Chimeric inhibitory signaling modulators

Abstract

Described herein are fusion proteins comprising an extracellular domain of an immune checkpoint receptor and a cytoplasmic domain of an activating receptor. Also described herein are fusion proteins comprising a cytoplasmic domain of an immune checkpoint receptor and an extracellular domain of an activating receptor. The present disclosure further provides methods of use of such fusion proteins.

Description

[0001] CHIMERIC INHIBITORY SIGNALING MODULATORS

[0002] CLAIM OF PRIORITY

[0003] This application claims the benefit of U.S. Provisional Application Serial No. 63 / 514,626, filed on July 20, 2023. The entire contents of the foregoing are incorporated herein by reference.

[0004] SEQUENCE LISTING

[0005] This application contains a Sequence Listing that has been submitted electronically as an XML file named “29539_0773WOl_ST26_SL.XML.” The XML file, created on July 18, 2024, is 94,999 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.

[0006] TECHNICAL FIELD

[0007] The present disclosure relates to fusion proteins for transforming inhibitory- signals to immune cell activating signals.

[0008] BACKGROUND

[0009] Natural killer (NK) cells are lymphocytes of the innate immune response characterized by their role in the destruction of tumor cells. Activation of NK cells depends on a balance between activating and inhibitory signals mediated by different receptors.

[0010] SUMMARY

[0011] Provided herein are fusion proteins comprising an extracellular domain of an immune checkpoint (IC) receptor and a cytoplasmic domain of DNAX accessory molecule- 1 (DNAM-1).

[0012] In some embodiments, the fusion proteins comprise the extracellular domain of the IC receptor at the N-terminal. In some embodiments, the fusion proteins comprise the cytoplasmic domain of DNAM1 at the C-terminal. In some embodiments, the extracellular domain of the IC receptor is fused directly to the cytoplasmic domain of DNAM-1, or via a linker and / or other intervening sequence, e.g., a transmembrane domain of DNAM1. In some embodiments, the cytoplasmic domain of DNAM-1 comprises the amino acid sequence of SEQ ID NO: 2, or a sequence at least 80% identical to SEQ ID NO: 2 as described herein. In some embodiments, the fusion proteins further comprise a transmembrane domain of DNAM1. In some embodiments, the transmembrane domain of DNAM-1 comprises the amino acid sequence of SEQ ID NO: 3, or a sequence at least 80% identical to SEQ ID NO: 3 as described herein. In some embodiments, the transmembrane domain of DNAM1 is positioned between the extracellular domain of the IC receptor and the cytoplasmic domain of DNAM-1.

[0013] In some embodiments, the IC receptor is selected from the group consisting of T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT). programmed cell death protein 1 (PD1). cytotoxic T-lymphocyte associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), T-cell immunoglobulin and mucin domain 3 (TIM3), Poliovirus receptor related immunoglobulin domain containing (PVRIG), and Cluster of differentiation (CD)96.

[0014] In some embodiments, the IC receptor is TIGIT. In some embodiments, the extracellular domain of TIGIT comprises the amino acid sequence of SEQ ID NO: 5, or a sequence at least 80% identical to SEQ ID NO: 5 as described herein. In some embodiments, the fusion proteins comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 6. In some embodiments, the fusion proteins comprise the amino acid sequence of SEQ ID NO: 6. In some embodiments, the fusion proteins comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 8. In some embodiments, the fusion proteins comprise the amino acid sequence of SEQ ID NO: 8.

[0015] In some embodiments, the IC receptor is PD1. In some embodiments, the extracellular domain of PD1 comprises the amino acid sequence of SEQ ID NO: 10, or a sequence at least 80% identical to SEQ ID NO: 10 as described herein. In some embodiments, the fusion proteins comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion proteins comprise the amino acid sequence of SEQ ID NO: 11. In some embodiments, the fusion proteins comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 13. In some embodiments, the fusion proteins comprise the amino acid sequence of SEQ ID NO: 13.

[0016] In some embodiments, the fusion proteins further comprise a purification tag. In certain embodiments, the purification tag is a FLAG tag, cMyc tag, His-tag, or biotin tag. In some embodiments, the fusion proteins further comprise one or more linkers.

[0017] Also provided herein are nucleic acid molecules encoding the fusion proteins described hereinabove. In some embodiments, the nucleic acid molecules comprise a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 45. In some embodiments, the nucleic acid molecules comprise a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 46.

[0018] Also provided herein are expression vectors comprising the nucleic acid molecules described hereinabove.

[0019] The present disclosure also provides isolated natural killer (NK) cells expressing the fusion proteins described hereinabove. Also provided herein are population or populations of such NK cells. In some embodiments, the NK cells further express one or more of CD16, NKP44, NKP46, and NKP30.

[0020] The present disclosure also provides isolated T cells expressing the fusion proteins described hereinabove. Also provided herein are population or populations of such T cells. In some embodiments, the T cells further express one or more of CD4 and CD8.

[0021] Also provided herein are methods for treating cancer in a subject in need thereof by administering to the subject a therapeutically effective amount of a population of the isolated NK cells described hereinabove and / or a population of the isolated T cells described hereinabove. In some embodiments, the methods further comprise administering to the subject one or more IC inhibitors, anti-tumor monoclonal antibodies, chimeric antigen receptor (CAR)-T cells, and / or CAR-NK cells. In some embodiments, the population of NK cells or T cells are administered intravenously.

[0022] Also provided herein are fusion proteins comprising an extracellular domain of DNAM-1 and a cytoplasmic domain of an IC receptor. In some embodiments, the fusion proteins comprise the extracellular domain of DNAM1 at the N-terminal. In some embodiments, the fusion proteins comprise the cytoplasmic domain of the IC receptor at the C-terminal. In some embodiments, the extracellular domain of DNAM-1 is fused directly to the cytoplasmic domain of the IC receptor, or via a linker and / or other intervening sequence, e.g., a transmembrane domain of the IC receptor. In some embodiments, the extracellular domain of DNAM-1 comprises the amino acid sequence of SEQ ID NO: 39, or a sequence at least 80% identical to SEQ ID NO: 39 as described herein.

[0023] In some embodiments, the IC receptor is selected from the group consisting of T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT). programmed cell death protein 1 (PD1). cytotoxic T-lymphocyte associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), T-cell immunoglobulin and mucin domain 3 (TIM3), Poliovirus receptor related immunoglobulin domain containing (PVRIG), and Cluster of differentiation (CD)96.

[0024] In some embodiments, the IC receptor is TIGIT. In some embodiments, the cytoplasmic domain of TIGIT comprises the amino acid sequence of SEQ ID NO: 40, or a sequence at least 80% identical to SEQ ID NO: 40 as described herein. In some embodiments, the fusion proteins further comprise a transmembrane domain of TIGIT. In some embodiments, the transmembrane domain of TIGIT comprises the amino acid sequence of SEQ ID NO: 41 , or a sequence at least 80% identical to SEQ ID NO: 41 as described herein. In some embodiments, the transmembrane domain of TIGIT is positioned between the extracellular domain of DNAM-1 and the cytoplasmic domain of TIGIT. In some embodiments, the fusion proteins comprise an amino acid sequence having at least 80% sequence identity' to the amino acid sequence of SEQ ID NO: 42. In some embodiments, the fusion proteins comprise the amino acid sequence of SEQ ID NO: 42. In some embodiments, the fusion proteins comprise an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 44. In some embodiments, the fusion proteins comprise the amino acid sequence of SEQ ID NO: 44.

[0025] In some embodiments, the fusion proteins further comprise a purification tag. In certain embodiments, the purification tag is a FLAG tag, cMyc tag, His-tag, or biotin tag. In some embodiments, the fusion proteins further comprise one or more linkers.

[0026] Also provided herein are nucleic acid molecules encoding the fusion proteins described hereinabove. In some embodiments, the nucleic acid molecules comprise a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 87.

[0027] Also provided herein are expression vectors comprising the nucleic acid molecules described hereinabove.

[0028] The present disclosure also provides isolated regulatory T (Treg) cells expressing the fusion proteins described hereinabove. Also provided herein are population or populations of such Treg cells. In some embodiments, the Treg cells further express one or more of CD4 and CD25.

[0029] Also provided herein are methods for treating graft-versus-host disease (GVHD) or an autoimmune disease in a subject in need thereof by administering to the subject a therapeutically effective amount of a population of the isolated Treg cells described hereinabove. In some embodiments, the population of Treg cells are administered intravenously.

[0030] Also provided herein are fusion proteins comprising the cytoplasmic and transmembrane domains of DNAM-1 fused to the C-terminal of the extracellular domain sequence of an immune checkpoint inhibitor.

[0031] In some embodiments, the immune checkpoint inhibitor is selected from TIGIT, PD1, CTLA4, LAG3, TIM3, CD96, and PVRIG.

[0032] Additionally, provided herein are methods of transforming an inhibitory signal from a ligand of an immune checkpoint overexpressed on a cancer cell to an immune cell activating signal comprising administering a fusion protein as described herein to the immune cell. In some embodiments, the immune cells is aNK cell, a T-cell, a T- reg cell, or a macrophage.

[0033] Further, provided herein are methods of preventing exhaustion of immune cells comprising administering to the immune cell a fusion protein as described herein, thereby enhancing the persistence of the immune cell.

[0034] Also provided herein are methods of maintaining the immunosuppressive function of a Treg cell, the methods comprising administering to said cell a DNAM1 - TIGIT fusion protein. Unless otherwise defined, 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 invention belongs. Methods and materials are described herein for use in the present invention; other, suitable methods and materials known in the art can also be used. The materials, methods, and examples are illustrative only and not intended to be limiting. All publications, patent applications, patents, sequences, database entries, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control.

[0035] Other features and advantages of the invention will be apparent from the following detailed description and figures, and from the claims.

[0036] DESCRIPTION OF DRAWINGS

[0037] FIG. 1 is a schematic showing potential ligands for fusion proteins that can convert inhibitory signal into stimulatory’ or activating signal.

[0038] FIGs. 2A-2B show the effect of TIGIT-DNAM1 and PD1-DNAM1 fusion proteins on cytotoxicity of NK92CIRB21cells. FIG. 2A is a graph showing effect of TIGIT-DNAM1 and PD1-DNAM1 fusion proteins on killing of cancer cells. FIG. 2B provides images of killing of PC-3 cells, plated as 400,000 cells, when incubated in the presence of 100,000 NK cells expressing DNAM1, TIGIT-DNAM1, or PD1- DNAM1.

[0039] FIG. 3 is a schematic showing activation of T-regs cells by interaction of PVR from cells that target T-Regs and DNAM1-TIGIT fusion protein expressed in T-regs.

[0040] FIG. 4 provides graphs showing induction of immune checkpoint receptor (1C) expression in NK92CIRB21cells exposed to increasing numbers of PC-3 (3. 4 or 5.105) cells for 4 days, then rested for 24 hours and analyzed by flow cytometry.

[0041] FIG. 5 provides graphs showing evolution by natural selection of IC knockout NK92CIRR21cell lines through repeated exposure to PC-3 cells. Represented are nextgeneration sequencing % KO for each immune checkpoint in the first and fourth generations of exposed NK92CIRB21cells.

[0042] FIG. 6 provides graphs showing log2 normalized expression of IC in NK92CIRB, NK92CIRB21, and NK92 under IL2 and / or IL21 (left panel); in NK92CIRB21after exposure to high numbers of breast cancer MDA-MB-231 cells for a week (central panel); and in NK92CIRBafter exposure to high numbers of breast cancer MDA-MB-231 cells for a week (right panel).

[0043] FIG. 7 provides graphs showing cytotoxicity profile of NK92CIRB21expressing signal converters TIGIT-DNAM1 and PD-1-DNAM1 compared to wild type DNAM1 against Pane- 1, (E:T= 0.3: 1), U251 (1 :1), A375 (1: 1), PC-9 (0.5: 1) and PC-3(0.3:l). The graph on bottom right (following the arrow) shows repeat cytotoxicity of NK92CIRB21previously exposed to PC-3 and resilience of cells expressing TIGIT- DNAM1.

[0044] FIG. 8 provides graphs showing PDL-1 and PVR surface expression profiles in cancer cell lines used.

[0045] FIG. 9 provides a chart showing Overton percentage of pAKT-positive NK92CIRB21cells expressing signal converters found in the sample above the control with or without 30 min of contact with PC-3 cancer cells.

[0046] FIG. 10 provides bar graphs showing impact of PVR and PD-L1 overexpression in PC-3 cells on the cytotoxicity profile of NK92cirb21 expressing signal converters.

[0047] FIG. 11 provides graphs showing anti-tumor response of NK cells. Left panel, a single injection of live NK92CIRB21cells (5.106), induced a substantial tumor delay of small PC-3 tumors (~70mm3) more than NK92CIRBin NSG mice (n=8 tumors). Right panel, injection of live NK92CIRD21cells in NSG mice bearing large PC-3 tumors (~275mm3), induced immediate tumor regression but no response after the second injection (n=8 tumors).

[0048] FIG. 12 provides graphs showing anti-tumor response of NK cells. Left panel, injection of live NK92CIRB21cells (5. 106red arrows) followed 2 hours later by IC inhibitors (anti-PDl (150 pg), anti-TIGIT (150 pg) and anti-CTLA-4 (75 pg) i.p, Black arrow s) in NSG mice bearing small PC-3 tumors (~65mm3) induced a prolonged tumor response extended by a second injection of NK cells and antibodies (n=8 tumors). Right panel, mice body weights remained stable during treatment suggesting the lack of toxicity from combination treatment (n=8 tumors).

[0049] FIG. 13 provides graphs showing anti-tumor response of NK cells expressing TIGIT-DNAM1 constructs. Left panel, NK92CIRB21cells expressing signal converter TIGIT-DNAM1 showed better anti-tumor response than NK92CIRB21, and combination with 200 pg anti-PDl further improved their effect. Right panel, TIGIT-DNAM1 expression and combination with antibodies did not affect body weight in mice (n=6 mice). Data are shown as the mean ± SD. ns represents no significant difference. * p<0.05, **p<0.01 ***p<0.001, ****p<0.0001.

[0050] FIG. 14 provides bar graphs showing effect of TIGIT-DNAM1 contract on cy totoxicity' of primary NK cells. Bar graphs show cytotoxicity of primary NK cells electroporated with TIGIT-DNAM1 construct against PC-3 cancer (32,000) cells, as compared to untouched primary NK cells, at a ratio of 1 : 1 (effector to target), at day 2 (left panel) and day 4 (right panel) after plating of the NK cells.

[0051] FIG. 15 is a bar graph showing effect of CD96-DNAM1, CTLA4-DNAM1, and LAG3-DNAM1 constructs on cytotoxicity' of NK cells.

[0052] DETAILED DESCRIPTION

[0053] The successes of adoptive NK cells and T cells armed with cytokines and chimeric antigen receptors in the clinic are encouraging but limited. Optimal activation of NK cell and T cell is not yet attained and would benefit from stronger cytokines signaling to prevent exhaustion, as also strategies to subvert immune checkpoint inhibition. The present disclosure provides fusion proteins for optimal activation of NK cells and T cells.

[0054] Paired receptors that interact with ligands of Nectin / Nectin-like (Necl)-5, (usually termed CD155 or PVR) and Nectin-2 (CD112), are expressed on different ty pes of tumor cells. These ligands are recognized by a group of receptors expressed on T and NK cells that exert opposing functions upon interaction with their ligands. These receptors include DNAX accessory molecule-1 (DNAM-1; CD226) and TIGIT (T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosinebased inhibitory motif (ITIM) domains). Whereas activation through DNAM-1 after recognition of CD155 or CD112 enhances NK cell-mediated cy totoxicity' against a wide range of tumor cells, TIGIT recognition of these ligands exerts an inhibitory' efiect on NK cells by diminishing IFN-y production and NK cell-mediated cytotoxicity.

[0055] In NK cells from patients with solid cancer and leukemia, it has been observed that a decreased expression of DNAM-1 may shift the balance in favor of the inhibitory' receptors TIGIT, further contributing to the diminished NK cell-mediated cytotoxic capacity observed in these patients. It has been speculated that the TIGIT pathway is upregulated in cancer patients and represents a novel target for checkpoint blockade immunotherapy. See Sanchez-Correa et al., Cancers (Basel) 2019 Jun 23;11(6):877.

[0056] On the other hand, upon ligation with its ligands CD155 (poliovirus receptor or nectin-like protein 5) or CD112 (nectin-2 or poliovirus receptor-related 2), DNAM- 1 transduces an activating signal for enhancing cell-mediated cytotoxicity and cytokine production through the phosphorylation of a conserved tyrosine in its cytoplasmic domain. See Sato et al., PNAS, May 19, 2021, 1 18 (21) e2021309118. Upon recognizing these ligands, DNAM-1 promotes NK cell-mediated elimination of transformed and virus -infected cells. See Zhang et al., J Exp Med 2015 Nov 16;212(12):2165-82. In vitro studies indicate that DNAM-1 triggers NK cell- mediated killing of tumor cells expressing CD155 and CD1 12, promotes costimulation of CD4 and CD8 T-cells, and mediates adhesion of monocytes to endothelial cells facilitating transendothelial migration. DNAM-1 also extends the range of target cells that can activate CD8 T-cell and NK cells. Consequently, it may be essential for immune surveillance against tumors and may promote activation of cytotoxic lymphocytes by nonprofessional antigen-presenting cells. It has been show n that DNAM-1 mediates NK cell recognition of freshly isolated ovarian carcinoma and neuroblastoma cells. In addition, DNAM-1 -deficient mice develop significantly more DNAM-1 ligand-expressing tumors than wildtype mice, indicating that DNAM-1 plays an important role in immunosurveillance during tumor development. See Morisaki et al., Anticancer Research 32 (6), June 2012: 2241-47. In sum, DNAM-1 is an activating / co-stimulatory receptor involved in the recognition and lysis of tumor cells, and TIGIT engagement inhibits NK cell function.

[0057] Chimeric proteins combining programmed cell death protein (PD)1 with chimeric antigen receptor (CAR) signaling domain that includes the CD28-CD3 domain or the 41BB domain are well known. However, these constructs are believed to be less optimal because the same domain is used in CAR-T or CAR-NK. and competition between the CARs in the tw o receptors could reduce the activity of both. In contrast, the fusion proteins described here are based on discovery by the present inventors that inhibitory signaling from ligands like Programmed death-ligand 1 (PD- Ll) and Poliovirus receptor (PVR) can be converted into stimulation or activation of immune cells by fusing the extracellular domain of immune checkpoint (IC) receptors (also referred to herein as immune checkpoints or ICs), like PD1 or TIGIT, to the cytoplasmic domain of activating receptors, like DNAX accessory molecule- 1 (DNAM1; CD226). Thus, in some embodiments, the fusion proteins of the present disclosure use DNAM1 signaling, which is different from CD28 signaling or 41BB signaling, and synergize (and not compete) with another CAR. These fusion proteins act as signal converters that can convert inhibitory signal from the extracellular domain of the IC receptor into stimulatory signal through the cytoplasmic domain of the activating receptor. Activation signaling from these signal converters and parallel deactivation signals from endogenous IC receptors prevent hyperactivation and exhaustion and can alleviate the toxicities of IC receptors. Thus, these fusion proteins act as signal converters that can enable an optimal and safe activation of NK cells and T cells and can benefit cancer immunotherapy. Accordingly, described herein are fusion protein that can enhance the safety and pan-anticancer effects of adoptive NK cells and T cells in cancer immunotherapies.

[0058] Release of activated NK cells from the bone marrow following inflammation or infection allows the migration of NK cells to affected tissues to kill abnormal cells and create inflammatory conditions in preparation for an adaptive immune response (56). However, NK cells' inflammatory response is temporary7and can only be sustained by cytokines secreted by other immune cells (64). Therefore, infusing self- sustaining NK cells that prolong inflammation and resist exhaustion in the target tissue without a proliferation risk is critical. Recent developments suggest that C AR- T cells could develop into lymphoma, leading to several patient deaths. Unlike NK adoptive immunotherapy, which allows allogeneic transfer with a reduced chance of allogeneic cancer transfer, CAR-T is always autologous, thus maintaining the risk. Additionally, autologous memory T-cells could expand indefinitely and have mechanisms to preserve their telomeres. IC blockade using anti-CTLA-4, anti-PDl, and anti-PDL-1 antibodies are now7standard cancer therapies. However, despite IC blockade's remarkable efficacy in certain tumors, it remains largely inefficient in many cancer patients. It is, therefore, critical to identify new IC blockade alternatives. Contact of NK92 cells with cancer cells triggers several ICs proportionally to cancer cell numbers (FIG. 4). IC contributions to exhaustion are not equal but synergistic. Ligands PVR and Nectin2 bind more efficiently to TIGIT than DNAM1 and inhibit T and NK cells, leading to exhaustion. PD-1, via its intracellular domain signaling, inhibits DNAM1 activation by recruiting Shp2 to inhibit its phosphorylation and TIGIT through disruption of DNAM1 and PVR interaction and DNAM1 dimerization. To subvert TIGIT and PD-1 inhibitory functions, we converted PVR, Nectin2, and PD-L1 inhibition into activation by tethering TIGIT or PD-1 extracellular domains to DNAM1 transmembrane and cytoplasmic domains. Wild-type DNAM1, TIGIT-DNAM1, and PD-1-DNAM1 expression enhanced NK92CIRB21killing of multiple cancer cell lines with different efficiencies (FIG. 7). pAKT was increased upon contact with cancer cells (FIG. 9). Without wishing to be bound by theory, the signal converters described herein can play two roles. In one, they compete with endogenous PD-1 and TIGIT for their ligands, and in a second role, they transmit an activation signal from PD-L1. PVR. Nectin2, Nectin3, and Nectin4. The protein expression of DNAM1, TIGIT-DNAM1, and PD-1-DNAM1 in NK92CIRB21was first based on the bicistronic co-expression of GFP which was similar in the three cell lines (FIG. 4). However, flow' cytometry analysis using anti- DNAM1. anti-TIGIT, and anti -PD-1 antibodies revealed lower expression of TIGIT - DNAM1 compared to DNAM1 and PD-1-DNAM1. Despite this. TIGIT-DNAM1 significantly enhanced NK92CIRB21anticancer activity against most cancer cell lines (FIG. 7, graphs on top panel and left and center of bottom panel) and allow ed better resistance to exhaustion (FIG. 7, graph on right of bottom panel). When the expression of TIGIT-DNAM1 was increased in TIGIT-DNAM1 ', we observed reduced PC-3 cell killing, suggesting that higher expression of certain signal converters based on DNAM1 could lead to exhaustion, probably by hyperactivation (FIG. 10). Canonical TIGIT and PD-1 are induced in NK92 after exposure to cancer cells (FIG. 4) and converge to dephosphorylate DNAM1 and inhibit the PI3K-AKT axis through multiple mechanisms (63). These counterbalancing inhibitory signals from endogenous ICs could prevent DNAM1 hyperactivation from signal converters. Indeed, overexpression of PD-L1 leads to increased killing of PC-3 / PD-L1 by DNAM1 and TIGIT-DNAM1. However, overexpression of PVR in PC-3 / PVR reduced killing in DNAM1 and TIGIT-DNAM1 to levels comparable to NK92CIRB21. suggesting activation from signal converters and inhibitions from endogenous TIGIT is equal, or DNAM1 signaling is hyperactivated.

[0059] PD-1-DNAM1. which does not bind to PVR, shows more activation than inhibition against PC-3 overexpressing PVR. This suggests that TIGIT-DNAM1 will be more efficacious after induction of PD-1 against tumors expressing high PD-L1. Conversely, PD-1-DNAM1 would be more efficacious after induction of TIGIT against tumors expressing PVR. However, the engineered overexpression of PD-L1 and PVR in PC-3 cells might be at levels far higher than those normally found in primary' human tumors.

[0060] Many patients resist IC inhibition or acquire resistance after an initial response. T-cells may develop resistance due to the absence of antigen, reduced infiltration, and exhaustion. There is no clear consensus on what an exhausted NK phenotype is. However, increased expression of inhibitory receptors such as PD-1, TIGIT, LAG3, and TIM3 is associated with a less active NK state. This is supported by our finding that anti-PD-1, anti-CTLA-4. and anti -TIGIT enhance NK92CIRB21antitumor effect in vivo (FIG. 12). Negative selection by evolution of ICs knockout in NK92CIRB21cells also suggests all Ics to be important factors in NK exhaustion (FIG. 5). It is not clear if IC inhibitors in the form of antibodies relieve immunosuppression by blocking every IC at the cell surface or do so only partially in which case a basal level of exhaustion is unavoidable. In the case of our signal converter technology, additional receptors are expressed at the cell surface, thereby competing for ligands with endogenous Ics and actively transmitting an activation signal that could counter the cell’s own ICs. This inhibition, in turn, may fine-tune the signal converter by preventing its hyperactivation. It is also possible that NK cells expressing these TIGIT-DNAM or PD1 -DNAM signal converters may, through antigen loss, select for cancer cells lacking PVR or PDL-1, respectively. In this case, the use of a mixed population of NK cells expressing separately the two signal converters may be more effective and also prevent hyperactivation if the two signal converters were in the same cell.

