiTE) with tumor-conditioned cytokine activity according to some embodiments of the invention, as illustrated in FIG. 1.EXAMPLE 2This example refers to the effect of the fusion of some embodiments of the invention on tumor growth. FIG. 2 shows average tumor volumes (mm3+SEM) in transgenic hTREM2 C57BL / 6J mice which were inoculated subcutaneously (s.c.) with MC38 on day 0 and were randomized on day 7 when the average tumor size reached 50 mm3. Mice were then treated intraperitoneally with isotype aRSV (lOOpg / dose, n=7). aRSV-IL2SK (lOOpg / dose. n=7) andaTREM2-IL2SK (lOOpg / dose, n=7) weekly for a total of two injections. Myeloid-targeted immunocytokine effectively abolished MC38 tumor growth in vivo.EXAMPLE 3[000495] The half-life of the fusion protein aTREM2-IL2SK according to some embodiments of the invention was assessed. FIG. 3 shows an ELISA analysis of aTREM2-IL2SK antibody concentration in serum from WT C57BL / 6J mice over time upon intraperitoneal administration (200 pg / dose). using recombinant human TREM2 and an HRP-conjugated anti -human IgG antibody. The myeloid-targeted immuncytokine exhibits reduced half-life (compared to naked anti-TREM2 alone, data not shown).EXAMPLE 4[000496] The effect of the fusion according to some embodiments of the invention os systemic toxicity was assessed. FIG. 4 shows probability of survival across time in transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 on day 0 and were randomized on day 7 when the average tumor size reached 50 mm3. Mice were then treated intraperitoneally with isotype control aRSV (lOOpg / dose, n=7), and aTREM2-IL2SK (lOOpg / dose, n=7) every73 days for a total of two injections. After the second injection, all mice in the treatment group died. FIG. 5 shows an ELISA analysis of IFNy levels in serum of WT C57BL / 6J mice which was collected 24 hours after intravenous administration of aTREM2 (200pg / dose, n=5), aTREM2- IL2SK (200pg / dose, n=45, aRSV (200pg / dose, n=5) or aRSV-IL2SK (200pg / dose, n=5), demonstrating systemic toxicity.EXAMPLE 5[000497] In order to identify a protease which is specific to the tumor niche and clusters with TAMs a single cell RNA sequencing was done. FIG. 6 shows single-cell RNA-sequencing analysis of macrophages in vivo represented as heatmap. These macrophages originate in spleen, tumor and peritoneum from WT C57BL / 6J mice (n=5), which were inoculated s.c. with the murine fibrosarcoma cell line, MCA205, on day 0, and were collected on day 10 when tumors reached an average tumor volume of 500mm3. The model was used for the identification of matrix metalloproteinases (MMPs) specific to tumor-associated macrophages. Specifically found was MMP 14 which binding sequence was used in the preparation of the masked configurations of some embodiments of the invention and as exemplified below.EXAMPLE 6[000498] To prove the MMP14 (identified as in Example 5), clusters with TREM2 in malignant tumors, various tumors were analyzed.[000499] bulk RNA-sequencing analysis of healthy (blue) versus tumor (red) tissues across 14 different tumor types from TCGA and GTEX (brain (n=1842), breast (n=1390), colon (n=637), esophagus (n=847), kidney (n=1043), liver(n=531). lung (n=1410). ovary (n=515), pancreas (n=350), prostate (n=646), skin (n=1281), stomach (n=623), testis (n=319), uterus (n=135)) as barplots, demonstrating that MMP14 and TREM2 are highly upregulated in malignant tumors.[000500] Based on these a number of fusion configurations were designed. FIG. 8 shows a schematic illustration of the design of the of MiTEs according to some embodiments of the invention. The aTREM2 antibody (blue / grey) is coupled by a linker element to mutant interleukin 2 “superkine” (orange) which binds to a specific blocking moiety (green), thereby inactivating the cytokine activity. This blocking moiety is connected through a MMP14-cleavable linker (here indicated as scissors) to the antibody.EXAMPLE 7[000501 ] This example shows that IL2SK activity' is dependent on TAM-specific protease cleavage. FIG. 9 shows optical density (OD) at 630 nm measuring embryonic alkaline phosphatase (SEAP) secretion from HEK Blue CD 122 / CD 132 reporter cells. Using this reporter cells, it was possible to quantify the bioactivity' of IL2SK after incubation with or without recombinant human TAM-specific MMP14. IL2SK activity' is dependent on TAM-specific protease cleavage in the tested variants. Likewise, FIG. 10 shows OD at 630 nm measuring SEAP secretion from HEK Blue CD122 / CD132 reporter cells. First, monocytes were isolated from blood and differentiated to tumor-associated macrophages across 7 days using 30 ng / mL hM-CSF cytokine and starting on day 5, additionally with 10 ng / ml hIL-4. The supernatant was then concentrated via ultracentrifugal columns. MiTEs were incubated in the presence of the concentrated supernatant overnight at 37 °C degrees, similar to the control condition using recombinant MMP14. IL2SK activity was measured via HEK Blue CD122 / CD132 reporter assay. The sectreome of human macrophages efficiently cleaved the MiTEs, demonstrating TAM-specificity. Hence the cleavage is dependent on MMP14 which is secreted to the medium from TAMs.EXAMPLE 8[000502] This example shows that the variants are functional in binding TREM2 expressing cells. Specifically, FIG. 11 shows mean fluorescence intensity (MFI) measured via FACS usingPE-conjugated anti-human IgG staining. First, either HEK parental or HEK cells overexpressing hTREM2 were treated for 4 h with indicated immunocytokine variants (20nM). MiTEs bind hTREM2 overexpressed in HEK cells similar to mAh aTREM2 in vitro. Indeed their binding was further characterized in vitro using recombinant human TREM2. FIG. 12 shows OD (450nm- 570nm) after a direct ELISA assay. Plates were coated with 0.5pg / ml recombinant human Trem2 overnight and binding was assessed using anti -human IgG conjugated with HRP the next day. MiTEs with and without cleavage bind recombinant human TREM2. Affinity of the indicated variants was determined by SPR. Results are shown in Figures 13 and 14.EXAMPLE 9[000503] This Example shows the effect of the variants on T cells proliferation. FIG. 15 shows mean fluorescence intensity (MFI-1) (normalized) of proliferation dye measurements via FACS. The proliferation of human T cells (n=3 donors) was measured after 4 days in the presence of 0.15 nM immunocytokines, which have or have not been pre-treated with MMP14. MiTEs activate robust human CD8 and CD4 T cell proliferation in a TME-specific manner. Also note, the IL2 activity is lowered when the blocking moiety is not cleaved.EXAMPLE 10[000504] This example shows the effect of the variants on tumor volume. FIG. 16 shows average tumor volume (mm3+SEM) in transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 on day 0 and were randomized on day 9 when the average tumor size reached 50 mm3. Mice were then treated intratumorally with isotype control aRSV (5pg / dose, n=3). aTREM2-IL2SK (5pg / dose, n=3), and all four immunocytokine variants individually (5pg / dose, n=3) every two days for a total of three injections. All variants show strong anti -tumor effects in vivo.[000505] FIG. 17 shows average tumor volumes (mm3+SEM) in transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 on day 0 and were randomized on day 8 when the average tumor size reached 50 mm3. Mice were then treated intratumorally with isotype control aRSV (5pg / dose, n=3), aPDl (40pg / dose, n=3), aTREM2 (5pg / dose, n=3), MiTE-144 (5pg / dose, n=3), aTREM2 (5pg / dose) together with aPDl (40pg / dose) (n=3), and MiTE-144 (5pg / dose) together with aPDl (40pg / dose) (n=3) every two days for a total of three injections. Each line represents one mouse. The immunocytokines outcompete combinatorial therapies of PD- 1 and Trem2 as well as their monotherapies in vivo. Based on tumor size, MiTE-144 has the strongest inhibition on tumor growth.EXAMPLE 11[000506] This example addresses the toxic effect of the fusion proteins according to some embodiments of the invention. FIG. 18A shows body weight [%] of WT C57BL / 6J mice after intraveneous administration of 50pg / dose of aRSV, aRSV-IL2SK. aTREM2, aTREM2- 1L2SK and all four variants of the immunocytokines (n=4 / group). Mice treated with the variants did not lose weight. FIG. 18B shows an ELISA analysis of IFNy levels in serum of WT C57BL / 6J mice which was collected 24 hours after intravenous administration of 50 pg / dose of aRSV, aRSV- IL2SK. aTREM2, aTREM2-IL2SK and all four variants of the immunocytokines individually (n=4 / group), demonstrating reduced systemic toxicity of the variants.[000507] Likewise, FIG. 19A shows an ELISA analysis measuring IFNy, IL6 and IL2 levels in serum of transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 and bear tumors with an average size above 100 mm3. These mice were treated intravenously with isotype control aRSV (200pg / dose, n=4-8), aTREM2 (200pg / dose, n= n=4-8), MiTE-76 or MiTE- 144 ( (200pg / dose, n=4-8) on day 10 and 13 for a total of two injections. FIG. 19B shows body weight [%] over time in mice treated as in FIG. 19 A.