Chimeric ilt receptor compositions and methods

Abstract

Provided are chimeric ILT receptors (CIRs) that include a targeting region from ILT2 or ILT4, a transmembrane domain, and an intracellular domain (ICD). The ICD includes a signaling region (e.g., CD3 zeta (CD3ζ), DAP10, DAP12) and optionally a costimulatory region (e.g., MyD88, a TIR domain, and the like). Also provided are nucleic acids (e.g., expression vectors) encoding a subject CIR, and genetically modified cells (e.g., immune cells such as NK cells, NK-T cells, T cells, iNKT cells, macrophages, and the like) expressing a subject CIR.

Description

Atty Docket No.: NKLT-002WO CHIMERIC ILT RECEPTOR COMPOSITIONS AND METHODS CROSS-REFERENCE

[0001] This application claims benefit of U.S. Provisional Patent Application No.63 / 516,288 filed July 28, 2023, and U.S. Provisional Patent Application No.63 / 607,881 filed December 8, 2023, which applications are incorporated herein by reference in their entirety. INCORPORATION BY REFERENCE OF SEQUENCE LISTING PROVIDED AS AN XML FILE

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “NKLT- 002WO_SEQ_LIST.xml” created on July 19, 2024 and having a size of 141,995 bytes. The contents of the Sequence Listing XML are incorporated by reference herein in their entirety. I. INTRODUCTION

[0003] Immune cell therapy is useful for specifically targeting diseased cells. This treatment can be potentially curative for both malignant and non-malignant conditions. For example, donor lymphocyte infusions, allogeneic T cells and allogeneic Natural Killer (NK) cells can be used to control the outgrowth of leukemias. Further, gene modification can direct the specificity of immune cells including T cells, Natural Killer (NK) cells, γd T cells, inducible NK-T cells and macrophages toward a target cell population for therapeutic purposes. For example, Chimeric antigen receptor- (CAR-) T cells can be used to redirect T cell specificity to tumor-associated cell surface molecules independent of Human Leukocyte Antigen (HLA) presentation of peptide antigens to a T cell receptor (TCR). CAR proteins can be engineered for expression other immune cells including NK cells, γ / d T cells, iNK-T cells and macrophages. In these cases, recognition of the target protein by a CAR focuses the cytotoxic potential of these cells that normally use so-called innate receptors to recognize foreign or diseased tissue. Numerous pre-clinical and clinical studies have demonstrated the utility of CAR technology expressed in T cells, NK cells, iNK-T cells and macrophages such that six therapies against B cell-derived tumors have received marketing approval.

[0004] Variation of CAR technology to alter the binding mechanism for target recognition can improve the affinity and specificity of an engineered cell for target proteins. For example, the antibody:antigen binding mechanism present on a CAR protein can be replaced with a receptor:ligand mechanism. An example of this is the engineering of the NKG-2D protein a modified or native protein for expression on immune cells with the purpose of recognizing the multiple ligands (MICA, MIC-B, ULBPs 1-5) for NKG-Atty Docket No.: NKLT-002WO 2D that may be expressed on diseased tissues in varying combinations. A further example described further in this patent is the expression of a Chimeric ILT Receptor or CIR that are engineered to target HLA-G expressed on tumors. CIR proteins are engineered with a binding moiety derived from ILT2 or ILT4 which are the principal receptors for the immunosuppressive HLA-G. The binding of the natural receptor domains for HLA-G (ILT2 or ILT4) improves the targeting for HLA-G which is expressed naturally as 7 protein isoforms derived from alternative mRNA splice products. An antibody / scFv CAR approach to targeting HLA would be limited by the loss of epitopes in one or more HLA-G isoforms.

[0005] T cells are the most commonly employed cell vehicle for targeted anti-cancer CAR therapy. They are generally employed as autologous, or patient-cell derived, products due to the risk of graft versus host disease with allogeneic T cell products. Cell types that lack TCR expression mitigate the risk of GvHD with autologous and can be produced with less expense and increased speed to patient. Increasingly, cell products for anti-cancer therapy are being developed from alternative cell types including NK cells, iNK-T cells and macrophage. NK and iNK-T CAR-based products frequently employ a CD28.CD3ζ or 4-1BB.CD3ζ signaling strategy to direct target- specific activation of the the cells frequently with the addition of mechanisms to promote IL-15 signaling. While these constructs do promote target-specific NK and iNK-T cell activation, the CAR mechanisms are designed to mimic T cell activation by APCs, particularly dendritic cells while NK cells and iNK-T cells are activated by mechanisms distinct from T cells. This raised the possibility that NK cell-based therapies with chimeric proteins may not be activated for optimal performance with traditional CAR-T cell construct designs. There is a need for improvement in NK cell activation particularly to promote the persistence of NK cell products to promote the eradication of a large tumor burden in patients and to maintain surveillance against tumor escape and relapse. II. SUMMARY

[0006] Variation of CAR technology to alter the binding mechanism for target recognition can improve the affinity and specificity of an engineered cell for target proteins. For example, the antibody:antigen binding mechanism present on a CAR protein can be replaced with a receptor:ligand mechanism. An example of this is the engineering of the NKG-2D protein a modified or native protein for expression on immune cells with the purpose of recognizing the multiple ligands (MICA, MIC-B, ULBPs 1-5) for NKG- 2D that may be expressed on diseased tissues in varying combinations. A further example described further herein is the expression of a Chimeric ILT Receptor (CIR) engineered to target HLA-G expressed on tumors. CIR proteins are engineered with aAtty Docket No.: NKLT-002WO binding moiety derived from ILT2 or ILT4 which are the principal receptors for the immunosuppressive HLA-G. The binding of the natural receptor domains for HLA-G (ILT2 or ILT4) improves the targeting for HLA-G which is expressed naturally as 7 protein isoforms derived from alternative mRNA splice products. An antibody / scFv CAR approach to targeting HLA would be limited by the loss of epitopes in one or more HLA-G isoforms.

[0007] CIR proteins are designed to replace the negatively signaling Immunoreceptor Tyrosine-based Inhibitory Motives (ITIM) of ILT2 or ILT4 with domains that activate immune cell signaling. Improvement in performance of non-T cell therapies may be achieved through alteration of the signaling domains present in the intracellular domain of a chimeric receptor such as a CIR and thereby the signal transduction pathways activated with target engagement. In some embodiments, the chimeric protein is a CIR containing binding elements derived from ILT2 or ILT4, each receptors for the HLA-G protein that is commonly expressed on tumors but not on normal tissues. In other embodiments, the chimeric protein is a CAR in which a single chain variable fragment (scFv) derived from an antibody gives specific targting for a tumor antigen.

[0008] Provided by the present disclosure is a genetically modified cell engineered to express a chimeric receptor protein that has affinity and specificity such that the modified cell can stimulate an immune response in a subject. For example, the chimeric receptor protein may target a protein expressed at high level in tumor tissue relative to untransformed, normal tissue and generate a cytotoxic or inflammatory response against the tumor. For the compositions and methods of the present disclosure, affinity and specificity are not maintained by use of antibody- or VhH-relationship or by binding of randomly generated peptides to a target antigen, but rather by use of ligand:receptor interactions where affinity and specificity are maintained by evolution. In some embodiments, the cell expressing a subject chimeric receptor protein is a NK cell. In some embodiments, the cell expressing the chimeric protein is an iNK-T cell. In some embodiments, the cell expressing the chimeric protein is an NK-T cell. In further embodiments, the cell expressing the chimeic protein are macrophages, γ / d T cells or α / β T cells. In each of these embodiments, the immune cell can be genetically-modified by introduction of a vector (e.g., multi-cistronic vector) by, e.g., transduction with a γ-retrovirus, lentivirus, adenovirus, adeno-associated virus, or DNA transfection of a transposon

[0009] For example, in some embodiments, the genetically modified cells express a chimeric receptor that has high affinity for HLA-G, a target protein that can exist on tumor tissues in one or more of seven known forms generated by alternative mRNA splicing and post-translational modifications. HLA-G naturally acts as an agent to suppressAtty Docket No.: NKLT-002WO immune responses through engagement with the negatively signaling Immunoglulin- like Transcript 2 (ILT2) and ILT4 receptors on the surface of immune cells. In these embodiments, immune cells such as T cells, NK cells, NK-T, or iNKT cells or macrophages are engineered such that recognition of active forms HLA-G by ILT2 or ILT4 instead generate activating signals. In some embodiments, the intracellular signalling elements of ILT2 or ILT4 are excised and replaced with ITAM-containing signalling domains of the CD3ζ chain (for DAP10 or DAP12) that drive immune cell activation and cytotoxicity. Such a protein is termed a ‘Chimeric ILT Receptor’ or ‘CIR’.

[0010] Thus, provided are chimeric ILT receptors (CIRs) [plus nucleic acids encoding them and genetically modified cells, such as immune cells, expressing them], which include a targeting region from ILT2 or ILT4, a transmembrane domain, and an intracellular domain (ICD), which includes a signaling region (e.g., CD3 zeta (CD3ζ), DAP10, DAP12) and optionally a costimulatory region (e.g., a TIR domain, a MyD88 protein, a domain that stimulates MyD88 signaling, CD28, 4-1BB, OX40, and the like). CD3ζ is naturally expressed in NK cells, γ / δ T cells and in iNK-T cells, but these cell types also express other ITAM-containing signaling adaptors specific for innate receptors that are not naturally expressed by most T cells. In some embodiments, the CD3ζ signaling is augmented by fusion with the signaling domain of DAP10 or DAP12 – each signaling adaptor proteins containing one ITAM that are naturally expressed by NK cells. The purpose of this addition is to enhance the cytotoxic potency of CIR or CAR-NK cells. In further embodiments, CD3ζ signaling is replaced by fusion with the signaling domain of DAP10 or DAP12. The purpose of this replacement is to optimize the persistence of NK cell anti-tumor potency.

[0011] The inventors appreciate that this approach provides an advantage over using an antibody-based targeting region (such as an scFv). An antibody-based targeting approach could cause a selection for tumor cells that express HLA-G isoforms that lack the targeted epitope – thus allowing a cancer to evade treatment. To the contrary, a subject ILT2 or ILT4 based chimeric ILT receptor should target many more, and perhaps all, HLA-G isoforms because ILT2 and ILT4 naturally bind those isoforms. This will greatly reduce, and perhaps eliminate, the ability of cancer cells to evade treatment by selection for a particular HLA-G isoform.

[0012] ILT2 and ILT4 are structurally similar in the extracellular region and are composed of four folded domains (D1, D2, D3 and D4) arranged in a distal-to-proximal fashion relative to the plasma membrane of the cell. HLA-G interacts with the D1 and D2 domains of ILT2 and ILT4 and these D1 and D2 domains can be separated from the rest of the ILT proteins while maintaining interaction with HLA-G. In some embodiments, ILT2 or ILT4 D3-D4 is replaced in the chimeric receptor fusion withAtty Docket No.: NKLT-002WO another extracellular domain that serves as a stalk and transmembrane domain to present ILT2 or ILT4 D1-D2 to HLA-G expressing target cells. Examples of stalk proteins to present D1-D2 are derived from CD28, CD8α, the CH2-CH3 region of IgG4, HER2 membrane proximal, and mGluR2. In other embodiments, the D3-D4 domains of ILT2 and ILT4 are simply deleted. As such, in some embodiments a subject chimeric ILT receptor includes D1-D2 of ILT2, but lacks D3-D4 of ILT2. In some embodiments a subject chimeric ILT receptor includes D1-D2 of ILT4, lacks D3- D4 of ILT4.

[0013] In CIR-NK constructs, signaling domains can be added to ITAM domain cytotoxicity domains to improve the survival, activation, persistence of cell activation, cytotoxicity and capacity to produce pro-inflammatory cytokines. Typically, these elements transduce signals that promote the PI3Kinase / AKT prosurvival pathway and the NF- κB pathway that promotes activation marker expression and cytokine release frequently in cooperation or synergy with the NF-AT pathway promoted by ITAM- domain signaling. In NK cells these signaling elements are termed coactivation signals. A canonical signaling element for many CAR constructs is 4-1BB. While 4- 1BB is expressed by activated NK, iNK-T and T cells, other members of the Tumor Necrosis Factor Receptor superfamily are also expressed and the signaling derived from alternate signaling domains are likely to give alternate phenotypes to cell products such as differential cytotoxicity, growth potential, cytokine production and survival.

[0014] In other embodiments, the signaling from 4-1BB to support coactivation can be replaced or supplemented by signaling from MyD88. This factor naturally acts as a signaling node downstream of innate signals from activated Toll-like Receptors (TLR) that are themselves activated with partial specificity by ligands derived from pathogens including RNA, DNA with altered methylation pattern and lipid or protein endotoxins. These innate signals are transduced strongly in responding cells by MyD88 activation. Further, MyD88 is a signaling node downstream of the IL-1 receptor superfamily of cytokines (including IL-18R and IL-33R) that are important activators of NK cells, iNK- T cells and T cells. Incorporation of the signaling Death Domain of MyD88 alone or in combination with TNF-R-derived signaling domains such as 4-1BB, HVEM or CD40 increased the target-specific potency of NK cells against AML. Signaling from MyD88 also increased the growth potential of NK cells intrinsically.

[0015] MyD88 signaling is naturally recruited to the plasma membrane by IL-1 family cytokine receptors and TLRs through Toll / Interleukin-1 Receptor / Resistance Protein (TIR) domains. Activation of signaling by both IL-18 receptor and TLRs is directed by conformational change in dimeric receptors leading to MyD88 oligomerization. In some embodiments, TIR domains can be fused as a coactivation domain with anAtty Docket No.: NKLT-002WO ITAM domain-containing cytotoxicity domain. The purpose of this fusion is to indirectly recruit native MyD88 to a CIR where it is inactive until activation and dimerization of the CIR by dimeric HLA-G. When expressed in NK cells, these fusions led to low tonic activity of the CIR in the absence of target cells, but high cytokine production in the presence HLA-G expressing target cells but not target cells lacking HLA-G expression.

[0016] Reagents, compositions, kits / systems, and methods related to chimeric ILT receptors are provided. For example, provided are methods of making genetically modified cells and methods of treatment (e.g., administering an immune cell, such as an NK cell, a T cell, or a macrophage, that expresses a subject CIR to an individual). III. BRIEFDESCRIPTIONOFTHEDRAWINGS

[0017] The following detailed description of embodiments of the invention will be better understood when read in conjunction with the appended drawings. It should be understood that the invention is not limited to the precise arrangements and instrumentalities of the embodiments shown in the drawings. The drawings illustrate certain embodiments of the technology and are not limiting. For clarity and ease of illustration, the drawings are not made to scale, and, in some instances, various aspects may be shown exaggerated or enlarged to facilitate an understanding of particular embodiments

[0018] FIG.1: Interaction of an ILT4 D1D2 CIR with HLA-G. (A) Cartoon depiction of the D1 and D2 domains of ILT4 fused as a chimeric hybrid with a stalk and transmembrane domain derived from a separate protein and further fused with an activating intracellular signaling moiety. Interaction of D1D2 with HLA-G initiates signaling to activate immune cells. Activation domains are contained in the intracellular domain (ICD) derived from signaling proteins that drive immune cell cytotoxicity, proliferation, persistence and release of cytokines. These domains can be one component driving cytotoxicity and activation or as chimeric domains that augment cytotoxicity and further drive cellular proliferation, persistence and production of pro-inflammatory cytokines.

[0019] FIG.2: Retroviral expression constructs of CIR forms with alternate intracellular signaling domains. Chart indicated retroviral constructs expressing ILT2 and ILT4 fusion proteins referred to as Chimeric ILT Receptors (CIRs). D1 through D2 indicate the encoding of extracellular domains D1 through D2 derived from native ILT4. STM refers to the stem (S), a linker domain linking extracellular D domains with the transmembrane spanning domain (TM) these are derived from CD8α (CD8a). Varying intracellular domain components driving activating signals are indicated.Atty Docket No.: NKLT-002WO

[0020] FIG.3: TNF-α production of CIR-NK cells containing alternative ITAM-containing cytotoxicity domains. (Upper) Tonic production of Tumor Necrosis Factor-α (TNF-α) by NK cells expressing only CD3ζ, DAP10, or DAP12 as ITAM-containing signaling domains. Cells were not cultured with target cells. (Lower) TNF-α production of CIR- NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0021] FIG.4: IFN-γ production of CIR-NK cells containing alternative ITAM-containing cytotoxicity domains. (Upper) Tonic production of Interferon-γ (IFN-γ) by NK cells expressing only CD3ζ, DAP10, or DAP12 as ITAM-containing signaling domains. Cells were not cultured with target cells. (Lower) IFN-γ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0022] FIG.5: Coculture of CIR-NK cells containing alternative ITAM-containing cytotoxity domains with KG1 target cells that do not express HLA-G. (Upper) KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ, DAP10, or DAP12 in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.

[0023] FIG.6: Coculture of CIR-NK cells containing alternative ITAM-containing cytotoxity domains with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ, DAP10, or DAP12 in their intracellular domains at an effector-to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0024] FIG.7: TNF-α production of CIR-NK cells containing multiple ITAM-containing cytotoxicity domains. (Upper) Tonic production of Tumor Necrosis Factor-α (TNF-α) by NK cells expressing only CD3ζ, DAP10 and DAP12 or DAP10 and DAP12 in combination with CD3ζ. Cells were not cultured with target cells. (Lower) TNF-α production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0025] FIG.8: IFN-γ production of CIR-NK cells containing multiple ITAM-containing cytotoxicity domains. (Upper) Tonic production of Interferon-γ (IFN-γ) by NK cells expressing only CD3ζ, DAP10 and DAP12 or DAP10 and DAP12 in combination with CD3ζ. Cells were not cultured with target cells. (Lower) IFN-γ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0026] FIG.9: Coculture of CIR-NK cells containing multiple ITAM-containing cytotoxicity domains with KG1 target cells that do not express HLA-G. (Upper) KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIRAtty Docket No.: NKLT-002WO (Mock) or CIRs expressing only CD3ζ, DAP10, or DAP12 or as a combination of DAP10 or DAP12 with CD3ζ in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.

[0027] FIG.10: Coculture of CIR-NK cells containing multiple ITAM-containing cytotoxicity domains with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ, DAP10, or DAP12 or as a combination of DAP10 or DAP12 with CD3ζ in their intracellular domains at an effector-to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0028] FIG.11: TNF-α production of CIR-NK cells containing a co-activation domain and an ITAM-containing cytotoxicity signaling domain. (Upper) Tonic production of Tumor Necrosis Factor-α (TNF-α) by NK cells expressing only CD3ζ or a chimera of CD3ζ with signaling elements from 4-1BB or HVEM. Cells were not cultured with target cells. (Lower) TNF-α production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0029] FIG.12: IFN-γ production of CIR-NK cells containing a co-activation domain and an ITAM-containing cytotoxicity signaling domain. (Upper) Tonic production of Interferon- γ (IFN-γ) by NK cells expressing only CD3ζ or a chimera of CD3ζ with signaling elements from 4-1BB or HVEM. Cells were not cultured with target cells. (Lower) IFN-γ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0030] FIG.13: Coculture of CIR-NK cells containing a co-activation domain and an ITAM- containing cytotoxicity signaling domain with KG1 target cells that do not express HLA-G. (Upper) KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or a chimera of CD3ζ with signaling elements from 4-1BB or HVEM in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.

[0031] FIG.14: Coculture of CIR-NK cells containing a co-activation domain and an ITAM- containing cytotoxicity signaling domain with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or aAtty Docket No.: NKLT-002WO chimera of CD3ζ with signaling elements from 4-1BB or HVEM in their intracellular domains at an effector-to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0032] FIG.15: TNF-α production of CIR-NK cells containing a co-activation domain and multiple ITAM-containing cytotoxicity signaling domains. (Upper) Tonic production of Tumor Necrosis Factor-α (TNF-α) by NK cells expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 in combination with signaling elements from 4-1BB. Cells were not cultured with target cells. (Lower) TNF-α production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0033] FIG.16: IFN-γ production of CIR-NK cells containing a co-activation domain and multiple ITAM-containing cytotoxicity signaling domains. (Upper) Tonic production of Interferon-γ (IFN-γ) by NK cells expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 in combination with signaling elements from 4-1BB. Cells were not cultured with target cells. (Lower) IFN-γ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0034] FIG.17: Coculture of CIR-NK cells containing a co-activation domain and multiple ITAM-containing cytotoxicity signaling domains with KG1 target cells that do not express HLA-G. (Upper) KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 in combination with signaling elements from 4-1BB in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.

[0035] FIG.18: Coculture of CIR-NK cells containing a co-activation domain and multiple ITAM-containing cytotoxicity signaling domains with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 in combination with signaling elements from 4-1BB in their intracellular domains at an effector-to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0036] FIG.19: TNF-α production of CIR-NK cells containing an ITAM-containing cytotoxicity domain and co-activation signaling elements derived from MyD88. (Upper) Tonic production of Tumor Necrosis Factor-α (TNF-α) by NK cells expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derivedAtty Docket No.: NKLT-002WO from the death domain of MyD88. Cells were not cultured with target cells. (Lower) TNF-α production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0037] FIG.20: IFN-γ production of CIR-NK cells containing an ITAM-containing cytotoxicity domain and co-activation signaling elements derived from MyD88. (Upper) Tonic production of Interferon-γ (IFN-γ) by NK cells expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88. Cells were not cultured with target cells. (Lower) IFN-γ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0038] FIG.21: Coculture of CIR-NK cells containing MyD88 as a co-activation domain and ITAM-containing cytotoxicity signaling domains with KG1 target cells that do not express HLA-G. (Upper) KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.

[0039] FIG.22: Coculture of CIR-NK cells containing MyD88 as a co-activation domain and ITAM-containing cytotoxicity signaling domains with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 in their intracellular domains at an effector- to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0040] FIG.23: TNF-α production of CIR-NK cells containing MyD88 and TNF-R derived co- activation domains and an ITAM-containing cytotoxicity signaling domain. (Upper) Tonic production of Tumor Necrosis Factor-α (TNF-α) by NK cells expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 with or without a further coactivation domain derived from 4-1BB or HVEM. Cells were not cultured with target cells. (Lower) TNF- α production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0041] FIG.24: IFN-γ production of CIR-NK cells containing MyD88 and TNF-R derived co- activation domains and an ITAM-containing cytotoxicity signaling domain. (Upper) Tonic production of Interferon-γ (IFN-γ) by NK cells expressing only CD3ζ or DAP12 orAtty Docket No.: NKLT-002WO a CD3ζ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 with or without a further coactivation domain derived from 4- 1BB or HVEM. Cells were not cultured with target cells. (Lower) IFN-γ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0042] FIG.25: Coculture of CIR-NK cells containing MyD88 and TNF-R derived co-activation domains and an ITAM-containing cytotoxicity signaling domain with KG1 target cells that do not express HLA-G. (Upper) KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derived from the death domain of MyD88 with or without a further coactivation domain derived from 4-1BB or HVEM in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.

[0043] FIG.26: Coculture of CIR-NK cells containing MyD88 and TNF-R derived co-activation domains and an ITAM-containing cytotoxicity signaling domain with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co- activation domain derived from the death domain of MyD88 in their intracellular domains at an effector-to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0044] FIG.27: TNF-α production of CIR-NK cells containing an ITAM-containing cytotoxicity signaling domain and a TIR containing co-activation domain. (Upper) Tonic production of Tumor Necrosis Factor-α (TNF-α) by NK cells expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derived from MyD88 or the TIR domain of the Interleukin 18 Receptor α chain (IL-18Rα), Toll-like Receptor (TLR) 2 or TLR3. Cells were not cultured with target cells. (Lower) TNF-α production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.

[0045] FIG.28: IFN-γ production of CIR-NK cells containing an ITAM-containing cytotoxicity signaling domain and a TIR containing co-activation domain. (Upper) Tonic production of Interferon-γ (IFN-γ) by NK cells expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derived from MyD88 or the TIR domain of the Interleukin 18 Receptor α chain (IL-18Rα), Toll-like Receptor (TLR) 2 or TLR3. Cells were not cultured with target cells. (Lower) IFN-γ production of CIR-NK cells 24 hours after coculture with Kasumi1 AML target cells.Atty Docket No.: NKLT-002WO

[0046] FIG.29: Coculture of CIR-NK cells containing an ITAM-containing cytotoxicity signaling domain and a TIR containing co-activation domain with KG1 target cells that do not express HLA-G. (Upper) KG1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co-activation domain derived from MyD88 or the TIR domain of the Interleukin 18 Receptor α chain (IL-18Rα), Toll- like Receptor (TLR) 2 or TLR3 in their intracellular domains at an effector-to-target ratio of 1:5. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with KG1 cells.

[0047] FIG.30: Coculture of CIR-NK cells containing an ITAM-containing cytotoxicity signaling domain and a TIR containing co-activation domain with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or CIRs expressing only CD3ζ or DAP12 or a CD3ζ chimera with DAP12 with or without a co- activation domain derived from MyD88 or the TIR domain of the Interleukin 18 Receptor α chain (IL-18Rα), Toll-like Receptor (TLR) 2 or TLR3 in their intracellular domains at an effector-to-target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0048] FIG.31: Retroviral expression constructs of CIR forms with alternate intracellular signaling domains. Chart indicated retroviral constructs expressing ILT4 (left) or ILT2 (right) D1 and D2 extracellular domains together with a CD8α stalk and transmembrane domain. Formats for the intracellular signaling domains are as indicated oriented from N-terminal (membrane proximal) to C-terminal (distal).

[0049] FIG.32: Viability of CIR-NK cells following transduction with CIR constructs with alternate signaling domains. Cell viability at 8 days post activation (5 days post- transduction) or at 14 days post-activation (11 days post-transduction) was assessed by exclusion of propidium iodide with costaining with Actinomycin D. Cells were transduced at greater than 80% and viability was high at each timepoint.

[0050] FIG.33: Expansion of CIR-NK cells in culture following transduction with CIR constructs with alternate signaling domains. Transduced CIR-NK cells were counted at day 5 post-activation and again at day 8 (upper) and day 14 to determine the relative proliferative ability of NK cells during the expansion period.

[0051] FIG.34: Expression level of transduced CIR proteins containing alternate signaling domains during expansion of NK cells. Expression of ILT4 CIR proteins was determined at Day 8 and Day 14 post-activation by flow cytometry with antibody toAtty Docket No.: NKLT-002WO ILT4 D1 / D2 and measured by the mean fluorescence intensity (MFI) (upper). The same cells were internally standardized by quantitation of a co-transduced Red Fluorescent Protein (lower).

[0052] FIG.35: IFN-γ production of CIR-NK cells containing alternate signaling domains. (Upper) Tonic production of Interferon-γ (IFN-γ) by 200,000 NK cells expressing only CD3ζ or a chimera of CD3ζ with DAP10 or DAP12 in combination with signaling elements from 4-1BB. Cells were not cultured with target cells. (Lower) Total IFN- Total IFN-production expressed on a per-cell basis.

[0053] FIG.36: IFN-γ production of CIR-NK cells containing alternate signaling domains when cocultured with target cells derived from an Acute Myeloid Leukemia (AML). (Upper) NK cells transduced with RFP alone (RFP) or CIR-NK cells were co-cultured with Kasumi1 target cells at an E:T ratio of 1:10 and IFN-γ levels were measured 24 hours later by ELISA (upper). (Lower) Total IFN-Total IFN-production expressed on a per-cell basis.

[0054] FIG.37: IFN-γ production of CIR-NK cells containing alternate signaling domains when cocultured with target cells derived from a solid tumor. (Upper) NK cells transduced with RFP alone (RFP) or CIR-NK cells were co-cultured with HT1376 bladder carcinoma target cells at an E:T ratio of 1:10 and IFN-γ levels were measured 24 hours later by ELISA (upper). (Lower) Total IFN-Total IFN-production expressed on a per-cell basis.

[0055] FIG.38: Short-term cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for two days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0056] FIG.39: 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells that express HLA-G1 and HLA-G5. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:40. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Molm13 cells.

[0057] FIG.40: Visual representation of 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with MOLM13 target cells that express HLA-G1 and HLA-G5. Molm13-GFP cells were cultured for 7 days with RFP-labelled NK cells expressing no CIR (RFP), or CIRs expressing the indicated signaling domains.Atty Docket No.: NKLT-002WO Images were taken in the Incucyte. Relative expansion of tumor is indicated by green fluorescence and NK cell expansion indicated by red fluorescence

[0058] FIG.41: Short-term cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Kasumi1 target cells that express HLA-G1 and HLA-G5. (Upper) Kasumi1 cells were cultured for two days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:20. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Kasumi1 cells.

[0059] FIG.42: 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Kasumi1 target cells that express HLA-G1 and HLA-G5. (Upper) Kasumi1 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:40. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with Kasumi1 cells.

[0060] FIG.43: Expression of HLA-G in cell lines derived from solid tumors. HLA-G expression was determined in dissociated HCT-116 colon carcinoma cells and in HT- 1376 bladder carcinoma cells by flow cytometry with the MEMG / 9 antibody. EGFR expression was determined as a separate control for integrity of the population.

[0061] FIG.44: 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with HT-1376-GFPffluc target cells that express HLA-G1, HLA-G2 and HLA-G5. (Upper) cells labelled with GFP were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with HT-1376 cells.

[0062] FIG.45: 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with HCT-116-GFPffluc target cells that do not express HLA-G. (Upper) HCT-116 cells labelled with GFP were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:10. Outgrowth of the tumor cells cultured alone is indicated for reference. (Lower) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with HCT-116 cells.

[0063] FIG.46: Expression of HLA-G in cell SU-8686 cells derived from a pancreatic ductal adenocarcinoma. HLA-G expression in was determined in dissociated SU8686 cells by flow cytometry with the MEMG / 9 antibody.Atty Docket No.: NKLT-002WO

[0064] FIG.47: 7-day cytotoxicity of CIR-NK cells containing alternate signalling domains cocultured with SU8686-GFPffluc target cells that express HLA-G1 and HLA-G2. (Upper) SU8686 cells labeled with GFP were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signalling domains at an effector to target ratio of 1:10.1G indicates a first-generation ILT2 CIR containing only a CD3ζ signalling domain. Outgrowth of the tumor cells cultured alone is indicated for reference. (Middle) Levels of NK cells and CIR-NK cells labelled with RFP was determined in coculture with SU8686 cells. (Bottom) Cocultures were harvested, and cells were quantitated by flow cytometry gating for CD56 expression (NK cells) and GFP expression (target cells).