[0061] Overall, the present disclosure describes IC signal converters that can convert IC inhibition into activation. We show this technology’s success using two major immune checkpoints in NK cells, PD-1 and TIGIT (as described in the '‘Examples” section; see, e.g., Examples 1-4), but this technology7may substitute other Ics. We chose the NK92 model to evaluate our technologies because the many genetic manipulations needed could not be easily done in primary NK cells. The NK92 model also offers the possibility of evaluating safety' switches and the ability of the cells to allow oncogenic signals to proceed. Together, the technology described here may improve the fitness, efficacy, and safety of genetically modified NK and possibly T cells cancer immunotherapy and warrant further investigations in clinical settings.

[0062] Also described herein are fusion proteins that comprise the extracellular domain of activating receptors, like DNAM1, fused to the cytoplasmic domain of IC receptors, like PD1 or TIGIT. These fusion proteins act as signal converters that can convert activation signal from the extracellular domain of the activating receptor into inhibitors’ signal through the cytoplasmic domain of the IC receptor.

[0063] Fusion Proteins

[0064] Described herein are fusion proteins that can act as signal converters. These fusion proteins are also referred to herein as chimeras, signal converters, and / or chimeric signal converters. The fusion proteins of the present disclosure can act as signal converters that can convert an inhibitory signal into a stimulatory or activating signal. Additionally, or in the alternative, described herein are fusion proteins acting as signal converters that can convert a stimulatory or activating signal into an inhibitory signal.

[0065] A stimulator}7signal (also referred to herein as activating signal or activation signal) can comprise cellular signaling (e.g., through activating receptors like DNAM1) that enhances or induces immune response. For example, stimulatory signal can comprise signaling (e.g., DNAM1 signaling) that enhances or induces cytotoxic response of immune cells (e.g., NK cells and / or T cells). An inhibitory signal can comprise cellular signaling (e.g., through immune checkpoint receptors) that inhibits immune response. For example, inhibitory signal can comprise signaling (e.g., IC receptor signaling, such as TIGIT signaling or PD1 signaling) that inhibits cytotoxic response of immune cells (e.g., NK cells and / or T cells). Additionally, or in the alternative, inhibitor}' signal can comprise cellular signaling (e.g., through immune checkpoint receptors) that induces (or maintains) immunosuppressive response. For example, inhibitory signal can comprise signaling (e.g., IC receptor signaling, such as TIGIT signaling or PD1 signaling) that induces or maintains immunosuppressive functions of Treg cells.

[0066] In some instances, a fusion protein of the present disclosure further comprises a purification tag (e.g., FLAG, cMyc tag (EQKLISEEDL (SEQ ID NO: 83)), His-tag, or biotin tag). Linkers

[0067] In some embodiments, one or more linkers is present between the different domains (e.g., between the extracellular domain and the cytoplasmic domain, between the extracellular domain and the transmembrane domain, and / or the transmembrane domain and the cytoplasmic domain). Linkers are described, for example, in Argos, Mol Biol 211:943-958, 1990; George and Heringa, Protein Eng 15:871-879, 2002; Chen et al.. Biotechniques 49:513-518, 2010; and Chen et al., Adv Drug Deliv Rev 65(10): 1357-1369, 2013. A linker for use in a fusion protein described herein can be a flexible linker, a rigid linker, and / or an in vivo cleavable linker.

[0068] For example, a linker for use in a fusion protein described herein can be a flexible linker. Flexible linkers are usually applied when the protein domains that need to be joined require a certain degree of movement or interaction. They are generally composed of small, non-polar (e.g., Gly) or polar (e.g., Ser or Thr) amino acids. The small size of these amino acids provides flexibility, and allows for mobility of the connecting functional domains. The incorporation of Ser or Thr can maintain the stability of the linker in aqueous solutions by forming hydrogen bonds with the water molecules, and therefore reduce the unfavorable interaction between the linker and the protein moieties. An example of the most widely used flexible linker is the sequence (Gly-Gly-Gly-Gly-Ser)n (SEQ ID NO: 84).

[0069] Additionally, or in the alternative, a linker for use in a fusion protein described herein can be a rigid linker. While flexible linkers have the advantage of connecting the functional domains passively and permitting a certain degree of movement, the lack of rigidity of these linkers can be a limitation. There are several examples in the literature where the use of flexible linkers resulted in poor expression yields or loss of biological activity. Under such situations, rigid linkers can be successfully applied to keep a fixed distance between the domains and to maintain their independent functions. Rigid linkers exhibit relatively stiff structures by adopting a-helical conformations or by containing multiple Pro residues. Examples of some rigid linkers are: (EAAAK)n (SEQ ID NO: 85) and (XP)n. with X designating any amino acid, preferably Ala, Lys, or Glu.

[0070] Additionally, or in the alternative, a linker for use in a fusion protein described herein can be an in vivo cleavable linker. Flexible and rigid linkers represent stable linkers that covalently j oin functional protein domains together to act as one molecule throughout the in vivo processes that the component protein(s) are involved in. This stable linkage between functional domains provides many advantages such as a prolonged plasma half-life (e.g., albumin or Fc-fusions). However, it also has several potential drawbacks, including, steric hindrance between functional domains, decreased bioactivity, and altered biodistribution and metabolism of the protein moieties due to the interference between domains. Under such circumstances, cleavable linkers are used to release free functional domains in vivo. This type of linker may reduce steric hindrance, improve bioactivity, or achieve independent actions / metabolism of individual domains of recombinant fusion proteins after linker cleavage. The design of in vivo cleavable linkers in recombinant fusion proteins is quite challenging. Unlike the versatility of crosslinking agents available for chemical conjugation methods, linkers in recombinant fusion proteins must necessarily be oligopeptides. For example, an in vivo cleavable disulfide linker (LEAGCKNFFPR|SFTSCGSLE) (SEQ ID NO: 86; the arrow indicates where cleavage occurs), based on the reversible nature of the disulfide bond, was designed for recombinant fusion proteins by Chen et al. (Biotechniques 49:513-518. 2010), and offered the advantage of generating a precisely constructed, homogeneous product by recombinant methods.

[0071] All of the fusion proteins described herein can be generated using standard molecular biological procedures, e.g., for manipulating and expressing recombinant DNA. See, e.g., Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) John Wiley & Sons (1995), and Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (June 15, 2012) and supplements thereof, and other standard laboratory manuals. The fusion proteins can be expressed, e.g., stably expressed, in an NK cell, a T cell, or a macrophage, e.g., a primary7or cultured NK cell, T cell, or macrophage. The cells are then infused into a subject, e g., a subject who has (e.g., has been diagnosed with) cancer and / or an autoimmune disease. (A) Fusion Proteins Converting Inhibitory Signal into Stimulatory Signal

[0072] In some embodiments, the fusion proteins of the present disclosure act as signal converters that can convert an inhibitory signal into a stimulatory or activating signal.

[0073] These fusion proteins (e.g., fusion proteins acting as signal converters that can convert an inhibitory signal into a stimulatory or activating signal) comprise the extracellular domain of an immune checkpoint receptor (IC receptor or IC) and the cytoplasmic domain of an activating receptor. Such fusion proteins act as signal converters by converting inhibitory signal from the extracellular domain of the IC receptor into stimulatory signal through the cytoplasmic domain of the activating receptor.

[0074] In some embodiments, the fusion protein comprises the extracellular domain of the IC receptor at the N-terminal. In some embodiments, the fusion protein comprises the cytoplasmic domain of the activating receptor at the C-terminal. In some embodiments, the extracellular domain of the IC receptor is fused to the cytoplasmic domain of the activating receptor. In some embodiments, the fusion protein further comprises the transmembrane domain of the activating receptor (e.g., the transmembrane domain of DNAM-1). The transmembrane domain of the activating receptor can be positioned between the extracellular domain of the IC receptor and the cytoplasmic domain of the activating receptor. In some embodiments, the transmembrane domain of the activating receptor is retained to preserve the activating receptor signaling. In some embodiments, the extracellular domain of the IC receptor is fused directly to the cytoplasmic domain of the activating receptor. In some embodiments, the extracellular domain of the IC receptor is fused to the cytoplasmic domain of the activating receptor via a linker and / or other intervening sequence. The intervening sequence can be a transmembrane domain of the activating receptor. For example, the extracellular domain of the IC receptor can be fused to the cytoplasmic domain of the activating receptor via the transmembrane domain of the activating receptor.

[0075] In some instances, a fusion protein of the present disclosure further comprises a purification tag (e.g., FLAG, cMyc tag, His-tag, or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., between the extracellular domain of the IC receptor and the cytoplasmic domain of the activating receptor, between the extracellular domain of the IC receptor and the transmembrane domain of the activating receptor, and / or between the transmembrane domain of the activating receptor and the cytoplasmic domain of the activating receptor).

[0076] The IC receptor of such fusion proteins can be one or more of TIGIT, PD 1 , CTLA4, LAG3, TIM3. PVRIG, and CD96. The activating receptor of such fusion proteins can be DNAM-1. Accordingly, a fusion protein descnbed herein (e.g.. a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal) can be one or more of: TIGIT-DNAM1, PD1-DNAM1, CTLA4- DNAM1. LAG3-DNAM1, TIM3-DNAM1, PVRIG-DNAM1, and CD96-DNAM1.

[0077] Upon binding of these fusion proteins (e.g., fusion proteins acting as signal converters that can convert inhibitory signal into stimulatory signal) to their respective immune checkpoint ligands (e.g., CD155, CD111, CD112, CD113, PDL1, PDL2, CD86, CD80, galectin-9, phosphatidylserine (PtdSer), high-mobility group protein Bl (HMGB1), CEACAM-1, FGL-1. Gal-3, LSECtin, and a-syn). the function of the fusion proteins is converted from inhibition (e.g.. by interaction of the IC receptor and its ligands) to stimulation or activation (see, e.g., the schematic provided at Figure 1). The conversion from inhibitory- signal to stimulatory or activating signal can be via engagement of DNAM1 signaling. For example. DNAM1 signaling can lead to phosphorylation of SLP-76 and Vavl, followed by the activation of PLCy2. ERK and AKT. AKT then phosphorylates FOXO1, which translocates from the nucleus to the cytoplasm, to be degraded and inactivated. DNAM1 signaling can promote expression of genes involved in degranulation and calcium mobilization, leading to activation of NK cells and T cells. Thus, conversion from inhibitory signal to stimulatory or activating signal via engagement of DNAM1 signaling can induce cytotoxic response of NK cells or T cells (e.g., trigger NK cell-mediated and / or T cell- mediated tumor cell killing). In some embodiments, expression of TIGIT and CD96 can lead to NK cell exhaustion, as both receptors compete with DNAM1 for binding to the same activating ligands.

[0078] These fusion proteins (e g., fusion proteins acting as signal converters that can convert inhibitory signal into stimulatory' signal) are designed to activate signaling of activating receptors (e.g., DNAM-1 signaling) in response to inhibition by immune checkpoint ligands. This opens the door to a vast array of applications in cancer immunotherapy. These signaling converters can replace immune checkpoint inhibitors (e.g.. antibodies) and eliminate their nonspecific effects and toxi cities by localizing the immune checkpoint signaling inhibition and conversion of signaling to immune cells only, without systemic toxicities and high costs of treatment. In some embodiments, these fusion proteins can be used in combination with other immunotherapies (e.g.. immune checkpoint inhibitors). These fusion proteins can be delivered using NK cells, T cells, CAR-T. CAR-NK, CAR-M, or any other immunotherapy protocol. They are intended to remove any inhibitory signaling associated with immune checkpoints and convert inhibition into activation that enhances immune cells and extends their cytotoxic potential.

[0079] Thus, the fusion proteins described here (e.g., fusion proteins acting as signal converters that can convert an inhibitory signal into a stimulatory signal) can be useful in treating disorders associated with abnormal apoptotic or differentiative processes, such as cellular proliferative disorders or cellular differentiative disorders (e.g., cancer, including both solid tumors and hematopoietic cancers). The method can include administering a therapeutically effective amount of NK cells and / or T cells expressing a fusion protein (e.g., fusion proteins acting as signal converters that can convert an inhibitory' signal into a stimulatory' signal) as described herein, to a subj ect who is in need of, or who has been determined to be in need of, such treatment.

[0080] TIGIT-DNAM1

[0081] A fusion protein (e.g., a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal) described herein can comprise the extracellular domain of T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor ty rosine-based inhibitory^ motif (ITIM) domains (TIGIT) and the cytoplasmic domain of DNAM1. Such a fusion protein can be referred to herein as TIGIT-DNAM1.

[0082] In some embodiments, a TIGIT-DNAM1 fusion protein comprises the extracellular domain of TIGIT at the N-terminal and / or the cytoplasmic domain of DNAM1 at the C-terminal. In some embodiments, the extracellular domain of TIGIT is fused to the cytoplasmic domain of DNAM1. In some embodiments, a TIGIT- DNAM1 fusion protein further comprises the transmembrane domain of DNAM1. The transmembrane domain of DNAM1 can be positioned between the extracellular domain of TIGIT and the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of TIGIT is fused directly to the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of TIGIT is fused to the cytoplasmic domain of DNAM1 via a linker and / or other intervening sequence. The intervening sequence can be the transmembrane domain of DNAM1. For example, the extracellular domain of TIGIT can be fused to the cytoplasmic domain of DNAMI via the transmembrane domain of DNAMI.

[0083] In some instances, a TIGIT-DNAM1 fusion protein further comprises a purification tag (e.g., FLAG, cMyc tag, His-tag, or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., between the extracellular domain of TIGIT and the cytoplasmic domain of DNAMI, between the extracellular domain of the TIGIT and the transmembrane domain of DNAMI, and / or the between the transmembrane domain and the cytoplasmic domain of DNAMI).

[0084] DNAMI is a 65 kDa transmembrane glycoprotein with 318 amino acids, including two immunoglobulin-like domains. Exemplary sequence of human DNAMI is provided at NCBI Reference Sequence: NP 006557.2 (encoded by NM_006566.4), and UNIPROT Accession No. Q15762. For example, full length human DNAMI can comprise the amino acid sequence of:

[0085] MDYPTLLLALLHVYRALCEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQ DSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQ GTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQ IDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGEN ETFVMRLTVAEGKTDNQYTLFVAGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTE SWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 1).

[0086] The amino acid sequence of the transmembrane and the cytoplasmic domains of DNAM-1 is shown in bold in SEQ ID NO: 1.

[0087] A TIGIT-DNAM1 fusion protein of the present disclosure can comprise the cytoplasmic domain of DNAMI that comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%, 83%. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%, 98%. 99%. or 100%) sequence identity to the amino acid sequence of: NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPK TRV (SEQ ID NO: 2).

[0088] A TIGIT-DNAM1 fusion protein of the present disclosure can comprise the transmembrane domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%. 83%. 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0089] GGTVLLLLFVISITTIIVIFL (SEQ ID NO: 3).

[0090] TIGIT is a 244 amino acid protein. Exemplary' sequences of human TIGIT are provided atNCBI Reference Sequence: NP 776160.2 (encoded by NM 173799.4), and UNIPROT Accession No. Q495A1. For example, full length human TIGIT can comprise the amino acid sequence of:

[0091] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVN WEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDG TYTGRIFLEVLESSVAEHGARFQIPLLGAMAATLVVICTAVIVVVALTRKKKALRIHSVE GDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRS LGNCSFFTETG (SEQ ID NO: 4).

[0092] A TIGIT-DNAM1 fusion protein of the present disclosure can comprise the extracellular domain of TIGIT that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0093] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISP

[0094] SFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARF QIP (SEQ ID NO: 5).

[0095] Thus, in some embodiments, a TIGIT-DNAMl fusion protein of the present disclosure comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,

[0096] 96%. 97%. 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0097] MMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVNWEQQDQLLAICNADLGWHISP

[0098] SFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDGTYTGRIFLEVLESSVAEHGARF QIPGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQ SMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 6).

[0099] ATIGIT-DNAM1 fusion protein of the present disclosure can also comprise a signal sequence. For example. aTIGIT-DNAMl fusion protein can comprise the signal peptide of TIGIT that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the amino acid sequence of:

[0100] MRWCLLLIWAQGLRQAPLASG (SEQ ID NO: 7).

[0101] Thus, in some embodiments, a TIGIT-DNAMl fusion protein of the present disclosure includes a signal peptide and comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0102] MRWCLLLIWAQGLRQAPLASGMMTGTIETTGNISAEKGGSIILQCHLSSTTAQVTQVN WEQQDQLLAICNADLGWHISPSFKDRVAPGPGLGLTLQSLTVNDTGEYFCIYHTYPDG TYTGRIFLEVLESSVAEHGARFQIPGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTES WDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 8).

[0103] In some embodiments, a TIGIT-DNAM1 fusion protein of the present disclosure is encoded by a nucleic acid molecule that comprises a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the nucleic acid sequence of:

[0104] GCTAGCGCCACCATGCGGTGGTGCCTGCTGCTGATCTGGGCCCAGGGCCTGCGGC

[0105] AGGCCCCCCTGGCCAGCGGCATGATGACCGGCACCATCGAGACCACCGGCAACAT

[0106] CAGCGCCGAGAAGGGCGGCAGCATCATCCTGCAGTGCCACCTGAGCAGCACCAC

[0107] CGCCCAGGTGACCCAGGTGAACTGGGAGCAGCAGGACCAGCTGCTGGCCATCTG

[0108] CAACGCCGACCTGGGCTGGCACATCAGCCCCAGCTTCAAGGACCGGGTGGCCCCC

[0109] GGCCCCGGCCTGGGCCTGACCCTGCAGAGCCTGACCGTGAACGACACCGGCGAG

[0110] TACTTCTGCATCTACCACACCTACCCCGACGGCACCTACACCGGCCGGATCTTCCT

[0111] GGAGGTGCTGGAGAGCAGCGTGGCCGAGCACGGCGCCCGGTTCCAGATCCCCGG

[0112] CGGCACCGTGCTGCTGCTGCTGTTCGTGATCAGCATCACCACCATCATCGTGATCT

[0113] TCCTGAACCGGCGGCGGCGGCGGGAGCGGCGGGACCTGTTCACCGAGAGCTGGG

[0114] ACACCCAGAAGGCCCCCAACAACTACCGGAGCCCCATCAGCACCAGCCAGCCCA

[0115] CCAACCAGAGCATGGACGACACCCGGGAGGACATCTACGTGAACTACCCCACCTT

[0116] CAGCCGGCGGCCCAAGACCCGGGTGTGATGACTCGAG (SEQ ID NO: 45). Upon binding of a TIGIT-DNAM1 fusion protein to its immune checkpoint ligands (e.g., CD155 (PVR), CD112 (PVRL2, nectin-2), and / or CD113) on cancer cells - e.g., as shown in FIG. 1 - the fusion protein can convert inhibitory signal (e.g., by interaction of TGIT and its ligands) to stimulatory or activating signal. The conversion from inhibitory signal to stimulatory or activating signal can be through DNAM1. Conversion from inhibitory signal to stimulatory or activating signal via engagement of the DNAM1 signaling can induce cytotoxic response of NK cells or T cells (e g., can trigger NK cell-mediated and / or T cell-mediated tumor cell killing). Thus. TIGIT-DNAM1 fusion proteins can be useful in treating disorders associated with abnormal apoptotic or differentiative processes, such as cellular proliferative disorders or cellular differentiative disorders (e.g., cancer, including both solid tumors and hematopoietic cancers). Such treatment methods can include administering a therapeutically effective amount ofNK cells and / or T cells expressing TIGIT- DNAM1 fusion proteins to a subject who is in need thereof.

[0117] PD1-DNAM1

[0118] A fusion protein (e.g., a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal) described herein can comprise the extracellular domain of programmed cell death protein 1 (PD1) and the cytoplasmic domain of DNAM1. Such a fusion protein can be referred to herein as PD1-DNAM1.

[0119] In some embodiments, a PD1-DNAM1 fusion protein comprises the extracellular domain of PD1 at the N-terminal and / or the cytoplasmic domain of DNAM1 at the C-terminal. In some embodiments, the extracellular domain of PD1 is fused to the cytoplasmic domain of DNAM1. In some embodiments, a PD1-DNAM1 fusion protein further comprises the transmembrane domain of DNAM1. The transmembrane domain of DNAM1 can be positioned between the extracellular domain of PD1 and the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of PD1 is fused directly to the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of PD1 is fused to the cytoplasmic domain of DNAM1 via a linker and / or other intervening sequence. The intervening sequence can be the transmembrane domain of DNAM1. For example, the extracellular domain of PD1 can be fused to the cytoplasmic domain of DNAM1 via the transmembrane domain of DNAM1.

[0120] In some instances, a PD1-DNAM1 fusion protein further comprises a purification tag (e.g., FLAG, cMyc tag, His-tag, or biotin tag). In some embodiments, one or more linkers is present betw een the different domains (e.g., between the extracellular domain of PD1 and the cytoplasmic domain of DNAM1, between the extracellular domain of PD1 and the transmembrane domain of DNAM1. and / or the between the transmembrane domain and the cytoplasmic domain of DNAM1).

[0121] A PD1-DNAM1 fusion protein of the present disclosure can comprise the cytoplasmic domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0122] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPK TRV (SEQ ID NO: 2).

[0123] A PD1-DNAM1 fusion protein of the present disclosure can comprise the transmembrane domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the amino acid sequence of:

[0124] GGTVLLLLFVISITTIIVIFL (SEQ ID NO: 3).

[0125] PD1 is a 288 amino acid protein. Exemplary' sequence of human PD1 is provided at NCBI Reference Sequence: NP 005009.2 (encoded by NM 005018.3), and UNIPROT Accession No. Q15116. For example, full length human PD1 can comprise the amino acid sequence of: MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS NTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVVG VVGGLLGSLVLLVWVLAVICSRAARGTIGARRTGQPLKEDPSAVPVFSVDYGELDFQ WREKTPEPPVPCVPEQTEYATIVFPSGMGTSSPARRGSADGPRSAQPLRPEDGHCSWP L (SEQ ID NO: 9).

[0126] A PD1-DNAM1 fusion protein of the present disclosure can comprise the extracellular domain of PD1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%,

[0127] 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0128] FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA

[0129] FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTERRAEVPTAHPSPSPRPAGQFQTLV (SEQ ID NO: 10).

[0130] Thus, in some embodiments, a PD1-DNAM1 fusion protein of the present disclosure comprises an amino acid sequence having at least 80% (e g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the amino acid sequence of:

[0131] FLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFSNTSESFVLNWYRMSPSNQTDKLAA FPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRARRNDSGTYLCGAISLAPKAQIKESL RAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGGTVLLLLFVISITTIIVIFLNRRRRRER RDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 11).

[0132] A PD1-DNAM1 fusion protein of the present disclosure can also comprise a signal sequence. For example, a PDl-DNAMl fusion protein can comprise the signal peptide of PD1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0133] MQIPQAPWPVVWAVLQLGWRPGW (SEQ ID NO: 12).