[000508] FIG. 19C shows the spleen weight [g] of mice treated as in FIG. 19A on day 16. No weight loss, no elevation in serum cytokine levels and no spleen enlargement, indicating no systemic toxicity and safe administration of the of MiTEs at high dosages.EXAMPLE 12[000509] This example shows the anti-tumorigenic effect of the variants. Figures 18A-B show average tumor volumes (mm3+SEM) in transgenic hTREM2 C57BL / 6J mice which were inoculated s.c. with MC38 on day 0 and were randomized on day 8 when the average tumor size reached 50 mm3. Mice were then treated intravenously with isotype control aRSV (200pg / dose, n=7-12), aPDl (200pg / dose, n=7-12), aTREM2 (200pg / dose, n=7-12), MiTE-144 (200pg / dose, n=7-12), aTREM2 (200pg / dose) together with aPDl (200pg / dose) (n=7-12), and MiTE-144 (200pg / dose) together with aPDl (200pg / dose) (n=7-12) on day 8, 10 and 13 for a total of three injections. On day 11, 5 mice per group were used for further analysis. The MiTEs abolish tumor growth more effectively than combinatorial therapies of PD-1 and TREM2 as well as their monotherapies in a systematic manner in vivo, without off-target effects and toxicity.EXAMPLE 13[000510] This example shows the effect of the fusion proteins of some embodiments of the invention on immune cells in the TME. This was done by single-cell RNA seq analyses of immune cells isolated on day 11 from MC38 tumors from mice which were intraveneously treated with isotype control aRSV (200pg / dose, n=5), aPDl (200pg / dose, n=5), aTREM2 (200pg / dose, n=5). MiTE-144 (200pg / dose, n=5), aTREM2 (200pg / dose. n=5) together with aPDl (200pg / dose. n=5). and MiTE-144 (200pg / dose, n=5) together with aPDl (200pg / dose, n=5) on day 8. 10. FIG. 21A shows a UMAP displaying identified cell clusters. FIG. 21B shows UMAPs displaying cells from each treatment group with over-lay ed cell densities. Treatment with MiTE-144 and MiTE- 144 together with aPDl shifts cell densities towards the T and NKT cell compartment. FIG. 21C shows frequencies of cell clusters among total immune cells in the tumors. Treatment with MiTE- 144 and MiTE-144 together with aPDl reprograms frequencies of immune cell populations. FIG. 21D shows the frequencies of selected cell populations in the T / NK(T) cell compartment. Tumors of mice treated with Variant 3 and Variant 3 together with aPDl have increased frequencies of two clusters of proliferating NKT cells, cytotoxic CD8 T cells and proliferating cytotoxic T cells. FIG. 21E shows a Dotplot and Violinplots displaying selected differentially expressed genes in NKT cells between the aRSV control and at least one treatment group. NKT cells isolated from tumors of mice treated with Variant 3 and Variant 3 together with aPDl show a gene signature of increased cytotoxicity and proliferation with reduced exhaustion / dysfunction. This includes increased expression of the genes Prfl (encoding Perforin- 1), Gzma. Gmzb (encoding Granzyme A and B) and Mki67 (encoding Ki-67). FIG. 21 F shows a Dotplot and Violinplots displaying selected differentially expressed genes in T cells between the aRSV control and at least one of treatment group. T cells isolated from tumors of mice treated with MiTE-144 and MiTE-144 together with aPDl show a gene signature of increased cytotoxicity and proliferation with reduced exhaustion / dysfunction. This includes increased expression of the genes Prfl (encoding Perforin- 1), Gzma, Gmzb (encoding Granzyme A and B) and Mki67 (encoding Ki-67).[00051 1 ] The results show a complete tumor eradication and molecular reprograming of the tumor immunne compratment of MiTE-144 and MiTE-144 together with aPDl with massive and unexpected effects on the NK and NKT compartment as well as CD8 and CD4 T cells. 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 that block activity of antigen presentation.[000512] Current T cell ICB or T cell focused immunocytokines, which are developed today and act in cis ( e.g. anti-PDl -immunocytokine, CD8-immunocytokine) mostly activate the cytotoxic CD8 T cell population in the TME and are limited through MHC I loss and other mechanisms tumor evolved to disarm CD8 T cells. MiTE-144 and MiTE-144 together with aPDl data activity shows, that unlike ICB or T cell focused immunocytokines, these molecules effect deeper and broader, more potent immune arms. MiTE-144 and MiTE-144 together with aPDl functions in a trans based mechanism, and shows a very dramatic effect on NK and NKT cells in the TME following treatment (30 fold more potent activity of the NK and NKT arm than standard ICB). While both MiTE and ICB activate the T cell arm the inventors' in vivo data demonstrates that NK / and NKT cells are responding much more potently then CD8 and bring the largest efficacy tumor killing effects. Importantly the NK and NKT cells are not only not inhibited by MHC-I loss but rather activated by such signals and hence can be very effective even in PD-1 unresponsive tumors including potential patients with mutations in the MHC-I pathway.EXAMPLE 14TAM and T cells interact closely in the TME across human cancer types[000513] To explore how macrophage-targeted cancer therapies could be improved, the key cell types that TAMs interact with in the TME were identified and their molecular interaction networks across human cancer types defined. The results uncover unique interaction circuits that can be harnessed to enhance the efficacy of TAM-directed therapies.[000514] First, to characterize macrophage spatial interactions in the TME, the inventors utilized subcellular resolution spatial transcriptomics to query the proximity of immune cells to macrophages. The inventors curated a spatial atlas of -1.86 million single cells from publicly available MERFISH datasets of human lung, breast, colon, and ovarian tumors41,42 (FIG. 22A- L, FIG. 29A-H). As transcript diffusion and technical noise from cell segmentation (even with membrane staining information) in spatial transcriptomic technologies limit the accuracy of the annotation, the inventors employed a deep learning model (ResolVI43) to address and correct for wrongly assigned transcripts in the data (Methods). Using this approach, the inventors identified the major immune populations as well as non-immune cell types across cancer types. Subsequently, the inventors computed the co-localization distances of roughly 10 microns (the size of an average cell) for the inventors’ cell types of interest and derived a cumulative distribution frequency on pairs of cells (FIG. 22C, F. I, L, S1E-H). The inventors found that TAMs are the main immune cell type that co-localizes with T cells within the TME, whereas other immune subsets are observed to be more distant from T cells. This was supported by the inventors’ CDFanalysis of infiltrating immune cells (FIG. 29E-H), which incorporated all major myeloid subsets, including TAMs, monocyte-derived macrophages (MoMacs), monocyte-like cells (Mon-like), monocytes (Mon), dendritic cells (DCs), and neutrophils. TAMs demonstrated the strongest spatial proximity to lymphocytes in breast, lung, and ovarian cancers. Interestingly, dendritic cells (DCs) appear to co-localize with T cells as frequently as TAMs in ovarian cancer. Overall, the inventors’ analysis revealed that T cells co-localize with TAMs in the TME across four major solid-tumor entities.[000515] To further study the molecular nature of TAM-T cell interactions, the inventors utilized a large publicly available single-cell RNA-sequencing (scRNA-seq) atlas containing human tumor samples (n = 123) and their adjacent healthy-like samples (n = 67) from breast, lung, and colon cancers44-47. After quality control, the inventors extracted 332,723 high quality singlecells of major immune populations from scVI integration as previously described47,48 and reannotated them using unsupervised graph-based clustering by major lineage markers (FIG. 29E). Given the information of tumor and adjacent healthy-like samples, the inventors applied MultiNicheNetR49 to compare the cell-cell communication within the major immune populations, thereby revealing a complex network of immune interactions enriched in the TME (FIG. 22M, N; FIG. 291; Table SI). Notably, the inventors observed a predominant interaction circuitry' between TAMs and T cells. These TAM- T cells interaction include chemokine axis involved in T cell recruitment (CXCL9-CXCR3. CCR8-CCL3), costimulatory and inhibitory interactions (CD80 / CD86-CTLA4, ICOSLG-ICOS; CD274-PDCD1 , SIRPA-TIGIT) as well as immunosupressive signals (TGFBR1-TGFB, LGALS1-CD69, LGALS3BP-ITGB1) which all were significantly upregulated in the TME. In summary', unbiased spatial and single cell analysis across human tumors