[0065] FIG.48: Visual representation of 7-day cytotoxicity of CIR-NK cells containing alternate signalling domains cocultured with SU8686 target cells that express HLA-G1 and HLA-G2. SU8686-GFP cells were cultured for 7 days with RFP-labelled NK cells (E:T of 1:5) expressing no CIR (RFP), or CIRs expressing the indicated signalling domains. Images representative of one donor’s NK cells were taken in the Incucyte. Relative expansion of tumor is indicated by green fluorescence and NK cell expansion indicated by red fluorescence.

[0066] FIG.49: 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells that express HLA-G1 and HLA-G5 following long term expansion. (Upper) Molm13 cells were cultured for seven days with NK cells derived from two donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at an effector to target ratio of 1:20. NK cells in the upper panel were expanded for the standard 14 days prior to coculture. Outgrowth of the tumor cells cultured alone is indicated for reference. Cells in the lower panel were cultured for 26 days to examine the persistence of enhanced cytotoxicity associated with CIR signaling.

[0067] FIG.50: Persistence of cytotoxicity of CIR-NK cells containing alternate signaling domains repeatedly cocultured with Molm13 target cells over 9 days. Molm13 cells were cultured with NK cells transduced to express RFP alone (RFP) or the indicated CIR-NK cells at an E:T ratio of 1:10. Co-cultures were allowed to expand for 9 days (1X, top left) or were reseeded with an identical amount of Molm13 target cells at day 2 only (2X, Top right), at day 2 and day 5 (3X, bottom left), and days 2, 5 and 7 (4X, bottom right).

[0068] FIG.51: Persistence of cytotoxicity of CIR-NK cells containing alternate signaling domains repeatedly cocultured with Molm13 target cells over 9 days. Molm13 cells were cultured with NK cells transduced to express RFP alone (RFP) or the indicated CIR-NK cells (and RFP) at an E:T ratio of 1:10. Co-cultures were allowed to expand for 9 days (1X, top left) or were reseeded with an identical amount of Molm13 targetAtty Docket No.: NKLT-002WO cells at day 2 only (2X, Top right), at day 2 and day 5 (3X, bottom left), and days 2, 5 and 7 (4X, bottom right).

[0069] FIG.52: Expansion of CIR-NK cells containing alternate signaling domains repeatedly cocultured with Molm13 target cells over 9 days. Molm13 cells were cultured with NK cells transduced to express RFP alone (RFP) or the indicated CIR-NK cells (and RFP) at an E:T ratio of 1:10. Co-cultures were allowed to expand for 9 days (1X, top left) or were reseeded with an identical amount of Molm13 target cells at day 2 only (2X, Top right), at day 2 and day 5 (3X, bottom left), and days 2, 5 and 7 (4X, bottom right).

[0070] FIG.53: Stress test of initial cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells. Molm13-GFPffluc were cultured for two days with NK cells derived from three donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at increasing effector to target ratio from 1:40 to 2:1. Outgrowth of tumor was measured at 2 days by quantitation of GFP fluorescence in the Incucyte. Outgrowth of the tumor cells cultured alone is indicated for reference. CIR-NK cells with an ILT4-derived extracellular domain are indicated on the left and ILT2-derived CIR-NK cells are represented on the right.

[0071] FIG.54: Stress test of 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells. Molm13-GFPffluc were cultured for seven days with NK cells derived from three donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at increasing effector to target ratio from 1:40 to 2:1. Outgrowth of tumor was measured at 2 days by quantitation of GFP fluorescence in the Incucyte. Outgrowth of the tumor cells cultured alone is indicated for reference. CIR-NK cells with an ILT4-derived extracellular domain are indicated on the left and ILT2-derived CIR-NK cells are represented on the right.

[0072] FIG.55: Stress test of initial expansion of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells. Molm13-GFPffluc were cultured for two days with NK cells derived from three donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at increasing effector to target ratio from 1:40 to 2:1. Outgrowth of NK cells was measured at 2 days by quantitation of RFP fluorescence in the Incucyte. CIR-NK cells with an ILT4-derived extracellular domain are indicated on the left and ILT2-derived CIR-NK cells are represented on the right.

[0073] FIG.56: Stress test of 7-day expansion of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells. Molm13-GFPffluc were cultured for seven days with NK cells derived from three donors expressing no CIR (RFP) or CIRs expressing the indicated signaling domains at increasing effector to target ratio from 1:40 to 2:1. Outgrowth of NK cells was measured at 2 days by quantitation of RFP fluorescence in the Incucyte. CIR-NK cells with an ILT4-derived extracellular domain are indicated on the left and ILT2-derived CIR-NK cells are represented on the right.Atty Docket No.: NKLT-002WO

[0074] FIG.57: 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with OE19-GFPffluc esophageal carcinoma target cells that express low levels of HLA-G1 and HLA-G2. (Left) OE19 cells labelled with GFP were cultured for seven days with NK cells derived from two donors expressing no CIR (Mock) or ILT4 CIRs expressing the indicated signaling domains at an effector to target ratio of 1:5 (left graph) or 1:10 (right graph).1G indicates a first-generation ILT4 CIR containing only a CD3ζ signaling domain. Outgrowth of the tumor cells cultured alone is indicated for reference.

[0075] FIG.58: Timeline of co-culture of CIR-NK cells containing alternate signaling domains with target cell cells following constant exposure to solid tumor target cells for 19 days. NK cells or CIR-NK cells labelled with RFP and derived from 2 donors were cocultured with HT-1376 target cells at an E:T ratio of 2:1. After 5 days of coculture, NK cells were harvested, counted and reseeded with fresh tumor target cells. This process was repeated two times and NK cells were cocultured with solid or leukemic target cells at the indicated E:T ratios for 7 days.

[0076] FIG.59: Expansion of CIR-NK cells containing alternate signaling domains during constant exposure to HT-1376 target cells for 19 days. NK cells or CIR-NK cells labelled with RFP and derived from 3 donors were cocultured with HT-1376 target cells at an E:T ratio of 2:1. After 5 days of coculture, NK cells were harvested, counted and reseeded with fresh tumor target cells. This process was repeated two times. Cell counts relative to the previous seeding are plotted to indicate the relative expansion of NK cells with each exposure to tumor target.

[0077] FIG.60: Cytotoxicity and expansion of CIR-NK cells containing alternate signaling domains against Molm13 cells following constant exposure to HT-1376 target cells for 19 days. NK cells or CIR-NK cells labelled with RFP and derived from 3 donors were cocultured with HT-1376 target cells at an E:T ratio of 2:1. After 5 days of coculture, NK cells were harvested, counted and reseeded with fresh HT-1376 cells. This process was repeated two times. After 19 days and four successive exposures to HLA-G expressing target cells, NK cells were cocultured with Molm13-GFPffluc target cells for 7 days at an E:T ratio of 1:10 (left) or 1:20 (right). Tumor cell expansion (upper graphs) and NK cell expansion (lower graphs) was measured by GFP and RFP fluorescence in the Incucyte.

[0078] FIG.61: Visual representation of 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with Molm13 target cells following 19 days of constant exposure to HLA-G expressing target cells. Molm13-GFP cells were cultured for 7 days with RFP-labelled NK cells (E:T of 1:10) expressing no CIR (Mock), or CIRs expressing the indicated signaling domains. Images representative of one donor’s NK cells were taken in the Incucyte. Relative expansion of tumor is indicated by greenAtty Docket No.: NKLT-002WO fluorescence and NK cell expansion of cells derived from donors 142 and 226 indicated by red fluorescence.

[0079] FIG.62: Cytotoxicity and expansion of CIR-NK cells containing alternate signaling domains against OE19 cells following constant exposure to HT-1376 target cells for 19 days. NK cells or CIR-NK cells labelled with RFP and derived from 3 donors were cocultured with HT-1376 target cells at an E:T ratio of 2:1. Following 5 days of coculture, NK cells were harvested, counted and reseeded with fresh HT-1376 cells. This process was repeated two times. After 19 days and four successive exposures to HLA-G expressing target cells, NK cells were cocultured with OE19-GFPffluc target cells for 7 days at an E:T ratio of 1:5 (left) or 1:10 (right). Tumor cell expansion (upper graphs) and NK cell expansion (lower graphs) was measured by GFP and RFP fluorescence in the Incucyte.

[0080] FIG.63: Visual representation of 7-day cytotoxicity of CIR-NK cells containing alternate signaling domains cocultured with OE19 target cells following 19 days of constant exposure to HLA-G expressing target cells. OE19-GFP cells were cultured for 7 days with RFP-labelled NK cells (E:T of 1:5) expressing no CIR (Mock), or CIRs expressing the indicated signaling domains. Images representative of one donor’s NK cells were taken in the Incucyte. Relative expansion of tumor is indicated by green fluorescence and NK cell expansion of cells derived from donors 142 and 226 indicated by red fluorescence.

[0081] FIG.64: Interferon-γ production from CIR-NK cells containing alternate signaling domains during constant exposure to HT-1376 target cells for 19 days. NK cells or CIR-NK cells labelled with RFP and derived from 3 donors were cocultured with HT- 1376 target cells at an E:T ratio of 2:1. After 5 days of coculture, NK cells were harvested, counted and reseeded with fresh tumor target cells. This process was repeated three times. Interferon-γ (IFN-γ) levels were determined at 48 hours following each successive seeding with the total levels divided by the number of NK cells at each seeding.

[0082] FIG.65: Control of Molm13-GFPffluc expansion in NSG mice with CIR-NK cells expressing alternative signaling domains. Molm13-GFPffluc cells were implanted into immunodeficient NSG mice and expanded for 5 days. NK cells (7.5E6 cells / mouse) were co-transduced with retroviruses encoding ONLRluc (for bioluminescence (BLI) detection) and CIR vectors (or Mock transduced) and were injected intravenously into NSG mice bearing Molm13 tumors. (Left) BLI for NK cells was measured 14 days post NK cell injection with coelenterazine as substrate. (Right) Total body BLI for Molm13- GFPffluc was measured 10 days post NK cell injection with luciferin as the substrate.

[0083] FIG.66: Control of Molm13-GFPffluc expansion in the bone marrow of NSG mice with CIR-NK cells expressing alternative signaling domains. Bone marrow was harvestedAtty Docket No.: NKLT-002WO from select groups of mice outlined in FIG.65 at 17 days post NK cell transplantation. Human CD45+cells were gated from total bone marrow cells and GFP+Molm13 tumor cells quantitated by flow cytometry.

[0084] FIG.67: Trafficking of CIR-NK cells to bone marrow of NSG mice and control of Molm13-GFPffluc expansion with CIR-NK cells expressing alternative signaling domains. Bone marrow was harvested from select mice at 17 days post NK cell transplantation. Human CD45+cells were gated from total bone marrow cells and GFP+Molm13 tumor cells and human NK cells (orangenanolantern (ONL)) quantitated by flow cytometry. Representative flow plots of bone marrow derived from group 2 (mock transduced NK cells with no CIR), group 3 (first generation signaling domain, ILT4 CIR-CD3ζ), group 7 (ILT4 CIR-BB.DAP10.TLR2) and group 11 (ILT2 CIR- BB.DAP10.TLR2) are displayed. IV. DETAILED DESCRIPTION

[0085] Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0086] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0087] Certain ranges are presented herein with numerical values being preceded by the term "about." The term "about" is used herein to provide literal support for the exact number that it precedes, as well as a number that is near to or approximately the number that the term precedes. In determining whether a number is near to or approximately a specifically recited number, the near or approximating unrecited number may be a number which, in the context in which it is presented, provides the substantial equivalent of the specifically recited number.

[0088] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to thoseAtty Docket No.: NKLT-002WO described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.

[0089] All publications and patents cited in this specification are herein incorporated by reference as if each individual publication or patent were specifically and individually indicated to be incorporated by reference and are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. The citation of any publication is for its disclosure prior to the filing date and should not be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.

[0090] It is noted that, as used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. As such, the articles “a” and “an” are used herein to refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Thus, for example, reference to “a cell” includes a plurality of such cells and reference to “the polypeptide” includes reference to one or more polypeptides and equivalents thereof known to those skilled in the art, and so forth. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.

[0091] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible. For example, it is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments pertaining to the invention are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub- combinations of the various embodiments and elements thereof are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.Atty Docket No.: NKLT-002WO

[0092] While the apparatus and method has or will be described for the sake of grammatical fluidity with functional explanations, it is to be expressly understood that the claims, unless expressly formulated under 35 U.S.C. §112, are not to be construed as necessarily limited in any way by the construction of "means" or "steps" limitations, but are to be accorded the full scope of the meaning and equivalents of the definition provided by the claims under the judicial doctrine of equivalents, and in the case where the claims are expressly formulated under 35 U.S.C. §112 are to be accorded full statutory equivalents under 35 U.S.C. §112.

[0093] The entirety of each patent, patent application, publication and document referenced herein hereby is incorporated by reference. Citation of patents, patent applications, publications and documents are not an admission that any of the foregoing is pertinent prior art, nor does it constitute any admission as to the contents or date of these publications or documents. Their citation is not an indication of a search for relevant disclosures. All statements regarding the date(s) or contents of the documents is based on available information and is not an admission as to their accuracy or correctness.

[0094] Modifications may be made to the foregoing without departing from the basic aspects of the technology. Although the technology has been described in substantial detail with reference to one or more specific embodiments, those of ordinary skill in the art will recognize that changes may be made to the embodiments specifically disclosed in this application, yet these modifications and improvements are within the scope and spirit of the technology.

[0095] The technology illustratively described herein suitably may be practiced in the absence of any element(s) not specifically disclosed herein. Thus, for example, in each instance herein any of the terms “comprising,” “consisting essentially of,” and “consisting of” may be replaced with either of the other two terms. The terms and expressions which have been employed are used as terms of description and not of limitation, and use of such terms and expressions do not exclude any equivalents of the features shown and described or portions thereof, and various modifications are possible within the scope of the technology claimed. The term “a” or “an” can refer to one of or a plurality of the elements it modifies (e.g., “a reagent” can mean one or more reagents) unless it is contextually clear either one of the elements or more than one of the elements is described. The term “about” as used herein refers to a value within 10% of the underlying parameter (i.e., plus or minus 10%), and use of the term “about” at the beginning of a string of values modifies each of the values (i.e., “about 1, 2 and 3” refers to about 1, about 2 and about 3). For example, a weight of “about 100 grams” can include weights between 90 grams and 110 grams. Further, when a listing of values is described herein (e.g., about 50%, 60%, 70%, 80%, 85% or 86%)Atty Docket No.: NKLT-002WO the listing includes all intermediate and fractional values thereof (e.g., 54%, 85.4%). Thus, it should be understood that although the present technology has been specifically disclosed by representative embodiments and optional features, modification and variation of the concepts herein disclosed may be resorted to by those skilled in the art, and such modifications and variations are considered within the scope of this technology.

[0096] Provided in this disclosure are chimeric ILT receptors (CIRs) that include a targeting region from ILT2 or ILT4, a transmembrane domain, and an intracellular domain (ICD) having a signaling region capable of transducing a signal, upon binding of said targeting region to HLA-G, into the interior of an immune effector cell (e.g., NK cell) to elicit effector cell function.

[0097] In some cases, the signaling region includes a costimulatory region with a MyD88 polypeptide. In some such cases, the signaling region includes a CD3ζ signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, or any combination thereof. For example, in some cases, the signaling region includes a CD3ζ signaling domain. In some cases, the signaling region includes a DAP10 signaling domain. In some cases, the signaling region includes a DAP12 signaling domain. In some cases, the costimulatory region also includes a 4-1BB costimulatory domain.

[0098] In some cases, the signaling region includes a DAP10 signaling domain or a DAP12 signaling domain. In some such cases, the signaling region also includes a CD3ζ signaling domain. In other such cases, the signaling region does not include a CD3ζ signaling domain. In some cases, the signaling region further includes a CD40, 4-1BB, or HVEM costimulatory domain.

[0099] Also provided are intracellular domain (ICD) polypeptides having a signaling region capable of transducing a signal in an immune effector cell to elicit effector cell function, wherein the signaling region comprises (i) a CD3ζ signaling domain, a DAP10 signaling domain, or a DAP12 signaling domain, and (ii) a costimulatory region that comprises a Toll / Interleukin-1 Receptor / Resistance Protein (TIR) domain. In some cases, the TIR domain is a TLR2 TIR domain, TLR3 TIR domain, or a IL18R1 TIR domain. In some cases, the signaling region includes the CD3ζ signaling domain. Also provided are CIRs that include such ICD polypeptides. 1. HLA-G

[0100] The non-classical MHC-I protein HLA-G is a major factor in the maintenance of immunological tolerance to maternal-fetal development [Kovats et al. Science (1990) 248:220, Ferreira et al, (2017) I 38:272]. Its normal expression is highest in theAtty Docket No.: NKLT-002WO extravillous trophoblasts of the fetal placenta where it functions to block activation and infiltration of maternal immune cells of most types, but particularly T cells and NK cells from the fetus which has a haploidentical MHC haplotype. It is maintained at much lower levels in other immunoprivileged tissues including the cornea, a subset of mesenchymal stem cells [Chapel et al. (2006) Blood 108:4257, Selmani et al (2008) Stem Cells 26:212] and endocrine pancreas [Le Discorede et al. (2003) Human Immunology 64:1039, Cirulli et al. (2006) Diabetes 55:1214]. HLA-G is expressed in a diverse set of solid tumor types and leukemias [Reviewed in Lin and Yan (2018) Front Imm.9:Art 2164] including melanoma [Paul et al, (1998) Proc Natl Acad Sci 95:4510], colorectal cancer, AML, ALL, renal cell carcinoma [Tronik-Le Roux et al,(2017) Mol. Oncol.11:1561], breast cancer and lung cancer. Its function in cancer is to directly evade immune attack, but HLA-G is also expressed in tolerogenic DC-10 dendritic cells that inhibit lymphocytic responses by suppressive cytokine secretion and activate Treg cells and myeloid derived suppressor cells (MDSC) to create an immunosuppressive tumor microenvironment [Reviewed in Carosella et al, Blood (2011) 118:6499, Gao et al, (2018) BBA 1869:278]. HLA-G can thereby be considered an important checkpoint mediator of tumor promotion.

[0101] The HLA-G gene produces multiple mRNA transcripts that encode at least seven different protein products [Ishitani et al (1992) Proc. Natl. Acad. Sci 89:3947, (HLA-G1 through G5 are SEQ ID NOs: 9, 15, 17, 19, 21 (protein) and encoding nucleotides are SEQ ID NOs: 8, 14, 16, 18, 20, respectively). HLA-G1 contains the α1-α2-α3 domain structure with an alpha helical peptide binding cleft and a transmembrane domain and short intracellular carboxy terminal domain (see Figure 1). This domain structure is canconical to MHC-I products. Other expressed splice products delete entire domains, for example HLA-G2 encodes α1, α3 and the transmembrane domain, deleting α2. HLA-G4 deletes the α3 domain and HLA-G3 encodes only the α1 domain. When expressed in M8 cells as transgenes, each of these forms of HLA-G was reported to exhibit immunosuppressive activity toward NK cell attack [Riteau et al (2001) J. Immunology 166:5018]. Secreted forms generated by alternative splicing include HLA-G5 and HLA-G6 which maintains the domain structure of HLA-G1 and G2 respectively but do not use the splice donor site for intron 4 and instead encode a short secreted peptide derived from intron 4. Similarly HLA-G7 uses a three amino acid peptide derived from intron 2. Further secreted forms of HLA-G1 are generated by cleavage at the transmembrane domain by matrix metalloproteinases to shed the cell surface of some HLA-G1 [Rizzo et al (2012) Mol Cell Biochem 381:243].

[0102] HLA-G exists in monomeric and oligomeric forms. Oligomers are chiefly dimers directed by disulphide linkages at Cys42 (in α1) or Cys 147 (in α2) [Gonen-Gross et al, (2005) J. Imm.175:4866, Boyson et al, Proc. Natl. Acad. Sci 99:16180]. EvidenceAtty Docket No.: NKLT-002WO exists that the dimeric form of HLA-G is the principal immunosuppressive form and that it adopts a kinked quaternary structure relative to that of native monomers [Shiroishi et al (2006) Proc Natl Acad Sci 103:10095, Clements et al (2005) Proc Natl Acad Sci 102:3360, Wang et al (2020) Cel and Mol. Imm.17:966].

[0103] The different HLA-G forms together create a challenge for CAR-based therapy that relies on binding of an antibody-derived scFv or VhH domain as the targeting agent. Because different splice forms delete epitopes for given antibodies, a selection is placed by CAR therapy for expression only of epitopes that are not recognized by the CAR’s binder while retaining immunosuppressive activity. Further, oligomerization can mask epitopes for an scFv due to structural change. As well, two of the commonly used antibody reagents for HLA-G 4H84 and 87G display crossreactivity to other HLA species that could lead to off-target, off-tumor targeting of CAR-T or CAR- NK cells [Attia et al (2021) Int. J. Mol. Sci 21:8678, Polakova et al (2004) Hum. Imm. 65:157, Swets et al (2018) Clin. Imm 194:80, Furukawa et al (2019) Int J. Mol. Sci 20:5947] 2. ILT2 and ILT4

[0104] HLA-G directs its immunosuppressive activity as a membrane bound ligand for inhibitory receptors Immunoglobulin-like transcript 2 (ILT2) and ILT4 (also called LIRB1 and LIRB2 or CD85j and CD85d respectively) on target immune cells [Colonna et al (1998) J. Immunology 160:3096 reviewed in Gao et al (2018) BBA 1869:278]. ILT2 (see Seq ID NO: 29)(an encoding nucleotide sequence is SEQ ID NO: 28) is expressed in a subset of Natural Killer cells, iNKT cells, T cells, B cells and dendritic cells. ILT4 (see Seq ID NO: 53) (an encoding nucleotide sequence is SEQ ID NO: 52) has a more broad expression pattern primarily in myeloid and stem cells including macrophage, myeloid derived suppressor cells (a population of less differentiated cells on the monocytic lineage), granulocytes including neutrophils, monocytes, hematopoietic stem cells and some neurons.

[0105] ILT2 has an extracellular domain structure consisting of four domains that have sequence and structural homology to Immunoglobulin domains (Ig domains) arranged in a column from membrane-distal D1 through to most membrane-proximal D4 followed by a transmembrane domain and an intracellular signaling domain that includes four iterated Immunoreceptor Tyrosine-based Inhibitory Motives (ITIMs). ILT4 has a similar extracellular and transmembrane architecture but only three ITIMs in its intracellular domain.

[0106] The D1 and D2 domains of ILT2 (see, e.g., Seq ID NO: 37 and 71) (SEQ ID NO: 36 provides a nucleotide sequence encoding SEQ ID NO: 37) and ILT4 (see, e.g., SEQ ID NO: 57 and 75)(SEQ ID NO: 56 provides a nucleotide sequence encoding SEQ IDAtty Docket No.: NKLT-002WO NO: 57) govern interaction with HLA-G and can be separated from the D3 and D4 domains [Donadi et al (2011) Cell. Mol. Life Sci.68:369, Morales (2007) 122:179, HoWanYin et al (2012) Cell. Mol. Life Sci.69:4041, Shiroishi et al (2006) Proc Natl Acad Sci 103:10095, Wang et al (2020) Cel and Mol. Imm.17:966]. Unlike activating interactions made by the CD3 complex with classical MHC-I and inhibitory and activating interactions made by KIR proteins of NK cells ILT2 and ILT4 do not bind with the α1-α2 domains that contain the peptide binding cleft, but instead interact with the membrane proximal α3 domain and with β2-microglobulin. ILT2 makes extensive contact with β2-M and relatively few contacts with α3 of HLA-G and requires β2-M association with HLA-G to maintain even a low affinity interaction. Conversely, ILT4 makes extensive contact with α3 and can maintain interaction with all known actively immunosuppressive forms of HLA-G, possibly excluding HLA-G3 / G7 that contains only the α1 domain.

[0107] ILT2 and ILT4 can interact with other MHC-I and MHCI-like proteins, notably HLA-A2, HLA-B, HLA-C, and HLA-F, CD1d and UL18. With the exception of UL18, a decoy MHC-I from cytomegalovirus [Wilcox et al (2002) BMC Struct. Biol 2:6], these are low affinity interactions with dissociation constants (KD) between 2 µM and 40 µM. Relevance for immunosuppressive signalling has not been demonstrated with affinities this weak. Similarly, interaction between ILT2 and ILT4 with monomeric forms of HLA-G are weak, in the µM range. However, dimeric HLA-G forms display high affinity (2-4 nM) interaction with ILT2 and ILT4 possibly due to display of further contact sites or, alternatively, due to an avidity effect reducing the off rate for ILT dissociation [Shiroishi et al, (2006) J. Biol. Chem 281: 10440, Gao et al (2020) Cell Mol Imm 17:966]. The dimeric forms of HLA-G are therefore most likely to be bioactive [Gonen-Gross et al, (2005) J. Imm.175:4866] and functional for immunosuppression in a tumor setting and further, are most likely to be relevant as a targeted molecule for cell-based immunotherapy by chimeric receptors.

[0108] ILT4 is a receptor for non-MHC ligands including Angiopoietin-like proteins 2 and 5 [Zheng et al (2012) Nature 485:656, Deng et al (2014) Blood 124:924] Regulation by soluble ANGPTL2 and ANGPTL5 is thought to provide a protective signal from bone marrow stroma for self-renewal and survival of ILT4-expressing hematopoietic stem cells. Interaction between ILT4 and ANGLPs is directed by the D1 domain in concert with the D4 domain of ILT4 and specific residues in either the D1 or D4 are essential to maintain high affinity interaction. Notably, mutation of tyrosine 96 to alanine reduced ANGPTL2 / 5 binding but did not reduce HLA-G1 interaction with full-length ILT4 [Deng et al (2014) Blood 124:924]. ILT4 interacts with moderate affinity to inhibitory Nogo receptor ligands derived from myelin [Atwal et al (2008) Science 322:967, Matsushita et al, (2014) J. Biol. ChemAtty Docket No.: NKLT-002WO 286:25739]. The mouse ortholog of ILT proteins, PIRB, is also found in subsets of neurons and may regulate axonal outgrowth by interaction with myelin-based MAG, Nogo and OMgp [US patent 20100047232] and Sema4a [Lu et al. (2018) Nat. Comm. 7:742]. High affinity interactions were characterized in the mouse ortholog for ILT4, PIRB and did not map to the HLA-G binding D1 and D2 domains, but rather to the membrane proximal domains of PIRB [Matsushita et al, (2014) J. Biol. Chem 286:25739]. 3. Chimeric receptors targeted by ILT proteins (i.e., a “Chimeirc ILT Receptor” or “CIR”)

[0109] Redirection of the cytotoxic specificity of T or NK cells can be controlled by engagement of an antigen-scFv (or TCR) interaction, but can also be controlled by a receptor-ligand pairing such that the receptor for the targeted ligand can be formed into a chimeric protein that can maintain a high affinity interaction with the cell while capably maintaining signal transduction to activate the immune cell (we use T cells and NK cells as an example hereafter). The receptor or portions of the receptor used to engage the target should be specific for the target, thereby preventing off-tumor targeting. Similar to scFv and other binders, high level expression of the target protein or ligand should be present on target tissue (for example, a tumor) relative to normal tissues. Cells that express the CIR on their surface, upon contact and ligation with HLA-G, signal through the signaling domain (e.g., CD3 zeta (CD3ζ) chain), inducing cellular activation. (i) Targeting region

[0110] The extracellular domains (D1-D4) of ILT2 (see, e.g., SEQ ID NO: 31, which includes D1-D4 plus the transmembrane region of ILT2; an encoding nucleotide sequence is SEQ ID NO: 30) can be engineered to target HLA-G expressing tumor cells and generate activating signal transduction in immune cells expressing a chimeric version of ILT2 that replaces the naturally inhibitory ITIM-containing ILT2 intracellular domain (ICD) with signaling components that drive activating signals.

[0111] Similarly, the extracellular domains (D1-D4) of ILT4 (see, e.g., SEQ ID NO: 55, which includes D1-D4 plus the transmembrane region of ILT4; nucleotides encoding are SEQ ID NO: 54) can be engineered to generate activating signals in immune cells by replacement of the ILT4 ICD with activation signaling moieties. Using the D1-D4 extracellular domain of ILT2 or ILT4 for the targeting region would create an ILT2 D1- D4 chimeric receptor (i.e., ILT2 D1-D4 CIR) or an ILT4 D1-D4 chimeric receptor (i.e., ILT4 D1-D4 CIR) (see Figure 4A). Thus, in some cases, the targeting region of aAtty Docket No.: NKLT-002WO subject chimeric ILT receptor (CIR) includes an ILT2 or ILT4 D1-D4 domain (which therefore targets HLA-G).

[0112] ILT4 maintains more contacts with α3 on the HLA heavy chain and can interact with free heavy chain forms of HLA-G while ILT2 D1 / D2 requires contact with β2-M and α3 to maintain interaction with HLA-G.

[0113] Because ILT2 and ILT4 maintain high affinity (KD low nM) interactions with dimeric HLA-G and low affinity (KDis µM for interaction with other MHC-I proteins including monomeric HLA-G and CD1d), specificity for tumors carrying high levels of HLA-G increases proportionately the amount of HLA-G in dimeric form and permits selection of tumor tissue over normal tissue expressing high levels of classical MHC-I, but little or no HLA-G. Mutation of HLA-G1 at positions Cys42 to Ser or Cys147 to Ser blocks HLA-G1 dimerization and severely reduces targeting by ILT2 or ILT4 CIR-T cells or CIR-NK cells.

[0114] It is appreciated by the inventors that HLA-G exists in several different isoforms. CARs which include an antibody-based targeting region (such as an scFv), would only be able to target HLA-G isoforms that include the epitope targeted by the antigen binding region (e.g., scFv). This could place a selection for tumor cells expressing HLA-G isoforms that lack the targeted epitope – thus allowing the cancer to evade treatment. To the contrary, a subject ILT2 or ILT4 based chimeric receptor protein (which targets HLA-G) should target many more, and perhaps all, HLA-G isoforms because ILT2 and ILT4 naturally bind those isoforms. Construction of an ILT2 D1 / D2 CIR and an ILT4 D1 / D2 CIR

[0115] The D1 and D2 domains are sufficient to direct binding of ILT2 and ILT4 to HLA-G while the D3 and D4 domains are likely to serve as a scaffold to display D1 and D2 to HLA-G [Shiroishi et al, (2006) J. Biol. Chem Apr 14; 281(15):10439-47]. In some embodiments, the D3 and D4 domains can be deleted from an ILT2 CIR or and ILT4 CIR and maintain functional interactions through the D1-D2 domains from ILT2 (see, e.g., Seq ID NO: 71) or ILT4 (see, e.g., Seq ID NO: 75) with HLA-G forms (see Figure 4A). Thus, in some cases, the targeting region of a subject ILT2 or ILT4 chimeric receptor will include the D1-D2 domains of ILT2 or ILT4 (see, e.g., SEQ ID NO: 71 for D1-D2 of ILT2 and SEQ ID NO: 75 for D1-D2 of ILT4), and in some such cases the targeting region will not include (i.e., will lack) the D3-D4 domains.