[0134] Thus, in some embodiments, a PD1-DNAM1 fusion protein of the present disclosure includes a signal peptide and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0135] MQIPQAPWPVVWAVLQLGWRPGWFLDSPDRPWNPPTFSPALLVVTEGDNATFTCSFS

[0136] NTSESFVLNWYRMSPSNQTDKLAAFPEDRSQPGQDCRFRVTQLPNGRDFHMSVVRA

[0137] RRNDSGTYLCGAISLAPKAQIKESLRAELRVTERRAEVPTAHPSPSPRPAGQFQTLVGG

[0138] TVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDD

[0139] TREDIYVNYPTFSRRPKTRV (SEQ ID NO: 13). In some embodiments, a PD1-DNAM1 fusion protein of the present disclosure is encoded by a nucleic acid molecule that comprises a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of:

[0140] GCTAGCGCCACCATGCAGATCCCCCAGGCCCCCTGGCCCGTGGTGTGGGCCGTGC TGCAGCTGGGCTGGCGGCCCGGCTGGTTCCTGGACAGCCCCGACCGGCCCTGGAA CCCCCCCACCTTCAGCCCCGCCCTGCTGGTGGTGACCGAGGGCGACAACGCCACC TTCACCTGCAGCTTCAGCAACACCAGCGAGAGCTTCGTGCTGAACTGGTACCGGA TGAGCCCCAGCAACCAGACCGACAAGCTGGCCGCCTTCCCCGAGGACCGGAGCC AGCCCGGCCAGGACTGCCGGTTCCGGGTGACCCAGCTGCCCAACGGCCGGGACT TCCACATGAGCGTGGTGCGGGCCCGGCGGAACGACAGCGGCACCTACCTGTGCG GCGCCATCAGCCTGGCCCCCAAGGCCCAGATCAAGGAGAGCCTGCGGGCCGAGC TGCGGGTGACCGAGCGGCGGGCCGAGGTGCCCACCGCCCACCCCAGCCCCAGCC CCCGGCCCGCCGGCCAGTTCCAGACCCTGGTGGGCGGCACCGTGCTGCTGCTGCT GTTCGTGATCAGCATCACCACCATCATCGTGATCTTCCTGAACCGGCGGCGGCGGC GGGAGCGGCGGGACCTGTTCACCGAGAGCTGGGACACCCAGAAGGCCCCCAACA ACTACCGGAGCCCCATCAGCACCAGCCAGCCCACCAACCAGAGCATGGACGACA CCCGGGAGGACATCTACGTGAACTACCCCACCTTCAGCCGGCGGCCCAAGACCCG GGTGTGATGACTCGAG (SEQ ID NO: 46).

[0141] Upon binding of a PD1-DNAM1 fusion protein to its immune checkpoint ligands (e.g., PDL1 and / or PDL2) on cancer cells - e.g., as shown in FIG. 1 - the fusion protein can convert inhibitory signal (e.g., by interaction of PD1 and its ligands) to stimulatory or activating signal. The conversion from inhibitory’ signal to stimulatory or activating signal can be through DNAM1. Conversion from inhibitory signal to stimulatory or activating signal via engagement of the DNAM1 signaling can induce cytotoxic response of NK cells or T cells (e.g., can trigger NK cell-mediated and / or T cell-mediated tumor cell killing). Thus, PD1-DNAM1 fusion proteins can be useful in treating disorders associated with abnormal apoptotic or differentiative processes, such as cellular proliferative disorders or cellular differentiative disorders (e.g., cancer, including both solid tumors and hematopoietic cancers). Such treatment methods can include administering a therapeutically effective amount of NK cells and / or T cells expressing PD1-DNAM1 fusion proteins to a subject who is in need thereof.

[0142] CTLA4-DNAM1

[0143] A fusion protein (e.g., a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal) described herein can comprise the extracellular domain of cytotoxic T-lymphocyte associated protein 4 (CTLA4) and the cytoplasmic domain of DNAM1. Such a fusion protein can be referred to herein as CTLA4-DNAM1.

[0144] In some embodiments, a CTLA4-DNAM1 fusion protein comprises the extracellular domain of CTLA4 at the N-terminal and / or the cytoplasmic domain of DNAM1 at the C-terminal. In some embodiments, the extracellular domain of CTLA4 is fused to the cytoplasmic domain of DNAM1. In some embodiments, a CTLA4-DNAM1 fusion protein further comprises the transmembrane domain of DNAM1. The transmembrane domain of DNAM1 can be positioned between the extracellular domain of CTLA4 and the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of CTLA4 is fused directly to the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of CTLA4 is fused to the cytoplasmic domain of DNAM1 via a linker and / or other intervening sequence. The intervening sequence can be the transmembrane domain of DNAM1. For example, the extracellular domain of CTLA4 can be fused to the cytoplasmic domain of DNAM1 via the transmembrane domain of DNAM1.

[0145] In some instances, a CTLA4-DNAM1 fusion protein further comprises a purification tag (e.g., FLAG, cMyc tag, His-tag, or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., betw een the extracellular domain of CTLA4 and the cytoplasmic domain of DNAM1. between the extracellular domain of CTLA4 and the transmembrane domain of DNAM1, and / or the between the transmembrane domain and the cytoplasmic domain of DNAM1).

[0146] A CTLA4-DNAM1 fusion protein of the present disclosure can comprise the cytoplasmic domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0147] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPK TRV (SEQ ID NO: 2).

[0148] A CTLA4-DNAM1 fusion protein of the present disclosure can comprise the transmembrane domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g.. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0149] GGTVLLLLFVISITTIIVIFL (SEQ ID NO: 3).

[0150] CTLA4 is a 223 amino acid protein. Exemplary sequence of human CTLA4 is provided at NCBI Reference Sequence: NP 005205.2 (encoded by NM_005214.5), and UNIPROT Accession No. P16410. For example, full length human CTLA4 can comprise the amino acid sequence of: MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVC EYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTI QGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDFLLWILAAVSSGLF FYSFLLTAVSLSKMLKKRSPLTTGVYVKMPPTEPECEKQFQPYFIPIN (SEQ ID NO: 14).

[0151] A CTLA4-DNAM1 fusion protein of the present disclosure can comprise the extracellular domain of CTLA4 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0152] KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY VIDPEPCPDSD (SEQ ID NO: 15).

[0153] Thus, in some embodiments, a CTLA4-DNAM1 fusion protein of the present disclosure comprises an amino acid sequence having at least 80% (e.g.. at least 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0154] KAMHVAQPAVVLASSRGIASFVCEYASPGKATEVRVTVLRQADSQVTEVCAATYMM GNELTFLDDSICTGTSSGNQVNLTIQGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIY VIDPEPCPDSDGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPI STSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 16).

[0155] A CTLA4-DNAM1 fusion protein of the present disclosure can also comprise a signal sequence. For example, a CTLA4-DNAM1 fusion protein can comprise the signal peptide of CTLA4 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of: MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFC (SEQ ID NO: 17).

[0156] Thus, in some embodiments, a CTLA4-DNAM1 fusion protein of the present disclosure includes a signal peptide and comprises an amino acid sequence having at least 80% (e.g.. at least 81%. 82%. 83%. 84%. 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0157] MACLGFQRHKAQLNLATRTWPCTLLFFLLFIPVFCKAMHVAQPAVVLASSRGIASFVC

[0158] EYASPGKATEVRVTVLRQADSQVTEVCAATYMMGNELTFLDDSICTGTSSGNQVNLTI

[0159] QGLRAMDTGLYICKVELMYPPPYYLGIGNGTQIYVIDPEPCPDSDGGTVLLLLFVISIT

[0160] TIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPT

[0161] FSRRPKTRV (SEQ ID NO: 18).

[0162] In some embodiments, a CTLA4-DNAM1 fusion protein of the present disclosure is encoded by a nucleic acid molecule that comprises a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of:

[0163] GCTAGCGCCACCATGGCCTGCCTGGGCTTCCAGCGGCACAAGGCCCAGCTGAACC TGGCCACCCGGACCTGGCCCTGCACCCTGCTGTTCTTCCTGCTGTTCATCCCCGTG TTCTGCAAGGCCATGCACGTGGCCCAGCCCGCCGTGGTGCTGGCCAGCAGCCGGG GCATCGCCAGCTTCGTGTGCGAGTACGCCAGCCCCGGCAAGGCCACCGAGGTGCG GGTGACCGTGCTGCGGCAGGCCGACAGCCAGGTGACCGAGGTGTGCGCCGCCAC CTACATGATGGGCAACGAGCTGACCTTCCTGGACGACAGCATCTGCACCGGCACC AGCAGCGGCAACCAGGTGAACCTGACCATCCAGGGCCTGCGGGCCATGGACACC GGCCTGTACATCTGCAAGGTGGAGCTGATGTACCCCCCCCCCTACTACCTGGGCAT CGGCAACGGCACCCAGATCTACGTGATCGACCCCGAGCCCTGCCCCGACAGCGAC GGCGGCACCGTGCTGCTGCTGCTGTTCGTGATCAGCATCACCACCATCATCGTGAT CTTCCTGAACCGGCGGCGGCGGCGGGAGCGGCGGGACCTGTTCACCGAGAGCTG GGACACCCAGAAGGCCCCCAACAACTACCGGAGCCCCATCAGCACCAGCCAGCC CACCAACCAGAGCATGGACGACACCCGGGAGGACATCTACGTGAACTACCCCACC

[0164] TTCAGCCGGCGGCCCAAGACCCGGGTGTGATGACTCGAG (SEQ ID NO: 47).

[0165] Upon binding of a CTLA4-DNAM1 fusion protein to its immune checkpoint ligands (e.g., CD80 and / or CD86) on cancer cells - e.g., as shown in FIG. 1 - the fusion protein can convert inhibitory signal (e.g., by interaction of PD1 and its ligands) to stimulatory or activating signal. The conversion from inhibitory signal to stimulatory or activating signal can be through DNAM1 . Conversion from inhibitory signal to stimulatory or activating signal via engagement of the DNAM1 signaling can induce cytotoxic response of NK cells or T cells (e.g., can trigger NK cell-mediated and / or T cell-mediated tumor cell killing). Thus, CTLA4-DNAM1 fusion proteins can be useful in treating disorders associated with abnormal apoptotic or differentiative processes, such as cellular proliferative disorders or cellular differentiative disorders (e.g., cancer, including both solid tumors and hematopoietic cancers). Such treatment methods can include administering a therapeutically effective amount of NK cells and / or T cells expressing CTLA4-DNAM1 fusion proteins to a subject who is in need thereof.

[0166] LAG3-DNAM1

[0167] A fusion protein (e.g., a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal) described herein can comprise the extracellular domain of lymphocyte-activation gene 3 (LAG3) and the cytoplasmic domain of DNAM1. Such a fusion protein can be referred to herein as LAG3-DNAM1.

[0168] In some embodiments, a LAG3-DNAM1 fusion protein comprises the extracellular domain of LAG3 at the N-terminal and / or the cytoplasmic domain of DNAM1 at the C-terminal. In some embodiments, the extracellular domain of LAG3 is fused to the cytoplasmic domain of DNAM1. In some embodiments, a LAG3- DNAM1 fusion protein further comprises the transmembrane domain of DNAM1. The transmembrane domain of DNAM1 can be positioned between the extracellular domain of LAG3 and the cytoplasmic domain of DNAM1 . In some embodiments, the extracellular domain of LAG3 is fused directly to the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of LAG3 is fused to the cytoplasmic domain of DNAM1 via a linker and / or other intervening sequence. The intervening sequence can be the transmembrane domain of DNAM1. For example, the extracellular domain of LAG3 can be fused to the cytoplasmic domain of DNAM1 via the transmembrane domain of DNAM1.

[0169] In some instances, a LAG3-DNAM1 fusion protein further comprises a purification tag (e.g.. FLAG. cMyc tag, His-tag. or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., between the extracellular domain of LAG3 and the cytoplasmic domain of DNAM1, between the extracellular domain of LAG3 and the transmembrane domain of DNAM1, and / or the between the transmembrane domain and the cytoplasmic domain of DNAM1). ALAG3-DNAM1 fusion protein of the present disclosure can comprise the cytoplasmic domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0170] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPK TRY (SEQ ID NO: 2).

[0171] A LAG3-DNAM1 fusion protein of the present disclosure can comprise the transmembrane domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0172] GGTVLLLLFVISITTIIVIFL (SEQ ID NO: 3).

[0173] LAG3 is a 525 amino acid protein. Exemplary sequence of human LAG3 is provided at NCBI Reference Sequence: NP 001401105.1 (encoded by NM 001414176. 1), and UNIPROT Accession No. P18627. For example, full length human LAG3 can comprise the amino acid sequence of:

[0174] MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLR RAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRL PLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQAS MTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFL PQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPA GVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLN ATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQE AQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLLLFLILGVLS LLLLVTGAFGFHLWRRQWRPRRFSALEQGIHPPQAQSKIEELEQEPEPEPEPEPEPEPEP EPEQL (SEQ ID NO: 19).

[0175] A LAG3-DNAM1 fusion protein of the present disclosure can comprise the extracellular domain of LAG3 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the amino acid sequence of:

[0176] LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHP

[0177] LAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWL

[0178] RPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSR PDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFN VSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLL VTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGK LLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGA AVYFTELSSPGAQRSGRAPGALPAGHL (SEQ ID NO: 20).

[0179] Thus, in some embodiments, a LAG3-DNAM1 fusion protein of the present disclosure comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0180] LQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLRRAGVTWQHQPDSGPPAAAPGHP LAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRLPLQPRVQLDERGRQRGDFSLWL RPARRADAGEYRAAVHLRDRALSCRLRLRLGQASMTASPPGSLRASDWVILNCSFSR PDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFLPQVSPMDSGPWGCILTYRDGFN VSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPAGVGTRSFLTAKWTPPGGGPDLL VTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLNATVTLAIITVTPKSFGSPGSLGK LLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQEAQLLSQPWQCQLYQGERLLGA AVYFTELSSPGAQRSGRAPGALPAGHLGGTVLLLLFVISITTIIVIFLNRRRRRERRDLF TESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 21).

[0181] A LAG3-DNAM1 fusion protein of the present disclosure can also comprise a signal sequence. For example, a LAG3-DNAMl fusion protein can comprise the signal peptide of LAG3 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%. 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0182] MWEAQFLGLLFLQPLWVAPVKP (SEQ ID NO: 22).

[0183] Thus, in some embodiments, a LAG3-DNAM1 fusion protein of the present disclosure includes a signal peptide and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the amino acid sequence of:

[0184] MWEAQFLGLLFLQPLWVAPVKPLQPGAEVPVVWAQEGAPAQLPCSPTIPLQDLSLLR RAGVTWQHQPDSGPPAAAPGHPLAPGPHPAAPSSWGPRPRRYTVLSVGPGGLRSGRL PLQPRVQLDERGRQRGDFSLWLRPARRADAGEYRAAVHLRDRALSCRLRLRLGQAS MTASPPGSLRASDWVILNCSFSRPDRPASVHWFRNRGQGRVPVRESPHHHLAESFLFL PQVSPMDSGPWGCILTYRDGFNVSIMYNLTVLGLEPPTPLTVYAGAGSRVGLPCRLPA GVGTRSFLTAKWTPPGGGPDLLVTGDNGDFTLRLEDVSQAQAGTYTCHIHLQEQQLN ATVTLAIITVTPKSFGSPGSLGKLLCEVTPVSGQERFVWSSLDTPSQRSFSGPWLEAQE AQLLSQPWQCQLYQGERLLGAAVYFTELSSPGAQRSGRAPGALPAGHLGGTVLLLLF VISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYV NYPTFSRRPKTRV (SEQ ID NO: 23).

[0185] In some embodiments, a LAG3-DNAM1 fusion protein of the present disclosure is encoded by a nucleic acid molecule that comprises a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of:

[0186] GCTAGCGCCACCATGTGGGAGGCCCAGTTCCTGGGCCTGCTGTTCCTGCAGCCCC TGTGGGTGGCCCCCGTGAAGCCCCTGCAGCCCGGCGCCGAGGTGCCCGTGGTGTG GGCCCAGGAGGGCGCCCCCGCCCAGCTGCCCTGCAGCCCCACCATCCCCCTGCAG GACCTGAGCCTGCTGCGGCGGGCCGGCGTGACCTGGCAGCACCAGCCCGACAGC GGCCCCCCCGCCGCCGCCCCCGGCCACCCCCTGGCCCCCGGCCCCCACCCCGCCG CCCCCAGCAGCTGGGGCCCCCGGCCCCGGCGGTACACCGTGCTGAGCGTGGGCC CCGGCGGCCTGCGGAGCGGCCGGCTGCCCCTGCAGCCCCGGGTGCAGCTGGACG AGCGGGGCCGGCAGCGGGGCGACTTCAGCCTGTGGCTGCGGCCCGCCCGGCGGG CCGACGCCGGCGAGTACCGGGCCGCCGTGCACCTGCGGGACCGGGCCCTGAGCT GCCGGCTGCGGCTGCGGCTGGGCCAGGCCAGCATGACCGCCAGCCCCCCCGGCA GCCTGCGGGCCAGCGACTGGGTGATCCTGAACTGCAGCTTCAGCCGGCCCGACCG GCCCGCCAGCGTGCACTGGTTCCGGAACCGGGGCCAGGGCCGGGTGCCCGTGCG GGAGAGCCCCCACCACCACCTGGCCGAGAGCTTCCTGTTCCTGCCCCAGGTGAGC CCCATGGACAGCGGCCCCTGGGGCTGCATCCTGACCTACCGGGACGGCTTCAACG TGAGCATCATGTACAACCTGACCGTGCTGGGCCTGGAGCCCCCCACCCCCCTGAC CGTGTACGCCGGCGCCGGCAGCCGGGTGGGCCTGCCCTGCCGGCTGCCCGCCGGC GTGGGCACCCGGAGCTTCCTGACCGCCAAGTGGACCCCCCCCGGCGGCGGCCCC GACCTGCTGGTGACCGGCGACAACGGCGACTTCACCCTGCGGCTGGAGGACGTG AGCCAGGCCCAGGCCGGCACCTACACCTGCCACATCCACCTGCAGGAGCAGCAG CTGAACGCCACCGTGACCCTGGCCATCATCACCGTGACCCCCAAGAGCTTCGGCA GCCCCGGCAGCCTGGGCAAGCTGCTGTGCGAGGTGACCCCCGTGAGCGGCCAGG AGCGGTTCGTGTGGAGCAGCCTGGACACCCCCAGCCAGCGGAGCTTCAGCGGCC CCTGGCTGGAGGCCCAGGAGGCCCAGCTGCTGAGCCAGCCCTGGCAGTGCCAGC TGTACCAGGGCGAGCGGCTGCTGGGCGCCGCCGTGTACTTCACCGAGCTGAGCA GCCCCGGCGCCCAGCGGAGCGGCCGGGCCCCCGGCGCCCTGCCCGCCGGCCACC TGGGCGGCACCGTGCTGCTGCTGCTGTTCGTGATCAGCATCACCACCATCATCGT GATCTTCCTGAACCGGCGGCGGCGGCGGGAGCGGCGGGACCTGTTCACCGAGAG CTGGGACACCCAGAAGGCCCCCAACAACTACCGGAGCCCCATCAGCACCAGCCA GCCCACCAACCAGAGCATGGACGACACCCGGGAGGACATCTACGTGAACTACCC CACCTTCAGCCGGCGGCCCAAGACCCGGGTGTGATGACTCGAG (SEQ ID NO: 48).

[0187] Upon binding of a LAG3-DNAM1 fusion protein to its immune checkpoint ligands (e.g., FGL-1, Gal-3, LSECtin, and / or a-syn) on cancer cells - e g., as shown in FIG. 1 - the fusion protein can convert inhibitory signal (e.g., by interaction of LAG3 and its ligands) to stimulatory or activating signal. The conversion from inhibitory signal to stimulatory’ or activating signal can be through DNAM1. Conversion from inhibitory' signal to stimulatory' or activating signal via engagement of the DNAM1 signaling can induce cytotoxic response of NK cells or T cells (e.g., can trigger NK cell-mediated and / or T cell-mediated tumor cell killing). Thus, LAG3-DNAM1 fusion proteins can be useful in treating disorders associated with abnormal apoptotic or differentiative processes, such as cellular proliferative disorders or cellular differentiative disorders (e.g., cancer, including both solid tumors and hematopoietic cancers). Such treatment methods can include administering a therapeutically effective amount ofNK cells and / or T cells expressing LAG3- DNAM1 fusion proteins to a subject who is in need thereof.

[0188] TIM3-DNAM1

[0189] A fusion protein (e.g., a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal) described herein can comprise the extracellular domain of T-cell immunoglobulin and mucin domain 3 (TIM3) and the cytoplasmic domain of DNAM1. Such a fusion protein can be referred to herein as TIM3-DNAM1.

[0190] In some embodiments, a TIM3-DNAM1 fusion protein comprises the extracellular domain of TIM3 at the N-terminal and / or the cytoplasmic domain of DNAM1 at the C-terminal. In some embodiments, the extracellular domain of TIM3 is fused to the cytoplasmic domain of DNAM1. In some embodiments, a TIM3- DNAM1 fusion protein further comprises the transmembrane domain of DNAM1. The transmembrane domain of DNAM1 can be positioned between the extracellular domain of TIM3 and the cytoplasmic domain of DNAM1 . In some embodiments, the extracellular domain of TIM3 is fused directly to the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of TIM3 is fused to the cytoplasmic domain of DNAM1 via a linker and / or other intervening sequence. The intervening sequence can be the transmembrane domain of DNAM1. For example, the extracellular domain of TIM3 can be fused to the cytoplasmic domain of DNAM1 via the transmembrane domain of DNAM1.

[0191] In some instances, a TIM3-DNAM1 fusion protein further comprises a purification tag (e.g.. FLAG. cMyc tag, His-tag. or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., betw een the extracellular domain of TIM3 and the cytoplasmic domain of DNAM1, between the extracellular domain of TIM3 and the transmembrane domain of DNAM1, and / or the between the transmembrane domain and the cytoplasmic domain of DNAM1). ATIM3-DNAM1 fusion protein of the present disclosure can comprise the cytoplasmic domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0192] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPK TRY (SEQ ID NO: 2).

[0193] ATIM3-DNAM1 fusion protein of the present disclosure can comprise the transmembrane domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0194] GGTVLLLLFVISITTIIVIFL (SEQ ID NO: 3).

[0195] TIM3 is a 301 amino acid protein. Exemplary sequence of human TIM3 is provided at NCBI Reference Sequence: NP 116171.3 (encoded by NM 032782.5), and UNIPROT Accession No. Q8TDQ0. For example, full length human TIM3 can comprise the amino acid sequence of:

[0196] MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGA CPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIP GIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLT QISTLANELRDSRLANDLRDSGATIRIGIYIGAGICAGLALALIFGALIFKWYSHSKEKI QNLSLISLANLPPSGLANAVAEGIRSEENIYTIEENVYEVEEPNEYYCYVSSRQQPSQPL GCRFAMP (SEQ ID NO: 24).

[0197] ATIM3-DNAM1 fusion protein of the present disclosure can comprise the extracellular domain of TIM3 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0198] SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNY WTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTP APTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDS GATIRIG (SEQ ID NO: 25). Thus, in some embodiments, a TIM3-DNAMl fusion protein of the present disclosure comprises an amino acid sequence having at least 80% (e.g.. at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0199] SEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGACPVFECGNVVLRTDERDVNY WTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIPGIMNDEKFNLKLVIKPAKVTP APTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLTQISTLANELRDSRLANDLRDS GATIRIGGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQ PTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 26).

[0200] A TIM3-DNAM1 fusion protein of the present disclosure can also comprise a signal sequence. For example, aTIM3-DNAMl fusion protein can comprise the signal peptide of TIM3 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0201] MFSHLPFDCVLLLLLLLLTRS (SEQ ID NO: 27).

[0202] Thus, in some embodiments, a TIM3-DNAMl fusion protein of the present disclosure includes a signal peptide and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0203] MFSHLPFDCVLLLLLLLLTRSSEVEYRAEVGQNAYLPCFYTPAAPGNLVPVCWGKGA CPVFECGNVVLRTDERDVNYWTSRYWLNGDFRKGDVSLTIENVTLADSGIYCCRIQIP GIMNDEKFNLKLVIKPAKVTPAPTRQRDFTAAFPRMLTTRGHGPAETQTLGSLPDINLT QISTLANELRDSRLANDLRDSGATIRIGGGTVLLLLFVISITTIIVIFLNRRRRRERRDLF TESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 28).

[0204] In some embodiments, a TIM3-DNAM1 fusion protein of the present disclosure is encoded by a nucleic acid molecule that comprises a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of:

[0205] GCTAGCGCCACCATGTTCAGCCACCTGCCCTTCGACTGCGTGCTGCTGCTGCTGCT

[0206] GCTGCTGCTGACCCGGAGCAGCGAGGTGGAGTACCGGGCCGAGGTGGGCCAGAA CGCCTACCTGCCCTGCTTCTACACCCCCGCCGCCCCCGGCAACCTGGTGCCCGTGT GCTGGGGCAAGGGCGCCTGCCCCGTGTTCGAGTGCGGCAACGTGGTGCTGCGGA CCGACGAGCGGGACGTGAACTACTGGACCAGCCGGTACTGGCTGAACGGCGACT TCCGGAAGGGCGACGTGAGCCTGACCATCGAGAACGTGACCCTGGCCGACAGCG GCATCTACTGCTGCCGGATCCAGATCCCCGGCATCATGAACGACGAGAAGTTCAAC CTGAAGCTGGTGATCAAGCCCGCCAAGGTGACCCCCGCCCCCACCCGGCAGCGG GACTTCACCGCCGCCTTCCCCCGGATGCTGACCACCCGGGGCCACGGCCCCGCCG AGACCCAGACCCTGGGCAGCCTGCCCGACATCAACCTGACCCAGATCAGCACCCT GGCCAACGAGCTGCGGGACAGCCGGCTGGCCAACGACCTGCGGGACAGCGGCGC CACCATCCGGATCGGCGGCGGCACCGTGCTGCTGCTGCTGTTCGTGATCAGCATCA CCACCATCATCGTGATCTTCCTGAACCGGCGGCGGCGGCGGGAGCGGCGGGACCT GTTCACCGAGAGCTGGGACACCCAGAAGGCCCCCAACAACTACCGGAGCCCCAT CAGCACCAGCCAGCCCACCAACCAGAGCATGGACGACACCCGGGAGGACATCTA CGTGAACTACCCCACCTTCAGCCGGCGGCCCAAGACCCGGGTGTGATGACTCGAG (SEQ ID NO: 49).