identified the proximal positioning of T cells with TAMs in the TME, including multiple ligand-receptor pairs associated with T cell recruitment and regulation.EXAMPLE 15Anti-TREM2-IL2SK immunocytokines induce systemic toxicity[000516] As TAMs are in close proximity' to T cells in the TME, the inventors aimed to develop a therapeutic strategy that can reprogram the inhibitory checkpoints of TAMs and activate the T cell compartments in a dual fashion. The inventors and others have demonstrated that TREM2 is a central myeloid inhibitory receptor and antagonizing TREM2 activity is a potential strategy' to reprogram myeloid cells in the TME25, 26, 36-40. Characterizing TREM2 expression in a bulk RNA seq dataset generated by the cancer genome atlas program (TCGA,https: / / www.cancer.gov / tcga), with over 10,000 patients across 14 different human cancer types in comparison to healthy tissues confirmed the notion that TREM2 is a tumor specific target highly upregulated in the TME as compared to healthy tissue (FIG. 30A). To target TREM2, the inventors developed an antagonistic anti-(human) TREM2 antibody (aTREM2) and utilized humanized TREM2 mice in which the murine trem2 gene was replaced by the human TREM2 locus (hTREM2 mice50) to test and characterize TREM2 antagonistic molecules. Following the selection of high affinity and specificity binders, the inventors applied scRNA-seq in order to define the functional impact of each molecule on TREM2 activity by using hTREM2 bone marrow derived macrophages stimulated with TAM programming conditions (M-CSF+IL-4). The inventors identified a molecule with similar activity to BMDM from mice of a TREM2 -knockout (KO) background, which completely abrogated programming of BMDM to TAMs (FIG. 30B-D). Cells treated with the inventors’ antagonistic anti-TREM2 antibody, but not binding control, prevented upregulation of the TAM program (Mmpl2 and Spp) and maintained a pro-inflammatory, monocytic program (Siglecl, Treml and Ifit2; FIG. 30D). To assess the impact of soluble TREM2 in human plasma on antibody binding, the inventors performed a competition assay using recombinant soluble TREM2, anti-TREM2 antibodies, and TREM2-overexpressing Jurkat cells. The results demonstrated that antibody binding was unaffected at physiological concentrations of soluble TREM2 (FIG. 30E). A reduction in binding was observed only at supraphy siological concentrations exceeding I O3M. Despite these drastic molecular effects indicating the strong reprogramming potential of anti-TREM2 treatment, anti-TREM2 monotherapy treatment of mice with MC38 tumors showed limited success in blocking tumor grow th, as assessed by tumor volume and tumor weight measurements (FIG. 30F. G). Similar results of poor efficacy despite effective molecular reprograming of TAMs were previously observed in the inventors’ own work26and the studies of Molega et al.39and Park et al.40, using different antagonistic murine anti-TREM2 molecules.[000517] To overcome the limitation of TAM-monotherapies, the inventors designed a myeloid-targeted immunocytokine, consisting of the inventors’ anti-TREM2 antibody fused to IL- 2 aiming to act on both TAM and effector lymphocyte compartments (FIG. 23A). To modulate IL- 2 receptor binding properties and enhance the anti -tumor activity of IL-2 while minimizing its protumor effects, the inventors incorporated a synthetic IL-2 variant (“Superkine” (SK)51). This variant is engineered to exhibit increased affinity for the IL-2RP subunit, resulting in a preferential expansion of effector T cells over Tregs, compared to wild-type IL-2. Notably, the Fc region of the inventors’ antibodies and immunocytokines was mutated (Fc-Null (N297A)) to abrogate FcyR binding and Fc-effector functions such as antibody-dependent cellular cytotoxicity (ADCC) andphagocytosis. Given the well-established role of IL-2 as a potent activator of lymphocytes52-54, the inventors further evaluated the potential of IL-2 for TIL recruitment and activation and analyzed TCGA data using the TIMER2.0 database to examine the correlation between IL-2 levels and immune cell infiltration across all TCGA cancers. This analysis revealed a strong association between elevated IL-2 expression and activated T and NK cell phenotypes, as opposed to naive states, across a wide range of human cancer types (FIG. 31 A). The anti-TREM2-IL-2SK immunocytokine showed high yields and purity as validated using SDS-PAGE analysis (FIG. 3 IB). In all following experiments, the inventors used an anti-RSV antibody targeting the Respiratory Syncytial Virus fusion protein as an isotype-matched control to account for nonspecific antibody effects. The inventors further confirmed the affinity and specificity of the anti- TREM2-IL-2SK molecule to recombinant TREM2 via SPR (FIG. 31C) and ELISA (FIG. 23B) as well as in a cell-based assay using HEK cells overexpressing human TREM2 by FACS (FIG. 3 ID). To validate the binding of the IL-2SK to IL-2RP, the inventors used a soluble recombinant IL2RP in a sandwich SPR setting (FIG. 31C), demonstrating that the enhanced binding affinity is preserved (aTREM2-IL2SK: KD = 4.94 x 1O10M, WT IL-2: KD = 2.80 x 107M). Moreover, the inventors measured the bioactivity of anti-TREM2-IL-2SK using the HEK Blue CD 122 / CD 132 reporter system, showing similar activation profile to recombinant IL-2 (FIG. 23C). Lastly, the inventors verified the inventors' molecule in a dual binding assay in vitro by engaging HEK cells overexpressing human TREM2. which were incubated with the inventors’ immunocytokine, and then excessively washed followed by incubation with HEK Blue CD 122 / CDI 32 reporter cells, demonstrating full activity of the Fab and IL-2SK components of the molecules in an in vitro coculture system. (FIG. 3 IE).[000518] After confirming the activity of anti-TREM2-IL-2SK in vitro, the inventors intraperitonealy (IP) administered the molecule to MC38-tumor-bearing mice in vivo comparing it to anti-RSV and anti-RSV-IL-2SK controls. However, treatment with two doses of 100 pg of anti-TREM2-IL-2SK or anti-RSV-IL-2SK resulted in mortality (n=7 / group) (FIG. 31F). To investigate the underlying cause of death, the inventors injected 200ug anti-TREM2-IL-2SK into naive mice. The inventors assessed the induction of cytokines and liver enzymes associated to systemic cytokine toxicities in response to anti-RSV, anti-RSV-IL-2SK, anti-TREM2, anti- TREM2-IL-2SK antibodies. Mice treated with anti-RSV-IL-2SK or anti-TREM2-IL-2SK experienced dramatic systemic toxicity' as assessed by elevated levels of the cytokines IFN-y, IL- 2, IL-6 (FIG. 23D) as well as ALT / AST levels in the serum (FIG. 23E).[000519] In summary', the inventors developed an anti-TREM2-IL-2SK immunocytokine to reprogram TAMs and activate T cells in the TME, showing effective reprogramming of TAMs andactivation of T cells in vitro. However, systemic administration in vivo was associated to lethal cytokine-dependent toxicity.EXAMPLE 16Development of on target cytokine activation modality dependent on TAM-specific protease[000520] To circumvent systemic toxicity, the inventors revised the inventors’ strategy to develop a IL-2SK domain that is inactive (pro-cytokine) in the periphery and is activated in proximity to TAMs. Recently, the concept of pro-cytokines, cytokines fused to a blocking domain via a protease cleavable tinker allowing tissue-specific cytokine activation has emerged as a strategy to limit systemic toxicities of cytokine monotherapies55-62. The inventors sought to incorporate a similar strategy for the anti-TREM2-immunocytokine by incorporating a protease- cleavable tinker between the antibody and a blocking moiety which masks the fused IL-2SK and functions as a tumor macrophage-specific switch.