[0116] In some embodiments, the targeting region (the region including the D1-D2 domains) of a subject ILT2 chimeric receptor includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the ILT2 sequence set forth in any one of SEQ ID Nos: 37, 70, 71, and 72, which sequences are as follows:Atty Docket No.: NKLT-002WO MHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKG QFPIPSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGN VTLQCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRC YAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 37) MHLPKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKG QFPIPSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGN VILQCDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRC YAYDSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 70) PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTAPWITRIPQELVKKGQFPI PSITWEHTGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVTL QCDSQVAFDGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAY DSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 71) PKPTLWAEPGSVITQGSPVTLRCQGGQETQEYRLYREKKTALWITRIPQELVKKGQFPI PSITWEHAGRYRCYYGSDTAGRSESSDPLELVVTGAYIKPTLSAQPSPVVNSGGNVILQ CDSQVAFDGFSLCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPSRRWWYRCYAY DSNSPYEWSLPSDLLELLVLG (SEQ ID NO: 72)

[0117] In some cases, the targeting region includes an amino acid sequence that has 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth in any one of SEQ ID Nos: 37, 70, 71, and 72. In some cases, the targeting region includes an amino acid sequence that has 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth in any one of SEQ ID Nos: 37, 70, 71, and 72. In some cases, the targeting region includes the amino acid sequence set forth in any one of SEQ ID Nos: 37, 70, 71, and 72.

[0118] In some cases, the targeting region (the region including the D1-D2 domains) of an ILT2 chimeric receptor includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 37. In some cases, the targeting region includes an amino acid sequence that has 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 37. In some cases, the targeting region includes an amino acid sequence that has 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 37. In some cases, the targeting region includes the amino acid sequence set forth as SEQ ID No: 37.

[0119] In some embodiments, the targeting region (the region including the D1-D2 domains) of a subject ILT4 chimeric receptor includes an amino acid sequence that has 80% orAtty Docket No.: NKLT-002WO more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the ILT4 sequence set forth in any one of SEQ ID Nos: 57, 74, and 75, which sequences are as follows: MTPIVTVLICLGLSLGPRTHVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRL YREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTG AYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSS RAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPG (SEQ ID NO: 57) MTPIVTVLICLGLSLGPRTRVQTGTIPKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRL YREKKSASWITRIRPELVKNGQFHIPSITWEHTGRYGCQYYSRARWSELSDPLVLVMTG AYPKPTLSAQPSPVVTSGGRVTLQCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSS RAIFSVGPVSPNRRWSHRCYGYDLNSPYVWSSPSDLLELLVPG (SEQ ID NO: 74) PKPTLWAEPDSVITQGSPVTLSCQGSLEAQEYRLYREKKSASWITRIRPELVKNGQFHI PSITWEHTGRYGCQYYSRARWSELSDPLVLVMTGAYPKPTLSAQPSPVVTSGGRVTL QCESQVAFGGFILCKEGEDEHPQCLNSQPHARGSSRAIFSVGPVSPNRRWSHRCYGY DLNSPYVWSSPSDLLELLVPG (SEQ ID NO: 75)

[0120] In some cases, the targeting region includes an amino acid sequence that has 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth in any one of SEQ ID Nos: 57, 74, and 75. In some cases, the targeting region includes an amino acid sequence that has 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth in any one of SEQ ID Nos: 57, 74, and 75. In some cases, the targeting region includes the amino acid sequence set forth in any one of SEQ ID Nos: 57, 74, and 75.

[0121] In some cases, the targeting region (the region including the D1-D2 domains) of an ILT4 chimeric receptor includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 57. In some cases, the targeting region includes an amino acid sequence that has 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 57. In some cases, the targeting region includes an amino acid sequence that has 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the sequence set forth as SEQ ID No: 57. In some cases, the targeting region includes the amino acid sequence set forth as SEQ ID No: 57.

[0122] For any of the above embodiments discussed in this section, in some cases the subject ILT2 or ILT4 chimeric receptor lacks a D3 and D4 domain (i.e., lacks a regionAtty Docket No.: NKLT-002WO corresponding to the D3-D4 domains of ILT2 (SEQ ID NO: 73) or ILT4 (SEQ ID NO: 76), respectively). For ILT2, the region with the D3-D4 domains is: PLDILIAGQFYDRVSLSVQPGPTVASGENVTLLCQSQGWMQTFLLTKEGAADDPWRLR STYQSQKYQAEFPMGPVTSAHAGTYRCYGSQSSKPYLLT (SEQ ID NO: 73). For ILT4, the region with the D3-D4 domains is: QPGPVMAPGESLTLQCVSDVGYDRFVLYKEGERDLRQLPGRQPQAGLSQANFTLGPV SRSYGGQYRCYGAHNLSSECSAPSDPLDILITGQIRGTPFISVQPGPTVASGENVTLLC QSWRQFHTFLLTKAGAADAPLRLRSIHEYPKYQAEFPMSPVTSAHAGTYRCYGSLNSD PYLLSHPSEPLEL (SEQ ID NO: 76).

[0123] In some cases, a subject CIR lacks an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with the sequence set forth as SEQ ID NO: 73. In some cases, a subject CIR lacks an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with the sequence set forth as SEQ ID NO: 76. In some cases, a subject CIR lacks an amino acid sequence having 85% or more (e.g., 90% or more, 95% or more, 98% or more, 99% or more, or 100%) sequence identity with the sequence set forth in any one of SEQ ID NOs: 73 and 76.

[0124] In some embodiments a linker may be fused as a chimera with D1-D2 domains from ILT2 or ILT4 to the plasma membrane and serves as a stalk that replaces D3 and D4 domains. In these embodiments, deletion of D3-D4 may prevent interaction of a CIR with proteins other than HLA-G that interact with native ILT2 or ILT4 through the D3 or D4 domains, for examples the interactions of ANGPTL2 and ANGPTL5 with ILT4 D4 and the interaction of nogo, Omgp and MAG with ILT4 D3-D4. Prevention of such interactions may reduce potentially toxic mistargeting of a CIR-expressing cell with non-tumor tissue such as bone marrow stroma, myelin and endothelium.

[0125] In some embodiments, the targeting region (the region including the D1-D2 domains) of a subject ILT2 chimeric receptor (an ILT2 CIR) includes domains D1-D4, and as such in some cases includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the ILT2 sequence set forth in SEQ ID NO: 94).

[0126] In some embodiments, the targeting region (the region including the D1-D2 domains) of a subject ILT4 chimeric receptor (an ILT4 CIR) includes domains D1-D4, and as such in some cases includes an amino acid sequence that has 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the ILT2 sequence set forth in SEQ ID NO: 96).Atty Docket No.: NKLT-002WO Stalk domains

[0127] In some embodiments, replacement of D3-D4 can be made with any protein or portion of a protein that properly displays the ILT2 or ILT4 D1-D2 binder in a context for HLA- G expressed on a separate cell. In certain embodiments a short polypeptide linker may form the linkage between the transmembrane domain and the intracellular domain of the chimeric ILT receptor. Thus, the chimeric ILT receptors may further comprise a stalk, that is, an extracellular region of amino acids between the extracellular domain and the transmembrane domain. The purpose of the stalk domain is to extend the D1 / D2 domains away from the plasma membrane and toward the target protein HLA-G. For example, the stalk may be a sequence of amino acids naturally associated with a selected transmembrane domain. In some embodiments, the CIR comprises a CD8α transmembrane domain, in certain embodiments, the CIR comprises a CD8α transmembrane domain together with additional amino acids on the extracellular portion of the transmembrane domain. In certain embodiments, a CIR comprises a CD8α transmembrane domain and a CD8α stalk (see SEQ ID NO: 43; an encoding nucleotide sequence is SEQ ID NO: 42). In a specific embodiment, a CD8α transmembrane domain comprises (or consists of) a sequence disclosed herein (see SEQ ID NO: 100). In another specific embodiment, a CD8α stalk comprises (or consists of) a sequence disclosed herein (see SEQ ID NO: 43, which includes a stalk and TM). The chimeric ILT receptor may further comprise a region of amino acids between the transmembrane domain and the cytoplasmic domain, which are naturally associated with the polypeptide from which the transmembrane domain is derived.

[0128] Examples of such chimeric stalk moieties include, but are not limited to membrane proximal portions of CD8α (see, e.g., SEQ ID NOs: 43 and 107), the CH2 / CH3 domains of IgG (e.g., IgG1, IgG4) (see, e.g., SEQ ID NOs: 51 and 98), the CH3 domain of IgG (e.g., IgG1, IgG4)(see, e.g., SEQ ID NO: 102), HER2, mGluR2, CD28 (see, e.g., SEQ ID NOs: 47 and 106) and CTLA4.

[0129] For example, in some cases the stalk domain of a subject CIR is selected from: an ILT2, ILT4, CD28, CH2 / CH3, CH3, and CD8α stalk domain. See, for example: VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLL LLFLILRHRRQ (SEQ ID NO: 39) (SEQ ID NO: 38 is an encoding nucleotide sequence), which includes an ILT2 stalk and TM domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACD (SEQ ID NO: 107), which includes a CD8α stalk domain;Atty Docket No.: NKLT-002WO PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYCNHRNRRRVCKCPR (SEQ ID NO: 43), which includes a CD8 stalk and TM domain; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKP (SEQ ID NO: 106), which includes a CD28 stalk; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWV (SEQ ID NO: 47), which includes a CD28 stalk and TM domain; VDKRVESKYGPPCPSCPAPPVAGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSQEDPE VQFNWYVDGVEVHNAKTKPREEQFQSTYRVVSVLTVLHQDWLNGKEYKCKVSNKGLP SSIEKTISKAKGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPE NNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKSLSLSLE L (SEQ ID NO: 98), which includes a CH2CH3 stalk; VDKRVESKYGPPCPGQPREPQVYTLPPSQEEMTKNQVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFFLYSRLTVDKSRWQEGNVFSCSVMHEALHNHYTQKS LSLSLEL (SEQ ID NO: 102), which includes a CH3 stalk; DPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKKDPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 51), which includes a CH2CH3 stalk and CD28 TM domain; VVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLL LLLFLILRHRRQ (SEQ ID NO: 59), which includes an ILT4 stalk and TM domain.

[0130] In some embodiments, the stalk of domain of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the stalk sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the stalk of domain of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the stalk sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the stalk of domain of a subject CIR includes the stalk sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.Atty Docket No.: NKLT-002WO

[0131] In some embodiments, the stalk of domain of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107. In some embodiments, the stalk of domain of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107. In some embodiments, the stalk of domain of a subject CIR includes the stalk sequence set forth in any one of SEQ ID NOs: 98, 102, 106, and 107.

[0132] Interaction of the D1 / D2 stalk-containing CIR with dimeric HLA-G will have the effect of dimerizing the intracellular signaling domains that in certain embodiments can stimulated ICD activation and cell signaling. Mutant forms of an ILT2 D1 / D2 CIR or an ILT4 D1 / D2 CIR

[0133] In further embodiments mutations may be made within the ILT2 or ILT 4 D1 or D2 domains contained within a CIR to increase the specificity of the CIR toward HLA-G over other potentially interacting proteins. For example, a mutation may be made to encode an amino acid other than tyrosine at the corresponding position of native amino acid 96 (Y96) of ILT4 SEQ ID NO: 53) or ILT2 (SEQ ID NO: 29) (e.g., Y96A). The effect of such a mutant form is to reduce potential interaction with ANGPTL2 and ANGPTL5 while retaining binding affinity for HLA-G. In yet another further embodiment a similar mutation may be placed in a full-length ILT4 CIR containing D1- D4 domains together with a mutation in domain D4 (at a position corresponding to tyrosine394 (Y394) of SEQ ID NO: 53) that further destabilizes interaction with ANGPTL2 and ANGPTL5. The corresponding position of ILT2 is tyrosine395 (Y395) of SEQ ID NO: 29.

[0134] As such, in some cases, a subject ILT4 CIR includes a mutation at an amino acid position corresponding to Y96 of SEQ ID NO: 53 (e.g., Y96A). In some cases, a subject ILT2 CIR includes a mutation at an amino acid position corresponding to Y96 of SEQ ID NO: 29 (e.g., Y96A). In some cases, a subject ILT4 CIR includes a mutation at an amino acid position corresponding to Y394 of SEQ ID NO: 53 (e.g., Y394A) (also see SEQ ID NOs: 60-61). In some cases, a subject ILT2 CIR includes a mutation at an amino acid position corresponding to Y395 of SEQ ID NO: 29 (e.g., Y395A). In some cases, a subject ILT4 CIR includes a mutation at an amino acid position corresponding to Y96 of SEQ ID NO: 53 (e.g., Y96A) and a mutation at an amino acid position corresponding to Y394 of SEQ ID NO: 53 (e.g., Y394A) (e.g., Y96A / Y394A). In some cases, a subject ILT2 CIR includes a mutation at an aminoAtty Docket No.: NKLT-002WO acid position corresponding to Y96 of SEQ ID NO: 29 (e.g., Y96A) and a mutation at an amino acid position corresponding to Y395 of SEQ ID NO: 29 (e.g., Y395A) (e.g., Y96A / Y395A).

[0135] Other embodiments along similar lines may limit interaction with classical HLA proteins or CD1 while retaining binding for HLA-G. These mutations may replace interacting sites with α3 domains that are specific to the heavy chains of these HLAs or on a surface bound to β2-M. (ii) Transmembrane (TM) region

[0136] A CIR may include a single-pass or multiple-pass transmembrane sequence (e.g., at the N-terminus or C-terminus of the chimeric protein, or within the protein, e.g., connecting the extracellular targeting region to the intracellular domain). Single pass transmembrane regions are found in certain CD molecules, tyrosine kinase receptors, serine / threonine kinase receptors, TGFβ, BMP, activin and phosphatases. Single pass transmembrane regions often include a signal peptide region and a transmembrane region of about 20 to about 25 amino acids, many of which are hydrophobic amino acids and can form an alpha helix. A short track of positively charged amino acids often follows the transmembrane span to anchor the protein in the membrane. Multiple pass proteins include ion pumps, ion channels, and transporters, and include two or more helices that span the membrane multiple times. All or substantially all of a multiple pass protein sometimes is incorporated in a chimeric protein. Sequences for single pass and multiple pass transmembrane regions are known and can be selected for incorporation into a chimeric protein molecule.

[0137] In some embodiments, the transmembrane domain is fused to the extracellular domain of the CIR. In some embodiments, the transmembrane domain is fused to the extracellular region and the intracellular region, thereby connecting the extracellular and intracellular regions to one another. In one embodiment, the transmembrane domain that naturally is associated with one of the domains in the CIR is used. In other embodiments, a transmembrane domain that is not naturally associated with one of the domains in the CIR is used. In some instances, the transmembrane domain can be selected or modified by amino acid substitution (e.g., typically changed to a hydrophobic residue) to avoid binding of such domains to the transmembrane domains of the same or different surface membrane proteins to minimize interactions with other members of the receptor complex.

[0138] Transmembrane (TM) domains may, for example, be derived from the alpha, beta, or zeta chain of the T cell receptor, CD3-ε, CD3 ζ, CD4, CD5, CD8, CD8α, CD9, CD16, CD22, CD28, CD33, CD38, CD64, CD80, CD86, CD134, CD137, ILT2, HER2, ILT4 or CD154 - or transmembrane regions containing functional variants thereof such asAtty Docket No.: NKLT-002WO those retaining a substantial portion of the structural, e.g., transmembrane, properties thereof can be used. See e.g., Kahlon et al. (2004) Cancer Res.64:9160-9166; Schambach et al. (2009) Methods Mol. Biol.506: 191-205; Jensen et al. (1998) Biol. Blood Marrow Transplant 4:75-83; Patel et al. (1999) Gene Ther.6:412; Song et al. (2012) Blood 119:696-706; Carpenito et al. (2009) Proc. Natl. Acad. Sci. USA 106:3360-5; Hombach et al. (2012) Oncoimmunology 1:458-66) and Geiger et al. (2001) Blood 98:2364-71.

[0139] Or, in some examples, the transmembrane domain may be synthesized de novo, comprising mostly hydrophobic residues, such as, for example, leucine, isoleucine, phenylalanine and valine. Examples of suitable CD8 stalk sequences, transmembrane sequences, and CD3ζ sequences are disclosed herein.

[0140] For example, in some cases the TM domain of a subject CIR is selected from: an ILT2 (see, e.g., SEQ ID NO: 39), ILT4 (see, e.g., SEQ ID NO: 59), CD28 (see, e.g., SEQ ID NOs: 47 and 104), and CD8 (see, e.g., SEQ ID NOs: 43 and 100) TM domain. See, for example: VVSGPSGGPSSPTTGPTSTSGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVILLLLLLL LLFLILRHRRQ (SEQ ID NO: 39), which includes an ILT2 stalk and TM domain; IYIWAPLAGTCGVLLLSLVITLYCNHRNRRRVCKCPR (SEQ ID NO: 100), which includes a CD8 TM domain; PAPRPPTPAPTIASQPLSLRPEACRPAAGGAVHTRGLDFACDIYIWAPLAGTCGVLLLSL VITLYCNHRNRRRVCKCPR (SEQ ID NO: 43), which includes a CD8 stalk and TM domain; FWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 104), which includes a CD28 TM domain; IEVMYPPPYLDNEKSNGTIIHVKGKHLCPSPLFPGPSKPFWVLVVVGGVLACYSLLVTVA FIIFWV (SEQ ID NO: 47), which includes a CD28 stalk and TM domain; DPAEPKSPDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDP EVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKAL PAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQP ENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSP GKKDPFWVLVVVGGVLACYSLLVTVAFIIFWV (SEQ ID NO: 51), which includes a CH2CH3 stalk and CD28 TM domain; andAtty Docket No.: NKLT-002WO VVSGPSMGSSPPPTGPISTPAGPEDQPLTPTGSDPQSGLGRHLGVVIGILVAVVLLLLLL LLLFLILRHRRQ (SEQ ID NO: 59), which includes an ILT4 stalk and TM domain.

[0141] In some embodiments, the TM domain of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the TM domain sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the TM domain of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the TM domain sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the TM domain of a subject CIR includes the TM domain sequence portion of the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.

[0142] In some embodiments, the stalk domain plus the TM domain (stalk / TM domain) of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the stalk domain plus the TM domain (stalk / TM domain) of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59. In some embodiments, the stalk domain plus the TM domain (stalk / TM domain) of a subject CIR includes an amino acid sequence having the amino acid sequence set forth in any one of SEQ ID NOs: 39, 43, 47, 51, and 59.

[0143] In some embodiments, the TM domain of a subject CIR includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the TM domain sequence set forth in any one of SEQ ID NOs: 100 and 104. In some embodiments, the TM domain of a subject CIR includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with the TM domain sequence set forth in any one of SEQ ID NOs: 100 and 104. In some embodiments, the TM domain of a subject CIR includes the TM domain sequence set forth in any one of SEQ ID NOs: 100 and 104. (iii) Intracellular Domain (ICD)Atty Docket No.: NKLT-002WO

[0144] As noted above, a subject chimeric ILT receptor (ILT2 or ILT4 based) includes an intracellular region (Intracellular domain or ICD) that replaces the natural intracellular portion of ILT2 or ILT4, which is inhibitory, with an ICD of a CAR, which is activating. As such, the ICD of a subject CIR (ILT2 version or ILT4 version) includes a “signaling region”, which has at least one signaling domain that causes activation of the cell, and can optionally include a “costimulatory region”, which can include one or more costimulatory domains. Signaling Region

[0145] Activation of T cells is directed by engagement of the T cell receptor with a peptide- MHC complex and activation of the CD3 signaling complex. Cell signaling is triggered by phosphorylation ITAM motives present on the CD3ζ, CD3d, CD3γ and CD3e components of this complex. CD3ζ contains three ITAMs that recruit the ZAP70 kinase that initiates a downstream signaling cascade to activate the T cell canonically through the activation of NF-AT. NK cells also express CD3ζ which acts as the intracellular signaling adaptor for the Natural cytotoxicity receptor (NCR) NKp46. Other NCRs such as CD94 / NKG2C engage with the ITAM-containing signaling adaptor DAP12 and NKG2D engages with the DAP10 signaling adaptor. DAP12 contains a single ITAM motif that can be phosphorylated by Syk or ZAP70 to activate NK cell signaling for cytotoxicity. It is notable that DAP10 does not contain a canonical ITAM domain but instead signals through mechanisms more similar to CD28 in T cells.

[0146] The “signaling region” (or “intracellular signaling domain”), e.g., of a CIR, refers to the part that participates in transducing the signal from binding (e.g., CIR) binding to a target molecule (HLA-G in the case of a subject CIR) into the interior of the immune effector cell to elicit effector cell function, e.g., activation, cytokine production, proliferation and / or cytotoxic activity, including the release of cytotoxic factors to the CIR-bound target cell, or other cellular responses elicited with target molecule binding to the extracellular CIR domain. Accordingly, the term “signaling region” (“intracellular signaling domain”) refers to the portion of a protein which transduces the effector function signal and that directs the cell to perform a specialized function. To the extent that a truncated portion of an intracellular signaling domain is used, such truncated portion may be used in place of a full-length intracellular signaling domain as long as it transduces the effector function signal. The term signaling region is meant to include any truncated portion of an intracellular signaling domain sufficient for transducing effector function signal. In some cases, the signaling region includes signaling motifs which are known as immunoreceptor tyrosine-based activation motifs (or “ITAMs”). The ITAM motives of CD3ζ are contained on the intracellular domain of, e.g., a canonical CAR. In some cases, the signaling region does not include an ITAM. InAtty Docket No.: NKLT-002WO some cases, the signaling region of a subject CIR includes an intracellular domain (e.g., CD3ζ) that includes an ITAM. In some cases, the signaling region of a subject CIR includes an intracellular domain (e.g., DAP10) that does not include an ITAM.

[0147] Examples of intracellular domain sequences that can be used in a signaling region of a subject CIR include those derived from an intracellular signaling domain of a lymphocyte receptor chain, a TCR / CD3 complex protein, an Fc receptor subunit, an IL-2 receptor subunit, CD3ζ, FcRγ, FcRβ, CD3γ, CD3δ, CD3ε, CD5, CD22, CD79a, CD79b, CD66d, CD278(ICOS), FcsRl, DAP10, and DAP12.

[0148] In some zeta (CD3ζ). the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a signaling region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 33. In some embodiments, the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 33. In some embodiments, the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 33. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 33.

[0149] In some embodiments, the signaling region (e.g., of a subject CIR) includes a DAP10 signaling domain (see, e.g., SEQ ID NO: 4), e.g., with or without a CD3ζ domain. In some embodiments, the signaling region (e.g., of a subject CIR) includes a DAP12 signaling domain (see, e.g., SEQ ID NO: 5), e.g., with or without a CD3ζ domain. In some embodiments, DAP10 of DAP12 (e.g., SEQ ID NO: 4, SEQ ID NO: 5) can supplement CD3ζ signaling by fusion. Examples are described herein where DAP10 or DAP12 replace or supplement CD3ζ alone or in combination with further signaling motives that drive coactivation of NK cells or costimulation of T cells.

[0150] In some embodiments, the signaling region (e.g., of a subject CIR) includes a DAP10 signaling domain (see, e.g., SEQ ID NO: 4). Thus, in some cases, the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a signaling region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 4. In some embodiments, the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 4. In some embodiments, the signaling region includes anAtty Docket No.: NKLT-002WO amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 4. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 4.

[0151] In some embodiments, the signaling region (e.g., of a subject CIR) includes a DAP12 signaling domain (see, e.g., SEQ ID NO: 23). Thus, in some cases, the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a signaling region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 23. In some embodiments, the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 23. In some embodiments, the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 23. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 23. Costimulatory Region

[0152] T cell activation is generated by antigen presenting cells (APC), typically dendritic cells that present peptide-MHC to receptive T cells. To prevent autoimmunity with MHC- peptide-TCR complexes with low affinity, costimulatory elements further stimulate T cells engaged with high affinity to presenting cells. These elements are typically derived from B7-H1 and B7-H2 ligation with CD28 on T cells and further by 4-1BB or OX40 interaction with their ligands once T cells are partially activated. The costimulation provided by CD28 or 4-1BB in combination with activation of ITAMs from the TCR / CD3 complex drives T cell differention into a fully activated state that supports proliferation and persistence to a memory cell state. Signaling derived from receptors that drive T cell costimulation can be incorporated into CIR or CAR products to support differentiation to a fully activated and persistent cell state. NK cells express 4-1BB, but not CD28 and are not typically stimulated by cell:cell contact with support cells but are activated directly by target cells.

[0153] Coactivation of NK cells is promoted by cytokines such as IL-12, IL-18, IL-21 and IL-1 within an inflammatory environment. IL-18 and IL-1 signaling is mediated by the cytoplasmic signaling node MyD88. Toll-like Receptors (TLR) activation by pathogenic ligands are also a potent NK cell activation mechanism employed by NK cells that is also directed downstream by MyD88.Atty Docket No.: NKLT-002WO

[0154] The examples below include the incorporation of signaling elements that drive NK cell activation to a degree further than costimulatory elements currently employed in CAR- T cell products and CAR-NK products that are derived from constructs designed for incorporation in T cells.

[0155] The costimulatory polypeptide may comprise one or more costimulatory signaling regions such as a truncated MyD88, 4-1BB or HVEM or a combination of these or other costimulatory motives. The costimulatory polypeptide may comprise one or more suitable costimulatory signaling regions that activate the signaling pathways activated by MyD88, 4-1BB or HVEM. Costimulating polypeptides include any molecule or polypeptide that activates the NF-κB pathway, MyD88 pathway, STAT5 pathway, STAT1 pathway, Akt pathway, and / or p38 pathway of tumor necrosis factor receptor (TNFR) family (i.e., CD40, RANK / TRANCE-R, OX40, 4-1BB) and CD28 family members (CD28, ICOS). More than one costimulating polypeptide or costimulating polypeptide cytoplasmic region may be expressed in the modified cells

[0156] In some embodiments, the ICD (e.g., of a subject CIR) also includes a costimulatory region. The costimulatory region includes at least one costimulatory domain (e.g., one, two, three, one or more, two or more, or three or more costimulatory domains). Examples of costimulatory domains include, but are not limited to: CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, and ICOS costimulatory domains. Examples of costimulatory domains include, but are not limited to: 4-1BB, OX40, ICOS, CD28, CD27, MyD88, IL-1Rα, HVEM, TRANCE, IL-1Rβ, CD70, IL-18Rα, CD40, IL-18Rβ, IL-33Rα, CD30, and IL-33Rβ. Examples of costimulatory domains include, but are not limited to: 4-1BB, OX40, ICOS, RANK, DAP10, DAP12, CD28, CD27, MyD88, IL-1Rα, HVEM, TRANCE, IL-1Rβ, CD70, IL-18Rα, CD40, IL-18Rβ, IL- 33Rα, CD30, and IL-33Rβ. In some cases, the costimulatory region includes one or more (e.g., one, two, three, one or more, or two or more) costimulatory domains selected from the group consisting of: CD28 (see, e.g., SEQ ID NO: 49), 4-1BB (see, e.g., SEQ ID NO: 35), and OX40 – or any combination thereof. In some cases, a CD28 costimulatory domain is used. In some cases, a 4-1BB costimulatory domain is used. In some cases, both a CD28 costimulatory domain and a 4-1BB costimulatory domain is used (i.e., they are both used). In some cases, a CD28 costimulatory domain and an OX40 costimulatory domain is used.

[0157] In some cases, the costimulatory region includes a truncated MyD88 polypeptide fused with signaling domains of receptor mediators of costimulation, such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS. In some cases, the costimulatory region includes a MyD88 polypeptide or a truncated MyD88 polypeptide and a costimulatory domain selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40, and DAP10.Atty Docket No.: NKLT-002WO

[0158] In some embodiments, the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a costimulatory region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49. In some embodiments, the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49. In some embodiments, the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 49.

[0159] In some embodiments, the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a costimulatory region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35 (4-1BB). In some embodiments, the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35. In some embodiments, the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35. In some embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 35.

[0160] In some embodiments, the ICD (e.g., of a subject chimeric ILT receptor (ILT2-version or ILT4-version)) includes a costimulatory region that includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35 and an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49. In some embodiments, the signaling region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35 and . an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49. In some embodiments, the signaling region includes an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 35 and an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 49. In someAtty Docket No.: NKLT-002WO embodiments, the signaling region includes the amino acid sequence set forth as SEQ ID NO: 35 and the amino acid sequence set forth as SEQ ID NO: 49.

[0161] In some embodiments, the signaling region (e.g., of a subject CIR) includes a CD3 zeta (CD3ζ) signaling domain and the costimulatory region includes a CD28 costimulatory domain. In some embodiments, the signaling region includes a CD3 zeta (CD3ζ) signaling domain and the costimulatory region includes a 4-1BB costimulatory domain. In some embodiments, the signaling region includes a CD3 zeta (CD3ζ) signaling domain and the costimulatory region includes a 4-1BB costimulatory domain and a CD28 costimulatory domain. In some embodiments, the signaling region includes a CD3 zeta (CD3ζ) signaling domain and the costimulatory region includes a CD28 costimulatory domain and an OX40 costimulatory domain.

[0162] In some embodiments, the signaling region (e.g., of a subject CIR) includes a DAP10 or DAP12 signaling domain (without a CD3ζ signaling domain) and the costimulatory region includes a CD28 costimulatory domain. In some embodiments, the signaling region includes a DAP10 or DAP12 signaling domain (without a CD3ζ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain. In some embodiments, the signaling region includes a DAP10 or DAP12 signaling domain (without a CD3ζ signaling domain) and the costimulatory region includes a 4- 1BB costimulatory domain and a CD28 costimulatory domain. In some embodiments, the signaling region includes a DAP10 or DAP12 signaling domain (without a CD3ζ signaling domain) and the costimulatory region includes a CD28 costimulatory domain and an OX40 costimulatory domain. In some embodiments, the signaling region includes a DAP12 signaling domain (without a CD3ζ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain. In some embodiments, the signaling region includes a DAP10 signaling domain (without a CD3ζ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain.