[0207] Upon binding of a TIM3-DNAM1 fusion protein to its immune checkpoint ligands (e.g., CEACAM-1, HMGB1, PtdSer, and / or galectin-9) on cancer cells - e.g., as shown in FIG. 1 - the fusion protein can convert inhibitor}’ signal (e.g., by interaction of TIM3 and its ligands) to stimulator}7or activating signal. The conversion from inhibitor}7signal to stimulator}7or activating signal can be through DNAM1. Conversion from inhibitory signal to stimulatory or activating signal via engagement of the DNAM1 signaling can induce cytotoxic response of NK cells or T cells (e.g., can trigger NK cell-mediated and / or T cell-mediated tumor cell killing). Thus, TIM3-DNAM1 fusion proteins can be useful in treating disorders associated with abnormal apoptotic or differentiative processes, such as cellular proliferative disorders or cellular differentiative disorders (e.g.. cancer, including both solid tumors and hematopoietic cancers). Such treatment methods can include administering a therapeutically effective amount ofNK cells and / or T cells expressing TIM3-DNAM1 fusion proteins to a subject who is in need thereof.

[0208] PVRIG-DNAM1

[0209] A fusion protein (e.g., a fusion protein acting as a signal converter that can convert an inhibitor}7signal into a stimulatory signal) described herein can comprise the extracellular domain of Poliovirus receptor related immunoglobulin domain containing (PVRIG) and the cytoplasmic domain of DNAM1. Such a fusion protein can be referred to herein as PVRIG-DNAM1.

[0210] In some embodiments, a PVRIG-DNAM1 fusion protein comprises the extracellular domain of PVRIG at the N-terminal and / or the cytoplasmic domain of DNAM1 at the C-terminal. In some embodiments, the extracellular domain of PVRIG is fused to the cytoplasmic domain of DNAM1. In some embodiments, a PVRIG-DNAM1 fusion protein further comprises the transmembrane domain of DNAM1. The transmembrane domain of DNAM1 can be positioned between the extracellular domain of PVRIG and the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of PVRIG is fused directly to the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of PVRIG is fused to the cytoplasmic domain of DNAM1 via a linker and / or other intervening sequence. The intervening sequence can be the transmembrane domain of DNAM1. For example, the extracellular domain of PVRIG can be fused to the cytoplasmic domain of DNAM1 via the transmembrane domain of DNAM1.

[0211] In some instances, a PVRIG-DNAM1 fusion protein further comprises a purification tag (e.g., FLAG, cMyc tag, His-tag, or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., between the extracellular domain of PVRIG and the cytoplasmic domain of DNAM1, between the extracellular domain of PVRIG and the transmembrane domain of DNAM1, and / or the between the transmembrane domain and the cytoplasmic domain of DNAM1).

[0212] A PVRIG-DNAM1 fusion protein of the present disclosure can comprise the cytoplasmic domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0213] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPK TRV (SEQ ID NO: 2).

[0214] A PVRIG-DNAM1 fusion protein of the present disclosure can comprise the transmembrane domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0215] GGTVLLLLFVISITTIIVIFL (SEQ ID NO: 3).

[0216] PVRIG is a 326 amino acid protein. Exemplary sequence of human PVRIG is provided at NCBI Reference Sequence: NP 076975.2 (encoded by NM_024070.3), and UNIPROT Accession No. Q6DKI7. For example, full length human PVRIG can comprise the amino acid sequence of:

[0217] MRTEAQVPALQPPEPGLEGAMGHRTLVLPWVLLTLCVTAGTPEVWVQVRMEATELSS FTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIRQWAPARQARWETQSSISL ILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDPGLSAPPTPAPILRADLAGIL GVSGVLLFGCVYLLHLLRRHKHRPAPRLQPSRTSPQAPRARAWAPSQASQAALHVPY ATINTSCRPATLDTAHPHGGPSWWASLPTHAAHRPQGPAAWASTPIPARGSFVSVENG LYAQAGERPPHTGPGLTLFPDPRGPRAMEGPLGVR (SEQ ID NO: 29).

[0218] A PVRIG-DNAM1 fusion protein of the present disclosure can comprise the extracellular domain of PVRIG that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity’ to the amino acid sequence of:

[0219] LVLPWVLLTLCVTAGTPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGA GGTTLAVLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEG SWEACGSLPPSSDPGLSAPPTPAPILRAD (SEQ ID NO: 30).

[0220] Thus, in some embodiments, a PVRIG-DNAM1 fusion protein of the present disclosure comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0221] LVLPWVLLTLCVTAGTPEVWVQVRMEATELSSFTIRCGFLGSGSISLVTVSWGGPNGA GGTTLAVLHPERGIRQWAPARQARWETQSSISLILEGSGASSPCANTTFCCKFASFPEG SWEACGSLPPSSDPGLSAPPTPAPILRADGGTVLLLLFVISITTIIVIFLNRRRRRERRDLF TESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 31).

[0222] APVRIG-DNAM1 fusion protein of the present disclosure can also comprise a signal sequence. For example, a PVRIG-DNAM1 fusion protein can comprise the signal peptide of PVRIG that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the amino acid sequence of:

[0223] MRTEAQVPALQPPEPGLEGAMGHRT (SEQ ID NO: 32).

[0224] Thus, in some embodiments, a PVRIG-DNAM1 fusion protein of the present disclosure includes a signal peptide and comprises an amino acid sequence having at least 80% (e.g.. at least 81%. 82%. 83%. 84%. 85%, 86%, 87%, 88%, 89%, 90%,

[0225] 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%. or 100%) sequence identity to the amino acid sequence of:

[0226] MRTEAQVPALQPPEPGLEGAMGHRTLVLPWVLLTLCVTAGTPEVWVQVRMEATELSS FTIRCGFLGSGSISLVTVSWGGPNGAGGTTLAVLHPERGIRQWAPARQARWETQSSISL ILEGSGASSPCANTTFCCKFASFPEGSWEACGSLPPSSDPGLSAPPTPAPILRADGGTVL LLLFVISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTRE DIYVNYPTFSRRPKTRV (SEQ ID NO: 33).

[0227] In some embodiments, a PVRIG-DNAM1 fusion protein of the present disclosure is encoded by a nucleic acid molecule that comprises a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of:

[0228] GCTAGCGCCACCATGCGGACCGAGGCCCAGGTGCCCGCCCTGCAGCCCCCCGAGC CCGGCCTGGAGGGCGCCATGGGCCACCGGACCCTGGTGCTGCCCTGGGTGCTGCT GACCCTGTGCGTGACCGCCGGCACCCCCGAGGTGTGGGTGCAGGTGCGGATGGA GGCCACCGAGCTGAGCAGCTTCACCATCCGGTGCGGCTTCCTGGGCAGCGGCAGC ATCAGCCTGGTGACCGTGAGCTGGGGCGGCCCCAACGGCGCCGGCGGCACCACC CTGGCCGTGCTGCACCCCGAGCGGGGCATCCGGCAGTGGGCCCCCGCCCGGCAG GCCCGGTGGGAGACCCAGAGCAGCATCAGCCTGATCCTGGAGGGCAGCGGCGCC AGCAGCCCCTGCGCCAACACCACCTTCTGCTGCAAGTTCGCCAGCTTCCCCGAGG GCAGCTGGGAGGCCTGCGGCAGCCTGCCCCCCAGCAGCGACCCCGGCCTGAGCG CCCCCCCCACCCCCGCCCCCATCCTGCGGGCCGACGGCGGCACCGTGCTGCTGCT GCTGTTCGTGATCAGCATCACCACCATCATCGTGATCTTCCTGAACCGGCGGCGGC GGCGGGAGCGGCGGGACCTGTTCACCGAGAGCTGGGACACCCAGAAGGCCCCCA ACAACTACCGGAGCCCCATCAGCACCAGCCAGCCCACCAACCAGAGCATGGACG ACACCCGGGAGGACATCTACGTGAACTACCCCACCTTCAGCCGGCGGCCCAAGAC CCGGGTGTGATGACTCGAG (SEQ ID NO: 50).

[0229] Upon binding of a PVRIG-DNAM1 fusion protein to its immune checkpoint ligand(s) (e.g., PVRL2) on cancer cells - e.g., as shown in FIG. 1 - the fusion protein can convert inhibitory signal (e.g., by interaction of PVRIG and its ligand(s)) to stimulatory or activating signal. The conversion from inhibitory signal to stimulatory or activating signal can be through DNAM1. Conversion from inhibitory7signal to stimulatory7or activating signal via engagement of the DNAM1 signaling can induce cytotoxic response of NK cells or T cells (e.g.. can trigger NK cell-mediated and / or T cell-mediated tumor cell killing). Thus, PVRIG-DNAM1 fusion proteins can be useful in treating disorders associated with abnormal apoptotic or differentiative processes, such as cellular proliferative disorders or cellular differentiative disorders (e.g., cancer, including both solid tumors and hematopoietic cancers). Such treatment methods can include administering a therapeutically effective amount of NK cells and / or T cells expressing PVRIG-DNAM1 fusion proteins to a subject who is in need thereof.

[0230] CD96-DNAM1

[0231] A fusion protein (e.g., a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal) described herein can comprise the extracellular domain of Cluster of differentiation (CD)96 and the cytoplasmic domain of DNAM1. Such a fusion protein can be referred to herein as CD96- DNAM1.

[0232] In some embodiments, a CD96-DNAM1 fusion protein comprises the extracellular domain of CD96 at the N-terminal and / or the cytoplasmic domain of DNAM1 at the C-terminal. In some embodiments, the extracellular domain of CD96 is fused to the cytoplasmic domain of DNAM1. In some embodiments, a CD96- DNAM1 fusion protein further comprises the transmembrane domain of DNAM1. The transmembrane domain of DNAM1 can be positioned between the extracellular domain of CD96 and the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of CD96 is fused directly to the cytoplasmic domain of DNAM1. In some embodiments, the extracellular domain of CD96 is fused to the cytoplasmic domain of DNAM1 via a linker and / or other intervening sequence. The intervening sequence can be the transmembrane domain of DNAM 1 . For example, the extracellular domain of CD96 can be fused to the cytoplasmic domain of DNAM1 via the transmembrane domain of DNAM1.

[0233] In some instances, a CD96-DNAM1 fusion protein further comprises a purification tag (e.g., FLAG, cMyc tag, His-tag, or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., between the extracellular domain of CD96 and the cytoplasmic domain of DNAM1, between the extracellular domain of CD96 and the transmembrane domain of DNAM 1, and / or the between the transmembrane domain and the cytoplasmic domain of DNAM1).

[0234] A CD96-DNAM1 fusion protein of the present disclosure can comprise the cytoplasmic domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0235] NRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPK TRV (SEQ ID NO: 2).

[0236] A CD96-DNAM1 fusion protein of the present disclosure can comprise the transmembrane domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0237] GGTVLLLLFVISITTIIVIFL (SEQ ID NO: 3).

[0238] CD96 (also known as T-cell surface protein tactile (TACT)) is a 585 amino acid protein. Exemplary sequence of human CD96 is provided at NCBI Reference Sequence: NP 937839. 1 (encoded by NM 198196.3), and UNIPROT Accession No.

[0239] P40200. For example, full length human CD96 can comprise the amino acid sequence of:

[0240] MEKKWKYCAVYYIIQIHFVKGVWEKTVNTEENVYATLGSDVNLTCQTQTVGFFVQM QWSKVTNKIDLIAVYHPQYGFYCAYGRPCESLVTFTETPENGSKWTLHLRNMSCSVS GRYECMLVLYPEGIQTKIYNLLIQTHVTADEWNSNHTIEIEINQTLEIPCFQNSSSKISSE FTYAWSVENSSTDSWVLLSKGIKEDNGTQETLISQNHLISNSTLLKDRVKLGTDYRLH LSPVQIFDDGRKFSCHIRVGPNKILRSSTTVKVFAKPEIPVIVENNSTDVLVERRFTCLL KNVFPKANITWFIDGSFLHDEKEGIYITNEERKGKDGFLELKSVLTRVHSNKPAQSDN LTIWCMALSPVPGNKVWNISSEKITFLLGSEISSTDPPLSVTESTLDTQPSPASSVSPARY PATSSVTLVDVSALRPNTTPQPSNSSMTTRGFNYPWTSSGTDTKKSVSRIPSETYSSSPS GAGSTLHDNVFTSTARAFSEVPTTANGSTKTNHVHITGIVVNKPKDGMSWPVIVAAL LFCCMILFGLGVRKWCQYQKEIMERPPPFKPPPPPIKYTCIQEPNESDLPYHEMETL (SEQ ID NO: 34).

[0241] A CD96-DNAM1 fusion protein of the present disclosure can comprise the extracellular domain of CD96 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0242] VWEKTVNTEENVYATLGSDVNLTCQTQTVGFFVQMQWSKVTNKIDLIAVYHPQYGF

[0243] YCAYGRPCESLVTFTETPENGSKWTLHLRNMSCSVSGRYECMLVLYPEGIQTKIYNLL

[0244] IQTHVTADEWNSNHTIEIEINQTLEIPCFQNSSSKISSEFTYAWSVENSSTDSWVLLSKGI

[0245] KEDNGTQETLISQNHLISNSTLLKDRVKLGTDYRLHLSPVQIFDDGRKFSCHIRVGPNK

[0246] ILRSSTTVKVFAKPEIPVIVENNSTDVLVERRFTCLLKNVFPKANITWFIDGSFLHDEKE GIYITNEERKGKDGFLELKSVLTRVHSNKPAQSDNLTIWCMALSPVPGNKVWNISSEK ITFLLGSEISSTDPPLSVTESTLDTQPSPASSVSPARYPATSSVTLVDVSALRPNTTPQPSN SSMTTRGFNYPWTSSGTDTKKSVSRIPSETYSSSPSGAGSTLHDNVFTSTARAFSEVPT TANGSTKTNHVHITGIVVNKPKDGM (SEQ ID NO: 35).

[0247] Thus, in some embodiments, a CD96-DNAM1 fusion protein of the present disclosure comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%.

[0248] 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0249] VWEKTVNTEENVYATLGSDVNLTCQTQTVGFFVQMQWSKVTNKIDLIAVYHPQYGF YCAYGRPCESLVTFTETPENGSKWTLHLRNMSCSVSGRYECMLVLYPEGIQTKIYNLL IQTHVTADEWNSNHTIEIEINQTLEIPCFQNSSSKISSEFTYAWSVENSSTDSWVLLSKGI KEDNGTQETLISQNHLISNSTLLKDRVKLGTDYRLHLSPVQIFDDGRKFSCHIRVGPNK ILRSSTTVKVFAKPEIPVIVENNSTDVLVERRFTCLLKNVFPKANITWFIDGSFLHDEKE GIYITNEERKGKDGFLELKSVLTRVHSNKPAQSDNLTIWCMALSPVPGNKVWNISSEK ITFLLGSEISSTDPPLSVTESTLDTQPSPASSVSPARYPATSSVTLVDVSALRPNTTPQPSN SSMTTRGFNYPWTSSGTDTKKSVSRIPSETYSSSPSGAGSTLHDNVFTSTARAFSEVPT TANGSTKTNHVHITGIVVNKPKDGMGGTVLLLLFVISITTIIVIFLNRRRRRERRDLFTE SWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYVNYPTFSRRPKTRV (SEQ ID NO: 36).

[0250] A CD96-DNAM1 fusion protein of the present disclosure can also comprise a signal sequence. For example, a CD96-DNAM1 fusion protein can comprise the signal peptide of CD96 that comprises an amino acid sequence having at least 80%

[0251] (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%. 89%. 90%. 91%. 92%.

[0252] 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0253] MEKKWKYCAVYYIIQIHFVKG (SEQ ID NO: 37).

[0254] Thus, in some embodiments, a CD96-DNAM1 fusion protein of the present disclosure includes a signal peptide and comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the amino acid sequence of:

[0255] MEKKWKYCAVYYIIQIHFVKGVWEKTVNTEENVYATLGSDVNLTCQTQTVGFFVQM QWSKVTNKIDLIAVYHPQYGFYCAYGRPCESLVTFTETPENGSKWTLHLRNMSCSVS GRYECMLVLYPEGIQTKIYNLLIQTHVTADEWNSNHTIEIEINQTLEIPCFQNSSSKISSE FTYAWSVENSSTDSWVLLSKGIKEDNGTQETLISQNHLISNSTLLKDRVKLGTDYRLH LSPVQIFDDGRKFSCHIRVGPNKILRSSTTVKVFAKPEIPVIVENNSTDVLVERRFTCLL KNVFPKANITWFIDGSFLHDEKEGIYITNEERKGKDGFLELKSVLTRVHSNKPAQSDN LTIWCMALSPVPGNKVWNISSEKITFLLGSEISSTDPPLSVTESTLDTQPSPASSVSPARY PATSSVTLVDVSALRPNTTPQPSNSSMTTRGFNYPWTSSGTDTKKSVSRIPSETYSSSPS GAGSTLHDNVFTSTARAFSEVPTTANGSTKTNHVHITGIVVNKPKDGMGGTVLLLLF VISITTIIVIFLNRRRRRERRDLFTESWDTQKAPNNYRSPISTSQPTNQSMDDTREDIYV NYPTFSRRPKTRV (SEQ ID NO: 38).

[0256] In some embodiments, a CD96-DNAM1 fusion protein of the present disclosure is encoded by a nucleic acid molecule that comprises a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the nucleic acid sequence of:

[0257] GCTAGCGCCACCATGGAGAAGAAGTGGAAGTACTGCGCCGTGTACTACATCATCC AGATCCACTTCGTGAAGGGCGTGTGGGAGAAGACCGTGAACACCGAGGAGAACG TGTACGCCACCCTGGGCAGCGACGTGAACCTGACCTGCCAGACCCAGACCGTGG GCTTCTTCGTGCAGATGCAGTGGAGCAAGGTGACCAACAAGATCGACCTGATCGC CGTGTACCACCCCCAGTACGGCTTCTACTGCGCCTACGGCCGGCCCTGCGAGAGC CTGGTGACCTTCACCGAGACCCCCGAGAACGGCAGCAAGTGGACCCTGCACCTG CGGAACATGAGCTGCAGCGTGAGCGGCCGGTACGAGTGCATGCTGGTGCTGTACC CCGAGGGCATCCAGACCAAGATCTACAACCTGCTGATCCAGACCCACGTGACCGC CGACGAGTGGAACAGCAACCACACCATCGAGATCGAGATCAACCAGACCCTGGA GATCCCCTGCTTCCAGAACAGCAGCAGCAAGATCAGCAGCGAGTTCACCTACGCC TGGAGCGTGGAGAACAGCAGCACCGACAGCTGGGTGCTGCTGAGCAAGGGCATC AAGGAGGACAACGGCACCCAGGAGACCCTGATCAGCCAGAACCACCTGATCAGC AACAGCACCCTGCTGAAGGACCGGGTGAAGCTGGGCACCGACTACCGGCTGCAC CTGAGCCCCGTGCAGATCTTCGACGACGGCCGGAAGTTCAGCTGCCACATCCGGG TGGGCCCCAACAAGATCCTGCGGAGCAGCACCACCGTGAAGGTGTTCGCCAAGC CCGAGATCCCCGTGATCGTGGAGAACAACAGCACCGACGTGCTGGTGGAGCGGC GGTTCACCTGCCTGCTGAAGAACGTGTTCCCCAAGGCCAACATCACCTGGTTCATC GACGGCAGCTTCCTGCACGACGAGAAGGAGGGCATCTACATCACCAACGAGGAG CGGAAGGGCAAGGACGGCTTCCTGGAGCTGAAGAGCGTGCTGACCCGGGTGCAC AGCAACAAGCCCGCCCAGAGCGACAACCTGACCATCTGGTGCATGGCCCTGAGCC CCGTGCCCGGCAACAAGGTGTGGAACATCAGCAGCGAGAAGATCACCTTCCTGCT GGGCAGCGAGATCAGCAGCACCGACCCCCCCCTGAGCGTGACCGAGAGCACCCT GGACACCCAGCCCAGCCCCGCCAGCAGCGTGAGCCCCGCCCGGTACCCCG CCACCAGCAGCGTGACCCTGGTGGACGTGAGCGCCCTGCGGCCCAACACCACCC CCCAGCCCAGCAACAGCAGCATGACCACCCGGGGCTTCAACTACCCCTGGACCAG CAGCGGCACCGACACCAAGAAGAGCGTGAGCCGGATCCCCAGCGAGACCTACAG CAGCAGCCCCAGCGGCGCCGGCAGCACCCTGCACGACAACGTGTTCACCAGCAC CGCCCGGGCCTTCAGCGAGGTGCCCACCACCGCCAACGGCAGCACCAAGACCAA CCACGTGCACATCACCGGCATCGTGGTGAACAAGCCCAAGGACGGCATGGGCGGC ACCGTGCTGCTGCTGCTGTTCGTGATCAGCATCACCACCATCATCGTGATCTTCCTG

[0258] AACCGGCGGCGGCGGCGGGAGCGGCGGGACCTGTTCACCGAGAGCTGGGACACC CAGAAGGCCCCCAACAACTACCGGAGCCCCATCAGCACCAGCCAGCCCACCAAC CAGAGCATGGACGACACCCGGGAGGACATCTACGTGAACTACCCCACCTTCAGCC GGCGGCCCAAGACCCGGGTGTGATGACTCGAG (SEQ ID NO: 51).

[0259] Upon binding of a CD96-DNAM1 fusion protein to its immune checkpoint ligands (e.g., CD155 or CD111) on cancer cells - e.g., as shown in FIG. 1 - the fusion protein can convert inhibitory signal (e.g., by interaction of CD96 and its ligands) to stimulatory or activating signal. The conversion from inhibitory signal to stimulatory or activating signal can be through DNAM1. Conversion from inhibitory' signal to stimulatory or activating signal via engagement of the DNAM1 signaling can induce cytotoxic response of NK cells or T cells (e.g., can trigger NK cell-mediated and / or T cell-mediated tumor cell killing). Thus, CD96-DNAM1 fusion proteins can be useful in treating disorders associated with abnormal apoptotic or differentiative processes, such as cellular proliferative disorders or cellular differentiative disorders (e.g., cancer, including both solid tumors and hematopoietic cancers). Such treatment methods can include administering a therapeutically7effective amount of NK cells and / or T cells expressing CD96-DNAM1 fusion proteins to a subject who is in need thereof.

[0260] Methods for Enhancing Cytotoxicity ofNK cells and / or T cells

[0261] In some embodiments, the fusion proteins described here (e.g., fusion proteins acting as signal converters that can convert an inhibitory' signal into a stimulatory signal) induces or increases the cytotoxicity of immune cells. For example, these fusion proteins can induce or increase the cytotoxicity of cytotoxic lymphocytes, such as, NK cells and / or T cells (e.g., cytotoxic T cells). NK cells and / or T cells can mediate their cytotoxic activity' via 2 distinct pathways.