[000521 ] scRNA-seq analysis of protease expression across diverse immune cell populations from the inventors’ human cancer atlas44-46 versus peripheral blood mononuclear cells (PBMCs)63 from 108 healthy donors and adjacent healthy-like tissue revealed that several proteases are enriched in TAMs (FIG. 24A, FIG. 32A). Overall, TAMs exhibited enhanced expression levels of proteases such as MMP14, MMP9, MMP19, ADAM9, and cathepsins (CTSB, CTSL, CTSK) compared to their counterparts in PBMCs or adjacent tissue. MMP14 stood out due to its high expression levels, specificity for TAMs, and minimal background activity in other immune cells , making it an ideal candidate for TAM-specific cytokine activation. This observation was further validated using a large single-cell atlas of 13,818,851 single cells, incorporating PBMCs (n=l,753), healthy tissues (n=147), tumor-adjacent samples (n=750) as well as tumors (n=2791) (FIG. 32B-C, Table S2). Consistent with previous reports, MMP14 is expressed to some extent by fibroblasts64. However, the inventors observed a marked upregulation of MMP14 in TAMs in tumor tissues, in comparison to healthy tissues and blood, characterized by both increased expression levels and a higher fraction of expressing cells. Next, the inventors analyzed bulk RNA sequencing dataset from over 10.000 patients across 14 different human cancer types from the TCGA research network (https: / / www.cancer.gov / tcga) and healthy human subjects tissues from Genotype-Tissue Expression (GTEx) Project (https: / / www.gtexportal.org), showing that MMP14 is significantly enriched in tumors compared to any healthy tissue (FIG. 32D). These findings are in line with similar observations by others showing the unique expression of MMP14 in tumor and tumor-draining lymph nodes65. Single-cell analysis of macrophages isolated from the TME, versus healthy tissue macrophages (peritoneum) or splenic monocytes from mice bearing MC38or MCA205 tumors, confirmed that the expression of MMP14 is conserved in mice and confined to TAM and absent from tissue macrophages and monocytes (FIG. 24B). Weighted co-expression correlation analysis of MMP14 and TREM2 in both human cancer and murine tumors (MC38 and MCA205) further showed that MMP14 has the strongest correlation with TREM2 in comparison to other proteases (FIG. 24C). Immunofluorescence analysis of matched human lung tumor and tumor-adjacent tissues confirmed enriched co-localization of TREM2 and MMP14 within the tumor compartment, with minimal overlap in adjacent tissue (FIG. 32E, F). Given this data, the inventors designed a new myeloid targeted therapeutic approach; myeloid-targeted immunocytokines, termed MiTEs. The inventors ultimately developed four immunocytokine variants: MiTE76, MiTE95, MiTE144, and MiTE208. each engineered with one or two human IL- 2SK molecules and one or two extracellular domains of human IL-2RP, which serve as blocking units due to their high affinity for IL-2SK. These blocking domains are linked via one or two cleavage sites that are specifically recognized and cleaved by the TAM-associated protease MMP14 (FIG. 24D). To promote efficient and stable heterodimerization of MiTE-144 and MiTE- 208, the inventors employed the knob-into-hole engineering strategy commonly used in bispecific antibodies66.[000522] Following verification of antibody integrity' by SDS-PAGE analysis (FIG. 32G), the inventors evaluated the efficiency of the inventors’ MMP14-sensitive linker (SGRSENIRTA) which the inventors selected based on the comprehensive substrate specificity’ screen67 demonstrating high hydrolysis efficiency and specificity for MMP 14. The inventors first conducted in vitro digestion assays for all four MiTE constructs, followed by SDS-PAGE analysis (FIG. 32H). All constructs demonstrated efficient cleavage upon MMP14 treatment, resulting in the release of the blocking domain. To assess substrate specificity, MiTE144 which contains a single MMP14 cleavage site, was incubated with MMP3, MMP7, MMP10, MMP11, or MMP14 (FIG. 321, J). MMP 14 induced robust, time-dependent cleavage, whereas the other MMPs exhibited little to no activity, or nonspecific digestion over time (FIG. 321, J).[000523] Next, the inventors evaluated the binding affinity and specificity’ of the MiTE molecules to human TREM2. All four MiTE designs exhibited high affinity and specificity comparable to the parental anti-TREM2 antibody, with no detectable impact from the incorporation of pro-cytokine domains (FIG. 33A-C). The inventors then assessed binding to recombinant IL-2RJ3 by comparing intact versus MMP14-digested MiTE molecules using SPR (FIG. 33D). Following cleavage, the synthetic IL-2SK domains displayed markedly enhanced binding affinity to IL-2R with dissociation constants (KD) of 1.26 x 109M (MiTE-76), 7.88 x 109M (MiTE-95), 2.6 x 109M (MiTE-144), and 14.52 x 109M (MiTE-208) compared to wild-type IL-2 (KD = 2.80 x | o7M). In their intact form, MiTEs showed no detectable binding to IL- 2R0. However, full binding activity was restored following pre-treatment with recombinant human MMP14, confirming successful protease-dependent activation.[000524] The inventors then evaluated the downstream activity' of the different MiTE designs using a HEK-Blue CD122 / CD132 in vitro reporter system, where the inventors observed a 3-log reduction in IL-2SK activity in several of the inventors’ leading blocking moiety designs. After incubation with recombinant human MMP14, IL-2SK bioactivity levels of the pro-cytokine forms of the MiTE were fully restored, matching the activity' of the non-blocked aTREM2-IL-2SK (FIG. 24E). To further validate these findings, the inventors investigated whether MiTE molecules can stimulate primary human T cells upon cleavage of the blocking moieties. Using flow cytometry to measure T cell proliferation, the inventors found that incubation of cells with non-cleaved (nonpretreated) MiTEs resulted in low CD4 and CD8 T cell proliferation (similar to control levels), whereas protease-treated MiTEs induced strong, dose-dependent proliferation, mimicking the effect of the (unmasked) anti-TREM2-IL-2SK or recombinant human IL-2 (FIG. 24F-G). All MiTE constructs induced robust T cell proliferation, but only following MMP14-mediated cleavage, confirming the requirement for protease activation. Notably, MiTE-144 and MiTE-208 exhibited lower background proliferation and activation across all T cell subsets in their uncleaved, pro-cytokine form compared to MiTE-76 and MiTE-95. This observation was further confirmed by assessing CD25 expression as an activation marker in both CD4 and CD8 T cells, where protease-activated MiTEs restored CD25 upregulation to levels comparable to positive control conditions (FIG. 33E-F). To evaluate the effects of MiTEs on Tregs, the inventors analyzed proliferation and activation states on CD69 expression in CD4+CD25high and CD4+CD251ow T cell subsets (FIG. 33G,H). All MiTE variants showed varying degrees of activity on Tregs. with MiTE144 exhibiting comparatively lower levels of proliferation and CD69 upregulation. Furthermore, unmasked MiTEs triggered strong IFN-y secretion in human T cells in vitro, whereas masked MiTEs elicited minimal response (FIG. 331).[000525] Importantly, in vivo administration of a single dose (50ug) of the inventors' MiTEs resulted in no observable toxicity, including no observed effects on body weight loss (FIG. 33J) nor elevated IFN-y cytokine serum levels, a cytokine commonly associated with toxicity7in cytokine-based therapies (FIG. 33K). When administered intratumorally (5ug / dosage), all four MiTE molecules demonstrated similar efficacy in abrogating MC38 tumor growth (FIG. 33L). Overall, these findings validate the successful development of trans-acting MiTE molecules with cytokine activity that is specifically activated by a TAM-associated protease. This design confersrobust tumor-restricted activation while minimizing off-target toxicity, supporting the therapeutic potential of MiTEs in targeted cancer immunotherapy.EXAMPLE 17MiTE robustly abrogates tumor growth without inducing systemic toxicities[000526] To evaluate any off-target toxicity and effects of the MiTE molecules in vivo, the inventors selected MiTE144, as it exhibited the lowest pro-cytokine IL-2 activity in vitro and in vivo, without significant reduction of on-target activity (FIG. 24F-G. FIG. 33E-K) and administered it intravenously at 200 pg / dose every three days in the syngeneic MC38 tumor model starting at day ten after tumor inoculation. Even at these therapeutic relevant concentrations and repeated doses, MiTE144 demonstrated no observed toxicities, with no observed effects on body weight (FIG. 25A), circulating cytokine levels (FIG. 25B), as well as ALT / AST enzymes levels in the serum (FIG. 25C), indicating an absence of systemic inflammation or liver toxicity. Additionally, no spleen enlargement was observed (FIG. 34A), and MiTE144 did not lead to an increase in total CD8 T cell or NK cell numbers per gram of spleen tissue (FIG. 34B), further demonstrating no observable off-target immune activation. To further characterize the in vivo profile of MiTE144. the inventors assessed its pharmacokinetics and biodistribution. MiTE144 displayed a slower elimination rate and extended half-life (11.88 h) compared to aTREM2-IL2SK (9.43 h), with an estimated clearance time of 60-72 hours (FIG. 34C). Biodistribution analysis revealed preferential accumulation of MiTE144 in the tumor, with limited uptake in non-target tissues , including brain, lung, kidney, spleen and liver (FIG. 34D). In addition, histopathological analyses of MiTE144 treatment in naive healthy mice did not induce inflammation in the liver and lungs as opposed to aTREM2-IL-2SK (FIG. 34E). These results collectively demonstrate that MiTE144 has no systemic activity and high systemic tolerability', reinforcing its potential as a safe and effective cytokine-based therapy for cancer treatment.