[0163] In some embodiments, the signaling region (e.g., of a subject CIR) includes a DAP10 or DAP12 signaling domain (fused with a CD3ζ signaling domain) and the costimulatory region includes a CD28 costimulatory domain. In some embodiments, the signaling region includes a DAP10 or DAP12 signaling domain (fused with a CD3ζ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain. In some embodiments, the signaling region includes a DAP10 or DAP12 signaling domain (fused with a CD3ζ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain and a CD28 costimulatory domain. In some embodiments, the signaling region includes a DAP10 or DAP12 signaling domain (fused with a CD3ζ signaling domain) and the costimulatory region includes a CD28 costimulatory domain and an OX40 costimulatory domain. In some embodiments, theAtty Docket No.: NKLT-002WO signaling region includes a DAP12 signaling domain (fused with a CD3ζ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain. In some embodiments, the signaling region includes a DAP10 signaling domain (fused with a CD3ζ signaling domain) and the costimulatory region includes a 4-1BB costimulatory domain.

[0164] Cells may include chimeric signaling polypeptides, including, for example, chimeric signaling polypeptides where a truncated MyD88 polypeptide has also been fused with signaling domains of receptor mediators of costimulation, such as, for example, 4- 1BB or HVEM. The fusion may incorporate other signaling domains such as those from CD40, CD27, CD28, 4-1BB, OX40, or ICOS. More than one costimulating polypeptide or costimulating polypeptide cytoplasmic region may be expressed in the modified cells.

[0165] Cells may include chimeric signaling polypeptides, including, for example, chimeric signaling polypeptides where a truncated MyD88 polypeptide has also been fused with signaling domains of receptor mediators of costimulation, such as, for example, CD40, CD27, CD28, 4-1BB, HVEM, TRANCE, RANK, OX40, or ICOS.

[0166] In some embodiments, a chimeric signaling polypeptide comprises cytoplasmic signaling regions from two costimulatory polypeptides, such as, for example, 4-1BB and CD28, or one, or two or more costimulatory polypeptide cytoplasmic signaling regions selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, or OX40,. In some embodiments, the chimeric signaling polypeptide comprises a MyD88 polypeptide or a truncated MyD88 polypeptide and a costimulatory polypeptide cytoplasmic signaling region selected from the group consisting of CD27, ICOS, RANK, TRANCE, CD28, 4-1BB, OX40.

[0167] Non-limiting examples of a 4-1BB, CD28, and OX40 costimulatory signaling domains can be found in U.S.20130266551, U.S. Pat. No.5,686,281; Geiger, T. L. et al., Blood 98: 2364-2371 (2001); Hombach A. et al., J Immunol 167: 6123-6131 (2001); Maher J. et al. Nat Biotechnol 20: 70-75 (2002); Haynes N. M. et al., J Immunol 169: 5780-5786 (2002); Haynes N. M. et al., Blood 100: 3155-3163 (2002); and in U.S. 2012 / 20148552, all of which are incorporated by reference herein for their teachings related to costimulatory domains.

[0168] Non-limiting examples of chimeric polypeptides useful for inducing cell activation, and related methods for inducing therapeutic cell activation including, for example, expression constructs, methods for constructing vectors, and assays for activity or function, may also be found in the following patents and patent applications: US2014- 0286987-A1; WO2014 / 151960; US2016 / 0046700; WO2015 / 123527; US2004 / 0209836; U.S. Patent No.7,404,950; WO2004 / 073641; US2011 / 0033388; U.S. Patent No.8,691,210; WO2008 / 049113; US2014 / 0087468; U.S. Patent No.Atty Docket No.: NKLT-002WO 9,315,559; WO2010 / 033949; US2011 / 0287038; WO2011 / 130566; US2016 / 0175359; WO2016 / 036746; WO2016 / 100241; US2017 / 0166877; WO2017 / 106185; and WO2018 / 208849 – each of which is incorporated by reference herein in its entirety, including all text, tables and drawings, for all purposes, including for purposes related to describing cell activation domains (e.g., cell signaling and costimulatory domains).

[0169] In some embodiments, cells are designed to provide constitutively active therapy. In some embodiments, genetically modified cells comprise a nucleic acid comprising a first polynucleotide encoding a Chimeric ILT2 or ILT4 Receptor (or CIR), and a second polynucleotide encoding a chimeric signaling polypeptide. In some embodiments, the second polynucleotide is positioned 5’ of the first polynucleotide. In some embodiments, the second polynucleotide is positioned 3’ of the first polynucleotide. In some embodiments, a third polynucleotide encoding a linker polypeptide is positioned between the first and second polynucleotides. Where the third polynucleotide is positioned 3’ of the first polynucleotide and 5’ of the second polynucleotide, the linker polypeptide, may remain intact following translation, or may separate the polypeptides encoded by the first and second polynucleotides during, or after translation. In some embodiments, the linker polypeptide is a 2A polypeptide (see elsewhere herein), which may separate the polypeptides encoded by the first and second polynucleotides during, or after translation. High level costimulation is provided constitutively through an alternate mechanism in which a leaky 2A cotranslational sequence (see elsewhere herein). is used to separate the CAR from the chimeric signaling polypeptide. Where the 2A separation is incomplete, for example from a leaky 2A sequence, most of the expressed chimeric signaling polypeptide molecules are separated from the chimeric antigen receptor polypeptide and may remain cytosolic, and some portion or the chimeric signaling polypeptide molecules remain attached, or linked, to the CAR.

[0170] By “constitutively active” is meant that the chimeric stimulating polypeptide’s cell activation activity is active even in the absence of an inducer. One method to generate constitutively active signalling is to tether the activation protein factor to the plasma membrane via a transmembrane domain or lipid targeting moiety Coactivation by MyD88 signaling

[0171] MyD88 (encoded by myeloid differentiation primary response gene 88) is a crucial mediator of signals downstream of several receptors, notably the Toll-like Receptors (TLRs) that direct a part of innate immune responses. MyD88 is also a principal mediator of signaling downstream of the Interleukin-1 family of receptors including the receptors for IL-1, IL-18 and IL-33. MyD88 contains two domains that direct its activity – an amino-terminal Death Domain that directs oligomerization into a complex that further directs downstream signaling through the NK-κB pathway for induced cytokineAtty Docket No.: NKLT-002WO production and coactivation of signals from separate ITAM-directed signals, the AKT growth and survival pathway and the Interferon Response Factor pathway. A carboxy terminal TIR domain directs recruitment to Toll-like Receptors and IL-1 family receptors through interaction with their TIR domains. Similar TIR-TIR interactions can recruit related signaling proteins that act as nodes, for example TRIF, to TLR3 and TLR4.

[0172] In some embodiments, a TIR domain from an IL1 family receptor or toll-like receptor (TLR) can be used to recruit MyD88 or TRIF signaling to an activated chimeric receptor (e.g., a CAR, a CIR) by direct fusion of a the TIR to the receptor as a coactivation domain, e.g., a “costimulatory region” can include a TIR domain (e.g., from IL1 family receptors or TLRs). As such, in some cases, the costimulatory region (e.g., of a CIR or CAR) includes a TIR domain from an IL1 family receptor or a TLR.

[0173] In some cases, the costimulatory region includes a TIR from TLR2. As such, in some cases, a costimulatory region includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 111. In some embodiments, a costimulatory region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 111. In some embodiments, a costimulatory region includes an amino acid an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 111. In some embodiments, a costimulatory region includes the amino acid sequence set forth as SEQ ID NO: 111.

[0174] In some cases, the costimulatory region includes a TIR from TLR3. As such, in some cases, a costimulatory region includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 113. In some embodiments, a costimulatory region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 113. In some embodiments, a costimulatory region includes an amino acid an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 113. In some embodiments, a costimulatory region includes the amino acid sequence set forth as SEQ ID NO: 113.

[0175] In some cases, the costimulatory region includes a TIR from IL-18R1 (IL-18 receptor alpha). As such, in some cases, a costimulatory region includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) withAtty Docket No.: NKLT-002WO SEQ ID NO: 109. In some embodiments, a costimulatory region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 109. In some embodiments, a costimulatory region includes an amino acid an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 109. In some embodiments, a costimulatory region includes the amino acid sequence set forth as SEQ ID NO: 109.

[0176] Any of the above TIR domains can be used in combination with any desired signaling domain, e.g. CD3ζ, DAP10, DAP12. In some case, a signaling region includes a CD3ζ signaling domain and a TIR from TLR2. In some case, a signaling region includes a CD3ζ signaling domain and a TIR from TLR3. In some case, a signaling region includes a CD3ζ signaling domain and a TIR from IL-18R1.In some case, a signaling region includes a DAP10 signaling domain and a TIR from TLR2. In some case, a signaling region includes a DAP10 signaling domain and a TIR from TLR3. In some case, a signaling region includes a DAP10 signaling domain and a TIR from IL- 18R1.In some case, a signaling region includes a DAP12 signaling domain and a TIR from TLR2. In some case, a signaling region includes a DAP12 signaling domain and a TIR from TLR3. In some case, a signaling region includes a DAP12 signaling domain and a TIR from IL-18R1.

[0177] Fusions of a truncated MyD88 polypeptide, lacking the TIR domain with the intracellular domain of CD40 to produce a chimeric polypeptide amplifies certain signals directed by MyD88. When T cells are transfected or transduced with nucleic acids that encode MC, in combination with a Chimeric Antigen Receptor (CAR), MC delivers potent costimulatory signals that enhance T and NK cell growth, persistence, and cytotoxic activity against cells specifically targeted by the CAR (Foster et al, Mol. Ther.25:2176 (2017), Duong et al., Mol. Ther. Onc.12:124 (2018), Collinson-Pautz et al, Leukemia 33:2195, Wang et al, Blood Adv.4:1950). MyD88 Fusions

[0178] In some embodiments, a truncated MyD88 polypeptide has also been fused with signaling domains of receptor mediators of costimulation, such as, for example, 4-1BB or HVEM. These chimeric signaling polypeptides generate different phenotypic outcomes in modified NK cells that express CIRs (CIR-NK cells), particularly in the cells’ cytotoxicity, growth potential and capacity to release cytokines upon engagement with their target.

[0179] Provided herein are chimeric truncated MyD88 polypeptides that, when expressed in, for example, CIR-NK cells, produce significantly fewer of certain toxic inflammatory cytokines such as TNF-α than CIR-NK cells that express an MyD88-CD40 chimericAtty Docket No.: NKLT-002WO polypeptide, while retaining potent or even enhanced tumor cell killing. Also provided are modified chimeric receptors, where the chimeric receptor polypeptide comprises truncated MyD88 polypeptides alone.

[0180] Also provided herein are immune cells, such as, for example, activated NK cells that express an chimeric signaling polypeptide. The activated cells may be used to increase the immune response against a disease, or to treat cancer by, for example, reducing the size of a tumor. Therapeutic courses of treatment using the activated NK cells and activated CIR-NK cells may be monitored by determining the size and vascularity of tumors by various imaging modalities (e.g. CT, bonescan, MRI, PET scans, Trofex scans), by various standard blood biomarkers (e.g. PSA, Circulating Tumor Cells), or by serum levels of various inflammatory, hypoxic cytokines, or other factors in the treated patient

[0181] In some embodiments a costimulatory region includes an MyD88 polypeptide (see, e.g., SEQ ID NO: 27). As such, in some cases, a costimulatory region includes an amino acid sequence having 80% or more sequence identity (e.g., 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 27. In some embodiments, a costimulatory region includes an amino acid sequence having 90% or more sequence identity (e.g., 95% or more, 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 27. In some embodiments, a costimulatory region includes an amino acid an amino acid sequence having 95% or more sequence identity (e.g., 96% or more, 97% or more, 98% or more, 99% or more, or 100%) with SEQ ID NO: 27. In some embodiments, a costimulatory region includes the amino acid sequence set forth as SEQ ID NO: 27. The MyD88 polypeptides of this paragraph can combined with other costimulatory molecules as part a costimulatory region. For example, in some cases with CD40. In some cases with 4-1BB. In some cases with HVEM (see, e.g., SEQ ID NO: 25). Any of these combinations (MyD88, MyD88-CD40, MyD88-4-1BB, MyD88-HVEM) can be used in combination with any desired signaling domain, e.g. CD3ζ, DAP10, DAP12. In some case, a signaling region includes a CD3ζ signaling domain and a MyD88, MyD88-CD40, MyD88-4-1BB, or MyD88-HVEM costimulatory region. In some case, a signaling region includes a DAP10 signaling domain and a MyD88, MyD88-CD40, MyD88-4-1BB, or MyD88-HVEM costimulatory region. In some case, a signaling region includes a DAP12 signaling domain and a MyD88, MyD88-CD40, MyD88-4- 1BB, or MyD88-HVEM costimulatory region. In some case, a signaling region includes a DAP12 signaling domain and a MyD88 costimulatory region. In some case, a signaling region (e.g., of a CIR) includes a CD3ζ signaling domain and a MyD88-4- 1BB costimulatory region.Atty Docket No.: NKLT-002WO CIR Variations

[0182] Provided are functional portions of the CIRs described herein. The term “functional portion” when used in reference to a CIR refers to any part or fragment of the CIR, which part or fragment retains the biological activity of the CIR of which it is a part (the parent CIR). Functional portions encompass, for example, those parts of a CIR that retain the ability to recognize the target (HLA-G) or target cells, or detect, treat, or prevent a disease, to a similar extent, the same extent, or to a higher extent, as the parent CIR. In reference to the parent CIR, the functional portion can comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%, 95%, or more, of the parent CIR. Also provided are ICD polypeptides. Such polypeptides can serve as in ICD as part of a CIR, but can also serve as and ICD as part of another chimeric receptor, such as a CAR.

[0183] The functional portion can comprise additional amino acids at the amino or carboxy terminus of the portion, or at both termini, which additional amino acids are not found in the amino acid sequence of the parent CIR. Desirably, the additional amino acids do not interfere with the biological function, e.g., recognize target cells, detect cancer, treat or prevent cancer, etc. More desirably, the additional amino acids enhance the biological activity, as compared to the biological activity of the parent CIR.

[0184] Included in the scope of the disclosure are functional variants or biological equivalent of the inventive CIRs disclosed herein. A functional variant can, for example, comprise the amino acid sequence of the parent polypeptide with at least one conservative amino acid substitution. Alternatively or additionally, the functional variants can comprise the amino acid sequence of the parent polypeptide with at least one non- conservative amino acid substitution. In this case, it is preferable for the non- conservative amino acid substitution to not interfere with or inhibit the biological activity of the functional variant. The non-conservative amino acid substitution may enhance the biological activity of the functional variant, such that the biological activity of the functional variant is increased as compared to the parent polypeptide.

[0185] Such biological variant (including functional portions thereof) can comprise synthetic amino acids in place of one or more naturally-occurring amino acids.

[0186] Such biological variant (including functional portions thereof) can be glycosylated, amidated, carboxylated, phosphorylated, esterified, N-acylated, cyclized via, e.g., a disulfide bridge, or converted into an acid addition salt and / or optionally dimerized or polymerized, or conjugated.

[0187] Such biological variant (including functional portions thereof) can be obtained by methods known in the art. The polypeptides may be made by any suitable method of making polypeptides or proteins. Suitable methods of de novo synthesizing polypeptides and proteins are described in references, such as Chan et al., FmocAtty Docket No.: NKLT-002WO Solid Phase Peptide Synthesis, Oxford University Press, Oxford, United Kingdom, 2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford University Press, Oxford, United Kingdom, 2001 and U.S. Pat. No.5,449,752. Also, polypeptides and proteins can be recombinantly produced using the nucleic acids described herein using standard recombinant methods. See, for instance, Sambrook et al, Molecular Cloning: A Laboratory Manual, 3rd ed., Cold Spring Harbor Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates and John Wiley & Sons, NY, 1994. 4. Safety switches

[0188] Genetically modified cells that express a subject chimeric ILT receptor (CIR) may also express a safety switch, also known as an inducible suicide gene or suicide switch, which can be used to eradicate the therapeutic cells in vivo if desired e.g. if GvHD develops. In some examples, therapeutic cells may trigger an adverse event, such as off-target toxicity due to a CIR, or a patient might experience a negative symptom during therapy using modified cells, or there may be side effects due to non-specific attacks on healthy tissue; or, sometimes, the therapeutic cells may no longer be needed, or the therapy is intended for a specified amount of time, for example, the therapeutic cells may work to decrease the tumor cell, or tumor size, and may no longer be needed. Thus it can be useful if genetically modified cells can also inducibly express a polypeptide which causes the cells to die, such as an inducible caspase-9 polypeptide. If there is a need, for example, to reduce the number of therapeutic cells, the switch can be triggered.

[0189] These switches respond to a trigger, such as a pharmacological agent, which is supplied when it is desired to eradicate the therapeutic cells, and which leads to cell death (e.g. by triggering necrosis or apoptosis). These agents can lead to de novo expression of a toxic gene product, but a more rapid response can be obtained if the genetically modified cells already express a protein which is switched into a toxic form in response to the agent.

[0190] In some embodiments, a safety switch is based on a pro-apoptotic protein that can be triggered by administering a trigger molecule (also referred to as a ligand inducer) to a subject. If the pro-apoptotic protein is fused to a polypeptide sequence which binds to the trigger molecule, delivery of this trigger molecule can bring two pro-apoptotic proteins into proximity such that they trigger apoptosis. For instance, caspase-9 can be fused to a modified human FK-binding protein which can be induced to dimerize in response to the pharmacological agent rimiducid (AP1903). The use of a safety switch based on a human pro-apoptotic protein, such as, for example, caspase-9 minimizesAtty Docket No.: NKLT-002WO the risk that cells expressing the switch will be recognized as foreign by a human subject’s immune system. Delivery of rimiducid to a subject can therefore trigger apoptosis of cells which express the caspase-9 switch.

[0191] Further non-limiting examples of chimeric polypeptides useful for inducing cell death or apoptosis may be found in the following patents and patent applications, each of which is incorporated by reference herein in its entirety for all purposes. U.S. Patent Application US2011 / 0286980; U.S. Patent 9,089,520; U.S. Patent Application US2014 / 0255360; U.S. Patent No.9,434,935; WO2014 / 16438; US2016 / 0151465; WO2014 / 197638; US2015 / 0328292; WO2015 / 134877; US2016 / 0166613; WO2016 / 100236; US2016 / 0175359; WO2016 / 100241; US2017 / 0166877; WO2017 / 106185; each of which is incorporated by reference herein in its entirety, including all text, tables and drawings, for all purposes. Details about some specific switches and approaches are also given below:

[0192] Inducible Caspase 9 (iC9): This proapoptotic switch includes a fusion of caspase-9 with FKBP12 or derivatives. It is latent in the absence of ligand but drives dimerization of the initiator caspase, caspase-9, from the intrinsic pathway for cell apoptosis. Dimerization leads to caspase-9 activation, cleavage and activation of the effector caspase, caspase-3, and rapid cell death by apoptosis. Inducible caspase-9 has particular utility as a safety switch in cell therapies to block toxic responses.

[0193] Caspase-9 switches: Examples are described in Di Stasi et al. (2011) supra; see also Yagyu et al. (2015) Mol Ther 23(9):1475-85; Rossigloni et al. (2018) Cancer Gene Ther doi.org / 10.1038 / s41417-018-0034-1; Jones et al. (2014) Front Pharmacol doi.org / 10.3389 / fphar.2014.00254; US patent 9,434,935; US patent 9,913,882; US patent 9,393,292; and patent application US2015 / 0328292.

[0194] The safety switch may comprise a modified Caspase-9 polypeptide having modified activity, such as, for example, reduced basal activity in the absence of the homodimerizer ligand. Modified Caspase-9 polypeptides are discussed in, for example, US patent 9,913,882 and US2015 / 0328292, supra, and may include, for example, amino acid substitutions at position 330 (e.g., D330E or D330A) or, for example, amino acid substitutions at position 450 (e.g., N405Q), or combinations thereof, including, for example, D330E-N405Q and D330A-N405Q. Caspase-9 polypeptide with lower basal activity have been described previously, e.g. in U.S. Patent Nos.9,434,935, 9,932,572 and 9,913,882, and U.S. Patent Application Nos. 62 / 668,223, 62 / 756,442, 62 / 816,799, 15 / 901,556, 15 / 888,948.

[0195] In some embodiments the safety switch may be, for example, iCasp9 discussed in Di Stasi et al. (2011) supra, which consists of the sequence of the human FK506-binding protein (FKBP12) (GenBank AH002818) with an F36V mutation, connected through a SGGGS linker to a modified human caspase 9 (CASP9) which lacks its endogenousAtty Docket No.: NKLT-002WO caspase activation and recruitment domain. The F36V mutation increases the binding affinity of FKBP12 to synthetic homodimerizers AP20187 and rimiducid.

[0196] FKBP12-allele specific binding by rimiducid: Rimiducid binds with high affinity (~0.1 nM) to the valine-36 allele of FKBP12 but with low affinity (~500 nM) to the wild-type phenylalanine-36 FKBP12 allele. Rapamycin and rapalogs can bind to either FKBP allele. Rimiducid has two identical, protein-binding surfaces arranged tail-to-tail, each with high affinity and specificity for the valine-36 form (known variously as FKBP12(F36V), FKBP12v36, FKBPV, FV36, or simply Fv). See Jemal et al., CA Cancer J. Clinic.58, 71-96 (2008); Scher & Kelly Journal of Clinical Oncology 11, 1566-72 (1993)). Two tandem copies of the protein may also be used in the construct so that higher-order oligomers are induced upon cross-linking by rimiducid. Attachment of one or more FV domains onto one or more cell signaling molecules that normally rely on homodimerization can convert that protein to a rimiducid-controlled switch. FKBP12 variants may also be used. Variants may bind to rapamycin, or rapalogs, but with less affinity to rimiducid than, for example, FKBP12v36. Examples of FKBP12 variants include those from many species, including, for example, yeast. In one embodiment, the FKBP12 variant is FKBP12.6 (calstablin).

[0197] The suicide switch may be controlled by a pharmaceutical composition comprising a trigger molecule (such as a dimerizing or multimerizing ligand). An effective amount of a pharmaceutical composition comprising the trigger molecule is an amount that achieves the desired result of killing the genetically-modified cells. The degree of killing may be high (e.g. over 60%, 70%, 80%, 85%, 90%, 95%, or 97%) or complete; conversely, sometimes only partial removal will be desired (e.g. under 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% of the genetically modified cells are killed). Thus genetically-modified may display a range of sensitivities to a trigger molecule. The trigger molecule may thus be used to eradicate only a portion of the cells (e.g. at least 10%) while permitting some of the cells (e.g. at least 10%) to survive. The concentration of the trigger molecule can be selected according to the desired balance of cell death and survival e.g. a higher concentration will be delivered if a higher proportion of cell eradication (or complete eradication) is desired.

[0198] These concentrations can be determined by simple dose-ranging experiments, monitoring levels of cell death in response to the trigger molecule. Any appropriate assay may be used to determine the percent of genetically modified cells that are killed. An assay may include the steps of obtaining a first sample from a subject before administration of the trigger molecule and obtaining a second sample from the subject after administration of the trigger molecule and comparing the number or concentration of therapeutic cells in the first and second samples to determine theAtty Docket No.: NKLT-002WO percent of therapeutic cells that are killed. One can empirically determine the effective amount of a particular composition presented herein without undue experimentation. 5. Chimeric antigen receptors

[0199] Chimeric antigen receptors (or CARs) are artificial receptors designed to convey antigen specificity to cells. They generally include an antigen-specific component, a transmembrane component, and an intracellular component selected to activate the cell. CAR-expressing cells may be used in various therapies, including cancer therapies.

[0200] A CAR is, for example, a chimeric polypeptide which comprises a polypeptide sequence that recognizes a target antigen (an antigen-recognition domain) linked to a transmembrane polypeptide and an intracellular domain polypeptide selected to activate the cell, and thereby provide specific immunity. The antigen-recognition domain may be a single-chain variable fragment (scFv), or may, for example, be derived from other molecules such as, for example, a T cell receptor or camelid VhH domain. The intracellular domain comprises at least one polypeptide which causes activation of the cell, such as, for example, but not limited to, CD3 zeta (CD3ζ), and, optionally, co-stimulatory molecules (for example, but not limited to, CD28, OX40 and 4-1BB).

[0201] Thus, in typical examples of CAR usage, cells are modified to express a CAR that comprises a single chain antibody variable fragment (scFv) fused with a transmembrane domain containing a linker region and an intracellular domain derived from the CD3 zeta component. In natural T cells and NK cells, signals from CD3zeta drive the initial activation of the T cell through signaling to the NF-ATc transcription factor. These signals drive targeted cell killing in cytotoxic T lymphocytes and synergize with costimulatory signaling pathways to drive the robust cell proliferation of T cell immune response. The genetically modified cells may be modified by transduction or transfection with a nucleic acid that expresses the CAR and a nucleic acid (the same or different) that comprises a polynucleotide that encodes a chimeric signaling polypeptide (see below). In other embodiments, a CAR is expressed without also expressing a chimeric signaling polypeptide.

[0202] Chimeric antigen receptors can be expressed in NK cells, iNKT cells or in macrophages to generate antigen specific cytotoxicity.

[0203] CARs include chimeric receptors that are derived from antibodies, but also include chimeric T cell receptors. These chimeric T cell receptors may comprise a polypeptide sequence that recognizes a target antigen, where the recognition sequence may be, for example, but not limited to, the recognition sequence derived from a T cell receptor or a scFv. The intracellular domain polypeptides are those that act to activate the TAtty Docket No.: NKLT-002WO cell. Chimeric T cell receptors are discussed in, for example, Gross & Eshar FASEB Journal (1992) 6:3370-3378, and Zhang et al., (2010) PLOS Pathogens 6:1-13. 8. Linker polypeptides

[0204] Where it is desired to encode two polypeptides in a single gene, such that they are encoded on a single transcript, the two polypeptides can be joined by a linker polypeptide. For instance, these may be included between MyD88 and CD40 in a MyD88-CD40 chimeric polypeptide, or between the costimulatory polypeptide cytoplasmic signaling region and the CD3ζ portion of a CAR or CIR. A linker can be positioned between any of the regions / domains described herein, where desired. For example, in some cases, a linker is positioned: between the TM domain and the signaling region or costimulatory region, between the ILT2 or ILT4 targeting region (e.g., D1-D2 domain) and the stalk, between a signaling region and a costimulatory region, between two costimularoty domains, between a costimulatory or signaling region and a T2A sequence, or any combination thereof.

[0205] Linker polypeptides include cleavable and non-cleavable linker polypeptides. Examples of linkers include, but are not limited to: SGR, GS, VD, and PRGSG (SEQ ID NO: 67). Additional linkers will be known to one of ordinary skill in the art and any convenient linker can be used.

[0206] Linker polypeptides include those for example, consisting of about 2 to about 30 amino acids e.g. furin cleavage site, (GGGGS)n. In some embodiments, the linker polypeptide consists of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 amino acids. In some embodiments, the linker polypeptide consists of about 18 to 22 amino acids. In some embodiments, the linker polypeptide consists of 20 amino acids.

[0207] Cleavable linkers include linkers that are cleaved by an enzyme in the modified cells. The enzyme may be exogenous to the cells, for example, an enzyme encoded by a polynucleotide that is introduced into the cells by transfection or transduction, either at the same time or a different time as the polynucleotide that encodes the linker. In some embodiments, cleavable linkers include linkers that are cleaved by an enzyme endogenous to the modified cells in the population, including, for example, enzymes that are naturally expressed in the cell, and enzymes encoded by polynucleotides native to the cell, such as, for example, lysozyme. The term “cleavable linker” also extends to a linker which is cleaved by any means, including, for example, non- enzymatic means, such as peptide skipping.

[0208] One advantage of a cleavable linker is that it permits an essentially fixed stoichiometric ratio of expression of two polypeptides (a 1:1 ratio if two mature polypeptides are linked by a single cleavable linker).Atty Docket No.: NKLT-002WO

[0209] The linker polypeptide may be a 2A-like sequence, which can be derived from many different viruses, including, for example, from the Thosea asigna insect virus. These sequences are sometimes also known as “peptide skipping sequences.” When this type of sequence is placed within a cistron, between two polypeptides that are intended to be separated, the ribosome appears to skip a peptide bond, in the case of Thosea asigna sequence; the bond between the Gly and Pro amino acids at the carboxy terminal “P-G-P” is omitted. This may leave two to three polypeptides, for example, an inducible chimeric pro-apoptotic polypeptide and a chimeric antigen receptor, or, for example, a marker polypeptide and an inducible chimeric pro- apoptotic polypeptide. When this sequence is used, the polypeptide that is encoded 5’ of the 2A sequence may end up with additional amino acids at the carboxy terminus, including the Gly residue and any upstream residues in the 2A sequence. The peptide that is encoded 3’ of the 2A sequence may end up with additional amino acids at the amino terminus, including the Pro residue and any downstream residues following the 2A sequence.

[0210] In some embodiments, the cleavable linker is a 2A polypeptide derived from porcine teschovirus-1 (P2A). In some embodiments, the 2A cotranslational sequence is a 2A- like sequence. In some embodiments, the 2A cotranslational sequence is T2A (thosea asigna virus 2A), F2A (foot and mouth disease virus 2A), P2A (porcine teschovirus-1 2A), BmCPV 2A (cytoplasmic polyhedrosis virus 2A) BmIFV 2A (flacherie virus of B. mori 2A), or E2A (equine rhinitis A virus 2A). In some embodiments, the 2A cotranslational sequence is T2A-GSG, F2A-GSG, P2A-GSG, or E2A-GSG. In some embodiments, the 2A cotranslational sequence is selected from the group consisting of T2A, P2A and F2A. In a specific embodiment, a 2TA comprises (or consists of) a sequence disclosed herein. comprises (consists of) a sequence disclosed herein (e.g., a sequence disclosed in the Examples below).