[0262] NK cells and / or T cells can mediate their cytotoxic activity by releasing lytic (or cytotoxic) secretory’ granules containing cytotoxins, such as perforin, granzymes (e.g., granzyme A, granzyme B, granzyme H, granzyme K, and / or granzyme M.27), and granulysin. Through the action of perforin, granzymes enter the cytoplasm of the target cell (e.g., cancer cell) and their serine protease function triggers the caspase cascade, which is a series of cysteine proteases that eventually lead to apoptosis (programmed cell death) of the target cell. This is called a "lethal hit” and allows to observe a wave-like death of the target cells. Due to high lipid order and negatively charged phosphatidylserine present in their plasma membrane, TC cells are resistant to the effects of their perforin and granzyme cytotoxins. See, e.g., Chang et al., Cellular and Molecular Life Sciences. 2017. 74 (3): 399-408; Rudd-Schmidt et al.. Nature Communications. 2019, 10 (1): 5396; Prager and Watzl, J Leukoc Biol. 2019,105: 1319-1329. In some embodiments, fusion proteins that act as signal converters and convert an inhibitory signal into a stimulatory signal (e.g., one or more of TIGIT-DNAM1, PD1-DNAM1. CTLA4-DNAM1, LAG3-DNAM1, TIM3- DNAM1, PVRIG-DNAM1, and CD96-DNAM1), can induce cytotoxicity of NK cells and / or T cells by inducing the expression of cytotoxins, such as perforin, granzymes (e.g., granzyme A, granzyme B, granzyme H, granzyme K, and / or granzyme M.27), and granulysin by the NK cells and / or T cells. For example, NK cells and / or T cells expressing these fusion proteins (or comprising expression constructs comprising nucleic acid molecules that encode these fusion proteins) can have increased expression (or release) of cytotoxins (e.g.. perforin, granzymes. and / or granulysin). as compared to control NK cells and / or T cells. Control NK cells and / or T cells can be NK cells and / or T cells that do not express these fusion proteins, or NK cells and / or T cells that do not comprise (e.g., cells that have not been transfected with) expression constructs comprising nucleic acid molecules encoding these fusion proteins.

[0263] A hallmark of the cytotoxicity of NK cells and / or T cells is degranulation, that is, the release of lytic granule contents (perforin, granzymes, and / or granulysin) onto the surface of the target cell (e.g., cancer cells). In some embodiments, fusion proteins as described herein that convert an inhibitory signal into a stimulatory signal (e g., one or more of TIGIT-DNAM1, PD1-DNAM1, CTLA4-DNAM1, LAG3- DNAM1, TIM3-DNAM1, PVRIG-DNAM1, and CD96-DNAM1), can induce degranulation of NK cells and / or T cells. For example, NK cells and / or T cells expressing these fusion proteins (or comprising expression constructs comprising nucleic acid molecules that encode these fusion proteins) can have increased degranulation, as compared to control NK cells and / or T cells. Degranulation can be assayed by degranulation assay, e.g., an assay using extemalization of CD107a as a marker of degranulation.

[0264] NK cells and / or T cells can also mediate their cytotoxic activity by engaging one or more death receptors. There are 3 different receptor / ligand systems that can mediate apoptosis upon activation: TNF (Tumor necrosis factor), binding to either TNF receptor (TNFR)-1 or -2; Fas ligand (FasL) engaging the CD95 (APO-l / Fas) receptor, and TNF-related apoptosis-inducing ligand (TRAIL), which can bind to different TRAIL receptors (see, e.g.. Peter ME, Krammer PH. Cell Death Differ. 2003, 10: 26-35; von Karstedt S, Montinaro A, Walczak H. Nat Rev Cancer. 2017, 17: 352-366). Cytotoxic lymphocytes (e.g., NK cells and / or T cells) can use all 3 receptor (e.g., death receptor) systems to kill target cells. NK cells and / or T cells can induce death receptor-mediated apoptosis by expressing TNF, TRAIL, and / or Fas ligand (FasL) to engage TNFR, TRAIL-R1 / -R2, or CD95 / Fas, respectively, on the surface of diseased cells (e.g., cancer cells).

[0265] In some embodiments, fusion proteins as described herein that convert an inhibitor}' signal into a stimulator}' signal (e.g., one or more of TIGIT-DNAM1, PD1- DNAM1, CTLA4-DNAM1, LAG3-DNAM1, TIM3-DNAM1, PVRIG-DNAM1, and CD96-DNAM1). can induce cytotoxicity of NK cells and / or T cells by inducing the expression and / or engagement of the death receptors (e.g., one or more of TNF, FasL, and TRAIL). For example, NK cells and / or T cells expressing these fusion proteins (or comprising expression constructs comprising nucleic acid molecules that encode these fusion proteins) can have increased expression and / or engagement of death receptors (e.g., one or more of TNF, FasL. and TRAIL), as compared to control NK cells and / or T cells. Control NK cells and / or T cells can be NK cells and / or T cells that do not express these fusion proteins, or NK cells and / or T cells that do not comprise (e.g., cells that have not been transfected with) expression constructs comprising nucleic acid molecules encoding these fusion proteins.

[0266] NK cell-mediated or T cell-mediated cellular cytotoxicity is needed for many approaches of immunotherapy against cancer. Antibody-based cancer therapies can depend on antibody-dependent cellular cytotoxicity (ADCC) mediated by NK cells or T cells. Chimeric antigen receptor (CAR)-T and CAR-NK cell therapies aim to directly target the activity of these cytotoxic cells against tumors and checkpoint inhibitor therapy enhances the activity of cytotoxic T cells and / or NK cells. Thus, in some embodiments, fusion proteins, as described herein, that convert an inhibitory signal into a stimulatory signal (e.g., one or more of TIGIT-DNAM1, PD1-DNAM1, CTLA4-DNAM1, LAG3-DNAM1, TIM3-DNAM1, PVRIG-DNAM1, and CD96- DNAM1), as described herein, can induce or increase NK cell-mediated or T cell- mediated killing of cancer cells. For example, NK cells and / or T cells expressing these fusion proteins (or comprising expression constructs comprising nucleic acid molecules that encode these fusion proteins) can induce increased killing or apoptosis of cancer cells, as compared to control NK cells and / or T cells. Thus. NK cells and / or T cells (or a population of NK cells and / or T cells) expressing these fusion proteins (e.g., one or more fusion proteins that can convert an inhibitory signal into a stimulatory or activating signal) can be used for treating cancer in a subject.

[0267] (B) Fusion Proteins Converting Stimulatory Signal into Inhibitory Signal

[0268] In some embodiments, the fusion proteins of the present disclosure act as signal converters that can convert a stimulatory signal into an inhibitory signal. These fusion proteins comprise the extracellular domain of an activating receptor and the cytoplasmic domain of an immune checkpoint receptor (IC receptor or IC). Such fusion proteins act as signal converters by converting stimulatory7or activation signal from the activating receptor into inhibitory7signal through the cytoplasmic domain of the IC receptor.

[0269] In some embodiments, the fusion protein comprises the extracellular domain of the activating receptor at the N-terminal. In some embodiments, the fusion protein comprises the cytoplasmic domain of the IC receptor at the C-terminal. In some embodiments, the extracellular domain of the activating receptor is fused to the cytoplasmic domain of the IC receptor. In some embodiments, the fusion protein further comprises the transmembrane domain of the IC receptor. The transmembrane domain of the IC receptor can be positioned between the extracellular domain of the activating receptor and the cytoplasmic domain of the IC receptor. In some embodiments, the transmembrane domain of the IC receptor is retained to preserve the IC receptor signaling. In some embodiments, the extracellular domain of the activating receptor is fused directly to the cytoplasmic domain of the IC receptor. In some embodiments, the extracellular domain of the activating receptor is fused to the cytoplasmic domain of the IC receptor via a linker and / or other intervening sequence. The intervening sequence can be a transmembrane domain of the IC receptor. For example, the extracellular domain of the activating receptor can be fused to the cytoplasmic domain of the IC receptor via the transmembrane domain of the IC receptor.

[0270] In some instances, a fusion protein of the present disclosure further comprises a purification tag (e g., FLAG, cMyc tag, His-tag, or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., between the extracellular domain of the activating receptor and the cytoplasmic domain of the IC receptor, between the extracellular domain of the activating receptor and the transmembrane domain of the IC receptor, and / or between the transmembrane domain of the IC receptor and the cytoplasmic domain of the IC receptor).

[0271] The IC receptor of such fusion proteins can be one or more of TIGIT, PD1, CTLA4, LAG3, TIM3, PVRIG, and CD96. The activating receptor of such fusion proteins can be DNAM-1. Accordingly, a fusion protein described herein (e.g., a fusion protein acting as a signal converter that can convert a stimulatory signal into an inhibitors’ signal) can be one or more of: DNAM1-TIGIT. DNAM1-PD1. DNAM1- CTLA4, DNAM1-LAG3, DNAM1-TIM3, DNAM1-PVRIG, or DNAM1-CD96.

[0272] Maintenance of inhibitory signaling through IC receptors (e.g., maintenance of TIGIT signaling) and inhibition of signaling through activating receptors (e.g., inhibition of DNAM1 signaling) is needed for immunosuppressive activities of regulatory T (Treg) cells. This can be achieved by suppression of FoxP3. See, e.g., Sato et al., Proc Natl Acad Sci USA 118 (21) e2021309118 (2021 May 25)). Therefore, Treg cells can be activated and / or their immunosuppressive function can be maintained by eliminating stimulatory signaling or by converting stimulatory signal into inhibitory signal through the fusion proteins described here (e.g.. fusion proteins acting as signal converters that can convert a stimulatory signal into an inhibitory signal). Thus, the fusion proteins described here (e.g., fusion proteins acting as signal converters that can convert a stimulatory signal into an inhibitory signal) can be useful in treating disorders associated with abnormal immune response, e.g., graft- versus-host disease or GVHD or autoimmune disease. The methods can include administering Treg cells expressing such fusion proteins to a subject who is in need thereof.

[0273] DNAM1-TIGIT

[0274] A fusion protein (e.g., a fusion protein acting as a signal converter that can convert a stimulatory signal into an inhibitory signal) described herein can comprise the extracellular domain of DNAM1 and the cytoplasmic domain of TIGIT. Such a fusion protein can be referred to herein as DNAM1 -TIGIT.

[0275] In some embodiments, a DNAM1 -TIGIT fusion protein comprises the extracellular domain of DNAM1 at the N-terminal and / or the cytoplasmic domain of TIGIT at the C-terminal. In some embodiments, the extracellular domain of DNAM1 is fused to the cytoplasmic domain of TIGIT. In some embodiments, a DNAM1- TIGIT fusion protein further comprises the transmembrane domain of TIGIT. The transmembrane domain of TIGIT can be positioned between the extracellular domain of DNAM1 and the cytoplasmic domain of TIGIT. In some embodiments, the extracellular domain of DNAM1 is fused directly to the cytoplasmic domain of TIGIT. In some embodiments, the extracellular domain of DNAM1 is fused to the cytoplasmic domain of TIGIT via a linker and / or other intervening sequence. The intervening sequence can be a transmembrane domain of TIGIT. For example, the extracellular domain of DNAM1 can be fused to the cytoplasmic domain of TIGIT via the transmembrane domain of TIGIT.

[0276] In some instances, a DNAM1 -TIGIT fusion protein further comprises a purification tag (e.g.. FLAG. cMyc tag, His-tag. or biotin tag). In some embodiments, one or more linkers is present between the different domains (e.g., between the extracellular domain of DNAM1 and the cytoplasmic domain of TIGIT, between the extracellular domain of the DNAM1 and the transmembrane domain of TIGIT, and / or between the transmembrane domain and the cytoplasmic domain of TIGIT).

[0277] A DNAM1 -TIGIT fusion protein of the present disclosure can comprise the extracellular domain of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0278] EEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAE RVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAA VPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKF PRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQ YTLFVA (SEQ ID NO: 39).

[0279] A DNAM1 -TIGIT fusion protein of the present disclosure can comprise the cytoplasmic domain of TIGIT that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity7to the amino acid sequence of:

[0280] LTRKKKALRIHSVEGDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDC AELHDYFNVLSYRSLGNCSFFTETG (SEQ ID NO: 40). ADNAM1-TIGIT fusion protein of the present disclosure can comprise the transmembrane domain of TIGIT that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the amino acid sequence of:

[0281] LLGAMAATLVVICTAVIV (SEQ ID NO: 41).

[0282] Thus, in some embodiments, a DNAM1 -TIGIT fusion protein of the present disclosure comprises an amino acid sequence having at least 80% (e g., at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the amino acid sequence of:

[0283] EEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQDSIAIFSPTHGMVIRKPYAE RVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQGTWQKVIQVVQSDSFEAA VPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQIDLLTYCNLVHGRNFTSKF PRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGENETFVMRLTVAEGKTDNQ YTLFVALLGAMAATLVVICTAVIVVVALTRKKKALRIHSVEGDLRRKSAGQEEWSPSA PSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRSLGNCSFFTETG (SEQ ID NO: 42).

[0284] A DNAM1-TIGIT fusion protein of the present disclosure can also comprise a signal sequence. For example, a DNAMl-TIGIT fusion protein can comprise the signal peptide of DNAM1 that comprises an amino acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%. 97%. 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0285] MDYPTLLLALLHVYRALC (SEQ ID NO: 43).

[0286] Thus, in some embodiments, a DNAM1 -TIGIT fusion protein of the present disclosure includes a signal peptide and comprises an amino acid sequence having at least 80% (e.g., at least 81%. 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the amino acid sequence of:

[0287] MDYPTLLLALLHVYRALCEEVLWHTSVPFAENMSLECVYPSMGILTQVEWFKIGTQQ DSIAIFSPTHGMVIRKPYAERVYFLNSTMASNNMTLFFRNASEDDVGYYSCSLYTYPQ GTWQKVIQVVQSDSFEAAVPSNSHIVSEPGKNVTLTCQPQMTWPVQAVRWEKIQPRQ IDLLTYCNLVHGRNFTSKFPRQIVSNCSHGRWSVIVIPDVTVSDSGLYRCYLQASAGEN ETFVMRLTVAEGKTDNQYTLFVALLGAMAATLVVICTAVIVVVALTRKKKALRIHSVE GDLRRKSAGQEEWSPSAPSPPGSCVQAEAAPAGLCGEQRGEDCAELHDYFNVLSYRS LGNCSFFTETG (SEQ ID NO: 44).

[0288] In some embodiments, a DNAM1-TIGIT fusion protein of the present disclosure is encoded by a nucleic acid molecule that comprises a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of:

[0289] ATGGACTACCCCACCCTGCTGCTGGCCCTGCTGCACGTGTACCGGGCCCTGTGCG AGGAGGTGCTGTGGCACACCAGCGTGCCCTTCGCCGAGAACATGAGCCTGGAGT GCGTGTACCCCAGCATGGGCATCCTGACCCAGGTGGAGTGGTTCAAGATCGGCA CCCAGCAGGACAGCATCGCCATCTTCAGCCCCACCCACGGCATGGTGATCCGGA AGCCCTACGCCGAGCGGGTGTACTTCCTGAACAGCACCATGGCCAGCAACAACA TGACCCTGTTCTTCCGGAACGCCAGCGAGGACGACGTGGGCTACTACAGCTGCA GCCTGTACACCTACCCCCAGGGCACCTGGCAGAAGGTGATCCAGGTGGTGCAGA GCGACAGCTTCGAGGCCGCCGTGCCCAGCAACAGCCACATCGTGAGCGAGCCCG GCAAGAACGTGACCCTGACCTGCCAGCCCCAGATGACCTGGCCCGTGCAGGCCG TGCGGTGGGAGAAGATCCAGCCCCGGCAGATCGACCTGCTGACCTACTGCAACC TGGTGCACGGCCGGAACTTCACCAGCAAGTTCCCCCGGCAGATCGTGAGCAACT GCAGCCACGGCCGGTGGAGCGTGATCGTGATCCCCGACGTGACCGTGAGCGACA GCGGCCTGTACCGGTGCTACCTGCAGGCCAGCGCCGGCGAGAACGAGACCTTCG TGATGCGGCTGACCGTGGCCGAGGGCAAGACCGACAACCAGTACACCCTGTTCG TGGCCCTGCTGGGCGCCATGGCCGCCACCCTGGTGGTGATCTGCACCGCCGTGA TCGTGGTGGTGGCCCTGACCCGGAAGAAGAAGGCCCTGCGGATCCACAGCGTGG AGGGCGACCTGCGGCGGAAGAGCGCCGGCCAGGAGGAGTGGAGCCCCAGCGCC CCCAGCCCCCCCGGCAGCTGCGTGCAGGCCGAGGCCGCCCCCGCCGGCCTGTGC GGCGAGCAGCGGGGCGAGGACTGCGCCGAGCTGCACGACTACTTCAACGTGCT GAGCTACCGGAGCCTGGGCAACTGCAGCTTCTTCACCGAGACCGGC (SEQ ID NO: 87).

[0290] By converting stimulatory signal into inhibitory signal, DNAM1-TIGIT fusion proteins can activate and / or maintain the immunosuppressive function of Treg cells. See, e.g., FIG. 3. Thus, DNAM1-TIGIT fusion proteins can be useful in treating disorders associated with abnormal immune response, e.g., graft-versus-host disease (GVHD) or autoimmune disease. Such treatment methods can include administering T-regulatory cells expressing DNAM1-TIGIT fusion proteins to a subject who is in need thereof.

[0291] Nucleic Acids and Expression Vectors

[0292] Also described herein are nucleic acid molecules encoding the fusion proteins of the present disclosure. For example, provided herein are: nucleic acid molecules that encode TIGIT-DNAM1 fusion proteins and comprise a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 45; nucleic acid molecules that encode PD1-DNAM1 fusion proteins and comprise a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 46; nucleic acid molecules that encode CTLA4-DNAM1 fusion proteins and comprise a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 47; nucleic acid molecules that encode LAG3-DNAM1 fusion proteins and comprise a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 48; nucleic acid molecules that encode TIM3- DNAM1 fusion proteins and comprise a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity7to the nucleic acid sequence of SEQ ID NO: 49; nucleic acid molecules that encode PVRIG- DNAM1 fusion proteins and comprise a nucleic acid sequence having at least 80% (e.g., at least 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 50; nucleic acid molecules that encode CD96- DNAM1 fusion proteins and comprise a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 51; and nucleic acid molecules that encode DNAM1- TIGIT fusion proteins and comprise a nucleic acid sequence having at least 80% (e.g., at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the nucleic acid sequence of SEQ ID NO: 87.

[0293] The nucleic acid molecules described here can comprise expression vectors that can be used for expression of the fusion proteins, e.g., in an NK cell, a T cell, or a Treg cell. For example, a nucleic acid molecule encoding TIGIT-DNAM1 can comprise expression vectors that can be used for expression of TIGIT-DNAM1 in an NK cell and / or a T cell; a nucleic acid molecule encoding PD1 -DNAM1 can comprise expression vectors that can be used for expression of PD1-DNAM1 in an NK cell and / or a T cell; a nucleic acid molecule encoding CTLA4-DNAM1 can comprise expression vectors that can be used for expression of CTLA4-DNAM1 in an NK cell and / or a T cell; a nucleic acid molecule encoding LAG3-DNAM 1 can comprise expression vectors that can be used for expression of LAG3-DNAM1 in an NK cell and / or a T cell; a nucleic acid molecule encoding TIM3-DNAM1 can comprise expression vectors that can be used for expression of TIM3-DNAM1 in an NK cell and / or a T cell; a nucleic acid molecule encoding PVRIG-DNAM1 can comprise expression vectors that can be used for expression of PVRIG-DNAM1 in an NK cell and / or a T cell; a nucleic acid molecule encoding CD96-DNAM1 can comprise expression vectors that can be used for expression of CD96-DNAM1 in an NK cell and / or a T cell; and a nucleic acid molecule encoding DNAM1-TIGIT can comprise expression vectors that can be used for expression of DNAM1-TIGIT in a Treg cell.

[0294] A nucleic acid molecule encoding the selected fusion protein can be inserted in an expression vector, to make an expression construct. For example, provided herein are: expression constructs comprising a nucleic acid molecule encoding TTGIT-DNAM1 that can be used for expression of TIGIT-DNAM1 in an NK cell and / or a T cell; expression constructs comprising a nucleic acid molecule encoding PD1-DNAM1 that can be used for expression of PD1-DNAM1 in an NK cell and / or a T cell; expression constructs comprising a nucleic acid molecule encoding CTLA4- DNAM1 that can be used for expression of CTLA4-DNAM1 in an NK cell and / or a T cell; expression constructs comprising a nucleic acid molecule encoding LAG3- DNAM1 that can be used for expression of LAG3-DNAM1 in an NK cell and / or a T cell; expression constructs comprising a nucleic acid molecule encoding TIM3- DNAM1 that can be used for expression of TIM3-DNAM1 in an NK cell and / or a T cell; expression constructs comprising a nucleic acid molecule encoding PVRIG- DNAM1 that can be used for expression of PVRIG-DNAM1 in an NK cell and / or a T cell; expression constructs comprising a nucleic acid molecule encoding CD96- DNAM1 that can be used for expression of CD96-DNAM1 in an NK cell and / or a T cell; and expression constructs comprising a nucleic acid molecule encoding DNAM1-TIGIT that can be used for expression of DNAM1-TIGIT in a Treg cell.

[0295] A number of suitable vectors are know n in the art, e.g., viral vectors including recombinant retroviruses, adenovirus, adeno-associated vims, lentivirus, herpes simplex virus-1, adenovirus-derived vectors; or recombinant bacterial or eukaryotic plasmids. For example, the expression construct can include a coding region for the fusion protein and one or more regulatory regions, e.g.. a promoter sequence (e.g.. a promoter sequence that restricts expression to a selected cell t pe, a conditional promoter, or a strong general promoter); an enhancer sequence; untranslated regulatory sequences (e.g., a 5'untranslated region (UTR), a 3'UTR); a poly adenylation site; and / or an insulator sequence, that direct expression of the fusion protein. Such sequences are known in the art, and the skilled artisan would be able to select suitable sequences. See, e.g., Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) John Wiley & Sons (1995), and Green and Sambrook, Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press (June 15, 2012) and supplements thereof, and other standard laboratory manuals.

[0296] Expression constructs can be administered in any biologically effective carrier, e.g., any formulation or composition capable of effectively delivering the component gene to cells in vivo. Viral vectors transfect cells directly; plasmid DNA can be delivered with the help of, for example, cationic liposomes (e.g., Lipofectin) or derivatized (e.g., antibody conjugated), poly lysine conjugates, gramicidin S, artificial viral envelopes or other such intracellular carriers, as well as direct injection of the gene construct or CaPCfi precipitation. In some embodiments, the nucleic acid is applied “naked’’ to a cell, i.e., is applied in a simple buffer without the use of any additional agents to enhance uptake. See, e.g., Current Protocols in Molecular Biology, Ausubel, F.M. et al. (eds.) Greene Publishing Associates, (1989), Sections 9. 10-9. 14 and other standard laboratory manuals.

[0297] NK Cells, T cells, and Treg Cells

[0298] The present methods include expressing, either stably or transiently, a fusion protein described herein (e.g., a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal (e.g., TIGIT-DNAM1, PD1- DNAM1, CTLA4-DNAM1, LAG3-DNAM1, TIM3-DNAM1, PVRIG-DNAM1, or CD96-DNAM1)), in an NK cell, e.g., a CD3-CD56+ lymphocyte; see, Cheng et al., Cellular & Molecular Immunology (2013) 10, 230-252. The NK cell can be a primary cell, e.g., derived from the peripheral blood of a subject and proliferated ex vivo, or can be a cultured NK cell. Thus, in some embodiments, the present disclosure provides NK cells expressing fusion proteins described hereinabove, such as, fusion proteins acting as signal converters that can convert inhibitory signal into stimulatory signal. For example, described herein are NK cells expressing one or more of TIGIT-DNAM1, PD1-DNAM1, CTLA4-DNAM1, LAG3-DNAM1, TIM3- DNAM1. PVRIG-DNAM1, and CD96-DNAM1. In some embodiments, the NK cells also express CD 16. In some embodiments, the NK cells express one or more of NKP44, NKP46, and NKP30.

[0299] When primary cells are used, allogeneic NK cells are preferred, as they were not exposed to immunosuppression and should be fully active. In preferred embodiments, the cells are obtained by performing apheresis on haploidentical related donors to collect peripheral blood leukocytes, which are then depleted of CD3+ cells before optional expansion and administration. See, e.g., Davis et al., Cancer J. 2015 Nov-Dec; 21(6): 486-491. Alternatively, the cells can be obtained from peripheral or cord blood cells, stem cells or even induced pluripotent stem cells (iPSCs); see Cheng et al.. Cellular & Molecular Immunology (2013) 10, 230-252.