[000527] To evaluate the in vivo efficacy of MiTE144, the inventors treated mice bearing syngeneic MC38 tumors with 200 pg of MiTE144 on days 8, 10, and 13 post-tumor implantation. Control groups received anti-RSV, anti-TREM2, or anti-RSV-IL-2SK masked antibodies, a construct containing the same blocking domain and MMP14-cleavable linker as MiTE 144, but fused to a non-targeting Fab. As previously observed, anti-TREM2 showed only minor efficacy while treatment with anti-RSV-IL-2SK-masked led to reduced tumor growth. Importantly, MiTE144 almost completely abrogated MC38 tumor grow th demonstrating synergistic effects in comparison to anti-RSV-IL-2SK-masked and anti-TREM2 monotherapies (FIG. 25D, FIG. 35A). The limited efficacy of aRSV-IL-2SK-masked, highlights the importance of TREM2 targeting forthe full therapeutic potential of MiTEs. Comparing the efficacy of MiTE144 with commonly used ICIs, showed that MiTE144 outperformed multiple ICI monotherapies, including anti-TREM2, anti-PD-1 (FIG. 25E, FIG. 35B) and anti-CTLA-4 (FIG. 25F, FIG. 35C), as well as the combinations of anti-CTLA-4 or anti-PD-1 with anti-TREM2, indicating a more robust immune activation via dual TAM-T cell targeting cytokine-based therapies than current checkpoint blockade strategies alone. Interestingly, combination of MiTE144 with anti-CTLA-4 (FIG. 25F, FIG. 35C), could even further enhance the efficacy of MiTE144 leading to complete eradication of MC38 tumors in 6 out of 7 mice.[000528] These findings were further validated in the syngeneic MCA205 tumor model (FIG. 25G, FIG. 35D), where MiTE144 exhibited similarly superior tumor control in comparison to anti- TREM2, anti-RSV-IL-2SK (masked) and RSV controls. Measurement of tumor weights across conditions reinforced these observations, confirming that MiTE144 consistently achieves strong control of tumor growth. Collectively, these results highlight the high therapeutic potential and safety profile of myeloid-targeted immunocytokines.EXAMPLE 18MiTE-based therapies robustly reprogram the tumor microenvironment[000529] To characterize the cellular composition and transcriptional states of immune cells within the TME and to understand the mechanism of action of MiTE144, the inventors assessed scRNA-seq profiles of 45,505 high-quality leukocytes sorted from 45 MC38 tumor-bearing hTREM2 mice on day 1 1 after tumor inoculation (FIG. 36A,B). Mice which were i.v. treated on day 8 and 10 with two injections of 200ug anti-RSV, anti-TREM2, MiTE144, anti-PD-1, anti- CTLA-4 and or combination therapies which included: anti-TREM2 + anti-PD-1, anti-TREM2 + anti-CTLA-4, MiTE144 + anti-PD-1 or MiTE144 + anti-CTLA-4 on day 8 and 10. Cluster annotation was performed by assessing the expression of top genes per cluster (Table S3) as well as the expression of canonical marker genes, allowing for the identification of major cell types and subpopulations (FIG. 26 A, B). The inventors identified 6 Macrophage clusters including different states of TAMs (hypoxia, interferon-response, Clq, antigen presentation, proliferating) and moMACs, two subsets of DCs and as well as granulocytes in the myeloid compartment. The inventors found four clusters of NK cells (early 1, early 2, late and proliferating NK cells), four subsets of T cells (early activation, effector, effector proliferating and effector exhausted) and three activation states of Tregs (early activation, effector and effector proliferating). Mapping the gene expression of MiTE-related targets onto distinct cell subsets confirmed high co-expression of (human) TREM2 and MmpI4 in TAMs in hTREM2 mice (FIG. 26C). Expression of interleukin-2 receptor subunits I12rb and I12rg was found across all T and NK cell subsets, while I12ra (CD25) was, as expected, highly enriched in regulatory T cells. Pdcdl (PD-1) and Ctla4 (CTLA-4) were mostly detected in different T cell and Tregs subsets.[000530] Subsequently, the inventors applied a deep generative model (MrVI) 68 and constructed a treatment-specific distance similarity network to reveal how different treatment conditions affect the immune landscape of the TME (FIG. 26D, FIG. 36C. Table S4). Treatments inculding MiTE144 (MiTE144 alone, MiTE144+anti-PD-l, and MiTE144+anti-CTLA-4) formed a distinct component cluster, suggesting a unique molecular signature associated with this therapy. Similarly, treatments involving anti-PD-1 (anti-PD-1 alone, anti-TREM2+anti-PD-l) as well as anti-CTLA-4 (anti-CTLA-4 alone. anti-TREM2+anti-CTLA-4) were closely related, highlighting a consistent immune response pattern induced by each individual immune checkpoint inhibitor. Correspondingly, the analysis of differentially expressed genes (DEGs) across all broad categories of immune cell subsets (Macrophages, DCs, NK cells, Tregs, T cells and Granulocytes) in response to different treatment conditions, compared to the control aRSV (padj < 0.05, LogFC > 1 or < -1), showed that MiTE144-based therapies (MiTE144 alone, MiTE144+anti-PD-l, and MiTEl 44+anti-CTLA-4) exhibit the most extensive transcriptional reprogramming, particularly in T and NK cells as well as macrophages (FIG. 26E, DEGs: Table S5-10). Macrophages (1,451 genes) and NK cells (2,018 genes) showed the highest DEG counts across treatments, highlighting their responsiveness to the therapeutic interventions. T cells (884 genes) and DCs (235 genes) also displayed significant transcriptional alterations, while granulocytes (19 genes) and Tregs (122 genes) exhibited less treatment-induced transcriptional reprogramming.[000531 ] To analyze the effects of different treatments on the myeloid compartment, the inventors assessed the proportional distribution of myeloid cell subsets across treatment groups (FIG. 26F, G, FIG. 36D). MiTEl 44-based treatments (MiTE144 alone, MiTEl 44+anti-PD-l, and MiTEl 44+anti-CTLA-4) induced the most substantial perturbation in the myeloid composition, leading to a marked reduction in TAMs associated to interferon response and DCs, while promoting an expansion of hypoxia-associated TAMs and granulocytes (FIG. 26F. G, FIG. 36D). Sub-clustering of granulocytes revealed that this population consists predominantly of neutrophils , with a smaller subset of basophils (FIG. 36E, F). MiTE144 and MiTE144 + anti-PD-1 treatment specifically expanded the neutrophil compartment (FIG. 36G), particularly enriching for neutrophils with an anti-tumor, beneficial T2 phenotype as described in Ng et al. 69 (FIG. 36H-J). In contrast, while anti-TREM2 treatment alone exhibited a trend towards expansion of TAMs associated to interferon response (FIG. 26G), anti-TREM2 (anti-TREM2 alone, anti-TREM2+anti- PD-1, and anti-TREM2+anti-CTLA-4) or ICI treatments (anti-PD-1 alone, anti-CTLA-4 alone)mostly exhibited a myeloid distribution comparable to the anti-RSV control. In line with the TME analyses, MiTE144 treatments led to an expansion of the number of CD45 immune cells as well as cell subsets such as monocytes, granulocytes indicative of an inflammatory response in tumordraining lymph nodes (tdLNs) as assessed by Flow Cytometry (FIG. 37 A, S9B).[000532] To better understand the treatment responses within the macrophage compartment in the TME. the inventors assessed Hotspot70 gene modules using the treatment-aware latent representation (z) from MrVI (FIG. 26H, Table S4) as well as DEGs expressed in macrophages (FIG. 261, J, Table S5). As expected, anti-CTLA-4 treatments (alone or in combination with anti- TREM2) induced the expression of a gene module associated to FcyR signaling and proliferation as well as a unique MrVI module expression pattern in combination with MiTE144 (FIG. 26H, I, Table S3, 4). MiTE-based treatments showed a strong pattern of MrVI module expression (FIG. 26H) and DEGs (FIG. 261, J, Table S4, 5). MiTE144 treatments induced upregulation of inflammatory, monocyte-like (1 and 2) and hypoxia associated gene modules (FIG. 26H) which was corroborated in an upregulation of genes such as S100a8, Hp, Ly6i (monocyte-like, inflammatory) and Argl, Nos2, Hmoxl (hypoxia) (FIG. 261). While macrophages from anti- TREM2 -treated groups (anti-TREM2 alone, anti-TREM2+anti-PD-l, and anti-TREM2+anti- CTLA-4) showed enrichment in a module associated to IFN response, this module and genes associated to interferon response (Ifitl, Ifit2, Ifit3b) were strongly reduced in MiTE144 treatment groups (FIG. 261, J). In addition, MiTE144 treatment reduced the expression levels of a suppressive module associated with hTREM2, as well as hTREM2 and Mmpl4 expression in Macrophages, while inducing enhanced expression levels of Treml and Trem3. These observations suggest that MiTE144 treatments induce a shift from suppressive TAMs towards potentially earlier and more inflammatory’ and monocytic cell states.