[0211] 2A-like sequences are sometimes “leaky” in that some of the polypeptides are not separated during translation, and instead, remain as one long polypeptide following translation. One theory as to the cause of the leaky linker, is that the short 2A sequence occasionally may not fold into the required structure that promotes ribosome skipping (a “2A fold”). In these instances, ribosomes may not miss the proline peptide bond, which then results in a fusion protein. To reduce the level of leakiness, and thus reduce the number of fusion proteins that form, a GSG (or similar) linker may be added to the amino terminal side of the 2A polypeptide; the GSG linker blocks secondary structures of newly-translated polypeptides from spontaneously folding and disrupting the ‘2A fold’. A leaky 2A sequence can be used, for example, so that the same encoded polypeptide can sometimes be directed to the cell surface but other times remain in the cytosol.Atty Docket No.: NKLT-002WO

[0212] In certain embodiments, a 2A linker includes the amino acid sequence of SEQ ID NO: 11. In certain embodiments, the 2A linker further includes a GSG amino acid sequence at the amino terminus of the polypeptide, in other embodiments, the 2A linker includes a GSGPR (SEQ ID NO: 68) amino acid sequence at the amino terminus of the polypeptide. Thus, by a “2A” sequence, the term may refer to a 2A sequence in an example described herein or may also refer to a 2A sequence as listed herein further comprising a GSG or GSGPR (SEQ ID NO: 68) sequence at the amino terminus of the linker.

[0213] In some embodiments, the linker, for example, the 2A linker, is cleaved in about 10, 20, 30, 40, 50, 60, 70, 75, 80, 85, 90, 95, 98, or 99% of the translated polypeptides.

[0214] Examples of suitable linker polypeptides (including T2A linkers) are disclosed herein. 6. Genetically Modified Cells

[0215] The genetically modified cells (cells such as immune cells that express, e.g., include a nucleic acid that encodes, a subject chimeric ILT receptor) may be any cells useful in cell therapy, e.g., immune cells. The cells may be, for example, natural killer (NK) cells, iNK-T cells, NKT cells, T cells, B cells, macrophages, peripheral blood cells, hematopoietic progenitor cells, or bone marrow cells. In some embodiments, the modified cells are NK cells, natural killer T cells (NKT cells / NK-T cells), or T cells.

[0216] Cells which are genetically modified as disclosed herein (cells such as immune cells that express, e.g., include a nucleic acid that encodes, a subject chimeric ILT receptor) are useful for administering to subjects who can benefit from receiving them e.g. who can benefit from donor lymphocyte administration. These subjects will typically be humans, so the methods will typically be performed using human cells. Sources of cells

[0217] Cells to be genetically modified may be autologous, syngeneic, or allogeneic. Allogeneic cells can be derived from any healthy donor, and syngeneic cells from any healthy donor who is appropriately related to the intended recipient. The donor will generally be an adult (at least 18 years old) but children are also suitable as cell donors (e.g. see Styczynski 2018, Transfus Apher Sci 57(3):323-330).

[0218] The term “autologous” means a cell derived from the same individual to which it is later administered. The term “allogeneic” refers to HLA or MHC loci that are antigenically distinct between the host and donor cells. Thus, cells from the same species can be antigenically distinct. The term “syngeneic” refers to cells that have genotypes that are identical or closely related enough to allow tissue transplant, or are immunologically compatible. For example, identical twins or close relatives can be syngeneic.Atty Docket No.: NKLT-002WO

[0219] The cells may be blood cells. For example, the source of the cells may be, for example, umbilical cord blood, bone marrow, or peripheral blood, and they may be peripheral blood mononuclear cells (PBMCs). These include lymphocytes (e.g. T cells, B cells, NK cells) or monocytes. The term “peripheral blood” as used herein, refers to cellular components of blood (e.g., red blood cells, white blood cells and platelets), which are obtained or prepared from the circulating pool of blood and not sequestered within the lymphatic system, spleen, liver or bone marrow. Umbilical cord blood is distinct from peripheral blood and blood sequestered within the lymphatic system, spleen, liver or bone marrow, and it refers to blood that remains in the placenta and in the attached umbilical cord after child birth. Cord blood often contains stem cells including hematopoietic cells.

[0220] A process for obtaining and expanding NK cells from a human is described in Cho & Campana (2009) Korean J Lab Med 29:89-96, Somanchi et al. (2011) J Vis Exp 48:2540 and in Wang et al (2020) Blood Adv.4:1950. A suitable process for obtaining T cells from a human is described in the published protocol which accompanied Di Stasi et al. (2011) N Engl J Med 365:1673-83 (‘The Protocol’). NK cells

[0221] NK cells, also known as natural killer cells or large granular lymphocytes (LGL), are cytotoxic lymphocytes critical to the innate immune system. The role of NK cells is analogous to that of cytotoxic T cells in the vertebrate adaptive immune response. NK cells provide rapid responses to virus-infected cells and respond to tumor formation.

[0222] T cells rely on priming interactions between the T-cell receptor (TCR) and MHC- peptide complexes on target cells as a necessary first step in T-cell activation. As a result, T cells can recognize a single antigen, and tumor cells may avoid T-cell recognition through mutations that significantly reduce antigen presentation. In contrast, NK cells are capable of recognizing a multitude of transformed and infected cells without being dependent on the presentation of a single antigen. Therefore, treatment with NK cells can bypass some of the resistance mechanisms to T-cell based therapy.

[0223] As innate cells, NK cells can secrete proinflammatory chemokines and cytokines to recruit and activate the body’s adaptive immune system, consisting of T and B cells, creating a second wave of durable antitumor response. Furthermore, NK cells are not associated with certain toxicities associated with CAR-T cell therapy such as cytokine release syndrome and central nervous system toxicity.

[0224] NK cells can be useful as a source for antigen or receptor-based directed cell therapy because of their innate cytotoxic mechanisms. NK cells comprise approximately 10- 15% of the lymphocytes in peripheral blood of a typical donor and can be readilyAtty Docket No.: NKLT-002WO purified, expanded and virally transduced. In instances of loss of the target of a directed cell therapy, for example HLA-G, on a cells within a tumor, an activated NK cell has alternative inate mechanisms to direct cytotoxic function including NKG2D, p46, p44, p30, DNAM and CD16 CD4+ and CD8+ T cells

[0225] A subject composition can include CD4+ and CD8+ T cells. Whereas the ratio of CD4+ cells to CD8+ cells in a leukopak is typically above 2, in some embodiments the ratio of genetically-modified CD4+ cells to genetically-modified CD8+ cells in a composition of the disclosure is less than 2 e.g. less than 1.5. Ideally there are more genetically-modified CD8+ T cells than genetically-modified CD4+ T cells in the composition i.e. the ratio is less than 1 e.g. less than 0.9, less than 0.8, less than 0.7, less than 0.6, or preferably even less than 0.5. Thus an overall procedure starting from donor cells and producing genetically-modified T cells ideally enriches for CD8+ cells T cells relative to CD4+ T cells. Preferably at least 60% of the genetically-modified T cells are CD8+ T cells, and more preferably at least 65%. Within the population of genetically-modified CD3+ T cells a preferred range for CD8+ T cells is between 55- 75% e.g. from 63-73%. The proportions of CD8+ and CD4+ T cells can easily be assessed by flow cytometry, and methods for sorting and counting CD4+ and CD8+ T cells are conventional in the art. Memory T cell subsets (see Mahnke et al. (2013) Eur J Immunol 43:2797-809)

[0226] A population of genetically-modified T cells can include terminal effector memory T cells (defined as CD45RA+CD45RO-CCR7- cells; ‘TEMRA’), T-effector memory cells (defined as CD45RA-CD45RO+CCR7- cells; ‘EM’), T-central memory cells (defined as CD45RA-CD45RO+CCR7+ cells; ‘CM’), and naïve T cells (defined as CD45RA+CD45RO-CCR7+ cells). These cells can be assessed by flow cytometry using the CD45RA / RO and CCR7 markers. Labelled reagents which recognise CCR7 and which can distinguish between the CD45RA and CD45RO isoforms are readily available from commercial suppliers.

[0227] An average leukopak typically contains ~20% each of terminal effector and T-effector memory cells. An overall procedure from donor cells to genetically-modified T cells may enrich for terminal effector memory T cells relative to T-effector memory cells.

[0228] In some embodiments, less than 60% of the genetically-modified T cells are naïve T cells e.g. less than 58%, preferably less than 55%, and more preferably less than 50%. Within the population of genetically-modified CD3+ T cells a preferred range for naïve T cells is between 30-60%, more preferably 42-49%, and most preferably from 43-46%. This proportion of naïve T cells has been seen to correlate with favourableAtty Docket No.: NKLT-002WO outcomes in T cell recipients. Naïve EM cells can be assessed by flow cytometry using the CD45RA / RO and CCR7 markers.

[0229] Within a population of genetically modified T cells, in addition to TEMRA, EM and naïve T cells, the proportion of T-central memory cells is generally <10%.

[0230] In some embodiments, a population of genetically-modified T cells in a composition comprises about 10% to about 40% CD4+ T cells and about 60% to about 90% CD8+ T cells. The population of genetically-modified CD3+ T cells can comprise about 15 % to about 40% CD4+ T cells and about 60% to about 85% CD8+ T cells, more preferably about 20% to about 40% CD4+ T cells and about 60% to about 80% CD8+ T cells. Genetic modification of cells

[0231] Cells are genetically modified by transferring an expression construct (e.g., encoding a subject chimeric ILT receptor) into them. Such transfer may employ viral or non-viral methods of gene transfer. This section provides a discussion of methods and compositions of gene transfer.

[0232] An expression vector can be introduced into a cell by various means. The term "transfection" and “transduction” are interchangeable and refer to the process by which an exogenous nucleic acid sequence is introduced into a eukaryotic host cell. Transfection (or transduction) can be achieved by any one of a number of means including electroporation, microinjection, gene gun delivery, retroviral infection, lipofection, superfection and the like.

[0233] Any appropriate method may be used to transfect or transform the cells (e.g. the T cells, NKT cells, or NK cells). Certain non-limiting examples are presented herein. In some embodiments, the viral vector is an SFG-based viral vector, as discussed in Tey et al. (2007) Biol Blood Marrow Transpl 13:913-24 and by Di Stasi et al., (2011) N Engl J Med 365:1673-83.

[0234] The cells can be transduced using a viral vector encoding polypeptides described herein. Suitable transduction techniques may involve fibronectin fragment CH-296. As an alternative to transduction using a viral vector, cells can be transfected with any suitable method known in the art such as with DNA encoding the relevant polypeptides e.g. using calcium phosphate, cationic polymers (such as PEI), magnetic beads, electroporation and commercial lipid-based reagents such as Lipofectamine™ and Fugene™. One result of the transduction / transfection step is that various donor cells will now be genetically-modified cells which can express the CIR and any other desired polypeptides.

[0235] In some embodiments, the viral vector used for transduction is the retroviral vector disclosed by Tey et al. (2007) Biol Blood Marrow Transpl 13:913-24 and by Di Stasi etAtty Docket No.: NKLT-002WO al. (2011) supra. This vector is based on Gibbon ape leukemia virus (Gal-V) pseudotyped retrovirus encoding an iCasp9 suicide switch and a ΔCD19 cell surface transgene marker (see further below – and see SEQ ID NOs: 12-13). It can be produced in the PG13 packaging cell line, as discussed by Tey et al. (2007) supra. Other viral vectors encoding the desired proteins can also be used. In some embodiments, retroviral vectors that can provide a high copy number of proviral integrants per cell are used for transduction.

[0236] After transduction / transfection, cells can be separated from transduction / transfection materials and cultured again, to permit the genetically-modified cells to expand. Cells can be expanded so that a desired minimum number of genetically-modified cells is achieved.

[0237] Genetically-modified cells can then be selected from the population of cells which has been obtained. The CIR may not be suitable for positive selection of desired cells, so in some embodiments, the genetically-modified cells should express a cell surface transgene marker of interest (see below). Cells which express this surface marker can be selected e.g. using immunomagnetic techniques. For instance, paramagnetic beads conjugated to monoclonal antibodies which recognise the cell surface transgene marker of interest can be used, for example, using a CliniMACS system (available from Miltenyi Biotec).

[0238] In an alternative procedure, genetically-modified cells are selected after a step of transduction, are cultured, and are then fed. Thus, the order of transduction, feeding, and selection can be varied.

[0239] The result of these procedures is a composition containing cells which have been genetically modified, and which can thus express the Chimeric ILT receptor (and any other desired polypeptides e.g. a costimulatory polypeptide, a suicide switch, a cell surface transgene marker, etc.). These genetically-modified cells can be administered to a recipient, but they might first be preserved (e.g. cryopreserved), optionally after further expansion, before being administered. Selectable Markers

[0240] Cells may be modified to express polypeptides whose expression can be identified in vitro or in vivo, thereby permitting selection of genetically-modified cells e.g. to separate them from unmodified cells. Such markers confer an identifiable change to the cell, permitting easy identification of cells containing the desired expression construct.

[0241] Inclusion of a drug selection marker aids in cloning and in the selection of transformants. For example, genes that confer resistance to neomycin, puromycin, hygromycin, DHFR, GPT, zeocin and histidinol are useful selectable markers.Atty Docket No.: NKLT-002WO Alternatively, enzymes such as Herpes Simplex Virus thymidine kinase (tk) are employed.

[0242] Immunologic surface markers containing the extracellular, non-signaling domains or various proteins (e.g. CD34, CD19, LNGFR) also can be employed, permitting a straightforward method for magnetic or fluorescence antibody-mediated sorting. These markers can be detected e.g. using a labelled antibody which binds to the protein.

[0243] The selectable marker employed is not believed to be important, so long as it is capable of being expressed simultaneously with the nucleic acid encoding a desired gene product e.g. a subject CIR. Moreover, the marker should ideally be a polypeptide which is not expressed by the initial (donor) cells, although difference in expression levels can be used in situations where the marker is indeed endogenous to the initial cells.

[0244] Ideally the marker is based on a human proteins as this minimises the risk that cells expressing the marker will be recognised as foreign by a human subject’s immune system (e.g. after they are administered therapeutically). For instance, where T cells are the desired type of cell, human CD proteins which are not naturally expressed by T cells can be used for this purpose.

[0245] The genetically modified cells provided herein may express a cell surface transgene marker, present on an expression vector that expresses a subject CIR, and / or, in some embodiments, present on an expression vector that encodes a protein other than the CIR, such as, for example a CAR, a pro-apoptotic polypeptide safety switch, or a costimulatory polypeptide.

[0246] In one embodiment, the cell surface transgene marker is a truncated CD19 (ΔCD19) polypeptide (Di Stasi et al. (2011) supra) that comprises a human CD19 truncated at amino acid 333 to remove most of the intracytoplasmic domain (see, e.g., SEQ ID NO: 12 (nucleotides) and SEQ ID NO: 13 (protein)). The extracellular CD19 domain can still be recognised (e.g. in flow cytometry, FACS or MACS) but the potential to trigger intracellular signalling is minimised. CD19 is normally expressed by B cells, rather than by T cells or NK cells, so selection of CD19+ cells permits genetically-modified cells (e.g. T cells, NK cells or NKT cells) to be separated from unmodified cells.

[0247] Another useful marker is CD34, which has a 16 amino acid minimal epitope that is useful as a marker.

[0248] By encoding a desired protein at the 5' end of an encoding gene, and a marker at the 3' end, the risk of selecting cells which do not have the desired polypeptide (e.g. due to premature termination of translation) is minimised. In this manner, expression of the marker and of the desired polypeptide run in parallel.Atty Docket No.: NKLT-002WO 7. Engineering expression constructs

[0249] Provided are nucleic acids that include a nucleotide sequence encoding a subject chimeric ILT receptor (CIR) (and optionally, other desired polypeptides such as chimeric antigen receptors, signaling polypeptides, safety switches, IL-15, etc.). In some cases, such a nucleic acid is an expression construct. Expression constructs for expressing the Chimeric ILT Receptor (and optionally, other desired polypeptides such as chimeric antigen receptors, signaling polypeptides, safety switches, IL-15, etc.) are provided herein. In some embodiments, one or more polypeptides is said to be “operably linked” to a promoter, which indicates that the promoter sequence is functionally linked to a second sequence, wherein the promoter sequence is in the correct location and orientation in relation to that second sequence to control RNA polymerase initiation and transcription of the DNA corresponding to the second sequence, whereby the resulting transcript encodes a polypeptide of interest.

[0250] A "promoter" is a DNA sequence recognized by the synthetic machinery of the cell, or introduced synthetic machinery, required to initiate the specific transcription of a gene. In some embodiments, the promoter is a developmentally regulated promoter i.e. a promoter that acts as the initial binding site for RNA polymerase to transcribe a gene which is expressed under certain conditions that are controlled, initiated by or influenced by a developmental program or pathway.

[0251] The term “expression construct” is any type of genetic construct containing a nucleic acid coding for gene products in which part or all of the nucleic acid encoding sequence is capable of being transcribed. The transcript can be translated into a protein, but it need not be. In certain embodiments, expression includes both transcription of a gene and translation of mRNA into a gene product. In other embodiments, expression only includes transcription of the nucleic acid encoding genes of interest. Expression vectors can contain a variety of control sequences, which refer to nucleic acid sequences necessary for the transcription and possibly translation of an operatively linked coding sequence in a particular host organism.

[0252] A“vector” is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, “vector construct,” “expression vector,” “expression construct,” and “gene transfer vector,” mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0253] In certain examples, a polynucleotide coding for the CIR is included in the same vector, such as, for example, a viral or plasmid vector, as a polynucleotide coding for a second polypeptide. This second polypeptide may be, for example (and as described elsewhere herein), a downregulator of endogenous proteins, a blocking antibody orAtty Docket No.: NKLT-002WO scFv for inhibitory receptors, a signaling polypeptide, an inducible suicide switch, or a marker polypeptide. In other examples added expressed transcription products may not encode proteins, but instead generate short hairpin RNA products designed to remove the expression of certain endogenous RNAs that encode unwanted proteins in the cell product.

[0254] A construct may be designed with one promoter operably linked to a nucleic acid comprising a polynucleotide coding for a fusion protein of the polypeptides, linked by a linker polypeptide (e.g. a cleavable linker polypeptide, such as a 2A polypeptide). In this example, the first and second polypeptides are produced during a single translation event, but they may then be separated. In other examples, the two polypeptides may be expressed separately from the same vector, where each nucleic acid comprising a polynucleotide coding for one of the polypeptides is operably linked to a separate promoter. In yet other examples, one promoter may be operably linked to the two polynucleotides, directing the production of two separate RNA transcripts, and thus two polypeptides; in one example, the promoter may be bi-directional, and the coding regions may be in opposite directions 5’-3’. Therefore, expression constructs discussed herein may comprise at least one, or at least two promoters.

[0255] In yet other examples, two polypeptides (such as, for example, the CIR and a marker protein) may be expressed in a cell using two separate vectors. The cells may be co- transfected or co-transformed with the vectors, or the vectors may be introduced to the cells at different times.

[0256] Any combinations of these approaches may be used, in order to achieve expression of desired polypeptides in a genetically modified cell.

[0257] In some embodiments, a nucleic acid construct is contained within a viral vector. In certain embodiments, the viral vector is a retroviral vector. In certain embodiments, the viral vector is an adenoviral vector or a lentiviral vector. It is understood that in some embodiments, a cell is contacted with the viral vector ex vivo, and in some embodiments, the cell is contacted with the viral vector in vivo. Thus, an expression construct may be inserted into a vector, for example a viral vector or plasmid. The steps of the methods provided may be performed using any suitable method; these methods include, without limitation, methods of transducing, transforming, or otherwise providing nucleic acid to the cell, described herein.

[0258] The particular promoter employed to control the expression of a polynucleotide sequence of interest is generally not of particular importance, so long as it is capable of directing the expression of the polynucleotide in a desired cell. Thus, where a human cell is targeted the polynucleotide sequence-coding region may, for example, be placed adjacent to and under the control of a promoter that is capable of being expressed in a human cell. Generally speaking, such a promoter might include eitherAtty Docket No.: NKLT-002WO a human or viral promoter. Promoters may be selected that are appropriate for the vector used to express the CIRs and other polypeptides provided herein.

[0259] In various embodiments, where, for example, the expression vector is a retrovirus, an example of an appropriate promoter is the Murine Moloney leukemia virus promoter. In other embodiments, the promoter may be, for example, the CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, β2-microglobulin, ribosomal protein 31, phosphoglycerate kinase, EF1α, ^- actin, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase can be used to obtain high-level expression of the coding sequence of interest. The use of other viral or mammalian cellular promoters which are well known in the art to achieve expression of a coding sequence of interest is contemplated as well, provided that the levels of expression are sufficient for a given purpose. By employing a promoter with well-known properties, the level and pattern of expression of the polypeptide of interest following transfection or transformation can be optimized.

[0260] In other embodiments the expression vector is a transposon such that the genetic elements encoding a CIR and associated marker proteins, coactivation proteins or inhibitors of endogenous factors or the tumor microenvironment a carried on a plasmid vector carrying elements recognized by a transiently coexpressed transposase. The action of the transposase is to catalyse the fusion of the transgenes carried between repeated elements recognized by the transposase with the cells genome. Examples of transposon systems that can be used in these embodiments are the Sleeping Beauty system and the Piggyback system. Promoter elements carried within the transposon direct transgene expression. The promoter may be, for example the CMV immediate early gene promoter, the SV40 early promoter, the Rous sarcoma virus long terminal repeat, β2-microglobulin, ribosomal protein 31, phosphoglycerate kinase, EF1α, ß-actin, rat insulin promoter and glyceraldehyde-3-phosphate dehydrogenase. The methods for introduction of the plasmids for the transposon and transposase to cells is transfection rather than viral transduction.

[0261] Promoters, and other regulatory elements, are selected such that they are functional in the desired cells or tissue. In addition, this list of promoters should not be construed to be exhaustive or limiting; other promoters that are used in conjunction with the promoters and methods disclosed herein.

[0262] It is understood that the order of the polynucleotides may vary and may be tested to determine the suitability of the construct for any particular method, thus, the nucleic acid may include the polynucleotides in varying orders, which also take into account a variation in the order of components.

[0263] In some embodiments, the cell is transfected or transduced with the nucleic acid that encodes two of the polynucleotides, and the cell also comprises a nucleic acidAtty Docket No.: NKLT-002WO comprising a polynucleotide coding for a third polypeptide and / or the cell also comprises a nucleic acid comprising a polynucleotide coding for the fourth polypeptide. In some embodiments, the cell is transfected or transduced with the nucleic acid that encodes three of the polynucleotides, and the cell also comprises a nucleic acid comprising a polynucleotide coding for the fourth polypeptide. For example, a cell may comprise a nucleic acid comprising the first, second and third polynucleotides, and the cell may also comprise a nucleic acid comprising a polynucleotide coding for a chimeric Caspase-9 polypeptide. Also, a cell may comprise a nucleic acid comprising the first, second and fourth polynucleotides, and the cell may also comprise a nucleic acid comprising a polynucleotide coding for a Chimeric ILT receptor, an scFv modulator of natural ILT2 function or Interleukin-15. 8. Methods for treating a disease

[0264] Also provided are methods of treatment or prevention of a disease where administration of cells (e.g., cells expressing a subject CIR) by, for example, infusion, may be beneficial. The cells may, for example, be used in regeneration, for example, to replace the function of diseased cells. The genetically-modified cells described herein may be used for cell therapy.

[0265] As used herein, the terms "treatment," "treating," and the like, refer to obtaining a desired pharmacologic and / or physiologic effect. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and / or may be therapeutic in terms of a partial or complete cure for a disease and / or adverse effect attributable to the disease. "Treatment," as used herein, covers any treatment of a disease in a mammal, particularly in a human, and includes: (a) preventing the disease from occurring in a subject predisposed to the disease or at risk of acquiring the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, i.e., arresting its development; and (c) relieving the disease, i.e., causing regression of the disease.

[0266] The terms "individual," "subject," and "patient" are used interchangeably herein, and refer to a mammal, including, but not limited to, human and non-human primates, including simians and humans; mammalian sport animals (e.g., horses); mammalian farm animals (e.g., sheep, goats, etc.); mammalian pets (dogs, cats, etc.); and rodents (e.g., mice, rats, etc.).

[0267] An "effective amount" or "sufficient amount" refers to an amount (e.g., an effective amount of cells) providing, in single or multiple doses, alone or in combination, with one or more other compositions (therapeutic agents such as a drug), treatments, protocols, or therapeutic regimens, a detectable response of any duration of time (long or short term), an expected or desired outcome in or a benefit to a subject of anyAtty Docket No.: NKLT-002WO measurable or detectable degree or for any duration of time (e.g., for minutes, hours, days, months, years, or cured).

[0268] The doses of an "effective amount" or "sufficient amount" for treatment (e.g., to ameliorate or to provide a therapeutic benefit or improvement) typically are effective to provide a response to one, multiple or all adverse symptoms, consequences or complications of a disease, one or more adverse symptoms, disorders, illnesses, pathologies, or complications, for example, caused by or associated with the disease, to a measurable extent, although decreasing, reducing, inhibiting, suppressing, limiting or controlling progression or worsening of the disease is also a satisfactory outcome.

[0269] Genetically-modified cells provided herein (i.e., cells expressing a subject CIR) can be used in methods for treating human subjects in need thereof, and can be used to prepare medicaments for treating such subjects. The cells will usually be delivered to the recipient subject by infusion.

[0270] The genetically-modified cells may be T cells, iNKT cells, macrophage or NK cells. A typical dose of T or NK cells for therapy in a subject is between 105-107cells / kg.

[0271] In general terms, genetically modified T and NK cells of the disclosure can be used in the same manner as known donor leukocyte infusion (DLI), but they have the added benefit of the CIR.

[0272] A recipient may undergo lymphodepletive conditioning prior to receiving the genetically-modified lymphocytes (and prior to receiving an allograft). Thus the recipient’s own α / β T cells (and B cells) can be depleted prior to receiving the genetically-modified T cells or NK cells.

[0273] The recipient may have a hematological cancer (such as a treatment-refractory hematological cancer) or an inherited blood disorder. For instance, the recipient may have acute lymphoblastic leukemia (ALL), acute myeloid leukemia (AML), severe combined immune-deficiency (SCID), Wiskott-Aldrich syndrome (WA), Fanconi Anemia, chronic myelogenous leukemia (CML), non-Hodgkin lymphoma (NHL), Hodgkin lymphoma (HL), or multiple myeloma.

[0274] The recipient of CIR-expressing T cells or NK cells may have non-hematological cancer expressing HLA-G. For instance, the recipient may have renal cell cancer (RCC), non-small cell lung cancer (NSCLC), colorectal cancer (CRC), breast cancer, neuroblastoma, hepatocellular cancer (HCC), ovarian cancer, endometrial cancer or prostate cancer.

[0275] Several cell-types can also be used in therapy, and include any cell administered to a patient for a desired therapeutic result. The therapeutic cells may be, for example, immune cells such as, for example, T cells, natural killer cells (NK cells), NK-T cells, B cells, tumor infiltrating lymphocytes, or macrophages, or a combination thereof; the therapeutic cells may be, for example, peripheral blood cells, hematopoietic progenitorAtty Docket No.: NKLT-002WO cells, bone marrow cells, or tumor cells. To further improve the tumor microenvironment to be more immunogenic, the treatment may be combined with one or more adjuvants (e.g., IL-12, checkpoint inhibitors, IDO inhibitors, etc.). In some embodiments, the cells may be delivered to treat a solid tumor, such as, for example, delivery of the cells to a tumor bed. In some embodiments, the cells may be delivered to treat a liquid tumor, such as, for example, delivery of the cells to treat a leukemia such as AML.

[0276] Also provided in some embodiments are nucleic acids which may be administered to a subject, thereby transforming or transducing target cells in vivo to form the genetically- modified cells in situ.

[0277] An effective amount of genetically-modified cells is administered. To determine if an effective amount of ligand or modified cells is administered, any means of assaying or measuring the number of target cells, or amount of target antigen, or size of a tumor may be used to determine whether the number of target cells, amount of target antigen or size of a tumor has increased, decreased, or remained the same. Samples, images, or other means of measurement taken before administration of the modified cells or ligand may be used to compare with samples, images, or other means of measurement taken after administration of the modified cells or ligand. Thus, for example, to determine whether the amount or concentration of cells expressing a target antigen has increased, decreased, or remained the same, a first sample may be obtained from a subject before administration of the ligand or modified cells, and a second sample may be obtained from a subject after administration of the ligand or modified cells. The amount or concentration of cells expressing the target antigen in the first sample may be compared with the amount or concentration of cells expressing the target antigen in the second sample, in order to determine whether the amount or concentration of cells expressing the target antigen has increased, decreased, or remained the same following administration of the ligand or modified cell.

[0278] The effective amount for any particular application can vary depending on such factors as the disease or condition being treated, the particular composition being administered, the size of the subject, and / or the severity of the disease or condition. One can empirically determine the effective amount of a particular composition presented herein.

[0279] In order to increase the effectiveness of the modified cells presented herein, it may be desirable to combine these compositions and methods with an agent effective in the treatment of the disease.

[0280] The administration of the pharmaceutical composition may precede, be concurrent with and / or follow the other agent(s) by intervals ranging from minutes to weeks. InAtty Docket No.: NKLT-002WO embodiments where the pharmaceutical composition and other agent(s) are applied separately to a cell, tissue or organism, one would generally ensure that a significant period of time did not expire between the times of each delivery, such that the pharmaceutical composition and agent(s) would still be able to exert an advantageously combined effect on the cell, tissue or organism. For example, in such instances, it is contemplated that one may contact the cell, tissue or organism with two, three, four or more modalities substantially simultaneously (i.e., within less than about a minute) with the pharmaceutical composition. In other aspects, one or more agents may be administered from substantially simultaneously, about 1 minute, to about 24 hours to about 7 days to about 1 to about 8 weeks or more, and any range derivable therein, prior to and / or after administering the expression vector. Yet further, various combination regimens of the pharmaceutical composition presented herein, and one or more agents may be employed.

[0281] Diseases that may be treated or prevented include diseases caused by viruses, bacteria, yeast, parasites, protozoa, cancer cells and the like. Exemplary diseases that can be treated and / or prevented include, but are not limited, to infections of viral etiology such as HIV, influenza, herpes, viral hepatitis, Epstein Barr, polio, viral encephalitis, measles, chicken pox, papillomavirus etc.; or infections of bacterial etiology such as pneumonia, tuberculosis, syphilis, etc.; or infections of parasitic etiology such as malaria, trypanosomiasis, leishmaniasis, trichomoniasis, amoebiasis, etc. Preneoplastic or hyperplastic states which may be treated or prevented using the pharmaceutical composition (transduced cells, expression vector, expression construct, etc.) include but are not limited to preneoplastic or hyperplastic states such as colon polyps, Crohn's disease, ulcerative colitis, breast lesions and the like.