[0300] Cultured NK cell lines are known in the art, e.g., including NK-92, KHYG-1 , NKL, NKG, NK-YS, YT, YTS and haNK-1 cells, as are methods of making new NK cell lines. NK-92 is a cytolytic cancer cell line that was immortalized ex vivo from NK cells from the blood of a subject suffering from a non-Hodgkins lymphoma. NK- 92 cells retain most of the activating receptors and cytolytic signaling pathways but lack the major inhibitory receptors displayed by normal NK cells, and do not express the Fc receptor CD16, and so cannot mediate antibody-dependent cellular cytotoxicity (ADCC). NK-92 cells are tumor-selective and non-immunogenic in humans. The NK- 92 cell line is described in Gong et al.. Leukemia. 8:652-8 (1994); Yan et al.. Clin Cancer Res. 4:2859-68 (1998); WO1998 / 49268 and U.S. 2002 / 0068044. NK-92 cells have been evaluated for potential therapeutic use in cancers, including hematological malignancies; see, e.g., Ljunggren and Malmberg, Nat Rev Immunol. 2007 May;7(5):329-39; Tonn et al., J Hematother Stem Cell Res. 2001 Aug;10(4):535-44; Klingemann, Cytotherapy. 2005;7(l): 16-22; Malmberg et al., Cancer Immunol Immunother. 2008 Oct;57(10): 1541-52. haNK is an NK-92 variant cell line that expresses the high-affinity Fc receptor FcyRIIIa (158V) and is in clinical development to be combined with IgGl monoclonal antibodies (mAbs). taNKs are targeted NK-92 cells that have been transfected with a gene that expresses a chimeric antigen receptor for a given tumor antigen. KHYG-1 cells were developed the blood of a patient with aggressive NK leukemia (Yagita et al., Leukemia (2000) 14, 922-930) that is IL-2 dependent and produces granzyme M. NKL cells were established from the peripheral blood of a patient with CD3-CD16+CD56+ large granular lymphocyte (LGL) leukemia (Robertson et al., Exp Hematol. 1996 Feb;24(3):406-15). NKG cells were established from the peripheral blood of a patient with rapidly progressive nonHodgkin's lymphoma (Cheng et al.. Cell Transplant. 2011:20(11-12): 1731-46). NK- YS cells were established from a patient with a leukemic-state nasal angiocentric natural killer (NK) cell lymphoma with systemic skin infiltration (Tsuchiyama et al., Blood. 1998 Aug 15:92(4): 1374-83). YT cells, a human NK-like leukaemia cell line, was established from cells in the pericardial fluid of a patient with acute lymphoblastic lymphoma (ALL) and thymoma (Yodoi et al., J Immunol 134: 1623- 1630 (1985)); Hamack et al., Anticancer Research 31(2):475-479 (2011)). YTS is a sub-clone of the NK cell leukemia line YT. All of these cell lines are commercially available. For additional information on NK cell lines, Klingermann et al.. Front. Immunol. 7:91 (2016); Dahlberg et al.. Front. Immunol. 6:605 (2015). The cells can be used as is, or modified, e.g., genetically modified as described in US7618817; US8034332 (NK-92 cells secreting cytokines including IL2); US8313943 (NK-92 cells expressing CD 16); WO 2015193411 (CAR-expressing nk-92 cells); and W02016160602 (NK-92 cells expressing FcR including CD16). Additional methods for generating and manufacturing cultured NK cells are known in the art; see, e.g., Chabannon et al., Front Immunol. 2016; 7: 504, which provides exemplary parameters for media, cytokines, and culture systems, inter alia.

[0301] The present methods include expressing, either stably or transiently, a fusion protein described herein (e.g., a fusion protein acting as a signal converter that can convert an inhibitory signal into a stimulatory signal (e.g., TIGIT-DNAM1, PD1- DNAM1. CTLA4-DNAM1, LAG3-DNAM1, TIM3-DNAM1, PVRIG-DNAM1, or CD96-DNAM1)), in a T cell, e.g., a CD3+CD8+ lymphocyte. The T cell can be a primary' cell, e.g., derived from the peripheral blood of a subject and proliferated ex vivo, or can be a cultured T cell. Thus, in some embodiments, the present disclosure provides T cells expressing fusion proteins described hereinabove, such as, fusion proteins acting as signal converters that can convert inhibitory' signal into stimulatorysignal. For example, described herein are T cells expressing one or more of TIGIT- DNAM1. PD1-DNAM1. CTLA4-DNAM1, LAG3-DNAM1, TIM3-DNAM1, PVRIG-DNAM1, and CD96-DNAM1. In some embodiments, the T cells also express CD3. In some embodiments, the T cells express CD8.

[0302] The present methods also include expressing, either stably or transiently, a fusion protein described herein (e.g., a fusion protein acting as a signal converter that can convert a stimulatory- signal into an inhibitory signal (e.g., DNAM1-TIGIT, DNAM1-PD1, DNAM1-CTLA4, DNAM1-LAG3, DNAM1-TIM3, DNAM1-PVRIG, or DNAM1-CD96)) in a Treg cell, i.e., a CD4+ / CD25+ T cell. Thus, in some embodiments, the present disclosure provides Treg cells expressing fusion proteins described hereinabove, such as, fusion protein acting as a signal converter that can convert a stimulatory signal into an inhibitory signal. For example, described herein are Treg cells expressing one or more of DNAM1-TIGIT, DNAM1-PD1, DNAM1- CTLA4, DNAM1-LAG3, DNAM1-TIM3, DNAM1-PVRIG, or DNAM1-CD96. The Treg cells can also express one or more of CD4 and CD25. The Treg cell can be a primary- cell, e.g., derived from the peripheral blood of a subject and proliferated ex vivo, or can be a cultured Treg cell.

[0303] When primary' Treg cells are used, ex-vivo expanded donor Treg cells, e.g., naturally occurring regulatory- T cells (nTregs) from peripheral blood, are preferred. In preferred embodiments, the cells are obtained from peripheral blood from a donor and expanded ex-vivo using methods known in the art: see, e.g., Dieckmann et al., J. Exp. Med. 193(1 1): 1303-1310 (2001) Chakraborty et al., Haematologica 98(4):533- 537 (2013); Hippen et al., Sci Transl Med. 2011 May 18; 3(83): 83ra41; and Taylor et al., Blood. 2002;99:3493-3499. Alternatively, the cells can be obtained from umbilical cord blood (see, e.g.. Brunstein et al., Blood. 2011 Jan 20; 117(3): 1061— 1070).

[0304] The NK cells, T cells, and Treg cells should be maintained according to good manufacturing practice (GMP) in GMP facilities. In some embodiments, the NK cells, T cells, or Treg cells are engineered to include a suicide gene, e.g., that allows for the cells expressing the gene to be killed by introduction of a specific and selective agent, as a safety measure to guard against tumorigenesis. A number of suicide gene systems are known in the art, including the cytosine deaminase gene, the varicella-zoster virus thymidine kinase gene, the nitroreductase gene, the Escherichia coli gpt gene, the E. coli Deo gene (see, e.g., Yazawa et al., World J. Surg. 2002 July; 26(7): 783-9), inducible caspase 9 (iCas9) (Di Stasi, N Engl J Med 365: 1673-1683 (2011) and Morgan, Molecular Therapy (2012); 20: 11-13), cytochrome P450, or the herpes simplex virus thymidine kinase (TK) gene, which can be wild-type or mutant TK gene (e.g., tk30, tk75, sr39tk). Cells expressing the TK protein can be killed using ganciclovir. See. e.g., WO 2016160602.

[0305] The NK cells, T cells, or Treg cells expressing a fusion protein as described herein, as w ell as any supplemental active agents for coadministration, can be incorporated into pharmaceutical compositions. Such compositions typically comprise the cells and a pharmaceutically acceptable carrier. A “pharmaceutically acceptable carrier" includes any and all solvents, antibacterial and antifungal agents, isotonic agents, and the like, compatible with pharmaceutical administration (Gennaro, 2000). Preferred examples of such carriers or diluents include, but are not limited to, water, saline, Ringer's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Supplementary7active compounds can also be incorporated into the compositions.

[0306] Sterile injectable solutions can be prepared by incorporating the active compound (e.g., NK cells, T cells, or Treg cells as described herein) in the required amount in an appropriate solvent with one or a combination of ingredients as required; preferably the solvent is already sterilized, or the formulation can be followed by sterilization. In some embodiments, the cells are cryopreserved.

[0307] Cancer Immunotherapy

[0308] The methods described herein include methods for the treatment of disorders associated with abnormal apoptotic or differentiative processes, such as, cellular proliferative disorders or cellular differentiative disorders, e.g., cancer, including both solid tumors and hematopoietic cancers. In some embodiments, the disorder is a solid tumor, e.g., breast, prostate, pancreatic, brain, hepatic, lung, kidney, skin, or colon cancer. Generally, the methods include administering a therapeutically effective amount of NK cells or T cells (or a population of NK cells or T cells) expressing a fusion protein (e.g., fusion proteins acting as signal converters that can convert an inhibitory signal into a stimulatory or activating signal), as described herein, to a subj ect who is in need of, or who has been determined to be in need of, such treatment. In some embodiments, a subject who is in need of (or who has been determined to be in need of) such treatment, is a subject who has, is diagnosed with, and / or shows the symptoms of one or more cellular proliferative disorders or cellular differentiative disorders (e.g., cancers). For example, a subject who is in need of (or who has been determined to be in need of) such treatment can be a subject who has one or more cancers, who is diagnosed with one or more cancers, and / or who shows the symptoms of one or more cancers. The subject can be a mammalian subject, such as, a human or a non-human veterinary subject (e.g., a cat, dog, rabbit, horse, goat, or Pig)

[0309] Treatment of cellular proliferative disorders or cellular differentiative disorders (e g., cancer) in a subject by the present methods can include administering a therapeutically effective amount of NK cells and / or T cells (or a population of NK cells and / or T cells) expressing a fusion protein (e g., fusion proteins acting as signal converters that can convert an inhibitory signal into a stimulatory or activating signal), as described herein, to the subject. Treatment by the present methods can result in increased death of cancer cells in a subject, as compared to before treatment (or as compared to a control subject, e.g., a subject with cancer who has not been treated by the present methods). Additionally, or in the alternative, treatment by the present methods can ameliorate at least one symptom of a cancer in a subject, as compared to before treatment (or as compared to a subj ect with cancer who has not been treated by the present methods). Additionally, or in the alternative, treatment of a subject by the present methods can result in one or more of the following in a subject, as compared to before treatment (or as compared to a subject with cancer who has not been treated by the present methods): reduction in tumor size or growth rate, reduction in risk or frequency of reoccurrence of cancer (or tumor), delay in reoccurrence of cancer, reduction in metastasis, increased survival, and / or decreased morbidity and mortality. In some embodiments, a subject before treatment by the present methods and / or a subject with cancer who has not been treated by the present methods can be referred to herein as a control subject or a control.

[0310] As used in this context, to ‘'treat’’ means to ameliorate at least one symptom of the disorder associated with abnormal apoptotic or differentiative processes. For example, a treatment can result in a reduction in tumor size or growth rate. Administration of a therapeutically effective amount of NK cells or T cells (or a population of NK cells or T cells) expressing a fusion protein (e.g.. fusion proteins acting as signal converters that can convert an inhibitory signal into a stimulator}7or activating signal), as described herein, for the treatment of a condition associated with abnormal apoptotic or differentiative processes can result in a reduction in tumor size or decreased growth rate, a reduction in risk or frequency of reoccurrence, a delay in reoccurrence, a reduction in metastasis, increased survival, and / or decreased morbidity and mortality, inter alia.

[0311] Examples of cellular proliferative and / or differentiative disorders include cancer, e.g., carcinoma, sarcoma, metastatic disorders or hematopoietic neoplastic disorders, e.g.. leukemias. A metastatic tumor can arise from a multitude of primary tumor types, including but not limited to those of prostate, colon, lung, breast and liver origin.

[0312] As used herein, the terms “cancer’’, “hyperproliferative” and “neoplastic’’ refer to cells having the capacity for autonomous growth, i.e., an abnormal state or condition characterized by rapidly proliferating cell growth. Hyperproliferative and neoplastic disease states may be categorized as pathologic, i.e., characterizing or constituting a disease state, or may be categorized as non-pathologic, i.e., a deviation from normal but not associated with a disease state. The term is meant to include all types of cancerous growths or oncogenic processes, metastatic tissues or malignantly transformed cells, tissues, or organs, irrespective of histopathologic type or stage of invasiveness. “Pathologic hyperproliferative’’ cells occur in disease states characterized by malignant tumor growth. Examples of non-pathologic hyperproliferative cells include proliferation of cells associated with wound repair.

[0313] The terms “cancer” or “neoplasms” include malignancies of the various organ systems, such as affecting lung, breast, thyroid, lymphoid, gastrointestinal, and genito-urinary tract, as well as adenocarcinomas which include malignancies such as most colon cancers, renal-cell carcinoma, prostate cancer and / or testicular tumors, non-small cell carcinoma of the lung, cancer of the small intestine and cancer of the esophagus.

[0314] The term '‘carcinoma’’ is art recognized and refers to malignancies of epithelial or endocrine tissues including respiratory system carcinomas, gastrointestinal system carcinomas, genitourinary system carcinomas, testicular carcinomas, breast carcinomas, prostatic carcinomas, endocrine system carcinomas, and melanomas. In some embodiments, the disease is renal carcinoma or melanoma. Exemplary carcinomas include those forming from tissue of the cervix, lung, prostate, breast, head and neck, colon and ovary'. The term also includes carcinosarcomas, e.g., which include malignant tumors composed of carcinomatous and sarcomatous tissues. An “adenocarcinoma” refers to a carcinoma derived from glandular tissue or in which the tumor cells form recognizable glandular structures.

[0315] The term “sarcoma” is art recognized and refers to malignant tumors of mesenchymal derivation.

[0316] Additional examples of proliferative disorders include hematopoietic neoplastic disorders. As used herein, the term “hematopoietic neoplastic disorders” includes diseases involving hyperplastic / neoplastic cells of hematopoietic origin, e g., arising from myeloid, lymphoid or ery throid lineages, or precursor cells thereof. Preferably, the diseases arise from poorly differentiated acute leukemias, e.g., erythroblastic leukemia and acute megakaryoblastic leukemia. Additional exemplary’ myeloid disorders include, but are not limited to, acute promyeloid leukemia (APML), acute myelogenous leukemia (AML) and chronic myelogenous leukemia (CML) (reviewed in Vaickus, L. (1991) Crit Rev. in Oncol. / Hemotol. 11 :267-97); lymphoid malignancies include, but are not limited to acute lymphoblastic leukemia (ALL) which includes B-lineage ALL and T-lineage ALL, chronic lymphocytic leukemia (CLL), prolymphocytic leukemia (PLL), hairy7cell leukemia (HLL) and Waldenstrom's macroglobulinemia (WM). Additional forms of malignant lymphomas include, but are not limited to non-Hodgkin lymphoma and variants thereof, peripheral T cell lymphomas, adult T cell leukemia / lymphoma (ATL), cutaneous T- cell lymphoma (CTCL), large granular lymphocytic leukemia (LGF), Hodgkin's disease and Reed-Stemberg disease. GVHD and Autoimmune Disease

[0317] Also described herein are methods for treatment of disorders associated with abnormal immune response, e.g., graft-versus-host disease (GVHD) or autoimmune diseases. The methods can include administering Treg cells (or a population of Treg cells) expressing a fusion protein (e.g., fusion proteins acting as signal converters that can convert a stimulatory or activating signal into an inhibitory signal), as described herein, to a subject who is in need thereof. For example, provided herein is a method of treating and / or preventing one or more of the following conditions (or diseases) in a subject in need thereof, by administering to the subject an effective amount of a population of Treg cells expressing a fusion protein of the present disclosure (e.g., fusion proteins acting as signal converters that can convert a stimulatory or activating signal into an inhibitory signal): multiple sclerosis (MS): lupus (or Systemic lupus erythematosus (SLE)); rheumatoid arthritis (RA) (e.g., rheumatoid factor positive RA or seropositive RA, rheumatoid factor negative RA or seronegative RA); Chronic inflammatory demyelinating polyneuropathy (CIDP); Myositis; Lupus Nephritis; other forms of MS- PPMS. SPMS: Myasthenia Gravis (MG); Antiphospholipid Syndrome; Thrombotic Thrombocytopenic Purpura (TTP); Ulcerative Colitis; Minimal Change Nephrotic Syndrome (MCNS); Aplastic Anemia; Autoimmune Glomerulopathies; Rheumatoid Arthritis (RA); Interstitial Lung Disease; Myasthenia Gravis (MG); Subepidermal Autoimmune Blistering Disease; Pulmonary Infectious Diseases; Acquired Hemophilia; Refractory Mixed Cryoglobulinemia; Primary Immune Thrombocytopenia; Graft Versus Host Disease (GVHD); Autoimmune Blistering Disease; Anti-Myelin Associated Glycoprotein (MAG) Polyneuropathy; Granulomatosis with Pollyangiitis (GPA); Neuromyelitis Optica; Systemic Lupus Erythematosus; Pemphigus; Post-Transplant Lymphoproliferative Disorders; Autoimmune Hemolytic Anemia; Cerebral Vasculitis; Microscopic Polyangiitis (MPA); and Idiopathic Nephritic Syndrome.

[0318] Allogeneic bone marrow transplantation (BMT) has been shown to be effective in hematologic malignancies and some solid tumors, but the high incidence of GVHD has limited the effectiveness and use of BMT. Tregs cells have shown efficacy in suppressing GVHD; see Olson et al., Blood. 2010 May 27;115(21):4293- 301; Sung and Chao, STEM CELLS TRANSLATIONAL MEDICINE, 2013;2:25- 32; Dieckmann et al., J. Exp. Med. 193(11): 1303— 1310 (2001) Chakraborty et al., Haematologica 98(4):533-537 (2013); Hippen et al., Sci Transl Med. 2011 May 18; 3(83): 83ra41; Taylor et al., Blood. 2002; 99:3493-3499; and Brunstein et al., Blood. 201 1; 1 17(3): 1061-1070).

[0319] Impairment of Treg functions or resistance of effector T cells to Tregs has been reported in many autoimmune diseases such as type-1 diabetes (T1D) (Brusko et al., Diabetes 2005;54(5): 1407-14). rheumatoid arthritis (van Amelsfort et al., Arthritis Rheum 2004;50(9):2775-85), multiple sclerosis (Fletcher et al.. J Immunol 2009;183(l l):7602-10), systemic lupus erythematosus (Lyssuk et al,. Adv Exp Med Biol 2007;601 :l 13-9) and psoriasis (Sugiyama et al., J Immunol 2005; 174(1): 164-73), as well as atopic disease (Singer et al., Front Immunol. 2014; 5: 46).

[0320] Tregs, e.g., CD4+CD25+, e.g.. CD4+CD25+CD127- Tregs (e.g., CD4+CD25hlghCD127-ICOS+ for atopy Tregs or CD4+CD25+CD127- CD62L+for GVHD), which are optionally FOXP3+ as well, expressing fusion proteins that can convert a stimulatory or activating signal into an inhibitory signal, can be used to reduce alloreactive T cells that are believed to mediate GVHD and autoimmunity and damage host tissues. In these embodiments, an effective amount of Treg cells expressing such fusion proteins (e.g., fusion proteins acting as signal converters that can convert a stimulatory or activating signal into an inhibitory signal) is an amount sufficient to decrease numbers of alloreactive T cells and decrease the self-immune response, e.g., by reduction of donor T cell proliferation and increased T cell apoptosis. See, e.g., Singer et al., Front Immunol. 2014; 5: 46; Riley et al., Immunity'. 2009 May; 30(5): 656-665.

[0321] In some embodiments, Treg cells (e.g.. CD4+CD25+ lymphocytes), which are optionally FOXP3+ as well, expressing fusion proteins that can convert a stimulatory or activating signal into an inhibitory signal, inhibit cytotoxic response of immune cells (e.g., NK cells and / or T cells). In these embodiments, an effective amount of Treg cells expressing fusion proteins (e.g., fusion proteins acting as signal converters that can convert a stimulatory or activating signal into an inhibitory signal) is an amount sufficient to decrease the numbers of cytotoxic immune cells and / or decrease the cytotoxic response of immune cells (e.g., NK cells and / or T cells). In some embodiments, Treg cells (e.g., CD4+CD25+ lymphocytes), which are optionally FOXP3+ as well, expressing fusion proteins that can convert a stimulatory or activating signal into an inhibitory signal, that induces (or maintains) immunosuppressive response. In these embodiments, an effective amount of Treg cells expressing fusion proteins (e.g., fusion proteins acting as signal converters that can convert a stimulatory or activating signal into an inhibitory signal) is an amount sufficient to induce (or maintain) immunosuppressive response.

[0322] Methods of Administration and Dosing

[0323] The methods include administration, preferably by intravenous infusion, of a therapeutically effective amount of the NK cells, T cells, or Treg cells described herein. A therapeutically effective dose can be determined empirically, e.g., based on animal experiments and clinical studies. In some embodiments, the methods include one or more infusions of at least IO4and up to I x 106, 5 x 106, 1 x 107. 5 x 107, l x 108, 3 x IO8, 5 x 108, 1 x 109, or 5 x 109cells per dose, e.g., between 1 billion and 3 billion cells, or any ranges between any two of the numbers, end points inclusive. The cells can be administered to a subj ect once or can be administered multiple times, e.g., once even’ 1, 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 or 23 hours, or once every 1. 2, 3, 4. 5, 6 or 7 days, or once every 1. 2, 3. 4. 5, 6, 7. 8, 9, 10 or more weeks during therapy, or any ranges between any two of the numbers, end points inclusive. See, e.g., Ljunggren and Malmberg, Nat Rev Immunol. 2007 May;7(5):329-39; Tonn et al., J Hematother Stem Cell Res. 2001 Aug; 10(4): 535 -44; Klingemann. Cytotherapy. 2005;7(l): 16-22; Malmberg et al.. Cancer Immunol Immunother. 2008 Oct;57(I O):1541 -52; Cheng et al., Cellular & Molecular Immunology (2013) 10, 230-252.

[0324] In some embodiments, before being infused into a subject the cells are treated so that they are no longer capable of proliferating, but retain cytotoxic activity. One way of achieving this state is by y irradiation, e.g., with 500 to 1000 cGy, or with 500, 1000, 2000, or 3000 cGy. Gamma irradiation of cells at doses of between about 750 and 1000 Grays, e.g., 750, 800, 850, 900 and 950 Grays, is considered to be sufficient for this purpose. Additional forms of radiation, including, for example, ultraviolet radiation, can also be used. Suitable sources to use for this purpose include, for example, a 137Cs source (Cis-US, Bedford, Mass.; Gammacell 40, Atomic Energy of Canada Ltd., Canada). Alternatively, the cells can include a suicide gene as described above. In some embodiments, before infusion of NK cells, T cells, or Treg cells, the subjects can be treated with a preparatory chemotherapy regimen, e.g., high cyclophosphamide and fludarabine (Hi-Cy [60 mg / kg x 2 days] / Flu [25 mg / m2x 5 days]), low cyclophosphamide (750 mg / m2) and methylprednisone (1000 mg / m2) or fludarabine alone (25 mg / m2x 5 days), and or with total body irradiation, e.g., a dose of 200-500, e.g., 400 cGy, radiation.

[0325] Combination Therapies

[0326] In some embodiments, NK cells or T cells expressing fusion proteins of the present disclosure (e.g., fusion proteins acting as signal converters that can convert an inhibitors’ signal into a stimulatory or activating signal) are used in combination with one or more immunotherapies. The immunotherapy can include, without limitation, immune checkpoint (IC) inhibitors (also referred to herein as checkpoint blocking agents or checkpoint inhibitors), cancer vaccines, dendritic cell vaccines, adaptive T cell therapies, and chimeric antigen receptor (CAR)-expressing immune effector cells (e.g.. CAR-T cells and / or CAR-NK cells). In some embodiments, the immunotherapy results in an increase in IFNy activity and / or levels. For example, NK cells or T cells expressing fusion proteins (e.g., fusion proteins acting as signal converters that can convert an inhibitory signal into a stimulatory or activating signal) can be administered as part of a therapeutic regimen that includes administration of one or more immunotherapies (e g., IC inhibitors, anti-tumor antibodies, CAR-T cells, and / or CAR-NK cells). The NK cells or T cells can be administered concurrently, e.g., substantially simultaneously or sequentially, with the immunotherapy, e.g., within 48, 24, 12, 6. 5, 4, 3, 2, or 1 hour, or within 45, 30, 20, or 15 minutes of administration of the immunotherapy.