[000533] In the DC cell compartment, MiTEl 44-based treatments induced genes associated to MHC I antigen presentation (e.g. H2-Q5, H2-Q6, H2-Q7, H2-K1) and development of cDCls (e.g. Irfl, Id2, Irf8, NT113), while downregulating monocyte-derived DC genes (e.g. SlOOal l, Ms4a6c, Fcgrl, Fcerlg) and interferon response genes (eg. Ifit2, Ifit3b) (FIG. 37C-D, Table S6). Interestingly. DCs isolated from tumors treated with MiTE144 alone and MiTE144 + anti-PD-1 showed reduced expression of genes associated to MHC-II presentation (eg. H2-Aa, H2-DMbl). This was not observed if MiTE144 w as combined with anti-CTLA-4 treatment.[000534] In summary , MiTEl 44 induces a strong and consistent reprogramming of the TME including the induction of inflammatory, more monocytic gene programs in TAMs and crosspresentation associated genes in DCs. Combination therapy with anti-CTLA-4 induces additional reprogramming effects.EXAMPLE 19MiTE limits NK and T cell exhaustion and enhances their cytotoxicity and proliferation in the TME[000535] Besides its strong effects on the myeloid compartment, the inventors observed that MiTE 144 treatment results in strong phenotypic and molecular reprogramming of the NK and T cell compartments in the TME and tdLNs (FIG. 27A. FIG. 38A-C). MiTE144 treatment alone or in combination with anti-PD-1 or anti-CTLA-4 induced activation and proliferation in NK cells, while reducing the frequencies of early NK activation states (FIG. 27B, C; FIG. 38A). Gene expression analysis revealed a broad molecular reprogramming of NK cells, characterized by the upregulation of late and activation (eg. Klrgl. Ctla4). cytotoxic (eg. Prfl (Perforin- 1), and proliferation genes (eg. Mki67, Mcm3, McM4). This was accompanied by downregulation of early, exhaustion and dysfunction-associated genes (eg. Cd27, Tigit, Tnfrsfl8) (FIG. 27D, E, Table S7). In line with the proliferative phenotype, total counts of NK cells were significantly elevated in the TME (FIG. 38B) and across all MiTE conditions in the tdLNs as assessed by Flow cytometry (FIG. 38C). The inventors’ findings suggest that MiTE144 treatments induce a shift from early toward a highly proliferative and cytotoxic NK phenotype allowing sustained NK cell-mediated anti-tumor immunity7. To further validate the inventors’ in vivo findings, the inventors performed an in vitro assay using human NK cells, demonstrating that cleaved MiTE144 robustly induces NK cell activation, as evidenced by elevated secretion of IFN-y, IL-2, and multiple cytotoxic effector molecules, including Granzyme A, Granzyme B, Perforin, FasL, and Granulysin (FIG. 38D).[000536] In the CD4 T cell compartment, despite the use of the IL-2SK w hich was designed to enhance effector T cell expansion over Treg activation in comparison to WT IL-251, MiTE 144 treatments slightly increased regulatory T cells (Tregs) frequencies in the TME (FIG. 27F) and drastically elevated Treg numbers in the tdLNs (FIG. 38F). However, this effect was effectively abrogated by combining anti-CTLA-4 treatment with MiTE144 which depleted Tregs as previously reported 71,72 (FIG. 27F, FIG. 38F).[000537] In the CD8 T cell compartment, MiTE144 induced an increase in CD8 T cell proliferation and T cell sternness (FIG. 27G) as well as total T cell count in the TME (FIG. 27H) and tdLNs (FIG. 38F). In particular, the inventors observed a shift toward increased CD8+T cell counts and elevated CD8+ / CD4+T cell ratios within the TME (FIG. 27H, FIG. 38G). These effects were even more pronounced in a combination treatment of MiTE144 with anti-CTLA-4. At the transcriptional level, MiTE144 promoted the upregulation of cytotoxic, proliferation and sternness associated genes (eg. Gzma, Mki67, Tcf7) and high levels of GITR protein expression in CD8 Tcells as assessed by flow cytometry, while simultaneously downregulating exhaustion-associated genes such as Tox (FIG. 27I-J, FIG. 38H, Table S8).[000538] Next, the inventors assessed if the TAM-T cell ligand receptor interactions which the inventors identified in human cancers (FIG. 22M, N) are reduced upon MiTE treatment. First, the inventors identified immunosuppressive immune interactions which are enriched in anti-RSV- treated controls compared to respective MiTE-treated tumors and cross-referenced these TAM-T cell interactions to human tumors. This comparison revealed that MiTEs and their combinatorial treatments with anti-PD-1 or anti-CTLA-4 disrupt multiple TAM-T cell immunosuppressive interaction axes, including Cd86-Ctla4, Pdcd-Cd48, and Ccr8-Ccl3 which are associated with T cell exhaustion and T cell recruitment. Additionally, they downregulate Lgalsl-Cd69. Lgals3bp- Itgbl, and Tgfbrl-Tgfbl interactions, potentialy reducing suppressive signaling, tissue retention, and persistent cell-cell contacts that sustain immune dysfunction within the TME (FIG. 27K).[000539] These results demonstrate that MiTE 144 treatment profoundly reprograms the tumor immune landscape, leading to enhanced NK cell activation and cytotoxicity, increased CD8+ T cell proliferation, and the induction of a stem-like T cell phenotype, while reducing exhaustion and immunosuppressive programs. Furthermore, the combination of MiTE144 with checkpoint blockade, especially anti-CTLA-4 amplifies these effects, by depleting Tregs, leading to a enhanced cytotoxic CD8 response and persistent tumor control.EXAMPLE 20MiTE treatment efficiently remodels the immune landscape of human patient-derived tumor fragments[000540] To investigate the effects of MiTE 144 on human tumors, the inventors treated patient-derived tumor fragments (PDTFs) 73 from five renal cell carcinoma (RCC) patients ex vivo with IgG controls, MiTE144, anti-PD-1, and combination of MiTE144 and anti-PD-1. Tumor fragments were cultured for 48 hours, after which CD45 ' immune cells were enriched and subjected to CITE-seq, enabling integrated transcriptomic and surface protein profiling (146 markers) of 93,087 high-quality single cells across all treatment conditions.[000541 ] The inventors applied totalVI74 to integrate transcriptomic and proteomic modalities, enabling unified analysis of mRNA and surface protein expression. Unsupervised clustering of the integrated latent space revealed a complex immune landscape, comprising diverse T cell states (CD4y CD8+, memory, cycling, dysfunctional, and regulatory T cells), distinct NK cell maturation states (early, intermediate, late), monocyte-like cells, dendritic cells (DCs), and tumor-associated macrophage subsets (TAMs, SATl-like TAMs, hypoxic TAMs) (FIG. 28A). Theinventors used canonical markers for cell type annotations using both mRNA and protein expressions, including surface markers such as TREM2, PD-1, CD25, ICOS, CD56 and CD132 (FIG. 28B, C, FIG. 39A).[000542] MiTE144 monotherapy and combination with anti-PD-1 induced striking changes in immune cell composition and distribution. Treatment enrichment density plots highlighted global shifts of immune cell populations (FIG. 28D). In order to denve patient-level statistics, the inventors first assessed the treatment response effect of each individual PDTF by deriving a global treatment response score (Methods) and the inventors removed a non-responder with minimal treatment effects across all conditions (FIG. 39B, C). The immune landscape compositional effects were evident across the rest of the patients, as shown by patient-specific immune cell type distributions (FIG. 28E). Quantitative assessment of cell type abundance demonstrated consistent expansion of CD8+cycling and memory T cells, as well as intermediate-stage NK cells in both MiTE144 and MiTE144+anti-PD-l conditions, while immunosuppressive subsets such as Tregs and hypoxic TAMs remained unchanged or decreased relative to the control (FIG. 28F,G, FIG. 39D). These results indicate a selective enrichment of pro-inflammatory lymphoid subsets without concurrent Treg expansion.[000543] Similar to the effects in the murine tumor model, MiTE144 and MiTE144+anti-PD- 1 treatments induced a coordinated immune activation program in both T and NK cells, marked by enhanced cytotoxicity, proliferation, and cytokine responsiveness (FIG. 40A, FIG. 28H). In T cells, treatment with MiTE144 resulted in robust upregulation of cytolytic effector genes, including PRF1, GZMA, and GZMB, with similar patterns observed in the combination treatment with anti- PD-1. This cytotoxic activation was accompanied by increased expression of cell cycle-associated genes such as MCM3. MCM4, MCM6. and CCND2. Importantly, expression of ICOS. CTLA4, and TOX markers associated with T cell exhaustion was reduced relative to control conditions. In NK cells, MiTE144 induced a similarly potent cytotoxic program, characterized by strong upregulation of PRF1, GZMB, GZMH, and lysosomal proteases CTSA and CTSD. Expression of proliferation-associated genes, including MCM3. MCM4, and CCND2, further indicated robust NK cell expansion upon treatment. MiTE144 also downregulated key exhaustion-related genes such as CXCR4, DUSP1, and TNFAIP3, which was further amplified in combination with anti- PD-1. CITE-seq protein profiling revealed upregulation of key activation and cytokine receptor components, including CD25 (IL-2Ra) and CD122 (IL-2 / IL-15RP) in both T cells and NK cells upon MiTE144 treatment (Fig S12B). Both treatments led to increased surface expression of activation markers CD69, NKG2D and Tim-3, reflecting enhanced NK cell activation (FIG. 40B). Notably, in contrast to the inventors’ observations in the murine tumors, PD-1 and LAG-3 proteinlevels were mildly increased in both T and NK cells across MiTE144 and combination treatments compared to control. However, PD-1 and LAG-3 protein expression was reduced under combination treatment with anti-PD-1, suggesting beneficial synergy of MiTE and anti-PD-1 treatments (FIG. 40B). Transcriptomic profiling revealed broad MiTE144-induced activation across immune subsets, including DCs, macrophages, Tregs, and cytotoxic lymphocytes, with conserved upregulation of inflammatory and effector programs (FIG. 40A). Cross-species global molecular comparisons with the inventors’ murine treatment data revealed a conserved immunostimulatory response between human and mouse immune programs (FIG. 40C), indicating that MiTE144 elicits consistent and translatable immune activation across species.