[0282] Cancers, including solid tumors and / or liquid tumors, which may be treated using the cells include, but are not limited to primary or metastatic melanoma, adenocarcinoma, squamous cell carcinoma, adenosquamous cell carcinoma, thymoma, lymphoma, sarcoma, lung cancer, liver cancer, non-Hodgkin’s lymphoma, Hodgkin’s lymphoma, leukemias (e.g., Chronic lymphocytic leukemia, Acute myeloid leukemia, Chronic myeloid leukemia, Acute lymphocytic leukemia), uterine cancer, breast cancer, prostate cancer, ovarian cancer, pancreatic cancer, colon cancer, multiple myeloma, neuroblastoma, NPC, bladder cancer, cervical cancer and the like.

[0283] Other hyperproliferative diseases that may be treated using the therapeutic cells and other therapeutic cell activation system presented herein include, but are not limited to rheumatoid arthritis, inflammatory bowel disease, osteoarthritis, leiomyomas, adenomas, lipomas, hemangiomas, fibromas, vascular occlusion, restenosis, atherosclerosis, pre-neoplastic lesions (such as adenomatous hyperplasia andAtty Docket No.: NKLT-002WO prostatic intraepithelial neoplasia), carcinoma in situ, oral hairy leukoplakia, or psoriasis.

[0284] Solid tumors from any tissue or organ may be treated using the present methods, including, for example, any tumor expressing a target antigen, for example, HLA-G, in the vasculature, for example, solid tumors present in, for example, lungs, bone, liver, prostate, or brain, and also, for example, in breast, ovary, bowel, testes, colon, pancreas, kidney, bladder, neuroendocrine system, soft tissue, boney mass, and lymphatic system. Other solid tumors that may be treated include, for example, glioblastoma, and malignant multiple myeloma.

[0285] Liquid tumors (leukemia and lymphoma, such as, e.g., Chronic lymphocytic leukemia, Acute myeloid leukemia, Chronic myeloid leukemia, and Acute lymphocytic leukemia) may be treated using the present methods, including, for example, any cancer (e.g., any leukemia or lymphoma) in which the cancer cells express a target antigen, for example, HLA-G.

[0286] Subjects may be given a zinc supplement to ensure that any zinc-dependent factors contained within a CIR or the cofactors expressed in a cell therapy product including a CIR have an adequate source of this ion to permit their full activity.

[0287] Also provided are methods of making the cells of the present disclosure. In some embodiments, such methods include transfecting or transducing cells with a nucleic acid or expression vector of the present disclosure (e.g., one encoding for a subject CIR). The term “transfection” is used to refer to the uptake of foreign DNA by a cell. A cell has been “transfected” when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rdedition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2ndedition, McGraw- Hill, and Chu et al. (1981 ) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material.

[0288] In some embodiments, a cell of the present disclosure is produced by transducing the cell with a viral vector encoding a CIR. In certain aspects, the polypeptide includes a CIR and the cell is a T cell, such that provided are methods of producing a CIR T cell. In some embodiments, such methods include activating a population of T cells (e.g., T cells obtained from an individual to which a CIR T cell therapy will be administered), stimulating the population of T cells to proliferate, and transducing the T cell with a viral vector encoding the polypeptide including the CIR. In some embodiments, an immune cell (e.g., T cells, NK cells, macrophages) is transduced with a retroviralAtty Docket No.: NKLT-002WO vector, e.g., a gamma retroviral vector, or lentiviral vector, or AAV encoding a CIR. In certain aspects, the immune cell T cells are transduced with a lentiviral vector encoding the polypeptide. In certain aspects, the polypeptide includes a CIR and the cell is an NK cell, such that provided are methods of producing a CIR NK cell (e.g., by using a viral vector such as an AAV, lentiviral, or retroviral vector). 9. General

[0289] The term “comprising” encompasses “including” as well as “consisting” e.g. a composition “comprising” X may consist exclusively of X or may include something additional e.g. X + Y.

[0290] The term “about” in relation to a numerical value x is optional and means, for example, x+10%.

[0291] The word “substantially” does not exclude “completely” e.g. a composition which is “substantially free” from Y may be completely free from Y. Where necessary, the word “substantially” may be omitted from the definition.

[0292] The term “between” with reference to two values includes those two values e.g. the range “between” 10 mg and 20 mg encompasses inter alia 10, 15, and 20 mg.

[0293] Unless specifically stated, a method comprising a step of mixing two or more components does not require any specific order of mixing. Thus components can be mixed in any order. Where there are three components then two components can be combined with each other, and then the combination may be combined with the third component, etc.

[0294] The various steps of methods may be carried out at the same or different times, in the same or different geographical locations, e.g. countries, and by the same or different people or entities.

[0295] The extent of similarity between two sequences can be based on percent sequence identity. "Sequence identity" herein means the extent to which two nucleotide or amino acid sequences are invariant. "Sequence alignment" means the process of lining up two or more sequences to achieve maximal levels of identity for the purpose of assessing the degree of similarity. Numerous methods for aligning sequences and assessing similarity / identity are known in the art such as, for example, the Cluster Method, wherein similarity is based on the MEGALIGN algorithm, as well as BLASTN, BLASTP, and FASTA. When using any of these programs, the settings may be selected that result in the highest sequence similarity. 10. Examples of Particular Nucleic Acid and Amino Acid Sequences

[0296] The following sections and tables include examples of polypeptide and nucleotide sequences coding for chimeric signaling polypeptides. It is understood that sequencesAtty Docket No.: NKLT-002WO of individual polypeptides provided in these examples, such as, for example, the truncated ILT4 D1-D2 polypeptides, co-stimulatory polypeptide cytoplasmic signaling regions, ITAM-containing cytotoxicity regions, IL-15 or safety switches may be used to construct other expression vectors that encode chimeric signaling polypeptides of the present embodiments. Sequences used in the experimental examples below include the following:

[0297] ILT4.CD3ζ Fragment SEQID #NucleotideSEQ ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTC T S IR T S A S P S Y L L GL L C Q L K A HAtty Docket No.: NKLT-002WO CACCTACGACGCCCTTCACATGCAAGCTCT TCCACCTCGT Linker 66 CCGCGGGGCAGTGGA 67 PRGSG P L FS K D Q IL E IFragmentSEQSEQ ID #NucleotideID #PeptideT S IR T S A S P S Y L L GL L CAtty Docket No.: NKLT-002WO Linker GGATCC GS I P L FS K D Q IL E IFragmentSEQSE ID #Nucleotide Q ID #PeptideT S IR T S A S P S Y L L GL L CAtty Docket No.: NKLT-002WO GGCACCTGTGGAGTCCTTCTGCTCAGCCTG GTTATTACTCTGTACTGTAATCACCGGAATC GCCGCCGCGTTTGTAAGTGTCCCAGG T Q P L FS K D Q IL E ISEQSEQ FragmentID #NucleotideID #PeptideT S IR T S A S P S Y LAtty Docket No.: NKLT-002WO Linker CAATTG QL CCCGCCCCAAGACCCCCCACACCTGCGCC GACCATTGCTTCTCAACCCCTGAGTTTGAGA SL GL L C I Q L K A H P L FS K D Q IL E I. . FragmentSEQNucleoti SEQ ID #deID #PeptideT S IR TAtty Docket No.: NKLT-002WO GCTCTCTTGAGGCCCAAGAGTACCGGCTGT GRYGCQYYSRARWSELS ACCGAGAGAAGAAGTCCGCCTCCTGGATCA DPLVLVMTGAYPKPTLSA CTAGGATCAGACCGGAGCTTGTGAAGAACG QPSPVVTSGGRVTLQCES P S Y L L GL L C T Q Q L K A H P L FS K D Q IL E IAtty Docket No.: NKLT-002WO TGCAAGTATTCATGATACAGTAGAAAATCTG ATCATCCTAGCAAACAACAGTTTGTCTTCTA ATGGGAATGTAACAGAATCTGGATGCAAAGFragmentSEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTC T S IR T S A S P S Y L L GL L C P E Q L KAtty Docket No.: NKLT-002WO AGGAGTACGATGTTTTGGACAAGAGACGTG NPQEGLYNELQKDKMAEA GCCGGGACCCTGAGATGGGGGGAAAGCCG YSEIGMKGERRRGKGHD AGAAGGAAGAACCCTCAGGAAGGCCTGTAC GLYQGLSTATKDTYDALH P L FS K D Q IL E IFragmentSEQID #Nucleotide SEQ ID #PeptideT S IR T S A S P S Y L L GL LAtty Docket No.: NKLT-002WO GGCCGTGCATACAAGAGGACTCGATTTCGC LLSLVITLYCNHRNRRRVC TTGCGACATCTATATCTGGGCACCTCTCGCT KCPR GGCACCTGTGGAGTCCTTCTGCTCAGCCTG V Q G Q L K A H P L FS K D Q IL E I

[0030] . - . 0 Fragment SEQSEQ IDNucleotideIDPeptideT SAtty Docket No.: NKLT-002WO TACAGACGGGCACAATTCCTAAGCCCACCC VITQGSPVTLSCQGSLEA TCTGGGCTGAGCCCGACTCAGTGATAACGC QEYRLYREKKSASWITRIR AGGGTTCTCCAGTTACCCTCTCTTGCCAAG PELVKNGQFHIPSITWEHT S A S P S Y L L GL L C P E I P L FS K D Q IL E IAtty Docket No.: NKLT-002WO AATGTGAGGAACTGGAGGAAAAAAATATTAA AGAATTTTTGCAGAGTTTTGTACATATTGTC CAAATGTTCATCAACACTTCT

[0305] ILT4.4-1BB.DAP10.CD3ζFragmentSEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTC T S IR T S A S P S Y L L GL L C P E IAtty Docket No.: NKLT-002WO Linker GTCGAC VD AGAGTGAAGTTCAGCAGGAGCGCAGACGC CCCCGCGTACCAGCAGGGCCAGAACCAGC Q L K A H P L FS K D Q IL E IFragmentSEQSEQ ID #NucleotideID #PeptideT S IR T S A S P S Y LAtty Docket No.: NKLT-002WO Linker CAATTG QL CCCGCCCCAAGACCCCCCACACCTGCGCC GACCATTGCTTCTCAACCCCTGAGTTTGAGA SL GL L C P E T Q P L FS K D Q IL E I4.4-1 . 12.C 3ζFragmentSEQNucleoti SEQ ID #deID #PeptideT S IR T SAtty Docket No.: NKLT-002WO ACCGAGAGAAGAAGTCCGCCTCCTGGATCA DPLVLVMTGAYPKPTLSA CTAGGATCAGACCGGAGCTTGTGAAGAACG QPSPVVTSGGRVTLQCES GCCAATTTCATATCCCGAGCATCACATGGG QVAFGGFILCKEGEDEHP S Y L L GL L C P E T Q Q L K A H PAtty Docket No.: NKLT-002WO ATGAGAATTTCCAAACCACATTTGAGAAGTA TTTCCATCCAGTGCTACTTGTGTTTACTTCTA AACAGTCATTTTCTAACTGAAGCTGGCATTC L FS K D Q IL E IFragmentSEQSEQ ID #NucleotideID #PeptideT S IR T S A S P S Y L L GL L CAtty Docket No.: NKLT-002WO ATGGCTGCAGGAGGTCCCGGCGCGGGGTC TGCGGCCCCGGTCTCCTCCACATCCTCCCT TCCCCTGGCTGCTCTCAACATGCGAGTGCG T L E T R P E L D Q L K A H P L FS K D Q IL E IAtty Docket No.: NKLT-002WO ATGACCCCTATAGTTACAGTCTTGATCTGTC TCGGGCTTAGCTTGGGACCTCGAACGCATG TACAGACGGGCACAATTCCTAAGCCCACCC T S IR T S A S P S Y L L GL L C T L E T R P E L DAtty Docket No.: NKLT-002WO AAAAGAGGAAGAAAAAAGTTGCTGTATATAT TTAAACAACCATTTATGAGACCAGTGCAAAC KRGRKKLLYIFKQPFMRP 4-1BB CACCCAAGAAGAAGACGGATGTTCATGCAG VQTTQEEDGCSCRFPEEE Q L K A H P L FS K D Q IL E I. y .FragmentSEQNucleot SEQ ID #ideID #PeptideT S IR T S A S P S Y LAtty Docket No.: NKLT-002WO TCTGCAATGCGAGTCCCAAGTGGCATTCGG TGGGTTCATACTCTGTAAAGAGGGCGAGGA CGAACATCCTCAGTGCCTTAATAGCCAACC L GL L C T L E T R P E L D T Q P L FS K D Q IL E IAtty Docket No.: NKLT-002WO AGTTACAAGTTATTTCACTTGAGTCCGGAGA TGCAAGTATTCATGATACAGTAGAAAATCTG ATCATCCTAGCAAACAACAGTTTGTCTTCTAFragmentSEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTC T S IR T S A S P S Y L L GL L C T L E T R P E L DAtty Docket No.: NKLT-002WO GCAGCAGCAGGAGGAGGCTGAGAAGCCTT TACAGGTGGCCGCTGTAGACAGCAGTGTCC CACGGACAGCAGAGCTGGCGGGCATCACC T Q Q L K A H P L FS K D Q IL E I. y . - . Fragment SEQ NucleotSEQ ID #ideID #PeptideT S IR T SAtty Docket No.: NKLT-002WO ACCGAGAGAAGAAGTCCGCCTCCTGGATCA DPLVLVMTGAYPKPTLSA CTAGGATCAGACCGGAGCTTGTGAAGAACG QPSPVVTSGGRVTLQCES GCCAATTTCATATCCCGAGCATCACATGGG QVAFGGFILCKEGEDEHP S Y L L GL L C T L E T R P E L D P EAtty Docket No.: NKLT-002WO AGAGTGAAGTTCAGCAGGAGCGCAGACGC CCCCGCGTACCAGCAGGGCCAGAACCAGC TCTATAACGAGCTCAATCTAGGACGAAGAG RVKF R ADAPAY Q L K A H P L FS K D Q IL E IFragmentSEQSE ID #Nucleotide Q ID #PeptideT S IR T S A S P S Y LAtty Docket No.: NKLT-002WO CCCGCCCCAAGACCCCCCACACCTGCGCC GACCATTGCTTCTCAACCCCTGAGTTTGAGA CCCGAGGCCTGCCGGCCAGCTGCCGGCGG PAPRPPTPAPTIASQPLSL RPEA RPAA AVHTRGL L C T L E T R P E L D V Q G Q L K A H PAtty Docket No.: NKLT-002WO ATGAGAATTTCCAAACCACATTTGAGAAGTA TTTCCATCCAGTGCTACTTGTGTTTACTTCTA AACAGTCATTTTCTAACTGAAGCTGGCATTC L FS K D Q IL E IFragmentSEQSEQ ID #NucleotideID #PeptideT S IR T S A S P S Y L L GL L CAtty Docket No.: NKLT-002WO ATGGCTGCAGGAGGTCCCGGCGCGGGGTC TGCGGCCCCGGTCTCCTCCACATCCTCCCT TCCCCTGGCTGCTCTCAACATGCGAGTGCG T L E T R P E L D P V E Q L K A H P L FS K D Q IL E IAtty Docket No.: NKLT-002WO AATGTGAGGAACTGGAGGAAAAAAATATTAA AGAATTTTTGCAGAGTTTTGTACATATTGTC CAAATGTTCATCAACACTTCTSESEQ Fragment QID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTC T S IR T S A S P S Y L L GL L C N F V Y V P K A EAtty Docket No.: NKLT-002WO GCCTGGTAGAGACGAACCGGAAGTCTTGCC TGTTCTTTCCGAGTCT Q L K A H P L FS K D Q IL E IFragmentSEQN SEQ ID #ucleotideID #PeptideT S IR T S A S P S Y LAtty Docket No.: NKLT-002WO TCTGCAATGCGAGTCCCAAGTGGCATTCGG TGGGTTCATACTCTGTAAAGAGGGCGAGGA CGAACATCCTCAGTGCCTTAATAGCCAACC L GL L C R Y E K S F K Y L Q L K A H P L FS K DAtty Docket No.: NKLT-002WO GCTTCCTAAAACAGAAGCCAACTGGGTGAA VHPSCKVTAMKCFLLELQ TGTAATAAGTGATTTGAAAAAAATTGAAGAC VISLESGDASIHDTVENLIIL CTTATTCAATCTATGCACATTGATGCTACTTT ANNSLSSNGNVTESGCKE IFragmentSEQNu SEQ ID #cleotideID #PeptideAT A TATA TTA A T TT AT T T T S IR T S A S P S Y L L GL L C E K K SI R RAtty Docket No.: NKLT-002WO TATGCAAAAGATTCAAGGTACATCATGCAGT IGAFRHKLQVALGSKNSV TCAACAAGCTATTGAACAAAATCTGGATTCC H ATTATATTGGTTTTCCTTGAGGAGATTCCAG Q L K A H P L FS K D Q IL E I

[0318] For the below constructs, see, e.g., Examples 6-7 below.

[0319] 241 ILT4,CD3ζ.2A’.IL-15 Fragment SEQSEQ ID #NucleotideID #PeptideV G R F RAtty Docket No.: NKLT-002WO AGAGTACCGGCTGTACCGAGAGAAGAAGTCCGC ARWSELSDPLVLVMTGAYPK CTCCTGGATCACTAGGATCAGACCGGAGCTTGT PTLSAQPSPVVTSGGRVTLQ GAAGAACGGCCAATTTCATATCCCGAGCATCACA CESQVAFGGFILCKEGEDEH V N P C T Q R L G TK N LP QS TA H T S

[0320] 242 ILT4.4-1BB.DAP10.CD3ζ.2A’.IL-15Atty Docket No.: NKLT-002WO Fragment SEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q G M Q R L G TKAtty Docket No.: NKLT-002WO Linker CCGCGGGGATCAGGT PRGSG N LP QS TA H T SILT4.4-1BB.DAP10.2A’.IL-15 FragmentSEQID #Nucleotide SEQ ID #PeptideAT A TATA TTA A T TT AT T T T V G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO AAAAGAGGAAGAAAAAAGTTGCTGTATATATTTAA ACAACCATTTATGAGACCA KRGRKKLLYIFKQPFMRPVQ 4-1BB GTGCAAACCACCCAA GAAGAAGACGGATGTTCATGCAGATTCCCAGAAG TTQEEDGCSCRFPEEEEGG M N LP QS TA H T S

[0322] 244 ILT4.DAP10.CD3ζ.2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K QAtty Docket No.: NKLT-002WO GAAGAACGGCCAATTTCATATCCCGAGCATCACA CESQVAFGGFILCKEGEDEH TGGGAGCATACTGGGCGCTACGGATGCCAGTAC PQCLNSQPHARGSSRAIFSV TATAGTCGCGCCAGGTGGTCCGAACTCAGCGAT GPVSPNRRWSHRCYGYDLN * P C T M Q R L G TK N LP QS TA H T SAtty Docket No.: NKLT-002WO GTTTTGTACATATTGTCCAAATGTTCATCAACACT TCT STOP TGA STOPILT4.DAP10.2A’.IL-15 FragmentSEQNucleotide SEQ ID # ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T M N LP QS TA H T SAtty Docket No.: NKLT-002WO ATCCTAGCAAACAACAGTTTGTCTTCTAATGGGA ATGTAACAGAATCTGGATGCAAAGAATGTGAGGA ACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAILT4.MyD88.DAP10.CD3ζ.2A’.IL-15 FragmentSEQID #Nucleotide SEQ ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T SL R E G D Q T FDAtty Docket No.: NKLT-002WO Linker GCATGC AC M Q R L G TK N LP QS TA H T S

[0325] 247 ILT4.MyD88.DAP10.2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K Q H VAtty Docket No.: NKLT-002WO CCGCTTGTCCTTGTCATGACGGGTGCTTACCCGA GPVSPNRRWSHRCYGYDLN AGCCAACCCTTTCAGCACAGCCGTCACCAGTAGT SPYVWSSPSDLLELLVPG* AACATCCGGCGGTAGGGTGACTCTGCAATGCGA P C T SL R E G D Q T FD M N LP QS TA H T SAtty Docket No.: NKLT-002WO ACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGA GTTTTGTACATATTGTCCAAATGTTCATCAACACT TCTILT4.4-1BB.DAP12.CD3ζ.2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q G K VAtty Docket No.: NKLT-002WO AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACCAGCAGGGCCAGAACCAGCTCTATAAC GAGCTCAATCTAGGACGAAGAGAGGAGTACGAT RVKFSRSADAPAYQQGQNQ R L G TK N LP QS TA H T S

[0327] 249 ILT4.4-1BB.DAP12.2A’.IL-15 FragmentSEQNucle SEQ ID #otideID #PeptideV G R F R K Q H V N P CAtty Docket No.: NKLT-002WO CCCGAGGCCTGGACTTCGCCTGCGATATATATAT DIYIWAPLAGTCGVLLLSLVIT TTGGGCTCCTCTGGCCGGTACCTGCGGCGTACT LYCNHRNRRRVCKCPR GCTCCTGTCACTGGTAATAACCCTGTATTGCAAT Q G K V N LP QS TA H T S

[0328] 250 ILT4.4-1BB.CD3ζ(1XX).2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K Q H V NAtty Docket No.: NKLT-002WO AGCCAACCCTTTCAGCACAGCCGTCACCAGTAGT AACATCCGGCGGTAGGGTGACTCTGCAATGCGA GTCCCAAGTGGCATTCGGTGGGTTCATACTCTGT P C T Q G Q R L G TK N LP QS TA H T SAtty Docket No.: NKLT-002WO

[0329] 251 ILT4.CD3ζ.2A’.IL-15 FragmentSEQNucleotide SEQ ID # ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TK N LP QS TA H T SAtty Docket No.: NKLT-002WO ATGCAAGTATTCATGATACAGTAGAAAATCTGATC ATCCTAGCAAACAACAGTTTGTCTTCTAATGGGA ATGTAACAGAATCTGGATGCAAAGAATGTGAGGAILT4.CD3ζ.P2A.MyD88.CD40.T2A’.IL-15 FragmentSEQID #Nucleotide SEQ ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TKAtty Docket No.: NKLT-002WO P2A 116GCAACGAATTTTTCCCTGCTGAAACAGGCAGGGGACGTAGAGGAAAATCCTGGTCCT 117 ATNFSLLKQAGDVEENPGPSL R E G D Q T FD E H R N LP QS TA H T S

[0331] 253 ILT4.DAP10.TLR2.2A’.IL-15Atty Docket No.: NKLT-002WO Fragment SEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T M V H K D I Y AAtty Docket No.: NKLT-002WO ATGAGAATTTCCAAACCACATTTGAGAAGTATTTC CATCCAGTGCTACTTGTGTTTACTTCTAAACAGTC ATTTTCTAACTGAAGCTGGCATTCATGTCTTCATT N LP QS TA H T SILT4.CD3ζ.P2A.MyD88.4-1BB.2A’.IL-15 FragmentSEQNucleotide SEQ ID # ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TKAtty Docket No.: NKLT-002WO CCTTCGACGCCCTTCACATGCAAGCTCTTCCACC TCGT SL R E G D Q T FD Q G N LP QS TA H T SAtty Docket No.: NKLT-002WO STOP TGA STOPILT4.CD3ζ.P2A.MyD88.HVEM.2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TKAtty Docket No.: NKLT-002WO LinkerATGCAT MHSL R E G D Q T FD R D N LP QS TA H T S

[0334] 256 ILT4.CD3ζ.P2A.IL18R1.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R FAtty Docket No.: NKLT-002WO TTACCCTCTCTTGCCAAGGCTCTCTTGAGGCCCA HIPSITWEHTGRYGCQYYSR AGAGTACCGGCTGTACCGAGAGAAGAAGTCCGC ARWSELSDPLVLVMTGAYPK CTCCTGGATCACTAGGATCAGACCGGAGCTTGT PTLSAQPSPVVTSGGRVTLQ H V N P C T Q R L G TK E L IE E P K LAtty Docket No.: NKLT-002WO ACTCAAGGTTCTGGAAGAACCTTCTTTACTTAATG CCTGCA N LP QS TA H T S

[0335] 257 ILT4.CD3ζ.P2A.TLR2.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO CACAGGAACAGAAGGAGAGTCTGTAAGTGCCCC CGG Q R L G TK V H K D I Y A N LP QS TA H T SAtty Docket No.: NKLT-002WO ACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGA GTTTTGTACATATTGTCCAAATGTTCATCAACACT TCTILT4.CD3ζ.P2A.TLR3.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TKAtty Docket No.: NKLT-002WO P2AGCAACGAATTTTTCCCTGCTGAAACAGGCAGGGGACGTAGAGGAAAATCCTGGTCCT ATNFSLLKQAGDVEENPGPH F FV Q KL P N LP QS TA H T S

[0337] 259 ILT4.CD3ζ.P2A.TLR5.T2A’.IL-15 Fragment SEQ NucleotSEQ ID #ideID #PeptideV G R F R K Q H VAtty Docket No.: NKLT-002WO CCGCTTGTCCTTGTCATGACGGGTGCTTACCCGA GPVSPNRRWSHRCYGYDLN AGCCAACCCTTTCAGCACAGCCGTCACCAGTAGT SPYVWSSPSDLLELLVPG* AACATCCGGCGGTAGGGTGACTCTGCAATGCGA P C T Q R L G TK N F N E VV Q HAtty Docket No.: NKLT-002WO T2A’GAGGGCAGGGGAAGTCTTCTAACATGCGGGGACGTGGAGGAAAATCCCGGGCCC EGRGSLLTCGDVEENPGPN LP QS TA H T SILT4.CD3ζ.P2A.TLR8.T2A’.IL-15 FragmentSEQID #Nucleotide SEQ ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO AGAGTGAAGTTCAGCAGGAGCGCAGACGCCCCC GCGTACCAGCAGGGCCAGAACCAGCTCTATAAC GAGCTCAATCTAGGACGAAGAGAGGAGTACGAT RVKFSRSADAPAYQQGQNQ R L G TK V V Q N II C T N LP QS TA H T SAtty Docket No.: NKLT-002WO

[0339] 261 ILT4.CD3ζ.MyD88.CD40.T2A’.IL-15 FragmentSEQNucl SEQ ID #eotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TK SL R E G D Q T FDAtty Docket No.: NKLT-002WO CGCTGTAGACAGCAGTGTCCCACGGACAGCAGA GCTGGCGGGCATCACCACACTTGATGACCCCCT GGGGCATATGCCTGAGCGTTTCGATGCCTTCATC E LH R N LP QS TA H T S

[0340] 262 ILT4.CD3ζ.MyD88.T2A’.IL-15 FragmentSEQNucleo SEQ ID #tideID #PeptideV G R F R K Q H V NAtty Docket No.: NKLT-002WO GGCGGTGGTCACACAGATGTTATGGATATGATCT CAATAGCCCGTACGTTTGGTCTAGCCCTAGTGAT CTTCTAGAGTTGCTTGTTCCAGGA P C T Q R L G TK SL R E G D Q T FD N LP QS TA H T SAtty Docket No.: NKLT-002WO ATCCTAGCAAACAACAGTTTGTCTTCTAATGGGA ATGTAACAGAATCTGGATGCAAAGAATGTGAGGA ACTGGAGGAAAAAAATATTAAAGAATTTTTGCAGAILT4.CD3ζ.MyD88.4-1BB.T2A’.IL-15 Fragment SEQID #NucleotideSEQ ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TKAtty Docket No.: NKLT-002WO Linker GTTAAC VN SL R E G D Q T FD Q G N LP QS TA H T S

[0342] 264 ILT4.CD3ζ.MyD88.HVEM.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV GAtty Docket No.: NKLT-002WO CGGGCACAATTCCTAAGCCCACCCTCTGGGCTG SPVTLSCQGSLEAQEYRLYR AGCCCGACTCAGTGATAACGCAGGGTTCTCCAG EKKSASWITRIRPELVKNGQF TTACCCTCTCTTGCCAAGGCTCTCTTGAGGCCCA HIPSITWEHTGRYGCQYYSR PK Q H V N P C T Q R L G TK SL R E G D Q T FDAtty Docket No.: NKLT-002WO TGTGTGAAAAGAAGAAAGCCAAGGGGTGATGTA GTCAAGGTGATCGTCTCCGTCCAGCGGAAAAGA CAGGAGGCAGAAGGTGAGGCCAC CVKRRKPRGDVVKVIVSVQR HVEM AGTCATTGAG KRQEAEGEATVIEALQAPPD N LP QS TA H T S

[0343] 265 ILT4.CD3ζ.IL18R1.T2A’.IL-15 FragmentSEQNuc SEQ ID #leotideID #PeptideV G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO CACAGGAACAGAAGGAGAGTCTGTAAGTGCCCC CGG Q R L G TK E L IE E P K L N LP QS TA H T SAtty Docket No.: NKLT-002WO

[0344] 266 ILT4.CD3ζ.TLR2.T2A’.IL-15 FragmentSEQNucle SEQ ID #otideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TK V H K D I Y AAtty Docket No.: NKLT-002WO GAAGGATTTTGGGTAAATCTGAGAGCTGCGATAA AGTCC N LP QS TA H T S

[0345] 267 ILT4.4-1BB.DAP10.TLR2.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO CACAGGAACAGAAGGAGAGTCTGTAAGTGCCCC CGG Q G M V H K D I Y A N LP QS TA H T SAtty Docket No.: NKLT-002WO

[0346] 268 ILT4.CD3ζ.TLR5.T2A’.IL-15 FragmentSEQNucleo SEQ ID #tideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q R L G TK N F N E VV Q HAtty Docket No.: NKLT-002WO TCTGACCTTAACAGTGCTCTCATCATGGTGGTGG TTGGGTCCTTGTCCCAGTACCAGTTGATGAAACA TCAATCCATCAGAGGCTTTGTACAGAAACAGCAG N LP QS TA H T S

[0347] 269 ILT4.CD3ζ.TLR8.T2A’.IL-15 FragmentSEQNucleotid SEQ ID #eID #PeptideV G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO CACAGGAACAGAAGGAGAGTCTGTAAGTGCCCC CGG Q R L G TK V V Q N II C T N LP QS TA H T SAtty Docket No.: NKLT-002WO

[0348] 270 ILT4.DAP12.TLR2.T2A’.IL-15 Fragment SEQ NucSEQ ID #leotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T K V V H K D I Y AAtty Docket No.: NKLT-002WO Linker CCGCGGGGATCAGGT PRGSG N LP QS TA H T S

[0349] 271 ILT4.4-1BB.DAP12.TLR2.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO Linker GGATCC GS Q G K V V H K D I Y A N LP QS TA H T SAtty Docket No.: NKLT-002WO