[0327] Currently approved immune checkpoint inhibitors include monoclonal antibodies (mAbs) that target the programmed cell death protein 1 (PD-l) / PD-Ll / 2 or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4) pathways, and agents targeting other pathways are in clinical development (including 0X40, Tim-3, and LAG-3) (See, e.g., Leach et al.. Science 271, 1734-1736 (1996); Pardoll, Nat. Rev. Cancer 12, 252-264 (2012); Topalian et al., Cancer Cell 27, 450-461 (2015); Mahoney et al., Nat Rev Drug Discov 14, 561-584 (2015)). The present methods can include administration of NK cells or T cells expressing fusion proteins (e.g.. fusion proteins acting as signal converters that can convert an inhibitory signal into a stimulatory or activating signal) described hereinabove, as part of a therapeutic regimen, that includes administration of one or more IC inhibitors, such as: anti- CD137 antibodies (BMS-663513); anti-PD-1 antibodies (including those described in US8008449, US9073994, and US20110271358, pembrolizumab, nivolumab, Pidilizumab (CT-011), BGB-A317, MEDI0680. BMS-936558 (ONO-4538)); anti- PDL1 or anti-PDL2 antibodies (e.g.. BMS-936559, MPDL3280A, atezolizumab. avelumab and durvalumab); or anti-CTLA-4 antibodies (e.g., ipilumimab or tremelimumab). See, e.g., Kruger et al., “Immune based therapies in cancer,” Histol Histopathol. 2007 Jun;22(6):687-96; Eggermont et al., “Anti-CTLA-4 antibody adjuvant therapy in melanoma,” Semin Oncol. 2010 Oct;37(5):455-9; Klinke DJ 2nd, “A multiscale systems perspective on cancer, immunotherapy, and Interleukin- 12,” Mol Cancer. 2010 Sep 15;9:242; Alexandrescu et al., “Immunotherapy for melanoma: current status and perspectives,” J Immunother. 2010 Jul-Aug;33(6):570-90; Moschella et al., “Combination strategies for enhancing the efficacy’ of immunotherapy in cancer patients.” Ann N Y Acad S ci. 2010 Apr;1194: 169-78;

[0328] Ganesan and Bakhshi, “Systemic therapy for melanoma,” Natl Med J India. 2010 Jan- Feb;23(l):21-7.

[0329] Alternatively, or in addition, the immunotherapy can include administration of a population of immune effector cells (e.g., T cells or Natural Killer (NK) cells) that can be engineered to express one or more Chimeric Antigen Receptors (CARs). CARs are hybrid molecules comprising three essential units: (1) an extracellular antigen-binding motif, (2) linking / transmembrane motifs, and (3) intracellular T-cell signaling motifs (Long A H, Haso W M, Orentas R J. Lessons learned from a highly - active CD22-specific chimeric antigen receptor. Oncoimmunology. 2013; 2 (4):e23621). Such T cells are referred to as CAR-T cells. See, e.g., US20180355052A1; WO2017117112A1; US20190309307; US20190292246; US 20190298772; US20190000880; US20150376296; Bollino and Webb,. “Chimeric antigen receptor-engineered natural killer and natural killer T cells for cancer.” Immunotherapy. Translational Research 2017;187:32-43; Fu and Tang, Recent Patents on Anti-Cancer Drug Discovery’, 14(1), 2019;14(l):60-69, DOI : 10.2174 / 1574892814666190111120908; Jurgens and Clarke, Nature Biotechnology 37:370-375 (2019); and references cited therein. In some embodiments, the CAR-T cells are autologous, i.e., derived from the same individual to whom it is later to be reintroduced. e.g., during therapy. In some embodiments, the CAR-T cells are non- autologous, i.e., derived from a different individual relative to the individual to whom the material is to be introduced. Alternatively, a T cell-engaging therapeutic agent, such as a bispecific or other multispecific agent, e g., antibody that is capable of recruiting and / or engaging the activity of one or more T cells, such as in a targetspecific manner, can be used (see US 20190292246).

[0330] A number of immunotherapies are known in the art. In some embodiments, these therapies may primarily target immunoregulatory cell types such as regulatory T cells (Tregs) or M2 polarized macrophages, e.g., by reducing number, altering function, or preventing tumor localization of the immunoregulatory cell types. For example, Treg-targeted therapy includes anti-GITR monoclonal antibody (TRX518), cyclophosphamide (e.g., metronomic doses), arsenic tri oxide, paclitaxel, sunitinib, oxaliplatin, PLX4720, anthracy cline-based chemotherapy, Daclizumab (anti-CD25); Immunotoxin eg. Ontak (denileukin diftitox); lymphoablation (e.g.. chemical or radiation lymphoablation) and agents that selectively target the VEGF-VEGFR signaling axis, such as VEGF blocking antibodies (e.g., bevacizumab), or inhibitors of VEGFR tyrosine kinase activity (e.g., lenvatinib) or ATP hydrolysis (e.g., using ectonucleotidase inhibitors, e.g., ARL67156 (6-N.N-Diethyl-D-p.y- dibromomethyleneATP trisodium salt), 8-(4-chlorophenylthio) cAMP (pCPT-cAMP) and a related cyclic nucleotide analog (8-[4-chlorophenylthio] cGMP; pCPT-cGMP) and those described in WO 2007135195, as well as mAbs against CD73 or CD39). Docetaxel also has effects on M2 macrophages. See, e.g.. Zitvogel et al., Immunity' 39:74-88 (2013). In another example, M2 macrophage targeted therapy includes clodronate-liposomes (Zeisberger, et al., Br J Cancer. 95:272-281 (2006)), DNA based vaccines (Luo, et al., J Clin Invest. 116(8): 2132-2141 (2006)), and M2 macrophage targeted pro-apoptotic peptides (Cieslewicz, et al., PNAS. 110(40): 15919-15924 (2013)). Immnotherapies that target Natural Killer T (NKT) cells can also be used, e.g., that support type I NKT over type II NKT (e.g., CDld type I agonist ligands) or that inhibit the immune-suppressive functions of NKT, e g., that antagonize TGF-beta or neutralize CDld.

[0331] Some useful immunotherapies target the metabolic processes of immunity, and include adenosine receptor antagonists and small molecule inhibitors, e.g., istradefylline (KW-6002) and SCH-58261; indoleamine 2, 3-di oxygenase (IDO) inhibitors, e.g.. Small molecule inhibitors (e.g., 1 -methyl-tryptophan (1MT), 1- methyl-d-tryptophan (DIMT), and Toho-1) or IDO-specific siRNAs, or natural products (e.g., Brassinin or exiguamine) (see, e.g., Munn, Front Biosci (Elite Ed). 2012 Jan l;4:734-45) or monoclonal antibodies that neutralize the metabolites of IDO, e.g., mAbs against N-formyl-kynurenine.

[0332] In some embodiments, the immunotherapies may antagonize the action of cytokines and chemokines such as IL-10, TGF-beta, IL-6, CCL2 and others that are associated with immunosuppression in cancer. For example, TGF-beta neutralizing therapies include anti-TGF-beta antibodies (e.g., fresolimumab, Infliximab, Lerdelimumab. GC-1008). antisense oligodeoxynucleotides (e.g.. Trabedersen), and small molecule inhibitors of TGF-beta (e.g. LY2157299), (Wojtowicz-Praga, Invest New Drugs. 21(1): 21-32 (2003)). Another example of therapies that antagonize immunosuppression cytokines can include anti-IL-6 antibodies (e.g., siltuximab) (Guo, et al., Cancer Treat Rev. 38(7): 904-910 (2012). mAbs against IL- 10 or its receptor can also be used, e.g.. humanized versions of those described in Llorente et al., Arthritis & Rheumatism, 43(8): 1790-1800, 2000 (anti-IL-10 mAb), or Newton et al., Clin Exp Immunol. 2014 Jul;177(l):261-8 (Anti-interleukin- 10R1 monoclonal antibody). mAbs against CCL2 or its receptors can also be used. In some embodiments, the cytokine immunotherapy is combined with a commonly used chemotherapeutic agent (e.g., gemcitabine, docetaxel, cisplatin, tamoxifen) as described in US8476246.

[0333] In some embodiments, immunotherapies can include agents that are believed to elicit 'danger" signals, e.g., “PAMPs” (pathogen-associated molecular patterns) or ■‘DAMPs’’ (damage-associated molecular patterns) that stimulate an immune response against the cancer. See, e.g., Pradeu and Cooper, Front Immunol. 2012, 3:287; Escamilla-Tilch et al., Immunol Cell Biol. 2013 Nov-Dec;91(10):601-10. In some embodiments, immunotherapies can agonize toll-like receptors (TLRs) to stimulate an immune response. For example. TLR agonists include vaccine adjuvants (e.g.. 3M- 052) and small molecules (e.g., Imiquimod, muramyl dipeptide, CpG, and mifamurtide (muramyl tripeptide)) as well as polysaccharide krestin and endotoxin. See, Galluzi et al., Oncoimmunol. 1(5): 699-716 (2012), Lu et al., Clin Cancer Res. Jan 1, 2011; 17(1): 67-76, US8795678 and US8790655. In some embodiments, immunotherapies can involve administration of cytokines that elicit an anti-cancer immune response, see Lee & Margolin, Cancers. 3: 3856-3893(2011). For example, the cytokine IL- 12 can be administered (Portielj e, et al.. Cancer Immunol Immunother. 52: 133-144 (2003)) or as gene therapy (Melero, et al.. Trends Immunol. 22(3): 113-115 (2001)). In another example, interferons (IFNs), e.g., IFNgamma, can be administered as adjuvant therapy (Dunn et al., Nat Rev Immunol. 6: 836-848 (2006)).

[0334] In some embodiments, immunotherapies can antagonize cell surface receptors to enhance the anti-cancer immune response. For example, antagonistic monoclonal antibodies that boost the anti-cancer immune response can include antibodies that target CTLA-4 (ipilimumab. see Tarhini and Iqbal, Onco Targets Ther. 3: 15-25 (2010) and US7741345 or Tremelimumab) or antibodies that target PD-1 (nivolumab, see Topalian, et al., NEJM. 366(26): 2443-2454 (2012) and WO2013 / 173223A1, pembrolizumab / MK-3475, Pidilizumab (CT-011)).

[0335] Some immunotherapies enhance T cell recruitment to the tumor site (such as Endothelin receptor- A / B (ETRA / B) blockade, e.g., with macitentan or the combination of the ETRA and ETRB antagonists BQ123 and BQ788, see Coffman et al., Cancer Biol Ther. 2013 Feb;14(2):184-92), or enhance CD8 T-cell memory7cell formation (e.g., using rapamycin and metformin, see, e.g., Pearce et al.. Nature. 2009 Jul 2:460(7251): 103-7: Mineharu et al., Mol Cancer Ther. 2014 Sep 25. ph: molcanther.0400.2014; and Berezhnoy et al., Oncoimmunology. 2014 May 14;3:e28811). Immunotherapies can also include administering one or more of: adoptive cell transfer (ACT) involving transfer of ex vivo expanded autologous or allogeneic tumor-reactive lymphocytes, e.g., dendritic cells or peptides with adjuvant; cancer vaccines such as DNA-based vaccines, cytokines (e.g., IL-2), cyclophosphamide, anti-interleukin-2R immunotoxins, Prostaglandin E2 Inhibitors (e.g., using SC-50) and / or checkpoint inhibitors including antibodies such as anti- CD137 (BMS-663513), anti-PDl (e.g., Nivolumab, pembrolizumab / MK-3475. Pidilizumab (CT-011)), anti-PDLl (e.g.. BMS-936559, MPDL3280A). or anti-CTLA- 4 (e.g., ipilumimab: see, e.g., Kruger et al., ‘’Immune based therapies in cancer,” Histol Histopathol. 2007 Jun;22(6):687-96; Eggermont et al., “Anti-CTLA-4 antibody adjuvant therapy in melanoma,” Semin Oncol. 2010 Oct;37(5):455-9: Klinke DJ 2nd, “A multiscale systems perspective on cancer, immunotherapy, and Interleukin-12,” Mol Cancer. 2010 Sep 15;9:242; Alexandrescu et al., “Immunotherapy for melanoma: current status and perspectives,” J Immunother. 2010 Jul-Aug;33(6):570-90; Moschella et al., “Combination strategies for enhancing the efficacy of immunotherapy in cancer patients,” Ann N Y Acad Sci. 2010 Apr; 1194: 169-78; Ganesan and Bakhshi, “Systemic therapy for melanoma,” Natl Med J India. 2010 Jan- Feb;23(l):21-7; Golovina and Vonderheide, “Regulatory T cells: overcoming suppression of T-cell immunity,” Cancer J. 2010 Jul-Aug; 16(4): 342-7. In some embodiments, the methods include administering a composition comprising tumor- pulsed dendritic cells, e.g., as described in W02009 / 114547 and references cited therein. See also Shiao et al., Genes & Dev. 2011. 25: 2559-2572.

[0336] For further information regarding immunotherapies that can be used in the present methods, see Christofi et al., Cancers (Basel). 2019 Sep 30; 11(10). pii: E1472; Demaria et al., Nature. 2019 Oct;574(7776):45-56; and Bastien et al., Seminars in Immunology 42 (2019) 101306, and references cited therein.

[0337] In some embodiments, NK or T cells expressing fusion proteins of the present disclosure are used in combination with an immunotherapy that comprises an antibody, e g., an anti-tumor antibody. The term “antibody” as used herein refers to an immunoglobulin molecule or immunologically active portion thereof, i.e., an antigen-binding portion. Examples of immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments, which retain the ability to bind antigen. Such fragments can be obtained commercially, or using methods known in the art. For example, F(ab)2 fragments can be generated by treating the antibody with an enzyme such as pepsin, a non-specific endopeptidase that normally produces one F(ab)2 fragment and numerous small peptides of the Fc portion. The resulting F(ab)2 fragment is composed of two disulfide-connected Fab units. The Fc fragment is extensively degraded and can be separated from the F(ab)2 by dialysis, gel filtration or ion exchange chromatography. F(ab) fragments can be generated using papain, a non-specific thiol-endopeptidase that digests IgG molecules, in the presence of a reducing agent, into three fragments of similar size: two Fab fragments and one Fc fragment. When Fc fragments are of interest, papain is the enzyme of choice because it yields a 50,00 Dalton Fc fragment; to isolate the F(ab) fragments, the Fc fragments can be removed, e.g.. by affinity purification using protein A / G. A number of kits are available commercially for generating F(ab) fragments, including the ImmunoPure IgGl Fab and F(ab’)2 Preparation Kit (Pierce Biotechnology. Rockford, IL). In addition, commercially available services for generating antigen-binding fragments can be used, e.g., Bio Express, West Lebanon, NH.

[0338] The antibody can be a polyclonal, monoclonal, recombinant, e.g., a chimeric, de-immunized or humanized, fully human, non-human, e.g., murine, or single chain antibody. In some embodiments the antibody has effector function and can fix complement. In some embodiments, the antibody has reduced or no ability to bind an Fc receptor. For example, the antibody can be an isotype or subtype, fragment or other mutant, which does not support binding to an Fc receptor, e.g., it has a mutagenized or deleted Fc receptor binding region. The antibody can be coupled to a toxin or imaging agent.

[0339] Therapeutic anti-tumor antibodies are also known in the art and include human, humanized and chimeric antibodies that bind to tumor antigens. The antibodies are typically monoclonal and can be, e.g., naked, conjugated, or bispecific. Specific examples include alemtuzumab, rituxumab, trastuzumab, ibritumomab, gemtuzumab, brentuximab, adotranstuzumab, blinatunomab, daratumumab and elotuzumab; abciximab; adalimumab; alefacept; basiliximab; belimumab; bezlotoxumab; canakinumab; certolizumab pegol; cetuximab; daclizumab; denosumab; efalizumab; elotuzumab; golimumab; inflectra; ipilimumab; ixekizumab; natalizumab; nivolumab; obinutuzumab; olaratumab; omalizumab; palivizumab; panitumumab; pembrolizumab; tocilizumab; secukinumab; and ustekinumab. A number of antibodies against cancer-related antigens are known; exemplary' antibodies are described in Tables 2-3 (Ross et al., Am J Clin Pathol 119(4):472-485, 2003). The method can be used, e.g., to treat a subject who has a cancer that the antitumor antibody has been approved to treat (e.g., NK cells or T cells in combination with trastuzumab for a subject who has breast cancer, with bemtuximab in a subject who has Hodgkin lymphoma, with daratumumab in a subject who has multiple myeloma, or with elotuzumab in a subject who has multiple myeloma).

[0340] IC inhibitors (also known as checkpoint blocking agents) are known in the art and include antibodies directed to CTLA-4 (e.g., ipilimumab, tremelimumab); PD-1 (e.g., nivolumab, pembrolizumab, BGB-A317); PD-L1 (e g., atezolizumab, avelumab and durvalumab). Exemplary Sequences

[0341] Provided hereinbelow are exemplary sequences for the various domains that make up the fusion proteins described herein. In some embodiments, the sequences used are at least 80% identical to the exemplary sequence as defined herein. In some embodiments, the sequences are at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100% identical.

[0342] To determine the percent identity of two sequences, the sequences are aligned for optimal comparison purposes (gaps are introduced in one or both of a first and a second amino acid or nucleic acid sequence as required for optimal alignment, and non-homologous sequences can be disregarded for comparison purposes). The length of a reference sequence aligned for comparison purposes is at least 80% (in some embodiments, about 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the length of the reference sequence) is aligned. The nucleotides or residues at corresponding positions are then compared. When a position in the first sequence is occupied by the same nucleotide or residue as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity7between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap. which need to be introduced for optimal alignment of the two sequences.

[0343] The comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For example, the percent identity between two amino acid sequences can be determined using the Needleman and Wunsch ((1970) J. Mol. Biol. 48:444-453) algorithm which has been incorporated into the GAP program in the GCG software package, using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

[0344] SEQUENCE TABLE

[0345] EXAMPLES

[0346] The invention is further described in the following examples, which do not limit the scope of the invention described in the claims.

[0347] Materials and Methods

[0348] Reagents

[0349] Chloroquine. Matrigel (cat# 126-2.5) (Sigma-Aldrich Co). Horse serum (HS), DMEM / F12, Lipofectamine 2000 and TRIzol (Life Technologies). Fetal bovine serum (FBS) (Atlanta Biologicals). RPMI 1640 (LONZA). Smartscribe and Blueprint Onestep RT-PCR Takara kit (Clontech Laboratories); Platinum SYBR Green qPCR (Invitrogen), PfuUltra DNA polymerase (Stratagene). Human TGFpi (Antigenix America Inc). IL2 was from MGH-DF / HCC Recombinant Protein Core (Boston, MA). Human IL-4, IL7, IL12, IL21 (Shenandoah Biotechnology Inc), LDH inhibitor (R)-GNE-140 (Medchemexpress LLC), L-(+)-Lactic acid, ca 85-90% aq. soln., (Thermo Scientific Chemicals). TLCV2 lentiviral vector (Plasmid #87360) and sleeping beauty vectors for establishing K562 feeder cell line pSBbi 41BBL-CD86 (Plasmid #183244), pSBbi G418R mbIL21 (Plasmid #194329) were all from Addgene.

[0350] Cells

[0351] HEK293T, NK92, PC-3, HepG2, MDA-MB-231 , Panc-1 , U87, K562, mouse mammary^ 4T1 cells were from ATCC, U251GM a gift from Dr. Samuel Rabkin. Dog melanoma CMGD-5 cells were obtained from Kerafast, LCC. Human primary hepatocytes were obtained from the Massachusetts General Hospital Cell resource core. Tumor cell lines PC-3, U251, Panc-1, MDA-MB-231, A375, PC-9, CMGD-5, K562 and 4T1 were cultured in complete RPMI1640 medium, HEK293T and HepG2 in complete DMEM / F12. NK92 and IL2-dependent derived cell lines were cultured in RPMI1640 with 100 lU / ml IL2, as described earlier30. All cell lines and assay cultures were maintained at 37°C and 5% CO2. All cell lines were used until a maximum passage of twenty. Mycoplasma contamination was monitored using MycoFluor mycoplasma detection kit (Molecular Probes).

[0352] Chimera CIRB21 construction

[0353] CIRB21 constructs were generated by adding IL21 receptor cytoplasmic domain to the CIRB chimera described earlier (29).

[0354] Immune checkpoint signal converters construction

[0355] Extracellular domains of human TIGIT from amino-acid 1-141 and human PD1 extracellular domain from amino-acid 1-171 were separately fused to the transmembrane and cytoplasmic domain of human DNAM1 from amino-acid 255- 336. Constructs were cloned in lentiviral vector CSCW-GFP at Nhel-Xhol.

[0356] Lentivirus production and NK cell lines transduction

[0357] HEK293T cells were transfected using Lipofectamine 2000 with 2.4 pg total DNA of pVSV (Clontech Laboratories), pCMVdr8.2dvrp Addgene (plasmid # 8455) or psPAX2 (Plasmid #12260) and the lentiviral construct CSCW-GFP or CSCW- mCherry vectors MGH Vector Core (Boston, MA) to express either CTRB, CIRB21, immune checkpoint converters, and other CRISPR guide RNA constructs cloned in TLCV2 plasmid, using the ratios 1: 0.4: 1, respectively. Six hours post-transfection, media was changed, and lentiviral supernatant was collected three days later and filtered through a 0.45um PES filter syringe. NK92 cells were infected by spinoculation at 1800g for 45 minutes at room temperature using an optimal multiplicity of infection (MOI) of 46 lentiviral particles per cell in a 2ml-Eppendorf tube containing 2xl05cells. Infected cells were plated in some experiments with IL2(100IU / ml) for two days and weaned off exogenous IL2 afterward.

[0358] Antibodies

[0359] All antibodies were used as specified by the manufacturer. NK cell markers expression was verified using mouse anti-human antibodies to CD45 -Brilliant violet (cat# 304049), CD25-PE-CY7 (cat# 385603), EGFR-APC (cat# 352905), NKG2D- APC (cat# 320808), NKP30-PE. NKP44-PE-CY7, NKP46-FITC. Granzyme B- FITC(cat# 515403), Perforin 1 -PE (cat# 353303), Interferon-y-APC (cat# 50251 1), TNF-al- APC-CY7 (cat# 502943), CD107- PE-CY7 (cat# 328618), PE anti-human CD274 (B7-H1, PD-L1, cat# 329705). APC anti-human CD155 (PVR cat# 337617), PE / Cyanine7 anti-human CD226 (DNAM1 cat# 338315). PE anti-AKT Phospho (Ser473, cat# 606553), FITC anti-CD86 (cat# 374203), APC-anti-CD28 (cat#302911) and CD69-PerCP-Cy5.5 (cat# 310926) were from BioLegend. CD56-PAC BLUE (cat# 580380) and CD122-PE (cat# BD 340254) were from BD biosciences.

[0360] Flow cytometry

[0361] For cell surface staining, the cells were harvested, washed twice with PBS, and then incubated with an appropriate amount of fluorochrome-conjugated antibodies for 30 min on ice in PBS 2% FBS, followed by two washes in PBS 2% FBS. For intracellular staining, cells were fixed for 10 minutes at room temperature using 4% paraformaldehyde in PBS, washed twice in PBS 2% FBS, and then permeabilized in 0.01% Saponin in PBS 2% FBS for 15 minutes at room temperature, followed by incubation with antibodies in the same buffer as recommended by manufactures for 30 minutes on ice followed by two washings with 0.01% Saponin (Sigma- Aldrich cat#84510) in PBS 2% FBS buffer and a final wash with PBS 2% FBS. Cells were then filtered through a cell strainer filter (Fisher Scientific, cat# 0877123) and analyzed by flow cytometry.

[0362] Cells were analyzed at the MGH Flow Cytometry Core facility using a BD 5 laser SORP FACS VANTAGE SE DIVA SYSTEM or FACSARIA (BD Biosciences). Mitochondrial mass was determined by flow cytometry using MITOVIEW GREEN (lOOnM) from Biotium. Inc (cat# NC1392183), incubated with cells at 37C for 15 minutes followed by two washes with PBS. FACS data and ^Median statistics were analyzed using FlowJo 10.8.1 software (Tree Star. Inc.).

[0363] Cytotoxic activity of NK92, NK92CIRBand NK92CIRB21cells

[0364] NK cell lines' anti-cancer effect was evaluated against U251, PC-3, Panc-1, MDA-MB-231, U87, HepG2, A375. PC-9 mouse mammary 4T1. and Dog melanoma CMGD-5. Cancer cells (32xl03) were first plated in triplicate in a 24-well plate 24 hours before adding NK92 (pre-stimulated with IL2, 1 OOIU / ml), NK92CIRB21or NK92CIRBat the indicated E / T ratio. Co-cultured cells were then incubated for 3 to 4 days. The viability of cancer cells was determined using a 0.2% crystal violet in a 2% alcohol solution, followed by extraction using 70% ethanol and an absorbance reading at 562nm. In other experiments, human primary hepatocyte cells were plated in 48- well at 105cells / well. NK cells were added at 2. 104cells and left for four days, after which cytotoxicity was determined using crystal violet. In other experiments. NK92CIRB21anticancer activity was compared to human primary NK cells expanded using K562 feeder cells (ratio 1 : 1) expressing membrane IL21, 4-1BBL, and CD86 at multiple ratios against PC-3 cells for four days. Cell viability and growth were determined with Trypan blue exclusion using a Luna II automated cell counter.