[000544] Overall, integrated analyses of murine and human tumor microenvironments demonstrate that MiTE molecules have the potential to reprogram the myeloid compartment, unleash robust NK and T cell effector responses, and enable effective tumor control (FIG. 281)EXAMPLE 21TREM2-targeting conditionally masked immunocytokines can be successfully produced in monovalent-IL2superkine and bivalent IL15 formats[000545] Recombinant TREM2-targeting immunocytokines can be produced with high yield and purity in two different formats, including bivalent IL15 format (FIGs 41A and 42A. E3C7- IL15-MV1) and a monovalent IL2-superkine format (FIGs 41 B and 42B, MiTE144). SDS-PAGE analysis of final recombinant protein product for each masked E3C7 immunocytokine prepared under reducing (R) and non-reducing (NR) conditions shows high purity with bands at the expected molecular weights.[000546] The sequence of the E3C7-IL15-MV1 immunocytokine, which is composed of two variable chains and two heavy chains (labeled E3C7_IgGl_N297A_IL15Ra_IL15_ MMP14clxl_IL15RB(6-208)), is as follows:[000547] Heavy Chains:QVQLQQPGAEPLKPGASVKMSCEASGYTFTNFWITWMKLRPGQGLEWIGDIYPGTGNT NYNEKFKSRATLTVDTSSTTAYMQLSSLTSEDSAVYYCAREAYYTNPGFAYWGHGTLV TVSSASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAV LQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPCPAPE LLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPR EEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLT VDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKETAAAKFERQHMDSITCPPPMS VEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDP ALVHQRPAPPSGGSGGGGSGGGSGGGGSGGNWVNVISDLKKIEDLIQSMHIDATLYTES DVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEE LEEKN1KEFLQSFVH1VQMF1NTSGGGGSSGFIANPVTAGGGGSYPYDVPDYASQFTCFY NSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQ KLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQ ASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDTQYEFQVRVKPLQG EFTTWSPWSQPLAFRTKPAA.[000548] Light Chains:DVLMTQSPLSLPVNLGDQASISCKSSQNIVHSNGNTYLEWYLQRPGQSPKLLIYKVSNRF SGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSV FIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYS LSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGECEXAMPLE 22Conditionally masked TREM2-targeting immunocytokines retain binding to TREM2[000549] E3C7-IL15-MV1 and MiTE-144 were incubated for 20h at 37°C in presence or absence of recombinant MMP-14. Subsequently, TREM2 binding capacity was measured either by ELISA, using plates coated with recombinant human TREM2 (FIG. 43A and FIG. 43C), and by cell surface binding assay utilizing Jurkat cell line engineered to express human TREM2 (FIG. 43B and FIG. 43D). A non targeted control immunocytokine (Pali-IL2SK) is shown for reference (FIG. 43A and FIG. 43C). The data demonstrate that TREM2 targeting immunocytokines in both E3C7-IL15-MV1 and MiTE144 format retain specific antigen binding to TREM2 in both the presence or absence of MMP14-mediated demasking.EXAMPLE 23Conditionally masked TREM2-immunocytokines conditionally are effectively demasked by rMMP14 in vitro[000550] MMP14-mediated demasking is evaluated by incubating immunocytokines with recombinant MMP14 under the indicated conditions and analyzing the resulting product usingSDS-PAGE under reducing conditions. The percent of specific demasking was quantified by calculating ratio of de-masked vs masked heavy chain. (FIG. 44A) 0.5 uM of each of the immunocytokines was incubated with increasing amount of MMP14 for 21h at 37°C; The data showed that both immunocytokine formats are effectively de-masked by lug of recombinant MMP14. (FIG. 44B) 0.5uM of immunocytokines were incubated with 0.45uM of MMP14 for indicated periods of time and compared to the complete digestion condition of 0.89uM for 21 hours at 37°C. The data showed that MiTE144 format is cleaved more rapidly than the E3C7-IL15-MV1 format.EXAMPLE 24MMP14 treatment restores cytokine activity of conditionally masked TREM2- immunocytokines[000551 ] E3C7-IL-15-MV1 (FIG. 45A) and MiTE-144 (FIG. 45B) were incubated for 20h at37C in presence or absence of recombinant MMP- 14. then diluted and subsequently added to the HEK Blue CD 122 / CD 132 (IL-2Rp / y) reporter cell line. Signal was acquired after overnight incubation. Recombinant human IL 15 and IL2 are included as reference controls. The data showed that MMP14-mediated de-masking induces a 10-15 fold increase in cytokine activity, resulting in activity comparable to the cytokine reference controls.EXAMPLE 25E3C7 immunocytokines show MMP14-dependent activation of primary immune cells[000552] Cryopreserved Peripheral Bone Marrow Cells (PBMCs) responded more strongly to immunocytokine with an MMP 14 cleaved mask than immunocytokine with intact mask. PBMCs from four donors were treated with masked or MMP 14 de-masked immunocytokine for 72 hours and concentrations O. lpM, IpM, lOpM, lOOpM, InM, lOnM, and lOOnM. (FIG. 46A) IFNy secretion was assessed through cell supernatant collection with values obtained from Nomic’s nELISA multiplex assay. (FIG. 46B) CD69 expression was assessed through flow cytometry gated on live CD3+, CD8+ T Cells. Data showed that the immunocytokines show dose dependent activity on primary human immune cells following MMP14-mediated demasking.EXAMPLE 26Conditionally masked E3C7-IL15-MV1 eliminates toxic systemic immune activation associated with non-masked IL15 immunocytokines[000553] Similar to our results with MiTE144 (Example 17, FIG. 25B)Masked E3C7-IL15- MV1 construct effectively limits systemic immune activation associated non-masked IL 15 immunocytokines in vivo. Female C57BL / 6 mice were injected twice at a 48 hour interval with the indicated dose of E3C7-IL15-MV1 or amatched non-masked E3C7-IL15 variant as areference control. Serum was collected 24 hours following the second dose and assessed for IFNg levels. The data show that administration of E3C7-IL15-MV1 did not result in systemic elevation of IFNg observed with non-masked controls even at any of the doses tested (even up to 1563pmol).EXAMPLE 27Alternative sequence for E3C7-IL-15sushi with masking moiety:[000554] Heavy chains (SEQ ID NO: 156):MGWSCIILFLVATATGVHSAYMQLSSLTSEDSAVYYCAREAYYTNPGFAYWGH GTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALT SGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKS CDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYASTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGKGGGGSGGGGSGGGGSITCPPPMSVEHADIWVKSYSLYSRERYICNSGFKRKAGTSSLTECVLNKATNVAHWTTPSLKCIRDPALVHQRPAPPGGGSGGGGSGGGGSGGGGSGGGSLQNWVNVISDLKKIEDLIQSMHIDATLYTESDVHPSCKVTAMKCFLLELQVISLESGDASIHDTVENLIILANNSLSSNGNVTESGCKECEELEEKNIKEFLQSFVHIVQMFINTSGGGGSSGRSENIRTAGGGGSAVNGTSQFTCFYNSRANISCVWSQDGALQDTSCQVHAWPDRRRWNQTCELLPVSQASWACNLILGAPDSQKLTTVDIVTLRVLCREGVRWRVMAIQDFKPFENLRLMAPISLQVVHVETHRCNISWEISQASHYFERHLEFEARTLSPGHTWEEAPLLTLKQKQEWICLETLTPDT QYEFQVRVKPLQGEFTTWSPWSQPLAFRTKPAALGKDT[000555] Lights chains (SEQ ID NO: 157):MGWSCIILFLVATATGVHSDVLMTQSPLSLPVNLGDQASISCKSSQNIVHSNGNTYLEWYLQRPGQSPKLLIYKVSNRFSGVPDRFSGSGSGTDFTLKISRVEAEDLGVYYCFQGSHVPYTFGGGTKLEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPR EAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACE VTHQGLSSPVTKSFNRGECREFERENCES1. 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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 which comprises a targeting moiety which binds a marker of a myeloid-derived suppressor cell (MDSC) attached to an immune cell-activating cytokine, wherein said immune cell-activating cytokine is masked by a masking agent, such that when said at least one fusion polypeptide is in a vicinity of an MSDC. said masking agent unmasks said immune cell-activating cytokine via an activity of an MSDC- specific protease so as to allow cytokine activity' of said immune cell-activating cytokine.