[0350] 272 ILT4.TLR2.DAP12.CD3ζ.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T V H K D I Y A K VAtty Docket No.: NKLT-002WO Linker GTCGAC VD Q R L G TK N LP QS TA H T S

[0351] 273 ILT4.TLR2.DAP12.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K Q H V NAtty Docket No.: NKLT-002WO GGCGGTGGTCACACAGATGTTATGGATATGATCT CAATAGCCCGTACGTTTGGTCTAGCCCTAGTGAT CTTCTAGAGTTGCTTGTTCCAGGA P C T V H K D I Y A K V N LP QS TA H T SAtty Docket No.: NKLT-002WO

[0352] 274 ILT4.DAP10.CD3ζ.P2A.TLR2.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T M Q R L G TKAtty Docket No.: NKLT-002WO P2AGCAACGAATTTTTCCCTGCTGAAACAGGCAGGGGACGTAGAGGAAAATCCTGGTCCT ATNFSLLKQAGDVEENPGPV H K D I Y A N LP QS TA H T S

[0353] 275 ILT4.4-1BB.DAP10.CD3ζ.P2A.TLR2.T2A’.IL-15 FragmentSEQNu SEQ ID #cleotideID #PeptideV G R F RAtty Docket No.: NKLT-002WO AGAGTACCGGCTGTACCGAGAGAAGAAGTCCGC ARWSELSDPLVLVMTGAYPK CTCCTGGATCACTAGGATCAGACCGGAGCTTGT PTLSAQPSPVVTSGGRVTLQ GAAGAACGGCCAATTTCATATCCCGAGCATCACA CESQVAFGGFILCKEGEDEH V N P C T Q G M Q R L G TKAtty Docket No.: NKLT-002WO P2AGCAACGAATTTTTCCCTGCTGAAACAGGCAGGGGACGTAGAGGAAAATCCTGGTCCT ATNFSLLKQAGDVEENPGPV H K D I Y A N LP QS TA H T S

[0354] 276 ILT4.DAP12.CD3ζ.P2A.TLR2.T2A’.IL-15 FragmentSEQNucleo SEQ ID #tideID #PeptideV G R F R K Q H VAtty Docket No.: NKLT-002WO TATAGTCGCGCCAGGTGGTCCGAACTCAGCGAT GPVSPNRRWSHRCYGYDLN CCGCTTGTCCTTGTCATGACGGGTGCTTACCCGA SPYVWSSPSDLLELLVPG* AGCCAACCCTTTCAGCACAGCCGTCACCAGTAGT P C T K V Q R L G TK V H K D I YAtty Docket No.: NKLT-002WO TGAACTGGACTTCTCCCATTTCCGTCTTTTTGATG LEWPMDEAQREGFWVNLRA AGAACAATGATGCTGCCATTCTCATTCTTCTGGA AIKS GCCCATTGAGAAAAAAGCCATTCCCCAGCGCTTC N LP QS TA H T S

[0355] 277 ILT4.4-1BB.DAP12.CD3ζ.P2A.TLR2.T2A’.IL-15 FragmentSEQNucl SEQ ID #eotideID #PeptideV G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO CACAGGAACAGAAGGAGAGTCTGTAAGTGCCCC CGG Q G K V Q R L G TK V H K D I Y AAtty Docket No.: NKLT-002WO Linker CCGCGGGGATCAGGT PRGSG N LP QS TA H T S

[0356] 278 ILT4.DAP10.P2A.TLR2.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO Linker GGATCC GS Q G M V H K D I Y A N LP QS TA H T SAtty Docket No.: NKLT-002WO ATGCAAGTATTCATGATACAGTAGAAAATCTGATC ATCCTAGCAAACAACAGTTTGTCTTCTAATGGGA ATGTAACAGAATCTGGATGCAAAGAATGTGAGGAILT4.4-1BB.DAP10.P2A.TLR2.T2A’.IL-15 FragmentSEQID #Nucleotide SEQ ID #PeptideATGACCCCTATAGTTACAGTCTTGATCTGTCTCG V G R F R K Q H V N P C T Q G MAtty Docket No.: NKLT-002WO Linker GTCGAC VD V H K D I Y A N LP QS TA H T S

[0358] 280 ILT4.DAP12.P2A.TLR2.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R FAtty Docket No.: NKLT-002WO TTACCCTCTCTTGCCAAGGCTCTCTTGAGGCCCA HIPSITWEHTGRYGCQYYSR AGAGTACCGGCTGTACCGAGAGAAGAAGTCCGC ARWSELSDPLVLVMTGAYPK CTCCTGGATCACTAGGATCAGACCGGAGCTTGT PTLSAQPSPVVTSGGRVTLQ H V N P C T K V V H K D I Y AAtty Docket No.: NKLT-002WO Linker CCGCGGGGATCAGGT PRGSG N LP QS TA H T S

[0360] ILT4.4-1BB.DAP12.P2A.TLR2.T2A’.IL-15 FragmentSEQSEQ ID #NucleotideID #PeptideV G R F R K Q H V N P C TAtty Docket No.: NKLT-002WO Linker GGATCC GS Q G K V V H K D I Y A N LP QS TA H T SAtty Docket No.: NKLT-002WO ATGCAAGTATTCATGATACAGTAGAAAATCTGATC ATCCTAGCAAACAACAGTTTGTCTTCTAATGGGA ATGTAACAGAATCTGGATGCAAAGAATGTGAGGAILT2.CD3ζ.2A’.IL-15 FragmentSEQD#Nuc SEQ I leotideID #PeptideATGACGCCGATTCTCACAGTTCTTATATGCCTGG Q G R F DT P C P G S P C T Q R L G TKAtty Docket No.: NKLT-002WO ATGAGAATTTCCAAACCACATTTGAGAAGTATTTC CATCCAGTGCTACTTGTGTTTACTTCTAAACAGTC ATTTTCTAACTGAAGCTGGCATTCATGTCTTCATT N LP QS TA H T S

[0362] 283 ILT2.4-1BB.CD3ζ.2A’.IL-15 FragmentSEQNucleoti SEQ ID #deID #PeptideATGACGCCGATTCTCACAGTTCTTATATGCCTGG Q G R F DT P C P G S P C T Q GAtty Docket No.: NKLT-002WO Linker GTCGAC VD Q R L G TK N LP QS TA H T S

[0363] 284 ILT2.4-1BB.DAP10.CD3ζ.2A’.IL-15 Fragment SEQSEQ ID #NucleotideID #PeptideQ G R F DT P C P G SAtty Docket No.: NKLT-002WO Linker CAATTG QL CCCGCGCCACGACCACCAACACCAGCCCCAACC ATTGCATCCCAGCCTTTGTCTCTCCGGCCCGAGG P C T Q G M Q R L G TK N LP QS TA H T SAtty Docket No.: NKLT-002WO

[0364] 285 ILT2.4-1BB.DAP10.2A’.IL-15 FragmentSEQNuc SEQ ID #leotideID #PeptideATGACGCCGATTCTCACAGTTCTTATATGCCTGG Q G R F DT P C P G S P C T Q G M N LP QSAtty Docket No.: NKLT-002WO TTGGGCTGTTTCAGTGCAGGGCTTCCTAAAACAG MHIDATLYTESDVHPSCKVTA AAGCCAACTGGGTGAATGTAATAAGTGATTTGAA MKCFLLELQVISLESGDASIH AAAAATTGAAGACCTTATTCAATCTATGCACATTG DTVENLIILANNSLSSNGNVT AT TA TTTATATA AAA T AT TT A E KE EELEEKNIKEFLQSILT2.4-1BB.DAP12.2A’.IL-15 FragmentSEQNucleotid SEQ ID #eID #PeptideATGACGCCGATTCTCACAGTTCTTATATGCCTGG Q G R F DT P C P G S P C T Q GAtty Docket No.: NKLT-002WO TATTTCCTCGGACGGCTCGTTCCCAGGGGAAGG GGCGCTGCCGAAGCAGCTACAAGAAAACAGCGA YFLGRLVPRGRGAAEAATRK DAP12 ATTACAGAGACCGAGTCTCCCTATCAGGAGTTGC QRITETESPYQELQGQRSDV N LP QS TA H T S

[0366] 87 ILT2.MyD88.DAP10.CD3ζ.2A’.IL-15 FragmentSEQNucl SEQ ID #eotideID #PeptideQ G R F DT P C P G S P C TAtty Docket No.: NKLT-002WO Linker GGATCC GS SL R E G D Q T FD M Q R L G TK N LP QS TA H T SAtty Docket No.: NKLT-002WO STOP TGA STOP

[0367] 288 ILT4.MyD88.DAP10.2A’.IL-15 Fragment SEQSEQ ID #NucleotideID #PeptideATGACGCCGATTCTCACAGTTCTTATATGCCTGG Q G R F DT P C P G S P C T SL R E G D Q T FDAtty Docket No.: NKLT-002WO CTGTGCGCACGCCCACGCCGCAGCCCCGCCCAA DAP10 GAAGATGGCAAAGTCTACATCAACATGCCAGGCA LCARPRRSPAQEDGKVYINM PGRG N LP QS TA H T S

[0368] 289 ILT2.MyD88.DAP12.CD3ζ.2A’.IL-15 Fragment SEQ NucleotideSEQ ID # ID #PeptideQ G R F DT P C P G S P C TAtty Docket No.: NKLT-002WO Linker GGATCC GS SL R E G D Q T FD K V Q R L G TK N LP QS TA H T SAtty Docket No.: NKLT-002WO STOP TGA STOP

[0369] 290 ILT2.MyD88.DAP12.2A’.IL-15 Fragment SEQSEQ ID #NucleotideID #PeptideATGACGCCGATTCTCACAGTTCTTATATGCCTGG Q G R F DT P C P G S P C T SL R E G D Q T FDAtty Docket No.: NKLT-002WO TATTTCCTCGGACGGCTCGTTCCCAGGGGAAGG GGCGCTGCCGAAGCAGCTACAAGAAAACAGCGA YFLGRLVPRGRGAAEAATRK DAP12 ATTACAGAGACCGAGTCTCCCTATCAGGAGTTGC QRITETESPYQELQGQRSDV N LP QS TA H T S

[0030] 9 ILT2.4-1BB.DAP10.P2A.TLR2.T2A’.IL-15 FragmentSEQNuc SEQ ID #leotideID #PeptideQ G R F DT P C P G S P C TAtty Docket No.: NKLT-002WO Linker GGATCC GS Q G M V H K D I Y A N LP QS TA H T SAtty Docket No.: NKLT-002WO ATGCAAGTATTCATGATACAGTAGAAAATCTGATC ATCCTAGCAAACAACAGTTTGTCTTCTAATGGGA ATGTAACAGAATCTGGATGCAAAGAATGTGAGGAExample Non-Limiting Aspects of the Disclosure

[0371] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure are provided below. As will be apparent to those of ordinary skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below. It will be apparent to one of ordinary skill in the art that various changes and modifications can be made without departing from the spirit or scope of the invention. 1. A chimeric receptor protein, comprising: (a) a targeting region, that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region, comprising a transmembrane amino acid sequence; and (c) an intracellular domain (ICD), comprising a signaling region capable of transducing a signal, upon binding of said targeting region to HLA-G, into the interior of an immune effector cell to elicit effector cell function, wherein the signaling region comprises a costimulatory region comprising a MyD88 polypeptide. 2. The chimeric receptor protein of 1, wherein the MyD88 polypeptide comprises an amino acid sequence having 85% or more sequence identity with the sequence of SEQ ID NO: 27. 3. The chimeric receptor protein of 1 or 2, wherein the MyD88 polypeptide is fused to a CD40, 4-1BB, or HVEM costimulatory domain. 4. The chimeric receptor protein of 3, wherein the MyD88 polypeptide is fused to a 4-1BB costimulatory domain.Atty Docket No.: NKLT-002WO The chimeric receptor protein of any one of 1-4, wherein the signaling region comprises a CD3ζ signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, or any combination thereof. The chimeric receptor protein of 4, wherein the signaling region comprises a CD3ζ signaling domain. The chimeric receptor protein of 1 or 2, wherein the signaling region comprises a DAP12 signaling domain. The chimeric receptor protein of 7, wherein the signaling region does not include a CD3ζ signaling domain. The chimeric receptor protein of 1 or 2, wherein the signaling region comprises a DAP12 signaling domain and a CD3ζ signaling domain. An intracellular domain (ICD) polypeptide, comprising a signaling region capable of transducing a signal in an immune effector cell to elicit effector cell function, wherein the signaling region comprises (i) a CD3ζ signaling domain, a DAP10 signaling domain, or a DAP12 signaling domain, and (ii) a costimulatory region that comprises a Toll / Interleukin-1 Receptor / Resistance Protein (TIR) domain. The ICD polypeptide of 10, wherein the TIR domain is a TLR2 TIR domain, TLR3 TIR domain, or a IL18R1 TIR domain. The ICD polypeptide of 10, wherein the TIR domain comprises an amino acid sequence having 85% or more sequence identity with the TLR2 TIR domain of SEQ ID NO: 111. The ICD polypeptide of 10, wherein the TIR domain comprises an amino acid sequence having 85% or more sequence identity with the TLR3 TIR domain of SEQ ID NO: 113. The ICD polypeptide of 10, wherein the TIR domain comprises an amino acid sequence having 85% or more sequence identity with the IL18R1 TIR domain of SEQ ID NO: 109. The ICD polypeptide of any of 10-15, wherein the signaling region comprises said CD3ζ signaling domain. The ICD polypeptide of any one of 10-15, wherein said costimulatory polypeptide is comprised by a chimeric receptor protein that comprises: (a) a targeting region, that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region, comprising a transmembrane amino acid sequence; and (c) the ICD polypeptide.Atty Docket No.: NKLT-002WO A chimeric receptor protein, comprising: (a) a targeting region, that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region, comprising a transmembrane amino acid sequence; and (c) an intracellular domain (ICD), comprising a signaling region capable of transducing a signal, upon binding of said targeting region to HLA-G, into the interior of an immune effector cell to elicit effector cell function, wherein the signaling region comprises a DAP10 signaling domain or a DAP12 signaling domain. The chimeric receptor of 17, wherein the DAP10 signaling domain comprises an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 4. The chimeric receptor of 17, wherein the DAP12 signaling domain comprises an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 4. The chimeric receptor of any one of 17-19, wherein the signaling region further comprises a CD3ζ signaling domain. The chimeric receptor of any one of 17-19, wherein the signaling region does not include a CD3ζ signaling domain. The chimeric receptor of any one of 17-21, wherein the signaling region further comprises a CD40, 4-1BB, or HVEM costimulatory domain. The chimeric receptor of any one of 17-21, wherein the signaling region further comprises a 4-1BB costimulatory domain. The chimeric receptor of 17, comprising the DAP12 signaling domain, wherein the signaling region does not include a CD3ζ signaling domain, and wherein the signaling region further comprises a 4-1BB costimulatory domain. The chimeric receptor of 24, wherein the DAP12 signaling domain comprises an amino acid sequence having 85% or more sequence identity with SEQ ID NO: 4. The chimeric receptor protein of any one of 1-25, wherein the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain. The chimeric receptor protein of 26, wherein the targeting region comprises a D3-D4 extracellular domain of ILT4. The chimeric receptor protein of 26, wherein the targeting region does not include a D3-D4 extracellular domain of ILT4, and comprises a stalk domain.Atty Docket No.: NKLT-002WO The chimeric receptor protein of any one of 1-25, wherein the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain. The chimeric receptor protein of 29, wherein the targeting region comprises a D3-D4 extracellular domain of ILT2. The chimeric receptor protein of 29, wherein the targeting region does not include a D3-D4 extracellular domain of ILT4, and comprises a stalk domain. The chimeric receptor protein of 28 or 31, wherein the stalk domain comprises a ILT2, ILT4, CD28, CH2 / CH3, CH3, or CD8α stalk domain. The chimeric receptor protein of any one of 1-33, wherein TM domain is an ILT2, ILT4, CD28, or CD8α TM domain. The chimeric receptor protein of any one of 1-25, wherein: the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain, the extracellular domain lacks an ILT4 D3-D4 extracellular domain, the chimeric receptor protein comprises a CD8α stalk domain, and the TM region is a CD8α TM. The chimeric receptor protein of any one of 1-25, wherein: the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain, the extracellular domain lacks an ILT2 D3-D4 extracellular domain, the chimeric receptor protein comprises a CD8α stalk domain, and the TM region is a CD8α TM. A nucleic acid, comprising a nucleotide sequence encoding the chimeric receptor protein of any one of 1-35. The nucleic acid of 36, wherein said nucleotide sequence is operably linked to a constitutive promoter. The nucleic acid of 36, wherein said nucleotide sequence is operably linked to an inducible promoter. The nucleic acid of any one of 36-38, wherein said nucleic acid is an expression vector. The nucleic acid of 39, wherein the expression vector is a retroviral vector, a lentiviral vector or a plasmid vector. A genetically modified cell, expressing the chimeric receptor protein of any one of 1-35. The genetically modified cell of 41, wherein the genetically modified cell is an immune cell. The genetically modified cell of 42, wherein the immune cell is a natural killer (NK) cell, an NK-T cell, a T cell, an iNKT cell, or a macrophage. The genetically modified cell of 42, wherein the immune cell is a natural killer (NK) cell.Atty Docket No.: NKLT-002WO A method of treatment, comprising administering the genetically modified cell of any one of 41-44 to an individual in need. The method of 45, wherein the genetically modified cell is autologous to the individual. The method of 45, wherein the genetically modified cell is allogeneic to the individual. The method of any one of 45-47, wherein the individual has cancer. The method of 48, wherein the individual has a solid tumor. The method of 48, wherein the individual has a liquid tumor. A method of producing a genetically modified cell, the method comprising: introducing the nucleic acid of any one of 36-40 into a cell, thus producing a genetically modified cell. The method of 51, wherein the genetically modified cell is an immune cell. The method of 52, wherein the immune cell is a natural killer (NK) cell, an NK- T cell, a T cell, an iNKT cell, or a macrophage. The method of 52, wherein the immune cell is a natural killer (NK) cell. The method of 52, wherein the immune cell is a T cell.Atty Docket No.: NKLT-002WO EXPERIMENTAL EXAMPLES

[0372] The following examples are provided for purposes of illustration only, and are not intended to be limiting unless otherwise specified. Thus, the invention should in no way be construed as being limited to the following examples, but rather should be construed to encompass any and all variations which become evident as a result of the teaching provided herein.

[0373] Without further description, it is believed that one of ordinary skill in the art can, using the preceding description and the following illustrative examples, make and utilize the present invention and practice the claimed methods. The following working examples therefore are not to be construed as limiting in any way the remainder of the disclosure.

[0374] General methods in molecular and cellular biochemistry can be found in such standard textbooks as Molecular Cloning: A Laboratory Manual, 3rd Ed. (Sambrook et al., HaRBor Laboratory Press 2001); Short Protocols in Molecular Biology, 4th Ed. (Ausubel et al. eds., John Wiley & Sons 1999); Protein Methods (Bollag et al., John Wiley & Sons 1996); Nonviral Vectors for Gene Therapy (Wagner et al. eds., Academic Press 1999); Viral Vectors (Kaplift & Loewy eds., Academic Press 1995); Immunology Methods Manual (I. Lefkovits ed., Academic Press 1997); and Cell and Tissue Culture: Laboratory Procedures in Biotechnology (Doyle & Griffiths, John Wiley & Sons 1998), the disclosures of which are incorporated herein by reference. Reagents, cloning vectors, cells, and kits for methods referred to in, or related to, this disclosure are available from commercial vendors such as BioRad, Agilent Technologies, Thermo Fisher Scientific, Sigma-Aldrich, New England Biolabs (NEB), Takara Bio USA, Inc., and the like, as well as repositories such as e.g., Addgene, Inc., American Type Culture Collection (ATCC), and the like. EXAMPLE 1. EXPRESSION OF ENGINEERED CIR CONSTRUCTS IN PRIMARY HUMAN NK CELLS

[0375] To demonstrate the utility of expression of chimeric receptors that augment NK cell signaling in a target-specific manner, γ-retroviruses were created target HLA-G through binding to its natural ILT receptors. A cartoon schematic of the resulting CIR constructs is depicted on Figure 1. In the generically depicted CIR construct, the D1 and D2 domains of ILT4 were fused with a CD8α stalk and transmembrane domain used to stably present the binder to the HLA-G target. All other constructs described in these examples contain the same extracellular components to permit unbiased cross- references for intracellular signaling domains that are altered between constructs. Recombinant DNA constructs were engineered in the SFG γ-retroviral vector (Rivere, Brose and Mulligan, Proc. Natl. Acad. Sci USA 15:6733-6737 (1995)) with whichAtty Docket No.: NKLT-002WO transgene expression is driven by the Moloney Murine Sarcoma Virus Long Terminal Repeat (LTR). A schematic of the constructs evaluated in this example and the following examples is depicted in Figure 2. In each embodiment of these constructs, DNA encoding a CIR was cloned 5’ to a gene encoding Interleukin-15 (IL-15), a growth factor important for sustained NK cell growth and survival that is provided in an autocrine fashion to support CIR-NK cells in culture. The cistrons encoding the ILT4 CIRs and IL-15 and CD19 were separated by the T2A cotranslational cleavage site derived from Thosea asigna virus or the P2A sequence derived from porcine teschovirus to permit separate protein expressing from individual mRNA molecules.

[0376] γ-Retroviruses were produced from these DNA constructs by transfection into HEK293 cells together with helper plasmids encoding reverse transcriptase and viral capsid and envelope proteins. These retroviral vectors were used to transduce primary human NK cells selected for CD56 expression from peripheral blood mononuclear cells (PBMCs) derived from 2 healthy donors. All NK cells used in comparative experiments were derived from the same donors. The efficiency of transduction was marked by expression of ILT4 present on the CIR, but not normally expressed by NK cells as detected by flow cytometry. Stability of transgene expression was monitored weekly.

[0377] Methods: DNA constructs were designed with SnapGene software and DNA sequences were synthesized from GBlock fragments provided by IDT Laboratories. Synthetic DNA fragments were cloned with standard cloning techiques for recombinant DNA assembly into the SFG DNA vector. EXAMPLE 2. ANTI-TUMOR EFFICACY WITH CIR-NK CELLS WITH ALTERED ITAM- CONTAINING CYTOTOXICITY DOMAINS

[0378] To generate tumor cell lines appropriate for evaluation of CIR target-specific cytotoxicity, derivatives of three Acute Myeloid Leukemia (AML) cell lines were created to express the marker protein Green Fluorescent Protein as a fusion with a separate marker, firefly luciferase (GFP-ffluc). KG1-GFPffluc cells did not express HLA-G protein detectable by flow cytometry or HLA-G1, HLA-G2 mRNA that encodes the most common membrane bound HLA-G isoforms determined by quantitative PCR. This cell line was used as a negative control for CIR-specific targeting and a reference for possible hyperactive innate NK cell cytotoxic activity that hypothetically could be produced by tonic CIR activity. Molm13-GFPffluc and Kasumi1- GFPffluc cells are AML lines that express low, but measurable levels of HLA-G1 protein and HLA-G1 and HLA-G5 mRNA. These cells were used to engage and activate CIR-NK cells.

[0379] To evaluate the relative signaling efficacy of CIR constructs containing alternative ITAM-containing signaling motives, primary human NK cells from two healthy donorsAtty Docket No.: NKLT-002WO were transduced with γ-retroviruses encoding the extracellular and stalk / transmembrane domain described in Example 1 and a CD3ζ, DAP10 or DAP12 intracellular domain. It is notable that DAP10 does not contain a canonical ITAM domain but instead signals through mechanisms similar to CD28 in T cells. No coactivation domain was present in the expressed CIR. A separate γ-retrovirus encoding Red Fluorescent Protein (RFP) was co-transduced to assess NK cell growth during coculture experiments. Mock transduced (cells that were manipulated identically as transduced cells, but without virus) and cells transduced with RFP without a CIR retrovirus were produced as negative controls.

[0380] 2 x 105 CIR-NK cells were cultured without a target to assess the degree of tonic NK cell activity that may be induced by signaling domains present on a CIR construct. Separately, 2 x 103 of the same NK cells were cocultered with HLA-G+ Kasumi1 cells at an effector to target (E:T) ratio of 1:5. Production of the cytokines Tumor Necrosis Factor-α (TNF-α) and Interferon-γ was determined to assess tonic and stimulated NK cell activity (Figures 3 and 4, respectively). Results were normalized to a cell number of 2 x 105 for comparison and assessment of NK cell stimulation.

[0381] Tonic secretion of TNF-α and IFN-γ was elevated in CIR-NK cells relative to NK cells lacking CIR expression. Following stimulation with Kasumi1 target cells, overall cytokine secretion was elevated in mock or RFP-transduced NK cells indicating innate targeting of Kasumi1 cells. CIR-NK cells were not further stimulated for TNF-α production, but ILT4.DAP12 CIR-NK cells were markedly elevated for IFN-γ production following Kasumi1 stimulation relative to CIR-NK cells expressing DAP10 or CD3ζ or control NK cells. IFN-γ production is a commonly used surrogate marker of NK cell activation. These results indicated that DAP12 may be a superior signaling moiety for the promotion of inflammation at a tumor site.

[0382] To assess the innate and target-specific cytotoxic potential of CIR-NK cells, cocultures were set up with HLA-G- KG1-GFPffluc cells (Figure 5) at and E:T ratio of 1:5 and HLA-G+ Molm13- GFPffluc cells at an E:T ratio of 1:10 (Figure 6). NK cell killing in cocultures was assessed over seven days by imaging of GFP fluorescence in an Incucyte microscope / incubator as an indicator of target cell outgrowth. Control cultures with NK cells expressing RFP but no CIR did not control KG1 cell outgrowth relative to cultures lacking NK cells (Tumor only) and innate activity of CIR-NK cells was low. CIR-NK cells containing DAP12 were capable of enhanced control of Molm13 cell outgrowth relative to CIR-NK cells expressing DAP10 or CD3ζ. NK cell proliferation over seven days was measured in the Incucyte by imaging of the RFP marker expressed in the NK cells. Enhanced CIR-NK cell proliferation was found with each of the CIR activation domains relative to NK cells expressing only RFP. TheseAtty Docket No.: NKLT-002WO results provided further indication that DAP12 provided superior NK cell performance relative to CD3ζ or DAP10 when expressed without a coactivation signaling moiety.

[0383] To assess whether inclusion of multiple ITAM-containing elements could further augment CIR-NK cell potency, constructs were prepared that included both DAP10 and CD3ζ or DAP12 and CD3ζ in the intracellular domain CIR-NK cell performance was compared with CIR-NK cells expressing each signaling element alone. Tonic and Kasumi1 target-stimulated cytokine production was compared and addition of CD3ζ signaling with DAP10 or DAP12 did not augment the production of TNF-α (Figure 7) or IFN-γ (Figure 8). Levels of IFN-γ were markedly lower in IFN.DAP12.CD3ζ CIR-NK cell cocultures with Kasumi1 relative to ILT4.DAP12 CIR-NK cell cocultures. Innate targeting of KG1 cells was not found in any of the CIR-NK cell cocultures (Figure 7), however HLA-G-specific cytotoxicity against Molm13-GFPffluc cells was enhanced by inclusion of CD3ζ with DAP10 or DAP12. These results indicated that addition of further ITAM domains to the intracellular signaling domain of a CIR can enhance killing performance of CIR-NK cells but that signaling to stimulate pro-inflammatory cytokine production is muted. EXAMPLE 3. EFFECT OF COACTIVATION SIGNALS ON CIR-NK CELL PERFORMANCE

[0384] 4-1BB (TNFRS9) and HVEM (TNFRS14) are members of the TNF Receptor superfamily and each can signal through the NF-κB and other pathways to promote cytokine gene transcription and cytokine release. These cooperate with ITAM-directed signaling pathways to promote the growth and survival of immune cells. These properties are termed costimulation in T cells and coactivation in NK cells. Retrovirus vectors were prepared that contain 4-1BB or HVEM together with the CD3ζ cytotoxicity domain and CIR-NK cells transduced with these constructs were evaluated relative to CIR-NK cells expressing CD3ζ signaling elements alone. Tonic NK cell signaling to production of TNF-α was not impacted by inclusion of 4-1BB or HVEM, but signaling to drive IFN-γ production was enhanced when 4-1BB was included but not when HVEM was included (Figure 12 and Figure 13). Production of TNF-α and IFN-γ by CIR-NK cells activated by coculture with Kasumi1 targets was not impacted by inclusion of 4-1BB or HVEM. Cytotoxicity against control KG1 cells that do not express HLA-G was also not impacted by 4-1BB or HVEM augmentation of CD3ζ signals in CIR-NK cells (Figure 13), but target-specific cytotoxicity against Molm13 cells was enhanced by 4-1BB / CD3ζ signaling relative to CD3ζ with or without HVEM augmentation (Figure 14). These results indicated that 4-1BB signaling augmented CIR-NK cell performance.Atty Docket No.: NKLT-002WO

[0385] These findings were extended by construction of retroviruses that included 4-1BB elements with DAP10 or DAP12 with or without inclusion of CD3ζ elements. Coactivation by 4-1BB reduced tonic production of TNF-α and IFN-γ when combined with DAP12 signaling with or without further CD3ζ signaling while DAP10 signaling to cytokine production was relatively unaffected by combination with 4-1BB (Figure 15, Figure 16). Target-specific cytokine production by DAP12-containing CIR-NK cells in cocultures with Kasumi1 cells was markedly enhanced by inclusion of 4-1BB with or without the further inclusion of CD3ζ. These results indicated that 4-1BB did not lead to overall hyperactivity of CIR-NK cells but increased the responsiveness of these cells to CIR activation by HLA-G when combined with DAP12 signaling. Cytotoxicity against HLA-G- KG1 cells was unaffected by 4-1BB inclusion with DAP10, DAP12 or CD3ζ alone or in combination (Figure 17). Target-specific cytotoxicity against Molm13 cells was improved by addition of 4-1BB with DAP10 and CD3ζ (ILT4.4- 1BB.DAP10.CD3ζ), but other combinations of DAP10 and DAP12 with 4-1BB did not improve cytotoxicity against Molm13 (Figure 18). EXAMPLE 4. EFFECT OF MyD88 COACTIVATION ON CIR-NK CELL PERFORMANCE

[0386] MyD88 is a cytoplasmic protein that is recruited to receptors including IL-1 type receptor and Toll-like receptors. Activation of these receptors by ligand-directed dimerization leads to MyD88 activation and downstream signaling through several pathways including Interferon Response Factors, MAPK, NF-κB, and the AKT growth and survival pathway. These signaling pathways are partially overlapping signaling by TNFR family proteins.