[0365] CRISPR knockout of immune checkpoints

[0366] CRISPR guide RNA target sites and flanking primers used for sequencing (Table 1) in PD-1, CTLA-4, TIGIT, LAG3, and TIM3 were identified using CHOPCHOP (31) software. Guide RNAs were cloned in TLCV2 vector at BsmbI sites. Lentiviral particles were obtained as described earlier and used to infect NK92CIRB21cells. Infected cells were selected with puromycin (2 pg / ml) for two days, and Cas9 was induced by Doxycycline (2 qg / ml) for one week. Indel mutations were confirmed by next-generation sequencing. Multiplexed sequencing of more than 100,000 DNA copies was produced for each amplicon. The sequencing error rate is less than 0.003 per base. Next-generation sequencing results were analyzed by the Massachusetts General Hospital Center for Computational and Integrative Biology and reported as the frequency of unique complementary sequence pairs, including clusters of sequences with overlapping ends, from each demultiplexed sample. Sequence pairs occurring less than 100 times were not reported.

[0367] TABLE 1. List of primers used for CRISPR knockout of human immune checkpoints

[0368] Impact of pre-exposure to PC-3 cells on NK92CIRB21cells immune checkpoints

[0369] PC-3 cells were plated as 3. 105, 4. 105, or 5. 105cells into 6-well plates. 48 hours later NK92CIRB21cells (N=2. 105) were added to PC-3 cells. After four days of exposure, NK92CIRB21cells were transferred to clean 75mm2flasks overnight. NK92CIRB21cells exposed to PC-3 were then analyzed by flow cytometry using BIOLEGEND antibodies against Tim3-PAC Blue (345041), LAG3-PE (369305), CTLA-4-PE CY7 (349913), TIGIT-APC (372705) and PDl-Alexa Fluor (329951). Data were analyzed as a percentage of the absolute positive population in the incrementally exposed and nonexposed NK92CIRB21cells stained by immune checkpoint antibodies above the corresponding negative control.

[0370] Evolution by selection of IC knockout NK92CIRB21cells exposed to PC-3 cells

[0371] To determine whether the CRISPR knockout of specific IC can provide a selective advantage to NK92CIRB21cells through multiple exposures to PC-3 cells. CRISPR knockout NK92CIRB21cell lines for PD-1, CTLA-4, TIGIT, TIM3. and LAG3 were added as (N=2.5.105) to PC-3 cells (N=5.105) plated overnight into 6-well plates. After three days of exposure, selected NK92CIRB21CRISPR cell lines were recovered and left to grow for two days. Three more subsequent exposures of the same NK cells were done sequentially against fresh PC-3 cells (N=2. 105) with a resting interval of two days between all four cycles of evolution. Following one week of recovery7, the genomic DNA of each immune checkpoint knockout NK92CIRB21cell line was prepared by alkaline lysis at 80 °C for 40 min, and the frequencies of gene knockout in the first and the evolved fourth generation of NK92CIRB21cells were determined from PCR-amplified amplicons using specific primers for each immune checkpoint gene target (Table 1). Next-generation sequencing results were analyzed as described earlier.

[0372] Expression profiles of cytotoxicity effectors in NK cells

[0373] Natural cytotoxicity receptors (NCRs): NKP30, NKP44, NKP46, cytolytic enzymes: Perforin-1, Granzyme-B, and cytokines: TNFa and INFy were quantified by qRT-PCR using the primers listed in Table 2. Transcriptomic analysis of RNA expression in NK92 activated by IL2, IL21, IL2+IL21, and in NK92CIRBand NK92CIRB21was done by RNAseq analysis using the HTG (Molecular Diagnostics, Tucson, AZ, USA) EdgeSeq Immuno-Oncology assay which quantitatively measures by Next-Generation sequencing the expression of 1392 human RNA transcripts involved in the innate and adaptive immune response to cancer. RNA was prepared from 1 million NK cells per sample. RNAseq data are expressed as Log2CPM sample to log2CPM NK92 activated by l OOIU / ml 1L2. Key protein expression profiles were also determined by flow cytometry' and western blots.

[0374] TABLE 2. List of primers used for qPCR Tumor Growth Delay Experiments

[0375] All experiments involving animals were approved by the IACUC at Massachusetts General Hospital. PC-3 cells were suspended in serum-free RPMI1640 containing 20% Matrigel and injected subcutaneously (s.c.) as 4xl06cells for PC-3 in a volume of 0.2 ml using a 0.5-inch 29-gauge needle and a 1 ml insulin syringe in 7- week-old (24-25g) male NOD.Cg-Prkdcscid I12rgtmlWjl / SzJ immunodeficient mice (Jackson Laboratory). Tumor areas (length x width) were measured twice a week using Vernier calipers (Manostat Corp., Switzerland), and tumor volumes were calculated based on Volume = n / 6 (length x width)3 2. Treatment with NK92CIRBor NK92CIRB21was initiated when the average tumor volume reached -100-200 mm3. Freshly prepared live NK cells were suspended in PBS and administered as weekly injections (5x106cells in 200ul per mouse) via the tail vein. Immune checkpoint antibodies anti-PDl (150ug), anti-TIGIT (150ug), and anti-CTLA-4 (75ug), all from Selleck chemicals LLC, were injected per mouse i.p., 2 hours after NK cells injection and again on day three post-NK cells injection. The same treatment was repeated seven days after the first injection of NK cells. In another experiment, NK92CIRB21cells expressing immune checkpoint signal converter TIGIT-DNAM were administered alone or with Anti-PDl (200ug / mouse, n=6), and again on day three post-NK cells injection.

[0376] Detection ofNK92clRB21in NSG mice peripheral blood

[0377] Live NK92CIRB21(5. 106cells) were injected via the tail vein. Animals were killed 17 hours later, and cardiac blood was collected, processed, and analyzed by flow cytometry using human-specific anti-CD45 and NK-expressed mCherry fluorescence.

[0378] Statistical Analysis

[0379] The statistical significance of differences was determined by a two-tailed student's test and a one-way ANOVA paired with Tukey’s multiple comparison test. All tests included comparisons to untreated samples or as indicated in the text. Statistical significance is indicated by *P<0.05, **P<0.01, ***P<0.001, ****P<0.001. Analyses were performed using Prism software version 8 (GraphPad Software). Example 1. PD1-DNAM1 and TIGIT-DNAM1 increased the cytotoxicity of NK92 cells

[0380] This example describes the effect of PD1-DNAM1 and TIGIT-DNAM1 fusion proteins on cytotoxicity of NK cells (NK92) against cancer cells.

[0381] Briefly, NK92CIRB21cells were transfected with PD1-DNAM1 construct, TIGIT-DNAM1 construct, full length DNAM1 (control), or were left unmodified. Prostate cancer cell line PC-3 was used as cancer cells. About 400.000 PC-3 cells were plated. Twenty-four hours after plating the PC-3 cells, unmodified NK92CIRB21cells or NK92CIRB21cells expressing TIGIT-DNAM1, PD1-DNAM1, or full length DNAM1 were added and left for 4 days with the PC-3 cells. After 4 days of incubation, the remaining surviving PC-3 cells were quantified using a crystal violet stain. The results are described in FIGs. 2A-2B.

[0382] As shown in FIGs. 2A-2B, the fusion protein constructs TIGIT-DNAM1 and PD1-DNAM1 markedly enhanced the cytotoxic effect of NK92CIRB21cells on the PC- 3 cancer cells (see, e.g., the reduced survival of the cancer cells in presence of NK cells expressing TIGIT-DNAM1 and PD1-DNAM1). As compared to full DNAM-1 sequence (used as control), PD1-DNAM1 and TIGIT-DNAM1, when expressed in NK92 cells, substantially increased the cytotoxicity of NK92 against cancer cells. The DNAM-1 control construct did not change the cytotoxicity of NK92 cells, suggesting that the fusion protein signal converters of the present disclosure are indeed transmitting an activation signal.

[0383] Example 2. Induction of the expression of immune checkpoint (IC) receptors and signal converters in NK92 cells

[0384] NK and T-cell activation is often associated with immune checkpoint receptor (IC receptor; also referred to herein as immune checkpoints or ICs) induction and exhaustion. We examined the major IC in NK92CIRB21after four days of exposure to increasing PC-3 cancer cell numbers. After exposure to cancer cells, NK92CIRB21cells were recovered and rested for 24 hours. Flow cytometry analysis revealed that PD-1 and TIGIT were the most induced immune checkpoints, with increased expression proportionally to PC-3 cell number (FIG. 4). While TIM3 was induced and remained stable at all ratios, CTLA-4 and LAG3 were induced at a lower PC-3 cell ratio but were reduced at higher PC-3 ratios. To understand the involvement of each of the major ICs during encounter of NK cells with cancer cells, we measured in NK92CIRB21cell line, the natural selection by the evolution of CRISPR knockout cell lines for each IC after multiple exposures to PC-3 for four generations (FIG. 5). The percentage of gene knockout was determined by NGS sequencing when the knockout cell lines were obtained (1stgeneration) and later after the fourth exposure to PC-3 cancer cells (4thgeneration). As determined by NGS, all IC showed a positive selection, with LAG3 KO being the most selected, with an enrichment of 23.1% (from 65.3 to 88.4%). TIGIT and TIM3 were equally selected with a further enrichment of 7% and 9%, respectively. CTLA-4 and PD-1 first generations were already highly enriched, with gene knockout approaching 87.5% and 72.3%, respectively. Despite these high initial ratios, the respective cell lines were selected further by ~3% and ~5% at the fourth generation. The outcome of these selections might have been different if a different target cancer cell line was used. However, all five major ICs appear to contribute to NK92CIRB21cell inhibition. Therefore, inhibition of these ICs will increase the fitness and resilience ofNK cells.

[0385] Untouched (e.g., non -transfected) NK92CIRB21, NK92CIRBand NK92 cells activated by both IL2 and IL21 or IL21 alone have higher intrinsic PD-1 and CTLA-4 RNA expression than NK92 stimulated by IL2 only with ~16-fold and 5.8-fold in NK92CIRD21, respectively (FIG. 6; left panel). However, only PD1 was significantly expressed at NK92CIRB21cell surface before contact with cancer cells (FIG.4). In other experiments, NK92CIRB21cells exposed to high numbers of breast cancer MDA- MB-231 cells for a week showed higher RNA expression of PD-1 (~92 to 104-fold), while CTLA-4. Lag3, Tim3 and TIGIT did not change (FIG. 6; central panel). Similar increases of PD-1 RNA approaching 7.7-fold were observed when exposing NK92CIRBto high numbers of MDA-MB-231 cells (FIG. 6; right panel).

[0386] Current IC inhibitors using therapeutic antibodies have achieved remarkable results in the clinic but remain ineffective for a large section of cancer patients. Current approaches are also passive, aiming only to block interactions between ICs and their ligands. We sought to create a new class of IC inhibitors that also convert their inhibitions into activations and compete with endogenous IC of immune cells for the same ligands on cancer cells. We engineered two chimeric signal converters based on TIGIT and PD-1 extracellular domains tethered to DNAM1 transmembrane and cytoplasmic domains (described hereinabove as TIGIT-DNAM1 and PD1- DNAM1. respectively). The signal converters were co-expressed with GFP in NK92CIRB21as it does not, like NK92, express detectable levels of DNAM1. TIGIT- DNAM1 can convert IC inhibition through interaction with the extracellular domain of TIGIT with PVR (CD155), PVRL2 (CD112), PVRL4 (nectin-4) and PVRL3 (CD113). TIGIT-DNAM1 can compete with wild-type DNAM1 for PVR and PVRL2. Similarly. PD-1-DNAM1 was engineered to convert the inhibition from ligands PDL-1 and PDL-2 through interaction with the extracellular domain of PD-1. Wild-type DNAM1, TIGIT-DNAM1, and PD-1-DNAM1 expression enhanced NK92CIRB21killing against multiple cancer cell lines with different efficiencies (FIG. 7; top panel). The enhanced killing of PC-3 cells was largely preserved after a second four-day exposure, with TIGIT-DNAM1 showing the most robust resistance to exhaustion (FIG. 7; bottom panel). The cytotoxicity' profile conferred by TIGIT- DNAM1 and PD-1-DNAM1 was largely correlated to the levels of ligands PD-L1 and PVR expression in the tested cancer lines (FIG. 8). Noticeably, PVR showed high expression in most cancer cell lines while PD-L1 expression was consistently lower. Downstream DNAM1 signaling is associated with AKT phosphorylation, which we measured using pAKT antibody by flow cytometry after exposure of NK cells to PC-3 cells for 30 minutes. However, the constitutive expression of DNAM1 constructs increased the baseline of pAKT activity compared to parental NK92CIRD21in the absence of contact with cancer cells by 39.6% in DNAM1, 17.4% in PD-1 -DNAM1, and 23% in TIGIT-DNAM1 (FIG. 9). Contact with cancer cells increased pAKT by a further 3.7% in TIGIT -DNAM1, 6.5% in PD-1-DNAM1, and 7.8% in DNAM1- expressing NK cells compared to NK92CIRB21. Therefore, the novel IC signal converters mediate an activation signal that enhances NK cell cancer activity through DNAM1 activation and AKT phosphorylation. We next examined how overexpressed ligands PVR and PD-L1 in PC-3 cells impact the cytotoxicity' mediated by signal converters. We also examined the impact of higher TIGIT-DNAM1+expression on the same cytotoxicity (FIG. 10). Counterintuitively, higher TIGIT- DNAM1+expression reduced the killing of PC-3 cells while the overexpression of ligand PD-L1 increased PC-3 / PD-L1 cells killing by all signal converters including by wild type DNAM1 and TIGIT-DNAM1+. However, overexpression of ligand PVR led to an opposite significant reduction of PC3 / PVR cells, killing, except for PD-1- DNAM1. which does not interact with PVR.

[0387] These results suggest that overactivation of signal converters by their overexpression or overexpression of their respective ligands may lead to exhaustion and that this exhaustion could be reduced through endogenous PD-1 and TIGIT inhibitory signaling of immune cells, which are known to reduce AKT phosphorylation. Therefore, a balance between pAKT activation and deactivation is obtained through signal converters and endogenous IC.

[0388] Example 3. Anti-tumor activity of live NK92CIRB21is enhanced by immune checkpoint inhibitors

[0389] Solid tumors constitute a significant challenge to many cancer immunotherapy modalities. We previously reported a better antitumor activity of irradiated NK92CIRBcells than NK92 expressing soluble IL2. Therefore, we assessed the antitumor activity of live NK92CIRB21compared to live NK92CIRBin NSG mice implanted subcutaneously with PC-3 tumors. When tumors were treated at volumes approaching 77 mm3, a single injection of live NK92CIRB21induced an immediate response followed by a nine-day tumor grow th delay versus a six-day tumor delay by NK92CIRBcompared to untreated tumors (FIG. 11, left panel). When using large tumors approaching 270 mm3size, comparable fast tumor response was seen after the infusions ofNK92CIRB21, but not with NK92CIRBand was follow ed by a tumor delay of 10 days (FIG. 11, right panel). However, a second infusion of NK cells seven days later did not engender a tumor response. To examine whether blocking ICs that are induced during the exposure of NK92CIRB21to PC-3 cancer cells could increase their antitumor effect in vivo, we injected anti-PDl, anti-CTLA-4, and anti-TIGIT antibodies (150, 75, and 150 pg / Kg ip.), two hours following NK cells infusions followed by another injection of antibodies, three days later. IC inhibitors injections alone did not exert any direct anti-tumor effect (FIG. 12, left panel) and were not toxic (FIG. 12, right panel). At day 28, NK92CIRB21cells alone caused a small tumor delay of four days compared to untreated tumors, w hile the combination of NK92CIRB21cells and antibodies led to 15 days tumor delay. This suggests that IC blockade is required for NK cells effectiveness despite their initial high cytoxicity and optimal activation. Mice's stable body weights suggest this combination of ICs was not toxic while being effective. The introduction of chimeric signal converter TIGIT- DNAM1 in NK92CIRB21enhanced its anti -tumor effect in vivo against PC-3 tumors (FIG. 13, left panel). This effect was further improved by the addition of anti-PDl Nivolumab (200 pg) alone, injected 2 hours post-NK cells injection and followed by another antibody injection three days later. TIGIT-DNAM1 expression did not induce significant toxicity in mice, as shown by body weight stability- (FIG. 13, right panel).

[0390] Example 4. TIGIT-DNAM1 increased the cytolytic activity of human primary NK cells

[0391] Human primary- NK cells were expanded using a feeder system expressing mbI121, CD86 and 41-BBL for two weeks and then frozen. Thawed primary NK cells were then grown with feeder cells for one w eek and then tw o w eeks with IL2 after which they were electroporated with the construct TIGIT-DNAM1 and grown in the presence of feeder cells for one week. The cytotoxicity of electroporated cells against PC-3 cancer (32.000) cells was compared to untouched (non-electroporated) primary NK cells at a ratio of 1 : 1 (effector to target) plated in a 24-well plate. Cytotoxicity assay of NK cells was stopped at day 2 and day 4 following plating of NK cells. The results are described in FIG. 14.

[0392] As shown in FIG. 14, TIGIT-DNAM1 significantly increased the cytolytic activity of human primary NK cells, as compared to untouched (non-transfected or non-electroporated) human primary NK cells.

[0393] Example 5. Effect of CD96-DNAM1, CTLA4-DNAM1, and LAG3-DNAM1 constructs on cytotoxicity of NK cells

[0394] Next, we tested the effect of some other fusion protein constructs - CD96- DNAM1, CTLA4-DNAM1, and LAG3-DNAM1 - on cytotoxicity- of NK cells. The results are described in FIG. 15.

[0395] As shown in FIG. 15, some constructs based on the intracellular domain of DNAM1 did not work as expected when used with extracellular domains from LAG3, CTLA-4, or CD96. This suggests that either the proteins are misfolded or that the transmission of signaling is not occurring.

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[0469] OTHER EMBODIMENTS

[0470] It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:1 . A fusion protein comprising an extracellular domain of an immune checkpoint (IC) receptor and a cytoplasmic domain of DNAX accessory molecule-1 (DNAM- 1).

2. The fusion protein of claim 1, wherein the extracellular domain of the IC receptor is fused to the cytoplasmic domain of DNAM-1.

3. The fusion protein of claim 1 or 2, wherein the cytoplasmic domain of DNAM-1 comprises the amino acid sequence of SEQ ID NO: 2.

4. The fusion protein of any one of claims 1-3, further comprising a transmembrane domain of DNAM1.

5. The fusion protein of claim 4, wherein the transmembrane domain of DNAM-1 comprises the amino acid sequence of SEQ ID NO: 3.

6. The fusion protein of claim 4 or 5, wherein the transmembrane domain of DNAM1 is positioned between the extracellular domain of the IC receptor and the cytoplasmic domain of DNAM-1.

7. The fusion protein of any one of claims 1-6, wherein the IC receptor is selected from the group consisting of T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor ty rosine-based inhibitory motif (ITIM) domains (TIGIT), programmed cell death protein 1 (PD1), cy totoxic T-lymphocyte associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), T-cell immunoglobulin and mucin domain 3 (TIM3), Poliovirus receptor related immunoglobulin domain containing (PVRIG), and Cluster of differentiation (CD)96.

8. The fusion protein of any one of claims 1 -7, wherein the IC receptor is TIGIT.

9. The fusion protein of claim 8, wherein the extracellular domain of TIGIT comprises the amino acid sequence of SEQ ID NO: 5.

10. The fusion protein of claim 8 or 9, wherein the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence ofSEQ ID NO: 6.1 1. The fusion protein of any one of claims 8-10, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 6.

12. The fusion protein of any one of claims 1-7, wherein the IC receptor is PD1.

13. The fusion protein of claim 12, wherein the extracellular domain of PD1 comprises the amino acid sequence of SEQ ID NO: 10.

14. The fusion protein of claim 12 or 13, wherein the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 11.

15. The fusion protein of any one of claims 12-14, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 11.

16. The fusion protein of any one of claims 1-15, further comprising a purification tag.

17. The fusion protein of claim 16, wherein the purification tag is a FLAG tag, cMyc tag. His-tag, or biotin tag.

18. The fusion protein of any one of claims 1-17, further comprising one or more linkers.

19. A nucleic acid molecule encoding the fusion protein of any one of claims 1-18.

20. The nucleic acid molecule of claim 19, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 45.

21. The nucleic acid molecule of claim 19, wherein the nucleic acid molecule comprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 46.

22. An expression vector comprising the nucleic acid molecule of any one of claims23. An isolated natural killer (NK) cell expressing the fusion protein of any one of claims 1-18.

24. The isolated NK cell of claim 23, wherein the NK cell further expresses one or more of CD 16, NKP44, NKP46, and NKP30.

25. An isolated T cell expressing the fusion protein of any one of claims 1-18.

26. The isolated T cell of claim 25, wherein the T cell further expresses one or more of CD4 and CD8.

27. A method for treating cancer in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a population of the isolated NK cell of claim 23 or 24, and / or a population of the isolated T cell of claim 25 or 26.

28. The method of claim 27, further comprising administering to the subject one or more IC inhibitors, anti-tumor monoclonal antibodies, chimeric antigen receptor (CAR)-T cells, and / or CAR-NK cells.

29. The method of claim 27 or 28, wherein the population of NK cells or T cells are administered intravenously.

30. A fusion protein comprising an extracellular domain of DNAX accessory molecule-1 (DNAM-1) and a cytoplasmic domain of an immune checkpoint (IC) receptor.

31. The fusion protein of claim 30, wherein the extracellular domain of DNAM-1 is fused to the cytoplasmic domain of the IC receptor.

32. The fusion protein of claim 30 or 31, wherein the extracellular domain of DNAM- 1 comprises the amino acid sequence of SEQ ID NO: 39.

33. The fusion protein of any one of claims 30-32, wherein the IC receptor is selected from the group consisting of T-cell immunoreceptor with immunoglobulin (Ig) and immunoreceptor tyrosine-based inhibitory motif (ITIM) domains (TIGIT), programmed cell death protein 1 (PD1), cytotoxic T-lymphocyte associated protein 4 (CTLA4), lymphocyte-activation gene 3 (LAG3), T-cellimmunoglobulin and mucin domain 3 (TIM3). Poliovirus receptor related immunoglobulin domain containing (PVRIG). and Cluster of differentiation (CD)96.

34. The fusion protein of any one of claims 30-33. wherein the IC receptor is TIGIT.

35. The fusion protein of claim 34, wherein the cytoplasmic domain of TIGIT comprises the amino acid sequence of SEQ ID NO: 40.

36. The fusion protein of claim 34 or 35, further comprising a transmembrane domain of TIGIT.

37. The fusion protein of claim 36, wherein the transmembrane domain of TIGIT comprises the amino acid sequence of SEQ ID NO: 41.

38. The fusion protein of claim 36 or 37, wherein the transmembrane domain of TIGIT is positioned between the extracellular domain of DNAM-1 and the cytoplasmic domain of TIGIT.

39. The fusion protein of any one of claims 34-38, wherein the fusion protein comprises an amino acid sequence having at least 80% sequence identity to the amino acid sequence of SEQ ID NO: 42.

40. The fusion protein of any one of claims 34-39, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO: 42.

41. The fusion protein of any one of claims 30-40, further comprising a purification tag.

42. The fusion protein of claim 41, wherein the purification tag is a FLAG tag. cMyc tag. His-tag, or biotin tag.

43. The fusion protein of any one of claims 30-42. further comprising one or more linkers.

44. A nucleic acid molecule encoding the fusion protein of any one of claims 30-43.

45. The nucleic acid molecule of claim 44, wherein the nucleic acid moleculecomprises a nucleic acid sequence having at least 80% sequence identity to the nucleic acid sequence of SEQ ID NO: 87.

46. An expression vector comprising the nucleic acid molecule of claim 44 or 45.

47. An isolated regulatory T (Treg) cell expressing the fusion protein of any one of claims 30-43.

48. The isolated Treg cell of claim 47, wherein the Treg cell further expresses one or more of CD4 and CD25.

49. A method for treating graft-versus-host disease (GVHD) or an autoimmune disease in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a population of the isolated Treg cell of claim 47 or 48.

50. The method of claim 49, wherein the population of Treg cells are administered intravenously.

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