2. The composition of matter of claim 1, wherein said MDSC is a tumor associated macrophage (TAM).

3. The composition of matter of claim 1 or 2, wherein said targeting moiety' is attached to said immune cell-activating cytokine directly.

4. The composition of matter of claim 1 or 2, wherein said targeting moiety is attached to said immune cell-activating cytokine indirectly.

5. The composition of matter of claim 4, wherein said targeting moiety is attached to said immune cell -activating cytokine via at least one linker.

6. The composition of matter of any one of claims 1-5. wherein said at least one fusion polypeptide is a homodimer or a homotrimer.

7. The composition of matter of any one of claims 1-5, wherein said at least one fusion polypeptide is a heterodimer or a heterotrimer.

8. The composition of matter of any one of claims 1-7, wherein said masking agent is attached to said at least one fusion polypeptide via a cleavable sequence of at least one protease specific to MDSC.

9. The composition of matter of claim 8, wherein said protease is a matrix metalloproteinases (MMP).

10. The composition of matter of claim 9, wherein said MMP is MMP14.

11. The composition of matter of any one of claims 1-10, wherein said marker is myeloid marker mediates a suppressor function of MDSCs.

12. The composition of matter of claim 11, wherein said targeting moiety is characterized by a binding activity and optionally an inhibitory activity of said suppressor function.

13. The composition of matter of any one of claims 1-12, wherein said marker is selected from the group consisting of TREM2, GPNMB. CSF1R. LILRB1 / ILT2. LILRB2 / ILT4, LILRB4, PDL1, PDL2, CLEVER1, SIRPA, SIGLEC-1, SIGLEC-7, SIGLEC-9, SIGLEC-10, SIGLEC-15, SLAMF7, MRC1 / CD206 , LGALS9 / Galectin-9, VSIG-4 and MARCO.

14. The composition of matter of any one of claims 1-12, wherein said marker is TREM2.

15. The composition of matter of any one of claims 1-14, wherein said cytokine is selected from the group consisting of Interleukin-2 (IL-2), Interferon-alpha (IFN-a), Granulocyte- Macrophage Colony-Stimulating Factor (GM-CSF), Interleukin- 12 (IL-12), Tumor Necrosis Factor-alpha (TNF-a). Interferon-gamma (IFN-y), Interleukin- 15 (IL-15), Interleukin- 18 (IL-18), Interleukin-21 (IL-21) and Granulocyte-Macrophage Colony-Stimulating Factor (GM-CSF).

16. The composition of matter of any one of claims 1-14, wherein said cytokine is Interleukin- 2 (IL-2).

17. The composition of matter of claim 16, wherein said cytokine is modified to have enhanced receptor selectivity to preferentially stimulate effector immune cells.

18. The composition of matter of any one of claims 1-16, wherein said fusion polypeptide activates lymphoid cells selected from the group consisting of T cells (e g., CD4 T cells, NKT and CD8 T cells) and NK cells.

19. The composition of matter of any one of claims 1-18, wherein said targeting moiety is an antibody.I ll20. A polynucleotide encoding the fusion polypeptide of any one of claims 1-19.

21. A pharmaceutical composition comprising as an active ingredient the fusion polypeptide or polynucleotide of any one of claims 1-20 and a pharmaceutically acceptable carrier or diluent.

22. A method of treating cancer in a subject in need thereof the method comprising administering to the subject a therapeutically effective amount of the pharmaceutical composition of claim 21, thereby treating cancer.

23. The pharmaceutical composition of claim 21 for use in treating cancer.

24. The method or composition for use of any one of claims 22-23, wherein said cancer is PD- 1, PD-L1 or CTLA-4-resistant.

25. The method or composition for use of any one of claims 22-23, wherein said cancer is MHC- 1 independent or dependent.

26. The method or composition for use of any one of claims 22-23. wherein said cancer is MHC- 1 dependent.

27. The method or composition for use of any one of claims 22-25, wherein said cancer is overexpressing TREM2 and optionally MMP14.

28. The method or composition for use of any one of claims 22-27, wherein said cancer is selected from the group consisting of brain cancer, breast cancer, colon cancer, esophagus cancer, kidney cancer , liver cancer, lung cancer, ovarian cancer, pancreatic cancer, prostate cancer, skin cancer, stomach cancer, testical cancer and cancer of the uterus.

29. The composition of matter, method, or composition for use of any one of claims 1-28, wherein the fusion polypeptide comprises:(i) an anti-TREM2 antibody, or a TREM2-binding portion thereof; and(ii) one or more cytokines; and, optionally,(iii) a cytokine mask, preferably, an IL2RB polypeptide sequence; preferably IL2RB(6-208) or IL2RB(6-97);wherein (i) and (ii) are covalently linked, directly or through a linker and / or a Sushi Domain of IL15Ra (SD); and (ii) and (iii) are linked by a cleavable or non-cleavable linker, preferably a protease-cleavable linker, preferably an MMP14, MMP12, or MMP11 protease-cleavable linker, or a combination thereof; and wherein, optionally, (i) and (iii) are covalently linked, directly or through a linker, and (ii) is linked by a cleavable or non-cleavable linker, and hence (ii) can be released.

30. The composition of matter or method of claim 29, wherein the anti-TREM2 antibody comprises at least one immunoglobulin heavy' chain and at least one immunoglobulin light chain, wherein the at least one heavy chain comprisesHC-CDR1 : GYTFTNFWHC-CDR2: IYPGTGNTHC-CDR3: AREAYYTNPGFAY; and the at least one light chain comprisesLC-CDR1 : QNIVHSNGNTYLC-CDR2: KVSLC-CDR3: FQGSHVPYT; wherein the antibody is optionally humanized.

31. The composition of matter or method of claim 29, wherein the fusion polypeptide is E3C7- IL15-MV1 or MiTE144.

32. The method or composition for use of any one of claims 22 to 31, further comprising administering an additional therapeutic agent or other therapy prior to, consecutively, and / or concurrently with the fusion polypeptide, wherein the additional therapeutic agent and the fusion polypeptide are in the same composition or in separate compositions.

33. The method or composition for use of claim 32, wherein the additional therapeutic agent or other therapy are selected from immune check point blockage therapy, an anti-cancer therapy, a chemotherapy, an anti-inflammatory agent, a cytokine, adoptive cell therapy, and radiotherapy.

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