[0387] Retroviral constructs encoding CIRs were created that added only the Death Domain signaling elements of MyD88 but not the receptor-engaging TIR domain as an element to replace 4-1BB coactivation together with CD3ζ, DAP12 or DAP12 / CD3ζ. Tonic production of TNF-α was only impacted by MyD88 inclusion in the combination of DAP12, and MyD88 signaling had little effect of target-specific TNF-α production (Figure 19). Tonic IFN-γ production by CIR-NK cells was higher when MyD88 was included with CD3ζ, DAP12 or DAP12 / CD3ζ (Figure 20). In contrast, MyD88 coactivation did not lead to enhanced IFN-γ or TNF-α production when CIR-NK cells were cocultured with Kasumi1 targets. Cytotoxicity against HLA-G-negative KG1 cells was not observed with or without MyD88 signaling, but enhanced NK cell proliferation was observed when MyD88 was included with each ITAM-containing cytotoxicity domain (ILT4.MyD88.CD3ζ, ILT4.MyD88.DAP12, ILT4.MyD88.DAP12.CD3ζ) (Figure 21). This finding indicated that MyD88 signaling stimulates CIR-NK cell growth in theAtty Docket No.: NKLT-002WO absence of CIR activation, and enhanced NK cell proliferation by MyD88 signaling was also observed in cocultures with HLA-G-expressing Molm13 targets (Figure 22). Cytotoxicity against Molm13 was markedly improved in CIR-NK cells containing MyD88 signaling elements when combined with CD3ζ, DAP12 or DAP12 / CD3ζ (Figure 22). These results indicated that MyD88 coactivation enhanced CIR-NK cell performance in terms of cytotoxicity and growth – each key elements of an effective cell therapy.

[0388] The effect of combining distinct coactivation signaling elements was evaluated in CIR- NK cells. Retroviral constructs were created that combine MyD88 with 4-1BB, HVEM or CD40 together with the CD3ζ cytotoxicity element in the intracellular signaling domain of the ILT4 CIR. CIR-NK cells expressing these so-called 3rd generation CIR constructs (ILT4.MyD88.4-1BB.CD3ζ, ILT4.MyD88.HVEM.CD3ζ, ILT4.MyD88.CD40.CD3ζ) were compared with CIR-NK cells with intracellular domains containing CD3ζ only, MyD88.CD3ζ, 4-1BB.CD3ζ and HVEM.CD3ζ. Tonic cytokine production was enhanced in all CIR-NK cells containing MyD88 coactivation elements. However, TNF-α production was only enhanced upon CIR engagement with HLA-G in Kasumi1 cell cocultures when the combination of MyD88.CD40.CD3ζ was contained in the intracellular domain of the CIR (Figure 23). IFN-γ production was enhanced by all MyD88 containing CIR-NK cells relative to their controls expressing CD3ζ alone, or 4-1BB.CD3ζ or HVEM.CD3ζ (Figure 24). These findings provided further support to the hypothesis that MyD88 signaling on chimeric receptors drive enhanced cytokine production.

[0389] Cytotoxicity against HLA-G-negative KG1 cells was not observed, but cocultures of these cells with all CIR-NK cells containing MyD88 signaling domains demonstrated enhanced proliferation relative to their controls that did not contain MyD88 elements (Figure 25). In cocultures with HLA-G-expressing Molm13 cells at an E:T ratio of 1:10 each MyD88-containing CIR-NK demonstrated markedly enhanced potency relative to their controls expressing only CD3ζ, 4-1BB.CD3ζ or HVEM.CD3ζ. These results further indicated that MyD88 signaling in combination with ITAM-domain cytotoxic signaling provides enhanced CIR-NK cell potency. EXAMPLE 5. RECRUITMENT OF MyD88 SIGNALING TO CIRs EXPRESSING TIR DOMAINS

[0390] Native MyD88 is recruited to IL-1 family receptors and TLRs by interaction between TIR domains on the receptor and a TIR domain on MyD88. Downstream signaling is directed by the separate MyD88 Death Domain that was incorporated in the CIRs described in Example 4. To recruit MyD88 signaling to a CIR domain, constructs wereAtty Docket No.: NKLT-002WO created in which CD3ζ cytotoxicity domains were fused with the TIR domain of the Interleukin 18-Receptor 1 (or α) chain (ILT4.IL18R1.CD3ζ). Further constructs were created that recruit MyD88 indirectly. The intracellular TIR domain of TLR2 interacts with the TIR domain of MAL which further interacts in a ternary complex with MyD88. A CIR construct was created that fused the TLR2 CIR with CD3ζ. A still further CIR construct fused the TIR domain of TLR3 with CD3ζ. The TIR domain of TLR3 does not interact with MyD88, but instead recruits TRIF through its TIR domain. TRIF signaling has overlapping and distinct downstream signaling pathways to MyD88. These constructs were expressed in CIR-NK cells and compared with CIR-NK cells containing CD3ζ alone and MyD88 as a direct fusion with CD3ζ.

[0391] Tonic cytokine production was low in CIR-NK cells expressing TIR domains, much lower than the ILT4.MyD88.CD3ζ direct CIR fusion, and TNF-α production in activated TIR-containing CIR-NK cell cocultures with Kasumi1 cells was not enhanced relative to direct ILT4.MyD88.CD3ζ cocultures (Figure 27). In contrast, IFN-γ was dramatically stimulated by CIR engagement of each TIR domain-containing CIR-NK cell coculture with Kasumi1 cells (Figure 28). Both innate killing activity (Figure 29) and target- specific killing of Molm13 cells (Figure 30) was not elevated by TIR domain-containing CIR-NK cells possibly due to insufficient expression to support cytotoxic signaling. MyD88 signaling is activated by oligomerization of the signaling death domains. The low tonic signaling for IFN-γ production and high inducibility by CIR-target engagement supports the hypothesis that TIR domain receptors are engaged by dimeric HLA-G and themselves dimerize. This creates CIR-NK cells that have low tonic activity and high inducible potential. EXAMPLE 6: COMPARISON OF ILT4 AND ILT2 BINDERS WITH ALTERNATE SIGNALING DOMAINS.

[0392] CIR constructs described in the previous examples that demonstrated the highest levels of enhanced potency when expressed in NK cells were selected and the ILT4 binder replaced with ILT2. The signaling domains described in this example are listed in Figure 31. Each construct contained a CIR and IL-15 separated by a 2A sequence and RFP encoding cistron to mark the cells and assess transduction efficiency. These constructs were transduced into NK cells to produce CIR-NK cells and the CIR expression level, viability of NK cells with different signaling capabilities, capacity to produce cytokines in the presence and absence of target and cytotoxicity against a range of targets expressing HLA-G was determined.

[0393] Viability of NK cells at day 8 and day 14 was determined by staining with Actinomycin D and propidium iodide such that the integrity of the cell membrane to exclude dye measured individual cell viability (Figure 32). CIR-NK cells containing each activationAtty Docket No.: NKLT-002WO domain demonstrated high viability >80% at day 14 that was comparable to control NK cells transduced to express only RFP. The growth potential of transduced cells was examined by counting populations at day 5, day 8 and day 14 and comparing the expanded level of cells at day 8 or day 14 relative to that at day 5 (Figure 33). All CIR- NK cells expressing an ILT4 binder expanded well through to day 14, but CIR-NK cells with an ILT2 binder and only a first-generation (1G) CAR (ILT2.CD3ζ), a 4-1BB domain or MyD88 domain linked to DAP10 and CD3ζ failed to expand significantly between days 8 and 14. Other ILT2 CIR-NK cells expanded readily. The mean levels of ILT4 CIR expression were determined in CIR-NK cells with each alternative signaling domain at day 8 and day 14 by flow cytometry with an antibody specific for the ILT4 D1 / D2 domain. CIR expression was high at day 8 (five days post- transduction) and generally stabilized to an appreciable mean fluorescence intensity (MFI) at day 14 (Figure 34). Overall transduction efficiency and stability was evaluated with the co-transduced RFP protein. Overall expression was lowest for MyD88- containing CIR constructs.

[0394] Despite low levels of CIR expression, CIR-NK cells with MyD88 linked to all domains readily produced tonic IFN-γ which is a good marker for signaling transduced through the NF-κB pathway following expansion to day 14 (Figure 35). Levels were elevated relative to a first-generation CAR (1G) lacking coactivation and comparable to constructs expressing 4-1BB, DAP12, DAP10 and recruiting endogenous MyD88 through fusion with the TIR domain of TLR2 in various combinations.

[0395] When cocultured with target cells expressing HLA-G (Kasumi1 AML cells (Figure 36) and HT-1376 bladder carcinoma cells (Figure 37)), CIR-NK cells expressing MyD88 demonstrated more potent production of IFN-γ than CIR-NK cells not containing MyD88 though all CIR-NK with coactivation domains demonstrated enhanced cytokine production relative to 1G CIR-NK cells. On a per-cell basis, levels of cytokine production upon CIR stimulation were enhanced relative to tonic levels (Figure 35).

[0396] ILT2 and ILT4 CIR-NK cells containing enhanced activation domains were potently cytotoxic against a range of HLA-G expressing target cells, but not against HLA-G- target cells. In short term (2-day) assays against Molm13 AML cells, about one-third of the targets were killed short term cytotoxicity most potent with MyD88.DAP10 activation domains linked with ILT2 or ILT4 (Figure 38). NK cell short-term expansion was variable with ILT2 CIR constructs but was most enhanced with MyD88.DAP12 fusions linked with ILT4 relative to other ILT4 containing constructs (Figure 38). In cocultures performed for 7-days, the efficacy of CIR-NK cells against AML target cells was dramatic. At NK cell (effector) to target ratios of 1:40, CIR-NK cells displayed serial killing activity that controlled the expansion of Molm13 cells relative to that observed with tumor cells alone (Figure 39 and Figure 40). Similar findings wereAtty Docket No.: NKLT-002WO observed when the same CIR-NK cell groups were cocultured with Kasumi1 cells that also express HLA-G but are somewhat harder to kill (Figures 41 and 42).

[0397] CIR-NK cells also demonstrated potent cytotoxicity and selectivity against target cell lines derived from solid tumors. Levels of HLA-G RNA were measured in HCT-116 cells derived from a human colon carcinoma and HT-1376 cells derived from a human bladder carcinoma. HCT-116 expressed low to no HLA-G RNA while HT-1376 expressed the HLA-G1, HLA-G2 and HLA-G5 isoforms. Cell surface expression of HLA-G was determined by flow cytometry with the MEMG / 9 antibody and HLA-G was readily detected in HT-1376 cells, but not HCT-116 cells (Figure 43). CIR-NK cell cytotoxicity was readily observed with HT-1376 cells (Figure 44) but not with HCT-116 cells (Figure 45).

[0398] SU8686 cells are derived from a pancreatic ductal adenocarcinoma (PDAC) and were previously noted to be particularly hard to kill with PSCA-targeted CAR-T cells despite robust expression of that target. SU8686 cells robustly express HLA-G1 on the cell surface (Figure 46) and isoform-specific QPCR detected abundant HLA-G1 (9359 copies / cell) and HLA-G2 (77,668 copies / cell) expression in SU8686 cells. While speculative, it is possible that the resistance of this cell line to targeted cell therapy was due to immunosuppression by HLA-G through ILT2. In cocultures of mock- transduced or CIR-NK cells with SU8686 cells little innate cytotoxicity was observed with the mock-transduced cells lacking CIR expression while ILT2 and ILT4 CIR-NK cells could recognize and target SU8686 cells effectively (Figures 47 and 48).

[0399] Enhanced signaling provided for effective CIR-NK cell cytotoxicity against SU8686. First generation (ILT2-CD3ζ) CIR-NK cells could not control SU8686 expansion in cocultures despite recognition of the HLA-G target and expansion of the NK cells while enhanced coactivation with the combination of 4-1BB, DAP10 and TLR2 (BB.DAP10.TLR2) was particularly effective for both tumor killing and cell expansion (Figures 47 and 48).

[0400] These findings demonstrated that CIR-NK cells with enhanced cytotoxicity domains are functional when linked with and ILT2 or an ILT4 binding domain for HLA-G. They also demonstrated that the CIR-NK cells are potent at high effector to target ratios of up to 1:40 demonstrating that many or most CIR-NK cells have so-called ‘serial killing’ ability. CIR-NK cells are also effective to control the outgrowth of target cells derived from solid tumors indicating that this technology has the potential for development as a therapy against a wide range of leukemia and solid tumor indications.Atty Docket No.: NKLT-002WO EXAMPLE 7. EXTENSION OF THE PERSISTENCE OF CIR-NK CELL POTENCY WITH ENHANCED ACTIVATION DOMAINS

[0401] NK cells have a limited functional life span in a natural setting, typically about 2 weeks, and are constantly replenished by hematopoiesis. As a cell therapy product, it is advantageous to maximize persistence of NK cell potency to treat a high tumor burden. To examine the persistence of CIR-NK cell functionality and evaluate the capacity of different activation domains to extend this potency, cocultures of ILT4 CIR- NK cells grown for a standard 14 days or extended for 26 days were prepared with Molm13 target cells at and E:T of 1:20. Activation domains contained the cytotoxicity domains of CD3ζ, DAP10 and DAP12 in varying combinations and were compared with the same combinations with 4-1BB coactivation or the TIR domain of TLR2 in varying amino-to-carboxy orientations of TLR2 relative to the cytotoxicity domains (Figure 49). Each of the CIR-NK cells was highly cytotoxic against Molm13 targets when expanded for 14 days. This potency was reduced in coculture containing ‘older’ first generation CIR-NK cells grown for 26 days and in several of the CIR-NK cells with alternative activation domains. CIR-NK cells containing 4-1BB in combination with DAP10 or DAP12 maintained potency at 26 days as did combinations of 4-1BB with DAP10 and TLR2 (BB.DAP10.TLR2).

[0402] In a further demonstration of the potency of enhanced signaling in CIR-NK cells to resist NK cell exhaustion, mock-transduced or CIR-NK cells were repeatedly challenged with Molm13 tumor target cells up to 4 times over a 9-day culture period (Figure 50). Innate cytotoxicity of mock-transduced NK cells was relatively robust against this target line and was exhibited when Molm13 were cultured only one time with NK cells and quickly exhausted if further Molm13 targets were added at day 2. First generation CIR-NK cells had reduced potency with 3 or more serial additions of tumor target. Enhanced signaling elements were differentiated with 3 or 4 additions of Molm13 target and it was evident that the BB.DAP10 and particularly the BB.DAP10.TLR2 combination of signaling elements with either an ILT2 or ILT4 ‘binder’ maintained NK cell potency with repeated tumor challenge. This finding was confirmed and extended in a similar experiment with a refined cohort of candidate CIR-NK cells. Again the combination of BB.DAP10.TLR2 on ILT2 and ILT4 CIRs exhibited the most effective maintenance of NK cell cytotoxicity (Figure 51) and proliferation (Figure 52) relative to other CIRs and NK cells transduced only with the RFP marker. The differentiation of CIR signaling domains was also examined in a ‘stress test’ of potency. Molm13 cells were cocultured with NK cells at increasing effector to target ratios (E:T) of 1:40 through to 2:1. Short term (2-day, Figure 53) and longer term (7- day, Figure 54) cytotoxicity was determined by quantitative imaging in the IncucyteAtty Docket No.: NKLT-002WO incubating microscope. Again, the combination of BB.DAP10 and BB.DAP10.TLR2 outperformed other combinations for cytotoxicity and for NK cell proliferation (Figures 54 and 55). It is interesting and surprising that cytotoxicity is enhanced so potently in NK cells with this combination of signaling nodes because none of 4-1BB, DAP10 or the TIR domain of TLR2 contain canonical sequence elements of ITAM domains that drive CD3ζ cytotoxicity in T cells.

[0403] These findings raise the potential for CIR-NK cells to have high persistence as a cell therapy in a patient setting. By extension, fusion of these activation domains with an scFv to produce a non-canonical CAR may increase the therapeutic potential of CAR- NK cells against targets other than HLA-G. EXAMPLE 8. LONG-TERM RESISTANCE TO CIR-NK CELL EXHAUSTION WITH ENHANCED ACTIVATION DOMAINS.

[0404] An effective cell therapy in a cancer patient should maintain potency for an extended period to overcome a potentially large tumor burden and to minimize the chance of tumor relapse. To model the efficacy of CIR NKs over a prolonged period in vitro, a challenge model was designed. OE19 cells are derived from an esophageal tumor and express relatively low levels of HLA-G1 (2080 RNA copies / cell) and HLA-G2 (899 copies / cell). Despite relatively low levels of HLA-G expression, ILT4 CIR-NK cells specifically target OE19 cells with CIR-NK cells harboring a BB.DAP10.TLR2 activation domain particularly effective at an E:T ratio of 1:10 (Figure 57).

[0405] To promote NK cell exhaustion over the course of three weeks, mock-transduced NK cells and CIR-NK cells were exposed to HT1376 cells (that are adherent and expressing high HLA-G levels) continually by coculture at day 0, careful harvesting and counting of semi-adherent NK cells at day 5 followed by repeated coculture of the NK populations with fresh HT1376 targets. This process was repeated at day 9 and day 14 of initial coculture (Figure 58). NK cell expansion was monitored with each round of serial coculture and NK cell proliferation was found to be most robust between the second and third rounds of HT1376 exposure and CIR-NK cells with BB.DAP10 activation domains most proliferative overall (Figure 59). Variation between donors for NK cells was wide.

[0406] For the fifth round of coculture, NK cells were cocultured with the same or different tumor targets expressing varying levels of HLA-G expression. In coculture with Molm13 target cells, CIR-NK cells containing BB.DAP10 or BB.DAP10.TLR2 were most effective for tumor control (Figures 60 and 61). Donor variation was wide and somewhat expected in this model system. NK cell expansion in coculture was most evident with the BB.DAP10 combination while expansion of BB.DAP10.TLR2 was relatively poor after 26 days of tumor exposure. Similar findings were observed whenAtty Docket No.: NKLT-002WO constantly tumor-exposed CIR-NK cells were cultured with OE19 solid tumor target cells (Figures 62 and 63). Cytokine production by first generation CIR-NK cells was lost following the first round of serial HT1376 exposure but was maintained with enhanced activation domains (Figure 64).

[0407] It was surprising that the ability of CIR-NK cells with the BB.DAP10.TLR2 combination to control Molm13 was robust in the absence of NK cell growth supporting the hypothesis that this combination of signaling elements promotes particularly potent cytotoxicity and maintains this cytotoxicity despite continual encounter with tumor target. EXAMPLE 9. ANTI-TUMOR EFFICACY OF CIR-NK CELLS WITH ENHANCED SIGNALING IN VIVO.

[0408] To determine the efficacy and dynamics of CIR-NK cell activity in vivo, a xenograft model was employed. NSG mice are immunodeficient due to the Non-obese diabetic (NOD) MHC haplotype, mutation at the Severe Combined Immunodeficiency (Scid) locus and nullizygosity for the IL-2 receptor γ-chain. This mouse model is severely limited in murine T, B and NK cell numbers and supports the engraftment of human cells. 2 x 105Molm13-GFPffluc cells were transplanted into NSG mice through the tail vein and permitted to engraft and establish for 5 days. Mice were then challenged with 7.5 x 106NK cells transduced to express Orangenanolantern fused with renilla luciferase (ONL-Rluc) that uses an orthogonal substrate from firefly luciferase (ffluc). Expansion of the tumor and NK cell grafts were monitored by bioluminescent imaging (BLI) with the orthogonal substrates over the course of 17 days (Figure 65). NK cells expressing only ONL-Rluc (Mock) failed to expand in vivo and first generation ILT4 CIR-NK cells similarly exhibited poor expansion. CIR-NK cells displayed robust initial expansion in vivo with the exception of ILT2 CIR-NK cells with the BB.DAP10.TLR2 combination. ILT4-BB.DAP10.TLR2 expanded well. Both ILT2 and ILT4 CIR-NK cells with the BB.DAP10.TLR2 activation domain controlled Molm13 expansion more effectively than control groups.

[0409] To examine the dynamics of NK cell trafficking to sites of tumor engraftment, mice were euthanized by day 17 and blood, spleen and bone marrow collected from select groups including two control populations (Mock and first generation CIR-NK cell) and the groups of ILT2 and ILT4 CIR-NK cells with BB.DAP10.TLR2. Molm13-GFPffluc cells were widely present in the femoral bone marrow of mice with mock-transduced or first generation CIR-NK cells but were absent in CIR-NK cells with enhanced BB.DAP10.TLR2 signaling (Figure 66). Further examination by flow cytometry revealed that mock transduced NK cells did not effectively engraft and traffic to bone marrow (Figure 67). First generation (CD3ζ) CIR-NK cells were abundant in the boneAtty Docket No.: NKLT-002WO marrow but failed to control Molm13. Conversely, ILT2 and ILT4 effectively trafficked to and had sufficient cytotoxicity to control the expansion of Molm13 in the bone marrow.

[0410] Molm13 cells are derived from an acute myeloid leukemia (AML), a tumor known to populate the bone marrow of patients and devastate hematopoiesis. The efficacy of CIR-NK cells with enhanced activation to traffic to bone marrow and target HLA-G expressing AML indicates that CIR technology in combination with these activation domains can offer an effective NK cell therapeutic option for AML patients.

[0411] Although the foregoing invention has been described in some detail by way of illustration and example for purposes of clarity of understanding, it is readily apparent to those of ordinary skill in the art in light of the teachings of this invention that certain changes and modifications may be made thereto without departing from the spirit or scope of the appended claims.

[0412] Accordingly, the preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. Moreover, nothing disclosed herein is intended to be dedicated to the public regardless of whether such disclosure is explicitly recited in the claims.

[0413] The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. In the claims, 35 U.S.C. §112(f) or 35 U.S.C. §112(6) is expressly defined as being invoked for a limitation in the claim only when the exact phrase "means for" or the exact phrase "step for" is recited at the beginning of such limitation in the claim; if such exact phrase is not used in a limitation in the claim, then 35 U.S.C. § 112 (f) or 35 U.S.C. §112(6) is not invoked.

Claims

Atty Docket No.: NKLT-002WO CLAIMS What is claimed is:

1. A chimeric receptor protein, comprising: (a) a targeting region, that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region, comprising a transmembrane amino acid sequence; and (c) an intracellular domain (ICD), comprising a signaling region capable of transducing a signal, upon binding of said targeting region to HLA-G, into the interior of an immune effector cell to elicit effector cell function, wherein the signaling region comprises a costimulatory region comprising a MyD88 polypeptide.

2. The chimeric receptor protein of claim 1, wherein the MyD88 polypeptide comprises an amino acid sequence having 85% or more sequence identity with the sequence of SEQ ID NO:

27.

3. The chimeric receptor protein of claim 1 or claim 2, wherein the MyD88 polypeptide is fused to a CD40, 4-1BB, or HVEM costimulatory domain.

4. The chimeric receptor protein of claim 3, wherein the MyD88 polypeptide is fused to a 4- 1BB costimulatory domain.

5. The chimeric receptor protein of any one of claims 1-4, wherein the signaling region comprises a CD3ζ signaling domain, a DAP10 signaling domain, a DAP12 signaling domain, or any combination thereof.

6. The chimeric receptor protein of claim 4, wherein the signaling region comprises a CD3ζ signaling domain.

7. The chimeric receptor protein of claim 1 or claim 2, wherein the signaling region comprises a DAP12 signaling domain.

8. The chimeric receptor protein of claim 7, wherein the signaling region does not include a CD3ζ signaling domain.

9. The chimeric receptor protein of claim 1 or claim 2, wherein the signaling region comprises a DAP12 signaling domain and a CD3ζ signaling domain.Atty Docket No.: NKLT-002WO 10. An intracellular domain (ICD) polypeptide, comprising a signaling region capable of transducing a signal in an immune effector cell to elicit effector cell function, wherein the signaling region comprises (i) a CD3ζ signaling domain, a DAP10 signaling domain, or a DAP12 signaling domain, and (ii) a costimulatory region that comprises a Toll / Interleukin-1 Receptor / Resistance Protein (TIR) domain.

11. The ICD polypeptide of claim 10, wherein the TIR domain is a TLR2 TIR domain, TLR3 TIR domain, or a IL18R1 TIR domain.

12. The ICD polypeptide of claim 10, wherein the TIR domain comprises an amino acid sequence having 85% or more sequence identity with the TLR2 TIR domain of SEQ ID NO:

111.

13. The ICD polypeptide of claim 10, wherein the TIR domain comprises an amino acid sequence having 85% or more sequence identity with the TLR3 TIR domain of SEQ ID NO:

113.

14. The ICD polypeptide of claim 10, wherein the TIR domain comprises an amino acid sequence having 85% or more sequence identity with the IL18R1 TIR domain of SEQ ID NO:

109.

15. The ICD polypeptide of any of claims 10-15, wherein the signaling region comprises said CD3ζ signaling domain.

16. The ICD polypeptide of any one of claims 10-15, wherein said costimulatory polypeptide is comprised by a chimeric receptor protein that comprises: (a) a targeting region, that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region, comprising a transmembrane amino acid sequence; and (c) the ICD polypeptide.

17. A chimeric receptor protein, comprising: (a) a targeting region, that targets HLA-G, comprising a D1-D2 extracellular domain of immunoglobulin-like transcript 2 (ILT2) or immunoglobulin-like transcript 4 (ILT4); (b) a transmembrane (TM) region, comprising a transmembrane amino acid sequence; and (c) an intracellular domain (ICD), comprising a signaling region capable of transducing a signal, upon binding of said targeting region to HLA-G, into the interior of an immune effector cell to elicit effector cell function,Atty Docket No.: NKLT-002WO wherein the signaling region comprises a DAP10 signaling domain or a DAP12 signaling domain.

18. The chimeric receptor of claim 17, wherein the DAP10 signaling domain comprises an amino acid sequence having 85% or more sequence identity with SEQ ID NO:

4.

19. The chimeric receptor of claim 17, wherein the DAP12 signaling domain comprises an amino acid sequence having 85% or more sequence identity with SEQ ID NO:

4.

20. The chimeric receptor of any one of claims 17-19, wherein the signaling region further comprises a CD3ζ signaling domain.

21. The chimeric receptor of any one of claims 17-19, wherein the signaling region does not include a CD3ζ signaling domain.

22. The chimeric receptor of any one of claims 17-21, wherein the signaling region further comprises a CD40, 4-1BB, or HVEM costimulatory domain.

23. The chimeric receptor of any one of claims 17-21, wherein the signaling region further comprises a 4-1BB costimulatory domain.

24. The chimeric receptor of claim 17, comprising the DAP12 signaling domain, wherein the signaling region does not include a CD3ζ signaling domain, and wherein the signaling region further comprises a 4-1BB costimulatory domain.

25. The chimeric receptor of claim 24, wherein the DAP12 signaling domain comprises an amino acid sequence having 85% or more sequence identity with SEQ ID NO:

4.

26. The chimeric receptor protein of any one of claims 1-25, wherein the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain.

27. The chimeric receptor protein of claim 26, wherein the targeting region comprises a D3-D4 extracellular domain of ILT4.

28. The chimeric receptor protein of claim 26, wherein the targeting region does not include a D3-D4 extracellular domain of ILT4, and comprises a stalk domain.

29. The chimeric receptor protein of any one of claims 1-25, wherein the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain.Atty Docket No.: NKLT-002WO 30. The chimeric receptor protein of claim 29, wherein the targeting region comprises a D3-D4 extracellular domain of ILT2.

31. The chimeric receptor protein of claim 29, wherein the targeting region does not include a D3-D4 extracellular domain of ILT4, and comprises a stalk domain.

32. The chimeric receptor protein of claim 28 or claim 31, wherein the stalk domain comprises a ILT2, ILT4, CD28, CH2 / CH3, CH3, or CD8α stalk domain.

33. The chimeric receptor protein of any one of claims 1-33, wherein TM domain is an ILT2, ILT4, CD28, or CD8α TM domain.

34. The chimeric receptor protein of any one of claims 1-25, wherein: the D1-D2 extracellular domain is an ILT4 D1-D2 extracellular domain, the extracellular domain lacks an ILT4 D3-D4 extracellular domain, the chimeric receptor protein comprises a CD8α stalk domain, and the TM region is a CD8α TM.

35. The chimeric receptor protein of any one of claims 1-25, wherein: the D1-D2 extracellular domain is an ILT2 D1-D2 extracellular domain, the extracellular domain lacks an ILT2 D3-D4 extracellular domain, the chimeric receptor protein comprises a CD8α stalk domain, and the TM region is a CD8α TM.

36. A nucleic acid, comprising a nucleotide sequence encoding the chimeric receptor protein of any one of claims 1-35.

37. The nucleic acid of claim 36, wherein said nucleotide sequence is operably linked to a constitutive promoter.

38. The nucleic acid of claim 36, wherein said nucleotide sequence is operably linked to an inducible promoter.

39. The nucleic acid of any one of claims 36-38, wherein said nucleic acid is an expression vector.

40. The nucleic acid of claim 39, wherein the expression vector is a retroviral vector, a lentiviral vector or a plasmid vector.Atty Docket No.: NKLT-002WO 41. A genetically modified cell, expressing the chimeric receptor protein of any one of claims 1- 35.

42. The genetically modified cell of claim 41, wherein the genetically modified cell is an immune cell.

43. The genetically modified cell of claim 42, wherein the immune cell is a natural killer (NK) cell, an NK-T cell, a T cell, an iNKT cell, or a macrophage.

44. The genetically modified cell of claim 42, wherein the immune cell is a natural killer (NK) cell.

45. A method of treatment, comprising administering the genetically modified cell of any one of claims 41-44 to an individual in need.

46. The method of claim 45, wherein the genetically modified cell is autologous to the individual.

47. The method of claim 45, wherein the genetically modified cell is allogeneic to the individual.

48. The method of any one of claims 45-47, wherein the individual has cancer.

49. The method of claim 48, wherein the individual has a solid tumor.

50. The method of claim 48, wherein the individual has a liquid tumor.

51. A method of producing a genetically modified cell, the method comprising: introducing the nucleic acid of any one of claims 36-40 into a cell, thus producing a genetically modified cell.

52. The method of claim 51, wherein the genetically modified cell is an immune cell.

53. The method of claim 52, wherein the immune cell is a natural killer (NK) cell, an NK-T cell, a T cell, an iNKT cell, or a macrophage.

54. The method of claim 52, wherein the immune cell is a natural killer (NK) cell.

55. The method of claim 52, wherein the immune cell is a T cell.

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